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1. We used electrophysiological and binding techniques to assess the presence of beta 1- and beta 2-adrenoceptors (beta 1AR and beta 2AR) in rat cardiac myocytes and to determine their ratio during aging. Experiments were performed in left ventricular myocytes enzymatically dissociated from the heart of 3-(young) or 22-month-old (old) Wistar Kyoto rats. 2. In patch-clamp experiments, myocytes from old rats showed a prolonged action potential duration (at -20 mV: 41.7 +/- 3.6 vs 26.2 +/- 3.1 ms; at -60 mV: 154.4 +/- 17.7 vs 87.1 +/- 6.9 ms, P < 0.05) and an augmented membrane capacitance (an index of cell size) (271.7 +/- 20.2 vs 164.3 +/- 14.6 pF, P < 0.05) compared to young rats. beta 2AR stimulation, achieved by superfusing myocytes with the selective beta 2AR agonist, zinterol (10 microM) or with (-)-isoprenaline (1 microM) in the presence of the selective beta 1AR antagonist, CGP 20712A (0.1 microM), significantly increased L-type calcium current (ICa,L) in rat ventricular myocytes. The percentage increase was similar in both young and old rats, either with zinterol (26.9 +/- 3.6% and 24.2 +/- 2.8%, respectively) or isoprenaline plus CGP 20712A (30.4 +/- 3.7% and 22.4 +/- 4.1%, respectively). Isoprenaline alone (beta 1AR and beta 2AR stimulation) caused a much smaller increase in ICa,L in old rats (58.4 +/- 12.1%) than in younger ones (95.3 +/- 8.1%) (P = 0.067). 3 The number of ,BAR mg-' protein, measured with saturation binding assays of the non selective ,betaAR antagonist [3H]-CGP 12177 was 1989.4+/-189.5 for 3- and of 1580.7 +/-161.5 for 22-month-old rats.Competition for [3H]-CGP 12177 binding by CGP 20712A gave biphasic curves which demonstrated two classes of binding sites. Densities (as percentages of total PAR density), and affinities for the two binding sites were: 80.4 +/- 2.2% (Ki = 6.6 +/- 1.3 nM) betaAR and 19.6 +/- 2.2% (Ki = 6.9 +/- 2.2 microM) beta2AR in young rats and 66.1 +/- 1.2% (Ki=8.3+/- 1.1 nM) beta1 AR and 33.9+/- 1.2% (Ki=5.2+/-0.6 PM) P2AR in old rats. Thebeta1AR/beta2AR ratio was significantly (P

The purpose of the present study was to assess the behavioral effects of the beta adrenergic agonist zinterol and to determine whether its actions were mediated by beta adrenergic receptors. Zinterol reduced response rate and increased reinforcement rate of rats under a differential-reinforcement-of-low-rate schedule in a dose-dependent manner; significant decreases in response rate and increases in reinforcement rate were observed at doses of 0.1–1 mg/kg. The effect of 0.3 mg/kg zinterol on this behavior was blocked by pretreatment with the beta adrenergic antagonist propranolol. Zinterol also reduced locomotor activity in a dose-dependent manner; significant reductions were observed at doses of 0.3–10 mg/kg. Similarly, the effect of 1 mg/kg zinterol on locomotor activity was antagonized by propranolol. These effects of zinterol were similar to those of other beta adrenergic agonists as well as those of antidepressant drugs. Although the site of action (central versus peripheral) of zinterol was not determined in the present study, an experiment was carried out to determine if zinterol could act centrally after peripheral administration. The ability of repeated, systemic administration of zinterol to reduce the density of beta adrenergic receptors in cerebral cortex and cerebellum was determined. Repeated treatment with a high dose of zinterol (10 mg/kg, IP) reduced the density of beta adrenergic receptors in these brain regions, suggesting that, at least under certain conditions, systemically administered zinterol did have access to the central nervous system.

We reported that inhibition of adenylate cyclase (AC)/cAMP by cell attachment to laminin (+LMN) or pharmacological (KT5720) inhibition of cAMP-dependent protein kinase (PKA) in cells not attached to LMN (-LMN-PKA), activates β2-AR stimulation of ICa,L via cPLA2 signaling. The present study determined the role of PKC and IP3-mediated Ca2+ release in β2-AR/cPLA2 signaling. As previously reported, 0.1 μM zinterol (β2-AR agonist) stimulation of L-type Ca2+ current (ICa,L) was unaffected by 10 μM AACOCF3 (cPLA2 inhibitor) in cells not attached to LMN (-LMN) but was significantly inhibited in +LMN and -LMN-PKA myocytes. Zinterol stimulation of ICa,L in -LMN-PKA myocytes was blocked by 5 μM U73122 (PLC inhibitor) and significantly inhibited by 4 μM chelerythrine (PKC inhibitor). Zinterol stimulation of ICa,L in -LMN myocytes was unaffected by inhibition of IP3-receptors (IP3Rs) by 2 μM 2-APB, but was significantly inhibited in +LMN and -LMN-PKA myocytes. Cells were cultured on LMN (24 hrs) with an adenovirus IP3 affinity trap to inhibit IP3-dependent Ca2+ signaling. Compared to control cells (β-gal), zinterol stimulation of ICa,L was significantly inhibited in cells infected with IP3 trap. Laser scanning confocal microscopy (fluo-4) revealed that zinterol stimulation of +LMN myocytes elicited local intracellular Ca2+ release events in 1 mM tetracaine (blocks RyR Ca2+ release), that were blocked by 2-APB. We conclude that inhibition of cAMP/PKA activates β2-AR stimulation of ICa,L via cPLA2 which is dependent on PKC and IP3-mediated Ca2+ signaling. These findings may be relevant to the remodeling of β-AR signaling in diseased (fibrotic) and/or aging atria, both of which exhibit decreases in AC activity.

To test the hypothesis that cardiac norepinephrine depletion related to heart failure alters contractile responses to beta-adrenergic agonists with a component of "indirect" action (acting by release of neuronal norepinephrine), we examined the inotropic potential of several pharmacologically distinct beta-agonists. Contractile responses to the nonselective beta-agonist isoproterenol, the beta 2-selective agonist zinterol, and the direct- and indirect-acting agonists dopamine and dopexamine were compared in isolated right ventricular trabeculae removed from failing, nonfailing innervated, and previously transplanted and, therefore, denervated nonfailing human hearts. In failing hearts, the contractile response to isoproterenol was significantly lower (41%) than that in nonfailing innervated hearts. The responses to the mixed agonists dopamine and dopexamine were even more attenuated in failing hearts, to a level 76-90% lower than those of nonfailing innervated hearts. In denervated, previously transplanted, nonfailing hearts, the contractile responses to the mixed agonists dopamine and dopexamine were 66-72% lower than those in the nonfailing innervated group, but the response to isoproterenol was not significantly different. The response to zinterol was not significantly different among the three groups. In subjects with severe heart failure, in vivo hemodynamic responses to dopexamine were compared with those of the direct-acting beta-agonist dobutamine. Responses to dopexamine and dobutamine were measured before and after prolonged continuous infusions of each drug. The response to dopexamine, but not to dobutamine, diminished over time. We conclude that a large component of the inotropic response to dopamine and dopexamine in human hearts is due to the ability of these agonists to promote the release of neuronal norepinephrine; when neuronal norepinephrine is depleted, indirect-acting agonists are less able to produce an inotropic response.

