Inactivation of 45Ca2+ uptake by prior depolarization of PC12 cells

Neuroscience Letters, 62 (1985) 377-381 Elsevier Scientific Publishers Ireland Ltd.

377

NSL 03693

INACTIVATION OF 45Ca2+ UPTAKE BY PRIOR DEPOLARIZATION OF PCI2 CELLS

D A V I D A. G R E E N B E R G * , C E L I A L. C A R P E N T E R and R O B E R T O. M E S S I N G

Department ( f Neurology, University ()f California, and Ernest Gallo Clinic and Research Center, San Francisco General Hospital, San Francisco, CA 94110 (U.S.A.) (Received May 27th, 1985; Revised version received August 19th, 1985; Accepted August 23rd, 1985)

Key words:

Ca ~ channel tion

45Ca2+ uptake

inactivation

PC12 cell

Ca 2+ channel agonist

depolariza-

45Ca2' uptake evoked by depolarization of PCI2 pheochromocytoma cells with K ÷ was reduced approximately 90"~, by prior depolarization in Ca2+-containing medium. Prior depolarization without added Ca 2 ~ reduced 45Ca3~ uptake by only about 20°/,,. The Ca 2~ channel agonists, BAY K 8644 and CGP 28392, had no effect on inactivation of 45Ca2+ uptake. These findings suggest that (I) voltage-gated Ca 2+ channels of PC12 cells undergo inactivation, (2) inactivation is Ca2+-dependent rather than voltage-dependent, and (3) Ca 2 ~ channel agonists do not promote Ca 2' flux by inhibiting Ca 2~ channel inactivation.

Voltage-gated Ca 2+ channels allow Ca 2+ to enter excitable cells, where it regulates neurotransmitter release, muscle contraction, ion channel gating and enzymatic reactions. The effects of Ca 2+ influx are limited by intracellular mechanisms for buffering and sequestering Ca 2+ and by a decline in inward Ca 2+ current. A major cause of this decline is Ca 2+ channel inactivation manifested by decay of Ca 2+ current during sustained depolarization and reduction of peak current with consecutive depolarizations [5]. Electrophysiologic studies reveal at least two modes of Ca 2+ channel inactivation, i.e. 'voltage-dependent' inactivation resulting from membrane depolarization [8, 1 1, 15] and 'Ca2+-dependent ' inactivation due to intracellular accumulation of Ca 2+ [6, 7, 18]. Ca 2~ channel function can be investigated by neurochemical methods based on measurement of 45Ca2+ uptake evoked by K '-induced depolarization of brain synaptosomes [16, 20] or cultured neural cells [9, 17, 19]. We and others have used the rat PC12 p h e o c h r o m o c y t o m a cell line, which expresses electrophysiologically demonstrable Ca 2+ currents [2], to study Ca 2+ channel physiology and pharmacology by the 45Ca2 + uptake method [10, 17, 19]. K +-stimulated 4 5 C a 2 + uptake into PC 12 cells exhibits properties of voltage-gated Ca 2+ flux, including dependence on [Ca2+], inhibition by Ca 2+ channel antagonists, and enhancement by Ca 2+ channel agonists [10, 17, 19]. *Author for correspondence at: Department of Neurology, University of California, Building 1. Room 101, San Francisco General Hospital, San Francisco, CA 94110, U.S.A.

