Carbonic Anhydrase, 5''-Nucleotidase, and 2'',3''-Cyclic Nucleotide-3''-Phosphodiesterase Activities in Oligodendrocytes, Astrocytes, and Neurons Isolated from the Brains of Developing Rats

The activities of three myelin-associated enzymes, carbonic anhydrase, 5'-nucleotidase, and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP), were measured in oligodendrocytes, neurons, and astrocytes isolated from the brain of rats 10, 20, 60, and 120 days old. The carbonic anhydrase specific activity in oligodendrocytes was three- to fivefold higher than that in brain homogenates at each age, and, at all the ages, low activities of this enzyme were measured in neurons and astrocytes. The oligodendrocytes and astrocytes from the brains of rats at all ages had higher activities of the membrane-bound enzyme 5'-nucleotidase than was observed in neurons. In oligodendrocytes from 10- and 20-day-old rats, the 5'-nucleotidase activity was two-to threefold the activity in the homogenates (i.e., relative specific activity = 2.0-3.0), and the relative specific activity of this enzyme in the oligodendrocytes declined to less than 1.0 at the later ages, concomitant with the accumulation of 5'-nucleotidase in myelin. The CNP activity was always higher in oligodendrocytes than in neurons, but not appreciably different from that in astrocytes from 20 days of age onward. The relative specific activity of CNP was highest in the oligodendrocytes from 10-day-old rats but was lower, at all ages, than we had observed in bovine oligodendrocytes. These enzyme activities in oligodendroglia are quite different in amount and developmental pattern from those reported previously for myelin.     

Alterations of Glial Cells in the Mouse Hippocampus During Postnatal Development

We investigated the postnatal alterations of neurons, astrocyte, oligodendrocyte, and microglia in the mouse hippocampal CA1 sector and dentate gyrus under the same conditions using immunohistochemistry. Neuronal nuclei (NeuN), Glial fibrillary acidic protein (GFAP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), and ionized calcium binding adaptor molecule 1 (Iba 1) immunoreactivity were measured in 1-, 2-, 4-, and 8-week-old mice. Total number of NeuN-positive neurons was unchanged in the mouse hippocampal CA1 sector and dentate gyrus from 1 to 8 weeks of birth. In contrast, a significant increase in the number of GFAP-positive astrocytes was observed only in the hippocampal CA1 sector of 1-week-old mice when compared with 8-week-old animals. Thereafter, total number of GFAP-positive astrocytes was unchanged in the hippocampal CA1 sector and dentate gyrus from 2 to 8 weeks of birth. For microglia, a significant increase in the number of Iba 1-positive microglia was observed in the hippocampal CA1 sector and dentate gyrus of 1-, 2-, and 4-week-old mice as compared with 8-week-old animals. On the other hand, a significant decrease in the area of expression of CNPase-positive fibers was observed in the hippocampal CA1 sector of 1- and 2-week-old mice as compared with 8-week-old animals. In dentate gyrus, a significant decrease in the area of expression of CNPase-positive fibers was found in 1-, 2-, and 4-week-old mice. Furthermore, our double-labeled immunostaining showed that brain-derived neurotrophic factor (BDNF) immunoreactivity was observed in GFAP-positive astrocytes and Iba 1-positive microglia in the hippocampal CA1 sector and dentate gyrus of 1- and 2-week-old mice. These results show that glial cells may play some role in the maintenance and neuronal functions of hippocampal CA1 pyramidal neurons and granule cells of dentate gyrus during postnatal development. Furthermore, our results demonstrate that glial BDNF may play an important role in the maturation of oligodendrocyte in the hippocampal CA1 sector and dentate gyrus during postnatal development. Thus, our findings provide valuable information on the developmental processes.     

