Opioid Peptides and Blood Pressure Control
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Opioid Peptides and Blood Pressure Control
Springer-Verlag Berlin Heidelberg New York London Paris Tokyo
Prof. Dr. med. K. O. Stumpe Dr. med. Karin Kraft Prof. med. A. I.
Faden Med. Univ.-Poliklinik WilhelmstraBe 35-37 5300 Bonn 1
11th Scientific Meeting of the International Society of
Hypertension Satellite Symposium· Bonn· September 6-7, 1986
ISBN-13:978-3-540-18935-0 e-ISBN-13:978-3-642-73429-8 DOl:
10.1007/978-3-642-73429-8
Ubrary of Congress Cataloging·in-Publication Data Opioid peptides
and blood pressure controll K. O. Stumpe (ed.).
p.em. Papers presented at a meeting held in Bonn, Sept. 6-7, 1986
as a satellite symposium to the 11th
Scientific Meeting of the International Society of Hypertension.
ISBN-13:978-3-540-l8935-0 (U.S.) 1. Blood
pressure--Regulation--Congresses. 2. Opioids--PhysiologicaI
effect--Congresses. 3. Hyper
tension--Pathophysiology--Congresses. I. Stumpe, K. O. (Klaus
Otto), 1938 -. II. International Society of Hypertension.
Scientific Meeting (11th: 1985 : Heidelberg, Germany)
[DNLM: 1. Blood Pressure--drug effects--congresses. 2.
Endorphins--pharmacology--congresses. 3.
Endorphins--physiology--congresses. 4. Hypertension-
physiopathology--congresses. WG 106 0611986] OP 109.065 1988
616.1'32061--dc19 DNLMIDLC for Ubrary of Congress 88-15945
CIP
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Copyright Law of September 9, 1965, in its version ofJune 24, 1985,
and a copyright fee must always be paid. Violations fall under the
posecution act of the German Copyright Law.
© Springer-Verlag Berlin Heidelberg 1988
The use of general descriptive names, trade names, trade marks,
etc. in this publication, even if the former are not especially
identified, is not to be taken as a sign that such names, as
understood by the Trade Marks and Merchandise Marks Act, may
accordingly be used freely by anyone.
Product Liability: The publisher can give no guarantee for
information about drug dosage and application thereof contained in
this book. In every individual case the respective user must check
its accuracy by consulting other phar maceuticalliterature.
2119/3140/543210
Contents
A. I. FADEN, K. KRAFf,andK.O. STUMPE. . . . . . . . . . . . . . . .
. . . . . . . . 1
Anatomy
Distribution of Opioid Peptides Functionally Related to the
Cardiovascular System
W. KUMMER, M. REINECKE, C. HEYM, and W. G. FORSSMANN . . . . . . .
. . . . . 5
Studies on Enkephalinergic Mechanisms in Cardiovascular Centers of
the Medulla Oblongata of the Rat and their Interactions with
Centrally Administered Neuropeptide Y
A. HARFSTRAND, K. FuxE, L. F. AGNATI, A. CINTRA, M. KALlA, and L.
TERENIUS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . 13
Multiplicity of Opioidergic Pathways Related to Cardiovascular
Innervation: Differential Contribution of All Three Opioid
Precursors
E. WEIHE, D. NOHR, W. HARTSCHUH, B. GAUWEILER, and T. FINK. . . . .
. . .. 27
Physiology
A. I. FADEN. . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . .. 53
Adrenergic Opioid Interaction in the Brain Stem: Role in
Cardiovascular Regulation
G. KUNos, andR. MOSQUEDA-GARCIA. . . . . . . . . . . . . . . . . .
. . . . . . .. 62
Influence of the Opioid System on Sympathetic Activity and the
Renin-Aldosterone System in Healthy Males
M. BRAMNERT, and B. HOKFELT .......... . . . . . . . . . . . . . .
. . . . .. 71
VI Contents
Role of Leu-morphin, an Opioid Peptide, in the Central Regulation
of Fluid Balance and Blood Pressure in Rats
H. IMuRA, K. NAKAO, T. YAMADA, H. bOH, S. SHIONO,~. SAKAMOTO, N.
MORII, A. SUGAWARA, Y. SAITO, andM. MUKOYAMA ...............
83
Endogenous Opioids in the Dorsal Vagal Complex and Resting
Cardiovascular Function in the Anesthetized Rat
A. H. HASSEN, and E. P. BROUDY . . . . . . . . . . . . . . . . . .
. . . . . . . . . .. 90
Influence of Opiate Peptides on Blood Pressure Regulation and on
Hypothalamic Blood Flow
W.DEJoNG,J.COXVANPuT,andP.SANDOR ...................... 98
Opioid Peptides in Human Adrenal Medulla: Their Role in the
Modulation of Catecholamine Secretion
E. BALDI, M. MAGGI, M. L. DE FEO, C. PUPILLI, C. SELLI, R.
ZIMLICHMAN, E. FORSBERG, V. CARLA, andM. MANNELLI . . . . . . . . .
. . . . . . . . . . . . .. 103
Cardiovascular Effects of Neuropeptide Yin the Caudal Ventrolateral
Medulla
I.M. MAcRAE,andJ.L. REID . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . .. 112
G.R. VANLoON,K. PIERZCHALA,L. V. BROWN, andD.R. BROWN. . . . . . .
.. 117
Pharmacology
Opioid Receptors in the Sympathetic Supply to Blood Vessels and the
Heart
B. SZABO, D. RAMME, andK. STARKE. . . . . . . . . . . . . . . . . .
. . . . . . . .. 129
Interactions of Opioid Peptides and Adrenergic Agents in the
Regulation of Blood Pressure
H. M. RHEE. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . .. 141
Effect of Opiate Receptor Blockade on the Cardiovascular and Plasma
Noradrenaline Response to Intravenous Tyramine in Man
P. M. G. BouLOux, A. GROSSMAN, and G. M. BESSER. . . . . . . . . .
. . . . . .. 150
Effects of Mu- and Delta-Opiate Receptor Agonists on Systemic and
Regional Hemodynamics in Conscious Rats
O. S. MEDVEDEV, E. R. MARTYNOVA, and A. HOQuE. . . . . . . . . . .
. . . . . .. 159
Retardment of Development of Hypertension in the Spontaneously
Hypertensive Rat by Long-Term Kappa-Opioid Receptor
Antagonism
J. DIEHL, K. KRAFT, andK. O. STUMPE. . . . . . . . . . . . . . . .
. . . . . . . . .. 168
Naltrexone Inhibits Alpha-Methydopa-Induced Hypotension in a
Dose-Dependent Manner
Contents VII
P.L.M.VANGIERSBERGEN,G.A.HEAD,andW.DEJoNG .............. 174
W. R. DIXON, and A. CHANDRA ..............................
183
Opioid Receptor Types at Noradrenergic Neurons and their Roles in
Blood Pressure Regulation
P. ILLES, andB. BUCHER .................................. ,
190
Effect of Opioids on Plasma Levels of Immunoreactive Atrial
Natriuretic Factor
J. GUTKOWSKA, B. BARANOWSKA, K. RAcz, R. GARCIA, G. THIBAULT, M.
CANTIN, andJ. GENEST ................................. 206
Production by Systemic Enkephalin of Hemodynamic Effects by
Afferent Modulation of Autonomic Nervous System Tone
T.D.GILEs,andG.E.SANDER ............................... 212
Pathophysiology and Clinical Aspects
Endogenous Opioids in the Pathophysiology of Shock: Sites of
Action, Autonomic Involvement, and Receptor Interactions
J.W. HOLADAY,D.S. MALCOLM,andJ.B. LONG . . . . . . . . . . . . . .
. . . . .. 221
Endogenous Opioids and Blood Pressure in Man
P. C. RUBIN. . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . .. 233
Effects of Hemorrhagic Shock on Plasma Met-enkephalin, Vasopressin,
Catecholamines, and Cardiovascular Functions in Intact and
Adrenalectomized Dogs
T. KIMURA, M. INOUE, K. MATSUI, K. OTA, M. SHon, and K. YOSHINAGA.
. . .. 236
Effect of Hypertension on the Response of Plasma Beta-Endorphin to
the Cold Pressor Test
R. FUKUNAGA,N. HANDA, S. YONEDA,K. KIMURA, andT. KAMADA . . . . . .
.. 247
Normalization by Clonidine of Reduced Plasma Beta-endorphin and
Leu-enkephalin Concentrations and Elevated Blood Pressure in Young
Patients with Mild Essential Hypertension
K. KRAFT, R. THEOBALD, R. KOLLOCH, and K. O. STUMPE . . . . . . . .
. . . . .. 253
VIII Contents
C. FARSANG .......................................... 260
Effect of Low Dosage of Naloxone on Clonidine-Induced Changes in
Blood Pressure, Catecholamines, Renin, and Aldosterone in Essential
Hypertension
M. BRAMNERT, andB. HOKFELT . . . . . . . . . . . . . . . . . . . .
. . . . . . . . .. 275
Effect of Lisinopril on Circulating Neuropeptides in Essential
Hypertensive Patients
S. BRANDMAN, W. T. WISEMAN, J.D. STEPHENS, C. LONG, D.R. GLOVER,
andM.J. VANDENBURG .. . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . .. 282
Endogenous Opioids and Reversal of Renovascular Hypertension
M. E. EDMUNDS, G. 1. RUSSELL, R. F. BING, H. THURSTON, andJ. D.
SWALES.. 287
Comparison of Pain Threshold as Assessed by Tooth Pulp Stimulation
in Normotensives with Different Hypertensive Hereditary Backgrounds
and in Borderline and Established Hypertensives
S. GHIONE, C. RosA, L. MEZZASALMA, andE. PANATTONI . . . . . . . .
. . . . .. 294
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . .. 299
A.!, FADEN, K. KRAFr, and K. O. STUMPE
Following the discovery of the pentapeptide enkephalins in 1975, a
number of endogenous opioid peptides and opiate receptors have been
identified. Endogenous opioids and opiate-receptor mechanisms have
been implicated in a variety of regulat ory and dysregulatory
functions including analgesia, cardiovascular regulation, shock,
hypertension, traumatic spinal cord and brain injury, stroke,
immune func tion, feeding behavior, diuresis, gastrointestinal
motility, and respiratory control, among others.
