Characteristics of two cancer cell lines derived from metastatic foci in liver and peritoneum of a patient with colon cancer

Three experiments were conducted to test the hypothesis that a vitamin D deficiency alters the immune responses of female broiler chicks. The control diet contained 800 IU of cholecalciferol (vitamin D3)/kg and the deficient diet was the same except without supplemental vitamin D3. The vitamin D deficiency status was established on the basis of a significantly lower blood ionized calcium or total serum calcium (75 to 85% of the control). Vitamin D-deficient chicks also had lower growth rate and bone ash. In Experiment 1 at 8 d of age, and Experiment 2 at 23 d of age, the cutaneous basophil hypersensitivity response as determined by the increase in interdigital skin thickness 20 h after a single injection of 100 microg phytohemagglutinin-P was significantly depressed in vitamin D-deficient chicks (62 to 64% of the control). Thymus weight, adjusted for body weight, was significantly lower in the vitamin D-deficient chicks at 24 d of age (61% of the control). Primary and secondary antibody responses against SRBC in vitamin D-deficient chicks were not different from the control. In Experiment 3, in 17-d-old chicks, vitamin D deficiency decreased the number of abdominal macrophages phagocytizing SRBC in vitro within 45 min from 14.7 to 10.1%. These results indicate that vitamin D deficiency depresses the cellular immune responses in young broiler chicks.     

Intestinal transport of calcium in rat biliary cirrhosis

The characteristics of intestinal calcium transport in chronic cholestasis remain largely unknown. Using an experimental model of biliary cirrhosis in the rat, we aimed to investigate changes in calcium transport at the jejunal and ileal levels. Two methods were used: 1) uptake of 45Ca in brush border membrane vesicles and 2) measurements of transepithelial fluxes of calcium in Ussing chambers. Thirty days postsurgery, cholestatic rats presented biliary cirrhosis, with normal growth, normal daily energy, and calcium intakes, but had depressed circulating levels of 25-(OH)-vitamin D2 and 1,25-(OH)-vitamin D3. Compared with sham-operated controls, 45Ca uptake ([Ca2+] = 0.03 mmol) measured in vesicles from cholestatic rats was decreased by 3-fold in the duodenojejunum, in concordance with a lower content in brush border membrane calmodulin. Other changes in brush border membrane composition included decreases in structural proteins, microvillous enzymes, and in triglyceride content. Transepithelial fluxes of calcium measured in the ileum ([Ca2+] = 1.2 mmol) revealed in controls a net basal secretion flux (Jnet = -30.4 +/- 8.1 mmol.h-1.cm-2) that was reduced by 3-fold (p < 0.05) in vitamin D-deficient rats (Jnet = -10.4 +/- 4.8 mmol.h-1.cm-2). In response to 25-(OH)-vitamin D2 treatment, calcium uptake rates increased by 40% in the jejunum, whereas in the ileum, the secretion flux returned to basal control levels. Oral administration of taurocholate or tauroursodeoxycholate (50 mmol) depressed almost completely calcium uptake capacity in the duodenojejunum. By complexing free calcium, tauroconjugated bile acids inhibited in vitro calcium uptake proportionally to their concentration in the medium (0-40 mmol). Our data indicate that, in rat biliary cirrhosis, transport capacity of calcium in the duodenojejunum is markedly reduced in association with vitamin D deficiency and alterations in brush border membrane composition.     

Sarcoplasmic reticular Ca2+ pump ATPase activity in congestive heart failure due to myocardial infarction