In fully developed androgen-induced hypertrophy of female mouse kidney, beta-adrenergic receptors per unit membrane protein were increased approx. 2.5-fold, as measured by the binding of [125I]iodocyanopindolol, with no change in apparent dissociation constants (Kd range 20-25 pM). Membrane protein relative to total kidney protein, Na+/K+-dependent ATPase (EC 3.6.1.3) and 5'-nucleotidase (EC 3.1.3.5) activities and cholesterol content per unit membrane protein did not differ significantly in preparations from control and treated animals. The binding of iodocyanopindolol to kidney membranes was characterized with respect to association and dissociation kinetics, and also in regard to the less-specific contributions of other major catecholamine or indolamine receptors, using mixtures of the corresponding specific competitors. beta 1-selective drugs, practolol and metoprolol, and beta 2-selective agents, IPS-339 and zinterol, were competed with iodocyanopindolol to assess the receptor type specificity, and the ensuing binding profiles were dissected by a nonlinear regression analysis as described by Munson, P.J. and Rodbard, D. (Anal. Biochem. (1982) 107, 220-239). Most of the androgen-induced beta-adrenergic receptors had the binding properties corresponding to beta 2-subtype. No consistent increase in the density of beta 1-adrenergic receptors could be shown.

beta-Adrenergic receptor subtypes were localized and differentiated in rat kidney slices by in vitro autoradiography using the nonselective beta-antagonist [125I]iodocyanopindolol in the presence of the selective agents betaxolol (beta 1) and zinterol (beta 2). [125I]Iodocyanopindolol binding to kidney sections in the presence of these agents could be differentiated into high- and low-affinity components as predicted by the subtype selectivity of the compounds. Autoradiography revealed that: beta-adrenergic receptors were highly concentrated within the renal cortex, especially in glomeruli and juxtaglomerular granule cells, and to tubular sites in the cortex and medulla; [125I]iodocyanopindolol labeling of the juxtaglomeruluar granule cells was abolished at lower concentrations (10(-8) M) of betaxolol than was the labeling of glomeruli (10(-6)--10(-4) M); and zinterol had little effect on labeling of juxtaglomerular granule cells and glomeruli unless high concentrations were used, whereas the tubular labeling in medulla was much more sensitive to incubation with zinterol. These results indicate that beta 1- and beta 2-adrenergic receptor subtypes are differentially distributed within the kidney: beta 1, predominantly contained in juxtaglomerular granule cells and glomeruli, and beta 2, predominantly in medullary tubules. In vitro autoradiography provides a useful means to examine different receptor populations in discrete tissue areas.

Changes of cyclic AMP levels were used to assess the specificity of the beta-adrenergic receptor in primary cultures of cells prepared from the intermediate lobe of rat pituitary gland. During a 4 min incubation, beta-adrenergic agonists led to a 4 to 6 fold stimulation of cyclic AMP concentration with the following order of potency (Kd values): zinterol (0.75 nM) greater than hydroxybenzylisoproterenol (1.0 nM) greater than (--)-isoproterenol (4.6 nM) greater than soterenol greater than (7.7 nM) greater than (--)-epinephrine (10 nM) greater than OPC 2009 (procaterol, 11 nM) much greater than (--)-norepinephrine (300 nM). The potent antagonists cyanopindolol, (--)-propranolol and hydroxybenzylpindolol reversed the stimulatory effect of (--)-isoproterenol at Kd values of 0.4-0.6 nM. Other beta-adrenergic antagonists had the following order of potency: pindolol = (--)-alprenolol = timolol (0.9-1.0 mM) much greater than metoprolol (100 nM) greater than dichloroisoproterenol (300 nM) greater than butoxamine (1100 nM). The beta 1-selective antagonist practolol had a low potency at 700 nM. The stereoselectivity of the receptor is indicated by the 400 to 70 fold higher potency of the (--)-isomers of isoproterenol, epinephrine and propranolol as compared to their (+)-stereoisomers. The data show that the beta-adrenergic receptor in the intermediate lobe of the rat pituitary gland is mainly of the bet 2-subtype. Study of this pure population of postsynaptic beta-adrenergic receptors where binding could be correlated with other parameters of cellular activity (cyclic AMP formation and alpha-MSH secretion) should yield useful information about the less accessible adrenergic systems of the brain.

The 125 I-labeled iodohydroxybenzylpindolol (IHYP) binding to β -receptors on brain microvessels is inhibited by isoproterenol, epinephrine and norepinephrine, with K i values of 2 × 10 −7 M, 2.5 × 10 −6 M and 1.2 × 10 −5 M, respectively. A modified Scatchard analysis of the inhibitory effects of practolol, metroprolol and zinterol on IHYP binding has shown that the proportion of β 2 -receptors in our preparation is about 80% of the total β -adrenergic receptor population. Our data indicate that the β -adrenergic receptors located on cerebral microvessels are of both β 1 and β 2 types, with a predominance of the β 2 type.

1 The chronotropic response and the variation in cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation induced by isoprenaline and six beta 2-selective agonists (fenoterol, salmefamol, soterenol, zinterol, salbutamol and formoterol) were analyzed on cultured heart cells of the rat. 2 The compounds elicited an enhancement of the frequency, but the time course of the variation of the beating rate was not identical for all of them. A rapid onset was observed for isoprenaline, zinterol and formoterol while it was slower for fenoterol, salmefamol and salbutamol. 3 In contrast with isoprenaline, the beta 2-selective agonists gave concentration-beating frequency curves which were not sigmoidal. Their effects extended up to a concentration of 5 to 6 orders of magnitude. Nevertheless, the concentration at which the maximal effect occurred and the intrinsic activities of the various compounds agrees better with the responses observed on guinea-pig atria than with those on trachea. 4 All the beta 2-selective agonists increased the accumulation of cyclic AMP in rat heart cells with a maximal effect at 10(-5)M or less. The effects of beta 2-agonists on cyclic AMP production showed some analogies with those on beating frequency of the heart cells. The increase in cyclic AMP accumulation induced by beta 2-agonists also corresponded to their chronotropic effects on guinea-pig atria. Thus, the correlation coefficient between the inverse of the log of the concentration producing the half maximal cyclic AMP accumulation in cultured heart cells and the pD2 values on guinea-pig atria was 0.93. 5 It is concluded that, in contrast to what was observed in other models, the beta 2-selective agonists induce an increase in the production of cyclic AMP in rat heart cells. Furthermore, the effects of the beta 2-agonists on cyclic AMP accumulation and on beating rate in the heart cells may correspond with their beta 1-adrenoceptor potencies.

We have shown that binding of 3H-dihydroalprenolol ([3H] DHA) to DDT1 MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [3H]DHA dissociation constants were 0.63 ± 0.15 nM (n=6) and 0.83 ± 0.04 nM (n=5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 ± 5,200 sites/ cell (n=6) and for membranes 468 ± 24 fmoles/mg protein (n=5). The order of agonist competition for the [3H]-DHA binding site of DDT1 cell membranes was isoproterenol (Ki = 0.20 ± 0.07 μM) > epinephrine (Ki = 0.4 ± 0.2 μM) > norepinephrine (Ki = 66.5 ± 5.15 μM) consistent with a β2-selective receptor interaction. Zinterol, a β2-selective antagonist, (Ki = 0.05 ± 0.01 μM) was 18x more effective than metoprolol, a β1-selective antagonist (Ki = 0.9 ± 0.1 μM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p>0.05) DDT1 cells possess a pure p...

