Somatostatin Receptor Gene Expression in Human Ocular Tissues: RT-PCR and Immunohistochemical Study

Abstract

purpose. Somatostatin (SST) analogues have been used to treat proliferative diabetic retinopathy, pseudotumor cerebri, thyroid orbitopathy, and cystoid macular edema. There is a paucity of published data in regards to cell-specific distribution of SST receptors (SSTR) in normal human eye tissues. Gene expression for all five known SSTRs in normal human ciliary body/iris, retina, choroid, and cultured retinal pigment epithelial (RPE) cells were studied.

methods. mRNA was isolated from human ocular tissues (iris/ciliary body, retina, and choroid) dissected from eight pairs of normal eyes (9–62 years) and from RPE cells grown in culture. RT-PCR was done for all five SSTRs in all analyzed tissues. Immunohistochemistry for SSTR1 and SSTR2 was performed on eight pairs of normal human eyes (28–74 years) imbedded in paraffin.

results. SSTR1 to 5 genes are expressed in retina, SSTR1 and SSTR2 genes in cultured RPE cells, and SSTR1, 2, and 4 in ciliary body and choroid. SSTR1 and SSTR2 immunoreactivity (-ir) was observed on a variety of cells within all analyzed tissues including cornea, iris, trabecular meshwork, Schlemm’s canal, ciliary processes, ciliary muscle, retina, choroid, cultured RPE cells, and optic nerve.

conclusions. SSTR genes are widely expressed in normal human eye tissues, with genes for SSTR1 and SSTR2 being the most widely expressed. Genes for all SSTRs are expressed in retina. SSTR1-ir and SSTR2-ir were observed in all analyzed ocular tissues. Detailed knowledge of SSTRs distribution and function in the human eye will result in a better understanding of their role in health and disease.

Somatostatin (SST) is a ubiquitously distributed cyclic neuropeptide that has diverse biological functions, the most important of which are neurotransmitter, antisecretory, and antiproliferative. ¹ SST-producing cells have been identified at high densities in a variety of normal human tissues including retina. ^{1 2 3} The presence of SST immunoreactive (-ir) cells was detected in the ganglion cell layer (GCL) and the innermost cell rows of the inner nuclear layer (INL) as well as on the cell processes in the inner plexiform layer (IPL) and nerve fiber layer (NFL) in fetal and adult human retinas. ^{3 4 5 6}  

The biological effects of SST are mediated by five high-affinity cell surface receptors (SSTR1–5) that have been detected in the eyes of various mammalian species by using ligand binding studies, in situ hybridization, and immunohistochemistry. ^{7 8 9 10 11 12 13 14 15} Lambooij and colleagues ¹⁵ detected SSTR2A-ir in the outer plexiform layer (OPL), INL, and retinal pigment epithelial (RPE) cells in normal human retinas as well as in thick-walled choroidal blood vessels. No published data exist on the presence and distribution of SSTR-ir cells in normal human cornea, ciliary body, iris, and choriocapillaris. 

Cloning of five SSTR subtypes has led to the development of subtype-selective agonists. Among those, SSTR2-specific SST analogs octreotide (OCT) and lantreotide have attracted significant attention in the past several years. They have been used as new diagnostic and treatment modalities for various hormone overproduction states and as adjunctive treatment for a variety of benign and malignant tumors. ¹⁶ The antiproliferative and antiangiogenic properties of OCT have been exploited in several clinical trials in the treatment of proliferative diabetic retinopathy (PDR), cystoid macular edema, thyroid orbitopathy, and pseudotumor cerebri. ^{16 17 18 19 20 21 22 23 24}  

Because of the possible role of SST and its analogues in the treatment of a variety of ocular diseases, we used RT-PCR to analyze gene expression for all five SSTRs in ciliary body/iris complex, retina, and choroid obtained from normal human donor eyes as well as from cultured human RPE. In addition, we used subtype specific antibodies (Ab) against SSTR1 and SSTR2 to analyze cell- and tissue-specific distribution of those receptors in normal human eye specimens. Basic knowledge of SSTR gene expression and their distribution in healthy human ocular tissues is important for understanding the role of somatostatin in normal eye physiology as well as for development of new therapeutic strategies in ophthalmology. 

