Nanosecond electric pulses: a novel stimulus for triggering Ca2+ influx into chromaffin cells via voltage-gated Ca2+ channels.

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca(2+) entry into the cells via L-type voltage-gated Ca(2+) channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca(2+) level ([Ca(2+)](i)) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca(2+) entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca(2+)](i) elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca(2+)](i) response of the cells, suggesting Ca(2+) influx occurs only via VGCCs. Lowering extracellular K(+) concentration from 5 to 2 mM or pulsing cells in Na(+)-free medium suppressed the pulse-induced rise in [Ca(2+)](i) in the majority of cells. Thus, both membrane potential and Na(+) entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca(2+) influx. However, activation of voltage-gated Na(+) channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca(2+)](i). These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na(+) transport across the plasma membrane. Whether Na(+) transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.

Basal and parathyroid hormone induced expression of the human 25-hydroxyvitamin D 1alpha-hydroxylase gene promoter in kidney AOK-B50 cells: role of Sp1, Ets and CCAAT box protein binding sites.

The regulation of the gene for renal 25-hydroxyvitamin D 1alpha- hydroxylase (1alpha(OH)ase; CYP27B1) by parathyroid hormone (PTH) under hypocalcemic conditions is fundamentally important for the maintenance of calcium and phosphate homeostasis. The molecular mechanism that underlies this hormonal response is of current interest and has been investigated in the present study by transfection analysis of the human 1alpha(OH)ase promoter in kidney AOK-B50 cells. We have shown that the first 305 bp of promoter can be induced by hormone in transient transfection assays and also within a chromatin environment when stably integrated. Mutagenesis of possible transcription factor binding sites within this promoter length has shown that three sites clustered within the region from -66 to -135 contribute to basal expression. A likely Sp1 and a CCAAT box site are particularly important for basal expression although these sites are not likely to functionally cooperate in a major way. Mutagenesis of the CCAAT box site consistently reduced PTH induction although mutagenesis of the Sp1, Ets and other possible binding sites in the 305 bp of promoter has no significant effect on the level of PTH induction. Other experiments showed that PTH induction but not basal expression was sensitive to the protein kinase inhibitor H89. We have therefore identified for the first time the sites in the 1alpha(OH)ase promoter responsible for basal expression and provide evidence for the role of a CCAAT box binding protein in a PTH mechanism of induction that involves an H89 sensitive step.

Modulation of intracellular Ca2+ levels in chromaffin cells by nanoelectropulses.

Exposing chromaffin cells to a single 5 ns, 5 MV/m pulse causes Ca(2+) influx and a rapid, transient rise in intracellular calcium concentration ([Ca(2+)](i)). A comparison of responses at room temperature versus 37°C revealed no effect of temperature on the magnitude of the increase in [Ca(2+)](i). The Ca(2+) 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 [Ca(2+)](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 10 Hz) or high (1 kHz) 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.

Facilitation of the purinergic contractile response of the guinea pig vas deferens by sodium orthovanadate.

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.