OBJECTIVE: Earlier studies have shown a depression in the sarcoplasmic reticular (SR) Ca2+ uptake and gene expression in Ca2+ pump ATPase protein in congestive heart failure subsequent to myocardial infarction. It is the objective of this study to understand further the mechanisms of depressed SR Ca2+ pump activity in the failing heart. METHODS: Heart failure in rats was induced by occluding the left coronary artery for 16 weeks and the viable left ventricle was processed for the isolation of SR membranes. Sham-operated animals were used as control. The characteristics of SR Ca2+ pump ATPase in the presence of different concentrations of K+, Ca2+ and ATP were examined and the purity of these membranes was monitored by determining the marker enzyme activities. In addition to measuring changes in cyclic adenosine monophosphate (cAMP) protein kinase and Ca(2+)-calmodulin induced phosphorylation, alterations in SR phospholipid composition as well as sulfhydryl (SH) group content were investigated. RESULTS: Ca(2+)-stimulated ATPase activity, unlike Mg(2+)-ATPase activity, was depressed in the left ventricular SR from failing hearts as compared to control. The decrease in Ca(2+)-stimulated ATPase activity was seen at different concentrations of Ca2+, K+ and ATP but no changes in the affinities of the enzyme for Ca2+ and ATP were evident. The SR Ca(2+)-stimulated ATPase activities in the presence of both cAMP-dependent protein kinase and Ca(2+)-calmodulin were markedly decreased in the failing hearts when compared to control preparations. Furthermore, the 32P incorporation in the presence of cAMP-dependent protein kinase or Ca(2+)-calmodulin was also reduced in the experimental heart SR membranes. The phospholipid composition of the SR membranes from the failing heart was markedly altered. No changes in SH-group or the degree of cross contamination with other membranes were apparent in the failing heart SR. CONCLUSIONS: These results suggest that abnormalities in membrane phospholipid composition and phosphorylation of the enzyme may partly explain the observed depression in SR Ca2+ pump ATPase activity in heart failure following myocardial infarction.     

Calbindin D28K regulation in precociously matured chick egg shell gland in vitro

Egg shell calcification in the hen uterus (egg shell gland, ESG) depends primarily on intestinal absorption of dietary Ca2+ as well as ESG Ca2+ transport into the shell. Intestinal Ca2+ absorption is linked to vitamin D-induced calbindin D28K (D28K) concentration. The ESG also contains D28K, and Ca2+ transport into the shell appears to be linked to D28K gene expression, but until this report, there was no direct proof that ESG D28K was or was not vitamin D-dependent. To address this issue, highly developed ESG from estradiol (E2)-injected, severely vitamin D-depleted chicks were cultured in serum-free medium with excellent viability. Addition of the vitamin D-hormone, 1,25(OH)2 vitamin D3 (1,25), to the culture medium increased ESG D28K levels as much as 70%. E2 alone had no effect, but E2 plus 1,25 further increased ESG D28K levels up to 160%. By contrast, progesterone (P4) prevented the 1,25-stimulated increase in D28K, while having no effect on basal D28K level. Of considerable interest, thapsigargin (THAPS), which increases intracellular Ca2+ concentration ([Ca2+]i) in many cell types, stimulated D28K synthesis in a concentration-dependent manner in the complete absence of 1,25 and independent of the [Ca2+] of the medium. These results are the first direct evidence that ESG D28K is under direct control of 1,25 and that both gonadal steroid hormones, E2 and P4, may be coregulators. Further, the effects of THAPS suggest that [Ca2+]i itself may also regulate D28K. This new in vitro model clearly represents a unique opportunity to study the regulation of the ESG calcium transport mechanism under stringently defined conditions.     

Calcium-binding protein and kidney 25-hydroxycholecalciferol-1-hydroxylase activity in turkey poults

A vitamin D-dependent calcium-binding protein (CaBP) has been found in the intestinal mucosa of turkeys (Meleagris gallopavo). This protein is similar, if not identical, to the corresponding one found in the intestine of the chicken (Gallus domesticus). The concentration of intestinal CaBP in the intestine of cholecalciferol-fed turkey poults was twice that found in the intestine of the domestic chicken of parallel age. Dietary calcium or phosphorus restriction resulted in a significant increase in intestinal CaBP. This increase was less pronounced than that obtained in chickens. The relationship between duodenal CaBP and dietary calcium appears to be linear. Cholecalciferol (vitamin D) deficiency or dietary calcium, but not phosphorus, restriction resulted in an increase of kidney 25 hydroxycholecalciferol-1-hydroxylase activity in vitro. The values of activity obtained in turkeys were higher than those obtained under the same experimental conditions in chicks. Bone ash was reduced by dietary calcium, phosphorus or cholecalciferol restriction. Plasma inorganic phosphorus was lower in either cholecalciferol- or phosphorus-restricted turkeys. Plasma calcium was significantly higher in the phosphorus-restricted turkeys than in the calcium-restricted or control turkeys.     

