Neuropeptide Y inhibition of calcium channels in PC-12 pheochromocytoma cells

Laura A. McCullough, Terrance M. Egan, and Thomas C. Westfall

Department of Pharmacological and Physiological Science, Saint Louis University Health Sciences Center, St. Louis, Missouri 63104

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We previously demonstrated, using rat PC-12 pheochromocytoma cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF), that neuropeptide Y (NPY) inhibits catecholamine synthesis as well as release. Inquiry into the mechanisms of these inhibitions implicated distinct pathways involving reduction of Ca2+ influx through voltage-activated Ca2+ channels. In the present investigation the effects of NPY on whole cell Ba2+ currents were examined to obtain direct evidence supporting the mechanisms suggested by those studies. NPY was found to inhibit the voltage-activated Ba2+ current in NGF-differentiated PC-12 cells in a reversible fashion with an EC50 of 13 nM. This inhibition was pertussis toxin sensitive and resulted from NPY modulation of L- and N-type Ca2+ channels. The inhibition of L-type channels was not seen with <1 nM free intracellular Ca2+ or when protein kinase C (PKC) was inhibited by chelerythrine or PKC-(19---31). Furthermore, the effect of NPY on L-type channels was mimicked by the PKC activator phorbol 12-myristate 13-acetate. These studies demonstrate that, in addition to inhibition of N-type Ca2+ channels, in NGF-differentiated PC-12 cells NPY inhibits L-type Ca2+ channels via an intracellular Ca2+- and PKC-dependent pathway.

nerve growth factor; omega -conotoxin GVIA; nifedipine; pertussis toxin

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

NEUROPEPTIDE Y (NPY) is a 36-amino acid peptide neurotransmitter that is colocalized and coreleased with catecholamines in the central and peripheral nervous systems. Peripherally, NPY acts to inhibit the release of catecholamines from sympathetic neurons (45), cultured superior cervical ganglion (SCG) cells (36), and PC-12 cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF) (6, 8). The mechanism of this action has been widely investigated, suggesting that the inhibition of catecholamine release involves the Y2-receptor subtype and inhibition of N-type voltage-gated Ca2+ channels (6, 36, 44). N-type Ca2+ current inhibition by NPY has been directly demonstrated in several systems, including SCG (11, 37), dorsal root ganglion (3), neuroblastoma (32), and nodose ganglion (47) neurons. This inhibition is pertussis toxin (PTX) sensitive (20, 32, 47) and is likely produced by the voltage-sensitive direct interaction of G protein subunits with the channel, a mechanism utilized by multiple inhibitory neurotransmitters (18).

We recently characterized a novel effect of NPY in NGF-differentiated PC-12 cells in addition to its inhibition of catecholamine release, a PTX-sensitive inhibition of catecholamine synthesis. In contrast to its effects on release, the inhibition of synthesis is mediated by inhibition of L-type Ca2+ channels through Y3-receptor activation (30, 31). Furthermore, the inhibition of synthesis is mimicked and occluded by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and attenuated by the selective PKC antagonist chelerythrine, suggesting that the reduction of L-type channel current by NPY is through the action of PKC (31).

In the present study we use electrophysiological techniques to definitively demonstrate the NPY-induced modulation of Ca2+ channels in NGF-differentiated PC-12 cells that was suggested by our previous studies on catecholamine synthesis and release. Specifically, we show that NPY produces PTX-sensitive inhibition of L- as well as N-type Ca2+ channels in these cells and provide evidence that the modulation of L-type channels involves an intracellular Ca2+- and PKC-dependent pathway. This study is the first to establish inhibition of neuronal L-type channels by NPY and to investigate the mechanism of this inhibition. In addition, in combination with previous studies, our work suggests that inhibition of different Ca2+ channel subtypes by NPY results in distinct physiological effects.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. Stock cultures of rat PC-12 pheochromocytoma cells (passages 19-30) were obtained from Dr. Steven Sabol (National Institutes of Health, Bethesda, MD) and grown in DMEM supplemented with 2 mM glutamine, 1 mM pyruvate, 5% FCS, 10% heat-inactivated horse serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone at 37°C in a humidified atmosphere containing 5% CO2 in air. Cells were passaged once per week with medium changes every 2-3 days. For electrophysiological studies, cells were plated onto 35-mm plates (Falcon Primaria) at a density of 2-2.5 × 105 cells/plate and differentiated with 50 ng/ml NGF for 4-7 days.

