The present study investigated the internalization behavior of the constitutively active mutant (CAM) N111G of angiotensin II type 1 (AT1) receptor and correlated the result with the mechanism of the constitutive activity of the mutant. The inverse agonist activity of valsartan, losartan, candesartan, and telmisartan was also examined by inositol phosphate (IP) accumulation study as well as receptor-internalization assay. Both wild-type (WT) and N111G mutant receptors were transiently expressed in COS-7 cells and the binding affinities towards the agonist and these four AT1 antagonists were determined. Production of total IP was measured in the presence and absence of the compounds. The agonist-induced receptor internalization of both WT and N111G mutant receptors was also investigated. Although the mutant showed similar binding characteristics with agonist and the antagonists used as WT, the internalization of the mutant was much lower (19.56 ± 2.87%) than that of the WT receptor (74.63 ± 1.00%). Internalization of the mutant significantly increased (63.22 ± 0.03%) in the presence of valsartan, which also showed significant inverse agonist activity in the N111G mutant. The results indicate that internalization of CAM N111G of the AT1 receptor is induced by the use of valsartan, which may be an important characteristic of inverse agonist activities of AT1 antagonists in N111G.
The aim of this study was to assess the effect of tadalafil (0.5, 2.5, and 10 mg/kg per day) on the progression of pulmonary arterial hypertension (PAH) in early treatment and on the survival rate in late treatment on the monocrotaline (MCT)-induced PAH rat model. Tadalafil was administered once daily to rats for 3 weeks from the day of MCT-injection or 21 days after the injection. With early treatment, tadalafil at 10 mg/kg per day prevented the development of PAH by maintaining mean pulmonary artery pressure within the normal range and attenuated right ventricular hypertrophy. With late treatment, tadalafil tended to increase the partial pressure of oxygen in arterial blood and dose-dependently improved the survival rate by 55%, 60%, and 70% at 0.5, 2.5, and 10 mg/kg per day, respectively, versus 40% in the MCT-control group. Both early and late treatments with tadalafil were associated with elevated lung cyclic guanosine monophosphate (cGMP). These results suggest that tadalafil relaxes pulmonary arteries by elevating cGMP in lungs and extend survival time by improving pulmonary hemodynamics even when treatment occurs in the late phase of PAH. Thus, it is expected that tadalafil may be an effective, once-daily treatment option in humans with PAH.
It was reported previously that non-steroidal anti-inflammatory drugs (NSAID)–induced gastric damage was markedly aggravated in rats during arthritis, and this response was mediated by the overproduction of nitric oxide (NO) derived from endothelial NO synthase (eNOS) in addition to inducible NO synthase (iNOS). The present study examined the gastric ulcerogenic response to cold-restraint stress in adjuvant arthritic rats, particularly in relation to NO/NOS isozymes. Exposure of normal rats to cold-restraint stress (13°C) produced slight gastric damage 3 h later, but the ulcerogenic response was markedly aggravated in arthritic rats. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) (a nonselective inhibitor of NOS) slightly increased the cold-restraint stress–induced gastric lesions in normal rats, but dose-dependently prevented the aggravation of these lesions in arthritic rats. The increased ulcerogenic response in arthritic rats was significantly suppressed by 1400W (a selective inhibitor of iNOS) and L-iminoethyl ornithine (L-NIO) (a selective inhibitor of eNOS), but not by NG-propyl-L-arginine (L-NPA) (a selective inhibitor of nNOS), and almost totally abolished by the co-administration of 1400W and L-NIO. The mucosal expression levels of eNOS and iNOS but not nNOS mRNAs were enhanced in arthritic rats compared with normal rats. The aggravation of stress-induced gastric lesions in arthritic rats was also significantly suppressed by pretreatment with glutathione. These results suggest that the gastric ulcerogenic response to cold-restraint stress is enhanced in arthritic rats, similar to that induced by NSAIDs, and this phenomenon may be causally associated with the upregulation of eNOS/NO in addition to iNOS/NO.
Baclofen, a GABAB-receptor (GABABR) agonist has been proposed to be useful as therapeutic agent for the management of gastro-esophageal reflux disease, but whether the compound acts directly at the lower esophageal sphincter (LES) remains to be elucidated. We performed the present study to assess the presence of GABABR in human LES. Western blot analysis showed that both proteins of GABAB1(a)/GABAB1(b) and GABAB2 subunits were present in the muscle layer of LES. Immunohistochemical findings showed that both GABAB1- and GABAB2-subunit proteins were located on the neurons within the myenteric plexus, and furthermore, both proteins were observed in the same neurons. Reverse transcriptase-polymerase chain reaction analysis also revealed the presence of mRNAs for both subunits of GABABR and also mRNAs for 6 isoforms of GABAB1 subunits, from GABAB1(a) to GABAB1(g), except GABAB1(d), in human LES. Thus, the functional GABABR-forming heterodimers with subunits of GABAB1 and GABAB2 are located on the myenteric neurons in human LES, suggesting that GABABR agonists and antagonists act at least, at the level of the peripheral nervous system.
