A.A. Bogomoletz Institute of Physiology, Ukrainian Academy of Sciences, Kiev, Ukraine ...... Conn PJ, Strong JA, Azhderian EM, Nairn AC, Greengard P, Kacz-.
Pfl/igers Arch (1992) 420:146-152
Journal of Physiology 9 Springer-Verlag1992
Parathyroid hormone enhances calcium current in snail neurones Simulation of the effect by phorbol esters P.G. Kostyuk, E.A. Lukyanetz, and A.S. Ter-Markosyan* A.A. BogomoletzInstitute of Physiology,Ukrainian Academyof Sciences, Kiev,Ukraine Received August 21, 1990/Receivedafter revision September 16, 1991/AcceptedOctober 8, 1991
Abstract. Effects of parathyroid hormone substance (PTH) on the voltage-activated calcium current (/ca) were studied on intraceUularly perfused neurones of the snail, H e l i x p o m a t i a , under voltage-clamp conditions. Application of 0.1 nM PTH produced a marked potentiation of the current. The effect developed slowly (60- 70 rain) and remained after removal of PTH. Potentiation could be observed in most neurones, but varied considerably from cell to cell; in some neurones/Ca was increased 2- to 3-fold. Addition of ethylenebis(oxonitrilo)tetraacetate (EGTA, 10 mM) to, or removal of adenosine 5'-triphosphate (ATE 2 mM) from the intracellular perfusing solution resulted in a suppression or attenuation of the potentiating effect. The effect could be reproduced by the synthetic 1 - 3 4 amino acid fragment of PTH. Extracellularly applied protein kinase-C (PK-C) activator phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 - 10 gM) produced a similar slow increase in Ica (up to 1.5- to 2-fold), while its inactive analogue (4aphorbol ester) had no effect on/Ca. The effects of PTH and PMA were not additive. PK-C inhibitors [1-(5-isoquinoline-sulphonyl)-2-methylpiperazine hydrochloride] (H-7, 100 IxM) and staurosporine (100 IxM) as well as calcium channel antagonists Cd 2+, verapamil, nifedipine and nimodipine depressed the effect of PTH. The chloride channel blocker 4,4'-diisothiocyanato-stilbene-2,2'disulphonic acid (DIDS, 1 raM) did not affect the potentiating action of PTH. Activation of the adelylate cyclase system also potentiated Ica in some neurones, but this effect had a different time course and was additive to the effect of PTH. A conclusion is made that activation of PK-C may mediate the slowly developing enhancement of /Ca by PTH. Key words: Parathyroid hormone - Protein kinase C Calcium channel - Snail neurone * Present address: Department of Physiology,State Medical Institute,
Erevan, Armenia Offprint requests to: P.G. Kostyuk
Introduction Parathyroid hormone (PTH) is one of the hormones which regulates calcium homeostasis in an animal organism. Its primary targets are considered to be bones, kidney and intestine [16]. It is known that in hyperthyroidism there is an abnormal accumulation of calcium in soft tissues, including brain [1] and muscle [15, 26]. PTH can penetrate the haemotoencephalic barrier: its content in cerebrospinal fluid of the rat was shown to be about one-third of that in the blood plasma [20]. This hormone was shown also to increase the flux of calcium into the cytosol of different cells by affecting its transmembrane transport or its intracellular redistribution [12, 18, 30]. Like other polypeptide hormones, PTH interacts with specific receptors on the plasma membranes of target ceils. This interaction initiates a cascade of intracellular events that includes, among others, the generation of secondary messengers. Cyclic adenosine monophosphate (cAMP) was first considered to be such messenger in the action of PTH on bone and kidney [16], now also other messengers are suggested [5, 19]. Although no special studies on the presence of PTH in invertebrates were carried out, nerve tissue from the snail H e l i x p o m a t i a was shown to bind this hormone [20]. Also, stimulation by PTH of calcium accumulation in snail neurones was reported by these authors. The preceding prompted us to investigate the mechanism by which PTH may affect the transmembrane transport of calcium in molluscan neurones, especially the possible triggering of calcium channel protein phosphorylation which is supposed to play an important role in the modulation of calcium permeability [9, 11].
