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PubMedDate not supplied

Comparative studies on rabbit corpus cavernosal contraction and relaxation. An in vitro study.

Erectile function (erection and detumescence) involves the complex interaction of direct neuronal stimulation of corporal smooth muscle, neurohumoral release of specific endothelial contractile and relaxant factors, and secondary modulation by a variety of putative neuropeptides and vasoactive modulators including nitric oxide. The specific aim of the current study was to determine the relative contribution of nitric oxide, adrenergic, purinergic, and cholinergic stimulation in the relaxant response to field stimulation. The results demonstrate that virtually all of the inhibitory effects of field-stimulated relaxation could be explained by the release of nitric oxide. L-NAME (L-NG-nitro arginine methyl ester, a competitive inhibitor of NO synthase) reduced field-stimulated relaxation by over 95% at all frequencies. Neither atropine nor propranolol (or the combination of the two) had any significant effect on field-stimulated relaxation. L-NAME blocked both field-stimulated relaxation and bethanechol-stimulated relaxation. However, methylene blue (a guanyl cyclase inhibitor) was significantly more potent at blocking bethanechol-stimulated relaxation than field-stimulated relaxation. Neither L-NAME nor methylene blue had any effect on nitroprusside (a direct liberator of NO) nor ATP-stimulated relaxation. Isoproterenol had only a minor inhibitory effect on phenylephrine-contracted tissue. These data suggest that 1) Methylene blue, which inhibits guanyl cyclase, is a relatively poor inhibitor of field-stimulated relaxation. 2) L-NAME is a potent inhibitor of NO synthesis and can in a dose-dependent fashion inhibit over 95% of field-stimulated relaxation. 3) Equipotent relaxation of corporal smooth muscle can be effected through pharmacologic stimulation with ATP (2 mM), nitroprusside (200 microM), and field stimulation (32 Hz).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed2003

Does cross-bridge activation determine the time course of myofibrillar relaxation?

The ability of force-generating cross-bridges to activate the thin filament in cardiac muscle was tested by studying the effects of initial force and [MgADP] on force relaxation kinetics in subcellular myofibrillar bundles prepared from left ventricles of the guinea pig. Relaxation was initiated by rapidly reducing the [Ca(2+)] from pCa 4.5 to 7.5. Initiating relaxation from lower force levels during pre-steady-state force development did not significantly accelerate the kinetics of the force decay compared to relaxations initiated from steady-state force development. This suggests that the force-generating cross-bridges which become formed during maximally Ca(2+)-activated steady-state contractions do not maintain thin filament activation for significant enough times after Ca(2+)-removal to exert a rate-limiting influence on force relaxation kinetics. Adding 2 mM MgADP to solutions slowed down relaxation kinetics approximately 4-fold. To differentiate whether these slower kinetics result from either (1) MgADP favoring accumulation of cross-bridges during the preceding contraction in a state of activating capability or (2) slow-down of cross-bridge turnover by the presence of the product MgADP during relaxation, the [MgADP] was either increased or removed at the time of Ca(2+)-removal. The addition of 2 mM MgADP to activating solutions (subsequent relaxation in the absence of MgADP) slowed-down the kinetics of the initial, slow, linear force decay following Ca(2+)-removal approximately 1.5-fold, suggesting that the high [MgADP] during contraction favors formation of cross-bridges which contribute in rate-limiting early relaxation kinetics by transiently sustaining thin filament activation. On the other hand, the addition of 2 mM MgADP to the relaxing solution (preceding Ca(2+)-activation in absence of MgADP) slowed-down the kinetics of the initial force decay approximately 3-fold, more similar to the kinetics observed in the continuous presence of 2 mM MgADP both before and after Ca(2+)-removal. This suggest that, despite some influence of cross-bridge activation, the main effect of MgADP on relaxation kinetics results from product inhibition of cross-bridge turnover. In summary, whereas under certain conditions (high [MgADP]) cross-bridge activation of the thin filament can weakly take part in rate-limiting relaxation kinetics induced by complete Ca(2+)-removal, cross-bridge activation does not influence relaxation kinetics under more physiologically normal conditions.

Actin Cytoskeleton↗
PubMed1993

Endothelium-dependent relaxation of porcine pulmonary arteries via 5-HT1C-like receptors.

