G protein-coupled prostaglandin receptor modulates conductive Na+ uptake in lung apical membrane vesicles

Somnath Mukhopadhyay, Asim K. Dutta-Roy, Gregor K. Fyfe, Richard E. Olver, and Paul J. Kemp

Lung Membrane Transport Group, Department of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY; and Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, United Kingdom

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Because G protein-regulated cation channels in type II pneumocytes constitute the most likely pathway for alveolar Na+ entry, we explored the hypothesis that a G protein-coupled prostaglandin (PG) E2 receptor controls perinatal lung alveolar Na+ transport. [3H]PGE2 binding to the alveolar apical membrane was trypsin sensitive and showed a rank order of competitive inhibition: PGE2 = PGE1 > PGD2 > PGF2alpha . Kinetic analysis demonstrated both high-affinity [dissociation constant (KD) = 2.1 ± 0.7 nM; maximal binding (Bmax) = 27 ± 7 fmol/mg protein] and low-affinity (KD = 28 ± 2 nM; Bmax = 265 ± 29 fmol/mg protein) binding sites. Modulation of high-affinity GTPase activity identified a similar potency order (IC50 = 11 mM for PGF2alpha vs. 10-50 µM for other PGs), suggesting that the receptors are G protein coupled. Finally, 1 µM PGE2 (approx IC25) increased conductive 22Na+ uptake into membrane vesicles only in the presence of 100 µM intravesicular GTP. The KD value for the high-affinity binding site together with the rank order of PG effect on ligand binding and G protein function places this PG receptor in the EP3 subtype, whereas Na+ uptake studies suggest that it helps maintain perinatal lung Na+ homeostasis.

sodium channel; prostaglandin E2 ; sodium ion

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

EFFICIENT LUNG ALVEOLAR Na+-channel function is crucial to survival. Epithelial Na+-channel "knockout" mice die of severe respiratory distress within 48 h of birth, with autopsy evidence of massive lung fluid overload (12). In humans, life-threatening neonatal respiratory distress commonly associates with lung alveolar and interstitial edema (2), implicating an underlying dysfunction of extra- or intracellular mechanisms regulating lung alveolar Na+ transport around the time of birth. After the characterization of the hormonal control (1) of this amiloride-inhibitable Na+-absorptive response (24), it is now imperative to understand the cellular mechanisms that regulate this absorptive process in order to work toward the development of therapeutic strategies for the treatment of lung alveolar fluid overload in the neonate.

Working toward this objective, we have recently identified low-conductance, amiloride-sensitive, Na+-selective channels in the freshly isolated mature end-gestation (term) fetal type II pneumocyte, which we propose may facilitate this Na+-driven fluid-absorptive role (8, 18). These channels are upregulated by polyunsaturated fatty acids and by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S; see Ref. 8a), implicating control via a G protein-linked pathway.

Although most available information supports the hypothesis that prostaglandins (PGs) play an important role in the regulation of ion transport in the first-trimester fetal lung by stimulating fluid secretion (4, 15, 19, 32), their effect on the fetal lung in late gestation has not been studied. However, evidence from the renal collecting tubule (an Na+-absorbing epithelium) would suggest that PGE2 is a likely candidate for control of alveolar fluid absorption. Using perfused, microdissected rabbit renal collecting tubule and adjacent thick ascending limb (containing cation channels electrophysiologically similar to perinatal mammalian lung; see Ref. 17), Grantham and Orloff (10) demonstrated a dual role for PGE2 in regulating Na+-linked water absorption in the collecting tubule and thick ascending limb: at low concentrations (10-9 M), PGE2 inhibited, whereas at relatively higher concentrations (>= 10-7 M), it stimulated the arginine vasopressin-mediated water-absorptive response. Higher concentrations of PGE2 (>= 10-7 M) also caused stimulation of adenylyl cyclase activity in both collecting tubule and thick limb cells (28). Collating this and related information, Smith (26) developed a unifying biochemical paradigm for the role of PGE2 in renal fluid transport.

