Rat Renal Arteries Research Articles

Design, synthesis, and evaluation of new series of Imperatorin analogs with potential vasodilatory activity

Two series of imperatorin analogs were synthesized based on our previous research and evaluated for their vasodilatation activities on in vitro rat mesenteric artery, basilar artery, and renal artery ring models. Target compounds were characterized by infrared, 1H NMR, and mass spectra. Most derivatives possessed significant vasodilatory activity on the mesenteric artery, and compound 3a exhibited favorable and broad vasodilatation activities on three kinds of rat artery ring models. The pharmacological results indicated that introducing nitrogen-contained ring in side chain or large steric hindrance at the distal end could increase the vasodilatory activity. Further, replacement of oxygen atom (–O–) in the skeleton of furocoumarin derivatives with nitrogen (–NH–) could cause the decrease of vasodilatory activity. The molecular docking also indicated that compound 3a showed a best affinity with α-1C receptor (PDB ID: 3G43). All these results suggested compound 3a would be a potential vasodilatory agent for hypertension.

Thermo-responsive hydroxybutyl chitosan hydrogel as artery intervention embolic agent for hemorrhage control

This work targeted to investigate the potential of thermo-responsive hydroxybutyl chitosan (HBC) hydrogel using as an embolic material for occlusion of selective blood vessels. HBC hydrogel was prepared via an etherification reaction between chitosan (CS) and 1, 2-butene oxide. The hydroxybutyl groups were introduced into CS backbone, which endowed HBC hydrogel with properties of porous structure, favorable hydrophilia and rapid sol-gel interconvertibility. The gelation temperatures and gelation time respectively decreased from 30.7°C to 11.5°C and 79.60±3.19s to 7.70±1.42s at 37°C, with HBC solutions viscoelasticity increased from 3.0% to 7.0%. HBC hydrogel exhibited noncytotoxic to mouse embryo fibroblasts (MEFs) and excellent hemocompatibility with red blood cells (RBCs). After injection HBC solution into rat renal arteries, HBC solution transformed into hydrogel and attached onto blood vessel inner wall tightly, giving immediate blood vessels embolization. Meanwhile, RBCs could aggregate around HBC hydrogel to form moderate coagulation, which was beneficial to avoid hydrogel migration with blood flow. Above results suggested that HBC hydrogel could be applied as a promising embolic agent for hemorrage in the interventional vascular embolization field.

The impaired vasodilatation in rat renal interlobar arteries after hypoxia/re‐oxygenation is endothelium independent

Renal vasculature plays an important role in renal ischemia/reperfusion injury. We showed recently that hypoxia/re‐oxygenation combined with low dose norepinephrine treatment increases the angiotensin II responses in interlobar arteries. Since angiotensin II activates constrictor and dilator systems we hypothesized an impaired vasodilatation in this context.To test the hypothesis rat interlobar arteries were studied by using wire myography. The dilatory capacity was investigated by application of acetylcholine (10‐9 to 10‐5 mol/l). Hypoxia (95% N2, 5% CO2 for 60 min) alone followed by re‐oxygenation (95% O2, 5% CO2, 10 min) did not affect vasodilatation. However, norepinephrine in a non‐constrictor dose (10‐9 mol/l) applied during hypoxia resulted in a decreased response to acetylcholine. The contribution of the nitric oxide system, COX‐products, and endothelial derived hyperpolarizing factor to reduced dilatation was tested by pretreatment with L‐NAME (10‐4 mol/l), indomethacin (10‐5 mol/l), TRAM‐34 (10‐6 mol/), and UCL1684 (10‐7 mol/l), respectively. L‐NAME decreased the acetylcholine response after hypoxia/re‐oxygenation stronger compared to control conditions, while indomethacin, TRAM‐34, and UCL1684 did not affect it. Endothelium‐independent effects were analyzed by using the NO‐donor SNP and simultaneous blocking of endothelium‐dependent NO production. SNP significantly reduces vasodilation after hypoxia+norepinephrine/re‐oxygenation (H+NE/R). Passive stiffness of arteries was not altered in our experimental setup.H+NE/R provokes an impairment of the dilatory response to acetylcholine in renal arteries. Our data suggest that the endothelial nitric oxide system, COX products, or TRAM‐34 and UCL1684 inhibitable potassium channels do not contribute to reduced dilatation. The results indicate changed dilatory signaling pathways in vascular smooth muscle cells.Support or Funding InformationThis study was financially supported by a grant of the German Research Council to A.P and P.B.P (FG 1368, DFG PA 479/10‐1 and PA 479/10‐2).

