Net Calcium Research Articles

Greater unidirectional calcium efflux from bone during metabolic, compared with respiratory, acidosis

There is a smaller net calcium efflux from bone in vitro during respiratory (increased PCO2) than metabolic (decreased [HCO3-] acidosis. This could be due to the elevated PCO2, which would lessen the driving force for mineral dissolution and increase the driving force for mineralization with respect to carbonated apatite in the bone mineral. To test this hypothesis, we injected neonatal mice with 45Ca and dissected the radiolabeled calvariae 24 h later. The live calvariae were then cultured for 24 h under conditions simulating respiratory acidosis (Resp, pH = 7.225 +/- 0.003, PCO2 = 87.5 +/- 0.1 mmHg), severe respiratory acidosis (SResp, pH = 7.072 +/- 0.004, PCO2 = 103.0 +/- 0.5 mmHg), metabolic acidosis (Met, pH = 7.212 +/- 0.003, HCO3- = 15.5 +/- 0.1 meq/l), or normal acid-base status (Ctl, pH = 7.452 +/- 0.003, PCO2 = 40.0 +/- 0.2 mmHg, HCO3- = 27.8 +/- 0.2 meq/l) and bidirectional net calcium flux (JCa) and unidirectional 45Ca release were determined. There was greater JCa from bone during Met than Resp, and JCa was not different from Met during SResp despite the latter having a significantly lower pH. There was greater unidirectional 45Ca release from bone during Met than Resp, SResp, or Ctl. There was a similar direct correlation between JCa and 45Ca efflux in the respiratory and metabolic groups. However, when calvarial osteoclast activity was inhibited with calcitonin,although there was again greater JCa and 45Ca release with a metabolic compared with respiratory acidosis, there was a greater proportion of 45Ca release than JCa from bone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis inhibits osteoblastic and stimulates osteoclastic activity in vitro.

Metabolic acidosis induces net calcium flux (JCa) from cultured neonatal mouse calvariae through physicochemical and cell-mediated mechanisms. To determine the role of osteoblasts in acid-induced JCa, collagen synthesis and alkaline phosphatase activity were assessed in calvariae incubated in reduced pH and bicarbonate medium, a model of metabolic acidosis (Met), and compared with controls (Ctl). Collagen synthesis fell from 30.5 +/- 1.1 in Ctl to 25.1 +/- 0.4% with Met, and alkaline phosphatase decreased from 403 +/- 25 in Ctl to 298 +/- 21 nmol Pi.min-1.mg protein-1 with Met. During acidosis JCa was correlated inversely with percent collagen synthesis (r = -0.743, n = 11, P = 0.009) and with alkaline phosphatase activity (r = -0.453, n = 22, P = 0.034). To determine the role of osteoclasts in acid-induced JCa, osteoclastic beta-glucuronidase activity was determined in Ctl and Met in the absence or presence of the osteoclastic inhibitor calcitonin (CT, 3 x 10(-9) M). Met increased beta-glucuronidase (5.9 +/- 0.2) compared with Ctl (4.6 +/- 0.3 micrograms phenolphthalein released.bone-1.h-1), whereas CT inhibited beta-glucuronidase in both Ctl and Met (3.1 +/- 0.2 and 3.5 +/- 0.3, respectively). During acidosis JCa was correlated directly with beta-glucuronidase activity (r = 0.683, n = 42, P less than 0.001). Thus the cell-mediated component of JCa during acidosis in vitro appears to result from a combination of inhibited osteoblastic and stimulated osteoclastic activity.

