Nelson N. Orie

Nitration of endogenous para-hydroxyphenylacetic acid and the metabolism of nitrotyrosine

Reactive nitrogen species, such as peroxynitrite, can nitrate tyrosine in proteins to form nitrotyrosine. Nitrotyrosine is metabolized to 3-nitro-4-hydroxyphenylacetic acid (NHPA), which is excreted in the urine. This has led to the notion that measurement of urinary NHPA may provide a time-integrated index of nitrotyrosine formation in vivo. However, it is not known whether NHPA is derived exclusively from metabolism of nitrotyrosine, or whether it can be formed by nitration of circulating para -hydroxyphenylacetic acid (PHPA), a metabolite of tyrosine. In the present study, we have developed a gas chromatography MS assay for NHPA and PHPA to determine whether or not NHPA can be formed directly by nitration of PHPA. Following the injection of nitrotyrosine, 0.5+/-0.16% of injected dose was recovered unchanged as nitrotyrosine, and 4.3+/-0.2% as NHPA in the urine. To determine whether or not NHPA could be formed by the nitration of PHPA, deuterium-labelled PHPA ([(2)H(6)]PHPA) was injected, and the formation of deuterated NHPA ([(2)H(5)]NHPA) was measured. Of the infused [(2)H(6)]PHPA, 78+/-2% was recovered in the urine unchanged, and approx. 0.23% was recovered as [(2)H(5)]NHPA. Since the plasma concentration of PHPA is markedly higher than free nitrotyrosine (approx. 400-fold), the nitration of high-circulating endogenous PHPA to form NHPA becomes very significant and accounts for the majority of NHPA excreted in urine. This is the first study to demonstrate that NHPA can be formed by nitration of PHPA in vivo, and that this is the major route for its formation.

Differential effects of vasopressin and norepinephrine on vascular reactivity in a long-term rodent model of sepsis.

Objective:There is escalating interest in the therapeutic use of vasopressin in septic shock. However, little attention has focused on mechanisms underlying its pressor hypersensitivity, which contrasts with the vascular hyporesponsiveness to catecholamines. We investigated whether a long-term rodent model of sepsis would produce changes in endogenous levels and pressor reactivity to exogenous norepinephrine and vasopressin comparable with those seen in septic patients. Design:In vivo and ex vivo animal study. Setting:University research laboratory. Subjects:Male adult Wistar rats. Interventions and Measurements:Fecal peritonitis was induced in conscious, fluid-resuscitated rats. Biochemical and hormonal profiles were measured at time points up to 48 hrs. Pressor responses to intravenous norepinephrine, vasopressin, and F-180, a selective V1 receptor agonist, were measured at 24 hrs. Contractile responses to these drugs were assessed in mesenteric arteries taken from animals at 24 hrs using wire myography. Comparisons were made against sham operation controls. Main Results:Septic rats became unwell and hypotensive, with a mortality of 64% at 48 hrs (0% in controls). Plasma norepinephrine levels were elevated in septic animals at 24 hrs (1968 ± 490 vs. 492 ± 90 pg/mL in controls, p = .003), whereas vasopressin levels were similar in the two groups (4.5 ± 0.8 vs. 3.0 ± 0.5 pg/mL, p = not significant). In vivo, the pressor response to norepinephrine was markedly reduced in the septic animals, but responses to vasopressin and F-180 were relatively preserved. In arteries from septic animals, norepinephrine contractions were decreased (efficacy as measured by maximum contractile response, Emax: 3.0 ± 0.3 vs. 4.7 ± 0.2 mN, p < .001). In contrast, the potency of vasopressin (expressed as the negative log of the concentration required to produce 50% of the maximum tension, pD2: 9.1 ± 0.04 vs. 8.7 ± 0.05, p < .001) and F-180 (pD2 8.2 ± 0.04 vs. 7.6 ± 0.02, p < .001) was enhanced (n ≥ 6 for all groups). Conclusions:This long-term animal model demonstrates changes in circulating vasoactive hormones similar to prolonged human sepsis, and decreased pressor sensitivity to norepinephrine. Ex vivo sensitivity to vasopressin agonists was heightened. This model is therefore appropriate for the further investigation of mechanisms underlying vasopressin hypersensitivity, which may include receptor or calcium-handling alterations within the vasculature.

