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Melatonin induces membrane conductance changes in isolated retinal rod receptor cells
Life Sciences, Vol. 60, No. 21, pp. 18E5-1869, 1997 Copyright Q 1997 Elscvicr Science Inc. Printed in the USA. All rights rcservcd 0024-3205/97 S17.00 t .oO
PII SOO243205(97)00150-1
ELSEVIER
MELATONIN INDUCES MEMBRANE CONDUCTANCE CHANGES IN ISOLATED RETINAL ROD RECEPTOR CELLS
B. Cosci, B. Longoni and P.L. Marchiafhva Dipartimento di Fisiologia e Biochimica, Universita di Pisa, Via S. Zeno, 3 1, Pisa, 56100 Italy
(Received in tinal form February 18, 1997)
Experiments were conducted to verity whether the neurohormone melatonin influences the membrane conductance of photoreceptors isolated from the frog retina. It has been fotmd that 20 @l melatonin decreases membrane conductances both in the linear and non linear ranges by CO.4nS. These actions are estimated to produce in dark adapted photoreceptors an increase of the response to a dim light induced change of the dark current of about 21%, i.e. from 1.3 to 1.62 mV/pA.
Key Words: melatonin, photoreceptors, conductance
The synthesis of melatonin by retinal photoreceptors reaches a peak in darkness (l), when receptor cells themselves undergo fln&nentaI membmne conductance processes cuhninatillg in the “dark current” (2). A possiile functional connection between these two events is strongly suggested by the simuhaneity of the light induced drop of both the dark current and the production of melatonin. In the present work we tried to investigate whether melatonin exerts any etibct on the membrane conductance of retinal photoreceptors and to discuss whether such efIbcts may be relevant for phototransduction Direct measuretmmts of membmne conductance, performed on suprachiasmatic neurons (3) have already shown that melatonin produces potassium conductance changes in accord with an inhibition of the spontaneous thing observed extracelhrlarly (4). Hem we confirm other Author’ Wings that photoreceptors possess relevant potassium conductances (5) adequate to be tested fbr a possible modulation by melatonin. In addition, photoreceptors are not electrically excitable and have different membrane conductance properties which may represent specifc substrates for the action of melatonin with respect to brain neurons. The present results indicate that photoreceptors activity may be modulated by this lipophilic molecule.
_______~__________~~______~~~~~~____~~~~~~~~____~~~~~~~~_~_~~~~~~~~~__~~~~~~~~~~_~~~~~~~
Correspondii
Author: P.L. Marchiafh~, Via San Zeno 3 1, Pisa 56100 Italy
1886
Melatonin and Membrane Conductance
Vol. 60, No. 21, 1997
Methods The experimental preparation consisted of rod receptor cells mechanically isolated horn the retina of rapidly decapitated f?ogs. Cells were introduced in a pet-l&ion chamber placed on an inverted Nikon microscope assembled for electrical recording under direct TV observation with white light. The dissected cells appeared deprived of the innermost portion of the inner segment, i.e. that below the nucleus, while the remain& portion of the cell, including a major part of the outer segment, appeared as in intact cells. A standard equipment for patch recording in whole cell configuration was used, with microelectrodes made from borosilicate glass capillaries (Clark Electromedical Instruments) having tip internal diameters of about 8 pm and resistance of 8-10 MS2. Except when otherwise stated, the bathing solution, maintained at 21°C, had the following composition, in mM: NaC1: 120; KCI: 2.4; CaC12: 2.5; MgC12: 1.2; Hepes: 10; Glucose:lO; pH adjusted to 7.6 by adding NaOH. The electrode tilling solution contained, in mM: KCI: 100; MgC12: 3.5; Hepes:lO; EGTA: 0.5; CaC12: 0.3; NaATP: 1.5; pH adjusted to 7.4 by addii KOH. Electrodes used to identity potassium currents were additionally backfilled just prior to their use with an equimolar CsCh solution substituting for KCl. This procedure delayed the initial cell dialysis by l-2 minutes. i.e. an interval suflicient to capture normal, control responses as in Fig l,A). Melatonin (N-acetyl-5-methoxytryptamine (97%), Aldrich-Chemie, Steinheim) was dissolved in 95% ethanol and diluted to tinal concentrations of lo,20 and 1OOpM,where the alcohol content was 0.02, 0.04 and 0.2%, respectively. Control experiments showed that these amounts of alcohol per se did not produced sign&ant effects. Data recording and analysis, and the elaboration of the illustrations were done with a PClamp 5.5 system (Axon Instruments, Inc.) and a MicroCal origin 3.01 (MicroCal Software, Inc.). Results Figure l.A represents a family of current recordings obtained by clamping a photoreceptor membrane potential from - 100 to 50 mV with 15 mV intervals. The currents amplitudes, measured at 40 and 480 milliseconds after the initial stepping voltage, are plotted against membrane potential in Figure 1,C Z and 2, respectively. Here the membrane conductance behaves linearly within a potential range from about -70 to 20 mV and its value was 5.12 x lo-” x 10“. Further hyperpolarization or depolarization beyond the linear range activated nonlinear membrane conductances in