Channels In Adrenal Chromaffin Cells Research Articles

N‐ and P/Q‐type Ca2+ channels in adrenal chromaffin cells

Ca2+ is the most ubiquitous second messenger found in all cells. Alterations in [Ca2+]i contribute to a wide variety of cellular responses including neurotransmitter release, muscle contraction, synaptogenesis and gene expression. Voltage-dependent Ca2+ channels, found in all excitable cells (Hille 1992), mediate the entry of Ca2+ into cells following depolarization. Ca2+ channels are composed of a large pore-forming subunit, called the alpha1 subunit, and several accessory subunits. Ten different alpha1 subunit genes have been identified and classified into three families, Ca(v1-3) (Dunlap et al. 1995, Catterall 2000). Each alpha1 gene produces a unique Ca2+ channel. Although chromaffin cells express several different types of Ca2+ channels, this review will focus on the Cav(2.1) and Cav(2.2) channels, also known as P/Q- and N-type respectively (Nowycky et al. 1985, Llinas et al. 1989b, Wheeler et al. 1994). These channels exhibit physiological and pharmacological properties similar to their neuronal counterparts. N-, P/Q and to a lesser extent R-type Ca2+ channels are known to regulate neurotransmitter release (Hirning et al. 1988, Horne & Kemp 1991, Uchitel et al. 1992, Luebke et al. 1993, Takahashi & Momiyama 1993, Turner et al. 1993, Regehr & Mintz 1994, Wheeler et al. 1994, Wu & Saggau 1994, Waterman 1996, Wright & Angus 1996, Reid et al. 1997). N- and P/Q-type Ca2+ channels are abundant in nerve terminals where they colocalize with synaptic vesicles. Similarly, these channels play a role in neurotransmitter release in chromaffin cells (Garcia et al. 2006). N- and P/Q-type channels are subject to many forms of regulation (Ikeda & Dunlap 1999). This review pays particular attention to the regulation of N- and P/Q-type channels by heterotrimeric G-proteins, interaction with SNARE proteins, and channel inactivation in the context of stimulus-secretion coupling in adrenal chromaffin cells.

L-type calcium channels in adrenal chromaffin cells: Role in pace-making and secretion

Voltage-gated L-type (Cav1.2 and Cav1.3) channels are widely expressed in cardiovascular tissues and represent the critical drug-target for the treatment of several cardiovascular diseases. The two isoforms are also abundantly expressed in neuronal and neuroendocrine tissues. In the brain, Cav1.2 and Cav1.3 channels control synaptic plasticity, somatic activity, neuronal differentiation and brain aging. In neuroendocrine cells, they are involved in the genesis of action potential generation, bursting activity and hormone secretion. Recent studies have shown that Cav1.2 and Cav1.3 are also expressed in chromaffin cells but their functional role has not yet been identified despite that L-type channels possess interesting characteristics, which confer them an important role in the control of catecholamine secretion during action potentials stimulation. In intact rat adrenal glands L-type channels are responsible for adrenaline and noradrenaline release following splanchnic nerve stimulation or nicotinic receptor activation. L-type channels can be either up- or down-modulated by membrane autoreceptors following distinct second messenger pathways. L-type channels are tightly coupled to BK channels and activate at relatively low-voltages. In this way they contribute to the action potential hyperpolarization and to the pace-maker current controlling action potential firings. L-type channels are shown also to regulate the fast secretion of the immediate readily releasable pool of vesicles with the same Ca 2+-efficiency of other voltage-gated Ca 2+ channels. In mouse adrenal slices, repeated action potential-like stimulations drive L-type channels to a state of enhanced stimulus-secretion efficiency regulated by β-adrenergic receptors. Here we will review all these novel findings and discuss the possible implication for a specific role of L-type channels in the control of chromaffin cells activity.

Destabilization of Na(v)1.7 sodium channel alpha-subunit mRNA by constitutive phosphorylation of extracellular signal-regulated kinase: negative regulation of steady-state level of cell surface functional sodium channels in adrenal chromaffin cells.

