tIstituto di Istologia, Universitd di Roma 'La Sapienza' and the

Journal of Physiology (1991), 433, pp. 631643 631With 7 figures

Printed in Great Britain

MODULATION OF VOLTAGE-ACTIVATED CHANNELS BYCALCITONIN GENE-RELATED PEPTIDE IN CULTURED RAT

NEURONES

BY CRISTINA ZONA,t DONATELLA FARINI,T ELEONORA PALMAtAND FABRIZIO EUSEBI*

From the *Dipartimento di Medicina Sperimentale, Universitd dell'Aquila, thetIstituto di Istologia, Universitd di Roma 'La Sapienza' and the tDipartimento di

Medicina Sperimentale e Scienze Biochimiche, Universitd di Roma 'Tor Vergata', Italy

(Received 22 January 1990)

SUMMARY

1. Whole-cell currents were recorded from cultures of dissociated neocorticalneurones of the rat. Rat a-calcitonin gene-related peptide (CGRP; 1 nM-1 jtM) causedsignificant dose-dependent decreases in the voltage-activated transient (A-current)and delayed rectifier K+ currents. Forskolin (10 nm-20 fSm) mimicked this effect.Peak K+ currents were gradually decreased after loading neurones with cyclic AMP(100 UM) through patch pipettes. CGRP was ineffective in, neurones loaded withcyclic AMP.

2. CGRP (0-5-2 /tM) increased cytosolic cyclic AMP concentration and this effectwas mimicked by forskolin (5-40 #M).

3. CGRP (0 1-1 ,SM) reduced high-threshold Ca2+ currents; as did forskolin(5-20 /LM) and cyclic AMP loaded into the neurones. In contrast, low-threshold Ca2+currents were not affected by any of these agents.

4. Voltage-activated Na+ currents were significantly reduced by both CGRP(0 1-1 JM) and forskolin (5-20 #M). A similar effect was observed when cells wereloaded with cyclic AMP.

5. We conclude that, in neocortical neurones, CGRP attenuates voltage-activatedcurrents by stimulating the intracellular cyclic AMP signalling system.

INTRODUCTION

Voltage-activated ion channels are phosphoproteins (Catterall, 1988; Rehm,Bidard & Lazdunski, 1988; Rossie & Catterall, 1988) that can be regulated byphosphorylation (Browning, Huganir & Greengard, 1985; Huganir, 1987). Amongextracellular messengers, such as hormones and neurotransmitters, the neuropeptideCGRP is a possible modulator of neuronal function since: (i) it is present at highconcentration in the nervous system (Goltzman & Mitchell, 1985); (ii) it is releasedfrom nerve cells by depolarization (Mason, Peterfreund, Sawchenko, Corrigan, Rivier& Vale, 1984); (iii) it may stimulate adenylate cyclase activity in the nervous tissue(Goltzman & Mitchell, 1985); (iv) it increases the cellular content of the secondmessenger cyclic AMP at post-synaptic elements (Laufer & Changeux, 1987; Eusebi,MS 8210

C. ZONA AND OTHERS

Farini, Grassi & Ruzzier, 1988); and (v) it may enhance phosphorylation of cellmembrane ion channels through the activation of cyclic AMP-dependent kinase(Miles, Greengard & Huganir, 1989).

Since the cortex contains a number of CGRP fibres (Ishida-Yamamaoto &Tohyama, 1989), and significant binding of CGRP was found in the rat cortex(Goltzman & Mitchell, 1985), we have investigated whether CGRP may affect thefunction of voltage-activated ion channels in cultured neocortical neurones of the rat.We show here that CGRP attenuates voltage-activated currents in parallel with theincrease in cytosolic cyclic AMP concentration. Thus, it is possible that in neocorticalneurones CGRP exerts its regulatory effect on the voltage-activated channelsthrough activation of the cyclic AMP second messenger system.

METHODS

Cell cultureDissociated cortical neurones from rat (Wistar) embryos at day 15 were prepared and cultured

as described elsewhere (Dichter, 1978; Dichter & Zona, 1989). The animals were killed bydecapitation. Experiments were performed at 25-26°C, on neocortical neurones cultured for 15-24days in Eagle's minimum essential medium (Gibco) additioned with 5% rat serum, 5'6 mM-glucose,2 mM-glutamine, 20 U/ml penicillin and streptomycin (Gibco). Electrophysiological recordingswere performed in a standard saline (see below) 30-60 min after washing. For more details, seeDichter & Zona (1989).

