Plasticity of hippocampal corticosteroid receptors during aging in the rat

OBJECTIVE: The purpose of this study was to describe and validate an image-quality phantom to be used in dental radiography for comparison of film and digitally acquired images. STUDY DESIGN: An aluminum block of 12 steps, with 7 holes in each step, was covered by acrylic blocks. This phantom was radiographed with Kodak Ultra-speed and Ektaspeed Plus films at 70, 65, and 60 kVp with the whole exposure range available. All together, 50 dental films were randomly sequenced and presented to 7 observers. The average number of perceptible holes from all steps was plotted against exposure for each tube voltage and film type, generating a modified perceptibility curve. The tentative optimum exposure level was determined from perceptibility curves in each experimental condition and compared with that determined by means of the standard aluminum stepwedge and the preset time of the x-ray machine. The density range of this phantom at the optimum exposure was compared with that of clinical dental radiographs. Validity of the phantom was evaluated according to the optimum exposure level from the modified perceptibility curves and the overall density range. Finally, the average maximum numbers of perceptible holes at the tentative optimum exposure level were compared for each tube voltage and film type. The statistical test used was a 2-way factorial analysis of variance. RESULTS: The exposure at the perceptibility curve peak approximated that obtained by means of the standard aluminum step-wedge and the time preset by the manufacturer. The overall density range at the perceptibility curve peak covered the clinical density range for each tube voltage and film type. There were no statistically significant differences between film types or among tube voltages. CONCLUSIONS: The x-ray attenuation range for this phantom seemed to approximate clinical conditions. In addition, differences in image quality could be quantitatively evaluated by means of the number of the holes seen in the phantom.     

Elevation of ubiquinone content by peroxisomal inducers in rat liver during aging

The development of muscle afferents in the tammar wallaby was examined to address whether proprioceptive input contributes to the marked asymmetry of the fore and hindlimb movement. Anatomical tracing with biocytin showed that the muscle afferents had reached the brachial motor horn by postnatal day (P1), but were less advanced in the lumbar region. Labelled cells lying outside the motor horn, presumably filled via gap junctions, were evident in the neonatal lumbar cord. By the 4th postnatal week, the afferent innervation of both brachial and lumbar cords became similar. Afferent discharges from stretching the biceps muscle could be recorded at birth, but not until P4 from the hindlimb gastrocnemius muscle. The discharges were predominantly phasic until P35 when tonic activity could also be recorded. Short latency spinal reflex responses superimposed upon a longer lasting potential were present in the brachial cord at birth, appearing in the lumbar cord at P4. By the 3rd postnatal week, spinal reflex became comparable in both segmental levels. The time course of muscle afferent development was compared to the progression of natural cell death in the lumbar cord. Sensorimotor connections were established towards the end of the rapid phase cell death as observed in other vertebrates.     

Heterologous regulation of muscarinic and beta-adrenergic receptors in rat cardiomyocytes in culture

Previous work indicated that hyperstimulation of muscarinic receptors brings about profound changes not only in the density of the muscarinic receptors, but also of the beta-adrenoceptors in rat heart atria in vivo. We have now investigated whether a similar receptor cross-regulation occurs in cardiomyocytes in vitro. Cardiomyocytes from 3-4 day old rats were exposed to chemical agents on days 5-6 in culture. Densities of muscarinic and beta-adrenergic receptors were measured according to the binding of N-[3H]methylscopolamine and [ H]CGP 12177, respectively, to cell surface membranes and cell homogenates. Exposure of cells to the muscarinic agonist carbachol (1 mmol/l) brought about a profound decrease in the number of muscarinic receptors. The number of beta-adrenoceptors displayed biphasic changes, being augmented after 24 h (by 20-45% on the cell surface and by 29% in the homogenate) and diminished after 48 h and 72 h (after 48 h, decrease by 44-75% on the cell surface and by 36% in the homogenate). These effects of carbachol were not prevented by dimethylaminopropyl-bis-indolylmaleimide, the inhibitor of protein kinase C. Exposure of cells to the beta-adrenoceptor agonist isoprenaline (0.1 mmol/l) strongly diminished the number of beta-adrenoceptors on the cell surface and in the homogenate. The density of muscarinic receptors on the cell surface was diminished by 24-43% after 24 h exposure to isoprenaline and unchanged after 48 h, whereas the concentration of muscarinic receptors in the homogenate was unchanged after 24 h and increased by 20% after 48 h. The isoprenaline-induced decrease in the density of cell surface muscarinic receptors could not be simulated by forskolin and was not abolished by the protein kinase A inhibitors Rp-cAMPS and HA-1004. Dibutyryl cyclic AMP diminished the density of cell surface muscarinic receptors more than that of the beta-adrenergic receptors. Our data reveal a novel phenomenon of a biphasic change (an increase followed by a loss) in the density of beta-adrenoceptors during exposure of cardiocytes to carbachol. Activation of beta-adrenoceptors brings about less conspicuous changes in the density of muscarinic receptors. The observed phenomena of receptor cross-regulation cannot be explained by simple activations of protein kinases A and C.     

