Suppression by protease-activated receptor-2 activation of gastric acid secretion in rats

Activation of protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, induces neurally mediated gastric mucus secretion accompanied by mucosal cytoprotection. In the present study, we investigated whether PAR-2 could modulate gastric acid secretion in rats. Messenger RNAs for PAR-2 and PAR-1 were detected in the gastric mucosa and smooth muscle. The PAR-2-activating peptide SLIGRL-NH(2), but not the inactive control peptide, when administered i.v., strongly suppressed gastric acid secretion in response to carbachol, pentagastrin or 2-deoxy-D-glucose in the rats with a pylorus ligation. The PAR-2-mediated suppression of acid secretion was resistant to cyclooxygenase inhibition or ablation of sensory neurons by capsaicin. Our results provide novel evidence that in addition to stimulating neurally mediated mucus secretion, activation of PAR-2 suppresses gastric acid secretion independently of prostanoid production or sensory neurons. These dual actions of PAR-2 would result in gastric mucosal cytoprotection.     

Protease-activated receptor-2-mediated contraction of urinary bladder is enhanced in cyclophosphamide-treated rats

Protease-activated receptor-2 (PAR-2) is activated by serine proteases, such as trypsin and mast cell tryptase. Recently, we have shown that activators of PAR-2 contract the rat urinary bladder mainly by stimulating release of prostaglandins (PGs) from the mucosal layer. In the present study, we investigated how the PAR-2-mediated responses are altered in rats with cyclophosphamide (CYP)-induced cystitis. The contractile responses to trypsin and PAR-2 activating peptide (PAR-2 AP; SLIGRL-NH₂) in the urinary bladders were augmented by treatment of rats with CYP. The contractile effects of these PAR-2 activators on the smooth muscles of the urinary bladder were also potentiated after induction of cystitis by CYP. On the other hand, CYP-induced cystitis significantly attenuated contractions produced by PGE₂ in the smooth muscles of the urinary bladder. The PAR-2-mediated contractions were significantly prevented by indomethacin or NS-398, an inhibitor of cyclooxygenase-2. Both trypsin and PAR-2 AP increased the release of PGE₂ from the urinary bladder mucosa and smooth muscle. CYP-induced cystitis enhanced the PAR-2 activators-induced PGE₂ releases from the urinary mucosa without affecting those from the smooth muscle of the urinary bladder. The PGE₂ releases were prevented by indomethacin or NS-398. The mRNAs for PAR-2 in the urinary bladder mucosa and smooth muscle preparations were not altered in CYP-induced cystitis. These results suggest that PAR-2-mediated responses were enhanced in bladders from CYP-treated rats. The enhancement of PAR-2-mediated contraction might be ascribed to the increased production of PGs and the altered sensitivity of smooth muscle to PAR-2 activators.     

Increased vascular permeability by a specific agonist of protease-activated receptor-2 in rat hindpaw

The present study examined the effect of intraplantar (i.pl.) administration of a selective agonist of protease-activated receptor (PAR)-2, SLIGRL-NH₂(PP6-NH₂), on vascular permeability in rat hindpaw. PP6-NH₂, administered i.pl. at 10–100 nmol per paw, enhanced vascular permeability and caused oedema formation in rat hindpaw. SLIGRL (PP6-OH) and trypsin, by i.pl. administration, also elicited an increase in vascular permeability, although i.pl. administration of the mixture of constituent amino acids of PP6-OH at an equivalent dose did not. The PP6-NH₂-induced increase in vascular permeability was abolished by repeated pretreatment with compound 48/80 to deplete bioactive amines in mast cells. These findings suggest that the activation of PAR-2 induces acute inflammation, at least partially, via mast cell degranulation in rat hindpaw.     

