α1-AR Subtypes Research Articles

Differential Distribution and Expression of α1-adrenergic Receptors in Low Shear Stress Induced Neointimal Formation

Low fluid shear stress in conduit arteries has emerged as a determinant of neointimal formation—an accumulation of vascular smooth muscle cells on the surface of the intima—the most challenging component of atherosclerosis to treat. α1-adrenergic receptor (α1-AR) antagonists have been well evaluated in vascular injury models yielding reduced neointimal formation. However, the α1-AR subtypes’ (α1A-AR, α1B-AR, α1D-AR) role in neointimal formation have not been determined under low shear stress conditions. α1-AR subtypes are expressed in various tissues and evoke different responses to the same ligand resulting in both adaptive and maladaptive responses. This study was designed to identify and characterize the α1-AR subtypes in low shear stress induced neointimal formation. An in vivo partial ligation mouse model was utilized to actuate low fluid shear stress and create neointimal formation in the left carotid artery in 14 days. The right carotid artery remained uninjured as the control. For tissue collection and processing, the left and right carotid arteries were harvested, snap-frozen in liquid nitrogen, and cryosectioned. Immunofluorescence staining was performed using primary antibodies for α1B-AR or α1A-AR and CD31 (endothelial cell marker). Confocal microscopy combined with processing and analysis was utilized for the detection, location, and distribution of cell markers. Histomorphometric analysis revealed significant structural changes, including the medial area and neointimal area, between the injured and non-injured arteries at the 14-day timepoint. Moreover, the injured arteries showed an increase in percent area stenosis, compared to the non-injured control. Histological observation of fluorescently labeled images revealed α1B-AR is densely distributed in the neointimal and medial area of the injured carotid arteries compared to the non-injured control. NucBlue (nuclear) staining indicated an increase in cell number in the medial and neointimal areas of the injured artery. Together, these results offer insight into changes in vascular α1-ARs under low shear stress conditions suggesting that α1B-AR may be upregulated in response to injury and contribute to neointimal formation. NIH HL155288. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Characteristics of α1-adrenoceptor antagonists-induced ejaculatory dysfunction on spontaneous seminal emission in rats.

Although α1-adrenoceptor (α1-AR) antagonists used to treat benign prostatic hyperplasia can cause ejaculation disorders, the aetiology of this adverse event is still controversial. Therefore, we investigated the effects of antagonists with different affinities for α1-AR subtypes on ejaculatory function and their mechanisms of action in normal rats. In the spontaneous seminal emission (SSE) test, systemically administered prazosin, terazosin, tamsulosin and naftopidil decreased the weight of ejaculated seminal material in a dose-dependent manner; the potency order was as follows: tamsulosin > terazosin > prazosin > naftopidil. The selective α1D-AR antagonist BMY7378 had no effect on SSE. Intrathecal tamsulosin and naftopidil did not inhibit SSE. Tamsulosin, the most potent, was ineffective as a single dose and significantly increased seminal vesicle fluid in rats treated for 2weeks but did not significantly change retrograde ejaculation. These results indicated that the difference in inhibitory potency of the five α1-AR antagonists against SSE was due to the involvement of α1A-AR subtypes. Our results further suggested that α1-AR antagonist-induced ejaculatory dysfunction at the peripheral level was mainly due to the loss of seminal emission, although some retrograde ejaculation may also be involved.

