Plasma levels of atrial natriuretic peptide under acute hypoxia in normal subjects

Respiration Physiology, 76 (1989) 79-92 Elsevier

79

RSP 01521

Plasma levels of atrial natriuretic peptide under acute hypoxia in normal subjects A. Kawashima, K. Kubo, K. Hirai, S. Yoshikawa, Y. Matsuzawa and T. Kobayashi First Department of Internal Medicine. Shinshu University School of Medicine. Matsumoto. Japan (Accepted for publication 7 January 1989) Abstract. To investigate whether the acute hypoxia can be a stimulus for atrial natriuretic peptide (ANP) secretion, plasma levels of ANP were determined under three different hypoxic conditions in six normal subjects. D ~ring 15% 02 breathing for 10 rain, no significant change in plasma ANP level was observed. Severe hypoxia induced by 10% 02 breathing increased the mean pulmonary arterial pressure (Ppa) by 11.6 mm Hg within 10 min (P < 0.01), accompanying a slight but significant rise in plasma ANP level of pulmonary artery (PA) from 24.3 ± 5.3 to 28.2 _+4.6 pgfml (P < 0.05). There was a tendency for the ANP level of PA to rise under hypoxic hypobaria at 515 Tort for 10 rain, followed by a decrease of that level during 100% 02 breathing under hypobaric condition. These changes, however, still remained in the normal range. No significant changes were observed both in right atrial pressure and in pulmonary capillary wedge pressure under any of the three hypoxic conditions. From these results we conclude that ANP can be released in response to the elevation of Ppa caused by acute hypoxia in normal subjects, but the changes in plasma ANP level may be too small to play a significant physiological role in hemodynamic responses to acute hypoxia.

ANP; Hemodynamics; Human; Hypoxia: Lung; Pulmonary artery pressure

Plasma levels of atrial natriuretic peptide (ANP) have recently been reported to be elevated in patients with respiratory disease, especially those with pulmonary hypertension (Adnot et al., 1987; Burghuber et al., 1988a,b). in addition, the presence of specific receptors for ANP in lung (Sakamoto et al., 1986) and the vasodilating properties of the synthetic ANP in pulmonary vasculature (Adnot et ai., 1988a,b) have been found in recent experimental studies. These findings strongly suggest that the ANP may be released in response to some pulmonary hemodynamic alterations and may play a significant pathophysiological role in the control of pulmonary circulation in hypoxic state. Atrial distension, which has been recognized to be a major stimulus for ANP secretion (Needleman and Greenwald, 1986; Ballermann and Brenner, 1986), may be Correspondence address: Akira Kawashima, M.D., First Department of Internal Medicine, Shinshu University School of Medicine, 3-I-I Asahio Matsumoto 390, Japan. 0034-5687/89/$03.50 @ 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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one of the factors responsible for elevated plasma ANP levels in some of conditions, in which the increased fight ventricular afterload leads to higher end-diastolic pressure resulting in a fight atrial distension. In fact, a strong positive correlation between plasma ANP level and mean fight atrial pressure has been observed (Burghuber et al., 1988a). However, pulmonary hypertension developing in respiratory disease is not always associated with an elevated fight atrial pressure. Some reports have shown that no correlation was observed between ANP levels and fight atrial pressure (Adnot et al., 1987; Burghuber etal., 1988b), suggesting that the elevated plasma ANP levels observed in these patients may be attributable to other factors than fight atrial distension. Pulmonary hypertension itself is expected to be a good candidate for these factors, since previous studies have demonstrated a significant positive correlation between plasma ANP levels and pulmonary arterial pressure (Adnot etal., 1987; Burghuber et al., 1988a,b). Therefore we wondered whether acute hypoxia, which causes an increase in pulmonary arterial pressure, can be a stimulus for ANP secretion in healthy subjects, not mediated by atrial distension. To test this hypothesis, we measured plasma levels of alpha-human ANP in blood obtained from the pulmonary artery and from the brachial artery in six normal subjects during hypoxic gas breathing of two different concentrations of oxygen, 15~o and 107/o. Additionally, to evaluate the effect of acute hypobaria on hemodynamics and ANP secretion, plasma levels of ANP were also measured following acute exposure to hypofiafic condition of 515 Torr, which was used as an inspired oxygen tension equivalent to the hypoxic test of 157/o oxygen. To our knowledge, this is the first report investigating the changes in plasma ANP level under acute hypoxic conditions in normal subjects.

