enzyme activities [26]. Some reports on a model of rabbit aortic endothelial cells have shown that estrogen
enhanced the activity of Ca
2 +
-activated K
+
channels [27]; other studies have demonstrated that acute estro-
gen administration in the canine coronary circulation induced epicardial coronary vasodilation through open-
ing of adenosine triphosphate ATP-sensitive potas- sium channels and calcium channel antagonism [28].
Thus, the relative contribution of EDHF in this benefi- cial effect of estrogen has not been clarified.
Our studies are the first to examine the involvement of EDHF in the protective effect of 17b-estradiol in
hypercholesterolemic rabbit carotid arteries, and to de- termine both the nature of EDHF and its target K
+
channels, in these arteries, by using selective blockers.
2. Methods
2
.
1
. Arterial ring preparation Male New Zealand rabbits 2.5 – 3 kg were assigned
randomly to four dietary groups for 8 weeks: control group, normal rabbit diet; control + estradiol group,
normal rabbit diet + 17b-estradiol di-undecylate intra- muscular i.m. injection, 700 mg per week; cholesterol
group, standard chow + 1 cholesterol; and choles- terol + estradiol group, 1 cholesterol chow + 17b-
estradiol di-undecylate i.m. injection, 700 mg per week. All animals were sacrificed by exsanguination after
anesthesia with sodium pentobarbital 30 mg kg
− 1
intravenously. The common carotid artery was iso- lated, placed in ice cold Krebs NaCl, 118.3 mM; KCl,
4.7 mM; CaCl
2
, 2.5 mM; MgSO
4
, 1.2 mM; KH
2
PO
4
, 1.2 mM; glucose, 11.1 mM; and NaHCO
3
, 24.9 mM; pH 7.4, cleaned of connective tissue and cut into
transverse rings 3 mm long. Special care was taken to avoid damage to the endothelium. Each ring was then
suspended vertically in the organ chamber volume, 25 ml between two stainless steel hooks, in Krebs solution
maintained at 37°C, and gassed with 95 oxygen and 5 carbon dioxide. One of the hooks was fixed to a
stand, while the other was attached to an isometric force transducer and the tension recorded on a Gould
polygraph model 272. Rings were initially stretched until the resting tension reached 3 g and then allowed
to equilibrate for 45 min; during this period, resting tension was continuously monitored and, if necessary,
readjusted to 3 g by further stretching.
2
.
2
. Determination of serum cholesterol and triglyceride concentrations
Blood samples were collected in tubes from each animal before excision of the aorta. After centrifuga-
tion at 2000 × g for 10 min, the total serum cholesterol and triglyceride concentrations were measured by a
standard enzymatic method cholesterol enzymatic PAP 61225 for cholesterol and 61236 for triglycerides; Bio
Merieux, France.
2
.
3
. Experimental protocols In a first set of experiments, rings were treated for 40
min with the nitric oxide NO synthase inhibitor N
v
- nitro-
L
-arginine methyl ester
L
-NAME 300 mM and the cyclooxygenase inhibitor indomethacin 10 mM.
They were then precontracted with phenylephrine 1 mM and exposed to increasing cumulative concentra-
tions of acetylcholine 3 nM – 30 mM. Some carotid artery rings were precontracted with elevated potassium
Krebs solution 50 mM, which was made by equimolar replacement of NaCl with KCl. In some experiments,
the endothelium was removed by gently rubbing the endothelial surface with a cannula. Removal of the
endothelium was confirmed by the absence of a relax- ant response to acetylcholine 3 mM.
To determine the nature of targeted K
+
channels, in the presence of
L
-NAME and indomethacin, either tetraethylammonium 10 mM, charybdotoxin 50 nM
alone or with apamin 50 nM, glibenclamide 10 mM or 4-aminopyridine 1 mM were added 40 min before
testing the acetylcholine ACh effect, to block, respec- tively, calcium-activated K
+
, ATP-sensitive K
+
and voltage-dependent delayed rectifier K
+
channels. In another set of experiments, in the presence of
L
-NAME and indomethacin, the rings were preincu- bated with hemoglobin 10 mM, a nitric oxide scav-
enger, LY83183 10 mM, a specific inhibitor of guanylate cyclase or clotrimazole 1 mM and an in-
hibitor of cytochrome P450-monooxygenase.
In all experiments, the phenylephrine concentration was adjusted in order to obtain a similar amount of
precontraction.
2
.
4
. Statistical analysis Relaxations were expressed as the percentage of the
contraction induced by 1 mM phenylephrine. All results are expressed as mean 9 S.E.M. Each concentration –
response curve was characterized by determining the pD
2
the negative log molar concentration of the drug inducing a half-maximal response and the maximal
response E
m
to drug. The results were analysed using Student’s t-test for paired observations. When more
than two groups were compared, statistical significance was assessed by an analysis of variance. If a significant
F value was found, Sheffe’s test was used to identify differences among groups. P B 0.05 was considered to
be a significant probability.
2
.
5
. Drugs Phenylephrine, acetylcholine, N
v
nitro-
L
-arginine methyl
ester, indomethacin,
tetraethylammonium, charybdotoxin, apamin, 4-aminopyridine, hemoglobin,
LY83183 and clotrimazole were purchased from Sigma Chemical Co. Glibenclamide was purchased from Re-
search Biochemicals Inc. 17b-Estradiol di-undecylate was purchased from Theramex. Stock solutions of
phenylephrine, acetylcholine,
L
-NAME, charybdotoxin, apamin and 4-aminopyridine were diluted in distilled
water. Clotrimazole, LY83183 and indomethacin were dissolved in ethanol. Glibenclamide was diluted in
dimethylsulphoxide. The final concentrations of all sol- vents used B 0.1 had no effects in preliminary ex-
periments. 17b-Estradiol di-undecylate was emulsioned in vegetable oil.
3. Results
