Estrogenic endocrine disruptive components interfere with calcium handling and differentiation of human trophoblast cells.

During development, calcium (Ca) is actively transported by placental trophoblasts to meet fetal nutritional and the skeletal mineralization needs. Maternal exposure to estrogenic pesticides, such as 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) and methoxychlor (MTC), has been shown to result in reproductive disorders and/or abnormal fetal development. In this study, we have examined the effects of exposure of trophoblastic cells to MTC and DTT, in comparison to 17beta-estradiol (E2) and diethylstilbestrol (DES), to test the hypothesis that cellular Ca handling is a target for these endocrine disruptive components. Treatment with DDT, MTC, DES, or E2 increased cellular Ca uptake, and the expression of trophoblast-specific human Ca binding protein (HCaBP) was down-regulated by both MTC and DDT. Treatment with MTC, DDT, and DES inhibited cell proliferation, induced apoptosis, and suppressed expression of several trophoblast differentiation marker genes. These effects were reversed by overexpression of metallothionein IIa, a gene highly responsive to cadmium and other metals. These results strongly suggest that trophoblast Ca handling functions are endocrinally modulated, and that their alteration by candidate endocrine disruptors, such as MTC and DDT, constitutes a possible pathway of the harmful effects of these components on fetal development.

The endothelin receptor antagonist, L-754,142 does not prevent cyclosporine A-induced osteopenia in rats.

Cyclosporine A (CsA) is a potent immunosuppressive agent widely used to prevent allograft rejection. In vivo administration of CsA is associated with the development of high-turnover osteopenia. Endothelin-1 (ET), a vasoconstrictive peptide, has been implicated in CsA-induced nephrotoxicity and hypertension. Recent evidence suggests that endothelin plays a pivotal role in bone metabolism. The present study was designed to investigate whether L-754,142 (ETRA), the combined endothelin A and B receptor antagonist, when given to rats, would favorably modify the bone loss caused by CsA. Fifty, 5-month-old male Sprague-Dawley rats were randomly divided into five groups of 10 rats each. The first group served as a basal control. The remaining four groups received, by daily gavage for 28 days, (1) a combined CsA and ETRA vehicle, (2) CsA, 10 mg/kg, (3) ETRA, 30 mg/kg, and (4) CsA, 10 mg/kg and ETRA, 30 mg/kg, respectively. Rats were weighed and venous blood was collected on days 0, 14, 28 for determination of BUN, creatinine, calcium, PTH, osteocalcin, and 1,25(OH)2 D. Tibiae, after double labeling, were removed following sacrifice for histomorphometry. Both CsA-treated rats and CsA/ETRA-treated rats demonstrated trabecular osteopenia with raised serum osteocalcin, and 1,25(OH)2D levels when compared to control animals (P < 0.05). Rats given CsA alone developed renal impairment, as shown by an increased BUN. The combination group did not develop renal impairment. The results suggest that endothelin may contribute to the development of CsA-induced nephrotoxicity, which was prevented by ETRA, but does not seem to play a role in CsA-induced osteopenia.

Direct microsensor measurement of nitric oxide production by the osteoclast.

Nitric oxide (NO) triggers marked osteoclast retraction which closely resembles that due to Ca2+. The effect of Ca2+ has been attributed to a stimulated release of NO. Here, we show for the first time, by direct measurement with a microsensor, that osteoclasts do indeed produce NO and that this production is enhanced by a high Ca2+. We also show that the Ca2+ ionophore, A23187, mimics the latter. Furthermore, osteoclasts on dentine produce more NO than osteoclasts on glass and NO release from dentine-plated osteoclasts is much less sensitive to stimulation by Ca2+. Finally, the microsomal Ca2+ store-depleting agent, thapsigargin, attenuates NO release only from osteoclasts on glass, suggesting that stored Ca2+ has the dominant effect in modulating NO release from non-resorbing cells. NO is a powerful inhibitor of bone resorption: a direct demonstration of its production is therefore strong evidence for a role in modulating osteoclast function.

Molecular and functional evidence for calcineurin-A alpha and beta isoforms in the osteoclast: novel insights into cyclosporin A action on bone resorption.

We provide the first molecular evidence for the presence of a functional serine/threonine phosphatase, calcineurin-A (CN-A), in the osteoclast. Polymerase chain reaction (PCR) of an osteoclast cDNA library, together with restriction mapping, revealed two isoform sequences, alpha and beta. We then examined the functionality of the detected CN-A by assessing the effect of a classical antagonist, cyclosporin A (CsA), in the osteoclast resorption (pit) assay. CsA (0.1 and 1 microg ml-1) potently inhibited bone resorption. The presence of lymphocytes, with or without prior exposure to CsA in vivo, failed to reverse the CsA-induced resorption-inhibition. Expectedly, CsA had no direct effect on cytosolic Ca2+ levels in fura-2-loaded osteoclasts. These studies are a prelude to further investigations into the possible role of CN-A in osteoclast regulation. Finally, mechanistic studies on the bone effects of CsA, a widely used immunosupressant, should proceed from these observations.

Vitamin D metabolism is altered in Asian Indians in the southern United States: a clinical research center study.

