Calcium-dependence of sugar transport in rat small intestine

The involvement of Ca2+ in the theophylline action on sugar transport was investigated in isolated rat small intestinal mucosa. Theophylline significantly increased cell water free sugar accumulation and reduced mucosal to serosal sugar fluxes both in the presence and absence of calcium, but the effects of theophylline were significantly less in calcium free media. In theophylline untreated tissues, calcium-deprived bathing solutions decreased tissue galactose accumulation and increased mucosal to serosal sugar flux. The calcium-channel blocker verapamil produced similar effects on intestinal galactose transport to those induced by low extracellular calcium activity. RMI 12330A and the calmodulin antagonist trifluoperazine abolished the theophylline-effects on intestinal galactose transport. Both drugs also affected sugar transport in basal conditions. These studies suggest that calcium might modulate sugar permeability across the basolateral boundary of rat enterocytes, and that its effect may be mediated by calmodulin.     

Fluid accumulation in mouse ligated intestine inoculated with Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin, when inoculated into the ligated intestinal loop of mice, caused marked distension due to fluid accumulation. The increase in weight of the intestinal loop was proportional to the log dose of enterotoxin within a range from 1 to 16 micrograms. The fluid accumulation was arrested by washing the loop with saline or by injection of the specific anti-enterotoxin serum into the loop 5 or even 30 min after inoculation of the enterotoxin. A significant increase in weight of the loop was found as early as 10 min after inoculation of the toxin. These results may suggest that entergotoxin is neither bound firmly to the mucosal membrane nor permeates into the cells of the intestinal wall. The mouse intestinal loop test is economical, simple to perform, and applicable for quantitative determination of the enteropathogenic activity of C. perfringens enterotoxin.     

Calcitonin stimulates adenylate cyclase activity, the accumulation of cyclic adenosine 3',5' monophosphate and the release of prostaglandin E2 in rat intestinal mucosa

Calcitonin stimulates intestinal fluid and electrolyte secretion by an unclear mechanism. The present results show that synthetic salmon calcitonin significantly stimulates adenylate cyclase activity in the membranes of rat intestinal mucosal cells. The effect of the hormone was in a dose-dependent manner for a dose range of 10(-9)-10(-7) M. The stimulated enzyme activity was followed by a progressive accumulation of cyclic adenosine 3',5' monophosphate and release of prostaglandin E2 in these cells during a 20 min experimental period of time. These results are discussed, and suggest that the formation of cyclic adenosine 3',5' monophosphate possibly mediates the action of calcitonin on intestinal cell functions.     

Fluid accumulation in mouse ligated intestine inoculated with Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin, when inoculated into the ligated intestinal loop of mice, caused marked distension due to fluid accumulation. The increase in weight of the intestinal loop was proportional to the log dose of enterotoxin within a range from 1 to 16 micrograms. The fluid accumulation was arrested by washing the loop with saline or by injection of the specific anti-enterotoxin serum into the loop 5 or even 30 min after inoculation of the enterotoxin. A significant increase in weight of the loop was found as early as 10 min after inoculation of the toxin. These results may suggest that entergotoxin is neither bound firmly to the mucosal membrane nor permeates into the cells of the intestinal wall. The mouse intestinal loop test is economical, simple to perform, and applicable for quantitative determination of the enteropathogenic activity of C. perfringens enterotoxin.     

Shedding of intestinal epithelium induced by chlorpromazine as mechanism inhibiting enterotoxin diarrhea

Chlorpromazine, in parallel with reduced fluid accumulation in ligated loops, interfered with the basement membrane and caused shedding of intestinal mucosal epithelium. This was examined by electron microscopy of rat ileum maintained both in vitro and in situ.     

Characterization of prostaglandin E receptors in canine small intestine using [3H] prostaglandin E1 binding.

