Immunohistochemical Localization of Somatostatin

Immunohistochemical Localization of Somatostatin

GEORGES PELLETIER

With 28 Figures

GUSTAV FISCHER VERLAG STUTTGART· NEW YORK· t 980

GEORGES PELLETIER, M.D., Ph.D.,

MRC Group in Molecular Endocrinology, Le Centre Hospitalier de l'Universite Laval Quebec GIV 4G2, Canada

CIP-Kurztitelaufnahme cler Deutschen Bibliothek Pelletier, Georges: Immunohistochemical localization of somatostatin / Georges Pelletier. - Stuttgart, New York: Fischer, 1980. (Progress in histochemistry and cytochemistry ; Vol. 12, No.3) ISBN 3-437-10614-7 (Stuttgart) ISBN 0-89574-085-0 (New York)

Progress in Histochemistry and Cytochemistry ISSN 0079-6336 © Gustav Fischer Verlag . Stuttgart . New York 1980 Aile Rechte vorbehalten Satz: Bauer & Bokeler, Denkendorf Druck und Einband: H. Laupp jun., TUbingen Printed in Germany

Contents 1 2 3

3.1 3.2

3.3

4

4.1 4.2 4.2.1

Introduction . . . . Discovery of somatostatin. Actions of somatostatin Effects of somatostatin on pituitary hormones secretion Effects of somatostatin on pancreas and gastrointestinal tract Neurotropic effects of somatostatin Distribution of somatostatin Immunohistochemical methods . . . Localization of somatostatin in the central nervous system Hypothalamus. . ....

1 1 2

4.2.2 4.2.3

2

4.3

4.2.4

3

4.4

3

4.5

4

4.6

4 5

5 5

6 7

Extra-hypothalamic regions Circumventricular organs . Non-mammalian vertebrate species Localization of somatostatin in the peripheral nervous system . . Localization of somatostatin in the pancreas . . . . . . Localization of somatostatin in the gastrointestinal tract Localization of somatostatin in other tissues Conclusion . References . . Subject index.

12 13

17

17 17 24

33 33

34 41

1 Introduction

During the last decade, the major advance in neuroendocrinology was the isolation and characterization of three hypothalamic hormones (thyrotropin-releasing hormone [TRH], luteinizing hormonereleasing hormone [LHRH] and somatostatin) involved in the control of adenohypophyseal hormones secretion. The rapid development of accurate techniques for the measurement of these hormones has permitted to establish that they were not restricted to the hypothalamus but were found in many extra-hypothalamic areas (BROWNSTEIN et al. 1975; JACKSON 1978). Even more surprisingly was the discovery that both TRH (MORLEY et al. 1977) and somatostatin (PELLETIER et al. 1976a) are found in anatomic locations not heretofore considered part of the nervous system, such as the pancreas and gastro-intestinal tract. Physiologists also were not long to demonstrate that somatostatin, besides its effects on the anterior pituitary secretion and behavior, could also exert an inhibitory action on a variety of peripheral hormones or secretory products (VALE et al. 1977). These reports about the numerous sites of action of somatostatin prompted morphologists and histochemists to search for the accurate localization and the clear identification of the structures responsible for somatostatin production in different tissues. It rapidly became obvious that somatostatin is produced not only by nervous tissue but also by typical endocrine cells. In this review, we have tried to summarize what is now known about somatostatin with a special emphasis on the morphology of

the cellular elements involved in the production and/or storage of this fascinating peptide.

2 Discovery of somatostatin

It has been long suggested that the secretion of anterior pituitary hormones including growth hormone (GH) is under the control of the hypothalamus (GREEN and HARRIS 1947; HARRIS 1955). The direct evidence of the existence of an extractable hypothalamic factor responsible for pituitary release of GH has been first reported in 1964 by DEUBEN and MEITES. In the following years, several investigators have observed GH-releasing activity (KRULICH et al 1968, 1972; WILBER et al. 1971; SANDMANN et al. 1972) as well as GH release-inhibiting activity (KRULICH et al. 1968, 1972; KRULICH and MCCANN 1969) based on the abilities of crude or partially purified hypothalamic extracts to modify the secretion of radioimmunoassayable GH. In 1971, while searching for evidence for GH-releasing factor (GRF), GUILLEMIN and his coworkers isolated from ovine hypothalamic extracts a factor which dramatically inhibited the secretion of immunoreactive GH from enzymaticalle dispersed rat anterior pituitary cells in vitro (VALE et al. 1972). This substance was subsequently identified and sequenced (BRAZEAU et al. 1973). The molecule is a Q-shaped chain of 14 amino acid units

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(Fig. 1). After its discovery, this GH release-inhibiting substance has been given a new name, somatostatin, which indicates its function in staying the growth of the body. Synthetic somatostatin prepared by solid-phase methodology was found to be equipotent with both native somatostatin and the synthetic reduced form of somatostatin (dihydro-somatostatin) (BRAZEAU et al. 1973; VALE et al. 1975). A similar structure for somatostatin of porcine origin was described by SCHALLY and coworkers (1976). H-Ala-Gly-CysLys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-The-SerCys-OH

Fig. 1. Structure of somatostatin

3 Actions of somatostatin 3.1 Effects of somatostatin on pituitary hormones secretion

The first effect of somatostatin was demonstrated on the release of GH by rat anterior pituitary cells in culture (BRAZEAU et al. 1973). The same inhibitory effects on GH secretion was also demonstrated in vivo. In the rat, somatostatin injected intravenously depressed basal levels of plasma GH (BRAZEAU et al. 1974a). In some species including dog, rat, sheep and baboon, somatostatin inhibited GH secretion acutely induced by all pharmacologic means, such as barbiturates, L-Dopa, arginine and morphine (BRAZEAU et al. 1974b; BROWN and VALE 1975; MARTIN et al. 1975; BRYCE et al. 1975; FERLAND et al.

1976) as well as by electrical stimulation of the hypothalamus or amygdala (MARTIN 1974) or catecholamines infusion into the third ventricle (KATo et al. 1974). In man, somatostatin inhibits GH responses to many physiological and pharmacological stimuli such as arginine, L-Dopa, insulin-induced hypoglycemia, exercice and sleep in normal man (SILER et al. 1973; MORTIMER et al. 1974; HALL et al. 1973) and in human subjects with GH-producing pituitary tumors, as in acromegaly (MORTIMER et al. 1974; HALL et al. 1973; YEN et al. 1974; BESSER and MORTIMER 1976). Somatostatin has also been shown to affect the secretion of other pituitary hormones. Results of administration of somatostatin to cultured rat pituitary cells indicate that it inhibits both the synthesis and the release of thyrotropin (TSH) (VALE et al. 1974, 1977). In man, somatostatin was shown to lower basal TSH secretion during onset of REM sleep (WEEKE et al. 1975) and in hypothyroid patients (LUCKE et al. 1975). In some experimental designs, somatostatin also decreases «in vitro» prolactin secretion (VALE et al. 1974, 1977.) The physiological importance of somatostatin in the regulation of GH and TSH secretion has recently been demonstrated in passive immunization studies. This treatment increased basal GH levels and enhanced the release of TSH due to cold or exogenous TRH administration (ARIMURA and SCHALLY 1976; ARIMURA et al. 1976; FERLAND et al. 1976).

