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. 2000 Mar 1;523(Pt 2):493–502. doi: 10.1111/j.1469-7793.2000.t01-3-00493.x

Lack of a role for cyclic nucleotide gated cation channels in lung liquid absorption in fetal sheep

R W J Junor *, A R Benjamin *, D Alexandrou *, S E Guggino *, D V Walters *
PMCID: PMC2269800  PMID: 10699091

Abstract

  1. Late gestation fetal sheep were chronically catheterised in uteroto allow measurement of the rate of production of lung liquid (Jv) from 132–143 days gestation (term, 147 days), and to test the hypothesis that cyclic nucleotide gated cation channels mediate a component of fetal lung liquid absorption.

  2. In eight experiments, 0·5 μg min−1 adrenaline caused a significant (P < 0·005) reduction in Jv from +18·12 ± 3·52 to −10·27 ± 5·26 ml h−1. Dichlorobenzamil (a blocker of cyclic nucleotide gated cation channels) at 1·5 × 10−5 M did not significantly inhibit the adrenaline-induced lung liquid absorption (Jv dichlorobenzamil, −5·77 ± 2·78 ml h−1; P> 0·1) when the data were grouped, but did exert a significant gestational effect (r= 0·90, P< 0·01). Subsequent addition of 10−4 M amiloride (a blocker of epithelial sodium channels) abolished the adrenaline-induced absorption of lung liquid (mean Jv amiloride, +6·45 ± 1·59 ml h−1; P< 0·01 relative to Jv adrenaline and P< 0·005 relative to Jv dichlorobenzamil).

  3. In seven experiments, 0·5 μg min−1 adrenaline caused a significant (P < 0·0005) reduction in Jv from +18·95 ± 2·98 to −10·08 ± 3·75 ml h−1. Amiloride (10−4 M) inhibited the adrenaline response (Jv amiloride, +5·46 ± 1·09 ml h−1; P< 0·005). However, subsequent addition of 1·5 × 10−5 M dichlorobenzamil had no additive effect to that of amiloride (Jv dichlorobenzamil, +4·58 ± 0·93 ml h−1; P> 0·1).

  4. In six experiments, the cGMP analogue 8-Br-cGMP at 10−4 M caused a significant (P < 0·05) reduction in Jv from +15·20 ± 2·81 to +11·63 ± 1·71 ml h−1. Amiloride (10−4 M) did not block the effect of 8-Br-cGMP (Jv amiloride, +14·00 ± 2·49 ml h−1; not significantly different from 8-Br-cGMP). Subsequent addition of 1·5 × 10−5 M dichlorobenzamil also did not block the effect of 8-Br-cGMP (Jv dichlorobenzamil, +11·37 ± 1·22 ml h−1; not significantly different from either Jv amiloride or Jv 8-Br-cGMP).

  5. We conclude that, in fetal sheep, neither adrenaline nor cGMP stimulate lung liquid absorption by actions on cyclic nucleotide gated cation channels, and that the effect of cGMP on fetal lung liquid secretion is minor and does not involve epithelial sodium channels. The effect of dichlorobenzamil, when given before amiloride, was probably due to an action on amiloride sensitive epithelial sodium channels.

Throughout fetal life, the mammalian lung is filled with liquid (lung liquid), which is essential for lung growth and development. Lung liquid is continuously secreted by the secondary active transport of Cl− into the lung lumen, a process powered by Na+,K+-ATPase (Strang, 1991). However, it is essential that lung liquid is resorbed at birth in order to allow postnatal gas exchange. The transition from lung liquid secretion to absorption at birth is stimulated by adrenaline (Brown et al. 1983), which via β adrenoreceptor activation (Walters & Olver, 1978; Warburton et al. 1987; Chapman et al. 1991) and cAMP (Walters et al. 1990), stimulates the active transport of Na+ out of the lung lumen (Olver et al. 1986).

