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The beta-adrenoceptor subtype that mediates adrenaline-induced relaxation was pharmacologically identified in smooth muscle cells of the isolated guinea-pig trachea. Adrenaline produced a concentration-dependent relaxation with a pD(2) value of 7.1. The concentration-response curve for adrenaline was shifted rightwards in a competitive fashion by the beta(1)-/beta(2)-nonselective antagonists propranolol and bupranolol, with pA(2) values of 8.85 and 8.97, respectively. Adrenaline-induced relaxation was not affected by the beta(1)-selective antagonists atenolol and CGP-20, 712A within the concentration ranges supposed to antagonize the beta(1)-subtype (atenolol, or=3x10(-6) M with a pA(2) value of 5.77. The concentration-response curve for adrenaline was also competitively antagonized by the beta(2)-selective antagonists butoxamine and ICI-118,551 with pA(2) values of 6.86 and 8.73, respectively. The pA(2) values of beta-adrenoceptor antagonists (propranolol, bupranolol, atenolol, butoxamine and ICI-118,551) tested against adrenaline were consistent with the values when tested against salbutamol, a beta(2)-selective adrenoceptor agonist. The present findings provide evidence that the relaxant response of the smooth muscle of the guinea-pig trachea to the adrenal medulla hormone, adrenaline, is mainly mediated through beta(2)-adrenoceptors.

Free radicals are involved in several diseases, including cancer, central nervous system alterations and inflammatory pathologies. Peroxidase is an oral enzyme implicated in the defence of oral cavity. It has been determined that flavonoids and lignans possess antioxidant and free radical scavenging either directly or indirectly, usually by means of increasing the secretion of free radicals scavenger enzymes. Larrea divaricata Cav. is a plant used in folk Argentine medicine for the treatment of cancer and inflammatory ailments. In this study, we have determined the effect and mechanism of action of an aqueous extract of the leaves of L. divaricata and NDGA on peroxidase secretion in female rat submandibular glands. The extract significantly increased the secretion and total peroxidase. % of secreted peroxidase (X +/- S.E.M.): extract maximum response: 150 +/- 10; % of total peroxidase (X +/- S.E.M.): extract maximum response: 1000 +/- 90. The effect of the extract on peroxidase secretion was mediated by beta1 adrenoceptors (% of secreted peroxidase: extract + atenol maximum response: 50 +/- 4 ). Meanwhile, NDGA produced a decrease in peroxidase secretion (peroxidase secreted: basal: 0.44 +/- 0.03; NDGA 2.5 x 10(-6) M: 0.20 +/- 0.02; prostaglandins E2 (PGE2) 10(-7)M: 1.32 +/- 0.5; NDGA + PGE2: 0.46 +/- 0.035), an effect that was exerted by the inhibition on prostaglandins synthesis.

1. The present study was carried out to pharmacologically identify the beta-adrenoceptor subtype that mediates isoprenaline-elicited relaxation in the isolated guinea-pig tracheal smooth muscle, to answer the question whether it is beta(1)- or beta(2)-subtype? 2. Isoprenaline as well as salbutamol, a well-known beta(2)-selective adrenoceptor agonist, produced a concentration-dependent relaxation with a pD(2) value of 8.12 vs. 7.54 for salbutamol. 3. Isoprenaline-elicited relaxation was not affected by beta(1)-selective antagonists, atenolol and CGP-20,712A, within the concentration ranges supposed to antagonize beta(1)-subtype: atenolol,or =10(-6) M; CGP-20,712A,or =10(-8) M. 4. By contrast, the concentration-response curves for isoprenaline as well as salbutamol were shifted rightwards in a competitive fashion by atenolol at the concentrationsor =3 x 10(-6) M. However, pA(2) values of atenolol against isoprenaline (5.86) and salbutamol (5.71) were consistent with the value corresponding to beta(2)- but not to beta(1)-subtype (around 7.00), and these values were not significantly different from each other. 5. Competitive antagonism of the relaxations to isoprenaline and salbutamol were also obtained with beta(2)-selective antagonists, butoxamine and ICI-118,551. Against isoprenaline and salbutamol, the pA(2) values of butoxamine (6.51 vs. 6.81) and ICI-118,551 (8.83 vs. 8.90) were substantially identical. Thus the primary mediation of beta(2)-receptor in the relaxations was strongly supported. 6. The present findings provide evidence that the beta-adrenoceptor which mediates isoprenaline-elicited relaxation of guinea-pig tracheal smooth muscle is essentially beta(2)- but not beta(1)-subtype. The present study also indicates the importance of using multiple receptor antagonists with different pA(2) values to pharmacologically identify the responsible receptor subtype in smooth muscle mechanical responses.

We previously reported that the central nucleus of the amygdala (CeA) plays a crucial role in the negative affective, rather than somatic, component of morphine withdrawal. However, numerous studies have reported that the central ascending noradrenergic system is implicated in morphine withdrawal syndrome, although the roles of the noradrenergic system within the CeA in the negative affective component remains less clear.The possible role of the noradrenergic system within the CeA in the negative affective component of morphine withdrawal was investigated.The extracellular noradrenaline level within the CeA during naloxone-precipitated morphine withdrawal was measured using an in vivo microdialysis experiment on unanesthetized and freely moving rats. The effects of microinjection of beta-adrenoceptor antagonists into the bilateral CeA on the naloxone-precipitated morphine withdrawal-induced conditioned place aversion (CPA) and somatic signs were examined.The extracellular noradrenaline level within the CeA was transiently elevated during morphine withdrawal. Intra-CeA injections of beta-adrenoceptor antagonists propranolol (30 nmol per side) and timolol (10 nmol per side) significantly attenuated the morphine withdrawal-induced CPA. Similarly, beta(1)-antagonist atenolol (30 nmol per side) or beta(2)-antagonist butoxamine (30 nmol per side) significantly attenuated the CPA. In contrast, they did not affect morphine withdrawal-induced somatic signs, except for propranolol.These results suggest that the activation of the noradrenergic system within the CeA contributes to naloxone-precipitated morphine withdrawal-induced CPA, rather than somatic signs, through beta(1)- and beta(2)-adrenoceptors.

Nebivolol is a highly selective beta(1) adrenoceptor blocker with additional vasodilating properties. Although it has been shown that the nebivolol-induced vasorelaxation is nitric oxide (NO) and cGMP dependent, the receptor that mediates these actions remains controversial, and serotonergic as well as beta-adrenergic pathways may be involved. Therefore, functional experiments investigating the receptor involved in nebivolol-induced vasorelaxation were performed in the rat aorta. Isolated aortic rings were exposed to cumulative concentrations of nebivolol. Nebivolol concentrations of 3 micromol/L and higher caused vasorelaxation, which was inhibited by the presence of the NO synthase inhibitor l-NNA (100 micromol/L), or by mechanical removal of the endothelium. Exposure of the vessel rings to the selective 5-HT(1A) antagonist NAN-190 (1 micromol/L) or the 5-HT(1/2) antagonist methysergide (1 micromol/L) did not influence nebivolol-induced vasorelaxation. Similarly, the incubation with the beta(2)-adrenoceptor antagonist butoxamine (50 micromol/L) did not prevent vasorelaxation. The selective beta(3)-adrenoceptor antagonist S-(-)-cyanopindolol (1 micromol/L), however, significantly counteracted the nebivolol-induced vasorelaxation. Furthermore, exposure of the aortic rings to cumulative concentrations of the beta(3) selective adrenoceptor agonist BRL37344 caused, like nebivolol, NO-dependent vasorelaxation that was antagonized by S-(-)-cyanopindolol. The results suggest that nebivolol-induced NO-dependent vasorelaxation is, at least in part, caused by a beta(3)-adrenoceptor agonistic effect.

Molecular mechanisms underlying the metamorphosis of larvae, e.g., ligand and receptor interaction, have to be determined and roles for the nervous system in marine invertebrates are not well understood. We report here that treatment of swimming larvae of the ascidian Ciona savignyi with noradrenaline or adrenaline promoted morphological changes in early metamorphosis, e.g., tail resorption. Antagonists of the beta-adrenergic receptor, propranolol, and the beta(1)-adrenergic receptor, metoprolol, inhibited the noradrenaline-induced tail resorption, while an antagonist of the alpha-adrenergic receptor, phentolamine, and of the beta(2)- adrenergic receptor, butoxamine, had no inhibitory effects. In addition, a selective agonist of the beta-adrenergic receptor, isoproterenol, the concentration of which was lower than the effective concentration of the neurotransmitters, facilitated tail resorption. Immunohistochemical studies, using an anti-dopamine-hydroxylase antibody, showed that neurotransmitters such as noradrenaline and adrenaline localized around the brain vesicle of the larvae during metamorphosis. The beta(1)-adrenergic receptor stained with antibodies was localized on the nervous system. Temporal expression of the beta(1)-adrenergic receptor was intense in the nervous system in the larvae competent for metamorphosis. We propose that interactions between noradrenaline or adrenaline and the beta(1)-adrenergic receptor in the nervous system mediate the process of metamorphosis of Ciona larvae.

