Hyaluronic acid and proteoglycan link protein 1 suppresses platelet­derived growth factor-BB-induced proliferation, migration, and phenotypic switching of vascular smooth muscle cells.

The development of atherosclerotic cardiovascular disease is associated with the phenotypic switching of vascular smooth muscle cells (SMCs) from a contractile to a synthetic state, leading to cell migration and proliferation. Platelet­derived growth factor­BB (PDGF­BB) modulates this de-differentiation by initiating a number of biological processes. In this study, we show that gene expression of hyaluronic acid (HA) and proteoglycan link protein 1 (HAPLN1) was upregulated during differentiation of human aortic SMCs (HASMCs) into a contractile state, but downregulated upon during PDGF-BB-induced dedifferentiation. This is the first study showing that the treatment of HASMCs with full-length recombinant human HAPLN1 (rhHAPLN1) significantly reversed PDGF-BB-induced decrease in the protein levels of contractile markers (SM22α, α-SMA, calponin, and SM-MHC), and inhibited the proliferation and migration of HASMCs induced by PDGF-BB. Furthermore, our results show that rhHAPLN1 significantly inhibited the phosphorylation of FAK, AKT, STAT3, p38 MAPK and Raf mediated by the binding of PDGF-BB to PDGFRß. Together, these results indicated that rhHAPLN1 can suppress the PDGF-BB-stimulated phenotypic switching and subsequent de-differentiation of HASMCs, highlighting its potential as a novel therapeutic target for atherosclerosis and other vascular diseases. [BMB Reports 2023; 56(8): 445-450].

Interaction of Synaptosomal-Associated Protein 25 with Neutral Sphingomyelinase 2: Functional Impact on the Sphingomyelin Pathway.

Neurotransmitter release is mediated by ceramide, which is generated by sphingomyelin hydrolysis. In the present study, we examined whether synaptosomal-associated protein 25 (SNAP-25) is involved in ceramide production and exocytosis. Neutral sphingomyelinase 2 (nSMase2) was partially purified from bovine brain and we found that SNAP-25 was enriched in the nSMase2-containing fractions. In rat synaptosomes and PC12 cells, the immunoprecipitation pellet of anti-SNAP-25 antibody showed higher nSMase activity than the immunoprecipitation pellet of anti-nSMase2 antibody. In PC12 cells, SNAP-25 was colocalized with nSMase2. Transfection of SNAP-25 small interfering RNA (siRNA) significantly inhibited nSMase2 translocation to the plasma membrane. A23187-induced ceramide production was concomitantly reduced in SNAP-25 siRNA-transfected PC12 cells compared with that in scrambled siRNA-transfected cells. Moreover, transfection of SNAP-25 siRNA inhibited dopamine release, whereas addition of C6-ceramide to the siRNA-treated cells moderately reversed this inhibition. Additionally, nSMase2 inhibition reduced dopamine release. Collectively, our results indicate that SNAP-25 interacts with nSMase2 during ceramide production, which mediates exocytosis and neurotransmitter release.

Effect of Robinia pseudoacacia Leaf Extract on Interleukin-1ß-mediated Tumor Angiogenesis.

BACKGROUND/AIM: Interleukin (IL)-1ß is a pro-inflammatory cytokine that has recently been established as a stimulator of angiogenesis via regulation of proangiogenic factor expression in the tumor microenvironment. This study aimed to demonstrate the inhibitory effects of Robinia pseudoacacia leaf extract (RP) on IL-1ß-mediated tumor angiogenesis. MATERIALS AND METHODS: Secreted embryonic alkaline phosphatase (SEAP) reporter gene assay, ex vivo and in vitro tube formation assay, western blot, and quantitative PCR were used to analyze the inhibitory effect of RP on IL-1ß-mediated angiogenesis. RESULTS: RP inhibited secretion of SEAP, blocked IL-1ß signaling, and inhibited IL-1ß-mediated angiogenesis in ex vivo and in vitro assays. RP inhibited nuclear translocation of NF-ĸB by suppressing phosphorylation of IL-1ß signaling protein kinases and inhibited mRNA expression of IL-1ß-induced pro-angiogenic factors including VEGFA, FGF2, ICAM1, CXCL8, and IL6. CONCLUSION: RP suppressed IL-1ß-mediated angiogenesis and, thus, could be a promising agent in anticancer therapy.

Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases.

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LCMS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 588.6 → 264.4 for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide.

