The somatostatin receptor 2 (sst2) agonist, MK-678, mimics the effect of SS14. A: L-VGCC current densities were larger when photoreceptors were recorded during the subjective night (CT 16–19; ▪) than during the subjective day (CT 4–7; •). B and C: application of MK-678 (100 nM) for 2 h prior to recordings had no effect during the subjective day (CT 4–7; ○; B) but caused a significant decrease in L-VGCC current density during the subjective night (CT 16–19; □; C). D: MK-678 significantly inhibited the maximum L-VGCC current density (I_max) during the subjective night but not the subjective day. Control (Con) CT 4–7: 3.9 ± 0.7 pA/pF, n = 11; control CT 16–19: 6.4 ± 0.8 pA/pF, n = 13; MK-678 CT 4–7: 5.4 ± 1.4 pA/pF, n = 13; MK-678 CT 16–19: 2.5 ± 1.0 pA/pF, n = 8. *P < 0.05.

The inhibitory effect of SS14 on L-VGCCs during the subjective night is mediated through the PTX-sensitive G protein. Cells were treated with PTX (200 ng/ml) for 20–24 h prior to recordings. A: treatment with PTX did not affect L-VGCC current density. B: treatment with SS14 for 2 h (○) decreased L-VGCC current density, but in the presence of PTX, SS14 did not affect L-VGCCs (•). C: treatment with PTX reversed the inhibitory effect of SS14 on the maximum L-VGCC current density (I_max). Control (Con): 6.6 ± 0.7 pA/pF, n = 15; PTX alone (PTX): 5.9 ± 1.8 pA/pF, n = 5; SS14 for 2 h (SS14): 3.5 ± 0.8 pA/pF, n = 22; treatment with SS14 in the presence of PTX (PTX + SS14): 6.0 ± 0.5 pA/pF, n = 15.

Intracellular calcium stores are present in cultured chicken cone photoreceptors. Dark-adapted cultured retinal cells were loaded with Fluo-4 AM. Treatment with thapsigargin triggered a surge of intracellular calcium followed by depletion of the intracellular calcium. Top: an example of a photoreceptor DIC image as well as 4 different fluo-4 images at different time points (2–5) following thapsigargin addition. Only elongated cone photoreceptors with oil droplets (as in circles) were analyzed. Bar = 3 μm. Bottom: an average from a group of photoreceptors. The arrow indicates the time when thapsigargin was applied. The normalized intensity represents the ratio of fluo-4 fluorescence intensity at any time point to basal fluo-4 fluorescence intensity (at time 0).

The inhibitory effect of SS14 on L-VGCCs during the subjective night requires intracellular Ca²⁺. A: the Ca²⁺ chealator BAPTA-AM (13 μM; ○) significantly decreased L-VGCC current density. B: Treatment with SS14 for 2 h (○) decreased L-VGCC currents, but BAPTA-AM reversed the inhibitory effect of SS14 on L-VGCCs (BAPTA-AM + SS14; •). C: the inhibitory effect of SS14 on the maximum L-VGCC current density was reversed by BAPTA-AM. Treatment with BAPTA-AM alone for 2 h significantly decreased the maximum L-VGCC current density (I_max). Treatment with 0.1% DMSO as controls (Con): 8.3 ± 0.9 pA/pF, n = 17; 13 μM BAPTA-AM dissolved in 0.1% DMSO (BAPTA-AM): 6.4 ± 0.8 pA/pF, n = 21; treatment with SS14 for 2 h (SS14): 5.4 ± 0.9 pA/pF, n = 11; treatment with SS14 in the presence of BAPTA-AM (SS14 + BAPTA-AM): 8.4 ± 0.8 pA/pF, n = 16; *P < 0.05.

