|Year : 1992 | Volume
| Issue : 1 | Page : 11-14
Effect of SH reagents on rod photoresponse of isolated frog retina
SA Salehi1, M Takagi2, T Shinozawa3, T Matsuura4
1 Division of Molecular Biology, Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Otsaka 560, Japan
2 Department of Biology, College of General Education, Osaka University, Toyonaka, Osaka 560, Japan
3 Department of Biological and Chemical Engineering, Gunma University, Kiryu, Gunma 376, Japan
4 Department of Physiology, Kinki University School of Medicine, Osakasayama-shi 589, Japan
Department of Biology. College of General Education, Osaka University, Toyonaka, Osaka 560
Source of Support: None, Conflict of Interest: None
Several SH reagents, N-ethylmaleimide (NEM), p-chloromercuribenzoic acid (PCMB), p-chloromercuribenzene sulphonate (PCMBS) and monoiodoacetic acid (MIAA) changed the wave form and the peak of the amplitude of the photoresponse remarkably. The effects of amino group modifying reagents, ethyl acetimidate (EA) and isethinyl acetimidate (ITA) on photoresponse were very slight. The possibility of a SH protein as cGMP-sensitive cation channel protein is discussed.
Keywords: Frog retina, Photoresponse, SH reagents, N-ethylmaleimide, Amino group modifying reagents
|How to cite this article:|
Salehi S A, Takagi M, Shinozawa T, Matsuura T. Effect of SH reagents on rod photoresponse of isolated frog retina. Indian J Ophthalmol 1992;40:11-4
|How to cite this URL:|
Salehi S A, Takagi M, Shinozawa T, Matsuura T. Effect of SH reagents on rod photoresponse of isolated frog retina. Indian J Ophthalmol [serial online] 1992 [cited 2020 May 30];40:11-4. Available from: http://www.ijo.in/text.asp?1992/40/1/11/24421
| Introduction|| |
The molecular mechanism of phototransduction in vertebrate photoreceptor cells was recently clarified; (1) the adsorption of light by rhodopsin decreases the concentration of 3',5' cyclic guanosine monophosphate (cGMP) in the rod outer segments (ROS) through the activation of (cGMP) phosphodiesterase , (2) the decrease of cGMP concentration closes the cation channels (in the plasma membrane of ROS) which is directly regulated by cGMP , and (3) the closure of cation channel in the ROS generates a hyperpolarized receptor potential . The detection of two common SH proteins (Mw of 250K and 1OOK) which reacted with 4 [ 3 H]N- Ethylmaleimide from the outside of frog ROS  and also bound cGMP , urged us to test the effect of the SH reagents on the rod photoresponse of the isolated frog retina.
| Material and methods|| |
Measurement of the photoresponse of the intact frog retina was done by the modification of the method used by Ussing and Zerraiin . The perfusate was a modified Conway's solution  containing in mM: NaCl 67; KCL 2.5; Ca gluconate 0.9; MgSo 4 1.2; Na 2 SO 4 0.6; NaHCO 3 25; Na 2 HPO 4 2.3; NaH 2 PO 4 0.7; and glucose 26. This was supplemented with 15 mM sodium asparate and the PH was adjusted to 7.4-7.6 (hereafter called normal physiological solution, NIPS). SH reagents and amino group modifying reagents (all the SH reagents were purchased from Sigma) were freshly dissolved in NPS, whenever needed, and added to the perfusate. The perfusate was continuously bubbled with a mixture of 98% oxygen and 2°o CO2, which also helped mixing the test reagents after being added. All experiments were performed under dim red light at 15-18° C
Bullfrogs (Rana catesbeiana) were kept on 12/12 hr:dark/light schedule for 1-3 weeks prior to use and fed Gaines Dog Food (Ajinomoto General Food Co., Tokyo) supplemented with Vitamin A. Frogs were dark adapted for at least 4-6 hr before killing. After killing the frogs by pithing, their eyes were enucleated. These were then hemisected, the cornea, lens and vitreous humor were discarded and the posterior half of the eyecup was cut into half in oxygenated NPS. The optic nerve was cut from the outside of the eyecup. The pigment epithelium was gently removed, the retina was then carefully detached and put, receptor side up, on a piece of filter paper with a hole of about 3 mm in its center. This was quickly clamped between the two halves of the perfusion chamber, each having a 3 mm hole, and sealed with vaseline. The chamber was kept inside a light tight box in total darkness for 30 min until the photoresponse threshold stabilized. Two 2% agar/KCL bridges were connecting the two compartments of the chamber to the two Calomel electrodes, one scleral to the retina and the other vitreal. The source of light was a photographic flash (Sunpack Co., Japan) from where the light was conveyed to the chamber by fiberoptics, the intensity of which was controlled by neutral density filters (Fuji Photo Film Co., Japan) The intensity of light at the surface of the chamber was 2 X 10  photons/um  flash and its duration was 5 msec. The intensity of light (log2 unattenuated = -5) was within the scotopic range, stimulating only the rod photoreceptors. The signals induced by flashes passed through a capacity compensating A-C amplifier, then through a data processor (ATAC 201, Niho Kohden) and finally recorded on chart by a pen recorder (Toa Denpa Co.).
