Maximal Relaxation Response Research Articles

Cytochrome P450 Activity and Endothelial Dysfunction in Insulin Resistance

Impaired endothelium-dependent relaxation attributable to nitric oxide/prostacyclin-independent factor (endothelium-dependent hyperpolarizing factor; EDHF) has been demonstrated in the small mesenteric arteries of insulin-resistant rats. The purpose of this study was to determine if modulation of the cytochrome P450 enzyme system would restore EDHF-mediated relaxation in insulin-resistant rats. Sprague-Dawley rats were randomized to control (n = 32) or insulin-resistant (n = 32) groups. Each group was further randomized to treatment (n = 48) or placebo (n = 16). Miconazole (3 days) and phenobarbital (3 and 14 days) achieved cytochrome P450 inhibition and induction, respectively. Following drug treatment, mean arterial pressure was measured and vascular function was assessed in small mesenteric arteries in vitro. Specifically, acetylcholine-induced relaxation alone and in the presence of indomethacin plus N-nitro-L-arginine (LNNA) or KCl was determined. Miconazole reduced the maximal relaxation in response to acetylcholine in control rats. Similarly, in the presence of LNNA plus indomethacin, acetylcholine-induced relaxation was impaired in the miconazole-treated control group versus the placebo group, whereas relaxation in the presence of KCl was unchanged. Miconazole did not affect relaxation in insulin-resistant arteries. In contrast, 3- and 14-day treatment with phenobarbital significantly improved acetylcholine-induced relaxation in insulin-resistant arteries. Likewise, acetylcholine-mediated relaxation in the presence of LNNA plus indomethacin was also improved after phenobarbital treatment, while relaxation in the presence of KCl was unchanged. Phenobarbital treatment did not affect the control group. Miconazole treatment increased the mean arterial pressure in control rats, while 14-day phenobarbital treatment normalized the mean arterial pressure in insulin-resistant rats. Cytochrome P450 induction results in the restoration of EDHF-mediated relaxation in small mesenteric arteries and the normalization of mean arterial pressure in insulin-resistant rats. Thus, endothelial dysfunction secondary to insulin resistance can be reversed by the induction of cytochrome P450.

Nitric oxide and sodium nitroprusside-induced relaxation of the human umbilical artery.

In the human umbilical artery (HUA) pre-contracted with the thromboxane mimetic U46619 or with 5-hydroxytryptamine (5-HT), (and pretreated with indomethacin (3 microM) to suppress the synthesis of prostanoids), authentic nitric oxide (NO) evoked concentration-dependent relaxation (pEC(50) 7.05 and 5.99, respectively). In contrast, sodium nitroprusside (SNP) induced relaxation only in U46619 pre-contracted HUA (pEC(50) 6.52). At high (>300 mmHg) vs low (<55 mmHg) oxygen tension the dose-response curves to NO- and SNP-induced relaxations were biphasic and shifted leftward. Preincubation of the arterial rings with the soluble guanylyl cyclase (sGC) inhibitor 1H[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ; 10 microM) shifted the concentration-response curve to NO, reduced the maximal relaxation response to NO (E(max) 71%) and to SNP (E(max) 10%). Pre-exposure of HUA rings to high extracellular K(+) (50 mM) reduced E(max) relaxation responses to NO (36%) and SNP (1%). Pretreatment of the HUA with the K(+) channel inhibitors, tetraethylammonium (TEA, 1 mM), 4-aminopyridine (4-AP, 0.5 mM), charybdotoxin (0.1 microM) or iberiotoxin (0.1 microM) increased the pEC(30) for NO and SNP and changed the shape of the dose-response curves from biphasic to monophasic. Pre-incubation of HUA rings with TEA (1 mM), 4-AP (0.5 mM) and ODQ (10 microM) significantly reduced the NO-induced maximal relaxation (E(max) 26%) but not the pEC(50) (5.60). These data indicate that SNP-induced relaxation in the HUA is primarily mediated via sGC-cyclic GMP whereas NO-induced relaxation also involves the activation of K(V) and K(Ca) channels and a cyclic GMP/K(+) channel-independent mechanism(s).

Vanadate-evoked relaxation of the perfused rat mesenteric vascular bed

Sodium orthovanadate (SOV) can contract smooth muscle; however, little is known about its effect on the vascular endothelium. We compared the vasorelaxant effects of acetylcholine (ACh) and SOV in the preconstricted, isolated perfused mesenteric vascular bed (MVB) of Sprague-Dawley rats. The maximal relaxation response evoked by SOV (40–45%) was lower than ACh (92–94%) but the IC 50 values were similar. At concentrations > 1 mM, SOV elevated the basal tone. Endothelial denudation resulted in a substantial reduction of relaxation responses to both agents, whereas either nitric oxide synthase (NOS) inhibitors or high KCl partially reduced the responses. A combination of NOS inhibitors along with either a calcium-activated potassium channel (K Ca) blocker, tetrabutylammonium (TBA), or high KCl inhibited the responses to a similar extent as endothelium denudation. Neither clotrimazole nor TBA attenuated ACh responses; however, maximal responses to SOV in the presence of TBA or clotrimazole were reduced. Indomethacin had no effect on responses to either agonists. These results indicate that like ACh, SOV-mediated vasorelaxation of the MVB involves recruitment of both endothelial derived hyperpolarizing factor (EDHF) and endothelial derived nitric oxide (NO) and not vasodilator eicosanoids. As the relaxation to SOV was dose-dependent at a low concentration range, it is likely that vanadate is involved in the regulation of total peripheral resistance.