Methods ~'ubjects. We studied 6 healthy male volunteers. Their mean (SE) age was 23.8 (0.7), range 21-26 years; height 174.5 (2.1), range 169-183 cm; and weight 68.5 (3.2), range 58-78 kg. All subjects were the students of Shinshu University. They were all nonsmoker, and none of them received any medication. They had mountain climbing experience above 3000 m without severe altitude sickness, and none had any history of cardiorespiratory disease. All underwent a careful medical evaluation before participation, and had normal results of physical examinations, routine laboratory tests, chest roentgenograms, electrocardiograms, and lung function tests. All su~bjectsgave written consent to participate as volunteers after they had been informed of all procedures and possible risks of this study. This study was approved by the Research Committee of our School of Medicine. No complication occurred during or after this study. Hemodynamic measurements. A 7-F Swan-Oanz thermodilution catheter was inserted through a left antecubital vein into the pulmonary artery for measurement of pulmonary arterial pressure (Ppa), pulmonary capillary wedge pressure (Pcw) and right atrial

81

ATRIAL NATRIURETIC PEPTIDE IN ACUTE HYPOXIA

pressure (Pra). Systemic arterial pressure (Psa) was obtained from a left brachial artery cannula. All pressures were continuously monitored using disposable transducer system (SCK-580, Nihon Koden Co., Tokyo, Japan), and recorded on an eight-channel recorder (WT-685G; Nihon Koden Co., Tokyo, Japan). Zero pressure was referenced to the midthoracic level, and calibration was performed using a mercury manometer. Mean pressures were obtained by electronic averaging. Cardiac output was measured by the thermodilution method using an Edwards model 9520 cardi~lc output computer (Edwards Laboratories, Santa Ana, CA). Cardiac outputs were measured in triplicate and averaged. Heart rate was monitored by the electrocardiograph. Arterial and mixed venous blood gases were measured on a Radiometer model ABL2 blood gas analyzer (Radiometer Co., Copenhagen, Denmark). These hemodynamic measurements were made in the supine position. From the directly measured parameters the following indices were derived: cardiac index (CI)(L" min- ~- m - ' ) = cardiac output/body surface area; pulmonary vascular resistance index (PVRI) (mm H g . L -~. rain.m2)= (mean P p a - mean Pcw)/Cl; systemic vascular resistance index (SVRi)(mm H g - L - ' . min. m:) = (mean Psa - mean Pra)/Ci; stroke volume index (SVIXmi'm -2) : CI/HR; oxygen delivery index (ml O 2 " m i n - I ' m -2) : arterial oxygen content (Cao2)x CI x 10; oxygen consumption (ml O2"min- ~" m- 2) : (Cao: - mixed venous oxygen content (C~o,)) x Ci x 10; and coefficient of oxygen delivery (COD) : Cao:/(Cao, - C~o: ).

Experimental protocol. This study was performed in Shinshu University Hospital, Matsumoto, Japan, at an altitude of 600 m. The barometric pressure in Matsumoto dunng the days of this study was approximately 700 Ton" whh small variation. This study was composed of three successive parts of experiments as outlined in fig. 1: experiment 1, moderate hypoxia for 10 min ir,duced by breathing a gas mixture of 15~, oxygen in nitrogen at a barometric pressure of 700 Torr; experiment 2, severe hypoxia for 10 min induced by 10°/o 0 , at the same pressure as in experiment 1; and experiment 3, hypoxic hypobaria for 10 min at a barometric pressure of 515 Ton (equivalent altitude: 3200 m, 21 ~o inspired O, fraction), followed by a 10 min period of hyperoxic hypobaria by breathing 100~o O2 at the s~ane pressure. Each experiment was preceded -.

Experiment 1

---,

Experiment2

.--,

Experiment 3

j

F

-

"

Decompression - ~

"1

t

I

I

'

t

t

J

J

t

t

R A : Room Air

Fig. i. E x p c r i m © n t a i

t

t

t ~

Inspired Oxygen Fraction : Hemodynamic MeasurP-~'~t & Sample Coll~)ction

15%, 10%, 1 0 0 % :

t

'

protocol.

