On the role of intracellular concentration of Ca2+ and H+ in thymocyte death after irradiation

AbstractThe role of intracellular Ca2+ and H+ concentrations in radiation-induced interphase death of rat thymocytes has been studied. In response to concanavalin A treatment in the Ca2+-containing medium, or to the CaCl2 treatment in the Ca2+-free medium, the [Ca2+]i rise in irradiated cells was as in the non-treated cells. No changes in the level of [Ca2+]i and pHi were found within l h after irradiation of thymocytes with a dose of 6 Gy. 15 μM 5-(N-ethyl-N-isopropyl)-amiloride. an inhibitor of Na+/H+ exchange, did not affect the DNA fragmentation. The fragmentation was prevented by 2–4 μM (1 -[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)]-2-[(2,4-dichlorophenyl)-methoxy]-ethyl)-1 -H-imidazoliumchloride, an inhibitor of calmodulin. The above data indicate that triggering of interphase death in irradiated thymocytes is not mediated by changes in either [Ca2+]i or pHi. Such changes seem to be involved in intermediate steps of the interphase death process

Nanomolar potency and selectivity of a Ca2+ release-activated Ca2+ channel inhibitor against store-operated Ca2+ entry and migration of vascular smooth muscle cells

BACKGROUND AND PURPOSE The aim was to advance the understanding of Orai proteins and identify a specific inhibitor of the associated calcium entry mechanism in vascular smooth muscle cells (VSMCs). EXPERIMENTAL APPROACH Proliferating VSMCs were cultured from human saphenous veins. Intracellular calcium was measured using fura‐2, whole‐cell current was recorded using patch‐clamp and cell migration quantified in modified Boyden chambers. Subcellular protein localization was determined by microscopy. Isometric tension was recorded from mouse aortic rings. KEY RESULTS Molecular disruption and rescue experiments indicated the importance of Orai1 in calcium entry caused by store depletion evoked passively or by platelet‐derived growth factor (PDGF), suggesting the presence of Ca2+ release‐activated Ca2+ (CRAC) channels like those of the immune system. The CRAC channel blocker, S66, was a potent inhibitor of the VSMC signals, IC50 26 nM, which was almost two orders of magnitude greater than with leucocytes. S66 had no effect on PDGF‐ and ATP‐evoked calcium release, overexpressed transient receptor potential canonical (TRPC)5 channels, native TRPC1/5‐containing channels, stromal interaction molecule 1 clustering, non‐selective cationic current evoked by store depletion and phenylephrine‐evoked aortic contraction. S66 reduced PDGF‐evoked VSMC migration while having only modest effects on cell proliferation and no effect on cell viability. CONCLUSIONS AND IMPLICATIONS The data suggest that Orai1 has a role in human VSMC migration, and that a CRAC channel inhibitor has high potency and selectivity for the associated calcium entry, suggesting a distinct characteristic of vascular CRAC channels and the potential for selective chemical suppression of vascular remodelling

Mechanism of action of novel NO-releasing furoxan derivatives of aspirin in human platelets

1. Incorporation of a nitric oxide (NO)-releasing moiety in aspirin can overcome its gastric side effects. 2. We investigated the NO-release patterns and antiplatelet effects of novel furoxan derivatives of aspirin (B8 and B7) in comparison to existing antiplatelet agents. 3. Cyclooxygenase (COX) activity was investigated in purified enzyme using an electron paramagnetic resonance-based technique. Concentration–response curves for antiplatelet agents±the soluble guanylate cyclase inhibitor, ODQ (50 μM) were generated in platelet-rich plasma (PRP) and washed platelets (WP) activated with collagen using turbidometric aggregometry. NO was detected using an isolated NO electrode. 4. The furoxan derivatives of aspirin (B8, B7) and their NO-free furazan equivalents (B16, B15; all 100 μM) significantly inhibited COX activity (P<0.01; n=6) in vitro and caused aspirin-independent, cGMP-dependent inhibition of collagen-induced platelet aggregation in WP. B8 was more potent than B7 (PRP IC(50)=0.62±0.1 μM for B8; 400±89 μM for B7; P<0.0001. WP IC(50)s=0.6±0.1 and 62±10 μM, respectively). The NO-free furazan counterparts were less potent antiplatelet agents (WP IC(50)s=54±3 μM and 62±10 μM, respectively; P<0.0001, B8 vs B16). Of the hybrids investigated, only B8 retained antiplatelet activity in PRP. 5. NO release from furoxan–aspirin hybrids was undetectable in buffer alone, but was accelerated in the presence of either plasma or plasma components, albumin (4%), glutathione (GSH; 3 μM) and ascorbate (50 μM), the effects of which were additive for B7 but not B8. NO generation from furoxans was greatly enhanced by platelet extract, an effect that could largely be explained by the synergistic effect of intracellular concentrations of GSH (3 mM) and ascorbate (1 mM). 6. We conclude that the decomposition of furoxan–aspirin hybrids to generate biologically active NO is catalysed by endogenous agents which may instil a potential for primarily intracellular delivery of NO. The blunting of the aspirin effects of furoxan hybrids is likely to be due to loss of the acetyl moiety in plasma; the observed antiplatelet effects are thereby primarily mediated via NO release. Compounds of this class might represent a novel means of inhibiting platelet aggregation by a combination of NO generation and COX inhibition

Stimulation of β-adrenoceptors inhibits store-operated channel currents via a cAMP-dependent protein kinase mechanism in rabbit portal vein myocytes