Dissociated hippocampal neurones from rat (Wistar) embryos at day 18 were prepared andcultured in BME basal medium (Gibco) as described (Toselli, Lang, Costa & Lux, 1989). Neuroneswere kept in culture for up to three weeks, but experiments were performed at 10-14 days afterplating.

Whole-cell recording

Using the whole-cell configuration of the patch-clamp method (Hamill, Marty, Neher, Sakmann& Sigworth, 1981) membrane currents were recorded from the cell soma, filtered at 3 kHz, digitizedby computer at 10 kHz, and stored on a hard disc. Leakage currents were subtracted automatically.Test pulses were usually applied at 1 s intervals. For more details see Dichter & Zona (1989).More than 300 neurones were studied in this work. They belonged to three main types of cells:

fusiform, pyramidal-like and multipolar. Most recordings were made from fusiform cells which hadspherical soma with a diameter of 20-26,um (Kriegstein & Dichter, 1983).

Mea8urement of cyclic AMP concentrationNerve cells were incubated with CGRP or forskolin and the incubation was terminated by adding

a solution of 95% ethanol and 0-1 % trichloroacetic acid. The neurones were then scraped off thedish and the cell suspension centrifuged for 30 min at 3000 r.p.m. Supernatants were lyophilizedand then reconstituted with H20. The cyclic AMP level was measured by radioimmunoassay(Steiner, Parker & Kipnis, 1972) after suitable dilution and acetylation. The radioimmunoassayhad a sensitivity of 2-3 x 10-16 M-cyclic AMP concentration, an intra-assay and inter-assaycoefficient of 10 and 20%, respectively. Protein content was determined in the precipitated pelletsby the method of Lowry, Rosembrough, Farr & Randall (1951). Values were expressed as pmolcyclic AMP/mg protein.

Solution8Experiments were carried out in a bath solution containing (in mM): NaCl, 120; KCl, 3; CaCl2,

2; MgCl2, 2; glucose, 20; HEPES/NaOH, 10; pH 7 3. When necessary, Na+, K+ and Ca2+ currentswere blocked by addition of tetrodotoxin (TTX; 1 ,4M), 4-aminopyridine (2 mM) and cadmium(200 /zM), respectively. The whole-cell recording pipette used for recording K+ currents contained(in mM): KCl, 120; CaCI2, 0 24; EGTA, 5; glucose, 30; ATP, 2; HEPES/KOH, 10; pH 7 3. When

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CGRP AND VOLTAGE-ACTIVATED CHANNELS

K+ currents had to be suppressed, the pipette solution consisted of (in mM): CsCl, 130; TEA-Cl, 20;MgCl2, 1; CaCl2, 0-24; EGTA, 5; ATP, 2; glucose, 10; HEPES/CsOH, 10; pH 7-3.Neurones were superfused with rat a-calcitonin gene-related peptide (CGRP; Bachem,

Switzerland), forskolin or bovine calcitonin (Sigma) dissolved at the desired final concentration innormal bath saline and applied by pressure ejection from pipettes with a tip diameter of 20-25 ,um.Micropipettes were positioned near the neurones (< 50 ,um) during each application and quicklyremoved to end the drug exposure. The delay of perfusion was ca 1-2 s. It was determined bymeasuring the interval between the application of a depolarizing saline solution containing 50 mm-KCl and the change in the membrane resting current.

