Elastase-2 Knockout Mice Display Anxiogenic- and Antidepressant-Like Phenotype: Putative Role for BDNF Metabolism in Prefrontal Cortex.

Several pieces of evidence indicate that elastase-2 (ELA2; chymotrypsin-like ELA2) is an alternative pathway to the generation of angiotensin II (ANGII). Elastase-2 knockout mice (ELA2KO) exhibit alterations in the arterial blood pressure and heart rate. However, there is no data on the behavioral consequences of ELA2 deletion. In this study, we addressed this question, submitting ELA2KO and wild-type (WT) mice to several models sensitive to anxiety- and depression-like, memory, and repetitive behaviors. Our data indicates a higher incidence of barbering behavior in ELA2KO compared to WT, as well as an anxiogenic phenotype, evaluated in the elevated plus maze (EPM). While a decrease in locomotor activity was observed in ELA2KO in EPM, this feature was not the main source of variation in the other parameters analyzed. The marble-burying test (MBT) indicated increase in repetitive behavior, observed by a higher number of buried marbles. The actimeter test indicated a decrease in total activity and confirmed the increase in repetitive behavior. The spatial memory was tested by repeated exposure to the actimeter in a 24-h interval. Both ELA2KO and WT exhibited decreased activity compared to the first exposure, without any distinction between the genotypes. However, when submitted to the cued fear conditioning, ELA2KO displayed lower levels of freezing behavior in the extinction session when compared to WT, but no difference was observed during the conditioning phase. Increased levels of BDNF were found in the prefrontal cortex but not in the hippocampus of ELA2KO mice compared to WT. Finally, in silico analysis indicates that ELA2 is putatively able to cleave BDNF, and incubation of the purified enzyme with BDNF led to the degradation of the latter. Our data suggested an anxiogenic- and antidepressant-like phenotype of ELA2KO, possibly associated with increased levels of BDNF in the prefrontal cortex.

Alternative pathways for angiotensin II generation in the cardiovascular system.

The classical renin-angiotensin system (RAS) consists of enzymes and peptides that regulate blood pressure and electrolyte and fluid homeostasis. Angiotensin II (Ang II) is one of the most important and extensively studied components of the RAS. The beneficial effects of angiotensin converting enzyme (ACE) inhibitors in the treatment of hypertension and heart failure, among other diseases, are well known. However, it has been reported that patients chronically treated with effective doses of these inhibitors do not show suppression of Ang II formation, suggesting the involvement of pathways alternative to ACE in the generation of Ang II. Moreover, the finding that the concentration of Ang II is preserved in the kidney, heart and lungs of mice with an ACE deletion indicates the important role of alternative pathways under basal conditions to maintain the levels of Ang II. Our group has characterized the serine protease elastase-2 as an alternative pathway for Ang II generation from Ang I in rats. A role for elastase-2 in the cardiovascular system was suggested by studies performed in heart and conductance and resistance vessels of normotensive and spontaneously hypertensive rats. This mini-review will highlight the pharmacological aspects of the RAS, emphasizing the role of elastase-2, an alternative pathway for Ang II generation.

Alternative pathways for angiotensin II generation in the cardiovascular system

The classical renin-angiotensin system (RAS) consists of enzymes and peptides that regulate blood pressure and electrolyte and fluid homeostasis. Angiotensin II (Ang II) is one of the most important and extensively studied components of the RAS. The beneficial effects of angiotensin converting enzyme (ACE) inhibitors in the treatment of hypertension and heart failure, among other diseases, are well known. However, it has been reported that patients chronically treated with effective doses of these inhibitors do not show suppression of Ang II formation, suggesting the involvement of pathways alternative to ACE in the generation of Ang II. Moreover, the finding that the concentration of Ang II is preserved in the kidney, heart and lungs of mice with an ACE deletion indicates the important role of alternative pathways under basal conditions to maintain the levels of Ang II. Our group has characterized the serine protease elastase-2 as an alternative pathway for Ang II generation from Ang I in rats. A role for elastase-2 in the cardiovascular system was suggested by studies performed in heart and conductance and resistance vessels of normotensive and spontaneously hypertensive rats. This mini-review will highlight the pharmacological aspects of the RAS, emphasizing the role of elastase-2, an alternative pathway for Ang II generation.

Spironolactone and hydrochlorothiazide exert antioxidant effects and reduce vascular matrix metalloproteinase-2 activity and expression in a model of renovascular hypertension.