Changes in expression of angiotensin receptor subtypes in the rat aorta during development

Quantitative autoradiography was used to characterize angiotensin AT1 and AT2 receptors, in the rat aorta at three developmental ages; embryonic day 18 (E18), and postnatal weeks 2 and 8. The expression of angiotensin receptors was higher in the aorta of E18 and 2-week-old rat. A major proportion of the angiotensin receptors expressed in the aorta at these two ages was AT2 (84 and 81% respectively). Conversely, in the aorta of 8-week-old rats, AT1 was the predominant angiotensin receptor subtype (71%). In 8-week-old rats, the AT2 subtype was also present (28%). In pre- and postnatal rats, [125I]Sar1-angiotensin II binding to AT1 receptors was sensitive to GTP gamma S whereas binding to AT2 receptors was not. AT2 receptors may serve an important role during stages of rapid growth of the aorta, and also have a significant function in the adult vasculature.     

Reduced Hippocampal Cell Differentiation in the Subgranular Zone of the Dentate Gyrus in a Rat Model of Type II Diabetes

It has recently been reported that diabetes mellitus is strongly associated with neurodegenerative and functional disorders of the central nervous system. In the present study, we investigated the changes in proliferating neurons in the dentate gyrus of type II diabetic rats using doublecortin (DCX), a marker of progenitors differentiating into neurons. At 4 weeks after birth, there were no differences in the blood glucose levels of Zucker diabetic fatty (ZDF) rats or Zucker lean control (ZLC) rats. DCX-immunoreactive neurons were detectable in the subgranular zone of the dentate gyrus in both the ZDF and ZLC rats; however, DCX immunoreactivity was higher in the ZLC rats than in the ZDF rats. At 12 weeks after birth, the blood glucose level was significantly increased by 400 mg/dl in the ZDF rats, but the blood glucose level in the ZLC rats was only slightly increased by 152.3 mg/dl. DCX immunoreactivity was significantly decreased in 12-week-old rats in comparison to 4-week-old rats. Some DCX-immunoreactive neurons were detectable in the subgranular zone of the dentate gyrus in the ZLC rats. However, only a few DCX-immunoreactive neurons were observed in the ZDF rats, and the DCX-immunoreactive neurons in the ZDF rats did not show fully developed processes. These results suggest that DCX-immunoreactive neurons were significantly decreased in an age-dependent manner and that DCX-immunoreactive neurons were also reduced in diabetic rats. In addition, the reduction in DCX-immunoreactive neurons in age matched rats may be associated with type II diabetes.     

Expression of p97/VCP and ubiquitin during postnatal development of the degenerating rat retina

In this study, we aimed to investigate the distribution pattern of ubiquitin and p97/VCP in the rat retina during postnatal development. Eyeballs from 1-, 4-, 10-, 36- and 72-week-old rats were examined by immunohistochemistry, and protein colocalization was determined by immunofluorescence microscopy. In the 1-week-old rat retina, p97/VCP was strongly expressed in the neuroblast layer, however no ubiquitin immunoreactivity was observed. p97/VCP immunoreactivity was present in the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment (IS) of the photoreceptor layer, and retinal pigment epithelium in the 4- and 10-week-old rat retinas. p97/VCP immunoreactivity increased significantly in the 10-week-old rat retinas. Ubiquitin was barely seen in the 4-week-old rat retinas, and ubiquitin expression was weak in the GCL and the IPL of the 10-week-old rat retinas. In the 36- and 72-week-old rats, the presence of ubiquitin was remarkable in the IS, INL, IPL and GCL, however, p97/VCP immunoreactivity was significantly decreased. Colocalization of ubiquitin and p97/VCP was also observed in the INL, IS, GCL and ONL of 36- and 72-week-old rat retinas. Our results indicate that p97/VCP immunoreactivity in the retina significantly decreases after rats reach 10 weeks of age, whereas ubiquitin immunoreactivity increases with aging. These results suggest that an altered expression pattern of p97/VCP and ubiquitin in the developing rat retina may associate with age-related retinal degeneration.     

