No abstract

Actions of growth-hormone-releasing hormone (GHRH) on single rat anterior pituitary cells were studied using indo-1 fluorescence to monitor changes in intracellular calcium, [Ca2+]i, and perforated-patch recording to measure changes in membrane potential and ionic currents. GHRH elevated [Ca2+]i in non-voltage-clamped cells by a mechanism that was dependent upon extracellular Na+ and Ca2+ and was blocked by the dihydropyridine Ca(2+)-channel blocker, nitrendipine. Resting cells had a fluctuating membrane potential whose a mean value depolarized by 9 mV in response to GHRH. The membrane-permeant cAMP analogue, 8-(4-chlorophenylthio)cAMP, mimicked the action of GHRH on membrane potential. Under voltage clamping, GHRH activated a small inward current (1-5 pA). Two types of response could be distinguished. The type I response had an inward current that was largest at more negative potentials (-90 mV), and the type II response had inward current that was larger at more positive potentials (-40 to -70 mV). Both types of response were reversible and blocked by removal of extracellular Na+. These results suggest that the rise in [Ca2+]i produced by GHRH in non-voltage-clamped cells results from the activation via cAMP of a Na(+)-dependent conductance, which depolarizes the cell and increases the Ca2+ influx through voltage-gated Ca2+ channels.

No abstract

1. To study mechanisms of receptor-operated Ca2+ influx in non-excitable cells, membrane currents of rat peritoneal macrophages were recorded using whole-cell cell-attached and outside-out configurations of the patch clamp technique. Under whole-cell recording conditions, ATP applied in micromolar concentrations elicited an inward current response when the bath solution contained Ba2+, Ca2+ or Na+ as the only permeant cations. 2. Increasing the Mg2+ concentration had an inhibitory effect on the ATP-induced inward current indicating that the active form of ATP responsible for the cation entry is ATP4-. The nucleotide potency order was ATP> ATPyS > ADP. UTP was completely ineffective (n = 19). The data obtained are consistent with the ATP receptor being of the P2Z type.3. The macrophage plasma membrane was impermeable to Tris+ during the ATP-induced current at ATP4-concentrations varying from 0 07 to 500 /M. At higher concentrations, ATP produced a large inward steady-state current, which could be attributed to membrane permeabilization. * 4. Activity of single channels was recorded when ATP was applied to the external surface of the patch membrane both in cell-attached and outside-out experiments. A specific property of the channels appeared to be the existence of at least four conductance sublevels. With 105 mm Ba2P as the permeant cation, the conductance sublevels were 3 5, 7, 10 and 15 pS. With 10 mM Ca2+ the sublevel conductances were equal to 4, 9, 13 and 17 pS. 5. The unitary conductance estimated from the whole-cell current noise analysis (3 5-4 5 pS for 105 mM Ba2+) was significantly lower than that obtained from single channel measurements at the main (3rd) current level, but it was very close to the conductance of the minimum (1st) level. 6. Extrapolated reversal potential values estimated from current-voltage curves for predominant conductance levels were equal to +40 and +26 mV for 105 mm Ba2+ and 10 mM Ca2+, respectively. The permeability ratios fell in the sequence: Pca: PBa: PK= 71: 29:1.Thus, ATP-activated channels in the macrophage membrane are rather selective for divalent vs. monovalent cations, with the predominant permeability being for Ca!+.Purinergic agonists affect various cellular processes such as elevation of intracellular Ca2+, alterations in ionic fluxes, cell division, vascular tone, cardiac and skeletal muscle contractility and platelet aggregation. In a number of cells, ATP and other nucleotides are known to produce an increase in inositol 1,4,5-trisphosphate followed by Ca2+release from the intracellular stores, and an elevation of intracellular Ca2+ due to the entry of extracellular Ca2+ through Ca2+-permeable ion channels by a mechanism that is not bound to phosphoinositide turnover. Direct activation of ionic channels by purinergic agonists has been described in various cell types: smooth muscle cells

No abstract