EFFECTS OF OKADAIC ACID AND ATP-GAMMA-S ON CELL LENGTH AND CA2+-CHANNEL CURRENTS RECORDED IN SINGLE SMOOTH-MUSCLE CELLS OF THE GUINEA-PIG TAENIA CECI

1 The effects of inhibiting phosphatase activity on Ca2+-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 Ca2+-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 Ca2+-channel currents in 7.5 mM Ba2+ (I(Ba)) 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-mu-M Cd2+. 4 Addition of the ATP analogue, adenosine 5'-0(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 I(Ba), 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-mu-M), a chemical inhibitor of phosphatase activity, produced similar effects when applied intracellularly. When OA (25-mu-M) was applied extracellularly the rate of rundown of I(Ba) was slowed. 7 Isoprenaline (1-mu-M) alone had no effect on I(Ba), but induced a small increase in I(Ba) in the presence of OA (25-mu-M). 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.