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Stimulation of Na+Ca2+ exchange by cholesterol in isolated cardiac sarcolemma vesicles
J Mol Cell Cardiol20
(Supplement
IV) (1988)
P-10
REMOVAL OF PHOSPHATIDYLINOSITOL FROM CARDIAC SARCOLBNMALMEMBRANESRESULTS IN A STIMULATION OF SODIUM-CALCIUM WCHANGE. G.N. Pierce, V. Panagia, M.G. Low*. Division of Cardiovascular Soienoesr St. Boniface Hospital Research Centre. Depts. of Physiology and Anatomy, University of Manitoba, Winnipeg, Canada; 'Dept. of Physiology h Cellular Biophysics, Columbia University, New York, NY, U.S.A. The treatment of cardiao sarcolemmal vesicles with phosphatidylinositol-specific phospholipase C (PI-specific PLase C) resulted in a speoifio depletion of PI and the release of 35 R and 100 K dalton proteins from the membrane. After treatment with the enzyme,- membranes were sepafated from the products og+the hydrolysis by centrifugation, suspended in Na. and-examined for Na+-Ca exchange. PI removal resulted in a stimulation of Nat-Cad+ exohansz by about 21%. V was inoreased in the treated preparations, apparent Km for Ca was unchanged. #m K+-ATPase activitr was not channed after PI deoletion from the membrane. Na' - Ca2+ exchange was also e&mined in an inside-out (IO) and in a mixed total population (TP) of vesicles. Na+-Ca exchange was stimulated 20% by PI-specifio PLase C treatment in the TP vesicles and by 8U%+in t&e IO vesicles. Our results demonstrate that i) hydrolysis of PI stimulates Na -Ca exchange directly or via possible release of an endogenous inhibitor protein* i$J PI is assymetrically distributed in the cardiac sarcolemmal membrane since N$ -C3+ exchange in IO vesicles was more responsive to the removal of PI, and iii) Na -Ca exchange may be functionally asymmetrical. Supported by the Manitoba Heart Foundation.
P-l 1
DEPENDENCE ON [Cali, [Cab, AND [Na]i, AND BLOCK BY 3’ 4’ DICHLOROBENZAMIL, OF OUTWARD NaCa EXCHANGE CURRENT IN GUINEA-PIG VENTRICULAR MYCCYTES. Mami Noda, Masakazu Nakao, R. Neal Shepherd, and David C. Gadsby. Laboratory of Cardiac Physiology, The Rockefeller Universky, New York, N.Y. 10021, U.S.A. Whole-cell currents were recorded at 36% in response to IOO-ms voltage pulses from -40 mV to potentials between -140 and +60 mV, in guinea-pig ventrtcular cells internally-dialyzed via wide-tipped pipettes. Internal and external sokrtions were designed to minimize ion channel currents and Na/K pump current but sustain outward N&a exchange current. [Nab was kept at 145 mM and all internal solutions included 10 mM MgATP, 40 mM HEPES @l-l 7.4) and 50 mM EGTA. Outward NalCa exchange current was activated by briefly raising [Cal, from nominally zero to millimolar levels. Its size and voftage dependence were determined by subtracting steady-state whole-cell currents elicited by voltage putses in the absence of external Ca from those recorded in the presence of 0.25 - 4.0 mM [Ca]o. Outward current was a saturable function of [Cali wfth apparent Km of 47 f 16 nM, and Hill coeffiiient, nH, of 1 .O, at +40 mV ([Na]l = 20 mM, ]Ca]o = 0.5 mM or 1 .O mu). With [Ca]t set at 50 nM, current was a saturable function both of [Ca]o ([Na]t = 20 mM), and of [Na]t ([Cal, = 0.5 or 1.0 mM), with apparent Km of 1.3 + 0.2 mM and 18 * 3 mM, and nH of 1.2 and 1.6, respectively, at +40 mV. A decay of extracel!ular-Ca-activated outward current, and an undershoot of holding current on withdrawal of extracellular Ca, often observed at tow (-20 mfvl) INali. were not seen at 100 mM fNa]t, suggesting that they reflected intracellular Na depletion. At a fted [Ca]i of 50 nM and [Na]t of 20 mM, outward-Na/Ca exchange curren~actiiatsd by 1 mM [Cab was-reduced by 3-48 f&f 3’,4’dichlorobenzamil (DCB) with apparent Km of 13 f 5 pM and nH of 1.5. Over a similar concantratttn range, DCB also inhibited steady-state [Calf-dependent inward current, presumably generated by Na/Ca exchange. Supported by NIH grant HL-14899; DCB was gift of Dr. G. Kaczomwskl, Merck, Sharp & Dohme, NJ.
