Control of calcium channels in neuroblastoma cells (N1E-115)

Experimental Gerontology, Vol. 25, pp. 247-253, 1990 Printedin the USA. All rightsreserved.

0531-5565/90 $3.00 + ,00 Copyright© 1990PergamonPressplc

CONTROL OF CALCIUM CHANNELS IN NEUROBLASTOMA CELLS (N1E-115)

P . K . T . PANG, R. WANG, L.Y. W u , E. KARPINSKI, J. SHAN and C.G. BENISHIN Department of Physiology, 7-55 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Abstract - - Neuroblastoma cells (N1E-115) were used as models of transient (T) and

long-lasting (L) Ca ++ channels: The whole cell version of the patch clamp technique was used to measure inward Ca ++ currents, and the fluorescent indicator, Fura-2, was used to measure changes in intracellular Ca ++. Cells were cultured and selected during recording so that predominately T or L channel currents were measured. T channel currents did not respond to dihydropyridine or parathyroid hormone, whereas L channel currents did. BAY-K-8644 increased and nifedipine decreased L channel currents. After a 15 mM KC1 challenge, cells with predominately T channels responded with a ffansient change in intracellular Ca ++, while cells with 9redominately L channels showed a sustained response. PTH inhibited the increase in intracellular Ca ++ in cells with L channels, but not in those with T channels. PTH may be an example of an endogenous calcium channel blocker, at least in neuroblastoma cells. Key Words: calcium channels, neuroblastoma cells, PTH

INTRODUCTION NUMEROUS INVESTIGATORShave demonstrated the importance of calcium as a second messenger in the regulation of cellular physiological processes. A n increase or decrease of intracellular free calcium concentration ([Ca]i) can be utilized as a signal to alter biochemical events in cells. The resting [Ca] i is in the range of 100-200 nM. However, extracellular concentration of ionic calcium ([Ca]o) is in the range of 1-1.5 mM. There is, therefore, a several-thousand-fold difference between [Ca]i and [Ca] o. Such a large concentration gradient can be maintained only if the cell membrane is relatively impermeable to calcium. How, then, is [Ca]i changed according to the physiological requirements of the cell in signal transduction? One answer is that there is an intracellular calcium store which is readily mobilized: The movement of calcium in and out o f such a storage pool will decrease or increase [Ca] i accordingly. There have been many studies on the regulation and mechanism of calcium exchange in such storage sites. In addition, there are active processes on ~ e cell membrane which will move calcium from the

Correspondence to: P.K.T. Pang. 247

248

P.K.T. PANG et al.

inside to the outside of the cell against the steep concentration gradient. Finally, there are special passages for calcium to move from the outside to the inside of the cell down the concentration gradient. These passages are called calcium channels, and they play an important role in the determination of [Ca] i. These channels are, therefore, tightly controlled, and an abnormality in calcium channel activity can result in various pathological conditions. The importance of such channels becomes obvious when one considers the recent development of the class of synthetic drugs called calcium channel blockers. These drugs can block the entry of calcium through calcium channels and are used extensively in the treatment of cardiovascular diseases. They are important therapeutic agents (Fleckenstein, 1983; Buhler and Kiowski, 1987), as well as research tools. The importance of calcium channels in physiological and pathophysiological regulation of [Ca] i is, therefore, well recognized. The authors have hypothesized that such an important cellular process must be precisely regulated by endogenous regulators. Hormones are likely candidates for this function (Pang, 1990). The authors have begun studies en the identification and characterization of endogenous calcium channel regulators. The hormones chosen for our initial investigations are the calcium regulatory hormones, with speci_~c emphasis on parathyroid hormone (PTH). There are two reasons for such a choice. First, in studies on the vascular action of PTH, it has been reported that PTH, in a manner similar to known synthetic calcium entry blockers, could inhibit calcium entry in vascular tissue stimulated by KCI, arginine vasopressin, and BAY-K-8644 (Schleiffer etal., 1979; Pang et eL, 1984, 1988). Paradoxically, PTH has a stimulatory effect on cardiac cells (Katoh etaL, 1981), probably through stimulation of calcium channels (Kondo e t a l . , 1988). These data suggest that PTH may be a good choice for studying the control of calcium channels by endogenous hormones. Secondly, PTH and other calcium regulating hormones such as calcitonin and vitamin D metabolites are well characterized in their roles for maintaining stable plasma calcium concentrations. It is known that these hormones regulate the absorption of calcium through the gut. the deposition and reabsorption of calcium in bone. and the excretion of calcium by the kidney. There is, however, little or no information available on the effect of hormones on the utilization of calcium. Since calcium is used so extensively by cells as a second messenger, the regulation of plasma calcium levels should also involve the regulation of calcium utilization by cells. Calcium-regulating hormones should, therefore, logically be involved in the regulation of cell membrane calcium channels and. hence, calcium utilization in the body, in addition to overall calcium absorption and excretion. In fact. PTH has been reported to increase calcium content in tissues such as heart (Bogin et alo, 1981), red blood cells (Zemel et al.. 1989), kidney (Bode and Uchikawa, 1978), and brain (Goldstein and Massry, t978). It is for the two preceding reasons that the authors chose PTH to study the effect of hormones on calcium channels in cells. MATERIALS AND METHODS In this communication, the effect of PTH on calcium channel activities is described in neuroblastoma cells (N1E-115). Calcium channel currents were measured directly using the whole cell version of the patch clamp technique. Inward calcium channel currents, with 20 mM Ba ~-~- as the charge carrier, were measured using a List-EPC-7 patch clamp amplifier. Calcium channel currents were corrected for leakage currents by digital subtraction of scaled, averaged hyperpolarizing pulses. To avoid contamination of inward calcium channel currents by other ionic currents, standard techniques were employed to block both K -~ and Na ~- channels, The bath solution was maintained at room temperature (20 to 22 °C). In order to ascertain that the

