K-channel blocking drugs induce histamine release and 45Ca uptake in isolated mast cells.

Histamine secretion and 45Ca uptake processes were studied in mast cells treated with four K+ channel blocking drugs in physiological saline and in media containing different ionic concentrations. Quinine, 4-aminopyridine and sparteine were effective as histamine-releasing agents when mast cells were incubated in physiologic saline solution. The dose-response profile obtained was in the range of 0.1-0.5 mM for quinine, 1-10 for 4-aminopyridine and 0.5-5 mM for sparteine and did not show significant differences between purified and unpurified mast cells. By contrast, tetraethylammonium (1-100 mM) did not induce histamine release. The presence of high K+ or Rb+ concentrations in the medium (Tris-K+ or Tris-Rb+, both at 150 mM) displaced the profile obtained to the right in cells stimulated with 4-aminopyridine or sparteine, but abolished histamine release induced by quinine. Additionally, all three K+ channel blockers increased 45Ca uptake in mast cells. The exact mechanism of the action of K+ channel blockers on mast cells is unknown. However, the fact that the drugs used were effective as histamine-releasing and 45Ca uptake promoters suggests both that mast cells might be endowed with a K+ channel activity and that the blockade of this should open certain calcium channels, leading to elevated intracellular Ca2+ levels which in turn activate mast cell secretion.     

Bone-like tissue formation by three-dimensional culture of MG63 osteosarcoma cells in gelatin hydrogels using calcium-enriched medium

The aim of this study was to investigate the effect of Ca(2+) concentration in culture medium on the promotion of osteogenesis by MG63 osteoblast-like cells and to prepare bone-like tissues by supplying Ca(2+)-enriched medium to MG63 cells immobilized in three-dimensional gelatin hydrogels. Human osteosarcoma MG63 cells were cultured on tissue culture dish under various Ca(2+) concentrations to evaluate the effect of Ca(2+) concentration on calcium deposition. When Ca(2+) concentration was 8 mM, the maximum calcium deposition was obtained at day 28. Then MG63 cells were entrapped in gelatin hydrogels cross-linked by transglutaminase and cultured for 28 days, either in a standard culture medium or in medium containing 8 mM Ca(2+). Effects of Ca(2+)-enriched medium on osteoblastic phenotype of MG63 cells in gelatin hydrogels were analyzed in terms of cell number, calcium deposition content, and alkaline phosphatase (ALP) activity. The characteristics of calcified gelatin hydrogels were evaluated by x-ray diffraction (XRD), histological analysis, and scanning electron microscopy (SEM). After 28 days of culture, no significant difference in cell numbers was found between the different culture conditions. However, calcium content of gelatin hydrogels with cells cultured in Ca(2+)-enriched media was significantly higher than that of hydrogels with cells cultured in standard Ca(2+) concentration medium. After 14 days of culture, ALP activity of cells cultured in Ca(2+)-enriched media was down-regulated compared with that of cells cultured in standard Ca(2+) concentration media. XRD analysis indicated the formation of hydroxyapatite in gelatin hydrogels cultured in the Ca(2+)-enriched media at day 14, and the XRD pattern of the composite at day 21 was almost similar to that of mouse tibia. Moreover, histological analysis and SEM analysis revealed that cross-sections of hydrogels cultured in Ca(2+)-enriched media had an organic/mineral layer structure analogous to that of mouse tibia.     

The effect of surface charge on hydroxyapatite nucleation

Both the charged surfaces and condition of supersaturated solutions are thought to be the important factors that greatly affect the nucleation of hydroxyapatite (HAp) onto the substrates. In the present study self-assembled monolayers (SAM) terminated with amino (NH2-SAM) and hydroxyl (OH-SAM) headgroups were employed to generate the positively and negatively charged surfaces, respectively. The soaking experiments for HAp nucleation were carried out in the supersaturated solutions (1.5 SBF) with Ca2+ and PO4(3-) ions concentrations 1.5 times higher than in simulated body fluid (SBF). The stability of solutions deeply depends on pH values if other factors (temperature, ionic series and their concentration, etc.) are fixed. Consequently, the opposite micropatterns of HAp films were observed on the UV-patterned SAM with NH2 and OH terminals in a stable solution (1.5 SBF, pH=7.2) where no particles formed and in an unstable solution (1.5 SBF, pH=7.6) where homogeneous nucleation occurred. The preferential nucleation of HAp was found on negative surface (OH-SAM) in a stable supersaturated solution, while selective deposition of HAp was formed on positive surface (NH2-SAM) in the unstable solution. Considering the latter results, it is conceivable that the electrostatic adhesion of microparticles formed in the solution dominated the formation of HAp in the unstable solution.     