OBJECTIVE: The sympathetic nervous system (SNS) is commonly activated in hypertension; however, the role of SNS activation in the pathogenesis of cardiovascular structural changes remains poorly defined. In particular, the effect of adrenergic stimulation on extracellular matrix (ECM) protein production by human cardiovascular cells is unknown. The present study thus investigated the direct effect of adrenergic stimulation on ECM protein production by cultured human vascular smooth muscle (VSM) cells. METHODS AND RESULTS: Exposing human VSM cells to norepinephrine increased collagen protein production by 42%, P < 0.01, when compared to control (unstimulated) cells. This effect was mediated by the alpha1-adrenoceptor, since it was inhibited by the selective alpha1-adrenoceptor antagonist; prazosin (2 micromol/l) and reproduced by the selective alpha1-adrenoceptor agonist; phenylephrine (10 micromol/l). In contrast, beta-adrenoceptor stimulation - isoprenaline (1 micromol/l) or norepinephrine (10 micromol/l) + prazosin (2 micromol/l) - inhibited collagen production by 12%, P < 0.01. This inhibitory effect was mediated via the beta1-adrenoceptor, since it was blocked by atenolol (beta1-adrenoceptor antagonist) but not butoxamine (beta2-adrenoceptor antagonist). Fibronectin, another ECM protein, was similarly regulated by alpha- and beta-adrenoceptor stimulation. Transforming growth factor beta1 (TGFbeta1) mRNA expression by human VSM cells was also significantly influenced by adrenergic stimulation, being increased by phenylephrine (alpha-agonist) and inhibited by isoprenaline (beta-agonist). CONCLUSIONS: These results uniquely demonstrate the capacity for adrenergic stimulation to directly modulate TGFbeta1 expression and ECM protein synthesis by the human cardiovascular system.

Recently, involvement of the sympathetic nervous system in bone metabolism has attracted attention. β2-Adrenergic receptor (β2-AR) is presented on osteoblastic and osteoclastic cells. We previously demonstrated that β-AR blockers at low dose improve osteoporosis with hyperactivity of the sympathetic nervous system via β2-AR blocking, while they may have a somewhat inhibitory effect on osteoblastic activity at high doses. In this study, the effects of butoxamine (BUT), a specific β2-AR antagonist, on tooth movement were examined in spontaneously hypertensive rats (SHR) showing osteoporosis with hyperactivity of the sympathetic nervous system. We administered BUT (1 mg/kg) orally, and closed-coil springs were inserted into the upper-left first molar. After sacrifice, we calculated the amount of tooth movement and analyzed the trabecular microarchitecture and histomorphometry. The distance in the SHR control was greater than that in the Wistar-Kyoto rat group, but no significant difference was found in the SHR treated with BUT compared with the Wistar-Kyoto rat control. Analysis of bone volume per tissue volume, trabecular number, and osteoclast surface per bone surface in the alveolar bone showed clear bone loss by an increase of bone resorption in SHR. In addition, BUT treatment resulted in a recovery of alveolar bone loss. Furthermore, TH-immunoreactive nerves in the periodontal ligament were increased by tooth movement, and BUT administration decreased TH-immunoreactive nerves. These results suggest that BUT prevents alveolar bone loss and orthodontic tooth movement via β2-AR blocking.

The expression and effects of β-adrenergic receptors (β-ARs) on the neurons of the bulbospinal rostral ventrolateral medulla (RVLM) have been limitedly examined to date. The objective of this study was to examine the expression of β1- and β2-ARs on the bulbospinal RVLM neurons electrophysiologically and histologically. To directly investigate whether RVLM neurons display sensitivity to metoprolol (a β1-AR antagonist), dobutamine (a β1-AR agonist), butoxamine (a β2-AR antagonist), and salbutamol (a β2-AR agonist), we examined changes in the membrane potentials of the bulbospinal RVLM neurons using the whole-cell patch-clamp technique during superfusion of these drugs. During metoprolol superfusion, 16 of the 20 RVLM neurons were hyperpolarized, and 5 of the 6 RVLM neurons were depolarized during dobutamine superfusion. During butoxamine superfusion, 11 of the 16 RVLM neurons were depolarized, and all of the 8 RVLM neurons were hyperpolarized during salbutamol superfusion. These results suggest the expression of β1- and β2-ARs on the RVLM neurons. To determine the presence of β1- and β2-ARs histologically, immunofluorescence examination was performed. Five metoprolol-hyperpolarized neurons were examined for β1-AR and tyrosine hydroxylase (TH) immunoreactivity. All of the neurons displayed β1-AR immunoreactivity, whereas three of the neurons displayed TH immunoreactivity. All of the five RVLM neurons that became depolarized during metoprolol superfusion and hyperpolarized during butoxamine superfusion displayed β1- and β2-AR immunoreactivity. Our findings suggest that β1-AR antagonists or β2-AR agonists may decrease blood pressure through decreasing the activity of the bulbospinal RVLM neurons.

Chronic visceral pain in patients with irritable bowel syndrome (IBS) has been difficult to treat effectively partially because its pathophysiology is not fully understood. Recent studies show that norepinephrine (NE) plays an important role in the development of visceral hypersensitivity. In this study, we designed to investigate the role of adrenergic signaling in visceral hypersensitivity induced by heterotypical intermittent stress (HIS). Abdominal withdrawal reflex scores (AWRs) used as visceral sensitivity were determined by measuring the visceromoter responses to colorectal distension. Colon-specific dorsal root ganglia neurons (DRGs) were labeled by injection of DiI into the colon wall and were acutely dissociated for whole-cell patch-clamp recordings. Blood plasma level of NE was measured using radioimmunoassay kits. The expression of β2-adrenoceptors was measured by western blotting. We showed that HIS-induced visceral hypersensitivity was attenuated by systemic administration of a β-adrenoceptor antagonist propranolol, in a dose-dependent manner, but not by a α-adrenoceptor antagonist phentolamine. Using specific β–adrenoceptor antagonists, HIS-induced visceral hypersensitivity was alleviated by β2 adrenoceptor antagonist but not by β1- or β3-adrenoceptor antagonist. Administration of a selective β2-adrenoceptor antagonist also normalized hyperexcitability of colon-innervating DRG neurons of HIS rats. Furthermore, administration of β-adrenoceptor antagonist suppressed sustained potassium current density (I K) without any alteration of fast-inactivating potassium current density (I A). Conversely, administration of NE enhanced the neuronal excitability and produced visceral hypersensitivity in healthy control rats, and blocked by β2-adrenoceptor antagonists. In addition, HIS significantly enhanced the NE concentration in the blood plasma but did not change the expression of β2-adrenoceptor in DRGs and the muscularis externa of the colon. The present study might provide a potential molecular target for therapy of visceral hypersensitivity in patents with IBS.

The properties of the beta1-/beta2-adrenoceptor partial agonist carteolol were investigated in atypical beta-adrenoceptors on the guinea pig gastric fundus. Carteolol induced concentration-dependent relaxation in this tissue (pD2 = 5.55, intrinsic activity = 0.94). However, a combination of the selective beta1-adrenoceptor antagonist atenolol (100 microM) and the selective beta2-adrenoceptor antagonist butoxamine (100 microM) produced only small rightward shifts in the concentration-response curves of carteolol in the gastric fundus (pD2 = 4.91, intrinsic activity = 0.94). In the presence of both atenolol (100 microM) and butoxamine (100 microM), the non-selective beta1-, beta2- and beta3-adrenoceptor antagonist (+/-)-bupranolol (10-100 microM) caused a concentration-dependent right-ward shift of the concentration-response curves for carteolol in the guinea pig gastric fundus. Schild plot analyses of the effects of (+/-)-bupranolol against carteolol gave the pA2 value of 5.29 and the Schild slope was not significantly different from unity. Furthermore, carteolol (10 microM) weakly but significantly antagonized the relaxant responses to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), a selective beta3-adrenoceptor agonist BRL37344 ((R*,R*)-(+/-)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxy-acetic acid sodium salt) and a non-conventional partial beta3-adrenoceptor agonist (+/-)-CGP12177A ([4-[3-[(1,1dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride) in the guinea pig gastric fundus. These results suggest that the partial agonistic effects of carteolol are mediated by atypical beta-adrenoceptors in the guinea pig gastric fundus.

