Concentration-dependent Contractions Research Articles

Pre- and post-junctional bradykinin B2 receptors regulate smooth muscle tension to the pig intravesical ureter.

Neuronal and non-neuronal bradykinin (BK) receptors regulate the contractility of the bladder urine outflow region. The current study investigates the role of BK receptors in the regulation of the smooth muscle contractility of the pig intravesical ureter. Western blot and immunohistochemistry were used to show the expression of BK B1 and B2 receptors and myographs for isometric force recordings. B2 receptor expression was consistently detected in the intravesical ureter urothelium and smooth muscle layer, B1 expression was not detected where a strong B2 immunoreactivity was observed within nerve fibers among smooth muscle bundles. On ureteral strips basal tone, BK induced concentration-dependent contractions, were potently reduced by extracellular Ca(2+) removal and by B2 receptor and voltage-gated Ca(2+) (VOC) channel blockade. BK contraction did not change as a consequence of urothelium mechanical removal or cyclooxygenase and Rho-associated protein kinase inhibition. On 9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2α (U46619)-precontracted samples, under non-adrenergic non-cholinergic (NANC) and nitric oxide (NO)-independent NANC conditions, electrical field stimulation-elicited frequency-dependent relaxations which were reduced by B2 receptor blockade. Kallidin, a B1 receptor agonist, failed to increase preparation basal tension or to induce relaxation on U46619-induced tone. The present results suggest that BK produces contraction of pig intravesical ureter via smooth muscle B2 receptors coupled to extracellular Ca(2+) entry mainly via VOC (L-type) channels. Facilitatory neuronal B2 receptors modulating NO-dependent or independent NANC inhibitory neurotransmission are also demonstrated.

In vitro Effects of the Organophosphorus Pesticide Malathion on the Reactivity of Rat Aorta

Background and Purpose: There is a remarkable paucity of studies analyzing the role of the endothelium-derived relaxing factors on the vascular effects of organophosphates. This study was carried out to evaluate the vascular effects of malathion and the role of nitric oxide (NO) and prostacyclin (PGI₂). Methods: Vascular reactivity measuring isometric forces in vitro (‘organ chambers') and flow cytometry (cells loaded with DAF-FM DA) were used. Results: In rat thoracic aorta segments contracted with phenylephrine (Phe) (10^-7 mol/l), malathion (10^-10 to 10^-5 mol/l) induced concentration-dependent relaxation in arteries with intact endothelium (n = 7; p < 0.05). Malathion-mediated relaxation was blocked by N-nitro-<smlcap>L</smlcap>-arginine methyl ester (<smlcap>L</smlcap>-NAME; 10^-4 mol/l), a nonspecific NO synthase inhibitor, and/or indomethacin (10^-5 mol/l), a nonspecific cyclooxygenase inhibitor (n = 10, p < 0.05). In thoracic aorta rings, with and without endothelium, Phe (10^-10 to 10^-5 mol/l) evoked concentration-dependent contraction, which was reduced in the presence of malathion. In rings with or without endothelium, incubated with malathion, <smlcap>L</smlcap>-NAME and indomethacin, the Phe-induced contraction was restored. The role of NO was confirmed using flow cytometry. Malathion evokes endothelium-dependent relaxation through the M₁ muscarinic receptor, since this relaxation was clearly blocked by atropine (M₁ and M₂ blocker) and pirenzepine (M₁ blocker), but was less blocked by gallamine (M₂ blocker) or 4-DAMP (M₃ blocker). Conclusions: These findings suggest that the organophosphate compound effects on vascular reactivity depend of NO and PGI₂.

The effects of methyl palmitate, a putative regulator from perivascular fat, on the contractility of pregnant human myometrium

AimsMethyl palmitate is thought to cause relaxation in vascular smooth muscle by opening voltage-activated potassium channels. We have tested the hypothesis that methyl palmitate, a putative regulator from perivascular fat, is an inhibitor of the contractility of human pregnant myometrium and that its effects might partially explain the higher incidence of dysfunctional labor in obese women compared to those with normal body mass indices. Main methodsStrips of myometrium obtained with informed consent from women undergoing elective cesarean section at term were mounted in organ baths. Strips stimulated with oxytocin (1nM) or KCl (30mM) were exposed to cumulatively increasing concentrations of methyl palmitate up to 10μM. Similar strips were exposed to cumulative addition of the potassium channel blockers 4-aminopyridine and tetraethylammonium. The contractility of the strips was monitored and analyzed using conventional methods. Key findingsMethyl palmitate failed to inhibit oxytocin- or KCl-induced contractions over the concentration range tested. In fact, it exerted a slight excitatory effect in the presence of KCl, though not in the presence of oxytocin. The contractility of naïve strips was unaltered by exposure to 1μM methyl palmitate. Both 4-aminopyridine and tetraethylammonium produced concentration-dependent contractions of human pregnant myometrium providing pharmacological evidence for the presence of voltage-activated potassium channels in this preparation. SignificanceOur findings do not support the hypothesis that methyl palmitate is an inhibitor of human pregnant myometrial contractility. Alternate hypotheses must be pursued to explain the higher incidence of dysfunctional labor in obese women.

