Passive Calcium Transport Research Articles

Alteration of Tight Junction Gene Expression by Calcium- and Vitamin D-Deficient Diet in the Duodenum of Calbindin-Null Mice

Calcium absorption is regulated by both active (transcellular) and passive (paracellular) pathways. Although each pathway has been studied, correlations between the two pathways have not been well elucidated. In previous investigations, the critical transcellular proteins, calbindin-D9k (CaBP-9k) and -D28k (CaBP-28k), were shown to affect other transcellular pathways by buffering intracellular calcium concentrations. The rate of paracellular calcium transport in the duodenum is generally determined by the expression of tight junction genes. In the present study, the effect of dietary calcium and/or vitamin D supplementation on the expression of tight junction genes (occludin, ZO-1 and claudin 2, 10b, 12 and 15) in the duodenum of CaBP-9k- and/or -28k-deficient mice was examined. With a normal diet, the expression of most tight junction genes in the duodenum was significantly increased in CaBP-9k knockout (KO) mice compared to wild-type (WT) animals. With a calcium- and vitamin D-deficient diet, tight junction gene expression was significantly decreased in the duodenum of the CaBP-9k KO mice. These findings suggest that expression of paracellular tight junction genes is regulated by transcellular CaBP proteins, suggesting that active and passive calcium transport pathways may function cooperatively.

Impaired body calcium metabolism with low bone density and compensatory colonic calcium absorption in cecectomized rats

An earlier study reported that cecal calcium absorption contributes less than 10% of total calcium absorbed by the intestine, although the cecum has the highest calcium transport rate compared with other intestinal segments. Thus, the physiological significance of the cecum pertaining to body calcium metabolism remains elusive. Herein, a 4-wk calcium balance study in cecectomized rats revealed an increase in fecal calcium loss with marked decreases in fractional calcium absorption and urinary calcium excretion only in the early days post-operation, suggesting the presence of a compensatory mechanism to minimize intestinal calcium wasting. Further investigation in cecectomized rats showed that active calcium transport was enhanced in the proximal colon but not in the small intestine, whereas passive calcium transport along the whole intestine was unaltered. Since apical exposure to calcium-sensing receptor (CaSR) agonists similarly increased proximal colonic calcium transport, activation of apical CaSR in colonic epithelial cells could have been involved in this hyperabsorption. Calcium transporter genes, i.e., TRPV6 and calbindin-D(9k), were also upregulated in proximal colonic epithelial cells. Surprisingly, elevated serum parathyroid hormone levels and hyperphosphatemia were evident in cecectomized rats despite normal plasma calcium levels, suggesting that colonic compensation alone might be insufficient to maintain normocalcemia. Thus, massive bone loss occurred in both cortical and trabecular sites, including lumbar vertebrae, femora, and tibiae. The presence of compensatory colonic calcium hyperabsorption with pervasive osteopenia in cecectomized rats therefore corroborates that the cecum is extremely crucial for body calcium homeostasis.

Passive transport pathways for Ca 2+ and Co 2+ in human red blood cells. 57Co 2+ as a tracer for Ca 2+ influx

