Roger J. Summers

Adrenoceptors and Their Second Messenger Systems

The catecholamines noradrenaline and adrenaline are produced in, and released from, nerve terminals and chromaffin tissue to produce a wide range of effects by activating multiple adrenoceptor subtypes. The classification of adrenoceptors into a and subtypes (Ahlquist, 1948), and later into aland a,adrenoceptors (Langer, 1974) and PIand @,-adrenoceptors (Arnold et al., 1966; Lands et al., 1967), was made on the basis of the rank orders of potency of a series of structurally related catecholamines in organ bath experiments. This approach, in some cases aided by the use of radioligand binding techniques, has provided evidence for at least three subtypes of a,-adrenoceptor (Han et al., 1987b), a,-adrenoceptor (Bylund, 1988), and @-adrenoceptor (Kaumann, 1989). Evidence for multiple adrenoceptor subtypes was regarded skeptically, particularly when based on receptor binding experiments. However, these approaches have been shown to be somewhat conservative with the advent of molecular biology techniques that have provided definitive structural information on many new subtypes (Dohlman et al., 199 1; Strosberg, 199 1). All of the adrenoceptors for which the sequences are known belong to the G protein-linked superfamily of receptors, have seven hydrophobic transmembrane-spanning regions, are proteins of between 402 and 560 amino acids in length (Dixon et al., 1986; Kobilka et al., 1987a-c; Cotecchia et al., 1988; Emorine et al., 1989; Raymond et al., 1990; Dohlman et al., 199 1 ; Strosberg, 199 l), and, in the case of well-characterized members such as the P,-adrenoceptor, have been shown to have specific regions that are critical for G protein coupling, agonist activation, ligand binding, protein kinase A (PKA) and C (PKC) phosphorylation, and P-adrenoceptor kinase phosphorylation (Dixon et al., 1988; Dohlman et al., 1991). Activation of these receptors by an agonist produces dissociation of GDP from the G protein. This is followed by association of GTP and the interaction of the activated G protein complex with an enzyme, such as adenylate cyclase or phospholipase C, to modulate levels of intracellular second messengers (Birnbaumer, 1 990). There are three major subgroups of adrenoceptors that have different classes of G protein linking them to their respective effector systems (Bylund, 1988). a,-Adrenoceptors are linked to phosphoinositide-specific phospholipase C (PI-PLC) by a G protein, probably G, (Smrcka et al., 199 l), a,-adrenoceptors to adenylate cyclase by Gi, and 0-adrenoceptors to adenylate cyclase by G,.

Cardiac effects of relaxin in rats

Relaxin is usually considered to be a hormone of pregnancy, but porcine relaxin has been shown to increase heart rate in rats. We investigated the cardiac effects of synthetic human gene-2 relaxin (hRlx-2) in vitro in isolated rat atria. Synthetic hRlx-2 produced concentration-dependent positive chronotropic effects in spontaneously beating right atria (EC50 [concentration required to produce 50% of the maximal response] = 0.09 [SE 0.03] nmol/l) and concentration-dependent positive inotropic effects in electrically driven left atria (EC50 = 0.31 [0.02] nmol/l). The potency of hRlx-2 is greater than that of endothelin, angiotensin II, and (-)-isoprenaline in isolated rat atria. Relaxin has powerful chronotropic and inotropic effects on the heart that are probably mediated through a direct action on relaxin receptors.

Expression of β3‐adrenoceptor mRNA in rat brain

The reverse transcription/polymerase chain reaction was used to demonstrate β3‐adrenoceptor mRNA in rat brain regions. Levels were highest in hippocampus, cerebral cortex and striatum and lower in hypothalamus, brainstem and cerebellum.

Characterization of postsynaptic α-adrenoceptors in rat aortic strips and portal veins

1 Postsynaptic α‐adrenoceptors in rat isolated aortic strips and portal veins have been examined using a number of agonist and antagonist drugs which have varying selectivity for α1‐ and α2‐adrenoceptors. 2 In both tissues (−)‐noradrenaline ((−)‐NA), (−)‐adrenaline ((−) Adr) (−)‐α‐methyl noradrenaline ((−)‐α‐Me‐NA) and (−)‐phenylephrine ((−)‐PE) were full agonists, while clonidine, oxymetazoline and (2‐(2,6‐dichlorophenyl)‐5,6‐dihydroimidazo(2,1,b) thiazole (44,549) were partial agonists. Guanfacine was a full agonist in aortic strips but only a partial agonist in portal veins. 3 In aortic strips, pA2 values for prazosin and yohimbine were not significantly different using (−)‐NA, (−)‐PE or guanfacine as the agonist, suggesting a single population of α‐adrenoceptors. The order of potency of the antagonists, prazosin = 2‐(β‐(4‐hydroxyphenyl)‐ethylaminomethyl)‐tetralone (BE2254) > phentolamine > yohimbine > rauwolscine, is indicative of an α1‐type of receptor. 4 In portal veins, the order of potency of the antagonists was prazosin > BE2254 > phentolamine > yohimbine > rauwolscine, again indicating an α1‐type of receptor. 5 The mean pA2 value for yohimbine was not significantly different in either tissue. However, mean pA2 values for prazosin, BE‐2254 and phentolamine were approximately one order of magnitude lower in portal veins than in aortic strips, suggesting that the receptors in the two tissues may not be identical.

