Influence of weaning and effect of post weaning dietary zinc and copper on electrophysiological response to glucose, theophylline and 5-HT in piglet small intestinal mucosa

This study aimed to examine how weaning and how dietary zinc and/or copper fed post weaning may affect the electrophysiological response to glucose and to chloride secretagogues in piglet small intestine in vitro. Study 1 included 54 piglets (six litters of nine piglets). One piglet from every litter was killed 1 day before weaning. The remaining 48 piglets were allocated at weaning (28 d) to four dietary zinc treatments and subsequently killed 1-2, 5-6 or 14-15 days after weaning. Study 2 included 48 piglets (six litters of eight piglets) allocated to four dietary treatments, consisting of high or low dietary zinc with or without high dietary copper. All piglets in study 2 were killed 5-7 days after weaning. The in vitro studies in Ussing chambers showed that weaning resulted in increased ileal glucose absorption as well as increased neuroendocrine-regulated (activated by 5-HT) and cAMP-dependent (activated by theophylline) chloride secretion. High zinc supplementation reduced the responses to 5-HT and theophylline. The study did not reveal any influence of copper on these parameters. It is concluded that the positive effect of zinc supplementation on diarrhoea in weaned piglets may be due to zinc reducing the intestinal mucosal susceptibility to secretagogues that activate chloride secretion.     

Serosal zinc attenuate serotonin and vasoactive intestinal peptide induced secretion in piglet small intestinal epithelium in vitro

This study addressed the mechanisms by which dietary zinc affects diarrhoea and aimed to study possible interactions between zinc status and the presence of zinc in vitro on secretagogue-induced secretion from piglet intestinal epithelium in Ussing chambers. In addition, it was studied from which side of the epithelium zinc would perform an effect and if copper caused similar effects. Twenty-four piglets (28 days of age) were weaned and fed diets containing 100 or 2500 mg zinc/kg (as ZnO) for 5 or 6 days (12 piglets per group). Intestinal epithelium underwent the following 5 treatments: zinc at the mucosal side (M(Zn)), zinc at the serosal side (S(Zn)), zinc at both sides (MS(Zn)), copper at both sides (MS(Cu)) or water at both sides (control). Provoked secretion in terms of short circuit responses to serotonin (5-HT) and vasoactive intestinal peptide (VIP) were measured. Zinc at the serosal or both sides of the epithelium reduced the 5-HT induced secretion (P<0.001); however, due to interactions (P=0.05) the effect of zinc in vitro was only present in the ZnO(100) group. The secretion caused by VIP was not affected by the diet (P=0.33), but zinc at the serosal side or both sides reduced the response to VIP (P<0.001). Copper reduced the 5-HT and VIP induced secretion to a larger extent than zinc. However, copper also disturbed intestinal barrier function as demonstrated by increased transepithelial conductance and increased short circuit current, which was unaffected by zinc. In conclusion, zinc at the serosal side of piglet small intestinal epithelium attenuated 5-HT and VIP induced secretion in vitro. These in vitro studies indicate that in vivo there will be no positive acute effect of increasing luminal Zn concentration on secretagogue-induced chloride secretion and that zinc status at the serosal side of the epithelium has to be increased to reduce secretagogue-induced chloride secretion and thereby diarrhoea.     

Serosal zinc attenuate serotonin and vasoactive intestinal peptide induced secretion in piglet small intestinal epithelium in vitro.