Changes in mechanism of PACAP-induced relaxation in longitudinal muscle of the distal colon of Wistar rats with age

Mechanisms of relaxation of longitudinal muscle of the distal colon induced by exogenously added pituitary adenylate cyclase activating peptide (PACAP) were studied in 2- to 30-week-old Wistar rats. Exogenous PACAP induced very significant relaxation of the longitudinal muscle in 2-week-old rats, but this effect decreased significantly with age. The cyclic AMP-cyclic AMP-dependent protein kinase (PKA) pathway and the tyrosine kinase-small conductance Ca2+-activated K+ channel (SK channel) pathway were found to be involved in the mechanism of PACAP-induced relaxation. In 2-week-old rats, PACAP-induced relaxation was significantly inhibited by tetrodotoxin (TTX). Since relaxation was also significantly inhibited by NG-nitro-L-arginine (N5-nitro-amidino-L-2,5-diamino-pentanoic acid: L-NOARG), the neurogenic effect of PACAP seems to be mediated mainly through nitric oxide neurons. In 8-week-old rats, L-NOARG and TTX had little effect on PACAP-induced relaxation, suggesting that the relaxant effect in 8-week-old rats is a direct action on longitudinal smooth muscle cells. Changes in the mechanisms of PACAP-induced relaxation with age were examined in the distal colon in relation to changes in the neurogenic and the direct effects of PACAP. The neurogenic effect in the exogenous PACAP-induced relaxation of the longitudinal muscle of the Wistar rat distal colon is dominant in tissue isolated from 2-week-old and lost in tissue isolated from 8-week-old rats.     

Ageing transition of superoxide dismutase in rat retina

To examine the relationship between retinal ageing and superoxide dismutase, the distribution and expression of the dismutase was studied in the retina of 2-year-old Sprague--Dawley albino rats with immunohistochemistry and immunochemical quantitative analysis. Eight-week-old Sprague--Dawley albino rats were used as controls. In 2-year-old rats, manganese superoxide dismutase (Mn-SOD) immunoreactivities in the photoreceptor inner segments, the outer nuclear layer and the inner plexiform layer were stronger than those in 8-week-old rats. Copper--zinc superoxide dismutase (CuZn-SOD) immunoreactivities in the outer nuclear layer and inner plexiform layer of 2-year-old rats were stronger than those in 8-week-old rats. Faint CuZn-SOD immunoreactivity became visible in the photoreceptor inner segments of 2-year-old rats, whereas no CuZn-SOD immunoreactivity was observed in 8-week-old rats. Our immunochemical quantitative analysis also showed an increase in the immunoreactivities of superoxide dismutases in the sensory retina with age. The transition of the dismutases may have some relationship with retinal ageing. © 1998 Chapman & Hall     

Neuron specific enolase: a marker for the early development of nerves and endocrine cells in the human lung

Neuron specific enolase (NSE), an isoenzyme of the glycolytic enzyme enolase, has been established by immunocytochemical means as a marker of morphological and functional maturation in central neurons and appears late in development. However, little is known about the presence of NSE in developing peripheral neurons and endocrine cells and its relationship to the development of classical neurotransmitters and peptides. We therefore investigated the appearance of NSE immunoreactivity in nerves and mucosal endocrine cells of the human respiratory tract in foetal, neonatal and adult life. NSE was found to be present in neuroblasts, nerve fibres and endocrine cells from the earliest period of gestation examined (8 weeks), before the appearance of acetylcholinesterase activity (10-12 weeks), dopamine-beta-hydroxylase (20 weeks), vasoactive intestinal polypeptide (20 weeks) or calcitonin (20 weeks). Bombesin-like immunoreactivity was found in a small proportion of mucosal endocrine cells as early as eight weeks in the foetal respiratory tract. These findings indicate that unlike central neurons and their processes, peripheral neurons of the lung contain NSE immunoreactivity well before full maturation and establishment of synaptic contact with end organs.     

