1 Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032; and 2 Department of Therapeutics, Institute of Cell Signaling, University Hospital of Nottingham, Nottingham NG7 2UH, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adenylyl cyclases are a nine-member family of differentially regulated enzymes responsible for the synthesis of cAMP. cAMP is an important second messenger that contributes to the regulation of airway smooth muscle tone. However, little is known regarding the expression and regulation of adenylyl cyclase isoforms in airway smooth muscle cells. Nondegenerate specific primers were designed for all nine known isoforms of human adenylyl cyclase. RT-PCR experiments were performed using total RNA extracted from whole human brain (positive control), whole rat brain (negative control), whole human trachea, human airway smooth muscle, and primary cultures of human airway smooth muscle cells. Seven of the nine known isoforms of adenylyl cyclase (isoforms I, III-VII, and IX) were expressed at the mRNA level in both human airway smooth muscle and primary cultures of human airway smooth muscle cells. Immunoblot and adenylyl cyclase functional assay indicated that isoform V is likely among the functionally predominant isoforms of adenylyl cyclase in human airway smooth muscle. These results suggest that multiple isoforms of adenylyl cyclase enzymes are coexpressed in human airway smooth muscle cells and that isoform V is among the functionally important isoforms.
reverse transcriptase-polymerase chain reaction; trachea; adenosine 3',5'-cyclic monophosphate; immunoblot; Sp-diasteromer of adenosine 3',5'-cyclic monophosphothioate; protein kinase A; protein kinase C
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
INCREASED cAMP mediates relaxation in airway smooth muscle by the activation of protein kinase A (PKA), which, in turn, activates a cascade of poorly understood pathways facilitating relaxation. These cellular targets of PKA result in decreased calcium levels and the phosphorylation of proteins favoring relaxation (8).
cAMP is synthesized by a nine-member family of adenylyl cyclase enzymes
that are divided into subfamilies based on their regulatory patterns in
response to products of other second messenger pathways (10). Group 1 includes isoforms I, III, and
VIII, which are stimulated by calcium/calmodulin (3, 5,
29). Group 2 includes isoforms II, IV, and VII, which
are regulated by G protein -subunits (4, 7, 11).
Group 3 includes isoforms V and VI, which are inhibited by
micromolar concentrations of calcium (16, 34), are
regulated by PKA (13) and protein kinase C (PKC; see Refs. 14, 17, 35), and are inhibited
by
-subunits (1). Group 4 includes
isoform IX, which is insensitive to calcium (23),
-subunits, or forskolin (32). Individual cell types
can simultaneously express multiple isoforms of adenylyl cyclases
(6, 19). Thus this allows for the complex regulation of
the net cellular synthesis of cAMP by second messenger products of
multiple other signaling pathways. It is unknown in airway smooth
muscle which isoforms of adenylyl cyclase are predominantly expressed,
how isoform-specific expression might be chronically regulated, or how
products of other second messenger pathways ultimately influence the
net synthesis of cAMP.
Adenylyl cyclase proteins are present in low levels in most cells (0.01-0.001% of membrane protein), making their immunodetection difficult (30). Thus most studies have relied on mRNA expression to determine organ- or cell-specific expression of different isoforms. Early mRNA studies relied on Northern blot analysis, which initially suggested that some isoforms had very limited expression (e.g., type III in neuroepithelium), but further studies using more sensitive detection techniques of ribonuclease protection and RT-PCR revealed a more diverse expression of most isoforms (28). Yet most of these mRNA surveys to date have relied upon tissue-wide distribution, with less information available regarding cell-specific expression within an organ (10, 24, 28). This is especially true in the lung where most adenylyl cyclase mRNA expression studies have evaluated mRNA in the whole lung (10, 28), which is a heterogeneous mixture of cell types of the respiratory, vascular, hematopoietic, and peripheral nervous systems. Little is known regarding the cell-specific expression of adenylyl cyclase isoforms in airway smooth muscle cells where the cellular levels of cAMP are a key determinant of airway smooth muscle tone. Studies of adenylyl cyclase mRNA expression in specific human cell types have been hampered to date by incomplete human cDNA sequence information for all nine human adenylyl cyclase isoforms. The current study was designed to first identify nondegenerate primers that would selectively amplify all nine known isoforms of human adenylyl cyclases. Subsequently, we sought to determine which mRNAs encoding isoforms of adenylyl cyclases are expressed in human airway smooth muscle cells using RT-PCR. Additionally, preactivation of PKA and PKC before adenylyl cyclase functional assays and immunoblot analyses offered insights into the likely functionally predominant isoforms of adenylyl cyclase proteins in human airway smooth muscle.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RNA Extraction
Total RNA from dissected human airway smooth muscle and total RNA from primary cultures of airway smooth muscle were used in these studies. Positive control total RNA from human whole brain, human whole trachea, and rat whole brain were commercially purchased (Clontech, Palo Alto, CA). These human brain and trachea RNAs were extracted from a pool of 70 male/female and 2 male tissue specimens, respectively. The whole human trachea RNA was extracted from the entire trachea, which included many cell types. Upon arrival, the RNA in ethanol was spun down at 15,000 g for 15 min to discard the ethanol supernatant, and the RNA pellet was washed with 70% ethanol and centrifuged again. The final pellet was dissolved in RNase-free water at 1 µg/µl and stored in aliquots atHuman airway smooth muscle was dissected from a region of trachea just
above the carina within 12 h of death. Tracheae were submerged in
cell culture medium during dissection under a dissecting microscope.