In PGF2 alpha-precontracted pulmonary arteries with intact endothelium, 5-hydroxytryptamine (5-HT, 1.0-100 nmol/l) caused a concentration-dependent reversible relaxation, at higher concentrations the contractile response prevailed. In endothelium-denuded vessels relaxation was absent. 5-HT-induced relaxation of precontracted pulmonary arteries was probably mediated by release of an endothelium-derived relaxing factor (EDRF). Preincubation of the arteries with methylene blue or NG-nitro-L-arginine (200 mumol/l) attenuated the relaxant effect. The 5-HT-induced relaxation was accompanied by an increase in cGMP. Indomethacin (3 mumol/l) did not influence the 5-HT-induced relaxation indicating that eicosanoids are not involved in the relaxant response to 5-HT. The 5-HT1C and 5-HT2 receptor agonist alpha-methyl-5-HT was as potent as 5-HT in inducing relaxation. The rank order of relaxant potency of the agonists investigated was alpha-methyl-5-HT > 5-HT > 5-methoxytryptamine > tryptamine > omega-methyl-5-HT > 5-carboxamidotryptamine > 2-methyl-5-HT > 5,6-dihydroxytryptamine > m-chlorophenylpiperazine > sumatriptan > 8-OH-DPAT. Phentolamine, pindolol and ICS 205-930 did not interfere with the relaxant effect. The 5-HT2 receptor antagonist ketanserin (1 mumol/l) inhibited the contractile response but did not alter vasodilatation. Apart from the blockade of the contractile effects, mesulergine, cyproheptadine and mianserin (0.1-3.0 mumol/l, each) induced a parallel shift to the right of the concentration-response curve for the relaxation induced by alpha-methyl-5-HT or 5-HT. Spiperone (0.3 mumol/l) exerted weak inhibitory effects on relaxation and contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

The effect of applied mechanical vibration on two different phases of rat papillary muscle relaxation.

Applying external mechanical vibration during the relaxation phase of rat papillary muscle decreases the duration of the first part of the relaxation phase. To elucidate the basic mechanism responsible for this shortening of the relaxation period, we applied a controlled vibration to isolated twitching rat papillary muscles during various phases in the relaxation of a twitch. The first part of the relaxation phase was accelerated when length perturbations were applied in the first part of the relaxation of a twitch, dependent on both amplitude and frequency of the perturbation. When vibrations were applied in the first half of the relaxation, the second phase of relaxation was slightly slower (about 20%), but when no vibrations were applied in the first phase, relaxation could be accelerated by applying vibration in the latter half of the relaxation phase. Thus, in the latter half of relaxation, the acceleration of relaxation depended upon perturbation events earlier during that twitch. This study indicates that vibration-induced acceleration of relaxation is due (at least in part) to an apparent increase in detachment rate of attached cross-bridges from the thin filament without substantial reattachment.

Animals↗
PubMed2006

Coupling between chemical reactivity and structural relaxation in pharmaceutical glasses.

PURPOSE: To test the hypothesis that the molecular motions associated with chemical degradation in glassy amorphous systems are governed by the molecular motions associated with structural relaxation. The extent to which a chemical process is linked to the motions associated with structural relaxation will depend on the nature of the chemical process and molecular motion requirements (e.g., translation of a complete molecule, rotational diffusion of a chemical functional group). In this study the chemical degradation and molecular mobility were measured in model systems to assess the degree of coupling between chemical reactivity and structural relaxation. The model systems included pure amorphous cephalosporin drugs, and amorphous molecular mixtures containing a chemically labile drug and an additive expected to moderate molecular mobility. METHODS: Amorphous drugs and mixtures with additives were prepared by lyophilization from aqueous solution. The physical properties of the model systems were characterized using optical microscopy and differential scanning calorimetry. The chemical degradation of the drugs alone and in mixtures with additives was measured using high-performance liquid chromatography (HPLC). Molecular mobility was measured using isothermal microcalorimetry to measure enthalpy changes associated with structural relaxation below T (g). RESULTS: A weak correlation between the rates of degradation and structural relaxation times in pure amorphous cephalosporins suggests that reactivity in these systems is coupled to molecular motions in the glassy state. However, when sucrose was added to one of the cephalosporin drugs stability improved even though this addition reduced T (g) and the relaxation time constant, tau(D)(beta), suggesting that there was no correlation between reactivity and structural relaxation in the cephalosporin mixtures. In contrast, the rate of ethacrynate sodium dimer formation in mixtures was more strongly coupled to the relaxation time constant, tau(D)(beta). CONCLUSIONS: These studies suggest that the extent to which chemical degradation is coupled to structural relaxation in glasses motions is determined by how closely the motions of the rate controlling step in chemical degradation are associated with structural relaxation. Moderate coupling between the rate of dimer formation for ethacrynate sodium in mixtures with sucrose, trehalose and PVP and structural relaxation constants suggests that chemical changes that require more significant molecular motion, and includes at least some translational diffusion, are more strongly coupled to the molecular motions associated with structural relaxation. The observation that sucrose stabilizes cefoxitin sodium even though it lowers T (g) and reduces the relaxation time constant, tau(D)(beta) is perhaps a result of the importance of other kinds of molecular motions in determining the chemical reactivity in glasses.

Calorimetry, Differential Scanning↗
PubMedDate not supplied

Mechanical loads and the isovolumic and filling indices of left ventricular relaxation.

