Suma I. Shimuta

Kinin B1 Receptor Deficiency Leads to Leptin Hypersensitivity and Resistance to Obesity

OBJECTIVE—Kinins mediate pathophysiological processes related to hypertension, pain, and inflammation through the activation of two G-protein–coupled receptors, named B1 and B2. Although these peptides have been related to glucose homeostasis, their effects on energy balance are still unknown. RESEARCH DESIGN AND METHODS—Using genetic and pharmacological strategies to abrogate the kinin B1 receptor in different animal models of obesity, here we present evidence of a novel role for kinins in the regulation of satiety and adiposity. RESULTS—Kinin B1 receptor deficiency in mice (B1−/−) resulted in less fat content, hypoleptinemia, increased leptin sensitivity, and robust protection against high-fat diet–induced weight gain. Under high-fat diet, B1−/− also exhibited reduced food intake, improved lipid oxidation, and increased energy expenditure. Surprisingly, B1 receptor deficiency was not able to decrease food intake and adiposity in obese mice lacking leptin (ob/ob-B1−/−). However, ob/ob-B1−/− mice were more responsive to the effects of exogenous leptin on body weight and food intake, suggesting that B1 receptors may be dependent on leptin to display their metabolic roles. Finally, inhibition of weight gain and food intake by B1 receptor ablation was pharmacologically confirmed by long-term administration of the kinin B1 receptor antagonist SSR240612 to mice under high-fat diet. CONCLUSIONS—Our data suggest that kinin B1 receptors participate in the regulation of the energy balance via a mechanism that could involve the modulation of leptin sensitivity.

Further evidence for the existence of two receptor sites for bradykinin responsible for the diphasic effect in the rat isolated duodenum

1 Low doses of bradykinin (below 10 nM), as well as of K+ (below 10 mM) induced relaxation, whereas higher doses caused contraction of the rat duodenum. 2 The relaxant responses induced by bradykinin and K+ were not affected by ouabain (1 μM), but pre‐incubation with 5.9 mM K+ abolished the responses to that ion but not those to bradykinin. 3 The contractile and relaxant components of the response to bradykinin (but not those to K+) increased with the time elapsed after mounting of the preparation, and this was due to stretching by the load of the recording system. 4 Specific and reversible desensitization (tachyphylaxis) was observed with the contractile response (but not the relaxation) induced by bradykinin. 5 Des‐Arg9‐bradykinin, an analogue specific for B1‐receptors, was much less active than bradykinin, and elicited only a contractile response. 6 Among four bradykinin potentiating peptides that were tested, potentiator C enhanced the relaxation only, whereas BPP5a and captopril potentiated only the contraction and BPP9a potentiated both types of response to bradykinin. 7 Our results support the hypothesis that the relaxant and contractile components of the rat duodenums response to bradykinin are due to actions at different receptor sites, which can be distinguished by their properties (desensitization) and their different apparent affinities for agonists and for potentiating peptides.

Functional rescue of a defective angiotensin II AT1 receptor mutant by the Mas protooncogene

Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT(1)/Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT(1) receptor, a non-functional AT(1) receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas. Cells single-expressing Mas or C18F/K20A did not show any binding for AngII. The co-expression of the wild-type AT(1) receptor and Mas showed a binding profile similar to that observed for the wild-type AT(1) expressed alone. Surprisingly, the co-expression of the double mutant C18F/K20A and Mas evoked a total recovery of the binding affinity for AngII to a level similar to that obtained for the wild-type AT(1). Functional measurements using inositol phosphate and extracellular acidification rate assays also showed a clear recovery of activity for AngII on cells co-expressing the mutant C18F/K20A and Mas. In addition, immunofluorescence analysis localized the AT(1) receptor mainly at the plasma membrane and the mutant C18F-K20A exclusively inside the cells. However, the co-expression of C18F-K20A mutant with the Mas changed the distribution pattern of the mutant, with intense signals at the plasma membrane, comparable to those observed in cells expressing the wild-type AT(1) receptor. These results support the hypothesis that Mas is able to rescue binding and functionality of the defective C18F-K20A mutant by dimerization.

Evidence for a regulatory site in the angiotensin II receptor of smooth muscle.

The homologous desensitization induced by angiotensin II analogues in the guinea-pig isolated ileum was studied. Desensitization assessed by the loss of response on repeated treatment showed [Sar1]angiotensin II to be a strong desensitizer whereas no desensitization to [Lys2]angiotensin II was detected. However, prolonged treatment with either analogue desensitized the tissue, indicating that [Lys2]angiotensin II-induced desensitization was reversed faster. A correlation was found between the degree of desensitization caused by repeated treatment and the time for half-relaxation after washout of the first treatment, but the relaxation after washout became faster in the desensitized state. In experiments designed to study competition between the agonistic and desensitizing properties of angiotensin II analogues, high concentrations of [Lys2]angiotensin II blocked the agonistic but not the desensitizing effect of lower concentrations of [Sar1]angiotensin II. It is concluded that desensitization is due to the interaction of angiotensin II with a regulatory site on the receptor.

