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The role of catecholamine receptors in cardiac energy metabolism is unknown. α1-adrenergic receptors (α1-ARs) have been identified to play a role in whole body metabolism but its role in cardiac en...

α1-Adrenergic receptors (ARs) are members of the G-Protein Coupled Receptor superfamily and with other related receptors (β and α2), they are involved in regulating the sympathetic nervous system through binding and activation by norepinephrine and epinephrine. Traditionally, α1-AR antagonists were first used as anti-hypertensives, as α1-AR activation increases vasoconstriction, but they are not a first-line use at present. The current usage of α1-AR antagonists increases urinary flow in benign prostatic hyperplasia. α1-AR agonists are used in septic shock, but the increased blood pressure response limits use for other conditions. However, with the advent of genetic-based animal models of the subtypes, drug design of highly selective ligands, scientists have discovered potentially newer uses for both agonists and antagonists of the α1-AR. In this review, we highlight newer treatment potential for α1A-AR agonists (heart failure, ischemia, and Alzheimer’s disease) and non-selective α1-AR antagonists (COVID-19/SARS, Parkinson’s disease, and posttraumatic stress disorder). While the studies reviewed here are still preclinical in cell lines and rodent disease models or have undergone initial clinical trials, potential therapeutics discussed here should not be used for non-approved conditions.

Despite standard therapies, heart failure patients have high rates of morbidity highlighting the need to develop alternative therapeutic approaches. Heart failure has been described as an energy-starved condition that is hypothesized to drive the pathological remodeling of the heart. Numerous studies have described the metabolic defects that occur when the heart fails and adaptive changes that take place to maintain the energy needed for the heart to function properly. In this review we will summarize the metabolic requirements of a normal heart and what happens during failure. We will also summarize the various metabolic therapeutic strategies that have been developed over the years to treat heart failure and their results from clinical trials.

The nomenclature of the Adrenoceptors has been agreed by the NC-IUPHAR Subcommittee on Adrenoceptors [60, 186]. Adrenoceptors, α1 The three α1-adrenoceptor subtypes α1A, α1B and α1D are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. -(-)phenylephrine, methoxamine and cirazoline are agonists and prazosin and doxazosin antagonists considered selective for α1- relative to α2-adrenoceptors. [3H]prazosin and [125I]HEAT (BE2254) are relatively selective radioligands. S(+)-niguldipine also has high affinity for L-type Ca2+ channels. Fluorescent derivatives of prazosin (Bodipy FLprazosin- QAPB) are used to examine cellular localisation of α1-adrenoceptors. α1-Adrenoceptor agonists are used as nasal decongestants; antagonists to treat symptoms of benign prostatic hyperplasia (alfuzosin, doxazosin, terazosin, tamsulosin and silodosin, with the last two compounds being α1A-adrenoceptor selective and claiming to relax bladder neck tone with less hypotension); and to a lesser extent hypertension (doxazosin, terazosin). The α1- and β2-adrenoceptor antagonist carvedilol is used to treat congestive heart failure, although the contribution of α1-adrenoceptor blockade to the therapeutic effect is unclear. Several anti-depressants and anti-psychotic drugs are α1-adrenoceptor antagonists contributing to side effects such as orthostatic hypotension. Adrenoceptors, α2 The three α2-adrenoceptor subtypes α2A, α2B and α2C are activated by (-)-adrenaline and with lower potency by (-)-noradrenaline. brimonidine and talipexole are agonists and rauwolscine and yohimbine antagonists selective for α2- relative to α1-adrenoceptors. [3H]rauwolscine, [3H]brimonidine and [3H]RX821002 are relatively selective radioligands. There are species variations in the pharmacology of the α2A-adrenoceptor. Multiple mutations of α2-adrenoceptors have been described, some associated with alterations in function. Presynaptic α2-adrenoceptors regulate many functions in the nervous system. The α2-adrenoceptor agonists clonidine, guanabenz and brimonidine affect central baroreflex control (hypotension and bradycardia), induce hypnotic effects and analgesia, and modulate seizure activity and platelet aggregation. clonidine is an anti-hypertensive (relatively little used) and counteracts opioid withdrawal. dexmedetomidine (also xylazine) is increasingly used as a sedative and analgesic in human [31] and veterinary medicine and has sympatholytic and anxiolytic properties. The α2-adrenoceptor antagonist mirtazapine is used as an anti-depressant. The α2B subtype appears to be involved in neurotransmission in the spinal cord and α2C in regulating catecholamine release from adrenal chromaffin cells. Although subtype-selective antagonists have been developed, none are used clinically and they remain experimental tools. Adrenoceptors, β The three β-adrenoceptor subtypes β1, β2 and β3 are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. Isoprenaline is selective for β-adrenoceptors relative to α1- and α2-adrenoceptors, while propranolol (pKi 8.2-9.2) and cyanopindolol (pKi 10.0-11.0) are relatively selective antagonists for β1- and β2- relative to β3-adrenoceptors. (-)-noradrenaline, xamoterol and (-)-Ro 363 show selectivity for β1- relative to β2-adrenoceptors. Pharmacological differences exist between human and mouse β3-adrenoceptors, and the 'rodent selective' agonists BRL 37344 and CL316243 have low efficacy at the human β3-adrenoceptor whereas CGP 12177 (low potency) and L 755507 activate human β3-adrenoceptors [88]. β3-Adrenoceptors are resistant to blockade by propranolol, but can be blocked by high concentrations of bupranolol. SR59230A has reasonably high affinity at β3-adrenoceptors, but does not discriminate between the three β- subtypes [320] whereas L-748337 is more selective. [125I]-cyanopindolol, [125I]-hydroxy benzylpindolol and [3H]-alprenolol are high affinity radioligands that label β1- and β2- adrenoceptors and β3-adrenoceptors can be labelled with higher concentrations (nM) of [125I]-cyanopindolol together with β1- and β2-adrenoceptor antagonists. Fluorescent ligands such as BODIPY-TMR-CGP12177 can be used to track β-adrenoceptors at the cellular level [8]. Somewhat selective β1-adrenoceptor agonists (denopamine, dobutamine) are used short term to treat cardiogenic shock but, chronically, reduce survival. β1-Adrenoceptor-preferring antagonists are used to treat cardiac arrhythmias (atenolol, bisoprolol, esmolol) and cardiac failure (metoprolol, nebivolol) but also in combination with other treatments to treat hypertension (atenolol, betaxolol, bisoprolol, metoprolol and nebivolol) [507]. Cardiac failure is also treated with carvedilol that blocks β1- and β2-adrenoceptors, as well as α1-adrenoceptors. Short (salbutamol, terbutaline) and long (formoterol, salmeterol) acting β2-adrenoceptor-selective agonists are powerful bronchodilators used to treat respiratory disorders. Many first generation β-adrenoceptor antagonists (propranolol) block both β1- and β2-adrenoceptors and there are no β2-adrenoceptor-selective antagonists used therapeutically. The β3-adrenoceptor agonist mirabegron is used to control overactive bladder syndrome. There is evidence to suggest that β-adrenoceptor antagonists can reduce metastasis in certain types of cancer [189].

