(Received for publication, November 3, 1994; and in revised form, January 23, 1995)
From the
In the presence of activated G, the
complex of heterotrimeric G proteins (
) stimulates adenylyl
cyclase (AC) in membranes prepared from cells expressing recombinant AC
II or AC IV. Conditional stimulation of AC by
has been
hypothesized to integrate cross-talk between G
- and
non-G
-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,
G
s, 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
conditionally stimulates AC in type II
cells. Consistent with this hypothesis, we found that the
-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
tripled the terbutaline-stimulated increase in AC activity.
In contrast,
inhibited AC activity in the absence of
terbutaline. The addition of purified G
blocked
-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
. These
observations support the overall hypothesis that conditional
stimulation of AC by
integrates cross-talk between signal
transduction systems in differentiated cells.
Extracellular signals affect intracellular cAMP mainly through
receptor-mediated activation of heterotrimeric G proteins that, in
turn, regulate adenylyl cyclase (AC) ()activity. In the
classically described G protein activation cycle, functionally specific
subunits dissociate from
complexes (
) 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,
stimulates AC activity in
membranes prepared from cells expressing recombinant AC II (1) or AC IV(2) . Because
conditionally
stimulates AC activity, it was hypothesized that stimulating receptors
coupled to classes of G proteins other than G
could elevate
cellular cyclic AMP, if G
-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
-adrenergic receptor (not coupled to G
)
gained the ability to increase cAMP when treated with an
-adrenergic receptor agonist(3) . It remains
to be determined whether
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
-coupled and non-G
-coupled regulation
of cAMP in cells containing only their native endogenous receptors,
G
s, and AC.
By Northern blot analysis, AC II is
expressed only in lung, olfactory, and brain tissue. If conditional
stimulation of AC II by can integrate cross-talk between
signal transduction systems in differentiated cells, then
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 -adrenergic agonists
and forskolin, increase type II cell cAMP
content(19, 20, 21, 22) . We have
previously shown that pertussis toxin (PTX) partially inhibits
-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
. We describe here the results of
experiments testing whether
potentiates
G
-stimulated AC activity in type II cells.
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).
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.
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.
Figure 4:
Effect
of on terbutaline-stimulated adenylyl cyclase activity in
type II cell membranes. Membranes were prepared from cultured type II
cells, incubated with purified
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 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.
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 -adrenergic agonists,
which increase type II cell cAMP (presumably through
G
-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
purinergic
agonists, which activate G protein classes other than G
.
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
-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
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
-adrenergic receptor. Our
results support the hypothesis that UK14304 potentiates
G
-stimulated AC activity through PTX-sensitive release
of
.
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 . Type II cells are known to express
pertussis toxin substrates of molecular mass consistent with that of
G
or G
(16, 26) , either of
which might serve as a source of
. It is not known what
agonists and receptors in type II cells are involved in the process.
One candidate is the adenosine/A
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
receptors, which are
coupled to G
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
. However,
there are conflicting reports that G
may partially
inhibit AC II(33) . It could be argued that tonic activation
might not release sufficient
to account for potentiation of
terbutaline-stimulated cAMP generation, because nanomolar
concentrations of
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
-mediated effects may be
quite different for endogenously derived
acting on native AC
in intact cells. In addition, intact cells may achieve high local
concentrations of
in relevant compartments containing AC.
To establish more conclusively that can stimulate AC
activity in type II cells, we measured the effect of exogenous
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,
significantly
enhanced the terbutaline-stimulated increase in AC activity relative to
basal levels. Preincubation of
with G
blocked this effect. Because exogenous G
also
inhibited AC activity in membranes treated with terbutaline alone (Fig. 4) (presumably by binding endogenous
), it seems
likely that endogenous
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, inhibited
membrane basal AC activity. These findings were not predicted by other
published observations. In experiments examining the effects of
on recombinant AC,
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
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
. A
second possibility, that
inhibits basal activity by binding
free G
, seems unlikely because
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 -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.