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  • 1
    ISSN: 1432-2013
    Keywords: Renal proximal tubule ; Intracellular calcium ; Calcium influx ; Primary culture ; Calcium store depletion ; 86Rb efflux ; Calcium ; sensitive potassium channels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Cytoplasmic calcium changes and calcium influx evoked by adenosine triphosphate (ATP) were investigated in primary cultures of rabbit proximal convoluted tubule cells. Extracellular ATP (50 μM) induced a biphasic increase of [Ca2+]i measured with the calcium probe fura-2. In the early phase, the mobilization of intracellular pools resulted in a transient increase of [Ca2+]i from 106±11 nM (n=36) to 1059±115% (n=29) of the resting level within 10 s. In the presence of external calcium, [Ca2+]i then decreased within 3 min to a sustained level (398±38%,n=8). Measurements of fura-2 quenching by external manganese revealed that this phase was the result of an increased Ca2+ uptake, blocked by lanthanum (10 μM) and verapamil (100 μM) but not by the nifedipin (25 μM). Internal calcium store depletion by ATP induced an increased calcium influx through lanthanum- and verapamil-sensitive, nifedipininsensitive calcium channels, located on the apical membrane of the cells. As indicated by86Rb+ efflux measurements, ATP activated a potassium efflux that was blocked by barium andLeiurus quinquestriatus hebraeus (LQH) venom (containing charybdotoxin) indicating the involvement of Ca2+-sensitive K+ channels. Moreover, in the presence of the LQH venom, the internal calcium stores were not replenished after being depleted by ATP. Our results indicate that an ATPevoked hyperpolarization of the plasma membrane leads to increased Ca2+ influx, which facilitates the replenishment of the internal stores.
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  • 2
    ISSN: 1433-2965
    Keywords: Bone mineral density ; Calcium ; Elderly ; Femoral neck ; Fracture ; Osteoporosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The efficacy of calcium (Ca) in reducing bone loss is debated. In a randomized placebo-controlled double-masked study, we investigated the effects of oral Ca supplements on femoral shaft (FS), femoral neck (FN) and lumbar spine (LS) bone mineral density (BMD), and on the incidence of vertebral fracture in vitamin-D-replete elderly. Ninety-three healthy subjects (72.1±0.6 years) were randomly allocated to three groups receiving 800 mg/day Ca in two different forms or a placebo for 18 months. Sixty-three patients (78.4±1.0 years) with a recent hip fracture were allocated to two groups receiving the two forms of Ca without placebo. FS BMD changes in Ca-supplemented non-fractured women were significantly different from those in the placebo group (+0.6±0.5% v −1.2±0.7%,p〈0.05). There was no difference in effect between the two forms of Ca. The changes of +0.7±0.8% v −1.7±1.6% in FN BMD of Ca-supplemented women and the placebo group did not reach statistical significance. In fractured patients, FS, FN and LS BMD changes were −1.3±0.8, +0.3±1.6 and +3.1±1.2% (p〈0.05 for the last). The rate of new vertebral fractures was 74.3 and 106.2 fractures per 1000 patient-years in Ca-supplemented non-fractured subjects and in the placebo group, respectively, and 144.0 in Ca-supplemented fractured patients. Thus, oral Ca supplements prevented a femoral BMD decrease and lowered vertebral fracture rate in the elderly.
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  • 3
    ISSN: 1432-1351
    Keywords: Aplysia ; Calcium ; Circadian ; Light ; Serotonin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The eye of the marine mollusk Aplysia californica contains an oscillator that drives a circadian rhythm of spontaneous compound action potentials in the optic nerve. Both light and serotonin are known to influence the phase of this ocular rhythm. The aim of the present study was to evaluate the role of extracellular calcium in both light and serotonin-mediated phase shifts. Low calcium treatments were found to cause phase shifts which resembled those produced by the transmitter serotonin. However, unlike serotonin, low calcium neither increased ocular cAMP levels nor could these phase shifts be prevented by increasing extracellular potassium concentration. Low calcium-induced phase shifts were prevented by the simultaneous application of the translational inhibitor anisomycin and low calcium treatment resulted in changes in [35S]methionine incorporation into several proteins as measured by a two-dimensional electrophoresis gel analysis. Finally, light treatments failed to produce phase shifts in the presence of low calcium or the calcium channel antagonist nickel chloride. These results are consistent with a model in which serotonin phase shifts the ocular pacemaker by decreasing a transmembrane calcium flux through membrane hyperpolarization while light-induced phase shifts are mediated by an increase in calcium flux.
