OLGA LARINA⁽¹⁾, Volodimir Manko^(2)
(1)DEPARTMENT OF HUMAN AND ANIMAL PHYSIOLOGY. I.FRANKO LVIV NATIONAL UNIVERSITY - LVIV . Ukraine
⁽²⁾Dept. Of Human and Animal Phisiology. I. Franko Lviv National University - Lviv. Ukraine

	[Endocrinology]	[Physiology]

INTRODUCTION

The Na⁺-Ca²⁺ exchanger is a countertransport mechanism located in the cell membrane. It was previously identified in heart muscle and in the squid giant axons more than thirty years ago (Reuter, Seitz, 1968; Baker et al., 1968). The Na⁺-Ca²⁺ exchanger was also found in cardiac muscle (Glitch et al., 1970), epithelia (Baker, 1972; Blaustein, 1974; Blaustein, Nelson, 1982), acinar cell plasma membrane (Bayerdoffer, Haase, Schultz, 1985; Grinkiv, Klevets, Shostakovskaya, 1988), salivary gland secretory cell membrane in Chironomus plumosus L. larva (Manko, Klevets, Fedirko, 1995; Klevets, Manko, Fedirko, 1995).

salivary gland secretory cell membrane Na⁺-Ca²⁺ exchange current in Chironomus plumosus L. larva was registered in response to the membrane hyperpolarization (Manko et al., 1995; Klevets et al., 1995). Slow inward current is evoked at E_Na=+54.6 mV and hyperpolarisating shifts (with duration 3-3.5 sec) of membrane potential from clamped level (-20 mV). It decreases at E_Na=0 mV and becomes outward at E_Na=-45 mV. Direction of current is determed by sign of Na⁺ transport motive force at clamped potential. This current depends on repolarising pulses value, [Ca²⁺]_e (Fedirko et al., 1995) and [Ca²⁺]_i that is why it was identified as current of Na⁺-Ca²⁺ exchange.

In spite of significant progress in the investigation of secretory cell membrane Na⁺-Ca²⁺ exchanger its structure and functional peculiarities is not thoroughly identified. So he investigation of these topics still remains actual. Full answer on questions about functional role of Na⁺-Ca²⁺ exchanger and its structure in certain tissue could be done only after discovery really specific blockators and modulators of Na⁺-Ca²⁺ exchange (Baker, 1986). That’s why investigations of blocators and mechanism of their effect on Na⁺-Ca²⁺ antiport system is inevitable step on the way to better understanding of exchanger’s structure and its functional role in secretory processes.

One of the well known modulators of Na⁺-Ca²⁺ exchange in different types of cells is level of pH. Cytoplasmic protons as previously reported is a potent inhibitor of Na⁺-Ca²⁺ exchange current in guinea-pig giant patch (Doering, Lederer, 1991 a,b). Moreover, Ca²⁺ transport mediated by Na⁺-Ca²⁺ exchange is very sensitive to change of pH (Doering, Lederer, 1993) and is modulated by it (Philipson et al., 1982). Alkalization of cytoplasmic solution from pH 7.2 to pH 8.0 evokes a large, biphasic increase in Na⁺-Ca²⁺ exchange current. A stepwise acidifying of intracellular pH (7.2 – 6.4) causes a biphasic, but monotonic decrease in Na⁺-Ca²⁺ exchange current in guinea-pig heart cells. In addition, the slowly developing block is dependent on the presence of Na⁺ on the intracellular side (Doering, Lederer, 1994).

Modulative effect of pH on Na⁺-Ca²⁺ exchange current was also discovered in secretory cells.

Preliminary studies of extracellular H⁺ effect on salivary gland secretory cell membrane Na⁺-Ca²⁺ exchanger in Chironomus plumosus L. larva showed, that acidifying of extracellular solution led to reduction of Na⁺-Ca²⁺ exchange current. Extracellular solution alkalization caused contrary effect (Klevets, Manko, Fedirko, 1996). In present report we examined the effect of intra- and extracellular H⁺ on salivary gland secretory cell membrane Na⁺-Ca²⁺ exchange and its dependence on Na⁺ and Ca²⁺ gradients.