N-methyl-D-aspartate promotes the survival of cerebellar granule cells in culture

Our previous studies on the survival-promoting influence of elevated concentrations of extracellular K+ ([K+]e) on cultured cerebellar granule cells led to the proposal that depolarization in vitro mimics the effect of the earliest afferent inputs received by the granule cells in vivo. This, in turn, might be mediated through the stimulation of excitatory amino acid receptors, in particular the N-methyl-D-aspartate-preferring subtype gating ion channels which are also permeable to Ca2+. Here we report that N-methyl-D-aspartate indeed has a dramatic effect on the survival in culture of cells derived from dissociated cerebella of 7-8-day-old rats and cultured in media containing 'low' [K+]e (5-15 mM). In addition to the visual inspection of the cultures, the effect of N-methyl-D-aspartate was quantitatively evaluated, using estimates related to the number of viable cells (determination of DNA and of reduction rate of a tetrazolium salt). Furthermore, proteins which are relatively enriched in either nerve cells (neuronal cell adhesion molecule, D3-protein and synaptin) or in glia (glutamine synthetase) were also measured. The findings showed that the rescue of cells by N-methyl-D-aspartate involved primarily nerve cells and that the survival requirement for N-methyl-D-aspartate, as for high K+, developed between 2 and 4 days in vitro. The effect depended on both the concentration of N-methyl-D-aspartate and the degree of depolarization of the cells: both the potency and the efficacy of N-methyl-D-aspartate were increased as [K+]e was raised from 5 to 15 mM, at which range K+ on its own has little if any influence on granule cell survival. These characteristics are consistent with the voltage-dependence of ion conductance through the N-methyl-D-aspartate receptor-linked channel. The most pronounced effect of N-methyl-D-aspartate was obtained in the presence of 15 mM K+, when cell survival approached that obtained in 'control' cultures (grown in 25 mM K+-containing media without N-methyl-D-aspartate), and the potency of N-methyl-D-aspartate (half-maximal effective concentration, EC50, about 20 microM) was similar to its known affinity in binding to cerebral membranes. The effect of N-methyl-D-aspartate was blocked by the specific receptor antagonist 2-amino-5-phosphonovalerate, which also reduced the limited survival of cells in cultures grown in 'low' K+ in the absence of N-methyl-D-aspartate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenosine relaxation of isolated vascular smooth muscle.

Adenosine relaxed hog carotid media strips contracted with norepinephrine (NE) and potassium (K+). Adenosine (3 X 10(-6)M) was more effective in relaxing the NE contractures than those produced by K+. In both cases, adenosine's efficacy decreased with increasing concentrations of the stimulating agent. A high adenosine concentration (1 X 10(-3)M) was necessary to elicit relaxation of completely depolarized (124 mM K+) media strips and equimolar concentrations of aminophylline caused greater relaxation than did adenosine. Adenosine inhibited the Ca2+ dose-response curves of strips stimulated with 20 mM and 30 mM K+ and its effect was dependent on the Ca2+ concentration. Neither 1 X 10(-6)M nor 1 X 10(-4)M adenosine produced any change in the cAMP content of vascular strips. Only at high concentrations did adenosine increase the cAMP content of vascular strips, but the increase was signficantly more than that observed with the same dose of aminophylline. The present results are consistent with the possibility that adenosine relaxes vascular smooth muscle by directly altering Ca2+ permeability and/or membrane potential; they do not support a role for cAMP in the adenosine-induced relaxation of vascular smooth muscle.     

