In vivo tissue specific modulation of rat insulin receptor gene expression in an experimental model of mineralocorticoid excess

Insulin receptor (IR) gene expression at the mRNA level was investigated in hindlimb skeletal muscle, epididymal adipose tissue and in the liver of rats exposed to prolonged in vivo administration of deoxycorticosterone acetate (DOCA). Following treatment, plasma insulin levels were reduced while glucose levels increased compared to values in control rats. DOCA-treated animals showed an increase in blood pressure and a reduction in body weight. This treatment also induced hypokalemia and decreased plasma protein levels. Sodium levels were unaffected. Moreover, no differences in DNA and protein content or in the indicator of cell size (protein/DNA) were observed in the skeletal muscle or adipose tissue of animals. In contrast, there was a clear increase in the protein and DNA contents of the liver with no change in the indicator of cell size. Northern blot assays revealed 2 major IR mRNA species of approximately 9.5 and 7.5 Kb in the 3 tissues from control animals. DOCA treatment induced no change in the levels of either RNA species in skeletal muscle. However, a decrease of approximately 22% was detected in the levels of both species in adipose tissue whereas the liver showed an increase of 64%. These results provide the first evidence for an in vivo tissue-specific modulation of IR mRNA levels under experimental conditions of mineralocorticoid excess.     

Partial saturation of rat liver Cd-binding proteins by Cd2+ in vivo

Liver Cd-binding proteins (Cd-BP) were isolated from rats chronically treated with 109Cd-labeled CdCl2 for ten days. Fractions purified using Sephadex G-75 and DEAE-Sephadex were characterized and found to be similar to those isolated by other investigators. Cd-binding was not saturated in any of the preparations and significant amounts of Cu and Zn were also found bound to the proteins. The percentage of saturation of Cd-BP1, and Cd-BP2 was independently determined by atomic absorption spectrometry and spectroscopy at 254 nm. These results indicate that the fraction of binding sites unoccupied by Cd on Cd-BP approaches 20% in vivo.     

In vivo stimulation of IL-12 secretion by subcutaneous low-dose IL-2 in metastatic cancer patients

Despite the well-demonstrated involvement of both interleukin 2 (IL-2) and interleukin 12 (IL-12) in the activation of host anti-cancer response, the knowledge of IL-2-IL-12 interactions has still to be better investigated. This study was performed to evaluate the effects of subcutaneous (s.c.) low-dose IL-2 on IL-12 secretion in metastatic cancer patients. The study included 19 evaluable metastatic renal cell cancer patients, who received s.c. low-dose IL-2 (6 MIU day(-1) for 6 days per week for 4 weeks) as a first-line immunotherapy of their metastatic disease. Serum levels of IL-12 were measured using an enzyme immunoassay on venous blood samples collected before the immunotherapy and at 1-week intervals. The clinical response consisted of partial response (PR) in four and stable disease (SD) in eight patients, whereas the other seven patients progressed. Mean serum levels of IL-12 observed in the overall patients significantly increased in response to IL-2 injection. Moreover, by evaluating IL-12 variations in relation to the clinical response, a marked significant increase in IL-12 mean values occurred in patients with response or SD, whereas the progressing patients showed a significant decline in IL-12 levels during IL-2 administration. Finally, IL-12 mean pretreatment values observed in patients who progressed were significantly higher than those seen in non-progressing patients. This study shows that low-dose IL-2 immunotherapy of cancer may stimulate the in vivo release of IL-12, and it would suggest that IL-2-induced IL-12 enhancement is associated with a favourable prognosis.     

