Mechanisms regulating the expression,self-maintenance,and signaling-function of the bradykinin B2 and B1 receptors

Bradykinin (BK) is a potent short-lived effector belonging to a class of peptides known as kinins. It participates in inflammatory and vascular regulation and processes including angioedema, tissue permeability, vascular dilation, and smooth muscle contraction. BK exerts its biological effects through the activation of the bradykinin B2 receptor (BKB2R) which is G-protein-coupled and is generally constitutively expressed. Upon binding, the receptor is activated and transduces signal cascades which have become paradigms for the actions of the Galphai and Galphaq G-protein subunits. Following activation the receptor is then desensitized, endocytosed, and resensitized. The bradykinin B1 (BKB1R) is a closely related receptor. It is activated by desArg(10)-kallidin or desArg(9)-BK, metabolites of kallidin and BK, respectively. This receptor is induced following tissue injury or after treatment with bacterial endotoxins such as lipopolysacharide or cytokines such as interleukin-1 or tumor necrosis factor-alpha. In this review we will summarize the BKB2R and BKB1R mediated signal transduction pathways. We will then emphasize the relevance of key residues and domains of the intracellular regions of the BKB2R as they relate to modulating its function (signal transduction) and self-maintenance (desensitization, endocytosis, and resensitization). We will examine the features of the BKB1R gene promoter and its mRNA as these operate in the expression and self-maintenance of this inducible receptor. This communication will not cover areas discussed in earlier reviews pertaining to the actions of peptide analogs. For these we refer you to earlier reviews (Regoli and Barabé, 1980, Pharmacol Rev 32:1-46; Regoli et al., 1990, J Cardiovasc Pharmacol 15(Suppl 6):S30-S38; Regoli et al., 1993, Can J Physiol Pharmacol 71:556-557; Marceau, 1995, Immunopharmacology 30:1-26; Regoli et al., 1998, Eur J Pharmacol 348:1-10).     

Mediator caused induction of a human bradykinin B1 receptor minigene: participation of c-Jun in the process

The bradykinin B1 receptor (BKB1R) gene is expressed in selected tissues such as lung and kidney. In these tissues it is expressed at a very low level until induced by inflammatory mediators. Our aim has been to understand the mechanism of this regulatory process. A human BKB1R minigene was constructed. It contained a 1.8 kb promoter, the entire exon I, 1.5 kb of intron I, the entire exon II and intron II, and the luciferase gene as a reporter. Transient transfection of the minigene into SV40-transformed IMR90 cells (IMRSV) resulted in a promoter activity which was activated by the mediators, lipopolysaccharide and (LPS) desArg(10)-kallidin. In contrast, these mediators did not induce the activity of the 1.8 kb promoter construct alone. Thus, motifs exclusive of the promoter such as 5'-UTR and/or intron regions are required for mediator-induced expression of this gene. Promoter activities of both the minigene and the 1.8 kb promoter construct were enhanced in a dose-dependent manner upon cotransfection with c-Jun. Furthermore, cotransfecting c-Jun with the minigene achieved the maximal promoter activity with no further increase in response to mediators. Conversely, the induction of the minigene promoter activity by mediators was abolished upon cotransfection with a dominant negative mutant of c-Jun. Other experiments suggest that multiple AP-1 sites are interactive with the c-Jun upregulation of this gene. Taken together, these results point to c-Jun as a key intermediary in the activation of the expression of this gene by mediators. However, participation of motifs outside of the promoter are necessary to obtain this inducible expression.     

