Distribution of myocardial beta-adrenoceptor subtypes and coupling to the adenylate cyclase in children with congenital heart disease and implications for treatment

In congestive heart failure, down-regulation of myocardial beta-adrenoceptors (beta-AR) due to an elevated sympathetic tone is well known. In infancy and childhood, heart failure is usually related to congenital heart disease (CHD). Therefore, 71 samples of right atrial tissue of infants and children with CHD undergoing cardiac surgery were studied for beta-adrenoceptor density and distribution of the beta 1-/beta 2-AR subtypes. In 49 cases, the coupling of the beta-AR to the adenylate cyclase (AC) was examined. In a further study of 19 myocardial samples, AC was selectively stimulated with beta 1- or beta 2-AR whereas the other subtype was blocked by an antagonist. The following results were obtained: (1) Infants and children with severe acyanotic or cyanotic CHD had severely reduced beta-AR densities. (2) In most of the cases, the beta-AR down regulation is beta 1-subtype selective, but in critically ill newborns with congenital aortic valve stenosis or transposition of the great arteries, there is additional significant beta 2-AR down-regulation. In Fallot patients treated with the beta-antagonist propranolol, a significant increased beta-AR number compared with untreated Fallot patients was found. (3) beta-Adrenoceptor reduction in CHD is correlated with elevated noradrenaline plasma levels, thus proving a sympathetic dysregulation. (4) In CHD with moderate hemodynamic load, beta 2-AR coupling to AC was markedly more efficient than beta 1-AR coupling. The small number of myocardial beta 2-AR produced most of the cyclic adenosine monophosphate. (5) In severe acyanotic and cyanotic CHD, a partial decoupling of the beta 2-AR to the AC occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of beta-adrenergic receptors of freshly isolated astrocytes and neurons from rat brain

The binding and characteristics of rat brain beta-adrenergic receptors (beta-AR) isolated from astrocytes and neurons were investigated. Equilibrium binding experiments demonstrated that beta-AR were more concentrated on astrocytes than on neurons isolated from forebrain, cerebral cortex and cerebellum. Inhibition experiments revealed that beta 1-AR and beta 2-AR were present in the two cell types. Isoproterenol revealed two interchangeable states of high and low affinity binding to both beta 1- and beta 2-AR in neurons. The high affinity binding sites were sensitive to guanylylimidodiphosphate (GppNHp). Similar results were found with other beta-AR agonists but not with salbutamol and salmeterol which recognized both affinity states of the neuronal beta 2-AR but only the low affinity state of beta 1-AR. In astrocytes only the low affinity state of beta-AR was observed.     

Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects

At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.     

Agonist interactions with chimeric and mutant beta1- and beta3-adrenergic receptors: involvement of the seventh transmembrane region in conferring subtype specificity

beta1- and beta3-adrenergic receptors (AR) are the predominant beta-AR subtypes in adipocytes, and analysis of native and recombinant beta-AR has revealed several pharmacological and biochemical differences between these subtypes. This study used chimeric and mutated rat beta-AR expressed in Chinese hamster ovary cells to examine the basis of certain characteristic differences in the agonist properties of catecholamines and prototypic beta3-AR agonists. The exchange of sequence beyond transmembrane (TM) region 6 between the beta-AR subtypes had dramatic and reciprocal effects on the affinity and efficacy of the prototypic beta3-AR agonists BRL 37,344 and CL 316,243, without affecting the interactions with catecholamines. Mutation of Phe350 and Phe351 in TM7 of the beta1-AR to Ala and Leu found in the beta3-AR was sufficient to allow activation by prototypic beta3-AR agonists. Interestingly, this mutation did not affect catecholamine action and it did not impair the ability of propranolol to block the actions of isoproterenol or the selective beta3-AR agonists. beta1-AR containing beta3-AR sequence from predicted TM5 through TM6 exhibited reduced affinity for catecholamines without altering agonist potency, suggesting enhanced coupling efficiency. Inclusion of the homologous beta1-AR sequence in the beta3-AR, however, did not produce reciprocal effects. These results are the first to define a major determinant of beta3-AR subtype-selective agonism in TM7 and demonstrate that the determinants of selective phenethanolamines, catecholamines, and propranolol action are distinct.     