Autophagy is essential for optimal cell function and survival, and the entire process accompanies membrane dynamics. Ceramides are produced by different enzymes at different cellular membrane sites and mediate differential signaling. However, it remains unclear which ceramide-producing pathways/enzymes participate in autophagy regulation under physiological conditions such as nutrient starvation, and what the underlying mechanisms are. In this study, we demonstrate that among ceramide-producing enzymes, neutral sphingomyelinase 2 (nSMase2) plays a key role in autophagy during nutrient starvation. nSMase2 was rapidly and stably activated upon starvation, and the enzymatic reaction in the Golgi apparatus facilitated autophagy through the activation of p38 MAPK and inhibition of mTOR. Moreover, nSMase2 played a protective role against cellular damage depending on autophagy. These findings suggest that nSMase2 is a novel regulator of autophagy and provide evidence that Golgi-localized ceramides participate in cytoprotective autophagy against starvation.

Dopamine transporter trafficking is regulated by neutral sphingomyelinase 2/ceramide kinase.

Dopamine (DA) reuptake is the primary mechanism to terminate dopaminergic transmission in the synaptic cleft. The dopamine transporter (DAT) has an important role in the regulation of DA reuptake. This study provides anatomical and physiological evidence that DAT recycling is regulated by ceramide kinase via the sphingomyelin pathway. First, the results show that DAT and neutral sphingomyelinase 2 (nSMase2) were successfully co-precipitated from striatal samples and were colocalized in the mouse striatum or PC12 cells. We also identified a protein-protein interaction between nSMase2 and DAT through in situ proximity ligation assay experiments in the mouse striatum. Second, dopamine (DA) stimulated the formation of ceramide and increased nSMase activity in PC12 cells, while treatment with a cell-permeable ceramide-1-phosphate (C1P) increased DA uptake. Third, we used inhibitors and siRNA to inhibit nSMase2 and ceramide kinase and observed the effects on DAT recycling in PC12 cells. Treatment with ceramide kinase inhibitor K1, or nSMase inhibitor GW4869, decreased DA uptake in PC12 cells, although the application of FB1, a ceramide synthase inhibitor, did not affect DA uptake. Transfection of nSMase2 and CERK siRNA decreased DAT surface level in PC12 cells. These results suggested that SM-derived C1P affects cell surface levels of DAT.

Nanoparticulation improves bioavailability of Erlotinib.

OBJECTIVES: Nanoparticulation using fat and supercritical fluid (NUFSTM) is a drug delivery platform technology enabling efficient and effective formulation of poorly soluble drugs. We performed experiments to examine whether NUFS™ could improve poor bioavailability and reduce fed-fasted bioavailability variances of erlotinib (Ert). METHODS: NUFS-Ert was prepared using NUFS™ technology; its physical properties were characterized, and drug release was measured. Furthermore, in vitro and in vivo efficacy tests and pharmacokinetic analysis were performed. RESULTS: NUFS-Ert nanoparticles had an average size of 250 nm and were stable for 2 months at 40 °C, 4 °C, and room temperature. The dissolution rate of NUFS-Ert increased in bio-relevant dissolution media. NUFS-Ert was more potent in inhibiting EGF signaling and in suppressing the proliferation of A549, a human non-small cell lung cancer cell line. Furthermore, A549 xenografts in BALB/c nude mice treated with NUFS-Ert regressed more efficiently than those in the mice treated with vehicle or Tarceva®. In addition, experimental lung metastasis was more efficiently inhibited by NUFS-Ert than by Tarceva®. The relative bioavailability of NUFS-Ert compared with that of Tarceva® was 550% and the ratio of the area under the concentration-time curve (AUC) of fed state to the AUC of fasted state was 1.8 for NUFS-Ert and 5.8 for Tarceva®. CONCLUSIONS: NUFS-Ert could improve poor bioavailability and reduce fed-fasted bioavailability variances of Ert. NUFS-Ert was more efficacious than Tarceva®.

mTOR inhibition by rapamycin protects against deltamethrin-induced apoptosis in PC12 Cells.

