Receptor-operated Ca Channels Research Articles

Inhibitory mechanisms of docosahexaenoic acid on carbachol-, angiotensin II-, and bradykinin-induced contractions in guinea pig gastric fundus smooth muscle

We studied the inhibitory actions of docosahexaenoic acid (DHA) on the contractions induced by carbachol (CCh), angiotensin II (Ang II), and bradykinin (BK) in guinea pig (GP) gastric fundus smooth muscle (GFSM), particularly focusing on the possible inhibition of store-operated Ca2+ channels (SOCCs). DHA significantly suppressed the contractions induced by CCh, Ang II, and BK; the inhibition of BK-induced contractions was the strongest. Although all contractions were greatly dependent on external Ca2+, more than 80% of BK-induced contractions remained even in the presence of verapamil, a voltage-dependent Ca2+ channel inhibitor. BK-induced contractions in the presence of verapamil were not suppressed by LOE-908 (a receptor-operated Ca2+ channel (ROCC) inhibitor) but were suppressed by SKF-96365 (an SOCC and ROCC inhibitor). BK-induced contractions in the presence of verapamil plus LOE-908 were strongly inhibited by DHA. Furthermore, DHA inhibited GFSM contractions induced by cyclopiazonic acid (CPA) in the presence of verapamil plus LOE-908 and inhibited the intracellular Ca2+ increase due to Ca2+ addition in CPA-treated 293T cells. These findings indicate that Ca2+ influx through SOCCs plays a crucial role in BK-induced contraction in GP GFSM and that this inhibition by DHA is a new mechanism by which this fatty acid inhibits GFSM contractions.

Pharmacological Studies on the Ca2+ Influx Pathways in Platelet-Activating Factor (PAF)-Induced Mouse Urinary Bladder Smooth Muscle Contraction.

Platelet-activating factor (PAF) not only acts as a mediator of platelet aggregation, inflammation, and allergy responses but also as a constrictor of various smooth muscle (SM) tissues, including gastrointestinal, tracheal/bronchial, and pregnancy uterine SMs. Previously, we reported that PAF induces basal tension increase (BTI) and oscillatory contraction (OC) in mouse urinary bladder SM (UBSM). In this study, we examined the Ca2+ influx pathways involved in PAF-induced BTI and OC in the mouse UBSM. PAF (10-6 M) induced BTI and OC in mouse UBSM. However, the PAF-induced BTI and OC were completely suppressed by extracellular Ca2+ removal. PAF-induced BTI and OC frequencies were markedly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors (verapamil (10-5 M), diltiazem (10-5 M), and nifedipine (10-7 M)). However, these VDCC inhibitors had a minor effect on the PAF-induced OC amplitude. The PAF-induced OC amplitude in the presence of verapamil (10-5 M) was strongly suppressed by SKF-96365 (3 × 10-5 M), an inhibitor of receptor-operated Ca2+ channel (ROCC) and store-operated Ca2+ channel (SOCC), but not by LOE-908 (3 × 10-5 M) (an inhibitor of ROCC). Overall, PAF-induced BTI and OC in mouse UBSM depend on Ca2+ influx and the main Ca2+ influx pathways in PAF-induced BTI and OC may be VDCC and SOCC. Of note, VDCC may be involved in PAF-induced BTI and OC frequency, and SOCC might be involved in PAF-induced OC amplitude.

Platelet-activating factor (PAF) enhances guinea pig detrusor smooth muscle contractile activities by stimulating voltage-dependent Ca2+ channels and store-operated Ca2+ channels

We investigated the extracellular Ca2+ influx pathways involved in platelet-activating factor (PAF)-enhanced guinea pig detrusor smooth muscle (DSM) contractile activities. One micromolar PAF-enhanced DSM contractile activities were completely inhibited by extracellular Ca2+ removal and strongly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors. PAF-enhanced DSM contractile activities remaining in the presence of verapamil (10 μM) were not inhibited by LOE-908 (30 μM, an inhibitor of receptor-operated Ca2+ channels (ROCCs)), but were almost completely inhibited by SKF-96365 (30 μM, an inhibitor of store-operated Ca2+ channels (SOCCs) and ROCCs). These results suggest that VDCCs and SOCCs are responsible for PAF-enhanced DSM contractile activities.