Glial-cell-line-derived neurotrophic factor enhances biosynthesis of substance P in striatal neurons in vitro

Glial-cell-line-derived neurotrophic factor (GDNF) is a novel trophic factor with potent trophic effects on several neuron populations in the central and peripheral nervous system. In the present study, we have investigated and compared the potential of dopamine and metamphetamine with that of the two striatal neurotrophic factors, viz., GDNF and neurotrophin-(NT)-4/5, to regulate substance P and its preprotachykinin-A mRNA in organotypic striatal slices from postnatal (day 10) rats. Incubation for 2 weeks with 10 ng/ml GDNF significantly increased substance-P-like immunoreactivity determined by radioimmunoassay. Similarly, the corresponding preprotachykinin-A mRNA increased after 1 and 2 weeks of incubation, as analyzed by in situ hybridization. NT-4/5 exhibited similar effects.The dopamine-releasing agent metamphetamine stimulated substance-P-containing neurons in 1-week-old striatal slices, whereas dopamine stimulated substance-P-like immunoreactivity in 1- and 2-week old striatal cultures. The effects of dopamine and GDNF were not additive. We conclude that substance-P-containing medium-sized spiny neurons in the striatum are under both dopaminergic and growth factor control by GDNF and NT-4/5, which are both synthesized in the striatum. This adds a previously unknown role to those that have been established for GDNF in the nigrostriatal system. Received: 9 March 1996 / Accepted: 14 June 1996     

Intrinsic neurons in the rat ovary: an immunohistochemical study

Previous studies have shown the presence of neuronal perikarya in the primate ovary, but not in the ovary from Sprague-Dawley rats. We report here that while such intrinsic neurons are indeed absent in this strain of rats, they can be visualized in the ovary from Wistar rats. The neurons, identified by their morphology and by the expression of NeuN (a neuron-specific nuclear protein), were detected at all postnatal intervals examined, from 14 h after birth to 50 days of age. While they were present in the ovarian hilum and medulla at all ages studied, neurons first appeared in the ovarian cortex during the juvenile period (postnatal days 10-20). In all cases, the size of the neuronal soma increased significantly during prepubertal development, reaching maximal values before puberty. Some neurons were catecholaminergic, as indicated by their content of immunoreactive tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. Some showed neuropeptide Y (NPY) immunoreactivity. TH-positive neurons were seen either in isolation or clustered in ganglion-like structures in both the ovarian cortex and medulla. These results indicate that ovarian neurons are not present in all strains of rats, but when present, the chemical phenotype of some of them is of a sympathetic nature, similar to that described in primates.     

Regulation of NAD- and NADP-linked isocitrate dehydrogenase by hydrocortisone in the brain and liver of male rats of various ages

The activity and hormonal regulation of NAD- and NADP-linked isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42, respectively) in the brain and liver of rats of various ages were investigated. The activity of NAD-linked isocitrate dehydrogenase of the brain was greater than cytoplasmic or mitochondrial NADP-linked isocitrate dehydrogenase. In contrast, the cytoplasmic NADP-isocitrate dehydrogenase of the liver predominates over both NAD- and mitochondrial NADP-isocitrate dehydrogenases at the three ages studied. The activity of NAD-isocitrate dehydrogenase increased in the brain (139%) and liver (17%) of rats upt o 33 weeks of age and decreased (57 and 39%, respectively) in old rats (85-week-old). The activity of cytoplasmic NADP-isocitrate dehydrogenase was maximum in immature (6-week-old) rat brain and decreased as the age of the rats increased; whereas, in liver, the activity of this enzyme was found to be maximum in adult rats (33-week-old). Brain mitochondrial NADP-isocitrate dehydrogenase activity increased (64%) in adult rats, but in liver it decreased (45 and 33% in 33- and 85-week-old rats, respectively). In both tissues, adrenalectomy and hydrocortisone treatment showed differential age-dependent response. Hydrocortisone-mediated induction of the level of enzymes was inhibited by actinomycin D.     

