Maximal Contractile Response Research Articles

The role of oxidant stress in angiotensin II-mediated contraction of human resistance arteries in the state of health and the presence of cardiovascular disease

Background Angiotensin II is a powerful vasoconstrictor and regulator of cardiovascular growth. Also, it increases formation of reactive oxygen species and contributes to vascular dysfunction. We investigated the role of oxidant stress in contraction of human resistance arteries to angiotensin II, in health and in the presence of cardiovascular disease. Methods and patients Studies of isometric contraction to angiotensin II, using human resistance arteries from healthy volunteers and patients, undergoing cardiac revascularization surgery, were performed by the broad-spectrum antioxidant agent vitamin C and superoxide dismutase mimetic TEMPOL. In the presence of vitamin C, the potency and the maximum contractile response were reduced in both patients and healthy volunteers. Addition of TEMPOL caused a decrease in angiotensin II-induced contraction only in the patients' group. Conclusions Our studies provide evidence for the role of oxidant stress in the contractile response of human resistance arteries to angiotensin II. In patients with cardiovascular disease, the superoxide anion may be the major species involved. In healthy subjects, other reactive oxygen species and the redox-independent vasoconstrictor action of angiotensin II predominate. Condensed abstract Increased formation of reactive oxygen species, due to angiotensin II, contributes to vascular dysfunction. We determined the oxidative reactivity of human resistance arteries to angiotensin II in healthy subjects and patients, undergoing cardiac revascularization surgery, using the broad-spectrum antioxidant agent, vitamin C, and superoxide dismutase mimetic, TEMPOL. There was a large decrease in potency and maximum of angiotensin II-induced contractile response noted in both groups with the former, while the latter reduced contraction only in the patients' group. Superoxide anion may play a major role in angiotensin II contractions of human resistance arteries in the presence of cardiovascular disease. In healthy subjects, other reactive species and the redox-independent pathways predominate.

Diazepam Attenuates Phenylephrine-Induced Contractions in Rat Aorta

In this in vitro study we examined the effects of diazepam on a phenylephrine-induced contraction in rat aorta and determined the associated cellular mechanism focusing on the endothelium-derived vasodilators. The concentration-response curves for phenylephrine and potassium chloride were generated in the presence or absence of diazepam. Phenylephrine concentration-response curves were generated from the endothelium-intact rings pretreated independently with N(W)-nitro-L-arginine methyl ester, PK 11195, tetraethylammonium, and indomethacin in the presence or absence of diazepam. Diazepam (7 x 10(-7) M) attenuated the phenylephrine-induced contraction in the endothelium-intact rings, whereas a large dose (5 x 10(-6) M) of diazepam attenuated the phenylephrine-induced contraction in the aortic rings with or without the endothelium. A pretreatment with the N(W)-nitro-L-arginine methyl ester completely abolished the diazepam (7 x 10(-7) M)-induced attenuation of the phenylephrine concentration-response curve, as well as the diazepam (5 x 10(-6) M)-induced attenuation of the maximal contractile response to phenylephrine. The N(W)-nitro-L-arginine methyl ester (10(-4) M)-induced contraction was enhanced in the rings pretreated with diazepam (5 x 10(-6) M). These results indicate that a supraclinical concentration of diazepam attenuates phenylephrine-induced contraction by increasing endothelial nitric oxide activity and directly affecting vascular smooth muscle.

Ultrafine particulate matter exposure attenuates mouse aortic relaxations

Particulate air pollution (PM) contributes to adverse cardiovascular events by yet unknown mechanisms. We tested the hypothesis that PM exposure altered endothelial regulation of systemic vascular tone. 6–10 week old male ICR mice were exposed to a single dose of 10, 30 or 100 μg of ultrafine PM or vehicle by intratracheal instillation. 24 hours later aortas were isolated and exposed to phenylephrine (PE), acetylcholine (ACH) or adenosine to probe their vascular responses. Constrictor responses to PE were not different between PM exposed and control aortas. PM exposure attenuated ACH relaxation in a dose-dependent manner. In endothelial-denuded aortas PM exposure increased the maximal contractile response (mN/mm2) to 10μM adenosine (control 1.39 ± 0.02 vs. PM-exposed 1.79 ± 0.11*). These results indicate that PM exposure altered endothelial-dependent and independent vascular responsiveness and may contribute to cardiovascular complications. This abstract dose not necessarily represents EPA policy. Supported by Phillip Morris Foundation # 567881.