Glycogen phosphorylase: developmental expression in rat liver

We studied the superficial abdominal reflexes of 83 normal men, using as stimuli a train of electrical pulses or a needle scratch. Electrical stimulation delivered to the midline of the abdominal wall evoked, almost symmetrically on both sides, two reflex discharges: an early response having an oligophasic wave form, and a late response of polyphasic wave form. The threshold of the early response significantly exceeded that of the late response. With repetitive stimulation, the late response generally revealed habituation. Electrical stimulation of the unilateral abdominal wall evoked two responses on the stimulated side, whereas it evoked only the late response on the contralateral side. A needle scratch on the unilateral abdominal wall evoked one reflex discharge with a long latency and a polyphasic wave form. This response occurred generally on the stimulated side and became habituated to repeated scratching. These observations suggest that the superficial abdominal reflexes elicited by electrical stimulation are composed of two reflex discharges with a different reflex arc. They appear to closely resemble the blink reflex. The response elicited by needle scratching is thought to correspond to the late response of the electrically elicited abdominal reflexes.     

Decreased expression and activity of P-glycoprotein in rat liver during acute inflammation

PURPOSE: Drug disposition is often altered in inflammatory disease. Although the influence of inflammation on hepatic drug metabolism and protein binding has been well studied, its impact on drug transport has largely been overlooked. The multidrug resistance (MDR) gene product, P-glycoprotein (P-gp) is involved in the active secretion of a large variety of drugs. Our goal was to ascertain the influence of acute inflammation (AI) on the expression and functional activity of P-gp. METHODS: AI was induced in rats through turpentine or lipopolysaccharide (LPS) administration. Expression of P-gp in liver was detected at the level of protein on Western blots using the monoclonal antibody C-219 and at the level of mRNA using an RNase protection assay. P-gp mediated transport activity was assessed by measuring the verapamil-inhibitable efflux of rhodamine 123 (R123) in freshly isolated hepatocytes. RESULTS: Turpentine-induced AI significantly decreased the hepatic protein expression of P-gp isoforms by 50-70% and caused a significant 45-65% reduction in the P-gp mediated efflux of R123. Diminished mRNA levels of all three MDR isoforms were seen. LPS-induced AI similarly resulted in significantly reduced levels and activity of P-gp in liver. Although differences in the constitutive levels of P-gp were seen between male and female rats, the influence of AI on P-gp expression and activity was not gender specific. CONCLUSIONS: Experimentally-induced inflammation decreases the in vivo expression and activity of P-gp in liver. This is the first evidence that expression of P-gp is modulated in response to experimentally-induced inflammation.     

The effect of aging on rat liver regeneration

The effect of age on hepatocyte mensuration and mitotic activity 48 h after partial hepatectomy was investigated in rats. Both age and partial hepatectomy had significant effects upon hepatocyte counts per microscopic field. The number of hepatocytes per microscopic field declined with age in the control groups of different advancing ages and in the experimental groups of advancing ages. There was essentially no mitotic activity in the livers of the control groups. However, mitotic counts were greatly increased in livers from those animals that were partially Hepatectomized; the increase in mitotic activity in the 13-month-old animals was double over that observed in both the very young and the very old.     