Prostaglandin E (PGE) receptors in canine small intestinal mucosal and muscle membrane preparations were labeled with [3H] PGE1. Saturable, high affinity binding of [3H] PGE1 was observed in both preparations. The density of binding sites (fmol/mg protein) was 39 for mucosal membranes and 60 for muscle membranes, with corresponding dissociation constants of 10.6 nM and 5.8 nM, respectively. [3H] PGE1 binding sites in both preparations showed stereospecificity and high affinity for natural PGE1 and PGE2, but not for I or F-type PGs. Synthetic PGEs such as misoprostol and enisoprost had lower affinity than PGE1 or PGE2. Several analogs of enisoprost bound weakly to the binding sites. A highly significant correlation (C.C. = 0.9) was demonstrated between mucosal and muscle binding potency for a series of enisoprost analogs. There was also a significant positive correlation between the receptor binding potency and rat diarrheagenic activity for these analogs. These results indicate that PGE receptors in canine intestinal mucosa and muscle can be directly studied with [3H] PGE1 binding. The mucosal and muscle PGE receptors may have similar ligand binding specificity. We speculate that these receptors are likely to be associated with the diarrheagenic activity of PGEs.     

Effects of indomethacin on intestinal secretion, prostaglandin E and cyclic AMP: evidence against a role for prostaglandins in cholera toxin-induced secretion.

The effects of indomethacin on intestine mucosal cAMP, intestinal fluid secretion, and mucosal and fluid PGE were studied in rabbits in vivo following challenge with cholera toxin. Indomethacin had no effect on cholera toxin-induced fluid secretion or cAMP accumulation. Inhibition of PGE synthesis was achieved by the administration of two but not one injection of indomethacin. These studies provide evidence against a role for PGE in mediating cholera toxin-induced secretion and point out the need to measure prostaglandin levels when using prostaglandin synthetase inhibitors in vivo.     

Pressure regulation in the microcirculation.

The results of direct pressure measurements are described which demonstrate that pressures in a certain fraction of mesenteric capillaries remain remarkably constant during large changes in systemic pressure. The results of isogravimetric studies, reported in the literature, are also described which indicate that this phenomenon may also occur in the intestine. The question is raised whether capillary pressures may therefore be regulated. Pressures recorded from mesenteric arterioles and capillaries are shown which indicate that maintenance of a constant capillary pressure is primarily the consequence of the vascular architecture peculiar to this tissue, and is merely a secondary reflection of mechanisms associated with flow regulation. The results of direct pressure measurements recorded in the microcirculation of intestinal muscle are also shown. These data indicate that capillary pressures in innervated, denervated, and xylocaine-treated intestinal muscle change in direct proportion to variations in arterial pressure. It is concluded that capillary pressures in the intestinal muscle layers are therefore not regulated, so that the observation that capillary pressures may be maintained is probably a phenomenon unique to the mesentery. Pressures recorded from capillaries in the mucosal villi are also shown and compared to capillary pressures measured in the microvasculature of mesentery and intestinal muscle. When systemic pressure was normal (107 +/- 10 mm Hg), capillary pressure in the mesentery averaged 30 to 33 mm Hg; capillary pressures in the intestinal muscle averaged 22 to 24 mm Hg; and capillary pressures in the mucosal villi averaged 13 to 15 mm Hg. These data suggest that mesenteric capillaries are primarily a filtering network; intestinal muscle capillaries are normally in fluid balance; whereas at rest mucosal capillaries are primarily absorptive. These pressures, recorded from the three major regions of the rat intestine, were used to calculate a weighted average for the whole organ. The calculated value, based on assumed values for relative capillary densities, was 17 mm Hg. This result compares favorably with data from whole organ, isogravimetric studies, and may clarify some of the apparent discrepancies between previous isogravimetric and servopressure studies.     

