Maximal Contractile Response Research Articles

Characterization of in vivo Function of Myosin light Chain Kinase in Internal Anal Sphincter Contraction

The spontaneous tone of the internal anal sphincter(IAS)primarily controls rectoanal continence. Multiple signal networks with or without MLCK have been implicated in IAS contractility, but the underlying regulatory mechanism and functional importance are incompletely understood. We analyzed mice with specific knockout of Mlck in adult smooth muscle and characterized in-vivo function of MLCK in IAS. Targeted deletion of Mlck in adult smooth muscle resulted in abnormal excretion. Mutant IAS displayed an abolished spontaneous tone, and impaired contractile response to KCl and bethanechol. This was compatible with reduction of RLC phosphorylation. The kinetics of force production showed that MLCK concentration in IAS was linearly correlated with basal tone, logarithmically correlated with the force induced by KCl or bethanechol. About 20% of MLCK was sufficient to produce a maximal contractile response to KCl and BCh, but a very weak spontaneous tone. This concentration dependency emphasized the importance of MLCK in IAS contractility. The differential dependency implied different activation features of MLCK during spontaneous tone formation and contractile response to stimuli. We thus concluded that MLCK and its RLC phosphorylation were required for spontaneous tone and depolarization- or agonist-induced contraction, possibly through different activation mechanisms.

Levosimendan preserves the contractile responsiveness of hypoxic human myocardium via mitochondrial K ATP channel and potential pERK 1/2 activation

This study investigated the role of levosimendan, a mitochondrial K ATP channel opener, during hypoxia–reoxygenation injury in human isolated tissue. The activation of preconditioning pathways, and the release of mitochondrial cytochrome c were determined. Human right atrial trabeculae were mounted in an organ bath, electrically paced and contractile force measured. Tissue was subjected to hypoxia–reoxygenation, and isoproterenol concentration–response experiments were performed as an index of contractile viability. The intracellular activities of Akt, ERK 1/2, P38, caspase 3, and cytochrome c were assayed by western blot. Following hypoxia–reoxygenation, the maximal contractile response of trabeculae to isoproterenol was significantly increased with levosimendan pretreatment compared to the hypoxia–reoxygenation control (0.88 ± 0.02 versus 0.60 ± 0.01 g, P < 0.01). This enhanced response was blocked by 5-hydroxydecoanate (0.54 ± 0.09 g, P < 0.01). A significant increase in both phosphorylated and total ERK 1/2 and P38 occurred at 60 min reoxygenation, compared to control tissue. No difference was observed in phosphorylated or total Akt, though there was a trend for increased levels in hypoxic tissue. Cytochrome c was detected at 60 min post reoxygenation, in both levosimendan treated and untreated tissue. No increase in cleaved-caspase 3 activity was observed. Our findings suggest that levosimendan preserves the contractile force to isoproterenol after hypoxia–reoxygenation, a response mediated via mK ATP channel activation. The significant increase in the activity of prosurvival mediators ERK 1/2 and P38 following hypoxia indicates a potential mechanism of action for levosimendan-induced cardioprotection.

The Role of Adrenergic and Angiotensinergic Systems in Vascular Effect of Alcoholic of Extract Trigonella foenum-graecum Seed in Diabetic Rats.

Considering the anti-diabetic potential of Trigonella foenum-graecum (TFG) and its beneficial effect on aortic reactivity of diabetic rats, this study was conducted to evaluate the effect of its alcoholic seed extract on aortic reactivity and also figure out mechanisms including the role of adrenergic and angiotensinergic systems in streptozotocin-diabetic rats. Male Wistar rats were divided into control, extract-treated control, diabetic, and extract-treated diabetic groups. Diabetes was induced by a single IP injection of streptozotocin (STZ; 60 mg/kg). Treatment groups received TFG extract (200 mg/kg; IP) every other day for 1 month. Contractile response of thoracic aorta to KCl and noradrenaline (NA) was then determined. For determination of the involvement of adrenergic and angiotensinergic systems, rings were incubated before the experiments with prazocin, propranolol, and/or captopril and then NA- and angiotensin I (Ag-I)-induced contractions were recorded. Diabetic state significantly increased maximum contractile response to KCl and NA (p < 0.01-0.005) comparing to control groups and treatment with TFG extract in diabetic group significantly improved these changes comparing to untreated diabetic group (p < 0.05-p < 0.01). On the other hand, pretreatment of prazosin and propranolol caused a significant reduction in contractile response of all groups (p < 0.05-0.001) meanwhile was no significant difference among the groups. In addition, pre-incubation with captopril caused a significant reduction in contractile response of TFG-treated diabetic group comparing untreated diabetic group (p < 0.05). Finally we concluded that alcoholic seed extract of TFG could improve some functional indices of the vascular system in diabetic state and angiotensinergic system is partly involved in this response.

