Effect of Rp diastereoisomer of adenosine 3′,5′ cyclic-monophosphothioate on the cAMP-dependent relaxation of smooth muscle

PII sooz4-3205(97)00589-4

EFFECT OF Rp DIASTEREOISOMER OF ADENOSINE 3’,5’ CYCLICMONOPHOSPHOTHIOATE ON THE CAMP-DEPENDENT RELAXATION OF SMOOTH MUSCLE Jose R. Perez-Vallina, Laboratorio

M.Pilar Revuelta,

Begoiia Cantabrana

de Farmacologia, Dpto. Medicina, 33006 Oviedo, Spain.

and Agustin

Hidalgo.

Facultad de Medicina.

(Received in final form May 29,1997)

Summary The effect of Rp diastereoisomer of adenosine 3’,5’-cyclic monophosphothioate (Rp-CAMPS) on relaxation elicited by histamine (l100 PM), forskolin (l-60 yM), papaverine (l-1 00 PM), vinpocetine (1-l 00 FM), rolipram (O.?-1 mM), Sp-CAMPS (1 O-300 FM), 8-BrcAMP (IO PM-I mM) and 8-BrcGMP (3 uM-1 mM) of the previous vanadate-induced contraction was assayed. The effect of Rp-CAMPS on the relaxing effect produced by forskolin, papaverine, vinpocetine, rolipram, Sp-cAMPS and 8-BrcAMP in KCI-induced tonic contraction was also assayed. Histamine, forskolin, papaverine, rolipram, Sp-cAMPS, 8-BrcAMP and 8-BrcGMP, but not vinpocetine, relaxed the vanadate-induced contractions in rat uterus incubated in medium lacking calcium plus EDTA in a concentrationdependent way. Rp-CAMPS (I-300 uM) had no effect on vanadate contraction. However, it antagonized the relaxation elicited by histamine and papaverine, but not that of forskolin, rolipram, Sp-CAMPS, 8-BrcAMP and 8-BrcGMP. Forskolin, papaverine, vinpocetine, rolipram and 8BrcAMP, but not Sp-cAMPS, relaxed the KCI-induced contraction. RpcAMPS antagonized the relaxation elicited by forskolin, papaverine and vinpocetine, but not that of rolipram and 8-BrcAMP. Our results suggest that: a) Rp-cAMPS is an effective PKA inhibitor that could be used to study the involvement of CAMP on drug-induced response in smooth muscle, and b) the effects of Sp-CAMPS, 8-BrcAMP and rolipram were independent of the activation of protein kinases. KS, Work

RgcAMPS,

8-BrcAMP, histamine, papaverinc, AMP-dependent

protein kinase, smooth muscle

contraction

Cyclic 3’, S-adenosine monophosphate (CAMP) is an important intracellular second messenger in many tissues and mediates the effect of multiple drugs and hormones. It is known that CAMP produces relaxation of smooth muscle by activation of CAMP- dependent protein kinase (PKA) which interferes with several processes involved in smooth muscle contraction (1,2). This kinase will phosphorylate proteins like MLCK. cAMP may also cause relaxation by lowering [Cai] by: 1 ) increased extrusion of Ca due to stimulation Corresponding

author: Dr. Agustfn Hidalgo. Farmacologla, Dpto. Medicina. Facultad de Medicina. c/Julian

Claveria 6.33006

Oviedo, Spain. Phone: 3485103551;

Fax: 3485232255.

