tdeuron,

Vol. 6, 557-563,

April,

1991, Copyright

0 1991 by Cell Press

Neuropeptide Inhibition of Voltage-Gated Channels Mediated by Mobilization of Intracellular CalciGm Richard H. Kramer,* Edwin S. Levitan+*

Leonard

K. Kaczmarek,+

*Howard Hughes Medical Institute and Center for Neurobiology and Columbia University College of Physicians and Surgeons New York, New York 10032 +Department of Pharmacology Yale University School of Medicine Yew Haven, Connecticut 06510

and

Behavior

Many neurotransmitters and hormones regulate secretion from endocrine cells and neurons by modulating voltage-gated Ca*+ channels. One proposed mechanism of neurotransmitter inhibition involves protein kinase C, activated by diacylgycerol, a product of phosphotidylinositol hydrolysis. Here we show that thyrotropinreleasing hormone (TRH), a neuropeptide that modulates hormone secretion from pituitary tumor cells, inhibits Ca*+ channels via the other limb of the phosphotidylinositol signaling system: TRH causes inositol trisphosphate-triggered Caz+ release from intracellular organelles, thus causing Caz+dependent inactivation of Ca*+ channels. Elevation of intracellular Ca*+ concentration is coincident with the onset of TRH-induced inhibition and is necessary and sufficient for its occurrence. The inhibition is blocked by introducing Ca*+ buffers into cells and mimicked by a variety of agents that mobilize Ca*+. Treatments that suppress protein kinase C have no effect on the inhibition. Hence inactivation of Ca*+ channels occurs not only as a result of Ca*+ influx through plasma membrane channels, but also via neurotransmitter-induced Ca*+ mobilization. This phenomenon may be common but overlooked because of the routine use of Ca*+ buffers in patch-clamp electrodes. Introduction Voltage-gated Ca2+ currents are modulated in many cell types, leading to a change in Ca2+ influx and altering Ca *+-dependent cellular processes. Several intracellular messengers are thought to mediate neurotransmitter modulation of Ca2+ currents in different cells. These include CAMP (for review, see Hess, 1990), cGMP (Paupardin-Tritsch et al., 1986), diacylglycerol (Strong et al., 1987; Rane et al., 1989), and subunits of G-proteins (Yatani and Brown, 1989). Another intracellular messenger known to regulate the activity of Ca*+ channels is Ca2+ itself. A series of studies by Roger Eckert and his colleagues (for review, see Eckert and Chad, 1984) showed that influx of Ca*+ through volt* Present Pittsburgh

address: School

Department of Medicine,

of Pharmacology, University of Pittsburgh, Pennsylvania 15261.

Calcium

age-gated Ca*+ channels and accumulation of Ca*+ in the cytoplasm lead to inactivation of those very same channels. While Ca*+-dependent inactivation of Ca*+ channels has been shown to serve a negative feedback function, the process has not been implicated in mediating neurotransmitter regulation of Ca*+ channels. In this paper we demonstrate that neurotransmitter inhibition of L-type Ca2+ channels in pituitary tumor (GH,) cells is indeed mediated by Ca*+ which is mobilized from intracellular organelles. The neuropeptide thyrotropin-releasing hormone (TRH) was used in this study. TRH induces hydrolysis of phosphatidylinositol, generating diacylglycerol, which activates protein kinase C (PKC), and inositol trisphosphate, which triggers Ca*+ mobilization from intracellular organelles (for reviews, see Gershengorn, 1986, 1989; Ozawa and Sand, 1986). TRH modulates various K+ currents in CH3 cells, including Ca*+activated K+ current (Dubinsky and Oxford, 1985; Ritchie , 1987), delayed rectifier K+ current (Dubinsky and Oxford, 1985), and inwardly rectifying K+current (Bauer et al., 1990). In addition, TRH enhances voltageindependent, divalent cation-permeable channel activity (Mason et al., 1988). Despite its myriad electrophysiological effects, it has been reported that TRH does not modulate voltage-gated Ca*+ current (Dubinsky and Oxford, 1985). However, a component of the TRH response is known todisappear rapidly,or“wash out,“ when ionic currents are recorded with the whole-cell version of the patch-clamp technique (Dubinsky and Oxford, 1985; Dufy et al., 1986, 1987). We therefore used the nystatin-perforated patch method (Horn and Marty, 1988; Korn and Horn, 1989) to voltage-clamp GH3 cells without dialyzing out cytoplasmic constituents and without introducing exogenous Ca*+ buffers. With this recording configuration we were surprised to find that TRH consistently inhibited Ca*+ current. Hence we could systematically study the mechanism of Ca*+ channel inhibition in cells with unperturbed cytoplasmic signaling systems.