Expression of β3‐adrenoceptor mRNA in rat tissues

1 This study examines the expression of β3‐adrenoceptor messenger RNA (β3‐AR mRNA) in rat tissues to allow comparison with atypical β‐adrenoceptors determined by functional and radioligand binding techniques. 2 A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of β3‐AR mRNA in rat tissues. 3 Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute β3‐AR mRNA. 4 The β3‐AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the β3‐AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of β3‐AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of β3‐AR. 5 Expression of adipsin in rat skeletal muscles coupled to very low levels of β3‐AR mRNA indicates that the observed β3‐AR may be due to the presence of intrinsic fat. β3‐AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical β‐AR demonstrated by functional and/or binding studies in muscle and in heart is not the β3‐AR. 6 By use of two different sets of primers for amplification of β3‐AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7 The detection of β3‐AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility β3‐AR may subserve other functions in the gastrointestinal tract. The absence of β3‐AR mRNA in rat heart or its presence with adipsin in skeletal muscle suggests that atypical β‐adrenoceptor responses in heart and skeletal muscle are unlikely to be mediated by β3‐AR.

Characterization and autoradiographic localization of β-adrenoceptor subtypes in human cardiac tissues

1 Receptor autoradiography using (—)‐[125I]‐cyanopindolol (CYP) was used to study the distribution of β‐adrenoceptor subtypes in human right atrial appendage, left atrial free wall, left ventricular papillary muscle and pericardium. 2 The binding of (—)‐[125I]‐CYP to slide‐mounted tissue sections of human right atrial appendage was time‐dependent (K1 = 4.11 ± 1.01 × 108 m−1 min−1, K−1 = 1.47 ± 0.25 × 10−3 min−1, n = 3), saturable (42.02 ± 2.96 pM, n = 4) and stereoselective with respect to the optical isomers of propranolol (pKD (—):8.97 ± 0.02, (+):6.88 ± 0.06, n = 3). 3 The proportions of β‐adrenoceptor subtypes were determined in slide‐mounted tissue sections using the antagonists CGP 20712A (β1‐selective) and ICI 118,551 (β2‐selective). In right atrial appendage and left ventricular papillary muscle 40% (34–45%) of the β‐adrenoceptors were of the β2‐subtype. 4 Images from X‐ray film and nuclear emulsion coated coverslips exposed to (—)‐[125I]‐CYP‐labelled sections showed an even distribution of β‐adrenoceptor subtypes over the myocardium of the right atrial appendage, left ventricular papillary muscle and left atrial free wall. Sections of pericardium exhibited predominantly β2‐adrenoceptors. β2‐Adrenoceptors were localized to the intimal surface of coronary arteries. 5 The selective β1‐adrenoceptor agonist R0363 and β2‐selective agonist procaterol produced concentration‐dependent inotropic responses in right atrial appendage strips. Responses to R0363 were antagonized by CGP 20712A (pKB = 9.29) suggesting an interaction with β,‐adrenoceptors. Responses to procaterol were antagonized by ICI 118,551 (pKB = 9.06) suggesting an interaction at β2‐adrenoceptors. 6 The finding that a significant proportion of human myocardial adrenoceptors are of the β2‐subtype has important clinical implications for the involvement of these receptors in the control of heart rate and force, and the autoradiographic evidence suggests other roles in the coronary vasculature and pericardium.

Autoradiographic analysis of receptors on vascular endothelium

Receptor autoradiography was used to examine the distribution of muscarinic cholinoceptors ([3H]QNB), alpha 2-adrenoceptors ([3H]rauwolscine), beta-adrenoceptors ([125I]CYP) and substance P receptors ([125I]BHSP) in rabbit aorta, pulmonary artery, rat aorta, dog aorta, splenic, renal and coronary arteries, bovine aorta and coronary arteries. Muscarinic cholinoceptors and alpha 2-adrenoceptors were not associated with endothelium in any of the blood vessels examined. Substance P receptors were found on endothelium in dog renal but not bovine coronary arteries, and beta-adrenoceptors were found on endothelium in dog coronary arteries but not bovine aorta. The results suggest that endothelium-dependent relaxation can result either from activation of receptors located directly on the endothelial cells or, as is the case for ACh, by an indirect mechanism via activation of receptors located on the vascular smooth muscle.

