Cytosolic free magnesium in cardiac myocytes: identification of a Mg2+ influx pathway

Regulation of intracellular Mg2+ activity in the heart is not well characterized. Cardiac myocytes were prepared as primary cultures from 7 day old chick embryo hearts and intracellular Mg2+ concentration [( Mg2+]i) was determined in single ventricular cells with mag-fura-2. Basal [Mg2+]i was 0.48 +/- 0.03 mM in normal culture medium. There was no correlation of basal [Mg2+]i with cellular contraction or intracellular [Ca2+]i (determined with fura-2). Cardiocytes cultured (16 hr) in low Mg (0.16 mM) media contained 0.21 +/- 0.05 mM Mg2+ which returned to normal levels when placed in Mg media with a refill time of 20 min. Basal [Ca2+]i (121 +/- 11 nM) and stimulated [Ca2+]i (231 +/- 41 nM) was similar to control cells. Verapamil, 25 microM, reversibly blocked Mg2+ refill. In conclusion, the basal [Mg2+]i of isolated cardiomyocytes is considerably below the Mg2+ electrochemical equilibrium allowing passive Mg2+ influx. The influx pathway for Mg2+ is inhibited by verapamil and appears to be independent of Ca2+ as assessed by fura-2.     

Basal intracellular free Mg2+ concentration in smooth muscle cells of guinea pig tenia cecum: intracellular calibration of the fluorescent indicator furaptra

Longitudinal muscle strips dissected from tenia cecum of guinea pig were loaded with the Mg2+ indicator, furaptra, and the relation between the fluorescent ratio signal (R) and cytoplasmic free Mg2+ concentration ([Mg2+]i) was studied in smooth muscle cells at 25 degrees C. After the application of ionophores (4-bromo-A23187, monensin, and nigericin), a small immediate offset of R (deltaRjump) was followed by a slow change in R (deltaRslow), which reached a steady level within 2-5 h. The deltaRjump was independent of Mg2+ concentration in solution ([Mg2+]o), and was thought to be unrelated to the change in [Mg2+]i. The direction of the deltaRslow depended on [Mg2+]o with a reversal at approximately 1 mM [Mg2+]o. The intracellular calibration curve was constructed from the steady levels of deltaRslow, and the dissociation constant was 5.4 mM. With the intracellular calibration curve and correction for the deltaRjump, basal [Mg2+], was estimated to be 0.98 +/- 0.05 mM (mean +/- SE, n = 12). When the same calibration was applied to A7r5 cells and rat ventricular myocytes, estimates of basal [Mg2+]i of these cells were 0.74 +/- 0.02 mM (n = 33) and 1.13 +/- 0.06 mM (n = 9), respectively. These results suggest that the basal [Mg2+] level is approximately 1 mM at least in some types of smooth muscle cells, as generally found in striated muscles.     

Thyroid status and beta-agonistic effects on cytosolic calcium concentrations in single rat cardiac myocytes activated by electrical stimulation or high-K+ depolarization.

The effects of the thyroid status on the cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were studied at rest and during contraction. The mean resting [Ca2+]i increased significantly from the hypothyroid (45 +/- 4 nM) through the euthyroid (69 +/- 12 nM) to the hyperthyroid condition (80 +/- 11 nM) at extracellular Ca2+ concentrations ([Ca2+]o) up to 2.5 mM. At [Ca2+]o above 2.5 mM the differences in [Ca2+]i between the groups became less. The amplitude of the Ca2+ transients became higher in all groups with increasing [Ca2+]o (1, 2.5 and 5 mM), and was highest at all [Ca2+]o in hyperthyroid myocytes. The beta-agonist isoprenaline elevated peak [Ca2+]i during contraction and increased the rate of the decay of the Ca2+ transients to a greater extent in hypothyroid myocytes than in hyperthyroid myocytes. Depolarization with high [K+]o induced a large but transient [Ca2+]i overshoot in hypothyroid myocytes, but not in hyperthyroid myocytes, before a new elevated steady-state [Ca2+]i was reached, which was not different between the groups. When isoprenaline was added to K+ o-depolarized myocytes after a steady state was reached, a significantly larger extra increase in [Ca2+]i was measured in the hypothyroid group (28%) compared with the hyperthyroid group (8%). It is concluded that in cardiac tissue exposed to increasing amounts of thyroid hormones (1) [Ca2+]i increases at rest and during contraction in cardiomyocytes and (2) interventions which favour Ca2+ entry into the cytosol [( Ca2+]o elevation, high [K+]o, beta-agonists) tend to have less impact on Ca2+ homoeostasis.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin

Single cell [Ca2+], studies were performed in chicken and rat osteoclasts loaded with fura-2 and exposed to a variety of treatments. Under resting conditions, basal [Ca2+]i, was 79.2 +/- 47.3 and 84.3 +/- 65.7 nM (averages +/- S.D.; n = 141 and 126) in the osteoclasts of the two species, respectively. Basal [Ca2+]i was stable in all rat and in approximately 80% of chicken osteoclasts. In the remaining 20%, spontaneous, irregular [Ca2+], fluctuations were observed (amplitude range: 50-200 nm over basal values). Increase of [Ca2+]o over the concentration of the Krebs-Ringer incubation medium (2 mM) induced rises of [Ca2+] in almost all cells investigated. [Ca2+] rises were already appreciable with 0.5 mM [Ca2+]o additions and reached high values with 4 mM additions: 390 +/- 113 and 364 +/- 214 nM [Ca2+], in rat and chicken osteoclasts, respectively (n = 122 and 101). Qualitatively, the responses to [Ca2+]o additions consisted of discrete [Ca2+]i transients, biphasic (an initial spike followed by a plateau), or monophasic (either the spike or the plateau). In a few chicken osteoclasts, the [Ca2+]i increase occurring after [Ca2+]o addition consisted of multiple, irregular fluctuations, similar to those observed in 20% of these cells under resting conditions. In individual osteoclasts subsequently exposed to multiple [Ca2+]o increase pulses, the type of the [Ca2+]i transient (mono- or biphasic) was maintained, and the size was dependent on the magnitude of the [Ca2+]o additions. Effects similar to those of [Ca2+]o were induced by the addition of Cd2+ or Ba2+ (but not La3+ or Mg2+) into the medium. The Cd2+ effect was maintained in part even in a Ca2+-free medium. Of various hormones and factors, parathormone, 1,25-dihydroxyvitamin D3, and prostaglandin E2 were inactive. In contrast, calcitonin was active in rat osteoclasts (which express numerous receptors). [Ca2+]i increases were small (19 +/- 17.9 nM; n = 21) when the hormone was administered alone; they were synergistic (severalfold potentiation) when the hormone was administered before or after [Ca2+]o. The [Ca2+]i effects of calcitonin were mimicked by 8Br-cAMP (31 +/- 26 nM; n = 12) when the nucleotide was administered alone; marked synergism when it was administered in combination with [Ca2+]o. This paper demonstrates for the first time that changes of [Ca2+]i are induced in osteoclasts by treatments with [Ca2+]o and calcitonin and can therefore be involved in intracellular mediation of the physiological effects of these two extracellular signals.     

Characterization of oscillations in cytosolic free Ca2+ concentration and measurement of cytosolic Na+ concentration changes evoked by angiotensin II and vasopressin in individual rat aortic smooth muscle cells. Use of microfluorometry and digital imaging

