The roles of cyclic adenosine monophosphate- and cyclic guanosine monophosphate-dependent protein kinase pathways in hydrogen peroxide-induced contractility of microvascular lung pericytes

BACKGROUND: Sepsis and posttraumatic inflammatory processes are accompanied by definite changes in microvascular permeability, particularly in the lung. These permeability changes may occur because of damaged regulatory mechanisms at the level of the capillary wall. Pericytes are adventitial cells located within the basement membrane of capillaries. These cells contain multiple cytoplasmic processes that envelope endothelial cells, and are consequently thought to stabilize capillary walls and participate in microcirculation and endothelial cell permeability. Data from this laboratory and other laboratories have confirmed that pericytes are contractile cells, adding to the evidence that pericytes may influence or help regulate capillary permeability. We have already determined that hydrogen peroxide (H2O2) causes dose-dependent relaxation in microvascular lung pericytes (MLPs) at 10 minutes and, conversely, dose-dependent contraction at 30 minutes. It is the aim of this study to determine the mechanism of this biphasic contractile response. Specifically, we will determine whether cyclic adenosine monophosphate (cAMP)- or cyclic guanosine monophosphate (cGMP)-dependent protein kinase intracellular pathways are responsible for the hydrogen peroxide-induced contractility of MLPs. METHODS: Rat MLPs were isolated by previously published protocol and cultured on collagen gel matrices. MLPs were pretreated with either ODQ, a soluble guanylate cyclase inhibitor (100 mumol/L), for 15 minutes; GKIP, a protein kinase G inhibitor (100 mumol/L), for 1 hour; SQ22536, an adenylate cyclase inhibitor (100 mumol/L), for 15 minutes; or H89, a protein kinase A inhibitor (10 mumol/L), for 1 hour. Hydrogen peroxide was then introduced to each MLP culture at 10 mumol/L, 100 mumol/L, and 1 mmol/L. After each of these treatments, the surface area of the collagen gels was digitally quantified at 10 and 30 minutes. RESULTS: SQ22536 attenuated both relaxation at 10 minutes and the contraction seen at 30 minutes for all concentrations of H2O2. H89 caused a marked basal relaxation and prevented the cells from contracting at 30-minute exposures to all concentrations of H2O2. Both ODQ and GKIP attenuated the relaxation at 10 minutes but had no affect on the later contraction. CONCLUSION: The cGMP-dependent protein kinase pathway is a mechanism for H2O2-induced relaxation of MLPs. Up-regulation of cAMP and cGMP is responsible for early H2O2-induced relaxation and late contraction. Protein kinase A (cAMP-dependent protein kinase pathway) may be an important intracellular signaling protein in the H2O2-induced contraction of MLPs or may be unable to down-regulate cAMP once inhibited. This evidence further supports the concept that there are separate intracellular pathways that regulate divergent cellular responses. This idea parallels the clinical concept of reversible and irreversible dysfunction of cellular processes in shock, and that the cellular dysfunction is initiated by separate intracellular pathways.     

Vascular endothelial growth factor modulates contractile response in microvascular lung pericytes

BACKGROUND: Pericytes are capillary support cells that may play a role in regulating permeability by their contractile responses. Vascular endothelial growth factor (VEGF) may play a role in the increased permeability found in sepsis and other inflammatory conditions. The purpose of this study was to evaluate the role of VEGF in regulating pericyte contraction. METHODS: Rat microvascular lung pericytes were isolated according to previously described methods and cultured on collagen gel matrices. Cells were exposed to VEGF (10, 100, and 1000 pg/mL) for varying time periods (0, 10, 30, 60, and 120 minutes). The gels were released and their contractile responses digitally quantified. RESULTS: At all doses, VEGF induced initial pericyte relaxation (contraction 85% to 90% of controls; P < .001). This was followed-up by increased and sustained contraction (107% to 120% of controls; P < .01). CONCLUSIONS: VEGF modifies the contractile response of microvascular lung pericytes. This mechanism may play a role in the increased permeability demonstrated in inflammatory states.     