The effects of pentoxifylline on rat erythrocytes of different age

Age-separated rat erythrocytes were exposed to pentoxifylline, a dimethylxanthine derivative which increases erythrocyte deformability. A comparison of drug-induced effects in young and old erythrocytes yielded age-specific alterations in: (1) accumulation of intracellular Ca2+; (2) membrane protein phosphorylation; (3) ATP concentrations; and (4) membrane associated protein kinase activity. The effect of Ca2+ accumulation and membrane protein phosphorylation appears to be biphasic. Low drug concentrations (0.5-2.5 mM) reduced intracellular Ca2+ and increased membrane protein phosphorylation, whereas higher concentrations (4.0-5.0 mM) increased Ca2+ levels and reduced membrane protein phosphorylation. Young cells exhibited increased ATP levels over the whole range of pentoxifylline tested; however, older erythrocytes demonstrated higher ATP levels at 5.0 mM drug only. Membrane-associated protein kinase activity was enhanced 10% in young erythrocytes at 1.0 mM pentoxifylline and decreased to 30% of control values at 4.0 and 5.0 mM drug. Protein kinase of old erythrocytes exhibited gradual inhibition over the entire drug concentration range. In general, younger erythrocytes appear to be more responsive to pentoxifylline exposure. Based on these studies, it appears that the ageing of the erythrocyte and loss of deformability in vivo may be a consequence of increased Ca2+ entry into the cell.     

Effects of magnesium on contractile activation of skinned cardiac cells.

1. In the presence of a slight buffering of the free [Ca2+] with 0.050 mM total EGTA cyclic contractions were induced by a Ca2+-triggered release of Ca2+ on skinned (sarcolemma-free) segments of single cardiac cells from rat ventricle. The threshold of the free [Ca2+] trigger was elevated when the free [Mg2+] was increased. 2. At a suprathreshold free [Ca2+] increasing the free [Mg2+] resulted in a decrease in frequency and in an increase in amplitude of the phasic contractions. Addition of caffeine at a specified interval after a cyclic contraction produced a larger contraction when free [Mg2+] was higher. It was concluded that an increase of free [Mg2+] increased the capacity and the rate of binding for Ca2+ by the sarcoplasmic reticulum (SR). 3. Small skinned fibres of skeletal muscle which were perfused with 10 mM caffeine yielded results similar to those obtained in skinned cardiac cells. It was concluded that the mechanism of action of free Mg2+ was similar in both preparations, but that the SR of skeletal muscle had a higher capacity and rate of binding for Ca2+ than the cardiac SR. 4. With a strong buffering of the free [Ca2+] with 4-0 mM total EGTA, a smaller tonic tension was developed for a given pCa in the presence of a higher free [Mg2+]. This result was nearly identical in skinned cells from cardiac and skeletal muscle tissue. 5. A decrease of the [MgATP2-] produced a tension in the skinned cardiac cells that were perfused in Ca2+ free media. The maximum tension was observed for [MgATP2-] 10(-5-50)M as in skinned fibres of skeletal muscle. A further decrease of [MgATP2-] resulted in a decrease of tension.     

Regulation of interleukin 2 production, interleukin 2 mRNA expression and intracellular glutathione levels in ex vivo derived T lymphocytes by lactate

The concentration of L-lactate in the blood plasma of higher vertebrates is about 1 mM but can be as high as 30 mM under certain physiological and pathological conditions or in the vicinity of glycolytically active cells including macrophages. Here we report that high but physiologically relevant concentrations of lactate increase the expression of interleukin 2 (IL 2)-specific mRNA and the production of IL 2 activity in cultures of mitogenically stimulated T cells. Lactate supports IL 2 production most effectively if added 0-8 h after T cell stimulation and only in cultures of CD4+ but not of CD8+ T cells. In contrast to the DNA synthesis activity in these cell cultures, IL 2 production is not augmented but rather inhibited by exogenous glutathione (GSH). Lactate causes a reduction of intracellular GSH levels, and lactate-containing cultures require accordingly higher extracellular cysteine concentrations than control cultures to achieve similar intracellular GSH levels. In view of the strong variations of extracellular lactate concentrations in vivo, our experiments suggest that lactate may be part of a previously unknown mechanism by which the metabolic microenvironment modulates gene expression in T cells.     

