A comparison of the influence of N-isopropyl and N-tert butyl substituents on the affinity of ligands for sinoatrial ?-adrenoceptors in rat atria and ?-adrenoceptors coupled to the adenylyl cyclase in kitten ventricle

We have recently suggested the existence in the heart of a ‘putative β4‐adrenoceptor’ based on the cardiostimulant effects of non‐conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block β1‐ and β2‐adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (−)‐[3H]‐CGP 12177A ((−)‐4‐(3‐tertiarybutylamino‐2‐hydroxypropoxy) benzimidazol‐2‐one) in rat atrium. We investigated (−)‐[3H]‐CGP 12177A for this purpose for two reasons, because it is a non‐conventional partial agonist and also because it is a hydrophilic radioligand. Increasing concentrations of (−)‐[3H]‐CGP 12177A, in the absence or presence of 20 μM (−)‐CGP 12177A to define non‐specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to β1‐ and β2‐adrenoceptors (pKD 9.4±0.1, Bmax 26.9±3.1 fmol mg‐1 protein) and higher concentrations bound to the ‘putative β4‐adrenoceptor’ (pKD 7.5±0.1, Bmax 47.7±4.9 fmol mg−1 protein). In other experiments designed to exclude β1‐ and β2‐adrenoceptors, (−)‐[3H]‐CGP 12177A (1–200 nM) binding in the presence of 500 nM (−)‐propranolol was also saturable (pKD 7.6±0.1, Bmax 50.8±7.4 fmol mg−1 protein). The non‐conventional partial agonists (−)‐CGP 12177A (pKi 7.3±0.2), (±)‐cyanopindolol (pKi 7.6±0.2), (−)‐pindolol (pKi 6.6±0.1) and (±)‐carazolol (pKi 7.2±0.2) and the antagonist (−)‐bupranolol (pKi 6.6±0.2), all competed for (−)‐[3H]‐CGP 12177A binding in the presence of 500 nM (−)‐propranolol at the ‘putative β4‐adrenoceptor’, with affinities closely similar to potencies and affinities determined in organ bath studies. The catecholamines competed with (−)‐[3H]‐CGP 12177A at the ‘putative β4‐adrenoceptor’ in a stereoselective manner, (−)‐noradrenaline (pKiH 6.3±0.3, pKiL 3.5±0.1), (−)‐adrenaline (pKiH 6.5±0.2, pKiL 2.9±0.1), (−)‐isoprenaline (pKiH 6.2±0.5, pKiL 3.4±0.1), (+)‐isoprenaline (pKi<1.7), (−)‐RO363 ((−)‐(1‐(3,4‐dimethoxyphenethylamino)‐3‐(3,4‐dihydroxyphenoxy)‐2‐propranol)oxalate, pKi 5.5±0.1). The inclusion of guanosine 5‐triphosphate (GTP 0.1 mM) had no effect on binding of (−)‐CGP 12177A or (−)‐isoprenaline to the ‘putative β4‐adrenoceptor’. In competition binding studies, (−)‐CGP 12177A competed with (−)‐[3H]‐CGP 12177A for one receptor state in the absence (pKi 7.3±0.2) or presence of GTP (pKi 7.3±0.2). (−)‐Isoprenaline competed with (−)‐[3H]‐CGP 12177A for two states in the absence (pKiH 6.6±0.3, pKiL 3.5±0.1; % H 25±7) or presence of GTP (pKiH 6.2±0.5, pKiL 3.4±0.1; % H 37±6). In contrast, at β1‐adrenoceptors, GTP stabilized the low affinity state of the receptor for (−)‐isoprenaline. The specificity of binding to the ‘putative β4‐adrenoceptor’ was tested with compounds active at other receptors. High concentrations of the β3‐adrenoceptor agonists, BRL 37344 ((RR+SS)[4‐[2‐[[2‐(3‐chlorophenyl)‐2‐hydroxy ‐ ethyl]amino]propyl]phenoxy]acetic acid, 6 μM), SR 58611A (ethyl{(7S)‐7‐[(2R)‐2 ‐ (3 ‐ chlorophenyl) ‐ 2 ‐ hydroxyethylamino] ‐ 5,6,7,...

