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PubMed1968

The release of 5-hydroxytryptamine from rabbit small intestine in vitro.

1. A method is described by which the lumen of intestinal segments may be perfused separately and simultaneously under identical conditions of perfusion pressure and temperature, and the rate of appearance of 5-hydroxytryptamine (5-HT) into the serosal fluid measured.2. 5-HT appeared in the serosal fluid from quiescent segments of rabbit intestine at a rate of about 4.0 ng/cm intestine/hr and about 14.0 ng/cm intestine/hr from segments performing active peristaltic movements.3. For both quiescent and active segments, when the perfusion fluid contained a monoamine oxidase inhibitor, nialamide (Niamid), the rate of appearance of 5-HT was increased and if, in addition, 5-HT was present in the perfusion fluid the rate of appearance of 5-HT in the serosal fluid was even greater.4. The rate of appearance of 5-HT from the intestine of rabbits pretreated with reserpine was about 0.8 ng/cm intestine/hr and Niamid only slightly increased this rate. However, if 5-HT as well as Niamid were present the rate of appearance of 5-HT was about 6.0 ng/cm intestine/hr.5. Possible explanations for these findings are discussed.

Animals↗
PubMed1970

The effect of adenosine triphosphate on the transmural potential in rat small intestine.

1. ATP either in the mucosal or serosal fluid caused a transient increase in the potential difference and short-circuit current across the wall of rat jejunum in vitro, the serosal solution becoming more positive.2. Similar responses were observed in the ileum and colon, and in in vivo preparations of small intestine.3. The response is relatively specific for ATP.4. The transient nature of the response is not due to rapid hydrolysis of extracellular ATP.5. High concentrations of K(+) in the mucosal medium, serosal ouabain or mucosal 2,4-dinitrophenol all inhibit the response without altering the time course.6. Significant inhibition was not observed in the presence of ADP or in Mg(2+)-free salines.7. The results are consistent with an intracellular action of ATP in the epithelium to stimulate net ion transport. The results do not demonstrate whether or not extracellular ATP can act as an energy donor for an electrogenic ion pump.8. Theophylline prolongs the time course of the response, and the involvement of the adenyl cyclase system cannot be excluded as an explanation for the findings.

Adenine Nucleotides↗
PubMed1974

The transamination of glutamate and aspartate during absorption in vitro by small intestine of chicken, guinea-pig and rat.

1. Procedures are described for direct measurement of the extent and rate of transamination of glutamate and aspartate over periods of up to 90 min, during absorption in vitro by the small intestine of chicken, guinea-pig, and rat.2. During absorption of dicarboxylic amino acids by rat small intestinal segments circulated through the lumen in vitro, alanine contributed up to 85% of the amino acids appearing in the fluid secreted at the serosal surface. In guinea-pig and chicken intestine, the proportion of alanine in the secreted amino acids did not exceed 60%.3. For the different species studied, a relationship was found between the extent to which the dicarboxylic amino acids were transaminated to alanine and the total amount of GPT found in other studies to be present in the intestinal mucosa. In both rat and guinea-pig small intestine, the proportion of alanine in the total amino acids appearing at the serosal surface was similar in the jejunum and ileum. The rate of appearance of alanine in serosal fluid was greater in the ileum than in the jejunum of the rat.4. Reasons are given for supposing that for all the species studied there is a limit to the capacity of the small intestinal mucosa to subject free dicarboxylic amino acids to transamination. It is concluded, however, that it is unlikely that this capacity will be exceeded under in vivo conditions.

Alanine↗
PubMed1975

Effect of bradykinin on transepithelial transfer of sodium and water in vitro.

