Effects of a ß2-adrenergic receptor blocker on experimental periodontitis in spontaneously hypertensive rats.

AIMS: Recent studies have reported a relationship between periodontal disease and hypertension, and previous evidence suggests that the sympathetic nervous system plays an important role in the control of bone metabolism. This study sought to evaluate the effect of the beta-2 adrenergic receptor (ß2-AR) blocker butoxamine on experimental periodontitis in a rat model. MATERIALS AND METHODS: Wistar-Kyoto and spontaneously hypertensive rats (n = 6 per group) were orally administered butoxamine 1 mg/kg/day and experimental periodontitis was induced by applying an orthodontic ligature wire. The rats were sacrificed after 4 weeks and the residual alveolar bone was measured using micro-computed tomography (micro-CT) imaging analysis software for histological analysis. KEY FINDINGS: Micro-CT imaging analysis showed a higher ratio of residual alveolar bone, BV/TV, and Tb.N in both Wistar-Kyoto and spontaneously hypertensive rats treated with butoxamine compared with the corresponding control rats. In histological analysis, compared with the Wistar-Kyoto and spontaneously hypertensive rat control groups, the corresponding butoxamine-treated groups showed a lower ratio of attachment level, lower values of osteoclast number and surface. SIGNIFICANCE: ß2-AR blockers maintained the alveolar bone mass and attachment level by suppressing osteoclast activity. Thus, ß2-AR blockers may be effective in preventing periodontitis.

Dynamic behavior of fully solvated beta2-adrenergic receptor, embedded in the membrane with bound agonist or antagonist.

Recently we predicted the 3D structure of the human beta2-adrenergic receptor (beta2AR) and of the binding site of several agonists and antagonists to beta2AR. These predictions (MembStruk and HierDock) included no explicit water and only a few lipid molecules. Here we include explicit H(2)O and an infinite lipid bilayer membrane in molecular dynamics (MD) simulations of three systems: apo-beta2AR, epinephrine-bound beta2AR, and butoxamine-bound beta2AR (epinephrine is an endogenous agonist, and butoxamine is a beta2AR selective antagonist). The predicted structures for apo-beta2AR and butoxamine-beta2AR are stable in MD, but in epinephrine-beta2AR, extracellular water trickles into the binding pocket to mediate hydrogen bonding between the catechol of epinephrine and Ser-204 on helix 5. The epinephrine-beta2AR structure shows dynamic flexibility with small, piston-like movements of helices 3 and 6 and transient interhelical hydrogen bonding between Ser-165 on transmembrane 4 and Ser-207 on transmembrane 5. These couplings and motions may play a role in protein activation. The apo-beta2AR shows less dynamic flexibility, whereas the antagonist-beta2AR structure is quite rigid. This MD validation of the structure predictions for G protein-coupled receptors in explicit lipid and water suggests that these methods can be trusted for studying the mechanism of activation and the design of subtype-specific agonists and antagonists.

Characterization of [3H]CGP 12177 binding to beta-adrenergic receptors in intact eel hepatocytes.

The aim of this study was to characterize [3H]CGP 12177 (CGP) binding to beta-adrenergic receptors in isolated hepatocytes of the European eel (Anguilla anguilla), in which the involvement of cAMP in epinephrine-induced glucose release has been previously observed. Specific binding of CGP was saturable, reversible, and linear as a function of cell number. Analysis of binding data suggested a single class of binding sites, with a Kd of 1.31 nM and a number of approximately 7000 beta-adrenergic receptors per cell. The potency order of specific inhibition of [3H]CGP binding was CGP > propranolol > or = alprenolol >> butoxamine > or = atenolol, while phentolamine and prazosin failed to significantly displace the tracer at concentrations up to 100 microM. The binding kinetics of CGP were closely related to its biological effect. In fact, the drug dose-dependently counteracted the enhancement of intracellular cAMP levels induced by epinephrine in isolated hepatocytes with a Kd of 1.06 nM. Moreover, it antagonized the hormone-induced stimulation of adenylyl cyclase activity in hepatic membranes as well as of glucose release from cells. These data clearly show that beta-adrenergic receptors are coupled to the adenylyl cyclase/cAMP transduction pathway in eel liver.

Involvement of beta 3-adrenoceptor in the relaxation response in guinea pig taenia caecum.

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.

Functional ß2-adrenoceptors in right atria isolated from footshock-stressed rats

The participation of ß2-adrenoceptors in the chronotropic response was evaluated in righ atria isolated from rats submitted to daily footshock stress for three days. Footshock increased both the sensitivity to salbutamol and the pA2 of butoxamine. These results provide additional support for the proposal that repeated footshock stress increases the functional of rat atrial postjunctional ß2-adrenoceptors

Functional beta 2-adrenoceptors in right atria isolated from footshock-stressed rats.

