Effect of glucocorticoids on the response of airway smooth muscle to catecholamine and resorcinol beta-agonists

The influence of hydrocortisone (11 beta, 17 alpha, 21-trihydroxy-pregn-4-ene-3,20-dione) or of methylprednisolone (6 alpha-methyl-11 beta, 17 alpha-21-trihydroxy-1,4-pregnadiene-3,20-dione) on the response of airway smooth muscle to a variety of beta-adrenergic bronchodilators was evaluated using incubated guinea pig tracheal rings, preconstricted with histamine. The adrenergic agonists chosen for this study included the nonselective beta 1- and beta 2-catecholamine, isoproterenol, the selective beta 2-catecholamine, rimiterol, and the selective beta 2-resorcinols, fenoterol and terbutaline. When the incubated rings were pretreated with 10-50 micrograms/mL of the steroids, there was a significant enhancement in smooth muscle sensitivity and reactivity to rimiterol and isoproterenol. Tracheal response to fenoterol or terbutaline, on the other hand, was not altered by the glucocorticoids. When used alone, neither steroid exerted an inotropic influence on the tracheal smooth muscle. The results of our study indicate that glucocorticoid enhancement of adrenergic bronchodilators is selective for catecholamines, and not for resorcinols.     

Beta adrenergic receptor repopulation of C6 glioma cells after irreversible blockade and down regulation

C6 glioma cells possess beta adrenergic receptors coupled with adenylate cyclase which can be irreversibly blocked by bromoacetylaminomethylpindolol (Br-AAM-pindolol), a beta adrenergic antagonist. With 1 microM Br-AAM-pindolol, more than 80% of beta adrenergic receptors, labeled by (3H)-dihydroalprenolol [3H)-DHA), were blocked. After this blockade, new beta adrenergic receptors were synthesized only during cell division. However, at cell confluency when the cell number was constant, turnover of beta adrenergic receptors was barely detectable. Cycloheximide (1 microgram/ml) inhibited cell growth as well as reappearance of beta adrenergic receptors. A 90% loss of beta adrenergic receptors in C6 glioma cells was obtained after down-regulation for 15 h with 10 microM isoproterenol, a beta adrenergic agonist. After removal of the agonist, recovery of beta-adrenergic-sensitive adenylate cyclase was complete within 2 to 3 days, whereas beta adrenergic receptors reached 90% of control value within 6 days. The half-life of the receptor recovery was 2 to 3 days. Pretreatment of C6 glioma cells by Br-AAM-pindolol and subsequent cell exposure to isoproterenol indicated that down regulation and recovery of unblocked beta adrenergic receptors did occur; however isoproterenol did not accelerate the biosynthesis of beta adrenergic receptors. The recovery of both biological response and beta adrenergic receptor occupancy was restored both in the presence or absence of cycloheximide (1 microgram/ml), a concentration which blocked 90% of protein synthesis. Our results suggest that reappearance of beta adrenergic receptors in C6 glioma cells, following isoproterenol-induced down regulation, was not due to synthesis of new receptors but to recycling of the beta adrenergic receptors.     

The bronchodilator acitivity of an 11-deoxyprostaglandin (AY 23 578).

The bronchodilator activity of AY-23 578 was studied in vivo and in vitro techniques. In the conscious guinea pig, aerosols of AY-23 578, prostaglandin (PGE2) E2, and isoproterenol afforded significant protection against histamine-induced convulsions. In the anesthetized guinea pig, where changes in tracheal pressure were taken as an index of bronchoconstriction, AY-23 578, PGE2, and isoproterenol were equipotent in inhibiting the bronchoconstriction induced by histamine. AY-23 578, PGE2, and isoproterenol reduced or prevented neostigmine-, prostaglandin F2alpha- or carbachol-induced increases in pulmonary resistance, and decreases in dynamic compliance in the anesthetized cat. The activities of the former two compounds were qualitatively similar but less potent than isoproterenol. In both the guinea pig and the cat, the aerosol administration of effective bronchodilator doses of AY-23 578 did not exhibit any significant cardiovascular effects. Both AY-23 578 and PGE2 caused relaxation of the isolated guinea pig tracheal strip; PGE2 was about six times more potent than AY-23 578. It is concluded that AY-23 578 is an effective bronchodilator in both the guinea pig and cat.     

