Somatostatin modulates voltage-gated K(+) and Ca(2+) currents in rod and cone photoreceptors of the salamander retina

Abstract

We investigated the cellular localization in the salamander retina of one of the somatostatin [or somatotropin release-inhibiting factor (SRIF)] receptors, sst(2A), and studied the modulatory action of SRIF on voltage-gated K(+) and Ca(2+) currents in rod and cone photoreceptors. SRIF immunostaining was observed in widely spaced amacrine cells, whose perikarya are at the border of the inner nuclear layer and inner plexiform layer. sst(2A) immunostaining was seen in the inner segments and terminals of rod and cone photoreceptors. Additional sst(2A) immunoreactivity was expressed by presumed bipolar and amacrine cells. SRIF, at concentrations of 100-500 nM, enhanced a delayed outwardly rectifying K(+) current (I(K)) in both rod and cone photoreceptors. SRIF action was blocked in cells pretreated with pertussis toxin (PTX) and was substantially reduced by intracellular GDP(beta)S. Voltage-gated L-type Ca(2+) currents in rods and cones were differently modulated by SRIF. SRIF reduced Ca(2+) current in rods by 33% but increased it in cones by 40%, on average. Both effects were mediated via G-protein activation and blocked by PTX. Ca(2+)-imaging experiments supported these results by showing that 500 nM SRIF reduced a K(+)-induced increase in intracellular Ca(2+) in rod photoreceptor terminals but increased it in those of cones. Our results suggest that SRIF may play a role in the regulation of glutamate transmitter release from photoreceptors via modulation of voltage-gated K(+) and Ca(2+) currents.

Fig. 1.

Retinal distribution of SRIF and somatostatin sst_2A immunoreactivity. a, A SRIF-immunoreactive amacrine cell body whose processes extend into both distal and proximal portions of the IPL. b,sst_2A receptor immunoreactivity that is located in both plexiform layers. Arrows indicate positive staining of photoreceptor bases. c, Absence of sst_2Aimmunoreactivity when antibody was preabsorbed with a blocking peptide.Labels indicate retinal layers: gcl, ganglion cell layer; inl, inner nuclear layer;ipl, inner plexiform layer; opl, outer plexiform layer; and pl, photoreceptor layer. Scale bar:a–c, 5 μm.

Fig. 2.

Effect of SRIF on delayed outward K⁺ current (I_K) in rods. A, SRIF increased voltage-activated K⁺ current. I_K was evoked by holding cells at −70 mV and applying depolarizing steps from −60 to +40 mV in 20 mV increments in control external solution (left) and after a 4 min exposure to 0.5 μm SRIF (stripedhorizontalbar; right). B,I–V relationship of I_Kobtained from the experiment described in A is shown. Inset, Somatostatin increasedI_K amplitude without substantially changing the current–voltage relation. ■, Control; ○, somatostatin. They-axis of the inset is normalized current. C, CTX reduced outward currents (left) but did not prevent a somatostatin-induced increase in I_K. D, I–Vrelationship of outward current in the presence of CTX (■), or CTX + somatostatin (▵); n = 3.

Fig. 3.

Sensitivity to PTX of SRIF effect onI_K. By the use of a voltage protocol similar to that in Figure 2, a family of I_K currents was recorded in rods pretreated for 16–20 hr with PTX (400 ng/ml).a, Exposure to 0.5 μm SRIF for 5 min failed to enhance I_K. b, There was no significant difference in the I–Vrelationship of I_K recorded in control solution and in the presence of SRIF. c, The histogram summarizes the effects of SRIF on I_K in control and PTX-pretreated rods. Numbers inparentheses in this and subsequent figures are the sample size.

Fig. 4.

The effect of GDP_βS on SRIF-induced changes in I_K. a, K⁺ currents were recorded with a patch pipette containing 500 μm GDP_βS in control solution and after a 5 min exposure to 0.5 μm SRIF.b, The I–V relationship ofI_K obtained from the experiments described in a indicates that SRIF failed to alterI_K in the presence of GDP_βS.c, The histogram summarizes SRIF-induced changes inI_K when the internal solution contained GTP (openbar) and when GTP was replaced by GDP_βS (hatchedbar) to block G-protein activation.

Fig. 5.

The inhibitory effect of SRIF on high voltage-activated Ca²⁺ currents in rods. The test solution contained 0 CaCl₂ and 20 mmBaCl₂ substituted for equimolar NaCl. a, A depolarizing step to 0 mV from a holding potential of −70 mV was applied to record whole-cell Ca²⁺ current in control external solution (1), after a 1 min exposure to 0.2 μm SRIF (2), and after a wash (3).b, The I–V relationship of Ca²⁺ currents evoked by depolarizing voltage steps from −50 to +50 mV in 10 mV increments in the absence (opencircles) and the presence of 0.2 μm SRIF (closedcircles) is shown. c, The time course of SRIF blockade was evaluated by applying 70 msec depolarizing pulses to a test potential of 0 mV from a holding potential of −70 mV each 5 sec. The time of SRIF application is shown by the hatchedhorizontalbar. Thenumbers correspond to the traces ina.

Fig. 6.

Excitatory effects of SRIF on the Ca²⁺ current in cones. A, Cones were held at −70 mV, and depolarizing pulses were applied from −40 to +40 mV in 10 mV steps. Responses were recorded in 20 mm Ba solution as described for Figure 5. Left, In control Ba Ringer's solution. Middle, In 0.5 μmSRIF. Right, After a wash in Ba Ringer's solution.B, Current–voltage plot of the data in Ais shown. C, A summary of the changes induced in the peak Ca current of rods and cones by SRIF is shown.Cntr, Control (○), wash (▵).

Fig. 7.

Effects of somatostatin on K⁺-induced Ca²⁺ accumulation in rods and cones. a, c, KCl (100 mm) was used to stimulate Ca²⁺ entry in the absence and the presence (hatchedhorizontalbar) of 0.5 μm somatostatin in rods (a) and cones (c). Somatostatin reduced Ca²⁺ accumulation in rods but increased it in cones. b, d, Histograms summarize the somatostatin-induced inhibition of Ca²⁺ accumulation in rods (b; n = 5) and its enhancement in cones (d; n = 5).Verticalbars show the mean values ± 1 SE.