Could a high-fat diet rich in unsaturated fatty acids impair the cardiovascular system?

Dyslipidemia results from consumption of a diet rich in saturated fatty acids and is usually associated with cardiovascular disease. A diet rich in unsaturated fatty acids is usually associated with improved cardiovascular condition. To investigate whether a high-fat diet rich in unsaturated fatty acids (U-HFD) - in which fatty acid represents approximately 45% of the total calories - impairs the cardiovascular system. Male, 30-day-old Wistar rats were fed a standard (control) diet or a U-HFD containing 83% unsaturated fatty acid for 19 weeks. The in vivo electrocardiogram, the spectral analysis of heart rate variability, and the vascular reactivity responses to phenylephrine, acetylcholine, noradrenaline and prazosin in aortic ring preparations were analyzed to assess the cardiovascular parameters. After 19 weeks, the U-HFD rats had increased total body fat, baseline glucose levels and feed efficiency compared with control rats. However, the final body weight, systolic blood pressure, area under the curve for glucose, calorie intake and heart weight⁄final body weight ratio were similar between the groups. In addition, both groups demonstrated no alteration in the electrocardiogram or cardiac sympathetic parameters. There was no difference in the responses to acetylcholine or the maximal contractile response of the thoracic aorta to phenylephrine between groups, but the concentration necessary to produce 50% of maximal response showed a decrease in the sensitivity to phenylephrine in U-HFD rats. The cumulative concentration- effect curve for noradrenaline in the presence of prazosin was shifted similarly in both groups. The present work shows that U-HFD did not impair the cardiovascular parameters analyzed.

In Vitro Antagonistic Properties of a New Angiotensin Type 1 Receptor Blocker, Azilsartan, in Receptor Binding and Function Studies

The angiotensin II (AII) antagonistic action of azilsartan (AZL) [2-ethoxy-1-{[2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl}-1H-benzimidazole-7-carboxylic acid] was investigated in radioligand binding and function studies. AZL inhibited the specific binding of ¹²⁵I-Sar¹-Ile⁸-AII to human angiotensin type 1 receptors with an IC₅₀ of 2.6 nM. The inhibitory effect of AZL persisted after washout of the free compound (IC(50) value of 7.4 nM). Olmesartan, telmisartan, valsartan, and irbesartan also inhibited the specific binding with IC₅₀ values of 6.7, 5.1, 44.9, and 15.8 nM, respectively. However, their inhibitory effects were markedly attenuated with washout (IC₅₀ values of 242.5, 191.6, >10,000, and >10,000 nM). AZL also inhibited the accumulation of AII-induced inositol 1-phosphate (IP1) in the cell-based assay with an IC₅₀ value of 9.2 nmol; this effect was resistant to washout (IC₅₀ value of 81.3 nM). Olmesartan and valsartan inhibited IP1 accumulation with IC₅₀ values of 12.2 and 59.8 nM, respectively. The activities of these compounds were markedly reduced after washout (IC₅₀ value of 908.5 and 22,664.4 nM). AZL was defined as an inverse agonist in an experiment by using a constitutively active mutant of human angiotensin type 1 receptors. In isolated rabbit aortic strips, AZL reduced the maximal contractile response to AII with a pD'₂ value of 9.9. The inhibitory effects of AZL on contractile responses induced by AII persisted after the strips were washed; these inhibitory effects were more potent than those of olmesartan. These results suggest that AZL is a highly potent and slowly dissociating AII receptor blocker. Its tight receptor binding might be expected to produce potent and long-lasting antihypertensive effects in preclinical and clinical settings.

