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INNOVATIVE METHODOLOGY

Arteriolar smooth muscle Ca²⁺ dynamics during blood flow control in hamster cheek pouch

¹The John B. Pierce Laboratory and Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut; and ²Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan

Submitted 28 December 2005 ; accepted in final form 1 February 2006

Intracellular calcium concentration ([Ca²⁺]_i) governs the contractile status of arteriolar smooth muscle cells (SMC). Although studied in vitro, little is known of SMC [Ca²⁺]_i dynamics during the local control of blood flow. We tested the hypothesis that the rise and fall of SMC [Ca²⁺]_i underlies arteriolar constriction and dilation in vivo. Aparenchymal segments of second-order arterioles (diameter 35 ± 2 µm) were prepared in the superfused cheek pouch of anesthetized hamsters (n = 18) and perifused with the ratiometric dye fura PE-3 (AM) to load SMC (1 µM, 20 min). Resting SMC [Ca²⁺]_i was 406 ± 37 nM. Elevating superfusate O₂ from 0 to 21% produced constriction (11 ± 2 µm) that was unaffected by dye loading; [Ca²⁺]_i increased by 108 ± 53 nM (n = 6, P < 0.05). Cycling of [Ca²⁺]_i during vasomotion (amplitude, 150 ± 53 nM; n = 4) preceded corresponding diameter changes (7 ± 1 µm) by 2 s. Microiontophoresis (1 µm pipette tip; 1 µA, 1 s) of phenylephrine (PE) transiently increased [Ca²⁺]_i by 479 ± 64 nM (n = 8, P < 0.05) with constriction (26 ± 3 µm). Flushing blood from the lumen with saline increased fluorescence at 510 nm by 45% during excitation at both 340 and 380 nm with no difference in resting [Ca²⁺]_i, diameter or respective responses to PE (n = 7). Acetylcholine microiontophoresis (1 µA, 1 s) transiently reduced resting SMC [Ca²⁺]_i by 131 ± 21 nM (n = 6, P < 0.05) with vasodilation (17 ± 1 µm). Superfusion of sodium nitroprusside (10 µM) transiently reduced SMC [Ca²⁺]_i by 124 ± 18 nM (n = 6, P < 0.05), whereas dilation (23 ± 5 µm) was sustained. Resolution of arteriolar SMC [Ca²⁺]_i in vivo discriminates key signaling events that govern the local control of tissue blood flow.

calcium photometry; microcirculation; vasomotion

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