The passive transport of calcium and cobalt and their interference were studied in human red cells using 45Ca and 57Co as tracers. In ATP-depleted cells, with the ATP concentration reduced to about 1 μM, the progress curve for 45Ca uptake at 1 mM rapidly levels off with time, consistent with a residual Ca-pump activity building up at increasing [Ca T] c to reach at [Ca T] c about 5 μmol (l cells) − 1 a maximal pump rate that nearly countermands the passive Ca influx, resulting in a linear net uptake at a low level. In ATP-depleted cells treated with vanadate, supposed to cause Ca-pump arrest, a residual pump activity is still present at high [Ca T] c. Moreover, vanadate markedly increases the passive Ca 2+ influx. The residual Ca-pump activity in ATP-depleted cells is fuelled by breakdown of the large 2,3-DPG pool, rate-limited by the sustainable ATP-turnover at about 40–50 μmol (l cells) − 1 h − 1 . The apparent Ca 2+ affinity of the Ca-pump appears to be markedly reduced compared to fed cells. The 2,3-DPG breakdown can be prevented by inhibition of the 2,3-DPG phosphatase by tetrathionate, and under these conditions the 45Ca uptake is markedly increased and linear with time, with the unidirectional Ca influx at 1 mM Ca 2+ estimated at 50–60 μmol (l cells) − 1 h − 1 . The Ca influx increases with the extracellular Ca 2+ concentration with a saturating component, with K ½(Ca) about 0.3 mM, plus a non-saturating component. From 45Ca-loaded, ATP-depleted cells the residual Ca-pump can also be detected as a vanadate- and tetrathionate-sensitive efflux. The 45Ca efflux is markedly accelerated by external Ca 2+, both in control cells and in the presence of vanadate or tetrathionate, suggesting efflux by carrier-mediated Ca/Ca exchange. The 57Co uptake is similar in fed cells and in ATP-depleted cells (exposed to iodoacetamide), consistent with the notion that Co 2+ is not transported by the Ca-pump. The transporter is thus neither SH-group nor ATP or phosphorylation dependent. The 57Co uptake shows several similarities with the 45Ca uptake in ATP-depleted cells supplemented with tetrathionate. The uptake is linear with time, and increases with the cobalt concentration with a saturating component, with J max about 16 μmol (l cells) − 1 h − 1 and K ½(Co) about 0.1 mM, plus a non-saturating component. The 57Co and 45Ca uptake shows mutual inhibition, and at least the stochastic Ca 2+ influx is inhibited by Co 2+. The 57Co and 45Ca uptake are both insensitive to the 1,4-dihydropyridine Ca-channel blocker nifedipine, even at 100 μM. The 57Co uptake is increased at high negative membrane potentials, indicating that the uptake is at least partially electrogenic. The 57Co influx amounts to about half the 45Ca influx in ATP-depleted cells. It is speculated that the basal Ca 2+ and Co 2+ uptake could be mediated by a common transporter, probably with a channel-like and a carrier-mediated component, and that 57Co could be useful as a tracer for at least the channel-like Ca 2+ entry pathway in red cells, since it is not itself transported by the Ca-pump and, moreover, is effectively buffered in the cytosol by binding to hemoglobin, without interfering with Ca 2+ buffering. The molecular identity of the putative common transporter(s) remains to be defined.

Gitelman Syndrome

Gitelman Syndrome

Direct stimulation of the transcellular and paracellular calcium transport in the rat cecum by prolactin

Prolactin (PRL) is reported to stimulate calcium absorption in the rat's small intestine. However, little is known regarding its effects on the cecum, a part of the large intestine with the highest rate of intestinal calcium transport. We demonstrated herein by quantitative real-time polymerase chain reaction and Western blot analysis that the cecum could be a target organ of PRL since cecal epithelial cells strongly expressed PRL receptors. In Ussing chamber experiments, PRL enhanced the transcellular cecal calcium absorption in a biphasic dose-response manner. PRL also increased the paracellular calcium permeability and passive calcium transport in the cecum, which could be explained by the PRL-induced decrease in transepithelial resistance and increase in cation selectivity of the cecal epithelium. PRL actions in the cecum were abolished by inhibitors of phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and RhoA-associated coiled-coil forming kinase (ROCK), but not inhibitors of gene transcription and protein biosynthesis. In conclusion, PRL directly enhanced the transcellular and paracellular calcium transport in the rat cecum through the nongenomic signaling pathways involving PI3K, PKC, and ROCK.