CALCIUMCHANNELSINNEUROBLASTOMACELLS

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F~6. 1. The effect of BAY-K-8644 on T and L channel currents. A. The effect of BAY-K-8644 on T channel currents. The I-V plot was taken from the data of one cell (holding potential - 80 mV). Both the amplitude and the I-V relationship of the inwm'dcurrent were not changed by BAY-K-8644. B. The amplification of L channel currents by BAY-K-8644. With a holding potential of - 4 0 mV and a threshold potential for activation of about - 30 mV, the peak amplitude of currents was +20 or + 10 mV (see text). BAY-K-8644 amplified the inward current by more than two times and produced a shift of the I-V plot towards more negative potentials by 20 mV. In both A and B, the leakage and capacitive currents have been subtracted.

change in calcium channel activities resulted in a detectable change in [Ca] i, the Fura-2 method (Grynkiewicz et al., 1985) was used to determine [Ca]i. Cells were incubated for 45 min in D M E M containing 10 I~M Fura-2 acetoxymethylester (Fura-2 AM) at room temperature. During the incubation, cells were kept in tile dark. The cells were gently washed five times with 5 m M K buffer (composition: 145 m M NaC1, 5 m M KC1, 1 m M MgC12, 10 m M glucose, 1 m M CaC12, 0.5 m M NaH2CO 4, 10 m M HEPES, p H 7.4) and kept in the same buffer. After about 5 rain, the coverslip with attached cells was placed in a 1-mL Sykes-Moore chamber on the stage o f an inverted microscope and fluorescence measurements were made with a Fluoroplex III spectrofluorimeter (Tracor Northern, Middleton, WI). Neuroblastoma cells were chosen because these cells can be easily maintained and studied using the patch clamp technique. The calcium channels have also been well characterized in these cells (Narahashi et al., 1987). There are two types of calcium channels w h i c h are distinguishable electrophysiol0gically and pharmacologically. The long-lasting, or L channel

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FIa, 2. The effect of nifedipine on T and L channel currents. A. The effect of nifedipine on T channel currents. The I-V plot was taken from the data of one cell (holding potential - 80 mV/. The inward current was activated at - 60 mV and was maximal at - i0 mV. After the control I-V plot was obtained, the addition of nifedipine changed neither the amplitude nor the I-V relationship of the inward current. B. The inhibition of L channel currents by nifedipine, The I-V plot was constructed from the data of another cell. The holding potential was set at - 4 0 mV. The L channel current appeared at - 20 mV and peaked at + 10 mV. Nifedipine decreased the amplitude of the channel current to 60% of the control value within 5 min. In both A and B, the leakage and capacitive currents have been subtracted.