Impairment of corpus cavernosal smooth muscle relaxation by glycosylated human haemoglobin

OBJECTIVE: To examine the effect of HbA1c, an isoform of glycosylated haemoglobin (GHb, a product of non-enzymatic reactions between elevated blood glucose and haemoglobin), on nitric oxide-mediated corpus cavernosal smooth muscle relaxation, and to categorize the mechanisms involved. MATERIALS AND METHODS: Corpus cavernosal tissue from Wistar rats (300-350 g body weight) was prepared for the measurement of isometric tension. After equilibration in Krebs solution gassed with 95% O2/5% CO2 at 37 degrees C for 90 min, optimal resting tension was applied. Tissue was precontracted with 1 micromol/L noradrenaline (NAd) and either relaxed with incremental doses of acetylcholine (ACh) or sodium nitroprusside (SNP). After washout, strips were again precontracted with NAd and then incubated with pyrogallol (100 micromol/L), 100 microL of haemoglobin or 100 microL of GHb in the presence of either L-arginine (100 micromol/L), indomethacin (10 micromol/L), allopurinol (100 micromol/L), deferoxamine (100 micromol/L), catalase (600 IU/mL), or superoxide dismutase (SOD) (120 IU/mL) before ACh- or SNP-induced relaxation responses were repeated. RESULTS: Haemoglobin and GHb significantly impaired the relaxation of rat corpus cavernosum to ACh in a dose-dependent manner. L-arginine reversed the impairment caused by Hb, but not GHb. A donor of superoxide anions, pyrogallol, mimicked this impairment to ACh when added to control strips. Catalase, deferoxamine, indomethacin and allopurinol had no significant effect on the impaired relaxation response to ACh, whilst L-arginine partially reversed it. SOD completely reversed the GHb-induced impaired relaxation; GHb did not alter the relaxation response to SNP. CONCLUSION: GHb significantly impairs endothelial NO-mediated corpus cavernosal relaxation in the rat, in vitro. This effect is caused partly by the generation of superoxide anions and the extracellular inactivation of NO.     

Altered endothelium-derived hyperpolarizing factor-mediated endothelial function in coronary microarteries by St Thomas' Hospital solution.

OBJECTIVES: We examined the effect of St Thomas' Hospital solution on endothelium-derived hyperpolarizing factor-mediated function in the porcine coronary microarteries with emphasis on the effect of temperature and washout time. METHODS: Microartery rings (diameter, 200-450 micrometers) were studied in myograph. The arteries were incubated in St Thomas' Hospital or Krebs solution (control) at 4 degrees C for 4 hours followed by 45 minutes (group Ia) or 90 minutes washout (group Ib) or at 22 degrees C for 1 hour followed by 45 minutes (group IIa) or 90 minutes washout (group IIb) and precontracted with -8.5 log M U 46619. The endothelium-derived hyperpolarizing factor-mediated relaxation to bradykinin was studied when endothelium-derived nitric oxide and prostaglandin I2 were inhibited with the presence of 7 micromol/L indomethacin and 300 micromol/L NG-nitro-L -arginine. RESULTS: After exposure to St Thomas' Hospital solution, the maximal endothelium-derived hyperpolarizing factor-mediated relaxation (percentage of the precontraction) was significantly reduced at either temperature after washout for 45 minutes (group Ia, 42.7% +/- 3.5% vs 69.0% +/- 5.3%; n = 9; P =.000; and group IIa, 12.3% +/- 1.6% vs 56.1% +/- 4. 4%; n = 8; P =.000) but fully recovered after washout for 90 minutes. The U46619-induced contraction force was also significantly reduced after washout for 45 minutes (P <.001) but fully recovered at 90 minutes. CONCLUSIONS: Under profound and moderate hypothermia, St Thomas' Hospital solution impairs endothelium-derived hyperpolarizing factor-mediated relaxation and smooth muscle contraction in the coronary microarteries. These effects exist during the reperfusion period for at least 45 minutes after exposure to St Thomas' Hospital solution and may account for the possible myocardial dysfunction during reperfusion.     

Pretreatment with phenoxybenzamine attenuates the radial artery's vasoconstrictor response to alpha-adrenergic stimuli

