Potential difference responses due to K+, Na+ and Cl- changes in bullfrog antrum with and without HCO3-

The effect of changing [K+], [Na+] and [Cl-] in nutrient solution was studied in bullfrog antrum with and without HCO3- in nutrient. In 25 mM HCO3- (95% O2/5% CO2) and in zero HCO3- (100% O2), nutrient pH was maintained at 7.3. Changing from 4 to 40 mM K+ or from 81 to 8.1 mM Cl- gave a decrease 10 min later in transmucosal PD (nutrient became more negative)--a normal response. These responses were less in zero than in 25 mM HCO3-. A decrease from 102 to 8 mM Na+ decreased PD (anomalous response of electrogenic NaCl symport). This effect was attenuated or eliminated in zero HCO3-. In contrast, change from 4 to 40 mM K+ gave initial anomalous PD response and change from 102 to 8 mM Na+, initial normal PD response with either zero or 25 mM HCO3-. Both responses were associated with (Na+ + K+)-ATPase pump and were greater in zero than in 25 mM HCO3-. Initial PD increases in zero HCO3- are explained as due to increase in the resistance of passive conductance and/or NaCl symport pathways. Thus, removal of HCO3- modifies conductance pathways of nutrient membrane.     

Subcellular localization of prostaglandin E2 binding sites in bovine adrenal medulla

Effect of changing [K+], [Na+] and [Cl-] in nutrient solution on potential difference (PD) and resistance was studied in bullfrog antrum with and without nutrient HCO3(-) but with 95% O2/5% CO2 in both cases. In both cases, changing from 4 to 40 mM K+ gave about the same initial PD maximum (anomalous response) which was followed by a decrease below control level. Latter effect was much less with zero than with 25 mM HCO3(-). Changing from 102 to 8 mM Na+ gave initial normal PD response about the same in both cases. However, 10 min later the change in PD with zero HCO3(-) was insignificant but with 25 mM HCO3(-) the PD decreased (anomalous response of electrogenic NaCl symport). PD maxima due to K+ and Na+ were largely related to (Na+ + K+)-ATPase pump. Changes in nutrient Cl- from 81 to 8.1 mM gave only a decrease in PD (normal response). Initial PD increases are explained by relative increases in resistance of simple conductance pathways and of parallel pathways of (Na+ + K+)-ATPase pump and Na+/Cl- symport. Removal of HCO3(-) and concurrent reduction of pH modify resistance of these pathways.     

Potential difference responses to secretory K+, Na+ and HCO3- changes in secreting and resting states of frog stomach in Cl(-)-free media

The effects of changes in secretory concentrations of K+, Na+ and HCO3- on transmucosal potential difference (PD) and resistance in Cl(-)-free (SO4(2-)) solutions were compared for secreting fundus and resting fundus of Rana pipiens. In the resting fundus experiments, histamine was not present in the nutrient solution and cimetidine was primarily used to obtain acid inhibition. Increase of K+ from 4 to 80 mM, decrease of Na+ from 156 to 15.6 mM and decrease of HCO3- from 25 to 5 mM gave, 10 min after the change, in the secreting fundus delta PD values of 39.7, -11.9 and 3.2 mV, respectively. In the resting fundus, 1.5 to 2 h after the addition of cimetidine, the same changes in secretory ion concentration gave delta PD values of 12.2, -5.6 and 1.5 mV, respectively. Replacement of cimetidine with SCN and without histamine yielded a delta PD somewhat lower than that in cimetidine, namely 9 mV for a K+ change from 4 to 80 mM. Subsequent addition of histamine with SCN present gave a delta PD of about 21 mV. The change in PD was attributed to histamine increasing the secretory membrane area, leading to an increase in K+ conductance. Another possibility is that histamine increases the K+ conductance per se.     

Evidence for HCO3- conductance pathways in nutrient membrane of bullfrog antrum

The effect of changing the nutrient HCO3- concentration on potential difference (PD) and resistance in bullfrog antrum bathing in CI- media was determined. Changes in HCO3- concentration were from 25 mM to several lower concentrations and back to 25 mM. A plot of /delta PD/ versus log [HCO3-] gave a linear relation for changes of HCO3- concentration from 25 down to 3.1 mM and back to 25 mM but deviated to some extent for changes to 1.6 mM. In these experiments, changes from higher to lower HCO3- concentrations gave a less rapid initial PD response than those in the reverse direction. This result eliminated H+ conductance pathways as being predominant. Experiments were done in which in the first part changes were made in nutrient solution from 5 percent CO2 and 25 mM HCO3- to 0.6 percent CO2 and 3 mM HCO3- and in the second part the same changes with a simultaneous changes of secretory solution from 5 percent to 10 percent CO2. The magnitude of PD decrease was greater by 4.5 mV in the second part. This result indicated that HCO3- conductance pathways rather than OH- conductance pathways are predominated . There was no evidence of HCO3-, OH-, and H+ conductance pathways in secretory membranes.     

