Identification of some plasma membrane proteins of cultured rat pigment epithelial cells by labeling with 125I

Cultured rat retinal pigment epithelial (PE) cells were labeled with 125I by lactoperoxidase (LPO)-catalyzed radioiodination. Examination of 125I-labeled cells by electron microscopic autoradiography suggested that 125I was incorporated into proteins at both the apical and basal surfaces of the PE cells, and into the extracellular matrix (ECM). Analysis of labeled cells by SDS-polyacrylamide gel electrophoresis and autoradiography showed that 125I was incorporated into at least 15 polypeptides. In order to determine which of these labeled proteins were derived from the plasma membrane. 125I-labeled cells were subjected to differential detergent extraction. Triton X-100 (2% v/v), which removed the cell plasma membrane, solubilized only three of the labeled proteins (152 000, 138 000, 123 000 daltons). SDS (0.25% w/v) completely removed cells from the tissue culture dish and solubilized all but four of the labeled proteins (225 000, 173 000, 87 000 and 72 100 daltons). When 125I-labeled PE cells were mechanically disrupted, and the resulting cell fractions separated by sucrose density gradient centrifugation, labeled proteins separated into two subcellular fractions. One fraction was especially enriched in the 152 000, 138 000 and 123 000 dalton labeled proteins, in addition to the plasma membrane marker enzymes Na+, K+-ATPase, and alkaline phosphodiesterase I. The second fraction was enriched in the 225 000, 196 000 and 173 000 dalton labeled proteins, the ECM proteins laminin and fibronectin, and the 43 000 actin band . It is proposed that 125I-labeled proteins in the former cell fraction are truly plasma membrane proteins, while those found in the latter cell fraction are a mixture of 125I-labeled ECM and basal plasma membrane proteins. The 152 000, 138 000 and 123 000 dalton labeled proteins of the plasma membrane fraction are glycoproteins and become firmly anchored to the Triton X-100 insoluble cytoskeleton when labeled cells are treated with concanavalin A.     

Quantitative light microscopic autoradiography of [H]hemicholinium-3 binding sites in the rat central nervous system: a novel biochemical marker for mapping the distribution of cholinergic nerve terminals

The distribution of specific [³H]hemicholinium-3 ([³H]HC-3) binding sites throughout the rat forebrain was studied by means of quantitative light microscopic autoradiography. Tissue sections were labeled with 2.5 nM[³H]HC-3, apposed to tritium-sensitive film for 2 months and analyzed by computer-assisted densitometry. Regions of intense [³H]HC-3 labeling include the caudate-putamen, nucleus accumbens, olfactory tubercle, amygdala, habenula and the granule cell layer of the dentate gyrus. Little or no specific binding was detected in the corpus callosum, a white matter region. This distribution of specific [³H]HC-3 binding sites is compatible with a selective labeling of central cholinergic nerve terminals.     

Brain palmitate incorporation in awake and anesthetized rats

Uniformly labeled [14C]palmitate was injected intravenously in awake and barbiturate-anesthetized rats, and arterial plasma radioactivity due to unesterified [14C]palmitate was determined on plasma samples removed at timed intervals up to the time of death. Overall brain radioactivity was determined by liquid scintillation spectroscopy, and regional brain radioactivity was determined by quantitative autoradiography. The transfer constant, k, for the unidirectional uptake of radiotracer palmitate into the brain at 4 h was calculated from the brain radioactivity and the integrated plasma radioactivity from injection to 4 h. The unidirectional palmitate uptake was calculated as the product of k and the plasma concentration of unesterified palmitate. Barbiturate anesthesia reduced regional palmitate transfer constants and unidirectional palmitate uptakes into different brain regions by 40-60%. Palmitate incorporation into the brains of awake rats at 4 h represents uptake into structural brain components which contain lipids. The results indicate that pentobarbital anesthesia reduces this rate of incorporation by about half.     

