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A discrete population of cells of the endocrine pancreas contains immunoreactive somatostatin as shown by immunofluorescence. These cells are different from those containing glucagon or insulin. This unexpected observation may be of physiopathological significance in the regulatory mechanisms involved in the secretion of glucagon and insulin.

The effect of subtotal pancreatectomy on the regulation of plasma glucose was studied in the chicken. The eventual appearance of an extra-pancreatic source of insulin and the effect of pancreatectomy on somatostatin containing cells of the intestine were also investigated. Over a period of 3-5 weeks, pancreatectomy decreased body weight gain, greatly impaired glucose tolerance and hindered the regulation of plasma glucose levels after a meal. A large deficit in the insulin release induced by a meal was noticed after pancreatectomy. Therefore, in the fed chicken, pancreatectomy results in the typical signs of diabetes. No extrapancreatic source of insulin could be found either by radioimmunoassay of extracts of proventriculus, gizzard, gizzard-duodenum junction and duodenum from controls or depancreatized chickens, or by immunohistological studies in the same organs plus pancreatic ducts. No glucagon producing cells could be found by immunohistological studies in the gastrointestinal tract. The size of the somatostatin cells from the gizzard-duodenum junction was negatively related to the magnitude of the glucose-induced insulin release, which suggests an increase in somatostatin cell activity in true insulin-deficient depancreatized chickens as observed in insulin deficient diabetic mammals.

Immunohistochemistry was employed to study the development of somatostatin-containing cells in the pancreas and duodenum of rat fetuses and of 1-7 day-old newborns. Immunoreactive cells were first detected in the pancreatic islets on day 17 of gestation and in the duodenum on day 18. Somatostatin cells were numerous in the pancreas and gut at term and in early postnatal stages. The development of somatostatin-containing cells in both pancreatic islets and duodenum was not affected by either fetal hypophysectomy on day 16 or intrauterine growth retardation induced by ligature of the main uterine vessels on day 17 of gestation.

Specific SRIF(1-14) fragments were synthetized on resin using conventional procedures. Rabbits received subcutaneously peptidyl resins in complete Freund adjuvant emulsion. The presence of antibodies was assessed by immunocytochemical and radioimmunological assays. 1. Peptidyl resins lead to antibodies production; their specificity depends on sequence and molecular configuration of the peptide on the resin. Anti-resin antibodies were not detected. 2. In the brain, SRIF(1-4) (in rat) and SRIF(10-13) (in garden-dormouse) can be demonstrated in neurophysins--positive cells of both paraventricular and supraoptic nucleus, but never in hypothalamic or extrahypothalamic SRIF(1-14)--positive neurophysin negative cells. 3. Endocrine cells of pancreatic islets contain SRIF(6-9) (in man) or SRIF(10-13) (in rat, mouse, garden-dormouse); generally, these cells are not detected by SRIF(1-14) anti-serum. Moreover, SRIF(10-13) positive cells are also detected by specific glucagon antibodies. However, it cannot be concluded that SRIF(10-13) antibodies reveal the common Thr-Phe-Thr-Ser fragment in the entire glucagon molecule. It is postulated that antibodies to several SRIF tetrapeptides reveal molecular fragments provided by the functional cleavage of an hypothetical prohormone or by the inactivation of SRIF(1-14) molecule in target cells.

Cells and fibers containing somatostain (SRIF) or SRIF-like peptides were detected immunocytochemically in the brain of the garden dormouse (Eliomys quercinus L.). The periventricular preoptic nucleus and the paraventricular nucleus encompass a component of the SRIF-immunoreactive hypothalamo-infundibular and hypothalamo-neurohypophyseal systems. The suprachiastmatic, ventromedial and arcuate nuclei contain a number of SRIF-positive cells and receive a rich SRIF innervation. The extrahypothalamic systems containing SRIF can be subdivided into the following groups: (1) Afferents, the cellular origin of which is not always clearly evident, i.e., fibers of the septum, the lateral preoptic area, the thalamus, the superior olivary nucleus, the mesencephalic gray matter, and the subfornical organ; (2) dispersed cells with short projections (neocortex, caudate nucleus, putamen); (3) scattered cells with short projections (nucleus accumbens) or innervating remote territories (nucleaus interpeduncularis); (4) vascular organ of the lamina terminalis, a neurohemal area comparable to the median eminence. These observations lead to a theory of a functional bipotentiality of the somatostatin molecule. Immunocytological results depend on the antisera employed, the type of fixation and the experimental conditions. Adrenalectomy is followed by an accumulation of immunoreactive material in all SRIF-containing systems of the brain. These results clearly indicate the SRIF participates in the function of the CRF-ACTH-adrenal axis. The endocrine disturbance induced by adrenalectomy appears to modify the activity of both the neurohormonal and neuromodulator components of the SRIF system.

