Ryanodine Receptor Blocker Research Articles

Enhanced pulsatile pressure accelerates vascular smooth muscle migration: implications for atherogenesis of hypertension

Clinical studies have suggested that pulsatile pressure is an independent risk factor for atherosclerosis. However, it is unknown whether enhanced pulsatile pressure per se directly accelerates vascular smooth muscle cell (VSMC) migration, an important process of atherosclerosis. Using our original Pressure-loading system with a Boyden chamber, we examined the direct effects of variable pressures and pulse rates on migration of rat aortic VSMCs in vitro. High pulse pressure (180/90 mmHg, pulsatile vs. 180 mmHg, static), high mean pressure (180/90 vs. 90/0 mmHg, with the same pulse pressure), wide pulse pressure (190/110 vs. 170/130 mmHg, with the same mean pressure), and high pulse rate (120 vs. 40 per min) significantly accelerated the VSMC migration (1.35, 2.38, 1.38 and 1.27-fold, respectively). The increase in intracellular calcium levels measured by fura-2/AM fluorescence was proportional to the magnitude of pressure loaded. The pressure-promoted VSMC migration was significantly inhibited by a phospholipase-C inhibitor U-73122 or a calmodulin inhibitor W-7. Inositol 1,4,5-trisphosphate receptor blockers 2-aminoethoxydiphenyl borate or xestospongin-C significantly inhibited the VSMC migration, whereas a ryanodine receptor blocker ryanodine had no effects. Furthermore, a calcium channel blocker (CCB), azelnidipine, and an angiotensin type-1 receptor blocker, olmesartan, also significantly inhibited the VSMC migration. These results provide direct evidence for the pro-atherogenic effects of enhanced pulsatile pressure and also suggest that the anti-atherogenic actions of CCBs and angiotensin type-1 receptor blockers are mediated in part by their direct inhibitory effects on VSMC migration in addition to their anti-hypertensive effects.

PLC‐dependent intracellular Ca2+ release was associated with C6‐ceramide‐induced inhibition of Na+ current in rat granule cells

In this report, the effects of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)), two types of main K(+) current [outward rectifier delayed K(+) current (I(K)) and outward transient K(+) current (I(A))], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01-100 microM, ceramide produced a dose-dependent and reversible inhibition of I(Na) without alteration of the steady-state activation and inactivation properties. Treatment with C(2)-ceramide caused a similar inhibitory effect on I(Na). However, dihydro-C(6)-ceramide failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent, 1,2-bis (2-aminophenoxy) ethane-N, N, N9, N9-tetraacetic acid, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with ryanodine receptor blocker induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide. Intracellular application of GTPgammaS also induced a gradual decrease in I(Na) amplitude, while GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Furthermore, the C(6)-ceramide effect on I(Na) was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C(6)-ceramide decreased cell viability and blocking I(Na) by tetrodotoxin did not mimic the effect of C(6)-ceramide, and inhibiting intracellular Ca(2+) release by dantrolene could not decrease the C(6)-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca(2+) release through the ryanodine-sensitive Ca(2+) receptor may be responsible for the C(6)-ceramide-induced inhibition of I(Na), which does not seem to be associated with C(6)-ceramide-induced granule neuron death.

Alcohol Induces Relaxation of Rat Thoracic Aorta and Mesenteric Arterial Bed

The aim of this study was to investigate the effect of alcohol on rat artery and its underlying mechanism. The tension of isolated Sprague-Dawley rat thoracic aortic rings and the pressure of rat mesenteric arterial beds perfused with different concentrations of alcohol (0.1-7.0 per thousand) were measured. At resting tensions, alcohol caused a concentration-dependent relaxation on endothelium-denuded aortic rings precontracted with KCl (6 x 10(-2) mol/L) or phenylephrine (PE, 10(-6) mol/L), and this effect was most evident on rings at a resting tension of 3 g. Alcohol induced much less vasodilation on endothelium-intact rings. Alcohol inhibited the CaCl(2)-induced contraction of endothelium-denuded aortic rings precontracted with KCl or PE. Incubation of rings with dantrolene (5 x 10(-5) mol/L), a ryanodine receptor blocker, or 2-aminoethyl diphenylborinate (7.5 x 10(-5) mol/L), an IP(3) receptor blocker, attenuated the vasodilating effect of alcohol on rings precontracted with PE. Alcohol also concentration-dependently relaxed rat mesenteric arterial beds precontracted with KCl (6 x 10(-2) mol/L) or PE (10(-5) mol/L), which was more potent on endothelium-denuded than on endothelium-intact beds. Alcohol has a vasodilating effect on rat artery depending on the resting tension. Both extracellular and intracellular Ca(2+) mobilization of vascular smooth muscle cells are involved in the vascular effect of alcohol.