Glycogenolysis stimulation by non-steroidal anti-inflammatories in the perfused rat liver is not accompanied by Ca2+ release

To better understand the characteristics of amyloid deposition in the choroid plexus, we examined autopsied brain by routine histology, immunohistochemistry, and electron microscopy in three group of patients: primary systemic amyloidosis (n = 7), cerebral amyloid angiopathy (CAA, n = 6), and controls (n = 3). Three of the CAA patients had Alzheimer's disease. Congophilic, birefringent amyloid deposits of the choroid plexus were seen in six of the seven cases of systemic light chain amyloidosis. Immunohistochemistry revealed that the deposited amyloids had reactivity for immunoglobulin light chain and amyloid P component. Accumulation of macrophages labeled with monoclonal antibodies against CD 68 and major histocompatibility complex class II antigens were observed around the massive amyloid deposits. The presence of approximately 10 nm amyloid fibrils along the epithelial basement membrane as well as in the vascular walls was ascertained by electron microscopy. In CAA, Congo red-positive amyloid deposits were consistently present in meningeal blood vessels and were often found in senile plaques of the cerebral parenchyma; congophilic amyloid deposits were absent in the choroid plexus. Choroid plexus epithelial cells exhibited immunostaining for beta amyloid precursor protein (APP) with N-terminal- and C-terminal-specific antibodies; in particular, consistent staining was obtained for the latter antibody. Immunoreactivity for amyloid beta protein (A beta) with monoclonal antibodies (6E10, 4G8) was often found in choroid plexus epithelial cells. These findings suggest that amyloid deposition of the choroid plexus depends on the major component protein in amyloidosis, and that the choroid plexus may produce APP and A beta protein although A beta amyloidosis is not evident in the choroid plexus.     

In vivo glycerol metabolism in the pregnant rat

Pregnant rats at 12 and 21 days of gestation and their virgin controls were injected intravenously with U-14C-glycerol and decapitated 1, 3, or 10 min later. The conversion of labelled glycerol to 14C-glucose was augmented in the 21-day pregnant rats. The disappearance of the newly formed 14C-glucuse from blood was faster in both 12- and 21-day pregnant rats than in their controls, being partially retained as liver 14C-glycogen. The greatest amount of radioactivity in all tissues appeared in the carcass hydrosoluble fraction. This amount was smaller in the pregnant rats. The reduced utilization of glycerol by extrahepatic tissues allowed the 21-day pregnant rats to dispose a greater amount of this substrate for gluconeogenesis.     

In vivo stimulation by tamoxifen of cathepsin D RNA level in breast cancer

We have previously shown that 3 weeks of treatment with tamoxifen, of patients with primary breast carcinomas, increased cytosolic cathepsin D protein in oestrogen receptor (ER) positive tumours [Maudelonde et al., Cancer 1989, 63, 1265-1270]. In order to investigate the mechanism of this increase and to eliminate a transient flare-up effect, we semi-quantified cathepsin D RNA levels by in situ hybridisation in 32 breast carcinomas from patients treated with tamoxifen for 3 weeks prior to surgery and in 35 breast cancer patients receiving no tamoxifen. We found that tamoxifen increased cathepsin D RNA level regardless of the ER status of the tumours. In ER positive tumours, tamoxifen increased the cathepsin D RNA level to the same extent as cytosolic cathepsin D protein but not in ER negative tumours. The induction of cathepsin D RNA by tamoxifen in ER positive tumours was probably due to its agonist activity, also observed in vitro in breast cancer cell lines. These results suggest that the cathepsin D gene is inducible by oestrogens in ER positive breast cancer as it is in breast cancer cell lines.     

In vivo tissue pO2 measurements in hamster skinfold by recessed pO2 microelectrodes and phosphorescence quenching are in agreement

