External Na-independent Ca extrusion in cultured ventricular cells. Magnitude and functional significance.

Abstract

The relative magnitudes and functional significance of Ca extrusion by Na-Ca exchange and by an Na.-independent mechanism were investigated in monolayer cultures of chick embryo ventricular cells. Abrupt exposure of cells in 0-Na., nominally 0-Ca. solution to 20 mM caffeine produced a large contracture (3 .94 ± 0.90 um of cell shortening) that relaxed with a tl, of 8.60 ± 1 .22 s. An abrupt exposure to caffeine plus 140 mM Na resulted in a contracture that was smaller in amplitude (1 .53 ± 0.50,um) and relaxed much more rapidly (t,, = 0.77 ± 0 .09 s) . An abrupt exposure to caffeine in 0-Na. solutions produced an increase in "Ca efflux that persisted for 20 s, and a net loss of Ca content, determined by atomic absorption spectroscopy (AAS), of^-4 nmol/mg protein, within 35 s. A comparable net loss of Ca was demonstrated in the presence of 100 jM [Ca]o. The abrupt exposure of cultured cells to 0 Na. in 1 .8 mM Ca produced a Ca uptake, estimated with 45 Ca, of 3.2 nmol/mg protein -15 s, but produced no increase in cell Ca content (AAS). In cells in which a 30% increase in Nai was produced by 5 min exposure to 10 -6 M ouabain, the abrupt exposure to 0 Na. produced a Ca uptake of 6 nmol/mg protein -15 s and an increase in Ca content (AAS) of 4 nmol/mg protein. We conclude that there is an Na.-independent mechanism for Ca extrusion in these cells, presumably a Ca-ATPase Ca pump, with a limited Ca transport capacity of no more than 2 nmol/mg protein -15 s. This is five times smaller than the demonstrated maximum capacity of the Na-Ca exchanger in these cells. The relaxation of twitch tension in these cells seems to be dependent primarily on sarcoplasmic reticulum uptake of Ca, with a secondary role provided by the Na-Ca exchanger. The Ca pump appears to contribute little to beat-to-beat relaxation .

Contributions of [Ca2+]i, [Pi]i, and pHi to altered diastolic myocyte tone during partial metabolic inhibition.

“…Layer cultures of spontaneously contracting ventricular myocardial cells were prepared from 10-d-old chick embryos with a modification of previously described techniques (22,23). Ventricles were minced and placed in Ca2+ -Mg2+ free Hanks' solution with 0.25 mg/ml of trypsin at 37°C for four cycles of 7 min each.…”

Alterations in cation homeostasis in cultured chick ventricular cells during and after recovery from adenosine triphosphate depletion.

“…Spontaneously contracting layers of chick embryo ventricular cells were prepared as described previously (7). Briefly, ventricles from 10-d-old embryos were minced and placed in Ca-Mg-free Hanks' solution.…”

“…Effects of interventions on contractile behavior of cultured cells were determined by measurement of the amplitude of motion of individual cells within the culture. A video motion detector system was utilized as described previously (7).…”

“…Atomic absorption spectroscopy methods were used to detect changes in cellular contents of Na, K, and Ca (7). Glass coverslips with an adherent layer of cells were washed for 30 s in 4°C in Hepes-buffered 140 mM choline Cl solution.…”

“…The details of this procedure have already been described (7). Quantities were analyzed per mg cell protein, determined by the…”