The effects on the β-adrenergic receptors of intact L6 muscle cells of exposure to agonists were investigated. Treatment of cells with isoproterenol decreased GTP-, isoproterenol-, and zinterol-stimulated adenylate cyclase activities, whereas exposure of cells to zinterol decreased only isoproterenol- and zinterol-stimulated activities. Although the effects of these two full agonists on GTP-stimulated adenylate cyclase activity were different, the time courses for development and reversal of the effects were similar. The decrease in agonist-stimulated adenylate cyclase activity observed in membranes prepared from cells previously exposed to isoproterenol or zinterol was due to both an increase in the concentration of agonist required for half-maximal activation and a decrease in the maximum level of activation. No effects on fluoride- or manganese-stimulated activities were observed following exposure of cells to either agonist. Decreases in GTP-stimulated adenylate cyclase activity were also observed following exposure of cells to epinephrine, Cc-25, fenoterol, norepinephrine, terbutaline, or metaproterenol, but not following incubation of cells with dobutamine or salmefamol. The results suggest that full agonists interacting with the same receptor can induce different changes in the components of the adenylate cyclase system. Desensitization following exposure to zinterol may result from modification of the receptor, whereas that following exposure to isoproterenol may be due to alterations in both the guanine nucleotide-binding protein and the receptor.

The effect of daily exercise on the coronary resistance vessel sensitivity to intracoronary infusion of several pharmacological agents was assessed in 12 conscious adult mongrel dogs. alpha-Adrenergic receptor agonists (norepinephrine and phenylephrine) significantly decreased coronary blood flow velocity. beta 2-Adrenergic receptor agonists (isoproterenol and zinterol) and a metabolic vasodilator (adenosine) significantly increased coronary blood flow velocity. These responses occurred without altering factors that influence myocardial metabolism. Daily exercise significantly enhanced the coronary vascular sensitivity to each of the pharmacological agents. These results suggest that a nonspecific potentiation to pharmacological activation occurs after daily exercise. After left stellate ganglionectomy, intracoronary infusions of each pharmacological agent had similar effects on coronary blood flow velocity as presented for the intact dogs; however, daily exercise did not enhance the coronary vascular sensitivity to the pharmacological agents. These results demonstrate the need for an intact nervous system for the vascular adaptations associated with daily exercise.

We have previously shown that, in the rat, ovarian beta-adrenergic receptor content varies during the time of puberty, with values first increasing and then decreasing abruptly on the afternoon of the first proestrus, i.e., at the time of the preovulatory surge of gonadotropins and prolactin (Prl). In the present study, experiments have been conducted to determine: 1) if hormones other than follicle stimulating hormone (FSH) that are known to be involved in regulating prepubertal ovarian function can mimic the facilitatory effect of FSH on progesterone (P) response of granulosa cells to beta-adrenergic stimulation; 2) if beta-adrenergic receptor content of granulosa cells is under hormonal regulation; and 3) whether the facilitatory effect of hormones on the P response to beta-adrenergic stimulation is due to an increased cyclic AMP response to receptor activation. A 48-h in vitro preexposure of granulosa cells from juvenile, 29-day-old ovaries to the pituitary hormones Prl, luteinizing hormone (LH), or FSH showed that only the latter was able to facilitate the subsequent P response to Zinterol, a beta2-adrenergic agonist. Follicle-stimulating hormone also increased basal P release. Of the two nonpituitary hormones examined, the luteinizing hormone-releasing hormone (LHRH) agonist D-(Ala6,Pro9)-LHRH-ethylamide (LHRH-A) failed to affect P responsiveness, whereas corticosterone enhanced both basal P release and P response to Zinterol. This effect was less pronounced than that of FSH. Luteinizing hormone, Prl and corticosterone decreased beta-adrenergic receptor content to different extents, with corticosterone being the most effective and LH the least (50% and 15% decrease, respectively); LHRH-A was ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)

In an attempt to study the involvement of an adrenergic mechanism in the control of ovarian function at puberty, the presence of β-adrenergic receptors wets demonstrated, and the receptors were characterized in pre- and peripubertal ovaries. Using [125I]iodohydroxybenzylpindolol (IHBP) as a ligand, a single class of high affinity, low capacity β-adrenergic sites was found in both granulosa cells and residual ovary. Analysis of Scatchard plots derived from saturation curves or kinetic analysis of association curves provided identical equilibrium dissociation constant (Kd) values for the ligand-receptor binding. Dissociation experiments showed that the ligand binding to the receptor had the presence of both loosely bound, easily displaceable and tight, nondisplaceable components. IHBP binding was inhibited by L-propranolol [inhibition constant (Ki) = 10.5 nM], isoproterenol (Ki = 696 nM), and zinterol, a β2-adrenergic agonist (Ki = 187 nM). Metoprolol, a β1-antagonist, exhibited a much higher Ki (5.3 μM). C...

The radioactive beta-adrenergic antagonist [3H] dihydroalprenolol (DHA) binds to particulate preparations of human myometrium in a manner compatible with binding to the beta-adrenergic receptor. The binding of DHA is rapid (attaining equilibrium in 12 minutes), readily reversible (half time = 16 minutes), high affinity (KD = 0.50 nM), low capacity (Bmax = 70 fmoles/mg of protein), and stereoselective ([-]-propranolol is 100 times as potent as [+]-ipropranolol in inhibiting DHA binding). Adrenergic agonists competed for DHA binding sites in a manner compatible with beta-adrenergic interactions and mirrored beta 2 pharmacologic potencies: isoproterenol greater than epinephrine much greater than norepinephrine. Studies in which zinterol, a beta 2-adrenergic agonist, competed for DHA binding sites in human myometrial particulate indicated that at least 87% of the beta-adrenergic receptors present are beta 2-adrenergic receptors. Binding of DHA to human myometrial beta-adrenergic receptors provides a tool which may be used in the examination of gonadal hormonal modification of adrenergic response in human uterus as well as in the analysis of beta-adrenergic agents as potentially useful tocolytic agents.

The activity of a series of nine β-adrenergic agonists was studied in the potassium cyanide (KCN; 5.00 mg/kg i.v.) and the compound 48/80 (0.50 mg/kg i.v.) lethality tests in rats. All compounds were found active in both tests. The ED50 values in mg/kg for protection against KCN-induced lethality were as follows: clenbuterol (0.0047), zinterol (0.0055), hexoprenaline (0.0093), fenoterol (0.012), isoproterenol (0.014), colterol (0.019), salbutamol (0.028), terbutaline (0.14), and metaproterenol (0.33). The obtained results illustrate that KCN antagonism in rats is a general property of β-adrenergic agonists and that their potency is in agreement with previously reported data for β-adrenergic stimulation. By increasing the dose levels, the protective activity of some of the compounds disappeared and LD50s at which lethality to KCN was restored could be calculated. The protective dose range, which is defined as the ratio of the LD50 and the ED50, greatly varies among the test compounds. Protection from compound 48/80 induced lethality was also obtained with all compounds. This protection occurred at higher dose levels than in the KCN test, but persisted at agonist doses that had lost protective effect against KCN. Protection from KCN-induced lethality seems to be due to an increased tissue perfusion and to occur as long as myocardial activity is not markedly changed from normal by the test compound.

