Platelets are small disc-shaped cell fragments which undergo a rapid transformation when they encounter vascular damage. They become more spherical and extrude pseudopodia, their fibrinogen receptors are activated, causing them to aggregate, they release their granule contents, and eventually form a plug which is responsible for primary haemostasis. Activation of platelets is also implicated in the pathogenesis of unstable angina, myocardial infarction and stroke. Here we show that platelets from mice deficient in the alpha-subunit of the heterotrimeric guanine-nucleotide-binding protein Gq are unresponsive to a variety of physiological platelet activators. As a result, G alpha(q)-deficient mice have increased bleeding times and are protected from collagen and adrenaline-induced thromboembolism. We conclude that G alpha(q) is essential for the signalling processes used by different platelet activators and that it cannot be replaced by G alpha(i) or the beta gamma subunits of the heterotrimeric G proteins. G alpha(q) may thus be a new target for drugs designed to block the activation of platelets.

Background and purpose: Hydrogen sulphide (H2S) is a labile, endogenous metabolite of cysteine, with multiple biological roles. The development of sulphide-based therapies for human diseases will benefit from a reliable method of quantifying H2S in blood and tissues. Experimental approach: Concentrations of reactive sulphide in saline and freshly drawn whole blood were quantified by reaction with the thio-specific derivatization agent monobromobimane, followed by reversed-phase fluorescence HPLC and/or mass spectrometry. In pharmacokinetic studies, male rats were exposed either to intravenous infusions of sodium sulphide or to H2S gas inhalation, and levels of available blood sulphide were measured. Levels of dissolved H2S/HS -were concomitantly measured using an amperometric sensor. Key results: Monobromobimane was found to rapidly and quantitatively derivatize sulphide in saline or whole blood to yield the stable small molecule sulphide dibimane. Extraction and quantification of this bis-bimane derivative were validated via reversed-phase HPLC separation coupled to fluorescence detection, and also by mass spectrometry. Baseline levels of sulphide in blood were in the range of 0.4-0.9 mM. Intravenous administration of sodium sulphide solution (2-20 mg·kg) or inhalation of H2S gas (50-400 ppm) elevated reactive sulphide in blood in a dose-dependent manner. Each 1 mg·kg -1 ·h -1 of sodium sulphide infusion into rats was found to be pharmacokinetically equivalent to approximately 30 ppm of H2S gas inhalation. Conclusions and implications:The monobromobimane derivatization method is a sensitive and reliable means to measure reactive sulphide species in whole blood. Using this method, we have established a bioequivalence between infused sodium sulphide and inhaled H2S gas.

We conclude that 1) endogenous adenosine building up during ischemia is cardioprotective, and 2) pretreatment with adenosine confers cardioprotection independent of hemodynamic effects. Whether pretreatment effects of adenosine subsequently modulate the effects of endogenous adenosine (through alterations in receptor population or sensitivity) or endogenous and exogenous adenosine represent additive compartments is unclear.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Hydrogen sulphide (H2S) is a biological mediator and a potential therapeutic agent. In animal studies, the metabolism and pharmacokinetics of H2S have been characterized. WHAT THIS STUDY ADDS• This study is first to demonstrate the pharmacokinetics of an intravenously administered H2S formulation in humans, and to characterize the exhaled H2S response in humans. INTRODUCTIONHydrogen sulphide (H2S) is an endogenous gaseous signaling molecule and potential therapeutic agent. Emerging studies indicate its therapeutic potential in a variety of cardiovascular diseases and in critical illness. Augmentation of endogenous sulphide concentrations by intravenous administration of sodium sulphide can be used for the delivery of H2S to the tissues. In the current study, we have measured H2S concentrations in the exhaled breath of healthy human volunteers subjected to increasing doses sodium sulphide in a human phase I safety and tolerability study. METHODSWe have measured reactive sulphide in the blood via ex vivo derivatization of sulphide with monobromobimane to form sulphide-dibimane and blood concentrations of thiosulfate (major oxidative metabolite of sulphide) via ion chromatography. We have measured exhaled H2S concentrations using a custom-made device based on a sulphide gas detector (Interscan). RESULTSAdministration of IK-1001, a parenteral formulation of Na2S (0.005-0.20 mg kg -1 , i.v., infused over 1 min) induced an elevation of blood sulphide and thiosulfate concentrations over baseline, which was observed within the first 1-5 min following administration of IK-1001 at 0.10 mg kg -1 dose and higher. In all subjects, basal exhaled H2S was observed to be higher than the ambient concentration of H2S gas in room air, indicative of on-going endogenous H2S production in human subjects. Upon intravenous administration of Na2S, a rapid elevation of exhaled H2S concentrations was observed. The amount of exhaled H2S rapidly decreased after discontinuation of the infusion of Na2S. CONCLUSIONExhaled H2S represents a detectable route of elimination after parenteral administration of Na2S.

Infarct size reduction in the rabbit via ischaemic preconditioning is reversible with the coadministration of glibenclamide.

Background and purpose: Sodium sulphide (Na2S) disassociates to sodium (Na + ) hydrosulphide, anion (HS -) and hydrogen sulphide (H2S) in aqueous solutions. Here we have established and characterized a method to detect H2S gas in the exhaled breath of rats. Experimental approach: Male rats were anaesthetized with ketamine and xylazine, instrumented with intravenous (i.v.) jugular vein catheters, and a tube inserted into the trachea was connected to a pneumotach connected to a H2S gas detector. Sodium sulphide, cysteine or the natural polysulphide compound diallyl disulphide were infused intravenously while the airway was monitored for exhaled H2S real time. , also caused exhalation of H2S gas. Conclusions and implications:This method has shown that significant amounts of H2S are exhaled in rats during sodium sulphide infusions, and the amount exhaled can be modulated by various pharmacological interventions.

We conclude that: (1) endogenous adenosine released from the myocardium during ischemia/reperfusion reduces infarct size by receptor-mediated mechanisms and (2) Ado-mediated cardioprotection is most pronounced during the early phase of reperfusion.

In vitro experimental models designed to study the effects of hypoxia and ischemia typically employ oxygen-depleted media and/or hypoxic chambers. These approaches, however, allow for metabolites to diffuse away into a large volume and may not replicate the high local concentrations that occur in ischemic myocardium in vivo. We describe herein a novel and simple method for creating regional hypoxic and ischemic conditions in neonatal rat cardiac myocyte monolayers. This method consists of creating a localized diffusion barrier by placing a glass coverslip over a portion of the monolayer. The coverslip restricts covered myocytes to a thin film of media while leaving uncovered myocytes free to access the surrounding bulk media volume. Myocytes under the coverslip undergo marked morphology changes over time as assessed by video microscopy. Fluorescence microscopy shows that these changes are accompanied by alterations in mitochondrial membrane potential and plasma membrane dynamics and eventually result in myocyte death. We also show that the metabolic activity of myocytes drives cell necrosis under the coverslip. In addition, the intracellular pH of synchronously contracting myocytes under the coverslip drops rapidly, which further implicates metabolic activity in regulating cell death under the coverslip. In contrast with existing models of hypoxia/ischemia, this technique provides a simple and effective way to create hypoxic/ischemic conditions in vitro. Moreover, we conclude that myocyte death is hastened by the combination of hypoxia, metabolites, and acidosis and is facilitated by a reduction in media volume, which may better represent ischemic conditions in vivo.