C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling

The purpose of the present experiments was to define the role of C-type natriuretic peptide (CNP) in the regulation of atrial secretion of atrial natriuretic peptide (ANP) and atrial stroke volume. Experiments were performed in perfused beating and nonbeating quiescent atria, single atrial myocytes, and atrial membranes. CNP suppressed in a dose-related fashion the increase in atrial stroke volume and ANP secretion induced by atrial pacing. CNP caused a right shift in the positive relationships between changes in the secretion of ANP and atrial stroke volume or translocation of the extracellular fluid (ECF), which indicates the suppression of atrial myocytic release of ANP into the paracellular space. The effects of CNP on the secretion and contraction were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). CNP increased cGMP production in the perfused atria, and the effects of CNP on the secretion of ANP and atrial dynamics were accentuated by pretreatment with an inhibitor of cGMP phosphodiesterase, zaprinast. An inhibitor of the biological natriuretic peptide receptor (NPR), HS-142-1, attenuated the effects of CNP. The suppression of ANP secretion by CNP and 8-BrcGMP was abolished by a depletion of extracellular Ca(2+) in nonbeating atria. Natriuretic peptides increased cGMP production in atrial membranes with a rank order of potency of CNP > BNP > ANP, and the effect was inhibited by HS-142-1. CNP and 8-BrcGMP increased intracellular Ca(2+) concentration transients in single atrial myocytes, and mRNAs for CNP and NPR-B were expressed in the rabbit atrium. From these results we conclude that atrial ANP release and stroke volume are controlled by CNP via NPR-B-cGMP mediated signaling, which may in turn act via regulation of intracellular Ca(2+).     

Protein kinase-dependent and Ca(2+)-independent cAMP inhibition of ANP release in beating rabbit atria

Regulation of atrial release of atrial natriuretic peptide (ANP) is coupled to changes in atrial dynamics. However, the mechanism by which mechanical stretch controls myocytic ANP release must be defined. The purpose of this study was to define the mechanism by which cAMP controls myocytic ANP release in perfused, beating rabbit atria. The cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine (IBMX) inhibited myocytic ANP release. The activation of adenylyl cyclase with forskolin inhibited ANP release, which was a function of an increase in cAMP production. Inhibitors for L-type Ca(2+) channels and protein kinase A (PKA) attenuated a minor portion of the forskolin-induced inhibition of ANP release. G?-6976 and KN-62, which are specific inhibitors for protein kinase C-alpha and Ca(2+)/calmodulin kinase, respectively, failed to modulate forskolin-induced inhibition of ANP release. The nonspecific protein kinase inhibitor staurosporine blocked forskolin-induced inhibition of ANP release in a dose-dependent manner. Staurosporine but not nifedipine shifted the relationship between cAMP and ANP release. Inhibitors for L-type Ca(2+) channels and PKA and staurosporine blocked forskolin-induced accentuation of atrial dynamics. These results suggest that cAMP inhibits atrial myocytic release of ANP via protein kinase-dependent and L-type Ca(2+)-channel-dependent and -independent signaling pathways.     

Attenuation of negatively regulated ANP secretion by calcium in hypertrophied atria

Abnormal intracellular Ca(2+)-handling has been described in various heart diseases associated with cardiac hypertrophy. The crucial role of Ca(2+) in the excitation-secretion coupling in atrial cardiomyocytes is not well established. To investigate modulation of atrial natriuretic peptide (ANP) secretion regulated by Ca(2+) in hypertrophied atria, responsiveness of stretch-induced ANP to Ca(2+) was studied using isolated perfused quiescent hypertrophied rat atria. Male Sprague-Dawley rats were given a single subcutaneous injection of 50 mg/kg monocrotaline (MCT) and were sacrificed at 5-6 weeks. In isolated perfused hypertrophied right atria from MCT rats, changes in atrial volume induced by increased atrial pressure caused proportional increases in mechanically stimulated extracellular fluid (ECF) translocation and stretch-induced ANP secretion. Stretch-induced ANP secretion was markedly increased by the depletion of extracellular Ca(2+). However, an accentuation of stretch-induced ANP secretion by Ca(2+) depletion was markedly attenuated in hypertrophied right atria, as compared to control right atria. Therefore, stretch-induced ANP secretion in terms of ECF translocation by Ca(2+) depletion in hypertrophied atria was significantly lower than in control right atria. However, no significant differences were observed between nonhypertrophied and control left atria. Depletion of extracellular Ca(2+) caused a decrease in intracellular calcium in single beating atrial myocytes, which was significantly attenuated in hypertrophied atrial myocytes. The results suggest that attenuation of Ca(2+)-induced negative regulation of ANP secretion in hypertrophied atria may be due to the disturbance of intracellular Ca(2+) regulation.     