Anomalous potential response and (Na+ + K+)-ATPase in in vitro frog gastric mucosa

In general, increasing K+ on the nutrient side decreases the transmucosal PD (nutrient becomes more negative) but after bathing the mucosa in zero K+ media for about 30 min, or longer, elevation of K+ on the nutrient side increases the PD, an anomalous effect. In Cl- media, increasing nutrient K+ from zero to 4 mM produces an increase in PD (an anomalous response) of 3.1 and 5.3 mV in 2 and 5 min, respectively. Ouabain (10(-3) M) to the nutrient side abolished the anomalous response as did removal of Na+ (choline for Na+) from bathing media. In SO4(2-) media (SO4(2-) for Cl-), a significant anomalous PD response was observed when K+ on the nutrient side was increased from zero to 1, 2 or 3 mM but not to higher K+ concentrations. In this case, ouabain also abolished the anomalous response. It is postulated, on the basis of the effects of ouabain and the use of choline media, that an electrogenic (Na+ + K+)-ATPase pump is present on the nutrient-facing membrane in which more Na+ than K+ are transported per cycle.     

ATP-dependent and DCCD-insensitive Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions

ATP-dependent Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions was not affected by the addition of 40 mM of K+, Na+ or HCO3- to the assay medium. Na+ and K+ did not alter the uptake even in the presence of a K+ ionophore, valinomycin (10 microM), or a H+/K+ exchanger, nigericin (10 microM), whereas in the presence of both of these ionophores, K+, but not Na+, reduced the Cl- uptake. Inhibitors of proton pump activity, N,N'-dicyclohexylcarbodiimide (1 mM) and 5-(N,N-hexamethylene)amiloride (40 microM), however, did not affect the Cl- uptake. These findings suggest the presence of a primary Cl- transport system probably associated with passive H+ flux in the brain plasma membranes.     

Effect of plasma [K+] on the DC potential and on ion distributions between CSF and blood.

Keeping the arterial pH at 7.4 and PaCO2 at 40 mmHg in eight anesthetized dogs, we acutely raised plasma potassium concentration from 3.4 to 8.2 meq/1, then allowed it to decay back to control levels. The cerebrospinal fluid (CSF)-blood electrical potential difference (pd) increased 13.2 mV per 10-fold increase in plasma [K+]. Again keeping arterial pH at 7.4 and PaCO2 at 40 mmHg, we elevated plasma [K+] in four dogs from 3.3 to 8.0 meq/1 and maintained this level for 6 h. We found 1) that the PD increased from a control value of +1.3 to +8.9mV, showing no tendency to decay over the 6 h; and 2) that the change in PD did not affect the distribution of Na+, K+, H+, Cl-, or HCO3- between blood and CSF over the 6 h. These results suggest that under these conditions the PD between CSF and blood may play no effective role in determining the distributions of these charged species by 6 h. These results are contrasted with recent findings which suggest that H+ and HCO3- are distributed according to passive forces between CSF and blood.     

