Stimulation of beta3-adrenoceptors relaxes rat urinary bladder smooth muscle via activation of the large-conductance Ca2+-activated K+ channels

We investigated the role of large-conductance Ca(2+)-activated K(+) (BK) channels in beta3-adrenoceptor (beta3-AR)-induced relaxation in rat urinary bladder smooth muscle (UBSM). BRL 37344, a specific beta3-AR agonist, inhibits spontaneous contractions of isolated UBSM strips. SR59230A, a specific beta3-AR antagonist, and H89, a PKA inhibitor, reduced the inhibitory effect of BRL 37344. Iberiotoxin, a specific BK channel inhibitor, shifts the BRL 37344 concentration response curves for contraction amplitude, net muscle force, and tone to the right. Freshly dispersed UBSM cells and the perforated mode of the patch-clamp technique were used to determine further the role of beta3-AR stimulation by BRL 37344 on BK channel activity. BRL 37344 increased spontaneous, transient, outward BK current (STOC) frequency by 46.0 +/- 20.1%. In whole cell mode at a holding potential of V(h) = 0 mV, the single BK channel amplitude was 5.17 +/- 0.28 pA, whereas in the presence of BRL 37344, it was 5.55 +/- 0.41 pA. The BK channel open probability was also unchanged. In the presence of ryanodine and nifedipine, the current-voltage relationship in response to depolarization steps in the presence and absence of BRL 37344 was identical. In current-clamp mode, BRL 37344 caused membrane potential hyperpolarization from -26.1 +/- 2.1 mV (control) to -29.0 +/- 2.2 mV. The BRL 37344-induced hyperpolarization was eliminated by application of iberiotoxin, tetraethylammonium or ryanodine. The data indicate that stimulation of beta3-AR relaxes rat UBSM by increasing the BK channel STOC frequency, which causes membrane hyperpolarization and thus relaxation.     

Different atypical β-adrenoceptors mediate isoprenalineinduced relaxation in vascular and non-vascular smooth muscles

Atypical β-adrenoceptors mediating smooth muscle relaxation were compared in several rat tissues including the distal colon, fundic strip, thoracic aorta and common carotid artery. Isoprenaline, CGP 12177 and BRL 37344 concentration-dependently relaxed longitudinal strips of the distal colon and fundus precontracted with carbachol (10⁻⁶M) as well as ring segments of the aorta and carotid artery precontracted with noradrenaline (10⁻⁷M). The rank order of potency was isoprenaline = BRL 37344 > CGP 12177 in the distal colon, isoprenaline = CGP 12177 > BRL 37344 in the aorta and carotid artery segments and isoprenaline > BRL 37344 > CGP 12177 in the fundic strip. Pretreatment with BRL 37344 induced a marked desensitization of the distal colon and fundic strips but not the aorta and carotid artery to isoprenaline. In the fundus and distal colon, pretreatment with CGP 12177 (10⁻⁴M) abolished the effect of isoprenaline. Cyanopindolol (⁻⁶M) shifted the isoprenaline curve to the right, without reducing the maximum response, in the distal colon and fundic strip — logK_B values were 7.44 ± 0.08 and 753 ± 0.10 in the distal colon and fundic strip respectively. The same concentration of cyanopindolol did not inhibit the relaxant effect of isoprenaline in the aorta and carotid artery segments. It was therefore concluded that atypical β-adrenoceptors in these preparations were not identical, indicating heterogeneity of atypical β-adrenoceptors.     

Further characterization of beta3-adrenoceptors in the guinea pig gastric fundus: stereoselectivity, beta-adrenoceptor alkylation, and structure-activity relationship

