Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557 - 0046
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ABSTRACT |
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Calmodulin (Cal) plays important roles for contractile activity in smooth muscles. Recently, two distinct Ca2+-binding protein superfamilies with sequence similarities to Cal have been identified in neuronal cells: neuronal Ca2+-binding proteins (NCBPs) and Cal-like Ca2+-binding proteins (CaBPs). Some NCBPs and CaBPs play significant roles for Ca2+-dependent cellular signaling in the nervous system. In gastrointestinal smooth muscles (GISMs), Cal functions as the regulator of contractile behavior and electrical rhythmicity. However, the molecular identification of NCBPs and CaBPs has not been elucidated in GISMs. Here, we have identified NCBPs and CaBPs expressed in GISMs and determined the expression levels of their transcripts by quantitative RT-PCR. Of 12 NCBPs, the transcripts for neuronal Ca2+ sensor 1, neural visinin-like proteins 1, 2, and 3, and K+ channel-interacting proteins 1 and 3 were detected in proximal colon, gastric fundus, gastric antrum, and jejunum. On the other hand, of seven CaBPs including alternatively spliced variants, only CaBP1L transcripts were detected in GISMs.
EF-hand motif; quantitative PCR; ion channel regulation; rhythmicity; contractile activity
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INTRODUCTION |
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IN SMOOTH MUSCLES, Ca2+ signals regulate contractile activity by the classic Ca2+-binding protein calmodulin (Cal) via regulation of Cal-binding proteins such as caldesmon and calponin (13, 40, 45). Cal-dependent protein kinase can also regulate ion channel activities (3, 14, 17, 21, 22). Recently, physiological and biochemical analyses have demonstrated novel properties and functions of Cal-superfamily proteins such as neuronal Ca2+-binding proteins (NCBPs) (7) and Cal-like Ca2+-binding proteins (CaBPs) (12). In neuronal cells, NCBPs regulate multiple target proteins and CaBPs functions as Cal-like proteins in some of Cal-regulatory processes. Szymanski et al. (42) showed that differences in Cal densities are involved in phasic and tonic behavior of smooth muscles (42). Therefore, identification of NCBP and CaBP components in gastrointestinal smooth muscles (GISMs) would provide important additional information concerning the regulation of contractile behavior and electrical rhythmicity.
NCBPs belong to the superfamily of EF-hand proteins and are implicated in multiple Ca2+-signaling pathways (7, 11). Of a large number of NCBP, the neuronal Ca2+ sensor 1 (NCS1), neural visinin-like proteins (NVPs), and K+ channel-interacting proteins (KChIPs) are widely but differentially expressed in the nervous system (4, 32, 41). Recent studies have suggested that NCBPs play a crucial function in regulation of neurotransmitter release (9), cyclic nucleotide metabolism (5, 6), regulation of receptor phosphorylation (30), and regulation of ion channels, such as P/Q-type Ca2+ channels and voltage-dependent potassium (Kv4) channels (2, 29, 31, 46).
CaBPs with high similarity to Cal (50-60%) also belong to the superfamily of EF-hand proteins; however, their function is poorly understood in contrast to NCBPs. To date, at least five members have been identified in mammalian neurons (12), and they can substitute for Cal in biochemical assays (39). CABP1 is widely expressed in the brain, whereas CABP 2-5 are specifically expressed in the retina (12). Two alternative spliced variants for CABP1 and CABP2 have been identified in neurons, respectively: CABP1L/1S and CABP2L/2S (12).
Of a large number of NCBPs and CaBPs, we report the molecular identification of NCS1, NVP1-3, KChIP1, KChIP3, and CABP1 both in phasic and tonic GISMs (proximal colon, gastric antrum, gastric fundus, and jejunum). Quantitative RT-PCR was further used to determine the relative expression levels of NCBPs and CaBPs.
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MATERIALS AND METHODS |
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Dissection of smooth muscles and smooth muscle cell preparations.
Adult BALB/c mice were killed by cervical dislocation according to the
Guiding Principles of Institutional Animal Care and Use Committee.
Preparation of GISM strips and cell isolation was performed as
previously reported (44). Cells were transferred to the
stage of a phase-contrast microscope and allowed to adhere to a glass
coverslip bottom for 20 min. Smooth muscle cells were differentiated by
their characteristic morphology. Single cells were collected through
applied suction by aspirating them into a wide-bore patch-clamp pipette
(borosilicate glass). Approximately 50 smooth muscle cells were
collected, flash-frozen in liquid nitrogen, and stored at 80°C
until use.