RESULTS

Cytosolic cyclic AMP levelIt has previously been shown that the neuropeptide CGRP causes an enhancement

of cellular cyclic AMP content by stimulating the adenylate cyclase system in musclecells (Laufer & Changeux, 1987; Eusebi et al. 1988). In order to see whether this alsooccurs in nerve cells, neocortical neurones, cultured for 15-24 days were treated witheither CGRP (0-5-2 /uM) or the activator of adenylate cyclase forskolin (5-40 /tM) for1-10 min. Absolute levels of cyclic AMP were then determined by radioimmunoassay.Addition of CGRP (1 uM) to neocortical neurone cultures caused a rapid increase incyclic AMP content which in about 3 min rose to a steady level about six timesgreater than the basal value. Forskolin (20 uM) also increased cytosolic cyclic AMP.A 6-fold increase in cyclic AMP was observed 20-30 s after drug application, and theeffect was maximal (21-fold the basal level on average in three experiments) 6-10 minlater. Both CGRP and forskolin increased the intracellular content of cyclic AMP ofneocortical neurones in a dose-dependent manner (Fig. 1). However, forskolin causedthe cells to accumulate more cyclic AMP than CGRP. This difference is even moremarked than shown in Fig. 1, where cyclic AMP levels were measured after 3 minincubations at which time the effect of forskolin is only 60% of maximal.

Voltage-activated potassium currentsNeocortical neurones equilibrated with a Na+ and Ca2+ current-suppressing

medium (see Methods), exhibit a fast transient K+ outward current (A-current)(Zona, Pirrone, Avoli & Dichter, 1988). This current is elicited by depolarizingvoltage steps from a holding potential of -80 mV, but is almost completelyinactivated by holding the cell at -40 mV (Zona et al. 1988; see Fig. 2A, B). Duringdepolarizing steps the A-current is followed by a delayed sustained outward phase,the delayed rectifier K+ current, which is more clearly observed when the A-currentis almost completely inactivated (Zona et al. 1988; e.g. Fig. 2B).

Effect of CGRPCGRP, at 0 5 /tM, significantly reduced the peak amplitude of the total K+ current

elicited by depolarizing the cell from -80 to + 40 mV, on average by 29 + 3(+ S.E.M., n = 15 neurones). A reduction of at least 15% was seen in every cell tested.The effect of the drug was evident within 20-40 s after the start ofCGRP application,although 2-3 min of continuous application were required to produce a maximaldecrease in the current. In most cells, as in Fig. 2, full recovery from the CGRP effectoccurred 1-3 min after the end of drug application. However, some nerve cells

633

634 C. ZONA AND OTHERS

exhibited only a slight recovery (up to ca 90% of the control current amplitude). Thiswas at least partially due to the slow run-down of the K+ current in internallydialysed neocortical neurones (see for instance Fig. 4).A typical example of the CGRP-induced decrease in K+ current is illustrated in

Fig. 2. In that cell the neuropeptide at 0 5 ,UM concentration inhibited the amplitude

L 12

10_

C*

0X5 1 1.5 2 * CGRP

CU~~ l

10 20 30 40 A ForskolinConcentration (#M)

Fig. 1. Enhancement of cytosolic cyclic AMP level as a function of CGRP and forskolintreatments in neocortical neurones. Abscissae, concentration of either CGRP (-) orforskolin (0). Ordinate, n-fold increase in cyclic AMP concentration as compared to thebasal level. Each point is the mean (+S.E.M.) of duplicate observations on four differentplates. Neurones were exposed to either CGRP or forskolin for 3 min. In this andsubsequent figures the neurones were cultured for 15 days, unless otherwise specified.

of the transient current (Fig. 2A, C) by ca 35% at + 30 mV; and the sustainedsteady-state current (Fig. 2A, B) by ca 30%. The rate of decay of the A-current wasnot affected by CGRP.The reduction in K+ current by CGRP occurred in the absence of any changes in

the voltage dependence of activation. Current-voltage curves were derived bymeasuring the peak K+ current in response to test pulses applied before and afterCGRP application (Fig. 2D). CGRP decreased the peak current amplitude withoutaltering the voltage threshold.The CGRP-induced effect on K+ currents was dose-dependent with a threshold

dose for an effect as low as 01 nm. For instance, at 1 nM-CGRP concentration, thetotal current size decreased on average, by 9+ 2% (four cells examined); at 10 nM,by 18+ 4% (five neurones examined); while at 100 nm, by 25 + 4% (four neuronesexamined). In an experiment, a 1*5-fold increase in cyclic AMP level was observedwhen neurones were exposed to 100 nM-CGRP for 4 min, indicating that even atrelatively low concentrations CGRP causes an elevation of cyclic AMP cellularcontent. CGRP at 1 gm concentration caused the same effect on the peak currents asat 0-5 ,eM.