Mucosal sodium and water transfer were measured in everted sacs of rat jejunum. 2. Bradykinin (7.86 X 10(-12)M), when present in both mucosal and serosal solutions, produced a biphasic effect on mucosal sodium and water transfer. When basal transfer was low a stimulation was observed whereas an inhibition of transfer was observed when basal transfer was high. Bradykinin at concentrations of 7-86 X 10(-11) and 7-86 X 10(-13) M produced qualitatively similar effects. 3. Inhibition of transfer was observed whether bradykinin was present in the mucosal, serosal or both solutions. Stimulation of transfer was observed only when bradykinin was present in the serosal solution. 4. Theophylline (1 mM), alone inhibited water transfer at high and intermediate levels of basal transfer, and significantly potentiated the inhibitory effect of bradykinin (7-86 X 10(-12)M) on water transfer at intermediate levels of control transfer. 5. Cyclic AMP (1 mM) inhibited water transfer when basal transfer was high. Dibutyryl cyclic AMP (1 mM) inhibited water transfer at all levels of basal transfer. Dibutyryl cyclic AMP (1 mM) and bradykinin (7-86 X 10(-12)M) together produced a significantly greater inhibition of water transfer than either agent alone, at intermediate basal transfer. 6. It was observed that the action of bradykinin upon sodium and water transfer consists of two different and opposing effects. It is possible that the inhibitory effect of bradykinin upon water transfer is related to increased cyclic AMP activity.

Animals↗
PubMed1976

The association between acidification and electrogenic events in the rat proximal jejunum.

1. Simultaneous measurement of hydrogen ion production, transmural potential difference and intermittent short-circuit current (SCC) was made in the rat proximal jejunum in vitro: similarly, potassium and sodium ion movements were measured to investigate the relationship of acidification to electrogenic events and associated ion movements in the jejunum.2. Acidification correlated significantly with the short-circuit current and both were inhibited by 10 mM serosal ouabain or 10 mM mucosal aminophylline. Both inhibitors had effects on net potassium movement but not on net sodium movement. Moreover, in isotope studies whereas 10 mM serosal ouabain reduced the J(ms) sodium flux, 10 mM mucosal aminophylline had no effect, i.e. aminophylline can reduce both short-circuit current and acidification without perceptibly altering the serosally directed sodium flux.3. In low-sodium buffers in which acidification still occurs although reduced, transmural potential differences occur of reversed polarity that are apparently unrelated to sodium diffusion potential differences (as evidenced by isotopic sodium efflux experiments) and which could be caused by hydrogen ion production. In low sodium buffers however the inhibitors have opposing effects, ouabain causing an increase and aminophylline a decrease in the reversed potential differences.4. A model for acidification (that of potassium rather than sodium ion exchange for the hydrogen ion and hydroxyl for chloride ion exchange) is proposed to explain the present experimental findings and other diverse observations in the literature: although either step might be the electrogenic step, acidification must be also considered as a component of the jejunal short-circuit current.

Aminophylline↗
PubMed1980

Effects of sugars, amino acids and inhibitors on electrolyte transport across hen colon at different sodium chloride intakes.

1. The isolated colonic mucosa from adult hens has been studied in vitro under short-circuit conditions. Colonic mucosa was prepared from hens fed either a NaCl-poor or a NaCl-rich diet. 2. The unidirectional transmucosal fluxes of chloride, K and Na were measured. For the high NaCl colon the fluxes of lysine were also measured. The effects of galactose, glucose, leucine, lysine, amiloride (10(-4) M) and acetazolamide (5 X 10(-4) M) on the short-circuit current (Isc) were examined. 3. The Isc of the high NaCl colon was stimulated by galactose, glucose, leucine and lysine in a manner which suggests that these substances were transported by the epithelium in co-transport with Na. The Isc was in most cases insensitive to amiloride (mucosal side), but moderately stimulated by acetazolamide (serosal side). The Isc was closely matched by the sum of the net transmucosal fluxes of chloride, lysine, K and Na. The effects of galactose, leucine and lysine on the Isc were maintained only when glucose was present on the serosal side of the preparations. 4. The Isc of the low NaCl colon depended on the presence of glucose on the serosal (or mucosal) side. It was not stimulated by galactose or by lysine, but leucine did stimulate when added to the mucosal solution. Adding amiloride on the mucosal side invariably eliminated or, in most cases, led to an inversion of the Isc. Acetazolamide markedly stimulated the Isc; and, when added after amiloride, acetazolamide caused the Isc to become positive again. The data on Isc and net fluxes of chloride, lysine, K and Na are consistent with the presence of a process of bicarbonate absorption or H ion secretion. 5. There is a net secretion of K across both the high and the low Na colon, which is highest in the former although both Isc and net absorption of Na are highest in the latter. 6. The results from both types of colon suggest that the transport of chloride differs with the functional status of tissues.