In Vitro Pharmacodynamic Profile of Loranthus ferrugineus: Evidence for Noncompetitive Antagonism of Norepinephrine-induced Vascular Contraction

Abstract The mode by which Loranthus ferrugineus methanol extract antagonizes and/or modulates norepinephrine-induced vasoconstriction was investigated in rat aortic rings. The vascular effects of three different concentrations of this extract were challenged against cumulative additions of norepinephrine. Phentolamine, a nonselective α-adrenoceptor antagonist, verapamil, an L-type calcium channel blocker, and papaverine, a phosphodiesterase inhibitor, were used in three different concentrations as positive controls. Log concentration-response curves and double-reciprocal plots were constructed for the extract and each vasorelaxant. To characterize antagonism reversibility, the norepinephrine maximum contractile effect was examined before extract addition to the aortic ring chamber and after its removal. Phentolamine shifted the norepinephrine log concentration-response curve to the right with no significant depression in the maximum response. Similar to verapamil and papaverine, the extract produced a rightward shift in norepinephrine log concentration-response curve and a significant drop in maximum response. The double-reciprocal plots showed comparable y-intercept values for all phentolamine concentrations, a characteristic of competitive antagonism. In contrast, different y-intercept values on double-reciprocal plots were obtained for each concentration of extract, verapamil, and papaverine, typical of noncompetitive antagonism. The norepinephrine maximum contractile response was approximately similar before the addition of extract and after its removal. The data collectively showed that L. ferrugineus methanol extract exerted its vascular effect by reversible noncompetitive antagonism of norepinephrine-induced vasoconstriction. These findings add to the understanding of the cardiovascular mechanisms by which L. ferrugineus, a plant traditionally used for the management of hypertension, elicits its action.

P38 Mitogen-Activated Protein Kinase (MAPK) Increases Arginase Activity and Contributes to Endothelial Dysfunction in Corpora Cavernosa from Angiotensin-II-Treated Mice

Angiotensin II (AngII) activates p38 mitogen-activated protein kinase (MAPK) and elevates arginase activity in endothelial cells. Upregulation of arginase activity has been implicated in endothelial dysfunction by reducing nitric oxide (NO) bioavailability. However, signaling pathways activated by AngII in the penis are largely unknown. We hypothesized that activation of p38 MAPK increases arginase activity and thus impairs penile vascular function in AngII-treated mice. Male C57BL/6 mice were implanted with osmotic minipumps containing saline or AngII (42 µg/kg/h) for 14 days and cotreated with p38 MAPK inhibitor, SB 203580 (5 µg/kg/day), beginning 2 days before minipump implantation. Systolic blood pressure (SBP) was measured. Corpus cavernosum (CC) tissue was used for vascular functional studies and protein expression levels of p38 MAPK, arginase and constitutive NO synthase (NOS), and arginase activity. Arginase expression and activity; expression of phospho-p38 MAPK, endothelial NOS (eNOS) and neuronal NOS proteins; endothelium-dependent and nitrergic nerve-mediated relaxations were determined in CC from control and AngII-infused mice. AngII increased SBP (22%) and increased CC arginase activity and expression (∼twofold), and phosphorylated P38 MAPK levels (30%) over control. Treatment with SB 203580 prevented these effects. Endothelium-dependent NO-mediated relaxation to acetylcholine was significantly reduced by AngII and this effect was prevented by SB 203580 (P < 0.01). AngII (2 weeks) did not alter nitrergic function. However, SB 203580 significantly increased nitrergic relaxation in both control and AngII tissue at lower frequencies. Maximum contractile responses for phenylephrine and electrical field stimulation were increased by AngII (56% and 171%, respectively) and attenuated by SB 203580 treatment. AngII treatment also decreased eNOS phosphorylation at Ser-1177 compared to control. Treatment with SB 203580 prevented all these changes. p38 MAPK inhibition corrects penile arginase activity and protects against erectile dysfunction caused by AngII.

Inhibition of p38 MAPK-dependent bronchial contraction after ozone by corticosteroids