Background —Nebivolol is a β 1 -selective adrenergic receptor antagonist with proposed nitric oxide (NO)–mediated vasodilating properties in humans. In this study, we explored whether nebivolol indeed induces NO production and, if so, by what mechanism. We hypothesized that not nebivolol itself but rather its metabolites augment NO production. Methods and Results —Mouse thoracic aorta segments were bathed in an organ chamber. Administration of nebivolol did not affect NO production. When nebivolol was allowed to metabolize in vivo in mice, addition of plasma of these mice caused a sustained 2-fold increase in NO release. Interestingly, coadministration of a selective β 2 -adrenergic receptor antagonist (butoxamine) prevented the response. Immunohistochemistry and Western blot analysis demonstrated the presence of β 2 - but not β 1 -adrenergic receptors on endothelial cells. In the absence of calcium, metabolized nebivolol failed to increase NO production, suggesting a role for calcium-dependent NO synthase. With digital fluorescence imaging, a rapid and sustained rise in endothelial cytosolic free Ca 2+ concentration was observed after administration of metabolized nebivolol, which also was abrogated by butoxamine pretreatment. Conclusions —In vivo metabolized nebivolol increases vascular NO production. This phenomenon involves endothelial β 2 -adrenergic receptor ligation, with a subsequent rise in endothelial free [Ca 2+ ] i and endothelial NO synthase–dependent NO production. This may be an important mechanism underlying the nebivolol-induced, NO-mediated arterial dilation in humans.

Intracerebroventricular (icv) injection of interleukin-1beta (IL-1beta) in rats induces elevated IL-6 levels in peripheral blood, exceeding those induced by iv or ip injection. Two hypotheses postulated to explain this phenomenon were tested. Mediation by peripheral sympathetic activation was excluded by showing that agents that blocked preganglionic cholinergic synapses (chlorisondamine), beta-adrenergic receptors (propanalol, butoxamine), and alpha-adrenergic receptors (phentolamine) did not prevent the IL-6 response. That the peripheral response was due to passage of the injected IL-1beta into blood from the brain was supported by several observations. Immunoreactive IL-1beta appeared in peripheral blood by 10 min after icv injection and remained constant between 10-100 min after injection; values after icv injection were virtually identical to those after iv injection at 60 and 80 min. Radioiodine-labeled IL-1beta appeared in blood as early as 5 min, and by phamacokinetic analysis was found to be transferred from the brain at a rate greater than 2% of brain content per min(-1). IL-1beta infused iv in a pattern mimicking brain to blood transfer induced IL-6 levels that were more than double the values induced by a single bolus injection and were not significantly different from the values observed after icv injection. Sustained levels of IL-1beta in blood over time contribute to the high peripheral IL-6 response. This was shown by administering the same total dose iv as a single bolus of 100 ng or in two doses of 50 ng 1 h apart. Rats given a divided dose had 6-10 times higher blood IL-6 levels at 2 h than those given a single injection. The high levels of IL-6 in blood after icv injection of IL-1beta are best explained by the reservoir function of the brain IL-1beta pool and the self-priming effect of IL-1beta in peripheral tissues.

With the rise in the field of neuroimmunomodulation research, there is increased recognition of the influence of the nervous system and neuropeptides in peripheral disease. The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) is a neuroimmunomodulatory agent that modulates production of proinflammatory cytokines and inhibits peripheral inflammation via actions on CNS receptors. We examined whether central alpha-MSH operates by inhibiting activation of the nuclear factor kappa B (NF-kappaB) that is essential to the expression of proinflammatory cytokines and development of inflammation in the periphery. Electrophoretic mobility shift assays of nuclear extracts from the murine foot pad injected with TNF-alpha demonstrated that centrally administered alpha-MSH does inhibit NF-kappaB activation. Western blot analysis revealed that this inhibition was linked to central alpha-MSH-induced preservation of expression of IkappaBalpha protein in the peripheral tissue. The NF-kappaB and IkappaBalpha effects were inhibited in mice with spinal cord transection. Intraperitoneal (i.p.) injection of the nonspecific beta-adrenergic receptor blocker propranolol, and of a specific beta2-adrenergic receptor antagonist, likewise prevented these effects of central alpha-MSH; blockade of cholinergic, alpha-adrenergic, or beta1-adrenergic receptors did not. Centrally administered alpha-MSH inhibited peripheral NF-kappaB activation and IkappaBalpha degradation even in mice with nonfunctional melanocortin 1 receptors (MC1R). These findings indicate that alpha-MSH can act centrally to inhibit NF-kappaB activation in peripheral acute inflammation via a descending neural pathway. The pathway involves beta2-adrenergic receptors, but does not require activation of MC1R within the brain.

It has been previously shown in anesthetized pigs that intravenous infusion of 2 microg/h of 17beta-estradiol primarily dilated renal, iliac and coronary circulations, while higher doses of the hormone were required to cause vasodilation also in the mesenteric vascular bed. In the same experimental model, a tonic beta2-adrenoceptor mediated vasodilation, which could be argued to attenuate the vasodilator effect of 17beta-estradiol, has been described. The present study was planned to investigate the role of beta2-adrenergic receptors in the hemodynamic responses of renal and mesenteric vascular beds to 17beta-estradiol. Changes in flow caused by intravenous infusion of 2 microg/h of the hormone at constant heart rate and aortic blood pressure in the left renal and superior mesenteric arteries were assessed using electromagnetic flowmeters. In six pigs, infusion of 17beta-estradiol caused an increase in renal blood flow, which averaged 12.1% of the control values, without affecting mesenteric blood flow. In the same pigs, after hemodynamic variables had returned to the baseline values, blockade of beta2-adrenergic receptors with butoxamine caused an increase in aortic blood pressure and an increase in renal and mesenteric resistance. The subsequent infusion of 17beta-estradiol elicited increases in renal and mesenteric blood flow which respectively averaged 19.6% and 12.8%. Therefore, the present study in anesthetized pigs have shown that the vasodilator responses of the renal and mesenteric circulations to 17beta-estradiol were attenuated and even masked by a tonic beta2-adrenoceptor mediated vasodilation. This indicates that some vasodilator effects elicited by normally used replacement doses of the hormone may not be apparent.

Abstract —Stimulation of vascular β-adrenoceptors leads to membrane hyperpolarization, presumably via the β-adrenoceptor/G s protein/adenylate cyclase signaling cascade; the ionic mechanisms of this phenomenon remain unclear. β-Adrenoceptor–mediated vascular relaxation is impaired with aging; however, little is known concerning whether β-adrenoceptor–mediated hyperpolarization is altered with aging. We sought to determine the ionic mechanisms of isoproterenol-induced hyperpolarization in the rat mesenteric resistance artery, as well as the age-related changes in isoproterenol-induced hyperpolarization and their underlying mechanisms. Isoproterenol-induced hyperpolarization was inhibited by high-K + solution and glibenclamide (10 −6 mol/L), an inhibitor of ATP-sensitive K + channels (K ATP ), but not by apamin, iberiotoxin, or charybdotoxin, inhibitors of Ca 2+ -activated K + channels. Isoproterenol-induced hyperpolarization was markedly less in aged rats (≥24 months) than in adults rats (12 to 20 weeks) (3×10 −6 mol/L; −3.1 versus −9.9 mV; P −9 g/mL), an activator of G s , evoked hyperpolarization only in adult rats. Hyperpolarization to forskolin, a direct activator of adenylate cyclase, was also reduced to some extent in aged rats (10 −5 mol/L; −8.8 versus −13 mV; P ATP opener, was comparable in both groups. These findings suggest that isoproterenol elicits hyperpolarization via an opening of K ATP in the rat resistance artery and that isoproterenol-induced hyperpolarization is attenuated in aged rats mainly because of a defective coupling of β-adrenoceptors to adenylate cyclase and partly because of a defect at the level of adenylate cyclase, but not because of an alteration of K ATP per se.

