To Fight or to Flee from Adrenoceptors

Adrenoceptors are seven transmembrane proteins belonging to the superfamily of G-protein coupled receptors (GPCRs). They respond to the endogenous ligands adrenaline and noradrenaline by inducing a cascade of physiological effects. These receptors have a central role in blood pressure regulation and smooth muscle relaxation and heart contraction. After Rhodopsin, they have become the most studied GPCR to date and are targets for the continuous development of agonists/antagonists. Alomone Labs offers an exhaustive list of antibodies related to adrenoceptors, enabling the never ending research done on adrenergic signaling. The wealth of information on these receptors is truly overwhelming; hence this very short review really glances over some of the important aspects of adrenergic signaling and its significance.

Introduction

Adrenaline and noradrenaline are two catecholamines (a monoamine) synthesized from tyrosine in the adrenal glands and secreted throughout the body. These catecholamines induce the “fight or flight” response occurring under stressful situations by exerting a cascade of physiological pathways via adrenergic receptors. Adrenergic receptors (or adrenoceptors) were originally categorized into α and β types. Following extensive pharmacological analysis, mainly consisting in radioligand binding studies, molecular analysis as well as their signaling properties, they are now divided into three major groups; α₁-, α₂– and β-adrenoceptors which are further divided.

This group of receptors belongs to the superfamily of G-protein coupled receptors (GPCRs) and therefore includes the classical features of GPCRs, namely a seven transmembrane spanning domain and the ability to couple a variety of G-proteins which ultimately determines the cellular output. In addition, adrenoceptors also undergo desensitization in order to regulate/terminate their signaling, a critical point which can be the source of many diseases. As will be elaborated below, adrenergic receptors are best known for their role in blood pressure regulation and cardiac functions as many agonists/antagonists have been and are being developed for the treatment of adrenoceptor signaling malfunctions.

α₁-Adrenoceptors

Three subtypes of α1 receptors have been described and identified to date: α_1A-, α_1B– and α_1D-adrenoceptors. All three receptors are known to couple G_q/11 and activate phospholipase C (PLC) which eventually leads to an increase in intracellular Ca²⁺. However, in the rat heart, α₁-adrenoceptors have been seen to couple G_s, thereby leading to an increase in intracellular cAMP levels¹⁵. Many GPCRs can form homo and heterodimers and/or oligomers²⁷. It also seems to be the case for α_1D-adrenoceptor, which is the only receptor belonging to this family, that when heterologously expressed, is found mostly in intracellular compartments, and largely nonfunctional^7,35. However, coexpression of α_1D– with β2-adrenergic receptor increases its cell surface expression suggesting that α_1D function and expression may involve heterodimerization³⁵. The α₁-adrenoceptors are responsible for mediating smooth muscle contraction. In the vascular system they are mainly responsible for controlling blood pressure¹⁰. In fact, hemorrhage, which decreases blood pressure, activates the baroreceptor reflex, adrenaline release and concomitantly vasoconstriction via α₁-adrenergic receptors. In general, antagonists of α₁-adrenoceptors lower blood pressure in hypertension (although not commonly used). Prazosin, a specific α₁-adrenergic receptor blocker is administered to patients suffering from high blood pressure and is therefore quite safe as it does not affect cardiac functions. In the history of the adrenergic receptor classification, prazosin sensitivity was used as a classification prerequisite; α_1H and α_1L for high and low sensitivity, respectively. It became apparent that the α_1H matches the sensitivity of α_1A-, α_1B-, and α_1D-adrenoceptors¹⁰. The low prazosin affinity receptor, α_1L-adrenoceptor, has been functionally detected in different tissues³⁴. However, no gene has been detected to date. It is believed that α_1L-adrenoceptor is a conformational state or a functional phenotype of the α_1A-adrenoceptor^13,26,34. Convincing evidence that this is in fact the case comes from a study in which α_1A-adrenoceptor knockout mice do not display the α_1L-adrenoceptor phenotype (the phenotype is apparent only when α_1A-adrenoceptor is present)^17,28. Agonists of these receptors are used for treating hypotension. These agonists reinforce the vasoconstrictive effects by a depolarizationmediated Ca²⁺ entry through L-type or T-type Ca²⁺ channels, or by releasing Ca²⁺ from intracellular stores⁸.

