Sao Paulo Med. J. vol.117 número5

Original Article

The e ffe ct of chronic nitric oxide inhibition on

vascular re activity and blood pre ssure in pre gnant rats

Medical Investigation Laboratory (LIM57), Obstetrics and Gynecology Department,

University of São Paulo, São Paulo, Brazil.

INTRODUCTION

Va rio us he mo dyna mic c ha ng e s c ha ra -c te riz e d b y d e -c re a se d , syste mi-c , va s-c ula r resistance and decreased blo o d pressure are a sso c ia te d w ith p re g na nc y.1 -4 The e xa c t physio patho lo gical mechanisms by which these vascular chang es take place are no t kno wn. Many facto rs such as decreased blo o d visco sity, changes in the pro stacyclin/ thro mbo xane ratio and a decrease in nitric oxide activity or synthesis are invo lved.5 , 6

N itric o xid e is no w kno w n to b e the princ ipa l c o mpo nent in EDRF (endo thelium-derivated relaxing facto r), which is released by a c etylc ho line stimulus.7 -9 This nitric o xide is pro duced by the vascular endo thelium and it regulates the vascular vaso relaxatio n to nus and b lo o d p re ssure .1 0 , 1 1 N itric o xid e a c ts b y stimulating so luble guanylate cyclase resulting in an increase in the intracellular co ncentratio n of cyclic guanosine monophosphate in the smooth muscle cells, which leads to vaso relaxatio n.1 2 -1 4 The impo rtance o f nitric o xide in co ntro lling blo o d pressure during pregnancy is unkno wn. Some studies suggest a greater relevance of nitric o xid e in p re g na nt tha n in no n-p re g na nt subjects,1 5 ,1 6 while o thers sho w the co nverse.1 7

ABSTRACT

Contex t: The exact mechanism invo lved in changes in blo o d pressure and peripheral vascular resistance during pregnancy is unkno wn.

O bjective: To evaluate the impo rtance o f endo thelium-derivated relaxing facto r (EDRF) and its main co mpo nent, nitric o xide, in blo o d pressure and vascular reactivity in pregnant rats.

Design: Clinical trial in experimentatio n animals.

Setting: University labo rato ry o f Pharmacology.

Sa m ple: Female W istar rats with no rmal blo o d pressure, weight (1 5 2 to 2 2 7 grams) and age (9 0 to 1 1 6 days).

Intervention: The rats were divided in to four groups: pregnant rats treated with L-NAME (13 rats); pregnant control rats (8 rats); virgin rats treated with L-NAME (10 rats); virgin control rats (12 rats). The vascular preparations and caudal blood pressure were obtained at the end of pregnancy, or after the administration of L-NAME in virgin rats.

M a in M ea surements: The caudal blo o d pressure and the vascular response to acetylcholine in pre-contracted aortic rings, both with and without endothelium, and the effect of nitric oxide inhibition, Nw-L-nitro-arginine methyl-ester (L-NAME), in pregnant and virgin rats. The L-NAME was administered in the drinking water over a 1 0 -day period.

Results: The blood pressure decreased in pregnancy. Aortic rings of pregnant rats were more sensitive to acetylcholine than those of virgin rats. After L-NAME treatment, the blood pressure increased and relaxation was blocked in both groups. The fetal-placental unit weight of the L-NAME group was lower than that of the control group.

Conclusion: Acetylcho line-induced vaso relaxatio n sensitivity was greater in pregnant rats and that blood pressure increased after L-NAME administration while the acetylcho line-induced vaso relaxatio n respo nse was blo cked.

The present study thus examines blo o d pressure during pregnancy in the presence and absence o f nitric o xide inhib ito rs, a nd the va sc ula r reactivity and fetal o utco me co nsequent o n this inhibitio n.

METHODS

Fema le W ista r ra ts with no rma l b lo o d pressure, weighing between 1 5 2 and 2 2 7 grams and aged 9 0 to 1 1 6 days were used. The rats were transferred to the labo rato ry o ne week befo re the experiments were started and during this time the y w e re ma inta ine d in a q uie t enviro nment under a cycle o f 1 2 ho urs dark to 1 2 ho urs light.

The animals were mated, placing a single male rat in each cage with three or four female rats. Vaginal smears were obtained daily; the day on which spermatozoids were found was considered to be the first day of pregnancy. Pregnancy in the rats under our conditions lasts 3 weeks.

The rats were divided in to fo ur gro ups: 1 . Pregnant rats treated with L-N AME (1 3 rats); 2 . Pregnant co ntro l rats (8 rats);

3 . Virgin rats treated with L-N AME (1 0 rats); 4 . Virgin co ntro l rats (1 2 rats).

The caudal blo o d pressure was measured in co nscio us rats previo usly maintained at 3 5 to 4 0 º C fo r 1 0 min. The rats were co vered with c o tto n tissue d uring b lo o d p re ssure measurements.

