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The role of naturally occurring human α1a-Adrenergic Receptor (α1aAR) genetic variants associated with cardiovascular disorders is poorly understood. Here, we present the novel findings that expression of human α1aAR-247R (247R) genetic variant in cardiomyoblasts leads to transition of cardiomyoblasts into a fibroblast-like phenotype, evidenced by morphology and distinct de novo expression of characteristic genes. These fibroblast-like cells exhibit constitutive, high proliferative capacity and agonist-induced hypertrophy compared with cells prior to transition. We demonstrate that constitutive, synergistic activation of EGFR, Src and ERK kinases is the potential molecular mechanism of this transition. We also demonstrate that 247R triggers two distinct EGFR transactivation-dependent signaling pathways: 1) constitutive Gq-independent β-arrestin-1/Src/MMP/EGFR/ERK-dependent hyperproliferation and 2) agonist-induced Gq- and EGFR/STAT-dependent hypertrophy. Interestingly, in cardiomyoblasts agonist-independent hyperproliferation is MMP-dependent, but in fibroblast-like cells it is MMP-independent, suggesting that expression of α1aAR genetic variant in cardiomyocytes may trigger extracellular matrix remodeling. Thus, these novel findings demonstrate that EGFR transactivation by α1aAR-247R leads to hyperproliferation, hypertrophy and alterations in cardiomyoblasts, suggesting that these unique genetically-mediated alterations in signaling pathways and cellular function may lead to myocardial fibrosis. Such extracellular matrix remodeling may contribute to the genesis of arrhythmias in certain types of heart failure.

We previously identified a naturally occurring human SNP, G247R, in the third intracellular loop of the α 1a -adrenergic receptor (α 1a -247R) and demonstrated that constitutive expression of α 1a -247R results in twofold increased cell proliferation compared with WT. In the present study we elucidate molecular mechanisms and signal transduction pathways responsible for increased cell proliferation unique to α 1a -247R, but not α 1a -WT, α 1b , or α 1d AR subtypes. We show that elevated levels of matrix metalloproteinase-7 (MMP7) and a disintegrin and metalloproteinase-12 (ADAM12) in α 1a -247R–expressing cells are responsible for EGF receptor (EGFR) transactivation, downstream ERK activation, and increased cell proliferation; this pathway is confirmed using MMP, EGFR, and ERK inhibitors. We demonstrate that EGFR transactivation and downstream ERK activation depends on increased shedding of heparin-binding EGF. Finally, we demonstrate that knockdown of MMP7 or β-arrestin1 by shRNAs results in attenuation of proliferation of cells expressing α 1a -247R. Importantly, accelerated cell proliferation triggered by the α 1a -247R is serum- and agonist-independent, providing unique evidence for constitutive active coupling to the β-arrestin1/MMP/EGFR transactivation pathway by any G protein-coupled receptor. These findings raise the possibility of a previously unexplored mechanism for sympathetically mediated human hypertension triggered by a naturally occurring human genetic variant.

Objective Hemorrhage continues to be a major problem after cardiac surgery despite the routine use of antifibrinolytic drugs, with striking inter-patient variability poorly explained by already known risk factors. The authors tested the hypothesis that genetic polymorphisms of inflammatory mediators and cellular adhesion molecules are associated with bleeding after cardiac surgery. Design Prospective, observational study. Setting Single, tertiary referral university heart center. Participants Adult patients undergoing aortocoronary surgery with cardiopulmonary bypass. Interventions Patients (n = 759) had 10 mL of blood drawn preoperatively and genomic DNA isolated then genotyped for 17 polymorphisms in 7 candidate genes: tumor necrosis factor, interleukins 1β and 6, interleukin 1 receptor antagonist, intercellular adhesion molecule-1 (ICAM-1), P-selectin and endothelial leucocyte adhesion molecule-1 (E-selectin). Multivariate analyses were used to relate clinical and genetic factors to bleeding and transfusion. Measurements and Main Results The 98G/T polymorphism of the E-selectin gene was independently associated with bleeding after cardiac surgery (p = 0.002), after adjusting for significant clinical predictors (patient size and baseline hemoglobin concentration). There was a gene dose effect according to the number of minor alleles in the genotype; carriers of the minor allele bled 17% (GT) and 54% (TT) more than wild type (GG) genotypes, respectively (p = 0.01). Carriers of the minor allele also had longer activated partial thromboplastin times (p = 0.0023) and increased fresh frozen plasma transfusion (p = 0.03) compared with wild type. Conclusions The authors found a dose-related association between the 98T E-selectin polymorphism and bleeding after cardiac surgery, independent of and additive to standard clinical risk factors.

We have reported that the alpha(1A)-adrenergic receptor (alpha(1A)AR) in rat-1 fibroblasts is a lipid raft protein. Here we examined whether disrupting lipid rafts by methyl-beta-cyclodextrin (MCD) sequestration of cholesterol affects alpha(1A)AR signaling. Unexpectedly, MCD increased alpha(1A)AR-dependent basal inositol phosphate formation and p38 mitogen-activated protein kinase activation in a cholesterol-dependent manner. It also initiated internalization of surface alpha(1A)AR, which was partially blocked by receptor inhibition. Binding assays revealed MCD-mediated increases in receptor agonist affinity as well as reciprocal decreases in inverse agonist affinity, a behavior that is usually interpreted as a shift toward the active receptor conformation. In untreated cells a fraction of the receptor was found to be present in preassociated receptor/G protein complexes, which rapidly dissociate upon receptor stimulation. Consistent with MCD-induced signaling, raft disruption resulted in an increase in receptor/G protein complexes. These results strongly suggest that lipid rafts constrain basal alpha(1A)AR activity; however, preassembled receptor/G protein complexes could still provide a mechanism for accelerating alpha(1A)AR signaling following stimulation.

Genetic variation plays an important role in chronic disease and in perioperative patient management. It was our intent in this short review to cite practical examples of relevant overlap between pharmacoge-nomics and clinical anesthesiology. The influence of genetics on patient safety is seen throughout the perioperative period, when evaluating patients with co-existing diseases in the pre-anesthesia clinic, when considering metabolism and efficacy of drugs intra-operatively, and at the time of postoperative pain management and clinical outcome assessment. The situations presented in this editorial provide compel-ling reasons for clinicians to embrace the partnership between genetics and anesthesiology.

It is unclear whether the regulatory distinction between non-identifiable and identifiable information--information used to determine informed consent practices for the use of clinically derived samples for genetic research--is meaningful to patients. The objective of this study was to examine patients' attitudes and preferences regarding use of anonymous and identifiable clinical samples for genetic research. Telephone interviews were conducted with 1,193 patients recruited from general medicine, thoracic surgery, or medical oncology clinics at five United States academic medical centers. Wanting to know about research being done was important to 72% of patients when samples would be anonymous and to 81% of patients when samples would be identifiable. Only 17% wanted to know about the identifiable scenario but not the anonymous scenario (i.e., following the regulatory distinction). Curiosity-based reasons were the most common (37%) among patients who wanted to know about anonymous samples. Of patients wanting to know about either scenario, approximately 57% would require researchers to seek permission, whereas 43% would be satisfied with notification only. Patients were more likely to support permission (versus notification) in the anonymous scenario if they had more education, were Black, less religious, in better health, more private, and less trusting of researchers. The sample, although not representative of the general population, does represent patients at academic medical centers whose clinical samples may be used for genetic research. Few patients expressed preferences consistent with the regulatory distinction between non-identifiable and identifiable information. Data from this study should cause policy-makers to question whether this distinction is useful in relation to research with previously collected clinically derived samples.