The autophagy pathway can be induced and upregulated in response to intracellular reactive oxygen species (ROS). In this study, we explored a novel pharmacotherapeutic approach involving the regulation of autophagy to prevent deltamethrin (DLM) neurotoxicity. We found that DLM-induced apoptosis in PC12 cells, as demonstrated by the activation of caspase-3 and -9 and by nuclear condensation. DLM treatment significantly decreased dopamine (DA) levels in PC12 cells. In addition, we observed that cells treated with DLM underwent autophagic cell death, by monitoring the expression of LC3-II, p62, and Beclin-1. Exposure of PC12 cells to DLM led to the production of ROS. Treatment with N-acetyl cysteine (NAC) effectively blocked both apoptosis and autophagy. In addition, mitogen-activated protein kinase (MAPK) inhibitors attenuated apoptosis as well as autophagic cell death. We also investigated the modulation of DLM-induced apoptosis in response to autophagy regulation. Pretreatment with the autophagy inducer, rapamycin, significantly enhanced the viability of DLM-exposed cells, and this enhancement of cell viability was partially due to alleviation of DLM-induced apoptosis via a decrease in levels of cleaved caspase-3. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), significantly increased DLM toxicity in these cells. Our results suggest that DLM-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against DLM-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 109-121, 2017.

JNK and p38 MAPK regulate oxidative stress and the inflammatory response in chlorpyrifos-induced apoptosis.

To investigate mechanisms of neuronal cell death in response to chlorpyrifos (CPF), a pesticide, we evaluated the regulation of ROS and COX-2 in human neuroblastoma SH-SY5Y cells treated with CPF. CPF treatment produced cytotoxic effects that appeared to involve an increase in ROS. In addition, CPF treatment activated MAPK pathways including JNK, ERK1/2, and p38 MAPK, and MAPK inhibitors abolished the cytotoxicity and reduced ROS generation. Our data demonstrate that CPF induced apoptosis involving MAPK activation through ROS production. Furthermore, after the CPF treatment, COX-2 expression increased. Interestingly, JNK and p38 MAPK inhibitors attenuated the CPF-induced COX-2 expression while an ERK1/2 inhibitor did not. These findings suggest that pathways involving JNK and p38 MAPK, but not ERK1/2, mediated apoptosis and are involved in the inflammatory response. In conclusion, the JNK and p38 MAPK pathways might be critical mediators in CPF-induced neuronal apoptosis by both generating ROS and up-regulating COX-2.

Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells.

Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders.

Rosiglitazone, a PPAR-γ agonist, protects against striatal dopaminergic neurodegeneration induced by 6-OHDA lesions in the substantia nigra of rats.

Rosiglitazone is a commonly prescribed insulin-sensitizing drug with selective agonistic activity at the peroxisome proliferator-activated receptor-γ (PPARγ). Previously, rosiglitazone was shown to attenuate dopaminergic cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD), an effect attributed to its anti-inflammatory properties. To elucidate the neuroprotective mechanisms of rosiglitazone, we investigated the effects of rosiglitazone on the expressions of striatal tyrosine hydroxylase (TH), cyclooxygenase-2 (COX-2) and glial fibrillary acidic protein (GFAP) in a 6-OHDA-lesioned rat PD model. Rosiglitazone (3 mg/kg) was administered intraperitoneally at 24 h and 30 min prior to the creation of an intranigral 6-OHDA lesion. A reduction in TH protein expression began at 3 days and a prominent decrease was observed at 7 days post-lesion, and decreases of dopamine (DA) levels began at 1 day post-lesion. In contrast, GFAP expression was significantly increased at 3 days and preserved for up to 7 days post-lesion and the patterns of GFAP expression was inversely correlated to changes in TH expression. Furthermore, COX-2 expression in the rostral striatum showed a significant increase at 6h post-lesion while that of the caudal striatum was increased at 12 h. In the 6-OHDA-lesioned model, the activation of PPARγ by rosiglitazone significantly prevented TH protein expression reductions, and inhibited 6-OHDA-induced microglia activation in striatum. In addition, rosiglitazone attenuated in production of both COX-2 and TNF-α expression. In contrast, rosiglitazone pretreatment led to greater increases in striatal GFAP expression than 6-OHDA alone and changes in the expression of this protein preceded the changes that were seen with TH expression. These results suggest that the neuroprotection observed with rosiglitazone treatment may be partially due to the attenuation of COX-2 production and the strengthening of astrocyte function. Our results provide insight into the neuroprotective mechanisms of rosiglitazone against 6-OHDA-induced neuronal damages.

Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos.

Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease.

Fluazinam targets mitochondrial complex I to induce reactive oxygen species-dependent cytotoxicity in SH-SY5Y cells.