The c a uda l b lo o d pre ssure a nd b o dy weight o f the pregnant rats were o btained at the end o f the acclimatizatio n perio d, and after the first and third weeks. In the virgin rats, the seco nd me a sure me nt w a s o b ta ine d a fte r L-N AME administratio n and after a similar perio d in the virgin co ntro l rats.

Nw-L-nitro -arginine methyl ester (L-N AME) inhib its nitric o xid e synthe sis a nd w a s administered diluted in the drinking water. During this treatment, the rats were caged individually. The w a te r w a s c ha ng e d d a ily a llo w ing d e te rmina tio n o f the vo lume d runk a nd calculatio n o f the amo unt o f the drug ingested. The co ncentratio n o f L-N AME in the water was between 3 5 to 6 0 mg %, resulting in a daily co nsumptio n o f L-N AME o f appro ximately 5 0 to 7 0 mg/ kg/ day.

L-N AME tre a tme nt la ste d 1 0 d a ys in pregnant and virgin rats. This treatment began o n day 1 0 o f pregnancy.

The rats were sacrificed at the end o f the

Ta ble 1 - Ca uda l blood pressure, w eight a nd a ge during a cclima tiza tion period in the four groups of ra ts

Pregnant L-NAME Pregnant control Virgin L-NAME Virgin control

Measurements n = 1 3 n = 8 n = 1 0 n = 1 2

Blood Pressure, mmHg 1 1 5 .8 6 (1 1 .1 3 ) 1 1 3 .2 8 (1 1 .8 7 ) 1 0 8 .7 5 (3 .9 0 ) 1 1 0 .4 1 (1 3 .4 6 )

Weight, grams 1 9 6 .2 3 (1 9 .5 7 ) 2 0 1 .5 0 (1 6 .1 7 ) 1 9 8 .8 0 (1 4 .6 5 ) 2 0 6 .9 1 (1 4 .3 0 )

Age, days 1 0 1 .3 8 (8 .2 6 ) 1 0 2 .1 2 (6 .5 2 ) 1 0 1 .0 0 (5 .0 8 ) 1 0 1 .1 6 (3 .8 6 )

* p < 0 .0 5 ; standard deviation given in parenthesis.

Ta ble 2 - Ca uda l blood pressure

Pregnant L-NAME Pregnant control Virgin L-NAME Virgin control

Period n = 13 n = 8 n = 10 n = 12

Acclimatization period 115.86 (11.13) 113.28 (11.87) 108.75 (3.90) 110.41 (13.46)

Week 1 (pregnant) 105.76 (12.61) 107.81 (15.22) -

-Week 3 (pregnant) or second measurement (virgin) 137.98 (13.07)* ߧ 98.43 (11.5)* ß 154.37 (14.80)* ß 109.89 (6.96)

* p < 0.05 compared with respective controls; ß p < 0.05 compared with the acclimatization period; § p < 0.05 compared with virgin rats;

treatment o r equivalent co ntro l perio d to perfo rm the vascular reactivity study. The pregnant rats (g ro up s 1 a nd 2 ) w e re sa c rific e d a fte r appro ximately 2 0 days o f pregnancy. The virgin rats were sacrificed after treatment with nitric o xide inhibito r in the drinking water.

The ra ts we re a ne sthe tiz e d with 1 0 % chlo ral hydrate administered subcutaneo usly. Laparo to my and tho raco to my were perfo rmed to o btain the tho racic ao rta, which was rapidly immersed in Krebs-Henseleit so lutio n. The ao rta was dissected and all co njunctive and adipo se tissues was remo ved. Two ring s, 0 .4 cm in diameter, were o btained fro m the ao rta and the endothelium was mechanically removed from one o f the pair. Each ring was suspended by a fine steel wire; and co nnected to an iso metric tensio n tra nsd uc e r (M yo g ra p h F6 0 ); w hic h w a s c o nnec ted to a N a rc o Tra c e 4 0 po lyg ra ph (N arco -Bio -Systems Inc., Texas, USA). The rings we re ma inta ine d in 1 5 ml Kre b s-He nse le it so lutio n at 3 7 °C ( range 3 6 .5 to 3 7 .5 ). A tensio n o f 0 . 5 g ra ms w a s ma inta ine d d uring a n equilibratio n perio d o f 6 0 min during which the bathing medium was changed every 2 0 minutes. The ao rtic rings were co ntracted by adding noradrenalin 10-7 M, a concentration that induces 6 0 -7 0 % o f the maximum co ntractio n, resulting in a tension from which the magnitude of subsequent vasorelaxation is highest. Progressively increased concentrations of acetylcholine were added to the preparatio ns. The percentage relaxatio n o f the co ntractio n was measured.

