ABSTRACT

1,25-Dihydroxyvitamin D₃(1,25-(OH)₂D₃) selectively enhances prolactin gene expression in GH₄C₁ clonal rat pituitary tumour cells. Because this effect requires extracellular Ca²⁺, we studied the effect of 1,25-(OH)₂D₃ on another Ca²⁺-dependent process, agonist-induced hormone secretion. Pretreatment with 1,25-(OH)₂D₃ (1 nmol/l) caused at least 25-fold sensitization of GH₄C₁ cells to the voltage-sensitive Ca²⁺ channel agonist BAY K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate) as a prolactin secretagogue. This inductive effect of 1,25-(OH)₂D₃ followed a similar time-course to the enhancement of prolactin production. 1,25-(OH)₂D₃ had no effect on basal or BAY K 8644-induced ⁴⁵Ca²⁺ uptake. The Ca²⁺-selective divalent cation ionophore 11,19,21-trihydroxy-4,6,8,12,14,18,20-heptamethyl-9-oxo-22-(tetrahydro-5 methyl-5-tetra hydro-5-(1-hydroxyethyl)-5-methyl-2-furanyl)-10,16-docosadienoic acid (ionomycin; 12 nmol/l–1·2 μmol/l) caused no significant increase in prolactin secretion in the absence of 1,25-(OH)₂D₃, but in cells treated with 1,25-(OH)₂D₃-(1 nmol/l), it increased prolactin secretion by 73% at 12 nmol/l and by a maximum of 98% at 0·12 μmol/l. These data demonstrate that vitamin D markedly enhances the responsiveness of GH₄C₁ functional pituitary tumour cells to two secretagogues which acts primarily through Ca²⁺-dependent mechanisms. They support the proposal that 1,25-(OH)₂D₃ acts in this cultured cell model either by effecting a redistribution of intracellular Ca²⁺ or by increasing the response of a Ca²⁺ -sensitive effector system, but not by enhancing agonist-induced Ca²⁺ uptake.

J. Endocr. (1988) 117, 293–298

ABSTRACT

Vitamin D may regulate pituitary function, as there are selective effects of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on gene expression in clonal pituitary tumour cells, and on TRH-induced TSH release in normal rat pituitary cells in vitro. The role of Ca²⁺ in 1,25-(OH)₂D₃-enhanced TSH release from primary rat pituitary cell cultures was investigated. Pretreatment with 10 nmol 1,25-(OH)₂D₃/l for 24 h augmented KCl (3–60 mmol/l)-induced TSH release over 1 h at all KCl concentrations greater than 7·5 mmol/l (P< 0·001), with a 76% enhancement of TSH release induced by 30 mmol KCl/l (P<0·001). The Ca²⁺ channel antagonist nifedipine (10 nmol/l–10 μmol/l) caused a concentration-dependent inhibition of KCl (60 mmol/l)-induced TSH secretion. Pretreatment with 1,25-(OH)₂D₃ enhanced KCl-induced release at all concentrations of nifedipine (P<0·001). The Ca²⁺ selective divalent cation ionophore ionomycin (1 nmol/l–1 μmol/l), and the Ca²⁺ channel agonist BAY K 8644 (10 nmol/l–1 μmol/l) increased prolactin secretion but did not increase TSH release, and 1,25-(OH)₂D₃ had no effect. At an extracellular Ca²⁺ concentration of less than 500 nmol/l, TRH-induced TSH release was observed only after treatment with 1,25-(OH)₂D₃ (P<0·01). As the extracellular Ca²⁺ concentration was increased, greater increments of TRH-induced TSH release were observed following pretreatment with 1,25-(OH)₂D₃ (P<0·01). However, the effect of 1,25-(OH)₂D₃ in the thyrotroph was independent of the pretreatment extracellular Ca²⁺ concentration. We have shown that 1,25-(OH)₂D₃ acts selectively on the thyrotroph to enhance in-vitro responsiveness to TRH and KCl. These data suggest that the action of 1,25-(OH)₂D₃ in the thyrotroph is to enhance intracellular signal transduction. They further support a permissive or regulatory role of vitamin D in the normal pituitary gland.