Activated Nuclear Metabotropic Glutamate Receptor mGlu5 Couples to Nuclear Gq/11 Proteins to Generate Inositol 1,4,5-Trisphosphate-mediated Nuclear Ca2+ Release

Recently we have shown that the metabotropic glutamate 5 (mGlu5) receptor can be expressed on nuclear membranes of heterologous cells or endogenously on striatal neurons where it can mediate nuclear Ca2+ changes. Here, pharmacological, optical, and genetic techniques were used to show that upon activation, nuclear mGlu5 receptors generate nuclear inositol 1,4,5-trisphosphate (IP3) in situ. Specifically, expression of an mGlu5 F767S mutant in HEK293 cells that blocks Gq/11 coupling or introduction of a dominant negative Galphaq construct in striatal neurons prevented nuclear Ca2+ changes following receptor activation. These data indicate that nuclear mGlu5 receptors couple to Gq/11 to mobilize nuclear Ca2+. Nuclear mGlu5-mediated Ca2+ responses could also be blocked by the phospholipase C (PLC) inhibitor, U73122, the phosphatidylinositol (PI) PLC inhibitor 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3), or by using small interfering RNA targeted against PLCbeta1 demonstrating that PI-PLC is involved. Direct assessment of inositol phosphate production using a PIP2/IP3 "biosensor" revealed for the first time that IP3 can be generated in the nucleus following activation of nuclear mGlu5 receptors. Finally, both IP3 and ryanodine receptor blockers prevented nuclear mGlu5-mediated increases in intranuclear Ca2+. Collectively, this study shows that like plasma membrane receptors, activated nuclear mGlu5 receptors couple to Gq/11 and PLC to generate IP3-mediated release of Ca2+ from Ca2+-release channels in the nucleus. Thus the nucleus can function as an autonomous organelle independent of signals originating in the cytoplasm, and nuclear mGlu5 receptors play a dynamic role in mobilizing Ca2+ in a specific, localized fashion.

Release‐enhancing pre‐synaptic muscarinic and nicotinic receptors co‐exist and interact on dopaminergic nerve endings of rat nucleus accumbens

Dopaminergic nerve endings in the corpus striatum possess nicotinic (nAChRs) and muscarinic cholinergic receptors (mAChRs) mediating release of dopamine (DA). Whether nAChRs and mAChRs co-exist and interact on the same nerve endings is unknown. We here investigate on these possibilities using rat nucleus accumbens synaptosomes pre-labeled with [(3)H]DA and exposed in superfusion to cholinergic receptor ligands. The mixed nAChR-mAChR agonists acetylcholine (ACh) and carbachol provoked [(3)H]DA release partially sensitive to the mAChR antagonist atropine but totally blocked by the nAChR antagonist mecamylamine. Addition of the mAChR agonist oxotremorine at the minimally effective concentration of 30 micromol/L, together with 3, 10, or 100 micromol/L (-)nicotine provoked synergistic effect on [(3)H]DA overflow. The [(3)H]DA overflow elicited by 100 micromol/L (-)nicotine plus 30 micromol/L oxotremorine was reduced by atropine down to the release produced by (-)nicotine alone and it was abolished by mecamylamine. The ryanodine receptor blockers dantrolene or 8-bromo-cADP-ribose, but not the inositol 1,4,5-trisphosphate receptor blocker xestospongin C inhibited the (-)nicotine/oxotremorine evoked [(3)H]DA overflow similarly to atropine. This overflow was partly sensitive to 100 nmol/L methyllycaconitine which did not prevent the synergistic effect of (-)nicotine/oxotremorine. Similarly to (-)nicotine, the selective alpha4beta2 nAChR agonist RJR2403 exhibited synergism when added together with oxotremorine. To conclude, in rat nucleus accumbens, alpha4beta2 nAChRs exert a permissive role on the releasing function of reportedly M(5) mAChRs co-existing on the same dopaminergic nerve endings.

