Large-scale expression of recombinant cardiac sodium-calcium exchange in insect larvae.

Recombinant bovine cardiac sodium-calcium exchange (NCX1) in a baculovirus construct was used to infect cabbage looper larvae (Trichoplusia ni). Infected larvae were homogenized and larvae membrane vesicles were purified. Western blot analysis indicated the presence of recombinant NCX1 protein in vesicles from infected larvae but not in controls. Vesicles from infected larvae expressed high levels of NCX1 activity (1.7 nmol Ca2+/mg protein/s) while vesicles from control larvae had no activity. NCX1 in larvae vesicles was bidirectional. Kinetic analysis yielded a Vmax of 3.6 nmol Ca2+/mg protein/s and a Km for Ca of 4.2 microM. NCX1 activity was inhibited by the exchange inhibitory peptide with an IC50 of 4 microM. These data demonstrate a novel and efficient method for the expression of large amounts of active recombinant NCX1 protein that has general application for expression and analysis of recombinant membrane proteins.

Localization of an exchange inhibitory peptide (XIP) binding site on the cardiac sodium-calcium exchanger.

The exchange inhibitory peptide (XIP; RRLLFYKYVYKRYRAGKQRG) is a potent inhibitor of cardiac Na-Ca exchange activity. This study attempted to identify the XIP binding site on the Na-Ca exchange protein. Bovine cardiac sarcolemmal vesicles were proteolyzed and fractionated by XIP-affinity column chromatography. A 24 kDa fragment was purified and subjected to amino acid sequence analysis. A negatively charged region of intracellular loop f of the Na-Ca exchange protein (IDDDIFEEDEN; aa 445-455) was identified. The affinity and specificity of XIP interaction with peptides IDDDIFEEDEN and GEDDDDDECGEE (another negatively charged region of the Na-Ca exchange protein) were examined. XIP cross-linked to peptide IDDDIFEEDEN but not GEDDDDDECGEE in a pH-dependent manner. Fluorescence titration binding studies indicated that binding of IDDDIFEEDEN with XIP was saturable (Kd=5 microM) while binding with GEDDDDDECGEE was not specific. These data suggest that amino acids 445-455 on Na-Ca exchange loop f are involved in XIP binding.

Identification of critical positive charges in XIP, the Na/Ca exchange inhibitory peptide.

The peptides XIP (RRLLFYKYVYKRYRAGKQRG) and C28R2 (LRRGQILWFRGLNRIQTQIRVVKAFRSS) correspond to the autoinhibitory domains of the Na-Ca exchanger and the plasma membrane Ca pump, respectively. An increase of ionic strength reduced the inhibition of exchange activity by XIP and C28R2, consistent with an important role for electrostatic interactions. Sulfosuccinimidyl acetate (SNA)-modified XIP did not inhibit Na-Ca exchange. Because SNA modifies lysines, we conclude that at least one of the positive charges at the XIP lysine positions (7, 11, or 17) is important for inhibition. 2CK-XIP (RRLLFYRYVYRCYCAGRQKG) has cysteines at 12 and 14 and only one lysine (at 19).2CK-XIP inhibited Na-Ca exchange; thus positive charges at 12 and 14 are not essential. SNA-modified 2CK-XIP did not inhibit; thus a positive charge at 19 is important. Iodoacetic acid-modified 2CK-XIP inhibits the Na-Ca exchanger but not the PM Ca pump. These results show that the structural determinants for inhibition of the Na-Ca exchanger and the PM Ca pump are different, that positive charges at 7, 11, or 17 (or some combination) are more important than positive charges at 12 and 14 for inhibition by XIP of the Na-Ca exchanger.

Plasmalogen and anionic phospholipid dependence of the cardiac sarcolemmal sodium-calcium exchanger.

Although plasmalogens are the predominant phospholipids of cardiac sarcolemma, their physiological role has not been forthcoming. Since the cardiac sarcolemmal sodium-calcium exchanger has been proposed to be regulated by anionic phospholipids, the roles of plasmalogens and anionic phospholipids as regulators of the sodium-calcium exchanger were explored. Reconstituted sodium-calcium exchange activity in plasmalogen-containing proteoliposomes was 10-fold higher than that in control proteoliposomes comprised of only diacyl phospholipids. Additionally, exchange activity in plasmalogen-containing proteoliposomes was regulated by anionic phospholipids. Thus, plasmalogens provide a critical lipid environment in which anionic phospholipids serve as boundary lipids for the regulation of the trans-sarcolemmal sodium-calcium exchanger.

