Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1804
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vivo and in vitro studies have demonstrated that somatostatin can influence motility and smooth muscle contractility of the stomach and colon. Recent studies have proposed that some of these effects may be mediated by somatostatin receptors (sst) directly on the smooth muscle cells. If this is correct, the sst receptor subtypes that are present are unknown. This study aimed to resolve these points. Because nucleotide sequences of guinea pig sst genes are unknown, we used sst subtype-specific primers based on comparisons of human and rat sst subtypes and performed RT-PCR of DNase I-treated total RNA from guinea pig total brain. PCR products were cloned in pCR II and sequenced and showed 87% (sst1), 90% (sst2), 90% (sst3), 99% (sst4), and 80% (sst5), respectively, nucleotide homology to the same region (transmembrane 4-6) of the human sst genes. Homology to rat sequences were lower. PCR products were obtained from first-strand cDNA derived from DNase I-treated RNA from dispersed guinea pig gastric and colonic smooth muscle cells. In gastric and colonic smooth muscle cells, we detected sst1-sst3 and sst5, and all were confirmed by sequencing. The presence of sst4 was shown by Southern blot analysis and hybridization with a guinea pig sst4-specific primer. RT-PCR from cultured colonic and gastric smooth muscle cells devoid of any neural elements gave identical results. These results demonstrate that in the guinea pig all five sst subtypes are present directly on gastric and colonic smooth muscle cells. Previous studies have suggested that a predominant sst3 subtype on gastric and a sst5 subtype on colonic muscle cells mediated somatostatin's contractile effects, but the finding here that all five sst subtypes exist on both of these cells suggests that other sst subtypes have only a small or no contractile effect, sst subtypes in guinea pig have a different pharmacological profile from rat or human sst, or these other sst subtypes have some yet undescribed physiological function in muscle cells.
colon; colonic motility; octreotide; cloning
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SOMATOSTATIN IS WIDELY distributed in the gastrointestinal tract, including in specific mucosal cells, in ganglia, and on neurons, some of which project into smooth muscle cells (14, 29). Somatostatin has been shown to influence colonic and gastric motility and contractility both in vivo and in vitro (2, 28, 30). Furthermore, previous studies have demonstrated that somatostatin can cause changes in contractility in isolated and dispersed guinea pig smooth muscle cells, suggesting somatostatin receptors exist on these cells. Specifically, the presence of somatostatin receptors on isolated and dispersed guinea pig gastric smooth muscle cells has been suggested by both binding studies and contraction-relaxation studies (15, 16, 27). Recently, the presence of functionally active somatostatin receptors on guinea pig descending colonic smooth muscle cells was supported by contraction studies (9). In that same study, various synthetic somatostatin analogs selective for one sst subtype over the other were used, and it was concluded that sst3 on guinea pig gastric smooth muscle cells and sst5 on guinea pig colonic smooth muscle cells mediate the contractile effect of somatostatin on these cells. However, conclusions from these data are limited by the fact that these synthetic somatostatin analogs have only moderate selectivity for one somatostatin receptor subtype over the other and species differences can occur in affinity for different subtypes (10). At present, there is no direct evidence of somatostatin receptor expression on guinea pig smooth muscle cells, and their detection is complicated by the fact that the structure of the guinea pig somatostatin receptors are unknown, even though guinea pig smooth muscle cells are widely used to study the cellular basis of hormones and/or neurotransmitters (26). Therefore, in the present study, we investigated whether sst receptor expression occurs in guinea pig colonic and gastric isolated smooth muscle cells and investigated which sst subtype was present after first determining specific sequences of the structure of guinea pig sst receptor genes.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Male guinea pigs (200-300 g) were obtained from the Small Animal
Section, Veterinary Resources Branch, National Institutes of Health.
HEPES was from Boehringer Mannheim Biochemicals (Indianapolis, IN).
Collagenase type II was from Worthington Biochemical (Freehold, NJ).
Soybean trypsin inhibitor was from Sigma Chemical (St. Louis, MO).
DMEM, PBS, first-strand cDNA synthesis kit,
EcoR I, and DNase I were from GIBCO
BRL (Gaithersburg, MD). RNeasy total RNA kit was from Qiagen
(Chatsworth, CA). Taq polymerase was
from Perkin-Elmer (Norwalk, CT), the
fmol DNA sequencing system was from
Promega (Madison, WI), and -actin primers were from Clontech (Palo
Alto, CA). TA Cloning kit was from Invitrogen (San Diego, CA). X-Omat films were from Kodak (Rochester, NY). Synaptophysin antibody was from
Zymed (San Francisco, CA). S-100 protein antibody and smooth muscle
actin antibody were from Biogenex (San Ramon, CA). [
-32P]ATP (3,000 Ci/mmol) and
[
-32P]dCTP (3,000 Ci/mmol) were purchased from DuPont NEN (Boston, MA).
Preparation of dispersed colonic and gastric smooth muscle cells.