OF la+-Ca2+ Y~HANGE By CHOLESTEROLIN ISOLATED CARDIAC ~AR~OLEMMA M.J.B. Kutryk and G.N. Pierce. Div. of Cardiovascular SCieWeS, St. Poniface Research Centre, Dept.. of Physiology, Univ. of Manitoba, Winnipeg, Canada The capacity of c$ole$ierol to alter sarcolemmal ion transport and the known exchanger $n t@ membrane lipid environment suggests that dependence of the Na -Ca cholesterol may modify sarcolemmal Na -Ca exchange. Modification of the cholesterol content of canine oardiao saroolemma was accomplished by incubatfon j$th phosphatidylcholine liposomes containing various amounts of cholesterol. Na -Ca exchange measured in cholesterol enriched sarco)emm3) vesicles was increased up to exchange was associated with an 48% over control values. The stimulation 95 Na -Ca increased affinity of the exchanger for Ca . Na+-Ca2+ exchange in cholesterol depleted membFaneS was decreased 15%. Thiq+depressed activity was associated with a decreased affinity of the exchanger for Ca . These eff&.s were not due to altered membrane uermeability or ohanxes in sarcolemmal bound.Ca . The StimUlating effect of cholesterol enrichment was-specific to the Na+-Cad+ exchange process SitICe saroolemmal Ca2+ pump activity was depressed by 40%. In situ oxidation of membrane cholesterol completely eliminated Naf-Cact exchange. The52 results suggest that membrane ChOlSSteFOl is intimately associated with Na+-Ca exchange and may interact with the exchange protein to modulate its aativity. Supported by the Medical Research Council of Canada and the Manitoba Medical Service Foundation.
P-12 ~PI&A','ON
.
s.40
(Supplement
IV) (1988)
P-10
REMOVAL OF PHOSPHATIDYLINOSITOL FROM CARDIAC SARCOLBNMALMEMBRANESRESULTS IN A STIMULATION OF SODIUM-CALCIUM WCHANGE. G.N. Pierce, V. Panagia, M.G. Low*. Division of Cardiovascular Soienoesr St. Boniface Hospital Research Centre. Depts. of Physiology and Anatomy, University of Manitoba, Winnipeg, Canada; 'Dept. of Physiology h Cellular Biophysics, Columbia University, New York, NY, U.S.A. The treatment of cardiao sarcolemmal vesicles with phosphatidylinositol-specific phospholipase C (PI-specific PLase C) resulted in a speoifio depletion of PI and the release of 35 R and 100 K dalton proteins from the membrane. After treatment with the enzyme,- membranes were sepafated from the products og+the hydrolysis by centrifugation, suspended in Na. and-examined for Na+-Ca exchange. PI removal resulted in a stimulation of Nat-Cad+ exohansz by about 21%. V was inoreased in the treated preparations, apparent Km for Ca was unchanged. #m K+-ATPase activitr was not channed after PI deoletion from the membrane. Na' - Ca2+ exchange was also e&mined in an inside-out (IO) and in a mixed total population (TP) of vesicles. Na+-Ca exchange was stimulated 20% by PI-specifio PLase C treatment in the TP vesicles and by 8U%+in t&e IO vesicles. Our results demonstrate that i) hydrolysis of PI stimulates Na -Ca exchange directly or via possible release of an endogenous inhibitor protein* i$J PI is assymetrically distributed in the cardiac sarcolemmal membrane since N$ -C3+ exchange in IO vesicles was more responsive to the removal of PI, and iii) Na -Ca exchange may be functionally asymmetrical. Supported by the Manitoba Heart Foundation.