In this report we have emphasized the significant influence of loading conditions on LV relaxation. Changes in inotropic state, ischemia, hypertrophy, and a host of other factors were not discussed herein. Ventricular asynchrony is likewise beyond the scope of this review, but it should be recognized that complex loading interactions among fibers within the LV wall influence relaxation and filling in ventricles that contract and relax nonsynchronously. Intact heart and isolated muscle experiments indicate that changes in preload do not influence relaxation rates when systolic pressure or total load remains constant. It appears, therefore, that the relaxation changes that occur with volume loading are primarily due to changes in systolic pressure or load. Indeed, an increase in systolic pressure may cause a substantial decrease in isovolumic relaxation rate, especially if there is a slow rise and a late peak in LV pressure. By contrast, an abrupt increase in late systolic load augments relaxation rate. Intact heart studies indicate that an abrupt load increment near the end of ejection results in premature and more rapid isovolumic relaxation, while an increase in left atrial pressure increases the filling rate; isolated muscle studies indicate that a load increment at the end of isometric relaxation causes an increase in isotonic relaxation rate. Thus, loading conditions during the periods of ejection, isovolumic relaxation, and filling can influence relaxation parameters and should be considered in clinical and experimental studies of LV relaxation.

Animals↗
PubMed2004

Proton T1 and T2 relaxivities of serum proteins.

In the present study, T(1) and T(2) of phantoms containing serum sets with varying amounts of proteins, serum samples with certain amounts of proteins, serum diluted by distilled water, and serum treated with iron were measured. In addition, T(1) and T(2) of phantoms containing normal serum, diluted serum, and albumin-doped serum were also measured. Relaxation rates were plotted versus protein concentrations. The slope of relation was taken as relaxivity. The T(1) relaxivities of proteins were ranged from 0.035 to 0.080 s(-1)(g/dl)(-1), whereas T(2) relaxivities were ranged from 0.24 to 0.68 s(-1)(g/dl)(-1). The T(1) and T(2) relaxivities of transferrin iron were 2.40 and 2.60 mM(-1)s(-1), respectively. The contributions of diamagnetic proteins and transferrin iron to the relaxation rate of serum were also calculated for each of diluted serum, normal and albumin-doped serum. The contributions and the average TP relaxivities(calculated by using individual relaxivities and the ratios of protein fractions in TP) were used for TP calculations. The agreement between the calculated TP and TP by autoanalyzer and also the agreement between average TP relaxivities and the TP relaxivities determined from dilution experiments show that the data of relaxivities are reliable. The results suggest that individual protein relaxivities explain the influence of serum TP composition on T(1) and T(2) relaxation times.

Blood Proteins↗
PubMed1985

31P NMR relaxation studies of the activation of the coenzyme phosphate of glycogen phosphorylase. The role of motion of the bound phosphate.

Spin-lattice and spin-spin relaxation rates (1/T1 and 1/T2) have been determined for the catalytically essential coenzyme phosphate at the active site of glycogen phosphorylase in both activated (R state) and inactive (T state) conformations of the enzyme. Dipolar contributions to 31P relaxation due to exchangeable protons on the phosphate group have been determined by measurement of relaxation rates at different concentrations of H2O and D2O, and field dependence studies have been performed to estimate the contribution of chemical shift anisotropy to the remaining 31P relaxation in D2O. At 109 MHz, dipolar relaxation from exchangeable protons was found to account for 50% of the spin-lattice relaxation for activated phosphorylase in 75% H2O, the remainder being due to chemical shift anisotropy. The spin-lattice relaxation rates in D2O for R-state glycogen phosphorylase are very similar to those measured for other proteins of very different size such as actin (Brauer, M., and B. D. Sykes, 1981, Biochemistry. 20:6767-6775), alkaline phosphatase (Coleman, J. E., I. D. Armitage, J. F. Chlebowski, J. D. Otvos, and A. J. M. S. Uiterkamp, 1979), and phosphoglucomutase (Rhyu, G. I., W. J. Ray, Jr., and J. L. Markley, 1984, Biochemistry. 23:252-260). In inactive (T state) phosphorylase the spin-lattice relaxation rates were almost an order of magnitude slower, while the spin-spin relaxation rates were essentially identical. These results have been analyzed by calculating the theoretically expected 31P relaxation rates in the presence of internal motions that are included in the relaxation calculation using the model-free approach of Lipari and Szabo (1982, J. Am. Chem. Soc. 104:4564-4559). The analysis suggests the coenzyme phosphate is relatively immobilized in the activated enzymic conformation, but in the inactive (Tstate) conformation it is considerably more mobile with a rotational correlation time one to two orders of magnitude smaller. Since the spin-lattice relaxation rate for the active R-state (immobilized) phosphate is similar to that observed in other phosphoenzymes of different size it is suggested that a librational motion on the nanosecond time scale may constitute a common spin-lattice relaxation pathway for phosphates in macromolecules. The consequences of phosphate motion in terms of recent suggestions concerning the environment and the catalytic role of the coenzyme phosphate are discussed.

Allosteric Regulation↗
PubMed2000

Mechanisms of nitric oxide-independent relaxations induced by carbachol and acetylcholine in rat isolated renal arteries.

