KCl-induced Contraction Research Articles

Abstract P058: Endothelial Dysfunction And Increased Contractility In Resistance Arteries Occurs Prior To Onset Of Hypertension In Schlager (BPH/2J) Mice

Genetically hypertensive mice (BPH/2J) were developed by Gunther Schlager more than 45 years ago, and these mice have neurogenic hypertension. Seven-weeks-old male BPH/2J mice present elevations in systolic blood pressure (SBP), and its highest value is observed in 20-weeks-old mice. Several studies have showed vascular dysfunction in these mice, but no one has demonstrated whether resistance artery dysfunction is present prior to the elevation in SBP. We hypothesized that hypercontractility and endothelial dysfunction is associated with the cause of hypertension in BPH/2J mice. We evaluated mesenteric resistance arteries (MRA) function from 5-weeks-old male BPH/2J and 9-weeks-old normotensive mice (BPN/3J), prior to the elevation of SBP. Intact-endothelium MRA were mounted in wire myographs. KCl-induced contraction (120 mM), or phenylephrine (PE) and acetylcholine (ACh) cummulative concentration curves were performed. Non-linear curve regressions were performed and maximum response (Rmax) was analyzed by Student t test ( p <0.05). BPH/2J mice presented increased KCl contraction (BPH/2J: 6.47±0.51 vs. BPN/3J: 2.95±0.13 mN/mm, n=4, p <0.05) and PE-induced contraction (Rmax: BPH/2J: 6.25±0.54 vs. BPN/3J: 4.23±0.28 mN/mm, n=4, p <0.05). ACh-dependent relaxation was decreased in BPH/2J (Rmax: BPH/2J: 76±5, n=4 vs. BPN/3J: 93±2%, n=3, p <0.05). ACh promoted contraction at high concentrations (>0.3 μM) in BPH/2J mice but not BPN/3J mice (Fig.1), suggesting increased cyclooxygenase-induced inflammation. These data reveal that vascular dysfunction contributes to the cause of hypertension in Schlager mice.

Neuropilin 2 Is a Novel Regulator of Distal Colon Contractility

Appropriate coordination of smooth muscle contraction and relaxation is essential for normal colonic motility. The impact of perturbed motility ranges from moderate, in conditions such as colitis, to potentially fatal in the case of pseudo-obstruction. The mechanisms underlying aberrant motility and the extent to which they can be targeted pharmacologically are incompletely understood. This study identified colonic smooth muscle as a major site of expression of neuropilin 2 (Nrp2) in mice and humans. Mice with inducible smooth muscle-specific knockout of Nrp2 had an increase in evoked contraction of colonic rings in response to carbachol at 1 and 4 weeks following initiation of deletion. KCl-induced contractions were also increased at 4 weeks. Colonic motility was similarly enhanced, as evidenced by faster bead expulsion in Nrp2-deleted mice versus Nrp2-intact controls. In length-tension analysis of the distal colon, passive tension was similar in Nrp2-deficient and Nrp2-intact mice, but at low strains, active stiffness was greater in Nrp2-deficient animals. Consistent with the findings in conditional Nrp2 mice, Nrp2-null mice showed increased contractility in response to carbachol and KCl. Evaluation of selected proteins implicated in smooth muscle contraction revealed no significant differences in the level of α-smooth muscle actin, myosin light chain, calponin, or RhoA. Together, these findings identify Nrp2 as a novel regulator of colonic contractility that may be targetable in conditions characterized by dysmotility.

H2S releasing sodium sulfide protects against pulmonary hypertension by improving vascular responses in monocrotaline-induced pulmonary hypertension

Pulmonary arterial hypertension is caused by complex structural and functional changes in the endothelial and smooth muscle cells of pulmonary arteries. Hydrogen sulfide (H2S), a gasotransmitter, can potentially treat pulmonary hypertension by relaxing the pulmonary arteries and decreasing bronchial pressure. Although the role of H2S in systemic circulation has been examined, the H2S levels in pulmonary arteries, the role of H2S in endothelium-dependent vasorelaxation and the L-cysteine/H2S pathway in monocrotaline-induced pulmonary arterial hypertension have not been investigated. The rats were divided into control, monocrotaline, monocrotaline + Na2S, and Na2S groups. The right ventricular pressure and hypertrophy were evaluated. KCl, acetylcholine, and L-cysteine responses were obtained in the main pulmonary arteries by wire myograph. H2S levels were measured in pulmonary arteries and lungs by methylene blue assay. Right ventricular pressure and hypertrophy were increased by monocrotaline and ameliorated by Na2S. The KCl-induced contractions and relaxing responses to acetylcholine and L-cysteine in pulmonary arteries and H2S production in the lungs and pulmonary arteries were significantly attenuated in the monocrotaline group and augmented in the monocrotaline + Na2S group. These findings suggest that H2S levels were reduced, and L-cysteine-induced and endothelium-dependent relaxations were impaired in the pulmonary arteries in monocrotaline-induced pulmonary arterial hypertension. The H2S donor, Na2S, prevented endothelial dysfunction and increased pulmonary artery pressure and hypertrophy. Also, Na2S enhanced the L-cysteine-mediated responses and restored the diminished H2S levels in pulmonary arteries and the lungs. The treatments targeting H2S might be beneficial for promoting vascular alterations, i.e. endothelial dysfunction and impaired H2S-mediated relaxation in pulmonary arterial hypertension.