Effect of tempol on altered angiotensin II and acetylcholine-mediated vascular responses in thoracic aorta isolated from rats with insulin resistance

The altered vascular responses to various vasopressors and relaxants have been well reported in various animal models of hypertension, insulin resistance and diabetes. Though the role of oxidative stress (increased superoxide levels) associated with these altered vascular responses in hyperglycemic/diabetic state is well documented, the role of the same remains to be largely unknown in vascular dysfunction coupled with prediabetic insulin resistant state. The objective of the present study was therefore to elucidate the role of free radicals particularly superoxides if any associated with vascular dysfunction in diet-induced insulin resistance of rats. In this regard, the effect of tempol (a membrane permeable superoxide dismutase mimetic/free radical scavenger) on the enhanced Ang II-induced contraction and impaired-ACh mediated relaxation in thoracic aorta of rats with insulin resistance was studied. Ang II-induced contraction and ACh-mediated relaxation responses were recorded isometrically in endothelium intact and denuded thoracic aortic ring preparations isolated from male Sprague–Dawley rats which were fed with either normal pellet diet (NPD) (control group) or high fat diet (HFD) (insulin resistant group) for 4 weeks. The HFD-fed rats exhibited characteristic features of insulin resistance syndrome viz., obesity, hyperinsulinaemia, mild hyperglycemia, hypertriglyceridemia, hypercholesterolemia, glucose intolerance and hypertension. Maximal contractile response ( E max) to Ang II was increased in endothelium intact aortic ring preparations obtained from HFD-fed rats as compared to NPD-fed control rats. Denudation of endothelium significantly increased Ang II-mediated E max responses in thoracic aortic rings of NPD-fed rats, whereas it produced only minimal alteration to the E max in the HFD-fed rats. In addition, ACh-mediated relaxation response was impaired in endothelium intact aortic rings isolated from HFD-fed rats. Tempol (30–300 μM) significantly and dose dependently inhibited enhanced vascular responses ( E max) of Ang II in endothelium intact, but not in endothelium denuded aortic ring preparations. Tempol (30 μM) reversed the impaired acetylcholine (ACh)-mediated relaxations in endothelium intact aortic ring preparations of HFD-fed rats. Endothelium independent vasorelaxations (EIV) to sodium nitroprusside (SNP) were similar for both NPD and HFD. In conclusion, our results indicate that superoxide radicals play crucial role in enhanced contractile and impaired vasodilatory responses to Ang II and ACh, respectively, in thoracic aortic rings isolated from diet-induced insulin resistant rats.

Effects of idarubicin and idarubicinol on rat coronary resistance and vasoconstrictor responsiveness of isolated aorta and mesentery

It has been hypothesized that coronary vasoconstriction is involved in the cardiotoxic action of anthracyclines. The purpose of this study was to determine whether an increase in coronary resistance induced by idarubicin (IDA) or its primary circulating metabolite idarubicinol (IDOL) is correlated with a decrease in vascular sensitivity to vasoconstrictor agonists. Coronary vasoconstriction was studied in single-pass perfused rat hearts after a 10-min infusion of 0.5 mg IDA or IDOL. In the endothelium-intact rat thoracic aorta and mesentery we measured the inhibition of phenylephrine (PE)- and KCl-induced contraction in the presence of IDA and IDOL, respectively. The increase in coronary vascular resistance evoked by IDOL (121%) exceeded that of IDA (75%). IDA (10-100 micromol/l) concentration-dependently diminished vascular sensitivity to PE and KCl due to a reduction in maximal contractile response (Emax), i.e. the antagonism by IDA of PE- or KCl-induced vasoconstriction was non-competitive, indicating a post-receptor cellular mechanism. These reductions of PE or KCl efficacy elicited by IDOL were significantly larger than those elicited by the corresponding doses of IDA. The decrease in efficacy of PE in the presence of IDA and IDOL was characterized by IC50 estimates of 44.3 and 30.7 mumol/l, respectively. With a 10-fold lower IC50, IDA inhibited the reactivity of small mesenteric arteries to noradrenaline with 10-fold higher potency. The correlation between the increase in coronary resistance and the decrease in vasoconstrictor responsiveness may suggest that these anthracyclines act through a common cellular mechanism.