Whole cell patch-clamp recordings. Voltage-clamp recordings were obtained using the whole cell patch-clamp technique (13). Borosilicate glass patch pipettes were coated with Sylgard (Dow Corning) and fire polished. The pipettes were filled with a solution containing 120 mM N-methyl-D-glucamine aspartate, 1 mM MgCl2, 20 mM tetraethylammonium chloride, 10 mM HEPES, 10 mM EGTA, 0-4.3 mM CaCl2, 4 mM Mg-ATP, and 1 mM Na-GTP (pH 7.4). Pipette resistances averaged 3-6 MOmega . Immediately before each experiment, PC-12 cells were dissociated and suspended in buffer containing 144 mM NaCl, 5.4 mM KCl, 5 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). An aliquot of the cell suspension was transferred to a polycarbonate-and-glass recording chamber positioned on the stage of an inverted microscope equipped with Hoffman-modulation optics. Currents through voltage-activated Ca2+ channels were measured using Ba2+ as the charge carrier in an external solution containing 144 mM NaCl, 10 mM CsCl, 10 mM BaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, 1 µM tetrodotoxin, and 300 µM anthracene-9-carboxylate (pH 7.4). Solutions containing the drugs of interest were applied by manually moving the cell, attached to the patch pipette, into the line of flow of solution exiting one of an array of six inlet tubes. This setup allows very rapid solution exchange and short duration (1-3 min) of drug application. Whole cell currents were recorded with an AxoPatch 200A amplifier (Axon Instruments) and digitized at 5 kHz with 16-bit accuracy with use of a MacADIOS II/16 board and Superscope II software (GW Instruments). Capacitive transients were canceled, and membrane currents were leak subtracted using a P/4 regimen. Voltage-activated Ba2+ currents were evoked by stepping the voltage every 20 s to 0 mV for 40 ms from a holding potential of -80 mV. For analysis of tail currents, the voltage was stepped to -40 mV for 40 ms after the step to 0 mV. Current-voltage curves were obtained using command voltage ramps, which changed the membrane voltage from -80 to +60 mV at a rate of 1.2 V/s.

Data analysis and statistics. Raw data were analyzed off-line with a Macintosh computer and IGOR software (Wavemetrics). Control and drug-modulated currents were compared by determining the total charge entry (pC) during the 40-ms depolarizing pulse through integration of the respective currents. Tail current amplitudes were measured at a point 5 ms after a repolarizing step from 0 to -40 mV, as described by Jones and Jacobs (23). Free Ca2+ concentrations were calculated using CHELATOR (41). Values are means ± SE. Statistical significance was determined using Student's two-tailed t-test or one-way ANOVA followed by Student-Newman-Keuls multiple comparisons test as appropriate. P <=  0.05 was considered significant.

Materials. NPY was purchased from Peninsula Laboratories (Belmont, CA) and American Peptide (Sunnyvale, CA). NGF was purchased from Collaborator Biomedical Products (Bedford, MA). Fetal bovine serum and horse serum were purchased from JRH Biosciences (Lenexa, KS). DMEM and penicillin, streptomycin, and fungizone were purchased from GIBCO BRL (Grand Island, NY). Chelerythrine chloride was purchased from RBI (Natick, MA). PKC-(19---31) was purchased from BIOMOL (Plymouth Meeting, PA). Nifedipine, omega -conotoxin GVIA (CgTX), PTX, PMA, and all other agents were purchased from Sigma Chemical (St. Louis, MO).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Unless otherwise specified, all experiments were done using an intracellular solution containing 10 mM EGTA and 4.3 mM CaCl2 to give a calculated free intracellular Ca2+ concentration ([Ca2+]i) of 100 nM (41), which is close to the resting [Ca2+]i in these cells (6). Currents through voltage-activated Ca2+ channels were measured using an extracellular solution containing Ba2+ as the charge carrier.

