Bromolasalocid (Ro 20-0006) is a calcium ionophore with antihypertensive activity that does not belong to any known class of antihypertensive agents. Bromolasalocid produces a relatively flat systolic blood pressure dose-response effect in the spontaneously hypertensive rat. An intensive cardiovascular evaluation of bromolasalocid at the highest dose used in the dose-response study showed full hemodynamic compensation; there was a significant decrease in both mean arterial blood pressure and peripheral resistance without a significant decrease in cardiac index. The antihypertensive action of bromolasalocid lasts many days after termination of dosing. Bromolasalocid is specifically antihypertensive and does not decrease arterial blood pressure in normotensive animals or in animal models of hypertensive cardiovascular disease with normal pulse pressures. Bromolasalocid is not a vasodilator and appears to mediate its antihypertensive action by restoring compliance of the large conduit arteries. Both the derived arterial compliance index and the blood pressure-pressor response to the carotid occlusion reflex are enhanced in the dog perinephritis model of hypertensive cardiovascular disease treated with bromolasalocid. Bromolasalocid appears to reverse the damage to cardiovascular tissue caused by prolonged hypertension via an action on calcium perturbations in large artery smooth muscle cells.
Neonatal liver or adult spleen was used as a source of B-lymphocytes in reconstituting lethally irradiated, syngeneic mice. Recipients were all given excess adult, syngeneic thymus cells and were immunized with dinitrophenylated bovine gamma globulin. The distribution of avidities of plaque-forming cells produced by immunized recipients of neonatal liver was highly restricted in comparison with animals reconstituted with adult spleen indicating a restriction of B-lymphocyte heterogeneity in the neonatal mouse.
It is now widely accepted that glucagon acts at the cell surface to activate adenylate cyclase, which, in turn, results in an increase in the intracellular concentration of cyclic AMP. In analogy with the situation in adipose tissue, it is proposed that cyclic AMP activates a lipase which results in an increased intracellular level of free fatty acids and fatty acyl CoA. The fatty acyl CoA inhibits the activity of acetyl CoA carboxylase and perhaps the mitochondrial tricarboxylate anion carrier as well, resulting in a reduced flow of carbon from acetyl CoA to long chain fatty acids. The mechanism for the control of the synthesis of malic enzyme is more tentative. It is suggested that fatty acyl CoA interacts with the reactions that control the synthesis of malic enzyme. Here the regulation of the lipogenic pathway may be analogous to bacterial systems where the product of a pathway regulates synthesis and activity of the pathway enzymes. Alternatively, the synthesis of malic enzyme may be directly affected by cyclic AMP or regulated by the intracellular demand for NADPH. Saturated fat in the diet and induction of the hepatic microsomal drug hydroxylating system both cause an increase in the activity of malic enzyme in rat liver without affecting fatty acid synthesis. Like lipogenesis, both desaturation of fatty acids and drug hydroxylation are systems that utilize NADPH. These questions and similar ones concerning the mechanism of action of thyroid hormone are currently being investigated in the author's laboratory.
The saturable component of transmural calcium transport in rat duodenum is transcellular, dependent on vitamin D, and can be evaluated by in situ gut loops or everted sacs. Vitamin D action at the molecular level can be studied by analyzing the response in terms of calcium-binding protein (CaBP; Mr congruent to 9000) biosynthesis to exogenous 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3). In vitamin D-replete animals, the CaBP response occurs within 1 h of intraperitoneal injection when the animals have been fed a high-calcium diet (III), but in 7 h if the animals have been fed a low-calcium diet(I). The latter response appears to be transcriptional, whereas the former seems posttranscriptional. In vitamin D-deficient animals, exogenous 1,25-(OH)2-D3 evokes a CaBP response that occurs 7-8 h after treatment and is transcriptional in nature. Calcium uptake by isolated duodenal cells can be stimulated by prior in vivo treatment with 1,25-(OH)2-D3. Peak response times parallel those found with CaBP biosynthesis, i.e., 3 h in cells from vitamin D-replete animals fed diet III, 7 h in cells from vitamin D-replete animals fed diet I, and 12 h in cells from vitamin D-deficient animal. Cycloheximide treatment appears to inhibit these responses. Moreover, everted sacs from vitamin D-replete animals fed diets III and I show an early and a delayed transport response, respectively. Studies with brush border membrane vesicles prepared from rat duodenum have shown calcium uptake to be vitamin D-dependent. Part of this uptake involves binding to the inner aspect of the membrane and may involve a high-affinity CaBP. Thus a major component of the action of vitamin D in stimulating calcium transport appears to involve protein synthesis. The time and molecular nature of these responses depend on the calcium intake and vitamin D status of the animals. A model of calcium movement through the intestinal cell is included.
