Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells

Abstract

Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, Vh=-80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell IBa could be fractionated into various subcomponents. Thus, IBa had a 25% fraction sensitive to 1 microM nifedipine (L-type channels), 21% sensitive to 1 microM omega-conotoxin GVIA (N-type channels), and 60% sensitive to 2 microM omega-agatoxin IVA (P/Q-type channels). The activation of IBa was considerably slowed down, and the peak current was inhibited upon superfusion with 10 microM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of IBa was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5-50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates.

PAC1 Receptor Activation by PACAP-38 Mediates Ca2+ Release from a cAMP-dependent Pool in Human Fetal Adrenal Gland Chromaffin Cells

“…However, a number of reports indicate that PACAP activates a Ca 2ϩ influx through various pathways, including Ca 2ϩ channels (29,(42)(43)(44). In chromaffin cells from adult human adrenal glands, voltage-dependent Ca 2ϩ channels have been described (45), but no electrophysiological studies have been performed in fetal cells. KCl depolarization, or application of nicotine, were able to induce an increase in [Ca 2ϩ ] i in human fetal chromaffin cells suggesting the presence of functional voltage-dependent Ca 2ϩ channels.…”

Altered regulation of calcium channels and exocytosis in single human pheochromocytoma cells

“…Endogenously released as well as exogenously applied ATP and opioids produce a strong modulatory effect on Ca 2+ channel currents in bovine [2,3,6,9,13], rat [20,23], guinea-pig [30] and human [14] chromaffin cells. Such modulation is mediated by purinergic receptors and µ and δ receptors coupled to G-proteins, and consists of a pronounced slowing down of current activation and a marked decrease in its peak amplitude.…”

“…Blockade induced by nifedipine accounted for 19±2.5% (n=18 cells), that by ω-conotoxin GVIA was 31±3% (n=21 cells), that by ω-agatoxin IVA (20 nM) was 8±1.2% (n=15 cells), and that by ω-agatoxin IVA (2 µM) was 48±5.5% (n=15 cells). For comparative purposes, the blockade of I Ba in normal human chromaffin cells obtained in a previous study [14] is also drawn. Observe that the blockade exerted by all compounds was quite similar in normal cells (white columns) and in pheochromocytoma cells (black columns).…”

“…ATP [9,10,13,23,30] and opioids [2,22,33], co-released with the catecholamines, cause the inhibition of Ca 2+ channel currents through L- [2,20], N-and P/Q-type channels [1,2,3,6,9], by a G-protein-coupled membrane-delimited pathway. This may constitute the basis for an autocrine-paracrine autoinhibitory mechanism of Ca 2+ entry, which has been shown to be present in different animal species, such as the bovine [3,6,9], rat [20,23], guinea-pig [30], as well as human [14] chromaffin cells. This control of Ca 2+ entry can thus serve to regulate the release of catecholamines into the circulation.…”

“…This current is also modulated by ATP and opiates in an autocrine-paracrine fashion [14]. In the light of these recent findings we pose the following questions:…”