Parathyroidectomy reduces 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the hypocalcemic vitamin D-deficient chick

To test the hypothesis that in the vitamin D-deficient state the activity of 25-hydroxyvitamin D3-1 alpha-hydroxylase (25-OHD3-1 alpha-hydroxylase) is modulated by parathyroid hormone and the plasma concentration of phosphate only in the presence of small amounts of 1,25-dihydroxyvitamin D3 (or some other metabolite of vitamin D), we measured the activity of this enzyme 24 h after parathyroidectomy (PTX) in frankly hypocalcemic, vitamin D-deficient chicks that were not supplemented with vitamin D or one of its metabolites. The otherwise predictable complications of PTX in this metabolic setting (hypocalcemia of increasing severity, tetany, moribundity, and death) were prevented by continuous intravenous administration of calcium (as a solution of calcium chloride/calcium gluconate 1:1) through a catheter in the external jugular vein placed at the time of PTX. The findings were as follows: (a) The activity of 25-OHD3-1 alpha-hydroxylase was significantly less in the parathyroidectomized group than in the sham-operated control chicks (P less than 0.001). (b) The reductive effect of PTX on the activity of this enzyme was significantly attenuated when hypophosphatemia was increased in severity by administration of glucose. (c) In the post-PTX state the activity of 25-OHD3-1 alpha-hydroxylase and plasma concentration of phosphate were significantly, inversely related (P less than 0.001). (d) In the sham-operated control group the activity of this enzyme and the plasma concentration of phosphate were not significantly correlated. These findings indicate that in the vitamin D-deficient state, both circulating parathyroid hormone and the plasma concentration of phosphate can significantly modulate the activity of 25-OHD3-1 alpha-hydroxylase in the absence of vitamin D or its metabolites. The findings also suggest that in the vitamin D-deficient state the plasma concentration of phosphate modulates the activity of this enzyme only when the concentration of circulating parathyroid hormone is not increased.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

In vivo treatment with calcitriol (1,25(OH)2D3) reverses age-dependent alterations of intestinal calcium uptake in rat enterocytes

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D3; [1, 25(OH)2D3] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)2D3 stimulation of Ca2+ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20-24-month-old rats given 1,25(OH)2D3 for 3 days (30 ng/100 g bw/day), calcitriol (10(-10) M, 3-5 minutes) stimulated Ca2&plus uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)2D3. When the aged rats were injected with 25(OH)D3, similar Ca2+ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1, 25(OH)2D3-dependent changes in Ca2+ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)2D3 stimulation of duodenal cell Ca2+ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)2D3 or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system.     

Phosphate transport in pig proximal small intestines during postnatal development: lack of modulation by calcitriol