BACKGROUND: Although the radial artery bypass conduit has excellent intermediate-term patency, it has a proclivity to vasospasm. We tested the hypothesis that brief pretreatment of a radial artery graft with the irreversible adrenergic antagonist phenoxybenzamine attenuates the vasoconstrictor response to the vasopressors phenylephrine and norepinephrine compared with the currently used papaverine/lidocaine. METHODS: Segments of human radial artery grafts were obtained after a 30-minute intraoperative pretreatment with a solution containing 20 mL of heparinized blood, 0.4 mL of papaverine (30 mg/mL), and 1.6 mL of lidocaine (1%). The segments were transported to the laboratory and placed into a bath containing Krebs-Henseleit solution and 10, 100, or 1000 micromol/L phenoxybenzamine or vehicle. The segments were tested in organ chambers for contractile responses to increasing concentrations of phenylephrine and norepinephrine (0.5-15 micromol/L). RESULTS: Contractile responses to 15 micromol/L phenylephrine in control radial artery segments averaged 44.2% +/- 9.1% of the maximal contractile response to 30 mmol/L KCl. Papaverine/lidocaine modestly attenuated contraction to 15 micromol/L phenylephrine (32.1% +/- 5.9%; P =.22), but 1000 micromol/L phenoxybenzamine completely abolished radial artery contraction (-7.2% +/- 4.4%; P <.001). The effect of 10 and 100 micromol/L phenoxybenzamine on attenuating vasocontraction was intermediate between 1000 micromol/L phenoxybenzamine and papaverine/lidocaine. Responses to 15 micromol/L norepinephrine in control radial artery segments averaged 54.7% +/- 7.5% of maximal contraction to 30 mmol/L KCl. Papaverine/lidocaine modestly attenuated the contraction response of radial artery segments (35.6% +/- 5.1%; P =.04). In contrast, 1000 micromol/L phenoxybenzamine showed the greatest attenuation of norepinephrine-induced contraction (-10.5% +/- 2.0%; P <.001). CONCLUSIONS: A brief pretreatment of the human radial artery bypass conduit with 1000 micromol/L phenoxybenzamine completely attenuates the vasoconstrictor responses to the widely used vasopressors norepinephrine and phenylephrine. Papaverine/lidocaine alone did not block vasoconstriction to these alpha-adrenergic agonists.     

Leadership, professional heroism, and the Eastern Association for the Surgery of Trauma: presidential speech at the 14th scientific assembly

BACKGROUND: In response to inflammation, endothelial cytoskeleton rearrangement, cell contraction, and intercellular gap formation contribute to a loss of capillary barrier integrity and resultant interstitial edema formation. The intracellular signals controlling these events are thought to be dependent on intracellular calcium concentration ([Ca2+]i). We hypothesized that, in human pulmonary microvascular endothelial cells, a thrombin-induced increase in permeability to albumin would be dependent on Ca2+i and subsequent actin cytoskeleton rearrangements. METHODS: Human lung microvascular endothelial cells, grown on 0.4 micromol/L pore membranes, were activated with 10 nmol/L human thrombin in Hank's balanced salt solution/0.5% fetal bovine serum. Select cultures were pretreated (45 minutes) with 4 micromol Fura-2/AM to chelate Ca2+i. Permeability was assessed as diffusion of bovine serum albumin/biotin across the monolayer. Similarly treated cells were stained with rhodamine-phalloidin to demonstrate actin cytoskeletal morphology. Separately, cells loaded 2 micromol Fura-2/AM were assessed at OD340/380nm after thrombin exposure to detect free Ca2+i. RESULTS: Intracellular Ca2+ levels increased 15-fold (2 seconds) and fell to baseline (10 minutes) after thrombin. Permeability increased 10-fold (30 minutes), and a shift from cortical to actin stress fiber morphology was observed. Chelation of Ca2+i diminished permeability to baseline and reduced the percentage of cells exhibiting stress fiber formation. CONCLUSION: Thrombin stimulates pulmonary capillary leak by affecting the barrier function of activated pulmonary endothelial cells. These data demonstrate a thrombin-stimulated increase in monolayer permeability, and cytoskeletal F-actin stress fibers were, in part, regulated by endothelial Ca2+i. This early, transient rise in Ca2+i likely activates downstream pathways that more directly affect the intracellular endothelial structural changes that control vascular integrity.     

Inhibition of heme oxygenase-1 in microvascular lung pericytes diminishes at high concentrations of an inflammatory mediator

Post-traumatic inflammation and sepsis induce changes in the lung microvasculature causing increased permeability. Pericytes, contractile cells positioned abluminally to endothelial cells, play a role in regulating this response. An in vitro model of microvascular lung pericytes (MLP) was used to investigate the effect of inhibiting heme oxygenase-1 (HO-1), a stress-induced enzyme, in the presence of varying levels of lipopolysaccharide (LPS), a mediator in the initiation of inflammation, on pericyte contractility. Rat MLP were cultured on collagen gel matrices. Cells were exposed to three concentrations of LPS in the presence of zinc protoporphyrin IX (ZnPP-9), a known inhibitor of HO-1. After 24 hours, the surface area of the collagen disks was quantified, thereby measuring pericyte contraction. ZnPP-9 caused a significant attenuation of the LPS-induced relaxation of the pericytes (P < or = 0.003). The effects of ZnPP-9, however, depended on the concentration of LPS to which the pericytes were exposed. Greater concentrations of LPS decrease the attenuating power of ZnPP-9. The inhibition of HO-1 diminished MLP relaxation triggered by LPS. The effect of ZnPP-9, however, is dependent on the concentration of LPS to which the MLP are exposed, indicating its saturation. ZnPP-9 may antagonize the microvascular response to trauma.     