The role of catecholamine receptors in cardiac energy metabolism is unknown. α(1)-adrenergic receptors (α(1)-ARs) have been identified to play a role in whole body metabolism but its role in cardiac energy metabolism has not been explored. We used freshly prepared primary adult mouse cardiomyocytes and incubated with either (14)C-palmitate or (14)C-glucose tracers to measure oxidation rates in the presence or absence of phenylephrine, an α(1)-AR agonist (with β and α(2)-AR blockers) under normal cell culture conditions. (14)CO(2) released was collected over a 10 min period in covered tissue culture plates using a 1M hyamine hydroxide solution placed in well cups, counted by scintillation and converted into nmoles/hr. We found that phenylephrine stimulated glucose oxidation but not fatty acid oxidation in adult primary cardiomyocytes. α(1)-AR stimulated glucose oxidation was blocked by the AMPK inhibitor, dorsomorphin dihydrochloride, and the PKC inhibitor, rottlerin. Ischemic conditions were induced by lowering the glucose concentration from 22.5mM to 1.375 mM. Under ischemic conditions, we found that phenylephrine also increased glucose oxidation. We report a direct role of α(1)-ARs in regulating glucose oxidation under normal and ischemic conditions that may lead to new therapeutic approaches in treating ischemia.

α1-adrenergic receptors are G-Protein Coupled Receptors that are involved in neurotransmission and regulate the sympathetic nervous system through binding and activating the neurotransmitter, norepinephrine, and the neurohormone, epinephrine. There are three α1-adrenergic receptor subtypes (α1A, α1B, α1D) that are known to play various roles in neurotransmission and cognition. They are related to two other adrenergic receptor families that also bind norepinephrine and epinephrine, the β- and α2-, each with three subtypes (β1, β2, β3, α2A, α2B, α2C). Previous studies assessing the roles of α1-adrenergic receptors in neurotransmission and cognition have been inconsistent. This was due to the use of poorly-selective ligands and many of these studies were published before the characterization of the cloned receptor subtypes and the subsequent development of animal models. With the availability of more-selective ligands and the development of animal models, a clearer picture of their role in cognition and neurotransmission can be assessed. In this review, we highlight the significant role that the α1-adrenergic receptor plays in regulating synaptic efficacy, both short and long-term synaptic plasticity, and its regulation of different types of memory. We will also present evidence that the α1-adrenergic receptors, and particularly the α1A-adrenergic receptor subtype, are a potentially good target to treat a wide variety of neurological conditions with diminished cognition.

The nomenclature of the Adrenoceptors has been agreed by the NC-IUPHAR Subcommittee on Adrenoceptors [58], see also [180]. Adrenoceptors, α1α1-Adrenoceptors are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. phenylephrine, methoxamine and cirazoline are agonists and prazosin and cirazoline antagonists considered selective for α1- relative to α2-adrenoceptors. [3H]prazosin and [125I]HEAT (BE2254) are relatively selective radioligands. S(+)-niguldipine also has high affinity for L-type Ca2+ channels. Fluorescent derivatives of prazosin (Bodipy PLprazosin- QAPB) are used to examine cellular localisation of α1-adrenoceptors. Selective α1-adrenoceptor agonists are used as nasal decongestants; antagonists to treat hypertension (doxazosin, prazosin) and benign prostatic hyperplasia (alfuzosin, tamsulosin). The α1- and β2-adrenoceptor antagonist carvedilol is used to treat congestive heart failure, although the contribution of α1-adrenoceptor blockade to the therapeutic effect is unclear. Several anti-depressants and anti-psychotic drugs are α1-adrenoceptor antagonists contributing to side effects such as orthostatic hypotension and extrapyramidal effects.Adrenoceptors, α2 α2-Adrenoceptors are activated by (-)-adrenaline and with lower potency by (-)-noradrenaline. brimonidine and talipexole are agonists and rauwolscine and yohimbine antagonists selective for α2- relative to α1-adrenoceptors. [3H]rauwolscine, [3H]brimonidine and [3H]RX821002 are relatively selective radioligands. There is species variation in the pharmacology of the α2A-adrenoceptor. Multiple mutations of α2-adrenoceptors have been described, some associated with alterations in function. Presynaptic α2-adrenoceptors regulate many functions in the nervous system. The α2-adrenoceptor agonists clonidine, guanabenz and brimonidine affect central baroreflex control (hypotension and bradycardia), induce hypnotic effects and analgesia, and modulate seizure activity and platelet aggregation. clonidine is an anti-hypertensive and counteracts opioid withdrawal. dexmedetomidine (also xylazine) is used as a sedative and analgesic in human and veterinary medicine with sympatholytic and anxiolytic properties. The α2-adrenoceptor antagonist yohimbine has been used to treat erectile dysfunction and mirtazapine as an anti-depressant. The α2B subtype appears to be involved in neurotransmission in the spinal cord and α2C in regulating catecholamine release from adrenal chromaffin cells.Adrenoceptors, ββ-Adrenoceptors are activated by the endogenous agonists (-)-adrenaline and (-)-noradrenaline. Isoprenaline is selective for β-adrenoceptors relative to α1- and α2-adrenoceptors, while propranolol (pKi 8.2-9.2) and cyanopindolol (pKi 10.0-11.0) are relatively β1 and β2 adrenoceptor-selective antagonists. (-)-noradrenaline, xamoterol and (-)-Ro 363 show selectivity for β1- relative to β2-adrenoceptors. Pharmacological differences exist between human and mouse β3-adrenoceptors, and the 'rodent selective' agonists BRL 37344 and CL316243 have low efficacy at the human β3-adrenoceptor whereas CGP 12177 and L 755507 activate human β3-adrenoceptors [88]. β3-Adrenoceptors are resistant to blockade by propranolol, but can be blocked by high concentrations of bupranolol. SR59230A has reasonably high affinity at β3-adrenoceptors, but does not discriminate well between the three β- subtypes whereas L 755507 is more selective. [125I]-cyanopindolol, [125I]-hydroxy benzylpindolol and [3H]-alprenolol are high affinity radioligands that label β1- and β2- adrenoceptors and β3-adrenoceptors can be labelled with higher concentrations (nM) of [125I]-cyanopindolol together with β1- and β2-adrenoceptor antagonists. [3H]-L-748337 is a β3-selective radioligand [474]. Fluorescent ligands such as BODIPY-TMR-CGP12177 can be used to track β-adrenoceptors at the cellular level [8]. Somewhat selective β1-adrenoceptor agonists (denopamine, dobutamine) are used short term to treat cardiogenic shock but, chronically, reduce survival. β1-Adrenoceptor-preferring antagonists are used to treat hypertension (atenolol, betaxolol, bisoprolol, metoprolol and nebivolol), cardiac arrhythmias (atenolol, bisoprolol, esmolol) and cardiac failure (metoprolol, nebivolol). Cardiac failure is also treated with carvedilol that blocks β1- and β2-adrenoceptors, as well as α1-adrenoceptors. Short (salbutamol, terbutaline) and long (formoterol, salmeterol) acting β2-adrenoceptor-selective agonists are powerful bronchodilators used to treat respiratory disorders. Many first generation β-adrenoceptor antagonists (propranolol) block both β1- and β2-adrenoceptors and there are no β2-adrenoceptor-selective antagonists used therapeutically. The β3-adrenoceptor agonist mirabegron is used to control overactive bladder syndrome.

