Article Figures & Data
Figures
- Figure 1.
Circadian modulation of the sensitivity of CNGCs to cGMP in cultured cone photoreceptors. Data were obtained from inside-out patch recordings immediately after patch excision at various times of day on the second day of DD. A, examples of concentration-response relationships in two patches excised during the subjective day (CT6–CT8) and during the subjective night (CT16–CT20), as indicated. Superimposed curves are nonlinear least squares fits to the Hill equation. B, ratio of the currents evoked by 20 μm cGMP and 200 μm cGMP in individual patches is inversely correlated with the K(1/2) determined from Hill equation fits to complete concentration-response curves. Each data point on this graph was from a different patch excised at various times of day. C, A statistically significant (*p < 0.05) circadian rhythm in cGMP sensitivity can be readily detected from the ratios of the currents evoked by 20 and 200 μm cGMP in excised inside-out patches.
- Figure 2.
Expression of transcripts encoding somatostatin receptor subtypes as determined by RT-PCR in retinal preparations highly enriched in cultured cone photoreceptors. Primers were designed to amplify the complete coding sequences, and products of the expected size were identified by sequencing. We observed that transcripts encoding sst2, sst3, sst4, and sst5 are expressed in cone-enriched retinal cell cultures.
- Figure 3.
Modulation of native cone CNGCs by SS14. The peptide was applied to cultured retinal cells 15 min or 2 h before patches were excised from cones. Sensitivity to cGMP was measured from the ratios of the currents evoked by 20 and 200 μm cGMP. A, B, Treatment with 100 nm SS14 for 15 min or 2 h decreased the sensitivity of CNGCs to cGMP during the subjective night in cones maintained on LD cycles (A) or on the second day of DD (B). In all figures, asterisks indicate *p < 0.05 or **p < 0.01 compared to controls shown by brackets.
- Figure 4.
Modulation of native cone CNGCs by SS28. The peptide was applied to cultured retinal cells 15 min or 2 h before patches were excised from cones. A, In retinal cells cultured under LD cycles, SS28 (100 nm) applied for 15 min or 2 h evoked a decrease in the sensitivity of CNGCs to cGMP during the subjective night. However, 15 min treatment with SS28 also caused an increase in channel sensitivity to cGMP early in the day (ZT4–ZT6), but not later in the day, after ZT6 (see data point with star). This increase in sensitivity effect was not seen after 2 h of SS28 treatment. B, The effects of SS28 are identical to those of SS14 in cells maintained in DD. Note that 15 min exposure to SS28 does not produce an increase in channel sensitivity to cGMP early in the subjective day in cells free-running in DD.
- Figure 5.
The effects of SS28 are mediated by multiple G-proteins. Cultured photoreceptors were treated with PTX (200 ng/ml) or with control medium for 16 h before application of 100 nm SS14 or SS28. Peptides were present for 15 min before patch excision. A, The effects of SS14 persist in cells pretreated with PTX. These data are from cells analyzed on the second day of DD, but the same result was obtained for cells in maintained in LD (data not shown). B, The nocturnal effect of SS28 also persists after PTX pretreatment. However, the increase of CNGC sensitivity to cGMP observed during the early day in cells maintained in LD cycles was completely blocked by PTX pretreatment.
- Figure 6.
The selective SRIF1 agonist MK-678 evokes a light-dependent increase in CNGC sensitivity during the early part of the day. In these experiments, MK-678 (100 nm) or vehicle was added to cultured cells 15 min before patch excision. A, Treatment with MK-678 evoked an increase in the sensitivity of CNGCs to cGMP during the early day (ZT4–ZT6) in cone photoreceptors cultured in LD cycles, but not in cells previously maintained in DD for 2 d before analysis. MK-678 differs from SS14 and SS28 in that it does not have any effect on the sensitivity of CNGCs to cGMP during the night. B, The effect of MK678 is completely blocked by PTX pretreatment.
- Figure 7.
Activation of PLC is sufficient and necessary to cause an increase in CNGC sensitivity to cGMP during the early day time. A, Application of the PLC activator m-3M3FBS (50 μm) causes an increase in the sensitivity of cone CNGCs to cGMP throughout the day and this effect can be observed in cells maintained in either LD and DD. B, Pretreatment with the PLC inhibitor ET-18-OCH3 (50 μm) can block the effects of MK-678 or m-3M3FBS on CNGCs observed during the day. C, Treating intact cells with the short-chain DAG analog 1,2-DiC8 (25 μm) during the day mimics the effect of m-3M3FBS, as it causes an increase in the sensitivity of CNGCs to cGMP. However, this effect is antagonized by simultaneous administration of the PKC inhibitor Ro-31–8220 (500 nm).
- Figure 8.
A live-cell imaging assay indicates that MK-678 causes activation of PLC during the early part of the day (ZT4–ZT6). Cultured cone photoreceptors were transfected with an expression vector encoding a PLCδ1-PH domain/GFP fusion protein, which dissociates from the plasma membrane with activation of PLC and cleavage of PtdIns[4,5]P2. Confocal micrographs of GFP fluorescence were taken from the same cells before and after application of MK-678. A, fluorescence observed in a photoreceptor during the early day (ZT4) in a cell maintained previously in LD. The graph below the micrograph shows fluorescence intensity as a function of distance along the arbitrarily drawn red line, indicating that the most intense fluorescence is in the periphery of the cell. B, Micrograph of the same cell 15 min after application of MK-678. The fluorescence intensity in the interior of the cell is markedly increased, as indicated by the higher plateau between the two peaks in the fluorescence intensity plot. C, D, A similar experiment in a cell observed later in the day, at ZT8 is shown before (C) and after (D) application of MK-678. Note that there is no rearrangement of fluorescence at that time of day. E, Quantitative results from several cells shows that MK-678 causes a statistically significant rearrangement of fluorescence, measured as a change in the fraction of signal in cell periphery, during the early part of the day (ZT4–ZT6), but not later in the day (after ZT6).