Materials and methods Voltage-clamp experiments were carried out on nonidentifiedisolated intracellularly perfused neurones of the snail, H. pomatia. The techniques of cell isolation, intracellularperfusionwith plastic pipette and voltage-clamphavebeen describedelsewhere[22]. The control solution
147 used for intracellular perfusion contained (in mM): CsC1 - 80, tris(hydroxymethyl)aminomethane (TRIS)aspartate - 40, adenosine 5'-triphosphate (ATP) (bisodium salt) - 2, MgC12 - 3; pH 7.3. The flee Ca 2+ concentration in these solutions was near 4~0-6 M as measured with a calcium-selective electrode (Orion). In some experiments the level of free Ca 2+ was specially stabilized at 10-6M by adding a Ca-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer to the perfusion solution. When EGTA was added to the solution, the same amount of TRIS-aspartate was removed. The extracellular solution contained (in mM): CaC12 - 20, MgC12 - 4, TRIS-C1 - 40, tetraethylammonium (TEA) bromide or chloride - 40, 4-aminopyrydine (4-AP) - 10; pH 7.6. The bath application system made it possible to produce several-fold exchange of the extracellular medium in a few seconds. The bovine PTH, synthetic rat PTH fragment 1-34, phorbol ester PMA (phorbol 12-myristate 13-acetate), and 4a-phorbol ester (4a-phorbol 12,13-didecanoate) were all from Sigma. PTH and the fragment were disolved in bidistilled water and stored in small portions (0.01 mM) at -20~ Phorbol esters stock solutions (1 mM) were prepared in dimethylsulphoxide (DMSO) and stored at - 5 ~ final concentrations of DMSO had no effect in control experiments. DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid) was from Sigma, nifedipine, nimodipine BAY K 8644 from Calbiochem, verapamil from Serva, 5-HT (serotonin creatinine phosphate) from Reanal, H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazinedihydrochloride] from Seikagaku Kogyo (Japan), staurosporine (antibiotic AM-2282) from Sigma. Transmembrane currents after analog substraction of linear capacitative and leakage currents were sampled with 100-1xs dwell time (12 bit AD converter) and stored on magnetic tape of the microcomputer for later analysis. All experiments were carried out at room temperature (19-21 ~
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Effects of PTH application on the calcium inward current In the experimental conditions specified (Cs + and TRIS + were the only intracellular monovalent cations and blockers o f outward current channels T E A and 4-AP were added to the extracellular solutions), the possibility of outward current activation (either potassium or hydrogen) was negligible. Together with the choice of the holding potential ( - 50 mV), this ensured that the transmembrane current evoked by depolarizing shifts of the membrane potential (to + 10 or +20 mV, 50 ms in duration, applied every 30 s) was predominantly carried through calcium channels, and in the following we refer to it as the calcium current (/ca). Nevertheless, special tests were carried out to exclude the participation of changes in other membrane conductances in the observed effects (see below). During the intracellular perfusion with the control s o lution, the current amplitude in all cells progressively decreased. This process of "wash-out" was extensively analysed in our [9, 10] and other laboratoties; the speed of the process could be lowered to some extent by additions of ATP and EGTA to the perfusion solution [2]. When applied in 0.1 nM concentration (lower concentrations were ineffective), P T H caused a marked, slowly developing increase in peak amplitude of the current. The time course of these changes is shown in Fig. 1 A (curve 1, arrow indicates time of substance application). The current amplitude remained increased for a long time (up to
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Fig. 1A, B. Action of bovine parathyroid hormone (PTH) and synthetic PTH fragment on calcium current (/Ca) in snail neurones. A Time course of changes in the peak/ca (normalized to its amplitude at the beginning of experiment) in 3 different cells. PTH (0.1 nM) application is marked by a r r o w . The cells were perfused with: 1, EGTA-free solution containing 2 mM ATP; 2, solution with 2 mM ATP and 10 mM EGTA; 3, solution without ATP or EGTA. B Time course of changes in the peak/ca caused by rPTH(1 -34). Moment of application is marked by a r r o w . Intracellular EGTA-free solution contained 2 mM ATP. Holding potential, - 50 mV; test potentials, +20 mV
2 - 3 h) even after removal of the hormone from the extracellular medium. Several experimental factors were found to affect the magnitude of the effect. Besides others (see below), time of the application during the intracellular perfusion was important. As a rule, the earlier the substance was applied, the more pronounced was its effect. The effect was maximally expressed when the substance was applied prior to, or simultaneously with, the beginning of cell perfusion: in the favourable conditions the current increased 2to 3-fold. The effect was observed in 21 out of 28 investigated cells. However, it did not occur if P T H was applied later then 30 min after beginning of cell perfusion. Heat-inactivated P T H substance did not affect/CaThe increase in the current amplitude was not accompanied by significant changes in its kinetics: though the
