Significant Contractile Response Research Articles

Increased inward rectifier K+ current of coronary artery smooth muscle cells in spontaneously hypertensive rats; partial compensation of the attenuated endothelium-dependent relaxation via Ca2+ -activated K+ channels.

Endothelium-dependent vasorelaxation is partly mediated by small-conductance (SK3) and intermediate-conductance Ca2+ -activated K+ channels (SK4) in the endothelium that results in endothelium-dependent hyperpolarization (EDH). Apart from the electrical propagation through myoendothelial gap junctions, the K+ released from the endothelium facilitates EDH by increasing inward rectifier K+ channel (Kir) conductance in smooth muscle cells. The EDH-dependent relaxation of coronary artery (CA) and Kir current in smooth muscle cells (CASMCs) of hypertensive animals are poorly understood despite the critical role of coronary flow in the hypertrophic heart. In spontaneously hypertensive (SHR) and control (WKY) rats, we found attenuation of the CA relaxation by activators of SK3 and SK4 (NS309 and 1-EBIO) in SHR. In isolated CASMCs, whole-cell patch-clamp study revealed larger IKir in SHR than WKY, whereas the myocytes of skeletal and cerebral arteries showed smaller IKir in SHR than WKY. While the treatment with IKir inhibitor (0.1mmol/L Ba2+ ) alone did not affect the WKY-CA, the SHR-CA showed significant contractile response, suggesting relaxing influence of the higher IKir in the CASMCs of SHR. Furthermore, the attenuation of NS309-induced relaxation of CA by the combined treatment with 0.1mmol/L Ba2+ was more prominent in SHR than WKY. Our study firstly shows a distinct increase of IKir in the CASMCs of SHR, which could partly compensate for the attenuated relaxation via endothelial SK3 and SK4.

Angiotensin Ⅱ induces the differentiation of mouse epicardial progenitor cells into vascular smooth muscle-like cells

Epicardial progenitor cells (EpiCs) have a crucial role in cardiac development and vasculature formation. Here we detected the expression of Angiotensin II (Ang II) receptors AT1 and AT2 on EpiCs and demonstrated that AngII could increase the expression of smooth muscle specific markers, including α-smooth muscle actin (α-SMA) and myosin heavy chain 11 (Myh11) in EpiCs. Moreover, the expression of α-SMA and Myh11 induced by Ang II was blocked by pretreatment of EpiCs with the AT1 receptor antagonist losartan, but not the AT2 receptor antagonist PD123319. We further showed that the AngII-induced cells showed significant contractile responses to carbachol. These results implied that AngII could effectively induce EpiCs to differentiate into vascular smooth muscle-like cells through the AT1 receptor.

MP30-12 SPONTANEOUS ACTIVITY OF THE HUMAN DETRUSOR DEPENDS ON THE RATIO BETWEEN CONTRACTILE RESPONSES TO NERVE STIMULATION AND ANTICHOLINERGIC EFFECTS OF PROTEIN KINASE C ACTIVATION