The heart has a reduced capacity to generate sufficient energy when failing, resulting in an energy-starved condition with diminished functions. Studies have identified numerous changes in metabolic pathways in the failing heart that result in reduced oxidation of both glucose and fatty acid substrates, defects in mitochondrial functions and oxidative phosphorylation, and inefficient substrate utilization for the ATP that is produced. Recent early-phase clinical studies indicate that inhibitors of fatty acid oxidation and antioxidants that target the mitochondria may improve heart function during failure by increasing compensatory glucose oxidation. Adrenergic receptors (α1 and β) are a key sympathetic nervous system regulator that controls cardiac function. β-AR blockers are an established treatment for heart failure and α1A-AR agonists have potential therapeutic benefit. Besides regulating inotropy and chronotropy, α1- and β-adrenergic receptors also regulate metabolic functions in the heart that underlie many cardiac benefits. This review will highlight recent studies that describe how adrenergic receptor-mediated metabolic pathways may be able to restore cardiac energetics to non-failing levels that may offer promising therapeutic strategies.

The role of α1-adrenergic receptors (α1-ARs) and their subtypes in metabolism is not well known. Most previous studies were performed before the advent of transgenic mouse models and utilized transformed cell lines and poorly selective antagonists. We have now studied the metabolic regulation of the α1A- and α1B-AR subtypes in vivo using knock-out (KO) and transgenic mice that express a constitutively active mutant (CAM) form of the receptor, assessing subtype-selective functions. CAM mice increased glucose tolerance while KO mice display impaired glucose tolerance. CAM mice increased while KO decreased glucose uptake into white fat tissue and skeletal muscle with the CAM α1A-AR showing selective glucose uptake into the heart. Using indirect calorimetry, both CAM mice demonstrated increased whole body fatty acid oxidation, while KO mice preferentially oxidized carbohydrate. CAM α1A-AR mice displayed significantly decreased fasting plasma triglycerides and glucose levels while α1A-AR KO displayed increased levels of triglycerides and glucose. Both CAM mice displayed increased plasma levels of leptin while KO mice decreased leptin levels. Most metabolic effects were more efficacious with the α1A-AR subtype. Our results suggest that stimulation of α1-ARs results in a favorable metabolic profile of increased glucose tolerance, cardiac glucose uptake, leptin secretion and increased whole body lipid metabolism that may contribute to its previously recognized cardioprotective and neuroprotective benefits.

The role of α₁-adrenergic receptors (ARs) in the regulation of cardiac hypertrophy is still unclear, because transgenic mice demonstrated hypertrophy or the lack of it despite high receptor overexpression. To further address the role of the α₁-ARs in cardiac hypertrophy, we analyzed unique transgenic mice that overexpress constitutively active mutation (CAM) α₁A-ARs or CAM α₁B-ARs under the regulation of large fragments of their native promoters. These constitutively active receptors are expressed in all tissues that endogenously express their wild-type counterparts as opposed to only myocyte-targeted transgenic mice. In this study, we discovered that CAM α₁A-AR mice in vivo have cardiac hypertrophy independent of changes in blood pressure, corroborating earlier studies, but in contrast to myocyte-targeted α₁A-AR mice. We also found cardiac hypertrophy in CAM α₁B-AR mice, in agreement with previous studies, but hypertrophy only developed in older mice. We also discovered unique α₁-AR-mediated hypertrophic signaling that was AR subtype-specific with CAM α₁A-AR mice secreting atrial naturietic factor and interleukin-6 (IL-6), whereas CAM α₁B-AR mice expressed activated nuclear factor-κB (NF-κB). These particular hypertrophic signals were blocked when the other AR subtype was coactivated. We also discovered that crossbreeding the two CAM models (double CAM α₁A/B-AR) inhibited the development of hypertrophy and was reversible with single receptor activation, suggesting that coactivation of the receptors can lead to novel antagonistic signal transduction. This was confirmed by demonstrating antagonistic signals that were even lower than normal controls in the double CAM α₁A/B-AR mice for p38, NF-κB, and the IL-6/glycoprotein 130/signal transducer and activator of transcription 3 pathway. Because α₁A/B double knockout mice fail to develop hypertrophy in response to IL-6, our results suggest that IL-6 is a major mediator of α₁A-AR cardiac hypertrophy.

It is well established that the gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, however, less is known about the reasons behind such global differences. MicroRNAs (miRNAs) are short non-coding RNAs that can target multiple molecules to regulate wide array of proteins in diverse pathways. The goal of the study was to profile alterations in miRNA expression using end-stage human heart failure samples with an aim to build signaling network pathways using predicted targets for the altered miRNA and to determine nodal molecules regulating individual networks. Profiling of miRNAs using custom designed microarray and validation with an independent set of samples identified eight miRNAs that are altered in human heart failure including one novel miRNA yet to be implicated in cardiac pathology. To gain an unbiased perspective on global regulation by top eight altered miRNAs, functional relationship of predicted targets for these eight miRNAs were examined by network analysis. Ingenuity Pathways Analysis network algorithm was used to build global signaling networks based on the targets of altered miRNAs which allowed us to identify participating networks and nodal molecules that could contribute to cardiac pathophysiology. Majority of the nodal molecules identified in our analysis are targets of altered miRNAs and known regulators of cardiovascular signaling. Cardio-genomics heart failure gene expression public data base was used to analyze trends in expression pattern for target nodal molecules and indeed changes in expression of nodal molecules inversely correlated to miRNA alterations. We have used NF kappa B network as an example to show that targeting other molecules in the network could alter the nodal NF kappa B despite not being a miRNA target suggesting an integrated network response. Thus, using network analysis we show that altering key functional target proteins may regulate expression of the myriad signaling pathways underlying the cardiac pathology.

