Federation Proceedings

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.
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.
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.
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.
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.
Male rats were made diabetic by intravenous administration of 75 mg/kg of streptozotocin and were fed, via a pair-feeding regimen, high-fat diets +/- 1,3-butanediol (BD) at 13.5 and 27% of the dietary calories for 30 days and 31 days, respectively. 1,3-Butanediol was added to the diets primarily as a replacement for fat. Food consumption and rat weight were recorded daily. Whole blood glucose concentrations were determined weekly. At sacrifice, liver, pancreas and epididymal fat pads were excised and blood samples were collected. Liver was analyzed for protein and lipid; pancreas was weighed and analyzed for insulin; fat pads were weighed and discarded; and blood was analyzed for glucose and lipid. The 13.5% BD diet increased the beta-hydroxybutyrate, acetoacetate and cholesterol concentrations, decreased the glucose concentration in blood, and increased the insulin content of the pancreas. The BD diets did not affect the concentrations of phospholipid, triglyceride, cholesterol and fatty acid in the liver; fatty acid concentrations in the blood; or the epididymal fat pad weight. The results suggest that BD produced a slight amelioration of the diabetic condition, which may have resulted from an increased capacity of the pancreas to synthesize insulin. In addition, the data provide further evidence suggesting that in the rat BD is oxidized to the ketone bodies, beta-hydroxybutyrate and acetoacetate.
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 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.
Research in the United States on synthetic sources of dietary calories was initiated in 1958 to develop high nutrient density food for extended manned space travel. Of many known compounds screened, 1,3-butanediol was the most promising. Small amounts in ester form with fatty acids exist in nature, and tests indicate a low acute oral and chronic toxicity similar to that of propylene glycol or glycerol. Multi-generation reproduction, teratological, and mutagen studies have revealed nothing detrimental. Following an adaptation period, 1,3-butanediol furnishes approximately 6 kcal/g if fed at levels not exceeding 20% in the diet of rats. Higher levels result in an impairment in growth and food utilization. In young animals, body fat stores appear to be lessened, as is resistance to the stress of extreme cold. However, dogs fed 20% 1,3-butanediol can maintain sustained muscular work on treadmills, but larger amounts can result in incoordination due to a narcotic effect common to glycols. Little research has been conducted on the behavioral effects of large doses. At present, 1,3-butanediol is used mainly as a solvent for food flavors. If the unpleasant taste problem can be overcome and if given FDA approval, 1,3-butanediol may have an increased role in our food supply as a functional food additive, preservative, and source of calories for man and animals.
A wide variety of receptors appear to be coupled to a phospholipase C (EC that hydrolyzes inositol lipids. This reaction is believed to provide a link between receptor activation and cellular Ca2+ mobilization. The mechanisms by which this occurs are believed to involve inositol 1,4,5-trisphosphate (1,4,5-IP3), which signals release of Ca2+ from the endoplasmic reticulum. In rat parotid acinar cells made permeable with saponin, 1,4,5-IP3 induced rapid release of sequestered Ca2+. In intact parotid cells, the concentration-response relationship for methacholine-induced IP3 formation was similar to the relationship for muscarinic receptor occupancy by methacholine. About 10-fold lower concentrations of methacholine were sufficient to increase cytosolic [Ca2+] and to activate secretion, indicating an excess IP3 forming capacity for the muscarinic receptor. The mechanisms for the coupling of receptors to IP3 formation were studied in pancreatic acinar cells made permeable electrically. In this preparation, nonhydrolyzable derivatives of GTP potentiated agonist-induced IP3 production, which suggests the involvement of a guanine nucleotide-dependent regulatory protein. The effects of agonists and guanine nucleotides were not altered by pretreating the acinar cells with cholera or pertussis toxins, which indicated that the regulatory protein linking receptors to IP3 formation is distinct from the ones involved in the regulation of adenylate cyclase.
Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase. Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis. Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain. Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate. This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes. Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound. This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase. The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.