Salmon and king crab trypsin stimulate interleukin-8 and matrix metalloproteinases via protease-activated receptor-2 in the skin keratinocytic HaCaT cell line

Occupational skin symptoms are prevalent among the workers of the seafood processing industry. In this study we investigate the role of salmon (Salmo salar) and king crab trypsin (Paralithodes camtschaticus) as inducers of inflammation in skin via secretion of inflammatory mediators. Human skin keratinocytes (HaCaT cells) were exposed to purified salmon and king crab trypsin. We observed that salmon trypsin enhanced the secretion of IL-8 and MMP-2 and crab trypsin enhanced the secretion of IL-8, MMP-2 and MMP-9 in a dose dependent manner. As protease activated receptors (PAR)-2 in skin are known to play an important role in physiology and pathology, we explored the involvement of these receptors in mediating the release of interleukin (IL)-8 and matrix metalloproteinase (MMP)-2 and -9 subsequent to exposure of skin keratinocytes to salmon and crab trypsin. In addition we observed that salmon and crab trypsin exhibit individual differences in stimulating the release of these inflammatory mediators. Finally, using specific small interfering RNA (siRNA) against PAR-2, we confirmed that the increase in secretion of IL-8, MMP-2 and MMP-9 in skin keratinocytes following exposure to salmon and crab trypsin was mediated via activation of PAR-2. These results suggest that exposure to proteases from the seafood may lead to inflammatory reactions in skin.     

Role of PGE(2) in protease-activated receptor-1, -2 and -4 mediated relaxation in the mouse isolated trachea

1. The potential mediator role of the prostanoid PGE(2) in airway smooth muscle relaxations induced by peptidic and proteolytic activators of PAR-1, PAR-2, PAR-3 and PAR-4 was investigated in carbachol-precontracted mouse isolated tracheal segments. 2. The tethered ligand domain sequences of murine PAR-1 (SFFLRN-NH(2)), PAR-2 (SLIGRL-NH(2)) and PAR-4 (GYPGKF-NH(2)), but not PAR-3 (SFNGGP-NH(2)), induced smooth muscle relaxation that was abolished by the non-selective cyclo-oxygenase (COX) inhibitor, indomethacin. The relative order for mean peak relaxation was SLIGRL-NH(2)>GYPGKF-NH(2) approximately amp; SFFLRN-NH(2)>SFNGGP-NH(2). 3. SFFLRN-NH(2), SLIGRL-NH(2) and GYPGKF-NH(2), but not SFNGGP-NH(2), induced significant PGE(2) release that was abolished by indomethacin. Like that for relaxation, the relative order for mean PGE(2) release was SLIGRL-NH(2)>GYPGKF-NH(2)>SFFLRN-NH(2)>SFNGGP-NH(2). 4. In dose-response studies, SLIGRL-NH(2) induced concentration-dependent increases in PGE(2) release (EC(50)=20.4 microM) and smooth muscle relaxation (EC(50)=15.8 microM). 5. The selective COX-2 inhibitor, nimesulide, but not the COX-1 inhibitor valeryl salicylate, significantly attenuated SLIGRL-NH(2)-induced smooth muscle relaxation and PGE(2) release. 6. Exogenously applied PGE(2) induced potent smooth muscle relaxation (EC(50)=60.3 nM) that was inhibited by the mixed DP/EP(1)/EP(2) prostanoid receptor antagonist, AH6809. SLIGRL-NH(2)-induced relaxation was also significantly inhibited by AH6809. 7. In summary, the results of this study strongly suggest that PAR-mediated relaxation in murine tracheal smooth muscle is dependent on the generation of the spasmolytic prostanoid, PGE(2). PAR-stimulated PGE(2) release appears to be generated preferentially by COX-2 rather than COX-1, and induces relaxation via activation of the EP(2) receptor.     

Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries

BACKGROUND AND PURPOSE

P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Although it is well-appreciated that the myocyte Ca²⁺ signalling system is composed of microdomains, little is known about the structure of the [Ca²⁺]_i responses induced by P2X receptor stimulation in vascular myocytes.

EXPERIMENTAL APPROACHES

Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca²⁺ signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP).

KEY RESULTS

RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP₃R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca²⁺]_i transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L⁻¹αβ-meATP triggered an abrupt Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum enriched with IP₃Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca²⁺ channels (VGCCs) or IP₃Rs suppressed the sub-plasmalemmal [Ca²⁺]_i upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP₃R inhibition on the sub-plasmalemmal [Ca²⁺]_i upstroke was attenuated following block of VGCCs.

CONCLUSIONS AND IMPLICATIONS

Depolarization of RVSMCs following P2X receptor activation induces IP₃R-mediated Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum, which is activated mainly by Ca²⁺ influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes.     