The Subtype Selectivity in Search of Potent Hypotensive Agents among 5,5-Dimethylhydantoin Derived α1-Adrenoceptors Antagonists

In order to find new hypotensive drugs possessing higher activity and better selectivity, a new series of fifteen 5,5-dimethylhydantoin derivatives (1–15) was designed. Three-step syntheses, consisting of N-alkylations using standard procedures as well as microwaves, were carried out. Crystal structures were determined for compounds 7–9. All of the synthesized 5,5-dimethylhydantoins were tested for their affinity to α1-adrenergic receptors (α1-AR) using both in vitro and in silico methods. Most of them displayed higher affinity (Ki < 127.9 nM) to α1-adrenoceptor than urapidil in radioligand binding assay. Docking to two subtypes of adrenergic receptors, α1A and α1B, was conducted. Selected compounds were tested for their activity towards two α1-AR subtypes. All of them showed intrinsic antagonistic activity. Moreover, for two compounds (1 and 5), which possess o-methoxyphenylpiperazine fragments, strong activity (IC50 < 100 nM) was observed. Some representatives (3 and 5), which contain alkyl linker, proved selectivity towards α1A-AR, while two compounds with 2-hydroxypropyl linker (11 and 13) to α1B-AR. Finally, hypotensive activity was examined in rats. The most active compound (5) proved not only a lower effective dose than urapidil but also a stronger effect than prazosin.

Inflammation induces α1-adrenoceptor expression in peripheral blood mononuclear cells of patients with complex regional pain syndrome

Persistent regional and systemic inflammation may promote pain and hyperalgesia in complex regional pain syndrome (CRPS). In this study, we investigated whether stimulation of α1-adrenoceptors (α1-AR) on peripheral blood mononuclear cells (PBMC) might contribute to this inflammatory state. PBMC were isolated from venous blood collected from 21 CRPS patients and 21 sex and age-matched controls. Lipopolysaccharide (LPS), a bacterial toxin, was administered to cultured PBMC for 24 h to trigger inflammation. Exposure to LPS resulted in heightened gene expression of α1-AR subtype B (α1B-AR) in PBMC of CRPS patients relative to controls. Interleukin (IL)-1β and IL-6 levels did not change when the α1-AR agonist phenylephrine was administered to naïve PBMC. However, α1-AR stimulation following LPS treatment increased IL-6 mRNA and protein levels in PBMC of patients and controls. To investigate the possible consequence of heightened IL-6 levels on immunoglobulin G antibody production, PBMC were stimulated with CD40 ligand and IL-21 to generate plasmablasts (B cells that secrete antibodies). This response was similar in patients and controls. Adding IL-6 to the cell culture medium increased plasmablast differentiation in controls and antibody production both in patients and controls. These findings suggest that the inflammatory cascade associated with elevated levels of IL-6 may generate α1B-AR expression in CRPS PBMC. A reciprocal interaction between heightened α1-AR expression in PBMC and IL-6 secretion may contribute to systemic inflammation and antibody production in CRPS.

Structural basis of agonist specificity of α1A-adrenergic receptor

α1-adrenergic receptors (α1-ARs) play critical roles in the cardiovascular and nervous systems where they regulate blood pressure, cognition, and metabolism. However, the lack of specific agonists for all α1 subtypes has limited our understanding of the physiological roles of different α1-AR subtypes, and led to the stagnancy in agonist-based drug development for these receptors. Here we report cryo-EM structures of α1A-AR in complex with heterotrimeric G-proteins and either the endogenous common agonist epinephrine or the α1A-AR-specific synthetic agonist A61603. These structures provide molecular insights into the mechanisms underlying the discrimination between α1A-AR and α1B-AR by A61603. Guided by the structures and corresponding molecular dynamics simulations, we engineer α1A-AR mutants that are not responsive to A61603, and α1B-AR mutants that can be potently activated by A61603. Together, these findings advance our understanding of the agonist specificity for α1-ARs at the molecular level, opening the possibility of rational design of subtype-specific agonists.

Alterations in expression of α1-adrenergic receptors possibly are involved in prevention of age-associated apoptosis in rat hippocampus by treadmill exercise.

