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.     

Refractoriness of cation transport in turkey erythrocytes to stimulation by cyclic adenosine 3':5'-monophosphate.

In turkey erythrocytes bidirectional fluxes of sodium and potassium develop a time-dependent refractoriness to stimulation by endogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP). The refractoriness of potassium influx and potassium outflux (both of which require extracellular sodium and potassium for stimulation by cyclic AMP) depends on the extracellular concentrations of sodium and potassium. In contrast, the refractoriness developed by sodium outflux (which does not require extracellular sodium or potassium for stimulation by cyclic AMP) does not depend on the extracellular concentrations of sodium or potassium. The refractoriness of these fluxes to cellular cyclic AMP reflects a decrease in the amount by which they can be maximally stimulated and appears to be proportional to the extent to which the transport system is utilized during the course of the incubation. Ouabain significantly reduces the rate at which cation transport in turkey erythrocytes becomes refractory to endogenous cyclic AMP. This effect of the glycoside is independent of the extracellular concentrations of sodium or potassium and does not correlate with how it alters the initial response of the transport systems to cyclic AMP.     

Unique regulation of anion/HCO3- exchangers by constitutive nitric oxide in rabbit small intestine

In the rabbit small intestine, there are three functionally different brush-border membrane (BBM) anion/HCO3- exchangers: 1) Cl/HCO3- exchange on the BBM of villus cells responsible for coupled NaCl absorption; 2) Cl/HCO3- exchange on the BBM of crypt cells possibly involved in HCO3- secretion; and 3) short-chain fatty acid (SCFA)/HCO3- exchange on the BBM of villus cells, which facilitates SCFA absorption. Although constitutive nitric oxide (cNO) has been postulated to alter many gastrointestinal tract functions, how cNO may specifically alter these three transporters is unknown. Inhibition of cNO synthase with NG-nitro-L-arginine methyl ester (L-NAME) 1) did not affect villus cell BBM Cl/HCO3 change, 2) stimulated crypt cell BBM Cl/HCO3- exchange, and 3) inhibited villus cell BBM SCFA/HCO3- exchange. D-NAME, an inactive analog of L-NAME, and L-N6-(1-iminoethyl)lysine, a more selective inhibitor of inducible NO, did not affect these transport processes. Kinetic studies demonstrated that 1) the mechanism of inhibition of crypt cell BBM Cl/HCO3- exchange is secondary to a decrease in the maximal rate of uptake of Cl, without an alteration in the affinity of the transporter for Cl, and 2) the mechanism of stimulation of villus cell BBM SCFA/HCO3- exchange is secondary to an increase in the affinity of the transporter for SCFA without an alteration in the maximal rate of uptake of SCFA. These results indicate that cNO uniquely regulates the three BBM anion/HCO3- transporters in the rabbit small intestine.     

Bioassays of APONIN-3 and -4 with rabbit erythrocytes

Rabbit erythrocytes in methanolic phosphate medium were used to bioassay the activity of authentic samples of methyl stearate and methyl palmitate (in 10% methanol:90% water, v/v), which had been identified as apparent oceanic naturally occurring cytolins (APONIN-3 and -4) produced by Nannochloris oculata. The two natural products are notable for cytolytic activity toward the unarmoured dinoflagellate, Gymnodinium breve Davis, an organism responsible for red tides consisting of harmful algal blooms in the Gulf of Mexico and along the eastern coast of the United States. Bioassays were done with heparinized rabbit blood. The absorbance at 540 nm was observed for 15 min in comparison with a sample treated with haemolysing agent. The results indicated that at reasonable concentrations of 1-10 ppm, neither was a haemolysin, although such concentrations caused cytolysis of G. breve cultures.     

