Ryanodine receptor dysfunction in hearts of streptozotocin-induced diabetic rats.

Studies have shown that evoked calcium release from sarcoplasmic reticulum is compromised in diabetic rat hearts. The present study was undertaken to determine whether this decrease might be ascribed to a reduction in expression and/or alteration in function of ryanodine receptor (RyR2) and whether changes could be minimized with insulin treatment. Hearts were isolated from 4- and 6-week streptozotocin (STZ)-induced diabetic, 4-week diabetic/2-week insulin-treated, and age-matched control rats. RyR2 mRNA and protein levels were determined using reverse transcription-polymerase chain reactions and polyacrylamide gel electrophoresis, respectively, whereas the functional integrity of RyR2 was assessed from their ability to bind [3H]ryanodine. RyR2 protein was unchanged with up to 6 weeks of untreated STZ-induced diabetes. Two weeks of insulin treatment initiated after 4 weeks of diabetes increased RyR2 mRNA levels by 42% and RyR2 protein levels by 45 to 61%. At equivalent amounts, RyR2 protein from 4-week STZ-induced diabetic rat hearts bound 9% less [3H]ryanodine than age-matched control rats (74.1 +/- 3.9 versus 67.4 +/- 3.4 fmol/microg RyR2), whereas that from 6-week STZ-diabetic rats bound 36% less than control rats (47.9 +/- 4.8 versus 74.2 +/- 4.5 fmol/microg RyR2, p < 0.05). RyR2 from insulin-treated animals bound significantly less [3H]ryanodine than control rats (65.2 +/- 4.9 fmol/microg RyR2, p < 0.05). Apparent affinity of ryanodine for RyR2 was similar among all groups (K(d) approximately 1.04 +/- 0.08 nM). Because expression did not change significantly but ryanodine binding decreased, these data suggest that the functional integrity of RyR2 is compromised in diabetic rat hearts, and these changes can be attenuated with 2 weeks of insulin treatment.

Effects of ryanodine on calcium sparks in cut twitch fibres of Rana temporaria.

1. Localized calcium release events (calcium sparks) were studied in voltage-clamped cut twitch fibres of Rana temporaria. 2. A histogram of thousands of spontaneous sparks displayed a monotonically decreasing amplitude distribution from the low to the high limit of > 7 DeltaF/F(0) units. 3. Several effects of low micromolar concentrations of ryanodine (0.4-2 microM) on spontaneous sparks, reproducing the agent's effects on single ryanodine receptor channel current in bilayers, were observed collectively for the first time in live fibres, namely (a) increases in spark frequency followed by (b) conversions of sparks into steady glows lasting tens of seconds, (c) occasional interruptions of the glows by brief gaps of darkness, and (d) abolition of sparks at the locations of the glows. The glow could reflect the incessant Ca(2+) flux through a single (or a few) calcium release channel locked in the semi-open state, which was allowed to make occasional transitions to the closed state but not to the fully open state. 4. Higher concentrations of ryanodine (> or = 20 microM) suppressed the spontaneous sparks effectively and permanently, presumably by deactivating the ryanodine receptors. 5. Depolarization-evoked sparks elicited with small pulses had higher frequencies and larger amplitudes than spontaneous sparks and were abolished by both concentrations of ryanodine. 6. With 1-2 microM ryanodine, however, a uniform non-sparking calcium release persisted during the pulse, with the globally averaged increase in fluorescence intensity being about half that of the control. A possible origin of this non-sparking release may be related to the structural coupling between the voltage sensors and the ryanodine receptors that can exist only in live fibres but not in the bilayer preparation.

Tectoridins modulate skeletal and cardiac muscle sarcoplasmic reticulum calcium-release channels.

