A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: Evidence for a calcium channel

Alan S. Fairhurst; Stanley A. Thayer; Jack E. Colker; David A. Beatty

doi:10.1016/0024-3205(83)90807-X

A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: Evidence for a calcium channel

Alan S. Fairhurst, Stanley A. Thayer, Jack E. Colker, David A. Beatty

Pharmacology

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29 Scopus citations

Abstract

The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [³H] -nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (K_D) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited K_D values of 0.2-0.3nM. The concentration of binding sites per mg. protein (B_max) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [³H] -nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5- trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 × 10^-5M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 × 10^-5M nitrendipine was seen on net ⁴⁵Ca uptake by HSR. These results suggest that [³H] -nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [³H] -nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [³H]- nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.

Original language	English (US)
Pages (from-to)	1331-1339
Number of pages	9
Journal	Life Sciences
Volume	32
Issue number	12
DOIs	https://doi.org/10.1016/0024-3205(83)90807-X
State	Published - Mar 21 1983

Bibliographical note

Funding Information:
This work was supported by a Grant-in-Aid from the California Heart Association (Orange County Chapter), by Hoffmann-La Roche Inc., by the U.S. Air Force Office of Scientific Research contract F49620-81-K0015, and through a Predoctoral Fellowship of the American Foundation for Pharmaceutical Education awarded to S.A.T.

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title = "A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: Evidence for a calcium channel",

abstract = "The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [3H] -nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (KD) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited KD values of 0.2-0.3nM. The concentration of binding sites per mg. protein (Bmax) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [3H] -nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5- trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 × 10-5M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 × 10-5M nitrendipine was seen on net 45Ca uptake by HSR. These results suggest that [3H] -nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [3H] -nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [3H]- nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.",

author = "Fairhurst, {Alan S.} and Thayer, {Stanley A.} and Colker, {Jack E.} and Beatty, {David A.}",

note = "Funding Information: This work was supported by a Grant-in-Aid from the California Heart Association (Orange County Chapter), by Hoffmann-La Roche Inc., by the U.S. Air Force Office of Scientific Research contract F49620-81-K0015, and through a Predoctoral Fellowship of the American Foundation for Pharmaceutical Education awarded to S.A.T.",

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T1 - A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum

T2 - Evidence for a calcium channel

AU - Fairhurst, Alan S.

AU - Thayer, Stanley A.

AU - Colker, Jack E.

AU - Beatty, David A.

N1 - Funding Information: This work was supported by a Grant-in-Aid from the California Heart Association (Orange County Chapter), by Hoffmann-La Roche Inc., by the U.S. Air Force Office of Scientific Research contract F49620-81-K0015, and through a Predoctoral Fellowship of the American Foundation for Pharmaceutical Education awarded to S.A.T.

PY - 1983/3/21

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N2 - The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [3H] -nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (KD) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited KD values of 0.2-0.3nM. The concentration of binding sites per mg. protein (Bmax) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [3H] -nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5- trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 × 10-5M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 × 10-5M nitrendipine was seen on net 45Ca uptake by HSR. These results suggest that [3H] -nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [3H] -nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [3H]- nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.

AB - The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [3H] -nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (KD) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited KD values of 0.2-0.3nM. The concentration of binding sites per mg. protein (Bmax) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [3H] -nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5- trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 × 10-5M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 × 10-5M nitrendipine was seen on net 45Ca uptake by HSR. These results suggest that [3H] -nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [3H] -nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [3H]- nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.

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A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: Evidence for a calcium channel

Abstract

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