Genomic medicine has provided new mechanistic understanding for many complex diseases over the last 5–10 years. More recently genomic approaches have been applied to the perioperative paradigm, facilitating identification of patients at high risk for adverse events, as well as those who will respond better/worse to specific pharmacologic therapies. The consistent biological theme emerging is that while inflammation is important in healing from surgical trauma, patients who are too robustly proinflammatory appear to be at higher risk for adverse perioperative events. Precise predictors of each adverse event are being elucidated so that corrective therapeutics can be instituted to improve outcomes in high-risk patients. While the field of perioperative genomics could be considered in its infancy, such approaches are the wave of the future.

The alpha(1a)-adrenergic receptor (alpha(1a)AR) occupies intracellular and plasma membranes in both native and heterologous expression systems. Based on multiple independent lines of evidence, we demonstrate the alpha(1a)AR at the cell surface occupies membrane rafts but exits from rafts following stimulation. In non-detergent raft preparations, basal alpha(1a)AR is present in low density membrane rafts and colocalizes with its G protein effectors on density gradients. Raft disruption by cholesterol depletion with methyl-beta-cyclodextrin eliminates these light rafts. To confirm the presence of the alpha(1a)AR in plasma membrane rafts, fluorescence resonance energy transfer measurements were used to demonstrate colocalization of surface receptor and the raft marker, cholera toxin B. This colocalization was largely lost following alpha(1a)AR stimulation with phenylephrine. Similarly, receptor stimulation causes exit of the alpha(1a)AR from light rafts within 3-10 min in contrast to the G proteins, which largely remain in light rafts. Importantly, this delayed exit of the alpha(1a)AR suggests acute receptor signaling and desensitization occur entirely within rafts. Interestingly, both confocal analysis and measurement of surface alpha(1a)AR levels indicate modest receptor internalization during the 10 min following stimulation, suggesting most of the receptor has entered non-raft plasma membrane. Nevertheless, activation does increase the rate of receptor internalization as does disruption of rafts with methyl-beta-cyclodextrin, suggesting raft exit enables internalization. Confocal analysis of surface-labeled hemagglutinin-alpha(1a)AR reveals that basal and stimulated receptor occupies clathrin pits in fixed cells consistent with previous indirect evidence. The evidence presented here strongly suggests the alpha(1a)AR is a lipid raft protein under basal conditions and implies agonist-mediated signaling occurs from rafts.

Reductions in federal support and clinical revenue jeopardize biomedical research and, in turn, clinical medicine.

Using an improved chromatin immunoprecipitation assay designed to increase immunoprecipitation efficiency, we investigated changes in RNA polymerase II (Pol II) density and carboxyl-terminal domain (CTD) phosphorylation during transcription of the cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and several androgen-responsive genes in LNCaP cells. As generally observed in higher eukaryotes, promoter proximal pausing of Pol II appeared to occur on the PPIA and GAPDH genes, but apparently not on the androgen-responsive genes PSA and NKX3-1. Unlike some mammalian studies, we found that the CTD of Pol II in promoter regions contains little phosphorylation at Ser-2 of the heptad repeat, suggesting that Ser-2 phosphorylation is not involved in polymerase exit from the promoter region. In contrast, Pol II near the promoter displayed high levels of Ser-5 phosphorylation, which decreased as polymerase transcribed beyond the promoter region of the PPIA and GAPDH genes. However, total Pol II levels appear to decrease as much or more, suggesting that Ser-5 phosphorylation is maintained. In support of this conclusion, a phosphoserine 5-specific antibody quantitatively immunoprecipitates native hyperphosphorylated Pol II, suggesting that all polymerase with phosphoserine 2 also contains phosphoserine 5. Given reports indicating that phosphoserine 5 is present during elongation in yeast, our data suggest that gross changes in CTD phosphorylation patterns during transcription may be more conserved in yeast and humans than recognized previously.

We identified nine naturally-occurring human single nucleotide polymorphisms (SNPs) in the alpha(1a)-adrenoceptor (alpha(1a)AR) coding region, seven of which result in amino acid change. Utilizing rat-1 fibroblasts stably expressing wild type alpha(1a)AR or each SNP at both high and low levels, we investigated the effect of these SNPs on receptor function. Compared with wild type, two SNPs (R166K, V311I) cause a decrease in binding affinity for agonists norepinephrine, epinephrine, and phenylephrine, and also shift the dose-response curve for norepinephrine stimulation of inositol phosphate (IP) production to the right (reduced potency) without altering maximal IP activity. In addition, SNP V311I and I200S display altered antagonist binding. Interestingly, a receptor with SNP G247R (located in the third intracellular loop) displays increased maximal receptor IP activity and stimulates cell growth. The increased receptor signaling for alpha(1a)AR G247R is not mediated by altered ligand binding or a deficiency in agonist-mediated desensitization, but appears to be related to enhanced receptor-G protein coupling. In conclusion, four naturally-occurring human alpha(1a)AR SNPs induce altered receptor pharmacology and/or biological activity. This finding has potentially important implications in many areas of medicine and can be used to guide alpha(1a)AR SNP choice for future clinical studies.

Alpha1a-adrenergic receptors (alpha1aARs) are present intracellularly and at the cell surface in cultured and natural cell models, where they are subject to agonist-mediated desensitization and internalization. To explore alpha1aAR trafficking, a hemagglutinin (HA)-tagged alpha1aAR/enhanced green fluorescent protein (EGFP) fusion protein was expressed in rat-1 fibroblasts and tracked by EGFP fluorescence and antibody labeling of surface receptors. Confocal analysis of antibody-labeled surface receptors revealed unexpected constitutive internalization in the absence of agonist stimulation. In partial agreement, the inverse agonist prazosin also caused a modest 20 +/- 2% increase in surface receptor levels, suggesting a partial block of constitutive internalization caused by decreased basal activation. However, prazosin was unable to prevent internalization of antibody-tagged surface receptors observed by confocal microscopy or cause obvious redistribution of intracellular receptor to the surface, suggesting that the alpha1aAR is internalizing even in a basal-inactive state. In contrast to the alpha1aAR, surface labeling of an HA-tagged alpha1b-EGFP fusion protein did not result in any apparent constitutive internalization. Constitutive internalization of the alpha1aAR seems to occur alongside reversible agonist-induced internalization, and both seem to involve clathrin-mediated endocytosis but not degradation in lysozymes. Surface receptor density must be maintained by recycling, because the protein synthesis inhibitor cycloheximide has no effect on total or surface receptor density in agonist-treated or untreated cells for 6 h. Constitutive agonist-independent trafficking of alpha1aARs may provide a novel mechanism by which an internal pool of alpha1aARs are maintained and recycled to allow continuous agonist-induced signaling.