Amiloride↗
PubMed1983

Rat endometrial bioelectric activity in vivo and in vitro: effects of adrenaline.

The transuterine potential difference (p.d.) generated by the endometrium was measured in vivo in anaesthetized, ovariectomized rats treated with 10 micrograms oestradiol-17 beta each day for 3 days. Intravenous injection of adrenaline induced transient hyperpolarization of the p.d.; sigmoid dose-dependency was observed over the range 2-40 micrograms/kg. Pre-treatment of the rats with syrosingopine shifted the dose-response curve to the left (enhancement) while combined propranolol (800 micrograms/kg) and phentolamine (800 micrograms/kg) shifted the dose-response curve to the right (depression). Isoprenaline gave similar quantitative changes in the p.d. in vivo which displayed a similar time course and sigmoid dose-dependency. Propranolol alone (800 micrograms/kg) shifted this dose-response curve significantly to the right. Addition of adrenaline or isoprenaline to the serosal solution bathing uteri incubated in vitro after removal from oestradiol-treated rats induced changes in endometrial bioelectric activity that exhibited sigmoid dose-response curves for the transuterine p.d. and short-circuit current (Isc). Propranolol significantly shifted the dose-response curves for adrenaline to the right. Only adrenaline caused small but significant decreases in the uterine resistance but these were not dose-dependent. Acetylcholine, 5-hydroxytryptamine, noradrenaline and phenylephrine had no effect on the transuterine p.d. in vivo or in vitro or on the Isc in vitro. No significant electrical changes could be induced by adrenaline or any of the other drugs across the uteri from untreated ovariectomized rats either in vivo or in vitro. The transluminal membrane p.d. of surface endometrial cells measured with micro-electrodes was depolarized by an addition of serosal adrenaline but only 41% of the cells successfully impaled responded in this way. The results indicate that endometrial cells under oestrogen stimulation possess electrogenic processes that can be modulated specifically by adrenaline through the mediation of a serosally sited beta-adrenoceptor. The physiological role of the mechanism has yet to be established.

Animals↗
PubMed1983

Influence of vasopressin and calcium on electrolyte transport across isolated colonic mucosa of the rat.

Vasopressin enhanced the absorption of water and Na+ across everted sacs of rat colon descendens but had no effect on absorption across the colon ascendens. The short-circuit current (Isc) and open-circuit potential difference (p.d.) across the colon descendens were dose-dependently decreased by vasopressin. Isc and p.d. across the colon ascendens were not altered by vasopressin. In the colon descendens the decrease in Isc and p.d. was significant at 1 microu. vasopressin/ml and reached a maximum at 1 mu./ml. Propranolol and phentolamine or naloxone did not alter the decrease in Isc and p.d. to a submaximal dose of vasopressin. Vasopressin increased the mucosal to serosal flux of Na+ and Cl- and decreased the serosal to mucosal flux of Cl- across short-circuited colon descendens. Consequently these changes increased the net flux of Na+ and Cl-. Adenylate cyclase activity in homogenates of the colon descendens was not altered by vasopressin. Omission of Ca2+ from the serosal bathing solution reversibly decreased Isc and p.d. and increased Na+ and Cl- absorption across the colon descendens in a similar way as did vasopressin. The results suggest that the effect of vasopressin on the colon descendens may be due to a decrease in intracellular Ca2+ activity.

Adenylyl Cyclases↗
PubMed1984

Effect of sodium on transport of weak electrolytes across rat jejunum in vitro.