1. In rat isolated renal artery segments contracted with 0.1 microM phenylephrine and in the presence of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME), carbachol and acetylcholine produced endothelium-dependent relaxations. The mechanisms underlying these relaxations were studied. 2. These relaxations were not affected by ODQ (1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one) or indomethacin. In arteries contracted with 20 - 30 mM K(+), L-NAME-resistant relaxations induced by carbachol and acetylcholine were virtually absent. 3. The Na(+)-K(+) ATPase inhibitor ouabain reduced these relaxations in a concentration-dependent manner. 4. In K(+)-free media, addition of K(+) (5 mM) produced 90. 5+/-3.9% (n=3) relaxation of phenylephrine-induced tone. This relaxation was endothelium-independent and ouabain-sensitive. 5. Tetraethylammonium (TEA), charybdotoxin (ChTX) and iberiotoxin (IbTX) reduced the sensitivity of carbachol-induced relaxations, but did not change the maximal response. These relaxations were not altered by 4-aminopyridine (4-AP), glibenclamide or apamin. Acetylcholine (1 microM)-induced relaxation was reduced by ChTX, but not by TEA or IbTX. 6. The cytochrome P450 inhibitor miconazole, but not 17-octadecynoic acid, reduced the sensitivity of carbachol-induced relaxations, without changing the maximal response. 7. In conclusion, in rat isolated renal arteries, acetylcholine and carbachol produced a non-NO/non-PGI(2) relaxation which is mediated by an endothelium-derived hyperpolarizing factor (EDHF). This factor does not appear to be a cytochrome P450 metabolite. The inhibition by ouabain of these relaxations suggests the possible involvement of Na(+)-K(+) ATPase activation in EDHF responses, although other mechanisms cannot be totally ruled out.

Acetylcholine↗
PubMed1998

Pharmacological characterization of kinin-induced relaxation of human corpus cavernosum.

OBJECTIVE: To characterize the kinin receptor subtype involved in the relaxation of human isolated corpus cavernosum (HCC) induced by bradykinin (BK), Lys-bradykinin (Lys-BK), Met-Lys-bradykinin (Met-Lys-BK) and des-Arg9-bradykinin, and to investigate whether the kinin-induced relaxation of HCC results from the stimulation of nonadrenergic, noncholinergic (NANC) neurons supplying the cavernosal tissue. MATERIALS AND METHODS: Excised HCC tissues were immediately placed in Krebs solution and kept at 4 degrees C until use (never > 24 h after removal). HCC was cut in strips of approximately 2 cm, suspended in a cascade system and superfused with oxygenated and warmed Krebs solution at 5 mL/min. After equilibration for approximately 90 min, noradrenaline (3 micromol/L) was infused to induce a submaximal contraction of the HCC strips. The release of cyclo-oxygenase products was prevented by infusing indomethacin (6 micromol/L). HCC strips were calibrated by injecting a single bolus of the nitrovasodilator glyceryl trinitrate (GTN) and the sensitivity of the tissues adjusted electronically to be similar. The agonists (kinins, histamine and acetylcholine) were injected as a single bolus (up to 100 microL) and the relaxation of HCC expressed as a percentage of the submaximal relaxation induced by GTN. RESULTS: Bradykinin, Lys-BK and Met-Lys-BK significantly relaxed the HCC tissues; on a molar basis, there was no statistical difference among the degrees of relaxation induced by these peptides. The B1 kinin receptor agonist des-Arg9-bradykinin had no effect on the HCC. The infusion of the B2 kinin receptor antagonist Hoe 140 (50 nmol/L) virtually abolished the relaxation induced by BK, Lys-BK and Met-Lys-BK without affecting those induced by acetylcholine and histamine. The infusion of the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester increased the tone of the HCC tissues and significantly reduced (P < 0.01) the relaxation induced by BK (74%), Lys-BK (90%), Met-Lys-BK (87%) and acetylcholine (89%) without affecting those induced by GTN. The subsequent infusion of L-arginine (300 micromol/L) partially reversed the increased tone and significantly (P < 0.01) restored the relaxation induced by BK, Lys-BK and Met-Lys-BK. The results were similar with the novel guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3,-alquinoxalin-1-one] which reduced by > 95% (P < 0.01) the relaxation induced by BK, Lys-BK, Met-Lys-BK, acetylcholine and GTN. The infusion of the sodium-channel blocker tetrodotoxin had no significant effect on the BK-, GTN- and acetylcholine-induced relaxation of HCC. CONCLUSION: This study clearly showed the existence of functional B2 kinin receptors in human erectile tissues that when activated lead to the release of NO and hence relaxation of the HCC tissues. As tetrodotoxin failed to affect the kinin-induced relaxation of HCC strips, it is likely that these peptides release NO from the endothelium of sinusoidal capillaries rather than from neuronal sources supplying the cavernosal tissue. Although tissue kallikreins and their components have been found in the male reproductive system, the physiopathological importance of these findings has yet to be elucidated.

Adolescent↗
PubMed1999

Impaired endothelium-dependent relaxation in mesenteric arteries of reduced renal mass hypertensive rats.

