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S B Liggett

Publications and source records attributed to S B Liggett.

At least 19 recordsLinked to original sources
PubMed2006

Overview of the pharmacogenetics of asthma treatment.

Asthma affects approximately 300 million individuals worldwide. Medications comprise a substantial portion of asthma expenditures. Despite the availability of three primary therapeutic classes of medications, there are a significant number of nonresponders to therapy. Available data, as well as previous pharmacogenetic studies, suggest that genetics may contribute as much as 60-80% to the interindividual variability in treatment response. In this methodologic review, after providing a broad overview of the asthma pharmacogenetics literature to date, we describe the application of a novel family-based screening algorithm to the analysis of pharmacogenetic data and highlight our approach to identifying and verifying loci influencing asthma treatment response. This approach seeks to address issues related to multiple comparisons, statistical power, population stratification, and failure to replicate from which previous population-based or case-control pharmacogenetic association studies may suffer. Identification of such replicable loci is the next step towards the goal of 'individualized therapy' for asthma.

Adrenergic beta-Agonists↗
PubMed2006

Pharmacogenetics of beta1-adrenergic receptors in heart failure and hypertension.

Currently it is generally accepted that an individual's genetic makeup can modify the efficacy of drug treatment or the risk of adverse reactions. Although not a new concept, the availability of human genome sequence and rapid genotyping at variable loci in drug targets or metabolizing genes has provided new opportunities for the field termed "pharmacogenetics". Somewhat surprisingly, multiple studies have shown the existence of common variants (polymorphisms) in members of the G-protein coupled receptor superfamily, which constitute around 50% of all the targets of currently prescribed drugs. The beta1-adrenergic receptors (beta1ARs) are interesting candidates for pharmacogenetic studies in two complex cardiovascular disease, heart failure and hypertension, since they mediate the effects of catecholamines in the sympathetic nervous system. These receptors are involved in the progression and treatment (beta-blockers therapy) of both diseases, and have polymorphisms that show altered function or regulation as compared to their allelic counterparts in recombinant expression systems and genetically modified mice. These results have prompted prospective and retrospective clinical studies examining whether polymorphisms of these genes are risk factors, disease modifiers, or predictors of b-blocker response in heart failure and hypertension. To date, it appears that beta1AR variants are very likely one genetic component that defines responsiveness to beta-blockers in heart failure and hypertension. Altogether, results are promising, but discrepancies between studies require resolution before these polymorphisms can be utilized in practice. With the goal of personalizing therapy based on an individual's genetic makeup, additional adequately powered, multiethnic, multi-drug studies will be needed.

Adrenergic beta-Antagonists↗
PubMed2001

Serine 232 of the alpha(2A)-adrenergic receptor is a protein kinase C-sensitive effector coupling switch.

alpha(2)-adrenergic receptors (alpha(2)AR) couple to multiple effectors including adenylyl cyclase and phospholipase C. We hypothesized that signaling selectivity to these effectors is dynamically directed by kinase-sensitive domains within the third intracellular loop of the receptor. Substitution of Ala for Ser232, which is in the N-terminal region of this loop in the alpha(2A)AR, resulted in a receptor that was markedly uncoupled ( approximately 82% impairment) from stimulation of inositol phosphate accumulation while the capacity to inhibit adenylyl cyclase remained relatively intact. In S232A alpha(2A)AR transfected cell membranes, agonist-promoted [(35)S]GTPgammaS binding was reduced by approximately 50%. Coexpression of modified G proteins rendered insensitive to pertussis toxin revealed that the S232A receptor was uncoupled from both G(i) and G(o). S232 is a potential PKC phosphorylation site, and whole cell phosphorylation studies showed that the mutant had depressed phosphorylation compared to wild type (1.3- vs 2.1-fold/basal). Consistent with S232 directing coupling to phospholipase C, PMA exposure resulted in approximately 67% desensitization of agonist-promoted inositol phosphate accumulation without significantly affecting inhibition of adenylyl cyclase. The dominant effect of mutation or phosphorylation at this site on inositol phosphate as compared to cAMP signaling was found to most likely be due to the low efficiency of signal transduction via phospholipase C vs adenylyl cyclase. Taken together, these results indicate that S232 acts as a selective, PKC-sensitive, modulator of effector coupling of the alpha(2A)AR to inositol phosphate stimulation. This represents one mechanism by which cells route stimuli directed to multifunctional receptors to selected effectors so as to attain finely targeted signaling.

