Transgenic overexpression of beta(2)-adrenergic receptors in airway smooth muscle alters myocyte function and ablates bronchial hyperreactivity.

beta(2)-Adrenergic receptors (beta(2)AR) act to relax airway smooth muscle and can serve to counteract hyperresponsiveness, although the effect may not be ablative even in the presence of exogenous agonist. Within this signaling cascade that ultimately transduces smooth muscle relaxation, a significant "spare receptor" pool has been hypothesized to be present in the airway. In order to modify the relationship between beta(2)AR and downstream effectors, transgenic mice (TG) were created overexpressing beta(2)AR approximately 75-fold in airway smooth muscle using a mouse smooth muscle alpha-actin promoter. While >90% of these receptors were expressed on the smooth muscle cell surface, the percentage of receptors able to form the agonist-promoted high affinity complex was less than that found with nontransgenic (NTG) cells (R(H) = 18 versus 36%). Nevertheless, beta(2)AR signaling was found to be enhanced. Intact airway smooth muscle cells from TG had basal cAMP levels that were greater than NTG cells. A marked increase in agonist-stimulated cAMP levels was found in the TG ( approximately 200% stimulation over basal) compared with NTG ( approximately 50% over basal) cells. Adenylyl cyclase studies gave similar results and also showed a 10-fold lower EC(50) for TG cells. Tracheal rings from TG mice that were precontracted with acetylcholine had an enhanced responsiveness (relaxation) to beta-agonist, with a 60-fold decrease in the ED(50), indicating that the enhanced signaling imposed by overexpression results in an increase in the coordinated function of the intact airway cells. In vivo studies showed a significantly blunted airway resistance response to the inhaled bronchoconstrictor methacholine in the TG mice. Indeed, with beta-agonist pretreatment, the TG mice displayed no response whatsoever to methacholine. These results are consistent with beta(2)AR being the limiting factor in the transduction system. Increases in the initial component of this transduction system (the beta(2)AR) are sufficient to markedly alter signaling and airway smooth muscle function to the extent that bronchial hyperresponsiveness is ablated, consistent with an anti-asthma phenotype.     

IL-1beta enhances beta2-adrenergic receptor expression in human airway epithelial cells by activating PKC

Protein kinase C (PKC)-activated signal transduction pathways regulate cell growth and differentiation in many cell types. We have observed that interleukin (IL)-1beta upregulates beta2-adrenergic receptor (beta2-AR) density and beta2-AR mRNA in human airway epithelial cells (e.g., BEAS-2B). We therefore tested the hypothesis that PKC-activated pathways mediate IL-1beta-induced beta-AR upregulation. The role of PKC was assessed from the effects of 1) the PKC activator phorbol 12-myristate 13-acetate (PMA) on beta-AR density, 2) selective PKC inhibitors (calphostin C and Ro-31-8220) on beta-AR density, and 3) IL-1beta treatment on the cellular distribution of PKC isozymes. Recombinant human IL-1beta (0.2 nM for 18 h) increased beta-AR density to 213% of control values (P < 0.001). PMA (1 microM for 18 h) increased beta-AR density to 225% of control values (P < 0.005), whereas Ro-31-8220 and calphostin C inhibited the IL-1beta-induced upregulation of beta-AR in dose-dependent fashion. PKC isozymes detected by Western blotting included alpha, betaII, epsilon, mu, zeta, and lambda/iota. IL-1beta increased PKC-mu immunoreactivity in the membrane fraction and had no effect on the distribution of the other PKC isozymes identified. These data indicate that IL-1beta-induced beta-AR upregulation is mimicked by PKC activators and blocked by PKC inhibitors and appears to involve selective activation of the PKC-mu isozyme. We conclude that signal transduction pathways activated by PKC-mu upregulate beta2-AR expression in human airway epithelial cells.     

Alveolar epithelial type I cells express beta2-adrenergic receptors and G-protein receptor kinase 2.