Although PG receptors have been identified in the airway (6, 13), their existence in lung alveoli (with different functional priorities such as surfactant secretion and Na+ absorption) has not been reported. We adopted the following stepwise strategy to test our hypothesis: 1) identification of putative PGE2-binding sites in the term fetal lung alveolar type II pneumocyte apical membrane by radioligand binding; 2) exploration of the PG effect on G protein function in this membrane (because PGs act via G proteins in other tissues; see Refs. 16 and 26); and, finally, 3) study of possible PGE2-mediated regulation of Na+ transport in the same preparation. High-affinity GTPase activity was measured as a marker of G protein function; this method of measurement and its usefulness in assessing tissue G protein turnover have been established already (20). The measurement of the rate of Na+ uptake into vesicles of type II pneumocyte apical membrane has been validated by our group as a marker of conductive Na+ transport (8); this method was utilized to study the possible effect of PGE2 on Na+ channels in the type II pneumocyte apical membrane.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

[gamma -32P]GTP (specific activity 10 Ci/mmol; Amersham, Bucks, UK), [3H]PGE2 (specific activity 171 Ci/mmol; Du Pont de Nemours), and 22Na (specific activity 15 Ci/mmol; Amersham) were the radioactive stocks purchased. Unlabeled PGE1, PGE2, PGD2, and PGF2alpha were purchased from Cascade (Reading, Berkshire, UK). All other reagents were of Sigma (Poole, Dorset, UK) analytical grade.

Methods

Isolation of type II pneumocyte apical membrane. Apical membrane was prepared from late-gestation (<3 days preterm) fetal guinea pig lung as previously described (8). Briefly, lung lobes were excised at 2nd/3rd generation bronchi, placed in ice-cold homogenization medium [250 mM sucrose and 10 mM triethanolamine (TRA), pH 7.6 with HCl, containing 200 µg deoxyribonuclease I, 2.6 mg aprotinin A, 1.0 mg leupeptin, 1.0 mg pepstatin A, and 17.5 mg 4-(2-aminoethyl)benzenesulfonyl fluoride per 100 ml of medium], and homogenized in a Waring blender followed by a Polytron emulsifier (5 min; 4°C for both). After this, differential centrifugation and Mg2+ precipitation (in 75 mM NaCl, 50 mM MgCl2, and 10 mM HEPES, pH 7.4 with NaOH) were employed to yield a membrane fraction that was enriched with the apical membrane marker alkaline phosphatase (approx 19-fold). This fraction was subsequently resuspended in transport buffer (250 mM mannitol and 10 mM TRA, pH 7.6 with HCl) and was stored at -70°C. All enzyme assays (for various contaminant membranes) were carried out as previously described. Markers for endoplasmic reticulum, mitochondria, and lysosomal membranes were not significantly enriched. Protein was estimated by the method of Bradford (3), with bovine serum albumin as the standard.

Identification of putative PGE2-binding sites. A [3H]PGE2 binding assay was performed to identify putative PGE2 receptors in the above preparation. The conditions of the assay have been described earlier (7). Briefly, membrane (50 µg protein) was incubated at 23°C with the indicated amounts of [3H]PGE2 (earlier experiments had established that optimum radiolabeled PG binding was achieved at room temperature). Incubations were performed in triplicate in buffer solution (250 mM mannitol, 10 mM TRA, and 2 mM MgCl2, pH 7.6 with HCl) in a total volume of 200 µl in the presence of 10 nM [3H]PGE2. Because <1% of [3H]PGE2 bound to the membrane, the concentration of free ligand remained essentially constant throughout the experimental period. Parallel experiments were run using a 1,000-fold excess of unlabeled PGE2 (10 µM) in the above incubation mixture to determine the nonspecific binding; this value was subtracted from the total binding to calculate the specific binding. At the end of the incubation, the mixture was vacuum filtered through glass microfilters (Whatman GF/C) that had been presoaked in assay buffer. Each assay tube and filter was washed with 10 ml of the buffer at 4°C. The filters were then dried and suspended in scintillation fluid for measurement of radioactivity. In initial experiments, the time course of association in binding was studied by incubating membrane (50 µg protein) with [3H]PGE2 (10 nM) in the absence (total binding) and in the presence (nonspecific binding) of 10 µM unlabeled PGE2. Subsequently, [3H]PGE2 was incubated with membranes at 23°C for 30 min. The maximum binding capacity (Bmax) and dissociation constant (KD) were obtained by an iterative fitting protocol (method of least spaces) using one- and two-site models (FigP; Biosoft, Cambridge, UK). Finally, the membrane was incubated with trypsin (5 µg or 53.5 Nalpha -benzoyl-L-arginine ethyl ester hydrolysing units/50 µg membrane in 200 µl buffer) as above. After the incubation, membranes were washed, and [3H]PGE2 binding activity was determined.

