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Adrenomedullin (AM) is a novel vasodilator peptide first purified from human pheochromocytoma by tracing its capacity to stimulate cAMP production in platelets. AM immunoreactivity is widely distributed in the central nervous system (CNS) and in the rat has been demonstrated by immunohistochemical techniques to be present in many neurons throughout the brain and spinal cord, as well as in some vascular endothelial cells and perivascular glial cells. Electron microscopy shows that the immunoreactivity is located mainly in the neuronal cytoplasm, but also occurs in the cell nucleus in some cells of the caudate putamen and olfactory tubercle. Biochemical analyses suggest that higher molecular forms, presumably precursor forms, may predominate over fully processed AM in some brain areas. The expression of AM immunoreactivity is increased in cortical neurons, endothelial cells, and perivascular processes after a simulation of ischemia by oxygen and glucose deprivation. Immunohistochemical, electrophysiological, and pharmacological studies suggest that AM in the CNS can act as a neurotransmitter, neuromodulator, or neurohormone, or as a cytoprotective factor in ischemic/hypoxic conditions, in addition to its vasodilator role.

Changes in the nitric oxide (NO) system of the rat cerebral cortex were investigated by immunohistochemistry, immunoblotting, NO synthase (NOS) activity assay, and magnetic resonance imaging (MRI) in an experimental model of global cerebral ischemia and reperfusion. Brains were perfused transcardially with an oxygenated plasma substitute and subjected to 30 minutes of oxygen and glucose deprivation, followed by reperfusion for up to 12 hours with oxygenated medium containing glucose. A sham group was perfused without oxygen or glucose deprivation, and a further group was treated with the NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) before and during perfusion. Global ischemia led to cerebrocortical injury as shown by diffusion MRI. This was accompanied by increasing morphologic changes in the large type I interneurons expressing neuronal NOS (nNOS) and the appearance of nNOS immunoreactivity in small type II neurons. The nNOS-immunoreactive band and calcium-dependent NOS activity showed an initial increase, followed by a fall after 6 hours of reperfusion. Inducible NOS immunoreactivity appeared in neurons, especially pyramidal cells of layers IV–V, after 4 hours of reperfusion, with corresponding changes on immunoblotting and in calcium-independent NOS activity. Immunoreactive protein nitrotyrosine, present in the nuclear area of neurons in nonperfused controls and sham-perfused animals, showed changes in intensity and distribution, appearing in the neuronal processes during the reperfusion period. Prior and concurrent L-NAME administration blocked the changes on diffusion MRI and attenuated the morphologic changes, suggesting that NO and consequent peroxynitrite formation during ischemia–reperfusion contributes to cerebral injury. J. Comp. Neurol. 443:183–200, 2002. © 2002 Wiley-Liss, Inc.