Studies of renal injury. I. Gentamicin toxicity and expression of basolateral transporters.

Gentamicin nephrotoxicity may arise in part from alterations in the expression of genes critical for renal proximal tubule metabolism. We tested the hypothesis that gentamicin suppressed the gene expression of the Na+/Ca2+ exchanger (NaCaX), glucose transporter 1 (GLUT1) and alpha 1-subunit of Na(+)-K(+)-ATPase (alpha 1-NKA) in renal tubules. The products of these genes mediate Na(+)-dependent Ca2+ efflux, glucose efflux and influx, and ATP-dependent Na+ efflux across tubular basolateral membranes, respectively. After 10 days of gentamicin intoxication (40 mg/kg ip, twice daily), levels of mRNAs encoding NaCaX and the cognate protein declined. GLUT1 mRNA levels increased, although GLUT1 protein levels were also reduced. Moreover, whereas alpha 1-NKA mRNA levels remained unchanged, alpha 1-NKA protein levels were also reduced. We suggest that the higher GLUT1 mRNA level is part of the stress response to tubular injury. However, regardless of the mRNA level, the most consistent effect of gentamicin was reduction of specific protein levels. We propose that failure to translate high levels of mRNA into proportionally high levels of protein, as in the case of GLUT1, may attenuate the expression of stress response gene products, and thus diminish the possibility of recovery in gentamicin intoxication.

A photoaffinity probe for the cardiac adenosine transporter.

We have developed and utilized a photoaffinity probe to identify the adenosine transporter in cardiac sarcolemmal (SL) vesicles. The probe is an azidosalicylate derivative of adenosine made by reacting adenosine with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA). Following synthesis and radiolabeling of the probe (ASA-adenosine), 125I-ASA-adenosine was purified by high pressure liquid chromatography. Iodine-125-ASA-adenosine, upon irradiation with uv light, covalently labeled a 65 kDa protein in bovine cardiac SL vesicles. Labeling of this protein was greatly diminished in the presence of nonradiolabeled adenosine, 5'-amino adenosine, or guanosine (inhibitors of purine nucleoside transport) but not by glucose (which does not inhibit transport). We conclude that the cardiac adenosine transporter is a protein with an apparent Mr of 65 kDa.

Eosin, a potent inhibitor of the plasma membrane Ca pump, does not inhibit the cardiac Na-Ca exchanger.

The Na-Ca exchanger and the sarcolemmal/plasma membrane (SL(PM)) Ca pump are the two major pathways for Ca transport to the extracellular space in many cells. In cardiac myocytes, the Na-Ca exchanger appears to be responsible for a greater portion of this Ca flux [Bassani, R. A., et al. (1992) J. Physiol. 453, 591-608]. However, the respective contributions of these two transporters are not as well-defined in all tissues (e.g., smooth muscle). We propose that eosin (tetrabromofluorescein) may be a useful tool for quantitatively determining the proportion of Ca transported by the Na-Ca exchanger vs the SL(PM) Ca pump in various cells. Eosin is the most potent inhibitor known for the SL(PM) Ca pump (IC50 approximately 0.3 microM in red blood cell inside-out vesicles); unlike the Na/K and H/K pumps, eosin does not compete with ATP for the SL(PM) Ca pump [Gatto, C., & Milanick, M. A. (1993) Am. J. Physiol. 264, C1577-C1586]. In the present study, we have shown that eosin was a potent inhibitor of the cardiac SL(PM) Ca pump (IC50 approximately 1 microM); in contrast, eosin (< or = 20 microM) did not inhibit the cardiac Na-Ca exchanger. In experiments where Ca was being transported by both the SL(PM) Ca pump and the Na-Ca exchanger simultaneously, eosin effectively eliminated the Ca pump-mediated transport. In addition, we show that eosin can permeate the human red cell membrane; cell permeability is an attractive feature for using eosin in whole cell studies. We conclude that eosin can be used for determining the role that the SL(PM) Ca pump plays in whole cell Ca homeostasis.

