Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes

Thirumalini Vaithianathan, Damodaran Narayanan, Maria T. Asuncion-Chin, Loice H. Jeyakumar, Jianxi Liu, Sidney Fleischer, Jonathan Jaggar, Alejandro Dopico

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Abstract

Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 ± 8, 334 ± 15, and 441 ± 27 pS in symmetric 300 mM Cs+ were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to ≤100 pS when Cs+ was replaced with Ca2+. The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NPo)-voltage curve was bell shaped, with NPo drastically decreasing when voltage was switched from -30 to -40 mV. NPo increased when intracellular calcium (Ca2+i) was raised within 0.1-100 μM to abruptly diminish with higher Ca2+ i. Thus maximal activity occurred within the Ca2+ i range found in rat cerebral artery myocytes under physiological conditions. NPo was reduced by ruthenium red (80 μM), increased monotonically by caffeine (0.1-5 mM) or ryanodine (0.05-5 μM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 μM), resistant to dantrolene (100 μM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume299
Issue number2
DOIs
StatePublished - Aug 1 2010

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Ryanodine Receptor Calcium Release Channel
Cerebral Arteries
Muscle Cells
Sarcoplasmic Reticulum
Dantrolene
Phenotype
Ruthenium Red
Ryanodine
Neomycin
Membranes
Digoxin
Caffeine
Fluorescent Antibody Technique
Smooth Muscle
Heparin
Real-Time Polymerase Chain Reaction
Protein Isoforms

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cell Biology

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Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes. / Vaithianathan, Thirumalini; Narayanan, Damodaran; Asuncion-Chin, Maria T.; Jeyakumar, Loice H.; Liu, Jianxi; Fleischer, Sidney; Jaggar, Jonathan; Dopico, Alejandro.

In: American Journal of Physiology - Cell Physiology, Vol. 299, No. 2, 01.08.2010.

Research output: Contribution to journalArticle

Vaithianathan, Thirumalini ; Narayanan, Damodaran ; Asuncion-Chin, Maria T. ; Jeyakumar, Loice H. ; Liu, Jianxi ; Fleischer, Sidney ; Jaggar, Jonathan ; Dopico, Alejandro. / Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes. In: American Journal of Physiology - Cell Physiology. 2010 ; Vol. 299, No. 2.
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