'

10 rain

82

A. KAWASHIMA et aL

by a 30 min base-line period, during which the subjects were allowed to breathe room air (barometric pressure: 700 Ton'). In experiments I and 2, the subjects breathed from a reservoir bag containing a hypoxic gas mixture through a tight-fitting face mask, in which two one-way flap valves were placed to prevent rehreathing and contamination by room air. Before and 10 rain after the start of the hypoxic gas breathing, the hemodynamic measurements were made and blood samples were drawn from the brachial artery and the pulmonary artery. In experiment 3, the subjects were decompressed in a 2.6 × 8 m cylindrical artificial climatic chamber in the Department of Environmental Physiology at Shinshu University. Temperature was maintained between 20 and 22 °C with humidity a constant 60%. Approximately 10 min were required to reach the hypobaric condition of 515 Ton., in which the inspired 0 : tension (Plo2) was equivalent to that in 15% O2 breathing at 700 Ton'. (Allowing 47 Ton" for water vapor and 0.21 as the percent of O2 present in room air, calculated Plo2 available at the barometric pressure of 515 Ton" was 98 Ton', which was almost the same as that available during 15~o O2 breathing at 700 Torr.) In addition, 100% O 2 was given to the subjects 10 min after arrival to evaluate the effect of hypobaria per se by relieving the hypoxic pulmonary vasoconstriction completely. Measurements of hemodynamic parameters and sampling of blood were made before decompression, 10 min after arrival and 10 rain after the start of the 100~o O2 breathing in hypobaric condition. During all experiments, the subjects were resting in a supine position. Analysis ofatrialnatriureticpeptide. Blood for analysis of ANP was obtained simullaneously from the pulmonary artery and from the brachial artery. These samples were drawn on chilled tubes containing EDTA-2K and aprotinin (500 kailikrein inactivator units/ml), placed on ice and centrifuged within 15 min. Plasma samples were stored at 30 °C until assayed. We measured plasma ANP level by radioimmunoassay, which was almost the same as previously described (Naruse et al., 1986) but more sensitive ~md specific for alpha-human-ANP, in brief, the extraction of ANP from plasma was performed by the Sep-Pak C~s cartridge with a recovery of 92.2 + 5.7%. The antiserum (Mitsubishi-Yuka Laboratory of Medical Science) was produced against the alphahANP (human, 1-28) and used at a final dilution of I : 5 × 104. The cross-reactivity of this antiserum was 100~o for alpha-hANP(l-28) and hANP(5-27), 44% for betahANP and 9% for alpha-rat ANP. The minimal detectable quantities were approximately 2 pg/tube in the present assay. The intra- and inter-assay coefficients of variations were 5.3~o (N = 8) and 9.2~o (N = 6), respectively. The resting levels of plasma ANP in the peripheral vein ranged from 13.4 to 49.5 pg/ml in 20 normal volunteers (mean + SD, 26.9 + 11. ! pg/ml). -

Statistical analysis. Data are expressed as the mean + SE. Statistical analysis was performed using Student's t test for paired data, and a P value of less than 0.05 was considered significant. Standard Pearson Correlation coefficients were calculated to assess the relation between two variables.

ATRIAL NATRIURETIC PEPTIDE IN ACUTE HYPOXIA

83

Results

Hemodynamics and bloodgas analyses. Experiment 1: These results are given in table 1. Saturation, tension and content of ox~ygen in both arterial and mixed venous blood showed significant decreases during 10 min period of 15% 02 breathing. Neither Paco~ nor pH of arterial blood changed in this experiment. Mean Ppa increased significantly from 14.8 _+0.3 to 18.3 +_0.6mm Hg ( P < 0.01). CI increased from 3.39_ 0.11 to 3.69_ 0.21 L . m i n - ~ - m -2, but the ,;.~hange was not significant. Calculated PVRI increased by nearly 60% (P < 0.05). There were no significant changes in other hemodynamic parameters including Psa, HR, Pew, Pra, and SVI. Both 02 delivery and 02 consumption declined minimally, and COD remained fairly constant in this experiment. Experiment 2: As shown in table 2, severe hypoxemia was induced by 10% O2 breathing (Pao~ = 35.6 +_ 1.4 mm Hg). In addition to the decreases in saturation, tension and content of oxygen both in arterial and in mixed venous blood, which were TABLE I Hemodynamics and blood gas analyses in experiment ! w