Extraction and Fractionation of Prokinetic Phytochemicals from Chrozophora tinctoria and Their Bioactivities.

Chrozophora tinctoria is an annual plant of the family Euphorbiaceae, traditionally used as a laxative, a cathartic and an emetic. A methanolic extract of Chrozophora tinctoria (MEC) whole plant and an n-butanol fraction of Chrozophora tinctoria (NBFC) were analyzed by gas chromatography–mass spectrometry (GC-MS) to detect the phytochemicals. MEC and NBFC were tested for in vitro anti acetylcholinesterase (AChE) potential. The effect of both samples on intestinal propulsive movement and spasmolytic activity in the gastrointestinal tract (GIT) was also studied. About twelve compounds in MEC and three compounds in NBFC were tentatively identified through GC-MS. Some of them are compounds with known therapeutic activity, such as toluene; imipramine; undecane; 14-methyl-pentadecanoic acid methyl ester; and hexadecanoic acid. Both NBFC and MEC samples were checked for acute toxicity and were found to be highly toxic in a dose-dependent manner, causing diarrhea and emesis at 1 g/kg concentration in pigeons, with the highest lethargy and mortality above 3 g/kg. Both the samples of Chrozophora tinctoria revealed significant (p ≤ 0.01) laxative activity against metronidazole (7 mg/kg) and loperamide hydrochloride (4 mg/kg)-induced constipation. NBFC (81.18 ± 2.5%) and MEC (68.28 ± 2.4%) significantly increased charcoal meal intestinal transit compared to distal water (41.15 ± 4.3%). NBFC exhibited a significant relaxant effect (EC50 = 3.40 ± 0.20 mg/mL) in spontaneous rabbit jejunum as compared to MEC (EC50 = 4.34 ± 0.68 mg/kg). Similarly, the impact of NBFC on KCl-induced contraction was more significant than that of MEC (EC50 values of 7.22 ± 0.06 mg/mL and 7.47 ± 0.57 mg/mL, respectively). The present study scientifically validates the folk use of Chrozophora tinctoria in the management of gastrointestinal diseases such as constipation. Further work is needed to isolate the phytochemicals that act as diarrheal agents in Chrozophora tinctoria.

Docosahexaenoic acid and eicosapentaenoic acid strongly inhibit prostanoid TP receptor-dependent contractions of guinea pig gastric fundus smooth muscle.

The inhibitory effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and linoleic acid (LA) on the contractions induced by five prostanoids and U46619 (a TP receptor agonist) were examined in guinea pig gastric fundus smooth muscle (GFSM). Tension changes were isometrically measured, and the mRNA expression of prostanoid receptors was measured by RT‐qPCR. DHA and EPA significantly inhibited contractions induced by the prostanoids and U46619, whereas LA inhibited those induced by prostaglandin D2 and U46619. The mRNA expression levels of the prostanoid receptors were TP ≈ EP3 >> FP > EP1. The inhibition by DHA, EPA, and LA was positively correlated with that by SQ 29,548 (a TP receptor antagonist) but not with that by L‐798,106 (an EP3 receptor antagonist). DHA and EPA suppressed high KCl‐induced contractions by 35% and 25%, respectively, and the contractions induced by the prostanoids and U46619 were suppressed by verapamil, a voltage‐dependent Ca2+ channel (VDCC) inhibitor, by 40%–85%. Although LA did not suppress high KCl‐induced contractions, it suppressed U46619‐induced contractions in the presence of verapamil. However, LA did not show significant inhibitory effects on U46619‐induced Ca2+ increases in TP receptor‐expressing cells. In contrast, LA inhibited U46619‐induced contractions in the presence of verapamil, which was also suppressed by SKF‐96365 (a store‐operated Ca2+ channel [SOCC] inhibitor). These findings suggest that the TP receptor and VDCC are targets of DHA and EPA to inhibit prostanoid‐induced contractions of guinea pig GFSM, and SOCCs play a significant role in LA‐induced inhibition of U46619‐induced contractions.

Prokinetic and Laxative Effects of Chrozophora tinctoria Whole Plant Extract.

