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Isoniazid and rifampicin are first-line anti-tubercular drugs. In a recent paper, Shen et al. provided interesting findings that rifampicin exacerbated isoniazid toxicity in human hepatocytes but not in rat hepatocytes. The main conclusion was that the difference in cytochrome P450 2E1 (CYP2E1) induction by rifampicin between rat and human hepatocytes accounted for the difference in exacerbation of isoniazid hepatotoxicity by rifampicin. 4-Nitrophenol hydroxylase (4-NP) activity was the only probe of CYP2E1 activity used in the paper. The authors presented data showing that rifampicin enhanced 4-NP activity and CYP2E1 mRNA expression in human hepatocytes, but not in rat hepatocytes. However, CYP3A also makes a significant contribution to 4-NP activity in humans and rats, which has been confirmed by both CYP3A-specific inducer and inhibitors. Rifampicin is a strong inducer of human CYP3A; thus, the increase in 4-NP activity in human hepatocytes could be due to the induction of CYP3A. Rifampicin did not increase 4-NP activities in rat hepatocytes, which could reflect a lack of the induction of rat CYP3A by rifampicin. Additionally, more experiments are needed to support the conclusion that rifampicin increased CYP2E1 mRNA expression in human hepatocytes because of the small sample size and the limitations of semi-quantitative RT-PCR. The study by Shen et al. suggests that another drug-metabolizing enzyme rather than CYP2E1 could be involved in the aggravation of isoniazid toxicity by rifampicin in human hepatocytes. [ABSTRACT FROM AUTHOR]

Background and Purpose: Although exogenous nitric oxide (NO) clearly modifies platelet function, the role and the source of endogenous NO in vivo remain undefined. In addition, endothelial NO synthase (NOS-3) critically regulates vessel tone but its role in modulating platelet function is unclear. In this paper we have investigated the roles of endogenous NO and NOS-3 in regulating platelet function in vivo and determined the functional contribution made by platelet-derived NO.Experimental Approach: We used a mouse model for directly assessing platelet functional responses in situ in the presence of an intact vascular endothelium with supporting in vitro and molecular studies.Key Results: Acute NOS inhibition by N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME) enhanced platelet aggregatory responses to thrombin and platelets were shown to be regulated primarily by NO sources external to the platelet. Elevation of endogenous NOS inhibitors to mimic effects reported in patients with cardiovascular diseases did not enhance platelet responses. Platelet responsiveness following agonist stimulation was not modified in male or female NOS-3(-/-) mice but responses in NOS-3(-/-) mice were enhanced by L-NAME.Conclusions and Implications: Platelets are regulated by endogenous NO in vivo, primarily by NO originating from the environment external to the platelet with a negligible or undetectable role of platelet-derived NO. Raised levels of endogenous NOS inhibitors, as reported in a range of diseases were not, in isolation, sufficient to enhance platelet activity and NOS-3 is not essential for normal platelet function in vivo due to the presence of bioactive NO following deletion of NOS-3. [ABSTRACT FROM AUTHOR]