The submucosa was removed, and the tracheal smooth muscle was carefully
dissected away from the underlying connective tissue. Dissected
tracheal muscle was rapidly frozen and stored at 80°C until
isolation of total RNA. Detailed characterizations of the primary
cultures of human airway smooth muscle cells have been reported
previously (9). Total RNA from dissected airway smooth
muscle and from primary cultures of human airway smooth muscle cells
was extracted by a modified Chomczynski and Sacchi method using TRIzol
reagent (Life Technologies, Gaithersburg, MD). For dissected airway
smooth muscle, 200-400 mg of muscle were homogenized in 3-6
ml of TRIzol reagent. Samples were homogenized at top speed using an
Ultra-Turrex homogenizer (Tekmar-Dohrmann, Cincinnati, OH) equipped
with a T-25 probe. Five cycles of homogenization occurred for 5-10
s each, with cooling of the samples on ice between cycles. The
homogenates from dissected airway smooth muscle were subjected to
centrifugation at 12,000 g for 10 min to remove nonsoluble cellular debris before the supernatant was transferred to a clean tube
with the addition of a 1:5 volume of chloroform. For cultured cells, 5 ml of TRIzol reagent were added directly to a 75-cm2 flask,
and the cells were transferred to tubes. After incubation at room
temperature for 10 min, 1:5 volume of chloroform was added. After
vigorous shaking and room temperature incubation for 3 min, both the
homogenate and cultured cell samples were centrifuged at 12,000 g for 15 min at 4°C to separate the phases. The upper aqueous phase containing the RNA was transferred to a clean tube to
which an equal volume of isopropyl alcohol was added. Centrifugation at
12,000 g for 10 min at 4°C yielded an RNA pellet that was
washed in 75% ethanol followed by centrifugation at 7,500 g
for 5 min at 4°C. The final resulting RNA pellet was air-dried and
redissolved in 100 µl of RNase-free water.
Adenylyl Cyclase Isoform-Specific Primer Design
Primers were designed using Oligo 5.0 software and were based on published GenBank sequences for human adenylyl cyclase isoforms (Table 1), except for isoform IV for which no human sequence was available. Isoform IV primers were designed based on a partial human GenBank sequence (AF088070) that has high homology to rat adenylyl cyclase type IV (M80633). Primer pairs were chosen to yield expected PCR products of 150-600 bp. All primers were purchased from Genosys (The Woodlands, TX) or Life Technologies with standard purification. All primers were dissolved in DNase- and RNase-free water at a stock concentration of 100 µM, further diluted to a working concentration of 10 µM, and stored at
|
RT-PCR
Total RNA from whole human brain was used as a positive control to ensure that RT-PCR yielded an appropriately sized PCR product for each human-specific adenylyl cyclase primer set. Rat whole brain total RNA was used as a negative control, since all human primer sets used except isoforms V and VIII have low homology to the respective rat adenylyl cyclase sequences. Additional controls included parallel tubes containing all reaction components except reverse transcriptase to ensure that PCR products did not arise from contaminating cDNA or genomic DNA. Parallel tubes that contained all reaction components except RNA were also included to ensure a lack of reagent contamination. RT-PCR experiments were performed with RNA isolated from at least three independent samples of dissected human tracheal smooth muscle and three independent samples of human primary cultured cells for each adenylyl cyclase primer set.DNase pretreatment, RT, and PCR were all performed on an RNA sample in a single tube. Unless indicated otherwise, 1 µg of total RNA from commercial sources (positive controls) or from dissected or primary cultured human airway smooth muscle was used in each tube. Initially, all RNA samples were treated with RNase-free DNase (Ambion, Austin, TX) for 1 h at 37°C to degrade contaminating DNA. Total RNA was incubated with RNase-free DNase (0.5 µl; 2 U/µl) in the same tube, and buffer was used for subsequent RT-PCR [1× concentration of proprietary reaction buffer (Access RT-PCR kit; Promega, Madison, WI), 1 mM MgSO4, 0.2 mM dNTPs, and 0.8 µM each primer] in a volume of 24 µl. Samples were then heated to 70°C for 5 min to inactivate the DNase enzyme, and, after cooling on ice, 0.5-µl aliquots each of AMV reverse transcriptase (5 U/µl) and Tfl DNA polymerase (5 U/µl) were added to each tube. First-strand cDNA synthesis was performed at 48°C for 45 min, at which time the reaction was heated to 94°C for 5 min to inactivate the reverse transcriptase.
PCR was immediately performed for 40 cycles in a thermal cycler (PT-200 MJ Research, Waltham, MA) equipped with a heated lid. Denaturation and extension temperatures/times of 95°C/30 s and 68°C/1 min were used, respectively. One-minute annealing times were used for all reactions at 52°C for isoforms I, II, and VIII, at 50°C for isoform III, at 55°C for isoforms IV, V, and VII, and at 58°C for isoforms VI and IX.
