Characterization of beta-adrenoceptor mediated smooth muscle relaxation and the detection of mRNA for beta1-, beta2- and beta3-adrenoceptors in rat ileum

Abstract

1. Functional and molecular approaches were used to characterize the beta-AR subtypes mediating relaxation of rat ileal smooth muscle. 2. In functional studies, (-)-isoprenaline relaxation was unchanged by CGP20712A (beta1-AR antagonist) or ICI118551 (beta2-AR antagonist) but shifted by propranolol (pKB=6.69). (+/-)-Cyanopindolol, CGP12177 and ICID7114 did not cause relaxation but antagonized (-)-isoprenaline relaxation. 3. BRL37344 (beta3-AR agonist) caused biphasic relaxation. The high affinity component was shifted with low affinity by propranolol, (+/-)-cyanopindolol, tertatolol and alprenolol. CL316243 (beta3-AR agonist) relaxation was unaffected by CGP20712A or ICI118551 but blocked by SR58894A (beta3-AR antagonist; pA2 = 7.80). Enhanced relaxation after exposure to forskolin and pertussis toxin showed that beta3-AR relaxation can be altered by manipulation of components of the adenylate cyclase signalling pathway. 4. The beta-AR agonist RO363 relaxed the ileum (pEC50=6.18) and was blocked by CGP20712A. Relaxation by the beta2-AR agonist zinterol (pEC50=5.71) was blocked by SR58894A but not by ICI118551. 5. In rat ileum, beta1-, beta2- and beta3-AR mRNA was detected. Comparison of tissues showed that beta3-AR mRNA expression was greatest in WAT>colon=ileum >cerebral cortex>soleus; beta1-AR mRNA was most abundant in cerebral cortex > WAT > ileum = colon > soleus; beta2-AR mRNA was expressed in soleus > WAT > ileum = colon > cerebral cortex. 6. These results show that beta3-ARs are the predominant beta-AR subtype mediating rat ileal relaxation while beta1-ARs may produce a small relaxation. The beta2-AR agonist zinterol produces relaxation through beta3-ARs and there was no evidence for the involvement of beta2-ARs in relaxation despite the detection of beta2-AR mRNA.

Figure 1

The effect of β₁/β₂ antagonists on the relaxation responses to (−)-isoprenaline in the rat isolated ileum precontracted with carbachol. Graph shows (−)-isoprenaline concentration response curves in the absence and presence of (A) propranolol (B) the β₁-AR antagonist CGP 20712A and (C) the β₂-AR antagonist ICI 118551. Inset (A) shows a Schild plot with a slope significantly less than unity. Note the lack of effect of CGP 20712A and the small rightward shift caused only by a high concentration of ICI 118551. Points show mean±s.e.mean (n=4) and are expressed as a percentage of maximum relaxation by papaverine (100 μM).

Figure 2

The effect of (±)-cyanopindolol (CYP) on the relaxation responses to (−)-isoprenaline in the rat isolated ileum precontracted with carbachol. Points show mean±s.e.mean and are expressed as a percentage of maximum relaxation by papaverine (100 μM) (n=4). Inset shows the Schild plot for (±)-CYP with slope 0.97 and pA₂ value of 7.98.

Figure 3

The effect of the stereoisomers of alprenolol (A, C and E) and tertatolol (B, D and F) on the relaxation responses to (−)-isoprenaline in the rat isolated ileum precontracted with carbachol. Shown are (−)-isoprenaline concentration response curves in the absence and presence of (A) (−)-alprenolol (B) (−)-tertatolol (C) (+)-alprenolol and (D) (+)-tertatolol. Points show mean±s.e.mean (n=4–6) and are expressed as a percentage of maximum relaxation by papaverine (100 μM). Slope values calculated from Schild plots for alprenolol (E) and tertatolol (F) show that the (−)-isomer in each case has a slope value not significantly different from unity while both (+)-isomers had slope values significantly less than unity. Slope values and pA₂ values were calculated and are shown in Table 1.

Figure 4

The effect of β₁- and β₂-AR selective agonists on relaxation in rat ileum precontracted with carbachol. Graph (A) shows relaxation with the β₂-AR agonist zinterol in the absence and presence of ICI 118551 and CGP 20712A. Also shown in (A) are the relaxation responses by the β₁-AR agonist RO363 in the absence and presence of CGP 20712A (n=4). Graph (B) shows the inhibition of zinterol relaxation by the β₃-AR antagonist SR 58894A in the presence of ICI 118551.

Figure 5

Mean concentration response curves for the relaxation of carbachol precontracted rat ileum by the atypical β-AR agonists (A) BRL 37344 and (B) SR 58611A. Both graphs show control concentration response curves and in the presence of (−)-propranolol (1 μM). Also shown are BRL 37344 concentration response curves in the presence of (C) (−)-tertatolol 3 μM and (+) tertatolol 10 μM; (D) (−)-alprenolol 3 μM and (+)-alprenolol 30 μM and (E) (±)-cyanopindolol 0.3 μM. Points are expressed as a percentage of maximum relaxation by papaverine (100 μM). Points show mean±s.e.mean (n=4–6). The pK_B values for all compounds were calculated and are shown in Table 2. Graph (F) shows a correlation plot of pK_B values for a range of compounds obtained against either (−)-isoprenaline concentration-response or BRL 37344 concentration-response curves. The regression line has a correlation coefficient of r=0.973, P=0.0011 and a slope of 0.863 (95% C.L. 0.58–1.14, n=6).

Figure 6

Mean concentration response curves for the relaxation of carbachol precontracted rat ileum by the β₃-AR agonist CL 316243. Graph (A) shows monophasic CL 316243 concentration-response curves in intact preparations and preparations with the mucosa removed. Graph (B) shows a control CL 316243 C-R curve and in the presence of the β₁-AR antagonist CGP 20712A 100 nM, the β₂-AR antagonist ICI 118551 100 nM and both antagonists in combination. Graph (C) shows the concentration dependent rightward shifts of the CL 316243 C-R curve with the β₃-AR antagonist SR 58894A. Graph (D) shows the Schild Plot (slope 95% C.I. 0.94–1.68) for SR 58894A. Points show mean±s.e.mean (n=4–6).

Figure 7

Effects of preincubation of rat ileal strips with forskolin for 45 min at 37°C on concentration-response curves to (A) isoprenaline and (B) CL 316243. Also shown (C) is the effect of preincubation with pertussis toxin (2 h, 37°C, n=4) on CL 316243 concentration-response curves.

Figure 8

Detection of β₁-AR, β₂-AR, β₃-AR and actin mRNA by RT/PCR. Cycle numbers were 27 for β-ARs and 16 for actin. Sizes of the PCR products were determined from ethidium bromide stained gels by comparison with 100 bp DNA ladder (Pharmacia). The blots were apposed to phosphorimager plates for 7 h prior to scanning.