Enantioselective separation and online affinity chromatographic characterization of R,R‐ and S,S‐fenoterol

rac-Fenoterol is a beta2-adrenoceptor agonist (beta2-AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R,R'- and S,S'-fenoterol (R-F and S-F). Here we report the separation of the R-F and S-F enantiomers and the evaluation of their binding to and activation of the beta2-AR. R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. Beta2-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human beta2-AR and standard membrane binding studies using the same membranes. The effect of R-F and S-F on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes. Chiral chromatography of rac-fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S-F and the more retained peak R-F. Frontal chromatography using membrane-bound beta2-AR as the stationary phase and rac-3H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-3H-fenoterol retention, while similar addition of S-F produced no change in rac-3H-fenoterol retention. The calculated dissociation constant of R-F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 +/- 55 nM (R-F) and 109,000 +/- 10,400 nM (S-F). In the cardiomyocytes, R-F increased maximum contractile response from (265 +/- 11.6)% to (306 +/- 11.8)% of resting cell length (P < 0.05) and reduced EC50 from -7.0 +/- 0.270 to -7.1 +/- 0.2 log[M] (P < 0.05), while S-F had no significant effect. Previous studies have shown that rac-fenoterol acts as an apparent beta2-AR/G(s) selective agonist and fully restores diminished beta2-AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac-fenoterol and that R-F is the active component of rac-fenoterol. Further evaluation of R-F will determine if it has enhanced selectivity and specificity for beta2-AR/G(s) activation and if it can be used in the treatment of congestive heart failure.

In Vivo and In Vitro Effects of Stellate Ganglion Blockade on Radial and Internal Mammary Arteries

To investigate the effects of stellate ganglion blockade (SGB) on the internal mammary (IMA) and radial arteries (RA) in patients undergoing coronary artery bypass graft (CABG) surgery with in vivo and in vitro studies. Prospective, randomized trial. University hospital. Thirty-seven patients undergoing CABG surgery. SGB was performed on 19 patients before anesthesia induction. Another group of 18 patients underwent surgery without SGB. Diameters of proximal RA, distal RA, and IMA were determined by Doppler ultrasonography before (T1) and after (T2) anesthesia induction. Control or blocked IMA and RA segments were obtained. Norepinephrine (NE) was applied to determine the contractile force of IMA and RA rings in a concentration-dependent manner. The maximal contractile response and the sensitivity of the vessels were compared. The diameters of IMA and distal RA were statistically larger in the SGB group than those in the control group at T2. NE-induced maximum contraction was higher in the blocked RA rings than those in the control RA and blocked IMA rings. The sensitivity of IMA segments to NE was higher than that of RA segments in the SGB group. The control and blocked IMA segments showed similar sensitivity to NE. The present results show that SGB not only increases distal RA and IMA diameters but is also associated with in vitro differences, the mechanism of which remains to be elucidated. Therefore, SGB might be considered as an alternative to topical and systemic vasodilators for reducing vasospasm in patients undergoing CABG.

Galangin protects pig detrusor nerves from repetitive field stimulation and anoxia/glucopenia injury

ObjectivesTo test the capability of the flavonoid galangin to protect pig urinary bladder from damage due to a period of repetitive field stimulation as well as a period of anoxia/glucopenia and reperfusion. MethodsSmooth muscle strips of the pig bladder were mounted for tension recording in small organ baths and the strips underwent either 1.5 hours of repetitive field stimulation at 32 Hz for 15 seconds every 5 minutes or under anoxia/glucopenia and reperfusion conditions. Galangin, at different concentrations, was added to the reperfusion Krebs solution to check the effect of this flavonoid compared with untreated strips under the same conditions. A group of experiments was performed to examine its possible underlying mechanisms. ResultsRepetitive field stimulation for 1.5 hours caused a progressive decrease in the maximal contractile response to electrical field stimulation (34% decrease). Galangin (10−7 M) partially prevented the progressive decrease in the contractile response. This effect was significantly reduced when verapamil was added to the solution. Galangin significantly improved the response of strips to electrical field stimulation under anoxia/glucopenia and reperfusion conditions compared with untreated tissues. ConclusionsGalangin has a protective effect on bladder contractility by an action that at least, in part, depends on l-type calcium channels. Furthermore, galangin protects detrusor nerves against the anoxia/glucopenic and reperfusion damage.

