©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for G-mediated Cross-talk in Primary Cultures of Lung Alveolar Cells
PERTUSSIS TOXIN-SENSITIVE PRODUCTION OF cAMP (*)

(Received for publication, November 3, 1994; and in revised form, January 23, 1995)

Mark S. Pian (1)(§) Leland G. Dobbs (2)

From the  (1)Cardiovascular Research Institute and the Departments of Pediatrics and (2)Medicine, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the presence of activated G, the beta complex of heterotrimeric G proteins (beta) stimulates adenylyl cyclase (AC) in membranes prepared from cells expressing recombinant AC II or AC IV. Conditional stimulation of AC by beta has been hypothesized to integrate cross-talk between G(s)- and non-G(s)-coupled regulation of cellular cAMP (Tang, W. J., and Gilman, A. G.(1991) Science 254, 1500-1503). Although [Medline] observations in cells expressing recombinant receptors, Gs, and AC support this hypothesis (Federman, A. D., Conklin, B. R., Schrader, K. A., Reed, R. R., and Bourne, H. R.(1992) Nature 356, 159-161), this mechanism has not been investigated in differentiated cells. Expression of AC II has been reported only in lung, olfactory, and brain tissue. We found that rat lung alveolar type II cells express AC II and IV. Therefore, we hypothesized that beta conditionally stimulates AC in type II cells. Consistent with this hypothesis, we found that the alpha(2)-adrenergic agonist UK14304 did not affect basal cAMP in type II cells but potentiated terbutaline-stimulated cAMP accumulation. Treatment of cells with pertussis toxin partially inhibited terbutaline-stimulated cAMP accumulation and completely inhibited the effects of UK14304. In type II cell membranes, purified beta tripled the terbutaline-stimulated increase in AC activity. In contrast, beta inhibited AC activity in the absence of terbutaline. The addition of purified G blocked beta-induced effects. In summary, type II cells expressing endogenous AC II and IV regulate cAMP accumulation and AC activity in a manner consistent with conditional stimulation by beta. These observations support the overall hypothesis that conditional stimulation of AC by beta integrates cross-talk between signal transduction systems in differentiated cells.


INTRODUCTION

Extracellular signals affect intracellular cAMP mainly through receptor-mediated activation of heterotrimeric G proteins that, in turn, regulate adenylyl cyclase (AC) (^1)activity. In the classically described G protein activation cycle, functionally specific alpha subunits dissociate from beta complexes (beta) to stimulate (via G) or inhibit (via G) AC activity. To date, seven distinct full-length(2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) and two partial sequence (11, 14, 15) cDNAs encoding mammalian AC have been described. The distinct regulatory characteristics of each of these types of AC suggest that regulation of cellular cAMP is far more complex than was appreciated when the classical activation cycle was first described.

In the presence of activated G, beta stimulates AC activity in membranes prepared from cells expressing recombinant AC II (1) or AC IV(2) . Because beta conditionally stimulates AC activity, it was hypothesized that stimulating receptors coupled to classes of G proteins other than G(s) could elevate cellular cyclic AMP, if G(s)-coupled receptors were also stimulated(1) . This hypothesis is supported by the results of experiments in which cells co-transfected with a mutationally activated G, recombinant AC II, and an alpha(2)-adrenergic receptor (not coupled to G(s)) gained the ability to increase cAMP when treated with an alpha(2)-adrenergic receptor agonist(3) . It remains to be determined whether beta can conditionally stimulate AC activity in differentiated cells. Were this found to be true, it would support the hypothesis that such a mechanism integrates cross-talk between G(s)-coupled and non-G(s)-coupled regulation of cAMP in cells containing only their native endogenous receptors, Gs, and AC.

By Northern blot analysis, AC II is expressed only in lung, olfactory, and brain tissue. If conditional stimulation of AC II by beta can integrate cross-talk between signal transduction systems in differentiated cells, then beta should potentiate G-stimulated AC activity in AC II-expressing cells isolated from these tissues. Our laboratory has identified several responses in lung alveolar type II cells that suggested to us that significant cross-talk among receptors may regulate AC activity and cAMP content in this cell type(16) .