A primitive nerve cell line (F7 clone), obtained by SV 40 transformation of mouse fetal hypothalamic cells, has been previously shown to share some properties with a progenitor cell for neurophysin synthesizing neuron. This paper describes the immunological detection, at the light microscope level, of somatostatin in this cell line. A SV 40 transformed neurosecretory clone, synthesizing neurophysin and vasopressin, was used as control.

Two distinct populations of endocrine cells in the chicken thymus display neurotensin and somatostatin immunoreactivity, respectively. Both cell types are few in number at hatching but proliferate rapidly during the first week. The neurotensin cells are 'Grimelius-positive' and 'Hellerström-Hellman-negative'. The somatostatin cells are 'Grimelius-negative' and 'Hellerström-Hellman-positive'. Both cell populations are non-argentaffin. The somatostatin-like material extracted from chicken thymus behaves immunochemically and chromatographically similar to synthetic ovine somatostatin, while the neurotensin-like material, from the thymus as well as from the gut, differs from synthetic bovine neurotensin in that it appears larger in size and more basic.

Using the immunoperoxidase method, neurons containing luteinizing hormone-releasing hormone (LHRH) and somatostatin (SRIF) were identified in the hypothalamus of growing ewe lambs between 3 days and 16 weeks of age. Both peptidergic neuronal systems were well developed in the early postnatal period. The centres producing LHRH were found to be situated in the anterior region of the hypothalamus and preoptic area. Immunoreactive (ir) LHRH perikarya in growing lambs were readily stained by the various anti-LHRH antibodies used and they were generally more numerous in young animals than in foetuses and adults. The concentration of irLHRH material stored in the median eminence (ME) increased with lamb age, reaching a maximum around 12 weeks of age. The centre producing somatostatin, situated in the anterior periventricular area of the hypothalamus, did not develop before the postpartum period. The irSRIF material in the ME was greatly depleted during the first neonatal days but regained its former level by 8 weeks of age. From 10 to 16 weeks of age, these stores in the ME were again depleted. The results show that the two neuronal systems investigated developed in a different way in the growing lamb. The secretory activity of these systems changed during subsequent periods of growth. The importance of these events in the processes of body growth and reproductive system maturation are still to be determined.

Indirect immunofluorescence technique, using an antisomatostatin antibody, has allowed us to detect immunoreactive fibers in neurohypophysis of the monkey Macaca irus. These fibers are located in stem and in neural lobe of the pituitary gland. They are thin fibers disposed around blood vessels. They are grouped together in areas more or less large, especially at the periphery of the neural lobe, under the glandular capsule and in the vicinity of the intermediate lobe. There are no sex-linked differences in the morphology as well as the distribution of these immunoreactive fibers.

Using the indirect immunofluorescent technique with anti-somatostatin serum, the distribution of scattered cells in the duct system of submandibular glands in the Monkey, Macaca irus has been assessed. In both males and females, these cells are located only in some portions of the duct system, e.g. striated ducts and excretory ducts. No immunoreactive cells were observed in the intercalated ducts or in secretory endpieces. The lymphatic node constantly adjacent to the submandibular gland did not contain immunoreactive cells. In the parotid glands, no immunoreactive cells to antisomatostatin immuneserum were ever observed.

By immunofluorescence, somatostatin-, glucagon- and insulin-containing cells are localized in serial sections of the pigeon pancreas. The distribution of the somatostatin immunofluorescent-cells corresponds to that of the D-cells (A1-cells), which are particularly numerous in this animal species. This observation, coupled with the finding of D, A and B-cells at the ultrastructural level, indicates that the D-cell is responsible for the secretion of somatostatin.

Using immunofluorescence somatostatin was localized in the forebrain of the Goldfish and Gambusia. Immunoreactive cell bodies were detected in the nucleus preopticus periventricularis (NPP), the nucleus lateralis tuberis (NLT) and a dorsomedial nucleus at the level of the posterior nucleus anterioris periventricularis (NAPv) and of the rostral part of the nucleus ventromedialis thalami (NVM). In the pituitary, immunoreactive terminals were restricted at the proximal pars distalis.

Indirect immunofluorescence technique with antisomatostatin serum has allowed us to detect somatostatin containing cells in the gastro-intestinal tract of three species of monkeys : Macacus irus, Papio hamadryas and Cercopithecus aethiops. These cells are localized only in the stomach, in the duodenum, and in the jejunum-ileum. No somatostatin cells were encountered in the esophagus and in the colon. The maximum of somatostatin positive cells is localized in the duodenum, as well in the Lieberkühn's glands as in the Brunner's glands. These cells are always separated from each other ; they were found only in glandular epithelium ; no immunopositive cells were observed in the surface epithelium. No morphological or numerical differences were noted between the three species of monkeys.