Psa (mm Hg) HR (beats. min- t ) Ppa (mm Hg) Pcw (mm Hgl I r a (mm Hg) CI ( L . m i n - t ' m - 2 ) PVR! (ram Hg" L ~ ~'min "m:) SVRI (mm H g . L - I ' m i n ' m 2) SVi ( m l . m - ' ) Sao= (°o) Pao= (mm Hg) Paco 2 (ram Hg) pHa Cao~ (vol °o) S~o= (°o) ! ~ o : (mm Hg) I ~ c o , (mm Hg) C~o, (vol °o) O 2 D. ( m l . m i n - " m -2) O , C. ( m l . m i n - ~.m -2)

COD

Room air

15~o oxygen

P value

95.7 58.2 14.8 9.8 5.2 3.39

96.7 ± 2.0 64.0 + 3.5 18.3 + 0.6 9.9 + 0.5 4.6 + 0.9 3.69 + 0.21 2.30 +_0.20 25.3 + 1.4 58.2 + 3.5 85.7 + 0.7 52.8 _+ 1.3 37.2 + 1.5 7.40 + 0.01 18.9 + 0.6 70.8 _+0.9 37.9 +_0.7 40.9 + 1.7 15.6 _+0.4 692 + 21

NS NS <0.01 NS NS NS

± 1.3 ~_~4.0 +_0.3 _+0.5 + 0.8 _+0.11

!.45 + 0.11

26.8 59.7 96.1 95.9 38.4 7.39 21.3 78.1 44.2

± 1.2 + 4.'J _+0.5 + 4.5

_+ !.3 _+ 0.01 + 0.7 + 0.9 _+0.9

41.7 + !.5

17.2 + 0.6 719 + 12 138 + 4

121 + 4

5.2 + 0.2

5.7 + 0.2

<0~05

NS NS <0.001 <0.001

NS NS <0.001 <0.001 <0.001 NS <0.01 NS NS NS

Definition of abbreviations: Psa = systemic arterial pressure; HR = heart rate; Ppa -- mean pulmonary arterial pressure; Pcw = pulmonary capillary wedge pressure; I r a -- right atrial pressure; C! = cardiac index; PVRI = pulmonary vascular resistance index; SVRi = systemic vascular resistance index; SVI = stroke volume index; Sao, = arterial oxygen saturation; pHa = arterial pH; Cao2 = arterial oxygen content; S~o: -- mixed venous oxygen saturation: C~o2 -- mixed venous oxygen content; 02 D. = oxygen delivery; O: C. -- oxygen consumption; COD -- coefficient of 02 delivery.

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A. K A W A S H I M A el al.

TABLE 2

Hemodynamics and blood gas analyses in experiment 2

Psa (ram Hg) HR ( b e a t s . r a i n - ~) Ppa (mm Hg) Pew (mm Hg) Pra (ram Hg) CI ( L ' m i n - ' "m - 2 ) PVR! (mm Hg. L- i. min.m 2) SVRI (ram Hg" L- ~. rain. m 2) SVI (ml. m - 2) Sao: (o~,) Pao2 (mm Hg) Paco2 (mm Hg)

pHa Cao: (vol 0,~,) S~o: (0o) Pro2 (ram Hg) Pvco: (mm Hg) C~o: (vol °o)

O: D. (ml.min-'.m- 2) O 2C.{ml'min ' . m : ) COD

Room air

10~o oxygen

P value

97.3 + 2.0 57.3 + 4.1 14.2 + 0.5 9.3 _+.0.6 5.1 + 1.0 3.33 + 0.12 1.46 + 0.1 ! 27.8 + 0.9 59.7 + 5.3 96.1 _+ 0.3 94.0 + 2.9 37.1 + 1.0 7.39 +_ 0.01 21,3 + 0.7 76.9 + 0.9 43.3 + 0.8 42.2 + 1.8 16.9 + 0.6 705 + 15 144+6 4.9 + 0.2

90.0 + 3.9 86.3 + 7.0 25.8 + 2.1 9.8 + !.4 4.1 + I.! 4.69 + 0.32 3.50 + 0.29 18.7 + 0.8 55.0 + 3.4 71.3 + 2.5 35.6 + 1.4 31.4 + 1.6 7.45 + 0.01 15.7 + 0.8 56.2 _+ 1.7 28.4 + 0.7 36.9 + 1.0 12.4 _+ 0.7 724_+ 21 152+9 4.8 + 0.2

