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Blocking effect of La3+ ions on transmembrane ionic current evoked by intracellular cyclic AMP injection in identified Helix pomatia neurons
137
Neuroscience Letters, 124 (1991) 137-139 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100032P NSL 07623
Blocking effect o f La 3 + ions on transmembrane ionic current evoked by intracellular cyclic AMP injection in identified Helix pomatia neurons Pavel V. Belan and Oleg Osipenko Department of General Physiology of Nervous System, A.A. Bogomoletz Institute of Physiology of the UkrainianAcademy of Sciences, Kiev
(u.s.s.R.) (Received 2 May 1990; Revised version received 13 December 1990; Accepted 17 December 1990)
Key words: Identified snail neurons; La 3 ÷ ion; Fluorescence measurement; Cytoplasmic Ca2÷ ion; cAMP Using the fluorescent probe fura-2 for measurements of the cytoplasmic concentration of free Ca 2÷ ions ([Ca]~,) in combination with conventional current- or voltage-clamp methods, we studied the effects of La 3+ ions on the cellular responses evoked by intracellular cAMP (adenosine 3',5'-cyclic monophosphate) injections into isolated Helixpomatia neurons. La 3÷ ions in submillimolar concentrations decreased cAMP-evoked [Ca]in transients. Transmembrane ionic currents evoked by cAMP injections were completely blocked by La 3+ ions (I.0 mM) in all neurons under investigation. La 3+induced effects essentially differ from each other in RPal (right parietal 1) and LPa3 (left parietal 3) neurons. La 3+ ions in milli- and submillimolar concentrations strongly affect different subcellular systems, especially, those which regulate an intracellular calcium concentration.
The inhibitory action of extracellular La 3+ ions on plasmatic membrane Ca-pumps [9], release of the neurotransmitters from neuroterminals [7], Ca 2+ ions transport into mitochondria [2] and different types of potential-operated [3] and receptor-operated calcium channels [6] were known. It was shown that La 3+ ions also affect other types of ionic channels, e.g. sodium potential-activated channels [10]. Using direct measurements of the intracellular calcium ion concentration, we focused our attention on studying the La 3+ ions effects on [Ca]in transients and currents (cAMP-currents) evoked by iontophoretic injection of cAMP to the snail neurons. Experiments were performed on isolated Helix pomatia neurons [8]. Isolation of cells, their loading with fura2/AM (Calbiochem) and dual wavelength technique of measurement of Ca 2+ ion intracellular concentration were done according to the previously described procedure [1, 5]. Triple-barrelled glass microelectrode was utilized for an intracellular injection and a voltage clamp. Two barrels, filled with 2.5 M KC1 were used for currentand voltage-clamp; the third barrel, filled with cAMP (adenosine 3',5'-cyclic monophosphate, 0.2 M, Serva), was used for iontophoretic cAMP injections. Correspondence: P.V. Belan, A.A. Bogomoletz Institute of Physiology, Ukrainian Academy of Sciences, Bogomoletz str., 4, GSP, Kiev-24, 252601, U.S.S.R.
The standard external solution had the following composition (in mM): NaC1, 100; KC1, 5; CaC12, 10; MgC12, 5; Tris-HCl or HEPES-NaOH (pH 7.4), 20. A corresponding amount of LaCI3 was added to the external solution (La-solution). Fig. 1A presents changes in the [Ca]in and in the membrane potential during cAMP injections to a neuron placed in the normal or in the La3+-containing solutions. In La-solution (0.7 mM) both [Ca]in transients and membrane depolarizations were considerably reduced. This effect was completely reversible and 10 min after replacement of La-solution by normal solution the initial values of both responses were restored. The lanthanum-induced inhibitions of cAMP-evoked effects were different in different neurons. In most of the cells studied La 3+ ions (1.0 mM) caused an almost complete block of cAMP-induced depolarization shift (current clamp) or ionic current (voltage clamp) responses (n =9) but [Ca]in transient decreased only partly (near 50-95%). Therefore, we carried out the following experiments on identified neurons. Typical responses of RPal (right parietal 1) neuron (voltage clamp) to cAMP injections and electrical stimulations in both the normal and La-containing solutions (1.0 mM) are presented in Fig. lB. Electrical stimulations produced an influx of Ca 2+ ions into the cell through potential-operated calcium channels and increase of [Cain] [5]. These stimulations were performed to control the efficiency of La 3+ ion
138
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decreased both inward and outward components of the cAMP-current, however, the block of outward component by lanthanum ions was less prominent (Fig. 2B). The I-V curves for both current components in the normal and La-solutions are shown in Fig. 2Bb. Fig. 2Aa shows typical registrations of cAMP-currents in LPa3 (left parietal 3) neuron in the normal (1) and La-solution (2). La-solution (0.5 mM) induced a significant decrease of cAMP-current amplitude (near 80%). After removal of La 3+ ions and restoration of the cAMP-current amplitude (not shown), a normal solution was changed by Cd 2+ ions containing solution (2.0 mM). In this solution the current amplitude decreased by 20% (Fig. 2Aa (3)). An addition of La 3+ ions (0.5 mM) to Cd2+-containing solution resulted in a further reduction of the current amplitude (Fig. 2Aa (4)).