Effect offorskolinThe action ofCGRP on the K+ currents in neocortical neurones could be mimicked

by forskolin (10-20 fM) which was as efficacious as CGRP (0 5-1 /tM) in reducing peakK+ currents. However, the decrease in the K+ current amplitude was faster. For

A Control CGRP Recovery

1 nAL10 ms

B=

C D 4

21 nAL

10 ms

-40 -20 0 20 40Vtest (mV)

Fig. 2. CGRP effects on whole-cell K+ currents. The CGRP concentration was 0 5 /SM. Allrecords (A-C) from one neurone. A, top, current responses during positive steps (bottomtraces) from a holding potential of -80 mV before (left), 150 s after starting CGRPapplication (middle), and 180 s after the end of perfusion (right). A, bottom, positive stepsof voltage from -80 mV to 0, + 10, + 20, + 30, and + 40 mV. B, top, total currentselicited by command voltage pulses (bottom traces) before (left), 180 s after startingCGRP application (middle) and 210 s after the end of perfusion (right), in the sameneurone held at -40 mV and under the same experimental conditions as in A. B, bottom,positive steps of voltage to same potentials as in A. C, A-current was obtained bysubtracting the current induced by a voltage step to 0 mV from a holding potential-40 mV from that elicited by a voltage step to the same potential from -80 mV. D, peakcurrent-voltage relationships of outward currents evoked by voltage steps from -80 mVto test potentials (Vtest) before (@) and after (0) CGRP application of 180 s. Each pointrepresents the mean value (±S.E.M.; n = 12) of determinations from different neurones.

instance, 30 s after application of 20 jsM-forskolin, the size of the K+ current elicitedby a step to + 30 mV was already maximally reduced by 30+4% (n = 5; cells heldat -80 mV) (e.g. Fig. 3A). K+ currents were not further reduced during long-lasting

635

C. ZONA AND OTHERS

forskolin applications (3-5 min). I-V curves in control and forskolin-treated neurones(Fig. 3B) show that forskolin, like CGRP, reduces only K+ current size. Theforskolin-induced effect was fully reversible in eleven out of fifteen neuronesexamined within 60 s after the end of drug application.

AControl 1 nA

10 m:

Forskolin

IS

4 T I (nA)B

-40 -20 0 20 40

Vtest (mV)

Fig. 3. Effect of forskolin (20 /M) on whole-cell K+ current in neocortical neurones. A, top,outward currents evoked by voltage steps from -80 mV to test potentials (Vtest; see tracesbelow) before (left) and after 30 s forskolin application. A, bottom, positive voltage stepsfrom -80 mV to Vtest of 0, + 10, + 20, and + 30 mV. B, current-voltage relationships ofthe peak currents before (@), 60 s after beginning forskolin application (U), and 120 safter the end of forskolin application (0). Points represent mean+S.E.M. of twelveexperiments from different cells.

Similarly to CGRP, the forskolin-induced effect on K+ currents was dosedependent. For instance at 100 nm concentration, forskolin reduced the current sizeby 15% (three experiments), while at 10 nm, by 7% (six experiments).

Effect of cyclic AMPWhen the recording pipette solution contained cyclic AMP (100 ftM), the peak K+

current was significantly reduced as compared with measurements in other cellswhen cyclic AMP was omitted. In these experiments, summarized in Fig. 4, the sizeof the current decreased over time by about 40 %, reaching a plateau 2-3 min afterthe membrane patch disruption. This indicates that a progressive increase in

636


cytosolic cyclic AMP caused an inhibition of the K+ current, which was similar tothat caused by either CGRP or forskolin.

In a number of experiments after adding cyclic AMP to the patch-pipette solution,following development of peak current attenuation, neurones were exposed to

XE<8

40.

U,c

0 1 2 3Time (min)

Fig. 4. Time course of the cyclic AMP-induced decrease in K+ current in neocorticalneurones. Points represent mean values (± S.E.M.) of eight experiments. The cyclic AMPconcentration in the patch-pipette solution was 100 gM. Cells were held at -80 mV andstepped to + 30 mV. Zero time indicates the time when the pipettes filled with a cyclicAMP-containing solution disrupted the membrane. Student's t test: P < 0 001 between0 5 and 3-5 min values in neurones loaded with cyclic AMP. *, control. *, cyclic AMP-treated neurones.