Na+/HCO3-co-transport in basolateral membrane vesicles isolated from rabbit renal cortex

Recent studies suggest that the major pathway for exit of HCO3- across the basolateral membrane of the proximal tubule cell is electrogenic Na+/HCO3- co-transport. We therefore evaluated the possible presence of Na+/HCO3- co-transport in basolateral membrane vesicles isolated from the rabbit renal cortex. Imposing an inward HCO3- gradient induced the transient uphill accumulation of Na+, and imposing an outward Na+ gradient caused HCO3- -dependent generation of an inside-acid pH gradient as monitored by quenching of acridine orange fluorescence, findings consistent with the presence of Na+/HCO3- co-transport. In the absence of other driving forces, generating an inside-positive membrane potential by imposing an inward K+ gradient in the presence of valinomycin caused net Na+ uptake via a HCO3- -dependent pathway, indicating that Na+/HCO3- co-transport is electrogenic and associated with a flow of negative charge. Imposing transmembrane Cl- gradients did not appreciably affect HCO3- gradient-stimulated Na+ influx, suggesting that Na+/HCO3- co-transport is not Cl- -dependent. The rate of HCO3- gradient-stimulated Na+ influx was a simple, saturable function of the Na+ concentration (Km = 9.7 mM, Vmax = 160 nmol/min/mg of protein), was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (I50 = 100 microM), but was inhibited less than 10% by up to 1 mM amiloride. We could not demonstrate a HCO3- -dependent or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive component of Na+ influx in microvillus membrane vesicles. This study thus indicates the presence of a transport system mediating electrogenic Na+/HCO3- co-transport in basolateral, but not luminal, membrane vesicles isolated from the rabbit renal cortex. Analogous to the use of renal microvillus membrane vesicles to study Na+/H+ exchange, renal basolateral membrane vesicles may be a useful model system for examining the kinetics and possible regulation of Na+/HCO3- co-transport.     

pH sensitivity of the Na+:HCO3- cotransporter in basolateral membrane vesicles isolated from rabbit kidney cortex.

HCO3- exit across the basolateral membrane of the kidney proximal tubule cell is mediated via an electrogenic Na+:HCO3- cotransporter. We have studied the effect of pH on the activity of this cotransport system in basolateral membrane vesicles isolated from rabbit renal cortex. At constant internal pH 6.0, increasing the external pH and [HCO3-] increased the rate of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 22Na+ influx into the vesicles. To determine the role of internal pH on the activity of the Na+:HCO3- cotransport system, the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange was measured in the absence of an initial pH and [HCO3-] gradient (pH(i) = pH(o), 5% CO2). Increasing the pH from 6.8 to 7.2 increased whereas, increasing the pH from 7.4 to 8.0 decreased the rate of 22Na+ influx via this exchange. Increasing pH at constant [HCO3-] (pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 10% CO2) reduced the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange. Increasing pH at constant [CO3(2-)](pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 60% CO2) was associated with reduced 22Na+ uptake. Decreasing the pH (pH(i) = pH(o) = 6.3, 60% CO2 versus pH(i) = pH(o) = 7.2, 5% CO2) was associated with a reduced rate of HCO3(-)-dependent Na(+)-Na+ exchange. We conclude that the Na+:HCO3- cotransporter displays a significant pH sensitivity profile with the cotransporter being more functional at pH 7.0-7.4 and less active at more acid or alkaline pH. In addition, the results suggest that the pH sensitivity arises at the inner surface of the basolateral membrane.     

Active chloride secretion by rabbit colon: calcium-dependent stimulation by ionophore A23187.

Addition of Ca ionophore, A23187, to the solution bathing the mucosal surface of descending rabbit colon resulted in a reversal of active C1absorption to active C1 secretion, a twofold increase in short-circuit current and a 40% increase in tissue conductance without affecting the rate of active Na absorption. These alterations in electrolyte transport are quantitatively similar to those previously observed in response to cyclic 3',5'-AMP (cAMP) (RA. Frizzell, M.J. Koch & S.G. Schulz, J. Membrane Biol. 27:297, 1976). When medium Ca concentration was reduced to 10(-6) M, the secretory response to A23187 was abolished but the response to cAMP was unaffected. The ionophore did not influence the cAMP levels of colonic mucosa. Addition of cyclic AMP to colonic strips preloaded with 45Ca elicited a reversible increase in Ca efflux from the tissue. These results suggest that an increase in intracellular Ca concentration stimulates colonic electrolyte secretion and that the secretory response to cAMP may be due, at least in part, to a release of Ca from intracellular stores.     