In cultured bovine adrenal chromaffin cells expressing Na(v)1.7 isoform of voltage-dependent Na(+) channels, treatment (> or = 6 h) with serum deprivation, PD98059, or U0126 increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by approximately 58% (t(1/2) = 12.5 h), with no change in the K(d) value. Immunoblot analysis showed that either treatment attenuated constitutive phosphorylation of extracellular signal-regulated kinase (ERK) 1 and ERK2 but not of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK) 1 and JNK2. The increase of [(3)H]STX binding and the attenuated phosphorylation of ERK1 and ERK2 returned to the control nontreated levels after the addition of serum or the washout of PD98059- or U0126-treated cells. Simultaneous treatment of serum deprivation with PD98059 or U0126 did not produce an additional increasing effect on [(3)H]STX binding, compared with either treatment alone. In cells subjected to either treatment, veratridine-induced maximum (22)Na(+) influx was augmented by approximately 47%, with no change in the EC(50) value; Ptychodiscus brevis toxin-3 enhanced veratridine-induced (22)Na(+) influx by 2-fold, as in nontreated cells. Serum deprivation, PD98059, or U0126 increased Na(+) channel alpha- but not beta(1)- subunit mRNA level by approximately 50% between 3 and 24 h; cycloheximide, an inhibitor of protein synthesis, increased alpha-subunit mRNA level and nullified additional increasing effect of either treatment on alpha-subunit mRNA level. Either treatment prolonged half-life of alpha-subunit mRNA from 17.5 to approximately 26.3 h without altering alpha-subunit gene transcription. Thus, constitutively phosphorylated/activated ERK destabilizes Na(+) channel alpha-subunit mRNA via translational event, which negatively regulates steady-state level of alpha-subunit mRNA and cell surface expression of functional Na(+) channels.

Serum deprivation-induced upregulation of voltage-dependent sodium channels in adrenal chromaffin cells: selective involvement of extracellular signal-regulated kinase pathway.

In the present study, we investigated whether activation of the MAPK family could regulate the cell surface expression of Na channels in cultured bovine adrenal chromaffin cells. The results suggest that constitutively activated ERK (but not p38 or JNK), by various extracellular stimuli, down-modulates the density of cell surface Na channels, which was mediated via the destabilization of Na channel alpha-subunit mRNA.

Differential facilitation of N- and P/Q-type calcium channels during trains of action potential-like waveforms.

Inhibition of presynaptic voltage-gated calcium channels by direct G-protein betagamma subunit binding is a widespread mechanism that regulates neurotransmitter release. Voltage-dependent relief of this inhibition (facilitation), most likely to be due to dissociation of the G-protein from the channel, may occur during bursts of action potentials. In this paper we compare the facilitation of N- and P/Q-type Ca(2+) channels during short trains of action potential-like waveforms (APWs) using both native channels in adrenal chromaffin cells and heterologously expressed channels in tsA201 cells. While both N- and P/Q-type Ca(2+) channels exhibit facilitation that is dependent on the frequency of the APW train, there are important quantitative differences. Approximately 20 % of the voltage-dependent inhibition of N-type I(Ca) was reversed during a train while greater than 40 % of the inhibition of P/Q-type I(Ca) was relieved. Changing the duration or amplitude of the APW dramatically affected the facilitation of N-type channels but had little effect on the facilitation of P/Q-type channels. Since the ratio of N-type to P/Q-type Ca(2+) channels varies widely between synapses, differential facilitation may contribute to the fine tuning of synaptic transmission, thereby increasing the computational repertoire of neurons.