Total RNA extraction and RT-PCR.
Total RNA was extracted from isolated GISM tissue and isolated cells
with the use of a TRIzol (Life Technologies, Gaithersburg, MD)
procedure and a SNAP total RNA isolation kit (Invitrogen, Carlsbad,
CA), respectively, as previously reported (44). Total RNA
was also isolated from brain and heart tissues. The SUPERSCRIPT II
RNase H (Life Technologies) and 200 µg/ml of random
hexamer (for tissues) were used to reverse transcribe the RNA sample.
The PCR amplification profile was as follows: a 15-s denaturation step
at 95°C and a 60-s primer extension step at 60°C using AmpliTag
Gold Taq DNA polymerase (PE Biosystems, Hayward, CA). In the
tissue- and cell-based RT-PCR, the amplification was performed for 30 and 40 cycles, respectively. Primers are listed in Table
1.
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Quantitative RT-PCR.
Real-time quantitative PCR was performed with the use of Syber Green
chemistry on an ABI 5700 sequence detector (PE Biosystems) (44). Regression analysis of the mean values of six
multiplex RT-PCRs for the log10-diluted cDNA was used to
generate standard curves. Unknown quantities relative to the standard
curve for a particular set of primers were calculated, yielding
transcriptional quantification of NCS subfamily gene products relative
to the endogenous standard (-actin). The reproducibility of the
assay was tested by ANOVA comparing repeat runs of samples, and mean values generated at individual time points were compared by Student's t-test.
Cloning of NCBP and CaBP members in murine colonic smooth muscles. Full-length cDNA fragments of NCS1, NVP1, NVP2, NVP3, KChIP1, KChIP3, and CABP1L were isolated from murine colonic smooth muscles by PCR-based cloning in the presence of the following gene-specific primers: NCS1: sense nt 1-20 and antisense nt 589-608, amplicon = 608 bp; NVP1: sense nt 1-20 and antisense nt 577-596, amplicon = 596 bp; NVP2: sense nt 138-157 and antisense nt 763-782, amplicon = 645 bp; NVP3: sense nt 231-250 and antisense nt 812-831, amplicon = 501 bp; KChIP1: sense nt 319-338 and antisense nt 990-1009, amplicon = 698 bp; KChIP3: sense nt 99-118 and antisense nt 883-902, amplicon = 804 bp; CABP1L: sense nt 24-43 and antisense nt 736-755, amplicon = 732 bp.
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RESULTS |
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Expression of NCBP superfamily transcripts in murine
gastrointestinal smooth muscles.
We first examined the expression levels of NCBP superfamily transcripts
(12 members) in murine GISMs (proximal colon, gastric antrum, gastric
fundus, and jejunum) by use of conventional RT-PCR: NCS1, guanylyl
cyclase-activating protein (GCAP) 1-2, NVP 1-3, KChIP
1-4, hippocalcin, and recoverin. Murine brain-derived cDNAs were used as positive controls for the various gene-specific primers to
test their ability to produce the correct amplicon. -Actin primers
were used to confirm that the products generated were representative of
RNA (498 bp) and not contaminated with genomic DNA (intron containing
708-bp band), because these primers were designed to span an intron as
well as two exons. This control serves the identical purpose as a cDNA
reaction lacking RT, however, it can be performed on the same RNA
preparation as the test reactions. This is extremely important for
isolated cell RNA preparations in which the low amount of RNA prevents
the synthesis of more than one cDNA reaction per cell preparation.
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Expression of CaBP superfamily transcripts in murine
gastrointestinal smooth muscles.
We examined the expression of CaBP superfamily transcripts CABP1, 2, 5, and 7 and caldendrin in murine GISMs by use of RT-PCR. As previously
reported by Haeseleer et al. (12), for CABP1 and CABP2,
two alternatively spliced variants have been isolated from the murine
cDNA library, and the longer/shorter variants are referred to as
CABP1L/1S and CABP2L/2S, respectively. We first designed primers for
the conserved regions in CABP1L/1S and CABP2L/2S, respectively (see
MATERIALS AND METHODS). As shown in Fig.
