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Abstract Background and purpose To help students develop a more integrated mindset, an integrated curricular model can facilitate the building of connections between the foundational and clinical sciences by presenting multi-disciplinary material within a cohesive framework in a single course. The main objective of this research was to assess the impact of interdisciplinary teaching on student performance in a skills course. Educational activity and setting A case study was presented and questions were embedded through an audience response system. Each of three groups of students (approximately 32 students per group) were divided into teams, and the scores were shown periodically to produce an atmosphere of friendly competition. The entire exercise lasted approximately 50 min. Findings Students found the pharmaceutical science and pharmacy practice faculty collaboration helpful in regards to reviewing for integrated exams. Student pharmacists were asked to provide one positive aspect of the course and one area for improvement. Twenty-two of 96 responders indicated that the integrated class session was the highlight of the course. Student pharmacists noted that they were able to recognize the integration between the basic and applied sciences. Students clearly favored this learning style over the didactic approach, as evidenced by the feedback. In the future, we plan to implement longer integrated cases more frequently to train future pharmacists as critical and integrative thinkers. Summary This integrated case study appeared to be effective in helping student pharmacists apply knowledge of various basic science disciplines to the applied sciences. [ABSTRACT FROM AUTHOR]

Vascular thiol redox state has been shown to modulate vasodilator functions in large conductance Ca2+-activated K+ channels and other related channels. However, the role of vascular redox in small resistance arteries is unknown. To determine how in vivo modulation of thiol redox state affects small resistance arteries relaxation, we generated a transgenic mouse strain that overexpresses thioredoxin, a small redox protein (Trx-Tg), and another strain that is thioredoxin-deficient (dnTrx-Tg). The redox state of the mesenteric arteries (MAs) in Trx-Tg mice is found to be predominantly in reduced state; in contrast, MAs from dnTrx-Tg mice remain in oxidized state. Thus, we created an in vivo redox system of mice and isolated the second-order branches of the main superior MAs from wild-type, Trx-Tg, or dnTrx-Tg mice to assess endothelium-dependent relaxing responses in a wire myograph. In MAs isolated from Trx-Tg mice, we observed an enhanced intermediate-conductance Ca2+-activated potassium channel contribution resulting in a larger endothelium-dependent hyperpolarizing (EDH) relaxation in response to indirect (acetylcholine) and direct (NS309) opening of endothelial calcium-activated potassium channels. MAs derived from dnTrx-Tg mice showed both blunted nitric oxide-mediated and EDH-mediated relaxation compared with Trx-Tg mice. In a control study, diamide decreased EDH relaxations in MAs of wild-type mice, whereas dithiothreitol improved EDH relaxations and was able to restore the diamide-induced impairment in EDH response. Furthermore, the basal or angiotensin II--mediated systolic blood pressure remained significantly lower in Trx-Tg mice compared with wild-type or dnTrx-Tg mice, thus directly establishing redox-mediated EDH in blood pressure control. [ABSTRACT FROM AUTHOR]

Acute increases in cellular protein O-linked N-acetyl-glucosamine (OGlcNAc) modification (O-GlcNAcylation) have been shown to have protective effects in the heart and vasculature. We hypothesized that D-glucosamine (D-GlcN) and Thiamet-G, two agents that increase protein O-GlcNAcylation via different mechanisms, inhibit TNF-α- induced oxidative stress and vascular dysfunction by suppressing inducible nitric oxide (NO) synthase (iNOS) expression. Rat aortic rings were incubated for 3h at 37°C with D-GlcN or its osmotic control L-glucose (L-Glc) or with Thiamet-G or its vehicle control (H2O) followed by the addition of TNF-α or vehicle (H2O) for 21 h. After incubation, rings were mounted in a myograph to assess arterial reactivity. Twenty-four hours of incubation of aortic rings with TNF-α resulted in 1) a hypocontractility to 60 mM K+ solution and phenylephrine, 2) blunted endothelium-dependent relaxation responses to ACh and substance P, and 3) unaltered relaxing response to the Ca2+ ionophore A-23187 and the NO donor sodium nitroprusside compared with aortic rings cultured in the absence of TNF-α. D-GlcN and Thiamet-G pretreatment suppressed the TNF-α-induced hypocontractility and endothelial dysfunction. Total protein OGlcNAc levels were significantly higher in aortic segments treated with D-GlcN or Thiamet-G compared with controls. Expression of iNOS protein was increased in TNF-α-treated rings, and this was attenuated by pretreatment with either D-GlcN or Thiamet-G. Dense immunostaining for nitrotyrosylated proteins was detected in the endothelium and media of the aortic wall, suggesting enhanced peroxynitrite production by iNOS. These findings demonstrate that acute increases in protein O-GlcNAcylation prevent TNF-α-induced vascular dysfunction, at least in part, via suppression of iNOS expression. [ABSTRACT FROM AUTHOR]