Des-Arg(10)-kallidin engagement of the B1 receptor stimulates type I collagen synthesis via stabilization of connective tissue growth factor mRNA

Expression of the kinin B1 receptor is up-regulated in chronic inflammatory and fibrotic disorders; however, little is known about its role in fibrogenesis. We examined human embryonic lung fibroblasts that constitutively express the B1 receptor and report that engagement of the B1 receptor by des-Arg(10)-kallidin stabilized connective tissue growth factor (CTGF) mRNA, stimulated an increase in alpha1(I) collagen mRNA, and stimulated type I collagen production. These events were not observed in B2 receptor-activated fibroblasts. In addition, B1 receptor activation by des-Arg(10)-kallidin induced a rise in cytosolic Ca(2+) that is consistent with B1 receptor pharmacology. Our results show that the des-Arg(10)-kallidin-stimulated increase in alpha1(I) collagen mRNA was time- and dose-dependent, with a peak response observed at 20 h with 100 nM des-Arg(10)-kallidin. The increase in CTGF mRNA was also time- and dose-dependent, with a peak response observed at 4 h with 100 nM des-Arg(10)-kallidin. The increase in CTGF mRNA was blocked by the B1 receptor antagonist des-Arg(10),Leu(9)-kallidin. Inhibition of protein synthesis by cycloheximide did not block the des-Arg(10)-kallidin-induced increase in CTGF mRNA. These results suggest that engagement of the kinin B1 receptor contributes to fibrogenesis through increased expression of CTGF.     

K317, R319, and E320 within the proximal C-terminus of the bradykinin B2 receptor form a motif important for phospholipase C and phospholipase A2 but not connective tissue growth factor related signaling

We showed previously that large domain exchanges between the bradykinin B2 (BKB2) and angiotensin II type 1a (AT1a) receptors can result in functional hybrids. However, when we proceeded to exchange the entire bradykinin B2 receptor (BKB2R) C-terminal tail with the AT1aR C-terminus, the hybrid, while continuing to bind BK and be endocytosed as wild type (WT) BKB2R, lost much of its ability to activate phosphatidylinositol (PI) turnover or the release of arachidonic acid (ARA). In this study, we constructed chimeric receptors within the proximal C-terminus between the BKB2R and AT1aR or bradykinin B1 receptor (BKB1R). The mutant and WT receptor cDNAs were stably transfected into Rat-1 cells. Also, point mutations were generated to evaluate the role of the individual residues within this region. These chimeric studies revealed that the proximal portion of the BKB2R C-tail is crucial for G protein-linked BKB2R functions. This region could not be swapped with the AT1aR to obtain a BK activated PI turnover or ARA release. Further studies demonstrated that the distal portion (325-330) of this region is exchangeable; however, the middle portion (317-324) is not. Small motif exchanges within this section identified the KSR and EVY motifs as crucial for G(alphaq), G(alphai) related signaling of the BKB2R. Point mutations then showed that the charged amino acids K317, R319, and E320 are the residues critical for linking to PI turnover and ARA release. However, these proximal chimeras showed normal receptor uptake. Interestingly, while apparently not activating G protein-linked signaling, the proximal tail AT1aR exchange mutant and the entire C-terminus exchange hybrid continued to cause a substantial bradykinin effected increase in connective tissue growth factor (CTGF) mRNA level, as WT BKB2R.     

Chimeric exchanges within the bradykinin B2 receptor intracellular face with the prostaglandin EP2 receptor as the donor: importance of the second intracellular loop for cAMP synthesis