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II1 Apr 2016MP30-12 SPONTANEOUS ACTIVITY OF THE HUMAN DETRUSOR DEPENDS ON THE RATIO BETWEEN CONTRACTILE RESPONSES TO NERVE STIMULATION AND ANTICHOLINERGIC EFFECTS OF PROTEIN KINASE C ACTIVATION Joseph Hypolite, Randall Meecham, and Anna Malykhina Joseph HypoliteJoseph Hypolite More articles by this author , Randall MeechamRandall Meecham More articles by this author , and Anna MalykhinaAnna Malykhina More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1244AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urinary bladder function includes the ability to store urine at low intravesical pressure followed by a subsequent voiding due to a rapid phasic contraction. Neurotransmitters released by electrical field stimulation (EFS) have been shown to modulate human detrusor smooth muscle (HDSM) contractility via protein kinase C (PKC)-dependent pathways, while potassium chloride (KCl) depolarization increases contractility via activation of voltage gated calcium channels. The objective of this study was to identify the factors predictive of spontaneous activity in HDSM under physiological conditions. METHODS Experiments were conducted in vitro using detrusor smooth muscle strips isolated from full thickness human bladder specimens (N=7, 35±5.5 yr). Contractile responses to EFS (80 volts, 32 Hz, 1ms duration), KCl (125 mM), carbachol (0.1-100 µM), and phorbol,12-13 dibutyrate (PDBu) 1µM, a PKC activator, were tested in organ bath studies. All contractions were normalized to the maximal KCl response taken as 100% (n=42). Data was analyzed by ANOVA with p=0.05 being statistically significant. RESULTS The maximal amplitude (peak force) of contractile responses of HDSM strips to EFS and carbachol was approximately 2-fold higher in comparison to KCl stimulation (232±15% and 236±17%, respectively, p=0.05 to KCl stimulation). Muscle strips in which the EFS/KCL force ratio was greater than 100% (119%±14.1, high-ratio group) did not show spontaneous contractions, whereas the strips with EFS/KCl ratio of 68±6.5% (low ratio group) revealed consistent spontaneous activity. Upon stimulation with carbachol, EC50 for high-ratio group was 0.35±0.021 µM, and 1.2±0.09 µM for the low EFS/KCl ratio, respectively (p=0.05). A significant contractile response of HDSM to PDBu was associated with increased tone in response to muscarinic stimulation but low incidence of spontaneous contractions. CONCLUSIONS The results of our study provide evidence that the ratio between the peak contractile responses of HDSM contractions to EFS versus KCl is predictive of spontaneous contractile activity present in the bladder wall. When EFS/KCl ratio is higher than 100%, the detrusor tends to be more quiet with no spontaneous contractions but higher sensitivity to muscarinic receptor stimulation. When the EFS/KCl ratio is lower than 100%, the detrusor shows more spontaneous activity and lower response to muscarinic activation. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e415-e416 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Joseph Hypolite More articles by this author Randall Meecham More articles by this author Anna Malykhina More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Bovine lateral saphenous veins exposed to ergopeptine alkaloids do not relax12

The ergot alkaloid ergovaline has demonstrated a persistent and sustained contractile response in several different vascular models. It was hypothesized that different alkaloids isolated from tall fescue (Lolium arundinaceum) will contribute to this contractile response differently. The objective was to compare contractile-response patterns of single additions of the ergoline alkaloids lysergic acid, lysergol, and ergonovine and the ergopeptine alkaloids ergotamine, ergocristine, ergocryptine, ergocornine, and ergovaline (provided as tall fescue seed extract). Lateral saphenous veins were collected from 6 Holstein steers (BW = 397 ± 28 kg) immediately after slaughter, sliced into cross-sections, and suspended in myograph chambers containing oxygenated Krebs-Henseleit buffer (95% O2/5% CO2; pH = 7.4; 37°C). Treatments were added at 0 min and buffer was replaced in 15-min intervals for a 120-min incubation. In addition to maximum tension and time to reach maximum tension, percent relaxation and rate of relaxation were determined following maximum tension for each treatment. All compounds tested produced significant contractile responses (P < 0.05). All ergoline alkaloids reached maximum response in less time (P < 0.05) than the remaining compounds and began to relax immediately after first buffer change. Lysergic acid had the greatest (P < 0.05) percent relaxation and ergonovine had the greatest (P < 0.05) rate of relaxation. The ergopeptine alkaloids ergovaline, ergotamine, ergocristine, ergocryptine, and ergocornine had slower developing contractile responses with a longer (P < 0.05) interval until maximum tension was achieved compared to the ergoline alkaloids. Maximal responses to all the ergopeptine alkaloids, however, all persisted for the 120-min duration with negligible relaxation occurring. The different classes of alkaloids differed greatly in the type of contractile response generated in the lateral saphenous vein. Persistence of contractile response is thought to be the primary contributing factor to the vasoconstriction observed in animals demonstrating signs of fescue toxicosis, where different ergot alkaloids can contribute differently.

Protein kinase C activation of a blebbistatin sensitive contractile component in the wall of hypertrophying mouse urinary bladder.

To examine the role of protein kinase C (PKC) and non-muscle myosin in regulation of wall tension in the hypertrophied urinary bladder. A partial urinary outflow obstruction was induced in the mouse. Tissue strips from sham operated controls and obstructed bladders were examined in vitro with quantitative gel electrophoresis, immunohistochemistry, and in vitro force recordings. Outlet obstruction (14-18 days) induced a significant growth of the bladder, 73 ± 6.13 mg compared to 19 ± 1 13 mg in sham operated controls. The hypertrophying bladder tissue had increased expression of non-muscle myosin B (SMemb) mainly localized to serosa and suburothelium. Direct activation of PKC with PDBu did not alter force in the control urinary bladder. In contrast, PDBu initiated a prominent and sustained contraction which had an increased sensitivity to the myosin type II inhibitor blebbistatin. PKC activates a significant contractile response in the wall of the hypertrophying urinary bladder, possibly supported by non-muscle myosin. This contractile component is not contributing to the physiological response to muscarinic stimulation, but might be separately regulated by other, yet unknown, mechanisms.

