Brain Research, 176 (1979) 365-368 (© Elsevier/North-HollandBiomedicalPress
365
Short Communications
Two effects of phosphodiesterase inhibitors on Limulus ventral photoreceptors
D. WESLEYCORSON, ALAN FEIN and JEFFREY SCHMIDT Laboratory o f Sensory Physiology, Marine Biological Laboratory, Woods Hole, Mass. 02543 and ( A. F.) Department of Physiology, Boston University School of Medicine, Boston, Mass. 02118 (U.S.A.)
(Accepted July [2th, 1979)
Recently, light has been found tc activate a phosphodiesterase responsible for cyclic nucleotide hydrolysis in frog rods (see review by Bitensky et al.1). The action spectrum of phosphodiesterase activation corresponds to the absorption spectrum of rhodopsin 6, and the speculation has been made that the reduction in cyclic nucleotide levels caused by activation of the phosphodiesterase may be involved in light and dark adaptation 1°. Previous studies by Wulff15 and Millera2 on Limulus photoreceptors indicate that in an invertebrate preparation, phosphodiesterase inhibitors reduce both the resting membrane potential and the receptor potential. We decided to investigate the possible role of cyclic nucleotides in excitation and/or adaptation in photoreceptors from the ventral eye of Limulus. Schmidt and Fein have reported that calcium blocking agents and phosphodiesterase inhibitors have similar effects on voltage-dependent conductances of ventral photoreceptors 14. In the study reported here, we have investigated whether known phosphodiesterase inhibitors had any direct effects on the light-activated currents elicited by dim flashes in voltage clamped ventral photoreceptors. Our findings suggest that phosphodiesterase inhibitors depress ventral photoreceptor sensitivity in a manner similar to light adaptation. In addition, the effects of the inhibitors on the voltage-dependent conductances a4 do not appear to be strongly linked to their effects on the light-activated conductance. Theophylline, isobutylmethylxanthine(IBMX) and papaverine suppress nucleotide hydrolysis in in vitro assays of rod outer segment phosphodiesterase activity3,9. All 3 inhibitors had qualitatively the same effect on ventral photoreceptors as described below. The methods of dissecting, viewing, stimulating and recording from the photoreceptor under voltage clamp have all been described in detail previously4,11. The preparation was suffused with artificial seawater (ASW) buffered at pH 7.0 with 10 mM Hepes, to which the inhibitors were added.
366 We sought to determine whether the reduction in the receptor potential described above reflects an actual reduction in the underlying light-induced current. As shown in Fig. 1, application of each of the 3 inhibitors produces a rapid and reversible depression of the inward currents elicited by dim test flashes. The desensitization is seen to occur without any appreciable shift in the baseline current recorded between test flashes. The inhibitors appear not to mimic the excitatory effect of light in that they do not produce an inward membrane current either transient or maintained. Under voltage clamp, plots of response amplitude vs light intensity in the region near threshold are well fitted by a line with a unit slope s. Light adaptation causes a simple translation of the response-intensity curve outward along the intensity axis 5,8. We measured response-intensity curves in the presence and absence of inhibitors in order to determine the similarity of inhibitor-induced desensitization to that induced by light. All response-intensity curves were seen to be well fitted by a line with unil slope. Those measured in the presence of inhibitors were found to be displaced out along the intensity axis, and the effect was reversible upon washing out the inhibitor. Phosphodiesterase inhibitors appear to depress sensitivity in a manner similar to light adaptation. The effects of the phosphodiesterase inhibitors on the voltage-dependent properties of the cell membrane in the d a r k were tested on the same cells, Fig. 2 shows currents elicited by 100 msec voltage d a m p command pulses in 10 mV steps in the presence and absence of 50 # M papaverine. The records contain an outward current during the c o m m a n d pulse and an outward tail current which occurs after the return of the membrane potential to its resting value. Both components of the currents are suppressed in 50 # M papaverine. Inhibitor-induced changes in the receptor potential are the
A
1 m M IBMX
t10na
B
5rnin 5raM Theophylline
50 HM P a p a v e r i n e Fig. 1. Effects of 3 phosphodiesterase inhibitors on inward currents elicited by very dim 20 msec test flashes of constant intensity. These are the result of successive application of each of the drugs in a single cell voltage clamped at its resting membrane potential.
