Differing roles of protein kinase C on the signal transduction of tumour necrosis factor-alpha and -beta on PANC-1 cells: in vitro autoradiographic investigation.

We investigated alterations in protein kinase C (PKC) activity of PANC-1 cells following treatment with tumour necrosis factor (TNF)-alpha or TNF-beta by an in vitro autoradiographic method. Binding studies performed on whole cells using [3H]phorbol-12,13-dibutyrate (PDBu) as a ligand revealed strong activation of PKC by TNFs within 30 min. The effect was similar to that seen after 30 min treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). After treatment for 24 h, TNF-beta caused a marked down-regulation of PKC similar to that seen after 24 h treatment with TPA; significant activation persisted, however, in the cells treated for 24 h with TNF-alpha. Our data suggest that PKC activation may play a more important role in the TNF-alpha signal transduction pathway than in that of TNF-beta.

Possible involvement of free radical scavenging properties in the action of tumor necrosis factor-alpha.

Constitutive production of hydroxyl radicals from four established cancer cell lines was detected as spin adducts of 5,5-dimethyl-l-pyroline-N-oxide (DMPO), using an electron spin resonance spectrometer. The generated hydroxyl radicals was decreased in three out of four cancer cell lines when incubated in vitro for 3 h with TNF-alpha No direct scavenging effect of TNF-alpha on hydroxyl radicals or superoxide anions was observed in the in vitro radical generation system. The modulation of intracellular reactive oxygen species of these cancer cells by adding menadione or CuDIPS to the culture medium changed the antiproliferative effect of TNF-alpha on the cells. The ultrastructural localization of the radical-generating sites in cancer cells was visualized using the diaminobenzidine/horseradish peroxide histochemical system at the electron microscopic level. The hydrogen peroxide-dependent formation of electron-dense materials localized at the mitochondrial membranes was decreased after the treatment of the cancer cells with TNF-alpha. These data indicate that the reduction of radical generation in cancer cells by TNF-alpha may be an early mechanism that contributes to the antiproliferative effect of this cytokine on some cancer cells.

Limited diffusibility of gene products directed by a single nucleus in the cytoplasm of multinucleated myofibres.

Two types of beta-galactosidase genes, whose products are distributed in the nucleus (N beta-gal) or cytoplasm (C beta-gal), were injected with fructose intramuscularly into the quadriceps of adult mice. Regionally restricted and overlapped distributions of both gene products were observed in the myofibres. These findings indicate that N beta-gal is incorporated into the nucleus responsible for its synthesis and that C beta-gal becomes located in the vicinity of the nucleus after its synthesis. This restricted location of C beta-gal in myofibres remained unchanged during the development of infant mouse muscle. Thus, the gene products directed by the nucleus of myofibres seem to show limited diffusibility, suggesting a universal localization of subcellular domains in myofibres.

Serotoninlike immunoreactivity in the cestode Hymenolepis diminuta.

Serotoninlike immunoreactivity in the cestode Hymenolepis diminuta was studied at the light microscope level by using an antibody specific to serotonin. The rostellum, the cerebral ganglia and commissure, and the strobila contained numerous process-free, unipolar and multipolar serotoninlike immunoreactive cells. The suckers contained a plexus of branching immunoreactive fibers. In the strobila the multipolar cell bodies were situated laterodorsal and lateroventral to the longitudinal nerve cords, from which neurites were directed to the contralateral and ipsilateral nerve cord to form up to three transverse commissures per proglottid. Secondary varicose branches passed anteriorly, posteriorly, and obliquely along the proglottids at the level of the deep longitudinal muscles. Other varicose multi-branching neurites passed centrifugally from the primary and secondary neurites, forming vertebratelike en passant or terminal varicosities on the deep longitudinal muscles with bulbous or spinose terminals at the level of the superficial longitudinal muscles, or in the cortical parenchyma. Serotoninlike immunoreactivity was seen on the external seminal vesicle, the sphincter and cirrus sac, and the proximal portion of the vagina. Varicose terminals were concentrated at the sphincter. The close association of serotoninlike immunoreactive terminals and varicosities with the longitudinal muscles gives credence to the concept that serotonin functions as a neuromuscular transmitter or modulator in the platyhelminths.

Distribution of reduced-nicotinamide-adenine-dinucleotide-phosphate diaphorase-positive cells and fibers in the cat central nervous system.

