Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons.

Multiple types of calcium channels have been found in neurons, but uncertainty remains about which ones are involved in stimulus-secretion coupling. Two types of calcium channels in rat sympathetic neurons were described, and their relative importance in controlling norepinephrine release was analyzed. N-type and L-type calcium channels differed in voltage dependence, unitary barium conductance, and pharmacology. Nitrendipine inhibited activity of L-type channels but not N-type channels. Potassium-evoked norepinephrine release was markedly reduced by cadmium and the conesnail peptide toxin omega-Conus geographus toxin VIA, agents that block both N- and L-type channels, but was little affected by nitrendipine at concentrations that strongly reduce calcium influx, as measured by fura-2. Thus N-type calcium channels play a dominant role in the depolarization-evoked release of norepinephrine.

ATP serves as a negative feedback inhibitor of voltage-gated Ca2+ channel currents in cultured bovine adrenal chromaffin cells.

Modulation of voltage-gated Ca(2+) channel current (I(Ca)) regulates secretion of catecholamines from adrenal chromaffin cells. Previous work demonstrated that I(Ca) can be augmented by phosphorylation to increase secretion or that inhibition of I(Ca) results in diminished catecholamine secretion. In the current manuscript, we show that stimulation of chromaffin cells results in the release of an "endogenous inhibitor" that suppresses I(Ca). The inhibition is due to the secretion of ATP, which is stored at high concentrations in secretory granules and is coreleased with catecholamines upon stimulation. The ATP exerts its actions through P(2 gamma) purinoceptors and inhibits both N- and P/Q-type Ca (2+) channels in a voltage-dependent manner but with different efficacies. Overall, we have identified and characterized a negative feedback pathway that may serve as an important regulatory mechanism for catecholamine secretion in chromaffin cells.

Differential activation of adenosine receptors decreases N-type but potentiates P-type Ca2+ current in hippocampal CA3 neurons.

Adenosine is released in the brain in significant quantities in response to increased cellular activity. Adenosine has been shown either to decrease synaptic transmission or to produce an excitatory response in hippocampal synapses, resulting in increased glutamate release. Previous reports have shown that adenosine or its analogs reduced Ca2+ current in dorsal root ganglion and hippocampal neurons. Here we show that the selective activation of adenosine receptor subtypes has different effects on Ca2+ channels from acutely isolated pyramidal neurons from the CA3 region of guinea pig hippocampus. Activation of A1 receptors inhibited primarily N-type Ca2+ current. In contrast, activation of A2b receptors resulted in significant potentiation of P-type but not N-type Ca2+ current. This potentiation could be inhibited by blocking the cAMP-dependent protein kinase. Because of the ubiquity of adenosine, the differential effects on Ca2+ channels of adenosine receptor subtype activation may have significant implications for neuronal excitability.

Omega-conotoxin GVIA blocks a Ca2+ current in bovine chromaffin cells that is not of the "classic" N type.

Previous studies have identified two components of whole-cell Ca2+ current in bovine chromaffin cells. The "standard" component was activated by single depolarizations, while "facilitation" could be activated by large prepulses or repetitive depolarizations. Neither current component was sensitive to changes in holding potential between -100 and -50 mV; thus neither appeared to be carried by N-type Ca2+ channels. We now report that the facilitation Ca2+ current is insensitive to omega-conotoxin GVIA (omega-CgTx), but that the toxin blocks approximately 50% of the standard Ca2+ current. In some cells the toxin blocks all of the standard Ca2+ current, in others about half of the current, while in others it has no effect. Kinetic differences in current activation are observed after toxin application. These results suggest that the standard component of chromaffin cell Ca2+ current is composed of two pharmacologically distinct channels-one is omega-CgTx sensitive and the other is not. Two kinetically distinct types of 14 pS Ca2+ channels that may correspond to the omega-CgTx-sensitive and -insensitive components were observed in single-channel experiments. Because omega-CgTx blocked Ca2+ channels that were not inactivated by a depolarized holding potential, the commonly used Ca2+ channel categorization scheme may be inadequate to describe the Ca2+ channels found in chromaffin cells.