A perfusion model of global cerebral ischemia was used for the immunohistochemical study of changes in the glutamate-nitric oxide (NO) system in the rat cerebellum and cerebellar nuclei during a 0–14 h reperfusion period after 30 min of oxygen and glucose deprivation, with and without administration of 1.5 mM N ω -nitro- l -arginine methyl ester ( l -NAME). While immunostaining for N -methyl- d -aspartate receptor subunit 1 (NMDAR1) showed no marked changes during the reperfusion period, neuronal NO synthase (nNOS) immunostaining increased in stellate and basket cells, granule cells and neurons of the cerebellar nuclei. However, global cerebellar nNOS concentrations determined by Western blotting remained largely unchanged in comparison with actin expression. Inducible NOS (iNOS) immunostaining appeared in Purkinje cells and neurons of the cerebellar nuclei after 2–4 h of reperfusion and intensified during the 6–14 h period. This was reflected by an increase in global cerebellar iNOS expression determined by Western blotting. Immunostaining for protein nitrotyrosine was seen in Purkinje cells, stellate and basket cells, neurons of the cerebellar nuclei and glial cells in controls, and showed a progressive translocation in Purkinje cells and neurons of the cerebellar nuclei from an initial perinuclear or nuclear location towards the periphery. At the end of the reperfusion period the Purkinje cell apical dendrites were notably retracted and tortuous. Prior and concurrent l -NAME administration eliminated nitrotyrosine immunostaining in controls and blocked or reduced most of the postischemic changes observed. The results suggest that while nNOS expression may be modified in certain cells, iNOS is induced after a 2–4 h period, and that changes in protein nitration may be associated with changes in cell morphology.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) and nitrotyrosine immunoreactivities were localized and semiquantitatively assessed in the cerebral cortex of aged rats by means of light microscopic immunocytochemistry and Western blotting, using a new series of specific polyclonal antibodies. In the aged rats the strongly nNOS-immunoreactive multipolar neurons found in layers II–VI of the cortex of young rats were seen in similar numbers, but showed varicose, vacuolated, and fragmented processes, with an irregular outline and loss of spines. A large number of more weakly nNOS-positive neurons, characterized by a ring of immunoreactive cytoplasm, and not seen in young rats, were observed in layers II–VI of aged rat cortex. While no iNOS-immunopositive neurons were found in the cortex of young rats, a large number of such neurons appeared throughout the aged rat cortex. Nitrotyrosine-positive cells outnumbered total NOS-positive neurons in the cortex of young rats, but this relation was inverted in the aged rats, although these showed a slight increase in the number and staining intensity of nitrotyrosine-positive cells. Western blots of brain extracts showed a several-fold increase in both nNOS- and iNOS-immunoreactive bands in the aged rat, but a less marked increase in nitrotyrosine-containing proteins. The results suggest that while nNOS and iNOS expression is substantially increased in the aged rat cortex, this is not necessarily accompanied by a proportionate increase in nitric oxide synthesis. The mechanisms underlying the increased expression of nNOS and iNOS, and the functional implications of this increase, require elucidation. Microsc. Res. Tech. 43:75–88, 1998. © 1998 Wiley-Liss, Inc.

The distribution of nitrergic neurons and processes in the esophagus of the cat and monkey was studied by light microscopic immunocytochemistry using a specific antibody against purified rat brain nitric oxide synthase and immunoperoxidase procedures. Immunoreactive nerve fibers were found pervading the myenteric plexus, submucous plexus and plexus of the muscularis mucosae, and particularly in the lower esophagus a few immunoreactive fibers entered the epithelium as free nerve endings, some of which derived from perivascular fibers. In the upper esophagus immunoreactive motor end-plates were found in the striated muscle. Thirty-forty-five percent of neuronal cell bodies found in the intramural ganglia and along the course of nerve fiber bundles were immunoreactive and were of the three morphological types earlier described. In the intramural ganglia immunoreactive nerve fibers formed a plexus in which varicose nerve terminals were in close relation to immunoreactive and non-immunoreactive neurons. The intramural blood vessels that crossed the different layers of the esophageal wall were surrounded by paravascular and perivascular plexuses containing immunoreactive nerve fibers. The anatomical findings suggest that nitric oxide is involved in neural communication and in the control of peristalsis and vascular tone in the esophagus. In the lower esophagus a few nitrergic nerve fibers are anatomically disposed to subserve a sensory-motor function.

Glucagon-like peptide-1 (GLP-1) has a sparse but well defined distribution in the rat brain where it is co-localized with glucagon-like immunoreactivity due to other fragments of the glucagon precursor. We have investigated the localization of GLP-1 receptors in rat brain using mono-125I-iodinated GLP-1(7-36) amide, the biologically active form of the peptide that occurs in brain, as the tracer for binding and autoradiographic studies of tissue sections. Displaceable binding of the label was sharply localized to discrete areas, being high in mamillary nuclei, the arcuate nucleus, nucleus of the solitary tract and the pretectal area, intermediate in the lateral septal nuclei, olfactory bulb, dorsal tegmental nuclei and the interpenduncular nucleus, and low in other regions. These results indicate areas where GLP-1(7-36) amide may have a role as a neurotransmitter or neuromodulator.