Determination of PG effect on G protein function. This followed an established procedure (20) that we have recently adpated for our preparation (21). The final concentrations (pH 7.5) in the GTPase assay reaction milieu were as follows: 1 mM 5'-adenylylimidodiphosphate, 1 mM ATP, 1 mM ouabain, 10 mM creatine phosphate, 2.5 U/ml creatine phosphokinase, 100 mM NaCl, 5 mM MgCl2, 2 mM dithiothreitol, 100 µM EDTA, 10 mM TRA, and 0.5 µM GTP with [gamma -32P]GTP (because the radioactive GTP needed to be added in trace amounts, it was necessary to make up the required concentration with unlabeled GTP). The final protein concentration was 0.1 µg/µl; the reaction was terminated at 20 min by the addition of 900 µl/tube of 5% (wt /vol) activated charcoal slurry in 20 mM phosphoric acid (pH 2.3). This protein concentration and time point were chosen on the basis of earlier experiments (21). Tubes were then centrifuged at 12,000 g for 20 min to pellet the charcoal together with the unhydrolyzed [gamma -32P]GTP and the now unlabeled GDP, whereas free 32Pi remained in the supernatant. Radioactivity in the supernatant was assayed by liquid scintillation counting. Low-affinity hydrolysis of [gamma -32P]GTP (not mediated by G proteins) was assessed by parallel incubation of membrane with excess (1 mM) GTP. The low-affinity activity was subtracted from the total to calculate high-affinity GTPase activity.

Determination of PG effect on ouabain-insensitive ATPase activity. ATPase activity in similarly prepared guinea pig lung apical membrane is mostly ouabain insensitive (14). Ouabain-insensitive ATPase activity was measured by Pi production at 37°C. Briefly, the assay was initiated by the addition of membrane protein (4 µg/ml) and terminated at 90 min (earlier experiments confirmed linearity at this concentration and time point) by the addition of 30% trichloroacetic acid and 1% bovine serum albumin. The reaction solution was held on ice for 20 min and centrifuged at 1,500 g for 10 min at 4°C, and the supernatant was assayed for Pi using the molybdate blue method (11). PG was added as appropriate just before the start of the reaction.

PGE2-mediated regulation of conductive Na+ transport. Apical membrane was revesiculated in the presence of G protein substrate (100 µM GTP or GTPgamma S), and after the imposition of an outwardly directed Na+ gradient, 1 µM PGE2 was added to extravesicular solution at the initiation of the transport assay as indicated by the conditions of the experiment. The detailed Na+ uptake experiment protocol has been extensively described by our group (8). Briefly, passing NaCl-loaded vesicles (125 mM) through 1-ml Dowex 50X8-100 columns enabled an outwardly directed Na+ gradient to be imposed across the vesicular membrane by resuspension in a low-concentration NaCl solution (125 µM). Such a gradient results in a negative intravesicular diffusion potential being established in those vesicles containing conductive Na+ pathways (channels), which in turn drives tracer 22Na+ uptake. Intra- and extravesicular 22Na+ were separated from each other by a further cation exchange step. Uptake was examined as an initial rate (at 2 min). Intravesicular 22Na+ content (uptake) was calculated as picomoles of 22Na+ per milligram of protein per minute. Data are presented as the ratio of uptake in the "experimental" group (e.g., in the presence of PG) to the uptake in the appropriate (vehicle) control.

Statistics and data presentation. Data are presented as means ± SE (when the number of experiments totaled three or more). Iterative fitting was performed on the entire data set, but, in some figures, only the means were plotted for clarity. Where relevant, differences between sets of experiments were studied by the Mann-Whitney test (2 tailed), with significance accepted at P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Identification of Putative PGE2-Binding Sites

