Stimulation Of Isc Research Articles

Apical regulation of nonselective cation channels by ATP in larval bullfrog skin

The apical membrane of larval bullfrog skin contains a nonselective cation channel that can be activated by apically applied amiloride and acetylcholine. In our search for other ligands that might activate this channel, ATP and other purinergics were tested. When ATP (10-1,000 microM) was added to the apical side of tadpole skin mounted in a modified Ussing chamber, there was a transient increase in short circuit current (Isc). The increase in Isc occurred with either Na or K as the dominant cation in the apical solution. The response was larger in a calcium-free Ringer. ADP and AMP had similar but smaller effects than ATP. Adenosine and UTP were without effect. The ATP response was blocked by W-7, atropine, curare, diltiazem, and suramin. These blockers also inhibit amiloride stimulation of Isc, suggesting that ATP activates a related transport pathway. Studies with analogs of ATP suggest that the ATP binding site in tadpole skin has characteristics in common with the P2x receptor found in other tissues. These results demonstrate that in addition to amiloride and acetylcholine, ATP stimulates cation transport at the apical membrane of larval amphibian skin epithelia.

Role of purinergic receptors in chloride secretion in Caco-2 cells.

Purinergic receptors play an important role in regulating Cl- secretion in epithelial cells. To explore further the role of these receptors in the intestine, we utilized the human intestinal epithelial cell line, Caco-2, grown on permeable membrane supports and assayed for Cl- secretion by measuring the short-circuit current (Isc). Stimulation of Isc by extracellular nucleotides could be detected by day 4 and increased by day 10 postseeding. The magnitude of stimulation of Isc at 10 microM in cells at day 10 was UTP > ATP > UDP > > 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) = ADP on the apical side and UTP = 2-MeS-ATP = ATP > ADP > > UDP on the basolateral side. Cross-desensitization studies suggested that two different receptors are expressed in the apical membrane, a P2U purinoceptor and a uridine nucleotide receptor. Two different receptors are also expressed in the basolateral membrane, a P2U receptor and another that reacts with both 2-MeS-ATP and ADP. This latter receptor has an unusual pharmacological profile, with a reactivity for 2-MeS-ATP > ADP but not for ATP. Responses to purinergic receptor agonists were inhibited by pretreatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, thapsigargin, or quinine. Thus we suggest that an increase in intracellular Ca2+ and subsequent opening of Ca(2+)-activated K+ channel play a role in increasing driving force for Cl- to exit across the apical membrane. The role of the cystic fibrosis transmembrane conductance regulator as a Cl- exit pathway on the apical membrane was also established.

Extracellular ATP modulates ion transport via P2Y purinoceptors in a middle-ear epithelial cell line.

Mucus and cellular debris are eliminated from the middle-ear cavity through the E-tube by the mucociliary system. Depth of the periciliary fluid layer is thought to be regulated by epithelial ion transport activity. Since impairment of the mucociliary system is a key step in the development of otitis media with effusion, we investigated the ion transport mechanisms of the middle-ear epithelium using the middle-ear MESV cell line. ATP has been shown to modulate ion transport as well as various cellular functions in several cell types via purinoceptors. In order to investigate a possible modulation of the transport activity of MESV cells, we evaluated short-circuit current (Isc) changes in response to specific stimulation of putative purinoceptors by ATP and its various analogs. ATP dramatically increased Isc, while adenosine had no effect, thus demonstrating the presence of P2 receptors according to the original classification by Burnstock. The rank order of potency of purinoceptor agonists for stimulation of Isc on the apical side (ATP > UTP > gamma-SATP >> beta-SADP > 2-methylthio-ATP, 2MeSATP > beta, gamma-methylene-ATP, beta,gamma-MeATP) and on the basolateral side (ATP > gamma-SATP > UTP >> beta-SADP > 2 MeSATP > beta,gamma-MeATP), along with studies using selective antagonists and intracellular calcium measurements are consistent with a P2Y receptor subtype. The ATP-induced increase in Isc was related to sodium transport. This modulation might be of importance in stress conditions such as inflammation.

Regulation of epithelial Na+ permeability by protein kinase C is tissue specific.

