The buffer barrier hypothesis, [Ca 2+ ] i homogeneity, and sarcoplasmic reticulum function in swine carotid artery

1 The goal of this study was to evaluate the buffer barrier hypothesis in an intact arterial smooth muscle. Specifically, we investigated the interrelationships between intracellular [Ca 2+ ] ([Ca 2+ ] i ) homogeneity and sarcoplasmic reticulum function in swine carotid artery. 2 We measured focal changes in [Ca 2+ ] i by exploiting the different characteristics of several [Ca 2+ ] i indicators: (1) aequorin, which can detect focal increases in [Ca 2+ ] i such as those that occur in the subplasmalemmal region ([Ca 2+ ] pm ); (2) fura‐2, which is primarily a measure of mean cytoplasmic [Ca 2+ ] ([Ca 2+ ] c ); and (3) force, which reflects increases in [Ca 2+ ] near the contractile apparatus. We then estimated the relative degree of [Ca 2+ ] i homogeneity with the aequorin/fura‐2 ratio. Finally, we inhibited sarcoplasmic reticulum Ca 2+ pumping with cyclopiazonic acid (CPA), an inhibitor of the sarco(endo)plasmic reticulum Ca 2+ ‐ATPase (SERCA). 3 We found that, after Ca 2+ depletion, the sarcoplasmic reticulum could be partially reloaded with Ca 2+ by manipulations that increased the aequorin signal relatively more than the fura‐2 signal. Complete reloading required large increases in the fura‐2 signal. These data suggest that increases in [Ca 2+ ] pm (as measured with aequorin) can partially reload the sarcoplasmic reticulum, but complete reloading required increases in [Ca 2+ ] c (as measured with fura‐2). Reloading could be partially inhibited by 10 μM CPA, indicating that SERCA function was important for reloading. 4 In unstimulated arteries, 10 μM CPA increased the fura‐2 signal without altering the aequorin signal, thereby decreasing the aequorin/fura‐2 ratio. Removal of extracellular Ca 2+ without CPA also reduced the aequorin/fura‐2 ratio. These data suggest that resting cells have a [Ca 2+ ] gradient with [Ca 2+ ] pm > [Ca 2+ ] c ; this gradient is maintained by SERCA function. 5 CPA slowed the decline in the fura‐2 signal observed when histamine stimulation was removed. This result is consistent with the concept of vectorial Ca 2+ efflux in which Ca 2+ pumping by SERCA reduces [Ca 2+ ] c after stimulation. 6 Ca 2+ depletion by prior treatment with 100 μM histamine and CPA transiently attenuated subsequent histamine‐induced aequorin and fura‐2 transients. The effect on contraction was smaller: a delay in contraction of approximately 10 s. These data suggest that histamine‐induced Ca 2+ release has at least a small role in the initial phase of contraction; however, other contractile mechanisms appear to be able to compensate for loss of Ca 2+ release with only modest changes in contraction kinetics. 7 These data suggest that there is a complex interrelationship between smooth muscle sarcoplasmic reticulum function and [Ca 2+ ] in at least two cytoplasmic compartments. [Ca 2+ ] pm and [Ca 2+ ] c can differentially regulate sarcoplasmic reticulum Ca 2+ filling; and sarcoplasmic reticulum function regulates [Ca 2+ ] pm and [Ca 2+ ] c .