The subtype of the beta-adrenergic receptor expressed in 3T3-L1 preadipocytes and adipocytes differentiated with dexamethasone and methylisobutylxanthine was determined by comparing the affinity of the receptors for epinephrine, norepinephrine, and beta-1 and beta-2 selective antagonist, 8-fold more avidly than adipocyte receptors. In contrast, adipocyte beta-receptors had a 10-fold higher affinity for epinephrine than for norepinephrine and complexed the beta-2 selective agonist zinterol with a 20-fold higher affinity than preadipocyte receptors. Hofstee plots and computer analyses of the binding data revealed that the populations of beta-1 receptors in preadipocytes and beta-2 receptors in adipocytes were nearly homogeneous. Preliminary characterizations of the beta-receptor phenotype in (nondifferentiating) 3T3-C2 cells treated with dexamethasone and methylisobutylxanthine and 3T3-422A adipocytes differentiated with insulin indicated that the expression of beta-2 receptors was not correlated with differentiation, but rather with exposure of the cells to dexamethasone and methylisobutylxanthine. The regulator of beta-receptor subtype was identified as the glucocorticoid analog, dexamethasone, by employing 3T3-L1 adipocytes which were stimulated to differentiate with methylisobutylxanthine and insulin. Detailed binding studies showed that under these conditions the adipocyte receptors retain beta-1 character. Subsequent treatment with 0.5 microM dexamethasone promoted the loss of beta-1 receptors, the appearance of beta-2 receptors, and a net 2- to 3-fold increase in the number of beta-receptors. Dexamethasone effected a complete switch from beta-1 to beta-2 subtype at concentrations as low as 2.5 nM while other steroids were ineffective below a concentration of 10 microM.

In the rat, the main source of adrenergic fibers innervating nonvascular ovarian tissue is the superior ovarian nerve (SON). To determine the influence of the SON on prepubertal ovarian function and, hence, on the time of puberty, several experiments were conducted. Transection of the SON in early juvenile rats (day 24) led to more than 60% depletion in ovarian norepinephrine (NE) content, but affected neither the timing of vaginal opening nor that of first ovulation. SON section, however, produced a 2-fold increase in beta-adrenergic receptor density, with no change in affinity, suggesting that hypersensitivity by denervation had developed. Removal of all adrenergic influences from ovarian granulosa cells by culturing the isolated cells in vitro produced a striking increase in beta-adrenergic receptor number, which was maximal at 72 h. In spite of this elevated receptor content, the beta-adrenergic agonist Zinterol did not induce progesterone release unless the cells were previously exposed for 48 h to a physiological concentration of FSH. Both NE and epinephrine down-regulated the receptors in a dose-related manner, without affecting receptor affinity. To test the hypothesis that denervation of the ovary results in greater steroidal sensitivity to adrenergic stimulation, a model was constructed in which cells were simultaneously exposed to FSH and NE or epinephrine (innervated cells) or exposed only to FSH (denervated cells) for 48 h. The response to adrenergic stimulation was subsequently measured by incubating the cells with Zinterol for 24 h. Zinterol was highly effective in eliciting progesterone release from the denervated cells, but failed to do so in innervated cells. Additionally, the agonist increased cAMP in the former cells, but it was ineffective in the latter, indicating that prior exposure to catecholamines desensitized the adenyl cyclase enzyme coupled to beta-adrenergic receptor sites. The results suggest that during prepubertal maturation, noradrenergic fibers reaching the ovary via the SON may modify ovarian steridogenic sensitivity to catecholamines, by regulating the number of functional beta-adrenergic receptors in the gland. They also indicate that the apparent failure of SON denervation to alter the maturation of ovarian steroidogenesis and the time of puberty may be due to development of compensatory hypersensitivity after the denervation.

This study describes a rapid in vitro assay for the order of potency of bronchodilator drugs using specific binding of (−)-[3H] dihydroalprenolol ([3H]DHA) to rat lung membranes. Under linear conditions with respect to tissue, specific binding of [3H]DHA showed saturability, rapid kinetics of association and dissociation of radioligand, and sterospecificity. Nanomolar (nM) concentrations for 50% inhibition (IC50±SE) for the bronchodilator drugs examined were as follows: albuterol, 1485±170; isoproterenol, 136±53; procaterol, 162±28; terbutaline, 3310±934; and zinterol, 51±8.3. A comparison of binding studies using rat lung tissue membranes and similar preparations of rat heart and skeletal muscle demonstrated that lung tissue had 7 to 8 times more receptor sites (Bmax) for [3H]DHA than heart or skeletal muscle. Adenyl cyclase activit of the rat lung membrane preparation almost doubled in the presence of (−)-isoproterenol. Displacement of specific (3H)DHA binding in membrane preparations may provide useful data for evaluating bronchodilator compounds.

beta-Adrenergic agents powerfully stimulate muscle growth in animals. Whether the mechanism of action involves a direct effect on muscle cell beta-receptors or is secondarily due to a beta-induced alteration in the hormonal environment is not known. To assess whether direct beta-receptor activation results in muscle protein accretion, we examined the effect of the beta-agonist zinterol on several anabolic processes in L8 muscle cells in culture. In vivo feeding of zinterol (26.5 ppm) to rats significantly increased muscle weight by 15%. In vitro, zinterol stimulated lactate release from L8 cells whereas propranolol inhibited this process, demonstrating that these cells have functional beta-receptors both before and after fusion. We measured several anabolic processes, in both serum-stimulated and quiescent cells, over a wide range of zinterol concentrations. Zinterol had no effect on protein or DNA synthesis, protein degradation, or rates of amino acid uptake. These data suggest that the in vivo muscle growth stimulation is either indirect or some in vivo requirements (e.g. tension and nerve interactions) are necessary for expression of the effect.

Catecholamines are known to have an inhibitory effect on oviductal smooth musculature by changing the adrenergic receptors activity. In order to further investigate the role of epinephrine and/or norepinephrine in oviduct, the distribution of beta-adrenergic receptors has been studied in the rat oviduct, using in vitro autoradiography and [125I]cyanopindolol (CYP), as radioligand. The specificity of the labelling and the characterization of receptor subtypes in different cell types was achieved by displacement of radioligand with increasing concentrations of zinterol, a beta-adrenergic agonist with preferential affinity for the beta 2-adrenoreceptor subtype and practolol, a beta-adrenergic antagonist that binds preferentially to beta 1-subtype. Quantitative estimation of ligand binding was achieved by densitometry. It was shown that the vast majority of beta-receptors were of the beta 2-subtype and were found in smooth muscle layers as well as in the epithelium. The latter localization suggests a role for epinephrine and/or norepinephrine on the oviductal epithelium.

The subclass of beta-adrenergic receptors mediating glycogenolysis in slices from cerebral cortex of the mouse, incubated in the presence of [3H]glucose, was identified by comparing the relative potencies of agonists and the inhibition constants of antagonists to those found on reference systems. (+/-)Isoprenaline, (-)adrenaline and (-)noradrenaline produced a concentration-related glycogenolysis with Kact values of 2.2 x 10(-8) M, 2.8 x 10(-7) M and 3.6 x 10(-7) M, respectively. Zinterol, a selective beta 2-adrenergic agonist, did not produce any glycogenolytic response even in a large concentration. Salbutamol, a predominantly beta 2-adrenergic receptor agonist, elicited in a very large concentration (10(-4) M) less than 40% glycogenolysis, an effect which was not related to stimulation of beta-adrenergic receptors. The predominantly beta 1-adrenergic receptor antagonists, practolol and metoprolol shifted the concentration-response curve to noradrenaline to the right, with apparent Ki values of 8.0 x 10(-7) M and 7.6 x 10(-8) M, respectively, close to those reported in the rat heart. These various data indicate that the glycogenolytic response is selectively mediated by beta 1-adrenergic receptors. Under experimental conditions which were strictly identical to those used to measure glycogenolysis, a saturable binding of [3H]dihydroalprenolol to the slices occurred with Kd and Bmax values consistent with corresponding values on cell free preparations. Whereas the Ki values of antagonists were similar on the two systems, the Kact values of agonists on glycogenolysis were 10 times less than the Ki values for the binding of [3H]dihydroalprenolol. This suggests that the maximal glycogenolytic response is elicited for a partial receptor occupancy.

