Changes In Contractile Responses Research Articles

Abstract 13: Vascular Chemerin, Particularly from PVAT, contributes to Norepinephrine and Serotonin-Induced Vasoconstriction and Vascular Stiffness in a Sex-Dependent Manner

The adipokine chemerin has been implicated in healthy blood pressure maintenance and adiposity-associated hypertension, evidenced by falls in mean arterial pressure after administration of an antisense oligonucleotide against chemerin in Dahl SS rats. Circulating levels of chemerin are positively associated, in humans, with hypertension and aortic stiffening—a blood pressure-independent risk factor of cardiovascular disease. While chemerin’s role in blood pressure is observed in whole animals, mechanisms of chemerin’s influence on vascular health and disease remain unknown. Chemerin causes direct vasoconstriction through its main receptor Chemerin1 and indirect vasoconstriction by amplifying electrical field stimulation-induced contraction. We identified chemerin production at the vasculature—the blood vessel and its perivascular adipose tissue (PVAT)—and are working to discover the mechanistic role of vascular chemerin in blood vessel function and disease. Using RNAScope®, QPCR, isometric contractility, western blot, and in vivo imaging, we test the hypothesis that vascular chemerin increases arterial contractility through Chemerin1 and plays a role in aortic stiffening. RNAScope® probes detected CMKLR1 expression in the tunica media and PVAT of the thoracic aorta. QPCR revealed Rarres2 expression was higher in the PVAT, and CMKLR1 expression was higher in the media. Chemerin1 antagonism via the selective inhibitor CCX832 (1μM) significantly reduced maximal contraction to both norepinephrine (NE) and serotonin (5-HT) (see figure), but not angiotensin II, in the male rat +PVAT groups. This supports that PVAT-derived chemerin plays a role in sympathetic stimulation of the vasculature. Interestingly, in females, this change in contractile response to NE and 5-HT was not observed. Western blot showed that females express significantly lower levels of chemerin protein in PVAT, suggesting PVAT as the primary source of active chemerin. Finally, male, but not female, genetic chemerin knockout Dahl SS rats have lower aortic arch pulse wave velocity than wild types (M WT: 3.7 ± .7 M KO: 1.4 ± .3 F WT: 2.6 ± .5, F KO: 1.5 ± .3 m/s, N=4-6/group), highlighting a role of chemerin in aortic stiffening. Thus, chemerin, sourced by the PVAT, plays a significant role in vascular contractility and aortic stiffening in males. Future work will determine if chemerin’s influence is amplified in adiposity-associated hypertension, where chemerin levels are elevated in both sexes.

The role of thiazole acetate derivatives on isolated heart and blood vessels in experimental rats

Background and objectivesThough various drugs are available in the market for cardiovascular diseases, search for an efficacious and patient friendly drug is an ongoing process. The effect of synthesized thiazole compounds on isolated rat heart and isolated rat blood vessel to determine their potential as newer therapeutic agents. Materials and methodsSix thiazole acetate compounds were examined for their function on isolated heart and blood arteries utilizing standardized experimental models after being characterized and identified in the laboratory. Anesthetized Wistar albino rats' hearts were removed and placed on a Langendorff setup. The percentage change in developed tension and heart rate owing to injecting each thiazole acetate compound in the developed tension and heart rate were seen and recorded in an isolated heart that was mounted in a retrograde fashion using Krebs Henseleit solution. Similarly, anesthetized rats' thoracic aortas were separated, and responses to the standard drug phenylephrine and different thiazole acetate derivatives were examined. The pharmacological effect of the specific thiazole compound was determined by the percentage change in contractile response. ResultEach of the six thiazole acetate derivatives was injected at 1 nM, 10 nM, 100 nM, 1 M, 10 M, and 100 M concentrations. All six (T1 to T6) chemicals investigated in the present study had no significant effect on developed tension or heart rate. There was a nosignificant change in the percentage contraction after injecting 1 nM 3 nM,10 nM, 30 nM, 100 nM, 300 nM, 1 M, 3 M, 10 M, and 30 M,100 M,300 M (cumulative dose) of T1, T3, T4, T5, and T6, while T2 (- ethyl-4-methyl-2-aminothiazole) showed contractile effect in the form of vasoconstrictor activity, which was 30 % of that produced by phenylephrine. Interpretation and conclusionThiazole acetate derivatives did not show any cardiovascular effects except ethyl-4-methyl-2-aminothiazole, which showed a slight vascular contractile response as compared to phenylephrine. A more thorough examination of the utilization of thiazole derivatives is required to establish their potential significance in the cardiovascular system.