Dispersed smooth muscle cells from guinea pig stomach were prepared as
previously described by Bitar and Makhlouf (4). The same method, with
the modifications described recently (9), was used to obtain dispersed
smooth muscle cells from guinea pig descending colon. Briefly, the
guinea pig descending colon was removed and cut longitudinally, the
mucosa was removed by gentle scraping, and the muscle layer was cut
transversely into 5- to 10-mm strips. The muscle strips were incubated
at 31°C for two successive 45-min periods in 15 ml of standard
incubation solution (4) containing 0.1% (wt/vol) collagenase. The
partially digested strips were successively washed with 50 ml of
collagenase-free standard incubation solution, and smooth muscle cells
were allowed to disperse spontaneously for 30 min and then were
harvested by filtration through a 500-µm mesh. Dispersed smooth
muscle cells were pelleted at 350 g
for 10 min and washed two times with PBS solution. Greater than 95% of
all intact cells excluded trypan blue. The isolated cells were lysed in
guanidine isothiocyanate solution (11) and stored at 70°C
until RNA extraction was performed.
Culture of colonic and gastric smooth muscle cells. A modification of the method described by Chijiiwa et al. (8) was used. After standard dispersion, smooth muscle cells were harvested by filtration, pelleted at 350 g for 10 min, and resuspended in DMEM. After a second centrifugation, the cells were washed once in DMEM medium [supplemented with 10% (wt/vol) fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 g/ml)] and finally resuspended in the same medium at a concentration of 2 × 105 cells/ml. Two milliliters of the cell suspension were plated in 35-mm-diameter well plates (Falcon, Oxnard, CA) and placed in a 5% CO2 incubator at 37°C. Medium was replaced every 3 days. After 10-14 days, the cells became confluent and were then used for RNA extraction and immunohistochemical studies.
RNA isolation. Total RNA extraction from guinea pig total brain, cerebellum, and isolated dispersed colonic or gastric smooth muscle cells was performed by the guanidine isothiocyanate-cesium chloride method (11). Total RNA from cultured colonic and gastric smooth muscle cells was extracted using the RNeasy total RNA kit (Qiagen).
RT-PCR.
All RNA samples were first treated with DNase I to remove genomic DNA
contamination from the total RNA preparations according to the
manufacturer's protocol (GIBCO BRL). First-strand cDNA for PCR was
synthesized from 1 µg of total RNA by reverse transcription either
using random hexamer or oligo(dT) primers according to the standard
protocol of the manufacturer (GIBCO BRL). Control experiments for
completeness of genomic DNA digestion were performed by
PCR without RT and by PCR for -actin for which the
primers were designed on either side of an intron so that genomic DNA contamination could be determined. PCR was carried out in a buffer at
pH 8.3, containing 10 mM Tris · HCl, 50 mM KCl, 1.0 mM MgCl2, 0.2 mM of each dNTP, 0.3 mM of each primer, and 2.5 units Taq DNA polymerase in a 50-µl reaction volume. PCR products were obtained either directly (one PCR) or by nested PCR. PCR primers were designed, with some degeneracy, by comparing human and rat somatostatin subtype-specific sequences and identifying areas of high homology and
specificity for each subtype. For all sst receptors, a sequence coding
for transmembrane regions four to six (TM4-TM6) was amplified. For
three receptors, sst1,
sst3, and
sst4, an additional sequence encoding from the first to the third or fourth transmembrane region was
amplified (Fig. 1). Nested PCR was used for
five of the sequences amplified:
sst1 (TM4-TM6),
sst3 (TM4-TM6),
sst4 (TM1-TM4),
sst4 (TM4-TM6), and
sst5 (TM4-TM6). Nested PCR
was not required for three sequences:
sst1 (TM1-TM4),
sst2 (TM3-TM7) and
sst3 (TM1-TM4). The specific
PCR primers used were as follows: for
sst1
(sst1 TM1-TM3),
5'-CAGCTGGGATGTTCCCCAATG-3' and
5'-AGCACAGTCAGACAGTAG-3'. For
sst1 (TM4-TM6), the first PCR
primers were 5'-GACCGCTATGTGGCTGTGGAGCACCC-3' and
5'-GGGTTGGCGCACGTGTTGGCATA-3', and then nested
amplification was performed with
5'-GTGGTGGTCTTCTCGGGAGTGCCCCG-3' and
5'-TGGTTGACAGTGGCATCGAG-3'. For
sst2 (TM3-TM7), the primers
used were 5'-GACCGCTACCTGGCTGTGGAGCACCC-3' and
5'-GGGTTGGCACAGCTGTTGGCATA-3'. For
sst3 (TM1-TM4), the primers used were 5'-CGGCACACGGCCAGCCCTTC-3' and
5'-GGCACTCCCGAGAAGACCACCAC-3'. For
sst3 (TM4-TM6), first PCR
primers were 5'-GACCGCTACCTGGCTGTGGAGCACCC-3' and
5'-GGGTTGGCACAGCTGTTGGCATA-3', and then nested
amplification was performed with
5'-GTGGTGGTCTTCTCGGGAGTGCCCCG-3' and
5'-TGGTTGACAGTGGCATCGAG-3'. For
sst4 (TM1-TM4), the first PCR
primers were 5'-CCCTCGACGCTGCCCCCCG-3' and
5'-GCCAGCCAGCGCGCAGGGC-3', and then nested amplification
was performed with 5'-GCGGGCATGGTCGCTATCCA-3' and
5'-CCACACGCCCAGGTTGATGAG-3'. For
sst4 (TM4-TM6), the first
primers were 5'-GACCGCTACCTGGCTGTGGAGCACCC-3' and
5'-GGGTTGGCACAGCTGTTGGCATA-3', and then nested
amplification was performed with 5'-ATCTTCGCAGACACCAGACC-3'
and 5'-ATCAAGGCTGGTCACGACGA-3'. For
sst5 (TM4-TM6), the first PCR
primers were 5'-GACCGCTACCTGGCTGTGGAGCACCC-3' and
5'-GGGTTGGCACAGCTGTTGGCATA-3', and then nested
amplification was performed with
5'-CTCTTGGTGTTCGCGGACGTGCAGGA-3' and
5'-GCCAGGTTGACGATGTTGACGGT-3'. All PCR reactions were
performed under the following conditions: an initial denaturing step of
3 min at 95°C followed by 30 cycles of 30-s denaturation at
95°C, 30-s annealing at 45-63°C, and 1-min extension at
72°C with a 10-min final extension at 72°C. Negative control
reactions were performed without template in the reaction mix. Human
-actin primers (Clontech) were used under identical conditions in
PCR reactions to verify the integrity of the cDNA template.
