Contraction Of Rat Mesenteric Artery Research Articles

Structurally diverse monoterpene indole alkaloids with vasorelaxant activities from the branches of Alstonia scholaris

Seven undescribed monoterpene indole alkaloids alstoscholarinines A‒G, along with nineteen known alkaloids, were isolated from the branches of Alstonia scholaris (L.) R. Br. The isolated alkaloids were classified into ten framework types. The structures of the undescribed alkaloids were elucidated by extensive spectroscopic analysis, ECD calculation, and single-crystal X-ray diffraction analysis. Alstoscholarinine A is an unreported and unusual monoterpene indole alkaloid incorporating three nitrogen atoms, characterized by a compact 6/5/6/6/6/5 hexacyclic system bearing a piperidine ring and a unique oxazolidine ring. Alstoscholarinine B represents the first naturally C-17 nor-isositsirikine-type alkaloid. Plausible biosynthetic pathways of alstoscholarinines A and B were proposed. All isolates were evaluated for their vasorelaxant activities against phenylephrine-induced contraction of rat mesenteric arteries. Among them, seven alkaloids showed significant vasorelaxant activities with EC50 values less than 10 μM. Importantly, the akuammicine-type alkaloids in this study showed much better vasorelaxant activities than other framework type alkaloids, indicating that this type of alkaloid may be a valuable source for the discovery of vasodilators. A preliminary structure-activity relationship for vasorelaxant activities of the isolated akuammicine-type alkaloids is also discussed.

Two new phenylpropanoids from the leaves of Rauvolfia vomitoria

ObjectiveStudy on the non-alkaloid chemical constituents with novel structure and their biological activities from the w-butanol fraction of the leaves of Rauvolfia vomitoria. MethodsThe w-butanol fraction of R. vomitoria was separated by silica gel, ODS, and Sephadex LH-20 chromatography column, as well as semi-preparative HPLC to obtain five compounds. Their structures were identified by extensive spectroscopic analysis, including HRESIMS, 1D and 2D NMR, and ECD analysis. All the isolated compounds were evaluated their AChE inhibitory activities by Ellman’s method with slight modification, their vasorelaxant activities against phenylephrine-induced contraction of rat mesenteric arteries, and their inhibitory activity of a-glucosidase employing pNPG as substrate. ResultsTwo new phenylpropanoids (1-2) along with three known compounds, methyl trans-3,4,5-trimethoxycinnamate (3), methyl cis-3,4,5-trimethoxycinnamate (4), and 3,4,5-trimethoxybenzoic acid methyl ester (5), were isolated from the leaves of R.vomitoria. The five isolated compounds did not show significant tested activity. ConclusionChemical investigation of the leaves of R. vomitoria led to the isolation and identification of two new phenylpropanoids (1–2), expanding the study of non-alkaloids in the genus of Rauvolfia and enriching the chemical diversity of this genus.

New phenylpropanoids and monoterpene alkaloids with vasorelaxant activities from the branches of Alstonia scholaris

Two new phenylpropanoids (1–2), one new nor-monoterpenoid alkaloid (3), one new monoterpene alkaloid (4), together with nine known compounds (5–13) were obtained from the branches of Alstonia scholaris. The structures of the undescribed compounds were determined by extensive spectroscopic analysis. Alkaloid 3 represented the first example of C-4 methylated nor-monoterpenoid alkaloids. A possible biosynthetic pathway for this new type of monoterpene alkaloids was proposed. All the isolates were evaluated for vasorelaxant activity against phenylephrine-induced contraction of rat mesenteric arteries. Compounds 1, 4, 9, 12, and 13 showed significant vasorelaxant activity with relaxation rates above 90% at 200 μM and exhibited moderate vasorelaxant activity with IC50 values ranging from 41.87 to 93.30 μM by further studies. It was the first report on the potential vasorelaxant activity of monoterpene alkaloids. Monoterpene alkaloids 3 and 4 may be served as the potential lead compounds for the discovery of vasodilators, due to their simple and optimizable structures.