Nondenaturing Polyacrylamide Gel, Restriction Endonucleases Analysis, and DNA Sequencing
PCR products were separated on nondenaturing 5-8% polyacrylamide gels (mono/bis = 39:1) using a 100-bp DNA ladder (Promega) as a DNA size standard and as an estimate of DNA concentrations. Gels were stained with ethidium bromide and visualized using a Fluor-S MultiImager (Bio-Rad, Hercules, CA). PCR products were isolated from gels by excising a gel slice and incubating overnight at 37°C in a minimal volume of water. Eluted DNA was quantified by ultraviolet (UV) spectrophotometry. Two approaches were used to confirm the identity of RT-PCR products. Restriction enzyme digests were performed using enzymes predicted to cut within the specifically amplified adenylyl cyclase sequence. Digestion maps were generated using WebCutter software. All restriction digests were performed for 2 h at 37°C in a final volume of 10 µl. Digested and undigested RT-PCR products were analyzed on 5-8% polyacrylamide gels. Second, specifically identified RT-PCR products were directly sequenced using automated sequence analysis at the DNA Core Facility of the Columbia University Cancer Center. Sequence analysis was visualized using EditView 1.0.1, and homologies to expected adenylyl cyclase sequences were confirmed using National Center for Biotechnology Information Blast Search.Preparation of Sense RNA for Adenylyl Cyclase Types II and VIII
For the two isoforms of adenylyl cyclase not identified in freshly dissected or cultured airway smooth muscle (isoforms II and VIII), we generated sense RNA templates of known quantity. By adding these templates of known quantities back to our RNA preparations, we could evaluate the following two issues: 1) we could be sure that the lack of RT-PCR products obtained with smooth muscle RNA was not the result of the presence of a nonspecific inhibitor of the RT-PCRs in these samples, and 2) by using serial dilutions of the control sense RNA template, we could determine the sensitivity of our RT-PCR and therefore know the lower limit of sensitivity for the detection of isoforms II and VIII in our samples.RT-PCR products for isoforms II and VIII were obtained from RT-PCRs from RNA of total human brain. These PCR products were gel purified, excised, and eluted in water overnight. Eluted cDNA then served as the template for a second round of PCR using Taq polymerase to obtain PCR products with "T-A" overhangs at each end. These PCR products were subcloned into pCR II-TOPO (Invitrogen, Carlsbad, CA) in which Sp6 and T7 RNA polymerase binding sites flank the insertion site. Plasmids were transformed into competent Escherichia coli, and isolated colonies were chosen. Aliquots of colonies were lysed at 94°C for 5 min in PCR buffer solution containing primers for either adenylyl cyclase II or VIII. Thirty cycles of PCR were initiated with the addition of Tfl DNA polymerase. Aliquots of PCRs were subjected to gel electrophoresis, and reactions containing products of appropriate size were subjected to another round of PCR using M13 forward and reverse primers, which yielded a linear template with Sp6 and T7 polymerase sites at each end of the adenylyl cyclase DNA fragment. Orientation was confirmed by restriction enzyme digests. The products of these PCRs were gel purified and used as the DNA template for the synthesis of sense RNA using an in vitro transcription kit (Ambion).
These newly synthesized sense RNA transcripts for adenylyl cyclases II
and VIII were quantitated by UV spectrophotometry and serially diluted
before storage at 80°C. These RNA sense transcripts for adenylyl
cyclase II or VIII were added to RNA samples of airway smooth muscle at
10-fold decreasing quantities (from 1 pg to 0.1 fg/reaction) to confirm
our ability to amplify this RNA control template in the presence of
airway smooth muscle total RNA and also to determine the lowest
quantity of RNA template that could be detected.
Additional Attempts to Amplify mRNA Encoding Adenylyl Cyclase II
Because of a recent report that adenylyl cyclase II mRNA was amplified by RT-PCR in human cultured airway smooth muscle cells (2), we performed additional experiments with the specific primers described in this previous report. The sense primer was 5'-acgtctcgagcgactacagccaggtcttat-3', and the antisense primer was 5'-gttgctcgagatatcatattgtggcttctgagc-3'. RT-PCR conditions were identical to those described above with an annealing temperature of 55°C. Parallel tubes were assembled containing 1 µg of total RNA extracted from whole human brain, human airway smooth muscle, and primary cultures of airway smooth muscle cells.Adenylyl Cyclase Assays and Pretreatments
Adenylyl cyclase activity was measured as previously described (12). PKC and PKA are known to selectively regulate certain isoforms of adenylyl cyclase. To determine if these isoforms are functionally important in airway smooth muscle, PKC or PKA were preactivated, using phorbol ester or the Sp-diastereomer of adenosine 3',5'-cyclic monophosphothioate (Sp-cAMPS), respectively, before forskolin-stimulated adenylyl cyclase activity was measured. Briefly, confluent cells in 24-well plates were washed one time with warm PBS (37°C) and resuspended in 100 µl of warm PBS. Cells were pretreated for 15 min with nothing, 1 µM phorbol 12-myristate 13-acetate, or 100 µM Sp-cAMPS. Fifty microliters of 3× adenylyl cyclase buffer (12) were added directly to the wells, and plates were incubated at 37°C for 10 min. The reactions were terminated by addition of 100 µl of stop buffer, and synthesized [32P]cAMP was quantitated as described previously (12).Immunoblot Analysis
Membranes were prepared from confluent cultured human airway smooth muscle cells grown in T-75 flasks and whole rat brain. Whole rat brain was used as a positive control for immunoblotting, since all known isoforms of adenylyl cyclase are expressed in brain. Cultured cells were washed two times with 10 ml of wash buffer (cold PBS containing 2 mM EDTA and 2 mM EGTA). From this point on, all preparations were kept at 4°C. Cells were scraped in 10 ml of wash buffer and subjected to centrifugation at 1,000 g for 5 min to pellet cells. The pellet was resuspended in 10 ml of extraction buffer [20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, and protease inhibitor cocktail tablets (EDTA free; Roche Molecular Biochemicals, Indianapolis, IN)] containing 10% sucrose. Cells were homogenized for three 15-s bursts using the top setting of high-speed cutting blades (T-25, Tissumizer Mark; Tekmar-Dohrmann). The homogenate was centrifuged at 750 g for 5 min at 4°C, and the supernatant was then subjected to centrifugation at 163,000 g for 1 h at 4°C. The final pellet was dissolved in a small volume (0.5-1.0 ml) of extraction buffer containing 1% SDS. An aliquot was diluted 1:10 with water for protein determination. Frozen whole rat brain was crushed in a cold mortar and pestle. The ground tissue was resuspended in 10 ml of cold extraction buffer containing 10% sucrose and homogenized and centrifuged as described above for cultured cells.Immunoblot analysis was performed as previously described (12) with minor modifications. Total protein (200-400 µg) was solubilized in sample buffer and subjected to electrophoresis through discontinuous 8% polyacrylamide gels. After overnight transfer to polyvinylidene difluoride filters, blocking, primary antibody, secondary antibody, and chemiluminescence detection were performed as described (12). Primary antibodies specific for adenylyl cyclase I-IV, V/VI, and VII-IX were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a dilution of 1:200 for 2 h at room temperature. Secondary antibodies (donkey anti-rabbit Ig-horseradish peroxidase for isoforms I-IV and V/VI; donkey anti-goat IgG-horseradish peroxidase for isoforms VII-IX) were used at a dilution of 1:3,000 for 90 min.