Materials and Methods

RNA Isolation

Eight pairs of normal human eyes (ages 9–62 years) were obtained from the Central Ohio Lions Eye Bank within 2 to 4 hours postmortem. Selected ocular tissues from each eye (cornea, ciliary body and iris, retina, and choroid) were promptly isolated under the dissecting microscope and immersed in Trizol reagent (∼100 mg tissue/1 ml Trizol reagent; GIBCO BRL, Life Technologies, Rockville, MD) and snap-frozen in liquid nitrogen to inhibit activity of endogenous RNAses. All cells were disrupted by using tissue homogenizer (10–15 seconds on ice), and total RNA was isolated by phenol: chloroform extraction (GIBCO BRL, LifeTechnologies) and precipitated with absolute ethanol. RNA samples were subsequently treated for 45 minutes with RNase-free DNase at 37°C (Message Clean; GeneHunter Corp., Nashville, TN), phenol:chloroform-extracted, ethanol-precipitated, and recovered in DEPC-treated water. RNA concentration was determined by spectrophotometric readings at 260 and 280 nm. In addition, RNA was extracted from three subsequent passages of cultured human RPE cells (ARPE-19 cell line; American Type Culture Collection [ATCC], Manassas, VA). Because of very high protein/RNA ratio, high-quality RNA could not be isolated from donor corneas. Corneal tissue was analyzed only by immunohistochemistry. 

Cell Culture

RPE cells (ATCC-2303) were purchased from ATCC (passage 21). Cells were grown in DMEM medium (GIBCO BRL, LifeTechnologies) with the addition of 10% heat-inactivated fetal bovine serum (FBS) and standard concentrations of streptomycin and penicillin. Cells were grown at 37°C with 5% CO₂ until 75% to 90% confluent. Three subsequent cell passages 22, 23, and 24 were harvested, and total RNA was isolated as described above. RPE cells used for immunohistochemistry were grown in four-chamber well slides (Fisher Scientific, Pittsburgh, PA) for at least 48 hours. 

RT-PCR

RT and PCR reactions were done by using SuperScript Preamplification System for First DNA strand cDNA synthesis as suggested by manufacturer (GIBCO BRL, Life Technologies). Random hexamer primers and 1 μg of total RNA were used in the RT step. Sequences of primers used in the PCR step for SSTR1–5 and c-abl ²⁵ are shown in Table 1 . From each RNA sample three separate cDNA samples were synthesized, and separate PCR reactions were performed for each SSTR gene. The following PCR programs were used: SSTR1: 94°C for 2 minutes followed by 33 cycles 94°C for 40 seconds, 62°C for 50 seconds, and 72°C for 1 minute and 30 seconds; SSTR2: 94°C for 2 minutes followed by 35 cycles 94°C for 45 seconds, 60°C for 1minute, and 72°C for 2 minutes and 30 seconds; SSTR3: 94°C for 2 minutes followed by 35 cycles 94°C for 50 seconds, 58°C for 50 seconds, and 72°C for 1 minute and 30 seconds; SSTR4: 94°C for 2 minutes followed by 35 cycles 94°C for 40 seconds, 62°C for 45 seconds, and 72°C for 1 minute; SSTR5: 94°C for 2 minutes followed by 30 cycles 94°C, 65°C for 5 seconds, and 72°C for 1 minute Each PCR reaction used 2 μl of each cDNA mix obtained in RT step. RT-PCR products were separated by electrophoresis in 1.5% to 2.0% agarose in 1× TEA buffer with ethidium bromide. 

