Vasoconstrictor effects of galanin and distribution of galanin containing fibres in three species of elasmobranch fish

ELSEVIER

Regulatory Peptides 58 (1995) 123-134

Vasoconstrictor effects of galanin and distribution of galanin containing fibres in three species of elasmobranch fish Elaine Preston a, Clare D. McManus a, Ann-Cathrine Jonsson b, Gillian P. Courtice a,* a School of Physiology and Pharmacology, University of New South Wales, Sydney, NSW 2052, Australia b Department of Zoophysiology, University ofGoteborg, Goteborg, Sweden Received 25 January 1995; revised 17 April 1995; accepted 19 April 1995

Abstract Galanin is found in perivascular sympathetic neurons in a wide range of vertebrate species. In placental mammals, galanin has either no effect on blood pressure, or weak depressor effects, but in other vertebrates it has been shown to be a potent pressor agent. To investigate how extensive the vasoconstrictor effects of galanin may be in the vertebrates, the vascular effects of galanin were tested in two species of shark, Heterodontus portusjacksoni, and Hemiscyllium ocellatum, and a ray, Rhinobatos typus. Nerve fibres showing immunoreactivity to galanin were located surrounding gut blood vessels, but were absent from branchial efferent arteries in all three species. Intravenous injection of galanin caused a significant rise in caudal arterial blood pressure in H. portusjacksoni and H. ocellatum, but no change in R. typus. Contraction of segments of pancreatico-mesenteric artery were measured in an organ bath also. Galanin (10 -6 M) caused 21-38% of the maximum K ÷ induced contraction in all species, but no response in efferent branchial arteries from R. typus. In conclusion, in three elasmobranchs, a galanin-like peptide is present in perivascular nerve fibres, and galanin causes differential vasoconstriction in vascular beds. These data extend the number of vertebrate groups in which galanin has been shown to be a vasoconstrictor peptide. Keywords: Galanin; Shark; Blood pressure; lmmunohistochemistry; Isolated vessel; Vasoconstriction

1. Introduction Galanin is a 29 amino acid peptide, first isolated in 1983 from porcine intestine [1]. Immunohistochemical studies have shown that it has a widespread distribution throughout the central and peripheral nervous systems of several species of vertebrates

* Corresponding author. Fax: +61 2 3851099.

(mammals: [2-6]; birds: [7]; reptiles: [8]; amphibians: [9-12]; fish: [13-17]). The physiological effects of galanin have been shown to be diverse. Of particular relevance to this paper are its effects on smooth muscle where it has been shown to cause contraction. For example, galanin causes smooth muscle contraction in the gut of the rat [5], the American opossum, Didelphis virginiana [18], the Cane toad, Bufo marinus [19], the Atlantic cod, Gadus morhua [15] and it causes potentiation of smooth muscle contraction due to electrical stimulation in rat vas deferens [20]. On the

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E. Preston et aL /Regulatory Peptides 58 (1995) 123-134

other hand, galanin has also been shown to inhibit smooth muscle contractility in some preparations (pigs: [21]; dogs: [22]; opossum: [23]). Despite these widespread effects on smooth muscle, galanin, until recently, was considered to have little effect on vascular smooth muscle and hence blood pressure in vertebrates. No pressor effect of intravenous galanin was evident in the dog [6,24], and a slight vasodepressor effect was demonstrated in the cat [25,26]. Central administration of galanin also resulted in a vasodepressor effect in rats [27]. In studies on isolated mammal vessels too, galanin had either no effect (rabbit femoral artery and vein, gastroepiploic artery, basilar artery, [28]) or caused vasodilatation (precontracted arterioles of guinea pigs, [29]). In contrast to these results in placental mammals, galanin has been shown to have potent vasopressor effects in other vertebrate species. In the Cane toad, B. marinus, intravenous administration of galanin caused a significant increase in blood pressure [30]. Likewise, galanin had pressor effects in two species of marsupial, the Brush-tailed possum, Trichosurus vulpecula [31] and the American opossum, D. virginiana [18]. In teleost fish also, galanin has been shown to contract isolated coeliac and mesenteric arteries from the Atlantic cod, G. morhua [15]. In light of these contrasting vascular responses to galanin in different vertebrate groups, the present study set out to assess the vascular actions of galanin in yet another vertebrate group, the elasmobranchs. Three species were investigated: the Port Jackson shark, Heterodontus portusjacksoni, the Epaulette shark, Hemiscyllium ocellatum, and the Giant Shovelnose ray, Rhinobatos typus. In all species, the distribution of galanin in perivascular nerve fibres was determined by immunohistochemistry, the blood pressure response to intravenous galanin was measured in whole anaesthetised animals, and contraction of isolated blood vessels to galanin was assessed in an organ bath.