The Use of Pharmacologic Agents to Study Mechanisms of Intestinal Calcium Transport ,

The mechanism of vitamin D-dependent intestinal calcium transport has been explored in experimental animals in vivo and in vitro with the aid of pharmacologic agents that inhibit steps in the translocation process. Glucocorticoids in vivo, but not in vitro, inhibit the mucosal-to-serosal flux (Jms) of calcium and thus reduce net calcium absorption. Chronic metabolic acidosis inhibits calcium transport in vivo through inhibition of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] production and by a direct effect in vitro on the enterocyte to decrease calcium Jms. Cellular functions that may be involved in the transport process have been inhibited in vitro, including brush border calcium uptake by calcium channel blockers; calmodulin-dependent Ca-activated ATPase by trifluoperazine; calcium binding to vitamin D-dependent calcium-binding protein (CaBP, calbindin) by theophylline and acidic lysosomal vesicle function by quinacrine, chloroquine and ammonium chloride. The results of these studies demonstrate the consequences of selectively inhibiting steps thought to be involved in calcium transport and suggest new directions for further research in elucidating mechanisms of cellular calcium transport.

Effects of amiloride on distal renal tubule sodium and calcium absorption: dependence on luminal pH.

The goal of our study was to examine the effects of amiloride on distal renal tubule calcium and sodium absorption at low and high luminal pH. In vivo microperfusion of distal convoluted tubules were performed on rats. Total Ca and Na concentrations in perfusion and collected fluids were measured by atomic absorption spectrometry. When tubules were perfused with low pH perfusate (pH = 5.6), net calcium absorption (JCa) averaged 5.4 pmol/min. With high pH perfusate (pH = 7.1), JCa was significantly higher averaging 8.9 pmol/min. (P less than 0.01). In contrast, net sodium absorption (JNa) was not affected by luminal pH (231 pmol/min. versus 237 pmol/min.). At low luminal pH, 10(-4) M amiloride analogue (5-N-methyl-N-isobutyl amiloride) (MIA) inhibited sodium absorption but stimulated calcium absorption. At high luminal pH MIA still inhibited Na absorption but it had no effect on calcium absorption. Thus, the natriuretic effect of amiloride appears to be independent of luminal pH, while the amiloride-stimulated distal calcium absorption is observed at low but not at high luminal pH.

Developmental changes in calcium kinetics in children assessed using stable isotopes.

Total exchangeable calcium pool size (TEP) and bone calcium accretion rate (Vo+) were measured using stable isotopes in healthy children and young adults. 42Ca or 46Ca was given intravenously to 10 children aged 10 months to 14 years and 3 women aged 23-33 years. Calcium kinetic parameters were determined using a two- or three-exponential curve of the resultant serum and urine tracer excesses. These data were compared with previously reported (radiotracer) kinetic studies of 21 children and 5 adults without known bone disease. Current results are comparable to those previously obtained, and the data from all studies were analyzed together. Total Vo+ was significantly greater in children aged 3-16 years than in adults (2.8 +/- 1.6 versus 0.7 +/- 0.2 g/day, p less than 0.01). Both TEP and Vo+ were significantly correlated to age independently of variations in body weight (p less than 0.01 for each). The ratio ko+ = Vo+/TEP was greater in children than adults (0.36 +/- 0.15 versus 0.12 +/- 0.03 day-1, p less than 0.001). These data demonstrate increased bone flow of calcium associated with increases in exchangeable calcium pools in children compared to adults. Vo+ and TEP may be maximum in early adolescence, associated with peak rates of net calcium accretion. The use of stable isotopes permits the safe evaluation of calcium kinetics in patients of all ages.

Paracellular Calcium Transport across the Small Intestine ,

Concentration and voltage dependence of unidirectional 45Ca transport measurements indicated that ∼60–70% of the mucosa-to-serosa calcium flux measured across the short-circuited rat duodenum, jejunum and ileum is paracellular, with only 30–40% of the mucosa-to-serosa calcium transport cellular. The calcium flux from serosa to mucosa was purely paracellular in all segments. Duodenal calcium serosal-to-mucosal flux was of the same order of magnitude as the mucosal-to-serosal paracellular movement. However, the serosal-to-mucosal flux of jejunum and ileum was twice as high. Therefore, net calcium absorption occurs only in the duodenum, whereas calcium is secreted in the jejunum and ileum by a passive paracellular route, presumably involving an anomalous solvent drag effect. Administration of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) led to an increase in the transcellular mucosa-to-serosa flux in the duodenum only. It also led to a stimulation of paracellular calcium flux in both directions in all three intestinal segments, with no change in net paracellular calcium absorption. Thus, the only vitamin D-related increase in calcium absorption was due to the increase in duodenal transcellular absorption. The mechanism by which 1,25-(OH)2D3 increased paracellular flux is not known but may have resulted from an osmotic effect on the intercellular spaces.