Predisposition for venoconstriction in the equine laminar dermis: implications in equine laminitis

Equine laminitis is a crippling condition associated with a variety of systemic diseases. Although it is apparent that the prodromal stages of laminitis involve microvascular dysfunction, little is known regarding the physiology of this vasculature. The aim of the present study was to determine the relative responses of equine laminar arteries and veins to the vasoconstrictor agonists phenylephrine (1 nM-10 microM), 5-HT (1 nM-10 microM), PGF2alpha (1 nM-100 microM), and endothelin-1 (1 pM-1 microM). We have determined that laminar veins were more sensitive, with respect to the concentration of agonist required to initiate a contractile response and to achieve EC(50), for all agonists tested. EC50 values, for veins and arteries, respectively, were 84+/-7 vs. 688+/-42 nM for phenylephrine, 35+/-6 vs. 224+/-13 nM for 5-HT, 496+/-43 nM vs. 3.0+/-0.6 microM for PGF2alpha, and 467+/-38 pM vs. 70.6+/-6.4 nM for endothelin-1. Moreover, when expressed as a percentage of the response to a depolarizing stimulus (80 mM potassium), the maximal contractile response of laminar veins exceeded that for the laminar arteries for each agonist. These results indicate that there may be a predisposition for venoconstriction within the vasculature of the equine digit. While this physiological predisposition for venoconstriction may be important in the regulation of blood flow during exercise, it also may help to explain why laminitis can result from a variety of pathological systemic conditions.

Enhanced cardiac function in mice overexpressing protein phosphatase Inhibitor-2

Protein phosphatase 1 (PP1) has been implicated in the control of cardiac function. Cardiac specific overexpression of the catalytic subunit, PP1c, results in hypertrophy and depressed contractility. To further address the role of PP1, transgenic mice (TG) were generated that overexpress in heart a functional COOH-terminally truncated form (amino acids 1-140) of the PP1 inhibitor-2 (I-2(140)). The TG hearts show increased levels of I-2(140) mRNA as well as protein and activity. No increase in absolute or relative heart weight was observed, nor any changes in gross pathology or increase in morbidity or mortality in the TG mice. Immunohistochemical and biochemical analyses revealed that expression of the I-2(140) protein is confined to cardiomyocytes where it is mainly localized in the cytosol. The total protein phosphatase (PP) activity was reduced by 80% in TG hearts as compared to wild-type littermates (WT). The PP1c mRNA level was the same in TG and WT, while the protein level was increased by approximately 7-fold in TG animals. The maximal rates of contraction (+dP/dt) and of relaxation (-dP/dt) were increased by 32% and 40%, respectively, in the intact catheterized TG mice compared to WT. However, the maximal contractile response to beta-adrenergic agonists was comparable in hearts from TG and WT mice. In isolated cardiomyocytes of TG mice, Ca2+transient amplitude was increased by 50% under basal conditions and by 60% upon rapid caffeine application. The phospholamban (PLB) protein level was unchanged whereas the basal phosphorylation of PLB at Ser(16) was significantly increased in TG hearts. These results indicate that I-2(140) overexpression results in decreased PP1 activity and enhanced contractility in the heart, underscoring the fundamental role of PP1 in cardiac function.