378

Prior depolarization of synaptosomes [16, 20] or clonal neuroblastoma cells [9] with elevated K ~ reduces 45Ca2+ uptake during a subsequent depolarization, and it has been proposed that this may correspond to Ca 2+ channel inactivation observed in electrophysioiogic preparations [9, 16, 20]. Despite the widespread use of the PC I2 cell line to investigate Ca `'+ channel function and drug interactions [10, 17, 19], little is known about Ca 2+ channel inactivation in these cells. Therefore, we investigated the effect of prior depolarization on K'-stimulated 45Ca2+ uptake into PC12 cells, the relative roles of depolarization per se and Ca 2+ influx in mediating this effect. and possible influences of drugs and toxins on inactivation of 45Ca2+ uptake. PC I2 monolayers, grown as described elsewhere [10], were pre-incubated at 2 2 C for 0-15 min in 2 ml of 5K buffer (5 mM KCI, 45 mM choline-Cl, 85 mM NaC1, 2 mM CaC12, 5 mM glucose and 50 mM HEPES; pH 7.4). For the remainder of the 15-min pre-incubation period, 5K buffer was replaced with buffer in which the concentration of KC1 was raised to 50 mM and choline-Cl was omitted (50K buffer). To assay 45Ca:+ uptake, pre-incubation buffer was replaced with 2 ml of 5K or 50K incubation buffer containing 0.75/~Ci of 45Ca2+ (17-36 mCi/mg Ca; New England Nuclear), and drugs or toxins where indicated. Cells were incubated at 22'C for 2-20 min, then rinsed rapidly 3 times with 2 ml of ice-cold 5K buffer. The 45Ca2 + content [10] and protein concentration [14] were assayed as previously described. Stimulated 45Ca2+ uptake was defined as the difference between uptake in 5K and 50K buffers. Results are expressed as means __+ S.D. from the indicated number of triplicate experiments. When cells were pre-incubated for 15 min in 5K buffer and then placed in either 5K or 50K buffer containing 45CAC!2, 45Ca2+ uptake was markedly enhanced at the higher (depolarizing) K ~ concentration (Fig. 1A). Pre-incubation for 15 min in 50K instead of 5K buffer reduced stimulated uptake by 88___4~o ( n = 1 4 ) and 82+3°~i (n = 3) at 2 and 20 min of incubation, respectively. Reduction of uptake following prior depolarization was not due to non-specific injury to cells since cell viability (assessed by trypan blue exclusion) and unstimulated (5K) 45Ca2+ uptake were unaltered by this treatment. To examine the time-course of inactivation of 45Ca2+ uptake, cells were pre-incubated for 0-15 min in 5K buffer and for the remainder of 15 min in 50K buffer. As the time of 50K pre-incubation increased, the ability of a subsequent 50K incubation to stimulate 45Ca2+ uptake declined, with half-maximal inactivation at about 1 min of 50K pre-incubation (Fig. 1B). To investigate the role of Ca 2+ influx in the inactivation o f 45Ca2+ uptake produced by prior depolarization of PC12 cells, prior depolarization was conducted in 50K buffer without added CaCI2. Under these conditions, the extent of inactivation of 45Ca2+ uptake was markedly diminished (Table I), suggesting that the observed effect of prior depolarization is Ca2+-dependent. Depression of 45Ca2+ uptake by prior depolarization could result not only from C a 2~ channel inactivation but also from activation of outward K ~ current, which promotes membrane repolarization [5]. This possibility was examined by conducting some experiments in the presence of the K ~ channel blocker, tetraethylammonium

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Fig. I. A: effect of 50K pre-incubation on 45Ca2+ uptake. Cells were pre-incubated in 5K (O, 0 ) or 50K (A, A) buffer for 15 min, then incubated with ~Ca 2~ in 5K (C), A) or 50K ( 0 , A) buffer for the indicated times. B: time-course of inactivation of45Ca 2~ uptake. Cells were pre-incubated in 50K buffer for the indicated times, then incubated for 2 min with 45Ca-" ' in 5K (A) or 50K (A) buffer. Inset: symbols ( I ) denote stimulated (50K minus 5K) uptake as a percent of stimulated uptake at 0 rain of 50K pre-incubation). The semilogarithmic plot was constructed by linear regression analysis. Data shown are means + S.E.M. from 3 experiments, each performed in triplicate; error bars are omitted where they do not extend beyond the symbol.

TABLE 1 EFFECT OF P R E - I N C U B A T I O N C O N D I T I O N S ON I N A C T I V A T I O N OF 4'Ca2~ UPTAKE INTO PCI2 ('ELLS Cells were pre-incubated for 15 min in 5 mM K' (5K) or 50 mM K + (50K) buffer with or without added drugs, toxins and CaCI?, then incubated for 2 rain in 5K or 50K buffer containing 2 mM 45CaC1~ (0.75 l~('i of 4~Ca-~ ' ). 'Percent inactivation' is the percent decrease in stimulated (50K minus 5K) 4~Ca2 ~ uptake produced by pre-incubation in 50K rather than 5K buffer. Values given are means + S.D. from 3 14 experiments, each performed in triplicate. AsTX, Anemonia suh'ata toxin 11: LqV, Leiurus qu#lquestrialtt.~ venom. *P < 0.001 relative to no drug, 2 mM CaCL, condition (one-way ANOVA). Additions to pre-incubation buffer

Percent

. . l)rug or toxin

. CaClz (mM)

