Glucagon-like peptide-1 (GLP-1) receptors are not overexpressed in pancreatic islets from patients with severe hyperinsulinaemic hypoglycaemia following gastric bypass.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) receptors are highly overexpressed in benign insulinomas, permitting in vivo tumour visualisation with GLP-1 receptor scanning. The present study sought to evaluate the GLP-1 receptor status in vitro in other pancreatic disorders leading to hyperinsulinaemic hypoglycaemia, specifically after gastric bypass surgery. METHODS: Fresh frozen pancreatic tissue samples (n=7) from six gastric bypass surgery patients suffering from hyperinsulinaemic hypoglycaemia were evaluated for GLP-1 receptor content using in vitro receptor autoradiography, and compared with normal pancreas and with pancreatic insulinoma tissues. RESULTS: GLP-1 receptor analysis of the pancreatic tissues, which histopathologically were compatible with nesidioblastosis and originated from post-bypass hypoglycaemic patients, revealed a mean density value of GLP-1 receptors in the islets of 1,483 ± 183 dpm/mg tissue. Pharmacological characterisation indicated the presence of specific GLP-1 receptors. The density of islet GLP-1 receptor in post-gastric bypass patients did not differ from that of normal pancreas (1,563 ± 104 dpm/mg tissue, n = 10). Receptor density in pancreatic acini was low in post-bypass and control conditions. In contrast, benign insulinomas showed a high density of GLP-1 receptors, with a mean value of 8,302 ± 1,073 dpm/mg tissue (n = 6). CONCLUSIONS/INTERPRETATION: In contrast to insulinoma, hyperinsulinaemic hypoglycaemia after gastric bypass surgery is not accompanied by overexpression of GLP-1 receptor in individual islets. Thus, patients with post-gastric bypass hyperinsulinaemic hypoglycaemia are not candidates for GLP-1 receptor imaging in vivo using radiolabelled exendin. These GLP-1 receptor data support the notion that the islet pathobiology of post-gastric bypass hypoglycaemia is distinctly different from that of benign insulinomas.

Cholecystokinin(CCK)-A and CCK-B/gastrin receptors in human tumors.

Cholecystokinin (CCK)-A and CCK-B/gastrin receptors were evaluated with in vitro receptor autoradiography in 406 human tumors of various origins using a sulfated 125I-labeled CCK decapeptide analogue 125I-(D-Tyr-Gly, Nle28,3l)-CCK 26-33 and 125I-labeled Leu15-gastrin as radioligands. CCK-B/gastrin receptors were found frequently in medullary thyroid carcinomas (92%), in small cell lung cancers (57%), in astrocytomas (65%), and in stromal ovarian cancers (100%). They were found occasionally in gastroenteropancreatic tumors, breast, endometrial, and ovarian adenocarcinomas. They were either not expressed or rarely expressed in colorectal cancers, differentiated thyroid cancers, non-small cell lung cancers, meningiomas, neuroblastomas, schwannomas, glioblastomas, lymphomas, renal cell cancers, prostate carcinomas, and the remaining neuroendocrine tumors (i.e., pituitary adenomas, pheochromocytomas, paragangliomas, and parathyroid adenomas). CCK-A receptors were expressed rarely in tumors except in gastroenteropancreatic tumors (38%), meningiomas (30%), and some neuroblastomas (19%). The identified CCK-A and CCK-B receptors were specific and of high affinity in the subnanomolar range. The rank order of potency of various CCK analogues was: sulfated CCK-8 = L-364,718 >> nonsulfated CCK-8 = L-365,260 > or = gastrin for CCK-A receptors and sulfated CCK-8 > gastrin = nonsulfated CCK-8 > L-365,260 > L-364,718 for CCK-B receptors. CCK-B receptors could also be selectively and specifically labeled with a newly designed nonsulfated 125I-(D-Tyr-Gly, Nle28,31)-CCK 26-33. Gastrin mRNA measured by in situ hybridization was present in most CCK-B receptor-positive small cell lung cancers, breast tumors, and ovarian tumors, representing the molecular basis of a possible autocrine growth regulation of these tumors. Gastrin and CCK mRNAs were lacking in medullary thyroid cancers. Thus, these results may have pathogenic, diagnostic, differential diagnostic, and therapeutic implications.

Expression and localization of somatostatin receptor SSTR1, SSTR2, and SSTR3 messenger RNAs in primary human tumors using in situ hybridization.

