Somatostatin-induced regulation of SST(2A) receptor expression and cellsurface availability in central neurons: role of receptor internalization

Abstract

To investigate the effects of somatostatin (somatotropin release-inhibiting factor, SRIF) on the regulation of SST(2A) receptors in mammalian brain, we examined how blockade of SRIF release or stimulation by the SRIF analog [d-Trp(8)]-SRIF would affect the expression and cell surface availability of SST(2A) receptors in rat brain slices. First, we measured the intensity of SST(2A) immunoreactivity, using quantitative light microscopic immunocytochemistry, and levels of SST(2A) mRNA, using semiquantitative RT-PCR, under conditions of acute SRIF release blockade. Incubation of slices from the claustrum or basolateral amygdala, two regions previously shown to contain high concentrations of SST(2A) receptors, in Ca(2+)-free Ringer's for 40 min induced a decrease in the intensity of SST(2A) receptor immunoreactivity and concentration of SST(2A) mRNA as compared with control values obtained in Ca(2+)-supplemented Ringer's. These effects were counteracted in a dose-dependent manner by the addition of 10-100 nm [d-Trp(8)]-SRIF to the Ca(2+)-free medium. Furthermore, both of these effects were abolished in the presence of the endocytosis inhibitors phenylarsine oxide or hyperosmolar sucrose, suggesting that they were dependent on receptor internalization. Electron microscopic immunogold labeling confirmed the existence of an agonist-induced internalization of SST(2A) receptors in central neurons. At a high (10 microm), but not at a low (10 nm), concentration of agonist this internalization resulted in a significant decrease in cell surface receptor density, irrespective of the presence of Ca(2+) in the medium. Taken together, these results suggest that ligand-induced endocytosis is responsible for rapid transcriptional (increase in SST(2A) expression) and trafficking (loss of cell surface receptors) events involved in the control of the somatostatinergic signal.

Fig. 1.

Light microscopic distribution of SST_2A receptor immunolabeling in claustrum slices incubated for 40 min at 37°C in Ringer's buffer (A), in Ca²⁺-free buffer (B), in Ca²⁺-free buffer containing 100 nm[d-Trp⁸]-SRIF (C), and in Ca²⁺-free buffer containing 100 nm[d-Trp⁸]-SRIF plus 10 μm PAO (D). A, After incubation in Ringer's, intense immunolabeling is observed over nerve cell bodies and their proximal dendrites (arrows). Punctate immunostaining typical of cross-sectioned immunoreactive dendrites is also evident (arrowheads).B, After incubation in Ca²⁺-free buffer, the intensity of SST_2A immunolabeling is reduced dramatically in both cell bodies and surrounding neuropil.C, Addition of 100 nm[d-Trp⁸]-SRIF to the Ca²⁺-free incubation medium almost totally reestablishes the level of SST_2A immunoreactivity to that seen in Ringer's controls. D, [d-Trp⁸]-SRIF-induced recovery of SST_2A immunoreactivity is totally prevented by addition of the endocytosis inhibitor PAO to the incubation medium. Scale bar, 100 μm.

Fig. 2.

A, Effect of exogenous SRIF on the intensity of SST_2A immunoreactivity in the claustrum. Slices were incubated in normal or Ca²⁺-free Ringer's or in Ca²⁺-free Ringer's containing [d-Trp⁸]-SRIF (10–100 nm). Density of immunoreactive signal was measured over individual labeled cells with computer-assisted densitometry. Note the substantially higher level of SST_2A immunoreactivity in the presence than in the absence of Ca²⁺ in the buffer and the increase in the intensity of the staining that followed agonist stimulation in Ca²⁺-free buffer. B, Effect of endocytosis blockers on the increase in SST_2Aimmunoreactivity induced by [d-Trp⁸]-SRIF in Ca²⁺-free medium. Slices were incubated in Ca²⁺-free Ringer's containing or not 100 nm [d-Trp⁸]-SRIF and in the presence or the absence of 10 μm PAO or 0.4m sucrose. Although without effect by themselves, both PAO and sucrose totally inhibited the SRIF-induced increase in SST_2A immunoreactivity. Values are the mean ± SEM from three animals.

Fig. 3.

PCR amplification of SST_2A mRNAs extracted from claustrum slices incubated for 40 min at 37°C in Ca²⁺-supplemented Ringer's (lane 1), in Ca²⁺-free Ringer's (lane 2), in Ca²⁺-free Ringer's containing 100 nm[d-Trp⁸]-SRIF (lane 3), in Ca²⁺-free Ringer's containing 100 nm [d-Trp⁸]-SRIF and 0.4 m sucrose (lane 4), and in Ca²⁺-free Ringer's containing 100 nm[d-Trp⁸]-SRIF and 10 μmPAO (lane 5). PCR reactions were performed on mRNAs reverse-transcribed with specific SST_2A receptor primers. The predicted size of amplified fragments was 373 bp (plasmid control). GAPDH mRNAs were transcribed in parallel (target size, 428 bp) and used as an internal standard for quantitation. SST_2A mRNA levels, expressed as a percentage of control (Ringer's buffer), are depicted above the corresponding gel bands. All values are the mean ± SEM of triplicate determinations from two independent experiments.

Fig. 4.

Electron microscopic localization of SST_2A receptors, using silver-enhanced immunogold in claustrum slices. Slices were incubated for 40 min at 37°C in Ca²⁺-supplemented Ringer's in the absence (A, control) or in the presence (B–D) of 10 μm[d-Trp⁸]-SRIF. A, Under control conditions a high proportion of immunogold particles is associated with dendritic plasma membranes (arrowheads).B, C, In the presence of 10 μm [d-Trp⁸]-SRIF the bulk of gold particles is intracellular. Several are seen in association with the membrane of endosomes (arrows).D, When [d-Trp⁸]-SRIF stimulation is performed in the presence of PAO, the distribution of immunoreactive SST_2A receptors is comparable to that seen in controls. Den, Dendrite; AT, axon terminal. Scale bars: A, D, 1 μm; B, C, 0.5 μm.

Fig. 5.

Effect of SRIF stimulation on the subcellular distribution of SST_2A receptors in dendrites of the claustrum and basolateral amygdala. Slices were incubated with or without [d-Trp⁸]-SRIF (10 nm–10 μm) in Ca²⁺-free or Ca²⁺-supplemented Ringer's. Dendrite-associated immunogold particles were counted and classified as either membrane-associated or intracellular; results are expressed as membrane-associated/total. In the absence of agonist, ∼60% of gold particles were associated with dendritic plasma membranes in both cerebral regions, irrespective of the Ca²⁺concentration in the medium. Addition of agonist induced a decrease in the percentage of receptors associated with plasma membranes, again both in the presence and absence of Ca²⁺. The agonist-induced decrease in surface receptors was prevented by adding 10 μm PAO to the incubation medium. Values are the mean ± SEM from three animals; **p < 0.01.