1. Isoproterenol relaxed KCl-precontracted rat bladder strips with a pD2 of 7.21 leaving a residual contractile response of 3.2% after 30 microM. The selective beta1-agonist, T-0509 (pD2 : 6.24, 10.1% residual contraction after 100 microM), beta2-agonist, terbutaline (pD2 : 5.43, 13.7% residual contraction after 100 microM), and beta3-agonists, BRL 37344A (pD2 : 6.60, 17.3% residual contraction after 100 microM), and SR 58611A (pD2 : 5.15, 34.0% residual contraction after 100 microM), also relaxed bladder strips. 2. The relaxant response to isoproterenol was weakly but significantly antagonized by 1 microM propranolol which produced a 3 fold shift of the concentration-response curve to the right, and significantly antagonized by the beta1-selective antagonist, metoprolol (10 microM, 3 fold shift), and the beta2-selective antagonist, butoxamine (100 microM, 6 fold shift). A combination of 10 microM metoprolol and 100 microM butoxamine caused a 15 fold shift of the concentration-response curve for isoproterenol to the right. Incubation with the beta3-antagonist, SR 59230A (1 microM), caused a 6 fold shift of the concentration response curve for isoproterenol to the right. 3. The non-conventional partial agonist, CGP 12177A, weakly relaxed KCl-precontracted bladder strips (pD2 : 3.31, 51.3% residual contraction after 300 microM); the relaxation was resistant to blockade by 1 or 10 microM propranolol. 4. In the presence of 200 microM propranolol, CGP 12177A (20 microM) or SR 59230A (10 microM) antagonized surmountably the relaxant effects of BRL 37344A. 5. The data suggest that rat urinary bladder body contains beta1, beta2, and beta3-adrenoceptors, all of which mediate relaxation.

We previously suggested that the existence of atypical beta/beta3-adrenoceptor with pA2-values for bupranolol, a non-selective beta-adrenoceptor antagonist, against BRL37344 and noradrenaline were 5.79 and 5.53 in guinea pig taenia caecum, respectively. We furthermore determined the affinity of bupranolol to subclassify atypical beta/beta3-adrenoceptor in rat oesophageal muscularis mucosae, because it is rich in atypical beta/beta3-adrenoceptor. BRL37344 and noradrenaline produced a concentration-dependent relaxation of rat oesophageal muscularis mucosae. The responses to BRL37344 and noradrenaline were resistant to 3x10(-6) M propranolol, 10(-4) M atenolol, and 10(-4) M butoxamine. However, bupranolol antagonized the responses to BRL37344 and noradrenaline in a concentration-dependent manner. Schild plot analyses of bupranolol against BRL37344 and noradrenaline gave pA2-values of 7.06 and 6.96, respectively. These results suggest that bupranolol can distinguish the difference in affinity between atypical beta/beta3-adrenoceptors in rat oesophageal muscularis mucosae and guinea pig taenia caecum. The difference in behavior of bupranolol confirms the existence of some atypical beta/beta3-adrenoceptors subtypes.

The subtype of beta-adrenergic receptors in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus was studied. Pigment of denervated melanophores in isolated, split caudal fins was preliminarily aggregated by incubating the specimens in a physiological saline containing 10 microM phentolamine and 30-100 microM verapamil or 2-10 nM melatonin, and the responses of the melanophores to a beta-adrenergic agonist added to the incubating medium were recorded photoelectrically. The beta-adrenergic agonists noradrenaline, adrenaline, isoproterenol, salbutamol and, dobutamine were all effective in evoking a dispersion of melanophore pigment in the presence of phentolamine and verapamil or melatonin. The pigment-dispersing effect of noradrenaline (beta 1-selective agonist) was inhibited by metoprolol (beta 1-selective antagonist), propranolol,- and butoxamine. Whereas, the effect of salbutamol (beta 2-selective agonist) was hardly inhibited by metoprolol, though it was considerably inhibited by propranolol and ICI-118551. It was estimated that beta 1- and beta 2-adrenergic receptors coexist at ratios of 8.6:91.4, in the melanophore of Tridentiger trigonocephalus, and 25:75, in the melanophore of Chasmichthys gulosus, through the analyses of Hofstee plots of the effects of the beta-adrenergic drugs. It was suggested that the relation between the pigment-dispersing effect of a beta-adrenergic agonist on the melanophores and the concentration of the drug follows mass action kinetics, when the effect is mainly caused by the activation of beta 2-adrenergic receptors of the melanophores. However, when it is mainly caused by the activation of beta 1-adrenergic receptors of the melanophores, the relation does not follow mass action kinetics.

Open-field behavior was compared between untreated, saline-treated, and butoxamine (a beta-2-adrenoceptor antagonist) treated (15 and 5 mg/kg body weight) male golden hamsters (Mesocricetus auratus). Butoxamine-treated males spent significantly more time self-grooming than saline-treated and untreated males. Especially, untreated males and very similar saline control males showed a clear time sequential structure of behavior with two axes: a grooming-grooming sequence and a highly organized flankmark followed by scrape axis. The degree of this organization was markedly decreased in the butoxamine-treated males, showing an increase in organization of the transition between grooming acts. In general, these males displayed a more stereotypic pattern of behavior than the other. To reveal a systematic relation between the flankmark-scrape response and the exposition to butoxamine and to keep the numbers of experimental animals low, mixed samples were created through consecutive summing of the individual transition matrices of six males treated with 5 mg/kg b.wt. butoxamine to the 15 mg/kg b.wt. sample. By analyzing all samples separately, a positive linear relation between the number of low dosed males in the samples and the degree of organization of the flankmark-scrape sequence was found. The results suggest that the analysis of the transitional structure of behavior during short-term challenges can considerably contribute to an estimation of the coping style and seems to be a more sensitive method than comparing frequencies of behavioral indicators of stress.

Mechanisms of relaxation induced by nerve stimulation were examined in isolated porcine iris sphincter muscle in reference to norepinephrine, nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) and the functional interaction of inhibitory and excitatory nerves. Changes in isometric tension were recorded in strips of the sphincter pupillae, which were stimulated by transmurally applied electrical pulses. The presence of neurons containing acetylcholinesterase and tyrosine hydroxylase (TH) was determined histochemically. Transmural electrical stimulation (0.5-20 Hz) produced a frequency-related contraction, which was reversed to a relaxation by atropine in prostaglandin F2alpha-contracted strips. The relaxant response was abolished by timolol and suppressed by metoprolol, a beta1-adrenoceptor antagonist, but was not influenced by butoxamine, a beta2-receptor antagonist. Norepinephrine-induced relaxations were also attenuated only by timolol and metoprolol. Treatment with NG-nitro-L-arginine, a NO synthase inhibitor, and [D-p-Cl-Phe6,Leu17]VIP, a VIP receptor antagonist, did not inhibit the neurogenic relaxation. Contractions induced by nerve stimulation were potentiated by timolol and physostigmine but not by the NO synthase inhibitor. In the sphincter muscle, cholinesterase- and TH-positive nerve fibers and bundles were histologically detected. It is concluded that porcine iris sphincter is innervated by cholinergic excitatory and adrenergic inhibitory nerves. The neurogenic relaxation is associated solely with activation of beta1 adrenoceptors by norepinephrine but is not mediated by NO or VIP.

The actions of different adrenoceptor antagonists on gastric potential difference (PD), electrical current (I) and resistance (R) were studied, using the voltage clamp technique. In an isolated gastric mucosal tissue, 5% ethanol was able to reduce the PD and I across the gastric mucosa. Direct incubation with propranolol 10–4 mol/l either from the mucosal or submucosal sides attenuated such effects. Intraperitoneal administration of propranolol (2.5–10 mg/kg), a nonselective β-adrenoceptor blocker with significant membrane-stabilizing activity, given 30 min before the preparation of the gastric tissue, not only alleviated the fall in PD and I across the gastric mucosa, but also increased the R of the stomach tissue. Butoxamine, a selective β2-antagonist, produced the similar but less significant effects in the same experimental setting. Metroprolol, a β1-adrenoceptor blocker, given by the similar doses did not produce significant effects. Nonselective β-adrenoceptor blocker, nadolol but not the β- and α-adrenoceptor blocker, labetalol, also significantly preserved the decrease of PD induced by ethanol, but to a lesser extent. These findings suggest that blockade of the β2-receptors in the gastric mucosa together with membrane-stabilizing activity could improve the integrity of the gastric mucosa, and these effects are probably acting through its direct action on the tissue.