Protein kinase C (PKC) is a major regulator of a broad range of cellular functions. Activation of PKC has been reported to stimulate Na+ transport across frog skin epithelium by increasing the apical Na+ permeability. This positive natriferic response has not been observed with other epithelial preparations, and could reflect the specific experimental conditions of different laboratories, or species or organ specificity of the response to PKC. In the present study, measurements were conducted with skins and urinary bladders from the same animals of two different species. The PKC activator TPA uniformly increased the transepithelial Na+ transport (measured as amiloride-sensitive short-circuit current, ISC, across skins from Rana temporaria and Bufo marinus, and inhibited ISC across bladders from the same animals. Inhibitors of PKC (staurosporine, H-7 and chelerythrine) partially blocked the TPA-induced stimulation of ISC across frog skin. The specificity of the PKC response by amphibian skin could have reflected an induction of moulting, similar to that observed with aldosterone. However, light micrographs of paired areas of frog skin revealed no evidence of the putative moulting. Separation of stratum corneum from the underlying stratum granulosum could be detected following application of aldosterone. We conclude that the effect of PKC on epithelial Na+ channels is organ, and not species specific. The stimulation of Na+ permeability in amphibian skin does not arise from sloughing of the stratum corneum. These observations are consistent with the hypothesis that the natriferic action arises from the calcium-independent isozyme of PKC previously detected in frog skin.

Regulation of electrogenic Na+ transport across leech skin.

The dorsal integument of the medical leech Hirudo medicinalis exhibits a marked amiloride-sensitive Na+ absorption. With 20 mM Na+ in the apical solution, the transepithelial short-circuit current (Isc) was approximately 40% higher than with 115 mM Na+, whereas the transepithelial potential (VT) with 20 mM Na+ was -35.7 +/- 4.5 and -20.6 +/- 2.6 mV with 115 mM Na+. Amiloride (100 microM) inhibition at 20 mM apical Na+ was also significantly larger than with 115 mM Na+ in the solution. Benzamil (100 microM) showed additional inhibition after amiloride. Large transient overshooting currents occurred only when 115 mM Na+ was added after some minutes of Na(+)-free apical solution. Addition of adenosine 3',5'-cyclic monophosphate (cAMP) to the serosal side in the presence of 115 mM apical Na+ nearly doubled Isc. This cAMP effect was reduced to only 20% in the presence of 20 mM Na+. Guanosine 3',5'-cyclic monophosphate (cGMP) slightly increased Isc, whereas ATP showed biphasic potency. Removal of calcium from the apical side resulted in a large stimulation of amiloride-sensitive Isc only in the presence of 115 mM Na+. When currents were activated with cAMP, a deprivation of Ca2+ modestly reduced the amiloride-sensitive Isc. The Na+ channel of leech integument was found highly selective for Na+ over other monovalent cations. The permeability ratio for Na+ over K+ was approximately 30:1; the selectivity relationship for the investigated cations was Na+ > Li+ > NH4+ > K+ approximately Cs+ approximately Rb+.

Distinct regulation of Na+ reabsorption and Cl- secretion by arginine vasopressin in the amphibian cell line A6.

The neurohypophysial peptide arginine vasopressin (AVP) increases Na+ absorption across A6 epithelia. In addition to the positive natriferic response, AVP increases net basolateral to apical Cl- flux. The time course of activation of electrogenic ion transport in A6 epithelia was examined by measuring transepithelial short-circuit current (ISC). Basolateral application of AVP (0.1 U/ml) or forskolin (10 microM) affects ISC in a biphasic manner. Shortly after addition of AVP, an early (transient) phase is observed in which ISC is rapidly stimulated, reaching a peak value at 1.4 +/- 0.1 min. A subsequent decrease in current is interrupted by a slower, late phase in which ISC reaches a peak 23 +/- 3 min after addition of AVP. The late increase in ISC is sustained over the remainder of the 40-min period of observation. The time course of ISC stimulation by forskolin is qualitatively similar. Replacement of external Cl- by aspartate lowers baseline transport nearly 40%, strongly blunts the early phase of ISC stimulation, and retains the late increase. Addition of amiloride (10 microM) to the apical bath before AVP or forskolin stimulation of ISC eliminates the late increase of ISC. Steady-state amiloride-insensitive ISC activated under these conditions was sensitive to apical application of the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (20 microM) and niflumic acid (100 microM). 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (1 mM) was not an effective inhibitor of this current. Basolateral bumetanide (100 microM) inhibited baseline ISC and reduced both the peak transient and steady-state amiloride-insensitive ISC.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterisation of volume-activated ion transport across epithelial monolayers of human intestinal T84 cells.

