Peter F. Brumbaugh

Regulation of serum 1alpha,25-dihydroxyvitamin D3 by calcium and phosphate in the rat

A new radioreceptor assay was used to quantify changes in serum concentration of 1alpha,25-dihydroxyvitamin D3 in rats with low calcium or low phosphate diets. Low availability of either ion elicits a fivefold increase in the circulating concentration of 1alpha,25-dihydroxyvitamin D3. The enhancement of 1alpha,25-dihydroxyvitamin D3 concentration in response to calcium deficiency is dependent on the presence of the parathyroid or thyroid glands (or both), suggesting that this effect is mediated by parathyroid hormone. In contrast, the response of phosphate deficiency is independent of these glands and may result from an action of low serum phosphate concentration or some factor associated with phosphate depletion on the renal synthesis of the 1alpha,25-dihydroxyvitamin D3 hormone.

Radioreceptor Assay for 1α,25-Dihydroxyvitamin D3

A competitive protein binding assay with a sensitivity of 80 picograms has been developed for 1α,25-dihydroxyvitamin D3, the hormonal form of vitamin D3. lα,25-Dihydroxyvitamin D3 displaced tritiated hormone from a cytosol-chromatin receptor preparation isolated from chick small intestine, providing a simple assay for the hormone. The concentration of lα, 25-dihydroxyvitamin D3 in human plasma, as determined by this assay, is approximately 6 nanograms per 100 milliliters; in patients with renal disease the concentration of this kidney-produced hormone is significantly lower.

Nuclear and cytoplasmic receptors for 1,25-dihydroxycholecalciferol in intestinal mucosa.

The apparent hormonal form of cholecalciferol, 1,25-dihydroxycholecalciferol (1,25-(OH)2-CC), was incubated with intestinal mucosa homogenates and whole intestinal tissue, in vitro. After 40–70 min, 1,25-(OH)2-CC was specifically associated with the nuclear chromatin fraction. This sterol remains bound to the cytosol fraction at 0°C and a dramatic movement to the nuclear chromatin occurs at 37°C indicating that the subcellular localization of the sterol is temperature dependent. Isolated intestinal cytosol, previously incubated with 1,25-(OH)2-CC, is required for transportation of the hormone to the intestinal chromatin fraction; cytosol fractions from other tissues are ineffective mediators of this sterol migration. It is concluded that the intestinal cytosol contains a specific receptor that functions to transport 1,25-(OH)2-CC to the nucleus, its probable site of action.

Nuclear and cytoplasmic binding components for vitamin D metabolites

Abstract Specific binding of 1α,25-dihydroxyvitamin D3 to macromolecular components of small intestinal nuclei and cytosol is demonstrated. The nuclear 1α,25-dihydroxyvitamin D3 complex can be extracted from chromatin by 0.3 M KCl and sediments at 3.7S in sucrose density gradients. The cytoplasmic 1α,25-dihydroxyvitamin D3-binding components also sediment at 3.7S, identically to the nuclear complex under the ultracentrifugation procedures employed. Macromolecular binding components with a high affinity for 25-hydroxyvitamin D3 (Kd = 4.5 × 10−9 M) were also identified in intestinal cytosol which differ from the 1α,25-hydroxyvitamin D3 receptor in that: 1) they sediment at 5–6S in sucrose gradients, 2) they are observed in organs other than the intestine, and 3) while they do bind 1α,25-dihydroxyvitamin D3 at higher concentrations than 25-hydroxyvitamin D3, they are not observed to transfer either 25-hydroxyvitamin D3 or 1α,25-dihydroxyvitamin D3 to the nucleus, in vitro .

lα,25-dihydroxyvitamin D3 receptor: competitive binding of vitamin D analogs

Abstract The intestinal nuclear receptor for lα,25-dihydroxyvitamin D 3 has been utilized to determine the ability of vitamin D-active sterols to compete with this hormone at the molecular level. 25-Hydroxyvitamin D 3 and lα-hydroxyvitamin D 3 must be present in 150 and 450 times the concentration respectively of lα,25-dihydroxyvitamin D 3 , in vitro , to displace the physiologic hormone. These data indicate that: i) superphysiologic levels of 25-hydroxyvitamin D 3 may simulate lα,25-dihydroxyvitamin D 3 and act directly on isolated target organs and ii) the biologic potency observed for low doses of lα-hydroxyvitamin D 3 , in vivo , is probably the result of 25-hidroxylation of the lα-derivative to form lα,25-dihydroxyvitamin D 3 .

