European Journal of Pharmacology, 211 (1992)383-391
3
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52259
Effect of flosequinan upon isoenzymes of phosphodiesterase from guinea-pig cardiac and vascular smooth muscle GiUian F r o d s h a m a n d R o b e r t B. J o n e s Boots Pharmaceuticals, Research Department, Nottingham NG2 3AA, U.K.
Received 17 July 1991,revised MS received 11 October 1991, accepted 5 November 1991
The effect of flosequinan and its sulphone metabolite BTS 53 554, on phosphodiesterase isoenzymes isolated from guinea-[ cardiac and vascular smooth muscle using DEAE-cellulose chromatography was investigated. Zaprinast and milrinone show peak I and peak III selectivity, and IBMX non-selective activity respectively, against both cardiac and vascular smooth mus~ isoenzymes, as expected for these reference inhibitors. Flosequinan and BTS 53 554 demonstrated non-selective inhibition wJ similar potency against both cardiac and vascular smooth muscle isoenzymes and, overall, were the least potent compoun tested. The high inhibitory concentrations observed (ICs0 peak III 660 p~M for cardiac tissue and 230/~M for vascular smo¢ muscle with flosequinan) relative to its clinically effective plasma concentration (10/~M) questions the relevance of phospho, esterase inhibition to the efficacy of flosequinan in heart failure. Phosphodiesterase isoenzymes; Cardiac muscle; Smooth muscle (aortic); Flosequinan; BTS 53 554; (Guinea-pig)
1. Introduction Flosequinan (BTS 49 465, 7-fluoro-l-methyl-3-methylsulphinyl-4-quinolone) (Davies et al., 1983)is a novel orally effective hypotensive agent with both arterial and venous vasodilator properties and a prolonged duration of action in man (Cowley et al., 1984). Flosequinan is under clinical investigation for use in hypertension and heart failure (Cowley et al., 1987; Cowley, 1991; Kessler and Packer, 1987). In preliminary studies with a commercially available mixed isoenzyme preparation from bovine heart (Frodsham et al., 1989) flosequinan appeared to be a non-selective inhibitor with only low potency when compared with the P D E III selective inhibitor milrinone, P D E V selective inhibitor zaprinast and nonselective inhibitor IBMX (Alousi et al., 1983; Bergstrand et al., 1977; Beavo et al., 1970), but the use Of a mixed isoenzyme preparation made interpretation of activity on individual isoenzymes difficult. A rise in both cAMP and cGMP would be expected as a consequence on non-selective phosphodiesterase inhibition
Correspondence to: (3. Frodsham, Boots Pharmaceuticals, Research Department, Nottingham NG2 3AA, U.K. Tel. 44.602.492 679, fax 44.602.492 614.
and increases in cAMP and cGMP are associated wi vasorelaxation in vascular smooth muscle (Katsuki m Murad, 1977; Hardman, 1984). In cardiac tissue ele~ tion in cAMP is associated with positive inotro[ activity (Evans, 1986) whereas elevation in cardi cGMP may reduce inotropic activity (Drummond a~ Severson, 1979). The purpose of the present study was to investig~ the effects of flosequinan upon isoenzymes of phosph diesterase, isolated from guinea-pig cardiac (left vent cle) and vascular smooth muscle (aorta). Since t sulphone metabolite of flosequinan, BTS 53 554 pharmacologically active (Davies et al., 1983) and m be responsible for a major part of the clinical respon to flosequinan (Wynne et al., 1985; Packer et al., 19~ its effect was also investigated. Although it has been reported to possess non-sek tive P D E inhibitory activity of low potency (Frodsh~ et al., 1989) recent evidence using right atrial pendage tissue (Weishaar et al., 1991) and in ventrk lar tissue (Perreault et al., in press) from heart faik patients suggests that flosequinan has little or no : otropic activity even in the presence of the adenyl~ cyclase stimulator forskolin. This contrasts with d~ obtained using the P D E III selective inhibitor rail none and the non-selective PDE inhibitor IBIV (Feldman et al., 1987).
384 The activity of flosequinan on PDE isoenzymes is discussed in relation to its therapeutic plasma concentration in man.
2. Materials and methods
2.1. Separation of isoenzymes ofphosphodiesterase Unless indicated otherwise all procedures were carried out at 4°C. Slight modifications of the methods of Thompson et al. (1979) and Silver et al. (1988) were used to separate the isoenzymes of phosphodiesterase from cardiac tissue. Hearts were removed from guinea-pigs (Dunkin Hartley, males, 400-600 g body weight) following cervical dislocation and the left ventricle and septum dissected and washed in ice-cold saline. Five hearts were used for each preparation (giving a total of 7.0-9.0 g tissue). Tissue was minced finely and homogenised in eight volumes (8 m l / g wet weight) of buffer containing 10 mM Tris acetate, pH 7.5, 2 mM MgC12, 1 mM dithiothreitol (DTT), 2000 units/ml aprotinin and 50 /xM phenylmethylsulphonyl fluoride (PMSF) for 3 × 10 s bursts using a Polytron PT-10 on setting No. 5. The resulting homogenate was sonicated (30 s / m l ) and centrifuged at 40000 × g for 30 min after which the supernatant fraction was applied to a DEAE-cellulose column (30 X 1.7 cm, bed volume 68 ml) previously equilibrated with 0.07 M sodium acetate buffer (pH 6.5). As with all other column buffers it contained 5 mM 2-mercaptoethanol. After application of the sample, the column was washed with three to four bed volumes of 0.07 M sodium acetate buffer, (pH 6.5). The phosphodiesterase isoenzymes were eluted (flow rate 0.5 m l / m i n ) using 200 ml of 0.2 M sodium acetate (pH 6.5) followed by a linear gradient of 0.2-0.6 M sodium acetate (pH 6.5) (450 ml). Fractions (11 ml) were collected and assayed for cAMP and cGMP phosphodiesterase activity at 1/xM substrate concentration. Calmodulin stimulation of cAMP hydrolysis was measured using 10 units calmodulin/10 /zM CaC12 and cGMP stimulation of cAMP hydrolysis by the inclusion of 0.5/xM cGMP. Appropriate fractions corresponding to peak activity were pooled and diluted with ethylene glycol to 30%. Aliquots were stored at - 2 0 ° C and activities and response to inhibitors were stable for over 3 months following preparation, Isoenzymes of phosphodiesterase were separated from guinea-pig aorta by a slight modification of the method of Hagiwara et al. (1984). Aortas were removed from guinea pigs (Dunkin Hartley, males, 300500 g body weight) and cleaned of adhering tissue, They were washed in ice-cold saline and then stored at
-80°C. Typically 70 aortas were used per preparati( (giving approximately 4.0 g tissue). The tissue was fir powdered at the temperature of liquid nitrogen using Braun Mikrodismembrator. The tissue powder was lowed to thaw in the presence of five volumes homogenisation buffer: 0.05 M Tris acetate (pH 6. containing 2.75 mM 2-mercaptoethanol, 1 mM MgCI 2000 units/ml aprotinin and 50 ~ M PMSF. All subs quent buffers contained 2.75 mM 2-mercaptoethan( Homogenisation was accomplished by 3 x 10 s burs with a Polytron PT-10 on setting No.7 and was faci tated by prior powdering of the tissue. The h mogenate was sonicated (30 s / m l ) and centrifuged 100000 × g for 60 min after which the supernata fraction was applied to a DEAE-cellulose column C × 1.7 cm, bed volume 45 ml) previously equilibrat~ with 0.05 M sodium acetate (pH 6.0). After applicati( of the sample the column was washed with three b~ volumes of 0.05 N sodium acetate (pH 6.0), followed 1 a linear gradient of 0.05-0.6 M sodium acetate (p 6.0), (400 ml) to elute the phosphodiesterase isoe zymes. Fractions (6 ml) were collected and assayed f, cAMP and cGMP phosphodiesterase activity using /zM substrate. Calmodulin stimulation of cGMP hydr lysis was measured as for cardiac enzymes. Approp~ ate fractions were treated as before except that wi smooth muscle preparations the enzymes were us~ within 3 weeks, during which time the response inhibitors was stable. The protein content of column fractions was mot toted by absorbance at 280 nm and the protein conte of pooled peak fractions assayed using the method Bradford (1976) with bovine serum albumin as stan ard.
2.2. Phosphodiesterase assay Phosphodiesterase activity was measured by tl method of Thompson et al. (1979) with the addition 0.005 /zCi [U-14C]guanosine or [U-14C]adenosine recovery markers for the column separation step. T] reaction mixture (0.4 ml final volume) contained . mM Tris HC1 (pH 8.0), 5 mM MgC12 and 1 mM D T The reaction was initiated by the addition of enzy13 diluted to give 15-20% substrate utilisation during t] 10 min incubation at 30°C. Typically the recovery [U-a4C]adenosine from the anion exchange column st~ was greater than 90% and [U-14C]guanosine great than 65%. Each sample was individually corrected f recovery. Under these conditions the assay was line with respect to time and enzyme concentration. F assessing phosphodiesterase activity in column fra tions the substrate ([3H]cAMP or [3H]cGMP)conce tration was 1 /zM.