Adrenergic receptors (ARs) are members of the G-protein-coupled receptor family, which includes alpha 1ARs, alpha 2ARs, beta 1ARs, beta 2ARs, beta 3ARs, adenosine, muscarinic, angiotensin, endothelin receptors, and many others that are responsible for a large variety of physiologic effects through G-protein coupling. This review focuses on alpha 1ARs and their regulation at both the mRNA and protein levels. Currently, three alpha 1AR subtypes have been characterized both pharmacologically and at the gene level: alpha 1aAR, alpha 1bAR, and alpha 1dAR. These are expressed in a species- and tissue-dependent manner. Mutagenesis approaches have been extremely valuable in the identification of key residues that govern alpha 1AR ligand binding and signaling. These studies reveal that alpha 1ARs have evolved an exquisitely sensitive regulation of their activity in which any disruption of the native structure has profound effects on subsequent function and effector coupling. Significant advances have also been made in the elucidation of signaling pathway components, resulting in the identification of novel pathways that can lead to pathologic conditions. Specific topics include mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and G-protein-coupled receptor cross-talk pathways. Within this context, recent studies identifying underlying transcriptional mechanisms involved in the regulation of the alpha 1AR subtypes are also discussed. Finally, given the potentially important role of alpha 1ARs in the vasculature, as well as in the pathology of many diseases, such as myocardial hypertrophy and benign prostatic hyperplasia, the clinical relevance of alpha 1AR distribution, pharmacology, and therapeutic intervention is reviewed.

Abstract —Cardiac G protein–coupled receptors that couple to Gα s and stimulate cAMP formation (eg, β-adrenergic, histamine, serotonin, and glucagon receptors) play a key role in cardiac inotropy. Recent studies in rodent cardiac myocytes and transfected cells have revealed that one of these receptors, the β 2 -adrenergic receptor (AR), also couples to the inhibitory G protein Gα i (activation of which inhibits cAMP formation). If β 2 ARs could be shown to couple to Gα i in the human heart, it would have important ramifications, because levels of Gα i increase with age and in failing human heart. Therefore, we investigated whether β 2 ARs in the human heart activate Gα i . By photoaffinity labeling human atrial membranes with [ 32 P]azidoanilido-GTP, followed by immunoprecipitation with antibodies specific for Gα i , we found that Gα i is activated by stimulation of β 2 ARs but not of β 1 ARs. In addition, we found that other Gα s -coupled receptors also couple to Gα i , including histamine, serotonin, and glucagon. When coupling of these receptors to Gα i is disrupted by pertussis toxin, their ability to stimulate adenylyl cyclase is enhanced. These data provide the first evidence that β 2 AR and many other Gα s -coupled receptors in human atrium also couple to Gα i and that abolishing the coupling of these receptors to Gα i increases the receptor-mediated adenylyl cyclase activity.

A DRENERGIC receptors (ARs) (classically (Xa, e~2, [31, [32) are plasma membrane-bound proteins involved in many aspects of general physiology and cardiovascular function? ARs are important in physiologic functions, such as myocardial rate and contractility, systemic and pulmonary vascular tone and blood flow, sedation/hypnosis, platelet aggregation, insulin secretion, and gut motility, as well as diseases such as congestive heart failure (CHF), hypertension, pheochromocytoma, benign prostatic hyperplasia, and possibly diabetes. ARs interact with the endogenous catecholamines norepinephrine (NE) and epinephrine (EPI), and several classes of drugs (predominantly adrenergic agonists and antagonists). 2,3 When occupied by agonists, these receptors couple to specific effector molecules via intermediary heterotrimeric guanine-nucleotide regulatory proteins (G proteins) (Fig 1). Specifically, [31-ARs and [32-ARs couple to the stimulatory G protein Gs, which activates the enzyme adenylyl cyclase (AC), resulting in increases in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) (Fig 2). Alpha2-ARs inhibit AC via coupling to the inhibitory G protein, Gi, and also activate a Na+/H + exchanger, resulting in ion fluxes in the cell. Alphal-AR agonist occupancy activates Gq, ultimately activating phospholipase C-13 isozymes, which hydrolyze membrane phosphoinositides into inositol trisphosphate (IP3) and diacyl glycerol (DAG). IP3 binds to IP3 receptors on the sarcoplasmic reticulum, resulting in increases in free cytosolic Ca ++ and ultimately in smooth muscle contraction (Fig 2). In cardiac myocytes, production of cAMP causes activation of protein kinase A (PKA), which phosphorylates calcium channels, causing them to open. Increased intracellular calcium with depolarization binds to the catalytic site on troponin C, resulting in freeing of actin from the actin/ tropomyosin complex; the net result is the ability of actin to form cross-bridges with myosin leading to myocyte contraction.

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied G protein-coupled receptors (GPCRs), resulting in GPCR desensitization. GRK2 is one of the better studied of the six known GRKs and phosphorylates several GPCRs. In a previous study, we documented that GRK2 and GRK3 phosphorylate purified and reconstituted rat substance P receptor (rSPR) [Kwatra et al. (1993) J. Biol. Chem. 268, 9161-9164]. Here, we characterize in detail GRK2-catalyzed phosphorylation of human SPR (hSPR) in intact membranes. GRK2 phosphorylates hSPR in urea-washed Sf9 membranes in an agonist-dependent manner with a stoichiometry of 19 +/- 1 mol of phosphate/mol of receptor, which increases slightly (1.3-fold increase) in the presence of G beta gamma. Kinetic analyses indicate that receptor phosphorylation occurs with a Km of 6.3 +/- 0.4 nM and a Vmax of 1.8 +/- 0.1 nmol/min/mg; these kinetic parameters are only slightly affected by G beta gamma [Km = 3.6 +/- 1.0 nM and Vmax = 2.2 +/- 0.2 nmol/min/mg]. The lack of a strong stimulatory effect of G beta gamma on GRK2-catalyzed phosphorylation of hSPR is surprising since G beta gamma potently stimulates GRK2-catalyzed phosphorylation of beta 2-adrenergic receptor and rhodopsin. Involvement of G beta gamma endogenously present in membranes is ruled out as a source of high levels of hSPR phosphorylation, since receptor phosphorylation was not affected by guanine nucleotides that suppress or enhance the release of endogenous G beta gamma. The present study determines, for the first time, the kinetics of phosphorylation of a receptor substrate of GRK2 in intact membranes. Further, our results identify hSPR as a unique substrate of GRK2 whose phosphorylation is strong even in the absence of G beta gamma.