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Fig. 1. Effects of extracellular La 3+ ions on cAMP-induced alterations in snail neurons. The m o m e n t s of injections are shown by circles. [Ca]in levels are shown on the upper traces. N R , normal Ringer solution. A: unidentified neuron. Current clamp mode. Membrane potential is shown on the lower traces. The start of solution changes are indicated by arrows. La 3+ concentration was 0.7 raM. B: R P a l neuron. Voltage clamp mode. Membrane current is shown on the lower traces. Holding potential - 7 5 mV. Two depolarization shifts (to - 1 0 mV) were produced between c A M P injections. The leakage current was not compensated. La 3+ concentration 1.0 raM.
influence on these channels. One can see that La 3+ ions blocked both the calcium transient produced by electrical stimulation and cAMP-dependent ionic current, however, the increase of [Ca]in induced by cAMP injection was up to 30% of the control value. It was shown by Kononenko et al. [4] that in Helix neurons the cAMP-induced current has a complex nature, and it is possible to distinguish an inward current component produced by Na + and Ca 2+ ions, and an outward current component generated by K + ion efflux. We studied the action of La 3+ ions on the outward component of the cAMP-current in RPal (Fig. 2B) and RPa3 (not shown) neurons. In these neurons substitution of the normal solution by the La-solution (0.5 mM)
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! 50s ! Fig. 2. Effects of La 3+ ions on cAMP-induced currents. A: LPa3 neuron. a - typical cAMP-induced current in: (1) normal Ringer solution, (2) La3+-containing solution (0.5 mM), (3) Cd2+-containing solution (2.0 mM), (4) both La 3+- and Cd2+-containing solution (0.5 and 2.0 m M , respectively). Holding potential - 6 5 inV. b - the current-voltage dependences of cAMP-induced current amplitudes for the currents in the solutions 1-4 (Aa). B: R P a l neuron, a - cAMP-induced currents in normal Ringer (NR) and La3+-containing (0.5 m M ) solutions. Holding potential - 4 5 mV. b - the current-voltage dependences were drafted on the m a x i m u m amplitudes of both inward and outward cAMP-induced current. Circles and triangles for normal inward and outward currents, rhombs and squares for inward and outward current in La-solution.
139
The half inhibition of cAMP-induced inward current was calculated from the results for 5 LPa3 neurons (60 _+20/tM). These studies indicate that extracellular La 3+ ions are effective (more potent than Cd 2+ ions) inhibitors of the cAMP-responses of Helix neurons. The addition of La 3+ ions to the extracellular solution strongly decreased the amplitudes of both inward and outward components of cAMP-currents (Figs. 1 and 2). The inward component was completely blocked (in 1.0 mM La 3+ solution) in all neurons under consideration, i.e. the blocking action of La 3+ ions on both Na + and Ca 2+ inward fluxes of the cAMP-current took place. Cyclic AMP-evoked [Ca]in transients were considerably reduced by La 3+ ions in all our experiments (Fig. 1), but in some neurons (for example in R P a l ) we found an incomplete block of the transients even after elimination of cAMP-current (Fig. 1B). These facts support our previous suggestions about two sources of calcium in cAMP-induced [Ca]in transients. These are as follows: calcium flow through the channels and calcium release from intracellular stores [1]. It was also found that La 3+ ions produced a much stronger block of the inward component of cAMP-current than its outward component in RPal neuron (Fig. 2B). Besides, in La-solution, both the outward component of the cAMP-current and [Ca]in transient decreased to equal extent (compare the reduction of [Ca]in transient in La-solution in Fig. 1B with the reduction of the outward current in the same solution in Fig. 2Ba). Thus, it is possible to suggest that the outward component in
RPal neuron is likely a calcium-activated potassium ionic current, which might depend on both inward calcium current and Ca2+-release from the intracellular stores [1] and therefore is incompletely blocked by La 3+ ions.