Testing of Efficacy of DNA-se Treatment

Genes for SSTR1, 3, 4, and 5 lack classical introns, and the SSTR2 gene has only one cryptic intron at the 3′ end of the coding segment, giving rise to two alternatively spliced isoforms, SSTR2A and SSTR2B. ²⁶ Absence of genomic DNA contamination of all DNA-se treated RNA samples was confirmed in two different ways: first, 5 μl of each RNA sample that was treated with DNase (before treatment with reverse transcriptase) was used in the PCR step with above-mentioned PCR conditions and primers used for c-abl amplification (Table 1) to test for presence of any PCR product that could be suggestive of the presence of genomic DNA contamination. No PCR products could be detected in any of the tested samples (Fig. 1A , lanes 2 through 5). Second, a portion of the c-abl gene was amplified by using cDNA from each sample. Primers used for this amplification originate in exons a1 and a2 of the c-abl gene spanning the first intron (Table 1) . The following PCR program was used: 94°C for 2 minutes and 30 cycles at 94°C for 30 seconds, 58°C for 50 seconds, and 72°C for 1 minute and 30 seconds. Amplification of c-abl cDNA yielded a 201-bp-long fragment that was present in all cDNA samples (Fig. 1B , lanes 1 through 4). Amplification of undigested genomic DNA would yield a 764-bp-long fragment that was uniformly absent in all analyzed cDNA samples (Fig. 1B , lanes 1 through 4). Genomic and cDNA c-abl fragments could easily be amplified using this PCR program. Figure 1B , lane 5, represents a sample that was made as a mixture of retinal c-DNA and human genomic DNA. Specificity of c-abl PCR products was confirmed by Southern blot (data not shown). 

Antibody Specificity Testing and Immunohistochemistry

The preparation of SSTR1 and SSTR2 Abs in rabbits was previously described. ²⁵ SSTR1 and SSTR2 Abs were produced by using synthetic peptide antigens, that is, N-terminal 57 amino acids of SSTR1 and N-terminal 45 amino acids of SSTR2. Specificity of SSTR1-Ab and SSTR2-Ab was tested by preabsorbing SSTR1 Ab and SSTR2 Ab with corresponding truncated SSTR1 and SSTR2 proteins that were used as antigens (for 3 hours at room temperature; final concentration of peptide was 50 μg/ml). Concentrations of preabsorbed Abs used for immunohistochemistry were corrected to account for dilution with blocking peptide. Antigen competition was performed on (1) two neuroblastoma cell lines in culture; (2) cultured RPE cells; and on (3) human eye sections. Neuroblastoma cell lines SKR1 and SY5Y express different SSTRs, that is, the SKR1 cell line expresses only SSTR1, and the SY5Y cell line expresses only SSTR2 (results for both cell lines were demonstrated by RT-PCR and confirmed by Southern blot; results not shown). 

Additionally, eight pairs of normal human eyes (obtained from the Central Ohio Lions Eye Bank within 2–4 hours postmortem; ages 28–74 years) were fixed in 4% buffered formalin, imbedded in paraffin, and serially cut on a microtome (thickness, 4 μm). Tissue sections were placed on charged slides, deparaffinized in xylene, and rehydrated. The slides were placed into preheated Antigen Retrieval solution (BioGenex, San Ramon, CA), microwaved for 10 minutes, and left in a sealed container for 15 minutes, followed by washing in phosphate-buffered saline (PBS) three times. Then slides were washed in OptiMax Wash Buffer (BioGenex) and incubated in Power Block (BioGenex) for 10 minutes. Slides were incubated overnight at 4°C with primary SSTR1 Ab and SSTR2 Ab (1:1000 and 1:2000 dilution in 3% BSA in PBS, respectively). Sections were washed with PBS three times andincubated for 1 hour at room temperature with rhodamine- or fluorescein-labeled polyclonal anti-rabbit Ab (in 1:250 dilution in 1% BSA in PBS; Vector Laboratories Inc., Burlingame, CA). Slides were again washed with PBS three times and cover-slipped with water-based imbedding media containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc.) for visualization of cell nuclei. Slides were viewed using Zeiss immunofluorescent microscope, and images were captured by using multiple-exposure system with DAPI, TRITC, or rhodamine filters with Smart Capture VP 1.4 software (Digital Scientific, Jersey City, NJ). 