Tyrosine phosphorylation regulates rapid endocytosis in adrenal chromaffin cells.

Secretion of neurotransmitter at the synapse and in secretory cells depends on the availability of vesicles for exocytosis. Rapid endocytosis is responsible for initiating local vesicle recycling and is essential during sustained neurotransmission. Although exocytosis is triggered by Ca(2+) influx and modulated by serine/threonine kinases, relatively little is known about the regulation of rapid endocytosis. Our data suggest that rapid endocytosis is controlled by tyrosine phosphorylation. Treatment of bovine adrenal chromaffin cells with tyrphostin 23, a protein tyrosine kinase inhibitor, dramatically slowed the time course of rapid endocytosis. In contrast, there was no effect on either the amount or rate of exocytosis. Application of orthovanadate, Zn(2+), or poly(Glu, Tyr) (4:1), each of which is a tyrosine phosphatase inhibitor, reversed the effect of tyrphostin 23 on rapid endocytosis. Thus rapid endocytosis, like exocytosis, is subject to regulation by intracellular signaling pathways.

Coexpression of cloned alpha(1B), beta(2a), and alpha(2)/delta subunits produces non-inactivating calcium currents similar to those found in bovine chromaffin cells.

Chromaffin cells express N-type calcium channels identified on the basis of their high sensitivity to block by omega-conotoxin GVIA (omega-CgTx GVIA). In contrast to neuronal N-type calcium currents that inactivate during long depolarizations and that require negative holding potentials to remove inactivation, many chromaffin cells exhibit N-type calcium channel currents that show little inactivation during maintained depolarizations and that exhibit no decrease in channel availability at depolarized holding potentials. N-type calcium channels are thought to be produced by combination of the pore-forming alpha(1B) subunit and accessory beta and alpha(2)/delta subunits. To examine the molecular composition of the non-inactivating N-type calcium channel, we cloned the alpha(1B) and accessory beta (beta(1b), beta(1c,) beta(2a), beta(2b), and beta(3a)) subunits found in bovine chromaffin cells. Expression of the subunits in either Xenopus oocytes or human embryonic kidney 293 cells produced high-threshold calcium currents that were blocked by omega-CgTx GVIA. Coexpression of bovine alpha(1B) with beta(1b), beta(1c), beta(2b), or beta(3a) produced currents that were holding potential dependent. In contrast, coexpression of bovine alpha(1B) with beta(2a) produced holding potential-independent calcium currents that closely mimicked native non-inactivating currents, suggesting that non-inactivating N-type channels consist of bovine alpha(1B), alpha(2)/delta, and beta(2a).

Calpains play a role in insulin secretion and action.

Studies of the genetic basis of type 2 diabetes suggest that variation in the calpain-10 gene affects susceptibility to this common disorder, raising the possibility that calpain-sensitive pathways may play a role in regulating insulin secretion and/or action. Calpains are ubiquitously expressed cysteine proteases that are thought to regulate a variety of normal cellular functions. Here, we report that short-term (4-h) exposure to the cell-permeable calpain inhibitors calpain inhibitor II and E-64-d increases the insulin secretory response to glucose in mouse pancreatic islets. This dose-dependent effect is observed at glucose concentrations above 8 mmol/l. This effect was also seen with other calpain inhibitors with different mechanisms of action but not with cathepsin inhibitors or other protease inhibitors. Enhancement of insulin secretion with short-term exposure to calpain inhibitors is not mediated by increased responses in intracellular Ca2+ or increased glucose metabolism in islets but by accelerated exocytosis of insulin granules. In muscle strips and adipocytes, exposure to both calpain inhibitor II and E-64-d reduced insulin-mediated glucose transport. Incorporation of glucose into glycogen in muscle also was reduced. These results are consistent with a role for calpains in the regulation of insulin secretion and insulin action.

Activation of nicotinic acetylcholine receptors augments calcium channel-mediated exocytosis in rat pheochromocytoma (PC12) cells.