Adenylate Cyclase Toxin↗
PubMed2001

The Ile164 beta(2)-adrenoceptor polymorphism alters salmeterol exosite binding and conventional agonist coupling to G(s).

beta(2)-adrenoceptors (beta(2)AR) are polymorphic at amino acid 164 (Thr or Ile) of the fourth transmembrane domain. In transfected fibroblasts, six agonists commonly used in the treatment of bronchospasm were studied. Isoproterenol, albuterol, metaproterenol, terbutaline, formoterol, and salmeterol displayed decreased binding affinities (K(i)s were 1.2-3.0-fold higher) and a significant degree of impaired maximal stimulation of adenylyl cyclase ( approximately 40%), was observed with all agonists for the Ile164 receptor. The ratios of signal transduction efficiencies (Tau function, Ile164/Thr164) varied from a low of 0.17 for terbutaline to 0.49 for salmeterol. In addition, Ile164 bound salmeterol at the exosite, as delineated in perfusion washout studies, at a decreased level (31+/-4.8% vs. 49+/-4.4% retained salmeterol, respectively, P=0.02). In cAMP production studies under perfusion conditions, this decreased exosite binding caused a approximately 50% decrease in the duration of action of salmeterol at Ile164 (t(1/2)=21.0+/-3.6 vs. 46.8+/-4.1 min for Thr164, P=0.001). The durations of action for isoproterenol and formoterol under similar perfusion conditions were not different between the two receptors. These in vitro results indicate the Ile164 polymorphic receptor represents a pharmacogenetic locus for the most commonly utilized agonists in the treatment of asthma with a unique phenotype for salmeterol.

Adrenergic beta-Agonists↗
PubMed2001

Modification of the beta 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy.

Depressed G-protein-coupled receptor (GPCR) signaling has been implicated as a component of the pathophysiology of a number of complex diseases including heart failure and asthma, and augmentation or restoration of signaling by various means has been shown to improve organ function. Because some properties of native GPCRs are disadvantageous for ectopic therapeutic expression, we utilized the beta(2)-adrenergic receptor (beta(2)AR) as a scaffold to construct a highly modified therapeutic receptor-effector complex (TREC) suitable for gene therapy. Altogether, 19 modifications were made to the receptor. The ligand-binding site was re-engineered in TM-3 so that a beta-hydroxylmethyl side chain acts as a proton donor for the binding of a novel ligand. In addition, sites critical for agonist-promoted down-regulation in the amino terminus and for phosphorylation by GPCR kinases, and protein kinases A and C, in the third intracellular loop and the carboxyl terminus of the receptor were altered. These modifications of the receptor resulted in depressed agonist-stimulated adenylyl cyclase activity (26.8 +/- 2.1 versus 41.4 +/- 8 pmol/min/mg for wild-type beta(2)AR). This was fully restored by fusing the carboxyl terminus of the modified receptor to G alpha(s) (43.3 +/- 2.7 pmol/min/mg). The fully modified fused receptor was not activated by beta-agonists but rather by a nonbiogenic amine agonist that itself failed to activate the wild-type beta(2)AR. This two-way selectivity thus provides targeted activation based on physiologic status. Furthermore, the TREC did not display tachyphylaxis to prolonged agonist exposure (desensitization was 1 +/- 5% versus 55 +/- 4% for wild-type beta(2)AR). Thus, despite extensive alterations in regions of conformational lability, the beta(2)AR can be tailored to have optimal signaling characteristics for gene therapy. As a general paradigm, TRECs for enhancement of other G-protein signaling appear to be feasible for modification of other pathologic states.

Animals↗
PubMed2001

Interactions between phospholamban and beta-adrenergic drive may lead to cardiomyopathy and early mortality.