Vasorelaxant and antiproliferative effects of berberine

The present study was intended to examine the relaxant effects of berberine in rat isolated mesenteric arteries. Berberine produced a rightward shift of the concentration–response curve to phenylephrine and significantly reduced the maximal contractile response to phenylephrine. Berberine (10 −7–3×10 −5 M) also relaxed the phenylephrine- and 9,11-dideoxy-11α,9α-epoxy-methanoprostaglandin F 2α-precontracted arteries with respective IC 50 values of 1.48±0.16×10 −6 and 2.23±0.22×10 −6 M. Removal of a functional endothelium significantly attenuated the berberine-induced relaxation (IC 50: 4.73±0.32×10 −6 M) without affecting the maximum relaxant response. Pretreatment with N G-nitro- l-arginine methyl ester ( l-NAME) or methylene blue reduced the relaxant effect of berberine, and l-arginine (10 −3 M) partially antagonized the effect of l-NAME. In contrast, pretreatment with 10 −6 M glibenclamide or 10 −5 M indomethacin had no effect. Berberine (10 −5 M) reduced over by 50% the transient contraction induced by caffeine or phenylephrine in endothelium-denuded rings bathed in Ca 2+-free Krebs solution. Pretreatment with putative K + channel blockers, such as tetrapentylammonium ions (1–3×10 −6 M), 4-aminopyridine (10 −3 M), or Ba 2+ (3×10 −4 M), significantly attenuated the berberine-induced relaxation in endothelium-denuded arteries. In contrast, tetraethylammonium ions (3×10 −3 M), charybdotoxin (10 −7 M) or glibenclamide (10 −6 M) were without effect. Berberine reduced the high-K +-induced sustained contraction and the relaxant response to berberine was greater in rings with endothelium (IC 50: 4.41±0.47×10 −6 M) than in those without endothelium (IC 50: 8.73±0.74×10 −6 M). However, berberine (10 −6–10 −4 M) did not affect the high-K +-induced increase of intracellular [Ca 2+] in cultured aortic smooth muscle cells. Berberine did not affect active phorbol ester-induced contraction in Ca 2+-free Krebs solution. In addition, berberine inhibited proliferation of cultured rat aortic smooth muscle cells with an IC 50 of 2.3±0.43×10 −5 M. These findings suggest that berberine could act at both endothelium and the underlying vascular smooth muscle to induce relaxation. Nitric oxide from endothelium may account primarily for the berberine-induced endothelium-dependent relaxation, while activation of tetrapentylammonium-, 4-aminopyridine- and Ba 2+-sensitive K + channels, inhibition of intracellular Ca 2+ release from caffeine-sensitive pools, or a direct relaxant effect, is likely responsible for the berberine-induced endothelium-independent relaxation. Mechanisms related to either Ca 2+ influx or protein kinase C activation may not be involved. Both vasorelaxant and antiproliferative effects may contribute to a long-term benefit of berberine in the vascular system.

Evidence against potassium as an endothelium‐derived hyperpolarizing factor in rat mesenteric small arteries

1. Endothelium-derived hyperpolarizing factor (EDHF) has recently been identified as potassium released from endothelial cells into the myo-endothelial space. The present study was designed to test this hypothesis. 2. In rat small mesenteric arteries, mounted in a wire myograph, relaxation to acetylcholine or potassium was not significantly changed following incubation with oxadiazolo-quinoxalin-1-one (ODQ, 4 microM) and indomethacin (10 microM, n = 9). 3. Maximal relaxations to acetylcholine occurred in all arteries, were maintained and were significantly greater (P < 0.01, n = 9) than the transient relaxations to potassium, which only occurred in 30-40% of vessels. 4. Removal of the vascular endothelium abolished relaxant responses both to potassium and acetylcholine (P < 0.005, n = 9). 5. Compared with responses in 5.5 mM potassium PSS, relaxation responses to added potassium in arteries maintained in 1.5 mM potassium PSS were more marked and were not dependent on the presence of an intact endothelium (n = 8). 6. Incubation with BaCl2 (50 microM) significantly inhibited the maximal relaxant response to potassium in the presence of an intact endothelium in 5.5 mM potassium PSS (P < 0.05, n = 4), but had no effect on relaxation of de-endothelialized preparations in 1.5 mM potassium PSS (n = 5). 7. Treatment with ouabain (0.1 mM) abolished the relaxant response to potassium in 1.5 mM potassium PSS (P < 0.001, n = 9), but only partly inhibited the maximal relaxant response to acetylcholine in 5.5 mM potassium PSS (P < 0.01, n = 5). 8. These data show that at physiological concentrations of potassium an intact endothelium is necessary for potassium-induced relaxation in rat mesenteric arteries. Furthermore, the response to potassium is clearly different to that from acetylcholine, indicating that potassium does not mimic EDHF released by acetylcholine in these arteries.