Mechanism of α1-Adrenergic Receptor-Induced Increased Contraction of Rat Mesenteric Artery in Aging Hypertension Rats

Introduction: Vasoconstriction is triggered by an increase in intracellular-free calcium concentration. Growing evidence indicates that contraction is also regulated by calcium-independent mechanisms involving RhoA-Rho kinase (ROCK), protein kinase C (PKC), and so on. In this study, we studied the changes of vascular reactivity as well as the underlying signaling pathways in aging spontaneously hypertensive rats (SHRs). Methods: The artery tension induced by α1-adrenergic receptor activator (α1-AR) phenylephrine (PE) was measured in the absence or presence of myosin light chain kinase (MLCK), PKC, and ROCK inhibitors. The α1-AR, PKC, ROCK, phosphorylation of myosin light chain (MLC), and PKC-potentiated phosphatase inhibitors of 17 kDa (CPI-17) of rat mesenteric arteries were analyzed at the mRNA level or protein level. Results: The vascular tension measurements showed that there was a significant increase in the mesenteric artery contraction induced by PE in old SHR. MLCK inhibitor ML-7 can similarly inhibit PE-induced vasoconstriction. PKC inhibitor GF109203X has the weakest inhibitory effect on PE-induced contraction in old SHR. At the presence of ROCK inhibitor H1152, PE-induced contraction was significantly reduced in young Wistar-Kyoto (WKY) rats, but this phenomenon disappeared in other rats. Furthermore, in old SHR the protein expression of α1-AR decreased and phosphorylation of MLC and CPI-17 were upregulated and MLC phosphatase (MLCP) activity was significantly lower. The expressions of PKC were upregulated in SHR and old rats. In addition, the expression of ROCK-1 was decreased and ROCK-2 was significantly upregulated with age in SHR. Conclusion: In aging hypertension, the expression/activity of PKC or ROCK-2/CPI-17 excessively increased, MLCP activity decreased and MLC phosphorylation enhanced, leading to increased α1-AR-induced vasoconstriction.

The inhibition effect of uncarialin A on voltage-dependent L-type calcium channel subunit alpha-1C: Inhibition potential and molecular stimulation

Cardiovascular diseases, such as hypertension and cardiac failure, have become the most major and global cause for threatening human health in recent years. Uncaria rhynchophylla as a traditional Chinese medicine is widely used to treat hypertension for a long history, whereas its medicinal effective components and potential action mechanism are uncertain. Therefore, twenty-four alkaloids (1–24) isolated from U. rhynchophylla were assayed for their relaxant effects against phenylephrine (Phe)-induced contraction of rat mesenteric arteries. Among them, we surprisingly found that uncarialin A (21) exhibited most potent relaxation effect against Phe-induced contraction (IC50 = 0.18 μM) in the manner of independent on endothelium-derived vasorelaxing factors and endothelium. All the experiments including measurement of Ca2+ in vascular smooth muscle cells (VSMCs) by fluorescence microscopy, whole-cell path clamp, molecular docking, and molecular dynamics, demonstrated that uncarialin A (21) could significantly inhibit L-type calcium channel subunit alpha-1C (Cav1.2) via the hydrogen bond interaction with amino acid residue Met1186, allowing the inhibition of Ca2+ inward current. Our results suggested that uncarialin A (21) could be served as a potential L-type Cav1.2 blocker in the effective treatment of cardiovascular diseases.

Trehalose Lipid Biosurfactant Reduced Cancer Cell Viability but Did not Affect the Isometric Contraction of Rat Mesenteric Arteries in vitro

Trehalose Lipid Biosurfactant Reduced Cancer Cell Viability but Did not Affect the Isometric Contraction of Rat Mesenteric Arteries in vitro

Mechanism of α1-adrenergic receptor-induced increased contraction of rat mesenteric artery in aging and hypertension

When α1 adrenergic receptor (α1-AR) binds to the agonist, the G protein-coupled receptor signal is activated, leading to vasoconstriction, a process associated with increased intracellular calcium concentration and activation of the myosin light chain kinase (MLCK) and protein kinase C (PKC)-mediated signaling pathways. In our previous experiments, α1-AR activator phenylephrine(PE) resulted in a significant increase in maximal contraction of the mesenteric artery in elderly rats. However, the expression of α1-AR decreased in these tissues. Therefore, the purpose of this study is to further elucidate the changing mechanism of α1-AR activation leading to vasoconstriction in aging and hypertension.