This study addressed the mechanisms by which dietary zinc affects diarrhoea and aimed to study possible interactions between zinc status and the presence of zinc in vitro on secretagogue-induced secretion from piglet intestinal epithelium in Ussing chambers. In addition, it was studied from which side of the epithelium zinc would perform an effect and if copper caused similar effects. Twenty-four piglets (28 days of age) were weaned and fed diets containing 100 or 2500 mg zinc/kg (as ZnO) for 5 or 6 days (12 piglets per group). Intestinal epithelium underwent the following 5 treatments: zinc at the mucosal side (M(Zn)), zinc at the serosal side (S(Zn)), zinc at both sides (MS(Zn)), copper at both sides (MS(Cu)) or water at both sides (control). Provoked secretion in terms of short circuit responses to serotonin (5-HT) and vasoactive intestinal peptide (VIP) were measured. Zinc at the serosal or both sides of the epithelium reduced the 5-HT induced secretion (P<0.001); however, due to interactions (P=0.05) the effect of zinc in vitro was only present in the ZnO(100) group. The secretion caused by VIP was not affected by the diet (P=0.33), but zinc at the serosal side or both sides reduced the response to VIP (P<0.001). Copper reduced the 5-HT and VIP induced secretion to a larger extent than zinc. However, copper also disturbed intestinal barrier function as demonstrated by increased transepithelial conductance and increased short circuit current, which was unaffected by zinc. In conclusion, zinc at the serosal side of piglet small intestinal epithelium attenuated 5-HT and VIP induced secretion in vitro. These in vitro studies indicate that in vivo there will be no positive acute effect of increasing luminal Zn concentration on secretagogue-induced chloride secretion and that zinc status at the serosal side of the epithelium has to be increased to reduce secretagogue-induced chloride secretion and thereby diarrhoea.     

Octreotide inhibits increases in short-circuit current induced in rat colon by VIP, substance P, serotonin and aminophylline.

We investigated the effect of octreotide (OCT), a stable somatostatin analog, (OCT) on changes in short-circuit current (Isc) induced by vasoactive intestinal peptide (VIP), aminophylline, serotonin (5-HT) and substance P. OCT significantly decreased basal Isc at a concentration of 10(-9) M; the maximum decrease in Isc was observed at 10(-6) M. OCT (10(-7) M) significantly inhibited the intestinal secretory response to all the secretagogues studied. The maximum Isc response was reduced when tissues were stimulated with VIP (184.9 +/- 18.0 vs. 119.7 +/- 14.1, P less than 0.05), 5-HT (135.1 +/- 14.4 vs. 79.5 +/- 13.4, P less than 0.05) and substance P (156.0 +/- 19.2 vs. 30.7 +/- 5.4, P less than 0.01). In the case of aminophylline, the concentration-response curve was shifted to the right but the maximum response was not reduced. Because VIP and aminophylline increase cAMP while 5-HT and substance P stimulate intestinal secretion principally by a calcium linked mechanism, we conclude that OCT inhibits Isc in rat colon by more than one mechanism.     

Zinc attenuates forskolin-stimulated electrolyte secretion without involvement of the enteric nervous system in small intestinal epithelium from weaned piglets

In a previous study, we found that secretagogue-stimulated electrolyte secretion was attenuated by dietary and serosal zinc in piglet small intestinal epithelium in Ussing chambers. Several studies show that the enteric nervous system (ENS) is involved in regulation of electrolyte and/or fluid transport in intestinal epithelium from many species. The aim of the present study is to examine the mechanisms behind the attenuating effect of zinc on electrolyte secretion and to study whether the ENS is involved in this effect of zinc in vitro. Twenty-four piglets (six litters of four piglets) were allocated randomly to one of two dietary treatments consisting of a basic diet supplemented with 100 mg zinc/kg (Zn(100)) or 2500 mg zinc/kg (Zn(2500)), as ZnO. All the piglets were killed at 5-6 days after weaning and in vitro experiments with small intestinal epithelium in Ussing chambers were carried out. Furthermore, zinc, copper, alkaline phosphatase (AP) and metallothionein (MT) in mucosa, liver, and plasma were measured. These measurements showed that zinc status was increased in the Zn(2500) compared to the Zn(100) fed piglets. The in vitro studies did not confirm previous findings of attenuating effects of dietary zinc and zinc in vitro on the 5-HT induced secretion. But it showed that the addition of zinc at the serosal side attenuated the forskolin (FSK) (cAMP-dependent) induced ion secretion in epithelium from piglets fed with Zn(100) diet. Blocking the ENS with lidocaine or hexamethonium apparently slightly reduced this effect of zinc in vitro, but did not remove the effect of zinc. Consequently, it is suggested that zinc attenuates the cAMP dependent ion secretion mainly due to an effect on epithelial cells rather than affecting the mucosal neuronal pathway.     

