E. Arilla

Characterization of somatostatin binding sites in cytosolic fraction of rat intestinal mucosa.

Specific binding sites for somatostatin have been characterized in cytosolic fraction of rat intestinal mucosa by using 125I-labelled Tyr11-somatostatin and a variety of physicochemical conditions. The binding depended on time, temperature and pH, and was reversible, saturable and specific. At apparent equilibrium, the specific binding of 125I-Tyr11-somatostatin was competitively inhibited by native somatostatin in the 1 nM-4 microM concentration range. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 0.07 microM) and low capacity (4.6 pmol/mg protein) and a class with low affinity (Kd = 1.05 microM) and high capacity (277 pmol/mg protein) at 25 degrees C. Somatostatin exhibited competitive inhibition of tracer binding, while neuropeptides such as neurotensin, substance P, Leu-enkephalin, and vasoactive intestinal peptide were ineffective. The presence of somatostatin binding sites in cytosolic fraction of intestinal mucosa, together with the known occurrence of somatostatin in D-cells and nerve endings in the small intestine, strongly suggest that this peptide may be involved in the physiology and physiopathology of intestinal epithelium.

Dopamine enhances somatostatin receptor-mediated inhibition of adenylate cyclase in rat striatum and hippocampus

Although there is evidence that suggests that dopamine (DA) has stimulatory effects on somatostatinergic transmission, it is unknown to date if DA increases the activity of the somatostatin (SS) receptor‐effector system in the rat brain. In this study, we evaluated the effects of the administration of DA and the DA D1‐like (D1, D5) receptor antagonist SCH 23390 and the D2‐like (D2, D3, D4) receptor antagonist spiperone on the SS receptor‐adenylate cyclase (AC) system in the Sprague‐Dawley rat striatum and hippocampus. An intracerebroventricular injection of DA (0.5 μg/rat) increased the number of SS receptors and decreased their apparent affinity in the striatum and hippocampus 15 hr after its administration. The simultaneous administration of the DA receptor antagonists SCH 23390 (0.25 mg/kg, ip) and spiperone (0.1 mg/kg, ip) before DA injection partially prevented the DA‐induced increase in SS binding. The administration of SCH 23390 plus spiperone alone produced a significant decrease in the number of SS receptors in both brain areas studied at 15 hr after injection, an effect that disappeared at 24 hr. The increased number of SS receptors in the DA‐treated rats was associated with an increased capacity of SS to inhibit basal and forskolin (FK)‐stimulated (AC) activity in the striatum and hippocampus at 15 hr after injection. This effect had disappeared at 24 hr. By contrast, basal and FK‐stimulated enzyme activities were unaltered after DA injection. No significant changes in the levels of the αI (αi1 + αi2) subunits were found in DA‐treated rats as compared with control rats. In addition, the immunodetection of the αi1 or αi2 subunits showed no significant changes in their levels in DA‐treated rats when compared with controls. DA injection also induced an increase in SS‐like immunoreactive content in the rat striatum but not hippocampus at 15 hr after administration and returned to control values at 24 hr. These results provide direct evidence of a functional linkage between the dopaminergic and somatostatinergic systems at the molecular level. J. Neurosci. Res. 48:238–248, 1997.

Somatostatin binding sites in cytosolic fraction isolated from rabbit antral and fundic gastric mucosa

Specific binding sites for somatostatin have been identified and characterized in cytosolic fraction of rabbit gastric mucosa at both antrum and fundus levels. The binding depended on time, temperature and pH, and was reversible and saturable. The stoichiometric data suggested the presence of two classes of binding sites: a class with high affinity (Kd = 26.7 and 37.0 nM in antrum and fundus, respectively) and low capacity (2.1 and 4.1 pmol somatostatin/mg protein in antrum and fundus, respectively), and a class with low affinity (Kd = 246.4 and 162.5 nM in antrum and fundus, respectively) and high capacity (134.1 and 110.9 pmol somatostatin/mg protein in antrum and fundus, respectively) at 25 degrees C and pH 7.4. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as Leu-enkephalin, neurotensin and substance P behaved as ligands with very low affinity.

