ATP and phenylephrine elicit different patterns of cytosolic Ca2+ transients in myocytes isolated from mouse vas deferens

Jozsef Somogyi, Richard D. Wassall & Thomas C. Cunnane, Oxford University, Department of Pharmacology, Mansfield Road, Oxford OX1 3QT, UK.

ATP and noradrenaline are released from postganglionic sympathetic neurones, which innervate the mouse vas deferens. The major excitatory receptors for ATP are P2X receptors, which when activated depolarize the plasma membrane. This depolarization opens voltage-gated Ca2+ channels and with contributions from internal Ca2+ stores triggers contraction. In contrast, noradrenaline predominantly activates α1 -adrenoceptors and acts through G protein-coupled receptors to initiate cellular Ca2+ waves and contraction. The aim of the present study was to characterize the spatiotemporal patterns of these Ca2+ transients (CTs) in isolated smooth myocytes taken from the vas deferens of the mouse.

Balb/c mice (8-10 weeks) were used. Smooth muscle cells were isolated using a technique adapted from Quayle et al. (1996). The Ca2+ indicator, Oregon Green® BAPTA-1 AM and a Perkin-Elmer Ultraview 21 confocal scanning unit were used for imaging. The cells were spread on poly-L-lysine-coated coverslips and perfused with Krebs solution ( mM: 118 NaCl, 25 NaHCO3, 1.13 NaH2PO4, 4.7 KCl, 1.3 MgCl2, 1.8 CaCl2, and 11.1 glucose , bubbled with 95% O2 / 5% CO2 to pH 7.4) at room temperature. Time series were analysed with ImageJ and Microsoft Excel softwares .

Freshly dissociated smooth muscle cells displayed localized, spontaneous CTs, which were abolished by simultaneous bath-application of ryanodine and caffeine (1 µM and 300 µM, respectively). When ATP (1 - 50 µM) was applied to a cell by rapid switching, whole-cell CTs were observed in 46 out of 47 cases. These CTs originated under the plasma membrane and travelled towards the centre of the cell within 200 - 250 ms. The application of NF449, a selective P2X1 receptor antagonist (10 µM), abolished the first, sub-plasmalemmal phase of the CTs initiated by ATP, and the average amplitude of the whole-cell CTs ((F peak-F rest)/F rest) was reduced from 0.9 ± 0.3 (n = 14), to 0.05 ± 0.05 (n = 15; P < 0.001, Wilcoxon-Mann-Whitney Rank Sum Test). Simultaneous bath-application of ryanodine and caffeine (1 µM and 300 µM, respectively) also reduced the amplitude of the ATP-induced whole-cell CTs ((F peak-F rest)/F rest) from 0.9 ± 0.2 (n = 6), to 0.5 ± 0.1 (n = 5; P < 0.05, Wilcoxon-Mann-Whitney Rank Sum Test), but did not affect the spatiotemporal pattern . Nifedipine (1 µM) had no effect and cells responded to ATP even when the external Ca2++ concentration was as low as 100 µM.

Phenylephrine (1-50µM), a selective a 1 -adrenoceptor agonist, was applied in the same manner as ATP. Expanding CTs occurred (in 53 of 55 cells), which originated from one or a few distinct sites directly below the plasma membrane. They travelled predominantly along the long-axis of the cell before the whole cell was invaded and a contraction commenced. These CTs were abolished by simultaneous bath-application of ryanodine and caffeine (1 m M and 300 µM, respectively) and prazosin, a selective α1-adrenoceptor antagonist (100 µM), but were unaffected by NF449.

These results show that acutely dissociated smooth muscle cells generate spontaneous CTs, which originate from internal stores, and that purinergic and adrenergic receptor signalling produce different spatiotemporal CT patterns.

Quayle, J.M., et al. (1996). J. Physiol, 494: (Pt 3), 715-726
Rasband, W.S,. (1997-2006). ImageJ. http://rsbinfonihgov/ij/ National Institutes of Health, Bethesda, Maryland, USA.