Taurochenodeoxycholate relaxes rat mesenteric arteries through activating eNOS: Comparing with glycochenodeoxycholate and tauroursodeoxycholate

The bile acids (BAs) and their conjugates have vascular activities and the serum levels of BAs and their conjugates are increased in liver diseases. In the present study, we examined the in vitro vasoactivities of BAs conjugates taurochenodeoxycholate (TCDC) (5–80µM), glycochenodeoxycholate (GCDC) (20–150µM) and tauroursodeoxycholate (TUDC) (20–150µM) in rat mesenteric arteries and thoracic aorta. The isometric tension of rat mesenteric arteries and thoracic aorta was recorded by using multi-wire myograph systems. TCDC induced significant concentration-dependent relaxation in endothelium-intact but not endothelium-denuded rat mesenteric arteries pre-contracted with phenylephrine (PE). TCDC also showed vasorelaxant effects on high K+ induced contraction in rat mesenteric arteries. L-NAME treatment inhibited TCDC-induced relaxation in mesenteric arteries pre-contracted with PE. Acute treatment with TCDC increased protein expression of P-eNOS (ser1177) in human umbilical vein endothelial cells. GCDC dose-dependently relaxed PE-induced vasoconstriction in both endotheium-intact and endothelium-denuded rat mesenteric arteries, but GCDC showed no effect on high K+-induced vasoconstriction. Both GCDC and TCDC showed no apparent relaxation on PE and high K+-induced vasoconstriction in rat thoracic aorta. TUDC showed no effect on PE and high K+-induced vasoconstriction in rat mesenteric arteries and thoracic aorta. The study demonstrates that TCDC relaxes rat mesenteric arteries through activating eNOS. TCDC might be the major BAs conjugate for vasorelaxation in vivo.

Facilitation of serotonin-induced contraction of rat mesenteric artery by ketamine.

Ketamine is an anesthetic with hypertensive effects, which make it useful for patients at risk of shock. However, previous ex vivo studies reported vasodilatory actions of ketamine in isolated arteries. In this study, we reexamined the effects of ketamine on arterial tones in the presence and absence of physiological concentrations of 5-hydroxytryptamine (5-HT) and norepinephrine (NE) by measuring the isometric tension of endothelium-denuded rat mesenteric arterial rings. Ketamine little affected the resting tone of control mesenteric arterial rings, but, in the presence of 5-HT (100~200 nM), ketamine (10~100 µM) markedly contracted the arterial rings. Ketamine did not contract arterial rings in the presence of NE (10 nM), indicating that the vasoconstrictive action of ketamine is 5-HT-dependent. The concentration-response curves (CRCs) of 5-HT were clearly shifted to the left in the presence of ketamine (30 µM), whereas the CRCs of NE were little affected by ketamine. The left shift of the 5-HT CRCs caused by ketamine was reversed with ketanserin, a competitive 5-HT2A receptor inhibitor, indicating that ketamine facilitated the activation of 5-HT2A receptors. Anpirtoline and BW723C86, selective agonists of 5-HT1B and 5-HT2B receptors, respectively, did not contract arterial rings in the absence or presence of ketamine. These results indicate that ketamine specifically enhances 5-HT2A receptor-mediated vasoconstriction and that it is vasoconstrictive in a clinical setting. The facilitative action of ketamine on 5-HT2A receptors should be considered in ketamine-induced hypertension as well as in the pathogenesis of diseases such as schizophrenia, wherein experimental animal models are frequently generated using ketamine.

