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Pflügers Archiv

, Volume 428, Issue 1, pp 9–16 | Cite as

Protein kinase A increases availability of calcium channels in smooth muscle cells from guinea pig basilar artery

  • Kirti Tewari
  • J. Marc Simard
Excitable Tissues and Central Nervous Physiology

Abstract

We studied single Ca2+ channels in smooth muscle cells from the basilar artery of the guinea pig using conventional patch-clamp techniques. With 40 mM or 90 mM Ba2+ as the charge carrier, a 23-pS inward current channel was observed in 46/187 cell-attached patches studied without the dihydropyridine, BAY K8644, in the pipette solution. At 0 mV, this channel exhibited short and long openings with time constants of 1.03 and 3.65 ms, respectively. The probability of channel opening was voltage dependent with half-activation occurring at +9.9 mV. In 14/26 patches tested, addition of 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) to the bath increased the probability of opening at -10 mV by a factor of 2.6, from 0.0272±0.0429 to 0.0695±0.0788 (P <0.01, paired t-test). Mean data from five patches fit to a Boltzmann function indicated that at positive potentials, the probability of opening increased by a factor of 1.7, from 0.352 to 0.600, whereas the voltage dependence, the number of channels, the number of open states, the time constants of the open states, and the proportion of time spent in each open state were unchanged. When BAY K8644 was added to the pipette solution, the 23-pS channel was observed in nearly all patches (62/66), but the voltage dependence of activation was shifted −15.3 mV compared to control. In some patches studied with 90 mM Ba2+, a 9-pS inward current channel also was observed and its activity also was increased significantly by 8-Br-cAMP. When membrane patches were excised from the cell and studied in an inside-out configuration, single-channel activity due to the 23-pS channel lasted 1–3 min before being irreversibly lost, regardless of the presence of BAY K8644 in the pipette or of 8-Br-cAMP plus Mg · ATP and leupeptin in the bath. Subsequent addition of the catalytic subunit of protein kinase A (PKACS) did not restore Ca2+ channel activity. Conversely, when patches were excised into a solution already containing 8-Br-cAMP plus Mg · ATP, leupeptin and PKACS, channel activity was prominent and generally lasted until the seal was lost, or until the experiment was terminated at 30–45 min, unless protein kinase inhibitor also was present, in which case channel lifetime was short. Our findings indicate that availability of the L-type Ca2+ channel in basilar artery smooth muscle cells is increased by activation of cAMP-dependent protein kinase A, and that the (or one of the) phosphoprotein(s) involved may not be membrane bound.

Key words

Ca2+ channel Smooth muscle cell Basilar artery 

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Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Kirti Tewari
    • 1
  • J. Marc Simard
    • 2
  1. 1.Department of PhysiologyUniversity of Maryland School of MedicineBaltimoreUSA
  2. 2.Department of Surgery (Neurological Surgery)University of Maryland School of MedicineBaltimoreUSA

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