Net Ca Research Articles

Interaction between Aluminum Toxicity and Calcium Uptake at the Root Apex in Near-Isogenic Lines of Wheat (Triticum aestivum L.) Differing in Aluminum Tolerance.

Aluminum (Al) is toxic to plants at pH < 5.0 and can begin to inhibit root growth within 3 h in solution experiments. The mechanism by which this occurs is unclear. Disruption of calcium (Ca) uptake by Al has long been considered a possible cause of toxicity, and recent work with wheat (Triticum aestivum L. Thell) has demonstrated that Ca uptake at the root apex in an Al-sensitive cultivar (Scout 66) was inhibited more than in a tolerant cultivar (Atlas 66) (J.W. Huang, J.E. Shaff, D.L. Grunes, L.V. Kochian [1992] Plant Physiol 98: 230-237). We investigated this interaction further in wheat by measuring root growth and Ca uptake in three separate pairs of near-isogenic lines within which plants exhibit differential sensitivity to Al. The vibrating calcium-selective microelectrode technique was used to estimate net Ca uptake at the root apex of 6-d-old seedlings. Following the addition of 20 or 50 [mu]M AlCl3, exchange of Ca for Al in the root apoplasm caused a net Ca efflux from the root for up to 10 min. After 40 min of exposure to 50 [mu]M Al, cell wall exchange had ceased, and Ca uptake in the Al-sensitive plants of the near-isogenic lines was inhibited, whereas in the tolerant plants it was either unaffected or stimulated. This provides a general correlation between the inhibition of growth by Al and the reduction in Ca influx and adds some support to the hypothesis that a Ca/Al interaction may be involved in the primary mechanism of Al toxicity in roots. In some treatments, however, Al was able to inhibit root growth significantly without affecting net Ca influx. This suggests that the correlation between inhibition of Ca uptake and the reduction in root growth may not be a mechanistic association. The inhibition of Ca uptake by Al is discussed, and we speculate about possible mechanisms of tolerance.

Inhibition of IP3 and IP3-dependent Ca2+ mobilization by cyclic nucleotides in isolated gastric muscle cells

The mechanisms by which cAMP and cGMP and agents that stimulate one (isoproterenol and nitroprusside) or both cyclic nucleotides (VIP) decrease cytosolic free Ca2+ ([Ca2+]i) and inhibit contraction were examined in dispersed, intact, and saponin-permeabilized gastric muscle cells. In these cells, the [Ca2+]i transient responsible for initial contraction is mediated by inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release (K. N. Bitar, P. G. Bradford, J. W. Putney, Jr., and G. M. Makhlouf, Science Wash. DC 232: 1143-1145, 1986, and J. Biol. Chem. 261: 16591-16596, 1986). In intact muscle cells, dibutyryl cAMP and all three relaxant agents inhibited contraction, [Ca2+]i, and net Ca2+ efflux (i.e., Ca2+ release) in a concentration-dependent fashion. In permeabilized muscle cells, cAMP, cGMP, and all three relaxant agents 1) inhibited cholecystokinin (CCK)-induced IP3 production (maximal 38-48%), 2) inhibited CCK- and IP3-induced Ca2+ efflux (maximal 55-59%) and contraction (maximal 59-66%), and 3) stimulated Ca2+ uptake (maximal 25-30%), in a concentration-dependent fashion. cAMP and cGMP were equipotent inhibitors of IP3 production and of CCK- and IP3-induced Ca2+ efflux and contraction, whereas cGMP was distinctly more potent as a stimulant of Ca2+ uptake. For all functions, maximal effects induced by cAMP and cGMP were similar to those induced by the three relaxant agents. Inhibition of Ca2+ release was the main determinant of inhibition of contraction; stimulation of Ca2+ uptake was relatively minor (< 5% of Ca2+ efflux). Decrease in IP3 production did not contribute to inhibition of Ca2+ efflux and contraction since inhibition of IP3-induced Ca2+ efflux was similar to inhibition of CCK-induced IP3-dependent Ca2+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for vitamin D-independent active calcium absorption in newborn piglets.