|
Cloning and sequencing. PCR products were initially sequenced directly and then cloned into the plasmid vector pCR II using the TA Cloning kit (Invitrogen), when corresponding to a sst sequence. Recombinant clones were identified by restriction enzyme digestion. DNA sequence of partial cDNA guinea pig sst clones was determined on both strands with the fmol DNA sequencing system (Promega).
Southern blot transfer.
Because of differences between the guinea pig, rat, and human sst
sequences, two guinea pig subtype-specific oligonucleotides were
synthesized for Southern blot hybridization studies; these were
5'-CGCAACAACGTGGAGGTGAC-3' and
5'-TGCTGGCCATTGGCCTGTGCT-3' for
sst1 and
sst4, respectively. Agarose gels
of guinea pig-sst PCR products were transferred to nitrocellulose
filters (42) and hybridized to
-32P-labeled guinea pig sst
subtype-specific oligonucleotides. Nitrocellulose filters were washed
at high stringency [final wash solution: 0.1× SSC (1×
SSC is 150 mM NaCl and 15 mM sodium citrate), 0.1% (wt/vol) SDS at
50°C], air-dried, and exposed to X-Omat film (Kodak) for 6-15 h.
Immunohistochemical analysis of cultured colonic and gastric smooth muscle cells. Formalin-fixed embedded cell blocks were prepared from cultured colonic and gastric smooth muscle cells using a thrombin-clot method. Five-micrometer sections were cut from each cell block and mounted on charged slides (Fisher Scientific, Pittsburgh, PA). The immunohistochemical procedure was carried out at room temperature for 2 h at a working dilution of 1:50. Results were visualized with a modified avidin-biotin technique, using 3,3'-diaminobenzidine as a chromogen. Samples were evaluated with antibodies to smooth muscle actin, S-100 protein, and synaptophysin.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning of partial gene sequences of guinea pig somatostatin
receptor subtypes.
After PCR with each of the eight sets of primers corresponding to the
sst receptor areas for each sst subtype shown in Fig. 1 and after the
PCR products were cloned, eight different cDNA clones were obtained
from the guinea pig brain total RNA (Fig. 1). For each sst subtype, one
guinea pig cDNA clone containing the receptor TM4-TM6 region was
obtained. Additional 5' regions of guinea pig
sst1,
sst3, and
sst4 were also cloned (Fig. 1). Specifically, we obtained a 459-bp
(sst1 TM1-TM3) and a 243-bp (sst1 TM4-TM6) fragment for
guinea pig sst1, a 513-bp
(sst2 TM3-TM7) fragment for
guinea pig sst2, a 321-bp
(sst3 TM1-TM4) and a 312-bp (sst3 TM4-TM6) fragment for
sst3, a 382-bp
(sst4 TM1-TM4) and a 333-bp
(sst4 TM4-TM6) fragment for
sst4, and a 303-bp
(sst5 TM4-TM6) fragment for
sst5 (Fig. 1). Comparison of the
nucleotide sequences of the guinea pig TM4-TM6 region to the
equivalent region of the human and rat sequences demonstrated a higher
degree of homology with the human than with the rat sequences in four
of the five sst subtypes (Fig. 2). The
highest homology (99%) existed for the nucleotide sequence of the
sst4 subtype TM4-TM6 (Fig. 2). An independently obtained partial cDNA clone from a different region of sst4 (TM1- TM4;
data not shown) also showed 99% homology to the human
sequence. In addition, the nucleotide sequence of guinea pig
sst1 (TM1-TM3) and guine a
pig sst3 (TM1-TM4) clones, compared with the respective human sst subtype, showed the same degree
of homology that was found between the TM4-TM6 region of the
two species (data not shown). The amino acid sequence of the guinea
pig TM4-TM6 nucleotide sequences showed a complete identity with the human homologous area in the case of
sst1 (Fig.
3), four amino acid changes for
TM4-TM6 of sst2 (96%
homology), and six changes for TM4-TM6 of
sst3 (94% homology) along with an
eight amino acid motif between TM5-TM6 that was absent in the
human sequence (Fig. 3). The guinea pig and human TM4-TM6
sst4 sequences differed in only
one amino acid (99% homology), whereas the TM4-TM6 sequence of
sst5 had 19 amino acid differences
(82% homology) (Fig. 3).
|
|
Expression of sst subtypes on freshly isolated gastric and colonic
smooth muscle cells.
Using first-strand cDNAs generated from total RNA preparations from
isolated gastric or colonic smooth muscle cells, we performed PCR with
the same primer pairs and with the same conditions used to obtain
guinea pig sst subtype-specific sequences from guinea pig brain, as
specified in MATERIALS AND METHODS.