Preconditioning improves cardioplegia-related coronary microvascular smooth muscle hypercontractility: role of KATP channels.

OBJECTIVES: The effect of preconditioning before hyperkalemic cardioplegia on the coronary smooth muscle remains to be elucidated. We tested the hypothesis that hypoxic preconditioning could protect coronary smooth muscle against subsequent hyperkalemic cardioplegia-induced coronary vasospasm and that this preconditioning effect could be mediated by K(ATP) channels. METHODS: Rat coronary arterioles (endothelium-denuded) were studied in a pressurized, no-flow, normothermic state. Simultaneous monitoring of luminal diameter and intracellular calcium concentration of vascular smooth muscle loaded with fura-2 was made with microscopic image analysis. All vessels were subjected to 60 minutes of hypoxic hyperkalemic cardioplegia (K(+) = 25.0 mmol/L) and were then reperfused. Six groups were studied: (1) controls, no precardioplegic intervention; (2) preconditioning, achieved with 10 minutes of hypoxia (PO2 < 30 mm Hg) and 10 minutes of reoxygenation; (3) preconditioning plus glibenclamide (10 micromol/L), achieved with 10 minutes of preconditioning in the presence of K(ATP) channel blocker glibenclamide; (4) pretreatment with K(ATP) channel opener pinacidil (100 micromol/L); (5) pretreatment with pinacidil (100 micromol/L) plus glibenclamide (10 micromol/L); and (6) pretreatment with glibenclamide (10 micromol/L) alone. RESULTS: Hypoxic preconditioning significantly (P <.01) reduced hyperkalemic cardioplegia-induced intracellular calcium concentration accumulation and prevented the hypercontractility during and after hyperkalemic cardioplegia compared with control vessels. Pinacidil provided effective microvascular protection similar to hypoxic preconditioning. These vasoprotective effects of preconditioning were significantly antagonized in glibenclamide-treated vessels. CONCLUSIONS: Hypoxic preconditioning can prevent coronary microvascular hypercontractility during and after subsequent cardioplegia by a K(ATP ) channel mechanism that regulates intracellular calcium concentration of the vascular smooth muscle.     

The in vitro effects of remifentanil and fentanyl on isolated human right atria and saphenous veins

OBJECTIVE: To determine the myocardial and vascular effects of remifentanil and fentanyl in human atria and saphenous veins. DESIGN: In vitro, prospective with repeated measures. SETTING: University research laboratory. INTERVENTIONS: The direct effects of remifentanil and fentanyl on the electrical stimulation-induced contraction of nonfailing human atrium and saphenous veins contracted with 5-hydroxytryptamine were studied. MEASUREMENTS AND MAIN RESULTS: In human atrial trabeculae, cumulative (10(-9)-10(-5) mol/L) added remifentanil had no effect on contractile force, compared with untreated muscles (p > 0.05). The force of contraction was significantly less than control values with concentrations of fentanyl ranging from 10(-8) to 10(-5) mol/L (p < 0.05). At the highest concentration (10(-5) mol/L), the inhibition by fentanyl of the electrical stimulation-induced contraction was 40.6% +/- 6.32%. In human saphenous vein strips preconstricted with 5-hydroxytryptamine, remifentanil (10(-8)-10(-5) mol/L) and fentanyl (10(-8)-10(-5) mol/L) produced "concentration-dependent" relaxation when compared with the control contraction value (p < 0.05). The IC(50) was similar with remifentanil and fentanyl and the E(max) of fentanyl was significantly higher than remifentanil (p < 0.05). The venodilatory effects of remifentanil and fentanyl were similar on veins with or without endothelium (p > 0.05). CONCLUSIONS: Remifentanil has no direct effect on the contraction of myocardium. Fentanyl inhibits the electrical stimulation-induced contraction in human right atrial muscles in vitro. Remifentanil and fentanyl produce "concentration-dependent" relaxation in human saphenous vein strips independent from the endothelium.     