Statistics
An unpaired two-tailed t-test was used to compare forskolin-stimulated adenylyl cyclase activities between control (untreated) and pretreated (either phorbol or Sp-cAMPS) cells. A P value of <0.05 was considered statistically significant. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Detection of Nine Adenylyl Cyclase Isoforms Using Type-Specific Primers
Results using whole tissue RNA preparations.
RT-PCR using total RNA from whole human brain yielded products of
expected sizes for each of the nine adenylyl cyclase primer sets
(positive controls; Figs.
1-3).
These RT-PCR products were subjected to both restriction enzyme digests
and direct sequencing to ensure that they represented an amplified
sequence of the intended adenylyl cyclase isoform. The expected size of
RT-PCR products, the specific restriction endonucleases that cleave at
specific internal sites, and the resulting sizes of digested DNA
fragments are presented in Table 2.
Direct sequencing of RT-PCR products amplified with primers specific
for adenylyl cyclases I-III and V-IX confirmed that these
products represented eight isoforms of human adenylyl cyclase.
|
|
|
|
Results using RNA isolated from freshly dissected or primary cultures of human airway smooth muscle. Identical RT-PCR results were obtained for all isoforms of adenylyl cyclase primers for RNA extracted from either freshly dissected or primary cultures of human airway smooth muscle. At least three independent RNA samples from each source were analyzed with each set of adenylyl cyclase primers. For adenylyl cyclases III-VII and IX, PCR products of expected size and correct identity were obtained from both sources of RNA using 1.0 µg total RNA/reaction. Adenylyl cyclase I was only detected when the amount of total RNA was increased to 5 µg/reaction. In contrast, no appropriate RT-PCR products were obtained using 1-5 µg of total RNA from either freshly dissected or primary cultures of human airway smooth muscle for either adenylyl cyclase II or VIII despite parallel reactions with RNA from whole human brain, which yielded RT-PCR products of expected size and identity. Additionally, the same smooth muscle RNA used in this series of studies did yield RT-PCR products using primers designed to amplify adenylyl cyclase III, attesting to the integrity of the RNA sample.
Parallel negative control tubes were analyzed in each RT-PCR in which either RT or RNA was left out of the reaction mixture. These negative controls yielded no RT-PCR products, suggesting that no cDNA or genomic DNA contamination was present in the RNA samples and that no cDNA contamination was introduced during assembly of RT-PCRs (Fig. 4).
|
Restriction enzyme digests and sequencing of PCR products. RT-PCR products for each adenylyl cyclase isoform were isolated from gels and subjected to both restriction enzyme digests and direct sequencing. RT-PCR products for isoforms I, III-VII, and IX were isolated from reactions using RNA from human cultured airway smooth muscle cells. RT-PCR products for isoforms II and VIII were isolated from reactions using RNA from whole human brain. All nine PCR products were digested into fragments of predicted sizes as shown in Table 2. Additionally, direct sequencing confirmed homology between the isolated RT-PCR products and their respective human adenylyl cyclase cDNA sequences, with homologies of 99-100%.
Addition of control sense templates of isoforms II and VIII to airway smooth muscle RNA. To confirm that the lack of RT-PCR products for isoforms II and VIII from airway smooth muscle RNA was not the result of nonspecific interference with amplification of these isoforms, control sense RNA transcripts were generated and in serially decreasing concentrations added to airway smooth muscle RNA. This was to ensure that our airway smooth muscle RNA preparations did not nonspecifically interfere with the ability of isoform II or VIII primers to amplify sense RNA in our reaction mixtures. Additionally, this strategy indicated the sensitivity of the RT-PCRs, since the control sense RNA transcripts were added in known quantities.
As shown in Fig. 5, 10-1,000 fg of control sense RNA input resulted in RT-PCR amplification of the expected 369-bp fragment despite the presence of 1 µg of total RNA from human airway smooth muscle in each tube. In contrast, no products were obtained with 1,000 fg of control sense RNA in the absence of reverse transcriptase, and no products were obtained with control sense RNA of <10 fg. Control tubes lacking the sense control but containing human airway smooth muscle RNA also yielded no products, confirming the experiments shown in Fig. 1 for adenylyl cyclase II.