The properties ofβ 1- andβ 2-adrenoceptors in right and left atria of rat heart, and their roles in mediating chronotropic and inotropic responses toβ-adrenoceptor agonists were examined. [125I](-)-pindolol (125IPIN) bound saturably and specifically to a single class of high affinity sites in homogenates of both right and left atria. Thek 1's for association in right and left atria were 6.5×109 l/mol-min and 2.3×109 l/mol-min respectively, while thek −1's for dissociation were 0.20 min−1 and 0.17 min−1. The kinetically determinedK D's were 75 pmol/l in right and 30 pmol/l in left atria and were similar to the equilibriumK D's determined from Scatchard analysis of saturation isotherms of specific125IPIN binding. Inhibition of125IPIN binding byβ-adrenoceptor antagonists was stereoselective and the order of potency was timolol > 1-propranolol > d-propranolol > sotalol. Inhibition byβ 1- andβ 2-adrenoceptor subtype selective antagonists yielded flat displacement curves with low Hill coefficients. Nonlinear regression analysis of displacement byβ 1-selective (practolol, atenolol and metoprolol) andβ 2-selective (ICI 118,551) antagonists gave estimates of the proportion ofβ 1- andβ 2-adrenoceptors present in rat atria. Right atria contained 67±4.2%β 2-adrenoceptors and 33±4.2%β 2-adrenoceptor, while left atria contained 67±2.8%β 1- and 33±2.8%β 2-adrenoceptors. Increases in the rate of spontaneously beating right atria and the force of electrically driven left atria caused byβ-adrenoceptor agonists were also measured. pA2 values for non-subtype selectiveβ-adrenoceptor antagonists in inhibiting isoprenaline-induced increases in rate and force were highly correlated withK D values determined for specific125IPIN binding. pA2 values forβ 1- andβ 2-selective antagonists in inhibiting isoprenaline-induced increases in rate and force correlated well with the pK D values of these drugs in binding toβ 1-adrenoceptors, but not with the pK D values in binding toβ 2-adrenoceptors. Dose-response curves for stimulation of both rate and force by theβ 2-selective agonists procaterol and zinterol were shifted to a much greater extent by selective blockade ofβ 1-adrenoceptors with metoprolol than by selective blockade ofβ 2-adrenoceptors with ICI 118,551, suggesting that these compounds caused their effects by activatingβ 1-adrenoceptors. These results suggest thatβ 1- andβ 2-adrenoceptors coexist in both left and right atria of rat heart in approximately a 2∶1 ratio, however onlyβ 1-adrenoceptors mediate the chronotropic and inotropic effects ofβ-adrenoceptor agonists.

Antibodies directed against a peptide corresponding to the second loop of the human beta2-adrenergic receptor were induced in rabbits by immunisation with the free peptide in complete Freund's adjuvant. The resulting antibodies were affinity-purified and shown to be monospecific for the target receptor. They were able to stimulate the L-type Ca2+ channels in whole-cell patch-clamp experiments on isolated adult guinea-pig cardiomyocytes. This effect was similar to that obtained by the specific beta2-adrenergic agonist zinterol. The antibody effects could be blocked with the specific beta2-adrenergic inverse agonist ICI118,551 but not with the neutral antagonist alprenolol. These results suggest that the antibodies recognise the active conformer of the beta2-adrenergic receptor.

Interruption of the ascending noradrenergic neurons of the locus coeruleus in the rat forebrain with 6-hydroxydopamine produced a progressive accumulation, proximal to the lesion, of tritiated dihydroalprenolol binding activity over 2 days. This accumulation could be blocked by interrupting the neurons closer to their cell bodies. Competitive binding studies with the beta 2 agonist Zinterol suggested that the accumulated beta-receptors were primarily of the beta 1 subtype. These results suggest that, in the rat brain, some beta 1-adrenoreceptors are located in presynaptic, noradrenergic locus coeruleus neurons and are transported in their axons.

β-adrenergic receptors have been classified into 2 subtypes based on differences in the specificity of action of natural catecholamines [1]. For β1-adrenergic receptors, epinephrine and norepinephrine were about equivalent. In contrase, for β2-adrenergic receptors, epinephrine was considerably more potent than norepinephrine. This initial classification had survived to the screening of a large number of synthetic agonists and antagonists of increasing selectivity, and 20 years after Land’s publication, it is still obvious that only 2 subtypes of β-adrenergic receptors are present in mammalian tissues. Until the last 8 years, β1- and β2-adrenergic receptors were essentially distinguished one from the other by pharmacological studies based on the relative efficiency of selected molecules: Among the most studied are the β1-selective antagonists practolol, metoprolol and atenolol, the β2-selective agonist zinterol and the β2 selective antagonist ICI 118551. More recently, efforts have been made to distinguish the receptors on the basis of chemical and/or immunological differences [2], but it is only very recently that the complete amino-acid sequence of both β1- and β2-adrenergic receptors had definitively established that β1- and β2-adrenergic receptors were distinct chemical entities. However, the two pharmacologically distinct β1- and β2-adrenergic receptors can be found in a single tissue, subserving sometimes distinct functions, and in some cases both subtypes have been found in the same cell [3].

Recent studies have shown that beta-adrenergic agents stimulate steroidogenesis and cyclic AMP formation in mouse Leydig cells in culture. To obtain information about the possible presence and the characteristics of a beta-adrenergic receptor in rat testicular interstitial cells, the potent beta-adrenergic antagonist [125I]cyanopindolol (CYP) was used as ligand. Interstitial cells prepared by collagenase dispersion from rat testis were incubated with the ligand for 2 h at room temperature. [125I]cyanopindolol binds to a single class of high affinity sites at an apparent KD value of 15 pM. A number of sites of 6,600 sites/cell is measured when 0.1 microM (-) propranolol is used to determine non-specific binding. The order of potency of a series of agonists competing for [125I]cyanopindolol binding is consistent with the interaction of a beta 2-subtype receptor: zinterol greater than (-) isoproterenol greater than (-) epinephrine = salbutamol much greater than (-) norepinephrine. In addition, it was observed that the potency of a large series of specific beta 1 and beta 2 synthetic compounds for displacing [125I]cyanopindolol in rat interstitial cells is similar to the potency observed for these compounds in a typical beta 2-adrenergic tissue, the rat lung. For example, the potency of zinterol, a specific beta 2-adrenergic agonist, is 10 times higher in interstitial cells and lung than in rat heart, a typical beta 1-adrenergic tissue. Inversely, practolol, a typical beta 1-antagonist, is about 50 times more potent in rat heart than in interstitial cells and lung.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro autoradiography of [125I]cyanopindolol ([125I]cyp) binding to sections of ovine pineal reveals a uniform distribution of beta-adrenergic receptors throughout the gland. Norepinephrine (NE) stimulated cyclic AMP production in pineal slices in both a time- and dose-dependent manner, producing a maximal two-fold increase. NE, isoproterenol (ISO) and epinephrine (E) stimulate cyclic AMP (cAMP) production with equal potency. NE stimulation of cAMP was totally blocked by propranolol (beta antagonist) but only partially blocked by practolol (a beta 1 preferential antagonist) indicating a mixed population of beta 1 and beta 2 receptor subtypes. Displacement of [125I]cyp binding by either practolol or zinterol (preferential beta 2 agonist) revealed IC50s of 1.3 x 10(-5) M and 9.95 x 10(-8) M respectively, confirming a mixed population of beta 1 and beta 2 receptors. A range of peptides previously localised within the pineal by immunocytochemistry were tested at a concentration of 10(-5) M for their effect on cyclic AMP production in pineal homogenates. Only vasoactive intestinal peptide (VIP) was effective showing a dose-dependent stimulation. ISO and VIP stimulation of cAMP were additive indicating action via independent receptors.

