oCRF and CRF (6-33) depress food but not water intake in the obese Zucker rat.

It has previously been demonstrated that oCRF and the CRF binding protein inhibitor CRF (6-33) reduce body-weight gain in obese Zucker rats. We investigated whether the reduction in body-weight is attributable to altered feeding and drinking behaviour. Obese Zucker rats were fitted with osmotic mini-pumps connected to i.c.v. cannulas. Vehicle, oCRF (5 microg/day) or CRF (6-33) (25 microg/day) were infused for 7 days and the animals observed for an additional 7 days. Body-weight and food and water-intake were recorded daily at 14.00 h. In agreement with published results, oCRF and CRF (6-33) significantly reduced body-weight gain in the obese Zucker rat. In addition, food intake was reduced, whereas water consumption was unaffected.

Isolation and characterization of a leech neuropeptide in rat brains: coupling to nitric oxide release in leech, rat and human tissues.

The osmoregulator peptide (leech osmoregulatory factor, LORF; IPEPYVWD) was first found in the leech central nervous system (CNS). Given the fact that certain peptides can be found in mammals and invertebrates, e.g., opioid, we examined rat brains to determine if LORF was present. This peptide was found and isolated by successive reversed-phase HPLC purification steps and characterized by electrospray mass spectrometry measurement. It was sequenced by Edman degradation and quantified in different tissues by ELISA. Our results demonstrate the presence of LORF in the hypothalamus, thalamus, and striatum (6 pmol/mg of protein extract) and in other brain areas at lower levels. This octapeptide is also present in the rat duodenum and liver (10 to 14 pmol/mg) and at lower levels in heart, lung, pancreas and caudal spinal cord (< 5 pmol/mg). The testes, adrenals and kidneys have the lowest levels of all the tissues examined (ca. 0.5 pmol/mg of protein). Furthermore, we also demonstrate that LORF is coupled to nitric oxide (NO) release in leech CNS, rat hypothalamus and human saphenous vein in a manner which is inhibited by a nitric oxide synthase inhibitor as well as an antibody directed toward LORF. The study demonstrates that LORF, and its function in relation to NO release, has been conserved over more than 400 million years of evolution.

Tachykinins with unusual structural features from a urodele, the amphiuma, an elasmobranch, the hammerhead shark, and an agnathan, the river lamprey.

Tachykinins were purified from extracts of gastrointestinal tissues of the urodele, Amphiuma tridactylum (three-toed amphiuma), and the elasmobranch Sphyrna lewini (hammerhead shark), and from the brain of the agnathan Lampetra fluviatilis (river lamprey). The amphiuma substance P (SP) (DNPSVGQFYGLM-NH2) contains 12 amino residues compared with 11 for mammalian SP and lacks the Arg/Lys-Pro-Xaa-Pro motif that is characteristic of NK1 receptor-selective agonists. Lampetra SP (RKPHPKEFVGLM-NH2) is identical to SP from the sea lamprey and the shark SP-related peptide (AKFDKFYGLM-NH2) is identical to dogfish scyliorhinin I. Amphiuma neurokinin A (NKA) (HKDAFIGLM-NH2) and lamprey NKA (HFDEFVGLM-NH2) contain 9 amino acid residues compared with 10 for mammalian NKA. The shark NKA-related peptide (ASGPTQAGIV10GRKRQKGEMF20VGLM-NH2) shows limited structural similarity to mammalian neuropeptide gamma and the teleost tachykinin, carassin but contains 24 rather than 21 amino acid residues. The data show that the primary structures of the tachykinins have been very poorly conserved during vertebrate evolution and that pressure has acted only to maintain the functionally important sequence -Phe-Xaa-Gly Leu-Met-NH2 at the COOH-termini of the peptides.

Novel tachykinins from the brain of the sea lamprey, Petromyzon marinus, and the skate, Raja rhina.

