Characterization of dihydropyridine-sensitive calcium channels in rat brain synaptosomes.

We examined the effects of dihydropyridine Ca2+-channel agonists on synaptosomal voltage-dependent Ca2+ entry and endogenous dopamine release. The (-) isomer of Bay K 8644 and the (+) isomer of Sandoz compound 202-791 were 100-1000 times more potent than their respective opposite enantiomers in enhancing Ca2+ uptake and dopamine release from striatal synaptosomes. The active isomer of each of these compounds increased Ca2+ entry and dopamine release to the same extent at a concentration of 1 nM. Fast-phase Ca2+ entry into synaptosomes isolated from cerebellum, cortex, and hippocampus was sensitive to nanomolar concentrations of Bay K 8644. No effect of Bay K 8644 was observed in synaptosomes isolated from brainstem. Bay K 8644 increased synaptosomal Ca2+ uptake and endogenous dopamine release from striatal synaptosomes only during the initial seconds of KCl-induced depolarization. The greatest increase was observed during the first second of depolarization. No effect was observed after greater than or equal to 5 sec of depolarization. Bay K 8644 did not alter Ca2+ uptake or dopamine release under resting conditions (5 mM KCl) or in response to KCl at greater than 15 mM. The activity of Bay K 8644 was also attenuated by lowering the concentrations of divalent cations in the incubation medium. Agonist activity was observed at Mg2+ concentrations greater than 500 microM (Ca2+ held at 100 microM) and Ca2+ concentrations greater than 100 microM (Mg2+ held at 1000 microM). These results suggest that the Ca2+ channels present in synaptosomes are sensitive to nanomolar concentrations of dihydropyridine agonists under a narrow range of experimental conditions.     

Binding of the phytotoxin zinniol stimulates the entry of calcium into plant protoplasts

Zinniol [1,2-bis(hydroxymethyl)-3-methoxy-4-methyl-5-(3-methyl-2-butenyloxy)benzene], a toxin produced by fungi of the Alternaria group, causes symptoms in plants that resemble those induced by the fungi. The phytotoxin binds to carrot protoplasts and isolated membranes in a saturable and reversible manner. Receptor occupancy stimulates entry of calcium into protoplasts. Zinniol can partially reverse the effects and binding of the calcium-channel blockers desmethoxyverapamil and bepridil. Selected cell lines that are insensitive to zinniol lose part of their binding capacity and sensitivity to the action of the agonist-like compound but are still able to bind calcium-channel blockers. We conclude that zinniol acts on calcium entry but that the targets of the toxin and of calcium-channel blockers are dissimilar, suggesting the occurrence of sites affected both by zinniol and by channel blockers and of sites affected only by zinniol.     

Cortisol stimulates whole body calcium uptake and the branchial calcium pump in freshwater rainbow trout

Exposure of rainbow trout to a reduced ambient calcium level (from 490 to 25 mumol Ca2+/l) caused hypocalcaemia and induced a rapid increase (within 1 h) in systemic cortisol levels. Under conditions of low environmental calcium concentrations, cortisol levels remained increased for at least 8 days. After this time the in-vitro Ca2+-transport capacity of branchial basolateral membrane vesicles was increased due to stimulation of Ca2+-ATPase activity, presumably as a result of chloride cell proliferation. Pituitary prolactin cells were unaffected by low ambient calcium levels. Fish kept in water containing 490 mumol Ca2+/l and treated with cortisol for 7 days displayed an increase in whole body calcium uptake and an enhancement of the branchial calcium transport capacity; concomitantly, hypercalcaemia was observed. We conclude that, in the rainbow trout, cortisol exerts hypercalcaemic effects by stimulating Ca2+ uptake from the water and that this effect forms an intrinsic part of the established mineralocorticoid action of cortisol in fish.     

Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle

Purified dihydropyridine-sensitive calcium channels from rabbit transverse-tubule membranes consist of three noncovalently associated classes of subunits: alpha (167 kDa), beta (54 kDa), and gamma (30 kDa). Cleavage of disulfide bonds reveals two distinct alpha polypeptides and an additional component, delta. The alpha 1 subunit, a 175-kDa polypeptide that is not N-glycosylated, contains the dihydropyridine binding site, cAMP-dependent protein kinase phosphorylation site(s), and substantial hydrophobic domain(s). alpha 2, a 143-kDa glycoprotein, has none of the properties characteristic of alpha 1 but binds lectins and contains about 25% N-linked carbohydrate. alpha 2 is disulfide-linked to delta, a 24- to 27-kDa glycopeptide. beta (54 kDa) contains a cAMP-dependent phosphorylation site but is not N-glycosylated and does not have a hydrophobic domain. gamma (30 kDa) has a carbohydrate content of about 30% and extensive hydrophobic domain(s). Precipitation with affinity-purified anti-alpha 1 antibodies or alpha 2-specific lentil lectin-agarose demonstrated that alpha 1 alpha 2 beta gamma delta behaves as a complex in the presence of digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, whereas the alpha 2 delta complex dissociates from alpha 1 beta gamma in the presence of Triton X-100. A model for subunit interaction and membrane insertion is proposed on the basis of these observations.     

Modulation of dihydropyridine-sensitive calcium channels in heart cells by fish oil fatty acids.

The highly unsaturated n-3 fatty acids from fish oils, eicosapentaenoic acid [EPA; C20:5 (n-3)] and docosahexanoic acid [DHA; C22:6 (n-3)], prevent the toxicity of high concentrations of the cardiac glycoside ouabain to isolated neonatal rat cardiac myocytes. Arachidonic acid [C20:4 (n-6)] lacks such protective action. The protective effect of the n-3 fatty acids is associated with their ability to prevent high levels of cytosolic free calcium from occurring in response to the ouabain. This in turn results, at least in part, from a 30% reduction in calcium influx rate induced by the n-3 fatty acids. This protective effect is simulated by nitrendipine, a dihydropyridine inhibitor of the L-type calcium channels in cardiac myocytes. Nitrendipine (0.1 mM) alone, however, inhibits myocyte contractility, as do verapamil (10 microM) and diltiazem (1.0 microM). EPA or DHA (5 microM) blocks the inhibitory effects of nitrendipine but not those of verapamil or diltiazem. Bay K8644, a known dihydropyridine agonist of L-type calcium channels, produces a ouabain-like effect that is also prevented by EPA or DHA. Specific binding of [3H]nitrendipine to intact myocytes is noncompetitively inhibited by EPA or DHA in a manner that reduces the number of high- and low-affinity binding sites (Bmax) and increases their affinities. The fish oil fatty acids prevent calcium overload from ouabain and Bay K8644. They also prevent a calcium-depleted state in the myocytes caused by the L-type calcium channel blocker nitrendipine. The protective effects of the n-3 fatty acids appear to result from their modulatory effects on nitrendipine-sensitive L-type calcium channels.     

Red light stimulates an electrogenic proton pump in Vicia guard cell protoplasts.

Stomatal opening in response to light has a component that matches the absorption spectrum of chlorophyll; however, the intervening sensory transduction steps are not well understood. To study this process, we illuminated Vicia faba guard cell protoplasts with red light and simultaneously recorded current flow across the plasma membrane, utilizing the patch clamp technique in the whole cell configuration. We report evidence that under voltage clamp conditions, red light (1 mmol of photons.m-2.S-1) stimulated an outward current. This response required ATP (2.5 mM) and orthophosphate (1 mM) at the cytoplasmic side of the membrane. Both red-light-stimulated currents and currents activated in the dark by the proton pump agonist fusicoccin (10 microM) were abolished by the protonophore carbonylcyanide m-chlorophenylhydrazone at 10 microM, indicating that these responses were carried by protons. Pump currents were inhibited by orthovanadate applied to the cytoplasmic side of the membrane (50% inhibition at 3.5 microM), implicating a H+ -ATPase. Elimination of the current by the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, in the presence of saturating concentrations of ATP, pointed to a requirement for photosynthetically active chloroplasts. We conclude that red light stimulates an electrogenic proton pump at the plasmalemma of Vicia guard cells and that chloroplasts modulate this response.     

Purified omega-conotoxin GVIA receptor of rat brain resembles a dihydropyridine-sensitive L-type calcium channel.