Extracellular calcium- and magnesium-mediated regulation of passive calcium transport across Caco-2 monolayers

The calcium-sensing receptor (CaR) is expressed on intestinal epithelial serosal membrane and in Caco-2 cells. In renal epithelium, CaR expressed on the basolateral membrane acts to limit excess tubular Ca 2+ reabsorption. Therefore, here we investigated whether extracellular calcium (Ca o 2+) can regulate active or passive 45Ca 2+ transport across differentiated Caco-2 monolayers via CaR-dependent or CaR-independent mechanisms. Raising the Ca o 2+ concentration from 0.8 to 1.6 mM increased transepithelial electrical resistance (TER) and decreased passive Ca 2+ permeability but failed to alter active Ca 2+ transport. The Ca o 2+ effect on TER was rapid, sustained and concentration-dependent. Increasing basolateral Mg 2+ concentration increased TER and inhibited both passive and active Ca 2+ transport, whereas spermine and the CaR-selective calcimimetic NPS R-467 were without effect. We conclude that small increases in divalent cation concentration elicit CaR-independent increases in TER and inhibit passive Ca 2+ transport across Caco-2 monolayers, most probably through a direct effect on tight junction permeability. Whilst it is known that the complete removal of Ca o 2+ lowers TER, here we show that Ca o 2+ addition actually increases TER in a concentration-dependent manner. Therefore, such Ca o 2+-sensitivity could modulate intestinal solute transport including the limiting of excess Ca 2+ absorption.

Prolactin is an important regulator of intestinal calcium transport

Prolactin has been shown to stimulate intestinal calcium absorption, increase bone turnover, and reduce renal calcium excretion. The small intestine, which is the sole organ supplying new calcium to the body, intensely expresses mRNAs and proteins of prolactin receptors, especially in the duodenum and jejunum, indicating the intestine as a target tissue of prolactin. A number of investigations show that prolactin is able to stimulate the intestinal calcium transport both in vitro and in vivo, whereas bromocriptine, which inhibits pituitary prolactin secretion, antagonizes its actions. In female rats, acute and long-term exposure to high prolactin levels significantly enhances the (i) transcellular active, (ii) solvent drag-induced, and (iii) passive calcium transport occurring in the small intestine. These effects are seen not only in pregnant and lactating animals, but are also observed in non-pregnant and non-lactating animals. Interestingly, young animals are more responsive to prolactin than adults. Prolactin-enhanced calcium absorption gradually diminishes with age, thus suggesting it has an age-dependent mode of action. Although prolactin's effects on calcium absorption are not directly vitamin D-dependent; a certain level of circulating vitamin D may be required for the basal expression of genes related to calcium transport. The aforementioned body of evidence supports the hypothesis that prolactin acts as a regulator of calcium homeostasis by controlling the intestinal calcium absorption. Cellular and molecular signal transductions of prolactin in the enterocytes are largely unknown, however, and still require investigation.

Prolactin-stimulated transepithelial calcium transport in duodenum and Caco-2 monolayer are mediated by the phosphoinositide 3-kinase pathway

Prolactin (PRL) has been shown to stimulate intestinal calcium absorption but the mechanism was still unknown. This study aimed to investigate the mechanism and signaling pathway by which PRL enhanced calcium transport in the rat duodenum and Caco-2 monolayer. Both epithelia strongly expressed mRNAs and proteins of PRL receptors. Ussing chamber technique showed that the duodenal active calcium fluxes were increased by PRL in a dose-response manner with the maximal effective dose of 800 ng/ml. This response diminished after exposure to LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Caco-2 monolayer gave similar response to PRL with the maximal effective dose of 600 ng/ml. By nullifying the transepithelial potential difference, we showed that the voltage-dependent paracellular calcium transport did not contribute to the PRL-enhanced flux in Caco-2 monolayer. In contrast, the calcium gradient-dependent paracellular transport and calcium permeability were increased by PRL. Effects of PRL on Caco-2 monolayer were abolished by PI3K inhibitors (LY-294002 and wortmannin), but not by inhibitors of MEK (U-0126) or JAK2 (AG-490). To investigate whether the PRL-enhanced paracellular transport was linked to changes in the epithelial charge selectivity, the permeability ratio of sodium and chloride (P(Na)/P(Cl)) was determined. We found that PRL elevated the P(Na)/P(Cl) in both epithelia, and the effects were blocked by PI3K inhibitors. In conclusion, PRL directly and rapidly stimulated the active and passive calcium transport in the rat duodenum and Caco-2 monolayer via the nongenomic PI3K-signaling pathway. This PRL-enhanced paracellular calcium transport could have resulted from altered charge selectivity.