Adrenergic receptors mediating prostaglandin production in the rabbit vas deferens

Contractile and prostaglandin E (PGE)-producing effects of adrenergic agonists were compared in the rabbit isolated vas deferens to determine which adrenergic receptor(s) potentially could mediate neural responses. Additionally, interactions among receptors were elucidated by comparing responses to norepinephrine, phenylephrine and isoproterenol to those in the presence of selective adrenergic agonists or antagonists. Norepinephrine increased the force of muscle contraction and the immunoassayable PGE concentrations in a concentration-dependent manner with EC50's of 55 +/- 8 and 112 +/- 39 microM, respectively. Propranolol (10 microM) enhanced the contractile effects of norepinephrine (p less than 0.01) whereas yohimbine (100 microM) or prazosin (1 microM) reduced norepinephrine-induced contractions and PGE production (p less than 0.01). Propranolol did not alter the PGE production induced by norepinephrine. Metoprolol (100 microM) also enhanced contractile effects of norepinephrine (p less than 0.05). The beta adrenergic agonist, isoproterenol (100 nM), decreased the contractile, but not the PGE-producing, effects of phenylephrine (p less than 0.001). Isoproterenol, given alone, increased PGE concentrations and inhibited electrically-induced force generation in a concentration-dependent manner. These results are consistent with the presence of alpha receptors on the vas deferens which mediate smooth muscle contraction and PGE generation. Beta receptors which mediate relaxation and PGE production also are present. Tentative identification of the beta receptor subtype revealed the presence of a beta 1 receptor.     

Prostacyclin synthesis elicited by cholinergic agonists is linked to activation of M2 alpha and M2 beta muscarinic receptors in the rabbit aorta

This study was conducted to investigate the subtypes of muscarinic receptors involved in the action of cholinergic agents on prostacyclin synthesis in the rabbit aorta. Prostacyclin production measured as 6-keto-PGF1 alpha was assessed after exposing the aortic rings to different cholinergic agents. Acetylcholine (ACh) (M1 and M2 agonist) (1-10 microM) and arecaidine proparagyl ester (APE) (M2 selective agonist) (1-10 microM) enhanced 6-keto-PGF1 alpha output in a concentration-dependent manner. A selective M1 receptor agonist, McN-A-343, at 1 microM-1 mM did not alter 6-keto-PGF1 alpha output. ACh- and APE induced increases in 6-keto-PGF1 alpha output were attenuated by the M1/M2 antagonist atropine (0.1 microM), M2 alpha antagonist (AF-DX 116), (0.1-1.0 microM), and by selective M2 beta antagonist, hexahydro-sila-difendiol (HHSiD) (0.1-1.0 microM), but not by the M1 antagonist pirenzepine (1.0 microM). 6-Keto-PGF1 alpha output elicited by ACh- or APE was not altered by the adrenergic receptor antagonists phentolamine and propranolol or by the nicotinic receptor blocker hexamethonium. Similarly, the arachidonic acid- or norepinephrine induced 6-keto-PGF1 alpha accumulation was not altered by these muscarinic receptor antagonists. Indomethacin, a cyclooxygenase inhibitor, prevented arachidonic acid, ACh- or APE induced 6-keto-PGF1 alpha output. Removal of the endothelium abolished the production of 6-keto-PGF1 alpha elicited by ACh, APE, bradykinin, and calcium ionophore A 23187, but not that induced by angiotensin II, K+ or norepinephrine. These data suggest that vascular prostaglandin generation elicited by cholinergic agonists is mediated via activation of M2 alpha and M2 beta but not M1 muscarinic receptors, which are most likely located on the endothelium.     