NS <0.05 <0.01 NS NS <0.01 <0.01 <0.001 NS <0.001 <0.001 <0.05 <0.001 <0.001 <0.001 <0.001 <0.05 <0.001 NS NS NS

For definition of abbreviations, see table I.

all greater than in experiment !, Paco, decreased significantly (P < 0.05). Arterial blood pH showed a significant increase in this experiment (P < 0.01). HR increased significantly (P < 0.05), but the decrease in SV! was slight and not significant, resulting in a 40~o increase in CI (P < 0.01). Mean Ppa also increased significantly from 14.2 + 0.5 to 25.8 + 2.1 mm Hg ( P < 0.01), mean Pew did not change, and PVR! increased by nearly 140°o (P < 0.01). In spite of the significant increase in Ci, mean Psa decreased from 97.3 + 2.0 to 90.0 _+ 3.9 mm Hg, resulting in a significant decrease in SVRI (P < 0.001). Both 02 delivery and consumption showed minimal increases, and COD remained constant as in experiment 1. Experiment 3: Data are summarized in table 3. Changes in blood gases under this hypoxic hypobaria were similar to those observed in experiment 1. Under this condition, consequently, hemodynamic parameters showed similar changes to those observed in experiment I. However, PVRI increased about twofold under hypoxic hypobaria (P < 0.01), which was 400~, greater than in experiment 1, so that the average of Ppa in hypoxic hypobaria was 2 mm Hg higher than that in experiment 1 despite identical amounts of Pro.,. The O~ delivery remained constant and 02 consumption decreased slightly, resulting in a significant increase in COD. 100% 02 breathing under this hypobaric condition caused significant increases in oxygen saturation, tension and

ATRIAL NATRIURETIC PEPTIDE IN ACUTE HYPOXIA

85

TABLE 3 Hemodynamics and blood gas analyses in experiment 3

Psa (mm Hg) HR (beats-min- I) Ppa (ram Hg) Pcw (ram Hg) Pra (ram Hg) CI (L-min- I-m-2) PVRI (ram Hg. L- ' -min-m 2) SVRI (mm H g . L - ' - m i n . m 2) SVI (ml-m- 2) Sao= (?~,) Pao, (ram Hg) Paco,(mm Hg) pHa Cao, (vol ~b) S~o2 (%) !~o= (ram Hg) l~co, (ram Hg) C'~o= (vol ?~)

700 Ton

515 Tort

Room air

Room air

97.3 + !.8 60.5 _+3.0 14.3 _+0.5 9.2 + 0.6 5.2 + 0.8 3.35 + 0.14 1.52 + 0.05 27.8 _+ 1.3 56.0 + 3.5 95.3 + 0.6 86.8 + 4.4 38A + 1.2 7.39 _-!-0.01 21.1 +_0.7 76.8 + 1.2 43.0 _+ 1.2 41.9 + 1.4 16.9 + 0.6

96.7 + 1.2 67.5 + !.8" 20.3 _+ 1.3"* 8.8 + 0.6 4.9 + 0.7 3.69 + 0.16"* 3.11 + 0.29** 24.7 4- !.3"* 54.7 + 2.2 86.4 +_0.3*** 52.7 + 0.8*** 36.8 _+0.9 7.41 + 0.01" 19.0 + 0.6*** 71.5 + 0.6** 37.9 + 0.8*** 39.2 + !.1 15.8

+ 0.6**

1007o oxygen 99.7 + 1.3 57.0 + 2.1 # # # 13.8 + 0.6 ¢ # 8.2 + 0.5* 4.5 + 0.7 3.18 + 0.14* * 1.79 + 0.09*# 30.3 + !.6***# 56.0 + 2.7 99.0 + 0.3"** # # 189.2 + 20.7**# # 38.9 + i.8 7.38 + 0.01 * 22.2 + 0.8**# * * 82.0 + 0.5*** * * 49.2 + 0.5*** * # 42.9 + 1.6" 18.1

+ 0.7*** # *

O 2 D. (ml. rain - ~. m - :) O , C. ( m l . m i n - S . m - ' )

704 _+ 23 137 _+ 5

700 + 29 121 _+ 6

702 _+ i 8 130 _+ 3

COD

5.1 + 0.3

5.8 + 0.2'

