The giant cardiac membrane patch method: stimulation of outward Na(+)‐Ca2+ exchange current by MgATP.

Abstract

SUMMARY1. A giant patch method was used to study the stimulatory effect of cytoplasmic MgATP on outward Na+-Ca2+ exchange current in inside-out cardiac membrane patches (1-10 GQ seals with 14-24 jum pipette tip diameters) excised from guineapig, rabbit and mouse myocytes.2. To establish the validity of the method with respect to structure, bleb formation was examined with electron microscopy and with confocal fluorescence light microscopy. The blebs, which form as the sarcolemma detaches, excluded intracellular organelles and transverse tubules. The blebbed cells contained normal sarcomeres, sarcoplasmic reticulum, triads and diads.3. To further establish the validity of the method for ion transport studies, measurements of Na+-K+ pump currents and charge movements are described briefly which demonstrate (i) free access to the cytoplasmic membrane side, (ii) MgATP dependence comparable to reconstituted pump (Kd, 94 Ium), (iii) fast, rigorous concentration control and (iv) Na+-K+ pump densities in the range of whole-cell densities.4. Stimulation of outward Na+-Ca2+ exchange current by MgATP attenuated exchange current decay during step increments of cytoplasmic sodium, shifted the secondary activation of outward exchange current by cytoplasmic calcium to lower free calcium concentrations and, particularly in mouse cardiac sarcolemma, induced cytoplasmic calcium-independent current.5. Upon removal of MgATP the stimulatory effect usually decayed with a t50 (halftime) of about 3 min. However, the reversal took place much more rapidly (t50, 5-20 s) in patches from individual guinea-pig and rabbit myocyte batches. When decay was rapid, secondary activation by cytoplasmic calcium was shifted to higher free cytoplasmic calcium concentrations (Kd, 10-65 gM-free calcium).6. With repeated applications of MgATP the rate and magnitude of the stimulatory effect progressively decreased.7. The Kd for MgATP of the initial rate of stimulation of outward exchange current was 3 mm or greater. When decay was rapid, the steady-state dependence of exchange current on MgATP also had a Kd of 3 mm or greater. MS 8777A. COLLINS, A. V. SOMLYO AND D. W. HILGEMANN 8. Stimulation of Na'-Ca2+ exchange current by MgATP occurred in the absence of cytoplasmic calcium with 9 mm-EGTA.9. The stimulatory effect of 2 mM-MgATP was not inhibited by up to 200 uM of the protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine (H7), or by peptide inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and calcium-calmodulin-dependent protein kinase II.10. The stimulatory effect of MgATP was not mimicked by MgATP-y-S, and it was not reversed by acid phosphatase, alkaline phosphatase or an isolated cardiac protein phosphatase. Further, the effect was not enhanced nor was decay of the effect prolonged by 2 /LM of the phosphatase inhibitor, okadaic acid.11. We conclude that stimulation of Na'-Ca2' exchange current in excised sarcolemmal patches by MgATP is not a calcium-dependent process and probably does not involve protein kin...

Effect of metabolic inhibitors and second messengers upon Na(+)‐H+ exchange in the sheep cardiac Purkinje fibre.

“…Similarly, the Na+-K+ pump in red blood cells (Proverbio & Hoffman, 1977) and in pancreatic fl-cells (Niki, Ashcroft & Ashcroft, 1989) (Allen & Orchard, 1986). A low affinity of the exchanger for ATPi would be similar to that reported recently for the ATPi sensitivity of the cardiac Na'-Ca2" exchanger by Hilgemann and co-workers (Collins, Somlyo & Hilgemann, 1992 Allen et al 1985). A role here for Pi seems unlikely, however, since it rises considerably during CN-application but not during DOG application (Allen et al 1985) Counillon & Pouysseger, 1993) has so far been shown not to be modulated by cAMPi to the extent that it is not directly phosphorylated by cAMP-dependent kinase (Fliegel et al 1992).…”

Phosphorylation-dependent Regulation of Cardiac Na+/Ca2+ Exchanger via Protein Kinase C

“…5). It should also be pointed out that MgATP-dependent stimulation of the cardiac NCX1 activity under patch conditions was suggested not to involve protein kinases (15), as briefly discussed in the Introduction. One possibility compatible with both the present and this previous result could be that phosphorylation of an unidentified ancillary factor(s) missing from the excised membrane patches, but not the direct phosphorylation of the NCX1 protein, is required for the PKC-dependent regulation of the NCX1 activity.…”

“…of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5, Suita, Osaka 565, Japan. hanced by protein phosphatase inhibitor okadaic acid (15), suggesting no direct effect of ATP or involvement of protein kinases. Consistent with these findings is a recent report that phosphorylation of cardiac NCX1 isoform was not detected in 32 P-labeled Chinese hamster ovary or COS cells that stably expressed the bovine cardiac NCX1 (14).…”

“…Na ϩ /Ca 2ϩ exchange in cardiomyocytes is highly modulated by several cytosolic factors; it is activated by cytosolic Ca 2ϩ and MgATP (10) but is inhibited by cytosolic Na ϩ (11), cytoplasmic H ϩ (12), and ATP depletion (13,14). MgATP has been shown to modulate the regulation by cytosolic Ca 2ϩ and Na ϩ in excised membrane patches from cardiomyocytes (15,16). These regulatory effects of Ca 2ϩ , Na ϩ , H ϩ , and ATP depletion are thought to be mediated by the cytoplasmic domain of the exchanger, because they are abolished by treating the cytoplasmic surface of the exchanger with chymotrypsin (10,12) or by deletion mutagenesis of the cytoplasmic domain (6,14).…”

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

“…This effect would occur in parallel with other factors that modulate NCX during changes in metabolism. For example, ATP depletion causes a significant decrease in cardiac NCX activity (19,20,30,31), and lactate accumulation not only increases cytosolic NADH but also mediates acidosis. Decreased pH inhibits NCX activity (32), which is fully reversible after pH normalization (33).…”