Izalpinin from fruits of Alpinia oxyphylla with antagonistic activity against the rat bladder contractility.

Alpinia oxyphylla (Zingiberaceae), an herbaceous perennial plant, its capsular fruit is commonly used in traditional Chinese medicine for the treatment of different urinary incontinence symptoms including frequency, urgency and nocturia. These symptoms are similar to the overactive bladder syndrome. In our lab, we found that the 95% ethanol extract of the capsular fruits exhibited significant anti-muscarinic activity. Some constituents in capsular fruits including flavonoids (e.g., izalpinin and tectochrysin), diarylheptanoids (e.g., yakuchinone A and yakuchinone B) and sesquiterpenes (e.g., nootkatone), are regarded as representative chemicals with putative pharmacological activities. This study aimed to evaluate the in vitro antagonistic actions of izalpinin on carbachol-induced contraction of the rat detrusor muscle. In vitro inhibition of rat detrusor contractile response to carbachol was used to study the functional activity of izalpinin. The isolated detrusor strips of rats were mounted in organ baths containing oxygenated Krebs' solution. The cumulative consecutive concentration-response curves to carbachol-evoked contractions in strips of rat bladder were obtained. Carbachol induced concentration-dependent contractions of isolated rat bladder detrusor strips. The vehicle DMSO had no impact on the contraction response. The contraction effects were concentration-dependently antagonized by izalpinin, with a mean EC50 value of 0.35 µM. The corresponding cumulative agonist concentration-response curves shifted right-ward. Izalpinin exhibits inhibitory role of muscarinic receptor-related detrusor contractile activity, and it may be a promising lead compound to treat overactive bladder.

Synergism of ochratoxin B and calcium-channel antagonist verapamil caused mitochondrial dysfunction

We examined the mechanism by which the ochratoxin B induced interaction with calcium-channel antagonist verapamil and mitochondrial dysfunction of the rat trachea in vitro experiment. The tracheas were cut into 2–3 mm wide rings and suspended in a tissue bath. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. Verapamil (1 × 10−6 M) produced a concentration-dependent contraction response in rat’s tracheal rings pre-contracted by acetylcholine. Incubation of rat’s tracheal rings with the ochratoxin B significantly potentiated the contraction responses of verapamil. Verapamil and OTB accelerate the overloading of Ca2+ in tracheal smooth muscle contributes the tissue toxicity as shown in electron microscopy and mitochondrial enzymes, through a mechanism that could involve perturbations of Ca2+ homeostasis. These results proved that ochratoxin B is a potential vasoconstrictor mycotoxin with the presence of calcium-channel antagonist. In conclusion, disturbance of Ca2+ homeostasis caused by OTA and plays a significant role in produces toxicity through mitochondrial enzyme inhibition.

Inhibition of smooth muscle force generation by focal adhesion kinase inhibitors in the hyperplastic human prostate.

Smooth muscle contraction may be critical for lower urinary tract symptoms (LUTS) in patients with benign prostate hyperplasia and requires stable anchorage of the cytoskeleton to the cell membrane. These connections are regulated by focal adhesion kinase (FAK). Here, we addressed the involvement of FAK in the regulation of smooth muscle contraction in hyperplastic human prostate tissues. Prostate tissues were obtained from radical prostatectomy. Expression of FAK and focal adhesion proteins was assessed by Western blot analysis and immunohistochemical stainings. Effects of the FAK inhibitors PF-573228 and Y-11 on contraction of prostate strips were examined in the organ bath. Expression of FAK and focal adhesion proteins (integrin-5α, paxilin, and c-Src) was detected by Western blot analysis in prostate samples. By double immunofluorescence staining with calponin and pan-cytokeratin, expression of FAK was observed in stromal and epithelial cells. Immunoreactivity for FAK colocalized with integrin-5α, paxilin, talin, and c-Src. Stimulation of prostate tissues with the α1-adrenergic agonist phenylephrine increased the phosphorylation state of FAK at Tyr³⁹⁷ and Tyr⁹²⁵ with different kinetics, which was blocked by the α1-adrenoceptor antagonist tamsulosin. Norepinephrine and phenylephrine induced concentration-dependent contractions of prostate strips. Both FAK inhibitors PF-573228 and Y-11 significantly inhibited norepinephrine- and phenylephrine-induced contractions. Finally, PF-573228 and Y-11 inhibited contractions induced by electric field stimulation, which was significant at the highest frequency. In conclusion, α1-adrenergic smooth muscle contraction or its regulation involves FAK in the human prostate. Consequently, FAK may be involved in the pathophysiology of LUTS and in current or future LUTS therapies.

