A primary role for kinin B1 receptor in inflammation, organ damage, and lethal thrombosis in a rat model of septic shock in diabetes.

Diabetes mellitus and septic shock increase the incidence of mortality by thrombosis. Although kinin B1 receptor (B1R) is involved in both pathologies, its role in platelet function and thrombosis remains unknown. This study investigates the expression, the inflammatory, and pro-thrombotic effects of B1R in a model of septic shock in diabetic rats. Sprague-Dawley rats were made diabetic with streptozotocin (STZ) (65 mg/kg, i.p.). Four days later, control and STZ-diabetic rats were injected with lipopolysaccharide (LPS) (2 mg/kg, i.p.) or the vehicle. B1R antagonist (SSR240612, 10 mg/kg by gavage) was given either acutely (12 and 24 h prior to endpoint analysis) or daily for up to 7 days. Moreover, a 7-day treatment was given either with cyclooxygenase (COX)-2 inhibitor (niflumic acid, 5 mg/kg, i.p.), non-selective COX-1 and COX-2 inhibitor (indomethacin, 10 mg/kg, i.p.), non-selective nitric oxide synthase (NOS) inhibitor (L-NAME, 50 mg/kg by gavage), iNOS inhibitor (1400W, 5 mg/kg, i.p.), or heparin (100 IU/kg, s.c.). The following endpoints were measured: edema and vascular permeability (Evans blue dye), B1R expression (qRT-PCR, western blot, flow cytometry), aggregation in platelet-rich plasma (optical aggregometry), and organ damage (histology). Rats treated with STZ, LPS, and STZ plus LPS showed significant increases in edema and vascular permeability (heart, kidney, lung, and liver) and increased expression of B1R in heart and kidney (mRNA) and platelets (protein). Lethal septic shock induced by LPS was enhanced in STZ-diabetic rats and was associated with lung and kidney damage, including platelet micro-aggregate formation. SSR240612 prevented all these abnormalities as well as STZ-induced hyperglycemia and LPS-induced hyperthermia. Similarly to SSR240612, blockade of iNOS and COX-2 improved survival. Data provide the first evidence that kinin B1R plays a primary role in lethal thrombosis in a rat model of septic shock in diabetes. Pharmacological rescue was made possible with B1R antagonism or by inhibition of iNOS and COX-2, which may act as downstream mechanisms.

Antioxidant/oxidant status and cardiac function in bradykinin B(1)- and B(2)-receptor null mice.

Kinin-vasoactive peptides activate two G-protein-coupled receptors (R), B(1)R (inducible) and B(2)R (constitutive). Their complex role in cardiovascular diseases could be related to differential actions on oxidative stress. This study investigated impacts of B(1)R or B(2)R gene deletion in mice on the cardiac function and plasma antioxidant and oxidant status. Echocardiography-Doppler was performed in B(1)R (B(1)R(-/-)) and B(2)R (B(2)R(-/-)) deficient and wild type (WT) adult male mice. No functional alteration was observed in B(2)R(-/-) hearts. B(1)R(-/-) mice had significantly lowered fractional shortening and increased isovolumetric contraction time. The diastolic E and A waves velocity ratio was similar in all mice groups. Thus B(1)R(-/-) mice provide a model of moderate systolic dysfunction, whereas B(2)R(-/-) mice displayed a normal cardiac phenotype. Plasma antioxidant capacity (ORAC) was significantly decreased in both B(1)R(-/-) and B(2)R(-/-) mice whereas the vitamin C levels were decreased in B(2)R(-/-) mice only. Plasma ascorbyl free radical was significantly higher in B(1)R(-/-) compared to WT and B(2)R(-/-) mice. Therefore, the oxidative stress index, ascorbyl free radical to vitamin C ratio, was increased in both B(1)R(-/-) and B(2)R(-/-) mice. Hence, B(1)R and B(2)R deficiency are associated with increased oxidative stress, but there is a differential imbalance between free radical production and antioxidant defense. The interrelationship between the differential B(1)R and B(2)R roles in oxidative stress and cardiovascular diseases remain to be investigated.

