Ca2+ signalling in rat vascular smooth muscle cells: a role for protein kinase C at physiological vasoconstrictor concentrations of vasopressin.
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 4beta-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 microM) and calphostin C (250 nM) completely blocked the stimulation of Ca2+ spiking by either PMA or AVP. alpha, beta, gamma, delta, epsilon, zeta and &lamdda; 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 microM 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 microM 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.
Fan
J
J
Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA.
Byron
K L
KL
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002273:Carcinogens / Q000494:pharmacology; D002478:Cells, Cultured; D008387:Marine Toxins; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D010080:Oxazoles / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014662:Vasoconstrictor Agents / Q000494:pharmacology*; D014667:Vasopressins / Q000494:pharmacology*
D002273:Carcinogens; D008387:Marine Toxins; D010080:Oxazoles; D014662:Vasoconstrictor Agents; D014667:Vasopressins; C059041:calyculin A; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium; D002118:Calcium