 

 

METHODS

Experiments were performed on secretory cells of isolated salivary glands in Chironomus plumosus L. larva. The Na⁺-Ca²⁺ exchange current (I_Na(Ca)) was registered by means of voltage-clamp method in conditions of intracellular perfusion in response to the membrane hyperpolarization from –20 to –60 mV by rectangular pulses (3.5 sec, 0.1 Hz).

Solution for intracellular perfusion contained (in mmol/l): Tris-HCl, 130.14; NaCl, 16; glucose, 5.55; pH 7.0. Physiological extracellular solution contained (in mmol/l): NaCl, 136.9; KCl, 5.36; CaCl2, 1.76; MgCl2, 0.49; Tris-HCl, 0.20; glucose, 5.55; pH 7.2.

Alterations in extracellular Na⁺ concentration ([Na⁺]_e) were provided with equimolar substitution of NaCl with Tris-HCl. Changes in pH were produced by addition of HCl (in case of acidification to pH 5.0) and Tris-HCl in case of alkalization to pH 8, 8.5, 9 and 10.

RESULTS

Effect H⁺ on functioning of salivary gland secretory cell Na⁺-Ca²⁺ exchanger in Chironomus plumosus L. larva was investigated in conditions of simultaneous changes of intra- and extracellular pH level and its changes by turn.

acidification of extracellular solution from pH 7.2 to pH 5.0 evoked decrease of I_Na(Ca) amplitude from (3.72+/-0.43)*10^-10 A to (2.68+/-0.29)*10^-10 A, that is by (25.99+/-5.23)% (P<0.05, n=7) and according to preliminary studies (Klevets et al., 1996). The influence of intracellular solution acidification to pH_i 5.0 was compared in seven preparations. It was somewhat variable from preparation to preparation. In four cases we observed insignificant reduction of Na⁺-Ca²⁺ exchange current amplitude, and I_Na(Ca) amplitude didn’t change in three cases. Thus Na⁺-Ca²⁺ exchange current insignificantly reduced at average by (10.48+/-7.85)%. Statistically unauthentic decrease of Na⁺-Ca²⁺ exchange current amplitude was also evoked by simultaneous reduction of intra- and extracellular solution pH level.

Fig.1 shows the effect of alkalization on Na⁺-Ca²⁺ exchanger functioning. Increase of extracellular solution pH level from pH 7.2 to pH 9.0 led to increase of I_Na(Ca) amplitude (Fig.1) from (8.35+/-1.41)*10^-10 A to (11.03+/-1.83)*10^-10 A (i.e. by (32.02+/-6.13)% ; p<0.01, n=7). This change was reversible when physiological level of pH_e was restorated. Alkalization of intracellular solution evoked significant decrease of I_Na(Ca) amplitude by (66.70+/-7.05)% (p<0.01, n=7). I_Na(Ca) amplitude achieved (10.98+/-2.42)*10^-10 A after restoration of physiological pH_i level.

Simultaneous intra- and extracellular solution alkalization caused significant reduction of the amplitude of inward Na⁺-Ca²⁺ exchange current (by (77.65+/-6.82)%; p<0.01, n=7). Moreover, in two cases we registered reversible changes of current direction from inward to outward. The outward current amplitude was (6.88+/-2.06)*10^-10 A.

Gradual intracellular solution alkalization to pH 8.0, 8.5, 9.0 and 10.0 evoked I_Na(Ca) reduction by (4.03+/-5.15), (30.72+/-10.18), (59.96+/-12.21) and (74.17+/-10.69)%, respectively (see Fig. 1 B). Moreover, in two cases from six (at pH_i 9.0 and 10.0) we registered outward phase of I_Na(Ca) with amplitude (2.73+/-1.00)*10^-10 and (3.45+/-1.36)*10^-10 A, respectively.

The conclusion from these studies is that intracellular solution alkalization leads to significant decrease of salivary gland secretory cell membrane inward Na⁺-Ca²⁺ exchange current amplitude in Chironomus plumosus L. larva and in several cases to change of its direction. These changes could be connected with character of interactions between Na⁺ and Ca²⁺ ions and Na⁺-Ca²⁺ exchanger molecule. That’s why we examined how the influence of alkalization on Na⁺-Ca²⁺ exchanger functioning depends on changes in Na⁺ and Ca²⁺ gradients.

