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SUMMARY

The pituitary prolactin levels were measured in the domestic duck (Anas platyrhynchos) and in juvenile gulls from a mixed population of herring and lesser black-backed gulls. In the domestic duck the pituitary prolactin levels increased on the 2nd and 3rd days of maintenance on 0·3 m-NaCl, but by the 5th day they had fallen appreciably below the control levels. Maintenance of gulls with 0·3 m-NaCl produced no change in pituitary prolactin levels after 5 days, but a marked fall in prolaction levels when the birds were maintained on 0·7 m-NaCl for 5 days. This difference in response between the two species may be related to their degree of adaptation to a marine environment.

SUMMARY

When injected intravenously, 20 i.u. ovine prolactin significantly enhanced the output of fluid from the minimally stimulated nasal salt-gland of ducks within 5 min. This effect persisted for a further 5 min. In ducks which were still producing nasal fluid 25 min. after prolactin administration, a second enhancement of secretion was observed which could be related to an indirect effect induced by a rise in the blood glucose concentration. The initial, rapid enhancement of secretion could not be attributed to changes in blood composition and it seems possible that prolactin may have a direct effect on the avian salt-gland.

SUMMARY

Ducks given corticotrophin (ACTH) i.m. for 5 days secreted significantly more nasal fluid in response to an i.v. injection of 0·5 m-NaCl. However, blood glucose and plasma potassium concentrations also increased in the birds given ACTH and when these changes in blood composition were produced by injecting glucose or KC1, an effect similar to that of ACTH was obtained, suggesting that glucocorticoids influence the salt gland indirectly rather than, or as well as, directly. The concentrations of Na⁺ and K⁺ in the nasal fluid were decreased by ACTH, an effect not mimicked by glucose or KC1, and this might suggest some direct influence on water movements in the salt gland. ACTH increased nasal secretion in response to a minimal stimulatory salt load approximately 15 min after i.v. injection and this increase coincided with a marked rise in blood glucose concentration.

Abstract

Transcription initiation in the insulin-like growth factor-I (IGF-I) gene is complex, involving multiple sites in two exons. While most transcripts are initiated in exon 1 in vivo, critical regulatory mechanisms are difficult to assess in intact animals. To examine the impact of insulin and growth hormone (GH) under more controlled conditions, we have studied the utilization of different exon 1 and exon 2 transcription-initiation sites in normal rat hepatocytes in primary culture. Normal rat hepatocytes were cultured for 48 h in serum-free medium, with insulin at 10⁻⁶ or 10⁻¹¹ m, and with or without human GH 200 ng/ml. Relative abundance of IGF-I transcripts was evaluated by the RNase-protection assay, using a probe which permitted identification of initiation in exon 1 (site 1 (−380 bp from the 3′ end of exon 1), site 2 (−343 bp), site 3 (−242 bp), sites 1 and 2 spliced, and site 4 (−32 bp)), as well as in exon 2. After normalization of signal intensity to adjust for differences in length of protected probe, the utilization of initiation sites in vitro was remarkably similar to that in vivo: 1, 14, 6, 23, 19 and 37% for sites 1, 2, 3, 1 and 2 spliced, 4 and exon 2 respectively in the cultured hepatocytes, compared with 1, 12, 8, 21, 18 and 40% for these sites in normal liver. Insulin alone increased transcripts initiated from exon 1, site 2 by over 3 times, and both sites 1 and 2 spliced and exon 2 transcripts by about 5 times. GH alone had similar effects, producing a 4–5 times increase in transcripts from these initiation sites. Addition of both insulin and GH had additive effects, increasing transcripts from exon 1, sites 2, 3 and 4 by 4–6 times, and from exon 1, sites 1 and 2 spliced, and exon 2 by over 8 times. Of the total IGF-I mRNA transcripts, 37% were initiated from sites 2 and/or sites 1 and 2 spliced, and 37% from exon 2. Analysis of the relative contribution of individual initiation sites revealed hormone-induced increases which were statistically significant only for exon 2, in the presence of insulin alone and in combination with GH.

In conclusion, in cultured hepatocytes, insulin or GH alone produced a coordinated increase in all exon 1 transcripts, and the effect of the combination of insulin and GH was additive for these transcripts. Exon 2 appeared to be more sensitive to insulin alone, and to GH in the presence of insulin, than exon 1. Since utilization of initiation sites in hepatocytes mimics that found in liver, this in vitro system should be useful for examining underlying transcriptional regulatory mechanisms.

Journal of Endocrinology (1996) 151, 215–223

ABSTRACT

The presence of insulin-like growth factors (IGFs) in blood is regulated by their association with specific IGF-binding proteins (IGFBPs). In turn, the level of IGFBPs in the blood is likely to depend on a dynamic equilibrium between peptide production and clearance to extravascular tissues or organ-specific degradation. Since circulating IGFBPs may largely derive from liver we have employed partial hepatectomy in the rat to study the clearance rate of endogenous IGFBPs from blood once a major site of production is removed. Adult male rats were partially hepatectomized and serum and the remaining liver removed between 30 min and 7 days after surgery. Ligand blot analysis revealed two major species of IGFBP, of 28–30 kDa and 40–44 kDa in sera from control rats or sham-operated rats respectively. The larger species corresponded in size to rat IGFBP-3, but the smaller form was not recognized by antisera against rat IGFBP-1, bovine IGFBP-2 or human IGFBP-5 following Western immunoblot. Following hepatectomy, the levels of both IGFBP forms in the serum declined within 30 min and were barely detectable after 3 h or 6 h. They began to increase again in serum 24 h following surgery. The reduction in IGFBPs following hepatectomy was not primarily due to degradation by specific proteases in serum. Circulating levels of insulin were increased fivefold 3 h after hepatectomy but subsequently returned to control values. The rise in insulin was accompanied by a significant (P < 0·05) reduction in circulating IGF-I after 3 h which persisted at 24 h. Glucose levels in serum showed a transient but non-significant reduction between 90 min and 6 h after hepatectomy. Total RNA was extracted from remnant liver and subjected to Northern blot hybridization with ³²P-labelled cDNAs encoding rat IGFBP-1, -2 or -3. Messenger RNA encoding IGFBP-1 was barely detectable in liver from control or sham-operated animals, but increased within 30 min of partial hepatectomy and peaked at 3 h. It subsequently declined and was again barely detectable after 24 h. No expression of IGFBP-2 or -3 mRNAs was found by Northern blot analysis in the liver of control animals or following partial hepatectomy. These results suggest that both IGF-I and IGFBPs in rat serum decreased rapidly following partial hepatectomy, and that this was due largely to the rapid clearance of the peptide and its binding proteins once the major source of production was removed. A rapid induction of IGFBP-1 in the remaining liver may be unrelated to the circulating IGFBPs since immunoreactive IGFBP-1 was not detected in rat serum.

