Mucosal Cyclic AMP Research Articles

Protein kinase C and intestinal fluid secretion: involvement of prostaglandin E 2 but not of 5-hydroxytryptamine

To determine the role of protein kinase C in the regulation of intestinal fluid transport, experiments were performed with the rat jejunum in vivo, using the active phorbol ester, 4-β-phorbol 12-myristate 13-acetate (PMA), as stimulator of protein kinase C. Intraluminally administered PMA dose dependently reversed the net fluid absorption to net fluid secretion and significantly increased prostaglandin E 2 (PGE 2) but not 5-hydroxytryptamine (5-HT) output into the lumen. Mucosal cyclic AMP levels remained unchanged by PMA. Indomethacin inhibited the increase in PGE 2 output and partially reduced the secretory response to PMA. Ketanserin was without effect whereas verapamil totally blocked the secretory response to PMA. It is concluded that intestinal fluid secretion, stimulated by activation of protein kinase C is partly mediated by PGE 2 release. PGE 2 may facilitate calcium entry rather than increase intracellular calcium through activation of cyclic AMP. Protein kinase C appears to play an important role as an intermediate in phosphoinositol hydrolysis, which is initiated by 5-HT, and finally induces fluid secretion via PGE 2.

Stimulation of bile acid active transport related to increased mucosal cyclic AMP content in rat ileum in vitro

The regulation of bile acid transport in rat ileum was studied in vitro using the adenylate cyclase stimulator forskolin, or 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Forskolin 20 μM as well as 100 μM IBMX enhanced mucosal cyclic AMP to 3-fold the control levels. As a physiological response, net fluid absorption in everted ileal sacs was reduced. Taurocholate (10–500 μM) transfer in everted perfused segments of rat ileum was measured using a three compartment dual label method suitable for measuring active transport. Transport asymmetry with absorption exceeding its counterflux by 26-fold, was measured at 500 μM taurocholate. Forskolin increased absorption of taurocholate still further, by 68%, and reduced the serosal to mucosal flux. Enhanced intracellular accumulation of taurocholate indicated a stimulatory action of forskolin on active transport at the mucosal brush-border membrane. In uptake studies, accumulation of taurocholate was enhanced by 100 μM IBMX also. Forskolin-induced uptake stimulation could also be shown for chenodeoxycholate and cholate. In the presence of the neuronal blocker tetrodotoxin, uptake stimulation was still effective. Results indicate that the ileal bile acid transporter is included within the group of sodium-dependent cotransporters of the rat small intestine which are subject to a cyclic AMP-related stimulation at the mucosal cellular level.

Inhibition of Cholera-Toxin-Stimulated Intestinal Secretion by CGS 9343B in Rats

In this study, the effect of CGS 9343B on cholera-toxin-stimulated intestinal secretion in rats was determined using in vivo isolated loops. This recently developed compound is a potent and specific inhibitor of calmodulin, but not of protein kinase C. At a luminal dose of 15 mg/kg, CGS 9343B has little effect on basal intestinal absorption but completely inhibited the secretory effects of cholera toxin. The less specific calmodulin inhibitor, trifluoperazine, has a similar antisecretory effect, but unlike with CGS 9343B, severe toxicity was noted at luminal doses of 7.5 mg/kg. In animals where two intestinal loops were created, one with cholera toxin alone and the other with CGS 9343B and cholera toxin, significant inhibition of secretion was observed in both loops, consistent with a systemic effect of this compound. Finally, both CGS 9343B and trifluoperazine inhibited choleratoxin stimulated increases in mucosal cyclic AMP content, whereas basal levels were unaffected. We conclude that CGS 9343B significantly inhibits choleratoxin-stimulated intestinal secretions, possibly by inhibition of calmodulin-dependent adenylate cyclase activity. Its lack of major toxicity at therapeutic doses makes this compound potentially useful for the treatment of enterotoxigenic diarrheal diseases.

