Eudistomin D and penaresin derivatives as modulators of ryanodine receptor channels and sarcoplasmic reticulum Ca2+ ATPase in striated muscle.

Eudistomin D (EuD) and penaresin (Pen) derivatives are bioactive alkaloids from marine sponges found to induce Ca(2+) release from striated muscle sarcoplasmic reticulum (SR). Although these alkaloids are believed to affect ryanodine receptor (RyR) gating in a "caffeine-like" manner, no single-channel study confirmed this assumption. Here, EuD and MBED (9-methyl-7-bromoeudistomin D) were contrasted against caffeine on their ability to modulate the SR Ca(2+) loading/leak from cardiac and skeletal muscle SR microsomes as well as the function of RyRs in planar bilayers. The effects of these alkaloids on [(3)H]ryanodine binding and SR Ca(2+) ATPase (SERCA) activity were also tested. MBED (1-5 µM) fully mimicked maximal activating effects of caffeine (20 mM) on SR Ca(2+) leak. At the single-channel level, MBED mimicked the agonistic action of caffeine on cardiac RyR gating (i.e., stabilized long openings characteristic of "high-open-probability" mode). EuD was a partial agonist at the maximal doses tested. The tested Pen derivatives displayed mild to no agonism on RyRs, SR Ca(2+) leak, or [(3)H]ryanodine binding studies. Unlike caffeine, EuD and some Pen derivatives significantly inhibited SERCA at concentrations required to modulate RyRs. Instead, MBED's affinity for RyRs (EC50 ∼ 0.5 µM) was much larger than for SERCA (IC50 > 285 µM). In conclusion, MBED is a potent RyR agonist and, potentially, a better choice than caffeine for microsomal and cell studies due to its reported lack of effects on adenosine receptors and phosphodiesterases. As a high-affinity caffeine-like probe, MBED could also help identify the caffeine-binding site in RyRs.

The Contractile Response to Oxytocin in Non-pregnant Rat Uteri Is Modified After the First Pregnancy.

During pregnancy, the uterus undergoes several modifications under the influence of hormonal and mechanical stimuli. We hypothesize that while most of these modifications are reverted during involution, some of the physiological properties of the uterus are permanently altered. To investigate this hypothesis, we conducted motility experiments to evaluate the contractility response of uterine tissue samples from non-pregnant virgin and proven breeder female rats to oxytocin (10-10 to 10-5 M). We found that the virgin tissue contracts more robustly than proven breeder tissue in the absence of oxytocin, yet with oxytocin, proven breeder samples displayed a significantly higher increase in activity. These results could depend on a more elevated expression of oxytocin receptor and/or on an alteration in the intracellular pathways affected by the activation of the oxytocin receptors. Here, we explored the impact of some structures involved in the management of intracellular calcium on the dose response to oxytocin recorded from virgin and proven breeder uterine strips. Specifically, we replicated the dose response experiments in low extracellular calcium (10 µM), in the presence of the intracellular calcium channel blocker ruthenium red (10 µM), and in the presence of the sarcoplasmic-endoplasmic reticulum calcium ATP-ase pump inhibitor, cyclopiazonic acid (10 µM). The results of these experiments suggest that also the expression of proteins that control intracellular calcium availability is affected by the experience of pregnancy. Molecular biology experiments will give us more detail on the magnitude of these expression changes.

Coupled gating of skeletal muscle ryanodine receptors is modulated by Ca2+, Mg2+, and ATP.

Coupled gating (synchronous openings and closures) of groups of skeletal muscle ryanodine receptors (RyR1), which mimics RyR1-mediated Ca(2+) release underlying Ca(2+) sparks, was first described by Marx et al. (Marx SO, Ondrias K, Marks AR. Science 281: 818-821, 1998). The nature of the RyR1-RyR1 interactions for coupled gating still needs to be characterized. Consequently, we defined planar lipid bilayer conditions where ∼25% of multichannel reconstitutions contain mixtures of coupled and independently gating RyR1. In ∼10% of the cases, all RyRs (2-10 channels; most frequently 3-4) gated in coupled fashion, allowing for quantification. Our results indicated that coupling required cytosolic solutions containing ATP/Mg(2+) and high (50 mM) luminal Ca(2+) (Ca(lum)) or Sr(2+) solutions. Bursts of coupled activity (events) started and ended abruptly, with all channels activating/deactivating within ∼300 µs. Coupled RyR1 were heterogeneous, where highly active RyR1 ("drivers") seemed open during the entire coupled event (P(o) = 1), while other RyR1s ("followers") displayed abundant flickering and smaller amplitude. Drivers mean open time increased with cytosolic Ca(2+) (Ca(cyt)) or caffeine, whereas followers flicker frequency was Ca(cyt) independent and more sensitive to inhibition by cytosolic Mg(2+). Coupled events were insensitive to varying lumen-to-cytosol Ca(2+) fluxes from ∼1 to 8 pA, which does not corroborate coupling of neighboring RyR1 by local Ca(2+)-induced Ca(2+) release. However, coupling requires specific Ca(lum) sites, as it was lost when Ca(lum) was replaced by luminal Ba(2+) or Mg(2+). In summary, coupled events reveal complex interactions among heterogeneous RyR1, differentially modulated by cytosolic ATP/Mg(2+), Ca(cyt), and Ca(lum,) which under cell-like ionic conditions may parallel synchronous RyR1 gating during Ca(2+) sparks.