Immunohistochemistry of Somatostatin . 3

3.2 Effects of somatostatin on pancreas

and gastrointestinal tract Soon after the characterization of somatostatin, there have been a few reports indicating that this peptide could lower plasma insulin and glucagon levels in baboons and humans (KOERKER et al. 1974; Mo RTIMER et al. 1974). In vitro, somatostatin has been shown to inhibit the basal secretion of insulin and glucagon (FuJIMOTO et al. 1974; JOHNSON et al. 1975; TURCOT-LEMAY et al. 1975) as well as the rise of insulin and glucagon stimulated by a variety of agents (JOHNSON et al. 1975; FUJIMOTO et al. 1974; TURCOT-LEMAY et al. 1975; FUJIMOTO 1975; GERICH et al. 1975). In fact, somatostatin differs from all other pancreatic agents in that it inhibits the secretion of insulin and glucagon induced by all known stimuli for each respective hormone (GERICH et al. 1974, 1976). The physiological importance of the inhibitory action of somatostatin on insulin and glucagon is still under investigation. Recently, BARDE et al. (1977) have shown by adding antibodies to somatostatin to isolated islets that neutralization of released somatostatin could induce a marked increase in the basal release of glucagon. These results suggest a tonic inhibiting action of somatostatin on glucagon cells. In juvenile type of diabetes mellitus, injection of somatostatin is capable of decreasing fasting and postprandial hyperglycemia up to 50 % in the absence of insulin (GERICH 1976; GUILLEMI and GERICH 1976; GERICH et al. 1974). A decrease of glucagon release is the proposed mechanism to explain the hypoglycemic action of somatostatin. Although it was

thought that somatostatin in combination with small dose of insulin might be advantageous over insulin alone in treatment of insulin-dependent diabetics, some controversy has been arisen over the importance of hypersecretion of glucagon in the controlled insulin-dependent diabetics (SHERWIN et al. 1976). An inhibitory effect of somatostatin on many peptides produced in the gut has been observed. Both in vitro and in vivo somatostatin has been found to inhibit gastrin secretion (BLOOM et al. 1974; HAYES et al. 1974). Somatostatin can also inhibit the following substances in vivo, secretin (BODEN et al. 1975), pepsin (GoMEZ-PAN et al. 1975), gastric inhibitory peptide (GIP) (PEDERSON et al. 1975). The physiological role of somatostatin in the regulation of these hormones and enzymes is still completely unknown. On the other hand, somatostatin has been used successfully in short term studies to strongly decrease the secretion of gastrinomas (BLOOM et al. 1974) and insulinomas (GERICH et al. 1976).

3.3 Neurotropic effects of somatostatin

The neurotropic action of somatostatin has been studied after the injection of this peptide into the cerebral ventricles. Injections of somatostatin into rats produced a variety of modifications. Among these effects, it has been shown to decrease spontaneous motor activity (SEGAL and MANDELL 1974), lengthen the anesthesia time of barbiturates (BROWN and VALE 1975) and induce «barrel rotation», sedation and hypothermia (COHN 1975; COHN and COH 1975). It thus seems that brain som-

4 . GEORGES PELLETIER

atostatin, besides its effects on the anterior pituitary secretion, can exert some other actions suggesting that, at some sites of the central nervous systems, it could act as a neurotransmitter. In agreement with this hypothesis, RENAUD et al. (1978) has recently reported that microiontophoretic application of somatostatin to neurons from many brain areas is associated with a decrease in neuronal excitability.

4 Distribution of somatostatin 4.1 Immunohistochemical methods

During the last few years, the accurate distribution of peptides has been made by the production of specific antibodies against synthetic or highly purified peptides. In all our studies on the localization of somatostatin, we have used primary antibodies generated in our laboratory or supplied by Dr. A. Arimura (New Orleans, LA). Rabbits were injected with synthetic somatostatin conjugated to either human y-Globulin or bovine thyroglobulin by the glutaraldehyde method (ARIMURA et al. 1975a; PELLETIER et al. 1977a). All the antisera used have been characterized and used in radioimmunoassay. They showed no cross-reactivity with any of the known brain, pituitary or pancreatic hormones. In order to study the central and peripheral distribution of somatostatin, a number of tissues including several regions of the gastrointestinal tract, pancreas, thyroid gland, pituitary gland, brain and spinal cord were obtained from

adult male rats, guinea pigs, frogs (Rana pipiens) and fishes (salvelinus jOntinalis). For both light and electron microscopic studies, the experimental animals were fixed by total vascular perfusion through the heart. The fixatives used were Bouin's fluid for light microscopy and 2 % glutaraldehyde or 4 % paraformaldehyde in 0.1 M cacodylate buffer for electron microscopy. Tissues fixed in Bouin's fluid were embedded in paraffin whereas those fixed for electron microscopy were embedded in Araldite. To study human tissues, nonpathologic autopsy specimens from brain and gastrointestinal tract from human subjects were fixed in Bouin's fluid for 24hr and embedded in paraffin. Fragments of human pancreas obtained after pancreatectomy were also treated for light and electron microscopy. For immunostaining at both light and electron microscopic levels, we have used the unlabeled antibody-enzyme method of STERNBERGER (1974). In brief, thick paraffin sections (light microscopy) or plastic ultrathin (electron microscopy) sections were successively exposed to 1 0 rabbit anti-somatostatin diluted to 1 : 500 to 1 : 3000, 2 0 goat anti-rabbit y-globulin diluted at 1 : 50, and 3 0 the peroxidaseantiperoxidase (PAP) complex diluted at 1 : 100. The peroxidase was detected in a medium containing diaminobenzidine and H 2 0 2 • To control the specificity of the localization observed, appropriate pairs of adjacent sections were incubated with primary antiserum and control antiserum (previously absorbed with excess of synthetic somatostatin or related peptides). In all our immunostaining experiments, only somatostatin was effective in blocking reaction.

Immunohistochemistry of Somatostatin . 5

4.2 Localization of somatostatin in the central nervous system

4.2.1 Hypothalamus

The distribution of somatostatin in the

rat central nervous system has been studied by radioimmunoassay (BROWNSTEIN et at. 1975; VALE et at. 1977). It has been shown that the hypothalamus contains the highest concentration of somatostatin. By immunocytochemistry and immunofluorescence, the highest concentration of nerve fibers staining for somatostatin have been found in the medial basal hypothalamus of the rat and guinea pig (PELLETIER et at. 1975a; DUBE et at. 1975; HOKFELT et at. 1974; PARSONS et at. 1976). In fact, most positive fibers appeared to be concentrated in the external zone of the me-

@

dian eminence (Figs. 2 and 5). Numerous immunostained fibers have also been localized in the ventromedial and arcuate nuclei. Positive fibers were also detected in the suprachiasmatic and periventricular nuclei. Somatostatin-containing cell bodies have only been observed in the periventricular nucleus within a narrow field extending only 1-2 mm from the midline (ELDE and PARSONS 1975; ALPERT et at. 1976; PELLETIER et at. 1977a; Fig. 3). Anteriorly, the small SRIF-containing perikarya are very close to the third ventricle, while those located more caudally are more laterally situated. In rats which had received an intraventricular injection of colchicine 48 hrs before sacrifice, an enhancement in the intensity of staining made possible the detection of a much larger numer of cell bodies (Fig. 4). On

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Fig. 2.A: Immunohistochemical localization of somatostatin in a coronal section of the median eminence of a normal rat. The reaction product (arrows) indicating the presence of somatostatin is concentrated in the ventral and lateral portions of the external zone. A few fibers can also be observed in the internal zone. B: Control section adjacent to that shown in A. Immunoabsorption with synthetic somatostatin has completely prevented immunostaining. V: Third ventricle. x 200.

