Br. J. Pharmacol. (1990), 101, 569-574 (D Macmillan Press Ltd, 1990 Evidence for the existence of 'atypical' fl-adrenoceptors (fl3-adrenoceptors) mediating relaxation in the rat distal colon in vitro

Daniel P. McLaughlin & 'Allan MacDonald

Department of Biological Sciences, Glasgow College, Cowcaddens Road, Glasgow G4 OBA

1 Experiments were carried out to characterize the adrenoceptors mediating relaxant responses in the rat distal colon. Three agonists were used: noradrenaline, and the fl3-adrenoceptor agonist BRL 37344. , and (±)cyanopindolol were tested as antagonists. Tone in the rat distal colon was induced with KCl (30-40mM) as a spasmogen, and relaxations of this KCl-induced tone produced by the agonists were measured. 2 Relaxant responses to noradrenaline that were obtained in the presence of propranolol (1 jM) were not antagonized by phentolamine (0.01 to 1 pM). Relaxant responses to isoprenaline that were obtained in the presence of phentolamine (1 pM) were antagonized in a concentration-dependent manner by propranolol (0.01 to 3,UM), although this antagonism was weak and non-competitive. Relaxant responses to BRL 37344 that were obtained in the presence of phentolamine (1 pM) were only weakly antagonized by high (1 pM) concentrations of propranolol. 3 Tachyphylaxis to BRL 37344 was observed, a second concentration-response curve being shifted to the right by 15 fold. Exposure of the tissues to BRL 37344 (1,M) between concentration-response curves also caused rightward shifts in the responses to noradrenaline (18 fold) and isoprenaline (19 fold) but not to papaverine. 4 In the presence of phentolamine (1upM) and propranolol (1p M), the rank order of potency of the agon- ists was: (-)-isoprenaline (1.0) > BRL 37344 (0.93) > (-)-noradrenaline (0.3). 5 Responses to BRL 37344 in the presence of phentolamine (1puM) and propranolol (1pM) were antago- nized by (±)-cyanopindolol (IpM), with an apparent pA2 value of 6.67. Responses to isoprenaline, under the same conditions, were antagonized in a competitive manner by (±)-cyanopindolol (0.1 to 10pM), with the slope of the Schild plot close to unity and a pA2 value of 7.12. 6 The resistance of the relaxant responses to antagonism by phentolamine and propranolol, along with the relatively high potency of the #3-adrenoceptor agonist BRL 37344 and the antagonism of 'resistant' responses by (±)-cyanopindolol would suggest that 'atypical' f6-adrenoceptors, similar to the fl3-adrenoceptors of rat adipocytes and other tissues, exist in the rat distal colon.

Introduction been presented to the British Pharmacological Society (McLaughlin & MacDonald, 1989). Responses to noradrenaline and isoprenaline which are resist- ant to blockade of classical a- and fl-adrenoceptors have been reported in a number of animal isolated tissues, such as Methods guinea-pig ileum (Wikberg, 1977; Bond et al., 1986; Bond & Clarke, 1987); rabbit stomach (Bristow et al., 1970); rabbit Tissue preparation colon (Gillespie & Khoyi, 1977); dog colon (Grivegnee et al., 1984); rat gastric fundus (Dettmar et al., 1986b; Kelly & Mac- Male Wistar rats (200-300g), fed ad libitum, were killed by a Donald, 1990) and guinea-pig gastric fundus (Coleman et al., blow to the head and cervical dislocation. The distal 6cm of 1987). So-called 'atypical',B-adrenoceptors mediating lipolysis the colon was removed and cleared of any faecal material by in rat adipocytes are also resistant to blockade by classical gentle squeezing of the colon with the fingers. Portions of fi-adrenoceptor antagonists (Stanton, 1972; Harms et al., colon were then immediately placed in cool Krebs physiologi- 1977) and in addition are characterized by the high selectivity cal saline solution (PSS). Segments (3 cm) of colon were sus- and potency of a novel group of agonists (Arch et al., 1984; pended in organ baths containing 30ml Krebs solution, at Wilson et al., 1984). It has been proposed that adipocyte a- 370C, bubbled continuously with 95% 02 and 5% CO2, adrenoceptors should be termed 'fi3-adrenoceptors' (Tan & under an initial tension of 1 g. The composition of the Krebs Curtis-Prior, 1983; Arch, 1989). The finding that selective solution was as follows (mM): NaCl 118, CaCl2 2.5, KCI 4:7, fl3-adrenoceptor agonists also have high potency