Angiotensin IV induced contractions in human jejunal wall musculature in vitro

Angiotensin II (AngII) has been reported to mediate contractile actions in rats and human jejunal wall musculature. However, except for one report showing the angiotensin IV (AngIV) contractile effects on the internal anal sphincter of rats, no data is available describing the action of AngIV on smooth muscle in human small intestine. The aim of this study was to investigate the expression and localization of the enzymes responsible to AngIV formation, as well as the receptor, and to elucidate the contractile function of AngIV in the muscular layer of human jejunum in vitro. Jejunal smooth muscle was taken from 23 patients undergoing Roux-en-Y gastric bypass surgery and was used to record isometric tension in vitro in response to AngIV alone and in the presence of losartan or PD123319. ELISA, western blot and immunohistochemistry were used to investigate the expression and localization of key components for AngIV formation: the enzymes aminopeptidases-A, B, M, and the AngIV receptor insulin-regulated aminopeptidase (IRAP). AngIV elicited concentration-dependent contraction in both longitudinal and circular smooth-muscle preparation. Presence of losartan abolished AngIV-induced contraction, but not PD123319. The main peptide AngII, as well as the enzymes aminopeptidases-A, B and M was detected in all muscle samples. Immunohistochemistry localized the enzymes and IRAP in the myenteric plexus between longitudinal and circular muscle layers. The present study indicates that all enzymes necessary for AngIV formation exist in human jejunal smooth muscle and that the contractile action elicited by AngIV is primarily mediated through the AngII type 1 receptor.

Dual effects of α2 -adrenoceptors in modulating myogenic tone in sheep isolated internal anal sphincter.

The role of α-adrenoceptors in promoting continence through modulation of sphincter tone has focused primarily on the effects of α1 -adrenoceptors. We have used three clinically available agents, which are selective for α2 -adrenoceptors, to investigate their role in contractile and neurogenic responses on the internal anal sphincter (IAS). IAS strips, which had spontaneously generated tone, were used to investigate the contractile effect of lofexidine, brimonidine, and dexmedetomidine on muscle tone in the presence or absence of subtype selective antagonists. The effect of brimonidine on the magnitude and time course of neurogenic responses generated by electrical field stimulation (EFS) was also examined. The affinity of test compounds at α1 - and α2 -adrenoceptors was established by competition binding with [3H]-prazosin and [3H]-RX821002. All agonists caused concentration-dependent contraction of the IAS and lofexidine demonstrated an enantiomeric difference in potency with a 10-fold difference between the (-) and (+) isomers. Responses to lofexidine and dexmedetomidine were inhibited in the presence of the α1 -adrenoceptor selective antagonist prazosin, but not in the presence of RX811059 (α2 -adrenoceptor selective antagonist); brimonidine responses were inhibited by RX811059 and, to a lesser extent, by prazosin. Brimonidine affected both magnitude and duration of neurogenic responses, which was reversed in the presence of RX811059. We conclude that α2 -adrenoceptors can mediate contraction of IAS, although this effect is most evident with efficacious imidazoline agonists rather than the most selective ligand. In addition, this receptor subtype can directly inhibit noradrenergic contractile responses to EFS and, indirectly, enhance nitrergic relaxatory responses.

Bradykinin induces NO and PGF2α production via B2 receptor activation from cultured porcine basilar arterial endothelial cells.

Our previous in vitro study demonstrated that bradykinin (BK) induced relaxation and contraction of porcine basilar artery (PBA) mediated via activation of endothelial B2 receptors. The main relaxing and contracting factors appeared to be nitric oxide (NO) and prostaglandin (PG) H2, respectively, but not thromboxane A2. After obtaining these findings, we succeeded in cultivating endothelial cells isolated from the PBA. Although PGH2 has different functionally active isoforms, including PGD2, PGE2, and PGF2α, we have not yet clarified which of them is responsible for BK-induced contraction. Therefore, we attempted to quantify NO and PG production from cultured porcine basilar arterial endothelial cells (PBAECs) and to identify which of the PGs was involved in this contraction. The cultured PBAECs produced NO spontaneously, and BK enhanced this production in a concentration-dependent manner. The NO synthase inhibitor Nω-nitro-L-arginine (L-NNA) and the B2 receptor antagonist HOE-140, but not the B1 receptor antagonist des-Arg(9), [Leu(8)]-BK, completely abolished it. In a functional study, PGD2, PGE2, and PGF2α induced concentration-dependent contractions in isolated porcine basilar arterial rings, the order of maximum contraction being PGF2α > PGE2 > PGD2. The cultured PBAECs produced PGD2, PGE2, and PGF2α spontaneously, and BK significantly enhanced the production of PGF2α, but not that of PGD2 and PGE2. The B2, but not B1, antagonist completely abolished the BK-enhanced production of PGF2α. These results suggest that BK induces production of NO and PGF2α simultaneously from PBAECs via B2 receptor activation.