Chrozophora tinctoria (Euphorbiaceae) has been used as an emetic, anthelminthic, and cathartic agent in traditional medicine. We used gas chromatography-mass spectrometry (GC-MS) to characterize the composition of ethyl acetate (EAC) and dichloromethane (DCMC) fractions from the whole Chrozophora tinctoria plant. EAC and DCMC fractions were evaluated for acetylcholinesterase (AChE) inhibitory activity and acute toxicity. Their effects on intestinal propulsive movement and spasmogenic activity of the gastrointestinal tract (GIT) muscle were also assessed. The compounds detected in both fractions were mostly fatty acids, with about seven compounds in EAC and 10 in DCMC. These included pharmacologically active compounds such as imipramine, used to treat depression, or hexadecanoic acid methyl ester, an antioxidant. Both EAC and DCMC fractions inhibited acetylcholinesterase (AChE) activity with IC50 values of 10 µg and 130 µg, respectively. Both the fractions were found to be toxic in a dose-dependent manner, inducing emesis at 0.5 g or higher and lethargy and mortality from 3–5 g upwards. Similarly, both of the fractions showed laxative activity in metronidazole- and loperamide-induced constipation models. EAC relaxed the intestinal muscle at a lower dose (1 mg/mL) than DCMC. Similarly, the EAC extract showed a significant relaxation effect (EC50 = 0.67 ± 0.15 mg/mL) on KCL-induced contraction in rabbit jejunum as compared to DCMC (EC50 = 5.04 ± 0.05 mg/kg). The present study strongly supports the folklore that this valuable plant is a cathartic agent. Further work is required to isolate and validate the bioactive compounds that act as diarrheal agents in Chrozophora tinctoria.

Chronic Effects of Loxapine, Iloperidone, Paliperidone on Mice Isolated Vas Deferens Contractility

Objective: Erectile dysfunction is a usual side effect of antipsychotic medications; this causes patients to avoid using drugs. The aim of this study to investigate the effects of iloperidone, paliperidone and loxapine on serotonin (5-HT), noradrenaline (NA), adenosine triphosphate (ATP) and potassium chloride (KCl)-induced contractions of the vas deferens in mice.Materials and Methods: The mice were randomly divided into experimental groups and treated by ip injection of drugs for 21 days. After the treatment, the effects of drugs were investigated on 5-HT, ATP, NA and KCl-induced contractile responses in the epididymal and prostatic portions of mice vas deferens strips. Results: 5-HT-induced contractile responses were significantly increased while ATP-induced contractile responses were significantly decreased in the both portions of the vas deferens obtained from the iloperidone, paliperidone and loxapine-treated groups. However, these drugs had no significant effect on NA- and KCl-induced contractions of mice vas deferens. Conclusion: These results showed that serotonin and ATP-induced contractions of vas deferens were affected by the chronic treatments of iloperidone, paliperidone, and loxapine. In mice chronically treated with these drugs, serotonergic and purinergic receptors may contribute to changes in vas deferens contractions that cause erectile dysfunction.

Chronic Effects of Atypic Antipsychotics on Mice Isolated Vas Deferens Contractility: An In vitro Study

Aims: Antipsychotic drugs are known to be commonly associated with sexual dysfunction. The aim of this study to investigate the effects of sertindole, asenapine and ziprasidone on serotonin (5-HT), noradrenaline (NA), adenosine triphosphate (ATP) and potassium chloride (KCl)-induced contractions of the isolated vas deferens in mice.
 Study design: All the drugs were administered intraperitoneally (i.p.) in a volume of 0.1 ml per 10 g body weight of mice.
 Place and Duration of Study: Department of Pharmacology, Kocaeli University, Animal Research Center, between May 2018 and August 2019.
 Methodology: The mice were randomly divided into groups (n=7) as follows: saline; sertindole 1.3 mg/kg; sertindole 2.5 mg/kg; asenapine 0.05 mg/kg; asenapine 0.075 mg/kg; ziprasidone 1 mg/kg; ziprasidone 2 mg/kg once daily for 21 days. Mice receiving only the vehicle (0.9% saline, i.p.) served as control group. After 21 days of treatment, the effects of drugs were investigated on 5-HT, NA, ATP and KCl-induced contractions of isolated vas deferens.
 Results: The results showed that serotonin-induced contractions of vas deferens were affected by the chronic treatments of sertindole, asenapine and ziprasidone, however these drugs had no significant effect on NA, ATP, and KCl-induced contractions of mice vas deferens.
 Conclusion: Serotonergic receptors may contribute to changes in vas deferens contractions in mice with chronic treatment of sertindole, asenapine and ziprasidone. Thus, our results may explain one of the causes of sexual dysfunction of sertindole, asenapine and ziprasidone.