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons II. Base influx

We used microelectrodes to determine whether the K/HCO3 cotransporter tentatively identified in the accompanying paper (Hogan, E. M., M. A. Cohen, and W. F. Boron. 1995. Journal of General Physiology. 106:821- 844) can mediate an increase in the intracellular pH (pHi) of squid giant axons. An 80-min period of internal dialysis increased pHi to 7.7, 8.0, or 8.3; the dialysis fluid was free of K+, Na+, and Cl-. Our standard artificial seawater (ASW), which also lacked Na+, K+, and Cl-, had a pH of 8.0. Halting dialysis unmasked a slow pHi decrease. Subsequently introducing an ASW containing 437 mM K+ and 0.5% CO2/12 mM HCO3- had two effects: (a) it caused membrane potential (Vm) to become very positive, and (b) it caused a rapid pHi decrease, because of CO2 influx, followed by a slower plateau-phase pHi increase, presumably because of inward cotransport of K+ and HCO3- ("base influx"). Only extracellular Rb+ substituted for K+ in producing the plateau-phase pHi increase in the presence of CO2/HCO3-. Mean fluxes with Na+, Li+, and Cs+ were not significantly different from zero, even though Vm shifts were comparable for all monovalent cations tested. Thus, unless K+ or Rb+ (but not Na+, Li+, or Cs+) somehow activates a conductive pathway for H+, HCO3-, or both, it is unlikely that passive transport of H+, HCO3-, or both makes the major contribution to the pHi increase in the presence of K+ (or Rb+) and CO2/HCO3-. Because exposing axons to an ASW containing 437 mM K+, but no CO2/HCO3-, produced at most a slow pHi increase, K-H exchange could not make a major contribution to base influx. Introducing an ASW containing CO2/HCO3-, but no K+ also failed to elicit base influx. Because we observed base influx when the ASW and DF were free of Na+ and Cl-, and because the disulfonic stilbene derivatives SITS and DIDS failed to block base influx, Na(+)-dependent Cl-HCO3 exchange also cannot account for the results. Rather, we suggest that the most straightforward explanation for the pHi increase we observed in the simultaneous presence of K+ and CO2/HCO3- is the coupled uptake of K+ and HCO3-.     

HCO3-secretion by bullfrog duodenum: dependence on nutrient Na+ during secretory stimulation

HCO3(-) secretion across in vitro duodenal mucosa of Rana catesbeiana was investigated under baseline conditions and during secretory stimulation. Baseline secretion was abolished by removal of CO2-HCO3(-)and reduced approximately 60% by removal of nutrient Na+, but was not sensitive to changes in Cl- or K+. Baseline secretion was not directly altered by exposure to 10(-3) M amiloride or 10(-3) M H2DIDS (dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the nutrient solution and only mildly reduced by acetazolamide. Following removal and restoration of Na+, recovery of secretion was impaired by exposure to acetazolamide (5 x 10(-4) M) or H2DIDS (5 x 10(-4) M) in the nutrient solution. Secretion stimulated by glucagon (10(-6) M) or 16,16-dimethyl prostaglandin E2 (10 microg.mL(-1)) was markedly attenuated by removal of Na+ or by exposure to H2DIDS, but secretion was not altered by acetazolamide (5 x 10(-4) M) or nutrient amiloride (1 mM). Thus, the HCO3(-) that is secreted under nonstimulated conditions derives partly from basolateral Na(+)-dependent uptake and partly from cellular CO2 hydration. Secretagogue-stimulated secretion by duodenal surface epithelium depends on stilbene-sensitive Na+(HCO3(-))n uptake across the basolateral membrane.     

Effects of barium and bicarbonate on glial cells of Necturus optic nerve. Studies with microelectrodes and voltage-sensitive dyes

We have studied the effects of Ba++, a known K+ channel blocker, on the electrophysiological properties of the glial cells of Necturus optic nerve. The addition of Ba++ reversibly depolarized glial cells by 25-50 mV; the half maximal deplorization was obtained with a Ba++ concentration of approximately 0.3 mM. In the presence of Ba++, the sensitivity of the membrane to changes in K+ was reduced and there was evidence of competition between K+ and Ba++ for the K+ channel. These effects, which were accompanied by a large increase in the input resistance of the glial cells, indicate that Ba++ blocks the K+ conductance in glial cells of Necturus optic nerve. With the K+ conductance reduced, we were able to investigate the presence of other membrane conductances. We found that in the presence of Ba++, the addition of HCO3- caused a Na+-dependent hyperpolarization that was sensitive to the disulfonic stilbene SITS (4-acetamido-4'-isothiocyanostilbene-2, 2'-disulfonic acid). Removal of Na+ resulted in a HCO3- -dependent, SITS-sensitive depolarization. These results are consistent with the presence in the glial membrane of an electrogenic Na+/HCO3- cotransporter in which Na+, HCO3-, and net negative charge are transported in the same direction. In Cl- -free solutions, the Ba++-induced depolarization increased, suggesting a small permeability to Cl-. Using voltage-sensitive dyes and a photodiode array for multiple site optical recording, the distribution of potential changes in response to square pulses of intracellularly injected current were recorded before and after the addition of increased and the decay of amplitude as a function of distance decreased. Such results indicate that Ba++ increases the membrane resistance more than the resistance of the intercellular junctions.     

The effects of various anions and cations on the regulation of pyruvate dehydrogenase complex activity from pig kidney cortex.