367 ASW before
,
z:
ASW + 50 tJ M P a p a v e r i n e
ASW after
I
zc=
150 n a
I|O0 rnv
'
iO0 rns'
I
I5na 100 m v
Fig. 2. Effect of papaverine on voltage-dependent currents in a voltage clamped cell in the dark. Outward tail currents are shown in expanded scale in the bottom row. The lower trace in each record shows the membrane potential, upward deflections corresponding to membrane depolarization. The upper trace of each record is the membrane current that results from the corresponding change in membrane potential in the lower trace. Outward currents shown as an upward deflection. result of the depression of both the light-induced conductance and the voltage-dependent conductances of the membrane. The outward currents elicited by depolarization would ordinarily hasten repolarization of the receptor potential 14. Of the 3 inhibitors, papaverine was the most potent, being effective at concentrations < 100 ffM while 1BMX and theophyIline were effective at concentrations > 1 raM. If a rise in the intracellular level of cyclic nucleotides is the primary effect of the phosphodiesterase inhibitors, then it may be that both the desensitization produced by the drugs and by light adaptation in these photoreceptors is associated with a rise in intracellular cyclic nucleotide levels. In the above context it is interesting to point out that a rise in intracellular calcium may be involved in light adaptation of ventral photoreceptors2, 7. It has been suggested that intracellular calcium and cyclic nucleotide levels are linked together in other systems la, and this may be the case in Limulus photoreceptors where they both might affect the sensitivity of the cell. We suggest that the effect of the inhibitors on the voltage-dependent conductances may have a different mechanism than that of the effect on the light-activated conductances. In some ceils we observed that the voltage-dependent conductances recovered completely after treatment with inhibitors, while the sensitivity only partially recovered or failed to recover at all. Also, the converse would sometimes happen: the light response would fully recover and the electrical characteristics would only partially recover. These two findings taken together suggest that the coupling between the effects on the light-activated conductance and the effects on the voltagedependent conductances is loose, if it exists at all. This work was supported by grants from the National Institute of Health and the Rowland Foundation, and a Grass Fellowship to J.S.
368 1 Bitensky, M. W., Miki, N., Keirns, J. J., Keirns. M., Baraban, J. M., Freeman, J., Wheeler, M. A.. Lacy, J. and Marcus, R. R., Activation of photoreceptor disk membrane phosphodiesterase by light and ATP, Advanc. Cyclic Nucleotide Res., 5 (1975) 2t3-240. 2 Brown, J. E., Brown, P. K. and Pinto, L. H., Detection of li~t-induced changes ofintraceltular ionized calcium concentration in Limulus ventral photoreceptors using Arsenazo III, J. Physiol. (Lond.), 267 (1977) 299-320. 3 Chader, G., Fletcher, R., Johnson, M. and Bensinger, R., Rod outer segment phosphodiesterase: factors affecting the hydrolysis of cyclic-AMP and CYclic-GMP, Exp.-Eye Res., 18 (1974) 509-515. 4 Fein, A. and Charlton, J. S., Enhancement and phototransduction in the ventral eye of Limulus, J. gen. Physiol., 69 (1977) 553-569. 5 Fein, A. and Charlton, J. S., Increased intracellular sodium mimics some but not all aspects of photoreceptor adaptation in the ventral eye of Limulus, ,/. gen. Physiol., 70 (1977) 601-620. 6 Keirns, J. J., Miki, N., Bitensky, M. W. and Keirns, M., Link between rhodopsin and disk membrane cyclic nucleotide phosphodiesterase-action spectrum and sensitivity to illumination, Biochemistry, 14 (1975) 2760-2766. 7 Lisman. J. E. and Brown, J. E., The effects of intracetlular Ca ~+ on the light response and on light adaptation in Limulus ventral receptors. In G. P. Arden (Ed.), The Visual System: Neurophysiology, Biophysics and their Clinical Applications, 1972, pp. 22-23. 