Inhibition of Cholera‐Toxin‐Stimulated Intestinal Secretion by CGS 9343B in Rats

In this study, the effect of CGS 9343B on cholera‐toxin‐stimulated intestinal secretion in rats was determined using in vivo isolated loops. This recently developed compound is a potent and specific inhibitor of calmodulin, but not of protein kinase C. At a luminal dose of 15 mg/kg, CGS 9343B has little effect on basal intestinal absorption but completely inhibited the secretory effects of cholera toxin. The less specific calmodulin inhibitor, trifluoperazine, has a similar antisecretory effect, but unlike with CGS 9343B, severe toxicity was noted at luminal doses of 7.5 mg/kg. In animals where two intestinal loops were created, one with cholera toxin alone and the other with CGS 9343B and cholera toxin, significant inhibition of secretion was observed in both loops, consistent with a systemic effect of this compound. Finally, both CGS 9343B and trifluoperazine inhibited choleratoxin stimulated increases in mucosal cyclic AMP content, whereas basal levels were unaffected. We conclude that CGS 9343B significantly inhibits choleratoxin‐stimulated intestinal secretion, possibly by inhibition of calmodulin‐dependent adenylate cyclase activity. Its lack of major toxicity at therapeutic doses makes this compound potentially useful for the treatment of enterotoxigenic diarrheal diseases.

Regional difference in intestinal adaptation after total colectomy as judged by the changes of mucosal Na-K ATPase, cyclic AMP, and transmural potential difference.

Intestinal adaptation and its regional difference after total colectomy were investigated in dogs by measuring mucosal Na-K ATPase, cyclic AMP, and transmural electric potential difference (PD). Twenty-four weeks after the total proctocolectomy, Na-K ATPase activity and PD increased significantly in all intestinal sites, whereas cyclic AMP showed no significant changes. The regional difference in the remaining intestine was examined in the jejunum, ileum, and interposed jejunum (neorectum). Na-K ATPase activity showed no significant regional difference, but the largest increase was found to occur in the ileum. PD also increased markedly in the ileum and there was significant difference between the ileum and other intestinal sites. These facts suggest that the increased active ion transport mediated by mucosal Na-K ATPase and transmural PD in the ileum is closely related to the intestinal adaptation occurring after total colectomy and indicates a greater potential of the ileum for adaptive compensation than either jejunum or neorectum.

Evidence for adenosine A 1 receptor action in rat jejunal mucosa

Cyclic AMP was elevated in rat jejunal mucosa in the presence of adenosine deaminase (2 mg/1) both alone or under conditions of stimulated mucosal cyclic AMP accumulation (forskolin 20 μM/papaverine 100 μM). The effects of adenosine deaminase were reversed by (−)-N 6-phenylisopropyl-adenosine (PIA, 10 μM). This action of PIA on mucosal cyclic AMP was antagonized by theophylline (1 mM) and was insensitive to tetrodotoxin (100 nM). The potency of PIA was 100-fold higher than that of 5′-N-ethylcarboxamido-adenosine (NECA). It is suggested that adenosine A 1 receptors are presemt in rat jejunal mucosal cells.

Inhibitory effect of somatostatin on cholera toxin-induced diarrhea and glycoenzyme secretion in rat intestine.

The effects of somatostatin on cholera toxin-induced secretory diarrhea and the appearance of glycoenzymes in the intestinal lumen and intestinal lymph were investigated in rat small intestine. After exposure to cholera toxin, marked fluid accumulation in the small intestinal tract and elevation of the jejunal mucosal cyclic AMP (cAMP) concentration were observed. The activity of alkaline phosphatase, aminopeptidase and sucrase increased in the intestinal lumen after toxin exposure. In intestinal lymph, alkaline phosphatase activity was increased after cholera toxin administration, while aminopeptidase activity remained unchanged. Somatostatin suppressed cholera toxin-induced secretory diarrhea, but it did not affect the elevated mucosal cAMP concentration. This peptide also inhibited the appearance of glycoenzymes in the intestinal lumen and lymph induced by cholera toxin administration. These results suggest that somatostatin exerts its inhibitory effects on cholera toxin-induced secretory diarrhea and on the appearance of glycoenzymes in the intestinal lumen and lymph by affecting processes beyond cAMP formation.

Proabsorptive properties of forskolin: Disposition of glycine, leucine and lysine in rat jejunum

The effects of forskolin on mucosal cyclic AMP levels and active transport of glycine, L-lysine and L-leucine were studied in rat jejunum in vitro. Furthermore, the effects on lysine and glycine incorporation into mucosal protein and on mucosal cell volume were investigated. Elevation of intestinal mucosal cyclic AMP to threefold control levels by 10 mumol 1(-1) forskolin was accompanied by increased absorption of glycine (+33%), L-leucine (+72%) and L-lysine (+188%), as determined in a three compartment model suitable to measure active transport. Increased intracellular accumulation could be demonstrated for lysine as a transport substrate. Accordingly, using a dual label method, calculated values for uphill transport of lysine at the site of the brush border membrane were markedly enhanced. Forskolin up to 10 mumol 1(-1) had no effects on the fraction of lysine or glycine incorporated into TCA-precipitable proteins of jejunal absorptive cells. Serosal to mucosal transfer, as well as basolateral entry into mucosal cells remained unchanged for all three amino acids. Likewise, intracellular fluid space, calculated from distribution spaces for 14C-inulin and 3H2O as well as the response of cellular volume to an osmotic gradient were not affected by forskolin. As comparable stimulatory effects of forskolin on active hexose transport were reported earlier, it is suggested that forskolin - known to inhibit sodium-coupled fluid absorption - may stimulate active transport by enhancing sodium availability for sodium dependent intestinal cotransporters in general.

