Effects of okadaic acid and ATPγS on cell length and Ca2+‐channel currents recorded in single smooth muscle cells of the guinea‐pig taenia caeci

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Abstract
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+(IBa) were recorded at potentials positive to −40mV, were maximal near 0mV and reversed near +60mV. Both the inward and outward flow of current was blocked by 100 μm Cd2+. 4 Addition of the ATP analogue, adenosine 5′-O(3-thio triphosphate) (ATPγS) (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 ATPγS. In particular, ATPγS 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 μm), a chemical inhibitor of phosphatase activity, produced similar effects when applied intracellularly. When OA (25 μm) was applied extracellularly the rate of rundown of IBa was slowed. 7 Isoprenaline (1 μm) alone had no effect on IBa, but induced a small increase in IBa in the presence of OA(25 μm). 8 Thus, our results indicate that (1) the contractions in ATPγS 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.