Specific binding of the calcium antagonist [3H]nitrendipine to subcellular fractions isolated from canine myocardium. Evidence for high affinity binding to ryanodine-sensitive sarcoplasmic reticulum vesicles

L. T. Williams, Larry Jones

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Abstract

[3H]Nitrendipine, a potent calcium antagonist, was used to determine the number of binding sites for dihydropyridine calcium antagonists in well characterized subcellular fractions prepared from canine myocardium. The distribution of high affinity [3H]nitrendipine binding sites was compared with the distribution of sarcolemma, sarcoplasmic reticulum, and mitochondrial markers. The highest density of [3H]nitrendipine binding sites (1.5 pmol/mg of protein) was in a subfraction enriched in ryanodine-sensitive sarcoplasmic reticulum vesicles. In contrast, the density of [3H]nitrendipine binding sites in the ryanodine-insensitive vesicles was less than 10% of that present in the ryanodine-sensitive vesicles. A preparation enriched in sarcolemmal vesicles had an intermediate density of binding sites. A crude particulate preparation and a mitochondrial preparation had 10 and 20% as many binding sites, respectively, as the ryanodine-sensitive sarcoplasmic reticulum vesicles. An analysis of the membrane markers in these preparations confirmed that the subcellular distribution of specific [3H]nitrendipine binding sites did not correlate with the distribution of sarcolemma markers, mitochondrial markers, or the protein phospholamban. The binding sites had a high affinity for [3H]nitrendipine (equilibrium dissociation constant of 0.2-0.4 nM) in both highly purified and crude particulate fractions. These findings indicate that dihydropyridine calcium antagonists bind to specific high affinity sites on membranes other than the sarcolemma and that they may act at sites associated with the ryanodine-sensitive sarcoplasmic reticulum.

Original languageEnglish (US)
Pages (from-to)5344-5347
Number of pages4
JournalJournal of Biological Chemistry
Volume258
Issue number9
StatePublished - 1983
Externally publishedYes

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Nitrendipine
Ryanodine
Subcellular Fractions
Sarcoplasmic Reticulum
Canidae
Myocardium
Binding Sites
Calcium
Sarcolemma
Membranes
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Specific binding of the calcium antagonist [3H]nitrendipine to subcellular fractions isolated from canine myocardium. Evidence for high affinity binding to ryanodine-sensitive sarcoplasmic reticulum vesicles",
abstract = "[3H]Nitrendipine, a potent calcium antagonist, was used to determine the number of binding sites for dihydropyridine calcium antagonists in well characterized subcellular fractions prepared from canine myocardium. The distribution of high affinity [3H]nitrendipine binding sites was compared with the distribution of sarcolemma, sarcoplasmic reticulum, and mitochondrial markers. The highest density of [3H]nitrendipine binding sites (1.5 pmol/mg of protein) was in a subfraction enriched in ryanodine-sensitive sarcoplasmic reticulum vesicles. In contrast, the density of [3H]nitrendipine binding sites in the ryanodine-insensitive vesicles was less than 10{\%} of that present in the ryanodine-sensitive vesicles. A preparation enriched in sarcolemmal vesicles had an intermediate density of binding sites. A crude particulate preparation and a mitochondrial preparation had 10 and 20{\%} as many binding sites, respectively, as the ryanodine-sensitive sarcoplasmic reticulum vesicles. An analysis of the membrane markers in these preparations confirmed that the subcellular distribution of specific [3H]nitrendipine binding sites did not correlate with the distribution of sarcolemma markers, mitochondrial markers, or the protein phospholamban. The binding sites had a high affinity for [3H]nitrendipine (equilibrium dissociation constant of 0.2-0.4 nM) in both highly purified and crude particulate fractions. These findings indicate that dihydropyridine calcium antagonists bind to specific high affinity sites on membranes other than the sarcolemma and that they may act at sites associated with the ryanodine-sensitive sarcoplasmic reticulum.",
author = "Williams, {L. T.} and Larry Jones",
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T1 - Specific binding of the calcium antagonist [3H]nitrendipine to subcellular fractions isolated from canine myocardium. Evidence for high affinity binding to ryanodine-sensitive sarcoplasmic reticulum vesicles

AU - Williams, L. T.

AU - Jones, Larry

PY - 1983

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N2 - [3H]Nitrendipine, a potent calcium antagonist, was used to determine the number of binding sites for dihydropyridine calcium antagonists in well characterized subcellular fractions prepared from canine myocardium. The distribution of high affinity [3H]nitrendipine binding sites was compared with the distribution of sarcolemma, sarcoplasmic reticulum, and mitochondrial markers. The highest density of [3H]nitrendipine binding sites (1.5 pmol/mg of protein) was in a subfraction enriched in ryanodine-sensitive sarcoplasmic reticulum vesicles. In contrast, the density of [3H]nitrendipine binding sites in the ryanodine-insensitive vesicles was less than 10% of that present in the ryanodine-sensitive vesicles. A preparation enriched in sarcolemmal vesicles had an intermediate density of binding sites. A crude particulate preparation and a mitochondrial preparation had 10 and 20% as many binding sites, respectively, as the ryanodine-sensitive sarcoplasmic reticulum vesicles. An analysis of the membrane markers in these preparations confirmed that the subcellular distribution of specific [3H]nitrendipine binding sites did not correlate with the distribution of sarcolemma markers, mitochondrial markers, or the protein phospholamban. The binding sites had a high affinity for [3H]nitrendipine (equilibrium dissociation constant of 0.2-0.4 nM) in both highly purified and crude particulate fractions. These findings indicate that dihydropyridine calcium antagonists bind to specific high affinity sites on membranes other than the sarcolemma and that they may act at sites associated with the ryanodine-sensitive sarcoplasmic reticulum.

AB - [3H]Nitrendipine, a potent calcium antagonist, was used to determine the number of binding sites for dihydropyridine calcium antagonists in well characterized subcellular fractions prepared from canine myocardium. The distribution of high affinity [3H]nitrendipine binding sites was compared with the distribution of sarcolemma, sarcoplasmic reticulum, and mitochondrial markers. The highest density of [3H]nitrendipine binding sites (1.5 pmol/mg of protein) was in a subfraction enriched in ryanodine-sensitive sarcoplasmic reticulum vesicles. In contrast, the density of [3H]nitrendipine binding sites in the ryanodine-insensitive vesicles was less than 10% of that present in the ryanodine-sensitive vesicles. A preparation enriched in sarcolemmal vesicles had an intermediate density of binding sites. A crude particulate preparation and a mitochondrial preparation had 10 and 20% as many binding sites, respectively, as the ryanodine-sensitive sarcoplasmic reticulum vesicles. An analysis of the membrane markers in these preparations confirmed that the subcellular distribution of specific [3H]nitrendipine binding sites did not correlate with the distribution of sarcolemma markers, mitochondrial markers, or the protein phospholamban. The binding sites had a high affinity for [3H]nitrendipine (equilibrium dissociation constant of 0.2-0.4 nM) in both highly purified and crude particulate fractions. These findings indicate that dihydropyridine calcium antagonists bind to specific high affinity sites on membranes other than the sarcolemma and that they may act at sites associated with the ryanodine-sensitive sarcoplasmic reticulum.

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