Specific PCR products for sst1,
sst2,
sst3, and
sst5 were obtained from both smooth muscle cell preparations, and the products were identical in
molecular weight to those obtained from guinea pig brain on ethidium
bromide staining (Fig.
4). Sequence
analysis of the PCR products demonstrated that products were identical
to the respective guinea pig sst subtypes, previously cloned and
sequenced from guinea pig brain. No PCR product was detected using
ethidium bromide staining after PCR when using specific guinea pig
primer pairs for sst4 (Fig. 4). No
PCR products were obtained when water or RT-negative samples were used
as a template in the same amplification reaction for any sst subtype
(Fig. 4). Furthermore, PCR using -actin primers demonstrated only a
838-bp product. This is a product expected when only cDNA is present.
Because this primer pair spans an intron, if a genomic DNA was present,
a larger PCR product would have been obtained.
|
Immunohistochemical detection of neural elements in cultured smooth
muscle cells.
Because of the possibility that sst subtypes in the freshly dispersed
smooth muscle preparations seen with RT-PCR in the above experiments
might exist on contaminating neural elements, we cultured the smooth
muscle cells for 10-14 days. Both cultured gastric and cultured
colonic smooth muscle cell preparations stained positive using a
monoclonal antibody to smooth muscle actin. The same cultured smooth
muscle cell preparations did not show any evidence of
immunohistochemical staining using antibodies to S-100 protein and
synaptophysin, two widely used proteins that identify neural tissue
(Fig. 5). These combined
immunohistochemical results support the lack of neural elements in our
cultured gastric and colonic smooth muscle cells.
|
Expression of sst subtypes on cultured gastric and colonic smooth
muscle cells.
When RT-PCR was performed on total RNA from cultured gastric and
colonic smooth muscle cells using guinea pig sst subtype-specific primer pairs, amplified products for
sst2,
sst3, and
sst5 subtypes were seen (Fig.
6). The amplified products had the same
molecular weight on ethidium bromide staining as that seen when brain
RNA was used with these primers. Similar to noncultured dispersed muscle cells, no evidence for sst4
was found. The lack of sst1 after
PCR and ethidium bromide staining in cultured cells raised the
possibility that the sst1 seen in
noncultured cells was from contaminating neural elements or that the
ethidium bromide staining was not sufficiently sensitive enough to
detect low amounts of the sst1 PCR
product. To distinguish these two possibilities, Southern blot analysis
was performed (Fig. 7). Furthermore, to determine whether the latter possibility (i.e., low
sst4 product) was a possible
explanation for not seeing evidence of the
sst4 subtype on ethidium bromide
staining after PCR on both cultured and noncultured cells, Southern
blot analysis was also performed for this subtype (Fig.
8).
|
|
|
Southern blot analysis of PCR products.
With the use of a -32P-labeled
oligonucleotide under conditions that only identified the
sst1 subtype RT-PCR product, the
sst1 subtype was shown to be
present in both cultured colonic and gastric smooth muscle cells (Fig.
7). With the use of a guinea pig
sst4-specific,
-32P-labeled oligonucleotide,
under conditions that identified only the
sst4 subtype (Fig. 8), both
dispersed and cultured gastric and colonic smooth muscle cell RT-PCR
products were found to possess the
sst4 subtype (Fig. 8).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The purposes of the present study were to determine whether somatostatin receptors are expressed on gastric and colonic smooth muscle cells and to define the somatostatin receptor subtypes present. The expression of somatostatin receptor subtypes on smooth muscle cells was investigated using an RT-PCR-based strategy. This was chosen because the amount of RNA in smooth muscle cells is low. Furthermore, the smooth muscle cells that are prepared using methods to limit contaminating elements from which the mRNA is extracted are limited. PCR amplification, because of its high sensitivity, can identify the presence of somatostatin receptor subtypes even when they are present in very low abundance. This approach was complicated by the fact that, although guinea pigs are one of the main laboratory animals used to study the effects of somatostatin and other hormones and/or neuropeptides on gastrointestinal smooth muscle motility (26), no guinea pig somatostatin receptor gene structure sequence was known. To overcome this limitation, we first determined the guinea pig cDNA structure sequences for all five guinea pig sst subtypes. To accomplish this, we assumed, as in other species (rat, human) (5-7, 33, 35, 36, 44, 45), that all five sst subtypes would be expressed in the guinea pig brain; therefore, we first performed RT-PCR using total RNA from guinea pig brain. After the human and rat sst sequences were analyzed, a number of sst subtype-specific primer pairs were designed from conserved areas that were specific for each sst subtype, and each was tested. If a single PCR product was seen on the ethidium bromide-stained gel and it was of the expected size, it was sequenced and the sequence was compared with that of the same sst subtype for the human and rat sequence. Generally, we performed further analysis (cloning into the plasmid vector pCR II) only when the nucleotide homology between the supposed guinea pig sst fragment and the human or rat sequence was higher than 75%. For this study, the complete coding sequence of each of the five guinea pig sst subtype mRNAs was not necessary; however, we obtained two-thirds or more of the structure of the coding region of sst1-4 and one-third of that for the guinea pig sst5. The guinea pig TM4-TM6 region, obtained for all five guinea pig sst subtypes, compared with the homologous area of human and rat nucleotide sst subtype structures, demonstrated the close homology between the guinea pig and the human and rat sequences (80-99%). These data confirm in the guinea pig what is known in other species (human, rat), that is, that the membrane-spanning helical domains and intracellular connection loops are highly conserved in different species (7, 36). Higher homology existed between guinea pig and human than between guinea pig and rat for the TM4-TM6 oligonucleotide structure for four of the five sst receptor subtypes, with only the guinea pig sst5 having a slightly higher homology for rat (84%) than for the human (80%) sequence. Recent studies demonstrated that the guinea pig is closer to human than to rat in the evolutionary scale, and this finding is consistent with the higher homology between guinea pig and human sst subtype sequences than with rat sequences (13). The homology at the amino acid level between guinea pig and human TM4-TM6 region is higher than that seen when the nucleotide sequences are compared from the same area. In fact, 100% identity existed for sst1; 96%, 94%, 99%, and 82% homologies were found for sst2, sst3, sst4, and sst5, respectively. This result, from comparison with the human and rat protein structure of sst subtypes (7, 36), is consistent with the conclusion that the sst1 subtype is the most highly conserved and the sst5 subtype is the most divergent sst subtype in the guinea pig.