Vascular reactivity was studied using do se-response curves and the percentage of relaxation induced by each do se o f acetylcho line, and by measurement o f the maximum respo nse and sensitivity to acetylcho line.

The maximum respo nse to acetylcho line wa s o b ta ined fro m the mea n a nd sta nda rd d e via tio n o f ma ximum re sp o nse fo r e a c h replicated experiment.

Ac etylc ho line sensitivity wa s estima ted using EC5 0 , i.e., the acetylcho line do se that pro duced 5 0 % o f the maximum co ntractio n in ea c h experiment. The mea ns a nd sta nda rd deviatio ns were calculated.

The drug s use d we re : no re pine phrine bitartrate, acetylcho line chlo ride and Nw

--Lnitro

-arginine methyl ester (L-NAME) (Sigma Chemical Co mpany); po tassium chlo rate, calcium chlo ride and chlo ral hydrate fro m Merck; mo no basic po tassium pho sphate (Carlo Erba); magnesium sulfate, glucose and sodium bicarbonate (Ecibra); and EDTA (Fischer Science Co mpany).

Se rum c re a tinine a nd uric a c id w e re measured using the Jaffé metho d, mo dified and a da pte d fo r a uto ma tio n (RA-1 0 0 0 Syste m, Technico n Instruments Co rpo ratio n, Tarryto wn, NY, USA).1 8 Uric acid was also measured using a chlo rimetric, enzymatic technique adapted fo r auto matio n.1 8 The left kidney was remo ved and fixed in fo rmalin fo r histo patho lo gical study.

The weight and number o f o ffspring and the p la c e nta l-fe tus unit w e ig ht w e re a lso o btained.

Sta tistic a l M e tho d s. The d a ta w e re analyzed statistically using variance analysis (one o r two way ANO VA, MANO VA), Student’s T test and the ManW hitney and Kruskal-W allis no n-parametric tests (SPSS/ PC 3 .0 + Microsoft Corp, 1 9 8 8 ; and STATISTICA fo r W indo wsTM, release 4 .3 , Statso ft Inc., 1 9 9 3 ).

RESULTS

Acclimatizatio n perio d. Blo o d pressure, weight and age were similar amo ng all gro ups during the acclimatizatio n perio d (Table 1 ).

Blo o d pressure during preg na nc y. The blo o d pressure in the pregnant rats decreased during pregnancy. The caudal blo o d pressures were 1 1 4 .4 4 mmHg (SD 1 1 .8 2 ), 1 0 6 .8 9 mmHg (SD 1 4 .4 0 ) and 9 8 .4 3 mmHg (SD 1 1 .9 6 ) in the first, seco nd and third weeks o f preg nancy, respectively (Table 2 ).

L-NAME effects on blood pressure. Drinking water containing L-NAME was administered over a 10-day period to pregnant and virgin rats. The quantities of L-NAME ingested were similar between groups. The pregnant group consumed 60.62 mg L-NAME/ kg/ day (SD 12.81) and the virgin group 61.05 mg/ kg/ day (SD 10.32) (Table 2).

the third week were sig nificantly altered as fo llo ws (Table 2 ):

a) the L-N AME pregnant gro up (1 3 7 .9 8 mmHg, SD 1 3 .0 7 ) was g reater than the preg nant co ntro l gro up (9 8 .4 3 mmHg, SD 1 1 .5 8 ); b) the L-N AME virgin gro up (1 5 4 .3 7 mmHg, SD

1 4 .8 0 ) was greater than the virgin co ntro l gro up (1 0 9 .8 9 mmHg, SD 6 .9 6 );

c) the L-N AME virgin gro up was greater than the L-N AME pregnant gro up;

d) the caudal blo o d pressures o f the L-N AME-treated gro ups were greater than tho se o f the co ntro l gro ups;

e) the caudal blo o d pressures o f the pregnant gro ups were lo wer than tho se o f the virgin gro ups, independent o f treatment.

The effects of L-NAME treatment on the blood pressures o f preg na nt a nd virg in ra ts were compared, using the differences in blood pressure between the acclimatizatio n perio d and final perio d. In the pregnant gro up, blo o d pressure decreased by 9 .3 8 mmHg and, in the L-NAME-treated group, blood pressure increased by 32.22 mmHg, an abso lute difference o f 4 1 .6 0 mmHg. In the virgin gro up blo o d pressure decreased by 0 .5 2 mmHg and, in the L-NAME-treated gro up, it increased by 45.62 mmHg, an absolute difference o f 4 6 .1 4 mmHg . These differences were no t statistically significant (Table 2 ).