Atypical form of the fourth criterion for transient entrainment

The typical fourth criterion for transient entrainment is defined when both a sudden shortening in conduction interval to and a distinct change in electrogram morphology at a bipolar recording site are demonstrated while performing overdrive pacing of a reentrant tachycardia from a single pacing site at two different constant rates. The purpose of this article was to test the hypothesis that if an intracardiac recording site showing both orthodromic and antidromic capture with entrainment pacing is located suitably distant from the circuit, sudden shortening in conduction interval to that site may occur without any significant change in the bipolar electrogram morphology (i.e., atypical form of the fourth criterion). Atrial overdrive pacing of orthodromic tachycardia was performed in 20 patients with either left anterior (12 patients) or left posterior (8 patients) accessory pathways. We investigated the effects of overdrive pacing from the proximal or distal coronary sinus, specifically effects on the electrogram interval and the electrogram morphology at the right atrial appendage. Overdrive pacing of orthodromic tachycardia from the proximal coronary sinus was performed in 10 of the 12 patients with left anterior accessory pathways; those 10 patients demonstrated the first entrainment criterion at the right atrial appendage site. Overdrive pacing of orthodromic tachycardia at still shorter cycle lengths demonstrated a sudden shortening in conduction interval to the right atrial appendage site. Despite shortening in conduction interval the morphology of the right atrial appendage electrogram was completely or almost identical to that during orthodromic tachycardia, indicating an atypical form of the fourth criterion. This criterion was not demonstrated in patients with left posterior accessory pathways. Thus, atypical fourth entrainment criterion was demonstrated during overdrive pacing of orthodromic tachycardia from the proximal coronary sinus only in patients with left anterior accessory pathways. Demonstration of atypical fourth criterion seems largely dependent on the location of the accessory pathway, the pacing, and the recording sites.     

Characterization of beta-adrenergic receptors of freshly isolated astrocytes and neurons from rat brain

The binding and characteristics of rat brain beta-adrenergic receptors (beta-AR) isolated from astrocytes and neurons were investigated. Equilibrium binding experiments demonstrated that beta-AR were more concentrated on astrocytes than on neurons isolated from forebrain, cerebral cortex and cerebellum. Inhibition experiments revealed that beta 1-AR and beta 2-AR were present in the two cell types. Isoproterenol revealed two interchangeable states of high and low affinity binding to both beta 1- and beta 2-AR in neurons. The high affinity binding sites were sensitive to guanylylimidodiphosphate (GppNHp). Similar results were found with other beta-AR agonists but not with salbutamol and salmeterol which recognized both affinity states of the neuronal beta 2-AR but only the low affinity state of beta 1-AR. In astrocytes only the low affinity state of beta-AR was observed.     

Three-step purification of glucocorticoid receptors from rat liver

A sensitive and specific method is described for the quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e]oxepin-3-acetic acid (oxepinac) in human plasma, urine and saliva. Oxepinac and internal standard are extracted from acidified plasma, urine or saliva, converted to the corresponding n-propyl esters and analysed by gas chromatography--mass fragmentography using selected ion monitoring. The method is accurate and precise over the range 100 microgram/ml to 1.0 ng/ml. The method has been applied to the analysis of plasma, urine and saliva from healthy volunteers receiving therapeutic doses of oxepinac.     

Negative cooperativity among beta-adrenergic receptors in frog erythrocyte membranes

(-)-[3H]Dihydroalprenolol, a potent competitive beta-adrenergic antagonist, has been previously documented to bind to the adenylate cyclase-coupled beta-adrenergic receptor sites in mammalian and non-mammalian tissues. Steady state binding of (-)-[3H]dihydroalprenolol to sites in frog erythrocyte membranes, a model system for adenylate cyclase-coupled beta-adrenergic receptors, displays characteristics consistent with negative cooperativity among the beta-adrenergic receptors: Scatchard plots are curvilinear with upward concavity and slopes of Hill plots are consistently less than 1.0. The existence of site-site interactions of the negatively cooperative type were demonstrated directly by the ability of unlabeled (-)-alprenolol to accelerate the dissociation of (-)-[3H]dihydroalprenolol under conditions were no rebinding of radioligand occurred. The dissociation rate of (-)-[3H]dihydroalprenolol alone is directly related to temperature and increases with increases in temperature from 4-37 degrees. (-)-[3H]Dihydroalprenolol dissociation is enhanced by unlabeled (-)-alprenolol at all temperatures studied; however, at 4 degrees, the time required to observe an enhancement of radioligand dissociated is greater than the time required for unlabeled (-)-alprenolol to occupy the empty receptor sites, suggesting that increased rigidity of the biomembrane at 4 degrees may be responsible for the absence of readily observable site-site interactions. The ability of a number of beta-adrenergic agonists and antagonists to induce negative cooperativity among the beta-adrenergic receptors was directly related to their affinity for the receptor sites rather than their intrinsic activity in the adenylate cyclase-coupled beta-adrenergic system. The ability to induce site-site interactions among the beta-adrenergic receptors occurs at physiological concentrations of beta-adrenergic agents, since occupancy of less than 10% of the receptor sites is sufficient to reduce receptor affinity. Changes in pH from 6.5 to 9.0 did not significantly alter the negatively cooperative site-site interactions among the receptor sites. The negatively cooperative phenomenon was also independent of Mg2+, Ca2+, and NaF concentrations in the buffer medium. The presence of guanyl-5'-yl imidodiphosphate, a nonhydrolyzable nucleotide analog which enhances adenylate cyclase stimulation (Vmax) by beta-adrenergic agonists and decreases the concentration of agonist required to half-maximally stimulate adenylate cyclase, did not alter the ability of either agonists or antagonists to induce negatively cooperative site-site interactions among the beta-adrenergic receptors.     