Although the underlying cause of Parkinson's disease (PD) is not well characterized, epidemiological studies suggest that exposure to agricultural chemicals is a risk factor for PD. Fluazinam (FZN) is a new active ingredient for the control of grey mould, belonging to the novel broad spectrum phenylpyridinamine fungicides. We used human neuroblastoma SH-SY5Y cells to investigate mechanisms of dopaminergic cell death in response to FZN. FZN treatment produced dose-dependent cytotoxicity, and decreased the tyrosine hydroxylase (TH) expression in SH-SY5Y cells. We provided evidence for the occurrence of oxidative stress and oxidative damage during FZN exposure on dopaminergic cells through the measurement of reactive oxygen species (ROS) in cells with DCFH-DA. The cytotoxic effects of FZN appear to involve an increase in ROS generation since pretreatment with N-acetyl cysteine (NAC), an anti-oxidant, reduced cell death. After FZN treatment, dopamine (DA) levels decreased in both cell and culture media, and oxidative effects of FZN were blocked by NAC pretreatment. We show that cell death in response to FZN was due to apoptosis since FZN exposure results in an increased in cytochrome c release into the cytosol and activated caspase-3 through p38 and JNK signaling. Furthermore, the blocking of p38 or JNK signaling inhibits FZN-induced cell death. Phosphorylation of mitogen-activated protein kinases precedes cytochrome c release and caspase-3 activation. This cellular response is characteristic of mitochondrial dysfunction. Therefore, we also investigated the effect of FZN on mitochondrial complex I activity in FZN-treated cell. Interestingly, we show that FZN inhibited the complex I activity. Thus in this study, we report a new mode of action by which the fungicide FZN could triggers apoptosis.

Reactive oxygen species and mitogen-activated protein kinase induce apoptotic death of SH-SY5Y cells in response to fipronil.

There are multiple lines of evidence showing that environmental toxicants including pesticides may contribute to neuronal cell death. Fipronil (FPN) is a phenylpyrazole insecticide that acts on insect GABA receptors. Although the action of FPN is restricted to insect neuronal or muscular transmitter systems, a few studies have assessed the effects of this neurotoxicant on neuronal cell death distinct from an insect. To determine the mechanisms underlying FPN-induced neuronal cell death, we evaluated the ability of this chemical to induce oxidative stress and studied the involvement of mitogen activated protein kinases (MAPKs) in FPN-induced apoptosis stress in human neuroblastoma SH-SY5Y (SH-SY5Y) cells. Exposure of SH-SY5Y cells to FPN led to the production of reactive oxygen species (ROS) and apoptotic cell death via activation of caspase-9 and caspase-3. Interestingly, the antioxidant, N-acetyl-cysteine (NAC) attenuated apoptotic cell death and ROS production induced by FPN. These results indicated that oxidative stress plays a central role in FPN-induced cytotoxicity. Mitochondrial complex I activity was also inhibited by FPN treatment. These finding indicate that FPN triggers intrinsic apoptosis via the mitochondrial signaling pathway that is initiated by the generation of ROS. Furthermore, FPN treatment induced phosphorylation of MAPK members. Activation of these protein kinases by FPN was involved in the onset of apoptosis as inhibitors specific to these kinases protect against FPN-induced cell death as well as ROS generation. Our data indicate that FPN-induced apoptosis is mediated primarily by the generation of ROS and activation of MAPK members followed by activation of the intrinsic apoptotic pathway.

Fluazinam-induced apoptosis of SH-SY5Y cells is mediated by p53 and Bcl-2 family proteins.

A number of epidemiological studies have demonstrated a strong association between the incidence of neurodegenerative disease and pesticide exposure. Fluazinam (FZN) is a preventative fungicide from the pyridinamine group that was introduced in the 1990 s and that quickly established itself as a new standard for the control of blight caused by Phytophthora infestans in potatoes. We used human neuroblastoma SH-SY5Y cells to investigate mechanisms of neuronal cell death in response to FZN and showed that FZN was cytotoxic to SH-SY5Y cells in a concentration- and time-dependent manner. Additionally, we showed that FZN treatment significantly decreased the neuron numbers including dopaminergic neurons and mitochondrial complex I activity. The cytotoxic effects of FZN were associated with an increase in reactive oxygen species (ROS) generation because pretreatment with N-acetyl cysteine, an anti-oxidant, reduced cell death. We showed that neuronal cell death in response to FZN was due to apoptosis because FZN increased cytochrome C release into the cytosol and activated caspase-3 through the accumulation of p53. FZN also reduced the levels of Bcl-2 protein but increased the levels of Bax. Our results provide insight into the molecular mechanisms of FZN-induced apoptosis in neuronal cells.

H89, an inhibitor of PKA and MSK, inhibits cyclic-AMP response element binding protein-mediated MAPK phosphatase-1 induction by lipopolysaccharide.