内源性一氧化碳对离体犬阴茎海绵体平滑肌的作用

目的:探讨内源性一氧化碳(CO)对离体犬阴茎海绵体平滑肌作用的影响。方法:利用水浴条件下阴茎海绵体肌条的张力测定技术,用CO合成的关键酶血红素氧合酶(HO)的诱导剂———氯高铁血红素诱导海绵体平滑肌生成内源性CO,观察CO对去氧肾上腺素(PE)诱导收缩的阴茎海绵体肌条作用的影响。结果:氯高铁血红素对10μmol/L PE诱导的肌条收缩具有浓度依赖性的松弛作用,10~100μmol/L氯高铁血红素对平滑肌肌条的松弛效应与空白对照相比明显升高(P<0.01)。用锌原卟啉-Ⅸ(ZnPP-Ⅸ)或亚甲蓝孵育处理后的肌条,氯高铁血红素的舒张作用明显减弱(P<0.01)。结论:内源性CO具有浓度依赖性松弛阴茎海绵体平滑肌的作用,其机制可能是通过CO环磷酸鸟苷途径作用所致。     

Alpha lipoic acid attenuates microvascular endothelial cell hyperpermeability by inhibiting the intrinsic apoptotic signaling

BACKGROUND: This study examined whether alpha lipoic acid (ALA), an antioxidant with anti-apoptotic properties, synthesized in mitochondria of endothelial cells, would inhibit intrinsic apoptotic signaling and microvascular endothelial cell hyperpermeability. METHODS: Rat lung microvascular endothelial cells were transfected with BAK (BH3) peptide (5 microg/mL) or active caspase-3 (5 microg/mL) and were pretreated with ALA (10 and 100 micromol/L). Hyperpermeability was determined using fluorescein isothiocyanate albumin-flux across the cells grown as monolayer. Reactive oxygen species (ROS) formation was determined using 123 dihydrorhodamine and mitochondrial membrane potential using JC-1. Cytochrome c levels and caspase-3 activity were determined using an enzyme-linked immunosorbent assay and a fluorometric assay, respectively. RESULTS: ALA (100 micromol/L) pretreatment attenuated BAK (BH3)-induced hyperpermeability and ROS formation. ALA restored BAK (BH3)-induced collapse in mitochondrial membrane potential and decreased BAK (BH3)-induced cytochrome c release and caspase-3 activity. CONCLUSIONS: These findings suggest that ALA attenuates BAK-induced monolayer hyperpermeability through the inhibition of ROS formation and intrinsic apoptotic signaling.     

Diazoxide provides protection to human myocardium in vitro that is concentration dependent

BACKGROUND: Diazoxide has been shown to confer significant myocardial protection in many experiments. This study was designed to assess its influence on the structural injury and functional recovery of human myocardium subjected to hypoxia/reoxygenation in vitro. METHODS: The isolated electrically driven human right atrial trabeculae, obtained during cardiac surgery, were studied. The tissue bath was oxygenated with 95% oxygen and 5% carbon dioxide, hypoxia being obtained by replacing oxygen with argon. The influence of diazoxide on atrial contractility was studied first. Next, the two trabeculae from one atrial appendage were studied simultaneously, adding diazoxide to the tissue bath 10 minutes before hypoxia in one, with another serving as a control. We tested 10(-4.5) mol/L and 10(-4) mol/L diazoxide in three sets of experiments testing 30, 60, and 90 minutes of hypoxia. We continued reoxygenation for 120 minutes (in 60-minute and 90-minute hypoxia experiments) and subsequently tested reaction to 10(-4) mol/L norepinephrine. Apart from continuous recording of the contraction force, we measured the troponin I release into the tissue bath after ischemia and reoxygenation. RESULTS: Diazoxide exerted a negative inotropic effect in human atrial muscle (pD(2)=3.96 +/- 0.18). Both concentrations of diazoxide studied resulted in better functional recovery of atrial trabeculae subjected to 30 minutes of hypoxia. With longer hypoxia, only the higher diazoxide concentration provided significant protection as assessed by contractility. After 120 minutes of reoxygenation, only diazoxide-treated muscle was viable enough to respond to norepinephrine. Only 10(-4) mol/L diazoxide resulted in lower troponin I release during hypoxia and reoxygenation. CONCLUSIONS: This study shows that diazoxide provides significant concentration-dependent protection against hypoxia/reoxygenation injury to human myocardium in vitro.     