The isoflavones tectoridin (TTR) and 3'-hydroxy TTR (3'-TTR) were isolated from an Ayurvedic herbal preparation Vacä and evaluated for their affinity and effect on ryanodine receptors (RyR) using junctional sarcoplasmic reticulum vesicles (JSRVs). In [(3)H]ryanodine displacement binding affinity assays, TTR and 3'-TTR exhibited IC(50) values of 17.3 +/- 1.3 microM (K(d) = 6.7 +/- 0.4 microM) and 6.6 +/- 1.4 microM (K(d) = 2.4 +/- 0.2 microM), respectively, for fast skeletal muscle RyR (RyR1) compared with an IC(50) value for ryanodine of 6.2 +/- 0.4 nM (K(d) = 2.4 nM). TTR demonstrated a 3-fold higher affinity for cardiac RyR (RyR2) [IC(50) value of 5.2 +/- 0.6 microM (K(d) = 0.95 +/- 0.3 microM)] than for RyR1. The displacement isotherms for both TTRs paralleled that for ryanodine, consistent with the notion that all three are likely binding to similar site(s) on the receptors. Calcium efflux from and calcium influx into JSRVs were used to measure function effects of TTRs on binding to RyR. In calcium efflux assays, TTR (up to 1 mM) enhanced the release of (45)Ca(2+) from JSRVs in a concentration-dependent manner (EC(50act) of 750 microM). Higher concentrations deactivated (partially closed) RyR1. 3'-TTR had similar effects, but was approximately 2-fold more potent, exhibiting an EC(50act) value of 480 microM. Using passive calcium influx assays, TTR activated and deactivated RyR1 in a time- and concentration-dependent manner. The aglycone tectorigenin also was effective in displacing [(3)H]ryanodine from RyR1 but not from RyR2. These results demonstrate that TTRs are capable of interacting at ryanodine binding sites to differentially modulate fast skeletal and cardiac calcium-release channels.

Ryanodine and inositol trisphosphate receptors are differentially distributed and expressed in rat parotid gland.

The present study examines the cellular distribution of the ryanodine receptor/channel (RyR) and inositol 1,4,5-trisphosphate receptor (InsP3R) subtypes in parotid acini. Using fluorescently labelled 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid glycyl-ryanodine (BODIPYtrade mark-ryanodine) and confocal microscopy, RyRs were localized primarily to the perinuclear region (basal pole) of the acinar cell. Ryanodine, Ruthenium Red, cAMP and cADP ribose (cADPR) competed with BODIPY-ryanodine, resulting in a reduction in the fluorescence signal. However, inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3] did not alter the binding of BODIPY-ryanodine. Using receptor-subtype-specific antisera, InsP3Rs (types I, II and III) were located predominantly in the apical pole of the parotid cell. The presence of these three subtypes was confirmed using reverse transcriptase PCR with RNA-specific oligonucleotide probes. Binding studies using a parotid cell-membrane fraction identified and characterized RyRs and InsP3Rs in terms of binding affinity (Kd) and maximum binding capacity (Bmax) and confirmed that cADPR displaces ryanodine from its binding sites. Ruthenium Red and 8-Br-cADP-ribose blocked Ca2+ release in permeabilized acinar cells in response to cADPR and cAMP or forskolin, whereas Ins(1,4,5)P3-induced Ca2+ release was unaffected. The localization of the RyRs and InsP3Rs in discrete regions endow broad areas of the parotid cell with ligand-activated Ca2+ channels. The consequences of the dual activation of the RyRs and InsP3Rs by physiologically relevant stimuli such as noradrenaline (norepinephrine) are considered in relation to Ca2+ signalling in the parotid gland.

Structure-function relationships among ryanodine derivatives. Pyridyl ryanodine definitively separates activation potency from high affinity.

Ryanodine derivatives are differentially effective on the two limbs of the ryanodine concentration-effect curve. This study comparing ryanodine, ryanodol, and pyridyl ryanodine and nine C10Oeq esters of them focuses on structure-function relations underlying their differential effectiveness. Ryanodol and pyridyl ryanodine had significantly lower affinities than ryanodine, but their EC50act values (concentration of ryanoid that induces one-half of full efficacy), potencies, and efficacies were not diminished in like fashion. Ryanodine and ryanodol were partial agonists, whereas pyridyl ryanodine was a full agonist, having a diminished deactivation potency. C10Oeq esterifications enhanced affinities and efficacies of the base ryanoids. The C10-Oeq ester derivatives of ryanodine and pyridyl ryanodine, but not those of ryanodol, lost their capacity to deactivate RyR1s. Thus, affinity differences among ryanoids clearly do not predicate functional differences as regards activation of Ca2+ release channels. The pyrrole carboxylate on the C3 of ryanodine is dispensable to ryanoid activation of Ca2+ release channels. Ryanodol lacks this ring, but it nevertheless effects substantial activation. Moreover, its C10-Oeq esters display full efficacy. The increased ability of all the C10-Oeq derivatives to release Ca2+ from the vesicles strengthens their role in directly impeding deactivation of RyR1, perhaps by interaction with some component within the transmembrane ionic flux pathway.

Ryanodine receptor expression is associated with intracellular Ca2+ release in rat parotid acinar cells.