It is well established that gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, but the reasons for such differences are not clear. MicroRNAs (miRNAs) are short noncoding RNAs that provide a novel mechanism for gene regulation. The goal of this study was to comprehensively test for alterations in miRNA expression using human heart failure samples with an aim to build signaling pathway networks using predicted targets for the miRNAs and to identify nodal molecules that control these networks. Genome-wide profiling of miRNAs was performed using custom-designed miRNA microarray followed by validation on an independent set of samples. Eight miRNAs are significantly altered in heart failure of which we have identified two novel miRNAs that are yet to be implicated in cardiac pathophysiology. To gain an unbiased global perspective on regulation by altered miRNAs, predicted targets of eight miRNAs were analyzed using the Ingenuity Pathways Analysis network algorithm to build signaling networks and identify nodal molecules. The majority of nodal molecules identified in our analysis are targets of altered miRNAs and are known regulators of cardiovascular signaling. A heart failure gene expression data base was used to analyze changes in expression patterns for these target nodal molecules. Indeed, expression of nodal molecules was altered in heart failure and inversely correlated to miRNA changes validating our analysis. Importantly, using network analysis we have identified a limited number of key functional targets that may regulate expression of the myriad proteins in heart failure and could be potential therapeutic targets.

While α(1)-adrenergic receptors (ARs) have been previously shown to limit ischemic cardiac damage, the mechanisms remain unclear. Most previous studies utilized low oxygen conditions in addition to ischemic buffers with glucose deficiencies, but we discovered profound differences if the two conditions are separated. We assessed both mouse neonatal and adult myocytes and HL-1 cells in a series of assays assessing ischemic damage under hypoxic or low glucose conditions. We found that α(1)-AR stimulation protected against increased lactate dehydrogenase release or Annexin V(+) apoptosis under conditions that were due to low glucose concentration not to hypoxia. The α(1)-AR antagonist prazosin or nonselective protein kinase C (PKC) inhibitors blocked the protective effect. α(1)-AR stimulation increased (3)H-deoxyglucose uptake that was blocked with either an inhibitor to glucose transporter 1 or 4 (GLUT1 or GLUT4) or small interfering RNA (siRNA) against PKCδ. GLUT1/4 inhibition also blocked α(1)-AR-mediated protection from apoptosis. The PKC inhibitor rottlerin or siRNA against PKCδ blocked α(1)-AR stimulated GLUT1 or GLUT4 plasma membrane translocation. α(1)-AR stimulation increased plasma membrane concentration of either GLUT1 or GLUT4 in a time-dependent fashion. Transgenic mice overexpressing the α(1A)-AR but not α(1B)-AR mice displayed increased glucose uptake and increased GLUT1 and GLUT4 plasma membrane translocation in the adult heart while α(1A)-AR but not α(1B)-AR knockout mice displayed lowered glucose uptake and GLUT translocation. Our results suggest that α(1)-AR activation is anti-apoptotic and protective during cardiac ischemia due to glucose deprivation and not hypoxia by enhancing glucose uptake into the heart via PKCδ-mediated GLUT translocation that may be specific to the α(1A)-AR subtype.

The importance of adult neurogenesis has only recently been accepted, resulting in a completely new field of investigation within stem cell biology. The regulation and functional significance of adult neurogenesis is currently an area of highly active research. G-protein-coupled receptors (GPCRs) have emerged as potential modulators of adult neurogenesis. GPCRs represent a class of proteins with significant clinical importance, because approximately 30% of all modern therapeutic treatments target these receptors. GPCRs bind to a large class of neurotransmitters and neuromodulators such as norepinephrine, dopamine, and serotonin. Besides their typical role in cellular communication, GPCRs are expressed on adult neural stem cells and their progenitors that relay specific signals to regulate the neurogenic process. This review summarizes the field of adult neurogenesis and its methods and specifies the roles of various GPCRs and their signal transduction pathways that are involved in the regulation of adult neural stem cells and their progenitors. Current evidence supporting adult neurogenesis as a model for self-repair in neuropathologic conditions, adult neural stem cell therapeutic strategies, and potential avenues for GPCR-based therapeutics are also discussed.

Previous studies demonstrated α₁-adrenergic receptors (ARs) increase STAT3 activation in transfected and non-cardiac primary cell lines. However, the mechanism used by α₁-ARs resulting in STAT3 activation is unknown. While other G-protein-coupled receptors (GPCRs) can couple to STAT3, these mechanisms demonstrate coupling through SRC, TYK, Rac, or complex formation with Gq and used only transfected cell lines. Using normal and transgenic mice containing constitutively active mutations (CAM) of the α(1A)-AR subtype, neonatal mouse myocytes and whole hearts were analyzed for the mechanism to couple to STAT3 activation. α₁-ARs stimulated time-dependent increases in p-SRC, p-JAK2, and p-STAT3 in normal neonatal myocytes. Using various kinase inhibitors and siRNA, we determined that the α(1A)-AR coupled to STAT3 through distinct and unique pathways in neonatal myocytes. We found that PKCϵ inhibition decreased p-ERK and p-Ser STAT3 levels without affecting p-Tyr STAT3. In contrast, we found that PKCδ inhibition affected p-SRC and p-JAK2 resulting in decreased p-Tyr and p-Ser STAT3 levels. We suggest a novel α(1A)-AR mediated PKCϵ/ERK pathway that regulates the phosphorylation status of STAT3 at Ser-727 while PKCδ couples to SRC/JAK2 to affect Tyr-705 phosphorylation. Furthermore, this pathway has not been previously described in a GPCR system that couples to STAT3. Given cell survival and protective cardiac effects induced by PKC, STAT3 and ERK signaling, our results could explain the neuroprotective and cardiac protective pathways that are enhanced with α(1A)-AR agonism.

alpha(1)-Adrenergic receptors (ARs) are not well defined in the central nervous system. The particular cell types and areas that express these receptors are uncertain because of the lack of high avidity antibodies and selective ligands. We have developed transgenic mice that either systemically overexpress the human alpha(1A)-AR subtype fused with the enhanced green fluorescent protein (EGFP) or express the EGFP protein alone under the control of the mouse alpha(1A)-AR promoter. We confirm our transgenic model against the alpha(1A)-AR knockout mouse, which expresses the LacZ gene in place of the coding region for the alpha(1A)-AR. By using these models, we have now determined cellular localization of the alpha(1A)-AR in the brain, at the protein level. The alpha(1A)-AR or the EGFP protein is expressed prominently in neuronal cells in the cerebral cortex, hippocampus, hypothalamus, midbrain, pontine olivary nuclei, trigeminal nuclei, cerebellum, and spinal cord. The types of neurons were diverse, and the alpha(1A)-AR colocalized with markers for glutamic acid decarboxylase (GAD), gamma-aminobutyric acid (GABA), and N-methyl-D-aspartate (NMDA) receptors. Recordings from alpha(1A)-AR EGFP-expressing cells in the stratum oriens of the hippocampal CA1 region confirmed that these cells were interneurons. We could not detect expression of the alpha(1A)-AR in mature astrocytes, oligodendrocytes, or cerebral blood vessels, but we could detect the alpha(1A)-AR in oligodendrocyte progenitors. We conclude that the alpha(1A)-AR is abundant in the brain, expressed in various types of neurons, and may regulate the function of oligodendrocyte progenitors, interneurons, GABA, and NMDA receptor containing neurons.

