NE-induced Contraction Research Articles

Mouse liver blood flow is regulated by hepatic stellate cells in response to the sympathetic neurotransmitter norepinephrine

BackgroundThere is no clear information on the regulation of liver blood flow by the autonomic nervous system. We conducted this study to investigate whether quiescent hepatic stellate cells (qHSCs) regulate liver blood flow in response to the sympathetic neurotransmitter norepinephrine (NE). MethodsqHSCs isolated from mice were cultured in Dulbecco's modified Eagle medium without fetal bovine serum for 1 day on collagen gel. NE-induced qHSC contraction was evaluated using the quantitative single-cell contraction measurement method that we had developed previously. For the measurement of liver perfusion pressure in situ, a buffer solution was perfused from the portal vein in mice. ResultsNE-induced a reversible contraction of qHSCs. This contraction was suppressed by the nonmuscle myosin II inhibitor blebbistatin, the myosin light chain kinase inhibitor ML-9, the Rho kinase inhibitor H-1152, the calmodulin inhibitor W-7, the store-operated calcium channel inhibitor YM-58483, and the IP3 receptor inhibitor xestospongin C. In contrast, the transient receptor potential C channel inhibitor SKF96365 did not affect the NE-induced contraction. ConclusionThese results suggest that qHSCs contract in response to NE. New & noteworthyThe present study provides direct evidence for the first time that norepinephrine (NE) induces a reversible contraction of isolated single quiescent hepatic stellate cells (qHSCs) and further suggests that the NE-mediated qHSC contraction participates in the regulation of liver blood flow in vivo.

Egg white hydrolysate modulates the renin-angiotensin system in mesenteric perivascular adipose tissue in doca-salt hypertensive rats and restores its anticontractile ability

Perivascular adipose tissue (PVAT) regulates vascular tonus with an anticontractile effect, which may be dysfunctional in hypertension. An Egg White Hydrolysate (EWH), a bioactive food, restores endothelium dysfunction and reduces blood pressure levels in malignant hypertension. We aimed to evaluate whether dietary supplementation with EWH interferes with the PVAT function in the mesenteric resistance arteries (MRA) of DOCA-salt hypertensive animals and the mechanisms involved. Male rats were divided into 4 groups and treated for 8 weeks. For 4 weeks, DOCA-salt or vehicle-treated rats (SHAM) were induced, and after that, some rats were co-treated with DOCA-salt or vehicle plus EWH (1 g/kg/day) for 4 weeks more. MRA and/or their PVAT (mPVAT) were used for functional, molecular, and biochemical analysis. The anticontractile effect of mPVAT in response to norepinephrine (NE) was observed only in MRA rings with PVAT of the SHAM group, while this effect was abolished in DOCA-salt rings. The EWH treatment did not change the anticontractile effect of mPVAT in SHAM but partially restored it in DOCA-salt rats. Acute aliskiren incubation reduced NE-induced contraction only in MRA with PVAT of DOCA-salt rats. EWH prevented the overactivation of the renin levels and ACE activity in mPVAT of DOCA-salt rats; while did not change AT1R expression, but increased AT2R expression. In mPVAT, EWH blocked the increase of MDA, ROS and pro-inflammatory cytokines observed in DOCA-salt rats. EWH improved PVAT dysfunction in MRA of severe hypertension. This beneficial effect could be mediated by an ameliorated redox balance, inflammatory state, and RAS axis.

Alkaloids from <i>Peganum harmala</i> attenuated contractile responses of vascular smooth muscle cells

Purpose: To investigate the contractile responses of vascular smooth muscle cells (VSMCs) to spasmogens after incubation with harmaline, harmine, and harmalol, which are alkaloids obtained from Peganum harmala L., a member of the Zygophyllaceae family.
 Methods: Contractile responses of VSMCs to norepinephrine (NE; 1 µmol/L) and potassium chloride (KCl; 60 mmol/L) were recorded in rat aortic ring preparations pre-incubated with 0.5, 1, 5 and 10 µmol/L of each alkaloid for 15 min. Responses were expressed as mean values of contractions in incubated preparations, relative to the recorded tension prior to treatment with alkaloids.
 Results: Pre- incubation with harmaline at concentration of 10 µmol/L significantly reduced contractile responses to NE by 69.0 ± 3.0 % (p < 0.00002), and decreased KCl-induced contraction by 34.0 ± 9.0 % (p < 0.05). Harmalol was the most effective in inhibiting contractions to KCl (48.0 ± 9.0 %, p < 0.01). However, harmalol produced relatively moderate inhibitory effects on NE-induced contractions (46.0 ± 4.0 %, p < 0.005), followed by harmine (52.0 ± 8.0 %, p < 0.02), but it did not significantly affect contractile responses to KCl.
 Conclusion: These results highlight the differential effects of pre-incubation with alkaloids from P. harmala and their potential effects on the prevention of VSMC spasms induced by either chemicals or stimuli that change the membrane potential.