Phenylephrine-Induced Contraction in Guinea Pig Thoracic Aorta Is Triggered by Stimulation of α1L-Adrenoceptors Functionally Coupled with Store-Operated Ca2+ Channels and Voltage-Dependent Ca2+ Channels.

We examined whether the α1L-adrenoceptor (AR), which shows low affinity (pA2 < 9) for prazosin (an α1-AR antagonist) and high affinity (pA2 ≈ 10) for tamsulosin/silodosin (α1A-AR antagonists), is involved in phenylephrine-induced contractions in the guinea pig (GP) thoracic aorta (TA). Intracellular signaling induced by α1L-AR activation was also examined by focusing on Ca2+ influx pathways. Tension changes of endothelium-denuded TAs were isometrically recorded and mRNA encoding α-ARs/Ca2+ channels and their related molecules were measured using RT-quantitative PCR. Phenylephrine-induced contractions were competitively inhibited by prazosin/tamsulosin, and their pA2 value were calculated to be 8.53/9.74, respectively. These contractions were also inhibited by silodosin concentration-dependently. However, the inhibition was not competitive fashion with the apparent pA2 value being 9.48. In contrast, phenylephrine-induced contractions were not substantially suppressed by L-765314 (an α1B-AR antagonist), BMY 7378 (an α1D-AR antagonist), yohimbine, and idazoxan (α2-AR antagonists). Phenylephrine-induced contractions were markedly inhibited by YM-254890 (a Gq protein inhibitor) or removal of extracellular Ca2+, and partially inhibited by verapamil (a voltage-dependent Ca2+ channel (VDCC) inhibitor). The residual contractions in the presence of verapamil were slightly inhibited by LOE 908 (a receptor-operated Ca2+ channel (ROCC) inhibitor) and strongly inhibited by SKF-96365 (a store-operated Ca2+ channel (SOCC) and ROCC inhibitor). Among the mRNA encoding α-ARs/SOCC-related molecules, α1A-AR (Adra1a)/Orai3, Orai1, and Stim2 were abundant in this tissue. In conclusion, phenylephrine-induced contractions in the GP TA can be triggered by stimulation of Gq protein-coupled α1L-AR, followed by activation of SOCCs and VDCCs.

Prostanoid TP receptor stimulation enhances contractile activities in guinea pig urinary bladder smooth muscle through activation of Ca2+ entry channels: Potential targets in the treatment of urinary bladder contractile dysfunction

AimsWe examined the potential stimulatory effects of U46619 (a prostanoid TP receptor agonist) and five prostanoids on the contractile activities of urinary bladder smooth muscle (UBSM), focusing on the role of the TP receptor and its associated Ca2+ influx routes to understand the roles of prostanoids in the regulation of UB contractile activity. Main methodsChanges in the basal tone and spontaneous contractile activity (amplitude and frequency) of isolated guinea pig UBSM were measured isotonically. The presence of TP receptors in UBSM was examined by RT-qPCR and immunofluorescence. Key findingsU46619, prostaglandin (PG) E2, PGF2α, and PGA2 enhanced UBSM basal tone and spontaneous contractile activities, which were measured as amplitudes and frequencies. The enhancing effects of U46619 were completely suppressed by SQ 29,548 (a TP receptor antagonist), which also partially suppressed the stimulating effects of other prostanoids. The expression of TP receptors in UBSMs was verified at the mRNA and protein level. The enhancing effects of U46619 completely disappeared in Ca2+-free solution. U46619-enhanced basal tone was completely suppressed by verapamil, an inhibitor of voltage-dependent Ca2+ channels (VDCCs), and verapamil strongly decreased the spontaneous contraction frequency. The spontaneous contractions remaining in the presence of verapamil were strongly suppressed by SKF-96365 (an inhibitor of receptor-operated Ca2+ channels (ROCCs)/store-operated Ca2+ channels (SOCCs)), but not by LOE-908 (an inhibitor of ROCCs). SignificanceProstanoids can enhance UBSM contractile activities and thus may be endogenous candidates for induction of detrusor overactivity. The TP receptor and TP-receptor-activated VDCCs/SOCCs are key molecules responsible for these effects.