Results TRH Inhibits

Ca2+ Current

Figure 1 shows the effect of TRH on voltage-gated inward current measured with the perforated patch method. Inhibition of inward currentwasobserved in 61 of 65 cells; on the average, 100 nM to 1 PM TRH reduced the current elicited with 40-50 mV depolarizing pulses from -40 mV by 31% f 7% (mean + SD). The percent inhibition was the same if Ca*+ or Ba2+ was used as the charge carrier (29% f 8% for Ca*+ [n = 211 versus 32% _+ 3% for Ba2+[n = 401). The inhibition lasted for the duration of TRH application (up to at least 2 min), was often fully reversible at low TRH concentrations (below 100 nM), and could be elicited repeatedly without decrement.

Neuron 558

Figure Current

C -‘~~

Control

&

(C-E) Effect of dihydropyridines on the TRH of 1 PM BAY K 8644 (D), and after replacement to 0 mV before (trace 1) and 30 s after (trace

1. TRH

Inhibition

of

L-type

Ca’+

(A) Inhibition of the sustained, but not the transient, component of Ca2+ channel current in a perforated patch-clamped CHI cell. Currents were elicited by 200 ms pulses from -80 mV to - 10 mV. Current elicited 30safter applicationof nM TRH was subtracted from current elicited before TRH application (Control) to yield the difference current. Calibrations: vertical, 80 pA; horizontal, 50 ms. (B) Current-voltage relation of L-type current measured beforeand 1 min afterapplication of 500 nM TRH. The cell was held E Nimodipine ,~w at -40 mV, and steady-state current was measured at the end of 100 ms pulses to c I !I the potentials indicated. The series resistance of the perforated patch was 30 MS1 and 70% compensated. Leak current was measured during 20 mV hyperpolarizing PA pulses from -60 mV and scaled for leak subtraction from currents elicited by depois larizing pulses. Note that the two currentvoltage curves meet at +80 mV, near the reversal potential of the inward current. inhibition. TRH (500 nM) was applied to the cell without BAY K 8644 (C), after addition of BAY K 8644 with 1 PM nimodipine (E). Currents elicited by 75 ms pulses from -40 mV 2) TRH. Trace 1 and trace 2 are superimposed in (E).

GH3 cells have T-type (low voltage-activated, rapidly inactivating) and L-type (high voltage-activated, slowly inactivating) Ca2+channels (Matteson and Armstrong, 1986; Simasko et al., 1988). The current inhibited by TRH exhibited little or no inactivation during a 100 ms depolarizing pulse with Ba*+ as the charge carrier (Figure IA; n = 4) and activated only at highly depolarized potentials (>-25 mV; Figure IB). A rapidly inactivating current component seen when the holding potential was hyperpolarized (-80 mV; Figure IA), but not when it was depolarized (-40 mV; Figure IC), was not affected by TRH, suggesting that L-and not T-type channels are modulated. Dihydropyridine drugs provided further evidence for L-type current inhibition. BAY K8644, adihydropyridineagonist, selectively augments L-type current and prolongs tail currents following depolarization (for review, see Bean, 1989). Figure ID shows that BAY K 8644 enhanced the magnitude of the TRH effect, while the percentageof current inhibited remained thesame as that for controls. TRH also reduced the tail current prolonged by BAY K 8644. The dihydropyridine antagonist nimodipine suppressed L-type current and eliminated the response to TRH. Hence the TRH inhibition of Ca2+ current is due specifically to modulation of L-type channels. We have previously shown that single L-type channels from GH3 cells are inhibited by TRH (Levitan and Kramer, 1990). Intracellular Ca*+ Mobilization Is Sufficient and Necessary for Inhibition Ca*+ channels in a wide variety of organisms are inactivated by intracellular Ca2+, which accumulates as a result of Ca2+ influx through the channels themselves