Alveolar type II cells secrete pulmonary surfactant, a complex mixture of lipids and proteins that helps to maintain alveolar inflation at low lung volume by lowering surface tension at the air-liquid interface. Several of the various chemical agents that stimulate type II cells to secrete surfactant (reviewed in (17) and (18) ), including beta-adrenergic agonists and forskolin, increase type II cell cAMP content(19, 20, 21, 22) . We have previously shown that pertussis toxin (PTX) partially inhibits beta-adrenergic agonist-stimulated secretion in type II cells but not secretion stimulated by forskolin or 8-bromo-cyclic AMP(16) . These results suggested to us that PTX-sensitive G proteins contribute to G-stimulated AC activity in type II cells, which is one expected characteristic of an effect mediated by conditional stimulation of AC by beta. We describe here the results of experiments testing whether beta potentiates G-stimulated AC activity in type II cells.


EXPERIMENTAL PROCEDURES

Isolation and Culture of Alveolar Type II Cells

We isolated alveolar type II cells from the lungs of adult male, specific pathogen-free Sprague-Dawley rats by elastase digestion and ``panning'' cells on IgG-coated bacteriologic plastic dishes(23) . Cells were cultured (5 times 10^4 cells/cm^2) on tissue culture plastic for 22 h at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in 10% CO(2), 90% air. More than 95% of the cells excluded the vital dye erythrosin B. In all experiments, <1% cellular lactic acid dehydrogenase (24) was released into the medium after cells were treated with control solutions, PTX, pertussis toxin B-oligomer (PTB), and/or agonists.

Northern Analysis

Poly(A) RNA (2.5 µg/lane) isolated from either rat lung homogenate or rat alveolar type II cells in culture was subjected to electrophoresis in 1% agarose/formaldehyde gels, transferred by positive pressure, and UV cross-linked to nylon filters (Duralon, Stratagene). Hybridizations with P-labeled cDNA probes (1 times 10^6 cpm/ml) were carried out for 18 h at 68 °C (QuikHyb, Stratagene), after which filters were washed under high stringency conditions and subjected to autoradiography using an intensifying screen for 4 days at -80 °C. For detection of AC II and AC IV, full-length inserts derived from a rat brain cDNA library were kindly provided by Dr. W. J. Tang (University of Texas Southwestern Medical Center). For detection of AC I, we used a 200-base pair polymerase chain reaction-generated fragment amplified from rat brain RNA, which was kindly provided by Dr. Randall Reed (Johns Hopkins University).

Measurement of cAMP

Type II cells in culture were washed with Dulbecco's modified Eagle's medium and incubated for 2 h in fresh Dulbecco's modified Eagle's medium with or without PTX or PTB (20 µg/ml). Cells were again washed, and control solution, terbutaline (10 µM), or UK14304 (10 µM) was added. After 5 min, cellular cAMP was extracted with 1 mM HCl at 4 °C, frozen immediately, and lyophilized. We measured cAMP by radioimmunoassay.

Measurement of Adenylyl Cyclase Activity

Type II cells were scraped from culture dishes in a solution of 10 mM TrisbulletHCl, pH 7.7, 1 mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin and were disrupted in a glass homogenizer. Membranes were pelleted by centrifugation at 100,000 times g for 1 h at 4 °C, resuspended in 10 mM Tris, pH 7.7, and stored at -70 °C. Adenylyl cyclase activity was measured in duplicate by the method of Alvarez and Daniels(25) . In experiments examining the effects of exogenous G protein subunits on AC activity, membrane preparations were preincubated with beta and/or G for 30 min at 4 °C. In some experiments, beta was first heat-inactivated at 100 °C for 10 min; in other experiments, a mixture of beta and G was preincubated for 10 min at room temperature prior to adding the membranes. The final concentration in the assay mixture of either beta or G was 100 nM. Reactions were initiated by adding the membranes to the other assay components and rapidly warming the mixture to 37 °C. To detect possible changes in cAMP phosphodiesterase activity, [^3H]cAMP was included in some reaction mixtures prior to adding membranes.