Modulation of Ca2+ channels by atrial natriuretic peptide in the bovine adrenal glomerulosa cell.

In the bovine adrenal glomerulosa cell, calcium influx through voltage-dependent calcium channels is critical to maintaining an aldosterone secretory response. In patch clamp, atrial natriuretic peptide (ANP) inhibits T-type calcium channel current yet stimulates L-type calcium channel current. In the present study the channel effects of ANP observed in the patch-clamp configuration were extended and related to populations of cells. We observed the following. (i) The effect of ANP on T-channel current resulted in the reduction in the open state probability. ANP decreased the mean open state duration from 14.2 to 1.8 ms/sweep. (ii) In the weakly depolarized cell stimulated by 8 mM K+, ANP reduced the level of aequorin luminescence (a measure of cytosolic calcium) and completely inhibited the stimulated rate of aldosterone secretion, returning it to prestimulation values. These effects are consistent with a decrease in net calcium channel influx and the reported inhibition of T-channel current. In contrast, the calcium channel blocker, nitrendipine, which at low dose selectively blocks L-type calcium channel flux, only slightly reduced luminescence, and partially inhibited the sustained secretory response. (iii) In the strongly depolarized cell, stimulated by 60 mM K+, ANP increased the level of aequorin luminescence consistent with an increase in net calcium channel influx and the reported stimulation of L-channel current. These results indicate that under physiological conditions the inhibition of T-type calcium channels may be involved in the inhibition of the aldosterone secretion induced by ANP.

Presynaptic calcium and serotonin-mediated enhancement of transmitter release at crayfish neuromuscular junction

Fura-2 microfluorometric measurements of calcium concentrations in excitor motor nerve terminals of the first walking leg of crayfish Procambrus clarkii were made during application of serotonin (5-HT) to examine whether changes in residual calcium concentration or calcium influx are responsible for the 5-HT-induced increase in transmitter release at this synapse. Calcium concentrations and EPSPs were monitored simultaneously during 0.5-Hz and moderate-frequency (3-16 Hz) stimulation before, during, and after a 5-min application of 5-HT at concentrations between 5 and 100 microM. We did not observe a significant increase in resting calcium concentration [Ca2+]i in presynaptic terminals during or after application of 5-HT despite simultaneously recorded excitatory junction potential (EJP) enhancement. Injection of the calcium buffer EGTA to concentrations sufficient to reduce significantly the buildup of frequency-dependent facilitation during trains of action potentials did not reduce 5-HT-mediated EJP enhancement. We therefore conclude that 5-HT does not release calcium from intracellular stores to an extent sufficient to overcome calcium buffering and removal systems. Spontaneous release of transmitter quanta was increased by 5-HT to the same extent in normal and increased-osmolarity saline in the absence of measured changes in [Ca2+]i. Thus, changes in residual calcium cannot explain 5-HT-induced synaptic enhancement. Increasing the action-potential-mediated calcium influx by broadening the spike or by increasing extracellular calcium increased evoked transmitter release and increased the accumulation of calcium during trains of action potentials. However, though 5-HT produced a comparable synaptic enhancement, we found no evidence for increased calcium accumulation during trains of action potentials, suggesting that net calcium influx, buffering, and removal are not affected by 5-HT. Therefore, we conclude that 5-HT's action does not result from effects on calcium metabolism and suggest that it acts on the neurosecretory apparatus to increase the effectiveness of calcium for stimulating release. Furthermore, based on its relatively constant effects on facilitated and unfacilitated action-potential-evoked release and spontaneous transmitter release, we conclude that 5-HT actions are independent of the concentration of calcium that is driving release.