Thromboxane A 2 modulates cyclic AMP relaxation and production in human internal mammary artery

Two forms of thromboxane A 2 (TP) receptors, TPα and TPβ receptors, have recently been cloned. These receptors regulate adenylate cyclase activity in two opposite ways: TPα receptors activate, whereas TPβ receptors inhibit adenylate cyclase and cAMP generation. The aim of this study was to examine the effects of the thromboxane A 2 analogue, U46619 (9,11-dideoxy-9α,11 α-methanoepoxy-prostaglandin F 2α), on forskolin-induced relaxation and cAMP accumulation in human internal mammary artery (IMA) and saphenous vein (SV). In organ baths, IMA rings precontracted with U46619 (3.10 −9 and 3.10 −8 M) were less sensitive to forskolin than rings precontracted with methoxamine (3.10 −6 M). In contrast, the sensitivity to forskolin was similar in SV rings contracted with the same concentrations of these agonists. U46619 reduced significantly the ten-fold increase in cAMP induced by forskolin in IMA but not in SV rings. Sensitivity and maximal relaxation in response to sodium nitroprusside were not altered in either IMA or SV. In summary, stimulation of TP receptors with the thromboxane A 2 analogue, U46619, inhibited the cAMP pathway of relaxation through the inhibition of cAMP synthesis in human IMA but not in SV. It is suggested that thromboxane A 2 may play a role in the control of muscle tone in IMA both by its potent contractile effect and by its inhibitory effect on the cAMP pathway of relaxation.

Heart preservation in HTK solution: role of coronary vasculature in recovery of cardiac function

Background. Poor myocardial tolerance to prolonged cold ischemia remains a major concern in heart transplantation. In this study, we estimated superiority of Histidine-Tryptophan-Ketoglutarate (HTK) over University of Wisconsin (UW) as a cardiac preservation solution.Methods. Isolated rat hearts were mounted on a Langendorff apparatus to estimate the baseline cardiac function. The hearts were arrested and stored at 4°C in UW and HTK solution for 8 hours, and then reperfused. The aortic flow, coronary flow, cardiac output, rate pressure product, and left ventricular dp/dt in the HTK group recovered significantly more than the UW group. The values of myocardial total adenine nucleotides and the adenosine triphosphate to adenosine diphosphate ratio were higher in the HTK than in the UW group. We also examined coronary vascular responsiveness using left coronary arteries dissected from the rat hearts before flushing, before storage, after storage, and after reperfusion.Results. The maximal relaxation response to acetylcholine was significantly higher in the HTK than in the UW group after reperfusion, although there were no significant differences at each stage before reperfusion. In addition, the endothelium-independent relaxation response to sodium nitroprusside in the HTK group was also well preserved after reperfusion.Conclusions. These results indicate that HTK is superior to UW solution for cardiac preservation. HTK protects coronary vasculature during preservation, which together with reperfusion might lead to improved functional cardiac recovery following preservation.

Age-related Changes in Airway Responsiveness to Phosphodiesterase Inhibitors and Activators of Adenyl Cyclase and Guanylyl Cyclase

The influence of animal age on the responsiveness of guinea-pig and rat isolated tracheal smooth muscle to the non-selective inhibitors of phosphodiesterase, theophylline and papaverine and to the adenylyl cyclase and soluble guanylyl cyclase activators, forskolin and sodium nitroprusside respectively, was examined. Significant age-related decreases in the potencies of theophylline and papaverine were observed across the age ranges in guinea-pig (2.8- and 3.4-fold decreases respectively) and rat (1.9- and 2.6-fold decreases respectively) trachea, suggesting age-related falls in the activity of phosphodiesterase in these tissues. However, maximum relaxant responses (Emax) to these agents were not altered with increasing animal age. The relaxant potency of sodium nitroprusside also decreased 4.4-fold across the age range in guinea-pig isolated trachea but not in rat isolated tracheal tissue, suggesting age-related falls in soluble guanylyl cyclase activity in guinea-pig trachea. In contrast, neither forskolin potency nor Emaxchanged significantly with increasing age in either guinea-pig or rat tracheal tissue. The present data indicate that ageing in both guinea-pigs and rats was associated with decreased relaxant potency of phosphodiesterase (PDE) inhibitors rather than to changes in adenylyl cyclase activity although reduced soluble guanylyl cyclase activity was also detected in the guinea-pig.

Pharmacological evidence for the 5-HT7 receptor mediating smooth muscle relaxation in canine cerebral arteries.