The role of 1,25-dihydroxycholecalciferol (calcitriol) for intestinal calcium (Ca2+) absorption was studied in newborn (< 1 week old) and weaned piglets (> 6 weeks old). In both groups, normal piglets and piglets suffering from inherited pseudo vitamin D-deficiency rickets, type I (PVDRI) were used. In this inherited disorder, renal production of calcitriol is absent. Plasma samples were assayed for calcitriol and total Ca, and dissociation constants (Kd) and maximum binding capacities (Bmax) of intestinal calcitriol receptors were determined under equilibrium conditions at 4 degrees C. Unidirectional Ca(2+)-flux rates were measured across stripped duodenal mucosae in Ussing chambers in the absence of electrochemical gradients. The plasma calcitriol concentrations of neonatal (26.5 +/- 7.1 pg/ml, n = 11; mean +/- SEM) and weaned PVDRI piglets (18.8 +/- 5.7 pg/ml, n = 8) were unphysiologically low and differed significantly from control animals (83.6 +/- 14.8 pg/ml, n = 8, and 86.9 +/- 9.6 pg/ml, n = 11, respectively). However, newborn PVDRI piglets had normal plasma Ca levels at least during the first days of life. They became hypocalcemic and developed clinical symptoms of rickets during the following weeks. In newborn PVDRI and control piglets, Bmax was significantly lower (84 +/- 28 fmol/mg protein and 127 +/- 55 fmol/mg protein, n = 9, respectively) than in weaned piglets (741 +/- 82 fmol/mg protein, n = 9, and 778 +/- 121 fmol/mg protein, n = 8, respectively). Significant net Ca(2+)-fluxes were found in both newborn PVDRI and control piglets (88.8 +/- 25.1 nmol.cm-2 x h-1, n = 6, and 86.5 +/- 10.5 nmol.cm-2 x h-1, n = 9, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium Buffering and Free Ca2+ in Rat Brain Synaptosomes

The effects of K+ depolarization and of stimulation by veratridine on apparent cytosolic free Ca2+ ([Ca2+]cyt) and net Ca2+ accumulation were measured in isolated rat brain presynaptic nerve terminals (synaptosomes). [Ca2+]cyt was determined with fura-2, and Ca2+ accumulation was measured with tracer 45Ca. [Ca2+]cyt was approximately 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K+]o was increased from the normal 5 mM to 30 or 50 mM, 45Ca uptake and [Ca2+]cyt both increased within 1 s. Both increases were directly related to [Ca2+]o for [Ca2+]o = 0.02-1.2 mM; however, the increase in 45Ca uptake greatly exceeded the increase in [Ca2+]cyt. With small Ca2+ loads (< or = 100 mumol/L of cell water, equivalent to the Ca2+ entry during a train of < or = 60 impulses), the 45Ca uptake exceeded the increase in [Ca2+]cyt by a factor of nearly 1,000. This indicates that approximately 99.9% of the entering Ca2+ was buffered and/or sequestered within approximately 1 s. With larger Ca2+ loads, a larger fraction of the entering Ca2+ was buffered; approximately 99.97% of the load was buffered with loads of 250-425 mumol/L of cell water. The ratio between the total Ca2+ entry and the increase in [Ca2+]cyt, the "calcium buffer ratio," beta, was therefore approximately 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca2+]cyt, which ranged between approximately 7,300:1 and 12,800:1. When the synaptosomes were activated with 10 microM veratridine ([Ca2+]o = 0.2-0.6 mM), 45Ca influx and [Ca2+]cyt increased progressively for approximately 10 s (beta = 2,700:1-3,050:1) and then leveled off.(ABSTRACT TRUNCATED AT 250 WORDS)

Compensatory downregulation of myocardial Ca channel in SR from dogs with heart failure