Ca ionophore-stimulated ion secretion in rabbit ileal mucosa: Relation to actions of cyclic 3′,5′-AMP and carbamylcholine

Summary Addition of the Ca ionophore, A23187 (0.5 g/ml) to the serosal side of stripped rabbit ileal mucosa, produced changes in ion transport qualitatively identical with those produced by cyclic 3,5-AMP (cAMP) and theophylline: an increase in short-circuit current and resistance, net secretion of Cl due both to a decrease in the unidirectional mucosa (m) to serosa (s) flux and an increase in the (s) to (m) flux, and net secretion of Na due to a decrease in (m) to (s) flux. Measurements of intracellular cAMP level demonstrated no change following incubation with the ionophore. Removal of Ca from the serosal bathing medium diminished the effects of A23187 but did not impair the action of theophylline. Furthermore, removal of Ca from both the mucosal and serosal bathing media by replacing it with Sr completely abolished the p.d. response to A23187. These results suggest that the ionophore elicits its secretory actions by increasing Ca influx into the epithelial cells. In a similar way, carbamylcholine and serotonin, secretagogues known to have no effect on intracellular cAMP level in intestinal mucosa, were shown to be dependent on extracellular Ca to produce their full electrical response (although, in the case of carbamylcholine at least, Sr can substitute for Ca). In contrast, the secretagogues vasoactive intestinal peptide and prostaglandin E₁, which raise cAMP concentration in intestinal mucosa, do not appear to require external Ca. It is interesting to speculate that Ca is an intracellular mediator of intestinal ion and water secretion and that some intestinal secretagogues may act as Ca ionophores.     

Substance P but not vasoactive intestinal peptide modulates immunoglobulin secretion in murine schistosomiasis.

Neuropeptides including SP and VIP modulate Ig secretion by in vitro stimulated lymphocyte cultures. It is not known whether these neuropeptides effect the B cell directly, or if they significantly alter humoral immune responses to pathogens. We have previously shown that granulomas derived from schistosome-infected mice contain immunoglobulin secreting B cells (ISC) as well as eosinophils that secrete substance P (SP) and vasoactive intestinal peptide (VIP). It therefore seemed plausible that B cells derived from infected animals might respond to these neuropeptides, and that such responses might effect immunoregulatory signals. In this study, we addressed these issues in the murine Schistosoma mansoni model, at the level of immunoglobulin secretion in single B cells. Spontaneous ISC were observed in both splenic and granuloma cell preparations. The addition of SP resulted in a dose-dependent reduction in the number and size of plaques (a 50% reduction was observed at 10(-9) M). This effect was blocked with SP antagonists. Similar results were observed in T cell-depleted cell cultures. VIP had no effect on ISC number or plaque size. We conclude that SP, but not VIP, decreases spontaneous ISC number and Ig secretion in short-term cultures of spleen and granuloma cells. SP appears to exert its effects at the level of single B cells through a receptor-mediated mechanism and may thus play an immunoregulatory role in schistosomiasis.     

Intestinal secretagogues increase cytosolic free Ca2+ concentration and K+ conductance in a human intestinal epithelial cell line