Ondansetron does not block paracetamol-induced analgesia in a mouse model of fracture pain.

BACKGROUND: The aim of this study was to assess any interaction between ondansetron and paracetamol on a model of post-fracture pain in mice. METHODS: In protocol A, after fracture of the tibia, mice were assigned to four groups: paracetamol 30 mg kg⁻¹, paracetamol 50 mg kg⁻¹, paracetamol 100 mg kg⁻¹, or a saline vehicle i.p. In protocol B, after fracture of the tibia, mice were randomized to receive either paracetamol (100 mg kg⁻¹) plus saline (vehicle), paracetamol (100 mg kg⁻¹) plus ondansetron (1 mg kg⁻¹), paracetamol (100 mg kg⁻¹) plus ondansetron (2 mg kg⁻¹), saline plus ondansetron (2 mg kg⁻¹), or saline plus saline i.p. Three tests were used to assess pain behaviour: von Frey filament application, hot-plate test, and a subjective pain scale. Rescue analgesia with morphine was administered as necessary. RESULTS: In protocol A, paracetamol (100 mg kg⁻¹)-treated animals had less mechanical nociception, thermal nociception, and a lower subjective pain scale rating, when compared with those receiving paracetamol at 30 or 50 mg kg⁻¹ or saline [ED50 paracetamol=46.3 (6.34) mg kg⁻¹]. No difference was found between paracetamol (30 mg kg⁻¹) and saline-treated animals. In protocol B, the mechanical withdrawal threshold, the thermal withdrawal latency, and the subjective pain scale were lower after injection of paracetamol (100 mg kg⁻¹)+saline, paracetamol (100 mg kg⁻¹)+ondansetron (1 mg kg⁻¹), and paracetamol (100 mg kg⁻¹)+ondansetron (2 mg kg⁻¹), whereas in mice receiving saline+ondansetron (2 mg kg⁻¹) or saline+saline, there was no difference. CONCLUSION: We found that paracetamol 100 mg kg⁻¹ blocked the development of hyperalgesia and allodynia after fracture pain and ondansetron did not modify the antinociceptive effect of paracetamol in this model.

Acute and sub-chronic oral toxicity studies of an aqueous stem bark extract of Pterocarpus soyauxii Taub (Papilionaceae) in rodents.

UNLABELLED: Pterocarpus soyauxii Taub (Papilionaceae) is used in Cameroonian traditional medicine and pharmacopoeia to treat hypertension, diabetes, gastrointestinal parasitizes and cutaneous diseases. AIM OF THE STUDY: The present investigation was carried out to evaluate the safety of an aqueous stem bark extract of Pterocarpus soyauxii by determining toxicity after acute and sub-chronic oral administration in male and female rodents. MATERIALS AND METHODS: The acute toxicity test was conducted in mice. An aqueous extract of barks was administrated by gavage in single doses of 2.5-12.5 g/kg. General behaviour and mortality were examined for up to 7 days. The sub-chronic toxicity test was performed in rats. The plant extract was administered by daily gavage of 150-600 mg/kg for 42 days. Body weight, food and water intakes were followed weekly. Haematological, biochemical and organ parameters were determined at the end of the 42-day administration. RESULTS: In the acute study in mice, oral administration of the aqueous extract of Pterocarpus soyauxii caused dose-dependent general behaviour adverse effects and mortality. The no-observed adverse effect level (NOAEL) of the extract was 5.0 g/kg. The lowest-observed adverse effect level (LOAEL) was 7.5 mg/kg. Mortality increased with the dose, LD(50) was>10.75 g/kg for the mouse. In the sub-chronic study in rats, daily oral administration of the aqueous extract of Pterocarpus soyauxii did not result in death or significant changes in haematological or biochemical parameters, excepted increased hepatic catalase activity (P<0.05) at the dose of 600 mg/kg. No alteration was observed in body weight, food and water intake. Liver, kidney, lung and pancreas histopathology did not reveal morphological alteration. CONCLUSIONS: The results showed that the aqueous stem bark extract of Pterocarpus soyauxii Taub had very low toxicity in oral acute high dose administration and no toxicity in oral sub-chronic low dose administration and indicate that the plant could be considered safe for oral medication.