Effects of a Relative-Frequency Elicitation Question on Likelihood Judgment Accuracy: The Case of External Correspondence

The sinoatrial (SA) node is the cardiac pacemaker and changes in its adrenergic-muscarinic phenotype have been postulated as a determinant of age-associated modifications in heart rate variability. To address this question, right atria were microdissected, the SA node area was identified by acetylcholinesterase staining, and, using a RT-PCR method, the accumulation of mRNA molecules encoding beta1- and beta2-adrenergic (beta1- and beta2-AR) and muscarinic (M2-R) receptor was quantified to define the proportion between beta-AR and M2-R mRNAs within the sinoatrial area of adult (3 months) and senescent (24 months) individual rat hearts. In adult hearts, the highest M2-R/beta-AR mRNA ratio was observed within the sinoatrial area compared with adjacent atrial myocardium, while in the senescent hearts, no difference was observed between sinoatrial and adjacent areas. This change was specific of the sinoatrial area since adult and senescent whole atrial or ventricular myocardium did not differ in their M2-R/beta-AR mRNA ratio, and was associated with a fragmentation of acetylcholinesterase staining of the senescent SA node. Quantitative changes in the expression of genes encoding proteins involved in heart rate regulation specifically affect the sinoatrial area of the senescent heart.     

Cardiac beta-adrenoceptors and adenylyl cyclase activity in human left ventricular hypertrophy due to pressure overload

The effect of left ventricular hypertrophy (LVH) due to chronic pressure overload on right atrial (RA) and left ventricular (LV) myocardial beta-adrenergic receptor (beta-AR) density and subtypes, adenylyl cyclase (AC) activity and ADP-pertussis toxin ribosylated proteins was investigated in humans with LVH due to aortic stenosis and in patients without LVH undergoing heart surgery for mitral stenosis or coronary artery disease taken as controls. Both groups presented normal systolic function or plasma catecholamine levels. In LVH and controls, beta-AR density was similar in RA (62 +/- 6 vs 77 +/- 12 fmol.mg-1 protein) and LV (39 +/- 7 vs 32 +/- 2 fmol.mg-1 protein). In LVH, beta 1-AR percentage was < than in controls in LV (35 +/- 11 vs 73 +/- 5%, P < 0.05) but not in RA (79 +/- 5 vs 73 +/- 8%). Basal AC activity in RA (19 +/- 4 vs 21 +/- 6 pmol.mg-1 protein) and LV (22 +/- 5 vs 27 +/- 3 pmol.mg-1 protein) was similar in LVH and in controls. Isoprenaline-induced stimulation of AC in RA was similar in LVH and in controls (51 +/- 18 vs 36 +/- 18%) but < in LV of LVH (7 +/- 6 vs 45 +/- 6%, P < 0.05). In the presence of ICI-118,551 (a beta 2-adrenoceptor antagonist), isoprenaline failed to induce any increase in cAMP in LVH. The quantification of ADP-pertussis toxin ribosylated proteins indicated a lower concentration of substrates in LV myocardial membranes from LVH. These data indicate that in LVH due to pressure overload, there is a down-regulation of beta 1-AR and an increase in beta 2-AR density. This is associated with alterations of the transmembrane signalling marked by a decreased capacity of isoprenaline to stimulate AC and an impaired expression of Gi proteins.     

Down-regulation of beta-adrenergic receptor following long-term monocular deprivation in cat visual cortex

To examine how adrenergic receptor binding is modified by experimental manipulation of sensory afferent, we carried out binding experiments (membrane fraction and in vitro autoradiography) for both alpha 2- and beta-adrenergic receptors in the brain of cats which had been deprived of vision in one eye. In the cerebral cortex of control animals, beta-adrenergic receptor (beta-AR) binding was found to be higher in the occipital regions than in other regions, while alpha 2-AR binding was relatively uniform. Monocular deprivation throughout the postnatal sensitive period (1-7 month of age) significantly decreased beta-AR binding in the visual cortex and lateral geniculate nucleus. Scatchard plot analysis in the visual cortex showed ca. 50% reduction in Bmax and little change in Kd. No significant difference was found in alpha 2-AR binding following monocular deprivation. Similar extent of down-regulation in beta-AR binding was confirmed in all layers of visual cortex using autoradiography.     