Inhibition of Ryanodine Receptors by 4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) in Canine Pulmonary Arterial Smooth Muscle Cells

Ryanodine is a selective ryanodine receptor (RyR) blocker, with binding dependent on RyR opening. In whole-cell studies, ryanodine binding can lock the RyR in an open-conductance state, short-circuiting the sarcoplasmic reticulum, which restricts studies of inositol-1,4,5-trisphosphate receptor (InsP3R) activity. Other RyR blockers have nonselective effects that also limit their utility. 4-(2-aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) blocks RyR-elicited Ca2+ increases in skeletal and cardiac muscle; yet, its actions on smooth muscle are unknown. Canine pulmonary arterial smooth muscle cells (PASMCs) express both RyRs and InsP3Rs; thus, we tested the ability of FLA 365 to block RyR- and serotonin-mediated InsP3R-elicited Ca2+ release by imaging fura-2-loaded PASMCs. Acute exposure to 10 mM caffeine, a selective RyR activator, induced Ca2+ increases that were reversibly reduced by FLA 365, with an estimated IC50 of approximately 1 to 1.5 microM, and inhibited by 10 microM ryanodine or 10 microM cyclopiazonic acid. FLA 365 also blocked L-type Ca2+ channel activity, with 10 microM reducing Ba2+ current amplitude in patch voltage-clamp studies to 54 +/- 6% of control and 100 microM FLA 365 reducing membrane current to 21 +/- 6%. InsP3R-mediated Ca2+ responses elicited by 10 microM 5-hydroxytryptamine (serotonin) in canine PASMCs and 100 microM carbachol in human embryonic kidney (HEK)-293 cells were not reduced by 2 microM FLA 365, but they were reduced by 20 microM FLA 365 to 76 +/- 9% of control in canine PASMCs and 52 +/- 1% in HEK-293 cells. Thus, FLA 365 preferentially blocks RyRs with limited inhibition of L-type Ca2+ channels or InsP3R in canine PASMCs.

Changes in the functioning of the electromechanical connection during tetanic contraction

The functioning of the electromechanical connection during tetanic contraction in frog skeletal muscle was studied. Analysis using caffeine, calcium-free medium, the ryanodine receptor blocker dantrolene, and the Ca-ATPase inhibitor thapsigargin showed that the initial increase in tetanus, as in twitch contractions, did not require the presence of calcium ions in the surrounding medium, which is in agreement with published data. Contraction was accompanied by activation of potential-dependent release of calcium from the sarcoplasmic reticulum. In contrast, the secondary rise phase and/or the duration of the tetanus plateau were critically dependent on the present of Ca2+ in the surrounding medium. Given that contraction in this situation was inhibited by dantrolene, activation of prolonged contraction was also mediated by calcium released from the sarcoplasmic reticulum, though ryanodine receptors were now activated not by changes in the membrane potential but by the influx of external calcium. Thus, external calcium plays a significant role in the formation of prolonged contractile responses, providing for longer-lasting maintenance of power in contracted muscles.

Complex interplay between glutamate receptors and intracellular Ca2+ stores during ischaemia in rat spinal cord white matter