Hypoglycaemic effects of Mammea africana (Guttiferae) in diabetic rats.

AIM OF THE STUDY: The stem bark of Mammea africana Sabine (Guttiferae) is used in African rain forest to treat various diseases, including diabetes mellitus. We investigated whether Mammea africana extract induced hypoglycaemic activity in rats. MATERIALS AND METHODS: We tested the effects of acute (5h) and sub-acute (21 days) oral administrations of the CH(2)Cl(2)-MeOH stem bark extract of Mammea africana (19-300 mg/kg body weight) on blood glucose levels of normal and streptozotocin (STZ)-induced type 1 diabetic rats. The effects were compared with those of glibenclamide. RESULTS: Acute administration reduced blood glucose in the diabetic rats only (33.87%, P<0.01). Sub-acute treatment for 21 days also reduced blood glucose level in diabetic rats (73.29%, P<0.01). A reduction or stabilization in total serum protein, triglyceride, cholesterol and alanine amino transferase levels was also observed. No effect was observed on body weight loss but food and water intakes were significantly reduced (P<0.01) in diabetic rats. The maximal anti-diabetic effect was obtained with the dose of 75 mg/kg and was more important than that of glibenclamide. CONCLUSION: It can be concluded that extracts of Mammea africana exhibited a significant anti-hyperglycaemic activity and improved the metabolic alterations in STZ-diabetic rats. These results provide a rationale for the use of Mammea africana to treat diabetes mellitus and hypercholesterolemia.

Opioid-induced hyperalgesia in a mice model of orthopaedic pain: preventive effect of ketamine.

BACKGROUND: The aim of this study was to assess the preventative effect of ketamine on the exaggerated postoperative pain observed in sufentanil-treated mice and its ability to improve the analgesic effectiveness of morphine during the postoperative period in an orthopaedic model of pain. METHODS: In this study, we assessed the effects of ketamine on sufentanil enhancement of pain behaviour induced by fracture and the effects of ketamine on postoperative morphine-induced analgesia. Three tests were used to assess pain behaviour: von Frey filament application, hot-plate test, and a subjective pain scale. RESULTS: When administered 1 day after surgery in mice treated with sufentanil on D0 (before surgery), morphine induced an analgesic effect as observed by the nociceptive threshold increase in saline- and ketamine-treated mice. Morphine was more effective in ketamine-treated (1 and 50 mg kg(-1)) mice. CONCLUSIONS: Our results suggest that pre-emptive use of ketamine is useful in orthopaedic surgery in this mice model to diminish short- and long-term hyperalgesia, but also to improve morphine effectiveness leading to a better mobilization and more rapid rehabilitation.

Hemodynamic and renal involvement of B1 and B2 kinin receptors during the acute phase of endotoxin shock in mice.

B1 kinin receptor (B1R) is up-regulated by endotoxins and thus may represent a therapeutic target in sepsis. We investigated the expression and role of B1R and B2R in the acute phase of lipopolysaccharide (LPS)-induced endotoxin shock in C57BL/6 mice (WT) and B1R and B2R knock out mice (B1KO, B2KO). B1R mRNA was enhanced from 6 to 48 h after LPS while B2R mRNA was further increased in B1KO. Maximal hypotension was found 24 h after LPS, and was more pronounced in B1KO, but was reduced in B2KO. Glomerular filtration rate was more reduced by LPS in B1KO than in WT and B2KO. Glycemia was reduced by LPS and particularly in B1KO and B2KO mice. Mortality was increased by LPS in B1KO. These data suggest that the up-regulated B1R plays, at least transiently, a significant beneficial role in acute LPS-induced hypotension. Conversely, supra activation of B2R could be also involved in the increased mortality observed in B1KO mice.

