Bichoy H. Gabra

Beneficial effect of chronic treatment with the selective bradykinin B1 receptor antagonists, R-715 and R-954, in attenuating streptozotocin-diabetic thermal hyperalgesia in mice

Kinins are important mediators of cardiovascular homeostasis, inflammation and nociception. Bradykinin (BK) B(1) receptors (BKB1-R) are over-expressed in pathological conditions including diabetes, and were reported to play a role in hyperglycemia, renal abnormalities, and altered vascular permeability associated with type 1 diabetes. Recent studies from our laboratory demonstrated that BKB1-R are implicated in streptozotocin (STZ)-diabetes-mediated hyperalgesia, since acute administration of the selective BKB1-R antagonists significantly and dose-dependently inhibited such hyperalgesic activity. In the present study, we examined the effect of chronic treatment of STZ-diabetic mice with the selective BKB1-R agonist desArg9bradykinin (DBK) and two specific antagonists R-715 and R-954, on diabetic hyperalgesia. Diabetes was induced in male CD-1 mice by injecting a single high dose of STZ (200mg/kg, i.p.) and nociception was assessed using the hot plate, plantar stimulation, tail immersion and tail flick tests. Drugs were injected i.p. twice daily for 7 days, starting 4 days after STZ. We showed that chronically administered R-715 (400 micrograms/kg) and R-954 (200 micrograms/kg), significantly attenuated the hyperalgesic effect developed in STZ-diabetic mice as measured by the four thermal nociceptive tests. Further, chronic treatment with DBK (400 micrograms/kg) produced a marked potentiation of the hyperalgesic activity, an effect that was reversed by both R-715 and R-954. The results from this chronic study confirm a pivotal role of the BKB1-R in the development of STZ-diabetic hyperalgesia and suggest a novel approach to the treatment of this short-term diabetic complication using BKB1-R antagonists.

Role of bradykinin B1 receptors in diabetes-induced hyperalgesia in streptozotocin-treated mice

Insulin-dependent diabetes mellitus (type-1 diabetes) is an inflammatory autoimmune disease associated with vascular permeability changes leading to many complications including nephropathy, retinopathy, hypertension, hyperalgesia and neuropathy. The bradykinin B(1) receptor was recently found to be upregulated during the development of the diabetes and to be involved in its complications. Kinins are known to be important mediators of a variety of biological effects including cardiovascular homeostasis, inflammation and nociception. In the present study, we studied the effect of the selective B(1) receptor agonist, des-Arg(9)-bradykinin, and its specific antagonists, Ac-Lys-[D-beta Nal(7), Ile(8)]des-Arg(9)-bradykinin (R-715) and Ac-Orn-[Oic(2), alphaMe Phe(5), D-beta Nal(7), Ile(8)]des-Arg(9)-bradykinin (R-954), on diabetic hyperalgesia. Diabetes was induced in male CD-1 mice by injecting a single high dose of streptozotocin (200 mg kg(-1), i.p.) and the nociception was assessed using the hot plate and the tail flick tests, 1 week following the injection of streptozotocin. Our results showed that induction of diabetes by streptozotocin provoked a marked hyperalgesia in diabetic mice expressed as about 11% decrease in hot plate reaction time and 26% decrease in tail flick reaction time. Following acute administration of R-715 (200-800 microg kg(-1), i.p.) and R-954 (50-600 microg kg(-1), i.p.), this hyperalgesic activity was blocked and the hot plate and tail flick latencies of diabetic mice returned to normal values observed in control healthy mice. In addition, the acute administration of des-Arg(9)-bradykinin (200-600 microg kg(-1), i.p.) significantly potentiated diabetes-induced hyperalgesia, an effect that was totally reversed by R-715 (1.6-2.4 mg kg(-1), i.p.) and R-954 (0.8-1.6 mg kg(-1), i.p.). These results provide a major evidence for the implication of the bradykinin B(1) receptors in the development of hyperalgesia associated with diabetes and suggest a novel approach to the treatment of this diabetic complication using the bradykinin B(1) receptor antagonists.