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of both Ca transport and Ca ATPase by the plasma membrane; 2) stimulating the Na-K pump, thereby lowering [NaJ, which in turn will enhance Ca efflux on Na-Ca exchange; 3) causing internal sequestration of Ca; and 4) inhibiting Ca influx, although this has not been demonstrated directly in the uterus (2). Rp diastereoisomer of adenosine cyclic 3’,5’-monophosphothioate (Rp-CAMPS) is a soluble membrane-permeable analogue of CAMP with enhanced metabolic stability and is not a substrate for CAMP phosphodiesterase (3). Contrary to the stereoisomer SpCAMPS that activates PKA, Rp-CAMPS behaves as an antagonist of CAMP and does not cause the release of the catalytic subunit of the enzyme, although it binds to the regulatory subunit of PKA (4,5). However, Rp-CAMPS also blocks the activation of PKA-dependent glycogenolysis induced by Sp-CAMPS and this suggests an inhibitory effect on PKA (6) Antagonism by Rp-CAMPS of CAMP-dependent enzyme activity or metabolic effects has been described In several tissues such as hepatocytes or parathyroid cells (5,7). The effects of Rp-CAMPS, however, on smooth muscle have not been documented The increase of Intracellular CAMP can be achieved through receptor activation or Inhibition of CAMP breakdown (8,9) Since the increase in CAMP levels in the cells feads to activation of PKA, the am of this work was to study the effect of Rp-cAMPS on CAMPdependent relaxation eLicIted by histamine, forskolin, papaverlne, vinpacetine, ralipfam and by CAMP analogs 8-bromoadenosine 3’:5’-cyclic monophosphate (~-&CAMP) and Spadenosine 3’,5’-cyclic monophosphothioate (Sp-CAMPS) (10) in isolated rat uterus In order to da this, the experiments were performed on vanadate-induced contractton in rat uterus incubated in medium lacking calcium plus EDTA. This contraction is relaxed by drugs that increase CAMP (? 1,12) and is thus suitable for studying the effect of Rp-CAMPS an smooth muscle relaxation elicited through CAMP-dependent mechanisms. For comparison, the effect of several drugs and their modification with Rp-CAMPS on KCI-induced tonic contraction in rat uterus have also been assayed. Materials and methods Animals used and method of preparation. Female Wistar rats, weighing 220-280 g and estrogenized with 1 mg/kg s. c. of estradiol benzoate 48 h before the experiment, were used. The animals were killed by decapitation and both uterine horns were extracted and cleaned of adherences. The preparations were vertically placed in 6 ml isolated organ baths, incubated in Locke Ringer and Locke Ringer calcium-free solutions at 32” C and bubbled with carbogen mixture (95% 0, and 5% CO,). The contractions were recorded through force-displacement transducers TRI 1 IO on a Letica 4006 polygraph. The composition (mM) of Locke-Ringer solution was: NaCl 154, KCI 5.63, CaCI, 2 16. MgCI, 2.10, NaHCO, 5.95 and glucose 5.55. In the same way, Locke Ringer was used without calcium in the presence of EDTA Vanadafe-induced contraction: The preparatrons were kept in Locke Ringer for 60 min. a? so/&ion that contained 3 mM EDTA. After 50 min, the solution was changed for another of equal composition cantaining 1 mM EDTA, in which it remained for the rest of the experiment (11). The load was 0.5 g.

the end of which the solution was substituted for a calcium-free

After 30 mln, vanadate (0.3 mM) was added and produced a tonic contractfan that was left to stabilize far 20-30 min. Once the contraction was stable, cumulative doses of

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histamine (180 pM), forskolin (160 PM), papaverine (l-l 00 FM), vinpocetine (l-300 FM), rolipram (0.1-l mM), 8-BrcAMP (10 uM - 1 mM), 8-BrcGMP (3 uM - 1 mM), Sp-CAMPS (lo-300 FM) or Rp-CAMPS (l-300 uM) were added, each being left to act the time necessary to produce the maximum effect. In other experiments, the effect of these drugs was assayed in the presence of Rp-CAMPS (10 and 100 PM). Thus, the Rp-CAMPS were added to the bath 30 min before vanadate. At this time, Rp-cAMPS produced its maximum effect (5). KC/-induced contraction: The incubation medium was Locke Ringer. Each uterine horn was subjected to a resting tension of 1 g and was allowed to equilibrate for at least 30-45 min before the drugs were added to the organ baths. Cumulative concentration-response curves of forskolin (0.16 FM), papaverine (3-60 PM), vinpocetine (3-60 FM), rolipram (O.l1 mM), 8-BrcAMP (I-IO mM), Sp-CAMPS (l-300 pM) or Rp-CAMPS (l-300 uM) were made when the tonic response of the KCI (60 mM)-induced contraction was stable (1 O-l 5 min). Each concentration was left until its effect was maximum (approximately 10 min). A 100% relaxation was obtained when the baseline of the recording was reached. In each preparation only one concentration-response curve was assayed. In the same experimental conditions, the modification of the relaxing effect by Rp-CAMPS was studied. The chemicals used were: vanadate (sodium orthovanadate), forskolin, histamine (histamine dihydrochloride), papaverine (papaverine hydrochloride), 8-BrcAMP (8bromoadenosine 3’,5’-cyclic monophosphate sodium salt) and 8-BrcGMP (8bromoguanosine 3’,5’-cyclic monophosphate sodium salt) Sigma (Madrid, Spain); RpCAMPS (Rp-adenosine 3’,5’-cyclic monophosphothioate, triethylamine salt) and Sp-cAMPS (Sp-Adenosine 3’,5’-cyclic monophosphothioate, triethylamine salt) Research Biochemical International (NaticK, USA); vinpocetine and rolipram Tockris Cookson (Bristol, UK); ethylenediaminetetraacetic acid (EDTA), Merck (Madrid, Spain). The drugs were dissolved in the incubation medium or DMSO (dimethylsulfoxide, Sigma). All concentrations are expressed as the final organ bath concentration. Statistical analysis of results: The results are expressed as the mean values + S.E.M. The statistical significance was evaluated with Bonferroni’s test and the values p
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TABLE 1. Drug concentrations that produced a 50% relaxation (I&) of rat uterus contractions induced with vanadate (0.3 mM) in calcium free solution and their modifications with Rp-CAMPS. Percentage of relaxation of the maximal concentration of drugs used in the previous contraction induced by vanadate (Emax %). DRUGS