Background and purpose: Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium-dependent relaxation and hyperpolarization in resistance arteries. Experimental approach: Rat and mouse mesenteric arteries were mounted in small vessel myographs for isometric force and smooth muscle membrane potential recording. Key results: Vasorelaxation to the HNO donor, Angeli's salt, was attenuated in both species by the soluble guanylate cyclase inhibitor (ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), the voltage-dependent K+ channel inhibitor, 4-aminopyridine (4-AP) and the HNO scavenger,l-cysteine. In mouse mesenteric arteries, nitric oxide (NO) synthase inhibition (withl-NAME, Nω-Nitro-L-arginine methyl ester) markedly attenuated acetylcholine (ACh)-mediated relaxation. Scavenging the uncharged form of NO (NO•) with hydroxocobalamin (HXC) or HNO withl-cysteine, or 4-AP decreased the sensitivity to ACh, and a combination of HXC andl-cysteine reduced ACh-mediated relaxation, as didl-NAME alone. ACh-induced hyperpolarizations were significantly attenuated by 4-AP alone and in combination withl-NAME. In rat mesenteric arteries, blocking the effects of endothelium-derived hyperpolarizing factor (EDHF) (charybdotoxin and apamin) decreased ACh-mediated relaxation 10-fold and unmasked a NO-dependent component, mediated equally by HNO and NO•, as HXC andl-cysteine in combination now abolished vasorelaxation to ACh. Furthermore, ACh-evoked hyperpolarizations, resistant to EDHF inhibition, were virtually abolished by 4-AP. Conclusions and implications: The factors contributing to vasorelaxation in mouse and rat mesenteric arteries are NO• = HNO > EDHF and EDHF > HNO = NO• respectively. This study identified HNO as an endothelium-derived relaxing and hyperpolarizing factor in resistance vessels. British Journal of Pharmacology (2009) 157, 540–550; doi:10.1111/j.1476-5381.2009.00150.x; published online 26 March 2009 This article is commented on by Martin, pp. 537–539 of this issue and is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: [ABSTRACT FROM AUTHOR]

Background and purpose: There are interactions between endothelin-1 (ET-1) and endothelial vascular injury in hyperhomocysteinemia (HHcy), but the underlying mechanisms are poorly understood. Here we evaluated the effects of HHcy on the endothelin system in rat carotid arteries. Experimental approach: Vascular reactivity to ET-1 and ETA and ETB receptor antagonists was assessed in rings of carotid arteries from normal rats and those with HHcy. ETA and ETB receptor expression was assessed by mRNA (RT-PCR), immunohistochemistry and binding of [125I]-ET-1. Key results: HHcy enhanced ET-1-induced contractions of carotid rings with intact endothelium. Selective antagonism of ETA or ETB receptors produced concentration-dependent rightward displacements of ET-1 concentration response curves. Antagonism of ETA but not of ETB receptors abolished enhancement in HHcy tissues. ETA and ETB receptor gene expressions were not up-regulated. ETA receptor expression in the arterial media was higher in HHcy arteries. Contractions to big ET-1 served as indicators of endothelin-converting enzyme activity, which was decreased by HHcy, without reduction of ET-1 levels. ET-1-induced Rho-kinase activity, calcium release and influx were increased by HHcy. Pre-treatment with indomethacin reversed enhanced responses to ET-1 in HHcy tissues, which were reduced also by a thromboxane A2 receptor antagonist. Induced relaxation was reduced by BQ788, absent in endothelium-denuded arteries and was decreased in HHcy due to reduced bioavailability of NO. Conclusions and implications: Increased ETA receptor density plays a fundamental role in endothelial injury induced by HHcy. ET-1 activation of ETA receptors in HHcy changed the balance between endothelium-derived relaxing and contracting factors, favouring enhanced contractility. British Journal of Pharmacology (2009) 157, 568–580; doi:10.1111/j.1476-5381.2009.00165.x; published online 9 April 2009 This article is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: [ABSTRACT FROM AUTHOR]

Background and purpose: Vascular ATP-sensitive potassium (KATP) channels are activated by cyclic AMP elevating vasodilators through protein kinase A (PKA). Direct channel phosphorylation is a critical mechanism, though the phosphatase opposing these effects is unknown. Previously, we reported that calcineurin, a Ca2+-dependent phosphatase, inhibits KATP channels, though neither the site nor the calcineurin isoform involved is established. Given that the type-2 regulatory (RII) subunit of PKA is a substrate for calcineurin we considered whether calcineurin regulates channel activity through interacting with PKA. Experimental approach: Whole-cell recordings were made in HEK-293 cells stably expressing the vascular KATP channel (KIR6.1/SUR2B). The effect of intracellular Ca2+ and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined. Key results: Constitutively active calcineurin Aα but not Aβ significantly attenuated KATP currents activated by low intracellular Ca2+, whereas calcineurin inhibitors had the opposite effect. PKA inhibitors reduced basal KATP currents and responses to calcineurin inhibitors, consistent with the notion that some calcineurin action involves inhibition of PKA. However, raising intracellular Ca2+ (equivalent to increasing calcineurin activity), almost completely inhibited KATP channel activation induced by the catalytic subunit of PKA, whose enzymatic activity is independent of the RII subunit. In vitro phosphorylation experiments showed calcineurin could directly dephosphorylate a site in Kir6.1 that was previously phosphorylated by PKA. Conclusions and implications: Calcineurin Aα regulates KIR6.1/SUR2B by inhibiting PKA-dependent phosphorylation of the channel as well as PKA itself. Such a mechanism is likely to directly oppose the action of vasodilators on the KATP channel. British Journal of Pharmacology (2009) 157, 554–564; doi:10.1111/j.1476-5381.2009.00221.x; published online 7 May 2009 This article is commented on by Tammaro, pp. 551–553 of this issue and is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: [ABSTRACT FROM AUTHOR]

