Indomethacin can downregulate the levels of inflammatory mediators in the hippocampus of rats submitted to pilocarpine-induced status epilepticus.

OBJECTIVE: Refractory status epilepticus is one of the most life-threatening neurological emergencies and is characterized by high morbidity and mortality. Additionally, the use of anti-inflammatory drugs during this period is very controversial. Thus, this study has been designed to analyze the effect of a low dose of indomethacin (a COX inhibitor) on the expression of inflammatory molecules. METHOD: The hippocampus of rats submitted to pilocarpine-induced long-lasting status epilepticus was analyzed to determine the expression of inflammatory molecules with RT-PCR and immunohistochemistry. RESULTS: Compared with controls, reduced levels of the kinin B2 receptors IL1ß and TNFα were found in the hippocampus of rats submitted to long-lasting status epilepticus and treated with indomethacin. CONCLUSIONS: These data show that low doses of indomethacin could be employed to minimize inflammation during long-lasting status epilepticus.

Indomethacin can downregulate the levels of inflammatory mediators in the hippocampus of rats submitted to pilocarpine-induced status epilepticus

OBJECTIVE: Refractory status epilepticus is one of the most life-threatening neurological emergencies and is characterized by high morbidity and mortality. Additionally, the use of anti-inflammatory drugs during this period is very controversial. Thus, this study has been designed to analyze the effect of a low dose of indomethacin (a COX inhibitor) on the expression of inflammatory molecules. METHOD: The hippocampus of rats submitted to pilocarpine-induced long-lasting status epilepticus was analyzed to determine the expression of inflammatory molecules with RT-PCR and immunohistochemistry. RESULTS: Compared with controls, reduced levels of the kinin B2 receptors IL1β and TNFα were found in the hippocampus of rats submitted to long-lasting status epilepticus and treated with indomethacin. CONCLUSIONS: These data show that low doses of indomethacin could be employed to minimize inflammation during long-lasting status epilepticus. .

[Development of specific protein labeling method by using small molecular probes].

Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions in living cells. In this review, we report a new protein labeling method that enables selective covalent modification of a tag-fused protein with small functional molecules. This method utilizes the specific interaction and rapid reaction between a short peptide tag and a molecular probe, which comprises the cysteine-containing short CA6D4x2 tag (CAAAAAADDDDGDDDD) and a tetranuclear Zn(II)-DpaTyr probe containing a reactive α-chloroacetyl moiety. This labeling system, so-called reactive tag system, was successfully applied to the fluorescence imaging of tag-fused GPCRs such as bradykinin receptor (B2R) and acetylcholine receptor (m1AchR) expressing on HEK293 cells. The utility of this labeling method was demonstrated in the function analyses of GPCRs, such as fluorescence visualization of the stimuli-responsive internalization of GPCRs and pH change in endosomes containing the internalized GPCRs.

Angiotensin II-mediated microvascular thrombosis.

Hypertension is associated with an increased risk of thrombosis that appears to involve an interaction between the renin-angiotensin system and hemostasis. In this study we determined whether angiotensin II-mediated thrombosis occurs in arterioles and/or venules and assessed the involvement of type 1 (AT1), type 2 (AT2), and type 4 (AT4) angiotensin II receptors, as well as receptors for endothelin 1 and bradykinin 1 and 2 in angiotensin II-enhanced microvascular thrombosis. Thrombus development in mouse cremaster microvessels was quantified after light/dye injury using the time of onset of the thrombus and time to blood flow cessation. Wild-type and AT1 receptor-deficient mice were implanted with an angiotensin II-loaded ALZET pump for 2 weeks. Angiotensin II administration in both wild-type and ATAT1 receptor-deficient mice significantly accelerated thrombosis in arterioles. Genetic deficiency and pharmacological antagonism of AT1 receptors did not alter the thrombosis response to angiotensin II. Isolated murine platelets aggregated in response to low (picomolar) but not high (nanomolar) concentrations of angiotensin II. The platelet aggregation response to angiotensin II depended on AT1 receptors. Antagonism of AT2 receptors in vivo significantly prolonged the onset of angiotensin II-enhanced thrombosis, whereas an AT4 receptor antagonist prolonged the time to flow cessation. Selective antagonism of either endothelin 1 or bradykinin 1 receptors largely prevented both the onset and flow cessation responses to chronic angiotensin II infusion. Our findings indicate that angiotensin II induced hypertension is accompanied by enhanced thrombosis in arterioles, and this response is mediated by a mechanism that involves AT2, AT4, bradykinin 1, and endothelin 1 receptor-mediated signaling.