After deparaffinization some slides were incubated with 3% H₂O₂ for 15 minutes at room temperature to inhibit endogenous peroxidase activity and processed for conventional immunohistochemistry using peroxidase-labeled polyclonal anti-rabbit Ab (DAKO Inc., Copenhagen, Denmark). Immunohistochemical binding was visualized by incubating tissue sections with diaminobenzidine (DAB) chromogen for up to 15 minutes; cells were then washed, counterstained, and cover-slipped. 

Neuroblastoma cell lines were grown in two-well chamber-slides in DMEM media (GIBCO BRL, LifeTechnologies) supplemented with 15% FBS and 100 U/ml penicillin and 100 μg streptomycin for at least 48 hours. SKR1, SY5Y, and RPE cells in culture were fixed for 10 minutes with ice-cold absolute methanol, washed three times with PBS, and incubated with 3% BSA in PBS for 30 minutes at room temperature. Then slides were treated in the same manner as tissue sections described above. 

Results

RT-PCR

Genes for various SSTRs are expressed in all analyzed ocular tissues (Table 2) . Genes for all five SSTR subtypes are expressed in normal retina (Fig. 1C) , with SSTR3 and SSTR5 genes being expressed only in retina. In spite of very high stringency conditions used within the PCR step to amplify SSTR5 (annealing temperature 65°C and annealing time of 5 seconds), several faint sidebands remained present throughout the PCR optimization process (SSTR5 lane, Fig. 1A ). Neither of those side bands hybridized with the SSTR5 probe during Southern blot. 

Only SSTR1 and SSTR2 genes are expressed in cultured RPE cells (Fig. 1D) . SSTR1, SSTR2, and SSTR4 gene expression was detected in ciliary body/iris complex and choroid (Figs. 1E 1F) . Specificity of all the above-mentioned RT-PCR results were confirmed with Southern blot analysis for each SSTR subtype (data not shown). 

Immunohistochemistry

Antibody Specificity Testing.

SSTR1-ir was detected only in SKR1 cells but not in the SY5Y cell line, whereas SSTR2-ir was detected in SY5Y cell line but not in the SKR1 cell line (Figs. 2A 2C) . In addition, SSTR1-ir in the SKR1 cell line as well as SSTR2-ir in the SY5Y cell line was completely competed out by preabsorbing SSTR1-Ab and SSTR2-Ab with truncated SSTR1 and SSTR2 proteins, respectively (Figs. 2B 2D) . The same competition was performed for both types of receptors in cultured RPE cells that express SSTR1-ir and SSTR2-ir (Figs. 3A 3C) . No immunoreactivity was observed in RPE cells when either SSTR1-Ab or SSTR2-Ab was preabsorbed with truncated SSTR1 or SSTR2 proteins, respectively (Figs. 3B 3D) . No SSTR1-ir or SSTR2-ir were observed in eye tissue sections incubated with preabsorbed SSTR1 Ab or SSTR2 Ab (Figs. 4A 4B) . In addition, no nonspecific binding was observed when slides were incubated with only secondary Ab, omitting primary Abs (data not shown). 

RPE Cells in Culture.

Intense SSTR1-ir and SSTR2-ir were observed on cell membranes and cytoplasm of individual RPE cells in culture (Figs. 3A 3C) . Actively dividing RPE cells showed increased cytoplasmic SSTR1-ir and SSTR2-ir compared with nondividing RPE cells. 

Cornea.

Neither SSTR1-ir nor SSTR2-ir was found in corneal epithelium. Moderate SSTR1-ir and SSTR-2-ir was observed on cell membrane and cytoplasm of stromal keratocytes (not shown). Corneal endothelial cells showed strong punctate SSTR1-ir and SSTR-2-ir in all specimens (Fig. 5) . 

Trabecular Meshwork and Schlemm’s Canal.

Moderate SSTR1-ir and SSTR-2-ir were present on trabecular endothelial cells in uveal and corneoscleral meshwork as well as on endothelial cells lining the Schlemm’s canal (Fig. 6) . 

Iris and Ciliary Body.