A na lysis o f the ma ximum re la xa tio n o btained with cumulative do ses o f acetylcho line revealed the fo llo wing (Fig. 1 and Table 3 ): a) Relaxatio n in ao rtic rings with endo thelium

fro m p re g na nt L-N AME-tre a te d ra ts w a s blo cked;

b) Relaxatio n in ao rtic rings with endo thelium fro m virgin L-NAME-treated rats was blo cked; c) Relaxatio n in ao rtic rings witho ut endo thelium

fro m pregnant rats was blo cked;

d) Relaxatio n in ao rtic rings witho ut endo thelium fro m virgin rats was blo cked;

e) Relaxatio n in ao rtic rings with endo thelium fro m virg in L-N A M E-tre a te d ra ts w a s co mpletely blo cked; so me relaxatio n o ccurred in the pregnant rats.

The sensitivity was evaluated using EC5 0 . O ur data revealed that the preg nant co ntro l

groups were more sensitive than the virgin control gro ups. In the pregnant gro up, the EC5 0 was 1 .4 x 1 0-8M, SD 1 .1 2 and in the virgin gro up it was 8 .1 2 x1 0-8 M, SD 1 0 .7 3 (p < 0 .0 5 ).

Renal histo patho lo gy. Renal histo patho lo gy was similar amo ng the fo ur gro ups.

Plasma uric acid and creatinine. Plasma creatinine and uric acid co ncentratio ns were similar amo ng the fo ur gro ups.

Fetal outcome. Analysis of the fetal outcome revealed the fo llo wing:

a) fetal weights in the L-NAME-treated, pregnant gro up (3 3 .8 1 g, SD 1 4 .7 6 ) were lo wer than in the co ntro l gro up (6 1 .0 3 g, SD 3 3 .7 6 ), altho ugh this was no t statistically significant (p = 0 .0 6 3 8 );

b) the number o f fetuses in the L-N AME-treated, pregnant gro up (9 .4 6 fetuses, SD 3 .4 5 ) was the same as in the co ntro l gro up (9 .8 7 fetuses, SD 3 .2 1 ) (p = 0 .7 5 0 1 );

c) the fe ta l-p la c e nta l unit w e ig hts o f the L-N AME-treated, preg nant g ro up (3 .8 9 g , SD 1 .6 5 ) were lo wer than tho se o f the preg nant c o ntro l g ro up (5 . 8 7 g , SD 1 . 9 2 ) (p = 0 .0 3 0 2 ) (Table 5 ).

DISCUSSION

Decreased blo o d pressure is characteristic of human and animal pregnancies. We confirmed this in W istar rats when they became pregnant; blo o d pressure was also lo wer at the end o f pregnancy. This fact has also been demo nstrated by Molnar & Hertelendy,19 Chu & Beilin,20 Ahokas et a l,1 6 Allen et a l.,1 7 Ao i et a l,2 1 Ba ylis & Davinso n22 and Baylis & Engels.23 O ther animal mo dels fo r hypertensio n, like the SHR, DO CA-salt and G o ldblatt rats, also sho w decreased blo o d pressure during pregnancy.2 1 ,2 4 -2 8

Decreased blo o d pressure is pro bably due to a decrease in peripheral vascular resistance a nd o f va sc ula r re a c tivity to va so a c tive substances.

to vaso active substances.2 9 -3 2 Rats thus appear to b e g o o d a nima l mo dels fo r the study o f hemo dynamic alteratio ns during pregnancy.

Blo o d pressure co ntro l is determined by ma ny fa c to rs. Endo thelium-derived rela xing facto r (EDRF), no w kno wn as nitric o xide, is o ne facto r that can mo dulate blo o d pressure. Thus, analysis o f the effect o f nitric o xide inhibito rs in rats may reveal the impo rtance o f nitric o xide in blo o d pressure co ntro l. In bo th preg nant and no n-preg nant rats, administratio n o f L-N AME in the drinking water increased blo o d pressure, revea ling the impo rta nc e o f nitric o xide in co ntro lling blo o d pressure, independently o f whether the animal is preg nant o r no t.

The mean blo o d pressure was higher in the virgin L-NAME-treated group than in the pregnant L-N A M E tre a te d -g ro up . This sug g e sts tha t although inhibition of an important vasodilatation fa c to r d id o c c ur, o the r p re ssure re d uc ing mechanisms avoided serious hypertension during pregnancy, po ssibly using the same mechanism that decreased blo o d pressure in the pregnant c o ntro l ra ts. If nitric o xide we re the o nly mechanism co ntro lling blo o d pressure, then the mean blo o d pressures o f pregnant and virgin rats wo uld be similar.

The co rrelatio ns between the quantity o f L-N AME ingested and blo o d pressure were no t always linear. W hen small quantities o f the drug

Ta ble 5 - Feta l outcome

Fetal outcome Pregnant L-NAME-treated Pregnant control

n = 1 3 n = 8

Total weight, grams 3 3 .8 1 (1 4 .7 6 ) 6 1 .0 3 (3 3 .7 6 )

Number of fetuses 9 .4 6 (3 .4 5 ) 9 .8 7 (3 .2 1 )

Weight of fetal-placental unit, grams 3 .8 9 (1 .6 5 )* 5 .8 7 (1 .9 2 )

* p < 0 .0 5 ; standard deviation given in parenthesis.

