Regulation of cytosolic free Ca2+ concentration in vascular smooth muscle cells by A- and C-kinases.
The cytosolic free Ca2+ concentration [( Ca2+]i) was measured in cultured human umbilical vein smooth muscle cells using fura-2 as a Ca2+ indicator and microscopic digital image analysis system. Activation of cells with histamine and vasopressin resulted in a prompt though transient rise in [Ca2+]i 10- to 12-fold higher than the resting [Ca2+]i. The [Ca2+]i then declined rapidly during the first 30-40 seconds after hormonal stimulation and then gradually decreased to near resting levels in 2-3 minutes in the continued presence of hormones. The magnitude of the increase in peak [Ca2+]i was similar in buffered salt solution containing 1.8 mM Ca2+, zero Ca2+, and zero Ca2+ buffered salt solution containing 10 mM La3+, suggesting that receptor-mediated increase in [Ca2+]i is primarily due to the release of Ca2+ from the intracellular stores. Addition of La3+ produced oscillations in [Ca2+]i in approximately half the cells in response to both hormones. Addition of 10 microM forskolin did not significantly affect the resting [Ca2+]i, the hormone-stimulated peak [Ca2+]i, or the time course of hormone-stimulated [Ca2+]i transients. These data suggest that mechanisms involved in A-kinase-mediated smooth muscle relaxation may be subsequent to the changes in [Ca2+]i. Activation of C-kinase by 1 microM 12 deoxyphorbol 13-isobutyrate-20 acetate (DPBA) did not affect the resting [Ca2+]i, though it attenuated the histamine and vasopressin-mediated peak elevation in [Ca2+]i. Since DPBA inhibited the peak [Ca2+]i response to both the hormones to the same extent, it would appear that C-kinase activation may uncouple the receptor-mediated activation of phospholipase C.
Sharma
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RV
Department of Anatomy, University of Iowa College of Medicine, Iowa City 52242.
Bhalla
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RC
D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology*; D011494:Protein Kinases / Q000502:physiology*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism; D014667:Vasopressins / Q000494:pharmacology
D001572:Benzofurans; D005456:Fluorescent Dyes; D010703:Phorbol Esters; D014667:Vasopressins; D005576:Colforsin; C047907:12-deoxyphorbol-13-isobutyrate-20-acetate; D006632:Histamine; D011494:Protein Kinases; D011493:Protein Kinase C; D002118:Calcium; D016257:Fura-2