Physiological consequences of beta-adrenergic receptor disruption

Activation of beta-adrenergic receptors (beta-ARs) in vivo is an important means by which animals regulate cardiac performance, vascular tone, lipid and carbohydrate metabolism, and behavior. The advent of targeted gene disruption in mice has led to significant advances in our understanding of the role that beta-AR subtypes play in these processes, and this technique has become an important tool for the study of G protein coupled receptors in general. To date, targeted disruption of both beta1- and beta3-ARs in mice has been reported. Mice lacking beta1-ARs are unresponsive to cardiac beta-AR stimulation, suggesting that neither beta2- nor beta3-ARs couple to inotropic or chronotropic responses in the mouse. Conversely, mice lacking beta3-ARs retain at least some adipose beta-AR responsiveness through remaining beta1- and beta2-ARs, suggesting that all three beta-AR subtypes mediate similar functions in this tissue. While these knockout models have been extremely valuable tools for revealing the roles that individual beta-ARs play in whole animal physiology, it is also useful to integrate the results of experiments derived from either transgenic overexpression of beta-ARs or purely pharmacological approaches to the study of beta-AR function in order to create a comprehensive model of beta-AR function in vivo.     

Reduced beta 1 receptor messenger RNA abundance in the failing human heart

Heart failure in humans is characterized by alterations in myocardial adrenergic signal transduction, the most prominent of which is down-regulation of beta 1-adrenergic receptors. We tested the hypothesis that down-regulation of beta 1-adrenergic receptors in the failing human heart is related to decreased steady-state levels of beta 1 receptor mRNA. Due to the extremely low abundance of beta 1 receptor mRNA, measurements were possible only by quantitative polymerase chain reaction (QPCR) or by RNase protection methods. Because the beta 1 receptor gene is intronless and beta 1 receptor mRNA abundance is low, QPCR yielded genomic amplification in total RNA, and mRNA measurements had to be performed in poly (A)(+)-enriched RNA. By QPCR the concentration of beta 1 receptor mRNA varied from 0.34 to 7.8 x 10(7) molecules/microgram poly(A)(+)-enriched RNA, and the assay was sensitive to 16.7 zeptomol. Using 100-mg aliquots of left ventricular myocardium obtained from organ donors (nonfailing ventricles, n = 12) or heart transplant recipients (failing ventricles, n = 13), the respective beta 1 mRNA levels measured by QPCR were 4.2 +/- 0.7 x 10(7)/micrograms vs. 2.10 +/- 0.3 x 10(7)/micrograms (P = 0.006). In these same nonfailing and failing left ventricles the respective beta 1-adrenergic receptor densities were 67.9 +/- 6.9 fmol/mg vs. 29.6 +/- 3.5 fmol/mg (P = 0.0001). Decreased mRNA abundance in the failing ventricles was confirmed by RNase protection assays in total RNA, which also demonstrated a 50% reduction in beta 1 message abundance. We conclude that down-regulation of beta 1 receptor mRNA contributes to down-regulation of beta 1 adrenergic receptors in the failing human heart.     

Mice expressing human but not murine beta3-adrenergic receptors under the control of human gene regulatory elements

Beta-adrenergic receptors (ARs) are expressed predominantly in adipose tissue, and beta3-selective agonists are effective anti-obesity drugs in rodents. Rodent and human beta3-ARs differ with respect to expression in white versus brown adipocytes as well as their ability to be stimulated by beta3-AR-selective agonists. Humans express beta3-AR mRNA abundantly in brown but not white adipocytes, while rodents express beta3-AR mRNA abundantly in both sites. To determine the basis for this difference, we have transgenically introduced 74 kilobases (kb) of human beta3-AR genomic sequence into gene knockout mice lacking beta3-ARs. Importantly, human beta3-AR mRNA was expressed only in brown adipose tissue (BAT) of transgenic mice, with little or no expression being detected in white adipose tissue (WAT), liver, stomach, small intestine, skeletal muscle, and heart. This pattern of expression differed from that observed in mice bearing a murine beta3-AR genomic transgene in which beta3-AR mRNA was expressed in both WAT and BAT, but not in other sites. Furthermore, we have transgenically introduced smaller human constructs containing -14.5 and -0.6 kb of upstream sequence into beta3-AR gene knockout mice. Both -14.5 and -0.6 kb constructs were expressed in BAT but not WAT. Thus, human but not murine cis-regulatory elements direct beta3-AR gene expression preferentially to brown adipocytes. Identification of responsible cis-regulatory element(s) and relevant trans-acting factor(s) should provide insight into mechanisms controlling human beta3-AR gene expression. In addition, the beta3-AR agonist, CGP-12177, stimulated oxygen consumption in mice expressing human but not murine beta3-ARs by 91% compared with only 49% in control beta3-AR gene knockout mice, demonstrating that the human beta3-AR can functionally couple with energy expenditure. These "humanized" mice should assist us in the development of drugs that may become effective anti-obesity agents in humans.     