Nongenomic Effects of Aldosterone in the Human Heart

Aldosterone exerts rapid "nongenomic" effects in various nonrenal tissues. Here, we investigated whether such effects occur in the human heart. Trabeculae and coronary arteries obtained from 57 heart valve donors (25 males; 32 females; 17 to 66 years of age) were mounted in organ baths. Aldosterone decreased contractility in atrial and ventricular trabeculae by maximally 34+/-3% and 15+/-4%, respectively, within 5 to 15 minutes after its application. The protein kinase C (PKC) inhibitor chelerythrine chloride, but not the mineralocorticoid receptor antagonists spironolactone and eplerenone, blocked this effect. Aldosterone also relaxed trabeculae that were prestimulated with angiotensin II (Ang II), and its negative inotropic effects were mimicked by hydrocortisone (at 10-fold lower potency) but not 17beta-estradiol. Aldosterone concentrations required to reduce inotropy were present in failing but not in normal human hearts. Previous exposure of coronary arteries to 1 micromol/L aldosterone or 17beta-estradiol (but not hydrocortisone) doubled the maximum contractile response (Emax) to Ang II. DeltaEmax correlated with extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (P<0.01). Spironolactone and eplerenone did not block the potentiating effect of aldosterone. Studies in porcine renal arteries showed that potentiation also occurred at pmol/L aldosterone levels but not at 17beta-estradiol levels <1 micromol/L. Aldosterone did not potentiate the alpha1-adrenoceptor agonist phenylephrine. In conclusion, aldosterone induces a negative inotropic response in human trabeculae (thereby antagonizing the positive inotropic actions of Ang II) and potentiates the vasoconstrictor effect of Ang II in coronary arteries. These effects are specific and involve PKC and ERK 1/2, respectively. Furthermore, they occur in a nongenomic manner, and require pathological aldosterone concentrations.

Modulation of the contractile responses of guinea pig isolated tracheal rings after chronic intermittent hypobaric hypoxia with and without cold exposure

Previous studies have documented that repetitive exposure to intermittent hypoxia, such as that encountered in preparation to high-altitude ascent, influences breathing. However, the impact of intermittent hypoxia on airway smooth muscle has not been explored. Ascents to high altitude, in addition to hypoxia, expose individuals to cold air. The objective of the present study is to examine the effect of chronic intermittent hypobaric hypoxia (CIH) and CIH combined with cold exposure (CIHC) on tracheal smooth muscle responses to various contractile and relaxant agonists. Experiments were performed on tracheal rings harvested from adult guinea pigs exposed either to CIH or CIHC [14 days (6 h/day) at barometric pressure of 350 mmHg with and without cold exposure of 5 degrees C] or to room air (normoxia). CIH and CIHC attenuated maximum contractile responses to ACh compared with normoxia. The maximum contractile response to histamine decreased with CIH, whereas CIHC restored the response back to normoxia. Both CIH and CIHC attenuated maximum contractile responses to 5-HT. Altered contractile responses after CIH and CIHC were independent of epithelium. Isoproterenol-induced relaxation was not altered by CIH, whereas it was enhanced after CIHC, and these responses were independent of the epithelium. The data demonstrate that intermittent exposure to hypoxia profoundly influences contractile response of tracheal smooth muscle, and cold exposure can further modulate the response, implying the importance of cold at high altitude.

Nitric oxide modulates angiotensin II-induced endothelial vasoconstrictor prostanoid release

This study investigated the modulation of angiotensin II-induced endothelial prostanoid release in rabbit aortic rings. Two cumulative dose response curves with 90-min washing interval were performed. Incubation with l-NG-nitroarginine methyl ester (l-NAME) 10−4 M increased angiotensin II maximal contractile response (Emax). This effect was reversed by indomethacin 10−5 M, diphenyliodinum 10−5 M, Tempol 10−5 M or ascorbic acid 10−4 M in both cumulative dose response curves and by SQ 29548 10−6 M in the second cumulative dose response curve. When segments were treated with tetraethylamonium 10−3 M but not with glibenclamide 10−5 M during the washing period, l-NAME recovered its ability to enhance the Emax in arteries incubated with SQ 29548. Conclusions: nitric oxide modulates angiotensin II-induced endothelial release of cyclooxygenase-dependent eicosanoids, one of which acts through thromboxane A2/prostaglandin H2 receptors and would decrease KCa channel activity. An increase in free radical production may account for the enhancement of such prostanoid release. Furthermore, it was found that in the present conditions, the release of the hyperpolarizing factor would improve in order to maintain the vascular tone.