Influence of Tilia tomentosa Moench Extract on Mouse Small Intestine Neuromuscular Contractility.

Functional gastrointestinal disorders (FGIDs) are characterized by abdominal pain, bloating and bowel disturbances. FGID therapy is primarily symptomatic, including treatment with herbal remedies. Flower extract of Tilia tomentosa Moench (TtM) is occasionally used as an anti-spasmodic in popular medicine. Since its effect on intestinal response is unknown, we evaluated the influence of TtM extract on small intestine contractility. Ileal preparations from C57BL/6J mice were mounted in organ baths to assess changes in muscle tension, following addition of TtM extract (0.5–36 μg/mL) or a vehicle (ethanol). Changes in contractile response to receptor- and non-receptor-mediated stimuli were assessed in ileal preparations pretreated with 12 μg/mL TtM. Alterations in the enteric nervous system neuroglial network were analyzed by confocal immunofluorescence. Increasing addition of TtM induced a marked relaxation in ileal specimens compared to the vehicle. Pretreatment with TtM affected cholinergic and tachykininergic neuromuscular contractions as well as K+-induced smooth muscle depolarization. Following incubation with TtM, a significant reduction in non-adrenergic non-cholinergic-mediated relaxation sensitive to Nω-Nitro-L-arginine methyl ester hydrochloride (pan-nitric oxide synthase inhibitor) was found. In vitro incubation of intestinal specimens with TtM did not affect the myenteric plexus neuroglial network. Our findings show that TtM-induced intestinal relaxation is mediated by nitric oxide pathways, providing a pharmacological basis for the use of TtM in FGIDs.

Characterization of rat tail lymphatic contractility and biomechanics: incorporating nitric oxide-mediated vasoregulation.

The lymphatic system transports lymph from the interstitial space back to the great veins via a series of orchestrated contractions of chains of lymphangions. Biomechanical models of lymph transport, validated with ex vivo or in vivo experimental results, have proved useful in revealing novel insight into lymphatic pumping; however, a need remains to characterize the contributions of vasoregulatory compounds in these modelling tools. Nitric oxide (NO) is a key mediator of lymphatic pumping. We quantified the active contractile and passive biaxial biomechanical response of rat tail collecting lymphatics and changes in the contractile response to the exogenous NO administration and integrated these findings into a biomechanical model. The passive mechanical response was characterized with a three-fibre family model. Nonlinear regression and non-parametric bootstrapping were used to identify best-fit material parameters to passive cylindrical biaxial mechanical data, assessing uniqueness and parameter confidence intervals; this model yielded a good fit (R2 = 0.90). Exogenous delivery of NO via sodium nitroprusside (SNP) elicited a dose-dependent suppression of contractions; the amplitude of contractions decreased by 30% and the contraction frequency decreased by 70%. Contractile function was characterized with a modified Rachev-Hayashi model, introducing a parameter that is related to SNP concentration; the model provided a good fit (R2 = 0.89) to changes in contractile responses to varying concentrations of SNP. These results demonstrated the significant role of NO in lymphatic pumping and provide a predictive biomechanical model to integrate the combined effect of mechanical loading and NO on lymphatic contractility and mechanical response.

Berberine restored nitrergic and adrenergic function in mesenteric and iliac arteries from streptozotocin-induced diabetic rats