Endothelin-1 (ET-1) is implicated in the development of endothelial dysfunction through the generation of reactive oxygen species by NADPH oxidase activation. Interleukin-10 (IL-10) is an antiinflammatory cytokine that stimulates nitric oxide production, decreases superoxide production, and restores endothelial integrity after vascular injury. In this study, we tested whether IL-10 attenuates ET-1-induced endothelial dysfunction by improving acetylcholine (ACh)-induced relaxation of cultured murine aortic rings. Aortic rings (2 mm long) of C57BL/6 mice were incubated in 2 mL DMEM containing 120 U/mL penicillin and 120 μg/mL streptomycin in the presence of one of 4 treatments: vehicle (deionized water), ET-1 (100 nmol/L), recombinant mouse IL-10 (300 ng/mL), or a combination of both ET-1 and IL-10. After incubation at 37 °C for either 1 or 6 h (short-term exposure) or 22 h (overnight exposure), rings were mounted in a wire myograph and stretched to a passive force of 5 mN. Endothelium-dependent vasorelaxation was assessed by constructing cumulative concentration-response curves to ACh (0.001-10 μmol/L) during 10 μmol/L phenylephrine (PE)-induced contraction. Short-term exposure of ET-1 did not result in an impairment of ACh-induced relaxation. Overnight exposure of aortic rings to ET-1 resulted in a statistically significant endothelial dysfunction characterized by a reduced maximal relaxation response to ACh compared with that of untreated rings (Emax 57% ± 3% versus 82% ± 4%). IL-10 treatment restored ACh-induced relaxation (Emax 77% ± 3%). Western blotting showed decreased eNOS expression in response to ET-1, whereas vessels treated with a combination of ET-1 and IL-10 showed increased expression of eNOS. Immunohistochemical analysis showed decreased eNOS expression in ET-1-treated vessels compared with those treated with both ET-1 and IL-10. We conclude that, in murine aorta, the antiinflammatory cytokine IL-10 prevents impairment in endothelium-dependent relaxation induced in response to long-term incubation with ET-1 via normalization of eNOS expression. L’endothéline-1 intervient dans le développement de la dysfonction endothéliale par l’intermédiaire de la production d’espèces réactives de l’oxygène activée par la NADPH oxydase. L’interleukine-10 (IL-10) est une cytokine antiinflammatoire qui stimule la production de la monoxyde d’azote synthase, diminue la production des superoxydes et rétablit l’intégrité endothéliale après un lésion vasculaire. Dans la présente étude, nous avons examiné si l’IL-10 atténue la dysfonction endothéliale induite par l’ET-1, en améliorant la relaxation induite par l’ACh d’anneaux aortiques murins cultivés. Des anneaux aortiques (longueur 2 mm) de souris C57BL6 ont été incubés dans 2 mL d’un milieu de DMEM contenant 120 U/mL de pénicilline et 120 µg/mL de streptomycine, en présence soit d’un véhicule (de l’eau désionisée), d’ET-1 (100 nmol/L), d’IL-10 recombinante de souris (300 ng/mL), ou d’ET-1 et d’IL-10, pendant 1 h et 6 h (exposition de courte durée) ou 22 h (exposition d’une journée), à 37 °C. Après l’incubation, les anneaux ont été montés sur un myographe et étirés jusqu’à une force passive de 5 mN. La vasorelaxation dépendante de l’endothélium a été évaluée par l’établissement de courbes concentration-réponse cumulatives à l’acétylcholine (ACh) (0.001-10 µmol/L) durant des contractions induites par la phényléphrine (PE) (10 µmol/L). L’exposition de courte durée à l’ET-1 n’a pas causé d’altération de la relaxation induite par l’ACh. L’exposition de longue durée des anneaux aortiques à l’ET-1 a entraîné une dysfonction endothéliale statistiquement significative caractérisée par une diminution de la relaxation maximale à l’ACh, comparativement à ce qui a été observé dans les anneaux non traités (Emax 57 % ± 3 % contre 82 % ± 4 % respectivement). Le traitement à l’IL-10 a rétabli la relaxation induite par l’ACh (Emax 77 % ± 3 %). Le buvardage Western a montré une diminution de l’expression de eNOS en réponse à l’ET-1, alors que les vaisseaux traités avec une combinaison d’ET-1 et d’IL-10 ont montré une augmentation de l’expression de eNOS. L’analyse immunohistochimique a indiqué une diminution de l’ [ABSTRACT FROM AUTHOR]