There is a biphasic response of intestinal calcium transport to 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3). The first or rapid response is by existng mature villus cells, whereas the slow second response is by maturing crypt cells. For both responses, [3H]1,25-(OH)2-D3 localizes in the nucleus before initiating the transport events. This localization is brought about by a specific cytoplasmic receptor, which has a molecular weight of 67,000, is highly specific for 1,25-(OH)2-D3, and has a Kd of 5 X 10(-11) M. Its essentiality for intestinal calcium transport response to 1,25-(OH)2-D3 can be demonstrated in neonatal rat pups. In cultured chick intestinal duodena calcium transport begins to appear within 4 h after the addition of 1,25-(OH)2-D3. The response of this calcium transport system to 1,25-(OH)2-D3 is totally blocked by cycloheximide in a reversible manner. Similarly, it is blocked by actinomycin D in a partially reversible manner. These results make it obvious that the rapid calcium transport response to 1,25-(OH)2-D3 involves nuclear activity and transcription of DNA into functional proteins. The exact nature of the transport proteins remains largely unknown except for the calcium-binding protein originally discovered by Wasserman and colleagues. The transport proteins are believed to operate at the brush border membrane surface to facilitate the transfer of calcium and phosphorus into the absorption cells.
Recent studies have shown that 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) stimulates the entry of calcium into the duodenal mucosal cell of the chick by a mechanism that does not require the synthesis of new protein. Using isolated brush border membrane vesicles (BBMV) from these cells, we have explored the mechanism by which 1,25-(OH)2-D3 acts. Administration of the hormone leads to an increase in calcium uptake into BBMV. This calcium uptake is a saturable process. Addition of the methyl ester of cis-vaccenic acid to BBMV in vitro leads to a specific increase in calcium uptake into vesicles from vitamin D-deficient chicks but not in those from 1,25-(OH)2-D3-treated chicks. Administration of 1,25-(OH)2-D3 leads to an increase in the de novo synthesis of phosphatidylcholine (PC) and an increase in the total PC content of the brush border membrane. It also increases the turnover of fatty acids into PC, which results in an increase in the content of polyunsaturated fatty acids in the PC fraction. These changes in lipid structure and turnover either precede in time or occur simultaneously with the change in calcium transport rate, and neither is blocked by the administration of cycloheximide. It is proposed that the primary mechanism by which 1,25-(OH)2-D3 regulates calcium transport across the luminal membrane of the enterocyte is by inducing a specific alteration in membrane PC content and structure, which leads to an increase in membrane fluidity and thereby to an increase in calcium transport rate.
Three studies of the role of 1,3-butanediol (BD) in human nutrition are described. Isocaloric substitution of BD for starch in the diets of volunteers caused less negative nitrogen balance. Ingestion of urea also decreased negative nitrogen balance, and the effect of BD plus urea in the diets seemed to be additive. No effects were detected on many blood parameters measured during and after the study, except that BD feeding decreased blood glucose significantly. 1,3-Butanediol was shown to be a nontoxic metabolite providing a source of calories for human nutrition. In a second study, ingestion of BD was shown to cause slight increases in serum insulin and growth hormone concentrations in the fasting state. We next studied the effects of prior ingestion of BD on serum insulin, growth hormone, glucose, and lipids during glucose tolerance tests. No significant differences in these parameters were noted when prior ingestion of sucrose or an isocaloric quantity of BD were compared. Possible mechanisms whereby ingestion of BD spared nitrogen and caused decreased blood glucose are discussed.
These studies have been designed to test whether 1,3-butanediol (BD) alleviates milk fat depression in lactating cows, to observe physiological changes in blood and rumen constituents when BD is fed to cows or growing cattle, and to test the effects of BD on growth rates and feed efficiency in growing cattle. In trials with lactating cows, milk fat percentage and total fat production were higher for cows fed BD than for controls. Feeding BD to either cows or growing cattle had no consistent effect on rumen pH or relative concentrations of rumen volatile fatty acids. 1,3-Butanediol feeding had little effect on blood glucose concentrations. Feeding more than 4% BD in diets sometimes caused increased concentrations of blood ketones. In trials where growing cattle were fed 4% BD, rates of gain and feed efficiency were at least as good as and often better than those of cattle fed the same diets without BD. Body composition was not significantly affected. 1,3-Butanediol can be utilized effectively as an energy source for cattle and causes no obvious problems with 4% in diets.
It has been demonstrated that administration of BD to rats results in rapid elevation ketone body concentrations of blood, urine, and tissue. In liver slices BD is metabolized almost quantitatively to acetoacetate and β hydroxybutyrate. Oxidation of a primary alcohol such as BD to a carboxylic acid implies the participation of an alcohol dehydrogenase. All of the data thus far obtained indicate that liver alcohol dehydrogenase (EC 22.214.171.124) is the enzyme primarily, if not solely, responsible for the initial oxidation of BD. The effect of NADH2 generated from BD oxidation on the lactate:pyruvate ratio is consistent with the predominantly cytosolic location of ADH. Tissues such as heart which have very low ADH activities produce minimal amounts of NADH2 and reduced flavoprotein when perfused with BD. The specificity of the ADH activity for NAD rules out the microsomal alcohol oxidizing system. Results of inhibitor experiments also favor the participation of ADH in BD metabolism. Although n butyraldoxime is known to affect both aldehyde oxidase and ADH activity in vivo, pyrazole appears to be specific for ADH, and it is effective in vivo and in vitro. The microsomal ethanol oxidizing system, however, is not sensitive to pyrazole inhibition. Thus the effects of pyrazole and of n butyraldoxime on BD oxidation can be ascribed to their inhibitory effect on the oxidation of BD to butyraldehyde in the cytosol. Furthermore, the BD dehydrogenase activity exhibits substrate inhibition at concentrations greater than 10 mM in a manner similar to that described for horse liver ADH. The suggested metabolic pathway for oxidation of 1,3 butanediol is presented.