Fetal lung liquid regulation by neuropeptides

Fetal lung liquid regulation by neuropeptides Michael G. Ross, M.D., Gore Ervin, Ph.D., Rosemary D. Leake, M.D., Paul Fu, M.D., and Delbert A. Fisher, M.D. Torrance and Los Angeles, California The fetal lung, filled with fluid during intrauterine life, is recognized as a site of fluid production, significantly contributing to amniotic fluid volume. To assess possible hormonal control of lung liquid production, we studied the effect of intravenous vasopressin or vasotocin on lung liquid production in chronically catheterized fetal sheep. Both vasopressin and vasotocin infusion resulted in a significant decrease in lung fluid production (38% and 40%, respectively) when compared to that in control animals receiving saline infusion. There was no significant change in lung fluid osmolality, sodium, or potassium. These findings suggest that lung liquid production in utero may be regulated by the fetus and that vasopressin or vasotocin may be of physiologic importance in lung fluid dynamics during the third trimester of ovine pregnancy and perhaps during parturition. (AM J OBSTET GVNECOl 1984;150:421-5.)

The fetal lung, previously thought to be a site of amniotic fluid reabsorption, is now recognized as a significant source of fluid production. 1 The respiratory tract of the ovine fetus is fluid filled throughout intrauterine life, containing approximately 33 ml of fluid per kilogram of body weight near term; during the third trimester some 300 ml of pulmonary fluid is secreted daily into the amniotic cavity.! Initiation of effective respiratory gas exchange in the newborn animal at the time of birth requires inhibition of pulmonary fluid secretion and resorption of alveolar fluid before or during parturition. Although previous reports have suggested that fetal cortisol" or catecholamines" affect lung fluid secretion, the actual regulation of fluid secretion and resorption in the lung is poorly understood. Arginine vasopressin is a neuropeptide involved primarily in the regulation of water homeostasis. Arginine vasopressin and arginine vasotocin, an evolutionary precursor of arginine vasopressin, have both been identified in the mammalian fetal pituitary gland." Arginine vasopressin has been shown to modulate renal free water clearance in the third-trimester fetal sheep and has been reported to increase maternal-to-fetal water flow across isolated guinea pig amnion.s- 6 Moreover, both arginine vasopressin and arginine From the Departments of Obstetrics and Gynecology and Pediatrics, Harbor/University of California (Los Angeles) Medical Center, University of California (Los Angeles) School ofMedicine, Torrance, and Cedars-Sinai Medical Center, Los Angeles. Supported by National Institutes ofHealth Grant No. HD-06335. Presented in part at the Thirty-first Annual Meeting of the Societyfor Gynecologic Investigation, San Francisco, California, March 21-24, 1984. Received for publication February 9,1984; revised May 9,1984; accepted May 14, 1984. Reprint requests: Michael G. Ross, M.D., Building A-17 Annex, Harbor/UCLA Medical Center, 1000 West Carson St., Torrance, CA 90509.

vasotocin block fetal-to-maternal water transfer across the sheep placenta in response to a maternal osmotic challenge.' Perks and Cassin," in a study of acutely exteriorized fetal goats, reported that arginine vasopressin may reduce lung fluid secretion. These authors measured fluid production indirectly by means of dye dilution; infused doses of arginine vasopressin ranged from 1.6 to 39 mD/kg . min-I; plasma arginine vasopressin concentrations were not measured. In the same study similar experiments were performed on the chronically catheterized sheep model with arginine vasopressin infusions of 7.5 to 20.5 m l.l/kg : min~l. However, the results of these experiments were not presented. In the present study we sought to further characterize the effect of neurohypophyseal peptides on fetal lung fluid secretion. With the use of the chronically catheterized sheep model, we directly measured the rate of production and composition of fetal pulmonary fluid in response to intravenous infusion of arginine vasopressin or arginine vasotocin. Methods

Animals and surgical procedures. Seven chronically prepared, pregnant, cross-bred ewes were used for these studies. Gestational ages at the time of experimentation ranged from 115 to 132 days (term 147 days). Gestational ages were based on induced ovulation with a 24-hour exposure to a ram during the mating period. Animals were maintained under controlled light and dark periods (12 hours light, 12 hours dark) and were housed individually in steel study cages both prior to and following operation. Food and water were made available freely except during the 24 hours prior to operation. Anesthesia was induced with intramuscular ketamine (800 mg) and atropine sulfate (1.2 mg) followed by a continuous intravenous infusion of ketamine (5 mg/min). The maternal abdomen was