Electrophysiological recordings of propagated compound action potentials (CAPs) and axonal Ca(2+) measurements using confocal microscopy were used to study the interplay between AMPA receptors and intracellullar Ca(2+) stores in rat spinal dorsal columns subjected to in vitro combined oxygen and glucose deprivation (OGD). Removal of Ca(2+) or Na(+) from the perfusate was protective after 30 but not 60 min of OGD. TTX was ineffective with either exposure, consistent with its modest effect on ischaemic depolarization. In contrast, AMPA antagonists were very protective, even after 60 min of OGD where 0Ca(2+) + EGTA perfusate was ineffective. Similarly, blocking ryanodine receptor-mediated Ca(2+) mobilization from internal stores (0Ca(2+) + nimodipine or 0Ca(2+) + ryanodine), or inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) release (block of group 1 metabotropic glutamate receptors with 1-aminoindan-1,5-dicarboxylic acid, inhibition of phospholipase C with U73122 or IP(3) receptor block with 2APB; each in 0Ca(2+)) were each very protective, with the combination resulting in virtually complete functional recovery after 1 h OGD (97 +/- 32% CAP recovery versus 4 +/- 6% in artificial cerebrospinal fluid). AMPA induced a rise in Ca(2+) concentration in normoxic axons, which was greatly reduced by blocking ryanodine receptors. Our data therefore suggest a novel and surprisingly complex interplay between AMPA receptors and Ca(2+) mobilization from intracellular Ca(2+) stores. We propose that AMPA receptors may not only allow Ca(2+) influx from the extracellular space, but may also significantly influence Ca(2+) release from intra-axonal Ca(2+) stores. In dorsal column axons, AMPA receptor-dependent mechanisms appear to exert a greater influence than voltage-gated Na(+) channels on functional outcome following OGD.

Androgens Induce Increases in Intracellular Calcium Via a G Protein—Coupled Receptor in LNCaP Prostate Cancer Cells

The receptor mechanism of testosterone-induced nongenomic Ca2+ signaling in prostate cancer cells is poorly understood. In this study we investigated androgen-induced intracellular Ca2+ increases in LNCaP human prostate cancer cells with Fura-2 as a Ca2+ probe. 5alpha-dihydrotestosterone (DHT) produced fast and transient increases in intracellular Ca2+ in LNCaP cells in a concentration-dependent manner. These effects were abolished by extracellular Ca2+ removal or pretreatment with L-type Ca2+ channel inhibitors (nifedipine, verapamil, and diltiazem). Pretreatment with endoplasmic reticulum ryanodine receptor blocker (procaine) or phospholipase C inhibitor (neomycin sulfate) did not alter DHT-induced Ca2+ influx. The concentration of Ca2+ was also increased by impermeable testosterone conjugated to bovine serum albumin. Neither an antagonist of intracellular androgen receptors (cyproterone acetate) nor a protein synthesis inhibitor (cycloheximide) affected this fast Ca2+ influx. Furthermore, the effect of DHT was abolished in cells incubated with a G protein inhibitor (pertussis toxin) and a nonhydrolyzable analog of guanosine triphosphate (guanosine 5-[beta-thio]disphosphate) but not in cells incubated with the tyrosine kinase inhibitor genistein. These results indicate that androgens induced an L-type calcium channel-dependent intracellular Ca2+ increase in LNCaP prostate cancer cells. The rapid responses triggered by DHT did not appear to be mediated through classic intracellular androgen receptors, c-Src kinase-androgen receptor complex, or sex hormone-binding globulin but through a G protein-coupled receptor in LNCaP prostate cancer cells. These results may provide a new explanation for progression of prostate cancer.

Nervous control of ciliary beating by Cl-, Ca2+ and calmodulin inTritonia diomedea

In vertebrates, motile cilia line airways, oviducts and ventricles. Invertebrate cilia often control feeding, swimming and crawling, or gliding. Yet control and coordination of ciliary beating remains poorly understood. Evidence from the nudibranch mollusc, Tritonia diomedea, suggests that locomotory ciliated epithelial cells may be under direct electrical control. Here we report that depolarization of ciliated pedal epithelial (CPE) cells increases ciliary beating frequency (CBF), and elicits CBF increases similar to those caused by dopamine and the neuropeptide, TPep-NLS. Further, four CBF stimulants (zero external Cl(-), depolarization, dopamine and TPep-NLS) depend on a common mode of action, viz. Ca(2+) influx, possibly through voltage-gated Ca(2+) channels, and can be blocked by nifedipine. Ca(2+) influx alone, however, does not provide all the internal Ca(2+) necessary for CBF change. Ryanodine receptor (RyR) channel-gated internal stores are also necessary for CBF excitation. Caffeine can stimulate CBF and is sensitive to the presence of the RyR blocker dantrolene. Dantrolene also reduces CBF excitation induced by dopamine and TPep-NLS. Finally, W-7 and calmidazolium both block CBF excitation by caffeine and dopamine, and W-7 is effective at blocking TPep-NLS excitation. The effects of calmidazolium and W-7 suggest a role for Ca(2+)-calmodulin in regulating CBF, either directly or via Ca(2+)-calmodulin dependent kinases or phosphodiesterases. From these results we hypothesize dopamine and TPep-NLS induce depolarization-driven Ca(2+) influx and Ca(2+) release from internal stores that activates Ca(2+)-calmodulin, thereby increasing CBF.