1. We investigated in the present study whether 5-HT is able to exert direct relaxant responses in canine basilar and middle cerebral arteries via the 5-HT7 receptor. 2. In arterial rings deprived of endothelium and pre-contracted with prostaglandin F2 alpha (2 microM), 5-HT, 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine, sumatriptan or alpha-methyl-5-HT produced further increase in tone and/or slight relaxation. Blockade of 5-HT1B 1D and 5-HT2A receptors with GR127935 (1 microM) and ketanserin (0.1 microM), respectively, antagonized the vasoconstrictor component of the response and unmasked a concentration-dependent relaxation to 5-HT, 5-CT and 5-methoxytryptamine; sumatriptan and alpha-methyl-5-HT remained inactive as relaxant agonists. The rank order of agonist potency in both arteries was 5-CT > 5-HT > 5-methoxytryptamine >> sumatriptan > or = alpha-methyl-5-HT. 3. In dog basilar artery, pre-incubated with GR127935 (1 microM) and ketanserin (0.1 microM) and precontracted with prostaglandin F2 alpha (2 microM), the 5-HT7 ligands, clozapine (1 microM), mesulergine (0.3 microM), methiothepin (3 nM), risperidone (3 nM), spiperone (1 microM) and LY215840 (10-100 nM), produced significant rightward shifts of the concentration-response curves for 5-HT and 5-CT. Only methiothepin and risperidone reduced significantly the maximum relaxant response (Emax), whilst the other drugs behaved as competitive antagonists with affinity values (pKB) that significantly correlated with their binding affinity (pKi) at recombinant 5-HT7 receptors. 4. These data disclosing the involvement of the 5-HT7 receptor in cerebrovascular relaxation may be strongly relevant in the light of: (1) the involvement of 5-HT in migraine; (2) the putative linkage between cephalovascular vasodilatation and migraine headache; and (3) the relatively high 5-HT7 receptor affinity of migraine prophylactic 5-HT antagonists.

Prolonged Hypothermic Cardiac Storage with Histidine-tryptophan-ketoglutarate Solution-Comparison with Glucose-insulin-potassium and University of Wisconsin Solutions

95 This study was designed to test the efficacy of histidine-tryptophan-ketoglutarate (HTK) solution after prolonged cold storage as compared with the conventional glucose-insulin-potassium (GIK) and University of Wisconsin (UW) solutions. Methods. (1). The hearts isolated from male Wistar rats were divided into three groups (n=6 per group) according to each preservation solution used. The hearts were arrested and stored in each solution for 6, 8, and 12 hrs at 4 °C. After storage, recovery of cardiac function and myocardial adenylate and water contents were evaluated. (2). Mechanical function of the epicardial coronary arteries (left main trunks) were compared between UW and HTK solutions. Coronary vascular responsiveness were examined at four predetermined time points: prior to flush and following flush, storage, and reperfusion. Results. (1). Cardiac function, which was sufficiently maintained in the HTK group up till 8 hrs of storage decreased below 50% following 8 hrs of storage in the UW and GIK groups. Although the myocardial ATP/ADP ratio in the HTK group was maintained above 50% up till 8 hrs of storage, those ratios in the GIK and UW groups declined to 15%. The values of myocardial energy charge in the HTK group were maintained above 60% up till 8 hrs of storage, however, those values in the UW and GIK groups declined to below 50% following 6 hrs of storage. The water contents following 12 hrs of storage were lower in the HTK and UW groups than in the GIK group. (2). Following reperfusion, the maximal relaxation response to acetylcholine in the HTK group was higher than in the UW group (p<0.05), and the endothelium-independent relaxation response to sodium nitroprusside in the HTK group was also well preserved. Discussion and Conclusion. In comparison with UW and GIK solutions, HTK solution, which sustains myocardial energy level and coronary artery function during preservation may lead to a better recovery of cardiac function; however, successful cold storage of the heart is highly energy-dependent, and a dramatic breakdown of myocardial energy level, which causes a crucial decline in the cardiac function, occurs between 8 and 12 hr of storage.

Differential Effects of Methanol on Rat Aortic Smooth Muscle

LI, W., B. T. ALTURA AND B. M. ALTURA. Differential effects of methanol on rat aortic smooth muscle. Alcohol 16(3) 221–229, 1998.—The effects of methanol on isolated segments of rat aorta were investigated. In the absence of any vasoactive agent, methanol (5–675 mM) failed to alter basal tension. In rat aortic rings precontracted with high K + (30 mM), methanol elicited a concentration-related relaxation at concentrations of from 5 to 675 mM. The K +-induced contraction in the presence of endothelium was more strongly inhibited by methanol than in the absence of endothelium. The effective concentration producing approximately 50% of the maximal relaxation response (ED 50) to methanol was about 96 mM. Methanol-induced relaxations could not be abolished either by 5 × 10 −5 M N-nitro- l-arginine methyl ester ( l-NAME) or N G-nitro- l-arginine ( l-NNA), both selective inhibitors of nitric oxide (NO) formation; these relaxations were not potentiated by addition of excess l-arginine. An inhibitor of prostanoid synthesis, indomethacin (10 −5 M), had no effects on methanol-induced relaxation. Removal of extracellular Ca 2+ ([Ca 2+] o) resulted in almost complete inhibition of the relaxant effects of methanol on rat aortic ring segments. Marked attenuation of the relaxation responses of intact arteries to methanol was obtained after buffering intracellular Ca 2+ ([Ca 2+] i) with 10 μM BAPTA-AM. In 5-hydroxytryptamine (5-HT, 2.5 μM)- or phenylephrine (PE, 0.1 μM)-precontracted rat aortic rings, methanol amplified contractile responses to 5-HT and PE; these increased responses were concentration dependent. No significant differences in these methanol potentiated responses were found between aorta with or without endothelial cells. The amplified rat aortic smooth muscle responses induced by methanol after PE could be modified only by phentolamine, an antagonist of PE, while responses to 5-HT could be inhibited by methysergide (an antagonist of 5-HT) and by phentolamine, diphenhydramine, and haloperidol. Pretreatment with 50, 200, and 500 mM methanol increased rat aortic contractile responses induced by 5-HT and PE. Our results suggest that: (a) acute methanol exposure relaxes rat aortic smooth muscle contractile responses induced by high K +, leading to vessel relaxation. This relaxation effect of methanol is endothelium-dependent, clearly Ca 2+ dependent, and independent of endogenous vasodilators such as acetylcholine, histamine, catecholamines, serotonin, or PG. (b) Methanol seems to increase potassium current by shifting the potential towards more negative values in depolarized vascular muscle cell membranes, probably inducing hyperpolarization of the cell membranes leading to a repolarization. (c) In contrast to the relaxant responses, methanol protentiates contractile response of rat aorta to 5-HT and PE.