2. Methods

2.1. Animals used in this study Eleven juvenile Port Jackson sharks, Heterodonms portusjacksoni, weight range 407-1500 g, were

captured in the coastal waters near Sydney, and maintained at the Sydney Aquarium, Darling Harbour. They were transported to holding tanks in the laboratory 1-2 days prior to experimentation. Eight adult Epaulette sharks, Hemiscyllium ocellatum, weight range 245-854 g, length range 46.5-69 cm, and eight juvenile Giant Shovelnose rays, Rhinobatos typus, weight range 180-204 g, length range 37-39 cm, were caught in hand nets over sandy areas of Heron Reef, MacKay/Capricorn section of the Great Barrier Reef Marine Park. The fish were removed immediately, and transferred from the net to a roofed 6 m square aquarium supplied with circulating seawater. Experiments on these latter two species were carried out at Heron Island Research Station, University of Queensland, Gladstone, Queensland.

2.2. Immunohistochemistry To determine the distribution of peripheral nerve fibres which may contain galanin, small tissue pieces were dissected from the efferent branchial arteries, the pancreatico-mesenteric artery, the coeliac artery, the mesenteric artery, gastric arteries and veins, intestinal arteries, the stomach, intestinal wall and the body muscle. Tissues were fixed by immersion in Zamboni's fixative for 24 h and then washed in 80% ethanol. The tissues were rinsed several times in dimethylsulfoxide (DMSO), then in phosphatebuffered saline (phosphate buffer; 0.1 M, pH 7.2) and stored in phosphate buffer containing 0.1% azide until processing. The tissue was transferred to phosphate buffer containing 30% sucrose and stored in this solution for at least 16 h before sectioning. The tissues were mounted in a tissue-tek mounting media, quick frozen in liquid nitrogen and sectioned in a cryostat. The 10 /xm sections were thaw-mounted onto poly(L-lysine) coated glass slides. Each section was incubated with a primary antibody for 16 h in a humid chamber. Two primary antibodies were used, both raised in rabbits against galanin (CRB-galanin, diluted 1:500, Cambridge Research Biochemicals, Cambridge, UK and P-galanin diluted 1:100, Peninsula Laboratories Inc, St. Helens, UK). The sections were then washed in phosphate buffer three times (10 min each) and incubated in a humid chamber for 1 h with a secondary antibody (swine-anti-rabbit-

E. Preston et aL / Regulatory Peptides 58 (1995) 123-134

FITC (fluorescein isothiocyanate) 1:10 or donkey anti-rabbit DTAF (dichlorotriazinyl amino fluorescein) 1:100, Jackson Immunoresearch Laboratories Inc., USA). The sections were again washed in phosphate buffer, mounted with carbonate buffer/glycerol (1:1, pH 8.5) and viewed in a Vanox fluorescence microscope.

2.3. Whole animal experiments Anaesthesia and surgical procedure All animals were anaesthetised by placing them in seawater containing 0.1% ethyl-m-aminobenzoate (MS222, Thomson and Joseph UK). Anaesthesia was maintained by placing the shark on an operating table and irrigating the gills with recirculating seawater containing 0.1% MS222 via a tube through the mouth. The seawater was either oxygenated (Sydney laboratory) or aerated (Heron Island Research Station). A flow rate of 1 - 2 l / m i n of seawater was found to maintain a stable level of anaesthesia and a constant blood pressure and heart rate during the experimental period. The sharks were covered in wet terry towelling material throughout the experiment to prevent desiccation of the skin. All experiments were carried out at room temperature between 22-24°C. Arterial blood pressure was recorded continuously via a catheter introduced into the caudal artery, the tip of which was advanced 5 cm into the dorsal aorta. The catheter containing heparinised dogfish saline (NaCI 296 mM, KC1 7.2 mM, CaCI 2.9 mM, MgC12 3.5 mM, Na2SO 4 5.7 mM, NaHCO 3 6.8 mM, urea 289 mM, TMAO 71 mM, [32]) was attached to a Statham (P23D) blood pressure transducer, and the signal monitored on either a Grass polygraph (Sydney) or a Graphtec chart recorder (WR7200, Graphtec Corp Japan) (Heron Island). In the Port Jackson sharks, the blood pressure signal was used also to trigger a linear display unit (Neurolog 701) to obtain a continuous measurement of pulse interval (PI), the interval between heart beats. In all species, drug and peptide administrations were made through a catheter in the caudal vein. To eliminate reflex chronotropic effects to the heart a single dose of atropine ( 2 0 0 / x g / k g : atropine sulfate, Astra Pharmaceuticals) was administered intravenously to all animals [33].