148 increased current seemingly inactivated faster than the control one, appropriate scaling resulted in their almost complete coincidence. The PTH substance contains according to radioimmuno assay analysis approximately 10o70 of pure PTH. Although it is extremely unlikely that some other component of the substance could induce the observed changes in/ca, similar experiments were repeated with pure synthetic 1-34 amino acid fragment of the hormone r F r H ( 1 - 34). Presented in Fig. 1 B is the time course of the current amplitude changes in one particular experiment. The fragment-induced potentiating effect was of somewhat shorter duration than that caused by the PTH substance (about 40-60min). Afterwards the current amplitude decreased rapidly. Also, the efficacy of the fragment action was lower: the increase in the current amplitude was observed in 7 out of 21 tested cells. The effective concentration of the fragment was 2 to 3 orders higher compared to that of PTH substance. The effect of the 1 - 34 fragment confirms the suggestion that the potentiating effect of PTH substance is in fact due to the action of the hormone; at the same time it indicates that this short fragment is not quite sufficient in substituting for the latter.
periments in which cells were perfused with ATP-containing solutions having an increased level of free Ca 2+ . Together with the above finding, this suggests a possible role of Ca-dependent metabolic processes in the potentiating effect of PTH.
Phorbol ester mimics the effect of PTH The features of the hormonal effect described - duration, dependence on ATP and intracellular calcium - all points to the possible involvement of a calcium/phospholipid-dependend protein kinase (protein kinase C, PK-C) in the phenomenon under study. Several reports have been published recently implicating the PK-C modulation of calcium conductance in nerve cells. DeRiemer et al. [7] observed enhancement of calcium currents in Aplysia bag cells under the effect of phorbol ester and PK-C, Doroshenko and Kostyuk [8] the same in nonidentified H. pomatia neurones under the effect of phorbol ester. To the contrary, Rane and Dunlap [29] observed attenuation of 1Ca in sensory neurones under the action of PK-C activator 1,2-oleoylacetylglycerol, and Hammond et al. [17] reported inhibition of/'Ca in identified H. aspersa neurones after intracellular injection of PK-C.
ATP dependence of the PTH effect
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In order to determine whether enhancement of Ica by the hormone depends on the intracellular metabolic processes, we investigated how intracellular ATP affects the action of PTH substance on /Ca. The time course of changes in current amplitude in one of these experiments is demonstrated in Fig. 1A. A cell perfused with ATPcontaining solution responded to the hormone application with usual increase in the current amplitude (Fig. 1A, curve 1). Removal of ATP from the perfusing solution (despite the maintenance of elevated Ca 2+ level because of absence of EGTA) resulted in a complete disappearance (Fig. 1 A, curve 3) or a marked attenuation of the effect. No potentiation of/Ca was observed even in case of very early application of the hormone. The necessity of ATP for development of the hormone effects indicates possible involvement of some energy-dependent process, presumably protein phosphorylation.
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Intracellular calcium and the effect of PTH
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We noticed that apart from the withdrawal of ATP, the presence of EGTA in the intracellular perfusion solution also significantly affected the magnitude of the PTH effect. In case of cell perfusion with ATP-containing solution containing a fixed elevated level of free Ca 2+ (10-6M) 0.1 nMPTH substance evoked a pronounced increase in the current amplitude, while in the case of 10 mM EGTA added (free Ca 2§ level below 10 -7 M) no potentiation of/Ca ( t w o cells) or its marked attenuation (two other cells) were observed. An example can be seen in Fig. 1 (compare curves 1 and 2). Obviously, intracellular Ca 2§ ions are necessary for the effect to develop. The largest increase in /Ca amplitude induced by the hormone were observed in those ex-
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149 To explore possible participation of PK-C in mediating the effect of P T H on the /Ca, the PK-C activator phorbol ester P M A [3] was used. The agent was added to the extracellular solution in concentration 0.1 - 10 ~tM. In 12 cells investigated, 9 cells responded with a slowly developing increase in the/Ca amplitude, up to about 1.5to 2-fold, 2 others displayed a clear stabilizing effect, and in a single case the stimulating effect was not observed. Both P M A and P T H induced identical changes in the potential dependence of Ica: the maximum of the current/voltage (I/V) curve became shifted towards more negative test potentials, as can be seen from examples presented on Fig. 2 B, D. The potentiating effect of PMA, like the effect of PTH, developed during 4 0 - 6 0 min after the drug application and was even more stable than the effect of P T H (see Fig. 3A). The inactive analogue of the P M A 4aphorbol ester, even in 3 orders higher concentrations had no effect o n / c a (not illustrated). Application of both P M A and P T H to the same cell did not show a notable additive effect, independently from the order in which the drugs were applied (compare date presented in Figs. 3 B - D ) . It should be noted that in cases of joint application of P T H and PMA, the rate of rise of 1Ca definitely increased (up to 2.5-times); however, the size of the final effect did not change.