3 2.3. Substrate kinetic studies
3. Results
For substrate kinetics experiments the concentration of cAMP and cGMP typically ranged from 0.1 to between 25 and 100 times the K m (K m = substrate concentration at which half-maximal reaction velocity is observed), A Hanes plot of [S]/v versus [S] (where v is reaction velocity and [S] is the substrate concentration) for the initial analysis of data from peaks I and III showed two apparently linear segments. These were analysed individually by non-linear regression of Michaelis-Menten plots using the Biosoft Enzfitter computer program. Two K~, and V.aax (maximum reaction velocity) values could be obtained in such cases, implying a high and low affinity site for hydrolysis of the substrate. Values for K m and Vmax were calculated for each isoenzyme type, two being calculated for peaks I and III where high and low affinity sites were demonstrated.
3.1. Isolation of multiple forms of cyclic nucleotide phc phodiesterase
2.4. Inhibitor studies For inhibitor studies the compounds to be tested were dissolved in DMSO (2.5% final assay concentration). Vehicle controls were included in all experiments. The concentration of inhibitor which produced 50% inhibition of enzyme activity (IC50) was determined graphically from concentration-response curves in which doses of drugs ranged from 0.1 to 3000/xM in half-log increments. All substrate kinetics and inhibitor assays were performed in triplicate and three separate preparations of phosphodiesterase were used from both cardiac and vascular tissue,
2.5. Materials [2,8-3H]cAMP (30-50 Ci/mmol), [8-3H]cGMP (1030 Ci/mmol), and [U-laC]adenosine (500-600 m C i / mmol) were purchased from Amersham International plc. [U-14C]guanosine (> 400 mCi/mmol) was purchased from Du Pont (LI.K.) Ltd. Calmodulin (from bovine brain), Ophiophagus hannah snake venom and aprotinin (from bovine lung) were purchased from Sigma Chemical Co. Dowex AG l-X8 200-400 mesh (Cl-form), disposable econo-columns, and protein assay kits were purchased from Bio-Rad Laboratories. DEAE-cellulose (DE52) was purchased from Whatman. All other reagents were purchased from either Sigma Chemical Co. or BDH and were Analar grade, Drugs were obtained from the following sources: IBMX (3-isobutyl-l-methylxanthine) (purchased from Sigma Chemical Co.); zaprinast (kindly donated by May & Baker); flosequinan, BTS 53 554 and milrinone (synthesised by Medicinal Chemistry, Boots Pharmaceuticals Research Department).
Using guinea-pig ventricle an elution profile simil to that obtained by Silver et al. (1988) was obtaine Three isoenzymes (fig. 1) were isolated having differe characteristics (table 1). The first isoenzyme (peak phosphodiesterase) eluted at 0.2 M sodium acetate a~ hydrolysed both cAMP and cGMP. High and low affi ity sites for cAMP and cGMP hydrolysis were show The Km'S were similar for both cAMP and cG/v though greater activity was demonstrated with cGi~ in contrast to the results obtained by Silver et al. (198 where cAMP activity was greatest. This isoenzyme w stimulated by Ca2+-calmodulin, the Vmax for cGI~ increasing by 6 fold (table 1). In the presence of Ca: -calmodulin there was only one high affinity site I cGMP hydrolysis. The second isoenzyme (peak II phosphodiestera~, eluted between 0.3-0.35 M sodium acetate and a peared to have a single affinity site with similar affin: for both cAMP and cGMP. This isoenzyme had low affinity for cAMP and cGMP than the other isoe zymes but a greater Vmax. It could be stimulated by ( /xM cGMP (fig. 1). In a separate experiment peak cAMP hydrolysis was stimulated maximally (7-8 fol by concentrations of cGMP of greater than 1 /x (single experiment). The third isoenzyme (peak III phosphodiestera~, was eluted at 0.4-0.5 M sodium acetate and show both high and low affinity sites for the hydrolysis cAMP. cGMP inhibited the hydrolysis of cAMP peak III phosphodiesterase (fig. 1) and in a separ~ experiment was shown to have an IC50 value of 2.2/z for this inhibition (single experiment), cGMP increas the K m for cAMP of this isoenzyme and it was possit to obtain K m and Vmax values for peak III phospho~ esterase with cGMP as substrate. The high affinity was the same for cGMP as for cAMP but greal activity was observed with cAMP. A relatively hi affinity for cGMP has been demonstrated previou: for peak 1II phosphodiesterase (Reeves et al., 1987). The elution profile obtained by DEAE-cellulc chromatography of vascular smooth muscle is shown fig. 2. Three phosphodiesterase isoenzymes were four The two peaks eluting at 0.1-0.2 and 0.3-0.4 M sodit acetate concentration both hydrolysed cGMP but h little cAMP hydrolytic activity. Calmodulin stimulation was not observed w: smooth muscle phosphodiesterase isoenzymes. Peat had high affinity for cGMP with a Kr, of 2.7 + 0.7 # (n = 3) compared with a K m of 25.4 + 3.1 /xM (n = for peak II and although cAMP hydrolysis could demonstrated it was too low to determine a K m vah
386 PDE
activity
(pmol/min/fraction)
Sodium
acetate
2500
/
(M) 0.6
/ /
2000
/
/
/
0.5
/
0.4
1500
0.3 1000
_
_
---/
/
/ 0.2
500
0.1
0 20
30
40
50
60
Fraction
.
.
70
80
0.0 90
100
11
number
Fig. 1. Representative DEAE-cellulose chromatogram of cAMP and cGMP phosphodiesterase activity from guinea-pig ventricle after elution 200 mM sodium acetate followed by a 200-600 mM sodium acetate gradient. Enzyme activity was measured as described in Materials a methods; (o) cAMP, ([]) cGMP, ( • ) cAMP plus Ca 2+-Calmodulin, (o) cAMP plus cGMP. ( - - - ) sodium acetate gradient. Fraction volun were 11 ml.
In c o n t r a s t , t h e t h i r d p e a k ( p e a k I I I ) w h i c h e l u t e d at 0 . 4 - 0 . 6 M s o d i u m a c e t a t e h a d a h i g h affinity for c A M P ( K m = 0.96 + 0.03 ~ M , n = 3) b u t n o s i g n i f i c a n t c G M P h y d r o l y t i c activity, c A M P h y d r o l y s i s by p e a k I I I was i n h i b i t e d by c G M P (IC50; 0.6 /xM, single e x p e r i m e n t ) ,
3.2. Effects of flosequinan, BTS 53 554 and reference phosphodiesterase inhibitors T h e e f f e c t s o f f i o s e q u i n a n , B T S 53 554, I B M X , milrinone and zaprinast upon cAMP and cGMP hydrolysis by c a r d i a c p h o s p h o d i e s t e r a s e isoenzymes are
s h o w n in t a b l e 2. I B M X w a s r e l a t i v e l y n o n - s e l e c t i b e i n g e q u i p o t e n t w h e n c G M P was u s e d as s u b s t r a t e d e m o n s t r a t i n g l o w e r p o t e n c y o n p e a k II p h o s p h o r e s t e r a s e w h e n c A M P was s u b s t r a t e . M i l r i n o n e d e m o strated a marked selectivity for the peak III isoenzyr w h e n c A M P was s u b s t r a t e a n d z a p r i n a s t s h o w e d s e k tivity for t h e p e a k I i s o e n z y m e r e g a r d l e s s o f substra type. F l o s e q u i n a n i n h i b i t e d all t h r e e i s o e n z y m e f o r n t h o u g h , o v e r a l l , b e i n g l e a s t e f f e c t i v e u p o n t h e peak i s o e n z y m e as w a s t h e s u l p h o n e m e t a b o l i t e B T S 53 5.' O f t h e a g e n t s t e s t e d , f l o s e q u i n a n a n d B T S 53 554 we the least potent inhibitors of phosphodiesterase act
TABLE 1 Apparent substrate affinities for isoenzymes of phosphodiesterase from guinea-pig ventricle. Isoenzyme Peak
Substrate type
A p p a r e n t K m (/xM) a
High affinity
Low affinity
Wmax (pmol/min per ~g protein) High Low affinity affinity
I
cAMP cGMP cGMP + CaCm b
1.3+0.2 1.4+-0.2 1.8 + 0.2
12.0+3.5 9.8+-1.3 c
5.7+_ 1.8 10.0+ 4.7 60.4 +-23.6
c
II
cAMP cGMP cAMP+0.5 IzM cGMP
d a a
31.3+-5.3 40.7+-0.3 24.7+-4.1
d d d
208 +-29.7 331 +-54.7 213 +- 16.4
III
cAMP cGMP cAMP+0.5/xM cGMP
1.6+-0.3 1.6+-0.5 3.4+-0.5
13.7_+1.5 38.7+-9.6 13.7+2.7
10.9+- 0.9 2.7+- 0.2 15.5+- 0.9
11.5+- 2.1 23.7+- 5.9
25.8+- 4.9 20.8+- 6.6 26.0+- 1.8
a The apparent K m (substrate concentration at which half-maximal velocity of hydrolysis occurs) was determined by non-linear regression anah of Michaelis-Menton plots using the Biosoft Enzfitter computer program. Phosphodiesterase activity was measured over cyclic nucleot concentrations typically ranging from 0.1 to between 25 and 100 times the anticipated Km. Values are the means±S.E.M, for triplic determinations from two or three preparations per isoenzyme, b Ca2+ concentration = 10 IzM; calmodulin (Cm)= 10 units, c In the presence Ca2+-calmodulin there was no indication of a low affinity site for cGMP hydrolysis, d No evidence of a high affinity site of hydrolysis.
3~ PDE
activity
(pmol/min/fraction)
S o d i u m a c e t a t e (M)
500
/
0.7
/ ./ / 400
/
0.6
/
"< 300
/
/
0.5
/
0.4
/
2OO
0.3 0.2
lO0 0.! . 20
30
40
50
.