Single ryanodine receptor channel basis of caffeine's action on Ca2+ sparks.

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca(2+) sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca(2+) level on both sides of the channel. Cytosolic Ca(2+) enhanced RyR2 caffeine affinity, whereas luminal Ca(2+) essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC(50) of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca(2+) spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca(2+) sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.

Ryanoids and imperatoxin affect the modulation of cardiac ryanodine receptors by dihydropyridine receptor Peptide A.

Ca(2+)-entry via L-type Ca(2+) channels (DHPR) is known to trigger ryanodine receptor (RyR)-mediated Ca(2+)-release from sarcoplasmic reticulum (SR). The mechanism that terminates SR Ca(2+) release is still unknown. Previous reports showed evidence of Ca(2+)-entry independent inhibition of Ca(2+) sparks by DHPR in cardiomyocytes. A peptide from the DHPR loop II-III (PepA) was reported to modulate isolated RyRs. We found that PepA induced voltage-dependent "flicker block" and transition to substates of fully-activated cardiac RyRs in planar bilayers. Substates had less voltage-dependence than block and did not represent occupancy of a ryanoid site. However, ryanoids stabilized PepA-induced events while PepA increased RyR2 affinity for ryanodol, which suggests cooperative interactions. Ryanodol stabilized Imperatoxin A (IpTx(A)) binding but when IpTx(A) bound first, it prevented ryanodol binding. Moreover, IpTx(A) and PepA excluded each other from their sites. This suggests that IpTx(A) generates a vestibular gate (either sterically or allosterically) that prevents access to the peptides and ryanodol binding sites. Inactivating gate moieties ("ball peptides") from K(+) and Na(+) channels (ShakerB and KIFMK, respectively) induced well resolved slow block and substates, which were sensitive to ryanoids and IpTx(A) and allowed, by comparison, better understanding of PepA action. The RyR2 appears to interact with PepA or ball peptides through a two-step mechanism, reminiscent of the inactivation of voltage-gated channels, which includes binding to outer (substates) and inner (block) vestibular regions in the channel conduction pathway. Our results open the possibility that "ball peptide-like" moieties in RyR2-interacting proteins could modulate SR Ca(2+) release in cells.

Trifluoperazine: a rynodine receptor agonist.

Trifluoperazine (TFP), a phenothiazine, is a commonly used antipsychotic drug whose therapeutic effects are attributed to its central anti-adrenergic and anti-dopaminergic actions. However, TFP is also a calmodulin (CaM) antagonist and alters the Ca(2+) binding properties of calsequestrin (CSQ). The CaM and CSQ proteins are known modulators of sarcoplasmic reticulum (SR) Ca(2+) release in ventricular myocytes. We explored TFP actions on cardiac SR Ca(2+) release in cells and single type-2 ryanodine receptor (RyR2) channel activity in bilayers. In intact and permeabilized ventricular myocytes, TFP produced an initial activation of RyR2-mediated SR Ca(2+) release and over time depleted SR Ca(2+) content. At the single channel level, TFP or nortryptiline (NRT; a tricyclic antidepressant also known to modify CSQ Ca(2+) binding) increased the open probability (Po) of CSQ-free channels with an EC(50) of 5.2 microM or 8.9 microM (respectively). This Po increase was due to elevated open event frequency at low drug concentrations while longer mean open events sustained Po at higher drug concentrations. Activation of RyR2 by TFP occurred in the presence or absence of CaM. TFP may also inhibit SR Ca uptake as well as increase RyR2 opening. Our results suggest TFP and NRT can alter RyR2 function by interacting with the channel protein directly, independent of its actions on CSQ or CaM. This direct action may contribute to the clinical adverse cardiac side effects associated with these drugs.

Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.

Flux regulation of cardiac ryanodine receptor channels.

The cardiac type 2 ryanodine receptor (RYR2) is activated by Ca2+-induced Ca2+ release (CICR). The inherent positive feedback of CICR is well controlled in cells, but the nature of this control is debated. Here, we explore how the Ca2+ flux (lumen-to-cytosol) carried by an open RYR2 channel influences its own cytosolic Ca2+ regulatory sites as well as those on a neighboring channel. Both flux-dependent activation and inhibition of single channels were detected when there were super-physiological Ca2+ fluxes (>3 pA). Single-channel results indicate a pore inhibition site distance of 1.2 +/- 0.16 nm and that the activation site on an open channel is shielded/protected from its own flux. Our results indicate that the Ca2+ flux mediated by an open RYR2 channel in cells (approximately 0.5 pA) is too small to substantially regulate (activate or inhibit) the channel carrying it, even though it is sufficient to activate a neighboring RYR2 channel.

Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP.

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca(2+) release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs(+) solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca(2+) release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca(2+)) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca(2+) (channel agonist) and counteracted by Mg(2+) (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca(2+) release is enhanced by high ATP-low Mg(2+) in the cytosol and by increased SR Ca(2+) load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.