Previous histochemical studies have suggested that reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase exists in distinct subsets of neurons that neither belong to a single transmitter type nor embrace all the neurons using a single transmitter. As a step toward establishing the role of this enzyme, the distribution of NADPH-diaphorase-positive neurons and fibers in the cat central nervous system was mapped by using a direct histochemical method. Heavily stained NADPH-diaphorase-positive neurons with many prominent cell processes were observed in the cerebral cortex, white matter, caudate nucleus, putamen, nucleus accumbens, septal nucleus, amygdala, anterior, lateral and posterior hypothalamic areas, dorsolateral part of the periaqueductal gray, superior colliculus, central tegmental field (Berman) (pedunculopontine tegmental area), dorsal tegmental nucleus, nucleus coeruleus, mesencephalic and pontine reticular formation, gigantocellular and magnocellular tegmental fields, nucleus facialis, and motor nucleus of the vagus. Moderately stained neurons with two or three prominent cell processes were observed in the nucleus of the diagonal band of Broca, globus pallidus, and substantia innominata. Medium-size, moderately stained neurons that had round large nuclei and no visible cell processes were found in the subthalamic nucleus, pontine gray, trapezoid body, and infratrigeminal, cochlear, and vestibular nuclei. Very dense NADPH-diaphorase-positive nerve terminal fields were seen in the olfactory tubercle, cortex, caudate nucleus, putamen, dentate gyrus, and interpeduncular nucleus. Intensely stained NADPH-diaphorase-positive nerve fibers were found in the stria terminalis, marginal region of the central tegmental field, dorsal tegmental nucleus, and spinal trigeminal tract as well as around the brachium conjunctivum. Although the staining of neurons and tracts was highly selective, they did not correspond to any single known neuronal or neurotransmitter type. Positive staining occurred in discrete subsets of neurons known to be associated with a variety of peptides and classical neurotransmitters. The functional significance of high NADPH diaphorase activity is unknown.

Electron microscopic study of mossy fiber endings in the hippocampal formation of rats after picrotoxin administration.

The hippocampal mossy fiber endings of rats were examined with electron microscopy, following the administration of picrotoxin. In the control group, many small round clear vesicles (40-60 nm in diameter) filled the nerve endings. Further, it was noted that large dense-core vesicles (80-100 nm in diameter) in small numbers were scattered among these small clear vesicles. After administration of picrotoxin, the large dense-core vesicles increased in number and accumulated next to the presynaptic membrane. These vesicles were often fused to the presynaptic membrane, in the form of omega-shaped profiles.

Dopamine metabolism in the striatum of hemiparkinsonian model rats with dopaminergic grafts.

To investigate dopamine (DA) levels as well as DA metabolism by which the striatal DAergic grafts may bring the functional recovery to hemiparkinsonian model rats, a microdialysis study was performed in the striatum, and an autoradiographic analysis for DA transporter was made. In hemiparkinsonian model rats, the concentrations of DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in striatal perfusates, decreased considerably (less than 5%, of control levels). In grafted rats that showed motor recovery, the concentration of DA recovered to almost control level, and DOPAC and HVA to about 20% of controls' suggesting that the rate of DA metabolism is low. L-DOPA loading to grafted rats induced a big release of DOPAC and HVA, thus the DOPAC/DA ratio was close to that of the controls'. Methamphetamine loading increased the concentration of DA but did not change the level of DOPAC and HVA. Haloperidol loading increased DA, DOPAC and HVA. [3H]mazindol binding that reflects the activity of the DA transporter decreased considerably in hemiparkinsonian model rats, but it reappeared more or less in grafted rats. Data indicated that in grafted striatum, the extracellular DA level is almost normal level while the rate of DA metabolism is low. By L-DOPA loading, the grafts show the capacity to synthesize, release and metabolize DA and then the DOPAC/DA ratio is normalized. Responses to methamphetamine and haloperidol, as well as the results of the autoradiographic study suggest that the grafts are under a good feedback regulation of DA metabolism.

A cerebral ischemia model produced by injection of microspheres via the external carotid artery in freely moving rats.