Calcium and Phosphorus Fluxes During Hemodialysis With Low Calcium Dialysate

We evaluated the acute effects of varying dialysate calcium concentration on plasma concentrations and dialyzer fluxes of calcium and phosphorus in adult hemodialysis patients. Seven individuals with stable end-stage renal failure were dialyzed 4 hours, three times weekly. The effects of dialysates containing 1.75, 1.25, or 0.75 mmol/L (70.1, 50.1, or 30.1 mg/L) of calcium were compared. Each patient was studied once at each bath calcium concentration. Compared with the predialysis mean value of 2.27 mmol/L (9.1 mg/dL), plasma total calcium concentration increased, remained constant, or decreased with the 1.75-, 1.25-, or 0.75-mmol/L calcium dialysates, respectively. The 0.75-mmol/L calcium dialysate did not cause signs or symptoms of hypocalcemia (and the plasma calcium concentration did not fall below 1.80 mmol/L [7.2 mg/dL]). Plasma phosphorus concentrations decreased equally from a predialysis mean value of 2.16 mmol/L (6.7 mg/dL), regardless of the dialysate calcium concentration. After 4 hours of treatment with the three different dialysates, the cumulative calcium fluxes were significantly different. With 1.75 mmol/L calcium, mean bodily calcium accumulation was 21.9 mmol (879 mg). With 1.25 mmol/L, there was no net calcium flux. With 0.75 mmol/L, mean patient calcium loss was 5.8 mmol (231 mg). Mean phosphorus removal after 4 hours was 32.5 mmol (1,006 mg) and was unaffected by dialysate calcium concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells

The effects of NH 4Cl on cytoplasmic free calcium concentration ([Ca 2+] i) and pH (pH i) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH 4Cl (10–40 mM) in the presence of 1 mM extracellular calcium ([Ca 2+] e) increased [Ca 2+] i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH 4Cl. In medium containing 0.1 μM [Ca 2+] e, or in 1 mM [Ca 2+] e medium containing 0.1 μM nitrendipine, the plateau was absent leaving only a transient [Ca 2+] i spike. NH 4Cl also increased pH i and this, like the [Ca 2+] i plateau, remained elevated during the continued presence of NH 4Cl. In medium containing only 0.1 μM [Ca 2+] e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH 4Cl induced repeated [Ca 2+] i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20–500 nM) caused a [Ca 2+] i rise but, after an additional exposure to NH 4Cl, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abollshed the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH 4Cl. Neither acetate removal nor methylamine increased [Ca 2+] i in medium containing 0.1 μM [Ca 2+] e, although in both cases pH i increased. We conclude that in bovine anterior pituitary cells NH 4Cl raises [Ca 2+] i by two independent pathways, increasing net calcium entry and mobilizing Ca 2+ from a TRH-insensitive calcium store.

Short-term exposure of rat pancreatic islets to human interleukin-1β increases cellular uptake of calcium

Interleukin-1 (IL-1) may be one of the effector molecules involved in the destruction of the pancreatic islet B cells resulting in insulin-dependent diabetes mellitus. Isolated islets exposed to IL-1 show an acutely increased substrate metabolism and insulin release, which is followed by a metabolic and functional suppression. Since an increased cellular uptake of calcium in the islets may be associated with both nutrient-induced insulin release and cell damage, the effects of recombinant IL-1β (rIL-β) on net cellular calcium uptake by isolated rat pancreatic islets were investigated. In short-term experiments the islets were exposed to 25 U/ml rIL-1β for 120 min in the presence of 1.7 mM or 16.7 mM glucose, or 16.7 mM glucose plus 5 mM verapamil. In these experiments rIL-1β induced an increase both in net cellular uptake of calcium and in insulin release only in the presence of 16.7 mM glucose. The stimulatory effect of rIL-1β at 16.7 mM glucose was blocked by verapamil. By long-term experiments, under tissue culture conditions in the presence of 11.1 mM glucose, islet net calcium uptake, insulin release and glucose oxidation were measured at different time points over a 24-h period. During the first 2 h of incubation 25 U/ml rIL-1β effected a significant increase of net calcium uptake, insulin release and glucose oxidation. However, after 4 – 5 h of incubation with the cytokine no such stimulatory effects were seen. After longer incubations with rIL-1β all the islet functions studied were suppressed. To further elucidate the potential role of cellular calcium uptake via voltage-dependent channels for the long-term deleterious effects of rIL-1β on islet function, isolated islets were incubated for 24 h with 25 U/ml rIL-1β in the absence and presence of 5 mM verapamil. Except for a partial protection against a decrease in islet DNA content, there was no effect of verapamil on the suppressive action of the cytokine on islet insulin content, insulin release, glucose oxidation or proinsulin-insulin and total protein biosynthesis. The combined data suggest that rIL-1β acutely increases insulin release by an increased substrate metabolism and cellular calcium uptake via voltage-dependent channels. In the suppressive phase of rIL-1β action on the pancreatic islet B cells, calcium uptake and insulin biosynthesis and release are apparently decreased by a deprived substrate metabolism.