the other hand, this treatment induced a marked decrease in the number of reactive fibers in the median eminence (Figs. 5 and 6) suggesting that migration of somatostatin from cell bodies to endings had been blocked. This origin of somatostatin found in the median eminence has been confirmed by lesion studies and deafferentiation experiments which have indicated that fibers endings found in the median eminence should come from the cell bodies found in the periventricular nuclei (BROWNSTEIN et al. 1977; ELDE and H6KFELD 1978; Fig. 7). Using immunoelectron microscopic 10-

calization of somatostatin, we have been able to definitely prove the neuronal nature of the positive fibers by demonstrating that this peptide was localized in granular vesicles in nerve endings (PELLETIER et al. 1974; Fig. 8). The diameter of these vesicles is about 80-110 nm and thus larger than that of LHRH-containing vesicles (70-95 nm). With the same approach, the peptide was found to be present within granular vesicles of some cell bodies in the periventricular nucleus. Most of the vesicles, but none of other organelles in a positive perikaryon, were labelled (Fig. 9). The diameter of these cyt-

Immunohistochemistry of Somatostatin . 7

Fig. 3. Localization of somatostatin in a coronal section through the anterior portion of the rat hypothalamic periventricular nucleus. Immunostained cell bodies (long arrows) as well as fibers (short arrows) are located close to the ependymal cells (E) bordering the third ventricle (V) - x 400.

Fig. 4. Effect of colchicine treatment on the immunostaining for somatostatinin in the hypothalamic periventricular nucleus. Both the intensity of staining and number of detectable cell bodies (arrows) are markedly increased (compare with Fig. 3). Fibers are almost completely absent. - x 400.

Figs 5 and 6. Coronal section through the median portion of the median eminence of an untreated (5) and colchicine - treated (6) rat. The treatment has markedly decreased the number of somatostatin-containing fibers. V: third ventricle. - x 160.

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GEORGES PELLETIER

Fig. 7 Immunohistochemical localization of somatostatin in the median eminence of a rat of which the anterior hypothalamic nuclei has been lesioned 10 days previously. Note the complete absence of reaction (compare with Fig. 5). V: third ventricle. - x 200.

oplasmic vesicles was also about 80 to 110 nm in diameter. A few vesicles were consistently weakly positive or negative. The absence of reaction in a small percentage of granules in the somatostatin-producing neurons remains to be explained.

Since the diameter of the somatostatin-containing vesicles is the same in the perikarya and nerve endings, it is suggested that this peptide is stored in cytoplasmic vesicles before being released into the vicinity of the fenestrated capillaries of

Fig. 8. Immunoelectron microscopic localization of somatostatin in the external zone of the rat median eminence. Nerve endings containing positive dense core vesicle (arrows) can be observed. Other nerve endings (NE) are completely unstained. PV: perivascular space. - x 12,000.

Immunohistochemistry of Somatostatin . 9

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GEORGES PELLETIER

Fig. 9. Immunoelectron microscopic localization of somatostatin in a neuronal cell body of a normal rat. A positive reaction is present in most dense core vesicles (long arrows) whereas a few ones remain completely unstained (shon arrows). N: nucleus. - x 20,000.

®

Immunohistochemistry of Somatostatin . 11

® Fig. 11. Localization of somatostatin in a coronal section through the arcuate nucleus of a human hypothalamus. Positive cell bodies (long arrows) and fibers (short arrow) can be observed. x 230.

the pituitary portal plexus. This hypothesis is supported by the observation that after colchicine treatment the increase in the concentration of somatostatin in perikarya is accompanied by a marked in-

crease in the amount of cytoplasmic vesicles (Fig. 10). In this case, the migration but not the synthesis of somatostatin is reduced by colchicine administration. In the human hypothalamus, somatos-

Fig. 10. Neuronal cell body of colchicine-treated rat. Note the marked incre~se in the number of immunoreactive dense core vesicles (long arrow) as compared to control (Fig. 9). Unlabelled vesicles are also observed (short arrow). L: lysosome. N: nucleus. - x 27,000.

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tatin-containing cell bodies appeared to have a different localization (DESY and PELLETIER 1977). They were found concentrated in the anterior portion of the arcuate nucleus in close proximity of the infundibular recess (Fig. 11.) No positive cell bodies could be detected in the periventricular nucleus. The distribution of somatostatin-producing perikarya appears thus different from that observed in the rat. Immunostained endings were detected in the neurovascular zone of the pituitary stalk which corresponds to the median eminence in the rat and guinea pig. The endings found in this area are likely involved in the release of somatostatin into the capillaries of the pituitary stalk. Interestingly, a large bundle of positive fibers was also observed extending from the level of the anterior portion of the paraventricular nucleus rostrally up to the posterior parts of the mammillary bodies caudally. This bilateral bundle was located close to the third ventricle ventrally to the paraventricular nucleus and medially to the ventro-medial nucleus. The functional role of this bundle which abruptly ends at the level of the mammillary bodies is still unknown. It may be tentatively speculated that somatostatin contained in these nerve fibers may act as a neurotransmitter to modulate the function of some hypothalamic neurons.

4.2.2 Extra-hypothalamic regions

Radioimmunoassay data (BROWNSTEIN et al. 1975; VALE et al. 1977) have ~hown that over ten times more immunoreactive somatostatin is found in the extrahypothalamic brain than in the hypothalamus

which contains however the highest concentration of somatostatin. The peptide is distributded largely throughout all regions of the central nervous system with high concentrations found in the preoptic region, amygdala, central grey mesencephalon, olfactory tubercle and spinal cord. There have been very few reports about the histological localization of extra-hypothalamic somatostatin. Using immunofluorescence, HOKFELT et a. (1974, 1976) and EWE and HOKFELT (1978) have reported the presence of positive fibers in the nucleus accumbens, the medial portion of the caudate nucleus, the olfactory tubercle, the amygdaloid complex and certain cortical areas. In addition, somatostatin fibers and terminals have been found in the parabrachialis nucleus and in the substantia gelatinosa of both the spinal nucleus of the trigeminal nerve and the dorsal horn of spinal cord. The terminals in the substantia gelatinosa of the spinal cord and medulla have probably their origin in cell bodies found in the dorsal root and trigeminal ganglia respectively. The exact localization of cell bodies which give rise to most of the other previously mentioned fibers is presently unknown, although somatostatin cell bodies have been observed in many areas such as the zone incerta, cortical amygdaloid nucleus and dentate gyrus. Deafferentiation of the medial basal hypothalamus as well as bilateral lesions of some hypothalamic nuclei did not produce any decrease in extra-hypothalamic somatostatin as measured radioimmunoassay (BROWNSTEIN et al. 1977; EPELBAUM et al. 1977). These results clearly indicate that extra,hyp~thalamic somatostatin is not produced by hypothalamic neurons and sup-

Immunohistochemistry of Somatostatin . 13

port the hypothesis that brain somatostatin have functions other than those directly related to the control of GH secretion. It has been suggested, on the basis of its depressant effect on the activity of central nervous system neurons (RENAUD et al. 1975), that somatostatin may also serve as a neurotransmitter or modulator of neuronal activity. So far, there has been no report about the ultrastructural localization of extrahypothalamic somatostatin. By radioimmunoassay, EPELBAUM et al. (1977) have described the presence of somatostatin in synaptosomes obtained from preoptic region and amygdala.