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380 (TEA). TEA failed to prevent the reduction in 45Ca2+ uptake produced by prior depolarization (Table I), indicating that activation of K + efflux was not the cause of this reduction. Sea anemone toxin and North African scorpion venom inhibit inactivation of voltage-gated Na- channels [4]. In contrast, prior depolarization of PC I2 cells in the presence of either of these agents failed to modify inactivation of 45Ca2÷ uptake (Table I). The Na ~ channel blocker, tetrodotoxin (TTX), also had little effect on the extent to which 45Ca2+ uptake was reduced by prior depolarization (Table I). Finally, we examined whether the Ca 2~ channel agonists BAY K 8644 and CGP 28392, which prolong Ca 2~ channel opening [3, 12, 13], might do so by inhibiting activation. At 1 #M, a concentration that maximally enhances Ca 2+ current [3], neither drug interfered with the reduction of 45Ca2+ uptake caused by prior depolarization (Table I). The major finding of this study is that prior depolarization of PC 12 cells results in Ca2+-dependent inactivation of depolarization-evoked 45Ca2+ uptake. In several respects, this phenomenon resembles the inactivation of voltage-gated Ca 2+ channels seen in electrophysiologic preparations [5]. One notable difference is the slow timecourse of inactivation of "sCa 2+ uptake in PC12, which has also been observed in clonal neuroblastoma cells [9], and is consistent with existing evidence for Ca 2+ channel subtypes differing in inactivation kinetics [1]. The failure of Ca 2+ channel agonists to alter the effect of prior depolarization suggests that these drugs do not enhance voltage-gated Ca 2+ flux by inhibiting channel inactivation. Our results indicate that the PC12 cell line may be useful in biochemical and pharmacologic investigations of conductance state-dependent C a 2 + channel function. Supported by Research Scientist Development Award AA00069 to D.A.G. and Research Fellowship Award AA05223 to R.O.M. Dr. Lawrence Toll supplied the original PC12 cell stocks, and drugs were provided by Miles Labs. (BAY K 8644) and Ciba-Geigy (CGP 28392). We thank Dr. Michael Charness for valuable discussions and for reviewing the manuscript. 1 Armstrong, C.M. and Matteson, D.R., Two distinct populations of calcium channels in a clonal line of pituitary cells, Science, 227 (1985) 65-67. 2 Brown, A.M., Camerer, H., Kunze, D.L. and Lux, H.D., Similarity of unitary Ca2. currents in three different species, Nature (London), 299 (1982) 156-158. 3 Brown, A.M,, Kunze, D.L. and Yatani, A., The agonist effect of dihydropyridines on Ca channels, Nature (London), 311 (1984) 570-572. 4 Catterall, W . A . , The molecular basis of neuronal excitability, Science, 223 (1984) 653-661. 5 Eckert, R. and Chad, J.E., Inactivation of Ca channels, Prog. Biophys. Molec. Biol.. 44 (1984) 215 267. 6 Eckert, R. and Ewald, D., Residual calcium ions depress activation of calcium-dependent current, Science, 216 (1982) 730-733. 7 Eckert, R. and Tillotson, D.L., Calcium-mediated inactivation of the calcium conductancein caesiumloaded giant neurones of Aplysia californica, J. Physiol. (London), 314 (1981) 265-280. 8 Fox, A.P., Voltage-dependent inactivation of a calcium channel, Proc. Natl. Acad. Sci., USA, 78 (1981) 953-956.

381 9 Freedman, S.B., Dawson, G., Villereal, M.L. and Miller, R.J., Identification and characterization of voltage-sensitive calcium channels in neuronal clonal cell lines, J. Neurosci., 4 (1984) 1453- 1467. 10 Greenberg, D.A., Carpenter, C.L. and Cooper, E.C., Stimulation of calcium uptake into PCI2 cells by the dihydropyridine agonist BAY K 8644, J. Neurochem., 45 (1985) 990-993. II Hagiwara, S., Ozawa, S. and Sand, O., Voltage clamp analysis-of two inward current mechanisms in the egg cell membrane of a starfish, J. Gen. Physiol., 65 (1975) 617-644. 12 Hess, P., Lansman, J.B. and Tsien, R.W., Different modes of Ca channel gating favoured by dihydropyridine Ca agonists and antagonists, Nature (London), 311 (1984) 538-544. 13 Kokubun, S., and Reuter, H., Dihydropyridine derivatives prolong the open state of Ca channels in cultured cardiac cells, Proc. Natl. Acad. Sci. USA, 81 (1984) 4824~4827. 14 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265- 275. 15 Lux, H.D. and Brown, A.M., Single channel studies on inactivation of calcium currents, Science, 225 (1984) 432-434. 16 Nachsen, D.A. and Blaustein, M.P., Some properties of potassium-stimulated calcium influx in presynaptic nerve endings, J. Gen. Physiol., 76 (1980) 709 728. 17 Stallcup, W.B., Sodium and calcium fluxes in a clonal nerve cell line, J. Physiol. (London), 286 (1979) 525 540. 18 Tillotson, D., Inactivation of Ca conductance dependent on entry of Ca ions in molluscan neurons, Proc. Natl. Acad. Sci. USA, 76(1979) 1497 1500. 19 Toll, L., Calcium antagonists: high-affinity binding and inhibition of calcium transport in a clonal cell line, J Biol. Chem., 257 (1982) 13189 13192. 20 Turner, T.J. and Goldin, S.M., Calcium channels in rat brain synaptosomes: identification and pharmacological characterization, J. Neurosci., 5 (1985) 841-849.