Somatostatin immunoreactivity in the cat adrenal medulla

Summary Somatostatin-like immunoreactivity was detected within the adrenal gland of the rat using specific monoclonal antibodies. Immunohistochemical studies demonstrated a few somatostatin-immunoreactive nerve fibers within the adrenal medulla. In addition, a large population of chromaffin cells in the cat adrenal medulla displayed intense somatostatin-like immunoreactivity. Similar cells were not observed in rat or guinea pig adrenal glands, although they were found in human material. The somatostatin-positive cells in the cat adrenal medulla often possessed short immunoreactive processes similar to those seen in somatostatin-immunoreactive paracrine cells of the gut. Characterization of the somatostatin-like immunoreactivity of the cat adrenal by high performance liquid chromatography and radioimmunoassay indicated that somatostatin-28 may account for over 90% of the observed immunoreactivity. It is suggested that somatostatin-28 may have a paracrine or endocrine role in the feline adrenal medulla.     

Large-molecular-weight somatostatin in human adrenal medullary tissue

Three human catecholamine-secreting adrenal medullary tumours, identified as phaeochromocytoma, were found to contain 774, 168, and 78 pmol/g of somatostatin-like immunoreactivity (SLI), compared to 40 pmol/g in a sample of normal human adrenal medulla. Sephadex-G50 gel-filtration chromatography of extracts from these tissues revealed SLI eluting in the position of somatostatin-14, somatostatin-28, and a peak eluting with a mol. wt. of about 5 K. After digestion of eluted material with clostridiopeptidase B, the predominant form of adrenal medullary SLI was found to elute in the position of a 7-K polypeptide.     

Conversion of somatostatin-28 to somatostatin-14 during maturation of epithelial cells in the porcine jejunum

Fractions of isolated epithelial cells were harvested from a segment of porcine jejunum by ten successive incubations with a chelating buffer. The cell fractions showed a progressive decrease in the activity of the brush-border enzymes, alkaline phosphatase and sucrase, with increasing incubation number but a progressive increase in the ability to incorporate labelled thymidine into DNA. Fractions enriched in cells from the crypt region (fractions 9 and 10) contained higher concentrations per mg protein of somatostatin-like immunoreactivity (1.8-fold), glucagon-like immunoreactivity (5.3-fold) and serotonin (3.0-fold) than fractions enriched in cells from the villus tip (fractions 1 and 2). Analysis of extracts of the fractions by gel filtration/radioimmunoassay showed that somatostatin-28 represented the predominant molecular form of somatostatin-like immunoreactivity in all cell fractions but the relative proportion of somatostatin-14 (and related metabolites) to somatostatin-28 was significantly higher (P less than 0.05) in fractions enriched in villus cells (fraction 1 and 2) than in fractions enriched in crypt cells (fractions 5-10). This result suggests that metabolism of somatostatin-28 to somatostatin-14 takes place during migration of the D cell from the crypt base to the villus tip. Heterogeneity in the somatostatin-14 region of the chromatograms indicates that the peptide may be further metabolized by the action of aminopeptidases.     

Reduced somatostatin-like immunoreactivity in the brain of LEC rats with hepatic encephalopathy.

Somatostatin-14-like immunoreactivity (S14LI) and somatostatin-28(1-12)-like immunoreactivity (S28(1-12)LI) in the brain of LEC (Long Evans Cinnamon) rats with hepatic encephalopathy were measured. Significant reduction of both S14LI and S28(1-12)LI was observed in the hypothalamus, medulla oblongata, striatum and spinal cord. Both of the immunoreactivities in the hypothalamus of these rats were approx. 50% of those in LEC rats without hepatic encephalopathy. The amounts of reduction of S14LI significantly correlated with those of reduction of S28(1-12)LI. No significant difference in gel chromatographic profiles of S14LI and S28(1-12)LI was observed between LEC rats with and without hepatic encephalopathy. These results suggest that the reduction of somatostatin-like immunoreactivity in LEC rats with hepatic encephalopathy may be caused by a decrease in production of prosomatostatin rather than altered degradation.     