Fig.2 demonstrates how the influence of intracellular solution alkalization to pH 9.0 on Na⁺-Ca²⁺ exchange current is effected by Na⁺ gradient. According to results shown in Fig.2 the decrease of [Na⁺]_e leads to reduction of I_Na(Ca) amplitude at pH_i 9.0. intracellular solution alkalization to pH 9.0 at [Na⁺]_e 136.9, 68.4 and 16.0 mmol/l caused significant reduction of I_Na(Ca) amplitude by (50.05+/-6.60), (78.72+/-5.39) and (96.48+/-3.52)%, respectively. Moreover, alkalization to pH_i 9.0 at [Na⁺]_e 68.4 and 16 mmol/l evoked in three and five cases from six (respectively) biphasic current changes. In these cases amplitude of outward phase was (1.09+/-0.09)*10^-10 and (2.22+/-0.38)*10^-10 A, respectively.

In order to investigate how intracellular alkalization effects on Na⁺-Ca²⁺ exchange current depends on Ca²⁺ gradient we changed extracellular Ca²⁺ concentration. Preliminary studies of [Ca²⁺]_e influence on Na⁺-Ca²⁺ exchange current at physiological levels of pH showed that increase of [Ca²⁺]_e from 1 to 10 mmol/l evoked hyperbolic elevation of I_Na(Ca) amplitude (Fedirko, Klevets, Manko, 1997). As we discovered in this study (see Fig.3) intracellular solution alkalization to pH 9.0 in conditions of extracellular Ca²⁺ concentration change from 1.76 to 10 mmol/l led to significant decrease of Na⁺-Ca²⁺ exchange current amplitude by (55.57+/-5.39) and (72.38+/-10.22)%, respectively. Moreover, in two cases from seven at [Ca²⁺]_e 1.76 mmol/l, pH_i 9.0 and in three cases at [Ca²⁺]_e 10 mmol/l and pH_i 9.0 the biphasic changes of Na⁺-Ca²⁺ exchange current were observed. Amplitudes of outward phases were (2.27+/-0.61)*10^-10 and (3.38+/-0.27)*10^-10 A, respectively.

 

 

DISCUSSION

 

We found that salivary gland secretory cell Na⁺-Ca²⁺ exchange in Chironomus plumosus L. larva is sensitive not only to changes of extracellular pH level but also to changes of intracellular pH level.

In particular reduction of extracellular pH level evoked decrease of I_Na(Ca) amplitude that conforms to preliminary studies (Klevets et al., 1996). This effect perhaps could be explained by the protonation of acid groups (COO^- ) of aminoacid residues, which participate in binding Na⁺ by exchanger molecule.

Acidification of intracellular solution caused insignificant decrease of Na⁺-Ca²⁺ exchange current. On our opinion, it is possible in conditions of absence of competition between Ca²⁺ and H⁺ for intracellular cation binding transport site of exchanger.

The fact that reduction of I_Na(Ca) amplitude at simultaneous intra- and extracellular acidification was statistically unauthentic, testify that functioning of Na⁺-Ca²⁺ exchanger in direct mode is more dependent on level of intracellular pH.

Obtained results show that intracellular solution alkalization leads to decrease of inward Na⁺-Ca²⁺ exchange current amplitude. Moreover, in several cases direction of I_Na(Ca) changes from inward to outward. These changes of I_Na(Ca) could be explained by change in dissociation-association processes of Na⁺ and Ca²⁺ with exchanger molecule. We suppose that intracellular solution alkalization leads to decrease of competition between H⁺ and Na⁺ for interior cation binding transport site. It aggravates sodium dissociation from and possibly simultaneously aggravates calcium association with this site. As result Na⁺-Ca²⁺ exchange begin to go along Ca²⁺ gradient but no along Na⁺ gradient. That is reason of decrease of I_Na(Ca) and change of its direction.

It is necessary to point out that changes of Na⁺-Ca²⁺ exchanger functioning in conditions of intracellular solution alkalization at decrease and liquidation of sodium gradient are more significant.

More significant changes of salivary gland secretory cell plasma membrane Na⁺-Ca²⁺ exchanger functioning were also evoked by intracellular solution alkalization at increase of Ca²⁺ gradient.

CONCLUSIONS

Results described above let us to suggest that steady-state level of Na⁺-Ca²⁺ exchange not always can be described in system n*D m _Na=D m _Ca, because electro-chemical gradient of H⁺ has considerable influence on this process.