Glycerol Phosphate Dehydrogenase, Glucose-6-Phosphate Dehydrogenase, and Lactate Dehydrogenase: Activities in Oligodendrocytes, Neurons, Astrocytes, and Myelin Isolated from Developing Rat Brains

Abstract: Glycerol phosphate dehydrogenase (GPDH), glucose-6-phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH) activities were determined in Oligodendrocytes, neurons, and astrocytes isolated from the brains of developing rats. The activity of each enzyme was significantly lower in both neurons and astrocytes than in Oligodendrocytes. The GPDH activity in Oligodendrocytes increased more than 4-fold during development, and at 120 days cells of this type had 1.4-fold the specific activity of forebrain homogenates. The G6PDH activities in Oligodendrocytes from 10-day-old rats were 1.4-fold the activities in the forebrain homogenates. The activities of this enzyme in Oligodendrocytes were progressively lower at later ages, such that at 120 days the cells had 0.8 times the specific activities of homogenates. The Oligodendrocytes had 0.6 times the homogenate activities of LDH at 10 days, and this ratio had decreased to 0.2 by 120 days. These enzymes were also measured in myelin isolated from 20-, 60-, and 120-day-old rats. By 120 days the specific activities of G6PDH and LDH in myelin were <8% of the respective activities in homogenates. The GPDH activity in myelin was, however, at least 20% the specific activity in the homogenates, even in the oldest animals. It is proposed that LDH could be used as a marker for oligodendroglial cytoplasm in subfractions of myelin and in myelin-related membrane vesicles.     

Real-time analysis of the interaction of a multiple-epitope peptide with antibodies against classical swine fever virus using surface plasmon resonance

The E2 envelope glycoprotein is the major immunodominant protein of classical swine fever virus (CSFV), and can induce neutralizing antibodies and protective immune responses in infected swine. We developed a tandem-repeat multiple-epitope recombinant protein that contains two copies of each of the regions of E2 spanned by residues 693-704, 770-780, and 826-843, coupled by two copies of the region spanned by residues 1446-1460 of the CSFV nonstructural protein NS2-3. The chemically synthesized gene was expressed in Escherichia coli as a fusion with glutathione S-8 (GST), named GST-BT21. After it was purified with Glutathione Sepharose 4B, we used Western blotting to characterize the construct and surface plasmon resonance to analyze its affinity and specific interaction with CSFV-positive serum. Purified GST-BT21 protein displayed excellent immunoreactivity with antiserum against CSFV (Tian et al., 2012), and surface plasmon resonance confirmed the specific affinity between BT21, but not GST, and antibodies in serum from animals infected with CSFV. Surface plasmon resonance is a sensitive and precise method for epitope evaluation, and it can be used to characterize the immunogenicity and functions of recombinant proteins.     

Comparison of serotonin and 5-methoxytryptamine immunoreactivity in rat raphe nuclei

We studied the immunoreactivity of 5-methoxytryptamine (MT) and 5-hydroxytryptamine (HT) in the raphe region of rats using specific polyclonal antibodies and the peroxidase/anti-peroxidase (PAP) technique. Overall, the patterns of the specific staining for these two antibodies were found to be the same in this region of the rat brain. The staining reaction was considerably less intense for MT than for HT. Specificity tests were performed using HT, MT and tryptamine (T) conjugates at concentrations of 5 X 10(-8) M for antibodies to HT and 2.5 X 10(-9) M for antibodies to MT. Although the distribution of HT-like and MT-like immunoreactivity broadly overlapped, the results obtained from adsorption-specificity tests confirmed the presence of specific MT staining in the rat raphe.     