It is believed that the ion channel responsible for fetal lung liquid absorption is the epithelial sodium channel (ENaC; see Garty & Palmer, 1997 for a review of ENaC) since adrenaline-induced lung liquid absorption is inhibited by the ENaC blocker amiloride (Olver et al. 1986) and also because homozygous mice, which are deficient in αENaC, die within hours of birth due to a failure to reabsorb lung liquid (Hummler et al. 1996). However, it has been consistently found that when lung liquid absorption is stimulated by adrenaline or by cAMP, the rate of lung liquid secretion does not fully return to control values in the presence of amiloride (Olver et al. 1986; Walters et al. 1990). This may indicate that adrenaline also stimulates an ion channel other than ENaC.

Indeed, we have recently shown that another class of ion channels unrelated to ENaC, i.e. cyclic nucleotide gated cation channels, which are present in the adult rat pulmonary epithelium (Schwiebert et al. 1997; Ding et al. 1997), mediate a substantial component of lung liquid absorption in sheep aged 6 months postpartum (Junor et al. 1999). Cyclic nucleotide gated cation channels were first discovered in the rod photoreceptor, where they mediate the sensory signal transduction to light (Fesenko et al. 1985), and can be blocked by 10−5 M dichlorobenzamil, a derivative of amiloride (Nicol et al. 1987).

Cyclic nucleotide gated cation channels are known to be directly gated by cAMP or cGMP (Biel et al. 1995; Finn et al. 1996). Cyclic AMP (Walters et al. 1990) and cGMP (Kabbani & Cassin, 1998) also decrease the rate of fetal lung liquid secretion, although it is not clear whether or not they do so by activating cyclic nucleotide gated cation channels. Therefore, experiments were performed to test the following hypotheses. (1) Adrenaline reduces the rate of fetal lung liquid secretion by acting via cyclic nucleotide gated cation channels, in addition to its well known effects on ENaCs. (2) cGMP reduces the rate of fetal lung liquid secretion by activating cyclic nucleotide gated cation channels or ENaCs.

METHODS

Surgical procedures

Pregnant ewes were operated on at 116–121 days gestation (term, 147 days) as previously described (Walters & Olver, 1978). Anaesthesia was induced with 15–20 ml of 5 % thiopentone sodium i.v. (Intraval sodium, Rhône Mérieux, UK) and artificial ventilation was commenced via an endotracheal tube with a 2:1 O2:N2O mixture (Manley MP3 ventilator, Blease Medical Equipment, UK), anaesthesia being maintained with 1.5-2 % halothane (Fluothane, Zeneca Ltd, UK).

Under aseptic conditions, mid-line laparotomy and hysterotomy were performed, and the fetal head was delivered. Two 0.9 % saline-filled silastic catheters (inner diameter 2.64 mm, outer diameter 4.88 mm, length 100 cm; Merck Ltd, UK) were inserted into the fetal trachea, one toward the lungs and one toward the larynx, and tied in place. Silastic catheters (inner diameter 1.02 mm, outer diameter 2.16 mm, length 150 cm) prefilled with heparinised normal saline (1250 units per ml; Multiparin, CP Pharmaceuticals Ltd, UK) were inserted into the fetal external jugular vein and common carotid artery towards the heart. The incisions were closed and the catheters were tunnelled subcutaneously to the flank of the ewe where they were exteriorised via a small skin incision. The open ends of the tracheal catheters were joined via a sterile glass T-piece, the side-arm being closed off with a short sterile plugged silastic tube. Thus an extracorporeal loop was formed through which the lung liquid could flow.

The exteriorised ends of the vascular catheters were connected to syringes and were wrapped in 70 % v/v ethanol-soaked sterile gauze swabs and placed in a sterile nylon bag on the ewe's back, as was the T-piece and the tracheal catheters. Postoperative analgesia was 2.5 mg buprenorphine (Temgesic, Reckitt and Coleman, UK) i.m. The ewe and fetus were given daily 3 ml (i.m.) and 0.5 ml (i.v.) of 250 mg ml−1 dihydrostreptomycin sulphate with 250 mg ml−1 procaine penicillin (Streptopen, Pitman-Moore, Cheshire, UK), respectively, for 3 days post-operatively. Thereafter the fetal vascular catheters were flushed every alternate day.