Asian Indians who immigrate to northern Europe have lower serum 25-hydroxyvitamin D [25(OH)D] than Caucasians, and they develop vitamin D deficiency, rickets, and osteomalacia. We investigated vitamin D metabolism, the effects of 25(OH)D3 on vitamin D metabolism and activity of 25(OH) D-24-hydroxylase, the rate-limiting enzyme for degradation of 25(OH)D, from cultured skin fibroblasts of Asian Indians and compared them with cultured skin fibroblasts of Caucasians in the southern United States. Normal subjects, ages 20-40 yr, were admitted to a metabolic ward for 2.5 days and given a daily diet containing 400 mg calcium and 900 mg phosphorus. Serum vitamin D, serum 25(OH)D, urinary calcium, and urinary phosphorus were significantly lower, whereas serum immunoreactive intact parathyroid hormone (PTH) and serum 1,25-dihydroxyvitamin D [1,25(OH)2D] were significantly higher in Asian Indians than in Caucasians. Administration of 25(OH)D3 increased serum 25(OH)D and urinary calcium but did not change serum PTH or serum 1,25(OH)2D in Asian Indians. In cultured skin fibroblasts, Emax and Vmax of 25(OH)D-24-hydroxylase activity were significantly higher in Asian Indians. In summary, in Asian Indians serum vitamin D and 25(OH)D are markedly reduced, altered vitamin D metabolism is only partially reversed by 25(OH)D3, and 25(OH)D-24-hydroxylase activity in cultured skin fibroblasts is markedly increased. Thus, Asian Indians residing in the U.S. are at risk for developing vitamin D deficiency, rickets, and osteomalacia.

Thyroid status and dietary fatty acids affect beta-adrenoceptor agonist stimulation of tension development in rat myocardium.

1. The inotropic responses to the beta-adrenoceptor agonists adrenaline, noradrenaline and isoprenaline were examined in papillary muscles isolated from hypothyroid rats and euthyroid controls that had been fed diets enriched in either n-6 or n-3 fatty acids. 2. In hypothyroid animals fed the n-6 diet, the maximum developed tension in the presence of isoprenaline was only 54% greater than resting tension compared to 160% in euthyroid animals. Maximum tension was 105% greater than resting in hypothyroid animals fed the n-3 diet compared to 399% in controls. Similar responses to adrenaline and noradrenaline were seen, i.e. maximum tension was significantly greater in both hypothyroid and euthyroid animals fed the n-3 diet, but tension was depressed in the hypothyroid state. 3. Binding of the beta-adrenoceptor antagonist [3H]-dihydroalprenolol to ventricular membranes was saturable and of high affinity, irrespective of thyroid state and diet. While binding site density (Bmax) was not affected by the hypothyroid state or diet, binding affinity (Kd) was higher in hypothyroid animals fed the n-6 diet. 4. The inotropic response to forskolin was the same in hypothyroid animals, irrespective of diet, but maximum developed tension was significantly greater in euthyroid animals fed the n-6 compared to the n-3 diet. The dose-response curve for forskolin was shifted to the right in hypothyroid animals fed the n-3 diet indicating a decrease in sensitivity. 5. These results indicate that the depressed contractility in the hypothyroid heart may be due in part to an altered lipid environment of the beta-adrenoceptor complex and that n-3 fatty acids can significantly increase maximum developed tension in the hypothyLroid state.

N-3 and N-6 fatty acids modulate the inotropic response to calcium in hypothyroid rat papillary muscle.

The calcium sensitivity of papillary muscles was enhanced at low [Ca2+] in euthyroid rats fed a diet enriched in n-3 fatty acids compared to rats fed diets high in n-6 and saturated (SAT) fatty acids. At the same time, the maximum developed tension was 44% lower in animals fed the n-3 diet compared to those fed the n-6 diet and 62% lower than the rats fed the SAT diet. In hypothyroid animals fed the n-3 diet, the inotropic response to added Ca2+ was only 60% of that in euthyroid controls and 50 and 65% of euthyroid controls in n-6 and SAT diet-fed animals, respectively. Although the response was again lower in n-3-fed animals, the differences among the diet treatments were not as great as those seen in euthyroid animals, and there were no apparent diet-dependent differences in sensitivity to Ca2+ in hypothyroid animals. The potency of the calcium-channel blocker nifedipine was diet-dependent in euthyroid animals, with the order of decreasing sensitivity being n-3 > n-6 > SAT. Papillary muscles were not as sensitive to nifedipine in hypothyroid animals, although n-3-fed animals again showed the greatest inhibition of tension development. On the other hand, nitrendipine-binding affinity was not different among euthyroid and hypothyroid animals fed the n-6 diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the extracts of Desmodium adscendens on anaphylaxis.

Desmodium adscendens, used by herbalists in Ghana for the treatment of asthma, is anti-anaphylactic in vitro. As the plant material is administered orally, in vivo studies of its anti-anaphylactic property were undertaken using the guinea-pig. The results show that both aqueous and ethanolic extracts of D. adscendens, when taken orally, reduce anaphylactic contractions, interfere with histamine-induced contractions, and reduce the amount of smooth muscle stimulating substances released from lung tissue of guinea pigs.