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The purpose of this study was to compare the effects of atriopeptin III (AP-III), vasoactive intestinal peptide (VIP), and ionomycin on Na and Cl influx and to correlate changes in transport with effects on intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 5'-cyclic monophosphate (cGMP) content of the tissue. In addition, the question of whether AP-III inhibits ion transport directly by acting on enterocyte receptors for AP-III or indirectly by stimulation of enteric nerves in the submucosa was also addressed. The results showed that AP-III, ionomycin, and bumetanide all inhibited the initial rate of Na and Cl influx, suggesting that they directly block Na-K-2Cl cotransport activity. VIP had no effect on unidirectional influx of Na and Cl. AP-III caused a fourfold increase in intracellular [cGMP] without any significant effect on [cAMP]. VIP stimulated [cAMP] by fourfold but had no effect on [cAMP]. Ionomycin had no effect on either [cAMP] or [cGMP]. Inhibition of transport by AP-III could not be blocked by tetrodotoxin (TTX), indicating that enteric nerves in the submucosa are not directly involved in mediating the effects of AP-III on epithelial ion transport. The observation that two classes of neuronal depolarizing agents (veratrine and scorpion venom) cause TTX-sensitive inhibition of basal ion transport establishes that NaCl absorption in flounder intestine is subject to regulation by enteric nerves located in the submucosa.

A heat-stable low-molecular-weight peptide isolated from porcine heart inhibited vasoactive intestinal polypeptide and ionomycin (a Ca ionophore) -stimulated Cl secretion across the rat ileum. The antisecretory effects of this peptide were resistant to the neuronal conduction blocker tetrodotoxin, suggesting that submucosal nerves were not involved in mediating its effects on Cl transport. Activity was found in extracts of the right and left atria and in the ventricles of the heart. The antisecretory effect was not mimicked by atrial natriuretic factor, brain natriuretic peptide, or by putative antisecretory factors (neuropeptide Y, somatostatin, enkephalin, and norepinephrine), suggesting that cardiac antisecretory peptide is a unique regulatory factor that may regulate Cl transport in the intestinal mucosa and other epithelia.

Summary The effects of several sulfamoyl benzoic acid derivatives on Na-K-Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC50 values) and order of potency of these derivatives were benzmetanide, 5×10-8m> bumetanide 3×10-7m>piretanide 3×10-6m>furosemide 7×10-6m> amino piretanide 1×10-5 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [3H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forKd and number of binding sites (n) were: bovine kidney,Kd=1.6×10-7,n=10.5 pmol/mg protein; winter flounder intestine,Kd 1.2×10-7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,Kd=5.3×10-7,n=20.4 pmol/mg protein. The estimatedKd for bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10-7m. The similarity in magnitudes of IC50 andKd for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mm concentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mm Cl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mm Cl concentrations showed that bothKd andn were lower at the higher Cl concentration (5mm Cl:Kd=5.29×10-7m,n=20.4 pmol/mg protein; 100mm Cl:Kd=2.3×10-7m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent with a competitive interaction with Cl.

The role that low molecular weight antifreeze glycopeptides play in the physiology of polar fishes has been an open question. In this study, we demonstrate that antifreeze glycopeptides are present in the bile as well as the intestinal fluid of antarctic fishes. Isolation of antifreeze glycopeptides from these fluids by DEAE ion exchange chromatography followed by polyacrylamide gel electrophoresis revealed the presence of only low molecular weight glycopeptides (6, 7 and 8). Removal of the gall bladder with subsequent occlusion of the common bile duct eliminated the transport of antifreezes into the intestine. This suggests that antifreeze glycopeptides enter the intestinal lumen by biliary secretion. Measurements of reabsorption, both in vivo and in vitro, indicate that antifreeze glycopeptides are not reabsorbed as intact molecules. Our results suggest that these glycopeptides are excreted. We conclude that low molecular weight antifreeze glycopeptides are necessary to prevent the intestinal fluid from freezing and provide the first clear evidence that low molecular weight antifreeze glycopeptides have a specific biological function in polar fishes.

