The effects of bradykinin agonists and antagonists on visceral polymodal receptor activities

The endogenous algesic agent bradykinin (BK) is a consistent stimulant for the polymodal receptor, a type of nociceptor. Two types of BK receptor, B1 and B2, have been proposed in smooth muscles by Regoli. The type of BK receptor mediating the BK response of the polymodal receptor was studied using 3 BK analogs, des-Arg9-BK (a B1 agonist), des-Arg9-[Leu8]-BK (a B1 antagonist), and [Thi5,8, D-Phe7]-BK (a B2 antagonist). Single- and multi-fiber activities from testicular polymodal receptors were recorded in vitro using testis-spermatic nerve preparations excised from dogs anesthetized with pentobarbital (30 mg/kg, i.v.). Neither des-Arg9-BK, des-Arg9-[Leu8]-BK, nor [Thi5,8,D-Phe7]-BK induced discharges in nociceptors at concentrations up to 9.4 x 10(-6) M. Des-Arg9-[Leu8]-BK (up to 9.4 x 10(-6) M) did not suppress responses to BK (9.4 x 10(-8 approximately -9) M), whereas [Thi5,8,D-Phe7]-BK (above 2.8 x 10(-7) M) suppressed the BK response in a concentration-dependent manner and shifted the concentration-response curve of BK to the right. It was ascertained that [Thi5,8,D-Phe7]-BK had no effect on responses to noxious heat and high K+ solution. These results suggest that the BK receptor mediating the nociceptor response to BK is of the B2 type.     

Bradykinin stimulates the production of cyclic GMP via activation of B2 kinin receptors in cultured porcine aortic endothelial cells

The production of endothelium-derived relaxing factor(s) in response to kinins was investigated in cultured porcine aortic endothelial cells. The production was estimated by the measurement of the accumulation of cyclic GMP, a response which can be attributed to activation of the soluble guanylate cyclase of the endothelial cells by endothelium-derived relaxing factor(s). Bradykinin increased markedly the levels of cyclic GMP in endothelial cells without affecting those of cyclic AMP. The bradykinin-stimulated production of cyclic GMP was transient and concentration-dependent. Kallidin (an agonist at B2-kinin receptors) but not des-Arg9 bradykinin and des-Arg10 kallidin (agonists at B1 kinin receptors) also increased, in a concentration-dependent manner, the content of cyclic GMP. The B2 kinin receptor antagonist, D-Arg0 [Hyp3, D-Phe7]bradykinin but not the B1 kinin receptor antagonists Leu8-des-Arg9 bradykinin and Leu9-des-Arg10 kallidin inhibited the production of cyclic GMP upon stimulation of the endothelial cells with either bradykinin or kallidin. Both the basal and kinin (bradykinin and kallidin)-stimulated productions of cyclic GMP were reduced by hemoglobin and potentiated by superoxide dismutase. Methylene blue also reduced kinin-stimulated production of cyclic GMP. These findings suggest that cultured porcine aortic endothelial cells possess B2 kinin receptors which are associated with the production and/or release of endothelium-derived relaxing factor(s). The endothelium-derived relaxing factor(s) produced in turn enhances the activity of soluble guanylate cyclase and induces the accumulation of cyclic GMP.     

Renal haemodynamic and sympathetic responses to head-up tilt in essential hypertension

Nine mild-to-moderate hypertensive patients (HT), aged 41 +/- 0.6 years (mean +/- SEM) and nine age-matched normotensive control subjects (NT) were tilted to 60 degrees for 10 min. During tilt, both systolic (S) blood pressure (BP) (p less than 0.01) and diastolic (D) BP (p less than 0.05) increased in HT, but not in NT. At supine rest renal blood flow was higher in HT than in NT and increased by 17% in HT during tilt, while a decrease of 13% was observed in NT (p less than 0.05). Renal vascular resistance was unchanged in HT during tilt, while a significant increase (p less than 0.01) was observed in NT. Arterial plasma noradrenaline increased in both groups (p less than 0.05) during tilt, significantly more in HT than in NT (p less than 0.05). No statistically significant difference was observed between the groups in renal catecholamine uptake or release. Our data indicate enhanced general sympathetic and circulatory responses to tilt in subjects with mild-to-moderate essential hypertension. However, the enhanced haemodynamic and sympathetic responses were not shared by hypertensive kidneys and renovascular resistance remained unaffected by tilt.     