Endothelium-dependent relaxation induced by hawthorn extract in rat mesenteric artery

The extract prepared from hawthorn (Crataegus fruit) was examined for its relaxant effect in rat isolated mesenteric arteries. Hawthorn extract induced concentration-dependent relaxation of the U46619-precontracted artery with an IC 50 of 0.22 ± 0.02 mg/ml. Removal of the functional endothelium reduced by approximately 85% the maximum relaxant response to hawthorn extract. Pretreatment of the arterial tissues with N G-nitro-L-arginine methyl ester (3–10 μM) or methylene blue (3–10 μM) inhibited the relaxation induced by hawthorn extract, while indomethacin (10 μM) had no effect. L-Arginine (3 mM) did not affect the relaxation induced by hawthorn extract but partially reversed the effect of 10 μM N G-nitro-L-arginine methyl ester. Iberiotoxin (100 nM) slightly but significantly inhibited the relaxant effect of hawthorn extract whilst glibendamide (3 μM) was ineffective. Glibenclamide at 3 μM reversed the relaxation induced by pinacidil. N G-nitro-L-arginine methyl ester and methylene blue markedly inhibited acetylcholine-induced relaxation in endothelium-intact arteries. Hawthorn extract also reduced the contraction induced by phenylephrine (1 μM) or high K + (60 mM) with respective IC 50 values of 0.13 ± 0.01 mg/ml and 0.11 ± 0.01 mg/ml. In high K −-contracted arteries, hawthorn extract induced only 55% of relaxation while it caused a complete inhibition of the U46619- or phenylephrine-induced contraction. These results suggest that hawthorn contains active components which cause vasorelaxation in rat isolated mesenteric arteries. Nitric oxide but not other endothelium-derived vasoaclive factors was probably involved in the relaxation induced by hawthorn extract.

Endothelial dysfunction in pulmonary arteries of patients with mild COPD.

To investigate whether endothelial dysfunction of pulmonary arteries (PA) is present in patients with mild chronic obstructive pulmonary disease (COPD) and to what extent it is related to the morphological abnormalities of PA, we studied 41 patients who underwent lung resection. Patients were divided into the following groups: nonsmokers (n = 7), smokers with normal lung function (n = 13), and COPD (n = 21). Endothelium-dependent relaxation mediated by nitric oxide was evaluated in vitro in PA rings exposed to cumulative concentrations of acetylcholine (ACh) and ADP. Structural abnormalities of PA were assessed morphometrically. PA of COPD patients developed lower maximal relaxation in response to ADP than both nonsmokers and smokers (P < 0.05 each) and a trend to reduced relaxation in response to ACh (P = 0.08). Maximal relaxation to ADP correlated with the degree of airflow obstruction (r = 0.48, P < 0. 01). Morphometrical analysis of PA revealed thicker intimas, especially in small arteries, in both smokers and COPD compared with nonsmokers (P < 0.05 each). We conclude that endothelial dysfunction of PA is already present in patients with mild COPD. In these patients, as well as in smokers with normal lung function, small arteries show thickened intimas, suggesting that tobacco consumption may play a critical role in the pathogenesis of pulmonary vascular abnormalities in COPD.

Responses to endothelium-dependent agonists in subcutaneous arteries excised from hypercholesterolaemic men.