Using radioimmunoassays for mammalian tachykinins, peptides with substance P-like immunoreactivity and neurokinin A-like immunoreactivity were identified in an extract of the brain of the longnose skate, Raja rhina (elasmobranch) but only a peptide with neurokinin A-like immunoreactivity was identified in the brain of the sea lamprey, Petromyzon marinus (agnathan). The primary structure of the skate peptide with substance P-like immunoreactivity (Ala-Lys-His-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH2) shows one amino acid substitution (Phe3-->His) compared with scyliorhinin I, previously isolated from dogfish brain and gut. The skate neurokinin A-related peptide (His-Lys-Leu-Gly-Ser-Phe-Val-Gly-Leu-Met-NH2) shows two substitutions (Thr3-->Leu and Asp4-->Gly) compared with mammalian neurokinin A. Although the COOH-terminus of the lamprey tachykinin (Arg-Lys-Pro-His-Pro-Lys-Glu-Phe-Val-Gly-Leu-Met-NH2) resembles neurokinin A, the presence of the strongly conserved Lys/Arg-Pro-Xaa-Pro motif at the NH2-terminus of the peptide indicates greater structural similarity with substance P. The additional arginine residue at position 1 in the peptide suggests that the lamprey is utilizing a site of posttranslational processing in the tachykinin precursor that is different from the equivalent site in mammalian and other lower vertebrate preprotachykinin(s).

Cardiovascular actions of dogfish urotensin II in the dogfish Scyliorhinus canicula.

Bolus injections of synthetic dogfish urotensin II (0.1-1.0 nmol) into the celiac artery of the conscious dogfish Scyliorhinus canicula (n = 8) resulted in sustained and dose-dependent increases in arterial blood pressure and pulse pressure. A maximum rise in mean arterial pressure of 10.5 +/- 1.2 mmHg (equivalent to 38.6 +/- 4.2% over mean basal values) and a maximum increase in pulse pressure of 3.9 +/- 0.8 mmHg was elicited by injection of 0.5 nmol of peptide. In comparison, a bolus injection of epinephrine (5 nmol) elicited a rise of 24.8 +/- 3.3% in mean arterial pressure. Bolus injection of 0.5 nmol synthetic goby (Gillichthys mirabilis) urotensin II under the same conditions did not elicit a significant hypertensive response. When dogfish urotensin II (0.5 nmol) was administered 3 min after an intra-arterial injection of phentolamine, the rise in arterial blood pressure was completely abolished. Dogfish urotensin II produced a dose-dependent contraction (pD2 = 6.58 +/- 0.07; n = 8) of isolated rings of vascular muscle prepared from the first afferent branchial artery of the dogfish. A maximum contractile force of 1.3 mN was produced by 10(-5) M peptide. The urotensin II-induced contraction of the vascular rings was unaffected by pretreatment with tetrodotoxin (1 microM) or indomethacin (14 microM). It is concluded that urotensin II has potent hypertensive activity in the dogfish that is mediated, at least in part, through release of catecholamines, but the sustained nature of the pressor response suggests that the peptide may have a direct action on the heart.

Neuropeptide Y potentiates contractile response to norepinephrine in skate coronary artery.

The skate is an extant representative of the first vertebrate group with a coronary circulation, the elasmobranch fish. Histochemical studies have revealed that skate coronary arteries are innervated by adrenergic nerves as well as by nerves showing neuropeptide Y (NPY)-like immunoreactivity. This study investigates the effect of NPY in the coronary system of an elasmobranch fish, a vertebrate that first evolved 450 million years before mammals and the mammalian coronary circulation. The responses of vascular ring preparations of the coronary artery from the longnose skate (Raja rhina) were measured using isometric force transducers. The main effect of NPY was a potentiation of the amplitude of the norepinephrine-induced contraction, leaving the pD2 value (-log 50% effective concentration) for norepinephrine unaffected. NPY per se occasionally contracted the coronary rings in higher concentrations. The potentiation response may not involve the endothelium and was abolished by pretreatment with tetrodotoxin. We conclude from this study that NPY potentiates norepinephrine-induced contraction in skate coronary artery via an indirect pathway.