.
.
60
0.0
70
80
90
Fraction number Fig. 2. Representative DEAE-cellulose chromatogram of cAMP and cGMP phosphodiesterase activity from guinea-pig aortic smooth muse after elution by a 50-700 mM sodium acetate gradient. Enzyme activity was measured as described in Materials and methods; (o) cAMP, (I cGMP. ( - - - ) sodium acetate gradient. Fraction volumes were 6 ml. % inhibition of enzyme activity
% inhihilion of enzyme activity
100"
100-
80
80"
60 "
61l "
40 "
40
20
20
0
0
-20
-20 10 ?
10 s
I 0 ~'
10.4
I 0~
102
II ?
.................. II)"
120 -
• .......................... I1l 4 10-.~
10 z
Molar cou,,'etilralion of drag
Molar concentration of drug
% inhihition
I0 s
of enzynne activity
% inhibition
of enzyme activity
100
c
I O0 "
80
80"
60"
60 " 4041l
20" 20 "
oi
oi i
I
IJ ~
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10"
10 r,
10.s
111.4
Mol:|r concentration of drug
IO ~
IO z
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
lO-g
10 -?
10-6
lo-s
10-4
1 0 .3
10 .2
Molar concentration of drug
Fig. 3. Effect of several agents upon phosphodiesterase (PDE) activity. (a) Peak I cGMP PDE from guinea-pig ventricle; (b) peak III cAMP P[ from guinea-pig ventricle; (c) peak I cGMP PDE from guinea-pig vascular smooth muscle; (d) peak III cAMP PDE from guinea-pig vascu smooth muscle. Substrate concentration in each case was 1.0 /~M. Each curve represents the meaned data generated from three separ~ concentration response curves (each obtained in triplicate) from three separate isoenzyme preparations except BTS 53 554 where n : (ventricle) and n = 1 (vascular smooth muscle). (©) Flosequinan, (o) BTS 53 554, ( • ) IBMX, ( / , ) zaprinast, ( • ) milrinone.
388 TABLE 2 Effect of agents upon isoenzymes of phosphodiesterase from guinea-pig ventricle. Agent
IC50 (/zM) a Peak I
Flosequinan BTS 53 554 b IBMX Milrinone Zaprinast
Peak II
Peak III
cAMP c
cGMP c
cAMP
cGMP
cAMP
cGMP
2200 ± 380 d 14% at l m M e 22 ± 3 440 _+ 130 12±0.3
2400 +_470 18% at 1 mM e 21 ± 3.4 235 _+55 b 14+ 1.3
2700 _+ 180 23% at 1 mM e 62 ± 12 820 _+93 240+32
600 ± 26 830±130 18 ± 0.6 290 ± 50 66+3.8
660 _+63 1000_+180 14 ± 1.3 5.7 ± 2.0 44% at 3 mM e
1200 _+250 37% at 1 mM e 15 _+1.7 395 ± 304 160±38
a IC5o values (concentration which inhibits substrate hydrolysis by 50%) were determined from concentration-response curves in which t concentration of agents ranged from 0.1 ~ M to 3 mM (half-log increments) except BTS 53 554 where the maximum concentration was 1 ml Three concentration-response curves were generated each in triplicate from three separate isoenzyme preparations, b Concentration-respor curves generated each in triplicate from two separate isoenzyme preparations, c Substrate concentration in the assay was 1.0 p~M. d Values given as means ± S.E.M. e Values are given as percentage inhibition where IC50 was not attained at the highest concentration tested.
ity, flosequinan being 116 and 171 times less potent than milrinone against peak III (cAMP) and zaprinast against peak I (cGMP) respectively (figs. 3a,b). The effects of flosequinan, BTS 53 554, IBMX, milrinone and zaprinast upon cAMP or cGMP hydrolysis by aortic smooth muscle phosphodiesterase isoenzymes are shown in table 3. The two isoenzyme peaks I and II were inhibited to a different extent by zaprinast which was 12.8 times more potent as an inhibitor of peak I than peak II. Both these isoenzyme forms were inhibited to a greater degree than was peak III. As with cardiac phosphodiesterase isoenzymes, milrinone was a selective peak III inhibitor and IBMX was relatively non-selective. Flosequinan and BTS 53 554 inhib±ted all three isoenzyme types, flosequinan with most potency against peak Ill and BTS 53 554 with equal potency on peaks II and III. Flosequinan and BTS 53 554 were the least potent of the compounds tested, TABLE 3 Effect of agents upon isoenzymes of phosphodiesterase from guinea-
pig vascular smooth muscle. Agent
IC50 (~.M) ~
Peak I
Peak II
cGMP c
cGMP c
1650+280
890+350
Peak III cAMP c 230+54
Flosequinan BTS 53 554 u
23% at 1 mM d
1000
900
IBMX
26+3.5
8±2.5
6.5+0.7
Milrinone
200 + 17
330 _+66
Zaprinast
2.5 +0.09
32+ 15
1.9 + 0.07
460+ 72
a iC50 values (concentration which inhibits substrate hydrolysis by 50%) were determined from concentration-response curves in which the concentration of agents ranged from 0.1 /zM to 1 mM (half-log increments) except BTS 53 554 where the maximum concentration was 1 mM. Three concentration-response curves were generated each in triplicate from three separate isoenzyme preparations. Values are given as means+S.E.M, b Concentration-response curve generated in triplicate from a single preparation, c Substrate concentration in the assay was 1.0/zM. d Value given as percentage inhibition where IC50 was not attained at the highest concentration tested,
flosequinan being 121 times less potent than milrinol against peak III and 660 times less potent than zap~ hast against peak I (figs. 3c, d respectively).
4. Discussion
All three isoenzymes of phosphodiesterase isolat~ from guinea-pigventricle hydrolysed cAMP and cGM peak I activity being stimulated by CaZ+-calmodul and peak II by cGMP. Peak III cAMP hydrolysis w inhibited by cGMP possibly due to a competitive effe between the two substrates since the affinity for hydr lysis of both cAMP and cGMP was the same in o study (K m = 1.6/xM), and the IC50 value for inhibiti~ of peak Ill PDE by cGMP was 2.2/.~M. The possibili of co-elution of PDE III and P D E IV has been rais4 by Reeves et al. (1987) when using DEAE-cellulo chromatography. Recently we have shown that usi~ FPLC with a Mono Q column, a small fourth pe, (rolipram sensitive P D E IV) can be distinguished fre P D E III (Frodsham et al., 1991). Flosequinan and B'~ 53 554 were weak inhibitors of PDE IV (IC50 values 1.8 and > 3.0 mM respectively) whereas the poten against the other isoenzymes were similar to that us±: DEAE-cellulose chromatography. In vascular smooth muscle three isoenzymes of PE activity were also isolated, peaks I and II hydrolysi: mainly cGMP and peak III cAMP. Peak I had a hil affinity and peak II a low affinity for cGMP hydrolys neither of which were stimulated by Ca 2 +-calmoduli Although this could have been caused by co-elution of the isoenzymes with calmodulin, the lack stimulation in the presence of additional Ca 2+ mak this unlikely. Preliminary separation of smooth m u s c PDE isoenzymes using FPLC with a Mono Q colun has demonstrated a fourth peak of activity eluti:
one
immediately prior to peak I which is only apparent the presence of Ca2÷-calmodulin and which may ha