Endothelium-dependent relaxation is not fully understood in volume-dependent models of hypertension. This study investigated the relaxation mediated by endothelium-derived nitric oxide (EDNO) and hyperpolarizing factor (EDHF) in superior mesenteric arteries from reduced renal mass hypertensive rats, an experimental model for volume-dependent hypertension. Hypertension was induced in male Wistar rats by subtotal nephrectomy and salt-loading (hypertensive group). The control group comprised rats that drank tap water after subtotal nephrectomy. Relaxation of isolated superior mesenteric arterial rings was investigated at the end of the 2-week study. In high K+-precontracted arterial rings, relaxation caused by acetylcholine (ACh) was markedly reduced in the hypertensive group compared with the findings for the control group (34+/-4% vs. 54+/-5% decrease in tension). In both groups, the relaxation was abolished by N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase. In phenylephrine-precontracted arterial rings, relaxation caused by ACh was also small in the hypertensive group, while it was large in the control group (49 +/- 5% vs. 96 +/- 2%). Superfusion of L-NAME inhibited most of the relaxation caused by ACh, but the arteries still exhibited relaxation. Apamin, a blocker of Ca-dependent K+ channel, together with L-NAME further inhibited the residual relaxation. The relaxation inhibited by apamin was also reduced in the hypertensive group. We conclude that the relaxation inhibited by L-NAME was mediated by EDNO, while that inhibited by apamin was mediated by EDHF. Endothelium-independent relaxation caused by nitroprusside and diazoxide was normal in the hypertensive group. The relaxation mediated by both EDNO and EDHF was depressed in the arteries of reduced renal mass hypertensive rats as the result of an arterial endothelial abnormality.

Acetylcholine↗
PubMed2001

Relevance of endothelium-derived hyperpolarizing factor in the effects of hypertension on rat coronary relaxations.

OBJECTIVES: To evaluate the relative participation of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) and prostanoids in the relaxing responses induced by acetylcholine and isoproterenol in isolated coronary arteries from adult Wistar- Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). DESIGN AND METHODS: Male adult WKY rats and SHR were used in the study. Segments from left coronary arteries (approximately 350-380 microm internal diameter and 2 mm long) were mounted in an isometric myograph and pre-contracted with serotonin. Dose-response curves to acetylcholine and isoproterenol were carried out in absence and presence of the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (LNAME), the inhibitor of the cyclo-oxygenase, indomethacin and KCI. Areas under the respective dose-response curves were used to calculate the approximate relative participation of NO, EDHF and prostanoids. RESULTS: Relaxations to either acetylcholine or isoproterenol were lower in SHR than in WKY rats. In WKY rats, presence of LNAME diminished (P< 0.05) relaxation to acetylcholine from 10(-9) to 10(-6) mol/l, and induced a contracting response at 10(-5) and 10(-4) mol/l of acetylcholine. Addition of indomethacin did not significantly affect dose-related relaxation to acetylcholine 10(-9) to 10(-6) mol/l in WKY rats, and reduced (P < 0.05) the contracting response observed at 10(-5) mol/l of acetylcholine. In SHR, addition of LNAME markedly reduced (P< 0.05) acetylcholine relaxations, but did not produce any contracting effect. Addition of indomethacin on top of LNAME slightly (P< 0.05) enhanced relaxing response to acetylcholine in SHR. Presence of LNAME in the media diminished (P < 0.05) relaxation to isoproterenol in both WKY rats and SHR. Addition of indomethacin on top of LNAME increased (P< 0.05) isoproterenol-relaxing response to levels similar to and higher than control conditions in WKY rats and SHR, respectively. Addition of KCI blunted both acetylcholine- and isoproterenol-relaxations in both groups. CONCLUSIONS: NO and EDHF are the main endothelium-derived relaxing factors underlying acetylcholine and isoproterenol relaxations in rat coronary arteries, respectively. EDHF reduction, and not only NO reduction play a key role in the diminished coronary relaxations induced by acetylcholine and isoproterenol in SHR. An arachidonic acid derivative with contracting activity released by acetylcholine and isoproterenol in a differential manner, could oppose the relaxing actions of NO and EDHF.

Acetylcholine↗
PubMed1989

Co-axial bioassay of a smooth muscle relaxant factor released from guinea-pig tracheal epithelium.

