Theoretical basis for the identification of allelic variants that encode drug efficacy and toxicity

Almost all drugs that produce a favorable response (efficacy) may also produce adverse effects (toxicity). The relative strengths of drug efficacy and toxicity that vary in human populations are controlled by the combined influences of multiple genes and environmental influences. Genetic mapping has proven to be a powerful tool for detecting and identifying specific DNA sequence variants on the basis of the haplotype map (HapMap) constructed from single-nucleotide polymorphisms (SNPs). In this article, we present a novel statistical model for sequence mapping of two different but related drug responses. This model is incorporated by mathematical functions of drug response to varying doses or concentrations and the statistical device used to model the correlated structure of the residual (co)variance matrix. We implement a closed-form solution for the EM algorithm to estimate the population genetic parameters of SNPs and the simplex algorithm to estimate the curve parameters describing the pharmacodynamic changes of different genetic variants and matrix-structuring parameters. Extensive simulations are performed to investigate the statistical properties of our model. The implications of our model in pharmacogenetic and pharmacogenomic research are discussed.     

Direct molecular haplotyping by melting curve analysis of hybridization probes: beta 2-adrenergic receptor haplotypes as an example

Direct determination of the association of multiple genetic polymorphisms, or haplotyping, in individual samples is challenging because of chromosome diploidy. Here, we describe the ability of hybridization probes, commonly used as genotyping tools, to establish single nucleotide polymorphism (SNP) haplotypes in a single step. Three haplotypes found in the beta 2-adrenergic receptor (β2AR) gene and characterized by three different SNPs combinations are presented as examples. Each combination of SNPs has a unique stability, recorded by its melting temperature, even when intervening sequences from the template must loop out during probe hybridization. In the course of this study, two haplotypes in β2AR not described previously were discovered. This approach provides a tool for molecular haplotyping that should prove useful in clinical molecular genetics diagnostics and pharmacogenetic research where methods for direct haplotyping are needed.     

Multiplex genotyping of the human beta2-adrenergic receptor gene using solid-phase capturable dideoxynucleotides and mass spectrometry

Previously, we established the feasibility of using solid phase capturable (SPC) dideoxynucleotides to generate single base extension (SBE) products which were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for multiplex genotyping, an approach that we refer to as SPC-SBE. We report here the expanding of the SPC-SBE method as a single-tube assay to simultaneously detect 20 single nucleotide variations in a model system and 3 single nucleotide polymorphisms (SNPs) in the human beta2-adrenergic receptor (beta2AR) gene. Twenty primers were designed to have a sufficient mass difference between all extension products for accurate detection of nucleotide variants of the synthetic templates related to the p53 gene. These primers were extended simultaneously in a single tube with biotin-ddNTPs to generate 3(')-biotinylated DNA products, which were first captured by streptavidin-coated magnetic beads and then released from the beads and analyzed with MALDI-TOF MS. This approach generates a mass spectrum free of primer peaks and their associated dimers, increasing the scope of multiplexing SNPs. We also simultaneously genotyped 3 SNPs in the beta2AR gene (5(')LC-Cys19Arg, Gly16Arg, and Gln27Glu) from the genomic DNA of 20 individuals. Comparison of this approach with direct sequencing and the restriction fragment length polymorphism method indicated that the SPC-SBE method is superior for detecting nucleotide variations at known SNP sites.     

Pharmacogenetic assessment of antipsychotic-induced movement disorders: contribution of the dopamine D3 receptor and cytochrome P450 1A2 genes

Tardive dyskinesia (TD) is characterized by involuntary movements predominantly in the orofacial region and develops in approximately 20% of patients during long-term treatment with typical antipsychotics. The high prevalence of TD and its disabling and potentially irreversible clinical course is an important shortcoming for treatment with typical antipsychotics. The studies presented in this article evaluate the role of single nucleotide polymorphisms in dopamine D3 receptor (DRD3) and CYP1A2 genes for propensity to develop TD in patients with schizophrenia. In theory, a combined pharmacogenetic analysis of pharmacokinetic and pharmacodynamic targets for antipsychotics should improve our ability to identify subpopulations that differ in drug safety profile. This information may in turn contribute to the design of more efficient clinical trials and thus expedite the development and regulatory approval of newer antipsychotic compounds.     

