Amelioration of cardiac function and activation of anti-inflammatory vasoactive peptides expression in the rat myocardium by low level laser therapy.

Low-level laser therapy (LLLT) has been used as an anti-inflammatory treatment in several disease conditions, even when inflammation is a secondary consequence, such as in myocardial infarction (MI). However, the mechanism by which LLLT is able to protect the remaining myocardium remains unclear. The present study tested the hypothesis that LLLT reduces inflammation after acute MI in female rats and ameliorates cardiac function. The potential participation of the Renin-Angiotensin System (RAS) and Kallikrein-Kinin System (KKS) vasoactive peptides was also evaluated. LLLT treatment effectively reduced MI size, attenuated the systolic dysfunction after MI, and decreased the myocardial mRNA expression of interleukin-1 beta and interleukin-6 in comparison to the non-irradiated rat tissue. In addition, LLLT treatment increased protein and mRNA levels of the Mas receptor, the mRNA expression of kinin B2 receptors and the circulating levels of plasma kallikrein compared to non-treated post-MI rats. On the other hand, the kinin B1 receptor mRNA expression decreased after LLLT. No significant changes were found in the expression of vascular endothelial growth factor (VEGF) in the myocardial remote area between laser-irradiated and non-irradiated post-MI rats. Capillaries density also remained similar between these two experimental groups. The mRNA expression of the inducible nitric oxide synthase (iNOS) was increased three days after MI, however, this effect was blunted by LLLT. Moreover, endothelial NOS mRNA content increased after LLLT. Plasma nitric oxide metabolites (NOx) concentration was increased three days after MI in non-treated rats and increased even further by LLLT treatment. Our data suggest that LLLT diminishes the acute inflammation in the myocardium, reduces infarct size and attenuates left ventricle dysfunction post-MI and increases vasoactive peptides expression and nitric oxide (NO) generation.

Functional and molecular characterization of kinin B1 and B 2 receptors in human bladder cancer: implication of the PI3Kγ pathway.

Kinins and their receptors have been recently implicated in cancer. Using functional and molecular approaches, we investigated the relevance of kinin B1 and B2 receptors in bladder cancer. Functional studies were conducted using bladder cancer cell lines, and human biopsies were employed for molecular studies. Both B1 des-Arg(9)-BK and B2 BK receptor agonists stimulated the proliferation of grade 3-derived T24 bladder cancer cells. Furthermore, treatment with B1 and B2 receptor antagonists (SSR240612 and HOE140) markedly inhibited the proliferation of T24 cells. Only higher concentrations of BK increased the proliferation of the grade 1 bladder cancer cell line RT4, while des-Arg(9)-BK completely failed to induce its proliferation. Real-time PCR revealed that the mRNA expression of kinin receptors, particularly B1 receptors, was increased in T24 cells relative to RT4 cells. Data from bladder cancer human biopsies revealed that B1 receptor expression was increased in all tumor samples and under conditions of chronic inflammation. We also show novel evidence demonstrating that the pharmacological inhibition of PI3Kγ (phosphatidylinositol 3-kinase) with AS252424, concentration-dependently reduced T24 cell proliferation induced by BK or des-Arg(9)-BK. Finally, the incubation of T24 cells with kinin agonists led to a marked activation of the PI3K/AKT and ERK 1/2 signaling pathways, whereas p38 MAP kinase remained unaffected. Kinin receptors, especially B1 receptors, appear to be implicated in bladder cancer progression. It is tempting to suggest that selective kinin antagonists might represent potential alternative therapies for bladder cancer.

The non-peptide kinin receptor antagonists FR 173657 and SSR 240612: preclinical evidence for the treatment of skin inflammation.

Peptide and non-peptide kinin receptor antagonists were evaluated in cutaneous inflammation models in mice. Topical and i.p. application of kinin B(1) and B(2) receptor antagonists caused a significant inhibition of the capsaicin-induced cutaneous neurogenic inflammatory response. The calculated mean ID(50) for Hoe140 and SSR240612 were 23.83 (9.14-62.14) nmol/kg and 0.23 (0.15-0.36) mg/ear, respectively. The I(max) observed for Hoe140, SSR240612, R-715, FR173657, and FR plus SSR were 61+/-5%, 56+/-3%, 65+/-10%, 48+/-8%, and 52+/-4%, respectively. Supporting these results, double B(1) and B(2) kinin receptors knockout mice showed a significant inhibition of capsaicin-induced ear oedema (42+/-7%). However, mice with a single deletion of either B(1) or B(2) receptors exhibited no change in their capsaicin responses. In contrast, all of the examined kinin receptor antagonists were unable to inhibit the oedema induced by TPA and the results from knockout mice confirmed the lack of kinin receptor signaling in this model. These findings show that kinin receptors are present in the skin and that both kinin receptors seem to be important in the neurogenic inflammatory response. Moreover, non-peptide antagonists were very effective in reducing skin inflammation when topically applied, thereby suggesting that they could be useful tools in the treatment of some skin inflammatory diseases.

Low level laser therapy modulates kinin receptors mRNA expression in the subplantar muscle of rat paw subjected to carrageenan-induced inflammation.