Ca2+-activated K+ channels (KCa), in particular, the small and intermediate KCa (SKCa and IKCa, respectively) channels, are key players in endothelium-derived hyper-polarizing factor (EDHF)-mediated relaxation in small arteries. Hypertension is characterized by an endothelial dysfunction, possibly via reduced EDHF release and/or function. We hypothesize that during angiotensin II (14 days)-induced hypertension (ANG II-14d), the contribution of SKCa and IKCa channels in ACh-induced relaxations is reduced due to decreased expression of SKCa and IKCa channel proteins in rat small mesenteric arteries (MAs). Nitric oxide- and prostacyclin-independent vasorelaxation to ACh was similar in small MAs of sham-operated and ANG II-14d rats. Catalase had no inhibitory effects on these relaxations. The highly selective SKCa channel blocker UCL-1684 almost completely blocked these responses in MAs of sham-operated rats but partially in MAs of ANG II-14d rats. These changes were pressure dependent since UCL-1684 caused a greater inhibition in MAs of I-day ANG IT-treated normotensive rats compared with ANG II-14d rats. Expression levels of both mRNA and protein SK3 were significantly reduced in MAs of ANG II-14d rats. The I + channel blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) resulted in comparable reductions in the relaxation responses to ACh in MAs of sham-operated and ANG II-14d rats. Relative mRNA expression levels of IK1 were significantly reduced in MAs of ANG II-14d rats, whereas protein levels of IK1 were not but tended to be lower in MAs of ANG II-14d rats. The findings demonstrate that EDHF-like responses are not compromised in a situation of reduced functional activity and expression of SK3 channels in small MAs of ANG II-induced hypertensive rats. The role of IK1 channels is less clear but might compensate for reduced SK3 activity. [ABSTRACT FROM AUTHOR]

Ca+-activated K+-channels (KCa) regulate vasomotor tone via smooth muscle hyperpolarization and relaxation. The relative contribution of the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation differs depending on vessel type and size. It is unknown whether these KCa channels are differentially distributed along the same vascular bed and hence have different roles in mediating the EDHF response. We therefore assessed the role of small-(SKCa), intermediate-(IKCa), and large-conductance (BKCa) channels in mediating acetylcholine-induced relaxations in both first- and fourth-order side branches of the rat superior mesenteric artery (MA1 and MA4, respectively). Two-millimeter segments of each MA were mounted in the wire myograph, incubated with Nω-nitro-ʟ-arginine methyl ester (ʟ-NAME, 100 μmol/l) and indomethacin (10 μmol/l), and precontracted with phenylephrine (10 μmol/l). Cumulative concentration-response curves to ACh (0.001–10 μmol/l) were performed in the absence or presence of selective KCa channel antagonists. Apamin almost completely abolished these relaxations in MA4 but only partially blocked relaxations in MA1. The selective IKCa channel blocker 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) caused a significantly greater inhibition of the ACh-induced relaxation in MA4 compared with MA1. Iberiotoxin had no inhibitory effect in MA4 but blunted relaxation in MA1. Relative mRNA expression levels of SKCa (rSK1, rSK3, and rSK4 = rIK1) were significantly higher in MA4 compared with MA1. BKCa (rBKα1 and rBKβ1 genes were similar in both MAI and MA4. Our data demonstrate regional heterogeneity in SKCa and IKCa function and gene expression and stress the importance of these channels in smaller resistance-sized arteries, where the role of EDHF is more pronounced. [ABSTRACT FROM AUTHOR]