The prostaglandin E2 (PGE(2)) EP2 receptor (EP2R) type is G protein coupled (GPCR) and links to Galphas. Through this receptor PGE(2) activates cAMP production. The bradykinin (BK) B2 receptor (BKB2R) is also a GPCR but links to Galphaq and Galphai and does not activate cAMP production in response to bradykinin. In an attempt to convert the BKB2R into a Galphas-linked adenylate cyclase-activating receptor we proceeded to make global and discrete motif replacements of the intracellular (IC) face of the BKB2R with the corresponding regions of the human EP2R. With this approach we produced hybrid receptors which, when stably transfected into wild type (WT) Rat-1 cells, bound BK but produced cAMP. Replacement of the second loop (IC2), third loop (IC3), the entire C terminus, and the distal C terminus resulted in receptors which bound BK. However, only the IC2 and IC3 exchanges resulted in cAMP-producing receptors. Of these two regions, the IC2 exchange was by far the better cAMP-generating receptor, producing cAMP at approximately 6.6-fold above WT BKB2R or approximately one fourth the amount produced by WT EP2R-transfected Rat-1 cells. Both human and rat EP2R and human beta2-adrenergic receptor exchanges of the IC2 produced equal quantities of cAMP. Focusing on the rBKB2R/hEP2R IC2 chimeras, the region consisting of residues 136-147 (BKB2R residue numbering) proved to contain a cAMP-generating motif. Within this region, the proximal six amino acids from the EP2R (HPYFYQ) at position 136-141 proved crucial for cAMP production (10-fold over WT BKB2R). The distal part of this region, the six residues at 142-147, played no role in cAMP production. On the other hand, the ALV motif of the BKB2R IC2, residues 133-135, proved important with respect to phosphatydilinositol (PI) turnover. Replacing the entire IC2 of BKB2R resulted in poor PI turnover, while including the AVL of BKB2R retained approximately half of the WT PI turnover. With respect to receptor uptake, all the IC2 mutants endocytosed as WT BKB2R (60% in 1h). However, the exchange of the distal and the whole C termini resulted in a marked drop in endocytosis (30% in 1h). These results demonstrate that the construction of a cAMP-producing BKB2/EP2 receptor hybrid is possible, with the IC2 region distal to DRYLALV proving important to Galphas linkage and the LALV motif within the IC2 of BKB2R and the region proximal to it proving important for Galphaq and Galphai linkage. Additionally, our results confirm the importance of the distal C terminus in determining receptor uptake.     

Regulation by interleukin-1beta of gene expression of bradykinin B1 receptor in MH-S murine alveolar macrophage cell line.

The effects of recombinant murine interleukin (IL)-1beta on gene expression of murine bradykinin B1 receptor (BDKRB1) in MH-S murine alveolar macrophage cell line were evaluated. BDKRB1 mRNA expression in MH-S cells was increased by IL-1beta (1, 3, and 10 ng/ml) in a time-dependent manner, peaking at 3-4 h by 100-1000 fold. IL-1beta (5 ng/ml, 24h) also induced significant binding to [3H]-des-Arg10-kallidin with a dissociation constant (Kd) of 2.95 nM and a maximal binding density (Bmax) of 670 sites/cell. Des-Arg10-kallidin (10 microM), a BDKRB1 agonist, increased intracellular calcium ion ([Ca2+]i) in IL-1beta (5 ng/ml, 24 h)-exposed cells, an increase not observed in the cells not exposed to IL-1beta. A significant increase of tumor necrosis factor (TNF)-alpha secretion occurred in the IL-1beta (5 ng/ml, 24 h)-exposed cells following addition of des-Arg10-kallidin (the IL-1beta-exposed group: 57. 8 +/- 13.7 vs. the vehicle-exposed group: 16.7 +/- 4.3 pg/ml, p < 0.05 after a 100 nM des-Arg10-kallidin for 8 h), with an optimal effect at 3-100 nM. These data suggest that IL-1beta may up-regulate BDKRB1-mediated functions of alveolar macrophages via an induction of BDKRB1 gene expression.     

Role of hydroxyl containing residues in the intracellular region of rat bradykinin B(2) receptor in signal transduction, receptor internalization, and resensitization