Experimental procedures

A recovery period of 1 week was allowed before commencing experiments. Fetal heart rate and blood pressure were monitored by a transducer connected to the arterial catheter (Washington PT400 and 400 MD4R, George Washington Ltd). Fetal blood parameters were determined approximately every hour during the experiments (Ciba Corning Diagnostics Corp., USA). Intravenous drug infusions (e.g. adrenaline) were given via the venous catheter.

Under aseptic conditions, the upper tracheal catheter was clamped and a sterilised mixing vessel was connected to the T-piece via a 50 cm long sterile silastic catheter (inner diameter 2.64 mm, outer diameter 4.88 mm). To prevent airborne infection of the lung liquid, an antibacterial filter (Minisart NML SM16534, Sartorius Ltd, UK) was connected to the air inlet of the mixing vessel. Lung liquid was mixed by gravity by repeatedly lowering and raising the mixing vessel below and above the level of the fetus. Further mixing was achieved between the vessel and a 50 ml sterile syringe from which aliquots of lung liquid were sampled. 125I-human serum albumin (1–5 μCi) was added as an impermeant tracer to act as a volume marker. After no less than 30 min (to ensure adequate mixing of the volume tracer) samples of lung liquid were taken at 6–8 min intervals, and the radioactivity in each weighed sample was measured (Wallac 1480 Wizard 3′, Wallac Oy, Turku, Finland) to determine the rate of lung liquid absorption in each intervention period (see below). Each experiment began with an observation of the resting lung liquid secretion rate and a further mixing period of 20 min was allowed after the administration of any drug before further samples of lung liquid were taken.

At the end of the experiment, all lung liquid in the mixing vessel was returned to the lungs, and antibiotics (0.5 ml Streptopen made up to 5 ml with normal saline) were administered i.v. to the fetus. The vascular catheters were flushed and filled with heparinised normal saline (1250 units per ml). The side-arm of the T-piece was sealed off with a short sterile plugged silastic tube. Repeat experiments on the same fetus were performed at least 48 h after the previous experiment. At the end of each series of experiments, the ewe and fetus were killed by barbituate overdose (Euthatal, Rhône Mérieux, UK).

Drugs

Adrenaline (adrenaline hydrochloride 1:1000; 1 mg ml−1) was purchased from Antigen Pharmaceuticals Ltd, Ireland, or from Martindale Pharmaceuticals Ltd, Essex, UK and 0.21 ml adrenaline were made up to 20 ml with 0.9 % w/v sodium chloride BP (Steripak, Cheshire, UK), prior to continuous intravenous infusion at 0.5 μg min−1 (2.9 ml h−1). 8-Br-cGMP (8-bromoguanosine 3′,5′-cyclic monophosphate sodium salt; Sigma Chemical Co., UK) was dissolved in 0.9 % w/v sodium chloride BP (Steripak, Cheshire, UK) to a volume of 2–5 ml, prior to administration into the lung liquid, to a give final concentration of 10−4 M. Dichlorobenzamil (benzamil-2′,4-dichloro HCl, a gift from Research Biochemicals International, MA, USA) was dissolved in dimethyl sulfoxide to 0.005 g ml−1. This solution was dissolved in normal saline to a volume of 2–5 ml, prior to administration into the lung liquid to a final concentration of 1.5 × 10−5 M. Amiloride (amiloride hydrochloride; Sigma Chemical Co., UK) was dissolved in 2–5 ml water for injections BP (Steripak, Cheshire, UK), prior to administration into the lung liquid to a final concentration of 10−4 M.

Fetal stability

Under control conditions, the mean ±s.e.m. arterial blood parameters were: PO2, 21.1 ± 1.1; PCO2, 45.3 ± 1.0; pH, 7.392 ± 0.008. There were no significant changes in blood parameters after the administration of adrenaline, 8-Br-cGMP, amiloride or dichlorobenzamil.