Beta2-Adrenergic agonists stimulate alveolar epithelial sodium (Na(+)) transport and lung fluid clearance. Alveolar type II (AT2) cells have been reported to express beta2-adrenergic receptors (beta2AR). Given the large surface area covered by alveolar type I (AT1) cells and their potential role in alveolar fluid removal, we were interested in learning if AT1 cells express beta2AR as well. Because beta2AR is potentially susceptible to desensitization by G-protein-coupled receptor kinase 2 (GRK2), we also undertook localization of GRK2. beta2AR and GRK2 expression was evaluated in whole lung, isolated alveolar epithelial cells (AECs), and AECs in primary culture, and was localized to specific AEC phenotypes by immunofluorescence techniques. beta2AR is highly expressed in AT1 cells. beta2AR mRNA increases with time in culture as AT2 cells transdifferentiate towards the AT1 cell phenotype. Immunoreactive GRK2 is seen in both AT1 and AT2 cells in similar amounts. These data suggest that both AT1 and AT2 cells may contribute to the increased alveolar Na(+) and water clearance observed after exposure to beta2 adrenergic agents. Both cell types also express GRK2, suggesting that both may undergo desensitization of beta2AR with subsequent decline in the stimulatory effects of beta2-adrenergic agonists over time.     

Preserved ventricular contractility in infarcted mouse heart overexpressing beta(2)-adrenergic receptors

Effects of cardiac specific overexpression of beta(2)-adrenergic receptors (beta(2)-AR) on the development of heart failure (HF) were studied in wild-type (WT) and transgenic (TG) mice following myocardial infarction (MI) by coronary artery occlusion. Animals were studied by echocardiography at weeks 7 to 8 and by catheterization at week 9 after surgery. Post-infarct mortality, due to HF or cardiac rupture, was not different among WT mice, and there was no difference in infarct size (IS). Compared with the sham-operated group (all P < 0.01), WT mice with moderate (<36%) and large (>36%) IS developed lung congestion, cardiac hypertrophy, left ventricular (LV) dilatation, elevated LV end-diastolic pressure (LVEDP), and suppressed maximal rate of increase of LV pressure (LV dP/dt(max)) and fractional shortening (FS). Whereas changes in organ weights and echo parameters were similar to those in infarcted WT groups, TG mice had significantly higher levels of LV contractility in both moderate (dP/dt(max) 4,862 +/- 133 vs. 3,694 +/- 191 mmHg/s) and large IS groups (dP/dt(max) 4,556 +/- 252 vs. 3,145 +/- 312 mmHg/s, both P < 0.01). Incidence of pleural effusion (36% vs. 85%, P < 0.05) and LVEDP levels (6 +/- 0.3 vs. 9 +/- 0.8 mmHg, P < 0.05) were also lower in TG than in WT mice with large IS. Thus beta(2)-AR overexpression preserved LV contractility following MI without adverse consequence.     

Phosphorylation of beta-arrestin2 regulates its function in internalization of beta(2)-adrenergic receptors

Beta-arrestins mediate agonist-dependent desensitization and internalization of G protein-coupled receptors. Previously, we have shown that phosphorylation of beta-arrestin1 by ERKs at Ser-412 regulates its association with clathrin and its function in promoting clathrin-mediated internalization of the receptor. In this paper we report that beta-arrestin2 is also phosphorylated, predominantly at residues Thr-383 and Ser-361. Isoproterenol stimulation of the beta(2)-adrenergic receptor promotes dephosphorylation of beta-arrestin2. Mutation of beta-arrestin2 phosphorylation sites to aspartic acid decreases the association of beta-arrestin2 with clathrin, thereby reducing its ability to promote internalization of the beta(2)-adrenergic receptor. Its ability to bind and desensitize the beta(2)-adrenergic receptor is, however, unaltered. These results suggest that, analogous to beta-arrestin1, phosphorylation/dephosphorylation of beta-arrestin2 regulates clathrin-mediated internalization of the beta(2)-adrenergic receptor. In contrast to beta-arrestin1, which is phosphorylated by ERK1 and ERK2, phosphorylation of beta-arrestin2 at Thr-383 is shown to be mediated by casein kinase II. Recently, it has been reported that phosphorylation of visual arrestin at Ser-366 prevents its binding to clathrin. Thus it appears that the function of all arrestin family members in mediating internalization of G protein-coupled receptors is regulated by distinct phosphorylation/dephosphorylation mechanisms.     