Distribution and activity of dogfish NPY and peptide YY in the cardiovascular system of the common dogfish.

Neuropeptide Y is present in sympathetic nerves in the mammalian cardiovascular system. This study has investigated the distribution of neuropeptide Y in the cardiovascular and gastrointestinal systems and the effect of dogfish neuropeptide Y and related peptides on cardiovascular tissue of an elasmobranch fish, the common dogfish (Scyliorhinus canicula). Neuropeptide Y-like immunoreactivity is present in varicose nerve fibers innervating dogfish gut and cardiovascular tissue and in endocrine cells of the dogfish spiral intestine. Dogfish neuropeptide Y, dogfish peptide YY, and porcine neuropeptide Y contract the dogfish afferent branchial artery in a concentration-dependent manner. The effect is not inhibited by the presence of tetrodotoxin or by removal of the endothelium. The mammalian Y1 receptor selective agonist [Leu31Pro34]NPY but not the mammalian Y2 receptor selective agonist neuropeptide Y-(13-36) peptide has vasoconstrictor properties in this system, suggesting that the receptor mediating the vasoconstriction resembles the mammalian Y1 receptor more than the Y2 receptor.

Structural characterization of neuropeptide Y from the brain of the dogfish, Scyliorhinus canicula.

A peptide of the pancreatic polypeptide (PP) family was isolated in pure form from the brain of an elasmobranch fish, Scyliorhinus canicula (European common dogfish). The primary structure of the peptide was established as: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu10-Gly-Ala-Pro-Ala-Glu-Asp- Leu-Ala-Lys- Tyr20-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Ile-Thr-Arg- Gln-Arg-Tyr-NH2. This sequence contains only two amino acid substitutions compared with pig neuropeptide Y (NPY) (Gly for Asp11 and Lys for Arg19), and two substitutions (Gly for Asp11 and Leu for Met17) compared with frog NPY. The amino acid sequence of NPY from dogfish brain is appreciably different from the neuropeptide Y-related peptide previously isolated from dogfish pancreas (five amino acid substitutions). The data indicate that evolutionary pressure to conserve the complete primary structure of neuropeptide Y has been very strong. It is suggested that the NPY-related peptide present in the pancreas of elasmobranch and teleost fish represents the piscine equivalent of mammalian peptide tyrosine tyrosine (PYY).

A C type natriuretic peptide is a vasodilator in vivo and in vitro in the common dogfish.

The effects of an elasmbranch cardiac C-type natriuretic peptide (dogfish CNP-22) on arterial blood pressure were investigated in vivo in chronically cannulated dogfish Scyliorhinus canicula and in vitro by a myographic technique using the distal part of the first branchial artery. In-vivo dogfish CNP-22 caused a dose-dependent reduction in mean arterial blood pressure which was much more potent than that of alpha-human ANP. In-vitro dogfish CNP-22 also caused a dose-dependent relaxation which was independent of the endothelium. These results are in marked contrast to those obtained in similar studies on other vertebrate species in which CNP exhibited only mild hypotensive effects compared to both atrial and brain natriuretic peptides. This study indicates the importance of using homologous peptides in determining the physiological role of natriuretic peptides in non-mammalian vertebrates.

Bombesin-like immunoreactivity in skates and the in vitro effect of bombesin on coronary vessels from the longnose skate, Raja rhina.

