Dihydropyridines (DHPs) generally have little effect on whole-cell calcium currents of neurons, even at concentrations far higher than those effective on muscle. Either neuronal calcium currents are much less sensitive to DHPs, or only a small proportion of the current is DHP-sensitive. We find that DHP agonists and antagonists act at low concentration on calcium currents in frog sympathetic neurons but that the effects are small even at optimal concentrations. The half-maximal dose (EC50) of the agonist Bay K 8644 is approximately 50 nM, and the effect of Bay K 8644 is blocked by 50% at approximately 300 nM nifedipine, from a holding potential of -80 mV. Nifedipine is more effective from a holding potential of -50 mV. These results suggest the presence of an L-type calcium current, with DHP sensitivity similar to L-currents in cardiac muscle. The predominant (greater than 90%) calcium current in frog sympathetic neurons is a DHP-resistant N-type current. However, high concentrations of DHPs (10 microM) partially block N-type calcium current, as well as voltage-dependent sodium and potassium currents.

We investigated the ability of angiotensin II (Ang II) or the stable analogue [Sar1]-Ang II to increase intracellular and extracellular free arachidonic acid in primary cultures of rabbit proximal tubular epithelial cells to better characterize the receptor subtype and orientation of phospholipase A2 (PLA2)-mediated signaling. Proximal tubular cells were labeled with [3H]arachidonic acid for 4 hours and then treated with Ang II or [Sar1]-Ang II. Lipids were extracted from labeled cells, separated by thin-layer chromatography, and quantified by liquid scintillation counting. Ang II (10 mumol/L, 1 minute) stimulated an increase in intracellular free [3H]arachidonic acid from 21.0 +/- 2.0 to 32.2 +/- 2.8 disintegrations per minute/microgram protein, an effect that was potentiated by EGTA. [Sar1]-Ang II stimulated a time- and concentration-dependent increase in [3H]arachidonic acid release from labeled cells. Release of [3H]arachidonic acid was maximal at 10 mumol/L [Sar1]-Ang II, with an EC50 of approximately 3 mumol/L. Ang II receptor antagonists caused concentration-dependent inhibition of [Sar1]-Ang II-stimulated [3H]arachidonic acid release with the following order of potency: CGP 42112 = PD 123319 > losartan. Furthermore, in proximal tubular epithelial cells grown on polyester membrane filters, the Ang II receptor that mediated arachidonic acid release was predominantly apical rather than basolateral. These observations are consistent with activation of a Ca(2+)-independent, apical PLA2 isoform in epithelial cells through an Ang II type 2 receptor subtype.

We have compared the abilities of extracellular ATP (acting via P2-purinergic receptors) and formylated peptides (FMLP) to stimulate both phospholipase D (PLD)-based signal transduction and primary granule (azurophilic) secretion in HL-60 cells induced to differentiate along the granulocytic pathway. In undifferentiated HL-60 cells, neither ATP nor FMLP elicited significant PLD activation or increased secretion despite the previously documented ability of ATP to stimulate large increases in polyphosphoinositide hydrolysis and Ca2" mobilization. Conversely, within 1 d after induction of granulocytic differentiation by dibutyryl cAMP, both ATP and FMLP induced large increases in azurophilic secretion and corresponding increases in PLD activity. ATP-activated PLD activity was nearmaximal within 1 d after dibutyryl cAMP treatment, while the FMLP-induced activity increased continuously over 4 d, with a maximal level twice that stimulated by ATP. Additional experiments characterized the activation ofPLD by receptor-independent pathways at different stages of differentiation; these included studies of phorbol ester action in intact cells and GTP7S action in electropermeabilized cells. An apparent role for guanine nucleotide-binding regulatory proteins in PLD regulation was also indicated by the significant reduction in FMLP-and ATP-stimulated PLD activity observed in cells pretreated with pertussis toxin. At all stages of differentiation, there was good correlation between the relative efficacies of ATP versus FMLP in stimulating both secretion and PLD activity. These data indicate: (a) that the receptor-regulated phospholipase D signaling pathway is induced during differentiation of myeloid progenitor cells; and (b) that differential activation of this signaling system by various Ca2+-mobilizing receptor agonists may underlie the differential regulation of secretion and other phagocyte functions by such agents. (J. Clin. Invest. 1991. 88:45-54.)