Altered muscarinic receptor subtype expression and functional responses in cyclophosphamide induced cystitis in rats

In the in vitro study, it was investigated whether the expression of muscarinic receptors and cholinergic responses were altered in the situation of experimental cystitis. Rats were treated with cyclophosphamide intraperitoneally and the bladders were excised 36–100 h later. Immunohistochemistry and immunoblotting showed all subtypes of the muscarinic receptor (M1–M5) to be present in the specimens from inflamed urinary bladders and controls. In the cyclophosphamide-treated rats, the expression of muscarinic M5 receptors was increased by more than 40 times ( p < 0.01; n = 8) both in the smooth muscle and the urothelium. Both the maximal contractile response to carbachol and to a high potassium concentration was approximately halved in cyclophosphamide-treated tissues, whereas the reduction was substantially greater in response to low carbachol concentrations (< EC 50). The administration of 4-DAMP inhibited the carbachol-induced contractile responses of inflamed strips less potently than of controls, whereas pirenzepine and methoctramine showed equipotency in the two groups. The nitric oxide synthase inhibitor l-NNA increased the contractile effect of carbachol in inflamed detrusor strips, while it had no effect in controls. Immunoblotting showed endothelial nitric oxide synthase (eNOS) to be up-regulated in cystitis, and immunohistochemistry revealed the change to occur in the urothelium and in the suburothelial layer. The alteration of cholinergic detrusor responses in cyclophosphamide-treated rats depends mainly on a general detriment of contractility but also on indirect effects possibly via nitric oxide synthesis. The most prominent histological alterations occurred in the urothelium in which muscarinic M5 receptors increased in particular. The study further underlines that the urothelium may play significant roles in the pathogenesis of urinary bladder disorders such as interstitial cystitis.

Fentanyl Attenuates α1B-Adrenoceptor–Mediated Pulmonary Artery Contraction

The authors tested the hypothesis that the intravenous anesthetic fentanyl would attenuate the pulmonary vasoconstrictor response to alpha1-adrenoceptor activation. They also investigated the alpha1-adrenoceptor subtypes that could potentially mediate this effect of fentanyl. Endothelium-denuded canine pulmonary arterial rings were suspended for isometric tension recording. Dose-response curves for the alpha1-adrenoceptor agonist phenylephrine were generated in the absence and presence of fentanyl. The effects of inhibiting alpha2 (rauwolscine), alpha1 (prazosin), alpha1A (5-methylurapidil), alpha1B (chloroethylclonidine), and alpha1D (BMY 7378) adrenoceptors on phenylephrine contraction were also investigated. Receptor "protection" studies were performed to investigate the specific role of alpha1B adrenoceptors in mediating fentanyl-induced changes in phenylephrine contraction. Finally, competition binding studies were performed in rat-1 fibroblasts stably transfected with human alpha1-adrenoceptor complementary DNAs corresponding to the alpha1A-, alpha1B-, or alpha1D-adrenoceptor subtypes to directly assess whether fentanyl can compete for the alpha1-adrenoceptor activation pocket. Fentanyl attenuated phenylephrine contraction in a dose-dependent fashion. Rauwolscine had no effect on phenylephrine contraction. Phenylephrine contraction was inhibited by prazosin and abolished by chloroethylclonidine but was relatively resistant to inhibition by 5-methylurapidil and BMY 7378. Pretreatment with fentanyl before exposure to chloroethylclonidine increased the maximal contractile response to phenylephrine compared to chloroethylclonidine pretreatment alone. Competition binding studies revealed that fentanyl binds to all three alpha1-adrenoceptor subtypes, with a fivefold greater affinity for the alpha1B-adrenoceptor compared with the alpha1D-adrenoceptor subtype. Phenylephrine-induced contraction is primarily mediated by alpha1B-adrenoceptor activation in canine pulmonary artery. Fentanyl attenuates phenylephrine contraction by binding to alpha1B adrenoceptors.