Exercise is assumed to attenuate age-related neuronal apoptosis, but the detailed mechanism(s) is not fully understood. α1-adrenergic receptors (ARs) can either trigger or suppress apoptosis, therefore, here we determined the impact of treadmill exercise on the expression of the apoptosis regulatory proteins as well as α1-AR subtypes α1A- and α1B-ARs, in order to elucidate a possible association between apoptosis and the hippocampal expression of α1-ARs in aged male rats. Twenty-one male Wistar rats were divided into 3groups (n=7): young control, aged sedentary, and aged+exercise. Western blot for α1A- and α1B-ARs as well as pro-(Bax and p53) and anti-apoptotic (Bcl2) proteins wasconducted. An 8-week regular moderate-intensity treadmill exercise intervention was carried out in exercise group. In aged rats, α1A-AR expression in the hippocampus was significantly increased, and exercise markedly prevented this event. While α1B-AR expression was no altered with aging, a marked reduction in α1B-AR level was detected in exercise group when compared to aged group. Furthermore, pro-apoptotic protein levels of Bax and p53 were upregulated and anti-apoptotic protein Bcl2 was downregulated in the aging hippocampus, but could be reversed by treadmill exercise. In the present research, exercise-induced reduction in α1A- and α1B-ARs was associated with an obvious downregulation of Bax/Bcl2 ratio in aged rats, suggesting that exercise may inhibit apoptosis through regulating α1-ARs, particularly α1A-AR. Our study suggests that manipulations attenuating α1-AR activity, including nonselective α1-adrenergic antagonists, may protect against hippocampal neurodegeneration in aging brains.

ADRA1A–Gαq signalling potentiates adipocyte thermogenesis through CKB and TNAP

Noradrenaline (NA) regulates cold-stimulated adipocyte thermogenesis1. Aside from cAMP signalling downstream of β-adrenergic receptor activation, how NA promotes thermogenic output is still not fully understood. Here, we show that coordinated α1-adrenergic receptor (AR) and β3-AR signalling induces the expression of thermogenic genes of the futile creatine cycle2,3, and that early B cell factors, oestrogen-related receptors and PGC1α are required for this response in vivo. NA triggers physical and functional coupling between the α1-AR subtype (ADRA1A) and Gαq to promote adipocyte thermogenesis in a manner that is dependent on the effector proteins of the futile creatine cycle, creatine kinase B and tissue-non-specific alkaline phosphatase. Combined Gαq and Gαs signalling selectively in adipocytes promotes a continual rise in whole-body energy expenditure, and creatine kinase B is required for this effect. Thus, the ADRA1A–Gαq–futile creatine cycle axis is a key regulator of facultative and adaptive thermogenesis.

Epinephrine evokes shortening of human airway smooth muscle cells following β2 adrenergic receptor desensitization.

Epinephrine (EPI), an endogenous catecholamine involved in the body’s fight-or-flight responses to stress, activates α1-adrenergic receptors (α1ARs) expressed on various organs to evoke a wide range of physiological functions, including vasoconstriction. In the smooth muscle of human bronchi, however, the functional role of EPI on α1ARs remains controversial. Classically, evidence suggests that EPI promotes bronchodilation by stimulating β2-adrenergic receptors (β2ARs). Conventionally, the selective β2AR agonism of EPI was thought to be, in part, due to a predominance of β2ARs and/or a sparse, or lack of α1AR activity in human airway smooth muscle (HASM) cells. Surprisingly, we find that HASM cells express a high abundance of ADRA1B (the α1AR subtype B) and identify a spontaneous “switch-like” activation of α1ARs that evokes intracellular calcium, myosin light chain phosphorylation, and HASM cell shortening. The switch-like responses, and related EPI-induced biochemical and mechanical signals, emerged upon pharmacological inhibition of β2ARs and/or under experimental conditions that induce β2AR tachyphylaxis. EPI-induced procontractile effects were abrogated by an α1AR antagonist, doxazosin mesylate (DM). These data collectively uncover a previously unrecognized feed-forward mechanism driving bronchospasm via two distinct classes of G protein-coupled receptors (GPCRs) and provide a basis for reexamining α1AR inhibition for the management of stress/exercise-induced asthma and/or β2-agonist insensitivity in patients with difficult-to-control, disease subtypes.