Effects of PK-C inhibitors Two PK-C inhibitors were tested in respect to their action on the potentiating effect of PTH. The isoquinoline sulphonamide derivative H-7 blocks several protein kinases; however it has been shown on Aplysia bag cell neurones [6] that in this case it blocks selectively PK-C without affecting the activity of PK-A. In our experiments extracellular application of 100 ~tM H-7 substantially depressed the potentiating effect of P T H (by 84+7%, n = 6). An example of such action is shown on Fig. 4A. At the same time H-7 did not affect the amplitude of Ica in control experiments. We also tested the effect of staurosporine which contrary to H-7 does to compete with ATP but interacts directly with the catalytic domain of PK. Therefore it is more convenient to use this inhibitor in vitro. In our in vitro experiments we used higher doses of staurosporine ( 3 0 - 1 0 0 ~tM). Cells from the same ganglion were separated into two groups one of which served as control. The experimental group was exposed to staurosporine for 1 - 3 h at 22~ The cells from control group (n = 4) responded to P T H in the usual way. In cells exposed to staurosporine the application of P T H evoked only a tran-
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Fig. 3A-D. Combined effects of PMA and PTH on/ca" Time course of changes in the peak Ica (expressed in % of maximal amplitude,/, /max) in 4 different cells caused by PMA (10 gM) and PTH (0.1 nM) application in different combinations. The moments of drug application are marked by arrows and horizontal bars. Holding potential -50mV; test potentials in A, B, D +10mV; C, 0mV. Intracellular EGTA-free solution contained 2 mM ATP
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t (min) 4A, B. Effects of protein kinase inbibitors on PTH effect. Time course of changes in peak Ica in 2 different ceils. Drug applications are marked by horizontal bars. A 100 ~M [1-(5'-isoquinoline-sulphonyl)2-metbylpiperazine hydrochloride] (H-7) was added after 0.1 nM PTH application. B Cell was previously incubated in 100 ~tM staurosporine for 3 b. Holding potential, -50 mV; test potentials, + 20 mV Fig.
150 sient increase of/Ca (see Fig. 4 B). After 30 min the amplitude of/Ca decreased by 52_+5% (n = 5).
Possible changes of calcium-dependent chloride conductance can be obviously excluded on the basis of experiments with calcium-buffered (10 -6 M) intracellular solutions. This concentration of free calcium is sufficient to activate the calcium-dependent chloride current [Icl(Ca)] and to make them independent on calcium influx from outside. However, the potentiating effect of PTH was well preserved in such conditions. Another way to block Io(Ca) is to introduce Ba 2+ instead of Ca 2+ in the cell [14]. As can be seen from Fig. 5D, replacement of Ca 2+ by Ba 2+ in the extracellular solution did not depress, but to the contrary largely potentiated the inward current already increased by PTH.
Possible effects of P T H on other types of membrane conductances Although there were little doubts that the changes in inward currents induced by PTH and PMA represent changes in Ica, special control experiments were performed to exclude possible changes in other membrane conductances. It should be noted that in rat osteoblasts PTH activate chloride conductance in a way very similar to that observed in our experiments [4]. Therefore we compared the effects of specific calcium channel antagonists (Cd 2§ verapamil, nifedipine and nimodipine) and chloride channel blocker DIDS on the recorded inward currents and their potentiation. As can be seen from recordings presented on Fig. 5 A - C calcium channel antagonists effectively and reversibly depressed the potentiating action of PTH; the recorded current became even smaller than the control one. The calcium channel agonist BAY K 8644 had no detectable effect (obviously because the activation of calcium channels was already maximal). Application of DIDS (0.5-1 mM) exerted no effect at all in a wide range of test potentials (from - 4 0 to + 90 mV, data from 6 cells). It should be noted in addition, that the maximal potentiating effects of PTH and PMA were observed at test potentials close to the chloride equilibrium potential (between - 13 and - 17 mV in our experimental conditions); this also indicates noninvolvement of chloride channels in the observed effects.
