Different contributions of calcium channel subtypes to electrical excitability of chromaffin cells in rat adrenal slices

We characterized the ionic currents underlying the cellular excitability and the Ca²⁺‐channel subtypes involved in action potential (AP) firing of rat adrenal chromaffin cells (RCCs) preserved in their natural environment, the adrenal gland slices, through the perforated patch‐clamp recording technique. RCCs prepared from adrenal slices exhibit a resting potential of ?54 mV, firing spontaneous APs (2–3 spikes/s) generated by the opening of Na⁺ and Ca²⁺‐channels, and terminated by the activation of voltage and Ca²⁺‐activated K⁺‐channels (BK). Ca²⁺ influx via L‐type Ca²⁺‐channels is involved in reaching threshold potential for AP firing, and is responsible for activation of BK‐channels contributing to AP‐repolarization and afterhyperpolarization, whereas P/Q‐type Ca²⁺‐channels are involved only in the repolarization phase. BK‐channels carry total outward current during AP‐repolarization. Blockade of L‐type Ca²⁺‐channels reduces BK‐current ~60%, whereas blockade of N‐ or P/Q‐type produces little effect. This study demonstrates that Ca²⁺ influx through L‐type Ca²⁺‐channels plays a key role in modulating the threshold potential from RCCs in situ.

     

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells: the role of MAPKs after angiotensin II stimulation

Angiotensin II (AII, 100 nM) stimulation of bovine adrenal chromaffin cells (BACCs) produced angiotensin II receptor subtype 1 (AT1)-mediated increases in extracellular regulated protein kinase 1/2 (ERK1/2) and stress-activated p38MAPK (p38 kinase) phosphorylation over a period of 10 min. ERK1/2 and p38 kinase phosphorylation preceded Ser31 phosphorylation on tyrosine hydroxylase (TOH). The inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) activation, PD98059 (0.1-50 microM) and UO126 (0.1-10 microM), dose-dependently inhibited both ERK2 and Ser31 phosphorylation on TOH in response to AII, suggesting MEK1/2 involvement. The p38 kinase inhibitor SB203580 (20 microM, 30 min) abolished Ser31 and Ser19 phosphorylation on TOH and partially inhibited ERK2 phosphorylation produced by AII. In contrast, 1 microM SB203580 did not affect AII-stimulated TOH phosphorylation, but fully inhibited heat shock protein 27 (HSP27) phosphorylation produced by AII. Also, 1 microM SB203580 fully inhibited Ser19 phosphorylation on TOH and HSP27 phosphorylation in response to anisomycin (30 min, 10 microg/mL). The results suggest that ERKs mediate Ser31 phosphorylation on TOH in response to AII, but p38 kinase is not involved. Previous studies suggesting a role for p38 kinase in the phosphorylation of Ser31 are explained by the non-specific effects of 20 microM SB203580 in BACCs. The p38 kinase pathway is able to phosphorylate Ser19 on TOH in response to anisomycin, but does not do so in response to AII.     

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells

1. Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. 2. In this study trypsin (30-50 micrograms/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. 3. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsin-sensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. 4. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3-10 micrograms/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.     

Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

This study examines the Cav1 isoforms expressed in mouse chromaffin cells and compares their biophysical properties and roles played in cell excitability and exocytosis. Using immunocytochemical and electrophysiological techniques in mice lacking the Cav1.3α1 subunit (Cav1.3(-/-) ) or the high sensitivity of Cav1.2α1 subunits to dihydropyridines, Cav1.2 and Cav1.3 channels were identified as the only Cav1 channel subtypes expressed in mouse chromaffin cells. Cav1.3 channels were activated at more negative membrane potentials and inactivated more slowly than Cav1.2 channels. Cav1 channels, mainly Cav1.2, control cell excitability by functional coupling to BK channels, revealed by nifedipine blockade of BK channels in wild type (WT) and Cav1.3(-/-) cells (53% and 35%, respectively), and by the identical change in the shape of the spontaneous action potentials elicited by the dihydropyridine in both strains of mice. Cav1.2 channels also play a major role in spontaneous action potential firing, supported by the following evidence: (i) a similar percentage of WT and Cav1.3(-/-) cells fired spontaneous action potentials; (ii) firing frequency did not vary between WT and Cav1.3(-/-) cells; (iii) mostly Cav1.2 channels contributed to the inward current preceding the action potential threshold; and (iv) in the presence of tetrodotoxin, WT or Cav1.3(-/-) cells exhibited spontaneous oscillatory activity, which was fully abolished by nifedipine perfusion. Finally, Cav1.2 and Cav1.3 channels were essential for controlling the exocytotic process at potentials above and below -10 mV, respectively. Our data reveal the key yet differential roles of Cav1.2 and Cav1.3 channels in mediating action potential firing and exocytotic events in the neuroendocrine chromaffin cell.     

Retinol activates tyrosine hydroxylase acutely by increasing the phosphorylation of serine40 and then serine31 in bovine adrenal chromaffin cells

Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of the catecholamines. It has been reported that retinol (vitamin A) modulates tyrosine hydroxylase activity by increasing its expression through the activation of the nuclear retinoid receptors. In this study, we observed that retinol also leads to an acute activation of tyrosine hydroxylase in bovine adrenal chromaffin cells and this was shown to occur via two distinct non-genomic mechanisms. In the first mechanism, retinol induced an influx in extracellular calcium, activation of protein kinase C and serine40 phosphorylation, leading to tyrosine hydroxylase activation within 15 min. This effect then declined over time. The retinol-induced rise in intracellular calcium then led to a second slower mechanism; this involved an increase in reactive oxygen species, activation of extracellular signal-regulated kinase 1/2 and serine31 phosphorylation and the maintenance of tyrosine hydroxylase activation for up to 2 h. No effects were observed with retinoic acid. These results show that retinol activates tyrosine hydroxylase via two sequential non-genomic mechanisms, which have not previously been characterized. These mechanisms are likely to operate in vivo to facilitate the stress response, especially when vitamin supplements are taken or when retinol is used as a therapeutic agent.     

Enhancement of insulin-induced PI3K/Akt/GSK-3beta and ERK signaling by neuronal nicotinic receptor/PKC-alpha/ERK pathway: up-regulation of IRS-1/-2 mRNA and protein in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells treated with nicotine (10 µm for 24 h), phosphorylation of Akt, glycogen synthase kinase‐3β (GSK‐3β) and extracellular signal‐regulated kinase (ERK)1/2 induced by insulin (100 nm for 10 min) was enhanced by ～ 62%, without altering levels of these protein kinases. Nicotine produced time (> 12 h)‐ and concentration (EC₅₀ 3.6 and 13 µm )‐dependent increases in insulin receptor substrate (IRS)‐1 and IRS‐2 levels by ～ 125 and 105%, without altering cell surface density of insulin receptors. In these cells, insulin‐induced tyrosine phosphorylation of IRS‐1/IRS‐2 and recruitment of phosphoinositide 3‐kinase (PI3K) to IRS‐1/IRS‐2 were augmented by ～ 63%. The increase in IRS‐1/IRS‐2 levels induced by nicotine was prevented by nicotinic acetylcholine receptor (nAChR) antagonists, the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetra‐acetic acid tetrakis‐acetoxymethyl ester, cycloheximide or actinomycin D. Nicotine increased IRS‐1 and IRS‐2 mRNA levels by ～ 57 and ～ 50%, and this was prevented by conventional protein kinase C (cPKC) inhibitor Gö6976, or ERK kinase inhibitors PD98059 and U0126. Nicotine phosphorylated cPKC‐α, thereby increasing phosphorylation of ERK1/ERK2, as demonstrated by using Gö6976, PD98059 or U0126. Selective activation of cPKC‐α by thymeleatoxin mimicked these effects of nicotine. Thus, stimulation of nAChRs up‐regulated expression of IRS‐1/IRS‐2 via Ca²⁺‐dependent sequential activation of cPKC‐α and ERK, and enhanced insulin‐induced PI3K/Akt/GSK‐3β and ERK signaling pathways.     