Somatostatin receptor gene expression of SSTR1, SSTR2, and SSTR3 subtypes was evaluated by in situ hybridization in 55 human primary tumors shown to contain a high density of somatostatin receptors in binding assays. All 55 tumors expressed at least one SSTR subtype. Of 55 somatostatin receptor-positive tumors, 46 had SSTR2 mRNA; all 46 were characterized as having receptors with a high affinity for the synthetic analogue octreotide. Of 55 tumors, 12 expressed SSTR1, and 14 expressed SSTR3 mRNA. The subtype SSTR1 was expressed alone in 4 cases, SSTR2 was expressed alone in 33 cases, and SSTR3 was expressed alone in one case. In 4 cases, all 3 SSTR were expressed simultaneously. The cases having SSTR1 mRNA were identified in binding experiments with 125I-labeled somatostatin-14 and -28 analogues rather than with 125I-[Tyr3]-octreotide. Whereas meningiomas, neuroblastomas, pituitary adenomas, small cell lung carcinomas, lymphomas, and breast tumors expressed primarily a high abundance of SSTR2, carcinoids, islet cell carcinomas, medullary thyroid carcinomas, and ovarian tumors had a mixed distribution of the somatostatin receptor subtypes. This is the first demonstration of the presence of SSTR1, SSTR2, and SSTR3 in primary human tumors using in situ hybridization. Since these somatostatin receptor subtypes probably mediate distinct somatostatin actions, it may be worthwhile to search for subtype-specific analogues to use for the treatment and diagnosis of these tumors.

Y(1)-mediated effect of neuropeptide Y in cancer: breast carcinomas as targets.

Overexpression of selected peptide receptors in human tumors has been shown to represent clinically relevant targets for cancer diagnosis and therapy. Neuropeptide Y (NPY) is a peptide neurotransmitter mediating feeding behavior and vasoconstriction. It has never been shown to be involved in human cancer. We show here, using in vitro receptor autoradiography, a NPY receptor incidence of 85% in primary human breast carcinomas (n = 95) and of 100% in lymph node metastases of receptor-positive primaries (n = 27), predominantly as Y(1) subtype, whereas non-neoplastic human breast expressed Y(2) preferentially. Y(1) mRNA was detected in Y(1)-expressing tumors by in situ hybridization, whereas Y(2) mRNA was found in normal breast tissue. The strong predominance of Y(1) in breast carcinomas compared with Y(2) in normal breast suggests that neoplastic transformation can switch the NPY receptor expression from Y(2) to Y(1) subtype. Moreover, in Y(1)-expressing human SK-N-MC tumor cells, an NPY-induced, dose-dependent inhibition of tumor cell growth of >40% was observed, suggesting a functional role of NPY in cancer, mediated by Y(1). NPY should therefore be added to the list of small regulatory peptides related to cancer. The high incidence of Y(1) in in situ, invasive, and metastatic breast cancers allows for the possibility to target them for diagnosis and therapy with NPY analogues.

Somatostatin and vasoactive intestinal peptide receptors in human mesenchymal tumors: in vitro identification.

Somatostatin and vasoactive intestinal peptide (VIP) have been shown to be of diagnostic and therapeutic interest in several types of human epithelial tumors expressing the respective receptor. The present study evaluates the presence of somatostatin and VIP receptors in 64 primary or metastatic human mesenchymal tumors. In vitro receptor autoradiography on cryostat sections was performed using 125I-labeled [Tyr3]-octreotide as well as 125I-labeled [Leu8,D-Trp22,Try25]-somatostatin-28 as radioligands for somatostatin receptors and 125I-labeled VIP as radioligand for VIP receptors. Somatostatin receptors were identified in bone and vascular/perivascular tumors (3 of 3 osteosarcomas, 1 of 1 giant cell tumor, 2 of 2 angiosarcomas, and 4 of 4 hemangiopericytomas), in 2 of 2 synovial sarcomas, in 2 of 5 histiocytomas, and in several muscle cell tumors (1 of 2 leiomyomas, 2 of 4 leiomyosarcomas, and 3 of 5 rhabdomyosarcomas) but were absent in 4 liposarcomas, 3 mesotheliomas, 3 chondrosarcomas, 10 Ewing sarcomas, 11 schwannomas, and 5 Wilms' tumors. VIP receptors were identified in 3 of 3 differentiated liposarcomas, 2 of 2 angiosarcomas, 4 of 4 hemangiopericytomas, 2 of 2 synovial sarcomas, 3 of 3 mesotheliomas, 5 of 5 Wilms tumors, as well as in 2 of 5 histiocytomas, 1 of 2 leiomyomas, 2 of 4 leiomyosarcomas, 3 of 3 intermediately differentiated rhabdomyosarcomas, and 1 of 3 osteosarcomas but not in chondrosarcomas, Ewing sarcomas, schwannomas, or undifferentiated rhabdomyosarcomas. The receptors were located on neoplastic cells. The somatostatin receptors were of high affinity and of high specificity for biologically active somatostatin analogues with high affinity for somatostatin-14 and somatostatin-28 as well as for octreotide, thus representing the sst2 subtype; in a few cases of tumors having somatostatin receptors with low affinity for octreotide, in situ hybridization techniques identified preferentially sst1 mRNA. These data suggest that human mesenchymal tumors may be targets for somatostatin and/or VIP receptor in vivo imaging; they may also be potential targets for somatostatin or VIP analogue therapy.

Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor subtypes in human tumors and their tissues of origin.

The evaluation of peptide receptors in man is needed not only to discover the physiological target tissues of a given peptide but also to identify diseases with a sufficient receptor overexpression for diagnostic or therapeutic interventions. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors have been evaluated in human tumors and in their tissues of origin using in vitro receptor autoradiography with 125I-VIP or 125I-acetyl-PACAP-27 in tissue sections. The VIP/PACAP receptor subtypes VPAC1, VPAC2, and PAC1 were evaluated in these tissues by determining the rank order of potencies of VIP and PACAP as well as VPAC1- and VPAC2-selective analogues. The VIP/PACAP receptors expressed in the great majority of the most frequently occurring human tumors, including breast (100% receptor incidence), prostate (100%), pancreas (65%), lung (58%), colon (96%), stomach (54%), liver (49%), and urinary bladder (100%) carcinomas as well as lymphomas (58%) and meningiomas (100%), are predominantly of the VPAC1 type. Their cells or tissues of origin, i.e., hepatocytes, breast lobules and ducts, urothelium, prostate glands, pancreatic ducts, lung acini, gastrointestinal mucosa, and lymphocytes, also predominantly express VPAC1. Leiomyomas predominantly express VPAC2 receptors, whereas paragangliomas, pheochromocytomas, and endometrial carcinomas preferentially express PAC1 receptors. Conversely, VPAC2 receptors are found mainly in smooth muscle (i.e., stomach), in vessels, and in stroma (e.g., of the prostate), whereas PAC1 receptors are present in the adrenal medulla and in some uterine glands. Whereas the very wide distribution of VIP/PACAP receptors in the normal human body is indicative of a key role of these peptides in human physiology, the high VIP/PACAP receptor expression in tumors may represent the molecular basis for clinical applications of VIP/PACAP such as in vivo scintigraphy and radiotherapy of tumors as well as VIP/PACAP analogue treatment for tumor growth inhibition.

Detection of somatostatin receptors in surgical and percutaneous needle biopsy samples of carcinoids and islet cell carcinomas.

Somatostatin (SS) receptor status was investigated in the tumor tissues from 62 patients with carcinoid tumors and 15 patients with islet cell carcinomas using receptor autoradiography techniques with two different iodinated somatostatin analogues as radioligands, a [Leu8, DTrp22, Tyr25]somatostatin-28 and a somatostatin octapeptide, Tyr3-octreotide. The carcinoid tumors were either primaries (n = 32) or metastases (n = 43), sampled as surgical specimens or as small needle liver biopsies. Fifty-four of 62 carcinoid patients had SS receptor-positive tumors (87%). All 15 islet cell carcinoma patients had positive tumors (4 primaries, 11 metastases), i.e., 3 vipomas, 3 insulinomas, 2 glucagonomas, 1 gastrinoma, 2 polyfunctional tumors, and 4 nonfunctioning tumors. Saturation and competition experiments on tissue sections revealed saturable, high affinity binding sites pharmacologically specific for bioactive SS analogues. In a majority of the tumors, the receptors were densely distributed and were always homogeneously found in the whole tumor. All except two tumors were labeled with both radioligands. Multiple liver metastases (n = 16) from three different patients were all shown to contain a comparable amount of receptors. SS receptors could be demonstrated even in very small tissue samples of liver metastases obtained by percutaneous liver biopsies (mean weight, 6.8 mg). The majority of the eight SS receptor-negative carcinoids were mainly bronchial carcinoids (n = 5), usually poorly differentiated. On the contrary, SS receptor-positive cases were never found to be anaplastic. All tumors except one from patients pretreated with octreotide (3 days to 3.8 years) were SS receptor positive. In the majority of carcinoids or islet cell carcinomas, the SS receptor status correlated with the in vivo biochemical response (hormone inhibition) to octreotide. These data demonstrate (a) the high prevalence of SS receptors in the primary tumors of both carcinoids and islet cell carcinomas, (b) their presence in metastases as well, (c) their continuous expression even during long term octreotide therapy, (d) the possibility of measuring SS receptors in percutaneous needle liver biopsies, and (e) the evidence of their functionality. This study therefore suggests that tumoral SS receptors may be the likely molecular basis for octreotide action and may be an important parameter for predicting the therapeutic efficacy of SS analogues in carcinoids and islet cell carcinomas.

Identification of somatostatin and gastrin receptors on enterochromaffin-like cells from Mastomys gastric tumors.