Ly 11.2, a newly defined T cell surface antigen, is present on two lymphocyte subpopulations playing a key role during leukemogenesis: the natural killer cell and possibly the target cells for malignant transformation by oncogenic agents. Antibodies to this cell surface marker should greatly help in the study, isolation, and characterization of these cells.
Cadmium can replace zinc and magnesium in alkaline phosphatase from Escherichia coli, which permits the characterization of the catalytically important metal-binding sites by 113Cd NMR. At pH 6.5, in the absence of phosphate, two equivalents of cadmium are bound in identical sites (A), one in each monomer. Either raising the pH or phosphorylation of Cd2AP (AP is apoalkaline phosphatase) results in migration of Cd(II) from the site A in one monomer to the opposite monomer to occupy a second site (B) adjacent to the A site in the first monomer, a site stabilized by phosphorylation or high pH. At pH 6.5 in the presence of phosphate, the 113Cd NMR spectrum of Cd6AP consists of three narrow resonances from three pairs of fully occupied sites, A, B, and C. The resonances at 153 and 70 ppm represent two metal sites (A and B) 3.9 A apart at each active center and adjacent to the serine phosphorylated during turnover. At this pH the enzyme exists almost exclusively as the covalent phosphoseryl form E-P with a 31P resonance at approximately 9 ppm. As the pH is raised a 31P signal from the noncovalent E.P complex appears at approximately 13 ppm. This is reflected in the 113Cd spectrum by a split of both the A- and B-site resonances into pairs, a set at 137 and 65 ppm for E.P, and 153 and 70 ppm for the E-P species. 113Cd-O-31P coupling of 30 Hz on the 31P resonance of E.P shows the noncovalently bound phosphate to be coordinated to one but not both metal ions at each active site. The resonance of E-P is not coupled and thus the phosphoseryl residue appears to shift out of the coordination sphere of the active site metal ion.
CL 115,347 orally (0.25-10 mg/kg) and topically (0.03 and 0.1 mg/kg) lowered blood pressure in a dose-dependent manner in conscious spontaneously hypertensive rats (SHR). Duration of action of the oral dose range was from 1 to more than 8 h and of the topical dose range, from more than 6 to more than 24 h. CL 115,347 was 100-200 times more potent orally and greater than 250 times more potent topically than l-prostaglandin (PG) E2. When 3 mg/kg was administered orally, CL 115,347 was also active in Dahl "S" salt-sensitive hypertensive rats, deoxycorticosterone acetate-salt hypertensive rats, aorta-coarcted renin-dependent hypertensive rats, normotensive rats, bilaterally nephrectomized SHR, and bilaterally ureteral-ligated SHR. CL 115,347 was also orally active at 0.1 mg/kg in normotensive rhesus monkeys and in renal hypertensive dogs at 1 mg/kg. CL 115,347 was as active as l-PGE2 in relaxing the rabbit ear arterial smooth muscle in vitro. In anesthetized dogs, CL 115,347 injected intra-arterially (0.5-10 micrograms) into the vascular bed being studied increased blood flow to femoral, carotid, coronary, superior mesenteric, and renal vascular beds. CL 115,347 decreased vasopressor responses induced by electrical stimulation of the spinal cord at T7-T9 but did not decrease the tachycardia induced by stimulation of the cardioaccelerator segments (C7-T1) in pithed SHR. CL 115,347 has a broad spectrum of antihypertensive activity in various animal models and probably exerts its major antihypertensive effects through relaxation of blood vessels.
Methods designed for estimation of the synthesis of plasma glucose are based on the transfer of 14C atoms from a selected precursor (substrate) such as lactate or alanine. This approach was shown to lead to an underestimation of the true synthesis of glucose because of the metabolic exchange of 14C atoms for 12C atoms in the hepatic oxaloacetate pool. From the incorporation of 14C atoms from intravenously infused [2-14C]acetate into plasma glucose, the extent of the metabolic exchange has been estimated. In normal dogs, metabolic exchange leads to an underestimation of plasma glucose synthesis from plasma lactate or alanine by a factor of 2.2 +/- 0.07, i.e., by 55%. In insulin-deprived diabetic dogs, the factor was found to be 1.8 +/- 0.05. In long-term fasted dogs, the factor may be higher than in the postabsorptive state, whereas treatment with methylprednisolone has no effect. The assumptions and sources of possible errors in the estimation of the extent of metabolic exchange are reviewed.