| Results and discussion|| |
[Figure - 1] shows that 1.0 mM of the SH reagent has marked effect on both peaks of the amplitude of response (Vmax) and the decay times. These effects corresponded to incubation time 5, 10, 15 minutes. The effects of 0.3 and 0.1 mM of these reagents were less than that of 1.0 mM (data not shown).
The effect of various concentrations of these reagents on the photoresponse of isolated and aspartate treated retina to flashes (2 X 10  photons/um  flash) at two minutes intervals is shown in [Figure - 2]. The term photoresponse used throughout this report refers to the amplitude of Pill component of the electroretinogram (ERG). Treatment with 1.0 mM NEM caused marked (60%) decrease of photoresponses in three minutes. In contrast, the effect of MIAA, was gradual, though total diminishing of the photoresponse resulted in 20 minutes after treatment. (n comparison to NEM and MIAA, the effect of 1.0 mM of PCMB and PCMBS were weaker, i.e. they respectively caused 600/0 and 50% decrease of the photoresponse in 20 minutes [Figure - 2]A. The effect of 0.3 mM of SH reagents was significant, but weaker than that of 1.0 mM. NEM and MIAA caused about 80% abolishment of photoresponse in 20 minutes, while PCMB and PCMBS caused 30% and 15% decrease in 20 min [Figure - 2]B and no further decrease even if incubated for 60 - min (data not shown). Except for 0.3 and 0.1 mM PCMBS, the above effects were irreversible, i.e. even when these reagents were discarded and the retina washed three times with NPS. the photoresponse did not recover. Concentrations below 0.1 mM of SH reagents had no decreasing effect on the photoresponse, but rather increased it. For instance, 0.03 mM of PCMBS caused almost 1.3 fold increase [Figure - 3]. The threshold concentration of SH reagents seemed to be at 0.1 mm.
Five mM of amino group modifying reagents, EA and ITA, were tested, because lower than this concentration had no effect on the photoresponse. Even this concentration of ITA had no effect, while 5 mM had a slight decreasing effect [Figure - 2]A.
The relative photoresponse was approximately proportional to the log concentration of the SH reagents [Figure - 3]. The addition of 3 mM NEM and MIAA completely abolished the photoresponse after 20 minutes incubation. While, higher concentrations (5 mM) of PCMB and PCMBS were required to abolish the response completely.
The results described above show that higher than 0.1 mM of SH reagents (NEM, PCMB, PCMBS and MIAA) has marked effect on the photoresponse and wave form of isolated frog retina. Lower concentrations (0.1 mM and below) , however, had no decreasing effect, but on the contrast, they increased the photoresponse to about 1.3 fold. This is in agreement with Cone, R.A. (personal communication; U.S. - Japan Joint Seminar on Molecular Mechanism in Visual Excitation. 1984. Honolulu.) who reported the increase of sodium influx by the addition of NEM less than 0.1 mM . Since his experiment was carried out by the method of Korenbrot and Cone  using isolated ROS, this influx probably caused by the opening of the cGMP sensitive cation channels in the ROS.
The Pill response was markedly affected by the addition of SH reagents. It is well known that the Pill response is composed of the fast Pill (= receptor potential ) and the slow Pill which arises from Muller cells  in response to the light-induced decrease in the extracellular potassium concentration in the photoreceptor layer ,..