the outward direction, like the fast rising and rapidly inactivating outward current (OIR) (Fig.l,A, upper traces) whereby the conductance value measured at 40 ms increased up to 1200 pS compared to 480 ms (580 pS). The average values obtained from 30 cells at 40 and 480 ms were 570 (+/-170) and 280 (+/-73) pS, respectively. The reversal potential of the OIR current was at -41 (+/-5.2) mV. By substituting TEA to 30 mM of extracellular sodium, the OIR current desappeared ahnost completely (Fig. l,A vz.B,upper traces), while the resulting all linear slope conductance decreased by about 62%, i.e. from 580 to 220 pS (Fig.l,C). Accordingly, the membrane resting potential was positively shifted by about 12 mV. These results indicate a principal role of potassium ions in the production of the OIR current (5), and its participation to the multiionic background currents. The addition of 20 pM melatonin to the bathing solution reduced membrane currents at all voltages, as in a typical cell illustrated in Fig.2. The IN relation obtained in the presence of melatonin (Fig.2,B, triangle symbols) intersects the control curve (square symbols) at about 0 mV and the membrane resting potential shifted by about -8 mV. The effects of melatonin were further tested in a series of trials in four cells, and the amplitude difference of the responses with 20 uM melatonin against their respective control responses are plotted in the diagram shown in Fig. 3. It is
Vol. 60, No. 21, 1997
Melatonin and Membrane Conductance
; time (ms) I
: ,B
,
0
400
o-
I -2o8OU
Fig. 1 Membrane currents of an isolated retinal rod cell recorded with patch electrodes in the whole cell configuration Membrane potential is clamped at various voltage steps from 50 to -100 mV. A: a set of control responses recorded with a ‘cesium’ electrode (see Methods) within the first minute after the patch recording had been established. B: the responses recorded from the same cell five minute after the application of the test solution C: IN functions with (curves 3 t 4) and without the test solutions (curves 1 & 2). Curves 1 & 3 and 2 L?z4 represent values measured at 40 and 480 ms, respectively.
---
--b-
control melatonin 40 ms
control melatonin 480 ms
Fig. 2 The effect of 2Ow melatonin on membrane conductance. A: superimposed current tracings recorded from the same cell before (heavy line) and after the addition of 20 PM melatonin (thin line). B: IN curves obtained from the same cell as in A, by measuring currents amplitudes at 40 and 480 ms.
1887
Melatonin and Membrane Conductance
ls88
Vol. 60, No. 21, 1997
IO-
%
o-10 -
-20 -=40 ms ____o____ 480 ms -30 Fig. 3 Plots of the currents amplitude obtained by subtracting control IN functions from their corresponding I/V curves in the presence of melatonin. Average and standard error of the data from four cells, obtained at 40 and 480 ms.
worth noting that the rnelatonin induced decrease of the current amplitudes measured between -20 and -70 mV corresponds to about 20% of the control response (cfr. Fii.2,B) . The most effective melatonin concentration was 20 p.h4, while 10 pM and 100 pM still produced a conductance decrease of about 3.9 and 9.2% respectively.
Discussion
The substitution of intracelhdar potassium with cesium and the addition of extracellular tetraetilammonium (TEA), hvo known blockers of potassium channels, produced remarkable changes of photoreceptor membrane currents thus comirming that our experimental conditions seem adequate to test both the effect of melatonin on membrane conductance and whether it consists of potassium permeability changes similar to those described in suprachiasmatic neurons (3). The addition of melatonin produces two main groups of results. Fit, a decrease of about 20% of the membrane conductance, both in the linear and non linear ranges. Second, the I/V function obtained in the presence of mclatonin intersects the control curve at near 0 potentir& indicating that all ionic components were a&&d, in proportion to their relative permeability at resting potential. The melatonin induced membrane conductance decrease which we have measured during illumination may represent a mechanism operative in the dark to alter the cell sensitivity to light within the potential range modulated by light (i.e. from about -25 to -70 mV, the dark and the light saturating potential, respectively). Fii 3 shows that in such voltage range melatonin induces a maximal current drop of about 10 PA, that is an about 20% conductance decrease (cfi. for example
Vol. 60, No. 21, 1997
Melatonin and Membrane Conductance
1889
Fig. 2,B at -70 mv). This percentage value may be slightly changed in the dark by the presence of the shunting parallel uxxluctanus across the! outer segment*. Thllq at nigh& melatonin may increase the photoreceptor voltage response to a dim light induced change of the dark current by 0.32 mV/pA ( ie. km 1.3 to 1.62 mV/pA). However the above hypothesis of a modulatory action of melatonin on the photoreceptor sensitivity to light should be fbrther substantiated by the knowledge of the physiological concentration of melatonin in the retina: an information presently UllkllOWll.