Forskolin-induced elevation of rat jejunal cyclic AMP levels and stimulation of active glucose transport in vitro.

The adenylate cyclase stimulator forskolin enhanced both mucosal cyclic AMP levels and D-aldohexose transport in rat jejunum in vitro in a concentration-dependent manner. With mucosal cyclic AMP elevated to 400% of basal values, active D-glucose transport was at 200% of control values. Transport parameters, as determined in a three compartment model using a dual label method, indicate a) increased "uphill" glucose transport at the brush border membrane with higher intracellular accumulation, b) unchanged serosal to mucosal glucose fluxes and passive permeabilities. Transport stimulating effects of forskolin were also present with D-galactose in the rat. In mouse jejunum active transport of D-glucose and 3-O-methylglucose was increased by forskolin 10 mumol/l. Phlorizin inhibited D-glucose transport as well as L-glucose transport in the rat were not changed by the persisting cyclic AMP elevation induced by forskolin. The results show a positive correlation of active intestinal hexose transport with a modest elevation of mucosal cyclic AMP. In accordance to current models of cyclic AMP-induced changes in intestinal fluid absorption, rat jejunal net fluid transport was reduced by 40% with 20 mumol/l forskolin.

Response of rat small intestinal active aldohexose transport to elevation of mucosal cyclic AMP by forskolin and 3-isobutyl-1-methylxanthine in vitro

The phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX) was able to elevate rat small intestinal cyclic AMP levels to 300% of basal values. Active jejunal D-glucose transport was enhanced parallel to the rise of intracellular cyclic AMP levels to 140% of control values at 100 mumol/l IBMX. Transport parameters, as determined in a three compartment model in vitro using a dual label method, indicate increased 'uphill' glucose transport at the site of the brush border membrane, higher intracellular accumulation of the sugar, with unchanged passive permeabilities. Phlorizin-inhibited D-glucose transport and L-glucose transfer in the rat were not affected by the persisting cyclic AMP elevation produced by IBMX. Stimulating effects could also be demonstrated with D-galactose as a substrate. IBMX 100 mumol/l also increased active D-glucose as well as 3-O-methylglucose transport in mouse jejunum. Stimulatory effects on intestinal hexose transport and mucosal cyclic AMP levels were also found with the adenylate-cyclase activator forskolin. In the present study, forskolin effects on jejunal mucosal cyclic AMP levels were enhanced in the presence of 100 mumol/l IBMX, resulting in a 20-fold increase compared to controls at 20 mumol/l forskolin. The concentration response for the effect of forskolin in the presence of 100 mumol/l IBMX on D-glucose transport did not produce a significant increase compared to transport stimulation with IBMX alone. At higher concentrations of forskolin however, glucose transport decreased to levels well below the IBMX controls. The elevation of cellular cyclic AMP levels had no effects on passive permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of cimetidine on cyclic nucleotides in human gastric mucosa.

1 Pre-drug concentrations of both nucleotides were similar in patients with or without peptic ulceration. 2 The effect of a singly infusion or repeated oral administration of cimetidine on human gastric mucosal content of cyclic AMP and cyclic GMP was studied. 3 Biopsies from the body of the stomach were taken at endoscopy from patients who were participating in a clinical trial of the value of cimetidine in the treatment of duodenal ulcer. Cyclic nucleotide determinations and histological examinations were performed on biopsies taken before and after cimetidine treatment. 4 Gastric mucosal content of cyclic AMP was significantly increased (P less than 0.01) 20 min after intravenous infusion of 200 mg cimetidine. There was also a significant increase (P less than 0.01) in gastric mucosal cyclic AMP following administration of the drug orally for 28 days. No alterations in gastric mucosal histology were observed following cimetidine treatment. 5 Gastric mucosal content of cyclic GMP was not altered by intravenous drug infusion, or by chronic treatment.