Various Nondigestible Saccharides Open a Paracellular Calcium Transport Pathway with the Induction of Intracellular Calcium Signaling in Human Intestinal Caco-2 Cells

Ingestion of soluble nondigestible saccharides increases calcium absorption, and it is suggested that paracellular calcium transport contributes to this effect. However, cellular mechanisms and the contribution of active transport have not been clarified. This study examined the effects of 4 nondigestible saccharides, difructose anhydride (DFA) III, DFAIV, fructooligosaccharides, and raffinose, on active and passive calcium transport, permeability of paracellular pathways, and intracellular calcium signaling in a human intestinal Caco-2 cell monolayer. Net, active, and passive calcium transport were evaluated using (45)Ca. Transepithelial electrical resistance (TEER) and transport of lucifer yellow were measured as indicators of paracellular passage in differentiated Caco-2 cell monolayers incubated with 0-100 mmol/L of the various saccharides. The changes in intracellular calcium ion concentrations ([Ca(2+)](i)) were measured by fura-2 loading before and after the addition of each saccharide (50 or 100 mmol/L). The addition of 100 mmol/L of each saccharide to the apical medium of the Caco-2 cells enhanced net calcium transport without any changes in active calcium transport. Relative TEER was dose dependently and reversibly decreased by the addition of saccharides, and the decreases in TEER were highly correlated with net calcium transport (P < 0.001). Basolateral application of the saccharides had a slight or no effect on indicators of the paracellular pathway. Each saccharide caused an immediate and dose-dependent rise in [Ca(2+)](i) in the cells. The 4 nondigestible saccharides increased net calcium transport in the cells via the paracellular route through tight junctions. The rise in [Ca(2+)](i) induced by these saccharides may be involved in the opening of tight junctions.

Effect of the ratio of casein phosphopeptides to calcium (w/w) on passive calcium transport in the distal small intestine of rats

Objective: We investigated the influence of different ratios between casein phosphopeptides and calcium (CPP/Ca) in intestinal lumen on passive Ca transport across the distal small intestine of rats in vitro. Methods: We measured the amounts of passive Ca absorbed across the everted distal small intestine of rats with the use of radioactive 45Ca as tracer. Four CPP/Ca ratios (5, 10, 15, and 20 w/w) and three mineral concentrations (4, 8, and 12 mmol/L) were tested on groups of six rats. Results: We found a significant effect of the CPP/Ca ratio on mineral absorption ( P < 0.001). The amounts of Ca absorbed increased from a ratio of 5 to a ratio of 15 and decreased with a ratio of 20, with a similar trend for every concentration tested. The more evident effect was found with the concentration of 12 mmol/L of Ca ( P < 0.001 to 0.05). Conclusion: The positive effect of CPPs on passive Ca absorption seems to depend on the relative amounts of both species in intestinal lumen. In this study, the ratio 15 was identified as the most efficient at increasing mineral transport. This evidence might be considered before suggesting CPP as a Ca dietary integrator, although more studies in vitro and in vivo are needed.

Acute effects of prolactin on passive calcium absorption in the small intestine by in vivo perfusion technique