(Eckert and Chad, 1984). In GH3 cells, L-type channels are highly sensitive to Ca2+-dependent inactivation (Kalman et al, 1988), whereas T-type channels are relatively insensitive (Armstrong and Kalman, 1988). A variety of agents that trigger release of Ca2+ from intracellular stores inhibited a fraction of L-type current similar to that affected by TRH (Figure 2). These agents inhibited the current in a Ca*‘-free, Ba*+-containing solution, eliminating the possibility that inhibition results from influx of extracellular Ca*+. Caffeine and ryanodine, which trigger Ca2+ release from a pool of intracellular Ca*+ distinct from the inositol trisphosphate-sensitive pool (Thayer et al., 1988; Burgoyne et al., 1989), reversibly inhibited the L-type current. Caffeine is an inhibitor of phosphodiesterase activity, which could lead to an elevation of CAMP, but CAMP increases, rather than decreases, L-type current in GH3 cells (Armstrong and Kalman, 1988; Kalman et al., 1988). lonomycin, a Ca2+ ionophore known to trigger intracellular Ca*+ release (Albert and Tashjian, 1984), also inhibited the current. Finally, thapsigargin, which mobilizes intracellular Ca2+ by blocking reuptake into the inositol trisphosphate-sensitive store (Thastrup et al., 1990), caused a similar inhibition. These results suggest that elevating the intracellular Ca*+ concentration ([Ca2+],) by causing internal release is sufficient for inducing L-type channel inhibition. Ca2+-dependent inactivation of Ca2+ current is prevented by loading GH3 cells with Ca2+ buffers, such as EGTA and BAPTA (Kalman et al., 1988). To test whether the TRH effect requires elevation of [Ca2+],, we recorded Ca*+current usingtheconventional whole-cell configuration of the patch-clamp technique (Hamill et al., 1981) and used the patch pipette to introduce

Neuropeptide 559

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inhibition

of Ca2’ Channels

u r .’ ‘; z !ij a

60

30 20 10

(8)

0

No Exogenous

0.1 mM EGTA

PERFORATED PATCH

WHOLE-CELL

Figure 3. Effect of Ca 2+ Buffering with Cal+ As the Charge Carrier

Figure Current

2. Effect

of

Ca2+-Mobilizing

Agents

on

Ca2+ Channel

Measurement

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L (5) (4)

1 (3

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Log TRH concentration

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during

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Inhibition

TodeterminehowthetimecourseoftheTRH-induced elevation of [Ca2+], is related to that

1.0 -

0.6 --

Current

TRH inhibition was normal. Hence strong buffering of Ca2+ blocks the response, suggesting that changes in [Ca2+], are necessary. TRH-induced inhibition of Ca2+ current was dosedependent, with a half-maximal response near 1 nM and a saturating response between 10 and 100 nM (Figure 4). The dose-response curve for the TRH inhibition was very similar to the previously reported relationship between TRH and Ca2+ release (Ritchie, 1987). Hence the TRH-induced elevation of [Ca2+]i is not only necessary and sufficient for Ca*+ channel inhibition, but also has the same concentration dependence.