Acute effects of D1- and D2-receptor agonist and antagonist drugs on somatostatin binding, inhibition of adenylyl cyclase activity and accumulation of inositol 1,4,5-trisphosphate in the rat striatum

A recent study carried out by our group demonstrated that exogenous dopamine increases the somatostatin (SS) receptor-effector system in the rat striatum. The present study examined the participation of the D1- and D2-dopaminergic systems in the modulation of the rat striatal SS receptor-effector system by use of the D1-receptor agonist and antagonist SKF 38393 and SCH 23390, respectively, and the D2-receptor agonist and antagonist bromocriptine and raclopride, respectively. In view of the rapid onset of dopamine action, the effect of dopaminergic agents on the SS mechanism of action were studied 3 h after their administration. SKF 38393 (4 mg/kg i.p.) or bromocriptine (2 mg/kg i.p.) administered to male Wistar rats increased the number of 125I-Tyr3-SMS receptors in the striatum (52 and 30%, respectively) without changing the affinity constant. The effect of SKF 38393 on 125I-Tyr3-SMS binding was antagonized by the D1-specific antagonist SCH 23390 (0.25 mg/kg i.p.) whereas the effect of bromocriptine was abolished by the D2-specific antagonist raclopride (5 mg/kg i.p.). No change in binding was produced when SKF 38393 or bromocriptine were added directly to the incubation medium. The acute systemic administration of SCH 23390 or raclopride alone had no effect on the binding of 125I-Tyr3-SMS to its receptors. The increase of the number of 125I-Tyr3-SMS receptor induced by SKF 38393 or bromocriptine was accompanied by an increase in the capacity of SMS 201-995 to inhibit basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity when compared to the control groups. In addition, the effect of SMS 201-995 on the mass accumulation of inositol 1,4,5-trisphosphate (IP3) was investigated. SKF 38393 as well as bromocriptine increased the capacity of SMS 201-995 to accumulate IP3 in the rat striatum although this effect was only statistically significant in the case of SKF 38393. These results suggest that the activation of D1 and D2 receptors increases the activity of the SS receptor-effector system, the effect being greater in the case of D1 receptors. These findings are consistent with a functional interaction between dopamine and SS in the rat striatum.

Somatostatin binding to dissociated cells from rat cerebral cortex

A method has been developed for the study of somatostatin (SS) binding to dissociated cells from rat cerebral cortex. Binding of [125I] [Tyr11]SS to cells obtained by mechanical dissociation of rat cerebral cortex was dependent on time and temperature, saturable, reversible and highly specific. Under conditions of equilibrium, i.e., 60 min at 25 degrees C, native SS inhibited tracer binding in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.60 +/- 0.08 nM with a maximal binding capacity of 160 +/- 16 fmol/mg protein. The binding of [125I] [Tyr11]SS was specific as shown in experiments on tracer displacement by the native peptides, SS analogues, and unrelated peptides.

Evidence for somatostatin binding sites in rabbit kidney

Specific binding sites for somatostatin have been identified in cytosolic fraction of rabbit kidney (cortex and outer medulla) using 125I-Tyr11-somatostatin. The binding was saturable and reversible, as well as time and temperature dependent. Optimal pH for binding was observed at about 7.4. Scatchard plots were compatible with the existence of two classes of binding sites: a first class with a high affinity (Kd = 40 nM) and a low binding capacity (2.0 pmol somatostatin/mg protein) and a second class with a low affinity (Kd = 222 nM) and a high binding capacity (114.3 pmol somatostatin/mg protein). Vasoactive intestinal peptide, neurotensin, substance P, Leu-enkephalin and vasopressin had practically no effect on somatostatin binding. The properties of these binding sites strongly support the concept that somatostatin could behave as a regulatory peptide on the rabbit kidney.