Beneficial effects of statins on cardiovascular diseases have been attributed to decreased generation of reactive oxygen species (ROS). We tested the hypothesis that atorvastatin protects against the development of hypertension by reducing levels of NADPH oxidase–derived ROS in two hypertensive animal models. Atorvastatin was given to mice chronically infused with angiotensin (Ang) II or to apolipoprotein E (ApoE)–deficient mice fed a high-fat diet. Increased mean blood pressure (MBP) demonstrated in both animal models was significantly suppressed by atorvastatin with reduced ROS production in the aorta. Treatment with atorvastatin did not alter the mRNA level of NOX1, a catalytic subunit of NADPH oxidase, but decreased the levels of other NOX isoforms, NOX2 and NOX4, in the ApoE-deficient mice fed a high-fat diet. In the Ang II–infused model treated with statin, only the NOX4 mRNA level was reduced. Membrane translocation of Rac1 was significantly reduced in the Ang II–infused mice treated with atorvastatin. Finally, atorvastatin administered to Ang II–infused mice lacking the Nox1 gene elicited an additional decline in MBP compared to Nox1-deficient mice treated with vehicle. Together, these findings suggest that reduced expression and activity of the isoforms of NADPH oxidase, involving NOX1, NOX2, and possibly NOX4, mediate the anti-hypertensive effect of atorvastatin.
Proteinase-activated receptor-2 (PAR2) triggers upregulation of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) formation in human alveolar epithelial A549 cells. This COX-2 upregulation appears to involve the Src / epidermal growth factor (EGF) receptor / p38 MAP kinase (p38MAPK) pathway and also the cAMP-response element–binding protein (CREB) pathway. Here, we investigated the roles of nuclear factor-κB (NF-κB)–related signals in the PAR2-triggered PGE2 release / COX-2 upregulation in A549 cells. The PAR2-triggered PGE2 release was clearly blocked by an inhibitor of the NF-κB pathway. Stimulation of PAR2 actually caused phosphorylation of inhibitor-κB, an indicator of NF-κB activation, an effect being blocked by inhibitors of MEK, phosphatidylinositol 3–kinase (PI3-kinase), and Akt, but little or not by inhibitors of p38MAPK and JNK. Stimulation of PAR2 also caused phosphorylation of Akt, an effect suppressed by inhibitors of PI3-kinase and MEK. Nonetheless, the PAR2-triggered upregulation of COX-2 was resistant to inhibitors of NF-κB, PI3-kinase, and Akt, but was attenuated by inhibitors of MEK and JNK. Stimulation of PAR2 induced phosphorylation of CREB, an effect abolished by an inhibitor of MEK but not inhibitors of p38MAPK and EGF receptor. These findings demonstrate that the MEK / ERK / PI3-kinase / Akt / NF-κB pathway is involved in PAR2-triggered PGE2 formation, but not upregulation of COX-2 that is dependent on activation of ERK/CREB and JNK in addition to p38MAPK.
Xanthorrhizol is a sesquiterpenoid from the rhizome of Curcuma xanthorrhiza. In our previous studies, xanthorrhizol suppressed cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, inhibited cancer cell growth, and exerted an anti-metastatic effect in an animal model. However, the exact mechanisms for its inhibitory effects against cancer cell growth have not yet been fully elucidated. In the present study, we investigated the growth inhibitory effect of xanthorrhizol on cancer cells. Xanthorrhizol dose-dependently exerted antiproliferative effects against HCT116 human colon cancer cells. Xanthorrhizol also arrested cell cycle progression in the G0/G1 and G2/M phase and induced the increase of sub-G1 peaks. Cell cycle arrest was highly correlated with the downregulation of cyclin A, cyclin B1, and cyclin D1; cyclin-dependent kinase 1 (CDK1), CDK2, and CDK4; proliferating cell nuclear antigen; and inductions of p21 and p27, cyclin-dependent kinase inhibitors. The apoptosis by xanthorrhizol was markedly evidenced by induction of DNA fragmentation, release of cytochrome c, activation of caspases, and cleavage of poly-(ADP-ribose) polymerase. In addition, xanthorrhizol increased the expression and promoter activity of pro-apoptotic non-steroidal anti-inflammatory drug–activated gene-1 (NAG-1). These findings provide one plausible mechanism for the growth inhibitory activity of xanthorrhizol against cancer cells.
Although liposome-encapsulated clodronate has been used as a means to deplete macrophages from certain tissues, target leukocyte subtypes within the kidney are not clearly known under normal and pathologic conditions. The present study was therefore conducted to examine the effects of liposome clodronate on renal infiltrating cell type following unilateral ureteral obstruction (UUO) and tried to correlate these changes to the mechanisms of early development of renal fibrosis. Renal infiltrating leukocyte subtypes and counts were determined by using multicolor flow cytometric analysis of cell suspensions from obstructed kidneys. UUO for 5 days elicited renal tubular apoptosis and renal fibrosis and showed 4-fold increase in renal leukocytes including monocytes/macrophages, dendritic cells, and T-cells. Repeated administration of liposome clodronate selectively depleted F4/80+ monocytes/macrophages and F4/80+ dendritic cells but not F4/80− dendritic cells or other cell types in both obstructed and non-obstructed kidneys. Tubular apoptosis and renal fibrosis were also significantly attenuated by liposome clodronate. Increased gene expression of TNF-α and TGF-β observed in obstructed kidneys were markedly attenuated by depletion of renal mononuclear phagocytes. These findings suggest that F4/80+ monocytes/macrophages and/or F4/80+ dendritic cells play a pivotal role in the development of obstruction-induced tubular apoptosis and renal fibrosis, possibly through TNF-α and TGF-β dependent mechanisms.