Effect of α1D-adrenoceptor blocker for the reduction of ureteral contractions.

Urolithiasis is a common urinary tract disease with growing prevalence. Alpha1-adrenoceptors (α1-ARs) are abundant in ureteral smooth muscle, distributed with different α1-AR subtypes. α1D-AR is the most widely distributed in the ureter. However, the effect of α1D-AR blockade on ureteric contraction remains unknown. We dissected smooth muscle tissues (3 mm×3 mm) from the rat bladder and human ureter, tied silk strips on both tissue ends, and measured contraction in an organ bath chamber. Contraction activity in ureteral smooth muscle cells (USMCs) was immunocytochemically examined using primary rat and human USMC cultures. Using the organ bath system, we determined the inhibitory effects of silodosin, tamsulosin, and naftopidil. Naftopidil significantly decreased contractility of rat bladder tissue; similar results were observed in human ureteral tissue. To confirm ex vivo experimental results in vitro , we examined the phosphorylation of myosin light chain (MLC), a marker of contractility, in a primary human USMC culture. The examined drugs decreased phospho-MLC levels in human USMCs; however, naftopidil profoundly increased MLC dephosphorylation. We studied the effects of naftopidil, an α1D-AR inhibitor, on the ureter. Compared with alpha-blockers, naftopidil significantly relaxed ureteral smooth muscle. Therefore, naftopidil could be an effective therapy for patients with ureteral stones.

Effect of the replacement of the o-methoxyphenyl moiety with nitrogen-containing aromatic rings within N-phenyl-piperazine and phenoxy-ethylamine-based 1,3-dioxo/oxathio/dithiolanes as α1 and 5-HT1A receptor ligands

In the present work, nineteen analogues of 1-[(2,2-Diphenyl-1,3-dioxolan-4-yl)methyl]-4-(2-methoxyphenyl)piperazine 5 and N-[2-(2-Methoxyphenoxy)ethyl]-2,2-diphenyl-1,3-dioxolane-4-methanamine 18 were synthesized. The compounds were tested for binding affinity at 5-HT1AR and α1-AR subtypes. They were also tested using functional assays as α1-AR antagonists and the most promising were tested for functional activity at 5-HT1AR, where they were shown to behave as agonists. The results highlight that the replacement of the 1,3-dioxolane ring with a 1,3-oxathiolane or a 1,3-dithiolane moiety leads to an overall reduction in in-vitro affinity at the α1-AR, while affinity, potency and efficacy were strongly enhanced at the 5-HT1A receptor. Overall, the nitrogen-containing aromatic moieties scarcely affect the affinity at the 5-HT1A receptor, while reducing potency and increasing efficacy. The oxidation of the sulphur atom in the 1,3-oxathiolane to give sulfoxides and solfones has a negative effect on affinity and potency at both receptor systems. Regardless of the effect on the other parameters, selectivity toward 5-HT1AR with respect to the α1-AR is often favoured, but never the contrary. The most striking result is the inversion of selectivity. In fact, while the lead 5 is 100-fold selective for α1-AR, the new derivatives, although to differing degrees, are selective for 5-HT1AR.

Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells.