CGRP. The neuropeptide (0-5ftM) did not influence peak K+ currents in sevenneurones loaded with cyclic AMP (0-1 mm in the patch-pipette). This suppression ofthe CGRP action in neurones loaded with cyclic AMP, suggests that the CGRP-induced peak K+ current attenuation may be mediated by the cyclic AMP-secondmessenger system.

CalcitoninSince in the rat cortex the presence of receptor sites for calcitonin have previously

been described (Goltzman & Mitchell, 1985), experiments were performed to seewhether the CGRP structural analogue calcitonin may affect peak K+ currents inneocortical neurones. Calcitonin at 1-5 /tM concentration was unable to mimic theCGRP effects on voltage-activated K+ currents in eight neurones from a cultureresponsive to CGRP. This indicates that, in neocortical neurones, the CGRP-inducedaction on the K+ currents is mediated through its own receptor.

Voltage-activated calcium currentAfter voltage-activated Na+ and K+ currents are pharmacologically inhibited,

depolarization of neocortical neurones from -80 to -20 mV and beyond elicit abiphasic Ca2+ current composed of a maintained low-voltage-activated (LVA)component, and a transient high-voltage-activated (HVA) component (Dichter &Zona, 1989).

637

C. ZONA AND OTHERS

Effect of CGRPCGRP (0-1-1 /,M) caused a decrease in the transient fast-inactivating Ca2`

current. The average decrease was 15 +4% (n = 6) after 3 min of CGRP (1 ,UM)application and was fully reversible in four neurones. The decrease in Ca2+ current

A

Con./rec.

CGRP 7

CGRP

CnrlRecovery0.4 nAL

20 ms

Vtest (mV)-20

-1

D20 -60

Con./rec.ForskolInForskolin

Corskolin/Rcvr

ontrol/Recovery

Vtest (mV)-20

-

\1-1

-2 t/ (nA) -2 t / (nA)

Fig. 5. Effects ofCGRP and of forskolin on voltage-activated Ca2+ current in cells culturedfor 21 days. CGRP and forskolin concentrations were respectively 0 5 JSM and 20 /LM. A,left and right: positive steps of voltage from -80 mV to -30 and 0 mV, eliciting currentsreported in B. B, left, top, three superimposed current traces evoked by voltage steps to-30 mV (control, in the presence ofCGRP and recovery). Bottom traces: CGRP-induceddecrease in the peak currents evoked by voltage steps from -80 mV to 0 mV (three tracesshow control, recovery and decrease caused by CGRP). The recovery was obtained 1 minafter the end of the perfusion. B, right, similar records showing greater reduction of theHVA current by forskolin. C and D, I-V relationship before (@), and 150 s after (U)beginning CGRP application (C), and before (@) and 60 s after (U) forskolin application(D). 0, show recovery 60 s after the end of drug applications. Points indicatemean + S.E.M. of eight experiments.

was independent of the membrane holding potential. A similar decrease was observedwhen nerve cells (n = 5) were held at either -40 or - 120 mV. In contrast, thesustained LVA Ca2+ current was not significantly affected by the CGRP treatment(e.g. Fig. 5B). No other changes in the electrical parameters of the Ca2+ current were

observed in CGRP-treated cells. Figure 5 C summarizes the effect of CGRP on HVACa2+ current.

B

C-60 20

638


Effect offor8kolinSimilarly to CGRP, forskolin (5-20 ,uM) did not produce any significant effect on the

LVA Ca2+ current, while it maximally reduced the HVA peak current on average by60+10% (n = 12) within 30-60 s exposure (Fig. 5B). A full recovery in the HVA

A

Control

0*4 nA

20 ms

B

Cyclic AMP

Fig. 6. Effect of cyclic AMP on Ca2+ current in a neurone cultured for 21 days. A, top:positive voltage step from -80 to -1O mV. Bottom: typical current response to thevoltage step (top) recorded with a pipette containing standard CS2+ solution (seeMethods), 180 s after membrane patch rupture. B, top: voltage step of same strength as inA, in another cell. Bottom: current response 180 s after membrane patch rupture, with apipette filled with a solution containing cyclic AMP (100 /uM).