Vasorelaxant Effects of Coriandrum SativumL. Extract on Rat Isolated Aorta

Aim: Coriander(Coriandrum sativum L.) is an annual plant belonging to the family Umbelliferae, which distributed in Central and Western Europe, India, Bangladesh, Thailand, China, and other Asian regions. Besides being edible, coriander is an important traditional medicine in India and China, and is used to treat circulatory disorders, such as respiratory, urinary tract, and skin diseases. In this study, we investigated the vasorelaxant effects of extracts from coriander and further studied to clarify their action mechanisms. Method: The aerial part of coriander, which analyzed the rutin content as its quality evaluand using a chromatographic method (HPLC), was cut and extracted with ethyl acetate or hot water. The extracts were concentrated under the reduced pressure. Vasorelaxant effects of these extracts were assessed on rat isolated aorta. The aorta was placed in a well-oxygenated bath of modified Krebs-Henseleit solution and the mechanical tension was measured isometrically. Results: These extracts showed vasorelaxant effects on aorta precontracted with 3×10-7 M norepinephrine (NE). The ethyl acetate extract showed biphasic vasorelaxation (fast and slowly developing relaxations) on isolated rat aortic rings with endothelium. Fast relaxation disappeared in deendothelialized or pre-administration of 10-4 M L-NG-monomethylarginine. Furthermore, the hot water extract showed only slowly developing relaxations independent in endothelium. After treatment with the hot water extract, NE-induced phasic vasoconstriction was not inhibited. While the hot water extract inhibited vasoconstrictions induced by a high concentration (60 mM) of K+ and also showed inhibitory effect on NE-induced vasoconstriction in the presence of nicardipine. Conclusion: These results suggest that the vasorelaxant effect of ethyl acetate extract of coriander on NE-induced vasoconstriction may be attributed to Nitric Oxide (NO) releasing dependent on endothelium. And the hot water extract of coriander showed vasorelaxant activities attributed to blocking of Ca2+ influx via voltage-depended Ca2+ channels (VDCs) and receptor-operated Ca2+ channels (ROCs), but not competing for the adrenergic receptor. Keywords: Coriandrum sativum; quality evaluation; vasorelaxant effects; aorta; endothelium; Ca2+ channel

Investigation of the mechanisms of Angelica dahurica root extract-induced vasorelaxation in isolated rat aortic rings.

BackgroundThe root of Angelica dahurica Bentham et Hooker (Umbelliferae) has been used as a traditional medicine for colds, headache, dizziness, toothache, supraorbital pain, nasal congestion, acne, ulcer, carbuncle, and rheumatism in China, Japan, and Korea. Interestingly, it has been used in the treatment of vascular diseases including hypertension. The aim of this study was to provide pharmacological evidence for the anti-hypertensive effect of A. dahurica by investigating the mechanism underlying its vasorelaxant effect.MethodsThe vasorelaxant effects of a 70 % methanol extract of the A. dahurica root (ADE) on rat thoracic aorta and its underlying mechanisms were assessed. Isolated rat aortic rings were suspended in organ chambers containing 10 ml Krebs-Henseleit (K-H) solution and placed between 2 tungsten stirrups and connected to an isometric force transducer. Changes in tension were recorded via isometric transducers connected to a data acquisition system.ResultsADE causes concentration-dependent relaxation in both endothelium-intact and endothelium-denuded aortic rings precontracted with phenylephrine (PE; 1 μM) or potassium (KCl; 60 mM) in K-H solution. And pre-treatment with ADE (1 mg/ml) inhibited calcium-induced vasocontraction of aortic rings induced by PE or KCl. However, ADE pre-treatment did not affect the contraction induced by PE or caffeine in Ca2+-free K-H solution.ConclusionsThese results suggested that the ADE has vasorelaxant effect and the vasorelaxant activity is mediated by endothelium-independent pathway that includes the blockade of extracellular calcium influx through the receptor-operated Ca2+ channel and voltage-dependent calcium channel pathways.