Summary β-Adrenergic receptors were demonstrated in placental membrane fractions of five mammalian species (rat, rabbit, guinea pig, sheep and human) with the radiolabelled antagonist (−)-[3H]-dihydroalprenolol ((−)-[3H]DHA). Identical membrane preparations showed a variable maximum binding capacity in different species, ranging from 70±6.7 in the term rat to 315 ±28.2 in the term rabbit. Binding affinity did not change with species and (−)-[3H]DHA binding was associated with a catecholamine-sensitive adenylate cyclase in each. In the rabbit placenta, β-adrenergic receptor subtypes were partially characterized and the ontogeny of the placental β-receptor demonstrated. Agonist competition for the (−)-[3H]DHA binding site showed the order of potency (−)-isoproterenol≫ (−)-epinephrine≫ (−)-norepinephrine characteristics of a β2 subtype. Guanine nucleotides (Gpp(NH)p) significantly reduced agonist affinity for the binding site and increased the Hill coefficient from 0.6 to approximately 1.0, as expected in a ‘coupled’ β-adrenergic receptor. Competition between (−)-[3]DHA and β-adrenergic receptor subtype selective agents was performed to define the β-receptor subtype populations. In contrast to the linear Hofstee plots obtained with classical agonists, curvilinear Hofstee plots were obtained with both metoprolol (β1 selective) and zinterol (β2 selective), indicating the presence of two distinct receptor subtype populations. A predominance of the β2 subtype was demonstrated with both agents in the term rabbit placenta (35 per cent β1, 65 per cent β2). β-Adrenergic receptor density in rabbit placentae from 16 to 30 days' gestation was determined. Protein and DNA content were also determined in these placentae. A period of growth by cellular hyperplasia from day 16 to day 25, during which DNA increased threefold and protein per DNA remained constant, was followed by a period of growth by cellular hypertrophy from day 25 to day 30, during which DNA remained constant and protein per DNA increased twofold. The β-adrenergic receptor density increased markedly with gestation (181±29 to 330±37 fmol/mg). There was no change in binding affinity with gestation. β-Receptor density per DNA remained constant during the period of cellular hyperplasia, increased twofold during early cellular hypertrophy (day 25 to day 27) and then remained constant to term (day 30). An adenylate cyclase coupled β-adrenergic receptor is present in the placentae of several mammalian species and changes in density in parallel with maturational events in one species, suggesting a role for catecholamines in placental function.

Beta adrenergic receptor binding was performed with 125I iodocyanopindolol on human cavernous tissue membrane fractions from normal tissue and transsexual procedures obtained postoperatively, as well as from postmortem sources. Isotherm binding studies on normal fresh tissues indicated that the receptor density was 9.1 fmoles/mg. with a KD of 23 pM. Tissue stored at room temperature for 4 to 6 hours, then at 4C in saline solution for 19 to 20 hours before freezing showed no significant changes in receptor density or affinity, and provided evidence for the stability of postmortem tissue obtained within the same time period. Beta receptor density of 2 cavernous preparations from transsexual procedures was not significantly different from normal control tissues, and showed that high concentrations of estrogen received by these patients had no effect on beta adrenergic receptor density. Displacement of 125iodocyanopindolol by 5 beta adrenergic agents demonstrated that 1-propranolol had the greatest affinity followed by ICI 118,551, zinterol, metoprolol and practolol. When the results of these displacement studies were subjected to Scatfit, non-linear regression line analysis, a single binding site was described. Based on the relative potency of the selective beta adrenergic agents it appears that these receptors were of the beta 2 subtype.

As previously shown, the beta-adrenergic agonists isoproterenol, epinephrine and norepinephrine stimulate prolactin (PRL) release from superfused rat anterior pituitary cell aggregates. In order to further characterize the beta-adrenergic response in this tissue preparation, the effects of various beta-adrenergic agents were investigated. The beta 2-agonist, zinterol, stimulated PRL release at concentrations more than 4 orders of magnitude lower than prenalterol, a beta 1-agonist with high potency in rat heart. The order of potency of the antagonists IPS 339 (beta 2), ICI 118.551 (beta 2), propranolol, sotalol, practolol (beta 1), metoprolol (beta 1) and H 35/25 for inhibition of beta-agonist-stimulated PRL release provided additional support for a beta 2-stimulatory effect. beta-Agonists were also capable of stimulating PRL release from superfused intact pituitaries. The beta-adrenergic response desensitized rapidly during prolonged exposure of the aggregates to beta-agonists.

The pharmacological and binding properties of four beta-adrenomimetic drugs with N-alkyl substitutions (isoprenaline, terbutaline, salbutamol and soterenol) were compared with those of four corresponding drugs with N-aralkyl substitutions (protokylol, ME 506, salmefamol and zinterol). BD-40 A, a very powerful beta 2-agonist with a related chemical structure, was also included in this study. The beta 1- and beta 2-activities of these drugs were determined on guinea-pig atria and trachea, their alpha-adrenolytic activity was measured on rat aorta and their affinities (Ki) for alpha 1- and alpha 2-adrenoceptors on rat cortical membranes were assessed using [3H]prazosin and [3H]yohimbine. In this group of beta-agonists, substitution of the N-alkyl by an N-aralkyl group had a variable effect on the beta 2-selectivity whereas alpha-adrenolytic properties were always enhanced. An increase of the affinities (Ki) for both alpha 1- and alpha 2-adrenoceptors was found but the effect was much more pronounced for alpha 1-adrenoceptors. These results indicated that the alpha-adrenolytic activity observed with the N-aralkyl beta-agonists was selective for alpha 1-adrenoceptors.

beta 1- and beta 2-Adrenergic receptors co-exist in the adult rat ventricle. We have employed radioligand binding and cell purification techniques to determine the cellular origin of these receptors. The beta-adrenergic antagonist ligand (+/-)-[125I] iodocyanopindolol binds to 2 X 10(5) receptors per purified adult rat cardiomyocyte, with a dissociation constant of 70 pM. The subtype-selective antagonists betaxolol (beta 1), practolol (beta 1), and zinterol (beta 2) compete for [125I]iodocyanopindolol-binding sites on intact myocytes in monophasic manners with dissociation constants of 46, 845, and 923 nM, respectively. [125I]iodocyanopindolol binding to membranes prepared from nonmyocyte elements of rat ventricle occurs with a dissociation constant of 43 pM and a capacity of 88 fmol/mg membrane protein. Computer analysis of competition of [125I]iodocyanopindolol binding by betaxolol, practolol, and zinterol in nonmyocyte membranes demonstrates biphasic curves that comprise binding to both beta 1- and beta 2-receptors. These data demonstrate that purified adult ventricular myocytes possess only beta 1-receptors, and that the beta 2-receptors found in rat ventricle are located on nonmyocyte cell types.

The inhibitory effects of catecholamines on uterine smooth muscle are known to be mediated through beta-adrenergic receptors. To investigate further the distribution of these receptors in the rat uterus, we utilized in vitro autoradiography using [125I]-cyanopindolol [CYP], a specific beta-receptor ligand that has equal activity for both beta 1- and beta 2-receptor subtypes. The specificity of the labeling and the characterization of receptor subtypes in different cell types were achieved by displacement of radioligand with increasing concentrations of zinterol, a beta-adrenergic agonist with preferential affinity for the beta 2-adrenoreceptor subtype, and practolol, a beta-adrenergic antagonist that binds preferentially to the beta 1-subtype. Quantitative estimation of ligand binding was performed by densitometry. It was shown that the vast majority of beta-adrenoreceptors were of the beta 2-subtype and were found in high concentration not only in the myometrium but also in the endometrial and serosal epithelia. Specific labeling was also observed in glandular elements. These results suggest that beta-adrenoreceptors might be involved in different functions in the uterus.