Ca2+ regulation of tight-junction permeability and structure in Necturus gallbladder

To explore the role of Ca2+ in tight-junction permeability, the Necturus gallbladder was exposed to varying Ca2+ concentrations and to the Ca2+ ionophore A23187 added to the mucosal side (1.9 X 10(-6) to 6.8 X 10(-5) M). Electrophysiological parameters measured in an Ussing-type chamber were correlated with tight-junction morphology revealed by freeze-fracture electron microscopy. In Ca2+-free bathing media, transepithelial resistance decreases and tight-junctional ultrastructure is fragmented. In 1.8 mM Ca2+ media, A23187 induces an initial drop in transepithelial resistance, followed by an increase in transepithelial resistance to a value 20% above base line. At peak response to A23187, NaCl diffusion potentials decrease. Freeze-fracture replicas reveal that the number of junctional strands increase pari passu with junctional depth. Both physiological and morphological changes were partially reversible. The initial decrease in transepithelial resistance coincided with a persistent hyperpolarization of the mucosal cell membrane potential difference and a decrease in the mucosal-to-serosal cell membrane resistance ratio. Thus A23187 alters both the transcellular and paracellular pathway, resulting in opposing effects on transepithelial resistance.     

Reversal of alpha 1-receptor mediated relaxation in intestinal smooth muscle

The alpha 1-receptor agonist phenylephrine relaxed longitudinal rabbit jejunal muscle contracted in vitro by low concentrations of barium ions (1 mM). When the Ba2+ concentration was increased to 10-15 mM the response to phenylephrine was a contraction, and at Ba2+ concentrations in between the high and low range this response was biphasic--a relaxation followed by a contractile phase. The alpha 2-receptor agonist clonidine did not affect the tone of the Ba2+ contracted preparation. When the muscle preparation was contracted by Sr2+ (1-20 mM) in the presence of Ca2+ (2.5 mM), phenylephrine relaxed it, and no contractile response to phenylephrine was observed. In the absence of extracellular Ca2+, 5 mM Ba2+ caused a contraction. Under these conditions phenylephrine had no effect on the tissue tone. When Ca2+ was added in a low concentration (0.2-2 mM), phenylephrine elicited a gradually increasing contractile response. At 5 mM Ca2+ the contractile response was replaced by the normal relaxation. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ was partially blocked by low concentrations of verapamil. In higher concentrations verapamil abolished the tissue tonus completely. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ could be reverted to the normal relaxation by the addition of 20 mM Mg2+. Increasing the K+ concentration from the normal 5.9 to 62.9 mM blocked the phenylephrine-induced relaxation. No contractile response to phenylephrine occurred. It is concluded that Ba2+ could reverse the response of alpha 1 receptor stimulation in rabbit jejunum from a relaxation to a contraction and that this contractile response was dependent on the presence of Ca2+.     

Elemental maps in human allantochorial placental vessels cells: 2. MgCl2 and MgSO4 effects.

Extracellular magnesium salts are known to interfere with ionic channels in the cellular membranes. The membrane potential, a regulator of vascular tone, is a function of the physiological activities of ionic channels (particularly, K+ and Ca2+ channels in these cells). These channels regulate the ionic distribution into these cells. Micro-Particule Induced X-ray Emission (PIXE) analysis was applied to determine the ionic composition of vascular smooth muscle cells (VSMC) and of vascular endothelial cells (VEC) in the placental human allantochorial vessels in a physiological medium (Hanks' solution) modified by the addition of 2 mM MgCl2 or 2 mM MgSO4 which block the calcium-sensitive K+ channels (K(Ca)), the ATP-sensitive K+ channels (K(ATP)) and the voltage-sensitive K+ (K(df)) and Ca2+ channels. In VSMC (media layer), the addition of MgCl2 induced no modification of the K, Cl, P, S and Ca concentrations but increased the Na and Mg concentrations and the addition of MgSO4 only significantly increased the Mg concentration, the other ion concentrations remaining constant. In endothelium (VEC), MgCl2 or MgSO4 addition implicated the same observations as in VSMC. These results confirmed the blockage of K(df), K(Ca), K(ATP) and Ca channels in VSMC and VEC by magnesium salts, the relationship between Mg2+ ions and internal Na and demonstrated the possible intervention of a Na+/Mg2+ exchanger.     