Pharmacogenetics in drug regulation: promise, potential and pitfalls

Pharmacogenetic factors operate at pharmacokinetic as well as pharmacodynamic levels-the two components of the dose-response curve of a drug. Polymorphisms in drug metabolizing enzymes, transporters and/or pharmacological targets of drugs may profoundly influence the dose-response relationship between individuals. For some drugs, although retrospective data from case studies suggests that these polymorphisms are frequently associated with adverse drug reactions or failure of efficacy, the clinical utility of such data remains unproven. There is, therefore, an urgent need for prospective data to determine whether pre-treatment genotyping can improve therapy. Various regulatory guidelines already recommend exploration of the role of genetic factors when investigating a drug for its pharmacokinetics, pharmacodynamics, dose-response relationship and drug interaction potential. Arising from the global heterogeneity in the frequency of variant alleles, regulatory guidelines also require the sponsors to provide additional information, usually pharmacogenetic bridging data, to determine whether data from one ethnic population can be extrapolated to another. At present, sponsors explore pharmacogenetic influences in early clinical pharmacokinetic studies but rarely do they carry the findings forward when designing dose-response studies or pivotal studies. When appropriate, regulatory authorities include genotype-specific recommendations in the prescribing information. Sometimes, this may include the need to adjust a dose in some genotypes under specific circumstances. Detailed references to pharmacogenetics in prescribing information and pharmacogenetically based prescribing in routine therapeutics will require robust prospective data from well-designed studies. With greater integration of pharmacogenetics in drug development, regulatory authorities expect to receive more detailed genetic data. This is likely to complicate the drug evaluation process as well as result in complex prescribing information. Genotype-specific dosing regimens will have to be more precise and marketing strategies more prudent. However, not all variations in drug responses are related to pharmacogenetic polymorphisms. Drug response can be modulated by a number of non-genetic factors, especially co-medications and presence of concurrent diseases. Inappropriate prescribing frequently compounds the complexity introduced by these two important non-genetic factors. Unless prescribers adhere to the prescribing information, much of the benefits of pharmacogenetics will be squandered. Discovering highly predictive genotype-phenotype associations during drug development and demonstrating their clinical validity and utility in well-designed prospective clinical trials will no doubt better define the role of pharmacogenetics in future clinical practice. In the meantime, prescribing should comply with the information provided while pharmacogenetic research is deservedly supported by all concerned but without unrealistic expectations.     

心脏中不同β肾上腺素受体亚型的信号体系及其病理生理意义

生理情况下,β肾上腺素受体(βAR)对心肌收缩和舒张活动起至关重要的作用;病理情况下,长期激动βAR可以诱发心肌细胞肥大、凋亡以及细胞坏死等心肌重塑性活动,从而参与了慢性心衰的发病过程。近十年以来,许多资料表明β1和β2肾上腺素受体亚型(β1AR和β2AR)共存于心脏中,且激动不同信号系统。短时间激动β1AR,使Gs蛋白-腺苷酸环化酶-环苷腺酸-蛋白激酶A(Gs-adenyly cyclase-cAMP-PKA)信号体系激活并广布于细胞内,而激动βAR则同时激活G1蛋白而产生空间及功能局限的cAMP信号;长时间激动β1AR和β2AR则对心肌细胞的命运产生不同影响：β1AR诱导细胞肥大和凋亡,β2AR促使细胞存活。β2AR的心肌保护作用是通过激活Gi蛋白-Gβγ-PI3K-Akt途径介导。但出乎意料,β1AR的心肌肥厚和凋亡效应并不依赖于经典的cAMP/PKA信号途径,而是激活钙,钙调素依赖性蛋白激酶Ⅱ(caMK Ⅱ)途径。用心肌特异性表达βAR亚型的转基因小鼠进行实验,进一步证实不同βAR亚型在调节心肌重塑和功能方面作用各异。βAR亚型作用不同的新观点不仅为β阻滞剂治疗慢性心衰提供了分子和细胞机制的依据,而且提出了选择性β1AR阻滞和β2AR激动联合治疗慢性心衰的新的治疗思路。     