Ta ble 3 - M ea n percenta ge rela x a tion induced by

increa sing doses of a cetylcholine in a ortic rings w ith endothelium

Acetylcholine

Groups 10-8M 3x 10-8M 10-7M 3x 10-7M 10-6 M 3x 10-6 M

Pregnant L-NAME 1.37 (3.98)* ß 3.25 (4.79)* ß 5.51 (6.73)* ߧ 8.46 (10.08)* ß 11.58 (13.33)* ß 13.83 (15.63)* ß

Pregnant control 35.68 (21.12)§ 58.45 (25.87)§ 66.42 (22.47)§ 73.05 (20.82)§ 76.05 (18.52)§ 76.05 (18.52)§

Virgin L-NAME -0.69 (1.36) -4.49 (7.65)* -3.01 (6.44)* -2.93 (7.21)* -1.75 (8.73)* -1.51 (10.01)*

Virgin control 20.25 (16.35) 39.50 (24.29)§ 61.16 (26.43)§ 77.45 (19.10)§ 87.96 (11.43)§ 90.29 (9.05)§

*p < 0.05 compared with concerning controls; ß p < 0.05 compared with virgin rats; § p < 0.05 compared with aorta without endothelium; the values are

the mean and standard deviations.

Ta ble 4 – M ea n percenta ge rela x a tion induced by increa sing doses of a cetylcholine in a ortic rings w ithout endothelium

Acetylcholine

G roups 1 0-8 M 3 x 1 0-8M 10-7M 3 x 1 0-7M 10-6 M 3 x 1 0-6 M

Pregnant L-NAME 0 .0 7 (2 .9 7 ) -0 .4 2 (4 .3 1 ) -0 .6 0 (5 .3 1 ) -0 .1 0 (7 .5 4 ) 0 .4 8 (9 .6 2 ) 0 .2 8 (1 0 .8 4 )

Pregnant control -4 .0 9 (2 .8 2 ) -5 .5 4 (3 .2 0 ) -6 .1 1 (3 .6 3 ) -6 .9 0 (4 .9 4 ) -6 .7 6 (5 .9 2 ) -7 .0 6 (6 .8 2 )

Virgin L-NAME -0 .8 8 (1 .2 7 ) -2 .5 4 (2 .5 3 ) -4 .3 4 (3 .4 2 ) -5 .9 9 (4 .6 8 ) -6 .4 5 (6 .9 1 ) -6 .6 2 (7 .1 1 )

Virgin control -1 .7 7 (3 .6 5 ) -1 .9 1 (6 .9 3 ) -2 .7 5 (9 .3 3 ) -2 .8 7 (1 3 .0 0 ) -4 .3 1 (1 4 .8 6 ) -2 .9 0 (1 6 .0 6 )

* p < 0 .0 5 compared with the respective control; ßp < 0 .0 5 compared with virgin rats; the values are the mean and standard

were ingested, a linear correlation was observed. Ho wever, with large amo unts, blo o d pressure reached a limit and stabilized (data no t sho wn). This limit was lo wer in the pregnant rats than in the virg in g ro up (data no t sho wn). This fact re info rc e s the id e a tha t o the r hyp o te nsive me c ha nisms a re p re se nt w hic h a vo id hypertensio n in the pregnant rats.

The blo o d pressure increased with the administratio n o f a nitric o xide inhibito r and was higher in the virgin gro up than in the pregnant gro up. Ho wever, the mean blo o d pressures in the two gro ups were no t statistically significant. These data suggest that nitric o xide do es no t play a mo re impo rtant ro le during pregnancy than during no n-pregnancy.

W e believe that during preg nancy many hypo tensive mechanisms are activated and that when o ne o f these is deactivated, there is a co mpensato ry increase in the o ther mechanisms, w hic h ma inta ins a d e q ua te , no rma l, b lo o d

pressure during g estatio n. Many hypertensive mechanisms are present in humans, pro viding pro tec tio n a g a inst hemo rrha g ic sho c k, a nd va rio us mec ha nisms ma y furnish pro tec tio n against hypertensio n during no rmal pregnancy. Thus, inhibitio n o f the mechanism may no t result in g reat variatio ns in blo o d pressure.