Methylprednisolone stimulation of guanylate cyclase activity in rat small intestinal mucosa: Possible role in electrolyte transport

A single, intramuscular dose of the glucocorticoid methylprednisolone (3 mg/100 g) significantly increased rat jejunal and ileal mucosal guanylate cyclase (GTP pyrophosphatelyase [cyclizing]; EC 4.6.1.2) activity and cyclic GMP concentration 6 h after treatment. This increase in guanylate cyclase activity was maximal 6 h after injection, was not associated with changes in mucosal cyclic AMP concentration, and occurred before any increase in NaK-activated adenosine triphosphatase activity could be detected. In contrast to the findings in the small intestine, no alterations in guanylate cyclase activity were present in the rat colon 6 or 72 h after methylprednisolone treatment. The increase in ileal guanylate cyclase activity and cGMP concentration coincided with increases in ileal short-circuit current, potential difference, net Cl secretion, and serosal-to-mucosal unidirectional Cl flux. In contrast, in the colon, where no changes in guanylate cyclase activity were observed, methylprednisolone did not alter electrolyte transport in vitro. A similar correlation was observed between various methylprednisolone doses, the increase in guanylate cyclase activity and the alteration in ileal short-circuit current and net Cl transport. The methylprednisoloneinduced increase in ileal guanylate cyclase activity was not prevented by pretreatment with the mineralocorticoid competitive inhibitor spironolactone, nor did the mineralocorticoid deoxycorticosterone acetate (0.5 mg/100 g) affect ileal or colonic mucosal guanylate cyclase activity. In addition, isolated enterocytes from methylprednisolone-treated animals exhibited significant increases in guanylate cyclase activity with the greatest percentage increase occurring in the crypt cell population. These results suggest that the changes in chloride transport which occurred in the small intestine 6 h after methylprednisolone administration may be due to alterations in guanylate cyclase activity, and further support the role of cGMP as an intracellular mediator of ileal chloride secretion.

Effect of prostaglandin on ion transport across isolated colonic mucosa

The effect of prostaglandins (PG) on colonic ion transport was investigated in in vitro experiments in the rat. Both PGE1 and PGA1 increased short-circuit current (Isc), potential difference, and mucosal cyclic AMP levels, but PGE1 was more potent than PGA1. 10(-4) M PGE1 inhibited active sodium transport across short-circuited colonic mucosa (6.1-1.3 microEq/hr/cm2). This effect, coupled with an increase in Isc, is consistent with prostaglandin stimulation of active anion secretion and with the recent suggestions that prostaglandins may be important intermediaries in the process by which several laxatives alter large-intestinal fluid and electrolyte movement.

Effect of somatostatin on ion transport in the rat colon.

The effect of somatostatin (SRIF) on ion transport was determined in the rat colon in vitro. SRIF produced a sustained decrease in the short circuit current (Isc) (-0.8 +/- 0.1 mueq/h x cm2) and increased net Cl absorption (0.9 +/- 0.3 mueq/h x cm2). The threshold effect of SRIF on Isc was observed at 6 nM. 10 microM serotonin decreased net Na absorption (-2.6 +/- 0.4 mueq/h x cm2), net Cl absorption (-3.6 +/- 0.5 mueq/h x cm2) and increased Isc (0.7 +/- 0.1 mueq/h x cm2); these changes were totally blocked by 0.1 microM SRIF. SRIF completely blocked net Cl secretion induced by 10 mM theophylline (-2.5 +/- 0.7 to +4.1 +/- 2.0 muq/h x cm2) and partially blocked theophylline-induced inhibition of net Na absorption (0.7 +/- 0.5 to 2.1 +/- 0.4 mueq/h x cm2). SRIF also blocked prostaglandin E1 (PGE1) induced increase in potential difference and Isc (P < 0.001). Mucosal cyclic AMP levels were increased by theophylline and PGE1 but not by serotonin. SRIF had no effect on basal or theophylline- and PGE1-stimulated cyclic AMP levels. These results indicate that SRIF blocks both cyclic AMP and noncyclic AMP mediated changes in ion secretion and suggest that SRIF is acting at a step in the secretory process beyond the formation of cyclic AMP.

Influence of vasoactive intestinal polypeptide on net water flux and cyclic adenosine 3',5'-monophosphate formation in the rat jejunum.