Localization of gonadotrophin-releasing hormone I, bradykinin and their receptors in the ovaries of non-mammalian vertebrates.

GnRH I and its receptors have been demonstrated in the ovaries of various vertebrates, but their physiological significance in reproductive cascade is fragmentary. Bradykinin is a potent GnRH stimulator in the hypothalamus. In the present study, the presence of GnRH I and its receptor, and bradykinin and its receptor in the ovaries of non-mammalian vertebrates were investigated to understand their physiological significance. GnRH I immunoreactivity in the ovaries of fish, frog, reptile and bird were mainly found in the oocyte of early growing follicles and granulosa cells and theca cells of previtellogenic follicles. Vitellogenic follicles showed mild GnRH immunoreactivity. GnRH I-receptor and bradykinin were localized in the same cell types of the ovaries of these vertebrates. The presence of GnRH I, GnRH I-receptor and bradykinin in the ovaries of these vertebrates was confirmed by immunoblotting. The presence of GnRH I mRNA was demonstrated in the ovary of vertebrates using RT-PCR. The ovaries of reptiles and birds showed significantly higher intensity of immunoreactivity for GnRH I-receptor as compared with the fish and amphibian. This may have a correlation with the higher yolk content in the ovary of reptile and bird. These results suggest the possibility of GnRH I and bradykinin as important regulators of follicular development and vitellogenesis in the vertebrate ovary.

Expression and localisation of kinin receptors in colorectal polyps.

Kinins increase vascular permeability as well as mitogenesis and proliferation, hence they have a potential to promote neoplasmatic transformation. In the present study we investigated the expression profile and localization of kinin B1 and B2 receptors in colorectal polyps. The biopsy samples from various polyps were obtained during endoscopy in tubular (n=18), villous (n=15) and hyperplasic polyps (n=15). The expression of genes encoding B1 and B2 was estimated by QRT-PCR TaqMan analysis. In second series B1 and B2 receptors were visualized by immunohistochemical staining in tissue specimens from colonic polyps and adjacent normal tissue. We found the highest expression of gene encoding B1 in tubular adenomas (1891 number of copies mRNA/microg total RNA+/-312 SE) which is significantly higher as compared with controls (683+/-197 SE, p<0.013). In contrast, the expression of gene for B2 was significantly increased in hyperplastic polyps (3852+/-936 SE) as compared with controls (843+/-263 SE, p<0.0016). In normal colon a well as in hyperplasic polyps B1 and B2 receptors were immunohistochemically localized in enterocytes, however in hyperplastic polyps the intensity of staining was more prominent for B2 comparing to the control group. In contrast, in tubular adenomas staining reaction for B1 was more intense than in control samples. Increased level of B1 in adenoma suggests that kinins may play a role in abnormal cellular transformation; whereas higher B2 level in hyperplasic polyp suggests its protective role. Our data may indicate that the overall effect of kinins on cellular proliferation depends on the relative level of B1 and B2 receptor expression.

Expression of metallopeptidases and kinin receptors in swine oropharyngeal tissues: effects of angiotensin I-converting enzyme inhibition and inflammation.

Angiotensin I-converting enzyme inhibitors (ACEi) cause both chronic and acute side effects, including rare but potentially life-threatening angioedema (AE). The main hypothesis to be tested in this study was that metallopeptidases and kinin receptors are present in oropharyngeal tissues and that their expression is modulated by ACEi and inflammation. Novel real-time polymerase chain reaction analysis was developed and allowed the relative quantification of tissue's gene expression for neprilysin, membrane-bound aminopeptidase P (mAPP), and both B1 and B2 kinin receptor subtypes in tongue, parotid gland, and laryngeal tissue (areas especially involved in the gravest clinical forms of AE) and in kidney in a porcine model (single injection or 7-day ACEi oral treatments applied or lipopolysaccharide injected as a positive inflammatory control). The results provide evidence of the expression and activities of kininases in oropharyngeal tissues in the swine. ACEi treatment modulated the expression of neutral endopeptidase and mAPP mRNA, but the corresponding enzyme activities and that of angiotensin I-converting enzyme (ACE) were generally stable in tissues. The 7-day ACEi treatment up-regulated both kinin receptor mRNAs in the oropharynx and the B1 receptor mRNA in the lingual vascular endothelium (immunohistochemistry). The inhibition of ACE in plasma is responsible for an accumulation of bradykinin and des-arginine9-bradykinin generated during activation of the contact system with glass beads. The expression of critical components of the kallikrein-kinin system in the oropharyngeal tissues supports the role of kinins in ACEi-induced AE.

