Adrenergic Receptors: Unique Localization in Human Tissues

Adrenergic Receptors: Unique Localization . -. in Human /issues .

I

Debra A. Schwinn Department of Anesthesiology Duke University Medical Center Durham, North Carolina 27710

1. Introduction Adrenergic receptors (ARs) are members of the much larger family of guanine nucleotide-binding proteins (G proteins) characterized by seven transmembrane domains (Fig. 1) (1). Stimulation of adrenergic receptors by the endogenous catecholamines epinephrine and norepinephrine results in increased myocardial chronotropy , inotropy , modulations in vascular tone, bronchodilation, and many endocrine processes. Adrenergic receptors have been classically subdivided into four major groups ( a , ,az. p,, p2) using pharmacological and physiological techniques. However, the advent of molecular biology has led to cloning of genes encoding nine distinct adrenergic receptor subtypes, namely, a I A I aD1 B, , a I c ,(Y2A, ( Y ~ B , azc,pl, p2, and p3 (Fig. 2) (2,3). Expression of adrenergic receptor subtypes individually in cell lines has facilitated definition of each subtype with regard to pharmacology, G protein coupling, and second messenger production. Mutagenesis of amino acids in specific locations in each receptor protein (by modifying nucleic acid sequences at the DNA level), has provided critical information regarding exact receptor sites important in ligand binding, G protein coupling, and receptor regulation. This process is called structure-function analysis ( 3 ) . In addition to pharmacology and second messenger coupling, another method of defining adrenergic receptor subtypes involves localization in A d u t r , ~n i n Pharrnocolnyi,. Volrimc 31 Copyright 0 IW4 by Academic Press. Inc. All rights of reproduction

In any form reserved

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Debra A. Schwinn

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Fig. 1 Schematic of guanine nucleotide (G protein)-coupled receptor in a cell. A hydrophilic ligand or drug circulating in the extracellular fluid binds to the transmembrane receptor (R).This initiates a change in conformation of the receptor enabling it to interact with an intermediary G protein (G). The LY sobunit of the heterotrimeric G protein then dissociates and interacts with the effector system (El. The effector system is usually an enzyme which catalyzes the formation of a second messenger, but it could also be a channel protein.

mammalian tissues. After all, a receptor-mediated physiological function cannot be assigned to a given subtype unless that receptor subtype is present in the tissue (or cell grouping) of interest. Hence, mammalian tissue distribution (usually in rat tissues) is an integral part of the difinition of a receptor. In this context, an interesting phenomenon was noted when cloned a,AR subtypes were initially defined. Specifically, the distribution of q A R subtypes appeared to be different in rat and rabbit tissues (4). Although minor species variations have been noted in adrenergic receptor physiology and pharmacology experiments over the last several decades, ADRENERGIC RECEPTORS

A

Alhquist

(original dnfiniuon)

"Classic" pharmacology ~ I A I D QIB

Q ~ C a 2 %B ~

%c

B1 8 2 83

Molecular pharmacology

Fig. 2 History of adrenergic receptor (AR) subtypes. Ahlquist first defined adrenergic

receptors in 1948 and divided them into two subtypes, a and p. In the 1%Os and 1970s. further subdivision into the four classic adrenergic receptors took place. With the advent of molecular biology tools, since 1986 nine genes or cDNAs encoding distinct adrenergic receptors subtypes have been discovered.