4.2.3 Circumventricular organs

The circumventricular organs represent a class of midline ependymal structures of which the best know representative is the median eminence (Fig. 12). Besides the

median eminence which is involved in the regulation of anterior pituitary secretion, the functions of the organum vasculosum of the lamina terminalis (OVLT), the subfornical organ the subcommissural organ and the area postrema are unknown. These organs share many anatomical characteristics: they all lack the bloodbrain barrier, they are in contact with cerebrospinal fluid and they are made up of specialized ependymal cells, subependymal cells and nerve endings (KOELLA and SUTSI 1967). Thus, it has been postulated that these structures could be involved in some communication between the systemic circulation and the cerebrospinal fluid. During the course of our studies on the localization of somatostatin in the rat and guinea pig, specific immunostaining was detected in all the periventricular organs (PELLETIER et al. 1975a; DUBE et al. 1975). In the OVLT positive fibers were detected close to the capillaries of the organum

AREA POSTREMA

MEDIAN EMINENCE

® Fig. 12. Topographical representation of the major circumventricular organs of the rat on a median sagittal section.

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GEORGES PELLETIER

vasculosum (Fig. 13). LHRH-containing fibers also found in the same area had a localization completely different from that of somatostatin (PELLETIER et al. 1976a, 1977c). In the subfornical organ, subcommissural organ and area postrema, a common pattern for the distribution of somatostatin was observed: a strong reaction was localized in the ependymal and subependymallayers and also in the vicinity of blood vessels (Fig. 14). In these organs, the staining was identical to that observed for LHRH (PELLETIER et al. 1976a). In the rat pineal gland, some staining was occasionally observed.

In OVLT, immunoelectron microscopy has shown that somatostatin was localized in dense core vesicles of a few nerve endings located in proximity of the basement membrane of the fenestrated capillaries (Fig. 15). As in the median eminence, all the dense core vesicles (diameter of about 90-120 nm) were labeled in a positive axon. This localizaton suggests that somatostatin might be released into the capillaries of the OVLT and then reach the general circulation. The origin of the somatostatin fibers found in the OVLT is completely unknown. The physiological meaning of this pathway is also still un-

® Fig. 13. Localization of somatostatin in the rat OVLT. Immunostained fibers (arrows) are concentrated close to the organum vasculosum (OV). - x 600.

Immunohistochemistry of Somatostatin . 15

® Fig. 14. Localization of somatostatin in the subcommissural organ. Reaction product can be observed in the ependymal layer (short arrows) bordering the third ventricle (Y). Immunostaining is also detected in proximity of blood vessels (long arrows). - x 470.

clear although it is known that somatostatin injected into the general circulation inhibits the secretion of insulin, glucagon and gastrin as previously discussed. In the other circumventricular organs, the ultrastructural localization of somatostatin has revealed a diffuse reaction in the cytoplasm of the ependymal and subependymal cells. Not infrequently, most of reaction product was located at one pole of the subependymal cells (Fig. 16). This reaction was not clearly associated with any organelles. Some cell processes were also found to be labeled mainly at proximity of the capillaries (Fig. 17). The

role of somatostatin as well as LHRH and TRH which have been found in large amounts in the circumventricular organs (PELLETIER et al. 1976a; KIZER et al. 1976) in these specialized organs is still completely unknown. The presence of somatostatin and LHRH throughout the cytoplasm of cells, which are not secretory neurons, may simply represent an uptake from the cerebrospinal fluid. In agreement with this hypothesis is a recent report from PATEL et al. (1977) describing the presence of somatostatin in the cerebrospinal fluid of man.

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c

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Immunohistochemistry of Somatostatin . 17

4.2.4 Non-mammalian vertebrate species

Besides mammalian, a variety of vertebrate species have been shQw l1 to possess a high content of brain' somatostatin. VALE et al. (1977) have described the presence of immunoreactive somatostatin in the brain of pigeon, /rag, catfish, torpedo (clasmabanch) and hagfish. DUBOIS et al. (1974) have reported the localization of the peptide in the median eminence of a bird (cock), an amphibian (Triton) and a teleost fish (trout). In the frog (Rana pipiens), we have found a high concentration of postive fibers in the median eminence with no signifant immunostaining in the other brain areas (Fig. 18). In the trout (Salvelinus fontinalis), somatostatin-containing fibers were observed in the infundibular-pituitary complex. They were located close to the capillaries of the neurohypophysis and also found deeply in the pro-, meso- and meta-adenohypophysis (Fig. 19). These observations suggest a direct neuronal control of adenohypophyseal hormone secretion in that species. 4.3 Localization of somatostatin in the

peripheral nervous system

Using indirect immunofluorescence .techniques, HOKFELT et al. (1976) have described the localization of somatostatin in neuronal cell bodies in spinal ganglia and fibers in the dorsal horn of the spinal cord. These somatostatin-containing cell bodies were small and constituted less

than 10% of all neuronal cell bodies in the ganglia. No somatostatin positive fibers seemed to be present in the ganglia. In the spinal cord, immunostained fibers which were mainly concentrated in the dorsal horn, but also observed in the ventral horn are probably originating from the small cell bodies observed in the spinal ganglia, as described for substance P (HOKFELT et al. 1976). In the gastro-intestinal tract, somatostatin-containing nerve fibers were observed in all parts of the small and large intestine with the highest concentration in the jejunum (HOKFELT et al. 1976). No reactive fibers were detected in the stomach. In the intestine, the positive fibers were mostly observed in the basal part of the lamina propria of the mucosa. They were also described around the ganglionic cells of the myenteric (Auerbach's) plexus. Since organotypic tissues of intestine contain immunoreactive material, it has been suggested by SCHULTZBERG et al. (1978) that somatostatin is synthesized by neurons intrinsic to the intestinal wall. However, the exact function of this somatostatinergic-like system in the regulation of the gastro-intestinal tract remains to be clarified. 4.4 Localization of somatostatin in the pancreas

Since somatostatin was shown to inhibit insulin and glucagon secretion, immunocytochemists rapidly became interested

Fig. 15. Immunoelectron microscope localization of somatostatin in the OVLT. Immunostaining is present in dense core vesicles (arrows) of nerve endings located in proximity of capillaries (C). Other nerve endings (NE) are completely negative. - x 16,000.

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Immunohistochemistry of Somatostatin . 19

in the localization of this peptide in pancreatic tissue. LUFf et al. (1974), using immunofluorescence, reported first the presence of somatostatin in certain cells of the rat pancreatic islets. These somatostatin cells were thought to belong to the A-cell system involved in the production of glucagon. It was later found that, in the human and rat pancreas, somatostatin was only observed in pancreatic cells staining with the Hellerstrom-Hellman argyrophilic impregnation method for D (or AI) cells (POLACK et al. 1975; HOKFELT et al. 1975a). Neither exocrine cells nor nerve fibers have been so far reported to stain for somatostatin. High concentration of somatostatin has also been measured by radioimmunoassay (ARIMURA et al. 1975b; VALE et al. 1976) and bioassay (VALE et al. 1977) in the rat pancreas. It has also been shown by several groups (POLACK et al. 1975; HOKFELT et al. 1975a; DUBOIS 1975; ORCI and UNGER 1975; PELLETIER 1977; PELLETIER et al. 1975b, 1976a; ORC! et al. 1976) that somatostatin-containing cells i. e. the D-cells, were located in close proximity of A-cells which produce glucagon: in the rat, guinea pig and mouse in which both A- and D-cells were found at the periphery of the islets whereas the insulin secreting cells (B-cells) were centrally located (Fig. 20). In some species, such as the horse in which the A-cells are more centrally located, the D-cells were also similarly distributed. In man, in addition to their gen-