Recently, a population of nerves has been described in the aganglionic mouse vas deferens, in which electrically evoked contractions were insensitive to high concentrations of the adrenergic neurone blocker, bretylium. In this paper, the pharmacology of this nerve-evoked contraction has been examined in more detail.Bretylium (20 μM) revealed, after 5 h exposure, a new residual neurogenic contraction (20 stimuli at 10 Hz) that was tetrodotoxin-sensitive.The muscarinic antagonist, cyclopentolate (0.1 and 1 μM), reduced this residual component and the inhibition was reversed by the acetylcholinesterase inhibitor, neostigmine (1 and 10 μM).Nicotine (30 μM) enhanced the residual component revealed by bretylium, suggesting that there are prejunctional nicotinic receptors (nAchRs) influencing acetylcholine (Ach) release.In the presence of prazosin (0.1 μM), a selective α1-adrenoceptor antagonist, and α,β-methylene ATP (1 μM), a purinergic agonist that desensitise P2X receptors, neostigmine increased the hump component of contraction and yohimbine (0.3 μM), an α2-adrenoceptor antagonist, enhanced both components of the electrically evoked stimulation. The contraction was blocked by cyclopentolate (1 μM).In the absence of bretylium, neostigmine alone increased the hump component of contraction in a frequency-dependent manner. This increase was reversed by atropine (1 μM) and cyclopentolate (1 μM) to control levels. However, in control experiments, atropine or cyclopentolate did not detectably influence the delayed neurogenic contraction.Ach (10 μM) induced a contraction in the mouse vas deferens, either when applied alone or in the presence of neostigmine.Thus, it has been demonstrated unequivocally that the mouse vas deferens is innervated by functional cholinergic nerves, whose action is terminated by cholinesterase. Furthermore, Ach release can be enhanced by activation of prejunctional nAchRs presumably located on the cholinergic nerve terminals.British Journal of Pharmacology (2005) 146, 927–934. doi:10.1038/sj.bjp.0706357; published online 19 September 2005 [ABSTRACT FROM AUTHOR]

1. This paper reports on a primary cell culture system that predominantly expresses native norepinephrine (NE) transporters (NETs), and is amenable to biophysical as well as biochemical analyses. 2. Previous research has identified human and rat placentas as rich sources of NET. We have exploited this to develop primary cultures of rat placental trophoblasts. NE uptake in these cultures is about 10 times higher when compared to 5HT uptake. The presence of NET protein is revealed by immunoblot analysis, while there is no detectable SERT protein. 3. NE transport in rat trophoblasts is sensitive to NET-specific antagonists, desipramine (DS) and nisoxetine (NX), but not to the dopamine-transporter (DAT) specific antagonist, GBR12909 or to the serotonin (5HT) transporter (SERT) specific antagonist paroxetine (PX). Drugs of abuse such as cocaine and amphetamine also inhibit NE transport in these cells. Together these results suggest that rat placental trophoblasts predominantly express NET over other monoamine transporters. 4. Patch-clamp analysis reveals that NETs in intact rat trophoblasts are electrogenic. Comparison of NE uptake with NE-induced currents suggests that these two modes of transporter activity are differentially regulated. [ABSTRACT FROM AUTHOR]