Targeted transgenic expression of beta(2)-adrenergic receptors to type II cells increases alveolar fluid clearance

Clearance of edema fluid from the alveolar space can be enhanced by endogenous and exogenous beta-agonists. To selectively delineate the effects of alveolar type II (ATII) cell beta(2)-adrenergic receptors (beta(2)-ARs) on alveolar fluid clearance (AFC), we generated transgenic (TG) mice that overexpressed the human beta(2)-AR under control of the rat surfactant protein C promoter. In situ hybridization showed that transgene expression was consistent with the distribution of ATII cells. TG mice expressed 4.8-fold greater beta(2)-ARs than nontransgenic (NTG) mice (939 +/- 113 vs. 194 +/- 18 fmol/mg protein; P < 0.001). Basal AFC in TG mice was approximately 40% greater than that in untreated NTG mice (15 +/- 1.4 vs. 10.9 +/- 0.6%; P < 0.005) and approached that of NTG mice treated with the beta-agonist formoterol (19.8 +/- 2.2%; P = not significant). Adrenalectomy decreased basal AFC in TG mice to 9.7 +/- 0.5% but had no effect on NTG mice (11.5 +/- 1.0%). Na(+)-K(+)-ATPase alpha(1)-isoform expression was unchanged, whereas alpha(2)-isoform expression was approximately 80% greater in the TG mice. These findings show that beta(2)-AR overexpression can be an effective means to increase AFC in the absence of exogenous agonists and that AFC can be stimulated by activation of beta(2)-ARs specifically expressed on ATII cells.     

Transgenic overexpression of cardiac A(1) adenosine receptors mimics ischemic preconditioning

The role of A(1) adenosine receptors (A(1)AR) in ischemic preconditioning was investigated in isolated crystalloid-perfused wild-type and transgenic mouse hearts with increased A(1)AR. The effect of preconditioning on postischemic myocardial function, lactate dehydrogenase (LDH) release, and infarct size was examined. Functional recovery was greater in transgenic versus wild-type hearts (44.8 +/- 3.4% baseline vs. 25.6 +/- 1.7%). Preconditioning improved functional recovery in wild-type hearts from 25.6 +/- 1.7% to 37.4 +/- 2.2% but did not change recovery in transgenic hearts (44.8 +/- 3.4% vs. 44.5 +/- 3.9%). In isovolumically contracting hearts, pretreatment with selective A(1) receptor antagonist 1, 3-dipropyl-8-cyclopentylxanthine attenuated the improved functional recovery in both wild-type preconditioned (74.2 +/- 7.3% baseline rate of pressure development over time untreated vs. 29.7 +/- 7.3% treated) and transgenic hearts (84.1 +/- 12.8% untreated vs. 42.1 +/- 6.8% treated). Preconditioning wild-type hearts reduced LDH release (from 7,012 +/- 1,451 to 1,691 +/- 1,256 U. l(-1). g(-1). min(-1)) and infarct size (from 62.6 +/- 5.1% to 32.3 +/- 11.5%). Preconditioning did not affect LDH release or infarct size in hearts overexpressing A(1)AR. Compared with wild-type hearts, A(1)AR overexpression markedly reduced LDH release (from 7,012 +/- 1,451 to 917 +/- 1,123 U. l(-1). g(-1). min(-1)) and infarct size (from 62.6 +/- 5.1% to 6.5 +/- 2.1%). These data demonstrate that murine preconditioning involves endogenous activation of A(1)AR. The beneficial effects of preconditioning and A(1)AR overexpression are not additive. Taken with the observation that A(1)AR blockade equally eliminates the functional protection resulting from both preconditioning and transgenic A(1)AR overexpression, we conclude that the two interventions affect cardioprotection via common mechanisms or pathways.     

