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R. Ravindra and R. S. Aronstam

Summary. Plasma membranes (1–2 mg protein) purified from the anterior pituitary lobes of adult male rats were incubated with 0·6 μmol [α-32P]guanosine 5′-triphosphate (GTP) l−1 in an ATP-regenerating buffer at 37°C for 60 min; during this incubation the [32P]GTP was hydrolysed and the nucleotide that was predominantly bound to the membranes was [32P]GDP. The release of [32P]GDP from the membranes was monitored at 37°C; the amount released was proportional to the protein concentration and increased as a function of time. 5′-Guanylylimidodiphosphate (Gpp(NH)p) increased [32P]GDP release by up to 30% at 0·1 μmol l−1. Although 10 nmol Gpp(NH)p l−1 had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP–GTP exchange.

Gonadotrophin-releasing hormone (GnRH) agonist and thyrotrophin-releasing hormone (TRH), at 0·1 μmol l−1 caused a maximum increase in the release of [32P]GDP of 31–38%. The GnRH agonist (0·1 μmol l−1) stimulated GDP release by 21%, 24%, 17% and 14% at 30 s, 1, 2 and 5 min, respectively. TRH (0·1 μmol l−1) stimulated GDP release by 38%, 30%, 17% and 16% at 30 s, 1, 2 and 5 min, respectively. A GnRH antagonist also stimulated [32P]GDP release, albeit less effectively than GnRH agonist; the antagonist did not inhibit agonist stimulation of GDP release. These results indicate that ligand binding to the GnRH and TRH receptors results in interaction of the receptor with a guanine-nucleotide-dependent transducer protein (G protein) and activation of GTP–GDP exchange. A receptor antagonist (at the GnRH receptor) has partial efficacy at eliciting this response. This GDP release represents an early biochemical marker of GnRH and TRH receptor stimulation.

Keywords: G proteins; pituitary; gonadotrophin-releasing hormone; thyrotrophin-releasing hormone; rat

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R. Ravindra and R. S. Aronstam

Colchicine and taxol, which are known to influence tubulin function, were used to delineate the possible role of tubulin in signal transduction in the anterior pituitary lobe. Anterior pituitary lobes, obtained from adult male rats, were processed by discontinuous sucrose gradient centrifugation to obtain plasma membranes. The low K m GTPase activity (EC 3.6.1.) was assayed in 5 μg membrane protein using [γ-32 P]GTP at 37°C in an ATP-regenerating buffer containing 1 μmol unlabelled GTP l−1. Ten nmol l−1 each of colchicine, lumicolchicine and taxol maximally stimulated the GTPase activity by about 40% (P < 0.05). A time-course study revealed that 100 nmol colchicine l−1 stimulated the enzyme activity by 55, 74, 89 and 53% at 5, 10, 20 and 30 min, respectively (P < 0.05); lumicolchicine (100 nmol l−1) stimulated the GTPase activity by 44, 36, 11 and 55% at 5, 10, 20 and 30 min, respectively (P < 0.05). Taxol (100 nmol l−1) stimulated the enzyme activity by 39 and 25% at 20 and 30 min, respectively (P < 0.05).

Gonadotrophin-releasing hormone (GnRH) and thyrotrophin-releasing hormone (TRH) stimulated the low K m GTPase activity in a concentration-dependent manner, by up to 40–60% (P < 0.05). In the presence of 100 nmol colchicine l−1, the ability of GnRH or TRH to stimulate the GTPase activity was inhibited. For example, at 1 nmol GnRH l−1, the enzyme activity was stimulated from 124 to 176 pmol min−1 mg−1 protein; in the presence of 100 nmol colchicine l−1, activity stimulated by GnRH (1 nmol l−1) was only 157 pmol min−1 mg−1 protein (P < 0.05). At 10 nmol TRH l−1 the enzyme activity was stimulated from 124 to 174 pmol min−1 mg−1 protein; in the presence of 100 nmol colchicine l−1, activity stimulated by TRH (10 nmol l−1) was only 155 pmol min−1 mg−1 protein (P < 0.05). GnRH or TRH stimulation of the enzyme activity was not affected in the presence of lumicolchicine. In the presence of taxol, the stimulation of the GTPase activity by either GnRH or TRH was inhibited. GnRH (1 nmol l−1) stimulated the GTPase activity from 124 to 150 pmol min−1 mg−1 protein; in the presence of 100 nmol taxol l−1, activity stimulated by GnRH (1 nmol l−1) was only 128 pmol min−1 mg−1 protein (P < 0.05); 1 nmol TRH l−1 stimulated the GTPase activity from 124 to 174 pmol min−1 mg−1 protein; in the presence of 100 nmol taxol l−1, activity stimulated by TRH (1 nmol l−1) was only 157 pmol min−1 mg−1 protein (P < 0.05). These results indicate that these drugs inhibit hormone-stimulated G protein GTPase activity as a result of their interaction with tubulin or G protein(s) or both.

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R. Ravindra, Kiran Bhatia and R. A. Mead

Summary. The present study reports steroid metabolism by corpora lutea (CL) obtained from skunks with diapausing embryos ('delay' CL) and with activated embryos (activated CL). CL from both reproductive periods were incubated with various radioactive precursors. Control incubations without any tissue or with 50 μl of packed skunk blood cells were also conducted simultaneously. Incubation of skunk CL with [3H]pregnenolone for 3 h resulted in 36% of the precursor accumulating as progesterone. Metabolism of [3H]dehydroepiandrosterone (DHEA) to androstenedione proceeded with approximately the same amount of product accumulating (34–46%) as was observed in the conversion of pregnenolone to progesterone. These results suggest that Δ5 isomerase, 3β-hydroxysteroid dehydrogenase, is the most prominent enzyme in skunk CL. Metabolism of [3H]pregnenolone to 17α-hydroxypregnenolone and [3H]progesterone to 17α-hydroxyprogesterone occurred at low rates (1–7%), suggesting the presence of C2l steroid 17α-hydroxylase in skunk CL. Aromatase activity, as estimated by measuring accumulation of oestradiol-17β from [3H]testosterone, was demonstrated in activated CL. These results suggest that skunk CL appear to metabolize steroids in a manner similar to CL of other mustelids such as the ferret and American badger.