The carbonic anhydrase inhibitor, acetazolamide, has been found to elicit a hypocalcemic response in acutely nephrectomized rats. This effect cannot be attributed to changes in pCCh, pH citrate, phosphate or protein. Administration of acetazolamide to parathyroidectomized-nephrectomized rats has been shown to inhibit the hypercalcemic response to parathyroid extract and dibutyryl cyclic 3',5'-AMP. The hypercalcemic response to epinephrine was not inhibited in the same preparation. These data are consistent with the hypothesis that cyclic 3',5'-AMP is the second messenger of parathyroid hormone in its action on bone, and that carbonic acid production may be a later, or possibly the final, messenger of the hormone.

The present study was conducted to show that the hypercalcemic and the vascular relaxing activities of PTH are two separable properties. During the hypotensive action of the synthetic fragment bovine (b) PTH-(1-34), plasma calcium levels were not significantly changed. Mild oxidation with hydrogen peroxide abolished the hypotensive and hypercalcemic actions of bPTH-(1-84). However, the same treatment on bPTH-(1-34) abolished only the hypotensive and not the hypercalcemic action. Analysis of the amino acid composition revealed only the oxidation of the methionines to methionine sulfoxides. The other amino acids remained unchanged. In addition, the analog with methionines replaced by norleucine, [Nle8,Nle18,Tyr34]bPTH-(1-34), was active in all the vascular assays, and these activities were unaffected by hydrogen peroxide treatment of the molecule. Perhaps the methionine sulfoxides in the hydrogen peroxide-treated bPTH-(1-34) affected the changes of the molecule in such a manner that the part of the molecule for the vascular action but not that for the hypercalcemic action was no longer accessible to the receptors of the target organs. The hypotensive pentapeptide, bPTH-(24-28), was not active in the hypercalcemic assay. All these data are consistent with our hypothesis that the vascular relaxing and the hypercalcemic actions of PTH are two separate properties of the molecule.

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The relative activities of adrenaline, noradrenaline and isoprenaline in producing hyperglycaemia and glycogenolysis in skeletal muscle have been studied in both fed and fasted rats, 1 hr after subcutaneous injection of the catechol amines. The relative hyperglycaemic activities of the three catechol amines depended greatly upon the prandial state of the rats and on the dose range used. In fed rats the a. relative potencies were in the descending order of potency, adrenaline-noradrenalineisoprenaline, irrespective of the dose range. Isoprenaline had no hyperglycaemic activity in fed rats even at doses as high as 2 mg/kg. In fasted rats the order of potency depended on the dose. At low doses (0.005 to 0.02 mg/kg) the descending v/ order was isoprenaline-adrenaline-noradrenaline. At higher doses (0.1 to 1 mg/kg) the descending order was adrenaline-isoprenaline-noradrenaline. The relative activities of the three catechol amines in causing glycogenolysis in muscle was independent of the dose range or the prandial state of the rats. Under all conditions the descending order of potency was isoprenaline-adrenaline-noradrenaline. The results are discussed with reference to Ahlquist's (1948) hypothesis of a-and /8-receptors and were consistent with the concept that, in the rat, liver glycogenolysis is mediated predominantly by a-receptors and muscle glycogenolysis mainly by /3-receptors. In general the hyperglycaemic response in the fed rat is mediated pre-/ dominantly by a-receptors and in the fasted rat the response is mainly due to the activation of P-receptors. A drug possessing both a-and P-receptor activity elicits an exception to this rule in the fasted rat. Several perturbing problems in the literature, particularly with regard to the hyperglycaemic activity of isoprenaline and to the difficulty in blocking the hyperglycaemic response, can now be explained in the light of these findings.

The effects of the carbonic anhydrase inhibitor acetazolamide on basal and parathyroid hormone (PTH)-induced bone metabolism were studied to evaluate the manner in which acetazolamide inhibits bone resorption. Half-calvaria from 5 to 6-day-old mice were cultured using the following treatments: control; acetazolamide (10, 33, or 100 microM); PTH (16.7 nM bovine PTH 1-34); acetazolamide + PTH. The effects of acetazolamide on PTH-induced cAMP accumulation and protein synthesis were determined. Media from bones cultured for 48 hours were analyzed for calcium to assess bone resorption, glucose to assess calvarial glucose utilization, and lactic acid to assess calvarial lactic acid release. Media were also assayed for beta-glucuronidase activity as an indicator of lysosomal enzyme release and for lactate dehydrogenase activity as an indicator of cytosolic enzyme release and cytotoxicity. Acetazolamide at 100 microM completely inhibited PTH-induced bone resorption. This inhibition did not appear to be due to cell death, as acetazolamide did not increase lactate dehydrogenase release. Acetazolamide had no effect on PTH-enhanced cAMP levels, indicating that receptor binding and adenylate cyclase activation were unaffected. Acetazolamide alone did not alter calvarial protein synthesis, but did significantly inhibit protein synthesis in the presence of PTH. PTH significantly enhanced calvarial glucose utilization, lactic acid release, and beta-glucuronidase release. Acetazolamide inhibited all of these PTH-induced parameters in a manner that roughly paralleled its inhibition of bone resorption; acetazolamide alone had no effect on the basal values. Our results indicate that acetazolamide inhibition of bone resorption in vitro may involve general alterations in hormonally stimulated bone cell metabolism secondary to carbonic anhydrase inhibition.