The effects of ion substitution and inhibitors on the transport of two representative weak electrolytes, benzoic acid and amphetamine, across short-circuited rat jejunum in vitro have been studied. The spontaneous asymmetries in the transport of the weak electrolytes were not dependent on the presence of either bicarbonate or chloride in the incubation medium. Net transport of the weak electrolytes was abolished when choline was substituted for sodium in the fluid bathing the mucosal surface of the tissue, but weak electrolyte transport was independent of the presence of sodium on the serosal side. Weak electrolyte transport was inhibited by ouabain and by orthovanadate. The effect of ouabain was abolished when potassium was substituted for sodium in the serosal fluid, and the effect of orthovanadate was potentiated in this condition. It was shown that the ability of the intestinal epithelium to discriminate between the ionized and non-ionized forms of weak electrolytes was not altered when choline was substituted for sodium in the incubation medium, indicating that the effect of sodium was associated with the driving force for weak electrolyte transport. Weak electrolyte transport was not abolished by raising the serosal potassium concentration, suggesting that the transport process was directly related with the function of the sodium pump, rather than correlated with gradients of electrical or chemical potential established as a consequence of pump activity.

Amphetamine↗
PubMed1984

The involvement of calcium in the intestinal response to secretagogues in the rat.

The involvement of Ca2+ in the regulation of intestinal secretion was investigated in stripped sheets of rat mid-intestine. Removal of serosal Ca2+ together with the addition of EGTA at concentrations of 0.5 and 1 mM inhibited the rise in short-circuit current (s.c.c.) induced by both acetylcholine and theophylline, a similar degree of inhibition being observed with both secretagogues. Ca2+-free serosal fluid with 0.5 mM-EGTA added reduced significantly the rises in s.c.c. induced by A23187, acetylcholine, 5-hydroxytryptamine, theophylline, dibutyryl cyclic AMP and prostaglandin E2, but not the increased s.c.c. associated with glucose absorption. The Ca2+ channel blocker verapamil produced similar results. The calmodulin antagonist trifluoperazine inhibited secretagogue action while its sulphoxide derivative was without effect at the same concentration. The intracellular Ca2+ antagonist TMB-8 reduced the increased s.c.c. observed with acetylcholine and dibutyryl cyclic AMP. The net Cl- secretion, but not the decreased mucosal-to-serosal Na+ flux, induced by acetylcholine was abolished in Ca2+-free conditions. There was no consistent effect on the reduction in the residual ion flux caused by acetylcholine. Absence of Ca2+ converted the stimulation of Cl- secretion induced by dibutyryl cyclic AMP observed under control conditions to an enhancement of net Na+ and Cl- absorption. It is concluded that intestinal secretagogues, whether they act through cyclic AMP or not, require both internal and external sources of Ca2+ if they are to produce their full effects. Moreover, it appears that the nature of the response to dibutyryl cyclic AMP depends on the prevailing Ca2+ concentration.

Acetylcholine↗
PubMed1985

Apical membrane potassium and chloride permeabilities in surface cells of rabbit descending colon epithelium.

The apical membranes of surface cells in the rabbit descending colon possess a significant ionic conductance in parallel to amiloride-blockable Na+ channels. The identity of the ion(s) responsible for the amiloride-insensitive conductance is unknown. The purpose of the present paper was to assess the permeability and net driving forces for K+ and Cl- across this membrane using conventional and ion-sensitive micro-electrode techniques. Intracellular Cl- activity (aiCl) averaged 23 +/- 2 mM with an equilibrium potential (ECl) of -38 +/- 2 mV. This value is less than previous estimates of the electromotive force (e.m.f.) of the amiloride-insensitive pathway (ca. -50 mV). Consequently, Cl- alone cannot account for the amiloride-insensitive conductance. Replacement of Cl- by gluconate in the serosal solution decreased aiCl to 17 +/- 2.8 mM. aiCl was lowered to approximately 1 mM by replacement in the mucosal bath or by replacement in both solutions. The results indicate a low Cl- conductance in the basolateral membrane, in agreement with previous electrophysiological studies of this epithelium. In contrast to Cl-, the chemical driving force for K+ was large enough to support the e.m.f. of the amiloride-insensitive pathway (K+ equilibrium potential, EK = -66 mV). The basolateral membrane potential (Vbl), EK and the intracellular K+ activity (aiK) were decreased in parallel following inhibition of the basolateral Na-K pump, providing evidence that Vbl is largely due to a K+ diffusion potential. In the presence of serosal 10(-4) M-ouabain, aiK appeared to remain above equilibrium and more than doubled after addition of Ba2+ to the serosal bath. Replacement of the mucosal bathing solution with KCl or gluconate Ringer solution largely restored Vbl and the transepithelial potential (VT) in tissues which had been previously treated with ouabain. The restoration of VT was decreased and the transepithelial resistance (RT) was increased by addition of tetraethylammonium to the mucosal bath. The above results suggest that there are at least four routes for ion movement across the apical membrane of rabbit colon surface epithelial cells. These are: (1) an amiloride-sensitive Na+ channel, (2) a K+ conductance, (3) electroneutral uptake of Cl- from lumen to cell interior and (4) an active K+ transport mechanism, also from lumen to cell interior.