Interaction of cardiac Na-Ca exchanger and exchange inhibitory peptide with membrane phospholipids.

We tested the hypothesis that the exchange inhibitory peptide (XIP) domain in the cardiac Na-Ca exchanger is a regulatory site under the control of the membrane lipid environment. We found that 125I-XIP bound to liposomes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) with peak binding at 1:1 PC/PS. No binding was observed in PC liposomes. XIP and pentalysine-inhibitable bovine sarcolemmal (SL) Na-Ca exchange activity was observed in reconstituted proteoliposomes composed of 1:1 PC/PS. Proteolysis of SL membranes resulted in a twofold stimulation of Na-Ca exchange activity, but the half-maximal inhibitory concentration (IC50) for XIP (3 microM) was not significantly changed, suggesting that the XIP binding site remained intact. In contrast, the IC50 for pentalysine was decreased from 500 to 150 microM in proteolyzed membranes. These data are consistent with a model of Na-Ca exchange regulation in which the endogenous XIP domain interacts either with another region of the exchange protein to induce an inactive conformational state or with membrane lipid to produce an active conformation.

Characterization of cardiac Na+/Ca(2+)-exchange by site-directed polyclonal antibodies.

Cardiac Na+/Ca(2+)-exchange is an integral membrane protein consisting of approx. 970 amino acids with as many as 12 membrane-spanning and 11 extramembranal regions (Nicoll, D.A., Lognoni, S. and Philipson, K.D. (1985) Science 250, 562-565). Based upon primary sequence information, 3 amino-acid sequences located in either extramembranal segment a or f, consisting largely of acidic amino-acids, were selected for the production of synthetic peptides. The peptides were cross-linked to carrier ovalbumin and used to generate site-directed polyclonal antibodies (sd-Ab). Western blot analysis of bovine cardiac sarcolemmal (SL) proteins demonstrated that sd-Ab against segment a and 1 against loop f recognized a 70 kDa protein and a lower molecular mass band at 55 kDa under reducing conditions. A different loop f sd-Ab failed to recognize the 70 kDa protein but did associate with a 120, 65 and 55 kDa protein under the same conditions. Under non-reducing conditions, antibodies to all three peptides recognized the 65 kDa protein. All sd-Ab were blocked by addition of their respective peptides and were not inhibited by either of the other peptides. A sd-Ab against loop f was immobilized to an affinity support matrix and used to immunoprecipitate detergent solubilized cardiac SL vesicle protein. Immunoprecipitated protein was reconstituted into proteoliposomes which demonstrated Na+/Ca(2+)-exchange activity. Immunoprecipitated protein cross-reacted with sd-Ab against all three peptides with bands at 120, 70 and 55 kDa on Western blots. Tryptic digests of native SL vesicles abolished recognition of segment a sd-Ab for SL proteins while having little or no affect on reactivity to the protein by both sd-Ab against loop f. Digestion of the SL vesicle protein with endoproteinase Arg C did not alter sd-Ab recognition. The results suggest that specific domains of the cardiac Na+/Ca(2+)-exchanger depending upon the conformation of the protein, may not be available for antibody binding. The 70 kDa polypeptide appears to include the N-terminal region of the protein and what is believed to be a large cytoplasmic extramembranal loop. Limited proteolysis by trypsin and endoproteinase Arg C yielded results consistent with the model which places the N-terminus of the protein on the extracellular surface and a large extramembranal segment (loop f) on the cytoplasmic side of the SL membrane.

Interactions of the exchange inhibitory peptide with Na-Ca exchange in bovine cardiac sarcolemmal vesicles and ferret red cells.