n

IC,, pM

Emax (%)

Histamine + Rp-CAMPS (10 yM) + Rp-CAMPS (100 pM)

12 4 5

3.4k1.2 8.3k4.7’ 6.5*3.0

94.1k2.1 81.&4.5’ 81.7k3.6’

Papaverine +RpcAMPS +RpcAMPS

(10 uM) (100 uM)

6 6 6

6.1k1.2 12.6jz5.3” 22.7*11.4”

97.5&l .2 85.3k2.9” 86.5*4.1

8-BrcAMP +RpcAMPS +RpcAMPS

(IO pM) (100 uM)

9 9 8

353*170 5162291 886*579

72.3k3.8 55.9zt6.4 55.2k3.0

8-BrcGMP +Rp-CAMPS +Rp-CAMPS

(10 uM) (100 pM)

6 6 7

323*167 362+225 254*143

58.6*1.9 55.4k2.16 58.9k3.5

Sp-CAMPS +Rp-CAMPS +Rp-CAMPS

(10 pM) (100 pM)

7 6 4

302*174 111*49 175*124

57.3k4.3 77.2k5.8’ 62.8k4.7

Forskolin +RpcAMPS +RpcAMPS

(10 uM) (100 pM)

11 6 6

8.9k3.5 9.7*3.4 8&3.5

81.4zt2.9 75.3ti.3 80.5k2.1

Each IC, value is the mean f 95% confidence limits for n experiments ~~0.05 vs histamine, *’ p
l

,RRcAMPS

10ym

Fig. 1 Redrawing of typical tracing showing the relaxant effect of histamine and its modification with RpcAMPS on tonic contraction of vanadate (OV 0.3 mM) in isolated rat uterus incubated in calcium free solution plus EDTA. Under the same experimental conditions the effect of vinpocetine and Sp-CAMPS are shown.

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873

60

-6

-5 Dw,log

-I

-3

[Ml

Fig.2 Concentration response curve with histamine (0) forskolin (m), papaverine (0), rolipram (A), B-BrcAMP (A), 8-BrcGMP ( 0 ), Sp-CAMPS (V) on tonic contraction of rat uterus incubated in calcium free solution and induced by vanadate (0.3 mM). The results are expressed as the percentage of relaxation of the previous contraction induced by vanadate. Each point is the mean f S.E.M. for at least 6 different experiments

Forskolin (0.1-8 PM), papaverine (3-80 mM) and 8-BrcAMP (l-10 mM) produced induced tonic contraction (Fig. 3 and 4). negligible. Its effect was not concentration produced at 10 min was similar to that at 2

PM), vinpocetine (3-80 FM), rolipram (0.1-l concentration-dependent relaxation on KCI The effect of Sp-CAMPS (l-300 pM) was or time-dependent because the relaxation h with 10 or 100 uM (Fig.3)