Perivascular adipose tissue contains functional catecholamines.

The sympathetic nervous system and its neurotransmitter effectors are undeniably important to blood pressure control. We made the novel discovery that perivascular adipose tissue (PVAT) contains significant concentrations of catecholamines. We hypothesized that PVAT contains sufficient releasable catecholamines to affect vascular function. High-pressure liquid chromatography, isometric contractility, immunohistochemistry, whole animal approaches, and pharmacology were used to test this hypothesis. In normal rat thoracic aorta and superior mesenteric artery, the indirect sympathomimetic tyramine caused a concentration-dependent contraction that was dependent on the presence of PVAT. Tyramine stimulated release of norepinephrine (NA), dopamine (DA) and the tryptamine serotonin (5-hydroxytryptamine [5-HT]) from PVAT isolated from both arteries. In both arteries, tyramine-induced concentration-dependent contraction was rightward-shifted and reduced by the noradrenaline transporter inhibitor nisoxetine (1 μmol/L), the vesicular monoamine transporter inhibitor tetrabenazine (10 μmol/L), and abolished by the α adrenoreceptor antagonist prazosin (100 nmol/L). Inhibitors of the DA and 5-HT transporter did not alter tyramine-induced, PVAT-dependent contraction. Removal of the celiac ganglion as a neuronal source of catecholamines for superior mesenteric artery PVAT did not significantly reduce the maximum or shift the concentration-dependent contraction to tyramine. Electrical field stimulation of the isolated aorta was not affected by the presence of PVAT. These data suggest that PVAT components that are independent of sympathetic nerves can release NA in a tyramine-sensitive manner to result in arterial contraction. Because PVAT is intimately apposed to the artery, this raises the possibility of local control of arterial function by PVAT catecholamines.

Purinergic receptor stimulation induces Ca2+ oscillations and smooth muscle contraction in small pulmonary veins (1175.6)

The small intrapulmonary veins (SPV) are thought to participate in the control of pulmonary blood flow and its excessive contraction has been associated with the development of pulmonary edema. However, the regulation of smooth muscle contraction by intracellular signaling in SPV is poorly understood. We used rat lung slices with phase‐contrast and confocal microscopy to investigate Ca2+ signaling and contraction during purinergic receptor activation in small (100‐250 µm) pulmonary veins. We found that ATP induced strong, concentration‐dependent, and reversible vein contraction. Vein contraction was accompanied by intracellular Ca2+ oscillations that propagated along the vein smooth muscle cell as Ca2+ waves. In the absence of extracellular Ca2+, ATP induced Ca2+ oscillations that were attenuated in time and transient vein contraction. The InsP3 receptor inhibitor 2‐APB inhibited the Ca2+ oscillations and contraction induced by ATP in absence of extracellular Ca2+. In contrast, Ca2+ oscillations and contraction were resistant to ryanodine. Vein contraction was inhibited by phospholipase Cβ inhibitor U73122 and by purinergic receptor antagonist suramin. These results suggest that ATP is a strong vasoconstrictor of SPV and that ATP activate purinergic P2Y receptors in vein smooth muscle to induce InsP3 receptor‐mediated Ca2+ oscillations and vein contraction.Grant Funding Source: American Heart Association

Contribution of ATP-Sensitive K+ Channels to Contraction of Rat Tail Artery is Significantly Reduced in Males but Not Females Rats in Streptozotocin-Induced Diabetes: Pharmacological Analysis

The aim of this article is to find out possible gender differences of participation of ATP-sensitive potassium channels in regulation of contractility in vascular smooth muscles of rats with diabetes. We used isolated tail artery of both genders rats with streptazotocin-induced diabetes (SID). After 3 weeks, we compared concentration-effects curves obtained with phenylephrine (0.1 to 300 μM) in the presence of 100 μM of diazoxid in control and SID rats of both genders. Diazoxide at 100 μM concentration significantly antagonizes concentration-dependent contraction of tail artery induced by phenylephrine in both male and female control rats. However, this antagonism disappeared in male but not female rats treated by streptozotocin. Our results indicate that SID leads to downregulation or functional malformation (lack of reaction to the opener) of ATP-sensitive potassium channels in males, but not in females rats. This observation has to be confirmed by immunochemistry analysis and taking into account in humans.

