| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Zoophysiology, Göteborg University, S-413 90 Göteborg, Sweden; and 2 Department of Zoology, University of Queensland, Brisbane, Queensland 4072, Australia
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Peptidergic mechanisms influencing the resistance of the gastrointestinal vascular bed of the estuarine crocodile, Crocodylus porosus, were investigated. The gut was perfused in situ via the mesenteric and the celiac arteries, and the effects of different neuropeptides were tested using bolus injections. Effects on vascular resistance were recorded as changes in inflow pressures. Peptides found in sensory neurons [substance P, neurokinin A, and calcitonin gene-related peptide (CGRP)] all caused significant relaxation of the celiac vascular bed, as did vasoactive intestinal polypeptide (VIP), another well-known vasodilator. Except for VIP, the peptides also induced transitory gut contractions. Somatostatin and neuropeptide Y (NPY), which coexist in adrenergic neurons of the C. porosus, induced vasoconstriction in the celiac vascular bed without affecting the gut motility. Galanin caused vasoconstriction and occasionally activated the gut wall. To elucidate direct effects on individual vessels, the different peptides were tested on isolated ring preparations of the mesenteric and celiac arteries. Only CGRP and VIP relaxed the epinephrine-precontracted celiac artery, whereas the effects on the mesenteric artery were variable. Somatostatin and NPY did not affect the resting tonus of these vessels, but somatostatin potentiated the epinephrine-induced contraction of the celiac artery. Immunohistochemistry revealed the existence and localization of the above-mentioned peptides in nerve fibers innervating vessels of different sizes in the gut region. These data support the hypothesis of an important role for neuropeptides in the control of the vascular bed of the gastrointestinal tract in C. porosus.
sensory neurotransmitters; somatostatin; vasoactive intestinal polypeptide; gut circulation; reptiles
| |
INTRODUCTION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
IN CROCODILIANS, THE RIGHT and left aortas emanating from the left and right side of heart, respectively, communicate at two points: the foramen of Panizza, a hole in the shared interaortic septum immediately outside the heart, and an anastomosis between the two vessels in the abdominal cavity proximal to the gut. During nonshunting conditions, no blood is ejected from the right ventricle into the left aortic arch, and the total flow in this aorta is due to a small net flow through the foramen of Panizza from the right aorta. During pulmonary-to-systemic shunting, blood is ejected from the right ventricle into the left aorta, and this blood flow in the left aorta, directed toward the gut, is therefore dependent on the magnitude of the shunt (1, 2, 11, 12, 17, 25). During nonshunting conditions, the majority of the celiac artery blood originates from the right aorta via the anastomosis (1, 19). The mechanisms regulating the arterial resistance and hence blood flow to the gastrointestinal tract have not been elucidated, although in the study by Karila et al. (19), which investigated the occurrence of neuropeptides in the cardiovascular system of the estuarine crocodile, Crocodylus porosus, several populations of peptidergic neurons in the anastomosis were found that may participate in the control of vessel tonus. Substance P (SP) and calcitonin gene-related peptide (CGRP) coexisted (100%) in one group of neurons, and tyrosine hydroxylase (TH, indicating adrenergic neurons), neuropeptide Y (NPY), and somatostatin coexisted to a high degree in another group. Vasoactive intestinal polypeptide (VIP)-, galanin-, and bombesin-like fibers were also present in the anastomosis. The cardiovascular effects of SP, NPY, and neurotensin were tested in vivo, and SP and NPY increased the flow through the anastomosis and celiac artery. The increase in celiac flow by SP agreed with results from a previous study by Axelsson et al. (1). Also bombesin has been shown to increase the celiac blood flow (13).
The aim of our study was to investigate fundamental regulatory effects of neuropeptides on arterial vascular resistance in the gastrointestinal canal of C. porosus and, by relating the responses to the known effects in other vertebrates, also to put it in an evolutionary perspective. In addition to immunohistochemical localization of peptide-containing neurons in different vessels of the gut, in situ perfusion of the mesenteric and celiac vascular beds and in vitro studies on isolated arteries were performed to increase the understanding of peptidergic control mechanisms of the gut vasculature in this species.
| |
MATERIALS AND METHODS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Estuarine crocodiles, Crocodylus porosus (body mass 0.2-1.5 kg), were purchased from the Edward River Crocodile Farm (Cairns, Australia) and flown to Brisbane. They were kept in 4-m-diameter outdoor tanks containing freshwater at 28°C and were fed on chicken necks weekly. Ethical clearance for this study was obtained from the University of Queensland Animal Experimentation Ethics Committee (approval nos. ZOO/688/95/ARC and ZOO/687/95/ARC).
Anatomy of Gut Circulation
In one animal, a corrosion cast of the gastrointestinal circulation was made to more closely examine the structure and location of the vasculature. The celiac and mesenteric arteries were cannulated as described below, and both vascular beds were thoroughly rinsed using Ringer solution. The casting material (Mercox resin, CL-2B) was simultaneously injected into the celiac and mesenteric arteries at physiological pressure and allowed to set overnight. The tissue was dissolved in 20% KOH with alternate rinses in HCl over 72 h. The cast was dissected and mounted onto scanning electron microscope stubs using copper print paste and sputter coated with gold (~50-70 nm coating). The microvasculature was viewed in a JEOL 6400 field emission electron microscope (15-kV voltage) and photographed on Ilford FP4 Plus film.Immunohistochemistry
The two animals (body mass 0.2 and 0.5 kg) used for histochemical studies were killed by injection of pentobarbital (1 ml/kg), and samples were collected from the mesenteric artery and small arteries supplying the stomach and intestine. The tissues were fixed overnight at 4°C in Zamboni's fixative (15% saturated picric acid, 2% formaldehyde in 0.1 M phosphate buffer, pH 7.2). The fixative was washed out with ethanol, and the preparations were subsequently dehydrated, xylene treated, and rehydrated. They were stored in PBS with 30% sucrose as a cryoprotectant until they were cut in 10-µm sections on a cryostat (7). To diminish unspecific staining, the sections were preincubated with 10% normal donkey serum in 0.1 M PBS (2% NaCl, 0.01% sodium azide; pH 7.2) for 30 min and then incubated with either one single or, to reveal possible colocalization, different combinations of primary antibodies (Table 1) for 2 days in a moist chamber at room temperature. The slides were washed in PBS, incubated with species-specific secondary antibodies for 1-2 h, washed, and finally mounted in buffered glycerol (pH 8.6). The secondary antibodies were purchased from JacksonImmuno Research and conjugated with CY 3, DTAF, AMCA, or biotin. The biotinylated antibodies were detected with streptavidin conjugated with AMCA. All antibodies were diluted with 0.1 M PBS (2% NaCl, 0.01% sodium azide, 10% normal donkey serum, pH 7.2). The preparations were viewed with a fluorescence microscope with filters for multiple labeling.