In past reports we illustrated the importance of Y131, Y322, and T137 within the intracellular (IC) face of the rat bradykinin B2 receptor (rBKB2R) for signal transduction and receptor maintenance (Prado et al. [1997] J. Biol. Chem. 272:14638-14642; Prado et al. [1998] J. Biol. Chem. 273:33548-33555). In this report, we mutate the remaining hydroxyl possessing residues located within the rBKB2R IC region. Exchange of S139A (IC2) or T239V (IC3) did not affect BK activated phosphatidylinositol (PI) turnover or receptor internalization. Chimeric exchange of the last 34 amino acids of BKB2R C-terminus with the corresponding 34 amino acids of the rat angiotensin II AT1a receptor (rAT1aR), both containing an S/T cluster, resulted in a mutant with normal endocytosis and BK activated PI turnover. A more selective chimera of these S/T clusters, with an exchange of BKB2R (333-351) with a rAT1aR fragment (326-342), resulted in a receptor with a retarded internalization but a normal BK activated PI turnover. Subsequent mutation of rBKB2R T344V showed little change in receptor uptake but a pronounced loss of BK activated PI turnover. The mutation of S335A, S341A, S348A, and S350A resulted in very poor receptor internalization and loss of activated PI turnover. Closer examination of this serine cluster illustrated that the replacement of S348A led to poor internalization; whereas the retention of S348 and mutation of S341A resulted in a receptor with a much greater internalization than WT. These and other results suggest that the presence of S348 promotes internalization while the presence of S341 dampens it. Conversely, S341 and S350 proved important for receptor signaling. In sum, our results illustrate that the distal C-terminus including its S/T cluster is important for both rBKB2R internalization and signal transduction. Individual S/T residues within this cluster appear involved in either signal transmission or receptor uptake capacity. However, replacement of the entire distal tail region with the corresponding rAT1aR sequence, also containing an S/T cluster, enables the BKB2R/AT1aR chimera to act in a very similar manner to wild type rBKB2R.     

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.     

Fever-like temperature modification differentially affects in vitro signaling of bradykinin B(1) and B(2) receptors

The bradykinin (BK) B(2) and B(1) receptors (B(2)R, B(1)R) belong to the rhodopsin-like G protein-coupled receptors (GPCRs) and are involved in (patho)physiological processes such as blood pressure regulation or inflammation. They mediate the effects of the pro-inflammatory peptides bradykinin/kallidin and desArg(9)-BK/desArg(10)-kallidin, respectively. Whereas the B(2)R is constitutively expressed and gets internalized upon activation, the B(1)R is especially induced by inflammatory mediators and responds to stimulation with increased surface receptor numbers. Stimulation of both receptors activates phospholipase Cβ (PLCβ) and mitogen activated protein kinase (MAPK) signaling. Because inflammatory processes are characterized by heat (fever), we analyzed the effect of increased temperature (41°C vs. 37°C) on B(1)R and B(2)R signaling in HEK 293 and IMR 90 cells. Our results show that signaling of both receptors is temperature-sensitive, however to a different extent and with regard to the investigated pathways. Comparing PLCβ activity and Ca(2+)-regulated signals, a temperature-dependent increase was only observed for B(1)R but not for B(2)R activation, whereas MAPK activities were doubled at 41°C for both receptors. Taken together, our findings suggest that the observed temperature sensitivity of B(1)R-induced PLCβ activation is B(1)R-specific. In contrast, the enhanced stimulation of MAPK activity under hyperthermic conditions appears to be a common phenomenon for GPCRs.     

Coulombic and hydrophobic interactions in the first intracellular loop are vital for bradykinin B2 receptor ligand binding and consequent signal transduction

The role of the first intracellular loop (IC1) in the function of the rat bradykinin B2 receptor (BKB2R) was probed. On the basis of the bovine rhodopsin X-ray structure, the BKB2R IC1 consists of six residues: (60)HKTNCT. Exchange of this sequence with the bradykinin B1 receptor IC1 (PRRQLN) resulted in a chimera which bound bradykinin and signaled as wild-type (WT) BKB2R. In contrast, a chimera containing the IC1 of rat angiotensin II type Ia receptor (AT1aR) (YMKLKT) did not bind BK nor signal in response to BK at a concentration as high as 5 microM. ELISA illustrated that this receptor was still processed and inserted into the plasma membrane. Employing portions of the IC1, we observed that (60)HKT of BKB2R could be exchanged as a group with either the BKB1R (PRR) or AT1aR (YMK) with no change in receptor binding or signaling activities. When only the YM of AT1aR replaced the HK of BKB2R, leaving the N-terminal portion of IC1 without a positively charged residue, binding and signaling were reduced by more than 70%. When only N63 was replaced with the corresponding leucine of AT1aR, binding and signaling were ablated. In fact, replacement of the entire IC1 with the AT1aR except for N63 resulted in binding and signaling as WT BKB2R. However, N63 could be replaced by glutamine (in BKB1R) or aspartate and continued to function as WT BKB2R. NMR data indicated that the BKB2R IC1 extends beyond the bovine rhodopsin prototype to include HKTNCTVAEI. When E68 was exchanged with a serine (in AT1aR), ligand binding decreased by 60% and PI turnover decreased by 69%. Molecular modeling points to a strict requirement for a hydrophilic residue at position 63 (N) at the middle of the IC1 and a Coulombic charge interaction between the positive charges (H60 and K61) at the N-terminus and a negative charge (E68) at the C-terminus of the IC1.     