Regulation of alpha1-adrenergic receptors (alpha1ARs) is a rapidly expanding field of study due to the involvement of these receptors in several human diseases. For example, upregulation of alpha1bAR mRNA/protein with agonist exposure has been implicated in myocardial hypertrophy. In order to understand mechanisms underlying these findings, initial studies of human alpha1bAR transcription have been completed and the primary promoter characterized. This review summarizes current knowledge regarding human alpha1bAR gene transcription and compares characteristics with other adrenergic receptor genes.

Depressed serum thyroid hormone concentrations from either preexisting chronic hypothyroidism or induced by physiologic stress may affect clinical outcome in the perioperative period. This article discusses the various forms of thyroid hormone, the effect of hypothyroidism on patients undergoing surgery, the general role of thyroid hormone supplementation in the perioperative period, and the use of triiodothyronine as a possible inotropic agent in the setting of cardiac surgery. In addition, we review alterations in thyroid hormone associated with brain death and the possible role of thyroid hormone supplementation in the setting of organ harvest procedures.

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non-selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32P or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

alpha 2-Adrenergic receptor (AR) subtype mRNA (alpha 2a, alpha 2b, alpha 2c) neuronal localization in human spinal cord has not been described. We therefore performed in situ hybridization to identify cell bodies at four levels of human spinal cord (cervical, thoracic, lumbar, sacral) containing alpha 2AR subtype specific mRNA. alpha 2AR mRNA is present in gray matter only (ventraldorsal; sacralcervicalthoracic = lumbar). In addition to alpha 2AR mRNA in cell bodies in thoracic and lumbar intermediolateral (sympathetic) and sacral intermediate (parasympathetic) cell columns (lamina VII), all levels in dorsal horn laminae I, II, V, and ventral horn lamina IX, we demonstrate alpha 2AR mRNA in dorsal horn laminae III and IV, and dorsal nucleus of Clarke, where alpha 2ARs have not been described. Previously unreported heterogeneity in alpha 2AR subtype distribution (alpha 2a and alpha 2bAR mRNA present, alpha 2cAR mRNA virtually absent) is found at all sites of alpha 2AR mRNA expression in human spinal cord, including locations known to mediate effects of alpha 2AR agonist drugs on nociception, autonomic function and motor tone. Cervical spinal cord demonstrates a predominance of alpha 2a mRNA signal, while thoracic, lumbar, and sacral spinal cord demonstrate an increasing predominance of alpha 2bAR mRNA. If confirmed at a protein level, these findings have profound implications for therapeutic strategies in managing human pain.

In heart failure, β-adrenergic receptor (βAR) stimulation desensitizes the receptor, uncouples the downstream Gαs protein, and diminishes signal transduction. We tested the hypotheses that haplotype-tagging single-nucleotide polymorphisms (htSNPs) within the Gαs gene (GNAS) (i) are functionally active and alter Gαs expression, (ii) influence survival after coronary artery bypass grafting (CABG), and (iii) interact with βAR SNPs.Amplification of GNAS intron 1 was followed by cloning, reporter assays, electrophoretic mobility shift assays, and western blots. In a pilot study, 185 patients on βAR blockade undergoing CABG were studied prospectively. The primary endpoint was cardiac-related mortality at 1 yr.Two htSNPs defined three common haplotypes with altered reporter activity, allele-specific transcription factor binding, and Gαs protein expression (highest in *3 carriers followed by *2 and *1 haplotypes, P=0.013). After CABG, mortality was GNAS diplotype-dependent: *3/*3: 0%; *3/*2: 2.4%; *3/*1: 2.9%; *2/*2: 4.5%; *2/*1: 9.1%; and *1/*1: 20.0% (P=0.004). While β(1)AR SNPs were not associated with mortality, β(2)AR Arg16 allele carriers were at higher risk than Gly16 allele carriers (P=0.008). Gene-gene interaction using gene-related risk alleles demonstrated the number of risk alleles to be independently associated with death (hazard ratio 2.3; 95% confidence interval: 1.5-3.5; P=0.0003). Carriers of the no-risk allele had higher maximum isoproterenol-stimulated adenylyl cyclase activities than risk allele carriers (P=0.003).Interactions in the βAR/Gαs pathway may be associated with altered mortality after CABG. This could reconcile previously inconclusive data regarding the effects of βAR SNPs on cardiovascular prognosis.

Myocardial β-adrenergic receptors (βARs) are important in altering heart rate, inotropic state, and myocardial relaxation (lusitropy). The β1AR and β2AR stimulation increases cyclic adenosine monophosphate concentration with the net result of myocyte contraction, whereas β3AR stimulation results in decreased inotropy. Downregulation of β1ARs in heart failure, as well as an increased β3AR activity and density, lead to decreased cyclic adenosine monophosphate production and reduced inotropy. The βAR antagonists are commonly used in patients with coronary artery disease and heart failure; however, perioperative use of βAR antagonists is controversial. Individual patient's response to beta-blocker therapy is an area of intensive research, and apart from pharmacokinetics, pharmacodynamics, and ethnic differences, genetic alterations have become more important in the last 20 years. The most common genetic variants in humans are single nucleotide polymorphisms (SNPs). There are 2 clinically relevant SNPs for the β1AR (Ser49Gly, Arg389Gly), 3 for the β2AR (Arg16Gly, Gln27Glu, Thr164Ile), and 1 for the β3AR (Trp64Arg). Although results are somewhat controversial, generally large datasets have the potential to show a relationship between βAR SNPs and outcomes such as development and progression of heart failure, coronary artery disease, vascular reactivity, hypertension, asthma, obesity, and diabetes. Although βAR SNPs may not directly cause disease, they appear to be risk factors for, and modifiers of, disease and the response to stress and drugs. In the perioperative setting, this has specifically been demonstrated for the Arg389Gly β1AR polymorphism with which patients with the Gly variant had a higher incidence of adverse perioperative events. Knowing that genetic variants play an important role, perioperative medicine will likely change from simple therapeutic intervention to a more personalized way of adrenergic receptor modulation.

The dynamic component of bladder outlet obstruction caused by benign prostatic hyperplasia (BPH) is regulated by alpha 1 adrenergic receptors (alpha 1-AR) located in the prostatic stroma. Recently two alpha 1-AR subtypes (alpha 1A, alpha 1B) have been identified in the human prostate by both functional and pharmacological assays. However, the presence of the alpha 1C subtype has not been evaluated, presumably due to the lack of availability of selective ligands for this receptor subtype. We have used molecular techniques to investigate the mRNA expression of all three alpha 1-AR subtypes in the human prostate. RNA extracted from the prostate gland of 15 patients was used in ribonuclease protection assays to identify the expression of three alpha 1-AR subtype mRNAs. Quantitative solution hybridization assays further identify the predominant subtype of alpha 1-AR mRNA to be the alpha 1C, which represents approximately 70% of the total alpha 1-AR mRNA in the human prostate. Furthermore, in situ hybridization localizes the alpha 1C AR mRNA predominantly to the stromal compartment. The identification of a predominant alpha 1-AR mRNA in human prostate identifies a potential need for subtype selective pharmaceutical agents. These agents could be very important clinically in the treatment of diseases such as BPH.