|
In Situ Perfusions
Crocodiles were killed instantaneously by a sharp blow to the head followed by transection of the spinal cord. The abdominal cavity was opened, and heparin was injected into the left ventricle. The celiac artery and, in six preparations, also the mesenteric artery were located and cannulated with polyethylene or polyurethane tubing (ID of 0.58-0.8 mm). The abdomen was isolated from the head and tail, giving an open venous outflow, and the spinal cord in this region was pithed. The preparation was immersed in saline (0.9% NaCl), and the cannulas were connected to constant-flow peristaltic pumps that perfused the preparation with crocodile Ringer solution (11). The inflow pressures in the two perfused vascular beds were measured using in-line pressure transducers calibrated against a static water column. The pressure signals were amplified on a Sensel AB 4 CHAMP amplifier and displayed on a Yokogawa LR 8100 recorder and simultaneously sampled with a personal computer using AD/DATA software (Dr. Peter Thorén, Karolinska Institute, Stockholm, Sweden); sampling frequency was 5 Hz, and mean values were stored every 10 s.To study effects on the somatic circulation, the tail was cut off posterior to the cloaca. The spinal cord of the tail was pithed, and the caudal artery was cannulated with polyurethane tubing (ID of 0.8 mm) and secured with silk. The preparation was immersed in saline and connected to a constant-flow peristaltic pump as described above.
Experimental Protocol
The flow from the peristaltic pumps gave an inflow pressure of between 3 and 8 kPa, depending on the intrinsic splanchnic vascular resistance of each animal. After each experiment, the volume pumped per minute was measured, giving the mean flow through each vascular bed. Pump rate was set to ~40 pulses/min. Thus both mean pressure and pulse rate were set to levels within the physiological range of C. porosus (12).Each peptide was injected in a bolus dose (0.1 ml/kg of body mass) into the inflow cannula. Initially, the peptides were given according to a dose-response scheme that revealed suitable doses producing reproducible responses. When these were found, only a submaximal dose was further used to avoid possible tachyphylactic effects. The peptides were injected in a randomized order. In each preparation, injection of the vehicle (0.9% NaCl) served as control. With the constant flow perfusion, changes in vascular resistance caused by peptide injections were recorded as changes in inflow pressure.
Some of the peptides caused an increase in motility of the gut wall,
masking the effects on vascular resistance by causing spikes of
increased pressure. Therefore, in three of the perfusion experiments,
tetrodotoxin (10
7 M) was
added to the Ringer solution to reduce gut motility and thereby
reveal vascular effects possibly hidden under these pressure spikes.
Isolated Vessel Ring Preparations
To investigate the peptidergic actions on the gut vasculature without the possible interference from gut motility, isolated ring preparations of the celiac and mesenteric arteries were made. The vessels were carefully mounted on stainless steel L-shaped hooks connected to Grass FT03 force transducers and placed in organ baths containing oxygenated crocodile Ringer solution. Caution was taken to minimize disturbance of the endothelial cell layer. The vessels were stretched until they corresponded to a 0.5- to 2-g load (5-20 mN), depending on arterial size and estimated to imitate physiological tension, and allowed to equilibrate for 30 min.Experimental Protocol
The vessels were activated twice with KCl (50 mM) and subsequently with epinephrine (106 M).
Epinephrine concentration-response curves were constructed before
testing the effects of the peptides. The peptides were given in a
randomized order, initially in a cumulative way with increasing
concentrations and later in single concentrations to avoid
tachyphylaxis. They were given either to the relaxed vessel or to an
epinephrine-precontracted
(107-106
M) vessel, depending on the peptide. Vasorelaxation was calculated as
percent of epinephrine precontraction level (100%). Possible synergistic effects of somatostatin and NPY on the epinephrine response
were also investigated in the celiac and mesenteric artery. Individual
responses to epinephrine and the peptide were compared with responses
to the combination of the two. The possible presence of intrinsic
inhibitory nitric oxide production was studied using the nitric oxide
synthase (NOS) inhibitor
NG-nitro-L-arginine
methyl ester (L-NAME;