1. Vasomotor function of the vascular endothelium was examined in human subcutaneous arteries excised from 8 hypercholesterolaemic and 7 normolipidaemic subjects. 2. Left gluteal skin biopsies were performed under local anaesthesia. Subcutaneous arteries were isolated and two vessels from each subject mounted in separate myographs. A 20 ml fasting blood sample was taken at the time of the biopsy. 3. Hypercholesterolaemic subjects had either never been treated with lipid lowering therapy or therapy had been stopped at least two weeks before the study (n = 2). At the time of the study total plasma cholesterol levels (control: 4.6+/-0.3 vs hypercholesterolaemic: 8.3+/-0.6 mmol l(-1): P < 0.01) were significantly elevated in hypercholesterolaemic subjects when compared with controls. 4. Full concentration-response curves to the vasoconstrictor noradrenaline and the vasodilators acetylcholine and substance P were constructed. A single point concentration-response to sodium nitroprusside (10 microM) was also obtained. Dilator responses were obtained in vessels pre-constricted with a submaximal concentration of noradrenaline. Vessels were then incubated for 30 min with either L- or D-arginine (10 microM) and the concentration-response curves to the three dilator agonists repeated in the presence of the amino acid. 5. Maximum relaxation responses to acetylcholine (control vs hypercholesterolaemic: 83.3+/-6.1% vs 47.4+/-13.5%; P < 0.05), but not to substance P or sodium nitroprusside, were dampened in the hypercholesterolaemic group when compared with controls. 6. Neither incubation with L-arginine nor D-arginine had any effect on maximum relaxation responses to acetylcholine in either the control group (pre L-arginine vs plus L-arginine: 83.3+/-6.1 vs 82.3+/-5.5%, pre D-arginine vs plus D-arginine: 98.9+/-1.2 vs 98.2+/-1.1%) or the hypercholesterolaemic group (pre L-arginine vs plus L-arginine: 47.4+/-13.5 vs 55.3+/-14.3%, pre D-arginine vs plus D-arginine: 43.3+/-13.6 vs 65.4+/-12.3%). 7. When results from the two study groups were pooled, the strongest predictor of maximum relaxation obtained to acetylcholine was apolipoprotein A1 (r = 0.67; P = 0.001). 8. In conclusion, relaxation responses mediated by the endothelium-dependent agonist acetylcholine, but not by substance P, are impaired in hypercholesterolaemic patients. L-Arginine did not improve the impaired relaxation responses to acetylcholine. We suggest that impaired endothelium-dependent relaxation is specific to acetylcholine and not to an abnormal L-arginine-nitric oxide pathway in subcutaneous arteries excised from this study group.

Autologously up-regulated Fc receptor expression and action in airway smooth muscle mediates its altered responsiveness in the atopic asthmatic sensitized state.

To elucidate the role of IgE-dependent mechanisms in inducing altered airway responsiveness in the atopic asthmatic state, the expression and actions of Fc receptor activation were examined in isolated rabbit tracheal smooth muscle (TSM) tissue and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. Relative to control tissues, the atopic asthmatic-sensitized TSM exhibited significantly increased maximal isometric contractility to acetylcholine (P < 0. 01) and attenuated maximal relaxation responses and sensitivity (i.e.,-log ED50) to isoproterenol (P < 0.005). These changes in agonist responsiveness in atopic sensitized TSM were ablated by pretreating the tissues with a blocking mAb to the low affinity receptor for IgE, FcepsilonRII (i.e., CD23) or by depleting the sensitizing serum of its immune complexes. Moreover, in complimentary experiments, exogenous administration of IgE immune complexes to naive TSM produced changes in agonist responsiveness that were qualitatively similar to those obtained in the atopic asthmatic-sensitized state. Extended studies further demonstrated that, in contrast to their respective controls, atopic asthmatic serum-sensitized human and rabbit TSM tissue and cultured cells exhibited markedly induced mRNA and cell surface expression of FcepsilonRII, whereas constitutive expression of the IgG receptor subtype, FcgammaRIII, was unaltered. Finally, the up-regulated mRNA expression of FcepsilonRII observed following exposure of TSM to atopic asthmatic serum or to exogenously administered IgE immune complexes was significantly inhibited by pretreating the tissues or cells with anti-CD23 mAb. Collectively, these observations provide evidence demonstrating that the altered agonist responsiveness in atopic asthmatic sensitized airway smooth muscle is largely attributed to IgE-mediated induction of the autologous expression and activation of FcepsilonRII receptors in the airway smooth muscle itself.

Preservation of vascular function in rat mesenteric resistance arteries following cold storage, studied by small vessel myography.

1. The use of isolated blood vessels to investigate the physiological and pharmacological control of the vasculature is limited by the requirement to use freshly isolated vessels. Hence, the aim of this study was to determine whether vascular smooth muscle and endothelial cell function could be preserved in resistance arteries by storing them in physiological salt solution (PSS) at 4 degrees C. 2. Third order mesenteric resistance arteries (mean internal diameter 237+/-6 microm) were dissected from the mesenteric bed of male Cob-Wistar rats. The vessel segments were mounted in a small vessel myograph for measurement of isometric tension, and equilibrated at their optimum resting force. Contractile responses to noradrenaline (NA; 1 x 10(-9) - 3 x 10(-5) M), phenylephrine (PE, 1 x 10(-9)-3 x 10(-5) M), potassium chloride (KCI; 2.5-140 mM) and endothelin (ET-1, 1 x 10(-11)-3 x 10(-7) M) and relaxant responses to acetylcholine (ACh; 1 x 10(-9) - 3 x 10(-5) M) and 3-morpholinosydnonimine (SIN-1; 1 x 10(-9) - 1 x 10(-4) M) were obtained in arteries, immediately after dissection (day 0) and following one to four days storage (day 1-day 4). 3. All arteries produced concentration-dependent contractions in response to each of the vasoconstrictors. There were no significant differences in the magnitude or sensitivity (pD2) of the vasoconstrictor responses between fresh and stored vessels. 4. Arteries precontracted with NA to approximately 80% of the maximum response, relaxed in a concentration-dependent manner in response to ACh and SIN-1. Vessel storage for up to three days resulted in no change in response to ACh or SIN-1. 5. Vessels analysed after four days of storage demonstrated a significant increase in sensitivity to ACh and SIN-1 (-logIC50 (M) values; ACh; day 0, 7.46+/-0.13 vs day 4, 7.97+/-0.11, P<0.01 and SIN-1; day 0, 4.87+/-0.10 vs day 4, 5.52+/-0.08, P<0.01). There was also a significant increase in the maximum relaxant response to ACh after four days of storage (% relaxation; day 0, 92.65+/-2.84 vs day 4, 100.36+/-0.36, P<0.05). 6. These results demonstrate that small resistance arteries remain viable if stored in PSS at 4 degrees C for up to four days, with no loss in endothelial cell function. The altered sensitivity to the vasodilators on day 4 suggests that vessels should only be stored for up to three days following dissection for analysis of functional responses.