The highly specific beta-adrenergic radio-ligand (+/-)-125 iodocyanopindolol (ICYP) was used to characterize the beta-adrenergic receptor subtype present in rat kidney. Binding of ICYP to membranes from rat kidney was of high affinity (KD = 68.9 pM) and saturable with 1.06 pmoles ICYP bound/g tissue wet wt at maximal occupancy of the sites. Analysis of inhibition of ICYP binding by beta 1- and beta 2-selective adrenergic drugs via pseudo-Scatchard ("Hoifstee') plots resulted in linear plots indicating the existence of a homogeneous population of beta-adrenergic receptors. From the resulting KD-values for practolol (2.2 microM), metoprolol (0.21 microM), zinterol (0.4 microM) and IPS 339 (0.046 microM) it is concluded that the beta-adrenergic receptor in rat kidney is of the beta 1-subtype. This subclassification is further supported by the fact that (-)-noradrenaline and (-)-adrenaline were equipotent in inhibiting ICYP binding. The beta-adrenergic agonists (-)-isoprenaline and zinterol bind to two distinct states of this beta 1-receptor, a high and low affinity state. GTP (10(-4) M) converts this heterogeneous binding into a homogeneous low affinity binding.

We determined the amount of beta 1- and beta 2-adrenoceptors in right and left atria and ventricles of rabbits. For this purpose inhibition of specific (-)-3H-dihydroalprenolol [(-)-3H-DHA] binding (5 nM) by beta 1-selective (practolol, metoprolol) and beta 2-selective (zinterol, IPS 339) adrenergic drugs was determined and analyzed by pseudo-Scatchard (Hofstee) plots. For both atria, inhibition of binding by the four selective beta-adrenergic drugs resulted in non-linear Hofstee plots, suggesting the coexistence of both beta-adrenoceptor subtypes. From these plots we calculated a beta 1:beta 2-adrenoceptor ratio of 72:28 for the right atrium and of 82:18 for the left. In contrast, only a very small amount of beta 2-adrenoceptors (approximately 5-7% of the total beta-adrenoceptor population) could be detected in the ventricles. For comparison we analyzed the inhibition of specific (-)-3H-DHA binding in tissues with homogeneous population of beta-adrenoceptors (beta 1:guinea pig left ventricle; beta 2: cerebellum of mature rats). For both tissues the four selective beta-adrenergic drugs showed linear Hofstee plots, demonstrating that in tissues with homogeneous beta-receptor population interaction of each drug with the receptor followed simple mass-action kinetics. We conclude that beta 1- and beta 2-adrenoceptors coexist in rabbit atria while the ventricles are predominantly endowed the beta 1-adrenoceptors.

The characteristics of beta-adrenergic receptors in human fetal lung were examined using the beta-adrenoceptor antagonist 3 H-dihydroalprenolol /DHA/. Steady-state binding was reached by 15 min at 25°C, and the association and dissociation rate constants were 0.0422 nM −1 min −1 and 0.0874 min −1 , respectively. From saturation experiments, B max of 82.0 ± 38 fmol/mg protein and K D =1.85 ± 0.92 nM were calculated. Inhibition of 3 H-DNA binding by beta-1 /metoprolol/ and beta-2 /zinterol, IPS-339, fenoterol/ selective drugs resulted in biphasic displacement curves with slope factors less than 1.0. Analysis of these curves revealed a beta-1: beta-2 ratio of 40:60 in human fetal lung. The presence of 3 H-DHA binding sites in early human lung may have a developmental importance which is not yet understood.

beta-Adrenoceptors of pig coronary arteries were investigated by the use of a new ligand, 125iodocyanopindolol (ICYP) in binding studies. Inhibition of ICYP binding by betaxolol (a selective beta 1-antagonist), zinterol (a selective beta 2-agonist) and ICI 118551 (a selective beta 2-blocking drug) resulted in non-linear Scatchard plots, suggesting that both beta-adrenoceptor subtypes are present in pig coronary arteries. Computer analysis of the data gave a beta 1:beta 2-adrenoceptor ratio of approximately 65:35.

The effects of beta-adrenoceptor agonists and antagonists on field-stimulated release of radioactivity from superfused guinea pig papillary muscles preincubated with 3H-noradrenaline were studied. Stimulation-evoked overflow of tritium was abolished in the absence of Ca2+ or the presence of tetrodotoxin. Isoprenaline (1 mumol/L) caused a slight facilitation of evoked overflow, whereas phentolamine (1 mumol/L) exerted a strong facilitatory action. However, when phentolamine (1 mumol/L) was present throughout superfusion, isoprenaline and the selective beta 2-adrenoceptor agonist, zinterol, caused concentration-dependent increases (half-maximal effects at 1 nmol/L). The effects of the agonists were inversely related to stimulation frequency. Furthermore, the concentration-response curve of isoprenaline was shifted to the right by the selective beta 2-adrenoceptor antagonist, ICI 118,551, but not by the selective beta 1-adrenoceptor antagonist, ICI 89,406. Schild-plot analysis revealed competitive antagonism and a pA2 value of 9.04 for ICI 118,551. Both ICI 118,551 and ICI 89,406, as well as beta-adrenoceptor antagonists with intrinsic sympathomimetic activity (pindolol and celiprolol; 1 mumol/L), had no effect on stimulation-evoked overflow of tritium (phentolamine present). It is concluded that guinea pig papillary muscles are endowed with prejunctional beta 2 adrenoceptors facilitating impulse-evoked noradrenaline release. The facilitation is markedly promoted by blockade of prejunctional alpha adrenoceptors.

Beta 2-adrenergic receptors comprise 40% of the total beta-receptor population in failing human right ventricles, as deduced from computer modeling of iodine-125 iodocyanopindolol-ICI 118,551 competition curves. A myocardial cell origin of at least some of the beta 2 subpopulation was demonstrated by documenting a beta 2-receptor mediated positive inotropic response to the selective beta 2 agonist zinterol. It is concluded that beta 2-adrenergic receptors are present on human ventricular myocardial cells.

We used radioligand binding methods to characterize beta-adrenergic receptors on endothelial cells cultured from adult human iliac vein (HIVE) and bovine fetal aorta (BFAE). For comparison, we also studied the well-characterized C6 glioma cell line (C6). Both human and bovine endothelial cells showed specific saturable binding of [125I]iodopindolol. There was no difference in the binding affinity (KD) of iodopindolol to membranes from the three cell types. However, the beta-receptor density (Bmax) was greater on HIVE cells and BFAE cells than on C6 cells. Displacement of ligand from HIVE and BFAE cells by zinterol or from BFAE cells by ICI 89,406 was consistent with binding to the beta 2-subtype. In contrast, displacement of ligand from C6 cells by zinterol or ICI 89,406 was consistent with binding to both beta 1- and beta 2-subtypes. Exposing BFAE cells in culture to 10 microM isoproterenol for 6 h resulted in a 55% decrease in Bmax without a change in KD. We conclude that 1) human and bovine endothelial cells in culture contain a substantial population of beta-adrenergic receptors, which are predominantly of the beta 2-subtype, and 2) endothelial beta-receptors exhibit downregulation by beta-agonists in culture.

