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High overexpression of somatostatin receptors in neuroendocrine tumors allows imaging and radiotherapy with radiolabeled somatostatin analogues. To ascertain whether a tumor is suitable for in vivo somatostatin receptor targeting, its somatostatin receptor expression has to be determined. There are specific indications for use of immunohistochemistry for the somatostatin receptor subtype 2A, but this has up to now been limited by the lack of an adequate reliable antibody. The aim of this study was to correlate immunohistochemistry using the new monoclonal anti-somatostatin receptor subtype 2A antibody UMB-1 with the gold standard in vitro method quantifying somatostatin receptor levels in tumor tissues. A UMB-1 immunohistochemistry protocol was developed, and tumoral UMB-1 staining levels were compared with somatostatin receptor binding site levels quantified with in vitro I-[Tyr]-octreotide autoradiography in 89 tumors. This allowed defining an immunohistochemical staining threshold permitting to distinguish tumors with somatostatin receptor levels high enough for clinical applications from those with low receptor expression. The presence of >10% positive tumor cells correctly predicted high receptor levels in 95% of cases. In contrast, absence of UMB-1 staining truly reflected low or undetectable somatostatin receptor expression in 96% of tumors. If 1% to 10% of tumor cells were stained, a weak staining intensity was suggestive of low somatostatin receptor levels. This study allows for the first time a reliable recommendation for eligibility of an individual patient for in vivo somatostatin receptor targeting based on somatostatin receptor immunohistochemistry. Under optimal methodological conditions, UMB-1 immunohistochemistry may be equivalent to in vitro receptor autoradiography.

The G(i)-coupled somatostatin 2A receptor (sst2A) mediates many of the neuromodulatory and neuroendocrine actions of somatostatin (SS) and is targeted by the SS analogs used to treat neuroendocrine tumors. As for other G protein-coupled receptors, agonists stimulate sst2A receptor phosphorylation on multiple residues, and phosphorylation at different sites has distinct effects on receptor internalization and uncoupling. To elucidate the spatial and temporal regulation of sst2A receptor phosphorylation, we examined agonist-stimulated phosphorylation of multiple receptor GPCR kinase sites using phospho-site-specific antibodies. SS increased receptor phosphorylation sequentially, first on Ser-341/343 and then on Thr-353/354, followed by receptor internalization. Reversal of receptor phosphorylation was determined by the duration of prior agonist exposure. In acutely stimulated cells, in which most receptors remained on the cell surface, dephosphorylation occurred only on Thr-353/354. In contrast, both Ser-341/343 and Thr-353/354 were rapidly dephosphorylated when cells were stimulated long enough to allow receptor internalization before agonist removal. Consistent with these observations, dephosphorylation of Thr-353/354 was not affected by either hypertonic sucrose or dynasore, which prevent receptor internalization, whereas dephosphorylation of Ser-341/343 was completely blocked. An okadaic acid- and fostriecin-sensitive phosphatase catalyzed the dephosphorylation of Thr-353/354 both intracellularly and at the cell surface. In contrast, dephosphorylation of Ser-341/343 was insensitive to these inhibitors. Our results show that the phosphorylation and dephosphorylation of neighboring GPCR kinase sites in the sst2A receptor are subject to differential spatial and temporal regulation. Thus, the pattern of receptor phosphorylation is determined by the duration of agonist stimulation and compartment-specific enzymatic activity.

The somatostatin receptor sst2A is highly expressed at the cellular surface of neuroendocrine and other human tumor cells, allowing somatostatin receptor-targeted scintigraphic tumor imaging and tumor therapy. In vitro information on tumoral somatostatin receptor expression is provided mainly by receptor autoradiography, based on binding of radiolabeled somatostatin analogs to tumoral somatostatin receptors. Recent availability of anti-sst2A antibodies opens the possibility of sst2A assessment in human tumors with immunohistochemistry. The aim of the present study was to evaluate the usefulness of several commercial anti-sst2A antibodies for this purpose in comparison with the extensively characterized antibody R2-88 and with receptor autoradiography. sst2A immunohistochemistry was performed in 64 formalin-fixed tumors known to frequently express sst2A (pancreatic, gastrointestinal, pulmonary neuroendocrine tumors, breast carcinomas, meningiomas, pituitary adenomas, neuroblastomas, medulloblastomas, pheochromocytomas, and paragangliomas); receptor autoradiography could be performed simultaneously in 37 of these cases. The commercial antibody SS-800 clearly identified sst2A and correlated excellently with R2-88: compared with R2-88, SS-800 immunohistochemistry generally yielded the same tissular and subcellular staining distribution. Results of R2-88 and SS-800 immunohistochemistry correlated excellently with those obtained with receptor autoradiography; compared with receptor autoradiography, immunohistochemistry with both R2-88 and SS-800 resulted in a slightly lower tumoral sst2A incidence, but a higher resolution, with frequent identification of heterogeneous sst2A distribution at high magnification. Finally, not only membranous, but also cytoplasmic, sst2A immunostaining was simultaneously observed with both antibodies in some tumors. In conclusion, the commercially available SS-800 antibody promises to be useful for the routine immunohistochemical assessment of sst2A in formalin-fixed human tumors.

