α2-Adrenergic receptors mediate part of the diverse biological effects of the endogenous catecholamines epinephrine and norepinephrine. Three distinct subtypes of α2-adrenergic receptors, α2A, α2B, α2C, have been identified from multiple species. Because of the lack of sufficiently subtype-selective ligands, the specific biological functions of these receptor subtypes were largely unknown until recently. Gene-targeted mice carrying deletions in the genes encoding for individual α2-receptor subtypes have added important new insight into the physiological significance of adrenergic receptor diversity. Two different strategies have emerged to regulate adrenergic signal transduction. Some biological functions are controlled by two counteracting α2-receptor subtypes, e.g., α2A-receptors decrease sympathetic outflow and blood pressure, whereas the α2B-subtype increases blood pressure. Other biological functions are regulated by synergistic α2-receptor subtypes. The inhibitory presynaptic feedback loop that tightly regulates neurotransmitter release from adrenergic nerves also requires two receptor subtypes, α2A and α2C. Similarly, nociception is controlled at several levels by one of the three α2-receptor subtypes. Further investigation of the specific function of α2-subtypes will greatly enhance our understanding of the relevance of closely related receptor proteins and point out novel therapeutic strategies for subtype-selective drug development.
- adrenergic receptors
- transgenic mice
- gene targeting
adrenergic receptors form the interface between the endogenous catecholamines epinephrine and norepinephrine and a wide array of target cells in the body to mediate the biological effects of the sympathetic nervous system. To date, nine distinct adrenergic receptor subtypes have been cloned from several species: α1A, α1B, α1D, α2A, α2B, α2C, β1, β2, and β3(11). For many of these receptors, their precise physiological functions and their therapeutic potential have not been fully elucidated. Only for β-adrenergic receptors have sufficiently subtype-selective ligands been developed that have helped to identify the physiological significance of β1-, β2-, and β3-receptors, some of which have entered clinical medicine. Selective agonists for the β2-adrenergic receptor play an important role in asthma therapy, whereas β1-receptor antagonists are first-line medication for patients with hypertension, coronary heart disease, or chronic heart failure (8, 20, 50). For α1-receptors, subtype-selective ligands that can diminish the symptoms of benign prostate hyperplasia without causing hypotension have just entered clinical therapy (33). Despite the fact that α2-adrenergic receptors serve a number of physiological functions in vivo and have great therapeutic potential, no sufficiently subtype-selective ligands are clinically available yet. Despite this fact, non-subtype-selective α2-receptor agonists like clonidine, medetomidine, and brimonidine are being used to treat patients with hypertension, glaucoma, tumor pain, postoperative pain, and shivering or to block the symptoms of sympathetic overactivity during drug withdrawal (66). Unfortunately, the fields of therapeutic application and unwanted side effects are overlapping, e.g., α2-receptor-mediated sedation is an important problem for treatment of hypertension. Severe side effects are one reason why α2-receptor agonists are only second-line antihypertensive agents. It is tempting to speculate that α2-receptor-mediated therapy could be greatly improved and advanced if receptor subtype-selective ligands were available. However, before developing specific ligands, the therapeutic targets have to be identified. Recently, transgenic and gene-targeted mouse models have added considerable information about individual adrenergic receptor subtypes (15, 25, 37, 39, 53, 54). This review focuses on the specific functions of the three α2-adrenergic receptor subtypes in mouse models carrying targeted deletions in the genes encoding for α2-receptors.
α2-ADRENERGIC RECEPTOR GENES
So far, three distinct genes have been identified from several species that encode for separate subtypes of α2-adrenergic receptors (11). From these genes, three α2-receptors are synthesized, termed α2A, α2B, and α2C. The pharmacological ligand binding characteristics of the α2A-subtype differ significantly between different species, thus giving rise to the pharmacological subtypes α2A in humans, rabbits, and pigs and α2D in rats, mice, and guinea pig (11). This species variation is at least in part due to a single amino acid variation in the fifth transmembrane domain of the α2A-receptor that renders this receptor less sensitive to yohimbine binding (34).
GENE-TARGETED MICE LACKING INDIVIDUAL α2-RECEPTOR SUBTYPES
Several mouse lines have been established by gene targeting that do not express functional α2-adrenergic receptors (2, 35, 36). All of these mice developed apparently normally, although mice lacking α2B-adrenergic receptors were not born at the expected Mendelian ratios, indicating that this receptor may play a role during embryonic development (13,35).