104 M).
Statistics
Data are presented as means ± SE for number (n) of animals indicated. Differences between the maximum response to the peptide and the preinjection value were evaluated using Wilcoxon's signed rank test for matched pairs, where a level of P < 0.05 was considered statistically significant. Effects on isolated vessels were tested using Wilcoxon's test or a Student's t-test when n was small (P < 0.05).Chemicals
The following drugs were used: epinephrine bitartrate, galanin (pig), gastrin-releasing peptide (GRP) (pig), L-NAME, neurokinin A (NKA) (mammalian), NPY (human), somatostatin (mammalian), SP (mammalian), and tetrodotoxin (all from Sigma Chemical, St. Louis, MO). CGRP (chicken) and VIP (chicken) were both from Peninsula Laboratories (Belmont, CA). Stock solutions of peptide dissolved in 0.9% NaCl containing bovine serum albumin (1 mg/ml) were made, and subsequently the drugs were diluted in saline. Tetrodotoxin was dissolved in an acetate buffer (pH 5) and subsequently diluted in saline. Composition of the crocodile Ringer solution was (in mM) 110.0 NaCl, 4.0 KCl, 2.8 CaCl · 2H2O, 1.4 MgSO4 · 7H2O, 2.6 NaH2PO4 · H2O, 238.0 NaHCO3, and 5.6 glucose. The Ringer was bubbled with oxygen (3% CO2) and during the experiments held at room temperature (26°C).The primary structures of crocodilian peptides are known in some cases (isolated from Alligator mississippiensis) (34, 35). We have tried to use peptides that are as similar as possible in amino acid sequence to the alligator homologues. Mammalian NKA, NPY, and somatostatin together with chicken VIP, all used in the present study, are identical to the alligator counterparts, whereas mammalian SP differs by one amino acid. Pig galanin is identical to alligator galanin in its NH2-terminal region (residues 1-22), which is considered the receptor binding domain of the peptide. Pig GRP differs in eight residues compared with alligator GRP, and the structure of crocodile CGRP is, as far as we know, presently unknown. The dissimilarities in primary structure between the presently used and the native peptides probably affect receptor binding affinities, which should be a consideration.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Anatomy of the Gut Vasculature
Posterior to the anastomosis, by definition, the right aorta continues as the dorsal aorta and the left aorta as the celiac artery. The mesenteric artery branches off the dorsal aorta just posterior to the anastomosis. At the microvascular level, imprints from sphincter-like structures were present at the base of arterial ramifications of 100 µm inner diameter or less (Fig. 1, B-D), thus indicating control sites for vascular resistance. The capillary system of the stomach mucosa was flat in appearance (Fig. 1E), distinct from the folded structure of the intestinal mucosa (Fig. 1F).
|
Immunohistochemistry
Immunoreactive nerve fibers were found at varying densities (see Table 2) in the mesenteric artery and in smaller vessels running to the stomach and the intestine, with all the listed antisera (Table 1). Most of the stained fibers were seen in the adventitiomedial border and in the adventitia. CGRP and SP were colocalized to 100% in the mesenteric artery and, at least to some extent, in the smaller vessels. A subpopulation of CGRP immunoreactive fibers also showed NKA immounoreactivity (Fig. 2). In the small vessels on the stomach, there was a complete overlap between the NPY and TH immunoreactivities. NPY also coexists with somatostatin, indicating that these nerve fibers contain all three substances. No coexistence was found between VIP and NPY.
|
|
In Situ Perfusions
The gastrointestinal circulation was perfused at a constant flow. In most preparations, the mesenteric inflow pressure was slightly oscillatory, whereas the celiac pressure had a more stable baseline. The mean flow through the arteries was 2.8 ± 0.4 ml/min for the mesenteric artery (n = 6) and 6.7 ± 0.5 ml/min for the celiac artery (n = 8) at a mean input pressure of 5.5 ± 0.7 and 4.8 ± 0.8 kPa, respectively. Several of the peptides induced stomach contractions, thus interfering with vascular pressure measurement. The addition of tetrodotoxin (107 M) to the Ringer
solution usually reduced, but did not abolish, these gut movements.
Effects of the injected peptides were seen within 1 min and generally
lasted 15-20 min. Initially, a dose of epinephrine
(1010 mol/kg) was given to
the preparation. An ensuing vasoconstriction, and thus increase in
inflow pressure, assured a viable and vasoactive preparation. When only
the vehicle (0.9% NaCl) was injected, no response was seen.
Tail Perfusions
The mean flow through the caudal artery was 5.2 ± 0.9 ml/min at a mean pressure of 5.4 ± 0.4 kPa (n = 9). Epinephrine, which was injected initially, caused a dose-dependent increase in the inflow pressure, whereas the vehicle itself did not cause any major changes in pressure.Isolated Vessel Preparations
Both the celiac artery and the mesenteric artery were contracted by 50 mM K+. Epinephrine caused concentration-dependent contractions of the celiac artery (n = 6), whereas the mesenteric artery showed various responses. In two preparations, epinephrine relaxed the vessels, an effect that could be reversed into a contraction by the
-antagonist sotalol (4.4 × 104 M), whereas three
preparations showed delayed intermittent contractions. Addition of the
NOS inhibitor L-NAME
(104 M,
n = 4) affected neither the basal
tonus of the vessels nor their response to epinephrine.
Summary of the Vascular Effects of the Various Peptides
The vascular effects of the various peptides are summarized in Table 3.
|
Effects of SP.
In seven of the preparations, injection of SP (100 pmol/kg,
n = 8) induced visible stomach
contractions. In the celiac vascular bed, the pressure usually
increased in spike surges, at first coinciding with contractions of the
gut wall. After a few minutes, most of the contractions had ceased, and
subsequently the pressure significantly decreased to a subbaseline
level (
0.3 ± 0.1 kPa) (Fig.
3A).