Low level laser therapy (LLLT) has been used clinically in order to treat inflammatory processes. In this work, we evaluated if LLLT alters kinin receptors mRNA expression in the carrageenan-induced rat paw edema. Experimental groups were designed as followed: A1 (Control-saline), A2 (Carrageenan-only), A3 (Carrageenan+laser 660 nm) and A4 (Carrageenan+laser 684 nm). Edema was measured by a plethysmometer. Subplantar tissue was collected for kinin receptors mRNA quantification by Real time-PCR. LLLT of both 660 and 684 nm wavelengths administrated 1 h after carrageenan injection was able to promote the reduction of edema produced by carrageenan. In the A2 group, B1 receptor expression presented a significantly increase when compared to control group. Kinin B1 receptor mRNA expression significantly decreased after LLLT's 660 or 684 nm wavelength. Kinin B2 receptor mRNA expression also diminished after both laser irradiations. Our results suggest that expression of both kinin receptors is modulated by LLLT, possibly contributing to its anti-inflammatory effect.

Genetic determinants of efficiency of magnetic laser therapy of essential hypertension.

The efficiency of magnetic laser therapy was evaluated in patients with essential hypertension (stages I and II). The role of angiotensinogen, angiotensin-converting enzyme, type II bradykinin receptor, and endothelial nitrogen oxide synthetase gene polymorphism in the realization of hypotensive effect of magnetic laser therapy was evaluated. The hypotensive effect of magnetic laser therapy depends on the polymorphism of the studied genes and is maximum in patients with MM polymorphism of angiotensinogen gene and DD polymorphism of angiotensin-converting enzyme gene. Additive interaction between angiotensin-converting enzyme and angiotensinogen genes in the formation of hemodynamic effects of magnetic laser therapy was detected.

Neuronal differentiation of P19 embryonal carcinoma cells modulates kinin B2 receptor gene expression and function.

Kinins are vasoactive oligopeptides generated upon proteolytic cleavage of low and high molecular weight kininogens by kallikreins. These peptides have a well established signaling role in inflammation and homeostasis. Nevertheless, emerging evidence suggests that bradykinin and other kinins are stored in the central nervous system and may act as neuromediators in the control of nociceptive response. Here we show that the kinin-B2 receptor (B2BKR) is differentially expressed during in vitro neuronal differentiation of P19 cells. Following induction by retinoic acid, cells form embryonic bodies and then undergo neuronal differentiation, which is complete after 8 and 9 days. Immunochemical staining revealed that B2BKR protein expression was below detection limits in nondifferentiated P19 cells but increased during the course of neuronal differentiation and peaked on days 8 and 9. Measurement of [Ca(2+)](i) in the absence and presence of bradykinin showed that most undifferentiated cells are unresponsive to bradykinin application, but following differentiation, P19 cells express high molecular weight neurofilaments, secrete bradykinin into the culture medium, and respond to bradykinin application with a transient increase in [Ca(2+)](i). However, inhibition of B2BKR activity with HOE-140 during early differentiation led to a decrease in the size of embryonic bodies formed. Pretreatment of differentiating P19 cells with HOE-140 on day 5 resulted in a reduction of the calcium response induced by the cholinergic agonist carbamoylcholine and decreased expression levels of M1-M3 muscarinic acetylcholine receptors, indicating crucial functions of the B2BKR during neuronal differentiation.

A novel encoded particle technology that enables simultaneous interrogation of multiple cell types.

The authors have developed a cellular analysis platform, based on encoded microcarriers, that enables the multiplexed analysis of a diverse range of cellular assays. At the core of this technology are classes of microcarriers that have unique, identifiable codes that are deciphered using CCD-based imaging and subsequent image analysis. The platform is compatible with a wide variety of cellular imaging-based assays, including calcium flux, reporter gene activation, cytotoxicity, and proliferation. In addition, the platform is compatible with both colorimetric and fluorescent readouts. Notably, this technology has the unique ability to multiplex different cell lines in a single microplate well, enabling scientists to perform assays and data analysis in novel ways.

Agonist-independent nuclear localization of the Apelin, angiotensin AT1, and bradykinin B2 receptors.

Signaling of the apelin, angiotensin, and bradykinin peptides is mediated by G protein-coupled receptors related through structure and similarities of physiological function. We report nuclear expression as a characteristic of these receptors, including a nuclear localization for the apelin receptor in brain and cerebellum-derived D283 Med cells and the AT(1) and bradykinin B(2) receptors in HEK-293T cells. Immunocytochemical analyses revealed the apelin receptor with localization in neuronal nuclei in cerebellum and hypothalamus, exhibiting expression in neuronal cytoplasm or in both nuclei and cytoplasm. Confocal microscopy of HEK-293T cells revealed the majority of transfected cells displayed constitutive nuclear localization of AT(1) and B(2) receptors, whereas apelin receptors did not show nuclear localization in these cells. The majority of apelin receptor-transfected cerebellum D283 Med cells showed receptor nuclear expression. Immunoblot analyses of subcellular-fractionated D283 Med cells demonstrated endogenous apelin receptor species in nuclear fractions. In addition, an identified nuclear localization signal motif in the third intracellular loop of the apelin receptor was disrupted by a substituted glutamine in place of lysine. This apelin receptor (K242Q) did not exhibit nuclear localization in D283 Med cells. These results demonstrate the following: (i) the apelin receptor exhibits nuclear localization in human brain; (ii) distinct cell-dependent mechanisms for the nuclear transport of apelin, AT(1), and B(2) receptors; and (iii) the disruption of a nuclear localization signal sequence disrupts the nuclear translocation of the apelin receptor. This discovery of apelin, AT(1), and B(2) receptors with agonist-independent nuclear translocation suggests major unanticipated roles for these receptors in cell signaling and function.