421

422 Ross et al. Am

opened via a midline incision, a sma ll h ysterotomy incision was made, an d a fe tal h ind lim b was extracted fro m th e u terus. A polyethylen e catheter (1.02 mm inside diameter , 1.78 mm outside dia meter) was pla ced in the fet al d or sal hind limb vein and th read ed to the inferior vena cava . Sim ilarly, a polyeth ylene catheter (0.51 mm inside diameter, 1.02 10m outside diameter) was threaded into the abdomi na l aorta via the hind limb artery. Prior to h yste rotomy closu re an in tra ut erine pla stic ca the ter (Corometr ics Medical Systems , In c., Wallin gford, Co nnect icut) was placed in the amnioti c cavity. A second h ysterot om y incision was mad e over the fetal neck. Fet al skin was incis ed vertically an d marsupialized to the uter ine incisio n. T he fet al trachea was blu ntl y dissected and incise d transversel y. A polyeth ylene ca theter (Fr. I I) with multiple ports was adva nced 7 cm into th e distal trach ea . The p roxim al trac hea was co m pletely occlu ded with a surgical ligatu re while the di stal trachea was ligat ed arou nd the cat he ter. The fetal skin and hysterotom y incision were closed . The mat ernal fem oral arte r y and vein were cathe terized with pol yethylene cat heters (Fr. 8) thread ed to th e abd ominal aorta and in ferior vena cava , resp ectively. All catheters were exteriorized to the maternal flank and placed in a cloth pouch sut u re d to the skin . T he ope n ends of the fe tal trach eal catheter and the amniotic fluid cathe ter were co n nected by stopcocks and exteriorized to the mat ernal flan k. This allowed a co ntinuous flow of lung secretions to amn iotic fluid , wh ile p roviding access to trach eal fluid during ex pe r ime nt s. Ani ma ls were allowe d at least 5 days of postoperati ve recovery, during which time chlo ra m p henico l (500 mg) was ad min istered twice da ily into the maternal vein and the am n iotic cavity. Study design. Eighteen ex pe ri me nts were performed in the seven an imals as follows : co ntrol (no rma l saline), n = 5; low-dose argini ne vaso pre ssin , n = 4; high-dose argi n ine vaso pres sin, n = 5; argini ne vasotocin, n = 4. Most an ima ls had th ree stud ies performed. T h e co ntrol stu dy was performed as foll ows: Prior to each study fet al arte rial pH was measured and the ex pe rimen tal protocol performed onl y if th e fe tal pH was > 7.30. The fetal am n iotic fluid catheter was closed and th e tr ach eal catheter was o pe ned to grav ity d rain age . Pulmonary volu mes were d rained d u ri ng a 40-min ute period with subsequent drainage assu med to represent active production of lung fluid (prior experiments in th ree an ima ls revealed tha t followin g an ini tial 30minute d r ain age period, there was an essentially lin ear flow of trach eal fluid fo r an additional 3 hours). Following th e prelimin ary 40-minute period (time 0) and throughout the remai nd er of the stu dy , fe tal lung flu id was d rain ed into glass tubes at lO-min ute int ervals.