The stereoselectivity of beta3-adrenoceptors, the effect of a beta-adrenoceptor alkylating agent, and the structure-activity relationship at beta3-adrenoceptors were investigated on the guinea pig gastric fundus. Isomeric activity ratios ((+)/(-)) for isomers of isoprenaline and noradrenaline were 20.9-fold and 43.7-fold, respectively, and were less than those obtained for activation of beta1- and beta2-adrenoceptors in the guinea pig atria and trachea, respectively. The concentration-response curves to the catecholamines ((-)-isoprenaline, (-)-noradrenaline, and (-)-adrenaline), the selective beta3-adrenoceptor agonist BRL37344 ((R*,R*)-(+/-)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium), and the nonconventional partial beta3-adrenoceptor agonist (+/-)-CGP12177A ((+/-)-[4-[3-[(1,1-dimethylethyl) amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride) were resistant to blockade by (+/-)-pindobind (10 microM), the beta-adrenoceptor alkylating agent. Furthermore, (+/-)-nadolol, which belongs to the aryloxypropanolamine class and has beta1- and beta2-adrenoceptor antagonistic characteristics, displays agonistic activity at beta3-adrenoceptors. These results indicate that pharmacological characteristics of the beta3-adrenoceptors of guinea pig gastric fundus differ from those of beta1- and beta2-adrenoceptors. (-)-Noradrenaline and (-)-adrenaline were more potent than dopamine and (-)-phenylephrine, respectively. In addition, dobutamine was 22-fold more potent than dopamine. These results suggest that the 4-hydroxyl group at the catechol ring and the beta-hydroxyl group and the large moiety on the alkylamine chain characterized efficacy at beta3-adrenoceptors.     

Characterization of putative beta-adrenoceptors in the myometrium of the pregnant ewe: correlation between the binding of [3H] dihydroalprenolol and the inhibition of myometrial contractility in vitro

beta-Adrenoceptors in the myometrium of the pregnant ewe were studied both functionally and by radioligand binding techniques using [3H] dihydroalprenolol (DHA). Spontaneous contractile activity in vitro was inhibited by beta-adrenoceptor agonists in a stereoselective manner; the order of potency suggested a beta 2-adrenoceptor was involved. The effects of salbutamol were antagonized competitively by propranolol but the antagonism demonstrated by atenolol and ICI 118,551 (beta 1- and beta 2-selective, respectively) was complex. DHA binding was saturable, rapidly reversible, stereoselective and appeared to occur to a single class of noninteracting sites with a Kd of 4.1 +/- 0.3 nM and a maximal capacity of 0.8 +/- 0.05 pmol/mg protein. Agonists demonstrated the same order of potency in competition for ligand binding sites as in inhibition of contractile activity. These data suggested that the DHA binding sites were part of the beta-adrenoceptor. Strong agonists occupied only 0.1% of all receptors to produce the full response. Competition experiments with antagonists produced complex curves which could be resolved into two components comprising approximately 70% and 30% of the total number of sites. It was suggested that these sites represented beta 2- and beta 1-adrenoceptors, respectively. The possibility of regulation of the relative numbers of beta 2- and beta 1-adrenoceptors in the myometrium was discussed.     

Discrepancies in lipolytic activities induced by beta-adrenoceptor agonists in human and rat adipocytes

A number of catecholamine and non-catecholamine beta-adrenoceptor agonists, including the lipolytically selective compound BRL 37344, were compared for lipolytic activity on human and rat adipocytes. On rat adipocytes, all compounds were full agonists, BRL 37344 being the most potent. On human adipocytes, only the catecholamines were full beta-adrenoceptor agonists. The other compounds were partial agonists, with intrinsic activities declining in the order fenoterol greater than salbutamol greater than clenbuterol greater than BRL 37344. This was the case with FFA- as well as with glycerol-production. Addition of 20 microM phentolamine did not enhance BRL 37344 activity. The isoprenaline- and BRL 37344-induced lipolysis on rat white adipocytes was stereoselectively antagonized by enantiomers of alprenolol, with atypical low potencies and stereoselectivity. It was concluded that (1) human and rat adipocyte beta-adrenoceptors mediating lipolysis are not essentially different, (2) partial agonism in human adipocytes is not explained by enhanced re-esterification and (3) BRL 37344 selectively stimulates rat adipocyte lipolysis.     

Stress-induced alteration in the lipolytic response to beta-adrenoceptor agonists in rat white adipocytes.