Expression of angiotensin I-converting enzymes and bradykinin B2 receptors in mouse inner medullary-collecting duct cells

We described in mouse inner medullary-collecting duct cells (mIMCD-3) the somatic and the N-domain ACE synthesis and its interaction with the kallikrein-kinin system co-localized in the same cells. We purified two ACE forms from culture medium, M1 (130 kDa) and M2 (N-domain, 60 kDa), and cellular lysate, C1 (130 kDa) and C2 (N-domain, 60 kDa). Captopril and enalaprilat inhibited the purified enzymes. The immunofluorescence studies indicated that ACE is present in the membrane, cytoplasm and in the cell nucleus. Kinin B1 and B2 receptors were detected by immunofluorescence and showed to be activated by BK and DesR9 BK, increasing the acidification rate which was enhanced in the presence of enalaprilat. The presence of secreted and intracellular ACE in mIMCD-3 confirmed the hypothesis previously proposed by our group for a new site of ACE secretion in the collecting duct.

Selectivity of bradykinin analogues for receptors mediating contraction and relaxation of the rat duodenum

1 Bradykinin produces a biphasic response in the rat duodenum that consists of a relaxation (pD2 = 8.44) followed by a contraction (pD2 = 6.91). 2 The B1 agonist des‐Arg9‐bradykinin produced a contraction (pD2 = 7.16) but no relaxation. Des‐Arg9‐[Leu8]‐bradykinin, which is a B1 antagonist in other systems produced contraction (pD2 = 7.65) in the rat duodenum. 3 Four bradykinin analogues that are preferential B2 agonists in other tissues had a biphasic effect with pD2 values in the range 7.22‐8.68 for relaxation and 6.26‐6.91 for contraction. 4 [Thi5,8,D‐Phe7]‐bradykinin, which is a B2 antagonist in most other systems produced relaxation in the rat duodenum, with a pD2 of 7.49. 5 It is concluded that the contractile component of the response to bradykinin in rat duodenum may be mediated by a subtype of the B1 receptor and the relaxant component by a receptor of the B2 subtype.

Angiotensin II Binding to Angiotensin I–Converting Enzyme Triggers Calcium Signaling

Angiotensin (Ang) I–converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system.

Role of the Cys18–Cys274 disulfide bond and of the third extracellular loop in the constitutive activation and internalization of angiotensin II type 1 receptor

An insertion of residues in the third extracellular loop and a disulfide bond linking this loop to the N-terminal domain were identified in a structural model of a G-protein coupled receptor specific to angiotensin II (AT1 receptor), built in homology to the seven-transmembrane-helix bundle of rhodopsin. Both the insertion and the disulfide bond were located close to an extracellular locus, flanked by the second extracellular loop (EC-2), the third extracellular loop (EC-3) and the N-terminal domain of the receptor; they contained residues identified by mutagenesis studies to bind the angiotensin II N-terminal segment (residues D1 and R2). It was postulated that the insertion and the disulfide bond, also found in other receptors such as those for bradykinin, endothelin, purine and other ligands, might play a role in regulating the function of the AT1 receptor. This possibility was investigated by assaying AT1 forms devoid of the insertion and with mutations to Ser on both positions of Cys residues forming the disulfide bond. Binding and activation experiments showed that abolition of this bond led to constitutive activation, decay of agonist binding and receptor activation levels. Furthermore, the receptors thus mutated were translocated to cytosolic environments including those in the nucleus. The receptor form with full deletion of the EC-3 loop residue insertion, displayed a wild type receptor behavior.

Calcium and sodium dependence of the biphasic response of the guinea-pig ileum to agonists

The isometric responses of the longitudinal smooth muscle of the guinea-pig ileum to acetylcholine and to histamine consist of a phasic contraction followed by a fade and a tonic contraction. At high agonist concentrations the fade is more pronounced and a period of lower tonus is observed in the early stage of the tonic component of the response. Experiments done in Ca2+-free medium indicate that the events responsible for the shape of the response take place in the absence of Ca2+ and of contraction. The phasic contraction was inhibited by hyperpolarization in low-Na+ medium. Small decreases in the external Na+ concentration caused a diminution of the fade. Reduction of the Na+ concentration during the early stages of the tonic response caused contraction whereas relaxation was observed at the late stages. It is proposed that the fade is dependent on a Na+-sensitive mechanism that is predominant at early but not at late stages of the responses to agonists.

Functional expression of kinin B1 and B2 receptors in mouse abdominal aorta

Previous studies have shown that the vascular reactivity of the mouse aorta differs substantially from that of the rat aorta in response to several agonists such as angiotensin II, endothelin-1 and isoproterenol. However, no information is available about the agonists bradykinin (BK) and DesArg(9)BK (DBK). Our aim was to determine the potential expression of kinin B(1) and B(2) receptors in the abdominal mouse aorta isolated from C57BL/6 mice. Contraction and relaxation responses to BK and DBK were investigated using isometric recordings. The kinins were unable to induce relaxation but concentration-contraction response curves were obtained by applying increasing concentrations of the agonists BK and DBK. These effects were blocked by the antagonists Icatibant and R-715, respectively. The potency (pD(2)) calculated from the curves was 7.0 +/- 0.1 for BK and 7.3 +/- 0.2 for DBK. The efficacy was 51 +/- 2% for BK and 30 +/- 1% for DBK when compared to 1 microM norepinephrine. The concentration-dependent responses of BK and DBK were markedly inhibited by the arachidonic acid inhibitor indomethacin (1 microM), suggesting a mediation by the cyclooxygenase pathway. These contractile responses were not potentiated in the presence of the NOS inhibitor L-NAME (1 mM) or endothelium-denuded aorta, indicating that the NO pathway is not involved. We conclude that the mouse aorta constitutively contains B(1) and B(2) subtypes of kinin receptors and that stimulation with BK and DBK induces contractile effect mediated by endothelium-independent vasoconstrictor prostanoids.