The omega-conotoxin GVIA (CTX) receptor has been purified 1900-fold to apparent homogeneity by monitoring both reversible binding of 125I-labeled CTX (125I-CTX) and photoincorporation of N-hydroxysuccinimidyl-4-azidobenzoate-125I-CTX (HSA-125I-CTX). Photoincorporation of HSA-125I-CTX into a 230-kDa protein exhibits a pharmacologic and chromatographic profile indicating that the 230-kDa protein is the CTX-binding subunit of the receptor. The pharmacologic specificity of 125I-CTX binding to the purified CTX receptor closely resembles that of the native membrane-bound form with respect to sensitivity towards CTX (Kd = 32 pM) and other peptide toxin antagonists. The purified CTX receptor comprises the 230-kDa protein (alpha 1) and four additional proteins with apparent molecular masses of 140 (alpha 2), 110, 70 (beta 2), and 60 (beta 1) kDa. This subunit structure closely resembles that of the 1,4-dihydropyridine-sensitive L-type calcium channel.     

Progesterone stimulates sulfate uptake in subcultured endometrial epithelial cells.

The effect of progesterone was studied on the sulfate entry in glandular epithelial cells of guinea-pig endometrium subcultured in bicameral chambers on matrix-coated filters in a chemically defined medium. At post-confluency (8 days of subculture), cells were treated with 10 nM estradiol alone or in association with various concentrations of progesterone. Optimal progesterone action was at a 16 h incubation time and a 10 nM hormonal concentration. Progesterone increased in a dose-dependent fashion the sulfate uptake specifically in glandular epithelial cells, preferentially from the basal surface. Progesterone effect on the sulfate uptake occurred only in estradiol-primed epithelial cells and was inhibited by the antiprogestin steroid RU-486. The progesterone-dependent increase in sulfate uptake was inhibited by the inhibitor of anion exchange, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). At physiological sulfate concentrations, progesterone essentially induces a high-affinity DIDS-sensitive transport system.     

Arachidonic acid stimulates glucose uptake in cerebral cortical astrocytes.

Arachidonic acid (AA) has recently been shown to influence various cellular functions in the central nervous system. Here we report that AA increases, in a time- and concentration-dependent manner, 2-deoxy-D-[1-3H]glucose ([3H]2DG) uptake in primary cultures of astrocytes prepared from the cerebral cortex of neonatal mice. This effect is mimicked by an unsaturated fatty acid such as linolenic acid, while palmitic and arachidic acids, two saturated fatty acids, are inactive. Pharmacological agents that increase the endogenous levels of AA by stimulating AA release (melittin) or by inhibiting its reacylation (thimerosal) also promote [3H]2DG uptake by astrocytes. We also report that norepinephrine (NE) stimulates the release of [3H]AA from membrane phospholipids, with an EC50 of 3 microM; this effect is accompanied, with a temporal delay of approximately 4 min, by the stimulation of [3H]2DG uptake, for which the EC50 of NE is 1 microM. Since the cerebral cortex, the brain region from which astrocytes used in this study were prepared, receives a massive noradrenergic innervation, originating from the locus coeruleus, the effects of NE reported here further stress the notion that certain neurotransmitters may play a role in the regulation of energy metabolism in the cerebral cortex and point at astrocytes as the likely targets of such metabolic effects.     

Oxyntomodulin stimulates intestinal glucose uptake in rats

BACKGROUND & AIMS: Enteroglucagon peptides have long been proposed as mediators of intestinal adaptation, including mucosal growth and nutrient absorptive capacity. The hypothesis that infusions of oxyntomodulin, a bioactive form of enteroglucagon, would stimulate glucose and amino acid uptake was tested and its effects were compared with those of glucagon. METHODS: Rats were infused intravenously via minipumps with either saline, rat oxyntomodulin (0.47 nmol x kg(-1) x h[-1]), or glucagon (0.88 nmol x kg(-1) x h[-1]) for 7 days, and plasma hormone levels were measured. At death, intestinal dimensions and brush border uptake of D-glucose and L-proline were measured using an in vitro everted sleeve technique. RESULTS: Plasma enteroglucagon and glucagon levels were increased 4- and 12-fold, respectively, but there were no effects on food intake, body weight, or intestinal dimensions. In contrast, oxyntomodulin and glucagon significantly stimulated total intestinal glucose uptake capacity by 44% and 53%, respectively, over controls. Oxyntomodulin most potently enhanced glucose uptake in the ileum (215%), whereas glucagon's greatest effect was in the jejunum (63%-85%). However, neither peptide affected proline uptake. CONCLUSIONS: These results support a new, specific action for oxyntomodulin in intestinal adaptation as a glucose uptake stimulator and confirm glucagon's role as a regulator of glucose uptake. (Gastroenterology 1997 Jun;112(6):1961-70)     