Ethnopharmacological relevancePerivascular neuropathy was reported to involve in the vascular disorders associated with diabetes. The dried rhizomes of Coptis chinensis Franch. (Latin name: Coptidis Rhizoma; common name: Huang Lian in China), used frequently in Traditional Chinese medicine to treat diabetes (Xiaoke), have been confirmed to possess beneficial effects on diabetic peripheral neuropathy by modern clinical and pharmacological studies. Berberine (BBR), the main effective component of Huang Lian in the treatment of diabetes, is reported to ameliorate diabetic central and peripheral neuropathy. However, the effects of BBR on nerve function of mesenteric and iliac arteries are unclear. Aim of the studyTo investigate the effects of BBR on the diabetes-induced changes in nitrergic and adrenergic function in mesenteric and iliac arteries. Materials and methodsIn this study, the animals were randomized into three groups: control rats, diabetic rats, and diabetic rats gavaged with BBR. We established diabetic rat model using intraperitoneal injection of streptozotocin (STZ, 55 mg kg−1). Two weeks after model establishment, those in the BBR-treated groups were gavaged with berberine chloride (Sichuan Xieli Fharmaceutical. Co., Ltd; 200 mg·kg−1·day−1) diluted in distilled water for another 2 weeks. The superior mesenteric artery and iliac artery were excised. Electric field stimulation (EFS) was used to induce arterial vasoconstriction and explore (1) the diabetes-induced changes in neurogenic function of the superior mesenteric artery and iliac artery; (2) the effects of BBR on neurovascular dysfunction in the early stage of STZ-induced diabetic rats. Nitric oxide (NO) and noradrenaline (NA) released from the nitrergic and adrenergic nerves were quantified using fluorescence assays and ELISA, respectively. ResultsEFS induced frequency-dependent vasoconstrictions in both superior mesenteric and iliac artery, and the contractile responses of arteries were abolished by 0.1 μmol·L−1 tetrodotoxin (TTX), or inhibited by 1 μmol·L−1 phentolamine or increased by 0.1 mmol·L−1 Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME). In superior mesenteric artery, but not in iliac artery, the changes of contractile responses with L-NAME were significantly decreased in diabetic rats, and NO release was less also. In contrast, in iliac artery of diabetic rats, but not in superior mesenteric artery, the changes of contractile responses with phentolamine were increased, and NA release was increased significantly. All these changes in diabetic rats on both superior mesenteric artery and iliac artery were reversed by treated with BBR. ConclusionsIn the STZ-induced early diabetic rats, neural control of mesenteric and iliac vasomotor tone are altered differently. The diminished nitrergic nerve in superior mesenteric artery and enhanced adrenergic nerve in iliac artery both contributed to increased vasocontrictor responses. All these changes in diabetic rats were reversed by BBR, suggesting a novel mechanism of BBR in balance of neural regulation of vascular tone.

310. Quercetin protects against the vascular contractile effect of placental secreted messengers released under placental hypoxia: an in-vitro model of preeclampsia

Introduction Placental hypoxia is a crucial factor in the pathogenesis of preeclampsia resulting in hypoxic stress. Upon this stress the placenta will release placenta secreted messengers (PSMs) which will cause maternal endothelial dysfunction. Previous research suggests that the antioxidant quercetin exerts anti-hypertensive effects and endothelial relaxation via NO release. Objective To determine the vascular effect of PSMs released under hypoxia and to study the protective effect of quercetin on this process. Methods In this study 44 non-complicated placentas (GA > 37 weeks) were used. PSMs were generated by exposing healthy term placentas to hypoxia. Chorionic arteries were incubated with these PSMs +/- quercetin 3 and 20 μM and contractile responses were recorded over a two-hour period. PSMs generated under normoxic conditions were used as control. Endothelial dysfunction induced by PSMs was assessed by analyzing changes in contractile responses to KCl (62.5 mM) before and after exposure to the PSMs. Results PSMs released under hypoxia induced an increased contraction in chorionic arteries compared to PSMs released in the presence of quercetin and normoxic conditions (39.1 μm ± 5.9 vs. quercetin 3 μM: 17.5 μm ± 1.6 vs. quercetin 20 μM: -8.5 μm ± 2.2 vs. normoxic: 0.5 μm ± 0.8, p Discussion Quercetin shows promising results for the protection against the PSMs released under preeclampsia-like stress conditions like placental hypoxia, which could eventually be used for prevention and/or treatment of preeclampsia.

340. Quercetin protects against the vascular contractile effect of placental secreted messengers released under placental hypoxia

Introduction Hypoxia is a crucial factor in the pathogenesis of preeclampsia resulting in hypoxic stress in the placenta. Upon this stress the placenta will release placenta secreted messengers (PSMs) which will cause maternal endothelial dysfunction. Previous research suggests that the antioxidant quercetin exerts anti-hypertensive effects and endothelial relaxation via NO release. Objective To determine the vascular effect of PSMs released under hypoxia and to study the protective effect of quercetin on this process. Methods In this study 44 non-complicated placentas (GA > 37 weeks) were used. PSMs were generated by exposing healthy term placentas to hypoxia. Chorionic arteries were incubated with these PSMs +/− quercetin 3 and 20 μM and contractile responses were recorded over a two-hour period. PSMs generated under normoxic conditions were used as control. Endothelial dysfunction induced by PSMs was assessed by analyzing changes in contractile responses to KCl (62.5 mM) before and after exposure to the PSMs. Results PSMs released under hypoxia induced an increased contraction in chorionic arteries compared to PSMs released in the presence of quercetin and normoxic conditions (39.1 μm ± 5.9 vs. quercetin 3 μM: 17.5 μm ± 1.6 vs. quercetin 20 μM: −8.5 μm ± 2.2 vs. normoxic: 0.5 μm ± 0.8, p Discussion Quercetin shows promising results for the protection against the PSMs released under preeclampsia-like stress conditions like placental hypoxia, which could eventually be used for prevention and/or treatment of preeclampsia.