Statistical Methods

Results are expressed as the means ± S.D. of duplicate samples. The experimental number represents the number of different type II cell isolations. Data were analyzed by one-way analysis of variance and a Newman-Keul's test.

Materials

Tissue culture medium and fetal bovine serum were obtained from the University of California Cell Culture Facility. Rats were purchased from Bantin-Kingman (Fremont, CA). We purchased terbutaline from Merrill-Dow (Cincinnati, OH), [2,8-^3H]cAMP, [alpha-P]ATP, and cAMP radioimmunoassay from DuPont NEN, and PTX and PTB from List Biochemical Labs Inc. (Campbell, CA). UK14304 was obtained from Research Biochemicals Inc. (Natick, MA). beta and G, each purified from bovine brain, were kindly provided by Dr. Henry Bourne (University of California, San Francisco).


RESULTS

Expression of Adenylyl Cyclase in Alveolar Type II Cells in Culture

Full-length cDNA probes for AC II or AC IV hybridized to 4.4- and 3.5-kilobase bands, respectively, of poly(A) RNA isolated from both lung and cultured alveolar type II cells (Fig. 1). A probe for AC I failed to hybridize to the same blots (not shown).


Figure 1: Expression of AC II and AC IV in lung and cultured type II cells. Samples (2.5 µg/lane) of poly(A) RNA were isolated from both rat lung and cultured alveolar type II cells. Northern blots were probed first for AC II, stripped, and reprobed for AC IV. Probe for AC I failed to hybridize to the same blots (not shown).



Inhibition of Terbutaline-stimulated cAMP Accumulation by Pertussis Toxin

Treatment of type II cells with terbutaline increased cAMP content 10-fold, as has been reported previously(21) . Treatment of cells with PTX did not alter basal cAMP content but inhibited the terbutaline-stimulated increase in cAMP by 56% (Fig. 2). Treatment of intact type II cells with PTX has previously been shown to ADP-ribosylate substrates of molecular mass characteristic of G protein alpha subunits(16, 26) . As a control, we tested the effects of PTB, which lacks the PTX subunit that catalyzes ADP-ribosylation. In contrast to PTX, PTB did not inhibit cAMP accumulation (Fig. 2).


Figure 2: Inhibition of terbutaline-stimulated cAMP accumulation by pertussis toxin. Cultured type II cells were incubated for 2 h with or without PTX or PTB and then treated for 5 min with control or agonist solutions as indicated. cAMP was measured as described under ``Experimental Procedures.'' Values represent the means ± S.D. of duplicate samples from the number of separate cell preparations indicated in parentheses. *, different from control cells, p < 0.025; , different from non-PTX-treated cells treated with terbutaline alone, p < 0.05.



Potentiation of Terbutaline-stimulated cAMP Accumulation by UK14304 and Inhibition by PTX

The finding that PTX inhibits terbutaline-stimulated cAMP accumulation in type II cells is consistent with the hypothesis that beta from PTX substrates potentiates G-stimulated cAMP accumulation. If so, beta derived from various sources, including activation of a G protein-coupled receptor not coupled to G(s), would be expected to augment G-stimulated cAMP accumulation. To test this hypothesis, we investigated the effect of the selective alpha(2)-adrenergic receptor agonist UK14304 on cAMP content. UK14304 alone did not affect cAMP content. However, UK14304 potentiated the terbutaline-stimulated increase in cAMP by 35% (Fig. 3). Furthermore, treatment with PTX completely blocked the UK14304-induced potentiation of cAMP accumulation (Fig. 3). In contrast to PTX, PTB did not block the effect of UK14304 (data not shown).


Figure 3: Potentiation of terbutaline-stimulated cAMP accumulation by UK14304. Cultured type II cells were treated, and cAMP was measured as described in Fig. 2and under ``Experimental Procedures.'' Values represent the means ± S.D. of duplicate samples from the number of separate cell preparations indicated in parentheses. *, different from control cells, p < 0.025; , different from non-PTX-treated cells treated with terbutaline alone, p < 0.05.