Oxalate transport via the sulfate/HCO3 exchanger in rabbit renal basolateral membrane vesicles

We evaluated the mechanism of oxalate transport in basolateral membrane vesicles isolated from the rabbit renal cortex. An outward HCO3- gradient induced the transient uphill accumulation of oxalate and sulfate, indicating the presence of oxalate/HCO3- exchange and sulfate/HCO3- exchange. For oxalate, sulfate, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, the K1/2 value for oxalate/HCO3- exchange was nearly identical to that for sulfate/HCO3- exchange, suggesting that both exchange processes occur via the same transport system. This was further supported by the finding of sulfate/oxalate exchange. Thiosulfate/sulfate exchange and thiosulfate/oxalate exchange were also demonstrated, but a variety of other tested anions including Cl-, p-aminohippurate, and lactate did not exchange for sulfate or oxalate. Na+ did not affect sulfate or oxalate transport, indicating that neither anion undergoes Na+ co-transport or Na+-dependent anion exchange in these membrane vesicles. Finally, we found that the stoichiometry of exchange is 1 sulfate or oxalate per 2 HCO3-, or a thermodynamically equivalent process. We conclude that oxalate, but not other organic or inorganic anions of physiologic importance, can share the sulfate/HCO3- exchanger in renal basolateral membrane vesicles. In series with luminal membrane oxalate/Cl- (formate) exchange, exchange of oxalate for HCO3- or sulfate across the basolateral membrane provides a possible transcellular route for oxalate transport in the proximal tubule.     

Electrogenic Na-independent HCO3 transport in OK cells.

We have shown previously that OK cells recover from an acid load in a medium nominally CO2-free by extruding H via a Na/H exchanger and a passive H-conductive pathway. In this work, the regulation of cell pH (pHi) was studied after addition or withdrawal of CO2/HCO3 (5% CO2, 95 mM HCO3, pH = 8) using the fluoroprobe BCECF. In the presence of Na and amiloride to inhibit Na/H exchange, the recovery of pHi after CO2 entry and CO2 exit were found to depend in part on HCO3 entry and exit, respectively. Efflux of H per se also contributed to restoring pHi after CO2 addition, whereas H influx may have played a smaller role to normalize pHi after CO2 removal. DIDS, 0.5 mM, significantly inhibited both recovery phases of pHi. Removal of Na failed to inhibit the recovery of pHi after CO2 addition and removal. Cl removal also failed to inhibit pHi recovery after CO2 removal. Cell depolarization in the presence of Na moderately stimulated the pHi recovery rate after CO2 addition whereas it markedly inhibited the normalization of pHi after CO2 removal. Cell depolarization in the absence of sodium had only a slight effect to increase pHi recovery after CO2 addition but markedly prevented the pHi recovery after CO2 removal. These results indicate that OK cells lack Na or Cl-dependent HCO3 transport systems. The OK cell possesses a novel stilbene-sensitive electrogenic HCO3 transport system that is involved in the regulation of cell pH.     

Enumeration and isolation of rabbit T and B lymphocytes by using antibody-coated erythrocytes.

Rosette formation with antibody-coated erythrocytes (Ab-E) was employed for the enumeration and isolation of rabbit B cells (Ig+T-) and T cells (Ig-T+). The cells bearing surface Ig (Ig+ cells) were enumerated by a direct immunocytoadhesion technique utilizing anti-rabbit IgG antibody-coated erythrocytes (Ab-E). To enumerate cells bearing thymus cell antigen (T+ cells), an indirect rosette technique was used in which lymphocytes were first sensitized with guinea pig anti-rabbit thymus cell antiserum and then rosetted with anti-guinea pig IgG Ab-E. To demonstrate the specificity of the anti-thymus cell antiserum, a 51Cr radioimmunoassay for counting rosettes was employed along with visual counting to enumerate Ig+ and T+ cells in lymph node cell populations. When Ig+ and T+ lymph node cells were rosetted simultaneously with sheep and human erythrocytes, no mixed rosettes (less than 1%) were observed. Ficoll-Hypaque gradient centrifugation was used to obtain purified Ig+T- and Ig-T+ cells by removing rosetted T+ and Ig+ cells, respectively. The purity of isolated Ig-T+ cells was indicated by 94 to 95% indirect rosetting with anti-thymus cell antiserum and by 0 to 3% direct rosetting with anti-rabbit IgG Ab-E. The purity of isolated Ig+T- cells was indicated by 90 t0 94% direct rosetting with anti-rabbit IgG Ab-E and by 2 to 3% indirect rosetting with anti-thymus cell antiserum. The percentage of Ig+T- and Ig-T+ cells were determined in peripheral blood and in various lymphoid organs. The isolated Ig+T- and Ig-T+ cells were also characterized by their responses to mitogens. Thus, nearly pure Ig+T- and Ig-T+ cells were isolated by "negative selection," which should minimize functional changes of the cells, and thereby facilitate the study of their biologic properties, e.g., their response to mitogens.     