The differences in the adrenergic receptors of proximal urethra between sexes.

The bladder and urethra work as a physiologically functional unit to facilitate continence in the storage and voiding phase. Sex differences have been found in the urethral contraction in response to α-adrenergic receptor activation. This study aimed to investigate the role of adrenergic receptors in the proximal urethra of male and female mice. Urinary bladder and proximal urethral smooth muscle (USM) samples from male and female C57BL/6 mice were isolated and mounted in an organ bath. Acetylcholine-induced contraction of the urinary bladder was compared in male and female mice. Phenylephrine and norepinephrine (NE) induced little contraction at a lower concentration, but a relaxing phase of female proximal USM was observed at a higher concentration. This contraction profile was inhibited by NG-nitro-L-arginine, lidocaine, and capsaicin. In addition, the NE-induced contraction was greater in the incubation of propranolol than that of L-NNA or lidocaine. These results suggested that the β-adrenoceptor may be the dominant receptor of female proximal USM, and the activity of calcitonin gene-related peptide sensory nerves and nitrergic nerves may pose an anti-contraction effect on the proximal urethra in female mice. β-adrenoceptor may be the dominant receptor of female proximal USM. The use of β-adrenergic receptor blocker agents might have the potential for the treatment of female voiding dysfunction.

Ageing-induced hypercontractility is related to functional enhancement of STIM/Orai and upregulation of Orai 3 in rat and human penile tissue

The role of STIM/Orai calcium entry system on vascular ageing has not been elucidated. We aimed to evaluate the influence of ageing on STIM/Orai signalling and its role on ageing-induced alterations of contractile function in rat corpus cavernosum (RCC) and human penile resistance arteries (HPRA) and corpus cavernosum (HCC). RCC was obtained from 3 months-old and 20 months-old animals. HPRA and HCC were obtained from organ donors of varied ages without history of erectile dysfunction. Aging was associated with enhanced norepinephrine (NE)- and thromboxane analogue (U46619)-induced contractions in RCC which were significantly inhibited by the STIM/Orai inhibitor, YM-58483 (20 μM). Other STIM/Orai inhibitor, 2-aminoethyldiphenylborate also reduced NE-induced contractions in RCC from aged rats. YM-58483 significantly reduced neurogenic contractions and potentiated neurogenic relaxations in RCC from aged rats. In HCC and HPRA, NE-induced contractions were significantly enhanced in older subjects (>65 years-old) but YM-58483 completely reversed ageing-related hypercontractility. Ageing did not modify STIM-1 and Orai1 protein expressions but Orai3 was significantly overexpressed in cavernosal tissue from old rats and older subjects. Contribution of STIM/Orai to cavernosal contraction increases with ageing together with increased expression of Orai3. Orai inhibition could be a potential therapeutic strategy to reduce ageing-related impact on vascular/erectile function.

The Role of Two-Pore Channels in Norepinephrine-Induced [Ca2+]i Rise in Rat Aortic Smooth Muscle Cells and Aorta Contraction.

Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca2+ release via two-pore channels (TPCs) localized in endolysosomal vesicles. The aim of the present work is to evaluate the role of TPCs in the action of norepinephrine (NE), angiotensin II (AngII), vasopressin (AVP), and 5-hydroxytriptamine (5-HT) on free cytoplasmic calcium concentration ([Ca2+]i) in smooth muscle cells (SMCs) isolated from rat aorta and on aorta contraction. To address this issue, the NAADP structural analogue and inhibitor of TPCs, NED 19, was applied. We have demonstrated a high degree of colocalization of the fluorescent signals of cis-NED 19 and endolysosmal probe LysoTracker in SMCs. Both cis- or trans-NED 19 inhibited the rise of [Ca2+]i in SMCs induced by 100 μM NE by 50–60%. IC50 for cis- and trans-NED 19 were 2.7 and 8.9 μM, respectively. The inhibition by NED 19 stereoisomers of the effects of AngII, AVP, and 5-HT was much weaker. Both forms of NED 19 caused relaxation of aortic rings preconstricted by NE, with relative potency of cis-NED 19 several times higher than that of trans-NED 19. Inhibition by cis-NED 19 of NE-induced contraction was maintained after intensive washing and slowly reversed within an hour of incubation. Cis- and trans-NED 19 did not cause decrease in the force of aorta contraction in response to Ang II and AVP, and only slightly relaxed aorta preconstricted by 5-HT and by KCl. Suppression of TPC1 in SMCs with siRNA caused a 40% decrease in [Ca2+]i in response to NE, whereas siRNA against TPC2 did not change NE calcium signaling. These data suggest that TPC1 is involved in the NE-stimulated [Ca2+]i rise in SMCs. Inhibition of TPC1 activity by NED 19 could be the reason for partial inhibition of aortic rings contraction in response to NE.