Effects of indomethacin on intestinal secretion, prostaglandin E and cyclic AMP: Evidence against a role for prostaglandins in cholera toxin-induced secretion

The effects of indomethacin on intestine mucosal cAMP, intestinal fluid secretion, and mucosal and fluid PGE were studied in rabbits in vivo following challenge with cholera toxin. Indomethacin had no effect on cholera toxin-induced fluid secretion or cAMP accumulation. Inhibition of PGE synthesis was achieved by the administration of two but not one injection of indomethacin. These studies provide evidence against a role for PGE in mediating cholera toxin-induced secretion and point out the need to measure prostaglandin levels when using prostaglandin synthetase inhibitors in vivo.     

Dynamics of fluid accumulation in intestinal segments

Intestinal obstruction occurring in human diseases or produced surgically in animal studies can produce fluid accumulation and intestinal distention. It was found that a quantitative theory for acute intestinal fluid accumulation could be derived and verified for a variety of experimental model systems. The contribution of intestinal secretagogues and distention-induced secretion may augment fluid accumulation in closed loop fluid accumulation experiments in animals. Criteria for stability and decompression of lumen volume were derived.     

Failure to reverse cholera toxin induced intestinal secretion by agents which decrease mucosal cAMP.

The feasibility of reducing intestinal secretion by the use of agents which decrease intestinal mucosal cAMP concentration has been investigated in the weanling pig and the rabbit. Three different agents for decreasing mucosal cAMP concentration were studied. The cyclic nucleotide phosphodiesterase activator, imidazole, significantly reduced mucosal cAMP concentrations only in the weanling pig. Intraluminal 2'-deoxyadenosine-3'AMP inhibited adenylate cyclase and caused a decrease in mucosal cAMP concentration in both the pig and the rabbit. The introduction of the heat-stable enterotoxin of Escherichia coli into pig jejunal segments also gave lowered mucosal cAMP concentrations. While these three agents effectively reduced cAMP concentrations in intestinal mucosa, they were ineffective in reducing the net fluid secretory effects of cholera toxin. Secretion caused by cholera toxin apparently persists independent of the temporary changes in cAMP concentration which can be induced by pharmacological agents.     

Activation of AMPK Inhibits Cholera Toxin Stimulated Chloride Secretion in Human and Murine Intestine

Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR), is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK), can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX) mediated diarrhea.Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK). In order to substantiate our findings on the whole tissue level, short-circuit current (SCC) was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops.CTX and forskolin (FSK) significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments.The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness.     

Actions of a leukocytic inflammatory substance on isolated tissue responses.

LIS had no action on the mechanical activity of isolated cat vascular, intestinal smooth, or cardiac muscle. No effect on platelet aggregation was observed, and PGF2alpha activity was absent in LIS preparations. Isolated lysosomal enzyme release was increased significantly when LIS was added to the incubation medium. This action may help to explain the inflammatory action of this naturally occurring material found in the inflamed synovial fluid of the canine knee joint.     

Smooth muscle overexpression of IGF-I induces a novel adaptive response to small bowel resection

Prior studies of intestinal adaptation after massive small bowel resection (SBR) have focused on growth factors and their effects on amplification of the gut mucosa. Because adaptive changes have also been described in intestinal smooth muscle, we sought to determine the effect of targeted smooth muscle growth factor overexpression on resection-induced intestinal adaptation. Male transgenic mice with smooth muscle cell overexpression of insulin-like growth factor I (IGF-I) by virtue of an alpha-smooth muscle actin promoter were obtained. SMP8 IGF-I transgenic (IGF-I TG) and nontransgenic (NT) littermates underwent 50% proximal SBR or sham operation and were then killed after 3 or 28 days. NT mice showed the expected alterations in mucosal adaptive parameters after SBR, such as increased wet weight and villus height. The IGF-I TG mice had inherently taller villi, which did not increase significantly after SBR. In addition, IGF-I TG mice had a 50% postresection persistent increase in remnant intestinal length, which was associated with an early decline and later increase in relative mucosal surface area. These results indicate that growth factor overexpression within the muscularis layer of the bowel wall induces significant postresection adaptive intestinal lengthening and a unique mucosal response. IGF-I signaling within the muscle wall may play an important role in the pathogenesis of resection-induced adaptation.     