Brazilin isolated from the heartwood of Caesalpinia sappan L induces endothelium-dependent and -independent relaxation of rat aortic rings

Brazilin is one of the major constituents of Caesalpinia sappan L with various biological activities. This study sought to investigate the vasorelaxant effect of brazilin on isolated rat thoracic aorta and explore the underlying mechanisms. Endothelium-intact and -denuded aortic rings were prepared from rats. The tension of the preparations was recorded isometrically with a force displacement transducer connected to a polygraph. The phosphorylation levels of ERK1/2 and myosin light chain (MLC) were analyzed using Western blotting assay. Application of brazilin (10-100 μmol/L) dose-dependently relaxed the NE- or high K(+)-induced sustained contraction of endothelium-intact aortic rings (the EC50 was 83.51±5.6 and 79.79±4.57 μmol/L, respectively). The vasorelaxant effect of brazilin was significantly attenuated by endothelium removal or by pre-incubation with L-NAME, methylene blue or indomethacin. In addition, pre-incubation with brazilin dose-dependently attenuated the vasoconstriction induced by KCl, NE or Ang II. Pre-incubation with brazilin also markedly suppressed the high K(+)-induced extracellular Ca(2+) influx and NE-induced intracellular Ca(2+) release in endothelium-denuded aortic rings. Pre-incubation with brazilin dose-dependently inhibited the NE-stimulated phosphorylation of ERK1/2 and MLC in both endothelium-intact and -denuded aortic rings. Brazilin induces relaxation in rat aortic rings via both endothelium-dependent and -independent ways as well as inhibiting NE-stimulated phosphorylation of ERK1/2 and MLC. Brazilin also attenuates vasoconstriction via blocking voltage- and receptor-operated Ca(2+) channels.

Mechanisms of Vasorelaxation Induced by Hexahydrocurcuminin Isolated Rat Thoracic Aorta.

This study was designed to examine the vasorelaxant effects of hexahydrocurcumin (HHC), one of the major natural metabolites of curcumin from Curcuma longa, on rat isolated aortic rings, and the underlying mechanisms. Isometric tension of the aortic rings was recorded using organ bath system. HHC (1 nM to 1 mM) relaxed the endothelium-intact aortic rings pre-contracted with PE and KCl in a concentration-dependent manner. Removal of the endothelium did not alter the effect of HHC-induced relaxation. In Ca(2+)-free Krebs solution, HHC significantly inhibited the CaCl2-induced contraction in high K(+) depolarized rings and suppressed the transient contraction induced by PE and caffeine in a concentration-dependent manner. HHC was also observed to relax phobal-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC), precontracted aortic rings in a concentration-dependent manner with EC50 values equivalent to 93.36 ± 1.03 μM. In addition, pre-incubation with propranolol (a β-adrenergic receptor blocker) significantly attenuated the HHC-induced vasorelaxation. These results suggest that the vasorelaxant effect of HHC is mediated by the endothelium-independent pathway, probably because of the inhibition of extracellular Ca(2+) influx through voltage-operated Ca(2+) channels and receptor-operated Ca(2+) channels, the inhibition of Ca(2+) mobilization from intracellular stores, as well as inhibition of PKC-mediated Ca(2+)-independent contraction. Moreover, HHC produces vasorelaxant effects probably by stimulating the β-adrenergic receptor.

Chronic Hypoxia Increases Intracellular Ca(2+) Concentration via Enhanced Ca(2+) Entry Through Receptor-Operated Ca(2+) Channels in Pulmonary Venous Smooth Muscle Cells.