TABLE 2. Drug concentrations that produced a 50% relaxation (I&,) of rat uterus contractions induced with KCI (60 mM) and their modifications with RpcAMPS. Percentage of relaxation of the maximal concentration of drugs used in the previous contraction induced bv KCI (Emax %) DRUGS

n

tC, IJM

Emax (%) 97.1*1.2 99.1*0.8 95.7Q.7

Forskolin + RpcAMPS + RpcAMPS

(10 uM) (30 pM)

13 8 9

0.55*0.12 1.49&0.64’ 3.75i1.53’

Papaverine + RpcAMPS + RpcAMPS

(10 pM) (30 uM)

11 7 8

12Q.2 16il .l*” 15*3.1

99.6f0.4 100 98.6&l .3

Vinpocetine + RpcAMPS + RpcAMPS

(10 pM) (30 pM)

11 7 6

8.9fl.4 12.7k3.3’ 11.1&2.5

93.5f1.6 94.7*3 92.2H.9

Each IC, value is the mean * 95% confidence limits for n experiments * peO.05 vs forskolin, ** pcO.05 vs papaverine, # peO.05 vs vinpocetine alone.

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874

r-h;:i:L;> KCI 60 mM( FordOlin

KClzO mM 1 Forrkolin [)rM]

L RpsAMPS

_I

BMI

30 pM

Sp-cAMPS WMI I

KC:60 mhl

Redrawing

Fig. 3 of typical tracing showing the relaxant

effect of forskolin and its modification

with Rp-CAMPS

on tonic contraction of KCI in isolated rat uterus. The effect of SpCAMPS and the relaxing effect of vinpocetine and rolipram are shown under the same experimental conditions.

Rp-CAMPS (I-300 FM) has no relaxant effect on contraction induced by vanadate. RpCAMPS (10 and 100 uM), added 30 min prior to addition of vanadate, shifted the concentration-response curves of relaxation elicited by histamine and papaverine to the right (Fig. 5) significantly increasing their respective IC,, values (Table 1). However. RpCAMPS did not modify the relaxation elicited by forskolin, rolipram, Sp-CAMPS and 8BrcAMP (Table 1). Rp-CAMPS (10 and 30 uM) antagonized the relaxation elicited by forskolin in a concentration dependent way. It antagonized the vinpocetine and papaverine relaxing effect with IO uM but not with 30 uM (Fig. 6, Table 2). Neither of the two doses of RpcAMPS assayed modified the relaxation produced with rolipram, 8-BrcAMP or Sp-CAMPS.

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875

80

Fig. 4 Concentration response curve with forskolin (V) papaverine (0) rolipram (A), vinpocetine m, 8BrcAMP (+), on tonic contraction of rat uterus induced by KCI (60 mM). The results are expressed as the percentage of relaxation of the previous contraction induced by KCI. Each point is the mean f S.E.M. for at least 6 different experiments.

20

0

I -7

I

I

I

I

-6

5

-4

-3

Dnrg,

log

1 -2

[Ml

The interaction between phosphodiesterase inhibitors and a PKA activator 8-BrcAMP has been assayed in other experiments. As shown in figure 7 the effect of 8-BrcAMP (1 mM) was increased in the presence of rolipram (0.1 mM) and vinpocetine (6 uM) at concentrations that produced a minimum relaxing effect.

100 -

-6

4

-5

-6

Histamine, log [M]

-5

-4

Papaverine, log [M] Fig. 5

Modification with Rp-cAfvlPS r# response curve by histamine ( l byvanadate in rat uterus incubated expressed as the percentage of vanadate. Each point is the mean

10 and A 100 pM) of the relaxing concentration ) and papavedne ( 0 ) on tonic contraction induced in calcium free solution plus EDTA. The results are relaxation of the previous contraction induced by f S.E.M. for at least 6 different experiments.

AAMP Derivatives and Smooth Muscle

-7

4

-5

-5

Papaverine,log [M]

Vinpocetins, log [M]

-5

Forskolin,log [Ml

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Fig. 6 Modification with Rp-CAMPS (U 10 and A 30 pM) of the relaxing concentration response curve with forskolin (e ), papaverine ( 0 ), vinpocetine ( 0 ) on tonic contraction of rat uterus induced by KCI (60 mM). The results are expressed as the percentage of relaxation of the previous contraction induced by KCI. Each point is the mean f S.E.M. for at least 6 different experiments.

