Unidirectional Calcium Influx Research Articles

Calcium transport across isolated gill epithelium of Carcinus

AbstractPreparations of posterior gills isolated from postmoult shore crabs (Carcinus mediterraneus) that had been acclimated to dilute seawater were used for calcium‐transport studies. Gills were perfused and bathed with saline of identical composition, with 45Ca2+ ions added exclusively to the bathing solution or the perfusate for measurement of unidirectional influx or efflux, respectively. The unidirectional influx of calcium (5.57 ± 0.95 μmol h−1 per g gill fresh weight) significantly exceeded the efflux (3.15 ± 1.02 μmol h−1 per g gill fresh weight), indicating a net transgill influx of calcium.Calcium influxes were inhibited when La3+ ions were added apically (IC50 = 7 × 10−6 mol 1−1) or basolaterally (IC50 = 1.6 × 10−6 mol 1−1). In addition, basolaterally applied La3+ ions inhibited effluxes with an IC50 of 3.0 × 10−7 mol 1−1. Amiloride had no significant effect on the unidirectional fluxes of calcium. Influxes of calcium gave a sigmoidal curve when plotted against the external concentration of calcium in the bath. The Hill coefficient (2.66 ± 0.20) suggested that there are at least two or three Ca2+ carrier systems that are active during the influxes of calcium. Moreover, apically added La3+ ions produced a concentration‐dependent inhibition of calcium influx, which is discussed in terms of inhibition of apically located calcium channels. By contrast, the inhibitory action of La3+ ions at the basolateral gill surfaces is discussed in terms of an effect on ATP‐dependent calcium transport and the Na+/Ca2+ exchanger. © 1994 Wiley‐Liss, Inc.

Involvement of Distinct G-Proteins in the Action of Vasopressin on Rat Glomerulosa Cells

We have previously shown that vasopressin (VP) induces breakdown of membrane phosphoinositides in adrenal glomerulosa cells. In the present study we demonstrate that the accumulation of inositol phosphates (IP) measured in the presence of arginine vasopressin (AVP) is reduced if the cells are incubated in a calcium-free medium. This effect cannot be accounted for by modification of VP binding, reduction of inositol lipid labeling, or stimulation of inositol, 1,4,5,-triphosphate 5-monophosphatase. It mainly affects phospholipase-C activity, since this enzyme is highly sensitive to calcium. Ionomycine and nifedipine, which, respectively, increase and decrease the intracellular calcium concentration, also, respectively, stimulate and inhibit IP accumulation. In membranes prepared from pertussis toxin (IAP)-treated cells, AVP stimulates inositol monophosphate accumulation to the same extent as in membranes derived from untreated cells. However, in intact cells, IAP decreases the inositol monophosphate accumulation. This decrease probably involves calcium influx, since we show that AVP stimulates a unidirectional calcium influx, which is completely blocked by IAP treatment. In rat adrenal glomerulosa cells, the AVP-stimulated secretion of aldosterone is mainly under the control of calcium, since a full inhibition of its secretion is observed under conditions in which the calcium influxes are completely suppressed despite a sustained accumulation of IP (calcium depletion or IAP treatment). Together, these results signify that VP acts on rat glomerulosa cells by two distinct mechanisms: calcium influx, which is IAP sensitive, and phosphoinositide turnover, which is IAP insensitive.

Dopaminergic reduction of intracellular calcium: The role of calcium influx

The effects of dopamine (DA) on 45Ca2+ ion movement and prolactin release in dispersed female rat anterior pituitary cells were studied to elucidate the mechanism for DA reduction of intracellular calcium levels. In 45Ca2+ prelabeled cells, DA inhibited fractional calcium efflux and prolactin release simultaneously and continuously in a concentration-dependent manner (IC50 20 nM DA). We then studied unidirectional calcium influx and observed haloperidol-reversible, concentration-dependent DA suppression of calcium influx into unlabeled cells. These data complement and extend reported fluorescent dye studies and suggest that dopamine primarily inhibits calcium influx, thereby reducing intracellular calcium levels, which leads to suppression of prolactin release and is manifest secondarily as a reduction in fractional 45Ca2+ efflux.

Dopamine inhibits calcium flux in the 7315a prolactin-secreting pituitary tumour

Cells of the 7315a prolactin-secreting tumour express biochemically normal cell-surface receptors for dopamine. However, dopamine inhibits prolactin release from these cells only when the basal rate of prolactin release is augmented by increasing the intracellular and/or extracellular calcium concentration of the tumour cells. This suggests that dopaminergic modulation of calcium ion flux could have a central physiological role in these neoplastic cells. In 7315a cells we examined the ability of dopamine to regulate 45Ca 2+ influx and fractional 45Ca 2+ efflux under conditions of enhanced calcium flux using the calcium channel activator, maitotoxin. It was observed that unidirectional calcium influx stimulated by maitotoxin was significantly inhibited by dopamine. Maitotoxin stimulated fractional efflux and prolactin release from the tumour cells and dopamine simultaneously inhibited both processes by a haloperidol-reversible mechanism. Therefore, in 7315a cells dopamine receptor activation is coupled to inhibition of calcium flux as at least one component in the regulation of prolactin release. These cells may provide further opportunity to study intracellular signalling mechanisms that are modulated by dopamine receptor activity.