Two distinct pathways for cAMP-mediated down-regulation of the beta 2-adrenergic receptor. Phosphorylation of the receptor and regulation of its mRNA level

We have studied cyclic AMP-mediated regulation of the beta 2-adrenergic receptor (beta 2AR). The effects of cAMP were assessed in Chinese hamster fibroblast (CHW) cells expressing either the wild type human beta 2AR receptor (CH-beta 2) or mutated forms of the receptor lacking the consensus sequences for phosphorylation by the cAMP-dependent protein kinase. Treatment of the CH-beta 2 cells with the cAMP analogue dibutyryl cAMP (Bt2cAMP) induces a time-dependent "down-regulation" of the number of beta 2AR. This down-regulation of the receptors is accompanied by a decline in the steady state level of beta 2AR mRNA. Moreover, the treatment with Bt2cAMP induces an increase in the phosphorylation level of the membrane-associated beta 2AR. Both the reduction in beta 2AR mRNA and the enhanced phosphorylation of the receptor are rapid and precede the loss of receptor. The down-regulation of beta 2AR induced by Bt2cAMP is concentration-dependent and mimicked by the other biologically active cyclic nucleotide analogue, 8-Br-cAMP, by forskolin, and by the phosphodiesterase inhibitor, isobutylmethylxanthine. In the CHW cell lines expressing receptors lacking the putative protein kinase A phosphorylation sites, the Bt2cAMP-induced phosphorylation of beta 2AR is completely abolished. In these cells the down-regulation of beta 2AR receptor number produced by cAMP is significantly slowed, whereas the reduction in beta 2AR mRNA level is equivalent to that observed in CH-beta 2 cells. These data indicate that there are at least two pathways by which cAMP may decrease the number of beta 2ARs in cells: one involves phosphorylation of the receptor by the cAMP-dependent protein kinase and the other leads to a reduction in steady state beta 2AR mRNA levels.     

Turning off the signal: desensitization of beta-adrenergic receptor function

Cellular responses to many hormones and neurotransmitters wane rapidly despite continuous exposure of cells to these stimuli. This phenomenon, termed desensitization, has been particularly well studied for the stimulation of cAMP levels by plasma membrane beta-adrenergic receptors (beta AR). The molecular mechanisms underlying rapid beta AR desensitization do not appear to require internalization of the receptors, but rather an alteration in the functioning of beta AR themselves that uncouples the receptors from the stimulatory G protein Gs. This uncoupling phenomenon involves phosphorylation of beta AR by at least two kinases, PKA and the beta AR kinase (beta ARK), which are activated under different desensitizing conditions. Receptor phosphorylation by the two kinases leads to desensitization of the receptor response via distinct biochemical mechanisms, and additional cytosolic factors appear to be involved in the case of beta ARK. Numerous experimental approaches have been used recently to elucidate the molecular details of this ubiquitous biological process.     