In the visual system, α₁-adrenoceptors are responsible for contracting the dilator papillae, thereby evoking dilation of the pupil and increasing the amount of light reaching the retina. Patients suffering from intraocular pressure can be treated by the administration of α₁-agonists, which may act by decreasing the blood flow in the area¹⁰.

Additional roles for α₁-adrenoceptors include the induction of sleep, mediated by post-synaptic α₁-adrenoceptors (and α₂) in noradrenergic afferents to the preoptic area²², constriction in the gastrointestinal tract, salivary secretion, bronchoconstriction and a predominant role in the genitourinary system, including prostate proper function, ejaculation and bladder contraction (which may involve α_1D-adrenoceptor)^9,10.

α₂-Adrenoceptors

Three α₂-adrenoceptor subtypes have been identified to date, and well characterized in different mammalian systems: α_2A-, α_2B-, and α_2C-adrenoceptors. α_2D-adrenoceptor, a fourth receptor was originally identified in rat and mouse. Today, it is largely accepted that this receptor is in fact α_2A-adrenoceptor displaying a slightly different pharmacological profile in these two organisms^23,29. Nevertheless, it seems that the complexity of this adrenergic receptor category is indeed complex as some organisms express multiple α₂-adrenoceptors; zebrafish and pufferfish express five and up to eight receptors although some may be gene duplicates⁶. All three receptors couple G_i/o thereby decreasing the activity of adenylyl cyclase. However, α₂-adrenoceptors could also couple G_s and consequently increase intracellular cAMP levels¹⁹. Interestingly, it was suggested that at low agonist concentrations, α₂-adrenoceptors decrease cAMP levels, whereas at high agonist concentrations, increased cAMP levels are observed^11,21. α_2A-, α_2B– and α_2C-adrenoceptors are widely distributed in the central nervous system and the periphery. The brain and its various regions express these receptors. In the periphery, the various subtypes can be found in platelets⁵, kidney³, gastric mucosa¹⁸, aorta, spleen, heart and liver³. This category of receptors participates in the proper physiological function of the cardiovascular system and the central nervous systems. Physiological roles attributed to these receptors include motor coordination, by α₂-adrenoceptors expressed in the cortex²⁰.

Not surprisingly, α₂-adrenoceptors have been characterized through pharmacological studies, but since specific pharmacological tools are not available, the specific function of each subtype remains to be deciphered¹⁹. Nevertheless, α_2A-adrenoceptors seem to mediate most of the classical functions of α₂-adrenoceptors such as the antihypertensive and bradycardic effects observed by the actions of α₂-agonists on the α_2A-adrenoceptor¹. α_2A-adrenoceptor exhibits a dominant role in regulating the cardiovascular system, demonstrated by knockout mice studies. These mice demonstrate high blood pressure, increased heart rate and a higher probability to develop heart failure⁴. α_2B-adrenoceptor is mostly post-synaptic as opposed to its α_2A– and α_2C-adrenoceptor counterparts. Also, α_2B-adrenoceptor mediates the contractions of rat pregnant uterus, whereas α_2A– and α_2C-adrenoceptors inhibit these contractions in late pregnancy¹⁶. α_2C-adrenoceptor is mainly localized to the CNS and may counteract α₂-agonist sedative effects in mice³¹. This receptor subtype may also have beneficial effects in various psychiatric disorders including stressdependent depression, schizophrenia, as well as drug withdrawal. α₂-adrenoceptor agonists are also being potentially used in the treatment of attention-deficit hyperactive disorder (ADHD)³³.

While the α_2A-adrenoceptor is the most abundant and is the most dominant pertaining to cellular functions, the α_2B– and α_2C-adrenoceptor subtypes can either contribute or counteract α_2A-adrenoceptor dependent processes in addition to their own independent roles.