Phorbol ester and calcium regulation of corticotrophin-releasing factor receptor 1 expression in a neuronal cell line

We have previously demonstrated that corticotrophin-releasing factor receptor 1 (CRF-R1) mRNA levels can be down-regulated via activation of the cyclic AMP pathway in CATH.a cells, a neuronal cell line. In this study, we show evidence for down-regulation of CRF-R1 mRNA levels via activation of the protein kinase C (PKC) and calcium second messenger pathways. Incubation of CATH.a cells with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, resulted in a time- and concentration-dependent down-regulation of CRF-R1 mRNA levels. Pretreatment with the inactive phorbol ester 4alpha-phorbol failed to influence significantly CRF-R1 mRNA levels. Incubation with carbachol, a cholinergic agonist known to activate PKC and increase intracellular calcium levels via phosphatidylinositol breakdown, also down-regulated CRF-R1 mRNA levels. Intracellular calcium levels were directly increased using A23187, a calcium ionophore, and thapsigargin, a calcium-ATPase inhibitor. Elevation of intracellular calcium content using either A23187 or thapsigargin significantly down-regulated levels of CRF-R1 mRNA. Furthermore, chelation of calcium with EGTA or blockade of voltage-dependent calcium channels with nifedipine inhibited agonist-mediated down-regulation of CRF-R1 mRNA levels. These results indicate that activation of PKC or calcium signal transduction pathways is sufficient to cause down-regulation of CRF-R1 mRNA levels and that calcium is required for agonist-mediated down-regulation of this receptor.     

Tocolytic therapy with fenoterol induces selective down-regulation of beta-adrenergic receptors in human myometrium

Tocolytic therapy with beta-adrenergic receptor agonists is a standard regimen to prevent preterm birth. Agonists exposure of beta-adrenergic receptors causes receptor desensitization in other organs, and this may limit the therapeutic value of beta-adrenergic receptor agonists. To study the effects of prolonged beta-adrenergic agonist treatment in human myometrium, we obtained biopsies during Caesarean section of 14 pregnant patients who had received fenoterol for at least 5 days and 14 untreated pregnant controls. The densities of total beta-adrenergic receptors, which are mainly of the beta 2-subtype as assessed by [125I]iodo-cyanopindolol binding in crude membrane fractions, were more than 50% smaller in women receiving fenoterol, whereas alpha 2-adrenergic receptor densities were similar. Gs and Gi G-protein alpha-subunit densities were unaltered as assessed by Western blotting and pertussis toxin-catalyzed [32P]ADP-ribosylation. beta-Adrenergic receptor kinase (beta ARK) activity, as determined using bovine rhodopsin as the substrate, was the same in the two groups. Adenylyl cyclase activities in the presence of guanine nucleotides, NaF, forskolin, or Mn+2 were also not altered by fenoterol treatment. The messenger RNA (mRNA) concentrations of beta 2-adrenergic receptors, beta ARK-I and glyceraldehyde-3-phosphate dehydrogenase (as a reference), as determined by quantitative PCR, were unaffected by fenoterol treatment. We conclude that tocolysis with fenoterol results in a selective down-regulation of myometrial beta-adrenergic receptors, which is not associated with a reduction in the respective mRNA concentrations or alterations of alpha 2-adrenergic receptors, Gs and Gi alpha-subunits, or beta ARK activity or mRNA.     

No effect of Trp64Arg beta3-adrenoceptor polymorphism on the plasma leptin concentration in Pima Indians

In rodents, administration of leptin promotes beta3-adrenergic stimulation of thermogenesis in brown adipose tissue. Conversely, administration of a beta3-adrenoceptor (beta3-AR) agonist decreases leptin mRNA expression and secretion, suggesting that leptin and sympathetic nervous system activity mediated through the beta3-AR comprise a negative-feedback loop. It has recently been proposed that a defect in the beta3-AR in humans may contribute to a resistance to the sympathetically mediated effects of leptin on thermogenesis and lipolysis, thus leading to obesity and type 2 diabetes mellitus. We thus hypothesized that the Trp64Arg variant in the human beta3-AR would be associated with elevated plasma leptin concentrations. We studied 101 healthy nondiabetic Pima Indians: 11 Arg64 homozygotes, 35 Trp64 homozygotes, and 55 heterozygotes. The fasting plasma leptin concentration as an absolute value or after adjustment for percent body fat and sex was not associated with the beta3-AR genotype. Thus, the data do not support an influence of the Trp64Arg variant on the plasma leptin concentration.     

