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D K Basu

Publications and source records attributed to D K Basu.

17 recordsLinked to original sources
PubMed1991

Regulation of free cytosolic Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. I. Efficiency of transport and interactions between cations.

Regulation of free cytosolic Ca2+ concentration in the rod outer segments (ROS) isolated from bovine retinas was examined with the fluorescent Ca(2+)-indicating dye fluo-3. In situ calibration of cytosolic fluo-3 was done in the presence of the Ca2+ ionophore A23187 and yielded a dissociation constant of 500 nM for the Ca(2+)-fluo-3 complex. Ca2+ influx in Ca(2+)-depleted ROS was completely abolished when internal Na+ was removed suggesting that Ca2+ influx exclusively occurred via Na-Ca-K exchange. The most striking observation was that Na-Ca-K exchange could mediate a rapid increase in cytosolic free Ca2+ over the most of the usable indicating range of fluo-3 (from 10 nM to 2 microM), even when exposed to free external Ca2+ concentrations as low as 10 nM. From a comparison between changes in free Ca2+ and changes in total Ca2+, we conclude that physiologically occurring changes in cytosolic free Ca2+ are mediated by exchange fluxes less than 1% of the maximal Na-Ca-K exchange flux. The Na-Ca-K exchanger could mediate both K(+)-dependent and K(+)-independent Ca2+ influx; Li+ caused a complete inhibition of K(+)-independent Ca2+ influx, but had no effect on K(+)-dependent Ca2+ influx. We examined the complex interactions of alkali cations with Ca2+ influx and discuss the results in terms of a three-site model for the Na-Ca-K exchanger (Schnetkamp, P. P. M. and Szerencsei, R. T. (1991) J. Biol. Chem. 266, 189-197). Ca2+ competed with one Mg2+ ion or two Na+ ions for binding to a common site. High K+ concentration greatly diminished the ability of Na+ and Mg2+ to compete with Ca2+ for this common site on the exchanger protein. As a result, high internal K+ induced a conformation of the exchange protein that kinetically favoured Ca2+ extrusion.

Aniline Compounds↗
PubMed1991

Regulation of intracellular free Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. II. Thermodynamic competence of transmembrane Na+ and K+ gradients and inactivation of Na(+)-dependent Ca2+ extrusion.

Regulation of cytosolic free Ca2+ in the physiologically relevant submicromolar range was measured in isolated intact bovine rod outer segments (ROS) with the intracellular Ca(2+)-indicating dye fluo-3. Changes in free Ca2+ were compared with changes in total Ca2+ measured with 45Ca fluxes and a good qualitative correlation was observed. Ca2+ homeostasis in isolated bovine ROS was exclusively mediated via the Na-Ca-K exchanger. Free cytosolic Ca2+ concentration was lowered by an increase in the inward Na+ gradient, was raised by an increase in external K+, and was raised by depolarization of the plasma membrane. The simplest stoichiometry consistent with these qualitative observations is 4Na:(1Ca + 1K). The individual K:Ca, Na:Ca, and K:Na coupling ratios were deduced from quantitative changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ and K+ gradients. The observed changes in free Ca2+ did not agree with changes in free Ca2+ calculated on the basis of the above fixed stoichiometry which may reflect the flexibility in the Ca:K coupling ratio observed before in flux experiments (Schnetkamp, P. P. M., Szerencsei, R. T., and Basu, D. K. (1991) J. Biol. Chem. 266, 198-206). The most dramatic discrepancy was observed for the Na:Ca coupling ratio: the expected very large changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ gradient were not observed. Rapid Na(+)-induced Ca2+ extrusion was unable to lower cytosolic free Ca2+ below 100 nM, even under nonequilibrium conditions and despite the observation that Ca2+ influx via reverse Na-Ca-K exchange readily occurred at a free external Ca2+ concentration of 20 nM. We conclude that the Na(+)-dependent extrusion mode of the Na-Ca-K exchanger occurs in a brief (20-s) burst of high maximal velocity transport followed by a nearly complete inactivation of transport. The importance of our findings for Ca2+ homeostasis in functioning rod photoreceptors is discussed.

Aniline Compounds↗
PubMed1991

Unidirectional Na+, Ca2+, and K+ fluxes through the bovine rod outer segment Na-Ca-K exchanger.

The properties of the Na-Ca exchanger in the plasma membrane of rod outer segments isolated from bovine retinas (ROS) were studied. Unidirectional Ca2+, Na+, and K+ fluxes were measured with radioisotopes and atomic absorption spectroscopy. We measured K+ fluxes associated with the Ca-Ca self-exchange mode of the Na-Ca exchanger to corroborate our previous conclusion that the ROS Na-Ca exchanger differs from Na-Ca exchangers in other tissues by its ability to transport K+ (Schnetkamp, P. P. M., Basu, D. K. & Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-C157). The Na-Ca-K exchanger was the only functional cation transporter in the plasma membrane of bovine ROS with an upper limit of a flux of 10(5) cations/ROS/s or a current of 0.01 pA contributed by other cation channels, pumps, or carriers; cation fluxes via the Na-Ca-K exchanger amounted to 5 x 10(6) cations/ROS/s or a current of 1 pA. Ca2+ efflux via the forward mode of the Na-Ca-K exchanger did not operate with a fixed single stoichiometry. 1) The Na/Ca coupling ratio was increased from three to four when ionophores were added that could provide electrical compensation for the inward Na-Ca exchange current. 2) The K/Ca coupling ratio could vary by at least 2-fold as a function of the external Na+ and K+ concentration. The results are interpreted in terms of a model that can account for the variable Ca/K coupling ratio: we conclude that the Ca2+ site of the exchanger can translocate independent of translocation of the K+ site, whereas translocation of the K+ site requires occupation of the Ca2+ site, but not its translocation. The results are discussed with respect to the physiological role of Na-Ca-K exchange in rod photoreceptors.