Adrenergic receptors (ARs) play an important role in the regulation of cardiac function. Cardiac inotropy is primarily regulated by beta(1)-ARs. However, alpha(1)-ARs may play an important role in inotropy during heart failure. Previous work has suggested that the alpha(1B)-AR modulates beta(1)-AR function in the heart. The potential role of the alpha(1A)-AR has not been previously studied. We used transgenic mice that express constitutively active mutant (CAM) forms of the alpha(1A)-AR or alpha(1B)-AR regulated by their endogenous promoters. Expression of the CAM alpha(1A)-AR or CAM alpha(1B)-AR had no effect on basal cardiac function (developed pressure, +dP/dT, -dP/dT, heart rate, flow rate). However, both alpha(1)-AR subtypes significantly decreased isoproterenol-stimulated +dP/dT. Pertussis toxin had no effect on +dP/dT in CAM alpha(1A)-AR hearts but restored +dP/dT to non-transgenic values in CAM alpha(1B)-AR hearts. Radioligand binding indicated a selective decrease in the density of beta(1)-ARs in both CAM mice. However, G-proteins, cAMP, or the percentage of high and low affinity states were unchanged in either transgenic compared with control. These data demonstrate that CAM alpha(1A)- and alpha(1B)-ARs both down regulate beta(1)-AR-mediated inotropy in the mouse heart. However, alpha(1)-AR subtypes are coupled to different beta-AR mediated signaling pathways with the alpha(1B)-AR being pertussis toxin sensitive.

alpha1-Adrenergic receptors (ARs) are well-known mediators of the sympathetic nervous system, are highly abundant in the brain, but are the least understood in the central nervous system. The particular cell types in the brain that contain these receptors or their functions are not known because of the lack of high avidity antibodies and selective ligands. We developed transgenic mice that endogenously overexpress the alpha1B-AR subtype fused with the enhanced green fluorescent protein (EGFP). Endogenous expression was obtained by using a 3.4 kb fragment of the mouse alpha1B-AR promoter. Using this model, we determined cellular localization of the alpha1B-AR throughout the brain. The alpha1B-AR-EGFP fusion protein is expressed in neurons throughout the brain and in the Purkinje cells of the cerebellum. The alpha1B-AR is also expressed in NG2 oligodendrocyte precursor cells in both neonatal cell cultures and in the adult cerebral cortex, but is weakly expressed in mature oligodendrocytes. The alpha1B-AR was not observed in astrocytes or in cerebral vascular smooth muscle, cell types previously suggested to contain alpha1-ARs. We conclude that the alpha1B-AR is highly abundant throughout the brain, predominately in neurons, and may be involved in the development of the oligodendrocyte. In adult NG2 cells, implicated in stem cell-like functions, the alpha1B-AR may also play a role. This is the first report of a transgenic tagged-GPCR approach to determine in vivo localization of a receptor.

alpha(1)-Adrenergic receptors (ARs) are known mediators of a positive inotropy in the heart, which may play even more important roles in heart disease. Due to a lack of sufficiently selective ligands, the contribution of each of the three alpha(1)-AR subtypes (alpha(1A), alpha(1B) and alpha(1D)) to cardiac function is not clearly defined. In this study, we used a systemically expressing mouse model that overexpresses the alpha(1B)-AR to define the role of this subtype in cardiac function.We used the mouse Langendorff heart model to assess changes in contractility under basal and phenylephrine-induced conditions.We find that a 50% increase of the alpha(1B)-AR in the heart does not change basal cardiac parameters compared to age-matched normals (heart rate, +/-dP/dT and coronary flow). However, the inotropic response to phenylephrine is blunted. The same results were obtained in isolated adult myocytes. The difference in inotropy could be blocked by the selective alpha(1A)-AR antagonist, 5-methylurapidil, which correlated with decreases in alpha(1A)-AR density, suggesting that the alpha(1B)-AR had caused a compensatory downregulation of the alpha(1A)-AR.These results suggest that the alpha(1B)-AR does not have a major role in the positive inotropic response in the mouse myocardium but may negatively modulate the response of the alpha(1A)-AR.

The alpha1-adrenergic receptors (alpha1ARs) play an important role in mediating sympathetic neurotransmission in peripheral organ systems; however, central alpha1ARs are not well characterized. Additionally, due to the lack of sufficiently subtype-selective drugs or high avidity antibodies, the contribution of each alpha1AR subtype to various central functions is currently unclear. Transcription regulation through alpha1AR subtypes in the CNS is also unknown. Of interest, transgenic mice that systemically overexpress the alpha1BAR show central symptoms that include age-progressive impaired mobility, neurodegeneration and susceptibility to epileptic seizure. To investigate the molecular basis of this phenotype, oligonucleotide microarray studies of whole brains of various ages were carried out to compare gene expression profiles between transgenic and normal brains. The results indicated changes in expression of apoptotic, calcium regulatory, neurodegenerative and genes involved in neurotransmission. Defects in regulation of intracellular calcium are known to play a role in cell death; thus, these genes may provide clues as to the molecular basis of alpha1BAR-induced neurodegeneration. Epilepsy is a disorder that can be caused by an imbalance between excitatory (e.g. glutamate) and inhibitory (e.g. GABA) signals. Microarray analysis of transgenic brains showed increased N-methyl-d-aspartate (NMDA) receptors and decreased GABAA, which were confirmed with immunohistochemistry, western blot and radioligand binding studies. The alpha1BAR also co-localized with the glutamatergic distribution, suggesting a glutamate imbalance as a molecular rationale for the epileptic seizures.