Voltage-independent Inhibition of P/Q-type Ca2+Channels in Adrenal Chromaffin Cells via a Neuronal Ca2+Sensor-1-dependent Pathway Involves Src Family Tyrosine Kinase

In common with many neurons, adrenal chromaffin cells possess distinct voltage-dependent and voltage-independent pathways for Ca(2+) channel regulation. In this study, the voltage-independent pathway was revealed by addition of naloxone and suramin to remove tonic blockade of Ca(2+) currents via opioid and purinergic receptors due to autocrine feedback inhibition. This pathway requires the Ca(2+)-binding protein neuronal calcium sensor-1 (NCS-1). The voltage-dependent pathway was pertussis toxin-sensitive, whereas the voltage-independent pathway was largely pertussis toxin-insensitive. Characterization of the voltage-independent inhibition of Ca(2+) currents revealed that it did not involve protein kinase C-dependent signaling pathways but did require the activity of a Src family tyrosine kinase. Two structurally distinct Src kinase inhibitors, 4-amino-5-(4-methylphenyl)7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP1) and a Src inhibitory peptide, increased the Ca(2+) currents, and no further increase in Ca(2+) currents was elicited by addition of naloxone and suramin. In addition, the Src-like kinase appeared to act in the same pathway as NCS-1. In contrast, addition of PP1 did not prevent a voltage-dependent facilitation elicited by a strong pre-pulse depolarization indicating that this pathway was independent of Src kinase activity. PPI no longer increased Ca(2+) currents after addition of the P/Q-type channel blocker omega-agatoxin TK. The alpha(1A) subunit of P/Q-type Ca(2+) channels was immunoprecipitated from chromaffin cell extracts and found to be phosphorylated in a PP1-sensitive manner by endogenous kinases in the immunoprecipitate. A high molecular mass (around 220 kDa) form of the alpha(1A) subunit was detected by anti-phosphotyrosine, suggesting a possible target for Src family kinase action. These data demonstrate a voltage-independent mechanism for autocrine inhibition of P/Q-type Ca(2+) channel currents in chromaffin cells that requires Src family kinase activity and suggests that this may be a widely distributed pathway for Ca(2+) channel regulation.

Short- and long-term differential effects of neuroprotective drug NS-7 on voltage-dependent sodium channels in adrenal chromaffin cells.

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited veratridine-induced (22)Na(+) influx via voltage-dependent Na(+) channels (IC(50)=11.4 microM). The inhibition by NS-7 occurred in the presence of ouabain, an inhibitor of Na(+),K(+) ATPase, but disappeared at higher concentration of veratridine, and upon the washout of NS-7. NS-7 attenuated veratridine-induced (45)Ca(2+) influx via voltage-dependent Ca(2+) channels (IC(50)=20.0 microM) and catecholamine secretion (IC(50)=25.8 microM). Chronic (>/=12 h) treatment of cells with NS-7 increased cell surface [(3)H]-STX binding by 86% (EC(50)=10.5 microM; t(1/2)=27 h), but did not alter the K(D) value; it was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, but was not associated with increased levels of Na(+) channel alpha- and beta(1)-subunit mRNAs. In cells subjected to chronic NS-7 treatment, (22)Na(+) influx caused by veratridine (site 2 toxin), alpha-scorpion venom (site 3 toxin) or beta-scorpion venom (site 4 toxin) was suppressed even after the extensive washout of NS-7, and veratridine-induced (22)Na(+) influx remained depressed even at higher concentration of veratridine; however, either alpha- or beta-scorpion venom, or Ptychodiscus brevis toxin-3 (site 5 toxin) enhanced veratridine-induced (22)Na(+) influx as in nontreated cells. These results suggest that in the acute treatment, NS-7 binds to the site 2 and reversibly inhibits Na(+) channels, thereby reducing Ca(2+) channel gating and catecholamine secretion. Chronic treatment with NS-7 up-regulates cell surface Na(+) channels via translational and externalization events, but persistently inhibits Na(+) channel gating without impairing the cooperative interaction between the functional domains of Na(+) channels.

