title: The buffer barrier hypothesis, [Ca2+]i homogeneity, and sarcoplasmic reticulum function in swine carotid artery.
abstract: 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.
authors.affiliation: Cardiovascular Division, Departments of Internal Medicine and Physiology, University of Virginia Health Science Center, Charlottesville, VA 22908, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Rembold
authors.forename: X L
authors.initials: XL
authors.lastname: Chen
mesh_terms: 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
chemical_list: D002021:Buffers; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007211:Indoles; D000331:Aequorin; D006632:Histamine; D002118:Calcium; D016257:Fura-2; C000543:cyclopiazonic acid