Induction of colostrogenesis in non-pregnant cows was used to evaluate the relationship between prolactin (PRL) and mammary immunoglobulin G1 (IgG1) receptor expression. Six of eleven non-pregnant, non-lactating Holstein cattle responded to a standard lactation induction protocol by development of elevated IgG1 concentrations in mammary secretions. In order to increase the diversity in PRL concentrations, two of the six cattle were treated with bromocriptine, and two others were treated with recombinant bovine PRL. Serum alpha-lactalbumin, serum PRL and mammary secretion IgG1 concentrations were measured throughout the experiment. Biopsies of mammary tissue were collected after induction of lactation, and after treatments to alter serum PRL. Immunohistochemistry was used to evaluate IgG1 receptor expression. Administration of recombinant bovine (rbPRL) was associated with increased lactogenic activity, decreased secretion IgG1 concentrations, and decreased IgG1 receptor expression. Decreased serum PRL, due to bromocriptine, was associated with decreased lactogenic activity and maintenance of IgG1 receptor expression. Results of this experiment are consistent with an effect of PRL in decreasing the expression of the bovine mammary IgG1 receptor at the onset of lactogenesis.
GM Barrington, TE Besser, CC Gay, WC Davis, JJ Reeves, TB McFadden and RM Akers
PJ Jenkins, TA Cross, LA Perry, SA Medbak, GM Besser and AJ Clark
Early descriptions of in vitro ACTH bioassays all emphasised the need to use extracted plasma samples due to interference by an unidentified component. The aim of these studies was to elucidate the effects of whole plasma on ACTH steroidogenic activity in vitro and to identify the responsible factor. A sensitive in vitro dispersed bovine adrenocortical cell bioassay was established. The addition of 10% ACTH-depleted human pooled plasma to the incubation media resulted in basal steroidogenesis equivalent to that achieved with 10(-9) M ACTH1-24 and potentiated the steroidogenic activity of 10(-9) M ACTH1-24 by 7.8-fold. This potentiation was dependent on the concentration of both ACTH and plasma in the media, but did not result from the mitogenic effect of plasma. A pituitary source was excluded and the potentiating activity was not extractable by Vycor glass. Column chromatography demonstrated two peaks of activity corresponding to molecular weights of 650 and 220x10(3) Da. These peaks did not correspond to the plasma binding of 125I-ACTH which resulted from non-specific binding to albumin. Lipoprotein-deficient serum had no effect on either basal or ACTH-stimulated steroidogenesis, but both were restored by the addition of purified lipoproteins. However, novel findings demonstrated a differential effect of low (LDL) and high (HDL) density lipoproteins on basal and ACTH-stimulated steroid production; thus, LDL exerted a greater effect on the former, whilst HDL potentiated the steroidogenic activity of added ACTH more than LDL. The addition of the lipoproteins to lipoprotein-deficient serum restored its basal and ACTH potentiating effects, the cholesterol concentrations of the chromatographic fractions exactly paralleling their ACTH potentiating effect. These findings suggest that not only are lipoproteins the plasma factor(s) which potentiates ACTH steroidogenic activity in in vitro bioassays, but also that they exert differential effects on basal and ACTH-stimulated steroid production.
A.C. Hale, J. Price, J.F. Ackland, I. Doniach, S. Ratter, G.M. Besser and L.H. Rees
The remission of Cushing's syndrome following surgical removal of a tumour containing bombesin-like immunoreactivity (BLI), but insignificant levels of ACTH, is described. However, an acid extract of the tumour tissue caused the release of ACTH from isolated rat anterior pituitary cells in vitro. These observations led to an investigation of the effects of synthetic C-terminal gastrin-releasing peptide (GRP(14-27)) on ACTH release from isolated rat anterior pituitary cells. GRP(14-27) (10-1000 ng/ml) directly stimulated the release of ACTH in vitro, whereas lower doses (10-1000 pg/ml), ineffective themselves in eliciting ACTH release, potentiated the CRF-mediated in-vitro release of ACTH.
E.S. Penny, R.L. Patience, A.M. Sopwith, J.A.H. Wass, G.M. Besser and L.H. Rees
Three forms of circulating immunoreactive human growth hormone-releasing factor (ir-hGRF) have been identified from a patient whose acromegaly was associated with a disseminated carcinoid tumour. This is the first known report of the molecular forms of ir-hGRF in human plasma. High performance liquid chromatography (HPLC) on a C3, wide pore reversed-phase column and gel filtration chromatography were used in conjunction with a sensitive radioimmunoassay (RIA). The greatly elevated concentration of the ir-hGRF in plasma from this patient was 25,000 ng/l (normal range <60 ng/l). Gel filtration (G50) chromatography of the plasma revealed a single peak which coeluted with synthetic hGRF-40. However, reversed-phase HPLC of Vycorextracted plasma resolved the ir-hGRF into three components, which coeluted with synthetic hGRF-40 (69%), hGRF-44 (22%) and hGRF-37 (9%). At present it is not clear if the three forms are natural variants or whether either or both hGRF-40 and hGRF-37 are cleavage products of hGRF-44.