Background: We have previously reported that in vitro interleukin-10 (IL-10) counteracts the endothelial dysfunction induced by endothelin (ET-1) in murine aortic rings. In this study we evaluated whether IL-10 modulates vascular ET-1 responses in vivo. We hypothesized that ET-1 reactivity is exacerbated in mice lacking IL-10 expression. Methods: Considering that vascular ET-1 release and prepro-ET-1 mRNA expression are enhanced upon stimulation with tumor necrosis factor alpha (TNF-α), C57BL6 and IL-10 deficient male mice were treated with human recombinat TNF-α (220 ng/Kg/day) or vehicle (saline) for 14 days. Subsequently, aortic rings were mounted in a myograph and contractile responses to endothelin-1 (ET-1, 10-10 to 10-7 M) were evaluated. Vascular ETA and ETB receptors gene expression was evaluated by RTPCR. Results: Responses to ET-1 were almost completed abrogated in vessels from wild-type mice infused with TNF-α or vehicle, as well as in IL-10 knock out mice infused with vehicle (% KCl-induced contraction, 1.3±0.9, 0.85±0.8, 0.74±0.42, respectively; n=4). However, contractile responses to ET-1 were enhanced (21.5±2.0%, n=4, p<0.05; Figure inserted) in IL-10 deficient mice infused with TNF-α. Vascular ETA receptor gene expression was significantly increased in vessels from IL-10 knockout mice (vehicle- and TNF-α-infused mice). No significant vascular ETB receptors mRNA expression were detected in neither of the groups. Conclusions: IL-10 counteracts both ET-1 mediated vascular responses and ETa receptor expression in vivo. [ABSTRACT FROM AUTHOR]

Reversible glutathionylation plays a critical role in protecting protein function under conditions of oxidative stress generally and for endothelial nitric-oxide synthase (eNOS) specifically. Glutathione-dependent glutaredoxin-mediated deglutathionylation of eNOS has been shown to confer protection in a model of heart damage termed ischemia-reperfusion injury, motivating further study of eNOS deglutathionylation in general. In this report, we present evidence for an alternative mechanism of deglutathionylation. In this pathway thioredoxin (Trx), a small cellular redox protein, is shown to rescue eNOS from glutathionylation during ischemia-reperfusion in a GSH-independent manner. By comparing mice with global overexpression of Trx and mice with cardiomyocyte-specific overexpression of Trx, we demonstrate that vascular Trx-mediated deglutathionylation of eNOS protects against ischemia-reperfusion-mediated myocardial infarction. Trx deficiency in endothelial cells promoted eNOS glutathionylation and reduced its enzymatic activity, whereas increased levels of Trx led to deglutathionylated eNOS. Thioredoxin-mediated deglutathionylation of eNOS in the coronary artery in vivo protected against reperfusion injury, even in the presence of normal levels of GSH. We further show that Trx directly interacts with eNOS, and we confirmed that Cys-691 and Cys-910 are the glutathionylated sites, as mutation of these cysteines partially rescued the decrease in eNOS activity, whereas mutation of a distal site, Cys-384, did not. Collectively, this study shows for the first time that Trx is a potent deglutathionylating protein in vivo and in vitro that can deglutathionylate proteins in the presence of high levels of GSSG in conditions of oxidative stress. [ABSTRACT FROM AUTHOR]