Challenges and recommendations for conducting epidemiological studies in the field of epilepsy pharmacogenetics

Epilepsy is one of the most prevalent neurological disorders, afflicting approximately 50 million Indians. Owing to affordability and easy availability, use of first-generation antiepileptic drugs (AEDs) is heavily encouraged for the treatment of epilepsy in resource-limited countries such as India. Although first-generation AEDs are at par with second-generation AEDs in terms of efficacy, adverse drug reactions (ADRs) are quite common with them. This could be attributed to the inferior pharmacokinetic parameters such as nonlinear metabolism, narrow therapeutic index and formation of toxic intermediates. In addition, epilepsy patients may differ in the pharmacokinetic and pharmacodynamic profiles, with about 1/3(rd) of the population failing to respond to treatment. A proportion of this interindividual variability in response may be explained by genetic heterogeneity in the activity and expression of the network of proteins such as metabolizing enzymes, transporters and targets of AEDs. Over the last two decades, a considerable effort has been made by the scientific community for unraveling this genetic basis of variable response to AEDs. However, there have been inconsistencies in such genetic association studies conducted across different territories of the world. There could be several reasons underlying the poor replicability of these studies, mainly nonuniform phenotypic definitions, poor sample size and interethnic variability. In the present review article, we provide an overview of heterogeneity in study designs for conducting pharmacogenetic studies. In addition, critical recommendations required for overcoming such challenges imposed by pharmacogenetic epidemiological studies have been briefly discussed.     

Norepinephrine- and epinephrine-induced distinct beta2-adrenoceptor signaling is dictated by GRK2 phosphorylation in cardiomyocytes

Agonist-dependent activation of G protein-coupled receptors induces diversified receptor cellular and signaling properties. Norepinephrine (NE) and epinephrine (Epi) are two endogenous ligands that activate adrenoceptor (AR) signals in a variety of physiological stress responses in animals. Here we use cardiomyocyte contraction rate response to analyze the endogenous beta(2)AR signaling induced by Epi or NE in cardiac tissue. The Epi-activated beta(2)AR induced a rapid contraction rate increase that peaked at 4 min after stimulation. In contrast, the NE-activated beta(2)AR induced a much slower contraction rate increase that peaked at 10 min after stimulation. Whereas both drugs activated beta(2)AR coupling to G(s) proteins, only Epi-activated receptors were capable of coupling to G(i) proteins. Subsequent studies showed that the Epi-activated beta(2)AR underwent a rapid phosphorylation by G protein-coupled receptor kinase 2 (GRK2) and subsequent dephosphorylation on serine residues 355 and 356, which was critical for sufficient receptor recycling and G(i) coupling. In contrast, the NE-activated beta(2)ARs underwent slow GRK2 phosphorylation, receptor internalization and recycling, and failed to couple to G(i). Moreover, inhibiting beta(2)AR phosphorylation by betaARK C terminus or dephosphorylation by okadaic acid prevented sufficient recycling and G(i) coupling. Together, our data revealed that distinct temporal phosphorylation of beta(2)AR on serine 355 and 356 by GRK2 plays a critical role for dictating receptor cellular events and signaling properties induced by Epi or NE in cardiomyocytes. This study not only helps us understand the endogenous agonist-dependent beta(2)AR signaling in animal heart but also offers an example of how G protein-coupled receptor signaling may be finely regulated by GRK in physiological settings.     

Desensitization of the isolated beta 2-adrenergic receptor by beta-adrenergic receptor kinase, cAMP-dependent protein kinase, and protein kinase C occurs via distinct molecular mechanisms.

Exposure of beta 2-adrenergic receptors (beta 2ARs) to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Phosphorylation of the beta 2AR by several distinct kinases plays an important role in this desensitization phenomenon. In this study, we have utilized purified hamster lung beta 2AR and stimulatory guanine nucleotide binding regulatory protein (Gs), reconstituted in phospholipid vesicles, to investigate the molecular properties of this desensitization response. Purified hamster beta 2AR was phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), or beta AR kinase (beta ARK), and receptor function was determined by measuring the beta 2AR-agonist-promoted Gs-associated GTPase activity. At physiological concentrations of Mg2+ (less than 1 mM), receptor phosphorylation inhibited coupling to Gs by 60% (PKA), 40% (PKC), and 30% (beta ARK). The desensitizing effect of phosphorylation was, however, greatly diminished when assays were performed at concentrations of Mg2+ sufficient to promote receptor-independent activation of Gs (greater than 5 mM). Addition of retinal arrestin, the light transduction component involved in the attenuation of rhodopsin function, did not enhance the uncoupling effect of beta ARK phosphorylation of beta 2AR when assayed in the presence of 0.3 mM free Mg2+. At concentrations of Mg2+ ranging between 0.5 and 5.0 mM, however, significant potentiation of beta ARK-mediated desensitization was observed upon arrestin addition. At a free Mg2+ concentration of 5 mM, arrestin did not potentiate the inhibition of receptor function observed on PKA or PKC phosphorylation. These results suggest that distinct pathways of desensitization exist for the receptor phosphorylated either by PKA or PKC or alternatively by beta ARK.     