Visualisation of transforming growth factor-beta 1, tissue kallikrein, and kinin and transforming growth factor-beta receptors on human clear-cell renal carcinoma cells.

Transforming growth factor-beta1 (TGF-beta1) has a biphasic effect on the growth of renal epithelial cells. In transformed cells, TGF-beta1 appears to accelerate the proliferation of malignant cells. The diverse cellular functions of TGF-beta1 are regulated by three high-affinity serine/threonine kinase receptors, namely TbetaRI, TbetaRII and TbetaRIII. The renal serine protease tissue kallikrein acts on its endogenous protein substrate kininogen to form kinin peptides. The cellular actions of kinins are mediated through B1 and B2 G protein-coupled rhodopsin receptors. Both kinin peptides and TGF-beta1 are mitogenic, and therefore may play an important role in carcinogenesis. Experiments were designed to immunolabel tissue kallikrein, TGF-beta1, TbetaRII, TbetaRIII and kinin receptors using specific antibodies on serial sections of normal kidney and clear-cell renal carcinoma (CCRC) tissue, which included both the tumour and the adjacent renal parenchyma. The essential result was the localisation of tissue kallikrein, kinin B 1 and B 2 receptors and TGF-beta1 primarily on the cell membranes of CCRC cells. In the distal and proximal tubules of the renal parenchyma adjacent to the carcinoma (RPTAC), immunolabelling for tissue kallikrein was reduced, but the expression of kinin B1 and B2 receptors was enhanced. Immunolabelling for TbetaRII and TbetaRIII was more pronounced in the proximal tubules of the tissue adjacent to the carcinoma when compared to the normal kidney. The expression of tissue kallikrein, kinin receptors, and TbetaRII and TbetaRIII may be relevant to the parenchymal invasion and metastasis of clear-cell renal carcinoma.

Localization of B1 bradykinin receptor mRNA in the primate brain and spinal cord: an in situ hybridization study.

The bradykinin 1 and 2 receptors (B1R, B2R) are important mediators of cardiovascular homeostasis, inflammation, and nociception. While B2R is constitutively expressed in many tissues, B1R expression is thought to be absent, but induced under proinflammatory conditions. However, recent data from knockout mice have indicated that B1R acts centrally to mediate nociception, a finding that suggests the constitutive presence of B1R in brain and/or spinal cord. The purpose of the present study was to further elucidate the physiological role of B1R by evaluating the localization of B1R mRNA in the nonhuman primate brain and spinal cord with in situ hybridization. Cryostat sections from monkey brain and spinal cord were hybridized with a [(35)S]-labeled riboprobe complementary to B1R mRNA, stringently washed, and apposed to film and emulsion. The results of these studies revealed the presence of B1R mRNA throughout the rostral-caudal extent of the brain and spinal cord. In particular, labeled cells were seen in the cerebral and entorhinal cortex, dentate gyrus, and pyramidal neurons of the hippocampus, in the thalamus, hypothalamus, amygdala, pontine nuclei, spinal cord, and dorsal root ganglion. Together the present findings offer detailed information about the distribution of B1R mRNA in the primate brain and spinal cord and demonstrate a basal level of expression in the primate nervous system. Moreover, these data provide a foundation for understanding the central actions of kinins and their putative role in mediating a number of processes, including pain and nociception.

Tissue kallikrein and bradykinin B2 receptors in the reproductive tract of the male rat.