Maximum vasodilatation occurred after 3.0 ± 0.6 min, and the effect
usually lasted ~15 min. In the mesenteric vascular bed, the pressure
decreased in four preparations, and gut motility obstructed measurement
in two other preparations. In the three tetrodotoxin-Ringer-perfused
preparations, the stomach contractions were reduced, but only one of
them showed a total inhibition, showing the underlying decrease in the
perfusion pressure as a cause of the vasodilatation. The gut
vasculature was sensitive at levels as low as 10 pmol/kg. The effect of
SP on the tail somatic vasculature (n = 7) was unclear. In isolated arterial ring preparations, SP in
concentrations of 10-100 nM did not affect the basal tonus or the
tonus of epinephrine-precontracted celiac
(n = 6) or mesenteric vessels
(n = 4). However, in three
preparations, a concentration of 1 µM induced strong vasoconstriction
of the celiac artery.
|
Effects of NKA. The same response was seen after NKA (100 pmol/kg, n = 7) injection as after SP: celiac inflow pressure initially increased and was correlated with stomach contractions, and, by the time the motility ceased, the underlying vasodilatation reduced the pressure to 0.5 ± 0.1 kPa (Figs. 3A and 4B). Maximum effect was obtained after 4.1 ± 1.9 min, and the dilatation lasted 15-25 min. Doses from 1 pmol/kg to 1 nmol/kg gave effects in a dose-dependent manner. The mesenteric inflow pressure (n = 5) showed no significant response to the injections. In the tail preparations (n = 7), no effects on somatic vasculature were obtained with doses of NKA between 100 pmol/kg to 100 nmol/kg. Likewise, NKA had no effect on isolated arteries (n = 5) at 10 nM to 1 µM.
|
Effects of CGRP.
CGRP (100 pmol/kg, n = 7) induced gut
contractions, although the effect was not as long lasting as after SP
or NKA injection. The ensuing vasodilatation was even more pronounced
than from the tachykinins; it caused a decrease in celiac perfusion
pressure (0.8 ± 0.3 kPa) with a maximum response occurring 5.4 ± 1.3 min after injection and usually lasting up to 25 min (Figs.
3A and 4A). The threshold dose that induced
an effect was 1 pmol/kg. In the two tetrodotoxin-Ringer preparations,
interference from gut motility was greatly reduced, thereby showing the
strong vasodilatation. Also, the mesenteric vascular bed showed a
significant vasodilatation (1.1 ± 0.4 kPa), with the nadir
6.7 ± 1.4 min after injection. CGRP (1 nmol/kg,
n = 5) had no significant effect on
the perfusion pressure in the tail preparations. In isolated vessels,
CGRP (10 nM) caused potent relaxation of the epinephrine-precontracted celiac artery (79.6 ± 2.8%,
n = 5) (see Fig.
6A). In the mesenteric artery
(n = 5), the response was more
difficult to interpret due to the transient epinephrine contractions.
Effects of VIP.
Injection of VIP (100 pmol/kg, n = 6)
caused a significant vasodilatation (0.5 ± 0.1 kPa) of the
celiac vascular bed, with a maximum effect after 3.1 ± 0.4 min and lasting ~15 min (Fig. 3A). The effects on the mesenteric
perfusions were variable (n = 4).
Doses lower than 100 pmol/kg did not cause any clear-cut effects on the
celiac vascular bed. The somatic vasculature of the tail was
significantly dilated by VIP (1 pmol/kg,
n = 8), although the responses were
very small in magnitude (0.06 ± 0.02 kPa) (Fig.
3C). In contrast, in the isolated
vessel preparation, VIP (10 nM) produced a marked vasodilatation
(89.3 ± 8.0%) of the epinephrine-precontracted celiac
artery (n = 5) (see Fig. 6B), whereas the mesenteric artery
preparations showed unclear responses.
Effects of somatostatin. Somatostatin (10 nmol/kg, n = 7) caused a significant vasoconstriction (+0.5 ± 0.2 kPa) of the perfused celiac vascular bed, with a peak response occurring 1.8 ± 0.3 min after injection (Figs. 3B and 5A). In two of the three perfused mesenteric vascular beds, a clear vasoconstriction was seen. No gut contractions were seen during the period of somatostatin exposure. The effect persisted for 10-20 min, and the lowest dose giving a vasoconstriction was 1 pmol/kg. Somatostatin in doses of 1-10 pmol/kg caused a significant vasoconstriction of isolated tail preparations (n = 6), as the perfusion pressure increased 0.3 ± 0.1 kPa following the injection (Fig. 3C). In isolated vessels, somatostatin (0.1-100 nM) did not affect the resting tonus of the celiac or mesenteric artery. However, when 10 nM was added before addition of epinephrine (100 nM), a significant potentiation of the epinephrine response compared with the control response was obtained (+156.7 ± 20.0%) in all four celiac artery preparations (n = 4) (Fig. 6C). In the mesenteric artery, no potentiation effect was seen (n = 3).
|
|
Effects of NPY. NPY (100 pmol/kg to 1 nmol/kg, n = 8) significantly increased the celiac perfusion pressure (+1.3 ± 0.6 kPa) with a maximum effect 1.8 ± 0.2 min after injection, which then lasted between 10 and 30 min (Fig. 3B). In the mesenteric vascular bed, no significance was obtained, although two preparations showed strong vasoconstriction (n = 5). In the perfused tail preparations, NPY (100 pmol/kg to 1 nmol/kg, n = 6) caused a significant increase of the perfusion pressure (+0.3 ± 0.1 kPa) and thus somatic vascular resistance (Fig. 3C). NPY (1-10 nM) did not affect the resting tonus of the isolated celiac (n = 6) or mesenteric (n = 3) arteries. Addition of the peptide (10 nM) before epinephrine (100 nM) had no significant effect on the epinephrine contraction.
Effects of GRP. Very forceful and occasionally long-lasting stomach contractions were induced by GRP (1 nmol/kg, n = 8), which also occurred in the tetrodotoxin-Ringer preparations. This activity caused strong oscillations of both the celiac and mesenteric perfusion pressures and hence made any interpretation of peptide vasoactivity impossible. GRP (1 pmol/kg to 1 nmol/kg) had no significant effects on the tail somatic vasculature (n = 6); GRP (10-100 nM) also did not have any effects on the isolated celiac (n = 5) or mesenteric (n = 3) arteries.