The effects of hypo-osmolarity upon transepithelial ion transport in human intestinal cell layers have been investigated. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a transient stimulation of inward short-circuit current (Isc). This transient stimulation of inward current by hypo-osmotic media was abolished by 100 mumol/l 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). After prestimulation of inward Isc by vasoactive intestinal peptide (VIP) or by combinations of carbachol and prostaglandin E1, hypo-osmotic exposure of the basal-lateral surfaces resulted in a further transient stimulation of Isc. The stimulation of Isc in these conditions was largely insensitive to DIDS inhibition. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a stimulation of loop-diuretic-insensitive 86Rb efflux across the basal-lateral surfaces. In addition, hypo-osmotic exposure of T84 cells is also associated with an increase in cytosolic Ca2+. It is concluded that the effects of hypo-osmotic exposure of T84 cells on secretory Isc are consistent with the activation of a DIDS-sensitive apical Cl- conductance and a basal-lateral K+ conductance. With prior activation of inward Isc by VIP via a cAMP-activated DIDS-insensitive apical Cl- conductance, augmentation of the secretory current by hypo-osmotic exposure is likely to result primarily from increased basal-lateral K+ current and loop-diuretic-sensitive Cl- uptake.

Variation in the electrical properties of cultured human proximal tubule cells

Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD), short-circuit current (Isc), and transepithelial specific resistance (RT). These electrical parameters were used to determine the degree of heterogeneity among independent isolates of human proximal tubule cell cultures. Seventeen independent isolates of cells were assessed, totaling 260 individual determinations of spontaneous electrical properties. On average, these cell monolayers displayed an apical-negative PD of 1.5 +/- 0.1 mV, an Isc of 2.7 +/- 0.2 microA/cm2, and an RT of 480 +/- 19 ohms x cm2. Each independent cell isolate, however, displayed electrical values within a narrow range, in some cases allowing isolates to be distinguished from one another. The individual isolates were also assessed for Na-coupled glucose transport, Na+,K(+)-ATPase activity, cAMP stimulation by parathyroid hormone (PTH), forskolin stimulation of Isc, and ouabain inhibition. With the exception of a strong correlation between Na+,K(+)-ATPase activity and Isc, these parameters, in contrast to electrical properties, were found to be consistent and did not reveal distinctions among the isolates. HPT cell cultures seem to consistently retain important features of proximal tubule differentiation while maintaining the variability, as demonstrated by electrical properties, that might be expected of cells isolated from a random population.

Low-affinity mixed acetylcholine-responsive receptors at the apical membrane of frog tadpole skin

The larval frog skin has a very high electrical resistance and a corresponding low rate of transepithelial ion transport. Amiloride, a blocker of sodium transport in adult skin, transiently stimulates rather than inhibits short-circuit current (Isc) across larval skin through nonselective cation channels. Acetylcholine (ACh) stimulates Isc like amiloride, although the response is more prolonged. Pretreatment with ACh markedly suppressed amiloride stimulation of Isc; amiloride pretreatment also suppressed ACh stimulation. Half-maximal stimulation of Isc by ACh occurred at 347 microM. Stimulation by ACh was inhibited by both d-tubocurarine [dissociation constant (Kd) = 57 microM] and atropine (Kd = 49 microM). The specific nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium and the specific muscarinic agonist oxotremorine-M both stimulated Isc and were blocked by either atropine or d-tubocurarine. Reciprocal desensitization and blocker cross-reactivity suggest that ACh activates the same population of receptors as amiloride. This ACh-responsive receptor has characteristics of both nicotinic and muscarinic receptors found in other tissues.

Requirement of HCO3- for Cl(-)-absorption in seawater-adapted eel intestine.