Calcium ion levels in resting and depolarized goldfish retinal ganglion cell somata and growth cones.

1. We have estimated free, intracellular calcium ion concentrations ([Ca]i) in isolated retinal ganglion cells of adult goldfish by ratio-imaging fura-2 emission intensity at two excitation wavelengths. Here we describe [Ca]i in these cells, both at rest and during depolarization by elevated levels of extracellular potassium ions ([K]o). 2. [K]o was varied between 5 and 60 mM in sodium-free, tetrodotoxin-containing salines. Ganglion cell membrane potential, measured with patch electrodes, fell with each increment of [K]o used, from approximately -70 mV in 5 mM K+ to approximately -20 mV in 60 mM K+. 3. In control saline, [Ca]i was roughly 120 nM in cell somata and at least twofold higher in their growth cones. [Ca]i increased in both somata and growth cones to as high as 1.5 microM in salines containing 60 mM K+. [Ca]i exceeded 1.5 microM in some cells in high-K+ salines, although these levels could not be quantified accurately with fura-2. 4. Increases in [Ca]i elicited by elevated [K]o persisted for the duration of the exposure to high-K+ saline and were blocked by replacement of most of the bath Ca2+ by Co2+. These increases in [Ca]i were also sensitive to dihydropyridine calcium-channel ligands, viz., enhanced by BAY K 8644 (3 microM) and antagonized by nifedipine (10 microM). 5. Partial recovery of control [Ca]i occurred when [K]o was reduced to 5 mM after exposure to high-K+ saline and in high-K+ saline when nifedipine was included. These results show that goldfish retinal ganglion cells can partially buffer intracellular Ca2+ in the absence of extracellular Na+ ions. 6. These results provide measurements of the changes in [Ca]i brought about by depolarization of goldfish retinal ganglion cells in Na(+)-free salines. In these salines, at least part of the increase in [Ca]i appears to result from Ca2+ influx through a voltage-activated, noninactivating calcium conductance in the somata and growth cones of these cells. These measurements complement whole-cell patch-clamp and vibrating microprobe recordings from the somata and neurites of these cells and also immunocytochemical studies and patch-clamp measurements in amphibian, reptilian, and mammalian retinal ganglion cells.     

Hydroxyapatite deposition by alternating soaking technique on poly(vinyl alcohol)-coated polyethylene films

A poly(vinyl alcohol) (PVA)-coating on polyethylene films, prepared by repetitive adsorption/drying in an aqueous PVA solution, accelerated hydroxyapatite (HAp) deposition by an altemate soaking in aqueous solutions containing Ca2+ and PO4(3-) ions. X-ray photoelectron spectra of the surface of the HAp-deposited film showed the presence of calcium and phosphorus of a suitable peak ratio for HAp formation. X-ray diffraction analyses also revealed peaks corresponding to HAp. Scanning electron microscopic observation showed the surface of the HAp layer to be smooth, with nano-ordered dotted threads in networks. A simple PVA coating on a surface will serve as a novel system for accelerated HAp formation via alternating soaking.     

Mechanism through which GABAA receptor modulates catecholamine secretion from bovine chromaffin cells.