Background and aim: Many neuroendocrine gastrointestinal tumours express receptors for the regulatory peptide somatostatin. Among the five existing somatostatin receptor (sst) subtypes, sst2A is the most frequently expressed in these tumours. However, little information is available about the cellular location of sst2A in corresponding non-neoplastic epithelial tissues. Methods: We searched for sst2A immunoreactive cells in non-neoplastic gastrointestinal tissues, and evaluated their number and immunohistochemical characteristics with neuroendocrine markers. Results: The gastric antrum showed numerous sst2A cells, situated in the epithelium, corresponding to gastrin containing neuroendocrine cells, while the gastric corpus was largely devoid of sst2A cells, including enterochromaffin-like cells. The remaining foregut, namely the duodenum and proximal jejunum, also contained a large number of sst2A cells, all being neuroendocrine cells and many of them characterised as gastrin cells. Sst2A cells were also detected in the midgut, in low numbers in the epithelium of the distal jejunum and ileum, but not in the appendix vermiformis, the caecum, or the hindgut, despite the large number of neuroendocrine cells present in this area. In addition, sst2A cells were found in the whole gastrointestinal tract in the myenteric and submucosal plexus. Conclusions: While sst2A receptors on antral gastrin cells presumably mediate somatostatin inhibition of gastrin secretion, the effects of somatostatin on motility and ion transport in the lower gastrointestinal tract may be mediated by sst2A receptors in the neural plexus. These data provide a molecular basis for the physiological actions of somatostatin in human gastrointestinal tissue.

International audience; Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) raise difficult therapeutic problems despite the emergence of targeted therapies. Somatostatin analogs (SSA) remain pivotal therapeutic drugs. However, the tachyphylaxis and the limited antitumoral effects observed with the classical somatostatin 2 (sst2) agonists (octreotide and lanreotide) led to the development of new SSA, such as the pan sst receptor agonist pasireotide. Our aim was to compare the effects of pasireotide and octreotide on cell survival, chromogranin A (CgA) secretion, and sst2 phosphorylation/trafficking in pancreatic NET (pNET) primary cells from 15 tumors. We established and characterized the primary cultures of human pancreatic tumors (pNETs) as powerful preclinical models for understanding the biological effects of SSA. At clinically relevant concentrations (1-10 nM), pasireotide was at least as efficient as octreotide in inhibiting CgA secretion and cell viability through caspase-dependent apoptosis during short treatments, irrespective of the expression levels of the different sst receptors or the WHO grade of the parental tumor. Interestingly, unlike octreotide, which induces a rapid and persistent partial internalization of sst2 associated with its phosphorylation on Ser341/343, pasireotide did not phosphorylate sst2 and induced a rapid and transient internalization of the receptor followed by a persistent recycling at the cell surface. These results provide the first evidence, to our knowledge, of striking differences in the dynamics of sst2 trafficking in pNET cells treated with the two SSAs, but with similar efficiency in the control of CgA secretion and cell viability.

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.