Activation of dihydropyridine-sensitive calcium channels and biphasic cytosolic calcium responses by angiotensin II in rat adrenal glomerulosa cells

The steroidogenic actions of angiotensin II (AII) and increased extracellular K+ concentrations [( K+]) in rat adrenal glomerulosa cells are selectively enhanced by the voltage-sensitive calcium channel agonist Bay K 8644 (BK 8644). The relationship between these effects of the dihydropyridine agonist and cytosolic calcium concentration [( Ca2+]i) was investigated in rat and bovine glomerulosa cells. In the rat glomerulosa cells, AII and increased [K+] elicited rapid elevations of [Ca2+]i with distinctive temporal characteristics. Whereas the [Ca2+]i response to [K+] declined to basal over 2-3 min, addition of 10 nM AII caused a biphasic increase in [Ca2+]i, with a rapid transient rise followed by a lower plateau phase that remained above basal for several minutes. BK 8644 alone did not affect [Ca2+]i, but at low concentrations (30 nM) increased the magnitude and duration of the [Ca2+]i response elicited by progressive elevation of extracellular [K+] to 12 mM. In AII-stimulated glomerulosa cells, 30 nM BK 8644 enhanced both phases of the cytosolic calcium response, with a more marked effect on the sustained plateau phase. In contrast to its prominent actions in rat glomerulosa cells, BK 8644 had no effect on AII-stimulated rises in [Ca2+]i in bovine glomerulosa cells, and only slightly enhanced their minor [Ca2+]i responses to potassium. These studies provide evidence that AII activates dihydropyridine-sensitive voltage-sensitive calcium channels in rat, but not bovine, adrenal glomerulosa cells. They also suggest that enhancement by BK 8644 of agonist-stimulated [Ca2+]i changes is responsible for its synergistic effects on aldosterone responses to potassium and AII in rat glomerulosa cells and emphasize the importance of the sustained phase of the cytosolic calcium signal in the steroidogenic action of AII.     

Phorbol ester increases the dihydropyridine-sensitive calcium conductance in a vascular smooth muscle cell line

In vascular smooth muscle, phorbol esters cause a slowly developing contraction and an associated transmembrane calcium flux, both of which are inhibited by dihydropyridine calcium channel antagonists. In the A7r5 cultured vascular cell line, we used the whole-cell voltage-clamp technique to identify voltage-dependent calcium conductances and investigate the effect of phorbol esters on that conductance having characteristic dihydropyridine sensitivity (slowly inactivating, high-threshold, "L-type"). With barium as the charge carrier, large-amplitude (100-800 pA) inward currents of two types were characterized by their kinetics and voltage dependence. With holding potential--80 mV, a rapidly inactivating, low-threshold current ("T-type") was activated by depolarizations above-40 mV and was maximal at -10 mV. With holding potential -30 mV, this component was inactivated, and a second slowly inactivating, high-threshold current was activated above -10 mV and was maximal at +10 to +20 mV. These currents are similar to the T-type and L-type currents previously described in vascular smooth muscle cells. When added to the bath, the active phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (100 nM) increased the slowly inactivating (L-type) current by 32 +/- 20% (n = 8, +/- SD). Phorbol-12,13-dibutyrate (100 nM) caused a similar effect, but the inactive phorbol, 4-alpha-phorbol (100 nM), did not. We conclude that at least two distinct calcium conductances are expressed in A7r5 vascular smooth muscle cells, and that the dihydropyridine-sensitive calcium conductance is acutely modulated by phorbol esters, presumably acting through stimulation of protein kinase C. Such modulation may play a role in increasing transmembrane calcium influx mediated by agonist-receptor interactions that lead to activation of protein kinase C and may help to sustain or amplify calcium-dependent cell responses.     

Mitochondrial calcium uptake

Calcium (Ca²⁺) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca²⁺ levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca²⁺ ([Ca²⁺]_i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca²⁺ uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca²⁺ uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca²⁺ uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca²⁺ influx into the mitochondria via the MCU is small relative to other cytosolic Ca²⁺ extrusion pathways; (ii) single MCU conductance is ∼6–7 pS (105 mM [Ca²⁺]), and MCU flux appears to be modulated by [Ca²⁺]_i, suggesting Ca²⁺ regulation of MCU open probability (P_O); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iv) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca²⁺ critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca²⁺ under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca²⁺]_i, mitochondrial Ca²⁺ uptake can increase 10- to 1,000-fold and begin to shape [Ca²⁺]_i dynamics.     

