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Identification and function of type-2 and type-3 ryanodine receptors in gut epithelial cells.

ABSTRACT: Reverse transcription-PCR (RT-PCR) techniques were used to identify the expression of ryanodine receptor (RyR) isoforms in gut epithelial cells. Restriction digest and sequence analysis of the PCR product showed the presence of RyR 2 and RyR 3. [3H]Ry binding studies on a microsome preparation, in a high-salt buffer, showed specific binding with an EC50 of 15 microM. In order to determine a potential functional role for these RyRs, we first characterized the response of the cells to acetylcholine. At all concentrations used acetylcholine induced sinusoidal cytosolic Ca2+ concentration ([Ca2+]i) oscillations. In response to 10(-4) M acetylcholine, levels of inositol 1,4,5-trisphosphate (InsP3) showed a peak of six times the basal level, at 30 s after stimulation. Application of caffeine alone failed to elicit a rise in cytosolic Ca2+. However, caffeine (5-50 mM) did rapidly and reversibly inhibit the acetylcholine-induced [Ca2+]i oscillations. The effects of Ry were more complex. Applied alone, Ry had no effect on the [Ca2+]i signal. When applied during agonist-evoked [Ca2+]i oscillations, Ry (10 microM) slowly blocked the response. In the continuous presence of Ry (10 microM) a short application of acetylcholine elicited a [Ca2+]i response that continued as oscillations even when the agonist was removed. The oscillations, in the presence of Ry (10 microM) but absence of agonist, were blocked either by removal of extracellular Ca2+ or by an application of a higher concentration of Ry (100 microM). These effects are consistent with the known use-dependence and dose-dependence for Ry action at the RyR. We conclude that the RyR 2 and RyR 3, identified by RT-PCR, play a central role in [Ca2+]i oscillations in gut epithelial cells.

SUBMITTER: Verma V

PROVIDER: S-EPMC1217789 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

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Project description: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 <or=100 pS when Cs(+) was replaced with Ca(2+). The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NP(o))-voltage curve was bell shaped, with NP(o) drastically decreasing when voltage was switched from -30 to -40 mV. NP(o) increased when intracellular calcium (Ca(2+)(i)) was raised within 0.1-100 microM to abruptly diminish with higher Ca(2+)(i). Thus maximal activity occurred within the Ca(2+)(i) range found in rat cerebral artery myocytes under physiological conditions. NP(o) was reduced by ruthenium red (80 muM), increased monotonically by caffeine (0.1-5 mM) or ryanodine (0.05-5 microM), 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 muM), resistant to dantrolene (100 muM), 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.

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Identification and function of type-2 and type-3 ryanodine receptors in gut epithelial cells.

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