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Biomedical subjects

R H Wasserman

Publications and source records attributed to R H Wasserman.

At least 37 records · Page 2Linked to original sources
PubMed1990

Enhancement of sulfhydryl group availability in the intestinal brush border membrane by deficiencies of dietary calcium and phosphorus in chicks.

The reactivity and availability of sulfhydryl (-SH) groups in brush border membranes (BBM) from chicks adapted to a calcium-deficient (low Ca) or a phosphorus-deficient (low P) diet were determined. The calbindin-D28K concentrations of the intestinal mucosa of the low Ca and low P groups were both increased approximately 2.5-fold, demonstrating that adaptation to the mineral deficiencies had occurred. By the Ellman reaction, a threefold increment in -SH groups in BBM from both mineral-deficient groups was noted. By using DACM (N-7-dimethylamino-4-methylcoumarin-3-yl maleimide), a fluorescent probe for -SH groups, it was observed that fluorescence development was considerably greater with BBM from the low Ca and low P groups than with BBM from the controls, whether measured in the absence or presence of sodium dodecyl sulfate (SDS). In the absence of SDS, the pseudo-first-order reaction rate constants, k', calculated from the fluorescence data, were greater than the control group values, but in the presence of SDS, the k' values for all groups were about the same. Similar changes in BBM-SH groups were previously observed when 1,25-dihydroxycholecalciferol was given to vitamin D-deficient chicks. The redox state of the sulfhydryl groups in enzymes and transport proteins is known to affect the level of their activity. The functional significance of the present observations concerning the -SH groups of chick intestinal BBM, particularly in relation to vitamin D-dependent calcium and phosphorus absorption, is not known but is under investigation.

Animals↗
PubMed1990

Relationship of membrane-bound sulfhydryl groups to vitamin D-stimulated uptake of [75Se]Selenite by the brush border membrane vesicles from chick duodenum.

The uptake of selenite by purified brush border membrane vesicles isolated from duodena of rachitic or vitamin D-treated chicks was studied by using radioactive selenite and a rapid filtration technique. Cholecalciferol treatment (500 IU at 72 h) significantly enhanced selenite uptake, a response that decreased when the vesicles were stored at room temperature for 2.5 h prior to the uptake measurement. Preincubation of the vesicles in 1.0 mmol/L H2O2 reduced [75Se]selenite uptake, indicating the involvement of oxidizable groups in the uptake reaction. Iodoacetic acid (IAA), a sulfhydryl-blocking reagent, at 1-2 mmol/L concentration eliminated the difference in selenite uptake due to cholecalciferol and had no effect on vesicles from rachitic animals. A higher concentration of IAA (10 mmol/L) enhanced selenite uptake manyfold and increased the absolute difference due to cholecalciferol treatment. Single intravenous doses of 100 IU cholecalciferol, 100 IU ergocalciferol, or 0.1 micrograms 1,25-dihydroxycholecalciferol also stimulated selenite uptake, suggesting a general response to vitamin D compounds. Normal animals given a single dose of 1,25-dihydroxycholecalciferol 12 h prior to killing also responded. Treatments that enhanced the uptake of [75Se]selenite also increased the amount of membrane-bound sulfhydryl groups, suggesting the involvement of membrane-bound sulfhydryl groups in the vitamin D response. A significant increase in selenite uptake by intravenous 1,25-dihydroxycholecalciferol occurred within 10 min. This rapid effect provides a new tool to probe early biochemical effects of vitamin D on intestinal epithelium.

Animals↗
PubMed1990

Recent studies on the biological actions of vitamin D on intestinal transport and the electrophysiology of peripheral nerve and cardiac muscle.