Global chimeric exchanges within the intracellular face of the bradykinin B2 receptor with corresponding angiotensin II type Ia receptor regions: generation of fully functional hybrids showing characteristic signaling of the AT1a receptor

The intracellular (IC) face of the G-protein coupled receptors (GPCR), bradykinin (BK) B2 and angiotensin (AT) 1a, is similar in sequence homology and in size. Both receptors are known to link to Galphai and Galphaq but differ markedly in a number of physiologic actions, particularly with respect to their hemodynamic action. We made single as well as multiple, global replacements within the IC of BKB2R with the corresponding regions of the AT1aR. When stably transfected into Rat-1 cells, these hybrid receptors all bound BK with high affinity. Single replacement of the intracellular loop 2 (IC2) or the distal 34 residues of the C-terminus (dCt) with the corresponding regions of AT1aR resulted in chimera, which turned over phosphotidylinositol (PI) and released arachidonic acid (ARA) as WT BKB2R. In contrast, incorporation of the AT1aR IC3 in a single replacement abolished signal transduction. However, the simultaneous exchange of IC2 and IC3 of BKB2R with AT1aR resulted in a receptor responding to BK with PI turnover and ARA release approximately 4-fold greater than WT BKB2R. Likewise, the simultaneous replacement of IC2 and dCt resulted in a 2.8- and 1.6-fold increase in PI turnover and ARA release, respectively. In contrast, the dual replacement of IC3 and dCt could not overcome the deleterious effects of the IC3 replacement, resulting in very low PI activation and ARA release. Replacement of all three IC domains (IC2, IC3, and dCt) resulted in PI closer to that of AT1aR than BKB2R. The uptake of the receptor chimeras was similar to that of WT BKB2R with the exception of the IC3/dCt dual mutant, which exhibited very poor internalization (18% at 60'). When transfected into Rat-1 cells, the AT1aR markedly increased the expression of connective tissue growth factor (CTGF) mRNA, while BK slightly decreased it. The dual IC2/dCt and triple IC2/IC3/dCt hybrids both upregulated CTGF mRNA in response to BK. These results show that the IC face of the BKB2R can be exchanged with that of AT1aR, producing hybrid receptors, which take on the functional characteristics of AT1aR. The characterization of the chimera with stepwise replacement of the IC domains should allow for assignment of specific roles to the individual loops and C-terminus in the signaling and internalization of the BKB2R and facilitate the generation of a receptor with BKB2R binding and AT1aR function.     

Na+/Ca2+ exchanger activity modulates connective tissue growth factor mRNA expression in transforming growth factor beta1- and Des-Arg10-kallidin-stimulated myofibroblasts