BACKGROUND AND PURPOSE: Increased oxidative stress and up-regulation of matrix metalloproteinases (MMPs) may cause structural and functional vascular changes in renovascular hypertension. We examined whether treatment with spironolactone (SPRL), hydrochlorothiazide (HCTZ) or both drugs together modified hypertension-induced changes in arterial blood pressure, aortic remodelling, vascular reactivity, oxidative stress and MMP levels and activity, in a model of renovascular hypertension. EXPERIMENTAL APPROACH: We used the two-kidney,one-clip (2K1C) model of hypertension in Wistar rats. Sham-operated or hypertensive rats were treated with vehicle, SPRL (25 mg.kg(-1).day(-1)), HCTZ (20 mg.kg(-1).day(-1)) or a combination for 8 weeks. Systolic blood pressure was monitored weekly. Aortic rings were isolated to assess endothelium-dependent and -independent relaxations. Morphometry of the vascular wall was carried out in sections of aorta. Aortic NADPH oxidase activity and superoxide production were evaluated. Formation of reactive oxygen species was measured in plasma as thiobarbituric acid-reactive substances. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry and immunohistochemistry. KEY RESULTS: Treatment with SPRL, HCTZ or the combination attenuated 2K1C-induced hypertension, and reversed the endothelial dysfunction in 2K1C rats. Both drugs or the combination reversed vascular aortic remodelling induced by hypertension, attenuated hypertension-induced increases in oxidative stress and reduced MMP-2 levels and activity. CONCLUSIONS AND IMPLICATIONS: SPRL or HCTZ, alone or combined, exerted antioxidant effects, and decreased renovascular hypertension-induced MMP-2 up-regulation, thus improving the vascular dysfunction and remodelling found in this model of hypertension.

Characterization of a local renin-angiotensin system in rat gingival tissue.

BACKGROUND: The systemic renin-angiotensin system (RAS) promotes the plasmatic production of angiotensin (Ang) II, which acts through interaction with specific receptors. There is growing evidence that local systems in various tissues and organs are capable of generating angiotensins independently of circulating RAS. The aims of this study were to investigate the expression and localization of RAS components in rat gingival tissue and evaluate the in vitro production of Ang II and other peptides catalyzed by rat gingival tissue homogenates incubated with different Ang II precursors. METHODS: Reverse transcription-polymerase chain reaction assessed mRNA expression. Immunohistochemical analysis aimed to detect and localize renin. A standardized fluorimetric method with tripeptide hippuryl-histidyl-leucine was used to measure tissue angiotensin-converting enzyme (ACE) activity, whereas high performance liquid chromatography showed products formed after the incubation of tissue homogenates with Ang I or tetradecapeptide renin substrate (TDP). RESULTS: mRNA for renin, angiotensinogen, ACE, and Ang II receptors (AT(1a), AT(1b), and AT(2)) was detected in gingival tissue; cultured gingival fibroblasts expressed renin, angiotensinogen, and AT(1a) receptor. Renin was present in the vascular endothelium and was intensely expressed in the epithelial basal layer of periodontally affected gingival tissue. ACE activity was detected (4.95 +/- 0.89 nmol histidyl-leucine/g/minute). When Ang I was used as substrate, Ang 1-9 (0.576 +/- 0.128 nmol/mg/minute), Ang II (0.066 +/- 0.008 nmol/mg/minute), and Ang 1-7 (0.111 +/- 0.017 nmol/mg/minute) were formed, whereas these same peptides (0.139 +/- 0.031, 0.206 +/- 0.046, and 0.039 +/- 0.007 nmol/mg/minute, respectively) and Ang I (0.973 +/- 0.139 nmol/mg/minute) were formed when TDP was the substrate. CONCLUSION: Local RAS exists in rat gingival tissue and is capable of generating Ang II and other vasoactive peptides in vitro.

Acute and chronic electrical activation of baroreceptor afferents in awake and anesthetized subjects.