Over the past 10 years, many studies have demonstrated a
relationship between endogenous opioids and the cardiovascular
system under both homeostatic and pathophysiological conditions.
Opioids and opiate receptors have been found in various
cardioregulatory sites within the brain and spinal cord, as well as
in peripheral tissues such as sympathetic ganglia, adrenal gland,
and heart. Both endogenous opioids and exogenous opiates have been
shown to produce potent cardiovascular effects following central
nervous system or systemic administration. Opiate-receptor
antagonists have been demonstrated to reverse hypotension from
sepsis, hypo volemia, and anaphylaxis; such studies have been used
to infer activity of endogenous opioid systems in shock. Changes in
tissue concentrations of endogenous opioids and! or opiate
receptors have been found after shock and hypertension, further
implying a role for opioid systems in the etiology of these
conditions. In addition, modification of opiate receptor
regulation, receptor binding, or opioid metabolism has also been
used to establish a potential role for endogenous opioid systems in
cardiovascular control and dyscontrol.
Although the relationship between opioids and cardiovascular
regulation has received increasing attention, there has not
previously been an international meeting devoted to this topic. In
September 1986, such a meeting was held in Bonn as a Satellite
Symposium of the International Society of Hypertension. Its purpose
was to permit anatomists, pharmacologists, physiologists, and
clinicians to interact in a critical analysis of the role of the
opioids on cardiovascular control in physiological and pathological
conditions. It was hoped that such a multidisciplinary symposium
would both serve as a state-of-the-art review and promote further
experimental and clinical research efforts in this area. Clearly,
we need to know far more about the interactions of opioids and the
cardiovascular system before establishing a clear role for the
opioid system in the physiology and pathophysiology of
cardiovascular con trol.
2 Introduction
We were most gratified by the participation of many outstanding
investigators in the scientific program. The present volume
contains the proceedings of this sym posium. It is our hope that
the book will serve both as reference for use within this field and
as a stimulus for further research.
Anatomy
w. KUMMER, M. REINECKE, C. HEYM, and W. G. FORSSMANN
Department of Anatomy, University of Heidelberg, 1m Neuenheimer
Feld 307, D-6900 Heidelberg, FRO
Introduction
Endogenous opioid peptides exert multiple modulatory effects in the
regulation of cardiovascular function at both central [11] and
peripheral sites [49]. A crucial basis for the understanding of the
complex mechanisms involved in this regulatory system is the
detailed knowledge of the morphological distribution of opioid
peptides. The morphological methods appropriate for this purpose
require antisera raised against the different opioid peptides and
the use of immunohistochemistry. However, dif ficulties arise from
the structural similarities of opioid peptides. To our present
knowledge, opioid peptides are cleavage products of three large
precursor molecules: a) proopiomeianocortin (POMC) processing
results in the production of endorphins, b) prodynorphin is the
precursor of neoendorphins and dynorphins, and c) proenkephalin
contains one copy of leu-enkephalin (LE) and several opioids
sharing the met-enkephalin (ME) sequence at their N-terminus.
However, the ME sequence is also part of endorphins, and LE
represents the N terminus ofneoendorphins and dynorphins [5, 21,
31, 32]. The multiple occurrence of the ME and LE fragment and
cross-reactions of most enkephalin (ENK) antisera with both ME and
LE complicate the interpretation of immunohistochemical studies.
Specific antisera raised against peptides characteristic for a
distinct precursor [e. g., beta-endorphin (END),
alpha-neo-endorphin (NEO) , met-enkephalin-arg-phe (MEAP)],
therefore, have to be used additionally. Thus, our
immunohistochemical study on the distribution of opioid peptides in
cardiovascular regulatory centers and in the sympathoadrenal system
is focussed mainly on precursor-specific peptides. The specificity
of the antisera used is presented elsewhere [25, 26]. The results
are compared with previous findings on ME and LE distributions and
the recent literature is included to present a survey on the
distribution of opioid peptides in the spinal cord and peripheral
nervous system with respect to cardiovascular function. The
morphol ogy of brain opioid systems has been extensively reviewed
recently [22, 33].
Paravertebral Ganglia
After colchicine treatment, principal neurons of the superior
cervical ganglion (SCG) and the stellate ganglion of the rat
exhibit moderate immunoreactive (IR)-MEAP.
6 W. Kummer et al.
b
t ·
0'
", , . j
, o'
f.
Fig. 1 a, b. Guinea pig stellate ganglion. a Dyn-IR principal
neurones and few immunorective fibres. b regional accumulation of
MEAP-IR pericellular fibres. Bar = 50 I!m
This is not the case in the same ganglia of the guinea pig. The
immunohistochemical findings are in accordance with data obtained
from high-performance liquid chromatography (HPLC) studies combined
with radioimmunoassay (RIA), showing a high MEAP content of rat SCG
when compared with guinea pig SCG [40]. Another cleavage product of
proenkephalin, ME, has been immunohistochemically localized in rat
SCG neurons [1, 2, 9, 37] . Alpha-NEO-IR neurons are visible in the
stellate ganglion of colchicine-treated rats, and both dynorphin
(DYN) A 1-17-IR and alpha NEO-IR neurons are visible in the guinea
pig stellate ganglion (Fig. 1 a) . These results correlate with the
demonstration of DYN A 1-17-IR and alpha-NEO-IR within
preganglionically denervated neurons of the guinea pig SCG and
within swollen nerve fibers at the proximal end of transected
postganglionic branches [27] . Prodynorphin processing in guinea
pig paravertebral sympathetic neurons was concluded from the marked
depletion of alpha-NEO-IR and DYN A 1-8-IR within heart extracts
follow ing treatment with the neurotoxin 6-hydroxydopamine
(6-0HDA) demonstrated by RIA and HPLC analysis [28, 49] . At
present, the alpha-NEO-IR material in rat sympathetic ganglia has
not been characterized by analytical biochemical methods.
Small intensely fluorescent (SIF) cells of paravertebral ganglia
display ENK-IR in most species, but not in rats [16, 37]. Both ME
and LE antisera were used, but due to cross-reacting properties of
the antisera with both enkephalins, with DYN, and with alpha-NEO it
is still unclear whether these pentapeptides in fact are present in
SIF cells [27,42] . In the guinea pig, peptides derived from
prodynorphin, i. e., DYN A 1- 17 and alpha-NEO, were
immunohistochemically demonstrated to occur in more SIF cells than
LE or ME [4, 42] . So far we have not detected
proenkephalin-related peptides in SIF cells of guinea pig
paravertebral ganglia using MEAP-specific anti bodies. ME-IR and
LE-IR varicosities terminating on principal neurons are a com mon
feature of mammalian paravertebral ganglia [14, 17, 27, 37, 42]. In
addition,
Distribution of Opioid Peptides Functionally Related to the
Cardiovascular System 7
MEAGL-IR-beaded fibers have been reported in the rat [15], and
MEAP-IR fiber baskets occur in human paravertebral ganglia [14]. In
the guinea pig stellate ganglion regional accumulations of ENK-IR
[42] and MEAP-IR fibers (Fig. 1 b) are striking. Costorage of
ME-IR, LE-IR, and MEAP-IR substances in pericellular varicosities
suggests proenkephalin processing in those perikarya which give
rise to these ENK-IR fibers. LE-IR [27] and MEAP-IR fiber baskets
are absent in the denervated guinea pig SCG, suggesting a
preganglionic origin ofthese fibers. ENK -IR nerve terminals of yet
unknown origin approach SIF cell clusters in rat paravertebral
ganglia [16]. In contrast to rat, DYN A 1-17-IR [27, 42] and
alpha-NEO-IR varicosities [27] can be found in guinea pig
paravertebral ganglia. They probably represent processes of SIF
cells as deduced from studies on denervated, axotomized, 6-0HDA- or
reserpine treated animals [27]. Alpha- and beta-END-IR elements
have not been detected in paravertebral ganglia [37] .
Prevertebral Ganglia
Less than 1 % of the neurons in the guinea pig inferior mesenteric
ganglion contain alpha-NEO-IR (Fig. 2a). Recently Jule et al. [20]
have detected numerous ENK-IR perikarya in the celiac ganglion of
colchicine-treated cats, but cross-reactivity of the applied
antibodies to DYN and alpha-NEO was not excluded. Most SIF cells of
guinea pig prevertebral ganglia exhibit alpha-NEO-IR ([4]; Fig.