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these differences were usually thought to be related to the level of receptor expression and not to the complete presence or absence of given subtypes. However, at the level of adrenergic receptor subsubtypes (i.e., a,*AR versus &,BAR),striking species variations apparently occur (4). This observation begs the question, What is the distribution of adrenergic receptor subtypes in human tissues? Because the answer to this question was first described in detail with a,ARs, it is fitting to examine the distribution of a,AR subtypes in human tissues. a,-Adrenergic receptors are classically present in brain, heart, liver, and spleen, as well as in many other tissues ( 5 ) . Pharmacological properties of a,ARs include an agonist potency series of epinephrin norepinephrine > phenylephrine % isoproterenol; the specific antagonist is prazosin (Table I). When activated, cu,ARs couple to the newly described and cloned G protein, Gq, a process which results in activation of phospholipase C (PLC) (6). PLC hydrolyzes phosphoinositide bisphosphate (PIP,) into two predominant products, inositol trisphosphate (IP,) and diacylglycerol (DAG). IP, binds to IP, receptors on the sarcoplasmic reticulum, causing release of calcium from intracellular stores. DAG activates protein kinase C, a process which leads to activation of various other proteins and potentially to regulation of receptor function. The primary physiological response to activation of a,AR subtypes is smooth muscle contraction. aIB. , ale), and Three distinct a,AR subtypes are known to exist ( a I A I D genes encoding all three subtypes have been cloned (7-9). Classically the aIAIDAR subtype has high affinity for the antagonists WB4101 and phentolamine as well as the agonist methoxamine, is partially sensitive to inactivation by the akylating agent chloroethylclonidine (CEC), and is present in several rat tissues including vas deferens, cerebral cortex, aorta, and spleen (Table 11) (5,lO). The (YIBARsubtype has relative low affinity

*

Table I General Properties of a,-Adrenergic Receptor Location Pharmacological properties Agonist potency series Selective antagonist Second messengers Physiology

Brain, heart, liver, spleen EPI 9 N E > PHE %= I S 0 Prazosin IP2 IP, and DAG Smooth muscle contraction --f

* EPI, Epinephrine; NE, norepinephrine; PHE, phenylephrine; 1S0, isoproterenol; IP,, inositol bisphosphate; IP,, inositol trisphosphate; DAG, diacylglycerol.

Debra A. Schwinn

336 Table I1 Pharmacological Properties of Cloned a,-Adrenergic Receptor Subtypes' Property

aIAIDAR

aIBAR

alCAR

High High High

Low Low High

High High High

High High High Partial

Low Low Low Yes

High Low Low Partial

Antagonist affinity

WB4101

Phentolamine Prazosin Agonist affinity Methoxamine Epinephrine Norepinephrine Inactivation by CEC

" Relative affinities are listed. CEC, chloroethylclonidine, an alkylating agent. to WB4101, phentolamine, and methoxamine, is sensitive to inactivation by CEC, and is present in rat liver, cerebral cortex, and heart (10). The alcAR subtype was not originally described pharmacologically and was discovered using molecular cloning techniques (8). It has properties quite similar to those of pharmacologically defined aIAAR, having affinity to WB4101, phentolamine, and methoxamine. However, the alcAR is partially sensitive to the alkylating properties of CEC (60-70% inactivated) and was not present in initial studies in rat tissues using Northern blot analysis (RNA level). [This receptor subtype has been subsequently found to be present in restricted locations in rat brain and heart using more sensitive techniques (1 l).] The name a I c was given to this a , A R subtype (8). Follow-up studies of the three cloned a,ARs using more recently described compounds such as ( + )niguldipine, 5-methylurapidil, benoxathian, and spiperone have suggested that the cloned a,,AR might actually be a distinct subtype called the alDAR based on subtle discrepancies between the cloned receptor and the previously pharmacologically defined aIA subtype from rat tissues (12). Until the debate is resolved, we shall continue to refer to this receptor as the alAIDAR. As described above, one of the classic methods to define a receptor subtype, in addition to ligand affinities, second messenger assays, and physiological effects, is determining the receptor distribution in rat tissues. As shown in Table 111, the cloned aIA,DAR is present in all of the rat tissues expected for the pharmacologically defined a,AAR subtype; the aIB receptor is also present in the expected rat tissues, whereas the alcAR subtype was not originally present in rat tissues by Northern analysis

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Adrenergic Receptors

Table 111 Distribution of a,-Adrenergic Receptor RNA in Rat Tissues by Northern Analysis Using Subtype-Selective Probes" Tissue Aorta Brain Hippocampus Brain stem Cerebellum Cerebral cortex Heart Kidney Liver Lung Spleen Vas deferens

~IA/DAR

+++

+++ ++ + +++ + -

+ ++ ++++

~IBAR

aicAR

c

+

++ ++ +++ +++ ++ ++++ ++

a Data from Schwinn et al. (4). aIcAR RNA has been described in more sensitive in siru hybridization assays in selected rat brain regions and is the only a l A R subtype present in rabbit liver by Northern analysis.

