Introduction

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Serotonin (5-hydroxytryptamine, 5-HT) is an endogenous amine involved in diverse biologic processes within the central and peripheral nervous system and the cardiovascular and gastrointestinal and respiratory systems (1). In addition to its role as a neurotransmitter, 5-HT has been implicated as a potential mitogen (4, 5) and was shown to have effects on morphogenesis and neuronal development (6). This diversity of actions is made possible because of the existence of specific 5-HT cell-surface receptor subtypes and their coupling to distinct intracellular messenger systems or ion channels. A family of serotonin receptors (5-HT-R) and their respective genes have recently been identified (7) based on operational, structural, and transductional data. New classification of 5-HT-Rs, based on operational, structural, and transductional data, has divided 5-HT-Rs into seven classes and at least ten subtypes (8). In addition to structural differences, there are important differences in coupling to intracellular effectors. For example, receptors belonging to the 5-HT₁-R family are thought to be negatively linked to adenylyl cyclase, whereas 5-HT₂-Rs are coupled via phosphoinositol hydrolysis signal and 5HT₃-R interacts directly with ion channels (8).

The distribution and localization of 5HT-Rs has been investigated extensively in the central nervous system, but there is much less data available on 5-HT-Rs in peripheral tissues. The lung is an important peripheral site for 5-HT action because this amine is known to produce contractile responses in both airway and vascular smooth muscle (9, 10). Further, inactivation of 5-HT in the pulmonary circulation by a carrier-active mechanism involving the endothelial cells is a well-established phenomenon (11).

The lung tissue is also known to be an important source of endogenous 5-HT, particularly in mast cells and pulmonary neuroendocrine cells (PNEC) (12, 13). The precise role of 5-HT synthesized and released from either the mast cells or PNEC is presently unknown, but may involve local effects such as bronchoconstriction, vasomotor tone, hypoxia signaling, and/or growth factor-like properties (14). In the present study we report expression and localization of the 5-HT receptor 2c subtype (5-HT_2c-R) in rat alveolar type II cells at the messenger RNA (mRNA) level using reverse transcriptase-polymerase chain reaction (RT-PCR) and nonisotopic in situ hybridization (NISH), and at the protein level using immunohistochemistry. Because type II pneumocytes play a critical role in lung physiology, 5-HT and its receptor(s) may be involved in modulation of type II cell homeostatic activities. This appears to be a new and previously unsuspected role for this amine in pulmonary physiology and pathophysiology.

	Materials and Methods

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Lung tissues for NISH studies and for immunohistochemistry were obtained from adult Wistar rats. The tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and 5-µm sections were placed on sialinated slides. Fetal type II cells (Day 20 fetal gestation) were isolated as described by Caniggia and colleagues (15). The adult type II pneumocytes were isolated using a modification of the method of Dobbs (16). Briefly, adult rats were anesthetized and the lungs removed in toto. The type II cells were separated from the alveolar basement membrane by incubation with porcine pancreatic elastase followed by removal of macrophages by differential adherence on Fc-coated Petri dishes (Fisher Scientific, Ottawa, ON, Canada). Type II cells were identified in cytospin samples and in culture dishes by histochemical staining for alkaline phosphatase, a specific marker of type II cells (17).

Isolated cells were plated in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 2 mM glutamine (GIBCO, Burlington, ON, Canada), 10 µg/ ml gentamycin, 100 µ/ml pencillin G, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at an initial seeding density of 10⁶ cells/cm² in 24-well culture dishes (Falcon Multiwell; Becton-Dickinson Labware, Lincoln Park, NJ).

For NISH and imunohistochemical studies, isolated type II cells were grown on Lab-Tek tissue culture slides (GIBCO) and fixed in 4% paraformaldehyde for 1 min. Total RNAs from lung tissue and pellets of isolated type II cells were prepared by the guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (18).