Recently, we reported that the chemokine (C-X-C motif) receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) heteromerize with α1A/B/D-adrenoceptors (ARs) and arginine vasopressin receptor 1A (AVPR1A) in recombinant systems and in rodent and human vascular smooth muscle cells (hVSMCs). In these studies, we observed that heteromerization between two receptor partners may depend on the presence and the expression levels of other partnering receptors. To test this hypothesis and to gain initial insight into the formation of these receptor heteromers in native cells, we utilized proximity ligation assays in hVSMCs to visualize receptor-receptor proximity and systematically studied how manipulation of the expression levels of individual protomers affect heteromerization patterns among other interacting receptor partners. We confirmed subtype-specific heteromerization between endogenously expressed α1A/B/D-ARs and detected that AVPR1A also heteromerizes with α1A/B/D-ARs. siRNA knockdown of CXCR4 and of ACKR3 resulted in a significant re-arrangement of the heteromerization patterns among α1-AR subtypes. Similarly, siRNA knockdown of AVPR1A significantly increased heteromerization signals for seven of the ten receptor pairs between CXCR4, ACKR3, and α1A/B/D-ARs. Our findings suggest plasticity of seven transmembrane helix (7TM) receptor heteromerization in native cells and could be explained by a supramolecular organization of these receptors within dynamic clusters in the plasma membrane. Because we previously observed that recombinant CXCR4, ACKR3, α1a-AR and AVPR1A form hetero-oligomeric complexes composed of 2–4 different protomers, which show signaling properties distinct from individual protomers, re-arrangements of receptor heteromerization patterns in native cells may contribute to the phenomenon of context-dependent GPCR signaling. Furthermore, these findings advise caution in the interpretation of functional consequences after 7TM receptor knockdown in experimental models. Alterations of the heteromerization patterns among other receptor partners may alter physiological and pathological responses, in particular in more complex systems, such as studies on the function of isolated organs or in in vivo experiments.

Wakefulness-Promoting Effects of Lateral Hypothalamic Area–Deep Brain Stimulation in Traumatic Brain Injury-Induced Comatose Rats: Upregulation of α1-Adrenoceptor Subtypes and Downregulation of Gamma-Aminobutyric Acid β Receptor Expression Via the Orexins Pathway

Previous studies have shown that deep brain stimulation (DBS) can improve the level of consciousness of comatose patients with traumatic brain injuries (TBIs). However, the most suitable targets for DBS are unknown, and the mechanisms underlying recovery remain to be determined. The aim of the present study was to assess the effects of lateral hypothalamic area-DBS (LHA-DBS) in comatose rats with TBIs. A total of 55 Sprague-Dawley rats were randomly assigned to 5 groups: the control group, TBI group, stimulated (TBI+LHA-DBS) group, antagonist (TBI+SB334867+LHA-DBS) group, and antagonist control (TBI+saline+LHA-DBS) group. The rats in the control group had undergone a sham operation and anesthesia, without coma induction. Coma was induced using a free-fall drop method. The rats in the stimulated group received bilateral LHA stimulation (frequency, 200 Hz; voltage, 2-4 V; pulse width, 0.1 ms) for 1 hour, with 5-minute intervals between subsequent stimulations, which were applied alternately to the left and right sides of the lateral hypothalamus. The comatose rats in the antagonist group received an intracerebroventricular injection with an orexins receptor type 1 (OX1R) antagonist (SB334867) and then received LHA-DBS. A I-VI consciousness scale and electroencephalography were used to assess the level of consciousness in each group of rats after LHA-DBS. Western blotting and immunofluorescence were used to detect OX1R expression in the LHA and α1-adrenoceptor (α1-AR) subtype and gamma-aminobutyric acid β receptor (GABABR) expression in the prefrontal cortex. In the TBI, stimulated, antagonist, and antagonist control groups, 5, 10, 6, and 9 rats were awakened. The electroencephalographic readings indicated that the proportion of δ waves was lower in the stimulated group than in the TBI and antagonist groups (P < 0.05). Western blotting and immunofluorescence analysis showed that OX1R expression was greater in the stimulated group than in the TBI group (P < 0.05). The expression of α1-AR was also greater in the stimulated group than in the TBI and antagonist groups (P < 0.05). In contrast, the GABABR levels in the stimulated group were lower than those in the TBI and antagonist groups (P < 0.05). A statistically significant difference was found between the antagonist and antagonist control groups. Taken together, these results suggest that LHA-DBS promotes the recovery of consciousness in comatose rats with TBIs. Upregulation of α1-AR expression and downregulation of GABABR expression in the prefrontal cortex via the orexins and OX1R pathways might be involved in the wakefulness-promoting effects of LHA-DBS.