Absence of diabetic hyperalgesia in bradykinin B1 receptor-knockout mice

Experimental evidence has shown that the inducible bradykinin (BK) B1 receptor (BKB1-R) subtype is involved in the development of hyperalgesia associated with type 1 diabetes. Selective BKB1-R antagonists inhibited, whereas selective agonists increased the hyperalgesic activity in diabetic mice in thermal nociceptive tests. Here we evaluate the development of diabetic hyperalgesia in a BKB1-R-knockout (KO) genotype compared to wild-type (WT) mice. The BKB1-R-KO mice were backcrossed for 10 generations to C57BL/6 genetic background before use in the experiments. Diabetes was induced by streptozotocin (STZ) and thermal nociception was assessed by the hot plate and tail immersion tests. The hyperalgesia observed in wild type mice was totally absent in the BKB1-R-KO mice. Furthermore, the selective BKB1-R agonist, desArg9BK, significantly increased the hyperalgesic activity in diabetic WT mice but had no effect on nociceptive responses in diabetic BKB1-R-KO mice. Taken together, the results confirm the crucial role of the BKB1-R, upregulated alongside inflammatory diabetes, in the development of diabetes-induced hyperalgesia.

Kinin B1 receptor antagonists inhibit diabetes-induced hyperalgesia in mice

Insulin-dependent diabetes mellitus (type 1 diabetes) is an inflammatory autoimmune disease associated with vascular permeability changes leading to many complications including nephropathy, retinopathy, neuropathy, hypertension and hyperalgesia. The bradykinin B(1) receptors (BKB(1)-R) were recently found to be upregulated alongside the development of type 1 diabetes and to be involved in its complications. Kinins are important mediators of a variety of biological effects including cardiovascular homeostasis, inflammation and nociception. In the present study, we studied the effect of a selective BKB(1)-R agonist desArg(9)-BK (DBK) and two selective receptor antagonists, the R-715 (Ac-Lys-[D-beta Nal(7), Ile(8)] desArg(9)-BK) and the R-954 (Ac-Orn-[Oic(2), alphaMe Phe(5), D-beta Nal(7), Ile(8)] desArg(9)-BK) on diabetic hyperalgesia. Type 1 diabetes was induced in male CD-1 mice via a single injection of streptozotocin (STZ, 200mg/kg, i.p.), one week before the test. Nociception, a measure of hyperalgesia, was assessed using the plantar stimulation (Hargreaves) and the tail-immersion tests. The induction of type 1 diabetes provoked a significant hyperalgesic activity in diabetic mice, causing an 11% decrease in plantar stimulation reaction time and 13% decrease in tail-immersion reaction time, compared to normal mice. Following acute administration of R-715 (100-600 microg/kg, i.p.), or R-954 (50-400 microg/kg, i.p.), the STZ-induced hyperalgesic activity was blocked in a dose-dependent manner and the hot plate and tail-immersion latencies of diabetic mice returned to normal values observed in control healthy mice. In addition, the acute administration of DBK (400 microg/kg, i.p.) significantly potentiated diabetes-induced hyperalgesia, an effect that was totally reversed by R-715 (1.6-2.4 mg/kg, i.p.) and R-954 (0.8-1.2mg/kg, i.p.). These results provide further evidence for the implication of the BKB(1)-R in type 1 diabetic hyperalgesia and suggest a novel approach in the treatment of this complication using the BKB(1)-R antagonists.

Enhanced dermal and retinal vascular permeability in streptozotocin-induced type 1 diabetes in Wistar rats: blockade with a selective bradykinin B1 receptor antagonist.

The vascular complications associated with type 1 diabetes are to some extent related to the dysfunction of the endothelium leading to an increased vascular permeability and plasma extravasation in the surrounding tissues. The various micro- and macro-vascular complications of diabetes develop over time, leading to nephropathy, retinopathy and neuropathy and cardiomyopathy. In the present study, the effect of a novel selective bradykinin B1 receptor (BKB1-R) antagonist, R-954, was investigated on the changes of vascular permeability in the skin and retina of streptozotocin (STZ)-induced type 1 diabetic rats. Plasma extravasation increased in the skin and retina of STZ-diabetic rats after 1 week and persisted over 4 weeks following STZ injection. Acute treatment with R-954 (2 mg/kg, bolus s.c.) highly reduced the elevated vascular permeability in both 1- and 4-week STZ-diabetic rats. These results showed that the inducible BKB1-R subtype modulates the vascular permeability of the skin and retina of type 1 diabetic rats and suggests that BKB1-R antagonists could have a beneficial role in diabetic neuropathy and retinopathy.

The kinin system mediates hyperalgesia through the inducible bradykinin B1 receptor subtype: evidence in various experimental animal models of type 1 and type 2 diabetic neuropathy.