Role of H(2)O(2) in hypertension, renin-angiotensin system activation and renal medullary disfunction caused by angiotensin II

BACKGROUND AND PURPOSE

Activation of the intrarenal renin-angiotensin system (RAS) and increased renal medullary hydrogen peroxide (H₂O₂) contribute to hypertension. We examined whether H₂O₂ mediated hypertension and intrarenal RAS activation induced by angiotensin II (Ang II).

EXPERIMENTAL APPROACH

Ang II (200 ng·kg⁻¹·min⁻¹) or saline were infused in Sprague Dawley rats from day 0 to day 14. Polyethylene glycol (PEG)-catalase (10 000 U·kg⁻¹·day⁻¹) was given to Ang II-treated rats, from day 7 to day 14. Systolic blood pressure was measured throughout the study. H₂O₂, angiotensin AT₁ receptor and Nox4 expression and nuclear factor-κB (NF-κB) activation were evaluated in the kidney. Plasma and urinary H₂O₂ and angiotensinogen were also measured.

KEY RESULTS

Ang II increased H₂O₂, AT₁ receptor and Nox4 expression and NF-κB activation in the renal medulla, but not in the cortex. Ang II raised plasma and urinary H₂O₂ levels, increased urinary angiotensinogen but reduced plasma angiotensinogen. PEG-catalase had a short-term antihypertensive effect and transiently suppressed urinary angiotensinogen. PEG-catalase decreased renal medullary expression of AT₁ receptors and Nox4 in Ang II-infused rats. Renal medullary NF-κB activation was correlated with local H₂O₂ levels and urinary angiotensinogen excretion. Loss of antihypertensive efficacy was associated with an eightfold increase of plasma angiotensinogen.

CONCLUSIONS AND IMPLICATIONS

The renal medulla is a major target for Ang II-induced redox dysfunction. H₂O₂ appears to be the key mediator enhancing intrarenal RAS activation and decreasing systemic RAS activity. The specific control of renal medullary H₂O₂ levels may provide future grounds for the treatment of hypertension.     

Perivascular adipose tissue-derived relaxing factors: release by peptide agonists via proteinase-activated receptor-2 (PAR2) and non-PAR2 mechanisms

BACKGROUND AND PURPOSE

We hypothesized that proteinase-activated receptor-2 (PAR2)-mediated vasorelaxation in murine aorta tissue can be due in part to the release of adipocyte-derived relaxing factors (ADRFs).

EXPERIMENTAL APPROACH

Aortic rings from obese TallyHo and C57Bl6 intact or PAR2-null mice either without or with perivascular adipose tissue (PVAT) were contracted with phenylephrine and relaxation responses to PAR2-selective activating peptides (PAR2-APs: SLIGRL-NH₂ and 2-furoyl-LIGRLO-NH₂), trypsin and to PAR2-inactive peptides (LRGILS-NH₂, 2-furoyl-OLRGIL-NH₂ and LSIGRL-NH₂) were measured. Relaxation was monitored in the absence or presence of inhibitors that either alone or in combination were previously shown to inhibit ADRF-mediated responses: L-NAME (NOS), indomethacin (COX), ODQ (guanylate cyclase), catalase (H₂O₂) and the K⁺ channel-targeted reagents, apamin, charybdotoxin, 4-aminopyridine and glibenclamide.

KEY RESULTS

Endothelium-intact PVAT-free preparations did not respond to PAR2-inactive peptides (LRGILS-NH₂, LSIGRL-NH₂, 2-furoyl-OLRGIL-NH₂), whereas active PAR2-APs (SLIGRL-NH₂; 2-furoyl-LIGRLO-NH₂) caused an L-NAME-inhibited relaxation. However, in PVAT-containing preparations treated with L-NAME/ODQ/indomethacin together, both PAR2-APs and trypsin caused relaxant responses in PAR2-intact, but not PAR2-null-derived tissues. The PAR2-induced PVAT-dependent relaxation (SLIGRL-NH₂) persisted in the presence of apamin plus charybdotoxin, 4-aminopyridine and glibenclamide, but was blocked by catalase, implicating a role for H₂O₂. Surprisingly, the PAR2-inactive peptides, LRGILS-NH₂ and 2-furoyl-OLRGIL-NH₂ (but not LSIGRL-NH₂), caused relaxation in PVAT-containing preparations from both PAR2-null and PAR2-intact (C57Bl, TallyHo) mice. The LRGILS-NH₂-induced relaxation was distinct from the PAR2 response, being blocked by 4-aminopyridine, but not catalase.