The role of calcitriol in the intestinal absorption of inorganic phosphate (Pi) during postnatal development was studied in newborn [<1 week postpartum (pp)], suckling (3-4 weeks pp), and weaned (>6 weeks pp) control piglets (con) and piglets suffering from inherited calcitriol deficiency (def). In addition, a number of def piglets were treated with vitamin D3 (def-D3). Regardless of age, plasma calcitriol concentrations in def piglets were unphysiologically low (16-21 pg/ml) and differed significantly from those in respective con animals (60-69 pg/ml) and vitamin D3-treated def piglets (50-56 pg/ml). However, newborn and suckling def piglets had normal Ca (approximately 3.0 mmol/liter) and Pi (approximately 2.8 mmol/liter) plasma levels. Def piglets became hypocalcemic (1.9 mmol/liter) and hypophosphatemic (1.9 mmol/liter) between 4-6 weeks pp. Treatment with vitamin D3 significantly increased plasma Ca (3.2 mmol/liter) and Pi (2.7 mmol/liter) levels in weaned def animals. Regardless of calcitriol status, net Pi flux rates (active Pi absorption, as determined with the in vitro Ussing-chamber technique) from the upper small intestines was maximal at birth [170-224 nmol/(cm2 x h)] and decreased by approximately 80% during the first week of life before remaining constant [30-50 nmol/(cm2 x h)] during the following development. In weaned def piglets, net Pi flux rates were significantly lower by about 80% compared with those in con animals. Treatment of def piglets with vitamin D3 had no effect in newborn and suckling animals but reconstituted net Pi flux rates to normal values at weaning age. Age-dependent and calcitriol-mediated changes in net Pi flux rates were paralleled by respective maximum velocity values of Na+-dependent Pi uptake across the brush border membrane of the enterocytes (newborn piglets, 1.9-2.2 nmol/(mg protein 10 sec); suckling piglets, 0.4-0.6 nmol/(mg protein x 10 sec); weaned piglets, 0.7, 0.3, and 0.7 nmol/(mg protein x 10 sec) in con, def, and def-D3 animals, respectively). These findings suggest that the apical Pi uptake represents the major rate-limiting step of the overall transepithelial Pi transport. At weaning, Na+/Pi transport across the intestinal brush-border membrane is clearly stimulated by calcitriol, but no significant effects of age or calcitriol on the Km values (0.5-0.7 mmol/liter) were observed. In conclusion, our findings reveal calcitriol-independent mechanisms for active intestinal Pi absorption during the neonatal and suckling periods. The onset of the classical calcitriol-dependent mechanism for active intestinal Pi absorption does not occur until weaning.     

The peroxisome proliferator nafenopin does not suppress hepatocyte apoptosis in guinea-pig liver in vivo nor in human hepatocytes in vitro

BACKGROUND: Vitamin D deficiency is a major risk factor for bone loss and fracture. Although hypovitaminosis D has been detected frequently in elderly and housebound people, the prevalence of vitamin D deficiency among patients hospitalized on a general medical service is unknown. METHODS: We assessed vitamin D intake, ultraviolet-light exposure, and risk factors for hypovitaminosis D and measured serum 25-hydroxyvitamin D, parathyroid hormone, and ionized calcium in 290 consecutive patients on a general medical ward. RESULTS: A total of 164 patients (57 percent) were considered vitamin D-deficient (serum concentration of 25-hydroxyvitamin D, < or = 15 ng per milliliter), of whom 65 (22 percent) were considered severely vitamin D-deficient (serum concentration of 25-hydroxyvitamin D, <8 ng per milliliter). Serum 25-hydroxyvitamin D concentrations were related inversely to parathyroid hormone concentrations. Lower vitamin D intake, less exposure to ultraviolet light, anticonvulsant-drug therapy, renal dialysis, nephrotic syndrome, hypertension, diabetes mellitus, winter season, higher serum concentrations of parathyroid hormone and alkaline phosphatase, and lower serum concentrations of ionized calcium and albumin were significant univariate predictors of hypovitaminosis D. Sixty-nine percent of the patients who consumed less than the recommended daily allowance of vitamin D and 43 percent of the patients with vitamin D intakes above the recommended daily allowance were vitamin D-deficient. Inadequate vitamin D intake, winter season, and housebound status were independent predictors of hypovitaminosis D in a multivariate model. In a subgroup of 77 patients less than 65 years of age without known risk factors for hypovitaminosis D, the prevalence of vitamin D deficiency was 42 percent. CONCLUSIONS: Hypovitaminosis D is common in general medical inpatients, including those with vitamin D intakes exceeding the recommended daily allowance and those without apparent risk factors for vitamin D deficiency.     