The activity of pyruvate dehydrogenase complex (PDC) purified from pig kidney cortex was found to be affected by various uni- and bi-valent ions. At a constant strength of 0.13 M at pH 7.8, K+, Na+, Cl-, HCO3- and HPO4(2-) had significant effects on the activity of PDC: Na+, K+ and HPO4(2-) stimulated, but HCO3- and Cl- inhibited. The stimulatory effect of Na+ was mediated by a change in the Vmax. of PDC only, whereas K+ produced an increase in Vmax. and a change in the Hill coefficient (h). The extent of stimulation produced by HPO4(2-)4 on the activity of PDC was dependent on the concentrations of K+ and Na+. Both cations at concentrations higher than 40 mM partially prevented the effect of HPO4(2-)4. Cl- and HCO3- anions decreased the Vmax. of the enzyme and increased the S0.5 for pyruvate. The effects of Na+, K+, Cl-, HPO4(2-) and HCO3- on the activity of PDC were additive. In the presence of 80 mM-K+, 20 mM-Na+, 10 mM-HPO4(2-), 20 mM-Cl- and 20 mM-HCO3- the activity of PDC was increased by 30%, the S0.5 for pyruvate was increased from 75 to 158 microM and h was decreased from 1.3 to 1.1. Under these conditions and at 1.0 mM-pyruvate, the activity of PDC was 80% of the maximal activity achieved in the presence of these ions and 4.5 mM-pyruvate. The present study suggests that PDC may operate under non-saturating concentrations for substrate in vivo.     

Normal and anomalous potential responses due to K+ changes in bullfrog antrum

The effect of changing the K+ concentration in the bathing media was studied in the bullfrog antrum. Usually increasing K+ on the nutrient side in standard Cl- -containing and Cl- -free solutions decreased the transmucosal potential difference (nutrient became more negative) - a normal effect. Similar results were obtained on the secretory side. Moreover, for K+ changes on the nutrient side in Cl- media, a plot of magnitude of delta V vs. log [K+] was linear for [K+] greater than 20 mM with slope of 27 mV per 10-fold change in [K+]. However, after bathing the mucosa in Cl- media with zero K+ for about 20 min, elevating the nutrient [K+] to 4 mM increased the potential difference (V) by 4.8 mV in 5 min and repeating the same sequence increased V by 6.9 mV in 5 min - both anomalous effects. Beyond 20 mM K+ the response was normal. In SO2-4 media, an anomalous potential difference of about 1 mV was obtained for changes from 0 to 3 or 6 mM nutrient K+. Ouabain (1 X 10(-3) M) in the nutrient solution abolished the anomalous response in Cl- and SO2-4 media. The normal response is attributed to passive, conductance pathways and the anomalous response because of the effect of ouabain, to a (Na+ + K+)-ATPase pump on the nutrient-facing membrane in which more Na+ than K+ ions are transported per cycle.     

Bicarbonate transport mechanisms in the Ambystoma kidney proximal tubule: transepithelial potential measurements

Modes of bicarbonate entry from tubule lumen to cell were examined in isolated Ambystoma proximal tubules, using determinations of transepithelial potential differences (V3). (1) Upon removal of luminal substrate, tubules first equilibrated in bilateral (lumen and bath) 94.72 mM Cl- and 10 mM HCO3- yielded a change in V3 between the experimental and control circumstances of +1.8 mV (delta V3). (2) The identical experiment conducted under the condition of symmetrical 4.72 mM Cl- produced a delta V3 of +7.6 mV. This reduction of luminal and bath Cl- generates an amplification of delta V3 by a factor of 4.4 and reflects a substantial increase in the paracellular Cl- shunt resistance. Ensuing experiments were conducted in bilateral nominally Cl(-)-free solutions and in the absence of luminal substrate. The experimental protocols are divided into several situations where HCO3- is removed from the lumen, bath, or lumen and bath; the HCO3- removal sequences are repeated in the presence of luminal SITS and then after SITS washout. 0.5 mM SITS (4-acetoamido-4-isothiocyanostilbene-2,2'-disulfonate) was applied exclusively to the luminal perfusate. (1) Removal of luminal HCO3- in the absence of SITS produces a delta V3 of -1.9 mV, whereas, in the presence of SITS, the delta V3 measures -1.3 mV. Subsequent removal of luminal HCO3- in the presence of bath HCO3- (in the presence of luminal SITS) yields a delta V3 of -1.0 mV. All of these measurements reflect a decrease in HCO3- current across the basolateral membrane Na+ (HCO3-)n co-transporter; the role of a possible Cl-/Anion- antiport cannot be assessed. (2) Removal of bath HCO3- in the absence of SITS yields a delta V3 of +1.5 mV, whereas, in the presence of SITS, the delta V3 value measures +1.2 mV. Subsequent removal of bath HCO3- in the absence of luminal HCO3- (in the presence of SITS) yields a delta V3 of +0.8 mV. These experiments are consistent with an increase in HCO3- current across the basolateral Na+(HCO3-)n co-transporter, do not rule out the possibility of an apical HCO3- conductance pathway, and diminish the likelihood of an apical Cl-/HCO3- antiport system.     