erally peripheral localization, A and D cells were also observed grouped together against capillary walls forming delineated cell cords within the islets. Finally, in the chicken embryo pancreas when there are no organized islets (BARDEN et al. 1979) A- and D-cells always showed a similar pattern of distribution (Fig. 21). This relationship between the anatomical distribution of A- and D-cells has led ORCI and UNGER (1975) to suggest that somatostatin could exert a greater inhibitory influence upon pancreatic glucagon secretion than in insulin secretion since B cells which are centrally distributed are not in proximity of D-cells. According to this hypothesis, there should be two anatomically and functionally different zones of the islets of Langerhans: a peripheral zone rich in A- and D-cells and a predominently central zone consisting mainly of B-cells. The peripheral zone could be involved in the acute regulation of glucagon and insulin secretion in response to various stimuli, whereas the central zone could be responsible for a less dynamic but more stable site of insulin release. The importance of this anatomical distribution in physiological and pathological conditions remains to be carefully investigated. In the other hand, it should be mentioned that the pancreatic polypeptide-containing cells have a distribution similar to that of A and D cells (PELLETIER and LECLERC, 1977; PELLETIER, 1977; PELLETIER et aI., 1977b; Fig. 20). Recently, somatostatin

Fig. 16. Immunoelectron microscopy of somatostatin in the subcommissural organ. A diffuse positive reaction (arrows) can be observed at one pole of a subependymal cell. N: nucleus. - x 25,000.

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c .. • I

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...

Immunohistochemistry of Somatostatin . 21

has been reported to inhibit the secretion a decreased production of insulin is acof the pancreatic polypeptide by the hu- companied by an increase in the concentration of somatostatin in pancreatic isman pancreas (MARCO et al. 1977). In an attempt to evaluate the possible lets. Although the role of somatostatin in role of somatostatin in the physiopatholo- physiopathology of diabetes remains to be gy of diabetes, the effect of experimental established, it is possible that insulin exdiabetes on the immunocytochemistry of erts a suppressive effect on somatostatinthe islet cell types has been studied (ORCI secreting cells Alternatively, hyperglyet al. 1976a, b; U GER et al. 1977; PELLE- caemia or increased glucagon production TIER, in preparation). In rats made diabet- could have a direct stimulatory effect on ic by the injection of streptozotocin which somatostatin cells. selectively destroys B-cells, the total voLocalization studies of somatostatin lume of somatostatin-containing cells per performed at the ultrastructural level have pancreas was about 2 to 3 times normal confirmed that the D-cell was the cell and the total number of these cells was al- type containing somatostatin. In the rat most twice normal (ORCI et al. 1976a, b; pancreas, immunostaining was confined PELLETIER, in preparation; Fig. 22). On .exclusively to the secretory granules of a the other hand, the volume and number few cells located at the periphery of the isof insulin cells was reduced in diabetic lets (PELLETIER et al. 1975b; GOLDSMITH et pancreas, whereas in the same diabetic al. 1975; Fig. 23). The A- and B-cells pancreases, the volume and number of were always unstained by the anti-somaglucagon cells was unchanged. In a few tostatin serum. The shape and the size of pancreases from human juvenile diabetics, positive granules were similar to those of similar findings were obtained. These im- D-cells. In monolayer culture of neonatal munocytochemical results were con- rat pancreas, somatostatin was also defirmed by PATEL and WEIR (1976) which tected in the secretory granules of cells showed that, in the streptozotocin-diabet- having the characteristics of D-cells (Ruic rat, the concentration of somatostatin FENER et al. 1975). Similar results were obper islet was markedly increased as com- tained in the human pancreas in which the pared to control animals. The concentra- detection of somatostatin resulted in a tion of glucagon was also found to be strong reaction in all the typical D-cells significantly increased in the treated rats. identifiable by the large diameter of their In spontaneously diabetic mice, PATEL et secretory granules (PELLETIER 1977; Fig. al. (1976) have reported an inverse rela- 24). These ultrastructural data strongly tionship between circulating levels of in- suggested that somatostatin was synthessulin and pancreatic somatostatin concen- ized by the D-cells and stored within setration. From these studies, it appears that cretory granules before being released in-

Fig. 17. Immunoelectron microscopic detection of somatostatin in the subfornical organ. Diffuse immunostaining is localized in many cell processes (arrows) situated close to a capillary (C). - x 10,000.

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GEORGES PELLETIER

/

Immunohistochemistry of Somatostatin . 23

I

Fig. 19. A: In a sagittal section through the trout pituitary, somatostatin-positive fibers (arrows) are seen between the pars nervosa (PN) and the pars distalis and also extending into the different divisions of the pars distalis (PD). B: Control section adjacent to that shown in A. Immunoabsorption with excess somatostatin has completely abolished the staining. - x 400.

to the extracellular space to act as local hormone and influence insulin, glucagon and pancreatic polypeptide secretion. This hypothesis was confirmed when modulation of somatostatin secretion by isolated islets could be demonstrated (BARDEN et al. 1976, 1977). Since gastrin had been occasionally reported to be present in pancreatic D-cells (LOMSKY et al. 1970; GREIDER and MCGUIGAN 1971; ERLANDSEN et al. 1976),

we became interested in the localization of this peptide in the rat and human pancreas. Using an antiserum (supplied by Dr. S. Bloom) that is capable of staining the gastrin cells strongly and specifically, we have been unable to detect gastrin in the human or rat pancreas (PELLETIER 1977). These negative findings are in agreement with recent reports (DUBOIS 1975; CREUTZFELDT et al. 1976) describing the absence of immunostaining for gastrin

Fig. 18. A: Localization of somatostatin in a sagittal section through the frog (rana pipiens) median eminence. Many positive nerve endings (arrows) are observed in proximity of capillaries, mainly in the caudal part of the infundibular. B: Control section adjacent to that shown in A. Previous immunoabsorption with synthetic somatostatin has completely prevented immunostaining. - x 400.

24 . GEORGES PELLETIER

PD.

in the cat and human pancreas. It thus appears so far that the D-cell is only involved in the production and release of somatostatin.

4.5 Localization of somatostatin in the gastro-intestinal tract

Substantial amount of somatostatin has also been found in the gastrointestinal tract (ARIMURA et al. 1975b; PATEL and REICHLIN 1978; KRONHEIM et al. 1976). High concentrations of somatostatin were measured in the stomach with significant

amounts of the peptide in the duodenum and jejunum. In the rat stomach, ARIMURA et al. (1975b) have found equivalent concentrations of somatostatin in both the body and pyloric antrum. Very little somatostatin was found in the cardiac portion of the stomach. Using immunofluorescence and various histochemical staining, POLAK et al. (1975) have been able to demonstrate that, in the mucosa of the upper gastrointestinal tract of pig, dog and man, the somatostatin-containing cells were predominantly localized in the midzone of the glands, although scattered cells could also be seen in the basal region

Fig. 20. Localization of insulin, glucagon, somatostatin and pancreatic polypeptide in consecutive sections of a normal rat pancreas. Glucagon-, somatostatin- and pancreatic polypeptide-containing cells are all located at the periphery of the islet. Insulin cells which are much more numerous are only observed in the central portion of the islet. - x 280.

Immunohistochemistry of Somatostatin . 25

AT I N

LVPEPTIDE

26 .

GEORGES PELLETIER

,

®

SOMA

PANCREATIC POLYPEPTIDE

Fig. 21. Localization of glucagon, insulin, somatostatin and pancreatic poplypeptide in consecutive sections of a 18 days-old chicken embryo pancreas. Note that glucagon and somatostatin cells have the same distribution. At this stage, there is no clear relationship between somatostatin celIs and insulin or pancreatic polypeptide cells. - x 320.