Hypoxic pulmonary hypertension (HPH) is characterized by pulmonary vascular remodeling. Intracellular Ca(2+)concentration ([Ca(2+)]i) is an essential signal for myocyte proliferation. Whether chronic hypoxia (CH) affects the basal [Ca(2+)]I and Ca(2+)entry through store- and/or receptor-operated calcium channels (SOCC, ROCC), and whether canonical transient receptor potential (TRPC) proteins are involved in CH-induced Ca(2+)influx and proliferation in pulmonary venous smooth muscle cells (PVSMCs) is examined. Rats were exposed to CH. PVSMCs were isolated from distal pulmonary veins. In freshly isolated PVSMCs, CH increased the basal [Ca(2+)]i; removal of Ca(2+)or application of SKF-96365 reversed the elevated [Ca(2+)]i, whereas nifedipine had no effect. Receptor-operated Ca(2+)entry (ROCE) was expressed in PVSMCs. In freshly isolated PVSMCs from CH rats, ROCE was enhanced, whereas store-operated Ca(2+)entry had no alteration. Furthermore, real-time polymerase chain reaction and western blotting showed that mRNA and protein expression level of TRPC6, but neither TRPC1 nor TRPC3, in pulmonary venous smooth muscle (PV) from CH rats and PVSMCs exposed to CH was greater than in normal PV and PVSMCs. The knockdown of TRPC6 in hypoxic PVSMCs with siRNA inhibited the enhanced ROCE and attenuated CH-induced PVSMCs proliferation. The enhanced Ca(2+)entry through ROCC, due to upregulated TRPC6, is a novel pathogenic mechanism contributing to the increased basal [Ca(2+)]iin PVSMCs and excessive PVSMC proliferation during the development of HPH.

Calcium affects OX1 orexin (hypocretin) receptor responses by modifying both orexin binding and the signal transduction machinery.

One of the major responses upon orexin receptor activation is Ca(2+) influx, and this influx seems to amplify the other responses mediated by orexin receptors. However, the reduction in Ca(2+) , often used to assess the importance of Ca(2+) influx, might affect other properties, like ligand-receptor interactions, as suggested for some GPCR systems. Hence, we investigated the role of the ligand-receptor interaction and Ca(2+) signal cascades in the apparent Ca(2+) requirement of orexin-A signalling. Receptor binding was assessed in CHO cells expressing human OX1 receptors with [(125) I]-orexin-A by conventional ligand binding as well as scintillation proximity assays. PLC activity was determined by chromatography. Both orexin receptor binding and PLC activation were strongly dependent on the extracellular Ca(2+) concentration. The relationship between Ca(2+) concentration and receptor binding was the same as that for PLC activation. However, when Ca(2+) entry was reduced by depolarizing the cells or by inhibiting the receptor-operated Ca(2+) channels, orexin-A-stimulated PLC activity was much more strongly inhibited than orexin-A binding. Ca(2+) plays a dual role in orexin signalling by being a prerequisite for both ligand-receptor interaction and amplifying orexin signals via Ca(2+) influx. Some previous results obtained utilizing Ca(2+) chelators have to be re-evaluated based on the results of the current study. From a drug discovery perspective, further experiments need to identify the target for Ca(2+) in orexin-A-OX1 receptor interaction and its mechanism of action.

Bradykinin Contracts Rat Urinary Bladder Largely Independently of Phospholipase C

Several receptor systems in the bladder causing detrusor smooth muscle contraction stimulate phospholipase C (PLC). PLC inhibition abolishes bladder contraction via P2Y6 but not that via M3 muscarinic receptors, indicating a receptor-dependent role of PLC. Therefore, we explored the role of PLC in rat bladder contraction by bradykinin. The PLC inhibitor U 73,122 [1-(6-[([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione] did not affect the bradykinin response to a significantly greater degree than its inactive analog U 73,343 [10 μM each; 1-(6-[-([17β]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-2,5-pyrrolidinedione], whereas the phospholipase D inhibitor butan-1-ol relative to its inactive control butan-2-ol caused a weak but significant inhibition (0.3% each). The cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone (300 μM) and the cyclooxygenase inhibitor indomethacin (10 μM) caused strong inhibition of the bradykinin response. The L-type Ca(2+) channel blocker nifedipine (10-100 nM) concentration-dependently caused strong inhibition, whereas only a small but significant inhibition was seen with SK&F 96,365 [10 μM; 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl], an inhibitor of receptor-operated Ca(2+) channels. Several protein kinase C inhibitors yielded an equivocal picture (inhibition by 10 μM bisindolylmaleimide I and 1 μM calphostin but not by 10 μM chelerythrine). The rho kinase inhibitor Y 27,632 [1-10 μM; trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide] caused a strong and concentration-dependent inhibition of the bradykinin response. Our data support that not only M3 but also bradykinin receptors cause bladder contraction by a largely PLC-independent mechanism. Both responses strongly involve L-type Ca(2+) channels and rho kinase, whereas only the bradykinin response additionally involves the phospholipase A2/cyclooxygenase pathway.