at 'atypical' NaHCO3 25, KH2PO4 1.2, MgSO4 1.2 and glucose 11.1. The adrenoceptors in the guinea-pig ileum (Bond & Clarke, 1988) Krebs solution contained cocaine (3 ,M) to block neuronal and the guinea-pig gastric fundus (Coleman et al., 1987) sug- uptake of the catecholamines and ascorbic acid (30M) and gests that f3-adrenoceptors similar to those present in rat adi- EDTA (30M) to prevent oxidation of the catecholamines. In pocytes may exist in gastrointestinal tissues. some experiments, hydrocortisone (301pM) was also included in The present study investigated responses to the catechol- the Krebs solution, to block extra-neuronal uptake of iso- amines, noradrenaline and -isoprenaline and the selective prenaline. Tissues were allowed to equilibrate for at least f3-adrenoceptor agonist, BRL 37344 (Arch et al., 1984) in the 30 min before experimental procedures were begun. rat distal colon. A preliminary account of these findings has Concentration-response curves to noradrenaline were con- structed in the presence of propranolol (1 pM) to allow investi- 'Author for correspondence. gation of the contribution of a-adrenoceptors to responses. 570 D.P. McLAUGHLIN & A. MAcDONALD

Concentration-response curves to isoprenaline were carried Results out in the presence of phentolamine (1 pM) to remove any pos- sible contribution from a-adrenoceptors. Responses to noradrenaline Concentration-response curves (CRCs) In the presence of propranolol (1 pM), noradrenaline relaxed KCl-induced tone in the rat distal colon, with a plCo of The relaxant action of agonists was determined by measuring 6.3 + 0.18 (n = 4). These relaxant responses to noradrenaline relaxations of KCI-induced tone evoked by addition of the were highly reproducible, CRCs being unchanged when con- agonists. Initially a CRC to KCI was constructed for each structed at hourly intervals over a period of 3 h (Table 1). tissue, to determine a concentration of KCI which gave The CRCs to noradrenaline were unaffected by phentola- approximately 75% of maximal tone. Concentrations of 30- mine (0.01 to 1 gm, Table 1). Also, in three experiments in 40 mm were added hypertonically to the organ baths by the presence of 1 iM propranolol, neither nor adding 0.3-0.5 ml of a 2 M KCl stock solution. UK 14,304 relaxed KCl-induced tone in the rat distal colon CRCs to adrenoceptor agonists were constructed by cumu- (in concentrations up to 10pUM). lative addition (0.5 log unit increments) to KCI-contracted strips at 1-2 min intervals until a stable response was observed. CRCs were constructed at 1 h intervals, allowing Responses to isoprenaline 45 min equilibration time for antagonists, which were added at the end of each CRC. In the presence of phentolamine (1 M), isoprenaline relaxed In experiments testing the effect of antagonists on the relax- KCl-induced tone in the rat distal colon, with a pIC50 of ations evoked by BRL 37344, only one CRC to BRL 37344 7.66 + 0.06 (n = 6). The relaxant responses to isoprenaline was constructed in any one tissue. In these cases paired were highly reproducible, CRCs being unchanged when con- experiments were carried out. If CRCs to isoprenaline were structed at hourly intervals over a period of 3h (Figure la). not significantly different in the paired tissues then one tissue The responses to isoprenaline were weakly antagonized in a was subsequently exposed to BRL 37344 in the presence of concentration-dependent manner by propranolol (0.01 to antagonist and a paired tissue exposed to BRL 37344 in the 3ym, Figure lb). A Schild plot (Figure lc) revealed that this absence of antagonist. The antagonist was present for 45min antagonism was non-competitive in nature, as demonstrated before construction of CRCs to BRL 37344. by the slope of the plot, which was significantly less than unity (0.44 + 0.18, 95% CL, n = 36). An apparent 'pA2' value for propranolol, calculated on the basis of the shift produced by Schild plots 1 uM propranolol, gave a value of 6.57 + 0.18 (n = 6). Agonist concentration-ratios (CRs) were determined from the Hydrocortisone (30pM) had no effect on the potency of iso- IC50 point on the CRC, with or without antagonist. The plot prenaline (pIC50 of 7.65 + 0.1, n = 8) or on the antagonism by of log (agonist concentration ratio - 