Immunohistochemistry of Somatostatin . 27

I'

@)S

to

A CREATIC tfOLYPEPTIDE

Fig. 22. Localization of insulin, glucagon, somatostatin and pancreatic polypeptide in consecutive sections of the pancreas of a streptozotocin-diabetic rat. The size of three islets (1-3) is markedly reduced (compare with Fig. 20). Somatostatin cells are increased in number, whereas glucagon and pancreatic polypeptide cells do not appear to be significantly modified. The number of insulin cells is markedly decreased. - x 280.

28 .

GEORGES PELLETIER

@ Fig. 23. Immunoelectron microscopic localization of somatostatin in a rat pancreatic islet. A positive reaction can be observed in the secretory granules (arrows) of a D-cell. Adjacent A- and Bcells are unstained. - x 10,000.

Immunohistochemistry of Somatostatin . 29

Fig. 24. Ultrastructural localization of somatostatin in a human pancreatic islet. The reaction product is concentrated in the secretory granules (arrows) with some diffusion in the cytoplasm. An adjacent B-cell is negative. - x 16,000.

30 .

GEORGES PELLETIER

@ Fig. 25. Immunohistochemical detection of somatostatin in the pyloric antrum of the human stomach. A few cells (arrows) show a strong immunostaining for somatostatin. - x 850.

and in the tips of the villi. The D-cell of the Wiesbaden classification appeared as the most likely source of somatostatin in the stomach and the upper gastrointestinal tract. A semi-quantitative evaluation of the somatostatin cells in the rat and human gastro-intestinal tract has been reported by PARSONS et al. (1976) who used an immunoperoxidase technique. In the human stomach, somatostatin cells were mostly concentrated in the body whereas in the rat stomach positive cells were equally found in the body and pyloric antrum. We have recently obtained different results in the human stomach where numerous immunostained endocrine cells were demonstrated in the antral region (unpublished data; Fig. 25). In both spe-

cies, immunostained cells were detected in all the portions of the gastro-intestinal tract with the highest concentration in duodenum. It is interesting to note that, in man, the concentration of somatostatin cells was higher in the rectum than in jeju'num, ileum and colon. In the rat stomach, we have observed that most somatostatin cells were located in the basal region of the villi of the pyloric antrum (PELLETIER et al. 1976b). They were very frequently observed in the neighborhood of the gastrin-secreting cells (Fig. 26). At the ultrastructural level, the reaction product indicating the presence of somatostatin was observed in the secretory granules and also to a lesser degree in the cytoplasm of a few cells situat-

Immunohistochemistry of Somatostatin . 31

@ Fig. 26. In the rat pyloric antrum, somatostatin represented by a dark reaction is localized in a few cells (arrows) mainly found at the base of the crypts. LP: lamina propria. - x 700.

ed in proximity of the basement membrane (PELLETIER et al. 1976b; Fig. 27). These positive cells had a round or elongated shape and did not seem to be in contact with the pyloric lumen. The diameter of the secretory granules was about 150-250 nm. Because of these ultrastructural characteristics, the somatostatin cell seems to correspond reasonably well to the endocrine cell type III (or intestinal D-cell) described by Forssmann et al. (1969). The ultrastructural data strongly suggest that, as in the endocrine pancreas, the D-cells of the stomach are responsible for synthesis and release of somatostatin. Since the gastric D-cells are located in close proximity of the gastrin-secreting

cells, it is likely that somatostatin act as a local hormone to inhibit gastrin secretion from G-cells. In this view, somatostatin may be analogous to other substances considered as local hormones, such as histamine and serotonin. In the other parts of the gastro-intestinal tract, there has been so far no report about the anatomical relationship between the somatostatin cells and the other cell types, such as secretin or GIP cells of which the secretion is inhibited by somatosatin. Recent observations of the number of G- and D-cells in the gastric mucosa of normal subjects and patients with duodenal ulceration have suggested that a deficiency of somatostatin might be responsible for hyperacidity 'and duodenal ulcer formation (PEARSE et

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Immunohistochemistry of Somatostatin . 33

al. 1977). More studies are however required to ascertain the physiological importance of somatostatin in the stomach.

4.6 Localization of somatostatin in other tissues

By immunofluorescence, HC>KFELT et al. (197Sb) have reporte& a sparse plexus of fibers staining for somatostatin in the rat posterior pituitary. So far, their results have not been confirmed. In the thyroid gland, a positive reaction for somatostatin has been described by HC>KFELT et al. (197Sa) and PARSONS et al. (1976). The reaction was localized in most but not all calcitonin-secreting cells. Using the immunoperoxidase technique at both light and electron microscopic level, we have not been able to demonstrate any specific immunostaining for somatostatin in both man and rat thyroid gland. By radioimmunoassay, no somatostatin could be detected in the rat thyroid gland (BRAZEAU, personal communication). In lymph nodes and in the thymus, positive cells were described by HC>KFELD et al. (1975b). Again, these results await further confirmation. No immunoreactive somatostatin has been detected in the liver, lung and kidney (PATEL and REICHLIN 1978). The discrepancy observed between several laboratories concerning the distribution of somatostatin in peripheral tissues other than the gut and pancreas still remains to be fully understood.

5 Conclusion On the basis of several recent works on the distribution and morphological localization of somatostatin, it now appears that this tetradecapeptide is widely distributed in the central nervous system and is also found in some epithelial cells of the pancreas and gastro-intestinal tract. The application of highly sensitive immunoelectron microscopic technique has led to the conclusion that somatostatin is synthesized by at least two different cell . types: hypothalamic neurons which have a morphology similar to that of typical secretory neurons and endocrine-like cells as observed in the pancreas and stomach (Fig. 28). Although somatostatin is produced by both neurons and non-neural cells, it remains to be conclusively demonstrated that cells of such diverse morphology and distribution share a common developmental origin from the neural ectoderm, as proposed by PEARSE (1969). In the central nervous system, somatostatin has probably two roles. First, as a secretion product synthesized by the hypothalamic neurosecretory cells and released into the capillaries of the pituitary portal plexus, it acts as an inhibitor of some pituitary hormones, especially GH and TSH. Secondly, its wide distribution in many extrahypothalamic areas and its direct effects on the electrical activity of neurons suggest that it may be a neurotransmitter or neuromodulator of which the role re-

Fig. 27. Immunoelectron microscopic localization of somatostatin in the rat pyloric antrum. The reaction product is almost exclusively found in the secretory granules (arrows) of a typical endocrine cell. N: nucleus. - x 20,000.

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SOMATOSTATIN·CONTAINING CELL TYPES

CNS

A&.NCREAS and GI TRACT

@ Fig. 28. Diagramatic representation of the two cell types involved in the production of somatostatin. In the central nervous system (CNS), somatostatin is found in the dense core vesicle of typical secretory neurons. In the pancreas and gastrointestinal (GI) tract, the peptide is stored in the secretory granules of endocrine cells.

mains to be clarified. In the pancreas and GI tract, the morphological localization of somatostatin has clearly established the close anatomical relationship between the somatostatin-producing cells and the cells of which the secretion is inhibited by the peptide. These data suggest that somatostatin could exert paracrine effects on the adjacent target cells. Somatostatin would then act as a local hormone directing its action to neighboring cells rather than via the bloodstream in manner truly endocnne.