CONCLUSIONS

Distinct ADRFs that may modulate vascular tone in pathophysiological settings can be released from murine PVAT by both PAR2-dependent and PAR2-independent mechanisms.     

ACE2, angiotensin-(1-7) and Mas receptor axis in inflammation and fibrosis

Recent advances have improved our understanding of the renin-angiotensin system (RAS). These have included the recognition that angiotensin (Ang)-(1-7) is a biologically active product of the RAS cascade. The identification of the ACE homologue ACE2, which forms Ang-(1-7) from Ang II, and the GPCR Mas as an Ang-(1-7) receptor have provided the necessary biochemical and molecular background and tools to study the biological significance of Ang-(1-7). Most available evidence supports a counter-regulatory role for Ang-(1-7) by opposing many actions of Ang II on AT₁ receptors, especially vasoconstriction and proliferation. Many studies have now shown that Ang-(1-7) by acting via Mas receptor exerts inhibitory effects on inflammation and on vascular and cellular growth mechanisms. Ang-(1-7) has also been shown to reduce key signalling pathways and molecules thought to be relevant for fibrogenesis. Here, we review recent findings related to the function of the ACE2/Ang-(1-7)/Mas axis and focus on the role of this axis in modifying processes associated with acute and chronic inflammation, including leukocyte influx, fibrogenesis and proliferation of certain cell types. More attention will be given to the involvement of the ACE2/Ang-(1-7)/Mas axis in the context of renal disease because of the known relevance of the RAS for the function of this organ and for the regulation of kidney inflammation and fibrosis. Taken together, this knowledge may help in paving the way for the development of novel treatments for chronic inflammatory and renal diseases.     

Proteinase-activated receptor-2 mediates hyperresponsiveness in isolated guinea pig bronchi

The mast cell serine protease tryptase has been implicated as a critical mediator of airway hyperresponsiveness in vitro and in vivo. We have previously demonstrated that tryptase promotes hyperresponsiveness in isolated guinea pig bronchi. In this study, we have investigated the potential role of tryptase-mediated activation of proteinase-activated receptor-2 (PAR-2) in promoting airway hyperresponsiveness. Ex vivo exposure of guinea pig bronchi to the PAR-2 agonists H(2)N-Ser-Leu-Ile-Gly-Arg-Leu-CONH(2) (SLIGRL) and t-cinnamoyl-H(2)N-Leu-Ile-Gly-Arg-Leu-O-CONH(2) (t-c-LIGRLO) (0.1-10 microM) induced a concentration-dependent increase of contractile response to histamine. Treatment with 10 microM SLIGRL or t-c LIGRLO for 45 min increased subsequent responsiveness to histamine (0.3mM) by 54+/-3% and 69+/-5%, respectively (P<0.05 vs. control). In contrast, the PAR-1 agonist peptide H(2)N-Ser-Phe-Leu-Leu-Arg-Asn-CONH(2) (SFLLRN) did not promote significant changes in the airway. Effects of the peptides were observed following at least a 30-min preincubation with the tissue. Coincubation with indomethacin or removal of epithelial cells is required for PAR-2-mediated hyperreactivity. The inactive analogue H(2)N-Leu-Ser-Ile-Gly-Arg-Leu-CONH(2) (LISGRL; 10 microM) failed to promote hyperresponsiveness. Neuropeptide antagonists blocked the effect of the PAR-2 agonists. Selective antagonists of NK1 (L-703,606), NK2 (L-659,877), and CGRP (alphaCGRP 8-37) provided additive inhibition of PAR-2-mediated hyperreactivity. Pretreatment of bronchi with capsaicin (0.8 microM) also prevented the effects of SLIGRL. These results demonstrate the potential involvement of tryptase-mediated activation of PAR-2 in promoting airway hyperresponsiveness. These results further demonstrate that the PAR-2-mediated response involves a neurogenic mechanism involving neuropeptide release.     