The neuropeptide substance P (SP) produces transient elevations in short-circuit current (Isc), a measure of active ion transport, across sheets of small intestinal mucosae from several animal species, but the ionic basis of this action remains unknown. The aim of this study was to test the hypothesis that SP promotes electrogenic anion secretion in the porcine proximal jejunum, an intestinal segment analogous to the human upper small intestine. Sheets of jejunal mucosa with attached submucosa responded to serosal (S), but not luminal (L) addition of 0.1 microM SP with a transient increase in Isc that was reduced in tissues pretreated with the Na(+)-K(+)-Cl- cotransport inhibitor bumetanide (10 microM) or bathed in media lacking Cl- or HCO3- ions. SP produced biphasic effects on transepithelial Na+ and Cl- fluxes; it initially stimulated a L-directed Na+ secretory flux during a 5-min period in which peptide-induced Isc elevations were maximum. The return of the Isc to base-line levels was temporally associated with an increase in L-directed Cl- transport. Both effects of SP were absent in tissues either pretreated with the neuronal conduction blocker tetrodotoxin (0.1 microM) or bathed in HCO3(-)-deficient media. Bumetanide abolished the Na+ secretory actions of SP, but did not affect peptide-induced Cl- secretion. pH-Stat titration experiments revealed that mucosal sheets alkalinized the L bathing medium at a rate twice that of the S medium. SP simultaneously increased and suppressed L and S alkalinization, respectively; this effect presumably represents HCO3- secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine is present in a majority of submucosal neurons which project to the intestinal epithelium. In this study, we examined the role of acetylcholine and the actions of cholinomimetic drugs, such as carbachol (CCH), on ion transport across muscle-stripped sheets of mucosa-submucosa from the proximal jejunum of weaned piglets. Serosal administration of CCH (10 nM-100 microM) produced rapid increases in short-circuit current (Isc) which were attributed to net Cl secretion. Acetylcholine, bethanechol and (4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride were partially effective in increasing Isc. Atropine and selective muscarinic cholinergic antagonists produced dextral shifts in the CCH concentration-effect relationship with an order of relative potency of 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) greater than atropine much greater than pirenzepine greater than 11-[[[2-(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one(AF-DX116). The muscarinic receptor blocker [3H]quinuclidinyl benzilate (QNB) bound specifically and saturably to two sites in the mucosa-submucosa having equilibrium dissociation constants of approximately 10 +/- 3 and 890 +/- 120 pM and Bmax = 7 +/- 3 and 47 +/- 9 fmol/mg protein, respectively. Selective cholinergic antagonists competed for [3H]QNB binding with a rank order of affinity of 4-DAMP greater than hexahydrosiladifenidol much greater than AF-DX 116 greater than or equal to pirenzepine. Specific [3H]QNB binding sites were autoradiographically localized in the jejunal wall to the epithelium, submucosa, and muscularis propria. Electrical transmural stimulation (10-300 pulses/10 sec, 0.5 msec duration, 60 V stimulus strength) delivered to mucosal sheets produced tetrodotoxin-sensitive Isc elevations which were proportional to the number of impulses delivered. Mucosal Isc responses to electrical stimulation were attenuated by 10 microM hexamethonium, 1 microM atropine or autotachyphylaxis to CCH. Tetrodotoxin, at 0.1 microM, produced a 20-fold increase in the secretory potency of CCH. These results suggest that acetylcholine released from intramural neurons in porcine proximal jejunum produces transepithelial Cl secretion. Its effects may be mediated through interactions with two populations of muscarinic cholinergic receptors, located on neuronal and non-neuronal cells within the intestinal mucosa and submucosa, which serve to inhibit and promote Cl secretion respectively.

Measurements of ion concentrations, freezing points and melting points of intestinal fluid were made for several Antarctic fishes and two North Atlantic species. These measurements indicated that plasma and intestinal fluid are nearly isosmotic. Freezing points of intestinal fluid were approximately 0·9 °C below the melting points, suggesting the presence of glycoprotein antifreeze within the intestinal fluid of the Antarctic fishes. Polyacrylamide gel electrophoresis and specific immunoprecipitation with glycoprotein antifreeze antibody confirmed the presence of appreciable quantities of antifreeze and showed that the major antifreeze fractions present in the intestinal fluid are low molecular weight glycopeptides.