OBJECTIVE: Lipopolysaccharide (LPS) stimulates the production of inflammatory cytokines and the amplification of immune responses via MAPK pathways. MAPK phosphatases (MKPs) feedback-regulate the activities of MAPKs to prevent excessive immunological functions. H89 has been used as an inhibitor of the protein kinase A (PKA) and mitogen- and stress-activated protein kinase (MSK) pathways. In view of the potential roles of PKA and MSK for MKP-1 induction and the ability of H89 to inhibit these kinases, this study examined the effect of H89 on MKP-1 induction by LPS and the role of cyclic-AMP response element binding protein (CREB) in the MKP-1 induction. RESULTS: H89 treatment inhibited increases in MKP-1 protein and mRNA levels, and gene transcription by LPS in Raw264.7 cells. Immunoblot, gel-shift, and chromatin-immunoprecipitation assays showed the activation of CREB by LPS, and the ability of H89 to inhibit it, suggesting that H89's inhibition of CREB may affect MKP-1 induction. In addition, H89 prevented the ability of LPS to induce other MKP genes (Dusp-2, 4, 8, and 16). Experiments using MAPK inhibitors showed that MAPKs are involved in CREB phosphorylation and MKP-1 induction, suggesting that CREB-mediated MKP-1 induction serves in part as a feedback-inhibitory loop of MAPKs. CONCLUSION: Our results demonstrate that H89 inhibits the activation of CREB and the CREB-mediated MKP-1 induction by LPS, which may result from its inhibition of PKA and MSK.

Modification of cardiovascular response of posterior hypothalamic adenosine A(2) receptor stimulation by adenylate cylase, guanylate cyclase and by K(ATP) channel blockade in anesthetized rats.

Cardiovascular inhibitory effects induced by posterior hypothalamic adenosine A(2) receptors and their modulation by nitric oxide were suggested by our previous report. In this experiment, we examined the modulation of cardiovascular effects of adenosine A(2) receptor stimulation by adenylate cyclase, guanylate cyclase and ATP-sensitive K(+) channel in the posterior hypothalamus. Posterior hypothalamic injection of drugs was performed in anesthetized, artificially ventilated male Sprague-Dawley rats. Injection of adenosine A(2) receptor agonist 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA; 1, 2 and 5 nmol) produced a dose-dependent decrease of blood pressure and heart rate. Pretreatment with adenosine A(2) receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 nmol) blocked the depressor and bradycardiac effects of CPCA (5 nmol). Pretreatments with adenylate cyclase inhibitor MDL-12330 (10 nmol) and guanylate cyclase inhibitor LY-83583 (5 nmol) attenuated the depressor and bradycardiac effects of CPCA (5 nmol). In addition, pretreatment with ATP-sensitive K(+) channel blocker glipizide (20 nmol) attenuated the depressor and bradycardiac responses of CPCA (5 nmol). These results suggest that posterior hypothalamic adenosine A(2) receptors play an inhibitory role in the central cardiovascular regulation and that both adenylate cyclase and guanylate cyclase mediate the depressor and bradycardiac actions of adenosine A(2) receptors. Also, ATP-sensitive K(+) channel mediates the posterior hypothalamic cardiovascular regulations of adenosine A(2) receptors.

The involvement of nitric oxide on the adenosine A(2) receptor-induced cardiovascular inhibitory responses in the posterior hypothalamus of rats.

This study was performed to investigate the putative relationship between nitric oxide (NO) and adenosine A(2) receptors on central cardiovascular regulation in the posterior hypothalamus of rats. Posterior hypothalamic injection of drugs was performed in anesthetized, artificially ventilated male Sprague-Dawley rats. Injection of adenosine A(2) receptor agonist 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA; 1, 2 and 5 nmol) produced a dose-dependent decrease of blood pressure and heart rate. Pretreatment with adenosine A(2) receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 nmol) blocked the depressor and bradycardiac effects of CPCA (5 nmol). Pretreatment with soluble guanylate cyclase inhibitor LY-83,583 (5 nmol) attenuated the depressor and bradycardiac effects of CPCA (5 nmol). In addition, pretreatment with NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (40 nmol) attenuated the depressor and bradycardiac responses of CPCA (5 nmol). These results suggest that adenosine A(2) receptor in the posterior hypothalamus plays an inhibitory role in central cardiovascular regulation and that NO participates in the inhibitory response induced by adenosine A(2) receptor stimulation in the posterior hypothalamus.