Effects of acute nicotine and mecamylamine administration on somatostatin concentration and binding in the rat brain

Since nicotine and somatostatin have regulatory effects on locomotor activity it was of interest to determine whether the receptors for somatostatin are modulated by the cholinergic nicotine-like effects. An i.v. dose of 0.3 mg/kg nicotine induced an increase in the concentrations of somatostatin-like immunoreactivity at 4 min in the parietal cortex and at 15 min in the hippocampus. These changes were associated with a significant increase in the total number of specific somatostatin receptors in the parietal cortex at 15 min and in the hippocampus at 30 min following injection. To determine if the above mentioned changes are related to the nicotine activation of central nicotine-like acetylcholine receptors, a cholinergic nicotinic blocking agent, mecamylamine, was administered before the nicotine injection. Pretreatment with mecamylamine (5.0 mg/kg i.v.) prevented the nicotine-induced changes in somatostatin level and binding in both brain areas. Mecamylamine alone had no observable effect on the somatostatinergic system. These results suggest that the somatostatinergic system can be regulated by nicotine-like acetylcholine receptors and may be involved in some of the behavioral central effects of nicotine.

Effect of somatostatin on the mass accumulation of inositol-1,4,5- trisphosphate in rat hypothalamus, striatum, frontoparietal cortex and hippocampus

Somatostatin-14 (SS) significantly increased inositol-1,4,5-trisphosphate (IP3) accumulation in rat hypothalamic, striatal, frontoparietal cortical and hippocampal slices. However, this stimulation of IP3 accumulation by SS was highest in the frontoparietal cortex and hippocampus. The effect was already significant with 0.01 microM in the frontoparietal cortex (P < 0.05) and hippocampus (P < 0.05) and the maximal accumulation was evident with 0.1 microM SS, in all areas studied. A concentration of 1 microM SS, lacked this effect in hypothalamus and striatum. SS rapidly increased IP3 accumulation in all brain areas studied. This effect was maximal at 15 s of incubation and decreased subsequently. At 60 s incubation, levels were still elevated in frontoparietal cortex and hippocampus but had returned to basal values in hypothalamus and striatum. Somatostatin-28 (SS-28) and the SS analogues, D-Trp8-D-Cys14 and SMS 201-995, also significantly stimulated IP3 accumulation although the effect of SMS 201-995 was greater than that of SS in the striatum in comparison with controls (P < 0.001 and P < 0.01, respectively). These results suggest that SS action at the hypothalamus, striatum, frontoparietal cortex and hippocampus is mediated at least in part by the accumulation of IP3, which may initiate intracellular processes responsible for some biological SS effects.

Somatostatin binding to a fresh rat astrocyte-enriched suspension.

Since there are conflicting reports regarding the effects of somatostatin (SS) on cyclic AMP levels in astrocytes derived from rat cerebral cortex and, to date, the SS binding to mature astrocytes is unknown, the present study has determined SS binding and its effect on cyclic AMP accumulation in a fresh astrocyte-rich suspension from rat cerebral cortex. 125I-Tyr11-SS binding was inhibited by SS in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.95 +/- 0.15 nM with a maximal binding capacity of 122 +/- 13 fmol/mg protein. Vasoactive intestinal peptide (VIP) stimulated cyclic AMP accumulation up to 2.3 times above the basal levels whereas SS had no effect. This effect at any of the VIP concentrations. Likewise, SS did not inhibit the stimulation of cyclic AMP accumulation provoked by other effectors such as isoproterenol and forskolin. In view of our results and those of other authors, SS receptor localized in astrocytes must be able to couple with signal transduction systems other than adenylate cyclase, in order to carry out its biological actions in the cell.

Long-term haloperidol treatment decreases somatostatin binding in rat brain

The effects of short and long-term haloperidol treatment on somatostatin concentration and specific binding in rat cerebral cortex and hippocampus were examined using the binding ligand 125I-Tyr1-somatostatin. Haloperidol treatment did not affect the concentration of somatostatin-like immunoreactivity in the two brain areas. Nevertheless, long-term, and not short-term, haloperidol treatment decreased the number of somatostatin receptors in the cerebral cortex and hippocampus. No significant differences in the apparent binding affinity values were seen after haloperidol treatment. When added at the time of the binding assay haloperidol 34.2 microM produced a 42% and 27% decrease in cerebrocortical and hippocampal membrane somatostatin receptors respectively.