2a), whereas DYN A-IR cell bodies are less frequent [4, 42].
MEAP-IR SIF cells occur rarely [4]. The question of coexistence of
pro en kephalin- and prodynorphin-derived opioid peptides in these
SIF cells has so far not been clarified. Only proenkephalin-related
peptides [ME, MEAP, met-enkephalin-arg-gly-Ieu, (MEAGL)] can be
identified immunohis tochemically in beaded nerve fibers within
large SIF cell clusters (Fig. 2b; [4, 15, 16,
~ ' a
" .. ,
Fig.2a,b. Consecutive sections of guinea pig inferior mesenteric
ganglion, a MEAP-IR fibers surrounding principal neurons and
penetrating a large SIF cell cluster; b Numerous NEO-IR nerve
fibers and SIF cells. Two principal neurons display NEO-IR (arrows)
. Bar = 50 !!m
8 W. Kummer et al.
37, 42]). In contrast, extremely dense networks of DYN-IR and
alpha-NEO-IR varicosities surround the principal neurons (Fig. 2a;
[7,42]). Degeneration experi ments carried out on guinea pig
inferior mesenteric ganglia reveal a peripheral (intestinal) origin
of these fibers, whereas the numerous ENK-IR [7,8] and MEAP IR
(Fig. 2b; [46]) fiber baskets stem from central (preganglionic)
neurons. Schultz berg et al. [37] observed a very dense
immunoreactive nerve terminal network in the guinea pig inferior
mesenteric ganglion after incubation with one beta-END anti serum
whereas negative results were obtained with two other beta-END
antisera. No additional evidence for the occurrence of POMC-derived
opioids in prevertebral ganglia has been presented as yet.
Blood Vessels
Our studies on the distribution of opioid peptides in the guinea
pig were carried out to identify this type of peptidergic
innervation in the perivascular plexus of many organs. Alpha-NEO-IR
and, less numerous, DYN A l-l7-IR varicosities are present mainly
around small arteries and arterioles, and sometimes related to
small veins. Skin vessels of guinea pig paw and snout are
innervated by LE-IR, DYN A l-8-IR, and alpha-NEO-IR fibers [47]. As
for large arteries, alpha-NEO-IR and DYN A l-l7-IR varicosities can
be observed along the guinea pig inferior mesenteric artery (Fig. 3
a), but not in perivascular nerves of the common carotid artery.
Differential supply with DYN-IR fibers even along a single artery
has been described recently by Morris et al. [30], who colocalized
DYN A l-l3-IR with both NPY-IR and VIP-IR in non noradrenergic
axons along a circumscribed segment of the guinea pig uterine
artery. The origin of perivascular DYN-IR and alpha-NEO-IR fibers
is still unclear, because both sympathetic [27] and sensory ganglia
[25, 47] of the guinea pig contain DYN-IR and alpha-NEO-IR
neurons.
a . ,
Fig. 3a, b. NEO-IR varicosities along a first-order branch of the
guinea pig inferior mesenteric artery (a), and within the guinea
pig atrial myocardium (b). Bar = 10 Itm
Distribution of Opioid Peptides Functionally Related to the
Cardiovascular System 9
Heart
Enkephalin-like material has been detected in heart extracts of
various mammals by different methods [19, 28, 46, 48, 49].
Surprisingly, in the guinea pig heart compara tively high ME-IR
levels were obtained in combined HPLC and RIA study [28] but almost
no ME-like activity was detected in the respective HPLC fraction
using the mouse vas deferens assay [48]. Immunohistochemically,
ENK-IR nerve fibers supply the coronary arteries of the guinea pig
heart [12, 34, 35] with a preference for the arterial system of the
atria. While no ENK-IR fibers seem to contact myocardiocytes or
nodal cells, they are, however, present in high numbers within the
intracardiac ganglia [34]. Prodynorphin-related peptides were
extracted from rat [38] and guinea pig heart [48, 49].
Immunohistochemical investigation of the guinea pig heart reveals
beaded alpha-NEO-IR nerve fibers mainly associated with arterioles
and small arteries. Fibers without a clear relationship to blood
vessels are only occasionally observed (Fig. 3 b). In general,
alpha-NEO-IR terminals are less numerous than those displaying
LE-IR. No immunoreactive fibers as yet have been observed after
incuba tion with antisera directed against DYN A 1-17. These
results correspond to biochem ical findings, revealing high levels
of alpha-NEO- and low concentrations of DYN A 1-17-like material in
guinea pig heart [48, 49], which is possibly due to the degradation
of DYN A 1-17 into smaller fragments. In the guinea pig, the marked
depletion of both extractable ENK-IR and prodynorphin-derived
peptide-IR in response to appli cation of the neurotoxin 6-0HDA
suggests a sympathetic origin of the respective nerve fibers
[28,49].
Adrenal Medulla
Opioid peptides are costored and coreleased with catecholamines
from adrenal medullary cells [29, 43]. Since the first
immunohistochemical demonstration ofENK IR in the adrenal medulla
[36], numerous studies have dealt with the distribution of ENK-IR,
MEAP-IR, and MEAGL-IR cell bodies and fibers within the adrenal
gland of various species and have revealed remarkable species
differences (see review [23]). Recently, the messenger RNA encoding
preproenkephalin has been detected in the rat adrenal medulla using
the in situ hybridization technique [3]. Endorphins are not common
to all species, but have been described in adrenal medullary cells
of pig, cow, dog, and man [10, 39]. RIA measurements of
fractionated bovine adrenal medullary cells indicated costorage of
several DYN A fragments with noradrenaline as well as costorage of
LE with adrenaline [29]. Recent work of our group [4] was focussed
on the guinea pig adrenal, which almost completely lacks typical
noradrenaline-contain ing cells [41]. Previously, DYN A 1-13-IR
cells were demonstrated in this organ [44]; however, the
immunoreactions obtained could be suppressed by preabsorption with
LE. After applying antisera specific for the C-terminus of DYN A
1-17 and alpha NEO, many cells display alpha-NEO-IR and few cells
exhibit DYN A-IR. Both immunoreactions cannot be blocked by ME or
LE. Investigations using immunos tained, plastic-embedded semithin
sections (0.5 !tm) reveal that LE-IR, ME-IR, and MEAP-IR are
coexistent with prodynorphin-related peptide-IR in some of
the
10 W. Kummer et al.
adrenal medullary cells. This indicates that no separate DYNergic
and ENKergic cell systems as proposed for the bovine adrenal
medulla [29] exist in the guinea pig.
Thoracolumbar Sympathetic Nuclei of the Spinal Cord
Preganglionic sympathetic neurons as identified by retrograde
labeling are richly supplied by ENK-IR fibers [13, 18]. Denervation
experiments in the rat revealed a supraspinal origin of these
fibers [18]. In agreement with findings on rat and sheep [33], we
observed an identical distribution pattern of MEAP-IR axons in the
guinea pig spinal cord, suggesting proenkephalin as the source of
the previously described ENK-IR material. Similarly, a previous
report on ENK-IR within perikarya of preganglionic sympathetic
neurons [6] has recently been substantiated by the use of antisera
directed against MEAGL [23]. From the ultrastructural appearance of
MEAGL-IR preganglionic fibers in the rat adrenal medulla,
coexistence ofproenke phalin-related peptides and acetylcholine
was suggested [24]. This hypothesis is strongly supported by the
simultaneous demonstration of MEAGL-IR and choline
acetyltransferase within identical neurons of thoracic sympathetic
nuclei [23]. Neither POMC-related nor prodynorphin-related peptides
have been described as yet within spinal cord sympathetic
nuclei.
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Studies on Enkephalinergic Mechanisms in Cardiovascular Centers of
the Medulla Oblongata of the Rat and their Interactions with
Centrally Administered N europeptide Y
A.lliRFSTRAND, K. FUXE, L.F. AGNATI, A. aNTRA, M. KALlA and L.
TERENIUS
Department of Histology, Karolinska Institutet, Box 60400, S-10401
Stockholm, Sweden
Introduction
In our previous work beta-endorphin, morphine, and
d-ala2-met-enkephalinamide in the nanomolar range were found to
produce preferential vasodepressor responses and bradycardia upon
intracisternal (i. c.) injection into the alpha-chloralose
anesthetized rat [1]. Leu- and met-enkephalin and alpha
neo-endorphin administered in the same way preferentially produced
vasopressor actions, which with the two latter peptides were
associated with bradycardia. Both the pressor and depressor
responses were counteracted by naloxone pretreatment i. c., but the
depressor actions were preferen tially sensitive to the blocking
activity of naloxone. These results indicated the existence of two
types of opiate receptors in central cardiovascular regulation,
both innervated by enkephalin immunoreactive (IR) terminals [2].