Electrical stimulation of baroreceptor afferents was used in the 1960's in several species, including human beings, for the treatment of refractory hypertension. This approach bypasses the site of baroreceptor mechanosensory transduction. Chronic electrical stimulation of arterial baroreceptors, particularly of the carotid sinus nerve (Hering's nerve), was proposed as an ultimate effort to treat refractory hypertension and angina pectoris due to the limited nature of pharmacological therapy available at that time. Nevertheless, this approach was abandoned in the early 1970's due to technical limitations of implantable devices and to the development of better-tolerated antihypertensive medications. More recently, our laboratory developed the technique of electrical stimulation of the aortic depressor nerve in conscious rats, enabling access to hemodynamic responses without the undesirable effect of anesthesia. In addition, electrical stimulation of the aortic depressor nerve allows assessment of the hemodynamic responses and the sympathovagal balance of the heart in hypertensive rats, which exhibit a well-known decrease in baroreflex sensitivity, usually attributed to baroreceptor ending dysfunction. Recently, there has been renewed interest in using electrical stimulation of the carotid sinus, but not the carotid sinus nerve, to lower blood pressure in conscious hypertensive dogs as well as in hypertensive patients. Notably, previous undesirable technical outcomes associated with electrical stimulation of the carotid sinus nerve observed in the 1960's and 1970's have been overcome. Furthermore, promising data have been recently reported from clinical trials that evaluated the efficacy of carotid sinus stimulation in hypertensive patients with drug resistant hypertension.

Acute and chronic electrical activation of baroreceptor afferents in awake and anesthetized subjects

Electrical stimulation of baroreceptor afferents was used in the 1960's in several species, including human beings, for the treatment of refractory hypertension. This approach bypasses the site of baroreceptor mechanosensory transduction. Chronic electrical stimulation of arterial baroreceptors, particularly of the carotid sinus nerve (Hering's nerve), was proposed as an ultimate effort to treat refractory hypertension and angina pectoris due to the limited nature of pharmacological therapy available at that time. Nevertheless, this approach was abandoned in the early 1970's due to technical limitations of implantable devices and to the development of better-tolerated antihypertensive medications. More recently, our laboratory developed the technique of electrical stimulation of the aortic depressor nerve in conscious rats, enabling access to hemodynamic responses without the undesirable effect of anesthesia. In addition, electrical stimulation of the aortic depressor nerve allows assessment of the hemodynamic responses and the sympathovagal balance of the heart in hypertensive rats, which exhibit a well-known decrease in baroreflex sensitivity, usually attributed to baroreceptor ending dysfunction. Recently, there has been renewed interest in using electrical stimulation of the carotid sinus, but not the carotid sinus nerve, to lower blood pressure in conscious hypertensive dogs as well as in hypertensive patients. Notably, previous undesirable technical outcomes associated with electrical stimulation of the carotid sinus nerve observed in the 1960's and 1970's have been overcome. Furthermore, promising data have been recently reported from clinical trials that evaluated the efficacy of carotid sinus stimulation in hypertensive patients with drug resistant hypertension.

Acute effect of amiodarone on cardiovascular reflexes of normotensive and renal hypertensive rats.

The aim of the present study was to evaluate the effect of amiodarone on mean arterial pressure (MAP), heart rate (HR), baroreflex, Bezold-Jarisch, and peripheral chemoreflex in normotensive and chronic one-kidney, one-clip (1K1C) hypertensive rats (N = 9 to 11 rats in each group). Amiodarone (50 mg/kg, iv) elicited hypotension and bradycardia in normotensive (-10 +/- 1 mmHg, -57 +/- 6 bpm) and hypertensive rats (-37 +/- 7 mmHg, -39 +/- 19 bpm). The baroreflex index (deltaHR/deltaMAP) was significantly attenuated by amiodarone in both normotensive (-0.61 +/- 0.12 vs -1.47 +/- 0.14 bpm/mmHg for reflex bradycardia and -1.15 +/- 0.19 vs -2.63 +/- 0.26 bpm/mmHg for reflex tachycardia) and hypertensive rats (-0.26 +/- 0.05 vs -0.72 +/- 0.16 bpm/mmHg for reflex bradycardia and -0.92 +/- 0.19 vs -1.51 +/- 0.19 bpm/mmHg for reflex tachycardia). The slope of linear regression from delta pulse interval/deltaMAP was attenuated for both reflex bradycardia and tachycardia in normotensive rats (-0.47 +/- 0.13 vs -0.94 +/- 0.19 ms/mmHg and -0.80 +/- 0.13 vs -1.11 +/- 0.13 ms/mmHg), but only for reflex bradycardia in hypertensive rats (-0.15 +/- 0.02 vs -0.23 +/- 0.3 ms/mmHg). In addition, the MAP and HR responses to the Bezold-Jarisch reflex were 20-30% smaller in amiodarone-treated normotensive or hypertensive rats. The bradycardic response to peripheral chemoreflex activation with intravenous potassium cyanide was also attenuated by amiodarone in both normotensive (-30 +/- 6 vs -49 +/- 8 bpm) and hypertensive rats (-34 +/- 13 vs -42 +/- 10 bpm). On the basis of the well-known electrophysiological effects of amiodarone, the sinus node might be the responsible for the attenuation of the cardiovascular reflexes found in the present study.