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The current study demonstrates that the mRNA encoding multiple isoforms of adenylyl cyclase (isoforms I, III-VII, and IX) is expressed in airway smooth muscle. The studies were performed in both freshly dissected human airway smooth muscle and primary cultures of human airway smooth muscle cells. Both preparations were evaluated, since even meticulously dissected airway smooth muscle is not a pure preparation of only airway smooth muscle, whereas the primary cultures of airway smooth muscle cells are a homogeneous cell population that expresses a smooth muscle cell phenotype. Conversely, we were concerned that expression of adenylyl cyclase mRNA may change when freshly isolated airway smooth muscle cells were established as a primary cell line and thus wanted to compare RNA from cultured airway smooth muscle with RNA isolated from freshly dissected human airway smooth muscle. We obtained identical results using RNA from fresh or cultured airway smooth muscle cells, suggesting that our results truly reflect mRNA expression in airway smooth muscle and that isoform-specific expression of adenylyl cyclases is maintained in primary cultures of airway smooth muscle cells. In an attempt to extend this study to the level of protein expression and function, both adenylyl cyclase functional assays and immunodetection of adenylyl cyclase proteins were performed, suggesting that adenylyl cyclase V may be among the functionally dominant isoforms of adenylyl cyclase in airway smooth muscle.
In the current study, we used a strategy of increasing cell-type purity in an effort to ensure that the detected adenylyl cyclase mRNA isoforms were actually expressed in smooth muscle cells and did not represent mRNA from other contaminating cell types. RNA from whole human brain served as a positive control to ensure that adenylyl cyclase-specific primers successfully amplified respective isoforms of human adenylyl cyclases. RNA from whole rat brain served as a negative control. We then used three different types of RNA preparations from the airway with increasing levels of purity of airway smooth muscle cells. First, RNA from whole human trachea was evaluated, followed by RNA from carefully dissected human tracheal smooth muscle, and finally RNA from primary cultures of human airway smooth muscle cells. Results were identical using RNA isolated from freshly dissected or primary cultures of human airway smooth muscle, but results differed in RNA isolated from whole human trachea. All isoforms except II and VIII were identified in airway smooth muscle, whereas all isoforms were identified in RNA from whole trachea. Moreover, isoform I was detected using only 1 µg of total RNA from whole trachea compared with needing 5 µg of total RNA in airway smooth muscle to obtain a product. Thus this suggests that adenylyl cyclases II and VIII identified in RNA of whole trachea are arising from cell types other than airway smooth muscle. Additionally, because isoform I was easier to detect in whole tracheal RNA compared with airway smooth muscle RNA, isoform I mRNA likely comprises a larger proportion of the total RNA in whole trachea as opposed to airway smooth muscle.
However, quantitative comparisons between tissues and between adenylyl cyclase isoforms must be made with extreme caution using the technique of RT-PCR. We did not attempt to determine which of the nine isoforms of mRNA encoding adenylyl cyclases are expressed in greatest abundance in the current study because of quantitative limitations of RT-PCR. The intensity of resulting bands from RT-PCRs cannot be directly related to the amount of starting mRNA in a preparation because of several technical limitations that limit the ability to compare band intensities and surmise predominant isoforms. These limitations include the fact that different primer pairs do not anneal with identical affinities to different mRNA targets during reverse transcription or to different cDNA targets during PCR. Moreover, RT and PCR do not occur with perfect annealing and amplification during each cycle, meaning that small differences between annealing and amplification efficiencies in the early rounds of PCR are logarithmically amplified after the typical 30-40 total cycles of PCR. Traditionally competitive templates of cDNA or, ideally, cRNA have been used during competitive PCR or competitive RT-PCR, respectively, to extend the quantitative capabilities of this technique.
This is the first study in any human tissue to systemically evaluate the expression of all nine known isoforms of adenylyl cyclase by RT-PCR. This is in part because the partial sequence for each of the human adenylyl cyclase isoforms has not been known. Currently, eight of the nine isoforms have a known cDNA sequence (21-23, 27, 31, 33) and GenBank direct submissions (U65473 and U65474). Only identification of human adenylyl cyclase IV was lacking. However, two unidentified human partial cDNA sequences with near-perfect homology to each other (AF088070 and AF086230) have 88% cDNA homology and 96% predicted protein homology to the 3'-end of the coding region of rat adenylyl cyclase IV. Although these previously unidentified human partial cDNA sequences might be expected to encode other members of this subfamily of human adenylyl cyclases (i.e., isoforms II or VII), human sequence AF088070 had only 81 and 82% homology to human adenylyl cyclases II (GenBank X74210) and VII (GenBank NM_001114), respectively. This close homology to rat adenylyl cyclase IV and lack of homology to related human adenylyl cyclase isoforms is very strong evidence that AF088070 represents a portion of human adenylyl cyclase IV.