MP19-09 NSC23766, A RAC GTPASE INHIBITOR, INHIBITS SMOOTH MUSCLE CONTRACTION IN THE HYPERPLASTIC HUMAN PROSTATE

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 2014MP19-09 NSC23766, A RAC GTPASE INHIBITOR, INHIBITS SMOOTH MUSCLE CONTRACTION IN THE HYPERPLASTIC HUMAN PROSTATE Thomas Kunit, Andrea Schreiber, Christian Gratzke, Beata Rutz, Raphaela Waidelich, Frank Strittmatter, Christian G. Stief, and Martin Hennenberg Thomas KunitThomas Kunit More articles by this author , Andrea SchreiberAndrea Schreiber More articles by this author , Christian GratzkeChristian Gratzke More articles by this author , Beata RutzBeata Rutz 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/10.1016/j.juro.2014.02.710AboutPDF 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) in patients with benign prostate hyperplasia (BPH). Although several options are available, medical therapy still remains a challenge, in particular with respect to the imminent demographic transition. Consequently, new strategies for inhibition of smooth muscle contraction in the lower urinary tract are of interest. Rac, a small monomeric GTPase, is an important regulator of smooth muscle tone outside the lower urinary tract. Here, we studied Rac inhibition and expression in the human prostate. METHODS Periurethral prostate tissues were obtained from patients undergoing radical prostatectomy (n=27). Effects of the Rac inhibitor, NSC23766 on adrenergic contraction were studied in the organ bath. Rac expression was examined by Western blot analysis, and immunofluorescence staining. RESULTS The α1-adrenoceptor agonist, phenylephrine (0.1-100 μM), induced concentration-dependent contractions of human prostate strips. NSC23766 (50 μM) inhibited phenylephrine-induced contractions (Fig.). This was significant at 0.3 μM phenylephrine (p<0.05 for NSC23766 vs. control), 1 μM (p<0.003), 3 μM (p<0.002), 10 μM (p<0.0002), and 30 μM (p<0.03). Similarly, NSC23766 (50 μM) inhibited noradrenaline-induced contractions (Fig.). This was significant at 0.3 μM (p<0.05) noreadrenaline, 1 μM (p<0.02), 3 μM (p<0.03), 10 μM (p<0.02), 30 μM (p<0.02), and 100 μM (p<0.03). In Western blot analysis revealed bands with the expected size for Rac1 in prostate tissue from each patient being included in this analysis. After immunofluorescence staining, immunoreactivity for Rac1 was observed in the prostate stroma, and in glands. Double labeling with calponin and pan-cytokeratin indicated localization in smooth muscle cells and in epithelial cells. CONCLUSIONS Rac is critical for prostate smooth muscle contraction. Regulation of smooth muscle tone in the lower urinary tract by Rac may be involved in pathophysiology of LUTS, and may represent a putative new target for therapy. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e192 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Thomas Kunit More articles by this author Andrea Schreiber More articles by this author Christian Gratzke More articles by this author Beata Rutz 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 Advertisement PDF downloadLoading ...

MP19-08 HONOKIOL, A CONSTITUENT OF MAGNOLIA SPEC., INHIBITS ADRENERGIC CONTRACTION OF HUMAN PROSTATE STRIPS AND INDUCES STROMAL CELL DEATH