Effect of aging on gastrin receptor gene expression in rat stomach

Broiler chickens were raised in separate rooms kept at temperatures of either 27 C or 16 C from 28 through 39 days of age. At the high temperature mouth breathing was recorded, but it was absent at the lower temperature. The number of dust particles in the air was greater in the warm rooms. More than 50% of the chickens in warm rooms had microscopic lesions in the bronchi of their lungs, whereas fewer than 5% of chickens in cold rooms had such lesions. Large dust particles were visible in some of the lesions. It was postulated that the increased incidence of lung lesions in chickens from warm rooms was due to mouth breathing rather than the higher dust levels in the air of these rooms.     

Silent synapses in the developing rat visual cortex: evidence for postsynaptic expression of synaptic plasticity

In the developing visual cortex activity-dependent refinement of synaptic connectivity is thought to involve synaptic plasticity processes analogous to long-term potentiation (LTP). The recently described conversion of so-called silent synapses to functional ones might underlie some forms of LTP. Using whole-cell recording and minimal stimulation procedures in immature pyramidal neurons, we demonstrate here the existence of functionally silent synapses, i.e., glutamatergic synapses that show only NMDA receptor-mediated transmission, in the neonatal rat visual cortex. The incidence of silent synapses strongly decreased during early postnatal development. After pairing presynaptic stimulation with postsynaptic depolarization, silent synapses were converted to functional ones in an LTP-like manner, as indicated by the long-lasting induction of AMPA receptor-mediated synaptic transmission. This conversion was dependent on the activation of NMDA receptors during the pairing protocol. The selective activation of NMDA receptors at silent synapses could be explained presynaptically by assuming a lower glutamate concentration compared with functional ones. However, we found no differences in glutamate concentration-dependent properties of NMDA receptor-mediated PSCs, suggesting that synaptic glutamate concentration is similar in silent and functional synapses. Our results thus support a postsynaptic mechanism underlying silent synapses, i.e., that they do not contain functional AMPA receptors. Synaptic plasticity at silent synapses might be expressed postsynaptically by modification of nonfunctional AMPA receptors or rapid membrane insertion of AMPA receptors. This conversion of silent synapses to functional ones might play a major role in activity-dependent synaptic refinement during development of the visual cortex.     

Differential expression of rat brain synaptic proteins in development and aging

We have previously reported the differential involvement of synaptic proteins in Alzheimer's disease (AD). As AD is an aging-associated disease, in the present study we examined the developmental and aging-related changes in synaptic proteins such as synaptophysin, synaptobrevin, synaptotagmin, synaptosomal-associated protein 25 (SNAP-25), syntaxin 1/HPC-1 and drebrin in the rat brain. Immunoblot analyses of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that the protein level of synaptophysin and synaptobrevin increased after birth, being highest at 24 weeks, and then decreased with aging. Synaptotagmin was detected at E19, with levels increasing after birth to 96 weeks. SNAP-25 levels were highest at 4 weeks, and then decreased with aging. Syntaxin 1/HPC-1 levels were high at E19 and 1 week, decreasing rapidly from 2 weeks onwards, and drebrin levels were highest at E19 and 1 week, and decreased during aging. The present results suggest that the expression of each synaptic protein is differentially regulated in development and aging.     