Heterodimerization of alpha 2A- and beta 1-adrenergic receptors

beta- and alpha(2)-adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the beta(1)-adrenergic receptor (beta(1)AR) and alpha(2A)-adrenergic receptor (alpha(2A)AR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged beta(1)AR and alpha(2A)AR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of alpha(2A)AR was found to induce substantial internalization of coexpressed beta(1)AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [(3)H]dihydroalprenolol binding to the beta(1)AR by various ligands revealed that beta(1)AR pharmacological properties were significantly altered when the receptor was coexpressed with alpha(2A)AR. Finally, beta(1)AR/alpha(2A)AR heterodimerization was found to be markedly enhanced by a beta(1)AR point mutation (N15A) that blocks N-linked glycosylation of the beta(1)AR as well as by point mutations (N10A/N14A) that block N-linked glycosylation of the alpha(2A)AR. These data reveal an interaction between beta(1)AR and alpha(2A)AR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors.     

ATP significantly increases P2Y2-dependent RANTES secretion and overexpression in human airway epithelial cells

Uncontrolled inflammation is frequently observed in human respiratory diseases. Extracellular ATP can induce a number of physiological phenomena via binding to purinergic receptors. In spite of the fact that ATP has long been known as a proinflammatory mediator in the airway, the signaling pathway mechanism is still unclear. Here we show that ATP increases RANTES secretion and overexpression in a time-dependent manner and siRNA-P2Y₂ significantly decreases RANTES secretion and overexpression. These results suggest that ATP can induce secretion and overexpression of the RANTES chemokine via a P2Y₂ Gαq coupled receptor-dependent manner. In addition, pharmacological inhibition of ERK1/2 MAPK by U0126 suppressed ATP/P2Y₂-induced RANTES overexpression in the human airway epithelium. These results show that RANTES secretion and overexpression are regulated by a P2Y₂ receptor and the ATP/P2Y₂ signaling complex may be critical for airway inflammation in respiratory diseases. Taken together, our investigation provides novel insight into the physiological functions of the P2Y₂ receptor and enhances our understanding of the inflamed microenvironment in the airway.     

Downregulation by a long-acting beta2-adrenergic receptor agonist and corticosteroid of Staphylococcus aureus-induced airway epithelial inflammatory mediator production

Although Staphylococcus aureus is a major cause of pulmonary infection, the role played by this bacterium in the induction of inflammation of human airway epithelial cells (HAEC) is poorly understood. In this study, we investigated the inflammatory response of HAEC to S. aureus soluble virulence factors and demonstrate that the combination of a long-acting beta2-adrenergic receptor agonist (salmeterol) with a glucocorticoid (fluticasone propionate) has an anti-inflammatory effect on HAEC. First, we demonstrate increased expression at both the mRNA and protein levels of interleukin (IL)-8, IL-6, and tumor necrosis factor (TNF)-alpha following incubation of HAEC in the presence of S. aureus soluble virulence factors and the increase of 1) the free nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) activities and 2) the phosphorylated (P-) extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), the P-c-Jun NH2-terminal kinase (JNK), and the P-isoform-alpha of the NF-kappaB inhibitor (IkappaB alpha). Next, when HAEC were preincubated with the combination of salmeterol and fluticasone propionate, the inflammatory response of HAEC was markedly attenuated in that levels of IL-8, IL-6, TNF-alpha, NF-kappaB, AP-1, P-ERK1/ERK2, P-JNK, and P-IkappaB alpha decreased significantly. These data emphasize the deleterious effect of S. aureus soluble virulence factors and suggest that the combination of a beta2-adrenergic receptor agonist with a glucocorticoid may attenuate the associated airway epithelial inflammation.     

Mediation of isoproterenol-induced thirst in rats by beta2-adrenergic receptors.

Isoproterenol-induced thirst in rats has been attributed to the activation of beta-adrenergic receptors. Since these receptors can be further differentiated pharmacologically into beta1 and beta2 types, experiments were performed using several beta-adrenergic agonists and antagonists to determine the receptor type initiating the isoproterenol-induced thirst. The beta1- and beta2-adrenergic antagonist, d,l-propranolol (1 mg/kg, ip), blocked the increase in water intake usually accompanying acute subcutaneous administration of isoproterenol (25 microgram/kg) to female rats. Since l-propranolol is known to stabilize membranes and to possess anesthetic-like properties, d-propranolol was also used. This isomer has little beta-adrenergic-blocking activity but possesses anesthetic-like activity. Administration of d-propranolol (1 mg/kg, ip) failed to affect the drinking response to acute administration of isoproterenol (25 microgram/kg). Practolol (125 mg/kg), a beta1-adrenergic antagonist with little anesthetic properties, also had no effect on water intake of isoproterenol-treated rats. Butoxamine, a selective beta2-adrenergic antagonist, attenuated the drinking response to isoproterenol. Salbutamol (150 microgram/kg), a beta2-adrenergic agonist, mimicked the effect of isoproterenol on water intake. These results are consistent with the suggestion that beta2-adrenergic receptors mediate the isoproterenol-induced thirst in rats.     