The actions and mechanism of GABAergic modulation of catecholamine secretion from isolated bovine chromaffin cells were investigated. The GABAA receptor agonist muscimol induced a fast rise in cytosolic [Ca2+]. The mean peak increase was 290 +/- 30 nM over basal levels. The increase in cytosolic [Ca2+] was abolished in the absence of extracellular [Ca2+] and was blocked by the GABAA antagonist bicuculline and the dihydropiridine nifedipine. Muscimol also elicited the release of catecholamines and increased the bisoxonol fluorescence indicating a cell depolarization. The [Ca2+] entry was well correlated with muscimol-evoked catecholamine secretion. When cells were treated with muscimol and a second secretagogue, a biphasic behavior was revealed. Muscimol enhanced the catecholamine release evoked by low concentrations of nicotine or K+, whereas release obtained at high concentrations of nicotine or K+ was actually inhibited. When the muscimol effect on membrane potential was studied in the presence of low K+ or nicotine concentrations, an enhancement of the bisoxonol fluorescence was observed. This effect was reversed at high concentrations of both K+ and nicotine. Measurement of 36Cl- fluxes showed an increase in membrane permeability to Cl- during muscimol stimulation. The influx or efflux in Cl- was dependent on membrane potential. In normal conditions, with a K+ concentration of 5.4 mM, a Cl- efflux was observed by both radiometric techniques, with 36Cl- and by the use of the chloride-sensitive fluorescent probe 6-methoxy-N-(3-sulphopropil)quinolinium, as indicator of intracellular Cl-. At high nicotine (20 mM) or K+ concentrations (105 mM) a Cl- influx was observed using 6-methoxy-N-(3-sulphopropil)quinolinium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and in vitro bioactivity of bredigite powders

Pure bredigite (Ca7MgSi4O16) powders are synthesized by the sol-gel method. The bredigite powders are composed of polycrystalline particles with dimensions of 1-10 micro m. The in vitro bioactivity of the bredigite powders are examined by evaluation of hydroxyapatite (HAp) formation ability in simulated body fluid (SBF) and the effect of ionic products from bredigite dissolution on osteoblast proliferation. The results showed that bredigite induced the formation of nanocrystalline HAp after soaking in SBF for 10 days. The Ca, Si, and Mg ions from bredigite dissolution at a certain concentration range stimulates osteoblast proliferation. Our study indicates that bredigite is bioactive and might be used for preparation of new biomaterials.     

Systematic strontium substitution in hydroxyapatite coatings on titanium via micro-arc treatment and their osteoblast/osteoclast responses

This study attempts to enhance the osseointegration of titanium implants by adopting a micro-arc treatment (MAT) capable of replacing calcium (Ca) with different percentages of strontium (Sr) in order to fabricate strontium-containing hydroxyapatite (Sr-HAp) coatings. Sr, regarded as a significant therapy promoting bone mass and bone strength, has a dual mechanism, enhancing osteoblast differentiation and inhibiting osteoclast differentiation. This study also investigates how Sr content affects the microstructure of and osteoblast/osteoclast growth on the coatings. Experimental results indicate that an increase in the Sr content in the electrolyte bath results in a greater degree of Sr substitution at Ca sites within the HAp phase, facilitating the formation of Sr-HAp coatings with Sr fully solid soluble in the HAp phase. Irrespective of the Sr content, most coatings are similar in porous morphology and pore size. Additionally, the Sr-HAp coating shows higher osteoblast compatibility than raw titanium metal and the HAp coating. Moreover, cell adhesion and proliferation after 48 h was greater than that after 4 h, indicating that Sr can stimulate osteoblast adhesion and proliferation. Further, Sr significantly inhibits osteoclast differentiation when the Sr-HAp coatings exceed 38.9 at.% Sr.     

Inhibitory effect of polymyxin B on catecholamine secretion from cultured bovine adrenal medullary cells

Incubation of cultured bovine adrenal medullary cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), was associated with increased secretion of catecholamine (CA) from the cells. Polymyxin B (PMB, 30-300 microM), a preferential inhibitor of protein kinase C, inhibited the TPA-induced secretion of CA. PMB also inhibited CA secretion induced by other secretagogues, the Ca2+ ionophore ionomycin (10 microM), 56 mM K+ or acetylcholine (ACh). Ionomycin, 56 mM K+ or ACh increased the concentration of intracellular free Ca2+ ([Ca2+]i) (measured using the fluorescent calcium indicator quin2), whereas TPA did not increase [Ca2+]i. PMB blocked the increase in [Ca2+]i induced by 56 mM K+ or ACh at concentrations similar to those inhibiting the secretion of CA. In contrast, PMB did not affect ionomycin-induced increase in [Ca2+]i. These results strongly suggest that CA secretion induced by TPA or ionomycin is mediated via activation of protein kinase C. The results further indicate that in 56 mM K+- or ACh-evoked CA secretion, PMB inhibits the secretion by blocking Ca2+ influx into the cells.     