Ca2+ dependent but PKC independent signalling mediates UTP induced contraction of rat mesenteric arteries.

Uridine triphosphate (UTP) can be released from damaged cells to cause vasoconstriction. Although UTP is known to act through P2Y receptors and PLC activation in vascular smooth muscle, the role of PKC in generating the response is somewhat unclear. Here we have used Tat-linked membrane permeable peptide inhibitors of PKC to assess the general role of PKC and also of specific isoforms of PKC in the UTP induced contraction of rat mesenteric artery. We examined the effect of PKC inhibition on UTP induced contraction, increased cytoplasmic Ca2+ and reduction of K+ currents and found that PKC inhibition caused a relatively small attenuation of contraction but had little effect on changes in cytoplasmic Ca2+. UTP attenuation of both voltage-gated (Kv) and ATP-dependent (KATP) K+ currents was abolished when intracellular Ca2+ was decreased from 100 to 20 nM. PKC inhibition reduced slightly the ability of UTP to attenuate Kv currents but had no effect on KATP current inhibition. In conclusion, both UTP induced contraction of mesenteric artery and the inhibition of Kv and KATP currents of mesenteric artery smooth muscle cells by UTP are relatively independent of PKC activation; furthermore, the inhibition of both Kv and KATP currents requires intracellular Ca2+.

Long-lasting physiological antagonism of calcitonin gene-related peptide towards endothelin-1 in rat mesenteric arteries and human coronary arteries

Endothelin-1 causes long-lasting contraction via endothelin type A receptor (ETAR) in isolated rat mesenteric arteries (RMA) that cannot be readily terminated by removing the agonist, or by adding the ETAR antagonist BQ123 or the NO donor sodium nitroprusside. It could be terminated by adding calcitonin-gene related peptide (CGRP), most likely because CGRP causes ET-1/ETAR dissociation. Here we investigated this phenomenon in human coronary microarteries (HCMA). We simultaneously verified the effects of CGRP in RMA and HCMA towards other vasoconstrictors, i.e., the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U46619 (9,11-dideoxy-11α,9α-epoxy-methano-prostaglandin F2α) and KCl. Long-lasting contraction (remaining after washing away the agonist) was observed for ET-1 in RMA, but not HCMA. Constrictions to phenylephrine, U46619 or KCl did not last upon washing. When added on top of ET-1-initiated contraction in RMA, CGRP effectively counteracted vasoconstriction, i.e., it caused full relaxation. Inhibitory effects of CGRP were also observed when briefly exposing RMA and HCMA to CGRP 1h before the addition of ET-1. Similar inhibitory effects of transient CGRP pre-incubation were seen towards phenylephrine, U46619 or KCl in RMA and HCMA. In conclusion, our data imply that CGRP, like ET-1, causes long-lasting effects that remain apparent up to 1h after its removal from the organ bath. Thus, in addition to the reported dissociation of ET-1/ETAR complexes, CGRP causes long-lasting non-selective arterial smooth muscle relaxation that may add to the neuropeptide being a physiological antagonist of arterial effects of ET-1. Long-lasting, washout-resistant ET-1/ETAR interaction does not occur in HCMAs.

Prostaglandin E2-prostanoid EP3 signal induces vascular contraction via nPKC and ROCK activation in rat mesenteric artery