Subsequent work has also demonstrated the existence of high
densities of the dynorphin IR nerve terminals and cell bodies
within the nucleus tractus solitarii (nTS) and in the nucleus
ambiguus [3,4]. The dynorphin synapses may inter alia operate via
kappa-opiate-receptors, and an injection of preferential
kappa-opiate agonists into the nucleus ambiguus and nTS produces
cardiovascular actions [5]. Both enkephalin and dynorphin peptides
and their associated opiate receptors may therefore be involved in
the regulation of the activity of the cardiovascular centers of the
medulla oblongata. In a recent study by Kalia et al. [6] it was
found that enkephalin IR terminals densely innervate the subnuclei
of the nTS receiving baroreceptor and chemoreceptor afferents
(dorsal strip, dorsal subnucleus, dorsal parasolitary region) and
also subnuclei receiving cardiac afferents (commissural nucleus of
the nTS) and gastrointestinal afferents (medial subnucleus of the
nTS). Substantial numbers of enkephalin IR nerve cell bodies have
also been observed in all the various subnuclei, including the
lateral respiratory subnuclei, which also receive sparse to
moderate enkephalin innervation [6].
These results indicated that there may be large numbers of
enkephalin IR inter neurons participating in the integration of
information in the cardiovascular, respira tory, and
gastrointestinal subnuclei of the nTS.
14 A. Hlirfstrand et al.
In the present paper, we have analyzed the codistribution of
enkephalin IR nerve terminals within the nTS, the dorsal motor
nucleus of the vagus, the nucleus ambi guus, and the C1 area in
relation to the distribution of [3H]etorphin and [3H]D-ala2-
D-Ieu5-enkephalin-binding sites, which represent markers for mu-
and delta-opiate receptors respectively [10], using
immunocytochemistry and receptor auto radiography.
Materials and Methods
Specific pathogen-free, 200- to 250-g male Sprague-Dawley rats
(ALAB, Stockholm, Sweden) were used.
Immunocytochemical Experiments
The rats underwent transcardiac perfusion with 150 ml 0.1 M sodium
phosphate buffer containing 4% (w/v) paraformaldehyde and 0.4%
(w/v) picric acid. The brain stem was then kept in the fixative for
4 h and then transferred to a 10% sucrose solution.
Twenty-micrometer-thick serial cryotome sections of the medulla
oblongata were made at levels 1 mm caudal to 2 mm rostral to the
obex. For further details of the immunocytochemical procedures, see
Fuxe et al. [14].
The location of enkephalin IR cell bodies, nerve terminals, and
preterminal proces ses was examined by the biotin-avidin
peroxidase method (Vecta Stain ABC, Vector Laboratories,
Burlington, California. The same procedure was also used to study
tyrosine hydroxylase (TH) IR in adjacent coronal sections.
Immunocytochemical analysis was combined with cytoarchitectural
identification of the various nuclear subgroups, performed on
adjacent sections using thionine staining [6]. For characteri
zation ofthe enkephalin antiserum used, see Schultzberg et al.
[11], forcharacteriza tion of the TH antiserum, see Hokfelt et al.
[12], and for purification of TH, see Markey et al. [13].
Receptor Autoradiographic Experiments
The rats underwent transcardiac perfusion with ice-cold sodium
chloride solution (0.9% w/v) under methohexital sodium anesthesia.
The medulla oblongata was dis sected out and frozen and coronal
14-J.tm-thick sections were made in a Leitz cryostat at various
rostrocaudal levels of the medulla oblongata, matching those taken
for immunocytochemistry. For further details, see Hiirfstrand et
al. [8]. In the [3H]etor phin-binding experiments the radioligand
concentration was 1.5 nm, and the sections were incubated with the
radioligand for 45 min at room temperature. Nonspecific binding was
defined as the binding in the presence of naloxone (2 J.tm) [10].
In the [3H]D-ala2-D-leu5 (DADL)-enkephalin experiments the
radioligand concentration was 10 nm. The binding procedure was
performed for 60 min at room temperature. Nonspecific binding was
defined as the binding in the presence of levalorphane (1 J.tm)
[10]. A tritium-sensitive sheet film eH Ultrofilm, LKB Stockholm,
Sweden),
Studies on Enkephalinergic Mechanisms in Cardiovascular Centers
15
was used. Exposure time was 4-6 weeks. The specific activity was 44
Cilmmol for the [3H]DADL-enkephalin and 36 Cilmmol for the
[3H]etorphin. Both ligands were purchased from NEN, United
States.
Physiological Experiments
Arterial blood pressure (ABP) and heart rate (HR) were recorded as
described [16, 17]. Briefly, alpha-chloralose anesthesia was
introduced by an injection into the lingual vein using a dose of
100 mg/kg after an initial anesthesia with halothane (3% in air).
Mean arterial blood pressure (MAP) and HR were measured by means of
a heparinized catheter positioned in the common carotid artery. The
catheter was connected to a Statham PC23DC transducer connected to
a Grass polygraph (Model 7).
Measurement of the respiration rate (RR) and indirect measurement
of tidal volume were carried out by means of an intraesophageal
catheter positioned at the mid-level of the mediastinum. This
catheter was also connected via a Statham trans ducer to the Grass
polygraph. The basal values were recorded for a 30-min period prior
to the i. c. treatment with the morphiceptine and/or neuropeptide
(NPY). The i. c. injections were made by means of a stereotaxic
device. All substances were dissolved in mock CSF and the injection
volume was 10 Ill. The body temperature was maintained at 370-37.5
0C, and each rat was used only once. Morphiceptine was purchased
from Peninsula (Belmont Ca., United States) and NPY from Bachem
(Bubendorf, Switzerland). In the statistical analysis Dunn's test
was used.
Results
Immunocytochemical Studies
In Fig. 1 a - f the distribution of enkephalin IR nerve terminals
is demonstrated within both the dorsal and ventral cardiovascular
areas of the medulla. In Fig. 1 c the distribution of the
enkephalin IR nerve cell bodies is also shown, since this section
was taken from an animal which had been treated with 120 mg
colchicine i. c. 48 h previously. The dense enkephalin innervation
of the part of the nTS medial to the TS is shown together with the
dense innervation of the dorsal motor nucleus of the vagus (mnX). A
sparse to moderate enkephalin innervation is shown in the lateral
subnuclei of the nTS as well as in the part of the reticular
formation extending from the nTS toward the Al and CI areas. In
Fig. 1 a-c a dense innervation by enkephalin IR terminals is also
found within the subtrigeminal part of the lateral reticular
nucleus. At these levels a moderate enkephalin innervation is also
found in the adjacent part of the caudal subnucleus of the nucleus
tractus spinalis nervi trigemini.
In Fig. 1 c enkephalin IR nerve cell bodies are found in large
parts of the reticular formation of the medulla oblongata. They are
usually scattered but aggregated within the Cl area.
In Fig. 2 (lower half) the distribution of enkephalin IR nerve cell
bodies is shown in great detail within the nTS and the area
postrema at a level - 0.2 mm caudal to obex.
16 A. Harfstrand et al.
A TS I PT
E ity is demonstrated at various rostrocaudal
dmnX levels of the medulla oblongata of the male rat. In c the
section is taken from a colchicine- treated rat (120 Jlg, i.
c.,
C3 48 h before killing). The level is indicated in the
- nSpVI lower left part of each figure and indicates the distance
in millimeters from the obex. The biotin-avidin immuno- peroxidase
procedure
Oi was used. Primaryanti- serum was diluted at
1.6 py 1: 1000. ap, area post- rema; TS, tractus sol-
Studies on Enkephalinergic Mechanisms in Cardiovascular Centers
17
B
-0.05
1.1. itarius; nXII, hypoglos- sal nucleus; cc, central canal; PT,
paratrigemi- F nal nucleus; nRtpc, par vocellular reticular nuc-
leus; LRt, lateral reticu lar nucleus; LRtPC, parvocellular part
of the lateral reticular nucleus; LRtS5, subtrigeminal part of the
lateral reticu larnucleus;py, pyrami dal tract; Oi, inferior
olive; nSp VC, caudal part of the nucleus trac tus spinalis nervi
trige mini; nSp VI, inter positus part of the nuc leus tractus
spinalis nervi trigemini; aA, 2.0 ambiguus nucleus
1mm
I
1mm
18 A. Hiirfstrand et at.
Enkepbalin IR nerve cell bodies are found in almost all subnuclei
of the nTS with the exception of the dorsolateral subnucleus. Large
numbers are also found in the external zone of the area postrema.
Within the dmnX, however, only enkephalin IR terminals are found
and the low density within the lateral subnuclei of the nTS
relative to the medially located subnuclei is also further
illustrated.
TR IV , ./.' I
ENK IV
200~m .
Fig. 2. Tyrosine hydoxylase and enkephalin-like immunoreactivity
are shown in adjacent coronal sections of the medulla oblongata at
the level of the area postrema. The biotin-avidin immunoperoxid
ase procedure was used. The TH antiserum was diluted 1: 1500 and
the enkephalin antiserum at 1:1000. The rat had been pretreated
with colchicine i.c. 48 h before killing (120 flg). Substantial
numbers of enkephalin IR nerve cell bodies are found in all the
subnuclei of the nTS and in the outer zone of the area postrema.