A number of results in the present study support the conclusion that somatostatin receptors are expressed directly on the gastric and colonic smooth muscle cells. First, the correct nucleotide sequence for sst receptors was generated by RT-PCR from dispersed isolated colonic and gastric smooth muscle cell preparations. Consistent with this result is the finding that somatostatin alters the contractility of isolated gastric and colonic smooth muscle cells in other studies using an identical preparation (9, 15). Second, because dispersed smooth muscle preparations may contain occasional contaminating neural elements (8), the smooth muscle cells were maintained in culture. After 10-14 days in culture, no neural elements were detected; however, somatostatin receptor expression on the cultured smooth muscle cells was demonstrated. The ability to culture these cells and obtain preparations free of contaminating neural elements will allow in the future a number of important studies to be performed, such as the determination of which promoter for the various receptor subtypes might be present in these smooth muscles, as was done recently for sst2 in a number of cell lines (21).
The second purpose of the present study was to identify the
somatostatin receptor subtypes present on the gastric and colonic smooth muscle cells. In a study using receptor-selective somatostatin analogs to assess changes in contractility (9), it was concluded that
sst3 and
sst5 on guinea pig gastric and
colonic smooth muscle cells, respectively, mediated the contractile
responses of native somatostatin, which can interact with all five sst
subtypes with high affinity (3, 23). However, in the present study, all five somatostatin receptor subtypes were detected in both isolated gastric and colonic smooth muscle cells. This discrepancy between results of the biological activity study (9) and from the PCR results
in the present study could have a number of possible explanations. First, it is possible that some of the sst subtypes could be detected at the mRNA level but are expressed in such low numbers that they cannot be detected by studies of contractility. The present results do
not allow reliable conclusions to be made about the relative amounts of
expression of the mRNAs for the different somatostatin receptors in the
gastric and colonic smooth muscle cells. The amount of PCR product may
be proportional not only to the amount of each subtype mRNA but also to
the efficiency of different primers and the condition of the mRNA of
the different subtypes, which could be differentially affected by the
isolated conditions used. Attempts to perform Northern blot analysis,
which would allow a direct comparison of the amount of mRNA of each
somatostatin receptor subtype, did not give reproducible results,
probably because of the low abundance of the sst mRNAs in these cells
and the limited number of dispersed cells that could be obtained (data not shown). Second, some sst subtypes, although present, might not be
coupled to transduction pathways, causing changes in smooth muscle cell
contractility, and therefore would not be detected during
contraction-relaxation studies. Third, the conclusions from the study
of biological activity (9) were obtained by comparing the relative
affinities of different synthetic somatostatin analogs in altering
contractility in guinea pig gastric and colonic smooth muscle cells
with those for the known relative affinities of these various analogs
for the sst subtypes in rat and human. This comparison assumes that the
somatostatin analogs have similar relative selectivity for the sst
subtypes in guinea pig, rat, and human. These conclusions may not be
valid if the pharmacology of guinea pig sst subtypes for these
different somatostatin analogs differs from that of the rat and human
sst subtypes. Fourth, the possibility exists that the somatostatin
receptor subtypes seen with PCR might not be on the isolated smooth
muscle cells but instead on a contaminating cell. This possibility
cannot be completely excluded. However, the fact that each somatostatin
receptor subtype could be detected on cultured gastric and colonic
smooth muscle cells that possessed no neural elements supports the
conclusion that these somatostatin receptor subtypes are on the smooth
muscle cells. Fifth, the possibility could be raised that the sst
subtypes found by PCR were actually due to contaminating genomic DNA
and not to the cDNA from the mRNA. This possibility is excluded by two
important controls. First, when no RT was included after the DNase
digestion and PCR was performed, no product was seen. Second, when PCR
for -actin was performed after DNase digestion and synthesis of the
cDNA, because primers spanning an intron were used, it could be
assessed whether a genomic DNA was present. None was detected in any of
the preparations, therefore excluding contamination by genomic DNA as a possibility.
