Jörgen Jensen

Evolution of vertebrate neuropeptides.

This review describes some of the most typical features in the evolution of neuropeptides. Neuropeptides are synthesized like other polypeptides and proteins, with an amino acid sequence determined by the DNA sequence of the corresponding gene. Mutations of bases in the coding regions of the DNA lead to changes in amino acid sequence, and explain the differences in amino acid sequence of a certain neuropeptide in different animal species. The more distantly related two species are, the more substitutions can be found in one and the same neuropeptide. The biologically active part of the neuropeptide is usually the most conserved part. Neuropeptides also form families of closely related peptides, where several members may occur in one animal species. This is due to gene or exon duplications followed by mutations. Gene splicing and posttranslational processing decides the gene product in a single cell. Difference in sequence may cause difference in function, but more often than not, members of a family appear to produce the same effect. Three neuropeptide families, the tachykinins, the neuropeptide Y family, and the vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide family will be described in more detail.

Neurotransmitters in the intestine of the atlantic cod, Gadus morhua

The effects of the putative neurotransmitters acetylcholine, adrenaline, adenosine, ATP, bombesin, 5-hydroxytryptamine, met-enkephalin, neurotensin, somatostatin, substance P and VIP have been investigated in the perfused intestine of the cod, Gadus morhua. The presence and distribution of the different types of nerves was investigated with immunohistochemistry and Falck-Hillarp fluorescence histochemistry. A spontaneous rhythmic activity of the perfused preparations usually occurred within a few minutes from the start of the experiment. This activity was diminished or abolished by addition of atropine, methysergide or tetrodotoxin to the perfusion fluid. Acetylcholine, 5-hydroxytryptamine or substance P caused a contraction of the intestinal wall. The response to acetylcholine was blocked by atropine but not by tetrodotoxin, while the response to 5-hydroxytryptamine was blocked by methysergide and usually also by tetrodotoxin. This indicates that the effect of acetylcholine is direct on the muscle cells, while the effect of 5-hydroxytryptamine may be at least partly via a second neuron. All adrenergic agonists (adrenaline, isoprenaline and phenylephrine) had a dominating inhibitory effect on the intestine. Experiments with antagonists showed that the inhibition is due to stimulation of both alpha-adrenoceptors and beta-adrenoceptors. ATP, adenosine and somatostatin also caused a relaxation of the intestinal wall, often followed by a contraction. Met-enkephalin produced variable responses, either a relaxation, a contraction or both. Bombesin caused a weak inhibition, if anything. Neurotensin and VIP did not visibly affect the intestinal motility. 5-HT-, substance P- and VIP-like immunoreactivity and catecholamine fluorescence were observed in the myenteric plexus, submucosa and muscle layers in all parts of the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulatory peptides and control of food intake in non-mammalian vertebrates

The current view of the control of food intake involves a central feeding system in the hypothalamus receiving input from peripheral systems. The presence of food in the gut stimulates the release of several regulatory peptides that control gut motility and secretion. Some of these peptides also act as feedback satiety signals, responsible for termination of a meal. Among the regulatory peptides suggested as peripheral satiety signals are cholecystokinin and gastrin releasing peptide. A more long-term peripheral regulation of food intake has also been postulated and leptin has been suggested as a regulator of food intake. Several regulatory peptides mediate orexigenic or anorexigenic effects in the central feeding system. Neuropeptide Y and galanin both act centrally and stimulate the intake of food, while corticotropin releasing factor reduces food intake. At present, most information about the regulation of food intake is gained from mammalian studies and these findings are used as a base for a discussion on the current knowledge of how regulatory peptides control appetite in non-mammalian vertebrates.

Tachykinins and intestinal motility in different fish groups

The presence and function of tachykinins were studied in the intestine of hagfish (Myxine glutinosa), lamprey (Lampetra fluviatilis), starry ray (Raja radiata), lungfish (Lepidosiren paradoxa), bichir (Polypterus senegalensis), and rainbow trout (Oncorhynchus mykiss), which represent different systematic groups of fish. Immunohistochemistry revealed cells containing substance P (SP)-like material in the intestine of lamprey and lungfish, and in the stomach of the ray. The intestinal motility was studied using isolated muscle strip preparations. SP had no effect on hagfish or lamprey intestine. In the other four species SP produced intestinal contractions. In ray, bichir, and lungfish the tachykinins may be released from endocrine cells and act, at least in the bichir and lungfish, directly onto the smooth muscle cells. In the rainbow trout intestine, where SP-like material may be released from both nerve fibres and endocrine cells, it is indicated that the contractile effect is in part direct upon the smooth muscle and in part via stimulation of cholinergic and serotonergic neurons.