In addition, a point mutation has been introduced into the α2A-receptor gene (α2-D79N) to evaluate the physiological role of separate intracellular signaling pathways of this receptor in vivo (38). The D79N mutation substitutes asparagine for an aspartate residue at position 79, which is predicted to lie within the second transmembrane region of the α2A-receptor and is highly conserved among G protein-coupled receptors. In vitro, the α2A-D79N receptor has been shown to be deficient in coupling to K+channel activation (76). However, in vivo this point mutation was found to be deficient in K+ current activation and Ca2+ channel inhibition (32). Surprisingly, the density of α2A-D79N receptors in the mouse brain was decreased to ∼20% of the normal level (38). Thus, in most (but not all) functional tests, the α2A-D79N receptor had characteristics resembling a functional “knockout” of the α2A-receptor (40). One important exception was the observation that the presynaptic inhibitory function of the α2A-D79N receptor was normal or only slightly blunted in intact tissues (2). Most likely, the decreased expression of α2A-D79N receptors in vivo rather than a selective defect in receptor signaling seems to be important for the “functional knockout.” At the presynaptic side, a high number of spare receptors is characteristic for α2-receptor function, i.e., activation of very few α2-receptors results in maximal presynaptic inhibition of transmitter release (1). Thus the reduced number of presynaptic α2A-D79N receptors may still be sufficient for presynaptic control, whereas the decreased receptor density may compromise receptor signal transduction at other sites with a smaller receptor reserve.
WHICH α2-RECEPTOR SUBTYPE IS THE PRESYNAPTIC REGULATOR?
α2-Adrenergic receptors were initially characterized as presynaptic receptors that serve as parts of a negative feedback loop to regulate the release of norepinephrine (71). Soon it was shown that α2-receptors are not restricted to presynaptic locations but also have postsynaptic functions (Fig.1 A). With the use of an array of pharmacological antagonists, the α2A-receptor was predicted to be the major inhibitory presynaptic receptor regulating release of norepinephrine from sympathetic neurons as part of a feedback loop (82). However, in some tissues, the α2C-receptors were considered to be in the inhibitory presynaptic receptor (55).
With the genetic deletion of individual α2-receptor genes in mice, this classification of the presynaptic autoreceptor subtype was challenged. In mice lacking the α2A-subtype, presynaptic feedback regulation was severely impaired but not abolished, indicating that indeed the α2A-receptor is the major autoreceptor in sympathetic neurons (Fig. 1 A) (2, 26). Most surprisingly, the α2C-receptor turned out to function as an additional presynaptic regulator in all central and peripheral nervous tissues investigated (Fig.1 A) (2, 9, 26, 70, 79, 80). However, the relative contributions of α2A- and α2C-receptors differed between central and peripheral nerves, with the α2C-receptor being more prominent in sympathetic nerve endings than in central adrenergic neurons. α2A- and α2C-receptors differ in their time course of expression after birth (65). While α2A-mediated autoinhibition of neurotransmitter release is already operative immediately after birth, the α2C-receptor function is established later in mice (65).
Furthermore, the α2-autoreceptor subtypes could be distinguished functionally: α2A-receptors inhibited transmitter release significantly faster and at higher action potential frequencies than the α2C-receptors (Fig. 1 B) (9, 26, 62). When α2A- and α2C-receptors were stably expressed together with N-type Ca2+ channels or with G protein-coupled inwardly rectifying K+ (GIRK) channels, no differences in the activation kinetics of these two receptor subtypes were detected at identical levels of receptor expression (10). However, when receptor GIRK channel deactivation after removal of norepinephrine was followed, the α2C-receptor was found to be active for a significantly longer time than the α2A-subtype irrespective of the level of receptor expression. This difference in α2-receptor deactivation kinetics could be explained by the higher affinity of norepinephrine for the α2C- than for the α2A-receptor subtype (10). This property makes the α2C-receptor particularly suited to control neurotransmitter release at low action potential frequencies (Fig. 1) (26). In contrast, the α2A-receptor seems to operate primarily at high stimulation frequencies in sympathetic nerves and may thus be responsible for controlling norepinephrine release during maximal sympathetic activation.
α2-Adrenergic receptors not only inhibit release of their own neurotransmitters (autoreceptors) but can also regulate the exocytosis of a number of other neurotransmitters in the central and peripheral nervous system. In the brain, α2A- and α2C-receptors can inhibit dopamine release in basal ganglia (9) as well as serotonin secretion in mouse hippocampal or brain cortex slices (61). In contrast, the inhibitory effect of α2-agonists on gastrointestinal motility was mediated solely by the α2A-subtype (63).