Significance: Potassium channels regulate the influx and efflux of K + ions in various cell types that generate and propagate action potential associated with excitation, contraction, and relaxation of various cell types. Although redox active cysteines are critically important for channel activity, the redox regulation of K + channels by thioredoxin (Trx) has not been systematically reviewed. Recent Advances: Redox regulation of K + channel is now increasingly recognized as drug targets in the pathological condition of several cardiovascular disease processes. The role of Trx in regulation of these channels and its implication in pathological conditions have not been adequately reviewed. This review specifically focuses on the redox-regulatory role of Trx on K + channel structure and function in physiological and pathophysiological conditions. Critical Issues: Ion channels, including K + channel, have been implicated in the functioning of cardiomyocyte excitation-contraction coupling, vascular hyperpolarization, cellular proliferation, and neuronal stimulation in physiological and pathophysiological conditions. Although oxidation-reduction of ion channels is critically important in their function, the role of Trx, redox regulatory protein in regulation of these channels, and its implication in pathological conditions need to be studied to gain further insight into channel function. Future Directions: Future studies need to map all redox regulatory pathways in channel structure and function using novel mouse models and redox proteomic and signal transduction studies, which modulate various currents and altered excitability of relevant cells implicated in a pathological condition. We are yet at infancy of studies related to redox control of various K + channels and structured and focused studies with novel animal models. Antioxid. Redox Signal. 00, 00-00.

Endothelial dysfunction is prevalent in chronic kidney disease. This study tested the hypothesis that transfusion of rat aortic endothelial cells (ECs) ameliorates endothelial dysfunction in a rat model of chronic kidney disease. Male Sprague-Dawley rats underwent sham surgery or 5/6 nephrectomy (Nx). Five weeks after Nx, EC (1.5 × 10(6) cells/rat) or vehicle were transfused intravenously. One week later, vascular reactivity of mesenteric artery was assessed on a wire myograph. Sensitivity of endothelium-dependent relaxation to acetylcholine and maximum vasodilation were impaired by Nx and improved by EC transfusion. Using selective pharmacological nitric oxide synthase isoform inhibitors, we demonstrated that the negative effect of Nx on endothelial function and rescue by EC transfusion are, at least in part, endothelial nitric oxide synthase mediated. Plasma asymmetric dimethylarginine was increased by Nx and decreased by EC transfusion, whereas mRNA expression of dimethylarginine dimethylaminohydrolases 1 (DDAH1) was decreased by Nx and restored by EC transfusion. Immunohistochemical staining confirmed that local expression of DDAH1 is decreased by Nx and increased by EC transfusion. In conclusion, EC transfusion attenuates Nx-induced endothelium-dependent vascular dysfunction by regulating DDAH1 expression and enhancing endothelial nitric oxide synthase activity. These results suggest that EC-based therapy could provide a novel therapeutic strategy to improve vascular function in chronic kidney disease.

TNF-α is a proinflammatory cytokine and is an important mediator of maternal endothelial dysfunction leading to preeclampsia. In this study, we tested whether IL-10 protects against TNF-α-induced endothelial dysfunction in murine aorta. In in vitro experiments, aortic rings of C57BL/6 female mice were incubated in Dulbecco's modified Eagle's medium in the presence of either vehicle (distilled H(2)O), TNF-α (4 nmol/l), or recombinant mouse IL-10 (300 ng/ml) or in the presence of both TNF-α and IL-10 for 22 h at 37°C. In in vivo experiments C57BL6/IL-10 knockout female mice were treated with saline or TNF-α (220 ng·kg(-1)·day(-1)) for 14 days. Aortic rings were isolated from in vitro and in vivo experiments and mounted in a wire myograph (Danish Myotech) and stretched to a tension of 5 mN. Endothelium-dependent relaxation was assessed by constructing cumulative concentration-response curves to acetylcholine (ACh, 0.001-10 μmol/l) during phenylephrine (10 μmol/l)-induced contraction. As a result, overnight exposure of aortic rings to TNF-α resulted in significant blunted maximal relaxing responses (E(max)) to ACh compared with untreated rings (22 ± 4 vs. 82 ± 3%, respectively). IL-10 knockout mice treated with TNF-α showed significant impairment in ACh responses (E(max)) compared with C57BL/6 mice treated with TNF-α (51 ± 3 vs. 72 ± 3%, respectively). Western blot analysis showed that endothelial nitric oxide synthase (eNOS) expression was reduced by TNF-α in in vitro and in vivo experiments, whereas IL-10 restored the eNOS expression. In conclusion, the anti-inflammatory cytokine IL-10 prevents impairment in endothelium-dependent vasorelaxation caused by TNF-α by protecting eNOS expression.