Heat shock response reduces intestinal permeability in septic mice: potential role of interleukin-10

Sepsis and other critical illnesses are associated with increased permeability of the intestinal mucosa. Loss of mucosal integrity may lead to multiple organ failure in these conditions. We tested the hypothesis that induction of the heat shock response reduces sepsis-induced increase in intestinal permeability. The heat shock response was induced in mice by intraperitoneal injection of 10 mg/kg sodium arsenite. Two hours later, at which time mucosal heat shock protein 72 levels were increased, sepsis was induced by cecal ligation and puncture (CLP) or sham operation was performed. Sixteen hours after sham operation or CLP, intestinal permeability was determined by measuring the appearance in blood of 4.4-kDa fluorescein isothiocyanate-conjugated dextran and 40-kDa horseradish peroxidase administered by gavage. Sepsis resulted in increased mucosal permeability for both markers, and this effect of sepsis was substantially reduced in mice treated with sodium arsenite. Plasma levels of the anti-inflammatory cytokine interleukin (IL)-10 were increased in septic mice pretreated with sodium arsenite, and the protective effect of sodium arsenite on intestinal permeability in septic mice was reversed by treatment with anti-IL-10 antibody. The present results suggest that sepsis-induced increase in mucosal permeability can be reduced by the heat shock response and that increased IL-10 levels may be involved in the protective effects of the heat shock response.     

Comparison of gastric and intestinal antisecretory and protective effects of prostacyclin and its stable thia-imino-analogue (Hoe 892) in conscious rats

The effects of prostacyclin (PGI2) and its stable thia-thimo-analogue (Hoe 892) on gastric and intestinal secretions and gastric mucosal lesions have been determined in conscious rats. Both PGI2 and Hoe 892 given subcutaneously (s.c.) reduced dose-dependent gastric acid secretion, the ID50 (dose producing 50% inhibition) being about 48.6 and 11.8 micrograms/kg, respectively. In contrast, intragastric (i.g.) PGI2 and Hoe 892 did not cause any change in gastric acid secretion at doses ranging from 1 to 100 micrograms/kg. Both PGI2 and Hoe 892 reduced significantly intestinal fluid secretion (antienteropooling activity). PGI2 and Hoe 892 given i.g. or s.c. reduced dose-dependent gastric ulcer formation induced by acidified aspirin (ASA), Hoe 892 being somewhat less potent than PGI2. Both PGI2 and Hoe 892 were equally effective against gastric mucosal necrosis induced by absolute ethanol and this effect was observed both after i.g. and s.c. administration of these agents. We conclude that stable thia-imino-PGI2 analogue, Hoe 892, has similar gastric and intestinal antisecretory and protective activity as PGI2 and may be useful in the prevention of gastric damage by various noxious agents.     

Response of glycine and glycyl-L-valine uptake parametrs across in vitro everted sacs of chicken gut to variations in oxygenation rate and fasting