Octo ber 15 . 1984 J O bste t Gyn eco l

At +60 m in utes an intravenous solution of 0.9% no rm al saline solu tio n was admin iste red via th e fe tal d o rsal vein at a constan t in fusio n ra te (0. 1 mil min) fo r 60 min utes . At +30, +90, a nd + 120 minutes, blood sa m ples (3.5 ml) for measu rem ent of a rgi nin e vasop ressin , arginine vasotocin , pH , P0 2, and Pco, were d rawn from the fetal arterial catheter, an d fetal mean blood pressu re an d heart rate wer e measured with a Beckman Dyno graph (Bec kma n In stru ments Inc., Fullert on , Califo rnia). After th e 120-minute fe tal blood sam ple was obta ine d , heparini zed mat e rn al blood (14 ml) d ra wn prior to the stu d y was ad mi nis tered to the fetus. At least 24 h ours elapsed between th e control and hormon e infusion studies. Hormon e infusion s were ad ministered in saline solu tio n as an intraveno us infusio n of 0.9% saline (0. 1 mil min) co ntai ni ng I m U/ kg . min " of argini ne vaso pressi n (Pitressin, Park e, Davis & Co ., Detroit, Michigan) , 2 mU/ kg . min - t of argi ni n e vaso pressin, or 8 n g/k g . min-I of argini ne vaso tocin (Bac hem Fine Chemicals, T orr ance, Califo rnia) . Trach eal fluid and blood sam pling were co nducted as in the co n trol stud ies. Fetal weights were estima ted by th e fo rm ula of Rob illard an d Wei tzma n." T racheal fluid osm olalit y was measured by freezi ng point depression with an Adva nced Instruments os mometer (Advanced Instruments, In c., Needham Heigh ts, Massachusetts). Tracheal fluid sod iu m an d potassium levels were mea sured by flam e photomet r y. Blood sa m ples fo r de te rmi na tion of argi nine vasopressin and ar ginine vaso tocin were collected in ch illed tubes co ntai n ing 30 J.d of 15% po tassi u m -ethyle ne d iamine -tetraace tic acid and im med iately placed o n ice . Plasma was pre pared by cent rifugation at 2000 rpm fo r 10 minutes at 4 0 C and sto red fro zen at -20 0 C until ex tracted for a rg in ine vasopressin and ar gin ine vasotocin measure me nts. Plasma was ex trac ted for argin ine vaso pressin with th e use of bentonite as described previou sly." A rg ini ne vasotocin extra ction was performed by mean s of silica columns (Se p Pak, Wa ters Associates , Inc., Milford, Massachusetts). In fusate an d plasma argin ine vasopressin and ar gin ine vasotocin levels were determined by ra d ioimm u noassay as previously described ." Statistics. All d ata are expressed as mean ± SEM . Arginine vaso pre ssin and arginine vaso toci n pla sm a levels, pH , Po 2, PC02, blood pressure, and heart rate were com pared between basa l (+30 mi nu tes) an d in fusion (mean of +90 and + 120 minutes) periods by Student's paired t test. Statistical an alyses of the effects of arginine vasopress in, argini ne vasotocin , or saline solu tion on tracheal fluid p ro ductio n, os mo lality, sod iu m, and pot assium du ri n g basal (0 to +60 minutes) and infusio n (+60 to + 120 minutes) periods were determined by paired t test. Co m parisons of th e effects of arginine vasopressin and ar gin ine vasotocin with th ose

Fetal lung liquid regulation by neuropeptides 423

Volume 150 Number 4

Table I. Fetal arterial blood values before and during infusion of arg inine vasopressin or arginine vasotocin* A rgini ne vasotocin

Arginine vasopressin

I

Basal

Value

7.33 :!: 23 :!: 49:!: 41 ±

pH Po. (mm Hg) Pco, (mm Hg) Blood pressure (torr) Heart rate (bp rn)

7.35 ± 25 :!: 46 ± 51 :!: 151 ±

0.02 I I

3

188 ± 12

I

Basal

Inf usion

7.34 24 46 55 151

7.32 ± 0.01 19 :!: I 50 ± 4

0.01 I

It 4t 9t

Infus ion

46 ± 6 ± 22

196

±

0.01

± It ± 2 ± 6t ±

lI t

*Values recorded as mean ± SEM. tS ignificantly different from basal values (p < 0.05).

Table II. Fetal tracheal fluid os mo lality, sodium, and potassium before and during infusion o f saline solut ion contr ol, arginine vasopressin , and arginine vasot ocin * A rginin e vasopressin

Control Basal

Osmolality (mo l/ L) Sodium (mEq/L) Potassium (mEq/L)

I

306 :!: 2 152 ± 3 4.5 ± 0.5

Infusion

Basal

307 :!: 2 153 :!: 8 4.6 :!: 0.3

300 :!: 2

155 ± 2 4.6 ± 0.2

I

A rginine vasotocin

Infusion

Basal

303 :!: 2

300 ± 5 147 ± 2 4.9 ± 0.3

158 ± 4

4.7

±

0.1

I

Infusion

303 ± 4 150 ± 2 4.9 ± 0.3

*Values recorded as mean ± SEM. of saline solution were pe rformed me asure-ment analysis of variance .