We analysed the sensitivity to beta-adrenoceptor agonists in epididymal adipose cells from rats submitted to a stress protocol previously reported to induce alterations in sensitivity to catecholamines in cardiac tissue from rats. Food intake and body weight were lower, whereas adipocytes basal lipolysis was higher (control: 0.59 +/- 0.04; stress: 1.00 +/- 0.11, micromol glycerol/100 mg total lipids/100 min) in stressed compared to control rats. The responses to isoprenaline (pD(2) control: 7.46 +/- 0.11; stress: 8.11 +/- 0.17), adrenaline (pD(2) control: 5.78 +/- 0. 20; stress: 6.13 +/- 0.18), and salbutamol (pD(2) control: 5.64 +/- 0.28; stress: 5.92 +/- 0.34) were sensitized, and the lipolytic responses to norepinephrine (pD(2) control: 6.98 +/- 0.13; stress: 6. 41 +/- 0.12) and to BRL37344 (pD(2) control: 8.43 +/- 0.19; stress: 7.54 +/- 0.21) were desensitized. Responses to the higher concentration (100 microm) of isoprenaline (control: 1.80 +/- 0.18; stress: 2.24 +/- 0.10 micromol glycerol/100 mg total lipids/100 min), epinephrine (control: 1.64 +/- 0.17; stress: 2.24 +/- 0.14 micromol glycerol/100 mg total lipids/100 min), salbutamol (control: 0.65 +/- 0.11; stress: 1.21 +/- 0.41 micromol glycerol/100 mg total lipids/100 min), and d-butyryl-cAMP (control: 1.59 +/- 0.17; stress: 2.72 +/- 0.25) were significantly enhanced in adipocytes from stressed rats. pD(2) or maximum response to CGP12177 were not altered. Supersensitivity to isoprenaline was abolished by 50 nm ICI118,551 but was not modified by 100 nm metoprolol. However, subsensitivity to norepinephrine and to BRL37344 was abolished by 100 nM metoprolol. Our results suggest that in epididymal adipocytes from stressed rats there is a desensitization of the response to adrenoceptor agonists mediated by beta(1)-adrenoceptors together with a sensitization of the response mediated by beta(2)-adrenoceptors. beta(3)-adrenoceptors seem to be resistant to the stress effect.     

autoregulatory role of endothelium-derived nitric oxide (NO) on Lipopolysaccharide-induced vascular inducible NO synthase expression and function

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.     

Similar atypical β-adrenergic receptors mediate in vitro rat adipocyte lipolysis and colonic motility inhibition

We investigated the putative common nature of the rat atypical β-adrenoceptors mediating white adipocyte lipolysis and proximal colon motility inhibition, using the non-selective antagonist alphenolol and agonist isoprenaline and the selective agonists SR 58611A and BRL 37344. Results in either isolated intestinal and fat tissues were consistent with: isoprenaline acting through both typical (β₁, β₂) and atypical β-adrenoceptors; SR 58611A and BRL 37344 acting solely through the latter. The identical pA₂ values obtained with alprenolol, irrespective of the tissue and the selective agonist (SR 58611A or BRL 37344) used, support the high functional homology of the atypical β-adrenoceptors in rat colon and adipocytes.     

Potential involvement of a propranolol-insensitive atypical beta-adrenoceptor the vasodilator effect of cyanopindolol in the human pulmonary artery.

The aim of our study was to examine whether non beta(1)-/beta(2)-adrenoceptors participate in the relaxation of the human pulmonary artery. For this purpose the vasodilatory effect of the non-conventional partial beta-adrenoceptor agonist cyanopindolol was examined. Cyanopindolol (1-300 microM), studied in the presence of the beta(1)-/beta(2)-adrenoceptor antagonist propranolol, relaxed the human pulmonary artery preconstricted with serotonin 1 microM in a concentration-dependent manner (maximally by about 80%). This effect was diminished by bupranolol 10 microM (an antagonist of beta(1)-beta(3)-adrenoceptors and the low affinity state of the beta(1)-adrenoceptor) and CGP 20712 10 microM (known to antagonize the low-affinity state of the beta(1)-adrenoceptor at high concentrations). In further experiments, the effect of beta-adrenoceptor ligands on the serotonin-induced vasoconstriction was examined. The concentration-response curve for serotonin was not affected by cyanopindolol 30 microM, bupranolol 10 microM and CGP 20712 10 microM but shifted to the right by cyanopindolol 100 and 300 microM; the serotonin 5-HT(2A) receptor antagonist ketanserin 0.3 microM abolished the maximum contraction elicited by serotonin. In conclusion, the present study reveals that the vasodilatory effect of cyanopindolol in the human pulmonary artery consists of two components, i.e. activation of a propranolol-insensitive atypical beta-adrenoceptor and antagonism against 5-HT(2A) receptors.     