The TH IR cell bodies are predominantly found within the medial
subnucleus ofthe nTS (mnTS) and within the dorsal subnuclei (dorsal
strip) (ds), dorsal subnucleus of the tractus solitarius (dnTS),
dorsal parasolitary region (dPSR), and area postrema. The
enkephalin IR nerve terminals appear as fine dots in the background
within all the various subnuclei of the tractus solitarius and
within the dmnX. dinPS, dorsolateral subnucleus of the tractus
solitarius; ni, interstitial subnucleus; vinTS, ventrolateral
subnucleus of the tractus solitarius; vnTS, ventral subnucleus of
the tractus solitarius; PVR, periventricular region; ncom,
commissural nucleus; cc, central canal; dmnX, dorsal motor nucleus
of the vagus; nRtpc, parvocellular reticular nucleus. Asterisks
indicate the same vessels
Studies on Enkephalinergic Mechanisms in Cardiovascular Centers
19
In Fig. 2 (upper half) the distribution of the catecholamine (CA)
nerve cell bodies as demonstrated by TH IR is shown in the nTS in
an adjacent section. There is an obvious codistribution with
enkephalin IR cell bodies within the medial subnucleus, the dorsal
strip, the dorsal subnucleus, and the dorsal parasolitary region.
However, the enkephalin IR cell bodies are found in much larger
numbers within the laterally located subnuclei and within the
intestitial nucleus of the tractus solitarius. Also in the area
postrema a substantial difference exists, since the TH IR cell
bodies are found to be located all over the area postrema and in a
high density.
In Fig. 3 the existence of substantial numbers of large enkephalin
IR nerve cell bodies is shown within the reticular
paragigantocellular nucleus, within the ventral part of the
gigantocellular reticular nucleus, and within the nucleus raphe
magnus. Also the dense enkephalin innervation of the nucleus
ambiguus is demonstrated. At this level the ventrolateral medulla
as seen in the adjacent section contains the adrenaline cell group
CI, which thus codistributes with large numbers of enkephalin IR
nerve cell bodies (see also Fig. 4). However, no evidence for
coexistence of enkephalin and TH IR has been found in this region,
using the occlusion method of Agnati et al. [18] . In Fig. 4 the
existence of a dense plexus of enkephalin IR nerve terminal within
the nucleus ambiguus is shown together with a further illustration
of the distribution of enkephalin cell bodies in the ventral
medulla (see also Fig. 1 e, f).
Fig. 3. Tyrosine hydroxylase and enkephalin IR are demons trated
in the ventrolateral medulla of the colchicine-tre ated rat (for
details, see text to previous figure legends). The
immunoreactivities are demon strated in adjacent sections at a
level + 1.8 mm rostral to obex. Both enkephalin and TH IR cell
bodies are demonstrated in the Cl area but without any obvious
codistribution within that area. The Cl area consists mainly of the
paragigantocellular reticu lar nucleus (PGi) and the reticular
gigantocellular nuc leus, ventral part (Giv). For other
abbreviations, see text to previous figures. Asterisks indi cate
the same vessels
TH 1:2500 .
i l~ *
EN~ 1:1000
) py J ./ Fig. 4. Enkephalin-like IR is
demonstrated in the ventral medulla with the ventral mid line
region and the nucleus ambiguus in a coronal section of the medulla
oblongata at a ros trocaudallevel + 1.8 mm rostral to obex. The
biotin-avidin peroxidase procedure was used. Large numbers of
enkephalin IR nerve cell bodies are found within the PGi and Giv as
well as in the ventral midline area mainly nucleus raphe magnus. A
dense enkephalin innerva tion is demonstrated in the nuc leus
ambiguus
Fig.5a-d. The nTS, the dmnX, and adjacent parts of the medulla
oblongata are shown in coronal section at the rostrocaudallevel -
0.20 mm caudal to obex following staining for nerve cell bodies
(thionin staining) (a) and following incubation with pH]D-ala2
-leus -enkephalin (DADL) (10 nm) and following incubation with
[3H]etorphin (ETO) (1 nm). It is shown that thereis dense labeling
of all the subnuclei of the nTS medial to the tractus solitarius
and moderate labeling in the dmnX following incubation with
[3H]DADL en Kephalin (10 nm). As for the incubation with PH]ETO the
labeling is mainly confined to the dorsal subnuclei of the nTS and
the periventricular region. Note the relative absence of labeling
in the dmnX. For abbreviations, see text to previous figures
Studies on Enkephalinergic Mechanisms in Cardiovascular Centers
21
3H - DADL 10nM
Receptor Autoradiographic Experiments
The results are summarized in Fig. 5 a, band 6. At the area
postrema level [3H]D-ala2-
D-leu5-enkephalin seems to label strongly the entire nTS with the
exception of the lateral subnuclei and the dmnX, while the
[3H]etorphin densely labels only the periventricular regions
surrounding the area postrema and the dorsal cardiovascular
subnuclei. The dmnX is only weakly labeled, which is true also for
the lateral
3H-ETO 1nM
3H-DADL 10nM
+1.7 Fig. 6. Visualization of [3Hletorphin (ETO) (1 nm), and
[3H1D-ala2-D-leu5-enkephalin (10 nm) binding in two paralle114-!lm
coronal sections of the rostral part (obex + 1.7 mm) of the medulla
oblongata of the rat. Note the intense [3H1ETO binding in the
lateral part of the nTS. TS, tractus solitarius; nA, nucleus
ambiguus
Studies on Enkephalinergic Mechanisms in Cardiovascular Centers
23
subnuclei. [3H]D-ala2-D-Ieu5 enkephalin also substantially labels
the area postrema. In Fig. 6a and b both [3H]etorphin and
[3H]D-ala2-D-leu5 enkephalin are shown to label strongly the
nucleus ambiguus. However, at this rostral level (1.7 mm rostral to
obex) only [3H]etorphin strongly labels the lateral subnuclei of
the nTS. Instead the dmnX and the medial subnuclei with adjacent
reticular formation and the ventral parasolitary region are
moderately labeled by both radioligands. Also both radioligands
only weakly label the ventrolateral medulla.
Functional Experiments
The results obtained in the studies on the interaction between NPY
and morphiceptin have demonstrated additive cardiovascular
responses obtained upon their coad ministration i.c. into the
a-chloralose-anesthetized male rat (Fig. 7.). Thus there appears to
develop an additive action, when morphiceptine and NPY are given
together in low doses, producing weak hypotensive responses by
themselves. No such interactions were observed with heart rate and
respiratory rate (Fig. 7).
Discussion
Overall the present study demonstrates a codistribution of
enkephalin IR nerve terminals and mu- and delta-opiate receptors
within the nTS, nucleus ambiguus, and CI area. This appears to be
especially true in the cardiovascular nuclei of the nTS, i. e.,
dorsal strip, dorsal parasolitary region, dorsal subnucleus and
adjacent periventricu lar region, and in the nucleus ambiguus that
may control the HR. The baroreceptor afferents are known to
terminate within the dorsal cardiovascular nuclei of the nTS.
Therefore, enkephalins released in these regions, probably reach
both the mu- and delta type of opiate receptors, both of which may
participate in modulation of baroreceptor reflex activity [24].
Thus, these results strongly support the view of the existence of
multiple opiate receptors involved in central cardiovascular
regulation [1, 5,20,21].
It seems possible that the two types of opiate receptors which have
been shown to overlap in many nuclei in the present study may
regulate the sensitivity of one another by a receptor-receptor
interaction [22] so that a more selective response can be obtained.
The results indicate that these subtypes of opiate receptors are
both reached by enkephalins.
It is of substantial interest that the lateral nuclei of the nTS,
which are involved with respiratory regulation, in the rostral
parts of the nTS are characterized by a high density of mu-opiate
receptors, while only a sparse plexus of enkephalin IR terminals
and a relatively low density of delta-opiate receptors are present.
Thus, in this region there is a mismatch between the pre- and
postsynaptic elements of enkephalin neurons. Furthermore the
mu-opiate receptors dominate. Such a mismatch has also been noted
in previous studies by Agnati et al. [23] in a correlation analysis
between the distribution of enkephalin IR and beta-endorphin IR
nerve terminals and the distribution of [3H]etorphin and of
[3H]d-ala2-d-Ieu5-enkephalin-binding sites in the tel- and
diencephalon. The mismatch was predominantly observed within
the
24 A. Harfstrand et al.
MAP
~ 10
0
-10
-20
Ba8al valuee: MAP HR RR n:
~
NPY 75pmol. 107t 6 427t16 81t6 6 ..... ~~~~:; Morphlceptln
1nmol
Fig. 7. Cardiovascular and respiratory effects of NPY and
morphiceptine are shown after their combined i. c. administration
in the alpha-chloralose-anesthetized male rat. The time curves are
shown in the left part in the figure. The values are given as means
± SEMs and taken as percentage of respective mean basal value. In
the right part of the figure the means ± SEMs are given for the
vasodepressor areas (D P A) and vasopressor areas (VP A) (upper
part), for the tachycardic (TCA) and the bradycardic (BPA) areas
(middle part), and for the tachypneic and bradypneic areas (lower
part) in arbitrary units calculated by an IBM XT (cardiovascular
software by GUNA Consult Stockholm, Sweden). The statistical
analysis was performed by means of Dunn's test for multiple
comparisons. • P < 0.05; *. P < 0.01
thalamus and hypothalamus. Based on these observations it was
suggested that in these areas enkephalin synapses may mainly
operate via volume transmission, i. e., that the opiate receptors
are reached by enkephalins, which have diffused from distant
enkephalin nerve terminals present in the same or adjacent regions.