5.4 + 0.1 *

For definition of abbreviations, see table I. *, P < 0.05; **, P < 0.01, *** P < 0.001 from baseline (700 Tort, room air). d, p < 0.05; 'p *, P < 0.01; * * *, P < 0.001 from hypobaric hypoxia (5 i 5 Tort, room air). c o n t e n t in both arterial a n d mixed venous blood, which exceeded those levels in baseline period (P < 0.01). T h e r e were significant decreases in H R , P p a a n d C! by 0 2 administration (P < 0.001, 0.01 a n d 0.01, respectively), whereas the m e a n P s a tended u p w a r d . T h e P r a a n d Pcw were unchanged. PVR! decreased significantly (P <'3.05) during 100% O , breathing, but did not reach the s a m e level as observed in baseline period. Similarly, SVRI u n d e r hyperoxic hypobaria was significantly greater than in baseline (P < 0.05). T h e O2 c o n s u m p t i o n returned t o w a r d the baseline level, whereas Oe delivery r e m a i n e d unchanged, resulting in a significant decrease in C O D .

Comparison of hemodynamic responses to three typos of acute hypoxia. Changes in various h e m o d y n a m i c parameters in each experiment are shown in fig. 2, expressed as percent o f baseline. As expected, increases in H R , C1 and P p a during 10% 0 2 breathing at 700 Ton" were significantly greater than t h o s e during 15 % 0 2 breathing at the sa~:e barometric pressure. Despite identical levels o f hypoxemia, the increase in Ppa during hypoxic hypobaria ( r o o m air breathing at 515 Ton") was significantly greater t h a n t h a t

86

A. KAWASHIMA et al.

[

%)

I

I

I: 15%Oxygen (700Toot) : lO%Oxygen (700Toot)

r--lr--1

+100

: Room air (515Torr) ::::::

r-it-1

I--II--I

-~o

!:i:i

!iiii I

Psa

Pra

Pew

S2

o

HR

CI

Ppa

-30 Fig. 2. Hemodynamic responses to three types of acute hypoxia (expressed as percent of baseline values). Bars denote + ISE. *, P < 0.05: **, P < 0.01; ***, P < 0.001.

APVRIIASaO= I

T

A

0,2

= 0.1 "~ ,.

T

I

,.......-..., .:.;.X.:,:. ,.,.,-..,-,.. +.,,,,..o.,,. .:':'F:qq"

o

,,,,.....,...

E E

'bb:':':':" , ....-...-.,.,,.,.,,....

!!!!!!i!i!i!i . . . . ._,_,_._,

15%

1096

515

T~T

Fig. 3. Increases in pulmonary vascular resistance index during three types of acute hypoxia divided by decrease in Sao:. 15°o, 15°,o O: breathing: !0~o, 10~ O2 breathing; 515 Ton., room air breathing at a barometric pressure of 515 Ton'; *, P < 0.05.

ATRIAL NATRIURETIC PEPTIDE IN ACUTE HYPOXIA

87

o-hANP

pg/mJ 50

40

)... PA

BA

.,5.:1:-

' 15% ' RA

• PA

,,~

PA

BA

30

20

10

o'

Expedme~ 1

Experiment 2

Expedmmt 3

Fig. 4. Changes in plasma levels of atrial natriuretic peptide. PA, pulmonary arterial blood; BA, brachial arterial blood. For definition of other abbreviations, see fig. 1.

during 15% 02 breathing at 700 Ton" (P < 0.05), while changes in HR and CI under these conditions were similar. Both Psa and Pra decreased slightly during 10% 02 breathing, but the changes of these parameters were not significantly different from those under other hypoxic conditions. Pcw changed minimallyunder all hypoxic conditions. When indexed to the change in arterial oxygen saturation (Sao,) as in fig. 3, the increase in PVRI per unit change in Sao, was more than 2 times greater under hypoxic hypobaria than under other hypoxic conditions (P < 0.05).