The slow Pill develops and rises very slowly in comparison to the fast Pill. Although our recording system with the A-C coupled amplifier and with the light flash stimulus was unfavourable to record the slow Pill, the slow component might. to a small extent, have been superimposed on the recordings. But, it seems to be difficult to assume that these SH reagents affect the Muller cell selectively. So far, in acute experiments, only the barium is known to suppress selectively the slow Pill  by decreasing the potassium conductance of Muller cell membrane . If the SH reagents had selectively affected the Muller cells, the fast Pill must have remained during the course of the experiments. As shown in [Figure - 3], the relative photoresponse was approximately proportional to the log concentration of the SH reagents, and 3 mM NEM, MIAA, 5mM PCMB, PCMBS completely abolished the photoresponse. Therefore, it will be safe to suppose that the site of the action of SH reagents in our experiments is the photoreceptors.
As shown in [Figure - 2]C, the effect of SH reagents on the photoresponse is biphasic, a sudden increase followed by a gradual decrease. This suggests that there might be two, high affinity and low affinity, sites for these reagents. The modification of high affinity site by low concentration of SH reagents causes an increase of the amplitude of photoresponse, presumably because of the opening of the ion channels in the plasma membrane of the ROS (Cone, R. A. 1984, personal communication). On the other hand the modification of low affinity site by high concentration of SH reagents causes a decrease of the amplitude of photoresponse, probably by closing the ion channels. The effects of NEM, MIAA and PCMB on all the tested criteria were irreversible. The reason could be that NEM and MIAA form convalent bonds  while PCMB forms mercaptide bonds  with SH groups. Contrary to that, the effect of PCMBS was partially reversible. This can be contributed to the fact that PCMBS penetrates very poorly and slowly (if at all) into the cell membrane sub there fore in the present experiments they might have reacted loosely with a few superficial SH groups which on washing might have been removed.
Comparing the effects of PCMB and PCMBS, which are very similar to each other , except their permeability, it is noticed that the effect of PCMB is irreversible and stronger that of PCMBS, while that of the latter being partly reversible. This suggest the presence of two types of photosensitive SH groups, one situated on the surface of the plasma membrane and the other located at the interior of the photoreceptor cells. Since penetration of PCMBS into the cell takes several hours  and in our experiments the average incubation time for the reading of the effect of these reagents is 30 min, this time is by no means enough for PCMBS to penetrate through the cell membrane and affect those intracellular components of the retina which are part of the phototransduction. Therefore it could be assumed that PCMBS might have affected the SH groups at the surface of the ROS and the affected SH groups have active role in phototransduction. Furthermore, our findings in the case of treatment of retina with [  H]NEM  shows that when [  H]NEM was applied on the intact retina, only 7 ROS integral membrane proteins were labeled, in contrast its application on disrupted retina resulted in the labeling of numerous proteins. This also suggests the presence of two types of SH groups, a superficial and a profound one, in the frog photoreceptor cells. We could not detect (by the labeling with [  H]NEM) the protein with Mw 63K which was reported as a cGMP sensitive cation channel protein by Cook et al  or with 66K which bound cGMP reported by us . This may be because of the presence of two types of (1-cis-diltiazem sensitive and insensitive) of cGMP sensitive cation channel proteins as suggested by Koch et al  and Pearce et al . Thus it is possible that some of these 7 proteins which have superficial SH groups might be related to ion channels.
Though amino group modifying reagents, EA and ITA, are commonly used for the modification of amino group of proteins and their reaction with these groups are believed to be very specific , they did not show any significant effect on the photoresponse.
The results of our experiments are compatible with the possibility that SH proteins which interact with cGMp in the plasma membrane of frog ROS participated in the phototransduction and propose a test by the method carried out by Fesenko et al .
| Acknowledgement|| |
The authors are highly grateful to Dr. I. Hanawa of Kobe University, School of Medicine for providing the amplifier. This work was supported by a Grant-in Aid for Special Project Research on (to TS. No. 01304060) from the Japanese Ministry of Education, Science and Culture.
| References|| |
Miki, N.. Keirns... F, Freeman, J. and Bitensky, M.: Regulation of cyclic nucleotide concentrations in photoreceptors: An ATP-dependent stimulation of cyclic nucleotide phosphodiesterase by light. Proc Natl. Acad. Sci. U. S. A., 70: 3820-3824, 1973.
