Na+/K+/Cl− cotransport is stimulated by a Ca++‐calmodulin–;mediated pathway in BALB/c 3T3 fibroblasts

In the present study, we investigated the role of intracellular Ca++ in the stimulation of the Na+/K+/Cl– cotransport in synchronized BALB/c 3T3 cells. The Na+/K+/Cl– cotransport was stimulated by the growth factors EGF, TGF‐α, IGF‐1, and IGF‐2, which do not activate protein kinase C, but do induce a transient increase in free cytoplasmic Ca++ In addition, direct activation of protein kinase C by the phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) did not affect the Na+/K+/Cl– cotransport activity of quiescent cells. The Na+/K+/Cl– cotransport was also stimulated by the above mitogens in cells pretreated with the phorbol ester TPA. This treatment led to a progressive decline in the activity of cellular protein kinase C. This result implies that cells deficient in protein kinase C may still support stimulation of the Na+/K+/Cl– cotransport. Taken as a whole, these findings suggest that the Na+/K+/Cl– cotransport is stimulated predominantly by a protein kinase C‐independent mechanism in BALB/c 3T3 fibroblasts. Both the intracellular Ca++ antagonist 8‐(N,N‐diethylamino)octyl‐3,4,5‐trimethoxybenzoate (TMB‐8) and two potent calmodulin antagonists, trifluoperazine (TFP) and chloropromazine(CP), blocked serum‐ and mitogen‐stimulated Na+/K+/Cl– cotransport. These results suggest that the Na+/K+/Cl– cotransport is stimulated by an increase of intracellular Ca++ and subsequently by a Ca++‐calmodulin‐mediated pathway in the synchronized BALB/c 3T3 fibroblasts.