PS-04-001 Aging increases contribution of Orai channels to contractile tone in rat and human corpus cavernosum

The contribution of calcium Orai channels to contractile responses in human corpus cavernosum and penile arteries has been proposed, especially in pathological conditions such as erectile dysfunction (ED). The aim of the present work was to evaluate the influence of aging on Orai channels contribution to cavernosal tissue contraction. The effect of the Orai channel inhibitor, YM-58483 (YM) was evaluated on contractions induced by norepinephrine (NE) in corpus cavernosum (CC) from young (3 months-old) and aged (20 months-old) rats. The effects of YM on endothelial relaxation and neurogenic contraction were also evaluated in tissues from aged rats. As well, the influence of YM on NE-induced contractions was determined in human corpus cavernosum (HCC) from organ donors (OD) without history of ED, which were divided into younger than 65 years (43.5±2.8 years, n=16) and older than 65 years (74.2±3.5 years, n=5). The expression of Orai channels in these HCCs was determined by Western blot.

Roles and mechanism of protein kinase D in vasoconstriction

Protein kinase D (PKD) is known to be implicated in a variety of cellular processes and physiological functions, including cell motility, cardiac contraction, angiogenesis and smooth muscle contraction through regulation of multiple molecules. Although these regulations are presumed to be closely associated with vasomotor activity, role of PKD in vascular function is unknown. Then, we investigated the role of PKD in systemic circulation and contraction of aorta in in rats. We also investigated precise role s of PKD isoforms using human aortic smooth muscle cells (HASMCs) with RNA interference.Material and methodsSystemic blood pressure (SBP) through femoral artery, cardiac output (CO) and systemic vascular resistances (SVR) were measured in Sprague‐Dawley rats. The hemodynamic changes in response to intravenous injections of PKD inhibitor, CRT0066101 were recorded. Isometric tension of aorta ring samples was measured to examine inhibitory effect of CRT0066101 on norepinephrine (NE)‐induced contraction. To explore mechanisms of PKD in vasomotor activity, phosphorylations of PKD, myosin targeting subunit‐1 (MYPT1), CPI‐17, regulatory myosin light chain (RLC) and heat shock protein 27 (HSP27) stimulated with NE were measured as an index of activation of the pathways in aorta rings. In HASMCs, simultaneous measurements of intracellular calcium concentrations and cell shortening with NE stimulation were performed. PKD1, 2, and 3 were knocked down by their specific siRNA respectively in HASMCs for measurements of phosphorylation of PKD, MYPT1, CPI‐17 and RLC and actin polymerization.ResultsInjection of CRT0066101 decreased SBP and SVR. CRT0066101 also inhibited NE‐induced contraction in aorta rings dose‐dependently. In the rings, NE increased phosphorylations of PKD, MYPT1, RLC and CPI‐17, which were attenuated by pretreatment of CRT0066101 except CPI‐17. NE did not increase HSP27 phosphorylation in the rings. In HASMCs, CRT0066101 inhibited NE‐induced cell shortening without affecting increased calcium concentrations. In HASMCs, increased phosphorylations of MYPT1 and RLC with NE were significantly inhibited by PKD1 but not PKD2, 3 knock down. NE did not affect actin polymerization in the cells. Collectively, attenuation of NE‐induced contraction of aorta rings with CRT0066101 was associated with decreased phosphorylations of MYPT1 and RLC, but not CPI‐17 and HSP27. In HASMCs, PKD1 knocked down or PKD inhibition attenuated NE‐induced phosphorylation of MYPT1 and RLC without affecting actin polymerization and intracellular calcium regulation.ConclusionsPKD1 may play roles in contraction of aorta and systemic circulation. The regulation of vasomotor activity by PKD1 may be associated with phosphorylation of MYPT1. Understanding of PKD may lead to novel regulation of circulation.Support or Funding InformationThis work was supported by Grant‐in‐Aid for Scientific Research (C) 17K11058 from Japan Society for the Promotion of ScienceThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

EFFECTS OF ROSUVASTATIN IN EXPERIMENTALLY-INDUCED CARDIAC ISCHEMIA AND ON AORTIC VASCULAR REACTIVITY.