Part of the functional differences between α2A- and α2C-receptors may be explained by their distinct subcellular localization patterns (Fig.2) (14, 47, 86, 87). In cultured sympathetic neurons from newborn mice, functional presynaptic α2-receptors develop to inhibit voltage-dependent Ca2+ channels and norepinephrine release (77,78). In sympathetic neurons, only the α2A-subtype but not the α2C-receptor contributed to inhibition of neurotransmitter release (81). Remarkably, inhibition of Ca2+ channels located on neuronal cell bodies and dendrites was mediated by both α2A- and α2C-receptors. Thus α2C-receptors in neurons may require a specific itinerary to guide their expression to axonal termini.
BLOOD PRESSURE REGULATION
α2-Receptors are involved in the control of blood pressure homeostasis at a number of locations (Fig.3). Nonselective activation of α2-receptors usually leads to a biphasic blood pressure response: after a short hypertensive phase that is more pronounced after rapid intravenous injection, arterial pressure falls below the baseline. After oral application of α2-agonists, the hypotensive action prevails and is being used to treat elevated blood pressure in hypertensive patients. Interestingly, the two phases of the pressure response are mediated by two different α2-receptor subtypes in vivo: α2B-receptors are responsible for the initial hypertensive phase, whereas the long-lasting hypotension is mediated by α2A-receptors (2, 35, 38). Thus the α2A-receptor is a therapeutic target for subtype-selective antihypertensive agents. The blockade of α2-receptors may be of therapeutic benefit in patients with atherosclerotic coronary arteries (3), whereas it is still unknown which α2-receptor subtype is responsible for the vasoconstriction in humans. An insertion/deletion polymorphism with decreased receptor desensitization of the α2B-receptor subtype is associated with an increased risk for acute coronary events (69).
Some evidence indicates that α2A-receptors also participate to a smaller degree in the vasoconstrictor action of α2-agonists in mice (38). Bolus injection of norepinephrine caused transient hypertension in wild-type mice and in α2B- and α2C-deficient mice but not in mice lacking the α2A-receptor (16). Vascular α2-receptor subtypes may be differentially distributed between vascular beds. When α2-agonists were injected into the carotid artery, most of the hypertensive response to α2-activation was mediated by the α2B-receptor (35), whereas injection into the femoral artery showed a blunted hypertensive effect in mice with the α2A-D79N receptor (38). In some arteries, α2-mediated vasoconstriction may even predominate over α1-receptor-induced contraction, and decreased α-receptor responsiveness may contribute to elevated blood flood in tissue inflammation, e.g., arthritis (45).
In addition to its role as a vasoconstrictor, the α2B-receptor seems to be required for the development of salt-sensitive hypertension (Fig. 3) (22, 41-43). Nephrectomy followed by Na+ loading has been established as a model of hypertension in mice (22). In this system, the development of hypertension depends on increased vasopressin release and sympathetic activation (21). Bilateral nephrectomy and saline infusion raised blood pressure in wild-type and in α2A- and α2C-receptor-deficient mice. However, in α2B-deficient animals a small fall in arterial pressure was observed (41). Recent experiments with α2B-antisense oligonucleotide injection into the lateral brain ventricle suggest that a central α2B-adrenergic receptor is necessary for induction of salt-dependent hypertension (31).
Under certain conditions, even the α2C-receptor subtype may contribute to vascular regulation: when kept below 37°C for a while, cutaneous arteries of the mouse tail show an α2C-receptor-dependent vasoconstriction that could not be observed when the vessel segments were incubated at body temperature (12). This finding may be of great therapeutic interest for the treatment of Raynaud's disease. Patients with Raynaud's phenomenon suffer from severe periods of vasoconstriction of their fingers and toes that are usually triggered by exposure to cold. Treatment of these patients with α2-adrenergic antagonists diminished the vasoconstriction (19). Interestingly, silent α2C-receptors may be translocated from an intracellular receptor pool to the cell surface on cooling (Fig. 2) (29). This phenomenon has been observed in human embryonic kidney (HEK-293) cells transfected with recombinant α2C-receptors: cooling of cells to 28°C evoked a redistribution of α2C-receptors from the Golgi apparatus to the plasma membrane within 1 h (29). Thus inhibition of α2C-receptors may prove an effective treatment for Raynaud's phenomenon.
In addition to these vascular and central neuronal mechanisms, renal α2-receptors may be involved in the long-term regulation of blood pressure and fluid and electrolyte homeostasis (48,49). Activation of renal vascular α2B-receptors may lead to an increase in medullary NO production and thus counteract the vasoconstrictor effects of norepinephrine in the renal medulla (90). Via this mechanism, α2B-receptors may be essential in the regulation of renal medullary blood flow and oxygen supply.