In this study we tested the hypothesis that the ryanodine-binding Ca-release channel activity and density of the sarcoplasmic reticulum (SR) terminal cisternae were decreased in congestive heart failure (CHF) that occurs spontaneously in doberman pinschers or experimentally with rapid ventricular pacing of mongrels. We used a novel, sensitive, and easy-to-perform microassay and demonstrated a 50% decrease in activity of the myocardial SR Ca pump and a 75% reduction in SR Ca-release channel activity in CHF. Decreases in Ca channel content were associated with increases in net Ca sequestration. 45Ca-release experiments from passively loaded SR terminal cisternae and ryanodine-binding studies confirmed a 53-68% downregulation of the Ca-release channel activity. As a consequence of release channel downregulation, there was partial restoration of net Ca sequestration activity in dogs with CHF and complete compensation in dogs with mild cardiac dysfunction. Deterioration of Ca cycling correlated with deterioration of myocardial performance, apparently due to decreased Ca-adenosinetriphosphatase (ATPase) pump and not Ca channel content. One-half the reduction in Ca-release activity could be attributed to decreased Ca sequestration and one-half to decreased Ca channel density. Downregulation of Ca channel content decreases the amplitude of the Ca cycle and maximizes the downregulation of Ca pumps that may occur. Although these adaptations may reduce cellular energy expenditure, they are likely to render the myocardium more susceptible to fatigue and failure.

Effects of (S)-nafenodone on 45Ca 2+ fluxes and contractions in rat isolated vascular smooth muscle

The effects of (S)-nafenodone, a new antidepressant, were studied on contraction and 45Ca 2+ fluxes in rat vascular smooth muscle. In isolated rat aorta (S)-nafenodone, 10 −7 − 10 −4 M, inhibited the contractions induced by 80 mM K KCl(IC 50 = 1.4 ± 0.4 × 10 −5 M) and 10 −5 M noradrenaline (IC 50 = 1.2 ± 0.2 × 10 −5 M). (S)-Nafenodone relaxed the contractions induced by both high K + and noradrenaline, this effect being independent of the presence of functional endothelium. It also inhibited the contractions induced by addition of CaCl 2 to Ca 2+-free high-K + solution ( IC 50 = 2.5 ± 0.9 × 10 −6 M) and the phasic contractions induced by noradrenaline in Ca 2+-free medium, but was a very weak relaxant of the contractions induced by phorbol 12-myristate-13-acetate in Ca 2+-free medium. In addition, (S)-nafenodone inhibited the spontaneous mechanical activity in portal vein segments (IC 50 = 1.4 ± 0.8 × 10 −6 M). (S)-Nafenodone inhibited the 45Ca 2+ uptake stimulated by high KCl or noradrenaline without altering 45Ca 2+ uptake in resting strips and decreased the net 45Ca 2+ content in aortic strips non-stimulated as well as stimulated by noradrenaline. In conclusion, (S)-nafenodone inhibited voltage- and agonist-stimulated Ca 2+ entry in isolated rat aortas. In addition, it decreased Ca 2+ content in both resting and noradrenaline-stimulated muscles, suggesting that it may deplete noradrenaline-sensitive intracellular Ca 2+ stores. As a consequence, (S)-nafenodone would reduce the concentration of intracellular free Ca 2+ available at the contractile apparatus for vascular smooth muscle contraction.

Role of protein kinase A in the regulation of cytosolic free calcium in human osteoblast-like SaOS-2 cells.

We have used wild-type and adenosine 3',5'-cyclic monophosphate (cAMP)-resistant mutant osteoblast-like SaOS-2 cells to investigate the role of protein kinase A (PKA) in the regulation of cytosolic free Ca2+ concentration ([Ca2+]i). Basal levels of [Ca2+]i were the same in wild-type (127 +/- 6.1 nM) and transfected (117 +/- 6.8 nM) SaOS-2 cells, although 45Ca2+ efflux was slower in the transfected cells. In wild-type cells, thapsigargin (TG, > or = 200 nM), an inhibitor of the Ca(2+)-ATPase activity of the endoplasmic reticulum, acutely increased [Ca2+]i (by up to 2-fold), which then returned promptly to basal [Ca2+]i. In cAMP-resistant cells, TG elicited a significantly greater acute rise in [Ca2+]i, which then decayed to an elevated plateau level. In mutant cells, high concentrations of dibutyryladenosine 3',5'-cyclic monophosphate, which overcome the PKA blockade, restored the changes in [Ca2+]i to the wild-type pattern. In cAMP-resistant, TG-blocked cells, ionomycin (or alpha-thrombin) induced a further elevation in [Ca2+]i, which then declined rapidly to the original basal level. We conclude that basal PKA activity is involved actively in regulation of [Ca2+]i in SaOS-2 cells by promoting Ca2+ efflux from the cell and, possibly, by inhibiting Ca2+ release from or stimulating net Ca2+ sequestration into the ER. We have also obtained evidence for an alternate Ca(2+)-triggered Ca2+ reuptake mechanism in SaOS-2 cells that is not dependent on either Ca(2+)-ATPase or PKA.