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Experimental protocol To assess whether tachyphylaxis occurred with repeated doses of galanin, in three Port Jackson sharks, three intravenous injections of galanin were administered (20 /xg/kg, 6 nmol/kg: porcine Galanin, Peninsula, USA). After the 1st dose, subsequent doses were given as soon as the blood pressure had returned to preinjection levels. One bolus dose of galanin (20 /xg/kg, 6 nmol/kg) was injected into the caudal vein of a further four Port Jackson sharks, seven Epaulette sharks and four Giant Shovelnose rays. A bolus dose of 10 /xg/kg (3 nmol/kg) galanin was also injected into a further two Port Jackson sharks. In each case, the cannula was flushed with 0.5-1 ml dogfish saline and the maximum change in mean blood pressure and the duration of the blood pressure response were measured. The duration of the response was recorded as the time taken for blood pressure to return to preinjection levels. To confirm that the vehicle itself had no effect, in two animals from each species an equivalent volume of dogfish saline was injected into the caudal vein.

2.4. Organ bath experiments All animals were anaesthetised in 0.1% MS222. Arterial segments from the pancreatico-mesenteric and efferent arteries were removed from four Port Jackson sharks, three of which had already been used in the whole animal study, four Epaulette sharks (of which three had been used in the whole animal study) and four Giant Shovelnose rays. When vessels were taken from animals which had already received intravenous galanin, at least 3 - 4 h elapsed before measurements were made in the organ bath. To remove the pancreatico-mesenteric artery, a midventral abdominal incision was made, the visceral organs moved aside and the coeliac trunk exposed caudal to the right side of the stomach and in close proximity to the bile duct and the hepatic portal vein. The pancreatico-mesenteric artery, a continuation of the coeliac artery caudal to the gastrohepatic branch, was removed and placed in a beaker of dogfish saline. The efferent branchial arteries were removed by exposing the roof of the buccal cavity and dissecting the arteries from the point of exit from the gills

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E. Preston et al. /Regulatory Peptides 58 (1995) 123-134

Fig. 1. (A-F) Galanin-like immunoreactive fibres innervate the blood vessels of the gut of the Epaulette shark (H. ocellatum), the Giant Shovelnose ray (R. typus) and the Port Jackson shark (H. portusjacksoni). (A) Nerve fibres in an artery supplying the spiral intestine with blood are labelled with P-gal in R. typus ( X 103). (B) CRB-gal labelled nerves in vessels of H. ocellatum ( x 206). (C) P-gal labelled nerves within a nerve bundle in the pancreatico-mesenteric vessels of R. typus ( × 206). (D) Nerve fibres immunoreactive to CRB-gal running up in the mucosa of the spiral intestine of H. ocellatum ( X 206). (E) A nerve bundle in the pancreatico-mesenteric vessels labelled with P-gal in R. typus (X 206). (F) P-gal labelled fibres innervate an artery supplying the intestine with blood in R. typus (X 206).