beta-Adrenergic stimulated lipolysis in pony adipocytes is exclusively via a beta2-subtype and is not affected by lactation

Catecholamines are important lipolytic agents in horses and ponies but the nature of the adrenergic receptor subtype distribution in their adipocytes is uncertain. A first objective was to identify the beta-adrenergic receptor subtype(s) present in adipocytes from horses and ponies. A second objective was to evaluate if the lipolytic responsiveness of isolated adipocytes to beta-adrenergic agonists is altered during lactation, a condition known to affect markedly maternal fat metabolism. Isoproterenol and salbutamol elicited strong lipolytic responses in adipocytes isolated from horse and pony subcutaneous adipose tissue. There were weak lipolytic responses to norepinephrine, dobutamine and BRL37344. The weak lipolytic response to NE compared to isoproterenol or salbutamol suggests an antilipolytic action from alpha2-adrenergic receptors. The relative order of potency for the beta-adrenergic agonists was isoproterenol>/=salbutamol>dobutamine=BRL37344. There was expression of beta2-adrenergic receptor mRNA in pony and horse adipose tissues, as estimated by relative RT-PCR, but no expression of mRNAs for beta1- or beta3-adrenergic receptors. Early lactation did not alter the lipolytic responses to beta-adrenergic agonists, nor the expression of beta2-adrenergic receptor mRNA. Thus, these results indicate a dominant if not exclusive presence of beta2-adrenergic receptors in pony and horse adipocytes that is not affected by lactation.     

Effects of prostaglandin E1 (PGE1) on the positive inotropic response of heart muscle to elevation of external Ca++-concentration, to increased driving frequency, and to paired stimulation.

Electrically driven left guinea pig atria were exposed to positive inotropic stimuli which are thought to be related to the turnover of calcium ions. For increasing contractibility, the following procedures were used: a) varying the concentration of CaCl2 in the bath fluid; b) stimulation at frequencies from 1.0 to 3.0 Hz; c) paired stimulation. Positive inotropic responses to the increase of the rate of stimulation and to paired stimulation were not affected by 0.1 microgram/ml tetrodotoxin (TTX). This excludes the adrenergic contribution to the positive inotropic effects observed. Actions of the positive inotropic stimuli were studied both in the absence and in the presence of 0.1--1.0--10.0--1000.0 ng/ml of PGE1-PGE1 in the highest concentration used increased contractile force. The inotropic stimulus-response curves were not affected by PGE1 at any concentration. This finding suggests there is no interaction between Ca ions and PGE1 in the contractile mechanism of the guinea pig heart muscle.     

Prostaglandin production in cultured gastric mucosal cells: role of cAMP on its modulation

The effect of cAMP on prostaglandin production may depend on cell types. To clarify the relationship between PG and cAMP, we examined arachidonate's effects on PG synthesis and intracellular cAMP accumulation in monolayers of rat gastric mucosal cells. These cells produced PGE2, PGI2 and thromboxaneA2 (TXA2) in amounts of 316 +/- 18, 100 +/- 7 and 30 +/- 5 pg per 10(5) cells in 10 min, respectively, in response to 10 microM arachidonic acid (AA). The production of these PG, however, leveled off subsequently. Cells initially exposed to AA responded poorly to a subsequent stimulation by AA. AA simultaneously stimulated intracellular cAMP accumulation; this stimulatory effect on cAMP production was abolished by the pretreatment with indomethacin. Nevertheless, the pretreatments with dibutyryl cAMP (0.1-5 mM) did not alter the amount of subsequent AA-induced PGE2 production. Furthermore, the preincubation with 1mM isobutyl methyl xanthine also failed to affect PGE2 synthesis, while it increased intracellular cAMP accumulation. Our studies suggest AA stimulates intracellular cAMP formation in cultured gastric mucosal cells, linked with conversion of AA to cyclooxygenase metabolites, AA-induced PG production is limited in these cells, and it seems, however, unlikely that intracellular cAMP modulates AA metabolism to PG.     