Animals↗
PubMed1991

The stoichiometry of Na-Ca+K exchange in rod outer segments isolated from bovine retinas.

Ca2+ extrusion in the outer segments of retinal rods (ROS) is mediated by a protein that couples both the inward Na+ gradient and the outward K+ gradient to Ca2+ extrusion. Na(+)-stimulated Ca2+ release from ROS requires internal K+ and is accompanied by release of internal K+, whereas a slow component of Na(+)-stimulated Ca2+ release does not require K+. In this paper we discuss our observations on the K+ transport via Na-Ca+ K exchange in bovine ROS, on the electrogenicity and stoichiometry of the ROS Na-Ca+ K exchanger, and on the mechanism on coupling Ca2+ to K+ via this protein. Finally, we discuss briefly the physiological implications of Na-Ca+ K exchange.

Animals↗
PubMed1989

Na+-Ca2+ exchange in bovine rod outer segments requires and transports K+.

Intact outer segments isolated from bovine retinas (bovine ROS) display a high activity of Na+-Ca2+ exchange, and Na+-Ca2+ exchange appears to be the only functional ion transporter present. Here we demonstrate for the first time that Na+-Ca2+ exchange requires and transports K+ from the following observations. 1) Na+-Ca2+ exchange in bovine ROS required the simultaneous presence of K+ and Ca2+ on one side of the membrane and the presence of Na+ on the other side. 2) Na+-stimulated Ca2+ release from bovine ROS was accompanied by an equally large release of K+. We used the electrogenic protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) as an added electrical shunt; in the intact rod cell, electrogenic Na+-Ca2+ exchange is shunted by K+ channels present in the rod inner segment. In the presence of FCCP, an inward Na+-Ca2+ exchange current was accompanied by an outward current of protons with a stoichiometry of 1 H+/Ca2+; in the absence of FCCP, no Na+-induced proton current was observed. Addition of FCCP did not uncouple Na+-induced K+ release from Na+-induced Ca2+ release. We conclude that Na+-Ca2+ exchange in bovine rod photoreceptors operates at an electrogenic stoichiometry of 4 Na+:(1 Ca2+ + 1 K+). In isolated ROS and in the absence of an external electrical shunt, Na+-Ca2+ exchange operated at an electroneutral stoichiometry of 3 Na+:(1 Ca2+ + 1 K+).

Animals↗
PubMed1987

Injury to rat hearts produced by an exogenous free radical generating system. Study into the role of arachidonic acid and eicosanoids.

This study was designed to evaluate the effect of an exogenous free radical generating system consisting of purine plus xanthine oxidase on the isolated rat heart and in particular to assess the possible contribution of arachidonic acid or its metabolites to toxicity produced by this drug combination. Purine plus xanthine oxidase produced a time-dependent depression in cardiac contractility which was associated with stimulated release of lactate dehydrogenase (LDH). Electron microscopic analysis revealed a distinct separation of the glycocalyx from the sarcolemmal membrane with no apparent intracellular defects. Purine plus xanthine oxidase was a potent stimulus for 6-keto-prostaglandin F1 alpha (6K-PGF1 alpha) synthesis but leukotriene production was undetectable under any condition. Eicosatetraynoic acid, which totally prevents the metabolism of arachidonic acid, accelerated the loss in force and increased LDH release invoked by purine plus xanthine oxidase, but produced no noticeable change in sarcolemmal ultrastructure. Cyclooxygenase inhibitors produced little influence although pretreatment with either acetylsalicylic acid or ibuprofen decreased contractility toward the end of purine plus xanthine oxidase perfusion. Nordihydroguarietic acid, a purported inhibitor of 5'-lipoxygenase accelerated the loss in force produced by purine plus xanthine oxidase. The nordihydroguarietic acid effects were associated with reduced 6K-PGF1 alpha efflux but LDH release was unaffected. We also examined whether modification of arachidonic acid release through changes in calcium concentration was associated with altered response to purine plus xanthine oxidase. Lowering the calcium concentration to 0.41 mM (from 1.25 mM control) reduced markedly 6K-PGF1 alpha, efflux as well as LDH release. Although the latter is suggestive of protection, hypocalcemic perfusion resulted in a greater loss in force due to free radical generation. Furthermore, cells from these hearts exhibited a greater degree of glycocalyx separation. Increasing the calcium concentration to 2.50 mM produced no further toxic manifestations in the response to purine plus xanthine oxidase, although the release of 6K-PGF1 alpha was increased. Our results suggest complex toxicity induced by an exogenously generated free radical system. The injury produced by this method is restricted to sarcolemmal changes, the latter being dependent on the external calcium concentration. The study further suggests that accumulation of intracellular unesterified arachidonic acid, which may result from peroxidation of membrane lipids, increases tissue injury caused by exogenous free radicals.

5,8,11,14-Eicosatetraynoic Acid↗