Summary A human intestinal epithelial cell line (Intestine 407) is known to retain receptors for intestinal secretagogues such as acetylcholine (ACh), histamine, serotonin (5-HT) and vasoactive intestinal peptide (VIP). The cells were also found to possess separate receptors for secretin and ATP, the stimulation of which elicited transient hyperpolarizations coupled to decreased membrane resistances. These responses were reversed in polarity at the K⁺ equilibrium potential. The hyperpolarizing responses to six agonists were reversibly inhibited by quinine or quinidine. By means of Ca²⁺-selective microelectrodes, increases in the cytosolic free Ca²⁺ concentration were observed in response to individual secretagogues. The time course of Ca²⁺ responses coincided with that of hyperpolarizing responses. The responses to ACh and 5-HT were abolished by a reduction in the extracellular Ca²⁺ concentration down to pCa 7 or by application of Co²⁺. Thus, in Intestine 407 cells, not only the intestinal secretagogues, which are believed to act via increased cytosolic Ca²⁺ (ACh, 5-HT and histamine), but also those which elevate cyclic AMP (VIP, secretin and ATP) induce increases in cytosolic Ca²⁺, thereby activating the K⁺ conductance. It is likely that the origin of increased cytosolic Ca²⁺ is mainly extracellular for ACh- and 5-HT-induced responses, whereas histamine, VIP, secretin and ATP mobilize Ca²⁺ from the internal compartment.     

Insulin, glucagon, and somatostatin secretion by cultured rat islet cell tumor and its clones

Cells derived from rat islet tumor and grown in culture (parent cells-RIN-m) and two clones obtained from them were used to study the effect of various secretagogues on insulin, glucagon, and somatostatin secretion. Parent cells secreted all three hormones in various quantities, while clone 5F secreted predominantly insulin and clone 14B secreted predominantly somatostatin. The secretory behavior of these cells were compared to each other and to that of normal islets. In general, as in the case of normal islets, insulin secretion was stimulated by calcium, potassium, tolbutamide, theophylline, and glucagon. It was inhibited by somatostatin. Glucagon secretion was stimulated by calcium, arginine, and theophylline. Somatostatin secretion was stimulated in clone 14B by arginine, tolbutamide, theophylline, and insulin. These cells differ from normal islets, in that they do not respond to glucose or arginine with increased insulin secretion. Also somatostatin failed to inhibit glucagon secretion. The similarity in insulin secretory responses of parent cells and clone 5F suggests that local or paracrine islet hormone secretion plays only a negligible role in the control of other hormone secretion in these cells.     

Soybean impairs Na(+)-dependent glucose absorption and Cl- secretion in porcine small intestine

Recent evidence indicates that soybean, which is widely used in animal nutrition, could directly alter intestinal ion and nutrient transport. However, the mechanisms involved are still unknown. The aim of the study was to investigate the effect of three differently treated soybean products on the glucose and Cl- transport capacity in porcine small intestine by the Ussing chamber technique. Jejunal and ileal piglet epithelial tissues were pre-incubated with extracts of raw soybean flour (RSF), heated soybean flour (HSF), or ethanol heat-treated soybean protein concentrate (SPC). The Na(+)-dependent glucose co-absorption capacity was then measured as an increase in the short-circuit current (ISC) after luminal addition of D-glucose. The effect of the soybean products on cAMP-dependent Cl- secretion was measured as the increase in ISC after the addition of the phosphodiesterase inhibitor, theophylline, while nervous regulation of Cl- secretion was investigated by the addition of the enteric neurotransmitters; 5-hydroxytryptamine (5-HT), substance P and vasoactive intestinal polypeptide (VIP). Incubation with RSF and HSF induced a 30% decrease of the Na(+)-dependent glucose absorption capacity in the jejunum. The effect was similar for RSF in the ileum. Theophylline-induced secretion was decreased by 30% after incubation with RSF, HSF and SPC but only in the jejunum. 5-HT-, substance P- and VIP-induced secretion were not altered by incubation with soybean extracts except in the HSF-incubated where the substance P-induced secretion was significantly reduced. In conclusion, soybean contains ethanol-sensitive heat-insensitive compounds impairing Na(+)-dependent glucose absorption in the jejunum and ileum, and ethanol- and heat-insensitive compounds causing an acute impairment of cAMP-dependent jejunal secretion.     