The effect of beta-carotene on the cytoplasmic membrane lipids of cells in rats under chronic gamma irradiation

Chronic gamma irradiation of rats at a dose rate of 12.9 rad/day for 155 days (total dose 20 Gy) did not change the cholesterol level in the brush border membrane of the small intestine. Maintenance of rats on a beta-carotene-enriched diet (daily throughout the irradiation period, at a diurnal dose of 3 mg/kg body weight produced an increase in cholesterol levels in the small intestinal brush border membranes. The phospholipid level after gamma irradiation alone did not change, but it was increased in gamma-irradiated rats fed with carotene, this increase being most pronounced for sphingomyelin. Beta-carotene decreased the cholesterol/phospholipid ratio in the brush border membrane of irradiated rats. Activation of lipid synthesis (as seen from inclusion of 2-14C-acetate in the epitheliocyte lipids of irradiated animals) and no influence of beta-carotene on the inclusion of the label in small intestine epitheliocytes of irradiated animals have been revealed. In the presynaptic membranes of brain nerve endings, chronic gamma irradiation of animals produced a deep fall of cholesterol and phospholipid levels. Also, a tendency was revealed for stimulation of transport of the neurotransmitter amino acids GABA and L-glutamate across the presynaptic membranes of nerve endings. Beta-carotene normalized the phospholipid and cholesterol levels in brain presynaptic membranes of irradiated rats, as well as decreased L-glutamate transport. It is assumed that the modifying and normalizing effects of beta-carotene on the lipid exchange of plasma membranes upon chronic irradiation of animals and permanent introduction of the drug are associated with the radioprotector activity of beta-carotene.     

PLA2 promotes fusion between PMN-specific granules and complex liposomes

Neutrophil stimulation results in the activation of a variety of phospholipases, including phospholipase A2 (PLA2), which releases arachidonic acid from the 2 position of membrane phospholipids, leaving a lysophospholipid. Because arachidonic acid is known to be a potent fusogen in vitro, we examined the effect of metabolism by PLA2 on the fusion of complex liposomes (liposomes prepared with a phospholipid composition similar to that found in neutrophil plasma membrane). We observed that PLA2 augmented the fusion of complex liposomes with each other as well as with specific granules isolated from human neutrophils, lowering the Ca2+ requirement for fusion by three orders of magnitude. Furthermore, although lysophospholipids inhibited fusion, the incorporation of arachidonic acid into liposome membranes overcame the inhibitory effects of the lysophospholipids. Thus with PLA2 and annexins we were able to obtain fusion of complex liposomes at concentrations of Ca2+ that are close to physiological. Our data suggest that the activation of PLA2 and the generation of arachidonic acid may be the major fusion-promoting event mediating neutrophil degranulation.     

Preservation of endogenous antioxidant activity and inhibition of lipid peroxidation as common mechanisms of antiatherosclerotic effects of vitamin E, lovastatin and amlodipine

OBJECTIVES: We sought to document the common mechanisms of the antiatherogenic effects of the cholesterol-lowering hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin, the dihydropyridine Ca2+ blocker amlodipine and the antioxidant vitamin E. BACKGROUND: Vitamin E, HMG-CoA reductase inhibitors and Ca2+ blockers each inhibit atherosclerosis in hypercholesterolemic animals. METHODS: New Zealand White rabbits were fed regular chow (Group A), chow with 1% cholesterol (Group B), 1% cholesterol diet plus lovastatin (Group C), 1% cholesterol diet plus vitamin E (Group D) or 1% cholesterol diet plus amlodipine (Group E) for 12 weeks. The extent of aortic atherosclerosis was measured by planimetry of the sudanophilic area. Malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were measured as indexes of lipid peroxidation and antioxidant activity, respectively. RESULTS: Group A rabbits showed no atherosclerosis, whereas Group B rabbits had 17.4 +/- 9.3% (mean +/- SD) of the aorta covered with atherosclerosis, and Groups C, D and E rabbits had significantly less atherosclerosis. Plasma SOD activity was lower in Group B than in Group A (6.9 +/- 1.1 vs. 12.8 +/- 1.5 U/ml, p < 0.01) and was preserved in the groups given lovastatin, vitamin E or amlodipine with a high cholesterol diet. The serum MDA level was higher in Group B rabbits than Group A rabbits (12.1 +/- 2.6 vs. 1.2 +/- 0.1 nmol/ml, p < 0.01) and increased minimally in rabbits given lovastatin, vitamin E or amlodipine with a high cholesterol diet. In in vitro experiments, both lovastatin and amlodipine preserved SOD activity and reduced the oxidizability of low density lipoproteins by rabbit leukocytes. CONCLUSIONS: This study suggests that a reduction in lipid peroxidation and preservation of SOD may be common mechanisms of antiatherosclerotic effects of lovastatin, vitamin E and amlodipine.     