The early post-natal development of the neuronal lysosome

Abstract— The hydrolysis of p-nitrophenyl-2-acetamido-2-deoxy-β-d -gluco-(I) and β-d -galacto-pyranoside (II) and of p-nitrophenyl-α-d -mannopyranoside (III) by neuronal cell bodies and glial cells isolated from the cerebral cortex of 18-day-old or adult rats was found to be equally efficient, with relative ratios of hydrolysis for I, II and III of approximately 10:1:0.5 in both cell types and at both ages. Homogenates of the neuronal cell bodies obtained from cerebral cortices of 3-, 8-, 12-, 18- and 32-day-old rats were subjected to differential centrifugation and the subcellular localization of N-acetyl-β-d -glucosaminidase (EC 3.2.1.30) hydrolysing (I)] was compared to that of the mitochondrial marker, succinate-INT- oxidoreductase (EC 1.3.99.1). A fraction in which N-acetyl-β-d -glucosaminidase exhibited maximal specific activity could be isolated at all ages, an observation indicating that the potential for active hydrolytic performance is incorporated into the neuronal lysosome very early post-natally. The specific activities of N-acetyl-β-d -glucosaminidase and succinate- INT-oxidoreductase reached their respective maxima at widely different times postnatally: at 10–12 days for the mitochondrial enzyme and at about 18 days for the glycosidase, a difference suggesting that in the cortical neuron lysosomes and mitochondria develop out of step. The mitochondrial, lysosomal and microsomal fractions obtained by differential centrifugation were subjected to equilibrium density centrifugation and the presence of two populations of N-acetyl-β-d -glucosaminidase-bearing particles was demonstrated. Although their presence was readily apparent in the neurons from 8- and 12-day old brains, it was difficult to discern their presence in the neurons from the 3- and the 18-day-old brains. In 8-day-old brains gradient fractions obtained from neurons containing N-acetyl-β-d -glucosaminidase of a specific activity up to 8-fold higher than that of the enzyme in the original neuronal homogenate were examined by electron microscopy and the concentration of numerous lysosomes and derivative bodies in these fractions was verified. Our present study demonstrates the capability of the immature and developing neuron to tightly couple the pace of its degradative processes to that of its highly efficient and highly selective synthetic activities.     

Catecholaminergic, cholinergic and peptidergic innervation of gut-associated lymphoid tissue in porcine jejunum and ileum

With its abundance of neurons and immunocytes, the gut is a potentially important site for the study of the interaction between the nervous and immune systems. Using immunohistochemical techniques, we tested the hypothesis that gut-associated lymphoid tissue in the porcine small intestine might receive catecholaminergic, cholinergic and peptidergic innervation. Antibodies against protein gene product (PGP) 9.5 were employed to detect neuronal membranes; antibodies against tyrosine hydroxylase (TH), type 2 vesicular monoamine transporter (VMAT-2) and choline acetyltransferase (ChAT) were used to detect catecholaminergic and cholinergic neurons; and antibodies to neuromedin U-8 (NMU-8), substance P (SP) and vasoactive intestinal peptide (VIP) were also used. PGP9.5-immunoreactive nerve fibers were observed between jejunal Peyer's patch (PP) follicles and in submucosal ganglia localized at the base of continuous ileal PP. Many ChAT-positive and a few TH-/VMAT-2-immunoreactive neurons or axons adjacent to jejunal and ileal PP were observed. Neurons and fibers from ganglia situated between or at the base of PP follicles manifested robust immunoreactivities to VIP and NMU-8; relatively less SP immunoreactivity was observed at these locations. All neuromedin-U 8-positive neurons observed exhibited immunoreactivity to ChAT as did some VIP-positive neurons. The specific chemical coding of enteric neurons in close apposition to jejunal and ileal PP and the differential localization of neuropeptides within the jejunal and ileal PP are indicative of neuroimmunomodulation at these sites.     

Presence of catecholamine-related enzymes in a subpopulation of primary sensory neurons in dorsal root ganglia of the rat.

The presence of enzymes (tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (D beta H)) and enzymatic activities (monoamino oxidase, (MAO)) related to catecholamine synthesis and degradation have been investigated in cervical, thoracic and lumbar dorsal root ganglia (DRG) of adult male rats using immunohistochemical and enzyme histochemical techniques, respectively. A small population (between 2-4%) of TH-like immunoreactive and MAO positive neurons was found. They were small in diameter (18 +/- 2 microns), predominate in lumbar DRG and did not display D beta H-like immunoreactivity. These sensory neurons which are likely catecholaminergic were insensitive to systemic administration of capsaicin and 6-hydroxydopamine. Colchicine administration caused an increase of TH-like immunoreactivity and MAO activity. Pargyline produced an increase in TH-like immunoreactivity and the disappearance of MAO activity. The possible dopaminergic nature of the subpopulation of DRG sensory neurons investigated in the present study is discussed.     