TGFβ/Smad4-Dependent and -Independent Regulation of Human Lens Epithelial Cells

Transforming growth factor (TGF)-beta can mediate fibrotic responses via Smad4-dependent and -independent signaling pathways. TGFbeta-induced transdifferentiation of lens epithelial cells to myofibroblasts and matrix contraction contribute to posterior capsule opacification (PCO) after cataract surgery. The primary objective of the study was to determine the importance of Smad4 in TGFbeta2-induced transdifferentiation, matrix contraction, and Smad signaling by human lens epithelial cells. The human lens cell line FHL 124 was used. Smad4 knockdown was achieved by using siRNA technology and efficiency tested at the message and protein level by real-time PCR and Western blots, respectively. Smad4 and Smad2/3 nuclear distribution after TGFbeta2 stimulation (10 ng/mL) was determined by immunocytochemistry. Gene expression of transdifferentiation markers, alpha-smooth muscle actin and fibronectin and the inhibitory Smad, Smad7, in the presence and absence of TGFbeta2 (10 ng/mL) was determined with real-time PCR. TGFbeta2-induced contraction was assessed with a patch contraction assay. Suspended bead array technology was used to determine pERK, pP38, and pJNK levels in response to TGFbeta2 over a 2-hour time course. SiRNA targeted against Smad4, successfully reduced expression of Smad4 message and protein after both the initial transfection period and removal of transfection conditions. These findings were confirmed by immunocytochemistry for Smad4, which showed a significant reduction in nuclear Smad4 after siSmad4 treatment relative to control siRNA in both the presence and absence of 10 ng/mL TGFbeta2. Smad4 knockdown cells exhibited TGFbeta2-induced Smad2/3 nuclear translocation which did not differ significantly from Smad4-expressing cells. Real-time PCR analysis showed significant induction of alphaSMA and fibronectin transcript in the Smad4-expressing cell line (control siRNA group). These inductions were suppressed in Smad4-knockdown cells. TGFbeta-induced mSmad7 expression in FHL 124 cells was unaffected by Smad4 knockdown. Smad4-expressing cells did not exhibit a significant contractile response after 24 hours of culture in the presence or absence of 10 ng/mL TGFbeta2. However, Smad4-knockdown cells demonstrated a significant reduction in patch area (i.e., contraction), after TGFbeta2 treatment. Bead array analysis showed that TGFbeta2 significantly increased pERK and pP38 levels relative to unstimulated control. No significant change was observed with pJNK. Smad4 is critical for the expression of transdifferentiation markers, alphaSMA, and fibronectin; in contrast, Smad4 signaling is not essential for TGFbeta-induced Smad7 expression. Smad4 does not appear to be necessary for TGFbeta-induced matrix contraction. TGFbeta does activate Smad-independent pathways in human lens epithelial cells. Therefore, TGFbeta2 regulation of transdifferentiation and matrix contraction appears to be mediated by distinct TGFbeta signaling pathways.

G020 Properties of pulmonary artery smooth muscle from task-1 knockout mice

In pulmonary artery (PA), compelling evidence have suggested that TWIK-related Acid Sensitive K+ -1 (TASK-1) channel contribute to the background K+ current that support the membrane potential (Em) of the smooth muscle cells. However, due to poor selectivity of its modulators, the role of TASK-1 channel in the functional regulation of tone in pulmonary arteries remains elusive. The purpose of this study was to investigate the properties of PA isolated from mice in which the TASK-1 and TASK-3 genes have been deleted (TASK1/3 knockout, KO), compared with their wild type controls (C57BL/6 mice, WT). Intra-PAs were isolated from adult male WT and KO mice. Vessels were mounted on a wire myograph for isometric tension recordings. PAs were also processed for gene expression studies using RT-PCR. The addition of 10mM KCl, the K+ channel modulators 4-AP (1mM) and levcromakalim (10μm), or the L-type calcium channel blocker nifedipine (1μm), did not elicit any significant contractile response in PAs from either KO (N=3) or WT mice (N=3), suggesting that the Em is not depolarised and that artery is fully dilated in both groups. The contractile response of PA to phenylephrine, serotonin and PGF2α did not show any difference between the two strains. Potency (pEC50) of the prostacyclin analog treprostinil to induce relaxation on preconstricted vessels was significantly higher in KO mice (6.7 ± 0.1, N = 3) than in WT mice (7.2 ± 0.1N = 4). RT-PCR confirmed TASK-1 expression in PA from WT but not KO mice whereas TASK-3 was not expressed in either WT or KO mouse PA. TASK-2, TASK-5, TWIK-2, Kv1.5 and Kv2.1 RNA levels did not show any significant alteration between WT (N = 3) or KO mice (N = 3). The PA isolated from KO mice does not display any resting tone or altered responses to the studied agonists. Surprisingly, response to treprostinil was increased, and TASK-1 absence in PA was not compensated by alteration of other K+ channels expression. The results thus far do not support a major physiological role for TASK-1 in mouse PA.

Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder

To assess the potential use of bone marrow stromal cell (BMSC)-seeded biodegradable scaffold for bladder regeneration in a canine model, by characterizing BMSCs and comparing them to bladder smooth muscle cells (SMCs) by immunohistochemistry, growth capability, and contractility. Bone marrow was taken by direct needle aspiration from the femurs of five beagle dogs for the in vitro study. Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation and cultivated in medium 199 with 10% fetal bovine serum. BMSCs were characterized by cell proliferation, in vitro contractility, immunohistochemical analysis, and the growth pattern on small intestinal submucosa (SIS) scaffolds compared to bladder SMC cultures from the same dogs. Another six dogs had a hemicystectomy and bladder augmentation with BMSC-seeded (two), bladder cells including urothelial cells plus SMC-seeded SIS (two) and unseeded SIS scaffolds (two). The six dogs were followed for 10 weeks after augmentation. In vitro BMSCs had a significant contractile response to calcium-ionophore, with a mean (sem) 36 (2)%, relative contraction (P < 0.01), which was similar to bladder SMCs but markedly different from fibroblasts. BMSCs also expressed alpha-smooth muscle actin by immunohistochemical staining and Western blotting, but did not express desmin or myosin. In vivo, both BMSC-seeded and bladder cell-seeded SIS grafts had solid smooth-muscle bundle formation throughout the graft. BMSCs had a similar cell proliferation, histological appearance and contractile phenotype as primary cultured bladder SMCs. SIS supported three-dimensional growth of BMSCs in vitro, and BMSC-seeded SIS scaffold promoted bladder regeneration in a canine model. BMSCs may serve as an alternative cell source in urological tissue engineering.

INCREASED CONTRACTILE RESPONSE TO PHENYLEPHRINE IN DETRUSOR OF PATIENTS WITH BLADDER OUTLET OBSTRUCTION: EFFECT OF THE α1A AND α1D-ADRENERGIC RECEPTOR ANTAGONIST TAMSULOSIN

Alpha1-adrenergic receptor (alpha1-AR) antagonists are effective for treating patients with lower urinary tract symptoms associated with bladder outlet obstruction (BOO). In humans up-regulation of alpha1-AR function in the detrusor in patients with BOO has been suggested but to our knowledge it is not yet confirmed. We investigated the effect of phenylephrine, an alpha1-AR agonist, on isometric tension in small detrusor muscle biopsies from patients with lower urinary tract symptoms and BOO compared with controls. Detrusor biopsies were obtained from 7 men with BOO undergoing prostatectomy and 7 undergoing cystectomy for bladder cancer (controls). Patients were characterized by symptom score and urodynamics. Isometric tension was measured in detrusor biopsies with an especially built mini myograph. Mean International Prostate Symptom Score +/- SEM in patients with BOO and controls were 22.3 +/- 2.3 and 4.0 +/- 0.8, respectively. Phenylephrine (10(-6) to 10(-3) M) induced a significant contractile response increase in detrusor biopsies from patients with BOO compared with controls at all concentrations. Tamsulosin (0.1 to 3.0 nM) inhibited phenylephrine induced contraction in a dose dependent manner. To our knowledge this functional study shows for the first time a highly significant increase in contractile force to phenylephrine in patients with BOO compared with controls. These results suggest up-regulation of alpha1-AR function in BOO since contractile responses were potently inhibited by the alpha1A/D-AR antagonist tamsulosin.

Different types of antagonism by losartan and irbesartan on the effects of angiotensin II and its degradation products in rabbit arteries.