Arachidonic acid is a physiological activator of the ryanodine receptor in pancreatic β-cells

Pancreatic β-cells have ryanodine receptors but little is known about their physiological regulation. Previous studies have shown that arachidonic acid releases Ca 2+ from intracellular stores in β-cells but the identity of the channels involved in the Ca 2+ release has not been elucidated. We studied the mechanism by which arachidonic acid induces Ca 2+ concentration changes in pancreatic β-cells. Cytosolic free Ca 2+ concentration was measured in fura-2-loaded INS-1E cells and in primary β-cells from Wistar rats. The increase of cytosolic Ca 2+ concentration induced by arachidonic acid (150 μM) was due to both Ca 2+ release from intracellular stores and influx of Ca 2+ from extracellular medium. 5,8,11,14-Eicosatetraynoic acid, a non-metabolizable analogue of arachidonic acid, mimicked the effect of arachidonic acid, indicating that arachidonic acid itself mediated Ca 2+ increase. The Ca 2+ release induced by arachidonic acid was from the endoplasmic reticulum since it was blocked by thapsigargin. 2-Aminoethyl diphenylborinate (50 μM), which is known to inhibit 1,4,5-inositol-triphosphate-receptors, did not block Ca 2+ release by arachidonic acid. However, ryanodine (100 μM), a blocker of ryanodine receptors, abolished the effect of arachidonic acid on Ca 2+ release in both types of cells. These observations indicate that arachidonic acid is a physiological activator of ryanodine receptors in β-cells.

FLA 365 as an inhibitor of ryanodine receptors and L‐type Ca 2+ channels in arterial myocytes

Ryanodine (RY) is a highly selective ryanodine receptor (RyR) blocker, but RY binding is dependent on RyR opening. In whole-cell studies, RY binding can lock the RyR in an open-conductance state, short-circuiting the sarcoplasmic reticulum (SR). This restricts studies of InsP3 receptor (InsP3R) activity. Other RyR blockers also have non-selective effects that also limit their utility. FLA 365 ([2,6-dichloro-4-dimethyl-aminophenyl] isopropylamine) blocks RyR elicited Ca2+ increases in skeletal and cardiac muscle, yet its actions on smooth muscle are unknown. Canine pulmonary arterial smooth muscle cells (PASMCs) express both RyRs and InsP3Rs; thus, we tested the ability of FLA 365 (FLA) to block RyR and InsP3R elicited Ca2+ release by imaging fura-2 loaded PASMCs. Acute exposure to 10 mM caffeine, a selective RyR activator, induced Ca2+ increases that were significantly reduced by 20 μM FLA, which was reversible. 10–100 μM FLA 365 reduced Ba2+ currents through L-type Ca2+ channels in patch voltage-clamp studies, similar to phenylalkylamines. 10 μM 5-HT, which activates InsP3Rs, induced Ca2+ increases of equivalent amplitude in the absence or presence of FLA. Thus, FLA blocks RyRs and L-type Ca2+ channels with limited impact on InsP3R signaling in PASMCs. NIH P20RR15518 from NCRR & HL49254 (JRH), PO1ES11269 & 2PO AR17605 (INP), HL10476, AI55462 & UM faculty fellowship (SMW).