Histamine-secreting enterochromaffin-like (ECL) cells of the gastric fundus of the Mastomys can develop into solid ECL cell tumors, either spontaneously or after induction by acid inhibition. We used this tumor tissue to perform in vitro receptor autoradiography for somatostatin (SS), gastrin, and substance-P, using, respectively, [125I]Tyr3-octreotide, [125I]gastrin-17, and [125I]Bolton-Hunter-labeled substance-P as radioligands. A high density of SS receptors was found in the nontumor fundic mucosa, where gastrin receptors were only barely detectable. However, in the group of spontaneously developing ECL cell tumors, a high density of SS and gastrin receptors was observed, homogeneously distributed in the tumor tissue. In addition, the loxtidine-induced ECL cell tumors expressed a high density of SS and gastrin receptors. The receptors were specific for the respective peptide and of high affinity, with a dissociation constant (Kd) of 0.90 nM for SS receptor and 0.87 nM for gastrin receptors. No substance-P receptors were detected on the ECL cell tumors, although they were present in the muscle layers of the Mastomys gastric fundus. These results demonstrate that ECL-derived tumors express receptors for both SS and gastrin. This observation is consistent with the proposal that there is substantial regulation of the histamine-producing ECL cell by SS and gastrin. The presence of gastrin receptors is compatible with a role for gastrin as a trophic factor in ECL cell hyperplasia and neoplasia. The expression of SS receptors may be of diagnostic and therapeutic relevance in the regulation of ECL function and neoplastic transformation.

Somatostatin (SRIH) messenger ribonucleic acid expression in human neuroendocrine and brain tumors using in situ hybridization histochemistry: comparison with SRIH receptor content.

Somatostatin (SRIH) receptors are expressed in a large number of neuroendocrine and brain tumors. To evaluate the potential of these tumors locally to produce the SRIH required to bind to SRIH receptors, we have investigated in 158 human tumors whether they express mRNA for SRIH, using in situ hybridization. Among 78 neuroendocrine tumors tested, 13 of 13 medullary thyroid carcinomas, 19 of 34 pheochromocytomas, 3 of 11 paragangliomas, 0 of 4 small cell lung cancers, 4 of 9 adrenal neuroblastomas, and 4 of 7 gastroenteropancreatic tumors contained SRIH mRNA. Among 47 central nervous system and meningeal tumors tested, including 23 meningiomas, 9 astrocytomas, 4 oligodendrogliomas, and 11 glioblastomas, none expressed SRIH mRNA. Unexpectedly, 16 of 33 ovarian tumors, including adenocarcinomas and borderline tumors, expressed SRIH mRNA. These results suggest that a large proportion of neuroendocrine tumors have the ability to express SRIH mRNA, whereas central nervous system tumors do not. The presence of SRIH mRNA in half of the tested ovarian tumors suggests that those tumors may be hormone producing and have neuroendocrine features. The presence of SRIH receptors in most of the neuroendocrine tumors together with the ability of many of those tumors to synthesize SRIH point toward an autocrine regulatory feedback mechanism of SRIH in these tissues.

Somatostatin receptors in human prostate and prostate cancer.

Benign as well as malignant human prostatic tissues were evaluated for their content of somatostatin (SRIH) receptors (SRIH-R). In vitro receptor autoradiography techniques on cryostat sections were performed using 125I-labeled [Tyr3]octreotide as well as 125I-labeled [Leu8,D-Trp22,Tyr25]SRIH-28 as radioligands. SRIH-R were identified in all normal and hyperplastic prostates in the smooth muscles of the stroma, whereas the glands did not express the receptors. Muscular nodules were strongly receptor positive as well. The receptors were of high affinity (Kd = 0.4 nmol/L) and high specificity for biologically active SRIH analogs; high affinity for SRIH-14, SRIH-28, and octreotide was detected, suggesting the presence of the SSTR2 receptor subtype. In situ hybridization studies confirmed the presence of SSTR2 messenger ribonucleic acid in these tissues. Primary prostate cancers did not have SRIH-R identified with 125I-labeled [Tyr3]octreotide. However, they were expressing SRIH-R identified with 125I-labeled [Leu8,D-Trp22,Tyr25]SRIH-28, with a high affinity for SRIH-14 and SRIH-28, but low affinity for octreotide. The receptors were located on tumoral cells. In situ hybridization studies revealed a preferential expression of SSTR1. Primary human prostate cancers, therefore, express a different SRIH-R subtype than benign prostate tissue. Several veins and the ganglion cells from the prostatic plexus in the surroundings of the tumors were expressing SRIH-R with high affinity for octreotide as well. These data suggest that the human prostate as well as prostate cancers may be targets for SRIH therapy; however, SRIH analogs with different selectivities for SRIH-R subtypes are required in each case.