Different changes of plasma membrane beta-adrenoceptors in rat heart after chronic administration of propranolol, atenolol and bevantolol

Recently, tissue segment binding method with a hydrophilic radioligand [(3)H]-CGP12177 was developed to detect plasma membrane beta-adrenoceptors in rat heart (Horinouchi et al., 2006). In the present study, propranolol (40 mg kg(-1) day(-1)), atenolol (40 mg kg(-1) day(-1)) and bevantolol (200 mg kg(-1) day(-1)) were administered to rats for 6 weeks, and the changes of plasma membrane beta-adrenoceptors and their mRNA expression in rat ventricle were examined. Chronic administration of propranolol increased the beta(1)-adrenoceptors but decreased the beta(2)-adrenoceptors without changing total amount of plasma membrane beta-adrenoceptors. Atenolol increased both plasma membrane beta(1)- and beta(2)-adrenoceptors, whereas bevantolol had no effect on the beta-adrenoceptor density and their subtype proportions. In contrast, the density of beta-adrenoceptors detected in conventional homogenate binding study was extremely low (approximately 60% of plasma membrane beta-adrenoceptors detected with the tissue segment binding method) and the effects of chronic administration of beta-adrenoceptor antagonists were not necessarily in accord with those at the plasma membrane beta-adrenoceptors. The mRNA levels of beta(1)- and beta(2)-adrenoceptors were not altered by propranolol treatment, while beta(1)-adrenoceptor mRNA significantly decreased after administration of atenolol or bevantolol without changing the level of beta(2)-adrenoceptor mRNA. The present binding study with intact tissue segments clearly shows that the plasma membrane beta(1)- and beta(2)-adrenoceptors of rat heart, in contrast to the homogenate binding sites and the mRNA levels, are differently affected by chronic treatment with three beta-adrenoceptor antagonists; up- and down-regulations of beta(1)- and beta(2)-adrenoceptors, respectively, by propranolol, and up-regulation of both the subtypes by atenolol, but no significant change in both the subtypes by bevantolol.     

The action of adrenoceptor agonists and antagonists on the guinea pig and dog trachea

The action of beta- and alpha-adrenoceptor agonists (isoprenaline, orciprenaline, noradrenaline, phenylephrine and ephedrine) and antagonists (propranolol, metipranolol, exaprolol, BL 445 and phentolamine) on the resting tension and cAMP level of the guinea pig and the mechanical and electrical activities of the dog trachea were studied. By activating beta 2-adrenoceptors, isoprenaline and orciprenaline relaxed the smooth muscle, elevated the membrane potential and attenuated the excitatory effect of histamine on membrane potential and muscle tension. Noradrenaline and phenylephrine, acting on alpha 1-receptors, did not affect the membrane potential and increased the basal tension of the dog trachea only insignificantly. Ephedrine, in high concentrations, however, hyperpolarized the smooth muscle membrane and relaxed the dog trachea, while it did not alter the cAMP level in the guinea pig preparations. It is, therefore unlikely that alpha 1-adrenoceptors play a major role in the excitation of the dog trachea under resting conditions whereas the participation of alpha 2-receptors in the mechanisms of adrenergic relaxation could not be ruled out. All the beta-adrenoceptor antagonists studied enhanced the action of low isoprenaline concentrations and competitively antagonized it in high concentrations. The order of their antagonistic potency in the guinea pig trachea was as follows: metipranolol greater than propranolol = exaprolol greater than or equal to BL 445. It was suggested that metipranolol and exaprolol are nonselective beta-adrenoceptor antagonists, similarly as propranolol, whereas BL 445 shown some beta 1-selectivity. In contrast to their antagonistic effects on the membrane activities and muscle tension, both histamine and isoprenaline increased the level of cAMP in smooth muscle cells and, when present simultaneously, their effect was additive. The mechanism of histamine-induced cAMP level elevation and the possible involvement of different subcellular compartments in the action of isoprenaline and histamine in relation to the contraction-relaxation cycle is discussed.     