It should be said that the better understanding of role pH level in functioning of Na⁺-Ca²⁺ exchanger in secretory cell plasma membrane needs additional research.

REFERENCES

Baker P.F. (1972). Transport and metabolism of calcium ions in nerve. Progress in Biophysics and Molecular Biology. 24:177-223.
Baker, P.F. (1986). The sodium-calcium exchange system / Calcium and cell. - Wiley, Chichester. P. 73-86.
Baker, P.F., Blaustein, M.P. (1968). Sodium-dependent uptake of calcium by crab nerve. Biochim. Biophisica acta. 150:167-170.
Bayerdoffer, E., Haase, W., Schulz, I. (1985). Countertransport in plasma membrane of rat pancreatic acinar cells. J. Membrane Biol. 87,2:107-119.
Blaustein, M.P. (1974). The interrelationship between sodium and calcium fluxes across cell membranes. Rev. Phisiol. Biochem. Pharmacol. 70:33-88.
Blaustein, M.P., Hodgkin, A.L. (1969). The effect of cyanide on the efflux of calcium from squid axons. J.Physiol. (London) 200:497-527.
Blaustein, M.P., Nelson, M.T. (1982). Sodium-calcium exchange: its role in regulation of cell calcium. Membrane transport of calcium / Eds.E.Carafoli. - London; New York: Acad. Press. P. 217-236.
Doering, A.E., Lederer, W.J. (1991a). Cytoplasmic acidity inhibits sodium-calcium exchange in cardiac cells. Biophysical Journal. 59:544a.
Doering, A.E., Lederer, W.J. (1991b). Voltage-dependent block of the Na-Ca exchanger in heart muscle examined using giant excised patches from guinea-pig cardiac myocytes. Annals of the N.Y. Academy of Sciences. 639:172-176.
Doering, A.E., Lederer, W.J. (1993). The mechanism by which cytoplasmic protons inhibit the sodium-calcium exchanger in guinea-pig heart cells. J. Phisiol. 466:481-499.
Doering, A.E., Lederer, W.J. (1994). The action of Na⁺ as a cofactor in the inhibition by cytoplasmic protons of the cardiac Na⁺-Ca²⁺exchanger in the guinea-pig. J. Phisiol. 480,1:9-20.
Fedirko, N., Klevets, M., Manko, V. (1997). Dependence of Na-Ca-exchange current amplitude of secretory cell membrane on extracellular Ca²⁺ concentration. Europ.Biophys.J. with Biophys.Letters. 26(1):79.
Glitsch, H.G., Reuter, H., Scholr H. (1970). The effect of internal sodium concentration on calcium fluxes in isolated guinea pig vesicles. J.Physiol. (London) 209:25-43.
Grinkiv, М., Klevets, М., Shostakovskaya, I. (1988). Role of calcium in extrusion of digestive enzymes by pancreatic acinar cells. J.Physiol. (Ukraine). 34(4):13-17.
Klevets, M., Manko, V., Fedirko, N. (1995). Investigation of secretory cell membrane Na-Ca exchange current. Reports of Ukrainian National Academy of Sciences. 11:123-126
Klevets, M., Manko, V., Fedirko, N. (1996). Dependence of sodium-calcium exchange through salivary gland secretory cell plasma membrane in Chironomus plumosus larva on pH of extracellular solution. Neurophysiology. (Ukraine). 28(4/5):193-196
Manko, V., Klevets, M., Fedirko, N. (1995). Identification of Na-Ca exchange current in secretory cell membrane. Pflugers Arch. J. Physiol. 430(4):R138.
Philipson, K.D., Bersohn, M.M., Nishimoto, A.Y. (1982). Effects of pH on Na⁺-Ca²⁺-exchange in canine cardiac sarcolemmal vesicles. Circ. Res. 50:287-293.
Reuter, H., Seitz, N. (1968). The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. J. Physiol.195:451-470.

Discussion Board

Any Comment to this presentation?

[ABSTRACT] [INTRODUCTION] [METHODS] [RESULTS] [IMAGES] [DISCUSSION] [CONCLUSIONS] [REFERENCES] [Discussion Board]

---

	[Endocrinology]	[Physiology]

OLGA LARINA, Volodimir Manko
Copyright © 1999-2000. All rights reserved.

Last update: 13/01/00