CYP epoxygenase-derived H2O2 is involved in the endothelium-derived hyperpolarization (EDH) and relaxation of intrarenal arteries

Reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are involved in the in endothelium-derived hyperpolarization (EDH)-type relaxant responses of coronary and mesenteric arterioles. The role of ROS in kidney vascular function has mainly been investigated in the context of harmful ROS generation associated to kidney disease. The present study was sought to investigate whether H2O2 is involved in the endothelium–dependent relaxations of intrarenal arteries as well the possible endothelial sources of ROS generation involved in these responses. Under conditions of cyclooxygenase (COX) and nitric oxide (NO) synthase inhibition, acetylcholine (ACh) induced relaxations and stimulated H2O2 release that were reduced by catalase and by the glutathione peroxidase (GPx) mimetic ebselen in rat renal interlobar arteries, suggesting the involvement of H2O2 in the endothelium-dependent responses. ACh relaxations were also blunted by the CYP2C inhibitor sulfaphenazole and by the NADPH oxidase inhibitor apocynin. Acetylcholine stimulated both superoxide (O2•−) and H2O2 production that were reduced by sulfaphenazole and apocynin. Expression of the antioxidant enzyme CuZnSOD and of the H2O2 reducing enzymes catalase and GPx-1 was found in both intrarenal arteries and renal cortex. On the other hand, exogenous H2O2 relaxed renal arteries by decreasing vascular smooth muscle (VSM) intracellular calcium concentration [Ca2+]i and markedly enhanced endothelial KCa currents in freshly isolated renal endothelial cells. CYP2C11 and CYP2C23 epoxygenases were highly expressed in interlobar renal arteries and renal cortex, respectively, and were co-localized with eNOS in renal endothelial cells. These results demonstrate that H2O2 is involved in the EDH-type relaxant responses of renal arteries and that CYP 2C epoxygenases are physiologically relevant endothelial sources of vasodilator H2O2 in the kidney.

Angiotensin-(1–7)-dependent vasorelaxation of the renal artery exhibits unique angiotensin and bradykinin receptor selectivity

Angiotensin-(1–7) [Ang-(1–7)] exhibits blood pressure lowering actions, inhibits cell growth, and reduces tissue inflammation and fibrosis which may functionally antagonize an activated Ang II-AT1 receptor axis. Since the vascular actions of Ang-(1–7) and the associated receptor/signaling pathways vary in different vascular beds, the current study established the vasorelaxant properties of the heptapeptide in the renal artery of male Wistar male rats. Ang-(1–7) produced an endothelium-dependent vasodilator relaxation of isolated renal artery segments pre-contracted by a sub-maximal concentration of phenylephrine (PE) (3×10−7M). Ang-(1–7) induced vasodilation of the rat renal artery with an ED50 of 3±1nM and a maximal response of 42±5% (N=10). The two antagonists (10−5M each) for the AT7/Mas receptor (MasR) [D-Pro7]-Ang-(1–7) and [D-Ala7]-Ang-(1–7) significantly reduced the maximal response to 12±1% and 18±3%, respectively. Surprisingly, the AT2R receptor antagonist PD123319, the AT1R antagonist losartan and B2R antagonist HOE140 (10−6M each) also significantly reduced Ang-(1–7)-induced relaxation to 12±2%, 22±3% and 14±7%, respectively. Removal of the endothelium or addition of the soluble guanylate cyclase (sGC) inhibitor ODQ (10−5M) essentially abolished the vasorelaxant response to Ang-(1–7) (10±4% and 10±2%, P <0.05). Finally, the NOS inhibitor LNAME (10−4M) reduced the response to 13±2% (p<0.05), but the cyclooxygenase inhibitor indomethacin failed to block the Ang-(1–7) response. We conclude that Ang-(1–7) exhibits potent vasorelaxant actions in the isolated renal artery that are dependent on an intact endothelium and the apparent stimulation of a NO-sGC pathway. Moreover, Ang-(1–7)-dependent vasorelaxation was sensitive to antagonists against the AT7/Mas, AT1, AT2 and B2 receptor subtypes.