Among all of the known proteins in the human genome, the G-protein-coupled receptors (GPCRs) may be the most versatile. They know how to manipulate the evolutionary forces that govern diversity by copying one ancestral gene and turning it into thousands of different proteins that can transduce light

Objective: Cardiac hypertrophy is closely associated with the development of cardiomyopathies that lead to heart failure. The α1B adrenergic receptor (α1-AR) is an important regulator of the hypertrophic process. Cardiac hypertrophy induced by systemic overexpression of the α1b-AR in a mouse model does not progress to heart failure. We wanted to explore potential gene expression differences that characterize this type of hypertrophy that may identify genes that prevent progression to heart failure. Methods: Transgenic and normal mice (B6CBA) representing two time points were compared; one at 2–3 months of age before disease manifests and the other at 12 months when the hypertrophy is significant. Age-matched hearts were removed, cRNA prepared and biotinylated. Aliquots of the cRNA was subjected to hybridization with Affymetrix chips representing 12 656 murine genes. Gene expression profiles were compared with normal age-matched controls as the baseline and confirmed by Northern and Western analysis. Results: The non-EST genes could be grouped into five functional classifications: embryonic, proliferative, inflammatory, cardiac-related, and apoptotic. Growth response genes involved primarily Src-related receptors and signaling pathways. Transgenic hearts also had a 60% higher Src protein content. There was an inflammatory response that was verified by an increase in IgG and κ-chained immunoglobulins by western analysis. Apoptosis may be regulated by cell cycle arrest through a p53-dependent mechanism. Cardiac gene expression was decreased for common hypertrophy-inducing proteins such as actin, collagen and GP130 pathways. Conclusions: Our results suggest a profile of gene expression in a case of atypical cardiac hypertrophy that does not progress to heart failure. Since many of these altered gene expressions have not been linked to heart failure models, our findings may provide a novel insight into the particular role that the α1BAR plays in its overall progression or regression.

alpha(1)-Adrenergic receptors (alpha(1A), alpha(1B), and alpha(1D)) are regulators of systemic arterial blood pressure and blood flow. Whereas vasoconstrictory action of the alpha(1A) and alpha(1D) subtypes is thought to be mainly responsible for this activity, the role of the alpha(1B)-adrenergic receptor (alpha(1B)AR) in this process is controversial. We have generated transgenic mice that overexpress either wild type or constitutively active alpha(1B)ARs. Transgenic expression was under the control of the isogenic promoter, thus assuring appropriate developmental and tissue-specific expression. Cardiovascular phenotypes displayed by transgenic mice included myocardial hypertrophy and hypotension. Indicative of cardiac hypertrophy, transgenic mice displayed an increased heart to body weight ratio, which was confirmed by the echocardiographic finding of an increased thickness of the interventricular septum and posterior wall. Functional deficits included an increased isovolumetric relaxation time, a decreased heart rate, and cardiac output. Transgenic mice were hypotensive and exhibited a decreased pressor response. Vasoconstrictory regulation by alpha(1B)AR was absent as shown by the lack of phenylephrine-induced contractile differences between ex vivo mesenteric artery preparations. Plasma epinephrine, norepinephrine, and cortisol levels were also reduced in transgenic mice, suggesting a loss of sympathetic nerve activity. Reduced catecholamine levels together with basal hypotension, bradycardia, reproductive problems, and weight loss suggest autonomic failure, a phenotype that is consistent with the multiple system atrophy-like neurodegeneration that has been reported previously in these mice. These results also suggest that this receptor subtype is not involved in the classic vasoconstrictory action of alpha(1)ARs that is important in systemic regulation of blood pressure.

Although agonist binding in adrenergic receptors is fairly well understood and involves residues located in transmembrane domains 3 through 6, there are few residues reported that are involved in antagonist binding. In fact, a major docking site for antagonists has never been reported in any G-protein coupled receptor. It has been speculated that antagonist binding is quite diverse depending upon the chemical structure of the antagonist, which can be quite different from agonists. We now report the identification of two phenylalanine residues in transmembrane domain 7 of the α1a-adrenergic receptor (Phe-312 and Phe-308) that are a major site of antagonist affinity. Mutation of either Phe-308 or Phe-312 resulted in significant losses of affinity (4–1200-fold) for the antagonists prazosin, WB4101, BMY7378, (+) niguldipine, and 5-methylurapidil, with no changes in affinity for phenethylamine-type agonists such as epinephrine, methoxamine, or phenylephrine. Interestingly, both residues are involved in the binding of all imidazoline-type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than phenethylamine-type agonists and may be more antagonist-like, which may explain their partial agonist properties. In modeling these interactions with previous mutagenesis studies and using the current backbone structure of rhodopsin, we conclude that antagonist binding is docked higher in the pocket closer to the extracellular surface than agonist binding and appears skewed toward transmembrane domain 7.

The regulation of the cellular distribution and intracellular signaling properties of the alpha(1B)- and alpha(1D)- adrenoceptor (alpha(1)-AR) subtypes was examined in stably transfected Rat 1 fibroblasts. In unstimulated cells, alpha(1B)-AR expression was noted primarily on the cell surface. Treatment with phenylephrine induced internalization of the alpha(1B)-AR and promoted association with arrestin 2. The internalized alpha(1B)-AR colocalized with the transferrin receptor, an endosomal marker. In unstimulated fibroblasts, the alpha(1D)-AR was detected in a perinuclear orientation and was colocalized with arrestin 2 in a compartment also containing the transferrin receptor. After treatment with prazosin, which exhibits inverse agonist properties, the alpha(1D)-AR was redistributed from intracellular sites to the cellular periphery and was no longer associated with the transferrin receptor or arrestin 2. alpha(1D)-AR-expressing cells exhibited a high degree of basal activity for both inositol phosphate formation and extracellular signal regulated kinase (ERK), which was reduced by treatment with prazosin. In these cells, phenylephrine induced a dose-dependent increase in inositol phosphate formation but had no effect on ERK activity. In alpha(1B) -AR-expressing cells, phenylephrine stimulated both inositol phosphate formation and ERK activity. These data show that: 1) there are differences in the cellular localization of the alpha(1)-AR subtypes; 2) the alpha(1B)-AR exhibits expected G protein-coupled receptor activity regarding cellular localization, agonist-mediated internalization, and coupling to second messengers; and 3) the alpha(1D)-AR is constitutively active and, as a result, is localized to intracellular compartments involved in receptor recycling.

Agonist-dependent activation of the α1-adrenergic receptor is postulated to be initiated by disruption of an interhelical salt-bridge constraint between an aspartic acid (Asp-125) and a lysine residue (Lys-331) in transmembrane domains three and seven, respectively. Single point mutations that disrupt the charges of either of these residues results in constitutive activity. To validate this hypothesis, we used site-directed mutagenesis to switch the position of these amino acids to observe, if possible, regeneration of the salt-bridge reverses that the constitutive activity of the single point mutations. The transiently expressed switch mutant receptor displayed an altered pharmacological profile. The affinity of selective α1b-adrenergic receptor antagonists for the switch mutant (D125K/K331D) was no different from the wild-type α1b-adrenergic receptor, suggesting that both receptors are maintaining similar tertiary structures in the cell membrane. However, there was a significant 4–6-fold decrease in the affinity of protonated amine receptor agonists and a 3–6-fold increase in the affinity of carboxylated catechol derivatives for the switch mutant compared with the wild-type α1b-adrenergic receptor. This pharmacology is consistent with a reversed charge at position 125 in transmembrane domain three. Interestingly, the ability of either a negatively or positively charged agonist to generate soluble inositol phosphates was similar for both types of receptors. Finally, the switch mutant (D125K/K331D) displayed similar basal signaling activity as the wild-type receptor, reversing the constitutive activity of the single point mutations (D125K and K331D). This suggests an ionic constraint has been reformed in the switch mutant analogous to the restraint previously described for the wild-type α1b-adrenergic receptor. These results strongly establish the disruption of an electrostatic interaction as an initial step in the agonist-dependent activation of α1-adrenergic receptors.