Immunohistochemical localization of somatostatin receptor sst2A in human pancreatic islets.

Somatostatin and octreotide inhibit endocrine pancreatic functions in man, via specific somatostatin receptors. However, the cellular distribution of the different somatostatin receptor subtype proteins has not been determined in the human pancreas. Here, the immunohistochemical distribution of the sst2A receptor was investigated using the sst2A receptor specific anti-peptide antibody R2-88 in cryostat as well as in formalin-fixed paraffin-embedded sections of human pancreatic tissue, and compared with insulin, glucagon and somatostatin immunostaining of adjacent sections. All pancreatic islets were immunostained with R2-88. Most islet cells were labeled: the sst2A receptors were present in insulin as well as glucagon producing cells, but were not detected in intra-islet vessels nor in adjacent acinar tissue. Absorption of the sst2A antibody with 100 nM of the antigen peptide abolished specific staining in tissue sections. Immunohistochemical staining with R2-88 correlated with the labeling observed after receptor autoradiography using the sst2-preferring radioligand, 125I-Tyr3-octreotide. Therefore, the clinical efficacy of octreotide on glucagon and insulin release can be explained by the presence of sst2A receptors in human A and B pancreatic islet cells. Moreover, absence of sst2A receptors in human acinar tissue suggests that the action of somatostatin on pancreatic exocrine secretion is mediated either indirectly or through a different somatostatin receptor subtype on acinar cells.

Human kidney as target for somatostatin: high affinity receptors in tubules and vasa recta.

Somatostatin (SRIH) receptors were identified in human kidneys by in vitro receptor autoradiography using 125I-[Tyr3]octreotide and 125I-[Leu8,D-Trp22,Tyr25]SRIH-28 as radioligands. Characterization of the binding demonstrated a single class of high affinity binding sites with a dissociation constant (Kd) of 0.5 nmol/L. Binding depended on GTP and magnesium and sodium concentrations. SRIH-14, SRIH-28, and octreotide were able to displace the radioligand in the high affinity range, whereas biologically inactive SRIH analogs or unrelated peptides were not. Microscopic localization of these receptors revealed binding over cortical and medullary areas. In the cortex, the receptors were located in the proximal tubules. No SRIH receptors were found in the glomeruli. In the medulla, the receptors were identified in high density in medullary vasa recta. A diffuse labeling of lesser density observed in the remaining medulla corresponded to collecting tubules. The receptor concentration was 32 fmol/mg protein in the renal cortex and 304 fmol/mg protein in the vasa recta. The receptors were undetectable in the rat kidney and are, thus, species dependent. SRIH receptors in the human kidney may be the molecular basis for the actions of SRIH on renal functions and may indicate a therapeutic potential for SRIH analogs in this tissue.

Subcellular distribution of somatostatin sst2A receptors in human tumors of the nervous and neuroendocrine systems: membranous versus intracellular location.

The distribution of the sst2A receptor was investigated, using immunohistochemistry, with the specific antipeptide antibody R2-88, in a total of 120 tumors of the nervous and the neuroendocrine systems, including small-cell lung carcinomas, medulloblastomas, neuroblastomas, pheochromocytomas, and paragangliomas. The great majority of the tumor samples, frozen or formalin-fixed, showed a positive immunohistochemical staining with R2-88, and an excellent correlation with receptor autoradiography using 125I[Tyr3]-octreotide. Whereas small-cell lung carcinomas and medulloblastomas had a predominantly plasma membrane staining, pheochromocytomas and neuroblastomas had variable ratios of cell surface and intracellular staining. Strikingly, a preferentially cytoplasmic staining was seen in tumors with a high level of somatostatin gene expression, whereas a more plasma membranous staining was seen in tumors lacking somatostatin messenger RNA. Specificity of both the plasma membrane and the cytoplasmic staining pattern was confirmed in immunoblots, which showed the immunoreactive receptor migrating as a characteristic 70-kDa broad band. In both immunohistochemical and immunoblotting experiments, staining was abolished by antibody blockade with 100 nM antigen peptide. These results describe, for the first time, the localization of the sst2A receptor protein in human small-cell lung carcinomas, medulloblastomas, neuroblastomas, and paragangliomas. Moreover, it is the first report investigating possible causes for distinct subcellular localizations of sst2A in human tissues. We show that the subcellular distribution of the receptor may be dependent on the surrounding somatostatin concentration, consistent with both the known effect of somatostatin to cause sst2A receptor internalization and an autocrine regulation of tumors by the peptide they produce. Moreover, our demonstration that the sst2A receptor can be identified in this group of tumors using simple immunohistochemical methods in formalin-fixed, paraffin-embedded material opens numerous diagnostic, therapeutic, and prognostic opportunities.