Calculation of lung liquid volume and the rate of absorption or secretion

Determination of lung liquid volume at time t (Vt) was calculated as described in Walters & Olver, 1978: Vt=Vs+ (X–R)/Ct, where Vs is the cumulative sum of the volume of lung liquid samples removed between t= 0 and time t, X is the amount of tracer initially added, R is the cumulative amount of tracer removed up to time t and Ct is the concentration of tracer in the lung liquid at time t.

The slope of a plot of Vt against t, calculated by least squares, gives the rate of net transepithelial liquid movement (Jv). Results are presented as Jv means ±s.e.m. under various conditions. Positive values of Jv indicate net secretion of liquid into the lumen of the lung. Negative values of Jv indicate net absorption of liquid from the lumen of the lung.

Statistical methods

The values of Jv (i.e. the regression coefficients) during the control period and after administration of each drug were compared using Student's unpaired t test in each experiment. Data from different experiments carried out under the same conditions were grouped and Student's paired or unpaired t test was carried out as appropriate, testing the hypothesis that there was no change in Jv between different intervention periods. All statistical tests were two-tailed unless explicitly stated otherwise. Statistical significance was taken as P < 0.05. Application of Bonferroni's correction for multiple t tests to the data in Tables 1 and 2 indicates that the P values attained initially remain significant.

Table 1.

The effects of 1.5 × 10−5m dichlorobenzamil followed by 10−4m amiloride on adrenaline (0.5 μg min−1)-induced absorption of fetal lung liquid (in ml h−1)

Jv control Jv adrenaline Jv dichlorobenzamil Jv amiloride
+18.12 ± 3.52 (8) −10.27 ± 5.26 (8)* −5.77 ± 2.78 (8) +6.45 ± 1.59 (8)†
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Here and in subsequent tables, values of transepithelial liquid movement (Jv) are given as means ±s.e.m. in ml h−1 with numbers of experiments in parentheses. Experiments were performed on three fetuses of 138 ± 1 days gestation.

*

P < 0.005 relative to Jv control, paired t test

†

P < 0.005 relative to Jv dichlorobenzamil and P < 0.01 relative to Jv adrenaline and P < 0.001 relative to Jv control, paired t test. Jv dichlorobenzamil was not significantly different from Jv adrenaline, paired t test.

Table 2.

The effects of 10−4m amiloride followed by 1.5 × 10−5m dichlorobenzamil on adrenaline (0.5 μg min−1i.v.)-induced absorption of fetal lung liquid (in ml h−1)

Jv control Jv adrenaline Jv amiloride Jv dichlorobenzamil
+18.95 ± 2.98 (7) −10.08 ± 3.75 (7)* +5.46 ± 1.09 (7)† +4.58 ± 0.93 (7)
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Experiments were performed on four fetuses of 138 ± 1 days gestation.

*

P < 0.0005 relative to Jv control, paired t test.

†

P < 0.005 relative to Jv adrenaline, and P < 0.005 relative to Jv control, paired t test. Jv dichlorobenzamil was not significantly different from Jv amiloride, paired t test.

RESULTS

The effect of dichlorobenzamil followed by amiloride on adrenaline-induced lung liquid absorption

In n= 8 experiments, performed on three fetuses at 138 ± 1 days gestation (range, 134–143 days), 0.5 μg min−1 adrenaline caused a significant reduction in Jv compared with the control period (P < 0.005, paired t test). In the group as a whole, 1.5 × 10−5 M dichlorobenzamil did not significantly inhibit the adrenaline-induced absorption of lung liquid (paired t test) but subsequent administration of 10−4 M amiloride abolished the adrenaline-induced absorption of lung liquid. Jv amiloride was significantly different from Jv adrenaline (P < 0.01, paired t test) and Jv dichlorobenzamil (P < 0.005, paired t test). Jv amiloride was also significantly less than Jv control (P < 0.001, paired t test) (see Table 1).