Factors that influence peak bone mass formation: a study of calcium balance and the inheritance of bone mass in adolescent females

We suggested that calcium may be an important determinant of peak bone mass. For further elucidation, calcium balances in adolescent females with different calcium intakes (270-1637 mg/d), and a 2-y intervention study of calcium supplementation were performed. Hereditary influences on bone status were also evaluated by comparing subjects' and parents' bone mass. The main determinant of calcium balance was calcium intake; net calcium absorption increased with intake and urinary calcium did not change. Adolescent females retained 200-500 mg Ca/d, suggesting that inadequate calcium intake may translate into inadequate calcium retention and a reduction in peak bone mass. There was a more pronounced increase in bone mass over time in the calcium-supplemented group (1640 mg Ca/d) than in the control group (750 mg Ca/d), but the differences between bone mass measurements were not statistically significant, possibly because of a type II error. By the age of 16 y daughters had accumulated 90-97% of the bone mass of their premenopausal mothers.

Mechanism of amphotericin B stimulation of net calcium efflux from neonatal mouse calvariae

Amphotericin B is a polyene antifungal agent that binds to membrane sterols, creating aqueous pores that permit ion fluxes sufficient to cause cell lysis. It has also been shown to alter ion transport in mammalian cells, including proton secretion from renal tubular cells. The latter effect can lead to distal renal tubular acidosis in patients treated for systemic fungal infections. Based on the understanding that osteoclast-mediated bone resorption is dependent on proton secretion, we examined the effect of amphotericin B on calcium efflux from neonatal mouse calvariae in organ culture. Amphotericin B (5 micrograms/ml) stimulated net calcium efflux from calvariae within 24 h to a level almost as great as that produced by a maximally effective concentration of parathyroid hormone. The stimulated calcium efflux was completely inhibited by both 10 ng/ml salmon calcitonin, a physiologic inhibitor of osteoclast activity, and 4 x 10(-4) M acetazolamide, a specific inhibitor of carbonic anhydrase, the enzyme necessary for substantial proton generation by osteoclasts. These results indicate a direct effect of amphotericin B on bone in vitro to stimulate osteoclast-mediated calcium efflux.

Compensatory increase in calcium extrusion activity of untreated lymphocytes from swine susceptible to malignant hyperthermia

SUMMARY We tested the hypothesis that lymphocytes from swine with susceptibility to malignant hyperthermia (mh) had calcium extrusion activity higher than unaffected swine. Cytoplasmic concentration of ionized calcium was determined by use of dual emission spectrofluorometry and measurement of the ratio of free to calcium-bound form of the fluorescent calcium dye indo-1. Net calcium accumulation and unidirectional calcium extrusion rate were dependent on intracellular calcium concentration. Calcium extrusion from calcium-loaded lymphocytes was monitored while calcium influx was inhibited by suspending the cells in calcium-free medium with a calcium chelator. Net calcium accumulation of untreated lymphocytes was monitored in calcium-replete medium. A novel method of calculation of ionized calcium was used. This method confirmed our previous findings of lower ionized calcium concentration (86 ± 40 and 370 ± 216 nmol/L; P < 0.01) and slower rates of calcium accumulation 39 ± 16 and 127 ± 52 nmol/L/min) in untreated lymphocytes from mh-susceptible swine compared with controls. These changes were attributable to calcium extrusion activity two- to three-fold higher in lymphocytes of mh-susceptible swine (154 ± 36 and 408 ± 47 nmol/L/min at 175 nmol/L; 972 ± 111 and 1,690 ± 505 nmol/L/min at 425 nmol/L). These data were compatible with our model of higher calcium extrusion activity being a compensatory adaptation of mh-susceptible swine lymphocytes to their hypersensitivity to stimuli that increase cytoplasmic calcium concentration.