Due to the lack of precise markers indicative of its occurrence and progression, coronary artery disease (CAD), the most common type of heart diseases, is currently associated with high mortality in the United States. To systemically identify novel protein biomarkers associated with CAD progression for early diagnosis and possible therapeutic intervention, we employed an iTRAQ-based quantitative proteomic approach to analyze the proteome changes in the plasma collected from a pair of wild type versus apolipoprotein E knockout (APOE −/−) mice which were fed with a high fat diet. In a multiplex manner ITRAQ serves as the quantitative ‘in-spectra’ marker for ‘cross-sample’ comparisons to determine the differentially expressed/secreted proteins caused by APOE knock-out. To obtain the most comprehensive proteomic datasets from this CAD-associated mouse model we applied both MALDI and ESI-based mass spectrometric (MS) platforms coupled with two different schemes of multidimensional liquid chromatography (2D-LC) separation. We then comparatively analyzed a series of the plasma samples collected at six and twelve weeks after the mice were fed with fat diets, where the 6-week or 12-week time point represents the early or intermediate phase of the fat-induced CAD, respectively. We then categorized those proteins showing abundance changes in accordance with APOE depletion. Several proteins such as the gamma and beta chains of fibrinogen, apolipoprotein B, apolipoprotein C-I, and thrombospondin-4 were among the previously known CAD markers identified by other methods. Our results suggested that these unbiased proteomic methods are both feasible and a practical means of discovering potential biomarkers associated with CAD progression.

Background— Human myocardial metabolism has been incompletely characterized in the setting of surgical cardioplegic arrest and ischemia/reperfusion. Furthermore, the effect of preexisting ventricular state on ischemia-induced metabolic derangements has not been established. Methods and Results— We applied a mass spectrometry–based platform to profile 63 intermediary metabolites in serial paired peripheral arterial and coronary sinus blood effluents obtained from 37 patients undergoing cardiac surgery, stratified by presence of coronary artery disease and left ventricular dysfunction. The myocardium was a net user of a number of fuel substrates before ischemia, with significant differences between patients with and without coronary artery disease. After reperfusion, significantly lower extraction ratios of most substrates were found, as well as significant release of 2 specific acylcarnitine species, acetylcarnitine and 3-hydroxybutyryl-carnitine. These changes were especially evident in patients with impaired ventricular function, who exhibited profound limitations in extraction of all forms of metabolic fuels. Principal component analysis highlighted several metabolic groupings as potentially important in the postoperative clinical course. Conclusions— The preexisting ventricular state is associated with significant differences in myocardial fuel uptake at baseline and after ischemia/reperfusion. The dysfunctional ventricle is characterized by global suppression of metabolic fuel uptake and limited myocardial metabolic reserve and flexibility after global ischemia/reperfusion stress in the setting of cardiac surgery. Altered metabolic profiles after ischemia/reperfusion are associated with postoperative hemodynamic course and suggest a role for perioperative metabolic monitoring and targeted optimization in cardiac surgical patients.

Over the last decades, tremendous advances have been made in understanding the genomic basis of a large number of chronic human diseases. Such advances were made possible by studying large numbers of patients over relatively long periods of time. It is increasingly recognized that acute robust stress stimulates pathways activated in chronic disease, facilitating mechanistic studies in much shorter time frames. A new field of molecular medicine, called perioperative genomics, uses robust surgical stress as a perturbation designed to explore such mechanisms. This new field is described briefly below.

OBJECTIVE To assess genetic variability in two serotonin-related gene polymorphisms (MAOA-uVNTR and 5HTTLPR) and their relationships to depression and adverse cardiac events in a sample of patients undergoing coronary artery bypass surgery. METHODS A total of 427 coronary artery bypass graft (CABG) patients were genotyped for two polymorphisms and assessed for depressive symptoms at three time points, in accordance with the Center for Epidemiological Studies-Depression (CES-D): preoperative baseline; 6 months postoperative; and 1 year postoperative. Logistic regression was used to assess the association between depressive symptoms (CES-D = >16), genotype differences, and cardiac events. Because MAOA-uVNTR is sex-linked, males and females were analyzed separately for this polymorphism; sexes were combined for the 5HTTLPR analysis. RESULTS Depressed patients were more likely than nondepressed patients to have a new cardiac event within 2 years of surgery (p < .0001); depressed patients who carry the long (L) allele of the 5HTTLPR polymorphism were more likely than the short/short (S/S carriers to have an event (p = .0002). Genetic associations with 6-month and 1-year postoperative depressive symptoms do not survive adjustment for baseline depressive symptoms. CONCLUSIONS A serotonin-related gene polymorphism--5HTTLPR--was associated with adverse cardiac events post CABG, in combination with depressive symptoms. Because depressed patients with the L allele of the 5HTTLPR polymorphism were more likely to have an event compared with the S/S carriers, combining genetic and psychiatric profiling may prove useful in identifying patients at the highest risk for adverse outcomes post CABG.

Benign prostatic hyperplasia (BPH) is a common cause of urinary outflow obstruction in aging men leading to lower urinary tract symptoms (LUTS). alpha(1)-Adrenoceptors (alpha(1)ARs) antagonists (blockers) have become a mainstay of LUTS treatment because they relax prostate smooth muscle and decrease urethral resistance, as well as relieving bladder LUTS symptoms. A review of key recent clinical trials suggests new insights into the role of specific alpha(1)AR subtypes in the treatment of LUTS.

Alpha-adrenergic receptor (AR) agonist drugs (e.g., epinephrine) are commonly used for upper airway procedures, to shrink the mucosa, retard absorption of local anesthetic agents, and improve visualization by limiting hemorrhage. Decongestant therapy often also includes alphaAR agonist agents, however overuse of these drugs (e.g., oxymetazoline) can result in chronic rhinitis and rebound increases in nasal secretion. Since current decongestants stimulate alphaARs non-selectively, characterization of alphaAR subtype distribution in human airway (nasal turbinate) offers an opportunity to refine therapeutic targets while minimizing side-effects. We, therefore, investigated alphaAR subtype expression in human nasal turbinate within epithelial, duct, gland, and vessel cells using in situ hybridization.Since sensitive and specific anti-receptor antibodies and highly selective alphaAR subtype ligands are currently unavailable, in situ hybridization was performed on sections of three human nasal turbinate samples to identify distribution of alphaAR subtype mRNA. Subtype specific (35)S-labelled mRNA probes were incubated with nasal turbinate sections, and protected fragments remaining after RNase treatment analyzed by light and darkfield microscopy.In non-vascular tissue alpha(1d) AR mRNA predominates, whereas notably the alpha(2c) is the only alphaAR subtype present in the sinusoids and arteriovenous anastamoses.Combined with the current understanding that AR-mediated constriction of nasal sinusoids underpins decongestant therapies that minimize secretions and shrink tissues for airway procedures, these findings suggest that alpha(2c) AR subtypes provide a novel selective target for decongestant therapy in humans.