P-l 1
DEPENDENCE ON [Cali, [Cab, AND [Na]i, AND BLOCK BY 3’ 4’ DICHLOROBENZAMIL, OF OUTWARD NaCa EXCHANGE CURRENT IN GUINEA-PIG VENTRICULAR MYCCYTES. Mami Noda, Masakazu Nakao, R. Neal Shepherd, and David C. Gadsby. Laboratory of Cardiac Physiology, The Rockefeller Universky, New York, N.Y. 10021, U.S.A. Whole-cell currents were recorded at 36% in response to IOO-ms voltage pulses from -40 mV to potentials between -140 and +60 mV, in guinea-pig ventrtcular cells internally-dialyzed via wide-tipped pipettes. Internal and external sokrtions were designed to minimize ion channel currents and Na/K pump current but sustain outward N&a exchange current. [Nab was kept at 145 mM and all internal solutions included 10 mM MgATP, 40 mM HEPES @l-l 7.4) and 50 mM EGTA. Outward NalCa exchange current was activated by briefly raising [Cal, from nominally zero to millimolar levels. Its size and voftage dependence were determined by subtracting steady-state whole-cell currents elicited by voltage putses in the absence of external Ca from those recorded in the presence of 0.25 - 4.0 mM [Ca]o. Outward current was a saturable function of [Cali wfth apparent Km of 47 f 16 nM, and Hill coeffiiient, nH, of 1 .O, at +40 mV ([Na]l = 20 mM, ]Ca]o = 0.5 mM or 1 .O mu). With [Ca]t set at 50 nM, current was a saturable function both of [Ca]o ([Na]t = 20 mM), and of [Na]t ([Cal, = 0.5 or 1.0 mM), with apparent Km of 1.3 + 0.2 mM and 18 * 3 mM, and nH of 1.2 and 1.6, respectively, at +40 mV. A decay of extracel!ular-Ca-activated outward current, and an undershoot of holding current on withdrawal of extracellular Ca, often observed at tow (-20 mfvl) INali. were not seen at 100 mM fNa]t, suggesting that they reflected intracellular Na depletion. At a fted [Ca]i of 50 nM and [Na]t of 20 mM, outward-Na/Ca exchange curren~actiiatsd by 1 mM [Cab was-reduced by 3-48 f&f 3’,4’dichlorobenzamil (DCB) with apparent Km of 13 f 5 pM and nH of 1.5. Over a similar concantratttn range, DCB also inhibited steady-state [Calf-dependent inward current, presumably generated by Na/Ca exchange. Supported by NIH grant HL-14899; DCB was gift of Dr. G. Kaczomwskl, Merck, Sharp & Dohme, NJ.
OF la+-Ca2+ Y~HANGE By CHOLESTEROLIN ISOLATED CARDIAC ~AR~OLEMMA M.J.B. Kutryk and G.N. Pierce. Div. of Cardiovascular SCieWeS, St. Poniface Research Centre, Dept.. of Physiology, Univ. of Manitoba, Winnipeg, Canada The capacity of c$ole$ierol to alter sarcolemmal ion transport and the known exchanger $n t@ membrane lipid environment suggests that dependence of the Na -Ca cholesterol may modify sarcolemmal Na -Ca exchange. Modification of the cholesterol content of canine oardiao saroolemma was accomplished by incubatfon j$th phosphatidylcholine liposomes containing various amounts of cholesterol. Na -Ca exchange measured in cholesterol enriched sarco)emm3) vesicles was increased up to exchange was associated with an 48% over control values. The stimulation 95 Na -Ca increased affinity of the exchanger for Ca . Na+-Ca2+ exchange in cholesterol depleted membFaneS was decreased 15%. Thiq+depressed activity was associated with a decreased affinity of the exchanger for Ca . These eff&.s were not due to altered membrane uermeability or ohanxes in sarcolemmal bound.Ca . The StimUlating effect of cholesterol enrichment was-specific to the Na+-Cad+ exchange process SitICe saroolemmal Ca2+ pump activity was depressed by 40%. In situ oxidation of membrane cholesterol completely eliminated Naf-Cact exchange. The52 results suggest that membrane ChOlSSteFOl is intimately associated with Na+-Ca exchange and may interact with the exchange protein to modulate its aativity. Supported by the Medical Research Council of Canada and the Manitoba Medical Service Foundation.
P-12 ~PI&A','ON
.
s.40