Cross-regulation between G-protein-coupled receptors. Activation of beta 2-adrenergic receptors increases alpha 1-adrenergic receptor mRNA levels.

Stimulation of DDT1 MF-2 vas deferens cells with epinephrine resulted in a time- and dose-dependent loss of alpha 1-adrenergic receptor-specific ligand binding. Regulation of alpha 1-adrenergic receptor mRNA was characterized. In monolayer culture, cells displayed 0.7 +/- 0.05 amol of alpha 1-adrenergic receptor mRNA/microgram of total cellular RNA. Epinephrine, which acts at both alpha 1- and beta 2-adrenergic receptors of DDT1 MF-2 cells, induced a short term (2-8 h) increase (50-70%) in the abundance of alpha 1-adrenergic receptor mRNA. Propranolol, a beta 2-adrenergic receptor antagonist, attenuated the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA but did not affect the decrease in alpha 1-adrenergic receptor-specific ligand binding. Phentolamine, an alpha 1-adrenergic receptor antagonist, did not attenuate the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA at 4 h but did block the decrease in alpha 1-adrenergic receptor-specific ligand binding. The half-life of the alpha 1-adrenergic receptor mRNA was approximately 7 h in untreated cells as well as in cells challenged with epinephrine. The epinephrine-promoted increase in alpha 1-adrenergic receptor mRNA was found to result from cross-regulation via beta 2-adrenergic receptors. Cholera toxin, forskolin, as well as the cyclic AMP analog CPT cAMP (8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate) increased the alpha 1-adrenergic receptor mRNA at 4 h, as did epinephrine in the presence of alpha 1-antagonists but not in the presence of a beta-adrenergic antagonist. This is the first report of heterologous up-regulation of mRNA levels of adrenergic receptors. Cross-regulation between alpha 1- and beta 2-adrenergic receptor-mediated pathways at 4 h occurs at the level of mRNA whereas later down-regulation of alpha 1-receptor mRNA and binding proceed via agonist activation of alpha 1-adrenergic receptors.     

Glycosylation of beta(1)-adrenergic receptors regulates receptor surface expression and dimerization

The beta(1)-adrenergic receptor (beta(1)AR) has one predicted site of N-linked glycosylation on its extracellular amino-terminus, but the glycosylation and potential functional importance of this site have not yet been examined. We show here that the beta(1)AR is glycosylated in various cell types and that mutation of the single predicted site of N-linked glycosylation (N15A) results in the formation of receptors that are not N-glycosylated. The beta(1)AR N15A mutant exhibited significantly decreased basal surface expression relative to the wild-type receptor but had no detectable deficits in ligand binding or agonist-promoted internalization. Co-immunoprecipitation experiments using Flag-tagged and HA-tagged receptors demonstrated that the beta(1)AR-N15A mutant receptor exhibits a markedly reduced capacity for dimerization relative to wild-type beta(1)AR. These data reveal that the beta(1)AR is glycosylated on Asn15 and that this glycosylation plays a role in regulating beta(1)AR surface expression and dimerization.     