Phytochemical study guided by the myorelaxant activity of the crude extract, fractions and constituent from stem bark of Hymenaea courbaril L.

Ethnopharmacological relevanceHymenaea courbaril L. (Caesalpinoideae) is used in Brazilian folk medicine to treat anemia, kidney problems, sore throat and other dysfunctions of the respiratory system, such as bronchitis and asthma, although such properties are yet to be scientifically validated. Aim of the studyIn order to give a scientific basis to support the traditional use of Hymenaea courbaril, this study was designed to evaluate antioxidant, myorelaxant and anti-inflammatory properties of the ethanol extract from stem bark and its fractions. The myorelaxant effect of astilbin, a flavonoid isolated from the bioactive ethyl acetate fraction (EAF), has also been evaluated. Material and methodsIn the present study ethanol extract from stem bark (EEHC) and fractions were analyzed using bioassay-guided fractionation. The following activities were investigated: antioxidant by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, myorelaxant on rat tracheal smooth muscle, and anti-inflammatory using ovalbumin-induced leukocytosis and airway hyperresponsiveness in rats. ResultsThe results of the present investigation show that the whole extract of Hymenaea courbaril and some of its fractions strongly scavenged DPPH radical. The extract showed myorelaxant activity on rat trachea, being EAF its highest efficient fraction. Bio-guided study allowed the isolation of astilbin, a well-known flavonoid. The activity induced by this compound indicates that it may be partly responsible for the myorelaxant effect of EAF. EAF reduced contractions that depended on divalent cation inflow through voltage-operated Ca2+ channels (VOCCs) or receptor-operated Ca2+ channels (ROCCs), but it was more potent to inhibit VOCC- than ROCC-dependent contraction induced by Ca2+ addition in ACh-enriched Ca2+-free medium. Oral pretreatment of antigen-challenged animals with EAF prevented airway hyperresponsiveness on KCl-induced contraction and reduced the number of total white cells, particularly eosinophils and neutrophils in bronchoalveolar lavage. ConclusionsThis study provided scientific basis that Hymenaea courbaril presents potential antioxidant, myorelaxant and anti-inflammatory actions, which support its use in folk medicine to treat inflammatory airway diseases.

Pathogenic Role of Store-Operated and Receptor-Operated Ca2+ Channels in Pulmonary Arterial Hypertension

Pulmonary circulation is an important circulatory system in which the body brings in oxygen. Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that predominantly affects women. Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness are the major causes for the elevated pulmonary vascular resistance (PVR) in patients with PAH. The elevated PVR causes an increase in afterload in the right ventricle, leading to right ventricular hypertrophy, right heart failure, and eventually death. Understanding the pathogenic mechanisms of PAH is important for developing more effective therapeutic approach for the disease. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC migration and proliferation which lead to pulmonary vascular wall thickening and remodeling. It is thus pertinent to define the pathogenic role of Ca2+ signaling in pulmonary vasoconstriction and PASMC proliferation to develop new therapies for PAH. [Ca2+]cyt in PASMC is increased by Ca2+ influx through Ca2+ channels in the plasma membrane and by Ca2+ release or mobilization from the intracellular stores, such as sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER). There are two Ca2+ entry pathways, voltage-dependent Ca2+ influx through voltage-dependent Ca2+ channels (VDCC) and voltage-independent Ca2+ influx through store-operated Ca2+ channels (SOC) and receptor-operated Ca2+ channels (ROC). This paper will focus on the potential role of VDCC, SOC, and ROC in the development and progression of sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in PAH.

Vasorelaxant effects on rat aortic artery by two types of indole alkaloids, naucline and cadamine

Two indole alkaloids, naucline from Nauclea officinalis and cadamine from Ochreinauclea maingayii, were individually evaluated for vasorelaxant effects on phenylephrine-precontracted aortic rings. Naucline induced concentration-dependent relaxation in aortic rings. Respective EC50 values of naucline on aorta rings with/without endothelium did not show a significant difference, indicating that naucline-induced relaxation was independent of the endothelium. In further experiments with various inhibitors, naucline was found to possess inhibitory effects on both voltage-dependent Ca(2+) channel (VDC)- and receptor-operated Ca(2+) channel (ROC)-dependent Ca(2+) influx in smooth muscle. Cadamine showed concentration-dependent relaxation in endothelium-intact aortic rings, which was inhibited by addition of L-NMMA, NOS inhibitor. These results suggested that the vasorelaxant effect of cadamine is partly mediated through the increased release of NO from endothelial cells. In addition to NO involvement, vasorelaxation induced by cadamine was also attributed to inhibition of both VDC- and ROC-dependent Ca(2+) influx.