Cells from goldfish and amphibian lymphoid organs, mainly leukocytes, express high affinity β -adrenergic receptors specific for β -adrenergic ligands (agonists: adrenaline, noradrenaline, terbutaline, and fenoterol; antagonists: CGP-12177, dihydroalprenolol, propranolol, atenolol, and butoxamine). The rank order of ligand potency does not allow their being classified into any known mammalian subtype. Among features that distinguish them from mammalian β 1 and β 2 -adrenoceptors is much lower affinity for (-)-CGP-12177, obtained in both saturation and kinetic experiments (about 25 nM for goldfish head kidney cells). The density of receptors on goldfish and anuran cells is organ-dependent and comparable to that estimated on mammalian leukocytes. The extraordinarily high receptor density on salamander splenic cells (about 183,000) correlates with the large size of urodele cells. The competition experiments on goldfish cells with propranolol and CGP-12177 suggest the existence of yet another binding site, which may be either another β -AR subtype, or a serotonergic receptor.

1. The presence and types of alpha and beta-adrenoceptors in the corpus cavernosum of the horse were studied in vitro by using selected ligands of adrenoceptors and isometric tension recording. 2. Noradrenaline and phenylephrine induced concentration-dependent contractions in corpus cavernosum preparations. B-HT 920 had no effect. 3. Phentolamine and prazosin produced a shift to the right of the dose-response curve of noradrenaline, while the alpha(2)-antagonist, rauwolscine had no effect on the response to noradrenaline. Phenylephrine-evoked contractions of corporal strips were significantly inhibited by the alpha(1)-adrenoceptor antagonist, prazosin. 4. Isoprenaline and salbutamol each relaxed precontracted corpus cavernosum preparations in a concentration-dependent manner; the isoprenaline effect was blocked by propranolol, practolol and butoxamine. The salbutamol effect was blocked by butoxamine. 5. These results suggest that presence of postjunctional alpha(1)-adrenoceptors in horse corpus cavernosum. There is also a heterogenous population of beta-adrenoceptors in this tissue, belonging to the beta(1) and beta(2) subtypes.

To understand the receptor subtypes responsible for beta-adrenoceptor-mediated relaxation of guinea pig taenia caecum, we investigated the effects of isoprenaline and salbutamol. Isoprenaline and salbutamol caused dose-dependent relaxation of the guinea pig taenia caecum. Propranolol, bupranolol and butoxamine produced shifts of the concentration response curves for isoprenaline and salbutamol. Schild regression analyses carried out for propranolol against isoprenaline and salbutamol gave pA2 values of 8.43 and 8.88, respectively. Schild regression analyses carried out for butoxamine against isoprenaline and salbutamol gave pA2 values of 6.46 and 6.68, respectively. Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 8.60 and 8.69, respectively. However, in the presence of 3 x 10(-4) M atenolol, 10(-4) M butoxamine and 10(-6) M phentolamine to block the beta1-, beta2- and alpha-adrenoceptor effects, respectively, Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 5.77 and 5.97, respectively. These results suggest that the relaxant responses to isoprenaline and salbutamol in the guinea pig taenia caecum are mediated by both the beta2- and the beta3-adrenoceptors.

1. 1. In femoral artery of 3-week-old rats, norepinephrine (NE) at 1 nM-0.1 μM concentration-dependently produced a contraction but, at higher concentrations (0.3–30 μM), NE relaxed the tissue. 2. 2. In aorta of the 3-week-old rat, NE elicited a contraction at 1 nM to 0.3 μM and a relaxation at 1 μM and higher. 3. 3. The magnitude of the NE-induced relaxation in the aorta was much less than that in the femoral artery. 4. 4. Atenolol (a β 1 -selective antagonist) modulated these NE responses in the femoral artery and butoxamine (a β 2 -selective antagonist) in the aorta. 5. 5. Subcutaneous application of propranolol and isoproterenol for 10 days in 4-week-old rats sensitized and desensitized the NE-induced relaxation, respectively. 6. 6. It is suggested that, in the young rat, β 1 -and β 2 -adrenoceptors contribute the NE-induced relaxation in the femoral artery and aorta, respectively, and that β-adrenoceptors may contribute to the limitation of NE-induced vasoconstriction in certain types of vascular beds.

Three experiments were conducted examining the contribution of beta-adrenergic receptors to stress-induced cholesterol increases. Rats were exposed to 3 90-min sessions of inescapable tailshock, or left undisturbed in their home cage. Propranolol, a nonselective beta-blocker, attenuated the stress-induced cholesterol increase when administered prior to the daily shock session. Atenolol, a beta-1 specific antagonist, also attenuated the stress-induced cholesterol increase. Butoxamine, a beta-2 specific antagonist, had no effect on the stress-induced cholesterol increases. Results are discussed in terms of catecholamine-stimulated free fatty acid (FFA) release as a potential mechanism for producing stress-induced hypercholesterolemia.

Adrenergic regulation of epididymal C1- currents was studied by the whole-cell patch-clamp technique using various a- and P-receptor agonists and antagonists in primary cultured rat cauda epididymal cells. Cl- currents could be activated with varying frequency by noradrenaline (primarily a- and P,-adrenoceptor-selective agonist, 1-5 gIM), isoprenaline (nonselective P-adrenoceptor agonist, 5 jLM), salbutamol (P,2-adrenoceptor-selective agonist, 2 ILM), and phenylephrine (a 1-adrenoceptor-selective agonist, 1-2 ILM). Noradrenaline alone elicited Cl- current activation in 85% of the cells examined. In the presence of phentolamine (nonselective a-adrenoceptor antagonist, 15 M), noradrenaline elicited Cl- current activation in 63% of the cells examined, whereas noradrenaline-induced activation was observed in 33% of the cells examined in the presence of both atenolol and butoxamine (P,- and ,3 2-adrenoceptor antagonists, respectively, 10 ;iM). In 27% of single cells examined, a second current activation in response to salbutamol was observed after the first response to phenylephrine. When the order of stimuli was reversed, dual activation was also observed in 22% of the single cells examined, indicating the presence of both a- and [Padrenoceptors in single epididymal cells. Profiles of time- and voltage-dependent CI- current upon activation by different adrenoceptor agonists exhibited characteristics similar to those previously reported for Ca 2+ and cAMP-activated Cl- currents, suggesting that regulation of epididymal Cl- conductances could be mediated by different adrenoceptor subtypes involving Ca 2+ and cAMP as intracellular second messengers.

Larval and adult Ambystoma tigrinum were subjected to acidosis by infusing lactic acid (2 μM·g-1) into the peritoneal cavity. Blood was sampled at intervals to establish the time-course of the ensuing acidosis. Both larvae and adults became significantly acidotic after 1 h. The larval acidosis was more pronounced (-4 pH units versus-2 pH units) than adults due to greater extracellular buffering capacity (higher [HCO3-]) in adults. Both forms recovered in about 8 h. Larvae showed a typical increase in circulating norepinephrine during the initial stages of the acidosis; adults did not, having significantly lower norepinephrine titer than larvae during the acidosis. Both larvae and adults showed transient increases in PO2 during the acidosis. The β1 and β2 antagonists, timolol and butoxamine respectively, (0.2 μg·g-1) were administered to separate groups of larvae. Butoxamine (β2) delayed the recovery from the acidosis by prolonging the increase in arterial PCO2 and reversing the recovery of [HCO3-]. Timolol (β1) did not delay recovery. We conclude that β2 receptors are involved in the catecholamine responses to acidosis in larvae. Catecholamines appear not to play the same role in adult acid-base disturbances as they seem to in larvae.

It has been shown that 1 h restraint shortens pentobarbital (PbNa)-induced sleeping time and that brain corticotropin-releasing hormone (CRH) is involved in the mechanism by which restraint shortens PbNa-induced sleeping time. The present study was designed to further examine the mechanism of the antagonistic effect of 1 h restraint on PbNa in rats. Intracerebroventricular (i.c.v.) administration of propranolol and metoprolol, but not butoxamine reversed the shortening of PbNa-induced sleeping time by 1 h restraint. The i.c.v. administration of phentolamine blocked the shortening of PbNa-induced sleeping time by restraint, while the same dose of phentolamine prolonged the sleeping time in unrestrained rats. Atropine did not affect the PbNa-induced sleeping time in restrained rats. These results suggest that in addition to CRH, the brain β 1 -adrenergic system is involved in the restraint stress-induced increase in arousal.