Gestation changes sodium pump isoform expression, leading to changes in ouabain sensitivity, contractility, and intracellular calcium in rat uterus.

Developmental and tissue‐specific differences in isoforms allow Na+, K+‐ATPase function to be tightly regulated, as they control sensitivity to ions and inhibitors. Uterine contraction relies on the activity of the Na+, K+ ATPase, which creates ionic gradients that drive excitation‐contraction coupling. It is unknown whether Na+, K+ ATPase isoforms are regulated throughout pregnancy or whether they have a direct role in modulating uterine contractility. We hypothesized that gestation‐dependent differential expression of isoforms would affect contractile responses to Na+, K+ ATPase α subunit inhibition with ouabain. Our aims were therefore: (1) to determine the gestation‐dependent expression of mRNA transcripts, protein abundance and tissue distribution of Na+, K+ ATPase isoforms in myometrium; (2) to investigate the functional effects of differential isoform expression via ouabain sensitivity; and (3) if changes in contractile responses can be explained by changes in intracellular [Ca2+]. Changes in abundance and distribution of the Na+, K+ ATPase α, β and FXYD1 and 2 isoforms, were studied in rat uterus from nonpregnant, and early, mid‐, and term gestation. All α, β subunit isoforms (1,2,3) and FXYD1 were detected but FXYD2 was absent. The α1 and β1 isoforms were unchanged throughout pregnancy, whereas α2 and α3 significant decreased at term while β2 and FXYD1 significantly increased from mid‐term onwards. These changes in expression correlated with increased functional sensitivity to ouabain, and parallel changes in intracellular Ca2+, measured with Indo‐1. In conclusion, gestation induces specific regulatory changes in expression of Na+, K+ ATPase isoforms in the uterus which influence contractility and may be related to the physiological requirements for successful pregnancy and delivery.

Device for Investigation of Mechanical Tension of Isolated Smooth Muscle Vessels and Airway Segments of Animals

For the purpose of testing and the search for new drug compounds, designed to heal many human diseases, it is necessary to investigate the deformation of experimental tissue samples under influence of these drugs. For this task a precision force sensor for measuring the mechanical tension, produced by isolated ring segments of blood vessels and airways was created. The hardware and software systems for the study of changes in contractile responses of the airway smooth muscles and blood vessels of experimental animals was developed.

Sildenafil treatment in a nonsevere hypertensive murine model lowers blood pressure without reducing fetal growth