Effect of beta on Adenylyl Cyclase Activity in Type II Cell Membranes

We measured both terbutaline-stimulated and basal AC activity in type II cell membranes treated with exogenous beta. beta nearly tripled the terbutaline-stimulated increase in AC activity (Fig. 4). In the absence of terbutaline, beta reduced basal AC activity by 70% (Fig. 5). Heat-inactivated beta did not affect either stimulated or basal AC activity (data not shown). As a control for the effects of beta, we measured the effect of adding G on AC activity. Incubation of membranes with G blocked the effect of beta on terbutaline-stimulated AC activity (Fig. 4) and substantially inhibited its effect on basal AC activity (Fig. 5). When added alone, G inhibited the terbutaline-stimulated increase in AC activity by nearly 85% relative to the increase in beta-treated membranes (Fig. 4) and had little effect on basal AC activity (Fig. 5). Addition of beta or G did not affect the recovery of [^3H]cAMP, indicating that cAMP phosphodiesterase activity was unaffected (data not shown).


Figure 4: Effect of beta on terbutaline-stimulated adenylyl cyclase activity in type II cell membranes. Membranes were prepared from cultured type II cells, incubated with purified beta and/or G, treated with terbutaline, and assayed for AC activity as described under ``Experimental Procedures.'' Values are expressed as the percentage of activity measured in membranes not treated with terbutaline (basal activity) and represent the means ± S.D. of duplicate samples from the number of separate cell preparations indicated in parentheses. *, different from control cells, p < 0.025.




Figure 5: Effect of beta on basal adenylyl cyclase activity in type II cell membranes. Experimental conditions were as described in the legend to Fig. 4, except membranes were not treated with terbutaline. Values are expressed as the rate of cAMP produced per mg of membrane protein and represent the means ± S.D. of duplicate samples from the number of separate cell preparations indicated in parentheses. *, different from control cells, p < 0.025.




DISCUSSION

Lung alveolar type II cells are critical for normal lung function. Type II cells synthesize and secrete pulmonary surfactant, a complex mixture of lipids and proteins that lowers surface tension and prevents alveolar collapse at low lung volume. From experiments in whole animals, isolated/perfused lungs, and primary cultures of type II cells, a variety of pharmacological stimuli of surfactant secretion have been identified (reviewed in (18) ). The physiologic correlates of these stimuli and the way signals from these stimuli are integrated are incompletely understood. The beta-adrenergic agonists, which increase type II cell cAMP (presumably through G(s)-mediated stimulation of AC), have been shown to stimulate secretion in type II cells both in primary culture (27) and in vivo(28, 29) . Type II cells also secrete surfactant in response to agents, such as P(2) purinergic agonists, which activate G protein classes other than G(s). The regulatory characteristics of AC isoforms II and IV enable them to integrate multiple signals(1, 3, 30) . Because type II cells express AC II and IV (Fig. 1), it seemed likely to us that beta-stimulated AC might integrate signals that regulate surfactant secretion.

PTX partially inhibits terbutaline-stimulated surfactant secretion (16) . This observation suggested to us that, under normal conditions, release of beta subunits enhances G-stimulated AC activity in type II cells. In the present study, we demonstrate that UK14304, which by itself has no effect on type II cell cAMP content, markedly increases the cAMP content of terbutaline-treated cells; this effect was blocked by PTX (Fig. 2). These results are similar to those observed by Federman et al.(3) in cells co-transfected with a mutationally activated G, recombinant AC II, and an alpha(2)-adrenergic receptor. Our results support the hypothesis that UK14304 potentiates G-stimulated AC activity through PTX-sensitive release of beta.