A previous study by our group has demonstrated that the selective AT1-receptor antagonist losartan behaves as a noncompetitive antagonist in rabbit isolated renal artery (RA). In the present investigation, the influence of losartan and irbesartan on the contractile effects of angiotensin II (AII) and its degradation products angiotensin III (AIII) and angiotensin IV (AIV) was determined in the rabbit isolated RA and femoral artery (FA). The arteries were set up in organ chambers and changes in isometric force were recorded. In both rabbit isolated RA and FA preparations, AII, AIII and AIV elicited significant contractile responses with a similar efficacy. These effects were impaired by the presence of functional endothelium in RA preparations but not in FA preparations. In both preparations studied, the effects of AII, AIII and AIV were influenced neither by the aminopeptidase-A and -M inhibitor amastatin (10 microM), nor by the aminopeptidase-B and -M inhibitor bestatin (10 microM). In endothelium-denuded FA preparations, preincubation with losartan (3-300 nM) antagonized AII-, AIII- and AIV-induced contractions in a competitive manner. However, in endothelium-denuded RA preparations, losartan depressed the maximal contractile responses induced by AII but not those induced by AIII and AIV. In the same preparations, preincubation of another selective AT1-receptor antagonist irbesartan (3-30 nM) concentration-dependently shifted AII and AIII curves to the right in an insurmountable manner. The reduction of the maximal response of AII is more potent when compared to that of AIII (47.7 +/- 1.51% vs. 66.7 +/- 1.88%, percentage of the initial maximal response; P < 0.05; n=5). The selective AT2-receptor antagonist PD123177 (1 microM) did not influence the responses to all three peptides in both RA and FA preparations. These heterogeneous antagonistic effects of the two AT1-receptor antagonists studied with respect to the contractile actions of AII, AIII and AIV suggest the possible existence of multiple, functionally relevant AT1-receptor subtypes in rabbit RA preparations.

AGE-RELATED CHANGES IN CONTRACTILE RESPONSES OF RABBIT LOWER URINARY TRACT TO ENDOTHELIN

AGE-RELATED CHANGES IN CONTRACTILE RESPONSES OF RABBIT LOWER URINARY TRACT TO ENDOTHELIN

Age-related changes in contractile responses of rabbit lower urinary tract to endothelin.

As there are significant amounts of endothelin (ET) receptors in the mammalian urinary tract, we investigated the pharmacological properties and localization of ET receptors in the rabbit lower urinary tract as a function of age. The characteristics of ET receptors in bladder dome, trigone and urethra of 6 weeks and 6 months old male rabbits were determined using muscle bath and autoradiographic techniques. ET-1 produces significant contractile responses in smooth muscle strips from bladder dome, trigone, and urethra in both 6 weeks and 6 months old rabbits. Although there was no significant difference in the maximum contractile response of urethral muscle strips to ET-1 between 6 weeks and 6 months old rabbits, the maximum responses to ET-1 were higher in both bladder dome and trigone of 6 weeks than 6 months old rabbits. A selective ETA receptor antagonist, BQ 610, shifted the concentration response curve to ET-1 to the right without decreasing maximal contractile responses in all regions from both age groups, whereas a selective ETB receptor antagonist, IRL 1038, had no significant effect on the contractile response in these tissues. Autoradiographic studies indicate that both ET receptor subtypes are expressed in bladder dome, trigone, and urethra with the ETA subtype being located only in the smooth muscle layers and the ETB subtype being located in both the urothelial and smooth muscle layers. Our data indicate the presence of region- and age-dependent differences in the contractile properties of ET receptors in the male rabbit lower urinary tract. Although both ETA and ETB receptor subtypes are present in the smooth muscle layers, the ETA receptor is the sub-type that is primarily involved in the mediation of contractions.

Hormonal variation of rat uterine contractile responsiveness to selective neurokinin receptor agonists.

Regulated uterine contractions are important in many reproductive functions such as sperm transport and embryo positioning during implantation. The role of classical neurotransmitters including acetylcholine and norepinephrine in regulating myometrial contractility has been well studied; however, the peripheral role of sensory neurotransmitters such as the neurokinins is less clear. The major neurokinins are substance P, neurokinin A, and neurokinin B, which predominantly activate neurokinin receptors (NK-Rs) 1, 2, and 3, respectively. This study utilized selective receptor agonists to examine the role of NK-Rs in uterine contractility. Uterine tissues, obtained from the major stages of the rat estrous cycle, were stimulated with selective NK-R agonists. Addition of each agonist resulted in a significant contractile response. However, the magnitude and nature of the response were dependent upon the stage of the estrous cycle, with responses to all agonists being significantly decreased in tissue from proestrus and estrus. Furthermore, the nature of NK3-R-mediated contraction was different in tissue from proestrus and estrus compared to metestrus and diestrus. The hormonal dependence of NK-R-mediated contractility was then examined in the ovariectomized estrogen-supplemented rat model. These studies confirmed that the magnitude and nature of uterine contractility in response to NK-R activation depend upon the hormonal environment.