Animals↗
PubMed1987

Steady-state analysis of ion fluxes in Necturus gall-bladder epithelial cells.

1. The steady-state fluxes of Na, K and Cl ions have been measured in Necturus gall-bladder epithelium by a technique that involves labelling the cells with tracer ions from the mucosal bath only, whilst the serosa is kept at low specific activity. After removing tracer, the efflux is followed into the serosal bath, revealing two exponential components. 2. The time constant of the fast component lies between 0.03 and 0.04 s-1 and corresponds to that of the extracellular space. The slow component closely matches the cellular efflux, with constants which lie between 0.14 and 0.46 X 10(-2) s-1. 3. Full unstirred-layer calculations have been performed to determine the specific activities in the mucosal solution, the cell and the corium (subepithelium). These involved measuring the diffusion coefficients of Na and Cl in the isolated corium: they are restricted by factors of 0.17 and 0.11. 4. The partial flux equations for this double-membrane system have been solved to obtain the cellular fluxes for all three ions. The results indicate that: (i) the net transcellular Na flux is 190 pmol cm-2 s-1, equivalent to the transepithelial salt flux during fluid secretion; (ii) the net transcellular K flux is effectively zero because this ion recirculates across the serosal membrane; (iii) the net transcellular Cl flux is 27 pmol cm-2 s-1, or 15% of the net transepithelial salt flux. 5. The permeability of the paracellular pathway to Cl is 1.65 X 10(-5) cm s-1 and the available driving forces will allow a maximum net electrodiffusive Cl transport of 10% through the shunt pathway. 6. 1:1 coupling of Na and Cl net fluxes at the mucosal membrane of this epithelium cannot be present, and processes other than simple electrodiffussion are required to effect net Cl transport by another route. 7. The serosal fluxes of K and Cl do not obey the flux-ratio equation. A component of these fluxes must be present which is neither active (pumped) nor passive (electrodiffusive and independent). If they are symmetrical in the steady state then the ratio of these exchange fluxes lies between 2:1 and 3:1 depending upon the size of the pump flux. They support the view that a mode of coupled K and Cl transport may be operating at the basolateral membrane of these cells.

Animals↗
PubMed1988

Luminal propionate-induced secretory response in the rat distal colon in vitro.

1. The stimulatory action of propionate on colonic electrolyte transport and involvement of the enteric reflex in this was studied in vitro using an Ussing chamber in the rat. The short-circuit current (Isc) and bidirectional fluxes of Na+ and Cl- were measured. Mucosa-submucosa preparations, containing the submucosal nerve, from the distal colon were used in most cases. 2. Mucosal application of propionate caused transient increases in the transmural potential difference, with the mucosal side negative, Isc and conductance. Serosal application of the acid had no effect. 3. Adaptation of the Isc response occurred when the acid was applied to the bathing solution cumulatively without washing out the first dose. If tissues were washed and held more than 20 min before the next application, the response was almost completely restored. 4. The increase in Isc in response to propionate was concentration dependent, with a 50% effective concentration of approximately 7 x 10(-5) M. 5. Two other short-chain fatty acids (SCFAs), n-butyrate and n-valerate, but not acetate, increased Isc when added to the mucosal bathing solution. 6. Bumetanide (3 x 10(-5) M) and the serosal chloride-free condition, but not amiloride (10(-4) M), inhibited the responses of Isc to propionate. Propionate-stimulated Cl- secretion resulted mainly from an increase in unidirectional serosal-to-mucosal Cl- movement. Propionate did not affect the Na+ flux. 7. Tetrodotoxin (10(-7) M), somatostatin (10(-7) M) and hexamethonium (10(-4) M) inhibited the propionate-evoked increase in Isc by 40, 70 and 30%, respectively. 8. Atropine (10(-5) M) also inhibited the Isc-increase response to propionate more than 90%. 9. Pre-treatment (2 min) of the mucosal surface with procaine (5 x 10(-4) M) inhibited the propionate-evoked increase in Isc by 90%. 10. The results suggest that luminal propionate transiently stimulated the colonic chloride secretory response that is not due to direct action on colonocytes, but due in large part to release of acetylcholine at neuro-colonocyte junctions, probably via an enteric reflex involving a mucosal sensory mechanism, cholinergic motor nerves and submucosal ganglia.