Changes in the distribution of immunoreactive cytochrome c and protein nitration were studied in the rat cerebral cortex after oxygen and glucose deprivation by bright field, confocal and electron microscopy. In control cerebral cortex, nitrotyrosine immunoreactivity indicating protein nitration was found mostly in the neuronal nuclear region, with only a small amount distributed in the cytosol, whereas cytochrome c immunoreactivity was found at the inner membrane and in the intermembrane space of the mitochondria. During the recovery phase after oxygen and glucose deprivation, cytochrome c immunoreactivity was released from the intermembrane space of swollen mitochondria into the surrounding cytosol. The cytosol now also displayed nitrotyrosine immunoreactivity, which had diminished in the nuclear region. Both immunoreactivities were dispersed throughout the soma and processes of the cortical neurons. These changes were largely prevented by the administration of cyclosporin A, which inhibits both the mitochondrial permeability transition and the neuronal isoform of nitric oxide synthase while blocking the induction of the inducible isoform. Ischemia/reperfusion injury increases the production of nitric oxide, reactive oxygen species and intracellular factors that damage the mitochondria and liberate apoptotic factors. We suggest that translocation of cytochrome c from the mitochondria to the cytosol, which has been shown to precede the mitochondrial permeability transition, could result from peroxynitrite-mediated nitration. This phenomenon is attenuated by cyclosporin A administration, suggesting a neuroprotective role for this agent.

Changes in the pattern of adrenomedullin expression in the rat cerebral cortex after ischemia–reperfusion were studied by light and electron microscopic immunohistochemistry using a specific antibody against human adrenomedullin (22-52). Animals were subjected to 30 min of oxygen and glucose deprivation in a perfusion model simulating global cerebral ischemia, and the cerebral cortex was studied after 0, 2, 4, 6, 8, 10 or 12 h of reperfusion. Adrenomedullin immunoreactivity was elevated in certain neuronal structures after 6–12 h of reperfusion as compared with controls. Under these conditions, numerous large pyramidal neurons and some small neurons were intensely stained in all cortical layers. The number of immunoreactive pre- and post-synaptic structures increased with the reperfusion time. Neurons immunoreactive for adrenomedullin presented a normal morphology whereas non-immunoreactive neurons were clearly damaged, suggesting a potential cell-specific protective role for adrenomedullin. The number and intensity of immunoreactive endothelial cells were also progressively elevated as the reperfusion time increased. In addition, the perivascular processes of glial cells and/or pericytes followed a similar pattern, suggesting that adrenomedullin may act as a vasodilator in the cerebrocortical circulation. In summary, adrenomedullin expression is elevated after the ischemic insult and seems to be part of CNS response mechanism to hypoxic injury.

The expression of neuronal nitric oxide synthase (nNOS) during the development of the rat cerebral cortex from embryonic day (E) 13 to postnatal day (P) 0 was analyzed by immunocytochemical procedures using a specific antibody against rat brain nNOS. Expression of nNOS was first seen on E14 in cells of Cajal–Retzius morphology located in the marginal zone. Neuronal NOS immunoreactivity persisted in this layer throughout the embryonic period and only began to decrease on E20, when neuronal migration is coming to an end. From E17 onwards, migrating neurons expressing nNOS were observed in the intermediate zone with their leading processes directed towards the cortical plate. At the same time, efferent nNOS-immunoreactive axons originating from cortical plate cells entered the intermediate zone. From E19 onwards, cells expressing nNOS and with the morphological characteristics of migrating cells were observed in and near the subventricular zone. Confocal analysis of double immunostaining for nNOS and glial fibrillary acidic protein or nestin showed no coexpression of nNOS and glial markers in these cells, suggesting that nNOS-positive cells leaving the subventricular zone were not glial cells. Commissural, callosal and fimbrial fibers were seen to express nNOS on E18 and E19. This expression decreased from E20 and was very weak on E21 and P0. The observations suggest that nitric oxide is synthesized during embryonic life in relation to maturational processes such as the organization of cerebral lamination, and is involved in controlling migrational processes and fiber ingrowth.