We determined the role of p38 mitogen-activated protein kinase (MAPK) in the increased airway smooth muscle (ASM) contractile responses following ozone and modulation by corticosteroids. Mice were exposed to air or ozone (3 ppm for 3 h) and isometric contractile responses of bronchial rings to acetylcholine (ACh) were measured using a myograph in the presence of p38 MAPK inhibitor, SB239063 (10⁻⁶ M) or dexamethasone (10⁻⁶ M). Because MAPK phosphatase (MKP)-1 is a negative regulator of p38 MAPK, we also studied these effects in MKP-1(-/-) mice. Bronchial rings from ozone-exposed wild-type and MKP-1(-/-) mice showed increased contractile responses, with a leftward shift of the dose-response curve in MKP-1(-/-) mice. SB239063 inhibited bronchial contraction equally in air- and ozone-exposed C57/BL6 and MKP-1(-/-) mice. Dexamethasone inhibited ACh-induced bronchial contraction in both air- and ozone-exposed C57/BL6 mice, but not in air- or ozone-exposed MKP-1(-/-) mice. ACh-stimulated p38 MAPK and heat shock protein (HSP)27 phosphorylation, as measured by Western blotting, and this effect was suppressed by SB239063 in C57/BL6 and MKP-1(-/-) mice, but not by dexamethasone in either air- or ozone-exposed MKP-1(-/-) mice. p38 MAPK plays a role in maximal ACh-induced isometric contractile responses and increased contractility induced by ozone. Dexamethasone inhibits ACh-induced ASM contraction through phosphorylation of p38 MAPK and HSP27.

Early Stages of In Situ Bladder Regeneration in a Rodent Model

Surgical removal of approximately 70% of the bladder (subtotal cystectomy [STC]) was used as a model system to gain insight into the normal regenerative process in adult mammals in vivo. Female F344 rats underwent STC, and at 2, 4, and 8 weeks post-STC, bladder regeneration was monitored via microcomputed tomography scans, urodynamic (bladder function studies) pharmacologic studies, and immunohistochemistry. Computed tomography imaging revealed a time-dependent increase in bladder size at 2, 4, and 8 weeks post-STC, which positively correlated with restoration of bladder function. Bladders emptied completely at all time points studied. The maximal contractile response to pharmacological activation and electrical field stimulation increased over time in isolated tissue strips from regenerating bladders, but remained lower at all time points compared with strips from age-matched control bladders. Immunostaining of the bladder wall of STC rats suggested a role for progenitor cells and cellular proliferation in the regenerative response. Immunostaining and the presence of electrical field stimulation-induced contractile responses verified innervation of the regenerated bladder. These initial studies establish the utility of the present model system for studying de novo tissue regeneration in vivo and may provide guidance with respect to optimization of intrinsic regenerative capacity for clinical applications.

Mechanisms of relaxant activity of the nitric oxide-independent soluble guanylyl cyclase stimulator BAY 41-2272 in rat tracheal smooth muscle

The soluble guanylyl cyclase is expressed in airway smooth muscle, and agents that stimulate this enzyme activity cause airway smooth muscle relaxation and bronchodilation. The compound 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator, but little is known about its effects in airway smooth muscle. Therefore, this study aimed to investigate the mechanisms underlying the relaxations of rat tracheal smooth muscle induced by BAY 41-2272. Tracheal rings were mounted in 10-ml organ baths for isometric force recording. BAY 41-2272 concentration-dependently relaxed carbachol-precontracted tracheal rings (pEC 50 = 6.68 ± 0.14). Prior incubation with the NO synthesis inhibitor l-NAME (100 μM) or the soluble guanylyl cyclase inhibitor ODQ (10 μM) caused significant rightward shifts in the concentration–response curves to BAY 41-2272. Sodium nitroprusside caused concentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 and completely inhibited by ODQ. In addition, BAY 41-2272 shifted to the right the tracheal contractile responses to either carbachol (0.01–1 μM) or electrical field stimulation (EFS, 1–32 Hz). BAY 41-2272 (1 μM) also caused a marked rightward shift and decreased the maximal contractile responses to extracellular CaCl2, and such effect was not modified by pretreatment with ODQ. In addition, BAY 41-2272 (up to 1 μM) significantly increased the cGMP levels, and that was abolished by ODQ. Our results indicate that BAY 41-2272 causes cGMP-dependent rat tracheal smooth muscle relaxations in a synergistic fashion with exogenous NO. BAY 41-2272 has also an additional mechanism independently of soluble guanylyl cyclase activation possibly involving Ca 2+ entry blockade.