The present study was performed to investigate the effects of kavain on the sleep-wake cycle in comparison with that of rilmazafone and diphenhydramine using sleep-disturbed rats. Electrodes for the electroencephalogram (EEG) and electromyogram (EMG) were implanted into Wistar rats. Total awake time, non-rapid eye movement (non-REM) sleep and rapid eye movement (REM) sleep were measured for 6 h. Kavain and rilmazafone showed a significant shortening in sleep latency, decreased awake time, and increased non-REM sleep time. On the other hand, significant shortening of the sleep latency was observed following the administration of diphenhydramine, while no effects were observed on the awake and non-REM sleep time. Moreover, kavain showed a significant increase in delta activity during non-REM sleep in sleep-disturbed rats, whereas a significant decrease in delta power during non-REM sleep was observed with rilmazafone. These results clearly indicate that kavain is a compound with not only hypnotic effects, but also sleep quality–enhancement effects.
The present study was designed to characterize the neurogenic contraction of rat radial artery. Electrical field stimulation (EFS) evoked frequency-dependent contraction that was abolished by tetrodotoxin (neuronal Na+ channel blocker), guanethidine (sympathetic neuron blocker), or phentolamine (α-adrenoceptor blocker). The α1-adrenoceptor antagonist prazosin inhibited endothelium-independent contractions to EFS, noradrenaline (NA), and the α1-adrenoceptor agonist phenylephrine. Rauwolscine, an α2-adrenoceptor antagonist, augmented nerve-mediated contractions and reduced sensitivity to NA and the α2-adrenoceptor agonist BHT-920. The β-adrenoceptor antagonist propranolol diminished EFS-elicited contractions, while sensitivity to NA was enhanced by propranolol. Relaxations evoked by isoproterenol, a β-adrenoceptor agonist, were abolished by propranolol. NG-Nitro-L-arginine (L-NOARG), a nitric oxide (NO) synthase inhibitor, increased both nerve-mediated and NA-induced responses in endothelium-intact, but not in endothelium-denuded arteries. Moreover, endothelium-dependent responses to BHT-920 and isoproterenol were modified by L-NOARG. Tetraethylammonium (TEA) or 4-amynopyridine, the Ca2+-activated (KCa) or voltage-dependent K+ (KV) channel blockers, respectively, enhanced the neurogenic contractions observed. TEA but not 4-amynopyridine increased NA-induced contractions. The ATP-sensitive K+ (KATP)–channel blocker glibenclamide failed to modify adrenergic contractions. Blockade of capsaicin-sensitive primary afferents increased EFS-induced contractions. In conclusion, adrenergic contractions are predominantly mediated by muscular α1-adrenoceptors, while endothelial α2- and β-adrenoceptors play a minor role. Presynaptic α2- and β-adrenoceptors cannot be precluded. Noradrenergic neurotransmission in rat radial artery seems to be modulated by both stimulation of endothelial NO, KCa, and KV channels and sensory C-fiber activation.
Platelet-derived growth factor (PDGF) signaling controls various physiological functions via two receptor subtypes: PDGF receptor (PDGFR) α and PDGFRβ. Nevertheless, our understanding of their roles is limited because of a lack of pharmacological tools to discriminate subtype-specific signaling. We developed a chimeric receptor by combining ligand-binding-domain truncated PDGFRβ with anti-fluorescein single chain antibody, expecting the control of PDGFRβ-specific signaling by oligomerized fluorescein as an artificial agonist. Results show that calcium mobilization, Cdc42 activation, and cell migration were elicited specifically by the artificial ligand in cells expressing the chimeric receptor. Our method is expected to be useful to understand the subtype-specific roles of PDGFRs in various cellular functions.
A pyrazolone compound acting as a formyl peptide receptor (FPR) 2/ALX–selective agonist has been reported, but its pharmacological activities on human FPRs (hFPRs) and mouse FPRs (mFprs) have not been well demonstrated. In this study, we found that this compound, designated as compound A, induced concentration-dependent calcium flux not only in Chinese hamster ovary (CHO) cells expressing hFPR2/ALX but also in cells expressing hFPR1, mFpr1, or mFpr2. It also induced the receptor internalization of hFPR1 and hFPR2/ALX and, accordingly, induced calcium influx and chemotactic responses in both human and mouse neutrophils. Our study revealed that compound A is in fact an FPR1 and FPR2/ALX dual agonist.