Enhanced G protein activation in immortalized lymphoblasts from patients with essential hypertension.

Epstein-Barr virus-immortalized B lymphoblasts obtained from hypertensive patients with enhanced Na+/H+ exchanger activity (HT cells) proliferate distinctly faster upon serum stimulation than those from normotensive controls with low exchanger activity (NT cells) (Rosskopf, D., E. Frömter, and W. Siffert. 1993. J. Clin. Invest. 92:2553-2559). Stimulation with platelet-activating factor (PAF) as well caused an enhanced proliferation of HT cells. In analyzing possible differences in signal transduction between the immortalized NT and HT lymphoblasts, we observed that cell stimulation with PAF and somatostatin caused a twofold higher increase in [Ca2+]i in HT than in NT cell lines. This difference was completely abrogated by pertussis toxin (PTX) treatment. Furthermore, PAF-stimulated formation of inositol 1,4,5-trisphosphate (IP3) was twofold enhanced in HT cell lines. On the other hand, PAF receptor density and affinity, total cellular phospholipase C activity, expression of PTX-sensitive G proteins, and control binding of the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), to membrane G proteins were not different in NT and HT cell lines. However, PAF- and mastoparan-stimulated binding of GTP gamma S to G proteins, which was fully PTX-sensitive, was 2.5-fold higher in HT than NT cell lines. These data suggest an enhanced receptor-mediated activation of PTX-sensitive G proteins despite unchanged receptor and G protein expression. Thus, this study not only suggests that enhanced signal transduction and cell proliferation are abnormalities in a certain group of patients with essential hypertension but also explains these findings as a result of an enhanced G protein activation in this common disorder.     

Increased adrenal renin in transgenic hypertensive rats, TGR(mREN2)27, and its regulation by cAMP, angiotensin II, and calcium.

The newly established rat strain TGR(mREN2)27 is a monogenetic model in hypertension research. Microinjecting the mouse Ren-2d renin gene caused it to become a stable part of the genome. The rats are characterized by fulminant hypertension, low plasma active renin, suppressed kidney renin, high plasma inactive renin, and high extrarenal transgene expression, most prominently in the adrenal cortex. Additionally, they exhibit significantly enhanced excretion of corticosteroids. Here we demonstrate that part of the plasma renin and most of the adrenal renin are transgene determined and that the adrenal renin is strongly activated. TGR(mREN2)27 adrenal cells may serve as a new tool to investigate the regulation and processing of Ren-2d-derived renin and its significance in hypertension and steroid metabolism. Adrenal renin in TGR(mREN2)27 is stimulated by 8-bromo-cAMP (8-Br-cAMP), angiotensin II (ANGII), and calcium. 8-Br-cAMP significantly stimulates active renin and prorenin release, as well as Ren-2d mRNA. Interestingly, within 60 min 8-Br-cAMP, ANGII, and calcimycin stimulate active renin, but not prorenin release. This indicates different intracellular pathways. An activated adrenal renin-angiotensin system in TGR (mREN2)27 as well as the lack of negative feedback on renin secretion by ANGII may be of pathophysiological significance in this hypertensive model.     

Arachidonic acid metabolism and urinary excretion of prostaglandins and thromboxane in rats with experimental diabetes mellitus