The participation of beta 2-adrenoceptors in the chronotropic response was evaluated in right atria isolated from rats submitted to daily footshock stress for three days. Footshock increased both the sensitivity to salbutamol and the pA2 of butoxamine. These results provide additional support for the proposal that repeated footshock stress increases the function of rat atrial postjunctional beta 2-adrenoceptors.

Characterization of beta adrenergic receptors in human cerebral arteries and alteration of the receptors after subarachnoid hemorrhage.

The nature of beta adrenergic receptors in human cerebral arteries was characterized and alteration of these receptors after subarachnoid hemorrhage was examined using a radioligand binding assay. The specific 3H-dihydroalprenolol, a beta adrenergic antagonist, binding to human cerebral arteries was saturable and of high affinity (KD = 12.3 nM) with a Bmax of 790 fmol/mg protein. Ki values and Hill coefficients of adrenergic agents for 3H-dihydroalprenolol were as follows; propranolol, 4.1 X 10(-8)M, 1.01; isoproterenol, 1.7 X 10(-6)M, 0.80; epinephrine, 8.3 X 10(-6)M, 0.48; norepinephrine, 2.3 X 10(-5)M, 0.45; metoprolol, 6.8 X 10(-8)M and 7.9 X 10(-6)M, 0.62; butoxamine, 2.2 X 10(-8)M and 2.1 X 10(-6)M, 0.43. The analysis of inhibition of specific 3H-dihydroalprenolol binding by these adrenergic agents suggests that human cerebral arteries contain a high density of beta adrenergic receptors and that the receptors are classified into two types, namely beta 1 and beta 2 adrenergic receptors. The calculated beta 1/beta 2 ratio from Hofstee plots was approximately 4/6. KD and Bmax of 3H-dihydroalprenolol binding to the cerebral arteries after subarachnoid hemorrhage were compared with those of control group. KD and Bmax of 3H-dihydroalprenolol binding of subarachnoid hemorrhage group were 13.9 nM and 1140 fmol/mg protein, respectively. The calculated beta 1/beta 2 ratio was approximately 6/4. These data suggest that the density of total beta adrenergic receptors increased without any significant change in the affinity after subarachnoid hemorrhage and that the increase of beta 1 adrenergic receptors was dominant.

Binding of [3H]-dihydroalprenolol and [3H]-acetobutolol to human blood platelets is not related to occupancy of beta-adrenoceptors.

Binding of [3H]-dihydroalprenolol to human platelet lysates is inhibited by (+/-)-propranolol and (+/-)-butoxamine, but less effectively by (+/-) practolol. (-)-Isoprenaline causes no significant inhibition of binding where stimulation of adenylate cyclase can be shown. Binding of [3H]-acetobutolol is also inhibited by (+/-)-propranolol. "Specific" binding of [3H]-dihydroalprenolol and [3H]-acetobutolol defined by (+/-) propranolol shows a non-classical saturation curve. 50% maximal binding is observed in the range 15-25 mM. The extent of "specific" binding is 2-fold greater for [3H]-dihydroalprenolol. Similar and rapid rates of binding of [3H]-dihydroalprenolol are observed at 4 degrees C and 20 degrees C. No stereoselectivity is observed for inhibition of [3H]-dihydroalprenolol binding by (+) and (-)-propranolol. Binding of [3H]-dihydroalprenolol and [3H]-acetobutolol may relate to the lipophilic character of these radioligands and does not represent interaction with beta-adrenoceptors.

Structure-activity relationships of some selected beta-adrenergic blocking agents--oxidation with N-bromosuccinimide.

A relationship between in vitro rate of oxidation by N-bromosuccinimide (NBS) and the pharmacologic activity (pA2) of different beta-adrenergic blockers for different blocking agent-tissue combinations has been studied. The rates of oxidation of the alcoholic group in the drugs by NBS, as well as their molecular conformations as represented by molecular models, were studied in order to determine requirements for selectivity and potency of action of beta-adrenergic blocking agents. Using data from all 7 drugs studied--both nonselective and selective blocking agents--no significant correlation between pA2 and -log k2 (k2 is the second order rate constant for the oxidative reaction) was found. If data from only the 4 nonselective agents were used (16 drug-tissue combinations), a correlation significant at p less than 0.01 was found. Hypotheses are presented to account for the selective action of some beta-adrenergic blocking agents.