CRACing the Cluster

Platelet-derived growth factor (PDGF) signaling is implicated in a wide range of diseases, and PDGF drives the pathological responses in many vascular disorders and fibrotic diseases such as atherosclerosis, restenosis, pulmonary arterial hypertension, and pulmonary fibrosis. In vascular smooth muscle cells (VSMC), PDGF is a potent mitogen which promotes proliferation and migration and contributes to pathological vascular remodeling. Recent studies have demonstrated a role for PDGF in stromal interaction molecule 1 (STIM1)/Orai1-mediated Ca2+ influx in VSMC.1 Regulation of cytosolic Ca2+ concentration ([Ca2+]cyt) is critical for many cellular processes. A rise in [Ca2+]cyt is a major trigger for VSMC proliferation and contraction. One of the major routes of Ca2+ entry into cells is through store-operated Ca2+ (SOC) channels. On depletion of Ca2+ from the stores (sarcoplasmic or endoplasmic reticulum, SR/ER), a Ca2+ deficiency signal is transmitted to the SOC channels in the plasma membrane via STIM1. This causes the SOC to open, allowing Ca2+ to flow into the cytosol, a process referred to as store-operated Ca2+ entry (SOCE). The cytosolic Ca2+ is then sequestered into the stores by the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA), thus replenishing the stores. Studies have demonstrated a role for Ca2+ release-activated Ca2+ (CRAC) channels in SOCE, with STIM1 and Orai1 as the major components.2 Article, see p 66 The majority of STIM1 is expressed in the SR/ER membrane, where it senses decreased Ca2+ concentration in the SR/ER when inositol 1,4,5-triphosphate (IP3)-mediated activation of IP3 receptor induces Ca2+ release. STIM1 then undergoes a conformational change allowing STIM1 to multimerize and translocate …

Alstiphyllanines I–O, ajmaline type alkaloids from Alstonia macrophylla showing vasorelaxant activity

Seven new ajmaline type alkaloids, alstiphyllanines I–O (1–7) were isolated from the leaves of Alstonia macrophylla together with six related alkaloids (8–13). Structures and stereochemistry of 1–7 were fully elucidated and characterized by 2D NMR analysis. A series of alstiphyllanines I–O (1–7) as well as the known ajmaline type alkaloids (8–13) showed that they relaxed phenylephrine (PE)-induced contractions against rat aortic ring. Among them, vincamedine (10) showed potent vasorelaxant activity, which may be mediated through inhibition of Ca2+ influx through voltage-dependent Ca2+ channels (VDCs) and/or receptor-operated Ca2+ channels (ROCs) as well as partially mediated the NO release from endothelial cells. The presence of substituents at both N-1 and C-17 may be important to show vasorelaxation activity.

Antispasmodic effects of a new kaurene diterpene isolated from Croton argyrophylloides on rat airway smooth muscle

The effects of rel-(1S,4aS,7S,8aS)-7-(1-vinyl)-tetradecahydro-1,4a-dimethylphenanthrene-7,8a-carbolactone-1-carboxylic acid (TCCA), a new ent-kaurene diterpene isolated from Croton argyrophylloides, on rat tracheal preparations were investigated. Tracheae were removed and cut into two-cartilage segments that were mounted in organ baths containing Tyrode's solution. TCCA reduced the contractions induced by electrical field stimulation, relaxed K(+)-induced contractions, and inhibited both phasic and tonic components of the K(+)- and ACh-induced contractions. TCCA reduced the serotonin-induced contraction, abolished that evoked by K(+) in the presence of epinephrine, and also reduced the ACh-induced contractions under Ca(2+)-free conditions. TCCA blocked contractions that depend on divalent cation inflow through voltage-operated Ca(2+) channels (VOCCs) and receptor-operated Ca(2+) channels (ROCCs), but had greater potency to block VOCC- than ROCC-dependent contractions or contractions induced by ACh in Ca(2+)-free conditions. TCCA relaxed the phorbol 12,13 dibutyrate (1 µm) induced contraction, but with slight potency. TCCA induces an antispasmodic effect through several mechanisms including blockade of either VOCCs (with greater potency) or ROCCs, blockade of IP(3)-induced Ca(2+) release from sarcoplasmic reticulum (with intermediate potency) and reduction of the sensitivity of contractile proteins to Ca(2+).