There is a circadian phase-dependent modulation of cone L-type voltage-gated calcium channels (L-VGCCs) by somatostain-14 (SS14). The current density-voltage (I-V) relationship was obtained from whole cell recordings on the 2nd day of constant darkness (DD). A: the L-VGCC currents were larger during the subjective night [circadian time (CT) 16–19; solid square] than during the subjective day (CT 4–7; solid circle). B: treatment with SS14 (500 nM) for 2 h prior to recordings (○) did not have significant effects on the current density during the day. C: treatment with SS14 for 2 h (□) significantly decreased the L-VGCC current density during the night compared with the control (▪). D: treatment with SS14 for 15 min prior to recordings (○) significantly inhibited the L-VGCCs during the subjective night. E: 2 representative traces recorded during the subjective night from the control (con) and SS14-treated (SS14 2h) groups show currents elicited from −65 to 0 mV. The maximum currents were obtained when the steps depolarized to 0 ∼10 mV. F: treatment with SS14 for 2 h or 15 min prior to recordings significantly decreased the maximum L-VGCC current density (I_max) elicited from −65 to 0 mV during the subjective night. The maximum current density of voltage step to 0 mV: control CT 4–7 (con CT 4–7): 5.0 ± 0.7 pA/pF, n = 24; control CT 16–19 (con CT 16–19): 7.4 ± 0.9 pA/pF, n = 21; treatment with SS14 for 2 h (SS14 for 2 h) CT 4–7: 5.5 ± 0.6 pA/pF, n = 26; SS14 for 2 h CT 16–19:3.4 ± 0.8 pA/pF, n = 26; treatment for 15 min control (con) CT 16–19: 7.9 ± 0.8 pA/pF, n = 14; treatment with SS14 for 15 min (SS14 for 15 min) CT 16–19: 5.2 ± 0.7 pA/pF, n = 15, * P < 0.05. G: the protein levels of L-VGCCα1D in cultures harvested during the subjective night (CT 17) were significantly higher than during the subjective day (CT 5) in controls. Treatment with SS14 (500 nM) for 2 h significantly decreased the L-VGCCα1D during the subjective night, *, P < 0.05.

The inhibitory effect of SS14 during the subjective night is mediated through somatostatin receptor 2 (sst2). A: application of the sst2 antagonist cyanamid-154806 (1 mM) for 2 h (○) had no effect on L-VGCCs compared with control (▪). B: treatment with SS14 for 2 h (○) significantly decreased L-VGCC current density, while cyanamid-154806 reversed the inhibitory effect of SS14 on L-VGCC current density (•). C: SS14 significantly decreased the maximum current density of L-VGCCs. Treatment with cyanamid-154806 did not have any effect on the maximum L-VGCC current density (I_max), but it significantly reversed the inhibitory effect of SS14 on L-VGCC maximum current density. Control (Con): 6.2 ± 0.6 pA/pF, n = 15; cyanamid-154806: 5.6 ± 0.7 pA/pF, n = 15; SS14: 3.3 ± 1.0 pA/pF, n = 9; cyanamid-154806 + SS14: 6.5 ± 1.0 pA/pF, n = 13.

Inhibition of PLC reverse the inhibitory effect of SS14 on L-VGCCs during the subjective night. A: there was a dose-dependent effect of ET-18-OCH₃, a phospholipase C (PLC) inhibitor, on L-VGCCs. Treatment with 25 μM ET-18-OCH₃ for 2 h (○) did not have a significant effect on L-VGCC current density, but 50 μM ET-18-OCH₃ (▵) increased L-VGCC current density (*P < 0.05). B: the inhibitory effect of SS14 (○) on L-VGCCs was reversed by 25 μM ET-18-OCH₃ (ET-18-OCH₃+SS14, •). C: treatment with 25 μM ET-18-OCH₃ alone (ET-18-OCH₃) had no significant effect on the maximum L-VGCC current density (I_max), but it significantly reversed the inhibitory effect of SS14 (ET-18-OCH₃ + SS14). Control (Con): 7.5 ± 0.7 pA/pF, n = 12; 25 μM ET-18-OCH₃ alone (ET-18-OCH₃): 8.8 ± 1 pA/pF, n = 9; SS14: 5.6 ± 0.8 pA/pF, n = 16; treatment with SS14 in the presence of ET-18-OCH₃ (SS14 + ET-18-OCH₃): 10.2 ± 0.9 pA/pF, n = 13; *P < 0.05.

The inhibitory effect of SS14 on L-VGCCs during the subjective night requires Ca²⁺ from intracellular stores. A: treatment with thapsigargin (in 0.05% DMSO) for 2 h (5 μM; ○) had no effect on the L-VGCC currents. B: treatment with SS14 for 2 h in the presence of thapsigargin (thapsigargin + SS14, •) reversed the inhibitory effect of SS14 (○) on L-VGCCs. C: SS14 significantly decreased the maximum L-VGCC current density (I_max), but thapsigarin reversed the inhibitory effect of SS14. Control (Con): 7.1 ± 0.9 pA/pF, n = 7; thapsigargin alone for 2 h (thapsigargin): 7.7 ± 1.3 pA/pF, n = 10; SS14 for 2 h (SS14): 4.6 ± 1.0 pA/pF, n = 8; treatment with SS14 in the presence of thapsigargin (SS14 + thapsigargin): 7.6 ± 0.9 pA/pF, n = 11; *P < 0.05.