Immunocytochemical localization of insulin- and somatostatin-like material in human breast tumors

Four types of human breast lesions and C3H mouse mammary adenocarcinomas (type A) were examined for the immunocytochemical localization of cells containing hormone-like substances. Insulin- or somatostatin-like immunoreactive material was observed in scattered single cells and nests of tumor cells in seven of eight infiltrating duct carcinomas, and in the majority of tumor cells from an anaplastic carcinoma. A few somatostatin-immunoreactive cells were observed in only one of seven fibroadenomas studied. No immunoreactive cells were observed in mouse adenocarcinomas or in human breast dysplasias. These results suggest that cells with hormone-like immunoreactivity may be a common feature in two types of malignant human breast tumors.     

Characterization of the somatostatin-like immunoreactivity extracted from an adrenal medullary tumour

Significant amounts of somatostatin-like immunoreactivity (SLI) were detected in the extract of a human catecholamine-secreting adrenal medullary tumour. After salt fractionation and reconstitution the major portion of SLI was purified by gel filtration and two HPLC steps; in all three systems it eluted in the position of somatostatin-14. The purified somatostatin-like peptide inhibited, in a dose-related manner, growth hormone release from stimulated perfused rat anterior pituitary cells in vitro. Amino acid analysis showed the purified peptide to have an identical composition to somatostatin found in other species.     

Ultrastructural distribution of somatostatin-14 and-28 in rat adrenal cells

Summary Previous studies have shown that somatostatin modulates angiotensin-induced aldosterone secretion by adrenal glomerulosa cells. This effect is mediated through specific receptors which do not show any preference for somatostatin-14 (S14) or the N-extended form somatostatin-28 (S28). The study of the distribution of ¹²⁵I-Tyr [Tyr⁰, DTrp⁸] S14-and ¹²⁵I-Tyr[Leu⁸, DTrp²², Tyr²⁵] S28-binding in frozen sections of the rat adrenal by autoradiography indicated that both peptides bind to similar loci. High concentrations of binding sites were observed in the zona glomerulosa, and low concentrations were detected in the medulla. At the ultrastructural level, immunocytochemistry after cryoultramicrotomy revealed endogenous S14-and S28-like immunoreactive material in zona glomerulosa and in medulla. In glomerulosa cells, immunoreactive material was localized at the plasma membrane level, in the cytoplasmic matrix, in the mitochondria, and in the nucleus. S14-and S28-like materials were detected in both epinephrine and norepinephrine-storing cells of the adrenal medulla. In these cells, the distribution of either immunoreactive product was similar; it was observed in cytoplasmic matrix, secretory granules and nucleus, but not at the plasma membrane level. In situ hybridization does not reveal somatostatin mRNA in zona glomerulosa or medulla. These results demonstrate that S14 and S28 bind to, and are taken up by zona glomerulosa and adrenal medullary cells, but are not produced by these cells.     

Somatostatin modulation of neurally mediated pepsinogen secretion from frog esophageal mucosa

Frog esophageal mucosa contains peptic glands which are innervated by cholinergic neurons. When incubated in a medium containing 1.5 mM CaCl₂, pepsinogen release from esophageal mucosa was increased by a high potassium concentration (55 mM KCl), 1,1-dimethyl-4-phenylpiperazinium (DMPP) or bethanechol. Whereas the response to bethanechol remained little changed, the response to high KCl concentrations or DMPP was abolished in the absence of Ca²⁺. The stimulatory effects of high KCl concentrations and DMPP were also eliminated by the presence of atropine or somatostatin. Furthermore, pepsinogen release in response to bethanechol was dose-dependently inhibited by somatostatin. Frog esophagus was found to contain somatostatin-like immunoreactivity, with a higher density at the end adjacent to the stomach. Chromatography of mucosa extract on Sephadex G-50 revealed a single peak of somatostatin-like immunoreactivity that coeluted with somatostatin-14. Immunohistochemical staining of the mucosa with peroxidase antiperoxidase technique demonstrated the presence of two varieties of somatostatin-like immunoreactivity-containing cells, one individually dispersed within the intercalated septa and the other in groups within the interlobular septa of the peptic glands. These results seem to indicate that somatostatin or somatostatin-like immunoreactivity may play a modulatory role in neurally mediated pepsinogen secretion in the frog esophagus.     