Bombesin-like immunoreactivity is present in nerve fibers projecting to the cardiovascular system, including the coronary arteries, and to the gastrointestinal canal, and in endocrine cells of the gut of skates belonging to the family Rajidae. Synthetic bombesin contracted isolated coronary rings from the longnose skate, Raja rhina, in a cumulative fashion. The contractile response was 84% of that of 60 mM potassium chloride. The pD2-value for bombesin was 8.83 (S.E.M. = 0.33; n = 15). Phentolamine, atropine and two substance P-antagonists increased the sensitivity to bombesin, while atenolol, sotalol, nifedipine, tetrodotoxin and two bombesin antagonists were devoid of significant effects. We conclude from this study that a bombesin-like peptide is present in nerves innervating the cardiovascular system and the gastrointestinal canal of skates of the family Rajidae, and that bombesin contracts coronary vessels in vitro via a direct mechanism and/or via mechanisms involving alpha-adrenergic and muscarinergic receptors.

Neuropeptide Y-related peptides from the pancreas of a teleostean (eel), holostean (bowfin) and elasmobranch (skate) fish.

Homologous peptides belonging to the pancreatic polypeptide (PP) family were isolated from the pancreas of a teleostean fish, the American eel (Anguilla rostrata), an holostean fish, the bowfin (Amia calva) and an elasmobranch fish, the skate (Raja rhina), and their primary structures were determined. The peptides show stronger homology to neuropeptide Y, particularly in their COOH-terminal regions, than to peptide YY or pancreatic polypeptide and contain an alpha-amidated COOH-terminal tyrosine residue. The skate peptide Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Asp10-Asp-Ala-Ala-Pro-Glu-Glu- Leu-Ala-Lys- Tyr20-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Ile-Thr-Arg- Gln-Arg-Tyr-NH2 represents the first member of the PP family to be isolated from a cartilaginous fish. The primary structure of the pancreatic PP family peptide has been more strongly conserved among the phylogenetically more ancient holostean and elasmobranch fishes than among the teleosts. A comparison of the primary structures of all PP family peptides supports the hypothesis and evolution has acted to conserve features of tertiiary structure in the molecules (e.g., the polyproline- and alpha-helices) rather than individual amino acid residues.

Bombesin-like immunoreactive material in the gut, and the effect of bombesin on the stomach circulatory system of an elasmobranch fish, Squalus acanthias.

The distribution, nature and amount of bombesin-like immunoreactivity (IR) in the gastrointestinal canal and its afferent vessels was investigated in the spiny dogfish (Squalus acanthias) together with the in vitro effect of synthetic bombesin on perfusion flow through the vascularly perfused dogfish stomach. Nerve fibres showing bombesin-like IR frequently occurred in the walls of the anterior mesenteric and coeliac arteries and the intrinsic vessels of the gut. Chromatographic studies revealed that multiple peaks of bombesin-like IR material were present in extracts of the spiny dogfish gastrointestinal vessels. Bombesin-like IR was also present in muscle and mucosal layers of the gut with higher levels in muscle compared with mucosa, and higher levels in the stomach than in the intestine and the rectum. Exogenous bombesin increased the flow through the vasculary perfused spiny dogfish stomach in a dose-dependent manner. Studies with tetrodotoxin and atropine showed that bombesin probably exerts its effect directly on the vascular musculature. It is concluded from this study that bombesin-like material is present in nerves innervating the gut circulatory system of the spiny dogfish. Bombesin may affect the blood-flow to the gastrointestinal canal, possibly via a direct effect on vascular smooth muscle.

Neuropeptides in the fish gut. An immunohistochemical study of evolutionary patterns.

The presence and distribution of bombesin-, enkephalin-, gastrin/cholecystokinin-, neuropeptide Y-, neurotensin-, somatostatin-, substance P-, and VIP-like immunoreactivities in gut nerves of representatives of nineteen cyclostome, elasmobranch and teleost species have been studied. The results have been correlated to results from previous studies in other species. Nerve plexuses showing bombensin-like, substance P-like and VIP-like immunoreactivity are commonly occurring, while other neuropeptides may have a more varied distribution. Tentative evolutionary patterns, and the possible function and importance of each peptide is discussed.