Endothelium-dependent and -independent vasorelaxation by a theophylline derivative MCPT: roles of cyclic nucleotides, potassium channel opening and phosphodiesterase inhibition

The vasorelaxation activities of MCPT, a newly synthesized xanthine derivative, were investigated in this study. In phenylephrine (PE)-precontracted rat aortic rings with intact endothelium, MCPT caused a concentration-dependent relaxation, which was inhibited by endothelium removed. This relaxation was also reduced by the presence of nitric oxide synthase inhibitor Nomega-nitro-L-arginine methylester (L-NAME, 100 microM), soluble guanylyl cyclase (sGC) inhibitors methylene blue (10 microM), 1 H-[1,2,4] oxidazolol [4,3-a] quinoxalin-1-one (ODQ, 1 microM), adenylyl cyclase (AC) blocker SQ 22536 (100 microM), ATP-sensitive K+ channel blocker (KATP) glibenclamide (1 microM), a Ca2+ activated K+ channels blocker tetraethylammonium (TEA, 10 mM) and a voltage-dependent potassium channels blocker 4-aminopyridine (4-AP, 100 microM). The vasorelaxant effects of MCPT together with IBMX (0.5 microM) had an additive action. In PE-preconstricted endothelium-denuded aortic rings, the vasorelaxant effects of MCPT were attenuated by pretreatments with glibenclamide (1 microM), SQ 22536 (100 microM) or ODQ (1 microM), respectively. MCPT enhanced cAMP-dependent vasodilator isoprenaline- and NO donor/cGMP-dependent vasodilator sodium nitroprusside-induced relaxation activities in endothelium-denuded aortic rings. In A-10 cell and washed human platelets, MCPT induced a concentration-dependent increase in intracellular cyclic GMP and cyclic AMP levels. In phosphodiesterase assay, MCPT displayed inhibition effects on PDE 3, PDE 4 and PDE 5. The inhibition % were 52 +/- 3.9, 32 +/- 2.6 and 8 +/- 1.1 respectively. The Western blot analysis on HUVEC indicated that MCPT increased the expression of eNOS. It is concluded that the vasorelaxation by MCPT may be mediated by the inhibition of phosphodiesterase, stimulation of NO/sGC/ cGMP and AC/cAMP pathways, and the opening of K+ channels.     

Pharmacologic characterization of cirazoline-activated inositol phospholipid hydrolysis in rat brain cortical slices

Interaction of cirazoline, an imidazoline derivative, with alpha 1-adrenoceptor coupled inositol phospholipid hydrolysis was characterized in rat brain cortical slices. Norepinephrine, a full alpha 1-agonist, and phenylephrine, a partial alpha 1-agonist, on inositol phospholipid hydrolysis were included for comparison. Norepinephrine produced a fourfold stimulation of inositol phospholipid hydrolysis, whereas cirazoline and phenylephrine caused only submaximal responses (40-60%) when compared with norepinephrine. The stimulation of inositol phospholipid hydrolysis by cirazoline was completely blocked by the alpha 1-adrenoceptor antagonist prazosin, but not by selective alpha 2- or beta-adrenoceptor antagonists. Furthermore, the norepinephrine dose-response curve was shifted to the right in the presence of cirazoline, without affecting the maximal response. These results suggest that cirazoline behaves as a partial agonist at brain alpha 1-adrenoceptors linked to inositol phospholipid hydrolysis.     