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 2014MP19-08 HONOKIOL, A CONSTITUENT OF MAGNOLIA SPEC., INHIBITS ADRENERGIC CONTRACTION OF HUMAN PROSTATE STRIPS AND INDUCES STROMAL CELL DEATH Martin Hennenberg, Daniel Herrmann, Andrea Schreiber, Anna Ciotkowska, Frank Strittmatter, Christian G. Stief, and Christian Gratzke Martin HennenbergMartin Hennenberg More articles by this author , Daniel HerrmannDaniel Herrmann More articles by this author , Andrea SchreiberAndrea Schreiber More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Frank StrittmatterFrank Strittmatter 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.2014.02.709AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Medical therapy of lower urinary tract symptoms (LUTS) targets smooth muscle contraction in the lower urinary tract, or prostate growth, which may both contribute to bladder outlet obstruction (BOO). Options comprise α1-blockers to induce prostate smooth muscle relaxation, and 5α-reductase inhibitors to reduce prostate growth. As an alternative, phytotherapy is widespread. Honokiol and Magnolol, two lignan constituents of Magnolia spec., are used in traditional Japanese medicine. However, their actions in the lower urinary tract are unknown. Objective: To examine the effects of Honokiol and Magnolol on contraction of human prostate tissue, and on viability of stromal cells. METHODS Prostate tissues were obtained from radical prostatectomy. Contractility was examined in organ bath studies. Contractions were induced by noradrenaline, by the α1-adrenergic agonist, phenylephrine, or by electric field stimulation (EFS). Effects on viability of stromal cells were assessed in cultured WPMY-1 cells (an immortalized line of human prostate stromal cells). Ki-67 mRNA was assessed by RT-PCR, and proliferation by a fluorescence EdU assay. RESULTS Noradrenaline and phenylephrine induced concentration-dependent contractions of prostate strips. Honokiol (100 μM) reduced noradrenaline-induced contractions, which was significant at 10 μM (p<0.02 for Honokiol vs. control), 30 μM (p<0.04), and 100 μM noradrenaline (p<0.04) (n=10 patients) (Figure). Honokiol reduced phenylephrine-induced contractions, which was significant at 3 μM (p<0.02), 10 μM (p<0.005), 30 μM (p<0.003), and 100 μM phenylephrine (p<0.02) (n=8) (Figure). Honokiol reduced EFS-induced contractions very slightly (n=9), while EFS-induced contractions were completely unchanged by solvent. In WPMY-1 cells, Honokiol (24 h) induced cell death (Figure, lower panel, left). Magnolol (100 μM) was without effects on contraction, viability, Ki-67, or proliferation (Figure, lower panel, right). CONCLUSIONS Honokiol may interfer simultaneously with the dynamic component of BOO by inhibition of prostate smooth muscle contraction, and with the static component by killing stromal cells. Whether or not Honokiol induces urodynamic effects or improvement of BOO can only be assessed in clinical studies. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e191-e192 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Martin Hennenberg More articles by this author Daniel Herrmann More articles by this author Andrea Schreiber More articles by this author Anna Ciotkowska More articles by this author Frank Strittmatter 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 ...

Calcium influx and release mechanism(s) in histamine-induced myometrial contraction in buffaloes

The present study was undertaken to characterize the presence of histamine H1R using molecular biology tools and unravel the influx and release mechanism(s) involved in calcium signalling cascades in histamine-induced myometrial contraction in buffaloes. The presence of H1R mRNA transcript and immunoreactive membrane protein in buffalo myometrium was confirmed by RT-PCR and Western blot analysis. Further, histamine produced concentration-dependent (1nM–10μM) contraction in buffalo myometrium with a potency of 7.13±0.11. When myometrial strips were pre-incubated either with Ca2+ free solution or with nifedipine, a L-type Ca2+ channel blocker, dose response curve (DRC) of histamine was significantly (P<0.05) shifted towards right with decline in maximal contraction (Emax). Reduction in Emax of histamine in the presence of nifedipine (55.75±3.10%) was significantly (P<0.001) greater than that in the presence of ruthenium red (93.61±3.43%), a blocker of IP3-gated and RyR-sensitive Ca2+ channels. Moreover, histamine produced only 26.87±1.99% of the maximum contraction in the presence of both nifedipine and CPA (blocker of sarco-endoplasmic reticulum Ca2+-ATPase). Interestingly, following concurrent exposure to U-73122 (a PL-C inhibitor) and nifedipine, the DRC of histamine was significantly (P<0.05) shifted towards left with increase in maximal contraction (126.30±3.36%). Our findings in buffalo uterus thus suggest that influx of extracellular calcium plays a major role in histamine-induced myometrial contraction, while release of intracellular calcium through calcium-release channels of sarcoplasmic reticulum has a minor role. A possible involvement of non-selective cation channels in histamine-induced myometrial contraction cannot be ruled out, and therefore requires further investigations.

Novel vasocontractile role of the P2Y14 receptor: characterization of its signalling in porcine isolated pancreatic arteries

The P2Y₁₄ receptor is the newest member of the P2Y receptor family; it is G(i/o) protein-coupled and is activated by UDP and selectively by UDP-glucose and MRS2690 (2-thiouridine-5'-diphosphoglucose) (7-10-fold more potent than UDP-glucose). This study investigated whether P2Y₁₄ receptors were functionally expressed in porcine isolated pancreatic arteries. Pancreatic arteries were prepared for isometric tension recording and UDP-glucose, UDP and MRS2690 were applied cumulatively after preconstriction with U46619, a TxA₂ mimetic. Levels of phosphorylated myosin light chain 2 (MLC2) were assessed with Western blotting. cAMP concentrations were assessed using a competitive enzyme immunoassay kit. Concentration-dependent contractions with a rank order of potency of MRS2690 (10-fold) > UDP-glucose ≥ UDP were recorded. These contractions were reduced by PPTN {4-[4-(piperidin-4-yl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthoic acid}, a selective antagonist of P2Y₁₄ receptors, which did not affect responses to UTP. Contraction to UDP-glucose was not affected by MRS2578, a P2Y₆ receptor selective antagonist. Raising cAMP levels and forskolin, in the presence of U46619, enhanced contractions to UDP-glucose. In addition, UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP levels. Removal of the endothelium and inhibition of endothelium-derived contractile agents (TxA₂, PGF(2α) and endothelin-1) inhibited contractions to UDP glucose. Y-27632, nifedipine and thapsigargin also reduced contractions to the agonists. UDP-glucose and MRS2690 increased MLC2 phosphorylation, which was blocked by PPTN. P2Y₁₄ receptors play a novel vasocontractile role in porcine pancreatic arteries, mediating contraction via cAMP-dependent mechanisms, elevation of intracellular Ca²⁺ levels, activation of RhoA/ROCK signalling and MLC2, along with release of TxA₂, PGF(2α) and endothelin-1.