Net calcium influx into rested rat cardiomyocytes

Net calcium influx into rested rat cardiomyocytes

Accumulation of calcium in substantia nigra lesions induced by status epilepticus. A microprobe analysis

Status epilepticus of sufficient duration (>30min) causes a unique lesion of substantia nigra pars reticulata (SNPR), and of globus pallidus (GP). This lesion, which encompasses a pan-necrotic destruction of neurons and glial cells seems to develop during ongoing seizures. We decided to investigate if the lesion is accompanied by net calcium accumulation. Seizures of 20 and 60 min duration were induced by the administration of flurothyl, and the tissue was frozen in situ either at the end of the seizure periods, or after recovery periods of 15 or 120 min. The total calcium and potassium contents of caudoputamen, neocortex, GP and SNPR were measured using particle induced X-ray emission (PIXE) in the microprobe mode. Seizures of 20 min duration did not cause net accumulation of calcium. When the duration of seizures was extended to 60 min the results varied depending on the location. In caudoputamen, which does not incur neuronal damage, no calcium accumulation was observed. In GP and SNPR, such a rise was unequivocally demonstrated, with calcium content increasing to about 150% of controls. The increase in calcium correlated to a decrease in potassium content. It is concluded that epileptic cell death occurs pari passu with accumulation of calcium although it cannot be stated that this accumulation is the cause of the cell death. It is speculated that seizures increase the permeability of the blood-brain barrier to calcium, and that enhanced blood to tissue transfer increases the calcium load of metabolically strained cells.

Mechanism of aluminum-induced calcium efflux from cultured neonatal mouse calvariae

Aluminum has been shown to increase unidirectional 45Ca efflux from prelabeled bones in vitro; whether aluminum affects net calcium efflux and, if so, by what mechanism has not been studied. To examine the effects of aluminum on net calcium flux from bone we cultured live and dead neonatal mouse calvariae with and without graded concentrations of aluminum (10(-8) to 10(-5) M). Aluminum induced a dose-dependent net calcium efflux from live bone after 24 h, but not 3 h, which was similar in magnitude to that produced by 10(-8) M parathyroid hormone. The normal calcium influx into dead bone was not altered by aluminum. Release of beta-glucuronidase, a lysosomal enzyme released by osteoclasts, increased after a 24-h incubation in aluminum-containing medium and was correlated with net calcium efflux. Calcitonin, an inhibitor of osteoclastic bone mineral dissolution, abolished the increase in beta-glucuronidase release and nullified the aluminum-induced net calcium efflux. Thus aluminum induces cell-mediated net calcium efflux from bone and increases beta-glucuronidase release. Calcitonin inhibits the increase in both calcium efflux and beta-glucuronidase release, suggesting that aluminum stimulates osteoclasts to release bone mineral.

Changes in the mitochondrial calcium influx and efflux properties are responsible for the decline in sperm calcium during epididymal maturation