Effects of age on morphology,protein synthesis and secretagogoue-evoked secretory responses in the rat lacrimal gland

This study investigated changes in the morphology and protein synthesis and protein and peroxidase secretion due to peptidergic and aminergic stimulation from rat lacrimal gland acinar cells of 3–5, 9, 12, 20 and 24 month old rats. There was a marked reduction in the presence of Golgi apparatus in the acinar cells of glands from the 24 month old rats coupled to dilatation and degeneration of rough endoplasmic reticulum, when compared to that in the acinar cells of glands from 3–5 and 12 month old rats. Following incorporation of tritiated leucine for 360 min (6 h), the amount of newly synthesised protein in acinar cells of the 12 month old rats was significantly (p < 0.01) higher than that in the acinar cells of 3–5 month old animals. However, at 20 months the amount of newly synthesised protein in these acinar cells was significantly (p < 0.01) reduced to less than that in acinar cells of both the 3–5 and 12 month old animals. Immunohistochemical and immunofluorescence studies identified the presence of substance P (SP), vasoactive intestinal peptide (VIP), histamine and 5-hydroxytryptamine (5-HT) in the lacrimal glands of 3–5 month old rats. Stimulation by either SP, VIP, histamine or 5-HT resulted in significant increases in total protein output and peroxidase release from acinar cells of the 3–5 month old rats. However, all responses to the secretagogues were reduced with ageing from 3–5 to 24 months of age. The results indicate that ageing is associated with alteration in the ability of acinar cells to synthesise and secrete proteins.     

Depletion of neuropeptides in rat parotid glands and declining atropine-resistant salivary secretion upon continuous parasympathetic nerve stimulation

In rats the parasympathetic auriculo-temporal nerve on one side was continuously stimulated at 40 Hz for 20-80 min in the presence of adrenergic blockers (dihydroergotamine and propranolol) +/- atropine. During the first 10 min this gave rise to a flow of saliva from the parotid gland that in the atropinized rats amounted to 35% of that found in rats not treated with atropine, while the protein and amylase outputs were 75% of those in non-atropinized rats. The atropine-resistant secretion of fluid and proteins declined to 5-10% of the initial value within 40 min but did not cease completely even after 80 min. The marked reduction in secretory responses was not due to desensitization or exhaustion of the gland cells. The nerve stimulation reduced the parotid gland content of vasoactive intestinal peptide (VIP) and substance P (SP) to approximately 60 and 25% of that of contralateral glands after 20 and 60 min, respectively. The probable explanation for the decline in secretory response seems to be depletion of non-adrenergic, non-cholinergic transmitter(s). The present results suggest that neuropeptides are involved in the regulation of salivary secretion but provide no direct evidence that either VIP or SP is responsible for the atropine-resistant salivary secretion.     

Electrical activity of an intestinal epithelial cell line: Hyperpolarizing responses to intestinal secretagogues

Summary Cultured epithelial cells (Intestine 407) derived from fetal human small intestine exhibited spontaneous oscillations of membrane potential between the resting level of about –20 mV and the activated level of about –75mV. The cells were hyperpolarized to the latter level in response to mechanical or electrical stimuli. The hyperpolarizing responses were also elicited by the application of intestinal secretagogues: acetylcholine, histamine, serotonin and vasoactive intestinal polypeptide (VIP). The spontaneous oscillation of membrane potential became prominent and long-lasting in the presence of acetylcholine, histamine, serotonin or VIP. These secretagogue-induced responses were mediated by individual independent receptors on the cell membrane. Muscarinic receptors were responsible for the acetylcholine response, and H₁-receptors for the histamine response. The cells also responded with a slow hyperpolarization to calcium ionophore A23187, which is known to induce intestinal secretion. The spontaneously occurring hyperpolarizing responses and those induced by stimuli were both due to an increase in the K⁺ conductance of the cell membrane. Since acetylcholine, histamine, serotonin and A23187 are known to promote mobilization of cellular Ca²⁺ ions in intestinal secretory cells, it is hypothesized that these electrical activities of the cell are closely related to the receptor stimulation which leads to the Ca²⁺-mediated intestinal secretion.     

