Thomas F. Mowles

GRF analogs and fragments : Correlation between receptor binding, activity and structure

GH-releasing activity in vitro was directly correlated with GRF receptor binding affinity for all hGRF analogs examined. hGRF(1-29)-NH2 analogs with Ala15-substitution (for Gly15) displayed 4-5 times higher affinity for the GRF receptor relative to hGRF(1-44)-NH2. Replacement of Gly15 with Sar15 resulted in a dramatic loss of activity and receptor binding. The present data supports the proposal that Ala15-substitution increases receptor affinity, and hence potency, due to increased amphiphilic alpha-helical interactions. Fragments of hGRF, representative of DPP-IV and trypsin-like cleavage, are inactive as a consequence of greatly diminished GRF receptor binding. These results provide a comprehensive analysis of the structural features required for both GRF receptor binding and activation.

The dopamine receptor of the rat mammotroph in cell culture as a model for drug action

Abstract Dopamine can act directly on pituitary cells to inhibit prolactin release. This action can be blocked by dopamine receptor blocking drugs such as haloperidol, sulpiride and other neuroleptic agents. Comparison of the properties of the mammotroph dopamine receptor with the adenylate cyclase linked dopamine receptor of the limbic forebrain reveals some obvious differences. For example, dopamine receptor stimulants such as S-584 and lergotrile mesylate are inactive in stimulating the adenylate cyclase preparations but are potent in inhibiting pituitary prolactin secretion. Such inhibition of prolactin secretion can be reversed by haloperidol or sulpiride. In contrast to these observations, sulpiride does not block dopamine stimulation of cAMP formation. In addition, dopamine, apomorphine or lergotrile mesylate have no effect on a pituitary adenylate cyclase preparation and dopamine fails to elevate cAMP in the intact cells in culture. Despite the similarity between these two dopamine sensitive systems with respect to a number of agonists and antagonists, the exceptions described suggest that the pituitary system with further study may offer some greater reliability as a predictive test for clinically useful agents. These results also suggest that the receptors for dopamine, like that for norepinephrine, are of two types, only one of which is coupled to adenylate cyclase.

Synthesis and biological activity of novel C-terminal-extended and biotinylated growth hormone-releasing factor (GRF) analogs

A series of novel hGRF(1-29)-NH2 analogs were synthesized and biotinylated. The immunological and biological activities of these analogs were then characterized. To distance the biotin moiety from the putative bioactive core, a C-terminal spacer arm consisting of -Gly-Gly-Cys-NH2 (-GGC) was added to hGRF(1-29)-NH2 (hGRF29) and analogs, with subsequent biotinylation performed at the cysteine residue. Neither addition of the C-terminal spacer arm nor biotinylation affected affinity of these analogs for GRF antibody. Relative to hGRF(1-44)-NH2 (hGRF44: potency = 1.0), the biotinylated analogs were equipotent in vitro to their nonbiotinylated, parent compounds: [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2 (4.7) = [Ala15]hGRF29-GGC-(tpBiocytin)-NH2 (3.9) greater than hGRF29-GGC-(tpBiocytin)-NH2 (0.8). Based upon cumulative GH release data in vivo (0-60 min postinjection), [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2, [Ala15]hGRF29-GGC-(tpBiocytin)-NH2, and hGRF29-GGC-(tpBiocytin)-NH2 displayed 8.6, 5.5, and 0.8 times, respectively, the potency of hGRF44. These in vivo potency values were not significantly different from the corresponding parent compounds (i.e., with or without the C-terminal spacer arm). In summary, biotinylated hGRF analogs have been developed that retain full immunoreactivity and potent bioactivity (in vitro and in vivo), thus permitting their use in GRF receptor isolation, ELISA, and histochemical procedures.

Chapter 19. Growth Hormone Releasing Factors (Somatocrinins)

Publisher Summary This chapter discusses the growth hormone releasing factors. Three growth hormone releasing factor (GRF) peptides, that are homologous from the amino-terminus, have been described in the chapter: GRF-OH, GRF-OH, and GRF-NH2. Although GRF-OH was the predominant peptide obtained from the tumors, GRF-NH2 has been confirmed as the primary structure on the basis of molecular cloning and DNA sequence analysis. The trivial name somatocrinin has been designated for GRF. Solid phase peptide synthesis has been successfully applied to the preparation of GRF found in human hypothalamus (hGRF). A series of peptides structurally unrelated to the somatocrinins have also been discussed in the chapter to elicit the growth hormone secretion specifically. In addition, hGRF-stimulated GH release is correlated with the stimulation of adenylate cyclase and cyclic adenosine monophosphate (cAMP) formation. Interactions between hGRF and other hormones have been studied in vitro and in vivo in animals and humans. The primary use of hGRF has been as a diagnostic tool in patients with growth deficiencies or in acromegalics. Its potential in growth promotion and improved milk production, feed utilization, and nutrient partitioning between fat and muscle in animals is currently being explored. Somatocrinin analogues with enhanced biological potency have been prepared and recombinant DNA methods of production of GRF are eagerly awaited so that larger quantities may become available more economically. These achievements are expected to facilitate more extensive testing in other human pathologies.

IMMUNIZATION AGAINST GROWTH HORMONE-RELEASING FACTOR SUPPRESSES IGF-I AND ABOLISHES OPIOID AGONIST INDUCED RELEASE OF GROWTH HORMONE IN LACTATING CATTLE

Publisher Summary This chapter elaborates the immunization against growth hormone-releasing factor (GRF). The effects of active immunization against GRF in lactating beef cows on concentrations of IGF-I, milk production, reproductive performance, and changes in GH following an opioid agonist and antagonist are studied. Immuno-neutralization of GRF resulted in lower milk yield in GRF than in human serum albumin (HAS) cows on day 86 and 96 pp. Interval from calving to elevated progesterone averaged 53 ± 0 and 72 ± 9 day in GRF and HSA cows, respectively. Immunization against GRF abolished the opioid agonist stimulated release of GH. Administration of FK33-824 elevated GH in HSA, but not in GRF cows and FK33-824 was effective in elevating GH only on day 47 and 119 pp in HSA cows. A single injection of naloxone failed to alter concentrations of GH in either GRF or HSA cows. In HSA cows, concentration, but not frequency, of GH release was greater on days 47 and 119 than on day 19. It is found that abolishment of GH pulses by active immunization against GRF suppressed IGF-I concentrations, lowered milk yield and reduced the interval from calving to oestrus.

CONTROL OF GROWTH HORMONE SECRETION DURING GESTATION AND LACTATION IN GILTS ACTIVELY IMMUNIZED AGAINST GROWTH HORMONE-RELEASING FACTOR

Publisher Summary This chapter describes the control of growth hormone (GH) secretion during gestation and lactation in gilts actively immunized against growth hormone-releasing factor (GRF). Changes in pattern of GH secretion during gestation and lactation, as well as possible opioid involvement in the control of GH during lactation, are not well understood in the pig. Immunization against growth hormone-releasing factor in the gilt suppresses episodic release of GH and inhibits release of GH in response to stimulation with an opioid agonist. Administration of naloxone during lactation suppressed GH in both GRF and HSA gilts. In HSA gilts, frequency of release and mean GH, respectively, were lower following naloxone than following saline. Naloxone lowered average GH in GRF gilts and however, frequency of GH release was not affected by naloxone. In summary, immuno-neutralization of GRF suppressed GH release during lactation, but failed to significantly alter GH during gestation. Administration of naloxone during lactation decreased GH in all gilts. The effect of naloxone on concentrations of GH in GRF-immunized gilts was independent of changes in frequency of release of GH.