Effect of LLLT Ga–Al–As (685 nm) on LPS-induced inflammation of the airway and lung in the rat

The purpose of this study was to investigate the effect of low level laser therapy (LLLT) on male Wistar rat trachea hyperreactivity (RTHR), bronchoalveolar lavage (BAL) and lung neutrophils influx after Gram-negative bacterial lipopolyssacharide (LPS) intravenous injection. The RTHR, BAL and lung neutrophils influx were measured over different intervals of time (90 min, 6 h, 24 h and 48 h). The energy density (ED) that produced an anti-inflammatory effect was 2.5 J/cm(2), reducing the maximal contractile response and the sensibility of trachea rings to methacholine after LPS. The same ED produced an anti-inflammatory effect on BAL and lung neutrophils influx. The Celecoxib COX-2 inhibitor reduced RTHR and the number of cells in BAL and lung neutrophils influx of rats treated with LPS. Celecoxib and LLLT reduced the PGE(2) and TXA(2) levels in the BAL of LPS-treated rats. Our results demonstrate that LLLT produced anti-inflammatory effects on RTHR, BAL and lung neutrophils influx in association with inhibition of COX-2-derived metabolites.

Estrogen potentiates vasopressin-induced contraction of female rat aorta by enhancing cyclooxygenase-2 and thromboxane function

To determine the roles of estrogen and constrictor prostanoids in vasopressin (VP)-induced contraction of female rat aorta, vascular reactivity to VP was determined in thoracic aortas of intact, ovariectomized, and ovariectomized + estrogen-replaced female rats in the presence of indomethacin (Indo), NS-398, SQ-29,548, or vehicle control. The effects of estrogen on vascular reactivity to the thromboxane A(2) analog U-46619 were also examined. Maximal contractile response to VP in intact female rats (5,567 +/- 276 mg/mg of aortic ring wt) was markedly attenuated by ovariectomy (2,485 +/- 394 mg; P < 0.001) and restored by estrogen replacement with 17beta-estradiol (5,059 +/- 194 mg; P > 0.1). Indo and NS-398 significantly attenuated maximal responses to VP in intact female rats to a similar extent [3,176 +/- 179 (P < 0.0001) and 3,258 +/- 152 mg (P < 0.0001), respectively]. Ovariectomy abolished and estrogen replacement restored the inhibitory effects of Indo, NS-398, and SQ-29,548. Contractile responses of rat aorta to U-46619 were significantly greater (P < 0.0001) in females (5,040 +/- 238 mg) than in males (3,679 +/- 96 mg). Ovariectomy markedly attenuated (3,923 +/- 84 mg; P < 0.01) and estrogen replacement restored (5,024 +/- 155 mg; P > 0.1) responses to U-46619 in female aortas. These data reveal that estrogen is an important regulator of the contractile responses of female rat aorta to VP, which appears to potentiate both cyclooxygenase-2 and constrictor prostanoid function in the vascular wall.

Effect of Glucagon-Like Peptide-1(7–36) and Exendin-4 on the Vascular Reactivity in Streptozotocin/Nicotinamide-Induced Diabetic Rats

We investigated the vascular effects of glucagon-like peptide-1 (GLP-1) and Exendin-4 in type 2 diabetic rat aortae. Studies were performed in a normal control group (NC) (0.2 ml i.p. saline, n = 10), streptozotocin (STZ)/nicotinamide diabetic control group (DC) (a single dose of 80 mg/kg STZ i.p. injection 15 min after administration of 230 mg/kg nicotinamide i.p.), GLP-1 (GLPC) control group (1 µg/kg twice daily i.p. for 1 month, n = 10), Exendin-4 control group (EXC) (0.1 µg/kg twice daily i.p. for 1 month, n = 10), GLP-1-treated diabetic group (GLPT) (1 µg/kg twice daily i.p. for 1 month, n = 10), and Exendin-4-treated diabetic group (EXT) (0.1 µg/kg twice daily i.p. for 1 month, n = 10). One month of GLP-1 and Exendin-4 treatment significantly decreased the blood glucose levels of diabetic rats (113 ± 2 mg/dl, p < 0.001, and 117 ± 1 mg/dl, p < 0.001, respectively versus 181 ± 9 mg/dl in the DC group). Sensitivity (pD₂) and maximum response (% Max. Relax) of acetylcholine-stimulated relaxations in the DC group (pD₂: 6.73 ± 0.12 and 55 ± 6, respectively) were decreased compared with the non-diabetic NC group (pD₂: 7.41 ± 0.25, p < 0.05, and 87 ± 4, p < 0.01). Treating diabetic rats with GLP-1, pD₂ values and with Exendin-4, Max. Relax %values of aortic strips to acetylcholine returned to near non-diabetic NC values (pD₂: 7.47 ± 0.15, p < 0.05, and 87 ± 3, p < 0.01, respectively). Maximal contractile responses (E_max) to noradrenaline in aortic strips from the diabetic DC group (341 ± 27 mg tension/mg wet weight) were significantly decreased compared with the non-diabetic NC (540 ± 66 mg tension/mg wet weight, p < 0.001) and the GLPT group (490 ± 25 mg tension/mg wet weight, p < 0.05). There were no significant differences in pD₂ values of aortic strips to noradrenaline from all groups. E_max to KCl in aortic strips from the DC group (247 ± 10 mg tension/mg wet weight, p < 0.01) was significantly decreased compared with non-diabetic NC group (327 ± 26 mg tension/mg wet weight). Treating diabetic rats with GLP-1 (GLPT), E_maxvalues of aortic strips to KCl returned to near non-diabetic NC values (271 ± 12 mg tension/mg wet weight). GLP-1 and (partially) Exendin-4 treatment could improve the increased blood glucose level and normalize the altered vascular tone in type 2 diabetic rats.