α1a Adrenergic receptors (α1aARs) are the predominant AR subtype in human vascular smooth muscle cells (SMCs). α1aARs in resistance vessels are crucial in the control of blood pressure, yet the impact of naturally occurring human α1aAR genetic variants in cardiovascular disorders remains poorly understood. To this end, we present novel findings demonstrating that 3D cultures of vascular SMCs expressing human α1aAR-247R (247R) genetic variant demonstrate significantly increased SMC contractility compared with cells expressing the α1aAR-WT (WT) receptor. Stable expression of 247R genetic variant also triggers MMP/EGFR-transactivation dependent serum- and agonist-independent (constitutive) hyperproliferation and agonist-dependent hypertrophy of SMCs. Agonist stimulation reduces contractility Using pathway-specific inhibitors we determined that the observed hyperproliferation of 247R-expressing cells is triggered via β-arrestin1/Src/MMP-2/EGFR/ERK-dependent mechanism. MMP-2-specific siRNA inhibited 247R-triggered hyperproliferation indicating MMP-2 involvement in 247R-triggered hyperproliferation in SMCs. β-arrestin1-specific shRNA also inhibited 247R-triggered hyperproliferation but did not affect hypertrophy in 247R-expressing SMCs, indicating that agonist-dependent hypertrophy is independent of β-arrestin1. Our data reveal that in different cardiovascular cells the same human receptor genetic variant can activate alternative modulators of the same signaling pathway. Thus, our findings in SMCs demonstrate that depending on the type of cells expressing the same receptor (or receptor variant), different target-specific inhibitors could be used to modulate aberrant hyperproliferative or hypertrophic pathways in order to restore normal phenotype.

Understanding the role of adrenergic receptors in iris biology has gained widespread interest due to the recently described intraoperative floppy iris syndrome sometimes encountered during cataract surgery. alpha(1)AR-mediated iris dilator smooth muscle contraction occurs via alpha(1a)ARs whereas alpha(1b)ARs mediate iris arteriolar contraction. Because alpha(1)AR antagonists are first-line therapy for benign prostatic hyperplasia and lower urinary tract symptoms, more elderly patients requiring cataract surgery now receive these drugs. After reviewing intraoperative floppy iris syndrome, strengths/weaknesses of supporting data, and reviewing iris biology, a case is made that rather than being drug specific (alpha(1)AR antagonists), intraoperative floppy iris syndrome may represent the "tip of the iceberg." Relaxed iris dilator muscle resistant to adrenergic agonists should be expected with clinical drugs shown to relax the iris dilator (e.g., antagonists at alpha(1)AR, endothelin-A, angiotensin receptors, nitric oxide donors such as nitrates), and/or diseases associated with endothelial dysregulation (e.g., congestive heart failure, diabetes, hypertension). Rather than a rare, unexpected, unpredictable syndrome due to one drug, a careful medical history should elucidate intraoperative floppy iris syndrome predisposition. Just as anticoagulants are discontinued prior to elective surgery, conservative management of elderly patients suggests discontinuation of drugs that relax iris dilator muscle, in consultation with the patient's primary physician, should be considered prior to cataract surgery.

Background— The inflammatory response triggered by cardiac surgery with cardiopulmonary bypass (CPB) is a primary mechanism in the pathogenesis of postoperative myocardial infarction (PMI), a multifactorial disorder with significant inter-patient variability poorly predicted by clinical and procedural factors. We tested the hypothesis that candidate gene polymorphisms in inflammatory pathways contribute to risk of PMI after cardiac surgery. Methods and Results— We genotyped 48 polymorphisms from 23 candidate genes in a prospective cohort of 434 patients undergoing elective cardiac surgery with CPB. PMI was defined as creatine kinase-MB isoenzyme level ≥10× upper limit of normal at 24 hours postoperatively. A 2-step analysis strategy was used: marker selection, followed by model building. To minimize false-positive associations, we adjusted for multiple testing by permutation analysis, Bonferroni correction, and controlling the false discovery rate; 52 patients (12%) experienced PMI. After adjusting for multiple comparisons and clinical risk factors, 3 polymorphisms were found to be independent predictors of PMI (adjusted P IL6 −572G>C; odds ratio [OR], 2.47), and 2 adhesion molecules: intercellular adhesion molecule-1 ( ICAM1 Lys469Glu; OR, 1.88), and E-selectin ( SELE 98G>T; OR, 0.16). The inclusion of genotypic information from these polymorphisms improved prediction models for PMI based on traditional risk factors alone (C-statistic 0.764 versus 0.703). Conclusions— Functional genetic variants in cytokine and leukocyte–endothelial interaction pathways are independently associated with severity of myonecrosis after cardiac surgery. This may aid in preoperative identification of high-risk cardiac surgical patients and development of novel cardioprotective strategies.

To test for an association between apolipoprotein E (APOE) genotypes and the occurrence of severe sepsis in an elective surgical cohort.Prospective, observational, single cohort study.Sixteen-bed surgical intensive care unit (ICU) at a university hospital.Patients were 343 patients with planned admission to the ICU after major elective noncardiac surgery.Blood samples, together with demographic data, baseline clinical data, and Acute Physiology and Chronic Health Evaluation II scores, were collected on admission to the ICU and on each subsequent ICU day. APOE genotyping was conducted using a polymerase chain reaction-based assay. The primary outcome was diagnosis of severe sepsis; secondary outcomes included time on mechanical ventilation, ICU length of stay, and ICU mortality.Severe sepsis was diagnosed in 34 of 343 patients (9.9%). Carriers of the APOepsilon3 allele (one or two copies) had a lower incidence of severe sepsis than patients with no APOepsilon3 allele (p = .014), with a relative risk of 0.284 (95% confidence interval 0.127-0.635). The protective effect of APOepsilon3 genotype on the incidence of severe sepsis remained significant (p.01) after adjusting for age, gender, or race in a logistic regression model. Supporting our findings, presence of the APOepsilon3 allele was also associated with fewer days spent in the ICU (p = .007). In contrast, APOE genotypes were not associated with duration of mechanical ventilation or ICU mortality.In an elective surgical cohort, presence of the APOepsilon3 allele is associated with decreased incidence of severe sepsis and a shorter ICU length of stay.

An immortalized human prostate stromal cell line (PS30) was previously established using recombinant retrovirus encoding human papillomavirus 16 gene products. In this study, we further characterize this stromal cell line for its potential use in a stromal-epithelial coculture model for prostate cancer prevention. Using reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and immunocytochemistry, we examined expression of androgen receptor (AR), vitamin D receptor (VDR), prostate-specific antigen (PSA), transforming growth factor-beta (TGF-beta), and insulin-like growth factors (IGF) families and their receptors, metalloproteinases (MMP) MMP-2 and MMP-9, as well as the cells' ability to respond to the synthetic androgen R1881. The PS30 stromal cells do not express PSA, confirming their stromal origin. They are positive for both AR messenger ribonucleic acid (mRNA) and protein; however, they do not respond to growth stimulation by the synthetic androgen R1881. The PS30 cells express mRNA for VDR, TGF-betas, IGFs and their receptors, as well as the MMPs. Moreover, they produce significant amounts of TGF-beta1, TGF-beta2, IGFBP-3, and MMP-2 proteins. Our observations confirm the use of PS30 for the study of stromal-epithelial interactions in the modulation of prostate carcinogenesis.