RT-PCR for 5-HT_2c-R

The 5-HT_2c-R complementary DNA (cDNA) fragment was synthesized using RT-PCR. The first-strand cDNA (19) was prepared from total RNA extracted from rat lung, brain, and type II cell cultures. According to the published information on 5-HT_2c-R (previously referred to as 1c) (20), we selected the regions 899 to 916 base pairs (bp) (on the first strand) and 1,327 to 1,310 bp (on the complementary strand), then synthesized two oligomers as the primers: 5' GCACCATGCAAGCTATCA 3' and 5' AGTTCTCCACCTGCATCT 3'.

For PCR reaction, the thermal cycle was 94°C for 30 s, 54°C for 60 s, and 72°C for 30 s. It was amplified for 35 cycles using first-strand cDNA as template, according to the protocol for DNA amplification from Perkin-Elmer (Norwalk, CT). A 430-bp rat 5-HT_2c-R cDNA fragment (from 899 to 1,327 bp) was amplified.

NISH and Immunohistochemistry Procedures

To generate an antisense RNA probe for 5-HT_2c-R, the 430-bp fragment was digested with restriction enzyme SstI and HindIII. Subsequently the SstI/HindIII 5-HT_2c-R fragment (186 bp, i.e., 1,050 to 1,235 bp) was subcloned into SP72 vector (at SstI and HindIII sites). We sequenced this 186-bp insert to confirm that it corresponded to 5-HT_2c-R cDNA fragment (sequencing data not shown). The vectors with inserts were linearized by restriction enzyme HindIII, and T7 RNA polymerase was used to synthesize nonradiolabeled RNA antisense probes (21).

The protocol for NISH was similar to that described previously (21). The immunohistochemistry for surfactant protein (SP) A used methods and procedures as described by Phelps and Harding (22). For correlation of mRNA hybridization signal with respective protein product, the sections were first immunostained with anti-SP-A rabbit polyclonal antibody (1:50 dilution) and reaction was visualized by the immunoperoxidase method according to Sternberger (23). Subsequently, NISH was carried out with digoxigenin-labled 5-HT_2c-R cRNA probe (19). Purified mouse monoclonal antibody (PharMingen, San Diego, CA) (1:200) was used for immunohistochemistry to localize 5-HT_2c-R in fetal and adult type II cell cultures. The incubation with primary antibody was followed by secondary goat antimouse immunoglobulin (Ig)G with peroxidase (Bio-Rad, Hercules, CA) and an immunopure metal- enhanced diaminobenzidine (DAB) substrate kit (Pierce, Rockford, IL). To test immunostaining specificity, the rat-brain synaptic plasma membrane preparation was used to block the specific 5-HT_2c-R antibody according to instructions from the manufacturer (24). The 5-HT_2c-R antibody was preincubated with rat-brain synaptic membranes at 37°C for 1 h, then applied to preparation of type II cells followed by peroxidase-conjugated goat antimouse IgG with peroxidase and DAB color developing. For immunofluorescence studies we used antimouse IgM conjugated with fluorescein isothiocyanate (FITC) (PharMingen) as a secondary antibody. In addition to routine immunofluorescence microscopy, we used laser confocal microscopy (Leitz TCS 4D confocal microscope with argon krypton laser excitation at 488 nm for FITC and a pinhole of 40 µm). Signals were analyzed using Scanware (Leica, Heerbrugg, Switzerland.).

	Results

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We used RT-PCR to detect gene expression of 5-HT_2c-R in extracts of cultures of isolated type II cells from fetal and adult animals as well as whole adult rat lung. In all these samples mRNA signal corresponding to approximately 430 bp was detected, with the same signal present in whole rat brain extract used as a positive control (Figure 1).

View larger version (67K): [in this window] [in a new window]   Figure 1.   RT-PCR for 5-HT2c-R using the RNA extracts from rat brain. Lane 2, positive control; lane 3, adult type II cells; lane 4, fetal type II cells; lane 5, adult rat whole lung extract. The 430-bp band (arrowhead) is present in each sample: X 174/HaeIII marker, lane 1.