Because of the heavy content of melanin in the pigment epithelium in iris and in ciliary processes, conventional immunohistochemistry was used to avoid significant artifacts related to autofluorescence of melanin in these two structures. Immunoreactive product was present in the form of the dark brown pigment (DAB). Weak SSTR1-ir was detected in sphincter and dilator iris muscles. No other cells were found to be SSTR1 immunoreactive. However, strong SSTR2-ir was present on fibrocytes and clump cells present in iris stroma, sphincter, and dilator muscles as well as on endothelial cells within iris blood vessels (Fig. 7) . SSTR1-ir or SSTR2-ir could not be reliably assessed for the posterior iris pigment epithelium and pigmented epithelium of the ciliary processes secondary to heavy pigmentation. Circular and longitudinal fibers of ciliary muscle showed intense cytoplasmic and membranous SSTR1-ir and SSTR2-ir (Fig. 8) . Intense SSTR1-ir and SSTR2-ir was also detected in the nonpigmented epithelium and on the endothelium of marginal capillaries of ciliary processes (Fig. 9) . 

Retina and Choroid.

SSTR1-ir and SSTR2-ir was detected across all cell and fiber layers in retina in the form of fine punctate staining on the membranes of outer and inner segments of rods and cones and individual cells in outer nuclear layer (ONL), INL, and GCL (Figs. 10A 10B 10C and 11A 11B 11C) . Because of the high cell density within ONL and INL, morphology of individual cells could not be reliably assessed (e.g., amacrine cells, Müller cells, and bipolar cells). Prominent SSTR1-ir and SSTR2-ir were also present in OPL, IPL, and NFL (Figs. 10A and 11A , 11B). Melanin and lipofuscin granules present within RPE cells demonstrated autofluorescence (orange-red). Very intense SSTR1-ir and SSTR2-ir (bright red) was abundantly present on the membranes and in the cytoplasm of RPE cells (Figs. 10B and 11A) . Distinct punctate SSTR1-ir and SSTR2-ir were present on the membrane and in the cytoplasm of endothelial cells in retinal vessels, including arterioles, venules, and capillaries (Figs. 10D and 11D) . The same pattern of strong SSTR1-ir and SSTR2-ir was present within endothelial cells, fibrocytes, and melanocytes within choriocapillaris and choroid (Fig. 12) . 

Optic Nerve.

Moderately strong SSTR1-ir and SSTR2-ir were observed in axons within the optic nerve. No SSTR1-ir or SSTR2-ir could be detected on glial cells within the nerve. Immunohistochemical results are summarized in Table 3 . 

Discussion

SSTRs are high-affinity cell surface receptors that belong to the family of G-protein–coupled receptors. ^{1 27} SSTRs have a broad expression pattern, and the individual receptors have both overlapping and tissue specific patterns of expression, with SSTR2 usually being the most widely expressed subtype. ^{1 16} SSTRs couple to a number of inhibitory signal transduction mechanisms, activation of which results in downregulation of processes such as secretion of growth factors, cellular proliferation, and differentiation in numerous mammalian cells. ^{1 2 27 28}  

In spite of growing interest in somatostatin analogues as new treatment modalities, surprisingly little is known about the distribution of SSTRs in the human eye and their functions. We, therefore, studied gene expression for all five SSTRs by RT-PCR and the distribution of SSTR1-ir and SSTR2-ir normal human ocular tissues. Our results demonstrate that genes for SSTRs are widely expressed in all analyzed tissues, with SSTR1 and SSTR2 genes being the most widely expressed, followed by SSTR4 gene (expressed in retina, choroid, and ciliary body/iris). SSTR3 and SSTR5 gene expression was detected only in the retina. Good correlation was detected in the distribution of SSTR1-ir and SSTR2-ir and SSTR1 and SSTR2 gene expression. Similar correlation between SSTR1 and SSTR2 gene expression and their immunohistochemical localization was shown in several SSTR-positive human tumors. ^{16 29 30} In the absence of immunohistochemical staining for SSTR3, SSTR4, and SSTR5, RT-PCR results confirmed by Southern blot are highly suggestive but not definitive proof of the presence of those receptor molecules in analyzed tissues. 