co-chromatographed with peak I previously using DEAE (unpublished data). Using these isoenzymes from Mono Q zaprinast was selective (approximately 10 fold) for the Ca2+-calmodulin insensitive peak whereas flosequinan was non-selective, Zaprinast has been reported (Lugnier et al., 1986) to inhibit the Ca2+-calmodulin insensitive form of cGMP PDE more than the Ca 2+ -calmodulin sensitive form (IC50 values with 1 /zM cGMP were 0.4 and 21.0/~M respectively). The ICs0 value obtained in this study for peak I PDE (2.5 p.M) lies between those reported by Lugnier et al. and could therefore be indicative of co-chromatography of the two peaks. If this is the case then the peak II eluted from DEAE in this study represents a form of cGMP phosphodiesterase with low affinity for cGMP rather like the peak II observed in cardiac muscle, In addition to substrate kinetic data several reference phosphodiesterase inhibitors were used to aid classification of isoenzymes and for comparison with the effects of flosequinan and BTS 53 554. Milrinone, zaprinast and IBMX displayed peak III, peak I and non-selective inhibitory profiles respectively in both ventricular and aortic smooth muscle, The substrate kinetic data obtained, combined with the selectivity observed with reference inhibitors demonstrated that typical isoenzymes from both cardiac and vascular smooth muscle had been isolated (with the possible exception of peak II from smooth muscle) (Hagiwara et al., 1984; Weishaar et al., 1 9 8 6 ; Silver et al., 1988). On this basis peaks I II and III from cardiac muscle have been classified as the Ca 2+calmodulin-dependent PDE (PDE I), cGMP-stimulated PDE (PDE II) and cGMP-inhibited PDE (PDE III) respectively. Peaks I, II and III isolated from vascular smooth muscle have been classified as cGMP specific PDE (PDE V), cGMP specific PDE with low substrate affinity (possibly PDE II) and cGMP-inhibited PDE (PDE III) respectively, Flosequinan and BTS 53 554 were found to be non-selective inhibitors of phosphodiesterase isoenzymes from both tissue types and overall were the least potent of the compounds tested. Data to support the view that flosequinan has nonselective PDE inhibitory activity of only low potency is also derived from in vitro experiments where elevation of tissue levels of cGMP and cAMP have been observed. In vascular smooth muscle, increases in both cAMP and cGMP are implicated in relaxation (Hardman, 1984). In rat aortic strips (at concentrations producing an approximate 90% relaxation) flosequinan at 1 mM increased cGMP 4 fold (Allcock et al., 1988) and cAMP by 38% (P < 0.001, Allcock, unpublished observations). However, flosequinan causes relaxation of noradrenaline or phorbol dibutyrate ester induced contractions in the presence of methylene blue (an in-
hibitor of soluble guanylate cyclase) in rat aortic stri (Yates and Holmes, 1988) and human resistance w sels (Richards et al., 1989) indicating that flosequin~ does not act by stimulation of soluble guanylate clase. In guinea-pig atria flosequinan had an EC40 val~ for inotropy of 650/xM, (Yates and Hicks, 1988) and chopped guinea-pig ventricle flosequinan at 650 al 6500 ~M produced significant increases in cAMP k els of 16 and 64% respectively (Yates, 1991). Small b non-significant increases in cGMP were also observ, at the EC40 value (24%) whereas a significant increa was observed at the 6500 ~M concentration (73~ When flosequinan and isoprenaline were given in col bination, greater increases in cAMP were observ than could be accounted for by the simple addition the two effects of flosequinan and isoprenaline alor Flosequinan is therefore able to produce small elex tions in both cAMP and cGMP in both vascular a: cardiac tissue although only at relatively high conce trations. There have been several reports in which elevatio in cyclic nucleotides were only apparent at concentl tions in excess of those required for PDE inhibitk vasorelaxation and inotropy (Weishaar et al., 19~ Lugnier et al., 1986; Pang, 1988; Kauffman et al., 19~ Weishaar et al., 1983; Silver, 1989). Such observatio have been explained on the basis of differential acc~ of drugs between isolated enzymes and whole tisst functional compartmentation and high variability basal cyclic nucleotide levels. A comparison between the in vitro pharmacologic responses for flosequinan, PDE inhibition and cli~ cally effective plasma concentrations are pertinent the discussion as to whether flosequinan exerts beneficial effects in the treatment in heart failure virtue of its PDE inhibitory properties. The PDE inhibitory activity of flosequinan can~ be correlated with its vasorelaxant activity (ICs0 P[ III 230 tzM (table 3)compared to ICs0 for vasorel~ ation of 65/zM (Yates and Holmes, 1988)) but can correlated with its weak inotropic activity (ICs0 P[ III 660/zM (table 2) compared to If40 for inotropy 650 /.LM (Yates and Hicks, 1988)). However in bc cases the concentrations required for PDE inhibiti are much higher than the therapeutic plasma level ( /~M; Packer et al., 1988). In view of this it is probat that there are alternative mechanisms involved in t clinical action of flosequinan. In right atrial appendage tissue from patients wJ heart failure (Weishaar et al., 1991) and in ventricu] tissue from patients with end-stage heart failure (Pq reault et al., in press), flosequinan has little or inotropic activity. In addition forskolin does not pote tiate the inotropic activity of flosequinan (Weishaar al., 1991), but does potentiate the inotropic activity
390 m i l r i n o n e a n d I B M X ( F e l d m a n et al., 1987) s u g g e s t i n g t h a t f l o s e q u i n a n acts via a d i f f e r e n t m e c h a n i s m c o m p a r e d to t h a t o f m i l r i n o n e o r I B M X . T h e r e f o r e , alt h o u g h in g u i n e a - p i g v e n t r i c l e t h e r e a p p e a r s to b e a c o r r e l a t i o n b e t w e e n i n o t r o p i c activity a n d P D E inhibition, t h e s i t u a t i o n in f a i l i n g h u m a n h e a r t is c u r r e n t l y less clear, I n c o n c l u s i o n , f l o s e q u i n a n a n d B T S 53 554 p o s s e s s n o n - s e l e c t i v e p h o s p h o d i e s t e r a s e i n h i b i t o r y activity o f low p o t e n c y in b o t h g u i n e a - p i g c a r d i a c a n d v a s c u l a r s m o o t h m u s c l e . T h e p r o f i l e o f i n h i b i t i o n is d i f f e r e n t to that observed with zaprinast and milrinone (PDE V a n d P D E I I I s e l e c t i v e i n h i b i t o r s r e s p e c t i v e l y ) a n d is s i m i l a r to t h e n o n - s e l e c t i v e P D E i n h i b i t o r I B M X . T h e d a t a h e r e , in a d d i t i o n to p r e v i o u s f i n d i n g s , s u g g e s t t h a t f l o s e q u i n a n m a y p r o d u c e p h a r m a c o l o g i c a l e f f e c t s at h i g h c o n c e n t r a t i o n s in i s o l a t e d a n i m a l tissues at l e a s t in p a r t by i n c r e a s i n g levels o f i n t r a c e l l u l a r cyclic n u c l e o t i d e s a n d in g u i n e a - p i g c a r d i a c tive P D E i n h i b i t i o n . T h e e f f e c t s studies must however be viewed since t h e y o c c u r at c o n c e n t r a t i o n s t h o s e o b s e r v e d clinically.
tissue by n o n - s e l e c o b s e r v e d in a n i m a l with some caution substantially above
Acknowledgements The authors gratefully acknowledge the excellent technical assistance provided by Miss Deborah Anthony, Mrs. Tracey North, Miss Christine Spinks and Mr. Gary Telford.
References Allcock, A.A., G. Frodsham and M.F. Sim, 1988, Effects of flosequinan, a novel arterioveneous dilating agent, on cGMP levels in rat isolated aortic strips, Br. J. Pharmacol. 94, 430P. Alousi, A.A., J.M. Canter, M.J. Montenaro, D.J. Fort and R.A. Ferrari, 1983, Cardiotonic activity of milrinone, a new and potent cardiac bipyridine, on the normal and failing heart of experimental animals, J. Cardiovasc. Pharmacol. 5, 792. Beavo, J.A, N.L. Rogers, O.B. Crofford, J.G. Hardman, E.W. Sutherland and E.V. Newman, 1970, Effects of xanthine derivatives on lipolysis and cAMP phosphodiesterase activity, Mol. Pharmacol. 6 (6), 597. Bergstrand, H., J. Kristofferson, B. Lundquist and A. Schurman, 1977, Effects of antiallergic agents, compound 48/80 and some reference inhibitors on the activity of partially purified human lung tissue cAMP and cGMP phosphodiesterases, Mol. Pharmacol. 13, 38. Bradford, H.M. 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding, Anal. Biochem. 72, 248. Cowley, A.J., R.D. Wynne and J.R. Hampton, 1984, The effects of BTS 49 465 on blood pressure and peripheral arteriolar and venous tone in normal volunteers, J. Hypertension. 2 (Suppl. 3), 547. Cowley, A.J., R.D. Wynne and J.R. Hampton, 1987, Flosequinan as a third agent for the treatment of hypertension: a placebo controlled double-blind study, Eur. J. Clin. Pharmacol. 33, 203.