안지오텐신 수용체 길항제 KR-31125의 특성에 관한 연구

KR-31125는 피리딜 이미다졸 시리즈 화합물로서 비펩타이드 안지오텐신 수용체 길항제로 새롭게 개발되었다. 동위원소 리간드를 사용한 재조합 수용체 결합실험과 기능성 토끼혈관실험 결과 기존 의약인 로자탄과 동등수준의 수용체 길항효과를 나타내었다. 이러한 KR-31125의 특징들은 제 1형 안지오텐신 수용체에 특이적으로 나타났으며(<TEX>$IC_{50}$</TEX>: <TEX>$19.72{\pm}2.65\;nM$</TEX>), 표준물질에 대한 대조실험 결과 제 2형 안지오텐신 수용체에 대한 결합친화력은 발견되지 않았다. 기능성 혈관실험에서 KR-31125가 안지오텐신에 의한 혈관수축 효과를 경쟁적으로 저하시켰지만 표준물질인 로자탄과는 달리 농도가 증가함에 따라 30-80% 정도의 최대 수축효과 감소가 관찰되어 로자탄과는 다른 분자작용 기전을 가진다고 판단된다. 제 1형 안지오텐신 수용체에 선택적으로 작용하는 것으로 나타난 KR-31125는 레닌-안지오텐신-알도스테론 시스템에 대한 연구 및 진단에 폭 넓게 활용될 수 있는 표지자 화합물로 가능성을 넓혀 줄 수 있을 것이라고 판단된다. KR-31125 (2-butyl-5-dimethoxymethyl-6-phenyl-7-methyl-3-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo[4,5-b]pyridine) is a potent inhibitor of angiotensin II type 1 (<TEX>$AT_1$</TEX>) receptors in human recombinant <TEX>$AT_1$</TEX> receptors and rabbit aorta. These in vitro studies revealed that KR-31125 inhibited specific [<TEX>$^{125}I$</TEX>] [<TEX>$Sar^1$</TEX>, <TEX>$Ile^8$</TEX>]-angiotensin II binding to human recombinant <TEX>$AT_1$</TEX> receptors in a concentration dependent manner with an <TEX>$IC_{50}$</TEX> value of <TEX>$19.72{\pm}2.65$</TEX> nM. However, no interaction with <TEX>$AT_2$</TEX> receptors was detected as displayed by the competition binding of [<TEX>$^{125}I$</TEX>] CGP 42112A to human recombinant <TEX>$AT_2$</TEX> receptor. The binding action was also confirmed as a competitive mode that was identical to the previously studied compound, losartan. In addition, KR-31125 caused a nonparallel shift to the right in the concentration response curves to angiotensin II with a 30-80% decrease in the maximum contractile responses (<TEX>$pK_B$</TEX>: 7.63). Compared to the previous studies with losartan that showed a parallel right shift in the maximum contractile responses to AII (<TEX>$pA_2$</TEX>: 7.59), KR-31125 presented a different mode of action with a similar potency to losartan. These results demonstrate that KR-31125 is a highly potent and <TEX>$AT_1$</TEX> selective angiotensin II receptor antagonist that can be applied to the fields of new diagnostic and research tools with upcoming in vivo study results.

Mechanisms underlying vascular effect of chronic resveratrol in streptozotocin‐diabetic rats

In this study, some underlying mechanisms responsible for the beneficial effect of chronic oral administration of resveratrol on aortic reactivity of streptozotocin (STZ)-diabetic rats were investigated. Male diabetic rats received resveratrol (10 mg/kg/day) for 8 weeks, 1 week after diabetes induction. Treatment of diabetic rats with resveratrol produced a hypoglycaemic effect and there were appropriate changes regarding serum lipids. Resveratrol also attenuated the increased malondialdehyde (MDA) content and reduced activity of superoxide dismutase (SOD) in liver and aortic tissues. Maximum contractile response of endothelium-intact aortic rings to KCl and phenylephrine (PE) was significantly lower in resveratrol-treated diabetic rats relative to untreated diabetics. Endothelium removal abolished the significant difference between resveratrol-treated and untreated diabetic groups regarding contractile response to KCl and PE. Meanwhile, endothelium-dependent relaxation to acetylcholine (ACh) was significantly higher in resveratrol-treated diabetic rats as compared to diabetic group and pretreatment with N(omega)-L-arginine methyl ester (L-NAME) and indomethacin (INDO) significantly attenuated these responses. Chronic treatment with resveratrol may prevent diabetes-related changes in vascular reactivity observed in diabetic rats directly and/or indirectly due to its hypoglycaemic and hypolipidaemic effects and attenuation of lipid peroxidation and through endothelial-derived factors.