E. Preston et aL / Regulatory Peptides 58 (1995) 123-134

to their convergence with the dorsal aorta. These vessels were also placed in a beaker of dogfish saline. Three 2 mm long segments of each artery were mounted on a modified Mulvaney myograph [34] in a 2 ml organ bath. The baths were continuously supplied either with room air (Heron Island) or 2% CO 2 in oxygen (Sydney). Two parallel pins were inserted into the lumen of the vessel. One pin was attached to a micro manipulator to allow precise adjustments of the resting tension. The other was attached to a Grass force transducer for measurement of isometric tension which was continuously monitored on a Grass polygraph (Sydney) or a Graphtec chart recorder (Heron Island). The vessels were left to equilibrate for 45-60 min before any measurements were carried out. The vessels were then stretched till an average steady tension of 1-3 mN was recorded. The vessels were exposed to a concentrated K ÷ solution (63 mM K +) and the maximum contraction measured. After all experimental measurements were made the vessels were exposed to the K + solution again and the response compared to the K ÷ induced contraction prior to experimentation.

Dose-response curves Porcine galanin was added to the organ bath containing 2 ml dogfish saline to produce cumulative galanin concentrations between 10 - 9 M and 10 - 6 M. Maximum tension was recorded at each concentration and expressed as a percentage of the maximum K ÷ induced contraction. Although a maximum response to galanin was not recorded in this range of concentrations, higher concentrations of galanin were not applied as the very large amounts of peptide necessary were not available.

2.5. Statistical analysis Data are presented as means + S.E.M. Statistical comparisons between two groups of data were made using a paired t-test. When more than two groups of data were compared, an analysis of variance with repeated measures was used. In all comparisons the 5% level of significance was accepted.

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3. Results 3.1. Immunohistochemistry Nerve fibres labelled with galanin-antibodies (GAL-IR) were found surrounding blood vessels (mainly arteries) of the gut and gut tissue in all three elasmobranch species. The number of fibres showing GAL-IR differed between the species. The density of immunoreactive fibres was partly dependent on the antibody, but mostly on the tissue. There were usually more fibres labelled with the CRB-galanin than with the P-galanin antibody. The mesenteric arteries of the Epaulette shark usually contained GAL-IR fibres (Fig. 1B, Fig. 2F). GAL-IR was also seen in blood vessels supplying the intestine and stomach (Fig. 1A, F). Fibres showing GAL-IR were seen surrounding the pancreatico-mesenteric arteries of all three species, with occasional GAL-IR seen in nerve trunks running through the tissues surrounding the pancreatico-mesenteric vessels, especially in R. typus (Fig. 1C,E). Fibres showing GAL-IR were predominantly found in the tunica adventitia which consists of collagen fibres with a longitudinal orientation and also containing fibroblasts. No nerve fibres could be seen in the tunica intima (endothelial layer), while nerve fibres innervating the mesenteric arteries were often found in the tunica media (muscle layer) (Fig. 2F). No GAL-IR fibres were found in the efferent branchial arteries from the three species examined. Fibres showing GAL-IR were found in the submucosa and mucosa (Fig. 1D) of the stomach and intestine, with numerous nerve fibres innervating the gland area of the stomach and the spiral valves of the intestine. No labelled endocrine cells were observed in any of the three species. Occasional blood vessels in the submucosa were innervated by fibres showing GAL-IR (Fig. 2B). Fibres showing GAL-IR were also found in the muscle layers of the gut but were less dense than in the mucosa. The arteries within the muscle layers were occasionally innervated by GAL-IR fibres (Fig. 2A, C,D,E,G).

3.2. Whole animal experiments Repeated doses of galanin in Port Jackson sharks A bolus dose of galanin administered intravenously caused a rise in blood pressure in the Port

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J

Fig. 2. ( A - G ) Galanin-like fibres in the blood vessels of the gut of the Epaulette shark (H. ocellatum), the Giant Shovelnose ray (R. typus) and the Port Jackson shark (H. portusjacksoni). (A) An artery in the muscle layer of the intestine is innervated by P-gal-like fibres in R. typus ( x 206). (B) Nerve fibres in an artery of the stomach submucosa are labelled with P-gal in H. ocellatum ( x 206). (C) An artery in the stomach muscle is innervated by P-gal-like nerve fibres in H. ocellatum ( x 206). (D) Artery in the muscle layer of the intestine in H. ocellatum is innervated with P-gal labelled nerve fibres ( x 206). (E) Arteries of the stomach muscle in H. ocellatum is innervated by P-gal-like fibres ( X 206). (F) Mesenteric vessels of H. ocellatum show galanin-like nerve fibres (CRB-gal) ( X 206). (G) CRB-gal-like nerve fibres in arteries of the post intestinal muscle layer of R. typus ( X 103).