Myocardial infarction (MI) continues to be a major public health problem in the world. The present study aimed to elucidate the protective effect of different doses of rosuvastatin and cilostazol, as well as their combination on isoprenaline-induced MI in rats as well as their effects on isolated aorta. Methods: I- Adult rats received different doses of rosuvastatin (1,2,5,10, & 20mg/kg), cilostazol (18mg/kg) and combination of rosuvastatin 1mg/kg and cilostazol 18mg/kg by oral gavage daily for 16 days, then rats were subcutaneously injected with two doses 24-h apart of 150mg/kg isoprenaline in the last two days. ECG pattern was monitored, myocardial injury markers (CK-MB and LDH) and inflammatory biomarker (CRP) were measured in serum. MDA, catalase and SOD were quantified in cardiac homogenates and heart tissue damage was examined by histopathology. II- Effects of rosuvastatin (0.2 µg/ml – 6.4 µg/ml), cilostazol (7.5 µg/ml – 480 µg/ml) and combination cilostazol (30 µg/ml) and rosuvastatin (0.2 µg/ml – 6.4 µg/ml) on NE-induced contraction in rabbit aortic strip were recorded. Each dose of either rosuvastatin or cilostazol were incubated for 10 min and 15min respectively then NE (0.5 µg/ml) was added and the contraction was recorded for one and half min. Results: I- Pretreatment with different doses of rosuvastatin (1,2,5 & 10), cilostazol 18mg/kg and the combination markedly ameliorated ISO-induced alterations in ECG, cardiac markers, inflammatory marker, oxidative markers and heart architecture. However, protection disappeared at higher dose of rosuvastatin 20mg/kg. II- Addition of either rosuvastatin (0.2-6.4 µg/ml), cilostazol (7.5-480µg/ml) or combination rosuvastatin (0.2-6.4 µg/ml) and cilostazol 30µg/ml produced a significant decrease in the height of NE-induced contraction in a dose dependent manner. Conclusion: I-This study provides evidence that rosuvastatin, cilostazol and their combination possess cardioprotective effect on isoprenaline-induced myocardial infarction. II- The drugs had vasorelaxant effect on aorta. Mechanism of drug action are discussed.

Perivascular Adipose Tissue's Impact on Norepinephrine-Induced Contraction of Mesenteric Resistance Arteries.

Background: Perivascular adipose tissue (PVAT) can decrease vascular contraction to NE. We tested the hypothesis that metabolism and/or uptake of vasoactive amines by mesenteric PVAT (MPVAT) could affect NE-induced contraction of the mesenteric resistance arteries.Methods: Mesenteric resistance vessels (MRV) and MPVAT from male Sprague-Dawley rats were used. RT-PCR and Western blots were performed to detect amine metabolizing enzymes. The Amplex® Red Assay was used to quantify oxidase activity by detecting the oxidase reaction product H2O2 and the contribution of PVAT on the mesenteric arteries' contraction to NE was measured by myography.Results: Semicarbazide sensitive amine oxidase (SSAO) and monoamine oxidase A (MAO-A) were detected in MRV and MPVAT by Western blot. Addition of the amine oxidase substrates tyramine or benzylamine (1 mM) resulted in higher amine oxidase activity in the MRV, MPVAT, MPVAT's adipocyte fraction (AF), and the stromal vascular fraction (SVF). Inhibiting SSAO with semicarbazide (1 mM) decreased amine oxidase activity in the MPVAT and AF. Benzylamine-driven, but not tyramine-driven, oxidase activity in the MRV was reduced by semicarbazide. By contrast, no reduction in oxidase activity in all sample types was observed with use of the monoamine oxidase inhibitors clorgyline (1 μM) or pargyline (1 μM). Inhibition of MAO-A/B or SSAO individually did not alter contraction to NE. However, inhibition of both MAO and SSAO increased the potency of NE at mesenteric arteries with PVAT. Addition of MAO and SSAO inhibitors along with the H2O2 scavenger catalase reduced PVAT's anti-contractile effect to NE. Inhibition of the norepinephrine transporter (NET) with nisoxetine also reduced PVAT's anti-contractile effect to NE.Conclusions: PVAT's uptake and metabolism of NE may contribute to the anti-contractile effect of PVAT. MPVAT and adipocytes within MPVAT are a source of SSAO.

Aged garlic extract exerts endothelium-dependent vasorelaxant effect on rat aorta by increasing nitric oxide production

BackgroundClinical trials have shown that aged garlic extract (AGE) is effective in reducing blood pressure of hypertensive patients. However, the mechanisms involved remain to be elucidated. PurposeThe aim of the present study was to investigate the vasorelaxant effect of AGE on the aorta and its mechanism of action in order to clarify the blood pressure-lowering action of AGE. MethodsThe vasorelaxant effect was evaluated in isolated rat aortic rings. After aortic rings were contracted by 3 × 10−6M norepinephrine (NE) for 30min, AGE and other test drugs were added to the aortic rings. All results were expressed as percentages of the maximal NE-induced contraction. ResultsAGE induced the concentration–dependent vasorelaxation of isolated rat aortic rings that had been precontracted with norepinephrine. The effect of AGE was severely impaired in aortic rings lacking endothelium. In addition, the effect of AGE was inhibited by a nitric oxide synthase (NOS) inhibitor and a nitric oxide (NO) scavenger. Moreover, AGE treatment of aorta significantly increased the NO production. When various constituents of AGE were tested, the vasorelaxation of aorta was observed only in the presence of L-arginine, a substrate of NOS. ConclusionAGE causes endothelium-dependent vasorelaxation of aorta via stimulation of NO production and that L-arginine in AGE serves as a key agent for NOS-mediated NO production.