Treatment of nonsevere hypertension during pregnancy is controversial. Sildenafil is a phosphodiesterase inhibitor that potentiates nitric oxide by promoting vasodilation. Nitric oxide plays a vital role in mediating the vascular adaptations during pregnancy. The objective of the study was to determine whether treatment with sildenafil during pregnancy would lower maternal systolic blood pressure without adversely affecting fetal growth. Females with nonsevere hypertension (endothelial nitric oxide synthase(+/-)) were cross-bred with normotensive wild-type males. At gestational day 1, pregnant dams were randomized to either sildenafil (0.4 mg/mL per day, comparable dose used in human pregnancy) or water for 3 weeks. Four groups were then generated: wild type (n= 7), wild type-sildenafil (n= 11), endothelial nitric oxide synthase(+/-) (n= 8), and endothelial nitric oxide synthase(+/-)sildenafil (n= 7). On gestational day 18, systolic blood pressure was measured. Dams were killed, fetal and placental weights were obtained, and carotidarteries were dissected to measure invitro vascular reactivity with awire-myography system. Responses to phenylephrine, L-NG-nitroarginine methyl ester, acetylcholine, and sodium nitroprusside were studied. Mean systolic blood pressure was elevated in the endothelial nitric oxide synthase(+/-) dams compared with wild-type controls (P= .03). Treatment with sildenafil decreased systolic blood pressure in the endothelial nitric oxide synthase(+/-)-treated dams compared with nontreated endothelial nitric oxide synthase(+/-) dams (P= .03). No differences were seen in the wild-type dams with or without sildenafil (P= .47). Fetuses from endothelial nitric oxide synthase(+/-) dams were smaller compared with wild-type controls (P < .001); however, when these endothelial nitric oxide synthase(+/-) dams were treated with sildenafil, fetal weight increased compared with the nontreated endothelial nitric oxide synthase(+/-) group (P < .001). No difference were seen in wild-type groups treated or not treated with sildenafil (P= .41). Placental weights were not significantly different among groups (endothelial nitric oxide synthase(+/-)sildenafil vs endothelial nitric oxide synthase(+/-) [P= .48]; wild-type-sildenafil vs wild type [P= .52]). Maximal vascular contraction induced by phenylephrine was blunted in endothelial nitric oxide synthase(+/-) dams treated with sildenafil compared with nontreated endothelial nitric oxide synthase(+/-) dams (P < .01). No change in contractile response was seen in wild-type groups treated or not treated (P= .53). When vessels were preincubated with L-NG-nitroarginine methyl ester, the contractile responses were similar among all groups (P= .54). In addition, maximal vascular relaxation induced by acetylcholine was improved in the endothelial nitric oxide synthase(+/-) dams treated with sildenafil compared with endothelial nitric oxide synthase(+/-) nontreated dams (P < .01). No change in relaxation response was seen in wild-type groups treated or not treated (P= .62). Sodium nitroprusside did not change the contractile response in any of the groups (P= .31). Pregnant dams deficient in endothelial nitric oxide synthase, a nonsevere hypertensive murine model, treated with sildenafil had lower maternal systolic blood pressure, increased fetal growth, and improvement in vascular reactivity. Treatment with sildenafil may be beneficial in pregnancies complicated by nonsevere hypertension.

The effect of week-long infusion of propranolol, via an osmotic minipump, on blood pressure, heart rate and vascular responses in spontaneously hypertensive rats.

Propranolol was infused in SHR subcutaneously for 7 days at two concentrations (either 3.75 or 7.5 mg kg-1 day) via a minipump. Mean blood pressure and heart rate measured under pentobarbitone anaesthesia on day 7 after implantation showed a significant dose-dependent decrease in both propranolol-treated groups. In the low-dose propranolol-treated rats, there was no change in contractile responses to phenylephrine over controls. In rats receiving the higher dose of propranolol there was a significant increase in the response to phenylephrine. There was no change in the relaxation response of any of the groups to isoprenaline. The results indicate that propranolol, while lowering blood pressure and heart rate, is also modifying the alpha-receptor response of the vascular wall in the spontaneously hypertensive rat.

Effect of Nandrolone Decanoate Treatment on Contractile Responses of Male Rats Skeletal Muscles

This study examine if the administration of different doses of an anabolic-androgenic steroids, nandrolone decanoate (ND), could produce changes in contractile responses of untrained rat fast- (edl) and slow- (soleus) twitch skeletal muscles. Male adult rats received weekly an intramuscular injection of sterile peanut oil (control; C), 0.75 mg/kg (Therapeutical-dose; TD), 1.5 mg/kg (low-dose; LD), 6 mg/kg (Intermediate-dose; ID) or 15 mg/kg (High-dose; HD) of ND during 8 weeks. Compared with respective C, although no significant effect was observed in slow and fast- twitch characteristics for TD, LD and ID rats, an increasing by 103% in the amplitude of fast- twitch of HD animals and no significant effect was observed in the time constant of relaxation. A prolongation in the time constant of relaxation was observed in slow- twitch fibers of HD rats and no significant modification was observed at the level of amplitude of contraction generated. Moreover, no significant effect was observed in K-contracture characteristics of slow- muscles of TD, LD, ID and HD rats, a progressive increase in the amplitude of K-contracture was observed in ID (17%) and HD (57%) of ND treated rats and no significant modification was observed in the kinetic of the contracture. These results suggest that the effect of ND treatment of male rats is muscle types and dose dependent and the edl muscles are more sensitive than soleus muscles to treatment.

