Desensitization of swine arterial smooth muscle to transplasmalemmal Ca2+ influx.
1. The effect of transplasmalemmal Ca2+ influx on the [Ca2+]i dependence of smooth muscle contraction was evaluated by measuring intracellular [Ca2+] (as estimated by aequorin), myosin phosphorylation, and isometric stress in swine carotid media. 2. Extracellular Ca2+ was removed by incubation in physiological saline with 1 mM-EGTA and no added CaCl2 for 20 min (termed EGTA treatment). In some preparations, intracellular Ca2+ was released by a brief (5 min) histamine stimulation while in this Ca2(+)-free EGTA solution (termed histamine treatment). 3. Restoration of extracellular CaCl2 to EGTA and histamine-treated preparations in the continued presence of histamine was associated with an initial large aequorin light transient. However, this light transient was not initially associated with an increase in myosin phosphorylation or rapid stress development, suggesting that the contractile apparatus was desensitized to aequorin-estimated myoplasmic [Ca2+]. The desensitization was temporary, and resolved by 10 min after restoration of extracellular CaCl2. 4. The light transient observed upon restoration of extracellular CaCl2 was smaller in preparations only EGTA treated when compared to preparations treated with both EGTA and histamine, suggesting that histamine treatment further desensitized the contractile apparatus. 5. The stress development rate was not slowed when histamine and extracellular CaCl2 were simultaneously added to EGTA-treated preparations, suggesting that the desensitization was only to transplasmalemmal Ca2+ influx (from extracellular CaCl2 readdition), and not intracellular Ca2+ release (from the histamine stimulation). 6. In EGTA and histamine-treated preparations, restoration of extracellular CaCl2 in the presence of 109 mM-KCl was associated with a larger aequorin light signal than was observed upon readdition of CaCl2 in the presence of histamine, suggesting that depolarization also further desensitized the contractile apparatus. 7. Depolarization of EGTA-treated preparations did not increase [Ca2+] or stress, suggesting that depolarization did not release intracellular Ca2+ stores. 8. No significant light transient was observed upon addition of extracellular LaCl3, suggesting that tissue damage or leakage of aequorin into the extracellular space was not the cause of the Ca2(+)-reintroduction light signal. 9. These data suggest that removal of extracellular CaCl2 desensitizes the contractile apparatus of smooth muscle to transplasmalemmal Ca2+ influx. This desensitization is only to readdition of extracellular Ca2+; the contractile apparatus still responds to intracellular Ca2+ release. The desensitization is increased by prior depolarization or brief histamine treatment (potentially by depleting intracellular Ca2+). The source of activator Ca2+ appears to affect the relationship between aequorin light and phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
Rembold
C M
CM
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.
D000331:Aequorin; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000502:physiology*; D009218:Myosins / Q000378:metabolism; D010766:Phosphorylation; D010783:Photometry; D013552:Swine
D000331:Aequorin; D004533:Egtazic Acid; D006632:Histamine; D000242:Cyclic AMP; D009218:Myosins; D002122:Calcium Chloride; D002118:Calcium