Dual wavelength microfluorometry was used to characterize the changes in cytosolic free Ca2+ concentration [( Ca2+]i) in individual cultured rat aortic vascular smooth muscle cells (VSMC). Angiotensin II (ANG II) at 10(-8) M induced a transient rise in [Ca2+]i from 43 +/- 2 to 245 +/- 23 nM, lasting for approximately 60 s (n = 42). In half of the population, discrete oscillations in [Ca2+]i of smaller amplitude occurred after the initial [Ca2+]i peak, with a period of 58 +/- 8 s and a maximum height of 132 +/- 24 nM. A similar oscillatory pattern was observed with arginine vasopressin (AVP). The oscillations depended upon the presence of extracellular Ca2+. Cytosolic free Na+ concentration ([Na+]i) in VSMC was also measured using the fluorescent Na+ probe sodium-binding benzofuran isophthalate. ANG II induced a gradual and sustained elevation of [Na+]i, from 24.0 +/- 6.2 to 36 +/- 9.7 mM. In response to AVP, [Na+]i rose to 41.0 +/- 11.6 mM. Video imaging of individual VSMC, with on-line ratio calibration of [Ca2+]i, revealed an inhomogeneous distribution of Ca2+ within the cell. [Ca2+] in the nucleus was invariably lower than in the cytoplasm in resting cells. In the cytoplasm, there were small regions in which [Ca2+] was elevated, or "hot spots." In Ca(2+)-containing medium, the initial rise in [Ca2+]i triggered by ANG II and AVP appeared to emanate from the hot spots and to spread evenly throughout the cytoplasm. Between [Ca2+]i oscillations, Ca2+ retreated back to the original hot spots. This study demonstrates the cellular and subcellular heterogeneity of [Ca2+]i both in resting VSMC and during stimulation by ANG II and AVP and reports the direct measurement of [Na+]i in VSMC. The results suggest an action of Ca2+ in both the initial and sustained phases of the response in VSMC and a link between changes in [Ca2+]i and [Na+]i.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Adenosine decreases intracellular free calcium concentrations in cultured vascular smooth muscle cells from rat aorta

Using an intracellularly trapped dye, quin 2, effects of adenosine on intracellular free calcium concentrations ([Ca2+]i) were recorded, microfluorometrically, using rat aortic medial vascular smooth muscle cells (VSMCs) in primary culture. Regardless of whether cells were at rest (in 5 mM K+), at K+-depolarization (in 55 mM K+) or at Ca2+ depletion (in Ca2+-free media), adenosine induced a rapid reduction of [Ca2+]i, following which there was a gradual increase to pre-exposure levels, in cells at rest and in the case of Ca2+ depletion. Only when the cells were depolarized (55 mM K+) did adenosine induce a new steady [Ca2+]i level, lower than the pre-exposure value. These findings indicate that decrease in [Ca2+]i by adenosine is one possible mechanism involved in the adenosine-mediated vasodilatation, and that adenosine decreases [Ca2+]i by direct extrusion, by sequestration, or by inhibiting the influx of Ca2+ into VSMCs.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

8-Bromoguanosine 3':5'-cyclic monophosphate decreases intracellular free calcium concentrations in cultured vascular smooth muscle cells from rat aorta

The effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br cGMP) on intracellular free calcium concentrations ([Ca2+]i) in cultured rat aortic vascular smooth muscle cells (VSMCs) loaded with fura-2 were recorded microfluorometrically. Irrespective of whether VSMCs were at rest (in 5 mM K+ PSS), under Ca2+ depletion (in Ca2+-free medium for 10 min) and K+ depolarization (in high K+ PSS), [Ca2+]i was actively reduced and reached a new and lower steady-state level with the application of 8-Br cGMP. This may be the first and direct evidence that cGMP, a putative mediator of various vasodilators, actively reduces [Ca2+]i in VSMCs.     

Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells

An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.     

LY294002, but not wortmannin,increases intracellular calcium and inhibits calcium transients in bovine and human airway smooth muscle cells

To characterize the effect that a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002, has on cytosolic calcium concentrations ([Ca2+]i), bovine airway smooth muscle cells (BASMC) and cultured human bronchial smooth muscle cells (HBSMC) were loaded with fura 2-AM, imaged as single cells and [Ca2+]i measured ratiometrically. LY294002 (50 microM) increased [Ca2+]i by 294+/-76 nM (P<0.01, n=13) and 230+/-31 nM (P<0.001, n=10) in BASMC and HBSMC, respectively, and increases occurred in the absence of extracellular calcium. In contrast, after pre-treatment with thapsigargin, LY294002 no longer increased [Ca2+]i. This calcium mobilization by LY294002 was associated with a significant functional effect since LY294002 also inhibited calcium transients to carbachol (45+/-23 nM), caffeine (45+/-32 nM), and histamine (20+/-22 nM), with controls of 969+/-190, 946+/-156, and 490+/-28 nM, respectively. Wortmannin, a different PI3-kinase inhibitor, neither increased [Ca2+]i nor inhibited transients. Also, LY294002 increased [Ca2+]i in the presence of wortmannin, U-73122, and xestospongin C. We concluded that LY294002 increased [Ca2+]i, at least in part, by mobilizing intracellular calcium stores and inhibited calcium transients. The effects of LY294002 on [Ca2+]i were not dependent on wortmannin-sensitive PI3-kinases, phospholipase C, or inositol trisphosphate receptors (IP3R). For BASMC and HBSMC, LY294002 has effects on calcium regulation that could be important to recognize when studying PI3-kinases.     

