RESUMO
Sex differences in the control of prolactin secretion are well documented. Sex-related differences in intrapituitary factors regulating lactotroph function have recently attracted attention. Sex differences in prolactinoma development are well documented in clinic, prolactinomas being more frequent in women but more aggressive in men, for poorly understood reasons. Kallikrein, the enzyme releasing kinins has been found in the pituitary, but there is no information on pituitary kinin receptors and their function. In the present work, we characterized pituitary bradykinin receptors (BRs) at the messenger RNA and protein levels in 2 mouse models of prolactinoma, Drd2 receptor gene inactivation and hCGß gene overexpression, in both males and females, wild type or genomically altered. BR B2 (B2R) accounted for 97% or more of total pituitary BRs in both models, regardless of genotype, and was present in lactotrophs, somatotrophs, and gonadotrophs. Male pituitaries displayed higher level of B2R than females, regardless of genotype. Pituitary B2R gene expression was downregulated by estrogen in both males and females but only in females by dopamine. Activation of B1R or B2R by selective pharmacological agonists induced prolactin release in male pituitaries but inhibited prolactin secretion in female pituitaries. Increased B2R content was observed in pituitaries of mutated animals developing prolactinomas, compared to their respective wild-type controls. The present study documents a novel sex-related difference in the control of prolactin secretion and suggests that kinins are involved, through B2R activation, in lactotroph function and prolactinoma development.
Assuntos
Neoplasias Hipofisárias , Prolactinoma , Animais , Feminino , Humanos , Cininas , Masculino , Camundongos , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Prolactina/metabolismo , Prolactinoma/genética , Prolactinoma/metabolismo , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo , Receptores da BradicininaRESUMO
Kinin B1 (B1R) and B2 receptors (B2R) and the transient receptor potential vanilloid 4 (TRPV4) channel are known to play a critical role in the peripheral neuropathy induced by paclitaxel (PTX) in rodents. However, the downstream pathways activated by kinin receptors as well as the sensitizers of the TRPV4 channel involved in this process remain unknown. Herein, we investigated whether kinins sensitize TRPV4 channels in order to maintain PTX-induced peripheral neuropathy in mice. The mechanical hyperalgesia induced by bradykinin (BK, a B2R agonist) or des-Arg9-BK (DABK, a B1R agonist) was inhibited by the selective TRPV4 antagonist HC-067047. Additionally, BK was able to sensitize TRPV4, thus contributing to mechanical hyperalgesia. This response was dependent on phospholipase C/protein kinase C (PKC) activation. The selective kinin B1R (des-Arg9-[Leu8]-bradykinin) and B2R (HOE 140) antagonists reduced the mechanical hyperalgesia induced by PTX, with efficacies and time response profiles similar to those observed for the TRPV4 antagonist (HC-067047). Additionally, both kinin receptor antagonists inhibited the overt nociception induced by hypotonic solution in PTX-injected animals. The same animals presented lower PKCε levels in skin and dorsal root ganglion samples. The selective PKCε inhibitor (εV1-2) reduced the hypotonicity-induced overt nociception in PTX-treated mice with the same magnitude observed for the kinin receptor antagonists. These findings suggest that B1R or B2R agonists sensitize TRPV4 channels to induce mechanical hyperalgesia in mice. This mechanism of interaction may contribute to PTX-induced peripheral neuropathy through the activation of PKCε. We suggest these targets represent new opportunities for the development of effective analgesics to treat chronic pain.
Assuntos
Hiperalgesia/metabolismo , Paclitaxel/toxicidade , Doenças do Sistema Nervoso Periférico/metabolismo , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Hiperalgesia/etiologia , Masculino , Camundongos , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Estimulação Física/efeitos adversos , Receptor B1 da Bradicinina/agonistas , Receptor B2 da Bradicinina/agonistas , Moduladores de Tubulina/toxicidadeRESUMO
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.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Neoplasias da Bexiga Urinária/enzimologia , Idoso , Idoso de 80 Anos ou mais , Biópsia , Antagonistas de Receptor B1 da Bradicinina , Antagonistas de Receptor B2 da Bradicinina , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor B1 da Bradicinina/agonistas , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tiazolidinedionas/farmacologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/enzimologia , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
BACKGROUND AND PURPOSE: Kinin B(1) and B(2) receptors have been implicated in physiological and pathological conditions of the urinary bladder. However, their role in overactive urinary bladder (OAB) syndrome following spinal cord injury (SCI) remains elusive. EXPERIMENTAL APPROACH: We investigated the role of kinin B(1) and B(2) receptors in OAB after SCI in rats. KEY RESULTS: SCI was associated with a marked inflammatory response and functional changes in the urinary bladder. SCI resulted in an up-regulation of B(1) receptor mRNA in the urinary bladder, dorsal root ganglion and spinal cord, as well as in B(1) protein in the urinary bladder and B(1) and B(2) receptor protein in spinal cord. Interestingly, both B(1) and B(2) protein expression were similarly distributed in detrusor muscle and urothelium of animals with SCI. In vitro stimulation of urinary bladder with the selective B(1) or B(2) agonist elicited a higher concentration-response curve in the SCI urinary bladder than in naive or sham urinary bladders. Cystometry revealed that treatment of SCI animals with the B(2) selective antagonist icatibant reduced the amplitude and number of non-voiding contractions (NVCs). The B(1) antagonist des-Arg(9) -[Leu(8) ]-bradykinin reduced the number of NVCs while the non-peptide B(1) antagonist SSR240612 reduced the number of NVCs, the urinary bladder capacity and increased the voiding efficiency and voided volume. CONCLUSIONS AND IMPLICATIONS: Taken together, these data show the important roles of B(1) and B(2) receptors in OAB following SCI in rats and suggest that blockade of these receptors could be a potential therapeutic target for controlling OAB.