The functional properties of the G protein-coupled P2Y1 receptor were investigated in Xenopus oocytes. Incubation of oocytes expressing either the human or turkey P2Y1 receptor with adenine nucleotide agonists resulted in an increase in Cl- current and activation of a novel cation current with an inwardly rectifying current-voltage relationship. Activation of either the human P2Y2 (P2U-purinergic) or M1 muscarinic receptor expressed in oocytes resulted in an increase in Cl- current similar to that observed in P2Y1 receptor-expressing oocytes but had no effect on cation current. P2 receptor agonists stimulated both the cation current and Cl- current in P2Y1 receptor-expressing oocytes with EC50 values and an order of potency (2-methylthioadenosine diphosphate > 2-methylthioadenosine triphosphate (2MeSATP) > ATP > UTP) that were similar to those previously observed for activation of phospholipase C in 1321N1 human astrocytoma cells stably expressing the human or turkey P2Y1 receptor. The P2Y receptor antagonists suramin and pyridoxal phosphate 6-azophenyl-2'-4'-disulfonic acid both shifted to the right the concentration-response relationship for 2MeSATP for stimulation of oocyte currents. Although injection of oocytes with either GDPbetaS (guanyl-5'-yl thiophosphate) or GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) resulted in loss of adenine nucleotide-promoted Cl- channel activation, neither guanine nucleotide altered the 2MeSATP-stimulated cation current. These data are consistent with the view that activation of the novel cation current by the P2Y1 receptor does not involve a G protein. Tail current analysis of the novel P2Y1 receptor-associated cation conductance revealed that the open channel current-voltage relationship was outwardly rectifying with a reversal potential of -38 mV for the turkey P2Y1 receptor and -36 mV for the human P2Y1 receptor. Replacement of Na+ with K+ ions in the bathing solution produced a shift in reversal potential to near zero mV, but significant outward rectification remained. The cation current was not permeable to either Ca2+ or Ba2+ and exhibited steady-state inactivation at holding potentials below -60 mV. These results indicate that the P2Y1 receptor exhibits both metabotropic properties and a novel G protein-independent ionotropic response when expressed in Xenopus oocytes.

The objective of this study was to investigate the effects of vasoactive intestinal peptide (VIP) and norepinephrine (NE) on Na and Cl transport across the isolated porcine gallbladder. Serosal addition of either VIP or secretin increased the short-circuit current (Isc). The half-maximal effect for VIP was 84.3 nM. The effect of VIP was mimicked by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). Replacement of Cl with gluconate nearly abolished the effect of 8-BrcAMP on Isc, whereas HCO3 replacement with N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid buffer had no effect. Transepithelial flux measurements indicated that 8-BrcAMP stimulates net Cl secretion and inhibits Na absorption. Norepinephrine inhibits VIP-stimulated changes in Isc as well as the basal Isc. NE does not, however, reverse the effects of 8-BrcAMP on Isc. The effects of NE are antagonized by yohimbine (alpha 2-adrenergic receptor antagonist) but not prazosin (an alpha 1-adrenergic receptor antagonist). VIP causes a 2.5-fold increase in cAMP content in the gallbladder epithelium. This increase is blocked by NE. Serosal tetrodotoxin did not inhibit the peptide effects, indicating that VIP receptors are localized on the epithelium. Depolarization of submucosal nerves with veratrine inhibited the basal Isc and was reversible with yohimbine. This result indicated that sympathetic nerve pathways regulate Na and Cl absorption in vitro.

Porcine gallbladder, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasma-like Ringer solution generates a serosal positive transepithelial potential of 4-7 mV and a short-circuit current (Isc) of 50-120 microA/cm2. Substitution of Cl with gluconate or HCO3 with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) results in a 50% decrease in Isc. Treatment with 1 mM amiloride (mucosal side) or 0.1 mM acetazolamide (both sides) causes 25-27% inhibition of the Isc. Mucosal addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits the Isc by 17%. Serosal addition of 0.1 mM bumetanide inhibits the Isc by 28%. Amiloride (1 mM) inhibits the net transepithelial fluxes of Na and Cl by 55 and 41%, respectively. Substitution of Cl with gluconate inhibits the net Na flux by 50%, whereas substitution of HCO3 with HEPES inhibits 85-90% of the net Na flux and changes Cl absorption to net secretion. Based on these results, it is hypothesized that Na and Cl transport across the apical membrane is mediated by two pathways, Na-H/Cl-HCO3 exchange and Na-HCO3 cotransport. Partial recycling of Cl and HCO3 presumably occurs through a Cl conductive pathway and Cl-HCO3 exchange, respectively, in the apical membrane. This results in net Na absorption, which accounts for most of the Isc observed under basal conditions. The effect of bumetanide on the basolateral membrane and the fact that Cl secretion occurs when HCO3 is absent suggests that Cl secretion involves a basolateral NaCl or Na-K-Cl cotransport system arranged in series with a Cl conductive pathway in the apical membrane.