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The effect of P T H is not mediated via cAMP-dependent pathways As it has been already mentioned in the Introduction, cyclic nucleotides are considered as mediators of the effect of PTH in several types of cells. To check this possibility in our case, we specially investigated the effect of PTH on snail neurones in which activation of the adenylate cyclase system regularly up-modulates/Ca [24]. These cells are located mainly in the ventral part of the pedal ganglion; 5-HT is a specific agonist of this process. Therefore we compared the effects of 5-HT and PTH on the same neurones. Superimposed records of / C a a r e presented in Fig. 6A, obtained in control and after addition of 5-HT (left panel, Fig. 6A). Then 5-HT was washed out, and after about 30min PTH was applied (centre panel,
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The results presented indicate that PTH can affect the voltage-operated calcium channels in neuronal membrane: it induces marked enhancement of/ca. The effect demonstrated all the properties necessary to relate it to the intracellular metabolism: it did not develop when ATP was removed from the perfusion solution, when the intracellular Ca 2§ ion concentration was lowered by EGTA addition or the hormone was added a long time after beginning of the cell perfusion. The voltage-operated calcium channels in neuronal membrane were shown to be modulated by presumable phosphorylation of the channel protein by different protein kinases: cAMP-dependent [10, 11, 24], cGMP-dependent [13, 28] and calcium/phospholipid-dependent [7, 8, 17, 29]. Though, previous data indicate that PTH may activate adenylate cyclase [16], the results obtained in our experiments are against involvement of the cyclic nucleotide-dependent protein kinase in the effect of PTH on calcium conductance in snail neurones. Potentiation of /Ca by activation of PK-A observed in definite snail neurones [24] was considerably shorter in duration, smaller in magnitude (about 20-40%), reversible and attenuated by an increase in intracellular calcium level (due to activation of corresponding phosphodiesterases). The results obtained agree well with the suggested involvement of inositol phospholipid metabolic pathway, namely, PK-C activation. Besides the understandable dependence on ATP, the enzyme activation depends on increased intracellular Ca 2§ concentration [25]. Indeed, the presence or absence of EGTA and the corresponding variation in the level of cytosolic free Ca 2§ markedly af-
PTH
6A, B. Comparison of the effects of PTH and 5-HT on calcium current. A Traces o f / C a recorded from the same cell in control conditions and after application of 10 ~tM serotonin creatinine phosphate (5-HT) or 0.1 ~tM rPTH(1-34). Current curves are presented in the time sequence of their recording. Intracellular solution contained 10 mM EGTA. B Time course of peak Ica changes in another cell. PTH (0.1 nM) and 5-HT (10 gM) applications are marked by horizontal bars. Intracellular EGTA-free solution contained 2 mM ATP. Holding potential, - 50 mV; test potentials, +20 mV
fected the magnitude of the PTH-induced effect. The potentiating action of PTH was especially well expressed when free calcium in the intracellular solutions was stabilized at the level of about 10 -6 M. Such levels of intracellular calcium can be reached in physiological conditions during repetitive activity of snail neurones [23]. It is also known that in an inactive state, the enzyme exists in a soluble form in cytoplasm, and its physical translocation from cytosol into membrane occurs only after the activation. The observed dependence of the effect on the time of the hormone application during intracellular perfusion (see also similar finding by DeRiemer et al. [7]) can be explained, if possible degradation or washout of the soluble enzyme or the corresponding second messengers from the cell are considered. It has been shown that protein phosphorylation by PK-C is relatively resistant to dephosphorylation [21]. Because of this, cellular effects of phosphorylation mediated by PK-C are suggested to last much longer than those mediated by cyclic nucleotide-dependent protein kinases. This is consistent with our observation of slow development and poor reversibility of the hormonal effect. Finally, one of the most