Projections and actions of tachykininergic, cholinergic, and serotonergic neurones in the intestine of the Atlantic cod

Abstract The native tachykinins cod neurokinin A and cod substance P, serotonin and acetylcholine have excitatory effects on the circular smooth muscle of the cod intestine. Furthermore, immunoreactivities to the cod tachykinins, serotonin and two markers for cholinergic neurones, viz. choline acetyltransferase and vesicular acetylcholine transporter, have been demonstrated in myenteric neurones of the cod intestine. In order to elucidate whether the neurones containing these substances project orally and thus might be involved in the ascending excitatory reflex of peristalsis, myotomy operations have been performed on the cod intestine. The immunoreactive areas of the myenteric plexus immediately oral and anal to the myotomy operations have been measured by using confocal laser scanning microscopy. Large accumulations of immunoreactivity to the tachykinins are found on the anal side of the myotomies, indicating oral projections of tachykininergic neurones. The areas immunoreactive to serotonin and choline acetyltransferase are of equal size on the oral and anal sides. Since the tachykinin containing neurones of the intestine project orally, and since cod neurokinin A and cod substance P have excitatory effects on circular smooth muscle, we conclude that tachykininergic neurones are involved in the ascending excitatory reflex of peristalsis in the cod intestine.

Primary structure, distribution, and effects on motility of CGRP in the intestine of the cod Gadus morhua

Calcitonin gene-related peptide (CGRP) was isolated from an extract of the intestine of the cod Gadus morhua. The primary structure of this 37-amino acid peptide was established as follows: ACNTA TCVTH RLADF LSRSG GIGNS NFVPT NVGSK AF-NH2. The peptide shows close structural similarities to other nonmammalian (3-4 amino acid substitutions) and mammalian (5-8 amino acid substitutions) CGRPs, and it contains the two residues Asp14 and Phe15 that seem to be characteristic for CGRP in nonmammalian vertebrates. Cod CGRP (10(-9)-10(-7) M) inhibited the motility of spontaneously active ring preparations from the cod intestine and was significantly (P < 0.05) more potent than rat alpha-CGRP. Neither prostaglandins nor nitric oxide is involved in the inhibitory response produced by cod CGRP, and the lack of effect of tetrodotoxin suggests an action of CGRP on receptors on the intestinal smooth muscle cells. The competitive CGRP antagonist human alpha-CGRP-(8-37) significantly (P < 0.05) reduced the response to cod CGRP. Immunohistochemistry demonstrated CGRP-immunoreactive neurons intrinsic to the intestine, and a dense innervation with immunoreactive nerve fibers was observed in the myenteric plexus and the circular muscle layer. Myotomy studies show that CGRP-containing nerves project orally and anally in the myenteric plexus, whereas nerve fibers in the circular muscle layer project mainly anally, indicating a role for CGRP in descending inhibitory pathways of the cod intestine.Calcitonin gene-related peptide (CGRP) was isolated from an extract of the intestine of the cod Gadus morhua. The primary structure of this 37-amino acid peptide was established as follows: ACNTA TCVTH RLADF LSRSG GIGNS NFVPT NVGSK AF-NH2. The peptide shows close structural similarities to other nonmammalian (3-4 amino acid substitutions) and mammalian (5-8 amino acid substitutions) CGRPs, and it contains the two residues Asp14 and Phe15 that seem to be characteristic for CGRP in nonmammalian vertebrates. Cod CGRP (10-9-10-7M) inhibited the motility of spontaneously active ring preparations from the cod intestine and was significantly ( P < 0.05) more potent than rat α-CGRP. Neither prostaglandins nor nitric oxide is involved in the inhibitory response produced by cod CGRP, and the lack of effect of tetrodotoxin suggests an action of CGRP on receptors on the intestinal smooth muscle cells. The competitive CGRP antagonist human α-CGRP-(8-37) significantly ( P < 0.05) reduced the response to cod CGRP. Immunohistochemistry demonstrated CGRP-immunoreactive neurons intrinsic to the intestine, and a dense innervation with immunoreactive nerve fibers was observed in the myenteric plexus and the circular muscle layer. Myotomy studies show that CGRP-containing nerves project orally and anally in the myenteric plexus, whereas nerve fibers in the circular muscle layer project mainly anally, indicating a role for CGRP in descending inhibitory pathways of the cod intestine.