Prostaglandin E2 (PGE2) is one major prostanoid produced under inflammatory situation. Although PGE2 is known to induce vascular contraction, its detailed mechanism remains unknown. In the present study, we investigated the signaling pathway underlying PGE2-induced smooth muscle contraction in rat mesenteric artery. PGE2 (0.3–30μM) concentration-dependently caused contraction in endothelium-denuded artery. RT-PCR showed that this artery expresses mRNAs for all four prostanoid EP receptors (prostanoid EP1–4). Among selective agonists for PGE2 receptors, only a prostanoid EP3 receptor agonist, ONO-AE-248 (0.3–30μM) induced contraction. Consistently, pretreatment with a prostanoid EP3 antagonist (L-798106, 1μM) significantly but not completely inhibited the PGE2-induced contraction. Interestingly, pretreatment with a prostanoid FP antagonist (AL8810, 1μM) or a TP antagonist (SQ29548, 10nM) also partially inhibited the PGE2-induced contraction. Since ONO-AE-248 (10μM) did not influence intracellular Ca2+ concentration in mesenteric artery, we next examined the involvement of Ca2+-independent contractile pathway including PKCs and ROCK in prostanoid EP3-mediated contraction. Pretreatment with bisindolyl-maleimide I (a general PKC inhibitor, 1μM), Ro-31-8425 (a conventional PKC and PKCε inhibitor, 1μM), rottlerin (a selective PKCδ inhibitor, 1μM) and Y-27632 (a ROCK inhibitor, 1μM) but not Go 6976 (a conventional PKC inhibitor, 1μM) attenuated 10μM ONO-AE-248-induced vascular contraction. In western blot analysis, we confirmed that the treatment with ONO-AE-248 (10μM, 30min) phosphorylated PKCδ (Thr505) and PKCε (Ser729). These results suggest that PGE2 induces vascular smooth muscle contraction via prostanoid EP3, FP and TP receptors in rat mesenteric artery. Prostanoid EP3-mediated contraction is ascribed to Ca2+-independent contractile pathway including PKCδ, ε and ROCK.

Selectivity and Specificity of Sphingosine-1-Phosphate Receptor Ligands: Caveats and Critical Thinking in Characterizing Receptor-Mediated Effects

Receptors for sphingosine-1-phosphate (S1P) have been identified only recently. Their medicinal chemistry is therefore still in its infancy, and few selective agonists or antagonists are available. Furthermore, the selectivity of S1P receptor agonists or antagonists is not well established. JTE-013 and BML-241 (also known as CAY10444), used extensively as specific S1P2 and S1P3 receptors antagonists respectively, are cases in point. When analyzing S1P-induced vasoconstriction in mouse basilar artery, we observed that JTE-013 inhibited not only the effect of S1P, but also the effect of U46619, endothelin-1 or high KCl; JTE-013 strongly inhibited responses to S1P in S1P2 receptor knockout mice. Similarly, BML-241 has been shown to inhibit increases in intracellular Ca2+ concentration via P2 receptor or α1A-adrenoceptor stimulation and α1A-adrenoceptor-mediated contraction of rat mesenteric artery, while it did not affect S1P3-mediated decrease of forskolin-induced cyclic AMP accumulation. Another putative S1P1/3 receptor antagonist, VPC23019, does not inhibit S1P3-mediated vasoconstriction. With these examples in mind, we discuss caveats about relying on available pharmacological tools to characterize receptor subtypes.

Mechanisms for perivascular adipose tissue-mediated potentiation of vascular contraction to perivascular neuronal stimulation: The role of adipocyte-derived angiotensin II

In rat mesenteric arteries we have recently found that perivascular adipose tissue (PVAT) promoted vasoconstriction to perivascular neuronal activation (by electrical field stimulation, EFS) through generation of superoxide. In this study, we examined the role of adipocyte-generated angiotensin II in PVAT-mediated potentiation of contraction to nerve stimulation. In rat mesenteric PVAT, the presence of angiotesinogen and angiotensin I-converting enzyme (ACE) mRNA was confirmed by RT-PCR. Immunohistochemical staining showed the presence of angiotensin II in mesenteric PVAT. In rat mesenteric arteries, treatment of the vessels with an ACE inhibitor (enalaprilat) or angiotensin II type 1 receptor antagonist (candesartan) reduced PVAT-mediated potentiation of EFS-induced contraction. Exogenously applied angiotensin II enhanced EFS-induced contraction in arteries without PVAT, but not in the arteries with intact PVAT. Chronic treatment with an ACE inhibitor quinapril (14 days) lowered blood pressure and alleviated the potentiation effects of PVAT in EFS-induced contraction. Mesenteric arteries from quinapril-treated group now exhibited the potentiation response to exogenously applied angiotensin II in arteries with intact PVAT to a comparable level as in arteries with PVAT removed. Treatment with hydralazine reduced blood pressure to the same level as quinapril treatment, but did not affect PVAT-associated potentiation of vasoconstriction to EFS and the response to exogenously applied angiotensin II in PVAT-intact arteries. These results showed that adipocyte-derived angiotensin II is critically involved in PVAT-mediated potentiation of EFS-evoked contraction in rat mesenteric arteries.

