Developmental Patterns of Catechol-O-Methyltransferase in Genetically Different Rat Strains: Enzymatic and Immunochemical Studies

Abstract: Catechol- O -methyltransferase (COMT) activity in the liver and kidneys of adult Fischer-344 (F-344) rats is only half of that in the same organs of Wistar-Furth (W-F) rats. The trait of low COMT activity in these animals is inherited in an autosomal recessive fashion. A comprehensive study of patterns of change in COMT activity during growth and development was performed to determine whether "temporal gene" effects might play a role in the inherited differences in enzyme activity present in adult animals. The COMT activity expressed per mg protein in liver and kidneys of newborn F-344 rats is only 50–60% of that in the same organs of W-F animals. The liver and the kidneys of newborn rats of both strains have COMT activity an order of magnitude higher than those in brain, heart, or blood. In addition, in both strains there are much larger increases in liver and kidney COMT activities during growth and development (5–10 fold) than in blood, brain, or heart (one- to twofold). Immunotitration with antibodies against rat COMT demonstrates that differences in immunoreactive COMT parallel differences in COMT activity, both between strains and within strains during growth and development. However, when the temporal patterns of change in enzyme activities in the liver and the kidneys of the two strains of rat are compared at multiple times during growth and development, no differences in the patterns are present. These results make it unlikely that temporal gene effects can explain the inherited differences in COMT activity in liver and kidneys of F-344 and W-F rats.     

Behavioral and physiological effects of chronic 2,450-MHz microwave irradiation of the rat at 0.5 mW/cm2

Adult male Long-Evans rats were intermittently exposed to 2450 MHz CW microwaves at an average power density of 0.5 mW/cm2 for 90 days. The resulting SAR was 0.14 W/kg (range 0.11 to 0.18 W/kg). The animals were exposed 7 h/day, 7 days/wk, for a total of 630 h in a monopole-above-ground radiation chamber while housed in Plexiglas holding cages. Daily measures of body mass and food and water intake indicated no statistically significant effects of microwave exposure. Monthly assessment of reactivity to electric footshock, levels of cholinesterase and sulfhydryl groups in blood, and 17-ketosteroids in urine revealed no reliable differences between 14 sham-exposed and 14 microwave-exposed rats. After the 90 days of exposure, seven rats, randomly chosen from each group, were assessed for open-field behavior, shuttlebox performance, and schedule-controlled (IRT schedule) lever pressing for food pellets. Statistically significant differences between microwave-exposed and sham-exposed rats were observed in shuttlebox performances and lever pressing. Post mortem measures of mass of several organs and microscopic examination of adrenal tissue revealed no differences between the two groups of animals.     

Nucleosomal organization of a BPV minichromosome containing a human H4 histone gene

Summary When the body temperature of rats is elevated to 42°C, four heat shock proteins, with the molecular weights of 70000, 71000, 85000, and 100000 (hsp 70, hsp 71, hsp 85, and hsp 100, respectively), are induced in various tissues of rats (Fujio et al., J Biochem 101, 181–187, 1987). Heat shock proteins are induced by various stresses other than heat in varieties of cultured cells, so we studied whether heat shock proteins are induced in intact rats by different treatments. Analysis of the translation products of poly(A) + RNA isolated from the livers of rats recovering from ischemia of the liver showed that mRNAs for hsp 70, hsp 71, and hsp 85 were induced. These hsp-mRNAs were also induced in the livers of rats 6 h after a partial hepatectomy, and had returned to control levels 24 h after the surgery. These results suggested that heat shock proteins have not only the function of protection against various stresses but also physiological functions in the normal growth and development of animals.     

A morphometric and ultrastructural study of lithium-induced changes in the medullary collecting ducts of the rat kidney

Summary Rats were given a lithium-containing diet (40 mmol/kg) to Study the effect of lithium on the structure of collecting ducts from the inner stripe of the outer medulla. The results show that there is a significant increase in the volume density of collecting ducts already after one week on this diet. The volume density of both intercalated and principal cells increases, whereas the volume density of mitochondria in the cytoplasm increases in the intercalated cells only. The increased volume of both principal and intercalated cells seems to be part of a general hyperplasia and hyperactivity of the collecting duct, which may in some way be related to the effects of lithium on vasopressinmediated water transport. The specific changes in the intercalated cells may be a consequence of the effects of lithium on distal nephron potassium and hydrogen ion transport in the distal nephron.     

Catecholaminergic innervation of GRF-containing neurons in the rat hypothalamus revealed by electron-microscopic cytochemistry

Summary The Catecholaminergic innervation of neurons containing growth hormone-releasing factor (GRF) was examined by use of a method which combined either 5-hydroxydopamine (5-OHDA) uptake or autoradiography after intraventricular injection of ³H-noradrenaline with immunocytochemistry for GRF in the same tissue sections at the electron-microscopic level. In the ventrolateral part of the arcuate nucleus of the rat hypothalamus a large number of immunonegative axon terminals were found to make synaptic contact with GRF-like immunoreactive (GRF-LI) cell bodies and processes. ³H-noradrenaline autoradiography or 5-OHDA-labeling combined with GRF immunocytochemistry revealed that axon terminals labeled with ³H-noradrenaline or 5-OHDA make synaptic contact with the GRF-LI nerve cell bodies and processes. These findings indicate that catecholamine-containing neurons innervate GRF neurons to regulate GRF secretion via synapses in the rat arcuate nucleus.     