The development of a sensitive radioimmunoassay (RIA) for C-terminally amidated forms of glucagon-like peptide-1 (GLP-1) is described. Rabbits immunized with GLP-1(7-36)amide conjugated to bovine serum albumin with glutaraldehyde produced antisera containing high-affinity antibodies directed against an epitope that included the free amidated C-terminus of the peptide. These antisera could be used in a sensitive RIA (detection limit 0.1 fmol/tube) that measured GLP-1(7-36)amide and GLP-1(1-36)amide equally. Total concentrations of amidated GLP-1 immunoreactivity in extracts of rat hypothalamus, pancreas and intestine were determined by RIA, and resolved into GLP-1(7-36)amide, GLP-1(1-36)amide and unidentified cross-reacting substances by HPLC. Whereas only GLP-1(7-36)amide could be identified in the hypothalamus, in amounts that represented 55-94% of total glucagon-like immunoreactivity (GLI), the pancreas produced chiefly GLP-1(1-36)amide, representing 0.8-3.4% of total GLI, and only trace or undetectable amounts of GLP-1(7-36)amide (0-0.36% of total GLI). This argues against any role of intrapancreatic GLP-1(7-36)amide in the secretion of insulin. In the terminal ileum total amidated GLP-1 immunoreactivity represented 27-73% of total GLI, and in five of six specimens only GLP-1(7-36)amide could be identified on HPLC, in amounts representing 13-17% of total GLI. Only one specimen of terminal ileum contained HPLC-identified GLP-1(1-36)amide (13% of total GLI) in addition to GLP-1(7-36)amide (31% of total GLI). Acid-ethanol extraction of peptide-free rat plasma with added GLP-1(7-36)amide gave recoveries of 91+/-SEM 2% in the range 20-200 pmol/l. Basal plasma amidated GLP-1 in six unanaesthetized rats was 4.1+/-1.1 pmol/l and rose to a maximum of 15.4+/-3.0 pmol/l 10 min after intragastric glucose 1 g/kg, illustrating the modest level of plasma responses of amidated forms of GLP-1.

Lithium salts, used in the treatment of affective disorders, may have adverse effects on glucose tolerance in man, and suppress glucose-stimulated insulin secretion in rats. To study the interaction of these effects with pre-existing diabetes mellitus, plasma glucose and insulin responses to lithium chloride were measured in male Wistar rats made diabetic with intraperitoneal streptozotocin, and in normal controls. In both normal and diabetic anaesthetized rats, intravenous lithium (4 mEq kg−1) caused a rise in plasma glucose. In absolute terms, the rise was greater in diabetic (5.2 mmol 1−1) than in normal rats (2.3 mmol l−1). Plasma insulin concentrations were reduced by lithium in normal rats, but the low insulin concentrations measured in the diabetic rats were not significantly changed. After intravenous glucose (0.5 g kg−1), lithium-treated diabetic rats showed a second rise in plasma glucose at 60–90 min without any insulin response, while normal rats showed typically reduced insulin responses and initial glucose disappearance rates. Intravenous glucose reduced plasma glucagon concentrations to a greater extent in normal than in diabetic rats, but lithium induced an equal rise in plasma glucagon in both groups, with a time-course similar to that of the hyperglycaemic effect. The hyperglycaemic action of lithium is greater in the hypoinsulinaemic diabetic rats and appears to involve a stimulation of glucagon secretion in both normal and diabetic animals., This work was supported by grants from the Dirección General de Investigacion Científica y Técnica (no. PM-89-0005) and from the Fundación Rodríguez Pascual.

Pancreatic polypeptide is often secreted by pancreatic endocrine tumors and is considered a marker for such tumors. To investigate the diagnostic value of this marker, we studied 323 patients with proved pancreatic endocrine tumors. We found plasma concentrations of pancreatic polypeptide to be elevated (more than 300 pmol per liter) in 144 patients (diagnostic sensitivity, 45 percent). However, plasma levels of pancreatic polypeptide can also be elevated in the absence of a pancreatic tumor. To ascertain whether the administration of atropine could distinguish between normal and tumor-associated polypeptide secretion, we studied 30 patients with pancreatic tumors and high plasma levels of pancreatic polypeptide, 18 patients without tumors who had elevated levels of pancreatic polypeptide, and eight normal controls. Polypeptide levels in the 18 patients without tumors were substantially lower than in the 30 patients with tumors. Atropine (1 mg intramuscularly) did not suppress polypeptide levels in patients with tumors, but did suppress plasma levels by more than 50 percent in all subjects without tumors. Thus, although its diagnostic sensitivity is low, pancreatic polypeptide appears to be a useful adjunctive marker of many pancreatic endocrine tumors, and the atropine suppression test can be used to distinguish normal from tumor-related secretion of the polypeptide. Identification of the type of pancreatic endocrine tumor still requires measurement of the hormone that is specific for the tumor.