Urinary prostaglandins (PGs) and thromboxane excretion, measured by radioimmunoassay, were examined in male Wistar rats made diabetic with streptozotocin, 70 mg/kg. In addition, immunoreactive (i) 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) production by aortic rings was studied as well as conversion of [14C] arachidonic acid (AA) by aortic rings, polymorphonuclear leukocytes, and macrophages isolated from diabetic and age-matched control rats. The profile of urinary eicosanoid excretion changed after induction of diabetes. iPGF2 alpha excretion transiently increased, reaching a peak at 7 days and declining to control values by 48 days. iPGE2 excretion declined with time after induction of diabetes while marked increases in i thromboxane B2 and i6-keto-PGF1 alpha excretion occurred within 48 h and were maintained for the duration of the study, up to 176 days. However, serum i thromboxane B2 levels were similar in control and diabetic rats. Formation of i6-keto-PGF1 alpha by aortic rings obtained from diabetic rats was approximately one-half that of aortic rings from control rats. Similarly, conversion of [14C]AA revealed a diminished capacity of aortic rings from diabetic rats to synthesize prostacyclin (PGI2) measured as 6-keto-PGF1 alpha. Conversion of [14C]AA by polymorphonuclear leukocytes and macrophages obtained from diabetic rats did not differ from those obtained from control rats. In conclusion, experimental diabetes mellitus is accompanied by temporal alterations in AA metabolism, the functional significance of which is unknown at this time.     

Vascular hyporesponsiveness to angiotensin II in rats with CCl4-induced liver cirrhosis

Background Portal hypertension is triggered by vasodilation due to impaired contraction of extrahepatic vessels. Angiotensin II type 1 (AT₁) receptor‐induced vasocontraction is mediated by G proteins and may be desensitized by recruitment of β‐arrestin‐2 to the receptor. In this study, we analysed the interaction of AT₁ receptors with β‐arrestin‐2 in the context of vascular hypocontractility in rats with CCl₄‐induced cirrhosis. Methods Micronodular liver cirrhosis in rats (n = 15) was induced by regular CCl₄ exposure. Age‐matched rats (n = 15) served as controls. Contractility of aortic rings was measured by myography. Protein expressions and phosphorylations were assessed by Western blot analysis, and AT₁ receptor interaction with β‐arrestin‐2 by co‐immunoprecipitation. Results Aortic rings from CCl₄ rats were hypocontractile to angiotensin II independent of nitric oxide synthases (Nω‐nitro‐l ‐arginine methyl ester 200 μM). Expression of the AT₁ receptor, Gα_q/11 and the contraction‐mediating effector Rho kinase was similar in aortas from both groups. Expression and AT₁ receptor binding of β‐arrestin‐2 were up‐regulated in aortas from CCl₄ rats. Stimulation of isolated aortas with exogenous angiotensin II caused recruitment of β‐arrestin‐2 in aortas from noncirrhotic rats, but no further interaction of AT₁ receptors with β‐arrestin‐2 was found in aortas from CCl₄ rats. While angiotensin II stimulation resulted in Rho kinase activation in aortas from noncirrhotic rats but not in aortas from CCl₄ rats, extracellular signal‐regulated kinase activation in response to angiotensin II was observed in aortas from both groups. Conclusions Vascular hyporesponsiveness to angiotensin II in CCl₄ rats is due to enhanced interaction of the AT₁ receptor with β‐arrestin‐2 and consecutively changed receptor function.     

Pharmacological characterization of the vascular muscarinic receptors mediating relaxation and contraction in rabbit aorta.

Studies were performed in the rabbit aortic rings, precontracted with norepinephrine, to determine the subtype(s) of muscarinic receptors involved in endothelium-dependent relaxation and contraction in the absence of endothelium elicited by cholinergic stimuli. Acetylcholine (ACh) and arecaidine propargyl ester (APE), a M2 and M3 agonist, produced a dose-dependent relaxation and contraction in endothelium-intact and endothelium-denuded rabbit aortic rings, respectively. Both of these responses were blocked by the muscarinic receptor antagonist atropine. M1 selective agonist McN-A-343 [4-[N-(3-chlorophenyl)carbamoyloxy]-2-butinyltrimethylammonium+ ++ chloride] did not produce any effect on the tone of precontracted aortic rings. ACh- and APE-induced relaxation in aortic rings with intact endothelium was selectively blocked by M3 receptor antagonists hexahydrosila-difenidol and p-fluoro-hexahydro-sila-difenidol (pA2 of 7.84 and 7.18) but not by M1 antagonist pirenzepine or M2 receptor antagonists AF-DX 116 [11-(2-[(diethylamino)methyl]- 1-piperidinyl]acetyl)-5, 11-dihydro-6H-pyrido-[2,3-b][1,4]-benzo-diazepin-6-one] and methoctramine. ACh- and APE-induced contraction was inhibited by M2 receptor antagonists AF-DX 116 and methoctramine (pA2 of 7.11 and 6.71) but not by pirenzepine, hexahydro-sila-difenidol or p-fluoro-hexahydro-sila-difenidol. ACh- and APE-induced relaxation or contraction were not altered by nicotinic receptor antagonist hexamethonium or cyclooxygenase inhibitor indomethacin. These data suggest that relaxation elicited by cholinergic stimulin in endothelium-intact aortic rings is mediated via release of endothelium-derived relaxing factor consequent to activation of M3 receptors located on endothelial cells, whereas the contraction in aortic rings denuded of their endothelium is mediated via stimulation of M2 receptors located on smooth muscle cells.     

