Inflammasome activity is essential for one kidney/deoxycorticosterone acetate/salt-induced hypertension in mice.

BACKGROUND AND PURPOSE: Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1ß and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1ß and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. EXPERIMENTAL APPROACH: Wild-type and inflammasome-deficient ASC(-/-) mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry. KEY RESULTS: 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro-caspase-1, and the cytokine, pro-IL-1ß, as well as protein levels of active caspase-1 and mature IL-1ß. Following treatment with 1K/DOCA/salt, ASC(-/-) mice displayed blunted pressor responses and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice. CONCLUSIONS AND IMPLICATIONS: Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1ß pathway as a potential therapeutic target in hypertension.

NOX1 deficiency in apolipoprotein E-knockout mice is associated with elevated plasma lipids and enhanced atherosclerosis.

Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are enzymes that generate reactive oxygen species (ROS). NOX2 activity in the vascular wall is elevated in hypercholesterolemia, and contributes to oxidative stress and atherogenesis. Here we examined the role of another NOX isoform, NOX1, in atherogenesis in apolipoprotein E-knockout (APOE(-/-)) mice fed a Western diet for 14 weeks. Although NOX1 mRNA expression was unchanged in aortas from APOE(-/-) versus wild-type mice, expression of the NOX1-specific organizer, NOXO1, was diminished, consistent with an overall reduction in NOX1 activity in APOE(-/-) mice. To examine the impact of a further reduction in NOX1 activity, APOE(-/-) mice were crossed with NOX1(-/y) mice to generate NOX1(-/y)/APOE(-/-) double-knockouts. NOX1 deficiency in APOE(-/-) mice was associated with 30-50% higher plasma very-low-density lipoprotein (VLDL)/LDL and triglyceride levels (P < 0.01). Vascular ROS levels were also elevated by twofold in NOX1(-/y)/APOE(-/-) versus APOE(-/-) mice (P < 0.05), despite no changes in expression of other NOX subunits. Although en face analysis of the descending aorta revealed no differences in plaque area between NOX1(-/y)/APOE(-/-) and APOE(-/-) mice, intimal thickening in the aortic sinus was increased by 40% (P < 0.05) in the double-knockouts. Moreover, NOX1 deficiency was associated with a less stable plaque phenotype; aortic sinus lesions contained 60% less collagen (P < 0.01), 40% less smooth muscle (P < 0.01), and 2.5-fold higher levels of matrix metalloproteinase-9 (P < 0.001) than lesions in APOE(-/-) mice. Thus, these data, which suggest a protective role for NOX1 against hyperlipidemia and atherosclerosis in APOE(-/-) mice, highlight the complex and contrasting roles of different NOX isoforms (e.g., NOX2 versus NOX1) in vascular pathology.

IL-1ß and IL-18: inflammatory markers or mediators of hypertension?

Chronic inflammation in the kidneys and vascular wall is a major contributor to hypertension. However, the stimuli and cellular mechanisms responsible for such inflammatory responses remain poorly defined. Inflammasomes are crucial initiators of sterile inflammation in other diseases such as rheumatoid arthritis and gout. These pattern recognition receptors detect host-derived danger-associated molecular patterns (DAMPs), such as microcrystals and reactive oxygen species, and respond by inducing activation of caspase-1. Caspase-1 then processes the cytokines pro-IL-1ß and pro-IL-18 into their active forms thus triggering inflammation. While IL-1ß and IL-18 are known to be elevated in hypertensive patients, no studies have examined whether this occurs downstream of inflammasome activation or whether inhibition of inflammasome and/or IL-1ß/IL-18 signalling prevents hypertension. In this review, we will discuss some known actions of IL-1ß and IL-18 on leukocyte and vessel wall function that could potentially underlie a prohypertensive role for these cytokines. We will describe the major classes of inflammasome-activating DAMPs and present evidence that at least some of these are elevated in the setting of hypertension. Finally, we will provide information on drugs that are currently used to inhibit inflammasome/IL-1ß/IL-18 signalling and how these might ultimately be used as therapeutic agents for the clinical management of hypertension.

Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke.

Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen-glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1ß and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1ß and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.

Reduction of cerebral infarct volume by apocynin requires pretreatment and is absent in Nox2-deficient mice.