Although SSTR1 and SSTR2 are membrane-associated receptors, we detected a significant amount of staining within the cytoplasm, in the peri-nuclear region and some nuclear staining in many immunoreactive cells. After binding their ligand, SSTR-ligand complexes undergo internalization. ³¹ Progressive intracytoplasmic and intranuclear translocation as well as DNA binding of radioactively labeled stable SST analogues seems to be cell and receptor dependent. ^{31 32 33 34} DNA sequence(s) that bind SST analogues as well as their role in the gene transcription process is very intriguing but poorly understood at present. 

Our results showed intense SSTR1-ir and SSTR2-ir on RPE cells, on outer and inner segments of photoreceptors, and on individual cells of ONL and INL as well as on cells in the GCL. Intense SSTR1-ir and SSTR2-ir was also noted within the OPL and IPL. SSTR1 and SSTR2 seem to be expressed on the same cell types across all retinal layers. Because of very high cell density in outer and inner nuclear layers, it was not possible to precisely define morphology of immunoreactive cells (i.e., bipolar cells, Müller glia, and horizontal cells) and their processes. Further double-labeling studies are necessary to precisely delineate subpopulations of SSTR1 and SSTR2 immunoreactive cells in the human retina. 

The exact physiologic role of SSTR1 and SSTR2 in visual signal processing in immunoreactive human retinal cells is currently unknown. Our results and results of Johnson’s study ¹⁴ show that SSTR1-ir and SSTR2-ir are much more widely distributed in human and monkey retinas than would be expected based on the cell density, distribution, or connectivity of SST-ir cells in retinas of both species. It was proposed that SST released by SST-ir cells could diffuse across retinal layers in a radial and tangential manner, affecting the retinal cells that do not directly synapse with SST-ir cells. ¹⁴ It is also possible that some of the widely distributed SSTR1 and/or SSTR2 could bind another ligand(s) such as cortistatin, as suggested by Siehler. ³⁵  

Experimental studies using SST and octreotide showed an inhibitory effect on the proliferation of human and murine endothelial cells in culture (cell lines HUV-EC-C and HECa10). ^{28 36 37} Strong SSTR1-ir and SSTR2-ir presence on endothelial cells within retinal vessels, choriocapillaris, and choroidal vessels may have critical implications for the development of future treatment modalities for PDR and choroidal neovascularization in age-related macular degeneration. 

Our results show more widespread distribution of SSTR2-ir in normal retina and choroid when compared with the distribution of SSTR2A-ir those tissues. ¹⁵ There are two possible explanations for this observation: first, SSTR2 Ab used in this study was raised against N-terminal 45 amino acids that are common to both SSTR2A and SSTR2B molecules, whereas Ab used by Lambooij and colleagues ¹⁵ was raised against 22-amino acid peptide located in the C-terminal region of the SSTR2A molecule. Therefore, more extensive immunoreactivity observed in our study is suggestive of the presence of SSTR2B in retinal and choroidal tissues that could not be detected by SSTR2A-Ab. Our immunohistochemistry results were also confirmed by the RT-PCR and Southern blot showing gene expression in those tissues, suggesting that our techniques are both sensitive and specific. Second, some of the differences could be explained by difference in sensitivity of the two immunohistochemical techniques, that is, immunofluorescence versus peroxidase method. 