Calcium Channel Blockade Mediates the Vasorelaxant Activity of Dichloromethane Extract from Roots of Oncidium cebolleta on Isolated Rat Aorta

Oncidium cebolleta belongs to the Orchidaceae family, and it is used in preparations in Mexican traditional medicine to treat several diseases. This research aimed to study the vasorelaxant activity of dichloromethane extract from the roots of Oncidium cebolleta (DEROc) on endothelium-intact and -removed rat aorta segments to determine its mode of vasorelaxant action. Several extracts (hexane, dichloromethane, and methanol) obtained from leaves and roots were evaluated on isolated rat aorta rings with and without endothelium to determine their vasorelaxant effect. Except for methanolic extract from leaves, all extracts induced a concentration-dependent vasorelaxant effect on endothelium-intact rat aorta rings pre-contracted with noradrenaline (NA, 0.1 µM). Dichloromethane extract from roots (DEROc) was the most potent and efficient, and the effect was endothelium-independent. Also, DEROc could relax KCl-induced contraction (80 mM) and inhibit the contraction induced by CaCl2-cumulative concentration on endothelium-denuded rings. However, DEROc was less potent than control nifedipine (L-type calcium channel blocker), but its effectiveness was similar. Contractions induced by NA and 5-HT [0.001 µM to 0.1 µM] were significantly reduced by DEROc. However, DEROc vasorelaxation was not reduced by TEA (potassium channel blocker, 10 mM) nor by ODQ (soluble guanylyl cyclase inhibitor, 0.1 µM) in endothelium-denuded aortic rings. Our functional results suggest that DEROc induces its relaxant effect by an endothelium-independent mechanism due to a Ca2+ channels blockade in rat aortic rings.

Sevoflurane and isoflurane inhibit KCl-induced, Rho kinase-mediated, and PI3K-participated vasoconstriction in aged diabetic rat aortas

BackgroundThe mechanism of volatile anesthetics on vascular smooth muscle (VSM) contraction in the setting of diabetes mellitus (DM) remains unclear. The current study was designed to determine the effects of sevoflurane (SEVO) and isoflurane (ISO) on phosphoinositide 3-kinase (PI3K) and Rho kinase (ROCK) mediated KCl-induced vasoconstriction in aged type 2 diabetic rats.MethodsKCl-induced (60 mM) contractions were examined in endothelium-denuded aortic rings from aged T2DM Otsuka Long-Evans Tokushima Fatty (OLETF) rats (65–70 weeks old), control age-matched nondiabetic Long-Evans Tokushima Otsuka (LETO) rats and young Wistar rats (6–8 weeks old). The effects of SEVO or ISO (1–3 minimum alveolar concentration, MAC) on KCl-induced vasoconstriction, as well as those of LY294002 (PI3K inhibitor) and Y27632 (ROCK inhibitor) were measured in aortic rings from the three groups using an isometric force transducer.ResultsKCl induced rapid and continuous contraction of aortic smooth muscle in the three groups, and the contraction was more obvious in OLETF rats.SEVO and ISO inhibited KCl-induced vasoconstriction in a concentration-dependent manner and were suppressed by LY294002 (10 µM) and Y27632 (1 µM). SEVO had a stronger inhibitory effect on the aortas of young Wistar rats than ISO, especially at 2 MAC and 3 MAC (P < 0.05). In aged rats, the inhibitory effect of ISO was stronger than that of SEVO, especially OLETF rats. There was no significant difference in the effects of different concentrations of ISO on arterial contraction among the three groups (P > 0.05). The effects of 1 MAC SEVO on Wistar rats and 3 MAC SEVO on OLETF rats, however, were noticeably and significantly different (P < 0.05).Compared with the control condition, LY294002 and Y27632 had the most noticeable effect on the KCl-induced contraction of aortic rings in OLETF rats (P < 0.01).ConclusionSEVO (3 MAC), ISO (1, 2, 3 MAC), LY294002 and Y27632 have more significant inhibitory effect on the contraction of vascular smooth muscle in aged T2MD rats. The mechanism of SEVO and ISO in vascular tension in T2DM is partly due to changes in PI3K and/or Rho kinase activity.