Downregulation of Thromboxane A2 Receptor Occurs Mainly via Nuclear Factor-KappaB Signaling Pathway in Rat Renal Artery.

Thromboxane A2 (TXA2) acts on TXA2 receptors (TP) to regulate renal artery blood flow and subsequently contributes to the pathogenesis of renal ischemia. The present study was designed to examine if nuclear factor-kappaB (NF-κB) signaling pathway is involved in the downregulation of TP receptors in rat renal artery. Rat renal artery segments were organ cultured for 6 or 24 h. Downregulation of TP receptors was monitored using myograph (contractile function), real-time PCR (receptor mRNA), and immunohistochemistry (receptor protein). Specific inhibitors (MG-132 and BMS345541) for NF-κB signaling pathway were used to dissect the underlying molecular mechanisms involved. Compared to fresh (noncultured) segments, organ culture of the renal artery segments for 24 h induced a significant rightward shift of U46619 (TP receptor agonist) contractile response curves (pEC50: 6.89 ± 0.06 versus 6.48 ± 0.04, P < 0.001). This decreased contractile response to U46619 was paralleled with decreased TP receptor mRNA and protein expressions in the renal artery smooth muscle cells. Specific inhibitors (MG-132 and BMS345541) for NF-κB signaling pathway significantly abolished the decreased TP protein expression and receptor-mediated contractions. In conclusion, downregulation of TP receptors in the renal artery smooth muscle cells occurs mainly via the NF-κB signaling pathway.

10 - Hydrogen Peroxide and Redox Control of Hypoxic Pulmonary Vasoconstriction: Identifying the Mitochondrial Redox Oxygen Sensor

The ability of the body to respond to changes in oxygen (O 2 ) is an integral part of both fetal development and adaptation to environmental hypoxia. Oxygen sensing is mediated by the homeostatic oxygen sensing system, which includes structures such as the resistance pulmonary arteries (PA), ductus arteriosus, carotid bodies and neuroepithelial bodies that function together to improve oxygen uptake and delivery. Within the PA, hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation, whereas systemic arteries dilate during hypoxia. It is hypothesized that subunit(s) of the mitochondrial electron transport chain (ETC) serve as a redox-sensitive O 2 sensor, and modulate vascular tone via release of reactive oxygen species (ROS). However, the identity of the specific subunits within the megacomplexes that accounts for O 2 sensing remains elusive. Hence, the ETC composition and function of mitochondria from rat pulmonary and renal artery (RA) smooth muscle cells (SMC) were compared. Relative to RASMC, PASMC exhibit increased Complex I activity under both normoxic and hypoxic conditions. Mass spectrometry analysis and gene expression array have identified seven candidate proteins/subunits within the mitochondrion and electron transport chain that are differentially expressed between PASMC and RASMC, including iron-sulfur subunits of Complexes I and III, and uncoupling proteins UCP2 and UCP3. We also measured hydrogen peroxide (H 2 O 2 ) flux in PASMC during the acute hypoxic response using a pH-sensitive, H 2 O 2 -specific vector under flow conditions, with increased intracellular calcium as a marker of vasoconstriction. Upon exposure to hypoxia, H 2 O 2 levels decrease immediately prior to the increase in intracellular calcium, supporting the theory that a decrease in mitochondrial ROS, and specifically H 2 O 2 , during hypoxia is the cellular signal mediating HPV. Further experiments aim to confirm the identity of the mitochondrial oxygen sensor using gene knockdown/silencing of candidate proteins. Identification of specific components of the oxygen sensing system may identify therapeutic targets for diseases in which oxygen-sensing is disordered, such as pulmonary arterial hypertension, chronic altitude sickness and chronic obstructive pulmonary disease.