Rapid inverse changes in alpha 1B- and beta 2-adrenergic receptors and gene transcripts in acutely isolated rat liver cells.

In vitro incubation of hepatocytes acutely isolated from adult male rats leads to a rapid conversion of the adrenergic activation of glycogenolysis from an alpha 1-receptor (alpha 1AR) to a beta 2-receptor (beta 2AR) mediated response within 4 h. In order to understand the underlying mechanism, we examined time-dependent changes in alpha 1- and beta 2-adrenergic activation of glycogenolysis and second messenger systems, the cellular density and affinity of alpha 1AR and beta 2AR, and the steady state levels of alpha 1BAR and beta 2AR mRNAs. Incubation of hepatocytes for 4 h resulted in a decrease in phosphorylase activation and inositol 1,4,5 trisphosphate accumulation in response to phenylephrine, a 40% decrease in alpha 1AR density, and a 70% decrease in alpha 1BAR mRNA levels. Incubation of hepatocytes for 4 h also resulted in the emergence of a phosphorylase response to isoproterenol, an increase in isoproterenol-induced but not in glucagon- or forskolin-induced cAMP accumulation, no significant change in beta 2AR density, and a twofold increase in beta 2AR mRNA levels. Exposure of cells to cycloheximide, 2 microM throughout the 4 h incubation, prevented the emergence of the phosphorylase response to isoproterenol and reduced beta 2AR densities, while the decrease in alpha 1AR density was not affected and the decrease in phosphorylase activation by phenylephrine was attenuated. The results indicate that dissociation of rat liver cells triggers a rapidly developing decrease in alpha 1BAR mRNA and increase in beta 2AR mRNA levels and corresponding inverse changes in the synthesis of alpha 1BAR and beta 2AR which account, at least in part, for the rapid conversion from alpha 1- to beta 2-adrenergic glycogenolysis.     

Structural basis of beta-adrenergic receptor function

Receptors that mediate their actions by stimulating guanine nucleotide binding regulatory proteins (G proteins) share structural as well as functional similarities. The structural motif characteristic of receptors of this class includes seven hydrophobic putative transmembrane domains linked by hydrophilic loops. Genetic analysis of the beta-adrenergic receptor (beta AR) revealed that the ligand binding domain of this receptor, like that of rhodopsin, involves residues within the hydrophobic core of the protein. On the basis of these studies, a model for ligand binding to the receptor has been developed in which the amino group of an agonist or antagonist is anchored to the receptor through the carboxylate side chain of Asp113 in the third transmembrane helix. Other interactions between specific residues of the receptor and functional groups on the ligand have also been proposed. The interaction between the beta AR and the G protein Gs has been shown to involve an intracellular region that is postulated to form an amphiphilic alpha helix. This region of the beta AR is also critical for sequestration, which accompanies agonist-mediated desensitization, to occur. Structural similarities among G protein-linked receptors suggest that the information gained from the genetic analysis of the beta AR should help define functionally important regions of other receptors of this class.     