BACKGROUND AND PURPOSE: Reactive oxygen species (ROS) derived from Nox2-containing reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity is reportedly detrimental in cerebrovascular disease. However, ROS generation by other Nox isoforms may have a physiological role. No Nox2-selective inhibitors have yet been identified, and thus it is unclear whether isoform non-selective Nox inhibitors would necessarily improve outcome after stroke. We assessed the effect of apocynin on cerebrovascular ROS production and also on outcome following cerebral ischaemia when administered either before ischaemia or after cerebral reperfusion. The involvement of Nox2-containing NADPH oxidase in the effects of apocynin was assessed using Nox2(-/-) mice. EXPERIMENTAL APPROACH: Transient cerebral ischaemia was induced by 0.5 h middle cerebral artery occlusion followed by 23.5 h reperfusion. Mice received apocynin (2.5 mg.kg(-1), i.p.) either 0.5 h before ischaemia or 1 h after reperfusion. In situ superoxide production after cerebral ischaemia-reperfusion was measured in brain sections of wild-type mice at 24 h using dihydroethidium fluorescence. KEY RESULTS: Treatment with apocynin 0.5 h before ischaemia reduced total infarct volume, neurological impairment and mortality in wild-type but not Nox2(-/-) mice. Conversely, treatment with apocynin 1 h after initiation of reperfusion had no protective effect. Cerebral ischaemia and reperfusion increased superoxide production in the brain at 24 h, and pretreatment but not posttreatment with apocynin reduced superoxide levels. CONCLUSIONS AND IMPLICATIONS: Apocynin improves outcome following stroke when administered before ischaemia in wild-type but not Nox2(-/-) mice.

The antioxidant tempol inhibits cardiac hypertrophy in the insulin-resistant GLUT4-deficient mouse in vivo.

Reactive oxygen species such as superoxide are implicated in cardiac hypertrophy, but their contribution to the cardiac complications of insulin resistance is unresolved. We tested the hypothesis that the antioxidant tempol attenuates cardiac hypertrophy in insulin-resistant mice. Mice with cardiac GLUT4 deletion (GLUT4-knockout), superimposed on global GLUT4 suppression (GLUT4-knockdown) were administered tempol for 4 weeks. Age-matched GLUT4-knockdown littermates were used as controls (14 mice/group). GLUT4-knockout mice exhibited marked cardiac hypertrophy: heart to body weight ratio was increased 61+/-7% and expression of the hypertrophic genes beta-myosin heavy chain and B-type natriuretic peptide (BNP) were elevated 5.5+/-0.7- and 6.2+/-1.5-fold relative to control, respectively. Pro-fibrotic pro-collagen III expression was also higher (3.8+/-0.7-fold) in the GLUT4-knockout myocardium (all p<0.001). Both gp91(phox) and Nox1 subunits of NADPH oxidase were also upregulated, 4.9+/-1.2- and 9.3+/-2.8-fold (both p<0.01). Tempol treatment significantly attenuated all of these abnormalities in GLUT4-knockout mice. Heart to body weight ratio was decreased, as was fold expression of beta-myosin heavy chain (to 3.8+/-0.8), BNP (to 2.5+/-0.5), pro-collagen III (to 1.9+/-0.4), gp91(phox) (to 0.9+/-0.3) and Nox1 (to 2.3+/-0.1, all p<0.05 versus untreated GLUT4-knockout mice). In addition, tempol upregulated ventricular expression of both thioredoxin-2 (confirming an antioxidant action) and glycogen synthase kinase-3beta (GSK-3beta). Tempol did not elicit any other significant changes in control mice. Cardiac superoxide generation, however, was not altered by GLUT4-knockout or tempol. In conclusion, tempol treatment reduced morphological and molecular evidence of cardiac hypertrophy in the GLUT4-knockout insulin-resistant mouse in vivo, even at doses insufficient to lower cardiac superoxide. Parallel reductions in pro-collagen III and NADPH oxidase have important implications for our understanding of the molecular basis of cardiac hypertrophy in the setting of insulin resistance. Antioxidants may offer new alternatives in this disorder.

Shear stress regulates endothelial nitric oxide synthase expression through c-Src by divergent signaling pathways.