Such a volume transmission could result in a high plasticity and a
long-term action [23]. Thus it should be considered that also in
the lateral nTS and other parts of the nTS
Studies on Enkephalinergic Mechanisms in Cardiovascular Centers
25
enkephalins may be released also in a paracrine fashion to reach
distant opiate receptors. It seems likely that the
[3H]etorphin-binding sites within the lateral nTS are reached by
enkephalins, since they appear to have a similar affinity to those
located within the medial nTS and the dorsal subnuclei and in the
nucleus ambiguus.
In contrast, the dmnX appears to be innervated by enkephalin
synapses which predominantly operate via the delta-opiate receptors
relative to mu-opiate receptors. These results further underline
the view that each enkephalin IR nerve terminal system may operate
with its own unique set of opiate receptors. It will be of
substantial interest also to evaluate how the kappa-opiate
receptors within the dorsal and ventral cardiovascular centers are
distributed in relation to the mu- and delta-opiate recep tors, in
order to possibly further understand interactions between these
three main types of opiate receptors in the brain and their
involvement in cardiovascular control.
In previous work we have demonstrated that a high density of
125I-NPY-binding sites exists within the dorsal subnuclei of the
nTS [8]. In the present study a high density of mu-opiate receptors
was also demonstrated at this site. It was therefore of substantial
interest to evaluate the cardiovascular effects of coadministered
NPY and morphiceptine given i. c. In the doses tested, NPY and
morphiceptine were both found to produce weak hypotensive actions
when given alone, and additivity was observed in the
coadministration experiments. Thus, unlike adrenaline and NPY,
which when given together centrally show no additivity and even
antagonistic interac tions with regard to their hypotensive
actions, the mu-opiate receptors and the NPY receptors appear to be
able to regulate cardiovascular responses without any obvious
interactions; that means neither antagonism nor enhancement of
their cardiovascular effects [25]. Thus, central NPY and mu-opiate
receptor mechanisms may show additivity in their ability to lower
arterial blood pressure.
Acknowledgments. This work was supported by a grant from the
Swedish Medical Research Council (04X-715). We are grateful to
Sylvia Oliphant for excellent secre tarial assistance.
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(1986) Receptor autoradiographical evidence for high densities of
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Multiplicity of Opioidergic Pathways Related to Cardiovascular
Innervation: Differential Contribution of All Three Opioid
Precursors
E. WEIHE*, D. NOHR*, w. HARTSCHUH**, B. GAUWEILER*, and T.
FINK*
• Department of Anatomy, Johannes Gutenberg-University, Mainz, FRG
** Department of Dermatology, University of Heidelberg, Heidelberg,
FRG
Introduction
The endogenous opioid family consists of the three precursors
proenkephalin (proenkephalin A), prodynorphin (proenkephalin B),
and proopiomelanocortin (POMC) , from which various opioid and
nonopioid peptides can be processed, apparently in a
tissue-specific manner (cf. Civelli et al. 1984; Goldstein 1984;
Herz 1984; Udenfriend and Kilpatrick 1984; Civelli et al. 1985;
Khachaturian et al. 1985; Kosterlitz 1985). Their distribution in
areas of the CNS which are involved in car diovascular regulation
is well documented. The biochemistry and functions of endoc rine
(pituitary and adrenal) opioids have also been well characterized
(cf. Millan and Herz 1985). The conception that endocrine and CNS
opioid peptides and receptors may play an important role in various
physiological and pathophysiological car diovascular regulatory
mechanisms is widely accepted (cf. Holaday 1983; McQueen 1983;
Holaday, this volume).
Peripheral neuronal, paracrine, and perhaps even nonadrenal
endocrine opioid systems related to the heart itself as well as to
the vasculature were investigated by biochemical and
immunohistochemical methods (McQueen 1983; North and Egan 1983;
Weihe and Reinecke 1983; Weihe et al. 1983; Vincent et al. 1984;
Xiang et al. 1984; Weihe et al. 1985, b, c; Bumstock 1986;
Hartschuh et al. 1986; Howells et al. 1986). Their molecular
identities, tissue-specific distribution, and precise actions in
cardiovascular regulation under resting or pathophysiological
conditions are still unclear. Differential presynaptic opioid
sympathoinhibitory receptors apparently playa crucial role in the
heart and in different vascular beds (cf. Starke 1977; Fukuda et
al. 1985; Krumins et al. 1985; Starke et al. 1985, Fuderet al.
1986; Illes et aI.1987).
The present study is based on the consideration that there may be
also differential processing and distribution of the various opioid
peptides in intrinsic or extrinsic efferent or afferent nerves
supplying the peripheral cardiovascular system. To investi gate
this hypothesis we envisaged a systematic immunohistochemical study
which was aimed to determine the preponderant molecular forms and
peripheral cardiovascular histotopography of the potential plethora
of peptides derived from the three opioid precursors. Particular
attention was paid to characterize peptides of the opioid family in
primary sensory afferents which are one source of cardiovascular
innervation. Their interrelation with nonopioid peptides, in
particular substance P, which are sensory transmitter candidates of
antidromic vasodilation and peripheral inflammat ory mechanisms
(cf. Salt and Hill 1983; Lundberg and Hokfelt 1986) will be
also
28 E. Weihe et al.
determined. Qur study concentrated on the guinea pig, but questions
of interspecies variations are also addressed.
Materials and Methods
Tissue Processing for Immunohistochemistry
Various tissues of several adult mammalian species (ten
guinea-pigs, five rats, four cats, two dogs, and three rabbits)
were fixed by perfusion or immersion with Bouin's solution. In some
cases a prefixation with a freshly prepared 4% paraformaldehyde/ 1
% glutaraldehyde solution was employed. Some tissues of pig, in
particular skin, were obtained within 15 min from a local slaughter
house and fixed by immersion in Bouin's solution. One isolated pig
heart (about 20 min postmortem) was perfused with the same
fixative. Human tissue (skin) was fixed by immersion in Bouin's
solution. Tissues were processed for immunohistochemistry using
various enzymatic (horseradish peroxidase) or immunofluorescence
methods as described (Weihe et al. 1984; 1985a; 1986).
Antisera
A plethora of commercial and donated antisera against various
opioid and nonopioid peptide sequences which are contained in the
three opioid precursors was used. 1. Polyclonal rabbit antisera
against proenkephalin (PRO-ENK)-opioid sequences:
Met-enkephalin (Immunonuclear); Met-enkephalyl-Arg-Phe,
Met-enkephalyl Arg-Gly-Leu, metorphamide, Leu-enkephalin, BAM 12 P
(Weber et al. 1983a, b); amidorphin (Seizinger et al. 1985).
2. Polyclonal rabbit antisera against prodynorphin (PRO-DYN)-opioid
sequences: Leu-enkephalin, dynorphin A 1-8, alpha-neoendorphin,
dynorphin B (cf. Weber et al. 1983a, b); alphalbeta-neoendorphin,
dynorphin A 1-17 (cf. Millan et al. 1986).
3. Polyclonal rabbit antisera against POMC-derived opioid peptide
~-endorphin (Immunonuclear, Peninsula) and against the non opioid
sequences adrenocor ticotrophic hormone (ACTH) (Weber et al.
1983a) and alpha-melanocyte stimulating hormone (alpha-MSH,
Immunonuclear, cf. Khachaturian et al. 1985) werde also used.
In addition, monoclonal mouse antibodies against the opioid message
sequence Tyr-Gly-Gly-Phe (Meo et al. 1983) and against
Leu-enkephalin (Seralab, Cuello et al. 1984) were employed.
Rabbit polyclonal antisera against calcitonin gene-related peptide
(CGRP) were obtained from Peninsula or Amersham and a monoclonal
rat antibody against subst ance P (SP) from Serotec. A rabbit
polyclonal antiserum against atrial natriuretic factor (ANF) was
also used (Arendt et al. 1985).
Working dilutions of polyclonal antisera in immunoenzymatic
procedures were in the range from 1 : 6000 to 1 : 80000. The
monoclonal SP antibody (ascites) was used in
Multiplicity of Opioidergic Pathways Related to Cardiovascular
Innervation 29
a dilution of 1 :50 to 1 : 400. The dilution of the monoclonal
antibody (ascites) against Leu-enkephalin varied from 1: 5000 to 1:
40000.
The specificities of all antisera were tested in various tissues
under our immunohis tochemical conditions by preabsorption with
homologous synthetic antigens and a plethora of heterologous
antigens having varying degrees of sequence homology (Weihe et al.
1986).
Sections were analyzed and photographed on a Leitz-Orthoplan light
microscope if not otherwise mentioned.
Results
Specificity of Antisera
In most cases the specificity of antisera was found to be
essentially similar to that determined by other authors or that
indicated on commercial data sheets.
The main specificity characteristics were: The antiserum against
Met-enkephalin (ME) cross-reacted with Leu-enkephalin (LE) and with
other peptide sequences containing either pentapeptide at the
N-terminus to a certain extent. The polyclonal antiserum against LE
also dit not fully differentiate LE-, ME-, or C-terminal extended
forms of either pentapeptides including heptapeptide (ME-RF),
octapep tide (ME-RGL), dynorphin A 1-17 (DYN A 1-17), DYN A 1-8,
DYN A 1-13, or alphalbeta-neoendorphin (alpha/~-NEO). Thus, the
antisera against the two pen tapeptides were to be expected to
stain PRO-ENK as well as PRO-DYN-opioid peptides to an extent which
could not be neglected.
