TIME-DEPENDENT CHANGES IN CA2+ SENSITIVITY DURING PHASIC CONTRACTION OF CANINE ANTRAL SMOOTH-MUSCLE

1. Relationships between cytosolic Ca2+ concentration ([Ca2+]cyt), myosin light chain (MLC) phosphorylation and muscle tension were examined in circular smooth muscle of canine gastric antrum. 2. Electrical slow waves induced a transient increase in [Ca2+]cyt and muscle tension. [Ca2+]cyt increased before the initiation of contraction and reached a maximum before the peak of the phasic contractions. Following the first Ca2+ transient, a second rise in [Ca2+]cyt was often observed. The second Ca2+ transient was of similar magnitude to the first, but only in some cases was this increase in [Ca2+]cyt associated with a second phase of contraction. Relaxation occurred more rapidly than the restoration of resting levels of [Ca2+]cyt. 3. Acetylcholine (ACh; 3 x 10(-7) M) increased the amplitude of Ca2+ transients, caused MLC phosphorylation and increased the force of contraction. The decay of contraction and MLC dephosphorylation preceded that of [Ca2+]cyt. 4. Increasing external K+ (to 25-40 mM) caused a sustained increase in [Ca2+]cyt, but little change in resting tension. This suggests that the Ca2+ sensitivity decreased as [Ca2+]cyt, increased. Increasing K+ to 59.5 mM further increased the level of [Ca2+]cyt, induced MLC phosphorylation and caused a transient contraction. When normal levels of K+ were restored, the rates of MLC dephosphorylation and relaxation exceeded the rate of decay in [Ca2+]cyt. 5. Removal of external Ca2+ in depolarized muscles decreased [Ca2+]cyt below the resting level without affecting resting tension. Readmission of Ca2+ to depolarized muscles caused force to develop at [Ca2+]cyt levels below the original resting level, suggesting that Ca2+ sensitivity was increased when the resting level of [Ca2+]cyt was decreased. 6. The phosphatase inhibitor, calyculin-A (10(-6) M), induced tonic contraction and MLC phosphorylation without an increase in [Ca2+]cyt. During these contractures, electrical activity caused transient increases in [Ca2+]cyt and phasic contractions which were superimposed upon the Ca2+-independent contracture. In the presence of calyculin-A, relaxation occurred in two phases. The initial, rapid phase of relaxation was not significantly affected by calyculin-A, but the slow phase was significantly decreased. 7. These results suggest that the relationship between [Ca2+]cyt, MLC phosphorylation and contraction changes as a function of [Ca2+]cyt in canine antral muscles. This may be due to a Ca2+- and time-dependent phosphatase that regulates the level of myosin phosphorylation.