The buffer barrier hypothesis, [Ca2+]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 [Ca2+] ([Ca2+]i) homogeneity and sarcoplasmic reticulum function in swine carotid artery. 2. We measured focal changes in [Ca2+]i by exploiting the different characteristics of several [Ca2+]i indicators: (1) aequorin, which can detect focal increases in [Ca2+]i such as those that occur in the subplasmalemmal region ([Ca2+]pm); (2) fura-2, which is primarily a measure of mean cytoplasmic [Ca2+] ([Ca2+]c); and (3) force, which reflects increases in [Ca2+] near the contractile apparatus. We then estimated the relative degree of [Ca2+]i homogeneity with the aequorin/fura-2 ratio. Finally, we inhibited sarcoplasmic reticulum Ca2+ pumping with cyclopiazonic acid (CPA), an inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). 3. We found that, after Ca2+ depletion, the sarcoplasmic reticulum could be partially reloaded with Ca2+ 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 [Ca2+]pm (as measured with aequorin) can partially reload the sarcoplasmic reticulum, but complete reloading required increases in [Ca2+]c (as measured with fura-2). Reloading could be partially inhibited by 10 microM CPA, indicating that SERCA function was important for reloading. 4. In unstimulated arteries, 10 microM CPA increased the fura-2 signal without altering the aequorin signal, thereby decreasing the aequorin/fura-2 ratio. Removal of extracellular Ca2+ without CPA also reduced the aequorin/fura-2 ratio. These data suggest that resting cells have a [Ca2+] gradient with [Ca2+]pm > [Ca2+]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 Ca2+ efflux in which Ca2+ pumping by SERCA reduces [Ca2+]c after stimulation. 6. Ca2+ depletion by prior treatment with 100 microM 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 Ca2+ 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 Ca2+ 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 [Ca2+] in at least two cytoplasmic compartments. [Ca2+]pm and [Ca2+]c can differentially regulate sarcoplasmic reticulum Ca2+ filling; and sarcoplasmic reticulum function regulates [Ca2+]pm and [Ca2+]c.
Rembold
C M
CM
Cardiovascular Division, Departments of Internal Medicine and Physiology, University of Virginia Health Science Center, Charlottesville, VA 22908, USA.
Chen
X L
XL
D000331:Aequorin; D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D017536:Carotid Artery, Common / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D006632:Histamine / Q000494:pharmacology; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D008955:Models, Cardiovascular; D009994:Osmolar Concentration; D012519:Sarcoplasmic Reticulum / Q000378:metabolism / Q000502:physiology*; D013552:Swine
D002021:Buffers; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007211:Indoles; D000331:Aequorin; D006632:Histamine; D002118:Calcium; D016257:Fura-2; C000543:cyclopiazonic acid