The Modulation of Motility of Pyloric Antral Smooth Muscles of Rat by Melatonin

Recently some researches have established that the melatonin, secreted by pineal gland, may evoke the changes of contractile responses on smooth muscles. We examined the effects of melatonin on the motility of rat pyloric antrum and which mechanism might be involved in the effects. Pyloric antral strips from the stomach of 20 Sprague-Dawley rats were prepared for isometric tension recording in organ bath. The strips were precontracted by acetylcholine and high-KCl solutions. In precontracted conditions the tensions were increased by accumulative application of melatonin (<TEX>$10^{-8}-10^{-4}$</TEX> M) dose-dependently, even in resting states. And the effects were almost disappeared when the concentrations of ACh were over than 10 <TEX>${\mu}M$</TEX>. The effects of melatonin were inhibited by pretreatment of 10 mM TEA and/or 10 <TEX>${\mu}M$</TEX> 4-AP and rarely affected by pretreatment of 1 mM TEA, 10 <TEX>${\mu}M$</TEX> glibenclamide and 10 <TEX>${\mu}M$</TEX> verapamil respectively. From these results it is concluded that the contractile responses of smooth muscles of rat pyloric antrum were enhanced by melatonin application and the mechanism might be concerned with the inhibition of some voltage-dependent potassium channels.

Developmental differences in the contractile response of isolated ovine tracheal smooth muscle cells.

Previous studies have shown developmental differences in smooth muscle tone of the airways. Differences in airway mechanics may be based upon cellular differences between animals of different ages. We developed a method [Driska et al. J Appl Physiol 86(1): 427-35, 1999] for isolating ovine tracheal smooth muscle cells and for measuring shortening velocity. This technique was used to study the change in contractile response of the airway smooth muscle (ASM) cell during development. Here we report differences between preterm (PT) (110-124 or 125-140 days post-conception), newborn (NB) (3-7 days postnatal) and adult (9-36 months) cells. These cells were compared with respect to morphometry, shortening velocity, and percent shortening. The neonatal cells were shorter and narrower than the adult cells. Maximum shortening velocity was faster for adult (45.1 microm/sec) than for neonatal cells (range 11.1-25.1 microm/sec). When velocity was normalized to the cell length, there was no difference between the adult and PT cells, but there was a significant difference between the NB (0.30 sec(-1)) and adult (0.54 sec(-1)) cells. The percent shortening did not show any significant difference with age. Within the neonatal groups, there were no significant differences in morphometry, shortening or velocity. To facilitate comparison between ASM tissues of different sizes with different sized cells, we also expressed percent shortening and velocities relative to a hypothetical 1 mm segment of tissue. Represented this way, the amount of shortening for all age groups was the same, but the predicted maximum velocity of the hypothetical PT tissue (125-140 days) was significantly greater than for NB.

Alteration in Contractile Responses in Human Detrusor Smooth Muscle from Obstructed Bladders with Overactivity

Introduction: In this study, we aimed to evaluate changes in contractile responses under in vitro conditions in detrusor overactivity (DO) in patients with bladder outflow obstruction (BOO). Materials and Methods: Detrusor strips obtained during open prostatectomy procedure from 16 patients with BOO related to benign prostate hyperplasia were evaluated under in vitro conditions. Patients were assigned to two groups as patients with (DO) and without (no DO) DO. Four detrusor strips were prepared from each bladder in dimensions of 2 × 10 mm, and were suspended in organ bath. Responses to carbachol (10^–8 to 10^–3M), electrical field stimulation (EFS) (0.5–32 Hz), single-dose adenosine 5′-triphosphate (ATP) (10^–3M) and KCl (120 mM) were recorded to evaluate the contractile responses. EFS responses were repeated in the presence of NG-nitro-L-arginine methyl ester (L-NAME; 10 µM) and L-NAME + indomethacin. All responses were expressed as mg tension developed per mg of bladder tissue. Data obtained were compared using independent t test and one-way ANOVA test. Values of p < 0.05 were accepted as statistically significant. Results: Of the 16 patients on whom open prostatectomy was performed because of BOO, 8 of the patients were determined as no DO and 8 as DO. There were no differences between groups regarding age and residual urine. We found statistically significant differences between groups regarding dimensions of prostate, maximum bladder capacity and maximum bladder pressure. In the comparison of cumulative dose of carbachol, it was seen that responses were higher in the DO group, but the differences were not statistically significant. In EFS application, contractile responses were found to increase significantly in the DO group. No changes were observed between groups for ATP and KCl. EFS responses were found to be significantly higher in presence of L-NAME + indomethacin in the no DO group; however, no difference was seen in the DO group. Conclusions: Detrusor contractile responses to EFS increased in patients with BOO in presence of overactivity. These changes in contractile responses are observed possibly as a result of deterioration in neuromodulation, rather than as a result of changes in purinergic or cholinergic receptor sensation or level. We suggest that a noncholinergic-nonpurinergic mechanism can have some effect on these changes.