Endogenous aminopeptidase N decreases the potency of peptide agonists and antagonists of the kinin B1 receptors in the rabbit aorta

The B(1) receptor for kinins is selectively stimulated by bradykinin-related fragments lacking the C-terminal arginine, des-arginine(9)-bradykinin (des-Arg(9)-BK), and Lys-des-Arg(9)-BK. The latter peptide is the optimal agonist at the human and rabbit receptor. The B(1) receptor is inducible as a function of inflammatory conditions in the vasculature. We studied the effect of endogenously expressed peptidases on the potency of ligands of this receptor in an established bioassay, the rabbit aorta contractility. The potency measured for agonists (EC(50)) or antagonists (pA(2) scale) in this assay was compared with the affinity of each agent determined using [(3)H]Lys-des-Arg(9)-BK binding competition in cultured aortic smooth muscle cells and with the competition K(i) for the hydrolysis of the aminopeptidase chromogenic substrate L-Ala-p-nitroanilide by smooth muscle cell membranes. The contractile potency of the agonist Lys-des-Arg(9)-BK is decreased by in situ metabolism, and aminopeptidase N mediates most of the distortion (inhibited by amastatin but not efficiently by puromycin). At the other end of the spectrum, the fully protected agonist Sar-[D-Phe(8)]des-Arg(9)-BK is not significantly potentiated by peptidase inhibitors. A similar distortion of apparent potency was observed for some peptide antagonists used in the contractility assay, B-10350 (Lys-Lys-[Hyp(3), Igl(5), d-Tic(7), CpG(8)]des-Arg(9)-BK) and Lys-[Leu(8)]des-Arg(9)-BK being intensely potentiated by amastatin treatment and effective L-Ala-p-nitroanilide competitors. N-Protected peptide antagonists or a nonpeptide antagonist of the B(1) receptor were not potentiated by amastatin. The coexpression of aminopeptidase N and the kinin B(1) receptor in rabbit arterial tissue is of interest for the inactivation of the high-affinity agonist Lys-des-Arg(9)-BK and for the design of hydrosoluble antagonist drugs.     

5-羟色胺2c受体拮抗剂减弱高血压大鼠血压及主动脉的收缩性

目的观察慢性颅室内给予选择性5-HT2C受体拮抗剂RS102221是否能阻止醋酸脱氧肾上腺皮质激素-盐(DOCA)高血压大鼠血压的发展,以证实中枢神经系统5-HT2C受体参与了血压的调节和高血压的病理。方法通过导管,用微型渗透泵以恒定速率脑室输注选择性5-HT2C受体拮抗剂RS102221,观察拮抗中枢神经5-HT2C受体后对醋DOCA高血压大鼠(n=6)血压的影响。此外检测了5-HT2C受体拮抗剂对DOCA大鼠主动脉等张收缩力的影响。结果脑室输注RS102221减低DOCA大鼠血压的发展。5-HT2c受体拮抗剂的治疗消除了DOCA大鼠和对照组大鼠主动脉环对5-HT的反应差异。结论中枢神经系统5-HT2C受体参与了DOCA大鼠血压的调节和高血压的病理,其主动脉5-HT受体亚型的改变可能是高血压病理之一。     

Melatonin counteracts the loss of agonist-evoked contraction of aortic rings induced by incubation