The Na-Ca exchange inhibitory peptide (XIP), which corresponds to residues 251-270 of the Na-Ca exchange protein, specifically inhibits exchange activity (Li, Z., Nicoll, D. A, Collins, A., Hilgemann, D. W., Filoteo, A. G., Penniston, J. T., Weiss, J. N., Tomich, J. M., and Philipson, K. D. (1991) J. Biol. Chem. 266, 1014-1020). We have found that XIP decreased Na+i-dependent Ca2+ uptake to 46 and 20% of control in mixed and inside-out bovine sarcolemmal (SL) vesicles, respectively, and to 22% of control in ferret red cell vesicles. XIP inhibited uptake in bovine SL vesicles after proteolytic digestion. XIP also inhibited Na+o-dependent Ca2+ efflux in bovine SL vesicles but did not inhibit Ca2+ uptake in reconstituted proteoliposomes. Extracellular XIP did not inhibit Ca2+ uptake into intact ferret red cells. Inhibition of uptake in bovine SL vesicles was reduced as the ionic strength was increased. 125I-labeled XIP (1 microM) was cross-linked to proteins of bovine SL vesicles, ferret red cell vesicles, and intact ferret red cells. Labeling of bands at approximately 75, 120, and 220 kDa (in bovine SL vesicles) and bands at 55 and 85 kDa (in ferret red cell vesicles) was detected. No cross-linking was detected in intact ferret red cells. We conclude that XIP inhibition is insensitive to proteolytic digestion and is partially dependent on charge association and conformation of the exchanger. XIP binds to and interacts with the intracellular side of the Na-Ca exchanger.

Myocardial glucose utilization. Failure of adenosine to alter it and inhibition by the adenosine analogue N6-(L-2-phenylisopropyl)adenosine.

The effects of adenosine and the nonmetabolizable adenosine analogue N6-(L-2-phenylisopropyl)adenosine (PIA) on glucose transport or metabolism were determined in purified myocardial sarcolemmal vesicles, isolated cardiocytes, and perfused hearts. Adenosine (100 microM) did not affect hexose transport in myocytes. Also, adenosine deaminase, added to metabolize adenosine to inosine, did not alter transport of hexose into myocytes regardless of whether or not insulin was present. In contrast, PIA effectively inhibited 3-O-methyl-D-glucose uptake in myocytes even during insulin stimulation. PIA inhibited D-glucose-specific transport in both rat and bovine cardiac sarcolemmal vesicles (Ki = 26 microM at [D-glucose] = 5 mM). However, insulin did not affect glucose transport in sarcolemmal vesicles, which implies that receptor-coupled processes probably are not intact in this preparation. Thus, inhibition of PIA may not be receptor mediated. Also, PIA inhibited binding of cytochalasin B to bovine cardiac sarcolemmal vesicles, which supports the idea that PIA inhibits glucose flux by binding to the glucose transporter. To determine if adenosine altered glucose metabolism rather than transport, we measured the rate of 3H2O production from metabolism of D-[2-3H]glucose in paced rat hearts ([D-glucose] = 5.5 mM, [pyruvate] = 0.2 mM) perfused with a range of PIA or adenosine concentrations with or without 0.01 microM insulin. Adenosine (0.01-100 microM) in the presence or absence of insulin increased coronary flow but did not change glycolytic rates. Similar results were obtained with PIA (no insulin) rather than adenosine in the perfusate. However, with glucose as the only exogenous substrate, 100 microM PIA inhibited glycolysis by approximately 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical characterization of exercise-trained porcine myocardium.

The purpose of this study was to determine whether cardiac biochemical adaptations are induced by chronic exercise training (ET) of miniature swine. Female Yucatan miniature swine were trained on a treadmill or were cage confined (C) for 16-22 wk. After training, the ET pigs had increased exercise tolerance, lower heart rates during exercise at submaximal intensities, moderate cardiac hypertrophy, increased coronary blood flow capacity, and increased oxidative capacity of skeletal muscle. Myosin from both the C and ET hearts was 100% of the V3 isozyme, and there were no differences between the myosin adenosine triphosphatase (ATPase) or myofibrillar ATPase activities of C and ET hearts. Also, the sarcoplasmic reticulum Ca(2+)-ATPase activity and Na(+)-Ca2+ exchange activity of sarcolemmal vesicles were the same in cardiac muscle of C and ET hearts. Finally, the glycolytic and oxidative capacity of ET cardiac muscle was not different from control, since phosphofructokinase, citrate synthase, and 3-hydroxyacyl-CoA dehydrogenase activities were the same in cardiac tissue from ET and C pigs. We conclude that endurance exercise training does not provide sufficient stress on the heart of a large mammal to induce changes in any of the three major cardiac biochemical systems of the porcine myocardium: the contractile system, the Ca2+ regulatory systems, or the metabolic system.