Assessment of the Effects of L- and N-Type Ca2+ Channel Blocking Drugs Using Canine Blood-Perfused Papillary Muscle Preparations

It is important to accurately and conveniently assess the effects of L- and N-type Ca(2+) channel blocking drugs, which are commonly used for treatment of hypertension, but no method is available to simultaneously assess the effects of them in the same preparation. We have therefore designed an ex vivo method to measure the changes in contractile response of anterior papillary muscle of right ventricle and myocardial interstitial norepinephrine (NE) level using canine blood-perfused papillary muscle preparations. Papillary muscle-developed tension (PMDT) induced by an electronic stimulator was measured with force transducer. Myocardial interstitial NE effluent was collected by microdialysis fiber, which was implanted at the base of the papillary muscle, and measured with high performance liquid chromatography. Cilnidipine, a typical L- and N-type Ca(2+) channel blocker, was used to prove the efficiency of this method. First, to assess the effects of drugs on L-type Ca(2+) channel, the changes in basal PMDT were measured. Cilnidipine and nicardipine, a selective L-type Ca(2+) channel blocker, but not omega-conotoxin GVIA (omega-CTX), a selective N-type Ca(2+) channel blocking peptide, decreased basal PMDT dose-dependently. Second, to assess the effects of drugs on N-type Ca(2+) channel, the changes in PMDT and myocardial interstitial NE level by intracardiac sympathetic ganglion stimulation were measured. Cilnidipine and omega-CTX, but not nicardipine, dose-dependently reduced sympathomimetic increases in PMDT and myocardial interstitial NE level. These results indicate that our method is efficient to assess the effects of various L- and N-type Ca(2+) channel blocking drugs in the same papillary muscle preparation.

Effect of magnesium sulfate on contractile force and intracellular calcium concentration in pregnant human myometrium

This study was undertaken to evaluate the effects of magnesium sulfate (MgSO4) on contractile force and increases in free intracellular calcium concentration ([Ca2+]i) in human myometrial strips from pregnant women. Simultaneous measurements of isometric tension and [Ca2+]i were measured in myometrial strips obtained at the time of cesarean delivery from pregnant nonlaboring women at term with the use of a fluorescence spectrometer equipped with a displacement force transducer. Changes in [Ca2+]i were measured with fura-2, a Ca(2+)-sensitive fluorescent probe. Myometrial strips were exposed to MgSO4 (5 or 10 mmol/L) for 5, 10, 20, and 30 minutes and observed for spontaneous contractions or stimulated with either oxytocin (OT; 0.1 micromol/L) or potassium chloride (KCl; 90 mmol/L). MgSO4 reduced spontaneous, OT, and KCl-evoked contractions and increases in [Ca2+]i in a time and concentration-dependent manner. After 20 minutes exposure to 5 mmol/L MgSO4, the OT-elicited changes in contractile response and [Ca2+]i were significantly decreased. MgSO4 did not change [Ca2+]i/force relationship of the responses to OT or KCl, or during spontaneous activity. At a pharmacologic concentration (5 mmol/L), MgSO4 inhibits contractile response and [Ca2+]i in pregnant human myometrial strips by a pattern that is consistent with both extra- and intracellular mechanisms. At a suprapharmacologic concentration (10 mmol/L), the more immediate effect of MgSO4 is consistent with an extracellular mechanism. MgSO4 does not appear to interfere at the level of the calcium-calmodulin interface, since the [Ca2+]i/force relationship was not changed.

Diabetes-related changes in contractile responses of stomach fundus to endothelin-1 in streptozotocin-induced diabetic rats

The contractile response of the stomach fundus to endothelin-1 (ET-1) was examined in streptozotocin (STZ)-induced diabetic rats. In STZ-diabetic rats (versus age-matched control rats) (a) ET-1 caused a longer-lasting contraction of stomach fundus strips, and (b) in the dose-response curve, the ET-1-induced contraction was significantly greater for a given concentration (3 x 10(-7) to 10(-7) M). Although repeated application of ET-1 led to desensitization, the desensitization was less pronounced in STZ-diabetic rats than in the controls. The density of the binding sites for [(125)I]-ET-1 was increased in the diabetic stomach fundus (versus the controls), but Kd values were similar between the two groups. The ET(B) receptor mRNA expression level was significantly increased in the diabetic stomach fundus. These results suggest that the diabetes-related enhancement of the ET-1-induced contraction of the stomach fundus may be due to an increase in the ET(B) receptor population.