1. P.M. IUVONE, The Ret& A model fbr Cell Biolo~v Studies, Part II, R. Adler and D. Farber (eds), l-72, Academic Press, Orlando, Florida (1986). 2. K.W. YAU and D.A. BAYLOR, Annu. Rev. Neurosci 12 289-327 (1989). 3. Z-G. JIANG, C.S. NELSON and C.N. ALLEN, Brain Res. w 125-132 (1995). 4. R MASON and A. BROOKS, Neurosci Lett.95 296-301(1988). 5. C.R. BADER, D. BERTRAND and E.A. SCHWARTZ, J. Physiot (Land.) 331 253-284 (1982). 6. P.M. IUVONE and J. GAN, J.Neurochem,~l18-124 (1994). 7. J .S. POPOVA and M.L. DUBOCOVICH, J. Neurochem. 64 130-8 (1995).
* Considering the photoreceptor membrane conductance in the linear range Gtd = 512 pS (taken from Fig.l,A) the sum of the external and internal segments conductanos (Gm + Gti) during illumination, where Gi,,,= Gm = 256 pS, with the addition of melatonin we have Gtacmltj= Gti . 0,80 + Gti .0,80 = 409 pS. Assuming in the dark GtidsrLj= Gm .2, than melatonin may reduce Gtti(dtij from 768 pS to G,oya~lr~ti)= Gi.t . 0,80 + (Gti.2) . 0,80 = 614 pS. Thus melatonin may resistance f%orn1.3 x 10’ Ohmto about 1.62 x 10’ Ohm. hxreaseasinglerodcelldarkmembmne
PII SOO243205(97)00150-1
ELSEVIER
MELATONIN INDUCES MEMBRANE CONDUCTANCE CHANGES IN ISOLATED RETINAL ROD RECEPTOR CELLS
B. Cosci, B. Longoni and P.L. Marchiafhva Dipartimento di Fisiologia e Biochimica, Universita di Pisa, Via S. Zeno, 3 1, Pisa, 56100 Italy
(Received in tinal form February 18, 1997)
Experiments were conducted to verity whether the neurohormone melatonin influences the membrane conductance of photoreceptors isolated from the frog retina. It has been fotmd that 20 @l melatonin decreases membrane conductances both in the linear and non linear ranges by CO.4nS. These actions are estimated to produce in dark adapted photoreceptors an increase of the response to a dim light induced change of the dark current of about 21%, i.e. from 1.3 to 1.62 mV/pA.
Key Words: melatonin, photoreceptors, conductance
The synthesis of melatonin by retinal photoreceptors reaches a peak in darkness (l), when receptor cells themselves undergo fln&nentaI membmne conductance processes cuhninatillg in the “dark current” (2). A possiile functional connection between these two events is strongly suggested by the simuhaneity of the light induced drop of both the dark current and the production of melatonin. In the present work we tried to investigate whether melatonin exerts any etibct on the membrane conductance of retinal photoreceptors and to discuss whether such efIbcts may be relevant for phototransduction Direct measuretmmts of membmne conductance, performed on suprachiasmatic neurons (3) have already shown that melatonin produces potassium conductance changes in accord with an inhibition of the spontaneous thing observed extracelhrlarly (4). Hem we confirm other Author’ Wings that photoreceptors possess relevant potassium conductances (5) adequate to be tested fbr a possible modulation by melatonin. In addition, photoreceptors are not electrically excitable and have different membrane conductance properties which may represent specifc substrates for the action of melatonin with respect to brain neurons. The present results indicate that photoreceptors activity may be modulated by this lipophilic molecule.