OBJECTIVE: Phosphorescence quenching has been used successfully to optically measure in vivo blood pO2 in the microvasculature. Optical measurements have also been made in some tissues, but it is not clear whether these results accurately reflect tissue pO2. METHODS: Recessed pO2 microelectrodes and the phosphorescence quenching technique were used simultaneously to measure in vivo tissue pO2 in hamster skinfold. The optical window for phosphorescence quenching was focused around the tips of microelectrodes that were positioned in tissue regions at least 100 microns from large microvessels. RESULTS: Mean tissue pO2 measured by recessed pO2 microelectrodes was 18.4 +/- 1.7 (SE) Torr, and mean tissue pO2 determined from the time course of phosphorescence decay was 18.8 +/- 2.0 Torr (no significant difference). The two tissue pO2 measurements agreed over a wide range, from 2 to 46 Torr (r = 0.93, 39 paired measurements from six sites in 3 animals). There was no systematic change in the microelectrode tissue pO2 during the period of light excitation used for the optical method. CONCLUSIONS: Under the conditions of our study, sufficient amounts of porphyrin dye leaked from the vasculature and diffused into tissue, allowing accurate measurements of tissue pO2 by the phosphorescence quenching technique. Furthermore, the optical method did not deplete significant amounts of O2 from tissue during light excitation.     

In vivo accumulation of iron on crocidolite is associated with decrements in oxidant generation by the fiber

Strategies have been developed to characterize tumor antigens recognized by cytolytic T lymphocytes (CTL). We use a genetic approach based on the transfection of HLA genes and cDNA libraries in COS cells to isolate the gene producing the antigenic peptide. The tumor-specific expression of this gene can be evaluated by cDNA synthesis and quantitative PCR amplification. Transfection of fragments of the isolated gene allows the identification of the region encoding the antigenic peptide. Peptides are synthesized and tested for their ability to sensitize target cells to lysis by the CTL.     

Regulation of K+ channel mRNA expression by stimulation of adenosine A2a-receptors in cultured rat microglia

Previous investigations suggest that the expression of K+ channels in cultured rat microglia is related to the activation status of these cells. Both, lipopolysaccharide (LPS) and agents that raise intracellular cyclic AMP have been shown to inhibit microglial proliferation. LPS also regulates the mRNA expression levels of K+ channels in cultured microglia, which led us to investigate possible regulatory interactions between K+ channels and adenosine A2a-receptors, which are coupled to the cAMP-signal transduction pathway. The selective adenosine A2a-receptor agonist CGS 21680 induced enhanced mRNA expression of both Kv1.3 and ROMK1, as well as an elevation of Kv1.3 protein. The selective adenosine A2a-receptor antagonist aminophenol (ZM 241385) and the nonselective antagonist 8-phenyltheophylline (8-PT) inhibited these effects. Elevations of cyclic AMP by use of dibutyryl cyclic AMP (dbcAMP), phosphodiesterase-inhibitor (RO 20-1724), forskolin, or cholera toxin (CTX), strongly enhanced Kv1.3-mRNA expression, but decreased ROMK1-mRNA levels. Results from experiments with actinomycin D suggest that K+ channel mRNA levels in cultured microglia were regulated by altered mRNA synthesis. Evidently, the CGS 21680-induced effects upon Kv1.3 were mediated via an increase in intracellular cyclic AMP, whereas ROMK1-mRNA expression appeared to be regulated by coupling of adenosine A2a-receptors to an alternative pathway, which involves activation of protein kinase C (PKC). It is concluded that the cyclic AMP second messenger system in microglia is not only involved in regulation of K+ channel activity, but also in regulation of de novo K+ channel synthesis.     

In vivo suppression of immune complex-induced alveolitis by secretory leukoproteinase inhibitor and tissue inhibitor of metalloproteinases 2

The pulmonary tree is exposed to neutrophil-derived serine proteinases and matrix metalloproteinases in inflammatory lung diseases, but the degree to which these enzymes participate in tissue injury remains undefined, as does the therapeutic utility of antiproteinase-based interventions. To address these issues, an in vivo rat model was examined in which the intrapulmonary deposition of immune complexes initiates a neutrophil-mediated acute alveolitis. In vitro studies demonstrated that rat neutrophils can release neutrophil elastase and cathepsin G as well as a neutrophil progelatinase, which was subsequently activated by either chlorinated oxidants or serine proteinases. Based on structural homologies that exist between rat and human neutrophil proteinases, rat neutrophil elastase and cathepsin G activities could be specifically regulated in vitro by recombinant human secretory leukoproteinase inhibitor, and rat neutrophil gelatinase activity proved sensitive to inhibition by recombinant human tissue inhibitor of metalloproteinases 2. When either of the recombinant antiproteinases were instilled intratracheally, in vivo lung damage as assessed by increased permeability or hemorrhage was significantly reduced. Furthermore, the coadministration of the serine and matrix metalloproteinase inhibitors almost completely prevented pulmonary damage while effecting only a modest decrease in neutrophil influx. These data support a critical role for neutrophil-derived proteinases in acute lung damage in vivo and identify recombinant human secretory leukoproteinase and recombinant human tissue inhibitor of metalloproteinases 2 as potentially efficacious interventions in inflammatory disease states.     