Potential use of cytokine therapy in poultry

The time course of incorporation of [14C]arachidonic acid and [3H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [14C]arachidonic acid and [3H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Regulation of rabbit erythrocyte Ca(2+)-pump sensitivity to calmodulin in experimental hyperlipidemia

Intracellular free calcium activity is in part determined by a calmodulin-regulated plasma membrane Ca(2+)-pump. Since changes in Ca2+ permeability have been implicated in atherosclerotic plaque formation, we initiated a lipid hyperalimentation protocol during which we measured various erythrocyte calcium flux parameters and early atheroma development. Adolescent New Zealand White rabbits were fed a diet with 0.5% cholesterol and 2.5% lard over a 3-month period. Plasma cholesterol and triacylglycerols increased on average 18.7- and 13.9-fold respectively, while erythrocyte membrane cholesterol content decreased 18% and total phospholipids by 54%. After 3 months of lipid hyperalimentation, 22% of the aortic arch was covered with large, early-stage, raised atheroma. Basal and calmodulin-activated (Ca2+ + Mg2+)-ATPase activities in erythrocyte membranes increased by 31% and 123%, respectively at 2 months, with a concomitant increase in calmodulin affinity (Km) from 15.6 to 4.2 nM. These differences were transient on account of changes in the control animals which exhibited a slowly developing sensitivity to calmodulin during maturation. Basal Ca2+ transport and passive Ca2+ permeability increased about 7-fold during the hyperlipidemic phase. This suggests that overt hyperlipidemia, leading to atherosclerotic plaque development, alters plasma membrane Ca2+ regulatory mechanisms including passive Ca2+ permeability. The changes in enzymatic function, membrane composition, and Ca2+ permeability seen in this red cell model system may be a reflection of early changes in cells that are directly involved in the development of atherosclerotic plaques.     

1,25-Dihydroxyvitamin D stimulation of specific membrane proteins in chick intestine

Vitamin D-deficient chicks were injected intracardially with physiological doses of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) and the formation of intestinal brush-border proteins was followed in vitro. Within 4 h of receiving the hormone the incorporation of radioactive leucine into at least two proteins in the brush-borders was increased. The apparent molecular weights of these proteins were 45 000 and 84 000. The change in the synthesis of these proteins was followed with time and compared with the concomitant changes in intestinal calcium transport. The relationship of these changes is such that there is a strong possibility that the proteins are involved in calcium absorption.     

Calcium transport by isolated brush border and basolateral membrane vesicles: role of essential fatty acid supplementation

Intestinal calcium transport is important in whole body calcium homeostasis and it is therefore of interest to understand the mechanism of absorption and its regulation by 1;25-dihydroxyvitamin D3 (1,25 (OH)2D3) (vitamin D). Significant changes in lipid composition of membranes have previously been shown in response to vitamin D3 administration. Deficiency in essential fatty acids (EFAs) may influence the vitamin D-dependent calcium absorption in the intestinal tract. The purpose of this study was to investigate the effect of unsaturated fatty acid supplementation on calcium transport. Simultaneous measurements of calcium transport, membrane fluidity and lipid structure have rarely been performed on the same preparation. Intestinal membrane vesicles were prepared using a novel procedure. Vesicles prepared from fish oil and evening primrose oil supplemented animals revealed the highest calcium transport over time as well as the highest degree of unsaturation as compared to those from animals which were unsupplemented or given sunflower or coconut oil. The relative content of polyunsaturated fatty acids in the intestinal membranes may change fluidity, enhance calcium transport and may influence the action of vitamin D3 on calcium absorption.     