The setting of a local tissue kallikrein kinin system (tKKS) within the reproductive organs of the male rat was investigated by analysing bradykinin subtype 2 receptor (B2R) gene expression and cellular distribution of B2R protein and the kinin-liberating protease tissue kallikrein (tK). Reverse transcription-polymerase chain reaction showed B2R expression in testis, epididymis and prostate from prepubertal and sexually mature rats. In mature testis, in situ hybridization and immunohistochemistry localized B2R mRNA and protein besides endothelial cells of blood vessels exclusively on pachytene spermatocytes and round and elongated spermatids. B2R expression within the seminiferous tubules was found to be dependent on the stage of the spermatogenic cycle. In pre-pubertal rat testis, B2R mRNA and protein were additionally located in peritubular cells. In the testis, specific staining for tK occurred in addition to endothelial cells of blood vessels on the acrosomal cap of round and elongated spermatids. This immunostaining was also stage-dependent. In the epididymis, tK was detected on epithelial cells near the apical surface. The stage-dependent specific expression of tK and bradykinin B2Rs in developing germ cells and peritubular cells suggests a potential role of the tKKS in the local regulation of spermatogenesis and seminiferous tubule function.

Cardiac angiotensin II type 2 receptor activates the kinin/NO system and inhibits fibrosis.

We have previously demonstrated that stimulation of the angiotensin (Ang) II type 2 receptor in vascular smooth muscle cells caused bradykinin production by activating kininogenase in transgenic mice. The aim of this study was to determine whether overexpression of AT2 receptors in cardiomyocytes attenuates Ang II-induced cardiomyocyte hypertrophy or interstitial fibrosis through a kinin/nitric oxide (NO)-dependent mechanism in mice. Ang II (1.4 mg/kg per day) or vehicle was subcutaneously infused into transgenic mice and wild-type mice for 14 days. The amount of cardiac AT2 receptor relative to AT1 receptor in transgenic mice was 22% to 37%. Ang II caused similar elevations in systolic blood pressure (by approximately 45 mm Hg) in transgenic mice and wild-type mice. Myocyte hypertrophy assessed by an increase in myocyte cross-sectional area, left ventricular mass, and atrial natriuretic peptide mRNA levels were similar in transgenic and wild-type mice. Ang II induced prominent perivascular fibrosis of the intramuscular coronary arteries, the extent of which was significantly less in transgenic mice than in wild-type mice. Inhibition of perivascular fibrosis in transgenic mice was abolished by cotreatment with HOE140, a bradykinin B2 receptor antagonist, or L-NAME, an inhibitor of NO synthase. Cardiac kininogenase activity was markedly increased (approximately 2.6-fold, P<0.001) after Ang II infusion in transgenic mice but not in wild-type mice. Immunohistochemistry indicated that both bradykinin B2 receptors and endothelial NO synthase were expressed in the vascular endothelium, whereas only B2 receptors were present in fibroblasts. These results suggest that stimulation of AT2 receptors present in cardiomyocytes attenuates perivascular fibrosis by a kinin/NO-dependent mechanism. However, the effect on the development of cardiomyocyte hypertrophy was not detected in this experimental setting.

Human B1 and B2 bradykinin receptors and their agonists target caveolae-related lipid rafts to different degrees in HEK293 cells.

To address the targeting of G protein-coupled receptors to caveolae-related lipid rafts (CLR), we studied the human B2 (B2R) and B1 (B1R) bradykinin receptor subtypes in HEK293 cells. CLR were enriched on the basis of their unique buoyant density and composition of cholesterol, caveolin-1, and flotillin-1 but not clathrin. CLR contained B2R and B1R as determined by both receptor immunoblotting and the increase in specific activity of receptor agonist binding to cells at both 4 and 37 degrees C when binding was followed by CLR enrichment. B2R was highly enriched in this fraction, whereas B1R was not enriched. Furthermore, acid washing of cells prior to cell disruption minimally affected the CLR-associated B2R agonist binding, whereas it dissociated a major portion of the CLR-associated B1R agonist binding. In addition, when agonist binding at 4 degrees C was followed by an increase in the temperature to 37 degrees C, B2R agonist binding in CLR transiently increased, and this increase was dependent on the C-terminal domain. On the other hand, B1R agonist binding remained unchanged and was independent of the C-terminal domain. Our results show that B2R is constitutively targeted to CLR in HEK293 cells and appears to shuttle the agonist through these domains, whereas B1R may be there by default.

Expression and location of the bradykinin B2 receptor in rat testis.