Effects of galanin. Galanin (1 nmol/kg, n = 6) significantly increased the celiac perfusion pressure (+0.5 ± 0.1 kPa), with a peak effect after 2.8 ± 0.6 min (Fig. 3B). No significant changes of the mesenteric vascular perfusion pressure were seen. Occasionally, gut contractions were associated with peptide injection. The responses were unaffected by tetrodotoxin-Ringer and lasted ~15 min. Galanin (10 pmol/kg to 1 nmol/kg) did not affect tail somatic vasculature (n = 6). Also, in the isolated vessel preparations, galanin (1-100 nM) was without any clear effects in the celiac (n = 5) or mesenteric (n = 3) arteries.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
This study investigates peptidergic regulation of gastrointestinal vascular resistance in crocodilians. The presence of immunoreactive fibers, in addition to the vasoactive properties of the different peptides, strongly suggests a regulatory function. In general, the vasoactive effects of the peptides in the gut circulation of C. porosus are similar to those observed for other vertebrates, further supporting an evolutionary conserved function of these transmitters (for review, see e.g., Ref. 28).
In the study by Karila et al. (19), fibers containing peptide-immunoreactivity were found in both autonomic ganglia and nerves associated with the cardiovascular system in the C. porosus. In the two arteries investigated, the anastomosis and the celiac artery, a few different populations of neurons were prominent: a group containing both SP and CGRP (which are possibly sensory fibers), a group of spinal autonomic nature in which TH, NPY, and somatostatin are coexisting, and fibers containing VIP, bombesin, or galanin. In the present study, we investigated the immunoreactive materials in the mesenteric artery but also in the smaller vessels of the gut region. The pattern of peptidergic innervation in these vessels was similar to that in the anastomosis and the celiac artery; a large number of VIP fibers are present, NPY and somatostatin coexist in adrenergic neurons, and CGRP is found together with SP but, in addition, also with NKA. Furthermore, we found neuronal NOS immunoreactivity, indicating inhibitory nitric oxide-producing fibers. In the isolated vessel preparations, the NOS inhibitor L-NAME was without effect; therefore, we could not demonstrate any tonic nitric oxide release in these arteries.
Addition of the tachykinin peptides SP and NKA to the perfusate initially induced gut motility to various degrees, which is a well-known effect of tachykinins also found in other vertebrates (see e.g., Ref. 16). These contractions increased the vascular pressure due to extravascular compression of the vessels. As the gut activity subsided, the extravascular compression decreased, showing the underlying vasodilatation caused by the tachykinins. Part of the vasodilatation could be due to reactive hyperemia as a consequence of the occlusion caused by the extravascular compression, but, considering the magnitude and the duration of the dilatation (lasting up to 20 min), it is likely that the peptides directly caused the vasodilatation. In addition, in the tetrodotoxin-perfused preparations in which motility was attenuated, the dilatation was even more clear-cut. This also agrees with the results from previous in vivo studies on the estuarine crocodile in which SP injections increased the celiac artery blood flow (1, 19). However, the isolated mesenteric and celiac arteries did not respond to the tachykinins in the doses used in the perfusion experiments, but, at higher concentrations, the vessels were instead contracted. The absence of a relaxation in these preparations could reflect a predominant location of receptors (neurokinin receptors) in the smaller-diameter vessels rather than in these large conduit arteries. In mammals, as in most other animals studied, it is known that the vascular resistance is controlled mainly in small-diameter arteries and arterioles (29). However, SP was found in varicose fibers innervating arteries of larger size. Another conceivable reason for the lack of relaxing effects could be a disrupted endothelium due to the handling of the vessels. Furthermore, because SP seems to be colocalized with CGRP (19), it is possible that the peptides have a synergistic effect on the vessels not revealed in these experiments. In situ injection of CGRP caused responses similar to those with the tachykinins, but the initial gut contractions were less pronounced and the ensuing vasodilatation was even more prominent and long lasting, an effect also seen in the mesenteric vascular bed. In addition, the isolated arteries precontracted with epinephrine were strongly relaxed by CGRP. Thus it appears that of the two peptides (SP and CGRP) found in perivascular (sensory?) fibers along crocodile gut vessels CGRP is the more potent vasodilator. In mammals, SP is often colocalized with CGRP in sensory fibers (30). CGRP typically produces long-lasting vasodilatation and is very potent, occasionally even more than SP (24, 33). SP, on the other hand, but not NKA, has been shown to be capable of regulating the vasodilator activity of CGRP (4, 33). Further experiments are needed to elucidate the possible interaction between SP and CGRP in the control of the crocodilian circulation.
Injection of VIP resulted in a reduced inflow pressure and thus a reduced resistance in the celiac vascular bed, without affecting the gut musculature. It also decreased the vascular resistance in the somatic circulation of the perfused tail. Furthermore, the isolated celiac artery precontracted with epinephrine was relaxed by VIP, demonstrating its effect even in larger conduit vessels. This effect on vessel tonus in C. porosus corresponds to numerous observations made in mammals and also in lower vertebrates such as fish (15, 18). It appears that this peptide is a "universal" vasodilator. The unclear effect in the mesenteric vasculature was not unique to VIP injection but was shown to be a more general feature in this circuit.
As expected, epinephrine caused a marked vasoconstriction of the celiac
and mesenteric vascular beds, revealing the presence of
-adrenoceptors, which are common to all vertebrates. As mentioned above, the adrenergic neurons associated with blood vessels also contain somatostatin and NPY, a combination also found in mammals (e.g., Ref. 22). This implies some kind of interaction among these
three neurotransmitters on release from their nerve endings. Injected
alone, somatostatin induced a marked vasoconstriction of the gut
circulatory system without affecting the activity of the
gastrointestinal wall. In mammalian vasculature, somatostatin has
either a vasoconstrictory effect (10, 22, 23) or no direct effect on
vessel tonus (26, 32). Somatostatin has also been shown to reduce the
contractile response of norepinephrine (10, 26). Similarly, NPY caused
a constriction of the vasculature in the perfused celiac preparations;
this is in contrast to the in vivo study in which NPY increased the
celiac artery blood flow (19). In addition, both somatostatin and NPY
increased the somatic vascular resistance of the perfused tail. It is
conceivable that this type of perivascular sympathetic neuron
containing TH, NPY, and somatostatin is present throughout the systemic
circulation of the crocodile, exerting a vasoconstrictory function.
