Discrimination of multiple binding sites for antagonists of the calcium release channel complex of skeletal and cardiac sarcoplasmic reticulum

M. M. Mack, I. Zimanyi, Isaac N Pessah

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Abstract

The mechanisms by which ruthenium red (RR), neomycin and FLA 365 ([2,6- dichloro-4-aminophenyl]isopropylamine) inhibit calcium channels of skeletal and cardiac sarcoplasmic reticulum (SR) are characterized. Neomycin and FLA 365 inhibit ryanodine-enhanced calcium release from skeletal SR vesicles in a dose-dependent manner. The apparent affinity of [3H]ryanodine is reduced in a dose-dependent manner by each inhibitor indicative of competitive mechanisms. Displacement studies with skeletal and cardiac SR demonstrate that the order of inhibitory potency is RR > neomycin > FLA 365 and RR > FLA 365 > neomycin, respectively. Neomycin is 100-fold less potent in cardiac SR and inhibition of [3H]ryanodine binding is biphasic in both tissues. Neomycin induces a greater proportion of [3H]ryanodine binding states recalcitrant to inhibition in cardiac SR. The ability of neomycin to increase the apparent affinity of [3H]ryanodine for its binding sites is potentiated by RR and attenuated by FLA 365. Kinetic binding studies reveal that increasing neomycin concentrations decreases the association of [3H]ryanodine as predicted for competitive inhibition. However, high (>>K(/1)) neomycin increases [3H]ryanodine binding affinity by slowing dissociation of the radioligand demonstrating that, like micromolar ryanodine, neomycin induces allosterism. Studies with combinations of antagonists demonstrate the existence of two nonoverlapping inhibitor recognition sites within the ryanoid site, one polycationic inhibitor site and one FLA 365 inhibitor site. These results suggest that aminoglycoside- induced muscle paralysis may be mediated by direct block of pre- and postsynaptic calcium release channels of endoplasmic reticulum.

Original languageEnglish (US)
Pages (from-to)1028-1037
Number of pages10
JournalJournal of Pharmacology and Experimental Therapeutics
Volume262
Issue number3
StatePublished - 1992

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Neomycin
Calcium Channel Blockers
Sarcoplasmic Reticulum
Ryanodine
Binding Sites
Ruthenium Red
Calcium Channels
Aminoglycosides
Paralysis
Endoplasmic Reticulum
FLA 365
Calcium
Muscles

ASJC Scopus subject areas

  • Pharmacology

Cite this

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abstract = "The mechanisms by which ruthenium red (RR), neomycin and FLA 365 ([2,6- dichloro-4-aminophenyl]isopropylamine) inhibit calcium channels of skeletal and cardiac sarcoplasmic reticulum (SR) are characterized. Neomycin and FLA 365 inhibit ryanodine-enhanced calcium release from skeletal SR vesicles in a dose-dependent manner. The apparent affinity of [3H]ryanodine is reduced in a dose-dependent manner by each inhibitor indicative of competitive mechanisms. Displacement studies with skeletal and cardiac SR demonstrate that the order of inhibitory potency is RR > neomycin > FLA 365 and RR > FLA 365 > neomycin, respectively. Neomycin is 100-fold less potent in cardiac SR and inhibition of [3H]ryanodine binding is biphasic in both tissues. Neomycin induces a greater proportion of [3H]ryanodine binding states recalcitrant to inhibition in cardiac SR. The ability of neomycin to increase the apparent affinity of [3H]ryanodine for its binding sites is potentiated by RR and attenuated by FLA 365. Kinetic binding studies reveal that increasing neomycin concentrations decreases the association of [3H]ryanodine as predicted for competitive inhibition. However, high (>>K(/1)) neomycin increases [3H]ryanodine binding affinity by slowing dissociation of the radioligand demonstrating that, like micromolar ryanodine, neomycin induces allosterism. Studies with combinations of antagonists demonstrate the existence of two nonoverlapping inhibitor recognition sites within the ryanoid site, one polycationic inhibitor site and one FLA 365 inhibitor site. These results suggest that aminoglycoside- induced muscle paralysis may be mediated by direct block of pre- and postsynaptic calcium release channels of endoplasmic reticulum.",
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AU - Zimanyi, I.

AU - Pessah, Isaac N

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N2 - The mechanisms by which ruthenium red (RR), neomycin and FLA 365 ([2,6- dichloro-4-aminophenyl]isopropylamine) inhibit calcium channels of skeletal and cardiac sarcoplasmic reticulum (SR) are characterized. Neomycin and FLA 365 inhibit ryanodine-enhanced calcium release from skeletal SR vesicles in a dose-dependent manner. The apparent affinity of [3H]ryanodine is reduced in a dose-dependent manner by each inhibitor indicative of competitive mechanisms. Displacement studies with skeletal and cardiac SR demonstrate that the order of inhibitory potency is RR > neomycin > FLA 365 and RR > FLA 365 > neomycin, respectively. Neomycin is 100-fold less potent in cardiac SR and inhibition of [3H]ryanodine binding is biphasic in both tissues. Neomycin induces a greater proportion of [3H]ryanodine binding states recalcitrant to inhibition in cardiac SR. The ability of neomycin to increase the apparent affinity of [3H]ryanodine for its binding sites is potentiated by RR and attenuated by FLA 365. Kinetic binding studies reveal that increasing neomycin concentrations decreases the association of [3H]ryanodine as predicted for competitive inhibition. However, high (>>K(/1)) neomycin increases [3H]ryanodine binding affinity by slowing dissociation of the radioligand demonstrating that, like micromolar ryanodine, neomycin induces allosterism. Studies with combinations of antagonists demonstrate the existence of two nonoverlapping inhibitor recognition sites within the ryanoid site, one polycationic inhibitor site and one FLA 365 inhibitor site. These results suggest that aminoglycoside- induced muscle paralysis may be mediated by direct block of pre- and postsynaptic calcium release channels of endoplasmic reticulum.

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