In previous studies, a number of different techniques (i.e., binding studies, Northern blot analysis, in situ hybridization, autoradiography, and RT-PCR) have been used to examine the distribution of the five sst subtypes in different human and rat tissues (6, 22, 25, 35, 41). The sst subtypes were reported to have tissue-specific localization with one subtype expressed on a given tissue and in other cases to have an overlapping pattern of distribution with different subtypes expressed on the same tissue (1, 6, 12, 35, 36). Specifically, in the gastrointestinal tract, somatostatin binding studies report binding sites in a number of different tissues and specific cell types (38, 39), whereas other studies using molecular methods have described the widespread presence of sst subtype mRNAs in rat intestine and pancreas (22, 24, 40). Only a few specific cellular localization studies in the gastrointestinal tract have been reported. Recently, the expression of sst subtypes, evaluated by RT-PCR, has been studied in the rat (34) and human enterochromaffin-like cells as well as in the colonic crypt epithelium (43). In rat enterochromaffin-like cells (34) and colonic crypt epithelium (43), the predominant expression of sst2 was found and sst2 activation accounted for the biological changes caused by somatostatin. Other sst subtypes, although present, were functionally not active (43). Recently, a study using in situ hybridization described results similar to the present study by showing the presence of all five sst subtypes on muscle layers from the whole rat gastrointestinal tract, including stomach and colon (22). Furthermore, in various pathological conditions such as on pancreatic endocrine tumors or carcinoid tumors, frequently all five sst subtypes are found to be present on these cells (19). However, the therapeutic actions of octreotide, such as inhibition of hormonal secretion, are due only to the presence of the sst2 and possibly the sst5 subtypes (17, 18, 20, 23, 32). These results, coupled with the findings in the present study, demonstrate that additional sst subtypes can exist in tissues that are not involved in the main biological actions assessed and therefore, at present, have an unknown function.
Recent studies have demonstrated that different sst receptor subtypes
can be coupled to different signaling cascades such as the activation
of protein tyrosine phosphates, inhibition of cGMP formation,
activation of phospholipase C-3, and inhibition of adenylate cyclase
(31, 37). Now that it is established that all five sst subtypes are
present on both gastric and colonic smooth muscle cells, with the
increased development of agonists highly selective for each sst
subtype, in the future it should be possible to explore the effect of
selective activation of each sst subtype on these different cellular
transduction pathways.
In conclusion, our study demonstrates that somatostatin receptors are expressed in both guinea pig gastric and colonic smooth muscle cells. Furthermore, this study demonstrates that all five somatostatin subtypes are present in smooth muscle cells from both tissues. These results differ from studies of contractile activity, which demonstrated evidence for only a sst3 subtype in gastric and a sst5 subtype in dispersed colonic smooth muscle cells, and suggest that the other four subtypes in each tissue likely have important functions in smooth muscle physiology, which may not be directly related to contractile responses.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Patti Fetsch and Dr. Andrea Abati of the Laboratory of Pathology, National Cancer Institute, National Institutes of Health for the immunohistochemical analysis.
![]() |
FOOTNOTES |
---|
During this study, V. D. Corleto was on leave from the Department of Cellular Biotechnology and Hematology, University La Sapienza, Rome, Italy. H. C. Weber is currently at the Section of Gastroenterology, Boston Medical Center, Boston University School of Medicine, Boston, MA.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. T. Jensen, NIH/NIDDK/DDB, Bldg. 10, Rm. 9C-103, 10 Center Dr. MSC 1804, Bethesda, MD 20892-1804 (E-mail: robertj{at}bdg10.niddk.nih.gov).
Received 9 September 1998; accepted in final form 1 April 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ain, K. B.,
K. D. Taylor,
S. Tofiq,
and
G. Venkataraman.
Somatostatin receptor subtype expression in human thyroid and thyroid carcinoma cell lines.
J. Clin. Endocrinol. Metab.
82:
1857-1862,
1997
2.
Atanassova, E.,
A. Bocheva,
and
K. Milenov.
Effect of cholecystokinin octapeptide and somatostatin on the mechanical and electrical activity of the colon of conscious dogs.
J. Gastrointest. Motil.
5:
57-62,
1993.
3.
Bell, G. I.,
K. Yasuda,
H. Kong,
S. F. Law,
K. Raynor,
and
T. Reisine.
Molecular biology of somatostatin receptors.
In: Somatostatin and Its Receptors, edited by D. J. Chadwick,
and G. Cardew. West Sussex, UK: Wiley, 1995, p. 65-88.
4.
Bitar, K. N.,
and
G. M. Makhlouf.
Receptors on smooth muscle cells: characterization by contraction and specific antagonists.
Am. J. Physiol.
242 (Gastrointest. Liver Physiol. 5):
G400-G407,
1982
5.
Bruno, J. F.,
Y. Xu,
J. Song,
and
M. Berelowitz.
Molecular cloning and functional expression of a brain-specific somatostatin receptor.
Proc. Natl. Acad. Sci. USA
89:
11151-11155,
1992[Abstract].
6.
Bruno, J. F.,
Y. Xu,
J. Song,
and
M. Berelowitz.
Tissue distribution of somatostatin receptor subtype messenger ribonucleic acid in the rat.
Endocrinology
133:
2561-2567,
1993[Abstract].
7.
Chadwick, D. J.,
and
G. Cardew
(Editors).
Somatostatin and Its Receptors. West Sussex, UK: Wiley, 1995, p. 1-274
8.
Chijiiwa, Y.,
K. S. Murthy,
J. R. Grider,
and
G. M. Makhlouf.
Expression of functional receptors for vasoactive intestinal peptide in freshly isolated and cultured gastric muscle cells.
Regul. Pept.
47:
223-232,
1993[Medline].
9.