Voltage-gated Ca(2+) channels of chromaffin cells are modulated by locally released neurotransmitters through autoreceptor-activated G-proteins. Clear evidence exists in favor of a Ca(2+) channel gating inhibition mediated by purinergic, opioidergic, and alpha-adrenergic autoreceptors. Few and contradictory data suggest also a role of beta-adrenergic autoreceptors (beta-ARs), the action of which, however, remains obscure. Here, using patch-perforated recordings, we show that rat chromaffin cells respond to the beta-AR agonist isoprenaline (ISO) by either upmodulating or downmodulating the amplitude of Ca(2+) currents through two distinct modulatory pathways. ISO (1 microm) could cause either fast inhibition (approximately 25%) or slow potentiation (approximately 25%), or a combination of the two actions. Both effects were completely prevented by propranolol. Slow potentiation was more evident in cells pretreated with pertussis toxin (PTX) or when beta(1)-ARs were selectively stimulated with ISO + ICI118,551. Potentiation was absent when the beta(2)-AR-selective agonist zinterol (1 microm), the protein kinase A (PKA) inhibitor H89, or nifedipine was applied, suggesting that potentiation is associated with a PKA-mediated phosphorylation of L-channels (approximately 40% L-current increase) through beta(1)-ARs. The ISO-induced inhibition was fast and reversible, preserved in cell treated with H89, and mimicked by zinterol. The action of zinterol was mostly on L-channels (38% inhibition). Zinterol action preserved the channel activation kinetics, the voltage-dependence of the I-V characteristic, and was removed by PTX, suggesting that beta(2)AR-mediated channel inhibition was mainly voltage independent and coupled to G(i)/G(o)-proteins. Sequential application of zinterol and ISO mimicked the dual action (inhibition/potentiation) of ISO alone. The two kinetically and pharmacologically distinct beta-ARs signaling uncover alternative pathways, which may serve the autocrine control of Ca(2+)-dependent exocytosis and other related functions of rat chromaffin cells.

β 2- adrenergic induced renal vasodilatation is greater in fetal than adult sheep. To determine a mechanism involved in this age related response, we characterized the β adrenoceptor in fetal and adult renal cortical membranes (30,000 xg fraction containing both vascular and tubular membranes) using radioligand binding studies. Specific binding of the non-selective β adrenergic antagonist 125I-pindolol (defined by 1 uM (-)-propranolol) was 80%. Receptor density from Rosenthal plots (Bmax, fmol/mg protein), inhibition constant (Ki, M), and % distribution of receptor subtypes using the β2 adrenergic antagonist, ICI 118, 551, from competition experiments (M±SE) are: Ki for (-)-isoproterenol and the β2 agonist zinterol were similar in both fetus and adult. However, stereoselectivity was greater in adults than fetuses: similar Ki for (-)-propranolol but Ki for (+)-propranolol less in fetus (6E−8M) (n=3) than adult (1E-7M)(n=3), (p

The ontogeny of β-adrenergic receptors (BAR) in lung was described in the rabbit, rat, sheep and guinea pig. Developmental increases in binding capacity were demonstrated in all species, for example, rabbit lung membrane bound (-)-[3H]-dihydroalprenolol, [3H]DHA to a single class of sites, was stereoselective, reversible and saturable; KD 1.78±0.30 nM. Binding at all ages was inhibited by agonists in the order of potency: Iso > Epi=Norepi characteristic of a β1AR subtype. Studies with metoprolol (β1) and zinterol (β2-selective) revealed 60% β1- and 40% β2adrenergic subtypes at all ages. Bmax increased 11.5 fold from day 25 of gestation to adulthood, increasing during the latter days of gestation and the early neonatal period, from 37±10 fmolemg-1 protein to 425±51 in the adult, m±SD. Gpp(NH)p decreased agonist affinity more effectively in adult than in fetal lung and we hypothesize that a “coupling” defect exists in fetal lung BAR. The sensitivity of adenylate cyclase activity to catecholamine was maximal in adult rabbit lung increasing with age from 23% at day 25 gestation to 255% stimulation in the adult. Pulmonary BAR numbers at term gestation were higher in animals which are relatively more mature at birth (guinea pig and sheep) as compared to less mature (rabbit and rat). In conclusion, β-adrenergic receptors appear to be a sensitive indicator of pulmonary maturation and the increase in BAR during development in mammalian lung and supports the role of β-adrenergic receptors in the regulation of pulmonary function.

β-Adrenergic stimulation of the placenta causes increased glycogenolysis and cAMP production. These effects are presumably mediated through the β-adrenergic receptor (βAR). In this study the βAR in rabbit placenta was characterized and shown to increase with gestational age. Binding of the β-adrenergic antagonist dihydroalprenolol, (-)[3H]DHA, to rabbit placental membranes was saturable to a single class of sites, KD=1.67±.07(nM), n=11 and Bmax=326±33 fmoles/mg. Agonist competition for the βAR was performed in the presence of the non-hydrolyzable GTP analog Gpp(NH)p (required for Hill coefficient = 1) and showed the potency order (-)iso ≫ (-)epi > (-)norepi characteristic of the β2 subtype. However, competition experiments with subtype specific agents metoprolol (β1) and zinterol (β2) indicated that the rabbit placenta contained approximately equal populations of βAR subtypes (51% β1, 49% β2). Rabbit placenta βAR/DNA increased 5.3 fold from day 16 to day 27 of gestation (410±120 fmoles/mg DNA to 2184±354 p

We reported that attachment of atrial myocytes to laminin (LMN) decreases adenylate cyclase (AC)/cAMP and increases β2-AR stimulation of L-type Ca2+ current (ICa,L). This study determined whether LMN enhances β2-AR signaling via a cAMP-independent mechanism, i.e. cPLA2 signaling. Atrial myocytes were plated on uncoated cover-slips (-LMN) or cover-slips coated with LMN (+LMN) (>2 hrs). As previously reported, 0.1 μM zinterol (β2-AR agonist) stimulation of ICa,L was larger in +LMN than -LMN myocytes. In +LMN myocytes, zinterol stimulation of ICa,L was inhibited by 10 μM AACOCF3 (cPLA2 inhibitor), pertussis toxin or 10 μM BAPTA-AM (intracellular Ca2+ chelator). Stimulation of ICa,L by fenoterol (β2-AR/Gs agonist) was smaller in +LMN than -LMN myocytes. Arachidonic acid (AA; 5 μM) stimulated ICa,L in -LMN and +LMN myocytes similarly. Inhibition of PKA by either 5 μM H-89 or 1 μM KT5720 in -LMN myocytes mimicked the effects of +LMN myocytes to enhance zinterol stimulation of ICa,L, which was blocked by AACOCF3. In contrast, H-89 inhibited fenoterol stimulation of ICa,L, which was unchanged by AACOCF3. Inhibition of ERK1/2 by 1 μM U-0126 inhibited zinterol stimulation of ICa,L in +LMN myocytes and -LMN myocytes in which PKA was inhibited (KT5720). Western blots showed that inhibition of PKA (KT5720) in -LMN myocytes markedly increased zinterol phosphorylation of ERK1/2. We conclude that inhibition of AC/cAMP/PKA by cell attachment to LMN or PKA by pharmacological agents in -LMN myocytes switches β2-AR signaling from predominantly Gs/AC/cAMP/PKA to Gi/ERK1/2/cPLA2/AA. These findings may be relevant to the remodeling of β-AR signaling in diseased (fibrotic) and/or aging atria, both of which exhibit decreases in AC activity.