Regulation of stretch-activated ANP secretion by chloride channels

This study was aimed to define roles of stretch-activated ion channels (SACs), especially Cl(-) channels, in regulation of atrial natriuretic peptide (ANP) secretion using isolated perfused beating atria. The volume load was achieved by elevating height of outflow catheter connected to isolated rat atria and the pressure load was achieved by decreasing diameter of outflow catheter. Both methods increased atrial contractility similarly although volume load was different (736microl for volume load vs. 129microl for pressure load). Atrial stretch by volume load markedly increased ECF translocation and ANP secretion but the pressure load slightly increased. The ANP secretion was positively correlated to workload generated by volume or pressure load. Treatment of atria with gadolinium, a blocker for SACs, attenuated the ECF translocation and the ANP secretion induced by volume load. A blocker for Ca2+-activated Cl(-) channel, niflumic acid (NFA), accentuated the ANP secretion induced by volume load whereas a blocker for swelling-activated Cl(-) channel, diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), attenuated the ANP secretion. The ANP secretion of hypertrophied atria by volume load was markedly reduced and the augmented effect of NFA on volume load-induced ANP secretion was not observed. These results indicate that Cl(-) channels may differently regulate stretch-activated ANP secretion.     

Postnatal changes in inhibitory effect of C-type natriuretic peptide on secretion of ANP

To define developmental changes in atrial natriuretic peptide (ANP) secretion and in the cross talk between C-type natriuretic peptide (CNP) and ANP, we performed experiments in isolated perfused nonbeating cardiac atria isolated from rabbits between 1 and 8 wk of age. Changes in atrial pressure resulted in increases in atrial volume that rose with age and reached the peak value at 4 wk. A rise in volume change increased ANP secretion with concomitant translocation of extracellular fluid (ECF) into the atrial lumen, which increased with age and reached the peak value at 4 wk. The positive relationship between stretch-induced ANP secretion and ECF translocation shifted upward and leftward with age. CNP suppressed stretch-induced ANP secretion in the 8-wk-old group but not in the 2- and 4-wk-old groups without differences in ECF translocation and atrial volume. Therefore, the ANP secretion in terms of ECF translocation was markedly suppressed by CNP in the 8-wk-old group but not in the 2- and 4-wk-old groups. The production of cGMP by CNP in atrial tissue membranes was markedly attenuated in young rabbits. However, 8-bromo-cGMP suppressed stretch-induced ANP secretion in 2- and 8-wk-old groups. Natriuretic peptide receptor-B mRNA was similar in both groups. Therefore, we conclude that the inhibitory effect of CNP on atrial ANP secretion is developmentally regulated, being absent during normal cardiac development in young animals and intact in adult animals.     

Regulation of ANP secretion by cardiac Na+/Ca2+ exchanger using a new controlled atrial model