Energy-dependent accumulation of glycine and glycyl-L-valine within the small intestinal mucosa in a chicken model of in vitro local oxygenation of the small intestinal preparation was studied. It has been shown that the most effective bilateral oxygenation significantly increase accumulation of glycyl-L-valine in the proximal segment as compared to that under oxygenation only from serosal surface both in the fed and 24-hour fasted chickens, whereas in other segments these differences was less apparent. This may be due to increased H+/ peptide cotransporter expression in the proximal segment. Thus the bilateral oxygenation probably may turn on an additional amount of already existing (but non-functional during serosal oxygenation) H+/ peptide co-transporters. Moreover, low glycine transporter expression may be the reason why supplemental oxygen (bilateral oxygenation) has no effect on glycine accumulation in the distal segment of fed chickens. A 48-hour fasting decreases glycyl-L-valine accumulation in the proximal (and medial) segments, possibly due to progressive decrease in villus height. It is concluded that: a) the accumulation rate of glycine was greater when presented as the glycyl-L-valine than when presented as the equivalent amount of free amino acid; b) the rates of accumulation of glycyl-L-valine are highest in the proximal segment, decrease in the medial segment and are the lowest in the distal segment; c) the serosal oxygenation is less effective than the mucosal and bilateral oxygenation, which markedly stimulates accumulation of nutrients in the intestinal mucosa; d) a 24-hour fasting increases glycyl-L-valine accumulation in the proximal segment only, while glycine uptake was increased in the distal segment.     

Effect of phloretin and phloridzin on properties of digestion and absorption in the rat small intestine

Experiments in vitro on everted sacs of rat small intestine have shown that phloretin (an inhibitor of basolatheral glucose GLUT2 transporter) added from mucosal side of the sacs decreases release of glucose from enterocytes into serosal fluid without changing glucose accumulation in tissue of the preparations. Addition of phloridzin (an inhibitor of Na⁺ and glucose co-transporter SGLT1) from mucosal side inhibited both glucose accumulation in the tissue and its release into serosal fluid. Unspecific effects of phloretin and phloridzin on activities of several digestive enzymes (in particular, alkaline phosphatase, amino peptidase, and glycyl-L-leucine dipeptidase) has been revealed in homogenates of the rat small intestine mucosa. In chronic experiments on rats, absorption of glycine from the isolated small intestinal loop was inhibited in the presence of phloretin in perfusate. The obtained results indicate that the experimental approach of inhibition of glucose absorption by phloretin used from mucosal side in vitro appears to give a significant overestimation of contribution of facilitated diffusion (with participation of the GLUT2 transporter inserted in the apical enterocyte membrane) to glucose transport across this membrane. Thus, the role of the GLUT2 transporter in the mechanism of glucose absorption in the small intestine under its physiological conditions does not seem to be as great as it is thought by the authors of the recently proposed hypothesis.     

Observations on “Detubulated” Muscle Fibres

GLYCEROL treatment of striated muscle^1–3 has been widely assumed to be a means of disconnecting the transverse tubular system from the surface membrane. Exposure of a sartorius muscle to Ringer plus 400 mM glycerol followed by a return to Ringer, reduces the membrane capacity of superficial fibres and the muscle is mechanically inactive even though the fibres can transmit action potentials; both these effects might be expected if the transverse tubular system was destroyed or disconnected from the extracellular fluid. We have studied this mechanical decoupling on isolated muscle fibres from the semitendinosus, examining the effects of glycerol concentration. Our results suggest that decoupling may be a complex process and in particular they show that under some circumstances both the depolarization and the loss of the twitch can be reversed by re-exposure to glycerol.     

Modulation by opiates of small intestinal prostaglandin E2 and 3',5'cyclic adenosine monophosphate levels and of indomethacin-induced ulceration in the rat.

We studied the effect of parenteral morphine and naloxone administration on intestinal mucosal Prostaglandin E2 (PGE2) and 3',5' cyclic adenosine monophosphate (cAMP) levels and on indomethacin-induced intestinal ulceration in the rat. Compared to the control group, morphine significantly decreased whereas naloxone markedly increased both PGE2 and cAMP mucosal levels, respectively. Morphine or naloxone alone did not cause mucosal injury. However, when given with indomethacin, morphine significantly potentiated the ulcerogenic effect of indomethacin while naloxone exerted a protective effect. These results suggest that opioid peptides may play a role in modulation of intestinal mucosal PGE2 and cAMP levels. In addition, enhancement of indomethacin-induced ulcer formation by morphine and amelioration by naloxone might be in part mediated through their effect on mucosal PGE2 and cAMP levels.