Pharmacology of Endothelium-Dependent and Independent Relaxation of Rabbit Aorta

The purpose of this study was to investigate several aspects of the acetylcholine-evoked relaxation of blood vessels: methodologic aspects; gender; storage; elevated glucose incubation; the signal transduction pathway; and effects of prolonged exposure. Furthermore, the relaxing effect of some adrenergic drugs was studied with regard to the possible dependence on endothelium and possible release of nitric oxide (NO).
Isolated rabbit thoracis aorta was used for isometric tension measurements; measurements of NO metabolites (Griess Reaction); and eNOS activity measurements (L-citrulline assay).
The acetylcholine-evoked relaxation of rabbit thoracic aorta was: independent of EDTA and ascorbic acid; the same whether the physiological salt solution (PSS) was oxygenated with 95% or 21 % O₂; independent of the phenylephrine-evoked precontractile tension; and did not differ in unstored or cold strored aorta.
It is concluded that the acetylcholine-evoked relaxation is uninfluenced by oxygen radicals, divalent metal ions, changes in oxygen tension, the degree of precontraction and storage of aorta in the cold.
Phenylephrine induced a steady state tension that was stable over a long time period. EDTA and ascorbic acid caused a decrease of the contraction with time. On the contrary in the presence of N^G-Nitro-L-arginine (L-NA) the contraction was increased and stable over a longer time period. NS 2028 and L-NA increased the phenylephrine-evoked tension equally in aorta from males and females.
It is concluded that phenylephrine-evoked precontraction of rabbit aorta causes a basal release of NO. The amount of basally released NO is the same independent of gender and it is inactivated by oxygen radicals and divalent metal ions.
The acetylcholine-evoked relaxation was uniform along the length of rabbit thoracic aorta in males but heterogeneous in females:a more pronounced relaxation was observed in the middel section of thoracic aorta. Some controversy is observed in the literature with respect to the role of gender in acetylcholine-evoked relaxation of rabbit aorta. the relaxation was higher in aorta from females compared to males (Sanchez et al. 1996) or the relaxation was independent og gender (Hayashi et al 1992). Our results lead to the conclusion, that the discrepancy observed in the literature is due to the heterogeneity of the female aorta.The acetylcholine-evoked relaxation was attenuated when aorta was incubated in PSS with elevated glucose. Ascorbic acid, indomethacin and L-arginine restored the acetylcholine-evoked relaxation. The same effect was observed with aorta incubated in normal glucose containing PSS. Furthermore, cycloheximide restored the attenuation observed in normal glucose incubated aorta.
It is concluded that prolonged incubation of aorta in normal and elevated glucose causes de novo synthesis of proteins, such a cyclooxygenase II; production of vasoconstricting prostanoids; generation of free radicals; and decreased NO availability.
The acetylcholine-induced relaxation was abolished by atropine, L-NA and in endothelium-denuded aorta. Indomethacin had no effect on the relaxation. NS 2028 attenuated the acetylcholine-evoked relaxation partially in phenylephrine precontracted aorta, but completely in KCl precontracted aorta. the NS 2028 resistant relaxation was inhibited in the presence og tetraethylammonium (TEA) and 4-aminopyridine (4-AP), but unaffected by glibenclamid.
It is concluded that the acetylcholine-evoked relaxation of rabbit aorta is dependent on: the endothelium; the activation of muscarinic receptors; independent of prostanoids; and mediatedn by NO alone. Th eNO released by acetlcholine stimulates a relaxation by activating soluble guanylate cyclase (sGC) and voltage dependent (K_V) and Ca²⁺ dependent (K_Ca) potassium channels in the smooth muscle.
Repeated addition of acetylcholine caused the development of tachyphylaxis. The relaxationevoked by a single concentration of acetylcholine was reversed slowly but completely within 3-4 h, i.e. fade. The fade was present even when aorta was pretreated with L-arginine, tetrahydrobiopterin (BH₄), zaprinast, superoxide dismutase (SOD) or physostigmine. Fade was also observed in the relaxation evoked by A23187, ATP, carbachol or oxotremine. On the contrary, SIN-1, YC-1 and 8-Br-cGMP induced a persistent relaxation in rabbit aorta. Acetylcholine added after the complete recovery of the A23187 induced relaxation caused a marked attenuation of the relaxation. Repeated addition of acetylcholine or A23187, after the complete recovery of the first relaxation, caused a marked attenuation of the evoked relaxation.
It is concluded that the fade of the acetylcholine-evoked relaxation is due to an effect on the part of the signal transduction pathway that is activated in the endothelium. The fade is not due to: inactivation of acetylcholine; muscarinic receptor desensitisation; limited NO synthesis due to the lack of substrate or cofactors; increased metabolism of NO or cGMP; production of vasoconstricting prostaglandins, and inhibition of sGC or cGMP dependent protein kinase (PKG).
The phenylephrine-evoked contraction induced in aorta that has been stored for a prolonged period, displayed an initial transient peak that decreased fast, i.e. an unstable contraction. This unstable contraction was changed into a stable steady state contraction in the presence of L-NA and aminoguanidine. A stable contraction was observed when aorta was stored for a prolonged period in cycloheximide, but not in polymyxin B.
It is concluded that prolonged storage of rabbit aorta causes the induction of iNOS. This induction is presumably due to gram positive bacteria that may be present in distilled water used to prepare the PSS.
UK14,304 induces a relaxation of phenylephrine precontracted rabbit aorta and a contraction of PGF_2α precontracted rabbit aorta. Furthermore, the contraction evoked by UK14,304 was abolished in the presence of prazosin. Rauwolscine relaxed phenylephrine precontracted but had no effect on PGF_2α precontracted rabbit aorta. Isoprenaline relaxed endothelium-denuded rabbit aorta. The relaxation was abolished by propranolol and ICI118551. Atenolol had no effect on the isoprenaline-evoked relaxation.
It is concluded that in rabbit aorta, UK14,304 is a partial α₁-adrenoceptor agonist and rauwolscine is an α₁ adrenoceptor antagonist. Isoprenaline activates β₂-adrenoceptors on the smooth muscle cells of rabbit aorta which induces a relaxation.