Measurement of calcium entry and exit in quiescent rat ventricular myocytes

Abstract

The aim of this work was to obtain the first quantitative measurements of Ca2+ influx and efflux in quiescent cardiac cells. The relationship between free and total Ca2+ was obtained during a caffeine application. This buffering curve was then used to calculate changes of total Ca2+ from measurements of free cytosolic [Ca2+] ([Ca2+]i) made with Indo-1. The rate of Ca2+ removal from the cytoplasm was calculated by differentiating total Ca2+ with respect to time. The dependence of d(total Ca2+)/dt on [Ca2+]i was hyperbolic. Inhibition of either Na+-Ca2+ exchange (by addition of 10 mmol l(-1) NiCl2 or removal of external Na+) or the sarcolemmal Ca2+-activated adenosine triphosphatase (Ca2+-ATPase) (with carboxyeosin) decreased the calculated efflux. In both cases, the main effect was on the apparent maximum rate (Vmax) with little effect on the Michaelis-Menten constant (Km). These results suggest that the Na+-Ca2+ exchange and Ca2+-ATPase have very similar affinities for [Ca2+]i and that their fractional contributions do not change over the systolic range of [Ca2+]i. Ca2+ influx was quantified in two ways. The first method was to extrapolate the curve relating Ca2+ efflux to [Ca2+]i to zero [Ca2+]i. This gave a value of 4.49+/-0.54 micromol l(-1) s(-1) which was reduced to zero by either removal of external Ca2+ or addition of Ni2+. In other experiments external Ca2+ was removed and the maximum rate of fall of total Ca2+ calculated as 2.53+/-0.93 micromol l(-1) s(-1). This approach can be used to provide a quantitative analysis of the control of resting [Ca2+]i.

Cardiac Basal Metabolism.

“…However, the extent of inhibition was small-only 8%-and may have been below the detection limit of Takaki et al If we accept this value and that of the microcalorimetrically-measured rate of heat production of quiescent rat trabeculae presented in Table 2 (50-100 mW g dry Ϫ1 [67,68]), the cost of maintaining the resting concentration of intracellular calcium ion would be about 1-2 mW g Ϫ1 . Note that this value is of the same order (0.5 mW g Ϫ1 ) as that calculated from the data of Choi et al [174], above, and proposed as the lower limit of the metabolic cost of achieving basal Ca 2ϩ homeostasis in quiescent mammalian myocardium.…”

“…The study of Choi et al [174] revealed a small contribution to resting Ca 2ϩ influx via nifedipine-sensitive (i.e., L-type) Ca 2ϩ channels. The magnitude of this component, at a whole-cell patch-clamp potential of Ϫ40 mV, amounted to about 0.8 M s Ϫ1 or about 0.08 mW g Ϫ1 .…”

“…The latter values can be used to estimate the cost of achieving Ca 2ϩ homeostasis in the resting myocyte. We use the data of Choi et al [174] who have estimated the rate of Ca 2ϩ influx in quiescent rat myocytes to be about 5 M/s. This influx is inferred to be countered entirely by the sarcolemmal Na ϩ -Ca 2ϩ exchanger and Ca 2ϩ -ATPase (in the ratio of 70 : 30).…”

Na + -Ca 2+ Exchange Activity Is Localized in the T-Tubules of Rat Ventricular Myocytes

“…Block of the exchanger might alter local Ca i , thus altering Ca 2ϩ -activated currents. However, Choi et al 15 have shown that even when Ca i is increased using caffeine (ie, to levels higher than those in the present study, in which Ca i was buffered with BAPTA), no resulting current was observed in rat ventricular myocytes when the exchanger was inhibited by Ni or by the absence of bathing Na ϩ and Ca 2ϩ , making this unlikely.…”

Reducing Ryanodine Receptor Open Probability as a Means to Abolish Spontaneous Ca 2+ Release and Increase Ca 2+ Transient Amplitude in Adult Ventricular Myocytes

“…25 influx in rat ventricular myocytes. 30 The latter possibility is suggested by recent work indicating that during adrenergic stimulation, the calcium current can activate Ca 2ϩ entry on …”