PD11-07 MIRABEGRON INHIBITS NEUROGENIC AND α 1 -ADRENERGIC HUMAN PROSTATE SMOOTH MUSCLE CONTRACTIONS, WITH SIMILAR PHARMACOLOGIC PATTERNS AS α 1 -BLOCKERS

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (PD11)1 Sep 2021PD11-07 MIRABEGRON INHIBITS NEUROGENIC AND α1-ADRENERGIC HUMAN PROSTATE SMOOTH MUSCLE CONTRACTIONS, WITH SIMILAR PHARMACOLOGIC PATTERNS AS α1-BLOCKERS Ru Huang, Yuhan Liu, Bingsheng Li, Ruixiao Wang, Alexander Tamalunas, Raphaela Waidelich, Frank Strittmatter, Christian G. Stief, and Martin Hennenberg Ru HuangRu Huang More articles by this author , Yuhan LiuYuhan Liu More articles by this author , Bingsheng LiBingsheng Li More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Alexander TamalunasAlexander Tamalunas More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001986.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Mirabegron inhibits detrusor smooth muscle contractions and is available for treatment of overactive bladder. Consequently, its effects on lower urinary tract symptoms (LUTS) suggestive of bengin prostatic hyperplasia (BPH) were subject of recent trials. However, available results are not yet conclusive, and effects of mirabegron on human prostate smooth muscle contraction are hardly characterized. Voiding symptoms suggestive of BPH may be driven by increased prostate smooth muscle tone. Here, we examined effects of mirabegron on contractions of human prostate tissues. METHODS: Human prostate tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath. Comparisons were performed between whole groups (one-way ANOVA), and at single frequencies and agonist concentrations (multivariate analysis). RESULTS: Electric field stimulation (EFS) induced neurogenic, frequence-dependent contractions, while agonists induced concentration-dependent contractions. Mirabegron (10 µM) inhibited EFS-induced contractions (p <0.001 for controls vs. mirabegron between whole groups), which was observed at different frequencies and reflected by reduced Emax values calculated by curve fitting (contractions in controls 84±11% of KCl-induced contractions, but 14±2% after mirabegron, p <0.01). Miragebron increased the EC50 values for noradrenaline (controls 0.55±0.32 µM, mirabegron 5.1±2.4 µM, p <0.004), resulting in rightshifts of concentration response curves for noradrenaline-induced contractions (p <0.001) and in reduced contractions up to 10 µM noradrenaline, but in recovery at higher noradrenaline concentrations and unchanged Emax values. Similarly, EC50 values for the α1-adrenergic agonist phenylephrine were increased by mirabegron (controls 7.4±4.1 µM, mirabegron 29.2±8.9 µM, p=0.05), resulting in rightshifts of concentration response curves for phenylephrine (p <0.001) and in reduced contractions up to 30 µM phenylephrine, but in recovery at higher phenylephrine concentrations and in unchanged Emax values. Mirabegron slightly reduced contractions induced by the thromboxane A2 analog U46619 (p <0.03), without changing EC50 values. Contractions induced by endothelin-1 remained unchanged by mirabegron. CONCLUSIONS: Mirabegron inhibits neurogenic and α1-adrenergic smooth muscle contractions in the human prostate. Surprisingly, the pharmacologic pattern is similar to that of α1-blockers, including rightshifts of α1-adrenergic concentration response curves, reduced Emax values for neurogenic contractions, and lacking or small effects on non-adrenergic contractions. Source of Funding: China Scholarship Council (CSC) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e200-e200 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ru Huang More articles by this author Yuhan Liu More articles by this author Bingsheng Li More articles by this author Ruixiao Wang More articles by this author Alexander Tamalunas More articles by this author Raphaela Waidelich More articles by this author Frank Strittmatter More articles by this author Christian G. Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement Loading ...

The inhibitory effect of trimetazidine on detrusor contractility - a potential repositioning of trimetazidine for the treatment of overactive bladder.

This study aimed to identify the effect of trimetazidine (TMZ), an antianginal drug, on detrusor smooth muscle (DSM) contractility and its possible mechanisms of action. We performed in-vitro contractility studies on isolated mouse DSM strips and investigated the effect of TMZ on Ca2+ levels in fura-2-loaded A7r5 cells. TMZ (300 or 1000 µM) inhibited carbachol (CCh)- and KCl-induced contractions and produced a concentration-dependent (10-1000 µM) relaxation in KCl-precontracted DSM strips. TMZ-induced relaxation was markedly decreased by BaCl2, an inward-rectifying K+ channel blocker, but was not altered by preincubation with tetraethylammonium, glibenclamide, 4-aminopyridine, propranolol, L-NAME or methylene blue. TMZ (300 or 1000 µM) reduced both the CaCl2-induced contraction of depolarized DSM strips under Ca2+-free conditions and the CCh-induced contraction of DSM strips preincubated with nifedipine in Ca2+-containing Krebs solution. Furthermore, TMZ (1000 µM) significantly decreased the Ca2+ levels in fura-2-loaded A7r5 cells. TMZ decreased DSM contractility and caused a concentration-dependent relaxation of the tissue possibly through its actions on Ca2+ transients and K+ channels. Our results provide preclinical evidence that TMZ would be a potential candidate to treat disorders related to the overactivity of the bladder.

Gentiopicroside Produces Endothelium-Independent Vasodilation by Deactivating the PI3K/Akt/Rho-Kinase Pathway in Isolated Rat Thoracic Aorta.