Assuntos
Receptor B1 da Bradicinina/fisiologia , Receptor B2 da Bradicinina/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Bexiga Urinária Hiperativa/fisiopatologia , Animais , Antagonistas de Receptor B1 da Bradicinina , Antagonistas de Receptor B2 da Bradicinina , Gânglios Espinais/fisiologia , Masculino , Contração Muscular , Ratos , Ratos Wistar , Receptor B1 da Bradicinina/agonistas , Receptor B2 da Bradicinina/agonistas , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/metabolismo , Bexiga Urinária/fisiologia , Bexiga Urinária Hiperativa/etiologia , Bexiga Urinária Hiperativa/metabolismoRESUMO
UNLABELLED: Kinins mediate their cellular effects through B1 (B1R) and B2 (B2R) receptors, and the activation of B2R reduces collagen synthesis in cardiac fibroblasts (CF). However, the question of whether B1R and/or B2R have a role in cardiac myofibroblasts remains unanswered. METHODS: CF were isolated from neonate rats and myofibroblasts were generated by an 84 h treatment with TGF-ß1 (CMF). B1R was evaluated by western blot, immunocytochemistry and radioligand assay; B2R, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and cyclooxygenases 1 and 2 (COX-1, and COX-2) were evaluated by western blot; intracellular Ca⺲ levels were evaluated with Fluo-4AM; collagen secretion was measured in the culture media using the picrosirius red assay kit. RESULTS: B2R, iNOS, COX-1 and low levels of B1R but not eNOS, were detected by western blot in CF. Also, B1R, B2R, and COX-2 but not iNOS, eNOS or COX-1, were detected by western blot in CMF. By immunocytochemistry, our results showed lower intracellular B1R levels in CF and higher B1R levels in CMF, mainly localized on the cell membrane. Additionally, we found B1R only in CMF cellular membrane through radioligand displacement assay. Bradykinin (BK) B2R agonist increased intracellular Ca²âº levels and reduced collagen secretion both in CF and CMF. These effects were blocked by HOE-140, and inhibited by L-NAME, 1400 W and indomethacin. Des-Arg-kallidin (DAKD) B1R agonist did not increase intracellular Ca²âº levels in CF; however, after preincubation for 1h with DAKD and re-stimulation with the same agonist, we found a low increase in intracellular Ca²âº levels. Finally, DAKD increased intracellular Ca²âº levels and decreased collagen secretion in CMF, being this effect blocked by the B1R antagonist des-Arg9-Leu8-kallidin and indomethacin, but not by L-NAME or 1400 W. CONCLUSION: B1R, B2R, iNOS and COX-1 were expressed differently between CF and CMF, and collagen secretion was regulated differentially by kinin receptor agonists in cultured CF and CMF.
Assuntos
Colágeno/metabolismo , Fibroblastos/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Receptores da Bradicinina/metabolismo , Animais , Ligação Competitiva/fisiologia , Western Blotting , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Inibidores de Ciclo-Oxigenase/farmacologia , Imuno-Histoquímica , Calidina/análogos & derivados , Calidina/farmacologia , Cininas/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptor B1 da Bradicinina/agonistas , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/metabolismo , Receptores da Bradicinina/agonistas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
In order to better understand the relationship between the primary structure of TsHpt-I - a bradykinin-potentiating peptide (BPP) isolated from the venom of the yellow scorpion Tityus serrulatus, with a non-canonical Lys residue prior to the conservative Pro-Pro doublet - and its cardiovascular effects, a series of ladder peptides were synthesized using the C-terminal portion of TsHpt-I as a template. All synthetic peptides having the Pro-Pro doublet at their C-terminal were able to potentiate the hypotensive effect of bradykinin. Conversely, only those analogues having Lys residue could induce a transient hypotension when intravenously administrated in male rats, indicating that the positive charge located toward the radical of this amino acid residue is crucial for this cardiovascular effect. Differently from all known BPPs, TsHpt-I acts as an agonist of the B(2) receptor and does not inhibit angiotensin-converting enzyme. The capacity of this peptide to activate this subtype of kinin receptor, releasing NO, was also affected by the absence of Lys' side-chain positive charge. Moreover, this study has demonstrated that the minimization of the primary structure of TsHpt-I does not significantly alter the biological effects of this native peptide, which could be of interest for biotechnological purposes.