EGTA or BAPTA into the cell (Figure 3). The TRH response consistently washed out within 15 min after establishing the whole-cell configuration. HenceTRH was applied quickly (5 min) after whole-cell break-in, and only responses to the first application were considered in Figure 3. When 10 mM BAPTAwas included in the pipette, TRH had almost no effect on the Ca2+ current. In contrast, when 0.1 mM EGTAwas used, the

0.6 --

on L-type

Responses observed in perforated patch-clamped cells with Baz+ as the charge carrierare shown for comparison. Current records from 3 representative cells are shown on top; group data are shown immediately below. Responses to 100 nM TRH were measured >5 min after establishment ofwhole-cell patch-clamp configuration. Data indicate first response to TRH; subsequent responses under whole-cell clamp exhibited washout as reported (Dubinsky and Oxford, 1985; Dufy et al., 1986). Data were taken from cells in which Ca2+ current was stable for at least 1 min before TRH application. Some cells exhibited rapid washout of Ca2+ current and were not used. Currents were elicited with pulses from -50 to 0 mV.

(A) Inhibition of Ca2+ channel current (BaZ’ as the charge carrier) by 100 nM TRH, 10 mM caffeine (CAFF), or 5 PM ionomycin (IONO). Currents were elicited with pulses from -50 to 0 mV before (CON) and 30 s after drug applications. Control and experimental currents are from a single CH, cell, with at least 3 min allowed between successive drug applications for L-type current recovery. (B) Group data of effects of Ca*+-mobilizing agents on L-type current. Ca*+ channel current was measured before and 30 s after application of 100 nM to 1 PM TRH, 10 mM caffeine, 1 PM ryanodine, 5 pM ionomycin,orl uM thapsigargin. Bars represent mean inhibition k SD, with number of experiments in parentheses.

: ‘Z .-‘Z AZ .E

10 ml4 BAPTA

-7

-6

I

of the

Figure 4. Dose-Response hibition of Ca*+ Channel

Ca2+ current

Curve of TRH InCurrent

Inhibition at variousTRH concentrations is normalized with respect to inhibition at 100 nM TRH. TRH was applied in order of increasing concentration (i.e., IO-l2 M, then 10-t’ M, etc.) for 20 s, with 3-4 min between successive applications. Inward currents were measured at the end of 100 ms depolarizing pulses from -40 to 0 mV. Data are from 7 cells and represent mean f SEM, with the number of cells indicated in parentheses.

Figure 5. Changes in [Ca2’], during the TRH Response (A) Measurements of basal [Caz’li immediately before and the peak change in Ca*+ (calcium transient) resulting from Ca2+ currents, with Ca*+ as the charge carrier. Currents result from 100 ms depolarizing pulses (-70 mV to -10 mV) given every 14 s. Note the large spike of elevated basal [Ca2+li coincident with inhibition of the CaZ+ current and Caz+ transient. CaZ+ current and Ca2+ transients recorded before (B) and during (C) the response to 500 nM TRH are shown. Currents in (B) and (C) elicited the transient marked with an asterisk (note different time scale).

TRH

0

50

100

150

200

Time (set)

“b-) I

I

40

i 100 msec

inhibition, [Ca*‘], was measured microspectrophotometrically in GH3 cells loaded with the Ca2+ indicator dye fura 2-AM, ‘while the cells were voltage-clamped using the perforated patch method. [Ca’+]i was monitored continuously, while Ca2+ currents were elicited every 14 s with depolarizing pulses. Figure 5A shows that TRH elevated basal [Ca*+], from 280 nM to 550 nM within 10 s, but the elevation was transient, with [Ca’+]i decaying to initial levels within 1 min. The onset of Ca*+ current inhibition also occurred within 10 s; however, the effect of TRH on the current was sustained, even outlasting the removal of TRH. Elevating [Ca’+]r to >400 nM for >I0 s by applying ionomycin (n = 2) also resulted in Ca*+ current inhibition that outlasted the elevation of [Ca’+]i. The bottom of Figure 5 shows Ca2+ currents and the resulting Caz+ transients before TRH (Figure 58) and 60 s after TRH (Figure 50, when basal [Ca”]i has returned to normal. The Ca2+ currents and transients were inhibited by45% and 47%, respectively, 60 s after TRH, and the inhibition only partially recovered following TRH removal. In 5 of 7 cells, TRH elevated basal [Ca2+], by >200 nM and inhibited the Ca*+ current by >25%. In the remaining 2 cells, TRH