BACKGROUND: Relieving the inhibition of sarcoplasmic reticular function by phospholamban is a major target of beta-adrenergic stimulation. Chronic beta-adrenergic receptor activity has been suggested to be detrimental, on the basis of transgenic overexpression of the receptor or its signaling effectors. However, it is not known whether physiological levels of sympathetic tone, in the absence of preexisting heart failure, are similarly detrimental. METHODS AND RESULTS: Transgenic mice overexpressing phospholamban at 4-fold normal levels were generated, and at 3 months, they exhibited mildly depressed ventricular contractility without heart failure. As expected, transgenic cardiomyocyte mechanics and calcium kinetics were depressed, but isoproterenol reversed the inhibitory effects of phospholamban on these parameters. In vivo cardiac function was substantially depressed by propranolol administration, suggesting enhanced sympathetic tone. Indeed, plasma norepinephrine levels and the phosphorylation status of phospholamban were elevated, reflecting increased adrenergic drive in transgenic hearts. On aging, the chronic enhancement of adrenergic tone was associated with a desensitization of adenylyl cyclase (which intensified the inhibitory effects of phospholamban), the development of overt heart failure, and a premature mortality. CONCLUSIONS: The unique interaction between phospholamban and increased adrenergic drive, elucidated herein, provides the first evidence that compensatory increases in catecholamine stimulation can, even in the absence of preexisting heart failure, be a primary causative factor in the development of cardiomyopathy and early mortality.

Adenylyl Cyclases↗
PubMed2001

Identification and functional characterization of alpha(2)-adrenoceptor polymorphisms.

For each alpha(2)-adrenoceptor subtype (alpha(2A), alpha(2B) and alpha(2C)), sequence variations within the coding region of each gene have been identified in humans. These result in substitutions or deletions of amino acids in the third intracellular loops of each receptor. This article summarizes the genetics and molecular biology of alpha(2)-adrenoceptor polymorphisms, including the consequences of each polymorphism on receptor signaling, as determined in transfected cells. These effects include alterations in G-protein coupling, desensitization and G-protein receptor kinase-mediated phosphorylation. Studies so far provide the mechanistic basis for future studies to investigate genetic risk factors and pharmacogenetics in pathophysiological conditions linked to alpha(2)-adrenoceptor function.

Base Sequence↗
PubMed2001

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.

Adenylyl Cyclases↗
PubMed2000

An asn to lys polymorphism in the third intracellular loop of the human alpha 2A-adrenergic receptor imparts enhanced agonist-promoted Gi coupling.

alpha(2A)-Adrenergic receptors (alpha(2A)AR) are presynaptic autoinhibitory receptors of noradrenergic neurons in the central and peripheral sympathetic nervous systems, which act to dynamically regulate neurotransmitter release. Signaling through the G(i)/G(o) family of G-proteins, the receptor subserves numerous homeostatic and central nervous system functions. A single nucleotide polymorphism of this receptor, which results in an Asn to Lys substitution at amino acid 251 of the third intracellular loop, was identified in the human population. The frequency of Lys-251 was 10-fold greater in African-Americans than in Caucasians, but was not associated with essential hypertension. To determine the consequences of this substitution, wild-type and Lys-251 receptors were expressed in CHO and COS-7 cells. Expression, ligand binding, and basal receptor function were unaffected by the substitution. However, agonist-promoted [(35)S]GTPgammaS binding was approximately 40% greater with the Lys-251 receptor. This enhanced agonist function was observed with catecholamines, azepines, and imidazolines albeit to different degrees. In studies of agonist-promoted functional coupling to G(i), the polymorphic receptor displayed enhanced inhibition of adenylyl cyclase (60 +/- 4. 4 versus 46 +/- 4.1% inhibition) and markedly enhanced stimulation of MAP kinase (57 +/- 9 versus 15- +/- 2-fold increase over basal) compared with wild-type alpha(2A)AR. The potency of epinephrine in stimulating inositol phosphate accumulation was increased approximately 4 fold with the Lys-251 receptor. Unlike previously described variants of G-protein-coupled receptors, where the minor species causes either a loss of function or increased non-agonist function, Lys-251 alpha(2A)AR represents a new class of polymorphism whose phenotype is a gain of agonist-promoted function.

Adenylyl Cyclase Inhibitors↗
PubMed2000

Polymorphic deletion of three intracellular acidic residues of the alpha 2B-adrenergic receptor decreases G protein-coupled receptor kinase-mediated phosphorylation and desensitization.