1. Isoproterenol caused relaxation of rat detrusor muscles contracted by electrical stimulations. Sodium nitroprusside (SNP) and methylene blue inhibited the relaxation induced by isoproterenol without affecting forskolin-induced relaxation. 2. In the presence of theophylline, zaprinast still potentiated isoproterenol-relaxation. In the presence of butoxamine, a selective β 2 -adrenoceptor inhibitor, but not metoprolol, a selective β 1 -adrenoceptor inhibitor, SNP still inhibited isoproterenol-relaxation. 3. SNP inhibited the relaxation to dobutamine, a β 1 -adrenoceptor agonist, but not that to terbutaline, a β 2 -adrenoceptor agonist. The relaxation to dobutamine was also inhibited by methylene blue. Further, in the presence of theophylline, zaprinast still potentiated dobutamine-relaxation. 4. Isoproterenol increased tissue level of cGMP, which was inhibited by propranolol. 5. These results suggest a possibility that, in rat detrusor muscles, isoproterenol causes relaxation due to increases in the level of both cAMP and cGMP.

beta-Adrenoceptors in the guinea pig taenia caecum were investigated by measuring relaxation responses to agonists and by a radioligand binding assay using [3H]CGP 12177. The rightward shift of the isoprenaline concentration-response curve was observed by butoxamine, a beta 2-selective antagonist, and the pA2 value for butoxamine was 6.46. In control preparations, catecholamines caused relaxation with the following rank order of potency: isoprenaline > adrenaline > noradrenaline. However, in the presence of 10(-6) M phentolamine, 3 x 10(-4) M atenolol and 10(-4) M butoxamine, the rank order of potency of the agonists was: isoprenaline > noradrenaline > adrenaline. CGP 12177 caused graded relaxation of the guinea pig taenia caecum, and this response was not influenced by 10(-6) M phentolamine, 3 x 10(-4) M atenolol, 10(-4) M butoxamine or 10(-6) M propranolol. The Scatchard plot of the specific [3H]CGP 12177 binding to microsomal fractions from the guinea pig taenia caecum showed two affinity sites of the receptor: high affinity (KD = 0.64 nM) and low affinity (KD = 142.21 nM) sites. The pKD value of the high affinity site of [3H]CGP 12177 was in agreement with its pA2 value, and that of the low affinity site was in agreement with its pD2 value. These results suggest that isoprenaline-, noradrenaline- and adrenaline-induced relaxations of the guinea pig taenia caecum predominantly involve beta 2- and beta 3-adrenoceptors, whereas CGP 12177-induced relaxation is mediated solely through beta 3-adrenoceptors.

In coronary arterial rings isolated from horse, norepinephrine (NE)(10(-7) - 10(-5) M) induced concentration-dependent contractions which were not influenced by endothelial denudation. Prazosin (alpha 1-antagonist) inhibited the contraction, but yohimbine (alpha 2-antagonist) did not, and propranolol (beta-antagonist) enhanced the contraction. Pretreatment with phentolamine (10(-5) M) (alpha-antagonist) converted the contraction induced by NE to relaxation in coronary rings precontracted with ONO11113 (thromboxane A2 derivative). The relaxation was not influenced by removal of the endothelium, and was inhibited by propranolol and atenolol (beta 1-antagonist) but not by butoxamine (beta 2-antagonist). These results suggest that in equine coronary arteries, the contractile response to NE is mediated by stimulation of alpha 1-adrenoceptors on the smooth muscle, and that stimulation of beta 1-adrenoceptors on the smooth muscle modifies the contraction by inducing relaxation.

We investigated effects of beta-adrenoceptor agonists (beta 1-selective: T-1583 and dobutamine, beta 2-selective: fenoterol, non-selective: isoproterenol) on urine outflow rate, blood pressure, heart rate, respiratory rate and rectal temperature. The drugs were applied into the paraventricular nuclei (PVN) of spontaneously hypertensive (SHR), Wistar-Kyoto (WKY) and Wistar rats. Fenoterol and isoproterenol markedly decreased the urine outflow rate, compared with T-1583 and dobutamine in the rats. There was no marked difference among the three strains in responsiveness to fenoterol and isoproterenol. The antidiuretic effects of fenoterol were inhibited by a beta 2-selective antagonist, butoxamine, more markedly than a beta 1-selective antagonist, atenolol, in SHR; and the inhibitory effects of these drugs were partial in WKY. In Wistar rats, the effect of fenoterol was inhibited by a non-selective beta-antagonist, timolol, but not by atenolol or butoxamine. A vasopressin antagonist (i.v.) did not diminish the antidiuretic effect of fenoterol. Fenoterol reduced the blood pressure in SHR and WKY, but not in Wistar rats. It was suggested that there were predominantly beta 2-adrenoceptors mediating antidiuresis in SHR. In WKY and Wistar rats, however, the beta-adrenoceptor subtypes mediating antidiuresis have yet to be determined. The ability of beta-adrenoceptor agonists to decrease urine outflow rates in SHR was not altered as compared to that in the control rats. beta-Adrenoceptor-mediated antidiuresis was not due to vasopressin release.

We have demonstrated that 100% oxygen inhalation is beneficial to zymosan-induced generalized inflammation, and reactive oxygen species may be involved in the protection of oxygen treatment. Other investigators suggest that reactive oxygen species may modulate the sympathetic nervous system activity and β2-adrenergic receptor (β2AR)-mediated pathway. Moreover, studies have demonstrated that β2AR agonists are beneficial to sepsis. Therefore, we assessed the effects of β2AR antagonist butoxamine on the protection of oxygen treatment against zymosan-induced generalized inflammation in mice. Mice were given oxygen treatment by exposure to 100% oxygen for 3 h starting at 4 and 12 h after zymosan injection, respectively. In the mortality study, survival was monitored for 7 days after zymosan injection in mice. At 24 h after zymosan injection, mice were killed, and blood sample and organs were harvested for analysis. We observed that 100% oxygen treatment prevented the abnormal changes in organ histopathology, lactate dehydrogenase and C-reactive protein in serum, inflammatory cytokines in serum and tissue, and arterial blood gas analysis and improved the survival rate in zymosan-challenged mice. We found that pretreatment with β2AR antagonist butoxamine partly abolished the protection of 100% oxygen inhalation. We also showed that zymosan induced the increase in serum 3'-5'-cyclic adenosine monophosphate (cAMP) and the decrease in tissue cAMP. However, oxygen treatment increased the cAMP levels in both serum and tissue, which were partly abolished by pretreatment with butoxamine. Thus, 100% oxygen inhalation may protect against zymosan-induced generalized inflammation in mice partly through activation of β2AR pathway and subsequently enhance cAMP levels in both serum and tissue.

In the present work, we sought to mimic the internal state changes in response to a predator threat by pharmacologically stimulating the brain circuit involved in mediating predator fear responses, and explored whether this stimulation would be a valuable unconditioned stimulus (US) in an olfactory fear conditioning paradigm (OFC). The dorsal premammillary nucleus (PMd) is a key brain structure in the neural processing of anti-predatory defensive behavior and has also been shown to mediate the acquisition and expression of anti-predatory contextual conditioning fear responses. Rats were conditioned by pairing the US, which was an intra-PMd microinjection of isoproterenol (ISO; β-adrenoceptor agonist), with amyl acetate odor—the conditioned stimulus (CS). ISO (10 and 40 nmol) induced the acquisition of the OFC and the second-order association by activation of β-1 receptors in the PMd. Furthermore, similar to what had been found for contextual conditioning to a predator threat, atenolol (β-1 receptor antagonist) in the PMd also impaired the acquisition and expression of OFC promoted by ISO. Considering the strong glutamatergic projections from the PMd to the dorsal periaqueductal gray (dPAG), we tested how the glutamatergic blockade of the dPAG would interfere with the OFC induced by ISO. Accordingly, microinjections of NMDA receptor antagonist (AP5, 6 nmol) into the dPAG were able to block both the acquisition, and partially, the expression of the OFC. In conclusion, we have found that PMd β-1 adrenergic stimulation is a good model to mimic predatory threat-induced internal state changes, and works as a US able to mobilize the same systems involved in the acquisition and expression of predator-related contextual conditioning.