Development and Dissipation of Ca 2+ Gradients in Adrenal Chromaffin Cells

We used pulsed laser imaging to measure the development and dissipation of Ca 2+ gradients evoked by the activation of voltage-sensitive Ca 2+ channels in adrenal chromaffin cells. Ca 2+ gradients appeared rapidly (<5 ms) upon membrane depolarization and dissipated over several hundred milliseconds after membrane repolarization. Dissipation occurred with an initial fast phase, as the steep gradient near the membrane collapsed, and a slower phase as the remaining shallow gradient dispersed. Inhibition of active Ca 2+ uptake by the endoplasmic reticulum (thapsigargin) and mitochondria (carbonylcyanide p-trifluoro-methoxyphenylhydrazone/oligomycin) had no effect on the size of Ca 2+ changes or the rate of gradient dissipation, suggesting that passive endogenous Ca 2+ buffers are responsible for the slow Ca 2+ redistribution. We used a radial diffusion model incorporating Ca 2+ diffusion and binding to intracellular Ca 2+ buffers to simulate Ca 2+ gradients. We included a 3D optical sectioning model, simulating the effects of out-of-focus light, to allow comparison with the measured gradients. Introduction of a high-capacity immobile Ca 2+ buffer, with a buffer capacity on the order of 1000 and appropriate affinity and kinetics, approximated the size of the Ca 2+ increases and rate of dissipation of the measured gradients. Finally, simulations without exogenous buffer suggest that the Ca 2+ signal due to Ca 2+ channel activation is restricted by the endogenous buffer to a space less than 1 μm from the cell membrane.

Regulation of Ca 2+ influx by a protein kinase C activator in chromaffin cells: differential role of P/Q- and L-type Ca 2+ channels

Phorbol esters reduce depolarization-evoked Ca 2+ influx in adrenal chromaffin cells, suggesting that voltage-sensitive Ca 2+ channels (VSCCs) are inhibited by protein kinase C-mediated phosphorylation. We now address the possibility that L- and P/Q-type Ca 2+ channel subtypes might be differentially involved in phorbol ester action. In bovine chromaffin cells, short-term (10 min) incubations with phorbol 12-myristate 13-acetate (PMA) inhibited early high K +-evoked rises in cytosolic free Ca 2+ concentration ([Ca 2+] i) and the early component of the depolarization-evoked Mn 2+ quenching of fura-2 fluorescence in a dose-dependent manner (IC 50: 18 and 7 nM; maximal inhibitions: 45 and 48%, respectively). The protein kinase C inhibitor staurosporine (100 nM) reverted the inhibitory action of PMA. PMA (0.1–1 μM) inhibited the early and late phases of the ionomycin (2 μM)-evoked [Ca 2+] i transients by 14–23%. ω-Agatoxin IVA, a blocker of P/Q-type Ca 2+ channels, inhibited high K +-evoked [Ca 2+] i rises in a dose-dependent fashion (IC 50=50 nM). In contrast, 0.1 μM ω-conotoxin GVIA, a blocker of N-type channels, was without effect. A sizeable (<45%) component of early Ca 2+ influx persisted in the combined presence of ω-agatoxin IVA (100 nM) and nitrendipine (1 μM). Simultaneous exposure to ω-agatoxin IVA and PMA inhibited both the early [Ca 2+] i transients and Mn 2+ quenching to a much greater extent than each drug separately. Inhibition of the [Ca 2+] i transients by nitrendipine and PMA did not significantly exceed that produced by PMA alone. It is concluded that phorbol ester-mediated activation of protein kinase C inhibits preferentially L-type VSCCs over P/Q type channels in adrenal chromaffin cells. However, the possibility cannot be ruled out that dihydropyridine-resistant, non-P/Q type channels might also be negatively regulated by protein kinase C. This may represent an important pathway for the specific control of VSCCs by protein kinase C-linked receptors, not only in paraneurones but presumably also in neurones and other excitable cells.

P–59 - Role of mitogen activated protein kinase in the regulation of the voltage-dependent sodium channels in adrenal chromaffin cells.

P–59 - Role of mitogen activated protein kinase in the regulation of the voltage-dependent sodium channels in adrenal chromaffin cells.

Protein kinase C-mediated down-regulation of voltage-dependent sodium channels in adrenal chromaffin cells.