Current Developments on the Role of α1-Adrenergic Receptors in Cognition, Cardioprotection, and Metabolism

The α1-adrenergic receptors (ARs) are G-protein coupled receptors that bind the endogenous catecholamines, norepinephrine, and epinephrine. They play a key role in the regulation of the sympathetic nervous system along with β and α2-AR family members. While all of the adrenergic receptors bind with similar affinity to the catecholamines, they can regulate different physiologies and pathophysiologies in the body because they couple to different G-proteins and signal transduction pathways, commonly in opposition to one another. While α1-AR subtypes (α1A, α1B, α1C) have long been known to be primary regulators of vascular smooth muscle contraction, blood pressure, and cardiac hypertrophy, their role in neurotransmission, improving cognition, protecting the heart during ischemia and failure, and regulating whole body and organ metabolism are not well known and are more recent developments. These advancements have been made possible through the development of transgenic and knockout mouse models and more selective ligands to advance their research. Here, we will review the recent literature to provide new insights into these physiological functions and possible use as a therapeutic target.

Role of α2-Adrenoceptor Subtypes in Suppression of L-Type Ca2+ Current in Mouse Cardiac Myocytes.

Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca2+ handling and contractility of cardiomyocytes. Cardiomyocyte-specific targeting of α2-AR would provide cardiac muscle-delimited stress control and enhance the efficacy of cardiac malfunction treatments. However, little is known about the specific contribution of the α2-AR subtypes in modulating cardiomyocyte functions. Herein, we analyzed the expression profile of α2A, α2B and α2C subtypes in mouse ventricle and conducted electrophysiological antagonist assay evaluating the contribution of these isoforms to the suppression of L-type Ca2+ current (ICaL). Patch-clamp electro-pharmacological studies revealed that the α2-agonist-induced suppression of ICaL involves mainly the α2C, to a lesser extent the α2B, and not the α2A isoforms. RT-qPCR evaluation revealed the presence of adra2b and adra2c (α2B and α2C isoform genes, respectively), but was unable to identify the expression of adra2a (α2A isoform gene) in the mouse left ventricle. Immunoblotting confirmed the presence only of the α2B and the α2C proteins in this tissue. The identified α2-AR isoform-linked regulation of ICaL in the mouse ventricle provides an important molecular substrate for the cardioprotective targeting.

Cardiac and Vascular α1-Adrenoceptors in Congestive Heart Failure: A Systematic Review.

As heart failure (HF) is a devastating health problem worldwide, a better understanding and the development of more effective therapeutic approaches are required. HF is characterized by sympathetic system activation which stimulates α- and β-adrenoceptors (ARs). The exposure of the cardiovascular system to the increased locally released and circulating levels of catecholamines leads to a well-described downregulation and desensitization of β-ARs. However, information on the role of α-AR is limited. We have performed a systematic literature review examining the role of both cardiac and vascular α1-ARs in HF using 5 databases for our search. All three α1-AR subtypes (α1A, α1B and α1D) are expressed in human and animal hearts and blood vessels in a tissue-dependent manner. We summarize the changes observed in HF regarding the density, signaling and responses of α1-ARs. Conflicting findings arise from different studies concerning the influence that HF has on α1-AR expression and function; in contrast to β-ARs there is no consistent evidence for down-regulation or desensitization of cardiac or vascular α1-ARs. Whether α1-ARs are a therapeutic target in HF remains a matter of debate.

Structural Insight into the Mechanism of 4-Aminoquinolines Selectivity for the alpha2A-Adrenoceptor.