Effect of sulodexide on vascular responses and liver mitochondrial function in diabetic rats.

This study investigates the effects of long-term treatment with sulodexide (SLX) on norepinephrine (NE)-induced contractions, acetylcholine(Ach)-induced relaxations, acute cyclooxygenase blockade by diclofenac (DIC) in isolated femoral arteries (FA) and the parameters of oxidative phosporylation in liver mitochondria. 15-weeks old Wistar rats were divided into four groups: control (C; injected with saline solution), treated control (C+SLX), diabetic (DM) and treated diabetic (DM+SLX). Diabetes was induced with a single i.v. dose of streptozotocin (STZ) 45 mg.kg(-1). SLX was administered i.p., at dose 100 IU.kg(-1) daily for 5 weeks. Vascular responses of isolated femoral arteries were measured using Mulvany-Halpern myograph. Respiratory function of the mitochondria was determined using voltamperometric method on oxygraph Gilson. In diabetic rats the amplitude of maximal response to NE was elevated. DIC pretreatment decreased the amplitudes of NE-induced contractions in all groups of rats. SLX treatment decreased sensitivity of FA to NE and caused higher relaxatory responses to Ach in C and DM. Oxygen consumption and phosphorylation rates ([QO(2)(S(3))], [QO(2)(S(4))] and (OPR)) and respiratory control ratio (RCR) were decreased in the mitochondria of DM rats. Mitochondria of C rats were not affected with SLX treatment. Administration of SLX in DM rats was associated with increase of RCR, other parameters were not affected. Our findings suggest that SLX treatment might be associated with vasculoprotective effects during diabetes and improvement of mitochondrial function.

Endothelial dysfunction in rats with ligature-induced periodontitis: Participation of nitric oxide and cycloxygenase-2-derived products

ObjectivesConsidering the evident relationship between periodontitis and cardiovascular diseases in humans, we aimed to study the in vitro vascular reactivity of aorta rings prepared from rats with ligature-induced periodontitis. MethodsSeven days after the induction of unilateral periodontitis, the animals were euthanised; rings were prepared from the descending abdominal aortas and mounted in tissue baths for the in vitro measurement of the isometric force responses to norepinephrine (NE) and acetylcholine (ACh), as well as in the presence of inhibitors of nitric oxide synthase (NOS) and cycloxygenase (COX) isoenzymes. Aortic COX and NOS gene expressions were analysed by RT-PCR, as well as protein COX-2 expression by Western blot. ResultsPeriodontitis resulted in significant alveolar bone loss and did not affect arterial pressure. However, both NE-induced contraction and ACh-induced relaxation were significantly decreased and related to the presence of endothelium. Diminished eNOS and augmented COX-2 and iNOS expressions were found in the aortas from rats with periodontitis, and the pharmacological inhibition of COX-2 or iNOS improved the observed vasomotor deficiencies. ConclusionsWe can thus conclude that periodontitis induces significant endothelial dysfunction in rat aorta which is characterized by decreased eNOS expression and mediated by upregulated iNOS and COX-2 products.

Abstract P627: The β3 Adrenergic Receptor is Partially Responsible for Anti-contractile Effect of Perivascular Adipose Tissue in Rat Mesenteric Resistance Arteries

In normal conditions, perivascular adipose tissue (PVAT) decreases contractile responses non-specifically in various vascular beds. This anti-contractile effect of PVAT is reduced in metabolic diseases and hypertension. The β3 adrenergic receptor (β3AR) is a G protein-coupled receptor expressed in adipocytes and involved in lipolysis and thermoregulation. We have previously demonstrated that chronic systemic infusion with a β3AR agonist induces white-to-brown adipose tissue remodeling and enhanced anti-contractile effects of PVAT via activation of cystathionine gamma lyase, enzyme involved in hydrogen sulfide synthesis. We hypothesized that the β3AR is directly mediating release of PVAT relaxing factors. Endothelium-intact mesenteric resistance arteries from adult male Wistar rats were used to measure contractile responses in the presence and absence of PVAT. In the absence of PVAT, the β3AR agonist CL316243 (1 nM-10 μM) did not directly induce relaxation of U46619-contracted arteries. In control conditions, norepinephrine (NE)-induced contraction was significantly reduced in the presence of PVAT. In contrast, incubation with the selective β3AR antagonist L-748337 (100 nM) led to a significant increase in NE-induced contraction in PVAT-intact arteries, while no change was observed in the absence of PVAT (figure). These data suggest that β3AR mediates the anti-contractile effect of PVAT on NE-induced contraction in resistance mesenteric arteries. Considering the structural and functional alterations of PVAT in hypertension, future studies may reveal a potential novel therapeutic approach via targeting of the PVAT β3AR pathway.