To investigate the possible role of the local tissue kallikrein-kinin system in spermatogenesis, we analyzed gene expression and cellular distribution of the bradykinin subtype-2 receptor (B(2) receptor) in the rat testis. Reverse transcription-polymerase chain reaction revealed B(2) receptor expression in testis and primary cultures of Sertoli cells and peritubular cells isolated from immature and mature rats. In situ hybridization of the B(2)-receptor mRNA showed intense labeling of cells on the base of the seminiferous tubule, whereas the autoradiographic signals gradually decreased toward the lumen. Immune histochemistry using testicular sections of pubertal and adult rats showed specific staining for the B(2)-receptor protein in cells of the adluminal compartment of the seminiferous tubules, especially on pachytene spermatocytes and spermatids. This immunostaining varied with the stages of the seminiferous cycle. The receptor protein was also observed on peritubular cells of pubertal rats. In conclusion, we demonstrated a stage-specific expression of the bradykinin B(2) receptor in different cells of the seminiferous tubules of the rat testis. The results point to a possible function of the tissue kallikrein-kinin system in the local regulation of spermatogenesis.

Bradykinin potentiation by ACE inhibitors: a matter of metabolism.

1. Studies in isolated cells overexpressing ACE and bradykinin type 2 (B(2)) receptors suggest that ACE inhibitors potentiate bradykinin by inhibiting B(2) receptor desensitization, via a mechanism involving protein kinase C (PKC) and phosphatases. Here we investigated, in intact porcine coronary arteries, endothelial ACE/B(2) receptor 'crosstalk' as well as bradykinin potentiation through neutral endopeptidase (NEP) inhibition. 2. NEP inhibition with phosphoramidon did not affect the bradykinin concentration-response curve (CRC), nor did combined NEP/ACE inhibition with omapatrilat exert a further leftward shift on top of the approximately 10 fold leftward shift of the bradykinin CRC observed with ACE inhibition alone. 3. In arteries that, following repeated exposure to 0.1 microM bradykinin, no longer responded to bradykinin ('desensitized' arteries), the ACE inhibitors quinaprilat and angiotensin-(1-7) both induced complete relaxation, without affecting the organ bath fluid levels of bradykinin. This phenomenon was unaffected by inhibition of PKC or phosphatases (with calphostin C and okadaic acid, respectively). 4. When using bradykinin analogues that were either completely or largely ACE-resistant ([Phe(8)psi(CH(2)-NH)Arg(9)]-bradykinin and [deltaPhe(5)]-bradykinin, respectively), the ACE inhibitor-induced shift of the bradykinin CRC was absent, and its ability to reverse desensitization was absent or significantly reduced, respectively. Caveolar disruption with filipin did not affect the quinaprilat-induced effects. Filipin did however reduce the bradykinin-induced relaxation by approximately 25-30%, thereby confirming that B(2) receptor-endothelial NO synthase (eNOS) interaction occurs in caveolae. 5. In conclusion, in porcine arteries, in contrast to transfected cells, bradykinin potentiation by ACE inhibitors is a metabolic process, that can only be explained on the basis of ACE-B(2) receptor co-localization on the endothelial cell membrane. NEP does not appear to affect the bradykinin levels in close proximity to B(2) receptors, and the ACE inhibitor-induced bradykinin potentiation precedes B(2) receptor coupling to eNOS in caveolae.

Detection of bradykinin and bradykinin-beta(2) receptors in the porcine endometrium during the estrous cycle and early pregnancy.

During the period of attachment of the trophectoderm to the uterine lumenal surface in the pig, there is an increase in uterine blood flow and a localized hyperemic response induced by the developing conceptuses. The presence of tissue kallikrein in the porcine uterine lumen suggests that the kallikrein-kinin system may be functional during pregnancy in the pig. The objective of the present study was to determine the concentration of bradykinin within the uterine lumen during the estrous cycle and early pregnancy as well as endometrial gene expression and cellular localization of the bradykinin beta(2) receptor. Concentration of bradykinin in uterine flushings was greatest during estrus (Day 0) and Days 12-18 of the estrous cycle. However, there was a 5- to 10-fold increase in bradykinin content in pregnant uterine flushings on Days 12-18 of pregnancy compared with the estrous cycle. Endometrial bradykinin beta(2) receptor gene expression was greatest on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy as gene expression decreased almost 6-fold on Days 5 and 10. Bradykinin beta(2) receptors were detected in the endometrial surface and glandular epithelium with greatest intensity of staining observed on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy. Results from the present study suggest that the kallikrein-kinin system plays a role in the establishment of pregnancy in the pig.

Identification of bradykinin receptors in clinical cancer specimens and murine tumor tissues.