Treatment of cultured bovine adrenal chromaffin cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), decreased [3H]saxitoxin ([3H]STX) binding in a concentration (IC50 = 19 nM)- and time (t1/2 = 4.5 h)-dependent manner. TPA (100 nM for 15 h) lowered the Bmax of [3H]STX binding by 53% without altering the KD value. Phorbol 12,13-dibutyrate (PDBu) also reduced [3H]STX binding, whereas 4 alpha-TPA, an inactive analogue, had no effect. The inhibitory effect of TPA was abolished when H-7 (an inhibitor of PKC), but not H-89 (an inhibitor of cyclic AMP-dependent protein kinase), was included in the culture medium for 1 h before and during TPA treatment. Simultaneous treatment with TPA in combination with either actinomycin D or cycloheximide, an inhibitor of protein synthesis, nullified the effect of TPA. TPA treatment also attenuated veratridine-induced 22Na+ influx but did not alter the affinity of veratridine for Na channels as well as an allosteric potentiation of veratridine-induced 22Na+ influx by brevetoxin. These results suggest that an activation of PKC down-regulates the density of Na channels without altering their pharmacological features; this down-regulation is mediated via the de novo synthesis of an as yet unidentified protein(s), rather than an immediate effect of Na channel phosphorylation.

The effect of racemic ketamine on the large conductance Ca +2-activated potassium (BK) channels in GH 3 cells

Recently, inhalation anesthetics have been reported to block BK channels in adrenal chromaffin cells. To determine if BK block was characteristic only of inhalation anesthetics or was also a property of other general anesthetics we examined the effects of ketamine, an intravenous general anesthetic which is structurally different than inhalation anesthetics. Cell-attached and excised patch single channel and standard whole cell recording techniques were used to examine the effect of racemic ketamine on the BK channel activity in GH 3 cells. When solutions containing 150 mM KCl are used in both the pipette and bath, the BK channels are characterized as a voltage-dependent channel with a unit conductance of 150–300 pS. Racemic ketamine (at clinically relevant concentrations; 2–500 μM) selectively blocked BK channels in a dose-dependent, reversible manner as evidenced by decreases in NP o (number of channels × open probability). This decrease was due to both a decrease in mean open time and an increase in the mean closed time but without a decrease in single-channel current amplitude. Ketamine shifts the P o vs voltage curve to higher potentials without a change in the slope of the voltage dependence. Ketamine also shifts the P o vs [Ca +2] relationship to higher Ca +2 concentrations. The IC 50 for the single-channel block by ketamine is 20.3 ± 15.9 μM. In an effort to confirm that the effect of ketamine was predominantly due to a block of the BK channels, standard whole cell techniques were utilized. As with the single-channel experiments, ketamine (2–500 μM) produced a dose-dependent, voltage-independent and reversible decrease in outward current with an IC 50 of 23.7 ± 11.5 μM. Addition of 100 μM ketamine to cells pretreated with the BK channel blocker, charybdotoxin (ChTX), did not result in a further decrease in outward current. These results demonstrate a selective effect of ketamine at clinically relevant concentrations which is consistent with results reported for inhalation anesthetics.

Inhibition of voltage-dependent Ca2+ channels via alpha 2-adrenergic and opioid receptors in cultured bovine adrenal chromaffin cells.

Adrenal chromaffin cells secrete catecholamines and opioids. The effects of these agents on whole-cell Ca2+ channel currents were studied, using bovine adrenal chromaffin cells kept in short term culture. Ca2+ channel currents recorded during voltage-clamp pulses from a holding potential of -80 mV to 0 mV were reversibly reduced by 10 microM epinephrine (in the presence of 1 microM propranolol) or 5 microM of the synthetic opioid, d-Ala2-d-Leu5-enkephalin (DADLE) by approximately 35% and 25%, respectively. The inhibitory action of epinephrine was mimicked by clonidine, reduced by yohimbine but not affected by prazosin. The DADLE-induced reduction of the Ca2+ channel current was antagonized by naloxone. The dihydropyridine (+)PN 200-110 (5 microM) reduced the Ca2+ channel current by approximately 40%; the Ca2+ channel current inhibited by (+)PN 200-110 was not further reduced by epinephrine. Intracellular infusion of guanosine-5'-O-(2-thiodiphosphate) and pretreatment of cells with pertussis toxin abolished the inhibitory effect of both epinephrine and DADLE. In membranes of adrenal chromaffin cells, four pertussis-toxin-sensitive G-proteins were identified, including Gi1, Gi2, Go1 and another Go subtype, possibly Go2. The data show that activation of alpha 2-adrenergic and opioid receptors causes an inhibition of dihydropyridine-sensitive Ca2+ channels in adrenal chromaffin cells. These inhibitory modulations are mediated by pertussis-toxin-sensitive G-proteins and may represent a mechanism for a negative feedback signal by agents released from the adrenal medulla.