Insulin causes airway hyper-reactivity

Background: We explored the acute effects of insulin and one possible mechanism underlying the acute contractile effects of insulin on isolated tracheal smooth muscle of guinea pig in vitro. Methods: Effects of increasing concentrations of histamine (10 −7 -10 −3 M), insulin (10 −7 -10 −3 M), insulin pretreated with a fixed concentration of indomethacin (10 −6 M) were studied on isolated tracheal tissue of guinea pig in vitro by constructing cumulative concentration response curves. The tracheal smooth muscle contractions were recorded with transducer on four channel oscillograph. Results: Histamine and insulin produced a concentration-dependent reversible contraction of isolated tracheal muscle of guinea pig. The mean±standard error of the mean of maximum amplitudes of contraction with histamine, insulin and insulin pretreated with indomethacin were 92.5±1.20 mm, 35±1.13 mm and 14.55±0.62 mm respectively. Indomethacin shifted the concentration-response curve of insulin to the right and downwards. Conclusions: Insulin has acute contractile effects on guinea pig airways, which were significantly inhibited by prostaglandin synthesis inhibitor indomethacin confirming the involvement of contractile prostaglandins in insulin-induced airway hyper-responsiveness.

Vasomotor effects of acetylcholine, bradykinin, noradrenaline, 5-hydroxytryptamine, histamine and angiotensin II on the mouse basilar artery.

ABSTRACTWe investigated the responsiveness of the mouse basilar artery to acetylcholine (ACh), bradykinin (BK), noradrenaline (NA), 5-hydroxytryptamine (5-HT), histamine (His) and angiotensin (Ang) II in order to characterize the related receptor subtypes in vitro. ACh and BK induced endothelium-dependent relaxation of precontracted arteries with U-46619 (a thromboxane A2 analogue). Atropine (a non-selective muscarinic receptor antagonist) and Nω-nitro-L-arginine (a NO synthase inhibitor, L-NNA) shifted the concentration-response curve for ACh to the right, whereas pirenzepine, methoctramine and pFHHSiD (muscarinic M1, M2 and M3 antagonists, respectively) had no significant effect. L-NNA and HOE140 (a B2 antagonist) shifted the concentration-response curve for BK to the right, whereas des-Arg9-[Leu8]-BK (a B1 antagonist) and indomethacin (a cyclooxygenase inhibitor) had no significant effect. NA failed to produce any vasomotor action. His and Ang II induced concentration-dependent contraction. Diphenhydramine (a H1 antagonist) shifted the concentration-response curve for His to the right, whereas cimetidine (a H2 antagonist) had no significant effect. Losartan (an AT1 antagonist) shifted the concentration-response curve for Ang II to the right, whereas PD123319 (an AT2 antagonist) had no significant effect. These results suggest that the H1 and AT1 receptor subtypes might play an important role in arterial contraction, whereas muscarinic receptor subtypes apart from M1, M2 and M3, and B2 receptors on the endothelium, might modify these contractions to relaxations.

