Cardiac expression of ryanodine receptor subtype 3; a strategic component in the intracellular Ca2+ release system of Purkinje fibers in large mammalian heart.

BACKGROUND: Three distinct Ca2+ release channels were identified in dog P-cells: the ryanodine receptor subtype 2 (RyR2) was detected throughout the cell, while the ryanodine receptor subtype 3 (RyR3) and inositol phosphate sensitive Ca2+ release channel (InsP3R) were found in the cell periphery. How each of these channels contributes to the Ca2+ cycling of P-cells is unclear. Recent modeling of Ca2+ mobilization in P-cells suggested that Ca2+ sensitivity of Ca2+induced Ca2+release (CICR) was larger at the P-cell periphery. Our study examined whether this numerically predicted region of Ca2+ release exists in live P-cells. We compared the regional Ca2+ dynamics with the arrangement of intracellular Ca2+ release (CR) channels. METHODS: Gene expression of CR channels was measured by qPCR in Purkinje fibers and myocardium of adult Yucatan pig hearts. We characterized the CR channels protein expression in isolated P-cells by immuno-fluorescence, laser scanning confocal microscopy, and 3D reconstruction. The spontaneous Ca2+ activity and electrically-evoked Ca2+ mobilization were imaged by 2D spinning disk confocal microscopy. Functional regions of P-cell were differentiated by the characteristics of local Ca2+ events. We used the Ca2+ propagation velocities as indicators of channel Ca2+ sensitivity. RESULTS: RyR2 gene expression was identical in Purkinje fibers and myocardium (6 hearts) while RyR3 and InsP3R gene expressions were, respectively, 100 and 16 times larger in the Purkinje fibers. Specific fluorescent immuno-staining of Ca2+ release channels revealed an intermediate layer of RyR3 expression between a near-membrane InsP3R-region and a central RyR2-region. We found that cell periphery produced two distinct forms of spontaneous Ca2+-transients: (1) large asymmetrical Ca2+ sparks under the membrane, and (2) typical Ca2+-wavelets propagating exclusively around the core of the cell. Larger cell-wide Ca2+ waves (CWWs) appeared occasionally traveling in the longitudinal direction through the core of Pcells. Large sparks arose in a micrometric space overlapping the InsP3R expression. The InsP3R antagonists 2-aminoethoxydiphenyl borate (2-APB; 3µM) and xestospongin C (XeC; 50µM) dramatically reduced their frequency. The Ca2+ wavelets propagated in a 5-10µm thick layered space which matched the intermediate zone of RyR3 expression. The wavelet incidence was unchanged by 2-APB or XeC, but was reduced by 60% in presence of the RyR3 antagonist dantrolene (10µM). The velocity of wavelets was two times larger (86±16µm/s; n=14) compared to CWWs' (46±10µm/s; n=11; P<0.05). Electric stimulation triggered a uniform and large elevation of Ca2+ concentration under the membrane which preceded the propagation of Ca2+ into the interior of the cell. Elevated Cai propagated at 150µm/s (147±34µm/s; n=5) through the region equivalent to the zone of RyR3 expression. This velocity dropped by 50% (75±24µm/s; n=5) in the central region wherein predominant RyR2 expression was detected. CONCLUSION: We identified two layers of distinct Ca2+ release channels in the periphery of Pcell: an outer layer of InsP3Rs under the membrane and an inner layer of RyR3s. The propagation of Ca2+ events in these layers revealed that Ca2+ sensitivity of Ca2+ release was larger in the RyR3 layer compared to that of other sub-cellular regions. We propose that RyR3 expression in P-cells plays a role in the stability of electric function of Purkinje fibers.

Effects of chronic in-vivo treatments with protease-activated receptor 2 agonist on endothelium function and blood pressures in mice.

Short-term treatments with protease-activated receptor 2-activating peptides (PAR2-AP) induce endothelium-dependent vasodilation and decrease blood pressure. In this study, we tested the effect of chronic in-vivo treatment with PAR2-AP on the blood pressure and endothelium function of mice. Male PAR2 wild-type (WT) and par2-deficient (KO) mice received subcutaneous infusions of either saline, low (PAR2-LD), or high (PAR2-HD) doses of 2-furoyl-LIGRLO-amide for 1 or 2 weeks. In each treatment group, endothelium function was assessed in isolated arteries. Blood pressure, heart rate, and locomotor activity were recorded by radiotelemetry, and levels of tumour nercrosis factor α (TNF-α) and interkeukin 1ß (IL-1ß) were measured in plasma samples by ELISA. The relaxation of WT aortas and mesenteric arteries induced by PAR2-AP was decreased by PAR2-LD and PAR2-HD. In mesenteric arteries, PAR2-LD and PAR2-HD decreased the relaxation induced by acetylcholine, but not by nitroprusside; in aortas, PAR2-LD and PAR2-HD caused differential decreases in the relaxations induced by acetylcholine and nitroprusside. Only PAR2-HD lowered systolic arterial pressures in WT, when compared with all of the other groups. TNF-α and IL-1ß plasma concentrations were not different among the groups. We conclude that the systolic blood pressure of unrestrained mice can be lowered by chronic in-vivo activation of PAR2; however, this effect is countered by receptor desensitization and the concomitant development of endothelium and vascular dysfunction.