Purinoceptor Signaling in Arterial Smooth Muscle is Regulated by G-Protein-Coupled Recptor Kinase-2

The regulation of arterial smooth muscle cell (SMC) contraction by adenine and uridine nucleotides, plays a key role in controlling systemic blood pressure. In SMCs, UTP activates P2Y receptors (subtypes 2/4/6), which couple via Gαq/11-proteins to stimulate phospholipase C, increasing IP3/Ca2+ concentrations and leading to SMC contraction. Continuous or repeated receptor stimulation reduces responsiveness to further stimulation, a process termed desensitization. Receptor desensitization is often regulated by G protein-coupled receptor kinases (GRKs), which phosphorylate receptors, enhancing their interaction with β-arrestins and uncoupling them from G-proteins.We investigated the regulation of receptors responding to UTP, which mediates concentration-dependent contraction in rat mesenteric arteries. To characterize adaptations that occur on repeated UTP additions, changes in IP3 and [Ca2+]i were assessed using single-cell imaging. Receptor desensitization was assessed by challenging mesenteric SMCs with an EC50 concentration of UTP (10 μM) for 30 sec before (R1) and after (R2) the addition of a maximal UTP concentration (Rmax, 100 μM, 30 sec) with 5 min washout periods. The change in R2 relative to R1 was used to characterize P2Y receptor desensitization. By extending the washout period after Rmax a time-dependent recovery of IP3/Ca2+ responses were observed. To evaluate the involvement of individual GRKs in this process, cells were transfected with catalytically-inactive, dominant-negative GRK mutants.Using IP3 generation to indicate receptor recovery (R2/R1%), over-expression of D110A,K220RGRK3 (34±4%), K215RGRK5 (38±7%), or K215RGRK6 (29±8%) caused similar reductions in IP3 levels to those in empty-vector-transfected cells (23±5%). In contrast, expression of D110A,K220RGRK2 (58±7%) markedly attenuated receptor desensitization (n=10-19 cells, >3 animals). Furthermore, siRNA-mediated knockdown (>75%) of GRK2 protein also attenuated agonist-induced receptor desensitization compared to control (68±8% versus 29±6%, respectively, n=8-12). In conclusion, this work implicates GRK2 as the pre-eminent GRK isoenzyme regulating UTP signaling in SMCs.

Superoxide anion mediates angiotensin II-induced potentiation of contractile response to sympathetic stimulation

Angiotensin II is known to potentiate vasoconstriction induced by electrical field stimulation (EFS), but the underlying mechanisms for this potentiation are not fully understood. This study was designed to investigate the role of superoxide anion in the potentiation effects of angiotensin II. Contraction of rat mesenteric arterial segments was induced by perivascular nerve stimulation with EFS, and superoxide production was measured with lucigenin-enhanced chemiluminescence. Extracellular signal-regulated kinase (ERK) phosphorylation was determined in cultured smooth muscle cells with Western blot. Angiotensin II concentration dependently potentiated the contraction of rat mesenteric arteries to EFS, which is frequency-dependent. This potentiation was blunted by an angiotensin AT 1 receptor antagonist (2-ethoxy-1-[[2′-(1 H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1 H-benzimidazole-7-carboxylic acid, CV-11974), NAD(P)H oxidase inhibitor (apocynin), superoxide dismutase (SOD) and its mimetic tiron, but not affected by angiotensin AT 2 receptor antagonist and inhibitors of xanthine oxidase, cytochrome P450, and cyclooxygenase. Angiotensin II increased superoxide production by mesenteric arteries, which was blunted by angiotensin AT 1 receptor antagonist CV-11974, and NAD(P)H oxidase inhibitor apocynin. Superoxide generating compound pyrogallol mimicked the effects of angiotensin II. Tyrosine kinase inhibitor (tyrphostin A25) and mitogen-activated protein kinase (MAPK)/ERK inhibitors (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio]butadiene (U 0126)) inhibited angiotensin II- and pyrogallol-induced potentiation of EFS-induced contraction, while inactive forms of these inhibitors did not show any inhibitory effects. In cultured smooth muscle cells from mesenteric arteries, angiotensin II and superoxide similarly induced ERK phosphorylation. These results showed that superoxide mediated angiotensin II-induced potentiation of contractile response to EFS and tyrosine kinase-MAPK/ERK activation was involved.

