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Modulation of spontaneous transient Ca2+-activated K+ channel currents by cADP-ribose in vascular smooth muscle cells

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Abstract

Transient local releases of Ca2+ from the sarcoplasmic reticulum activate nearby Ca2+-activated K+ channels to produce spontaneous transient outward current (STOC) in smooth muscle cells. We examined if cADP-ribose, an endogenous mediator of Ca2+ release channels of the sarcoplasmic reticulum, could modify STOC activity. In freshly isolated rat tail arterial cells, cADP-ribose (5 μM) increased STOC frequency significantly from 308±26.2 to 398.8±28.8 per minute. The average current at a test potential of −20 mV was increased significantly from 47.8±0.7 to 101.1±0.7 pA in the presence of cADP-ribose. The cell permeant antagonist 8-bromo-cADP-ribose (50 μM) reduced significantly the STOC frequency to 52.5±7.5 per minute and the average current to 24.7±0.1 pA. The STOCs were inhibited significantly by ryanodine (1 μM) and charybodotoxin (150 nM). These findings suggest the presence of basal cADP-ribose activity in resting vascular smooth muscle cells and that STOC activity is stimulated by cADP-ribose.

Introduction

Spontaneous transient outward currents (STOCs) are Ca2+-activated K+ channel currents activated by local Ca2+ transients arising from opening of ryanodine-sensitive Ca2+ release channels in the sarcoplasmic reticulum Benham and Bolton, 1986, Nelson et al., 1995. STOCs may contribute to regulation of vascular tone by hyperpolarization of vascular smooth muscle cells. It is noted that agents that deplete Ca2+ from the sarcosplamic reticulum also inhibit STOCs Benham and Bolton, 1986, Nelson et al., 1995. cADP-ribose, a metabolite of nicotinamide-adenine dinucleotide, is an endogenous regulator of Ca2+-induced Ca2+ release. It induces Ca2+ release from ryanodine-sensitive intracellular stores independent of inositol 1,4,5-trisphosphate in a variety of tissues (Lee, 1997). An enzymatic pathway for the production of cADP-ribose is present in vascular tissues (Li et al., 2000). cADP-ribose has been shown to activate directly reconstituted ryanodine receptors from arterial smooth muscle (Li et al., 2001). In permeabilized smooth muscle cells, cADP-ribose stimulates Ca2+ release from the sarcoplasmic reticulum Kuemmerle and Makhlouf, 1995, Kannan et al., 1996. Since ryanodine receptors are functionally coupled to Ca2+-activated K+ channels in vascular smooth muscle (Perez et al., 1999), we examined if cADP-ribose could modulate STOC activity in freshly isolated cells from the rat tail artery.

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Materials and methods

Fresh single cells from tail arteries of 12–14-week-old male rats (Charles Rivers) were isolated using a method developed in our laboratory (Bolzon and Cheung, 1989). The arteries were incubated in a Ca2+-free medium containing 0.02% collagenase, 0.1% papain, and 4 mM dithiothreitol for 90–120 min at 37 °C. Single cells obtained by this method are relaxed and contract in response to stimulation by a wide variety of agonists. Patch electrodes of resistance 2–6 MΩ were used for recording of whole

Results

The STOC frequency was 308.0±26.2 per minute (n=15) in control cells. Most of the STOCs (72.0±2.0%) were of amplitudes less than 200 pA. In the presence of cADP-ribose (5 μM), the STOC frequency increased significantly to 398.8±28.8 per minute (n=17). The STOCs, even in the presence of cADP-ribose, were inhibited significantly by ryanodine (1 μM; n=3) (Fig. 1A) and by charybdotoxin (150 nM; n=2, data not shown). Signal averaging of the traces showed an overall increase in current amplitude in

Discussion

cADP-ribose mobilizes intracellular Ca2+ in a wide variety of cells by sensitizing ryanodine-sensitive Ca2+ release channels of the sarcoplasmic reticulum to Ca2+ (Lee, 1997). It activates directly reconstituted ryanodine receptors from vascular smooth msucle cells (Li et al., 2001). Since STOC is triggered by Ca2+ release from ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum, we tested if cADP-ribose could modify STOC activity in vascular smooth muscle cells. We observed a

Acknowledgements

This study was supported by the Heart and Stroke Foundation of Ontario. The experiments were carried out with the technical assistance of Ms. L. Xu.

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