Vitamin D, with parathyroid hormone and calcitonin, is an essential factor in the homeostatic regulation of systemic calcium in most vertebrate species. Targets for this aspect of vitamin D action, through its biologically active metabolites, are primarily the intestine, kidney and bone. Each of these tissues or organs are stimulated by 1,25(OH)2D3 to increase the transport calcium into the extracellular fluid compartment when plasma calcium levels are below normal and/or when there is a greater need for calcium to meet the requirements of physiological processes, such as growth, gestation and lactation. During such periods, the efficiency of the absorption of calcium from the intestine increases, the resorption of calcium salts from bone is stimulated, and the efficiency of the reabsorption of filtered calcium by the renal tubule is increased. In addition to the homeostatic function of vitamin D, there is an increasing amount of evidence that vitamin D has important effects on tissues and organs other than those concerned with calcium homeostasis. With regard to the intestinal epithelial system, the genomic effect of 1,25(OH)2D3 was shown several years ago when the de novo synthesis of a specific vitamin D-induced calcium-binding protein (CaBP, calbindin-D) was demonstrated. In our view, this appears to be an essential factor in the well-documented enhancement of calcium absorption by vitamin D. The function of calbindin-D, a high affinity calcium-binding protein, in the absorptive process is not precisely known but currently considered to act as an intracellular facilitator of the diffusion of calcium from the microvillar pole of the enterocyte to the basal-lateral membrane. There is evidence that vitamin D influences another step in the absorptive process. This step appears to be associated with the entrance of luminal calcium into the enterocyte, the first step in the transepithelial transport process. This response appears to occur relatively early (1 h or less) after 1,25(OH)2D3 is given to vitamin D-deficient animals, whereas the de novo synthesis of transport proteins has a much longer lag time (about 4 h). The in vitro absorption studies of Nemere et al (1984) and the in vivo experiments of our group (Wasserman et al, 1982) accentuate this point. However, the more rapid reaction, i.e., the possible modification of the permeability properties of the brush border membrane, does not result in a substantive increase in overall calcium absorption unless the enterocyte had been "primed" by previous exposure to vitamin D. The "priming" reaction might represent the synthesis of CaBP or some other intracellular component.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

On the molecular mechanism of intestinal calcium transport.

The intestinal absorption of calcium is certainly a complex process, dependent on several factors of which vitamin D, via 1,25(OH)2D3, is the major controlling hormone. The efficiency of calcium absorption is a function of calcium status and calcium need. As the body's demand for calcium increases, the process commonly termed, adaptation, is activated in which the synthesis of 1,25(OH)2D3 from precursor is increased, resulting in the stimulation of the rate of calcium absorption. The increased demand for calcium might result from the ingestion of a diet deficient in calcium, from growth, pregnancy, lactation and egg shell formation in the laying hen. Accomapanying the change in calcium absorptive efficiency are molecular modifications of the transporting enterocytes, some mentioned herein and elsewhere (Wasserman & Chandler, 1985; Wasserman, 1980; Wasserman et al., 1984). Highly correlated with the rate of calcium absorption under a wide variety of conditions is the concentration of the vitamin D-induced calcium-binding protein, calbindin-D28K (avian type) and calbindin-D9K (mammalian intestinal type). The role of calbindin-D in this transport process is not precisely known but is considered to act at the present time as a cytosolic facilitator of Ca2+ diffusion from the brush border membrane to the basolateral membrane. In addition to the induction of calbindin-D synthesis, 1,25(OH)2D3 exerts other effects on the intestinal epithelium that can have consequences on the calcium absorptive process. Some of these effects are summarized in Figure 14. Vitamin D-dependent reactions might be either direct effects of 1,25(OH)2D3 or indirect effects due to elevated intracellular Ca2+ concentrations. These include changes in the fluidity of the brush border membrane, an increase in microvillar alkaline phosphatase-low affinity Ca-activated ATPase activity, an association of calmodulin with the 105 kD brush border cytoskeletal protein and, following calbindin D synthesis, the binding of calbindin D to a 60 kD brush border protein and to microtubules. The latter has been suggested to be related to the proposed transfer of Ca2+ by an endocytotic-exocytotic mechanism. In addition, a vitamin D-dependent intestinal membrane calcium-binding protein has been identified (Kowarski & Schachter, 1980). Playing into this multi-component system is a stimulation of cyclic nucleotide synthesis by 1,25(OH)2D3 which, through activation of cyclic nucleotide-dependent protein kinases, might modify membrane Ca2+ "channels" by phosphorylation reactions.4+ Intracellular organelles, i.e., the endoplasmic reticulum, mitochondria, the Golgi apparatus, are potent sequesters of Ca2+ and could contribute to the protection of the cell from excessively high Ca2+ concentrations by transiently storing absorbed Ca2+.

Animals↗
PubMed1989

Uptake of 75Se-selenite by brush border membrane vesicles from chick duodenum stimulated by vitamin D.