Allen at al17 demo nstrated greater increase in blo o d pressure in virgin rats than in pregnant rats when both were treated with NG -monomethyl-L-arginine (LN MMA). O ther investigato rs have fo und co ntrary data.1 5 ,1 6 ,1 9

W e o bserved that the sensitivity o f ao rtic rings fro m pregnant co ntro l rats to acetylcho line was g reater than that o f the virg in rats. This pro vides co nfirmatio n o f the majo r effect o f EDRF in ao rtic rings fro m pregnant rats, which may be due to an increase in nitric oxide release or effect, due to the release o f ano ther EDRF, due to increased sensitivity and quantity o f muscarine recepto rs fo r acetylcho line, o r due to increased

Figure 1 - Percentage relaxation as a function of increasing doses of acetylcholine in aorta rings with

sensitivity of smooth muscle to cyclic AMP. W einer at al3 3 o btained similar results.

St-Lo uis & Scio tte2 7 o btained disco rdant data, altho ugh these autho rs did no t o bserve greater sensitivity o r an increase in the maximum response in pregnant rats, compared to virgin rats. In the gro ups o f rats treated with L-N AME, the relaxatio n effect was blo cked. This suggests that nitric o xide is the principal facto r respo nsible fo r the relaxatio n o f ao rtic rings by acetylcho line and subsequent co ntro l o f blo o d pressure.

In the virgin rats, the L-N AME treatment co mpletely blo cked relaxatio n. In the pregnant rats, residual relaxatio n was still present. This data suggests reduced efficiency o f L-N AME in inhibiting the release o f EDRF during pregnancy, which may result fro m the lo wer abso rptio n o r higher dilutio n o f L-N AME, o r fro m the presence o f a no the r EDRF, like EDHF (e nd o the lium-derivated hyperpo larizing facto r).3 4

A comparison between pregnant and virgin rats is difficult, as the precise mechanism fo r L-N AME actio n, and its pharmaco kinetics and pha rma c o dyna mic s during pre g na nc y, a re unkno wn. Thus, c a utio n is nec essa ry when co mparing virgin and pregnant rats.

Treatment with L-N AME had a detrimental effect o n fetal o utco me. O ur data sho wed a d e c re a se in fe ta l-p la c e nta l unit w e ig ht. Yallampalli & G arfield3 5 and Baylis & Engels2 3 have also sho wn a po o r fetal o utco me. This may result fro m the direct actio n o f the drug o r fro m increased blo o d pressure.

The true functio n o f nitric o xide during pregnancy is controversial. O ur data suggest that EDRF is increased during pregnancy, pro bably by ano ther EDRF, the actio n o f which co uld no t be blo cked by L-N AME. This wo uld explain the residual relaxatio n seen in pregnant rats, the similar differences in relaxatio n after L-N AME in the two g ro ups, the lo wer increase in blo o d pressure in the pregnant rats, and the similar differenc es in b lo o d pressure a fter L-N AME tre a tme nt in the two g ro ups. Ho we ve r, the p o ssib ility o f d iffe re ntia l a b so rp tio n a nd hemo dilutio n o f the drug canno t be co mpletely excluded.

CONCLUSION

A c e tylc ho line -ind uc e d va so re la xa tio n sensitivity is greater in pregnant rats than in virgin rats and that, after L-NAME administratio n, blo o d p re ssure inc re a se s, a c e tylc ho line -ind uc e d vaso relaxatio n respo nse is blo cked and fetal o utco me is detrimentally affected.

REFERENCES

1. Wilso n M, Mo rganti AO, Zervo udakis I, et al. Blo o d pressure, renin-aldo stero ne system and sex stero ids thro ugho ut no rmal pregnancy. Am J Med 1980;68:97-104.

2. Macgillivray I, Ro se GA, Ro we B. Blo o d pressure surveys in pregnancy. Clin Sci 1969;37:395-8.

3. Mo utquin JM, Rainville C, Giro ux L, et al. A pro spective study o f blo o d pressure in pregnancy: prediction of pre-eclampsia. Am J Obstet Gynecol 1985;151:91-6.

4. Kahhale S, Zugaib M. Fisio pato lo gia da pré-eclâmpsia. In: Síndro mes Hipertensivas na Gravidez. 1st ed. São Paulo : Atheneu; 1995:15-30. 5. Walsh SW. Pre-eclampsia: an imbalance in placental pro stacyclin and

thro mbo xane pro ductio n. Am J Obstet Gyneco l 1985;152:335-40. 6. Friedman AF, Taylo r NY, Ro berts JM. The physio patho lo gy o f

pre-eclampsia. Clin Perinat 1991;18:661-70.

7. Furchgo tt RF, Zawadzki JV. The o bligato ry ro le o f endo thelial cells in the relaxatio n o f arterial smo o th muscle by acetylcho line. Nature 1980;288:373-6.

8. Furchgo tt RF. The ro le o f the endo thelium respo nse o f vascular smo o th muscle to drugs. Ann Rev Pharmaco l To xico l 1984;24:175-97. 9. Furchgo tt VN, Vanho utte PM. Endo thelium-derivated relaxing and

co ntracting facto rs. FASEB J 1989;3:2007-18.