Modulation by bradykinin and nitric oxide of angiotensin II-induced apoptosis in a vascular smooth muscle cell phenotype.

There is evidence for a clinical benefit of ACE inhibitors or AT1 antagonists in cardiovascular diseases with deleterious smooth muscle cells (SMC) apoptosis. We have previously shown that angiotensin II (Ang II) induces a phenotype-dependent SMC apoptosis. We asked whether bradykinin (BK) and nitric oxide (NO) could modulate Ang II-induced SMC apoptosis. BK alone did not induce significant apoptosis in either spindle (Sp-SMC) or epithelioid (Ep-SMC) SMC phenotypes cultured in serum reduction, but phenotype-dependently, reduced cell proliferation. Pretreatment with BK partly impaired Ang II-induced reduction of Ep-SMC culture viability and partly prevented apoptotic features. Pretreatment with sodium nitroprusside completely prevented all Ang II-induced deleterious effects in Ep-SMC, i. e. reduction of culture viability, Annexin V binding, nuclear condensation and cell fragmentation. These findings indicate that the BK-NO system may phenotype-dependently modulate SMC survival and in particular may oppose, mostly by NO, Ang II-induction of apoptosis in the Ep-SMC phenotype.

Angiotensin II induces phenotype-dependent apoptosis in vascular smooth muscle cells.

Angiotensin II regulates vascular structure through growth and apoptosis, with implications in pathophysiology. Subtypes of vascular smooth muscle cells with specific morphology, growth, or apoptotic features have been isolated. Here, we investigated the effects of angiotensin II on apoptosis of 2 morphologically different rat aortic smooth muscle cell phenotypes. Spindle and epithelioid cell lines cultured under low serum conditions were stimulated by angiotensin II. Responsiveness was evaluated by calcium signaling. In both phenotypes, an angiotensin II type 1 receptor-mediated transient intracellular calcium peak arose from intracellular pools. However, a sustained nifedipine-sensitive calcium entry occurred specifically in epithelioid cells. Angiotensin II did not impair spindle cell survival, whereas a delayed reduction in cell number occurred in epithelioid cells. Cell death through apoptosis was characterized by cellular and nuclear morphology. Consistently, DNA fragmentation, evaluated by biochemical quantification, nuclei staining, and ladders, and caspase 3-like activity were promoted by angiotensin II in epithelioid cells. Kinetics of annexin V binding showed that apoptosis was a delayed process. Angiotensin II-induced apoptosis of epithelioid cells was prevented by angiotensin II type 1 but not type 2 receptor antagonists and was inhibited by a calcium chelator or calcium antagonist. Conversely, epithelioid cell apoptosis could be induced by a calcium ionophore. Thus, the death signaling promoted by angiotensin II in epithelioid cells involves type 1 receptor-mediated calcium entry. These data suggest that angiotensin II can promote angiotensin II type 1 receptor-mediated apoptosis in vascular smooth muscle cells, depending on their phenotype. This process may play a role in vascular remodeling in cardiovascular diseases.

Bradykinin-induced inhibition of cell proliferation and tyrosine kinase activity in rat mesangial cells.

The relationship between cell proliferation, protein tyrosine phosphorylation, phosphotyrosine kinase activity and bradykinin receptor activation in rat mesangial cells was investigated. We demonstrated that bradykinin (BK), through the B2 receptor, induced a dose-dependent inhibition of mesangial cell proliferation stimulated by fetal calf serum. We next found that BK induced a dose-dependent inhibition of phospho-tyrosine kinase activity. Treatments with pertussis-toxin, inhibition of phospholipase C and protein kinase C inhibitors and chelation of free cytosolic calcium did not change the bradykinin-induced inhibition of phosphotyrosine kinase. Western blot analysis of phosphotyrosinated proteins demonstrated that BK reduced tyrosine phosphorylation of several proteins among which we identified the 125-focal adhesion kinase. Taken together, these data suggest for the first time that, in proliferating rat mesangial cells, B2 receptor stimulation is able to induce, via a pertussis insensitive pathway, the inhibition of tyrosine kinase activity and mesangial cell proliferation.

