Modulation of Ca2+ channels by cyclic nucleotide cross activation of opposing protein kinases in rabbit portal vein.

Cyclic nucleotides are known to modify voltage-gated (L-type) Ca2+ channel activity in vascular smooth muscle cells, but the exact mechanism(s) underlying these effects is not well defined. The purpose of the present study was to investigate the modulatory role of the cAMP- and cGMP-dependent protein kinase (PKA and PKG, respectively) pathways in Ca2+ channel function by using both conventional and perforated-patch-clamp techniques in rabbit portal vein myocytes. The membrane-permeable cAMP derivative, 8-bromo cAMP (0.1 to 10 micromol/L), significantly increased (14% to 16%) peak Ba2+ currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba2+ currents (23% to 31%). In contrast, 8-bromo cGMP inhibited Ba2+ currents at all concentrations tested (0.01 to 1 mmol/L). Basal Ca2+ channel currents were significantly inhibited by the PKA blocker 8-Bromo-2'-O-monobutyryladenosine-3',5'-monophosphorothioate, Rp-isomer (Rp 8-Br-MP cAMPS, 30 micromol/L) and enhanced by the PKG inhibitor beta-Phenyl-1,N2-etheno-8-bromoguanosine-3',5'-monophosphorothioate, Rp-isomer (Rp-8-Br PET cGMPS, 10 nmol/L). In the presence of Rp 8-bromo PET cGMPS (10 to 100 nmol/L), both 8-bromo cAMP (0.1 mmol/L) and 8-bromo cGMP (0.1 mmol/L) enhanced Ba2+ currents (13% to 39%). The excitatory effect of 8-bromo cGMP was blocked by Rp 8-bromo MB-cAMPS. Both 8-bromo cAMP (0.05 mmol/L) and forskolin (10 micromol/L) elicited time-dependent effects, including an initial enhancement followed by suppression of Ba2+ currents. Ba2+ currents were also enhanced when cells were dialyzed with the catalytic subunit of PKA. This effect was reversed by the PKA blocker KT 5720 (200 nmol/L). Our results suggest that cAMP/PKA stimulation enhances and cGMP/PKG stimulation inhibits L-type Ca2+ channel activity in rabbit portal vein myocytes. Our results further suggest that both cAMP and cGMP have a primary action mediated by their own kinase as well as a secondary action mediated by the opposing kinase.

[1]  Kim Cooper,et al.  Low access resistance perforated patch recordings using amphotericin B , 1991, Journal of Neuroscience Methods.

[2]  E. Arnold,et al.  Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP. , 1994, The American journal of physiology.

[3]  K. Sanders,et al.  Modulation of Ca2+ current in canine colonic myocytes by cyclic nucleotide-dependent mechanisms. , 1996, The American journal of physiology.

[4]  T. Ishikawa,et al.  Regulation of Ca2+ channels by cAMP and cGMP in vascular smooth muscle cells. , 1993, Circulation research.

[5]  G. Isenberg,et al.  Calcium channel current of vascular smooth muscle cells: extracellular protons modulate gating and single channel conductance , 1994, The Journal of general physiology.

[6]  T. Lincoln,et al.  Towards an understanding of the mechanism of action of cyclic AMP and cyclic GMP in smooth muscle relaxation. , 1991, Blood vessels.

[7]  J. Corbin,et al.  Relaxation of vascular and tracheal smooth muscle by cyclic nucleotide analogs that preferentially activate purified cGMP-dependent protein kinase. , 1988, Molecular pharmacology.

[8]  T. Schneider,et al.  Calcium channels: Structure, function, and classification , 1994 .

[9]  H. Yokoshiki,et al.  Regulation of Ca2+ channel currents by intracellular ATP in smooth muscle cells of rat mesenteric artery. , 1997, The American journal of physiology.

[10]  R. Cox,et al.  GTP requirement for isoproterenol activation of calcium channels in vascular myocytes. , 1995, The American journal of physiology.

[11]  N. Sperelakis,et al.  Regulation of L-type calcium channels of vascular smooth muscle cells. , 1995, Journal of molecular and cellular cardiology.

[12]  T. Lincoln,et al.  Regulation of the expression of cyclic GMP-dependent protein kinase by cell density in vascular smooth muscle cells. , 1994, Journal of Vascular Research.

[13]  D. Øgreid,et al.  Studies of cGMP analog specificity and function of the two intrasubunit binding sites of cGMP-dependent protein kinase. , 1986, The Journal of biological chemistry.

[14]  E. Degerman,et al.  Type III cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE3 gene family). , 1995, Cellular signalling.

[15]  F. Franciolini,et al.  Chloride channels of biological membranes. , 1990, Biochimica et biophysica acta.

[16]  J. Corbin,et al.  Direct evidence for cross-activation of cGMP-dependent protein kinase by cAMP in pig coronary arteries. , 1992, The Journal of biological chemistry.

[17]  F. Hofmann,et al.  Phosphorylation of cGMP-dependent protein kinase increases the affinity for cyclic AMP. , 1986, European journal of biochemistry.

[18]  U. Walter,et al.  Characterization of Sp-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole- 3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells. , 1991, The Biochemical journal.

[19]  M. Morad,et al.  Characteristics of calcium currents in rabbit portal vein myocytes. , 1992, The American journal of physiology.

[20]  B. Albat,et al.  Voltage-gated calcium channel currents in human coronary myocytes. Regulation by cyclic GMP and nitric oxide. , 1997, The Journal of clinical investigation.

[21]  M A Portman,et al.  Maturational changes in respiratory control through creatine kinase in heart in vivo. , 1992, The American journal of physiology.

[22]  W. Sterling Edwards,et al.  Blood Vessels , 1959 .

[23]  H. Liu,et al.  Cyclic nucleotides regulate the activity of L-type calcium channels in smooth muscle cells from rat portal vein. , 1997, Journal of molecular and cellular cardiology.

[24]  T. Lincoln,et al.  Phosphorylation of the Inositol 1,4,5-Trisphosphate Receptor , 1996, The Journal of Biological Chemistry.

[25]  O. McManus.  Calcium-activated potassium channels: Regulation by calcium , 1991, Journal of bioenergetics and biomembranes.

[26]  T. Mcdonald,et al.  Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. , 1994, Physiological reviews.

[27]  J. Simard,et al.  Sodium nitroprusside and cGMP decrease Ca2+ channel availability in basilar artery smooth muscle cells , 1996, Pflügers Archiv.

[28]  W. Stec,et al.  Specificity of cyclic GMP activation of a multi-substrate cyclic nucleotide phosphodiesterase from rat liver. , 2005, European journal of biochemistry.

[29]  T. Ishikawa,et al.  Intracellular divalent cations block smooth muscle K+ channels. , 1993, Circulation research.

[30]  J. P. Huggins,et al.  Inhibition of cyclic GMP‐dependent protein kinase‐mediated effects by (Rp)‐8‐bromo‐PET‐cyclic GMPS , 1995, British journal of pharmacology.

[31]  J. Polson,et al.  Cyclic nucleotide phosphodiesterases and vascular smooth muscle. , 1996, Annual review of pharmacology and toxicology.

[32]  C. Fenoglio-Preiser,et al.  Regulation of L-type calcium channels by cyclic nucleotides and phosphorylation in smooth muscle cells from rabbit portal vein. , 1994, Journal of vascular research.

[33]  C. Murakata,et al.  K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. , 1987, Biochemical and biophysical research communications.

[34]  T. Lincoln,et al.  cGMP-dependent protein kinase mediates the reduction of Ca2+ by cAMP in vascular smooth muscle cells. , 1990, The American journal of physiology.