Cyclic GMP stimulates Na+/Ca2+ exchange in vascular smooth muscle cells in primary culture.

We examined the effect of cGMP on Na+/Ca2+ exchange in rat aortic smooth muscle cells (VSMCs) in primary culture. The intracellular Ca2+ concentration [( Ca2+]i) was raised by adding ionomycin to VSMCs incubated at high extracellular pH (pH0) (pH0 = 8.8) and high extracellular Mg2+ (Mg2+0) (Mg2+0 = 20 mM), conditions that inhibit activity of the sarcolemmal Ca2+ pump. 45Ca2+ efflux observed under these conditions was mostly extracellular Na+ (Na+0)-dependent and thus presumably catalyzed by the Na+/Ca2+ exchanger. Brief treatment of VSMCs with 8-bromo-cGMP or atrial natriuretic peptide increased this Na+0-dependent 45Ca2+ efflux by about 50%. The 8-bromo-cGMP treatment did not significantly influence total cell Na+, membrane potential, and cell pH. Conversely, when VSMCs were loaded with Na+ and then exposed to a Na+0-free medium, the rate of 45Ca2+ uptake into VSMCs increased as cell Na+ increased. Prior treatment of VSMCs with 8-bromo-cGMP accelerated 45Ca2+ uptake by up to 60% without influencing Na+ loading itself. Treatment of VSMCs with 25 microM 2,5-di-(tert-butyl)-1,4-benzohydroquinone, an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, induced a transient elevation of [Ca2+]i. 8-Bromo-cGMP stimulated the rate of recovery phase of this Ca2+ transient measured in the high pHo/high Mg2+o medium. All these results indicate that cGMP stimulates Na+/Ca2+ exchange in VSMCs.     

Increased [Mg2+]o reduces Ca2+ influx and disruption of mitochondrial membrane potential during reoxygenation

Increase in extracellular Mg2+ concentration ([Mg2+]o) reduces Ca2+ accumulation during reoxygenation of hypoxic cardiomyocytes and exerts protective effects. The aims of the present study were to investigate the effect of increased [Mg(2+)](o) on Ca2+ influx and efflux, free cytosolic Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i), Ca2+ accumulation in the presence of inhibitors of mitochondrial or sarcoplasmatic reticulum Ca2+ transport, and finally mitochondrial membrane potential (Delta(psi)m). Isolated adult rat cardiomyocytes were exposed to 1 h of hypoxia and subsequent reoxygenation. Cell Ca2+ was determined by 45Ca2+ uptake, and the levels of [Mg2+]i and [Ca2+]i were determined by flow cytometry as the fluorescence of magnesium green and fluo 3, respectively. Ca2+ influx rate was significantly reduced by approximately 40%, whereas Ca2+ efflux was not affected by increased [Mg2+]o (5 mM) during reoxygenation. [Ca2+]i and [Mg2+]i were increased at the end of hypoxia, fell after reoxygenation, and were unaffected by increased [Mg2+]o. Clonazepam, a selective mitochondrial Na+/Ca2+ exchange inhibitor (100 microM), significantly reduced Ca2+ accumulation by 70% and in combination with increased [Mg2+]o by 90%. Increased [Mg2+]o, clonazepam, and the combination of both attenuated the hypoxia-reoxygenation-induced reduction in Delta(psi)m, determined with the cationic dye JC-1 by flow cytometry. A significant inverse correlation was observed between Delta(psi)m and cell Ca2+ in reoxygenated cells treated with increased [Mg2+]o and clonazepam. In conclusion, increased [Mg2+]o (5 mM) inhibits Ca2+ accumulation by reducing Ca2+ influx and preserves Delta(psi)m without affecting [Ca2+]i and [Mg2+]i during reoxygenation. Preservation of mitochondria may be an important effect whereby increased [Mg2+]o protects the postischemic heart.     