6 References ALPERT, L. c., BRAWER, J. R., PATEL, Y. c., REICHLIN, S.: Somatostatinergic neurons in anterior hypothalamus: immunohistochemical localization. - Endocrinology 98, 225-258 (1976). ARIMURA, A., SATO, H., COY, D. H., SCHALLY, A. V.: Radioimmunoassay for GH release inhibiting hormone. - Proc. Soc. expo BioI. Med. 148, 784-789 (1975a). ARIMURA, A., SATO, H., DUPONT, A., NISHI, N., SCHALLY, A. V.: Somatostatin: abundance of immunoreactive hormone in rat stomach and pancreas. - Science 189, 1007-1009 (1975b).

Immunohistochemistry of Somatostatin . 35

ARIMURA, A., SCHALLY, A. V.: Increase in basal and thyrotropin-releasing hormone (TRH) stimulated secretion of thyrotropin (TSH) by passive immunization with antiserum to somatostatin in rats. - Endocrinology 98, 1069-1072 (1976). ARIMURA, A., SMITH, W. D., SCHALLY, Av.: Blockage of the stress-induced decrease in blood GH by anti-somatostatin serum in rats. - Endocrinology 98,540-543 (1976). BARDEN, N., ALVARADO, G., COTE, J. P., DuPONT, A., Cyclic AMP-dependent stimulation of somatostatin secretion by isolated rat islets of Langerhans. - Biochem. biophys. Res. Commun. 71,840-843 (1976). BARDEN, N., LAVOIE, M., DUPONT, A., COTE, J., COTE, J. P., Stimulation of glucagon release by addition of antisomatostatin to islets of Langerhans in vitro. - Endocrinology 101, 635-639 (1977). BARDEN, N., DUBE, D., COTE, J. P., LECLERC, R., PELLETIER, G.: Immunohistochemical characterization of monolayer cell cultures of embryonic chicken pancreas and measurement of somatostatin release. - J. Histochem. Cytochem. (1979) in press. BESSER, G. M., MORTIMER, C. H.: Clinical neuroendocrinology. - In: Frontiers in Neuroendocrinology (L. MARTINI and W. F. GANONG, eds.), pp. 227-254 - Raven Press, New York 1976. BLOOM, S. R., MORTIMER, C. H., BESSER, G. M., HALL, R., GOMEZ-PAN, A., Roy, V. M., RUSSELL, R. C. G., COy, D. H., KASTIN, A. J., SCHALLY, A. V.: Inhibition of gastrin and gastric acid secretion by growth hormone release-inhibiting hormone. - Lancet 2, 1106-1113 (1974). BODEN, G., SIVITZ, M. c., OWEN, G. E.: Somatostatin suppresses secretion and pancreatic exocrine secretion. - Science 190, 163-164, 1975. BRAZEAU, P., VALE, W., BURGUS, R., LING, N., BUTCHER, M., RIVIER, J., GUILLEMIN, R., Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. - Science 179, 77-79 (1973). BRAZEAU, P., RIVIER, J., VALE, W., GUILLEMIN, R.: Inhibition of growth hormone secretion

in the rat by synthetic somatostatin. - Endrocrinology 94, 184-187 (1974a). BROWN, M., VALE, W.: Growth hormone release in the rat: effects of somatostatin and thyrotropin-releasing factor. - Endocrinology 97, 1151-1156 (1975a). BRAZEAU, P., VALE, W., GUILLEMIN, R.: Transhypothalamic effects of drugs of abuse on the secretion of pituitary humans. - In: Narcotics and the hypothalamus (E. ZIMMERMAN and R. GEORGE eds). pp. 109-119. Raven Press, New York 1974b. BROWN, M., VALE, W.:Central nervous system effects of hypothalamic peptides. - Endocrinology 96, 1333-1336 (1975b). BROWNSTEIN, M., ARIMURA, A., FERNANDEZDURANGO, R., SCHALLY, A. V., PALKOVITS, M., KIZER, J. S.: The effect of hypothalamic deafferentiation of somatostatin-like activity in the rat brain. - Endocrinology 100, 246-249 (1977). BROWNSTEIN, M., ARIMURA, A., SATO, H., SCHALLY, A. V., KIZER, J. S.: The regional distribution of somatostatin in the rat brain. - Endocrinology 96, 1431-1461 (1975). BRYCE, P., YEH, M., FUNDERBUCK, c., TODD, H., HERTELENDY, F.: Studies on growth hormone secretion. VII. Effects of somatostatin on plasma GH, insulin and glucagon in sheep. - Diabetes 24, 842-850 (1975). COHN, M. L.: Cyclic AMP, thyrotropin-releasing factor and somatostatin: key factors in the regulation of the duration of narcosis. In: Molecular Mechanisms of Anesthesia (B. R. FINK, ed.), pp. 485-500. - Raven Press, New York 1975. COHN, M. L., COHN, M. Barrel rotation induced by somatostatin in the non-lesioned rat. - Brain Res. 96, 138-141 (1975). CREUTZFELDT, W., CREUTZFELDT, c., ARNOLD, R.: Gastrin-producing cells. - In: Endocrinology of the Gut, W. Y. CHEY and F. P. BROOKS, eds.), pp. 35-62 - Charles B. Slack, Inc., Thorofare, N. J. 1975. DESY, L., PELLETIER, G.: Immunohistochemical localization of somatostatin in the human hypothalamus. - Cell. Tiss. Res. 184, 491-497 (1977). DEUBEN, R. R., MEITES, J.: Stimulation of pituitary growth hormone release by a hypothal-

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amic extract in vitro. - Endocrinology 74, 408-414 (1964). DUBE, D., LECLERC, R., PELLETIER, G., ARIMURA, A., SCHALLY, A V.: Immunohistochemical detection of growth hormone release inhibiting hormone (somatostatin) in the guinea pig brain. - Cell Tiss. Res. 161,385-392 (1975). DUBOIS, M. P.: Immunoreactive somatostatin is present in discrete cells of the endocrine' pancreas. - Proc. nat. Acad. Sci. (Wash.) 72, 1340-1343 (1975). DUBOIS, M. P., BARRY, J., LEONARDELLI, J: Mise en evidence par immunofluorescence et repartition de la somatostatine (SRIF) dans I' eminence mediane des vertebres (Mammiferes, oiseaux, amphibiens, poissons). - C. R. Acad. Sci. (Paris) 279, 1899-1902 (1974). ELDE, R., HOKFELT, T.: Distribution of hypothalamic hormones and other peptides in the brain. - In: Frontiers in Neuroendocrinology (W. F. GANONG and L. MARTINI, eds.), pp. 1-33 - Raven Press, New York 1978. ELDE, R. P., PARSONS, J A: Immunocytochemical localiztion of somatostatin in cell bodies of the rat hypothalamus. - Amer. J Anat. 144,541-548 (1975). EPELBAUM, J, WILLOUGHBY, I. 0., BRAZEAU, P., MARTIN, J B.: Effects of brain lesions and hypothalamic deafferentiation on somatostatin distribution in the rat brain. - Endocrinology 101, 1494-1502 (1977). ERLANDSEN, S. L., HEGRE, O. P., PARSONS, J A MCERAY, R. c., ELDE, R. P.: Pancreatic islet cell hormones: distribution of cell types in the islet and evidence for the presence of somatostatin and gastrin within the D-cells. - J Histochem. Cytochem. 24, 883-897 (1976). FERLAND, L., LABRIE, F., COY, D. H., COY, E. J., SCHALLY, A. V.: Inhibition by six somatostatin analogs of plasma GH levels stimulated by thyamylal-morphine in the rat. MoL eel!. Endocrino!. 4,79-88 (1976). FERLAND, L., LABRIE, F., JOBIN, M., ARIMURA, A., SCHALLY, A. V.: Physiological role of somatostatin in the control of growth hormone and thyrotropin secretion. - Biochem. biophys. Res. Commun. 68, 149-156 (1976).