Ultrastructure and distribution of somatostatin-like immunoreactive neurons and nerve fibres in the coeliac ganglion of cats

Summary Somatostatin-like immunoreactivity was localized in nerve cell bodies and nerve terminals in the cat coeliac ganglion. Two types of somatostatin-immunoreactive cell bodies were revealed, the first being large (diameter 35 m), numerous and weakly labelled, where—as the second was considerably smaller (diameter 10.4 m), sparsely distributed and heavily stained. The immunoreactive nerve terminals were in synaptic contact with many immunonegative large neurons and dendrites. However, in a few cases, somatostatin-immunoreactive nerve terminals could also be observed on the surface of lightly stained neurons. Transection of vagal or mesenteric nerve failed to affect the distribution or density of somatostatin-like immunoreactive nerve terminals. These results demonstrate the existence of a synaptic input to the principal neurons of the coeliac ganglion of the cat by somatostatin-containing nerve terminals and suggest that this peptide may act as a neuromodulator or neurotransmitter. It is proposed that somatostatin-positive neurons provide intrinsic projections to other somatostatin-positive and to somatostatin-negative neurons throughout the coeliac ganglion, thereby creating a complex interneuronal system.     

The intestinal mucosa preferentially releases somatostatin-28 in pigs

We studied the molecular forms of somatostatin-like immunoreactivity (SLI), newly released from isolated perfused preparations of the porcine antrum, stomach, pancreas and upper small intestine: Perfusion effluents were concentrated by Sep-Pak C-18 adsorption, eluted with ethanol, dessicated, and subjected to gel filtration with subsequent radioimmunoassays for somatostatin-14 and N-terminal somatostatin-28 immunoreactivity. All the SLI newly released from the stomach and antrum eluted at the position of somatostatin-14, and such was also the case for more than 95% of the SLI newly released from the pancreas, while 68 -/+ 7% and 75 -/+ 8% of the SLI newly released from the isolated perfused jejunum and ileum, respectively, corresponded to somatostatin-28. By reverse phase HPLC the identity of these peptides with synhetic somatostatin-14 and -28 was established.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Somatostatin Precursor in the Rat Striatum: Changes After Local Injection of Kainic Acid

The molecular forms of somatostatin contained in the rat striatum were separated by size-exclusion HPLC. Three major peaks of somatostatin-like immunoreactivity (SLI) were resolved. Two peaks cochromatographed with synthetic somatostatin-14 (SS-14) and somatostatin-28 (SS-28), respectively. One peak exhibited a higher molecular weight (about 10,000) and may contain a proform of somatostatin. Local injection of the neurotoxin kainic acid (1 microgram) into the left striatum resulted in a persistent decrease (65-85%) of all three forms of somatostatin. In the contralateral--not injected--striatum a decrease of SLI was also observed which was maximal (45%) after 2 days and was largely abolished after 7 days. This decrease of SLI in the contralateral striatum, however, was due mainly to a decrease of SS-14 and SS-28 but not of the putative proform. Our data suggest that kainic acid causes a destruction of somatostatin-containing perikarya in the injected striatum, whereas in the contralateral striatum increased release with subsequent inactivation of SS-14 and SS-28 takes place. The putative somatostatin proform may serve as neurochemical marker for somatostatin-containing perikarya in the striatum.     

Presence of somatostatin-28 like immunoreactivity in the human pancreas

Specific antisera against somatostatin-28 were prepared by absorption of somatostatin-28 antisera with sepharose 4B-somatostatin-14. Indirect immunofluorescence techniques using somatostatin-14 antisera and specific antisera against somatostatin-28 were carried out to elucidate the time of occurrence of somatostatin-28 in the fetal pancreatic islets and to ascertain whether somatostatin-28 was present in the adult pancreatic islets or not, and further to examine whether cells reacting with specific antisera against somatostatin-28 are identical to those reacting with somatostatin-14 antisera or not. Somatostatin-28 like immunoreactivity occurred in the fetal pancreatic islets at 11th week's gestation and was found in all fetal pancreatic islets examined in the present study. It was also found in the adult pancreatic islets. Furthermore, cells reacting with specific antisera against somatostatin-28 in the fetal and adult pancreatic islets were identical to those reacting with somatostatin-14 antisera. Thus, the present study elucidated the presence of somatostatin-28 like immunoreactivity in the human pancreas. However, it could not be decided whether cells reacting with somatostatin-28 antisera contain either only somatostatin-28 or both somatostatin-28 and somatostatin-14; in other words, whether somatostatin-14 is produced from somatostatin-28 or not, since somatostatin-14 antisera had a cross-reactivity to both somatostatin-14 and somatostatin-28.     