Modulation of tracheal smooth muscle responses in inducible T-cell kinase knockout mice

It has been shown that the Tec family nonreceptor tyrosine kinase inducible T cell kinase (ITK) plays a role in the activation of naïve T cells and in the differentiation of T helper (T H2)-type cells producing cytokines in a model of allergic inflammation, thereby possibly indirectly mediating hyperresponsivenes of airway smooth muscle tone. Using excised tracheae from wild type (WT) mice and those lacking ITK, we conducted a series of in vitro experiments in which isometric smooth muscle tones were assessed in response to several agonists to determine whether the absence of ITK would affect the responsiveness of tracheal smooth muscle cells. The resulting change in contractile responses was evaluated by measuring agonist cumulative concentration-response curves (CCRC). Our results indicate that the cholinergic agonist acetylcholine (ACh) and its analog carbachol (CCh) exhibited comparable CCRC profiles in contracting isolated tracheae from both WT and ITK −/− mice, with no alteration in their efficacies. However, the EC 50 values for the two agonists were found to be significantly higher in ITK −/− tracheae than in those from WT mice, suggesting an alteration of the potencies of these cholinergic agonists in the trachea of ITK −/− mice. Moreover, we found that the depolarizing agent potassium chloride (KCl) had a significantly lower efficacy in contracting ITK −/− tracheae compared to those from WT mice. This difference in KCl efficacy was abolished in the presence of a calcium (Ca 2+) voltage-dependent channel (VDC) agonist, Bay K8644, suggesting a modulation of the KCl induced permeability of VDC Ca 2+ channels in the trachea of ITK −/− mice. Taken together, these results suggest that the presence of ITK may play a modulating role in the pharmacomechanical as well as in the electromechanical coupling of airway smooth muscle contraction.

Expression of several cytoskeletal proteins in ovine cerebral arteries: developmental and functional considerations.

Cytoskeleton proteins play important roles in regulating vascular smooth muscle (VSM) contraction and relaxation. We tested the hypotheses that the expression levels of several of these proteins change significantly during the course of development, and that these changes contribute to age-related changes in contractile responses. In cerebral arteries from 95-day (d) gestation and 140-d fetus, newborn lambs, and adult sheep, by Western immunoblot (n= 5 for each age) we quantified the relative expression of alpha-actin, alpha-tubulin, cyclophilin A, and proliferating cell nuclear antigen (PCNA). In addition, we examined middle cerebral artery tension responses to phenylephrine (PHE) stimulation in the absence or presence of cytochalasin D (3 x 10(-7)m) and nocodazole (3 x 10(-6)m), inhibitors of alpha-actin and alpha-tubulin polymerization, respectively. The expression levels of alpha-actin and cyclophilin A varied little during the course of development. In contrast, alpha-tubulin expression was approximately 2.5-fold greater in both fetal age groups as compared to adult. Also, as compared to adult and as expected, expression of PCNA was several-fold greater in cerebral arteries of the 95-d fetus (x8), 140-d fetus (x 5), and newborn (x 3). In both adult and fetal middle cerebral artery, cytochalasin D-induced inhibition of actin polymerization decreased PHE-induced contraction, to approximately 60 and approximately 40% of control, respectively (despite no significant change in expression level). In contrast, alpha-tubulin inhibition by nocodazole showed little effect on PHE-induced tension (in spite of the age-related decrease in expression). In conclusion, expression levels of alpha-actin, a thin filament protein involved in contraction, remained relatively constant during the course of development, as did the effects of inhibition of its polymerization on contractility. In contrast, alpha-tubulin, important in intracellular protein trafficking, showed a significant age-related decrease in expression and played a relatively minor role in contractility. The present studies suggest that other cytoskeletal structural proteins and/or elements of pharmaco-mechanical coupling are important to developmental differences in cerebrovascular contractility. In addition, the relatively constant expression levels of alpha-actin and cyclophilin A with development, suggest that these are useful internal standards for studies of cytosolic protein expression.