Hemodynamic and autonomic mechanisms underlying the cardiovascular defense reaction elicited by electrical stimulation of the dorsolateral periaqueductal gray region were evaluated in pentobarbital sodium-anesthetized Sprague-Dawley rats. Stimulation of this area produced transient increases in mean arterial pressure and more sustained increases in heart rate, hindlimb blood flow, and plasma catecholamine levels. The pressor component of the defense reaction was due entirely to increased total peripheral vascular resistance; elevations in cardiac output occurred only at the end of the stimulation period when blood pressure had returned to resting values. The hindlimb vasodilatory response included distinct early and late components. The late component was produced by epinephrine-mediated activation of beta 2-receptors because it was abolished by bilateral adrenalectomy or treatment with butoxamine, a beta 2-receptor selective antagonist. In contrast, the early component was only partially reduced by adrenalectomy or butoxamine treatment. During conditions of constant flow provided by a pump-perfused hindlimb preparation, the early hindlimb vasodilatory response was eliminated and a neurogenic vasoconstrictor response revealed. This finding suggests that the early hindlimb blood flow response may result from shunting of blood from highly vasoconstricted vascular beds to relatively less constricted hindlimb vasculature.

We have studied the contribution of epinephrine to experimentally induced urethral inflammation in the rat. Inflammation was induced by inserting latex strips into the urethra. The effects of various experimental procedures were assessed according to a 4-point scale based on histological findings. The results showed that 0.5 mg/kg epinephrine decreased the severity of catheter-induced urethral inflammation. This effect was blocked by the alpha 2-adrenergic agonist, yohimbine, but not by alpha 1 (prazosin), beta 1 (M32 MTC), or beta 2 (butoxamine) antagonists. The results suggest that the suppressive effect of epinephrine is mediated by action at the alpha 2 adrenergic receptor.

The effect of beta 2-adrenoceptor agonists and interleukin-4 (IL-4) on the CD23 expression on, and release from, the human promonocytic cell line, U 937, was investigated. As assessed by flow cytometry, incubation of U 937 cells in the presence of salbutamol, fenoterol or IL-4 induced a concentration- and time-dependent increase in CD23 expression, that was maximal after 48 hr and followed by a decrease thereafter. In addition, salbutamol potentiated the effect of IL-4, the optimal concentration of the drug being a function of the concentration of this cytokine. This synergy between IL-4 and beta 2-adrenoceptor agonists was also observed for the release of the soluble form of CD23. The effect on CD23 expression of salbutamol and fenoterol, but not of IL-4, was blocked in the presence of D,L-propranolol (1 microM) or butoxamine (1 microM). The alpha-adrenoceptor agonist, norepinephrine (1 microM), was ineffective in inducing CD23 expression or potentiating the one evoked by IL-4. Salbutamol down-regulated the expression of Fc gamma RI (CD64) and Fc gamma RII (CD32) whereas IL-4 was ineffective. Only when added together at the onset of the culture did salbutamol and IL-4 induce, after 48 hr, the expression of the monocyte marker, CD14. The expression of CD18 was up-regulated in response to salbutamol either alone or in combination with IL-4, this cytokine alone being inefficient. These data suggest that IL-4 and beta 2-adrenoceptor agonists induce differentiation of U 937 cells into monocyte-like cells.

1. The specificity of betaxolol, a beta-adrenoceptor antagonist, for beta 1- and beta 2-adrenoceptors was compared with that of other beta-antagonists, atenolol, ICI-118551, butoxamine and (+/-)-propranolol, in the bovine trachea and heart by competitive interaction with [3H]-CGP12177 as a radioligand. 2. The radioligand Kd values were 0.75 +/- 0.12 and 1.60 +/- 0.11 nM in the trachea and heart, respectively, and the Bmax values were 34.00 +/- 4.41 and 21.54 +/- 2.94 fmol mg-1 protein, respectively. 3. Using ICI-118551, we determined the ratio of beta 1:beta 2-adrenoceptors in the trachea and heart to be approximately 29:71 and 56:44, respectively. 4. In the trachea, a beta 2-predominant tissue, betaxolol and atenolol were more selective for beta 1-adrenoceptor binding sites than beta 2-adrenoceptor binding sites, whereas ICI-118551 and butoxamine were more selective for beta 2-adrenoceptor binding sites. 5. The beta 1-selectivity of betaxolol was 2.2 and 2.7 fold higher than that of atenolol in the bovine trachea and heart. These findings suggest that betaxolol may be useful in the treatment of hypertension, cardiac arrhythmia and angina pectoris.

We investigated the inhibitory effects of β₁- or β₂-adrenoceptor (AR) antagonists on salivary amylase secretion produced by various emetic agents, such as cisplatin, apomorphine, and lithium chloride (LiCl), or the non-emetic agent β(½)-AR agonist isoprenaline in rats. We also determined the inhibitory effect of metoclopramide, a dopamine D₂-receptor antagonist, on increases in the salivary amylase activity induced by apomorphine or granisetron, a 5-HT(3)-receptor antagonist, on LiCl-induced increased salivary amylase activity. Isoprenaline (0.01 mg/kg, s.c.) produced an increase in salivary amylase and the increase was inhibited by the β(½)-AR antagonist propranolol (5 mg/kg, s.c.) and β₁-AR antagonist atenolol (2 mg/kg, s.c.) but not by the β₂-AR antagonist butoxamine (8 mg/kg, s.c.). The increased amylase activity induced by cisplatin (15 mg/kg, i.v.), apomorphine (3 mg/kg, s.c.), or LiCl (120 mg/kg, i.p.) was inhibited significantly by atenolol (2 mg/kg, s.c.) but not by butoxamine (8 mg/kg, s.c.). In addition, increases in amylase activities induced by apomorphine and LiCl were inhibited significantly by metoclopramide (10 mg/kg, i.v.) and granisetron (3 mg/kg, i.v.), respectively. These results suggest that salivary amylase secretion induced by various emetogens is involved in β₁-adrenoceptor activity and that salivary amylase activity is useful to detect emetogens with no direct β₁-AR activation in rats, a species that does not exhibit vomiting.

Objective To evaluate the effect of the inotropes epinephrine, dopamine and dobutamine on expression of endothelial adhesion molecules and on neutrophil adhesion to endothelial cells under dynamic conditions. Methods Endothelial cells were obtained by collagenase digestion of human umbilical cord veins. Endothelial monolayers were pre-incubated with one of the chosen inotropes, with or without butoxamine, and exposed to interleukin-1. The monolayers were then incubated with fluorescence-labelled anti-human monoclonal antibodies directed against the endothelial adhesion molecules ICAM-1, E-selectin or VCAM-1. Expression of endothelial adhesion molecules was analysed by flow cytometry after pre-incubation of endothelial monolayers with one of the chosen inotropes, with or without butoxamine, and after exposure to interleukin-1. To evaluate the neutrophil adherence, the endothelium was placed on a horizontal shaker-incubator and overlayered with neutrophils. Then, non-adherent neutrophils were removed, and cells were completely dissociated. Finally, neutrophils and endothelial cells were counted by flow cytometry. Results The expression of E-selectin on endothelium following stimulation with interleukin-1 is attenuated by the inotropes dopamine or dobutamine, but not by epinephrine. The addition of butoxamine does not modify the expression of E-selectin following stimulation with interleukin-1 and pre-incubation with one of the chosen inotropes. The decrease in neutrophil adhesion to endothelium following stimulation with interleukin-1 and addition of inotropes is antagonised by the β-blocker butoxamine. Conclusion In contrast to the modulation of E-selectin expression on endothelium, the effect of inotropes on neutrophil adhesion to endothelium is regulated by the expression of adhesion molecules on PMNs and mediated by the β-adrenoceptor.

1. Short-circuit current (SCC) technique was used to study the adrenoceptors involved in the electrogenic chloride secretion by cultured cauda epididymal epithelium of rats. Stimulation of the epithelium with noradrenaline (primarily beta 1-adrenoceptor selective agonist), salbutamol (beta 2-adrenoceptor selective agonist) and adrenaline (non-selective beta-adrenoceptor agonist) led to a rise in SCC. At a low chart-speed (2 mm min-1), the response profile to these agonists consisted of a peak followed by a sustained response considerably higher than the basal SCC. 2. The EC50s (doses of agonist producing 50% maximum response) of noradrenaline, salbutamol and adrenaline were 300, 115 and 10 nM respectively. Pretreating the tissues with 1 microM atenolol (beta 1-selective antagonist) and 10 microM butoxamine (beta 2-selective antagonist) shifted the dose-response curves of noradrenaline (shifted EC50 = 4000 nM) and salbutamol (shifted EC50 = 1050 nM) to the right. Atenolol (1 microM) and butoxamine (10 microM) shifted the dose-response curve of adrenaline to the right with new EC50s of 30 nM and 115 nM, respectively. 3. The rapidly rising phase of the SCC response to noradrenaline and adrenaline observed at low chart-speed consisted of a brief and transient retraction followed by a rebound increase in SCC. At a high chart-speed (1 mm s-1), the retraction and rebound phenomenon manifested as a fast initial spike which could be blocked by phentolamine (non-specific alpha-adrenoceptor antagonist) in a dose-dependent fashion. Similar initial spikes were observed when the tissues were stimulated with phenylephrine (alpha-selective agonist) but not with isoprenaline (non-selective beta-agonist) or forskolin (activator of adenylate cyclase). The response of the initial spike triggered by noradrenaline was dose-dependent and the EC50 was 2000 nM.4. The present study showed that the electrogenic chloride secretion by rat epididymis could be stimulated by (alphaxi-, beta131- and beta2-adrenoceptor agonists. The al-mediated response had a faster onset and more transient action than the 3-counterpart. It is postulated that epididymal chloride secretion might be regulated by neural (noradrenaline-mediated) and humoral (adrenaline-mediated) controls and that the stimulus-secretion coupling mechanisms might involve both Ca2+(alpha-mediated response) and adenosine 3':5'-cyclic monophosphate (beta-mediated response) as intracellular second messengers.