8 Lisman, J. E. and Brown, J. E., Effects of intraceltular injection of calcium buffers on light adaptation in Limulus ventral photoreceptors, J. gen. Physiol., 66 (1975) 489-506. 9 Miki, N., Keirns, J. J., Marcus, F. R., Freeman. J. and Bitensky, M. W., Regulation of cyclic nucleotide concentrations in photoreceptors: an ATP dependent stimulation of cyclic nucleotide phosphodiesterase by light, Proc. nat. Acad. Sci. i Wash. J. 70 (1973) 3820-3824. 10 Miki, N., Baraban, J. M., Keirns, J. J., Boyce, J. J. and Bitensky, M. W.. Purification and properties of light-activated cyclic nucleotide phosphodiesterase of rod outer segments. J. biol. Chem.. 16 (1975) 6320-6327. I 1 Millecchia, R. and Mauro, A., The ventral photoreceptor cells of Limulus. 111. A voltage-clamp study, J. gen. Physiol., 54 (1969} 331-351. 12 Miller, W. H., Cyclic nucleotides and photoreception, Exp. Eye Res., 16 (1973) 357-363. 13 Rasmussen, H. and Goodman. D. P. B., Relationships between calcium and cyclic nucleotides in cell activation, Physiol. Rev., 57 (19771 421-509. 14 Schmidt, J. A. and Fein, A., Effects of external calcium and calcium blocking agents on the photoresponse in Limulus ventral photoreceptors, Neurosci. Abstr.. 742 (1978) 237. 15 Wuiff, V. J.. The effect of cyclic AMP and aminophylline on Limulus lateral eye retinular cells, Vision Res., 13 (1973) 2335-2344.
365
Short Communications
Two effects of phosphodiesterase inhibitors on Limulus ventral photoreceptors
D. WESLEYCORSON, ALAN FEIN and JEFFREY SCHMIDT Laboratory o f Sensory Physiology, Marine Biological Laboratory, Woods Hole, Mass. 02543 and ( A. F.) Department of Physiology, Boston University School of Medicine, Boston, Mass. 02118 (U.S.A.)
(Accepted July [2th, 1979)
Recently, light has been found tc activate a phosphodiesterase responsible for cyclic nucleotide hydrolysis in frog rods (see review by Bitensky et al.1). The action spectrum of phosphodiesterase activation corresponds to the absorption spectrum of rhodopsin 6, and the speculation has been made that the reduction in cyclic nucleotide levels caused by activation of the phosphodiesterase may be involved in light and dark adaptation 1°. Previous studies by Wulff15 and Millera2 on Limulus photoreceptors indicate that in an invertebrate preparation, phosphodiesterase inhibitors reduce both the resting membrane potential and the receptor potential. We decided to investigate the possible role of cyclic nucleotides in excitation and/or adaptation in photoreceptors from the ventral eye of Limulus. Schmidt and Fein have reported that calcium blocking agents and phosphodiesterase inhibitors have similar effects on voltage-dependent conductances of ventral photoreceptors 14. In the study reported here, we have investigated whether known phosphodiesterase inhibitors had any direct effects on the light-activated currents elicited by dim flashes in voltage clamped ventral photoreceptors. Our findings suggest that phosphodiesterase inhibitors depress ventral photoreceptor sensitivity in a manner similar to light adaptation. In addition, the effects of the inhibitors on the voltage-dependent conductances a4 do not appear to be strongly linked to their effects on the light-activated conductance. Theophylline, isobutylmethylxanthine(IBMX) and papaverine suppress nucleotide hydrolysis in in vitro assays of rod outer segment phosphodiesterase activity3,9. All 3 inhibitors had qualitatively the same effect on ventral photoreceptors as described below. The methods of dissecting, viewing, stimulating and recording from the photoreceptor under voltage clamp have all been described in detail previously4,11. The preparation was suffused with artificial seawater (ASW) buffered at pH 7.0 with 10 mM Hepes, to which the inhibitors were added.