NMDA receptor-mediated differential laminar susceptibility to the intracellular Ca2+ accumulation induced by oxygen-glucose deprivation in rat neocortical slices

Slices of somatosensory cortex taken from immature rats on postnatal day (P)7-14 were labeled with fura-2. Intracellular Ca2+ concentration ([Ca2+]i) was monitored in identified pyramidal cells as the ratio of fluorescence intensities (RF340/F380) during oxygen-glucose deprivation. The RF340/F380 ([Ca2+]i) of individual pyramidal cells was monitored in each of the cortical layers II-VI simultaneously. Neurons in all neocortical layers exhibited significant increases in [Ca2+]i that varied with the duration of oxygen-glucose deprivation. Individual neurons responded to oxygen-glucose deprivation with abrupt increases in [Ca2+]i after various latencies. The ceiling level of the [Ca2+]i increase differed from cell to cell. Neurons in layer II/III showed significantly greater increases in [Ca2+]i than those in layers IV, V, or VI. Kynurenic acid, a nonselective glutamate receptor antagonist, and bicuculline, a selective gamma-aminobutyric acid (GABA)A receptor antagonist, suppressed the intracellular Ca2+ accumulation induced by oxygen-glucose deprivation in all neocortical layers examined. After kynurenic acid, but not after bicuculline, there was no longer a differential [Ca2+]i increases in layer II/III. Both 2-amino-5-phosphonopentanoic acid (AP5), a selective N-methyl-D-aspartate (NMDA) receptor antagonist, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, strongly suppressed the intracellular Ca2+ accumulation induced by oxygen-glucose deprivation in all layers. The laminar difference in terms of the [Ca2+]i increases was abolished by AP5, but not by CNQX. These results indicate that layer II/III cells are the most prone to oxygen-glucose deprivation-induced intracellular Ca2+ accumulation, and that this is primarily mediated by NMDA receptors. Thus, layer II/III neurons would be more likely to suffer cellular Ca2+ overload and excitotoxicity during ischemia than layer IV-VI cells. Such a differential laminar vulnerability might play an important role in determining the pathological characteristics of the immature cortex and its sequelae later in life.     

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes

The vesicle-associated membrane protein (VAMP) family is essential to vesicle-mediated protein transport. Three mammalian isoforms, VAMP-1, VAMP-2, and cellubrevin, play a role in protein transport to the plasma membrane. In this study, we describe a new rat VAMP-1 isoform produced by alternative pre-mRNA splicing. Only one VAMP-1 isoform dominates in each tissue. Analysis of the nucleotide sequence for the newly discovered isoform, VAMP-1b, reveals that its expression is determined by whether an intron is retained or removed. The predicted amino acid sequences for the VAMP-1 isoforms differ at the carboxy-terminal end of the protein. A similar process has been described for VAMPs in Drosophila melanogaster and suggests a conserved function for the carboxy-terminal domain that can be modulated.     