Binding of 10 nM [3H]PGE2 was time dependent and saturable, with equilibrium being attained within 30 min of incubation, which was the time point chosen for all subsequent equilibrium binding experiments (data not shown). Addition of a large excess of unlabeled PGE2 (1,000-fold) to the reaction mixture at the beginning of the incubation reduced the binding of [3H]PGE2 to the membrane to ~40% of the total (see Fig. 2A), indicating the competitive nature of [3H]PGE2 binding. The binding isotherm shown in Fig. 1A is well fitted by a two-site model with affinities (KD) of 2.11 ± 0.70 and 27.83 ± 1.99 nM and Bmax of 27.46 ± 6.89 and 265.44 ± 29.13 fmol/mg protein. Separate theoretical isotherms using these calculated parameters are also shown for information. Transformation of the data to yield a Scatchard plot (Fig. 1B) clearly shows the presence of two binding sites. Incubation of the lung apical membrane with trypsin (53.5 U/50 µg protein in 200 µl buffer) completely abolished specific [3H]PGE2 binding (data not shown). The specificity of [3H]PGE2 binding was investigated by adding increasing concentrations (0.001-2.5 µM) of unlabeled PGs E1, E2, D2, and F2alpha to the reaction mixture. Fitting the dose-response data using the Hill equation (which was plotted on a linear concentration axis; Fig. 2, A-D) showed that PGE2 and PGE1 had similar affinites (Fig. 2, A and B) for the receptor (IC50 for PGE2 self-inhibition = 47.4 ± 19.0 nM; IC50 for PGE1 inhibition of PGE2 binding = 35.4 ± 6.0 nM). However, PGD2, although able to inhibit PGE2 binding (Fig. 2C), did so with reduced affinity (IC50 = 412 ± 143 nM). PGF2alpha clearly produced minimal inhibition (Fig. 2D), and IC50 values could not be reliably calculated from the data.


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Fig. 1.   Binding isotherm (A) and corresponding Scatchard plot (B) of [3H]prostaglandin (PG) E2 binding to type II pneumocyte apical membrane. Membrane (50 µg protein) was incubated with varying concentrations of [3H]PGE2 (1-10 nM) for 30 min at 23°C. Binding was calculated from the specific activity (dpm/pmol) of the radioligand at known concentrations of [3H]PGE2. An iterative fitting protocol (method of least spaces) using 1- and 2-site models (FigP; Biosoft) was utilized. The data fit well with a 2-site model, which predicts a high-affinity binding site with a dissociation constant (KD) of 2.11 ± 0.70 nM and maximum binding capacity (Bmax) of 27.46 ± 6.89 fmol/mg protein and a low-affinity binding site with a KD value of 27.83 ± 1.99 nM and Bmax of 265.44 ± 29.13 fmol/mg protein. Separate theoretical isotherms using these parameters are also shown in A (dotted lines).


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Fig. 2.   Specificity of [3H]PGE2 binding to type II pneumocyte apical membrane. Membrane (50 µg protein) was incubated with 10 nM [3H]PGE2 together with varying concentrations of PGE2 (A), PGE1 (B), PGD2 (C), and PGF2alpha (D) for 30 min at 23°C. [3H]PGE2 binding was determined as described in Methods. Results are expressed as a percentage of total [3H]PGE2 bound (n >=  3). Iterative fitting protocol (method of least spaces) with 1- and 2-site models (FigP; Biosoft) was utilized.

PG Effect on G Protein Function

The dose responses of PGE1, PGE2, PGD2, and PGF2alpha on high-affinity Galpha -GTPase activity was studied between 1 nM and 1 mM (Fig. 3). With the use of the Hill equation, calculated IC50 values for inhibition of GTPase activity by PGE2 (29.3 ± 12.2 µM), PGE1 (24.9 ± 14 µM), and PGD2 (40.0 ± 17.2 µM) are more than two orders of magnitude larger than that of PGF2alpha (11.3 ± 9.2 mM). The IC50 values for PGE2 and PGE1 are similar, whereas the IC50 for PGD2 is greater than that for either PGE2 or PGE1 (although the difference did not achieve significance). The IC50 for PGF2alpha is greatly different (P < 0.05) from all of the other PGs. This rank order of potency for inhibition of Galpha -GTPase activity is in agreement with that obtained in the binding studies. The Hill coefficient of all the curves was significantly less than one (manifest as shallow log dose-response curves). This is compatible with two populations of G protein-linked receptors, each exhibiting an affinity about one order of magnitude different from the other.


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Fig. 3.   Concentration-dependent inhibition of high-affinity GTPase activity by PGE2 (A), PGE1 (B), PGD2 (C), and PGF2alpha (D). High-affinity GTPase activity was assessed as described in text. PGs were added to the reaction mixture 1 min before the start of the reaction. The reaction was activated by the addition of lung membrane preparation. All 4 PGs tested inhibited high-affinity GTPase activity in a dose-dependent manner. The IC50, calculated from the Hill equation, for GTPase activity modulation by PGs E1 and E2 is lower than that for PGD2 but is >500-fold below the IC50 for PGF2alpha (n >=  3), suggesting a relation between binding site (Fig. 2) and G protein modulation specificity. Iterative fitting protocol (method of least spaces) with 1- and 2-site models (FigP; Biosoft) was utilized.