1.0) versus log propranolol (apparent pA2 of 6.48 + 0.09, n = 8). [antagonist] (Arunlakshana & Schild, 1959) was analysed by linear regression. Antagonism was considered to be competi- Responses to BRL 37344 tive if the slope of the regression line was not significantly different from unity. In these cases a mean pA2 value was In the presence of phentolamine (1,UM) thefl3-adrenoceptor obtained from individual estimates using the equation agonist BRL 37344 relaxed KCI-induced tone in the rat distal = - - colon, with a pIC50 of 7.33 + 0.12 (n 4). Tachyphylaxis to pA2 = log(agonist concentration ratio 1) log[antagonist] BRL 37344 was observed, a second CRC being shifted to the after first verifying that there was no significant regression of right by 15 fold, when compared to the control CRC (Figure pA2 on antagonist concentration (MacKay, 1978). 2a). The responses to BRL 37344 were not antagonized by low In cases where the slope of the Schild plot was significantly concentrations of propranolol (0.01 to 0.1 pM), but were different from unity, or in cases where only one concentration antagonized to some extent by 1 pM propanolol (mean 8 fold of antagonist was tested, 'apparent' pA2 values were estimated shift of IC50 values, P < 0.05, Figure 2b). An apparent pA2 from a single antagonist concentration and the above equa- value for propranolol, calculated on the basis of the shift pro- tion. duced by 1 JM propranolol, gave a value of 6.39. In the presence of phentolamine (1,pM) and propranolol Drugs used (1pM), exposure of the tissues to BRL 37344 (1pM) for 3min approximately mid-way between CRCs to noradrenaline and The following were dissolved in distilled water, with the excep- isoprenaline produced significant (paired t test, P < 0.01) tion of hydrocortisone and (± )-cyanopindolol which were dis- mean rightward 18 and 19 fold shifts of the CRCs to nor- solved in absolute ethanol and 0.1M tartaric acid, respectively: and isoprenaline, respectively (Figures 3a and 3b). BRL 37344 (sodium-4[2[2-hydroxy-2(3-chlorophenyl)ethyl- amino]propyl]phenoxyacetate) (Beecham Research Labor- cirazoline Table 1 Effect of phentolamine on noradrenaline-induced atories, Great Burgh, Epsom); hydrochloride relaxations in rat distal colon in the presence of propranolol (Synthelabo); cocaine hydrochloride (Thornton and Ross (1pM) Ltd.); (±)-cyanopindolol (Sandoz); hydrocortisone-21-acetate (Sigma); (-)-isoprenaline (+)-bitartrate (Sigma); (-)-nor- Phentolamine pIC50 of adrenaline (+)-bitartrate (arterenol) (Sigma); papaverine (pM) noradrenaline hydrochloride (Sigma); phentolamine mesylate (Ciba-Geigy); time time (± )-propranolol hydrochloride (Sigma); UK 14,304 (5-bromo- CRC No. controls treated controls treated 6[2-imazolin-2-yl-amino]quinoxaline) bitartrate (Pfizer). 0 0 6.55 ± 0.02 6.30 ± 0.18 2 0 0.01 6.43 ± 0.08 6.35 ± 0.19 Statistical analysis 3 0 0.1 6.51 + 0.10 6.37 + 0.15 1.0 6.50 + 0.03 6.33 + 0.08 Results are expressed as mean + s.e.mean with the number of 4 0 determinations, n, given in parentheses with the exception of Four concentration-response curves (CRCs) to noradrenaline regression line slopes which are expressed as slope + 95% were constructed at hourly intervals, allowing 45 min equili- confidence limits. Statistical significance between two data sets bration with each of the concentrations of phentolamine in was tested by either Student's t test or a paired t test. A prob- the treated tissues. Values shown are means + s.e.mean for 6 ability level of P < 0.05 was considered statistically significant. tissues in each case. 'ATYPICAL' fi-ADRENOCEPTORS IN RAT DISTAL COLON 571

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Figure 2 Effect of BRL 37344 on KCl-induced tone in the rat distal colon in the presence of 1 fM phentolamine. (a) Time controls for responses to BRL 37344. Curves represent first (El, n = 4) and second (M, n = 4) concentration-response curves, constructed 1 h apart. (b) C-) The effect of propranolol, 0.01 iM (a, n = 4), 0.1 FM (0, n = 4) and 1 FM (A, n = 4) on control (O.' n = 4) concentration-response curves to BRL 37344. Tone was raised by the addition of KCI (30-40mM) to 0) the organ baths. Error bars have been omitted from the figure for 0 clarity. In each case, standard errors were less than 15%.