Not so EEZE: the 'EDHF' antagonist 14, 15 epoxyeicosa-5(Z)-enoic acid has vasodilator properties in mesenteric arteries

P-450 metabolites, including the epoxyeicosatrienoic acids, are likely candidates for endothelial derived hyperpolarising factor (EDHF). In the present study, we confirm that the stable analogue 11-nonyloxyundec-8(Z)-enoic acid is a vasodilator of murine vessels. However, we also show that the 'epoxyeicosatrienoic acid receptor' antagonist 14,15 EEZE similarly dilates murine vessels contracted with U46619, prostaglandin F2alpha or methoxamine, but not with endothelin-1 or potassium. We suggest that 14,15 EEZE is a partial agonist for the epoxyeicosatrienoic acids/EDHF receptor. These results illustrate an important pharmacological property of this antagonists, which is being increasingly used to study the nature of EDHF.     

Stx2 Induces Differential Gene Expression and Disturbs Circadian Rhythm Genes in the Proximal Tubule

Shiga toxin-producing Escherichia coli (STEC) causes proximal tubular defects in the kidney. However, factors altered by Shiga toxin (Stx) within the proximal tubules are yet to be shown. We determined Stx receptor Gb3 in murine and human kidneys and confirmed the receptor expression in the proximal tubules. Stx2-injected mouse kidney tissues and Stx2-treated human primary renal proximal tubular epithelial cell (RPTEC) were collected and microarray analysis was performed. We compared murine kidney and RPTEC arrays and selected common 58 genes that are differentially expressed vs. control (0 h, no toxin-treated). We found that the most highly expressed gene was GDF15, which may be involved in Stx2-induced weight loss. Genes associated with previously reported Stx2 activities such as src kinase Yes phosphorylation pathway activation, unfolded protein response (UPR) and ribotoxic stress response (RSR) showed differential expressions. Moreover, circadian clock genes were differentially expressed, suggesting Stx2-induced renal circadian rhythm disturbance. Circadian rhythm-regulated proximal tubular Na⁺-glucose transporter SGLT1 (SLC5A1) was down-regulated, indicating proximal tubular functional deterioration, and mice developed glucosuria confirming proximal tubular dysfunction. Stx2 alters gene expression in murine and human proximal tubules through known activities and newly investigated circadian rhythm disturbance, which may result in proximal tubular dysfunctions.     

Proteinase‐activated receptor‐2 (PAR2) and mouse osteoblasts: Regulation of cell function and lack of specificity of PAR2‐activating peptides

1. Using synthetic proteinase‐activated receptor‐2 (PAR₂)‐activating peptides (PAR₂APs) corresponding to the tethered ligand domain of the extracellular N‐terminus of PAR₂ to mimic the actions of activating proteinases and using primary cultures of calvarial osteoblasts derived from both wild‐type (WT) and PAR₂‐null (KO) mice, we investigated the potential role of PAR₂ in regulating osteoblast function. 2. Primary calvarial osteoblasts from WT and KO mice were evaluated for their growth kinetics and mineralization in the absence of PAR₂ agonists and for their responses in a variety of functional assays to the PAR₂APs Ser‐Leu‐Ile‐Gly‐Arg‐Leu‐amide (SLIGRL‐NH₂) and 2‐furoyl‐Leu‐Ile‐Gly‐Arg‐Leu‐Orn‐amide (2‐fLIGRLO‐NH₂), as well as to trypsin. 3. In contrast with WT cells, PAR₂‐KO osteoblasts did not exhibit increased collagen Type I mRNA expression in response to SLIGRL‐NH₂. When grown in serum‐containing medium, KO cells increased in number more rapidly than WT cells, an effect that could be attributed to decreased apoptosis rather than increased proliferation. Surprisingly, in both WT and KO osteoblasts, the two PAR₂APs induced mobilization of intracellular calcium stores. Similarly, the PAR₂APs inhibited serum deprivation‐induced apoptosis and parathyroid hormone‐, 1,25‐dihydroxyvitamin D₃‐ or interleukin‐11‐induced mineralization in WT and KO cells. 4. We conclude that PAR₂ plays a role in osteoblast survival and collagen Type I mRNA induction and that osteoblasts can respond to the PAR₂APs via both PAR₂‐dependent and ‐independent mechanisms.     