Beta 3-adrenergic receptor is involved in feeding regulation in chicks

We examined whether the brain beta 3-adrenergic receptor (B3-AR) is involved in the feeding regulation of chicks. Intracerebroventricular (ICV) injection of BRL37344, a B3-AR agonist, reduced food intake of chicks under ad libitum, but not fasting, feeding conditions. The ICV injection of BRL37344 did not affect chick posture or locomotion activity suggesting that BRL37344 inhibited feeding without induction of sleep-like behavior as caused by norepinephrine. Furthermore, the rectal temperature increased following the ICV injection of BRL37344. Intraperitoneal administration of BRL37344 did not reduce food intake under ad libitum feeding condition. The present study demonstrated that the brain B3-AR is involved in the inhibition of feeding in chicks. We also suggested that activation of the brain affects the energy metabolism in chicks.     

Comparison of the Lipolytic Effects of Norepinephrine and BRL 37344 in Rat Brown and White Adipocytes

The lipolytic effects of norepinephrine (a non-selective β-agonist) and BRL 37344 (a selective β₃-agonist) were compared in isolated rat brown and white adipocytes. Norepinephrine and BRL 37344 maximally stimulated lipolysis in brown and white adipocytes, approximately 10 times above basal values. However, adipocyte sensitivity for BRL 37344 was greater than that for norepinephrine, particularly in brown adipocytes [the EC₅₀ values (nM) for BRL 37344 and norepinephrine were 5 ± 1 and 103 ± 31 in brown adipocytes (P <0.01) versus 56 ± 9 and 124 ± 17 in white adipocytes (P <0.05), respectively]. On the other hand, the lipolytic effects of norepinephrine were totally blocked by 20–40 times superior concentrations of propranolol or bupranolol in brown as well as in white adipocytes. In contrast, the lipolytic effects of BRL 37344 were fully inhibited by concentrations of propranolol or bupranolol that were 200–1000 superior to the β₃ agonist concentration. The results demonstrate that: (1) the (β₃-agonist BRL 37344 is as effective as norepinephrine for maximally stimulating lipolysis in rat brown and white adipocytes, (2) both adipocyte types are more sensitive to the lipolytic effects of BRL 37344 than to those of norepinephrine, (3) although bupranolol is a better antagonist than propranolol on BRL 37344-stimulated lipolysis, it cannot be considered as a specific β₃-antagonist, (4) brown adipocytes are 10 times more sensitive than white adipocytes to the lipolytic effects of BRL 37344, suggesting an important role of β₃-receptors in brown adipose tissue.     

Estrogen Induces Nitric Oxide-Mediated Vasodilation of Human Mammary Arteriesin Vitro

Human internal mammary arteries (IMA) are relatively protected from atherosclerosis. Estrogen plays a protective role in cardiovascular disease. It causes in vitro and in vivo vasodilatation, but the mechanisms are contradictory. To investigate the in vitro vasomotor effect of estrogen on IMA and the role of endothelium, we studied 30 IMA segments harvested from 10 men during coronary artery bypass grafting surgery. Patients with diabetes mellitus, hypercholesterolemia, hypertension, and smoking were excluded. Twenty IMA rings had intact endothelium ((+)Endo) and 10 rings were denuded of endothelium ((-)Endo). Vasomotor response of each ring was expressed as the percentage of maximal response to norepinephrine (NE). Acetylcholine (10(-8)-10(-5) M) given to (+)Endo and (-)Endo rings induced vasorelaxation of 72 +/- 30.4% and vasoconstriction of 48.5 +/- 20.1%, respectively. 17-Beta-estradiol (10(-8)-10(-5) M) given after maximal precontraction with NE induced marked relaxation in (+)Endo (80.9 +/- 39.2%), but no significant vasomotor effect in (-)Endo rings (P < 0.0001). Vasorelaxation to 17-beta-estradiol (10(-6) M) in (+)Endo rings was 64.5 +/- 18.4 and 8.6 +/- 8.4%, before and after 15-min treatment with nitric oxide synthase inhibitor, L-nitroarginine methyl ester, respectively (n = 14, P < 0.0001). Tamoxifen (10(-6) M) decreased 17-beta-estradiol (10(-7) M)-induced relaxation by 71%. In conclusion, 17-beta-estradiol induces endothelium-dependent NO-mediated vasodilation of human mammary arteries in vitro. This response is mediated through estrogen receptors.     