Mechanisms of calcium transport across the basolateral membrane of the rabbit cortical thick ascending limb of Henle's loop.

Although net Ca2+ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca2+ entry step across the luminal membrane is mediated by Ca2+ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca2+ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca2+ ([Ca2+]i) with fura-2. The resting [Ca2+]i in this segment was 49.8 +/- 4.5 nmol/l. Neither Na+ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca2+]i, indicating that a Na+/Ca2+ exchanger in the basolateral membrane may not contribute in any major way to [Ca2+]i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na+/Ca2+ exchanger has been reported. In this segment, Na+ removal from the bath increased cell Ca2+ from 148.6 +/- 6.4 nmol/l to 647.6 +/- 132.0 nmol/l, confirming the documented fact. [Ca2+]i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca2+]i. When the bath pH was made alkaline, [Ca2+]i was also increased. This response was abolished when Ca2+ was eliminated from the bath, indicating that the Ca2+ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca2+]i induced by an alkaline bath was inhibited by increased the bath K+ from 5 nmol/l to 50 mmol/l, suggesting that the Ca2+ entry system is voltage-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitonin acutely increases net 45Ca uptake and alters alkaline phosphatase specific activity in human osteosarcoma cells

Altnough the primary skeletal action of exogenous calcitonin is to inhibit bone resorption, calcitonin also has effects on bone formation. In-vitro data indicate that the latter may include direct effects on bone cells of osteoblastic lineage. In the current studies, we examined the effects of calcitonin on cyclic adenosine monophosphate (cAMP) and PGE 2 synthesis and 45Ca uptake in human osteosarcoma cells, specifically, TE-85 cells and subpopulations of SaOS-2 cells with low-, intermediate-, and high-steady-state levels of skeletal alkaline phosphatase (ALP) activity. Since previous in-vivo studies had shown that calcitonin could acutely decrease skeletal ALP activity in rat periosteal osteoblasts, we also measured the effects of calcitonin treatment on ALP specific activity. Neither salmon nor human calcitonin altered the net synthesis of cAMP or PGE 2 by SaOS-2 cells, but human calcitonin gene-related peptide increased both ( P < .001 and P < .005, respectively). Both salmon and human calcitonin had short-term effects to alter ALP activity in TE-85 and SaOS-2 cells. The effects were different in SaOS-2 subpopulations with different pretreatment ALP levels. Four hours of exposure to salmon calcitonin had dose-dependent, biphasic effects on ALP levels in SaOS-2 cells with intermediate pretreatment ALP levels, increasing ALP at doses between 0.16 and 1.6 nmol/L ( P < .005) and decreasing ALP at higher concentrations ( P < .05). Both salmon and human calcitonin, but not human calcitonin gene-related peptide, also had short-term effects to increase net 45Ca uptake by SaOS-2 cells; these effects were dose-dependent and long-lasting. Salmon calcitonin (8 pmol/L) increased net 45Ca uptake within 10 minutes (242% of control, P < .001), and the effect was still apparent after 4 hours of continuous exposure (151% of control, P < .01); similar effects were seen with TE-8t cells. The effect of calcitonin to increase net 45Ca uptake by SaOS-2 cells could be obeserved in (Fitton-Jackson-modified) Biggers-Gwatkin-Jones culture medium (BGJ b) containing 1.5 or 2.0 mmol/L Ca, but not in medium containing 0.5 or 1.0 mmol/L Ca, or in the presence of 10 μmol/L verapamil.

Ca2+ cycling between sarcoplasmic reticulum and mitochondria in rabbit cardiac myocytes.