TNF-alpha and IL-1beta increase pericyte/endothelial cell co-culture permeability

BACKGROUND: Pericytes (PC) have a unique synergistic relationship with microvascular endothelial cells (MVEC) in the regulation of capillary permeability. This study investigates the effect of TNF-alpha, IL-1beta, and IL-6 on the microvasculature by measuring changes in PC contractility, and also, albumin permeability across MVEC/PC co-cultures. MATERIALS AND METHODS: Semi-permeable inserts were plated first with rat lung MVEC and then PCs (on the fourth day) at a ratio of 10:1 MVEC/PC. On day 5, 50 ng/ml of TNF-alpha, IL-1beta, and IL-6 were added with or without a secretory phospholipase A(2)-IIA (sPLA(2)-IIA) inhibitor for 24 h. After treatments, albumin clearances were quantified. For measuring contractility, PCs were cultured on collagen matrices and exposed for 24 h to TNF-alpha, IL-1beta, and IL-6 at 1 ng/ml, 10 ng/ml, and 50 ng/ml with/without inhibitors for sPLA(2)-IIA, phospholipase A(2) (PLA(2)), and cyclooxygenase-II (COX-II). After treatments, the surface area of the collagen disks was digitally quantified. RESULTS: TNF-alpha and IL-1beta significantly increased albumin clearance in MVEC/PC co-cultures (P < 0.05) and induced dose-dependent relaxation of PCs (P < 0.05). PC relaxation was completely attenuated with the sPLA(2)-IIA and pLA(2) inhibitors; the COX-II inhibitor provided partial blockade. IL-6 had no effect on PC contractility or permeability. CONCLUSION: TNF-alpha and IL-1beta directly increased microvascular permeability in co-cultures. They also induced relaxation of PCs through a sPLA(2)-IIA dependent mechanism. Interestingly, IL-6 had no effect, although its presence in high levels has been demonstrated in inflamed lungs. These findings may help elucidate the significance of PC in regulating the capillary response to various pro-inflammatory cytokines.     

The effects of vasopressin on endotoxin-induced attenuation of contractile responses in human gastroepiploic arteries in vitro

We studied the effects of vasopressin on contraction in normal and endotoxin-treated human gastroepiploic arterial rings in vitro. In this tissue, vasopressin (50-500 pg/mL) produced concentration-dependent, endothelium-independent contractions. Vasopressin also potentiated the contraction elicited by 1.0 micromol/L norepinephrine (NE) in both the presence and absence of endothelium. Endotoxin (10 microg/mL) attenuated the 1.0 micromol/L NE-induced contractions, and this attenuation was reversed by 300 micromol/L N(G)-nitro-L-arginine-methyl ester (L-NAME) and by 300 micromol/L N(G)-nitro-L-arginine (L-NoArg). After 12 h endotoxin treatment, the vasopressin-induced contraction was attenuated, and the enhancing effect of vasopressin was diminished. However, both before and after endotoxin, the enhancement produced by vasopressin was larger than the vasopressin-contraction itself. An antagonist of the vasopressin V1 receptor, 1.0 micromol/L beta-mercapto-[beta,beta-cyclopentamethylenpropionyl1,O-MeTyr2+ ++,Arg8]-vasopressin, and an antagonist of V1 + V2 receptor receptor, 1.0 micromol/L des-Gly9-[beta-mercapto-beta,beta-cyclopentamethylenepropionyl1 ,O-Et-Tyr2,Val,Arg8]-vasopressin, each diminished the vasopressin-induced enhancement of the NE contraction. IMPLICATIONS: The results of our study suggest that, in addition to its direct vasoconstrictor effect, vasopressin strongly enhances the responses to norepinephrine through V1-receptor stimulation and that vasopressin could find a role in the management of endotoxin-induced vasodilation.     

The effect of elevated homocysteine levels on adrenergic vasoconstriction of human resistance arteries: the role of the endothelium and reactive oxygen species