PGE2 Effect on Lung Membrane is G Protein Specific

Fatty acids at micromolar concentrations can alter the physical characteristics of membrane to exert a possibly nonspecific effect on membrane enzymes (30). Because PGE2 belongs to the oxygenated C20 fatty acid family, the extent of this effect was assessed by comparing possible PGE2 inhibition of ouabain-insensitive ATPases with inhibition of high-affinity GTPase activity produced by PGE2. PGE2 (1 µM) attenuated the GTPase activity by 32.0 ± 4.0% (n = 3) and the ouabain-insensitive ATPase activity by only 2.1 ± 0.2% (n = 3), indicating that the effect of PGs was specific to G protein GTPase activity.

PGE2-Mediated Regulation of Na+ Transport

Figure 4 shows that extravesicular PGE2 (1 µM) or intravesicular GTP (100 µM) in isolation produced no significant change in the initial rate of 22Na+ uptake (n = 7). In combination, however, these concentrations of PGE2 and GTP significantly increased the initial rate of 22Na+ uptake 1.38 ± 0.09-fold above control (P < 0.05; n = 7), indicating that PGE2 stimulation of apical Na+ channels is G protein dependent. The differences between the effect of PGE2 and GTP in combination and that of PGE2 or GTP alone were also significant (P < 0.05; n = 7). There was no significant difference between the increase in the initial rate of 22Na+ uptake achieved with intravesicular GTPgamma S (100 µM) and that achieved with PGE2 and GTP in combination (n = 7), suggesting that PGE2 could maximally activate the G protein-dependent component of Na+ transport through conductive pathways.


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Fig. 4.   PGE2 stimulates the initial rate of conductive 22Na+ uptake into type II pneumocyte apical membrane vesicles. Apical membrane was vesiculated in the presence of Mg2+ (1 mM). Results are presented as proportional increase in the initial rate of 22Na+ uptake compared with control (means + SE; n = 7). As observed earlier (see text), intravesicular guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S; 100 µM) consistently stimulated conductive 22Na+ uptake by causing irreversible G protein activation (P < 0.05). Intravesicular GTP (100 µM) and/or extravesicular PGE2 (1 µM) was added as indicated. GTP and PGE2 alone had no significant effect on 22Na+ uptake. In combination, they significantly increased 22Na+ uptake. A significant difference is observed between the effects of 1) PGE2 and GTP + PGE2 and 2) GTP and GTP + PGE2 (P < 0.05).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This paper confirms the presence of a high-affinity PGE2-binding site on the type II pneumocyte apical membrane of the term guinea pig fetus. Kinetic analysis of [3H]PGE2 binding to type II pneumocyte apical membrane shows that this binding is saturable and reversible. The ligand affinity is similar (i.e., within 1 and 10 nM for the high-affinity site) to that observed in plasma membranes of liver, smooth muscle, etc. (for review, see Ref. 5), although the binding capacity is lower, resembling, for example, rabbit erythrocyte membrane where PGI2 and PGE1 purportedly modulate red cell deformability, controlling oxygen delivery to tissue through capillaries (7). Our data are, however, also consistent with the existence of a G protein-linked binding site of lower affinity. This may or may not be a separate entity; further molecular studies are required for its characterization.

Because PGE2, PGE1, PGD2, and PGF2alpha have well-characterized Na+-absorptive roles in the kidney (26), we investigated their relative potencies in the lung alveolar apical membrane by studying their ability to displace [3H]PGE2 from the putative receptor site. Although PGE2 and PGE1 were approximately equally effective in competing with [3H]PGE2, PGD2 showed a diminished effect, followed by PGF2alpha . We identified a similar differential in the PG effect on high-affinity GTPase activity inhibition in the same preparation: PGE2 and PGE1 are roughly equipotent, whereas PGD2 and PGF2alpha (in that order) show an apparently diminished effect on inhibition of G protein turnover. This match between rank order of ligand binding and G protein turnover inhibition is further suggestive of the presence of a functional PG receptor working via G proteins in the fetal alveolar type II cell apical membrane.