Action Potentials↗
PubMed1990

Active potassium absorption in rat distal colon.

1. Active potassium (K+) absorption in rat distal colon was investigated by measuring mucosal-to-serosal (JK, ms) and serosal-to-mucosal (JK, sm) 42K+ fluxes (mu equiv h-1 cm-2) across isolated stripped mucosa under short-circuit conditions in normal and dietary Na-depleted animals. As previously demonstrated, removal of Na+ from both mucosal and serosal solutions bathing the normal colon slightly increased net K+ absorption as a result of inhibition of JK, sm without affecting JK, ms, while in the Na-depleted group net K+ secretion (-0.54 +/- 0.11) was converted to a marked net K+ absorption (1.68 +/- 0.30, P less than 0.001). 2. In both groups of animals in Na(+)-free Ringer solution, JK, ms exhibited saturable and linear components, while JK, sm was a linear function of [K+]. Estimated affinity constants (mM) for saturable net K+ absorption were similar in normal (0.52 +/- 0.12) and Na-depleted (0.67 +/- 0.11) animals; however, there was a greater than 3-fold increase in the saturable flux (Jmax) from 0.54 +/- 0.04 in the normal colon to 1.78 +/- 0.08 mu equiv h-1 cm-2 in Na-depleted animals. 3. Mucosal orthovanadate (100 microM) inhibited JK, ms in both normal (control, 0.66 +/- 0.05 vs. orthovanadate, 0.36 +/- 0.03 mu equiv h-1 cm-2, P less than 0.001) and Na-depleted animals (control 1.20 +/- 0.13 vs. orthovanadate 0.77 +/- 0.07 mu equiv h-1 cm-2, P less than 0.01) without affecting JK, sm or the short-circuit current. In the Na-depleted group mucosal omeprazole or SCH28080 (100 microM), inhibitors of gastric K(+)-H(+)-ATPase, insignificantly or slightly reduced (by 10%) JK, ms respectively; in contrast, mucosal ouabain (1 mM) markedly inhibited JK, ms (control, 1.61 +/- 0.16 vs. ouabain, 0.83 +/- 0.98 mu equiv h-1 cm-2, P less than 0.001). 4. Mucosal Na+ appeared to be a competitor of K+ uptake across the apical membrane. 5. These results indicate that dietary Na-depletion increases electroneutral K+ absorption by increasing its transport capacity and suggest that the mechanism of this active K+ absorption process may involve an apical K(+)-ATPase with properties that are unlike the gastric K(+)-H(+)-ATPase but similar, in part, to Na(+)-K(+)-ATPase.

Adenosine Triphosphatases↗
PubMed1991

Effects of calcitriol on stimulation of ion transport in pig jejunal mucosa.