BML‐241 fails to display selective antagonism at the sphingosine‐1‐phosphate receptor, S1P3

The thiazolidine carboxylic acid, BML-241, has been proposed as a lead compound in development of selective antagonists at the sphingosine-1-phosphate receptor (S1P3), based on its inhibition of the rise in intracellular calcium concentrations ([Ca2+]i) in HeLa cells overexpressing S1P receptors. We have studied the antagonistic properties of BML-241 for the S1P(3) receptor in more detail. Chinese hamster ovary (CHO) cells stably transfected with the S1P3, S1P2 or alpha(1A)-adrenoceptors were used to investigate the effect of BML-241 on increases in [Ca2+]i mediated via different receptors. CHO-K1 cells were used to study ATP-induced [Ca2+]i elevations. Effects on S1P3 -mediated inhibition of forskolin-induced cAMP accumulation and on binding to alpha(1A)-adrenoceptors were also investigated. In addition, the effect of BML-241 on contractions of rat mesenteric artery induced by phenylephrine was studied in an organ bath. High concentrations of BML-241 (10 microM) inhibited the rise in [Ca2+]i induced by S1P3 and S1P2 receptor stimulation; lower concentrations were ineffective. This high concentration of BML-241 also inhibited [Ca2+]i increases via P2 (nucleotide) receptor or alpha(1A)-adrenoceptor stimulation. Moreover, BML-241 (10 microM) inhibited alpha(1)-adrenoceptor-mediated contraction of rat mesenteric artery but did not displace [3H]-prazosin from alpha(1A)-adrenoceptors in concentrations up to 100 microM. BML-241 (10 microM) did not affect the S1P3 -mediated decrease of forskolin-induced cAMP accumulation. We conclude that BML-241 is a low potency, non-selective inhibitor of increases in [Ca2+]i, rather than a specific antagonist at the S1P3 receptor.

Vascular endothelin ET B receptor-mediated contraction requires phosphorylation of ERK1/2 proteins

In cardiovascular diseases, endothelin type B (ET B) receptors in arterial smooth muscle cells are upregulated. The present study revealed that organ culture of rat mesenteric artery segments enhanced endothelin ET B receptor-mediated contraction paralleled with increase in the receptor mRNA and protein expressions. The endothelin ET B receptor-mediated contraction was associated with increase in phosphorylation of extracellular regulation kinase 1 and 2 (ERK1/2) proteins and elevated levels of intracellular calcium. The elevation curve of intracellular calcium consisted of two phases: one rapid and one sustained. Inhibition of ERK1/2 phosphorylation by SB386023 or blockage of calcium channels by nifedipine significantly reduced the endothelin ET B receptor-mediated contraction ( P < 0.05) and decreased the sustained phase of intracellular calcium level, but not the rapid phase. Thus, phosphorylation of ERK1/2 proteins and elevation of intracellular calcium level are required for endothelin ET B receptor-mediated contraction in rat mesenteric artery.

