Calmodulin Stimulation of Plasmalemmal Ca ²⁺-Pump of Canine Aortic Smooth Muscle

Abstract

Plasma-membrane-enriched fractions of canine aortic smooth muscle possess an ATP-supported Ca²⁺ accumulation which has an absolute requirement for Mg²⁺ and a high affinity for Ca²⁺ (Km ∼ 0.5 µM). The rate of ATP-supported Ca²⁺ transport is not affected by several calmodulin antagonists, but is stimulated by exogenously added calmodulin. The maximal effect of calmodulin on the rate of ATP-dependent Ca²⁺ transport (at 5.0 µM Ca²⁺) occurs at 10 µg/ml calmodulin and represents an approximately 3-fold stimulation. This calmodulin stimulation of Ca²⁺ transport does not require pretreatment of the membranes by EGTA and is an intrinsic property of the plasma membranes. A high-affinity Ca²⁺-ATPase (K_m for Ca²⁺ ∼0.5 µM) is also present in the aortic smooth muscle plasma membrane. This high-affinity Ca²⁺-ATPase does not require Mg²⁺ for catalytic activity, but is in fact inhibited by increasing Mg²⁺ concentrations. Calmodulin at concentrations effective for the stimulation of the ATP-dependent Ca²⁺ transport has no effect on the high-affinity Ca²⁺-ATPase activity or on the basal ATPase activity stimulated by 5 mM Mg²⁺ or Ca²⁺. Our results indicate that isolated plasma membranes of canine aortic smooth muscle contain no endogenous calmodulin. The ability of exogenously added calmodulin to stimulate the rate of ATP-dependent Ca²⁺ transport by vascular smooth muscle plasma membranes suggests that calmodulin may play a role in lowering the cytoplasmic concentration of ionized calcium during vasodilatation. An Mg²⁺-independent, but not an Mg²⁺-dependent high-affinity Ca²⁺-ATPase, was identified in the plasma membranes. This may be separate from the plasmalemmal Ca²⁺-pump.