Cowley, A.J., 1991, Clinical efficacy of flosequinan in heart failm Am. Heart. J. 121,983. Davies, R.V., J. Fraser, K.J. Nichol, R. Parkinson, M.F. Sim aJ D.B. Yates, 1983,'UK Patent 2047691B. Drummond, G.I. and D.L. Severson, 1979, Cyclic nucleotides al cardiac function, Circ. Res. 44, 145. Evans, D.B., 1986, Modulation of cAMP: mechanism for positi inotropic action, J. Cardiovasc. Pharmaeol. 8 (Suppl. 9), $22. Feldman, M.D., L. Copelas, J.K. Gwathmey, P. Phillips, S.E. W~ ren, F.J. Schoen, W. Grossman and J.P. Morgan, 1987, Deficie production of cyclic AMP: pharmacologic evidence of an impc tant cause of contractile dysfunction in patients with end-sta heart failure, Circulation 75, 331. Frodsham, G., K. Dickinson, S.L. Hayes, R.B. Jones and D.B. Yat~ 1991, Effect of flosequinan and BTS 53 554 upon isoenzymes phosphodiesterase (PDE)isolated from guinea-pig ventricle usi Mono-Q FPLC, Br. J. Pharmacol. 104, 172P. Frodsham, G., R.B. Jones and M.F. Sim, 1989, Effects of flo~ quinan, a novel arteriovenous dilating agent, and its metaboli on bovine heart phosphodiesterase activity, Br. J. Pharmacol. 733P. Hagiwara, M., T. Endo and H. Hidaka, 1984, Effects of vinpoceti on cyclic nucleotide metabolism in vascular smooth musc Biochem. Pharmacol. 33, 453. Hardman, J.G., 1984, Cyclic nucleotides and regulation of vascul smooth muscle, J. Cardiovasc. Pharmacol. 6 (Suppl. 4), $639. Katsuki, S. and F. Murad, 1977, Regulation of adenosine cyc 3',5'-monophosphate and guanosine cyclic 3',5'-monophosph~ and contractility in bovine heart tracheal smooth muscle, M, Pharmacol. 13, 330. Kauffman, R.F., K.W. Schenck, B.G. Unerback, V.G. Crowe a~ M.L. Cohen, 1987, In vitro vascular relaxation by new inotroI agents: relationship to phosphodiesterase inhibition and cyc neucleotides, J. Pharmacol. Exp. Ther. 242, 864. Kessler, P.D. and M. Packer, 1987, Haemodynamic effects of BTS 465, a new long-acting systemic vasodilator drug in patients wi severe congestive heart failure, Am. Heart. J. 113, 137. Lugnier, C., P. Schoeffter, A. Le Bec, E. Strouthou and J.C. Stocl~ 1986, Selective inhibition of cyclic nucleotide phosphodiesteras of human, bovine and rat aorta, Biochem. Pharmacol. 35, 1743 Packer, M., P.D. Kessler, R.S. Porter, N. Medina, M. Yushak a~ S.S. Gotlieb, 1988, Do plasma levels of flosequinan correlate wi its haemodynamic effects in severe chronic heart failure?, J. A Coll. Cardiol. 11, Suppl. A. 42A. Pang, D.C., 1988, Cyclic AMP and cyclic GMP phosphodiesteras~ Target for drug development, Drug Dev. Res. 12, 85. Perreault, C.L., N.L. Hague, I.M. Hunneyball, M.F. Sim and J. Morgan, Lack of inotropic response to flosequinan in ventricul muscle from patients with end-stage heart failure, Circulation press). Reeves, M.L., B.K. Leigh and P.J. England, 1987, The identificati, of a new cyclic nucleotide phosphodiesterase activity in hum and guinea-pig cardiac ventricle: implications for the mechanL, of action of selective phosphodiesterase inhibitors, Biochem. 241,535. Richards, N.T., L. Poston and P.J. Hilton, 1989, Flosequinan-i duced vasodilation of human resistance vessels is associated wi decreased calcium sensitivity, Br. J. Pharmacol. 98, 734P. Silver, P.J., 1989, Biochemical aspects of inhibition of cardiovascul low (Km) cyclic adenosine monophosphate phosphodiestera~ Am. J. Cardiol. 63, 2A. Silver, P.J., L.T. Hamel, M.H. Perrone, R.G. Bentley, C.R. Busho~ and D.B. Evans, 1988, Differential pharmacologic sensitivity cyclic nucleotide phosphodiestera~se isoenzymes isolated fro cardiac muscle, arterial and airway smooth muscle, Eur. J. Ph~ macol. 150, 85. Thompson, W.J., W.L. Terasaki, P.M. Epstein and S.J. Strada, 19,
3 Assay of cyclic nucleotide phosphodiesterase and resolution of multiple molecular forms of the enzyme, Adv. Cycl. Nucl. Res. 10, 69. Weishaar, R.E., M. Quade, D. Boyd, J. Schenden, S. Marks and H.R. Kaplan, 1983, The effect of several "new and novel" cardiotonic agents on key subcellular processes involved in the regulation of myocardial contractility, Drug Dev. Res. 3, 517. Weishaar, R.E., M. Quade, J.A. Schenden, D.K. Boyd and D.B. Evans, 1985, Studies aimed at elucidating the mechanism of action of CI-914, a new cardiotonic agent, Eur. J. Pharmacol. 119, 205. Weishaar, R.E., S.D. Burrows, D.C. Kobylarz, M.M. Quade and D.B. Evans, 1986, Multiple molecular forms of cyclic nucleotide phosphodiesterase in cardiac and smooth muscle and in platelets: isolation, characterisation and effects of various reference phosphodiesterase inhibitors and cardiotonic agents, Biochem. Pharmacol. 35,787.
Weishaar, R.E., A.M. Wallace, M.L. Kirker, V.A. Ferraris and M Sim, 1991, Comparative effects of flosequinan and milrinone the contractile activity of cardiac muscle fibres from patients wi and without heart failure, Br. J. Pharmacol. 104, 18P. Wynne, R.D., E.L. Crampton and I.D. Hind, 1985, The pharmaco netics and haemodynamics of BTS 49 465 and its major metal: lite in healthy volunteers, Eur. J. Clin. Pharmacol. 28, 659. Yates, D.B. 1991, Pharmacology of flosequinan, Am. Heart. J. 1~ 974. Yates, D.B. and G.R. Hicks, 1988, Effects of flosequinan on guin~ pig atria and on cardiac variables in the anaesthetised dog, Br. Pharmacol. 95, 711P. Yates, D.B. and J.R. Holmes, 1988, Effect of flosequinan and otl~ vasorelaxants on rat aortic contractions stimulated by nt adrenaline and phorbol ester, Br. J. Pharmacol. 95, 819P.
3
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52259
Effect of flosequinan upon isoenzymes of phosphodiesterase from guinea-pig cardiac and vascular smooth muscle GiUian F r o d s h a m a n d R o b e r t B. J o n e s Boots Pharmaceuticals, Research Department, Nottingham NG2 3AA, U.K.
Received 17 July 1991,revised MS received 11 October 1991, accepted 5 November 1991
The effect of flosequinan and its sulphone metabolite BTS 53 554, on phosphodiesterase isoenzymes isolated from guinea-[ cardiac and vascular smooth muscle using DEAE-cellulose chromatography was investigated. Zaprinast and milrinone show peak I and peak III selectivity, and IBMX non-selective activity respectively, against both cardiac and vascular smooth mus~ isoenzymes, as expected for these reference inhibitors. Flosequinan and BTS 53 554 demonstrated non-selective inhibition wJ similar potency against both cardiac and vascular smooth muscle isoenzymes and, overall, were the least potent compoun tested. The high inhibitory concentrations observed (ICs0 peak III 660 p~M for cardiac tissue and 230/~M for vascular smo¢ muscle with flosequinan) relative to its clinically effective plasma concentration (10/~M) questions the relevance of phospho, esterase inhibition to the efficacy of flosequinan in heart failure. Phosphodiesterase isoenzymes; Cardiac muscle; Smooth muscle (aortic); Flosequinan; BTS 53 554; (Guinea-pig)
1. Introduction Flosequinan (BTS 49 465, 7-fluoro-l-methyl-3-methylsulphinyl-4-quinolone) (Davies et al., 1983)is a novel orally effective hypotensive agent with both arterial and venous vasodilator properties and a prolonged duration of action in man (Cowley et al., 1984). Flosequinan is under clinical investigation for use in hypertension and heart failure (Cowley et al., 1987; Cowley, 1991; Kessler and Packer, 1987). In preliminary studies with a commercially available mixed isoenzyme preparation from bovine heart (Frodsham et al., 1989) flosequinan appeared to be a non-selective inhibitor with only low potency when compared with the P D E III selective inhibitor milrinone, P D E V selective inhibitor zaprinast and nonselective inhibitor IBMX (Alousi et al., 1983; Bergstrand et al., 1977; Beavo et al., 1970), but the use Of a mixed isoenzyme preparation made interpretation of activity on individual isoenzymes difficult. A rise in both cAMP and cGMP would be expected as a consequence on non-selective phosphodiesterase inhibition
Correspondence to: (3. Frodsham, Boots Pharmaceuticals, Research Department, Nottingham NG2 3AA, U.K. Tel. 44.602.492 679, fax 44.602.492 614.
and increases in cAMP and cGMP are associated wi vasorelaxation in vascular smooth muscle (Katsuki m Murad, 1977; Hardman, 1984). In cardiac tissue ele~ tion in cAMP is associated with positive inotro[ activity (Evans, 1986) whereas elevation in cardi cGMP may reduce inotropic activity (Drummond a~ Severson, 1979). The purpose of the present study was to investig~ the effects of flosequinan upon isoenzymes of phosph diesterase, isolated from guinea-pig cardiac (left vent cle) and vascular smooth muscle (aorta). Since t sulphone metabolite of flosequinan, BTS 53 554 pharmacologically active (Davies et al., 1983) and m be responsible for a major part of the clinical respon to flosequinan (Wynne et al., 1985; Packer et al., 19~ its effect was also investigated. Although it has been reported to possess non-sek tive P D E inhibitory activity of low potency (Frodsh~ et al., 1989) recent evidence using right atrial pendage tissue (Weishaar et al., 1991) and in ventrk lar tissue (Perreault et al., in press) from heart faik patients suggests that flosequinan has little or no : otropic activity even in the presence of the adenyl~ cyclase stimulator forskolin. This contrasts with d~ obtained using the P D E III selective inhibitor rail none and the non-selective PDE inhibitor IBIV (Feldman et al., 1987).
384 The activity of flosequinan on PDE isoenzymes is discussed in relation to its therapeutic plasma concentration in man.