Vasoconstrictive effects of native tachykinins in the rainbow trout, Oncorhynchus mykiss

The role of trout substance P (tSP) and neurokinin A (tNKA) in cardiovascular regulation was investigated in the rainbow trout, Oncorhynchus mykiss, in vivo and in vitro. In vivo, the coeliac arterial and ventral aortic relative blood flows were measured with Doppler flow probes, and blood pressure was measured via a cannula inserted into the dorsal aorta. tSP (0.1 and 1 nmol kg-1) and tNKA (1 nmol kg-1) increased both systemic and coeliac vascular resistances, leading to hypertension and bradycardia. In addition, cardiac output was decreased. The mammalian NK1 tachykinin receptor antagonist CP-96,345 did not affect the responses to tSP or tNKA. In vitro perfusions of the dorsal aortic and coeliacomesenteric vascular beds were performed using peristaltic pumps. The dorsal aortic vascular resistance was dose-dependently increased following infusion of the two peptides (pD2 values 7.6 +/- 0.1 and 7.3 +/- 0.1 for tSP and tNKA, respectively). Tetrodotoxin did not affect the tSP-induced hypertension. Increases in coeliac vascular resistance caused by tSP was correlated with stomach contractions when measurement of intragastric pressure was made using an inserted balloon. In conclusion, native SP and NKA are potent vasoconstrictors of rainbow trout vasculature, a property quite unusual to tachykinins compared with the vasodilation normally seen in mammals.

Effects of cod bradykinin and its analogs on vascular and intestinal smooth muscle of the Atlantic cod, Gadus morhua

The effects of [Arg(0),Trp(5),Leu(8)]-BK (cod [Arg(0)]BK) on vascular preparations from branches of the cod celiac artery and on longitudinal smooth muscle preparations from the cod intestine were investigated. Cod [Arg(0)]BK (3 x 10(-8) M) caused a relaxation of the celiac artery precontracted with adrenaline. The relaxation was abolished by the cyclooxygenase inhibitor indomethacin, suggesting that the effect is mediated through the release of prostaglandins, but there was no evidence for the involvement of leukotrienes or nitric oxide in the response. In the intestinal preparations, cod [Arg(0)]BK produced concentration-dependent contractions (pD(2) = 8.28 +/- 0.16). Experiments with N-terminally and C-terminally truncated analogs and with alanine-substituted analogs of cod [Arg(0)]BK demonstrate that the central amino acid Gly(4) and the C-terminal amino acids Leu(8) and Arg(9) are the most important in determining the conformation of the peptide that interacts with the receptor. The results indicate that the ligand binding properties of the cod BK receptor are considerably different from the receptor present in trout tissues and may resemble those of the mammalian B(2) receptor more closely.

Structure-activity relationships of trout bradykinin ([Arg0,Trp5,Leu8]-bradykinin])

Abstract Trout bradykinin ([Arg0,Trp5,Leu8]-BK) produces sustained and concentration–dependent contractions of isolated longitudinal smooth muscle from trout stomach, although mammalian BK is without effect. Circular dichroism studies have demonstrated that trout BK, unlike mammalian BK, does not adopt a stable β-turn conformation, even in the presence of sodium dodecyl sulfate (SDS) or trifluoroethanol. The myotropic actions of a series of analogs in which each amino acid in trout BK was replaced by either alanine or the corresponding D-isomer were investigated. The peptides with Ala4, D-Pro3, D-Trp5, D-Ser6, and D-Pro7 substitutions were inactive and did not act as antagonists of trout BK. The analog with [Ala5] was a weak partial agonist. The substitution (Arg0 → Ala) led to >50-fold decrease in potency but, in contrast to the importance of Phe8 in both BK and desArg9-BK in activating the mammalian B2 and B1 receptors respectively, substitutions at Leu8 in trout BK had only a minor effect on potency. Antagonists to the mammalian B2 receptor generally contain a D-aromatic amino acid at position 7 of BK but the analog [Arg0,Trp5,D-Phe7,Leu8]-BK was a weak agonist at the trout receptor. Similarly, the potent nonpeptide mammalian B2 receptor antagonist FR173657 was without effect on the action of trout BK. These data suggest the hypothesis that the receptor binding conformation of trout BK is defined by the central region (residues 3–7) of the peptide but is adopted only upon interaction with the receptor. The bioactive conformation is probably stabilized by an ionic interaction between Arg0 in the peptide and an acidic residue in the receptor.

Co-Release of Substance P and Neurokinin A From the Atlantic Cod Stomach

The function of tachykinins in the control of gastric motility in the cod, Gadus morhua, was studied using native cod substance P ([Lys1, Arg3, Ile3]SP) and cod neurokinin A ([Ile3, Asn4]NKA). Both cod SP and NKA produced contractions of the vascularly perfused cod stomach, SP being almost 6 times more potent than NKA (pD2-values 7.05 +/- 0.06 and 6.28 +/- 0.09, respectively). The release of tachykinins from the cod stomach was measured in radioimmunoassay, using specific antibodies for the two cod tachykinins. Stimulation of the stomach motility by electrical stimulation of the vagus nerve or infusion of acetylcholine increased the amounts of SP and NKA released into the vascular perfusate. The results suggest that both tachykinins are involved in the excitatory response of the cod stomach produced by vagal and cholinergic stimulation.