Ca2+ current occurred 1-3 min after the end of exposure. In addition, similarly toCGRP, forskolin decreased only the peak HVA current without altering the voltagethreshold for current activation, the potential at which current was maximal, orthe rate of inactivation (see Fig. 5B and D).

Effect of cyclic AMPIn order to see whether an elevation in cytosolic cyclic AMP could directly affect

the Ca2+ current, recordings were carried out with cyclic AMP (100 /LM) in the whole-cell patch-pipette. While LVA Ca2+ currents were not significantly different in cellsloaded with cyclic AMP for 2-5 min compared to untreated neurones, depolarizationswere unable to evoke fast inactivating Ca2+ currents ca 3 min after membrane patchdisruption in seven cells examined (e.g. Fig. 6).

Effect offorskolin on hippocampal neuronesIt has been elsewhere demonstrated that forskolin enhances the peak Ca2+ current

in hippocampal neurones (Gray & Johnston, 1987). This is in contrast to the resultsabove shown in neocortical neurones and might be caused by the experimentalconditions used, including cell culture and drug perfusion procedures. In order todemonstrate that these discrepancies reflect a genuine differential regulation of

639

C. ZONA AND OTHERS

voltage-activated Ca2+ currents by adenylate cyclase system, experiments werecarried out in cultured hippocampal neurones exposed to forskolin (5-20 jtM).Forskolin was found to significantly and consistently increase peak Ca2+ currents by12% on average (± 4 %; nine pyramidal neurones), in agreement to what was

A B C

TTX

~CRP ForskolinControl Control/Recovery Control/Recovery

1 nAL2.5 ms

D Vtest (mV) E Vtest (mV)-40 0 -40 0

-4 (nA) I (nA)

Fig. 7. Effects of CGRP (1 4uM) and forskolin (20 /tM) on Na+ currents recorded fromneurones cultured for 14 days. A, top: voltage steps from -80 to -10 mV. Bottom:control current and after blocking with TTX (1 ,UM). B, top: V,est as in A. Bottom: decreaseand recovery of Na+ current caused by CGRP applied for 150 s. Recovery record wastaken 100 s after the end of perfusion. C, top: Vtest as in A and B. Bottom: forskolin-induced decrease in the peak current 30 s after application. Recovery 70 s after the endof perfusion. D and E, I-V relationships in neurones treated for 180 s with CGRP (D) orfor 60 s with forskolin (E). Control, (@); and during drug application (U); recovery (0)ca 80 s after the end of drug perfusion. Number of experiments, 10 (D) and 8 (E).

observed previously in hippocampal neurones (Gray & Johnston, 1987), but incontrast to the results obtained here from neocortical neurones under the sameexperimental conditions.

Voltage-activated sodium currentWhen voltage-activated K+ and Ca21 currents were pharmacologically suppressed,

depolarizations of neocortical neurones elicited Na+ currents that could be fullyblocked by TTX (e.g. Fig. 7A). Following exposure to CGRP (1 tam; 3 min) these Na+currents decreased in size by ca 17 %, and recovered to the original values 1-3 minafter the end of drug exposure (Fig. 7B). On the other hand, the voltage threshold

640


for current activation, the potential at which current was maximal, and the rate ofinactivation were all virtually unaltered by the neuropeptide (Fig. 7D). Forskolin(5-20 #M) mimicked CGRP (0 1-1 JtM) in reducing the Na+ current (Fig. 7 C, E) butit was more efficacious and faster than CGRP, producing a maximal effect within30-40 s after drug addition. For instance currents elicited by a step from -80 to-10 mV, decreased on average by 258 +3.0% (n = 12) 30 s after application offorskolin (10 /M) and by 17-6±+ 1-7% (n = 12) 2 min after exposure to CGRP (0-5 ftM).Furthermore, the size of Na+ currents from nerve cells (n = 8) loaded with cyclicAMP (100 fM in the patch pipette solution) for 20-60 s were 36 +4% smaller thanthe Na+ currents in untreated neurones.