1. The effects were studied of vasoactive intestinal polypeptide (VIP), theophylline, and morphine on net water flux and cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels in the jejunum of anaesthetized rats in vivo and of VIP and morphine on adenylate cyclase activity in rat epithelial cell membranes in vitro. 2. Infusion of VIP (0.1-2 x 10(-9) mol/min/kg) dose dependently caused a reversal from net water absorption to net secretion; 2 x 10(-9) mol/min/kg enhanced the mucosal cyclic AMP content by 67%. 3. Theophylline (5 mg/ml, intraluminally) enhanced the effect of intra-arterial infusion of VIP (2 x 10(-9) mol/min/kg) as to net water secretion and increase in mucosal cyclic AMP content. 4. Pretreatment with morphine (5 mg/kg, s.c.) did not influence the effects of VIP on net water flux and on mucosal cyclic AMP content. 5. Atropine (2 mg/kg, s.c.) also failed to reduced the effect of VIP (0.4 x 10(-9) mol/min/kg) on net water flux. 6. Stimulation of adenylate cyclase activity was a function of VIP concentration over a range of 1 x 10(-10)-1 x 10(-7) M. Morphine (up to 1 x 10(-3) M) failed to influence stimulation of adenylate cyclase by VIP. 7. The finding that low doses of VIP, which already have an effect on net water flux, fail to increase cyclic AMP levels makes it likely that other mediators besides cyclic AMP are involved in the effect of VIP on net water flux. Some of the present results, however, support the assumption that VIP stimulates intestinal fluid secretion by increasing mucosal cyclic AMP levels.

Interaction of laxatives with enzymes of cyclic AMP metabolism from human colonic mucosa.

The mechanism by which laxatives such as dioctyl sodium sulfosuccinate and ricinoleic acid evoke colonic fluid secretion has been suggested to involve mucosal cyclic AMP. Ricinoleic acid and dioctyl sodium sulfosuccinate were tested for their capacity to modulate the key enzymes of cAMP-metabolism--adenylate cyclase and cAMP-phosphodiesterase--in human colonic mucosa. Both laxatives were ineffective stimuli of human colonic adenylate cyclase. In contrast to ricinoleic acid, dioctyl sodium sulfosuccinate was a competitive inhibitor of soluble cAMP-phosphodiesterase activity. These experiments suggest that the cathartic properties of dioctyl sodium sulfosuccinate in human colonic mucosa might be mediated by cyclic AMP via inhibition of soluble phosphodiesterase activity.

Inhibition by morphine of prostaglandin E1-stimulated secretion and cyclic adenosine 3',5'-monophosphate formation in the rat jejunum in vivo.

1 The effects were studied of prostaglandin E1 (PGE1), theophylline and morphine on net water flux and mucosal cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels in the jejunum of anaesthetized rats in vivo. 2 Infusion of PGE1 (3.2 micrograms/min, i.a.) caused a reversal from net water absorption to net secretion and enhanced the mucosal cyclic AMP content by 54%. 3 Theophylline (5 mg/ml, intraluminal) similarly produced a reversal from net water absorption to net secretion and increased mucosal cyclic AMP content by 54%. Additional intra-arterial infusion of PGE1 resulted in a massive increase in net water secretion and an increase in mucosal cyclic AMP content by about 200%. 4 Pretreatment with morphine (10 mg/kg, s.c.) reduced the effect of PGE1 on net water flux and completely inhibited its effect on the mucosal cyclic AMP content. Naloxone (10 mg/kg, s.c.) abolished both effects of morphine. 5 A good correlation (r = 0.99) was demonstrated between mucosal cyclic AMP levels and net water flux. 6 The present results demonstrate that PGE1 stimulates intestinal fluid secretion by increasing mucosal cyclic AMP levels. The antidiarrhoeal effect of morphine can be explained by its inhibition of the PGE-mediated increase in cyclic AMP levels, which, in turn, leads to a reduction in intestinal secretion.

Laxatives and human colonic mucosal cyclic AMP

Laxatives and human colonic mucosal cyclic AMP

The effect of prostacyclin on intestinal ion transport in the rat

The actions of PGI 2 and PGE 2 on electrically monitored ion transport in rat jejunum and colon have been determined both in vivo and in vitro . Whilst PGE 2 was shown to induce a marked change in ion transport PGI 2 was relatively ineffective. The ability of the prostanoids to influence ion transport is related to their capacity to change mucosal cyclic AMP levels since in isolated small intestinal enterocytes PGE 2 caused a marked stimulation in cyclic AMP levels whilst PGI 2 had little effect. In colonic mucosal scrapes PGE 2 was more effective than PGI 2 in stimulating changes in cyclic AMP levels. It appears doubtful that PGI 2 plays a direct role in the regulation of intestinal ion transport.

Prostaglandins and the human mucosal alimentary cyclic AMP system

Prostaglandins and the human mucosal alimentary cyclic AMP system