This study was undertaken to determine the role of calcium ion, a key regulator of the intensity and form of motility in mature demembranated sperm, in the development of motility during passage through the bovine epididymis. Cellular calcium levels in bovine caput and cauda epididymal spermatozoa were measured with three different techniques. 45Ca2+ uptake measurements revealed that net calcium uptake and Ca2(+)-Ca2+ exchange in caput spermatozoa were about 2 to 3 times higher than in caudal spermatozoa. Intracellular free calcium determination with the calcium fluorophore Fura 2 showed that the levels were 6 times higher in caput spermatozoa. The values for caput and caudal sperm were 875 +/- 55 nM (n = 15) and 155 +/- 6 nM (n = 24), respectively. Total cellular calcium levels quantitated by atomic absorption were 626 +/- 30 (n = 48) and 304 +/- 19 (n = 46) ng/10(8) sperm in caput and caudal epididymal sperm, respectively. At least one of the reasons for the high calcium content of caput epididymal sperm is the result of a higher rate and extent of mitochondrial calcium accumulation in caput compared to caudal sperm. Mitochondrial calcium uptake rates measured in digitonin permeabilized cells revealed uptake rates 2- to 3-fold higher in caput compared to caudal sperm. However, mitochondrial calcium efflux rates were identical in caput and caudal epididymal sperm. The efflux rates in both cell types were unaffected by external sodium levels but were found to be proportional to pH. Alkalinization or acidification of internal pH of intact sperm resulted in a corresponding lowering or elevation of cytoplasmic free calcium levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Conditioned medium from ras oncogene-transformed nih 3T3 cells induces bone resorption in vitro

Tumor-associated hypercalcemia is due, in part, to enhanced osteoclastic bone resorption induced by soluble factors elaborated from malignant cells. ras transformation of NIH 3T3 cells results in a 50-fold induction of cathepsin L mRNA and secretion of the corresponding protein. Since cathepsin L is an acid proteinase we asked whether conditioned medium from these cells would directly increase calcium release from bone in vitro. We tested conditioned medium obtained after 72 h culture of NIH 3T3 ras-transformed cells (DT) or nontransformed NIH 3T3 cells (3T3) and identical medium not exposed to cells (Ctl). Incubation of either live or dead neonatal mouse calvaria for 48 h in DT-conditioned medium increased calcium release compared to bones incubated with 3T3 medium. In both states the increased calcium release with DT medium was blocked by 0.25 mM E-64, a general cysteine proteinase inhibitor, and 1 microM Z-Phe-Ala-CH2F, a specific inhibitor of cathepsin L activity. Thus, conditioned medium from ras-transformed cells enhances calcium release in both live and dead bone. Since cathepsin L is the major protein secreted by these cells and the effect of DT-conditioned medium is blocked by a specific inhibitor of cathepsin L, these studies suggest that this acid proteinase acts directly on bone mineral to enhance net calcium release.

Recent advances in calcium transport by the kidney.

In the normal adult human, net intestinal calcium absorption and renal calcium excretion are similar in steady-state conditions. 7 Approximately 15 mg/kg (1,000 mg) of calcium are ingested in food each day; 80% of ingested calcium or approximately 11 mg/kg appears in the feces (Fig 1). The fraction of calcium absorbed by the intestine is approximately 30 % to 40 % of amount present in the lumen. Some calcium (the endogenous fecal calcium) is excreted into the intestine via intestinal and pancreatic secretions. Thus, the net calcium absorbed is the difference between the total amount actually absorbed and the amount excreted via endogenous fecal calcium. The calcium absorbed (4 mg/kg) enters the plasma and extracellular fluid (ECF) space. It equilibrates between the ECF and labile and stable bone pools and, to a smaller extent, between ECF and intracellular calcium pools. In a state of calcium balance, the amounts of calcium entering via the intestinal absorption route and the amounts excreted via the kidney are the same. The amount of calcium filtered by the glomeruli is approximately 10 g/d. Because only a fraction of this (1% to 3 %) appears in the urine, it follows that a considerable amount of calcium is reabsorbed in the tubules. Calcium balance is maintained through the action of parathyroid hormone and 1,25-dihydroxyvitamin D3 (1,25-[OH],D,) acting on target tissues such as the intestine, bone and kidney.8,9 The interactions of parathyroid hormone and the vitamin D endocrine system in response to changes in serum calcium concentrations are shown in Fig 2. This review does not attempt to exhaustively cover all aspects of calcium handling by the kidney but will discuss some recent findings concerning the nature of calcium pumps (Ca2+-pump) and exchangers and their distribution in different parts of the nephron. For more exhaustive reviews of the renal handling of calcium by different parts of the kidney, the reader is referred to several excellent recent reviews.‘O-14