Journal of Endocrinology (1989) 121, 441–450

ABSTRACT

The hormone 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) has been shown to selectively enhance agonist-induced TSH release in the rat thyrotroph in vitro. The interaction of 1,25-(OH)₂D₃ with tri-iodothyronine (T₃) and cortisol was studied in primary cultures of dispersed anterior pituitary cells. TRH (1 nmol/l)-induced TSH release over 1 h was enhanced by 70% (P<0·01) following exposure to 10 nmol 1,25-(OH)₂D₃/l for 24 h. Pretreatment with T₃ (1 pmol/l–1 μmol/l) for 24 h caused a dose-dependent inhibition of TRH-induced TSH release. Net TRH-induced TSH release was inhibited by 85% at T₃ concentrations of 3 nmol/l or greater. Co-incubation with 1,25-(OH)₂D₃ resulted in enhanced TRH-induced TSH release at all T₃ concentrations tested (P<0·001). The increment of TRH-induced TSH release resulting from 1,25-(OH)₂D₃ pretreatment was equivalent in the presence or absence of maximal inhibitory T₃ concentrations. At 1 nmol T₃/1, there was a two- to threefold relative increase in 1,25-(OH)₂D₃-enhanced TRH-induced TSH release. Incubation with cortisol (100 pmol/l–100 nmol/l) had no effect on basal or TRH-induced TSH release, nor did it alter 1,25-(OH)₂D₃-enhanced TRH-induced TSH release when added 24 h before, or at the time of addition of 1,25-(OH)₂D₃. Actinomycin D and α-amanitin abolished 1,25-(OH)₂D₃-enhanced TSH secretion.

These data demonstrate that the action of 1,25-(OH)₂D₃ in the thyrotroph required new RNA transcription, and was not affected by cortisol. In the presence of T₃, the response of the thyrotroph to TRH induced by 1,25-(OH)₂D₃ was increased. We have shown that 1,25-(OH)₂D₃ has significant effects on the action of TRH and T₃ in vitro. These findings support the proposal that 1,25-(OH)₂D₃ may modulate TSH secretion in vivo.

Journal of Endocrinology (1989) 121, 451–458

Abstract

1,25-Dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is active in primary dispersed and clonal pituitary cells where it stimulates pituitary hormone production and agonist-induced hormone release. We have studied the effect of 1,25-(OH)₂D₃ on thyrotropin-releasing hormone (TRH) binding in clonal rat pituitary tumour (GH₃) cells. Compared with vehicle-treated cells, 1,25-(OH)₂D₃ (10 nmol/l) increased specific [³H]MeTRH binding by 26% at 8 h, 38% at 16 h, 35% at 24 h and reached a maximum at 48 h (90%). In dose–response experiments, specific [³H]MeTRH binding increased with 1,25-(OH)₂D₃ concentration and reached a maximum at 10 nmol/l. Half-maximal binding occurred at 0·5 nmol 1,25-(OH)₂D₃/l. The vitamin D metabolite, 25-OH D₃, increased [³H]MeTRH binding but was 1000-fold less potent than 1,25-(OH)₂D₃. In equilibrium binding assays, treatment with 10 nmol 1,25-(OH)₂D₃/l for 48 h increased the maximum binding from 67·4 ± 8·8 fmol/mg protein in vehicle-treated cells to 96·7 ± 12·4 fmol/mg protein in treated cells. There was no difference in apparent K _d (1·08 ± 0·10 nmol/l for 1,25-(OH)₂D₃-treated and 0·97 ± 0·11 nmol/l for vehicle-treated cells). Molecular investigations revealed that 10 nmol 1,25-(OH)₂D₃/l for 24 h caused an 8-fold increase in TRH receptor-specific mRNA. Actinomycin D (2 μg/ml, 6 h) abrogated the 1,25-(OH)₂D₃-induced increase in [³H]MeTRH binding. Cortisol also increased [³H]MeTRH binding but showed no additivity or synergism with 1,25-(OH)₂D₃. TRH-stimulated prolactin release was not enhanced by 1,25-(OH)₂D₃. We conclude that the active vitamin D metabolite, 1,25-(OH)₂D₃, caused a time- and dose-dependent increase in [³H]MeTRH binding. The effect was vitamin D metabolite-specific and resulted from an upregulation of the TRH receptor. Further studies are needed to determine the functional significance of this novel finding.