3, CABP1 signals alone were detected in
GISMs examined, whereas signals for CABP2, 5, and 7 and caldendrin were
not detectable. The signals of CABP1 and 7 and caldendrin were detected
in the brain, and CABP2, 4, and 5 transcripts were specifically
expressed in the retina as previously reported (12). To
show that PCR reactants were free from contamination, no template
control experiments were performed (Fig. 3, NTC). In humans, the
COOH-terminal sequences of CABP5 are identical to those of CABP3
(12). In the present study, we show that the nucleotide
sequences of amplified products for CABP5 have ~90% homology to
those of human CABP3. Because primers were designed in a region of
100% identity between CABP3 and 5, amplification should detect both of
these transcripts. Because no amplicon was detected, this suggests that
CABP3 is also not expressed in murine GISMs. Similar results were
obtained from at least three separate experiments. The specificity of
each PCR product was confirmed by DNA-sequence analysis.
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Quantitative determination of NCBP and CaBP superfamilies in murine
gastrointestinal smooth muscles.
The ABI 5700 genetic analyzer (PE Biosystems) was used for accurate
quantification of steady-state transcript levels by RT-PCR. Total RNA
was prepared from murine brain, heart, proximal colon, gastric antrum,
gastric fundus, and jejunum tissue. These preparations, however,
contain smooth muscle cells and other minor cell types (e.g.,
interstitial cells of Cajal, macrophages, and fibroblasts) that will
contribute to the quantitative measurement. RNA was reverse transcribed
to cDNA, and steady-state transcripts were determined relative to an
endogenous control housekeeping gene (-actin). Therefore, the data
are expressed as relative to
-actin. The relative transcriptional
expression of NCBP and CaBP members is shown in Figs.
5 and 6,
respectively. Note that the scale for all NCBP and CaBP expression data
in GISMs is kept consistent to allow comparison of relative expression
levels, and all expression data are expressed as means ± SE.
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DISCUSSION |
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The present study has demonstrated that mouse GISMs express transcripts for several members of NCBPs and CaBPs. Of 12 NCBPs, NCS1, NVP1, NVP3, KChIP1, and KChIP3 transcripts were detected in GISMs (proximal colon, gastric antrum, gastric fundus, and jejunum) at different levels (Fig. 5). Of seven CaBPs, including alternatively spliced variants, the longer variant of CaBP1, CABP1L alone was expressed in GISMs at similar levels (Figs. 4 and 6). This is the first report of NCBP and CaBP expressions in smooth muscles. Accumulating evidence indicated that both NCBPs and CaBPs could substitute for or potentiate Cal functions and modulate the multiple Ca2+-signaling pathways in the nervous system [reviewed by Burgoyne and Weiss (7); see also Ref. 12]. In addition, it has been reported that some NCBPs interact with multiple enzyme targets and ion channels and possess cytoskeletal-binding activity (25, 28, 35).
Szymanski et al. (42) reported that the contents of Cal and Cal-binding proteins are different between phasic and tonic smooth muscles. In GISMs, contraction is mediated by the initial intracellular Ca2+ concentration ([Ca2+]i) transient, resulting in Ca2+/calmodulin-dependent activation of myosin light chain (MLC) kinase, phosphorylation of MLC20, and interaction of actin and myosin, whereas relaxation is mediated by cAMP- and/or cGMP-dependent protein kinase, which inhibit the initial [Ca2+]i transient and reduce the sensitivity of MLC kinase to [Ca2+]i. Interestingly, Schaad et al. (35) showed that NCS1 represents an ideal switch for neurons that need to respond rapidly to slight alternation in [Ca2+]i because a transient change in [Ca2+]i from 0.1 to 1.5 µM produces a shift from 10 to 90% in the Ca2+ saturation and activity of NCS1, whereas Cal needs a much higher rise in [Ca2+]i from 1 to 30 µM (35). These data suggest that Cal and NCS1 could provide a double range of Ca2+-sensing activities. Differential expression of NCBPs and CaBPs could be tailored to the requirements of individual smooth muscles.