2. Materials and methods
2.1. Separation of isoenzymes ofphosphodiesterase Unless indicated otherwise all procedures were carried out at 4°C. Slight modifications of the methods of Thompson et al. (1979) and Silver et al. (1988) were used to separate the isoenzymes of phosphodiesterase from cardiac tissue. Hearts were removed from guinea-pigs (Dunkin Hartley, males, 400-600 g body weight) following cervical dislocation and the left ventricle and septum dissected and washed in ice-cold saline. Five hearts were used for each preparation (giving a total of 7.0-9.0 g tissue). Tissue was minced finely and homogenised in eight volumes (8 m l / g wet weight) of buffer containing 10 mM Tris acetate, pH 7.5, 2 mM MgC12, 1 mM dithiothreitol (DTT), 2000 units/ml aprotinin and 50 /xM phenylmethylsulphonyl fluoride (PMSF) for 3 × 10 s bursts using a Polytron PT-10 on setting No. 5. The resulting homogenate was sonicated (30 s / m l ) and centrifuged at 40000 × g for 30 min after which the supernatant fraction was applied to a DEAE-cellulose column (30 X 1.7 cm, bed volume 68 ml) previously equilibrated with 0.07 M sodium acetate buffer (pH 6.5). As with all other column buffers it contained 5 mM 2-mercaptoethanol. After application of the sample, the column was washed with three to four bed volumes of 0.07 M sodium acetate buffer, (pH 6.5). The phosphodiesterase isoenzymes were eluted (flow rate 0.5 m l / m i n ) using 200 ml of 0.2 M sodium acetate (pH 6.5) followed by a linear gradient of 0.2-0.6 M sodium acetate (pH 6.5) (450 ml). Fractions (11 ml) were collected and assayed for cAMP and cGMP phosphodiesterase activity at 1/xM substrate concentration. Calmodulin stimulation of cAMP hydrolysis was measured using 10 units calmodulin/10 /zM CaC12 and cGMP stimulation of cAMP hydrolysis by the inclusion of 0.5/xM cGMP. Appropriate fractions corresponding to peak activity were pooled and diluted with ethylene glycol to 30%. Aliquots were stored at - 2 0 ° C and activities and response to inhibitors were stable for over 3 months following preparation, Isoenzymes of phosphodiesterase were separated from guinea-pig aorta by a slight modification of the method of Hagiwara et al. (1984). Aortas were removed from guinea pigs (Dunkin Hartley, males, 300500 g body weight) and cleaned of adhering tissue, They were washed in ice-cold saline and then stored at
-80°C. Typically 70 aortas were used per preparati( (giving approximately 4.0 g tissue). The tissue was fir powdered at the temperature of liquid nitrogen using Braun Mikrodismembrator. The tissue powder was lowed to thaw in the presence of five volumes homogenisation buffer: 0.05 M Tris acetate (pH 6. containing 2.75 mM 2-mercaptoethanol, 1 mM MgCI 2000 units/ml aprotinin and 50 ~ M PMSF. All subs quent buffers contained 2.75 mM 2-mercaptoethan( Homogenisation was accomplished by 3 x 10 s burs with a Polytron PT-10 on setting No.7 and was faci tated by prior powdering of the tissue. The h mogenate was sonicated (30 s / m l ) and centrifuged 100000 × g for 60 min after which the supernata fraction was applied to a DEAE-cellulose column C × 1.7 cm, bed volume 45 ml) previously equilibrat~ with 0.05 M sodium acetate (pH 6.0). After applicati( of the sample the column was washed with three b~ volumes of 0.05 N sodium acetate (pH 6.0), followed 1 a linear gradient of 0.05-0.6 M sodium acetate (p 6.0), (400 ml) to elute the phosphodiesterase isoe zymes. Fractions (6 ml) were collected and assayed f, cAMP and cGMP phosphodiesterase activity using /zM substrate. Calmodulin stimulation of cGMP hydr lysis was measured as for cardiac enzymes. Approp~ ate fractions were treated as before except that wi smooth muscle preparations the enzymes were us~ within 3 weeks, during which time the response inhibitors was stable. The protein content of column fractions was mot toted by absorbance at 280 nm and the protein conte of pooled peak fractions assayed using the method Bradford (1976) with bovine serum albumin as stan ard.
2.2. Phosphodiesterase assay Phosphodiesterase activity was measured by tl method of Thompson et al. (1979) with the addition 0.005 /zCi [U-14C]guanosine or [U-14C]adenosine recovery markers for the column separation step. T] reaction mixture (0.4 ml final volume) contained . mM Tris HC1 (pH 8.0), 5 mM MgC12 and 1 mM D T The reaction was initiated by the addition of enzy13 diluted to give 15-20% substrate utilisation during t] 10 min incubation at 30°C. Typically the recovery [U-a4C]adenosine from the anion exchange column st~ was greater than 90% and [U-14C]guanosine great than 65%. Each sample was individually corrected f recovery. Under these conditions the assay was line with respect to time and enzyme concentration. F assessing phosphodiesterase activity in column fra tions the substrate ([3H]cAMP or [3H]cGMP)conce tration was 1 /zM.
3 2.3. Substrate kinetic studies
3. Results
For substrate kinetics experiments the concentration of cAMP and cGMP typically ranged from 0.1 to between 25 and 100 times the K m (K m = substrate concentration at which half-maximal reaction velocity is observed), A Hanes plot of [S]/v versus [S] (where v is reaction velocity and [S] is the substrate concentration) for the initial analysis of data from peaks I and III showed two apparently linear segments. These were analysed individually by non-linear regression of Michaelis-Menten plots using the Biosoft Enzfitter computer program. Two K~, and V.aax (maximum reaction velocity) values could be obtained in such cases, implying a high and low affinity site for hydrolysis of the substrate. Values for K m and Vmax were calculated for each isoenzyme type, two being calculated for peaks I and III where high and low affinity sites were demonstrated.
3.1. Isolation of multiple forms of cyclic nucleotide phc phodiesterase
2.4. Inhibitor studies For inhibitor studies the compounds to be tested were dissolved in DMSO (2.5% final assay concentration). Vehicle controls were included in all experiments. The concentration of inhibitor which produced 50% inhibition of enzyme activity (IC50) was determined graphically from concentration-response curves in which doses of drugs ranged from 0.1 to 3000/xM in half-log increments. All substrate kinetics and inhibitor assays were performed in triplicate and three separate preparations of phosphodiesterase were used from both cardiac and vascular tissue,
2.5. Materials [2,8-3H]cAMP (30-50 Ci/mmol), [8-3H]cGMP (1030 Ci/mmol), and [U-laC]adenosine (500-600 m C i / mmol) were purchased from Amersham International plc. [U-14C]guanosine (> 400 mCi/mmol) was purchased from Du Pont (LI.K.) Ltd. Calmodulin (from bovine brain), Ophiophagus hannah snake venom and aprotinin (from bovine lung) were purchased from Sigma Chemical Co. Dowex AG l-X8 200-400 mesh (Cl-form), disposable econo-columns, and protein assay kits were purchased from Bio-Rad Laboratories. DEAE-cellulose (DE52) was purchased from Whatman. All other reagents were purchased from either Sigma Chemical Co. or BDH and were Analar grade, Drugs were obtained from the following sources: IBMX (3-isobutyl-l-methylxanthine) (purchased from Sigma Chemical Co.); zaprinast (kindly donated by May & Baker); flosequinan, BTS 53 554 and milrinone (synthesised by Medicinal Chemistry, Boots Pharmaceuticals Research Department).
Using guinea-pig ventricle an elution profile simil to that obtained by Silver et al. (1988) was obtaine Three isoenzymes (fig. 1) were isolated having differe characteristics (table 1). The first isoenzyme (peak phosphodiesterase) eluted at 0.2 M sodium acetate a~ hydrolysed both cAMP and cGMP. High and low affi ity sites for cAMP and cGMP hydrolysis were show The Km'S were similar for both cAMP and cG/v though greater activity was demonstrated with cGi~ in contrast to the results obtained by Silver et al. (198 where cAMP activity was greatest. This isoenzyme w stimulated by Ca2+-calmodulin, the Vmax for cGI~ increasing by 6 fold (table 1). In the presence of Ca: -calmodulin there was only one high affinity site I cGMP hydrolysis. The second isoenzyme (peak II phosphodiestera~, eluted between 0.3-0.35 M sodium acetate and a peared to have a single affinity site with similar affin: for both cAMP and cGMP. This isoenzyme had low affinity for cAMP and cGMP than the other isoe zymes but a greater Vmax. It could be stimulated by ( /xM cGMP (fig. 1). In a separate experiment peak cAMP hydrolysis was stimulated maximally (7-8 fol by concentrations of cGMP of greater than 1 /x (single experiment). The third isoenzyme (peak III phosphodiestera~, was eluted at 0.4-0.5 M sodium acetate and show both high and low affinity sites for the hydrolysis cAMP. cGMP inhibited the hydrolysis of cAMP peak III phosphodiesterase (fig. 1) and in a separ~ experiment was shown to have an IC50 value of 2.2/z for this inhibition (single experiment), cGMP increas the K m for cAMP of this isoenzyme and it was possit to obtain K m and Vmax values for peak III phospho~ esterase with cGMP as substrate. The high affinity was the same for cGMP as for cAMP but greal activity was observed with cAMP. A relatively hi affinity for cGMP has been demonstrated previou: for peak 1II phosphodiesterase (Reeves et al., 1987). The elution profile obtained by DEAE-cellulc chromatography of vascular smooth muscle is shown fig. 2. Three phosphodiesterase isoenzymes were four The two peaks eluting at 0.1-0.2 and 0.3-0.4 M sodit acetate concentration both hydrolysed cGMP but h little cAMP hydrolytic activity. Calmodulin stimulation was not observed w: smooth muscle phosphodiesterase isoenzymes. Peat had high affinity for cGMP with a Kr, of 2.7 + 0.7 # (n = 3) compared with a K m of 25.4 + 3.1 /xM (n = for peak II and although cAMP hydrolysis could demonstrated it was too low to determine a K m vah
386 PDE
activity
(pmol/min/fraction)
Sodium
acetate
2500
/
(M) 0.6
/ /
2000
/
/
/
0.5
/
0.4
1500
0.3 1000
_
_
---/
/
/ 0.2
500
0.1
0 20
30
40
50
60
Fraction
.
.
70
80
0.0 90
100
11
number
Fig. 1. Representative DEAE-cellulose chromatogram of cAMP and cGMP phosphodiesterase activity from guinea-pig ventricle after elution 200 mM sodium acetate followed by a 200-600 mM sodium acetate gradient. Enzyme activity was measured as described in Materials a methods; (o) cAMP, ([]) cGMP, ( • ) cAMP plus Ca 2+-Calmodulin, (o) cAMP plus cGMP. ( - - - ) sodium acetate gradient. Fraction volun were 11 ml.