Stimulus-secretion coupling in bovine parathyroid cells. Dissociation between secretion and net changes in cytosolic Ca2+

The relationship between the concentration of cytosolic free Ca2+ ([Ca2+]i) and secretion of parathyroid hormone (PTH) was investigated in isolated bovine parathyroid cells using the fluorescent Ca2+ indicator, quin 2. Increasing the concentration of extracellular Ca2+ from 0.5 to 2.0 mM caused a 3-fold increase in [Ca2+]i (from 183 +/- 4 to 568 +/- 21 nM) which was associated with a 2-4-fold decrease in secretion of PTH. Decreasing extracellular Ca2+ to about 1 microM caused a corresponding fall in [Ca2+]i to 60-90 nM. Extracellular Ca2+-induced changes in [Ca2+]i were not affected by omission of extracellular Na+. Depolarizing concentrations of K+ (30 mM) depressed [Ca2+]i at all concentrations of extracellular Ca examined, and this was associated with increased secretion of PTH. Ionomycin (0.1 or 1 microM) increased [Ca2+]i at extracellular Ca2+ concentrations of 0.5, 1.0, and 2.0 mM, but inhibited secretion of PTH only at Ca concentrations near the "Ca2+ set point" (1.25 microM). In contrast, dopamine, norepinephrine (10 microM each), and Li+ (20 mM) potentiated secretion of PTH without causing any detectable change in [Ca2+]i. The results obtained with these latter secretagogues provide evidence for a mechanism of secretion which is independent of net changes in [Ca2+]i. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) did not alter [Ca2+]i or secretion of PTH at low (0.5 mM) extracellular Ca2+ concentrations. At 2.0 mM extracellular Ca2+, however, TPA (20 nM or 1 microM) depressed [Ca2+]i and potentiated secretion of PTH. The addition of TPA prior to raising the extracellular Ca2+ concentration reduced the subsequent increase in [Ca2+]i. The results show that the effects of TPA on secretion in the parathyroid cell are not readily dissociated from changes in [Ca2+]i and suggest that some TPA-sensitive process, perhaps involving protein kinase C, may be involved in those mechanisms that regulate [Ca2+]i in response to changes in extracellular Ca2+.     

Ryanodine Inhibits Caffeine-Evoked Ca2+ Mobilization and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

The effects of ryanodine, a selective inhibitor of the Ca(2+)-induced Ca2+ release mechanism, on caffeine-evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5-40 mM) caused a concentration-dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg(2+)-free medium containing 0.2 mM EGTA. Ryanodine (5 x 10(-5) M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 x 10(-7) - 5 x 10(-5) M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2+/Mg(2+)-free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin-permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.     

Intracellular Ca2+ requirement for activation of the Na+/H+ exchanger in vascular smooth muscle cells

The role for intracellular Ca2+ in modulating activity of the Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. Na+/H+ exchange was activated by four distinct stimuli: 1) phorbol 12-myristate 13-acetate, 2) thrombin, 3) cell shrinkage, and 4) intracellular acid loading. [Ca2+]i was independently varied between 40 and 200 nM by varying the bathing Ca2+ from 10 nM to 5.0 mM. Thrombin-induced intracellular Ca2+ transients were blocked with bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). In the absence of stimulators of Na+/H+ exchange, varying [Ca2+]i above or below the basal level of 140 nM did not activate Na+/H+ exchange spontaneously. However, varying [Ca2+]i did affect stimulus-induced activation of Na+/H+ exchange. Activation of the exchanger by phorbol 12-myristate 13-acetate was blunted by reduced intracellular Ca2+ (half-maximal activity at 50-90 nM [Ca2+]i), consistent with a Ca2+ requirement for protein kinase C (Ca2+/phospholipid-dependent enzyme). Activation of the exchanger by thrombin in protein kinase C-depleted cells was also sensitive to reduced intracellular Ca2+ (half-maximal activity at 90-140 nM [Ca2+]i) and was increased 40% by raising [Ca2+]i to 200 nM. Activation of the exchanger by cell shrinkage or intracellular acid loads was not significantly affected over the range of [Ca2+]i tested. Thus, altered [Ca2+]i does not itself affect Na+/H+ exchange activity in vascular smooth muscle but instead modulates activation of the transporter by particular stimuli.     