To study the degradation of neurotensin in plasma in vitro, fresh human plasma was incubated with neurotensin in the presence and absence of the peptidase inhibitors pepstatin A, EDTA, PMSF and aprotinin. The half-time of disappearance of neurotensin at 37 degrees C was calculated to be 226 min in vitro as opposed to 1.4 min in vivo when measured by radioimmunoassay with a C-terminally directed neurotensin antiserum. Both gel filtration and reversed phase high-pressure liquid chromatography (HPLC) showed that the main degradation product of neurotensin in human plasma in vitro was chromatographically and immunologically identical to neurotensin 1-8 and HPLC also demonstrated the formation of neurotensin 1-11. The loss of neurotensin incubated in human plasma in vitro was greatly reduced by EDTA but not by the other peptidase inhibitors tested. In this respect peptidase(s) responsible for the degradation of neurotensin in plasma differ from those present in brain homogenates. EDTA may be of importance in the preservation of neurotensin in plasma samples.

Regulatory peptides are likely to have a role in the control of net intestinal fluid transport. PHI is a peptide recently isolated from porcine duodenum which has been shown to have sequence homologies with other peptides of the glucagon-secretin family. We have studied the effect of intravenous infusion of synthetic PHI on net intestinal fluid transport in the rat small intestine. During PHI infusion net absorption was reduced in the duodenum and jejunum and net secretion was observed in the ileum. Thus synthetic PHI appears to be capable of strongly stimulating intestinal secretion in the rat.

The truncated form of glucagon-like peptide-1 (TGLP-1, or proglucagon 78-108), secreted by the mammalian intestine, has potent pharmacological activities, stimulating insulin release and inhibiting gastric acid secretion. We have characterized high-affinity receptors for this peptide in rat isolated fundic glands. Scatchard analysis of binding studies using mono-125I-TGLP-1(7-36) amide as tracer showed a single class of binding site of Kd (4.4 +/- (SE) .08) x 10(-10) M, with a tissue concentration of 1.0 +/- 0.1 fmol sites/microgram DNA. Whole GLP-1 was approximately 700 times less potent in displacing tracer, while human GLP-2 and pancreatic glucagon produced no significant displacement at concentrations up to 10(-6) M. The data support a physiological role for TGLP-1 in the regulation of gastric acid secretion.

Although the purified porcine enteroglucagons glicentin and oxyntomodulin inhibit pentagastrin-stimulated gastrin acid secretion when given parenterally to rats, it is not known whether the postprandial rise in endogenous enteroglucagons is capable of exerting a similar effect. We have used the alpha-glucosidase inhibitor acarbose in combination with a sucrose- and starch-rich semisynthetic diet over 8 days to bring about a mean increase of 89 pmol/l in the fasting plasma enteroglucagon concentration in rats, without significantly affecting plasma gastrin concentrations. There was no significant suppression of pentagastrin-stimulated gastric acid secretion in the acarbose-treated rats, suggesting that endogenous enteroglucagons do not act as physiological inhibitors of gastric acid secretion.

Molecular forms of the glucagon-like peptides (GLP) encoded by the human preproglucagon gene were analysed by chromatography combined with specific radioimmunoassays to the synthetic peptides. Whereas extracts of human pancreas and a glucagonoma contained a large proglucagon cleavage product possessing both GLP-1 and GLP-2 immunoreactivities, extracts of human intestine contained products corresponding to free GLP-1 and a small amount ofchromatographically distinct GLP-2 immunoreactivity. It is concluded that post-translational processing of proglucagon differs in pancreas and intestine, so that the C-terminal portion of the molecule is cleaved to liberate free GLP-1 in the intestine. Further processing or degradation results in loss especially of GLP-2 immunoreactivity.