(4). Because some of the original experiments suggesting linkage of the pharmacologically defined a,AAR to a calcium channel were performed in rabbit aorta, we next investigated the distribution in rat tissues using Northern analysis. To our surprise, the a,,AR subtype was present in most rabbit tissues studied including kidney, spleen, heart, lung, and vascular tissues such as aorta and ear artery, but was conspicuously absent in rabbit liver (4). This is in contrast to the rat where the aIBARis the only subtype present in liver. In the rabbit, however, the C Y , ~ AisR the only a , A R subtype present in liver. Further, the a,Al,AR subtype (which was present in many rat tissues) was virtually absent in the rabbit tissues studied. These data led us to then examine the distribution of a,ARs in human tissues.

II. Methods Used to Study Receptor Distribution To study the tissue distribution of receptors in human tissues, several methods can be used. Ligand binding (interaction of ligands or drugs with receptor protein) and autoradiography are classically used but require subtype-selective Iigands which are not currently available for a,ARs. Labeled antibodies (immunohistochemistry techniques) are also fre-

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quently used but require receptor-selective antibodies which are not yet available for a,ARs. This leaves molecular techniques since genes encoding each a,AR subtype are available. Although the polymerase chain reaction is frequently used to amplify DNA, this technique may be too sensitive for examining receptor distribution in human tissues since the presence of a single copy (or very few copies) of a gene may not correlate with sufficient receptor protein concentrations to influence physiological function. Hence the predominant subtype present in a given tissue is most important for initially screening human tissues. Ribonuclease (RNase) protection assays (also called solution hybridization when performed in a quantitative fashion) are RNA-RNA hybridization assays which are sensitive but also very specific in defining the presence of RNA encoding a receptor subtype in tissues. Moreover, in situ hybridization techniques can be used to further identify the presence of RNA in specific cells in tissue sections. Although determining the presence of RNA in a tissue does not guarantee that receptor protein will be present in identical concentrations, the absence of RNA does suggest that a given receptor subtype is not present in the tissue of interest. However, in general, with a few notable exceptions, RNA expression tends to correlate with receptor protein expression levels. Because RNase protection assays require a species match of probe, it was necessary to obtain portions of human genes encoding each a,AR subtype. We have cloned portions of a,AR cDNAs over the last several years and so were in a unique position to be able to perform these experiments. Human a,AR probes ranged in length from 0.3 to 0.6 kilobases (kb). In general, RNase protection assays are performed by incubating RNA made from human tissues with a radiolabeled RNA probe generated from a human receptor cDNA fragment. Once hybridization occurs, then RNase is added to the assay; digestion of all single-stranded (nonhybridized) RNA then occurs. The resulting double-stranded protected RNA fragment is electrophoretically separated on an agarose gel and exposed to X-ray film or phosphorimager screens. Bands detected using laser densitometry (for autoradiograms) or directly from the phosphorimager are then normalized for size and incorporation of radiolabeled probe.