The functional effect of activating Ca2+-permeable neuronal nicotinic acetylcholine receptors (nAChRs) on vesicle secretion was studied in PC12 cells. Single cells were patch-clamped in the whole-cell configuration and stimulated with either brief pulses of nicotine to activate the Ca2+-permeable nAChRs or with voltage steps to activate voltage-dependent Ca2+ channels. Membrane capacitance was used as a measure of vesicle secretion. Activation of nAChRs by nicotine application to cells voltage clamped at -80 mV evoked secretion. This secretion was completely abolished by nicotinic antagonists. When the cells were voltage clamped at +20 mV in the presence of Cd2+ to block voltage-activated Ca2+ channels, nicotine elicited a small amount of secretion. Most interestingly, when the nAChRs were activated coincidentally with voltage-dependent Ca2+ channels, secretion was augmented approximately twofold over the secretion elicited with voltage-dependent Ca2+ channels alone. Our data suggest that Ca2+ influx via nAChRs affects Ca2+-dependent cellular functions, including vesicle secretion. In addition to the secretion evoked by nAChR activation at hyperpolarized potentials, we demonstrate that even at depolarized potentials, nAChRs provide an important Ca2+ entry pathway underlying Ca2+-dependent cellular processes such as exocytosis.

Action potential waveforms reveal simultaneous changes in ICa and IK produced by 5-HT in rat dorsal raphe neurons.

Action potentials were recorded from serotonergic dorsal raphe (DR) neurons acutely isolated from the adult rat brain. Action potential waveforms were used as command potentials for whole-cell patch-clamp studies to investigate the Ca2+ and K+ currents underlying action potentials and the modulatory effects of 5-Hydroxytryptamine (5-HT) on them. These data were compared with currents elicited by using rectangular voltage steps of the type commonly used in voltage-clamp experiments. In the same cell, 5-HT simultaneously augmented K+ currents and inhibited Ca2+ currents. Experimental conditions were chosen which allowed us to examine the action of 5-HT on K+ and Ca2+ currents simultaneously or in isolation; 5-HT produced a larger inhibition of calcium current during an action potential waveform compared with that measured by using rectangular steps of voltage. A possible explanation for this finding is that the maximal inhibition is seen immediately after a voltage jump and then decreases with time. Action potentials are, in general, so brief that little time-dependent relief of block is observed. Most of the inhibition of Ca2+ current resulted from a direct effect on Ca2+ channels rather than a shortening of the action potential. The inhibition of Ca2+ current by 5-HT also decreased the Ca(2+)-activated K+ currents. These results suggest that 5-HT reduces DR neuron excitability by the simultaneous activation of K+ channel currents open at the resting potential and the suppression of Ca2+ channel currents.

Activation of purinergic receptors by ATP inhibits secretion in bovine adrenal chromaffin cells.

Autoinhibition is a common mechanism observed in neurons to regulate neurotransmission. Released neurotransmitter interacts with presynaptic autoreceptors to inhibit subsequent release. The requisite elements for autoinhibition are present in chromaffin cells: secretory granules contain millimolar levels of ATP which is coreleased with catecholamines upon stimulation and the cells express purinergic receptors. We were interested to determine whether autoinhibition produced by ATP binding to purinergic receptors plays an important role in catecholamine release from chromaffin cells. In these studies, short depolarizations were used to elicit transmitter release measured by membrane capacitance. We find that stimulation of chromaffin cells results in the release of endogenous ATP which may suppress Ca(2+) channel currents and secretion. In the presence of a maximal concentration of ATP, both the amount of secretion and the maximal rate of release are about half that observed in the absence of ATP. ATP-mediated inhibition of secretion was blocked by Reactive Blue-2 suggesting the involvement of P(2Y) purinergic receptors. Prepulses to positive potentials that relieve the Ca(2+) channel block largely relieve the inhibition of secretion. Furthermore, when secretion is plotted as a function of Ca(2+) influx there is no apparent change in the relationship between control cells and those stimulated in the presence of ATP and prepulses. These results suggest that ATP diminishes secretion by inhibiting Ca(2+) influx into the cells. Our results indicate that feedback inhibition by ATP, mediated primarily by Ca(2+) channels, may be an important regulator of catecholamine release in chromaffin cells.

