The mechanism of action of GTP on Ca2+ efflux from rat liver microsomal vesicles

Dawson, Alan P.; Hills, G. J.; Comerford, J G

doi:10.1042/bj2440087

“…It was recently suggested that guanosine triphosphate (GTP) could activate Ca2+ release from endoplasmic reticulum in non-muscle cells (Chueh & Gill, 1986;Dawson et al, 1987). In skinned vascular smooth muscle Saida & van Breemen (1987) reported that GTP enhances the InsP3-induced Ca2 + release from sarcoplasmic reticulum, and they suggested that guanine nucleotide-binding proteins are involved in the mechanism of Ca2+ -release.…”

Section: Introductionmentioning

confidence: 99%

Properties of intracellular calcium stores in pregnant rat myometrium

Kanmura

¹

,

Missiaen

²

,

Casteels

³

1988

British J Pharmacology

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“…The preparation of rat liver microsomes and the measurements of Ca2+ uptake and release using a Ca2+-sensitive electrode were as described by Dawson et al (1987). Aluminium was solubilized by mixing AICl3 (10 mM) and sodium citrate (50 mM) for at least 2 h prior to use.…”

mentioning

confidence: 99%

The perturbation, by aluminium, of receptor-generated calcium transients in hepatocytes is not due to effects of Ins(1,4,5)P3-stimulated Ca2+ release or Ins(1,4,5)P3 metabolism by the 5-phosphatase and 3-kinase

Shears¹,

Dawson²,

Loomis-Husselbee³

et al. 1990

Biochemical Journal

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The perturbation, by aluminium, of receptorgenerated calcium transients in hepatocytes is not due to effects of Ins(1,4,5)P3-stimulated Ca2+ release or Ins(1,4,5)P3 metabolism by the 5-phosphatase and 3-kinaseThe pursuit of the mechanism underlying toxic effects of Al3+ has led to the demonstration that A13+ may compete for Mg2+-binding sites on certain enzymes even when the free Mg2+ concentration is as much as 108-fold in excess (Miller et al., 1989). Moreover, a recent publication in this journal has highlighted the possibility of an interaction between aluminium and the phosphoinositide-mediated calcium signalling pathway (Schofl et al., 1990); in hepatocytes, hormone-stimulated repetitive Ca2+ transients (Woods et al., 1986), were perturbed by superfusion of the cells with as little as 5-10 FuM-Al3+ (as A13+ citrate). Amongst possible explanations for these effects are the proposals that Ins(1,4,5)P3 might bind A13+ particularly avidly, thereby affecting Ins(1,4,5)P3 metabolism or Ca2+ mobilization (Birchall & Chappell, 1988; Sch6fl et al., 1990). Therefore we decided to look at possible effects of aluminium on (i) calcium movements in isolated rat liver microsomes, and (ii) Ins(1,4,5)P3 metabolism by the 5-phosphatase and 3-kinase. It should be emphasized that in view of the uncertainties surrounding the kinetics of Al3+ exchange between various chelators (Miller et al., 1989), the media used in our experiments were pre-equilibrated for several hours in plastic containers.The preparation of rat liver microsomes and the measurements of Ca2+ uptake and release using a Ca2+-sensitive electrode were as described by Dawson et al. (1987). Aluminium was solubilized by mixing AICl3 (10 mM) and sodium citrate (50 mM) for at least 2 h prior to use. Rat liver microsomes were preincubated for 2 h at 0-4 'C with 300 /uM-Al3+/1.5 mM-citrate (to give 10 /LM-Al3+ in the final assay medium) and, separately, 0.5 mM-Ins(1,4,5)P3 with 5 mM-Al3+/25 mM-citrate for up to 5 h. Al3+-pretreated microsomes behaved exactly like control microsomes (i.e. plus citrate alone) in terms of Ca2+ uptake, GTP-enhancement of Ins(1,4,5)P3 sensitivity and Ins(1,4,5)P3-stimulated Ca2+ release. Similarly, Al3+-pretreated Ins(1,4,5,)P3 behaved just like control Ins(1,4,5)P3.For the studies of Ins(1,4,5)P3 metabolism, 1 mM-AlC13 was stirred with 25 mM-sodium citrate for 3-4 h. Ins(1,4,5)P3 5-phosphatase was assayed in 0.5 ml aliquots of buffer containing 2 /tM-[3H]Ins(1,4,5)P3 (1 nCi/ml), 100 mM-KCl, 1 mM-MgSO4, 10 mM-Hepes (pH 7.2 with KOH) and saponin (0.2 mg/ml). Ins(1,4,5)P3 3-kinase was assayed in similar buffer, except for the addition of 5 mM-Na2ATP plus 5 mM-MgSO4. Either 40 ,tM-Al3+ (or the equivalent citrate concentration as the control) was added to each of the buffers, which were preincubated overnight at 0-4 IC before being brought to 37°C and mixed with 25 ,ul of a 1 % (w/v) liver homogenate prepared in 0.25 M-sucrose/5 mmHepes (pH 7.2 with KOH). Ins(1,4,5)P3 metabolism was then analysed as described by Shears (1989). There was no sign...