Kv7 Channel Activation Underpins EPAC-Dependent Relaxations of Rat Arteries.

To establish the role of Kv7 channels in EPAC (exchange protein directly activated by cAMP)-dependent relaxations of the rat vasculature and to investigate whether this contributes to β-adrenoceptor-mediated vasorelaxations. Isolated rat renal and mesenteric arteries (RA and MA, respectively) were used for isometric tension recording to study the relaxant effects of a specific EPAC activator and the β-adrenoceptor agonist isoproterenol in the presence of potassium channel inhibitors and cell signaling modulators. Isolated myocytes were used in proximity ligation assay studies to detect localization of signaling intermediaries with Kv7.4 before and after cell stimulation. Our studies showed that the EPAC activator (8-pCPT-2Me-cAMP-AM) produced relaxations and enhanced currents of MA and RA that were sensitive to linopirdine (Kv7 inhibitor). Linopirdine also inhibited isoproterenol-mediated relaxations in both RA and MA. In the MA, isoproterenol relaxations were sensitive to EPAC inhibition, but not protein kinase A inhibition. In contrast, isoproterenol relaxations in RA were attenuated by protein kinase A but not by EPAC inhibition. Proximity ligation assay showed a localization of Kv7.4 with A-kinase anchoring protein in both vessels in the basal state, which increased only in the RA with isoproterenol stimulation. In the MA, but not the RA, a localization of Kv7.4 with both Rap1a and Rap2 (downstream of EPAC) increased with isoproterenol stimulation. EPAC-dependent vasorelaxations occur in part via activation of Kv7 channels. This contributes to the isoproterenol-mediated relaxation in mesenteric, but not renal, arteries.

Fasudil evokes vasodilatation of rat mesenteric vascular bed via Ca2+ channels and Rho/ROCK pathway

As a Rho kinase (ROCK) inhibitor, fasudil has been used in clinical trials of several cardiovascular diseases. This study was to investigate the vasorelaxant effect of fasudil on resistance arterial rings including mesenteric, renal, ventral tail and basilar artery. We also examined the potential mechanisms of its vasodilatory action using mesenteric artery rings.A DMT multiwire myograph system was used to test the tension of isolated small arteries. K+ channel blockers, NO-cGMP pathway blockers and Ca2+-free physiological salt solution (PSS) were employed to verify the underlying mechanisms.Fasudil (10−7–10−4M) relaxed four types of small artery rings pre-contracted by 60mmol/l KCl (pEC50: 6.01±0.09, 5.47±0.03, 5.54±0.04, and 5.72±0.10 for mesenteric, renal, ventral tail and basilar artery rings, respectively). Pre-incubation with fasudil (1, 3, or 10μmol/l) attenuated KCl (10–60mmol/l) and angiotensin II (Ang II; 1μmol/l)-induced vasoconstriction in mesenteric artery rings. Fasudil at the concentration of 10−6mol/l showed different relaxant potency in endothelium intact (pEC50:6.01±0.09) or denued (5.75±0.06) mesenteric artery. The influx and release of Ca2+ were inhibited by fasudil. In addition, fasudil could block the increased phosphorylation level of myosin light chain (MLC) and myosin-binding subunit of myosin phosphatase (MYPT1) induced by Ang II. However, pretreatment with various K+ channel blockers did not affect the relaxant effects of fasudil remarkably.The present results demonstrate that fasudil has a vasorelaxant effect on isolated rat resistance arteries, including mesenteric, renal, ventral tail and basilar artery, and may exert its action through the endothelium, Ca2+ channels, and the Rho/ROCK pathway.

