Ca2+ signalling in rat vascular smooth muscle cells:: a role for protein kinase C at physiological vasoconstrictor concentrations of vasopressin

1. Physiological vasoconstrictor concentrations of Arg(8)-vasopressin (AVP, 10-100 pw) stimulate oscillations (spikes) in cytosolic free Ca2+ concentration ([Ca2+](1)) in A7r5 rat vascular smooth muscle cells. These Ca2+ spikes are dependent on L-type voltage-sensitive Ca2+ channels and increase in frequency with increasing AVP concentration. The signal transduction pathway responsible for this effect was examined in fura-2-loaded A7r5 cell monolayers. 2. The serine/threonine phosphatase inhibitor calyculin A (5 nM) sensitized A7r5 cells to AVP, resulting in the stimulation of Ca2+ spiking by 1-10 pM AVP. Calyculin A alone did not stimulate Ca2+ spiking. 3. The protein kinase C (PRC) activator 4 beta-phorbol 12-myristate 13-acetate (PMA, 100 pM to 200 nM), also stimulated Ca2+ spiking and this effect was additive with a submaximal concentration of AVP (50 pM). The PKC inhibitors Re-31-8220 (1 mu M) and. calphostin C (250 nM) completely blocked the stimulation of Ca2+ spiking by either PMA or AVP. 4. alpha, beta, gamma, delta, epsilon, zeta and lambda isoforms of PKC were detected in A7r5 cells by Western blot analysis. Time-dependent redistribution of PKC-alpha, -delta and -epsilon isoforms between the membrane and cytosolic fractions occurred in response to 100 pM AVP. Pretreatment for 24 h with 1 mu M PMA downregulated expression of PKC-alpha, and -delta, but not PKC-epsilon, and prevented the Ca2+ spiking responses to either 1 nM PMA or 100 pM AVP. Neither the release of intracellular Ca2+ by 1 mu M AVP nor the increase in [Ca2+](1) in response to elevated extracellular [K+] was prevented by the PMA pretreatment. 5. We conclude that PKC activation is a necessary step in the signal transduction pathway linking low concentrations of AVP to Ca2+ spiking in A7r5 cells.