1. Shortening and intracellular Ca2+ (Ca2+i) transients were measured in isolated rabbit ventricular myocytes during paired contractures induced by rapid application of 10 mM caffeine. 2. Caffeine-induced contractures relax despite maintained presence of caffeine. In control solution, a second phasic caffeine contracture failed to appear, unless the sarcoplasmic reticulum (SR) was refilled by a series of electrically stimulated twitches during the interval between caffeine exposures. 3. The relaxation of caffeine-induced contractures in 0 Na(+)-0 Ca2+ solution has previously been shown to rely on mitochondrial Ca2+ uptake and sarcolemmal Ca2(+)-ATPase. Thus, a second caffeine contracture (T2) while still in 0 Na(+)-0 Ca2+ was greatly reduced compared to the first one (T1). However, the amplitude of T2 increased exponentially with the time interval, attaining a maximum of approximately 50% of T1 for an interval of 180-300 s, with a time constant (tau) of 41.2 s. Similar results were found for Ca2+i transients (tau = 45 s). 4. Inhibition of the mitochondrial Ca2+ uptake by the oxidative phosphorylation uncoupler, FCCP during T1 dramatically depressed T2. On the other hand, inhibition of the sarcolemmal Ca2(+)-ATPase (by increasing extracellular Ca2+ concentration, [Ca2+]o) resulted in increase of T2. Spermine inclusion during T1 also increased T2, possibly by an increase of mitochondrial Ca2+ uptake. 5. We conclude that Ca2+ taken up by mitochondria during the decline of T1 moves back to the SR after caffeine is removed, with a tau approximately 40 s. 6. Partial intracellular Na+ depletion by prolonged (3 min) perfusion with 0 Na(+)-0 Ca2+ solution before T1 (a) accelerated relaxation and [Ca2+]i decline during T1, and (b) slowed, but did not abolish, the recovery of T2 as the interval was increased. This effect was particularly pronounced when choline was used instead of Li+ as the Na+ substitute. 7. We further conclude that the mitochondrial Na(+)-Ca2+ antiporter influences the rate of net Ca2+ uptake by mitochondria and is also important in Ca2+ efflux from mitochondria during rest.

Calcium mass transfer in CAPD: the role of convective transport

One hundred and fifty calcium (Ca) balance studies were performed in 50 patients treated with CAPD using dialysate with a 1.75 mmol/l (7 mg/dl) Ca content, in order to calculate the peritoneal balance of Ca by measuring the Ca in all the effluent for a 24-h period, and looking at the influence of serum ionized Ca and the ultrafiltration rate in the calcium balance. Of the 150 balance studies, 77 were made using four exchanges of dialysate per day and 73 using three exchanges per day. The serum ionized Ca was 1.17 +/- 0.09 mmol/l, the ultrafiltration 844 +/- 723 ml/day and the peritoneal Ca transfer 39 +/- 46 m/day. The net Ca abortion with four exchanges was less than that with three exchanges per day. There was a strong negative correlation between the peritoneal Ca absorption and the ultrafiltration (r = -0.7, P < 0.00001) and with the ionized Ca (r = -0.49, P < 0.0001). Thirty-three peritoneal balance studies showed a negative Ca balance and in all 33 cases ultrafiltration was greater than 350 ml/day. We conclude that the peritoneal balance of Ca depends not only on the serum ionized Ca, but also on ultrafiltration. The lesser Ca gain observed with four dialysis exchanges per day is due to greater ultrafiltration rates present in this setting.

Hypercalciuria and urinary saturation measurements in climacteric women

Fifteen percent (20/130) of a group of climacteric women on diets of their choice had urinary calcium (Ca) levels exceeding 4 mg/kg per day. Most of these hypercalciuric subjects had a daily Ca intake of 0.4–0.5 g. Their bone turnover rates were raised and high Ca absorption was observed in 4 cases. Serum Ca and total protein and glomerular filtration rates were normal in all the hypercalciuric patients. The calcium/creatinine (Ca/Cr) ratio (mg 1 −1/mg 1 −1, fasting, 09:00–10:00 h) was measured in 72 climacteric women, 35 of whom (49%) had ratios > 0.1. The latter defines a relative hypercalciuria as compared with premenopausal Ca excretion levels. Only 5 of the 35 subjects had calciuria levels above 4 mg/kg per day. The Ca/Cr ratio cannot replace daily urinary Ca measurements for the screening of subjects in whom calciuria may exceed net Ca absorption. Urinary saturation measurements were carried out in 70 women. Supersaturation was observed only in the case of Ca oxalate (CaOx) among several calcium salts usually found in urinary stones. CaOx supersaturation was observed in 95% of the hypercalciuric subjects and in 48% of the rest of the women investigated. The relatively high frequency of CaOx supersaturation can be attributed in part to the decreased excretion of citrate associated with ovarian failure. Oestrogen replacement therapy increased citrate excretion and lowered the level of CaOx supersaturation. Ca supplementation (1 g Ca/day) reduced the degree of supersaturation as a result of the concurrent reduction in oxalate excretion.