Melamine Impairs Renal and Vascular Function in Rats.

Melamine incident, linked to nephrotoxicity and kidney stone in infants previously exposed to melamine-contaminated milk products, was unprecedentedly grave in China in 2008 as little was known about the mechanistic process leading to renal dysfunction in affected children. This study investigates whether neonatal ingestion of melamine leads to renal and vascular dysfunction in adulthood; and whether ingestion of melamine in pregnant rats leads to renal dysfunction in their offspring. A combination of approaches employed includes functional studies in rat renal arteries, renal blood flow measurement by functional magnetic resonance imaging, assay for pro-inflammatory and fibrotic biomarkers, immunohistochemistry, and detection of plasma and renal melamine. We provide mechanistic evidence showing for the first time that melamine reduces renal blood flow and impairs renal and vascular function associated with overexpression of inflammatory markers, transforming growth factor-β1, bone morphogenic protein 4 and cyclooxygenase-2 in kidney and renal vasculature. Melamine also induces renal inflammation and fibrosis. More importantly, melamine causes nephropathies in offsprings from pregnant rat exposed to melamine during pregnancy, as well as in neonatal rat exposed to melamine afterbirth, thus supporting the clinical observations of kidney stone and acute renal failure in infants consuming melamine-contaminated milk products.

Contribution of K(+) channels to endothelium-derived hypolarization-induced renal vasodilation in rats in vivo and in vitro.

We investigated the mechanisms behind the endothelial-derived hyperpolarization (EDH)-induced renal vasodilation in vivo and in vitro in rats. We assessed the role of Ca(2+)-activated K(+) channels and whether K(+) released from the endothelial cells activates inward rectifier K(+) (Kir) channels and/or the Na(+)/K(+)-ATPase. Also, involvement of renal myoendothelial gap junctions was evaluated in vitro. Isometric tension in rat renal interlobar arteries was measured using a wire myograph. Renal blood flow was measured in isoflurane anesthetized rats. The EDH response was defined as the ACh-induced vasodilation assessed after inhibition of nitric oxide synthase and cyclooxygenase using L-NAME and indomethacin, respectively. After inhibition of small conductance Ca(2+)-activated K(+) channels (SKCa) and intermediate conductance Ca(2+)-activated K(+) channels (IKCa) (by apamin and TRAM-34, respectively), the EDH response in vitro was strongly attenuated whereas the EDH response in vivo was not significantly reduced. Inhibition of Kir channels and Na(+)/K(+)-ATPases (by ouabain and Ba(2+), respectively) significantly attenuated renal vasorelaxation in vitro but did not affect the response in vivo. Inhibition of gap junctions in vitro using carbenoxolone or 18α-glycyrrhetinic acid significantly reduced the endothelial-derived hyperpolarization-induced vasorelaxation. We conclude that SKCa and IKCa channels are important for EDH-induced renal vasorelaxation in vitro. Activation of Kir channels and Na(+)/K(+)-ATPases plays a significant role in the renal vascular EDH response in vitro but not in vivo. The renal EDH response in vivo is complex and may consist of several overlapping mechanisms some of which remain obscure.

Two-photon imaging of endothelin-1-mediated intracellular Ca2 + handling in smooth muscle cells of rat renal resistance arteries

AimsEndothelin-1 (ET-1) is a potent vasoconstrictor which regulates the physiology of cardiorenal system. The aim of this study was to evaluate ET-1-mediated elevation of intracellular Ca2+ in smooth muscle cells (SMC) of renal resistance arteries. Main methodsIn in vitro studies of primary SMC, which were isolated from rat renal microvessels, the levels of intracellular Ca2+ were calculated from the ratio of emissions at 340 and 380nm after loading cells with Fura 2-AM dye. In ex vivo studies we used two-photon imaging of renal resistance arteries excised from rat kidneys and loaded with fluorescent Ca2+ indicator Fluo-4 AM. Key findingsThe two-photon imaging demonstrates that treatment of isolated rat renal resistance arteries with ET-1 causes a rapid increase of intracellular Ca2+ concentration in smooth muscle vasculature of these vessels. These ex vivo observations are in accordance with in vitro findings indicating that ET-1 mediates activation of TRPC channels and increases the level of intracellular Ca2+ in cultured SMC to 510±83nM. SignificanceET-1-mediated elevation of intracellular Ca2+ is strongly linked to renal microvascular contraction and is crucial for ET-1-induced contraction of SMC. The two-photon imaging of intracellular Ca2+ in intact SMC of rat renal resistance arteries is a powerful technique which allows the detailed ex vivo analysis of intracellular Ca2+ handling by ET-1, an important player in hypertension-related kidney diseases.