NPY inhibits voltage-activated Ba2+ current in NGF-differentiated PC-12 cells. Our hypothesis is that the NPY-induced inhibition of catecholamine release and synthesis is associated with inhibition of Ca2+ influx through voltage-gated channels. To test this hypothesis, we first determined the effect of NPY on whole cell Ba2+ current. Figure 1 shows an example of the inhibition of voltage-activated Ba2+ current by NPY. This inhibition was often accompanied by an increase in the time to peak of the current (Fig. 1A), which most likely reflected a change in channel activation kinetics, and was not associated with a shift in the current-voltage relationship (Fig. 1B). An identical pattern of kinetic slowing often accompanies G protein-mediated inhibition of Ca2+ current in neuronal tissues (18). The concentration-response curve for NPY-induced (0.1-300 nM) inhibition exhibited an EC50 of 13 nM and an average maximal effect of 35% inhibition (Fig. 1C).


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Fig. 1.   Neuropeptide Y (NPY) inhibits voltage-activated Ba2+ current in nerve growth factor-differentiated PC-12 cells. A: inhibition by 100 nM NPY of current elicited by a 40-ms depolarizing pulse to 0 mV from a holding potential of -80 mV. B: inhibition by 100 nM NPY of current elicited by a voltage ramp from -80 to +60 mV at a rate of 1.2 V/s. C: cumulative concentration-response relationship for NPY inhibition of Ba2+ current. Percent inhibition was determined here and in subsequent figures by comparing control and drug-modulated total charge entry during depolarizing pulse as described in MATERIALS AND METHODS. Values are means ± SE from 3-6 cells. Curve was fit using dose-response fitting routine of Origin (Microcal Software). Calculated EC50 for inhibition by NPY was 13 nM.

NPY inhibits L- and N-type Ca2+ channels. Inhibition of neurotransmitter release by NPY has previously been associated with inhibition of N-type Ca2+ channels in several neuronal systems (7, 44, 47). In contrast, our studies on the mechanism of NPY inhibition of catecholamine synthesis suggested that inhibition of L-type Ca2+ channels was involved. Therefore, the next series of studies was designed to demonstrate this implied inhibition of L-type channels in addition to inhibition of N-type Ca2+ channels.

The Ca2+ current in NGF-differentiated PC-12 cells is carried primarily by N- and L-type Ca2+ channels. The N-type channel blocker CgTX (500 nM) irreversibly inhibited the total current by 42 ± 4% whereas the L-type channel blocker nifedipine (1 µM) produced a reversible inhibition of 24 ± 4%. The combination of CgTX and nifedipine blocked the total current by 57 ± 10%. NPY (100 nM) was found to produce additional, although reduced, Ba2+ current inhibition after blockade of L-type channels by nifedipine or N-type channels by CgTX (Fig. 2), suggesting that NPY causes inhibition of both Ca2+ channel subtypes.


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Fig. 2.   NPY inhibits L- and N-type Ca2+ channels. A: sample traces demonstrating that 100 nM NPY produces further inhibition of current remaining after blockade of L-type Ca2+ channels with 1 µM nifedipine (Nif) or N-type Ca2+ channels with 500 nM omega -conotoxin GVIA (CgTX). B: summary of results from 5-7 cells showing percent inhibition (mean ± SE) by nifedipine or CgTX alone (control) and with NPY (+NPY). * Significantly different from control, P < 0.05.

The actions of NPY in NGF-differentiated PC-12 cells, including inhibition of catecholamine synthesis and release and inhibition of cAMP accumulation, are PTX sensitive (6, 8, 30). In the present study we tested the PTX sensitivity of the observed inhibition of N- and L-type Ca2+ channels by incubating cells in PTX (50 ng/ml) for 18 h. NPY no longer produced significant Ba2+ current inhibition after PTX pretreatment (Fig. 3), demonstrating the involvement of Gi or Go protein subtypes in N- and L-type channel inhibition, consistent with our previous findings.


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Fig. 3.   NPY inhibition of Ca2+ channels is pertussis toxin (PTX) sensitive. Cells were incubated with PTX (50 ng/ml) for 18 h. A: sample traces obtained from a PTX-pretreated cell demonstrating that NPY (100 nM) does not produce significant current inhibition. B: summary of results showing percent inhibition (mean ± SE) by NPY without (-PTX, n = 24) and with PTX pretreatment (+PTX, n = 5). * Significantly different from -PTX, P < 0.05.