Role of large conductance calcium-activated potassium channels in vascular hyporesponsiveness in rats with obstructive jaundice

Objective To evaluate the role of large conductance calcium-activated potassium (BKCa) channels in vascular hyporesponsiveness in rats with obstructive jaundice. Methods Eighteen male Sprague-Dawley rats, weighing 180-200 g, were randomly divided into 3 groups (n = 6 each) using a random number table: control group (group C), sham operation group (group S), and bile duct ligation group (group BDL). Obstructive jaundice was produced by common bile duct ligation.At 7 days after surgery, blood samples were collected for determination of the levels of serum total bilirubin (TBL), direct bilirubin (DBL), indirect bilirubin (IBL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Thoracic aortic rings were prepared, and the endothelium was removed.The aortic rings were sequentially perfused with different concentrations of norepinephrine (NE) and sodium nitroprusside (SNP), and the maximum amplitude of contraction and dilatation of aortic rings was recorded.The aortic rings were then perfused with BKCa channel blocker Chtx with the final concentration of 10-7 mol/L, followed by perfusion with different concentrations of NE and SNP again, and the maximum amplitude of contraction and dilatation of aortic rings was recorded under each concentration.The percentage of maximum contraction and dilatation (maximum amplitude after Chtx administration÷maximum amplitude before Chtx administration×100%) was calculated. Results Compared with C and S groups, the levels of TBL, DBL, IBL, ALT and AST in serum were significantly increased, the maximum amplitude of NE-induced contraction of aortic rings was decreased, and the percentage of the maximum NE-induced dilatation of aortic rings was increased, the maximum amplitude of SNP-induced contraction of aortic rings was increased, and the percentage of the maximum SNP-induced dilatation of aortic rings was decreased in group BDL. Conclusion Excessive opening of BKCa channels may be involved in the mechanism of vascular hyporesponsiveness in rats with obstructive jaundice. Key words: Jaundice, obstructive; Large-conductance calcium-activated potassium channels; Aorta, thoracic

Erythropoietin Reverses Sepsis-Induced Vasoplegia to Norepinephrine Through Preservation of α1D-Adrenoceptor mRNA Expression and Inhibition of GRK2-Mediated Desensitization in Mouse Aorta.

We investigated the effect of erythropoietin (EPO) posttreatment on survival time and vascular functions in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture. After 20 ± 2 hours of sepsis, thoracic aorta was isolated for assessing its reactivity to norepinephrine (NE) and acetylcholine (ACh). We also measured the tissue nitric oxide (NO) level, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), G protein-coupled receptor kinase 2 (GRK2), and α1D adrenoceptor messenger RNA (mRNA)/protein expression. In septic mice, EPO moderately improved the survival time from 19.68 ± 0.75 to 34.7 ± 3.2 hours. Sepsis significantly decreased the aortic contractile response to NE along with reduced α1D mRNA and protein expression. Erythropoietin significantly preserved the α1D receptor expression and restored NE-induced contractions to control levels in septic mice. Further, it attenuated the aortic α1D receptor desensitization in sepsis which was evident from reduced GRK2 mRNA expression. Accordingly, a selective GRK2 inhibitor markedly restored the contractile responses to NE in sepsis. Erythropoietin treatment attenuated iNOS mRNA expression and iNOS-induced overproduction of NO, but improved endothelium-dependent relaxation to ACh associated with increased eNOS mRNA expression. In conclusion, EPO seems to reverse sepsis-induced vasoplegia to NE through the preservation of α1D adrenoceptor mRNA/protein expression, inhibition of GRK2-mediated desensitization, and attenuation of NO overproduction in the mouse aorta.

Echinacoside induces rat pulmonary artery vasorelaxation by opening the NO-cGMP-PKG-BKCa channels and reducing intracellular Ca2+ levels.