111. localization of Receptors in Human Tissue Preliminary results of RNase protection experiments with all three a,AR subtype and human tissues are shown in Table IV. The predominant subtype of a,AR RNA in human tissues studied is the aIcAR (13). This is in striking contrast to the expression of this receptor subtype in tissues

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Adrenergic Receptors

Table IV Distribution of a,-Adrenergic Receptor RNA in Human Tissues: Preliminary Results Using Ribonuclease Protection Assays Tissue Aorta Cerebellum Cerebral cortex Heart Kidney Liver Spleen Vena cava

++

+I-

++ +

+/-

+I-

+

+I-

+ ++ + + ++ + ++

+/-

+I-

+++ +++ +++ +/-

++++ + +

from other mammals such as rat and rabbit. In general, a,,AR RNA predominates in human liver, heart, vena cava, cerebellum, and cerebral cortex; alBAR RNA predominates in kidney and spleen, whereas the aIAIdR predominates in aorta. This distribution suggests that some selectivity may be obtained using a ,AR subtype-selective agonists and antagonists to treat various human diseases. In addition to general localization of a,AR subtype RNA in human tissues, we have also studied individual human tissues where a,ARs play an important role as therapeutic agents. For example, benign prostatic hypertrophy (BPH) is a condition where enlargement of the prostate gland in males can lead to urinary retention. Classically, therapy for BPH requires surgical resection of portions of the prostate gland. However, recent therapy with a,AR antagonists has provided symptomatic relief for many patients. The main problem with a,AR antagonist therapy relates to nonspecific a,AR blockade, resulting in hypotension and dizziness. If the exact subtype of a,AR associated with BPH could be identified, then a,AR subtype-selective agents could be developed for targeted therapy with minimal side effects. BPH is a stromal disease (involved in increased bulk of prostate smooth muscle), as opposed to an epithelial disease (such as prostate cancer). We used RNase protection assays to determine the predominate a,AR RNA in human prostate and then used in situ hybridization to localize the subtype present in stromal tissue. Clearly the predominate alAR RNA present in human prostate is the aIc (70-75% of the total), and on tissue sections this subtype is localized to the stroma (14). Smaller amounts of a I A IRNA D are also present in human prostate, fol-

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lowed by very minimal amounts of aIBARRNA. Hence, although a,AR therapy for BPH can initially be aimed at the aIcAR subtype, it important to remember that there are two components to BPH, static and active; these components may involve more than one a,AR subtype. In addition, it will be important to investigate using ligand binding and selective antibodies whether this relationship between a,AR subtypes holds up at the protein level. The prostate is not the only human tissue where a,ARs have important clinical significance. In the cardiovascular system, a,ARs play an important role in vascular tone, mediate myocardial inotropy (albeit far less than PARS), and may be involved in anesthetic-induced myocardial arrhythmias. Acute therapy for hypotension in the operating room or intensive care unit with an a,AR subtype-selective agonist could potentially provide restoration of blood pressure without renal vascular compromise if the subtype of a,AR differs in small resistance vessels and renal artery. Indeed, a mapping of adrenergic receptor subtypes in many human tissues should provide a more rational use of receptor subtype-selective drugs currently being developed and may enhance understanding of the mechanism of human diseases such as hypertension, diabetes, and congestive heart failure.

IV. Summary The final point to be made is that RNA studies are only the first step in localizing the distribution of adrenergic receptors in human tissues. Although RNA levels tend to correlate well with receptor protein expression in many tissues, this must be confirmed with studies aimed at receptor protein. Selective antibodies are being developed currently by various researchers and selective ligands by several pharmaceutical companies. In the next few years, not only should it be possible to confirm or modify results of adrenergic receptor subtype distribution studies, it also should be possible to design and test specific hypotheses related to adrenergic receptor diseases in whole animal models with newly developed subtypeselective ligands. Because species heterogeneity in adrenergic receptor tissue distribution exists, final testing of adrenergic receptor subtypeselective drugs will have to occur in humans. This is a potentially exciting possibility for anesthesiologists, for what better clinical laboratory is there than the operating room? Hence, anesthesiologists are in a key position to help redefine human adrenergic physiology once new adrenergic receptor subtype-selective agents become available.