Incubation of aortic rings in a culture medium produces phenomena similar to those observed with aging, i.e. oxidative stress and inflammation leading to increased nitric oxide (NO)-mediated dilation and decreased arterial sensitivity to vasoconstrictor agents. We evaluated whether melatonin protects aortic rings from such a decrease in vasoreactivity. Two concentrations of melatonin were used: 10(-8) M, EC50 for vascular MT1-MT2 receptors, and 10(-5) M, reported as anti-oxidant. Anti-oxidant capacity, inducible nitric oxide synthase (iNOS) expression and isometric contraction of thoracic aorta rings (Wistar rats) evoked by norepinephrine (NE) were assessed. Three days of incubation of aortic rings induced iNOS expression and a fall in NE-evoked contraction. When melatonin was added to the organ bath, it (10(-5) M) increased (+96%, P < 0.05), but did not restore (compared with freshly isolated rings) NE-evoked contraction. Three days of treatment with melatonin increased (10(-8) M, +99%) or restored (10(-5) M, +216%) NE-evoked contraction (compared with freshly isolated rings). The beneficial effects of 10(-8) and 10(-5) M melatonin on NE-evoked contraction were abolished in the presence of luzindole (2 x 10(-6) M, a melatonin receptor antagonist). The incubation-induced increase in iNOS expression was reduced following 3 days of melatonin administration (10(-8) and 10(-5) M). Melatonin (10(-5) M) increased catalase activity (6550 +/- 256, P < 0.05 vs. nontreated fresh aortic rings 5554 +/- 444 nmol min(-1) mg protein(-1)). In conclusion, melatonin counteracts the incubation-induced loss of agonist-evoked contraction of aortic rings by a specific receptor-mediated phenomenon involving iNOS expression; at higher melatonin concentrations, an anti-oxidant effect is probably also involved.     

Endothelial regulation of cyclic GMP and vascular responses in hypertension

The mechanism whereby endothelial modulation of drug-induced vascular responses might change during hypertension was examined. Acetylcholine (ACh) (1 microM) induced maximal relaxation of aortic ring segments with intact endothelium from both Wistar-Kyoto, normotensive rats (WKY) and spontaneously hypertensive rats (SHR) at 5 to 6 weeks of age. At 15 to 18 weeks of age the relaxation response to ACh was reduced in rings from both SHR and WKY (to a greater extent in SHR) and was attenuated even more in the deoxycorticosterone acetate (DOCA)-salt hypertensive rat. The contractile responses of aortic preparations to norepinephrine (NE) (0.1 microM) were similar between 5-6-week-old and 15-18-week-old WKY, but were increased in 15-18-week-old SHR compared to 5-6-week-old SHR. Endothelial cell removal increased contractile responses to NE to a greater extent in WKY than SHR but this did not affect that seen in DOCA-salt hypertensive rats. Methylene blue treatment increased contractions of aortic rings with intact endothelium from 15-18-week-old WKY and SHR to the level detected in rubbed arteries, but it did not affect the NE-induced constriction of intact aortic rings from DOCA-salt hypertensive rats. Basal cyclic GMP concentrations in intact aortic rings were not different between SHR and WKY at 5 to 6 weeks of age. The basal aortic cyclic GMP was unchanged in WKY at 15 to 18 weeks of age, but decreased in SHR and in DOCA-salt hypertensive rats of the same age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood pressure and vascular reactivity to endothelin-1, phenylephrine, serotonin, KCl and acetylcholine following chronic alcohol consumption in vitro

Ethanol has been reported to cause hypertension, the mechanism of which is unknown. Therefore, the effect of chronic ethanol consumption on vascular responsiveness and blood pressure was investigated. Systolic blood pressure was recorded weekly by tail-cuff method. Aortic rings from rats fed chow ad libitum or pair-fed liquid diets containing either ethanol (7.2% v/v) or isocaloric carbohydrate for 4 weeks were placed in organ chambers for isometric tension measurement. There was a mild but significant elevation of the systolic blood pressure in the alcohol-fed rats by week 1 compared to baseline measurements and this remained higher. No significant changes in reactivity of rat isolated aortas to phenylephrine, serotonin, endothelin-1 (ET-1) and KCl were seen in chronic ethanol consumption. In addition, the sensitivity (i.e. pD2) of alcohol-fed aortic rings to the vasoconstrictors was also unchanged compared to controls. Chronic ethanol consumption, however, increased relaxation to acetylcholine with increased pD2 values, but did not alter relaxation to sodium nitroprusside, a cyclic guanosine monophosphate (cGMP)-dependent direct smooth muscle dilator. The results indicate that chronic ethanol consumption significantly potentiates endothelium-dependent relaxations in aortic rings, probably through interference with the production and/or the release of nitric oxide (NO) or adaptive alterations in muscarinic receptors on the endothelial cells, and that increased vascular responsiveness to several vasoconstrictors is not a mechanism responsible for the blood pressure elevation in the chronic alcohol consumption in rats.     