In vitro and in vivo detection of somatostatin receptors in pheochromocytomas and paragangliomas.

Fifty-one adrenal pheochromocytomas and 14 paragangliomas were evaluated for somatostatin (SRIH) receptor content with in vitro autoradiography on tissue sections from surgically removed tumors, using iodinated 125I[Tyr]3 octreotide as radioligand. Thirty-seven of 51 pheochromocytomas were SRIH receptor positive (73%), as well as 13 of 14 paragangliomas (93%). These SRIH receptors were of high affinity, pharmacologically specific for SRIH and localized on the tumor tissue. Using in vivo imaging techniques with radiolabeled SRIH analogs, paragangliomas could be visualized in five patients, as well as pheochromocytomas in two of three patients. All tumors tested subsequently in vitro (n = 7) were shown to contain SRIH receptors. A majority of pheochromocytomas were also shown to have a high tumoral SRIH content as measured by immunohistochemical techniques. Detection of SRIH messenger RNA in pheochromocytomas by in situ hybridization indicated that the SRIH was produced in the tumors. A weak inverse correlation was observed between SRIH receptor status and tumoral SRIH content, suggesting that SRIH receptors may be down-regulated by high levels of endogenous SRIH in some tumors. There was no correlation between the SRIH receptor status and sex, age, tumor size, benign vs. malignant tumor, or urinary metanephrine excretion. These tumors were also analyzed for allelic losses on various chromosomes and showed significant loss of heterozygosity (LOH) on chromosomes 1p, 3p, 17p, and 22q. All eight tumors with LOH on chromosome 1p were SRIH receptor positive (100%), whereas only 6 of 11 tumors without LOH on 1p (55%) were SRIH receptor positive, suggesting a correlation between LOH on 1p and SRIH receptor positive status. SRIH receptors thus represent a consistent pathobiochemical marker for most of these adrenal and extra-adrenal tumors. In addition, these receptors may be of potential interest for the in vivo localization of these tumors.

Immunohistochemical detection of somatostatin sst2a receptors in the lymphatic, smooth muscular, and peripheral nervous systems of the human gastrointestinal tract: facts and artifacts.

The cellular distribution of the somatostatin sst2A receptor protein was investigated in the lymphatic, smooth muscular, and nervous components of the human gastrointestinal tract using subtype-specific antibody R2-88 for immunohistochemical staining of cryostat and formalin-fixed, paraffin-embedded tissue sections. Germinal centers of intestinal lymphatic follicles were immunostained, exhibiting a predominantly plasma membrane localization of the receptor. Similarly, nerve fibers and cells in the submucosal and myenteric plexus were stained for sst2A. Antibody preabsorption with 100 nmol/L antigen peptide abolished staining in all of these tissues, and immunohistochemical staining correlated with the labeling observed after receptor autoradiography using the sst2-preferring radioligand 125I-[Tyr3]octreotide. Cytoplasmic immunostaining was detected in gastrointestinal smooth muscle cells and was inhibited by antibody pre-absorption with antigen peptide. However, 125I-[Tyr3]octreotide autoradiography was negative, and Western blots showed no band at the usual 70-90 kDa location for sst2A. Instead, a band was observed at 205 kDa. This band comigrated with the rabbit myosin standard, which was also stained with R2-88, although antibody sensitivity for myosin was less than 0.002% of that for the sst2A receptor. Rigorous computer-based sequence analysis demonstrated the peptide sequence chosen for antibody production was unique. Moreover, standard sequence alignment protocols were unable to identify the sequences in myosin responsible for the observed reactivity with the R2-88 antiserum. The observed cross-reactivity emphasizes the need for extensive controls to prove the specificity of immunostaining for such low abundance proteins as receptors even when the peptide sequence chosen for antibody production is unique. This study demonstrates for the first time the presence of specific sst2A receptor protein by immunohistochemistry in the human gastrointestinal lymphatic and nervous components, but not in gastrointestinal circular and longitudinal smooth muscle.

Potent somatostatin undecapeptide agonists selective for somatostatin receptor 1 (sst1).