The acute effect of intraperitoneally administered prolactin (0.2, 0.4, and 0.6 mg/kg body weight) on passive calcium transport in duodenum, proximal jejunum, and ileum of sexually mature female Wistar rats was investigated by using an in vivo perfusion technique. Test solution containing (in mM) NaCl, 100; KCl, 4.7; MgSO4, 1.2; CaCl2, 20; D-glucose, 11; sodium ferrocyanide (Na4Fe(CN)6), an index of net water transport, 20; and 0.7 microCi 45CaCl2 (1 Ci = 37 GBq) was perfused through the 10-cm intestinal loop for 60 min. Results showed that 0.4 mg prolactin/kg body weight significantly increased duodenal net Ca absorption (net Ca) from 23.81 +/- 1.84 to 30.56 +/- 1.57 mmol/g dry weight (p < 0.05) by stimulating the lumen to plasma calcium flux (CaL-P). The jejunum responded to 0.2, 0.4, and 0.6 mg prolactin/kg body weight by reversing from net Ca absorption of 18.60 +/- 1.70 mmol/g dry weight to net secretion of -3.30 +/- 1.56, -10.39 +/- 2.21, and -11.79 +/- 2.04 mmol/g dry weight (p < 0.01), respectively, as a result of a dose-dependent increase in plasma to lumen calcium flux (CaP-L). Calcium fluxes in the ileum on the other hand did not respond to prolactin. There was a close correlation between net water flux and net calcium flux in all three intestinal segments under basal condition regardless of the luminal sodium concentration. However, this correlation was lost after prolactin administration, which while having no effect on net water flux, altered the duodenal and jejunal calcium fluxes. By varying the luminal concentration of sodium, it was found that the stimulatory effect of 0.4 mg prolactin/kg body weight on the duodenal CaL-P was reduced when compared with control, i.e., 17.84 +/- 0.91 vs. 26.64 +/- 1.05 mmol/g dry weight at a sodium concentration of 180 mM, and 14.48 +/- 0.99 vs. 20.12 +/- 1.34 mmol/g dry weight at a sodium concentration of 140 mM. At a sodium concentration of 80 mM, the prolactin effect was absent. Since duodenal Na(+)-K+ ATPase activity was increased by prolactin from 3.77 +/- 0.16 to 4.95 +/- 0.30 mumol Pi.mg-1 protein.h-1 (p < 0.05), sodium dependency of the prolactin-enhanced lumen to plasma calcium flux may be related to both sodium-induced water flow and calcium-sodium exchange across the basolateral membrane. Thus, it was postulated that under basal condition, net calcium transport in the small intestine occurred with the sodium-induced water transport along the paracellular pathway. However, after prolactin administration, this association was lost. Prolactin-enhanced lumen to plasma calcium flux in the duodenum was sodium dependent and involved the Na(+)-K+ ATPase activity. In the proximal jejunum, prolactin stimulated plasma to lumen calcium flux, but the mechanism was not known.

Acute effects of prolactin on passive calcium absorption in the small intestine by in vivo perfusion technique

The acute effect of intraperitoneally administered prolactin (0.2, 0.4, and 0.6 mg/kg body weight) on passive calcium transport in duodenum, proximal jejunum, and ileum of sexually mature female Wistar rats was investigated by using an in vivo perfusion technique. Test solution containing (in mM) NaCl, 100; KCl, 4.7; MgSO4, 1.2; CaCl2, 20; D-glucose, 11; sodium ferrocyanide (Na4Fe(CN)6), an index of net water transport, 20; and 0.7 microCi 45CaCl2 (1 Ci = 37 GBq) was perfused through the 10-cm intestinal loop for 60 min. Results showed that 0.4 mg prolactin/kg body weight significantly increased duodenal net Ca absorption (net Ca) from 23.81 +/- 1.84 to 30.56 +/- 1.57 mmol/g dry weight (p < 0.05) by stimulating the lumen to plasma calcium flux (CaL-P). The jejunum responded to 0.2, 0.4, and 0.6 mg prolactin/kg body weight by reversing from net Ca absorption of 18.60 +/- 1.70 mmol/g dry weight to net secretion of -3.30 +/- 1.56, -10.39 +/- 2.21, and -11.79 +/- 2.04 mmol/g dry weight (p < 0.01), respectively, as a result of a dose-dependent increase in plasma to lumen calcium flux (CaP-L). Calcium fluxes in the ileum on the other hand did not respond to prolactin. There was a close correlation between net water flux and net calcium flux in all three intestinal segments under basal condition regardless of the luminal sodium concentration. However, this correlation was lost after prolactin administration, which while having no effect on net water flux, altered the duodenal and jejunal calcium fluxes. By varying the luminal concentration of sodium, it was found that the stimulatory effect of 0.4 mg prolactin/kg body weight on the duodenal CaL-P was reduced when compared with control, i.e., 17.84 +/- 0.91 vs. 26.64 +/- 1.05 mmol/g dry weight at a sodium concentration of 180 mM, and 14.48 +/- 0.99 vs. 20.12 +/- 1.34 mmol/g dry weight at a sodium concentration of 140 mM. At a sodium concentration of 80 mM, the prolactin effect was absent. Since duodenal Na(+)-K+ ATPase activity was increased by prolactin from 3.77 +/- 0.16 to 4.95 +/- 0.30 mumol Pi.mg-1 protein.h-1 (p < 0.05), sodium dependency of the prolactin-enhanced lumen to plasma calcium flux may be related to both sodium-induced water flow and calcium-sodium exchange across the basolateral membrane. Thus, it was postulated that under basal condition, net calcium transport in the small intestine occurred with the sodium-induced water transport along the paracellular pathway. However, after prolactin administration, this association was lost. Prolactin-enhanced lumen to plasma calcium flux in the duodenum was sodium dependent and involved the Na(+)-K+ ATPase activity. In the proximal jejunum, prolactin stimulated plasma to lumen calcium flux, but the mechanism was not known.