Increased mRNA expression of the B1 and B2 bradykinin receptors and antinociceptive effects of their antagonists in an animal model of neuropathic pain

We examined the role of B1 and B2 bradykinin receptors in promoting neuropathic hypersensitivity following peripheral nerve injury. Forty eight-hours following chronic constriction injury to a rat sciatic nerve there was an increased expression of B2 receptor mRNA in the lumbar dorsal root ganglia ipsilateral to the site of nerve injury. At 14 days following surgery there was also an ipsilateral increase of B1 receptor mRNA as well as a contralateral increased expression of B2 receptor mRNA. Increased expression of both receptors also coincided with analgesic effects of their antagonists. While HOE-140, a potent B2 receptor antagonist was analgesic at both time points tested, the B1 receptor antagonist des-Arg(9), [Leu(8)]-BK had an analgesic effect only at 14 days. The results support the concept that peripheral nerve injury is associated with local inflammation and that bradykinin, acting on both of its receptors promotes pain hypersensitivity.     

Stress-induced alterations of somatodendritic 5-HT1A autoreceptor sensitivity in the rat dorsal raphe nucleus — in vitro electrophysiological evidence

Summary— Somatodendritic 5-HT_1A autoreceptors play a key role in the control of the electrical and metabolic activity of serotoninergic neurons in the dorsal raphe nucleus. These neurons also possess intracellular glucocorticoid receptors which may be involved in the well established modulation of serotonin (5-hydroxytryptamine, 5-HT) metabolism by corticosterone in stressed animals. The possible mediation by somatodendritic 5-HT_1A autoreceptors of such corticosterone-dependent changes in serotoninergic neuron activity was investigated using an in vitro electrophysiological approach. 5-HT_1A autoreceptor-mediated inhibition of the firing of serotoninergic neurons was examined in brain stem slices from rats whose serum corticosterone concentrations had been markedly increased (+ 100–200%) by two different stressful conditions. Immobilization for 30 or 90 min (restraint stress) did not modify the concentration-dependent inhibition of the firing of serotoninergic neurons by the 5-HT_1A receptor agonist ipsapirone. In contrast, placing the rats in novel uncontrolled environmental conditions for 16 h significantly reduced the cell response to ipsapirone, indicating a decreased sensitivity of somatodendritic 5-HT_1A autoreceptors. Such a change was not observed in adrenalectomized rats subjected to the same stressful conditions. These data show that some forms of stress can reduce the 5-HT_1A autoreceptor-dependent inhibitory control of the electrophysiological activity of serotoninergic neurons in the dorsal raphe nucleus. Both the nature and duration of stress seem to be critical factors for triggering the (corticosterone-dependent) mechanism(s) responsible for the functional desensitization of 5-HT_1A autoreceptors in stressed rats.     

Circadian systemic haemodynamics in borderline and mild hypertension

Circadian variations in blood pressure (BP), stroke volume (SV), heart rate (HR), cardiac output (CO) and total peripheral resistance (TPR) were determined by a pulse contour method from the intra‐arterial pulse wave in 32 normotensive (NT), 32 borderline hypertensive (BHT) and 31 hypertensive (HT) middle‐aged men. Daytime averages were used as the reference levels. The nocturnal decrease in BP and HR were similar in the three groups. In the night, SV did not change in the NT group, but was increased in the BHT and HT groups. The nocturnal increase in SV may reflect reduced venous capacity causing increased cardiac filling. As a consequence of the difference in SV, the nocturnal CO fall was diminished in the HT group as compared with the NT group. Moreover, TPR had a tendency to decrease in the HT group, which may be considered as a baroreflex response to buffer the expected rise in BP. Five years later, 25 NT, 24 BHT and 19 HT subjects were reassessed using casual BP measurements. In the NT and BHT groups, six and 17 subjects, respectively, had progressed to hypertension. In a logistic regression model for those who became HT, the nocturnal increase in SV was a significant predictor for future hypertension. In conclusion, the results suggest that circadian systemic haemodynamics may be altered before BP is markedly elevated, and that haemodynamic studies might be useful in predicting the development of sustained hypertension.     