R-BrcAMP

8-BrcAMP

[mM]

0 KCI 60 rnM

kl

[mb!]

60 mM

1 Rp-CAMPS 10 PM

CAM:

0

:kn j-l_.

Rollpram 0.1 mM

KCI ~IJ mm

IVinpocetlne

, mm 6 PM

KCI 60 mbl Fig. 7 Redrawing of typical tracing showing the relaxant effect of 8BrcAMP and its modification with Rp-CAMPS on tonic contraction of KCI (60 mM) in isolated rat uterus. The effect of rolipram and vinpocetine added 10 min before 8-BrcAMP are shown under the same experimental conditions.

Discussion The contraction induced with vanadate on rat uterus in medium lacking calcium EDTA is produced by blocking the (Ca” + Mg”)-ATPase of the plasma membrane releasing the intracellular calcium and, hence, stimulating the calmodulin activity. contraction is relaxed with inhibitors of intracellular calcium release (e.g. TMB8 sodium dantrolene), calmodulin inhibitors (calmidazolium, W-7 and nonsteroidal

plus and This and anti-

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CAMP Derivatives and Smooth Muscle

inflammatory drugs) and drugs that increase intracellular cAMP (e.g. isoprenaline) 13).

877

(11, 12,

The KCI-induced tonic contraction is maintained by calcium influx through voltagedependent calcium channel (14, 15) and is relaxed by drugs that inhibit calcium influx (15, 16) or increase of intracellular CAMP (17, 18, 19). The drugs assayed produce a CAMPdependent inhibitory effect in many tissues and this is well documented. However, the results obtained by using Rp-CAMPS suggest that some of these drugs also produce CAMP-independent effects. Histamine produced concentration-dependent relaxation on vanadate-induced contraction in rat uterus in medium lacking calcium plus EDTA and this effect is antagonized with Rp-cAMPS This suggests that the relaxation produced by histamine is CAMP-dependent as is generally assumed (1, 2, 20, 21). The activator of adenylyl cyclase forskolin produced relaxation of both KCI- and vanadate-induced contraction in rat uterus. Although this drug increased CAMP and produced cAMPdependent relaxation in smooth muscle (22, 23) our results suggest that in the present experimental conditions, forskolin also produced CAMP-independent relaxation. Rp-CAMPS antagonized, in a concentration-dependent manner, the relaxation produced by forskolin on KCI contraction. This suggests a CAMP-dependent effect, as expected. However, the relaxation on vanadate contraction is not antagonized by RpCAMPS, which suggests a CAMP-independent effect. On the other hand, the inhibitor of PKA TPCK (12, 24, 25) and the inhibitors of adenylyl cyclase 2’,3’- and 2’-5’dideoxyadenosine (26) also antagonized the relaxation produced by forskolin on KCI but not on vanadate contraction (unpublished observations). This supports the CAMPindependent effect of forskolin on vanadate contraction. This seems to be possible because: a) the activation of adenylyl cyclase by forskolin requires divalent ions (27) and the vanadate contraction is obtained in the presence of EDTA, b) several cAMPindependent effects of forskolin have been described (28, 29) and c) CAMP-independent relaxation of rat uterus with P-agonist and forskolin has been documented (2, 30, 31, 32). The phosphodiesterase inhibitors assayed show a different behaviour. Papaverine relaxed both KCI- and vanadate-induced contractions and Rp-cAMPS antagonized its effect. This suggests the involvement of CAMP in the relaxation elicited by this nonspecific inhibitor of phosphodiesterases as occurs in other systems (1, 8, 9). Vinpocetine, an inhibitor of phosphodiesterase type I (33, 34) produced concentrationdependent relaxation of KCI-induced contraction and its effect is significantly antagonized with 10 uM but not with higher concentrations of Rp-cAMPS. This suggests a partial contribution of cAMP to its relaxant effect. The calcium antagonist.effect of vinpocetine (35) could contribute to its relaxant effect. On the other hand, vinpocetine did not relax the vanadate contraction (see Fig. 1) which suggests that under these experimental conditions (absence of extracellular calcium) vinpocetine produced CAMP- and calciumindependent effects. The increase of contraction produced with 10 and 30 uM could be related to inhibition of Na’ current as has been described in cortical neurons (36). The inhibitor