Homologous and heterologous induction of the human bradykinin B1-receptor and B1-receptor localisation along the rat nephron.

The kinin B1-receptor which is absent or expressed at very low levels under physiological conditions is strongly induced under inflammatory conditions. It has been shown that B1-receptor induction during inflammation involves interleukin-1beta (IL-1beta) production and activation of nuclear factor-kappaB (NF-kappaB). Since bradykinin (BK), the B2-receptor agonist induces IL-1beta expression and activates NF-kappaB, we have analysed the effect of B2-receptor activation in cultured human lung fibroblasts cells on B1-receptor expression by a semiquantitative RT-PCR analysis. Treatment with BK resulted in a significant increase in the expression of B1-receptor mRNA which was abolished by a specific B2-receptor antagonist. This result suggests that B2-receptor activation can prime the expression of B1-receptors. Although the renal localisation of the B2-receptor has been thoroughly studied, nothing is known about the distribution of the B1-receptor in the kidney. Using a combination of microdissection and a semiquantitative RT-PCR/Southern blot analysis we showed the absence of B1-receptors under physiological conditions in 10 microdissected rat nephron segments. However, 18 h LPS-treatment induced significant expression of the B1-receptor in all, but one segment. These studies provide the first molecular basis for the observed changes in renal haemodynamics after B1-agonist infusion in animal kidney models.

Effect of bradykinin on tyrosine kinase and phosphatase activities and cell proliferation in mesangial cells.

We investigated the relationship between protein tyrosine phosphorylation and bradykinin (BK) receptor activation in rat mesangial cells (MC). Stimulation of the B2 receptor resulted in a dual effect consisting of an independent activation and inhibition of tyrosine kinase activity (TKA). The activation was rapid and transient, reaching a peak value at 30 s whereas the inhibition was observed at 5 min and persisted up to 10 min. Treatments with pertussis-toxin and U73122 showed that only the BK-induced stimulation of TKA is dependent on phospholipase C activation via a pertussis-toxin sensitive G-protein. In addition, BK induced an increase in tyrosine phosphatase activity. Western-blot analysis demonstrated that the dual effect of BK on TKA was associated with both an increase and a decrease in tyrosine phosphorylation of the p125-focal adhesion kinase (FAK). Moreover, BK was able to reduce the maximal stimulated MC cell proliferation induced by fetal calf serum. These data show that in rat MC, B2 receptor stimulation activates and inhibits two independent tyrosine kinase signaling pathways associated with tyrosine phosphorylation of p125-FAK that might be implicated in MC proliferation.

Serotonin metabolism in rat mesangial cells: involvement of a serotonin transporter and monoamine oxidase A.

BACKGROUND: Serotonin is one of the factors regulating mesangial cell proliferation, and convergent evidence supports its involvement in the development of glomerulonephritis. In this study, we identified a serotonin transporter and the amine-degrading enzyme monoamine oxidases (MAOs) in mesangial cells, and we studied their involvement in serotonin degradation. METHODS: MAOs were characterized in membrane preparations and intact mesangial cells by enzyme assay using [14C]5-hydroxytryptamine and [14C]beta-phenylethylamine as specific substrates for MAO-A and MAO-B, respectively, and by Western blot analysis. The expression of a serotonin transporter was determined by [14C]5-hydroxytryptamine uptake experiments and Western blot. Mesangial cell proliferation was measured by BrdU incorporation. RESULTS: Quantitation of the MAO isoforms by enzyme assay and Western blot analysis showed that MAO-A was largely predominant in mesangial cells, accounting for approximately 90% of the total enzyme population. The MAO substrate [14C]serotonin was transported into mesangial cells by a saturable uptake system (Vmax 310 +/- 36 pmol/30 min/mg protein; Km 5.9 +/- 1.4 microM) displaying the pharmacological properties of a serotonin transporter. The expression of a serotonin transporter was confirmed by Western blot analysis. MAO activity measured in intact cells showed that after accumulation into mesangial cells, [14C]serotonin was metabolized by MAO-A. Finally, serotonin-mediated mesangial cell proliferation was significantly increased after irreversible MAO inhibition. CONCLUSIONS: Our results suggest that serotonin concentration and function in glomeruli may be regulated in part by its transport into mesangial cells and degradation by MAO-A.