1. Active sodium (Na+) and chloride (Cl-) fluxes were studied in vitro in Ussing-type chambers with stripped jejunal mucosa of piglets which suffered from pseudo-vitamin D deficiency rickets, type I. The piglets are devoid of renal calcitriol (1,25-dihydroxy vitamin D3) production and have only small amounts of calbindin in their jejunal enterocytes. 2. In the presence of 0.01 mM-indomethacin non-stimulated short-circuit current (Isc), transepithelial potential difference (PD), tissue conductance (Gt) and unidirectional Na+ (JNa) and Cl- fluxes (JCl) were not affected by the low calcitriol (LC) concentration in plasma. 3. Adding 10 mM-theophylline to the serosal solution in the presence of 0.01 mM-indomethacin caused significantly greater increases in Isc in LC mucosa than in mucosa of vitamin D3-treated and control piglets with normal calcitriol (NC) concentrations in plasma. Omission of indomethacin significantly increased Isc stimulation provoked by theophylline with LC and NC mucosa. The increase, however, was significantly greater with LC than with NC mucosa. 4. Omission of calcium (Ca2+) from the serosal bathing solution significantly depressed Isc stimulation by 10 mM-theophylline in indomethacin-treated LC and NC mucosa. But depression was greater with LC than with NC mucosa. 5. Blocking Ca2+ entry into the cytosol by adding either 0.1 mM-TMB-8 or 0.5 mM-d,l-verapamil to the serosal bathing solution abolished the difference in Isc response to theophylline between indomethacin-treated LC and NC mucosa due to greater depression of Isc in LC than in NC mucosa. 6. The combined effects of theophylline and A23187 on Isc stimulation were calcitriol dependent. In the presence of indomethacin this dependence was only significant when A23187 was given prior to theophylline. In the absence of indomethacin the combined effects of A23187 and theophylline on Isc were always significantly greater in LC than in NC mucosa, irrespective of the order of adding the two agents. 7. Addition of theophylline stimulated net Na+ and Cl- secretion in indomethacin-treated LC and NC mucosa. The increases of net Na+ and Cl- fluxes fully accounted for the rise of Isc with NC mucosa but accounted only partly for the increase in Isc with LC mucosa. This resulted in significant increase in theophylline-stimulated residual ion flux (JR) in LC mucosa which probably resulted from enhanced secretion of bicarbonate.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Tetraethylammonium-sensitive apical K+ channels mediating K+ secretion by turtle colon.

1. Apical membrane K+ channels in turtle colon were identified and characterized using current fluctuation analysis. 2. Under short-circuit conditions in NaCl-Ringer solution, the power density spectrum (PDS) of the short-circuit current (Isc) sometimes exhibited a clearly discernible Lorentzian component, indicating spontaneous fluctuations produced by a population of apical ion channels. The Lorentzian component had a characteristic corner frequency (fc) which averaged 10.2 +/- 0.9 Hz (mean +/- S.E.M., n = 20). 3. The power of the spontaneous fluctuations was enhanced (So increased) by manoeuvres that depolarize the apical membrane electrical potential (Va). Discernible fluctuations were enhanced or induced by raising the serosal K+ concentration ([K+]s = 50-115 mM, Na+ replacement), by clamping the transepithelial potential (Vt) to serosa-positive values, or by blocking basolateral K+ channels with Ba2+. 4. Mucosal amiloride (100 microM) attenuated the spontaneous fluctuations observed in NaCl-Ringer solution but had no effect in the presence of serosal high K+, indicating that amiloride did not block the K(+)-permeable channels but these channels resided in the same cells as the amiloride-sensitive Na+ channels. 5. Raising the mucosal K+ concentration attenuated spontaneous fluctuations. 6. In the presence of serosal high K+ and mucosal amiloride, the spontaneous fluctuations were often accompanied by a reversed Isc consistent with K+ secretion. These conditions were used to test the effects of putative channel blockers. 7. Mucosal Ba2+ and tetraethylammonium (TEA+) were effective inhibitors of the spontaneous fluctuations and the reversed Isc. At a concentration of 10 mM, TEA+ was maximally effective but the TEA+ analogues tetramethylammonium (TMA+) and tetrapropylammonium (TPrA+) were much less effective. Mucosal Rb+ or Cs+ did not inhibit at a concentration of 10 mM. 8. Mucosal lidocaine (200 microM), quinidine (200 microM), or diphenylamine-2-carboxylate (DPC, 1 mM) had little or no effect on the spontaneous fluctuations and reversed Isc. Quinine (100 microM), 4-aminopyridine (1 mM), and apamin (100 nM) were also without effect. 9. Mucosal TEA+ (10 mM) abolished the active secretory K+ flux measured in the presence of serosa-positive transepithelial potentials. 10. These experiments identified a population of TEA(+)-sensitive, apical K+ channels which mediate active K+ secretion in turtle colon. Sensitivity to external TEA+ distinguishes these channels from basolateral K+ channels in turtle colon and demonstrates similarity to apical K+ channels in mammalian colon.

