Plasma membrane Ca2+ transport: Antagonism by several potential inhibitors

Abstract

Inside-out vesicles prepared from human red blood cells took up Ca2+ by an active transport process. Membranes from the same red blood cells displayed Ca2+-activated, Mg2+-dependent adenosine triphosphatase activity. Both the initial rate of Ca2+ transport and the (Ca2+ + Mg2+)-adenosine triphosphatase activity were increased approximately twofold by the calcium binding protein, calmodulin. Activities in the absence of added calmodulin were termed basal activities. Calmodulin-activated Ca2+ transport and adenosine triphosphatase activities could be antagonized in a relatively selective fashion by the phenothiazine tranquilizer drug, trifluoperazine. High concentrations of trifluoperazine also inhibited basal Ca2+ transport and adenosine triphosphatase activity. By contrast, calmodulin binding protein from beef brain selectively antagonized the effect of calmodulin on Ca2+ transport with no inhibition of basal activity. Ruthenium red antagonized calmodulin-activated and basal activity with equal potency. The results demonstrate that although phenothiazines can act as relatively selective antagonists of calmodulin-induced effects, other effects are possible and cannot be ignored. Calmodulin-binding protein may be a useful tool in the analysis of calmodulin functions. Ruthenium red probably interacts with Ca2+ pump adenosine triphosphatase at a site not related to calmodulin.

α-Amylase Secretion by Single Barley Aleurone Layers

“…Ruthenium red is known to block Ca2+ transport (9,19), and Ca"+ was verified by a switch to 0 Ca2" and return to high Ca2". The aleurone layer was exposed to a I to 2 min pulse of ruthenium red (5 mg ml-') by immersion of the flow-through chamber in this solution.…”

“…4 Experiments with ruthenium red also provide convincing evidence that the effects of Ca2" are on the secretory process. This compound has been shown to be a potent inhibitor of Ca2" uptake and efflux by animal cells and mitochondria (9,19). Ruthenium red also interferes with Ca2' efflux from aleurone layers of barley, although it is without effect on the release of Mg2+ from this tissue.…”

Thyrotropin-releasing hormone mobilizes Ca2+ from endoplasmic reticulum and mitochondria of GH3 pituitary cells: characterization of cellular Ca2+ pools by a method based on digitonin permeabilization.

“…The latter could be the indirect result of mitochondrial inhibition (e.g., altered ATP levels) or, alternatively, the result of direct effects on ER to release Ca2+ as suggested by Shoshan et al for sarcoplasmic reticulum (19). Trifluoperazine acted selectively to deplete Ca2+ from pool I. Ca2+ ATPases have been correlated with Ca2+ pump activity (20) and these may be TFP sensitive (21). A TFPsensitive Ca2`ATPase activity is present in GH3 microsomes and ATP-dependent Ca2+ uptake into pool I in GH3 cells permeabilized with 0.003% digitonin is TFP sensitive as well (unpublished results).…”

A high affinity Ca2+‐ATPase in C57 black mouse liver plasma membranes

“…Then, effect of trifluoperazine on the high affinity Ca2*-ATPase was studied. Trifluoperazine up to 100/aM, a concentration inhibiting 100% of the (Ca 2* + Mg2+)-ATPase of erythrocyte [16], caused no significant inhibition of the enzyme activity ( fig.3). Higher concentrations of the drug inhibited the enzyme activity.…”

“…The membrane may have contained a sufficient amount of calmodulin for satisfying its requirement for the enzyme. Trifluoperazine, a calmodulin-antagonist, inhibits the Ca2*-pumping ATPase in erythrocyte membrane [16]. This effect is thought to be because of binding of the drug to calmodulin.…”