Effect of N-(Biphenylyl-Methyl)Imidazole, a Type 1 Angiotensin II Receptor Inhibitor,on the Co ntractile Function of the Rat Corpus cavernosum

The effect of N-(biphenylyl-methyl)imidazole, losartan potassium, a newly developed antihypertensive type 1 angiotensin II receptor antagonist on the rat erectile function, was studied. Sexually active 9-week-old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were given losartan 60 mg/kg intraperitoneal injections. Mean blood pressure (MBP) dropped significantly in both SHR and WKY rats (for SHR: from 140 ± 8 to 114 ± 5 mm Hg, p < 0.05, n = 8; for WKY: from 113 ± 7 to 79 ± 9 mm Hg, p < 0.05, n = 8). On the contrary, the intracavernous pressure (ICP) of SHR and WKY rats did not differ significantly from that of the corresponding controls receiving saline injections (p > 0.05, n = 8 for each group). For the chronic study, the rats were fed with losartan 30 mg/kg/day for 30 days. MBP decreased significantly in SHR but not in WKY rats (for SHR: from 137 ± 7 to 113 ± 5 mm Hg, p < 0.05, n = 8; for WKY: from 110 ± 6 to 107 ± 5 mm Hg, p > 0.05, n = 8). The ICP of the losartan-treated rats was not significantly different from that of control rats (p > 0.05, n = 8 for each group). In contrast, WKY rats receiving guanethidine 1 mg/kg/day for 30 days showed significantly decreased ICP. Angiotensin II (10^–9–10^–5 M) and losartan (10^–9– 10^–5 M) did not induce significant contractile responses of the cavernosal strip when tested in vitro. On the other hand, methoxamine 10^–4 M induced good contractile responses. In conclusion, the present study demonstrated that angiotensin II did not cause significant change in the contractile status of rat corpus cavernosum. Correspondingly, the type 1 angiotensin II inhibitor effectively lowered blood pressure but did not affect cavernosal contractile function, thus is useful clinically in the treatment of hypertensive disorders without significant detrimental effects on male sexual function.

CHARACTERIZATION OF CULTURED BLADDER SMOOTH MUSCLE CELLS: ASSESSMENT OF IN VITRO CONTRACTILITY

The contractile properties of in vitro cultured bladder smooth muscle cells (SMC) are unknown. This study characterized the in vitro contractile response of human and rat bladder SMC to several pharmacological agonists known to induce in vivo contraction of intact bladder muscle. Human and rat bladder SMC were seeded separately within attached collagen lattices. Contractility of SMC was analyzed by measuring alterations in lattice diameter after exposure and release to the following contractile agonists: carbachol (10(-7)-10(-3) microM), calcium-ionophore (10 microM), lysophosphatidic acid (LPA) (1 microM), endothelin (0.1 microM), KCl (3.33 mmicroM) angiotensin II (10 microM), and serotonin (100 microM). Results were recorded as a mean reduction of the lattice diameter. In addition, immunohistochemical analysis for phenotypic markers of smooth muscle cell differentiation was performed on bladder SMC cultured within collagen lattices. Human palmar fascia fibroblasts, which have been previously well characterized by in vitro contractility and immunohistochemistry, were tested in parallel and used as controls for all the above experiments. Human SMC had significant contractile responses to calcium-ionophore (31% +/- 4 relative percent contraction, p <0.05), LPA (34% +/- 4, p <0.05), and endothelin (37 +/- 5%, p <05). There was no significant contraction in response to carbachol, angiotensin II, KCl, or serotonin. Rat bladder SMC had a similar contractile response but did not contract in response to endothelin. In contrast to human and rat bladder SMC, fibroblasts did not contract to calcium-ionophore. In vitro cultured bladder SMC demonstrate loss of contractile response to normal in vivo pharmacologic agonists. Both human and rat bladder SMC can be distinguished in vitro from fibroblasts based upon their lack of contractile response to calcium- ionophore. These results demonstrate the ability to further characterize cultured bladder SMC with in vitro contractility. Further characterization is essential if we are to advance our understanding of the clinical applicability of in vitro studies utilizing cultured bladder SMC.