Plasma levels ofatrial natriuretic peptide. The results are given in fig. 4. In experiment l, the mean ANP level in plasma from the pulmonary artery (PA) elevated slightly from 27.0 + 6.2 to 28.9 + 5.8 pg/ml, whereas that from the brachial artery (BA) decreased from 26.4 + 6.4 to 22.1 + 5.0 pg/ml. These changed had no statistical significance, in experiment 2, the ANP level in PA elevated significantly from 24.3 + 5.3 to 28.2 + 4.6 pg/ml (P < 0.05), accompanying the similar elevation of ANP level in BA from 22.6 + 5.8 to 25.9 + 5.7 pg/ml (NS). In experiment 3, the ANP level both in PA and in BA showed similar tendency to elevate from 25.4 + 4.8 to 28.5 + 5.7 and from 18.1 + 3.2 to 24.3 + 4.5 pg/ml, respectively, under hypoxic hypobaria. During 100% 02 breathing under hypobaric condition, the ANP level in PA decreased from 28.5 + 5.7 to 20.7 + 6.0 pg/ml, whereas that in BA remained unchanged. No significantcorrelation was observed between plasma ANP levels and either Ppa or PVRI under any ofthe three hypoxic conditions.

88

A. KAWASHIMA et al.

Discussion In the present study, a slight but statistically significant rise in plasma ANP levels obtained from the pulmonary artery was observed under the severe hypoxic condition induced by 10% 02 breathing, under which Pao, was reduced to 35.6 + 1.4 mm Hg. Since the ANP levels returned almost to the baseline levels within a recovery period of 30 rain, this change was thought to be the result of hypoxic challenge. Some hemodynamic parameters, such as HR, Ppa, CI, or PVRI, showed a significant increase during 10% 0 2 breathing, whereas both Pra and Pew remained unchanged, suggesting that atrial distension was not responsible for the rise in ANP levels. Acute hypoxia has been reported to be a potent stimulus for ANP secretion in recent experimental studies using isolated per[used heart (Baertschi etal., 1986) or anesthetized pigs (Adnot et al., 1988a). In the latter study, hypoxic ventilation with 1i ~o 02 for 15 rain resulted in a nearly twofold increase in plasma ANP levels. Although atrial pressures were not measured, atrial distension seemed not to account for the finding in the latter study, since hypoxia has been reported to have no effect either on the Pra or on Pew (Harris and Heath, 1977; Groves etai., 1987) as observed in our study. Therefore, our results coupled with these experimental studies suggest to us that acute severe hypoxia can be a stimulus for ANP secretion in normal subjects by some mechanism not related to atrial distension. Several possibilities may exist to explain this phenomenon. Recent reports have shown a significant positive correlation between plasma ANP level and Ppa (Adnot et al., 1987); Burghuber et aL, 1988a,b). In the present study, only one subject showed a decrease in ANP level under hypoxic hypobaria, whereas ANP level decreased in half of the subjects during 15 % O: breathing. The only hemodynamic measurement that was notably different between these two hypoxic conditions was Ppa, as shown in fig. 2. Although a statistical significance was absent, the average ofplasma ANP levels showed a tendency to increase with the elevation of Ppa under hypoxic hypobaria at 515 Ton', and then to decrease with the fall of Ppa during 100% O 2 breathing under the same hypobaric condition. These findings suggest that ANP can be released in response to the elevation of Ppa, but the mechanism transmitting these pressure changes to ANP secreting myocyte in right atrium is unclear at present Other possible explanations for the rise in plasma ANP levels under severe hypoxia were as follows: First, increased HR might cause the ANP secretion, since the elevated plasma AN P level has been reported in patients with supraventricular tachycardia (Roy et al., 1987). Cardiac pacing at rate faster than sinus rhythm (130-200 beats/rain) has also been shown to cause a significant rise in plasma ANP level in coronary sinus blood (Crozier et al., 1986; Obata et al., 1987). Compared with these reports, however, the increase in HR observed in our study seems to be rather small. Second, ifthe degradation rate of ANP in the systemic circulation was reduced by hypoxia, plasma ANP levels could rise without any increase in ANP secretion. At present, however, there is no evidence of such effect of hypoxia. In our preliminary study, in which simultaneous sampling from the brachial artery and the central vein was performed in another normal