The myocardial interstitium is important in regulating cardiac function. Between the atrial lumen and the pericardial space are transmural pathways, and movement of interstitial fluid (ISF) through these pathways is one of the main driving forces regulating translocation of substances from the interstitium into the blood. To define how ISF translocation from the interstitial space into the luminal space is regulated by each component of atrial hemodynamics, we devised a new rabbit atrial model in which each physical parameter could be controlled independently. Using this system, we also defined the physiological role of the cardiac Na(+)/Ca(2+) exchanger on secretion of atrial natriuretic peptide (ANP) by depletion of extracellular Na(+) ([Na(+)](o)). Increases in stroke volume and atrial end-systolic volume increased ISF translocation and ANP secretion. However, an increase in atrial rate did not influence ISF translocation but, rather, increased ANP secretion. Gradual depletion of [Na(+)](o) caused gradual increases in ANP secretion and intracellular Ca(2+) ([Ca(2+)](i)), which were blocked in the presence of Ca(2+)-free buffer and Ni(2+), but not in the presence of KB-R7943, diltiazem, mibefradil, caffeine, or monensin. Amiloride and its analog blocked an increase in ANP secretion but not an increase in [Ca(2+)](i) by [Na(+)](o) depletion. Therefore, we suggest that ANP secretion and ISF translocation may be differently controlled by each physical factor. These results also suggest that the increase in ANP secretion in response to [Na(+)](o) depletion may involve inhibition of Na(+)/Ca(2+) and Na(+)/H(+) exchangers but not an increase in [Ca(2+)](i).     

Osmoregulation of atrial myocytic ANP release: osmotransduction via cross-talk between L-type Ca2+ channel and SR Ca2+ release

Hyperosmolality has been known to increase ANP release. However, its physiological role in the regulation of atrial myocytic ANP release and the mechanism by which hyperosmolality increases ANP release are to be defined. The purpose of the present study was to define these questions. Experiments were performed in perfused beating rabbit atria. Hyperosmolality increased atrial ANP release, cAMP efflux, and atrial dynamics in a concentration-dependent manner. The osmolality threshold for the increase in ANP release was as low as 10 mosmol/kgH2O (approximately 3%) above the basal levels (1.55 +/- 1.71, 17.19 +/- 3.11, 23.15 +/- 5.49, 54.04 +/- 11.98, and 62.00 +/- 13.48% for 10, 20, 30, 60, and 100 mM mannitol, respectively; all P < 0.01). Blockade of sarcolemmal L-type Ca2+ channel activity, which increased ANP release, attenuated hyperosmolality-induced increases in ANP release (-13.58 +/- 4.68% vs. 62.00 +/- 13.48%, P < 0.001) and cAMP efflux but not atrial dynamics. Blockade of the Ca2+ release from the sarcoplasmic reticulum, which increased ANP release, attenuated hyperosmolality-induced increases in ANP release (13.44 +/- 7.47% vs. 62.00 +/- 13.48%, P < 0.01) and dynamics but not cAMP efflux. Blockades of Na+-K+-2Cl- cotransporter, Na+/H+ exchanger, and Na+/Ca2+ exchanger had no effect on hyperosmolality-induced increase in ANP release. The present study suggests that hyperosmolality regulates atrial myocytic ANP release and that the mechanism by which hyperosmolality activates ANP release is closely related to the cross-talk between the sarcolemmal L-type Ca2+ channel activity and sarcoplasmic reticulum Ca2+ release, possibly inactivation of the L-type Ca2+ channels.     

T-type calcium currents in rat cardiomyocytes during postnatal development: contribution to hormone secretion

T-type Ca(2+) channels have been suggested to play a role in cardiac automaticity, cell growth, and cardiovascular remodeling. Although three genes encoding for a T-type Ca(2+) channel have been identified, the nature of the isoform(s) supporting the cardiac T-type Ca(2+) current (I(Ca,T)) has not yet been determined. We describe the postnatal evolution of I(Ca,T) density in freshly dissociated rat atrial and ventricular myocytes and its functional properties at peak current density in young atrial myocytes. I(Ca,T) displays a classical low activation threshold, rapid inactivation kinetics, negative steady-state inactivation, slow deactivation, and the presence of a window current. Interestingly, I(Ca,T) is poorly sensitive to Ni(2+) and insensitive to R-type current toxin SNX-482. RT-PCR experiments and comparison of functional properties with recombinant Ca(2+) channel subtypes suggest that neonatal I(Ca,T) is related to the alpha(1G)-subunit. Atrial natriuretic factor (ANF) secretion was measured using peptide radioimmunoassays in atrial tissue. Pharmacological dissection of ANF secretion indicates an important contribution of I(Ca,T) to Ca(2+) signaling during the neonatal period.     