Further pharmacological evidence of nuclear factor-kappa B pathway involvement in bradykinin B1 receptor-sensitized responses in human umbilical vein

Bradykinin (BK) B(1) receptors are thought to exert a pivotal role in maintaining and modulating inflammatory processes. They are not normally present under physiological situations but are induced under physiopathological conditions. In isolated human umbilical vein (HUV), a spontaneous BK B(1) receptor up-regulation and sensitization process has been demonstrated. Based on pyrrolidine-dithiocarbamate inhibition, it has been proposed that this phenomenon is dependent on nuclear factor-kappaB (NF-kappaB) activation. The aim of this study was to further evaluate the NF-kappaB pathway involvement on BK B(1) receptor sensitization in isolated HUV, using several pharmacological tools. In 5-h incubated rings, either the I-kappaB kinase inhibitor 3-(4-methylphenylsulfonyl)-2-propenenitrile (Bay 11-7082) or the proteasome activity inhibitor Z-Leu-Leu-Leu-CHO (MG-132) inhibited the development of the BK B(1) receptor-sensitized contractile responses. Furthermore, pro-inflammatory cytokine interleukin-6 (IL-6) produced a leftward shift of the concentration-response curve to the BK B(1) receptor agonist, whereas anti-inflammatory cytokines interleukin-4 (IL-4) and tumor growth factor-beta1 (TGF-beta1) produced a rightward shift of the responses to des-Arg(9)-BK in our preparations. Taken together, these results point to NF-kappaB as a key intermediary in the activation of the expression of BK B(1) receptor-sensitized responses in HUV and support the role of inflammatory mediators in the modulation of this process.     

Intrathecal administration of NTS1 agonists reverses nociceptive behaviors in a rat model of neuropathic pain

Chronic neuropathic pain arising from peripheral nerve damage is a severe clinical issue where there is a major unmet medical need. We previously demonstrated that both neurotensin (NT) receptor subtypes 1 (NTS1) and 2 (NTS2) are involved in mediating the naloxone‐insensitive antinociceptive effects of neurotensin in different analgesic tests including hotplate, tail‐flick, and tonic pain. However, the role of these receptors in neuropathic pain management has been poorly investigated. In the present study, we therefore examined whether intrathecal delivery of NTS1 agonists was effective in reducing neuropathic pain symptoms in rats. Neuropathy was induced by sciatic nerve constriction (CCI model), and the development of mechanical allodynia and thermal hyperalgesia on the ipsi‐ and contralateral hind paws was examined 3, 7, 14, 21, and 28 days post‐surgery. CCI‐operated rats exhibited significant increases in thermal and mechanical hypersensitivities over a 28‐day testing period. Spinal injection of NT to CCI rats alleviated the behavioral responses to radiant heat and mechanical stimuli, with a maximal reversal of 91% of allodynia at 6 μg/kg. Intrathecal administration of the NTS1‐selective agonist, PD149163 (30–90 μg/kg) also produced potent anti‐allodynic and anti‐hyperalgesic effects in nerve‐injured rats. Likewise, heat hyperalgesia and tactile allodynia produced by CCI of the sciatic nerve were fully reversed by the NTS1 agonist, NT69L (5–25 μg/kg). Altogether, these results support the idea that the NTS1 receptor subtype is involved in pain modulation, and the potential use of NTS1 agonists for the treatment of painful neuropathies.     