Catecholamine release from bovine chromaffin cells: the role of sodium-calcium exchange in ouabain-evoked release

Spontaneous catecholamine (CA) release from bovine chromaffin cells maintained in primary tissue culture has been measured after pre-loading the cells with [3H]noradrenaline. Ouabain inhibited 86Rb+ uptake and increased 3H release in a concentration-dependent manner during a 60 min incubation period. Low external Na+ (5 mM: Li+ substitution) also increased 3H release. Whereas the 3H-releasing action of ouabain was maintained, the Li(+)-evoked release decreased with time. The effects of both ouabain and low Na+ solution on 3H release were completely inhibited by removal of Ca2+ from the external medium even though in Ca2(+)-free solution ouabain further inhibited 86Rb+ uptake into the cells. Readmission of Ca2+ to Na(+)-loaded cells (10-4 M-ouabain in Ca2(+)-free-1 mM-EGTA solution for 60 min) markedly increased the release of 3H. In the additional presence of diphenylhydantoin (DPH, 10-4 M) 3H release was significantly less on Ca2+ readmission. The 3H release from Na(+)-loaded cells was proportional to the concentration of Ca2+ readmitted. The 3H release was further increased from Na(+)-loaded cells in response to Ca2+ readmission when [Na+]o was lowered from 149 to 5 mM (Li+, choline+, Tris+ or sucrose substitution) though Li+ was less effective than the other Na+ substitutes. Potassium removal from the external medium significantly inhibited the 3H release evoked by Ca2+ readmission to Na(+)-loaded cells, even when [Ca2+]o was greater than normal (7.5 mM) or if Ca2+ was readmitted in low [Na+]o solution. Rb+, Cs+ or Li+ could substitute for K+ with the order of potency: Rb+ greater than or equal to K+ greater than Cs+ greater than Li+. A slight increase of external K+ (10.8 mM) potentiated the 3H release from Na(+)-loaded cells on Ca2+ readmission, but a higher concentration of K+ (149.4 mM) had the opposite action. The data is consistent with the hypothesis that ouabain-evoked CA release from bovine chromaffin cells is, in part, a consequence of an internal Na(+)-dependent Ca2+ influx. The evidence also suggests that there is Na(+)-Ca2+ competition at the external arm of the exchanger together with a monovalent cation activation site.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

Resistance of retinal extracellular space to Ca2+ level decrease: implications for the synaptic effects of divalent cations.

Ion-sensitive microelectrodes were used to measure the variations of [Ca2+]o induced by application of low Ca2+ media in the superfused eyecup preparation of the Pseudemys turtle. The aim of the experiments was to evaluate the possibility, suggested by previous studies, that in the deep, sclerad, layers of the retina [Ca2+]o may remain high enough to sustain chemical synaptic transmission even after prolonged application of low-Ca2+ saline. It was found that, at depths of 100-200 micron from the vitreal surface, [Ca2+ ]o did not fall below 1 mM even after application for periods of 30-60 min of nominally Ca2+-free media, and it was >0.3 mM after 30-min application of media containing EGTA and with a Ca2+ concentration of 1 nM. Previous studies in isolated salamander photoreceptors have shown that a reduction of [Ca2+ ]o to 0.3-1.0 mM may result in a paradoxical increase of Ca2+ influx into synaptic terminals due to the reduced screening of negative charge on the external face of the plasma membrane. On the basis of these results, the persistence or enhancement of synaptic transmission from photoreceptors to horizontal cells observed in various retinas treated with low-Ca2+ media may be accounted for within the classical Ca2+-dependent theory of synaptic transmission without invoking a Ca2+-independent mechanism.