Mechanism of L-type channel inhibition. In many systems, neurotransmitter inhibition of L-type Ca2+ channels is lost when free intracellular Ca2+ is buffered to very low levels with the Ca2+ chelators EGTA or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (14, 22, 24, 28, 40). This finding suggests that Ca2+ is required in the pathway responsible for inhibition of these channels. Using pipette solutions containing 10 mM EGTA with 4.3 mM CaCl2 (~100 nM free intracellular Ca2+) or 10 mM EGTA without CaCl2 (<1 nM free intracellular Ca2+), we tested for the presence of such a mechanism in our observed NPY-induced inhibition of L-type channels (41).

With ~100 nM free intracellular Ca2+, nifedipine applied in the presence of NPY had little effect, indicating that NPY had inhibited a significant portion of the current carried by L-type Ca2+ channels (Fig. 4, A and B). In contrast, with <1 nM free intracellular Ca2+, nifedipine inhibited Ba2+ current to the same degree in the absence or presence of NPY, indicating that NPY no longer produced inhibition of L-type channels (Fig. 4, C and D). It is noteworthy, however, that NPY still inhibited a portion of the total current at <1 nM free intracellular Ca2+ (Fig. 4C), indicating that intracellular Ca2+ is not required for the inhibition of N-type channels.


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Fig. 4.   Buffering free intracellular Ca2+ concentration ([Ca2+]i) to <1 nM prevents NPY-induced inhibition of L-type Ca2+ channels. A and C: peak current as a function of time during application of 1 µM nifedipine alone or in presence of 100 nM NPY with ~100 nM (A) or <1 nM (C) free intracellular Ca2+. Insets: sample traces obtained during each of the test conditions. B and D: summaries of results from 4-5 cells showing nifedipine-sensitive (L-type) component of total charge entry (mean ± SE) in absence (-NPY) or presence (+NPY) of NPY with ~100 nM (B) or <1 nM free intracellular Ca2+ (D). * Significantly different from -NPY, P < 0.05.

To more directly demonstrate NPY-induced modulation of L-type Ca2+ channels, we tested its effect on the slow tail current induced by the L-type channel agonist BAY K 8644. This drug prolonged tail current deactivation in every cell tested, increasing by more than fourfold the amount of current measured 5 ms after a repolarizing step from 0 to -40 mV (Fig. 5A). This enhanced portion of the tail current selectively represents current through L-type Ca2+ channels (23). With ~100 nM free intracellular Ca2+, NPY reversibly inhibited the BAY K 8644-enhanced slow tail current (Fig. 5B) by an average of ~50% (Fig. 5C). However, with <1 nM free intracellular Ca2+, NPY had no significant effect on BAY K 8644-enhanced tail currents (Fig. 5C). These results demonstrate that the inhibition of L-type Ca2+ channels by NPY is mediated by an intracellular Ca2+-dependent mechanism.


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Fig. 5.   NPY produces Ca2+-dependent inhibition of BAY K 8644-enhanced tail currents. A: BAY K 8644 (1 µM) slows deactivation of tail currents elicited by a repolarizing step from 0 to -40 mV. B: NPY (300 nM) reduces BAY K 8644-induced slow tail current (~100 nM free intracellular Ca2+). Series resistance compensation was 60-80%. bullet , Time at which tail currents were measured. C: summary of results obtained in presence of 1 µM BAY K 8644 from 3 cells with ~100 nM (+Ca2+) and <1 nM free intracellular Ca2+ (-Ca2+) showing tail current inhibition by 300 nM NPY (mean ± SE) as measured 5 ms after repolarizing step. * Significantly different from +Ca2+, P < 0.05.

