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* From the Division of Pulmonary and Critical Care Medicine, Michael Reese Hospital, University of Illinois at Chicago, Chicago, IL, and National Cancer Institute, National Institutes of Health, Bethesda, MD.
Correspondence to: J.I. Sznajder MD, FCCP, Department of Medicine, Michael Reese Hospital and Medical Center, 2929 S Ellis Ave, Baum-101, Chicago, IL 60616
Alveolar epithelial
Na,K-adenosine triphosphatase (ATPase) plays an important role in
lung edema clearance.1
Na,K-ATPase is an integral
membrane protein consisting of the 
subunit, which has catalytic
activity and ion binding sites,2
and the ß subunit,
which plays a regulatory role contributing to the stability of the
complex and its insertion in the plasma membrane.3
Dopamine (DA) upregulates the Na,K-ATPase in the alveolar
epithelium and increases lung liquid clearance,4
but the
mechanisms of this regulation have not been elucidated. The
mitogen-activated protein kinase (MAPK), also known as
extracellular-signal-regulated kinase (ERK) cascade, is a major
signaling system by which cells transduce extracellular signals into
intracellular responses.5
In mammalian cells ERK proteins,
ERK1 and ERK2, also known as p44/p42 respectively, are activated by a
variety of hormones, growth factors, and peptides.6
Little
is known about ERK activation by catecholamines and we are not aware of
studies about ERK activation in alveolar type II (ATII) cells. Thus, we
tested whether ERK proteins are activated by DA in ATII cells and
whether this activation is involved in the upregulation of the
Na,K-ATPase in these cells.
Experimental Design and Results
ATII cells were isolated from adult pathogen-free Sprague Dawley male rats (weight approximately 225 g) using the method by Dobbs et al.7 Total cellular RNA from ATII cells was isolated by using RNeasy total RNA kit (QIAGEN; Valencia, CA), as described by the manufacturer. The reverse transcriptase reaction was performed (using the Superscript Preamplification System) (GIBCO-BRL; Grand Island, NY) and G3PDH was used as control in the polymerase chain reaction reactions. Western Blot analysis with total protein was performed using the ECL-plus detection kit (Amersham) as recommended by the manufacturer. For determination of the ERK activity, the cells were starved for 16 to 20 h prior to treatment with agonists and antagonists. The ERK activity was determined as described in the "p44/p42 MAP kinase assay kit manual" (New England Biolabs).
Incubation of ATII cells with 1 µM DA resulted in a rapid
stimulation (within 2 to 10 min) of ERK activity, measured as
phosphorylation of the ERK substrate Elk-1. To study whether the
activation of ERK proteins by DA plays a role in the regulation of the
Na,K-ATPase, subconfluent day 2 ATII cells were serum starved and
treated with the following: (1) control; (2) 1 µM DA; and (3) 50 µM
of the specific MEK inhibitor PD98059, 2 h prior to DA
stimulation, at times ranging between 1 and 30 h. RT-polymerase
chain reaction analysis of total RNA showed that, while the
1-messenger RNA (mRNA) levels did not change
significantly in the DA-stimulated cells, there was an increase in the
ß1-mRNA levels after 6 h of DA stimulation that was
maximal between 12 and 18 h. This increase was mediated by ERK
activation, as the MEK inhibitor PD98059 completely blocked the
DA-induced increase in ß1-mRNA. Western blot analysis of
ATII cell homogenates showed that Na,K-ATPase ß1-subunit
protein increased between 24 and 30 h, following DA stimulation.
As with the ß1 mRNA, the increase in ß1
protein was inhibited in the presence of the MEK inhibitor PD98059.
To determine which dopaminergic receptor mediates the activation of ERK by DA in ATII cells, we treated the cells with specific agonists and antagonists of the D1 and D2 receptors. Our results showed that D2 but not D1 antagonists blocked the DA activation of ERK proteins. Moreover, inhibition of D1 receptors resulted in a further increase in ERK activation by DA. These results indicate that D2 receptors are involved in the DA-mediated ERK activation and that D1 receptors activation has actually an antagonistic effect on the ERK activation.
Ras proteins are membrane-bound guanine nucleotide-binding proteins that mediate ERK activation in different cell types.8 To determine whether Ras proteins are involved in the ERK activation by DA, ATII cells were treated with a peptide corresponding to the SH3 binding sequence of the Ras guanine nucleotide exchange factor SOS (Calbiochem). The SH3-binding region of SOS has strong affinity for the N-terminal SH3 domain of the adaptor protein Grb29 blocking the interaction SOS/Grb2 and thereby preventing Ras activation. Preincubation of ATII cells with the SH3-binding peptide, 10 to 20 µM prior to DA stimulation did not block ERK activation by DA. Under the same conditions, ERK activation by epidermal growth factor was completely blocked in the presence of the SH3-binding peptide. These results suggest that the DA-mediated ERK activation in ATII cells is a Ras-independent mechanism.
Raf1 is a ser/thr kinase that participates in ERK activation.6 Although Raf1 is a Ras efector, it can be activated by a Ras-independent mechanism.8 To determine whether Raf1 kinase is involved in the DA-mediated ERK activation in ATII cells, we performed a Raf1 assay on cells treated with DA, using syntide-2 as substrate.10 Our results show that DA stimulated Raf1 kinase activity in ATII cells and that this activation was Ras independent. The involvement of Raf1 kinase in the DA-ERK pathway was confirmed by treatment of the cells with forskolin, a protein kinase A activator, as forskolin completely blocked the activation of ERK by DA. Treatment with the protein kinase A inhibitor adenosine-3', 5'-cyclic monophosphothioate showed the opposite effect.
It has been shown that ERK proteins can be activated by protein kinase C (PKC) and be mediators of the multiple effects of PKC, such as differentiation and proliferation.11 To investigate whether PKC is a component of the signal transduction pathway DA-ERK, we treated ATII cells with the PKC inhibitors bisyndolilmaleimide (1 µM) and calphostin-C (100 nM) prior to DA stimulation. We observed that bisindolylmaleimide blocked the activation of ERK by DA, whereas calphostin-C, an inhibitor of diacylglycerol-dependent PKC isoforms, did not inhibit the stimulation of ERK by DA, suggesting that atypical PKC isoforms may be involved in the DA-ERK pathway in ATII cells.
Discussion
Previous studies have demonstrated that DA increases lung liquid clearance by upregulating the Na,K-ATPase.4 Our data suggest that Na,K-ATPase upregulation can be due in part to increased synthesis of the Na,K-ATPase, via a mechanism that involves activation of MAPK/ERK proteins. In the present study, we have in part elucidated a pathway involved in the DA stimulation of ERK activity in ATII cells, which includes D2 receptors, Raf1 kinase, and atypical PKC but is independent of Ras activation.
Our results suggest the existence of a novel pathway of activation of ERK/MAPK in ATII cells. Conceivably, this pathway is an important mechanism in the DA-mediated transcriptional/translational regulation of the Na,K-ATPase in ATII cells and thus, lung edema clearance.
Footnotes
Supported in part by grants HL-48129, American Heart Association grant 96012890, NRSA (KMR), and the Research and Education Foundation of the Michael Reese Medical staff.
References
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