Inhibition of cGMP accumulation in mesangial cells by bradykinin and tyrosine kinase inhibitors.

We examined the effect of bradykinin (BK) on the accumulation of cGMP of the mesangial cell (MC), a smooth muscle-like cell of the renal glomerulus. BK caused a time- and concentration dependent reduction of the cGMP concentration. In addition, BK inhibited total protein tyrosine kinase (PTK) activity. Two tyrosine kinase inhibitors (TKI) genistein and tyrphostin also reduced the cGMP concentration. The inhibition of BK and TKI were not additive. The inhibition of PTK by BK, mediated through activation of the B2-receptor, was unaffected by inhibitors of Gi/o proteins, phospholipase C, protein kinase C, cyclooxygenase and Ca2+ release from intracellular stores. Only IBMX a broad spectrum inhibitor of phosphodiesterases (PDE) and 8-methoxymethyl IBMX a specific type-1 PDE inhibitor prevented the inhibitory effects of BK and TKI indicating the involvement of type-1 PDE. In addition, BK had no effect on soluble guanylate cyclase (sGC) and nitric oxide synthase activity. In freshly isolated glomeruli, which represent the physiological environment of MC, BK also reduced the cGMP concentration. Like in MC, the inhibitory effect was suppressed by IBMX. These data demonstrate that BK suppresses a PTK-dependent pathway of cGMP production in rat MC at a level downstream of NO synthase and sGC. It is suggested that BK and TKI inhibitors decrease cGMP levels by preventing tyrosine phosphorylation of type-1 PDE activity, thereby leading to enzyme activation.

Effects of lysophosphatidic acid on proliferation and cytosolic Ca++ of human adult vascular smooth muscle cells in culture.

Lysophosphatidic acid (LPA) is a lipid mediator generated by activated platelets and having various effects on numerous cell types. We investigated some effects of 1-oleyl LPA on vascular smooth muscle cells cultured from adult human normal arteries. At micromolar concentrations, LPA induced a mitogenic effect ([3H]-thymidine incorporation and cell proliferation) on quiescent cells, without an additional growth factor being required. This effect was equipotent to that of 10% fetal calf serum, and it was accompanied by early (5 minutes) and late (1-3 hours) phosphorylation of mitogenactivated protein kinase. LPA inhibited cell migration through collagen coated membranes, with or without platelet-derived growth factor BB as chemoattractant. LPA induced a typical biphasic Ca2+ signal response made up of a rapid first phase due to Ca2+ release from intracellular stores followed by a second wave due to external Ca2+ influx. These findings support the proposal that LPA released from activated platelets is a mediator for smooth muscle cell response at the site of vessel injury in humans.

Renal bradykinin receptors: localisation, transduction pathways and molecular basis for a possible pathological role (review).

Kinins are biologically active peptides that exert their effects by activating two seven transmembrane G-protein coupled receptors termed B1 and B2 which have only about 36% of homology. The major kinin peptide under physiological conditions, bradykinin (BK), modulates renal haemodynamics and function. Under physiological conditions most BK effects involve bradykinin B2-receptors. Studies on the intra-cellular transduction pathways, the regulation of the expression and the localisation of these receptors along the nephron, as well as the first studies on transgenic mice models, have allowed to better define the role of these receptors under physiological and pathological conditions. The role of the renal B1-receptor, induced in a variety of pathologies related to inflammation, is poorly understood. Recent investigations on the molecular mechanism of B1-receptor induction and its detailed renal localisation have shown that under inflammatory conditions this kinin receptor might be of importance. B2-receptors are suggested to be involved in part of the renoprotective effects of angiotensin converting enzyme (ACE)-inhibitors in insulin-dependent diabetes. However, ACE-inhibitor treatment, resulting also in an increased B1-agonist concentration might result in homologous induction and activation of the B1-receptor.