β-adrenoceptors

β-adrenoceptors have been classified into β₁, β₂ and β₃ subtypes. Activation of β₁-adrenoceptor leads to its coupling to G_s and subsequently to the increase in intracellular cAMP levels. β₂– and β₃-adrenoceptors couple both G_s and G_i rendering the β₂-adrenoceptor signaling in cardiac cells more complicated³⁶. Although all three receptors are expressed in the heart, and share expression in various tissues, β₁-adrenoceptor is mostly expressed in the heart and regulates key events in the kidney. β₂-adrenoceptor is mainly found in the airway smooth muscle (i.e. the respiratory tract) while β₃-adrenoceptor is largely distributed in adipose tissue where it stimulates the mobilization of lipids from white fat cells and increases thermogenesis in brown fat cells^2,14. The central role and involvement of β₁-adrenoceptor in proper heart function is demonstrated by the fact that patients suffering from heart failure exhibit a decrease in β₁-adrenoceptor cell surface expression which is partly due to the internalization of the receptor as well as its desensitization²⁵.

Evidence regarding mutations and coding single nucleotide polymorphisms (cSNPs) is growing concerning their effect and incidence in genes encoding β-adrenoceptors. The polymorphisms inadvertently affect coupling and desensitization rates of the receptor. cSNPs have been observed for β₁-adrenoceptors which affect the risk of cardiovascular disease as well as the response to treatment (namely antagonists)³². A missense mutation in the β₂-adrenoceptor gene is strongly associated with obesity². Also, a polymorphic form of the β₃-adrenoceptor is also associated with obesity as well as diabetes (reviewed in ref. 2).

Activation of β-adrenoceptors activates pathways that eventually lead to receptor desensitization. The mechanisms engaged in turning off the signaling response include phosphorylation of the receptors. Desensitization of β-adrenergic receptors could be homologous, where solely activated receptors are phosphorylated; this requires GPCR kinase 2 and 3 (GRK2, GRK3). Heterologous desensitization is non-specific and non-activated receptors are also phosphorylated; this phosphorylation requires the activity of PKA and occurs rapidly following activation of the receptor. The phosphorylation of the β-adrenergic receptors eventually leads to the uncoupling of the receptor from the G-protein and its internalization via clathrin-coated pits where it is dephosphorylated and either recycled back to the cell surface or degraded²⁴.

β-adrenoceptor agonists (β-agonists) were, for the longest time, administered to patients suffering from asthma in order to induce bronchodilation. At some point it became clear that some β-agonists also have the ability to increase muscle mass and decrease body fat¹². Although this characteristic of β-agonists can be practical in the treatment of disorders like muscular dystrophy, β-agonists have also become abusive drugs in competitive bodybuilders and athletes wanting to improve their performance³⁰.

The interest and research in adrenoceptors is still intensive after so many years. Alomone Labs offers antibodies to Adrenoceptors: Anti-α_1A-Adrenergic Receptor (extracellular) Antibody (#AAR-015), Anti-α_1B-Adrenergic Receptor (extracellular) Antibody (#AAR-018), the labeled version, Anti-α_1B-Adrenergic Receptor (extracellular)-ATTO Fluor-488 Antibody (#AAR-018-AG), Anti-α_1D-Adrenergic Receptor (extracellular) Antibody (#AAR-019), Anti-α_2B-Adrenergic Receptor (extracellular) Antibody (#AAR-021), Anti-α_2C-Adrenergic Receptor (extracellular) Antibody (#AAR-022), Anti-β₂-Adrenergic Receptor (extracellular) Antibody (#AAR-016) and Anti-β₃-Adrenergic Receptor (extracellular) Antibody (#AAR-017). Alomone Labs adrenergic receptor antibodies enable the detection of these receptors using various applications such as western blot, immunohistochemistry, immunocytochemistry, direct and indirect flow cytometry (see the adjoined figures).

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