The antisera against ME-RF or ME-RGL did not cross-react with
PRO-DYN sequences or with POMC sequences and therefore could be
regarded as being table to stain PRO-ENK sequences rather
specifically. The antiserum against metorphamide (METOR) was very
specific since no cross-reactions with other opioid peptides could
be observed. The amidorphin (AMID OR) antiserum recognized neither
PRO-DYN nor POMC sequences, but cross-reacted with peptide F. It
therefore could also be regarded as being able to stain the PRO-ENK
family specifically.
The antisera against DYN A 1-17, DYN A 1-8, and NEO did not
cross-react with any PRO-ENK or POMC-opioid sequences. They did not
recognize the penta peptides and therefore appear to be very
specific in staining only the PRO-DYN family. In addition they
appeared to discriminate different dynorphins and neoendor
phins.
The monoclonal antibody against LE did not discriminate between ME
and LE. Since it did not recognize larger molecular forms of the
pentapeptides it was found to be very specific for the two
pentapeptides as described (Cuello et al. 1984). The alpha MSH
antiserum cross-reacted with des-acetyl-alpha-MSH but not with
ACTH. The beta-endorphin antiserum (Peninsula) showed some
cross-reactivity with beta lipotropins and with Met-enkephalin
although the commercial data sheet indicated zero cross-reaction
with ME in radioimmunoassay (RIA). Immunoreactions obtained with
the beta-endorphin antiserum were partly but not completely
preabsorbable with up to 10 !-tmol beta-lipotropin or ME. The
monoclonal antibody against SP did not recognize opioid
peptides.
30 E. Weihe et al.
Histotopography of Immunoreactivities
Paravertebral and Prevertebral Sympathetic Ganglia of Guinea
Pig
In stellate ganglia immunoreactive(IR)-ME-RF, IR-ME-RGL, IR-AMIDOR,
and IR-peptide F were equally present in fibers and varicosities,
surrounding about one third of principal ganglionic cells. The
intraganglionic distribution of these codistri buted IR -opioid
peptides overlapped with that of the equally frequent IR-ME and IR
LE (Fig. la, Table 1). As a rule, the density of IR-METOR or IR-BAM
12 P
Table 1. Distribution of selected IR derivatives of the three
opioid precursors in extrinsic and intrinsic neurons and paracrine
systems related to peripheral cardiovascular innervation in guinea
pig
Sympathetic ganglia (stellate) Principal neurons Paraganglionic
cells Fibers Axosomatic contacts
(mesenteric) Principal neurons Paraganglionic cells Fibers
PRO-ENK PRO-DYN PRO-ENKI POMC PRO-DYN
ME-RGL METORDYN NEO LE MSH* ~-END
- /+ +++ +++ ++ ++ +++ ++ ++
++++ +++ ++++ ++ +++
+++ +++ ++ +++ +++ ++ ++ +++ ++
-/+ +++ ++++ +++ ++++ +++ ++
Fig. la, b Adjacent sections of guinea pig stellate ganglion, a
Immunoreactive LE in fibers and endings surrounding a major
population of non-IR principal ganglionic cells and in a group of
para ganglionic cells (arrow), x 150. b Immunoreactive-alpha-MSH
is present in large-diameter intraganglionic fibers but absend from
cells, x 150
Multiplicity of Opioidergic Pathways Related to Cardiovascular
Innervation 31
Table 1. (Continued)
ME-RGL METORDYN NEO LE MSH* ~-END
Sensory (spinal and trigeminal) ganglia
Small cells +++ +++ +++ Intermediate cells + + + -/+ Large cells
-/+ -/+ ++++ - Fibers ++ ++ ++ ++++ -
Somatik nerve trunks -/+ + + ++ +++
Vagal nerve -/+ -/+ -/+ ++ +++
Phrenic nerve -/+ -/+ ++ +++
Heart (intrinsic ganglia) Ganglionic cells -/+ NT Paraganglionic
cells -/+ ++ ++ +++ NT Fibers + -/+ ++ ++ +++ +++ NT
(chemoreceptor glomera) Paraganglionic cells -/+ + + +++ NT Fibers
-/+ -/+ +++ -/+ NT
(neuroeffector junctions) Aorta, pulmonary trunk + + ++ NT Coronary
arteries + + ++ NT Microvasculature + + ++ NT Myoendocrine atrial
cells -/+ -/+ NT Conduction system + + ++ NT
(nonneuroeffector fibers) Interstitial and paravascular + + ++ +++
NT
Neurovascular fibers in Skin + + + ++/+++ ++++ NT Mucocutaneous
junctions + + + ++/+++ ++++ NT Skeletal muscle + +++ NT Joint
tissues NT NT + ++ NT Digestive system
Tongue + -/+ -/+ +++ ++++ NT Salivary glands ++++ NT ++++ ++ NT
Esophagus ++ ++ ++ +++ + NT GEP system +++ ++ +++ ++++ + ?
Urogenital tract +++ +++ +++ +++ ++ -/+ Respiratory tract +++ ++ NT
Lymphatic system + + NT Adrenal gland +++ + ++ ++ ++++ +++ NT
Intrinsic neurons GI tract +++ + ++ ++ ++++ + +
Paracrine cells Epithelium of GI tract +++ Respiratory epithelium
+++
Subjective rating of relative densities of IR fibers or IR cells:
-, absent; -/+, very low; +, low; + +, moderate; +++, high; + +++,
very high. (?) presence questionable; NT, not tested; note
thatIR-MSH* occupied myelinated fibers (nonvaricose) larger than A
delta whereas IR-opioid peptides were restricted to C and also some
A delta fibers (varicose). For more details, the constellation of
other IR peptides, and comparison with other species see text
32 E. Weihe et al.
surrounding a significantly minor proportion of principal
ganglionic cells was lower. In mesenteric cells was lower. In
mesenteric ganglia, the vast majority of principal ganglionic cells
were innervated by an extremely dense plexus containing IR-ME-RF,
IR-ME-RGL, IR-AMIDOR (peptide F), IR-ME, and IR-LE as well as IR-SP
and IR-CGRP. IR-METOR was less frequent. In comparison with
IR-PRO-ENK-sequ ences, IR-PRO-DYN sequences (alpha/beta-neo, DYN A
1-17, DYN A 1-8) were less frequent in stellate but almost equally
frequent in mesenteric ganglia. Innervation of the ganglionic
microvasculature was a regular finding. IR-PRO-ENK and IR-PRO DYN
sequences were codistributed, but coexistence in individual fibers
or endings could not be assessed unequivocally. Relatively
infrequent, and apparently merely trespassing, IR-SP and IR-CGRP
fibers, but no IR-primary ganglionic cells, were present in
stellate and mesenteric ganglia whereas mesenteric ganglia
contained abundant IR-SP and IR -CG RP fibers which not only
trespassed but also targeted non IR ganglionic cells.
Paraganglionic cells in stellate ganglia contained IR-ME, IR-LE,
IR-ME-RGL, IR-DYN A 1-17, IR-DYN A 1-8, and IR-alphalbeta-NEO but
apparently no IR METOR (Fig. 1 a). It was not unequivocally clear
whether IR-PRO-ENK and IR PRO-DYN sequences coexisted in
individual paraganglionic cells. Almost all para ganglionic cells
in the abdominal paraganglia in the vicinity of mesenteric
sympathetic ganglia contained IR-PRO-ENK sequences (ME, LE, ME-RGL,
AMID OR) but apparently no IR-METOR, which was restricted to
paraganglionic fibers. The IR PRO-DYN sequences (DYN A 1-17, DYN A
1-8, alpha/beta-NEO) were clearly present in about one-half of the
paraganglionic cells. The staining produced by the monoclonal
LE-antibody was equivalent to that of the polyclonal antisera
against ME or LE. No clear evidence was obtained for the presence
of opioid immunoreactivity in principal ganglionic cells. Some of
them showed faint staining with the alpha/beta NEO antisera, but
this was not drastically different from background staining to
allow classification as being unequivocally specific at the current
stage of our investigations using non-colchicine-treated animals.
In some lumbar paravertebral ganglia we observed some principal
ganglionic cells faintly stained with the antiserum against
ME-RGL.
Some IR-beta-endorphin fibers were seen in stellate ganglia.
However, this immunoreactivity was partly pre absorbable with ME.
Large-diameter fibers contain ing IR-alpha-MSH were visualized
trespassing stellate and prevertebral sympathetic ganglia. No
IR-MSH principal ganglionic or paraganglionic cells were observed
(Fig.1b).
Sensory ganglia
In guinea-pig, in all segmental dorsal root ganglia and in
trigeminal ganglia, mainly small cells « 20 !lm), but also some
cells of intermediate (20-40 !lm) diameter, contained coexisting
IR-PRO-DYN sequences (LE, alpha/beta-NEO, DYN A 1-17), but no
IR-PRO-ENK-sequences. The staining pattern of the monoclonal
antibody against the opioid message sequence was equal.
Interestingly, the antiserum angainst ME produced no staining in
the sensory cells in spite of its potential cross reactivity to
LE. The frequency of opioid-IR cells varied depending on the
segmental level. The highest number of opioid-IR cells was in the
lumbosacral ganglia (Fig. 2).