SSTR1-ir and SSTR2-ir were also detected on nonpigmented ciliary epithelium, on marginal capillaries of ciliary processes, and on endothelial cells in trabecular meshwork and in Schlemm’s canal, suggesting their role in the aqueous fluid homeostasis in the human eye. The role of somatostatinergic signaling system in the production and/or absorption of aqueous fluid in primates is unknown. In the nonpigmented epithelium of rabbit ciliary processes, SST was shown to modulate aqueous fluid production by affecting adenylate cyclase activity and concentration of intracellular Ca²⁺. ^{38 39 40}  

In summary, by using RT-PCR, SSTR1–5 gene expression has been detected in iris/ciliary body, retina, RPE cells, and choroid of the human eye. By using newly developed SSTR1 and SSTR2 Ab, SSTR1-ir and SSTR2-ir were detected in cornea, trabecular meshwork and Schlemm’s canal, iris, ciliary body, retina, choroid, and optic nerve. Further studies are necessary to clarify the exact biological functions of SSTR1 and SSTR2 on immunoreactive cells in the human eye. Detailed knowledge of SSTRs distribution and function in the human eye will lead to a better understanding of their role in health and disease. 

 Supported in part by the Central Ohio Lions Eye Association, The Bremer Fund, The George and Miriam Mikesell Research Fund, The Jacob and Florence Moses Fund, and National Institutes of Health Grant ROI CA 64177.

 Submitted for publication January 12, 2001; revised April 2, 2001; accepted April 19, 2001.

 Commercial relationships policy: N.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked“ advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Primer Sequences Used for RT-PCR and Sizes of PCR Products for Both Genomic DNA (gDNA) and cDNA as Templates

Gene	Primers	gDNA (bp)	cDNA (bp)	Accession Number
SSTR1	5′ 751 CGC TGG CTG GTG GGC TTC GTG TTG 3′	481	481	M81829
	5′ 1231 CGC CGC CGG ACT CCA GGT TCT CAG 3′
SSTR2	5′ 82 CAT GGA CAT GGC GGA TGA G 3′	1112	1112	M81830
	5′ 1193 CTC AGA TAC TGG TTT GGA G 3′
SSTR3	5′ 623 GCG AGC CGG CTT CAT CAT CTA CAC 3′	517	517	M96738
	5′ 1139 GAC CCG GCC GTT CAT CTC CTT C 3′
SSTR4	5′ 715 TGG TCG GCA GTC TTC GTG GTC TAC 3′	516	516	L14856
	5′ 1230 CTT GCG GCC GGG TTC TGG T 3′
SSTR5	5′ 534 GCG GCC TGG GTC CTG TCT CT 3′	627	627	D16827
	5′ 1160 CCC CCG CCT GCA CTC TCA C 3′
c-abl	5′ 450 TTC AGC GGC CAG TAG CAT CTG ACT T 3′	764	201	M14752
	5′ 650 TGT GAT TAT AGC CTA AGA CCC GGA G 3′

Figure 1.

 

(A) Results of c-abl PCR done with DNase-treated RNA samples (no RT treatment); retina, RPE, choroid, and ciliary body (lanes 2 to 5, respectively). Lane 1, a positive control; c-abl PCR done with retinal c-DNA. (B) c-abl RT-PCR done with cDNA samples; retina, RPE, choroid, and ciliary body (lanes 1 to 4, respectively; 201-bp fragment). Lane 5, a c-abl PCR product obtained from mixture of retinal cDNA and human genomic DNA (210- and 764-bp fragments). SSTRs gene expression in retina (C), RPE cells (D), choroid (E), and ciliary body/iris (F). DNA marker of 100-bp is loaded in each gel (intense band represents 600 bp; arrow).

Table 2.

 

SSTRs Gene Expression in Ocular Tissues Analyzed

Tissue	SSTR1	SSTR2	SSTR3	SSTR4	SSTR5
RET	+ (16/16)	+ (16/16)	+ (16/16)	+ (16/16)	+ (16/16)
RPE	+ (3/3)	+ (3/3)	− (0/3)	− (0/3)	− (0/3)
CH	+ (16/16)	+ (16/16)	− (0/16)	+ (15/16)	− (0/16)
CB/IR	+ (15/16)	+ (16/16)	− (0/16)	+ (14/16)	− (0/16)

 RET, retina; RPE, cultured retinal pigment epithelium; CH, choroid; CB/IR, ciliary body and iris. Numbers in parentheses represent the number of positive RT-PCR results. All results were obtained in triplicate. +, presence of the band in the gel; −, absence.

Figure 2.