Insulin‐like Growth Factors (IGF) I and II Utilize Different Calcium Signaling Pathways in a Primary Human Parathyroid Cell Culture Model

In most cell types, influx of calcium (Ca2+) induces a growth or secretory response. The opposite occurs in parathyroid (PTH), cells where there is an inverse relationship between intracellular Ca2+ concentration and PTH secretion. We have examined the effects of calcium channel and metabolism modulators on insulin-like growth factors (IGFs) in a parathyroid cell culture model. Cell cultures were prepared from 9 patients undergoing operation for hyperparathyroidism. Following adhesion, the cells were transferred to serum-free medium and dosed with IGF I, II +/- ethyleneglycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), nifedipine, nickel, 2-aminoethoxy-diphenylborate (2-APB), or dantrolene. Proliferation (96 hours) was assessed by measuring tritiated thymidine incorporation and PTH release (1 and 3 hours) assayed by IRMA. Both IGF I and II increased DNA synthesis to 162.8% +/- 10.6% (SEM) and 131.1% +/- 7.7%, respectively (P < 0.05). EGTA at 0.2 mmol (ionized Ca2+ 0.2 mmol) did not affect the response to both IGFs. EGTA at 2 mmol (ionized Ca2+ 0 mmol) reduced the DNA synthesis of IGF I and II to 29% and 26%, respectively (P < 0.05). Nifedipine and nickel (nonspecific Ca2+ channel blocker) were equally potent in negating the mitogenic effects of both IGFs. 2-APB (IP3R blocker) reduced the basal DNA synthesis to 51.3% +/- 8.4% but had no effect on either IGF. Dantrolene (ryanodine receptor blocker) negated IGF II induced mitogenisis (74.2% +/- 6.7%) and partially inhibited IGF I mitogenesis (123% +/- 6%) (P < 0.05). The rate of PTH secretion was greater after IGF II stimulation than after IGF I stimulation. IGFs I and II induce mitogenesis by different calcium signaling pathways. These data suggest that parathyroid cells may utilize different calcium signaling pathways to distinguish growth factors and serum calcium changes.

Eugenol-induced contractions of saponin-skinned fibers are inhibited by heparin or by a ryanodine receptor blocker

The effects of eugenol on the sarcoplasmic reticulum (SR) and contractile apparatus of chemically skinned skeletal muscle fibers of the frog Rana catesbeiana were investigated. In saponin-skinned fibers, eugenol (5 mmol/L) induced muscle contractions, probably by releasing Ca(2+) from the SR. The Ca(2+)-induced Ca(2+) release blocker ruthenium red (10 micromol/L) inhibited both caffeine- and eugenol-induced muscle contractions. Ryanodine (200 micromol/L), a specific ryanodine receptor/Ca(2+) release channel blocker, promoted complete inhibition of the contractions induced by caffeine, but only partially blocked the contractions induced by eugenol. Heparin (2.5 mg/mL), an inositol 1,4,5-trisphosphate (InsP3) receptor blocker, strongly inhibited the contractions induced by eugenol but had only a small effect on the caffeine-induced contractions. Eugenol neither altered the Ca(2+) sensitivity nor the maximal force in Triton X-100 skinned muscle fibers. These data suggest that muscle contraction induced by eugenol involves at least 2 mechanisms of Ca(2+) release from the SR: one related to the activation of the ryanodine receptors and another through a heparin-sensitive pathway.

Sphingosine releases Ca 2+ from intracellular stores via the ryanodine receptor in sea urchin egg homogenates

Various reports have demonstrated that the sphingolipids sphingosine and sphingosine-1-phosphate are able to induce Ca 2+ release from intracellular stores in a similar way to second messengers. Here, we have used the sea urchin egg homogenate, a model system for the study of intracellular Ca 2+ release mechanisms, to investigate the effect of these sphingolipids. While ceramide and sphingosine-1-phosphate did not display the ability to release Ca 2+, sphingosine stimulated transient Ca 2+ release from thapsigargin-sensitive intracellular stores. This release was inhibited by ryanodine receptor blockers (high concentrations of ryanodine, Mg 2+, and procaine) but not by pre-treatment of homogenates with cADPR, 8-bromo-cADPR or blockers of other intracellular Ca 2+ channels. However, sphingosine rendered the ryanodine receptor refractory to cADPR. We propose that, in the sea urchin egg, sphingosine is able to activate the ryanodine receptor via a mechanism distinct from that used by cADPR.