Prophylaxis by taurine in the hearts of cardiomyopathic hamsters

Abstract Cardiomyopathic Syrian hamsters develop necrotic lesions consequent to calcium overload when 60 days old onwards. Taurine, given as 0.1 m solution in place of drinking water for one month prior to killing to animals of initial age 35 days, decreased the severity of subsequently developing cardiac lesions by 40%. Calcium concentration in the heart was decreased by 57%. Magnesium and iron concentrations were unaltered. Taurine given in a similar manner for 4 months still had a protective effect, decreasing lesion severity by 21% and calcium concentration by 35%. Magnesium concentrations were increased by 12%. Compared to random-bred animals, cardiomyopathic hamsters at 1 and 2 months of age have the same concentrations of calcium, magnesium and iron in the quadrants of the heart, except in the left ventricle, which has significantly higher concentration of calcium. Calcium concentrations are 70, 1320 and 2100% higher respectively in 1-month, 2-month and 5-month-old animals. Five-month-old animals differ slightly but significantly in iron (17% decrease) and magnesium concentrations (17% increase). Cardiomyopathic hamsters have insignificant differences in β-adrenergic receptor density compared to random-bred animals and have a significantly higher rate of taurine influx.

1,25-Dihydroxyvitamin D3: mode of action in intestine and parathyroid glands, assay in humans and isolation of its glycoside from Solanum malacoxylon.

Vitamin D3 is metabolized first to 25-hydroxyvitamin D 3 (25-OH-D3) and then to several dihydroxylated products (Fig. 1). The most biologically active of these metabolites (Haussler et al., 1971), la,25-dihydroxyvitamin D3 (1,25-(OH)2D3), is formed in the kidney (Fraser and Kodicek, 1970). The action of vitamin D 3 to mobilize calcium and phosphate from sites such as intestine and bone is thought to be mediated by the 1,25-(OH)2D 3 sterol and, therefore, this metabolite is considered to be the hormonal form of vitamin D (DeLuca, 1974). Furthermore 1,25-(OH)2D3 qualifies as a renal hormone controlling calcium and phosphate metabolism since its biosynthesis is regulated by the calcium and phosphorus status of animals (DeLuca, 1974) and its biochemical action on the target intestine resembles the molecular functioning of classic steroid hormones in their respective target organs (Haussler, 1974). The primary regulated step in the formation of 1,25-(OH)2D3 from the parent vitamin is the la-hydroxylation of 25-OH-Da (Tucker et al., 1973), and the renal la-hydroxylase (la-OHase) enzyme has become the focal point of investigations of the dietary, physiological, and pathological conditions that affect the biosynthesis of 1,25-(OH)2D a. Fig. 1 illustrates that the formation of 1,25-(OH)2Da from 25OH-Da is controlled by calcium (Boyle et al., 1971), parathyroid hormone (PTH) (Garabedian et al., 1972), and phosphate (Tanaka and DeLuca, 1973). Evidence suggests that hypocalcemia stimulates PTH secretion which, in turn, increases the production of 1,25-(OH)zD a (Hughes et al., 1975). Thus, PTH, rather than calcium, may be the dominant modulator of the la-OHase with respect to calcium

Taurine decreases lesion severity in the hearts of cardiomyopathic hamsters.

Cardiomyopathic Syrian hamsters develop necrotic lesions consequent upon calcium overload from 60 days of age onward. Taurine, given as a 0.1 M solution in place of drinking water for one month prior to sacrifice of animals of initial age 35 days, decreased the severity of subsequently developing cardiac lesions by 40%. Calcium concentration in the heart was decreased by 57%. Magnesium and iron concentrations were unaltered. Taurine given in a similar manner for 4 months had a protective effect, decreasing lesion severity by 21% and calcium concentration by 35%. Magnesium concentrations were increased by 12%. Compared to random-bred animals, cardiomyopathic hamsters at one and two months of age have the same concentrations of calcium, magnesium and iron in the quadrants of the heart, except in the left ventricle, which has significantly higher concentration of calcium. Calcium concentrations are 70%, 1320% and 2100% higher respectively in one month, two month and five month old animals. Five month old animals differ slightly but significantly in iron (17% decrease) and magnesium concentrations (17% increase). Cardiomyopathic hamsters have insignificant differences in beta-adrenergic receptor density compared to random-bred animals and have a significantly higher rate of taurine influx.