10. Mo ncada S, Higgs A. The L-arginine-nitric o xide pathway. N Engl J Med 1993;329:2002-12.

11. Mo ncada S, Palme r RMJ, Higgs EA. Nitric o xide : physio lo gy, patho physio lo gy and pharmaco lo gy. Pharmaco l Rev 1991;43:109-42. 12. Waldman SA, Murad F. Bio chemical mechanism underlying vascular

smo o th muscle relaxatio n: the guanylate cyclase-cyclic GMP system. J Cardio vasc Pharmaco l 1988;12(5):S115-8.

13. Palmer RMJ, Mo ncada S. A no vel citrulline-fo rming enzyme implicated in the fo rmatio n o f nitric o xide by vascular endo thelial cells. Bio chem Bio phys Res Co mmun 1989;158:348-52.

14. Palmer RMJ, Rees DD, Ashto n DS, Mo ncada S. L-arginine is the physiological precursor for the formation of nitric oxide in endothelial-dependent relaxation. Biochem Biophys Res Commun 1988;153:1251-6. 15. Nathan L, Cuevas J, Chaudhuri G. The ro le o f nitric o xide in the altered vasc ular re ac tivity o f p re gnanc y in the rat. Br J Pharm ac o l 1995;114:955-60.

16. Ahokas RA, Sibai BM. Endothelium-derivated relaxing factors inhibition augments vascular angio tensin-II reactivity in the pregnant rat hind limb. Am J Obstet Gyneco l 1992;167:1053-8.

17. Allen R, Castro L, Aro ra C, et al. Endo thelium-derivated relaxing facto r inhibitio n and the presso r respo nse to no repinephine in the pregnant rat. Obstet Gyneco l 1994;83:92-6.

18. Whelto n A, Watso n AJ, Ro ck RC. Nitro gen metabo lites and renal functio n. In: Burtis CA, Ashwo o d ER, edito rs. Tietz Textbo o k o f Clinical Chemistry. 2nd ed. Philadelphia: Saunders; 1994:1536-9.

pregnancy-induced refracto riness to vaso presso r agents. Am J Obstet Gyneco l 1992;166:1560-7.

20. Chu ZM, Beilin LJ. Mechanisms o f vaso dilatatio n in pregnancy: studies o f the ro le o f pro staglandins and nitric o xide in changes o f vascular reactivity in the in situ blo o d-perfused mesentery o f pregnant rats. Br J Pharmaco l 1993;109:322-9.

21. Ao i W, Gab le D, Cleary RE, Yo ung PCM, Weinb erger MH. The antihypertensive effect o f pregnancy in spo ntaneo usly hypertensive rats. Pro c So c Exp Bio l Med 1976;153:13-5.

22. Baylis C, Davinso n J. The Urinary System. In: Hytten F, Chamberleined G, edito rs. Clinical Physio lo gy in Obstetrics. Oxfo rd: Blackwell Scientific 1990:245-302.

23. Baylis C, Engels K. Adverse interactio ns between pregnancy and the new mo del o f systemic hypertensio n pro duced by chro nic blo ckade o f endo thelium-derivated relaxing facto r (EDRF) in rats. Clin Exper Hyper Pregnancy 1992; b(11):117-29.

24. Aho kas RA, Mercer BM, Sibai BM. Enhanced endo thelium-derivated relaxing facto r activity in pregnant spo ntaneo usly hypertensive rats. Am J Obstet Gyneco l 1991;165:801-7.

25. Lindheimer MD, Katz AI, Ko eppen BM, Ordo nez NG, Oparil S. Kidney functio n and so dium handling in the pregnant spo ntaneo usly hypertensive rat. Hypertensio n 1983;5:498-506.

26. Ljungblad U, Karlsso n K, Lundgren Y. The effect o f pregnancy o n heart size and blo o d pressure in renal hypertensive rats. Clin Exper Hyper pregnancy 1983;b2:61-74.

27. St-Lo uis J, Scio tte B. Pro staglandin- o r endo thelium-mediated vaso dilatatio n is no t invo lved in the blunted respo nse o f blo o d vessels to vaso co nstricto rs in pregnant rats. Am J Obstet Gyneco l 1992;166:684-92.

28. Takeda T. Experimental study o n the blo o d pressure o f pregnant hyp e rte nsive rats: I: Effe c ts o f p re gnanc y o n the c o urse o f experimentally and spo ntaneo usly hypertensive rats. Jap Circulatio n J 1964;28:49-54.

29. Paller MS. Mechanism o f decreased presso r respo nsiveness to angio tensin II, no repinefrine and vaso pressine in pregnant rats. Am J Physio l 1984;247:H100-8.

30. Co nrad KP. Renal hemo dynamics during pregnancy in chro nically catheterized, co nscio us rats. Kidney Int 1984;26:24-9.

