Immunoblockade of endogenous glucagon-like peptide-1 by monoclonal antibodies in conscious rats: effect on the insulin response to intragastric glucose.

The physiological action of endogenous active forms of glucagon-like peptide-1 (GLP-1) on the insulin response to intragastric glucose was studied in conscious male Wistar rats by immunoblockade with two monoclonal antibodies directed against different epitopes of GLP-1(7-36)amide. Plasma concentrations of intraperitoneally injected monoclonal antibodies were determined before and during each experiment by an enzyme-linked immunosorbent assay (ELISA) specific for GLP-1-binding antibodies. Three hours after injection of the two monoclonal antibodies, the plasma insulin response (area under the curve) following intragastric glucose 1 g/kg was reduced to a mean level (mean +/- SEM) of 60%+/-8% (n = 11) of control responses previously determined in the same rats, and the time course of the response showed almost no increase in insulin during the first 10 minutes, reaching a maximum of 45.1+/-4.6 microU/mL at 30 minutes, in contrast to the rapid increase of the control response to a maximum of 64.5+/-5.1 microU/mL at 15 minutes. Total C-terminally amidated GLP-1 measured by radioimmunoassay (RIA) of acid ethanol-extracted plasma increased from a mean basal level of 10+/-2 pmol/Lto a peak of 31+/-5 pmol/L at 15 minutes in the control experiments, while basal and response levels greater than 100 pmol/L were recorded after antibody treatment. The increase of plasma glucose was reduced in the presence of the antibodies, peaking at a mean of 9.7+/-0.3 mmol/L at 30 minutes, compared with 11.8+/-0.5 mmol/L at 30 minutes in the control experiments. The action of GLP-1 appears particularly important for the early insulin response to ingested glucose, and the unexpected effect of the antibodies on the glucose response may point to a net promoting effect of GLP-1 on intestinal glucose absorption.

Distribution of catecholaminergic afferent fibres in the rat globus pallidus and their relations with cholinergic neurons.

The topographical distribution of catecholaminergic nerve fibres and their anatomical relationship to cholinergic elements in the rat globus pallidus were studied. Peroxidase-antiperoxidase and two-colour immunoperoxidase staining procedures were used to demonstrate tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase (ChAT) immunoreactivities, combined with acetylcholinesterase (AChE) pharmacohistochemistry. TH immunoreactive nerve fibres were seen to enter the globus pallidus from the medial forebrain bundle. The greatest density of such fibres was found in the ventral region of the globus pallidus, which was also characterized by the greatest density of ChAT immunoreactive neurons. TH immunoreactive nerve fibres showed varicose arborizations and sparse boutons, which were occasionally seen in close opposition to cholinergic structures. In all regions of the globus pallidus, there were also larger, smooth TH immunoreactive nerve fibres of passage to the caudate putamen. A smaller number of DBH immunoreactive nerve fibres and terminal arborizations were found in the substantia innominata, internal capsule and in the globus pallidus bordering these structures. A few PNMT immunoreactive nerve fibres in the substantia innominata and internal capsule did not enter the globus pallidus. Electron microscopy revealed TH immunoreactive synaptic profiles in the ventromedial area of the globus pallidus corresponding to the nucleus basalis magnocellularis of Meynert (nBM). These made mainly symmetrical and only a few asymmetrical synaptic contacts with dendrites containing AChE reaction product. The results indicate that cholinergic structures in the nBM are innervated by dopaminergic fibres and terminals, with only a very small input from noradrenergic fibres.

Presence of calcitonin gene-related peptide in intraepithelial nerve fibers and motor end-plates of the cat esophagus: a light and electron microscopic study.

The morphology and distribution of the motor end-plates in the striated muscle and the terminal nerve fibers in the epithelium of the wall of the esophagus, which contain calcitonin gene-related peptide, were studied by light and electron microscopic immunocytochemistry, Varicose immunoreactive nerve fibers arising from the subepithelial plexus were seen to penetrate into the epithelium where they ended in terminal boutons. These nerve fibers lost their Schwann cells just at the point of penetration into the epithelium. Characteristically, the epithelial cells of the spinous layer showed prominent tonofilaments in the part of the cytoplasm in contact with the immunoreactive nerve varicosities, but membrane specializations between these structures were not observed. In the striated muscle of the esophageal wall there were small, elliptical, immunoreactive motor end-plates, which contained a small number of axonal clear vesicles and mitochondria. They were associated with relatively short and rarely branched junctional folds, reduced postjunctional surfaces and few organelles in the underlying sarcoplasm, features characteristic of the neuromuscular junctions of slow-fatiguing red muscle fibers. The two types of immunoreactive nerve endings, epithelial and muscular, presumably participate in afferent and efferent limbs respectively of the neural control of esophageal motility. The relationship between immunoreactive nerve terminals and epithelial cells in the spinous layer exhibiting prominent tonofilaments allowed us to speculate about the existence of two different patterns of reception to sensory stimuli. The intraepithelial fibers that end in the middle layer of the epithelium could be related to mechanoreceptor reflexes, while those that end in the upper layer may be related to thermoreceptor reflexes or facilitate information about the chemical and other characteristics of foods.