Chimeric receptors of the beta2-adrenergic receptor in which the extracellular loops were replaced with the corresponding amino acids of the alpha1a-adrenergic receptor were generated to measure changes in alpha1-antagonist affinity. Although no changes in alpha1-antagonist affinity were measured in the beta2/alpha1a chimeras, a decreased IC50 (10-fold) for agonists as compared with wild type beta2 control was found because of the replacement of the third extracellular loop (EX3). These agonist high affinity changes were because of a greater proportion of high affinity sites (2-fold) that were convertible to low affinity sites with guanosine 5'-3-O-(thio)triphosphate. Adenylate cyclase activity evoked by the EX3 chimera showed commensurate increases in the basal signal transduction as well as the isoproterenol-stimulated potency, suggesting constitutive activity. However, unlike other constitutively active adrenergic receptor mutants in which the mutation causes G protein-independent changes, the mechanism of the EX3 chimera seems to be attributable to a greater ease with which the active ternary complex is formed because of a higher affinity/coupling of the G protein. Although the changes because of EX3 are indirect and most likely affect helical packing, they support an emerging hypothesis that G protein-coupled receptors have evolved their structure-function relationships to constrain the receptor in an inactive state.

A cysteine-to-phenylalanine mutation of residue 116 in the third transmembrane domain of the beta2-adrenergic receptor caused selective constitutive activation of Na+/H+ exchange through a pathway not involving cAMP. This selectivity was identified by comparing binding and signaling characteristics of wild-type (WT) versus C116F mutant receptors transiently transfected into COS-1 cells. Indicating constitutive activity, ligand binding to the C116F mutant showed a 78-fold higher than WT affinity for isoproterenol and a 40-fold lower than WT affinity for ICI 118551. Although agonist-independent activation of cAMP production was not exhibited by the C116F mutant, a constitutive stimulation of the Na+/H+ exchanger (NHE1) was observed. This was identified by measuring either basal intracellular pH (pHi) or rate of pHi recovery from cellular acid load. Due to a higher rate of H+ efflux through NHE1, C116F transfectants exhibited a significantly higher pHi (7.42) than did WT transfectants (7.1). Furthermore, the rate of pHi recovery from acid load facilitated by NHE1 was 2.1-fold faster in mutant transfectants than in WT transfectants. The lower rate seen in the WT case was stimulated by epinephrine, and the higher rate seen in the mutant case was inhibited by ICI 118551. These findings, which show that a C116F mutation of the beta2-adrenergic receptor evokes selective constitutive coupling to NHE1 over cAMP, form the basis of our prediction that multiple and distinct activation states can exist in G protein-coupled receptors.

The α1-adrenergic receptor (α1AR) subtypes, α1AAR and α1BAR, have differential effects in the heart and central nervous system. Long-term stimulation of the α1AAR subtype prolongs lifespan and provides cardio- and neuro-protective effects. We examined the lifespan of constitutively active mutant (CAM)-α1BAR mice and the incidence of cancer in mice expressing the CAM form of either the α1AAR (CAM-α1AAR mice) or α1BAR. CAM-α1BAR mice have a significantly shortened lifespan when compared with wild-type (WT) animals; however, the effect was sex dependent. Female CAM-α1BAR mice lived significantly shorter lives, while the median lifespan of male CAM-α1BAR mice was not different when compared with that of WT animals. There was no difference in the incidence of cancer in either sex of CAM-α1BAR mice. The incidence of cancer was significantly decreased in CAM-α1AAR mice when compared with that in WT, and no sex-dependent effects were observed. Further study is warranted on cancer incidence after activation of each α1AR subtype and the effect of sex on lifespan following activation of the α1BAR. The implications of a decrease in cancer incidence following long-term α1AAR stimulation could lead to improved treatments for cancer.

Recently a number of mutations have been found in vitro which maintain alpha 1-adrenergic receptors (ARs) in a partially activated form. We have previously identified two amino acid residue positions in the alpha 1b-adrenergic receptor (AR), Cys128 and Ala204, one in each of the third and fifth transmembrane segments, that constitutively activate the receptor when substituted for a phenylalanine or valine, respectively [Perez et al. (1996) Mol. Pharmacol. 49, 112-122; Hwa et al. (1996) J. Biol. Chem. 271, 7956-7964]. Another mutation analyzed previously, Ala293Glu, located in the third intracellular loop, also constitutively activates the receptor [Kjelsborg et al. (1992) J. Biol. Chem. 267, 1430-1433]. All three mutations displayed similar manifestations of constitutive activity such as higher binding affinity and potency for agonists as well as higher basal signal transduction as predicted by the revised ternary complex model of receptor activation. We hypothesized that the individual mutations because of their critical location alter the conformation of the transmembrane helices such that mimicry occur that partially conforms to the activated state, R*. To explore whether these potential conformations are independent, we combined these three mutations in all possible permutations. The combined triple mutation displays 700-fold higher binding affinity for (-)-epinephrine and 20-fold higher basal IP3 release than the wild-type receptor. We also observed that each mutation contributed independently and synergistically to both receptor agonist binding and activation with the combined mutations basal activity exceeding that of the fully-stimulated wild-type receptor. There was also a direct correlation between epinephrine's binding affinity and the degree of constitutive activity. Because the mutations affect different transmembrane domains, these results are consistent with a mechanism that helical movement acts in a concerted fashion in agonist-induced activation, a synergism predicted if multiple helix movement is involved in receptor activation.