Role of beta 2-adrenergic receptors on coronary resistance during exercise

The effects of regional alpha- and specific beta 2-adrenergic receptor blockade on measurements of late diastolic coronary resistance (LDCR) and mean coronary blood flow velocity (CBFV) during exercise were examined in 14 conscious adult mongrel dogs. Specific beta 2-adrenergic receptor blockade (ICI 118.551) significantly decreased CBFV and increased LDCR by blockade of beta 2-vasodilator tone independent of alpha-adrenergic receptor-mediated tone and independent of altering myocardial metabolism. alpha-Adrenergic receptor blockade (phentolamine, 1 mg) significantly increased CBFV and decreased LDCR by blocking sympathetically mediated vasoconstrictor tone. There was no significant difference in the magnitude of response between alpha- and beta 2-adrenergic receptor blockade. These results demonstrate that alpha- and beta 2-adrenergic receptors have a significant and evidently equal influence on CBFV and LDCR during exercise. Four weeks of daily exercise and left stellate ganglionectomy (LSGx) prevented phentolamine-induced vasodilation but not ICI 118.551-induced vasoconstriction. This suggests that daily exercise and LSGx significantly decreased the alpha-adrenergic receptor-mediated vasoconstrictor tone on the coronary circulation, resulting in an apparently greater role for the coronary vascular beta 2-adrenergic receptor on the control of CBFV and LDCR during exercise.     

Phenotypic effects of overexpression of PKC beta 1 in rat liver epithelial cells

We have used a previously described retroviral expression vector pMV7-PKC beta 1 to develop derivatives of two rat liver epithelial cell lines, K16 and K22, that stably express about tenfold-higher PKC activity than control cells. Despite these high levels of PKC, these cells did not exhibit gross morphologic changes, anchorage-independent growth, or tumorigenicity. K16PKC-4 and K22PKC-2, two lines with the highest PKC enzyme activity, were studied further in terms of several responses to the phorbol ester tumor promoter TPA. When treated with 100 ng/ml of TPA, the control K16MV7 and K22MV7 cells displayed a slight change in morphology, whereas the K16PKC-4 and K22PKC-2 cells displayed a marked change in morphology. Northern blot analyses demonstrated that TPA induced increased levels of fos, myc, phorbin, and ODC RNAs in control K16MV7 and K22MV7 cells, with maximum induction occurring at about 0.5, 1, 8, and 8 h, respectively. In K16PKC-4 and K22PKC-2 cells, TPA induction of phorbin and ODC RNAs was markedly enhanced, but this was not the case for myc and fos RNAs. In addition, the levels of myc RNA were constitutively higher in both K16PKC-4 and K22PKC-2 cells than in the control cells. Taken together, these results provide direct evidence that PKC plays a critical role in modulating the expression of myc, phorbin, and ODC RNAs. On the other hand, overexpression of PKC beta 1 is not itself sufficient to cause cell transformation.     

Mammalian beta 1- and beta 2-adrenergic receptors. Immunological and structural comparisons

Beta 1- and beta 2-adrenergic receptors, pharmacologically distinct proteins, have been reported to be structurally dissimilar. In the present study three techniques were employed to compare the nature of mammalian beta 1- and beta 2-adrenergic receptors. Antibodies against each of the receptor subtypes were raised separately. Polyclonal antisera against beta 1-receptors of rat fat cells were raised in mice, and antisera against beta 2-receptors of guinea pig lung were raised in rabbits. Receptors purified from rat fat cells (beta 1-), S49 mouse lymphoma cells (beta 2-), and rat liver (beta 2-) were probed with these antisera. Each anti-receptor antisera demonstrated the ability to immunoprecipitate purified receptors of both beta 1- and beta 2- subtypes. The mobility of beta-receptors subjected to polyacrylamide gel electrophoresis was probed using antireceptor antibodies and nitrocellulose blots of the gels. Fat cell beta 1-adrenergic receptors display Mr = 67,000 under reducing conditions and Mr = 54,000 under nonreducing conditions, as previously reported (Moxham, C. P., and Malbon, C. C. (1985) Biochemistry 24, 6072-6077). Both beta 1- and beta 2-receptors displayed this same shift in electrophoretic mobility observed in the presence as compared to the absence of disulfide bridge-reducing agents, as detected both by autoradiography of the radiolabeled receptors and by immunoblotting of native receptors. Finally, isoelectric focusing of purified radioiodinated beta 1- and beta 2-adrenergic receptors revealed identical isoelectric points. These data are the first to provide analyses of immunological, structural, and biochemical features of beta 1- and beta 2-subtypes in tandem and underscore the structural similarities that exist between these pharmacologically distinct receptors.