BK large conductance Ca2+-activated K+ channel deficient mice are not resistant to hypotension and display reduced survival benefit following polymicrobial sepsis

Nitric oxide-mediated activation of large conductance calcium-activated potassium (BK) channels is considered an important underlying mechanism of sepsis-induced hypotension. Indeed, the nonselective K-channel inhibitor, tetraethylammonium chloride (TEA), has been proposed as a potential treatment to raise blood pressure in septic shock by virtue of its ability to inhibit BK channels. As experimental evidence has so far relied on pharmacological inhibition, we examined the effects of channel deletion using BK&agr; subunit knockout (&agr;−/−, Slo−/−) mice in two mouse models of polymicrobial sepsis, namely, intraperitoneal fecal slurry and cecal ligation and puncture. Comparison was made against TEA treatment in wild-type (WT) mice. Following slurry, BK&agr;−/− and WT mice developed similar degrees of hypotension over 10 h with no difference in cardiac output as assessed by echocardiography between groups. Tetraethylammonium chloride raised blood pressure significantly in septic WT mice, but had no effect on survival. However, following cecal ligation and puncture, a significantly reduced survival was seen in both BK&agr;−/− mice and (high-dose) TEA-treated WT mice compared with untreated WT animals. In conclusion, the BK channel does not appear to be integral to sepsis-induced hypotension but does affect survival through other mechanisms. The pressor effect of TEA may be related to effects on other potassium channels.

Inhibition of vascular adenosine triphosphate-sensitive potassium channels by sympathetic tone during sepsis.

Objective:Excessive opening of the adenosine triphosphate-sensitive potassium channel in vascular smooth muscle is implicated in the vasodilation and vascular hyporeactivity underlying septic shock. Therapeutic channel inhibition using sulfonylurea agents has proved disappointing, although agents acting on its pore appear more promising. We thus investigated the hemodynamic effects of adenosine triphosphate-sensitive potassium channel pore inhibition in awake, fluid-resuscitated septic rats, and the extent to which these responses are modulated by the high sympathetic tone present in sepsis. Temporal changes in ex-vivo channel activity and subunit gene expression were also investigated. Design:In vivo and ex vivo animal study. Setting:University research laboratory. Subjects:Male adult Wistar rats. Interventions and Measurements:Fecal peritonitis was induced in conscious, fluid-resuscitated rats. Pressor responses to norepinephrine and PNU-37883A (a vascular adenosine triphosphate-sensitive potassium channel inhibitor acting on the Kir6.1 pore-forming subunit) were measured at 6 or 24 hrs, in the absence or presence of the autonomic ganglion blocker, pentolinium. The aorta and mesenteric artery were examined ex vivo for 86rubidium efflux as a marker of adenosine triphosphate-sensitive potassium channel activity, and for adenosine triphosphate-sensitive potassium channel subunit gene expression using quantitative reverse transcription-polymerase chain reaction. Main Results:A total of 120 rats (50 sham-operated controls, 70 septic) were included. Septic rats became hypotensive after 12 hrs, with a 24-hr mortality of 51.7% (0% in controls). At 6 hrs, there was an attenuated pressor response to norepinephrine (p < .01) despite blood pressure being elevated (p < .01). PNU-37883A had no pressor effect, except in the presence of pentolinium (p < .01). Kir6.1 subunit mRNA increased significantly in the mesenteric artery while 86rubidium efflux was increased in both the aorta and mesenteric artery at 24 hrs. Conclusions:Despite evidence of increased adenosine triphosphate-sensitive potassium channel activity in sepsis, it appears to be inhibited in vivo by high sympathetic tone. This may explain, at least in part, the reduced efficacy of adenosine triphosphate-sensitive potassium channel blockers in human septic shock. (Crit Care Med 2012; 40:–1268)

Stoking Up BKCa Channels in Hemorrhagic Shock: Which Channel Subunit Is Really Fueling the Fire?

