The effects of staurosporine (ST), a widely used protein kinase C (PKC) inhibitor, were examined on Kv1.3 channels stably expressed in Chinese hamster ovary (CHO) cells using the whole-cell and excised inside-out configurations of the patch clamp technique. In whole-cell recordings, ST, at external concentrations from 300 nM to 10 microM, accelerated the rate of inactivation of Kv1.3 currents and thereby reduced the current at the end of the depolarizing pulse in a concentration-dependent manner with an IC50 of 1.2 microM. The actions of ST were unaffected by pretreatment with another selective PKC inhibitor, chelerythrine, or by including the PKC pseudosubstrate peptide inhibitor, PKC 19-36, in the intracellular solution. Rp-cAMPS, a specific protein kinase A inhibitor, included in intracellular solution did not affect the effects of ST. Furthermore, the same effects of ST on Kv1.3 were also observed in excised inside-out patches when applied to the internal face of the membrane. These effects were completely reversible upon washing. Current-voltage relations for Kv1.3 currents at the end of voltage steps indicated that ST reduced Kv1.3 currents over a wide voltage range. The blockade exhibited a shallow voltage dependence between -10 mV and +40 mV, increasing at more positive potentials. ST had no effect on the voltage dependence of steady-state inactivation. It reduced the tail current amplitude and slowed the deactivation time course, resulting in a crossover phenomenon. These results suggest that the action of ST on Kv1.3 is independent of PKC and PKA inhibition. ST blocks the open state of Kv1.3 channels to produce an apparent acceleration of the inactivation rate.
The effect of histamine on contractile and electric activity was studied in the isolated stomach muscle strips of 138 cats. Histamine dose-dependently produced tonic and phasic contractions of the muscle preparations from the fundus and the corpus but only phasic contraction of the antral muscle preparations. The frequency of gastric slow waves (SWs) was also increased dose-dependently by histamine. The responses of muscle contractions and gastric SW frequency to histamine were completely blocked by pretreatment with pyrilamine (10(-6) M) and were significantly inhibited by atropine (10(-5) M) but not by cimetidine (10(-5) M), hexamethonium (10(-5) M), phentolamine (10(-5) M), or propranolol (10(-5) M). The inhibition by pyrilamine was competitive. Although atropine inhibited the effect of histamine significantly, it could not completely block the effect of histamine even at a high concentration (3 x 10(-5) M). It is concluded that histamine may participate in the regulation of gastric motility in the cat by acting on the H1 receptor to cause the release of acetylcholine and also other contractile substance(s).
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