We produced an improved microembolism model of cerebral focal ischemia by injection of 1000-2000 microspheres (50 +/- 5 microns diameter) via a tube retrogradely inserted into the right external carotid artery in freely moving rats. The group injected with 2000 spheres showed a much more severe mortality rate as well as neurological signs than did the 1000-sphere group. Brain water content of the 2000-sphere group was examined and found to show an increase from 4 to 24 h after embolization in the right hemisphere, indicating serious brain edema. Severe neurological signs and individual deaths by embolization were most likely related to the extent of development of brain edema. Examination of learning behavior by shuttle-box avoidance revealed partial but significant impairment of learning in the 1000-sphere group. Autoradiographic studies for muscarinic acetylcholine receptors and protein kinase C binding sites were conducted. Both these binding sites decreased in number, but protein kinase C seems to be more susceptible to ischemic injury than muscarinic acetylcholine receptors. The observation was considered to be closely related with an impairment of learning. The present study suggests that our microembolism model in freely moving rats is useful for investigations of the early phase and late phase of cerebral ischemia.

Localization of receptors for atrial natriuretic polypeptide (ANP) in the glomerulus: in vitro electron microscopic autoradiographical investigation using 125I-labeled ANP.

The localization of receptors for atrial natriuretic polypeptide (ANP) in the glomerulus of the rat was studied by electron microscopic autoradiography using 125I-labeled ANP. A total of 1,134 silver grains counted in 30 glomeruli were distributed as follows: processes of the podocytes (36.4%), cell bodies of the podocytes (14.8%), basement membrane (12.3%), endothelial cells (6.3%), mesangial cells (7.7%) and others, for example the vascular spaces (22.5%). This finding indicates that ANP binding sites are mainly localized on the foot processes of the podocytes in the glomerulus.

Properties of [3H]bunazosin binding in rat kidney.

Properties of alpha 1-adrenoceptor binding in the renal cortex of rats were studied with [3H]bunazosin as the radiolabeled ligand. Both temperature and incubation time influenced bunazosin receptor binding. Subcellular distribution of [3H]-bunazosin binding revealed that the membrane fraction (30,000 x g pellet) had the largest proportion of total binding sites. Pretreatment of membrane preparation at 56 degrees C for 30 minutes greatly decreased the specific binding of [3H]-bunazosin, though there was no significant effect of the pretreatment at 24 degrees C and 37 degrees C for 30 minutes. Among various cations, some divalent ions such as Cu++ and Zn++ greatly decreased bunazosin binding, whereas monovalent ions had no effect on specific binding. Results of Scatchard analysis suggested that the rat renal cortex membrane has single binding sites with an apparent dissociation constant of 0.38 +/- 0.06 nmol/L, though the rat medulla membrane has no potent binding activity with bunazosin. The displacement study revealed that various adrenergic agents inhibit [3H]bunazosin binding in dose-dependent fashion; the rank order of potencies was bunazosin prazosin > phentolamine >> dl-norepinephrine > clonidine, yohimbine >> pindolol, propranolol. These findings reveal that bunazosin has specific receptor binding in the rat renal cortex, indicating that alpha 1-adrenoceptors exist in the rat renal cortex.

Regional responses of rat brain muscarinic cholinergic receptors to immobilization stress.

The effect of immobilization stress (IM-stress) on the muscarinic cholinergic (m-Ch) receptor binding was determined in 8 brain regions using [3H]quinuclidinyl benzilate (QNB). IM-stress produced an increase in specific QNB binding in the septum, striatum, hippocampus and pons + medulla oblongata. Scatchard analysis revealed that IM-stress produced an increase in the affinity of m-Ch receptors in the septum, hippocampus and pons + medulla oblongata, but did not alter the maximum number of binding sites (Bmax). In the striatum, an increase in specific QNB binding was due to both the increase in Bmax and reduction of the dissociation constant (Kd). The present study suggests that IM-stress induces supersensitivity of postsynaptic m-Ch receptors probably due to a decrease in presynaptic cholinergic activities in the septum, striatum, hippocampus and pons + medulla oblongata. As the m-Ch receptors in the striatum and pons + medulla oblongata are affected by IM-stress, further studies of the m-Ch neural system must be performed under stressful situations in these regions as well as in the septum, hippocampus and cerebral cortex.

Time course of changes in lipid peroxidation, pre- and postsynaptic cholinergic indices, NMDA receptor binding and neuronal death in the gerbil hippocampus following transient ischemia.