Fast exocytosis mediated by T- and L-type channels in chromaffin cells: distinct voltage-dependence but similar Ca<Superscript>2+</Superscript>-dependence

Expression, spatial distribution and specific roles of different Ca(2+) channels in stimulus-secretion coupling of chromaffin cells are intriguing issues still open to discussion. Most of the evidence supports a role of high-voltage activated (HVA) Ca(2+) channels (L-, N-, P/Q- and R-types) in the control of exocytosis: some suggesting a preferential coupling of specific Ca(2+) channel subunits with the secretory apparatus, others favoring the idea of a contribution to secretion proportional to the expression density and gating properties of Ca(2+) channels. In this work we review recent findings and bring new evidence in favor of the hypothesis that also the LVA (low-voltage-activated, T-type) Ca(2+) channels effectively control fast exocytosis near resting potential in adrenal chromaffin cells of adult rats. T-type channels recruited after long-term treatments with pCPT-cAMP (or chronic hypoxia) are shown to control exocytosis with the same efficacy of L-type channels, which are the dominant Ca(2+) channel types expressed in rodent chromaffin cells. A rigorous comparison of T- and L-type channel properties shows that, although operating at different potentials and with different voltage-sensitivity, the two channels possess otherwise similar Ca(2+)-dependence of exocytosis, size and kinetics of depletion of the immediately releasable pool and mobilize vesicles of the same quantal size. Thus, T- and L-type channels are coupled with the same Ca(2+)-efficiency to the secretory apparatus and deplete the same number of vesicles ready for release. The major difference of the secretory signals controlled by the two channels appear to be the voltage range of operation, suggesting the idea that stressful conditions (hypoxia and persistent beta-adrenergic stimulation) can lower the threshold of cell excitability by recruiting new Ca(2+) channels and activate an additional source of catecholamine secretion.     

Voltage-dependent calcium entry in confluent bovine capillary endothelial cells.

Confluent bovine capillary endothelial cells display, when examined for voltage-dependent calcium entries using cell-attached channel recordings, two types of Ca2+ channels (4 and 23.5 pS in 110 mM Ba2+) both sensitive to the dihydropyridine Ca agonist BAY K 8644. In contrast to isolated cells, confluent cells display no T-type, low threshold activity, and Ca currents were typically only elicited at very depolarized potentials. In these cells, voltage-dependent calcium entries will only be made operative by substances able to shift their activation towards the resting potential.     

Role of voltage-gated calcium channels in potassium-stimulated aldosterone secretion from rat adrenal zona glomerulosa cells

The mineralocorticoid aldosterone plays an important role in the regulation of plasma electrolyte homeostasis. Exposure of acutely isolated rat adrenal zona glomerulosa cells to elevated K(+) activates voltage-gated calcium channels and initiates a calcium-dependent increase in aldosterone synthesis. We developed a novel 96-well format aldosterone secretion assay to rapidly evaluate the effect of known T- and L-type calcium channel antagonists on K(+)-stimulated aldosterone secretion and better define the role of voltage-gated calcium channels in this process. Reported T-type antagonists, mibefradil and Ni(2+), and selected L-type antagonist dihydropyridines, inhibited K(+)-stimulated aldosterone synthesis. Dihydropyridine-mediated inhibition occurred at concentrations which had no effect on rat alpha1H T-type Ca(2+) currents. In contrast, below 10 microM, the L-type antagonists verapamil and diltiazem showed only minimal inhibitory effects. To examine the selectivity of the calcium channel antagonist-mediated inhibition, we established an aldosterone secretion assay in which 8Br-cAMP stimulates aldosterone secretion independent of extracellular calcium. Mibefradil remained inhibitory in this assay, while the dihydropyridines had only limited effects. Taken together, these data demonstrate a role for the L-type calcium channel in K(+)-stimulated aldosterone secretion. Further, they confirm the need for selective T-type calcium channel antagonists to better address the role of T-type channels in K(+)-stimulated aldosterone secretion.     