PURPOSE: This study investigated the effect of elevated homocysteine levels on adrenergic contraction of human resistance arteries and tested the hypothesis that homocysteine-induced generation of reactive oxygen species contributes to vascular reactivity changes. METHODS: Small (<200 microm) subcutaneous arteries were cannulated and pressurized in an arteriograph chamber that allowed the measurement of lumen diameter. Two arteries from the same patient were obtained; one was perfused and superfused (intraluminal pressure = 50 mm Hg) with physiologic saline solution (control, n = 6), and the other was perfused and superfused with physiologic saline solution plus 200 micromol/L homocysteine (HC, n = 6); the reactivity to adrenergic stimulation was assessed. Another group of arteries was incubated in 200 micromol/L homocysteine plus 1200 U/mL superoxide dismutase and 120 U/mL catalase (HC + SC, n = 6), and the reactivity to norepinephrine was determined. The vasoreactivity of homocysteine was further assessed in intact (n = 6) and denuded (n = 6) arteries that were precontracted with an intermediate concentration of norepinephrine and homocysteine (20-200 micromol/L) added to the bath while the lumen diameter was continuously recorded. RESULTS: Sensitivity to norepinephrine was diminished in HC arteries, which increased the median effective concentration (EC(50)) from 0.24 +/- 0.06 micromol/L in control arteries to 0.65 +/- 0.10 micromol/L in HC arteries (P <.01). Homocysteine also caused concentration-dependent vasodilation of arteries contracted with an intermediate concentration of norepinephrine that was greater in intact than denuded arteries, with the half-maximum responses occurring at 61 +/- 6 micromol/L (intact) and 90 +/- 11 micromol/L (denuded; P <.05). There was no significant difference in sodium nitroprusside sensitivity between control and homocysteine arteries (EC(50) = 61 +/- 3 nmol/L vs 50 +/- 19 nmol/L; P >.05) or in sensitivity to acetylcholine (EC(50) = 19 +/- 7 nmol/L vs 12 +/- 3 nmol/L; P >.05). Arteries in the presence of superoxide dismutase and catalase had similarly impaired reactivity to norepinephrine as did homocysteine arteries (EC(50), 0.58 +/- 0.15 micromol/L; P >.05 vs HC, P <.01 vs control). CONCLUSION: An elevated homocysteine level in vitro diminishes adrenergic contraction, with a differential endothelial versus smooth muscle influence that appears unrelated to the generation of reactive oxygen species.     

The inhibitory role of melatonin on isolated guinea-pig urinary bladder: an endogenous hormone effect

OBJECTIVE: To investigate the effects of melatonin, an endogenous hormone, on acetylcholine and KCl-induced contractions of isolated guinea-pig detrusor muscle. MATERIALS AND METHODS: Detrusor smooth muscle strips isolated from guinea-pig bladders were placed in an organ bath containing physiological saline at 37 degrees C and pH 7.4, constantly bubbled with 95% oxygen and 5% CO2. The effects of cumulatively applied melatonin on the acetylcholine- and KCl-induced contractions of isolated bladder strips were examined using isometric contraction measurements. RESULTS: Melatonin (100 and 300 micromol/L) significantly inhibited the peak amplitude of both acetylcholine (10 micromol/L) and KCl (30 mmol/L)-induced contraction of the isolated bladder strips (P < 0.05). Similarly, melatonin caused a significant reduction in the contractile frequency induced by KCl (eight strips) in a concentration-dependent manner, while having no significant effect on the frequency of contractile response to acetylcholine, even at the highest concentration (300 micromol/L) used (P = 0.58, 14 strips). CONCLUSIONS: These results suggest that melatonin inhibits acetylcholine- and KCl-induced contractions in isolated bladder strips from guinea pigs. The endogenous nature of melatonin, with its low side-effect profile, makes it a potentially useful agent to be considered in the medical management of the overactive bladder.     

Nitric oxide donating aspirins: novel drugs for the treatment of saphenous vein graft failure

BACKGROUND: A new class of nitric oxide donating aspirin (NO-ASA) drugs may increase the therapeutic impact of aspirin in saphenous vein coronary artery bypass grafting (CABG) not only through the inhibition of thrombosis but also through a reduction of vasospasm and inhibition of vascular smooth muscle cell (VSMC) proliferation (effects that are inhibited by NO but not ASA). In order to test this proposal the effect of three NO-ASA drugs (NCX4040, NCX4050, and NCX4060) on in vitro relaxation and cyclic guanosine monophosphate (cGMP) formation in the human isolated saphenous vein and the proliferation of human VSMCs was investigated. METHODS: Saphenous vein segments were obtained from 30 patients undergoing CABG (median age, 59 years; range, 49 to 68). The effect of the NO-ASA adducts, ASA alone, and sodium nitroprusside (NO donor) were investigated on (1) relaxation of phenylephrine-stimulated contraction using an organ bath, (2) cyclic guanosine monophosphate (cGMP) formation using an enzyme-linked immunosorbent assay, and (3) the proliferation of VSMCs derived from saphenous vein using bromo-deoxyuridine (BRDU) incorporation. RESULTS: All three NO-ASA adducts (at concentrations that inhibited responses by 50% [IC50s] between 1 micromol/L and 100 micromol/L) and nitroprusside (at IC50s between 0.5 and 10 micromol/L) elicited relaxation of isolated human saphenous vein, promoted cGMP formation, and inhibited VSMC proliferation whereas ASA alone (up to 100 micromol/L) had no effect on any variable. CONCLUSIONS: These data indicate that the NO-ASA adducts by virtue of their capacity to release NO and stimulate guanylyl cyclase may be useful not only in the prevention of thrombosis following CABG but also the reduction of saphenous vein graft spasm and neointima formation.     