Hemolysis of rabbit erythrocytes induced by cigarette smoke

Cigarette smoke has been found to induce the hemolysis of rabbit erythrocytes. The particulate phase had more profound effect than the gas phase. Neither free radical scavengers such as ascorbic acid, uric acid and water-soluble vitamin E analogue nor antioxidant enzymes such as catalase and superoxide dismutase suppressed the cigarette smoke-induced hemolysis, suggesting that free radicals, hydrogen peroxide, and superoxide were not the active species.     

Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.     

Transport ATPases in the erythrocytes of rats acclimatized to intermittent altitude hypoxia

The activity of Na+/K+- and Ca2+-ATPase and some allosteric properties of Na+/K+-ATPase were studied in whole erythrocytes and their membrane preparations (ghosts) from rats exposed to intermittent altitude hypoxia (10 and 24 exposures, 8 h/day in an altitude chamber, stepwise up to an altitude of 7,000 m). Ca2+-ATPase activity was increased both in whole erythrocytes and ghosts after the first phase of acclimatization (10 exposures). In a standard incubation medium (containing 3 mmol.l-1 MgCl2 ), Na+/K+-ATPase activity in the ghosts was also increased after the initial phase of acclimatization whereas in whole erythrocytes Na+/K+-ATPase was only decreased in the regression phase. At high MgCl2 concentrations (12 mmol.l-1) changes of Na+/K+-ATPase activity both in whole erythrocytes and in the ghosts followed similar time course with a pronounced increase in the first phase of acclimatization (10 exposures) followed by an abrupt drop (24 exposures) and then by a gradual normalization in the regression phase. Sensitivity of the enzyme to mounting MgCl2 concentrations was increased in the ghosts at the end of acclimatization and was decreased in whole erythrocytes during acclimatization and especially in the regression phase. It has been suggested that chronic altitude hypoxia leads to the alteration of cooperative interaction of the Na+/K+-ATPase subunits in the erythrocyte membrane and accumulation of some factor in the cells inhibiting this enzyme.     

Effect of 3-isobutyl-1-methylxanthine on HCO3- transport in turtle bladder. Evidence for electrogenic HCO3- secretion

Ouabain-treated turtle bladders bathed on both surfaces by identical HCO3-/CO2-containing, Cl- -free Na+ media exhibit a short-circuit current (Isc) and transepithelial potential (p.d.) serosa electronegative to mucosa. Addition of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, rapidly reverses the direction of the Isc and p.d. The IBMX-induced reversal of Isc and p.d. is (1) dependent on the presence of HCO3- (and CO2) in the serosal bathing fluid, (2) independent of Na+ and other ions in the bathing medium, (3) decreased by inhibitors of carbonic anhydrase or oxidative metabolism, (4) increased by the serosal addition of cyclic AMP or the disulfonic stilbene, SITS. The results constitute evidence that the reversed Isc elicited by IBMX represents electrogenic secretion of HCO3-.     

Studies in the mechanism of phenylethylamine uptake by rabbit erythrocytes

The mechanism of phenylethylamine (PEA) uptake by in vitro rabbit erythrocyte preparations involve both a larger component of passive diffusion and an Na+-dependent facilitated transport system. This is reflected by the fast rate and lack of saturation of PEA erythrocyte uptake, and by the decrease in PEA tissue/medium ratio when the amine was incubated in an Na+-free medium or in the presence of ouabain. Results obtained after the addition of iodoacetamide or by the use of glucose-free medium suggest the Na+-K+ gradient as the driving force responsible for operation of the carrier mechanism. Preliminary results indicate that amphetamine PEA share a similar mechanism for their uptake by rabbit erythrocytes. At the levels normally present in rabbit blood (c. 1 X 10(-8)M), about one-third of the PEA is found in the serum and only a very small portion of it (less than 1%) is present as the "biologically active" non-ionized amine. Most of the remaining phenylethylamine is bound to plasma protein or inside the erythrocytes.