Dose effects of naloxone on fixed morphine dependence: simultaneous behavioral and 2-deoxyglucose study in the rat

We have simultaneously studied regional cerebral glucose utilization (RCGU) and behavior during naloxone precipitated morphine withdrawal. For RCGU studies, 25 brain regions were analyzed that previously had been shown to participate in morphine withdrawal. Four established behavioral signs of morphine withdrawal were recorded: wet shakes, jumping, weight loss, and autonomic signs. Using a 10(4) range of naloxone dose (0.0005-5.0 mg/kg), dose dependent effects were found for 3 behaviors: jumping, weight loss and autonomic signs. The incidence of wet shakes did not correlate with naloxone dose. Increases in RCGU in several specific brain sites were also naloxone dose dependent. Naloxone dose dependent increases in RCGU during precipitated morphine withdrawal may be divided into 3 classes of responses: Class I structures (paraventricular, ventromedial, and lateral hypothalamus) exhibited their largest RCGU increases between 0.005 and 0.05 mg/kg of naloxone; Class II structures (preoptic areas, basal ganglia, anterior and intralaminar thalamic nuclei, mammillary nuclei, and certain midbrain regions) showed gradual RCGU increases across the 10(4) range of naloxone dose; and, Class III structures (diagonal band, medial and lateral septum, and the central amygdaloid nucleus) displayed large RCGU increases across 0.5-5.0 mg/kg of naloxone. Regression analysis of RCGU vs behavior showed correlations between Class I responses and autonomic signs (P less than 0.010); weight loss was correlated with all 3 classes of naloxone dose dependent RCGU responses during withdrawal (P less than 0.05). The strong positive correlation among these RCGU increases and certain morphine withdrawal behaviors supported the use of RCGU measurements in specific brain sites as a sensitive and objective biochemical indicator of the presence and severity of morphine dependence. In addition, this study demonstrates that changes in RCGU in different brain regions are heterogeneous with respect to naloxone dose and appear reproducibly along a continuum from mild to severe withdrawal.     

Autoradiographic demonstration of peripheral adenosine binding sites using [3H]NECA

Autoradiographic localization of 5'-N-ethylcarboxamido[3H]adenosine ([3H]NECA) binding sites revealed a heterogeneous labeling of guinea-pig intestine with heavy labeling over the enteric ganglia and in clusters over the mucosa; a low level of label was homogeneously distributed over the muscularis externa. Under the conditions employed, no binding sites were revealed using [3H]-N6-cyclohexyladenosine ([3H]CHA), although both [3H]CHA and [3H]NECA binding sites were localized over comparable areas of rat brain. The relationship of the [3H]NECA binding sites to extracellular adenosine receptors is discussed.     

Image guided interstitial laser thermotherapy: a canine model evaluated by magnetic resonance imaging and quantitative autoradiography

Summary Objective. To determine the applicability and safety of a new canine model suitable for correlative magnetic resonance imaging (MRI) studies and morphological/pathophysiological examination over time after interstitial laser thermotherapy (ILTT) in brain tissue.Material and methods. A laser fibre (Diode Laser 830nm) with an integrated temperature feedback system was inserted into the right frontal white matter in 18 dogs using frameless navigation technique. MRI thermometry (phase mapping i.e. chemical shift of the proton resonance frequency) during interstitial heating was compared to simultaneously recorded interstitial fiberoptic temperature readings on the border of the lesion. To study brain capillary function in response to ILTT over time quantitative autoradiography was performed investigating the unidirectional blood-to-tissue transport of carbon-14-labelled alpha amino-isobutyric acid (transfer constant K of AIB) 12, 36 hours, 7, 14 days, 4 weeks and 3 months after ILTT.Results. All laser procedures were well tolerated, laser and temperature fibres could be adequately placed in the right frontal lobe in all animals. In 5 animals MRI-based temperature quantification correlated strongly to invasive temperature measurements. In the remaining animals the temperature fibre was located in the area of susceptibility artifacts, therefore, no temperature correlation was possible. The laser lesions consisted of a central area of calcified necrosis which was surrounded by an area of reactive brain tissue with increased permeability. Quantitative autoradiography indicated a thin and spherical blood brain barrier lesion. The magnitude of K of AIB increased from 12 hours to 14 days after ILTT and decreased thereafter. The mean value of K of AIB was 19 times (2 times) that of normal white matter (cortex), respectively.Conclusion. ILTT causes transient, highly localised areas of increased capillary permeability surrounding the laser lesion. Phase contrast imaging for MRI thermomonitoring can currently not be used for reliable temperature readings in vivo. The suggested new canine model proved to be safe, accurate, easy to use, and provides clinical, radiographic, pathological and physiological correlations.     