Gentiopicroside (GPS), a main active secoiridoid glucoside derived from the roots of perennial herbs in the Gentianaceae family, has antispasmodic and relaxant effects. However, the vasorelaxant effects of GPS on aortic rings and the molecular mechanisms involved in these effects are not yet clear. Therefore, we investigated whether GPS inhibits phenylephrine- (PE-) or KCl-induced contractions in isolated rat thoracic aortic rings. The present study found that GPS produced a dose-dependent relaxation in aortic rings precontracted with PE or KCl and significantly reduced CaCl2-, narciclasine- (Rho-kinase activator-), and phorbol-12,13-diacetate- (PKC activator-) induced vasocontractions. Pretreatment with NG-Nitroarginine methyl ester hydrochloride (L-NAME, NOS inhibitor), methylene blue (sGC inhibitor), indomethacin (COX inhibitor), 4-aminopyridine (KV channel inhibitor), and glibenclamide (KATP channel inhibitor) had no influence on the vasorelaxant effect of GPS, while BaCl2 (Kir channel inhibitor), tetraethylammonium chloride (KCa channel inhibitor), ruthenium red (RYR inhibitor), and heparin (IP3R inhibitor) significantly reduced GPS-induced vasorelaxation. Moreover, GPS pretreatment remarkably inhibited the influx of Ca2+ in vascular smooth muscle cells stimulated using KCl or PE-containing CaCl2 solution. Western blot analysis confirmed that GPS treatment inhibited PE-induced increases in the protein levels of p-Akt, p-myosin light chain (MLC), and p-myosin-binding subunit of myosin phosphatase 1 (MYPT1) in the aortic rings. Additionally, the vasorelaxation activity of GPS was attenuated upon pretreatment with LY294002 (PI3K/Akt inhibitor), Y27632 (Rho-kinase inhibitor), and verapamil (L-type Ca2+ channel inhibitor). These findings demonstrate that GPS exhibits endothelium-independent vasorelaxant effects through inhibition of voltage-dependent, receptor-operated, and inositol triphosphate receptor (IP3R)/ryanodine receptor- (RYR-) mediated Ca2+ channels as well as the PI3K/Akt/Rho-kinase signaling pathway.

Purinergic smooth muscle contractions in the human prostate: estimation of relevance and characterization of different agonists

Non-adrenergic prostate smooth muscle contractions may account for the limited effectiveness of α1-adrenoceptor antagonists, which are the first-line option for medical treatment of voiding symptoms suggestive of benign prostatic hyperplasia. In non-human prostates, purinergic agonists induce contractions reaching similar magnitudes as α1-adrenergic contractions. However, evidence for the human prostate is highly limited, and pointed to much weaker purinergic contractions. Here, we examined contractions of different purinergic agonists in human prostate tissues. Tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath, and expression of purinergic receptors was studied by RT-PCR. Electric field stimulation (EFS)–induced contractions amounted to 104% of KCl-induced contractions (95% CI: 84–124%). From all tested agonists, only ATP induced concentration-dependent contractions, reaching an average maximum of 18% (12–24%) of KCl. Maximum tensions following application of other agonists averaged to 7.1% of KCl for α,β-methylene-ATP (1.8–12.4%), 3.9% for β,γ-methylene-ATP (2.0–5.4%), 3.1% for 2-methylthio-ATP (− 0.1–6.3%), and 5.1% for ATPγS (1.0–9.2%). Responses were not affected by the P2X antagonist NF023 or the P2Y antagonist PPADS. mRNA expression of P2X1-4 correlated with expression of a marker for catecholaminergic nerves, although neither ATP, NF023, nor PPADS changed EFS-induced contractions. Correlation between expression of receptors and the smooth muscle marker calponin was not observed. Our findings point to a low relevance of purinergic contractions in the human prostate, compared to other contractile stimuli in the human prostate and compared to purinergic contractions in non-human prostates. Purinergic contractions in the human prostate are not sensitive to NF023 or PPADS.

Cardioprotective Solutions Exposure For 1 Hour in Hypoxia and Low Temperatures Affects Vascular Reactivity Differently.