Bradykinin (BK) has multiple pathophysiologic functions such as induction of vascular permeability and mitogenesis, and it triggers the release of other mediators such as nitric oxide in inflammatory and cancer tissues. To explore the pathophysiologic roles of BK in tumor, we examined the distribution of BK B2 receptors in human adenocarcinoma (lung, stomach), lymphoma (lymph node), hepatoma, squamous cell carcinoma (lung) and carcinoid (duodenum), and in mouse colon adenocarcinoma 38 (C-38) and sarcoma 180 (S-180) tumor tissues. Immunohistochemical staining of tumor tissues with an anti-BK B2 receptor antibody, or autoradiography with the B2 receptor antagonist [125I]HOE 140 (D-Arg-[Hyp Thi D-Tic Oic8]-BK) and the B2 receptor agonist [3H]BK indicated the presence of B2 receptors in all human tumor cells and murine S-180 and C-38 cells. Specific binding of [3H]HOE 140 was observed in S-180 cells with a Kd of 2.1 nM. Binding of [125I]HOE 140 to S-180 cells was competed by an excess amount (20-100 times) of nonradiolabeled HOE 140 or BK, but not by BK B1 receptor agonist des-Arg9-BK. These results provide direct evidence that the BK B2 receptor is expressed in human cancer and experimental murine tumors, which suggests a potential role for BK in inducing pathologic signal transduction in cancer growth and progression, nitric oxide production and vascular permeability enhancement in tumors. BK antagonists may thus have applications in the modulation of cancer growth and in paraneoplastic syndromes.

Characterization of bradykinin receptors in a human osteoblastic cell line.

Bradykinin receptor subtypes linked to prostaglandin release have been assessed in a human osteosarcoma cell line with osteoblastic phenotype (MG-63). Bradykinin (BK; 1 micromol/l) caused a burst of prostaglandin E(2) release that was maximal at 10 min. When the effect on the burst of PGE(2) and PGI(2) release by a variety of kinins and kinin analogues was assessed, the following rank order of response was found: Lys-BK>BK> or =Met-Lys-BK>Ile-Ser-BK>[Tyr(8)]-BK> or =[Hyp(3)]-BK>>>des-Arg(9)-BK=des-Arg(10)-Lys-BK=des-Arg(1)-BK, [Thi(5,8),D-Phe(7)]-BK=Sar-[D-Phe(8)]-des-Arg(9)-BK=Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK. The rapid effect of BK on PGE(2) and PGI(2) release was unaffected by des-Arg(9)-[Leu(8)]-BK, des-Arg(10)-[Leu(9)]-Lys-BK and des-Arg(10)-[Hoe 140], but strongly inhibited by Hoe 140 in a concentration-dependent manner. When the incubation time was extended to 48 h, it was found that des-Arg(9)-BK and des-Arg(10)-Lys-BK caused a delayed enhancement of the formation of PGE(2). When PGE(2) formation was assessed in 24-h experiments, the following rank order of response was obtained: Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK>>BK=Lys-BK>>des-Arg(10)-Lys-BK>Sar[D-Phe(8)]-des-Arg(9)-BK>des-Arg(9)-BK. The stimulatory effect of BK at 24 h was unaffected by des-Arg(9)-[Leu(8)]-BK, des-Arg(10)-[Leu(9)]-Lys-BK and des-Arg(10)-[Hoe 140] but inhibited by Hoe 140. The stimulatory effect of des-Arg(10)-Lys-BK in 24-h experiments was inhibited by des-Arg(9)-[Leu(8)]-BK, des-Arg(10)-[Leu(9)]-Lys-BK and des-Arg(10)-[Hoe 140]. Similarly, the stimulatory effects of Sar[D-Phe(8)]-des-Arg(9)-BK and Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK was inhibited by des-Arg(10)-[Hoe 140]. The following rank order of response was seen for inhibition of [3H]-BK binding to MG-63 cells: Lys-BK=BK=Hoe 140>>>>>>des-Arg(10)-Hoe 140=des-Arg(10)-Lys-BK=des-Arg(9)-BK=Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK. Using [3H]-des-Arg(10)-Lys-BK, the following rank order of response for inhibition of binding was seen: des-Arg(10)-Lys-BK=Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK>des-Arg(10)-Hoe 140>des-Arg(9)-BK=Lys-BK=BK=Hoe 140. MG-63 cells expressed mRNAs for BK B1 and B2 receptors, as assessed by RT-PCR. These data indicate that the human osteoblastic osteosarcoma cell line MG-63 is equipped with functional BK receptors of both B1 and B2 receptor subtypes. The B2 receptors are linked to a burst of prostanoid release, whereas the B1 receptors mediate a delayed prostaglandin response, indicating that the two receptor subtypes are linked to different signal transducing mechanisms or that the molecular mechanisms involved in prostaglandin release are different.