Synthesis and anti-inflammatory structure-activity relationships of thiazine-quinoline-quinones: inhibitors of the neutrophil respiratory burst in a model of acute gouty arthritis.

Sixteen new thiazine-quinoline-quinones have been synthesised, plus one bicyclic analogue. These compounds inhibited neutrophil superoxide production in vitro with IC(50)s as low 60 nM. Compounds with high in vitro anti-inflammatory activity were also tested in a mouse model of acute inflammation. The most active compounds inhibited both neutrophil infiltration and superoxide production at doses 2.5 micromol/kg, highlighting their potential for development as novel NSAIDs.

Sesquiterpene dialdehydes inhibit MSU crystal-induced superoxide production by infiltrating neutrophils in an in vivo model of gouty inflammation.

A hallmark feature of monosodium urate (MSU) crystal-induced inflammation in gouty arthritis is the infiltration of activated neutrophils into the joint. Therefore inhibition of neutrophil superoxide production is a rational target for treating inflammation in gout. The natural product polygodial and related sesquiterpene dialdehyde analogs were tested in vitro and in vivo for their ability to inhibit neutrophil infiltration and superoxide production in response to MSU crystal stimulation. Polygodial and other sesquiterpene dialdehydes exhibited dose-dependent inhibition of MSU-induced superoxide production in the micromolar and submicromolar ranges. Inhibition of superoxide production was dependent on the presence of the dialdehyde functional groups and was sensitive to blockade with the thiol-containing amino acid cysteine. Polygodial, 6-hydroxypaxidal and sesquiterpene 2 inhibited both neutrophil infiltration and neutrophil superoxide production in an MSU crystal-induced mouse model of gouty inflammation. Together, these data highlight the potential of sesquiterpene dialdehydes for development as anti-inflammatory agents for the treatment of neutrophil-driven inflammatory diseases including gout.

An antiproliferative bis-prenylated quinone from the New Zealand brown alga Perithalia capillaris.

Bioactivity-directed isolation work on the endemic New Zealand brown alga Perithalia capillaris, seeking anti-inflammatory compounds, led to a new bis-prenylated quinone ( 4). This compound inhibited superoxide production by human neutrophils in vitro (IC 50 2.1 microM), but was more potent at inhibiting proliferation of HL60 cells (IC 50 0.34 microM). Two related bis-prenylated phenols were also isolated, one known ( 2) and one new ( 5), with weaker biological activities. This report extends the examples of bis-prenylated phenols as chemotaxonomic markers for brown algae of the order Sporochnales.

E/Z-rubrolide O, an anti-inflammatory halogenated furanone from the New Zealand ascidian Synoicum n. sp.

Bioassay-directed fractionation of extracts of a Synoicum n. sp. ascidian from New Zealand led to the isolation of the principal anti-inflammatory component, which was identified by spectroscopic methods as a new member of the rubrolide family, rubrolide O (1), existing as a mixture of E/Z isomers.

Anti-inflammatory thiazine alkaloids isolated from the New Zealand ascidian Aplidium sp.: inhibitors of the neutrophil respiratory burst in a model of gouty arthritis.

Ascidiathiazones A (3) and B (4), two new tricyclic thiazine-containing quinolinequinone alkaloids, were isolated from the New Zealand ascidian Aplidium species. Both compounds inhibited the in vitro production of superoxide by PMA-stimulated human neutrophils in a dose-dependent manner with IC50 1.55 +/- 0.32 and 0.44 +/- 0.09 microM, respectively. In vivo inhibition of superoxide production by peritoneal neutrophils in a murine model of gout was observed for both compounds with oral doses of 25.6 micromol/kg. Ascidiathiazone A (3) was synthesized in four steps from 8-hydroxyquinoline-2-carboxylic acid.

Anti-inflammatory sesquiterpene-quinones from the New Zealand sponge Dysidea cf. cristagalli.

The inhibition of superoxide production by human neutrophils has been used to screen New Zealand's unique biota for anti-inflammatory natural products. Bioactivity-directed isolation on an extract of the sponge Dysidea cf. cristagalli led to a new sesquiterpene-quinone (4) with anti-inflammatory activity, plus acetylated hydroquinone (3). These compounds inhibited superoxide production in vitro with IC50's of 3 microM (3) and 11 microM (4).