Prostaglandins--modulation of adrenergic nervous system.

Prostaglandins (PGs) affect vascular tone by a direct action on the vascular smooth muscle and by influencing vascular reactivity to adrenergic simuli and several vasoactive substances. Thus, in the isolated Tyrode's perfused rabbit renal, mesenteric and splenic vasculature PGE2 inhibited adrenergically induced vasoconstriction. Since the vasoconstrictor responses to renal nerve stimulation were enhanced by the blockade of PG synthesis and were reduced by stimulation of PG synthesis with arachidonic acid, this suggests that PGE2 functions as an inhibitory modulator of the adrenergic nervous system. However, our demonstration that PGE2 enhanced adrenergically induced vasoconstriction in the renal and mesenteric vasculature of the rat, but had opposite effects in the rat splenic vasculature indicates that the modulatory-effect of PGE-compounds on the adrenergic neuromuscular junction is species dependent and varies in different vascular beds within the same species. Prostaglandins, the release of which is evoked by several vasoactive substances including angiotensins, kinins, and adenine nucleotides, may also contribute to the regulation of vascular tone by either opposing or amplifying the vascular actions of vasoactive substances.     

Effects of prostaglandin E2 and lipopolysaccharide on osteoclastogenesis in RAW 264.7 cells

INTRODUCTION: Prostaglandins (PGs) can act on both hematopoietic and osteoblastic lineages to enhance osteoclast formation. METHODS: We examined PGE2 stimulated osteoclastogenesis in RAW 264.7 cells and the role of endogenous PGE2 in lipopolysaccharide (LPS) stimulated osteoclastogenesis. RESULTS: RANKL (1-100 ng/ml) increased formation of osteoclasts, defined as tartrate resistant acid phosphatase multinucleated cells, with peak effects at 30 ng/ml. Addition of PGE2 (0.01-1.0 microM) to RANKL (30 ng/ml) dose dependently increased osteoclast number 30-150%. Use of NS-398 (0.1 microM) or indomethacin (Indo, 1.0 micro M) to block endogenous PG synthesis had little effect on the response to RANKL alone but significantly decreased the response to PGE2. Addition of LPS (100 ng/ml) to RANKL increased osteoclast number 50%, and this response was significantly decreased by NS-398 and Indo. RANKL and PGE2 produced small, additive increases in COX-2 mRNA levels, while LPS produced a larger increase. PG release into the medium was not increased by RANKL and PGE2 but markedly increased by LPS. CONCLUSION: We conclude that RANKL stimulated osteoclastogenesis can be enhanced by PGE2 and LPS though direct effects on the hematopoietic cell lineage and that these effects may be mediated in part by induction of COX-2 and enhanced intracellular PG production.     

Platelet-activating factor stimulates production of prostaglandin E2 in murine osteoblast-like cell line MC3T3-E1.

We found that platelet-activating factor (PAF) stimulated the production of prostaglandin (PG) E2 in MC3T3-E1 cells in a time- and dose-dependent manner. 1.0 microM PAF gave a maximal stimulation of PGE2 production by MC3T3-E1 cells after a 4 hr PAF-treatment. Furthermore, the PAF-induced PGE2 production was abolished by the pre-treatment of the cells with a PAF receptor antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine, which occupied the same receptor site as PAF. These results suggest that PAF stimulates the PGE2 synthesis through a PAF receptor mediated pathway. Possibly PAF modulates bone metabolism by stimulating PGE2 synthesis.     