Characterization of β1- and β2-adrenoceptors in rat skeletal muscles

Abstract Binding studies with (−)-[ 125 I]cyanopindolol (ICYP) were conducted to characterize β-adrenoceptors in plantaris and soleus muscles of rats (male, 250–300 g). The distribution of β 1 - and β 2 -adrenoceptors in different muscle fiber types, identified in serial sections by succinic dehydrogenase (SDH) staining, was studied by autoradiography. The densities of binding sites (B max , mol/mg protein) were 5.4 ± 0.9 (mean ± SEM) in plantaris and 11.5 ± 2.0 in soleus muscle. In plantaris muscle, monophasic competition curves were observed when binding experiments were performed using CGP 20712A (50 pM to O.SmM), a β 1 -adrenoceptor selective antagonist, or ICI 118,551 (50 pM to 20,μM), a β 2 -adrenoceptor selective antagonist, to compete for ICYP binding. Analysis with LIGAND revealed a single binding site with a K D value of 2.41 ± 0.56 nM (mean ± SEM) for ICI 118,551 and 8.93 ± 3.00 μM for CGP 20712A, indicating the presence of a homogeneous population of β 2 -adrenoceptors. In soleus muscle, competition curves were biphasic with 16–21% β 1 -adrenoceptors. Autoradiographic studies supported the findings from binding studies with membrane homogenates. The ICYP binding pattern was associated closely with the muscle fiber types identified by SDH staining. Propranolol-resistant binding sites were observed, and these sites were associated with muscle fibers positive to SDH staining.

[3H]-rauwolscine binding to alpha 2-adrenoceptors in the mammalian kidney: apparent receptor heterogeneity between species.

1 Binding of the α2‐adrenoceptor antagonist [3H]‐rauwolscine was characterized in membrane preparations from the kidneys of mouse, rat, rabbit, dog, and man. 2 In all species, binding reached equilibrium within 45 min and dissociated at a single exponential rate after addition of phentolamine 10 μM. 3 Saturation studies showed that the affinity of [3H]‐rauwolscine was similar in all species (2.33‐3.03 nM) except man where it was significantly higher (0.98 nM). Marked differences were seen in the density of binding sites, increasing in the order: man < dog < rabbit < rat < mouse. In all cases, Hill coefficients were not significantly different from unity. 4 [3H]‐rauwolscine binds with low affinity (KD > 15 nM) to membranes prepared from guinea‐pig kidney. The low affinity binding is not due to the absence of particular ions in the incubation medium or to receptor occupation by endogenous agonist. 5 The binding in all species was found to be stereoselective with respect to the isomers of noradrenaline. However, differences were seen in the characteristics of agonist interactions with the binding site both between isomers and between species. 6 Marked differences in affinity of particular α‐adrenoceptor antagonists were observed for α2‐adrenoceptors labelled by [3H]‐rauwolscine. These differences were most evident with the α1‐adrenoceptor selective antagonist prazosin which displayed inhibition constants (Ki values) of 33.2, 39.5, 261, 570 and 595 nM in rat, mouse, dog, man and rabbit, respectively. 7 Differences are apparent in the characteristics of α2‐adrenoceptors labelled by [3H]‐rauwolscine between species and it is suggested that the differences observed for α1‐selective antagonists such as prazosin may be related to binding to additional sites in the vicinity of the α2‐adrenoceptor.

Autoradiographic localization and function of β-adrenoceptors on the human internal mammary artery and saphenous vein

1 Receptor autoradiography with (−)‐[125 I]‐cyanopindolol (CYP) was used to study the distribution of β1 and β2‐adrenoceptor subtypes in the human internal mammary artery and saphenous vein. 2 Images from X‐ray film and nuclear emulsion coated coverslips, exposed to [125 I]‐CYP labelled sections, showed a high density of β2‐adrenoceptors localized to the endothelium of the internal mammary artery and fewer β2‐adrenoceptors on the smooth muscle. 3 The function of β‐adrenoceptors in ring preparations of the internal mammary artery was investigated in organ bath studies. (−)‐Isoprenaline produced concentration‐dependent relaxation of phenylephrine contracted rings. The potency and maximal effects of (−)‐isoprenaline were not influenced by the presence of the endothelium. 4 Images of [125 I]‐CYP binding to the saphenous vein, from X‐ray film and nuclear emulsion coated coverslips, showed localization of β2‐adrenoceptors to the outer smooth muscle and not to the endothelium. 5 Relaxation of mammary artery and saphenous vein to (−)‐isoprenaline is mediated via β2‐adrenoceptors located on the smooth muscle. Endothelial β2‐adrenoceptors, although present on the internal mammary artery, mediate other functions.