Selective in vivo stimulation of stress-activated protein kinase in different rat tissues by immobilization stress

The quaternary structure of Lumbricus terrestris hemoglobin was investigated by small-angle x-ray scattering (SAXS). Based on the SAXS data from several independent experiments, a three-dimensional (3D) consensus model was established to simulate the solution structure of this complex protein at low resolution (about 3 nm) and to yield the particle dimensions. The model is built up from a large number of small spheres of different weights, a result of the two-step procedure used to calculate the SAXS model. It accounts for the arrangement of 12 subunits in a hexagonal bilayer structure and for an additional central unit of clylinder-like shape. This model provides an excellent fit of the experimental scattering curve of the protein up to h = 1 nm-1 and a nearly perfect fit of the experimental distance distribution function p(r) in the whole range. Scattering curves and p(r) functions were also calculated for low-resolution models based on 3D reconstructions obtained by cryoelectron microscopy (EM). The calculated functions of these models also provide a very good fit of the experimental scattering curve (even at h > 1 nm-1) and p(r) function, if hydration is taken into account and the original model coordinates are slightly rescaled. The comparison of models reveals that both the SAXS-based and the EM-based model lead to a similar simulation of the protein structure and to similar particle dimensions. The essential differences between the models concern the hexagonal bilayer arrangement (eclipsed in the SAXS model, one layer slightly rotated in the EM model), and the mass distribution, mainly on the surface and in the central part of the protein complex.     

Long-lasting effects of cholinergic stimulation of the amygdaloid complex in the rat.

Assigned 104 male Sprague-Dawley albino rats to 4 groups: (a) unoperated control, (b) cannulated control, (c) carbachol injections, and (d) eserine injections. Injection of carbachol into the amygdaloid complex caused EEG seizures and behavioral convulsions. After convulsions and abnormal EEG had disappeared, impaired acquisition of a 1-way active-avoidance response and facilitated acquisition of a 2-way shuttle-box avoidance response persisted. There was normal acquisition of an appetitive visual discrimination task, but no improvement in 1-way active-avoidance acquisition following daily handling that facilitated acquisition in controls. Eserine injections into the amygdala produced a deficit in 1-way avoidance acquisition similar to that produced by carbachol, without altering EEG or inducing convulsions. It is suggested that the behavioral changes were due to altered amygdaloid synaptic function which elevated the Ss' reactivity to noxious stimuli. (20 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

In vivo stimulation-induced release of manganese in rat amygdala

The role of manganese ions in neural functions is poorly understood because of the low level of manganese in the brain. An excess of this ion is associated with neurological disorders such as extrapyramidal symptoms. We demonstrated that manganese may be taken up by piriform neurons (tertiary olfactory neurons) after release from the terminals of secondary olfactory neurons, in which 54Mn taken up by the soma may be anterogradely transported [A. Takeda, Y. Kodama, S. Ishiwatari, S. Okada, Manganese transport in the neural circuit of Rat CNS, Brain Res. Bull. (45) (1998) 149-152]. Here we demonstrate for the first time that 54Mn previously taken up into the amygdala is released with neurotransmitters into the extracellular space during stimulation with high K+. The results suggest that the role of manganese ions in the amygdala, and probably in the olfactory system, is dynamically linked to neural signalling processes.     

AT1 receptor-mediated stimulation by angiotensin II of rat aortic fibronectin gene expression in vivo

Fibronectin plays an important role in various vascular diseases. A subpressor (200 ng kg-1 min-1) or pressor (1000 ng kg-1 min-1) dose of angiotensin II was continuously infused into rats by osmotic minipump for various times, to investigate the effects on aortic fibronectin gene expression. In rats infused with a subpressor dose of angiotensin II in which blood pressure was normal for 3 days, aortic fibronectin mRNA levels started to increase by 1.4 fold at 12 h and reached the maximal levels (increased by 3.1 fold) at 3 days. Treatment with TCV-116 (3 mg kg-1 day-1), a non-peptide selective AT1 receptor antagonist, completely inhibited the angiotensin II-induced increase in aortic fibronectin mRNA, while hydralazine (10 mg kg-1 day-1) did not block this effect. Similar results were also obtained for a pressor dose of angiotensin II. Thus, angiotensin II directly stimulates aortic fibronectin gene expression in vivo, which is mediated by the AT1 receptor but not by blood pressure.     

