Autoradiographic characterization of [3H]inositol (1,4,5) trisphosphate and [3H]inositol (1,3,4,5) tetrakisphosphate binding sites in human brain

Autoradiographic techniques were used to investigate the characteristics of tritiated inositol(1,4,5)trisphosphate ([3H]IP3) and inositol (1,3,4,5) tetrakisphosphate ([3H]IP4) binding to human brain. In brain sections [3H]IP3 exhibited a two-site binding with KD values of 87 nM and 9.3 microM respectively for the higher and lower affinity sites. [3H]IP4 also bound to two sites with KD values of 43 nM and 1.4 microM, respectively. With the conditions fixed in this study, [3H]IP3 and [3H]IP4 autoradiography in the cortex, caudate, hippocampus and cerebellum were performed. The most prominent [3H]IP3 binding among these regions was found in the cerebellum, particularly in the molecular layer. Within the hippocampus, the subiculum and the CA1 region showed much more prominent binding than the other subfields. [3H]IP4, binding was fairly homogeneous in the regions studied, with the exception of a slightly higher binding in the molecular layer of the cerebellum.     

In vivo [11C]flumazenil-PET correlates with ex vivo [3H]flumazenil autoradiography in hippocampal sclerosis

By using [11C]flumazenil-positron emission tomography ([11C]FMZ-PET), we have previously shown that reductions of central benzodiazepine receptors (cBZRs) are restricted to the hippocampus in mesial temporal lobe epilepsy (mTLE) caused by unilateral hippocampal sclerosis (HS). Receptor autoradiographic studies on resected hippocampal specimens from the same patients demonstrated loss of cBZRs that was over and above loss of neurons in the CA1 subregion. Here, we report the first direct comparison of in vivo cBZR binding with [11C]FMZ-PET and ex vivo binding using [3H]FMZ autoradiography. We applied a magnetic resonance imaging-based method for partial volume effect correction to the PET images of [11C]FMZ volume of distribution ([11C]FMZ Vd) obtained in 10 patients with refractory mTLE due to unilateral, histologically verified HS. Saturation autoradiography was performed on the hippocampal specimens obtained from the same patients, allowing calculation of receptor availability ([3H]FMZ Bmax). After correction for partial volume effect, [11C]FMZ Vd in the body of the epileptogenic hippocampus was reduced by a mean of 42.1% compared with normal controls. [3H]FMZ Bmax, determined autoradiographically from the same hippocampal tissue, was reduced by a mean of 42.7% compared with control hippocampi. Absolute in vivo and ex vivo measurements of cBZR binding for the body of the hippocampus were significantly correlated in each individual. Our study demonstrates that reduction of available cBZR on remaining neurons in HS can be reliably detected in vivo by using [11C]FMZ-PET after correction for partial volume effect.     

In vivo uptake of [3H]nimodipine in focal cerebral ischemia: modulation by hyperglycemia

Cell membrane depolarization and tissue acidosis occur rapidly in severely ischemic brain. Preischemic hyperglycemia is recognized to increase ischemic tissue acidosis and the present studies were undertaken to correlate depolarization and tissue acidosis during acute focal cerebral ischemia and hyperglycemia. We used a dual-label autoradiography method to simultaneously measure the in vivo distribution of [3H]nimodipine and [14C]DMO (5,5-dimethyl-2,4-oxazolidinedione) in brain to identify regions of ischemic depolarization and measure regional net tissue pH. Regional cerebral blood flow (CBF) was measured in separate studies. Measurements were made 30 minutes after combined middle cerebral artery and ipsilateral common carotid artery occlusion in normoglycemic and hyperglycemic rats. Tissue pH in the ischemic cortex was depressed to 6.76 +/- 0.11 in normoglycemic rats (n = 12) and 6.57 +/- 0.13 in hyperglycemic rats (n = 12), with significantly greater acidosis in the hyperglycemic group (P < 0.001). In contrast the ratio of [3H]nimodipine uptake in the ischemic cortex relative to the contralateral nonischemic cortex was significantly greater in normoglycemic (1.83 +/- 0.45) than hyperglycemic (1.40 +/- 0.50) rats (P < 0.05). Within this region of ischemic cortex CBF was 31 +/- 22 mL/100 g in normoglycemic rats (n = 8) and 33 +/- 22 mL/100 g/min in hyperglycemic rats (n = 9). Cerebral blood flow did not differ between these two groups in any region. Thus hyperglycemia reduced the extent of ischemic depolarization within the cortex during the first 30 minutes of focal cerebral ischemia. This effect may be related to the increased tissue acidosis or to other factors that may lessen calcium influx and preserve cellular energy stores in the ischemic cortex of the hyperglycemic rats.     