_______~__________~~______~~~~~~____~~~~~~~~____~~~~~~~~_~_~~~~~~~~~__~~~~~~~~~~_~~~~~~~
Correspondii
Author: P.L. Marchiafh~, Via San Zeno 3 1, Pisa 56100 Italy
1886
Melatonin and Membrane Conductance
Vol. 60, No. 21, 1997
Methods The experimental preparation consisted of rod receptor cells mechanically isolated horn the retina of rapidly decapitated f?ogs. Cells were introduced in a pet-l&ion chamber placed on an inverted Nikon microscope assembled for electrical recording under direct TV observation with white light. The dissected cells appeared deprived of the innermost portion of the inner segment, i.e. that below the nucleus, while the remain& portion of the cell, including a major part of the outer segment, appeared as in intact cells. A standard equipment for patch recording in whole cell configuration was used, with microelectrodes made from borosilicate glass capillaries (Clark Electromedical Instruments) having tip internal diameters of about 8 pm and resistance of 8-10 MS2. Except when otherwise stated, the bathing solution, maintained at 21°C, had the following composition, in mM: NaC1: 120; KCI: 2.4; CaC12: 2.5; MgC12: 1.2; Hepes: 10; Glucose:lO; pH adjusted to 7.6 by adding NaOH. The electrode tilling solution contained, in mM: KCI: 100; MgC12: 3.5; Hepes:lO; EGTA: 0.5; CaC12: 0.3; NaATP: 1.5; pH adjusted to 7.4 by addii KOH. Electrodes used to identity potassium currents were additionally backfilled just prior to their use with an equimolar CsCh solution substituting for KCl. This procedure delayed the initial cell dialysis by l-2 minutes. i.e. an interval suflicient to capture normal, control responses as in Fig l,A). Melatonin (N-acetyl-5-methoxytryptamine (97%), Aldrich-Chemie, Steinheim) was dissolved in 95% ethanol and diluted to tinal concentrations of lo,20 and 1OOpM,where the alcohol content was 0.02, 0.04 and 0.2%, respectively. Control experiments showed that these amounts of alcohol per se did not produced sign&ant effects. Data recording and analysis, and the elaboration of the illustrations were done with a PClamp 5.5 system (Axon Instruments, Inc.) and a MicroCal origin 3.01 (MicroCal Software, Inc.). Results Figure l.A represents a family of current recordings obtained by clamping a photoreceptor membrane potential from - 100 to 50 mV with 15 mV intervals. The currents amplitudes, measured at 40 and 480 milliseconds after the initial stepping voltage, are plotted against membrane potential in Figure 1,C Z and 2, respectively. Here the membrane conductance behaves linearly within a potential range from about -70 to 20 mV and its value was 5.12 x lo-” x 10“. Further hyperpolarization or depolarization beyond the linear range activated nonlinear membrane conductances in the outward direction, like the fast rising and rapidly inactivating outward current (OIR) (Fig.l,A, upper traces) whereby the conductance value measured at 40 ms increased up to 1200 pS compared to 480 ms (580 pS). The average values obtained from 30 cells at 40 and 480 ms were 570 (+/-170) and 280 (+/-73) pS, respectively. The reversal potential of the OIR current was at -41 (+/-5.2) mV. By substituting TEA to 30 mM of extracellular sodium, the OIR current desappeared ahnost completely (Fig. l,A vz.B,upper traces), while the resulting all linear slope conductance decreased by about 62%, i.e. from 580 to 220 pS (Fig.l,C). Accordingly, the membrane resting potential was positively shifted by about 12 mV. These results indicate a principal role of potassium ions in the production of the OIR current (5), and its participation to the multiionic background currents. The addition of 20 pM melatonin to the bathing solution reduced membrane currents at all voltages, as in a typical cell illustrated in Fig.2. The IN relation obtained in the presence of melatonin (Fig.2,B, triangle symbols) intersects the control curve (square symbols) at about 0 mV and the membrane resting potential shifted by about -8 mV. The effects of melatonin were further tested in a series of trials in four cells, and the amplitude difference of the responses with 20 uM melatonin against their respective control responses are plotted in the diagram shown in Fig. 3. It is
Vol. 60, No. 21, 1997
Melatonin and Membrane Conductance
; time (ms) I
: ,B
,
0
400
o-
I -2o8OU
Fig. 1 Membrane currents of an isolated retinal rod cell recorded with patch electrodes in the whole cell configuration Membrane potential is clamped at various voltage steps from 50 to -100 mV. A: a set of control responses recorded with a ‘cesium’ electrode (see Methods) within the first minute after the patch recording had been established. B: the responses recorded from the same cell five minute after the application of the test solution C: IN functions with (curves 3 t 4) and without the test solutions (curves 1 & 2). Curves 1 & 3 and 2 L?z4 represent values measured at 40 and 480 ms, respectively.