Improvement of insulin sensitivity and cardiovascular outcomes in the JCR:LA-cp rat by D-fenfluramine.

Obese male rats of the JCR:LA-cp strain are insulin resistant, normoglycaemic, hypertriglyceridaemic, and atherosclerosis-prone. Such rats were treated from 6 to 39 weeks of age with 5 mg x kg(-1) x day(-1) of D-fenfluramine. The treatment normalised food intake, after 20 weeks of age, to that of lean control animals. At 39 weeks, treated rats weighed about 650 g compared to 800 g for untreated cp/cp rats and 400 g for +/+ controls. Fasting plasma glucose and triglyceride levels were not significantly affected; however, fasting insulin concentrations were lower and the size and volume density of the hyperplastic islets of Langerhans were markedly reduced. The severity of raised atherosclerotic lesions on the aortic arch was decreased by 39% (p < 0.01). Concomitantly, the occurrence of mature, scarred ischaemic myocardial lesions was virtually abolished (p < 0.01). Severe food restriction of the obese rats to normalise body weights to those of lean controls reduced plasma insulin and triglyceride concentrations at 26 weeks of age, but without a significant reduction in the frequency of myocardial lesions. Rats (with established insulin resistance) were treated from 6 to 12 weeks of age with 2.5 mg x kg(-1) x day(-1) of D-fenfluramine. Insulin-mediated glucose turnover during a euglycaemic insulin clamp was strongly increased (p < 0.05). Rats treated from 3 weeks of age (before development of the insulin resistance) showed a significant delay in the development of hyperinsulinaemia and a reduced postprandial increase in plasma insulin. In contrast, restriction of food to that consumed by rats treated with D-fenfluramine did not decrease post-absorptive hyperinsulinaemia. D-fenfluramine treatment markedly improved the maximum relaxant response of aortic rings to acetylcholine, indicating improvement of the defective endothelium-derived relaxation factor system. A matched-food restriction regimen had no effect on vascular relaxation. D-fenfluramine treatment thus improved insulin sensitivity and had anti-atherosclerotic and cardioprotective effects in the presence of continuing obesity and hyperlipidaemia. The results are consistent with the protection of the function and integrity of the vessel wall associated with a decreased hyperinsulinaemia. The results emphasise the importance of focussing treatment of the metabolic syndrome (obesity/insulin resistance/hyperlipidaemia) on improving insulin sensitivity and glycaemic control rather than on the simple normalisation of body weight.

Digital Analysis of the Pharmacological Effects of in vitro Ischemia on Rabbit Corpus cavernosum

Purpose: Regulation of corporal smooth muscle tone is essential for the initiation of penile erection. In recent years, in vitro isometric tension studies using isolated corpus cavernosal tissue have been used extensively to investigate the mechanisms regulating corporal smooth muscle tone and tension. In the present study, we utilized digital analysis of contractile data generated from investigation of contractile and relaxation responses of isolated rabbit corpus cavenosum to various forms of stimulation. Digital analysis of the contractile and relaxation data allows quantitation of both maximal and mean rates of tension change, and time elapsed to maximal response. Rates of tension changes may provide additional important information regarding cellular events that mediate corporal tone and tension changes. Methods: Sexually mature male New Zealand White rabbits were used. Each corpus cavernosum was dissected sharply from the removed penis, then two longitudinal strips were prepared for isometric tension studies and placed in individual baths. Tension was monitored continually using an 8-channel Grass Polygraph. The Grass PolyVIEW system simultaneously converted analog signals to digital information and stored data using a 486 PC computer. Each corporal strip was prestimulated with 300 µmol/l phenylephrine to produce a maximal contraction, then field stimulation (FS), carbachol and nitroprusside were applied consecutively to determine relaxation effects. This procedure was repeated after strips were deprived of glucose and oxygen (in vitro ischemia) for 1 h. The following parameters were quantitated for all responses: maximal tension change; maximal and mean rates of tension change, and time to maximal response. Results: Effects of 1-hour in vitro ischemia on rabbit corporal tissue were as follows: (1) an 85% decrease in contractile response to phenylephrine, and (2) a marked increase in rate of contractile response to phenylephrine. Phenylephrine precontracted strips exhibited: (3) no change in relaxant response to FS; (4) increased relaxant responses to carbachol and nitroprusside; (5) no change in rates of relaxation in response to FS; (6) increased rates of relaxation in response to carbachol and nitroprusside, (7) no change in elapsed time to maximal relaxation in response to FS, and (8) decreased elapsed time to maximal relaxation in response to carbachol and nitroprusside. Conclusion: Digital analysis of the data generated facilitates recording, reviewing and analyzing of in vitro isometric tension studies on rabbit corpus cavernosum. Digital analysis allows quantification of additional parameters that have important implications for determination of mechanisms by which specific pathological processes occur.