Although the effect of dichlorobenzamil relative to adrenaline was not significant when grouping the data, dichlorobenzamil did have a significant effect in older (see Fig. 1), but not in younger, fetuses (no significant effect achieved from 134–138 days gestation, n= 5, significance achieved from 140–143 days gestation, n= 3, P < 0.001; comparison of regression lines by unpaired t tests). Furthermore, the magnitude of the effect of dichlorobenzamil increased with gestational age: a plot of the difference between Jv adrenaline and Jv dichlorobenzamil in each experiment against gestational age, yields a significant relationship (n= 8, r= 0.90, P < 0.01) (Fig. 2). The relationship is also significant when data from one animal are analysed in this way (n= 5, r= 0.97, P < 0.01).

Figure 1. Effect of dichlorobenzamil followed by amiloride in the presence of adrenaline.

Figure 1

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Example experiment, performed on a fetus at 143 days gestation, showing the effects of dichlorobenzamil (1.5 × 10−5 M) followed by amiloride (10−4 M) on adrenaline-induced (0.5 μg min−1i.v.) absorption of lung liquid. Here and in subsequent figures, Jv values for each observation period, in ml h−1, are given above the regression line for each period. During the control period, secretion of lung liquid was occurring (i.e. lung liquid volume was increasing with respect to time). Adrenaline caused reabsorption of lung liquid. Dichlorobenzamil partially blocked the effect of adrenaline and amiloride had an additive effect to that of dichlorobenzamil. *P < 0.001 relative to Jv control, **P < 0.001 relative to Jv adrenaline, ***P < 0.001 relative to Jv dichlorobenzamil.

Figure 2. The gestational effect of 1.5 × 10−5 M dichlorobenzamil alone (i.e. in the absence of amiloride) on adrenaline-induced (0.5 μg min−1i.v.) absorption of fetal lung liquid.

Figure 2

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Ordinate, change in Jv due to dichlorobenzamil, i.e. Jv dichlorobenzamil - Jv adrenaline for each experiment. r= 0.90, n= 8, *P < 0.01.

The effect of amiloride followed by dichlorobenzamil on adrenaline-induced lung liquid absorption

In seven experiments, performed on four fetuses at 138 ± 1 days gestation (range, 135–143 days gestation), 0.5 μg min−1 adrenaline caused a significant (P < 0.0005, paired t test) reduction in Jv compared with the control period. Amiloride (10−4 M) inhibited the adrenaline effect (P < 0.005, paired t test). Jv amiloride was also significantly less than Jv control (P < 0.005, paired t test). However, subsequent administration of 1.5 × 10−5 M dichlorobenzamil had no significant additive effect relative to that of amiloride (paired t test). Furthermore, dichlorobenzamil did not exert an additive effect to that of amiloride in any single experiment (comparison of regression lines by unpaired t test) (Table 2 and Fig. 3).

Figure 3. Effect of amiloride followed by dichlorobenzamil in the presence of adrenaline.

Figure 3

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Example experiment performed on a fetus at 135 days gestation, showing the effects of 10−4 M amiloride followed by 1.5 × 10−5 M dichlorobenzamil on adrenaline (0.5 μg min−1i.v.)-induced absorption of lung liquid. During the control period, secretion of lung liquid was taking place. Adrenaline caused reabsorption of lung liquid. Amiloride blocked the effect of adrenaline, but dichlorobenzamil had no additive effect to that of amiloride. *P < 0.001 relative to Jv control, **P < 0.001 relative to Jv adrenaline. Jv dichlorobenzamil was not significantly different from Jv amiloride.