PA

elevated basal [Ca*+]i by 50-100 nM and inhibited the current by90% of PKC activity in GH3 cells (Winicov and Gershengorn, 1988). Down regulation of PKC (Ballester and Rosen, 1985) by prolonged treatment (24 hr) with the phorbol ester PKC activator 12-0tetradecanoyl-phorbol-13-acetate (TPA) did not affect the percentage of current inhibited by TRH, but did reduce the basal current density. Acute application of TPA (100 nM for l-5 min) neither inhibited L-type current, nor reduced the TRH response (n = 5). A diacylglycerol PKC activator, I-oleoyl-2-acetylglycerol (OAC), does reduce L-type current (Marchetti and Brown, 1988), but this effect was not blocked by 1 PM staurosporine (n = 3), and OAG may act directly on Ca2+ channels (Hockberger et al., 1989). Thus we conclude that PKC is not involved in the TRH inhibition of Ca2+channels, but may regulate the long-term activity or number of Ca2+ channels in GH3 cells. It is unlikely that arachidonic acid or its metabolites mediate the TRH response because arachidonic acid increases the voltage-gated Ca*+ current in GH3 cells (Vacher et al., 1989).

Discussion We have shown that TRH inhibits specifically L-type Ca*+ current in GHS cells. This inhibition is highly sensitive to the recording configuration; it is evident with perforated patch clamp recordings, but it disappears when conventional whole-cell clamp technique is used, suggesting that inhibition requires soluble cytoplasmic constituents. The inhibition is blocked by intracellular application of Ca2+ chelators and is mimicked by Ca2’-releasing agents such as caffeine and ionomycin. The dose of TRH required for releasing internal Ca*+ is equivalent to the dose required for Ca2+ channel inhibition, and the onset of inhibition is coincident with a rise in [Ca2+],. Hence the evidence is compelling that intracellular Ca*+ mediates the TRH-induced inhibition of L-type channels. The molecular mechanism whereby intracellular Ca2+ regulates L-type Ca2+ channels remains to be elucidated. Our findings are consistent with an indirect Ca*+dependent modification of L-type channels (e.g., de-

N 13 6 5 6 and inhibition PKC. Current TRH reduction

by TRH density of Ca2+

phosphorylation; Armstrong and Eckert, 1987) rather than with a direct effect of Ca2+ on channels, because the TRH-induced inhibition outlasts the elevation of [Ca2+],. An alternative mechanism implicated in Ca*+ channel regulation involves PKC. In chick dorsal root ganglion neurons, PKC activators mimic the effect of neurotransmitters that inhibit voltage-gated Ca2+ current (Rane and Dunlap, 1986) and a variety of PKC inhibitors block Ca2+ current inhibition (Rane et al., 1989). In GH3 cells, TRH does activate PKC, and a component of the electrophysiological response that washes out under whole-cell patch-clamp conditions can be reinstated by internal perfusion with purified PKC (Dufy et al., 1987). However, our results show that the TRH inhibitionof L-typeCa2+current isunaffected bythree treatments that almost completely inhibit PKC activity: H7 treatment, staurosporine treatment, and down regulation by prolonged treatment with phorbol ester. In addition, acute treatment with phorbol esters does not mimic the effect of TRH. Hence we conclude that PKC is not involved in the TRH-induced inhibition. TRH triggers a biphasic secretion of peptides that is correlated with, and dependent upon, a biphasic elevation of [Ca”]i (Albert and Tashjian, 1984). This consists of an initial “spike” of [Ca2+],, due to inositol trisphosphate-triggered intracellular Ca*+ mobilization, followed by a prolonged “plateau,” involving Ca*+ influx (Albert and Tashjian, 1984; Gershengorn, 1986,1989; Benham, 1989). Since we have shown that TRH inhibits Ca2+ channels, the prolonged plateau of [Ca”]i cannot be due to an increased voltage-gated Ca*+ conductance. It must have another immediate cause: possibilities include enhanced Ca2+ influx through Ca*+-permeable cation channels (Mason et al., 1988), or an increase in action potential frequency. It is possible that the amount of Ca2+ influx per action potential is reduced because of inhibition of L-type current, but the accumulated intracellular Ca2+ is increased because of the increased frequency of action potentials. It is interesting to note that TRH produces opposite effects on two paths of Ca*+ entry into the cytoplasm: Ca2+-permeable channels in intracellular organelles are activated, and plasma membrane Ca2+ channels are inhibited. This opposing pattern of Ca2+ regulation may be an important homeostatic mecha-