FORSSMANN, W. G., ORCI, L., PICTEL, R., RENALD. A. E., ROUILLER, c.: The endocrine cells in the epithelium of the gastrointestinal mucosa of the rat. - J Cell Bio!. 40, 692-715 (1969). FUJIMOTO, W. Y.: Somatostatin inhibition of glucose-, tulbutamid-, theophylline-, cytochelasin B-, and calcium-stimulated insulin release in monolayer cultures of cat endocrine pancreas. Endocrinology 97, 1494-1500 (1975). FUJIMOTO, W. Y., ENSINCK, J W., WILLIAMS, R. H.: Somatostatin inhibits insulin and glucagon release by monolayer cell cultures of rat endocrine pancreas. - Life Sci. 15, 1999-2004 (1974). GERICH, J E., LOVINGER, R., GODSKY, G. M.: Inhibition by somatostatin of ,~lucagon and insulin release from the perfused rat pancreas in response to arginine, isoproterenol and theophylline: evidence for a preferential effect on glucagon secretion. - Endocrinology 96, 749-754 (1975). GERICH, J E.: Somatostatin and the endocrine pancreas. - In: Hypothalamus and the endocrine pancreas: Hypothalamus and endocrine functions (F. LABRIE, J MEITES and G. PELLETIER, eds), pp. 127-143 - Plenum Press. New York 1976. GERICH, J E., CHARLES, M. A, GRODSKY, G. M.: Regulation of pancreatic insulin and glucagon secretion. - Ann. Rev. Physio!. 38, 353-388 (1976). GERICH, J E., Lorenzi, M., SCHNEIDER, KARAM, J H., RIVIER, J, GUILLEMIN, R., FARSHAM, P.: Effects of somatostatin on plasma glucose and glucagon levels in human diabetes mellitus: Pathophysiologic and therapeutic implications. - New Eng!. J Med. 291, 544-547 (1974). GOLDSMITH, P. c., ROSE, J c., ARIMURA, A, GANONG, W. F.: Ultrastructural localization of somatostatin in pnacreatic islets of the rat. - Endocrinology 97, 1061-1064 (1975). GOMEZ-PAN, A.: Direct inhibition of gastric acid and pepsin secretion by growth hormone release-inhibiting hormone in cats. Lancet 4, 888-890 (1975). GREIDER, M. H., MCGUIGAN, J E.: Cellular localization of gastrin in the human pancreas.

Immunohistochemistry of Somatostatin . 37

- Diabetes 20,389-396 (1971). GREEN, J. D., HARRIS, G. W.: The neurovascular link between the neurohypophysis and adenohypophysis. - J. Endocrino!. 5, 136-146 (1947). GUILLEMIN, R., GERICH, J. E.: Somatostatin: physiological and clinical significance. Ann. Rev. Med. 27,379-388 (1976). HALL, R., BESSER, G. M., SCHALLY, A. V., COY, D. H., EVERED, P., GOLDIE, P. J., KASTIN, A. J., McNEILLY, A. 5., MORTIMER, C. H., TUNBRIDGE, W. M. G., PHENEKOS, c., WEIGHTMAN, D.: Growth hormone: its action in normal subjects and in acromegaly. Lancet 2, 581-584 (1973). HAYES, J. R., JOHNSON, D. G., KOERKER, D., WILLIAMS, R. H.: Inhibition of gastrin release by somatostatin in vitro. - Endocrinology 96, 1374-1376 (1975). HARRIS, G. W.: Neural control of the pituitary gland. - Arnold, London 1955. HOKFELT, T., EFENDIC, 5., HELLERSTROM, c., JOHANSSON, 0., LUFT, R. ARIMURA, A.: Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special references to the AI-cells of the pancreatic islets and to the hypothalamus. - Acta Endocri. (Kbh.) 80 (supp!. 200), 5-41 (1975a). HOKFELT, T, EFENDIC, 5., JOHANSSON, 0., LUFT, R., ARIMURA, A.: Immunohistochemical localization of somatostatin (growth hormone release-inhibiting factor) in the guinea pig brain. - Brain Res. 80, 165-169 (1974). HOKFELT, T, ELDE, R. P., JOHANSSON, 0., LUFT, R., NILSSON, G., - ARIMURA, A.: Immunohistochemical evidence for separate populations of somatostatin- containing and substance P-containing primary apparent neurons in the rat. - Neuroscience 1, 131-136 (1976). HOKFELT, T, JOHANSSON, 0., EFENDIC, 5., LUFT, R., ARIMURA, A.: Are there somatostatin-containing nerves in the rat gut? Immunohistochemical evidence for a new type of peripheral nerves. - Experientia (Basel) 31,852-854 (1975b). JACKSON, I. M. P.: Extrahypothalamic and phylogenetic distribution of hypothalamic

peptides. - In: The Hypothalamus (5. REICHLIN, R. J. RALDESSARINI and J. B. MARTIN, eds) pp. 217-231 - Raven Press, New York 1978. JOHNSON, D. G., ENSINCK, J. W., KOERKER, D., PALMER, J., GOODNER, C. J.: Inhibition of glucagon and insulin secretion by somatostatin in the rat pancreas perfused in situ. Endocrinology 96, 370-374 (1975). KATO, Y., CHIHARA, K., OHGO, 5., IMuRA, H.: Inhibiting effect of somatostatin on growth hormone release induced by isoprenaline or chlorpromazine in rats. - J. Endocrino!. 62, 687-688 (1974). KIZER, J. 5., PALKOVITS, M., BROWNSTEIN, M. J.: Releasing factors in the circumventricular organs of the rat brain. - Endocrinology 98, 311-319 (1976). KOERKER, D. J., RUCH, W., CHIDECKEL, E., PALMER, J., GOODNER C. J., ENSINCK, J., GALE, C. c.: Somatostatin: hypothalamic inhibitors of the endocrine pancreas. - Science 184,482-484 (1974). KRONHEIM, 5., BERELOWITZ, M., PIMOSTONE, B. L.: A radioimmunoassay for growth hormone release-inhibiting hormone: method and quantitative tissue distribution. - Clin. Endocrino!. 5,619-626 (1976). KRULICH, L., DHARIVAL, A. P. 5., MCCANN, S. M.: Stimulatory and inhibitory effects of purified hypothalamic extracts on growth hormone release from rat pituitary in vitro. Endocrinology 83, 783-790 (1968). KRULICH, L., ILLNER, c., FAWCETT, C. P., QUIJADA, M., MCCANN, S. M.: Dual hypothalamic regulation of growth hormone secretion. - In: Growth and growth hormone (A. PECILE and MULLER, E. E., eds) pp. 306-316 - Excerpta Medica, Amsterdam 1972. KRULICH, L., MCCANN, S. M.: Effect of GHreleasing factor on the release and concentration of GH in pituitaries incubated in vitro. - Endocrinology 85, 319-324 (1969). KUELLA, W. P., SUTIN, J.: Extra blood-brainbarrier brain structures. - Int. Rev. Neurobio!. 10,31-55 (1967). LOMSKY, R., LANGER, F., VORTEL, V.: Immunohistochemical demonstration of gastrin in mammalian islets of Langerhans. - Nature (Lond.) 223,618-619 (1970).

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