In this study, we defined the signaling cascade responsible for increased eNOS mRNA expression in response to laminar shear stress. This pathway depends on the tyrosine kinase c-Src because shear induction of eNOS mRNA is blocked by the c-Src inhibitors PP1 and PP2, as well as an adenovirus encoding kinase inactive c-Src. After activation of c-Src, this pathway diverges. One arm is responsible for the short-term (6 hour) increase in eNOS mRNA. This involves a transient, 1-hour increase in eNOS transcription, as detected by nuclear run-on, that is dependent on activation of Ras and is blocked by adenoviral infection with dominant negative Ras. Downstream of Ras, MEK1/2 and ERK1/2 are important in this pathway, as 2 inhibitors of MEK1/2, PD98059 and UO126, completely prevented this early increase in eNOS mRNA. ERK1/2 was rapidly phosphorylated in response to shear, and this was prevented by c-Src and Ras inhibition. Further, Raf is phosphorylated in response to shear stress, and this is prevented by c-Src inhibition, suggesting that Raf may transduce the signal between Ras and ERK1/2. The second arm of the pathway linking activation of c-Src to eNOS expression involves stabilization of eNOS mRNA by shear stress. This response to shear is completely abrogated by the c-Src inhibitor PP1 but not altered by Ras or MEK1/2 inhibition. Thus, c-Src plays a central role in modulation of eNOS expression in response to shear stress via divergent pathways involving a short-term increase in eNOS transcription and a longer-term stabilization of eNOS mRNA.

Induction of endothelial NO synthase by hydrogen peroxide via a Ca(2+)/calmodulin-dependent protein kinase II/janus kinase 2-dependent pathway.

We have recently demonstrated that hydrogen peroxide (H(2)O(2)) is an extremely potent stimulus of endothelial NO synthase (eNOS) gene expression. The present study was designed to identify the signaling mechanisms mediating this response. Induction of eNOS expression by H(2)O(2) was found to be Ca(2+) dependent, inasmuch as it was blocked by BAPTA-AM. Further studies have indicated that Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) plays a critical role in mediating this response. Immunocytochemical staining with an anti-CaM kinase II antibody confirmed the expression of CaM kinase II in cultured bovine aortic endothelial cells. H(2)O(2) induced autophosphorylation of CaM kinase II and increased the activity of the enzyme, as assessed by an in-gel kinase assay. A specific inhibitor for CaM kinase II, KN93, and a calmodulin antagonist, W-7, attenuated eNOS induction by H(2)O(2). Further studies have indicated that janus kinase 2 is important in mediating increased eNOS expression in response to H(2)O(2) and likely is downstream from CaM kinase II. In conclusion, these data provide the first evidence that CaM kinase II plays a critical role in endothelial redox signaling. Regulation of eNOS via this pathway may represent an important vascular adaptation to oxidant stress.

Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide.

Diverse stimuli, including shear stress, cyclic strain, oxidized LDL, hyperglycemia, and cell growth, modulate endothelial nitric oxide synthase (eNOS) expression. Although seemingly unrelated, these may all alter cellular redox state, suggesting that reactive oxygen intermediates might modulate eNOS expression. The present study was designed to test this hypothesis. Exposure of bovine aortic endothelial cells for 24 hours to paraquat, a superoxide (O(2)(-*))-generating compound, did not affect eNOS mRNA levels. However, cotreatment with paraquat and either Cu(2+)/Zn(2+) superoxide dismutase or the superoxide dismutase mimetic tetrakis(4-benzoic acid)porphyrin chloride increased eNOS mRNA by 2.3- and 2.2-fold, respectively, implicating a role for H(2)O(2). Direct addition of 100 and 150 micromol/L H(2)O(2) caused increases in bovine aortic endothelial cell eNOS mRNA that were dependent on concentration (ie, 3.1- and 5.2-fold increases) and time, and elevated eNOS protein expression and enzyme activity, accordingly. Nuclear run-on and 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole-chase studies showed that H(2)O(2) caused a 3.0-fold increase in eNOS gene transcription and a 2.8-fold increase in eNOS mRNA half-life. Induction of eNOS by H(2)O(2) was not affected by the hydroxyl radical scavenger DMSO, mannitol, or N-tert-butyl-alpha-phenylnitrone, but it was inhibited by the antioxidants N-acetylcysteine, ebselen, and exogenously added catalase. Unlike H(2)O(2), the 4.0-fold induction of eNOS by shear stress (15 dyne/cm(2) for 6 hours) was not inhibited by N-acetylcysteine or exogenous catalase. In conclusion, H(2)O(2) increases eNOS expression through transcriptional and post-transcriptional mechanisms. Although H(2)O(2) does not mediate shear-dependent eNOS regulation, it is likely to be involved in regulation of eNOS expression in response to other physiological and/or pathophysiological stimuli.