Journal of Endocrinology (1993) 137, 271–280

ABSTRACT

Two experiments were conducted with ovariectomized and hypothalamo-pituitary disconnected (HPD) ewes to ascertain the pattern of inputs, to the pituitary gland, of gonadotrophin-releasing hormone (GnRH) necessary for the full expression of an oestrogen-induced LH surge. The standard GnRH replacement to these sheep was to give pulses of 250 ng (i.v.) every 2 h; at the onset of experimentation, pulses were given hourly. In experiment 1, groups of sheep (n = 7) were given an i.m. injection of 50 μg oestradiol benzoate, and after 10 h the GnRH pulse frequency or pulse amplitude was doubled. Monitoring of plasma LH concentrations showed that a doubling of pulse frequency produced a marked increase in baseline values, whereas a doubling of amplitude had little effect on the LH response. In a second experiment, ovariectomized HPD sheep that had received hourly pulses of GnRH for 16 h after an i.m. injection of oil or 50 μg oestradiol benzoate were given either a 'bolus' (2·25 μg GnRH) or a 'volley' (500 ng GnRH pulses 10 min apart for 30 min, plus a 500 ng pulse 15 min later). Both groups then received GnRH pulses (250 ng) every 30 min for the next 13 h. Oestrogen enhanced the LH responses to the GnRH treatments, and the amount of LH released was similar in ovariectomized HPD ewes given oestrogen plus bolus or volley GnRH treatments and ovariectomized hypothalamopituitary intact ewes given oestrogen.

These results suggest that the oestrogen-induced LH surge is initiated by a 'signal' pattern of GnRH secretion from the hypothalamus.

Journal of Endocrinology (1989) 122, 127–134

Abstract

Plasma follistatin (FS) concentrations were determined after castration (n=5) or sham castration (n=4) of mature rams. Both treatments resulted in a prolonged increase in FS between 7 and 19 h after surgery, which returned to pretreatment concentrations by 24 h. Tumour necrosis factor-α (TNF-α), a sensitive marker of an acute-phase response, was undetectable in plasma, indicating that the FS response was not induced by trauma due to surgery. In a second experiment, injection of castrated rams (n=4) with ovine recombinant interleukin-1β, an acute-phase mediator, resulted in a sustained rise in FS concentrations within 4 h of injection. Plasma TNF-α concentrations increased transiently within 1 h of interleukin-1β injection, indicating that an acute-phase response had been initiated. Plasma follicle-stimulating hormone (FSH) concentrations were significantly decreased at 8 and 24 h after interleukin-1β injection, strongly suggestive of an inhibitory effect of increased FS concentrations on the secretion of FSH. Injection of castrated rams (n=2) with a control preparation of recombinant interleukin-2 did not induce an acute-phase response, and plasma FS and FSH concentrations were unaffected. These data show that the testis is not a major source of circulating FS, that the increase in circulating FS following sham castration/castration is not due to an acute-phase response, but that conversely FS concentrations are modulated by the acute-phase mediator, interleukin-1β.

Journal of Endocrinology (1996) 151, 119–124

ABSTRACT

A non-peptide, orally effective, vasopressin (AVP) V₁ receptor antagonist 1-{1-[4-(3-acetylaminopropoxy) benzoyl]-4-piperidyl}-3,4-dihydro-2(1H)-quinolinone (OPC-21268) has recently been described. This paper reports the in-vitro and in-vivo characterization of OPC-21268 binding to vasopressin receptors in rat liver and kidney.

OPC-21268 caused a concentration-dependent displacement of the selective V₁ receptor antagonist radioligand, ¹²⁵I-labelled [d(CH₂)₅,sarcosine⁷]AVP to V₁ receptors in both rat liver and kidney medulla membranes. The concentration of OPC-21268 that displaced 50% of specific AVP binding (IC₅₀) was 40±3 nmol/l for liver V₁ and 15±2 nmol/l for kidney V₁ receptors (mean ± s.e.m.; n = 3). OPC-21268 had little effect on the selective V₂ antagonist radioligand [³H]desGly-NH₂ ⁹[d(CH₂)₅,d-Ile²,Ile⁴] AVP binding to V₂ receptors in renal medulla membranes (IC₅₀ >0·1 mmol/l).

After oral administration to rats, OPC-21268 was an effective V₁ antagonist in a time- and dose-dependent manner. Binding kinetic studies showed that OPC-21268 acted as a competitive antagonist at the liver V₁ receptor in vitro and in vivo, in addition to its in-vitro competitive effects at the renal V₁ receptor. OPC-21268 shows promise as an orally active V₁ antagonist.

Journal of Endocrinology (1993) 138, 259–266