Therapeutics to treat human heart failure and the identification of proteins associated with heart failure are still limited. We analyzed α1-adrenergic receptor subtypes in human heart failure and performed proteomic analysis on more uniform samples to identify novel proteins associated with human heart failure. Six failing hearts with end-stage dilated cardiomyopathy and four non-failing heart controls were subjected to proteomic analysis. Out of 48 identified proteins, 26 proteins were redundant between samples. 10 of these 26 proteins were previously reported to be associated with heart failure. Of the newly identified proteins, we found several muscle proteins and mitochondrial/electron transport proteins, while novel were functionally similar to previous reports. However, we also found novel proteins involved in functional classes such as β-oxidation and G-protein coupled receptor signaling and desensitization not previously associated with heart failure. We also performed radioligand-binding studies on the heart samples and confirmed a large loss of β1-adrenergic receptors in end-stage dilated cardiomyopathy but also found a selective decrease in the α1A-adrenergic receptor subtype not previously reported. We have identified new proteins and functional categories associated with end-stage dilated cardiomyopathy. We also report that similar to the previously characterized loss of β1-adrenergic receptors in heart failure, there is also a concomitant loss of α1A-adrenergic receptors, which are considered cardioprotective proteins.

The α1ARs are important mediators of sympathetic nervous system responses, particularly those involved in cardiovascular homeostasis, such as arteriolar smooth muscle constriction and cardiac contraction.1 2 In addition, α1ARs have more recently been implicated in the pathogenesis of cardiac hypertrophy, in ischemia-induced cardiac arrhythmias, and in ischemic preconditioning.1 3 Like other ARs, α1ARs are activated by the catecholamines, norepinephrine and epinephrine. They are intrinsic membrane glycoproteins and are members of the GPCR superfamily. Over the past 10 to 15 years, data initially based on functional, radioligand, and biochemical studies have accumulated, indicating that the α1ARs are a heterogeneous group of distinct but related proteins. This conclusion has been confirmed with the molecular cloning of three distinct α1-receptor subtypes, although until recently discrepancies between the properties of the cloned expressed receptors and those characterized pharmacologically and biochemically have led to confusion in the classification of α1-receptor subtypes and their coupled effector responses. As detailed in the present review, much of this confusion has now been clarified for the three cloned α1ARs. These and other recent insights into the molecular structure, function, and signaling of α1ARs, the control of α1AR-gene expression, and pharmacological evidence for additional α1AR subtypes will be reviewed here. For additional information, the reader is also referred to several previous reviews of α1ARs.4 5 6 7 Functional studies of AR responses, particularly from the laboratories of McGrath8 and Ruffolo,9 provided the initial evidence that there may be subtypes of α1ARs. These studies indicated that postjunctional responses mediated by α1ARs could not be explained adequately on the basis of a single population of receptors. This concept was further advanced …

We have identified previously two amino acids, one in each of the fifth and sixth transmembrane segments of both the alpha1a-adrenergic receptor and the alpha1b-adrenergic receptor (AR), that account almost entirely for the selectivity of agonist binding by these receptor subtypes (Hwa, J., Graham, R. M., and Perez, D. M. (1995) J. Biol. Chem. 270, 23189-23195). Thus reversal of these two residues, from those found in the native receptor of one subtype to those in the other subtype, produces complementary changes in subtype selectivity of agonist binding. Here we show that mutating only one of these residues in either the alpha1b-AR or the alpha1a-AR to the corresponding residue in the other subtype (Ala204 --Val for the alpha1b; Met292 --Leu for the alpha1a-AR) results in chimeras that are constitutively active for signaling by both the phospholipase C and phospholipase A2 pathways. This is evident by an increased affinity for agonists, increased basal phospholipase C and phospholipase A2 activation, and increased agonist potency. Although mutation of the other residue involved in agonist binding selectivity, to the corresponding residue in the other subtype (Leu314 --Met for the alpha1b-AR; Val185 --Ala for the alpha1a-AR) does not alter receptor binding or signaling, per se, when combined with the corresponding constitutively activating mutations, the resulting chimeras, Ala204 --Val/Leu314 --Met ( alpha1b-AR) and Val185 --Ala/Met292 --Leu ( alpha1a-AR), display wild type ligand binding and signaling. A simple interpretation of these results is that the alpha1a- and alpha1b-ARs possess residues that critically modulate isomerization from the basal state, R, to the active state R*, and that the native receptor structures have evolved to select residues that repress active state isomerization. It is likely that the residues identified here modulate important interhelical interactions between the fifth and sixth transmembrane segments that inhibit or promote receptor signaling.

Many tissues of the body cannot only repair themselves, but also self-renew, a property mainly due to stem cells and the various mechanisms that regulate their behavior. Stem cell biology is a relatively new field. While advances are slowly being realized, stem cells possess huge potential to ameliorate disease and counteract the aging process, causing its speculation as the next panacea. Amidst public pressure to advance rapidly to clinical trials, there is a need to understand the biology of stem cells and to support basic research programs. Without a proper comprehension of how cells and tissues are maintained during the adult life span, clinical trials are bound to fail. This review will cover the basic biology of stem cells, the various types of stem cells, their potential function, and the advantages and disadvantages to their use in medicine. We will next cover the role of G-protein coupled receptors in the regulation of stem cells and their potential in future clinical applications.

The alpha 1-adrenergic receptors activate a phospholipase C enzyme by coupling to members of the large molecular size (approximately 74 to 80 kilodaltons) G alpha h family of guanosine triphosphate (GTP)-binding proteins. Rat liver G alpha h is now shown to be a tissue transglutaminase type II (TGase II). The transglutaminase activity of rat liver TGase II expressed in COS-1 cells was inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) or by alpha 1-adrenergic receptor activation. Rat liver TGase II also mediated alpha 1-adrenergic receptor stimulation of phospholipase C activity. Thus, G alpha h represents a new class of GTP-binding proteins that participate in receptor signaling and may be a component of a complex regulatory network in which receptor-stimulated GTP binding switches the function of G alpha h from transglutamination to receptor signaling.

The role of α(1)-adrenergic receptors (α(1)ARs) in cognition and mood is controversial, probably as a result of past use of nonselective agents. α(1A)AR activation was recently shown to increase neurogenesis, which is linked to cognition and mood. We studied the effects of long-term α(1A)AR stimulation using transgenic mice engineered to express a constitutively active mutant (CAM) form of the α(1A)AR. CAM-α(1A)AR mice showed enhancements in several behavioral models of learning and memory. In contrast, mice that have the α(1A)AR gene knocked out displayed poor cognitive function. Hippocampal brain slices from CAM-α(1A)AR mice demonstrated increased basal synaptic transmission, paired-pulse facilitation, and long-term potentiation compared with wild-type (WT) mice. WT mice treated with the α(1A)AR-selective agonist cirazoline also showed enhanced cognitive functions. In addition, CAM-α(1A)AR mice exhibited antidepressant and less anxious phenotypes in several behavioral tests compared with WT mice. Furthermore, the lifespan of CAM-α(1A)AR mice was 10% longer than that of WT mice. Our results suggest that long-term α(1A)AR stimulation improves synaptic plasticity, cognitive function, mood, and longevity. This may afford a potential therapeutic target for counteracting the decline in cognitive function and mood associated with aging and neurological disorders.