E. Preston et al. // Regulatory Peptides 58 (1995) 123-134

129

p, 2] (s)

1 v

BP 15] o~

(mmHg)

Ist dose

5 3nmol galanin

2min

Fig. 3. Traces of pulse interval (PI) and arterial blood pressure (measured in the caudal artery) from an anaesthetised 580 g Port Jackson shark, in the presence of atropine. Galanin causes no change in PI, but an increase in arterial blood pressure which returns to preinjection levels within 5 min.

Jackson shark but had no effect on pulse interval (Fig. 3). Injection of an equivalent volume of dogfish saline caused no significant change in blood pressure. On occasions, a transient rise in blood pressure lasting less than 20 s was associated with injection time; however, the maximum change in blood pressure in response to galanin always occurred after this transient effect. In three Port Jackson sharks, three intravenous injections of galanin were administered sequentially, each one given as soon as the blood pressure had returned to preinjection levels after the previous dose. In all three animals the blood pressure response was reduced in response to the second and third dose of galanin compared to the first dose (Fig. 4, A N O V A P = 0.07, n = 3). The first injection of galanin caused an increase in mean blood pressure of 3.1 _ 0.95 mmHg, with a duration of 9 -t- 1.3 min, which was larger than the second (0.87 + 0.27 mmHg, 7.3 +__1.6 min) and the third ( 0 . 6 7 _ 0.44 mmHg, 4.7 + 2.9 min).

2nd dose

3r'd dose

Fig. 4. The mean changes in mean arterial blood pressure in three Port Jackson sharks after three consecutive doses of galanin. Second and third doses caused a smaller change in mean blood pressure compared with the first injection.

sure returned to preinjection levels in 6.0-t-0.73 min. A similar rise in blood pressure was seen when a 10 / x g / k g dose of galanin was injected into two Port Jackson sharks (1.8 and 1.9 mmHg, duration 8 and 9 min). In seven Epaulette sharks, with resting mean blood pressure of 9.94 + 1.2 mmHg, single doses of galanin (6 n m o l / k g ) caused a significant increase in mean blood pressure of 5.29 -t- 1.5 m m H g returning to preinjection levels in 5.45 + 1.8 min ( P = 0.006). In the four Giant Shovelnose rays, no statistically significant changes in blood pressure were recorded in response to galanin (0.86-t-1.25 mmHg) ( P > 0.5, n = 4). Within the four rays, a slight increase in mean blood pressure was measured in two animals, and a slight decrease in two.

20 ~

15

"

10

Single doses of galanin in all three species The blood pressure responses to galanin varied among the species investigated (Fig. 5). Single injections of galanin (20 / z g / k g , 6 n m o l / k g ) in six Port Jackson sharks with resting mean caudal arterial blood pressure of 8.8 -t- 1.2 mmHg, caused a significant increase in mean blood pressure of 2.58 + 0.49 m m H g (paired t-test, P = 0.003, n = 6). Blood pres-

0 Fig. 5. Mean arterial blood pressures measured in the caudal artery immediately prior to a dose of galaain ( [] ) and at the peak of the pressor response after administration of galanin (hatched) in three species of elasmobranch. ( P < 0.01 = * * ). Galanin caused a significant increase in blood pressure in two of the three species.

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E. Prestonet al. / RegulatoryPeptides58 (1995) 123-134 mo

A

g e~

o o

50 g ~D C_

¢ o i 10-9

mo

i 10-8 [galanEn]

i 10 - 7 M

i 10 - 6

B

6B), increasing to 38 ___16% of the maximum K ÷ induced contraction at 10 - 6 M. The efferent branchial arteries from the Port Jackson shark and the Epaulette shark showed no significant contraction in a concentrated K + solution. Consequently, no measurements in response to galanin were made. In the Giant Shovelnose ray, the efferent branchial arteries contracted in response to the concentrated K + solution. However, no change in tension was recorded at concentrations of galanin up to 10 - 6 M (Fig. 6B). Comparisons of K ÷ contractions at the beginning and end of the experiments showed no significant decline in the responsiveness of the vessel over the experimental period.