---
--b-
control melatonin 40 ms
control melatonin 480 ms
Fig. 2 The effect of 2Ow melatonin on membrane conductance. A: superimposed current tracings recorded from the same cell before (heavy line) and after the addition of 20 PM melatonin (thin line). B: IN curves obtained from the same cell as in A, by measuring currents amplitudes at 40 and 480 ms.
1887
Melatonin and Membrane Conductance
ls88
Vol. 60, No. 21, 1997
IO-
%
o-10 -
-20 -=40 ms ____o____ 480 ms -30 Fig. 3 Plots of the currents amplitude obtained by subtracting control IN functions from their corresponding I/V curves in the presence of melatonin. Average and standard error of the data from four cells, obtained at 40 and 480 ms.
worth noting that the rnelatonin induced decrease of the current amplitudes measured between -20 and -70 mV corresponds to about 20% of the control response (cfr. Fii.2,B) . The most effective melatonin concentration was 20 p.h4, while 10 pM and 100 pM still produced a conductance decrease of about 3.9 and 9.2% respectively.
Discussion
The substitution of intracelhdar potassium with cesium and the addition of extracellular tetraetilammonium (TEA), hvo known blockers of potassium channels, produced remarkable changes of photoreceptor membrane currents thus comirming that our experimental conditions seem adequate to test both the effect of melatonin on membrane conductance and whether it consists of potassium permeability changes similar to those described in suprachiasmatic neurons (3). The addition of melatonin produces two main groups of results. Fit, a decrease of about 20% of the membrane conductance, both in the linear and non linear ranges. Second, the I/V function obtained in the presence of mclatonin intersects the control curve at near 0 potentir& indicating that all ionic components were a&&d, in proportion to their relative permeability at resting potential. The melatonin induced membrane conductance decrease which we have measured during illumination may represent a mechanism operative in the dark to alter the cell sensitivity to light within the potential range modulated by light (i.e. from about -25 to -70 mV, the dark and the light saturating potential, respectively). Fii 3 shows that in such voltage range melatonin induces a maximal current drop of about 10 PA, that is an about 20% conductance decrease (cfi. for example
Vol. 60, No. 21, 1997
Melatonin and Membrane Conductance
1889
Fig. 2,B at -70 mv). This percentage value may be slightly changed in the dark by the presence of the shunting parallel uxxluctanus across the! outer segment*. Thllq at nigh& melatonin may increase the photoreceptor voltage response to a dim light induced change of the dark current by 0.32 mV/pA ( ie. km 1.3 to 1.62 mV/pA). However the above hypothesis of a modulatory action of melatonin on the photoreceptor sensitivity to light should be fbrther substantiated by the knowledge of the physiological concentration of melatonin in the retina: an information presently UllkllOWll.
1. P.M. IUVONE, The Ret& A model fbr Cell Biolo~v Studies, Part II, R. Adler and D. Farber (eds), l-72, Academic Press, Orlando, Florida (1986). 2. K.W. YAU and D.A. BAYLOR, Annu. Rev. Neurosci 12 289-327 (1989). 3. Z-G. JIANG, C.S. NELSON and C.N. ALLEN, Brain Res. w 125-132 (1995). 4. R MASON and A. BROOKS, Neurosci Lett.95 296-301(1988). 5. C.R. BADER, D. BERTRAND and E.A. SCHWARTZ, J. Physiot (Land.) 331 253-284 (1982). 6. P.M. IUVONE and J. GAN, J.Neurochem,~l18-124 (1994). 7. J .S. POPOVA and M.L. DUBOCOVICH, J. Neurochem. 64 130-8 (1995).
* Considering the photoreceptor membrane conductance in the linear range Gtd = 512 pS (taken from Fig.l,A) the sum of the external and internal segments conductanos (Gm + Gti) during illumination, where Gi,,,= Gm = 256 pS, with the addition of melatonin we have Gtacmltj= Gti . 0,80 + Gti .0,80 = 409 pS. Assuming in the dark GtidsrLj= Gm .2, than melatonin may reduce Gtti(dtij from 768 pS to G,oya~lr~ti)= Gi.t . 0,80 + (Gti.2) . 0,80 = 614 pS. Thus melatonin may resistance f%orn1.3 x 10’ Ohmto about 1.62 x 10’ Ohm. hxreaseasinglerodcelldarkmembmne