Effect of diflubenzuron on the incorporation of UDP-N-acetyl-[3H]glucosamine (UDP-[3H]NAGA) to chitin in permeabilized, and isolated integuments from the newly molted American cockroach Periplaneta americana

1. Diflubenzuron (DFB) was found to inhibit the incorporation of UDP-N-acetyl-[3H]glucosamine (UDP-[3H]NAGA) to chitin in permeabilized and isolated integument from newly molted American cockroach. 2. The favorable experimental conditions demonstrating the effect of diflubenzuron were: 10 mM phosphate, low calcium concentration (10(-6) M-10(-8) M), high potassium concentration (> 100 mM), and high pH (> or = 7). 3. The action of diflubenzuron was completely erased by preincubating the isolated integument with valinomycin, FCCP, or A23187. 4. By lowering the external pH to 5.2, it was also possible to reduce the rate of UDP-[3H]NAGA incorporation to the extent that DFB's effect was no longer recognizable. 5. Both Cs+ and Rb+ could replace K+ in maintaining a high level of chitin synthesis and the inhibitory action of DFB under the optimum conditions.     

Specific transfer of 3H from D-[3-3H]gluconic acid into L-tartaric acid in vitaceous plants

Transfer of 3H from D-gluconic acid, specifically labelled with 3H at C-2 or C-3 and 14C at C-1, C-2, or C-3, 4, to L(+)-tartaric acid was examined in leaves and berries of Vitis labrusca cv Delaware and in leaves of Parthenocissus quinquefolia. 3H located at C-3 of D-gluconic acid was highly conserved in this transfer, yielding a 3H/14C ratio between 3.3 and 14 in the light and between 11 and 22 in the dark. These experiments strongly suggest that a portion of the 3H present in L(+)-tartaric acid may have been transferred from D-gluconic acid to L(+)-tartaric acid, possibly via NADP[3H] through a redox process involving reduction of L-xylo-2-hexulosonate (2-keto-L-idonate). Both [3H]-tartaric acid and [14C]tartaric acid synthesized in grape leaves from D-[3-3H, 2-14C]gluconic acid, or [3-3H, 3,4-14C]gluconic acid were characterized as L(+)-chiral form exclusively, the naturally occurring from of tartaric acid.     

Autoradiographic study on [3H]-[D-Ala2]-deltorphin-I binding sites in the rat brain

Previous biochemical and pharmacological studies have shown that [D-Ala2]-deltorphin-I (DADTI) has a high affinity and selectivity for delta-opioid receptors. In this study, designed to provide morphological details, the distribution of DADTI binding sites was examined by autoradiography on coronal, sagittal and horizontal frozen sections of adult rat brain. The sections were incubated with tritiated DADTI solution and exposed for 12 weeks to a 3H-sensitive film. DADTI labelling clearly demonstrated selective and high affinity binding sites of delta-opioid type in several brain regions, including olfactory system, neostriatum, nucleus accumbens, and cortical layers I-II and V-VI.     

Dexamethasone: increased weights and decreased [3H] estradiol retention of uterus, vagina and pituitary in the ovariectomized rat

Ovariectomized rats given 100 mug dexamethasone per day for 5 days had significantly heavier dry weights for uterus, vagina and pituitary, indicating a growth promoting activity of dexamethasone on these tissues in which estrogen normally promotes growth changes. The dexamethasone treated animals also retained significantly less [3H]estradiol per mug dry weight of tissue for uterus, vagina and pituitary. When[3H]estradiol retention was examined in vitro for the nuclear fraction, a significant decrease in retention was found for uterus, vagina and pituitary but not for hypothalamus or cerebral cortex. The decreased ability to bind [3H]estradiol, shown by the estrogen target tissues of the dexamethasone-treated rats, along with the increased growth of the estrogen target tissues, demonstrates that these tissues were able to show trophic responses even when greater levels were one-third of normal. Dexamethasone-treated animals tested for sexual receptivity in the presence or absence of progesterone priming did not show induction of facilitation of sexual receptivity. However, estrogen plus progesterone injections induced sexual receptivity in the presence of dexamethasone. When dexamethasone was combined with a dosage of estrogen, which by itself did not induce sexual receptivity, there was a significant response with 6 to 10 animals showing a low level of receptivity. Thus, dexamethasone can apparently synergize with estrogen to facilitate sexual receptivity.     