In c o n t r a s t , t h e t h i r d p e a k ( p e a k I I I ) w h i c h e l u t e d at 0 . 4 - 0 . 6 M s o d i u m a c e t a t e h a d a h i g h affinity for c A M P ( K m = 0.96 + 0.03 ~ M , n = 3) b u t n o s i g n i f i c a n t c G M P h y d r o l y t i c activity, c A M P h y d r o l y s i s by p e a k I I I was i n h i b i t e d by c G M P (IC50; 0.6 /xM, single e x p e r i m e n t ) ,
3.2. Effects of flosequinan, BTS 53 554 and reference phosphodiesterase inhibitors T h e e f f e c t s o f f i o s e q u i n a n , B T S 53 554, I B M X , milrinone and zaprinast upon cAMP and cGMP hydrolysis by c a r d i a c p h o s p h o d i e s t e r a s e isoenzymes are
s h o w n in t a b l e 2. I B M X w a s r e l a t i v e l y n o n - s e l e c t i b e i n g e q u i p o t e n t w h e n c G M P was u s e d as s u b s t r a t e d e m o n s t r a t i n g l o w e r p o t e n c y o n p e a k II p h o s p h o r e s t e r a s e w h e n c A M P was s u b s t r a t e . M i l r i n o n e d e m o strated a marked selectivity for the peak III isoenzyr w h e n c A M P was s u b s t r a t e a n d z a p r i n a s t s h o w e d s e k tivity for t h e p e a k I i s o e n z y m e r e g a r d l e s s o f substra type. F l o s e q u i n a n i n h i b i t e d all t h r e e i s o e n z y m e f o r n t h o u g h , o v e r a l l , b e i n g l e a s t e f f e c t i v e u p o n t h e peak i s o e n z y m e as w a s t h e s u l p h o n e m e t a b o l i t e B T S 53 5.' O f t h e a g e n t s t e s t e d , f l o s e q u i n a n a n d B T S 53 554 we the least potent inhibitors of phosphodiesterase act
TABLE 1 Apparent substrate affinities for isoenzymes of phosphodiesterase from guinea-pig ventricle. Isoenzyme Peak
Substrate type
A p p a r e n t K m (/xM) a
High affinity
Low affinity
Wmax (pmol/min per ~g protein) High Low affinity affinity
I
cAMP cGMP cGMP + CaCm b
1.3+0.2 1.4+-0.2 1.8 + 0.2
12.0+3.5 9.8+-1.3 c
5.7+_ 1.8 10.0+ 4.7 60.4 +-23.6
c
II
cAMP cGMP cAMP+0.5 IzM cGMP
d a a
31.3+-5.3 40.7+-0.3 24.7+-4.1
d d d
208 +-29.7 331 +-54.7 213 +- 16.4
III
cAMP cGMP cAMP+0.5/xM cGMP
1.6+-0.3 1.6+-0.5 3.4+-0.5
13.7_+1.5 38.7+-9.6 13.7+2.7
10.9+- 0.9 2.7+- 0.2 15.5+- 0.9
11.5+- 2.1 23.7+- 5.9
25.8+- 4.9 20.8+- 6.6 26.0+- 1.8
a The apparent K m (substrate concentration at which half-maximal velocity of hydrolysis occurs) was determined by non-linear regression anah of Michaelis-Menton plots using the Biosoft Enzfitter computer program. Phosphodiesterase activity was measured over cyclic nucleot concentrations typically ranging from 0.1 to between 25 and 100 times the anticipated Km. Values are the means±S.E.M, for triplic determinations from two or three preparations per isoenzyme, b Ca2+ concentration = 10 IzM; calmodulin (Cm)= 10 units, c In the presence Ca2+-calmodulin there was no indication of a low affinity site for cGMP hydrolysis, d No evidence of a high affinity site of hydrolysis.
3~ PDE
activity
(pmol/min/fraction)
S o d i u m a c e t a t e (M)
500
/
0.7
/ ./ / 400
/
0.6
/
"< 300
/
/
0.5
/
0.4
/
2OO
0.3 0.2
lO0 0.! . 20
30
40
50
.
.
.
60
0.0
70
80
90
Fraction number Fig. 2. Representative DEAE-cellulose chromatogram of cAMP and cGMP phosphodiesterase activity from guinea-pig aortic smooth muse after elution by a 50-700 mM sodium acetate gradient. Enzyme activity was measured as described in Materials and methods; (o) cAMP, (I cGMP. ( - - - ) sodium acetate gradient. Fraction volumes were 6 ml. % inhibition of enzyme activity
% inhihilion of enzyme activity
100"
100-
80
80"
60 "
61l "
40 "
40
20
20
0
0
-20
-20 10 ?
10 s
I 0 ~'
10.4
I 0~
102
II ?
.................. II)"
120 -
• .......................... I1l 4 10-.~
10 z
Molar cou,,'etilralion of drag
Molar concentration of drug
% inhihition
I0 s
of enzynne activity
% inhibition
of enzyme activity
100
c
I O0 "
80
80"
60"
60 " 4041l
20" 20 "
oi
oi i
I
IJ ~
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10"
10 r,
10.s
111.4
Mol:|r concentration of drug
IO ~
IO z
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
lO-g
10 -?
10-6
lo-s
10-4
1 0 .3
10 .2
Molar concentration of drug
Fig. 3. Effect of several agents upon phosphodiesterase (PDE) activity. (a) Peak I cGMP PDE from guinea-pig ventricle; (b) peak III cAMP P[ from guinea-pig ventricle; (c) peak I cGMP PDE from guinea-pig vascular smooth muscle; (d) peak III cAMP PDE from guinea-pig vascu smooth muscle. Substrate concentration in each case was 1.0 /~M. Each curve represents the meaned data generated from three separ~ concentration response curves (each obtained in triplicate) from three separate isoenzyme preparations except BTS 53 554 where n : (ventricle) and n = 1 (vascular smooth muscle). (©) Flosequinan, (o) BTS 53 554, ( • ) IBMX, ( / , ) zaprinast, ( • ) milrinone.
388 TABLE 2 Effect of agents upon isoenzymes of phosphodiesterase from guinea-pig ventricle. Agent
IC50 (/zM) a Peak I
Flosequinan BTS 53 554 b IBMX Milrinone Zaprinast
Peak II
Peak III
cAMP c
cGMP c
cAMP
cGMP
cAMP
cGMP
2200 ± 380 d 14% at l m M e 22 ± 3 440 _+ 130 12±0.3
2400 +_470 18% at 1 mM e 21 ± 3.4 235 _+55 b 14+ 1.3
2700 _+ 180 23% at 1 mM e 62 ± 12 820 _+93 240+32
600 ± 26 830±130 18 ± 0.6 290 ± 50 66+3.8
660 _+63 1000_+180 14 ± 1.3 5.7 ± 2.0 44% at 3 mM e
1200 _+250 37% at 1 mM e 15 _+1.7 395 ± 304 160±38
a IC5o values (concentration which inhibits substrate hydrolysis by 50%) were determined from concentration-response curves in which t concentration of agents ranged from 0.1 ~ M to 3 mM (half-log increments) except BTS 53 554 where the maximum concentration was 1 ml Three concentration-response curves were generated each in triplicate from three separate isoenzyme preparations, b Concentration-respor curves generated each in triplicate from two separate isoenzyme preparations, c Substrate concentration in the assay was 1.0 p~M. d Values given as means ± S.E.M. e Values are given as percentage inhibition where IC50 was not attained at the highest concentration tested.
ity, flosequinan being 116 and 171 times less potent than milrinone against peak III (cAMP) and zaprinast against peak I (cGMP) respectively (figs. 3a,b). The effects of flosequinan, BTS 53 554, IBMX, milrinone and zaprinast upon cAMP or cGMP hydrolysis by aortic smooth muscle phosphodiesterase isoenzymes are shown in table 3. The two isoenzyme peaks I and II were inhibited to a different extent by zaprinast which was 12.8 times more potent as an inhibitor of peak I than peak II. Both these isoenzyme forms were inhibited to a greater degree than was peak III. As with cardiac phosphodiesterase isoenzymes, milrinone was a selective peak III inhibitor and IBMX was relatively non-selective. Flosequinan and BTS 53 554 inhib±ted all three isoenzyme types, flosequinan with most potency against peak Ill and BTS 53 554 with equal potency on peaks II and III. Flosequinan and BTS 53 554 were the least potent of the compounds tested, TABLE 3 Effect of agents upon isoenzymes of phosphodiesterase from guinea-
pig vascular smooth muscle. Agent
IC50 (~.M) ~
Peak I
Peak II
cGMP c
cGMP c
1650+280
890+350
Peak III cAMP c 230+54
Flosequinan BTS 53 554 u
23% at 1 mM d
1000
900
IBMX
26+3.5
8±2.5
6.5+0.7
Milrinone
200 + 17
330 _+66
Zaprinast
2.5 +0.09
32+ 15
1.9 + 0.07
460+ 72
a iC50 values (concentration which inhibits substrate hydrolysis by 50%) were determined from concentration-response curves in which the concentration of agents ranged from 0.1 /zM to 1 mM (half-log increments) except BTS 53 554 where the maximum concentration was 1 mM. Three concentration-response curves were generated each in triplicate from three separate isoenzyme preparations. Values are given as means+S.E.M, b Concentration-response curve generated in triplicate from a single preparation, c Substrate concentration in the assay was 1.0/zM. d Value given as percentage inhibition where IC50 was not attained at the highest concentration tested,
flosequinan being 121 times less potent than milrinol against peak III and 660 times less potent than zap~ hast against peak I (figs. 3c, d respectively).