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux

We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter.     

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.     

Ion activities in the lateral intercellular spaces of gallbladder epithelium transporting at low external osmolarities

The ion activities in the lateral spaces of the unilateral preparation of the gallbladder of Rana catesbiana were measured by double-barrelled ion-selective microelectrodes. The bladders were bathed in a saline solution with a low osmolarity (62 mOsm) containing, in mM: 27 Na+, 27 Cl-, 2 K+, 1 Ca++, 4 HCO3-. Working at reduced osmolarities had the advantage of an increased volume transport and of widened intercellular spaces. The reference barrel recorded an electrical potential of +2.7 mV in the spaces; they contained a solution similar to the external solution. The electrodes recorded a Na+ concentration of 27 mM, a K+ concentration of 1.7 mM, a Ca++ concentration of 0.69 mM and a Cl- concentration of 28.5 mM. In the spaces there was a lower resistance between the tip of the electrode and the serosal bath than that recorded with the tip in the lumen, and injection of fluorescent dye (11 A diameter) via the electrodes did not stain the cells. The concentrations in the secretion were similar to those in the spaces. The intracellular compartment had an apparent K+ concentration of 95 mM, and the concentrations of Na+ and Cl- were both about 5 mM. These data indicate that when the gallbladder is bathed with hypotonic solutions and is transporting fluid at approximately three or four times the normal rate, there are no significant osmotic gradients between the lumen and the lateral spaces. It is suggested that transcellular transport of water is implemented by a combination of high osmotic permeabilities across both mucosal and serosal cell membranes and low reflection coefficients (for K+ salts) at the serosal cell membranes.     

The regulation of intracellular pH in monkey kidney epithelial cells (BSC-1). Roles of Na+/H+ antiport, Na+-HCO3(-)-(NaCO3-) symport, and Cl-/HCO3- exchange

Using the pH-sensitive absorbance of 5 (and 6)-carboxy-4',5'- dimethylfluorescein, we investigated the regulation of cytoplasmic pH (pHi) in monkey kidney epithelial cells (BSC-1). In the absence of HCO3-, pHi is 7.15 +/- 0.1, which is not significantly different from pHi in 28 mM HCO3-, 5% CO2 (7.21 +/- 0.07). After an acid load, the cells regulate pHi in the absence of HCO3- by a Na+ (or Li+)-dependent, amiloride-inhibitable mechanism (indicative of Na+/H+ antiport). In 28 mM HCO3-, while still dependent on Na+, this regulation is only blocked in part by 1 mM amiloride. A partial block is also observed with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (1 mM). With cells pretreated with DIDS, 1 mM amiloride nearly totally inhibits this regulation. Cl- had no effect on pHi regulation in the acidic range. In HCO3(-)-free saline, Na+ removal leads to an amiloride-insensitive acidification, which is dependent on Ca2+. In 28 mM HCO3-, Na+ (and Ca2+) removal led to a pronounced reversible and DIDS-sensitive acidification. When HCO3- was lowered from 46 to 10 mM at constant pCO2 (5%), pHi dropped by a DIDS-sensitive mechanism. Identical changes in pHo (7.6 to 6.9) in the nominal absence of HCO3- led to smaller changes of pHi. In the presence but not in the absence of HCO3-, removal of Cl- led to a DIDS-sensitive alkalinization. This was also observed in the nominal absence of Na+, which leads to a sustained acidification. It is concluded that in nominally bicarbonate-free saline, the amiloride-sensitive Na+/H+ antiport is the predominant mechanism of pHi regulation at acidic pHi, while being relatively inactive at physiological values of pHi. In bicarbonate saline, two other mechanisms effect pHi regulation: a DIDS-sensitive Na+-HCO3- symport, which contributes to cytoplasmic alkalinization, and a DIDS-sensitive Cl-/HCO3- exchange, which is apparently independent of Na+.     