Acute effect of amiodarone on cardiovascular reflexes of normotensive and renal hypertensive rats

The aim of the present study was to evaluate the effect of amiodarone on mean arterial pressure (MAP), heart rate (HR), baroreflex, Bezold-Jarisch, and peripheral chemoreflex in normotensive and chronic one-kidney, one-clip (1K1C) hypertensive rats (N = 9 to 11 rats in each group). Amiodarone (50 mg/kg, iv) elicited hypotension and bradycardia in normotensive (-10 ± 1 mmHg, -57 ± 6 bpm) and hypertensive rats (-37 ± 7 mmHg, -39 ± 19 bpm). The baroreflex index (deltaHR/deltaMAP) was significantly attenuated by amiodarone in both normotensive (-0.61 ± 0.12 vs -1.47 ± 0.14 bpm/mmHg for reflex bradycardia and -1.15 ± 0.19 vs -2.63 ± 0.26 bpm/mmHg for reflex tachycardia) and hypertensive rats (-0.26 ± 0.05 vs -0.72 ± 0.16 bpm/mmHg for reflex bradycardia and -0.92 ± 0.19 vs -1.51 ± 0.19 bpm/mmHg for reflex tachycardia). The slope of linear regression from deltapulse interval/deltaMAP was attenuated for both reflex bradycardia and tachycardia in normotensive rats (-0.47 ± 0.13 vs -0.94 ± 0.19 ms/mmHg and -0.80 ± 0.13 vs -1.11 ± 0.13 ms/mmHg), but only for reflex bradycardia in hypertensive rats (-0.15 ± 0.02 vs -0.23 ± 0.3 ms/mmHg). In addition, the MAP and HR responses to the Bezold-Jarisch reflex were 20-30 percent smaller in amiodarone-treated normotensive or hypertensive rats. The bradycardic response to peripheral chemoreflex activation with intravenous potassium cyanide was also attenuated by amiodarone in both normotensive (-30 ± 6 vs -49 ± 8 bpm) and hypertensive rats (-34 ± 13 vs -42 ± 10 bpm). On the basis of the well-known electrophysiological effects of amiodarone, the sinus node might be the responsible for the attenuation of the cardiovascular reflexes found in the present study.

Transcriptional regulation of glucose-6-phosphatase catalytic subunit promoter by insulin and glucose in the carnivorous fish, Sparus aurata.

Increase in glucose-6-phosphatase catalytic subunit (G6Pase, G6pc) transcription enhances hepatic glucose production in non-insulin-dependent diabetes mellitus (NIDDM). The fact that carnivorous fish is an alternative model to study NIDDM led us to clone and characterise the first G6pc promoter region reported for fish and non-mammalian animals. The 5'-flanking region of G6pc from gilthead sea bream (Sparus aurata) was isolated by chromosome walking. With SMART RACE-PCR, the transcription start site was located 106 base pairs (bp) upstream of the translational start. Transfection analysis in HepG2 cells located a functional promoter in the 850 bp 5'-flanking isolated fragment (positions -770 to +80 relative to the transcription start). Sequential 5'-deletion analysis of the promoter fragment revealed that a core functional promoter for basal transcription is comprised within the 190 bp upstream of the transcription start site. In vivo, glucose and insulin reduced G6Pase mRNA levels in the fish liver. Transfection experiments in HepG2 cells showed that insulin repressed S. aurata G6pc under high-glucose conditions. Synergistic activation of piscine G6pc promoter was induced by cotransfection with expression plasmids for hepatocyte nuclear factor-4alpha (HNF-4alpha) and peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1alpha). No direct relationship was found between PGC-1alpha coactivation of HNF-4alpha transactivation and the repressive effect of insulin. Interestingly, insulin hardly affected G6pc promoter activity in the absence of glucose, suggesting that a reduced capacity of insulin-dependent repression of piscine G6pc may lead to insulin resistance in carnivorous fish.