An approach to chemoprevention of common forms of epithelial cancer, during the period of preneoplasia, is described. Vitamin A and its synthetic analogs (retinoids) are potent agents for control of cell differentiation in many epithelial tissues. Direct effects of retinoids on normal and preneoplastic cell differentiation can be measured in organ culture. In experimental animals, deficiency of dietary retinoids enhances susceptibility to chemical carcinogenesis. Natural retinoids, fed at high dietary levels, have some ability to prevent chemical carcinogenesis in epithelial tissues of bronchi, trachea, stomach, uterus, and skin of experimental animals. However, natural retinoids have limited usefulness for chemoprevention of cancer because of inadequate tissue distribution and excessive toxicity. Synthetic retinoids have been made and shown to be more potent and less toxic for prevention of cancer in animals. Several structural modifications of the ring and terminal portions of the retinoid molecule have significant biological activity; modification of the side chain has been more difficult. The potential future usefulness of this approach to cancer prevention in man will depend on further synthetic modification of the retinoid molecule.
113Cd NMR has been used to determine the structures of the multiple metal-binding sites in the two major isoproteins of metallothionein from mammalian livers (rabbits, calf, and human) and from Scylla serrata hepatopancreas. The native protein isolated from the livers of rabbits that had been subjected to repeated injections of 113CdCl2 contains an appreciable amount of Zn2+ in addition to 113Cd2+, ranging from 2 to 3 g-atoms of a total metal content of 7 g-atoms/mol of protein. The native Zn2+ can be replaced in vitro with 113Cd2+ to give a 113Cd NMR spectrum consisting of eight distinct multiplets in the chemical shift range of 604-670 ppm. The multiplet structure is due to 113Cd-113Cd scalar coupling arising from two-bond interactions between 113Cd2+ ions linked to one another by bridging cysteine thiolate ligands. Analysis of the 113Cd spectra by selective homonuclear 113Cd decoupling techniques showed that both isoproteins of rabbit liver metallothionein contain two separate metal clusters, one containing Cd2+ ions (cluster A) and the other containing three (cluster B). Structures for the clusters are proposed that account for the 113Cd chemical shift and spin coupling data and the participation of all 20 cysteine residues in metal ligation. The 113Cd NMR spectrum of 113Cd2+-reconstituted human liver metallothionein is remarkably similar to that of the rabbit and the analysis confirms that it contains the same two-cluster arrangement. Native calf liver metallothionein was found to contain copper and Zn2+ and the in vitro exchange with 113Cd2+ selectively replaces only the Zn2+. The reconstituted protein contains 3.9 g-atoms of 113Cd2+ and 2.6 g-atoms of copper and the 113Cd NMR spectrum showed four major multiplets with identical chemical shifts to the resonances previously assigned to the four-metal cluster A. This result was confirmed by homonuclear decoupling experiments. The 2.6 g-atoms of electron spin resonance-silent copper in this sample is presumably selectively bound to the three-metal cluster B sites. Both isoproteins of metallothionein isolated from 113Cd2+-injected mud crabs (S. serrata) contain only 113Cd2+. The analysis of the 113Cd NMR spectra show that the total of six metals bound per mole of crab metallothionein-1 (MT-1) and MT-2 are arranged in two separate three-metal clusters.
Top-cited authors
Michael Sporn
  • Geisel School of Medicine at Dartmouth
Burton Altura
  • State University of New York Downstate Medical Center
B.T. Altura
  • State University of New York Downstate Medical Center
Floyd Elliott Bloom
  • The Scripps Research Institute
Alfred L Goldberg
  • Harvard Medical School