Inhibition of calcium currents in cultured myenteric neurons by neuropeptide Y: evidence for direct receptor/channel coupling.

Single channel recordings from rat myenteric plexus neurons demonstrated the presence of two categories of Ca2+ channels. One type of Ca channel had a slope conductance of 27 pS and was sensitive to dihydropyridines while the other channel type had a conductance of 14 pS and was dihydropyridine-insensitive. The 14 pS channel was mostly inactivated at a holding potential of -40 mV, while the 27 pS channel was much more resistant to depolarized holding potentials. A majority of whole-cell current was reprimed by the use of negative holding (-90 mV) potentials, when compared to that obtained at a holding potential of -40 mV. These properties are consistent with N- and L-type Ca channels previously described. In general, the inactivating part of the whole-cell Ca2+ current, selectively reprimed by negative holding potentials, was inhibited by neuropeptide Y (NPY). Depolarization-induced [Ca2+]i transients assessed using fura-2 showed that the inhibitory effects of nitrendipine and NPY were additive. The effects of NPY were abolished by pertussis toxin pretreatment. Single-channel experiments showed that neither the 14 nor the 27 pS Ca channel currents were inhibited by the addition of NPY outside the patch pipette. These results suggest that NPY modulates N-type Ca2+ channels selectively in these neurons and that an easily diffusible second messenger does not appear to participate in receptor/channel coupling.

Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion.

To facilitate the study of trophic interactions between mesencephalic dopaminergic neurons and their target cells, clonal hybrid cell lines have been developed from rostral mesencephalic tegmentum (RMT) of the 14-day-old embryonic mouse employing somatic cell fusion techniques. Among the hybrid cell lines obtained, one contains a high level of dopamine (DA), another predominantly 3,4-dihydroxyphenylalanine (DOPA), and a third no detectable catecholamines. The hybrid nature of the cell lines is supported by karyotype analysis and by the expression of adhesion molecules as assessed by aggregation in rotation-mediated cell culture. The DA cell line shows neuronal properties including catecholamine-specific histofluorescence, neurite formation with immunoreactivity to neurofilament proteins, and large voltage-sensitive sodium currents with the generation of action potentials. In contrast to the pheochromocytoma cell line (PC12), the dopamine content of the DA hybrid cell line is depleted by low concentrations of N-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Does myxomatosis still regulate numbers of rabbits (Oryctolagus cuniculus Linnaeus, 1758) in the United Kingdom?

Myxomatosis now kills a much smaller proportion of rabbit populations than in the past, while remaining an important regulatory factor, as shown experimentally. On two separate occasions, experimental reduction of the prevalence of the disease (by reducing infestations of the main vector, the rabbit flea) led to significant increases in numbers of rabbits surviving the winter.

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.

Excitatory and inhibitory actions of isoflurane in bovine chromaffin cells.

Isoflurane, a halogenated volatile anesthetic, is thought to produce anesthesia by depressing CNS function. Many anesthetics, including isoflurane, are thought to modulate and/or directly activate GABA(A) receptors. Chromaffin cells are known to express functional GABA(A) receptors. We previously showed that activation of the GABA(A) receptors, with specific agonists, leads to cellular excitation resulting from the depolarized anion equilibrium potential. In this study, our goal was to determine whether isoflurane mimicked this response and to explore the functional consequences of this activation. Furthermore, we sought to study the actions of isoflurane on nicotinic acetylcholine receptors (nAChRs) as they mediate the "sympathetic drive" in these cells. For these studies the Ca(2+)-indicator dye fura-2 was used to assay [Ca(2+)](i). Amperometric measurements were used to assay catecholamine release. We show that bovine adrenal chromaffin cells were excited by isoflurane at clinically relevant concentrations. Isoflurane directly activated GABA(A) receptors found in chromaffin cells, which depolarized the cells and elevated [Ca(2+)](i). Application of isoflurane directly to the chromaffin cells elicited catecholamine secretion from these cells. At the same time, isoflurane suppressed activation of nAChRs, which presumably blocks "sympathetic drive" to the chromaffin cells. These latter results may help explain why isoflurane produces the hypotension observed clinically.

