Excitation-contraction coupling was studied in mammalian cardiac cells in which the opening probability of L-type calcium (Ca2+) channels was reduced. Confocal microscopy during voltage-clamp depolarization revealed distinct local transients in the concentration of intracellular calcium ions ([Ca2+]i). When voltage was varied, the latency to occurrence and the relative probability of occurrence of local [Ca2+]i transients varied as predicted if Ca2+ release from the sarcoplasmic reticulum (SR) was linked tightly to Ca2+ flux through L-type Ca2+ channels but not to that through the Na-Ca exchanger or to average [Ca2+]i. Voltage had no effect on the amplitude of local [Ca2+]i transients. Thus, the most efficacious "Ca2+ signal" for activating Ca2+ release from the SR may be a transient microdomain of high [Ca2+]i beneath an individual, open L-type Ca2+ channel.

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SUMMARY1. The mechanisms that control release of Ca2+ from the sarcoplasmic reticulum (SR) of guinea-pig ventricular cells were studied by observing intracellular calcium concentration ([Ca2+]i transients) and membrane currents in voltage-clamped guinea-pig ventricular myocytes perfused internally with Fura-2.2. Sarcolemmal Ca2+ current was identified through the use of tetrodotoxin (TTX) and Ca2+ channel antagonists (verapamil) and agonists (Bay K 8644). 6. When a holding potential of -68 mV and TTX (30 /bM) were used, the most negative pulse potential at which activation of an inward current occurred was -49 mV while changes in [Ca2+]i occurred at -43 mV.7. Ryanodine-sensitive increases in [Ca2+]i elicited by repolarization (tail transients) were maximal for repolarization to 0 mV. Smaller changes in [Ca2+]i than maximal were elicited by repolarization to both more positive and more negative potentials than 0 mV. The peak amplitude of the verapamil-sensitive tail currents elicited by repolarization increased continuously as the membrane was repolarized to potentials more negative than 60 mV.8. Increasing depolarizing pulse duration beyond 10-20 ms did not increase the amplitude of the [Ca2+]i transient, but prolonged it.9. The experimental results are compared to the predictions of two theories on the

A key question in hypertension is: How is long-term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain-like compound (EOLC) and induces salt-dependent hypertension mediated by Na + /Ca 2+ exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na + pump α1 (α1 +/-) or α2 (α2 +/-) catalytic subunits. Both low-dose ouabain (1-100 nM; inhibits only α2) and high-dose ouabain (≥1 µM; inhibits α1) elevate myocyte Ca 2+ and constrict arteries from α1 +/-, as well as α2 +/-and wild-type mice. Nevertheless, only mice with reduced α2 Na + pump activity (α2 +/-), and not α1 (α1 +/-), have elevated blood pressure. Also, isolated, pressurized arteries from α2 +/-, but not α1 +/-, have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB-R7943) normalize myogenic tone in ouabain-treated arteries. Only the NCX blockers normalize the elevated myogenic tone in α2 +/-arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt-dependent and ouabain-induced hypertension. Thus, chronically reduced α2 activity (α2 +/-or chronic ouabain) apparently regulates myogenic tone and long-term blood pressure whereas reduced α1 activity (α1 +/-) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt-dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing α2 Na + pump activity and promoting Ca 2+ entry via NCX in myocytes.