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  • 4
    ISSN: 1432-1912
    Keywords: Px purinoceptors ; Calcium ; Magnesium ; Zinc ; Rat vas deferens ; α,β-methyleneATP binding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In this study we have examined the effect of metal cations (as their chloride salts) on the binding of [3H]α,β-methylene ATP ([3H]αβmeATP) to rat vas deferens membranes using a vacuum filtration receptor binding assay. Whereas NaCl and KCl (0.01 and 30 mM) did not affect total binding of 1 nM [3H]αβmeATP, several divalent and trivalent cation salts markedly increased binding. The trivalent cation salts, FeCl3 and AlCl3 (0.1 to 100 μM), produced the greatest increases in total binding of [3H]αβmeATP, however, their effects were most probably due to precipitation of the radioligand. In contrast, several divalent cations, at concentrations between 1 μM and 1–10 mM, increased total binding of [3H]αβmeATP to rat vas deferens by between 87% and 215% while having no effect on either filter binding or non specific binding. The following pEC50 values for potentiating binding of the radioligand were obtained: ZnCl2 (5.44), MnCl2 (4.52), CaCl2 (4.17), CoCl2 (4.06), MgCl2 (3.67) and BaCl2 (3.10). Both EDTA and EGTA (0.01–1 mM) inhibited the binding of the radioligand. The effects of ZnCl2, CaCl2 and MgCl2 were examined in saturation studies. In the absence of added divalent cations, [3H]αβmeATP labelled both high (pKd = 9.15) and low (pKd = 7.06) affinity binding sites. The affinity of the radioligand for its high affinity sites was increased by 3 mM CaCl2 (pKd = 9.56) and by 30 μM ZnCl2 (pKd = 9.46) but not by 3 mM MgCl2. The Bmax of the low affinity site for [3H]αβmeATP was increased (approximately 4 fold) by both 3 mM MgCl2 and 30 μM ZnCl2 but not by 3 mM CaCl2. The selective effect of CaCl2 on the high affinity binding sites enabled these sites to be labelled in the presence of 3 mM CaCl2 using a low concentration of [3H]αβmeATP (1 nM); the sites exhibited the binding characteristics expected of the P2x purinoceptor. The selective effect of MgCl2 on the low affinity binding sites enabled these sites to be labelled in the presence of 3 mM MgCl2 and using a high concentration of [3H]αβmeATP (100 nM). A comparison of the binding characteristics of the high and low affinity sites for [3H]αβmeATP revealed several other differences, in addition to their cation selectivity. First, the adenine analogues ADP, αβmeATP and adenosine tetraphosphate possessed between 13 and 62 fold higher affinity for the high affinity [3H]αβmeATP binding sites than for the low affinity binding sites. Secondly, GTP-γ-S and pyrophosphate were selective ligands for the low affinity [3H]αβmeATP binding sites possessing approximately 43 and 1995 fold, respectively, higher pIC50 values at the low affinity sites than at the high affinity sites. Finally, treatment of the membranes with 0.01–1 mM N-ethyl maleimide increased low affinity binding of the radioligand while not affecting binding to the high affinity sites. The binding characteristics of the low affinity sites suggest that they do not equate with functional P2x purinoceptors; their identity remains to be determined. There was evidence for heterogeneity of both the high and low affinity sites for [3H]αβmeATP since competition curves to several nucleotide and polyphosphate compounds displayed Hill slopes less than unity. In conclusion the present study has demonstrated that cations have a marked effect on the binding of [3H]αβmeATP in rat vas deferens. Of particular interest was the ability of CaCl2 to increase the affinity of the radioligand for its high affinity sites enabling these sites to be selectively labelled, while the ability of MgCl2 to increase the Bmax of the low affinity binding sites enabled these sites to be selectively labelled.
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  • 5
    ISSN: 1432-1912
    Keywords: α1A-adrenoceptors ; α1B-adrenoceptors ; Rat kidney ; Inositol phosphates ; G protein ; Calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have compared the coupling mechanisms of rat renal α1A- and α1B-like adrenoceptors to inositol phosphate formation. The experiments were performed in parallel in native renal tissue preparations and in those where α1B-adrenoceptors had been inactivated by treatment with 10 μmol/l chloroethylclonidine for 30 min at 37°C; renal slices were used in most experiments but isolated renal cells were also used in some cases. The Ca2+ chelating agent, EGTA (5 mmol/l), reduced noradrenaline-stimulated inositol phosphate formation in native but enhanced it in chloroethylclonidine-treated renal slices. The inhibitory effect of EGTA was not mimicked by 100 nmol/l nifedipine. Inactivation of 87% of cellular Gi by 16–20 h treatment with 500 ng/ml pertussis toxin did not significantly affect noradrenaline-stimulated inositol phosphate formation in isolated renal cells but abolished the inhibitory effect of chloroethylclonidine. The adenylate cyclase activator, forskolin (20 μmol/l), inhibited noradrenaline-stimulated inositol phosphate formation in native and chloroethylclonidine-treated slices, and the inhibitory effects of chloroethylclonidine treatment and forskolin were additive. We conclude that in rat kidney inositol phosphate formation via α1B-like adrenoceptors may involve the influx of extracellular Ca2+ and a pertussis toxin-sensitive G-protein but is insensitive to inhibition by forskolin. In contrast α1A-like adrenoceptor-mediated inositol phosphate formation does not require the presence of extracellular Ca2+ or of Gi and is sensitive to inhibition by forskolin. In comparison to published data from other model systems we further conclude that the signaling mechanisms of α1-adrenoceptor subtypes may depend on their cellular environment.
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