Reactivity of Small Intramyocardial Arteries from Atherosclerotic and Non-Atherosclerotic Human Hearts

Little is known about how the vascular reactivity of the coronary microcirculation is affected by upstream atherosclerotic disease. We have examined, with a wire myograph, the responses of intramyocardial arteries from hearts in which the epicardial vessels were either free of atherosclerotic lesions (non-diseased group) or were affected by atherosclerosis (diseased group). Vasodilator responses of preconstricted vessels to substance P (84.1 ± 12.6 compared to 42.0 ± 19.7%) were less in vessels from the diseased group (p < 0.05). In contrast, the relaxation to bradykinin (70.2 ± 21.2 compared to 100.6 ± 7.9%) was increased in vessels from the diseased group (p < 0.05). The dilator responses to acetylcholine, adenosine diphosphate, histamine and sodium nitroprusside showed no significant differences between arteries from each group. 5-Hydroxytryptamine was without any significant vasodilator effect in arteries from either group. Assessment of contractile function revealed that the responses to 5-hydroxytryptamine, acetylcholine, U46619, endothelin-1 and L-N^G-monomethylarginine in each group were not significantly different. Histamine, noradrenaline and dopamine were without any significant contractile response. These results demonstrate that upstream atherosclerosis does not confer any global impairment of endothelium-dependent vasorelaxant responses or smooth muscle hyperreactivity to vasoconstrictors in the arteries that penetrate the myocardium.

The involvement of nitric oxide synthase in the effect of histamine on guinea-pig airway smooth muscle in vitro.

The influence of histamine on nitric oxide synthase (NOS) in the development of airway smooth muscle hyperresponsiveness to histamine was investigated in vitro. In the absence of histamine, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) had no significant effect on the basal tone. However, precontraction of the tissues with histamine (0.3 microM) resulted in a significant contractile response to L-NAME in the preparations with intact epithelium. Removal of the epithelial layer decreased the responses to L-NAME. L-arginine could partially reverse the contraction produced by L-NAME. L-NAME enhanced the maximal response to histamine, but the sensitivity of the tracheal smooth muscle to histamine was not affected. These results suggest that, in the airway, histamine can activate NOS, resulting in the release of nitric oxide. The latter may be regarded as a local negative modulator to maintain the tissue in a physiological homeostasis.

Calcitonin gene-related peptide stimulates a positive contractile response in rat ventricular cardiomyocytes.

Calcitonin gene-related peptide (CGRP) elicits marked positive inotropic and chronotropic actions in the atria of several mammals. The second-messenger substance cyclic AMP and activation of L-type calcium channels have been implicated in these actions, but CGRP failed consistently to stimulate a contractile response in ventricular tissue obtained from various mammals. We assessed the actions of CGRP using isolated ventricular cardiomyocytes obtained from adult rats. Maximum changes in cell length (dL) of isolated cardiomyocytes during electrically stimulated (0.5 Hz) contractions were determined with adenosine deaminase (2.5 U/ml). In these conditions, CGRP produced a potent concentration-dependent positive contractile response that became maximal 4 min after initial stimulation. CGRP increased amplitude of cellular contractions maximally at a 1-nM concentration to a value 21.4% greater than that obtained without peptide. The EC50 value for the response was 31 pM. At concentrations greater than 1 nM, amplitude of the cellular contractile response decreased rapidly. The CGRP2-selective agonist, [cys ACM2,7] CGRP, increased the amplitude of cellular contractions maximally at 500 nM to a value 19.8% greater than that obtained without peptide. EC50 for this response was 6 nM. Salmon calcitonin (< or = 100 nM) did not elicit a significant contractile response. The fragment, CGRP8-37, a selective antagonist at the CGRP1 receptor subtype, while devoid of agonist activity, was a potent competitive antagonist of the positive contractile action of CGRP (pA2 value = 7.95). CGRP, present at maximally effective concentration (1 nM), when combined with isoprenaline ISO 100 pM-1 microM, elicited a greater increase in contractile amplitude than that elicited by ISO 100 pM-1 microM without CGRP. CGRP 1 nM combined with low concentrations of extracellular calcium ion < or = 4 mM produced a greater increase in contractile amplitude than that elicited by calcium ion < or = 4 mM without CGRP, but this additive effect was abolished in the presence of higher concentrations of extracellular calcium ion (> 4 mM). The cyclic AMP antagonist, Rp-cyclic AMPS (< or = 200 microM), did not inhibit the contractile response to CGRP 1 nM, but inhibited the contractile responses to ISO 100 nM and secretin 20 nM significantly and in a concentration-dependent manner. Diltiazem < or = 1 microM, a selective antagonist of L-type calcium channels, also failed to inhibit the contractile response to CGRP 1 nM but inhibited the contractile responses to ISO 100 nM and secretin 20 nM significantly and in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered pulmonary microvascular reactivity after total cardiopulmonary bypass