Development of a technique for in situ studies of calcium absorption in the intestine of rats

The aim of the present study was to develop (feasibility, reliability, reproducibility) a technique for the in situ measurement of intestinal calcium absorption in the rat. An intestinal loop (duodenum + proximal jejunum) was perfused both by the intraluminal and vascular routes. A solution of NaCl 155 mM and CaCl 2 1.25 mM containing 45Ca was perfused intraluminally at a flow rate of 0.2 ml/min and the 45Ca appearing in the venous effluent was determined to estimate calcium absorption. This technique was used to study the effect of a 10-day period of calcium supplementation on calcium absorption. The animals received enterally either 5 or 30 mg/day per kg BW. The results showed that intestinal calcium transport was enhanced when rats were given the calcium supplement for 10 days prior to the experiment (99.3 ± 2.5 ( n = 5) versus 36.6 ± 3.6 ( n = 5) nmol/min per kg BW, P < 0.001). This study indicates that dietary calcium supplement enhances calcium absorption probably by increasing the passive transport of calcium in the small intestine.

Maximal calcium extrusion capacity and stoichiometry of the human red cell calcium pump.

1. The uphill calcium efflux through calcium-saturated pumps in intact red cells was investigated with the aid of a new method, in initial conditions of uniform ionophore A23187-induced calcium distribution among the cells. The method is based on findings by Tiffert, García-Sancho & Lew (1984) which show that cobalt can suddenly arrest passive calcium transport by the ionophore and expose, without noticeable interference, uphill calcium extrusion by the pump. The results comprise methodological aspects and questions concerning interactions between inner pump sites, ATP and Ca2+, and the calcium: ATP stoichiometry of the calcium-saturated pump. 2. Ionophore-induced calcium influx was set to be far in excess of the maximal calcium pump capacity. This secured a uniform calcium distribution among the cells, and Ca2+ equilibration by 2 min or less of calcium permeabilization. Cobalt was added between 15 s and 5 min after ionophore addition. The calcium and ATP content of the cells was followed during ionophore-induced influx and cobalt-exposed efflux. 3. The external cobalt concentrations required to block completely ionophore-mediated calcium transport were similar or only marginally higher than those of calcium. 4. The reproducibility of independent cobalt-exposed calcium efflux measurements from single blood samples was within an 8% range. 5. During cobalt-exposed calcium efflux, the calcium content of subpopulations of cells, with and without active Ca2+-sensitive K+ channels, investigated by post-incubation of samples in low-K+, thiocyanate (SCN-) media (modified from García-Sancho & Lew, 1988a), was similar. This is consistent with the maintenance of a uniform calcium distribution among the cells during uphill calcium extrusion. 6. Cobalt-exposed calcium efflux was similar in the interval from 15 s to 5 min after calcium permeabilization although cell ATP levels had fallen by over 50% in that period. Therefore, cell ATP concentrations within the physiological range do not seem to be regulatory for calcium-saturated pumps in the intact red cell. 7. All cobalt-exposed calcium efflux curves were linear in time, at least until total cell calcium contents reached levels below 100 mumol/l cells. This suggests that internal calcium is not inhibitory for calcium-saturated efflux in intact cells in the 0.1-1 mmol/l cells range. 8. The cobalt-exposed calcium fluxes were in the range from 4 to 24 mmol/(1 cells.h) for fresh cells and from 10 to 18 mmol/1 cells. h) for samples from the Blood Bank.(ABSTRACT TRUNCATED AT 400 WORDS)

The inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin, activates platelets by selective mobilization of calcium as shown by protein phosphorylations

We have studied the activation of human blood platelets by the inflammatory and tumor-promoting sesquiterpene lactone, thapsigargin. The effect of thapsigargin was compared with other common agonists (calcium ionophore A23187, phorbol ester TPA and thrombin). Platelet aggregation, serotonin release, raised cytoplasmic free calcium level and phosphorylation of platelet proteins was examined in platelet-rich plasma and washed platelet suspension. In contrast to A23187 and thrombin, the platelet activation induced by thapsigargin developed slowly, with maximal response obtained after 2–3 min. Both the thapsigargin- and the A23187-induced serotonin releases were synergistically increased by TPA. Studies of the phosphorylation of platelet proteins revealed that thapsigargin and A23187 equally well induced a selective phosphorylation of two proteins with apparent molecular masses of 20 kDa and 47 kDa. These proteins, which are substrates of myosin light-chain kinase and protein kinase C respectively, are known to be involved in platelet activation. The thapsigargin-induced platelet aggregation and serotonin release was completely inhibited by class I (nimodipine), class II (verapamil) and class III (diltiazem) calcium-channel blockers. The inhibitory activity of nimodipine was abolished by the corresponding 1,4-dihydropyridine calcium-channel agonist, BAY K 8644. These results shows that the thapsigargin-induced platelet activation is mediated by an increase in the cytoplasmic free calcium level, presumably obtained by stimulation of the passive calcium transport through specific channels. These thapsigargin-sensitive channels should predominantly be located in the membranes of intracellular calcium stores rather than in the plasma membrane, because removal of extracellular calcium by EGTA had only an insignificant effect on the thapsigargin-induced rise in cytoplasmic free calcium level.

Influence of glucocorticoids on calcium absorption in different segments of the rat intestine.

Glucocorticoids enhance the movement of fluid and sodium in the duodenum, thereby resulting in an increase in the passive transport of calcium. Since passive transport of calcium predominates in the distal intestinal segments, the influence of glucocorticoids on calcium and fluid transport in the duodenum, mid-jejunum, ileum, and colon of the rat was studied. Calcium transport and fluid absorption was determined by the in vivo ligated loop technique. One segment was ligated in each animal. Under either normal osmolarity or hypertonic conditions, administration of cortisone stimulated intestinal fluid absorption in each segment of the small intestine, but not in the colon. Since the final fluid sodium concentration was not altered, cortisone enhanced net sodium absorption in proportion to the increase in fluid transport. Glucocorticoids inhibited the active transport of calcium in the proximal regions of the small intestine by bidirectional changes in calcium flux. However, the inhibitory effect was not apparent under the conditions where passive transport predominates. The stimulation of passive transport in the mid-jejunum overcomes the inhibition of active transport. In the ileal region, glucocorticoids increased active transport of calcium by 165%, whereas the enhancement of calcium transport under conditions where passive transport predominated reached 217%. This result indicates that both active and passive transport of calcium were enhanced by glucocorticoid treatment. In the colon, glucocorticoids increased active transport by 170%. However, the magnitude of the increase in calcium transport was less under conditions where passive transport predominates (129%), indicating that glucocorticoids stimulate the active transport of calcium in the colon with no appreciable stimulation of passive transport.