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subject, the difference in ANP level between them did not change during 10% 02 breathing, suggesting that hypoxia may not reduce the degradation rate of ANP in the systemic circulation. Compared ~,Ath the results in previous experimental studies (Baertschi et al., 1986; Adnot et ai., 1988a), the rise in plasma ANP levels observed in the present study was exceedingly small. Also, the ANP levels under the three different hypox[c conditions were similar and still remained within the normal range. Recent reports investigating the effects of synthetic ANP on pulmonary hemodynamics have shown that the infusion rate required to reduce pulmonary arterial pressure significantly was at least 0.01 pg/kg/min, which increased circulating ANP by 2 to 3-fold (Adnot et al., 1988a,b). From these findings, it seems unlikely that such a small rise in ANP levels as observed in our study could have any regulatory effect on pulmonary pressor response to acute hypoxia. However, a greater rise in ANP levels similar to those previously reported may be obtained by employing a much longer duration of hypoxia or a much lower concentration of oxygen than used in this study, but this cannot be tested. If ANP is involved in the regulatory mechanism of pulmonary circulation under hypoxia, this peptide should be removed and degraded in the pulmonary vascular bed. Therefore, if so, the plasma ANP levels in the pulmonary vein should be significantly lower than those in the pulmonary artery. In the present study no significant differences were observed between the plasma ANP levels simultaneously obtained from the pulmonary artery and from the brachial artery. If we can exclude the possibility that ANP was released from the left atrium or from the pulmonary vascular bed under hypoxic condition, our findings support those of previous workers who have suggested that ANP degradation in the pulmonary circulation has little clinical significance (Crozier et al., 1986; Matsubara et al., 1987). Another interesting finding in our study was that hypoxic hypobaria at 515 Ton. caused greater increase in Ppa than 15~/o O2 breathing at 700 Ton" despite identical amounts of calculated P'o,; that is, when indexed to the change in Sao,, hypoxic hypobaria caused more than 2 times greater increase in PVRI than identical hypoxia in almost normobaric condition. There may be two possible mechanisms for this phenomenon. First, the exposure to hypoxic hypobaria was preceded by the hypoxic gas breathing of 15% and then 10~/o O,, so that the hypoxic pulmonary vasoconstriction under hypoxic hypobaria might be potentiated by repeated intermittent hypoxia. Second, hypobaria itself might potentiate the hypoxic pulmonary vasoconstriction. It has been reported that there was a progressive rise in the Ppa with repeated exposure to the same hypoxic stimulus in anesthetized dogs ventilated alternately with air and 10% 02 (Unger etal., 1977). On the contrary, Chen et al. (1985) have shown that in normal physiologic circumstances the initial response to hypoxia was maximal and was not potentiated by repeated hypoxic stimulation. Furthermore, t~vo populations of dogs have been identified, one with a strong initial response to hypoxic challenge and the other with a weak initial response that becomes stronger with time (Miller and Hales, 1980). In any case, there has been a controversy as to whether the intermittent hypoxia potentiates the hypoxic pulmonary vasoconstriction, thus it seems difficult to attribute

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the greater increase in PVRI under hypobaric condition to the potentiating effect of intermittent hypoxia. We previously investigated the effects of barometric pressure on lung fluid balance in awake sheep. Hirai et al. observed that lung lymph flow significantly increased under normoxic hypobaria and emphasized the role of bubble formation during decompression (Hirai et aL, 1988). Levine et al. noted a smaller but significant rise in Ppa under normoxic hypobaria (Levine et al., 1988). Based on these studies, we therefore speculate that the greater increase in PVRI under hypoxic hypobaria observed in the present study may be attributed to the altered pulmonary pressor response to hypoxia caused by a reduction in barometric pressure. This speculation may be supported by the finding that the PVRI during 100% 0 2 breathing at 515 Torr was significantly higher than that in baseline period at 700 Torr. In summary, we observed that severe hypoxia induced by 10~o 0 2 breathing for 10 min caused a slight but statistically significant rise in plasma ANP level in pulmonary arterial blood. Although whether such a small change as observed in our study can play a significant role in regulation of pulmonary pressor response to acute hypoxia requires another independent study, it can be stated that acute hypoxia may be a stimulus for ANP secretion without atrial distension also in man.

Acknowledgements.We express our gratitude to Mr. Fumihiko Kurimoto (Mitsubishi-Yuka Laboratory of Medical Science, Tokyo, Japan) for the assistance in performing the radioimmunoassay used in this study. We also thank the following participating doctors for their advice and cooperation: Akio Sakai, Masao Fukushima, Keisaku Fujimoto, Hikaru Yagi, Shire Shinozaki, Tomonobu Koizumi, Shigeru Koyama, and Koji Asano. This study was supported in part by Grants for Scientific Research No. 61480194 and No. 62480203 from the Ministry of Education, Science and Culture of Japan.

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