Differential induction of functional B1-bradykinin receptors along the rat nephron in endotoxin induced inflammation.

BACKGROUND: Under physiological conditions, the effects of kinins in the kidney are mainly mediated by the bradykinin B2-receptor, whereas the kinin B1-receptor is strongly induced under inflammatory conditions in a variety of tissues. Knowledge of the distribution of the B1-receptor along the nephron is of importance since the B1-receptor might replace B2-receptors under these conditions. METHODS: Using a RT-PCR/Southern blot approach allowing relative quantification of mRNA levels, ten different microdissected rat nephron segments were analyzed for the presence of the B1- and B2-receptor before and after endotoxin treatment to induce experimental inflammation. The functionality of the expressed receptors was assessed by kinin-induced intracellular calcium ([Ca2+]i) mobilization in microdissected nephron segments. RESULTS: While under physiological conditions no B1-receptor mRNA could be detected, after 18 hours of treatment with bacterial lipopolysaccharide (LPS) the expression of B1-receptor mRNA was strongly induced in the efferent arteriole, the medullary and inner medullary thin limb, and in the distal tubule. Moderate expression was found in the glomerulus, proximal convoluted and straight tubules, and in the medullary thick ascending limb. Small but detectable expression was observed in the cortical collecting duct. The induction of B1-receptor mRNA expression resulted in functional receptor expression, since increases in [Ca2+]i were observed upon B1-agonist stimulation. LPS treatment also increased the expression of B2-receptor mRNA in all nephron segments except in the glomerulus, the inner medullary thin limb and the outer medullary collecting duct. However, no related changes in B2-agonist induced rises in [Ca2+]i were found. CONCLUSIONS: These studies show a functional induction of the B1-kinin receptor along the rat nephron, which should be taken in account to address the effects of kinins under inflammatory conditions in the kidney.

The B1-agonist [des-Arg10]-kallidin activates transcription factor NF-kappaB and induces homologous upregulation of the bradykinin B1-receptor in cultured human lung fibroblasts.

The bradykinin B1-receptor is strongly upregulated under chronic inflammatory conditions. However, the mechanism and reason are not known. Because a better understanding of the mechanism of the upregulation will help in understanding its potential importance in inflammation, we have studied the molecular mechanism of B1-receptor upregulation in cultured human lung fibroblasts (IMR 90) in response to IL-1beta and the B1-agonist [des-Arg10]-kallidin. We show that treatment of human IMR 90 cells by IL-1beta stimulates the expression of both B1-receptor mRNA and protein. The latter was studied by Western blot analysis using antipeptide antibodies directed against the COOH-terminal part of the human B1-receptor. We furthermore report the novel observation that the B1-receptor is upregulated by its own agonist which was completely blocked by the specific B1-antagonist [des-Arg10-Leu9]-kallidin, indicating an upregulation entirely mediated through cell surface B1-receptors. The increased population of B1-receptors was functionally coupled as exemplified by an enhancement of the B1-agonist induced increase in free cytosolic calcium. Upregulation by the B1-agonist was blocked by a specific protein kinase C inhibitor. B1-agonist-induced upregulation was correlated to the induction of transcription factor nuclear factor kappaB (NF-kappaB) which efficiently bound to the NF-kappaB-like sequence located in the promoter region of the human B1-receptor gene. This correlation was further confirmed by reporter gene assays which showed that this NF-kappaB-like sequence, in the B1-receptor promoter context, could contribute to IL-1beta and DLBK-induced B1-receptor transcription activation, and by the effect of NF-kappaB inhibitor pyrrolidinedithiocarbamate which diminished both B1-receptor upregulation and NF-kappaB activation. NF-kappaB is now recognized as a key inflammatory mediator which is activated by the B1-agonist but which is also involved in B1-receptor upregulation.