Sustained pulmonary vasoconstriction as experienced at high altitude can lead to pulmonary hypertension (PH). The main purpose of this study is to investigate the vasorelaxant effect of echinacoside (ECH), a phenylethanoid glycoside from the Tibetan herb Lagotis brevituba Maxim and Cistanche tubulosa, on the pulmonary artery and its potential mechanism. Pulmonary arterial rings obtained from male Wistar rats were suspended in organ chambers filled with Krebs-Henseleit solution, and isometric tension was measured using a force transducer. Intracellular Ca(2+) levels were measured in cultured rat pulmonary arterial smooth muscle cells (PASMCs) using Fluo 4-AM. ECH (30-300 μmol/L) relaxed rat pulmonary arteries precontracted by noradrenaline (NE) in a concentration-dependent manner, and this effect could be observed in both intact endothelium and endothelium-denuded rings, but with a significantly lower maximum response and a higher EC50 in endothelium-denuded rings. This effect was significantly blocked by L-NAME, TEA, and BaCl2. However, IMT, 4-AP, and Gli did not inhibit ECH-induced relaxation. Under extracellular Ca(2+)-free conditions, the maximum contraction was reduced to 24.54%±2.97% and 10.60%±2.07% in rings treated with 100 and 300 μmol/L of ECH, respectively. Under extracellular calcium influx conditions, the maximum contraction was reduced to 112.42%±7.30%, 100.29%±8.66%, and 74.74%±4.95% in rings treated with 30, 100, and 300 μmol/L of ECH, respectively. After cells were loaded with Fluo 4-AM, the mean fluorescence intensity was lower in cells treated with ECH (100 μmol/L) than with NE. ECH suppresses NE-induced contraction of rat pulmonary artery via reducing intracellular Ca(2+) levels, and induces its relaxation through the NO-cGMP pathway and opening of K(+) channels (BKCa and KIR).

MP31-12 TAMSULOSIN IMPROVES MEMORY FUNCTION BY ACTIVATING NMDA RECEPTORS IN RAT HIPPOCAMPUS

INTRODUCTION AND OBJECTIVES: Prostate smooth muscle contraction may be critical for pathophysiology and therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostate hyperplasia. Although application of a1-blockers represents the goldstandard for medical treatment, their effects are limited to max. 50%. The reasons are unknown, while only little is known about contributions of nonadrenergic mediators to prostate contraction. In vivo, different mediators probably do not occur separately, but rather act simultaneously on smooth muscle cells. Here, we compared different contractile mediators with norepinephrine, and adressed their cooperative effects in contraction of human prostate smooth muscle. METHODS: Prostate tissues were obtained from radical prostatectomy (n1⁄472 patients). Contractility of prostate strips was assessed in an organ bath. Receptor expression was detected by Western blot analysis and fluorescence staining. RESULTS: Maximum endothelin-1 (ET-1)(0.1-3 mM), endothelin-2 (ET-2)(0.1-3 mM), and norepinephrine (NE)(0.1-100 mM) induced contractions were of similar magnitude (ET-1 116 23% of KCl-induced contraction; ET-2 86 17%; NE 117 18%). Maximum contractions by the thromboxane analogue, U46619 (10 mM) amounted on average around 50% of NE-induced contraction (63 9% of KCl). ET-1or ET-2-induced contractions were not affected by the a1-blocker tamsulosin (300 nM), while NE-induced contraction was virtually completely abolished and U46619-induced contraction was slightly inhibited. Dopamine (0.1-300 mM) and serotonin (0.1-300 mM) induced only very slight contractions (22 4% and 9 2% of KCl) of prostate strips, which may be of minor or local relevance. Carbachol and uridine adenosine tetraphosphate did not induce contractions. After maximumNE-induced precontraction, ET-1 did not further increase tension. In contrast, contractile effects of ET-1 (3 mM) and U46619 (10 mM) were additive, so that tension after combined application of ET-1 and U46619 (172 25% of KCl) was higher than maximumNE(100 mM) induced tension (p<0.05). Endothelin receptor A and B were detectable by Western blot in all samples. Double fluorescence staining suggests expression in stromal smooth muscle cells. CONCLUSIONS: NE-induced contraction of human prostate smooth muscle can be exceeded by cooperative actions of endothelins and thromboxane. These contractions may even occur under treatment with a1-blockers. Such contributions of non-adrenergic mediators might explain the limited efficacy of a1-blockers.

MP31-10 ADDITIVE CONTRACTIONS BY THROMBOXANE A2 AND ENDOTHELIN-1 ARE STRONGER THAN NOREPINEPHRINE-INDUCED CONTRACTIONS IN HUMAN PROSTATE SMOOTH MUSCLE