Cal also regulates the gating and/or trafficking of a number of ion channels and transporters [reviewed by Saimi and Kung (34)]: the types 1 and 2 inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1 and 2) (26, 48), the types 1 and 3 ryanodine receptors (RyR1 and 3) (8, 15), the type 4 transient receptor potential channel (TRPC4) (43), Na2+-Ca2+ exchanger (33), L-type Ca2+ channel (47, 50), and small (SK)- and large-conductance (BK) Ca2+-activated K+ channels (3, 16, 18, 36). Recent studies have shown that NCS1 and KChIPs functionally regulate the activities of P/Q-type Ca2+ channels and/or A-type K+ channels (Kv4) in nervous and cardiovascular systems (23, 27, 46) and CaBP1 modulates the functions of P/Q-type Ca2+ channels and IP3R in different manners compared with Cal (24, 49). In GISMs, the activities of SK and Kv are regulated by Cal kinase II (1, 21, 22), suggesting that NCBPs and CaBPs could also contribute to myogenic regulation of gastrointestinal motility. In the nervous system, an A-type potassium channel mediated by Kv4.3 and KChIP3 has a key role in pacemaker control, and diverse KChIP3 expression tunes variable firing rates in the different types of neurons by transcriptional control of K+-channel genes (27). The present study, therefore, suggests that the differential expression patterns of KChIPs may contribute to the diverse electrical excitability among GISMs. Walker et al. (44) showed that of the seven Ca2+-permeable cation channel members (TRPC), transcripts for TRPC4, 6, and 7 were expressed in GISMs at different expression levels. Moreover, release of Ca2+ from the sarcoplasmic reticulum regulates smooth muscle contractile activity, and the participation of RyR and IP3R complexes are involved in differences in the sensitivity to the pump inhibitors for Ca2+ release (10). NCBPs and CaBPs may therefore play significant roles for mechanical coupling of these intracellular Ca2+ regulators. Together, the diverse expression patterns for NCBPs and CaBPs in GISMs with different phenotypes may allude to their specific regulatory roles via various ion channels and/or transporters and Ca2+-release mechanisms.
NVP1 expression increases cAMP and cGMP levels via direct or indirect regulation of adenylyl cyclase and guanylate cyclase activities, respectively (5, 6). In the present study, NVP1 and 3 transcripts were significantly expressed in GISMs at different expression levels. In smooth muscles, NO signals are transduced through the production of cGMP and activation of protein kinase G. NO-mediated pathways play crucial roles for many physiological processes of the GI tract, such as relaxation of sphincters, gastric accommodation, and receptive relaxation during feeding (38). A recent study (37) showed that nitrergic relaxations occur via a cGMP-activated ryanodine-sensitive mechanism in the longitudinal gastric fundus, resulting in the modulation of Ca2+ spark activity by cGMP. Moreover, stretch-dependent K+ channels encoded by TREK-1 contribute to maintenance of relaxation of GISM cells and regulation by NO-dependent mechanisms (19, 20). Together, NVPs might be involved in myogenic regulation via nitrergic inhibitory pathways in GISMs.
NCS1 has been shown to associate with membrane organelles such as the Golgi apparatus and some cytoskeletal elements (28). Therefore, some NCBPs and CaBPs could function as the regulators of protein trafficking. The possible interaction between NCBPs/CABPs and the cytoskeleton will be important to determine in smooth muscles for investigating functions of NCBPs and CaBPs as posttranslational modulators. Moreover, the KChIP3 homolog downstream regulatory element antagonist modulator works as a nuclear transcriptional factor directly regulated by Ca2+ binding (41). Further investigations (e.g., subcellular localization in GISMs) may lead to an understanding of the role for NCBPs and CaBPs of protein assembly, transcriptional regulation, and trafficking in GISMs.
In summary, several members of NCBPs and CaBPs mRNAs are expressed in GISMs at various levels. Transcriptional expression levels, although usually a good indicator of protein expression, may not accurately correlate with functionally assembled gene products. Although we have not determined the functional significance for these accessory and regulatory proteins, nor determined their protein expression levels, determining the transcriptional expression pattern in both phasic and tonic GISMs will aid investigators in this task.
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ACKNOWLEDGEMENTS |
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We thank G. C. Amberg for preparation of murine gastrointestinal smooth muscle cells. We also thank L. Miller for technical support of molecular biology.
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FOOTNOTES |
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The nucleotide sequence data reported in this paper (murine KChIP1 and NVP2) have been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases with accession nos. AB07504 and AB079893, respectively.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41315.
Present address for S. Ohya: Dept. of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City Univ., 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.
Address for reprint requests and other correspondence: B. Horowitz, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, MS352/Anderson Medical Bldg., Reno, NV 89557-0046 (E-mail: burt{at}physio.unr.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.
May 29, 2002;10.1152/ajpgi.00101.2002
Received 14 March 2002; accepted in final form 29 May 2002.
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