Nonendothelial NO blunts sympathetic response of normotensive rats but not of SHR.

The inhibitory role of NO on sympathetic-induced contraction of resistance vessels of spontaneously hypertensive rats (SHR) has not been defined. Accordingly, we investigated the effect of endothelial removal or NO synthase inhibition on vasoconstrictor responses to sympathetic stimulation or phenylephrine in perfused mesenteric beds isolated from normotensive rats (NR) and SHR. Electrical stimulation (10 to 64 Hz) of perivascular nerves elicited a frequency-dependent increase in perfusion pressure that was greater in preparations from SHR (maximal effect: 223.4+/-8.4 versus 117.6+/-10.3 mm Hg in NR, n=6, P<0.001), and endothelium removal did not affect these responses in arteries from NR but caused a significant shift to the left of the frequency-response curve in arteries from SHR. In arteries with endothelium, inhibition of NO synthase with N(G)-nitro-L-arginine (L-NNA, 50 micromol/L) augmented the vasoconstrictor responses to sympathetic stimulation in both NR and SHR preparations. In preparations that had the endothelium removed, however, L-NNA potentiated only the responses to sympathetic stimulation of NR arteries. Vasoconstrictor responses to phenylephrine was potentiated by endothelium removal and in the presence of L-NNA only when the endothelium was intact in both NR and SHR arteries. The number of NADPH-diaphorase-positive cells in the superior mesenteric sympathetic ganglion of SHR was significantly less compared with that of NR. In conclusion, these data suggest a prejunctional inhibitory action of non-endothelial-derived NO, most probably neuronal-derived NO, on sympathetic-mediated vasoconstriction in NR arteries. In contrast, enhancement of the sympathetic-mediated vasoconstriction in SHR arteries elicited by L-NNA can be attributed to inhibition of endothelial-derived NO.

Cardiac sympathetic overactivity and decreased baroreflex sensitivity in L-NAME hypertensive rats.

The present study evaluated the possible changes in the autonomic control of heart rate in the hypertensive model induced by the inhibition of nitric oxide synthase. Rats were treated with N(G)-nitro-L-arginine methyl ester (L-NAME group) in the drinking water during 7 days, whereas control groups were treated with tap water (control group) or with the N(G)-nitro-D-arginine methyl ester (D-NAME group), an inactive isomer of the L-NAME molecule. The L-NAME group developed hypertension and tachycardia. The sequential blockade of the autonomic influences with propranolol and methylatropine indicated that the intrinsic heart rate did not differ among groups and revealed a sympathetic overactivity in the control of heart rate in the L-NAME group. The spectral density power of heart rate, calculated using fast-Fourier transformation, indicated a reduced variability in the low-frequency band (0.20-0.60 Hz) for the L-NAME group. The baroreflex sensitivity was also attenuated in these animals when compared with the normotensive control or D-NAME group. Overall, these data indicate cardiac sympathetic overactivity associated with a decreased baroreflex sensitivity in L-NAME hypertensive rats.

Effect of captopril on neurally induced contraction and relaxation of mesenteric arteries of renal hypertensive rats.

The effect of captopril treatment on neurally induced vasoconstrictor and vasodilator responses was examined in the isolated mesenteric arterial bed from normotensive and one-kidney, one clip hypertensive (1K1C) rats. In isolated mesenteric beds, electrical field stimulation (EFS) of perivascular nerves at basal tone induced a frequency-dependent increase in perfusion pressure that was greater in preparations from hypertensive rats compared with those from normotensive rats. Captopril treatment was associated with a decrease in vasoconstrictor responses in the hypertensive group compared with its non-treated control. Responses to norepinephrine (320 ng) were greater in hypertensive than normotensive groups; captopril reduced this response only in the hypertensive group. In preconstricted mesenteric arteries perfused with solutions containing guanethidine (5 microM) and atropine (1 microM), EFS elicited a frequency-dependent decrease in perfusion pressure that was abolished by tetrodotoxin (1 microM). Vasodilator responses to EFS were not affected by captopril treatment, although they were smaller in the hypertensive group. Acetylcholine (10 ng) induced similar decreases in perfusion pressure of normotensive and 1K1C groups; captopril did not influence these responses. These results indicate that captopril treatment does not affect the reduced neurogenic vasodilation but normalizes the augmented sympathetic-mediated vasoconstrictor responses of mesenteric resistance vessels of chronic 1K1C hypertensive rats.

