Comparison of mechanisms of action of luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix and LHRH agonist triptorelin on the gene expression of pituitary LHRH receptors in rats

Abstract

The mechanisms through which luteinizing hormone (LH)-releasing hormone (LHRH) antagonists suppress pituitary gonadotroph functions and LHRH-receptor (LHRH-R) expression are incompletely understood. Consequently, we investigated the direct effect of LHRH antagonist cetrorelix in vitro on the expression of the pituitary LHRH-R gene and its ability to counteract the exogenous LHRH and the agonist triptorelin in the regulation of this gene. We also compared the effects of chronic administration of cetrorelix and triptorelin on the LHRH-R mRNA level and gonadotropin secretion in ovariectomized (OVX) and normal female rats. The exposure of pituitary cells in vitro to 3-min pulses of 1 nM LHRH or 0.1 nM triptorelin for 5 h increased the LHRH-R mRNA level by 77-88%. Continuous perfusion of the cells with 50 nM cetrorelix did not cause any significant changes, but prevented the stimulatory effect of LHRH pulses on the receptor mRNA expression. In OVX rats, 10 days after administration of a depot formulation of cetrorelix, releasing 100 microg of peptide daily, the elevated LHRH-R mRNA level was decreased by 73%, whereas daily injection of 100 microg of triptorelin caused a 41% suppression. In normal female rats, cetrorelix treatment suppressed the LHRH-R mRNA level by 33%, but triptorelin increased it by 150%. The highly elevated serum LH levels in OVX rats and the normal LH concentration of cycling rats were rapidly and completely suppressed by cetrorelix. Triptorelin decreased the serum LH in OVX rats to the precastration level, but had no effect on basal LH in normal rats. Our results confirm that LHRH antagonists, such as cetrorelix, inhibit the gene expression of pituitary LHRH-R indirectly, by counteracting the stimulatory effect of LHRH. A rapid suppression of serum LH by LHRH antagonists would be advantageous in the treatment of sex hormone-dependent tumors and other conditions.

Figure 1

In vitro effects of LHRH, triptorelin, cetrorelix, and their combinations on LHRH-R expression and LH secretion from rat pituitary cells in the superfusion system. (a) LHRH-R mRNA levels after an exposure of the cells to 3-min pulses of 1 nM LHRH and 0.1 nM triptorelin for 5 h, a continuous perfusion with 50 nM cetrorelix for 5 h, and a coperfusion with 50 nM cetrorelix and 1 nM LHRH or 0.1 nM triptorelin. **, P < 0.01 vs. control; Tripto, triptorelin; Cetro, cetrorelix. (b) Reverse transcription (RT)-PCR products of the pituitary LHRH-R mRNA and β-actin mRNA after separation by agarose gel electrophoresis and staining with ethidium bromide. M, 100-bp DNA molecular weight marker; lanes 1 and 2, exposed to LHRH pulses; lanes 3 and 4, perfused with cetrorelix; lanes 5 and 6, perfused with cetrorelix and exposed to LHRH pulses simultaneously; lanes 7–9, untreated control cells. (c) LH responses of pituitary cells to 3-min pulses of 0.1 nM triptorelin (T, arrows) at 30-min intervals during the continuous superfusion with 50 nM cetrorelix for 5 h (horizontal bar).

Figure 2

Effects of a single dose of cetrorelix, triptorelin, and their combinations on the pituitary LHRH-R mRNA expression and serum LH concentration in normal rats. (a) LHRH-R mRNA level; (b) RT-PCR products of the pituitary LHRH-R mRNA and β-actin mRNA, after separation by agarose gel electrophoresis and staining with ethidium bromide. M, 100-bp DNA molecular weight marker; lanes 1 and 2,100 μg of cetrorelix; lanes 3 and 4, 10 μg of triptorelin; lanes 5 and 6, 100 μg of cetrorelix and 10 μg of triptorelin; lanes 7 and 8, 100 μg of triptorelin; lanes 9 and 10, 100 μg of cetrorelix and 100 μg of triptorelin; lanes 11 and 12, untreated controls. (c) Serum LH concentration. +, P < 0.05 vs. 10 μg of triptorelin; ++, P < 0.01 vs. corresponding triptorelin-treated only; **, P < 0.01 vs. control; Tripto, triptorelin; Cetro, cetrorelix.

Figure 3

Pituitary LHRH-R mRNA expression and serum LH concentration during and after treatment for 10 days with a depot formulation of cetrorelix pamoate, releasing 100 μg/day or repeated daily injection of 100 μg of triptorelin. (a) LHRH-R mRNA level after the treatment. (b) RT-PCR products of the pituitary LHRH-R mRNA and β-actin mRNA after separation by agarose gel electrophoresis and staining with ethidium bromide. M, 100-bp DNA molecular weight marker; lanes 1–3, cetrorelix-treated; lanes 4–6, triptorelin-treated; lanes 7–9, untreated control. (c) Serum LH during the treatment. **, P < 0.01 vs. control or 0 h; ++, P < 0.01 vs. corresponding value on day 1; Tripto, triptorelin; Cetro, cetrorelix.

Figure 4

Pituitary LHRH-R mRNA expression and serum LH in OVX rats during and after the treatment for 10 days with a depot formulation of cetrorelix pamoate, releasing 100 μg/day or repeated daily injection of 100 μg of triptorelin. (a) LHRH-R mRNA level. (b) RT-PCR products of the pituitary LHRH-R mRNA and β-actin mRNA after separation by agarose gel electrophoresis and staining with ethidium bromide. M, 100-bp DNA molecular weight marker; lanes 1–3, cetrorelix-treated; lanes 4–6, triptorelin-treated; lanes 7–9, untreated control. (c) Serum LH during the treatment. **, P < 0.01 vs. control or 0 h; +, P < 0.05; ++, P < 0.01 vs. corresponding value on day 1; Tripto, triptorelin; Cetro, cetrorelix.