NMR measurement of cytosolic free calcium, free magnesium, and intracellular sodium in the aorta of the normal and spontaneously hypertensive rat

We have utilized multinuclear NMR spectroscopy to examine the relationship between cytosolic free Ca2+ ([Ca2+]in), free Mg2+ ([Mg2+]in) and intracellular Na+ ([Na+]in) levels of the intact thoracic aorta and primary hypertension using the Wistar-Kyoto and Sprague-Dawley rats as controls and the spontaneously hypertensive rat as a model for genetic hypertension. Cytosolic free [Ca2+] was measured using 19F NMR of the intracellular Ca2+ indicator 5,5'-difluoro-1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, free [Mg2+] using the 31P resonances of intracellular ATP, and intracellular [Na+] by 23Na NMR in combination with the extracellular shift reagent dysprosium tripolyphosphate. We have found that both the [Na+]in and [Ca2+]in levels were significantly increased in the hypertensive animals relative to normotensive controls (p less than 0.01). Mean systolic blood pressures (using tail cuff method) of control and hypertensive rats were 123 +/- 8 mm Hg (mean +/- 2 S.E., n = 7) and 159 +/- 6 mm Hg (mean +/- 2 S.E., n = 7), respectively. [Na+]in and [Ca2+]in were 21.9 +/- 6.4 mM (mean +/- 2 S.E., n = 7) and 277 +/- 28 nM (mean +/- 2 S.E., n = 5) for the spontaneously hypertensive rats versus 10.1 +/- 1.8 mM (mean +/- 2 S.E., n = 7) and 151 +/- 26 nM (mean +/- 2 S.E., n = 5) for control rats, respectively. A slight difference observed between intracellular free Mg2+ levels in hypertensives (180 +/- 38 microM, mean +/- 2 S.E., n = 4) and controls (246 +/- 76 microM, mean +/- 2 S.E., n = 4) was not statistically significant (p greater than 0.1). These data indicate alterations in the cell membrane ion transport function of the aortic smooth muscle in primary hypertension.     

Platelet activating factor raises intracellular calcium ion concentration in macrophages

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.     

Sphingolipids regulate [Mg2+]o uptake and [Mg2+]i content in vascular smooth muscle cells: potential mechanisms and importance to membrane transport of Mg2+

Sphingolipids have a variety of important signaling roles in mammalian cells. We tested the hypothesis that certain sphingolipids and neutral sphingomyelinase (N-SMase) can regulate intracellular free magnesium ions ([Mg2+]i) in vascular smooth muscle (VSM) cells. Herein, we show that several sphingolipids, including C2-ceramide, C8-ceramide, C16-ceramide, and sphingosine, as well as N-SMase, have potent and direct effects on content and mobilization of [Mg2+]i in primary cultured rat aortic smooth muscle cells. All of these sphingolipid molecules increase, rapidly, [Mg2+]i in these vascular cells in a concentration-dependent manner. The increments of [Mg2+]i, induced by these agents, are derived from influx of extracellular Mg2+ and are extracellular Ca2+ concentration-dependent. Phospholipase C and Ca2+/calmodulin/Ca2+-ATPase activity appear to be important in the sphingolipid-induced rises of [Mg2+]i. Activation of certain PKC isozymes may also be required for sphingolipid-induced rises in [Mg2+]i. These novel results suggest that sphingolipids may be homeostatic regulators of extracellular Mg2+ concentration influx (and transport) and [Mg2+]i content in vascular muscle cells.