Young and Old Sickle Cell Disease Transgenic Mice Present Underactive Bladder

Urinary bladder dysfunction is rarely spontaneously reported by sickle cell disease (SCD) patients to their caregivers. With increasing survival of these patients, physicians may expect that urinary complaints increase in association with classical urological disorders associated with advanced age. Nocturia has long been attributed to constant increased urinary volumes in SCD. As part of the renal complications of sickling, renal medullary infarcts lead to decreased ability to concentrate urine, yielding higher daily urinary volumes, compensatory polydipsia and, eventually, the need for nocturnal bladder voiding. For comparison, the effects of alterations on bladder function have been better characterized in diabetic bladder dysfunction (DBD). It has been suggested that DBD comprehends so-called “early” and “late” phases of the disease. In the early phase of DBD, the bladder is hyperactive, leading to nocturia and urge incontinence. Later in the course of the disease, the detrusor smooth muscle becomes atonic, leading to an underactive bladder. Moreover, Berkeley sickle cell disease (SS) transgenic mice display several clinical manifestations similar to those seen in humans. Thus, the aim of this study was to evaluate the contractile mechanisms of isolated DSM (in vitro) and urinary function by cystometry (in vivo) in young (early) and old (late) SS mice. Young (3 month) old (1 year and 3 month) SS mice and young (3 month) C57BL/6 mice (control) tissues were removed and placed in Krebs solution. The strips were mounted in isolated organ baths. Cumulative concentration-response curves to muscarinic agonist, carbachol (CCh; 0.01-100 μM), hyperpolarizing solution, chloride potassium (KCl; 1-300mM) and frequency-response curves for electrical field stimulation (EFS; 1-32Hz) were obtained in DSM. Cystometry was used to evaluate the urinary function in vivo. The cumulative addition of the CCh (10nM-100µM) produced concentration dependent contractions in strips of isolated DSM. The maximal contractions induced by CCh were markedly lower in bladder muscle from the young-SS mice (0.58 ± 0.11 mN/mg of tissue; n=7; P<0.05) and old-SS mice (0.37 ± 0.11 mN/mg of tissue; n=7; P<0.05) when compared with the control mice (1.44 ± 0.23 mN/mg of tissue; n=5). No differences in the potency (pEC50) values for CCh were found between control (5.92 ± 0.05; n=5), young-SS (5.80 ± 0.06; n=7) and old-SS (5.69 ± 0.10; n=7) mice. Similarly, the cumulative addition of chloride potassium (KCl: 1–300 mM) produced concentration-dependent contractions in strips of isolated DSM. The maximal contractions induced by KCl in the DSM were significantly reduced in the young-SS mice (0.74 ± 0.12 mN/mg of tissue; n=7; P<0.05) and old-SS mice (0.50 ± 0.07 mN/mg of tissue; n=7; P<0.05), when compared to the control mice (1.11 ± 0.21 mN/mg of tissue; n=5). No differences in the potency (pEC50) values for KCl were found between control (1.29 ± 0.10; n=5), young-SS (1.20 ± 0.15; n=4) and old-SS (1.13 ± 0.27; n=7) mice. EFS-induced contractions in DSM of young-SS and old-SS mice were significantly decreased in the higher frequencies (8, 16 and 32 Hz), compared to control mice. In the cystometric study, control mice showed regular micturition cycles with rare non-voiding contractions. Interestingly, young- and old-SS mice showed atypical void patterns, characterized by the incapacity to produce characteristic bladder contractions to empty bladder. This result is in accordance with the data obtained in the functional in vitro study and is the first to show the underactive bladder in SCD mice. Our data demonstrate that DSM from young and old SS mice exhibit impaired contractile responses to the muscarinic agonist, KCl, and EFS indicating that SS mice (young or old) have an atonic detrusor smooth muscle, resulting in underactive bladder.Financial Support: FAPESP/CNPq/INCTS Disclosures:No relevant conflicts of interest to declare.

Age-dependent contribution of Rho kinase in carbachol-induced contraction of human detrusor smooth muscle in vitro

Activation of muscarinic receptors on the detrusor smooth muscle is followed by contraction, which involves both myosin light chain kinase (MLCK) and Rho kinase (ROCK). The aim of this study was to determine the relative contributions of MLCK and ROCK to carbachol-induced contraction of human detrusor smooth muscle in vitro. Detrusor smooth muscle strips were prepared from the macroscopically unaffected bladder wall of patients underwent cystectomy. The strips were fixed in an organ bath, and carbachol or KCl-induced isometric contractions were measured by force transducers. Addition of carbachol (0.4-4 μmol/L) into the bath induced concentration-dependent contractions of detrusor specimens, which was completely abolished by atropine (1 μmol/L). Pre-incubation of detrusor specimens with either the MLCK inhibitor ML-9 or the ROCK inhibitors HA1100 and Y-27632 (each at 10 μmol/L) significantly blocked carbachol-induced contractions as compared to the time-control experiments. Moreover, MLCK and ROCK inhibition were equally effective in reducing carbachol-induced contractions. The residual carbachol-induced contractions in the presence of both MLCK and ROCK inhibitors were significantly smaller than the contractions obtained when only one enzyme (either MLCK or ROCK) was inhibited, suggesting an additive effect of the two kinases. Interestingly, ROCK-mediated carbachol-induced contractions were positively correlated to the age of patients (r=o.52, P<0.05). Both MLCK and ROCK contribute to carbachol-induced contractions of human detrusor smooth muscle. ROCK inhibitors may be a new pharmacological approach to modulate human bladder hyperactivity.