Effects of cholera toxin on the potential difference and motor responses induced by distension in the rat proximal small intestine in vivo

Cholera toxin (CT) may induce uncontrolled firing in recurrent networks of secretomotor neurons in the submucous plexus. This hypothesis was tested in chloralose-anesthetized rats in vivo. The secretory reflex response to graded intestinal distension was measured with or without prior exposure to luminal CT. The transmural potential difference (PD) was used as a marker for electrogenic chloride secretion. In controls, distension increased PD, and this response was reduced by the neural blocker tetrodotoxin given serosally and the vasoactive intestinal peptide (VIP) receptor antagonist [4Cl-d-Phe(6),Leu(17)]VIP (2 mug.min(-1).kg(-1) iv) but unaffected by the serotonin 5-HT(3) receptor antagonist granisetron, by the nicotinic receptor antagonist hexamethonium, by the muscarinic receptor antagonist atropine, or by the cyclooxygenase inhibitor indomethacin. Basal PD increased significantly with time in CT-exposed segments, an effect blocked by granisetron, by indomethacin, and by [4Cl-d-Phe(6),Leu(17)]VIP but not by hexamethonium or atropine. In contrast, once the increased basal PD produced by CT was established, [4Cl-d-Phe(6),Leu(17)]VIP and indomethacin had no significant effect, whereas granisetron and hexamethonium markedly depressed basal PD. CT significantly reduced the increase in PD produced by distension, an effect reversed by granisetron, indomethacin, and atropine. CT also activated a specific motility response to distension, repeated cluster contractions, but only in animals pretreated with granisetron, indomethacin, or atropine. These data are compatible with the hypothesis that CT induces uncontrolled activity in submucous secretory networks. Development of this state depends on 5-HT(3) receptors, VIP receptors, and prostaglandin synthesis, whereas its maintenance depends on 5-HT(3) and nicotinic receptors but not VIP receptors. The motility effects of CT (probably reflecting myenteric activity) are partially suppressed via a mechanism involving 5-HT(3) and muscarinic receptors and prostaglandin synthesis.     

Abnormalities of caerulein- and carbamylcholine-stimulated pancreatic enzyme secretion in the obese Zucker rat

The secretory function of the exocrine pancreas has been studied in dispersed pancreatic acini from obese and homozygous lean Zucker rats at 6 and 22 wk. No abnormality was found in acini from young rats. Acini from 22 wk obese and lean rats were equally responsive to secretagogues which stimulate cAMP, i.e. vasoactive intestinal peptide (VIP) and secretin. By contrast, there was a reduction in the maximum responsiveness to caerulein and carbamylcholine in acini from obese rats. These latter secretagogues act through mobilization of intracellular Ca2+. Since obese animals are insulin resistant and amylase release is modulated by insulin, the role of insulin resistance in the secretory defect was then investigated. A group of 22 wk obese rats received treatment with Ciglitazone (a drug which reduces insulin resistance in obese laboratory animals) for 4 wk before the secretion study. Despite the expected reduction in insulin resistance there was no improvement of the secretory defect seen with caerulein and carbamylcholine stimulation. Thus, the secretory abnormality in the exocrine pancreas of adult obese Zucker rats does not appear to be directly associated with insulin resistance. Furthermore, the secretory defect is linked to those secretagogues which induce Ca2+-independent phosphoinositide hydrolysis and Ca2+ mobilization in the target cell.     