See related article, pages 493–502 Large conductance calcium-activated potassium channels (BKCa) are abundantly expressed in smooth muscle cells (SMCs) lining the blood vessel wall. They are composed of an α-subunit (Slo) and a modulatory β1-subunit, which serves to maintain the normal high voltage- and Ca2+-sensitivity of the pore-forming α-subunit (reviewed in1,2). In the vasculature, BKCa operate by limiting Ca2+ entry and arterial contraction by repolarizing SMCs and closing voltage-dependent Ca2+ channels previously opened by pressure or vasoconstrictor agents.1 BKCa can also mediate cellular hyperpolarization and vasorelaxation as a result of spontaneous transient outward currents (STOCs) activated by the localized release of micromolar concentrations of Ca2+ (Ca2+ sparks) from ryanodine receptors located in the sarcoplasmic reticulum (SR).1 Moreover, increased frequency of Ca2+ sparks may underlie activation of BKCa by endogenous vasodilators,1 though other mechanisms undoubtedly contribute.2,3 Genetic experiments also highlight BKCa as important regulators of vascular tone and blood pressure. Deletion of the α-subunit in mice results in membrane depolarization, a complete lack of STOCs, and attenuates cGMP relaxation in isolated blood vessels.4 On the other hand, deletion of β1 impairs the coupling of Ca2+ sparks to the activation of hyperpolarizing BKCa currents and enhances agonist-induced vasoconstriction without affecting nitric oxide (NO) mediated vasorelaxation.3 Knockout of both genes leads to systemic hypertension, though in BKCa β1–null mice this is more pronounced3,4 suggesting physical interactions of the β1-subunit with other proteins, possibly other ion-conducting pores.5 Moreover, depending on the hypertensive model, β1-subunit expression can either increase2,6 or decrease.7 The latter might argue that β1 acts as a compensatory mechanism to limit development of hypertension. Consistent with this, a gain-of-function mutation in the human β1-subunit gene …

Comparative Effect of Type 1 and Type 2 Diabetes Mellitus on Vascular Responses of Rat Thoracic Aorta to Potassium Ion Channel Openers

Background: Diabetes mellitus is associated with many cardiovascular dysfunction and impairment of potassium channel function. Aim: We compared the vascular reactivity in aorta from streptozotocin -induced and Goto-Kakizaki (GK) diabetic rat sto potassium channel openers. Methodology: Diabetes mellitus (DM) was induced in Sprague Dawley rats by intraperitoneal injection of streptozotocin (STZ) at 65 mg/kg body weight. After four weeks of DM, vascular reactivity of the aortic rings from STZ -induced Sprague Dawley and age-matched GK and control rats to phenylephrine, acetylcholine, levcromakalim and naringeninwas studied using standard organ bath procedure. Results:The phenylephrine-induced contraction was significantly (P<0.05) increased in

Attenuated vascular responsiveness to K+ channel openers in diabetes mellitus: the differential role of reactive oxygen species.

The current study examined the responsiveness of blood vessels from diabetic rats to K+ channel openers and explored whether ROS might be involved in any changes. Responses were measured in aortic rings isolated from four weeks streptozotocin (65 mg/kg)-induced diabetic rats. Relaxation to levcromakalim (ATP-sensitive potassium channel KATP opener, 10(-9)-10(-5) mol/l) and (+/-)-naringenin (large conductance calcium-activated channel BKCa opener, 10(-8)-10(-3) mol/l) were recorded in phenylephrine (1 µmol/l) pre-contracted segments in the absence and presence of superoxide dismutase (SOD, 100 µmol/l) and apocynin (an antioxidant and inhibitor of NADPH oxidase, 100 µmol/l). Contractions to phenylephrine (10(-9)-10(-5) mol/l) and relaxation to acetylcholine (ACh, 10(-9)-10(-5) mol/l) were also recorded. Relaxation curves for levcromakalim, naringenin and ACh for the diabetic group were shifted to the right (p < 0.05) compared with the control. Contractions to phenylephrine were enhanced in the diabetic group (p < 0.01). SOD restored the ACh response but not those of K+ channel openers. On the other hand, apocynin restored the relaxation to naringenin but had no effect on both levcromakalim and ACh responses. The results suggest that both KATP and BKCa activities are attenuated in diabetes mellitus and that ROS appears to contribute only to the change in BKCa function.