Degradation and conversion of somatostatin in normal and diabetic rats in vivo and in vitro

Plasma somatostatin-like immunoreactivity in the portal and jugular veins of streptozotocin diabetic rats was compared with that in normal control rats. In the diabetic group, somatostatin levels in the portal (p less than 0.05) and jugular (p less than 0.01) veins were both elevated compared with those in the control group. Moreover, the degree of elevation was greater in the jugular vein than in the portal vein. To further investigate the role of the liver in the clearance of somatostatin-28 in vivo, 2 micrograms of somatostatin-28 was administered as a bolus into the external jugular vein of intact and functionally hepatectomized rats. The mean half-time of somatostatin-28 was significantly longer in intact diabetic rats than in controls (p less than 0.05). The functional hepatectomy did not cause a significant difference in the half-time in diabetic rats but made it longer in control rats. These results suggest that the longer half-time of somatostatin-28 in diabetic rats in vivo is due to its slower hepatic clearance. The hepatic clearance of somatostatin-28 and somatostatin-14 was further studied in vitro using a recirculating liver perfusion method. The hepatic clearance of 1.2 nM of either somatostatin-28 or somatostatin-14 was significantly lower in diabetic rats than in controls (p less than 0.01). This indicates that elevated plasma somatostatin levels in diabetic rats are caused at least in part by decreased hepatic clearance of somatostatin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcitonin gene-related peptide (CGRP)-like immunoreactivity in scattered chromaffin cells and nerve fibers in the adrenal gland of rats

Summary The present immunohistochemical study reveals that a small number of chromaffin cells in the rat adrenal medulla exhibit CGRP-like immunoreactivity. All CGRP-immunoreactive cells were found to be chromaffin cells without noradrenaline fluorescence; from combined immunohistochemistry and fluorescence histochemistry we suggest that these are adrenaline cells. In addition, all CGRP-immunoreactive cells simultaneously exhibited NPY-like immunoreactivity. CGRP-chromaffin cells were characterized by abundant chromaffin granules with round cores in which the immunoreactive material was densely localized. These findings suggest the co-existence of CGRP, NPY and adrenaline within the chromaffin granules in a substantial number of chromaffin cells.Thicker and thinner nerve bundles, which included CGRP-immunoreactive nerve fibers, with or without varicosities, penetrated the adrenal capsule. Most of them passed through the cortex and entered the medulla directly, whereas others were distributed in subcapsular regions and among the cortical cells of the zona glomerulosa. Here the CGRP-fibers were in close contact with cortical cells. A few of the fibers supplying the cortex extended further into the medulla. The CGRP-immunoreactive fibers in the medulla were traced among and within small clusters of chromaffin cells and around ganglion cells. The CGRP-fibers were directly apposed to both CGRP-positive and negative chromaffin cells, as well as to ganglion cells. Immunoreactive fibers, which could not be found close to blood vessels, were characterized by the presence of numerous small clear vesicles mixed with a few large granular vesicles. The immunoreactive material was localized in the large granular vesicles and also in the axoplasm. Since no ganglion cells with CGRP-like immunoreactivity were found in the adrenal gland, the CGRP-fibers are regarded as extrinsic in origin. In double-immunofluorescence staining for CGRP and SP, all the SP-immunoreactive fibers corresponded to CGRP-immunoreactive ones in the adrenal gland. This suggests that CGRP-positive fibers in the adrenal gland may be derived from the spinal ganglia, as has been demonstrated with regard to the SP-nerve fibers.     

Tissue-specific posttranslational processing of pre-prosomatostatin encoded by a metallothionein-somatostatin fusion gene in transgenic mice

The somatostatins are neuropeptides of 14 and 28 amino acids that inhibit the release of growth hormone and other hypophyseal and gastrointestinal peptides. These neuropeptides are cleaved posttranslationally from a common precursor, pre-prosomatostatin. We report here the production and processing of pre-prosomatostatin by transgenic mice carrying a metallothionein-somatostatin fusion gene. The most active site of somatostatin production, as determined by hormone concentrations in the tissues, is the anterior pituitary, a tissue that does not normally synthesize somatostatin-like peptides. Anterior pituitary processed pre-prosomatostatin almost exclusively to the two biologically active peptides, somatostatin-14 and somatostatin-28, whereas the liver and kidney synthesized much smaller quantities of predominantly a 6000 dalton somatostatin-like peptide. The growth of the transgenic mice was normal despite high plasma levels of the somatostatin-like peptides. These studies indicate that proteases which cleave prosomatostatin to somatostatin-28 and somatostatin-14 are not specific to tissues that normally express somatostatin.     