The effect of chronic treatment with trandolapril on cyclic AMP-and cyclic GMP-dependent relaxations in aortic segments of rats with chronic heart failure.

1 Characteristics of cyclic GMP- and cyclic AMP-mediated relaxation in aortic segments of rats with chronic heart failure (CHF) and the effects of chronic treatment with an angiotensin I converting enzyme (ACE) inhibitor, trandolapril, were examined 8 weeks after coronary artery ligation. 2 Cardiac output indices of coronary artery-ligated and sham-operated rats were 125+/-8 and 189+/-10 ml min(-1) kg(-1), respectively (P<0.05), indicating the development of CHF at this period. 3 The maximal relaxant response of aortic segments to 10 microM acetylcholine in rats with CHF and sham-operated rats was 64.0+/-5.7 and 86.9+/-1.9%, respectively (P<0.05), whereas the relaxant response to sodium nitroprusside (SNP) remained unchanged. Tissue cyclic GMP content in rats with CHF was lower than that of sham-operated rats. 4 In endothelium-intact segments of rats with CHF, the maximal relaxant response to 10 microM isoprenaline (44.5+/-6.7%) was lower that sham-operated rats (81.3+/-2.5%, P<0.05) and the concentration-response curve for NKH477, a water-soluble forskolin, was shifted to the right without a reduction in the maximal response. Isoprenaline-induced relaxation of aortic segments was attenuated by NG-nitro-L-arginine methyl ester (L-NAME) in sham-operated rats, but not in rats with CHF. Relaxation to 30 microM dibutyryl cyclic AMP in rats with CHF (26.8+/-2.7%) was lower than that in sham-operated rats (63.4+/-11.8%, P<0.05). 5 Trandolapril (3 mg kg(-1) day(-1)) was orally administered from the 2nd to 8th week after the operation. Aortic blood flow of rats with CHF (38.5+/-3.6 ml min(-1)) was lower than that of sham-operated rats (55.0+/-3.0 ml min(-1)), and this reduction was reversed (54.1+/-3.4 ml min(-1)) by treatment with trandolapril. The diminished responsiveness described above was normalized in the trandolapril-treated rat with CHF (i.e., the maximal relaxation to acetylcholine, 94.7+/-1.0%; that to isoprenaline, 80.5+/-2.8%; that to dibutyryl cyclic AMP, 54.7+/-6.2%). However, aortic segments of trandolapril-treated rats with CHF, L-NAME did not attenuate isoprenaline-induced relaxation and the tissue cyclic GMP level was not fully restored, suggesting that the ability of the endothelium to produce NO was still partially damaged. 6 The results suggest that vasorelaxation in CHF, diminished mainly due to dysfunction in endothelial nitric oxide (NO) production and cyclic AMP-mediated signal transduction, was partially restored by long-term treatment with trandolapril. The mechanism underlying the restoration may be attributed in part to prevention of CHF-induced endothelial dysfunction.

The Bronchorelaxant Effect of Helicidine, a Helix Pomatia Extract, Involves Prostaglandin E2 Release

Helicidine is a biological extract prepared from the snail Helix pomatia L. and used in man as an anti-tussive agent. However, its mechanisms of action are not fully defined. In this study, we have investigated a possible relaxant effect of helicidine on guinea-pig airway smooth muscle and evaluated the role of prostanoids and airway epithelium in this relaxation. H. pomatia extract (0.001–1 mg/ml) induced a dose-dependent relaxation of guinea-pig trachea pre-contracted with histamine both in the presence and absence of tracheal epithelium. No significant difference in dose-dependency or magnitude of the relaxation was observed between tracheal segments with or without epithelium (maximal relaxant response of 35 ± 7 and 25 ± 7.5%, respectively). Relaxation of the trachea induced by H. pomatia extract (0.001–1 mg/ml) was inhibited by pretreatment with the cyclooxygenase inhibitor, indomethacin, both in the presence or absence of tracheal epithelium. H. pomatia extract (1 mg/ml) induced a marked and significant increase in prostaglandin E 2 release in tracheal segments with and without epithelium. These results indicate that helicidine possesses a bronchorelaxant activity which is independent of epithelium integrity and which is partly mediated by the release of the relaxant prostanoid, prostaglandin E 2. The origin of prostaglandin E 2 production in the airways remains to be defined.