The ryanodine receptor mediates intracellular Ca2+ mobilization in muscle and nerve, but its physiological role in nonexcitable cells is less well defined. Like adenosine 3',5'-cyclic monophosphate and inositol 1,4,5-trisphosphate, cyclic ADP-ribose (0.3-5 microM) and ADP (1-25 microM) produced a concentration-dependent rise in cytosolic Ca2+ in permeabilized rat parotid acinar cells. Adenosine and AMP were less effective. Ryanodine markedly depressed the Ca2+-mobilizing action of the adenine nucleotides and forskolin in permeabilized cells and was likewise effective in depressing the action of forskolin in intact cells. Cyclic ADP-ribose-evoked Ca2+ release was enhanced by calmodulin and depressed by W-7, a calmodulin inhibitor. A fluorescently labeled ligand, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indac ene-3-propionic acid-glycyl ryanodine, was synthesized to detect the expression and distribution of ryanodine receptors. In addition, ryanodine receptor expression was detected in rat parotid cells with a sequence highly homologous to a rat skeletal muscle type 1 and a novel brain type 1 ryanodine receptor. These findings demonstrate the presence of a ryanodine-sensitive intracellular Ca2+ store in rat parotid cells that shares many of the characteristics of stores in muscle and nerve and may mediate Ca2+-induced Ca2+ release or a modified form of this process.

Structural components of ryanodine responsible for modulation of sarcoplasmic reticulum calcium channel function.

Comparative molecular field analysis (CoMFA) was used to analyze the relationship between the structure of a group of ryanoids and the modulation of the calcium channel function of the ryanodine receptor. The conductance properties of ryanodine receptors purified from sheep heart were measured using the planar, lipid bilayer technique. The magnitude of the ryanoid-induced fractional conductance was strongly correlated to specific structural loci on the ligand. Briefly, electrostatic effects were more prominent than steric effects. The 10-position of the ryanoid had the greatest influence on fractional conductance. Different regions of the ligand have opposing effects on fractional conductance. For example, steric bulk at the 10-position is correlated with decreased fractional conductance, whereas steric bulk at the 2-position (isopropyl position) is correlated with increased fractional conductance. In contrast to fractional conductance, the 3-position (the pyrrole locus) had the greatest influence on ligand binding, whereas the 10-position had comparatively little influence on binding. Two possible models of ryanodine action, a direct (or channel plug) mechanism and an allosteric mechanism, were examined in light of the CoMFA. Taken together, the data do not appear to be consistent with direct interaction between ryanodine and the translocating ion. The data appear to be more consistent with an allosteric mechanism. It is suggested the ryanoids act by inducing or stabilizing a conformational change in the ryanodine receptor that results in the observed alterations in cation conductance.

Amiodarone instilled into the canine pericardial sac migrates transmurally to produce electrophysiologic effects and suppress atrial fibrillation.

INTRODUCTION: We investigated whether amiodarone delivered into the pericardial sac exerted an effect on atrial and ventricular refractoriness, impulse generation, and conduction and on induced atrial fibrillation. METHODS AND RESULTS: All animals were anesthetized with alpha-chloralose. After a sternotomy, the pericardium was opened and cradled to produce a "container" of approximately 75 mL. Part I experimental animals received amiodarone, 0.5, 1.0, or 5.0 mg/mL, dissolved in 3 mL polysorbate 80 and 5% dextrose in water (D5W) instilled into their pericardial sac for 3-hour intervals. Part II experimental animals received either 1.0 or 5.0 mg/mL of amiodarone. Control dogs received a pericardial solution of 3 mL polysorbate 80 in D5W. Pre- and postinstillation electrophysiologic studies were performed. In part I, the increase in sinus cycle length, 1:1 AV conduction, and effective refractory period (ERP) of atrium, right ventricular (RV) and left ventricular epicardium, and RV endocardium were significantly greater in animals receiving amiodarone compared with controls. Amiodarone concentrations in the tissue samples were highest in the superficial sites of the atria, sinoatrial node, and ventricular epicardial samples and lowest in the interventricular septum. Only trace concentrations of amiodarone and no desethylamiodarone were found in the blood samples. In part II, atrial ERP significantly increased in the animals receiving amiodarone, and the number of episodes of sustained atrial fibrillation that could be induced decreased. CONCLUSIONS: Amiodarone instilled into the pericardial sac migrates transmurally to produce significant electrophysiologic effects at superficial sites and appears to suppress electrically induced atrial fibrillation.