Transforming growth factor (TGF)-beta and des-Arg(10)-kallidin stimulate the expression of connective tissue growth factor (CTGF), a matrix signaling molecule that is frequently overexpressed in fibrotic disorders. Because the early signal transduction events regulating CTGF expression are unclear, we investigated the role of Ca(2+) homeostasis in CTGF mRNA expression in TGF-beta1- and des-Arg(10)-kallidin-stimulated human lung myofibroblasts. Activation of the kinin B1 receptor with des-Arg(10)-kallidin stimulated a rise in cytosolic Ca(2+) that was extracellular Na(+)-dependent and extracellular Ca(2+)-dependent. The des-Arg(10)-kallidin-stimulated increase of cytosolic Ca(2+) was blocked by KB-R7943, a specific inhibitor of Ca(2+) entry mode operation of the plasma membrane Na(+)/Ca(2+) exchanger. TGF-beta1 similarly stimulated a KB-R7943-sensitive increase of cytosolic Ca(2+) with kinetics distinct from the des-Arg(10)-kallidin-stimulated Ca(2+) response. We also found that KB-R7943 or 2',4'-dichlorobenzamil, an amiloride analog that inhibits the Na(+)/Ca(2+) exchanger activity, blocked the TGF-beta1- and des-Arg(10)-kallidin-stimulated increases of CTGF mRNA. Pretreatment with KB-R7943 also reduced the basal and TGF-beta1-stimulated levels of alpha1(I) collagen and alpha smooth muscle actin mRNAs. These data suggest that, in addition to regulating ion homeostasis, Na(+)/Ca(2+) exchanger acts as a signal transducer regulating CTGF, alpha1(I) collagen, and alpha smooth muscle actin expression. Consistent with a more widespread role for Na(+)/Ca(2+) exchanger in fibrogenesis, we also observed that KB-R7943 likewise blocked TGF-beta1-stimulated levels of CTGF mRNA in human microvascular endothelial and human osteoblast-like cells. We conclude that Ca(2+) entry mode operation of the Na(+)/Ca(2+) exchanger is required for des-Arg(10)-kallidin- and TGF-beta1-stimulated fibrogenesis and participates in the maintenance of the myofibroblast phenotype.     

Modulation by bradykinin of angiotensin type 1 receptor-evoked RhoA activation of connective tissue growth factor expression in human lung fibroblasts

The mechanisms regulating the opposing physiological actions of bradykinin (BK) and angiotensin II (AngII) are not well understood. Here we investigate signaling interactions between these two effectors. Connective tissue growth factor (CTGF) expression in IMR-90, human lung fibroblasts, is used as the endpoint target. In these cells the BK B2 receptor (BKB2R) is expressed constitutively, while no binding of AngII is detected. An inducible expression system is used to insert AngII receptor 1 (AT1R) and to obtain a signal level in response to AngII at the magnitude of BK. AngII and BK activate G protein-coupled targets, arachidonate release from cellular phospholipid stores, and intracellular phosphatidylinositol turnover equally. Both activate ERK, JNK, and p38 equally. However, AngII activates, whereas BK inactivates, RhoA. AngII induces a rapid (1 h) CTGF mRNA expression. RhoA siRNA and RhoA activation inhibitor, Y-27632, markedly reduce the AngII effect. Simultaneous treatment with BK and AngII attenuates the AT1R action. Additionally, BK in the absence of AngII lowers CTGF mRNA expression below basal levels over a span of 4 h. An AT1R/BKB2R chimera lacking heterotrimeric G protein coupling continues to activate MAP kinases to the same extent as wild-type (WT) AT1R and BKB2R. However, the increase of CTGF mRNA expression by this mutant is low, almost identical with that obtained by the simultaneous treatment of the WT AT1R-expressing cells with BK and AngII. In this context the chimeric receptor displays the characteristics of both receptors. These data demonstrate that, in human lung fibroblasts, BK modulates the action of AngII through the small G protein RhoA, but in a Galphai/Galphaq-independent manner.     

Role of phosphoinositide 3-kinase in the nonselective cation channel activation by endothelin-1/endothelinB receptor