In vitro and in vivo studies of drug residue accumulation in pigmented tissues

A procedure was developed to enable ready isolation of melanin granules from pigmented tissues of the bovine eye. The granules were used in a simple in vitro test to model the potential for a range of veterinary drugs to accumulate in melanin-containing tissues such as hair and the choroid/pigmented retinal epithelium (choroid/PRE) of the eye. The beta-agonists clenbuterol and salmeterol, but not salbutamol, showed appreciable binding, as did the beta-blockers propranolol and carazolol and the tranquillizers azaperone and xylazine. All of the natural and synthetic growth promoters tested gave rise to significant binding (17 beta-estradiol, testosterone, alpha-zeranol, diethylstilbestrol and 19-nortestosterone) but progesterone and 17 alpha-trenbolone bound to a lesser extent. To provide a preliminary indication of the validity of the model, animals were treated with clenbuterol for 21 d, to enable the assessment of accumulation in vivo. Clenbuterol was detected in choroid/PRE and hair at high concentrations from the last day of treatment (1779 ng g-1 and 424 ng g-1, respectively) until the end of the study period, 63 d later (512 ng g-1 and 483 ng g-1, respectively). These studies clearly indicate the wider potential for pigmented tissue analysis in monitoring for the use of veterinary drugs (particularly unlicensed substances) in food producing animals. Hair analysis may offer particular advantages for on-farm monitoring and in providing historic information.     

Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15

Proliferation of memory-phenotype (CD44hi) CD8+ cells induced by infectious agents can be mimicked by injection of type I interferon (IFN I) and by IFN I-inducing agents such as lipopolysaccharide and Poly I:C; such proliferation does not affect naive T cells and appears to be TCR independent. Since IFN I inhibits proliferation in vitro, IFN I-induced proliferation of CD8+ cells in vivo presumably occurs indirectly through production of secondary cytokines, e.g., interleukin-2 (IL-2) or IL-15. We show here that, unlike IL-2, IL-15 closely mimics the effects of IFN I in causing strong and selective stimulation of memory-phenotype CD44hi CD8+ (but not CD4+) cells in vivo; similar specificity applies to purified T cells in vitro and correlates with much higher expression of IL-2Rbeta on CD8+ cells than on CD4+ cells.     

Co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in the cortical thick ascending limb cell of the rat kidney. Inhibition of hormone-dependent cAMP accumulation by extracellular Ca2+

The Ca2+-sensing receptor protein and the Ca2+-inhibitable type 6 adenylyl cyclase mRNA are present in a defined segment of the rat renal tubule leading to the hypothesis of their possible functional co-expression in a same cell and thus to a possible inhibition of cAMP content by extracellular Ca2+. By using microdissected segments, we compared the properties of regulation of extracellular Ca2+-mediated activation of Ca2+ receptor to those elicited by prostaglandin E2 and angiotensin II. The three agents inhibited a common pool of hormone-stimulated cAMP content by different mechanisms as follows. (i) Extracellular Ca2+, coupled to phospholipase C activation via a pertussis toxin-insensitive G protein, induced a dose-dependent inhibition of cAMP content (1.25 mM Ca2+ eliciting 50% inhibition) resulting from both stimulation of cAMP hydrolysis and inhibition of cAMP synthesis; this latter effect was mediated by capacitive Ca2+ influx as well as release of intracellular Ca2+. (ii) Angiotensin II, coupled to the same transduction pathway, also decreased cAMP content; however, its inhibitory effect on cAMP was mainly accounted for by an increase of cAMP hydrolysis, although angiotensin II and extracellular Ca2+ can induce comparable release of intracellular Ca2+. (iii) Prostaglandin E2, coupled to pertussis toxin-sensitive G protein, inhibited the same pool of adenylyl cyclase units as extracellular Ca2+ but by a different mechanism. The functional properties of the adenylyl cyclase were similar to those described for type 6. The results establish that the co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in a same cell allows an inhibition of cAMP accumulation by physiological concentrations of extracellular Ca2+.