Apamin-sensitive, non-nitric oxide (NO) endothelium-dependent relaxations to bradykinin in the bovine isolated coronary artery: no role for cytochrome P450 and K+.

Since cytochrome P(450)-derived metabolites of arachidonic acid and K(+) have been implicated in endothelium-derived hyperpolarizing factor (EDHF)-dependent responses, the aim of this study was to determine whether such factors contribute to non-nitric oxide (NO), endothelium-dependent relaxation to bradykinin (BK) in bovine isolated coronary artery. In rings of artery contracted with U46619 and treated with indomethacin (3 microM) and N(G)-nitro-L-arginine (L-NOARG; 100 microM), relaxation to BK (0.01 nM-0.3 microM) was blocked by approximately 60% after inhibition of K(+) channels with either high extracellular K(+) (high [K(+)](o); 15 - 67 mM) or apamin (0.3 microM). Ouabain (1 microM), an inhibitor of Na(+)/K(+)-ATPase, decreased the sensitivity to BK without affecting the maximum response. In L-NOARG-treated rings, ouabain had no further effect on the relaxation to BK. An inhibitor of inward-rectifying K(+) channels, Ba(2+) (30 microM), had no effect on relaxations to BK in the absence or presence of either L-NOARG or ouabain. KCl (2.5 - 10 mM) elicited small relaxations ( approximately 20%) that were abolished by nifedipine (0.3 microM) and ouabain. Both the high [K(+)](o)/apamin-sensitive relaxation to BK, and the relaxation to the K(ATP) channel-opener, levcromakalim (0.6 microM), were unaffected by the cytochrome P(450) inhibitor, 7-ethoxyresorufin (10 microM), or by co-treatment with a phospholipase A(2) inhibitor, arachidonyl trifluoromethyl ketone (AACOCF(3); 3 microM) and a diacylglycerol (DAG)-lipase inhibitor, 1, 6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267; 30 microM). The non-NO/high [K(+)](o)-insensitive, approximately 40% relaxation to BK was, however, abolished by these treatments. Therefore, neither cytochrome P(450)-derived metabolites of arachidonic acid nor K(+) appear to mediate the EDHF-like relaxation to BK (i.e the non-NO, high [K(+)](o)/apamin-sensitive component) in bovine coronary arteries. Cytochrome P(450)-derived metabolites may be released at higher BK concentrations to act in parallel with NO and the high [K(+)](o)/apamin-sensitive mechanism.

Modulation of expression of endothelial nitric oxide synthase by nordihydroguaiaretic acid, a phenolic antioxidant in cultured endothelial cells.

Retrospective epidemiological studies have suggested that antioxidant therapy may decrease cardiovascular morbidity and mortality rates, although the mechanisms for this effect remain unclear. In the present study, we demonstrate that selective antioxidants can enhance expression of endothelial nitric oxide synthase (eNOS). We found that the antioxidants nordihydroguaiaretic acid (NDGA), catechol, glutaryl probucol, and N-acetylcysteine increased eNOS expression in cultured bovine aortic endothelial cells (BAECs). NDGA seemed to be the most potent of the phenolic antioxidants, producing a 3-fold increase in eNOS mRNA. This effect of NDGA was enhanced by nonphenolic antioxidants such as N-acetylcysteine and ascorbic acid. Nuclear run-on studies indicated that NDGA increased eNOS transcription. A similar increase in eNOS protein content was observed with Western blot analysis after treating BAECs or human aortic endothelial cells with NDGA. Exposure of BAECs to NDGA enhanced NO production, as measured by electron paramagnetic resonance spin trapping and eNOS activity, as measured by [14C]arginine-to-[14C]citrulline assay. Methylation of the phenolic hydroxyl groups completely inhibited the NDGA effect on eNOS mRNA levels. This effect of NDGA was not due to inhibition of lipoxygenase because cis-5,8,11,14-eicosatetraynoic acid did not alter eNOS expression. We conclude that antioxidants may not only increase the bioactivity of nitric oxide but also enhance expression of the eNOS enzyme. Such an effect may prove useful in conditions such as hypertension and atherosclerosis, in which nitric oxide production and/or biological activity is impaired.