Background and Purpose— Stroke represents a significant cause of morbidity and mortality after cardiac surgery. Although the risk of stroke varies according to both patient and procedural factors, the impact of genetic variants on stroke risk is not well understood. Therefore, we tested the hypothesis that specific genetic polymorphisms are associated with an increased risk of stroke after cardiac surgery. Methods— Patients undergoing cardiac surgery utilizing cardiopulmonary bypass surgery were studied. DNA was isolated from preoperative blood and analyzed for 26 different single-nucleotide polymorphisms. Multivariable logistic regression modeling was used to determine the association of clinical and genetic characteristics with stroke. Permutation analysis was used to adjust for multiple comparisons inherent in genetic association studies. Results— A total of 1635 patients experiencing 28 strokes (1.7%) were included in the final genetic model. The combination of the 2 minor alleles of C-reactive protein (CRP; 3′UTR 1846C/T) and interleukin-6 (IL-6; −174G/C) polymorphisms, occurring in 583 (35.7%) patients, was significantly associated with stroke (odds ratio, 3.3; 95% CI, 1.4 to 8.1; P =0.0023). In a multivariable logistic model adjusting for age, the CRP and IL-6 single-nucleotide polymorphism combination remained significantly associated with stroke ( P =0.0020). Conclusions— We demonstrate that common genetic variants of CRP (3′UTR 1846C/T) and IL-6 (−174G/C) are significantly associated with the risk of stroke after cardiac surgery, suggesting a pivotal role of inflammation in post–cardiac surgery stroke.

Considerable progress has been made in understanding the pathophysiology of perioperative stress responses and their impact on the cardiovascular system; however, researchers are just beginning to unravel genetic and molecular determinants that predispose to increased risk for postoperative cardiovascular adverse events. A new field, coined perioperative genomics, aims to apply functional genomic approaches to uncover the biological reasons why similar patients can have dramatically different clinical outcomes after surgery. For the perioperative physician, such findings may soon translate into prospective risk assessment incorporating genomic profiling of markers important in inflammatory, thrombotic, vascular, and neurologic responses to perioperative stress, with implications ranging from individualized additional pre-operative testing and physiological optimization, to perioperative decision-making, choice of monitoring strategies, and critical care resource utilization. We review current knowledge regarding genomic technologies in perioperative cardiovascular disease characterization and outcome prediction, as well as discuss future trends/challenges for translating integrated "omic" information into daily clinical management of the surgical patient.

Summary. Background: Postoperative bleeding remains a common, serious problem for cardiac surgery patients, with striking inter-patient variability poorly explained by clinical, procedural, and biological markers. Objective: We tested the hypothesis that genetic polymorphisms of coagulation proteins and platelet glycoproteins are associated with bleeding after cardiac surgery. Patients/methods: Seven hundred and eighty patients undergoing aortocoronary surgery with cardiopulmonary bypass were studied. Clinical covariates previously associated with bleeding were recorded and DNA isolated from preoperative blood. Matrix Assisted Laser Desorption/Ionization, Time-Of-Flight (MALDI-TOF) mass spectroscopy or polymerase chain reaction were used for genotype analysis. Multivariable linear regression modeling, including all genetic main effects and two-way gene-gene interactions, related clinical and genetic predictors to bleeding from the thorax and mediastinum. Results: Nineteen candidate polymorphisms were assessed; seven [GPIaIIa−52C>T and 807C>T, GPIbα 524C>T, tissue factor−603A>G, prothrombin 20210G>A, tissue factor pathway inhibitor−399C>T, and angiotensin converting enzyme (ACE) deletion/insertion] demonstrate significant association with bleeding (P

Advances in medicine and clinical care often begin with astute observations by clinicians. Such is the case with the recently described intraoperative floppy iris syndrome (IFIS). This syndrome, occurring rarely during cataract surgery, was identified by Chang and Campbell in May 2005. Intraoperative floppy iris syndrome is characterized by iris billowing and undulation during phacoemulsification cataract extraction, iris prolapse, and progressive intraoperative miosis, all conditions that potentially increase the risk of intraoperative complications. Although IFIS was initially associated by Chang and Campbell with the 1-adrenergic receptor ( 1AR) antagonist tamsulosin, 1 more recent reports suggest that it may be associated with all 4 commercially available 1AR antagonists (alfuzosin, doxazosin, tamsulosin, and terazosin). Because 1AR antagonists are increasingly prescribed to elderly patients for treatment of lower urinary tract symptoms (most commonly as nonsurgical therapy for benign prostatic hyperplasia, but sometimes to women as well), IFIS may be encountered more frequently by ophthalmologists. Although the overall incidence of complications was low in the Chang and Campbell study, the presence of this syndrome can make cataract surgery more difficult. One of the most critical elements of safe cataract surgery is adequate pupil dilation. In IFIS, the mechanical pupil stretching of the miotic pupil is ineffective as it snaps back to its original size. Iris hooks or expansion rings before capsulorrhexis may be helpful in these cases; therefore, it would be helpful to predict who would be at risk preoperatively. But what are the underlying mechanisms responsible for IFIS? 1-Adrenergic receptor agonists such as phenylephrine are commonly used as agents to induce mydriasis. Because 3 1AR subtypes exist ( 1a, 1b, and 1d), 3 understanding the physiologic role of 1AR subtypes in iris biology has gained increasing interest over the last decade. Recent studies at molecular, protein, and physiologic levels have demonstrated that 1AR-mediated iris dilator smooth muscle contraction occurs via the 1aAR subtype. In contrast, 1bARs mediate iris arteriolar contraction, and 2aARs are present on iris presynaptic nerve terminals. Because 1aAR and 1dARs are key targets in treating lower urinary tract symptoms, and because these drugs reach all tissues in the body, it would be expected that 1AR antagonists would inhibit iris dilator smooth muscle contraction, producing relaxation and miosis.