-8.5 -7.5 -6.5 -5.5 -4.5 -3.5 -2.5 log [Prop] (M) Effect of( )-cyanopindolol on responses to isoprenaline and BRL 37344 under conditions of cx- and Figure 1 (a) Time control concentration-response curves to iso- fi-adrenoceptor blockade prenaline (Isop) in the rat distal colon in the presence of 1 FM phentol- was - amine. Tone raised by the addition of KCI (30-40mM) to the In the presence of phentolamine (1 gM) and propranolol (1 pM), organ baths. Each of the concentration-response curves was con- responses to BRL 37344 were antagonized by the f6- structed at hourly intervals: the first (El. n = 6), second (I, n = 6), adrenoceptor antagonist (±)-cyanopindolol (1pM), with an third (0. n = 6) and fourth curves (A, n = 6) are shown. (b) The effect apparent pA2 value calculated from the single concentration of propranolol, in concentrations 0.01 pM (-, n = 6), 0.1 pM (0, = n = 6), 0.3pM (A, n = 6), 1pM (0, n = 12) and 3pM (A, n = 6) on of cyanopindolol of 6.67 + 0.25 (n 7). Also, cyanopindolol control (El, n = 12) concentration-response curves to isoprenaline in (0.1 to 10pM) antagonized responses to isoprenaline in a the rat distal colon is shown. Error bars have been omitted for clarity. concentration-dependent manner (Figure 4a). The antagonism Standard errors were less than 15% in each case. (c) Schild plot of the of the action of isoprenaline was competitive in nature, since antagonism of isoprenaline action by propranolol (Prop) in the rat the slope of the Schild plot was not significantly different from distal colon. unity (1.03 + 0.17, 95% CL, n = 12, Figure 4b). The pA2 value for cyanopindolol was 7.12 + 0.06 (n = 12). The difference between the pA2 values for cyanopindolol against BRL 37344 and isoprenaline was statistically significant (P < 0.05, Student's t test). Cyanopindolol itself had no effect on the The same experimental procedure, however, had no effect on degree of tone induced by KCl (tone in cyanopindolol-treated the relaxant effect of papaverine on KCl-induced tone in the strips as % of control, n = 4; 0.1 M, 89.7 + 7; 1 pM, 105 + 10; rat distal colon (Figure 3c). 1OpM, 109 ± 7).