Assuntos
Bradicinina/metabolismo , Oligopeptídeos/química , Receptor B2 da Bradicinina/agonistas , Venenos de Escorpião/química , Sequência de Aminoácidos , Animais , Hipotensão/induzido quimicamente , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Oligopeptídeos/fisiologia , Ratos , Ratos Wistar , Análise de Sequência de Proteína , Relação Estrutura-AtividadeRESUMO
The kinin system can contribute distinctly to the sensory changes associated with different models of nerve injury-induced neuropathic pain. This study examines the roles of kinin B(1) and B(2) receptor-operated mechanisms in alterations in nociceptive responses of rats submitted to unilateral L5/L6 spinal nerve ligation (SNL) injury. Behavioural responses to ipsilateral hind paw stimulation with acetone (evaporation-evoked cooling), radiant heat (Hargreaves method) or von Frey hairs revealed that SNL rats developed long-lasting cold allodynia (from Days 3 to 40 post-surgery, peak on Day 6), heat hyperalgesia (stable peak from Days 9 to 36) and tactile allodynia (stable peak from Days 3 to 51). SNL rats manifested nocifensive responses to intraplantar injections on Day 12 of the selective B(1) receptor agonist des-Arg(9)-bradykinin (DABK) and augmented responses to the selective B(2) receptor agonist bradykinin (BK; each at 0.01-1nmol/paw). Systemic treatment of SNL rats with des-Arg(9)-Leu(8)-BK or HOE 140 (peptidic B(1) and B(2) receptor antagonists, respectively; 0.1-1mumol/kg, i.p.) selectively blocked responses triggered by DABK and BK (1nmol/paw) and alleviated partially and transiently established cold allodynia, heat hyperalgesia and (to a lesser extent) tactile allodynia. Western blot analysis revealed enhanced expression of kinin B(1) and B(2) receptor protein in ipsilateral L4-L6 spinal nerve and hind paw skin samples collected on Day 12 after SNL surgery. These results indicate that peripheral pronociceptive kinin B(1) and B(2) receptor-operated mechanisms contribute significantly to the maintenance of hind paw cold and mechanical allodynia and heat hyperalgesia induced by L5/L6 SNL in rats.
Assuntos
Neuralgia/fisiopatologia , Receptor B1 da Bradicinina/fisiologia , Receptor B2 da Bradicinina/fisiologia , Nervos Espinhais/fisiologia , Animais , Comportamento Animal , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B1 da Bradicinina , Antagonistas de Receptor B2 da Bradicinina , Modelos Animais de Doenças , Vias de Administração de Medicamentos , Lateralidade Funcional , Hiperalgesia/fisiopatologia , Ligadura , Masculino , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Ratos , Ratos Wistar , Receptor B1 da Bradicinina/agonistas , Receptor B2 da Bradicinina/agonistas , Nervos Espinhais/efeitos dos fármacos , Nervos Espinhais/lesões , Fatores de TempoRESUMO
This work aimed to functionally characterize the mechanisms underlying the relaxation induced by bradykinin (BK) in the rat carotid artery. Vascular reactivity experiments, using standard muscle bath procedures, showed that BK (0.1 nmol/L-3 mumol/L) induced relaxation of phenylephrine-pre-contracted rings in a concentration-dependent manner. Endothelial removal strongly attenuated BK-induced relaxation. HOE-140, the selective antagonist of bradykinin B(2) receptors concentration-dependently reduced the relaxation induced by BK. Pre-incubation of endothelium-intact rings with L-NAME (100 micromol/L), a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME (100 micromol/L), a selective inhibitor of the eNOS or 7-nitroindazole (100 micromol/L), the selective inhibitor of nNOS, reduced BK-induced relaxation. Conversely, 1400 W (10 nmol/L), a selective inhibitor of iNOS, did not alter the relaxation induced by BK. Surprisingly, indomethacin (10 micromol/L) a non-selective inhibitor of cyclooxygenase (COX) increased BK-induced relaxation in endothelium-intact but not denuded rings. Neither SQ29548 (3 micromol/L), a competitive antagonist of PGH(2)/TXA(2) receptors nor AH6809 (10 micromol/L), an antagonist of PGF(2alpha) receptors significantly altered the relaxation induced by BK in endothelium-intact rings. The combination of SQ29548 and AH6809 increased BK-induced relaxation. The present study shows that the vasorelaxant action displayed by BK in the rat carotid is mediated by endothelial B(2) receptors and the activation of the NO pathway. The major finding of this work is that it demonstrated functionally that endothelial-derived vasoconstrictor prostanoids (probably PGH(2), TXA(2) and PGF(2alpha)) counteract the vasorelaxant action displayed by BK.