Use‐dependent inhibition of the skeletal muscle ryanodine receptor by the suramin analogue NF676

The skeletal muscle Ca2+ release channel, the ryanodine receptor, is activated by the trypanocidal drug suramin via the calmodulin-binding site. As calmodulin activates and inhibits the ryanodine receptor depending on whether Ca2+ is absent or present, suramin analogues were screened for inhibition of the ryanodine receptor. Up to 300 microM, the novel suramin analogue, 4,4'-(carbonyl-bis(imino-4,1-phenylene-(2,5-benzimidazolylene)carbonylimino))-bis-benzenesulfonic acid disodium salt (NF676) was not able to significantly inhibit the basal [3H]ryanodine binding. However, kinetic analysis of the high affinity [3H]ryanodine binding elucidates a time-dependent increment of inhibition by NF676, which is indicative for an open channel blocker. Moreover, the ryanodine receptor was much more sensitive towards inhibition by NF676 when preactivated with caffeine or the nonhydrolysable ATP analogue, adenylyl-imidodiphosphate. Nonetheless, the suramin activated ryanodine receptor was not susceptible towards high-affinity NF676 inhibition, indicating an allosteric hindrance between the binding sites of suramin and NF676. In the line of this finding, NF676 per se was not capable to elute the purified ryanodine receptor from a calmodulin-Sepharose, but it prevented the elution by suramin. Other than suramin, NF676 did not inhibit the Ca2+ ATPase of the sarcoplasmic reticulum. However, suramin-induced Ca2+ release from sarcoplasmic reticulum was completely abrogated by preincubation with NF676. Taken together, we conclude from these data that NF676 represents a novel lead compound as a potent use-dependent blocker of the skeletal muscle ryanodine receptor via an allosteric interaction with the suramin-binding site.

8-isoprostaglandin E2 activates Ca2+-dependent K+ current via cyclic AMP signaling pathway in murine renal artery

The inhibitory pathway of 8-isoprostaglandin E2 was investigated in murine renal arterial smooth muscle. K+ current was augmented in a concentration-dependent fashion, with an average increase of 123±28% (n=6) following application of 10−5 M 8-isoPGE2. This augmentation was observed in the presence of 4-aminopyridine (4-AP, 10−3 M) but not that of charybdotoxin (ChTx, 10−7 M). Fluorimetric recordings showed marked concentration-dependent increase of cytosolic Ca2+ levels by 8-isoPGE2, while an enzyme-linked immunosorbent assay (ELISA)-based cyclic AMP assay showed increased cAMP levels by 10−7 M 8-isoPGE2 challenge. The isoprostane-induced augmentation was prevented by the ryanodine receptor blocker ruthenium red (10−5 M) or the adenylate cyclase blocker SQ 22536 (10−4 M). The protein kinase A (PKA) inhibitor H89 (10−5 M) inhibited resting K+ currents (78±5%, n=5) but did not prevent 8-isoPGE2 from augmenting the remaining K+ current. We conclude that 8-isoPGE2 enhances Ca2+-dependent K+ currents in murine renal artery through a cAMP-dependent pathway which may involve internally sequestered Ca2+.

Ryanodine receptor-mediated interference of neuronal cell differentiation by presenilin 2 mutation

Neuronal cell differentiation alterations induced by mutant presenilin 2 (PS2) were investigated in transgenic mice expressing wild-type or mutant-type PS2. Progressive increases in differentiation and marker protein expression were found in neuronal cells expressing wild-type PS2, whereas these processes were much perturbed in mutant-type PS2 with elevated ryanodine-receptor (RyR) expression and intracellular calcium levels. Moreover, dantrolene, a blocker of RyR reduced the PS2 mutation-induced interference of cell differentiation and calcium release, but caffeine, an activator of RyR, exacerbated PS2 mutation-induced interference with cell differentiation. Our results indicate that mutant PS2 inhibits normal neuronal cell differentiation and that RyR-mediated calcium overrelease may be a significant factor.