Resveratrol wine polyphenol relaxes rat renal artery in diabetic rats: the role of smooth muscle voltage-sensitive potassium channels

Resveratrol wine polyphenol relaxes rat renal artery in diabetic rats: the role of smooth muscle voltage-sensitive potassium channels

Inflammation and hypoxia linked to renal injury by CCAAT/enhancer-binding protein δ

Tubulointerstitial hypoxia plays a critical role in the pathogenesis of kidney injury, and hypoxia-inducible factor (HIF)-1 is a master regulator of cellular adaptation to hypoxia. Aside from oxygen molecules, factors that modify HIF-1 expression and functional operation remain obscure. Therefore, we sought to identify novel HIF-1-regulating genes in kidney. A short-hairpin RNA library consisting of 150 hypoxia-inducible genes was derived from a microarray analysis of the rat renal artery stenosis model screened for the effect on HIF-1 response. We report that CCAAT/enhancer-binding protein δ (CEBPD), a transcription factor and inflammatory response gene, is a novel HIF-1 regulator in kidney. CEBPD was induced in the nuclei of tubular epithelial cells in both acute and chronic hypoxic kidneys. In turn, CEBPD induction augmented HIF-1α expression and its transcriptional activity. Mechanistically, CEBPD directly bound to the HIF-1α promoter and enhanced its transcription. Notably, CEBPD was rapidly induced by inflammatory cytokines, such as IL-1β in a nuclear factor-κB-dependent manner, which not only increased HIF-1α expression during hypoxia, but was also indispensable for the non-hypoxic induction of HIF-1α. Thus our study provides novel insight into HIF-1 regulation in tubular epithelial cells and offers a potential hypoxia and inflammation link relevant in both acute and chronic kidney diseases.

A vasoconstrictor response to COX-1-mediated prostacyclin synthesis in young rat renal arteries that increases in prehypertensive conditions.

This study aimed to determine whether prostacyclin (PGI2) functions as an endothelium-derived contracting factor (EDCF) in young rat renal arteries, and, if so, we wanted to examine the underlying mechanism(s) and how it changes in prehypertensive conditions. Vessels from Wistar-Kyoto (WKY) and prehypertensive spontaneously hypertensive rats (SHRs) of 25-28 days of age were isolated for functional and biochemical analyses. Result showed that following NO synthase (NOS) inhibition PGI2 and the thromboxane-prostanoid (TP) receptor agonist U-46619 evoked contractions in young WKY renal arteries that were similar to those in prehypertensive SHRs. Meanwhile, the endothelial muscarinic receptor agonist ACh evoked an endothelium-dependent contraction under NOS-inhibited conditions and a production of the PGI2 metabolite 6-keto-PGF1α; both were sensitive to cyclooxygenase (COX) and/or COX-1 inhibition but higher in prehypertensive SHRs than in young WKYs. Interestingly, in WKY renal arteries PGI2 did not evoke relaxation even after TP receptor antagonism that diminished the contraction evoked by the agonist. Indeed, PGI2 (IP) receptors were not detected in the vessel with Western blot. Moreover, we noted that treatment with the nonselective COX inhibitor indomethacin, which was started at the prehypertensive stage, blunted the elevation of systolic blood pressure and reduced the heart-to-body ratio in SHR within 2 mo of treatment. These results demonstrate that due to scarcity of IP receptors, PGI2, which is derived mainly from COX-1-mediated metabolism, acts as an EDCF in young WKY renal arteries, and it increases in prehypertensive conditions. Also, our data revealed that COX inhibition starting from the prehypertensive stage has an antihypertensive effect in young SHRs.