However, the contradictory responses to NPY between the present and the in vivo study are confusing. Maybe the dilatation of the gut
vasculature in vivo involves a reflex resulting from an NPY effect
elsewhere in the body.
In the isolated celiac artery preparation, somatostatin itself did not affect the resting tonus of the vessel. However, when added to the organ bath before epinephrine, it potentiated the epinephrine-induced contraction compared with control. This effect could not be seen in the mesenteric artery preparation. NPY did not affect the resting tonus of the arteries, and it did not cause any clear-cut effects of the epinephrine-induced contraction. As mentioned before, a concentration of receptors at the microcirculatory level may be the reason for this lack of direct effect of the peptides on the isolated vessels. The vasoconstriction caused by somatostatin and NPY in the perfused gut preparation could be due to a direct stimulation of such receptors or due to an additional modulation of adrenergic neuronal activity. The in vitro result suggests a modulatory capacity of somatostatin. In other vertebrates, a lot more studies have been made about the vasoactive effects of NPY than of somatostatin. NPY is a potent modulator of sympathetic activity with both prejunctional inhibitory and postjunctional stimulatory action. In some vessels, it directly causes vasoconstriction (see, e.g., Refs. 6, 27). In contrast, in the shark Squalus acanthias, NPY decreases the resistance of the celiac vascular bed (J. Kågström, S. Holmgren, and M. Axelsson, unpublished observations), but, in another elasmobranch (the skate Raja rhina), NPY potentiates the effect of norepinephrine (3). The role of somatostatin and NPY in crocodilian adrenergic fibers is not clear, although both a direct stimulation of the vasculature and a neuronal modulatory action are feasible.
Nerve fibers containing immunoreactivity like bombesin, a peptide closely related to GRP, have been found in the gut musculature of crocodilians (5, 13, 36). Bombesin was shown to have an excitatory effect on intestinal smooth muscle of the caiman, Caiman crocodylus crocodylus. Furthermore, it is involved in vascular control, by increasing the resistance in the perfused lung of the caiman and by increasing the celiac blood flow in vivo in the estuarine crocodile (13). In the present study, we investigated the role of GRP in the control of gut and somatic vascular resistance. However, the vascular effect of GRP was unclear. It induced strong and long-lasting contractions of the gastrointestinal musculature that interfered with measurement of the vascular pressure. In half of the preparations, a reduced vascular resistance succeeded the gut activity, but whether this dilatation was peptide induced or the result of a reactive compensatory effect is not clear. Neither the somatic tail vasculature nor the isolated artery was affected by GRP. Thus it cannot be concluded whether GRP has a direct effect on the vascular tonus in estuarine crocodile or not. The bombesin-induced increase in celiac blood flow in vivo (13) could be due to shunting of blood from the pulmonary circulation into the left aorta and thus into the celiac artery.
Although galanin often is found in vascular neurons in various vertebrates, not much is known about its vasoactive properties. The arterial responses have been diverse, showing either no effect (9), a vasodilatation (21), or a vasoconstriction (14, 20, 31). In the snake Elaphe obsoleta, galanin was colocalized with NPY in adrenergic nerves (8), suggesting a vasoconstrictory or possibly a neuromodulatory role. In the perfused gut of the estuarine crocodile, galanin increased the inflow pressure in most of the preparations, demonstrating an increased vascular resistance. Although no coexistence between galanin and TH or NPY has been found, it is possible that galanin can cooperate with sympathetic nerves in the control of vascular tone. In some of the preparations, the peptide also induced gut motility, which probably contributed to the increased pressure. No effects on isolated vessels or somatic vasculature were seen, which further complicates the picture of galanin as a vasoeffector.
In conclusion, the present results demonstrate a vasoactive capacity of several neuropeptides found in perivascular fibers in C. porosus. The ability of neuropeptides to affect the vascular resistance of the perfused gut preparation suggests a function in the control of gastrointestinal blood flow. In most cases, the actions of the peptides are similar to those found in other vertebrates, such as vasodilatation caused by the common sensory neurotransmitters SP, NKA, and CGRP, and vasoconstriction by somatostatin and NPY are reminiscent of mammalian sympathetic neuropeptide activity. Thus neuropeptidergic regulation of vascular tone and hence blood flow probably are early evolved mechanisms and present in most vertebrates.
Perspectives
Numerous immunohistochemical studies have revealed the presence of different neuropeptides in lower vertebrates, such as teleosts, elasmobranchs, amphibians, and reptiles, and indicate the ancient origin of these regulatory systems. By studying and comparing earlier evolved vertebrate species, we can get a picture of the fundamental mechanisms involved in peptide function. The overall increasing evidence of peptidergic neurotransmitters in the cardiovascular system of mammals, as well as in nonmammalian species, implies a significant role in the control of arterial physiology. The present study shows that the vascular effects of the different peptides in C. porosus resemble those found in mammals and, by inference, their vasoactivities are properties already evolved in the ancient reptiles. Their regulatory role probably involves interactions with other transmitters and various receptor subtypes. To clarify the nature of these interactions, the use of specific receptor antagonists is imperative. However, presently, it is difficult to find peptide-receptor antagonists that work properly in nonmammalian species. Another problem with studying nonmammals is that the native peptide often is not available, and we are constrained to use peptides from other species. As these problems are gradually overcome by the sequencing and synthesis of native peptides and their receptors and by the development of new potent antagonists, we will be able to elucidate in detail the role of the neuropeptides in cardiovascular control in different vertebrates.| |
ACKNOWLEDGEMENTS |
|---|
We thank Christina Hagström and Lina Daddow for help with the photographs and electron microscopy, respectively.
| |
FOOTNOTES |
|---|
This study was supported by grants from the Australian Research Council (ARC) (a small ARC to C. E. Franklin), Adlerbertska Research Foundation, the Hierta-Retzius Foundation, Göteborgs Kungliga Vetenskaps-och Vitterhets-Samhälle, the Wennergren Centre, and the Swedish Natural Science Research Council. M. Axelsson was the recipient of a Univ. of Queensland Visiting Zoologist award at the time of this study.