Corleto, V. D.,
C. Severi,
D. H. Coy,
G. Delle Fave,
and
R. T. Jensen.
Colonic smooth muscle cells possess a different subtype of somatostatin receptor from gastric smooth muscle cells.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G689-G697,
1997
10.
Coy, D. H.,
and
J. E. Taylor.
Development of somatostatin agonists with high affinity and specificity for the human and rat type 5 receptor subtype.
In: Proceedings of the 15th American Peptide Symposium, edited by J. P. Tam,
and T. P. Kaumaya. Dordrecht, The Netherlands: Kluwer Academic, 1999, p. 559-560.
11.
Davis, L. G.,
W. M. Kuehl,
and
J. F. Battey.
Basic Methods in Molecular Biology. Norwalk, CT: Appleton & Lange, 1994, p. 1-777.
12.
Day, R.,
W. Dong,
R. Panetta,
J. Kraicer,
M. T. Greenwood,
and
Y. C. Patel.
Expression of mRNA for somatostatin receptor (sstr) types 2 and 5 in individual rat pituitary cells. A double labeling in situ hydridization analysis.
Endocrinology
136:
5232-5235,
1995[Abstract].
13.
D'Erchia, A. M.,
C. Gissi,
G. Pesole,
C. Saccone,
and
U. Arnason.
The guinea-pig is not a rodent.
Nature
381:
597-600,
1996[Medline].
14.
Elde, R.,
T. Hökfelt,
O. Johansson,
M. Schultzberg,
S. Efendic,
and
R. Luft.
Cellular localization of somatostatin.
Metabolism
27:
1151-1159,
1978[Medline].
15.
Gu, Z. F.,
V. D. Corleto,
S. A. Mantey,
D. H. Coy,
P. N. Maton,
and
R. T. Jensen.
Somatostatin receptor subtype 3 mediates the inhibitory action of somatostatin on gastric smooth muscle cells.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G739-G745,
1995
16.
Gu, Z. F.,
T. Pradhan,
D. H. Coy,
S. Mantey,
N. W. Bunnett,
R. T. Jensen,
and
P. N. Maton.
Actions of somatostatin on gastric smooth muscle cells.
Am. J. Physiol.
262 (Gastrointest. Liver Physiol. 25):
G432-G438,
1992
17.
John, M.,
W. Meyerhof,
D. Richter,
B. Waser,
J. C. Schaer,
H. Scherubl,
J. Boese-Landgraf,
P. Neuhaus,
C. Ziske,
K. Molling,
E. O. Riecken,
J. C. Reubi,
and
B. Wiedenmann.
Positive somatostatin receptor scintigraphy correlates with the presence of somatostatin receptor subtype 2.
Gut
38:
33-39,
1996[Abstract].
18.
Jonas, S.,
M. John,
J. Boese-Landgraf,
R. Haring,
G. Prevost,
F. Thomas,
S. Rosewicz,
E. O. Riecken,
B. Wiedenmann,
and
P. Neuhaus.
Somatostatin receptor subtypes in neuroendocrine tumor cell lines and tumor tissues.
Langenbecks Arch. Chir.
380:
90-95,
1995[Medline].
19.
Kolby, L.,
B. Wangberg,
H. Ahlman,
S. Jansson,
E. Foressell-Aronsson,
J. D. Erickson,
and
O. Nilsson.
Gastric carcinoid with histamine production, histamine transporter and expression of somatostatin receptors.
Digestion
59:
160-166,
1998[Medline].
20.
Kolby, L.,
B. Wangberg,
H. Ahlman,
L. E. Tisell,
M. Fjalling,
E. Forssell-Aronsson,
and
O. Nilsson.
Somatostatin receptor subtypes, octreotide scintigraphy, and clinical response to octreotide treatment in patients with neuroendocrine tumors.
World J. Surg.
22:
679-683,
1998[Medline].
21.
Kraus, J.,
M. Woltje,
N. Schonwetter,
and
V. Hollt.
Alternative promoter usage and tissue specific expression of the mouse somatostatin receptor 2 gene.
FEBS Lett.
428:
165-170,
1998[Medline].
22.
Krempels, K.,
B. Hunyady,
A. M. O'Carroll,
and
E. Mezey.
Distribution of somatostatin receptor messenger RNAs in the rat gastrointestinal tract.
Gastroenterology
112:
1948-1960,
1997[Medline].
23.
Lamberts, S. W. J.,
A. J. van der Lely,
W. W. de Herder,
and
L. J. Hofland.
Octreotide.
N. Engl. J. Med.
334:
246-254,
1996
24.
Laws, S. A.,
A. C. Gough,
A. A. Evans,
M. A. Bains,
and
J. N. Primrose.
Somatostatin receptor subtype mRNA expression in human colorectal cancer and normal colonic mucosae.
Br. J. Cancer
75:
360-366,
1997[Medline].
25.
Le Romancer, M.,
Y. Cherifi,
S. Levasseur,
J. P. Laigneau,
G. Peranzi,
P. Jais,
M. J. Lewin,
and
F. Reyl-Desmars.
Messenger RNA expression of somatostatin receptor subtypes in human and rat gastric mucosa.
Life Sci.
58:
1091-1098,
1996[Medline].
26.
Makhlouf, G. M.,
and
J. R. Grider.
Receptors for gut peptides on smooth muscle cells of the gut.
In: Handbook of Physiology: The Gastrointestinal System. Bethesda, MD: Am. Physiol. Soc., 1989, p. 281-289.