Kinetic characterization and radiation-target sizing of the glucose transporter in cardiac sarcolemmal vesicles.

Stereospecific glucose transport was assayed and characterized in bovine cardiac sarcolemmal vesicles. Sarcolemmal vesicles were incubated with D-[3H]glucose or L-[3H]glucose at 25 degrees C. The reaction was terminated by rapid addition of 4 mM HgCl2 and vesicles were immediately collected on glass fiber filters for quantification of accumulated [3H]glucose. Non-specific diffusion of L-[3H]glucose was never more than 11% of total D-[3H]glucose transport into the vesicles. Stereospecific uptake of D-[3H]glucose reached a maximum level by 20 s. Cytochalasin B (50 microM) inhibited specific transport of D-[3H]glucose to the level of that for non-specific diffusion. The vesicles exhibited saturable transport (Km = 9.3 mM; Vmax = 2.6 nmol/mg per s) and the transporter turnover number was 197 glucose molecules per transporter per s. The molecular sizes of the cytochalasin B binding protein and the D-glucose transport protein in sarcolemmal vesicles were estimated by radiation inactivation. These values were 77 and 101 kDa, respectively, and by the Wilcoxen Rank Sum Test were not significantly different from each other.

Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles.

Na-Ca exchange activity in bovine cardiac sarcolemmal vesicles was stimulated up to 10-fold by preincubating the vesicles with 1 microM FeSO4 plus 1 mM dithiothreitol (DTT) in a NaCl medium. The increase in activity was not reversed upon removing the Fe and DTT. Stimulation of exchange activity under these conditions was completely blocked by 0.1 mM EDTA or o-phenanthroline; this suggests that the production of reduced oxygen species (H2O2, O2-.,.OH) during Fecatalyzed DTT oxidation might be involved in stimulating exchange activity. In agreement with this hypothesis, the increase in exchange activity in the presence of Fe-DTT was inhibited 80% by anaerobiosis and 60% by catalase. H2O2 (0.1 mM) potentiated the stimulation of Na-Ca exchange by Fe-DTT under both aerobic and anaerobic conditions; H2O2 also produced an increase in activity in the presence of either FeSO4 (1 microM) or DTT (1 mM), but it had no effect on activity by itself. Superoxide dismutase did not block the effects of Fe-DTT on exchange activity; however, the generation of O2-. by xanthine oxidase in the presence of an oxidizable substrate stimulated activity more than 2-fold. Hydroxyl radical scavenging agents (mannitol, sodium formate, sodium benzoate) did not attenuate the stimulation of activity observed with Fe-H2O2. Exchange activity was also stimulated by the simultaneous presence of glutathione (GSH; 1-2 mM) and glutathione disulfide (GSSG; 1-2 mM). Neither GSH nor GSSG was effective by itself and either 0.1 mM EDTA or o-phenanthroline blocked the effects on transport activity of the combination of GSH + GSSG. Treatment of the GSH and GSSG solutions with Chelex ion-exchange resin to remove contaminating transition metal ions reduced (by 40%) the degree of stimulation observed with GSH + GSSG. Full stimulating activity was restored to the Chelex-treated GSH and GSSG solutions by the addition of 1 microM Fe2+; Cu2+ was less effective than Fe2+ whereas Co2+ and Mn2+ were without effect. In the presence of 1 microM Fe2+, GSH alone produced a slight increase in transport activity, but this was markedly enhanced by the addition of Chelex-treated GSSG. The results indicate that stimulation of exchange activity requires the presence of both a reducing agent (DTT, GSH, O-.2, or Fe2+) and an oxidizing agent (H2O2, GSSG, and perhaps O2) and that the effects of these agents are mediated by metal ions (e.g. Fe2+).(ABSTRACT TRUNCATED AT 400 WORDS)