B2 kinin receptor upregulation by cAMP is associated with BK-induced PGE2 production in rat mesangial cells.

In the rat mesangial cell (MC), activation of the bradykinin B2 receptor (B2R) by bradykinin (BK) is associated with both phospholipase C (PLC) and A2 (PLA2) activities and with inhibition of adenosine 3',5'-cyclic monophosphate (cAMP) formation leading to cell contraction. Because cAMP plays an important role in the regulation of gene expression in general, we investigated the effect of increasing the intracellular cAMP concentration ([cAMP]i) in mesangial cells on the B2 mRNA expression, on the density of B2 receptor binding sites, on the BK-induced increase in both the free cytosolic Ca2+ concentration ([Ca2+]i), and in the prostaglandin E2 (PGE2) production. Forskolin, PGE2, and cAMP analog, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), were used to increase [cAMP]i. Twenty-four-hour treatment with forskolin, PGE2, and 8-BrcAMP resulted in significant increases in B2 receptor binding sites, which were inhibited by cycloheximide. The maximum B2 receptor mRNA expression (160% above control) was observed in cells treated during 24 h with forskolin and was prevented by actinomycin D. In contrast, the D-myo-inositol 1,4,5-trisphosphate (IP3) formation and the BK-induced increase in [Ca2+]i, reflecting activation of PLC, were not affected by increased levels of [cAMP]i. However the BK-induced PGE2 release, reflecting PLA2 activity, was significantly enhanced. These data bring new information regarding the dual signaling pathways of B2 receptors that can be differentially regulated by cAMP.

Cyclosporine A-induced increase in glomerular cyclic GMP in rats and the involvement of the endothelinB receptor.

1. A transient two fold increase in the cyclic GMP content was observed in rat freshly isolated glomeruli 6 to 9 h after a single subcutaneous injection of 20 mg kg-1 cyclosporine A (CsA) in conscious animals. 2.In vitro stimulation with endothelin 3 (ET-3) of isolated glomeruli obtained from CsA-untreated rats resulted in a dose-dependent increase in cyclic GMP content. The increase observed with 10 nM ET-3 was similar to that observed in glomeruli isolated 9 h after in vivo CsA administration. 3. The rise in glomerular cyclic GMP content after in vivo CsA injection was prevented by in vivo treatment with L-NAME (10 mg kg-1) or by in vitro calcium deprivation of the incubation medium. 4. The stimulating effects of CsA on glomerular cyclic GMP content were inhibited by in vivo administration of the ETB receptor antagonist BQ-788 (2 mg kg-1) but not by the ETA receptor antagonist BQ-123 (2 mg kg-1). 5. The maximum increase in glomerular cyclic GMP content induced in vitro by acetylcholine (100 microM) and by ET-3 (100 nM) was slightly lower (approximately by 20-25%; P < 0.05) in glomeruli from CsA-treated rats than in glomeruli from untreated rats. In contrast, the maximum increase achieved with 1 microM sodium nitroprusside was similar in both groups. 6. A single subcutaneous injection of CsA did not significantly alter the glomerular mRNA expression of constitutive endothelial NO synthase (eNOS), as evaluated by RT-PCR, whereas the mRNA expression of the inducible NO synthase (iNOS), which follows pretreatment with lipopolysaccharide, was prevented. 7. These results indicate that in vivo administration of a single dose of cyclosporine A transiently increases the cyclic GMP content of freshly isolated glomeruli, and that activation of ETB receptors and stimulation of the NO pathway are involved in this process. Furthermore, a single administration of CsA does not impair eNOS mRNA expression and only slightly reduces NO-dependent glomerular cyclic GMP production.