N‐methyl‐d‐aspartate receptor‐mediated modulations of the anti‐allodynic effects of 5‐HT1B/1D receptor stimulation in a rat model of trigeminal neuropathic pain

Previous studies showed that triptans and other 5‐HT_1B/1D‐receptor agonists attenuate hyper‐responsiveness to mechanical stimulation of the face in a rat model of trigeminal neuropathic pain, probably by activating 5‐HT_1B/1D‐receptors on primary afferent nociceptive fibers. We now tested whether blockade of post‐synaptic receptors for the excitatory amino acid glutamate released by these fibers would increase this action. We thus evaluated whether (±)1‐hydroxy‐3‐aminopyrrolidine‐2‐one (HA‐966), an antagonist at the glycine/d ‐serine site of N‐methyl‐d ‐aspartate (NMDA)‐receptors, would potentiate the anti‐allodynic action of dihydroergotamine and zolmitriptan in rats with chronic constriction injury to the infraorbital nerve (CCI‐ION). Complementary studies were performed with other NMDA‐receptor ligands and in rats with chronic constriction injury to the sciatic nerve (CCI‐SN) for comparison. Injury was produced by loose ligatures of the nerves. Responsiveness to mechanical stimulation (vibrissae or hindpaw territories) with von Frey filaments was used to evaluate allodynia 2 weeks after nerve ligature. Rats received NMDA‐receptor ligands or saline 20 min before dihydroergotamine (25–100μg/kg, i.v.) or zolmitriptan (25–100μg/kg, s.c.). HA‐966 (2.5 mg/kg, s.c.), inactive on its own, enhanced the anti‐allodynic effects of dihydroergotamine (eightfold increase) and zolmitriptan (threefold increase) in CCI‐ION rats, but these drugs exerted no effects in allodynic CCI‐SN rats. NMDA‐receptor blockade by memantine (5 mg/kg, i.p.) also enhanced, whereas activation at glycine/NMDA site by d ‐cycloserine (3 mg/kg, i.p.) reduced the anti‐allodynic properties of zolmitriptan in CCI‐ION rats. Combined administration of NMDA‐receptor antagonist and 5‐HT_1B/1D‐receptor agonist may be a promising approach for alleviating trigeminal neuropathic pain.     

Activation of G proteins by neuropeptide Y and gamma-aminobutyric acid(B) receptor agonists in rat cerebral cortical membranes through distinct modes of action

We investigated the pharmacological profile of MEN 11270, or H-D-Arg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7gamma-10 alpha), a conformationally constrained derivative of the B2 kinin receptor antagonist Icatibant. MEN 11270 bound with high-affinity to the B2 kinin receptor constitutively expressed by WI38 human fibroblasts, inhibiting 3H-bradykinin (BK) with a pKi value of 10.3 +/- 0.08 (n = 5). The rank order of affinity of several peptide and nonpeptide antagonists was also assessed: Icatibant (pKi = 10.6) approximately MEN 11270 (pKi = 10.3) approximately B9430 (pKi = 10.0) > B9858 (pKi = 8.0) > FR173657 (pKi = 7.6) > WIN64338 (pKi = 7.2) > Lys-[des-Arg9, Leu8]-BK (pKi < 6) > [des-Arg9,Leu8]-BK (pKi < 5). MEN 11270 showed a low affinity in inhibiting 3H-Lys-[des-Arg9]-BK binding at the human B1 kinin receptor constitutively expressed by the same cells (pKi 6.0 +/- 0.33; n = 3). MEN 11270 showed no binding affinity (pIC50 < 5.5) at 29 different receptors and ion channels. In the human umbilical vein contraction assay, MEN 11270, shifted the concentration-response curve to BK to the right in a concentration-dependent manner (pA2 8.14 +/- 0.22, n = 7). The Schild plot was linear (slope 0.95 +/- 0.11), consistent with a competitive antagonism. In the same bioassay, MEN 11270 (10 microM) did not affect the concentration-response curve to the B1 agonist Lys-[des-Arg9]-BK nor the contractile responses elicited by noradrenaline or serotonin. These findings indicate MEN 11270 as an antagonist at the human B2 kinin receptor, with potency and selectivity comparable to those of the linear peptide antagonist, supporting the hypothesis that a constrained C-terminal beta-turn conformation preserves a high affinity for the interaction of Icatibant with the B2 kinin receptor.