Characterization of Merkel cells and mechanosensory axons of the rat by styryl pyridinium dyes

Summary The epidermal Merkel cells and their sensory innervation serve tactile sensation in vertebrates. In this study the fluorescent cationic mitochondrial dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), which has recently been used as a vital stain for motor and autonomic nerve terminals, was tested for its ability to stain Merkel cells and sensory fibers in the snout of the rat. Brightly-fluorescent structures resembling Merkel cells as well as nerve fibers and their terminations were evident in whole mounts of the vibrissal follicle. Unilateral denervation of the vibrissal follicles soon after birth resulted in a staining pattern remarkably similar to that obtained after labelling of the Merkel cells selectively with the fluorescent marker quinacrine, but all fiber staining was abolished. Likewise, in the separated epidermis of other skin regions, including the hairy and glabrous skin of the nose, the staining pattern revealed by 4-Di-2-ASP was indistinguishable from that obtained by quinacrine fluorescence. These results indicate that certain styryl pyridinium dyes may be used as vital stains for epidermal Merkel cells as well as cutaneous mechanosensory axons.     

Twenty-four-hour variations in subcellular structures of rat type II alveolar epithelial cells

Summary Subcellular structures of type II alveolar epithelial cells in the rat lung were analyzed at six evenly spaced times over 24 h (light period: 06.00 h–18.00 h), using a morphometric technique. The cell volumes were maximal at 16.00 h and minimal at 08.00 h. The volume and surface densities of rough endoplasmic reticulum and mitochondria were low during the light period, and high during the dark period. Morphometric parameters of multivesicular bodies did not significantly fluctuate over 24 h, but they increased from 04.00 h to 08.00 h. The volume densities of lamellar bodies increased from 16.00 h to 20.00 h, and decreased from 00.00 h to 08.00 h. The change in numerical densities of lamellar bodies was inversely correlated to that in the volume densities. As shown by electron microscopy, small lamellar bodies predominated at 08.00 h, larger lamellar bodies increasing at 16.00h. Composite bodies often appeared at 08.00 h and 12.00 h. Type II cells thus appear to fluctuate, showing three phases over 24 h: formation, accumulation and secretion of lamellar bodies. In particular, it is noteworthy that the accumulation stage occurs during the resting phase of the rat, whereas the secretion stage occurs during its body-active phase.     

Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry

Interest in the biological behavior of a growing number of elements, along with increasing recognition of the importance of interactions among them, demands a versatile and reliable technique for multielement analysis of biological samples. Significant improvements over the sensitivity achieved with conventional inductively coupled plasma (ICP) optical emission spectrometries have been realized with the introduction of quadrupole mass spectrometry (MS) for detection of ions in the plasma. The hybrid technique of ICP-MS promises to be a method of rapid multielement analysis, at detection limits that approach or surpass those of other technologies. However, the application of ICP-MS to analyses of biological interest is truly in its infancy. Here we report the use of ICP-MS for the determination of more than 30 elements of biological interest in a tissue and a biological fluid (rat liver and serum, respectively). Experimental values of the elements serve as a basis for discussion of analytical protocols, performance criteria, and certain problems peculiar to ICP-MS.     

Induction of hepatic drug metabolizing enzymes by DL-methionine in rats

A single intraperitoneal injection of DL-methionine (500 mg/kg body wt.) to adult male Wistar rats was shown to significantly induce all the components of the hepatic microsomal mixed function oxidase system such as NADPH cytochrome C reductase activity, cytochromes P-450 and b₅, as well as activities of drug metabolizing enzymes such as aminopyrine demethylase and uridine 5′ -diphosphate-glucuronosyltransferase. Combined administration of nicotinamide (250 mg/kg body wt.) and DL-methionine (500 mg/kg body wt.) was shown to bring about an additional increase (25-30%) in the activities of these enzymes as compared to their induction on independent administration of the two endobiotics. In rats bearing Yoshida sarcoma (ascites) tumour as well as in normal rats injected with serum from tumour bearing animals, the decreased activities of hepatic mixed function oxidases could be restored to their normal levels by administration of DL-methionine (500 mg/kg body wt.) to these rats. Whereas actinomycin D (1 mg/kg body wt.) had no effect on the increased incorporation of [¹⁴C] labelled leucine into microsomal proteins following administration of nicotinamide, the enhanced incorporation of the label following DL-methionine administration was completely inhibited by the same dose of actinomycin D. Administration of cycloheximide (0·5 mg/kg body wt.) to rats could completely inhibit the increased incorporation of [¹⁴C] leucine into hepatic microsomal proteins following independent administration of nicotinamide and DL-methionine. Similar inhibitory pattern with actinomycin D and cycloheximide was also demonstrated in case of induction of NADPH cytochromeC reductase activity by both these endobiotics.     

Effects of postnatal treatment with naloxone on plasma gonadotropin,prolactin, testosterone and testicular functions in male rats

Opioid peptides are implicated in the control of gonadotropin and prolactin secretion. The role of opioid antagonist naloxone and its effects on plasma gonadotropin, prolactin, testosterone levels and testicular hyaluronidase, acid phosphatase, [³H]uridine and thymidine incorporation, RNA, DNA and protein concentrations were evaluated in rats after administration of naloxone beginning day 1 through 21 and autopsied on 45, 60 and 90 days of age. Plasma gonadotropin and testosterone levels were significantly elevated after naloxone treatment. Testicular hyaluronidase and acid phosphatase activity increased till 60 days post treatment and declined thereafter. Concentrations of RNA and protein did not change significantly but the concentration of DNA declined at 45 and 60 days of age. These results suggest that endogenous opioid peptides exert regulatory influence on gonadotropin secretion which in turn control the testicular function in the male rat.