Section: Discussionsupporting

confidence: 67%

Validity of (−)‐[³H]‐CGP 12177A as a radioligand for the ‘putative β₄‐adrenoceptor’ in rat atrium

Sarsero

Molenaar

Kaumann

1998

“…One line of evidence for atypical P-ARs in the rat heart is the finding that a series of fl-AR partial agonists related to pindolol cause positive chronotropic effects at concentrations greatly in excess of their affinities at PI and f2-ARs (Kaumann et al, 1979;Kaumann, 1989). Mediation of these effects would presumably be via receptors present in the sinoatrial (SA) node.…”

Section: Design Of Oligonucleotide Primers For Pcrmentioning

confidence: 99%

Expression of β₃‐adrenoceptor mRNA in rat tissues

Evans

Papaioannou

Bonazzi

et al. 1996

1 This study examines the expression of f3-adrenoceptor messenger RNA (fl3-AR mRNA) in rat tissues to allow comparison with atypical f,-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 fl3-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 fl3-AR mRNA. 4 The fl3-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 fl3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of P3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of f3-AR.5 Expression of adipsin in rat skeletal muscles coupled to very low levels of fl3-AR mRNA indicates that the observed fl3-AR may be due to the presence of intrinsic fat. fl3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical fl-AR demonstrated by functional and/or binding studies in muscle and in heart is not the fl3-AR. 6 By use of two different sets of primers for amplification of fl3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7 The detection of f3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility fl3-AR may subserve other functions in the gastrointestinal tract. The absence of fl3-AR mRNA in rat heart or its presence with adipsin in skeletal muscle suggests that atypical f,-adrenoceptor responses in heart and skeletal muscle are unlikely to be mediated by fl3-AR.

“…To address this question we decided to compare the properties of the third cardiac fi-adrenoceptor with those of another atypical fi-adrenoceptor in the same species. We chose the rat heart and colon because in this species there is both in vitro (Kaumann et al, 1979) and in vivo (Malinowska & Schlicker, 1996) evidence for a third cardiac /3adrenoceptor (assessed with non-conventional partial agonists) and a colonic atypical fl-adrenoceptor that greatly resembles the #3 subtype (McLaughlin & MacDonald, 1990; reviewed in Kaumann, 1993 andPietri-Rouxel &Strosberg, 1995). To define a fi3-adrenoceptor we followed the three criteria established by Arch & Kaumann (1993):…”

Section: Introductionmentioning

confidence: 99%

Differences between the third cardiac β‐adrenoceptor and the colonic β₃‐adrenoceptor in the rat

Kaumann

Molenaar

1996

151

171

1 The heart of several species including man contains atypical JJ-adrenoceptors, in addition to coexisting /3k-and fl2-adrenoceptors. We now asked the question whether or not the third cardiac fladrenoceptor is identical to the putative P3-adrenoceptor. We compared the properties of the third cardiac fl-adrenoceptor with those of fl3-adrenoceptors in isolated tissues of the rat. To study the third cardiac fl-adrenoceptor we used spontaneously beating right atria, paced left atria and paced left ventricular papillary muscles. As a likely model for putative fl3-adrenoceptors we studied atypical f1-adrenoceptors of the colonic longitudinal muscle precontracted with 30 mM KCl. We used fl3-adrenoceptor-selective agonists, antagonists and non-conventional partial agonists (ie high-affinity blockers of both f#l-and f2-adrenoceptors known to exert also stimulant effects through /3-adrenoceptors). 7 Cyanopindolol in the presence of 200 nM (-)-propranolol relaxed the colon with a pD2 of 7.0 and a maximum effect of 40% compared to (-)-isoprenaline. As expected from a partial agonist, cyanopindolol antagonized the relaxant effects of both BRL 37344 and CL 316243 with a pKp=7.6 and (-)-noradrenaline with a pKp = 7.4. 8 The following 3-adrenoceptor-selective agonists were potent colonic relaxants (pD2 values between parentheses): BRL 37344 (9.1), ZD 2079 (7.0), CL 316243 (9.0) and SR 58611A (8.2). The relaxant effects of these agonists were only marginally affected by 200 nM (-)-propranolol, not blocked by 3 Mm CGP 20712A or 3 Mm ICI 118551, and blocked by SR 59230A 3 Mm (pKB=6.9-7.5), 1 MM (-)-bupranolol (pKB= 6.2-6.4) and 2 gM (-)-propranolol (pKB= 6.3 -6.5). 9 The colonic relaxation caused by the nanomolar concentrations of the #3-adrenoceptor-selective agonists and the non-conventional partial agonists (-)-CGP 12177 and cyanopindolol and their relative resistance to blockade by antagonists with high affinity for fI-and f12-adrenoceptors but blockade by the 133-adrenoceptor selective SR 59230A agree with the hypothesis that the receptors involved are P3-adrenoceptors. On the other hand, the failure of micromolar concentrations of 113-adrenoceptor-selective agonists to produce cardiac stimulation or affect the cardiostimulant effects of (-)-CGP 12177 is inconsistent with the hypothesis that the third cardiac fl-adrenoceptor is f3. Additionally, the selective blockade of the colonic putative fl3-adrenoceptor compared to the third cardiac ,B-adrenoceptor by SR 59230A, as well as the blockade of cardiac but not colonic receptors by CGP 20712A is also inconsistent with an identical putative fl3-adrenoceptor in colon and heart. We conclude that in the rat the third cardiac fl-adrenoceptor is different from the colonic f13-adrenoceptor.