Guanylate cyclase activators influence reactivity of human mesenteric superior arteries retrieved and preserved in the same conditions as transplanted kidneys

INTRODUCTION: This study sought to investigate the mechanisms of relaxation induced by the (nitric oxide (NO)-independent soluble guanylyl cyclase (sGC) stimulators 3-[5'-hydroxymethyl-2'-furyl]-1-benzylindazole (YC-1) in human mesenteric arteries relaxed and precontracted with 1 micromol/L 5-hydroxytryptamine (serotonin). MATERIAL AND METHODS: Human mesenteric arteries obtained during kidney retrieval were preserved in the same conditions as transplanted kidneys. All experiments were performed after reperfusion with Krebs buffer in 37 degrees C and 100% oxygen exposure. RESULTS: In endothelium-intact rings, YC-1 (0.001 to 30 mmol/L) caused concentration-dependent relaxation (pEC(50): 6.59 +/- 0.12), which shifted to the right in endothelium-denuded rings. The sGC inhibitor 1H- [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ 10 mmol/L) partially attenuated the maximal responses to YC-1 (E(max) = 51.30% +/- 3.70%; n = 6) and displaced its curve to the right in intact and denuded vessels. Both, the NO synthesis inhibitor N-nitro-L-arginine methyl ester (100 mmol/L) and the NO scavenger carboxy-2-[4-carboxyphenyl]-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (100 mmol/L) significantly reduced YC-1 relaxation. The sodium pump inhibitor ouabain (1 micromol/L) produced a greater decrease in the vasodilator response of YC-1 (E(max) = 18.7% +/- 4.55%; n = 9). ODQ (10 micromol/L) plus 1 mumol/L ouabain abolished the relaxant response of YC-1 (E(max) = 9.4% +/- 2.94%, n = 9). CONCLUSIONS: This study demonstrated that sodium pump stimulation by YC-1 as an additional mechanism of sGC activation independent of cGMP relaxed human mesenteric artery, including blockade of Ca(2+) influx. Furthermore, this study suggested an ability of NO to mediate relaxation of resistance-like arteries through the activation of soluble guanylate cyclase and K(+) channels.     

Impaired endothelium-derived hyperpolarizing factor-mediated relaxation in porcine pulmonary microarteries after cold storage with Euro-Collins and University of Wisconsin solutions

BACKGROUND: Endothelium plays an important role in mediating the function of transplanted organs. The widely used University of Wisconsin solution impairs the endothelium-derived hyperpolarizing factor-mediated relaxation in coronary arteries, but little is known about effects of lung preservation on endothelium-derived hyperpolarizing factor-mediated endothelial function. This study examined the effect of organ preservation solutions on the endothelium-derived hyperpolarizing factor-mediated relaxation in the pulmonary microarteries (diameter 200 to 450 microm). METHODS: Two segments (1 as control) from the same microartery were allocated in 2 chambers of a myograph. After incubation with hyperkalemia (potassium 115 mmol/L), University of Wisconsin, or Euro-Collins solution (at 4 degrees C for 4 hours), the endothelium-derived hyperpolarizing factor-mediated relaxation was induced by bradykinin (-10 to -6.5 log M, n = 8) or calcium ionophore (A(23187), -9 to -5.5 log M, n = 7) in U(46619) (-7.5 log M) precontracted rings in the presence of indomethacin (7 micromol/L), N(G)-nitro-L-arginine (300 micromol/L), and oxyhemoglobin (20 micromol/L). RESULTS: Exposure to hyperkalemia and storage with Euro-Collins or University of Wisconsin solution significantly decreased the relaxation to bradykinin (51.9 +/- 8.4% vs 60.3 +/- 6.1%, P =.02 or 49.3 +/- 7.3% vs 65.2 +/- 3.5%, P =.04) or A(23187) (12.5 +/- 0.02% vs 33.8 +/- 0.07%, P =.02 or 13.2 +/- 0.03% vs 31.0 +/- 0.05%, P =.03%). CONCLUSIONS: Endothelium-derived hyperpolarizing factor plays an important role in porcine pulmonary microarteries, and the endothelium-derived hyperpolarizing factor-mediated relaxation is impaired when the lung is preserved with University of Wisconsin or Euro-Collins solution. This impairment may affect the lung function during the reperfusion period after lung transplantation.