1. The ability of guinea-pig trachea to release an epithelium-derived relaxant factor (EpDRF) was assessed in a co-axial bioassay system. 2. Histamine (100 microM) and methacholine (25 microM) caused endothelium-dependent relaxation of rat isolated aorta, presumably via the release of endothelium-derived relaxant factor (EDRF). In contrast, endothelium-denuded rat aorta did not relax in response to these agents. 3. EDRF release was detected in response to methacholine in a co-axial bioassay system, consisting of intact rabbit aorta tube (EDRF donor) and endothelium-denuded rat aorta strip (assay preparation). These results indicated the transfer of EDRF from a donor to an assay preparation, thereby validating the co-axial bioassay method. 4. Substitution of endothelium-intact rabbit aorta tube by epithelium-intact guinea-pig tracheal tube tissue in co-axial assemblies, still allowed the assay preparation to relax in response to histamine or methacholine. Removal of the intact tracheal tube from the system, or removal of the epithelium from the donor tracheal tube in co-axial preparations, abolished such relaxant responses. These observations are consistent with histamine- or methacholine-induced release of an epithelium-derived relaxant factor (EpDRF) from the trachea. 5. In the co-axial assembly comprising intact guinea-pig trachea and endothelium-denuded rat aorta, histamine and methacholine produced concentration-dependent, EpDRF-induced aortic relaxation. Mean concentrations of histamine and methacholine producing 50% of the maximum relaxation (EC50) were 39.8 microM and 2.7 microM respectively. Histamine-induced relaxation was inhibited in the presence of mepyramine (2 microM) and responses to methacholine were inhibited by atropine (0.1 microM). 6. Methylene blue (50 microM) had no effect on such relaxant responses, indicating that EpDRF does not activate guanylate cyclase. Furthermore, the cyclo-oxygenase inhibitor indomethacin (5 microM), the cyclo-oxygenase/lipoxygenase inhibitor BW 755C (150 microM) and the leukotriene receptor antagonist FPL 55712 (10 microM) each failed significantly to alter EpDRF-mediated relaxation of vascular smooth muscle suggesting that EpDRF is not a prostanoid. Platelet activating factor (Pat) failed to cause relaxation of endothelium-denuded rat aorta, indicating that this mediator was also not EpDRF. 7. EpDRF was also released from human bronchial segments. 8. This study provides direct evidence for the release of an EpDRF from non-diseased airway tissue and further suggests that healthy airway reactivity to spasmogens is modulated by the release of an endogenous protective, spasmolytic substance. The bronchial hyperreactivity of asthma may be partly caused by attenuated production of such an inhibitory signal.

Animals↗
PubMed1995

Impaired endothelium-dependent relaxation in isolated resistance arteries of spontaneously diabetic rats.

1. Previous studies have shown that endothelium-dependent relaxation in the aorta of spontaneously diabetic bio bred rats (BB) is impaired. 2. We have investigated noradrenaline (NA) contractility, endothelium-dependent acetylcholine (ACh) and bradykinin (BK) relaxation, and endothelium-independent sodium nitroprusside (SNP) relaxation in mesenteric resistance arteries of recent onset BB rats and established insulin treated BB rats, compared to their age-matched non diabetic controls. 3. There was no significant difference in the maximum contractile response or sensitivity to noradrenaline in either of the diabetic groups compared to their age-matched controls. 4. Incubation with the nitric oxide synthetase inhibitor NG-nitro-L-arginine (L-NOARG) resulted in a significant increase in maximum contractile response to noradrenaline in the recent onset age-matched control group (P < 0.05). Analysis of the whole dose-response curve (using ANOVA for repeated measures with paired t test) showed a significant left-ward shift following the addition of L-NOARG (P < 0.001). A similar but less marked shift (P < 0.01) was evident in vessels from recent onset diabetics. An overall shift in both sensitivity and maximum response was also evident in the age-matched non diabetic controls of the insulin-treated group (P < 0.05). However, by contrast, there was no significant change in sensitivity in the insulin-treated diabetic rats. 5. ACh-induced endothelium-dependent relaxation was significantly impaired in the recent onset diabetic rats compared to their age-matched controls (47 +/- 11% versus 92 +/- 2%, P < 0.05, n = 6), and in the insulin treated diabetic rats (34 +/- 5% versus 75 +/- 6%, P < 0.05, n = 6). The relaxation responses to BK also were significantly impaired in the diabetic rats compared to their age-matched controls (recent onset: 20 +/- 3% versus 72 +/- 7%, P < 0.05, n = 6; insulin treated: 12 +/- 9% versus 68 +/- 7%, P < 0.05, n = 7). 6. Incubation with either the nitric oxide synthetase substrate, U-arginine, or the free radical scavenging enzyme superoxide dismutase (150 mu ml-1) failed to improve the attenuated response of acetylcholine-induced relaxation in the diabetic vessels. 7. Endothelium-dependent relaxation mediated by ACh and BK was significantly attenuated in both the diabetic and control vessels after incubation with L-NOARG. 8. Pretreatment with a cyclo-oxygenase inhibitor, indomethacin, significantly enhanced the relaxation to ACh in both the recent onset and insulin treated diabetic rats (42 +/- 10%, n = 7 versus 64 +/- 7%, n = 7, P < 0.05, and 40 +/- 5%, n = 7 versus 65 +/- 9%, n = 6, P < 0.05). 9. Following endothelium removal, there was a marked impairment in endothelium-dependent relaxation responses to ACh and BK in both the diabetic and control vessels. 10. Incubation with the thromboxane A2 receptor antagonist SQ29548, did not significantly improve the ACh endothelium-dependent relaxation response in the diabetic vessels. 11. Endothelium-independent relaxation to sodium nitroprusside was significantly impaired in the first group of diabetic vessels studied; however, subsequent studies showed no impairment of the sodium nitroprusside response in the diabetic vessels. 12. In conclusion, the ability of the endothelium to regulate vascular contractility is reduced in recent onset diabetic vessels, and significantly impaired in established insulin treated diabetics. Relaxation to the endothelium-dependent vasodilators ACh and BK was impaired in both the recent onset and the established insulin treated diabetics, and the ACh response was significantly improved following pretreatment with indomethacin, suggesting a role for a cyclo-oxygenase-derived vasoconstrictor. Preliminary studies with a thromboxane A2, receptor antagonist, SQ29548 did not significantly improve the impaired relaxation to ACh, indicating that the vasoconstrictor prostanoid is not thromboxane A2.