α2-Agonists are potent analgesics, and they can potentiate the analgesic effect of opioids (75, 85, 88). Recent data indicate that all three α2-receptor subtypes are involved in the regulation of pain perception in the mouse (Fig.4).
The α2A-receptor mediates the antinociception induced by systemically applied α2-agonists, including clonidine and dexmedetomidine (18, 74). Compared with control mice, α2-agonists were completely ineffective as an antinociceptive agent in the tail immersion or substance P test in α2A-D79N mice (27). The α2A-D79N mutation also blocked the synergy seen in wild-type mice between α2-agonists and delta-opioid agonists (74). Interestingly, α2A-receptor-deficient mice showed a reduced antinociceptive effect to isoflurane (30). However, not all α2-receptor agonists required functional α2A-receptors for their antinociceptive effect (Fig. 4). The imidazoline/α2-receptor ligand moxonidine caused spinal antinociception that was at least partially dependent on α2C-receptors (17).
Surprisingly, nitrous oxide, which is used as a potent inhalative analgesic during anesthesia, requires the α2B-subtype for its antinociceptive effect (Fig. 4) (23, 60). Supraspinal opioid receptors and spinal α2B-receptors are involved in the analgesic pathway for nitrous oxide. Activation of endorphin release in the periaqueductal gray by nitrous oxide stimulates a descending noradrenergic pathway that releases norepinephrine onto α2B-receptors in the dorsal horn of the spinal cord (89). In mice lacking α2B-receptors, the analgesic effect of nitrous oxide was completely abolished (60).
α2-Agonists are used in the postoperative phase or in intensive care as sedative, hypnotic, and analgesic agents (44, 66). The sedative effects of α2-agonists in mice are solely mediated by the α2A-receptor subtype (32). α2A-D79N mice showed no sedative response to the α2-agonist dexmedetomidine (32). In contrast, mice lacking the α2B- or α2C-receptors did not differ in their sedative response from wild-type control mice (27, 59). Similarly, the anesthetic-sparing effect of α2-agonists was completely abolished in α2A-D79N mice (32).
The hypnotic effect of α2-agonists is most likely mediated in the locus ceruleus. Neurons of the locus ceruleus express α2A-adrenergic receptors at very high density (84). Furthermore, α2A-antisense oligonucleotide injection into the locus ceruleus in rats attenuated the sedative effects of exogenous α2-agonists (46).
Because of their widespread distribution in the central nervous system, α2-receptors affect a number of behavioral functions (5, 56, 57, 67). In particular, the α2C-receptor subtype has been demonstrated to inhibit the processing of sensory information in the central nervous system of the mouse (for a recent review, see Ref. 64). Activation of α2-receptors also resulted in locomotor inhibition. While direct activation of α2-receptors by dexmedetomidine did not alter spontaneous motor activity in α2C-receptor-deficient mice (59),d-amphetamine stimulated locomotor activity to a greater extent in α2C-deficient mice than in wild-type mice (58).
Mice overexpressing α2C-receptors were impaired in spatial and nonspatial water maze tests, and an α2-antagonist fully reversed the water maze escape defect in these mice (4-6). The α2-agonist dexmedetomidine increased swimming distance more effectively in wild-type mice than in α2C-receptor-deficient mice (4). Activation of α2C-receptors disrupts execution of spatial and nonspatial search patterns, whereas stimulation of α2A- and/or α2B-receptors may actually improve spatial working memory in mice (7). It may be concluded that novel agonists devoid of α2C-receptor affinity can modulate cognition more favorably than non-subtype-selective drugs.
Altered startle reactivity and attenuation of the inhibition of the startle reflex by an acoustic prepulse have been observed in schizophrenia, and disrupted prepulse inhibition has frequently been used as an animal model for drug antipsychotic drug development. Interestingly, α2C-receptor-deficient mice had enhanced startle responses, diminished prepulse inhibition, and shortened attack latency in the isolation-aggression test (57). Thus drugs acting via the α2C-receptor may have therapeutic value in disorders associated with enhanced startle responses and sensorimotor gating deficits, such as schizophrenia, attention deficit disorder, posttraumatic stress disorder, and drug withdrawal. In addition to the α2C-subtype, the α2A-receptor has an important role in modulating behavioral functions. Experiments using gene-targeted mice indicate that the α2A-receptor may play a protective role in some forms of depression and anxiety, and this receptor may mediate part of the antidepressant effects of imipramine (67). Thus α2A- and α2C-receptors complement each other to integrate central nervous system function and behavior.