Stimulation of ANP secretion by 2-Cl-IB-MECA through A(3) receptor and CaMKII

Adenosine is a potent mediator of myocardial protection against hypertrophy via A(1) or A(3) receptors that may be partly related to atrial natriuretic peptide (ANP) release. However, little is known about the possible involvement of the A(3) receptor on ANP release. We studied the effects of the A(3) receptor on atrial functions and its modification in hypertrophied atria. A selective A(3) receptor agonist, 2-chloro-N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide (2-CI-IB-MECA), was perfused into isolated, beating rat atria with and without receptor modifiers. 2-CI-IB-MECA dose-dependently increased the ANP secretion, which was blocked by the A(3) receptor antagonist, but the increased atrial contractility and decreased cAMP levels induced by 30muM 2-CI-IB-MECA were not affected. The 100muM 2-(1-hexylnyl)-N-methyladenosine (HEMADO) and N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide (IB-MECA), A(3) receptor agonist, also stimulated the ANP secretion without positive inotropy. The potency for the stimulation of ANP secretion was 2-CI-IB-MECA>IB-MECA=HEMADO. The inhibition of the ryanodine receptor or calcium/calmodulin-dependent kinase II (CaMKII) attenuated 2-CI-IB-MECA-induced ANP release, positive inotropy, and translocation of extracellular fluid. However, the inhibition of L-type Ca(2+) channels, sarcoplasmic reticulum Ca(2+)-reuptake, phospholipase C or inositol 1,4,5-triphosphate receptors did not affect these parameters. 2-CI-IB-MECA decreased cAMP level, which was blocked only with an inhibitor of CaMKII or adenylyl cyclase. These results suggest that 2-CI-IB-MECA increases the ANP secretion mainly via A(3) receptor activation and positive inotropy by intracellular Ca(2+) regulation via the ryanodine receptor and CaMKII.     

Modulation of Ca2+ channels by atrial natriuretic peptide in the bovine adrenal glomerulosa cell.

In the bovine adrenal glomerulosa cell, calcium influx through voltage-dependent calcium channels is critical to maintaining an aldosterone secretory response. In patch clamp, atrial natriuretic peptide (ANP) inhibits T-type calcium channel current yet stimulates L-type calcium channel current. In the present study the channel effects of ANP observed in the patch-clamp configuration were extended and related to populations of cells. We observed the following. (i) The effect of ANP on T-channel current resulted in the reduction in the open state probability. ANP decreased the mean open state duration from 14.2 to 1.8 ms/sweep. (ii) In the weakly depolarized cell stimulated by 8 mM K+, ANP reduced the level of aequorin luminescence (a measure of cytosolic calcium) and completely inhibited the stimulated rate of aldosterone secretion, returning it to prestimulation values. These effects are consistent with a decrease in net calcium channel influx and the reported inhibition of T-channel current. In contrast, the calcium channel blocker, nitrendipine, which at low dose selectively blocks L-type calcium channel flux, only slightly reduced luminescence, and partially inhibited the sustained secretory response. (iii) In the strongly depolarized cell, stimulated by 60 mM K+, ANP increased the level of aequorin luminescence consistent with an increase in net calcium channel influx and the reported stimulation of L-channel current. These results indicate that under physiological conditions the inhibition of T-type calcium channels may be involved in the inhibition of the aldosterone secretion induced by ANP.     

A Ca(v)3.2/syntaxin-1A signaling complex controls T-type channel activity and low-threshold exocytosis

T-type calcium channels represent a key pathway for Ca(2+) entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca(v)3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca(v)3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca(v)3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca(2+) channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.     