of cAMP phosphodiesterase

type IV rolipram (33, 34) inhibits smooth

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muscle contractions of bovine, guinea-pig and mouse traquea, porcine bronchus and human pregnant myometrium (37, 38). In these tissues, a good correlation between phosphodiesterase and contractility inhibition has been observed. However, in our experiments, although rolipram relaxed KCI and vanadate contraction, these effects are CAMP-independent because: a) the concentration ranges necessary to relax both contractions (0.1-I mM) are much higher in comparison to those that inhibit CAMP phosphodiesterase (I&,: 1-2 uM, 33, 39) and b) Rp-CAMPS did not modify the relaxant effect of rolipram. The stereoisomer of Rp-CAMPS, Sp-CAMPS is an activator of PKA (7, 10). This drug produces CAMP-dependent metabolic effects in hepatocytes and parathyroid cells (5, 7) which are antagonized with Rp-CAMPS. Sp-CAMPS also produces relaxation of vascular smooth muscle (40) but higher concentrations are needed for it. In our experiments, SpCAMPS relaxed the vanadate contraction up to 57.3+4.2% and KCI contraction up to only 16.3k2.9 % with 300 uM. The effect on KCI contraction was not concentration or timedependent because the inhibition was similar with all concentrations assayed in times ranging between 10 min and 2 h. The effect of Sp-CAMPS is PKA-independent because Rp-CAMPS did not modify it. The other PKA activator 8-BrcAMP (11) relaxed both vanadate and KCI contraction, However, the maximal relaxation was 72.3k3.8 % on vanadate and 29.9k4.6 % on KCI, and concentrations of 1 and 10 mM, respectively are needed. Their relaxant effects also seem to be PKA-independent because they were not antagonized by Rp-CAMPS, and not mediated by PKG as occurs in vascular smooth muscle (41). On the other hand, the relaxation induced by 8-BrcGMP is much lower than that produced in rat aorta. In this tissue 100 uM of 8-BrcGMP relaxed the phenylephrine-induced contraction by 100% (42). This suggests tissue differences in the effect of 8-BrcGMP. The inhibitor effect of 8BrcGMP in rat uterus could be related to a reduction of intracellular free calcium (43) because KCI and vanadate increase intracellular calcium in smooth muscle (44-46). The results obtained with Sp-CAMPS and 8-BrcAMP suggest that: a) PKA is not directly activated by these drugs in the present experimental conditions, b) they are, probably, poorly permeable CAMP analogs (47) in intact rat uterus preparations, and c) their effects presumably could occur in plasma membrane cells because the effect of Sp-CAMP is not time-dependent and the effect of 8-BrcAMP is further increased with rolipram (whose effect was unmodified with Rp-CAMPS) than with vinpocetine. On the other hand, the effects of CAMP analogs different to those produced by endogenous CAMP have been reported by several authors (48-52). Rp-cAMPS behaved as a PKA inhibitor and significantly antagonized the CAMP-PKArelaxation elicited by histamine and papaverine on vanadate contraction and that produced by forskolin, papaverine and vinpocetine on KCI-induced contraction. In some experiments, the antagonism obtained with 100 uM of Rp-CAMPS was no greater than the one with 10 or 30 FM. Similar observations have been described in other experimental conditions, for example, antagonism of: a) glucagon-induced glycogenolysis in rat hepatocyte (6), b) diclofenac-induced relaxation in rat uterus (12) and c) phenylephrine - induced relaxation in rat aorta (40). A possible explanation is that concentrations of Rp-CAMPS (I& on PKA: 0.9 uM (4)) higher than 10 uM could inhibit CAMP- phosphodiesterase in consequence decrease CAMP degradation (6). Thus, accumulation of CAMP occurs and could partially counteract the Rp-CAMPS inhibition of PKA. For this, and also for economical reasons,

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concentrations

of Rp-cAMPS

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879

higher that 30 uM should not be assayed.

In summary, Rp-cAMPS is an effective PKA inhibitor that could be used to examine the involvement of cAMP in drug-induced response on smooth muscle. Acknowledgement This work was supported by the Spanish Dire&on y Tecnica (Project Ref. PB 92-l 128).

General de lnvestigacion

Cientifica

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