Analysis of the Mechanisms Underlying the Vasorelaxant Action of Coptisine in Rat Aortic Rings

The aim of the present study was to evaluate the vasorelaxant effects of coptisine and its possible mechanisms in isolated rat aortic rings. Coptisine was evaluated on isolated rat aortic rings precontracted with norepinephrine (NE) and KCl. The mechanisms were evaluated in the presence or absence of specific pharmacological inhibitors. Coptisine (1 ~ 200 μM) relaxed NE (1 μM) or KCl (60 mM) induced sustained contraction with pEC(50) values of 4.49 ± 0.48 and 4.85 ± 0.57 in a concentration dependent manner. Pretreatment with coptisine (10, 50 or 100 μM) also inhibited concentration-response curves to NE and KCl. The vasorelaxant effect of coptisine was attenuated significantly by endothelium removal, and incubation with Nω-nitro-L-arginine methyl ester (L-NAME, 100 μM), methylene blue (10 μM) and indomethacin (5 μM) partially reduced the vasorelaxant effect of coptisine. In endothelium-denuded rings, the vasorelaxant effect of coptisine was reduced significantly by 4-aminopyridine (4-AP, 100 μM), but not glibenclamide (10 μM) ortetraethylammonium (TEA, 5 mM). Coptisine also reduced NE-induced transient contraction in Ca(2+)-free solution, and inhibited contraction induced by increasing external calcium in Ca(2+)-free medium plus 60 mM KCl. It was concluded that coptisine induced both endothelium-dependent and -independent relaxation in rat aortic rings. The NO-cGMP mediated pathway may be involved in the endothelium-dependent relaxation and in the activation of voltage-dependent K(+) channels, contributing in part to the endothelium-independent relaxation bycoptisine. Coptisine also blocks extracellular Ca(2+) influx by interacting with both voltage- and receptor-operated Ca(2+) channels.

Muscular effects of orexin A on the mouse duodenum: mechanical and electrophysiological studies

Orexin A (OXA) has been reported to influence gastrointestinal motility, acting at both central and peripheral neural levels. The aim of the present study was to evaluate whether OXA also exerts direct effects on the duodenal smooth muscle. The possible mechanism of action involved was investigated by employing a combined mechanical and electrophysiological approach. Duodenal segments were mounted in organ baths for isometric recording of the mechanical activity. Ionic channel activity was recorded in current- and voltage-clamp conditions by a single microelectrode inserted in a duodenal longitudinal muscle cell. In the duodenal preparations, OXA (0.3 μM) caused a TTX-insensitive transient contraction. Nifedipine (1 μM), as well as 2-aminoethyl diphenyl borate (10 μM), reduced the amplitude and shortened the duration of the response to OXA, which was abolished by Ni(2+) (50 μM) or TEA (1 mM). Electrophysiological studies in current-clamp conditions showed that OXA caused an early depolarization, which paralleled in time the contractile response, followed by a long-lasting depolarization. Such a depolarization was triggered by activation of receptor-operated Ca(2+) channels and enhanced by activation of T- and L-type Ca(2+) channels and store-operated Ca(2+) channels and by inhibition of K(+) channels. Experiments in voltage-clamp conditions demonstrated that OXA affects not only receptor-operated Ca(2+) channels, but also the maximal conductance and kinetics of activation and inactivation of Na(+), T- and L-type Ca(2+) voltage-gated channels. The results demonstrate, for the first time, that OXA exerts direct excitatory effects on the mouse duodenal smooth muscle. Finally, this work demonstrates new findings related to the expression and kinetics of the voltage-gated channel types, as well as store-operated Ca(2+) channels.