c u r r e n t , is a c t i v a t e d at m o r e p o s i t i v e p o t e n t i a l s a n d s h o w s a p e a k i n w a r d c u r r e n t a b o v e + 10 m V . It is a c t i v a t e d a n d i n h i b i t e d b y t h e d i h y d r o p y r i d i n e s . B A Y - K - 8 6 4 4 , a n d n i f e d i p i n e , r e s p e c t i v e l y . T h e t r a n s i e n t , or T c h a n n e l c u r r e n t , is a c t i v a t e d at m o r e n e g a t i v e p o t e n t i a l s a n d s h o w s a p e a k i n w a r d c u r r e n t at - 2 0 m V . It is n o t i n f l u e n c e d b y B A Y - K - 8 6 4 4 o r n i f e d i p i n e . I n m o s t n e u r o b l a s t o m a cells, b o t h t y p e s o f c h a n n e l s are p r e s e n t at t h e s a m e time. I n t h e i r l a b o r a t o r y , the a u t h o r s h a v e b e e n able m c u l t u r e cells to e x p r e s s p r e d o m i n a t e l y T o r L c h a n n e l s . Cells c u l t u r e d in the n o r m a l m e d i u m s h o w e d p r e d o m i n a t e l y , T c h a n n e l s , a n d cells e x p o s e d to 2 % D M S O f o r 1 m o n t h s h o w e d p r e d o m i n a t e l y L c h a n n e l s . F o r the c o n v e n i e n c e o f d e s c r i p t i o n , t h e s e cells are c a l l e d T o r L c h a n n e l cells i n this paper. In o r d e r to b e sure t h a t t h e T a n d L c h a n n e l s s h o w t h e s a m e e l e c t r o p h y s i o l o g i c a l a n d p h a r m a c o l o g i c a l f e a t u r e s as h a v e b e e n r e p o r t e d ( Z e m e l et al., 1989), t h e s e cells w e r e first c h a r a c t e r i z e d .

251

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FIc. 3. [Ca] i in neuroblastoma cells which have been depolarized with 15 mM KC1. [Ca]i has been measured using the Fura-2 method. The ratio of the emitted fluorescence at the two excitation wavelengths (340 and 380 nM) is an indication of [Ca] i. A. Changes in [Ca] i due to KC1 stimulation in L channel cells. B. Changes in [Ca]i due to KC1 stimulation in T channel cells.

RESULTS T channel currents were inactivated in approximately 15 ms. As can be seen from the I-V relationship curve, the peak inward current appeared around - 20 mV (Fig. 1A). BAY-K-8644 had no effect on this current. The L channel cells, which were grown in 2% DMSO, exhibited inward currents which showed little or no inactivation up to 100 ms. In the I-V relationship curve, the peak current appeared around + 10 mV (Fig. 1B). These currents were increased by BAY-K-8644 (Fig. IB). Figure 2A shows that nifedipine (0.1 mM) had no effect on T channel currents. Figure 2B shows the inhibitory effect of a similar concentration of nifedipine on L channel currents. Depolarization of the T and L channel cells with KC1 resulted in an increase in [Ca] i as determined by the Fura-2 method. Figure 3A shows the KCl-stimulated long-lasting increase in [Ca] i in L channel, and Fig. 3B shows the transient increase in [Ca] i in T channel cells. The authors are, therefore, confident that the cells studied contained channels similar to those reported by others as T and L channels. The effects of the active synthetic bovine PTH fragment containing the N-terminal 1-34 amino acids [bPTH-(1-34)] (Bachem) were then investigated. In T channel cells, the T channels TABLE. I. EFFECTSOF bPTH-(I-34) ON T AND L CHANNEL CURREm'S T Channels (n = 8)

Control bPTH-(1-34) BAY-K-8644

L Channels (n = 9)

Vpe,~k (mV)

]peak (pA)

Vpeak (mV)

Ipe,,k (pA)

- 1 2 . 5 ± 2.5 - 1 1 . 3 ± 2.3 - 1 8 . 8 -4- 2.3

181 - 34 170 ± 37 177 ± 36

3.3 ± 3.3 - 4 . 4 ± 4.7 -5 ± 5

45 ± 6 32 ± 5 a 58 ± 8 b

abPTH-(1-34) (1)xM) significantly decreased L channel currents (p < "0.05, Student's t-test). bBAY-K-8644 (5 p,M) significantly increased L channel currents after inhibition by bPTH-(1-34) (p < 0.05, Student's t-test).

252

P,K.T. PANG et al. TABLE 2, EFFECT OF P T H ON K C I - s T I M U L A T E D INCREASEIN INTRACELLULAR FREE CALCIUMCONCENTRATIONS IN ~7~

Cells with T channels Cells with L channels

KCI (15 mM)

PTH (1 g~g/ml) + KCl (15 raM)

100 100

99.0 + 3.3 (n = 4) 12.1 -+ 10.0 (n = 5)*

*Statistically significant (p < 0.05. Student's t-test).