A family of analogues of des-AA(1,2,5)-[DTrp(8)/D2Nal(8)]-SRIF that contain a 4-(N-isopropyl)-aminomethylphenylalanine (IAmp) at position 9 was identified that has high affinity and selectivity for human somatostatin receptor subtype 1 (sst1). The binding affinities of des-AA(1,2,5)-[DTrp(8),IAmp(9)]-SRIF (c[H-Cys-Lys-Phe-Phe-DTrp-IAmp-Thr-Phe-Thr-Ser-Cys-OH], CH-275) (7), des-AA(1,5)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (CH-288) (16), des-AA(1,2,5)-[Tyr(7),DTrp(8),IAmp(9)]-SRIF (23), and des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-SRIF (25) are about (1)/(7), (1)/(4), (1)/(125), and (1)/(4) that of SRIF-28 (1) to sst1, respectively, about (1)/(65), (1)/(130), <(1)/(1000), and <(1)/(150) that of 1 to sst3, respectively, and about or less than (1)/(1000) that of 1 to the other three human SRIF receptor subtypes. A substitution of DTrp(8) by D2Nal(8) in 7 to yield des-AA(1,2,5)-[D2Nal(8),IAmp(9)]-SRIF (13) and in 16 to yield des-AA(1,5)-[Tyr(2),D2Nal(8),IAmp(9)]-SRIF (17) was intended to increase chemical stability, selectivity, and affinity and resulted in two analogues that were less potent or equipotent with similar selectivity, respectively. Carbamoylation of the N-terminus as in des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (27) increased affinity slightly as well as improved selectivity. Monoiodination of 25 to yield 26 and of 27 to yield 28 resulted in an additional 4-fold increase in affinity at sst1. Desamination of the N-terminus of 17 to yield 18, on the other hand, resulted in significant loss of affinity. Attempts at reducing the size of the ring with maintenance of selectivity failed in that des-AA(1,4,5,13)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (33) and des-AA(1,4,5,6,12,13)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (34) progressively lost affinity for all receptors. Both des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (27) and des-AA(1,2,5)-[DCys(3),DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (29) show agonistic activity in a cAMP assay; therefore, the structural basis for the agonist property of this family of analogues is not contingent upon the chirality of the Cys residue at position 3 as shown to be the case in 18-membered ring SRIF octapeptides. None of the high affinity structures described here showed receptor antagonism. We have prepared the radiolabeled des-AA(1,2,5)-[DTrp(8),IAmp(9),(125)ITyr(11)]-SRIF ((125)I-25) and des-AA(1,2,5)-[DTrp(8),IAmp(9), (125)ITyr(11)]-Cbm-SRIF ((125)I-27), used them as in vitro tracers, and found them to be superior to des-AA(1,5)-[(125)ITyr(2),DTrp(8),IAmp(9)]-SRIF ((125)I-16) for the detection of sst1 tumors in receptor autoradiography studies.

Potent and long-acting corticotropin releasing factor (CRF) receptor 2 selective peptide competitive antagonists.

We present evidence that members of the corticotropin releasing factor (CRF) family assume distinct structures when interacting with the CRF(1) and CRF(2) receptors. Predictive methods, physicochemical measurements, and structure-activity relationship studies have suggested that CRF, its family members, and competitive antagonists such as astressin [cyclo(30-33)[DPhe(12),Nle(21),Glu(30),Lys(33),Nle(38)]hCRF((12-41))] assume an alpha-helical conformation when interacting with their receptors. We had shown that alpha-helical CRF((9-41)) and sauvagine showed some selectivity for CRF receptors other than that responsible for ACTH secretion(1) and later for CRF2.(2) More recently, we suggested the possibility of a helix-turn-helix motif around a turn encompassing residues 30-33(3) that would confer high affinity for both CRF(1) and CRF(2)(2,4) in agonists and antagonists of all members of the CRF family.(3) On the other hand, the substitutions that conferred ca. 100-fold CRF(2) selectivity to the antagonist antisauvagine-30 [[DPhe(11),His(12)]sauvagine((11-40))] did not confer such property to the corresponding N-terminally extended agonists. We find here that a Glu(32)-Lys(35) side chain to side chain covalent lactam constraint in hCRF and the corresponding Glu(31)-Lys(34) side chain to side chain covalent lactam constraint in sauvagine yield potent ligands that are selective for CRF(2). Additionally, we introduced deletions and substitutions known to increase duration of action to yield antagonists such as cyclo(31-34)[DPhe(11),His(12),C(alpha)MeLeu(13,39),Nle(17),Glu(31),Lys(34)]Ac-sauvagine((8-40)) (astressin(2)-B) with CRF(2) selectivities greater than 100-fold. CRF receptor autoradiography was performed in rat tissue known to express CRF(2) and CRF(1) in order to confirm that astressin(2)-B could indeed bind to established CRF(2) but not CRF(1) receptor-expressing tissues. Extended duration of action of astressin(2)-B vs that of antisauvagine-30 is demonstrated in the CRF(2)-mediated animal model whereby the inhibition of gastric emptying of a solid meal in mice by urocortin administered intraperitoneally at time zero is antagonized by the administration of astressin(2)-B but not by antisauvagine-30 at times -3 and -6 h while both peptides are effective when given 10 min before urocortin.

Somatostatin receptor scintigraphy in central nervous system tumors: role of blood-brain barrier permeability.