Regulation of catecholamine release and tyrosine hydroxylase in human adrenal chromaffin cells by interleukin-1β: role of neuropeptide Y and nitric oxide

Adrenal chromaffin cells synthesize and secrete catecholamines and neuropeptides that may regulate hormonal and paracrine signaling in stress and also during inflammation. The aim of our work was to study the role of the cytokine interleukin-1β (IL-1β) on catecholamine release and synthesis from primary cell cultures of human adrenal chromaffin cells. The effect of IL-1β on neuropeptide Y (NPY) release and the intracellular pathways involved in catecholamine release evoked by IL-1β and NPY were also investigated. We observed that IL-1β increases the release of NPY, norepinephrine (NE), and epinephrine (EP) from human chromaffin cells. Moreover, the immunoneutralization of released NPY inhibits catecholamine release evoked by IL-1β. Moreover, IL-1β regulates catecholamine synthesis as the inhibition of tyrosine hydroxylase decreases IL-1β-evoked catecholamine release and the cytokine induces tyrosine hydroxylase Ser40 phosphorylation. Moreover, IL-1β induces catecholamine release by a mitogen-activated protein kinase (MAPK)-dependent mechanism, and by nitric oxide synthase activation. Furthermore, MAPK, protein kinase C (PKC), protein kinase A (PKA), and nitric oxide (NO) production are involved in catecholamine release evoked by NPY. Using human chromaffin cells, our data suggest that IL-1β, NPY, and nitric oxide (NO) may contribute to a regulatory loop between the immune and the adrenal systems, and this is relevant in pathological conditions such as infection, trauma, stress, or in hypertension.     

Lysophosphatidic acid-LPA1 receptor-Rho-Rho kinase-induced up-regulation of Nav1.7 sodium channel mRNA and protein in adrenal chromaffin cells: enhancement of 22Na+ influx, 45Ca2+ influx and catecholamine secretion

In cultured bovine adrenal chromaffin cells, chronic (> or = 24 h) treatment with lysophosphatidic acid (LPA) augmented veratridine-induced 22Na+ influx via Na(v)1.7 by approximately 22% (EC(50) = 1 nmol/L), without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated channel. LPA enhanced veratridine (but not nicotine)-induced 45Ca2+ influx via voltage-dependent calcium channel and catecholamine secretion. LPA shifted concentration-response curve of veratridine for 22Na+ influx upward, without altering the EC(50) of veratridine. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced 22Na+ influx by twofold in non-treated and LPA-treated cells. Whole-cell patch-clamp analysis showed that peak Na+ current amplitude was greater by 39% in LPA (100 nmol/L for 36 h)-treated cells; however, I-V curve and steady-state inactivation/activation curves were comparable between non-treated and LPA-treated cells. LPA treatment (> or = 24 h) increased cell surface [3H]saxitoxin binding by approximately 28%, without altering the K(d) value; the increase was prevented by cycloheximide, actinomycin D, or Ki16425, dioctylglycerol pyrophosphate 8:0 (two inhibitors of LPA(1) and LPA3 receptors), or botulinum toxin C3 (Rho inhibitor), Y27632 (Rho kinase inhibitor), consistent with LPA(1) receptor expression in adrenal chromaffin cells. LPA raised Nav1.7 mRNA level by approximately 37%. Thus, LPA-LPA(1) receptor-Rho/Rho kinase pathway up-regulated cell surface Nav1.7 and Nav1.7 mRNA levels, enhancing veratridine-induced Ca2+ influx and catecholamine secretion.