Background: The phosphorylation of myosin light chain (MLC) maintains the contracted state of vascular smooth muscle. Dephosphorylation results in relaxation and is determined by the activity of myosin light chain phosphatase (MLCP), which is negatively regulated by Rho kinase., Methods: We tested whether an increased Rho kinase activity, and hence a decreased contribution of MLCP, results in an increased contractility of small fourth-order mesenteric arteries (MA) during the early onset of angiotensin II (Ang II)-induced hypertension (Ang II-14d)., Results: Calcium sensitivity was similar, but contractile tension in response to [Ca]ex (5 mmol/l) in endothelium-denuded and depolarized MA was greater, in Ang II-14d rats compared to sham-operated normotensive (SHAM) and Ang II-1d. The Rho kinase inhibitor Y-27,632 caused a significantly greater inhibition of the contractile response to various agents (phenylephrine, norepinephrine, U46,619 and K) in MA of Ang II-14d compared to SHAM. Protein expression levels of the GDP/GTP exchange factor PDZ-RhoGEF, which co-immunoprecipitated with RhoA, were increased in MA of Ang II-14d compared to SHAM. RhoA translocation was greater in U46,619 (1 micromol/l)-stimulated MA of Ang II-14d compared to SHAM. Expression levels of Rho kinase beta were higher in MA of Ang II-14d. The MLCP inhibitor calyculin A (100 nmol/l) caused a greater contraction in MA of SHAM compared to Ang II-14d. Phosphorylation of the target subunit of MLCP (MYPT1) was enhanced in U46,619-stimulated MA of Ang II-14d compared to SHAM., Conclusion: This is the first study demonstrating enhanced PDZ-RhoGEF/RhoA/Rho kinase signaling during hypertension at the level of resistance-sized arteries. This enhanced signaling leads to increased MLCP phosphorylation, resulting in vascular hyper-reactivity.

ANG II stimulates the production of reactive oxygen species and activates proinflammatory cytokines leading to endothelial dysfunction. We hypothesized that the anti-inflammatory cytokine IL-10 counteracts the impairment in endothelium-dependent ACh relaxation caused by ANG II. Aortic rings of C57BL/6 mice were incubated in DMEM in the presence of vehicle (deionized H(2)O), ANG II (100 nmol/l), recombinant mouse IL-10 (300 ng/ml), or both ANG II and IL-10 for 22 h at 37 degrees C. After incubation, rings were mounted in a wire myograph to assess endothelium-dependent vasorelaxation to cumulative concentrations of ACh. Overnight exposure of aortic rings to ANG II resulted in blunted ACh-induced vasorelaxation compared with that shown in untreated rings (maximal response = 44 +/- 3% vs. 64 +/- 3%, respectively; P<0.05). IL-10 treatment significantly restored this impairment in relaxation (63 +/- 2%). In addition, the NADPH oxidase inhibitor apocynin restored the impairment in relaxation (maximal response = 76 +/- 3%). Western blotting showed increased gp91(phox) expression (a subunit of NADPH oxidase) in response to ANG II. Vessels treated with a combination of ANG II and IL-10 showed decreased expression of gp91(phox). Immunohistochemical analysis showed increased gp91(phox) expression in ANG II-treated vessels compared with those treated with combined ANG II and IL-10. We found that the anti-inflammatory cytokine IL-10 prevents impairment in endothelium-dependent vasorelaxation in response to long-term incubation with ANG II via decreasing NADPH oxidase expression.