Intracellular IFN-γ and IL-2 expression monitoring as surrogate markers of the risk of acute rejection and personal drug response in de novo liver transplant recipients

Biomarker monitoring is needed in transplantation to reflect individual response to immunosuppressive drugs and graft outcome. We evaluated intracellular expression and soluble production of interferon-(IFN)-γ and interleukin-(IL)-2 as predictive biomarkers of acute rejection (AR) and personal drug response. Pharmacokinetic–pharmacodynamic profiles were determined in 47 de novo liver recipients treated with tacrolimus, mycophenolate mofetil and prednisone. Of the 47 patients, AR occurred in nine. There were no differences in drug concentrations between rejectors and non-rejectors. A pre-transplantation cut-off value of 55.80% for %CD8⁺–IFN-γ⁺ identified patients at high risk of AR with a sensitivity of 75% and a specificity of 82%. In the first week post-transplantation, patients with a % inhibition for soluble IFN-γ, %CD8⁺–IFN-γ⁺ and %CD8⁺–IL2⁺ lower than 40% developed AR, showing low susceptibility to immunosuppressive drugs. Therefore, effector-T-cell response monitoring may help physicians to identify personal response to treatment and patients at high risk of AR.     

Temporal dynamics of inotropic, chronotropic, and metabolic responses during beta1- and beta2-AR stimulation in the isolated, perfused rat heart

During the beta-adrenergic receptor (beta-AR)-mediated stress response in the heart, the relations between functional responses and metabolism are ill defined, with the distinction between beta1- and beta2-AR subtypes creating further complexity. Specific outstanding questions include the temporal relation between inotropic and chronotropic responses and their metabolic correlates. We sought to elucidate the relative magnitudes and temporal dynamics of the response to beta1- and beta2-AR stimulation and the energy expenditure and bioenergetic state related to these responses in the isolated perfused rat heart. Inotropic [left ventricular developed pressure (LVDP) and dP/dt], chronotropic [heart rate (HR)], and metabolic responses were measured during beta1- (n = 9; agonist: norepinephrine) and beta2- (n = 9; agonist: zinterol) AR stimulation. Myocardial oxygen consumption (MVO2) was measured using fiber-optic oximetry, and high-energy phosphate levels and intracellular pH were measured using 31P NMR spectroscopy. A multiple-dose protocol was used, with near-maximal beta-AR stimulation at the highest doses. In both beta1 and beta2 groups, there were dose-dependent increases in LVDP, dP/dt, HR, and MVO2. The inotropic response showed more rapid onset, washout, and variation during dose than did the chronotropic response and was closely correlated with MVO2. This suggests that the myocardial bioenergetic state is more closely related to the inotropic response than to the chronotropic response. In addition, beta1-AR stimulation resulted in a greater magnitude and rate of onset of inotropic and MVO2 responses than did beta2-AR stimulation during maximal stimulation. However, a similar decrease in intracellular energy charge was seen in the two groups, consistent with a greater rate of oxidative phosphorylation during beta1- than during beta2-AR stimulation.     

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.     

Isoproterenol response following transfection of the mouse beta 2-adrenergic receptor gene into Y1 cells.

The beta 2-adrenergic receptor (beta 2AR) gene was isolated from a mouse genomic library, sequenced and shown to share 93% identity with the hamster beta 2AR cDNA at the amino acid level. This mouse beta 2AR genomic clone was transfected into the Y1 mouse adrenal cortex tumor cell line. Northern blot and S1 nuclease analysis showed that the beta 2AR-transfected cells expressed an mRNA of the appropriate size to encode the receptor. Membrane receptor number and affinities for various beta-adrenergic agonists demonstrated that the transfected clone encoded a beta 2AR protein product. Incubation of the transfected Y1 cells, which do not normally possess beta 2AR, with the beta 2AR agonist, isoproterenol, resulted in an increase in the rate of steroid secretion by these cells as well as a rapid change in cell morphology. This response was fully blocked by the beta 2AR antagonist, propranolol. Prolonged incubation of the cells with isoproterenol resulted in agonist insensitivity and an 80% reduction in membrane receptor number.