Design,synthesis and in vitro evaluation of (R)-4-(2-(tert-butylamino)-1-hydroxyethyl)-2-(hydroxymethyl)phenyl hydrogen phenylboronate: A novel salbutamol derivative with high intrinsic efficacy on the β2 adrenoceptor

We tested a set of boron containing arylethanolamine derivatives on the human and guinea pig β₂ adrenoceptor (β₂AR) 3-D structures by docking methodology. The compound with the highest affinity based on docking analysis, (R)-4-(2-(tert-butylamino)-1-hydroxyethyl)-2-(hydroxymethyl)phenyl hydrogen phenylboronate (boronterol) was synthesized, characterized and tested in guinea pig tracheal rings at basal tone and with histamine-induced contractions. Boronterol was at least eightfold more potent than salbutamol as a smooth muscle relaxant drug (judged by the EC₅₀ values) and showed a similar maximal relaxant effect as isoproterenol. ICI118,551 showed competitive antagonism on the relaxing effect of boronterol. These results suggest the β₂AR agonist action of boronterol.     

Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)gamma agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1beta-stimulated rat chondrocytes: evidence for PPARgamma-independent inhibition by 15-deoxy-Delta12,14prostaglandin J2

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1alpha and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1beta, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)gamma agonists. Real-time PCR analysis showed that IL-1beta induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1alpha and PGE2 peaked 24 hours after stimulation with IL-1beta; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Delta12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 microM), with more potency on PGE2 level than on 6-keto-PGF1alpha level (-90% versus -66% at 10 microM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 microM. Inhibitory effects of 10 microM 15d-PGJ2 were neither reduced by PPARgamma blockade with GW-9662 nor enhanced by PPARgamma overexpression, supporting a PPARgamma-independent mechanism. EMSA and TransAM analyses demonstrated that mutated IkappaBalpha almost completely suppressed the stimulating effect of IL-1beta on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-kappaB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-kappaB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARgamma through inhibition of the NF-kappaB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.     

High affinity binding of reovirus type 3 to cells that lack beta adrenergic receptor activity

A previous report demonstrated both immunological crossreactivity and structural similarity between the mammalian beta adrenergic receptor and the cell surface receptor for the reovirus type 3 (14). We now demonstrate that reovirus type 3 can bind selectively and with high affinity to cells that lack beta adrenergic receptor activity (L-cells). The present study was also designed to determine what effect reovirus binding has on beta adrenergic receptor function in cells (DDT1) that possess an intact ligand binding site. Based on computer analysis of reovirus competitive inhibition curves, the apparent dissociation binding constants (Kd) for reovirus binding to DDT1 and L-cells are 0.1 nM and 0.25 nM, respectively. High affinity [125I]-iodocyanopindolol (CYP) binding to beta adrenergic receptors can also be demonstrated in DDT1 cells but not in L-cells. In agreement with these ligand binding studies, adenylate cyclase activity is stimulated by the beta receptor agonist isoproterenol in DDT1 cell membranes but not in L-cell membranes. In addition, isoproterenol increases cAMP levels in DDT1 cells but not in L-cells. Neither reovirus serotype stimulates cAMP levels in either cell line, nor do they influence beta-adrenergic agonist stimulation of cAMP in DDT1 cells. These results argue against identity of the receptors for reovirus type 3 and beta adrenergic ligands.     

Beta-adrenergic receptor subtypes mediating lipolysis in porcine adipocytes. Studies with BRL-37344, a putative beta3-adrenergic agonist

The beta3-selective adrenergic receptor ligand BRL 37344 (BRL) was used to differentiate the presence and functional role of beta-adrenergic receptor (betaAR) subtypes in pig tissues. BRL did not stimulate adenylyl cyclase in membrane preparations or increase lipolysis from pig adipocytes. In contrast to some species, BRL appears to be a poor agonist for the pig betaAR and is not a useful betaAR ligand. Based on displacement of [3H]dihydroalprenolol binding, BRL exhibited a 100-fold selectivity for pig betaAR subtypes in adipose and skeletal muscle membranes. The high affinity site was proposed to be the beta2AR. When used as an antagonist, BRL blockade of the high affinity site did not interfere with isoproterenol-stimulated lipolysis but did inhibit adenylyl cyclase activation. Results indicate that the high affinity betaAR (betaAR) is not linked to lipolysis, possibly due to intracellular compartmentalization. Therefore, betaAR subtypes may have function-specific effects.     

