Focal [Ca2+]i increases detected by aequorin but not by fura-2 in histamine- and caffeine-stimulated swine carotid artery.
1. We hypothesized that the homogeneity of intracellular [Ca2+] ([Ca2+]i) varies and is regulated in arterial smooth muscle. 2. We evaluated this hypothesis by exploiting the different characteristics of several [Ca2+]i indicators: (1) aequorin, which theoretically can measure focal increases in [Ca2+]i, (2) fura-2, which is predominantly a measure of mean cytoplasmic [Ca2+], and (3) myosin light chain phosphorylation and force, which reflect increases in [Ca2+] near the contractile apparatus. 3. From the differences in the observed aequorin and fura-2 signals, we developed an index of the relative degree of [Ca2+]i homogeneity as the ratio of the aequorin signal and fura-2 signal. 4. Stimulation with intermediate concentrations of histamine (1 and 10 microM) or high [K+]o (25 and 40 mM) increased [Ca2+]i and contractile stress. Relative [Ca2+]i homogeneity, estimated from the aequorin/fura-2 ratio, remained similar to levels observed in unstimulated tissues. 5. Higher concentrations of histamine (100 microM) also increased [Ca2+]i and stress, but the aequorin/fura 2 ratio declined , indicating increased [Ca2+]i homogeneity. Similarly, the aequorin/fura-2 ratio decreased when extracellular Ca2+ was removed. 6. Stimulation with histamine in low extracellular [Ca2+] transiently increased [Ca2+]i and the aequorin/fura-2 ratio. Similarly, in tissues treated with low extracellular [Ca2+], restoration of extracellular Ca2+ transiently increased both [Ca2+]i and the aequorin/fura-2 ratio. Although both of these experiments demonstrated a transient decrease in [Ca2+]i homogeneity, only histamine stimulation led to increased myosin light chain phosphorylation and force. These results indicate that the focal increases in [Ca2+]i observed with histamine stimulation and Ca2+ restoration occurred in different cellular regions. 7. Addition of caffeine (20 mM) increased [Ca2+]i and [cAMP], but this was not accompanied by sustained increased myosin light chain phosphorylation or contraction. Phosphorylation of myosin light chain kinase did not appear to underlie the lack of increase in myosin light chain phosphorylation. Rather, caffeine induced a sustained increase in the aequorin/fura-2 ratio, suggesting that caffeine inhibits smooth muscle contraction by localizing increases in [Ca2+]i to a region distant from the contractile apparatus. 8. These data suggest that there can be transient and sustained focal increases in [Ca2+]i. Aequorin detected increased [Ca2+]i in small regions of the cytoplasm during release from and refilling of the intracellular Ca2+ store and with caffeine stimulation. Dual use of aequorin and fura-2 permits determination of relative [Ca2+]i homogeneity in smooth muscle.
Rembold
C M
CM
Department of Internal Medicine, University of Virginia Health Science Center, Charlottesville 22908, USA.
Van Riper
D A
DA
Chen
X L
XL
D000331:Aequorin; D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002339:Carotid Arteries / Q000166:cytology* / Q000187:drug effects; D004594:Electrophysiology; D016257:Fura-2; D006632:Histamine / Q000494:pharmacology*; D007202:Indicators and Reagents; D008564:Membrane Potentials / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D013552:Swine
D002120:Calcium Channel Agonists; D007202:Indicators and Reagents; D002110:Caffeine; D000331:Aequorin; D006632:Histamine; D011188:Potassium; D002118:Calcium; D016257:Fura-2