Brush border membrane vesicles were isolated from mucosal homogenates of duodena from normal, rachitic and vitamin D-treated rachitic chicks using a discontinuous sucrose gradient, and further purified by glycerol gradient centrifugation. In vitro uptake of 75Se-selenite by purified brush border membrane vesicles was studied using a rapid filtration technique. The time course of 75Se uptake was non-linear; rapid initial binding was followed by a gradual decrease in the rate of uptake until an equilibrium value was reached at 60-120 min. The initial binding at 36 s was not affected by selenite concentration in the incubation buffer, while the fractional rate of uptake between the 36 s and 2 min time periods was clearly lower with 1 mM Se than with 4-100 microM Se. 75Se uptake did not show any dependency on the external Na-gradient, nor could it be inhibited by other anions (arsenate, phosphate). Treatment of rachitic chicks either with cholecalciferol (500 Iu, 72 h) or with 1,25(OH)2-cholecalciferol (0.5 microgram given 16 h prior to isolation of the vesicles) significantly enhanced 75Se uptake. A threefold excess of mannitol in the outside buffer reduced 75Se uptake by vesicles from vitamin D-deficient and D-treated chicks 60% and 35% respectively, but had no effect on vesicles from vitamin D-treated chicks preloaded with 75Se. Neither saponin treatment nor excess cold selenite could release the label from the vesicles preloaded with 75Se. These data are compatible with the hypothesis that selenite easily crosses the brush border membrane into the intravesicular space and, once inside, is tightly bound by the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Calcium-dependent translocation of calbindin-D28k from intestine to blood.

Calbindin-D (vitamin D-induced calcium-binding protein; CaBP) is known to be present in blood at concentrations which vary directly with levels in the intestinal mucosa. Employing a sensitive radioimmunoassay and sampling mesentery venous blood, the present experiments demonstrated a direct relationship between intestinal calcium absorption and serum CaBP. Solutions containing 150 mM NaCl and 45Ca-labeled calcium chloride (5 or 20 mM) were placed in the lumen of ligated duodenal preparations in situ and mesentery venous blood sampled with time. The concentration of absorbed 45Ca in serum was maximal at 5 min, followed by a significant increase in mesentery CaBP maximizing at 15-20 min. Elevation of serum CaBP was not observed when calcium in the dosing solution was omitted or replaced by either glucose or glycine. The possible transfer of absorbed calcium from the enterocyte to the circulation as a CaBP complex was ruled out by calculations revealing that considerably more calcium was transferred than could be accounted for by the low and high affinity binding sites on the protein. It is proposed that vitamin D-dependent enhanced transcellular calcium transport constitutes a stimulus for the increased release of intestinal CaBP into the circulation.

Animals↗
PubMed1987

The effect of disodium ethane-1-hydroxy-1,1-diphosphonate on the metabolism of calcitriol in chicks.

Decreased intestinal absorption of Ca2+ occurs in response to treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP). The effect is due to decreased 1-hydroxylation of calcidiol (25-hydroxycholecalciferol) in the kidney. In an attempt to establish whether impairment of vitamin D metabolism at steps beyond kidney hydroxylation occurs due to treatment with EHDP, chicks were depleted of vitamin D and were treated with calcitriol (1,25-dihydroxycholecalciferol) as their sole source of the vitamin. The chicks were then divided into two groups, one being treated with EHDP while the second group served as control. Intestinal absorption of Ca2+ in the EHDP-treated group was found to be impaired, along with decreases in concentrations of calbindin D28K (the 28,000-Mr vitamin D-dependent Ca2+-binding protein). When the chicks were dosed with [3H]calcitriol, significantly lower concentrations of the sterol were detected in the duodena of EHDP-treated birds. Measurement of levels of receptors for calcitriol in duodena showed no difference between groups, but levels of calcitriol in sera were considerably lower in the EHDP-treated group along with the elevated biliary and urinary excretion of glucuronidated conjugates. It is therefore concluded that treatment with EHDP results in increased catabolism of calcitriol in addition to the known suppression of the renal production of the hormone.

Animals↗
PubMed1987

Chicken intestinal 28-kilodalton calbindin-D: complete amino acid sequence and structural considerations.

The complete primary structure of the cholecalciferol-induced chicken intestinal 28-kDa calbindin-D is reported. It is a single-chain polypeptide consisting of 261 amino acid residues (computed Mr = 30,042) and is blocked at the amino terminus. Tryptic digestion of the S-pyridylethylated protein followed by HPLC peptide mapping and automated sequence determination provided the bulk of the sequence information. Subsequent chymotryptic and Staphylococcus aureus V8 protease cleavages yielded the sequences of several additional regions as well as extensive overlapping of the tryptic peptides. The primary structure shows six homologous regions of sequence based on the EF-hand concept of calcium binding, four of which are predicted to actually bind calcium. Aside from these regions, there is no overall structural identity or apparent similarity with the mammalian calbindins (9 kDa), calmodulin, or troponin C. It is predicted that the secondary structure of 28-kDa calbindin-D is significantly different from the other proteins of this class, which bind four calcium atoms.