4. Discussion
All three isoenzymes of phosphodiesterase isolat~ from guinea-pigventricle hydrolysed cAMP and cGM peak I activity being stimulated by CaZ+-calmodul and peak II by cGMP. Peak III cAMP hydrolysis w inhibited by cGMP possibly due to a competitive effe between the two substrates since the affinity for hydr lysis of both cAMP and cGMP was the same in o study (K m = 1.6/xM), and the IC50 value for inhibiti~ of peak Ill PDE by cGMP was 2.2/.~M. The possibili of co-elution of PDE III and P D E IV has been rais4 by Reeves et al. (1987) when using DEAE-cellulo chromatography. Recently we have shown that usi~ FPLC with a Mono Q column, a small fourth pe, (rolipram sensitive P D E IV) can be distinguished fre P D E III (Frodsham et al., 1991). Flosequinan and B'~ 53 554 were weak inhibitors of PDE IV (IC50 values 1.8 and > 3.0 mM respectively) whereas the poten against the other isoenzymes were similar to that us±: DEAE-cellulose chromatography. In vascular smooth muscle three isoenzymes of PE activity were also isolated, peaks I and II hydrolysi: mainly cGMP and peak III cAMP. Peak I had a hil affinity and peak II a low affinity for cGMP hydrolys neither of which were stimulated by Ca 2 +-calmoduli Although this could have been caused by co-elution of the isoenzymes with calmodulin, the lack stimulation in the presence of additional Ca 2+ mak this unlikely. Preliminary separation of smooth m u s c PDE isoenzymes using FPLC with a Mono Q colun has demonstrated a fourth peak of activity eluti:
one
immediately prior to peak I which is only apparent the presence of Ca2÷-calmodulin and which may ha
co-chromatographed with peak I previously using DEAE (unpublished data). Using these isoenzymes from Mono Q zaprinast was selective (approximately 10 fold) for the Ca2+-calmodulin insensitive peak whereas flosequinan was non-selective, Zaprinast has been reported (Lugnier et al., 1986) to inhibit the Ca2+-calmodulin insensitive form of cGMP PDE more than the Ca 2+ -calmodulin sensitive form (IC50 values with 1 /zM cGMP were 0.4 and 21.0/~M respectively). The ICs0 value obtained in this study for peak I PDE (2.5 p.M) lies between those reported by Lugnier et al. and could therefore be indicative of co-chromatography of the two peaks. If this is the case then the peak II eluted from DEAE in this study represents a form of cGMP phosphodiesterase with low affinity for cGMP rather like the peak II observed in cardiac muscle, In addition to substrate kinetic data several reference phosphodiesterase inhibitors were used to aid classification of isoenzymes and for comparison with the effects of flosequinan and BTS 53 554. Milrinone, zaprinast and IBMX displayed peak III, peak I and non-selective inhibitory profiles respectively in both ventricular and aortic smooth muscle, The substrate kinetic data obtained, combined with the selectivity observed with reference inhibitors demonstrated that typical isoenzymes from both cardiac and vascular smooth muscle had been isolated (with the possible exception of peak II from smooth muscle) (Hagiwara et al., 1984; Weishaar et al., 1 9 8 6 ; Silver et al., 1988). On this basis peaks I II and III from cardiac muscle have been classified as the Ca 2+calmodulin-dependent PDE (PDE I), cGMP-stimulated PDE (PDE II) and cGMP-inhibited PDE (PDE III) respectively. Peaks I, II and III isolated from vascular smooth muscle have been classified as cGMP specific PDE (PDE V), cGMP specific PDE with low substrate affinity (possibly PDE II) and cGMP-inhibited PDE (PDE III) respectively, Flosequinan and BTS 53 554 were found to be non-selective inhibitors of phosphodiesterase isoenzymes from both tissue types and overall were the least potent of the compounds tested. Data to support the view that flosequinan has nonselective PDE inhibitory activity of only low potency is also derived from in vitro experiments where elevation of tissue levels of cGMP and cAMP have been observed. In vascular smooth muscle, increases in both cAMP and cGMP are implicated in relaxation (Hardman, 1984). In rat aortic strips (at concentrations producing an approximate 90% relaxation) flosequinan at 1 mM increased cGMP 4 fold (Allcock et al., 1988) and cAMP by 38% (P < 0.001, Allcock, unpublished observations). However, flosequinan causes relaxation of noradrenaline or phorbol dibutyrate ester induced contractions in the presence of methylene blue (an in-
hibitor of soluble guanylate cyclase) in rat aortic stri (Yates and Holmes, 1988) and human resistance w sels (Richards et al., 1989) indicating that flosequin~ does not act by stimulation of soluble guanylate clase. In guinea-pig atria flosequinan had an EC40 val~ for inotropy of 650/xM, (Yates and Hicks, 1988) and chopped guinea-pig ventricle flosequinan at 650 al 6500 ~M produced significant increases in cAMP k els of 16 and 64% respectively (Yates, 1991). Small b non-significant increases in cGMP were also observ, at the EC40 value (24%) whereas a significant increa was observed at the 6500 ~M concentration (73~ When flosequinan and isoprenaline were given in col bination, greater increases in cAMP were observ than could be accounted for by the simple addition the two effects of flosequinan and isoprenaline alor Flosequinan is therefore able to produce small elex tions in both cAMP and cGMP in both vascular a: cardiac tissue although only at relatively high conce trations. There have been several reports in which elevatio in cyclic nucleotides were only apparent at concentl tions in excess of those required for PDE inhibitk vasorelaxation and inotropy (Weishaar et al., 19~ Lugnier et al., 1986; Pang, 1988; Kauffman et al., 19~ Weishaar et al., 1983; Silver, 1989). Such observatio have been explained on the basis of differential acc~ of drugs between isolated enzymes and whole tisst functional compartmentation and high variability basal cyclic nucleotide levels. A comparison between the in vitro pharmacologic responses for flosequinan, PDE inhibition and cli~ cally effective plasma concentrations are pertinent the discussion as to whether flosequinan exerts beneficial effects in the treatment in heart failure virtue of its PDE inhibitory properties. The PDE inhibitory activity of flosequinan can~ be correlated with its vasorelaxant activity (ICs0 P[ III 230 tzM (table 3)compared to ICs0 for vasorel~ ation of 65/zM (Yates and Holmes, 1988)) but can correlated with its weak inotropic activity (ICs0 P[ III 660/zM (table 2) compared to If40 for inotropy 650 /.LM (Yates and Hicks, 1988)). However in bc cases the concentrations required for PDE inhibiti are much higher than the therapeutic plasma level ( /~M; Packer et al., 1988). In view of this it is probat that there are alternative mechanisms involved in t clinical action of flosequinan. In right atrial appendage tissue from patients wJ heart failure (Weishaar et al., 1991) and in ventricu] tissue from patients with end-stage heart failure (Pq reault et al., in press), flosequinan has little or inotropic activity. In addition forskolin does not pote tiate the inotropic activity of flosequinan (Weishaar al., 1991), but does potentiate the inotropic activity
390 m i l r i n o n e a n d I B M X ( F e l d m a n et al., 1987) s u g g e s t i n g t h a t f l o s e q u i n a n acts via a d i f f e r e n t m e c h a n i s m c o m p a r e d to t h a t o f m i l r i n o n e o r I B M X . T h e r e f o r e , alt h o u g h in g u i n e a - p i g v e n t r i c l e t h e r e a p p e a r s to b e a c o r r e l a t i o n b e t w e e n i n o t r o p i c activity a n d P D E inhibition, t h e s i t u a t i o n in f a i l i n g h u m a n h e a r t is c u r r e n t l y less clear, I n c o n c l u s i o n , f l o s e q u i n a n a n d B T S 53 554 p o s s e s s n o n - s e l e c t i v e p h o s p h o d i e s t e r a s e i n h i b i t o r y activity o f low p o t e n c y in b o t h g u i n e a - p i g c a r d i a c a n d v a s c u l a r s m o o t h m u s c l e . T h e p r o f i l e o f i n h i b i t i o n is d i f f e r e n t to that observed with zaprinast and milrinone (PDE V a n d P D E I I I s e l e c t i v e i n h i b i t o r s r e s p e c t i v e l y ) a n d is s i m i l a r to t h e n o n - s e l e c t i v e P D E i n h i b i t o r I B M X . T h e d a t a h e r e , in a d d i t i o n to p r e v i o u s f i n d i n g s , s u g g e s t t h a t f l o s e q u i n a n m a y p r o d u c e p h a r m a c o l o g i c a l e f f e c t s at h i g h c o n c e n t r a t i o n s in i s o l a t e d a n i m a l tissues at l e a s t in p a r t by i n c r e a s i n g levels o f i n t r a c e l l u l a r cyclic n u c l e o t i d e s a n d in g u i n e a - p i g c a r d i a c tive P D E i n h i b i t i o n . T h e e f f e c t s studies must however be viewed since t h e y o c c u r at c o n c e n t r a t i o n s t h o s e o b s e r v e d clinically.
tissue by n o n - s e l e c o b s e r v e d in a n i m a l with some caution substantially above
Acknowledgements The authors gratefully acknowledge the excellent technical assistance provided by Miss Deborah Anthony, Mrs. Tracey North, Miss Christine Spinks and Mr. Gary Telford.
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