UNLABELLED: Somatostatin receptors are expressed in meningiomas and low-grade gliomas, raising the hope that scintigraphy with 111In-DTPA-D-Phe1-octreotide might be helpful in the in vivo localization, differential diagnosis and postoperative/postradiotherapy brain tumor follow-up. METHODS: Indium-111-DTPA-D-Phe1-octreotide scintigraphy and brain scintigraphy using 99mTc-DTPA as a nonspecific tracer for blood-brain barrier integrity were simultaneously performed in 60 patients with CNS tumors using dual-isotope acquisition mode SPECT. For 23 patients, the scintigraphic findings were also compared with in vitro somatostatin receptor autoradiography of surgical biopsy specimens. RESULTS: In meningiomas (located outside the blood-brain barrier), the somatostatin receptor scan showed all tumors and scintigraphic signal intensity correlating with in vitro SSR density positive in all meningiomas. Less contrast was seen on 99mTc-DTPA scans. In all tumors inside the blood-brain barrier, the 111In-DTPA-D-Phe1-octreotide scan visualized the tumors with a disrupted blood-brain barrier, as seen by 99mTc-DTPA scintigraphy. Discrepancies, however, were observed between somatostatin receptor scintigraphy and in vitro receptor autoradiography. CONCLUSION: Combined somatostatin receptor and 99mTc-DTPA scintigraphy may be helpful for noninvasive differentiation between meningiomas and other CNS tumors. False-negative scans were observed as a result of shielding by the intact blood-brain barrier. Interpretation of negative and positive somatostatin receptor scans in CNS tumors must therefore be done with caution.

Immunohistochemical localization of somatostatin receptor sst2A in human gut and lung tissue: possible implications for physiology and carcinogenesis.

Many neuroendocrine gastrointestinal and lung tumors express sst2A somatostatin receptors. Because the cellular location of sst2A in the corresponding non-neoplastic tissue is unknown, we searched for sst2A immuno-reactive cells and characterized their type in these tissues using a highly specific sst2A antibody (R2-88). Epithelial sst2A cells, identified as neuroendocrine, gastrin-producing cells, were found in large numbers in the antrum and the duodenum, but not in the gastric corpus. They were also present in the proximal jejunum, rarely noted in the distal jejunum and ileum, and absent in the large intestine and the appendix vermiformis. Moreover, sst2A cells were found abundantly in the neural plexus. sst2A receptors on antral gastrin cells could mediate somatostatin inhibition on gastrin secretion, whereas those in the neural plexus could mediate somatostatin effects on motility and ion transport in the lower gastrointestinal tract. Rare sst2A cells in bronchi and bronchioles located basally and parabasally in the gastrointestinal epithelium were detected that could represent stem/progenitor cells. It is currently not clear whether and which of the identified sst2A cells are at the origin of sst2A-positive neuroendocrine gut or lung tumors.

Expression of somatostatin receptors in normal, inflamed, and neoplastic human gastrointestinal tissues.

The multiple actions of somatostatin are mediated by specific membrane-bound receptors present in all somatostatin target tissues, such as brain, pituitary, pancreas, gastrointestinal tract, and kidney. For instance, in the human gastrointestinal tract, three different types of tissue compartments express somatostatin receptors: the gastrointestinal mucosa, the peripheral nervous system, and the gut-associated lymphoid tissue, where the receptors are preferentially located in germinal centers. In all these cases, somatostatin binding is of high affinity and specific for bioactive somatostatin analogues. Somatostatin receptors are also expressed in pathological states, such as cancers. A particular abundance is found in neuroendocrine tumors of the gastrointestinal tract. Ninety percent of the carcinoids and a majority of islet cell carcinomas, including their metastases, usually have a high density of somatostatin receptors. Several different somatostatin-receptor subtypes can be expressed by these tumors, the SSTR2 subtype being the most frequently and abundantly expressed. The somatostatin receptors in tumors are identified with in vitro-binding methods, molecular biology techniques, or in vivo-imaging techniques; the latter allow the precise localization of the tumors and their metastases in the patients. Because somatostatin receptors in human gastroenteropancreatic tumors are functional, their identification can be used to predict the therapeutical efficacy of octreotide to inhibit excessive hormone release. Of differential diagnostic importance is the fact that other pathological processes in the gastrointestinal tract may be associated with a high density of somatostatin receptors. Ninety percent of lymphomas, including those with intestinal involvement express somatostatin receptors. Furthermore, a moderate number of colorectal carcinomas contain somatostatin receptors, whereas exocrine pancreatic carcinomas do not. Finally, an increased expression of SS receptors in nonneoplastic conditions, such as in intestinal veins in inflammatory bowel disease, has been recently observed. These observations demonstrate the ability of the human body to regulate SS receptors in a wide number of tissues and conditions.