Amino Acid Sequence↗
PubMed1987

Calbindin-D in peripheral nerve cells is vitamin D and calcium dependent.

The vitamin D-induced calcium-binding protein calbindin-D (CaBP) was localized immunohistochemically in some but not all of the cell bodies and axons within the intestinalis nerve of the chicken. Unlike other nerve tissue thus far examined, the CaBP content of the intestinalis nerve was decreased in vitamin D deficiency and increased in chicken adapted to a calcium-deficient diet. These changes are qualitatively similar to the pattern of response of enterocytes. The inclusion of calcium-containing solutions within the duodenal lumen caused, directly or indirectly, a decrease in the amount of CaBP in this nerve in a dose-dependent manner. The exact role of CaBP in intestinalis nerve cells is unknown but may be in the regulation of intracellular ionic Ca2+ concentrations during excitation, although other functions of CaBP cannot be excluded.

Animals↗
PubMed1985

Lead-binding properties of intestinal calcium-binding proteins.

The bovine and chick vitamin D-induced intestinal calcium-binding proteins (CaBP) bind lead. Bovine CaBP binds 2 atoms of lead/molecule, and chick CaBP binds 4 atoms of lead per molecule and these values are identical to those for calcium binding. 45Calcium-displacement studies indicate significantly higher affinities for lead than for calcium for both proteins. All evidence indicates that lead is bound to the 4 high affinity calcium-binding sites on chick CaBP and to the corresponding 2 high affinity sites on bovine CaBP, and that binding of lead to sulfhydryl groups is, relatively, not significant. Calmodulin, troponin C, and oncomodulin also bind lead with high affinities and in preference to calcium, indicating that lead binding is a general property of proteins belonging to the troponin C superfamily of calcium-binding proteins.

Animals↗
PubMed1985

Intestinal absorption of arsenate in the chick.

The intestinal absorption of arsenate(As(V)) has been investigated in the chick by means of the in situ ligated duodenal loop technique. By this procedure, it was observed that arsenate is rapidly and essentially completely absorbed (80-95%) from the lumen at As(V) concentrations up to 5 mM, declining to about 50% absorption at 50 mM. Transfer from the intestinal lumen to the mucosal cells at low As(V) concentration (0.1 mM) is rapid, while transfer from the mucosal cells to the body occurs more slowly. At stable As(V) concentrations greater than 1 mM, fractional mucosal cell accumulation of As(V) remains constant, while fractional transfer to the body declines. However, total mucosal accumulation of As(V) and that transferred to the body increase in a linear logarithmic fashion from 0.05 to 5 mm As(V). The results indicate that As(V) readily penetrates both the mucosal and serosal surfaces of the epithelial membrane. Furthermore, arsenate and phosphate do not appear to share a common transport pathway in the duodenum and no evidence was obtained for any interaction between the two at this level. Vitamin D3 administration to rachitic chicks was effective in significantly elevating duodenal arsenate absorption, acting primarily to enhance serosal transport.

Administration, Oral↗
PubMed1984

Effect of phosphate on the intestinal absorption of lead (203Pb) in chicks.

The effect of phosphate on the intestinal absorption of lead (203Pb2+) was examined in chicks. Absorption was determined by the in situ ligated duodenal loop technique. In one approach, diets differing in phosphate content were fed to 2-week-old chicks for a period of 7 days. With respect to the control group (1.06% P), a severe phosphate deficiency (0.16% P) decreased growth, CaBP synthesis and 203Pb absorption; a moderate phosphate deficiency (0.33% P) resulted in an increase in CaBP production and 203Pb absorption; and the high phosphate diet (2.12%) gave mean values for CaBP synthesis and 203Pb absorption intermediate between those from the chicks fed the 0.33 and 1.06% diets. 203Pb absorption was highly correlated with CaBP concentrations. In another approach, phosphate in varying concentrations was added directly to the dosing solution. In one study, phosphate addition (0.1 mM) depressed 203Pb absorption in rachitic and cholecalciferol-treated chicks. When the data were expressed in terms of absorption of the soluble 203Pb present in the intestinal lumen at the end of the absorption period, an effect of phosphate in addition to the precipitation of the insoluble lead salt was uncovered. In an experiment with normal chicks, it was observed that phosphate in the dosing solution at concentrations of 1 and 2 mM partially and significantly reversed the inhibitory effect of lower concentrations of phosphate (0.01 mM and 0.1 mM). These data demonstrate the complexity of the phosphate-lead interaction, in addition to directly showing an effect of dietary phosphate on intestinal lead absorption.