The ability of PGs to displace [3H]PGE2 has been recently utilized to classify EP receptors (receptors apparently specific for PGE2; see Ref. 5). Although not studied here, we have previously shown that ~70% of the basal high-affinity GTPase activity in this preparation is pertussis toxin insensitive. Interestingly, an EP receptor has been shown to be coupled via the pertussis toxin-insensitive G protein Gq (possibly one of the functionally important G proteins in our system; see Ref. 21) in bovine adrenal glands (22). Our observed rank order (PGE2 = PGE1 > PGD2 > PGF2alpha ) for [3H]PGE2 displacement is characteristic of the EP3 subtype (5). Although EP3 receptor mRNA is expressed in many tissues, it is of relevance that expression is significant in both kidney and lung (31).

Finally, we studied the effect of stimulating this G protein-linked PG receptor on Na+ transport into apical membrane vesicles. It is appropriate at this point to mention the caveat that agonist-dependent stimulation of the initial rate of 22Na+ uptake into vesicles represents data obtained from a pure system and may not replicate events at the complex level of the cells and in the animal. The system allows us, however, to study more specifically the regulation of conductive Na+ transport in type II pneumocytes while generating data that we expect will be interpretable in due course in the context of a functionally integrated preparation.

In apical membrane vesicles, PGE2 (1 µM) or GTP (100 µM) on their own had no significant effect on the initial rate of 22Na+ uptake compared with control. In combination, however, they produced a 38% increase in this rate. Interestingly, this is not significantly different from the increase in initial rate achieved by irreversible G protein activation alone with the nonhydrolyzable GTP analog GTPgamma S. Because all PG receptors are G protein linked, it follows that stimulation of the alveolar prostanoid receptor is capable of fully activating the G protein-mediated upregulation of lung alveolar Na+ transport. Thus (as for the renal Na+ absorptive process), PGE2 receptors may prove to be an important determinant of overall perinatal alveolar fluid homeostasis, facilitating the clearance of excess lung fluid in preparation for birth.

Although inhibition of G protein activity with PGs (23) and other agonists (9) has been previously observed, we are the first to note the apparent association of such inhibition with an upregulation of cellular function. G protein-mediated signal transduction is not a simple linear process but involves intricate cross talk between different G protein-coupled pathways and between G protein-coupled pathways and other signal transduction systems (29). Gbeta gamma dimer could regulate this interaction between G protein and the Na+ channel, since maintaining the Galpha -GTP state will hinder reassociation of the heterotrimeric G protein and could facilitate possible Gbeta gamma -mediated stimulation of Na+ transport. Also, both stimulation and inhibition of G proteins regulate surfactant secretion in the type II pneumocyte (25), and further studies are necessary to relate the observations of downregulation of high-affinity GTPase activity and stimulation of Na+-channel function. We (21) and other workers (9) have recently observed similar G protein turnover inhibition induced by polyunsaturated fatty acids. Although others have discussed possible mechanisms by which such an effect on G proteins could be of functional importance by being transiently inhibitory (thus allowing participation in a feedback regulatory process), we have explored its relation to concomitant upregulation of cellular function.

Neonatal respiratory distress syndrome continues to have a high mortality and morbidity despite the introduction of surfactant treatment (27). Novel therapeutic strategies directed toward lung alveolar fluid overload (a problem that coexists with surfactant depletion in respiratory distress syndrome) are likely to improve survival and reduce morbidity. A clear understanding of the cellular pathways that regulate Na+-driven absorption, like that described here of PG receptors, of lung fluid at birth is of crucial importance in the formulation of such strategies.

    ACKNOWLEDGEMENTS

We thank Craig Carr, Prof. Milligan (Biochemistry, Glasgow), Alan Monaghan, Debbie Baines, Mark Clunes, Lorraine Gambling, Craig Gould (Child Health, Dundee), and Margaret Gordon and Fiona Campbell (Rowett Research Institute, Aberdeen) for laboratory and/or statistical assistance.

    FOOTNOTES

This work was supported by Wellcome Trust Grants 042224/Z/94/Z and 039124/Z/4A. S. Mukhopadhyay was a recipient of the Wellcome Advanced Research Training Fellowship from the Wellcome Trust, UK.

Current addresses: G. F. Fyfe, Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., PO Box 3333, New Haven, CT 06510-6026; P. J. Kemp, Dept. of Physiology, Worsley Medical and Dental Building, University of Leeds, Leeds LS2 9JT, UK.

Address for reprint requests: S. Mukhopadhyay, Lung Membrane Transport Group, Dept. of Child Health, Ninewells Hospital and Medical School, Univ. of Dundee, Dundee DD1 9SY, UK.

Received 7 August 1997; accepted in final form 16 January 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 274(4):L567-L572
1040-0605/98 $5.00 Copyright © 1998 the American Physiological Society




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