The role of HCO3-/CO2 buffer in Cl- absorption was examined in the in vitro perfused eel intestine adapted to seawater. Cl- absorption, expressed as short-circuit current (Isc), was measured in either 20 mM HCO3-/1% CO2 Ringer or HEPES Ringer, pH 8.0. Unilateral (mucosal or serosal) substitution of HCO3-/CO2 with HEPES/O2 was without effect on Isc and transepithelial voltage (Vt), whereas bilateral removal of HCO3-/CO2 reduced Isc and Vt by 50%, indicating that the presence of HCO3-/CO2 buffer at one side of the epithelium is sufficient to keep Cl- absorption at the maximum rate. We examined in further detail the individual components of the HCO3-/CO2 system that stimulates Cl- absorption. We found that, in tissues bathed with HEPES Ringer, addition of 1% CO2 to the luminal or serosal solution (final pH = 7.6 in the chamber) had no effect on Isc and Vt, while both electrical parameters could be restored to control values by unilateral (luminal or serosal) substitution of HEPES Ringer with 20 mM HCO3-/1% CO2 Ringer or 20 mM HCO3- alone. Stimulation of Isc induced by unilateral (luminal or serosal) HCO3-/CO2 was inhibited by luminal or serosal 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) (0.25 mM) or by serosal Na+ removal, whereas amiloride (1 mM), luminal or serosal, had no effect. Acetazolamide (0.1 mM, both sides) inhibited stimulation of Isc induced by luminal addition of HCO3-/CO2, whereas it was without effect when HCO3-/CO2 was added serosally or bilaterally.(ABSTRACT TRUNCATED AT 250 WORDS)

Purinergic receptor activation of Cl- secretion in T84 cells.

The regulation by ATP of Cl- secretion in T84 cells grown on filters was investigated by measuring short-circuit current (Isc = net Cl- secretion). ATP (greater than or equal to 10 microM) added to the basolateral side markedly stimulated Isc both in the presence and absence of forskolin-activated Isc. Fluorescence microscopy of cells loaded with the Ca2+ indicator fura-2 showed that ATP stimulated a transient increase in intracellular free Ca2+ concentration [Ca2+]i. The augmentation of forskolin-stimulated Isc by ATP was at least partly caused by mobilization of Ca2+ from an internal store because prior depletion of the store using ionomycin prevented the response. The activity sequence for stimulation of Isc in the presence of forskolin was adenosine 5'-O-(3-thiotriphosphate) = 5'-adenylylimidodiphosphate (AMP-PNP) greater than ATP greater than ADP greater than AMP, suggesting the presence of a P2 purinergic receptor. Neither beta, gamma-methyleneadenosine 5'-triphosphate nor alpha, beta-methyleneadenosine 5'-triphosphate increased the Isc. Stimulation of Isc by ATP in the absence of forskolin was at least partly due to the breakdown of ATP to AMP and adenosine, which act at P1 receptors to stimulate Isc, since 1) inhibition of the ecto-phosphohydrolase 5'-nucleotidase by alpha, beta-methylene-ADP partially inhibited stimulation of Isc by ATP, 2) the adenosine receptor antagonists caffeine and 8-phenyltheophylline markedly inhibited the ATP-stimulated Isc, and 3) AMP-PNP, a weakly hydrolyzable analogue of ATP, caused a much smaller increase in Isc compared with ATP. Adenosine had no effect on [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Ion transport by cultures of human tracheobronchial submucosal glands

Acini of human tracheobronchial submucosal glands were isolated by enzymatic disaggregation, and, when plated on flasks coated with human placental collagen (HPC) in media containing Ultroser G serum substitute (USG) and a variety of growth factors (GF), they became confluent after 14-20 days. The cells were then isolated by trypsinization and replated in media containing USG and GF at 10(6) cells/cm2 on porous-bottomed inserts coated with HPC. Confluent monolayers formed on day 1 after replating and were studied on day 10. Transepithelial resistance and short-circuit current (Isc) were 578 +/- 89 omega.cm2 and 12.9 +/- 1.9 microA/cm2 (means +/- SE, n = 23 cell sheets). The potency sequence for stimulation of Isc by mediators was methacholine greater than bradykinin greater than isoproterenol approximately or equal to phenylephrine. Amiloride decreased baseline Isc by 42 +/- 9% (n = 6 cell sheets) but had little effect on the Isc response to mediators. Diphenylamine-2-carboxylic acid, however, had no effect on baseline Isc but markedly inhibited the Isc response to all mediators. These results show that submucosal gland cells from human trachea can be grown in culture to produce epithelial sheets of high resistance, which secrete Cl in response to bradykinin and alpha- and beta-adrenergic and cholinergic agents.