Tritiated 2-deoxy-D-glucose: A high-resolution marker for autoradiographic localization of brain metabolism

The technique for autoradiographic localization of 2-deoxy-D-glucose (2DG) uptake has become a useful method for observing alterations of functional brain activity resulting from experimental manipulation. Autoradiographic resolution is improved using tritiated ([³H]) rather than carbon-14 ([¹⁴C])2DG, due to the lower energy and shorter path of tritium emissions. In addition, lower 2DG uptake by white matter relative to gray matter is exaggerated in the [³H]2DG autoradiographs due to the greater absorption of tritium emissions by lipids. Using [³H]2DG, it is possible to observe differential metabolic labeling in various individual nuclei or portions of nuclei that is unresolvable using [¹⁴C]2DG in the awake, normal animal. Heterogeneous patterns of 2DG uptake seen only with [³H]2DG are found in the nucleus accumbens, the anterior portion of the basolateral nucleus of the amygdala, specific nuclei of the inferior olivary complex, various hypothalamic regions, and a region straddling the border of the medial and lateral habenular nuclei. The lamination of differential 2DG uptake in the hippocampus is better localized using [³H]2DG. Autoradiographic resolution of labeled 2DG is further improved when the brain is perfused prior to frozen sectioning, due perhaps to selective fixation and retention of intracellular labeled 2-deoxy-glycogen. A series of [³H]2DG autoradiographs are presented together with views of the Nissl-stained sections that produced the autoradiographs.     

Intraspinal distribution and reaction in the grey matter with tetanus toxin of intracisternally injected anti-tetanus toxoid F (ab′)2 fragments

Anti-tetanus toxoid F(ab′)₂ fragments were purified using immune-affinity chromatography on tetanus toxoid-Sepharose. Fragments were labeled with¹²⁵I. Labeled or non-labeled fragments were injected into the intrathecal space of rats. The labeled fragments were found in the spinal cord outside but not inside neurons.Tetanus toxin was injected into a muscle and 36 h later labeled fragments were injected intracisternally. After another 24 h the label was elevated in the spinal cord half segments giving neural supply to the injected muscle and in these half-segments the label was concentrated around some α-motoneurons.[¹²⁵I]Tetanus toxin was injected into a muscle and at different times thereafter non-labeled fragments were injected intracisternally. The development of hindlimb rigidity but not the accumulation of [¹²⁵I]tetanus toxin in α-motoneurons was prevented by early intracisternal injection of fragments. Injection of fragments after the appearance of hindlimb rigidity did not revert the rigidity but prevented the further development of symptoms.It is concluded that an action of tetanus toxin inside α-motoneurons is of no importance for the development of motor symptoms in clinical tetanus. The data suggest that in order to evoke spinal symptoms of toxicity tetanus toxin has to reach interneurons by transneuronal migration. In the very early stages of clinical tetanus the intrathecal injection of fragments may be useful.     

Nerve stump length-dependent loss of acetycholinesterase activity in endplate regions of rat diaphragm

Light microscopy autoradiographs of the rat hypothalamic median eminence were prepared after injection of high specific activity tritiated GABA and GABA structural analogs.Following intracardiac injection of labeled GABA with short (15 min) survival time, a dense accumulation of silver grains was observed over the external layer of the median eminence. The silver grains appeared much less numerous and randomly scattered over the internal layer. No conspicuously labeled cells could be detected in the median eminence.A similar pattern of labeling was observed after 10 min in vitro incubation of the median eminence with a low concentration(2.5 × 10^?7M) of labeled GABA. Clusters of silver grains were also visible over the external layer following intraventicular injection of labeled GABA. In this latter case, however, other sites of labeling were revealed over the internal and ependymal layers.The dense labeling over the external layer with tritiated GABA was partially reduced by a simultaneous intracardiac injection of a 50-fold excess of non-radioactive cis-aminocyclohexane car?ylic acid — a reported preferential substrate for GABA neuronal uptake — but it was not displaced by a 2000-fold excess of non-radioactive β-alanine — a reported specific substrate for GABA glial uptake.Intracardiac injection of triated β-alanine led to a faint and even labeling over the entire median eminence with no preferential accumulation of silver grains over the various layers. Following intraventricular injection of labeled β-alanine the tanycytes and their processes as well as numerous glial cells appeared heavily labeled.These results suggested that there exist cell elements in the external layer of the hypothalamic median eminence which are capable of accumulating exogenous GABA according to its neuronal uptake characteristics. Although the exact nature of these cells is not readily apparent at this stage of our investigations, these findings led us to speculate that there might be a subpopulation of GABAergic nerve endings in the vicinity of the primary plexus capillaries.