Ca2+]i changes in guinea pig tracheal smooth muscle cells in culture: effects of Na+ and ouabain

The objective of this work was to confirm that the contractile effects of ouabain and Na(+)-free solutions in guinea pig tracheal rings are associated with increments in the cytosolic free Ca2+ concentration ([Ca2+]i) in cultured tracheal smooth muscle (TSM) cells. Cultured cells were alpha-actin positive. Histamine (50 microM) and Na(+)-free solution elicited a transient increase in [Ca2+]i, while the responses to thapsigargin (1 microM) and ouabain (1 mM) were long lasting. However, carbachol (10, 200, and 500 mM) and high K(+)-solution produced no effect on [Ca2+]i, suggesting that cultured guinea pig TSM cells display a phenotype change but maintain some of the tracheal rings physiological properties. The transient rise in [Ca2+]i in response to the absence of extracellular Na+ and the effect of ouabain may indicate the participation of the Na(+)/Ca2+ exchanger (NCX) in the regulation of [Ca2+]i.     

Effect of oestradiol 17 beta on prostaglandin biosynthesis by the uterus of ovariectomized rat and guinea pig

In vitro prostaglandin biosynthesis by uteri of ovariectomized rats and guinea pigs treated or untreated with oestradiol 17 beta, administered subcutaneously, was measured by R.I.A. of PGF2 alpha and PGE2. Incubations with [1-14C] arachidonic acid were also performed and labelled metabolites were analyzed by TLC. The main metabolite in rats was 6 keto PGF1 alpha and in decreasing order of magnitude, PGF2 alpha and PGE2. In guinea pig PGF2 alpha was the main product. Ovariectomy in rats completely changed the pattern of synthetized prostanoids : PGI2 production was doubled when compared to cycling rats and PGE2 increased 10 fold. PGF2 alpha values were similar to the mean value measured during the cycle. OE2 treatment almost completely inhibited PGI2 synthesis and reduced PGE2 by half. Total PG synthesis in OE2 treated animals was decreased by 5 fold when compared to spayed rats. Endogenous PGF2 alpha synthesis was slightly stimulated. In the guinea pig OE2 treatment of ovariectomized animals increased the total synthesis from 50 per cent. PGF2 alpha was always the main metabolite. In conclusion OE2 regulation of uterine PG synthesis is depending on the animal species and cannot be explained by a unique effect on the cyclooxygenase, but rather by an interplay on the various enzymes of the arachidonic acid cascade.     

Bile duct ligation induced acute inflammation up-regulates cyclooxygenase-2 content and PGE2 release in guinea pig gallbladder smooth muscle cell cultures

INTRODUCTION: This study examines hypotheses that BDL induces increased guinea pig gallbladder smooth muscle PGE2 release by up-regulation of COX-2. METHODS: BDL, Sham and Control Hartley guinea pig gallbladders were placed in cell culture, grown to confluence and underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacylin Synthase, actin, caldesmon, vinculin, meta-vinculin and tropomyosin and were assayed for basal release of 6-keto-PGF(1alpha), PGE2 and TxB2 by EIA. RESULTS: BDL did not alter content of smooth muscle cytoskeletal proteins. BDL for 48 h increased smooth muscle cell release of PGE2 and 6-keto-PGF(1alpha) by 3-fold or more when compared to the Control and Sham groups. Western Blot analysis showed increased content of COX-2 in the BDL group. CONCLUSIONS: BDL for 48 h markedly increased endogenous guinea pig smooth muscle cell PG release, which was due to increased COX-2 synthesis.