The expression of bradykinin B(1) receptors on primary sensory neurones that give rise to small caliber sciatic nerve fibres in rats.

The bradykinin B(1) receptor has been considered as an important mediator for inflammatory pain. In the present study, we have investigated the fibre types of sciatic nerve primary sensory neurones that express B(1) receptors by retrograde tracing in combination with immunohistochemical staining, or double-immunohistochemical staining. Approximately 12% of the A-fibre dorsal root ganglion neurones, retrogradely labelled from an intra-sciatic nerve injection of fluorescein isothiocyanate-conjugated cholera toxin B subunit, were B(1) receptor-immunoreactive. Over 70% of the small diameter dorsal root ganglion neurones, retrogradely labelled from an intra-sciatic nerve injection of tetramethylrhodamine isothiocyanate-conjugated wheat germ agglutinin, were B(1) receptor-immunoreactive. Over 50% of the (predominantly non-peptidergic) C-fibre dorsal root ganglion neurones, retrogradely labelled from an intra-sciatic nerve injection of fluorescein isothiocyanate-conjugated Bandeiraea simplicifolia isolectin B4, were B(1) receptor-immunoreactive. When calcitonin gene-related peptide, which is contained mainly in small caliber C- and A(delta)-fibre primary afferents, and B(1) receptors were stained with a double-immunofluorescent method, over 80% of the calcitonin gene-related peptide-positive dorsal root ganglion neurones were B(1) receptor-immunoreactive. From these results we suggest that B(1) receptors are predominantly expressed by small diameter primary afferent neurones that give rise to sciatic nerve fibres, which include both peptidergic and non-peptidergic C-fibres and A(delta)-fibres. Since peripheral nociceptive information is primarily transmitted by C- and A(delta)-fibres, B(1) receptors may be involved in the modulation of nociceptive transduction or transmission.

Localization of bradykinin B(2) receptor in the follicles of porcine ovary and increased expression of matrix metalloproteinase-3 and -20 in cultured granulosa cells by bradykinin treatment.

We have recently shown that not only bradykinin, but also all components for the production of bradykinin, can be detected within the follicle of porcine ovaries. To elucidate the relevance of the intrafollicular bradykinin-producing system to its physiological role, we investigated the distribution of bradykinin receptor (B(2)R) mRNA and the protein in porcine ovaries. A cDNA encoding porcine B(2)R was first cloned from a porcine uterus cDNA library. The receptor mRNA was scarcely detected in the ovary by Northern blot analysis. Polymerase chain reaction analysis with total RNAs isolated from the ovary and from granulosa cells of small and large follicles demonstrated the ovarian expression of B(2)R mRNA. The B(2)R protein was detected by Western blot analysis in extracts of isolated granulosa cells. In situ hybridization of B(2)R mRNA and immunohistochemical analysis of the protein revealed that the receptor is expressed in the theca and granulosa cells of all growing follicles. The effect of bradykinin on the expression of some matrix metalloproteinase (MMP) genes was examined using isolated granulosa cells. Bradykinin treatment induced MMP-3 and MMP-20 gene expression to an extreme degree. The expression of MT1-MMP was also affected by bradykinin treatment. These results suggest that MMPs play a role in follicle rupture during ovulation. The present study provides new information regarding the mechanisms of bradykinin-induced ovulation in porcine ovaries.

Basal expression of bradykinin B(1) receptor in the spinal cord in humans and rats.

The bradykinin B(1) receptor has been considered as a receptor induced by tissue injury and inflammation mainly in the peripheral tissues. In the present study, we have investigated whether there is a basal expression in the spinal cord by both reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining methods. Southern blotting of the DNA reverse-transcribed from human and rat spinal cord mRNA and amplified by polymerase chain reaction (PCR) showed a substantial basal B(1) receptor expression in both human and rat spinal cord. Immunohistochemical staining demonstrated B(1)-positive neurons in the spinal cord dorsal horn, suggesting that the B(1) receptor is constitutively expressed by spinal neurons.