Pituitary adenylate cyclase-activating polypeptide stimulates calcium mobilization in amphibian pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily. Although PACAP is a potent stimulator of adenylate cyclase activity in the adenohypophysis, the precise target cells for PACAP in the anterior pituitary remain unknown. The aim of the present study was to investigate whether PACAP could stimulate calcium mobilization in individual cells of the pituitary and to determine the type of cells that responded to PACAP. Enzymatically dispersed frog distal pituitary cells were plated on photoetched coverslips and cultured for 3-7 days. The cells were loaded with the fluorescent calcium indicator indo-1, and changes in intracellular calcium concentrations ([Ca2+]i) were monitored using dual wavelength microfluorimetry. The individual cells were localized with the aid of the alpha/numeric grid of the coverslips and identified retrospectively by immunofluorescence. Approximately 45% of GH and PRL cells and 25% of ACTH and TSH cells responded to PACAP (10(-5) M) ejection by an elevation of [Ca2+]i. Only 16% of gonadotropes were stimulated by PACAP. The time course of [Ca2+]i variations showed three different patterns: transient spikes, sustained stimulations, and oscillatory responses. In addition, heterogenous responses were observed within each cell type. These data provide evidence for the involvement of calcium mobilization in the mechanism of action of PACAP on pituitary cells. The results also indicate that in frogs, PACAP may stimulate the secretory activity of GH and PRL cells and, to a lesser extent, ACTH, TSH, and gonadotrope cells.     

Effects of calcium channel blockers on calcium uptake in rat aortic valve allografts.

BACKGROUND. The life span of human aortic valve allografts is finite, and many fail because of cusp rupture or calcification. Subcellular changes occurring in aortic valves in response to transplantation include the uptake of calcium. This study uses a heterotropic rat aortic valve transplant model to determine whether the calcium channel blockers diltiazem and verapamil might attenuate leaflet calcification. METHODS AND RESULTS. The 60 rats studied were divided into the following groups: 1) control: valves from normal, unoperated F1 generation of Lewis and Brown Norway cross (LBNF1) rats; 2) control: valves from syngeneic transplant combinations (Lewis/Lewis); 3) control: valves from allogeneic transplant combinations (LBNF1/Lewis, donor/recipient); 4) experimental: valves from allogeneic strain combinations treated with 30 mg/kg per day diltiazem; 5) experimental: valves from allogeneic strain combinations treated with 30 mg/kg per day verapamil. Drugs or saline (controls) were administered with osmotic pumps placed subcutaneously 2 days before transplantation. Animals were killed 3 weeks later, and the valves were harvested and prepared for calcium analysis. Energy-dispersive x-ray microanalysis was used to measure the calcium in a section of one leaflet from each valve studied. Paired t tests showed that allograft valves treated with diltiazem or verapamil contained significantly less calcium than allograft controls treated with saline (p < 0.001). When all five groups were subjected to one-way ANOVA, the valves in the allograft control group contained significantly more calcium than all other groups. All other groups were not different from each other. CONCLUSIONS. The calcium channel blockers verapamil and diltiazem were effective in preventing early calcification that occurs in aortic valves after transplantation. Thus, these agents might play a role in prolonging the life of human aortic valve allografts.     

Beta-adrenergic receptor stimulates L-type calcium current in adult smooth muscle cells.

The hormonal regulation of L-type calcium current was investigated in freshly isolated tracheal smooth muscle cells using the whole-cell configuration of the patch-clamp technique. Isoproterenol stimulated the L-type calcium current 2.6-fold through beta-adrenoceptors. Dialysis of these cells with cyclic AMP, cyclic AMP analogues or the catalytic subunit of cyclic AMP kinase had no effect on basal or isoproterenol-stimulated calcium current. The calcium current was stimulated and inhibited by dialysis of the cells with GTP gamma S and GDP beta S, respectively. Evidently, in some smooth muscle cells the beta-adrenoceptor couples directly to L-type calcium channels via a G protein.     