Avian cecum: role of glucose and volatile fatty acids in transepithelial ion transport

In the fowl cecum in vitro, the influence of glucose and the three most prevalent naturally occurring volatile fatty acids (acetate, propionate, butyrate) on short-circuit current (Isc), electrical resistance, and transport of Na and Cl was determined. When glucose, acetate, or butyrate was present, ion transport was characterized by electrogenic Na absorption, greater than 65% of which was amiloride inhibitable, and Cl secretion, which also was electrogenic. Isc could be completely accounted for by net fluxes of Na and Cl. When glucose, acetate, or butyrate (10 mM both sides) was included in the incubation medium, cecal tissue maintained its Isc and a constant rate of net Na absorption and Cl secretion for a 5-h period. When no substrate was present or propionate was included in the medium, a marked fall in Isc and net Na and Cl fluxes was seen. Glucose caused an increase in Isc when added only to the serosal side. As 3-O-methylglucose (not metabolized) was not effective in stimulating Isc of the cecum (serosal or mucosal addition), it appeared that glucose increased Isc by acting as an energy substrate for active Na transport. Acetate and butyrate appeared to be equally effective in stimulating Na transport and Isc when placed on either side of the membrane. When the preparation was supplied with glucose (serosal side) and acetate was added to the mucosal side, no further stimulation of Isc occurred. Thus it appeared that acetate and butyrate were acting as substrates for active Na transport rather than stimulating Na transport by some other mechanism such as a cotransport with Na.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemotactic peptide effects on intestinal electrolyte transport

The bacterial-derived chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) increases short-circuit current (Isc) and arachidonic acid metabolism (AAM) in rabbit ileum and distal colon. Serosal (s) or mucosal (m) addition of fMLP transiently increases Isc. Half-maximally effective dose and maximal increases in Isc were 32 nM and 84 microA/cm2 in ileum and 234 nM and 80 microA/cm2 in colon, respectively. Piroxicam, a cyclooxygenase inhibitor, diminished the Isc response by 97% in colon and 69% in ileum. Changes in Isc were dependent on Cl- and HCO3- in the bathing media. In ileum, fMLP inhibited m-to-s 36Cl- fluxes and stimulated s-to-m 36Cl- fluxes. These changes in Cl- flux were also inhibited by piroxicam. fMLP stimulated prostaglandin E2 (PGE2) release in intact tissue and in isolated subepithelial components. Increased tissue adenosine 3',5'-cyclic monophosphate levels were detected in intact tissue but not in isolated components. Previous desensitization of ileum to PGE1 inhibited fMLP stimulation of Isc in ileum by 88%. Desensitization to fMLP in ileum failed to alter the effect of PGE1 (10 microM) on Isc. In isolated microsomal membranes of ileal enterocytes, fMLP binding sites could not be demonstrated, suggesting that fMLPs action was initially mediated via stimulation of nonepithelial cell cyclooxygenase activity. The above results indicate that fMLP stimulates net secretion in both ileum and colon probably by the activation of AAM.

Ca2+ entry through the apical membrane reduces antidiuretic hormone-induced hydroosmotic response in toad urinary bladder

The role of Ca2+ in the regulation of antidiuretic hormone(ADH)-induced water permeability of the apical membrane of the toad urinary bladder was examined. The effects of modifying Ca2+ entry through the apical membrane of toad urinary bladders on the hydroosmotic water flow (phi H2O) and short circuit current (Isc) were measured. In most experiments the bladders were treated with small amounts of Ag+ (10(-7) mol/l) on the apical side. This treatment was used because previous experiments indicate that it markedly increases alkali-earth cation fluxes through an amiloride-insensitive cation channel in the apical membrane of the urinary bladder. Moreover, when Ca2+ is the major cation in the apical solution of these Ag(+)-treated bladders, Isc is mostly due to Ca2+ entry through the apical membrane. Ag+ increased Isc and simultaneously inhibited phi H2O in bladders perfused with Ca2+ solutions on the apical side. Addition of La3+ to the apical solution reversed the stimulation of Isc and the inhibition of phi H2O produced by Ag+. When bladders were perfused with Ca2(+)-free solutions on the apical side, addition of Ag+ did not inhibit phi H2O while the stimulation of cation movements through the amiloride-insensitive cation channel persisted. In bladders perfused with apical Ca2+ solutions and treated with chlorophenyl thio-cyclic adenosine monophosphate (ClPheS-cAMP) the addition of Ag+ did not inhibit phi H2O while it still increased Isc. Finally, addition of Ca2+ to the apical solution of bladders not treated with Ag+ reduced phi H2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Forskolin-induced HCO3- current across apical membrane of the frog corneal epithelium