DISCUSSION

The results demonstrate that the neuropeptide CGRP causes an appreciable andreversible attenuation of both the transient (A-current) and the delayed rectifierK+ currents, and, to a lesser extent, of the HVA Ca2+ current and of the voltage-activated Na+ current. This current attenuation is dose dependent, parallels a rise inthe cytosolic level of cyclic AMP and is fully developed when cyclic AMP cellularcontent is increased by ca 6-fold. Calcitonin does not mimic CGRP action on K+currents, indicating that the CGRP effects are mediated through its own receptors inneocortical neurones. Forskolin, as well as cyclic AMP present in the recordingpipette, mimic CGRP in decreasing both K+ and Na+ currents, and are much moreefficacious than the neuropeptide in attenuating HVA Ca2+ currents. Furthermore,the addition of cyclic AMP to the patch pipette solution suppresses the action ofCGRP on the K+ currents.

All these data taken together suggest that the neuropeptide CGRP may regulatethe function of voltage-operated channels in rat neocortical neurones by actingthrough the cyclic AMP second messenger system. The mechanism of the currentattenuation is not yet clear, but might involve reduction of open probability and/ora decrease in the conductance of the single channels.

Considerable evidence has accumulated that voltage-dependent channel proteinsare phosphorylated by the cyclic AMP-dependent kinase system (Catterall, 1988;Krueger, 1989; Levitan, 1985; Rehm et al. 1988; Rossie & Catterall, 1988). Inparticular, it has been reported that the voltage-dependent Na+ channel oc-subunitis phosphorylated at several sites by cyclic AMP-dependent protein kinase (Catterall,1988). We have now shown that agents such as CGRP, cyclic AMP and forskolin,which are expected to stimulate phosphorylation, cause a significant decrease in thevoltage-activated Na+ current amplitude. This, together with the inhibition of thechannel-mediated Na+ influx observed by Costa & Catterall (1984) after cyclic AMP-dependent phosphorylation, suggests that phosphorylation of the Na+ channelprotein may regulate its function. Furthermore, we describe similar action of CGRP,forskolin and cyclic AMP on K+ voltage-dependent currents, suggesting that theyalso may be regulated via a cyclic AMP-dependent phosphorylation. An inhibition ofK+ currents by cyclic AMP has been described in both invertebrate (Strong &Kaczmarek, 1987) and sensory neurones (Dunlap, 1985).

In related experiments CGRP was found to enhance Ca2+ currents in rat dorsal21 PHY 433

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C. ZONA AND OTHERS

root ganglion neurones (Ryu, Gerber, Murase & Randic, 1988) and in amphibianheart myocytes (Ono, Delay, Nakajima, Irisawa & Giles, 1989), while cellular cyclicAMP elevation was reported to increase the activity of voltage-dependent Ca2+channels in hippocampal neurones (Gray & Johnston, 1987; data confirmed in thispaper). Moreover, an upward modulation of the Ca2+ current by cyclic AMP-dependent phosphorylation has been described in several other cell systems (for areview see Byerly & Hagiwara, 1988). By contrast, in the present paper we haveshown a CGRP-induced inhibition of HVA Ca2+ currents, that is mimicked, withmore efficacy, by forskolin and by loading nerve cells with cyclic AMP. Our result isin agreement with a downward modulation of the Ca2+ currents by cyclic AMPobserved in smooth muscle (Saida & van Breemen, 1985) and in rat sympatheticneurones (Horn & McAfee, 1980). It is possible that this differential regulation ofvoltage-activated Ca2+ currents by second messenger systems reflects differences inthe phosphorylation sites of the channel proteins and/or in the biochemical cellmachinery involved in channel protein regulation (Sumikawa & Miledi, 1989).

In conclusion, the results presented suggest the possibility that the activity ofvoltage-activated membrane channels involved in determining neuronal excitabilitymight be regulated in neocortical neurones by CGRP-impinging fibres, possiblythrough cyclic AMP-dependent phosphorylations. Thus, from the present data, thepeptide CGRP might be considered a candidate modulator of nerve cell activity inthe cortex.

We are indebted to Professor Ricardo Miledi and to Dr Carlo G. Caratsch for critical reading ofthe manuscript; and to Dr Tomoyuki Takahashi for discussions. The present work was supportedby grants from FIDIA (to F. E.) and from Ministero Pubblica Istruzione.

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