A potential limitation of the evaluation of adenylyl cyclase isoform expression by RT-PCR is that this technique may be so sensitive as to amplify mRNA of low abundance that may encode for a physiologically insignificant amount of a particular isoform of adenylyl cyclase protein. Characterization of the expression of protein levels of various adenylyl cyclase isoforms in native cells is difficult because of a limited selection of commercially available antibodies and the difficulty in detecting a protein expressed in native tissues at relatively low levels (0.01-0.001% of membrane protein; see Ref. 28). In an attempt to gain insights into the likely functionally predominant isoforms of adenylyl cyclase protein in airway smooth muscle, we used two approaches. First, immunoblot detection was attempted for all nine isoforms using commercially available antibodies. Second, the known selective effects of PKA and PKC activation on certain adenylyl cyclase isoforms was exploited to implicate potentially functionally important isoforms. Adenylyl cyclase proteins I-V/VI and VIII were detected by immunoblot analysis in membranes prepared from whole rat brain, but only isoform V/VI was detected in airway smooth muscle. This suggests that isoforms V and/or VI may be one of the predominant adenylyl cyclases expressed and is consistent with our RNA results where mRNA encoding isoforms II and VIII was not detected and increased amounts of input RNA (5 µg) were necessary to detect isoform I in cultured airway smooth muscle. The failure to detect adenylyl cyclase proteins III and IV may suggest that despite the expression of mRNA in airway smooth muscle, the relative expression of protein is low. Conversely, it is also possible that the affinity of the antibodies for isoforms III and IV is less than that of the antibody for isoform V/VI or that the antibody specificity for the human isoforms of isoforms III and IV is low, accounting for our negative immunoblots in human airway smooth muscle despite detection in rat brain. We were unable to detect proteins for isoforms VII and IX in airway smooth muscle or rat brain, suggesting that either 1) these proteins are expressed at low levels in both tissues, 2) the primary antibodies are of poor quality or low affinity, or 3) the antibodies have low cross-species reactivity (antibody for VII was designed against mouse, and antibody for IX was designed against human). Therefore, immunoblot analysis does not allow for conclusions to be drawn about the expression of isoforms VII and IX in human cultured airway smooth muscle.
Because isoforms V/VI were easily detected at both the mRNA and protein levels, we next performed functional assays to further support their important functional expression. Pretreatment of human cultured airway smooth muscle cells with phorbol ester, an agent activating PKC, which, in turn, is known to acutely activate isoforms II, V, and VII (15, 17, 35) and inhibit isoform VI (18), resulted in an increase in forskolin-stimulated activity. Because isoform II was not detected at the mRNA or protein level, these functional data would suggest that isoforms V or VII may be the functionally predominant isoforms in cultured human airway smooth muscle. This was further supported in adenylyl cyclase functional assays in which cells were pretreated with Sp-cAMPS to activate PKA (known to acutely inhibit isoforms V and VI; see Ref. 13), which resulted in decreased forskolin-stimulated adenylyl cyclase activity. Taken together, these functional studies, along with mRNA and immunoblot analyses, suggest that isoform V may be a functionally dominant isoform of adenylyl cyclase (although an important functional role for isoforms VII or IX cannot be ruled out). Despite these functional insights, it should be noted that a full understanding of the effects of acute activation of PKA or PKC on all isoforms of adenylyl cyclase is lacking.
Most previous studies of adenylyl cyclase isoform expression have relied on Northern blot analysis of RNA extracted from whole organs (10, 28). Although Northern blot analysis is likely to identify the most abundant adenylyl cyclase mRNAs expressed, these studies are hampered by not knowing from which cell type the mRNA originated. For example, the lung is reported to express isoforms II-VII and IX (10, 28), but it is unknown in these Northern blot studies which cell types are expressing these mRNAs, since RNA extracted from whole lung contains RNA from pulmonary, vascular, neural, and hematopoietic cells. To understand unique cell type-specific regulation of adenylyl cyclase isoforms, directed evaluation of isoform-specific expression needs to be evaluated in individual cell types.
A limited number of studies have evaluated the expression of adenylyl cyclase mRNA isoforms in smooth muscle, and even fewer studies have evaluated their expression in airway smooth muscle. Northern blot analysis has been used to evaluate adenylyl cyclase mRNA expression in vascular, gut, and uterine smooth muscle. Adenylyl cyclase III but not I or VIII was identified in rat aortic smooth muscle (36), whereas guinea pig ileal smooth muscle was found to express isoforms I and IV (26) by Northern blotting. Uterine smooth muscle was found to express isoforms II-VI and IX in humans and II-VII and IX in rats by Northern analysis (20). Using the more sensitive technique of RT-PCR, we recently demonstrated mRNA for isoforms II-IX plus a unique splice variant for isoform IV in rat uterine smooth muscle (6).
The current study agrees in part with a recent study in cultured human airway smooth muscle cells in which four isoforms of adenylyl cyclase mRNA were identified (2). RT-PCR employing predominantly degenerate primers was used to evaluate adenylyl cyclase mRNA expression. Isoforms VI and IX were identified using nonselective degenerate primers, and isoforms II and VII were identified using isoform-specific primers. In contrast, in the current study, we did not identify mRNA for isoform II in RNA isolated from either cultured or freshly isolated airway smooth muscle despite using the same primers and PCR conditions. Additionally, in the present study, a second pair of isoform II primers did not identify isoform II mRNA from either cultured or freshly isolated airway smooth muscle. Both sets of primers for isoform II yielded RT-PCR products of expected sizes using RNA from whole brain or using a positive control template for isoform II, suggesting that both primer sets were capable of amplifying isoform II using the RT-PCR conditions employed in the present study. It is unclear why our results with isoform II differ from this previous study (2), but our results in cultured cells concur with our results in freshly isolated airway smooth muscle.
The current study does not agree with a previous study in primary cultures of guinea pig airway smooth muscle cells in which immunoblot analysis identified the expression of adenylyl cyclase II protein (25). This previous study did not attempt to evaluate the expression of other adenylyl cyclase isoforms but differs from the current study in that type II was not identified in RNA from freshly dissected or primary cultures of human airway smooth muscle cells. This suggests a possible species difference in the expression of adenylyl cyclase isoforms in airway smooth muscle.