Adrenergic Receptors

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References I . Berkowitz, D. E., and Schwinn. D. A. (1991). New advances in receptor pharmacology. Curr. Opin. Anesthesiol. 4, 486-496. 2. Schwinn, D. A. Adrenoceptors as models for G protein-coupled receptors: Structure/ function/regulation. Br. J . Anaesrh. 71, 77-85. 3. Schwinn, D. A , , Caron, M.G . . and Lefkowitz. R. J . (1991). The P-adrenergic receptor as a model for molecular structure-function relationship in G protein-coupled receptors. In "The Heart and Cardiovascular System: Scientific Foundations" (H. A. Fozzard, E. Haber, R. B. Jennings, A. M. Katz. and H. E. Morgan, eds.), 2nd Ed.. pp. 1657-1684. Raven, New York. 4. Schwinn, D. A , , Page, S. O., Middleton, J . , Lorenz. W.. Liggett. S. B . . Yamamoto, K., Caron, M. G., Lefkowitz, R. J . . and Cotecchia, S. (1991). The afc-adrenergic receptor: Characterization of signal transduction pathways and mammalian tissue heterogeneity. Mol. Pharmacol. 40,619-626. 5 . McGrath, J . C., Brown, C. M.. and Wilson, V . G. (1989). Alpha-adrenoceptors: A critical review. Med. Res. Rev. 9 . 407-533. 6. Wu, D.. Katz, A., Lee. C., and Simon. M. 1. (1992). Activation of phospholipase C by a,-adrenergic receptors is mediated by the a subunits of Gq family. J . Biol. Chem. 267. 25798-25802. 7. Cotecchia, S., Schwinn, D. A.. Randall. R. R.. Lefkowitz, R. J.. Caron, M. G., and Kobilka, B. K. (1988). Molecular cloning and expression of the cDNA for the hamster alpha-1-adrenergic receptor. Proc. Narl. A u l d . Sci. U . S . A . 85. 7159-7163.

8 . Schwinn, D. A., Lomasney, J . W., Lorenz, W.. Szklut, P. J., Yang-Feng, T . L.. Caron. M. G., Lefkowitz, R . J., and Cotecchia, S. (1990). Molecular cloning and expression of the cDNA for a novel a,-adrenergic receptor subtype. J . Biol. Chem. 265,8183-8189. 9. Lomasney, J . W.. Cotecchia, S . . Lorenz. W., Leung, W.-Y.. Schwinn, D. A., YangFeng. T. L., Brownstein, M.,Lefkowitz. R. J.. and Caron, M. G. (1991). Molecular cloning and expression of the cDNA for the a,,-adrenergic receptor, the gene for which is located on human chromosome 5. J. Biol. Chem. 266. 6365-6369. 10. Ruffolo, R. R.. Jr., Nichols, A. J., Stadel, J. M.. and Hieble, J . P. (1991). Structure and function of a-adrenoceptors. Pharmacol. Rev. 43. 475-504. 11. McCune, S. K.. Voigt, M. M..and Hill, J . M. (1992). Developmental expression of the alpha-I A, alpha-1B and alpha-1C adrenergic receptor subtype mRNAs in the rat brain. Abstr. Soc. Neurosci. 18. 196.2. 12. Schwinn, D. A.. and Lomasney, J . W. (1992). Pharmacological characterization of cloned a,-adrenergic receptor subtypes: Selective antagonists suggest the existence of a fourth subtype. Eur. J . Pharmncol. 227, 433-436. 13. Price, D. T., Lefkowitz, R. J.. Caron. M. G., and Schwinn, D. A. (1993). Alpha ,-adrenergic receptor mRNA expression in human tissues. FASEB J . 7 , A141. 14. Price, D. T., Schwinn, D. A,. Lomasney. J . W., Allen, L . E., Caron, M. G.. and Lefkowitz, R. J. (1993). Identification. quantification, and localization of mRNA for three distinct alpha,-adrenergic receptors subtypes in human prostate. J . Urol. 150, 546-55 1.