Binding of neuropeptides to their receptors usually results in internalization of receptor-ligand complexes. This process serves a crucial role in receptor downregulation, resensitization, and transmembrane signaling. It has mainly been investigated in cells ectopically expressing recombinant receptors. In the present study, we investigated whether rat central neurons and astrocytes naturally expressing somatostatin (SRIF) receptors internalized this neuropeptide. We demonstrated that 29% of cortical and 45% of hippocampal neurons in culture expressed the SRIF receptor sst2A and that 40–50% of the neurons internalized fluorescent SRIF. Similarly, an important proportion of astrocytes expressed sst2A (up to 60% in cortical cultures) and internalized fluo-SRIF. Competition experiments using the sst2/sst5-preferring agonist SMS 201-995 (octreotide) showed that a subpopulation of neurons internalized fluo-SRIF via sst2 and/or sst5 receptors, but that others also did so via other subtypes. Fluo-SRIF labeling was barely competed for by the sst1-selective agonist CH-275, indicating that sst1 was unlikely to be mediating SRIF internalization in hippocampal and cortical neurons. Given the paucity of sst5 receptors in cerebral cortex and hippocampus and the poor yield of sst4 internalization in transfected cells, we conclude that sst2 and sst3 subtypes are the most likely to be responsible for SRIF internalization in our culture systems. Synapse 38:177–186, 2000. © 2000 Wiley-Liss, Inc.

Although in situ hybridization has been used to examine the distribution of messenger RNA for somatostatin receptor subtypes (sst) in human tumors, the cellular localization of sst1 and sst2A receptors has not been reported. In this study, we describe the cellular localization of human sst1 and sst2A receptor proteins in both cryostat- and paraffin-embedded sections of 25 human tumor tissues using two recently developed polyclonal antibodies. Six somatostatin (SS) receptor (SSR) positive tumors (two gastrinomas, three carcinoids, one pheochromocytoma) and one SSR negative tumor (renal cell carcinoma), selected by positive and negative SSR autoradiography, respectively, were studied by both immunohistochemistry and Western blot analysis. The six SSR positive tumors expressed sst2A, while 4 of 5 expressed sst1 as well. The SSR negative tumor did not express either sst1 or sst2A. Western blot analysis of wheat germ agglutinin purified membrane proteins confirmed the presence of the sst1 and sst2A glycosylated receptors. The paraffin-embedded sections gave best information with respect to the subcellular localization. Sst1 immunoreactivity was observed both on the membrane and in the cytoplasm, while sst2A showed predominantly membrane-associated immunoreactivity. This subcellular distribution of sst1 or sst2A receptors was confirmed in paraffin-embedded sections of 8 additional intestinal carcinoids, 5 gastrinomas and 5 pheochromocytomas. Sst1 receptors were detected in 7 out of 8 carcinoids, in all gastrinomas, and in 4 out of 5 pheochromocytomas, while 6 out of 8 carcinoids, all gastrinomas, and 3 out of 5 pheochromocytomas expressed sst2A receptors. In conclusion, sst1 and sst2A receptors show a differential subcellular localization in human SSR positive tumors. The use of SSR subtype selective antibodies to detect the subcellular distribution of SSR subtypes in individual tumor cells is an important step forward to understand more about the pathophysiological role of the different SSR subtypes in human tumors.

Glyphosate (N-phosphonomethyl-glycine) is the most widely used herbicide in the world: glyphosate-based formulations exhibit broad-spectrum herbicidal activity with minimal human and environmental toxicity. The extraordinary success of this simple, small molecule is mainly attributable to the high specificity of glyphosate for the plant enzyme enolpyruvyl shikimate-3-phosphate synthase in the shikimate pathway, leading to the biosynthesis of aromatic amino acids. Starting in 1996, transgenic glyphosate-resistant plants were introduced, thus allowing application of the herbicide to the crop (post-emergence) to remove emerged weeds without crop damage. This review focuses on mechanisms of resistance to glyphosate as obtained through natural diversity, the gene-shuffling approach to molecular evolution, and a rational, structure-based approach to protein engineering. In addition, we offer a rationale for the means by which the modifications made have had their intended effect.

THE neuropeptide somatostatin inhibits secretion from electrically excitable cells in the pituitary, pancreas, gut and brain1. In mammalian pituitary tumour cells somatostatin inhibits secretion through two distinct pertussis toxin-sensitive mechanisms2–5. One involves inhibition of adenylyl cyclase6, the other an unidentified cyclic AMP-independent mechanism that reduces Ca2+ influx7,8 by increasing membrane conductance to potassium9,10. Here we demonstrate that the predominant electrophysiological effect of somatostatin on metabolically intact pituitary tumour cells is a large, sustained increase in the activity of the large-conductance Ca2+- and voltage-activated K+ channels (BK). This action of somatostatin does not involve direct effects of Ca2+, cAMP or G proteins on the channels. Our results indicate instead that somato-statin stimulates BK channel activity through protein dephosphorylation.