Assuntos
Bradicinina/farmacologia , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/fisiologia , Vasodilatação , Animais , Bradicinina/análogos & derivados , Antagonistas de Receptor B2 da Bradicinina , Compostos Bicíclicos Heterocíclicos com Pontes , Inibidores Enzimáticos/farmacologia , Ácidos Graxos Insaturados , Hidrazinas/farmacologia , Iminas/farmacologia , Técnicas In Vitro , Indazóis/farmacologia , Indometacina/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos Wistar , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/fisiologia , Xantonas/farmacologiaRESUMO
This study analyzed bradykinin (BK)-evoked contractile responses in the mouse colon under normal and inflammatory conditions. BK and the preferential B(2) receptor agonists Hyp(3)-BK, Lys-BK, Met-Lys-BK and Tyr(8)-BK produced a marked and concentration-related contraction of the normal mouse colon, whereas the selective B(1) receptor agonist des-Arg(9)-BK had no effect. BK-induced contraction was concentration-dependently antagonized (in a non-competitive manner) by both B(2) receptor antagonists Hoe 140 and FR173657, but not the B(1) receptor antagonist des-Arg(9)-[Leu(8)]-BK. Analysis of the possible mechanisms implicated in the contractile responses of BK in the mouse colon revealed the involvement of the neural release of acetylcholine, the activation of L- and N-type voltage-gated calcium channels, and the release of neuropeptides, prostanoids and leukotrienes. The contraction induced by BK was markedly increased in preparations obtained from TNBS-treated mice. The up-regulation of B(2) receptors following the induction of colitis was confirmed with binding studies using [(3)H]-BK, which revealed a marked increase in B(2) receptor densities, without alterations of affinity. We provide convincing evidence on the relevance of B(2) receptors in the mouse colon under normal conditions, as well as under an inflammatory profile of colitis. Selective B(2) receptor antagonists might well represent rational therapeutic options for treating inflammatory bowel diseases.
Assuntos
Colite/fisiopatologia , Colo/fisiologia , Contração Muscular/efeitos dos fármacos , Receptor B2 da Bradicinina/química , Acetilcolina/metabolismo , Animais , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo N/metabolismo , Colite/induzido quimicamente , Colite/metabolismo , Colo/metabolismo , Relação Dose-Resposta a Droga , Calidina/farmacologia , Leucotrienos/metabolismo , Masculino , Camundongos , Neuropeptídeos/metabolismo , Prostaglandinas/metabolismo , Quinolinas/farmacologia , Receptor B2 da Bradicinina/agonistas , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
We have previously reported that exogenous bradykinin activates immature dendritic cells (DCs) via the bradykinin B(2) receptor (B(2)R), thereby stimulating adaptive immunity. In this study, we show that these premises are met in a model of s.c. infection by Trypanosoma cruzi, a protozoan that liberates kinins from kininogens through its major protease, cruzipain. Intensity of B(2)R-dependent paw edema evoked by trypomastigotes correlated with levels of IL-12 produced by CD11c(+) dendritic cells isolated from draining lymph nodes. The IL-12 response induced by endogenously released kinins was vigorously increased in infected mice pretreated with inhibitors of angiotensin converting enzyme (ACE), a kinin-degrading metallopeptidase. Furthermore, these innate stimulatory effects were linked to B(2)R-dependent up-regulation of IFN-gamma production by Ag-specific T cells. Strikingly, the trypomastigotes failed to up-regulate type 1 immunity in TLR2(-/-) mice, irrespective of ACE inhibitor treatment. Analysis of the dynamics of inflammation revealed that TLR2 triggering by glycosylphosphatidylinositol-anchored mucins induces plasma extravasation, thereby favoring peripheral accumulation of kininogens in sites of infection. Further downstream, the parasites generate high levels of innate kinin signals in peripheral tissues through the activity of cruzipain. The demonstration that the deficient type 1 immune responses of TLR2(-/-) mice are rescued upon s.c. injection of exogenous kininogens, along with trypomastigotes, supports the notion that generation of kinin "danger" signals is intensified through cooperative activation of TLR2 and B(2)R. In summary, we have described a s.c. infection model where type 1 immunity is vigorously up-regulated by bradykinin, an innate signal whose levels in peripheral tissues are controlled by an intricate interplay of TLR2, B(2)R, and ACE.