To localize 5-HT_2c-R mRNA expression at the cellular level we used antisense RNA probe and NISH protocol. In adult type II cell cultures there was a strong positive 5-HT_2c-R mRNA signal localized within the cytoplasm, whereas the nuclei showed no reactivity (Figure 2A). The specificity of NISH reaction was confirmed using the same type of sample but hybridized with a sense probe, resulting in negative signal in type II cell preparations (Figure 2C). In cultures of fetal type II cells, the positive signal was localized mostly in the perinuclear area with a weak staining of the adjacent cytoplasm (Figure 2B). To identify alveolar type II cells in sections of rat lung, we first used immunocytochemistry with an anti-SP-A antibody. Alveolar type II cells showed positive cytoplasmic immunostaining for SP-A (Figure 3A). NISH performed on the same sections using antisense 5-HT_2c-R probe showed positive signal colocalized in type II cells with SP-A immunoreactivity (Figure 3B).

View larger version (88K): [in this window] [in a new window]   Figure 2.   NISH for 5-HT2c-R on type II cell cultures. Using antisense probe in cultured adult type II cells, there is a strong signal localized in the cytoplasm (A); in fetal type II cell culture (B) the signal is weaker and localized in the perinuclear area. In adult type II cell culture using sense probe (C) there is no signal confirming specificity of hybridization reaction. Original magnification: ×400.Figure 3. (A) Immunohistochemical localization of SP-A (brown) in alveolar type II cells (arrowheads). (B) NISH for 5-HT2c-R in the same area as in A showing 5-HT2c-R mRNA expression (purple-blue) in the same type II cells (arrowheads). Original magnification: ×250.Figure 4. Immunolocalization of 5-HT2c-R in cultured adult type II cells. (A) Incubation with primary antibody against 5-HT2c-R shows strong positive signal localized on cell membrane or perimembranous area. (B) Negative control. Preincubation with rat-brain synaptic plasma membrane abolished specific staining for 5-HT2c-R. Original magnification: ×1,000.Figure 5. Same as Figure 4, but using fetal rat type II cell cultures. Immunostaining with primary antibody against 5-HT2c-R shows positive signal localized in the perinuclear region (A). Negative control incubated with rat-brain synaptic plasma membranes shows no signal (B). Original magnification: ×400.Figure 6. Immunofluorescence confocal microscopy localization of 5-HT2c-R in cultured adult and fetal type II cells. In adult type II cells the positive fluorescence shows membranous or submembranous localization (A). In fetal type II cells the positive fluorescence is localized in the perinuclear region (B). Original magnification: ×1,000.

To localize 5-HT_2c-R at the protein level, we used specific monoclonal antibody against 5-HT_2c-R on type II cell preparations. In samples of adult rat type II cells, positive immunoreactivity for 5-HT_2c-R was localized mostly in the plasma membrane (Figure 4A). The specificity of immunoreactivity was confirmed by a blocking experiment in which immunoreactivity was abolished by preincubation of 5-HT_2c-R antibody with rat-brain synaptic membrane preparation (Figure 4B).

The immunolocalization of 5-HT_2c-R in rat fetal type II cells differed from that of type II cells from adult rats in that the positive signal was localized mostly in the perinuclear cytoplasm (Figure 5A). This reaction was abolished in a blocking experiment (Figure 5B) as shown for adult type II cell preparation.

The cellular localization and topography of 5-HT_2c-R in type II cells was further examined using immunofluorescence and laser confocal microscopy. The samples of adult rat type II cells showed strong immunofluorescence signal for 5-HT_2c-R on the plasma membrane with minimal nuclear immunoreactivity (Figure 6A). In contrast, fetal rat type II cells showed strong perinuclear immunoreactivity with minimal membrane immunostaining (Figure 6B).