Intracellular localization of tyrosine hydroxylase immunoreactivity in the rat adrenal chromaffin and pheochromocytoma cells

The localization of tyrosine hydroxylase (TH) immunoreactivity in rat adrenal chromaffin and pheochromocytoma (PC12) cells was investigated by immunoelectron microscopy using monoclonal and polyclonal antisera against TH purified from rat adrenal medulla. Strong TH immunoreactivity was found uniformly in the granules of the adrenaline cells; the immunoreactivity was visible mainly within the periphery, but not in the clear space of the granules of the noradrenaline cells. In the PC12 cells, strong TH immunoreactivity was also observed uniformly in the granules. In addition, TH immunoreactivity was seen in the cytoplasm, the ribosomes attached to the endoplasmic reticulum and the free ribosomes of both the rat adrenal chromaffin and PC12 cells. These results suggest that TH may be localized in the granules, cytoplasm and ribosomes of rat adrenal chromaffin and PC12 cells.     

Gamma-aminobutyric acid (GABA) immunoreactivity in the mouse adrenal gland

Gamma-aminobutyric acid (GABA) immunoreactivity was revealed by immunocytochemistry in the mouse adrenal gland at the light and electron microscopic levels. Groups of weakly or faintly GABA immunoreactive chromaffin cells were often seen in the adrenal medulla. By means of immunohistochemistry combined with fluorescent microscopy, these GABA immunoreactive chromaffin cells showed noradrenaline fluorescence. The immunoreaction product was seen mainly in the granular cores of these noradrenaline cells. These results suggest the co-existence of GABA and noradrenaline within the chromaffin granules. Sometimes thick or thin bundles of GABA immunoreactive nerve fibers with or without varicosities were found running through the cortex directly into the medulla. In the medulla, GABA immunoreactive varicose nerve fibers were numerous and were often in close contact with small adrenaline cells and large ganglion cells; a few, however, surrounded clusters of the noradrenaline cells, where membrane specializations were formed. Single GABA immunoreactive nerve fibers, and thin or thick bundles of the immunoreactive varicose nerve fibers ran along the blood vessels in the medulla. The immunoreaction deposits were observed diffusely in the axoplasm and in small agranular vesicles of the GABA immunoreactive nerve fibers. Since no ganglion cells with GABA immunoreactivity were found in the adrenal gland, the GABA immunoreactive nerve fibers are regarded as extrinsic in origin.     

Somatostatin as a mediator of the effect of neurotensin on pentagastrin-stimulated acid secretion in rats

Neurotensin and somatostatin have both been shown to inhibit gastric acid secretion, but no interaction between these peptides has been demonstrated. To determine whether somatostatin might be a mediator of neurotensin's effect on pentagastrin-stimulated gastric acid secretion, we performed the following three experiments. First, we collected 0.2-ml samples of portal venous blood as frequently as every 5 min, and we confirmed a significant release of somatostatin-like immunoreactivity into portal venous blood during neurotensin-induced inhibition of acid secretion. This release of somatostatin-like immunoreactivity and inhibition of acid secretion were only seen in pentobarbital-anesthetized rats, but no sustained release of somatostatin-like immunoreactivity or inhibition of acid secretion occurred in urethane-anesthetized animals. In the second experiment, we analyzed portal plasma by high pressure liquid chromatography, and found that portal somatostatin-like immunoreactivity in blood collected during neurotensin infusion was composed of a single peak corresponding to somatostatin-14. In the third experiment, we found that infusion of antibody to somatostatin prevented neurotensin from inhibiting pentagastrin-stimulated acid secretion. Taken together, these data show that somatostatin, possibly from the stomach itself, is a necessary mediator of neurotensin's inhibitory effect in pentobarbital-anesthetized rats.