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Abstract: Exposing chromaffin cells to a single 5ns, 5 MV/m pulse causes Ca2+ influx and a rapid, transient rise in intracellular calcium concentration ([Ca2+]i). A comparison of responses at room temperature versus 37°C revealed no effect of temperature on the magnitude of the increase in [Ca2+]i. The Ca2+ transient, however, was shortened in duration almost twofold at 37°C, indicating that the rate of recovery was temperature-sensitive. Temperature also affected the interval required for a second pulse to elicit another maximal rise in [Ca2+]i, which was shorter at the higher temperature. In addition, a second pulse applied 5s after the first pulse was sufficient to cause cells at room temperature to become refractory to subsequent stimulation. At 37°C, cells became refractory after 5 pulses regardless of whether pulse delivery was at low (1 and 10Hz) or high (1kHz) rates. When refractory, cells showed no signs of swelling or uptake of the impermeant dye YO-PRO-1. These results demonstrate that temperature plays a role in determining how chromaffin cells respond to and become refractory to nanoelectropulses. They also indicate that despite the ultra-short duration of the pulses, pronounced effects on cell excitability result from the application of only very few pulses. [Copyright &y& Elsevier]

Experiments were carried out to examine the effects of protein tyrosine kinase (PTK) and protein tyrosine phosphatase inhibitors on the purinergic contraction of the guinea pig vas deferens. Sodium orthovanadate (SOV) produced a robust increase of the amplitude of contractions evoked by both neurogenic electrical field stimulation and exogenous ATP. This effect of SOV was concentration- and time-dependent, as well as, reversible and reproducible. Genistein, a PTK inhibitor, but not its inactive structural analog daidzein, inhibited the SOV-induced facilitation of the purinergic contraction. Another PTK inhibitor, 2,5-dihydroxycinnamic acid methyl ester, which is structurally unrelated to genistein, also inhibited the facilitation effects of SOV. Although an application of as low as 3 microM of these inhibitors significantly decreased the effect of SOV, other PTK inhibitors, namely, butein, levandustin C, and thyrphostin 23, were less effective even at concentrations of 100 microM. Western blot experiments showed that the facilitation of the purinergic contraction by SOV is associated with a prominent increase in the level of tyrosine phosphorylation of proteins with estimated molecular sizes of 180 and 123 kDa, which was reversed in the presence of genistein. Evidence is also presented that argue against the possibility that inhibition of the Na(+)/K(+)-ATPase or ATPases, responsible for the clearance of ATP is involved in the SOV-induced facilitation of the purinergic contraction. Together, these results suggest that the responsiveness of the smooth muscle of the vas deferens to the actions of ATP is modulated via a previously unidentified mechanism, which may involve protein tyrosine phosphorylation.