Ca(2+)-activated K(+) channels (K(Ca)), in particular, the small and intermediate K(Ca) (SK(Ca) and IK(Ca), respectively) channels, are key players in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in small arteries. Hypertension is characterized by an endothelial dysfunction, possibly via reduced EDHF release and/or function. We hypothesize that during angiotensin II (14 days)-induced hypertension (ANG II-14d), the contribution of SK(Ca) and IK(Ca) channels in ACh-induced relaxations is reduced due to decreased expression of SK(Ca) and IK(Ca) channel proteins in rat small mesenteric arteries (MAs). Nitric oxide- and prostacyclin-independent vasorelaxation to ACh was similar in small MAs of sham-operated and ANG II-14d rats. Catalase had no inhibitory effects on these relaxations. The highly selective SK(Ca) channel blocker UCL-1684 almost completely blocked these responses in MAs of sham-operated rats but partially in MAs of ANG II-14d rats. These changes were pressure dependent since UCL-1684 caused a greater inhibition in MAs of 1-day ANG II-treated normotensive rats compared with ANG II-14d rats. Expression levels of both mRNA and protein SK3 were significantly reduced in MAs of ANG II-14d rats. The IK(Ca) channel blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) resulted in comparable reductions in the relaxation responses to ACh in MAs of sham-operated and ANG II-14d rats. Relative mRNA expression levels of IK1 were significantly reduced in MAs of ANG II-14d rats, whereas protein levels of IK1 were not but tended to be lower in MAs of ANG II-14d rats. The findings demonstrate that EDHF-like responses are not compromised in a situation of reduced functional activity and expression of SK3 channels in small MAs of ANG II-induced hypertensive rats. The role of IK1 channels is less clear but might compensate for reduced SK3 activity.

Ca+ -activated K+ -channels (KCa) regulate vasomotor tone via smooth muscle hyperpolarization and relaxation. The relative contribution of the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation differs depending on vessel type and size. It is unknown whether these KCa channels are differentially distributed along the same vascular bed and hence have different roles in mediating the EDHF response. We therefore assessed the role of small- (SKCa), intermediate- (IKCa), and large-conductance (BKCa) channels in mediating acetylcholine-induced relaxations in both first- and fourth-order side branches of the rat superior mesenteric artery (MA1 and MA4, respectively). Two-millimeter segments of each MA were mounted in the wire myograph, incubated with Nomega-nitro-L-arginine methyl ester (L-NAME, 100 micromol/l) and indomethacin (10 micromol/l), and precontracted with phenylephrine (10 micromol/l). Cumulative concentration-response curves to ACh (0.001-10 micromol/l) were performed in the absence or presence of selective KCa channel antagonists. Apamin almost completely abolished these relaxations in MA4 but only partially blocked relaxations in MA1. The selective IKCa channel blocker 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) caused a significantly greater inhibition of the ACh-induced relaxation in MA4 compared with MA1. Iberiotoxin had no inhibitory effect in MA4 but blunted relaxation in MA1. Relative mRNA expression levels of SKCa (rSK1, rSK3, and rSK4 = rIK1) were significantly higher in MA4 compared with MA1. BKCa (rBKalpha1 and rBKbeta1) genes were similar in both MA1 and MA4. Our data demonstrate regional heterogeneity in SKCa and IKCa function and gene expression and stress the importance of these channels in smaller resistance-sized arteries, where the role of EDHF is more pronounced.

In resistance-sized arteries, a chronic increase in blood flow leads to increases in arterial structural luminal diameter and arterial wall mass. In this review, we summarize recent evidence that outward remodeling of resistance arteries 1) can help maintain and restore tissue perfusion, 2) is not intimately related to flow-induced vasodilatation, 3) involves transient dedifferentiation and turnover of arterial smooth muscle cells, and 4) is preceded by increased expression of matricellular proteins, which have been shown to promote disassembly of focal adhesion sites. Studies of experimental and physiological resistance artery remodeling involving differential gene expression analyses and the use of knockout and transgenic mouse models can help unravel the mechanisms of outward remodeling.

