Analogues of somatostatin bind selectively to brain somatostatin receptor subtypes.

Somatostatin (SRIF) is a neurotransmitter that produces its multiple effects in the CNS through interactions with membrane-bound receptors. Subtypes of SRIF receptors are found in the CNS that are distinguished by their sensitivities to the cyclic hexapeptide MK-678, such that SRIF1 receptors are sensitive to MK-678 and SRIF2 receptors are insensitive to MK-678. In the present study, we further examined the selectivities of a series of structurally diverse SRIF analogues for SRIF receptor subtypes. SRIF receptors were labeled by 125I-Tyr11-SRIF, which has indistinguishable affinities for SRIF receptor subtypes. The inhibition by MK-678 was incomplete, indicating this peptide is highly selective for a subtype of SRIF receptor that we have termed the SRIF1 receptor. The binding of 125I-MK-678 to SRIF1 receptors was monophasically inhibited by SRIF, the octapeptides (such as SMS-201-995), and the hexapeptides (such as MK-678), consistent with the highly selective labeling of a subtype of SRIF receptor. In contrast, the smaller CGP-23996-like analogues did not inhibit 125I-MK-678 binding to SRIF1 receptors. The binding of 125I-CGP-23996 to SRIF receptors was inhibited by SRIF and the octapeptides with Hill coefficients of less than 1, indicating that 125I-CGP-23996 labels multiple SRIF receptor subtypes. The hexapeptides and CGP-23996-like compounds produced only partial inhibitions of 125I-CGP-23996 binding, which were additive, indicating selective interactions of these compounds with the different receptor subpopulations labeled by 125I-CGP-23996. 125I-Tyr11-SRIF binding and 125I-CGP-23996 binding to SRIF receptors were likewise only partially affected by 100 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a concentration that completely abolishes specific 125I-MK-678 binding to SRIF1 receptors. The component of 125I-CGP-23996 labeling that was sensitive to GTP gamma S was also MK-678 sensitive. Thus, two subpopulations of SRIF receptors exist in the CNS. The SRIF1 receptor is sensitive to cyclic hexapeptides such as MK-678 and to GTP gamma S but insensitive to smaller CGP-23996-like compounds. The SRIF2 receptor is sensitive to the CGP-23996-like compounds and can be selectively labeled by 125I-CGP-23996 in the presence of high concentrations of the hexapeptides or GTP gamma S because, unlike the SRIF1 receptor, the SRIF2 receptor is insensitive to these agents. The SRIF receptor subtype-selective peptide analogues will be useful in the future characterization of the functions mediated by SRIF receptor subtypes in the CNS.

Effect of cyclosporine on steroidogenesis in rat Leydig cells.

Cyclosporine induces hypoandrogenism in adult male rats. In order to assess whether this effect of CsA may be due to a direct inhibitory effect on Leydig cell function, CsA (0, 50, 500, and 5000 ng/ml) was added to a collagenase-dispersed mixed Leydig cell preparation and incubated with and without hCG (0, 0.1, 0.3, 1.0, 3.0, and 10.0 ng/ml). Testosterone (T) production, mitochondrial cholesterol side chain cleavage (CSCC) and microsomal 17,20-desmolase enzyme activities in Leydig cells were determined after 3 hr of incubation. In the absence of CsA, stimulation of T production was maximal (about 16-fold) with 1.0 ng/ml hCG. With 50 and 500 ng/ml CsA there were no changes in either the hCG-stimulated T levels or the two enzymatic activities. However, 5000 ng/ml CsA significantly (P less than 0.05) reduced the hCG (1 ng/ml)-stimulated T levels, CSCC and 17,20-desmolase activities. The high dosage of CsA (5000 ng/ml) also caused a significant decrease in cell viability (P less than 0.05) during the incubation period. These effects of CsA were not due to cremophor EL, the CsA vehicle. This in vitro data indicate that high dosages of CsA (greater than or equal to 5000 ng/ml) appear to have a cytotoxic effect on rat Leydig cells that results in a decrease in T production. However, lower doses of CsA (less than 500 ng/ml) do not have any direct inhibitory effect on the rat Leydig cells, suggesting that the hypoandrogenic effect of in vivo CsA in rats is not due to any direct effect on the testis.

Effects of cyclosporine on the hypothalamic-pituitary-gonadal axis in the male rat: mechanism of action.

In the intact adult male rat, cyclosporine (CsA) induces a significant decrease in serum testosterone (T), serum LH, and intratesticular T. To elucidate the mechanism of action of this CsA-induced hypogonadotropic hypogonadism, castrated male rats were treated with oral CsA (30 mg/kg.day) or vehicle alone, and serum LH was measured after 1, 2, and 4 weeks of treatment. Surprisingly, serum LH was higher in these CsA-treated castrated rats at 1, 2, and 4 weeks than in castrated controls. To provide insight into the cause of this increase in serum LH, a T implant (8 mm) was inserted (at week 5 of treatment) in both CsA-treated and control castrated animals, and serum LH was measured 1 week after insertion of the T implant. Serum LH levels decreased to control values after insertion of the T implant. Subsequently, two other groups of rats received 8-mm T implant at the time of castration and were then treated up to 4 weeks with either CsA or vehicle alone. Serum LH showed a significant decrease in these CsA- plus T-treated animals compared to the vehicle- plus T-treated animals. A GnRH stimulation test performed after 2 weeks of CsA/vehicle treatment showed a significant increase in serum LH in all the rats 30 min after GnRH administration (1, 10, 30, and 100 ng/100 g BW, ip), indicating a normal pituitary response. The increase in LH after exogenous GnRH treatment was more significant in CsA-treated intact rats than in the controls. There was no difference in creatinine clearance between intact and castrate T-treated rats regardless of whether they received CsA. These studies indicate that the hypogonadotropic hypogonadism induced by CsA in intact male rats is mediated through the hypothalamic-pituitary axis, primarily at its hypothalamus end, does not seem to be due to the nephrotoxicity of CsA, and is modulated by T and/or its metabolites.