Address for reprint requests: J. Kågström, Dept. of Zoophysiology, Göteborg Univ., Medicinaregatan 18, S-413 90 Göteborg, Sweden.
Received 17 January 1997; accepted in final form 23 February 1998.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
1.
Axelsson, M.,
R. Fritsche,
S. Holmgren,
and
D. J. Grove.
Gut blood flow in the estuarine crocodile, Crocodylus porosus.
Acta Physiol. Scand.
142:
509-516,
1991[Medline].
2.
Axelsson, M.,
S. Holm,
and
S. Nilsson.
Flow dynamics of the crocodile heart.
Am. J. Physiol.
256 (Regulatory Integrative Comp. Physiol. 25):
R875-R879,
1989
3.
Bjenning, C.,
S. Holmgren,
and
A. P. Farrell.
Neuropeptide Y potentiates contractile response to norepinephrine in skate coronary artery.
Am. J. Physiol.
265 (Heart Circ. Physiol. 34):
H661-H665,
1993
4.
Brain, S. D.,
and
T. J. Williams.
Substance P regulates the vasodilator activity of calcitonin gene-related peptide.
Nature
335:
73-75,
1988[Medline].
5.
Buchan, A. M. J.,
V. Lance,
and
J. M. Polak.
Regulatory peptides in the gastrointestinal tract of Alligator mississippiensis.
Cell Tissue Res.
231:
439-449,
1983[Medline].
6.
Burnstock, G.
Integration of factors controlling vascular tone
overview.
Anesthesiology
79:
1368-1380,
1993[Medline].
7.
Costa, M.,
R. Buffa,
J. B. Furness,
and
E. Solcia.
Immunohistochemical localization of polypeptides in peripheral autonomic nerves using whole-mount preparations.
Histochemistry
65:
157-165,
1980[Medline].
8.
Davies, P. J.,
and
J. A. Donald.
The distribution and colocalization of neuropeptides in perivascular nerves innervating the large arteries and veins of the snake, Elaphe obsoleta.
Cell Tissue Res.
269:
495-504,
1992[Medline].
9.
Ekblad, E.,
R. Håkansson,
F. Sundler,
and
C. Wahlestedt.
Galanin: neuromodulatory and direct contractile effects on smooth muscle preparations.
Br. J. Pharmacol.
86:
241-246,
1985[Medline].
10.
Fischer, L.,
S. Auberson,
C. Bretton,
and
J. S. Lacroix.
Adrenergic and non-adrenergic vasoconstrictor mechanisms in the human nasal mucosa.
Rhinology
31:
11-15,
1993[Medline].
11.
Franklin, C. E.,
and
M. Axelsson.
The intrinsic properties of an in situ perfused crocodile heart.
J. Exp. Biol.
186:
269-288,
1994[Abstract].
12.
Grigg, G. C.,
and
K. Johansen.
Cardiovascular dynamics in Crocodylus porosus breathing air and during voluntary aerobic dives.
J. Comp. Physiol. [A]
157:
381-392,
1987.
13.
Holmgren, S.,
M. Axelsson,
J. Jensen,
G. Aldman,
K. Sundell,
and
A.-C. Jönsson.
Bombesin-like immunoreactivity and the effect of bombesin in the gut, circulatory system and lung of the caiman, Caiman crocodylus crocodylus, and the crocodile, Crocodylus porosus.
Exp. Biol.
48:
261-271,
1989[Medline].
14.
Holmgren, S.,
R. Fritsche,
P. Karila,
I. Gibbins,
M. Axelsson,
C. Franklin,
G. Grigg,
and
S. Nilsson.
Neuropeptides in the Australian lungfish Neoceratodus forsteri: effects in vivo and presence in autonomic nerves.
Am. J. Physiol.
266 (Regulatory Integrative Comp. Physiol. 35):
R1568-R1577,
1994
15.
Jensen, J.,
M. Axelsson,
and
S. Holmgren.
Effects of substance P and vasoactive intestinal polypeptide on gastrointestinal blood flow in the Atlantic cod Gadus morhua.
J. Exp. Biol.
156:
361-373,
1991
16.
Jensen, J.,
and
S. Holmgren.
The gastrointestinal canal.
In: Comparative Physiology and Evolution of the Autonomic Nervous System, edited by S. Nilsson,
and S. Holmgren. New York: Harwood Academic, 1994, p. 119-167.
17.
Jones, D. R.,
and
G. Shelton.
The physiology of the alligator heartleft aortic flow patterns and right-to-left shunts.
J. Exp. Biol.
176:
247-269,
1993[Abstract].
18.
Kågström, J.,
and
S. Holmgren.
VIP-induced relaxation of small arteries of the rainbow trout, Oncorhynchus mykiss, involves prostaglandin synthesis but not nitric oxide.
J. Auton. Nerv. Syst.
63:
68-76,
1997[Medline].
19.
Karila, P.,
M. Axelsson,
C. E. Franklin,
R. Fritsche,
I. L. Gibbins,
G. C. Grigg,
S. Nilsson,
and
S. Holmgren.
Neuropeptide immunoreactivity and co-existence in cardiovascular nerves and autonomic ganglia of the estuarine crocodile, Crocodylus porosus, and cardiovascular effects of neuropeptides.
Regul. Pept.
58:
25-39,
1995[Medline].
20.