IntroductionHeart preservation benefits cardiac performance after operations decreasing morbidity but the contribution of the vascular reactivity has been neglected.ObjectiveWe evaluated whether cardioprotective solutions, Krebs-Henseleit (KH), Bretschneider-HTK (BHTK), St. Thomas No. 1 (STH-1), and Celsior (CEL), affect vascular reactivity.MethodsAortic rings from Wistar rats were used in two protocols. First, the rings were exposed to BHTK, STH-1 or CEL for 1 hour of hypoxia at 37 °C. Second, the rings were exposed to 10 °C or 20 °C for 1 hour under hypoxia. After treatment, the rings were immersed in KH at 37 °C, endothelial integrity was tested and concentration-response curves to phenylephrine were performed.ResultsIn the first protocol, the solutions did not damage the endothelium; CEL and BHTK reduced KCl-induced contractions but not STH-1; only CEL and BHTK reduced vascular reactivity; there was a positive correlation between Rmax and KCl concentration. At 20 °C, 1 hour under hypoxia, the solutions produced similar KCl-induced contractions without endothelial damage. CEL, BHTK and STH-1 decreased vascular reactivity. At 10 °C, STH-1 increased reactivity but CEL and BHTK decreased. After 1 hour under hypoxia in CEL or BHTK solutions, reactivity was similar at different temperatures. At 20 °C, endothelial damage after exposure to STH-1 produced more vasoconstriction than CEL and BHTK. However, at 10 °C, endothelial damage after CEL and BHTK exposure elicited more vasoconstriction while STH-1 showed a small vasoconstrictor response, suggesting endothelial damage.ConclusionSTH-1 decreased reactivity at 20 °C and increased at 10 °C. CEL promoted greater endothelial modulation at 10 °C than at 20 °C, while STH-1 promoted higher modulation at 20 °C than at 10 °C. Vascular tone was reduced by CEL and BHTK exposure, also depending on the KCl concentration.

Vascular reactivity in old spontaneously hypertensive stroke-prone rats- effects of in vitro ischemia/reperfusion injury

Objectives: Peripheral arterial disease affects mainly the aorta and lower extremities and commonly results in tissue ischemia. Medical interventions to restore blood flow can induce reperfusion injury. We investigated the effects of both hypertension and ageing on vascular graft responsiveness under normoxia and ischemia/reperfusion (IR) conditions. Methods: Hemodynamic changes and aortic morphometry were studied in 18-month-old spontaneously hypertensive stroke prone (SHRSP, n=8) and age-matched normotensive Wistar (control, n=8) rats. Whereas thoracic aortic rings of the control- and SHRSP groups were immediately mounted in organ bath chambers (normoxia-group), aortic segments in the control-IR and SHRSP-IR groups underwent cold ischemic preservation, prior to mounting. Results: Significantly increased intima-media thickness, intima-media cross-sectional area, and lumen normalized to body weight were observed in SHRSP displaying a systolic blood pressure of 205±16 mmHg compared to controls 124±8 mmHg, p < 0.05. Phenylephrine-induced vasoconstriction was greater in aorta of SHRSP compared to controls. IR injury increased contractile responses to phenylephrine in both control and SHRSP compared to the corresponding groups, which was further increased in SHRSP-IR compared to control-IR (as percentage of KCl-contraction: SHRSP-IR 167±26% vs. control-IR 101±5%, p < 0.05). Compared to controls, vasoconstrictive response to high K+-induced depolarization was significantly reduced in SHRSP, control-IR, and SHRSP-IR. Additionally, KCl-induced contraction was reduced in SHRSP-IR compared to control-IR (SHRSP-IR 0.7±0.1g vs. control-IR 2.5±0.1g, p < 0.05). IR reduced maximum endothelium-dependent relaxation to acetylcholine in the normoxia control group, whereas it had no effect in the aortic rings from SHRSP. Conclusions: IR increases the susceptibility of aortic rings to vascular smooth muscle contractile dysfunction in 18-month-old hypertensive rats.

Antispasmodic effects of the essential oil of Croton zehnteneri, anethole, and estragole, on tracheal smooth muscle

Croton zehntneri is a plant well adapted to the semi-arid climate of northeastern region of Brazil. The essential oil of C. zehntneri (EOCz) has been described to have several pharmacologic properties, including effect on airflow resistance of in vivo respiratory system. For this reason, we investigated the hypothesis that EOCz and its major constituents, anethole and estragole, have antispasmodic activity on tracheal muscle. In tracheal rings of Wistar rats, maintained in Krebs-Henseleit's solution, EOCz, anethole and estragole inhibited contractions induced by 60mM [K+], ACh (10μM), Ba2+ and Phorbol dibutirate (1 μM). For EOCz, anethole and estragole, the IC50 for inhibition of KCl-induced contractions were 145.8 ± 14.8, 89.9 ± 7.4 and 181.0 ± 23.3 μg/mL, respectively, and for ACh-induced contraction, they were 606.1 ± 122.0, 160.5 ± 33.0 and 358.6 ± 49.2 μg/mL. Pharmacodynamic efficacy was maximal in all cases. These data in Ba2+-induced contraction and the differential IC50 suggested that blockade of Voltage Dependent Calcium Channels (VDCC) is a component of the mechanism of action of the three agents. Evaluation of the direct effect of anethole, on VDCC, showed inhibition of the Ca2+ current through this type of channel. These results show that EOCz and the constituents have antispasmodic activity and the mechanism includes blockade of VDCC channels.