Backgroundα2A-adrenoceptor (AR) is a potential target for the treatment of degenerative diseases of the central nervous system, and α2A-AR agonists are effective drugs for this condition. However, the lack of high selectivity for α2A-AR subtype of traditional drugs greatly limits their clinic usage.MethodsA series of homobivalent 4-aminoquinolines conjugated by two 4-aminoquinoline moieties via varying alkane linker length (C2-C12) were characterized for their affinities for each α2-AR subtype. Subsequently, docking, molecular dynamics and mutagenesis were applied to uncover the molecular mechanism.ResultsMost 4-aminoquinolines (4-aminoquinoline monomer, C2-C6, C8-C10) were selective for α2A-AR over α2B- and α2C-ARs. Besides, the affinities are of similar linker length-dependence for each α2-AR subtype. Among all the compounds tested, C10 has the highest affinity for α2A-AR (pKi=−7.45±0.62), which is 12-fold and 60-fold selective over α2B-AR and α2C-AR, respectively. Docking and molecular dynamics suggest that C10 simultaneously interacts with orthosteric and “allosteric” sites of the α2A-AR. The mutation of F205 decreases the affinity by 2-fold. The potential allosteric residues include S90, N93, E94 and W99.ConclusionThe specificity of C10 for the α2A-AR and the potential orthosteric and allosteric binding sites proposed in this study provide valuable guidance for the development of novel α2A-AR subtype selective compounds.

A positive feedback loop between alpha1-adrenoceptors and inflammatory cytokines in keratinocytes

A positive feedback loop between inflammatory cytokines and alpha1-adrenoceptors (α1-AR) (a target of the sympathetic nervous system neurotransmitter norepinephrine) influences inflammatory responses in immune cells. This cross-talk between the sympathetic nervous system and immune system may play a role in promoting chronic inflammation. Emerging evidence shows that α1-AR interact with inflammatory cytokines in keratinocytes, and this epidermal adrenergic signalling may contribute to skin inflammatory responses following injury, disease or stress. In this study, utilizing an in vitro approach, we hypothesized that α1-AR interact in a positive feedback loop with inflammatory mediators in keratinocytes. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) was used to induce an inflammatory state in cultured keratinocytes. TNFα increased interleukin (IL)-1β, IL-6, IL-8 and nerve growth factor (NGF) production and gene expression levels of α1-AR subtype B (α1B-AR). Additional stimulation of α1-AR further increased IL-6 levels, while maintaining high levels of IL-8 and decreasing levels of IL-1β and NGF. Our results suggest that reciprocal influences between α1-ARs and inflammatory cytokines may play a role in normal inflammatory responses. However, if unchecked, this cycle could contribute to pathology (e.g. chronic inflammatory diseases, chronic pain conditions, and stress-induced cancer progression).

Upregulation of α2-adrenoceptor synthesis in SHR cardiomyocytes: Recompense without sense – Increased amounts, impaired commands

Aimsto investigate α2-AR subtype distribution and the relationship between receptor amounts and their functionality in normotensive and spontaneously hypertensive rats. Methodsexperiments were performed on left ventricular cardiomyocytes isolated from Wistar rats and SHR (2–2.5 months). Molecular routine tools (RT-PCR, Western blotting, immunocytochemistry) were used for semi-quantitative estimation of α2-AR subtypes. Fluorescence of both the Ca2+-dependent and NO-sensitive probes were used to define functionality of α2-AR, evaluated by changes in the dynamics of spontaneous Ca2+-transients and NO production in cardiomyocytes in response to the α2-AR agonist application. Resultspercentage of the three known α2-AR subtypes in Wistar and SHR cardiomyocytes is not principally different. Total amounts of α2A-AR subtype in SHR increases, for both the sarcolemmal and intracellular receptor pools. Total number of α2B-AR is also significantly higher in hypertensive rats with an increase in the sarcolemmal, but not the intracellular immunoreactivity. For α2C-AR subtype, no significant differences between Wistar and SHR were identified, despite the fact that its amounts in cardiomyocytes are somewhat higher than the other two subtypes. Notwithstanding the increased expression of α2-AR subtypes in SHR, α2-AR-agonist guanabenz was ineffective in suppression of spontaneous Ca2+-transients, as well as the lowering of free calcium levels in the cytosol. Guanabenz-induced NO synthesis is well correlated with the Ca2+-loading into sarcoplasmic reticulum and actually decreased in SHR cardiomyocytes. Conclusiondata indicate α2-AR dysfunction and ineffectiveness of α2-AR-mediated signaling pathways in this model of cardiovascular pathologies. Results can be used for clinical practice for more effective control of cardiovascular functions in various disease states.