The vascular kallikrein-kinin system contributes to about one third of flow-dependent dilation in mice carotid arteries, by activating bradykinin B2 receptors coupled to endothelial nitric oxide (NO) release. Because the bradykinin/NO pathway may mediate some of the effects of angiotensin II AT2 receptors, we examined the possible contribution of AT2 receptors to the kinin-dependent response to flow. Changes in outer diameter after increases in flow rate were evaluated in perfused arteries from wild-type animals (TK+/+) and in tissue kallikrein-deficient mice (TK-/-) in which the presence of AT2 receptor expression was verified. Saralasin, a nonselective angiotensin II receptor antagonist, impaired significantly flow-induced dilation in TK+/+, whereas it had no effect in TK-/- mice. In both groups, blockade of AT1 receptors with losartan or candesartan did not affect the response to flow. Inhibition of AT2 receptors with PD123319 reduced significantly flow-induced dilation in TK+/+ mice, but had no significant effect in TK-/- mice. Combining PD123319 with the bradykinin B2 receptor antagonist HOE-140 had no additional effect to AT2 receptor blockade alone in TK+/+ arteries. Flow-dependent-dilation was also impaired in AT2 receptor deficient mice (AT2-/-) when compared with wild-type littermates. Furthermore, HOE-140 significantly reduced the response to flow in the AT2+/+, but not in AT2-/- mice. In conclusion, this study demonstrates that the presence of functional AT2 receptors is necessary to observe the contribution of the vascular kinin-kallikrein system to flow-dependent dilation.

Objective: To test whether membrane-bound angiotensin I-converting enzyme (t-ACE) is involved in arterial remodeling, we applied unilateral carotid artery (CA) ligation and studied uterine arteries (UA) before, during, and after pregnancy in t-ACE-/- and t-ACE+/+ mice. RESULTS- In CA of t-ACE-/- mice, blood pressure, outer diameter (D), and medial cross-sectional area (mCSA) were reduced, whereas blood flow (BF) and the number of medial cells (mC) were not modified. In the ligated CA, mCSA and number of mC were increased while outer D and distensibility were reduced. These changes were significantly less pronounced in t-ACE-/- than t-ACE+/+ mice. In UA of t-ACE-/- mice, D was larger and mCSA was unaltered. At term pregnancy, D and mCSA of the UA were reversibly increased. Structural changes of UA during and after pregnancy were comparable in both strains., Conclusions: t-ACE contributes to arterial structure and remodeling. It plays a major role in hyperplastic inward remodeling of the CA imposed by blood flow cessation, but it is not essential for outward hypertrophic and subsequent inward hypotrophic remodeling of the UA during and after pregnancy.

Objective: Tissue kallikrein (TK) participates in acute flow-induced dilatation (FID) of large arteries. We investigated whether TK deficiency blunts FID and alters chronic flow-related arterial structural and functional changes in resistance-sized muscular arteries., Methods and Results: Vasomotor responses and structural parameters were determined in uterine arteries isolated from nonpregnant, 18- to 19-day pregnant, and 7-day postpartum TK-/- and TK+/+ littermate mice. In TK-/- mice, values of diameter, medial cross-sectional area (CSA), myogenic tone, and dilatation in response to acetylcholine were comparable to those values in TK+/+ mice, but FID (0 to 100 microL/min) was significantly reduced (55+/-4% versus 85+/-4% in TK+/+ mice). In both mouse strains, pregnancy resulted in significant increases in diameter and medial CSA and in the Nw-nitro-l-arginine methyl ester-sensitive component of FID. By 7 days after pregnancy, uterine arterial diameter and CSA values no longer differed from nonpregnant values, and FID was markedly reduced in TK-/- and TK+/+ mice., Conclusions: These observations (1) confirm at the level of resistance arteries the key role of TK in FID and (2) indicate that TK deficiency does not compromise arterial remodeling and changes in the contribution of NO to FID during and after pregnancy.