OTHER PHYSIOLOGICAL FUNCTIONS AND PHARMACOLOGICAL TARGETS
α2-Receptors are involved in the regulation of body temperature as well as seizure threshold. Activation of central α2A-receptors causes a powerful antiepileptogenic effect in mice (28). Two receptor subtypes, α2A and α2C, may be involved in the hypothermic action of α2-agonists (27, 59). Another important function of α2-agonists is their inhibitory effect on intraocular pressure. The α2-agonists apraclonidine and brimonidine are currently being used to lower intraocular pressure in patients with glaucoma (52, 68). In adipose tissue, α2-receptors inhibit lipolysis (72, 73) and α2-receptors are potential targets for the treatment of obesity. Mice expressing human α2-receptors in fat tissue in vivo, in the absence of β3-adrenergic receptors, developed high-fat diet-induced obesity (83). However, the precise role of individual α2-receptor subtypes in the control of lipolysis is unknown at present.
Genetic deletion of α2-adrenergic receptor subtype genes in mice has greatly enhanced our understanding of the physiological functions and therapeutic potential of individual α2-receptor subtypes. α2-Adrenergic receptors are important regulators of sympathetic tone, neurotransmitter release, blood pressure, and intraocular pressure. α2-Receptor activation causes sedation and potent analgesia. Further potential therapeutic functions may be unraveled with the help of mouse models with deleted α2-receptor genes. Before these genetic animal models were available, it was hypothesized that each biological function of α2-receptors would be mediated by one receptor subtype. Thus it was reasonable to assume that novel subtype-specific pharmacological agonists or antagonists would be of great therapeutic value because of their reduced potential for α2-receptor-mediated side effects. However, with more and more studies of the α2-receptor physiology in gene-targeted mice being published, the situation became more complicated than initially anticipated. Indeed, only a few biological functions of α2-receptors were found to be mediated by one single α2-adrenergic receptor subtype. Examples are the hypotension or sedation caused by α2A-receptor activation.
For other α2-receptor-mediated functions, two different strategies seem to have emerged to regulate adrenergic signal transduction: some biological functions are controlled by two counteracting α2-receptor subtypes, e.g., α2A-receptors decrease sympathetic outflow and blood pressure, whereas the α2B-subtype increases blood pressure by direct vasoconstriction. In contrast, the inhibitory presynaptic feedback loop that tightly regulates neurotransmitter release from adrenergic nerves requires two receptor subtypes, α2A and α2C, with similar but complementary effects. Similarly, pain perception is controlled at several levels of by one of the three α2-receptor subtypes.
The fact that more than one receptor subtype may be involved in regulating one particular physiological function does not limit the therapeutic potential of novel subtype-selective drugs for α2-adrenergic receptors. However, it emphasizes that knowledge of the spectrum of in vivo biological effects is mandatory before making precise predictions about the in vivo effects of subtype-specific drugs. For treatment of hypertension, a selective α2A-receptor agonist without affinity for the α2B-receptor might be advantageous. As α2B-receptors counteract the hypotensive effect of α2A-receptor activation, a selective α2A-agonist could be given at a lower dose to achieve similar blood pressure lowering with reduced sedative side effects. In addition, a combination of agonistic and antagonistic properties may become desirable, for instance, for antihypertensives, e.g., α2A-agonist and α2B-antagonist. The primary target for α2-mediated pain modulation would be the α2B-receptor. As illustrated by the potent analgesic effect of nitrous oxide, α2B-receptor activation might be a very promising analgesic strategy. Whether α2C-receptors are equally effective in inhibiting pain pathways in the spinal cord has to be tested in future studies (17). The main advantage of α2B- or α2C-receptor-specific agonists for antinociception would be their lack of sedative side effects compared with nonselective α2-agonists that also stimulate α2A-receptors. In addition, they would not cause respiratory depression and addiction, which are two major problems associated with opioid therapy. In anesthesia and intensive care, the availability of pairs of subtype-selective agonists and antagonists might be of great benefit (66). α2A-Receptor-mediated sedation that can be rapidly reversed by a selective α2-antagonist may be used in future human anesthesia (as it is already being used with non-subtype-selective agonists/antagonists in veterinary anesthesia). Finally, mice lacking all α2-receptor subtypes will also be essential tools to determine the function of imidazoline receptors and the potential of future imidazoline receptor drugs (24,51).
Further investigation of the specific function of α2-subtypes will greatly enhance our understanding of the relevance of closely related receptor proteins and point out novel therapeutic strategies for subtype-selective drug development.
Our work has been supported by the Deutsche Forschungsgemeinschaft.
Address for reprint requests and other correspondence: L. Hein, Institut für Pharmakologie und Toxikologie, Universität Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany ().
- Copyright © 2002 the American Physiological Society