The Ca2+-dependent second messenger responsible for the NPY-induced inhibition of L-type channels suggested by our previous studies on catecholamine synthesis is PKC, since this effect of NPY was mimicked and occluded by the PKC activator PMA and prevented by the selective PKC inhibitor chelerythrine (31). Indeed, several groups have reported that PKC activation by phorbol esters inhibits Ca2+ influx into PC-12 cells (5, 9, 15, 27, 33). We used two selective inhibitors of PKC, chelerythrine (17) and the pseudosubstrate peptide PKC-(19---31) (21) to test for the involvement of this enzyme. Preincubation (>15 min) of cells with chelerythrine or intracellular dialysis (>10 min) with PKC-(19---31) prevented the subsequent NPY-induced inhibition of L-type channels, as demonstrated by equality of the nifedipine-sensitive (L-type) current in the absence and presence of NPY (Fig. 6). In addition, application of the PKC agonist PMA produced inhibition of the total Ba2+ current (Fig. 7A), which was not seen after treatment with chelerythrine or PKC-(19---31) (Fig. 7, B and C). Furthermore, PMA did not cause additional current inhibition in the presence of nifedipine (Fig. 7D), suggesting that the inhibition is limited to L-type channels in agreement with the results of others (5).


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Fig. 6.   Protein kinase C (PKC) inhibitors chelerythrine and PKC-(19---31) prevent inhibition of L-type Ca2+ channels by NPY. A: nifedipine-sensitive (L-type) subtraction currents in absence (-NPY) and presence (+NPY) of 100 nM NPY in control and PKC inhibitor-treated cells. Traces were obtained by subtraction of currents recorded before and after nifedipine application. Scale bars, 50 pA, 10 ms. B: summary of results showing percentage of control nifedipine-sensitive (L-type) component of total charge entry remaining in presence of NPY (mean ± SE) without PKC inhibitors (n = 5), after preincubation (>15 min) with 10 µM chelerythrine (n = 4), or after intracellular dialysis (>10 min) with 50 µM PKC-(19---31) (n = 5). * Significantly different from NPY alone, P < 0.05.


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Fig. 7.   Activation of PKC by phorbol 12-myristate 13-acetate (PMA) produces inhibition of L-type Ca2+ channels. A, B, and C: plots of peak current as a function of time during application and washout of 1 µM PMA in control, chelerythrine-pretreated, and PKC-(19---31)-dialyzed cells, respectively. Insets: sample traces obtained in absence and presence of PMA. Scale bars, 100 pA, 20 ms. D: summary of results from 4-5 cells showing percent inhibition by PMA (means ± SE) in absence and presence of 1 µM nifedipine. * Significantly different from PMA alone, P < 0.05.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The results of this study show that NPY produces PTX-sensitive inhibition of L- and N-type voltage-activated Ca2+ channels in NGF-differentiated PC-12 cells. Although NPY-mediated inhibition of N-type channels is a well-accepted concept (20, 32, 47), the present study is the first to definitively demonstrate NPY-mediated inhibition of neuronal L-type channels and to investigate the mechanism of this effect. Furthermore, these results provide strong support for our previous hypotheses on the pathways of NPY-induced modulation of catecholamine neurotransmission: modulation of release through inhibition of N-type channels and modulation of synthesis through inhibition of L-type channels (31).

The inhibition of L-type channels by NPY was lost after the removal of CaCl2 from the intracellular recording solution. This, in combination with the presence of 10 mM EGTA, reduces the calculated free [Ca2+]i to <1 nM. Although many enzymes are Ca2+ dependent, the results of our previous studies, in which the NPY-induced inhibition of catecholamine synthesis was mimicked and occluded by PMA and prevented by chelerythrine, suggested that PKC was the most likely mediator of L-type channel inhibition (31). In the present study we used the selective PKC inhibitors chelerythrine and PKC-(19---31), both of which prevented the inhibitory effect of NPY on L-type Ca2+ channels. In addition, the PKC activator PMA mimicked the NPY-induced inhibition of L-type channels. The results of these experiments, in conjunction with our previous studies (31), provide strong evidence that NPY inhibits L-type Ca2+ channels in NGF-differentiated PC-12 cells through the action of PKC. L-type channel and catecholamine synthesis inhibition by NPY are PTX sensitive, consistent with the observation that the stimulation of phospholipase C by several other neurotransmitters is transduced through beta gamma -subunits of Gi/Go (PTX-sensitive) rather than Gq (PTX-insensitive) proteins (10).