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) in Chinese hamster ovarian cells expressing endothelin(B) receptor (CHO-ET(B)R). These channels can be discriminated using the Ca(2+) channel blockers, LOE 908 and SK&F 96365. LOE 908 is a blocker of NSCC-1 and NSCC-2, whereas SK&F 96365 is a blocker of NSCC-2. In this study, we investigated the possible role of phosphoinositide 3-kinase (PI3K) in the ET-1-induced activation of NSCCs in CHO-ET(B)R using wortmannin and LY-294002, inhibitors of PI3K. ET-1-induced Ca(2+) influx was partially inhibited in CHO-ET(B)R pretreated with wortmannin or LY-294002. In contrast, addition of wortmannin or LY-294002 after stimulation with ET-1 did not suppress Ca(2+) influx. The Ca(2+) channels activated by ET-1 in wortmannin- or LY-294002-treated CHO-ET(B)R were sensitive to LOE 908 and resistant to SK&F 96365. In conclusion, NSCC-2 is stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated independently of the PI3K pathway. Moreover, PI3K seems to be required for the initiation of the Ca(2+) entry through NSCC-2 but not for its maintenance.     

Hormone-specific combinations of isoforms of adenylyl cyclase and phosphodiesterase in the rat liver

Since many isoforms of adenylyl cyclase and adenosine 3', 5'-monophosphate (cAMP) phosphodiesterase have been cloned, it is likely that receptors of each hormone have a specific combination of these isoforms. Types I, III and VIII adenylyl cyclases are reported to be stimulated by Ca(2+)-calmodulin, type I phosphodiesterase by Ca(2+)-calmodulin, but types IV and VII (cAMP-specific) phosphodiesterases by Co2+. In the present study, we examined different effects of Ca2+ and Co2+ on hormone-induced cAMP response in the isolated perfused rat liver.The removal of Ca2+ from the perfusion medium (0 mM CaCl(2 ) + 0.5 mM EGTA) did not affect glucagon (0.1 nM)-responsive cAMP but reduced secretin (1 nM)-, vasoactive intestinal polypeptide (VIP, 1-10 nM)- and forskolin (1 microM)-responsive cAMP considerably. The addition of 1 mM CoCl2 reduced glucagon- and secretin-responsive cAMP considerably, forskolin-responsive cAMP partly, did not affect 1 nM VIP-responsive cAMP, but enhanced 10 nM VIP-responsive cAMP. Forskolin- and VIP-responsive cAMP was greater in the combination (0 mM CaCl(2) + 0.5 mM EGTA + 3 mM CoCl2) than in the Ca(2+)-free perfusion alone.These results suggest that secretin, VIP1 and VIP2 receptors are linked to Ca(2+)-calmodulin-sensitive adenylyl cyclase; glucagon receptor to Ca(2+)-calmodulin-insensitive adenylyl cyclase; VIP1 receptor to Ca(2+)-calmodulin-dependent phosphodiesterase; glucagon, secretin and VIP2 receptors to cAMP-specific phosphodiesterase, respectively, in the rat liver.     

Calcium-dependent inhibition of adrenal TREK-1 channels by angiotensin II and ionomycin

Bovine adrenocortical cells express bTREK-1 K(+) (bovine KCNK2) channels that are inhibited by ANG II through a Gq-coupled receptor by separate Ca(2+) and ATP hydrolysis-dependent signaling pathways. Whole cell and single patch clamp recording from adrenal zona fasciculata (AZF) cells were used to characterize Ca(2+)-dependent inhibition of bTREK-1. In whole cell recordings with pipette solutions containing 0.5 mM EGTA and no ATP, the Ca(2+) ionophore ionomycin (1 μM) produced a transient inhibition of bTREK-1 that reversed spontaneously within minutes. At higher concentrations, ionomycin (5-10 μM) produced a sustained inhibition of bTREK-1 that was reversible upon washing, even in the absence of hydrolyzable [ATP](i). BAPTA was much more effective than EGTA at suppressing bTREK-1 inhibition by ANG II. When intracellular Ca(2+) concentration ([Ca(2+)](i)) was buffered to 20 nM with either 11 mM BAPTA or EGTA, ANG II (10 nM) inhibited bTREK-1 by 12.0 ± 4.5% (n=11) and 59.3 ± 8.4% (n=4), respectively. Inclusion of the water-soluble phosphatidylinositol 4,5-bisphosphate (PIP(2)) analog DiC(8)PI(4,5)P(2) in the pipette failed to increase bTREK-1 expression or reduce its inhibition by ANG II. The open probability (P(o)) of unitary bTREK-1 channels recorded from inside-out patches was reduced by Ca(2+) (10-35 μM) in a concentration-dependent manner. These results are consistent with a model in which ANG II inhibits bTREK-1 K(+) channels by a Ca(2+)-dependent mechanism that does not require the depletion of membrane-associated PIP(2). They further indicate that the Ca(2+) source is located in close proximity within a "Ca(2+) nanodomain" of bTREK-1 channels, where [Ca(2+)](i) may reach concentrations of >10 μM. bTREK-1 is the first two-pore K(+) channel shown to be inhibited by Ca(2+) through activation of a G protein-coupled receptor.     