The pharmacology of the behavioral and hypothermic responses of rats to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)

The hypothermic and motor behavioural responses to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) have been investigated in the rat. The dose-effect relationship showed that hypothermia appeared at a lower dose than a definite motor syndrome. The hypothermic response to 8-OH-DPAT was attenuated following depletion of 5-hydroxytryptamine (5-HT) by repeated intraperitoneal (IP) administration of parachlorophenylalanine (200 mg/kg) or by injection of 5,7-dihydroxytryptamine (5,7-DHT, 100 g) into the region of the third ventricle; the motor behavioural response produced simultaneously was not. Indeed, after 5,7-DHT, it was increased. Quipazine (1 mg/kg, IP) antagonised the hypothermic response and facilitated the motor behaviour. Clenbuterol (2.5 mg/kg, IP) increased both hypothermic and motor responses. (±)-propranolol was without effect on the simple hypermotility produced by 8-OH-DPAT, although it is known to antagonise the hypothermic and stereotyped motor responses. It is concluded that 8-OH-DPAT probably produces its hypothermic effects by actions at 5-HT receptors located presynaptically on 5-HT neurones, while the stereotyped components of the serotonin syndrome appear to be mediated by post-synaptic receptors.     

Age-related differences and roles of endothelial nitric oxide and prostanoids in angiotensin II responses of isolated, perfused mesenteric arteries and veins of rats

We examined whether or not cyclo-oxygenase products of arachidonic acid and endothelium-derived relaxing factor (nitric oxide, NO) regulate the vascular response to angiotensin II differently with aging or development. For this purpose angiotensin II responses of isolated, perfused rat mesenteric vascular beds were compared between rats aged 4 weeks and 32 weeks. Angiotensin II increased perfusion pressure in arteries and veins of both rats aged 4 weeks and 32 weeks. In the arteries of rats aged 32 weeks the increase was slight, and less than that in rats aged 4 weeks. In contrast, the veins showed similar increases in perfusion pressure in rats aged 4 weeks and 32 weeks. Indomethacin, an inhibitor of cyclo-oxygenase, at 5×10⁻⁶ M depressed the increase in perfusion pressure only in the arteries of rats aged 32 weeks. N^G-nitro-

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-arginine methyl ester (

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-NAME), an inhibitor of nitric oxide (NO) synthase, applied at 5×10⁻⁶ M in the presence of indomethacin enlarged the perfusion pressure increase in the arteries of both rats aged 4 weeks and 32 weeks, while it failed to modify that in the veins. After removal of the endothelium from the blood vessels, the perfusion pressure responses in arteries were increased in both rats aged 4 weeks and 32 weeks, whereas those in veins were not affected. Regardless of the endothelium being intact or removed, the increase in arterial perfusion pressure of rats aged 32 weeks all but disappeared with 5×10⁻⁶ M furegrelate, an inhibitor of thromboxane A₂ synthase, and with a combined application of furegrelate and 10⁻⁶ M SQ29,548, a blocker of thromboxane A₂/prostaglandin H₂ receptors. These results indicate the following: in rat mesenteric vascular beds the angiotensin II response in the arteries appears to diminish with aging or development, whereas that in the veins does not change. The NO released from the endothelium regulates the arterial response but vasodilating prostanoids have no role in the response. Moreover, in the arteries of rats aged 32 weeks, vasoconstricting prostanoids, such as prostaglandin H₂ and thromboxane A₂, seem to play a role in angiotensin II-induced vasoconstriction. With aging or development, and depending on the type of blood vessel, NO and prostanoids appear to modify the angiotensin II response differently.     

Di(2-ethylhexyl)phthalate-induced renal oxidative stress in rats and protective effect of selenium

This study was designed to examine the oxidative stress potential of di(2-ethylhexyl)phthalate (DEHP) on rat kidney and to evaluate possible protective effect of selenium (Se) status. Se deficiency (SeD) was produced in 3-week old Sprague?Dawley rats by feeding them ≤ 0.05 Se mg/kg diet for 5 weeks; Se supplementation group (SeS) was on 1 mg Se/kg diet. DEHP treated groups received 1000 mg/kg dose by gavage during the last 10 days of the feeding period. Activities of antioxidant selenoenzymes [glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), thioredoxin reductase (TrxR)], catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST); concentrations of total glutathione (GSH), thiols and thiobarbituric acid reactive substance (TBARS) levels were measured. DEHP treatment was found to induce oxidative stress in rat kidney, as evidenced by significant decreases in GPx1 (~20%) and SOD (~30%) activities and GSH levels (~20%), along with marked decrease in thiol content (~40%) and increase in TBARS (~30%) levels. The effects of DEHP was more pronounced in SeD rats, whereas Se supplementation was protective by providing substantial elevations of GPx1 and GPx4 activities and GSH levels. These findings emphasized the critical role of Se as an effective redox regulator and the importance of Se status in protecting renal tissue from the oxidant stressor activity of DEHP.     