CARDIOVASCULAR ACTIONS OF THE VENOM FROM THE IRUKANDJI (CARUKIA BARNESI) JELLYFISH: EFFECTS IN HUMAN, RAT AND GUINEA‐PIG TISSUES IN VITRO AND IN PIGS IN VITRO

1. We have investigated the cardiovascular pharmacology of the crude venom extract (CVE) from the potentially lethal, very small carybdeid jellyfish Carukia barnesi, in rat, guinea-pig and human isolated tissues and anaesthetized piglets. 2. In rat and guinea-pig isolated right atria, CVE (0.1-10 microg/mL) caused tachycardia in the presence of atropine (1 micromol/L), a response almost completely abolished by pretreatment with tetrodotoxin (TTX; 0.1 micromol/L). In paced left atria from guinea-pig or rat, CVE (0.1-3 microg/mL) caused a positive inotropic response in the presence of atropine (1 micromol/L). 3. In rat mesenteric small arteries, CVE (0.1-30 microg/mL) caused concentration-dependent contractions that were unaffected by 0.1 micromol/L TTX, 0.3 micromol/L prazosin or 0.1 micromol/L omega-conotoxin GVIA. 4. Neither the rat right atria tachycardic response nor the contraction of rat mesenteric arteries to CVE were affected by the presence of box jellyfish (Chironex fleckeri) antivenom (92.6 units/mL). 5. In human isolated driven right atrial trabeculae muscle strips, CVE (10 microg/mL) tended to cause an initial fall, followed by a more sustained increase, in contractile force. In the presence of atropine (1 micromol/L), CVE only caused a positive inotropic response. In separate experiments in the presence of propranolol (0.2 micromol/L), the negative inotropic effect of CVE was enhanced, whereas the positive inotropic response was markedly decreased. 6. In anaesthetized piglets, CVE (67 microg/kg, i.v.) caused sustained tachycardia and systemic and pulmonary hypertension. Venous blood samples demonstrated a marked elevation in circulating levels of noradrenaline and adrenaline. 7. We conclude that C. barnesi venom may contain a neural sodium channel activator (blocked by TTX) that, in isolated atrial tissue (and in vivo), causes the release of transmitter (and circulating) catecholamines. The venom may also contain a 'direct' vasoconstrictor component. These observations explain, at least in part, the clinical features of the potentially deadly Irukandji syndrome.

Heteromultimeric Kv1 Channels Contribute to Myogenic Control of Arterial Diameter

Inhibition of vascular smooth muscle (VSM) delayed rectifier K+ channels (K(DR)) by 4-aminopyridine (4-AP; 200 micromol/L) or correolide (1 micromol/L), a selective inhibitor of Kv1 channels, enhanced myogenic contraction of rat mesenteric arteries (RMAs) in response to increases in intraluminal pressure. The molecular identity of K(DR) of RMA myocytes was characterized using RT-PCR, real-time PCR, and immunocytochemistry. Transcripts encoding the pore-forming Kvalpha subunits, Kv1.2, Kv1.4, Kv1.5, and Kv1.6, were identified and confirmed at the protein level with subunit-specific antibodies. Kvbeta transcript (beta1.1, beta1.2, beta1.3, and beta2.1) expression was also identified. Kv1.5 message was approximately 2-fold more abundant than that for Kv1.2 and Kv1.6. Transcripts encoding these three Kv1alpha subunits were approximately 2-fold more abundant in 1st/2nd order conduit compared with 4th order resistance RMAs, and Kvbeta1 was 8-fold higher than Kvbeta2 message. RMA K(DR) activated positive to -50 mV, exhibited incomplete inactivation, and were inhibited by 4-AP and correolide. However, neither alpha-dendrotoxin or kappa-dendrotoxin affected RMA K(DR), implicating the presence of Kv1.5 in all channels and the absence of Kv1.1, respectively. Currents mediated by channels because of coexpression of Kv1.2, Kv1.5, Kv1.6, and Kvbeta1.2 in human embryonic kidney 293 cells had biophysical and pharmacological properties similar to those of RMA K(DR). It is concluded that K(DR) channels composed of heteromultimers of Kv1 subunits play a critical role in myogenic control of arterial diameter.