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Inositol-1,4,5-triphosphate releases calcium from skinned cultured smooth muscle cells. Biochem Biophys Res Commun

Department of Pharmacology, University of Miami School of Medicine, Miami, Florida 33101 USA
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 08/1985; 130(1):270-4. DOI: 10.1016/0006-291X(85)90412-7
Source: PubMed

ABSTRACT

We examined the effects of inositol-1,4,5-trisphosphate on 45Ca uptake and 45Ca efflux in the saponin skinned primary cultured rat aortic smooth muscle cells. 10 microM inositol-1,4,5-trisphosphate induced a rapid (half time less than 10 sec) and large quantity of Ca release in both 45Ca uptake and 45Ca efflux in the skinned cells preloaded with 1 microM free Ca. Dose response curves showed that 100 microM inositol-1,4,5-trisphosphate produced a maximal Ca release of 97.3% of the MgATP dependent 45Ca uptake or 289 mumoles/liter cells, which was much greater than the maximal caffeine induced Ca release and would be sufficient to produce maximal tension.

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    ABSTRACT: A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established . The Saponin-treated cells could be stained with trypan blueand incorporated more "Ca" than the nontreated cells under the same conditions . At low free Ca" concentration, >85% of"Ca" uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased"Ca" efflux . In the skinned cells, caffeine increased "'Ca" efflux, whereas norepinephrine did not. The caffeine-releasable"Ca" uptake fraction in theskinned cellsappeared at 3 x 10'M Ca", increased gradually with the increase in free Ca" concen- tration, and reached a plateau at 1 x 10-5MCa". The "Ca' uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 x 10-5 M Ca" and increased monotonically with increasing free Ca"concentration. The results suggest that the caffeine-sensitive Ca' store, presumably the sarco- plasmic reticulum, plays a physiological role by releasing Ca' in response to norepinephrine or caffeine and by buffering excessive Cat+. The"Ca" uptake by mitochondria appears too insensitive to be important under physiological conditions .
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    ABSTRACT: A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established. The saponin-treated cells could be stained with trypan blue and incorporated more 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, greater than 85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 X 10(-7) M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 X 10(-5) M Ca2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 X 10(-5) M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The 45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions.
    Preview · Article · Apr 1986 · The Journal of General Physiology
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    ABSTRACT: The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.
    Preview · Article · Jun 1986 · Biochemical Journal
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