PTX also inhibits terbutaline-stimulated cAMP accumulation in type II cells in the absence of UK14304. Therefore, it appears that the terbutaline-stimulated response is partially mediated by the release of beta. Type II cells are known to express pertussis toxin substrates of molecular mass consistent with that of G(i) or G(o)(16, 26) , either of which might serve as a source of beta. It is not known what agonists and receptors in type II cells are involved in the process. One candidate is the adenosine/A(1) receptor system. In type II cells, addition of exogenous adenosine deaminase has been shown to augment cellular responses to secretory agonists. These observations have led others to suggest that A(1) receptors, which are coupled to G(i) in some cells, are tonically stimulated by endogenous sources of adenosine(31) .

It is also not clear which G proteins are involved in the effects we have observed. Recent reports that G does not inhibit AC II (32) might explain why PTX does not increase basal cAMP in type II cells, despite possible tonic activation of G(i). However, there are conflicting reports that G may partially inhibit AC II(33) . It could be argued that tonic activation might not release sufficient beta to account for potentiation of terbutaline-stimulated cAMP generation, because nanomolar concentrations of beta appear to be needed to augment AC activity(1, 32) . However, these concentrations pertain to the effects of solubilized G protein subunits on recombinant AC; the concentration dependence of beta-mediated effects may be quite different for endogenously derived beta acting on native AC in intact cells. In addition, intact cells may achieve high local concentrations of beta in relevant compartments containing AC.

To establish more conclusively that beta can stimulate AC activity in type II cells, we measured the effect of exogenous beta or G on both basal and terbutaline-stimulated AC activity in type II cell membranes. As predicted by the expression in type II cells of message for AC types II and IV and by our results in intact cells, beta significantly enhanced the terbutaline-stimulated increase in AC activity relative to basal levels. Preincubation of beta with G blocked this effect. Because exogenous G also inhibited AC activity in membranes treated with terbutaline alone (Fig. 4) (presumably by binding endogenous beta), it seems likely that endogenous beta contributes to full expression of terbutaline-stimulated AC activity in type II cell membranes. This observation adds further support to a similar interpretation of experiments in which PTX inhibited terbutaline-stimulated cAMP accumulation in whole cells.

In contrast to its effect on terbutaline-stimulated type II cell membranes, beta inhibited membrane basal AC activity. These findings were not predicted by other published observations. In experiments examining the effects of beta on recombinant AC, beta directly inhibits only AC I(34) . By Northern blot analysis, we did not detect mRNA for AC I in either rat lung or isolated type II cells. Of course, mRNA levels may not accurately reflect the abundance of previously translated protein(13) , and, therefore, our results do not exclude the possibility that AC I may be present in type II cells. However, comparing our results to those previously reported may not be straightforward, because the effects of beta on AC have generally been measured in membranes obtained from cells overexpressing recombinant AC rather than in membranes from cells expressing a mixture of native enzymes. One possible explanation for our results is that type II cells express a previously unrecognized AC isoform that is functionally similar to AC I in that it is inhibited by beta. A second possibility, that beta inhibits basal activity by binding free G, seems unlikely because beta should then prevent, rather than enhance, the terbutaline-stimulated increase in AC activity.

In summary, lung alveolar type II cells appear to provide a naturally occurring example of the interpathway cross-talk previously demonstrated only in broken or whole cell models in which the necessary component parts were purposefully assembled by expression of recombinant cDNA. Our findings in type II cells support the hypothesis that AC II and IV can integrate beta-mediated cross-talk to regulate cAMP-dependent processes in cells containing their native complement of receptors, regulatory proteins, and effectors. In addition, these results support a role for the regulatory characteristics of individual AC in the expression of phenotype-specific functions in normally differentiated cells.


FOOTNOTES

*
This work was supported in part by Grants HL-02157 and HL-24075 from the National Heart, Lung, and Blood Institute of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Cardiovascular Research Inst., University of California, San Francisco, CA 94143-0130. Tel.: 415-476-1509; Fax: 415-476-3586.

(^1)
The abbreviations used are: AC, adenylyl cyclase; G, alpha subunits of heterotrimeric G proteins; beta, beta complexes of heterotrimeric G proteins; PTX, pertussis toxin; PTB, pertussis toxin B-oligomer.


ACKNOWLEDGEMENTS

We are grateful to Dr. H. Bourne for helpful discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.