Extrinsic regulation of T-type Ca(2+) channel expression in chick nodose ganglion neurons

Functional expression of T-type Ca(2+) channels is developmentally regulated in chick nodose neurons. In this study we have tested the hypothesis that extrinsic factors regulate the expression of T-type Ca(2+) channels in vitro. Voltage-gated Ca(2+) currents were measured using whole-cell patch clamp recordings in E7 nodose neurons cultured under various conditions. Culture of E7 nodose neurons for 48 h with a heart extract induced the expression of T-type Ca(2+) channels without any significant effect on HVA currents. T-type Ca(2+) channel expression was not stimulated by survival promoting factors such as BDNF. The stimulatory effect of heart extract was mediated by a heat-labile, trypsin-sensitive factor. Various hematopoietic cytokines including CNTF and LIF mimic the stimulatory effect of heart extract on T-type Ca(2+) channel expression. The stimulatory effect of heart extract and CNTF requires at least 12 h continuous exposure to reach maximal expression and is not altered by culture of nodose neurons with the protein synthesis inhibitor anisomycin, suggesting that T-type Ca(2+) channel expression is regulated by a posttranslational mechanism. Disruption of the Golgi apparatus with brefeldin-A inhibits the stimulatory effect of heart extract and CNTF suggesting that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Heart extract- or CNTF-evoked stimulation of T-type Ca(2+) channel expression is blocked by the Jak/STAT and MAP kinase blockers, AG490 and U0126, respectively. This study provides new insights into the electrical differentiation of placode-derived sensory neurons and the role of extrinsic factors in regulating the functional expression of Ca(2+) channels.     

Synthesis and biological activity of 3,4-dihydroquinazolines for selective T-type Ca2+ channel blockers

We have synthesized 3,4-dihydroquinazoline derivatives for the potent and selective T-type Ca(2+) channel blockers and evaluated for their inhibitory activities against two subtypes T-type Ca(2+) channels and N-type Ca(2+) channels. Among them, 5b (KYS05044, IC(50)=0.56+/-0.10 microM) was identified as potent T-type Ca(2+) channel blocker with in vitro selectivity profile at meaningful level (T/N-type, SI=>100).     

Role of calcium channels in cadmium-induced disruption of cortisol synthesis in rainbow trout (Oncorhynchus mykiss)

The mechanisms of toxicity of cadmium (Cd(2+)) in adrenal steroidogenesis were investigated in vitro in adrenocortical cells of rainbow trout (Oncorhynchus mykiss). Toxicity of Cd(2+) was increased in absence of extracellular Ca(2+), but was prevented in Ca(2+)-supplemented medium. Pretreatment of cells with BAY K8644 (BAY), an agonist of voltage-dependent calcium channels, increased the Cd(2+)-mediated inhibition of ACTH-stimulated secretion but not pregnenolone (PREG)-stimulated secretion. Nicardipine, an antagonist of voltage-dependent calcium channels, also increased the inhibition of adrenocorticotropic hormone (ACTH)-stimulated secretion by Cd(2+). These results suggest that opening of voltage-dependent calcium channels with BAY may allow Cd(2+) entry at the same time as calcium, thus increasing toxicity of Cd(2+), however voltage-dependent calcium channels may not be the only way of entry into adrenocortical cells. The influx of Cd(2+), measured as intracellular Cd(2+) using Fluo-3 in PREG-stimulated adrenocortical cells, was significantly enhanced by the stimulation. These results suggest that the deleterious effect of Cd(2+) on cortisol steroidogenesis may be enhanced when the endocrine stress response is triggered.     

Dopaminergic modulation of axon initial segment calcium channels regulates action potential initiation

Action potentials initiate in the axon initial segment (AIS), a specialized compartment enriched with Na(+) and K(+) channels. Recently, we found that T- and R-type Ca(2+) channels are concentrated in the AIS, where they contribute to local subthreshold membrane depolarization and thereby influence action potential initiation. While periods of high-frequency activity can alter availability of AIS voltage-gated channels, mechanisms for long-term modulation of AIS channel function remain unknown. Here, we examined the regulatory pathways that control AIS Ca(2+) channel activity in brainstem interneurons. T-type Ca(2+) channels were downregulated by dopamine receptor activation acting via protein kinase C, which in turn reduced neuronal output. These effects occurred without altering AIS Na(+) or somatodendritic T-type channel activity and could be mediated by endogenous dopamine sources present in the auditory brainstem. This pathway represents a new mechanism to inhibit neurons by specifically regulating Ca(2+) channels directly involved in action potential initiation.