important evidence in favour of PK-C activation being the main step in the effect development is that the effect could be mimicked by the tumour-promoting phorbol ester but not by its inactive analoque. Contrary to physiological activators of PK-C, phorbol ester is hardly degraded [25]; this may explain the more stable potentiation o f / c a under its action. Our findings with PK-C dependent up-regulation of calcium channels in H. pomatia neurones agree with those by DeRiemer et al. [7] and Strong et al. [31] and contradict those by Hammond et al. [17] obtained on some identified neurones. In the latter study, PK-C activation mediated the inhibitory influence on the calcium current of cholecystokinin application. Probably, some specific features of the interaction between PK-C and calcium channels may exist is certain snail neurones. l The described effect of PTH is another example of receptor-mediated modulation of/Ca in nerve cells involv1 Recently a paper appeared by Wang et al. [32] describing effective potentiation of calcium currents by PTH in rat ventricular and smooth muscle cells. In this case the effect seems to be mediated by cAMPdependent mechanisms
152
ing PK-C. The results presented in this communication and those obtained earlier show that the calcium channels in molluscan neurones can be regulated through two different metabolic pathways - cyclic nucleotides and inositol phospholipids. It is not clear yet whether this double regulation may affect one and the same channel or different types of channels. The two types of channel regulations may be expressed differently in different neurones, adding more flexibility to their functional activity. There are no data yet if PTH-like substance is synthetized in molluscs and plays a physiological role in the function of its nervous system. It should be noted that recently widely distributed PTH immunoreactivity has been detected in vertebrate brain [27], indicating possible important role of PTH-like substances in brain functioning.
14.
15.
16.
17.
18.
19.
References 20. 1. Arieff AI, Massry SG (1974) Calcium metabolism of brain in acute renal failure: effect of uremia, hemodialysis and parathyroid hormone. J Clin Invest 53:387-392 2. Byerly L, Yazejian B (1986) Intracellular factors for the maintenance of calcium currents in perfused neurones from the snail, Lymnea stagnalis. J Physiol (Lond) 370:631-650 3. Castagna M, Takai Y, Kaibushi K, Sano K, Kikkawa U, Nishizuka Y (1982) Direct activation of calcium activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 257:7847- 7851 4. Chesnoy-Marchais D, Fritsch J (1989) Chloride current activated by cyclic AMP and parathyroid hormone in rat osteoblasts. Pflttgers Arch 415:101 - 104 5. Civitelly R, Reid IR, Westbrook S, Avioli LV, Hruska RA (1988) PTH elevates inositol polyphosphates and diacylglycerol in a rat osteoblast-like cell line. Am J Physiol 255:E660-E667 6. Conn PJ, Strong JA, Azhderian EM, Nairn AC, Greengard P, Kaczmarek LK (1989) Protein kinase inhibitors selectively block phorbol-ester- or forskolin-induced changes in excitability of Aplysia neurons. J Neurosci 9:473-479 7. DeRiemer SA, Strong JA, Albert KA, Greengard P, Kaczmarek LK (1985) Enhancement of calcium current in Aplysia neurons by phorbol ester and protein kinase. C Nature 313:313-316 8. Doroshenko PA, Kostyuk PG (1987) Enhancement of calcium current in the somatic membrane of snail nerve cells by phorbol ester. Biol Membr (Moscow) 4:1160-1163 9. Doroshenko PA, Kostyuk PG, Martynyuk AE (1982) Intracellular metabolism of adenosine 3,5-cyclic monophosphate and calcium current in perfused neurones of Helix pomatia. Neuroscience 7:2125-2134 I 0. Doroshenko PA, Kostyuk PG, Martynyuk AE, Vorobetz ZD (1984) Intracellular protein kinase and calcium inward current in perfused neurones of the snail Helix pomatia. Neuroscience 11:263 - 267 11. Eckert R, Chad JE, Kalman D (1986) Enzymatic regulation of calcium current in dialyzed and intact molluscan neurones of Aplysia californica. J Physiol (Paris) 81:318-324 12. Fraser, CL, Sarnacki P, Budayr A (1988) Evidence that parathyroid hormone-mediated calcium transport in rat brain synaptosomes is independent of cyclic adenosine monophosphate. J Clin Invest 81:982-988 13. Gerschenfeld HM, Hammond C, Paupardin-Tritsch D (1986) Mod-
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