A polymorphic variant of the human alpha(2B)-adrenergic receptor (alpha(2B)AR), which consists of a deletion of three glutamic acids (residues 301-303) in the third intracellular loop was found to be common in Caucasians (31%) and to a lesser extent in African-Americans (12%). The consequences of this deletion were assessed by expressing wild-type and the Del301-303 receptors in Chinese hamster ovary and COS cells. Ligand binding was not affected, although a small decrease in coupling efficiency to the inhibition of adenylyl cyclase was observed with the mutant. The deletion occurs within a stretch of acidic residues that is thought to establish the milieu for agonist-promoted phosphorylation and desensitization of the receptor by G protein-coupled receptor kinases (GRKs). Agonist-promoted phosphorylation studies carried out in cells coexpressing the alpha(2B)ARs and GRK2 revealed that the Del301-303 receptor displayed approximately 56% of wild-type phosphorylation. Furthermore, the depressed phosphorylation imposed by the deletion was found to result in a complete loss of short term agonist-promoted receptor desensitization. Thus the major phenotype of the Del301-303 alpha(2B)AR is one of impaired phosphorylation and desensitization by GRKs, and thus the polymorphisms renders the receptor incapable of modulation by this key mechanism of dynamic regulation.

Adenylyl Cyclases↗
PubMed2000

alpha 2A/alpha 2C-adrenergic receptor third loop chimera show that agonist interaction with receptor subtype backbone establishes G protein-coupled receptor kinase phosphorylation.

The alpha(2A)-adrenergic receptor (AR) undergoes rapid agonist-promoted desensitization due to phosphorylation by G protein-coupled receptor kinases (GRKs) 2 and 3 at serines in the third intracellular loop of the receptor. In contrast, the alpha(2C)AR fails to display such desensitization or phosphorylation, which has been presumed to be due to this receptor lacking GRK phosphorylation sites. However, the alpha(2C)AR has multiple serines and threonines in putative favorable motifs within its third intracellular loop. We considered that the conformation of the third intracellular loop imposed by agonists binding to the transmembrane-spanning domains could be the basis of this subtype-specific property, rather than the presence or absence of phosphoacceptors per se. To address this, alpha(2A)/alpha(2C) third loop chimeric receptors were constructed. In whole cell phosphorylation studies, the alpha(2A) with the alpha(2C) third loop receptor underwent agonist-promoted phosphorylation while the alpha(2C) with the alpha(2A) third loop receptor did not, indicating that the agonist interaction with the parent receptor backbone establishes the phosphorylation phenotype. We postulated then that agonists with diverse structures that distinctly interact with alpha(2)AR should display different degrees of phosphorylation independent of receptor activation. Indeed, several full and partial agonists were identified, which evoked phosphorylation that was not related to intrinsic activity as established by [(35)S]guanosine 5'-3-O-(thio)triphosphate binding. Taken together, it appears that phosphorylation of the alpha(2)AR evoked by agonist is highly sensitive to the conformation of the third intracellular loop induced/stabilized by agonist to such an extent that these properties dictate the extent of phosphorylation of the loop when phosphoacceptors are present, and are the basis for subtype-specific phosphorylation.

Adrenergic alpha-Agonists↗
PubMed2000

Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness.

The human beta(2)-adrenergic receptor gene has multiple single-nucleotide polymorphisms (SNPs), but the relevance of chromosomally phased SNPs (haplotypes) is not known. The phylogeny and the in vitro and in vivo consequences of variations in the 5' upstream and ORF were delineated in a multiethnic reference population and an asthmatic cohort. Thirteen SNPs were found organized into 12 haplotypes out of the theoretically possible 8,192 combinations. Deep divergence in the distribution of some haplotypes was noted in Caucasian, African-American, Asian, and Hispanic-Latino ethnic groups with >20-fold differences among the frequencies of the four major haplotypes. The relevance of the five most common beta(2)-adrenergic receptor haplotype pairs was determined in vivo by assessing the bronchodilator response to beta agonist in asthmatics. Mean responses by haplotype pair varied by >2-fold, and response was significantly related to the haplotype pair (P = 0.007) but not to individual SNPs. Expression vectors representing two of the haplotypes differing at eight of the SNP loci and associated with divergent in vivo responsiveness to agonist were used to transfect HEK293 cells. beta(2)-adrenergic receptor mRNA levels and receptor density in cells transfected with the haplotype associated with the greater physiologic response were approximately 50% greater than those transfected with the lower response haplotype. The results indicate that the unique interactions of multiple SNPs within a haplotype ultimately can affect biologic and therapeutic phenotype and that individual SNPs may have poor predictive power as pharmacogenetic loci.