31. Blizzard DA, Fo lk TG. Reso urce-sharing in rat gestatio n: ro le o f m ate rnal c ard io vas c ular he m o d ynam ic s . Am J Phys io l 1990;250:R1299-307.

32. Metcalfe J, Sto ck MK, Barro n DH. Maternal physio lo gy during gestatio n.

In: Kno bil E, Neil JD, Ewing LL, et al. The Physio lo gy o f repro ductio n. New Yo rk: Raven Press;1988:2145-76.

33. Weiner C, Martinez E, Zhu LK, Gho dsi A, Chesnut D. In vitro release o f e ndo the lium -de rivate d re laxing facto r b y ace tylcho line is increased during the guinea pig pregnancy. Am J Obstet Gyneco l 1989;161:1599-605.

34. Feleto u M, Vanho utte PM. Endo thelium-dependent hyperpo larizatio n o f canine co ro nary smo o th muscle. Br J Pharmaco l 1988;93:515-24. 35. Yallampalli C, Garfield RE. Inhibitio n o f nitric o xide synthesis in rats

during pregnancy pro duces signs similar to tho se o f pre-eclampsia. Am J Obstet Gyneco l 1993;169:1316-20.

N ilton Hideto Ta k iuti - MD, MSc, Department of O bstetrics and G yneco lo gy, University o f São Paulo Medical School and Medical Investigation Laboratory (LIM 5 7 ).

M a ria Helena Ca telli Ca rva lho - Pro fesso r,

Pharmaco lo gy Department, Bio medical Science Institute o f São Paulo University.

Soubhi Ka hha le - MD and Professor, Department of

O bstetrics and G yneco lo gy, University o f São Paulo Medical School and Medical Investigation Laboratory (LIM 5 7 ).

Dorothy N igro - Pro fesso r, Pharmaco lo gy Department, Bio medical Science Institute o f São Paulo University.

Herm es V ieira Ba rbeiro - Technical assistant, Pharmaco lo gy Department, Bio medical Science Institute o f São Paulo University, Brazil.

M a rcelo Zuga ib - MD, Pro fesso r and Chairman, Department o f O bstetrics and G yneco lo gy, University o f São Paulo Medical Scho o l and Medical Investigatio n Labo rato ry (LIM 5 7 ).

Sources of funding: CNPQ - Brazilian Financial

Suppo rt fo r Po stgraduate Research

Conflict interest: N o t declared

La st received: 2 6 February 1 9 9 9

Accepted: 9 April 1 9 9 9

Address for correspondence:

N ilto n Hideto Takiuti

R. Francisco Leitão , 4 6 9 , 1 1 º andar, cj. 1 1 0 9 São Paulo / SP – Brasil - CEP 0 5 4 1 4 -0 2 0 E-mail: nilto n@ o bstetricia.hcnet.usp.br

RESUMO

Contex to: O s mecanismo s envo lvido s na diminuição da pressão arterial e da resistência vascular periférica da gravidez não são to talmente esclarecido s, assim co mo a real impo rtância do fato r relaxante derivado do endo télio (Endo thelium-derivated relaxing facto rs - EDRF), um impo rtante mo dulato r do tô nus vascular. O bjetivo: O trabalho avalio u a impo rtância do EDRF e seu principal co mpo nente, ó xido nitrico , na prenhez das ratas. Tipo de estudo: Experimentação clínica em animal. Loca l:

Labo rató rio Universitário de Farmaco lo gia. Va riá veis estuda da s: Fo ram avaliado s, em ratas W istar no rmo tensas, virgens o u prenhes, a influência da inibição crô nica da síntese de ó xido nítrico , utilizando o Nw-nitro -L-arginina (L-NAME) diluído na água ingerida pelo s animais durante 1 0 dias e co mparadas co m o s respectivo s grupo s co ntro les. Fo ram analisado s as repercussõ es da prenhez e do L-NAME so bre a pressão arterial, reatividade vascular da ao rta a acetilco lina in vitro e resultado s perinatais. Resulta dos: A pressão arterial caudal diminui no final da prenhez. A sensibilidade da ao rta das ratas prenhes à acetilco lina fo i maio r que nas virgens. O tratamento co m L-NAME determino u aumento da pressão arterial caudal em ambo s o s grupo s de animais e determino u blo queio no relaxamento induzido pela acetilco lina, de mo do semelhante a retirada mecânica do endo télio da ao rta. O grupo de ratas prenhes que ingeriram L-NAME apresento u peso da ninhada e do peso de cada feto meno r que o grupo co ntro le. Conclusões: Demo nstramo s maio r sensibilidade para acetilco lina na gestação , co ntribuindo co m a diminuição da pressão arterial e um blo queio no relaxamento induzido pela acetilco lina apó s administração de L-NAME.