~E o u

4. Discussion

5o

I

~D

0

'~t 10 -`9

- - A ~ A i 10-8 [Galanln]

• i 10 - 7

i 10-6

M

Fig. 6. (A) Contractile responses of the pancreatico-mesenteric artery of the Port Jackson shark ([3) and the Epaulette shark (O) in response to increasing concentrations of galanin. (B) Response of the pancreatico-mesentericartery ( A) and the efferent branchial ( • ) arteries of the Giant Shovelnose ray to increasing concentrations of galanin. In both Fig. 4A and 13contraction is expressed as % of maximum contraction recorded in response to 117 mM KCI. 3.3. Organ bath experiments Galanin caused contraction of isolated segments of the pancreatico-mesenteric artery in all species. In the Port Jackson shark, the segments of pancreaticomesenteric artery (n = 10) contracted to galanin at concentrations greater than 10 -7 M and reached 25 + 6% of the K + induced contraction at 10 - 6 M (Fig. 6A). Rings of pancreatico-mesenteric artery from Epaulette sharks (n = 7) contracted at concentrations above 10 -8 M. At 10 - 6 M the vessel reached 21 + 6% of the maximum K ÷ contraction (Fig. 6A). Vessel rings from the pancreatico-mesenteric artery of the Giant Shovelnose ray contracted to galanin at concentrations greater than 10 - 7 M (n = 5) (Fig.

The neuropeptide galanin has been shown to have potent vasopressor effects in several vertebrate species, including the Cane toad, B. marinus and the marsupials, the Brush-tailed possum, T. vulpecula and the A m e r i c a n opossum, D. virginiana [18,30,31,35]. In addition, galanin contracts isolated vessels from a teleost fish, the cod, G. morhua [15]. In the present study, a vasopressor response to galanin has been demonstrated in animals from a different vertebrate class, the elasmobranchs. Galanin caused a significant increase in blood pressure in two species of sharks, H. portusjacksoni and H. ocellatum. In a third species, R. typus, galanin caused contraction of isolated vessels, although a significant rise in blood pressure was not recorded in anaesthetised animals. Thus, it appears that contraction of vascular smooth muscle by galanin is a common phenomenon throughout the vertebrates. In light of these results, it is interesting that the placental mammals are the only vertebrates studied so far that have yet to show vasoconstriction in response to galanin in any species. In the present study, galanin was shown to cause contraction of isolated segments of pancreaticomesenteric arteries in all three species. This result is supported by the finding that fibres showing GAL-IR were found, using both antisera, innervating the smooth muscles of the blood vessel wall of the pancreatico-mesenteric arteries. The nerves were located usually in the tunica adventitia, though in some

E. Preston et al. /Regulatory Peptides 58 (1995) 123-134

mesenteric arteries GAL-IR nerve fibres were found in the tunica media. It is likely that the two antibodies used recognize different parts of the galanin molecule, since one (CRB-galanin) showed a greater density of staining than the other. In addition, the presence of GAL-IR fibres in the mucosa of the three elasmobranchs suggests that galanin may be involved in the mucosal functions of these species. In other species, galanin has been shown to influence secretory mechanisms, for example acid secretion in the gut (see [36]). Vessels from the Giant Shovelnose ray showed a variable response to galanin. Contraction of the pancreatico-mesenteric artery occurred in response to galanin, while no effect was measured using the efferent branchial arteries (see Fig. 6). Differential responses in different vascular beds may explain the different blood pressure responses found in the four anaesthetised rays studied. In two animals there was a vasodepressor response and in the remaining two animals a vasopressor response. In the other two elasmobranchs, the Port Jackson and the Epaulette sharks, the efferent branchial arteries were mounted in the organ bath, but no contraction could be elicited by the concentrated K + solution. Thus no response to galanin could be determined. The inability of the efferent arteries to contract to K ÷ in the two species may be explained in part by differences in the size of the tunica adventitia in efferent vessels and gut vessels. The tunica adventitia in the pancreaticomesenteric vessels, and in other blood vessels outside the gut wall is very prominent and nerve fibres containing GAL-IR are a consistent feature. In the efferent arteries, the tunica adventitia is thin, and nerves containing GAL-IR are absent. Differential effects of galanin on vascular beds have been demonstrated in other species also. In the brush-tailed possum, the overall vasopressor response to galanin has been attributed to increases in vascular resistance in the adrenal gland, kidneys, spleen, skin, gastrointestinal tract and remaining carcass, with no significant changes occurring in the heart, lung, liver and muscle vascular beds [35]. In the cat, in which galanin causes a slight vasodepressor response, differential changes in resistance in various vascular beds contributed to this response [35]. In the Australian lungfish also, galanin has been shown to cause an increase in vascular resistance in

