Susanne Holmgren

The control of gut motility.

Gut motility in non-mammalian vertebrates as in mammals is controlled by the presence of food, by autonomic nerves and by hormones. Feeding and the presence of food initiates contractions of the stomach wall and subsequently gastric emptying, peristalsis, migrating motor complexes and other patterns of motility follow. This overview will give examples of similarities and differences in control systems between species. Gastric receptive relaxation occurs in fish and is an enteric reflex. Cholecystokinin reduces the rate of gastric emptying in fish as in mammals. Inhibitory control of peristalsis is exerted, e.g. by VIP, PACAP, NO in fish and amphibians, while excitatory stimuli arise from nerves releasing tachykinins, acetylcholine or serotonin (5-HT). In crocodiles, we have found the presence of the same nerve types, although the effects on peristalsis have not been studied. Recent studies on signal transduction in the gut smooth muscle of fish and amphibians suggest that external Ca2+ is of great importance, but not the only source of Ca2+ recruitment in tachykinin-, acetylcholine- or serotonin-induced contractions of rainbow trout and Xenopus gastrointestinal smooth muscle. The effect of acetylcholine involves reduction of cAMP-levels in the smooth muscle cells. It is concluded that, in general, the control systems in non-mammalian vertebrates are amazingly similar between species and animal groups and in comparison with mammals.

Bombesin-, gastrin/CCK-, 5-hydroxytryptamine-, neurotensin-, somatostatin-, and VIP-like immunoreactivity and catecholamine fluorescence in the gut of the elasmobranch, Squalus acanthias

SummaryThe presence of peptides and 5-hydroxytryptamine (5-HT) in neurons and endocrine cells in the gastrointestinal tract of the spiny dogfish, Squalus acanthias, was investigated by means of immunohisto-chemistry, and the distribution of catecholamines by use of the Falck-Hillarp fluorescence-histochemical technique. Bombesin-like immunore-activity was present in numerous nerves in all layers and all parts of the gut, and also in endocrine cells in the mucosa throughout the stomach, rectum and intestine. VIP-like immunoreactivity occurred in an abundance of nerve fibres and in nerve cell bodies in all parts of the gut except the oesophagus, while 5-HT-like immunoreactivity was found sparsely in nerve fibres and more frequently in endocrine cells throughout the gut. Gastrin/CCK-like immunoreactivity was present in numerous nerve fibres in the rectum, but only in scattered fibres in the other parts of the gut. Endocrine cells showing gastrin/CCK-like immunoreactivity were present in the intestine only. Somatostatin-like immunoreactivity occurred in both nerve fibres and endocrine cells of the stomach and intestine, but only in nerves in the rectum. Neurotensin-like immunoreactivity was confined to endocrine cells of the intestine. Falck-Hillarp fluorescence histochemistry revealed 5-HT in endocrine cells and catecholamines in nerve fibres (and possibly also in endocrine cells) throughout the gut. Bombesin-, VIP-, gastrin/CCK- and somatostatin-like immunoreactivities and catecholamine fluorescence were present in nerve fibres of the rectal gland and, with the exception of gastrin/CCK-like immunoreactivity, also in nerve bundles in the walls of the coeliac and mesenteric arteries. The findings of the present study form an anatomical basis for the assumption that several of the neuropeptides and amines could function as neurotransmitters or neuromodulators in the gut of Squalus.

Development of enteric and vagal innervation of the zebrafish (Danio rerio) gut

The autonomic nervous system develops following migration and differentiation of precursor cells originating in the neural crest. Using immunohistochemistry on intact zebrafish embryos and larvae we followed the development of the intrinsic enteric and extrinsic vagal innervation of the gut. At 3 days postfertilization (dpf), enteric nerve cell bodies and fibers were seen mainly in the middle and distal intestine, while the innervation of the proximal intestine was scarcer. The number of fibers and cell bodies gradually increased, although a large intraindividual variation was seen in the timing (but not the order) of development. At 11–13 dpf most of the proximal intestine received a similar degree of innervation as the rest of the gut. The main intestinal branches of the vagus were similarly often already well developed at 3 dpf, entering the gut at the transition between the proximal and middle intestine and projecting posteriorly along the length of the gut. Subsequently, fibers branching off the vagus innervated all regions of the gut. The presence of several putative enteric neurotransmitters was suggested by using markers for neurokinin A (NKA), pituitary adenylate cyclase‐activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide, serotonin (5‐hydroxytryptamine, 5‐HT), and calcitonin gene‐related peptide (CGRP). The present results corroborate the belief that the enteric innervation is well developed before the onset of feeding (normally occurring around 5–6 dpf). Further, the more detailed picture of how development proceeds at stages previously not examined suggests a correlation between increasing innervation and more regular and elaborated motility patterns. J. Comp. Neurol. 508:756–770, 2008.