Fesenko, E. E.. Kolesnikov. S.S. and Lyubarsky, A.L Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment. Nature 313: 310-313. 1985.
Tomita T: Electrical activity of vertebrate photoreceptors. O. Rev. Biophys.. 3: 179-222. 1970
Shinozawa. T. Salehi S.A. and Takagi. M. Labeling of frog rod outer segment proteins with [H]N-ethylmaleimide. sci Rep . Col. Gen. Educ. Osaka University. 34. 23-27, 1985.
Shinozawa, T. Sokabo. M.. Terada. S.. Matsusaka, H. and Yoshinozawa. T: Detection of cyclic GMP binding protein and ion channel activity in frog rod outer segments J. Bichem.. 102:281-290. 1987
Shinozawa. T. Terada. S.. Matsuska. H. and Yamashita, S : Detection of Ca2+ -dependent cyclic GMP binding protein in frog rod outer segments. FEES Lett., 219: 293-295, 1987.
Ussing, H.H. and Zerraiin. K.: Active transport as the source of electrical current in the short circuited isolated from skin Acta. Physiol. Scand., 23: 109-127, 1951.
Hanawa. I.. Ando. H. and Takahashi, K: Enhancement of visual cell response after illumination in the isolated frog retina. Exp. Eve Res.. 32: 719-727. 1981.
Korenbrot. J.I. and Cone. R.A.: Dark ionic flux and the effects of light in isolated rod outer segments J. Gen. physiol . 6n. 20-45, 1972.
Faber, D.: Analysis of the slow transretinal potential in response to light. Ph.D. Thesis. SUNY and Buffalo N. Y.. 1969.
Oakley, B. IL: Measurement of light-induced transient changes in extracellular potassium ion concentration in the frog retina. Ph. D. Dissertation, University of Michigan. 1975.
Matsuura, T. Miller. W.H. and Tomita, T.: Cone-specific c-wave in the turtle retina . Vision Res . 18: 767-775. 1978.
Fujimoto, M, and Tomita. T: Reconstruction of the slow Pill from the rod potential. Invest Ophthalm. Visual Sci., 18. 1090-1093. 1979.
Bolnick. D. A., Walter A.E and Sillan. A. J : Barium suppresses, slow Pill in perfused bullfrog retina. Vision Res.. 19 : 1117-1119. 1979.
Matsuura. T: Effect of barium on separately recorded fast and slow PHI responses in bullfrog retina. Experientia. 40: 817-819. 1984.
Benesch. R. and Benesch. R. E. The chemistry of the Bohr effect. I The reaction of N-ethylmaleimide with the oxygen-linked acid groups of hemoglobin. J. Biol. Chem- 236: 405-411, 1969.
Motais. R. and Sola. F: Characteristics of a sulfhydryl group essential for sodium exchange diffusion in beef erythrocyte J Physic, . 233: 423-4338, 1973.
Van Steveninnck, J.. Weed, R. I and Rolhstein. A.. Localization of erythrocyte membrane sulfhydryl groups essential for glucose transport. J. Gen. Physiol.. 48: 617-632. 1965.
Cook, N J., Hanke. W. and Kaupp. U. B. Identification, purification and functional reconstitution of the cyclic GMP dependent channel from rod photoreceptors. Proc. Will. Acad. Sci. U S.A.. 84: 585-589. 1987.
Koch, K-W.. Cook. N.J. and Kaup. U.B . The cGMP dependent channel of vertebrate rod photoreceptors exists in two forms of different cGMP sensitivity and pharmacological behaviour. J. Biol. Chem.. 262: 1441514421, 1987.
Pearce. L. B. Calhoon, R.D. Burn PR. Burn. PR., Vincen. A. and Goldin S.M.: Two functionally distinct forms of guanosine cyclic 3. 5'-phosphate stimulated cation channels in a bovine rod photoreceptor disk preparation. Biochemistry. 27 4396-4406. 1988
Plapp. B. V.: Enhancement of the activity of horse liver alcohol dehydrogenase by modification of amino groups at the active sites. U Bio. Chem.. 245 1727-1735. 1970.
[Figure - 1], [Figure - 2], [Figure - 3]