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“…Therefore we decided to look at possible effects of aluminium on (i) calcium movements in isolated rat liver microsomes, and (ii) Ins(1,4,5)P3 metabolism by the 5-phosphatase and 3-kinase. It should be emphasized that in view of the uncertainties surrounding the kinetics of Al3+ exchange between various chelators (Miller et al, 1989), the media used in our experiments were pre-equilibrated for several hours in plastic containers.The preparation of rat liver microsomes and the measurements of Ca2+ uptake and release using a Ca2+-sensitive electrode were as described by Dawson et al (1987). Aluminium was solubilized by mixing AICl3 (10 mM) and sodium citrate (50 mM) for at least 2 h prior to use.…”

mentioning

confidence: 99%

On the use of EGTA to assess the effect of Ca2+ on liver microsomal glucose-6-phosphatase

Werve¹,

Vidal²

1990

Biochemical Journal

5

1

0

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The perturbation, by aluminium, of receptorgenerated calcium transients in hepatocytes is not due to effects of Ins(1,4,5)P3-stimulated Ca2+ release or Ins(1,4,5)P3 metabolism by the 5-phosphatase and 3-kinaseThe pursuit of the mechanism underlying toxic effects of Al3+ has led to the demonstration that A13+ may compete for Mg2+-binding sites on certain enzymes even when the free Mg2+ concentration is as much as 108-fold in excess (Miller et al., 1989). Moreover, a recent publication in this journal has highlighted the possibility of an interaction between aluminium and the phosphoinositide-mediated calcium signalling pathway (Schofl et al., 1990); in hepatocytes, hormone-stimulated repetitive Ca2+ transients (Woods et al., 1986), were perturbed by superfusion of the cells with as little as 5-10 FuM-Al3+ (as A13+ citrate). Amongst possible explanations for these effects are the proposals that Ins(1,4,5)P3 might bind A13+ particularly avidly, thereby affecting Ins(1,4,5)P3 metabolism or Ca2+ mobilization (Birchall & Chappell, 1988; Sch6fl et al., 1990). Therefore we decided to look at possible effects of aluminium on (i) calcium movements in isolated rat liver microsomes, and (ii) Ins(1,4,5)P3 metabolism by the 5-phosphatase and 3-kinase. It should be emphasized that in view of the uncertainties surrounding the kinetics of Al3+ exchange between various chelators (Miller et al., 1989), the media used in our experiments were pre-equilibrated for several hours in plastic containers.The preparation of rat liver microsomes and the measurements of Ca2+ uptake and release using a Ca2+-sensitive electrode were as described by Dawson et al. (1987). Aluminium was solubilized by mixing AICl3 (10 mM) and sodium citrate (50 mM) for at least 2 h prior to use. Rat liver microsomes were preincubated for 2 h at 0-4 'C with 300 /uM-Al3+/1.5 mM-citrate (to give 10 /LM-Al3+ in the final assay medium) and, separately, 0.5 mM-Ins(1,4,5)P3 with 5 mM-Al3+/25 mM-citrate for up to 5 h. Al3+-pretreated microsomes behaved exactly like control microsomes (i.e. plus citrate alone) in terms of Ca2+ uptake, GTP-enhancement of Ins(1,4,5)P3 sensitivity and Ins(1,4,5)P3-stimulated Ca2+ release. Similarly, Al3+-pretreated Ins(1,4,5,)P3 behaved just like control Ins(1,4,5)P3.For the studies of Ins(1,4,5)P3 metabolism, 1 mM-AlC13 was stirred with 25 mM-sodium citrate for 3-4 h. Ins(1,4,5)P3 5-phosphatase was assayed in 0.5 ml aliquots of buffer containing 2 /tM-[3H]Ins(1,4,5)P3 (1 nCi/ml), 100 mM-KCl, 1 mM-MgSO4, 10 mM-Hepes (pH 7.2 with KOH) and saponin (0.2 mg/ml). Ins(1,4,5)P3 3-kinase was assayed in similar buffer, except for the addition of 5 mM-Na2ATP plus 5 mM-MgSO4. Either 40 ,tM-Al3+ (or the equivalent citrate concentration as the control) was added to each of the buffers, which were preincubated overnight at 0-4 IC before being brought to 37°C and mixed with 25 ,ul of a 1 % (w/v) liver homogenate prepared in 0.25 M-sucrose/5 mmHepes (pH 7.2 with KOH). Ins(1,4,5)P3 metabolism was then analysed as described by Shears (1989). There was no sign...

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The mechanism of action of GTP on Ca2+ efflux from rat liver microsomal vesicles

Cited by 60 publications

References 22 publications

Properties of intracellular calcium stores in pregnant rat myometrium

Properties of intracellular calcium stores in pregnant rat myometrium

The perturbation, by aluminium, of receptor-generated calcium transients in hepatocytes is not due to effects of Ins(1,4,5)P3-stimulated Ca2+ release or Ins(1,4,5)P3 metabolism by the 5-phosphatase and 3-kinase

On the use of EGTA to assess the effect of Ca2+ on liver microsomal glucose-6-phosphatase

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