Cytoskeletal control of calcium absorption across the rat small intestine

1. 1. The effect of colchicine, cytochalasin-B and procaine on calcium transport across the rat small intestine was investigated. The results obtained show the following: 2. 2. Colchicine and cytochalasin-B at different concentrations inhibited significantly ( P < 0.001) calcium accumulation in rat intestinal cells, whereas procaine at different concentrations increased significantly ( P < 0.001) calcium accumulation in the rat small intestine. 3. 3. Unidirectional influx of calcium across the rat small intestine was significantly inhibited ( P <0.01) in the presence of colchicine and cytochalasin-B in the preincubation medium. Procaine, on the other hand, caused a significant increase ( P < 0.01) in the unidirectional influx of calcium across the rat intestinal cells. 4. 4. The cell water content was not altered in the presence of the different drugs indicating that the changes in calcium transport across the rat intestinal cells are not due to alterations in the structure of the cell membrane.

IgE receptor-activated calcium permeability pathway in rat basophilic leukemia cells: measurement of the unidirectional influx of calcium using quin2-buffered cells

The intracellular calcium indicator and buffer quin2 has been used to generate a large calcium buffering capacity in the cytoplasm of rat basophilic leukemia cells. Above 3 mM intracellular quin2, there is no further increase in the initial rate of antigen-induced 45Ca uptake, suggesting that 45Ca buffering by quin2 is now sufficient to prevent the immediate efflux of 45Ca from the cells. Thus, the initial rate of 45Ca uptake should reflect the true unidirectional influx of calcium that occurs when immunoglobulin E (IgE) receptors are aggregated by antigen. The antigen-induced calcium permeability pathway appears to be saturable, with a Km of about 0.7 mM and a Vmax of 0.9 nmol of calcium (10(6) cells)-1 min-1. Although net 45Ca uptake reaches a plateau a few minutes after antigen stimulation, the increase in plasma membrane permeability is maintained for at least an hour, provided that receptors for IgE remain aggregated. The initial rate of 45Ca influx correlates well with the subsequent secretion of [3H]serotonin in response to different concentrations of antigen. Both 45Ca uptake and [3H]serotonin secretion are maximal when only 10% of the receptors are occupied with antigen-specific IgE. Thus, 45Ca influx correlates more closely with secretion than with the number of IgE receptors aggregated by antigen.

Cysteamine-induced duodenal ulcer: Effects on calcium absorption across rat duodenum

1. 1. The effect of cysteamine-induced duodenal ulcers on calcium transport across rat duodenum was investigated. 2. 2. Intracellular calcium accumulation measured after 24 hr and 3 days of cysteamine injection showed a significant increase ( P < 0.001) in the duodenal strips isolated after 3 days with no change noticed in those isolated after 24 hr, although the morphological changes in both were very similar. 3. 3. The relationship between increasing calcium concentration in the incubation medium and intracellular calcium concentration is a saturable process that conforms to the Michaelis-Menten type of kinetics. 4. 4. The average maximal flux ( V max) increased from 8.93 nmole/hr-gdw in normal to 12.5 nmole/hr-gdw in 3-day-ulcerated rats, with no apparent change in the Michaelis constant ( K t) (0.8 mM). 5. 5. Unidirectional influx of calcium across the mucosal membrane was significantly increased ( P < 0.001) in 3-day-ulcerated duodenum suggesting that the increase in calcium transport could be due to the activation of the active step at the mucosal border.

The kinetics of calcium influx into rat liver slices

1. 1. The unidirectional influx of calcium across rat liver slices is a carrier-mediated process which displays saturation kinetics. 2. 2. The presence of Mg 2+ in the incubation medium competitively inhibits calcium influx into rat liver slices. 3. 3. Metabolic inhibitors such as ouabain and 2,4-dinitrophenol at a concentration of 1 × 10 −4 and 2.5 × 10 −4 M respectively, inhibited significantly ( P < 0.001) calcium influx across the liver slices. 4. 4. Calcium influx is dependent on the presence of sodium in the extracellular medium, and is significantly reduced ( P < 0.01) when sodium concentration in the preincubation solution is reduced to zero.

Asymmetric calcium influx and efflux at maternal and fetal sides of the guinea-pig placenta: kinetics and specificity.