The role of dicarboxylic anion transport in the slower Ca2+ uptake in fetal cardiac sarcoplasmic reticulum.

Sarcoplasmic reticulum- (SR-)mediated Ca2+ transport is slower in the fetal heart compared with the adult. Virtually all previous studies of cardiac SR Ca2+ transport were performed in the presence of oxalate, a dicarboxylic anion that is cotransported with Ca2+ in skeletal muscle SR. If anion transport is developmentally regulated in cardiac SR, this could explain, in part, the previously reported results. The purposes of this study were to establish the presence of an SR dicarboxylic anion transport process in the rabbit heart and to determine if the perinatal changes in SR Ca2+ transport occur in a dicarboxylic anion-dependent and/or independent manner. In isolated fetal and adult rabbit cardiac SR membranes, we measured Ca2+ ATPase rates and 45Ca2+ uptake in the presence of the dicarboxylic anions maleate and succinate compared with the zwitterionic buffer PIPES, to which cardiac SR is essentially impermeable. We also measured 14C-succinate uptake by fetal and adult SR membranes. Anion-independent Ca2+ ATPase activity and net 45Ca2+ uptake were significantly lower in the fetal SR membranes than in the adult. Maleate and succinate increased the Ca2+ ATPase rates in the fetal and adult SR, but the effect was significantly greater in the adult. Maleate and succinate stimulated earlier attainment of maximal net Ca2+ uptake in the fetal and adult SR, suggesting that these dicarboxylic anions stimulated the rate of Ca2+ accumulation. Maleate and succinate significantly increased the maximal net Ca2+ uptake in the adult SR, but not in the fetus. The percentage of stimulation of Ca2+ uptake by maleate and succinate was similar in the fetal and adult SR.(ABSTRACT TRUNCATED AT 250 WORDS)

The enhancement of Ca 2+ efflux from sarcoplasmic reticulum vesicles by urea

Urea, in nondenaturing concentrations, inhibited Ca 2+ uptake by sarcoplasmic reticulum vesicles with no concomitant effect on ATP hydrolysis. This inhibition was antagonized by 5 m m oxalate and 20 m m orthophosphate. At concentrations of 0.2 to 1.0 m, urea induced an increase in the Ca 2+ efflux from preloaded vesicles diluted in a medium at pH 7.0 containing 2 m m ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid, 0.1 m m orthophosphate, and 0.1 m m MgCl 2. The urea-induced efflux was arrested by ligands of the (Ca 2+-Mg 2+) ATPase, namely, K +, Mg 2+, Ca 2+, and ADP, and by ruthenium red and the polyamines spermine, spermidine, and putrescine. In the case of polyamines a dissociation between the effect on the efflux and the net Ca 2+ uptake was observed, as only the efflux could be blocked by the drugs. Glycine betaine, trimethylamine- N-oxide, and sucrose antagonized the effects of urea on both the net Ca 2+ uptake and the rate of Ca 2+ efflux.