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 2015MP31-10 ADDITIVE CONTRACTIONS BY THROMBOXANE A2 AND ENDOTHELIN-1 ARE STRONGER THAN NOREPINEPHRINE-INDUCED CONTRACTIONS IN HUMAN PROSTATE SMOOTH MUSCLE Martin Hennenberg, Alice C. Acevedo, Alexander Tamalunas, Yiming Wang, Beata Rutz, Frank Strittmatter, Raphaela Waidelich, Christian G. Stief, and Christian Gratzke Martin HennenbergMartin Hennenberg More articles by this author , Alice C. AcevedoAlice C. Acevedo More articles by this author , Alexander TamalunasAlexander Tamalunas More articles by this author , Yiming WangYiming Wang More articles by this author , Beata RutzBeata Rutz More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , and Christian GratzkeChristian Gratzke More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1365AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate smooth muscle contraction may be critical for pathophysiology and therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostate hyperplasia. Although application of α1-blockers represents the goldstandard for medical treatment, their effects are limited to max. 50%. The reasons are unknown, while only little is known about contributions of non-adrenergic mediators to prostate contraction. In vivo, different mediators probably do not occur separately, but rather act simultaneously on smooth muscle cells. Here, we compared different contractile mediators with norepinephrine, and adressed their cooperative effects in contraction of human prostate smooth muscle. METHODS Prostate tissues were obtained from radical prostatectomy (n=72 patients). Contractility of prostate strips was assessed in an organ bath. Receptor expression was detected by Western blot analysis and fluorescence staining. RESULTS Maximum endothelin-1 (ET-1)- (0.1-3 μM), endothelin-2 (ET-2)- (0.1-3 μM), and norepinephrine (NE)- (0.1-100 μM) induced contractions were of similar magnitude (ET-1 116±23% of KCl-induced contraction; ET-2 86±17%; NE 117±18%). Maximum contractions by the thromboxane analogue, U46619 (10 μM) amounted on average around 50% of NE-induced contraction (63±9% of KCl). ET-1- or ET-2-induced contractions were not affected by the α1-blocker tamsulosin (300 nM), while NE-induced contraction was virtually completely abolished and U46619-induced contraction was slightly inhibited. Dopamine (0.1-300 μM) and serotonin (0.1-300 μM) induced only very slight contractions (22±4% and 9±2% of KCl) of prostate strips, which may be of minor or local relevance. Carbachol and uridine adenosine tetraphosphate did not induce contractions. After maximum NE-induced precontraction, ET-1 did not further increase tension. In contrast, contractile effects of ET-1 (3 μM) and U46619 (10 μM) were additive, so that tension after combined application of ET-1 and U46619 (172±25% of KCl) was higher than maximum NE- (100 μM) induced tension (p<0.05). Endothelin receptor A and B were detectable by Western blot in all samples. Double fluorescence staining suggests expression in stromal smooth muscle cells. CONCLUSIONS NE-induced contraction of human prostate smooth muscle can be exceeded by cooperative actions of endothelins and thromboxane. These contractions may even occur under treatment with α1-blockers. Such contributions of non-adrenergic mediators might explain the limited efficacy of α1-blockers. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e359 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Martin Hennenberg More articles by this author Alice C. Acevedo More articles by this author Alexander Tamalunas More articles by this author Yiming Wang More articles by this author Beata Rutz More articles by this author Frank Strittmatter More articles by this author Raphaela Waidelich More articles by this author Christian G. Stief More articles by this author Christian Gratzke More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Non-steroidal anti-inflammatory drugs attenuate the vascular responses in aging metabolic syndrome rats.

Metabolic syndrome (MS) and aging are low-grade systemic inflammatory conditions, and inflammation is a key component of endothelial dysfunction. The aim of this study was to investigate the effects of non-steroidal anti-inflammatory drugs (NSAIDs) upon the vascular reactivity in aging MS rats. MS was induced in young male rats by adding 30% sucrose in drinking water over 6, 12, and 18 months. When the treatment was finished, the blood samples were collected, and aortas were dissected out. The expression of COX isoenzymes and PLA2 in the aortas was analyzed using Western blot analysis. The contractile responses of aortic rings to norepinephrine (1 μmol/L) were measured in the presence or absence of different NSAIDs (10 μmol/L for each). Serum levels of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) in control rats were remained stable during the aging process, whereas serum IL-6 in MS rats were significantly increased at 12 and 18 months. The levels of COX isoenzyme and PLA2 in aortas from control rats increased with the aging, whereas those in aortas from MS rats were irregularly increased with the highest levels at 6 months. Pretreatment with acetylsalicylic acid (a COX-1 preferential inhibitor), indomethacin (a non-selective COX inhibitor) or meloxicam (a COX-2 preferential inhibitor) decreased NE-induced contractions of aortic rings from MS rats at all the ages, with meloxicam being the most potent. Acetylsalicylic acid also significantly reduced the maximum responses of ACh-induced vasorelaxation of aortic rings from MS rats, but indomethacin and meloxicam had no effect. NSAIDs can directly affect vascular responses in aging MS rats. Understanding the effects of NSAIDs on blood vessels may improve the treatment of cardiovascular diseases and MS in the elders.