Acetylcholine↗
PubMed1995

The inhibitory effect of 3-amino-1,2,4-triazole on relaxation induced by hydroxylamine and sodium azide but not hydrogen peroxide or glyceryl trinitrate in rat aorta.

1. In this study we investigated the role of catalase in relaxation induced by hydroxylamine, sodium azide, glyceryl trinitrate and hydrogen peroxide in isolated rings of rat aorta. 2. Hydrogen peroxide (1 microM-1 mM)-induced concentration-dependent relaxation of phenylephrine (PE)-induced tone in endothelium-containing rings. In endothelium-denuded rings, however, higher concentrations (30 microM-1 mM) of hydrogen peroxide were required to produce relaxation. The endothelium-dependent component of hydrogen peroxide-induced relaxation was abolished following pretreatment with N(O)-nitro-L-arginine methyl ester (L-NAME, 30 microM). L-NAME (30 microM) had no effect, however, on hydrogen peroxide-induced relaxation in endothelium-denuded rings. 3. Pretreatment of endothelium-denuded rings with catalase (1000 u ml-1) blocked relaxation induced by hydrogen peroxide (10 microM-1 mM). The ability of catalase to inhibit hydrogen peroxide-induced relaxation was partially blocked following incubation with 3-amino-1,2, 4-triazole (AT, 50 mM) for 30 min and completely blocked at 90 min. 4. Pretreatment of endothelium-denuded rings with methylene blue (MeB, 30 microM) inhibited relaxation induced by hydrogen peroxide (10 microM-1 mM), sodium azide (1-300 nM), hydroxylamine (1-300 nM) and glyceryl trinitrate (1-100 nM) suggesting that each acted by stimulation of soluble guanylate cyclase. 5. Pretreatment of endothelium-denuded rings with AT (1-50 mM, 90 min) to inhibit endogenous catalase blocked relaxation induced by sodium azide (1-300 nM) and hydroxylamine (1-300 nM) but had no effect on relaxation induced by hydrogen peroxide (10 microM-1 mM) or glyceryl trinitrate (1-100 nM). 6. In a cell-free system, incubation of sodium azide (10 microM-3 mM) and hydroxylamine (10 microM-30 mM) but not glyceryl trinitrate (10 microM-1 mM) with catalase (1000 u ml-1) in the presence of hydrogen peroxide (1 mM) led to production of nitrite, a major breakdown product of nitric oxide. AT (1-100 mM) inhibited, in a concentration-dependent manner, the formation of nitrite from azide in the presence of hydrogen peroxide. 7. These data suggest that metabolism by catalase plays an important role in the relaxation induced by hydroxylamine and sodium azide in isolated rings of rat aorta. Relaxation appears to be due to formation of nitric oxide and activation of soluble guanylate cyclase. In contrast, metabolism by catalase does not appear to be involved in the relaxant actions of hydrogen peroxide or glyceryl trinitrate.

Amitrole↗
PubMed2003

Exercise training preserves endothelium-dependent relaxation in brachial arteries from hyperlipidemic pigs.

We tested the hypothesis that exercise training (Ex) attenuates the effects of hyperlipidemia on endothelial function by enhancing NO-mediated vasorelaxation in porcine brachial (Br) arteries. Adult female pigs were fed a normal-fat (NF) or high-fat (HF) diet for 20 wk. Four weeks after initiation of the diet, pigs underwent Ex or remained sedentary (Sed) for 16 wk. Relaxation to ACh was impaired by HF (P = 0.03). The combination of HF and Sed impaired ACh-induced relaxation more than HF or Sed alone (P = 0.0002). Relaxation to high doses of bradykinin (BK) was impaired by HF (P = 0.0002). Ex significantly improved ACh-induced relaxation (P = 0.01) and tended to improve relaxation to BK (P = 0.38). To determine the mechanism(s) by which HF and Ex affected relaxation to ACh and BK, relaxation was assessed in the presence of N(G)-nitro-l-arginine methyl ester (l-NAME; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), or l-NAME + Indo. In the presence of l-NAME, Indo, or l-NAME + Indo, ACh-induced relaxation was no longer different between HF and NF arteries; however, relaxation remained greater in Ex than in Sed arteries. In the presence of l-NAME or Indo, BK-induced relaxation was no longer altered by HF but was enhanced by Ex. In the presence of l-NAME + Indo, BK-induced relaxation was enhanced by HF and Ex. These data indicate that hyperlipidemia impairs ACh- and BK-induced relaxation by impairing NO- and PGI(2)-mediated relaxation. Ex attenuates the effects of HF by enhancing a vasodilator mechanism independent of NO and PGI(2).