by

repeat-

Results

Sam ples of infusate solu tions were assayed fo r arginine vaso pressin and argin ine vasot ocin to determine actual dosages administered during the infusion periods. Low-dose arginine vasopressin was infused at a rate of 0.9 mU/kg . min-I; the hi gh-dose arginine vasopressin infusion rate was 2.0 mU /kg . min-I. Arginine vasot ocin was administered at a rat e of 7.8 ng/ kg . min-I. Plasma arginine vaso tocin concen tration s increas ed from basal levels of 2.6 ± 0.6 to >7.4 pg/ml (u p per limit of assa y, p < 0.05) durin g argin in e vaso tocin infusion . There were significa nt inc reases in plasma argi n ine vasopressin during both low-dose (2. 1 ± 0.6 to 12.7 ± 2.0 poU /ml, p < 0.0 5) and h igh-dose (1.6 ± 0.1 to 11.6 ± 1.0 poU/ml, p < 0. 05) infusions . Despite the high-dose arginine vaso p ress in infusion being more th an twice the low-dose in fusi on rat e, there was no significant difference in steady-state plasma arginine vasopressin levels . The results of th e low- an d high-dose arg in ine vasopressin infusion s were therefore combined for the purpose of analysis. There were minimal changes in pH , Pcoj, and POz du ring the infusion periods of arginine vasopressin and arg in ine vasotocin as compared with basal periods (Table I). The trend, however, was toward increased pH and POz and decreased Pco, values. There was a significant increase in mean blood pressure with a con-

co mita nt sign ificant de cre ase in mean fetal heart rate during both the arginine vasopressin and the arginine vasotocin infusions. There were n o sign ifica nt changes in pH , blood gases, heart rat e, or blood pressure dur in g the co n tro l infusions. Tracheal fluid product ion di d not cha nge significantly during saline solution in fu sion (1.27 to 1.09 mll 10 min, p > 0.05). In co nt rast, there were significant (p < 0.05) decreases in trac h eal fluid production during both arginine vasot ocin (-40%, 2.21 to l. 32 mll 10 min) and arginine vasopressin (- 38%; 2.11 to 1.30 ml/lO min) infusions . Moreover, the effects of argi ni ne vaso pressin and arginine vaso tocin on tracheal flu id production were significant when com pared to th e saline solution control study by ana lysis of variance (Fig. I). As show n in Table II, th ere were no significan t cha nges in tracheal fluid os mo lality (306 ± 2 to 307 ± 2), sod iu m (152 ± 3 to 153 ± 8), or potassium (4.5 ± 0 .5 to 4.6 ± 0.3 ) concentrati on s durin g any of the studies (values reported in text are for saline infusion). Comment

Amnio tic fluid represents a d yn am ic fluid pool with input from the fetal lungs and kidneys and outflow via fet al swallow ing and tran samniot ic fluid absorption. Earl y experiments in which fe tal sheep tracheal occlusion led to abnormal distention of fetal lung provided evidence that lung liquid is secreted by the fetal lung. Chronic in utero tracheal cathe terization studies re-

424

Ross at al.

October 15, 1984 Am J O bstet Gyne col

+20 +10

w

o

Z

10

C)


ConirOI

~ - 20 ~ -30

VP

- 40

-50

VT

o +20 +40 +60 TIME (Minutes)

-60 -40 -20

Fig. 1. Percent change (mean ± SEM) in tracheal fluid volume

before and during infusion of saline solution control, arginine vasopressin (VP) , or arginine vasotocin (VT) .

vealed that the ovine fetus produces lung fluid at a rat e of 4.3 rnl/kg : hr", a tot al of a p p roxi ma tely 300 nil/day for a 3 kg fetus." Studies of elect rochemical gradients across the fet al pulmon ary epithelium!' h ave su ggested th at an active ch loride pump is resp onsible for the production o f fet al pulmonary flu id. The co mposition of lung flu id has been show n to differ sign ificantl y fro m th at of fetal plasm a or pulmona ry lymph, with increased co ncent ra tions of pot assium and chl oride and signifi cantly decreased protein, pH, bicarbonate , an d calcium.' In the present study we used a ch ro nic su rgical preparat ion in wh ich we measured pulmonar y fluid production directly. The fetus was stu died in an un stressed co nd ition as evide nce d by arterial pH >7.30 and low ba sal arginine vasopressin concentrations. The basal tracheal fluid secretory rates measured during this stu dy are con sistent with those of previous reports," confi rming th e validi ty of o ur model. The re was no significant cha n ge in lung liquid production during the seco n d hou r of our cont ro l stu d y. Shortterm ( I hour) fetal infusion of argi nine vaso pressin and arginine vasotocin produced significant decreases in lung fluid production (Fig. I) . There were no changes in lung fluid osmolality or sod iu m or pot assium co ncentration s, suggesting that th e effect of argi n ine vaso pressin or arginine vasotocin is to decrease the rate of fluid production rather th an to co ncen trate the fluid. Arginine vaso pressin has previously been sho wn to act at the fetal kidney to reduce free water clearance.! Although arginine vasotocin has been reported in th e mammalian fetal pituitary gland ,4 this is the first study d ocumenting an effect o f arginine vasotocin on pulmonary fluid production. Intravenous administration of arginine vaso press in and arginine vasotocin to the fetus produced respective plasma hormone concentrations less than those resultin g from endogenous secret ion in response to hernor-