Animals↗
PubMed1984

Gastrointestinal absorption of lead in chicks: involvement of the cholecalciferol endocrine system.

The role of dietary calcium and phosphorus in modifying the intestinal absorption of lead and also the effect of lead ingestion on the metabolism of cholecalciferol were studied in chicks. The efficiency of absorption of 203Pb and 47Ca was increased when the animals were fed a low calcium diet and treated with cholecalciferol. The synthesis of the vitamin D-induced calcium-binding protein (CaBP) was correspondingly increased. When the chicks were depleted of vitamin D and repleted with 1,25-dihydroxycholecalciferol [1,25(OH)2D3] as their only source of the vitamin, the absorption of both 47Ca and 203Pb was unaffected by dietary calcium levels, and no change in CaBP levels occurred. Low dietary intake of phosphorus resulted in an increase in 47Ca and 203Pb absorption and in CaBP synthesis when the animals were treated with cholecalciferol. However, when the birds were repleted with 1,25(OH)2D3, the intestinal absorption of 47Ca and of 203Pb was increased, as well as the intestinal CaBP levels. Intracardial injection of increasing doses of 1,25(OH)2D3 to rachitic chicks resulted in a concomitant increase in 203Pb absorption in a manner that correlated with the degree of synthesis of CaBP. Ingestion of lead by the chicks was found to impair growth and renal production of 1,25(OH)2D3, resulting in lowered circulating and intestinal content of the hydroxylated metabolites of cholecalciferol.

Animals↗
PubMed1984

Membrane-associated vitamin D-induced calcium-binding protein (CaBP): quantification by a radioimmunoassay and evidence for a specific CaBP in purified intestinal brush borders.

The vitamin D-induced intestinal calcium-binding protein (CaBP) was quantitated in membranous components of the intestinal mucosa by a specific and sensitive RIA. Inclusion of detergent (Triton X-100) in extraction buffer and in the RIA system was required to release and measure membrane-associated CaBP. Purified brush borders were shown to contain CaBP with a specific activity (micrograms per mg protein) about 12% of that in the total homogenate. By transferring proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose blots electrophoretically, CaBP was immunologically detected in brush borders from vitamin D3-treated chicks, but not in those from vitamin D3-deficient chicks. CaBP was also detected in isolated brush border membrane vesicles by the gel electrophoresis-blot transfer technique. Brush border CaBP was inaccessible to proteolytic hydrolysis by trypsin unless trypsinized in the presence of detergent. CaBP-binding substances were found to be present in purified brush borders, using the gel overlay technique. A specific binding protein with a mol wt in the range of 50,000-70,000 daltons was identified, as well as an avid CaBP binder at less than 14,000 mol wt. These observations provide evidence for the association of a significant fraction of total intestinal CaBP with brush borders in vivo, which might have physiological relevance.

Animals↗
PubMed1984

Early and direct effect of 1,25-dihydroxycholecalciferol on calcium uptake by duodena of rachitic chicks.

Injection of 1,25 dihydroxycholecalciferol (1,25(OH)2D3, 10 micrograms) directly into the in situ ligated duodenal loop of rachitic chicks significantly elevated the tissue accumulation of 47Ca within 20-30 min. The transfer of 47Ca from lumen to blood, during the same time period, was not increased nor was there any measurable intestinal calcium-binding protein synthesized. Lesser amounts of 1,25(OH)2D3 (1 or 5 micrograms) did not result in any statistically significant elevation of 47Ca tissue accumulation, nor did they have any effect on 47Ca transfer from lumen to blood (transmural). Ten micrograms of 1,24R,25(OH)3D3 was similarly effective in elevating tissue accumulation, whereas 24R,25(OH)2D3 and 25(OH)D3 were not. These results provide additional evidence for an early and direct action of 1,25(OH)2D3 in altering intestinal epithelial membrane transport prior to the induction of synthesis of specific transport proteins.

24,25-Dihydroxyvitamin D 3↗