Interrelation of cortisone and 1,25 dihydroxycholecalciferol on intestinal calcium and phosphate absorption.

The interrelation of glucocorticoids and 1,25 dihydroxycholecalciferol (1,25(OH)2D3) on intestinal calcium and phosphate absorption was investigated. The active and passive transport of calcium and phosphate was evaluated by the in situ intestinal loop technique. Administration of cortisone resulted in a decrease of the luminal fluid and an increase of the luminal calcium and phosphate concentration. Under active transport conditions, administration of cortisone resulted in a decrease of net calcium absorption through two mechanisms: (1) depressed vitamin D-dependent calcium absorption, (2) increased vitamin D-independent calcium backflux. The enhancement of bidirectional phosphate flux by cortisone was independent of 1,25(OH)2D3. An enhancement of water movement by cortisone resulted in an increase of luminal calcium and phosphate concentration which favors the passive diffusion of these ions. Enhanced calcium diffusion by cortisone compensates for the inhibitory effect of cortisone on vitamin D-dependent calcium transport. However, enhanced phosphate diffusion by cortisone is additive to the effect of 1,25(OH)2D3.

Effect of hypophysectomy and 1,25-dihydroxyvitamin D on duodenal calcium absorption.

Intestinal active and passive transport of calcium were studied in hypophysectomized (HX) and intact rats using the in vivo duodenal loop technique. In the vitamin D-supplemented condition, hypophysectomy resulted in a decrease in serum 1,25-dihydroxyvitamin D [1,25-(OH)2D]. Hypophysectomy prevented a gain in body weight and decreased intestinal mucosal weight and total calcium absorption. When the data were expressed per unit mucosal wet weight, duodenal active calcium transport was not different in the HX and intact groups, but passive transport was persistently decreased by hypophysectomy. Administration of bovine GH to the HX rats did not change the mucosal mass, but enhanced both active and passive duodenal transport to calcium. Vitamin D depletion for 6 weeks decreased serum 25-hydroxy-vitamin D and 1,25-(OH)2D levels in both intact and HX rats to about the same level. After bovine GH and 1,25-(OH)2D3 replacement, the calcium absorption studies suggest that 1) 1,25-(OH)2D3 enhances intestinal calcium passive transport as well as active transport in intact and HX rats; 2) GH enhances both active and passive transport of calcium in the presence of sufficient quantities of 1,25-(OH)2D; 3) this latter effect is independent of the metabolism of vitamin D; and 4) a decrease in mucosal mass is one of the factors that results in decreased calcium absorption after hypophysectomy.

Calmodulin Levels and Divalent Cation Pump Activities in Kidneys of Streptozotocin-Diabetic Rats

Levels of calmodulin and activities of calcium and magnesium ATPases were determined in renal tissues of age-matched male rats after increasing durations of the following conditions: untreated streptozotocin (STZ) induced diabetes; STZ-diabetic rats which received daily insulin (NPH) treatment beginning 24 hours after STZ administration; and STZ-diabetic rats which began receiving NPH after having endured untreated diabetes for 1, 2, and 3 weeks. Results were compared with those of age-matched control animals. Calmodulin levels were the same in renal tissues from all groups of rats as were magnesium-ATPase activities. Calcium-ATPase activities were significantly higher than control activities after 2 weeks of untreated STZ-diabetes and in tissues from all rats that received insulin treatment which was instituted after 1, 2 and 3 weeks of STZ-diabetes. Calcium ATPase activities of tissues from rats which received insulin treatment 24 hrs after STZ injections were no different from control values. The data indicate that prolonged deficiency of insulin results in insufficient passive transport of calcium from urine into tubular cells. Thus, the increased activity of calcium-ATPase on the contraluminal membrane of kidney cells represents a futile attempt to conserve calcium.