Fetal 8-Br-cGMP experiments

In four experiments performed on three fetuses at 137 ± 2 days gestation (range, 132–140 days), 10−4 M 8-Br-cGMP caused a significant (P < 0.05, paired t test) reduction in the rate of lung liquid secretion (Table 3 and Fig. 4). This effect persisted for at least 4 h. Statistical analysis of the pooled data showed no change in effect over this time period. Therefore we were able to proceed to test whether this effect could be blocked by interventions (i.e. amiloride and dichlorobenzamil) within this time period. Six experiments were performed on three fetuses at 136 ± 2 days gestation (range, 131–142 days). 8-Br-cGMP (10−4 M) again caused a significant reduction in Jv (P < 0.05, paired t test). Analysis of the group data showed that 10−4 M amiloride did not block the effect of 8-Br-cGMP (Jv amiloride not significantly different from Jv 8-Br-cGMP, paired t test). Furthermore, detailed analysis within each experiment showed that amiloride did not inhibit the effect of 8-Br-cGMP in five of the six experiments (comparison of regression lines by unpaired t test), although it did exert a block in one (P < 0.001). Subsequent administration of 1.5 × 10−5 M dichlorobenzamil was also unable to block the effect of 8-Br-cGMP (Jv dichlorobenzamil not significantly different from either Jv amiloride or Jv 8-Br-cGMP, paired t test). Furthermore, dichlorobenzamil never exerted an additive effect to that of amiloride in any single experiment (comparison of regression lines by unpaired t test) (Table 4 and Fig. 5). By pooling the data from all ten 8-Br-cGMP experiments, we investigated the possibility of a gestational effect of 8-Br-cGMP, but could find no evidence for such an effect (Fig. 6).

Table 3.

The effect of 10−4m 8-Br-cGMP on the rate of fetal lung liquid secretion (in ml h−1), over 3–4 h

Jv control Jv 8-Br-cGMP
+17.41 ± 2.58 (4) +10.85 ± 1.96 (4)*
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Experiments were carried out on three fetuses of 137 ± 2 days gestation.

*

P < 0.05 relative to Jv control, paired t test.

Figure 4. Effect of 8-Br-cGMP on lung liquid secretion.

Figure 4

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Example experiment performed on a fetus at 136 days gestation, showing the effect of 10−4 M 8-Br-cGMP on the resting rate of lung liquid secretion. After a control period of observation, 8-Br-cGMP was added into the lung liquid, and its effect observed for 3 h. *Jv control significantly different from Jv 8-Br-cGMP, P < 0.001 comparison of regression lines, unpaired t test).

Table 4.

Results from experiments in which the effect of 10−4m 8-Br-cGMP on the rate of lung liquid secretion (in ml h−1) was tested for its susceptibility to block by amiloride (10−4 m) and dichlorobenzamil (1.5 × 10−5m)

Jv control Jv 8-Br-cGMP Jv amiloride Jv dichlorobenzamil
+15.20 ± 2.81(6) +11.63 ± 1.71 (6)* +14.00 ± 2.49 (6) +11.37 ± 1.22 (6)
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*

P < 0.05 relative to Jv control, paired t test. Jv amiloride and Jv dichlorobenzamil were not significantly different from each other or from Jv 8-Br-cGMP, paired t test.

Figure 5. Effect of amiloride followed by dichlorobenzamil in the presence of 8-Br-cGMP.

Figure 5

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Example experiment performed at 133 days gestation showing the effect of 10−4 M 8-Br-cGMP, followed by 10−4 M amiloride and then 1.5 × 10−5 M dichlorobenzamil on the rate of lung liquid secretion. *8-Br-cGMP significantly reduced the resting rate of secretion (P < 0.01, comparison of regression lines by t test). Dichlorobenzamil had no significant effect relative to amiloride (P > 0.1), and amiloride had no significant effect relative to 8-Br-cGMP (P > 0.05).

Figure 6. The lack of a gestational effect of 8-Br-cGMP.

Figure 6

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Data plotted as the reduction in the rate of lung liquid secretion due to 10−4 M 8-Br-cGMP versus gestational age for each experiment. The effect of 8-Br-cGMP was calculated as Jv control –Jv 8-Br-cGMP. No gestational dependency was found (n= 10, r= 0.07, P > 0.1).

DISCUSSION

In the lungs of sheep aged 6 months, we have previously shown that 1.5 × 10−5 M dichlorobenzamil has an additive effect to that of 10−4 M amiloride (Junor et al. 1999), indicating that cyclic nucleotide gated cation channels are responsible for a component of lung liquid absorption. In the experiments presented in this paper, dichlorobenzamil and amiloride were given at the same concentrations used previously, which would have allowed detection of cyclic nucleotide gated cation channel activity, if it were present, in the fetal lung.