Recombinant Interleukin-8 Induces Changes in Cytosolic Ca2+ in Human Neutrophils

Activation of polymorphonuclear leukocytes (PMNL) by most soluble stimulants is associated with a marked increase in cytosolic free Ca2+ ([Ca2+]i). Interleukin-8 (IL-8), a monocyte-derived neutrophil chemotactic factor and potent neutrophil-activating cytokine, effectively enhanced the resting free [Ca2+]i within human PMNL in a dose-dependent manner (maximal effect with 100 ng/mL). The increase in [Ca2+]i was substantially (55%) inhibited in the absence of extracellular Ca2+. Thus, the increase was due to extra- and intracellular cooperative mobilization of Ca2+, as supported by the reduced effect of IL-8 on [Ca2+]i after quenching with Mn2+. Granulocyte-macrophage colony-stimulating factor and interferon-gamma failed to induce a change in [Ca2+]i, suggesting that they may operate through different signal pathways. Pretreatment with Bordetella pertussis toxin largely inhibited the IL-8-induced change in [Ca2+]i. Thus, IL-8-induced cooperative mobilization of intra- and extracellular Ca2+ leads to a net Ca2+ influx into the cytoplasm through a process mediated by a guanosine triphosphate-binding protein.

Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex

The effects of aluminum on the concentration-dependent kinetics of Ca(2+) uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-tolerant Atlas 66 and Al-sensitive Scout 66. Seedlings were grown in 100 μM CaCl2 solution (pH 4.5) for 3 d. Subsequently, net Ca(2+) fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating Ca(2+)-selective microelectrode. The kinetics of Ca(2+) uptake into cells of the root apex, for external Ca(2+) concentrations from 20 to 300 μM, were found to be quite similar for both cultivars in the absence of external Al; Ca(2+) transport could be described by Michaelis-Menten kinetics. When roots were exposed to solutions containing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 μM total Al), a strong correlation was observed between Al toxicity and Al-induced inhibition of Ca(2+) absorption by root apices. For Scout 66, exposure to Al immediately and dramatically inhibited Ca(2+) uptake over the entire Ca(2+) concentration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca(2+) uptake by Al. For example, exposure of Scout 66 roots to increasing Al levels (from 0 to 10 μM) caused the K m for Ca(2+) uptake to increase with each rise in Al concentration, from approx. 100 μM in the absence of Al to approx. 300 μM in the presence of 10 μM Al, while having no effect on the V max. The same Al exposures had little effect on the kinetics of Ca(2+) uptake into roots of Atlas 66. The results of this study indicate that Al disruption of Ca(2+) transport at the root apex may play an important role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca(2+)-transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution.

Mechanisms for the positive inotropic effect of alpha 1-adrenoceptor stimulation in rat cardiac myocytes.

alpha 1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+ sensitivity and action potential prolongation, which can increase net Ca2+ entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2-0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+ entry and differentiate between the two mechanisms. At 22-23 degrees C the specific alpha 1-adrenoceptor agonist methoxamine (100 microM) prolonged action potential duration at 50% repolarization from 55 +/- 2 to 81 +/- 5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3 +/- 0.6 to 7.8 +/- 1 microns (n = 18). Reduction of the transient outward current and other K+ currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52 +/- 6 to 98 +/- 12 msec and increased unloaded cell shortening from 2.9 +/- 0.4 to 6.6 +/- 0.6 microns (n = 4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70-500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3 +/- 0.7 to 4.97 +/- 0.8 microns at pulse potentials positive to -40 mV. Similar alpha 1-adrenoceptor responses were observed at 35 degrees C during action potential and voltage-clamp experiments, which could be blocked by 10 microM prazosin. In myocytes loaded with the Ca2+ indicator indo-1, alpha 1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+ transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+ transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to alpha 1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+ current reduction. In addition, modulation of K+ channels could occur independent of changes in contractility and/or [Ca2+]i.

Rapid calcium exchange for protons and potassium in cell walls of Chara

ABSTRACTNet fluxes of Ca2+, H+ and K+ were measured from intact Chara australis cells and from isolated cell walls, using ion‐selective microelectrodes. In both systems, a stimulation in Ca2+ efflux (up to 100 nmol m−2 s−1, from an influx of ∼40 nmol m−2 s−1) was detected as the H+ or K+ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K+ 0.2 to 10mol m−3, respectively). A Ca2+ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca2+ efflux (pH 5.2) is similar to a reported pH threshold for acid‐induced wall extensibility in a closely related characean species. Application of NH4+ to intact cells caused prolonged H+ efflux and also transient Ca2+ efflux. We attribute all these net Ca2+ fluxes to exchange in the wall with H+ or K+. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca2+ in the cell wall and the effect of Ca2+ exchanges on the measured fluxes of other ions, including bathing medium acidification by H+ efflux, are discussed.