Ontogeny of the gut motility control system in zebrafish Danio rerio embryos and larvae

SUMMARY Using digital motion analysis, the ontogeny of the cholinergic, tachykinin and pituitary adenylate cyclase-activating polypeptide (PACAP) control systems was studied in zebrafish Danio rerio larvae, in vivo. For the first time we show that the regular propagating anterograde waves that occur in the zebrafish larval gut before and around the onset [at 5–6 days post fertilization (d.p.f.)] of feeding are modulated by acetylcholine or atropine, PACAP and NKA (neurokinin A). At 3 d.p.f., when no spontaneous motility has developed, application of acetylcholine did not affect the gut. However, at 4 d.p.f., acetylcholine increased and atropine reduced the frequency of propagating anterograde waves. At 5 d.p.f., NKA increased and PACAP reduced the wave frequency. This suggests that both excitatory and inhibitory pathways develop at an early stage in the gut, independent of exogenous feeding. Immunohistochemistry established the presence of gut neurons expressing PACAP and NKA in the proximal part of the developing gut from the first stage investigated (2 d.p.f.) and before regular motility was observed. 1 d.p.f. (PACAP) or 2 d.p.f. (NKA) stages later the whole gut was innervated. This supports physiological results that gut motility is under neuronal control during the period when regular motility patterns develop.

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)

Respiratory and cardiovascular responses to hypoxia in the Australian lungfish.

Simultaneous measurements of pulmonary blood flow (qPA), coeliacomesenteric blood flow (qCoA), dorsal aortic blood pressure (PDA), heart rate (fH) and branchial ventilation frequency (fv) were made in the Australian lungfish, Neoceratodus forsteri, during air breathing and aquatic hypoxia. The cholinergic and adrenergic influences on the cardiovascular system were investigated during normoxia using pharmacological agents, and the presence of catecholamines and serotonin in different tissues was investigated using histochemistry. Air breathing rarely occurred during normoxia but when it did, it was always associated with increased pulmonary blood flow. The pulmonary vasculature is influenced by both a cholinergic and adrenergic tonus whereas the coeliacomesenteric vasculature is influenced by a beta-adrenergic vasodilator mechanism. No adrenergic nerve fibers could be demonstrated in Neoceratodus but catecholamine-containing endothelial cells were found in the atrium of the heart. In addition, serotonin-immunoreactive cells were demonstrated in the pulmonary epithelium. The most prominent response to aquatic hypoxia was an increase in gill breathing frequency followed by an increased number of air breaths together with increased pulmonary blood flow. It is clear from the present investigation that Neoceratodus is able to match cardiovascular performance to meet the changes in respiration during hypoxia.

Neuropeptides in the fish gut

SummaryThe 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 bombesinlike, 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.

The Comparative physiology of regulatory peptides

Part 1 Distribution and function of regulatory peptides: atrial natrieretic peptides localization, structure, function and phylogeny, Manfred Reinecke Bombesin, S.R.Vigna and M.C.Thorndyke gastrin/CCk-related peptides: comparative aspects, A.Jonsson neurotensin, R.E.Carraway and M.Reinecke opioid peptides: comparative peripheral mechanisms, G.B.Stefano substance P and other tachykinins, J.Jensen vasoactive intestinal peptide, R.Dimaline pancreatic peptides, E.M.Plisetskaya peptides in invertebrates, M.C.Thorndyke. Part 2 Integrated physiological functions: gut mobility, S.Holmgren gut secretion, A.Jonsson and S.Holmgren the cardiovascular system, J.Morris co-existence and co-function, I.Gibbins biosynthesis of regulatory peptides: evolutionary aspects, J.M.Conlon.

Gastrin/CCK-like immunoreactivity in endocrine cells and nerves in the gastrointestinal tract of the cod, Gadus morhua, and the effect of peptides of the gastrin/CCK family on cod gastrointestinal smooth muscle.

Immunohistochemistry shows the presence of one or several gastrin/cholecystokinin (CCK)-like peptides in the gastrointestinal tract of the Atlantic cod, Gadus morhua. Immunoreactive (IR) endocrine cells are present mainly in the lower part of the cardiac and in the pyloric stomach, with a concentration to the inner curvature, and in the proximal part of the intestine, especially in the area of the pyloric caeca, after which the population density of IR cells decreases anally. IR nerve fibers are found in the myenteric plexus and muscle layers of the stomach only. Radioimmunoassays, performed with two C-terminal specific gastrin/CCK antisera, confirm the immunohistochemical findings. The highest levels of IR material are found in the mucosa of the pyloric caeca, the proximal intestine, and the pyloric part of the stomach. The smooth muscle layers contain IR material in a decreasing gradient toward the rectum. The excitatory effect of gastrin/CCK-related peptides on gut smooth muscle is investigated in isolated strip preparations of the gut wall. Of the peptides tested, cerulein is the most potent, followed by CCK 8 and gastrin 17, while gastrin 5 is the least effective. Sulphated CCK 8 gives a higher amplitude of the response but in the same concentration range as nonsulphated CCK 8. It is concluded that one or several gastrin/CCK-like peptides are present in the gut of the cod and may be involved in the nervous and endocrine control of the cod gut motility.