Forskolin (and other Cl- secretagogues) does not affect the very small Na(+)-originated short-circuit current (Isc) across frog corneal epithelium bathed in Cl- free solutions. However, forskolin in combination with increased PCO2 bubbling of the solutions (5-20% CO2) stimulated Isc proportionally to PCO2 to a maximum of approximately 8 microA/cm2. This current could be eliminated and reinstated by sequentially changing the gas composition of the bubbling to 100% air and 20% CO2-80% air. The same effects were observed when PCO2 changes were limited to the apical-side solution. Stroma-to-tear HCO3- movement was deemed unlikely, since the increase in Isc was observed with a HCO3(-)-free solution on the stromal side and CO2 gassing limited to the tear side. From the effects of ouabain and tryptamine, at least 80% of the Isc across the basolateral membrane can be accounted for by the Na+ pump current plus K+ movement from cell to bath. Methazolamide also inhibited Isc. Current across the apical membrane cannot be attributed to an electronegative Na(+)-HCO3- symport given the insensitivity of Isc to a disulfonic stilbene and the fact that stroma-to-tear Na+ fluxes did not increase on stimulation of Isc. The tear-to-stroma Na+ flux also remained unaltered, negating an increased apical bath-to-cell Na+ flow. The forskolin-20% CO2 manipulation produced a depolarization of the intracellular potential, a reduction in the apical-to-basolateral resistance ratio, and a decrease in transepithelial resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of calcium in mediating action of carbachol in T84 cells

To examine the role of calcium in mediating carbachol's action in secretory epithelia, we simultaneously measured intracellular free [Ca] [( Ca]i) and transepithelial chloride transport in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value [( Ca]'i) normalized to control. Chloride transport was measured as the short-circuit current (Isc) with a voltage clamp. Monolayers were pretreated with cyclic AMP to augment the response of Isc to carbachol, a procedure that did not qualitatively change the response of the monolayer to carbachol. The carbachol-induced changes in Isc appeared to be dependent on the increase in [Ca]i. First, carbachol caused both Isc and [Ca]'i to increase in parallel. Isc increased from 32 +/- 5 to 70 +/- 9 microA and then declined to 57 +/- 16 microA while [Ca]'i increased from 72 +/- 14 to 156 +/- 22 nM and then declined to 133 +/- 45 nM. Second, the carbachol-induced increases in Isc and [Ca]'i were correlated. The greater the hormone-stimulated rise in [Ca]'i, the higher the increase in Isc. Third, carbachol's stimulation of Isc was blunted by preventing the calcium spike with the cellular calcium buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Although the carbachol-induced increase in [Ca]'i appeared necessary for the increase in Isc, it was not clear if carbachol's action was solely the result of an increase in [Ca]'i. Increasing [Ca]'i with ionomycin, although causing Isc and [Ca]'i to increase in parallel, failed to increase Isc to the levels observed with carbachol. These experiments suggest that although the carbachol-induced increase in Isc is dependent on the increase in [Ca]i, the hormone may activate a second process that increases the sensitivity of the calcium-activated transport process to changes in [Ca]i.

Coupling of volume and Na+ transport in frog skin epithelium

Whole skins and isolated epithelia were bathed with isotonic media (- 244 mOsm) containing sucrose or glucose. The serosal osmolality was intermittently reduced (to - 137 mOsm) by removing the nonelectrolyte. Transepithelial and intracellular electrophysiological parameters were monitored while serosal osmolality was changed. Serosal hypotonicity increased the short-circuit current (Isc) and the basolateral conductance, hyperpolarized the apical membrane (Φmc), and increased the intracellular Na+ concentration. The increases in apical conductance and apical Na+ permeability (measured from Goldman fits of the relationship between amiloride-sensitive current and Φmc) were not statistically significant. To verify that the osmotically induced changes in Isc were mediated primarily at the basolateral membrane, the basolateral membrane potential of the experimental area was clamped close to 0 mV by replacing the serosal Na+ with K+ in Cl−-free media. The adjoining control area was exposed to serosal Na+. Serosal hypotonicity produced a sustained stimulation of Isc across the control, but not across the adjoining depolarized tissue area. The current results support the concept that hypotonic cell swelling increases Na+ transport across frog skin epithelium by increasing the basolateral K+ permeability, hyperpolarizing the apical membrane, and increasing the electrical driving force for apical Na+ entry.