nism for allowing prolonged Ca2+-dependent secretion while preventing excessiveaccumulation of intracellular Ca*+. The mechanism of TRH-induced changes in spike activity is somewhat controversial. The TRH response consists of an initial hyperpolarization (inhibition) followed by a long-lasting increase in the spontaneous firing frequency (hyperexcitability) of GH3 cells (Dufy et al., 1979). The initial hyperpolarization is generated by activation of Ca*+-dependent K+channels resulting from Ca*+ release from internal stores (Dubinsky and Oxford, 1985; Ritchie, 1987). The subsequent hyperexcitability has been attributed to inhibition of various outward currents, including the delayed rectifier K+ current (Dubinskyand Oxford, 1985) and the inwardly rectifying K+ current (Bauer et al., 1990). We have found that TRH transiently increases, and then inhibits, the Ca2+-activated K+ current activated during voltage-clamp depolarizations (Kramer and Levitan, 1989, Biol. Bull., abstract). We suggest that the inhibition of L-type Ca*+ current leads to reduced Ca2+ influx during each action potential, which results in reduced activation of Ca*+-activated K+channels during the hyperexcitability phase. This decrease in K+ conductance could reduce spike afterhyperpolarizations and contribute to enhanced excitability. Several neurotransmitters inhibit Ca2+ channels in neurons, muscle, and secretory cells (Tsien et al., 1988). In several cases the same transmitters that inhibit Ca2+ channels also induce phosphatidylinositol hydrolysis and mobilize intracellular Ca2+ (Fisher and Agranoff, 1987; Thayer et al., 1988). It is possible that mobilization of intracellular Ca2+ is involved in some of these responses, but has been prevented because of the inclusion of Ca2+ buffers in whole-cell patch electrodes. Investigation of these responses using the perforated patch method might reveal a mediatory role for the mobilization of intracellular Ca*+.

Outward K+ currents were blocked by replacement of K’with internal Cs+ (see solution B, above), and/or by application of IO mM external TEA. In addition, the usual chargecarrier, Ba2+, also blocks some K+ channels. In experiments in which Cs’was used in the patch pipette, this ion appeared to replace internal K’ quickly and completely, such that a noninactivating inward current appeared upon depolarization from - 40 to 0 mV as soon as the R, had dropped below about 50 MD. The inward current showed little additional increase with time and was little affected by TEA, suggesting that K+ replacement was nearly complete (see also Korn and Horn, 1989). When internal K+ was not replaced, external TEA and Ba2+ were effective in blocking K+ currents, revealing an inward current with kinetics and magnitude similar to those observed with Cs+ replacement. To test the effectiveness of the TEA further, 1 uM nimodipine (n = 3) or 0.5 mM La” (n = 3) was applied to block the inward current, revealing a very small outward current (