Brief (5 min) bilateral carotid occlusion in the gerbil produces forebrain ischemia resulting, as previously reported, in almost complete neuronal loss in the CA1 region of the hippocampus; this neuronal destruction occurs between the 4th and 7th day post-ischemia. Various hippocampal biochemical indices were measured from just after such ischemia to 21 days of recirculation, and the temporal pattern of changes compared with that of cell loss. The level of thiobarbiturate reacting substances (TBARS), a measure of lipid peroxidation, was greatly elevated at 30 min after ischemia, rapidly returned to normal levels (by 60 min), but was again elevated on days 4-14. The beginning of this second period of elevation correlated closely with the onset of neuronal loss and the very abrupt and large (to about 32%) decrease in specific N-methyl-D-aspartate (NMDA) binding sites, measured with radioactive CPP. The number of muscarinic binding sites, measured with radioactive quinuclidinyl benzilate, showed an even greater decrease (to 13%) at 21 days post-ischemia, but the decrease was delayed (starting at day 7) and much more gradual than the loss in NMDA binding. In neither case was there any change in binding affinity at any time studied. Acetylcholine (ACh) concentrations were initially greatly decreased (to about 15% at 5 min), transiently increased (to about 130% at 30 min), and then decreased again (to about 15% at 60 min), after which gradual recovery occurred and was completed by day 14. Since no inhibition of choline acetyltransferase activity was observed at any time, the reversible depression in ACh must depend upon some factor other than loss of this key synthetic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase-positive neurons in cat cerebral white matter.

Two populations of neurons in the cat cerebral white matter were detected using histochemistry for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity. One type was bipolar in shape with two cell processes extending in opposite directions, existed mainly in the subcortical areas and was oriented parallel to fiber bundles. The second type had 4 or 5 very long, prominent and varicose cell processes radiating in various directions. They were round or polygonal in shape and formed networks in the white matter of the frontoparietal area. NADPH-diaphorase-positive neurons were also examined by the modified Golgi-Cox silver impregnation method. With this impregnation method, the same two morphological types could be detected but the detailed morphology of these particular populations of neurons was revealed much more fully by NADPH-diaphorase enzyme histochemistry than by the silver impregnation method.

Ultrastructure of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive neurons in the cat cerebral cortex, amygdala and caudate nucleus.

The ultrastructure of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive neurons in cat cerebral cortex, amygdala and caudate nucleus was investigated by electron microscopy using a modified method applicable to aldehyde-fixed tissues. These NADPH diaphorase-positive neurons were morphologically similar to neurons immunohistochemically positive for somatostatin. They had large amounts of electron-dense formazan reaction products scattered through the whole cytoplasm but not in the mitochondria or nucleus. Similar electron-dense reaction products were visible in the dendrites of these neurons. The results indicate that NADPH diaphorase histochemistry is a useful method for the ultrastructural examination of particular groups of neurons.

The distribution of somatostatin-immunoreactive neurons and fibers in the rat cerebral cortex: light and electron microscopic studies.

The distribution of somatostatin (SOM)-immunoreactive neurons in rat motor and somatosensory cortices was studied by immunohistochemistry at both light and electron microscopic levels. Three types of SOM-positive neurons were described. Type A cells are large to medium in size, multipolar in shape and have 3-5 long primary cell processes. Type B cells are medium to small, fusiform in shape and have two primary processes. These two subtypes show abundant subcellular organelles and are heavily stained throughout their cytoplasm. Type C cells are small, ovoid or fusiform in shape and are lightly stained. These previously undescribed cells are the largest SOM-immunoreactive population in the cortex. Ultrastructurally they have few subcellular organelles and only a patchy immunostaining in the cytoplasm. SOM-immunoreactive neurons occur in all cortical layers except I, and are most numerous in layer V of the somatosensory and motor cortex. In SOM-positive dendrites, electron-dense immunoreactive/peroxidase end product is primarily associated with microtubules. Fine deposits also occur deep to the postsynaptic membrane asymmetric synapses making contact with such dendrites.

Alterations of muscarinic cholinergic receptors in the hippocampal formation of stressed rat: in vitro quantitative autoradiographic analysis.

Alterations in muscarinic cholinergic (mACh) binding sites in the hippocampal formation of rats after immobilization stress (IM-stress) lasting 0.5 or 3 h were quantitated using a computer-assisted image analysis system on autoradiograms following labeling of frozen sections with [3H]quinuclidinyl benzilate (QNB). The concentration of acetylcholine (ACh) in the rat hippocampus after IM-stress was also measured and showed no significant change compared to control. IM-stress for 0.5 h produced significant increases in the concentration of mACh binding sites in the whole hippocampal formation and in two subdivisions studied (CA1 plus CA2 and dentate gyrus); after 3 h of stress, the increase remained significant only in the dentate gyrus. These findings suggest that IM-stress induce a hypersensitivity of the septo-hippocampal cholinergic system and the 'up-regulation' of postsynaptic mACh receptors is more lasting in the dentate gyrus than in the hippocampus as a whole.