However, in the presence of either adrenaline or 8-Br-cGMP, dichlorobenzamil had no additive effect to that of amiloride. Since all the cyclic nucleotide gated cation channels so far reported are gated either by cAMP or by cGMP (Biel et al. 1995; Finn et al. 1996), it may be concluded that cyclic nucleotide gated cation channels are not involved in fetal lung liquid absorption. Thus it is evident that the epithelial mechanisms for lung liquid absorption change during postnatal development, as first proposed by Ramsden et al. (1992).

As amiloride and dichlorobenzamil are chemically similar, the possibility of some overlap in their actions (i.e. that dichlorobenzamil could affect ENaCs, and that amiloride could affect cyclic nucleotide gated cation channels) could not be ignored. However, it has been reported that the KI of amiloride for the olfactory-type cyclic nucleotide gated cation channels is > 2 × 10−4 M and that amiloride is only effective when applied to the intracellular side of the membrane (Frings et al. 1992). It is therefore unlikely, in the experiments reported here, that amiloride would have had an appreciable effect on cyclic nucleotide gated cation channels, as it was administered extracellularly at 1 × 10−4 M. In the case of dichlorobenzamil, we were unable to find data concerning its effect on ENaCs, and therefore the possibility of imperfect selectivity of dichlorobenzamil led us to administer it before and after amiloride.

When dichlorobenzamil was given before amiloride in the presence of adrenaline, it partially blocked adrenaline-induced absorption of lung liquid in a gestationally dependent manner; something which it did not do when given after amiloride. Since dichlorobenzamil exerted no additive effect to that of amiloride, we conclude that cyclic nucleotide gated cation channels are not functionally present. Therefore, the effect of dichlorobenzamil (when given before amiloride) on adrenaline-induced absorption of fetal lung liquid, must be due to its actions on ENaCs (i.e. it has amiloride-like effects), and not due to actions on cyclic nucleotide gated cation channels. This also explains the gestational effect of dichlorobenzamil, which is very similar to that of amiloride as described in Olver et al. (1986). Indeed, a simple calculation (using the values in Table 1) reveals that 1.5 × 10−5 M dichlorobenzamil causes 37 % inhibition of adrenaline-induced absorption of fetal lung liquid, relative to 10−4 M amiloride, which is roughly equivalent to giving 10−6 M amiloride (see dose-response curve in Olver et al. 1986).

From the data in Tables 1 and 2 it can be seen that, even after adrenaline-induced lung liquid absorption was blocked by amiloride, the rate of secretion did not return fully to control values. This phenomenon was also reported by Olver et al. (1986) and by Walters et al. (1990). A suitable explanation for this finding is not provided by the data presented here, but it is possible that it is due to a partial inhibition of Cl− channels by adrenaline/cAMP, perhaps involving apical membrane G proteins. This speculation is based on the observations that the active transport of Cl− diminished during adrenaline infusions (Olver et al. 1986), and that apical membrane Cl− channels can be inhibited by G proteins (Kemp et al. 1993).

cGMP results in the context of other work in this field

Kabbani & Cassin (1998) studied the effects of 1 h long i.v. infusions of 300–500 μg min−1 of 8-Br-cGMP into the left pulmonary artery of late gestation chronically catheterised fetal sheep. 8-Br-cGMP had no effect on the rate of secretion during the infusion (hour 1). However, in the hour following the infusion (hour 2), the rate of secretion decreased 70 % compared with control. During hour 3, the effect had diminished and the rate of secretion was only 44 % less than the control value. In contrast, we found that 8-Br-cGMP only led to a 30 % reduction in the rate of secretion (combining results from Tables 3 and 4) and furthermore, the effect was immediate and persisted for up to 4 h (the maximum length of observation). It is likely that these differences are explained by the different experimental protocols used, i.e. in the doses and routes of administration of 8-Br-cGMP.

Other reports of the effects of cGMP on fetal lung liquid secretion are not currently available as far as we are aware, although some hormones known to act via cGMP (e.g. acetylcholine, nitric oxide and atrial natriuretic factor) have also been shown to decrease the rate of fetal lung liquid secretion (Castro et al. 1989; Cummings, 1995; Woods et al. 1996; Cummings, 1997).

