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T. Lei, M. Buchfelder, R. Fahlbusch, and E. F. Adams


Growth hormone releasing peptide (GHRP-6) is a synthetic hexapeptide which specifically stimulates secretion of growth hormone (GH) by pituitary somatotrophs. The precise intracellular mechanism by which this is achieved has not been deciphered although it is known to involve protein kinase C (PKC) and Ca2+ but to be cAMP-independent. We have used cell cultures of human pituitary somatotrophinomas to demonstrate powerful effects of GHRP-6 on membrane phosphatidylinositol (PI) turnover, a second messenger system which leads to activation of PKC and mobilisation of intracellular Ca2+ reserves. Incubation of somatotrophinoma cells with GHRP-6 led to a dose-dependent stimulation of rate of PI turnover. GH secretion was increased in parallel. Effects were discernable after only 15 minutes incubation and rose to a maximum at 2 hours. PI turnover was stimulated by GHRP-6 in 8 of 8 tumours examined, effects ranging from 2.1–7.9 fold increases. Stimulation of GH secretion by GHRP-6 was independent of presence of gsp oncogenes, emphasising the cAMP-independent nature of its effects. These results provide evidence that the GH-stimulatory effects of GHRP-6 are achieved through activation of the PI second messenger system and thus support earlier findings that PKC and Ca2+ play central roles in mediating the effects of GHRP-6.

(PI) into diacylglycerol (DAG) and inositol phosphates (Nishizuka, 1988; Berridge and Irvine, 1989), raising the possibility that GHRP-6 acts by stimulating this intracellular second messenger system. To test this hypothesis, we have examined whether GHRP-6 is able to promote increased PI turnover in human pituitary somatotrophinoma cells in culture.

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P S Mountford, M R Brandon, and T E Adams


Stably transfected cell lines expressing the α subunit, β subunit and α/β heterodimer of ovine (o)FSH have been established following the transfection of Chinese hamster ovary cells with α and β subunit cDNA expression vectors. In the absence of the α subunit, FSH β subunit polypeptides were inefficiently secreted and displayed a short intracellular half-life, while free α subunits were readily secreted in the absence of the β subunit. Cotransfection of oFSH α and β subunit cDNAs led to heterodimer assembly and secretion. While alteration of the nucleotide sequence flanking the β subunit AUG initiation codon did not appreciably enhance heterodimer biosynthesis and secretion, the replacement of the 5′ untranslated and signal peptide-coding regions of the β subunit cDNA with the corresponding sequences from an oGH cDNA clone was associated with a twofold increase in oFSH heterodimer secretion. The recombinant oFSH had a higher molecular weight than pituitary-derived oFSH, and was more acidic than the native hormone when analysed using isoelectric focusing, suggesting a greater degree of sialylation of the recombinant hormone. A comparison of the activities of the recombinant and native hormones in the porcine testis radioreceptor assay and in the in vitro Sertoli cell bioassay revealed that the recombinant oFSH displayed enhanced biological activity in the Sertoli cell assay when compared with the native hormone.