27.
McHenry, L.,
K. S. Murthy,
J. R. Grider,
and
G. M. Makhlouf.
Inhibition of muscle cell relaxation by somatostatin: tissue-specific, cAMP-dependent, pertussis toxin-sensitive.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G45-G49,
1991
28.
McKeen, E. S.,
W. Feniuk,
and
P. P. Humphrey.
Mediation by SRIF1 receptors of the contractile action of somatostatin in rat isolated distal colon; studies using some novel SRIF analogues.
Br. J. Pharmacol.
113:
628-634,
1994[Abstract].
29.
Messenger, J. P.
Immunohistochemical analysis of neurons and their projections in the proximal colon of the guinea-pig.
Arch. Histol. Cytol.
56:
459-473,
1993[Medline].
30.
Milenov, K.,
and
E. Atanassova.
Effects of cholecystokinin octapeptide and somatostatin on the motility and release of [3H]acetylcholine in canine colon.
Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol.
106C:
337-342,
1993.
31.
Murthy, K. S.,
D. H. Coy,
and
G. M. Makhlouf.
Somatostatin receptor-mediated signaling in smooth muscle. Activation of phospholipase C-3 by G
and inhibition of adenylyl cyclase by G
il and G
o.
J. Biol. Chem.
271:
23458-23463,
1996
32.
Nilsson, O.,
L. Kolby,
B. Wangberg,
A. Wigander,
H. Billig,
L. William-Olsson,
M. Fjalling,
E. Forssell-Aronsson,
and
H. Ahlman.
Comparative studies on the expression of somatostatin receptor subtypes, outcome of octreotide scintigraphy and response to octreotide treatment in patients with carcinoid tumours.
Br. J. Cancer
77:
632-637,
1998[Medline].
33.
O'Carroll, A. M.,
S. J. Lolait,
M. König,
and
L. C. Mahan.
Molecular cloning and expression of a pituitary somatostatin receptor with preferential affinity for somatostatin-28.
Mol. Pharmacol.
42:
939-946,
1992[Abstract].
34.
Prinz, C.,
G. Sachs,
J. H. Walsh,
D. H. Coy,
and
S. V. Wu.
The somatostatin receptor subtype on rat enterochromaffin-like cells.
Gastroenterology
107:
1067-1074,
1994[Medline].
35.
Raulf, F.,
J. Perez,
D. Hoyer,
and
C. Bruns.
Differential expression of five somatostatin receptor subtypes, SSTR1-5, in the CNS and peripheral tissue.
Digestion
55:
46-53,
1994[Medline].
36.
Reisine, T.,
and
G. I. Bell.
Molecular properties of somatostatin receptors.
Neuroscience
67:
777-790,
1995[Medline].
37.
Reisine, T.,
D. Woulfe,
K. Raynor,
H. Kong,
J. Heerding,
J. Hines,
M. Tallent,
and
S. Law.
Interaction of somatostatin receptors with G proteins and cellular effector systems.
In: Somatostatin and Its Receptors, edited by D. J. Chadwick,
and G. Cardew. West Sussex, UK: Wiley, 1995, p. 160-186.
38.
Reubi, J. C.,
U. Horisberger,
B. Waser,
J. O. Gebbers,
and
J. Laissue.
Preferential location of somatostatin receptors in germinal centers of human gut lymphoid tissue.
Gastroenterology
103:
1207-1214,
1992[Medline].
39.
Reubi, J. C.,
J. Laissue,
B. Waser,
U. Horisberger,
and
J. C. Schaer.
Expression of somatostatin receptors in normal, inflamed, and neoplastic human gastrointestinal tissues.
Ann. NY Acad. Sci.
733:
122-137,
1994[Abstract].
40.
Rossowski, W. J.,
Z. F. Gu,
M. S. Akarca,
R. T. Jensen,
and
D. H. Coy.
Characterization of somatostatin receptor subtypes controlling rat gastric acid and pancreatic amylase release.
Peptides
15:
1421-1424,
1994[Medline].
41.
Schaer, J. C.,
B. Waser,
G. Mengod,
and
J. C. Reubi.
Somatostatin receptor subtype sst1, sst2, sst3, and sst5 expression in human pituitary, gastroentero-pancreatic and mammary tumors: comparison of mRNA analysis with receptor autoradiography.
Int. J. Cancer
4:
530-537,
1997.
42.
Southern, E. M.
Detection of specific sequences among DNA fragments separated by gel electrophoresis.
J. Mol. Biol.
98:
503-517,
1975[Medline].
43.
Warhurst, G.,
N. B. Higgs,
H. Fakhoury,
A. C. Warhust,
J. Garde,
and
D. H. Coy.
Somatostatin receptor 2 mediates somatostatin inhibition of ion secretion in rat distal colon.
Gastroenterology
111:
325-333,
1996[Medline].
44.
Yamada, Y.,
S. R. Post,
K. Wang,
H. S. Tager,
G. I. Bell,
and
S. Seino.
Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney.
Proc. Natl. Acad. Sci. USA
89:
251-255,
1992[Abstract].
45.
Yasuda, K.,
S. Rens-Domiano,
C. D. Breder,
S. F. Law,
C. B. Saper,
T. Reisine,
and
G. I. Bell.
Cloning of a novel somatostatin receptor, SSTR3, coupled to adenylylcyclase.
J. Biol. Chem.
276:
20422-20428,
1992.