Identification of a key region of kinin B(1) receptor for high affinity binding of peptide antagonists

To investigate the molecular basis for the specificity of ligand recognition in human kinin B(1) (B(1)R) and B(2) (B(2)R) receptors, we constructed a series of chimeric receptors by progressively replacing, from the N to the C terminus, the human B(2)R domains by their B(1) counterparts. The chimeric construct possessing the C-terminal tail and the transmembrane domain VII (TM VII) of the B(2)R (construct 6) displayed 7- and 20- fold decreased affinities for the B(1) agonist [(3)H]desArg(10)-kallidin (desArg(10)-KD) and the B(1) antagonist [(3)H]desArg(10)-[Leu(9)]-KD respectively, as compared with the wild-type B(1)R. Moreover, the substitution of the B(1) TM VII by its B(2) homologue TM increased the affinity for the pseudopeptide antagonists, Hoe140 and NPC 567. High affinity for desArg(10)-KD binding was fully regained when the B(2) residue Thr(287) was replaced in construct 6 by the corresponding B(1) Leu(294) residue. When the B(2) residue Tyr(295) was exchanged with the corresponding B(1) Phe(302), high affinity binding for both agonist and antagonist was recovered. Moreover, the L294T and F302Y mutant B(1)R exhibited 69- and 6.5-fold increases, respectively, in their affinities for the B(2) receptor antagonist, Hoe140. Therefore we proposed that Leu(294) and Phe(302) residues, which may not be directly involved in the binding of B(1)R ligands and, hence, their Thr(287) and Tyr(295) B(2) counterparts, are localized in a receptor region, which plays a pivotal role in the binding selectivity of the peptide or pseudopeptide kinin ligands.     

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of β-arrestin1 and PP2A with noninternalized NK(1)R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.     

IP(3)-mediated Ca(2+) signals in human neuroblastoma SH-SY5Y cells with exogenous overexpression of type 3 IP(3) receptor

Human neuroblastoma SH-SY5Y cells, predominantly expressing type 1 inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), were stably transfected with IP(3)R type 3 (IP(3)R3) cDNA. Immunocytochemistry experiments showed a homogeneous cytoplasmic distribution of type 3 IP(3)Rs in transfected and selected high expression cloned cells. Using confocal Ca(2+) imaging, carbachol (CCh)-induced Ca(2+) release signals were studied. Low CCh concentrations (< or = 750 nM) evoked baseline Ca(2+) oscillations. Transfected cells displayed a higher CCh responsiveness than control or cloned cells. Ca(2+) responses varied between fast, large Ca(2+) spikes and slow, small Ca(2+) humps, while in the clone only Ca(2+) humps were observed. Ca(2+) humps in the transfected cells were associated with a high expression level of IP(3)R3. At high CCh concentrations (10 microM) Ca(2+) transients in transfected and cloned cells were similar to those in control cells. In the clone exogenous IP(3)R3 lacked the C-terminal channel domain but IP(3)-binding capacity was preserved. Transfected cells mainly expressed intact type 3 IP(3)Rs but some protein degradation was also observed.We conclude that in transfected cells expression of functional type 3 IP(3)Rs causes an apparent higher affinity for IP(3). In the clone, the presence of degraded receptors leads to an efficient cellular IP(3) buffer and attenuated IP(3)-evoked Ca(2+) release.