Alpha 2-receptors mediate catecholamine-stimulated acid secretion in Amphiuma jejunum

The receptors mediating adrenergic stimulation of acid secretion by Amphiuma jejunum were characterized in this study using alpha- and beta-adrenergic agonists and antagonists. Isolated segments of jejunum were mounted in Ussing chambers and bathed in Cl- -free (SO4(2-] medium. Shortcircuit current (Isc) and acid secretion (JH) were recorded, the latter by measuring the rate of alkalinization of the serosal medium. The beta-adrenergic receptor antagonist, propranolol (10(-4) M), had no effect on the Isc and JH stimulated by norepinephrine (NE). The alpha 2-adrenergic agonists, clonidine and UK-14,304, mimicked the effect of NE, with effective concentrations providing 50% maximal delta Isc of 2.0 X 10(-7) and 9.0 X 10(-8) M, respectively. NE added subsequently produced no greater stimulation. In contrast, the alpha 1-adrenergic agonists, phenylephrine and methoxamine, produced little stimulation of JH and Isc; NE added subsequently stimulated the Isc. The alpha 1-adrenergic antagonist prazosin had no effect on the NE-induced Isc or JH, whereas the alpha 2-adrenergic antagonist yohimbine inhibited the NE-stimulated Isc with a half-maximal effective concentration of 3.5 X 10(-7) M. Yohimbine (10(-4) M) reduced the NE-stimulated Isc by 88%, whereas the spontaneous Isc was reduced by only 12%. These results demonstrate that alpha 2-adrenergic receptors on the basolateral membrane of Amphiuma enterocytes mediate NE-enhanced, but not spontaneous, intestinal acid secretion.

Electrogenic sodium absorption in rabbit cecum in vitro.

Regional variations in the ion transport properties of the colon may have significant physiological and pathophysiological implications. However, only limited studies have been performed in cecum, which comprises 50% of the macrosurface of the rabbit colon. In vitro under short-circuit conditions, cecum actively absorbed Na and Cl (JnetNa = 5.6 +/- 0.3, JnetCl = 1.5 +/- 0.3 mu eq.cm-2.h-1) with a short-circuit current (Isc) of 6.29 +/- 0.2 mu eq.cm-2.h-1.Cl substitution with sulfate decreased both JnetNa and Isc by 1.3 mu eq/cm2.h-1.HCO3 removal decreased both JnetNa and Isc 3.3 mu eq.cm-2.h-1. This effect was due primarily to removal of serosal HCO3. There was both a linear correlation between JNanet and Isc (r = 0.845) and a concentration-dependent stimulation of Isc by increasing [Na] in the bathing media. However, 10(-4) M amiloride did not significantly alter either Isc or JnetNa. In contrast, 10(-4) M phenamil, an amiloride analogue highly specific for the Na channel, significantly blocked both Isc and JnetNa. The sulfhydryl reagent PCMBS increased Isc; this response was reversed by phenamil. Electrogenic Cl secretion was stimulated by 1 mM theophylline, 10(-4) M 8BrcAMP and 10(-4) M 8BrcGMP. None of the secretagogues inhibited JnetNa. Epinephrine (5.5 microM) increased JnetNa from 5.9 +/- 1.3 to 7.8 +/- 1.1 (P = 0.02) and JnetCl from 0.1 +/- 1.2 to 2.0 +/- 0.8 (P NS) mu eq.cm-2.h-1. Studies of pH stat demonstrated an epinephrine-stimulated increase in Jm-sHCO3 without a change in Js-mHCO3. Thus, cecum exhibits a distinct type of electrogenic Na electrogenic Na absorption which is partially dependent on the presence of Cl and HCO3, not blocked by amiloride but by phenamil. Because of its large surface area and its novel mechanism of electrogenic Na transport, the cecum exerts an important regulatory role in colonic fluid and electrolyte balance.