T1686 Gastric Clasp and Lower Esophageal Circular Muscle Fibers From GERD Patients With Barrett's Esophagitis Have a Decreased Contractile Response to Cholinergic Stimulation

Background: We recently showed that the gastric sling/clasp muscle complex does not contribute to the high pressure zone in GERD patients indicating a role in pathophysiology. Aim: We determined the cholinergic contractile response in clasp, sling and lower esophageal circular (LEC) fibers. Methods: Stomach and esophagi were obtained from 17 human transplant donors: 11 with no GERD history, 4 with probable GERD (proton pump inhibitor use) and 2 with definite GERD (Barrett's esophagitis). The contractile response to increasing carbachol concentrations was determined. Muscarinic receptor density was measured by subtype selective immunoprecipitation of [3]H-QNB binding. Results: Clasp and LEC fibers from definite GERD have decreased maximal contractile response. Contractility is increased in sling fibers of both definite and probable GERD. Concentrations of carbachol higher than 100 μM induce relaxations that are decreased in clasp fibers of probable and definite GERD. Relaxations are greater in sling fibers from definite GERD and higher in LEC from probable and definite GERD. Total and M2 receptor density is statistically lower in the GERD than non-GERD specimens in both clasp and sling fibers and M3 density is statistically lower in GERD than non-GERD clasp fibers. Conclusion: This suggest that a myogenic defect in clasp fibers may be involved in GERD pathophysiology in the organ donors with Barrett's esophagitis. The greater contractile response in the sling fibers from GERD donors suggest a possible compensatory mechanism for the lack of contractility of the clasp muscle fibers.

Indomethacin enhances reactivity to vasopressin in normotensive and hypertensive rats, while PGE2 receptor blockade only attenuates reactivity in normotensive rats.

Previously, we reported that in female (F) rats 17‐β‐estradiol and aortic coarctation‐induced hypertension (ACIH) increase release of thromboxane (TXA2) and prostacyclin from mesenteric arterioles (MA), and that constrictor responses of aorta to vasopressin (VP) are reduced ~40% by TP receptor antagonist SQ 29,548 (SQ). Thus, the roles of TXA2 and PGE2 in constrictor responses to VP were studied in MA. F Sprague‐Dawley rats (14–16 wks age) underwent ACIH (hypertensive; HT) or sham (normotensive; NT). Rats were sacrificed 12–14 days post‐ACIH and reactivity to VP was measured in MA (150–200 μm dia.) using isometric myography. Data are means ± S.E. (n = 4–5 rats/group). Maximal contractile responses of MA to VP did not differ between NT (9.97 ± 0.75 mN/mm) and HT (11.77 ± 1.40). SQ (40 μM) did not alter responses in NT (9.16 ± 1.53) or HT (12.69 ± 1.77). In separate rats, maximal responses to VP did not differ between NT and HT. AH6809, a PGE2 receptor blocker (EP1, 2, 3) reduced maximal responses of MA from NT (8.95 ± 0.36 vs. 10.24 ± 0.63) but not HT (9.73 ± 0.81 vs. 9.36 ± 0.61). Indomethacin (non‐specific COX inhibitor; 10 μM) increased contractions to VP in NT and HT (11.74 ± 0.84 and 12.75 ± 0.79, respectively). While TXA2 potentiates constrictor responses of aorta to VP, the role of TXA2 in reactivity of MA to VP is less certain. However, it appears that PGE2 may also play a role in modulating MA responses to VP in NT rats. (NIH:HL‐080402)

Seeding of vascular smooth muscle cells confers enhanced pharmacological responses to tissue engineered blood vessels in a large animal model

These studies evaluated the impact of VSM cell seeding and bioreactor preconditioning on the phenotype and function of tissue engineered blood vessels (TEBV) following 4 months of implantation in vivo. Autologous endothelial progenitor cells (EPC) and vascular smooth muscles cells (VSM) were collected from female sheep and differentiated into mature EC and VSMC in culture. Five TEBV were seeded with both endothelial cells (EC) and VSM, and another 5 grafts seeded with EC only. All grafts were preconditioned to pulsatile flow and physiologic pressure for 2 weeks in a bioreactor. TEBV were implanted in female sheep as an interposition graft of the carotid or femoral artery. The patency rate was 100%, with no aneursymal degeneration or anasomotic stenosis. Vessel wall cells of dual‐seeded grafts had increased immuno‐positive staining to α‐actin (p=0.05) and smooth muscle myosin (p=0.05), as well as increased overall cellularity (p=0.01), when compared to EC only‐seeded grafts. Significantly increased steady‐state maximal contractile responses were observed in the dual‐seeded grafts in response to stimulation with phenylephrine (PE, 10 μM; p=0.01) and 5‐Hydroxytryptamine (5‐HT;10 μM; p=0.01), but not KCl. Maximal contractions to PE and 5‐HT were ≈20% and 15%, respectively, of that observed for the native vessel, respectively. This work represents an important first step toward creation of more native‐like TEBV.