Effect of dopamine on amylase secretion from guinea pig pancreatic acinar cells in vitro

Dopamine has been shown to effect pancreatic flow, protein output and amylase secretion in a variety of species. However, there is conflicting evidence regarding the role of dopamine on amylase release in vitro. Specific studies were conducted to evaluate the effect of dopamine and to compare its effects with other substances on basal- and secretagogue-stimulated amylase secretion in a guinea pig dispersed pancreatic acinar cells preparation. Dopamine (10(-6) M) induced a small, but significant (P less than 0.05) increase of amylase secretion. Established secretagogues (10(-6) M) including bombesin, cholecystokinin-octapeptide (CCK-8) and carbachol as anticipated induced significantly larger responses. Other substances tested (10(-6) M) including thyrotropin-releasing hormone (TRH) and muscimol were without effect. Complete dose-response studies (10(-11)-10(-3) M) in the presence of bombesin, CCK-8 and carbachol revealed that dopamine does not affect amylase release in response to these secretagogues. These findings suggest that dopamine is a weak stimulant of amylase secretion in vitro, and that it may therefore play a minor role in regulation of pancreatic enzyme secretion. Several factors including vascular, hormonal and neural have been implicated in regulation of pancreatic exocrine secretion. In particular, autonomic nervous system activity, notably cholinergic, has been shown to affect the secretory status of the pancreatic acinar cell. In addition, several biologically active peptides including bombesin, cholecystokinin (CCK), secretin, vasoactive intestinal peptide (VIP), substance P, gastrin and stimulation of cholinergic (muscarinic) receptors with carbachol have been shown to stimulate pancreatic enzyme secretion both in vivo and in vitro. Certain controversy regarding the role of the sympathetic nervous system in regulation of pancreatic exocrine secretion does exist. For example, several studies with agonists and antagonists of noradrenergic and dopaminergic receptor subtypes suggest a stimulatory effect on pancreatic fluid, electrolyte and enzyme secretion. However, these responses are species-specific and variations inherent to the model have been described. Dopamine administration has been shown to stimulate pancreatic bicarbonate and enzyme secretion in a variety of species including mice, dogs, and man. Radioligand binding studies with 3H-dopamine have revealed the presence of high- and low-affinity dopamine binding sites in dog pancreatic acinar cells. Stimulation of these receptors has been correlated with dose-dependent increases in intracellular cAMP levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of the amyloid precursor protein ectodomain shedding by the 5-HT4 receptor and Epac

The serotonin 5-hydroxytryptamine (5-HT4) receptor is of potential interest for the treatment of Alzheimer's disease because it increases memory and learning. In this study, we investigated the effect of zinc metalloprotease inhibitors on the amyloid precursor protein (APP) processing induced by the serotonin 5-HT4 receptor in vitro. We show that secretion of the non-amyloidogenic form of APP, sAPPalpha induced by the 5-HT4(e) receptor isoform was not due to a general boost of the constitutive secretory pathway but rather to its specific effect on alpha-secretase activity. Although the h5-HT4(e) receptor increased IP3 production, inhibition of PKC did not modify its effect on sAPPalpha secretion. In addition, we found that alpha secretase activity is regulated by the cAMP-regulated guanine nucleotide exchange factor, Epac and the small GTPase Rac.     

Regulation mechanisms of intestinal secretion: implications in nutrient absorption

Intestinal secretion is a normal phenomenon, indispensible to solubilize and dilute nutrients and to maintain fluidity in the intestinal lumen. Enterotoxins and certain drugs may disrupt the proabsorptive status maintained by the small intestine under physiologic conditions. Hormones found in nervous and specialized intestinal enterochromaffin cells are responsible, in part, for secretion of fluid into the lumen. Afferent vagal nerve impulses mediated by 5-hydroxytryptamine (serotonin; 5-HT), vasoactive intestinal peptide (VIP) and substance P are the major agents of secretory stimulation. Toxins from pathogenic bacteria, especially some strains of E. coli and V. cholerae, trigger a secretory response and a chain of events involving cGMP and cAMP which result in chloride secretion, coupled to sodium and fluid efflux into the lumen. If secretion is unchecked by natural mechanisms or medications, the consequences are diarrhea, with potential dehydration, hyponatremia and ultimately death. Introduction of absorbable nutrients in the intestinal lumen has a major antisecretory action, both by a nutrient-gene interaction and by proabsorptive hormone expression. In additon, during the absorptive process water is carried into the enterocyte together with solutes. Hydrolysis-resistant peptides of dietary origin and ingested soluble fiber may also have a proabsorptive effect. The gastrointestinal system has a variety of antisecretory or proabsorptive hormonal and protein agonists that balance the outflow of fluid and electrolytes. The more extensively studied are neuropeptide Y/peptide YY (NPY/PYY) and the antisecretory factor (AF). Nitric oxide (NO), a short-lived second messenger, has a major role in secretion by activating cGMP. The intracellular concentration of NO may regulate the absorptive/secretory status of the small intestine, either stimulating absorption or inducing secretion. Specifically targeted 5-HT receptor antagonist drugs and other pharmacologic agents have been clinically tried for the treatment of severe diarrhea, drug-induced malabsorption and reversal of cellular damage.     