Abstract Both insulin-dependent (type 1) and insulin-independent (type 2) diabetes are complex disorders characterized by symptomatic glucose intolerance due to either defective insulin secretion, insulin action or both. Unchecked hyperglycemia leads to a series of complications among which is painful diabetic neuropathy, for which the kinin system has been implicated. Here, we review and compare the profile of several experimental models of type 1 and 2 diabetes (chemically induced versus gene-prone) and the incidence of diabetic neuropathy upon aging. We discuss the efficacy of selective antagonists of the inducible bradykinin B1 receptor (BKB1-R) subtype against hyperalgesia assessed by various nociceptive tests. In either gene-prone models of type 1 and 2 diabetes, the incidence of hyperalgesia mostly precedes the development of hyperglycemia. The administration of insulin, achieving euglycemia, does not reverse hyperalgesia. Treatment with a selective BKB1-R antagonist does not affect basal nociception in most normal control rats, whereas it induces a significant time- and dose-dependent attenuation of hyperalgesia, or even restores nociceptive responses, in experimental diabetic neuropathy models. Diabetic hyperalgesia is absent in streptozotocin-induced type 1 diabetic BKB1-R knockout mice. Thus, selective antagonism of the inducible BKB1-R subtype may constitute a novel and potential therapeutic approach for the treatment of painful diabetic neuropathy.

Inhibition of Type 1 Diabetic Hyperalgesia in Streptozotocin-Induced Wistar versus Spontaneous Gene-Prone BB/Worchester Rats: Efficacy of a Selective Bradykinin B1 Receptor Antagonist

Insulin-dependent type 1 diabetes (T1D) is linked to a series of complications, including painful diabetic neuropathy (PDN). Several neurovascular systems are activated in T1D, including the inducible bradykinin (BK) B1 receptor (BKB1-R) subtype. We assessed and compared the efficacy profile of a selective BKB1-R antagonist on hyperalgesia in 2 models of T1D: streptozotocin (STZ) chemically induced diabetic Wistar rats and spontaneous BioBreeding/Worchester diabetic-prone (BB/Wor-DP) rats. Nociception was measured using the hot plate test to determine thermal hyperalgesia. STZ diabetic rats developed maximal hyperalgesia (35% decrease in their hot plate reaction time) within a week and remained in such condition and degree for up to 4 weeks postinjection. BB/Wor-DP rats also developed hyperalgesia over time that preceded hyperglycemia, starting at the age of 6 weeks (9% decrease in the hot plate reaction time) and stabilizing over the age of 16 to 24 weeks to a maximum (60% decrease in the hot plate reaction time). Single, acute subcutaneous administration of the selective BKB1-R antagonist induced significant time- and dose-dependent attenuation of hyperalgesia in both STZ diabetic and BB/Wor-DP rats. Thus, selective antagonism of the inducible BKB1-R subtype may constitute a novel and potential therapeutic approach for the treatment of PDN.

Implication of the bradykinin receptors in antigen‐induced pulmonary inflammation in mice

The involvement of bradykinin (BK) receptors in the allergic inflammation associated with airway hyper‐reactivity (AHR) was evaluated by means of the selective bradykinin B1 receptor (BKB1‐R) antagonists R‐715 (Ac‐Lys‐[D‐βNal7, Ile8]desArg9‐BK) and R‐954 (Ac‐Orn[Oic2, α‐MePhe5, D‐βNal7, Ile8]desArg9‐BK) or the selective bradykinin B2 receptor (BKB2‐R) antagonist HOE‐140 (D‐Arg0‐Hyp3‐Thi5‐D‐Tic7‐Oic8‐BK). Cellular migration and AHR were examined 24 h after the second ovalbumin (OA) challenge. R‐715 (10–500 μg kg−1) and R‐954 (1–100 μg kg−1) injected intravenously (i.v.), 5 min prior to aerosol OA challenges, decreased by approximately 50% the induced lung eosinophilia in OA‐sensitized mice but did not reduce AHR. HOE‐140 (1 μg kg−1) administered in the same manner, decreased mononuclear cell and eosinophil infiltration in the bronchoalveolar lavage fluid (BALF) of OA‐sensitized mice. Moreover, treatment of OA‐sensitized mice with HOE‐140 (100 μg kg−1) completely abolished the AHR to carbachol. The BKB1‐R agonist desArg9‐BK (DBK; 10–1000 μg kg−1) administered intratrachealy to normal mice had no effect on the basal cell counts recovered in BALF nor on the plasma extravasation, while the BKB2‐R selective agonist BK (20 μg kg−1) stimulated mononuclear cell migration, neutrophilia and plasma extravasation in normal mouse lungs. Such effects were inhibited by HOE‐140 (10 μg kg−1). Our results suggest that the airway inflammatory response induced by antigen challenge in mice is mediated by stimulation of both BKB1‐R and BKB2‐R.