In summary, we have identified mRNA encoding for the expression of multiple isoforms of adenylyl cyclase in human airway smooth muscle. Results were identical in RNA isolated from freshly dissected and primary cultures of human airway smooth muscle. Immunoblot and functional assays suggest that isoform V is one of the functionally significant isoforms of adenylyl cyclase in human airway smooth muscle. These results suggest that the synthesis of cAMP in an airway smooth muscle cell is the net result of the activities of multiple isoforms of uniquely regulated adenylyl cyclases.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: C. W. Emala, Dept. of Anesthesiology, College of Physicians and Surgeons of Columbia Univ., 630 W. 168th St., PH 5, New York, NY 10032 (E-mail: cwe5{at}columbia.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 April 2000; accepted in final form 23 May 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bayewitch, ML,
Avidor-Reiss T,
Levy R,
Pfeuffer T,
Nevo I,
Simonds WF,
and
Vogel Z.
Inhibition of adenylyl cyclase isoforms V and VI by various subunits.
FASEB J
12:
1019-1025,
1998
2.
Billington, CK,
Hall IP,
Mundell SJ,
Parent J,
Panettieri RA,
Benovic JL,
and
Penn RB.
Inflammatory and contractile agents sensitize specific adenylyl cyclase isoforms in human airway smooth muscle.
Am J Respir Cell Mol Biol
21:
597-606,
1999
3.
Cali, JJ,
Zwaagstra JC,
Mons N,
Cooper DM,
and
Krupinski J.
Type VIII adenylyl cyclase A Ca2+ calmodulin-stimulated enzyme expressed in discrete regions of rat brain.
J Biol Chem
269:
12190-12195,
1994
4.
Chen, J,
DeVivo M,
Dingus J,
Harry A,
Li J,
Sui J,
Carty DJ,
Blank JL,
Exton JH,
Stoffel RH,
Inglese J,
Lefkowitz RJ,
Logothetis DE,
Hildebrandt JD,
and
Iyengar R.
A region of adenylyl cyclase 2 critical for regulation by G protein subunits.
Science
268:
1166-1169,
1995[ISI][Medline].
5.
Choi, EJ,
Xia Z,
and
Storm DR.
Stimulation of the type III olfactory adenylyl cyclase by calcium and calmodulin.
Biochemistry
31:
6492-6498,
1992[ISI][Medline].
6.
Emala, CW,
Kumasaka D,
Hirshman CA,
and
Lindeman KS.
Adenylyl cyclase messenger ribonucleic acid in myometrium: splice variant of type IV.
Biol Reprod
59:
169-175,
1998
7.
Federman, AD,
Conklin BR,
Schrader KA,
Reed RR,
and
Bourne HR.
Hormonal stimulation of adenylyl cyclase through Gi-protein subunits.
Nature
356:
159-161,
1992[ISI][Medline].
8.
Hakonarson, H,
and
Grunstein MM.
Regulation of second messengers associated with airway smooth muscle contraction and relaxation.
Am J Respir Crit Care Med
158:
S115-S122,
1998
9.
Hall, IP,
and
Kotlikoff M.
Use of cultured airway myocytes for study of airway smooth muscle.
Am J Physiol Lung Cell Mol Physiol
268:
L1-L11,
1995
10.
Hanoune, J,
Pouille Y,
Tzavara E,
Shen T,
Lipskaya L,
Miyamoto N,
Suzuki Y,
and
Defer N.
Adenylyl cyclases: structure, regulation and function in an enzyme superfamily.
Mol Cell Endocrinol
128:
179-194,
1997[ISI][Medline].
11.
Hellevuo, K,
Berry R,
Sikela JM,
and
Tabakoff B.
Localization of the gene for a novel human adenylyl cyclase (ADCY7) to chromosome 16.
Hum Genet
95:
197-200,
1995[ISI][Medline].
12.
Hotta, K,
Emala CW,
and
Hirshman CA.
TNF- upregulates Gi
and Gq
protein expression and function in human airway smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
276:
L405-L411,
1999
13.
Iwami, G,
Kawabe J,
Ebina T,
Cannon PJ,
Homcy CJ,
and
Ishikawa Y.
Regulation of adenylyl cyclase by protein kinase A.
J Biol Chem
270:
12481-12484,
1995
14.
Jacobowitz, O,
Chen J,
Premont RT,
and
Iyengar R.
Stimulation of specific types of Gs-stimulated adenylyl cyclases by phorbol ester treatment.
J Biol Chem
268:
3829-3832,
1993
15.
Jacobowitz, O,
and
Iyengar R.
Phorbol ester-induced stimulation and phosphorylation of adenylyl cyclase 2.
Proc Natl Acad Sci USA
91:
10630-10634,
1994
16.
Katsushika, S,
Chen L,
Kawabe JI,
Nilakantan R,
Halnon NJ,
Homcy CJ,
and
Ishikawa Y.
Cloning and characterization of a sixth adenylyl cyclase isoform: types V and VI constitute a subgroup within the mammalian adenylyl cyclase family.
Proc Natl Acad Sci USA
89:
8774-8778,
1992[Abstract].
17.
Kawabe, J,
Iwami G,
Ebina T,
Ohno S,
Katada T,
Ueda Y,
Homcy CJ,
and
Ishikawa Y.
Differential activation of adenylyl cyclase by protein kinase C isoenzymes.