Pulmonary vascular resistance is frequently elevated after cardiac operations in which cardiopulmonary bypass is used. In our study of the possible contribution of altered pulmonary microvascular reactivity to this condition, sheep were heparinized, cannulated via the aorta and right atrium, and placed on total cardiopulmonary bypass. After 90 minutes of total cardiopulmonary bypass and pulmonary arterial occlusion, the sheep were removed from cardiopulmonary bypass, and their lungs were perfused normally for 60 minutes. Noninstrumented animals were used as controls. To evaluate the effect of 90 minutes of extracorporeal circulation without reduced pulmonary perfusion, we studied additional sheep after they underwent right heart bypass with a pump-oxygenator. Pulmonary microarterial vessels (130 to 230 microns in diameter) from each group were examined in vitro in a pressurized (20 mm Hg), no-flow state with video microscopic imaging and electronic dimension analysis. After preconstriction of vessels with the thromboxane A2 analog U46619 by 30% to 40% of the baseline diameter, vasoactive drugs were applied extraluminally. Serotonin caused control microvessels to dilate. In the presence of the nitric oxide synthetase inhibitor NG-methyl-L-arginine, this was converted to a significant contractile response. Acetylcholine alone had minimal effect on control vessels. However, in the presence of the cyclooxygenase inhibitor indomethacin, acetylcholine caused a significant relaxation response. After total cardiopulmonary bypass and pulmonary reperfusion, pulmonary microvessels contracted significantly when exposed to acetylcholine and serotonin, compared with respective control responses. Both these contractile responses were inhibited in the presence of indomethacin. Endothelium-independent responses to sodium nitroprusside and U46619 and dilation responses to adenosine were not altered after cardiopulmonary bypass. Extracorporeal circulation with continued pulmonary arterial perfusion (right heart bypass group) had no effect on microvascular responses. In conclusion, total cardiopulmonary bypass with associated reduced pulmonary perfusion causes significant alterations of endothelium-dependent pulmonary microvascular responses because of the increased release of a constrictor prostanoid substance and possibly because of reduced release of endothelium-derived relaxing factor.

Effect of cimetidine on the histamine and dimaprit induced responses on the guinea pig ileum during development

1. 1. In ileal segments obtained from 1, 15, 30, 45 and 60-day-old guinea pigs, histamine produced a dose-dependent significant contractile response from a threshold concentration of 3.2 × 10 −8 M. 2. 2. The mean contractile responses (at 10 −5 M) of the ileum from the 1, 15, 30 and 45-day-old guinea pigs compared to that from the 60-day-old ones were 73.79, 92.30, 94.07 and 94.41% respectively. 3. 3. Dimaprit (at 10 −5 M) had no effect on the ileum of the 1-day-old guinea pigs, while it produced a significant contractile response on the ileum of the guinea pigs at all other ages. 4. 4. The mean contractile responses of the ileum from the 15, 30 and 45-day-old guinea pigs compared to that from the 60-day-old ones were 74.44, 66.66 and 76.94% respectively. 5. 5. Cimetidine (at 10 −5, 3.2 × 10 −5 and 10 −4 M) significantly potentiated the histamine-induced contraction on the ileum of the 1-day (from 10 −7 to 3.2 × 10 −6 M) and the 15-day-old guinea pigs (at 10 −8 M). 6. 6. On the contrary, cimetidine (at 3.2 × 10 −5 M) significantly inhibited the contractile response of the ileum to dimaprit. 7. 7. In conclusion, the guinea pig ileum appears increasingly sensitive to histamine from the newborn to the 60-day-old ones, but its sensitivity to dimaprit, not detectable in the newborn, fluctuates during development. 8. 8. Cimetidine produces a potentiating effect, which declines with age, on the histamine-induced contraction, while it inhibits the dimaprit contractile response on the ileum.