Phenotype of T cell clones obtained from bronchial biopsies and peripheral blood from three asthmatics

Muscarinic receptor kinase activity previously described in intact CHO cells transfected with human m3-muscarinic receptor cDNA (CHO-m3 cells) [Tobin, A.B and Nahorski, S.R (1993) J. Biol. Chem. 268, 9817-9823] was found to be associated, at least in part, with a crude membrane fraction of CHO-m3 cell lysates. Phosphorylation of the m3-muscarinic receptor was agonist dependent, reaching a maximum after 10 min exposure to carbachol (1 mM) and was completely blocked by atropine (10 microM). m3-Muscarinic receptor phosphorylation was insensitive to Zn2+ (0.1 mM) and heparin (1 microgram/ml), concentrations that inhibit endogenous beta-adrenergic receptor kinase activity present in CHO-m3 cells strongly suggesting that the m3-muscarinic receptor kinase is distinct from beta-adrenergic receptor kinase. A role for protein kinase C can also be eliminated on the basis that the potent protein kinase C inhibitor, Ro-318220 (1 microM), had no effect on agonist-mediated m3-muscarinic receptor phosphorylation. Further, the inability of calcium (300 microM), cAMP (0.2 mM) and cGMP (0.2 mM) to elevate the basal phosphorylation state of m3-muscarinic receptors eliminates a role for protein kinases regulated by these second messengers. Finally, agonist mediated phosphorylation appears to be independent of G-protein activation as both GDP-beta-S (500 microM) and GTP-gamma-S (100 microM) did not influence m3-muscarinic receptor phosphorylation.     

The unique nature of the serine interactions for alpha 1-adrenergic receptor agonist binding and activation

Activation of the beta2- and alpha2-adrenergic receptors (AR) involves hydrogen bonding of serine residues in the fifth transmembrane segment (TMV) to the catechol hydroxyls of the endogenous agonists, epinephrine and norepinephrine. With the beta2-AR both Ser204 and Ser207 but not a third TMV serine (Ser203) are required for binding and full agonist activity. However, with the alpha2a-AR only one of two TMV serines (Ser204, equivalent to Ser207 in the beta-AR) appears to contribute partially to agonist-binding and activation. Because the alpha1a-AR uniquely contains only two TMV serines, this subtype was used to systematically evaluate the role of hydrogen bonding in alpha1-AR activation. Binding of epinephrine or its monohydroxyl congeners, phenylephrine and synephrine, was not decreased when tested with alanine- substitution mutants that lacked either Ser188 (Ser188--> Ala) or Ser192 (Ser192-->Ala). With the substitution of both serines in the double mutant, Ser188/192-->Ala, binding of all three ligands was significantly reduced (10- 100-fold) consistent with a single hydrogen bond interaction. However, receptor-mediated inositol phosphate production was markedly attenuated only with the Ser188-->Ala mutation and not with Ser192-->Ala. In support of the importance of Ser188, binding of phenylephrine (meta-hydroxyl only) by Ser192-->Ala increased 7-fold over that observed with either the wild type receptor or the Ser188-->Ala mutation. Binding of synephrine (para-hydroxyl only) was unchanged with the Ser192-->Ala mutation. In addition, when combined with a recently described constitutively active alpha1a-AR mutation (Met292-->Leu), only the Ser188-->Ala mutation and not Ser192-->Ala relieved the high affinity binding and increased agonist potency observed with the Met292-->Leu mutation. A simple interpretation of these findings is that the meta-hydroxyl of the endogenous agonists preferentially binds to Ser188, and it is this hydrogen bond interaction, and not that between the para-hydroxyl and Ser192, that allows receptor activation. Furthermore, since Ser188 and Ser192 are separated by three residues on the TMV alpha-helix, whereas Ser204 and Ser207 of the beta2-AR are separated by only two residues, the orientation of the catechol ring in the alpha1-AR binding pocket appears to be unique and rotated approximately 120 degrees to that in the beta2-AR.