Postnatal maturation and hypoxic acclimatization modulate PKG‐mediated activation of BK channels in ovine cerebral arteries

NO‐dependent relaxation of ovine cerebral arteries is enhanced during postnatal maturation but is attenuated by acclimatization to high altitude. Because NO‐mediated vasorelaxation is mediated by activation of PKG, which in turn activates large conductance BK channels, we hypothesized that the effects of both maturation and chronic hypoxia on NO‐dependent relaxation are mediated by modulation of PKG effects on BK channels. In middle cerebral arteries from normoxic and chronically hypoxic fetal and adult sheep, iberiotoxin (IBTX, an inhibitor of BK channels) enhanced the maximum contractile response to 5HT ≈10% in both age groups, but 8pCPT‐cGMP (8pCPT, a PKG activator) decreased this response ≈15% in fetal and ≈20% in adult arteries. Addition of 8pCPT followed by IBTX produced responses similar to controls in both age groups. In arteries treated with cumulative log dose increases of IBTX before or after incubation with 8pCPT, IBTX significantly enhanced basal tone ≈40% in both age groups. Preincubation with 8pCPT attenuated the contractile responses to IBTX by ≈70% in fetal and ≈90% in adult arteries. Addition of 8pCPT following maximum responses to IBTX decreased tone ≈30% in fetal and ≈40% in adult arteries. These results indicate that PKG enhances BK channel activity more in adult than fetal arteries, and markedly less in hypoxic than normoxic arteries.Supported by USPHS Grants HL54120, HD31266, &amp; HL64867.

Pharmacological characterization of KR-30988, a novel non-peptide AT1 receptor antagonist, in rat, rabbit and dog.

The pharmacological profile of KR-30988, a non-peptide AT1-selective angiotensin receptor antagonist, has been investigated by use of a variety of experimental models in-vitro and in-vivo. KR-30988 inhibited the specific binding of [125I][Sar1, Ile8]-angiotensin II to the recombinant AT1 receptor from man with a potency similar to that of losartan (IC50 values, the concentrations of drugs displacing 50% of specific binding, 13.6 and 12.3 nM, respectively), but did not inhibit the binding of [125I]CGP 42112A to recombinant AT2 receptor from man (IC50 >10 microM for both drugs). Scatchard analysis showed that KR-30988 interacted competitively with recombinant AT1 receptor from man in the same manner as losartan. In functional studies with rat and rabbit aorta, KR-30988 noncompetitively inhibited the contractile response to angiotensin II (pD2, = -log EC50 (where EC50 is the dose resulting in 50% of a reference contraction), 8.64 and 7.73, respectively) with a 20-85% decrease in the maximum contractile responses, unlike losartan. In pithed rats intravenous KR-30988 resulted in a non-parallel shift to the right of the dose-pressor response curve to angiotensin II (ID50 value, the dose inhibiting the pressor response to angiotensin II by 50%, 0.09 mg kg(-1)) with a dose-dependent reduction in the maximum responses; in this antagonistic effect KR-30988 was 20 times (approx.) more potent than losartan (ID50 1-74 mg kg(-1)). In conscious renal hypertensive rats oral administration of KR-30988 produced a dose-dependent and long-lasting (>24 h) anti-hypertensive effect; the potency was six times that of losartan (ED30 values, the dose reducing mean arterial blood pressure by 30 mmHg, 0.48 and 2.97 mg kg(-1), respectively). In conscious furosemide-treated dogs oral administration of KR-30988 produced a dose-dependent and long-lasting (>8 h) hypotensive effect with a rapid onset of action (time to Emax, the maximum effect, 1-2 h); KR-30988 was eight times more potent than losartan (ED20, the dose reducing mean arterial blood pressure by 20 mm Hg, 1.04 and 7.96 mg kg(-1), respectively). These results suggest that KR-30988 is a potent, orally active selective AT1 receptor antagonist with a mode of insurmountable antagonism.