Order ofpotency ofagonists under conditions ofclassical Discussion cx- and ,f-adrenoceptor blockade The action of catecholamines on the rat distal colon was not In the presence of phentolamine (1pM) and propranolol (1p M), mediated by a-adrenoceptors, since (a) the responses to nor- the pIC50 s of isoprenaline, BRL 37344 and noradrenaline adrenaline were resistant to the classical a-adrenoceptor were, respectively, 6.85 + 0.18 (12), 6.82 + 0.16 (4) and antagonist phentolamine and (b) the selective al- and 6.33 + 0.08 (6), giving a rank order of potency of: isoprenaline a2-adrenoceptor agonists cirazoline (van Meel et al., 1981) and (1.0) > BRL 37344 (0.93) > noradrenaline (0.3). UK 14,304 (Cambridge, 1981) were devoid of any relaxant 572 D.P. McLAUGHLIN & A. MAcDONALD

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c Figure 4 (a) The effect of cyanopindolol, in concentrations 0.1 JiM 120 - (a, n = 4), 1lM (O. n = 4) and 10pM (A, n = 4) on control concentration-response curves n = 4) to 01) (El, isoprenaline in the rat C 100 - distal colon in the presence of 1 AM phentolamine and 1 M propran- 0 olol. Tone was raised by the addition of KCI (30-40mM) to the organ Co 80- baths. (b) Schild plot of the antagonism of isoprenaline action by Ne0 cyanopindolol. 60- x 40 similarly low pA2 value (6.6) for propranolol against the lipo- Co I/u lytic effect of isoprenaline was reported (Wilson et al., 1984) 20 - 0-o and therefore it seems possible that the receptors mediating relaxation of the colon and lipolysis are similar. -7 -6 -5 -4 -3 -2 Non-competitive antagonism of responses to catechol- log [Papaverine] (M) amines may result from failure to block extraneuronal uptake processes for the catecholamines (Furchgott, 1967). This possi- Figure 3 The effect of exposure of the tissues to 1 FM BRL 37344 for bility was ruled out in the present study since both the a period of 3min between concentration-response curves to agonists. responses to isoprenaline and the antagonism by propranolol In each case, first (EO. n = 6) and second (U, n = 6) concentration- were similar in the presence and absence of the extraneuronal response curves are shown. Tone was raised by the addition of KC1 uptake blocker hydrocortisone (Iversen & Salt, 1970). (30-40mM) to the organ baths and phentolamine (1 pM) and propra- Relaxation of the rat distal colon mediated by the selective nolol (1 pM) were present throughout. (a) Noradrenaline (NA), (b) Iso- prenaline (Isop) and (c) papaverine. fl3-adrenoceptor agonist BRL 37344 was only weakly antago- nized by propranolol, the apparent 'pA2' value of 6.39 being similar to the reported pA2 values for propranolol against action. Inhibitory postjunctional a1-adrenoceptors have pre- ,f3-adrenoceptor agonists stimulating lipolysis of rat white viously been shown to exist in the rat distal colon (Dettmar et adipocytes (Wilson et al., 1984). This again suggests that the al., 1986a) and the failure to show their presence in the present receptors mediating relaxation in the rat distal colon and the study is probably related to the KCI-induced depolarization, receptors mediating lipolysis in adipocytes may be similar. since cx-adrenoceptor-mediated inhibitory effects in intestinal The relative potency of BRL 37344 under conditions of a- smooth muscle involve abolition of spontaneous spike dis- and P-adrenoceptor blockade, isoprenaline (1.0) > BRL 37344 charge and hyperpolarization (Bulbring, 1954; 1957) and are (0.93) > noradrenaline (0.3), compares with rat adipocyte not seen if the tissue is depolarized sufficiently to block spike f?3-adrenoceptors where BRL 37344, or its methyl ester BRL generation (Magaribuchi & Kuriyama, 1972). Thus the 35135, were equipotent with or more potent than isoprenaline present study does not rule out the presence of inhibitory a- (Arch et al., 1984; Wilson et al., 1984). A similar high potency adrenoceptors in this preparation, but their effects are not of selective,3-adrenoceptor agonists was found in guinea-pig seen in the presence of KCl-induced tone. gastric fundus (Coleman et al., 1987) and guinea-pig ileum The relaxations to the catecholamines were also elicited in (Bond & Clarke, 1988). the presence of propranolol (1 pM), conditions under which In the present study BRL 37344 caused tachyphylaxis to one would assume that classical f-adrenoceptors had been itself and also to noradrenaline and isoprenaline. This was not blocked. Propranolol did produce a shift of the isoprenaline simply a non-specific effect on all relaxant responses in the rat CRC but the antagonism was non-competitive (slope of Schild distal colon since responses to papaverine, which acts by inhi- plot less than unity) and weak with an apparent pA2 of 6.57 bition of phosphodiesterase (Bar, 1974), were unaffected. compared with 8.2-8.8 for