Pharmacological and Immunocytochemical Characterization of Subtypes of Alpha-1 Adrenoceptors in Dog Aorta1
- 1Department of Biomedical Sciences, McMaster University (A.M.L., H.L.-C., C.Y.K., E.E.D.), Hamilton, ON L8N 3Z5, Canada and 2Research Service, (151), Edward Hines Jr. VA Hospital (R.D.B.), Hines, Illinois
Abstract
In this study, the effects of nine alpha-1 adrenoceptor antagonists [prazosin, WB 4101 (WB), chloroethylclonidine (CEC), 5-methylurapidil (5-MU), BMY 7378 (BMY), MDL 73005EF (MDL73), MDL 72832 (MDL72), RS 17053 (RS) and SK&F 105854 (SKF)] were studied on contractile responses to phenylephrine (PE) of the endothelium-denuded dog aorta in vitro. All antagonists, except CEC, 5-MU and RS, produced concentration-dependent competitive inhibition of contractile responses of the aorta to PE. The rightward shift of the concentration-response curves of PE yielded constant pKBvalues with increasing antagonist concentrations in most cases allowing a single pooled value to be determined: for prazosin, a pKBof 8.99 ± 0.11 (n = 20,KB of 1.03 nM); for WB, a pKB of 8.75 ± 0.08 (n = 23,KB of 1.76 nM); for BMY, a pKBof 7.21 ± 0.13 (n = 13,KB of 62 nM); for MDL72, a pKBof 7.95 ± 0.15 (n = 12,KB of 11.2 nM); and for SK&F 105854, a pKB of 5.82 ± 0.08 (n = 15,KB of 1.52 μM). For MDL73, pKBvalues decreased with antagonist concentration: 7.88 ± 0.06 at 10 nM, 7.56 ± 0.28 at 100 nM and 6.92 ± 0.18 at 1000 nM, which suggests the presence of more than one receptor subtype. CEC (10 and 100 μM) almost completely inhibited responses to PE; lower concentrations had no significant effect. 5-MU (10–300 nM) and RS (3–300 nM) were ineffective antagonists in this tissue. Because WB, a highly selective alpha-1D andalpha-1A adrenoceptor subtypes inhibitor, blocked PE responses (with less affinity than foralpha-1A adrenoceptors), and 5-MU and RS, which are selective blockers for alpha-1A adrenoceptor, were ineffective, we conclude that alpha-1A adrenoceptors are absent in the dog aorta. The effects of the less selective MDL72 were inconsistent with actions at alpha-1B oralpha-1D adrenoceptors. Although WB shifted the PE concentration-response curve to the right, the abilities of BMY, MDL73 and SKF to inhibit competitively PE contraction were of lower affinity compared with expectations for interaction with alpha-1D adrenoceptors; they are not the predominant subtype. The complete inhibition of PE responses by CEC suggests that the dog aorta contains the alpha-1B adrenoceptor subtype. In immunocytochemical studies of the expression of alpha-1B adrenoceptor, all cells apparently expressed this protein. Moreover, Western blot studies of the microsomal fractions confirmed the presence ofalpha-1B adrenoceptors. In the dog aorta, the alpha-1 adrenoceptors predominantly resemblealpha-1B rather than alpha-1D adrenoceptors as reported in the rat aorta.
Footnotes
-
Send reprint requests to: E.E. Daniel, PhD, Professor Emeritus, Room 4N51, Department of Biomedical Sciences, McMaster University, Hamilton ON L8N 3Z5, Canada.
-
↵1 Supported by the Heart and Stroke Foundation of Ontario.
-
↵2 Recipient of the Career Investigator award of the Heart and Stroke Foundation of Ontario.
- Abbreviations:
- BMY
- BMY 7378 or {8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl)- 8-azaspirol[4,5]decane-7,9-dione
- Bmax
- maximum concentration of bound ligand per mg membrane protein
- CEC
- chloroethylclonidine
- DMA
- dog mesenteric artery
- EC50
- concentration for 50% maximum response
- IC50
- 50% inhibitory concentration
- KB
- calculated antagonist dissociation constant in functional studies
- KD
- dissociation constant in saturation ligand binding studies
- Ki
- dissociation constant in competition ligand binding studies
- MDL72
- MDL 72832, {8-[4-(1,4-benzodioxan-2-ylmethylamino)butyl]-8-azaspirol[4,5]decane-7,9-dione HCl
- MDL73
- MDL 73005EF, {8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]-8-azaspirol[4,5]decane-7,9-dione HCl
- MIC2
- microsomal fraction used for binding
- RS
- RS-17053 or N-[2-(-cyclopropyl methoxy phenoxy) ethyl]-5-chloro-α, α-dimethyl-1H-indole-3-ethanamine HCl
- SKF
- SK&F 105854 or 7-chloro-2-bromo-3,4,5,6-tetrahydro-4-methylfurol[4,3,2-ef]-3 benzapine
- WB
- WB 4101 or 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane
- 5-MU
- 5-methylurapidil
- PE
- phenylephrine
-
- Received August 21, 1997.
- Accepted January 30, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