Flavonoid galangin prevents smooth muscle fatigue of pig urinary bladder

There is increasing evidence that the generation of free radicals plays a role in the development of bladder dysfunction. Flavonoids are a group of polyphenolic compounds with broad pharmacological activity. In the present study, the protective effects of the flavonoid galangin on the progressive decrease of bladder smooth muscle contractile responses during repetitive field stimulation (RFS; a model for muscular fatigue) were demonstrated. Pig detrusor strips were mounted for tension recording in organ baths aand were subjected to RFS for 90 min at 32 Hz for 15 s every 5 min. The strips were then washed four times with fresh buffer and allowed a period of recovery for 90 min. The 90 min of RFS caused a progressive decrease in maximal contractile response to electrical field stimulation and to muscarinic agonist-induced contractions (34% and 46% decrease, respectively). Galangin (10(-7) M) prevented the decrease in contractile smooth muscle response of strips to electrical field stimulation during RFS compared with untreated tissues. The antioxidant activity of galangin was assessed by measuring its ability to inhibit the lipid peroxidation induced by iron and ascorbate in rat liver microsomes (IC50 1.7+0.12x10(-6) M). If the data are confirmed in-vivo, exogenously administered galangin may be a new approach in the prevention and/or treatment of bladder dysfunction.

Quercetin 3,7-dimethyl ether: a vasorelaxant flavonoid isolated from Croton schiedeanus Schlecht.

The vasorelaxant profile of quercetin 3,7-dimethyl ether, a flavonoid isolated from Croton schiedeanus Schlecht (Euphorbiaceae), was assessed in aortic rings isolated from Wistar rats. To gain insight into its structure-activity relationship, we compared this substance with quercetin 3,4',7-trimethyl ether (ayanin), another flavonoid isolated from this plant, quercetin 3,3',4',7-tetramethyl ether, a flavonoid synthesized by us, and quercetin. In addition we examined the interaction of quercetin 3,7-dimethyl ether with the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway. According to their pEC50 values (concentration producing a 50% inhibition of the maximal contractile response) to phenylephrine-induced precontraction in rat isolated aorta, the potency order was quercetin 3,7-dimethyl ether > quercetin > quercetin 3,4',7-trimethyl ether > quercetin 3,3',4',7-tetramethyl ether (4.70+/-0.18; 3.96+/-0.07; 3.64+/-0.02; 3.11+/-0.16). The relaxant effect of quercetin 3,7-dimethyl ether was significantly decreased by the removal of endothelium as well as by methylene blue, an inhibitor of guanylyl cyclase, and by N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME), an NO-synthase inhibitor. Therefore, quercetin 3,7-dimethyl ether has a NO/cGMP pathway-related profile, with increased vasorelaxant activity due to hydroxylation at positions 3 and 4 of the B ring. In addition, methylation at positions 3 and 7 with respect to quercetin of the C and A rings, respectively, seems to further enhance the vasorelaxant activity of quercetin 3,7-dimethyl ether.

Terpinen-4-ol: mechanisms of relaxation on rabbit duodenum

The effect of terpinen-4-ol was studied on rabbit duodenum in-vitro. Terpinen-4-ol induced relaxation of the basal tonus (IC50 170.2 (95% confidence interval, 175-204) microM) with a maximal relaxant response of 180.4+/-3.9% (n=6) of the contraction induced by 60 mM [K(+)]. The maximal relaxation induced in control conditions was not affected (P>0.05) by pretreatment of the tissues with phentolamine (50 microM) or propranolol (10 microM), N(G) nitro-L-arginine methyl ester (L-NAME; 1 mM), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 100 microM), hexamethonium (1 mM), tetrodotoxin (1 microM), the mixture charybdotoxin-apamin (1 microM), glibenclamide (10 microM), 4-aminopyridine (10 microM) or tetraethyl-ammonium (100 microM). In addition, terpinen-4-ol completely relaxed tissues precontracted with 60 mM [K(+)] solutions (IC50 325.9 (245.1-433.1) microM) and also blocked (IC50 154.7 (117.7-191.7) microM) the phasic component of this contraction. At a concentration of 195 and 650 muM it reduced by 41.3+/-3.4% and 75.4+/-3.1%, respectively the maximal contractile response to Ca(2+) in depolarized duodenum. Terpinen-4-ol completely blocked the component of carbachol-induced contraction, which was resistant to nifedipine (100 microM) pretreatment or to a Ca(2+)-free solution. These data show that terpinen-4-ol relaxes intestinal smooth muscle and suggest that this effect is myogenic in nature and depends on calcium antagonism.