Dynamic of the GRF-induced GH response in genetically obese Zucker rats: influence of central and peripheral factors

To determine the time onset of the growth hormone (GH) alteration in the genetically obese rat, we studied the in vivo and in vitro rat growth hormone releasing factor (rGRF(1-29)NH2)-induced GH secretion in 6- and 8-week-old lean and obese male Zucker rats. Under sodium pentobarbital anesthesia, rGRF(1-29)NH2 (GRF) was injected intravenously at two doses: 0.8 and 4.0 micrograms/kg b.w. Basal serum GH concentrations were similar in lean and obese age-matched animals. The GH response to both GRF doses tested was unchanged in 6-week-old obese rats as compared to their lean litter mates. In contrast, a significant decrease of the GH secretion in response to 4.0 micrograms/kg b.w. GRF was observed in the 8-week-old obese rats. The effect of GRF (1.56, 6.25 and 12.5 pM) was further studied in vitro, in a perifusion system of freshly dispersed anterior pituitary cells of lean and obese Zucker rats. Basal GH release was similar in the 6-week-old animal group. In contrast, it was significantly decreased in 8-week-old obese rats as compared to their lean litter mates. Stimulated GH response to 1.56 and 6.25 pM GRF was significantly greater in the 6-week-old obese group than in the age-matched control group. In contrast, the GH response to all GRF concentrations tested was significantly decreased in the 8-week-old obese rats as compared to their respective lean siblings. In 8-week-old obese rats, a decrease of GH pituitary content and an increase of hypothalamic somatostatin (SRIF) concentration were observed. Insulin and free fatty acid serum were significantly increased in 8-week-old obese rats. In contrast, lower insulin-like growth factor I serum levels were observed in the obese animals as compared to their lean litter mates. Finally, to further clarify the role of the periphery in the inhibition of GH secretion observed in the 8-week-old fatty rats, we exposed cultured pituitary cells of 8-week-old lean animals to 17% serum of their obese litter mates. A significant decrease of GRF-stimulated GH secretion of lean rat pituitary cells exposed to the obese serum was noted (P less than 0.05). This study demonstrates that, in the obese Zucker rat, an alteration of the GH response to GRF is evident by the 8th week of life. This defective GH secretion could be related to peripheral and central abnormalities.     

Mast cells contain spleen-type prostaglandin D synthetase

Prostaglandin D synthetase activity in the cytosol (100,000 x g, 1-h supernatant) fraction of peritoneal mast cells of adult rats (105.0 nmol/min/mg protein) was the highest among such activities in various rat tissues and cells. As judged by the absolute requirement for glutathione for the reaction (Km = 300 microM), the Km value for prostaglandin H2 (200 microM), and insensitivity of the activity to 1 mM 1-chloro-2,4-dinitrobenzene, the enzyme in mast cells was similar to rat spleen prostaglandin D synthetase and differed from rat brain prostaglandin D synthetase or glutathione S-transferase, all of which catalyze the isomerase reaction from prostaglandin H2 to prostaglandin D2. In immunotitration analyses, the activity in mast cells showed a titration curve exactly identical with that of the purified spleen-type enzyme and almost completely absorbed by an excess amount of antibody against this enzyme, but it remained unchanged after incubation with antibodies against the brain-type enzyme and glutathione S-transferase isozymes thus far purified. In Western blot after two-dimensional electrophoresis of crude extracts of mast cells, a single immunoreactive spot was observed with antibody against the spleen-type enzyme at the same position as that of the purified enzyme (Mr = 26,000, pI = 5.2). Furthermore, the immunoreactive protein obtained from mast cells showed the same peptide fingerprints as those of the purified spleen-type enzyme, after partial digestion with Staphylococcus aureus V8 protease or trypsin. In immunoperoxidase staining, the immunoreactivity of the spleen-type enzyme was found in the cytosol of tissue mast cells in various organs such as thymus, intestine, stomach, and skin of adult rats. These findings indicate that prostaglandin D2 is produced by the spleen-type synthetase in mast cells of various tissues.