Unidirectional influx of calcium across maternal and fetal sides of the syncytiotrophoblast was investigated in the guinea-pig placenta by using a rapid (less than 30 s) paired-tracer dilution technique. Experiments were performed in an in situ placenta artificially perfused through the umbilical vessels or in an isolated placenta in which both the maternal and fetal circulations were perfused. At equimolar Ca2+ concentrations, unidirectional calcium influx was always significantly lower on the maternal side than on the fetal side. Saturation kinetics were observed: on the fetal side the estimated Km was 1.8 +/- 0.7 mM and Vmax was 1.66 +/- 0.28 mumol/min X g (mean +/- S.E. of mean) and on the maternal side Km ranged from 0.18 to 1.15 mM and Vmax ranged from 0.12 to 0.59 mumol/min X g. When the inhibition of calcium influx was investigated on the fetal side of the trophoblast by using competing cations, the following sequence was observed: Ba2+ greater than Ca2+ congruent to Ni2+ greater than Sr2+ greater than Mg2+ congruent to Li+. Efflux of 45Ca2+ from the trophoblast into the ipsilateral circulation (backflux) was rapid (20-100% in 6 min) and asymmetric since the fetal:maternal ratio was 1.35 +/- 0.11 (mean +/- S.E. of mean) in the presence of 0.1 mM-Ca2+. In the dually perfused placenta, transplacental transfer (6 min) of 45Ca2+ varied over a wide range (0-80%); however, it was similar to that of the extracellular reference tracer, 22Na+, in either maternal-to-fetal or fetal-to-maternal directions. It is suggested that this is a consequence of the 'leakiness' of the dually perfused placenta since the transplacental transfer of 22Na+ and D-[3H]mannitol (or L-[14C]glucose) measured simultaneously were also variable but similar. Transplacental transfer of 45Ca2+ could not be used to characterize specific calcium-transport mechanisms, whereas highly sensitive trophoblast uptake measurements were provided by the single-circulation, paired-tracer technique. Our findings suggest the presence of a specific carrier-mediated transport system for calcium on both maternal and fetal surfaces of the trophoblast. The asymmetries in unidirectional influx into the trophoblast and rapid backflux indicate a mechanism by which the net transfer of calcium from the maternal to the fetal circulation is maintained in favour of the fetus.

Undirectional calcium and nucleotide fluxes in cardiac sarcoplasmic reticulum. II. Experimental results

Unidirectional calcium influx and efflux were evaluated in cardiac sarcoplasmic reticulum (SR) by 45Ca-40Ca exchange at steady state calcium uptake in the absence of calcium precipitating anions. Calcium efflux was partitioned into a pump-mediated efflux and a parallel passive efflux by separately measuring passive efflux referable to the steady state. Unidirectional and net ATP-ADP fluxes were measured using [3H]-ATP----ADP and [3H]-ADP----ATP exchanges. Methods are presented that take into account changing specific activities and sizes of the nucleotide pools during the measurement of nucleotide fluxes. The contribution of competent and incompetent vesicles to the unidirectional and net nucleotide fluxes was evaluated from the specific activity of these fluxes in incompetent vesicles and from the fraction of vesicles that were incompetent. The results indicate that, in cardiac SR, unidirectional calcium fluxes are larger than the unidirectional nucleotide fluxes contributed by competent vesicles. Because the net ATPase rate of competent vesicles is similar to the parallel passive efflux, it appears that cardiac SR Ca-ATPase tightly couples ATP hydrolysis to calcium transport even at static head, with a coupling ratio near 1.0.

The effect of local anesthetics on calcium transport across the rat and turtle small intestine

1. 1. Procaine at different concentrations enhanced significantly ( P < 0.01) calcium accumulation in rat intestinal cells, whereas the same concentrations of procaine inhibited significantly ( P < 0.01) calcium uptake by the turtle small intestine. 2. 2. Unidirectional calcium influx across the rat small intestine was significantly enhanced ( P < 0.001) by the presence of procaine in the preincubation medium. However, procaine had no effect on calcium influx across the turtle intestinal cells. 3. 3. The cell water content and the cell volume were not altered by preincubating the intestinal tissues with procaine in both animals.

A simple technique for the measurement of unidirectional calcium influx at the mucosal surface of organ-cultured embryonic chick duodenum

Unidirectional calcium influx can be measured by application of a lilter paper blotter saturated with a 45Ca-labeled buffer solution to the mucosal surfaces of slitopen embryonic chick duodena which had been cultured mucosal side up on stainless-steel grids. The results obtained with this simple technique support the contention of a direct involvement of a vitamin D-induced calcium-binding protein in the transport of calcium across the mucosal surface. Permeability and inulin space measurements validate the method for measurement of calcium influx at initial velocity and obviate concern for “unstirred layer” effects. Kinetic analyses indicate a two step influx process, one saturable at about 5 m m calcium and the other essentially unsaturable.