1 Belan, P.V., Mironov, S.L., Osipenko, O.N. and Tepikin, A.V., The effect of iontophoretic cAMP injection on the changes in intracellular calcium concentration and transmembrane currents in snail neurons, Neurophysiology (Kiev), 21 (1988) 396-403. 2 Fiskum, G. and Lemninger, A.L., Mitochondrial regulation of intracellular calcium. In B.A. Croc (Ed.), Calcium and Cell Function, Vol. 2, Academic press, New York, 1982, pp. 39-80. 3 Hagiwara, S. and Byerly, L., Calcium channel, Am. Rev. Neurosci., 4 (1981) 69-125. 4 Kononenko, N.I., Kostyuk, P.G. and Shcherbatko, A.D., Properties of cAMP-induced transmembrane current in mollusc neurons, Brain Res., 376 (1983) 239-245. 5 Kostyuk, P.G., Mironov, S.L., Tepikin, A.V. and Belan, P.V., Cytoplasmic free Ca in isolated snail neurons as revealed by fluorescent probe Fura-2: mechanisms of Ca recovery after Ca load and Ca release from intracellular stores, J. Membr. Biol., 110 (1989) 1118. 6 Negulscu, P.A. and Machen, T.E., Release and reloading of intracellular Ca stores after cholinergic stimulation of the parietal cells, Am. J. Physiol., 23 (1988) 497-504. 7 Rubin, R.P., Calcium and Cellular Secretion, Plenum, New York, 1982, 276 pp. 8 Sakharov, D.A. and Salanki, J., Physiological and pharmacological identification of neurons in the central nervous system of Helix pomatia L., Acta. Physiol., 35 (1969) 19-30. 9 Schatzmann, H.J., Membrane Transport of Calcium, Academic press, London, 1982, 266 pp. 10 Vogel, W., Calcium and lanthanum effects at the nodal membrane, Eur. J. Physiol. (Pfliig Arch.), 350 (1974) 25-39.
Neuroscience Letters, 124 (1991) 137-139 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100032P NSL 07623
Blocking effect o f La 3 + ions on transmembrane ionic current evoked by intracellular cyclic AMP injection in identified Helix pomatia neurons Pavel V. Belan and Oleg Osipenko Department of General Physiology of Nervous System, A.A. Bogomoletz Institute of Physiology of the UkrainianAcademy of Sciences, Kiev
(u.s.s.R.) (Received 2 May 1990; Revised version received 13 December 1990; Accepted 17 December 1990)
Key words: Identified snail neurons; La 3 ÷ ion; Fluorescence measurement; Cytoplasmic Ca2÷ ion; cAMP Using the fluorescent probe fura-2 for measurements of the cytoplasmic concentration of free Ca 2÷ ions ([Ca]~,) in combination with conventional current- or voltage-clamp methods, we studied the effects of La 3+ ions on the cellular responses evoked by intracellular cAMP (adenosine 3',5'-cyclic monophosphate) injections into isolated Helixpomatia neurons. La 3÷ ions in submillimolar concentrations decreased cAMP-evoked [Ca]in transients. Transmembrane ionic currents evoked by cAMP injections were completely blocked by La 3+ ions (I.0 mM) in all neurons under investigation. La 3+induced effects essentially differ from each other in RPal (right parietal 1) and LPa3 (left parietal 3) neurons. La 3+ ions in milli- and submillimolar concentrations strongly affect different subcellular systems, especially, those which regulate an intracellular calcium concentration.
The inhibitory action of extracellular La 3+ ions on plasmatic membrane Ca-pumps [9], release of the neurotransmitters from neuroterminals [7], Ca 2+ ions transport into mitochondria [2] and different types of potential-operated [3] and receptor-operated calcium channels [6] were known. It was shown that La 3+ ions also affect other types of ionic channels, e.g. sodium potential-activated channels [10]. Using direct measurements of the intracellular calcium ion concentration, we focused our attention on studying the La 3+ ions effects on [Ca]in transients and currents (cAMP-currents) evoked by iontophoretic injection of cAMP to the snail neurons. Experiments were performed on isolated Helix pomatia neurons [8]. Isolation of cells, their loading with fura2/AM (Calbiochem) and dual wavelength technique of measurement of Ca 2+ ion intracellular concentration were done according to the previously described procedure [1, 5]. Triple-barrelled glass microelectrode was utilized for an intracellular injection and a voltage clamp. Two barrels, filled with 2.5 M KC1 were used for currentand voltage-clamp; the third barrel, filled with cAMP (adenosine 3',5'-cyclic monophosphate, 0.2 M, Serva), was used for iontophoretic cAMP injections. Correspondence: P.V. Belan, A.A. Bogomoletz Institute of Physiology, Ukrainian Academy of Sciences, Bogomoletz str., 4, GSP, Kiev-24, 252601, U.S.S.R.