Base Sequence↗
PubMed2000

A four amino acid deletion polymorphism in the third intracellular loop of the human alpha 2C-adrenergic receptor confers impaired coupling to multiple effectors.

The alpha(2)-adrenergic receptors (alpha(2)ARs) play a critical role in modulating neurotransmitter release in the central and peripheral sympathetic nervous systems. A polymorphism of the alpha(2)AR subtype localized to human chromosome 4 (the pharmacologic alpha(2C)AR subtype) within an intracellular domain has been identified in normal individuals. The polymorphism (denoted Del322-325) is because of an in-frame 12-nucleic acid deletion encoding a receptor lacking Gly-Ala-Gly-Pro in the third intracellular loop. To delineate the functional consequences of this structural alteration, Chinese hamster ovary cells were permanently transfected with constructs encoding wild-type human alpha(2C)AR and the polymorphic receptor. The Del322-325 variant had decreased high affinity agonist binding (K(H) = 7.3 +/- 0.95 versus 3.7 +/- 0.43 nm; %R(H) = 31 +/- 4 versus 49 +/- 4) compared with wild-type indicating impaired formation of the agonist-receptor-G protein complex. The polymorphic receptor displayed markedly depressed epinephrine-promoted coupling to G(i), inhibiting adenylyl cyclase by 10 +/- 4.3% compared with 73 +/- 2.4% for wild-type alpha(2C)AR. This also was so for the endogenous ligand norepinephrine and full and partial synthetic agonists. Depressed agonist-promoted coupling to the stimulation of MAP kinase ( approximately 71% impaired) and inositol phosphate production ( approximately 60% impaired) was also found with the polymorphic receptor. The Del322-325 receptor was approximately 10 times more frequent in African-Americans compared with Caucasians (allele frequencies 0.381 versus 0.040). Given this significant loss of function phenotype in several signal transduction cascades and the skewed ethnic prevalence, Del322-325 represents a pharmacoethnogenetic locus and may also be the basis for interindividual variation in cardiovascular or central nervous system pathophysiology.

Adrenergic alpha-Agonists↗
PubMed2000

Polymorphisms of the beta(2)-adrenergic receptor determine exercise capacity in patients with heart failure.

The beta(2)-adrenergic receptor (beta(2)AR) exists in multiple polymorphic forms with different characteristics. Their relevance to heart failure (HF) physiology is unknown. Cardiopulmonary exercise testing was performed on 232 compensated HF patients with a defined beta(2)AR genotype. Patients with the uncommon Ile164 polymorphism had a lower peak VO(2) (15.0+/-0.9 mL. kg(-1). min(-1)) than did patients with Thr164 (17.9+/-0.9 mL. kg(-1). min(-1), P<0.0001). The percentage achieved of predicted peak VO(2) was also lower in patients with Ile164 (62. 3+/-4.5% versus 71.5+/-5.1%, P=0.045). The relative risk of a patient having a VO(2) </=14 mL. kg(-1). min(-1) who had Ile164 was 8.0 (P=0.009). Catheterization-based invasive exercise testing revealed depressed changes in the exercise-induced cardiac index, systemic vascular resistance, stroke volume, and VO(2) in patients with Ile164. The polymorphisms at position 16 also impacted exercise capacity: peak VO(2) for Arg16 versus Gly16 was 17.0+/-0.8 versus 15. 6+/-0.5 mL. kg(-1). min(-1), respectively (P=0.03). Because the polymorphisms at loci 16 and 27 can occur together, 4 homozygous combinations exist. Patients with Arg16/Glu27 had the highest percentage achieved of predicted peak VO(2) (75. 7+/-6.4%), whereas those with Gly16/Gln27 had the lowest (55.3+/-2. 8%, P=0.0032). The above findings were not confounded by baseline clinical characteristics, including beta-blocker usage. We conclude that the beta(2)AR polymorphisms Ile164, Gly16, and the combination of Gly16 and Gln27 are associated with depressed exercise performance in HF and represent a genetically determined factor in the pathophysiology of HF.

Alleles↗