Cross-modulation of synaptic plasticity by beta-adrenergic and 5-HT1A receptors in the rat basolateral amygdala

Neurotransmitter receptors are often colocalized in a neuron with other receptors, and activation of one receptor can either amplify or antagonize the response to a colocalized receptor. The aim of this study was to investigate the cross-regulation of synaptic transmission by beta-adrenergic and serotonin 1A (5-HT1A) receptors and to elucidate their underlying mechanisms. Stimulation of presynaptic beta-adrenergic receptors with isoproterenol (Iso) in the basolateral amygdala resulted in a long-lasting increase in synaptic transmission. This effect was mimicked by forskolin, an activator for adenylyl cyclase and a cAMP analog. In addition, the effect of forskolin was blocked by catalytic and regulatory site antagonists for cAMP-dependent protein kinase (PKA), indicating a PKA-mediated mechanism. Application of 5-HT depressed the synaptic transmission and blocked Iso- and forskolin-induced potentiation. The effect of 5-HT was mimicked by the selective 5-HT1A agonist 8-hydroxy-dipropylaminotetralin and was blocked by the selective 5-HT1A antagonist 1-(2-methoxyphenyl)-4[4-(2-phthalimido)butyl]piperazine, indicating its mediation by 5-HT1A receptors. To determine the locus of interaction, Sp-cAMPS, a membrane-permeable activator of PKA, was applied, and the potentiation produced by Sp-cAMPS was completely blocked in slices pretreated with 5-HT. These results suggest that the interaction between the intracellular signaling pathways activated by 5-HT1A and beta-adrenergic receptors occurs at a step downstream from cAMP production.     

Regulation of intracellular free Mg2+ concentration in isolated rat hearts via beta-adrenergic and muscarinic receptors

Regulation of the intracellular free magnesium concentration ([Mg2+]i) was investigated in isolated rat hearts, using 31P-nuclear magnetic resonance (31P-NMR). [Mg2+]i was found to be slowly and significantly decreased during prolonged application of isoproterenol (ISO) through beta-adrenergic receptor stimulation, and restored by subsequent washouts. The ISO-induced decrease in [Mg2+]i was antagonized by addition of a muscarinic receptor agonist, carbachol (CCh). In the presence of atropine, CCh did not exert this effect. A water-soluble forskolin derivative, NKH477, which directly activates adenylate cyclase, also caused a decrease in [Mg2+]i, which could be antagonized by CCh, but a greater concentration was required as compared to the ISO case. The manner of [Mg2+]i regulation mimicked those noted for the action potential duration and the Ca2+ channel current, in which cAMP is known to act as a second messenger. Even in the presence of a Ca2+ channel blocker, verapamil, [Mg2+]i was reversibly decreased by ISO. Changes in the intracellular ATP concentration demonstrated any clear correlation with changes in [Mg2+]i. These results suggest that [Mg2+]i can be controlled by a balance of sympathetic and parasympathetic activities. cAMP may play a key role in the [Mg2+]i regulation via beta-adrenergic and muscarinic receptors, although some other metabolic pathways also appear to be involved. Hormonally induced changes in [Mg2+]i have possible clinical significance.     

Lack of coordinate control of ferritin and transferrin receptor expression during rat liver regeneration

Cytomegalovirus (CMV) is the most important infectious agent in transplant recipients. The critical step in the pathogenesis of CMV infection is the reactivation of latent virus, which is affected by the immunosuppressive therapy and/or alloantigenic stimulation. The clinical effects of CMV infection include CMV disease (syndrome), an immunosuppressed state, and allograft injury. Recently, the incidence of serious CMV diseases after transplant has been decreased, probably due to the advance in the method for rapid diagnosis, the ganciclovir administration, and the effective prevention of CMV diseases. Seronegative or filtered blood products, CMV immune globulin, and prophylactic or preemptive therapy with ganciclovir appear contribute to the improvement in the prophylaxis for CMV diseases after transplant. Antigenemia-guided early treatment may be promising for the effective prevention of CMV diseases after transplant.     

Endomorphin-stimulated [35S]GTPgammaS binding in rat brain: evidence for partial agonist activity at mu-opioid receptors

Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at mu-opioid receptors. In the present study, the effect of endomorphin-1 on mu receptor-coupled G proteins was compared with that of the mu agonist DAMGO by using agonist-stimulated [35S]GTPgammaS binding in rat brain. [35S]GTPgammaS autoradiography revealed a similar localization of endomorphin-1- and DAMGO-stimulated [35S]GTPgammaS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [35S]GTPgammaS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [35S]GTPgammaS binding. Differences in maximal stimulation of [35S]GTPgammaS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [35S]GTPgammaS binding revealed a lower apparent Bmax value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [35S]GTPgammaS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the mu-opioid receptor in brain.