Effects of okadaic acid and ATP gamma S on cell length and Ca(2+)-channel currents recorded in single smooth muscle cells of the guinea-pig taenia caeci.

Abstract

1. The effects of inhibiting phosphatase activity on Ca(2+)-channel currents and cell shortening in single cells of the guinea-pig taenia caeci were investigated by whole-cell voltage clamp and video recording techniques. 2. Ca(2+)-channel currents were isolated by use of pipette solutions containing Cs, tetraethylammonium and adenosine triphosphate (ATP) (3 mM). Ca2+ or Ba2+ (7.5 mM) in the bathing solution acted as the charge carrier during inward current flow. 3. Ca(2+)-channel currents in 7.5 mM Ba2+ (IBa) were recorded at potentials positive to -40 mV, were maximal near 0 mV and reversed near +60 mV. Both the inward and outward flow of current was blocked by 100 microM Cd2+. 4. Addition of the ATP analogue, adenosine 5'-O(3-thiotriphosphate) (ATP gamma S) (1 mM) to the pipette solution (containing 3 mM ATP) caused cell shortening to 23 +/- 2% (n = 5) of their initial length within 5 min. Control cells (containing 4 mM ATP) did not contract during recording periods up to 60 min in duration. 5. IBa, recorded 1-2 min after membrane rupture, was 134 +/- 19 (n = 13) pA, compared with 209 +/- 25 (n = 5) pA in control cells, otherwise there were no significant time-dependent effects of ATP gamma S. In particular, ATP gamma S did not prevent the decrease in amplitude, nor the acceleration of inactivation when Ca2+ (7.5 mM) replaced Ba2+ as the permeating ion. 6. Okadaic acid (OA) (50 microM), a chemical inhibitor of phosphatase activity, produced similar effects when applied intracellularly. When OA (25,microM) was applied extracellularly the rate of rundown of 'Ba was slowed. 7. Isoprenaline (1 microM) alone had no effect on 'Ba, but induced a small increase in IBa in the presence of OA (25 microM). 8. Thus, our results indicate that (1) the contractions in ATP gamma S and OA may well arise from the activation of a kinase which phosphorylates myosin at low concentrations of Ca2 +, and (2) changes in the state of phosphorylation of Ca2+ channels, or associated proteins, in the taenia caeci modulate their function, but probably not via mechanisms involving cyclic AMP-dependent protein kinases.