classical 01J-adrenoceptors medi- Tachyphylaxis to BRL 35135 has also been reported in ating atrial stimulation and 8.3-8.6 for 82-adrenoceptors guinea-pig gastric fundus (Coleman et al., 1987). The mecha- mediating tracheal relaxation (Wilson et al., 1984). However a nism of the tachyphylaxis in the present study and in the 'ATYPICAL' fi-ADRENOCEPTORS IN RAT DISTAL COLON 573 study by Coleman et al. (1987) is unclear but the cross- partial agonist effect of at f3-adrenoceptors and the desensitization may be evidence that BRL 37344 and the cate- antagonistic effect of cyanopindolol at 'atypical' f- cholamines are acting at the same site. adrenoceptors in the gut. Pindolol analogues may also act as Responses to isoprenaline in the presence of a- and 6- partial agonists at rodent f3-adrenoceptors (Engel et al., adrenoceptor blockade with phentolamine and propranolol 1981). However, there was no evidence in the present study for were antagonized in a competitive manner by cyanopindolol. a partial agonist effect of cyanopindolol at the concentrations Cyanopindolol has previously been shown to be a potent used (up to 10pM, approximately 100 x pKa at the 'atypical' competitive antagonist at propranolol-resistant 'atypical' f6- fl-adrenoceptors in this study). adrenoceptors in guinea-pig ileum (Blue et al., 1989) and the Recently, a human gene has been isolated that encodes a reported pA2 of 7.63 is similar to that obtained in the present product referred to as the "f3-adrenoceptor" (Emorine et al., study (7.12). Thus the receptor mediating responses to iso- 1989). Investigation of the properties of this receptor in prenaline in rat distal colon appears to be similar to the Chinese Hamster ovary cells transfected with the gene showed 'atypical' fl-adrenoceptor present in guinea-pig ileum. A Schild low affinities for classical fi-adrenoceptor antagonists and a regression was not carried out for cyanopindolol against BRL high potency of BRL 37344. It is likely, therefore, that this 37344 but the single concentration of cyanopindolol tested receptor represents a separate class of fl-adrenoceptor which (1 pUM) gave an apparent pA2 of 6.67 which was significantly includes the f3-adrenoceptors of adipocytes, myocardium and lower than the value obtained with isoprenaline as agonist. gastrointestinal smooth muscle. It has been suggested that Lower pA2 values for fl-adrenoceptor antagonists when BRL these receptors may represent intermediate stages in evolution 37344 or analogues were the agonists than when isoprenaline towards classical fl- and fl2-adrenoceptors (Bond & Clarke, was the agonist have previously been found (Stock & Sudera, 1988; Zaagsma & Nahorski, 1990). 1988; Jones et al., 1989; Arch, 1989), suggesting that the inter- In conclusion, the resistance of catecholamine-induced action of the BRL compounds with the f3-adrenoceptor may relaxations to propranolol, the relatively high potency of the differ from that of isoprenaline. It has been suggested that the fl3-adrenoceptor agonist, BRL 37344, and the competitive bulky N-substituent of the BRL compounds may bind to an antagonism of propranolol-resistant responses by cyano- accessory site and reduce the affinity of antagonists for the pindolol suggest that the rat distal colon contains 'atypical' main binding site (Arch, 1989). /J-adrenoceptors similar to the fl3-adrenoceptors in rat adi- There is evidence that some partial adrenoceptor agonists pocytes and other tissues. The physiological significance of acting on human myocardial tissue, designated unconven- such receptors in the gastrointestinal tract is at present tional and including (-)-pindolol, cause part of their effects unknown, although it has been shown that their activation by through heart adrenoceptors which are neither alJ- nor selective agonists can affect the motility of rat proximal colon /32-adrenoceptors (Walter et al., 1984; Kaumann, 1989). These in vivo (Manara et al., 1989). agonist effects are seen at concentrations greater than are necessary to produce, blockade of and fl,- f2-adrenoceptors, UK14,304 bitartrate and cirazoline hydrochloride were gifts from Dr are resistant to conventional fl-blockade and have been attrib- P.W. Dettmar of Reckitt and Colman plc, Dansom Lane, Hull. BRL uted to an action at a third heart fi-adrenoceptor, termed f3 37344 was kindly supplied by Dr M.A. Cawthorne, Beecham Phar- (Kaumann, 1989). It may be that the cardiac fl3-adrenoceptors maceuticals Research Division, Great Burgh, Epsom. D.P.McL. was are similar to the fl-adrenoceptors of the rat distal colon and supported by a Glasgow College Research Studentship. The authors other tissues and that there is some relationship between the are also grateful to the College for financial support.

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