Evidence for mediation by endothelium-derived hyperpolarizing factor of relaxation to bradykinin in the bovine isolated coronary artery independently of voltage-operated Ca2+ channels.

1. The role of endothelium-derived hyperpolarizing factor and voltage-operated Ca2+ channels in mediating endothelium-dependent, NG-nitro-L-arginine (L-NOARG; 100 microM) -resistant relaxations to bradykinin (BK), was examined in isolated rings of endothelium-intact bovine left anterior descending coronary artery. 2. Rings of artery were contracted isometrically to approximately 40% or their respective maximum contraction to 125 mM KCl Krebs solution (KPSSmax) with the thromboxane A2-mimetic, U46619. Relaxations to BK and the endothelium-independent NO donor, S-nitroso-N-acetylpenicillamine (SNAP), were normalized as percentages of reversal of the initial contraction to U46619. All experiments were carried out in the presence of indomethacin (3 microM). 3. BK caused concentration-dependent relaxations [sensitivity (pEC50) 9.88 +/- 0.05; maximum relaxation (Rmax), 103.3 +/- 0.5%] in U46619-contracted rings of bovine coronary artery. L-NOARG (100 microM) caused a significant (P < 0.01) 3 fold reduction in the sensitivity to BK (pEC50, 9.27 +/- 0.11) without affecting the Rmax (101.8 +/- 2.3%). A similar, significant 3 fold reduction in sensitivity to BK with no change in Rmax was observed after treatment with oxyhaemoglobin (20 microM; pEC50, 9.18 +/- 0.13, P < 0.001) or a combination of oxyhaemoglobin (20 microM) and L-NOARG (100 microM; pEC50, 9.08 +/- 0.10, P < 0.001). Oxyhaemoglobin (20 microM) either alone or in combination with L-NOARG (100 microM) caused an approximate 600 fold decrease in the sensitivity to SNAP. 4. The L-type voltage-operated Ca2+ channel inhibitor, nifedipine (0.3 microM-3 microM), reduced the maximum contraction (Fmax) to isotonic 68 mM KCl Krebs solution (103.5 +/- 2.0% KPSSmax) by 85-90% (P < 0.001); yet, the highest concentration of nifedipine (3 microM) caused only a small but significant reduction in both the sensitivity and Fmax to U46619. By contrast, nifedipine (3 microM) had no effect on the relaxation response to BK. Furthermore, a combination of nifedipine (3 microM) and L-NOARG (100 microM) had no further inhibitory effects on relaxations to BK (pEC50, 8.79 +/- 0.10; Rmax, 101.7 +/- 2.4%) than did L-NOARG (100 microM) alone (pEC50, 9.05 +/- 0.12; Rmax, 99.62 +/- 1.19). Also, nifedipine (0.3 microM and 3 microM) had no effect on the maximum relaxation to the K+ channel opener, levcromakalim (0.3 microM). 5. In the presence of nifedipine (0.3 microM to control contractions induced by high KCl) and isotonic 68 mM KCl Krebs solution (to inhibit K+ channel activity), relaxations to BK (pEC50, 9.42 +/- 0.10; Rmax, 93.9 +/- 1.8%) were similar to those observed in normal Krebs solution (pEC50, 9.58 +/- 0.09; Rmax, 98.4 +/- 0.8%). However, in the presence of 68 mM KCl Krebs solution the inhibitory effect of L-NOARG (100 microM) on relaxations to BK (pEC50, 8.53 +/- 0.20; Rmax, 31.0 +/- 11.3%) was markedly greater than that in normal KCl Krebs solution (pEC50, 9.12 +/- 0.08; Rmax, 91.5 +/- 2.0%). Similar treatment with 68 mM KCl Krebs had no effect on relaxations to the NO donor, SNAP, yet abolished the response to the K+ channel opener, levcromakalim (0.3 microM). 6. In summary, this study has shown that (1) NO synthesis in response to BK in bovine coronary artery endothelial cells in situ is likely to be abolished by L-NOARG, (2) NO-independent relaxations to BK are markedly attenuated by 68 mM KCl-containing Krebs, which, in the absence of L-NOARG, had no effect, (3) nifedipine blocked contractions to a maximum-depolarizing stimulus (KCl) yet had no effect on NO-independent relaxations to BK, and (4) maximum relaxations to levcromakalim were abolished by 68 mM KCl Krebs but were not affected by nifedipine. Therefore, we hypothesize that if smooth muscle hyperpolarization is involved in non-NO-, endothelium-dependent relaxation in bovine coronary arteries contracted with U46619, then it can accomplish this via a mechanism which does not i

Endothelium-dependent relaxation to the B1 kinin receptor agonist des-Arg9-bradykinin in human coronary arteries.