Multiplicity of Opioidergic Pathways Related to Cardiovascular
Innervation 33
Fig. 2a, b Adjacent sections oflumbosacral dorsal root ganglion L5
of guinea pig. a Immunoreactive DYN A 1-17 in mainly small diameter
ganglionic cells and fibers, x 360. b Immunoreactive SP in small
and medium-sized ganglionic cells and fibers, x 360. Identical
cells labeled with IR-DYN and IR-SP are marked by arrows. Normarsky
optics (Zeiss Axiophot)
Also centrally and peripherally directed processes and
small-diameter fibers in the C or A delta-range contained
IR-PRO-DYN sequences. A part of them supplied the ganglionic
microvasculature. The monoclonal antibody against LE visualized
almost the same number of cells and fibers as the other antisera
against prodynorphin sequences. Almost all large (> 40 !lm)
cells and myelinated, but not unmyelinated, fibers contained
IR-alpha-MSH whereas other IR-POMC pep tides were absent (Fig.
3).
In a substantial, but not total, number of small and also some
medium-sized ganglionic cells IR-PRO-DYN sequences, IR-SP and
IR-CGRP were found to coexist (Fig. 2a, b). There were also
nonopioid IR cells containing IR-SP or IR-CGRP and vice versa, as
revealed by analyzing adjacent sections. Nonopioid-IR cells
containing IR-SP or IR-CGRP were mainly of medium or large
diameter. The overall number of opioid IR cells was lower than of
those containing IR-SP or IR-CGRP. IR-MSH was found to be
independent of opioid-IR or SP/CGRP-IR cell and fiber
populations.
In rats neither IR-PRO-DYN sequences nor IR-PRO-ENK sequences could
be detected unequivocally. Lumbosacral dorsal root ganglia of cat
contained equal
34 E. Weihe et aI.
Fig. 3. Immunoreactive alpha-MSH in the majority of large diameter
cells and fibers of cervical dorsal root ganglion C4 of guinea pig,
x 180
numbers of IR-PRO-DYN sequences in mainly small but also
medium-sized cells. In ganglionic cells of cervical dorsal root
ganglia of rabbits IR-LE and IR-DYN A 1-17 were present.
Pig trigeminal ganglia contained some IR-LE, but also some
IR-ME-RGL cells. Coexistence patterns in non-guinea-pig species
have not yet been analyzed. IR-MSH was found in the sensory ganglia
of all species investigated, and it was always localized in most of
the large cells and larg-diameter myelinated fibers. The
immunostaining of sensory ganglionic cells was successfully
performed without colchicine treatment.
Peripheral Somatic and Visceral Nerve Trunks
It was very difficult to visualize opioid-IR fibers in peripheral
nerves because their diameter is very small, mainly in the
nonmyelinated range. In contrast the population of SP-IR and
CGRP-IR fibers, which were in the larger-diameter range (A delta),
was visualized more easily. Thick myelinated alpha-MSH-IR fibers
were distinguished without any difficulty. The most suitable
antiserum for visualization of opioid-IR fibers was the polyclonal
antiserum against LE, which, however, cross-reacted with several
PRO-ENK and PRO-DYN sequences. LE-IR fibers were seen in the
cervical and thoracic vagal nerve, in the phrenic nerves, in the
trigeminal branches, and in the brachial plexus, and in the sciatic
nerves of guinea pig. The vasa nervorum appeared to be innervated
with IR-LE fibers. Results with other species were equivocal and
will be published elsewhere.
Multiplicity of Opioidergic Pathways Related to Cardiovascular
Innervation 35
Heart and Macrovasculature
Immunoreactive LE was the predominant opioid immunoreactivity which
could be visualized in guinea pig heart. IR-LE nerve fibers were
sparse and present in small diameter fibers supplying coronary
arteries, some intramural vessels, endocardium, valves, epicardium,
and pericardium. IR-LE was present in neuroeffctor and non
neuroeffector fibers (Table 1). Some IR-LE was found in guinea pig,
dog, rabbit, and pig sinoatrial node. It was also present in nerves
supplying coronary vessels of pig and dog. The situation in the cat
was equivocal.
Immunoreactive LE predominated in intrinsic cardiac ganglia where
numerous IR LE fibers and endings surrounding ganglionic cells
were seen in guinea pig, dog, and pig (Fig. 4a, b). Equivocal faint
staining of some ganglionic cells was observed. IR ME-RGL in some
cardiac nerves was rare. Some intracardiac ganglia contained IR
NEO and IR-DYN A fibers which were also seen in a few coronary
nerves of guinea pig and cat.
Paraganglionic cells of various cardiac ganglia contained IR-ME,
-LE, and -DYN A 1-8 particularly in guinea pig, but also in cat
(Fig. 4 b). Guinea pig paraganglionic cells of aortic, pulmonary,
and coronary cardiac glomera as well as small cells in the carotid
body contained IR-ME, IR-LE, IR-DYN A 1-8, and IR-alpha-NEO.
Results with other species and antisera were equivocal.
Immunoreactive LE small-diameter fibers exhibiting some
varicosities were regul ary seen in the thoracic aorta, pulmonary
trunk, and carotid artery of guinea pig, cat, and dog (Fig. 4c).
IR-LE fibers were denser adjacent to cardiac, aortic, or carotid
glomera. Some of the IR-LE fibers penetrated the outer media; also
contacts to the vasa vasorum were seen. IR-SP or IR-CGRP fibers
were much more frequent in these locations. IR-MSH was regularly
seen in numerous large-diameter myelinated fibers of paravascular
nerves whereas other IR-POMC peptides were not visualized .
b c
. .
Fig. 4a-c Guinea pig intrinsic cardiac ganglia and
macrovasculature: a Immunoreactive LE in varicose fibers and
endings surrounding juxtasinunodal intrinsic ganglionic cells, x
750. b Immunoreactive DYN A 1-8 in a paraganglionic cell (arrow) of
a juxtacoronary intrinsic ganglion and in a few fiber profiles, x
750. c Immunoreactive LE small-diameter fibers in the
media-adventitia border of juxtacardiac ascending aorta, x
750
36 E. Weihe et al.
Somatic and Mucocutaneous Tissues
In the skin and in various mucocutaneous tissues (nose, ear, and
anogenital regions), in striated muscle, and in joint tissues IR-LE
was the predominant opioid IR form in nerves supplying the
vasculature which could be clearly visualized (Table 1). In skin,
some IR-DYN A 1-8 and IR-NEO fibers were observed to be associated
with the subepidermal microvasculature and other cutaneous blood
vessels. IR-LE was regu larly seen in close contact with papillary
venules in skin and mucocutaneous tissues. It was also present in
association with the microvasculature of some striated muscles and
of various joint tissues. This pattern was observed in guinea pigs
and to a certain extent also in pig and cat. In these locations
codistribution with some of the much more frequent IR-SP and
IR-CGRP fibers was obvious. There were many large diameter fibers
containing IR-MSH in these tissues and in all species investigated
including human skin (Fig. 5).
Vasculature of the Digestive System
Several molecular forms of opioid peptides were visualized
throughout the digestive tract. The tongue of guinea pig, dog, and
in particular cat was extremely rich in IR-LE fibers supplying
arteries, veins, and the microvasculature, including that of
subepithe lial areas and of seromucous glands (Fig. 6). The
vasculature of the submandibular
.. '
6
Fig. 6. Immunoreactive LE in abundant small diameter varicose
fibers supplying cat intraling ual artery (tangential-longitudinal
section), x 160
Multiplicity of Opioidergic Pathways Related to Cardiovascular
Innervation 37
v
7
Fig. 7. Immunoreactive LE in small-diameter fibers supplying the
microvasculature (venule, v) of guina pig submandibular gland, x
2000
\ .~ ,
8
glands was also richly innervated with IR-LE fibers in guinea pig,
cat, and dog (Fig. 7). In guinea pigs numerous IR-ME-RGL
varicosities were seen in the salivary glands and their
microvasculature whereas IR-PRO DYN sequences were absent in this
location.
Opioidergic fibers were seen throughout the gastroenteropancreatic
(GEP) sys tem. Numerous fibers containing IR-PRO-ENK sequences
(ME, LE, ME-RGL, ME-RF, AMIDORIPEPTIDE F) and IR-PRO-DYN sequences
(DYN A 1-17, DYN A 1-8, DYN B, NEO) supplied the vasculature -
including the microvascula ture - of the lamina propria,
submucosa, and muscle cell layers in esophagus, stomach, duodenum,
jejunum, ileum, and colon of guinea pig and rat (Fig. 8, Table 1).
The guinea pig gastrointestinal (GI) tract seemed to contain more
IR-PRO-DYN sequences than that of the rat . Duodenum of cat and dog
contained IR-PRO-ENK and IR-PRO-DYN proportions similar to guinea
pig. Intrinsic ganglia of the entire digestive tract contained
IR-PRO-ENK and IR-PRO-DYN sequences in many fibers and perikarya.
Interestingly, IR-METOR was almost restricted to ganglia of the
myenteric plexus where it was present in numerous perikarya and
fibers. IR-DYN A 1-8 predominated in perikarya of the submucous
plexus whereas the IR-PRO-ENK sequences showed so