Mercia de Paula Lima

Tonin expression in the rat brain and tonin-mediated central production of angiotensin II.

Tonins are serine proteinases mainly found in the rat submandibular gland, which are capable of generating the pressor octapeptide angiotensin II (Ang II) not only from the classical substrate angiotensin I but also from the synthetic tetradecapeptide (AG(1-14)) and from angiotensinogen. In this work, tonin expression levels were evaluated in astrocytes and brain areas of the rat. By two different techniques (ribonuclease protection assay and reverse transcription-polymerase chain reaction), we could verify the presence of tonin mRNA in astrocytes and in the thalamus of the rat brain. Sequencing of the amplified brain cDNA determined it to be identical to that found in the submandibular gland. Central microinjection of tonin produced a transient (10-20 min) elevation of blood pressure and heart rate and induced water and saline intake within the first 10 min after injection. Urinary volume and salt excretion increased within 7 h after tonin injection. These effects were partially blocked by previously administered losartan, indicating that tonin effectively induced a central Ang II formation. Our data suggest that tonin may be an alternative pathway to Ang II generation in the brain and could participate in the physiological effects exerted by Ang II such as water and saline intake and blood pressure elevation.

Increased activity of the renin-angiotensin and sympathetic nervous systems is required for regulation of the blood pressure in rats fed a low-protein diet.

•  What is the central question of this study? Is the increase in blood pressure observed in rats fed a low protein diet due to overactivation of sympathetic and renin angiontensin systems? •  What is the main finding and its importance? The data show an increase in the expression of angiotensin II type 1 receptors and an incrased sympathetic activity in this experimental model, suggesting that both systems are contributing to the high blood pressure observed in these animals.

Increased blood pressure and water intake in transgenic mice expressing rat tonin in the brain

Abstract Tonin is a serine proteinase of the kallikrein family that can produce angiotensin II directly from angiotensinogen. To clarify the importance of this enzyme for central nervous control of the cardiovascular system, we generated transgenic mice, TGM(rTon), that express rat tonin in astrocytes. These mice present high levels of tonin mRNA and activity specifically in the brain. As a consequence, TGM(rTon) develop increased blood pressure and water intake. Lisinopril, an ACE inhibitor, is less hypotensive for transgenic mice than for control animals. The AT1 receptor antagonist candesartan equally lowers blood pressure in transgenic and in control mice. Plasma angiotensin II, but not angiotensin I, is increased in TGM(rTon) compared to the wild type, suggesting release of the peptide from the brain into the circulation. However, AT1 receptors are desensitized in this transgenic model, as demonstrated by a blunted pressor response to intravenous application of angiotensin II. In conclusion, tonin in the brain may represent an alternative pathway for angiotensin II generation with effects on the cardiovascular system.

Angiostatic activity of human plasminogen fragments is highly dependent on glycosylation

To assess the importance of carbohydrate moieties to the anti‐angiogenic activity of plasminogen fragments, we cloned the fragment corresponding to amino acids Val79 to Thr346 (Kint3–4) that presents the three glycosylation sites. The activity of glycosylated and unglycosylated Kint3–4 was tested in murine sponge implant model. We observed a significant decrease in the neovascularization on the sponge after treatment with Kint3–4 by histological examination and determination of the hemoglobin levels. The effects were more intense with the glycosylated than the unglycosylated protein. 99mTechnecium‐labeled red blood cells confirmed the inhibition of cell infiltration in the implanted sponge. Studies using melanoma B16F1 implanted in a mouse demonstrated that treatment with glycosylated Kint3–4 (0.15 nmol/48 h) during 14 days suppresses tumor growth by 80%. The vascular endothelial growth factor mRNA levels on the tumor were reduced after treatment. Kint3–4 is a potent plasminogen fragment that has been found to inhibit tumor growth. (Cancer Sci 2009)

Characterization of the renal renin-angiotensin system in transgenic mice that express rat tonin

Introduction: Tonin is an enzyme that is able to generate angiotensin II (Ang II) from angiotensin I (Ang I) or directly from angiotensinogen. Our goal was to characterize the renal renin-angiotensin system in transgenic mice that express rat tonin (TGM`(rTon)). Materials and methods: Mice were euthanized and the kidneys removed for analysis. Tonin activity was evaluated by radioimmunoassay and angiotensin I-converting enzyme (ACE) activity by HPLC. Tonin, ACE and angiotensin II-converting enzyme (ACE2) expression was analyzed by Western blotting. Results: Tonin activity was significantly increased in TGM`(rTon) compared to their respective wild-type (WT) littermates (1.7 ± 0.21 vs 0.11 ± 0.02 nmol of Ang II/min/mg of protein). Tonin activity had a strong positive correlation with tonin expression in both TGM`(rTon) and their respective wild-type littermates. The ACE activity and expression levels of 65-kDa N-domain angiotensin I-converting enzyme isoform were significantly increased in the TGM`(rTon) when compared with WT. ACE2 expression levels were statistically significantly higher in the TGM`(rTon) when compared with WT. Angiotensin 1–7 (Ang(1–7)) and Ang I levels were significantly lower in the TGM`(rTon). Conclusions: We suggest that the environment of tonin abundance may increase N-domain ACE activity liberated by a secretase able to cleave somatic ACE.

Antinociceptive response in transgenic mice expressing rat tonin

Angiotensin II (Ang II) may be produced directly from angiotensinogen by tonin. Studies have demonstrated that Ang II and its metabolite Ang-(1-7) produce antinociception in pain animal models. The aim of the present study was to determine whether the transgenic mice that express rat tonin (TGM(rTon)) show altered nociceptive behavior and investigate the possible involvement of angiotensin metabolites. Nociception was evaluated using the thermal tail-flick and chemical acetic acid writhing tests, and the drugs were administered by intracerebroventricular and subcutaneous pathways, respectively. Probabilities less than 5% (P<0.05) were considered to be statistically significant (t test; ANOVA/Bonferronis test). The results demonstrate that the transgenic mice showed an antinociceptive effect in the tail-flick and acetic acid writhing tests. In addition, it was observed that losartan, an AT₁ receptor antagonist and A-779 (D-Ala7-Ang-(1-7)), a Mas receptor antagonist attenuated the antinociceptive behavior. Our data suggest that the Ang II produced in TGM(rTon) induces antinociception via the AT₁ receptor, while the Ang-(1-7) produced from Ang II induced antinociception via the Mas receptor.

Antiserum for insulin radioimmunoassay generated by an insulin derivative devoid of hypoglycemic activity

A tetraiodinated derivative of bovine insulin, prepared at pH 1 with stable iodine, was unable to cause signs of hypoglycemia in doses up to 2.4 micrograms/g in fasting mice, when native insulin caused 100% mortality. In neutral and acidic solutions, in absence of chaotropic agents, it behaved as the monomer, and could be separated from less iodinated, active species, that appeared as dimers, by conventional gel filtration. To generate antibodies in guinea-pigs, the tetraiodinated insulin was injected in doses three times higher than native insulin, without any harm to recipient animals. The induced antiserum was compared with antiserum generated by conventional methods in radioimmunoassay (RIA) of native insulin, and parallel curves were obtained.