The standard external solution had the following composition (in mM): NaC1, 100; KC1, 5; CaC12, 10; MgC12, 5; Tris-HCl or HEPES-NaOH (pH 7.4), 20. A corresponding amount of LaCI3 was added to the external solution (La-solution). Fig. 1A presents changes in the [Ca]in and in the membrane potential during cAMP injections to a neuron placed in the normal or in the La3+-containing solutions. In La-solution (0.7 mM) both [Ca]in transients and membrane depolarizations were considerably reduced. This effect was completely reversible and 10 min after replacement of La-solution by normal solution the initial values of both responses were restored. The lanthanum-induced inhibitions of cAMP-evoked effects were different in different neurons. In most of the cells studied La 3+ ions (1.0 mM) caused an almost complete block of cAMP-induced depolarization shift (current clamp) or ionic current (voltage clamp) responses (n =9) but [Ca]in transient decreased only partly (near 50-95%). Therefore, we carried out the following experiments on identified neurons. Typical responses of RPal (right parietal 1) neuron (voltage clamp) to cAMP injections and electrical stimulations in both the normal and La-containing solutions (1.0 mM) are presented in Fig. lB. Electrical stimulations produced an influx of Ca 2+ ions into the cell through potential-operated calcium channels and increase of [Cain] [5]. These stimulations were performed to control the efficiency of La 3+ ion
138
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4
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decreased both inward and outward components of the cAMP-current, however, the block of outward component by lanthanum ions was less prominent (Fig. 2B). The I-V curves for both current components in the normal and La-solutions are shown in Fig. 2Bb. Fig. 2Aa shows typical registrations of cAMP-currents in LPa3 (left parietal 3) neuron in the normal (1) and La-solution (2). La-solution (0.5 mM) induced a significant decrease of cAMP-current amplitude (near 80%). After removal of La 3+ ions and restoration of the cAMP-current amplitude (not shown), a normal solution was changed by Cd 2+ ions containing solution (2.0 mM). In this solution the current amplitude decreased by 20% (Fig. 2Aa (3)). An addition of La 3+ ions (0.5 mM) to Cd2+-containing solution resulted in a further reduction of the current amplitude (Fig. 2Aa (4)).
b
l.,aCla
A
• [Cae'lln, /zM 4~
. LaCIo
~an
-.3 -6
|m
,
-9 .-12 lm,nA
Fig. 1. Effects of extracellular La 3+ ions on cAMP-induced alterations in snail neurons. The m o m e n t s of injections are shown by circles. [Ca]in levels are shown on the upper traces. N R , normal Ringer solution. A: unidentified neuron. Current clamp mode. Membrane potential is shown on the lower traces. The start of solution changes are indicated by arrows. La 3+ concentration was 0.7 raM. B: R P a l neuron. Voltage clamp mode. Membrane current is shown on the lower traces. Holding potential - 7 5 mV. Two depolarization shifts (to - 1 0 mV) were produced between c A M P injections. The leakage current was not compensated. La 3+ concentration 1.0 raM.