Platelet-free calcium and vascular calcium uptake in ethanol-induced hypertensive rats

This study examined the effect of moderate ethanol intake on systolic blood pressure, platelet cytosolic free calcium, aortic calcium, and rubidium-86 uptake in Wistar-Kyoto rats. Twelve Wistar-Kyoto rats, aged 6 weeks, were given 5% ethanol in drinking water the first week followed by 10% ethanol in drinking water for the next 6 weeks. Twelve control animals were given regular tap water. Systolic blood pressure in the ethanol-treated rats was significantly higher (p less than 0.05) than that in controls after 1 week and remained higher throughout the study. At 13 weeks of age, platelet cytosolic free calcium and calcium uptake by aortas were significantly higher (p less than 0.001) in ethanol-treated animals as compared with those in controls. Ethanol intake did not affect aortic ouabain-sensitive 86Rb uptake. The in vitro effect of ethanol on calcium-45 and 86Rb uptake was also investigated in aortas of untreated Wistar-Kyoto rats at 13 weeks of age. In vitro ethanol (2.5-20 mmols/l) did not significantly affect 45Ca and 86Rb uptake in rat aortas. The increases in systolic blood pressure, platelet cytosolic free calcium, and vascular calcium uptake suggest that increases in cytosolic free calcium and calcium uptake mechanisms are associated with ethanol-induced hypertension.     

Recombinant human thyrotropin stimulates thyroid function and radioactive iodine uptake in the rhesus monkey.

The administration of bovine TSH to stimulate thyroid radioactive iodine uptake to detect functioning thyroid tissue in man after surgery for thyroid cancer is rarely, if ever, used, due to allergic reactions and/or the development of TSH antibodies. Human (h) TSH would be far less likely to induce allergic reactions or TSH antibodies. Recombinant hTSH (rec-hTSH) was produced by a line of Chinese hamster ovary cells that had been transfected with cDNA for the two subunit proteins that comprise hTSH. The present study was carried out to determine the half-life of rec-hTSH in the monkey and its ability to stimulate thyroid function. The half-life of rec-hTSH after iv administration was approximately 63 min for the rapid phase and 326 min for the slow phase. After three daily im injections of 2 U rec-hTSH to two monkeys, serum T4 concentrations increased several-fold, and serum T3 increased 2-3 times above basal values. The 6 and 20 h thyroid 123I uptakes doubled after rec-hTSH administration. These results demonstrate the biological efficacy of rec-hTSH administered to the monkey and strongly suggest that rec-hTSH will be effective in stimulating thyroid function in man.     

Decreased intestinal calcium absorption in vivo and normal brush border membrane vesicle calcium uptake in cortisol-treated chickens: evidence for dissociation of calcium absorption from brush border vesicle uptake.

The influence of cortisol on intestinal calcium transport was studied in isolated duodenal loops and brush border membrane (BBM) vesicles of vitamin D-deficient or replete chickens. Four- to five-week-old vitamin D-deficient cockerels were dosed intraperitoneally with 1 microgram of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] alone 15 hr before sacrifice or in combination with 1, 3, or 5 mg of cortisol 24 and 48 hr before sacrifice. After a 1-microgram dose of 1,25-)OH)2D3 the in situ intestinal ligated loop technique revealed a 60% increase in calcium absorption compared to control birds (P less than or equal to 0.001). However, the administration of cortisol in various doses (3 and 5 mg) to chickens given 1,25-(OH)2D3 resulted in significant decreases in intestinal calcium transport in vivo (P less than or equal to 0.05; P less than or equal to 0.05). When intestinal BBM vesicles were prepared from birds treated in a manner identical with that described above, there was no observable difference between calcium uptake in BBM vesicles of the 1,25-(OH)2D3-treated birds and that of the cortisol plus 1,25-(OH)2D3-treated birds. 1,25-(OH)2D3-treated and 1,25-(OH)2D3 plus cortisol-treated chicks had intestinal BBM vesicle uptakes that were significantly greater than those of vitamin D-deficient controls (P less than or equal to 0.02; P less than or equal to 0.025). These data show that in vivo intestinal calcium transport may be markedly reduced in the presence of normal intestinal BBM vesicle calcium uptake. This suggest that factors other than BBM calcium uptake (e.g., protein synthesis or contraluminal membrane events) play an important role in the movement of calcium from the intestinal lumen into the bloodstream and extracellular fluid of the organism.