	Discussion

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The main finding of this study is the identification and localization of 5-HT_2c-R in alveolar type II cells of both fetal and adult rat lungs. Although 5-HT_2c-R shares many similarities with other 5-HT receptors, especially those in group 2 (7, 8), we used antisense RNA probe synthesized from 1,050 to 1,235 bp, close to the 3' end of the coding region. According to Julius and coworkers (7, 20), this region is relatively unique and therefore unlikely to cross-hybridize with other 5-HT₂-R subtypes. At the mRNA level, gene expression for 5-HT_2c-R was identified by RT-PCR in extracts of isolated type II cell cultures as well as extracts of whole lung. Using NISH, strong signal for 5-HT_2c-R mRNA was localized in the cytoplasm of adult rat type II cells; whereas in fetal type II cells, the signal was weaker and was localized mostly in the perinuclear areas. At the receptor protein level, the pattern of 5-HT_2c-R localization was investigated by immunohistochemistry. In adult rat type II cells, 5-HT_2c-R immunoreactivity was localized primarily in the plasma membrane; whereas in fetal type II cells, positive immunoreactivity was found in the perinuclear region. There are several possible explanations for these findings. The differences in the localization of 5-HT_2c-R between fetal and adult type II cells could reflect developmental regulation. It should be noted, however, that in fetal rat brain, 5-HT-Rs appear fully functional and play a role in neuronal development (25). The other possibility could involve intracellular trafficking of receptor protein with translocation of receptors between the cell surface and endosomes (26). This situation may be analogous to that reported for muscarinic acetylcholine receptor (ACh-R), where agonist-induced intracellular receptor trafficking appears to be cell type- and receptor subtype- dependent (27). Significant agonist-induced internalization of receptor was observed in fibroblast (HeLa) and epithelial (HT29) cell lines, both of which express the m3 subtype of ACh-R (26). Although it is presently not known whether the differences in 5-HT_2c-R localization in type II cells are agonist-induced, it is well established that the distribution and frequency of 5-HT immunoreactive PNEC as well as 5-HT concentration in lung tissues show striking changes during development (28, 29). In most species, PNEC are more numerous in fetal/neonatal lungs than in those of adults (28). In the rat lung, however, the principal source of 5-HT appears to be mast cells, with significantly higher concentrations of 5-HT reported in the adult as compared with the neonate (29). Further studies are required to determine the precise mechanism of 5-HT_2c-R trafficking and its expression in alveolar type II cells during lung development.

The alveolar type II cells are involved in a variety of important functions in the lung, including surfactant synthesis, ion transport, and alveolar repair following injury (30). Whether 5-HT and its receptor are involved in any of these processes is currently speculative. Earlier studies suggested that 5-HT may inhibit amiloride-sensitive Na⁺ absorption in respiratory epithelium (31). These authors suggested that because 5-HT is an indoleamine structurally similar to amiloride, its role may involve local regulations of Na⁺ absorption, complementing the action of mineralocorticoids (31). The possible involvement of 5-HT_2c-R in ion transport is strengthened by the observation that this receptor is highly expressed at the apical membrane of chorioid plexus epithelial cells, known to be involved in the production and secretion of cerebrospinal fluid (22, 32). Further, mRNA derived from brain expressed in Xenopus oocyte model (normally not responsive to 5-HT) shows increased Cl conductances after application of 5-HT (33, 34). In the same model, 5-HT_2c-R expressed from cloned DNA was shown to mediate the closing of coexpressed rat-brain K⁺ channels in a Ca²⁺-independent manner (34).

The effects of 5-HT as a mitogen and/or growth factor have recently been documented as a part of neuromodulator substances acting via G protein-coupled receptor signaling pathway (4, 5, 7). Specifically, 5-HT has been shown to increase DNA synthesis in rat pulmonary vascular smooth-muscle cells in culture (35). These effects, however, are mediated via 5-HT_2a-R subtype. Of interest are studies with NIH 3T3 cells, where introduction of functional 2HT-_2a-R and 2-HT_2c-R results in generation of transformed foci at high frequency (36). The long-term maintenance of the transformed state requires continued activation of these 5-HT receptors, indicating that they may represent conditional proto-oncogenes (7). Although the direct effects of 5-HT on alveolar type II cell proliferation have not yet been investigated, we speculate that its actions in the alveolus could involve regulation of epithelial cell turnover as well as repair after injury.

Finally, our findings are consistent with a notion that the expression of different 5-HT-R subtypes in lung cells underlies the diversity of pulmonary responses induced by 5-HT released locally. Because lung parenchyma is a source of a variety of regulatory peptides, amine, and other bioactive substances (14), the direct effects as well as their interactions are likely to be multiple and complex. The full characterization of 5-HT-Rs in lung cells and their role in pulmonary function in normal and disease states await future investigations.