Impact of the Potential Antitumor Agent 2-(4-Hydroxyphenyl) Amino-1,4-Naphthoquinone (Q7) on Vasomotion Is Mediated by the Vascular Endothelium, But Not Vascular Smooth Muscle Cell Metabolism.

Vasomotion is defined as rhythmic oscillations in arterial diameter that regulate the blood flow and blood pressure. Because antitumor treatment may impair vascular functions and increase the blood pressure, we sought to evaluate whether a new naphthoquinone derivative, postulated as an antitumor agent, manifests adverse effects on vascular function. In this article, we evaluated the toxicity of 2-(4-hydroxyphenyl) amino-1,4-naphthoquinone (Q7) and its effects on vascular vasomotion in 3 models of vascular structure: endothelial cells, aortic ring, and smooth muscle cells. Although showing nontoxic effects, Q7 inhibited the formation of capillary-like structures of the EA.hy926 endothelial cell line grown on Matrigel. In exvivo experiments with aortic rings precontracted with phenylephrine (PE, 10-6 M), Q7 (10-5 M) significantly (P < 0.05) reduced vascular rhythmic contractions induced by the acetylcholine (ACh; 10-7-10-5 M), whereas sodium nitroprusside (a nitric oxide donor; 10-8 M) recovered the vasomotion. Furthermore, Q7 (10-5 M) did not decrease KCl-induced vascular rhythmic contractions in the aortic rings precontracted with BaCl2 (a nonselective K+ channel blocker; 10-3 M). Vascular smooth muscle cells (A7r5) preincubated with Q7 (10-5 M) for 3 hours also demonstrated a reduced glucose uptake. However, the Adenosine Triphosphate content was unaffected, suggesting that the rapid reduction in vasomotion observed in vascular reactivity experiments did not involve cellular metabolism but may be due to faster mechanisms involving endothelial nitric oxide and K+ channels leading to oscillations in intracellular Ca2+. In summary, the naphthoquinone derivative Q7 presents low cytotoxicity yet may alter the endothelial cell response and vasomotion in the absence of changes in smooth muscle cell metabolism.

Cardiovascular changes in genetic absence epileptic WAG/Rij rats: Effects of chronic ethosuximide treatment

Purpose: The aim of this study was to investigate the effects of chronic ethosuximide (ETX) treatment on absence seizures and cardiovascular parameters in WAG/Rij rats with genetic absence epilepsy.Materials and Methods: Eight-weeks old, male Wistar and WAG/Rij rats were divided into four groups (n=20): Wistar control, Wistar ETX, WAG/Rij control and WAG/Rij ETX. ETX groups received chronic ETX treatment (oral, 300 mg/kg/day) for 3 months. At the end of the 3-month-treatment period; the total and mean duration, also number of spike wave discharges (SWDs) were evaluated using EEG recordings. Mean arterial blood pressure (MAP) and heart rate (HR) measurements were performed. Results: ETX treatment significantly decreased the duration and frequency of SWDs in WAG/Rij rats. MAP in WAG/Rij control group was markedly higher than Wistar control group. In Wistar ETX group, HR was significantly slower than Wistar control group. KCl-induced contraction response enhanced in Wistar ETX group and diminished in WAG/Rij control group compared to Wistar control group. Conclusion: Increased MAP and vascular reactivity in WAG/Rij rats. ETX treatment did not alter cardiovascular parameters in WAG/Rij rats whereas the treatment decreased the HR and vascular reactivity without affecting MAP in Wistar rats. T-type Ca++ channels may play a role in these changes.

Assessment of Polygonum odoratum Lour. Leaf Extract on Rat's Ileum Contraction and the Mechanisms Involved.

Vietnamese coriander (Polygonum odoratum Lour.) is a plant native to northern Thailand. The biological activities of P. odoratum Lour. extract (POE) include antibacterial, antiviral, and expectorant. However, the effect of POE on intestinal smooth muscle motility is unclear. The aim of this study was to evaluate the relaxant effects of POE on isolated rat ileum. Propranolol (1 μM), calcium chloride (1-20 mM), and Nω-nitro-l-arginine methylester (l-NAME, 100 μM) were used to investigate the mechanisms of action. The results showed that POE (0.01-5 mg/mL) reduced KCl-induced contraction. In addition, POE (1 mg/mL) reduced the contraction by propranolol and l-NAME and attenuated CaCl2-induced contractions. Our results indicate that the relaxation effect of POE on ileum contractions seems to involve nitric oxide and β-adrenergic pathways, and blockade of calcium influx. These findings provide a pharmacological basis for the traditional use of POE to treat gastrointestinal disorders such as irritable bowel syndrome or diarrhea.