Enalapril prevents aortic hyperreactivity and remodelling in one-kidney, one-clip hypertensive rats without reducing arterial pressure.

1. The present study was designed to evaluate the blood pressure-independent effects of angiotensin-converting enzyme (ACE) inhibition on cardiovascular structure and function in one-kidney, one-clip (1K1C) hypertensive rats. 2. The study was conducted in four groups of rats: (i) uninephrectomized normotensive rats (1K); (ii) 1K1C hypertensive rats; (iii) 1K rats treated with enalapril; and (iv) 1K1C rats treated with enalapril. Enalapril treatment (20 mg/kg per day, p.o.) was started after surgery to induce hypertension or nephrectomy and continued for 5 weeks. 3. The increase in blood pressure of 1K1C rats was associated with activation of cardiac and aortic, but not plasma, ACE activity and with hypertrophy of both heart and aorta. No difference in cardiac output and in vitro systolic function was observed among the groups. Hypertrophied aorta isolated from 1K1C rats displayed increased sensitivity to phenylephrine (PE) and unaltered responses to both acetylcholine (ACh) and sodium nitroprusside compared with the 1K group. 4. Enalapril treatment effectively inhibited plasma and tissue ACE activity in 1K1C and 1K rats. Enalapril did not prevent the development of hypertension and cardiac hypertrophy nor did it change haemodynamic parameters in 1K1C rats. However, enalapril prevented the increase in aortic media thickness and cross-sectional area and restored the hypersensitivity to PE in aortic rings of 1K1C rats. The endothelium-dependent response to ACh was enhanced by enalapril in the aorta of 1K but not 1K1C rats. 5. These results suggest a role for activated local angiotensin II generation in aortic but not cardiac hypertrophy secondary to 1K1C hypertension.

Tratamento da osteoartrite erosiva pelo fosfato de cloroquina / Treatment of erosive osteoarthritis with phosphate of cloroquine

Os autores apresentam uma avaliação terapeutica em 25 pacientes com diagnóstico clínico e radiológico de osteoartrite erosiva (OEA), nos quais foi utilizado o fosfato de cloroquina, na dose de 150mg/dia, por um período de 12 meses. O tratamento foi considerado eficaz em 21 pacientes (84por cento), com boa tolerabilidade medicamentosa

Cardiovascular effects of clonidine-like drugs in pithed rabbits.

Administration (3 to 100 microg/kg IV) of clonidine, rilmenidine, and an imidazoline derivative, 2-(2-chlorophenylamino)imidazoline, in pithed nonstimulated rabbits caused a dose-dependent increase in mean arterial pressure without affecting heart rate. Prazosin (0.1 mg/kg IV) almost abolished the pressor responses to 2-(2-chlorophenylamino)imidazoline, partially inhibited those induced by clonidine, but failed to affect those elicited by rilmenidine. In contrast, yohimbine (1 mg/kg IV) blunted the pressor responses of the 3 drugs. In sympathetically stimulated pithed rabbits, 2-(2-chlorophenylamino)imidazoline induced only pressor effects, whereas clonidine and rilmenidine caused a transient pressure increase followed by a dose-dependent depressor effect. Yohimbine abolished the depressor effect of both drugs, which may have involved presynaptic alpha(2)-adrenoceptors. In conclusion, peripheral effects of 2-(2-chlorophenylamino)imidazoline and clonidine involved at least alpha(1)- and alpha(2)-adrenoceptor activation, whereas pressor and depressor effects of rilmenidine were mediated by alpha(2)-adrenoceptors.

Purification and substrate specificity of an angiotensin converting elastase-2 from the rat mesenteric arterial bed perfusate.