influence on these channels. One can see that La 3+ ions blocked both the calcium transient produced by electrical stimulation and cAMP-dependent ionic current, however, the increase of [Ca]in induced by cAMP injection was up to 30% of the control value. It was shown by Kononenko et al. [4] that in Helix neurons the cAMP-induced current has a complex nature, and it is possible to distinguish an inward current component produced by Na + and Ca 2+ ions, and an outward current component generated by K + ion efflux. We studied the action of La 3+ ions on the outward component of the cAMP-current in RPal (Fig. 2B) and RPa3 (not shown) neurons. In these neurons substitution of the normal solution by the La-solution (0.5 mM)
J 0.1
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Vm.mV
]:o° L2 0 0 s
B
Ica2'~.oaw
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! 50s ! Fig. 2. Effects of La 3+ ions on cAMP-induced currents. A: LPa3 neuron. a - typical cAMP-induced current in: (1) normal Ringer solution, (2) La3+-containing solution (0.5 mM), (3) Cd2+-containing solution (2.0 mM), (4) both La 3+- and Cd2+-containing solution (0.5 and 2.0 m M , respectively). Holding potential - 6 5 inV. b - the current-voltage dependences of cAMP-induced current amplitudes for the currents in the solutions 1-4 (Aa). B: R P a l neuron, a - cAMP-induced currents in normal Ringer (NR) and La3+-containing (0.5 m M ) solutions. Holding potential - 4 5 mV. b - the current-voltage dependences were drafted on the m a x i m u m amplitudes of both inward and outward cAMP-induced current. Circles and triangles for normal inward and outward currents, rhombs and squares for inward and outward current in La-solution.
139
The half inhibition of cAMP-induced inward current was calculated from the results for 5 LPa3 neurons (60 _+20/tM). These studies indicate that extracellular La 3+ ions are effective (more potent than Cd 2+ ions) inhibitors of the cAMP-responses of Helix neurons. The addition of La 3+ ions to the extracellular solution strongly decreased the amplitudes of both inward and outward components of cAMP-currents (Figs. 1 and 2). The inward component was completely blocked (in 1.0 mM La 3+ solution) in all neurons under consideration, i.e. the blocking action of La 3+ ions on both Na + and Ca 2+ inward fluxes of the cAMP-current took place. Cyclic AMP-evoked [Ca]in transients were considerably reduced by La 3+ ions in all our experiments (Fig. 1), but in some neurons (for example in R P a l ) we found an incomplete block of the transients even after elimination of cAMP-current (Fig. 1B). These facts support our previous suggestions about two sources of calcium in cAMP-induced [Ca]in transients. These are as follows: calcium flow through the channels and calcium release from intracellular stores [1]. It was also found that La 3+ ions produced a much stronger block of the inward component of cAMP-current than its outward component in RPal neuron (Fig. 2B). Besides, in La-solution, both the outward component of the cAMP-current and [Ca]in transient decreased to equal extent (compare the reduction of [Ca]in transient in La-solution in Fig. 1B with the reduction of the outward current in the same solution in Fig. 2Ba). Thus, it is possible to suggest that the outward component in
RPal neuron is likely a calcium-activated potassium ionic current, which might depend on both inward calcium current and Ca2+-release from the intracellular stores [1] and therefore is incompletely blocked by La 3+ ions.
1 Belan, P.V., Mironov, S.L., Osipenko, O.N. and Tepikin, A.V., The effect of iontophoretic cAMP injection on the changes in intracellular calcium concentration and transmembrane currents in snail neurons, Neurophysiology (Kiev), 21 (1988) 396-403. 2 Fiskum, G. and Lemninger, A.L., Mitochondrial regulation of intracellular calcium. In B.A. Croc (Ed.), Calcium and Cell Function, Vol. 2, Academic press, New York, 1982, pp. 39-80. 3 Hagiwara, S. and Byerly, L., Calcium channel, Am. Rev. Neurosci., 4 (1981) 69-125. 4 Kononenko, N.I., Kostyuk, P.G. and Shcherbatko, A.D., Properties of cAMP-induced transmembrane current in mollusc neurons, Brain Res., 376 (1983) 239-245. 5 Kostyuk, P.G., Mironov, S.L., Tepikin, A.V. and Belan, P.V., Cytoplasmic free Ca in isolated snail neurons as revealed by fluorescent probe Fura-2: mechanisms of Ca recovery after Ca load and Ca release from intracellular stores, J. Membr. Biol., 110 (1989) 1118. 6 Negulscu, P.A. and Machen, T.E., Release and reloading of intracellular Ca stores after cholinergic stimulation of the parietal cells, Am. J. Physiol., 23 (1988) 497-504. 7 Rubin, R.P., Calcium and Cellular Secretion, Plenum, New York, 1982, 276 pp. 8 Sakharov, D.A. and Salanki, J., Physiological and pharmacological identification of neurons in the central nervous system of Helix pomatia L., Acta. Physiol., 35 (1969) 19-30. 9 Schatzmann, H.J., Membrane Transport of Calcium, Academic press, London, 1982, 266 pp. 10 Vogel, W., Calcium and lanthanum effects at the nodal membrane, Eur. J. Physiol. (Pfliig Arch.), 350 (1974) 25-39.