J Biol Chem
269:
16554-16558,
1994
18.
Lai, HL,
Yang TH,
Messing RO,
Ching YH,
Lin SC,
and
Chern Y.
Protein kinase C inhibits adenylyl cyclase type VI activity during desensitization of the A2a-adenosine receptor-mediated cAMP response.
J Biol Chem
272:
4970-4977,
1997
19.
Manolopoulos, VG,
Liu J,
Unsworth BR,
and
Lelkes PI.
Adenylyl cyclase isoforms are differentially expressed in primary cultures of endothelial cells and whole tissue homogenates from various rat tissues.
Biochem Biophys Res Commun
208:
323-331,
1995[ISI][Medline].
20.
Mhaouty-Kodja, S,
Bouet-Alard R,
Limon-Boulez I,
Maltier JP,
and
Legrand C.
Molecular diversity of adenylyl cyclases in human and rat myometrium.
J Biol Chem
272:
31100-31106,
1997
21.
Nomura, N,
Miyajima N,
Sazuka T,
Tanaka A,
Kawarabayasi T,
Sato S,
Nagase T,
Seki N,
Ishikawa K,
and
Tabata T.
Prediction of the coding sequences of unidentified human genes. I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) deduced by analysis of randomly sampled cDNA clones from human immature myeloid cell line KG-1.
DNA Res
1:
27-35,
1994[Medline].
22.
Parma, J,
Stengel D,
Gannage MH,
Poyard M,
Barouki R,
and
Hanoune J.
Sequence of a human brain adenylyl cyclase partial cDNA: evidence for a consensus cyclase specific domain.
Biochem Biophys Res Commun
179:
455-462,
1991[ISI][Medline].
23.
Paterson, JM,
Smith SM,
Harmar AJ,
and
Antoni FA.
Control of a novel adenylyl cyclase by calcineurin.
Biochem Biophys Res Commun
214:
1000-1008,
1995[ISI][Medline].
24.
Pieroni, JP,
Miller D,
Premont RT,
and
Iyengar R.
Type 5 adenylyl cyclase distribution.
Nature
363:
679-680,
1993[ISI][Medline].
25.
Pyne, NJ,
Moughal N,
Stevens PA,
Tolan D,
and
Pyne S.
Protein kinase C-dependent cyclic AMP formation in airway smooth muscle: the role of type II adenylate cyclase and the blockade of extracellular-signal-regulated kinase-2 (ERK-2) activation.
Biochem J
304:
611-616,
1994[ISI][Medline].
26.
Rivera, M,
and
Gintzler AR.
Differential effect of chronic morphine on mRNA encoding cyclase isoforms: relevance to physiological sequela of tolerance/dependance.
Mol Brain Res
54:
165-169,
1998[ISI][Medline].
27.
Stengel, D,
Parma J,
Gannage MH,
Roeckel N,
Mattei MG,
Barouki R,
and
Hanoune J.
Different chromosomal localization of two adenylyl cyclase genes expressed in human brain.
Hum Genet
90:
126-130,
1992[ISI][Medline].
28.
Sunahara, RK,
Dessauer CW,
and
Gilman AG.
Complexity and diversity of mammalian adenylyl cyclases.
Annu Rev Pharmacol Toxicol
36:
461-480,
1996[ISI][Medline].
29.
Tang, WJ,
Krupinski J,
and
Gilman AG.
Expression and characterization of calmodulin-activated (type I) adenylyl cyclase.
J Biol Chem
266:
8595-8603,
1991
30.
Taussig, R,
and
Gilman AG.
Mammalian membrane-bound adenylyl cyclases.
J Biol Chem
270:
1-4,
1995
31.
Villacres, EC,
Xia Z,
Bookbinder LH,
Edelhoff S,
Disteche CM,
and
Storm DR.
Cloning, chromosomal mapping, and expression of human fetal brain type I adenylyl cyclase.
Genomics
16:
473-478,
1993[ISI][Medline].
32.
Yan, SZ,
Huang ZH,
Andrews RK,
and
Tang WJ.
Conversion of forskolin-insensitive to forskolin-sensitive (mouse-type IX) adenylyl cyclase.
Mol Pharmacol
53:
182-187,
1998
33.
Yang, B,
He B,
Abdel-Halim SM,
Tibell A,
Brendel MD,
Bretzel RG,
Efendic S,
and
Hillert J.
Molecular cloning of a full-length cDNA for human type 3 adenylyl cyclase and its expression in human islets.
Biochem Biophys Res Commun
254:
548-551,
1999[ISI][Medline].
34.
Yoshimura, M,
and
Cooper DM.
Cloning and expression of a Ca2+-inhibitable adenylyl cyclase from NCB-20 cells.
Proc Natl Acad Sci USA
89:
6716-6720,
1992[Abstract].
35.
Yoshimura, M,
and
Cooper DMF
Type-specific stimulation of adenylyl cyclase by protein kinase C.
J Biol Chem
268:
4604-4607,
1993
36.
Zhang, J,
Sato M,
Duzic E,
Kubalak SW,
Lanier SM,
and
Webb JG.
Adenylyl cyclase isoforms and vasopressin enhancement of agonist-stimulated cAMP in vascular smooth muscle cells.
Am J Physiol Heart Circ Physiol
273:
H971-H980,
1997
37.
Zimmermann, G,
and
Taussig R.
Protein kinase C alters the responsiveness of adenylyl cyclases to G protein and
subunits.
J Biol Chem
271:
27161-27166,
1996