Fig. 1. Effects of K⁺ gradient reduction and non-selective blockade of K⁺ channels on sildenafil-induced relaxation of the rabbit basilar artery. Concentration-dependent relaxations to sildenafil in endothelin-1-precontracted arteries (control), KCl-precontracted arteries, and endothelin-1-precontracted arteries preincubated with tetraethylamonium. Data are means ± S.E.M.

Fig. 2. Effects selective blockade of K⁺ channels on sildenafil-induced relaxation of the rabbit basilar artery. Concentration-dependent relaxations to sildenafil in endothelin-1-precontracted arteries (control), and endothelin-1-precontracted arteries preincubated with 4-aminopyridine (voltage-dependent K⁺ channel blocker), glibenclamide (ATP-sensitive K⁺ channel blocker) or iberiotoxin (Ca²⁺-activated K⁺ channel blocker). Data are means ± S.E.M.

Fig. 3. Detection of amplified products corresponding to Kv1 mRNA in rabbit basilar artery. RT–PCR screening detected mRNA encoding Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6 in basilar artery (BA) and brain (positive control), but not in liver (negative control). Expected product sizes are given in base pairs (bp) and can be checked against the ladder lane spanning from 100 bp to 700 bp in one hundred steps. Expression of the ubiquitous β-actin is a positive control of RT–PCR in the different rabbit tissues. Blank lane is a control PCR with primers but without cDNA.

Fig. 4. Detection of Kv1 proteins in rabbit basilar artery. Representative Western blots in basilar artery (BA), brain (positive control) and liver (negative control) probed with antibodies to Kv1.1, Kv1.2, Kv1.4 and β-actin (loading control). Protein expression of Kv1.1, Kv1.2 and Kv1.4 was detected in basilar artery and brain but not in liver.

Fig. 5. Typical records showing reversible inhibition by sildenafil of vasoconstriction to CaCl₂ in rabbit basilar artery. (A) Two consecutive concentration-dependent contractions to CaCl₂ show reproducibility of responses; (B) abolished response to CaCl₂ in the presence of 100 µM sildenafil, followed by concentration-dependent contraction to CaCl₂ after sildenafil washout. Contractions to CaCl₂ were obtained in Ca²⁺-free, highly depolarizing (50 mM KCl) medium and are compared with previous response to 50 mM KCl in the same artery.

Fig. 6. Effects of sildenafil on CaCl₂-induced contraction of the rabbit basilar artery. Concentration-dependent contractions to CaCl₂ in control conditions and during incubation with increasing sildenafil concentrations. Data are means ± S.E.M.

Fig. 7. Effects of zaprinast on CaCl₂-induced contraction of the rabbit basilar artery. Concentration-dependent contractions to CaCl₂ in control conditions and during incubation with low and high zaprinast concentrations. Data are means ± S.E.M.

Fig. 8. Effects of selective L-type Ca²⁺ channel activation on sildenafil-induced inhibition of contraction to CaCl₂ in the rabbit basilar artery. Concentration-dependent contractions to CaCl₂ in control conditions, during incubation with the highest sildenafil concentration, and during incubation with sildenafil plus increasing Bay K 8644 concentrations. Data are means ± S.E.M.

Fig. 9. Effects of sildenafil on Bay K 8644-induced contraction of the rabbit basilar artery. Concentration-dependent contractions to Bay K 8644 in control conditions and during incubation with increasing sildenafil concentrations. Data are means ± S.E.M.

Fig. 10. Effects of zaprinast on Bay K 8644-induced contraction of the rabbit basilar artery. Concentration-dependent contractions to Bay K 8644 in control conditions and during incubation with low and high zaprinast concentrations. Data are means ± S.E.M.

Oligonucleotides used for RT–PCR analysis

Pharmacological parameters for the concentration–response curves to sildenafil, CaCl₂ and Bay K 8644 in the rabbit basilar artery

Values averaged from ‘n’ arteries. EC₅₀ (mean with 95% confidence limits) is half-maximal effective concentration. E_max (mean ± S.E.M.) is maximum relaxant effect expressed as percent of active tone in precontracted arteries, or maximum contractile effect expressed as percent of previous contraction to 50 mM KCl. ET-1, endothelin-1.