Voltage-dependent inactivation of a calcium channel.

The inactivation of Ca currents in unfertilized eggs of the marine polychaete Neanthes arenaceodentata was investigated by using a voltage clamp technique. These Ca currents do not appear to be masked by other currents in the voltage range studied. Inactivation increased monotonically with increasing depolarization and occurred at potentials more negative than the inward Ca current. Currents elicited by depolarization at different Ca concentrations had approximately the same time course of inactivation, even though the size of the currents varied by almost an order of magnitude. There was complete inactivation in solutions containing Ca, Sr, or Ba (all permeant) for depolarizations beyond -30 mV absolute; the time course of the inactivation process was similar for all three permeant ions. Increasing depolarizations toward the expected equilibrium potential for Ca, Ba, or Sr did not produce any lessening of the inactivation. Thus, it appears that the inactivation seen in Neanthes eggs is a purely voltage-dependent phenomenon.

Comparison of N- and P/Q-type voltage-gated calcium channel current inhibition.

Activation of N- and P/Q-type voltage-gated calcium channels triggers neurotransmitter release at central and peripheral synapses. These channels are targets for regulatory mechanisms, including inhibition by G-protein-linked receptors. Inhibition of P/Q-type channels has been less well studied than the extensively characterized inhibition of N-type channels, but it is thought that they are inhibited by similar mechanisms although possibly to a lesser extent than N-type channels. The aim of this study was to compare the inhibition of the two channel types. Calcium currents were recorded from adrenal chromaffin cells and isolated by the selective blockers omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (400 nM). The inhibition was elicited by ATP (100 microM) or intracellular application of GTP-gamma-S. It was classified as voltage-sensitive (relieved by a conditioning prepulse) or voltage-insensitive (present after a conditioning prepulse). The voltage-insensitive inhibition accounted for a 20% reduction of both currents, whereas the voltage-sensitive inhibition reduced the N-type current by 45% but the P/Q-type current by 18%. However, the voltage dependence of the inhibition, the time course of relief from inhibition during a conditioning prepulse, and the time course of reinhibition after such a prepulse showed few differences between the N- and P/Q-type channels. Assuming a simple bimolecular reaction, our data suggest that changes in the kinetics of the G-protein/channel interaction alone cannot explain the differences in the inhibition of the N- and P/Q-type calcium channels. The subtle differences in inhibition may facilitate the selective regulation of neurotransmitter release.

Multiple Ca2+ currents elicited by action potential waveforms in acutely isolated adult rat dorsal root ganglion neurons.

Ca2+ entry into different diameter cell bodies of dorsal root ganglion (DRG) neurons depolarized with action potential (AP) waveform commands was studied using the whole-cell patch-clamp technique and pharmacological probes. We have previously shown that Ca2+ current expression in DRG neuron cell bodies depends on cell diameter. In small diameter DRG neurons, L- and N-type Ca2+ currents usually accounted for most Ca2+ entry during APs as determined by blockade with nimodipine and omega-conotoxin GVIA (omega-CgTx). In medium- diameter DRG neurons, T-type Ca2+ currents accounted for 29% or 54% of Ca2+ entry in cells held at -60 mV or -80 mV, respectively, based on blockade by amiloride. T-type Ca2+ currents did not usually contribute to Ca2+ entry in large diameter DRG neurons. An amiloride/omega-CgTx/nimodipine-resistant Ca2+ current was prominent in medium diameter DRG neurons, while L- and N-type Ca2+ currents played a relatively small role in Ca2+ entry. In all DRG neuron sizes, AP-generated currents were large in amplitude, resulting in significant Ca2+ entry. APs with slower rates of repolarization increased Ca2+ entry. In DRG neurons that expressed T-type Ca2+ currents, the duration of Ca2+ current entry during APs was prolonged, and this prolongation was reduced by amiloride. Thus, antagonists selective for different Ca2+ channels produced different patterns of blockade of AP-generated Ca2+ entry in different diameter DRG cell bodies. Selective Ca2+ channel modulation by neurotransmitters might be expected to have similar effects.