Localization of nitric oxide synthase in the adult rat brain.

The distribution of the immunoreactivity to nitric oxide synthase has been examined from rostral to caudal areas of the rat central nervous system using light microscopy. Endogenous nitric oxide synthase was located using a specific polyclonal antiserum, produced against affinity purified nitric oxide synthase from whole rat brain, following the avidin-biotin peroxidase procedure. Immunoreactive cell bodies and processes showed a widespread distribution in the brain. In the telencephalon, immunoreactive structures were distributed in all areas of the cerebral cortex, the ventral endopiriform nucleus and claustrum, the main and accessory olfactory bulb, the anterior and posterior olfactory nuclei, the precommisural hippocampus, the taenia tecta, the nucleus accumbens, the stria terminalis, the caudate putamen, the olfactory tubercle and islands of Calleja, septum, globus pallidus and substantia innominata, hippocampus and amygdala. In the diencephalon, the immunoreactivity was largely found in both the hypothalamus and thalamus. In the hypothalamus, immunoreactive cell bodies were characteristically located in the perivascular-neurosecretory systems and mamillary bodies. In addition, immunoreactive nerve fibres were detected in the median eminence of the infundibular stem. The mesencephalon showed nitric oxide synthase immunoreactivity in the ventral tegmental area, the interpeduncular nucleus, the rostral linear nucleus of the raphe and the dorsal raphe nucleus. Immunoreactive structures were also found in the nuclei of the central grey, the peripeduncular nucleus and substantia nigra pars lateralis, the geniculate nucleus and in the superior and inferior colliculi. The pons displayed immunoreactive structures principally in the pedunculopontine and laterodorsal tegmental nuclei, the ventral tegmental nucleus, the reticulotegmental pontine nucleus, the parabrachial nucleus and locus coeruleus. In the medulla oblongata, immunoreactive neurons and processes were detected in the principal sensory trigeminal nucleus, the trapezoid body, the raphe magnus, the pontine reticular nuclei, the supragenual nucleus, the prepositus hypoglossal nucleus, the medial and spinal vestibular nuclei, the dorsal cochlear nucleus, the medullary reticular field, the nucleus of the solitary tract, the gracile and cuneate nuclei, the dorsal nucleus of the vagus nerve and the oral, interpolar and caudal parts of the spinal trigeminal nucleus. In the cerebellum, the stellate and basket cells showed immunoreactivity, which was also seen in the basket terminal fibres of the Purkinje cell layer. Isolated immunoreactive Purkinje cells were found in the vermis and parafloccular regions of the cerebellum. In the granular layer of the cerebellum, the granular cells and glomeruli were also immunoreactive. Numerous positive varicose nerve fibres and occasional neurons were also found in the lateral and interposed cerebellar nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

Subcellular localization of the inositol 1,4,5-trisphosphate receptor, P400, in the vestibular complex and dorsal cochlear nucleus of the rat.

The subcellular localization of the inositol 1,4,5-trisphosphate receptor protein, P400, was studied in the vestibular complex, an area to which Purkinje cells project, as well as in neurons of the dorsal cochlear nucleus and in ectopic Purkinje cells of adult rat brain. The receptor was demonstrated by electron microscopical immunocytochemistry using the avidin-biotin peroxidase complex procedure, with the monoclonal antibody 4C11 raised against mouse cerebellar inositol 1,4,5-trisphosphate receptor protein. Immunoreactivity was found in preterminal fibres and terminal boutons in the nuclei of the vestibular complex, generally associated with the subsurface systems and stacks or fragments of smooth endoplasmic reticulum. Ectopic Purkinje cells and cartwheel cells of the dorsal cochlear nucleus also displayed immunoreactivity, but this was much less intense in the latter. The results of the present study suggest that this receptor protein, involved in the release of Ca2+, is located in sites that enable it to influence the synthesis, transport and release of neurotransmitters.