Journal of Endocrinology (1995) 147, 397–404

Aromatase inhibitors have been increasingly used in boys with growth retardation to prolong the duration of growth and increase final height. Multiple important roles of oestrogen in males point to potential adverse effects of this strategy. Although the deleterious effects of aromatase deficiency in early childhood and adulthood are well documented, there is limited information about the potential long-term adverse effects of peripubertal aromatase inhibition. To address this issue, we evaluated short-term and long-term effects of peripubertal aromatase inhibition in an animal model. Peripubertal male Wistar rats were treated with aromatase inhibitor letrozole or placebo and followed until adulthood. Letrozole treatment caused sustained reduction in bone strength and alteration in skeletal geometry, lowering of IGF1 levels, inhibition of growth resulting in significantly lower weight and length of treated animals and development of focal prostatic hyperplasia. Our observation of adverse long-term effects after peripubertal male rats were exposed to aromatase inhibitors highlights the need for further characterisation of long-term adverse effects of aromatase inhibitors in peripubertal boys before further widespread use is accepted. Furthermore, this suggests the need to develop more selective oestrogen inhibition strategies in order to inhibit oestrogen action on the growth plate, while beneficial effects in other tissues are preserved.

ABSTRACT

Indirect evidence has suggested that the kidney is a major organ of clearance for osteocalcin, a circulating marker of osteoblast function. The objectives of the present study were (1) to confirm the role of the kidney in osteocalcin clearance (2) to quantify the contribution of extrarenal sites and (3) to investigate the renal mechanism(s) of osteocalcin clearance. Plasma osteocalcin levels, osteocalcin plasma clearance rate (PCR) and plasma production rate (PPR) were determined in oophorectomized (OX) and uninephrectomized oophorectomized (UOX) sheep. The osteocalcin renal extraction efficiency (REE) and the effective renal plasma flow (ERPF) were measured, and the osteocalcin renal clearance rate (RCR) was calculated.

The osteocalcin PCR was reduced significantly in UOX compared with OX sheep (2·0±0·1 (n = 9) vs 2·5±0·1 litres/h (n = 44); P < 0·0005). In UOX sheep with plasma creatinine levels ≤ 130 μmol/l, the osteocalcin REE was 9±1·3% and the osteocalcin RCR was 50–91% of osteocalcin PCR (n = 4). In UOX sheep with plasma creatinine levels in the range 100–440 μmol/l, there was a linear relationship between osteocalcin PCR and ERPF; the osteocalcin RCR was related to the osteocalcin PCR (RCR = 0·9 × PCR −0·50). Intravenous infusion of the synthetic glucocorticoid triamcinolone acetonide (TA) in UOX sheep led to marked decrements in plasma osteocalcin levels and the osteocalcin PPR, and a significant increase in the osteocalcin PCR. These changes were accompanied by a 44% increase in ERPF. During i.v. infusion of ¹²⁵I-labelled osteocalcin in three UOX sheep, the urinary excretion of trichloroacetic acid-precipitable radioactivity represented 27% (range 22–31%) of the amount cleared by the kidney. Bio-Gel P6 chromatography of urine suggested the presence of intact ¹²⁵I-labelled osteocalcin and at least one radiolabelled osteocalcin fragment.

These findings confirm that the kidney is the major site of osteocalcin clearance and show that extrarenal sites also make an appreciable contribution. ERPF is an important determinant of the osteocalcin PCR. Augmentation of the ERPF by TA may mediate the induction of osteocalcin clearance by this glucocorticoid. In the UOX sheep, urinary excretion of intact osteocalcin may account for up to 30% of renal osteocalcin clearance.

Journal of Endocrinology (1991) 130, 213-221