Immunocytochemical methods were applied to camel pituitaries. ACTH, beta-lipotropin (LPH), LH beta, GH, TSH, and PRL immunoreactive cells were identified. They were located on different parts of anterior pituitary and had different organization and shape. In addition, somatostatin-like immunoreactivity was seen in cells situated at the top of pituitary cleft specially in a cone-shaped extension of the intermediate lobe. The role and the eventual origin of these cells have yet to be established and their occurrence related to age and life conditions.

Three sites of somatostatin-synthesizing perikarya, or a related antigen, were determined by immunofluorescence in the hypothalamus of the tadpole, Alytes obstetricans (Amphibia, Anura). Two sites of neurosecretory perikarya were localized in the preoptic nuclei of the anterior hypothalamus; the axons extended either to the anterior diencephalon or to the median eminence and the pituitary. The third site was found in the posterior hypothalamus. These neurosecretory cells showed a strong immunofluorescent reaction; their axons all terminated at the level of the median eminence. Somatostatin cells were only found in intact or hypophysectomized tadpoles given somatotropin (STH). The strong reaction observed in hypophysectomized tadpoles was possibly due to the loss of the terminal portion of the neurosecretory pathway (median eminence and pituitary) by which the agent is transported to the site of discharge.

The ontogenic development of some hypothalamic neuropeptides: luteinizing hormone releasing hormone (LHRH); somatostatin (SRIF) and neurophysin (NF) and their localization in the hypothalamus of fetuses in different stages of the fetal life were studied by immunoperoxidase method. It was found that differentiation of the neurons which produce the examined hormones begins in the midstage of pregnancy. LHRH is stored in the nerve terminals of the median eminence (ME) and organum vasculosum of the lamina terminalis (OVLT) since 72 day of gestation and its amount gradually increases with the development of the embryo. In this stage a few immunoreactive (ir) LHRH perikarya appear but they are most numerous in the last days of pregnancy (110 day). They are localized in the most anterior periventricular parts of the hypothalamus, area preoptica, diagonal band of Broca and very rare in the medial-basal hypothalamus. Somatostatin is produced in the separate neuronal system and appears in the last days of fetal life. Neurophysin is present in both magnocellular nuclei in 72 day-old fetuses, but at the end of gestation it is seen also in some preoptico-septal region.

The immunocytological detection of adrenocorticotrophic hormone (ACTH) and somatotropin release inhibitor factor (SRIF) like immunoreactivity was carried out on tumour cells from bronchial brush smears in 39 cases of lung tumours. Results obtained were compared with the cytological and histological diagnosis and confirmed the high incidence of ACTH synthesis by malignant bronchial carcinoma cells: the same phenomenon also seems to occur for somatostatin. The concomitant detection of ACTH and SRIF like immunoreactivity seems to be highly suggestive of small cell carcinoma and indicates that the immunocytological detection of hormones carried out at the same time as cytological examination can improve the accuracy of the diagnosis.

The tetradecapeptide, somatostatin (SRIF), is a potent inhibitor on pituitary hormone release, by a direct effect. The immunocytological method was used with the aim of localizing SRIF at the cellular and subcellular levels. Rat pituitaries were fixed and frozen. Ultrathin sections obtained by cryoultramicrotomy, were incubated with anti-SRIF serum. The antigen-antibody reaction was detected by 4-chloro-1-naphthol. SRIF immunoreactivity was observed in somatotrophs, thyreotrophs and prolactin cells, but not in corticotrophs or gonadotrophs. Im immunoreactive cells, SRIF was found in the cytoplasmic matrix, in the secretory granules and in the nucleus distributed primarily in the euchromatin, in the vicinity of the heterochromatin regions. SRIF immunoreactivity was also observed at the plasma membrane. No immunoreactivity was observed when nonimmune serum or anti-SRIF serum incubated with SRIF was used. No modification was observed when anti-SRIF serum incubated with gonadoliberin, thyroliberin or vasopressin was used. These data (1) provide immunocytological evidence for the presence of somatostatin in pituitary gland, and (2) indicate the presence of SRIF peptide in the somatotrophs, thyreotrophs and prolactin cells.