Fungal membrane responses induced by plant defensins and thionins

Treatment of hyphae of Neurospora crassa with antifungal plant defensins, i.e. Rs-AFP2 and Dm-AMP1 isolated from radish and dahlia seed, respectively, induced a rapid K+ efflux, Ca2+ uptake, and alkalinization of the incubation medium. The Rs-AFP2-induced alkalinization of the incubation medium could be inhibited with G-protein inhibitors. alpha-Hordothionin, an antifungal thionin from barley seed, caused a sustained increased Ca2+ uptake at subinhibitory concentrations but only a transient increased uptake at inhibitory concentrations. alpha-Hordothionin also caused increased K+ efflux and alkalinization of the medium, but these fluxes occurred more rapidly compared to those caused by plant defensins. Furthermore, alpha-hordothionin caused permeabilization of fungal hyphae to the non-metabolite alpha-aminoisobutyric acid and, in addition, altered the electrical properties of artificial lipid bilayers, consistently leading to rupture of the lipid bilayers. The plant defensins did not form ion-permeable pores in artificial membranes and did not exhibit substantial hyphal membrane permeabilization activity. Our results are consistent with the notion that thionins inhibit fungal growth as a result of direct protein-membrane interactions, whereas plant defensins might act via a different, possibly receptor-mediated, mechanism.     

Differential rate of cholesterol efflux from the apical and basolateral membranes of MDCK cells

Epithelial cells contain two distinct membrane surfaces, the apical and basolateral plasma membranes, which have different lipid and protein compositions. In order to assess the effect of the compositional differences of the apical and basolateral membranes on their ability to undergo cholesterol efflux, MDCK cells were radiolabeled with [3H]cholesterol and grown as a polarized monolayer on filter inserts, that separate the upper apical compartment from the lower basolateral compartment. The rate of cholesterol efflux from the basolateral membrane into media containing HDL in the basolateral compartment was 6.3%/h +/-0.7, whereas HDL-mediated efflux from the apical membrane was approximately 3-fold slower (1.9%/h +/-0.3). In contrast, Fu5AH cells, which do not form distinct polarized membrane domains, had a similar rate of HDL-mediated cholesterol efflux into the apical and basolateral compartments. Similar to HDL, other cholesterol acceptors, namely LDL, bovine serum albumin, and a lipid emulsion, also showed a decreased rate of cholesterol efflux from the apical membrane surface versus the basolateral membrane. Compared to the basolateral membrane, the apical membrane was also found to be more resistant to cholesterol oxidase treatment, to bind less HDL, and to take up less cholesterol from the medium. In conclusion, cholesterol efflux occurred less readily from the apical membrane than from the basolateral membrane for all types of acceptors tested. These results suggest that differences in the composition of the apical and basolateral membrane lead to a relative decrease in cholesterol desorption from the apical membrane and hence a reduced rate of cholesterol efflux.     

Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein

A line of mice deficient in vitamin D binding protein (DBP) was generated by targeted mutagenesis to establish a model for analysis of DBP's biological functions in vitamin D metabolism and action. On vitamin D-replete diets, DBP-/- mice had low levels of total serum vitamin D metabolites but were otherwise normal. When maintained on vitamin D-deficient diets for a brief period, the DBP-/-, but not DBP+/+, mice developed secondary hyperparathyroidism and the accompanying bone changes associated with vitamin D deficiency. DBP markedly prolonged the serum half-life of 25(OH)D and less dramatically prolonged the half-life of vitamin D by slowing its hepatic uptake and increasing the efficiency of its conversion to 25(OH)D in the liver. After an overload of vitamin D, DBP-/- mice were unexpectedly less susceptible to hypercalcemia and its toxic effects. Peak steady-state mRNA levels of the vitamin D-dependent calbindin-D9K gene were induced by 1,25(OH)2D more rapidly in the DBP-/- mice. Thus, the role of DBP is to maintain stable serum stores of vitamin D metabolites and modulate the rates of its bioavailability, activation, and end-organ responsiveness. These properties may have evolved to stabilize and maintain serum levels of vitamin D in environments with variable vitamin D availability.