Stimulation of cellular free Ca2+ elevation and inhibition of store-operated Ca2+ entry by kazinol B in neutrophils

Kazinol B, a natural isoprenylated flavan, stimulated the [Ca(2+)](i) elevation in the presence or absence of Ca(2+) in the medium. Treatment with chymotrypsin or phorbol 12-myristate 13-acetate to shedding of L: -selectin had no effect on subsequent kazinol B-induced Ca(2+) response. Upon initial cyclopiazonic acid (CPA) treatment in the absence of external Ca(2+), the subsequent [Ca(2+)](i) rise followed by challenge with kazinol B was greatly diminished. The ryanodine receptor blockers, 8-bromo-cyclic ADP-ribose and ruthenium red did not affect kazinol B-evoked Ca(2+) release from internal stores. However, the inhibitors of sphingosine kinase, dimethylsphingosine, but not dihydrosphingosine, inhibited kazinol B-induced Ca(2+) release. Kazinol B-induced [Ca(2+)](i) rise was not affected by two nitric oxidase inhibitors, N-(3-aminomethyl)benzylacetamidine (1400W) and 7-nitroindazole, cytochalasin B and Na(+)-deprivation. This response was slightly attenuated by 2-aminoethyldiphenyl borate (2-APB), a D: -myo-inositol 1,4,5-trisphosphate (IP(3)) receptor blocker, and by genistein, a general tyrosine kinase inhibitor. However, the Ca(2+) response was greatly diminished by two actin filament reorganizers, calyculin A and jasplakinolide, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), an inhibitor of phosphoinositide 3-kinase, N-(3-aminomethyl)benzylacetamidine (SB 203580), the p38 mitogen-activated protein kinase inhibitor, 1-[6-[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, and by 0.3 mM La(3+) or Ni(2+). Kazinol B did not evoke any appreciable Ba(2+) and Sr(2+) entry into cells. The Ca(2+) entry blockers, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), but not cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A), inhibited a kazinol B-induced [Ca(2+)](i) rise. Kazinol B had no effect on the pharmacologically isolated plasma membrane Ca(2+)-ATPase activity. In a Ca(2+)-free medium, kazinol B inhibited the subsequent Ca(2+) addition, resulting in robust entry in CPA- and formyl peptide-activated cells. Kazinol B produced a concentration-dependent reduction in the mitochondrial membrane potential. These results indicate that kazinol B stimulates Ca(2+) release from internal Ca(2+) store, probably through the sphingosine 1-phosphate and IP(3) signaling, and activates external Ca(2+) influx mainly through a non-store-operated Ca(2+) entry (non-SOCE) pathway. Inhibition of SOCE by kazinol B is probably attributable to a break in the Ca(2+) driven force of mitochondria.

Activation of chloride currents in murine portal vein smooth muscle cells by membrane depolarization involves intracellular calcium release.

The present study describes the first characterization of Ca(2+)-activated Cl(-) currents (I(ClCa)) in single smooth muscle cells from a murine vascular preparation (portal veins). I(ClCa) was recorded using the perforated patch version of the whole cell voltage-clamp technique and was evoked using membrane depolarization. Generation of I(ClCa) relied on Ca(2+) entry through dihydropyridine-sensitive Ca(2+) channels because I(ClCa) was abolished by 1 microM nicardipine and enhanced by raising external Ca(2+) concentration or by application of BAY K 8644. I(ClCa) was characterized by the sensitivity to Cl(-) channel blockers and the effect of altering the external anion on reversal potential. Activation of I(ClCa) after membrane depolarization was dependent on Ca(2+) release from intracellular stores. Thus the amplitude of I(ClCa) was diminished by the SR-ATPase inhibitor cyclopiazonic acid, the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate (2-APB), and the ryanodine receptor blocker tetracaine. The degree of inhibition produced by the application of 2-APB and tetracaine together was significantly greater than the effect of each agent applied alone. In current-clamp mode, injection of depolarizing current elicited a biphasic action potential, with the later depolarization being sensitive to niflumic acid (NFA; 10 microM). In isometric tension recordings, NFA inhibited spontaneous contractions. These data support a role for this conductance in portal vein excitability.