Identification of ω‐Conotoxin Binding Sites on Adrenal Medullary Membranes: Possibility of Multiple Calcium Channels in Chromaffin Cells

Binding of 125I-omega-conotoxin GVIA and [3H]nitrendipine to membranes from bovine adrenal medulla was investigated to test for the presence of N- and L-type Ca2+ channels in adrenal chromaffin cells. Saturable, high-affinity binding sites for 125I-omega-conotoxin and [3H]nitrendipine were detected in a membrane fraction from adrenal medulla. [3H]Nitrendipine binding sites were found to have a KD of 500 +/- 170 pM and a Bmax of 26 +/- 11 pmol/g of protein. 125I-omega-Conotoxin binding sites had a KD of 215 +/- 56 pM and a Bmax of 105 +/- 18 pmol/g of protein, about four times the number of sites found for [3H]nitrendipine. 125I-omega-Conotoxin binding was potently inhibited by unlabeled toxin and Ca2+ but was unaffected by dihydropyridines, verapamil, and diltiazem. [3H]Nitrendipine binding was not affected by omega-conotoxin, whereas it was inhibited by other dihydropyridines. Bay K 8644 potentiated K+-evoked cytosolic Ca2+ transients measured by fura-2 fluorescence, and this potentiation was completely blocked by nifedipine. In contrast, omega-conotoxin had no effect on Bay K 8644-evoked Ca2+ transients. Thus, the binding sites for omega-conotoxin and for nitrendipine appear to be different. The results confirm the presence of L-type Ca2+ channels and open the possibility of N-type Ca2+ channels as the omega-conotoxin binding sites in chromaffin cell membranes.

Large depolarization induces long openings of voltage-dependent calcium channels in adrenal chromaffin cells

Single Ca2+-channel currents in bovine adrenal chromaffin cells were studied with the patch-clamp technique using Ba2+ as the charge carrier. Depolarizing pulses to voltages less than +10 mV from holding voltage of -60 mV elicited short openings with a mean life time of less than 1 msec. Depolarization to more positive voltages elicited longer openings with a mean life time of about 3 msec in addition to the short openings similar to those observed at less positive voltages. Following large depolarizing prepulses, 2 types of "tail" openings, one with a mean duration of less than 1 msec and the other with a mean duration of 4 msec, were observed. In the presence of a dihydropyridine BAY K 8644, openings with a mean duration of more than 12 msec were present. Depolarization-induced long openings and BAY K 8644-produced long openings differed in the first latency and open-time properties. The results could be explained in terms of multiple open states of one type of Ca2+ channel. A kinetic model with at least 2 open states is required to explain activation of Ca2+ channels in chromaffin cells.

Gamma-Aminobutyric acid receptor channels in adrenal chromaffin cells: a patch-clamp study.

We have studied membrane channels activated by gamma-aminobutyric acid (GABA) in adrenal medullary chromaffin cells by using patch-clamp techniques. These channels share many properties with GABA-receptor channels in the central nervous system. They are chloride-selective, blocked by the GABA antagonist bicuculline, and reversibly desensitized at high GABA concentrations. The dose-response curve has a slope of 2 in the Hill plot, indicating a bimolecular binding reaction of GABA to the receptor. Single-channel currents display multiple conductance states as do glycine-activated chloride channels in mouse spinal neurons. Gating properties of GABA-activated channels, as described by a sequential model for agonist-activated channels, are similar to gating properties in central neurons. GABA-induced currents are potentiated by diazepam, indicating that anxiolytic drugs like the benzodiazepines might be involved in the regulation of anxiety states in the peripheral nervous system.