Amiloride↗
PubMed1995

Dipeptide transport and hydrolysis in isolated loops of rat small intestine: effects of stereospecificity.

1. Isolated jejunal loops of rat small intestine were perfused by a single pass of bicarbonate Krebs-Ringer solution containing either D- or L-phenylalanine or one of eight dipeptides formed from D- or L-alanine plus D- or L-phenylalanine. 2. At 0.5 mM L-phenylalanyl-L-alanine increased serosal phenylalanine appearance to forty times the control rate giving a value similar to that found with 0.5 mM free L-phenylalanine. No serosal dipeptide could be detected. 3. Perfusions with the two mixed dipeptides with N-terminal D-amino acids (D-alanyl-L-phenylalanine and D-phenylalanyl-L-alanine) gave rise to the appearance of intact dipeptides in the serosal secretions although there were substantial differences in their rates of absorption and subsequent hydrolysis. 4. L-Alanyl-D-phenylalanine was absorbed from the lumen three to five times as fast as L-phenylalanyl-D-alanine. At 1 mM L-alanyl-D-phenylalanine transferred D-phenylalanine across the epithelial layer at more than seven times the rate found with the same concentration of the free D-amino acid. 5. Perfusions with D-alanyl-D-phenylalanine or D-phenylalanyl-D-alanine showed that these two dipeptides are poor substrates for both transport and hydrolysis by the rat small intestine. 6. Analysis of mucosal tissue extracts after perfusion with the two mixed dipeptides with N-terminal D-amino acids revealed that both dipeptides were accumulated within the mucosa and suggested that exit across the basolateral membrane was rate limiting for transepithelial dipeptide transport.

Alanine↗
PubMed2003

Polarized distribution of HCO3- transport in human normal and cystic fibrosis nasal epithelia.

The polarized distribution of HCO3- transport was investigated in human nasal epithelial cells from normal and cystic fibrosis (CF) tissues. To test for HCO3- transport via conductive versus electroneutral Cl-/HCO3- exchange (anion exchange, AE) pathways, nasal cells were loaded with the pH probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and mounted in a bilateral perfusion chamber. In normal, but not CF, epithelia, replacing mucosal Cl- with gluconate caused intracellular pH (pHi) to increase, and the initial rates (Delta pH min-1) of this increase were modestly augmented (approximately 26 %) when normal cells were pretreated with forskolin (10 microM). Recovery from this alkaline shift was dependent on mucosal Cl-, was insensitive to the AE inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS; 1.5 mM), but was sensitive to the cystic fibrosis transmembrane conductance regulator (CFTR) channel inhibitor diphenylamine-2-carboxylate (DPC; 100 microM). In contrast, removal of serosal Cl- caused pHi to alkalinize in both normal and CF epithelia. Recovery from this alkaline challenge was dependent on serosal Cl- and blocked by H2DIDS. Additional studies showed that serosally applied Ba2+ (5.0 mM) in normal, but not CF, cells induced influx of HCO3- across the apical membrane that was reversibly blocked by mucosal DPC. In a final series of studies, normal and CF cells acutely alkaline loaded by replacing bilateral Krebs bicarbonate Ringer (KBR) with Hepes-buffered Ringer solution exhibited basolateral, but not apical, recovery from an alkaline challenge that was dependent on Cl-, independent of Na+ and blocked by H2DIDS. We conclude that: (1) normal, but not CF, nasal epithelia have a constitutively active DPC-sensitive HCO3- influx/efflux pathway across the apical membrane of cells, consistent with the movement of HCO3- via CFTR; and (2) both normal and CF nasal epithelia have Na+-independent, H2DIDS-sensitive AE at their basolateral domain.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