Previous investigations into the role of PKC in Ca2+ channel modulation have produced conflicting results. Although PKC activation has been reported to inhibit Ca2+ channels in adrenal chromaffin cells (38, 42) and several other cell types (4, 14, 16, 25, 39), PKC activation enhances Ca2+ channel currents in other systems (43, 48, 49). However, the evidence supporting Ca2+ channel inhibition by PKC in our model system, PC-12 cells, is consistent and compelling. Indeed, several independent investigators, using 45Ca2+ influx and [Ca2+]i-imaging techniques, demonstrated that PKC activation inhibits Ca2+ channels in PC-12 cells (9, 15, 27, 33). In addition, an electrophysiological study has been performed that confirms that PKC activation by PMA inhibits L-type, but not N-type, Ca2+ channels in NGF-differentiated PC-12 cells (5). Therefore, our hypothesis of PKC-mediated inhibition of L-type channels in PC-12 cells is well supported by our previous and present experiments as well as by the work of other investigators. The explanation for the complex actions of PKC on Ca2+ channels is unclear, but it may involve different subtypes or forms of the channels or, alternatively, the actions of additional mediators, kinases, phosphatases, or G proteins that are present at various levels in different cell types.

In our present as well as previous studies, we have used NGF-differentiated PC-12 cells as a sympathetic neuronal model. In rat SCG neurons, NPY also inhibits Ca2+ channels (11, 37). As opposed to its effects in NGF-differentiated PC-12 cells, in SCG neurons NPY did not produce further inhibition after CgTX application (11) and did not inhibit tail currents prolonged by the L-type channel agonist (+)-(S)-202-791 (37), suggesting that its effects were limited to inhibition of N-type channels. However, the recording conditions used in the SCG studies (high intracellular BAPTA or EGTA, no added Ca2+) may not be optimal for the observation of Ca2+-dependent L-type channel inhibition by NPY.

Catecholamine synthesis and release are two separate but related processes that influence the level of catecholaminergic neurotransmission in the central and peripheral nervous systems. Our studies have revealed that NPY, which is coreleased with catecholamines, can act to regulate both of these processes. In addition, NPY appears to utilize different mechanisms to modulate the two processes, with inhibition of N-type Ca2+ channels coupled to inhibition of catecholamine release and inhibition of L-type Ca2+ channels coupled to inhibition of catecholamine synthesis. Because different NPY receptor subtypes are responsible for inhibition of catecholamine release (Y2) and synthesis (Y3), it is likely that different NPY receptor subtypes are responsible for the inhibition of the different Ca2+ channel subtypes. We recently performed experiments which suggest that this is indeed the case (29).

The importance of N-type Ca2+ channels in the stimulation of neuronal transmitter release has been well demonstrated (19, 34), and numerous neurotransmitters in addition to NPY have been found to inhibit transmitter release through inhibition of this channel subtype (18). In contrast, in adrenal chromaffin cells, L-type Ca2+ channels play a dominant role in secretion (1, 26). Although the function of L-type channels in neurons is less well characterized, depolarization-induced stimulation of transmitter synthesis (31) and gene transcription (2, 12, 35) are processes that depend on L-type channel activity. Therefore, the demonstration that NPY and other transmitters can modulate L- as well as N-type Ca2+ channels suggests that they can induce long- and short-term alterations in neuronal function.

In summary, this investigation is the first to establish NPY-induced inhibition of neuronal L-type voltage-gated Ca2+ channels in addition to inhibition of N-type channels. The finding that the reduction of L-type channel current is PTX sensitive and mediated through an intracellular Ca2+- and PKC-dependent pathway is in agreement with our investigations into the mechanism of catecholamine synthesis inhibition by NPY. This study, in combination with our previous work, provides strong evidence supporting the hypothesis that NPY acts through distinct pathways to modulate catecholamine synthesis and release and thereby regulate sympathetic neuronal function.

    ACKNOWLEDGEMENTS

We thank Drs. V. A. Chiappinelli and K.-W. P. Yoon for critical reading of the manuscript.

    FOOTNOTES

This study was supported by National Institutes of Health Grants HL-26319, HL-60260, and 5-T32-GM-08306 (to T. C. Westfall) and HL-56236 (to T. M. Egan).

Address for reprint requests: L. A. McCullough, Dept. of Pharmacological and Physiological Science, Saint Louis University Health Sciences Center, 1402 South Grand Blvd., St. Louis, MO 63104.

Received 9 October 1997; accepted in final form 22 January 1998.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 274(5):C1290-C1297
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