Potential-dependent inward currents in single isolated smooth muscle cells of the rat ileum.

1. Calcium (ICa) and sodium (INa) currents were studied in single smooth muscle cells freshly isolated from both the newborn (1-3 days old) and adult rat ileum, using the patch-clamp technique (whole-cell configuration). 2. Under conditions when INa was blocked, two components of ICa, low-voltage activated or ICa,low and high-voltage activated or ICa,high, were observed in the newborn rat ileal cells. ICa,high and ICa,low have differing voltage ranges of activation and steady-state inactivation and time courses of recovery from inactivation. Potential dependence of ICa,low was much steeper and shifted toward negative membrane potential than that for ICa,high (slope factors and the potential of half-maximal inactivation were 13.6 and -60.6 and 8.8 and -49 mV for ICa,low and ICa,high, correspondingly). 3. Nifedipine at the high concentration of 30 microM exerted no effect on ICa,low and only slightly suppressed ICa,high, decreasing its peak to 0.81 +/- 0.04 (n = 7) at the holding potential of -80 mV and to 0.66 +/- 0.05 (n = 3) at -50 mV. ICa,high was suppressed significantly by Cd2+ ions, while ICa,low was more sensitive to Ni2+ ions. 4. Results presented here suggest that the properties of high-voltage-activated (HVA) Ca2+ channels in the rat small intestine are quite different to those described for L-type Ca2+ channels found in other smooth muscles. It is proposed that HVA Ca2+ channels are similar to N-type Ca2+ channels. 5. Comparison of Ca2+ currents in newborn and adult rat ileal cells showed that the contribution of ICa,low to the net Ca2+ current was negligible in adults, whereas the properties of HVA Ca2+ channels were similar in the neonatal and adult animals. 6. INa, studied in nominally Ca(2+)-free physiological salt solution, activated in the voltage range between -50 and -40 mV and reached its peak at -10 mV. INa was blocked in a dose-dependent manner by TTX with an apparent dissociation constant of 4.5 nM. 7. INa decay was monoexponential in the voltage range studied and its time constant decreased monotonically with membrane depolarization from 4.7 +/- 0.2 ms (n = 6) at -30 mV to 0.51 +/- 0.03 ms (n = 7) at 20 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Halothane and isoflurane effects on Ca2+ fluxes of isolated myocardial sarcoplasmic reticulum.

To elucidate better the differential myocardial depressant actions of halogenated volatile anesthetics, anesthetic-induced changes in Ca2+ accumulation, release, and Ca-ATPase (Ca2+ pump) activity of isolated canine cardiac sarcoplasmic reticulum (SR) vesicles were examined. An initial crude microsomal fraction of homogenized canine ventricle was subfractionated on a discontinuous sucrose gradient after Ca2+ loading in the presence of phosphate. Junctional SR (JSR) enriched with terminal cisternae was identified by its content of an electrophoretically verified approximately 450-kDa protein, the Ca(2+)-release channel (CaRC). When the CaRC of JSR was blocked by 1 microM ruthenium red (RR), the rate of Ca2+ uptake increased 47% as measured spectrophotometrically using the Ca-sensitive dye antipyrylazo III. A second fraction was identified as primarily longitudinal SR (LSR) based on its trace content of 450-kDa protein and 11% increase of Ca2+ uptake with RR. Halothane (0.75-2.5%) or isoflurane (2.5-4%) decreased net Ca2+ accumulation rate by either LSR or JSR, and the decrease in uptake rate of JSR was only partially reversed by addition of 1 microM RR (27% increase for isoflurane, 7% increase for halothane). Both halothane and isoflurane increased JSR ATP consumption as measured by a coupled-enzyme assay. 45Ca2+ efflux from passively loaded SR vesicles was then determined to verify that the decreased net uptake rate was due to enhanced Ca2+ efflux from vesicles. Both anesthetics increased passive Ca2+ efflux from SR vesicles in which the CaRC was blocked by 10 microM RR as well as those in which Ca2+ release via the CaRC was activated by 10 microM Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)