 

SSTR1-ir in SKR1 cells in culture before (A) and after (B) preadsorbing SSTR1 Ab with SSTR1 peptide. SSTR2-ir in SY5Y cells in culture before (C) and after (D) preadsorbing SSTR2 Ab with blocking SSTR2 peptide. Magnification,× 400.

Figure 3.

 

SSTR1-ir in RPE cells in culture before (A) and after (B) incubating primary SSTR1 Ab with blocking SSTR1 peptide. SSTR2-ir in RPE before (C) and after (D) preabsorbing SSTR2 Ab with blocking SSTR2 peptide. Magnification,× 400.

Figure 5.

 

SSTR2-ir in human corneal endothelial cells. Nuclei of endothelial cells are blue (DAPI), and immunoreactivity is presented as red, fine punctate staining of cell membranes and cytoplasms. AC, anterior chamber. Magnification, ×400.

Figure 6.

 

SSTR2-ir (green staining) in endothelial cells of Schlemm’s canal and trabecular meshwork cells. Orange, cell nuclei; SC, Schlemm’s canal. Magnification, ×400.

Figure 7.

 

Strong SSTR2-ir in iris sphincter, dilator, iris stroma, and endothelial cells of iris blood vessels. Magnification,× 100.

Figure 9.

 

SSTR2-ir of the ciliary processes. Immunoreactivity is present in nonpigmented epithelium (arrow) and endothelium of blood vessels as a brown product. Magnification,× 200.

Figure 10.

 

(A through C) SSTR1-ir in retina. RPE, retinal pigment epithelium; PR, photoreceptors; ONL, outer nuclear layer; INL, inner nuclear layer. (D) SSTR1-ir in retinal blood vessels is present in endothelial cell. Red blood cells within the vessel lumen show autofluorescence. Magnification, (A) ×200; (B and D) ×400; and (C)× 1000.

Figure 11.

 

(A through C) SSTR2-ir in the retina. RPE, retinal pigment epithelium; PR, photoreceptors; ONL, outer nuclear layer; INL, inner nuclear layer. SSTR2-ir is present as a bright red staining (A and B) or as a green staining (C). (D) SSTR2-ir in endothelial cells of retinal blood vessels. Magnification, (A and D) ×400; (B and C)× 1000.

Figure 12.

 

SSTR2-ir in choriocapillaris. SSTR2-ir is present as a punctate red staining on the endothelial cells, choroidal fibrocytes, and melanocytes. Magnification, ×400.

Table 3.

 

Intensity of SSTR1-ir and SSTR2-ir in Intraocular Tissues

Tissue/Cell Type	SSTR1-ir	SSTR2-ir
Cornea
Epithelium	−	−
Stromal keratocytes	++	++
Endothelial cells	+++	+++
Trabecular meshwork	++	++
Iris
Stroma	−	+++
Dilator muscle	+	+++
Constrictor muscle	+	+++
Endothelial cells	−	+++
Ciliary body
Nonpigmented ep.	+++	+++
Ciliary muscle	++	++
Endothelial cells	++	++
Retina
RPE	+++	+++
Photoreceptors	++	++
INL	++	++
IPL	++	++
ONL	++	++
OPL	++	++
GCL	++	++
NFL	++	++
Endothelial cells	++	++
Choroid
Endothelial cells	+++	+++
Fibrocytes	++	++
Melanocytes	++	++
Optic nerve
Axons	++	++
Glial cells	−	−

 +, weak; ++, moderate; +++, strong; RPE, retinal pigment epithelium; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer.

Figure 4.

 

Negative tissue controls: retinal tissue (cell nuclei are stained with DAPI). Slides were incubated with SSTR1-Ab (A) and SSTR2-Ab (B) that were preabsorbed with their blocking peptides. Magnification, ×200.

Figure 8.

 

SSTR2-ir in ciliary muscle (punctate green staining). Nuclei are orange-red. Magnification,× 200.

The authors thank Central Ohio Lions Eye Bank for providing ocular tissues and Florinda Jaynes for her superb technical assistance.