Effect of colchicine and taxol on stimulation of G protein GTPase activity in anterior pituitary lobe of rats by gonadotrophin- and thyrotrophin-releasing hormones

in Reproduction

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 Km 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 Km 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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    Society for Reproduction and Fertility

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