Enrichment, solubilization, and partial characterization of digitonin-solubilized muscarinic receptors derived from canine ventricular myocardium

A. S. Manalan, D. K. Werth, L. R. Jones, A. M. Watanabe

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

The subcellular distribution of cardiac muscarinic receptors was defined in canine ventricular myocardium, and receptors were solubilized from subcellular fractions enriched in muscarinic receptor content. The subcellular location of muscarinic receptors in cardiac tissue was determined by measurement of the distribution of [3H](±)quinuclidinyl benzilate-binding activity in particulate fractions isolated from canine ventricular myocardium. Based upon excellent correlation between [3H](±)quinuclidinyl benzilate binding and activity of the sarcolemmal Na+,K+-ATPase throughout the subcellular fractions, muscarinic receptors appeared to be localized to sarcolemma in canine ventricular myocardium. Therefore, membrane fractions enriched in sarcolemma were used as a source of cardiac muscarinic receptors for solubilization. Treatment of membrane vesicle fractions with digitonin (0.6%) resulted in solubilization of [3H](±)quinuclidinyl benzilate-binding activity with an extraction yield of 25-35%. Criteria of pharmacological specificity and stereospecificity established the identity of the solubilized binding activity of muscarinic receptors. Solubilization of muscarinic receptors was documented by demonstration of hydrodynamic behavior consistent with molecularly dispersed material. Upon glycerol gradient centrifugation, digitonin-solubilized muscarinic receptors from cardiac tissue sedimented with an apparent sedimentation coefficient of 9S. Pharmacological characterization of the digitonin-solubilized receptors revealed 8- to 39-fold reductions in affinities for muscarinic antagonists compared to the affinities exhibited by receptors in the membrane-bound state. Substantially greater reductions in agonist affinities (reduction of at least 700-fold for all agonists studied) suggested selective loss of ability of the digitonin-solubilized receptors to exhibit high affinity agonist interactions. In contrast to membrane-bound receptors, digitonin-solubilized receptors also demonstrated a loss of guanine nucleotide regulation, as well as steep agonist:radioligand competition curves with slope factors of 1.0, suggesting a homogeneous population of agonist-binding sites. Interpreted within the context of a model of state interconversion for membrane-bound receptors, the results suggested that either muscarinic receptors of a single state were selectively solubilized, or that solubilization induced conversion of all receptors to a single low affinity state, possibly by removal of constituents necessary for assumption of a high affinity agonist conformation.

Original languageEnglish (US)
Pages (from-to)664-676
Number of pages13
JournalCirculation research
Volume52
Issue number6
DOIs
StatePublished - Jan 1 1983

Fingerprint

Digitonin
Muscarinic Receptors
Canidae
Myocardium
Quinuclidinyl Benzilate
Membranes
Sarcolemma
Subcellular Fractions
Pharmacology
Muscarinic Antagonists
Guanine Nucleotides
Hydrodynamics
Centrifugation
Glycerol
Binding Sites

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Enrichment, solubilization, and partial characterization of digitonin-solubilized muscarinic receptors derived from canine ventricular myocardium. / Manalan, A. S.; Werth, D. K.; Jones, L. R.; Watanabe, A. M.

In: Circulation research, Vol. 52, No. 6, 01.01.1983, p. 664-676.

Research output: Contribution to journalArticle

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abstract = "The subcellular distribution of cardiac muscarinic receptors was defined in canine ventricular myocardium, and receptors were solubilized from subcellular fractions enriched in muscarinic receptor content. The subcellular location of muscarinic receptors in cardiac tissue was determined by measurement of the distribution of [3H](±)quinuclidinyl benzilate-binding activity in particulate fractions isolated from canine ventricular myocardium. Based upon excellent correlation between [3H](±)quinuclidinyl benzilate binding and activity of the sarcolemmal Na+,K+-ATPase throughout the subcellular fractions, muscarinic receptors appeared to be localized to sarcolemma in canine ventricular myocardium. Therefore, membrane fractions enriched in sarcolemma were used as a source of cardiac muscarinic receptors for solubilization. Treatment of membrane vesicle fractions with digitonin (0.6{\%}) resulted in solubilization of [3H](±)quinuclidinyl benzilate-binding activity with an extraction yield of 25-35{\%}. Criteria of pharmacological specificity and stereospecificity established the identity of the solubilized binding activity of muscarinic receptors. Solubilization of muscarinic receptors was documented by demonstration of hydrodynamic behavior consistent with molecularly dispersed material. Upon glycerol gradient centrifugation, digitonin-solubilized muscarinic receptors from cardiac tissue sedimented with an apparent sedimentation coefficient of 9S. Pharmacological characterization of the digitonin-solubilized receptors revealed 8- to 39-fold reductions in affinities for muscarinic antagonists compared to the affinities exhibited by receptors in the membrane-bound state. Substantially greater reductions in agonist affinities (reduction of at least 700-fold for all agonists studied) suggested selective loss of ability of the digitonin-solubilized receptors to exhibit high affinity agonist interactions. In contrast to membrane-bound receptors, digitonin-solubilized receptors also demonstrated a loss of guanine nucleotide regulation, as well as steep agonist:radioligand competition curves with slope factors of 1.0, suggesting a homogeneous population of agonist-binding sites. Interpreted within the context of a model of state interconversion for membrane-bound receptors, the results suggested that either muscarinic receptors of a single state were selectively solubilized, or that solubilization induced conversion of all receptors to a single low affinity state, possibly by removal of constituents necessary for assumption of a high affinity agonist conformation.",
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