In normal and L-Dopa treated rabbits and mice, combined immunochemical methods, photonic histological methods for endocrine cells and ultrastructural methods were used to elucidate ultrastructure and properties of somatostatin cells of the antral mucosa. In normal rabbits, immunoreactive cells giving no fluorescence with Falck's technic, they corresponded neither to serotonin cells nor gastrin cells; they were unreactive with Fontana, Hellerström-Hellmann, Sevier-Munger and Mac Conaill methods but very slightly stained with Grimelius methods. In L-Dopa treated animals somatostatin cells gave formaldehyde induced fluorescence (they were included in GIC cells, thus in Apud group), exhibited a good reaction with Grimelius and Sevier-Munger methods. In order to carry out the alternate semi-thin/thin section procedure (semi-thin sections for immunofluorescence or immunoenzymatic detection and serial thin sections counter-stained for conventional ultrastructure studies), immunological treatment were performed on M.F.F.--glutaraldehyde fixed small fragments of mucosa before inclusion in Epon 812 or, after inclusion, on semi-thin sections. We succeeded in identifying ultrastructurally somatostatin cells. They displayed round or ovoïd shaped secretory granules, and three constant typical structures: numerous microfilaments--light and homogenous granules, often seeming like lipids---granules made up by coarsely filamentous cores surrounded by a large empty halo. Somatostatin cells seemed different of X cells because of their predominant localisation in the antral mucosa (in the rabbit X cells were predominantly in the fundus) and because of the lack of nuclear microfilaments; they also seemed ultrastructuraly different of D1 cells.

Several antisera, specific to the neuropeptides of Vertebrates, were applied to frontal sections of the planarian Dugesia lugubris. Five of them enabled to show specific neurones: anti-sera anti-SRIF, anti-neurophysines, anti-ACTH 17-39, anti-ACTH4-10 and anti-angiotensin II. The positive perikarya were situated mainly at the margin of the cerebroïd ganglia and the nerve cords, at all levels of the animal. Nerves processes were detected in the central nervous system and in the surrounding areas.

The appearance, time and distribution of somatostatin in the pancreas, gastro-intestinal tract and hypothalamus were studied comparatively in human foetuses aged 6--32 weeks, by immuno-cytochemistry and radioimmunoassay. Somatostatin was detected by both methods in all segments including the colon. The first cells were observed, and somatostatin was present in measurable amounts at 8 weeks in pancreas, duodenum and intestine, while the peptide was detected at 12 weeks in antrum and colon, at 14 weeks in fundus, and at 16 weeks in hypothalamus. Subsequently, the largest cell population was located in the pancreas, where peptide concentration and age were positively correlated (P less than 0.01, r = 64). From 15 to 21 weeks of age, the mean somatostatin concentration in pancreas (12.4 +/- 1.84 ng/mg) was clearly higher than in hypothalamus (0.05 +/- 0.02 ng/mg) or in any segment of the gut, where values ranged from 0.36 +/- 0.06 (fundus) to 4.74 +/- 0.83 ng/mg (duodenum). The early appearance time of somatostatin, and its specific distribution with preferential location in the pancreas, suggest that the peptide may play a major role for the development of the foetal digestive tract, and that it may be involved in the regulation of other endocrine secretions, especially in the pancreas.

Immunocytological tests reveal the presence of a somatostatin-like substance in perikarya and axons in the brain of the giant slug Limax maximus L. Controls carried out on adjacent sections with absorbed antiserum or different antibodies raised against several biologically active peptides of vertebrates (ACTH-17-39, alpha- and beta endorphin, alpha- and beta MSH, methionin-enkephalin, TRH) demonstrate the specificity of the "staining". However, some cells are both somatostatin- and FMRF-amide-positive. In the cerebral ganglia, the right Z-area cells, responsible for the synthesis of the maturation hormone (MH) are strongly somatostatin-positive. These results suggest a similarity between the MH and the somatostatin-like material contained in the Z-area cells. The simultaneous presence of two peptides in one and the same cell, the nature (elementary granules or soluble product) of the material, and its site of release are discussed.

Indirect immunofluorescence technique with anti-somatostatin serum has allowed us to detect somatostatin containing cells in the Langerhans' islets of three species of monkeys (Macacus irus, Papio hamadryas, Cercopithecus aethiops). These cells are in close relationship with another cells, but occupy a more central position in the islets. Thus, the distribution of somatostatin-containing cells in the monkey's endocrine pancreas has not a similar pattern as in rodent's pancreas; they have a comparable topography as in the human islets but these cells are always more elongated than in the human islets. Only slight morphological differences are observed in the three species of monkeys.

Evidence is presented that somatostatin-containing cells are present in the gastro-intestinal tract of the dog. Thus immuno-fluorescent cells were detected by the use of antiserum to cyclic somatostatin. These cells were mainly encountered in the antral mucosa and in the neighbourhood of gastrin-producing cells. No cross reaction was observed between gastrin and somatostatin. It is suggested that locally produced somatostatin controls gastrin secretion, and, more generally, that somatostatin-containing cells, multifocally distributed, modulates secretion of a large number of glands.