Involvement of bradykinin B1 and B2 receptors in relaxation of mouse isolated trachea

1. The aim of the present study was to investigate the effects of bradykinin and [des-Arg⁹]-bradykinin and their relaxant mechanisms in the mouse isolated trachea.
2. In the resting tracheal preparations with intact epithelium, bradykinin and [des-Arg⁹]-bradykinin (each drug, 0.01–10 μM) induced neither contraction nor relaxation. In contrast, bradykinin (0.01–10 μM) induced concentration-dependent relaxation when the tracheal preparations were precontracted with methacholine (1 μM). The relaxation induced by bradykinin was inhibited by the B₂ receptor antagonist, D-Arg⁰-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]-bradykinin (Hoe 140, 0.01–1 μM) in a concentration-dependent manner whereas the B₁ receptor antagonist, [des-Arg⁹,Leu⁸]-bradykinin (0.01–1 μM), had no inhibitory effect on bradykinin-induced relaxation. [des-Arg⁹]-bradykinin (0.01–10 μM) also caused concentration-dependent relaxation after precontraction with methacholine. The relaxation induced by [des-Arg⁹]-bradykinin was concentration-dependently inhibited by the B₁ receptor antagonist, [des-Arg⁹,Leu⁸]-bradykinin (0.01–1 μM), whereas the B₂ receptor antagonist, Hoe 140 (0.01–1 μM) was without effect.
3. In the presence of the cyclo-oxygenase inhibitor, indomethacin (0.01–1 μM), the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin were inhibited concentration-dependently.
4. Two nitric oxide (NO) biosynthesis inhibitors N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) and N^G-nitro-L-arginine (L-NOARG, 100 μM) had no inhibitory effects on the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin. Neither did the selective inhibitor of the soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) inhibit the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin.
5. Prostaglandin E₂ (PGE₂, 0.01–33 μM) caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. Indomethacin (1 μM) and ODQ (10 μM) exerted no inhibitory effects on the relaxation induced by PGE₂.
6. The NO-donor, sodium nitroprusside (SNP; 0.01–100 μM) also caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. ODQ (0.1–1 μM) concentration-dependently inhibited the relaxation induced by SNP.
7. These data demonstrate that bradykinin and [des-Arg⁹]-bradykinin relax the mouse trachea precontracted with methacholine by the activation of bradykinin B₂-receptors and B₁-receptors, respectively. The stimulation of bradykinin receptors induces activation of the cyclo-oxygenase pathway, leading to the production of relaxing prostaglandins. The NO pathway is not involved in the bradykinin-induced relaxation. The relaxation caused by NO-donors in the mouse trachea is likely to be mediated via activation of soluble guanylate cyclase.

     

Valproic acid and active unsaturated metabolite (2-en): transfer to mouse liver following human therapeutic doses

The transfer of valproic acid (VPA, 2-propylpentanoic acid) and its unsaturated active metabolite (2-en, 2-propyl-2-pentenoic acid) from plasma to liver has been studied in the mouse between 2 min and 6 h following oral administration of 50 mg of the sodium salts per kg body weight. Transfer of both compounds was extremely rapid. Liver concentrations of VPA were higher than those in plasma, while liver concentrations of 2-en were lower than those in plasma. The low hepatic levels of 2-en may be explained by extensive plasma protein binding of this metabolite. The liver/plasma concentration ratios were concentration-dependent, indicating the presence of active transport mechanisms and/or saturation of plasma protein binding. Our results indicate that 2-en should be further studied in regard to its potential for the induction of liver toxicity. The desirable low level of 2-en reached in the liver, seen together with previously observed favourable-anticonvulsant profile and low teratogenicity, would indicate that this compound may be a valuable alternative antiepileptic agent.