Acetylcholine↗
PubMed2004

Relaxation induced by acetylcholine involves endothelium-derived hyperpolarizing factor in 2-kidney 1-clip hypertensive rat carotid arteries.

Acetylcholine induced relaxation in a concentration-dependent way in isolated phenylephrine-contracted carotid artery rings from normotensive two-kidney (2K) and hypertensive two-kidney one-clip (2K-1C) rats. In the presence of the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NOARG, 100 micromol/l), the relaxation stimulated with acetylcholine was blocked in 2K arteries. However, in 2K-1C arteries, the relaxation was only partially inhibited. Indomethacin (3 micromol/l) had no effect in both groups. In 2K arteries, the combination of L-NOARG and indomethacin had similar effects to L-NOARG alone. On the other hand, in 2K-1C arteries, indomethacin further inhibited the maximum effect induced by acetylcholine. Endothelium-dependent relaxation induced by acetylcholine was markedly reduced in 2K arteries contracted with 90 mmol/l KCl, and it was abolished in 2K-1C arteries. The remaining response to acetylcholine in 2K arteries was blocked by L-NOARG. Thus, in addition to NO, a relaxing factor sensitive to extracellular K+ changes in the membrane potential contributes to endothelium-dependent relaxation in 2K-1C rat carotid artery. On the other hand, in arteries from 2K rats, only NO is involved in the relaxation induced by acetylcholine. The combination of 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 3 micromol/l), indomethacin (3 micromol/l) and L-NOARG (100 micromol/l) reduced the relaxation induced by acetylcholine in arteries from 2K-1C rats contracted with phenylephrine. On the other hand, in 2K arteries, the relaxation induced by acetylcholine was abolished. The combination of ODQ and K+ channel blockers charybdotoxin (100 nmol/l), apamin (500 nmol/l) and 4-aminopyridine (1 micromol/l) abolished the relaxation induced by acetylcholine in 2K and 2K-1C carotid arteries. These data indicate that the endothelium-derived relaxing factors that contribute to relaxation induced by acetylcholine are different in 2K and 2K-1C arteries. In 2K arteries, the only factor is NO, which involves the activation of K+ channels and the cGMP pathway. However, in 2K-1C arteries, the relaxation induced by acetylcholine is dependent on NO in addition to another factor, which is insensitive to indomethacin, but also activates the K+ channels and the cGMP pathway, presumably by membrane hyperpolarization through endothelium-derived hyperpolarizing factor.

Acetylcholine↗
PubMed1974

Incomplete relaxation between beats after myocardial hypoxia and ischemia.

Recovery from hypoxia has been shown to prolong cardiac muscle contraction, particularly the relaxation phase. The present studies were designed to examine whether incomplete relaxation between beats can result from this prolongation of contraction and relaxation in isolated muscle after hypoxia and in the canine heart after both hypoxia and acute ischemia. The relationship between heart rate and the extent of incomplete relaxation is emphasized in view of the known enhancement of the velocity of contraction caused by increasing heart rate. The extent of incomplete relaxation during 10-s periods of pacing at increasing rates was examined before and after hypoxia in isometric cat right ventricular papillary muscle (12-120 beats/min) and in the canine isovolumic left ventricle (120-180 beats/min). Incomplete relaxation was quantified by measuring the difference between the lowest diastolic tension or pressure during pacing and the true resting tension or pressure determined by interruption of pacing at each rate. In eight cat papillary muscles (29 degrees C), there was significantly greater incomplete relaxation 5 min after hypoxia at rates of 96 and 120 beats/min (P < 0.02 vs. before hypoxia). In seven canine isovolumic left ventricles, recovery from hypoxia and higher heart rates also resulted in incomplete relaxation. Incomplete relaxation before hypoxia at a rate of 180 beats/min was 0.8+/-0.5 cm H(2)O and at 5 min of recovery from hypoxia was 12.6+/-3.5 cm H(2)O (P < 0.01). 12 hearts were subjected to a 1.5-3-min period of acute ischemia and fibrillation. There was significant incomplete relaxation at a rate of 140 beats/min for 5 min after defibrillation and reperfusion. These data indicate that incomplete relaxation is an important determinant of diastolic hemodynamics during recovery from ischemia or hypoxia. The extent of incomplete relaxation appears to be a function of the rate of normalization of the velocity of relaxation and tension development after ischemia or hypoxia, the heart rate, and the magnitude of developed tension or pressure.

Animals↗