Karila, P.,
A.-C. Jönsson,
J. Jensen,
and
S. Holmgren.
Galanin-like immunoreactivity in extrinsic and intrinsic nerves to the gut of the Atlantic cod, Gadus morhua, and the effect of galanin on the smooth muscle of the gut.
Cell Tissue Res.
271:
537-544,
1993[Medline].
21.
Kotecha, N.,
and
T. O. Neild.
Vasodilatation and smooth muscle membrane potential changes in arterioles from the guinea-pig small intestine.
J. Physiol. (Lond.)
482:
661-667,
1995
22.
Lacroix, J. S.,
S. Auberson,
D. R. Morel,
E. Theodorsson,
T. Hökfelt,
and
J. M. Lundberg.
Vascular control of the pig nasal mucosa: distribution and effects of somatostatin in relation to noradrenaline and neuropeptide Y.
Regul. Pept.
40:
373-387,
1992[Medline].
23.
Long, J. B.,
D. D. Rigamonti,
K. Dosaka,
J. M. Kraimer,
and
A. Martinez-Arizala.
Somatostatin causes vasoconstriction, reduces blood flow and increases vascular permeability in the rat central nervous system.
J. Pharmacol. Exp. Ther.
260:
1425-1432,
1992
24.
Lundberg, J. M.
Pharmacology of cotransmission in the autonomic nervous system: Integrative aspects on amines, neuropeptides, adenosine triphosphate, amino acids and nitric oxide.
Pharmacol. Rev.
48:
113-178,
1996[Medline].
25.
Malvin, G. M.,
J. W. Hicks,
and
E. R. Greene.
Central vascular flow patterns in the alligator Alligator mississipiensis.
Am. J. Physiol.
269 (Regulatory Integrative Comp. Physiol. 38):
R1133-R1139,
1995
26.
Maynard, K.,
I. Saville,
and
V. L., and G. Burnstock.
Somatostatin modulates vascular sympathetic neurotransmission in the rabbit ear artery.
Eur. J. Pharmacol.
196:
125-131,
1991[Medline].
27.
Morris, J. L.,
I. L. Gibbins,
P. J. Kadowitz,
H. Herzog,
D. L. Kreulen,
N. Toda,
and
A. Claing.
Roles of peptides and other substances in cotransmission from vascular autonomic and sensory neurons.
Can. J. Physiol. Pharmacol.
73:
521-532,
1995[Medline].
28.
Morris, J. L.,
and
S. Nilsson.
The circulatory system.
In: Comparative Physiology and Evolution of the Autonomic Nervous System, edited by S. Nilsson,
and S. Holmgren. New York: Harwood Academic, 1994, p. 193-246.
29.
Mulvany, M. J.,
and
C. Aalkjaer.
Structure and function of small arteries.
Physiol. Rev.
70:
921-961,
1990
30.
Otsuka, M.,
and
K. Yoshioka.
Neurotransmitter functions of mammalian tachykinins.
Physiol. Rev.
73:
229-308,
1993
31.
Preston, E.,
C. D. Mcmanus,
A.-C. Jönsson,
and
G. P. Courtice.
Vasoconstrictor effects of galanin and distribution of galanin containing fibres in three species of elasmobranch fish.
Regul. Pept.
58:
123-134,
1995[Medline].
32.
Sieber, C. C.,
P. G. Mosca,
and
R. J. Groszmann.
Effect of somatostatin on mesenteric vascular resistance in normal and portal hypertensive rats.
Am. J. Physiol.
262 (Gastrointest. Liver Physiol. 25):
G274-G277,
1992
33.
Vincent, M. B.,
L. R. White,
T. Elsas,
G. Qvigstad,
and
O. Sjaastad.
Substance-P augments the rate of vasodilation induced by calcitonin gene-related peptide in porcine ophthalmic artery in vitro.
Neuropeptides
22:
137-141,
1992[Medline].
34.
Wang, Y.,
and
J. M. Conlon.
Neuroendocrine peptides (NPY, GRP, VIP, somatostatin) from the brain and stomach of the alligator.
Peptides
14:
573-579,
1993[Medline].
35.
Wang, Y.,
F. O'Harte,
and
J. M. Conlon.
Structural characterization of tachykinins (neuropeptide 
, neurokinin A, and substance P) from a reptile, Alligator mississippiensis.
Gen. Comp. Endocrinol.
88:
277-286,
1992[Medline].
36.
Yamada, J.,
V. J. M. Campos,
N. Kitamura,
A. C. Pacheo,
T. Yamashita,
and
N. Yanaihara.
An immunohistochemical study of the endocrine cells in the gastrointestinal mucosa of the Caiman latirostris.
Arch. Histol. Jpn.
50:
229-241,
1987[Medline].
This article has been cited by other articles:
|
|
 |
|
  F. Seebacher and R. S. James Plasticity of muscle function in a thermoregulating ectotherm (Crocodylus porosus): biomechanics and metabolism Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2008; 294(3): R1024 - R1032. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Y.-L. Yu, C.-H. Pon, H.-C. Ku, C.-T. Wang, and Y.-H. Kao A preprogalanin cDNA from the turtle pituitary and regulation of its gene expression Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2007; 292(4): R1649 - R1656. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Shahbazi, S. Holmgren, D. Larhammar, and J. Jensen Neuropeptide Y effects on vasorelaxation and intestinal contraction in the Atlantic cod Gadus morhua Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2002; 282(5): R1414 - R1421. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Platzack, Y. Wang, D. Crossley, V. Lance, J. W. Hicks, and J. M. Conlon Characterization and cardiovascular actions of endothelin-1 and endothelin-3 from the American alligator Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2002; 282(2): R594 - R602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Wang, M. Axelsson, J. Jensen, and J. M. Conlon Cardiovascular actions of python bradykinin and substance P in the anesthetized python, Python regius Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2000; 279(2): R531 - R538. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