Hormone-stimulated receptor internalization and desensitization occur widely in the G protein-coupled receptor (GPCR) family. A critical first step in both these processes is thought to be receptor phosphorylation, a reaction which has been extensively characterized in cell culture. However, little is known about GPCR phosphorylation in vivo. The somatostatin (SS) receptor subtype (sst)2A is widely distributed in human neuroendocrine tumors, and SS analogs are commonly used to target this receptor for both therapy and diagnosis. In cultured pituitary cells sst2A is rapidly phosphorylated and internalized after hormone binding. The aim of the present study was to go one crucial step further and characterize the phosphorylation state of this receptor in human neuroendocrine tumors using a newly developed gel-shift assay. The receptor from a somatostatinoma was completely phosphorylated. In contrast, only unphosphorylated sst2A was present in human tumors that were not exposed to autocrine stimulation. Both in vivo and in cultured cells, the phosphorylation state of the sst2A receptor was correlated with its subcellular localization: phosphorylated receptor was mostly intracellular, whereas unphosphorylated receptor was localized at the cell surface. These results are the first to demonstrate ligand-stimulated GPCR phosphorylation in human tissue in situ, providing a crucial step toward a better understanding of receptor regulation in vivo. Analysis of sst2A phosphorylation promises to provide a sensitive indicator of the effectiveness of SS analogs in diagnostic and therapeutic situations in tumor patients.

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.

Binding of neuropeptides to their receptors usually results in internalization of receptor-ligand complexes. This process serves a crucial role in receptor downregulation, resensitization, and transmembrane signaling. It has mainly been investigated in cells ectopically expressing recombinant receptors. In the present study, we investigated whether rat central neurons and astrocytes naturally expressing somatostatin (SRIF) receptors internalized this neuropeptide. We demonstrated that 29% of cortical and 45% of hippocampal neurons in culture expressed the SRIF receptor sst(2A) and that 40-50% of the neurons internalized fluorescent SRIF. Similarly, an important proportion of astrocytes expressed sst(2A) (up to 60% in cortical cultures) and internalized fluo-SRIF. Competition experiments using the sst(2)/sst(5)-preferring agonist SMS 201-995 (octreotide) showed that a subpopulation of neurons internalized fluo-SRIF via sst(2) and/or sst(5) receptors, but that others also did so via other subtypes. Fluo-SRIF labeling was barely competed for by the sst(1)-selective agonist CH-275, indicating that sst(1) was unlikely to be mediating SRIF internalization in hippocampal and cortical neurons. Given the paucity of sst(5) receptors in cerebral cortex and hippocampus and the poor yield of sst(4) internalization in transfected cells, we conclude that sst(2) and sst(3) subtypes are the most likely to be responsible for SRIF internalization in our culture systems.

Genes for five somatostatin receptor subtypes, designated sst1-5, have been cloned and shown to belong to the seven transmembrane domain receptor family. The sst2 mRNA transcript is alternatively spliced to generate two related receptor products (sst2A and sst2B) which differ in their carboxylterminal sequence whereas each of the other genes is transcribed to give a single unique receptor protein. The six sst receptor subtypes all bind SRIF14, SRIF28 and the cortistatins with high affinity but vary in their affinity for analogs, such as octreotide. Although the tissue distribution of sst mRNAs has been extensively examined, much less is known about the cellular distribution of the individual receptor proteins. Recent studies with sst subtype specific antibodies have localized individual sst receptors to specific cell types within the rat gastrointestinal tract, pancreas, pituitary and brain. Furthermore, sst receptors have recently been identified in human tumors by immunocytochemistry, providing a significantly improved method for sst receptor detection. All six sst receptor subtypes are linked to guanine nucleotide binding proteins (G proteins) and lead to inhibition of adenylyl cyclase following hormone binding. The sst receptors also regulate a variety of different effectors via G proteins, including calcium and potassium channels and serine and tyrosine phosphatases. In addition to signalling, two other processes are activated by hormone binding: receptor desensitization and receptor internalization. The extent to which these occur seems to vary for the different receptor subtypes. Recent studies have shown that the sst2A receptor is rapidly phosphorylated upon hormone binding, suggesting that this phosphorylation may be responsible for the desensitization and/or internalization of this receptor. The importance of receptor regulation in cellular responsiveness to somatostatin and for receptor detection as well as the molecular mechanisms by which these processes occur provide important areas for future investigations.