Tumor necrosis factor α induces α1B-adrenergic receptor expression in keratinocytes

Keratinocytes produce cytokines and nerve growth factor (NGF) as part of a repair response to injury, disease or stress, and express alpha1-adrenoceptors (α1-AR). The expression of these receptors is elevated in some inflammatory diseases and chronic pain conditions. In this study, we investigated whether inflammatory signalling affects α1-AR expression in keratinocytes in vitro. Tumor necrosis factor α (TNFα) was administered to human keratinocytes, after which the levels of other key pro-inflammatory cytokines and NGF were measured. The production of these cytokines and NGF increased in cells treated with TNFα compared to untreated cells. Furthermore, exposure to TNFα increased gene expression of the α1-AR subtype B in keratinocytes. Our results suggest that inflammatory cytokines released during injury stimulate α1-AR expression in keratinocytes. The up-regulation of α1-AR may amplify the adrenergic sensitivity of these cells to catecholamines released during sympathetic nervous system activation after injury which, in turn, could heighten the inflammatory response.

Abstract 579: Involvement of the Alpha 1A-Adrenergic Receptor in the Cardiac Adaptation to Physiological Stress

The ‘fight or flight’ response, involving sympathetic nervous system activation and catecholamine release, is central to survival. Resulting activation of adrenergic receptors (ARs) drives physiological responses, such as positive inotropy, which to date has been attributed solely to β 1 -AR/Gs/adenylyl cyclase/protein kinase A pathway-activation. We previously demonstrated that overexpression of the α 1A -AR is cardioprotective in rodents in the setting of pathological cardiac stress; a finding in agreement with studies of α 1A -AR inactivation. However, it remains unknown if α 1A -ARs also protect the heart from physiological stress. Using a tamoxifen-inducible, cardiomyocyte-specific α 1A -AR knockdown mouse model, we evaluated cardiac responses to swim exercise. Treatment of adult (8-12 week) mice with 4-OH tamoxifen (Tam) resulted in a 7.4 fold decrease (p&lt;0.001) in cardiac α 1A -AR mRNA levels, with no compensatory changes in expression of β 1 -, β 2 - or α 2 -AR subtypes. A 90 minute, twice daily, four-week swim challenge induced eccentric cardiac hypertrophy, with Tam- and vehicle-treated mice displaying similar increases in heart weight/tibia length (mean increase: 1.4 and 1.6mg/mm, respectively; p&lt;0.001 vs sedentary mice) and left ventricular chamber radius (mean increase: 0.16 and 0.17mm, respectively; p&lt;0.001 vs sedentary mice) with no change in wall thickness. Consistent with physiological hypertrophy, Igf-1 mRNA increased 1.7 and 0.7 fold (p=0.004 vs sedentary mice), respectively, with no increase in Nppb , Myh7 or Acta1 mRNA. Cardiac function, assessed by micromanometry and echocardiography, showed increases in dP/dt min/EDV (mean: -117 vs -150 mmHg/s/μL; p=0.011), end-diastolic volume (mean: 76.9 vs 68.0μL; p=0.038) and end-systolic volume (mean: 29.4 vs 21.9μL; p=0.018), as well as a decrease in ejection fraction (mean: 63 vs 68%; p=0.046) in Tam- compared with vehicle-treated mice, respectively. Thus, despite a similar hypertrophic response, mice with cardiomyocyte-specific α 1A -AR inactivation showed an impaired contractile response to a sustained swim challenge. These findings are consistent with cardiomyocyte α 1A -ARs playing an important role in maintaining cardiac contractility in response to physiological stress.