Background To evaluate the possible protective effect of sympatholytic medications with respect to neutrophil function, we evaluated the influence of a nonselective β-blocker medication on the interaction of neutrophils and epinephrine after cardiopulmonary bypass. Therefore, we studied the importance of adrenoceptors for the immunomodulation of neutrophils by catecholamines in vitro. Methods First, we investigated the modulation of neutrophils from healthy volunteers, after stimulation with n-formyl-l-methionyl-l-leucyl-l-phenylalanin (FMLP) in the presence of epinephrine with or without the addition of one of the following adrenergic receptor antagonists: atenolol, butoxamine, pindolol, prazosin, or RS79984. The second part included an investigation of the modulation of neutrophils from patients after operative coronary revascularization with or without extracorporeal circulation after stimulation with FMLP and addition of epinephrine. After loading with anti-CD62l or anti-CD11b antibodies or dihydrorhodamine, the expression of CD62l and CD11b and generation of oxidative free radicals were assessed by flow cytometry. Results The suppression of oxidative free radical generation, inhibition of CD62l downregulation after stimulation with FMLP, and suppression of CD11b upregulation after FMLP stimulation from epinephrine were all mediated by β 2 -adrenoceptors. After cardiac surgery with cardiopulmonary bypass, epinephrine inhibited the CD62l downregulation, the suppression of CD11b upregulation, and the generation of oxidative free radicals after FMLP stimulation. The pre-operative administration of β-blockers abolished the immunomodulatory effects of epinephrine on CD62l and CD11b expression and the generation of oxidative free radicals. Conclusion The immunomodulatory effects of epinephrine on neutrophils remained unchanged irrespective of cardiopulmonary bypass and could contribute to the detrimental effects of epinephrine after heart surgery. The preoperative administration of nonselective β-blockers abolished the immunomodulatory effects of epinephrine in vitro and in patients, and it enhanced the immunocompetence of neutrophils in a context of increased catecholamine levels.

A study was undertaken to determine the presence and distribution of α- and β-adrenoceptors in the sheep bladder body and base. In the bladder body, noradrenaline and isoproterenol induced relaxation which was significantly inhibited by propranolol, pafenolol and butoxamine. In the presence of propranolol (10 −5 M), noradrenaline induced a small contraction, as well as phenylephrine, but B-HT 920 failed to cause any effect on the bladder body. In the bladder base, noradrenaline caused a contraction that was significantly inhibited by prazosin but not by yohimbine. Phenylephrine also induced a contractile response in this structure which was inhibited by prazosin. Isoproterenol caused a relaxation that was significantly inhibited by propranolol and pafenolol but not by butoxamine. Relaxation was mediated by both β 1 and β 2 -adrenoceptors in the detrusor muscle and by β 1 -adrenoceptors in the bladder base. α 1 -adrenoceptors contributed to maintain the detrusor tone and contract the bladder base.

In isolated liver from fed rats, simultaneously single-pass-perfused via both the hepatic artery (80 mmHg, 30-35% flow) and the portal vein (10 mmHg, 70-65% flow), adrenaline was infused either singly or jointly via the hepatic artery or the portal vein in the absence or presence of the alpha 1-blocker prazosin and the beta 2-blocker butoxamine. It was found that: (1) arterial adrenaline caused increases in glucose and lactate output which were slower in onset, smaller in peak height but longer in duration than did portal adrenaline; (2) arterial adrenaline elicited a much more pronounced decrease in flow and increase in pressure in the ipsilateral vessel than did portal adrenaline, and arterial, but not portal, adrenaline elicited qualitatively similar alterations also in the contralateral vessel; (3) arterial adrenaline caused metabolic changes mainly via alpha 1-receptors, with beta 2-receptors playing a permissive role via haemodynamic alterations, whereas portal adrenaline acted only via alpha 1-receptors; (4) arterial adrenaline decreased arterial flow via alpha 1-receptors counteracted via beta 2-receptors and operated on portal flow as portal adrenaline only via alpha 1-receptors; and (5) arterial adrenaline was extracted to a far greater extent than portal adrenaline. The results indicate that the hepatic artery and the portal vein can function as independent sites of hormonal signal input, which interact by complex, still undefined, mechanisms.

Objectives Propranolol inhibited pancreatic cancer cell proliferation by blocking signaling through the beta-adrenoceptor. We hypothesized that propranolol may suppress pancreatic cancer cell growth through induction of apoptosis. Methods The beta-adrenoceptor antagonist propranolol, beta1-adrenoceptor antagonist metoprolol, and beta2-adrenoceptor antagonist butoxamine were used to induce apoptosis in PC-2 cells. The mRNA and protein expression of beta1- and beta2-adrenoceptors was analyzed using reverse transcriptase-polymerase chain reaction and Western blot. The apoptotic index was determined using Hoechst 33342 fluorescent staining, TUNEL, and annexin V and fluorescein isothiocyanate/propidium iodide flow cytometry assay. The expression of caspase 3, caspase 9, and caspase 8 was analyzed using Western blotting. Results PC-2 cell line expressed mRNA and protein for both of beta1- and beta2-adrenoceptors. The Hoechst staining, TUNEL, and flow cytometry assay documented that the 3 drugs increased the number of apoptotic cells; the rate of apoptosis was the highest using butoxamine followed by propranolol, whereas the least was using metoprolol. beta-Adrenoceptor antagonists therapy affected caspase 3 and caspase 9 expression. Conclusions The rate of apoptosis in PC-2 cells was higher after treatment with butoxamine than propranolol, suggesting that propranolol induces apoptosis in PC-2 cells via the beta2-adrenoceptors principally. Our data could be useful for developing beta-adrenoceptor antagonists for inducing apoptosis in pancreatic cancer cells.

BACKGROUND Proinflammatory cytokines increase in cerebrospinal fluid (CSF) after subarachnoid hemorrhage (SAH). Recent evidence suggested that beta-adrenoceptor antagonist could reduce proinflammatory cytokines. We conducted the present study to examine whether beta-adrenoceptor antagonists would reduce proinflammatory cytokine concentrations after SAH in rats. METHODS In Experiment 1, to investigate the time course of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), rats were randomized into groups: 1, 3, 6, and 12 h after SAH or sham operation. CSF and blood samples were obtained at each time point. In Experiment 2, to investigate the effects of beta-adrenoceptor antagonists on the IL-6 and TNF-alpha concentrations, rats were randomized into groups: 1) control group: SAH + normal saline, 2) propranolol group: SAH + propranolol, 3) metoprolol group: SAH + metoprolol, and 4) butoxamine group: SAH + butoxamine (beta(2)-adrenoceptor antagonist). CSF and blood samples were obtained 6 h after SAH. IL-6 and TNF-alpha concentrations in samples were measured. RESULTS In Experiment 1, CSF IL-6 concentrations in the SAH groups increased markedly and peaked at 6 h after SAH, whereas CSF TNF-alpha concentrations in the SAH groups were consistently low. In Experiment 2, CSF IL-6 concentrations in the propranolol and butoxamine groups were significantly lower compared with those in the control group (P < 0.01 and P < 0.05 for each group). Plasma IL-6, CSF TNF-alpha, and plasma TNF-alpha concentrations were comparable in all four groups. CONCLUSIONS CSF IL-6 concentrations increased in the acute stage of SAH and beta-adrenoceptor antagonists with a beta(2)-adrenoceptor blocking action suppressed this elevation of IL-6 concentrations after SAH in rats.