Endothelial potentiation of relaxation response to ascorbic acid in rat and guinea pog thoracic aorta

The role of the endothelium was evaluated in the relaxation of rat and guinea pig aortic rings induced by ascorbic acid. Ascorbic acid relaxed rat and guinea pig aortic rings that were previously contracted with submaximal dose of phenylephrine (PE), in a concentration dependent manner. Removal of the endothelium significantly reduced the sensitivity but not the magnitude of the response to ascorbic acid. Methylene blue, but not propranolol, blocked the endothelial augmentation of vascular relaxation to ascorbic acid. Vessels precontracted with potassium chloride (high K⁺ were also relaxed by ascorbic acid. Methylene blue also inhibited the response to ascorbic acid in the intact vessels precontracted with high K⁺. A23187 and acetylcholine, but not ADP, variably caused endothelium dependent component relaxation in guinea pigs, whereas all of these three probes constantly caused it. In Ca²⁺-free medium, Ca²⁺-induced contraction of high K⁺-depolarized rat aorta was inhibited by the presence of ascorbate, which was more pronounced in endothelium intact rings than in endothelium denuded ones. PE-induced contraction in the presenced of different concentrations of ascorabte reduced both the sensitivity and the maximal contractile force in rat aorta. Ascorbic acid (0.125-32 mM) did not change the pH in the medium. From these findings, it is speculated that 1) receptor- and potential-operated Ca²⁺ channeld may be modulated by ascorbate, 2) endothelium has a significant role in promoting relaxation induced by ascorbic acid.     

Effects of autonomic drugs on contractions of rat seminal vesicles in vivo

Various autonomic drugs were placed on the peritoneal covering of the seminal vesicles of anaesthetized rats. Adrenaline (which stimulates the alpha-, beta 1- and beta 2-adrenoceptors) and phenylephrine (an alpha-stimulating agent) produced a sudden increase in tonus and in the amplitude and frequency of contractions. Phentolamine (an alpha-blocker) prevented these effects, whereas propranolol (a beta 1- and beta 2-blocker) did not. Phentolamine also abolished the seminal vesicle response to electrical stimulations. Terbutaline (a beta 2-stimulating agent) did not affect the spontaneous activity. There were no differences between the effects of terbutaline alone and those of terbutaline in the presence of propranolol. Moreover, propranolol did not block the contractile response of the gland to adrenaline or to electrical stimulation. These results indicate that alpha-adrenergic receptors are present in the muscle cell membrane of the rat seminal vesicle. The effects of acetylcholine were similar to those produced by adrenaline or phenylephrine although of smaller magnitude. Atropine prevented the effects of acetylcholine, indicating that they are of the muscarinic type.     

O2 and rat pulmonary artery tone: effects of endothelium, Ca2+, cyanide, and monocrotaline

The present study examines the influence of the endothelium (E), Ca2+ concentration, cyanide and monocrotaline (MCT) pretreatment on the responses of isolated rat hilar pulmonary arterial rings (PA) to hypoxia. In PA precontracted with phenylephrine, hypoxia induced an initial E-dependent relaxation phase followed by an E-independent transient contraction and a final relaxation. An increase in Ca2+ concentration from 1.5 to 2.5 mM produced an E-dependent reduction in tone generation under O2 and a significant enhancement of the hypoxia-elicited initial relaxation and the transient contractile responses. Addition of cyanide (0.1 mM) to precontracted PA produced a transient contraction similar to that caused by hypoxia. Preincubation with cyanide led to inhibition of tone generation and abolition of the contraction to hypoxia. However, the final relaxation response to hypoxia was not inhibited by cyanide. Thus, hypoxia produces an E-independent contraction via a mechanism that appears also to be activated by cyanide, and this response is not altered by MCT. The endothelium alters the response to hypoxia in a Ca(2+)-dependent manner.