Bradykinin Stimulates MMP-2 Production in Guinea Pig Tracheal Smooth Muscle Cells

The implication of bradykinin (BK) receptors in the release of the matrix metalloproteinase-2 (MMP-2; gelatinase A) was studied in guinea pig tracheal smooth muscle cells (GP-TSMC). Bradykinin (10−8–10−4 M) induced a time- and concentration-dependent upregulation of MMP-2 production from cultured GP-TSMC. Pretreatment of the GP-TSMC with the bradykinin B2 receptor (BKB-R) antagonist Hpp-HOE-140 (Hpp-D-Arg0-Hyp3-Thi5-D-Tic7-Oic8-BK; 10−8–10−4 M) significantly inhibited the BK-stimulated upregulation of MMP-2 in GP-TSMC in a concentration-related manner. Conversely, GP-TSMC pretreated with the selective bradykinin B1 receptor (BKB1-R) antagonist R-954 (Ac-Orn[Oic2, α-MePhe5, D-βNal7, Ile8]desArg9BK; 10−8–10−4 M) did not show any change in the response to BK. Moreover, the selective BKB2-R agonist Lys0BK (kallidin; 10−8–10−4 M) stimulated whereas the selective BKB1-R agonist desArg9BK (DBK; 10−8–10−4 M) had no effect on MMP-2 release from GP-TSMC. Further, the nonselective cyclooxygenase (COX) enzyme inhibitor indomethacin (IND; 10−5 M), the glucocorticosteroid dexamethasone (DEX; 1 ng/mL) and the protein synthesis inhibitors, cycloheximide (CHX; 10−6 M) and actinomycin D (ACT-D; 10−8 M) also inhibited BK-induced MMP-2 release from GP-TSMC. These results provide the first evidence for the involvement of BK in the release of MMP-2 from airway smooth muscle cells through activation of the BKB2-R. Such response is mostly mediated by the induction of COX and the subsequent production of endogenous prostaglandins (PGs). It could therefore be suggested that MMP-2 might play a role in the process of airway remodeling.

Des-Arg9-bradykinin increases intracellular Ca2+ in bronchoalveolar eosinophils from ovalbumin-sensitized and -challenged mice.

The effects of the selective bradykinin B1 receptor agonist, des-Arg9-bradykinin and the bradykinin B2 receptor agonist, bradykinin were studied on the intracellular free Ca2+ concentration ([Ca2+]i) in murine bronchoalveolar lavage cells from control and ovalbumin-sensitized mice using fura-2 microfluorimetry. The bronchoalveolar lavage cells of control mice, which were predominantly alveolar macrophages, showed an increase in [Ca2+]i in response to bradykinin (1 microM) but not to des-Arg9-bradykinin (1 microM), indicating the presence of functional bradykinin B2 receptors and the absence of B1 receptors. Such elevation in [Ca2+]i induced by bradykinin was totally inhibited by the selective bradykinin B2 receptor antagonist, D-Arg0-Hyp3-Thi5-D-Tic7-Oic8-bradykinin (HOE-140; 10 microM). In contrast, bronchoalveolar lavage cells from ovalbumin-sensitized and -challenged mice significantly responded to both bradykinin and des-Arg9-bradykinin, indicating the presence of both functional bradykinin B1 and B2 receptors. Eosinophils exhibited higher response to des-Arg9-bradykinin (1 microM; 485% increase in [Ca2+]i) compared to bradykinin (1 microM; 163% increase in [Ca2+]i). This des-Arg9-bradykinin-induced [Ca2+]i increase was markedly inhibited by the selective bradykinin B1 receptor antagonist, Ac-Lys-[D-betaNal7, Ile8]des-Arg9-bradykinin (R-715; 10 microM). Des-Arg9-bradykinin neither modified the basal [Ca2+]i in lymphocytes nor in mononuclear cells from ovalbumin-sensitized and challenged mice, while bradykinin produced a [Ca2+]i increase in both cell types. Our results further support the implication of the inducible bradykinin B1 receptors in airway inflammatory response in ovalbumin-sensitized and challenged mice.