Possible explanations for the effects of 8-Br-cGMP on fetal lung liquid secretion

It is unlikely that the effect of 8-Br-cGMP on lung liquid secretion was due to stimulation of Na+ absorption since it was not blockable by amiloride or dichlorobenzamil. This conclusion is strengthened by Cummings (1997) who found that nitric oxide, when delivered directly into the lung liquid, caused a 50 % reduction in the rate of secretion, even in the presence of amiloride. Furthermore, there is a wealth of literature showing that cGMP inhibits amiloride-sensitive Na+ absorption in various epithelial cells (Cantiello & Ausiello, 1986; Light et al. 1990; Chevalier et al. 1996; Kelley et al. 1998).

A reduction in the rate of lung liquid secretion by 8-Br-cGMP could also have been due to an attenuation of Cl− secretion. However, the characteristic effect of cGMP is to stimulate Cl− secretion in numerous epithelial cells (Field et al. 1978; Forte et al. 1993; Darvish et al. 1995; Taylor & Baird, 1995; Kamosinska et al. 1997; Schwiebert et al. 1997; Rolfe et al. 1997).

Since cGMP is the intracellular mediator for NO (endothelium-derived relaxing factor) it is possible that cGMP was exerting its actions by haemodynamic effects. Kabbabi & Cassin (1998) thought this unlikely because although 8-Br-cGMP increased pulmonary blood flow, this was not accompanied by a simultaneous decrease in the rate of lung liquid secretion – the two effects were temporally dissociated by 1 h. Other reports also indicate that pulmonary blood flow and lung liquid secretion are unrelated (Cassin et al. 1964; Walters & Olver, 1978; Iwamoto et al. 1979; Perks et al. 1990, 1991, 1993; Wallace et al. 1990; Cummings et al. 1995; Woods et al. 1997).

It is possible that 8-Br-cGMP reduced lung liquid secretion by inhibiting Na+,K+-ATPase. Guo et al. (1998) found that 8-Br-cGMP had no effect on Na+,K+-ATPase in cultured alveolar type II cells. However, in mouse proximal tubule cells nitric oxide inhibits Na+,K+-ATPase, via cGMP formation (Guzman et al. 1995). In rat kidney, atrial natriuretic peptide, acting via cGMP, inhibits Na+,K+-ATPase (Scavone et al. 1995). It has also been reported that cGMP inhibits Na+,K+-ATPase in brain endothelial cells (Pontiggia et al. 1998). Therefore, this mechanism seems the most likely explanation for the reduction in the rate of fetal lung liquid secretion observed in the presence of 8-Br-cGMP.

An upregulation of the cGMP pathway in the lung epithelium at birth has been described (Kawai et al. 1995; Bloch et al. 1997; Sanchez et al. 1998), and at first sight it would appear that as cGMP reduces the rate of lung liquid secretion, it has a physiological role in clearing the lungs of liquid at birth. However, since the driving force for both lung liquid secretion and absorption is provided by Na+,K+-ATPase (Strang, 1991), the actual effect of cGMP would be to inhibit lung liquid absorption (assuming the mechanism proposed above is correct). This is contrary to what is required at birth (a time at which Na+,K+-ATPase in the lung is upregulated; Bland & Boyd, 1986), and would suggest that the effect of cGMP on lung liquid secretion in fetal life may not be physiological. The upregulation of the cGMP pathway at birth may therefore be related to other pulmonary epithelial functions.

Conclusions

The effect of adrenaline on fetal lung liquid secretion does not involve cyclic nucleotide gated cation channels. Although cGMP reduces the rate of lung liquid secretion, its effect is minor and does not involve cyclic nucleotide gated cation channels or ENaC. Thus, cyclic nucleotide gated cation channels are not involved in lung liquid absorption in fetuses, in contrast to their previously reported role in sheep aged 6 months.

Acknowledgments

We are grateful to the Wellcome Trust for supporting this work.

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