rhage or h ypoxia." Although the concentrations of arginine vasopressin in the high- a nd low-dose infusate s were sign ifican tly d ifferent, there was no significant difference in fetal arginine vasop ressin pla sm a levels, suggestin g the presence o f a n augmented clearance of argin ine vasopressin in res po nse to increased plasma levels. We noted minimal changes in fetal pH, P0 2 , and Pc0 2 despite a sign ifica n t inc rease in mean blood pressu re and a decrease in he art rate during both argin ine vasopressin and arg in ine vaso tocin infusions. These cha n ges are cons iste n t with the repon of Iwamoto e t al. 13 in wh ich simil ar plasma levels of a rgin ine vasopressin resulted in co m para ble hemod ynamic chan ges. The trend toward increased pH and P0 2 may represent in cr eased placental blood flow. The mech anism (s) by wh ich argin ine vasop ressi n and arginine vaso tocin reduce pulmonary fluid production is not presentl y known . It is unl ikely that th e action of th ese neu ropeptides is seco ndary to pulmonary hemod ynamic cha n ges, since Iwamo to et al. " reponed that similar plasma level s of argin ine vasopressin d o not sig n ifica n tly cha nge pulmonary blood flow. It is pos sible that argi ni ne vasopressin may in h ibit the active chl oride pump responsible fo r pulmonary fluid secretion. Wh ate ver th e mec ha nis m, the p resent re sults suggest th at lung liquid production in ut ero ma y be re gulated by the fetus. Kitterman et al." have shown th at tracheal fluid production d ecreases prior to th e onset o f lab or, being inversely co rrelated with the fet al plasma cortisol level, Othe r in vestigators have reponed th at ,B-adren ergi c agents decrease lung fluid formati on in fetal lambs," suggesting th at endogenous catec ho lami nes ma y be the stimulus for the reduction in lung fI uid production. It is possible that ,B-adrenergicre ceptor stimulation d ecreases lung fluid production via changes in pulmonary vascula r re sistan ce or stimulation of arginine vaso pressin rel ease. In addition to reducing secr etion, arginine vaso p re ssin may co n tr ibute to the resorption of fetal lung liqu id at birth , Arginine vaso p ress in has been reported to ind uce the forma tion of epithelial water pores." Egan et al.," stu dy ing ch anges in alveolar permeability in the lamb at birth , sugges ted th at spontaneous venti lation was associated with the openin g of water-filled cylin d rical pores. The open ing of water pores, accompan ied by a de crease in fluid secr etion , wou ld create a significant colloid osmotic gradient across the pulmonary epithelium, favoring the resorption of alveolar liquid into the pulmonary interstitium. An increase in pulmonary lymph How' " has been noted in newborn lambs at th e onset of spo n taneo us vent ila tion . The effect o f arginine vaso pressin o n lung liquid secre tio n and reabsorption ma y explain, in part, the increased incidence of respiratory distress occu rrin g in in fants del ivered by cesa rean section as co m pared to

Fetal lung liquid regulation by neuropeptides

Volume 150 Number 4

those delivered vaginally." Bland et al." reported that the extravascular lung water volume at birth is greater in rabbits born by cesarean section than in those born vaginally. Plasma arginine vasopressin has been shown to be significantly elevated in infants delivered vaginally as compared to those delivered by cesarean section.'? The release of arginine vasopressin during vaginal delivery may be physiologically important for the inhibition oflung fluid secretion and the resorption of alveolar fluid occurring at birth. The results of this study suggest that the neurohypophyseal peptides may have important physiologic roles in the control of ovine lung fluid secretion in utero and at birth. By affecting water transport at the pulmonary epithelium, renal tubules, chorioamnion and placenta, arginine vasopressin and arginine vasotocin may be primary modulators of fetal water homeostasis.

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