G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity

Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K(+) currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2-250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein-coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein-coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone.

Protein kinase Cβ mediates downregulated expression of glucagon-like peptide-1 receptor in hypertensive rat renal arteries.

Glucagon-like peptide-1 (GLP-1) exerts its actions via activating GLP-1 receptor (GLP-1R). Our previous study showed a reduced GLP-1R expression in spontaneously hypertensive rat (SHR) renal arteries. The present study investigated the mechanisms underlying GLP-1R downregulation in hypertension. Intrarenal arteries of normotensive Wistar-Kyoto rat (WKY) and SHR were suspended in the myograph for force measurement. GLP-1R expression was evaluated by both immunofluorescence and western blotting. Protein kinase Cα (PKCα), PKCβ, PKCδ, and total PKC levels were assayed by western blotting. Immunofluorescence revealed reduced GLP-1R level in SHR renal arteries compared with WKY renal arteries. GLP-1R agonist exendin-4 induced concentration-dependent relaxations in WKY arteries, which mainly depended on the presence of endothelium. GLP-1R antagonist exendin 9-39 inhibited this relaxation in WKY arteries, whereas the relaxations were significantly less in SHR arteries. Ex-vivo treatment with PKC inhibitor GFX, PKCα and PKCβ inhibitor Gö6976, and PKCβ inhibitor hispidin but not PKCδ inhibitor rottlerin improved the impaired relaxations and restored the diminished GLP-1R expression in SHR arteries. Furthermore, PKCβ level was greater in SHR than WKY arteries, with no difference in PKCα, PKCδ, or total PKC expressions between two rat strains. Treatment with PKC-activating agent phorbol-12-myristate-13-acetate attenuated exendin-4-induced relaxations and reduced GLP-1R expression in WKY arteries, which were reversed by GFX, Gö6976, or hispidin. More relevantly, immunofluorescence of human renal arteries also showed a reduced GLP-1R level in hypertensive patients. The present results provide novel evidence that the reduced GLP-1R expression in SHR renal arteries is most likely mediated through PKCβ upregulation; the latter probably contributes to the impaired GLP-1R-mediated vasorelaxations in hypertension.

Synthesis and vasorelaxation evaluation of novel biphenyl–furocoumarin derivatives

A series of novel biphenyl–furocoumarin derivatives were synthesized and evaluated for their vasorelaxant activities in vitro against rat mesenteric artery, basilar artery, and renal artery ring models pre-contracted by high KCl. The results showed that compounds 12b, 13b, 13d, 14b, and 14d exhibited good broad spectrum vasorelaxant activities on three kinds of rat artery ring models compared to imperatorin. Noteworthy, most of the compounds had potent good selectivity against mesenteric artery in which compound 14c exhibited highest vasodilation activity (pEC50 = 6.44, Emax = 105.8 %). These were suggested that the introduction of biphenyl in imperatorin was useful to improve vasodilation activities. The preliminary structure–activity relationships studies revealed that larger halogen substituent R1 on phenyl ring and smaller nitrogenous base R2 were favorable for vasorelaxant activities. In addition, the CoMFA model showed acceptable correlative and predictive abilities (r2 = 0.875, q2 = 0.502).

Antagonism of Ca2+ influx via L-type Ca2+ channels mediates the vasorelaxant effect of Catharanthus roseus-derived vindorosine in rat renal artery.