Previously we have described the properties of store-operated channel currents (SOCs) in freshly dispersed rabbit portal vein smooth muscle cells. In addition to Ca(2+) store depletion these SOCs could also be activated by α-adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)-dependent mechanism. In the present study we have investigated the effect of β-adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole-cell recording the selective β-adrenoceptor agonist isoprenaline reduced the current evoked by cyclopiazonic acid (CPA, sarcoplasmic/endoplasmic reticulum ATPase inhibitor) by over 85%. With cell-attached patch recording, bath application of isoprenaline produced a pronounced inhibition of SOC activity evoked by either CPA or the acetoxymethyl ester form of BAPTA (BAPTA-AM). SOC activity evoked by CPA, the DAG analogue, 1-oleoyl-acetyl-sn-glycerol (OAG) or the phorbol ester, phorbol-12,13-dibutyrate (PDBu) was also markedly inhibited by the adenylate cyclase activator, forskolin, and the cell-permeable non-hydrolysable analogue of cyclic adenosine monophosphate (cAMP), 8-Br-cAMP. With inside-out patches, bath application of PDBu evoked channel currents with similar properties to SOCs which were inhibited by over 90% by a catalytic subunit of protein kinase A (PKA) and by 8-Br-cAMP. Moreover bath application of PKA inhibitors, H-89, KT5720 and an inhibitory peptide to quiescent cell-attached or inside-out patches, activated channel currents with similar properties to SOCs. These data suggest that in rabbit portal vein myocytes, stimulation of β-adrenoceptors inhibits SOC activity via a cAMP-dependent protein kinase signal transduction cascade. In addition it is concluded that constitutive PKA activity has a profound inhibitory effect on SOC activity in this vascular preparation

Monovalent cation (MC) current in cardiac and smooth muscle cells: regulation by intracellular Mg(2+) and inhibition by polycations

1. Previously we have described a monovalent cation (MC) current that could be unmasked by the removal of extracellular divalent cations in vascular smooth muscle cells (SMC) and cardiac myocytes, but specific and potent inhibitors of MC current have not been found, and the mechanism of its intracellular regulation remains obscure. 2. Here we show that small MC current is present in intact cells and could be dramatically up-regulated during cell dialysis. MC current in dialyzed cells strongly resembled monovalent cation current attributed to Ca(2+) release-activated Ca(2+)-selective (CRAC) channels, but its activation did not require depletion of Ca(2+) stores, and was observed when the cells were dialyzed with, or without BAPTA. 3. Intracellular free Mg(2+) inhibits MC current with K(d)=250 μM. 4. Extracellular (but not intracellular) spermine effectively blocked MC current with K(d) =3–10 μM, while store-operated cations (SOC) channels and capacitative Ca(2+) influx were not affected. 5. Spermine effectively inhibited MC current-induced SMC depolarization, and prevented Ca(2+) paradox-induced vascular contracture. 6. Both, MC and SOC currents were inhibited by 2-aminoethoxydiphenyl borate (2-APB). 7. It is concluded that MC current could be regulated by intracellular Mg(2+), and low concentrations of extracellular spermine could be used to discriminate it from SOC current, and to assess its role in cellular function

Reducing QT liability and proarrhythmic risk in drug discovery and development

Drug-induced torsades de pointes (TdP), a rare, life-threatening, polymorphic, ventricular tachycardia associated with prolongation of the QT interval, has been the main safety reason for the withdrawal of medicines from clinical use over the last decade. Most often, drugs that prolong the action potential and delay ventricular repolarization do so through blockade of outward (repolarizing) currents, predominantly the rapid delayed rectifying potassium current, IKr. While QT interval prolongation is not a safety concern per se, in a small percentage of people, it has been associated with TdP, which either spontaneously terminates or degenerates into ventricular fibrillation. Furthermore, recent data suggest that shortening of the QT interval may also be a new safety issue waiting to surface. This review article summarizes the presentations given at a symposium entitled ‘Reducing QT liability and proarrhythmic risk in drug discovery and development’, which was part of the Federation of the European Pharmacological Societies congress, Manchester, UK, 13–17 July 2008. The objective of this symposium was to assess the effects of implementing the latest regulatory guidance documents (International Conference on Harmonization S7A/B and E14), as well as new scientific and technical trends on the ability of the pharmaceutical industry to reduce and manage the QT liability and associated potential proarrhythmic risk, and contribute to the discovery and development of safer medicines. This review outlines the key messages from communications presented at this symposium and attempts to highlight some of the key challenges that remain to be addressed

hERG-related drug toxicity and models for predicting hERG liability and QT prolongation

BACKGROUND: hERG K(+) channels have been recognized as a primary antitarget in safety pharmacology. Their blockade, caused by several drugs with different therapeutic indications, may lead to QT prolongation and, eventually, to potentially fatal arrhythmia, namely torsade de pointes. Therefore, a number of preclinical models have been developed to predict hERG liability early in the drug development process. OBJECTIVE: The aim of this review is to outline the present state of the art on drug-induced hERG blockade, providing insights on the predictive value of in vitro and in silico models for hERG liability. METHODS: On the basis of latest reports, high-throughput preclinical models have been discussed outlining advantages and limitations. CONCLUSION: Although no single model has an absolute value, an integrated risk assessment is recommended to predict the pro-arrhythmic risk of a given drug. This prediction requires expertise from different areas and should encompass emerging issues such as interference with hERG trafficking and QT shortening