366 We sought to determine whether the reduction in the receptor potential described above reflects an actual reduction in the underlying light-induced current. As shown in Fig. 1, application of each of the 3 inhibitors produces a rapid and reversible depression of the inward currents elicited by dim test flashes. The desensitization is seen to occur without any appreciable shift in the baseline current recorded between test flashes. The inhibitors appear not to mimic the excitatory effect of light in that they do not produce an inward membrane current either transient or maintained. Under voltage clamp, plots of response amplitude vs light intensity in the region near threshold are well fitted by a line with a unit slope s. Light adaptation causes a simple translation of the response-intensity curve outward along the intensity axis 5,8. We measured response-intensity curves in the presence and absence of inhibitors in order to determine the similarity of inhibitor-induced desensitization to that induced by light. All response-intensity curves were seen to be well fitted by a line with unil slope. Those measured in the presence of inhibitors were found to be displaced out along the intensity axis, and the effect was reversible upon washing out the inhibitor. Phosphodiesterase inhibitors appear to depress sensitivity in a manner similar to light adaptation. The effects of the phosphodiesterase inhibitors on the voltage-dependent properties of the cell membrane in the d a r k were tested on the same cells, Fig. 2 shows currents elicited by 100 msec voltage d a m p command pulses in 10 mV steps in the presence and absence of 50 # M papaverine. The records contain an outward current during the c o m m a n d pulse and an outward tail current which occurs after the return of the membrane potential to its resting value. Both components of the currents are suppressed in 50 # M papaverine. Inhibitor-induced changes in the receptor potential are the
A
1 m M IBMX
t10na
B
5rnin 5raM Theophylline
50 HM P a p a v e r i n e Fig. 1. Effects of 3 phosphodiesterase inhibitors on inward currents elicited by very dim 20 msec test flashes of constant intensity. These are the result of successive application of each of the drugs in a single cell voltage clamped at its resting membrane potential.
367 ASW before
,
z:
ASW + 50 tJ M P a p a v e r i n e
ASW after
I
zc=
150 n a
I|O0 rnv
'
iO0 rns'
I
I5na 100 m v
Fig. 2. Effect of papaverine on voltage-dependent currents in a voltage clamped cell in the dark. Outward tail currents are shown in expanded scale in the bottom row. The lower trace in each record shows the membrane potential, upward deflections corresponding to membrane depolarization. The upper trace of each record is the membrane current that results from the corresponding change in membrane potential in the lower trace. Outward currents shown as an upward deflection. result of the depression of both the light-induced conductance and the voltage-dependent conductances of the membrane. The outward currents elicited by depolarization would ordinarily hasten repolarization of the receptor potential 14. Of the 3 inhibitors, papaverine was the most potent, being effective at concentrations < 100 ffM while 1BMX and theophyIline were effective at concentrations > 1 raM. If a rise in the intracellular level of cyclic nucleotides is the primary effect of the phosphodiesterase inhibitors, then it may be that both the desensitization produced by the drugs and by light adaptation in these photoreceptors is associated with a rise in intracellular cyclic nucleotide levels. In the above context it is interesting to point out that a rise in intracellular calcium may be involved in light adaptation of ventral photoreceptors2, 7. It has been suggested that intracellular calcium and cyclic nucleotide levels are linked together in other systems la, and this may be the case in Limulus photoreceptors where they both might affect the sensitivity of the cell. We suggest that the effect of the inhibitors on the voltage-dependent conductances may have a different mechanism than that of the effect on the light-activated conductances. In some ceils we observed that the voltage-dependent conductances recovered completely after treatment with inhibitors, while the sensitivity only partially recovered or failed to recover at all. Also, the converse would sometimes happen: the light response would fully recover and the electrical characteristics would only partially recover. These two findings taken together suggest that the coupling between the effects on the light-activated conductance and the effects on the voltagedependent conductances is loose, if it exists at all. This work was supported by grants from the National Institute of Health and the Rowland Foundation, and a Grass Fellowship to J.S.