Amphotericin B enhanced anomalous potential difference response to changes in aqueous K+ in frog cornea

An increase in aqueous K+ from 0 to 4 mM increased the potential difference (anomalous response of electrogenic (Na+ + K+)-ATPase antiport) by 1.1 mV in Cl(-)-free solutions compared to 6.8 mV in Cl- solutions. With amphotericin B added to the tear solution in Cl(-)-free solutions, the anomalous PD response for the addition of 4 mM K+ to the aqueous solution was about 20 mV, significantly greater than in Cl- solutions. This anomalous response was inhibited by ouabain. These data support the electrogenicity of the (Na+ + K+)-ATPase pump. It is also evident that, for the pump to respond, Na+ should readily enter the cell. This may be accomplished experimentally, either across the basolateral membrane in Cl- solutions or across the apical membrane in Cl(-)-free solutions with amphotericin B present in the tear solution.     

Effect of short-chain fatty acids on cyclic 3',5'-guanosine monophosphate-mediated colonic secretion

Short chain fatty acids (SCFA) prevent and reverse cyclic 3',5'-adenosine monophosphate (cAMP) but not Ca(2+)-mediated Cl- secretion. Mucosal [HCO3-]i has an opposite effect on these secretagogues. We examined whether SCFA and [HCO3-]i affect cyclic 3',5'-guanosine monophosphate (cGMP)-induced secretion. Stripped segments of male Sprague-Dawley rat (Rattus norvegicus) proximal and distal colon, and cultured T84 cells were studied in Using chambers, and pHi and [HCO3-]i were determined. Mucosal [cGMP] was measured in proximal colon. In T84 cells, the increase in Cl- secretion (measured as Isc) induced by mucosal 0.25 microM Escherichia coli heat-stable enterotoxin (STa) was prevented/reversed by bilateral 50 mM Na+ butyrate (71%/73%), acetate (58%/76%), propionate (68%/73%) and (poorly metabolized) isobutyrate (80%/79%). In proximal colon in HCO3- Ringer, basal Cl- secretion was not affected by [HCO3-]i or 25 mM butyrate. Mucosal 0.25 microM STa decreased net Na+ and Cl- absorption. Bilateral but not mucosal 25 mM SCFA reversed STa-induced effects on Na+ absorption and Cl- secretion. Bilateral and mucosal 25 mM SCFA but not [HCO3-]i prevented STa-induced Cl- secretion and increases in mucosal [cGMP]. STa did not produce Cl- secretion in distal colon. It was concluded that SCFA but not [HCO3-]i can prevent and reverse cGMP-induced colonic Cl- secretion.     

Analysis of hyposmolarity-induced taurine efflux pathways in the bullfrog sympathetic ganglia.

Hyposmolarity-induced taurine release was dependent on the decrease in medium osmolarity (5-50%) in the satellite glial cells of the bullfrog sympathetic ganglia. Release of GABA induced by hyposmolarity was much less than that of taurine. Omission of external Cl- replaced with gluconate totally suppressed taurine release, but only slightly suppressed GABA release. Bumetanide and furosemide, blockers of the Na+/K+/2Cl- cotransport system, inhibited taurine release by about 40%. Removal of external Na+ by replacement with choline, or omission of K+, suppressed taurine release by 40%. Antagonists of the Cl-/HCO3 exchange system, SITS, DIDS and niflumic acid, significantly reduced taurine release. The carbonic anhydrase inhibitor, acetazolamide, reduced the taurine release by 34%. Omission of external HCO3 by replacement with HEPES caused a 40% increase in the hyposmolarity-induced taurine release. Hyposmolarity-induced GABA release was not affected by bumetanide or SITS. Chloride channel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and N-phenylanthranilic acid (DPC), practically abolished taurine release. Blockers of K+ channels, clofilium and quinidine, had no effect on the taurine release. The hyposmolarity-induced taurine release was considerably enhanced by a simultaneous increase in external K+. GABA was not mediated by the same transport pathway as that of taurine. These results indicate that Cl- channels may be responsible for the hyposmolarity-induced taurine release, and that Na+/K+/2Cl- cotransporter and Cl-/HCO3 exchanger may contribute to maintain the intracellular Cl- levels higher than those predicted for a passive thermodynamic distribution in the hyposmolarity-induced taurine release.