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Dark voltage and light responses of isolated retinal rods of Rana esculenta were investigated by employing the whole-cell patch-clamp technique. When the recording pipette was filled with a medium devoid of nucleotides, a spontaneous hyperpolarization of the dark voltage partly due to a diffusional loss of cGMP and its precursor GTP and a retardation in the recovery of the light responses was observed. The larger part of the retardation of the light responses was prevented by 1 mM ATP. Addition of GTP attenuated the hyperpolarization, but did not abolish it completely. When the nitric-oxide-releasing substance sodium nitroprusside plus GTP was applied, the tendency of hyperpolarization disappeared and a stable dark voltage or even a slight depolarization was measured during the whole-cell recording period. Similar results were also obtained when GTP was given in combination with either EGTA or IBMX which are both known to interfere with the cGMP regulating enzymes in retinal rods. In addition to its effects on the dark voltage, an acceleration of the recovery phase of the light responses by sodium nitroprusside was also observed. Our observations strongly suggest that sodium nitroprusside activates guanylate cyclase in photoreceptors, as it does in other tissues, but we cannot exclude with certainty an effect on the phosphodiesterase.

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Isolated vertebrate retinas bathed in circulating Ringer solution cannot regenerate all of their bleached visual pigments. When dioleoyl-lecithin vesicles containing certain retinol congeners are added to the Ringer solution, such retinas begin to regenerate pigment immediately. The visual pigment of a bleached perfused retina can now be restored fully, making the isolated retina an independent unit for study. Loposomes can protect oxygen-sensitive, lipid-soluble substances and deliver them to living cells.

Single frog rods consisting of the outer segment and the ellipsoid were investigated by the whole-cell patch-clamp technique. When the recording pipette was filled with a simple intracellular medium containing potassium as the principal cation, a slow increase in dark voltage (hyperpolarization) associated with a decay of the photoresponses was observed. The hyperpolarization started at a dark voltage of -27 ± 8 mV, followed an exponential course, and leveled out at -52 ± 6 mV. The time constant was proportional to the access resistance of the preparations. With a pipette medium containing a 0.5 or 1.0 /iM cGMP, the initial dark voltage was shifted to more positive values and the tendency of hyperpolarization was clearly attenuated. Similar results were obtained with 1 mM GTP. The effects of GDP and of ATP were less significant. In experiments with 1 mM GTP plus 1 mM ATP, the dark voltage behaved as in experiments with only GTP. The stabilizing action of GTP was amplified by EGTA so that with 1 mM GTP plus 1 mM free EGTA the dark voltage was stable at a level of -15 mV. It is concluded that the preparations lose intracellular components such as cGMP and GTP by diffusion into the recording pipette and that the losses are prevented or reduced when the pipette medium contains these nucleotides in nearly physiological concentrations. For the internal transmitter cGMP, the results suggest that its free concentration does not exceed 1 ^M.

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Abstract— The dark current of retinal rods is suppressed for an extended period when their rhodopsin is bleached. An 8% bleach completely suppresses the current for 8 min and after 35 min it is fully recovered. The dark current can recover fully from a bleaching flash without any rhodopsin being regenerated. Moreover the recovery can be hastened either by lowering the activity of calcium ions surrounding the rods or by regenerating the rhodopsin. The recovery of the dark current following these bleaches showed zero order kinetics, regardless of whether the recovery was hastened by low calcium, 11‐cis retinaldehyde or not. If all the rhodopsin is bleached in the retina, the dark current does not recover; the addition of 11‐cis retinaldehyde, but not all‐trans retinaldehyde, to the bleached retina causes the dark current to begin its recovery as early as 10 min after the addition with zero order kinetics (1.3% min‐1). In two of three similar experiments, the dark current recovered 100%. When the recovery rate of the dark current from the retina showing the earliest response is compared with the rate of the regeneration of rhodopsin in the plasma and disc membranes, the dark current begins its recovery after the plasma membrane rhodopsin is fully regenerated and the disc rhodopsin is half regenerated. When the disc rhodopsin is fully regenerated, the dark current is recovered 75%, and 20 min later it is completely recovered.