368 1 Bitensky, M. W., Miki, N., Keirns, J. J., Keirns. M., Baraban, J. M., Freeman, J., Wheeler, M. A.. Lacy, J. and Marcus, R. R., Activation of photoreceptor disk membrane phosphodiesterase by light and ATP, Advanc. Cyclic Nucleotide Res., 5 (1975) 2t3-240. 2 Brown, J. E., Brown, P. K. and Pinto, L. H., Detection of li~t-induced changes ofintraceltular ionized calcium concentration in Limulus ventral photoreceptors using Arsenazo III, J. Physiol. (Lond.), 267 (1977) 299-320. 3 Chader, G., Fletcher, R., Johnson, M. and Bensinger, R., Rod outer segment phosphodiesterase: factors affecting the hydrolysis of cyclic-AMP and CYclic-GMP, Exp.-Eye Res., 18 (1974) 509-515. 4 Fein, A. and Charlton, J. S., Enhancement and phototransduction in the ventral eye of Limulus, J. gen. Physiol., 69 (1977) 553-569. 5 Fein, A. and Charlton, J. S., Increased intracellular sodium mimics some but not all aspects of photoreceptor adaptation in the ventral eye of Limulus, ,/. gen. Physiol., 70 (1977) 601-620. 6 Keirns, J. J., Miki, N., Bitensky, M. W. and Keirns, M., Link between rhodopsin and disk membrane cyclic nucleotide phosphodiesterase-action spectrum and sensitivity to illumination, Biochemistry, 14 (1975) 2760-2766. 7 Lisman. J. E. and Brown, J. E., The effects of intracetlular Ca ~+ on the light response and on light adaptation in Limulus ventral receptors. In G. P. Arden (Ed.), The Visual System: Neurophysiology, Biophysics and their Clinical Applications, 1972, pp. 22-23. 8 Lisman, J. E. and Brown, J. E., Effects of intraceltular injection of calcium buffers on light adaptation in Limulus ventral photoreceptors, J. gen. Physiol., 66 (1975) 489-506. 9 Miki, N., Keirns, J. J., Marcus, F. R., Freeman. J. and Bitensky, M. W., Regulation of cyclic nucleotide concentrations in photoreceptors: an ATP dependent stimulation of cyclic nucleotide phosphodiesterase by light, Proc. nat. Acad. Sci. i Wash. J. 70 (1973) 3820-3824. 10 Miki, N., Baraban, J. M., Keirns, J. J., Boyce, J. J. and Bitensky, M. W.. Purification and properties of light-activated cyclic nucleotide phosphodiesterase of rod outer segments. J. biol. Chem.. 16 (1975) 6320-6327. I 1 Millecchia, R. and Mauro, A., The ventral photoreceptor cells of Limulus. 111. A voltage-clamp study, J. gen. Physiol., 54 (1969} 331-351. 12 Miller, W. H., Cyclic nucleotides and photoreception, Exp. Eye Res., 16 (1973) 357-363. 13 Rasmussen, H. and Goodman. D. P. B., Relationships between calcium and cyclic nucleotides in cell activation, Physiol. Rev., 57 (19771 421-509. 14 Schmidt, J. A. and Fein, A., Effects of external calcium and calcium blocking agents on the photoresponse in Limulus ventral photoreceptors, Neurosci. Abstr.. 742 (1978) 237. 15 Wuiff, V. J.. The effect of cyclic AMP and aminophylline on Limulus lateral eye retinular cells, Vision Res., 13 (1973) 2335-2344.