Role of cyclic adenosine 3',5'-monophosphate-dependent protein kinase in hormone-stimulated beta-endorphin secretion in AtT20 cells.

Secretion of beta-endorphin from mouse pituitary AtT20 cells is stimulated by a variety of compounds that raise intracellular cAMP and Ca2+. To investigate the role of cAMP-dependent protein kinases in secretion, AtT20 cells were transfected with an expression vector coding for a regulatory (R) subunit of cAMP-dependent protein kinase containing mutations in both cAMP-binding sites. Expression of the mutant regulatory subunit in stable transformants (RAB cells) results in a dominant inhibition of cAMP-dependent protein kinase activity. Isoproterenol (1 microM) or analogs of cAMP stimulated beta-endorphin secretion from AtT20 cells, but failed to stimulate secretion in RAB cells expressing the mutant R subunit. Secretion in response to CRF (100 nM) was inhibited by 80% in these mutant clones, whereas the secretory response to vasoactive intestinal peptide (VIP; 100 nM) or phorbol ester (100 nM phorbol myristate acetate) was not inhibited by the R subunit mutation. Intracellular cAMP was elevated in response to CRF (11- to 15-fold), isoproterenol (5- to 10-fold), and VIP (4- to 8-fold) in RAB cells. Similar concentrations of VIP were required to evoke beta-endorphin secretion in either RAB cells or AtT20 cells. As with most secretagogues, VIP-induced secretion was inhibited in the presence of either EGTA or a voltage-sensitive Ca2+ channel antagonist, PN200-110. The secretory response to VIP was unaffected by down-regulation of protein kinase-C. These results suggest that CRF and isoproterenol work via cAMP-dependent protein kinase to activate beta-endorphin secretion, whereas VIP can act by a different mechanism that does not involve cAMP-dependent protein kinase or protein kinase-C.     

Effects of growth hormone releasing factor on pancreatic secretion in vivo and in vitro

Growth hormone releasing factor (GRF), a 44-residue peptide originally isolated from human pancreatic tumors, shows structural similarities to the members of the secretin-vasoactive intestinal peptide (VIP) peptides. This study was designed to determine the effects of human GRF (hGRF-(1-44] on pancreatic secretion in vivo in conscious dogs and in vitro in dispersed rat pancreatic acini. GRF given i.v. in graded doses in dogs caused a small but significant stimulation of pancreatic HCO3- and protein outputs and potentiated secretin- and cholecystokinin (CCK)-induced pancreatic HCO3- but not protein secretion. When given together with somatostatin, GRF failed to reverse the inhibitory action of this peptide on HCO3- and protein responses to secretin plus CCK in dogs. Studies in vitro dispersed rat pancreatic acini showed that GRF added to the incubation medium of these acini caused an increase in basal amylase release and shifted to the left the amylase dose-response curve to caerulein and urecholine but failed to affect the amylase response to VIP. This study indicates that GRF in vivo stimulates basal and augments secretin- or CCK-induced pancreatic HCO3- secretion and that this is probably due to direct stimulatory action of the peptide on pancreatic secretory cells.