A soluble angiotensin (Ang) II-generating enzyme has been purified to homogeneity from the rat mesenteric arterial bed (MAB) perfusate by a combination of gel filtration and affinity chromatographies. The enzyme is a glycoprotein of 28.5 kDa (SDS-PAGE), whose N-terminal sequence is identical with that of the rat pancreatic elastase-2; therefore the enzyme will henceforth be referred to as rat MAB elastase-2. When Ang I was used as the substrate, the enzyme specifically released Ang II and the dipeptide His-Leu (Km=36 microM; Kcat=1530 min-1). The catalytic efficiency (Kcat/Km=42.5 min-1 microM-1) of this reaction was comparable to those of other known Ang I-converting enzymes. The proteolytic specificity of the purified enzyme toward mellitin, oxidized insulin B chain, somatostatin-14 and renin substrate tetradecapeptide suggested that the enzyme-substrate interaction was defined by an extended substrate binding site, typical of elastases-2 of pancreatic origin. According to the sensitivity of the rat MAB elastase-2 to various inhibitors this enzyme could be described as a member of the chymostatin-sensitive group of Ang II-forming serine proteases. The localization and biochemical properties of this enzyme suggest that it might play a role in the regional control of vascular tonus.

Role of non-nitric oxide non-prostaglandin endothelium-derived relaxing factor(s) in bradykinin vasodilation

The most conspicuous effect of bradykinin following its administration into the systemic circulation is a transient hypotension due to vasodilation. In the present study most of the available evidence regarding the mechanisms involved in bradykinin-induced arterial vasodilation is reviewed. It has become firmly established that in most species vasodilation in response to bradykinin is mediated by the release of endothelial relaxing factors following the activation of B2-receptors. Although in some cases the action of bradykinin is entirely mediated by the endothelial release of nitric oxide (NO) and/or prostacyclin (PGI2), a large amount of evidence has been accumulated during the last 10 years indicating that a non-NO/PGI2 factor accounts for bradykinin-induced vasodilation in a wide variety of perfused vascular beds and isolated small arteries from several species including humans. Since the effect of the non-NO/PGI2 endothelium-derived relaxing factor is practically abolished by disrupting the K+ electrochemical gradient together with the fact that bradykinin causes endothelium-dependent hyperpolarization of vascular smooth muscle cells, the action of such factor has been attributed to the opening of K+ channels in these cells. The pharmacological characteristics of these channels are not uniform among the different blood vessels in which they have been examined. Although there is some evidence indicating a role for KCa or KV channels, our findings in the mesenteric bed together with other reports indicate that the K+ channels involved do not correspond exactly to any of those already described. In addition, the chemical identity of such hyperpolarizing factor is still a matter of controversy. The postulated main contenders are epoxyeicosatrienoic acids or endocannabinoid agonists for the CB1-receptors. Based on the available reports and on data from our laboratory in the rat mesenteric bed, we conclude that the NO/PGI2-independent endothelium-dependent vasodilation induced by BK is unlikely to involve a cytochrome P450 arachidonic acid metabolite or an endocannabinoid agonist.

Role of non-nitric oxide non-prostaglandin endothelium-derived relaxing factor(s) in bradykinin vasodilation.

The most conspicuous effect of bradykinin following its administration into the systemic circulation is a transient hypotension due to vasodilation. In the present study most of the available evidence regarding the mechanisms involved in bradykinin-induced arterial vasodilation is reviewed. It has become firmly established that in most species vasodilation in response to bradykinin is mediated by the release of endothelial relaxing factors following the activation of B2-receptors. Although in some cases the action of bradykinin is entirely mediated by the endothelial release of nitric oxide (NO) and/or prostacyclin (PGI2), a large amount of evidence has been accumulated during the last 10 years indicating that a non-NO/PGI2 factor accounts for bradykinin-induced vasodilation in a wide variety of perfused vascular beds and isolated small arteries from several species including humans. Since the effect of the non-NO/PGI2 endothelium-derived relaxing factor is practically abolished by disrupting the K+ electrochemical gradient together with the fact that bradykinin causes endothelium-dependent hyperpolarization of vascular smooth muscle cells, the action of such factor has been attributed to the opening of K+ channels in these cells. The pharmacological characteristics of these channels are not uniform among the different blood vessels in which they have been examined. Although there is some evidence indicating a role for KCa or KV channels, our findings in the mesenteric bed together with other reports indicate that the K+ channels involved do not correspond exactly to any of those already described. In addition, the chemical identity of such hyperpolarizing factor is still a matter of controversy. The postulated main contenders are epoxyeicosatrienoic acids or endocannabinoid agonists for the CB1-receptors. Based on the available reports and on data from our laboratory in the rat mesenteric bed, we conclude that the NO/PGI2-independent endothelium-dependent vasodilation induced by BK is unlikely to involve a cytochrome P450 arachidonic acid metabolite or an endocannabinoid agonist.