131

the lungs, but a decrease in resistance in the coeliac artery [14]. The net result in this species is no overall change in dorsal aorta blood pressure in response to 1 nmol/kg galanin [14]. Differential vascular effects are a feature of other vasoconstrictor peptides also [37-40]. In the pithed rat, for example neuropeptide Y has been shown to cause vasoconstriction in the spleen, kidney, pancreas and mesentery while causing dilation of the lung vasculature [39]. In anaesthetised cats, a low dose of endothelin causes an increase in resistance of renal vessels and a decrease in resistance of small intestinal vessels [40]. It appears that such regional differences are a feature of the vascular response to galanin in elasmobranchs also. Such regional effects may allow for control of regional blood flow in response to different physiological demands. The mechanism underlying the pressor effects of galanin is most likely to be a direct effect of galanin on the vasculature, rather than a potentiation of catecholamine induced vasoconstriction. Experiments in arterial strips from a teleost show no potentiation of the constriction induced by adrenaline [15]. In toads too, galanin does not appear to enhance contraction due to adrenaline in isolated mesenteric arteries although it causes direct contraction (McManus and Courtice, unpublished observations). Such a mechanism was not tested in the elasmobranchs studied here. The vasopressor response to galanin in the Port Jackson shark appeared to desensitise with repeated applications of galanin (see Fig. 4). These results are in contrast to the results obtained in other vertebrate species. Repeated injections of porcine galanin were equally effective in raising blood pressure in the American opossum, D. virginiana [18] as they were in the Cane toad, B. marinus (Preston and Courtice, unpublished observations). In the cat also, no desensitisation to the vasodepressor effect which is evident in this species, could be demonstrated (Ulman, personal communication), although the inhibitory effect of galanin on cardiac vagal slowing was easily desensitised [26]. In the present experiments porcine galanin was used for injection. To date, the amino acid sequence for galanin, in the elasmobranch species studied here, has not been determined. However, it is likely that similar responses will be elicited to porcine

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galanin as to the native galanin for each species since there seems to be a high homology in the galanin amino acid sequence between species from different vertebrate groups. The galanin amino acid sequence determined from pig, rat, cow, sheep, chicken, man [41] and alligator [42] show that the first fifteen amino acids are identical. Wang and Conlon [43] have reported that in dogfish and two species of teleost fish the first fifteen amino acids are identical also. However, there seems to be differences at amino acid 15 in the trout (Jenssen, personal communication) and differences at amino acid 6 and 15 in the sequence of tuna galanin, although only partial sequencing has been completed in this species [44]. Since it is the N-terminal end of the peptide which is highly conserved, and is the part of the molecule responsible for most biological activity [45-47], it is likely that the vasopressor responses to porcine galanin demonstrated in this study are representative of the responses to the native galanin species. Further, comparisons of galanin from different species on physiological responses show similar results. Ulman and co-workers [48] showed that human, rat and porcine galanin had the same vasodepressor effects in cats, while porcine and human galanin have similar pressor responses in the Cane toad (unpublished observations). In addition, both rat and porcine galanin have been reported to have the same effects on inhibiting the insulin response to glucose in the rat [49].

Acknowledgements This study was supported by the Australian Research Council. We are grateful to New South Wales State Fisheries Department for collection of Port Jackson sharks, and to the Sydney Aquarium, Darling Harbour for maintenance of the sharks prior to experimentation. We would like to thank the director and staff of the Heron Island Research Station for allowing us to use their facilities, and Dr. M. Bennett, Dr. S. Bennett, Dr. L.C. Llewellyn and John and Lisa Chopin for their assistance in capture of the animals and in the laboratory at Heron Island. We would also like to thank Lena Nyman and Parvin Korckchi for their technical assistance with immunohistochemistry and photography.

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