Post-ischemic administration of bifemelane hydrochloride prohibits ischemia-induced depletion of the muscarinic M1-receptor and its mRNA in the gerbil hippocampus.

Parallel determinations of muscarinic cholinergic M1 receptor (M1-R) binding and of M1-R mRNA levels were carried out in the gerbil hippocampus 14 days after 5 min of transient ischemia. Both were reduced in the ischemic tissue to about 50% of the levels found in sham-operated controls, indicating that the late loss of M1-R is probably dependent on decreased synthesis. Three administrations of bifemelane hydrochloride (15 mg/kg, i.p., just after ischemia and 6 and 12 h later) completely prevented neuronal death in the hippocampus and ischemia-induced losses of hippocampal M1-R and its mRNA. Since vascular dementia may depend upon the ischemia-induced losses in cholinergic communication in the hippocampus, these findings suggest that it may be possible to prevent its occurrence by post-ischemic treatment with bifemelane hydrochloride.

The cholinergic system of the human hindbrain studied by choline acetyltransferase immunohistochemistry and acetylcholinesterase histochemistry.

A map of cholinergic cells of the human brainstem identified by immunohistochemistry of choline acetyltransferase (ChAT) is presented, along with a map of acetylcholinesterase (AChE)-containing cells and fibers. ChAT-positive structures belong to 4 brainstem systems: the cranial motor nuclei; the parabrachial complex; the reticular system; and the vestibular system. All motor nuclei of the cranial nerves, as well as the nucleus supraspinalis, are ChAT-positive. The positively staining structures of the parabrachial system include the nucleus tegmentali pedunculopontinus, and the nuclei parabrachialis medialis and lateralis. Nuclei of the reticular system containing some ChAT-positive cells include the nucleus reticularis pontis oralis and caudalis, the nucleus reticularis tegmenti pontis, the nucleus reticularis gigantocellularis, the nucleus reticularis lateralis and the formatio reticularis centralis (medulla). Structures of the vestibular and auditory systems which contain some ChAT-positive cells include the nucleus vestibularis lateralis, and the nuclei olivaris superioris medialis and lateralis. All ChAT-positive structures stain strongly for AChE. AChE-positive, ChAT-negative structures were noted in several sensory systems. The substantia nigra, locus coeruleus and raphe nuclei, known to contain non-cholinergic cells, also stain positively. The significance of the AChE-positive, ChAT-negative staining in most structures remains to be determined. A knowledge of the cholinergic systems of human brain may be important to an understanding of the pathology of a number of diseases.

Quantitative electron microscopic analysis of postnatal development of zinc-positive nerve endings in the rat amygdala using Timm's sulphide silver technique.

The distribution and quantitation of zinc during postnatal development of the rat amygdala were investigated by light and electron microscopy with Timm's sulphide silver method. The adult rat amygdala could be divided into basolateral, basomedial, central, cortical, intercalated, lateral and medial subnuclei on the basis of Timm's plus Toluidine blue staining. Only a very weakly positive Timm's reaction could be observed in newborns, and a positive reaction was seen on the 5th postnatal day. The reaction became stronger with development, and reached adult levels by the 30th postnatal day. Electron microscopically, we investigated the basolateral subnucleus of the amygdala. The electron-dense deposits of silver grains from the Timm's reaction were only seen in nerve fibers endings containing many small clear vesicles. The results are similar to those described for mossy fiber endings in the rat hippocampus and in boutons of other telencephalic structures. The number of deposits of silver grains increased with age, and reached that of the adult between the 20th and 30th postnatal days. A significant increase in the number of silver grains per micron 2 area of the Timm's-positive nerve terminals occurred between the 10th and 20th postnatal days. Based on the available literature, these findings suggest that zinc exists in nerve fiber endings in the rat amygdala and that the amount increases with postnatal age. The findings are consistent with an important role for zinc in synaptic transmission.

Monoclonal antibody that binds to both the prenatal and postnatal astroglia in rodent cerebellum.

A monoclonal antibody (MAb), generated by immunizing mice with homogenized guinea pig cerebellum, labeled cerebellar astroglia including perikarya, radial fibers and veil-like processes in adult rats, mice and guinea pigs. Cell bodies and processes of the immature radial glia in the ventricular neuroepithelium of fetal mice cerebellum were definitely stained by the MAb on the 14th day of gestation. The astroglial components continued to show selective immunoreactivity to the MAb after the 14th day of gestation.