Des-Arg9-bradykinin (des-Arg9-BK) caused endothelium-dependent relaxations in human, isolated coronary arteries which upregulated with in vitro incubation time. Relaxations to des-Arg9-BK were inhibited by the B1 receptor antagonist, des-Arg9-[Leu3]-BK (pK(B), 6.14 +/- 0.11) but were unaffected by the B2 receptor antagonist, Hoe-140. Therefore, this is the first demonstration that human coronary arteries possess endothelial B1 receptors which mediate endothelium-dependent relaxation and appear to be synthesized de novo during the incubation period.

Endothelium-dependent relaxations mediated by inducible B1 and constitutive B2 kinin receptors in the bovine isolated coronary artery.

1. Rings of bovine left anterior descending coronary artery (LAD) were contracted with the thromboxane A2-mimetic, U46619 (1-30 nM), to approximately 40% of their maximum contraction to 125 mM KCl Krebs solution (KPSSmax) for comparison of responses to the B1 and B2 kinin receptor agonists, des-Arg9-bradykinin (des-Arg9-BK) and bradykinin (BK), respectively. Relaxation responses were normalized as percentages of the initial U46619-induced contraction level, while contractile responses were expressed as percentages of KPSSmax. 2. After 6 h of in vitro incubation in Krebs solution at 37 degrees C, des-Arg9-BK (pEC50, 8.00 +/- 0.08; maximum response (Rmax), 93.9 +/- 1.9%) and BK (pEC50, 9.75 +/- 0.07; Rmax, 100.1 +/- 0.7%) caused endothelium-dependent relaxations in precontracted rings of bovine LAD which were competitively and selectively antagonized by the B1 receptor antagonist, des-Arg9-[Leu8]-BK (pA2, 6.27 +/- 0.11) and the B2 receptor antagonist Hoc-140 (pA2, 9.63 +/- 0.14), respectively. 3. At 3 h of in vitro incubation, the sensitivity (pEC50, 7.45 +/- 0.10) and Rmax (84.6 +/- 3.3%) to des-Arg9-BK were significantly less than those obtained in the same tissues at 6 h (pEC50, 7.94 +/- 0.06; Rmax, 91.4 +/- 2.5%), whereas endothelium-dependent relaxations to BK and ACh were unaffected by incubation time. 4. Relaxation responses to des-ARg9-BK, but not BK, at both 3 h and 6 h were significantly attenuated by the protein synthesis inhibitors, cycloheximide (30 and 100 microM) and actinomycin D (2 microM). 5. At 6 h, the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NOARG, 100 microM), caused a significant 2 fold decrease in pEC50 (9.58 +/- 0.03) but had no effect on Rmax for BK. For des-Arg9-BK, L-NOARG (100 microM) caused a marked and significant decrease in both the pEC50 and Rmax and revealed contractions to low concentrations of des-Arg9-BK. In both cases, L-NOARG inhibition was reversed in the presence of L-arginine (10 mM). 6. At 6 h removal of the endothelium abolished relaxation responses to des-Arg9-BK and BK, and for des-Arg9-BK, but not BK, unmasked concentration-dependent contractions (pEC50, 7.57 +/- 0.09; Rmax, 83.4 +/- 9.1%). The sensitivity of contractions to des-Arg9-BK increased slightly from 3 h (pEC50, 7.37 +/- 0.08) to 6 h (pEC50, 7.62 +/- 0.12) of in vitro incubation; however, there was a small but significant depression in the maximum response over this time (Rmax, 126.8 +/- 8.5% and 103.3 +/- 8.6% for 3 h and 6 h of incubation respectively). 7. In conclusion, the bovine LAD contains inducible B1 and constitutive B2 endothelial cell kinin receptors, both of which mediate endothelium-dependent relaxation partly via the release of NO. B1 receptors were also present on the smooth muscle layer of the bovine LAD.