were first identified and bPTH-(1-34) was then added to the bath. No effect was observed up to 10 rain. Lanthanum, a nonspecific divalent ion blocking calcium channels, was added and the T current was completely blocked, as expected. In L channel cells, the L channels were again first identified and bPTH-(l-34) was added. The L channel activity was inhibited. To show that the decrease in channel activity was not due to a normal channel rundown phenomenon. BAY-K-8644 was subsequently added to these inhibited cells. L channel inward currents were reestablished. Addition of lanthanum again completely blocked these currents, indicating that these are indeed calcium channel currents. These data are summarized in Table 1. To show that the effect on channel activities could be correlated with changes in intracellular free ealcium concentration, the effect of bPTH-(1-34) on [Ca] i was determined in T and L channel cells with the Fura-2 method. In cells with predominately T channels. KC1 increased [Ca]~ and bPTH-(1-34) had no effect on the increase. However. in the cells with predominatety k channels, KC1 also increased [Ca]i and this increase was inhibited by bPTH-(1-34). Table 2 summarizes the data. DISCUSSION The data strongly suggest that PTH can inhibit calcium channels in neuroblastoma cells. This effect is specific for the L, but not the T channels, as in the case of the synthetic dihydropyridines. More importantly, these channel inhibitory effects can be correlated with the effect of PTH on the stimulated increase of [Ca] i by cell depolarization. This correlation indicates the functional significance of such a channel inhibitory effect. Measurement of channel activity with the patch clamp method is conducted under rather abnormal conditions. The sodium and potassium channels are usually inhibited with tetrodotoxin and TEA. The patch pipette contains EGTA, and [Ca]i is continuously decreased. Instead of calcium, barium at a high concentration is added to the external medium as the charge carrier. An effect on the channels under such artificial conditions is difficult to interpret in terms of the normal functions of the cells. A similar effect on [Ca]i by the test substance would provide strong confirmation of the ultimate effect of the substance, such as an effect on [Ca]i through its effect on calcium channels. In many studies, only patch clamp data or [Ca]i were given. Data on [Call alone wi/1 not necessarily reflect effects on channels. The present studies also strongly support the hypothesis that cell membrane calcium channels are so important in the regulation of [Ca]i that there must be endogenous calcium channel regulators. Hormones, especially those involved in plasma calcium level regulation, are good candidates. PTH is such a hormone. It will be important to extend these studies to other calcium regulatory hormones and other hormones in general. PTH may not be the only calcium channel modulator. It may be one of many to be identified.

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REFERENCES BOGIN, E., MASSRY, S.G., and HARARY, I. Effect of parathyroid hormone on heart cells. J. Clin. Invest. 67, 1215-1227, 1981. BORLE, A.B. and UCH1KAWA, T. Effects of parathyroid hormone on the distribution and transport of calcium in cultured 16dney ceils. Endocrinology 102, 1725-1732, 1978. BUHLER, F.R. and KIOWSKI, W. Calcium antagonists in hypertension. J. Hypertens. 5(Suppl. 3), $3-S10, 1987. FLECKENSTEIN, A. History of calcium antagonists. Circ. Res. 52(Suppl. 1), 113-116,1983. GOLDSTEIN, D.A. and MASSRY, S.G. Effect of parathyroid hormone administration and its withdrawal on brain calcium and electroencephalogram. Miner. Electrolyte Metab. 1, 84-91, 1978. GRYNKIEWICZ, G., POENIE, M., and TSIEN, R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440-3450, 1985. KATOH, Y., KLEIN, K.L., KAPLAN, R.A., SANBORN, W.G., and KUROKAWA, K. Parathyroid hormone has a positive inotropic action in the rat. Endocrinology 107, 2252-2254, 1981. KONDO, N., SHIBATA, S., TENNER, T.E., JR., and PANG, P.K.T. Electromechanical effects of bPTH-(1-34) on rabbit sinus node cells and guinea pig papillary muscles. J. Cardiovasc. Pharmacol. 11, 619-625, 1988. NARAHASHI, T., TSUNOO, A., and YOSHII, M. Characterization of two types of calcium channels in mouse neuroblastoma cells. J. Physiol. 383, 231-249,1987. PANG, P.K.T. Recent advances in the study of the vascular action of parathyroid hormone. In: Proceedings of the First International Conference on New Actions of Parathyroid Hormone, Kobe, Japan, 1990. PANG, P.K.T., YANG, M.C.M., and SHAM, J.S.K. Parathyroid hormone and calcium entry blockade in a vascular tissue. Life Sci. 42, 1385-1400, 1988. PANG, P.K.T., ZHANG, R.H., and YANG, M.C.M. The hypotensive action of parathyroid hormone in chicken. J. Exp. ZooL 232, 691-697, 1984. SCHLEIFFER, R., BERTHELOT, A., and GAIRARD, A. Action of parathyroid extract on arterial blood pressure and on contraction and 4SCa exchange in isolated aorta of the rat. Eur. J. Pharmacol. 58, 163-167, 1979. ZEMEL, M.B., BEDFORD, B.A., STANDLEY, P.R., and SOWERS, J.R. Saline infusion causes rapid increase in parathyroid hormone and intracellular c~cium levels. Am. J. Hypertens. 2, 185'-187, 1989.