Catharanthus roseus is a traditional herbal medicine used in Asian and African countries for the treatment of various diseases including hypertension. The present study examined possible cellular mechanisms for the relaxation of rat renal arteries induced by vindorosine extracted from C. roseus. Intrarenal arteries were isolated from 200-300 g male Sprague-Dawley rats and treated with different pharmacological blockers and inhibitors for the measurement of vascular reactivity on a Multi Myograph System. Fluorescence imaging by laser scanning confocal microscopy was utilized to determine the intracellular Ca(2+) level in the vascular smooth muscles of the renal arteries. Vindorosine in micromolar concentrations relaxes renal arteries precontracted by KCl, phenylephrine, 11-dideoxy-9α,11α-epoxymethanoprostaglandin F2α, and serotonin. Vindorosine-induced relaxations were unaffected by endothelium denudation or by treatment with the nitric oxide synthase inhibitor N (G)-nitro-L-arginine methyl ester hydrochloride, the guanylyl cyclase inhibitor 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one, the cyclooxygenase inhibitor indomethacin, or K(+) channel blockers such as tetraethylammonium ions, glibenclamide, and BaCl2. Vindorosine-induced relaxations were attenuated in the presence of 0.1 µM nifedipine (an L-type Ca(2+) channel blocker). Vindorosine also concentration-dependently suppressed contractions induced by CaCl2 (0.01-5 mM) in Ca-free 60 mM KCl solution. Furthermore, fluorescence imaging using fluo-4 demonstrated that 30 min incubation with 100 µM vindorosine reduced the 60 mM KCl-stimulated Ca(2+) influx in the smooth muscles of rat renal arteries. The present study is probably the first report of blood vessel relaxation by vindorosine and the possible underlying mechanisms involving the inhibition of Ca(2+) entry via L-type Ca(2+) channels in vascular smooth muscles.

Abstract 335: Prostaglandin I 2 Receptor Blockade Contracts Rat Renal Arteries and Facilitates Endothelium-Dependent Contractions.

Endothelium-dependent contractions are augmented in human hypertension and in isolated preparations, including renal arteries, from hypertensive animals. Renal vasomotor tone is essential in the regulation of arterial blood pressure and renal arteries are highly sensitive to vasoconstrictor prostanoids including prostacyclin, identified as endothelium-derived contracting factor (EDCF) activating thromboxane prostanoid (TP) receptors. However, the mechanisms underlying increased EDCF responses in hypertension are not completely resolved. Isometric tension was measured, in wire myographs, in renal arterial rings from spontaneously hypertensive (SHR) and age (38-52 weeks)-matched Wistar Kyoto (WKY) rats. Responses were compared under control conditions and after incubation with indomethacin (10 -5 mol/L, cyclooxygenase inhibitor), ketanserin (10 -6 mol/L, 5-HT 2 receptor antagonist), CAY10441 (10 -6 mol/L, prostaglandin I 2 [IP] receptor antagonist), and S18886 (10 -7 mol/L, TP receptor antagonist). All experiments were performed in the presence of L-NAME (3 x 10 -4 mol/L) to exclude effects of endothelium-derived NO. Surprisingly, CAY10441 (10 -10 to 10 -6 mol/L) contracted renal arterial rings from both strains; the contraction at the highest concentration was significantly larger in SHR (15±0.7 mN) than in WKY (10±1.5 mN) preparations ( n =10, P &lt;0.05). This phenomenon was blunted by indomethacin, ketanserin, but not S18886. After washout and re-incubation, EDCF responses to acetylcholine (10 -8 to 10 -4 mol/L) were similar between SHR and WKY preparations in the presence of CAY10441 ( E max 72.6±5.3% and 67.4±3.8% KCl, n =8), but significantly larger than under control conditions ( n =4-8, P &lt;0.05). S18886 prevented EDCF responses in the absence but not in the presence of CAY10441. Contractions to prostacyclin [in the presence of indomethacin] were facilitated by CAY10441 in rings from both strains and no longer prevented by S18886, although the TP receptor antagonist abolished contractions to U46619 (10 -11 to 3 x 10 -6 mol/L). These data suggest that: a) inhibitory IP receptors reduce renal vasomotor tone; b) IP and TP receptors form heterodimers; and c) endogenous serotonin contributes to the action of prostacyclin in rat renal arteries.