Adrenergic response patterns in vas deferens isolated from rats under diurnal rhythms: Influence of stress

The aim of this study was to verify, by means of functional methods, whether the circadian rhythm changes adrenergic response patterns in the epididymal half of the vas deferens isolated from control rats as well as from rats submitted to acute stress. The experiments were performed at 9:00 a.m., 3:00 p.m., 9:00 p.m., and 3:00 a.m. The results showed a light–dark dependent variation of the adrenergic response pattern on organs isolated from control as well as from stressed rats. In the control group, only the phenylephrine sensitivity was changed throughout the circadian rhythm. Under the stress condition, both norepinephrine and phenylephrine response patterns were changed, mainly during darkness. The maximal contractile response to both α- and β-agonist and α 1-agonist was increased in the dark phase, corresponding to high plasmatic concentrations of endogenous melatonin. The vas deferens isolated from stressed rats during the light phase simultaneously incubated with exogenous melatonin showed the same pattern of response obtained in the dark phase, thus indicating a peripheric action of melatonin on this organ. Therefore, the circadian rhythms are important to the adrenergic response pattern in rat vas deferens from both control and stressed rats. In conclusion, we suggest a melatonin modulation on α 1-postsynaptic adrenergic response in the rat vas deferens.

TGF-β potentiates airway smooth muscle responsiveness to bradykinin

The molecular mechanisms by which bradykinin induces excessive airway obstruction in asthmatics remain unknown. Transforming growth factor (TGF)-beta has been involved in regulating airway inflammation and remodeling in asthma, although it is unknown whether TGF-beta can modulate bradykinin-associated bronchial hyperresponsiveness. To test whether TGF-beta directly modulates airway smooth muscle (ASM) responsiveness to bradykinin, isolated murine tracheal rings were used to assess whether TGF-beta alters ASM contractile responsiveness to bradykinin. Interestingly, we found TGF-beta-treated murine rings (12.5 ng/ml, 18 h) exhibited increased expression of bradykinin 2 (B(2)) receptors and became hyperreactive to bradykinin, as shown by increases in maximal contractile responses and receptor distribution. We investigated the effect of TGF-beta on bradykinin-evoked calcium signals since calcium is a key molecule regulating ASM excitation-contraction coupling. We reported that TGF-beta, in a dose- (0.5-10 ng/ml) and time- (2-24 h) dependent manner, increased mRNA and protein expression of the B(2) receptor in cultured human ASM cells. Maximal B(2) receptor protein expression that colocalized with CD44, a marker of membrane cell surface, occurred after 18 h of TGF-beta treatment and was further confirmed using fluorescence microscopy. TGF-beta (2.5 ng/ml, 18 h) also increased bradykinin-induced intracellular calcium mobilization in fura-2-loaded ASM cells. TGF-beta-mediated enhancement of calcium mobilization was not attenuated with indomethacin, a cyclooxygenase inhibitor. These data demonstrate for the first time that TGF-beta may play a role in mediating airway hyperresponsiveness to bradykinin seen in asthmatics by enhancing ASM contractile responsiveness to bradykinin, possibly as a result of increased B(2) receptor expression and signaling.