Background: Post-cardiac surgery renal dysfunction is a common, serious, multifactorial disorder, with interpatient variability predicted poorly by preoperative clinical, procedural, and biological markers. Therefore, we tested the hypothesis that selected gene variants are associated with acute renal injury, reflected by a serum creatinine level increase after cardiac surgery. Methods: One thousand six hundred seventy-one patients undergoing aortocoronary surgery were studied. Clinical covariates were recorded. DNA was isolated from preoperative blood; mass spectrometry was used for genotype analysis. A model was developed relating clinical and genetic factors to postoperative acute renal injury. Results: A race effect was found; therefore, Caucasians and African Americans were analyzed separately. Overall, clinical factors alone account poorly for postoperative renal injury, although more so in African Americans than Caucasians. When 12 candidate polymorphisms were assessed, 2 alleles (interleukin 6 −572C and angiotensinogen 842C) showed a strong association with renal injury in Caucasians ( P 50% decrease in renal filtration when they present together). Using less stringent criteria for significance (0.01 > P > 0.001), 4 additional polymorphisms are identified (apolipoproteinE 448C [ϵ4], angiotensin receptor1 1166C, and endothelial nitric oxide synthase [eNOS] 894T in Caucasians; eNOS 894T and angiotensin-converting enzyme deletion and insertion in African Americans). Adding genetic to clinical factors resulted in the best model, with overall ability to explain renal injury increasing approximately 4-fold in Caucasians and doubling in African Americans ( P Conclusion: In this study, we identify genetic polymorphisms that collectively provide 2- to 4-fold improvement over preoperative clinical factors alone in explaining post-cardiac surgery renal dysfunction. From a mechanistic perspective, most identified genetic variants are associated with increased renal inflammatory and/or vasoconstrictor responses.

Alpha1-adrenoceptors are G-protein-coupled receptors that bind catecholamines. Sixteen distinct human alpha1A-adrenoceptor isoforms have been identified from human tissues, including five full-length and 11 truncated versions. An updated scheme for the identification of alpha1A-adrenoceptor splice variants is proposed. Given the established roles of alpha1-adrenoceptors in benign prostatic hyperplasia, myocardial hypertrophy and other cardiovascular disorders, elucidation of the biological significance of the signaling diversity and potential pharmacological roles of alpha1A-adrenoceptor splice variants are important areas of future research.

Recent data suggest novel functional roles for cerebellar involvement in a number of neurologic diseases. Function of cerebellar neurons is known to be modulated by norepinephrine and adrenergic receptors. The distribution of adrenergic receptor subtypes has been described in experimental animals, but corroboration of such studies in the human cerebellum, necessary for drug treatment, is still lacking. In the present work we studied cell-specific localizations of alpha1 adrenergic receptor subtype mRNA (alpha 1a, alpha 1b, alpha 1d), and alpha2 adrenergic receptor subtype mRNA (alpha 2a, alpha 2b, alpha 2c) by in situ hybridization on cryostat sections of human cerebellum (cortical layers and dentate nucleus). We observed unique neuron-specific alpha1 adrenergic receptor and alpha2 adrenergic receptor subtype distribution in human cerebellum. The cerebellar cortex expresses mRNA encoding all six alpha adrenergic receptor subtypes, whereas dentate nucleus neurons express all subtype mRNAs, except alpha 2a adrenergic receptor mRNA. All Purkinje cells label strongly for alpha 2a and alpha 2b adrenergic receptor mRNA. Additionally, Purkinje cells of the anterior lobe vermis (lobules I to V) and uvula/tonsil (lobules IX/HIX) express alpha 1a and alpha 2c subtypes, and Purkinje cells in the ansiform lobule (lobule HVII) and uvula/tonsil express alpha 1b and alpha 2c adrenergic receptor subtypes. Basket cells show a strong signal for alpha 1a, moderate signal for alpha 2a and light label for alpha 2b adrenergic receptor mRNA. In stellate cells, besides a strong label of alpha 2a adrenergic receptor mRNA in all and moderate label of alpha 2b message in select stellate cells, the inner stellate cells are also moderately positive for alpha 1b adrenergic receptor mRNA. Granule and Golgi cells express high levels of alpha 2a and alpha 2b adrenergic receptor mRNAs. These data contribute new information regarding specific location of adrenergic receptor subtypes in human cerebellar neurons. We discuss our observations in terms of possible modulatory roles of adrenergic receptor subtypes in cerebellar neurons responding to sensory and autonomic input signals, and review species differences in cerebellar adrenergic receptor expression.

We provide a comprehensive overview of the role of alpha1-adrenergic receptors (alpha1ARs) as critical mediators of lower urinary tract symptoms (LUTS) and pathophysiology in benign prostatic hyperplasia (BPH), and we review the pharmacological antagonists of alpha1ARs.A review was performed of pertinent studies in the literature relating to the pathophysiology of LUTS and BPH, focusing on the role of alpha1ARs, and of clinical trial and practice data evaluating the different agents that inhibit these receptors.Further characterization of the alpha1AR gene family indicates that 3 receptor subtypes exist in humans. Their different distribution between urinary tract and cardiovascular tissues has provided a strategy for the development of improved therapeutic agents. Since excessive activity of the alpha1aAR and alpha1dAR subtypes appears to be a common feature in symptomatic BPH and alpha1aARs are enriched in prostatic tissue, drugs that demonstrate high alpha1aAR selectivity have attracted attention. Tamsulosin, which has high affinity for alpha1aAR and alpha1dAR subtypes but not for alpha1bAR, shows efficacy similar to the nonsubtype selective agents terazosin and doxazosin. It is associated with fewer cardiovascular side effects, although it has some ejaculatory side effects. The nonsubtype selective agent alfuzosin also demonstrates efficacy and offers an enhanced side effect profile, particularly minimizing hypotension. Other agents with super selective specificity for the alpha1aAR subtype are under investigation.Further advances in the treatment of LUTS associated with BPH may depend not only on receptor subtype selectivity, but also on other pharmacokinetic and pharmacodynamic factors.

Background Cardiopulmonary bypass (CPB) has been implicated in the development of organ injury associated with cardiac surgery. At the molecular level, CPB is accompanied by a pronounced proinflammatory response including an increase in plasma interleukin (IL)-6. The IL-6 has been shown to be increased in rheumatoid arthritis, a chronic inflammatory disease, where it has been implicated in decreasing G protein-coupled receptor kinases (GRKs) in peripheral blood mononuclear cells. Since IL-6 is substantially increased after CPB, the study tested whether the increase of IL-6 during CPB leads to a decrease of GRKs in mononuclear cells. This is important because GRKs regulate the function of G protein-coupled receptors involved in inflammation. Methods Fifteen patients had blood withdrawn before CPB, 2 h after CPB, and on postoperative day one (POD1). Plasma IL-6 concentrations were determined by enzyme-linked immunosorbent assay. The GRK protein expression and activity were determined by Western blot and phosphorylation of rhodopsin using [gamma-(32)P] adenosine triphosphate, respectively. Results Plasma IL-6 increased over 20-fold after CPB and remained increased on POD1. Cytosolic GRK activity in mononuclear cells decreased by 39 +/- 29%; cytosolic GRK2 and membrane-bound GRK6 decreased by 90 +/- 15 and 65 +/- 43%, respectively. The GRK activity and expression of GRK2/GRK6 on POD1 returned to basal levels in many but not all patients. Conclusions The CPB causes a profound decrease in mononuclear cell GRKs, and the recovery of these kinases on POD1 is quite variable. The significance of the variable recovery of GRKs after CPB and their potential role as a marker of clinical outcome deserves further investigation.