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

摘要

class="enumerated" style="list-style-type:decimal">Physiological vasoconstrictor concentrations of Arg⁸-vasopressin (AVP, 10–100 pM) stimulate oscillations (spikes) in cytosolic free Ca²⁺ concentration ([Ca²⁺]i) 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.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.The protein kinase C (PKC) activator 4β-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 Ro-31–8220 (1 μM) and calphostin C (250 nM) completely blocked the stimulation of Ca²⁺ spiking by either PMA or AVP.α, β, γ, δ, ε, ζ and λ isoforms of PKC were detected in A7r5 cells by Western blot analysis. Time-dependent redistribution of PKC-α, -δ and -ε isoforms between the membrane and cytosolic fractions occurred in response to 100 pM AVP. Pretreatment for 24 h with 1 μM PMA downregulated expression of PKC-α and -δ, but not PKC-ε, and prevented the Ca²⁺-spiking responses to either 1 nM PMA or 100 pM AVP. Neither the release of intracellular Ca²⁺ by 1 μM AVP nor the increase in [Ca²⁺]i in response to elevated extracellular [K⁺] was prevented by the PMA pretreatment.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.