Nicotine-stimulated release of [3H]norepinephrine from fetal rat locus coeruleus cells in culture

Acute nicotine administration stimulated [3H]norepinephrine ([3H]NE) release from cultured fetal locus coeruleus (LC) cells. The effect was concentration dependent, with an EC50 of 0.9 microM, and was abolished by removal of calcium from, or addition of tetrodotoxin (500 nM) to, the assay buffer. Other nicotinic receptor agonists stimulated [3H]NE release, with the rank order of potency being (+)-epibatidine > (-)-nicotine > 1,1-dimethyl-4-phenylpiperazinium (DMPP). Whereas (-)-nicotine and (+/-)-epibatidine exhibited equal maximal responses, DMPP was a partial agonist and (-)-cytisine had no agonist activity. Nicotine-stimulated release of [3H]NE was blocked by nicotinic receptor antagonists, with an order of potency of mecamylamine > lobeline > cytisine > methyllycaconitine > dihydro-beta-erythroidine. The pharmacological profile of this nicotinic receptor is largely consistent with that described previously for an alpha4beta2 subunit combination, although discrepancies in the efficacies of agonists were observed. No additivity in NMDA- and nicotine-stimulated [3H]NE release was observed, suggesting a common signal transduction mechanism. However, the pharmacological characteristics of MK-801 blockade of nicotine-induced responses were not consistent with those of an NMDA receptor. We therefore conclude that nicotine directly releases [3H]NE from LC cells and does not act indirectly via activation of glutamate release.     

Characterization of [3H]staurosporine binding in protein kinase C-II purified from rat brain

The type II protein kinase C (PKC-II) densely present in mammalian brain plays functional roles in CNS. We examined the characteristics of [3H]staurosporine binding to PKC-II purified from rat brain, compared to [3H]phorbol 12, 13-dibutyrate (PDBu) binding. In brief, [3H]staurosporine binding increased by phosphatidylserine (PtdSer) in a concentration-dependent manner and the binding was enhanced by Ca2+ and phorbol 12-myristate 13-acetate (PMA). In the presence of Ca2+, PMA and PtdSer, Bmax of these bindings markedly increased, but KD did not change. These characteristics of binding were similar to [3H]PDBu binding to PKC-II. Although [3H]PDBu binding was not affected by protein kinase inhibitors such as staurosporine, H-7, K-252a and K-252b, [3H]staurosporine binding was inhibited by these inhibitors. [3H]staurosporine binding was inhibited by several ATP analogues, but was not by guanine nucleotides. PtdSer-induced increase in [3H]PDBu binding was inhibited by Zn2+, but Zn2+ induced increase in [3H]staurosporine binding as well as PtdSer and/or Ca2+. Staurosporine would thus appear to bind to a domain different from phorbol ester-binding one in PKC, interactions between both domains may regulate kinase activity, and 1 mol staurosporine and 4 mol phorbol ester may bind to 1 mol PKC-II.     

Dopamine transporter development in postnatal rat striatum: an autoradiographic study with [3H]WIN 35,428

The dopamine transporter mediates the reinforcing effects of cocaine, thus playing a central role in human cocaine addiction, and perhaps providing the mechanism for inducing the effects of prenatal cocaine exposure. This possibility has stimulated growing interest in the normal and abnormal development of this transporter. [3H]WIN 35,428 is a cocaine analog that is useful for studying the distribution and density of the dopamine transporter in striatum and other brain regions. The postnatal development of the dopamine transporter in the rat striatum was measured by quantitative autoradiography with [3H]WIN 35,428. Dopamine transporter levels were low at birth, increased through day 15, followed by much more rapid growth in late postnatal development. The majority of the transporter sites appeared after day 15. Lateral to medial and anterior to posterior gradients in transporter density were established early during development, and there was also an early concentration of transporter in striosomes that became difficult to identify by day 15. Differences between the developmental patterns described here and studies using other ligands for the dopamine transporter suggest there are significant differences in the transporter binding sites for these drugs. These differences in transporter ligand binding characteristics may reflect developmental changes in post-translational modification of the transporter and/or changes in the functional activity rather than simply the presence of the transporter.     

Effects of alpha-D- and beta-L-glucose pentaacetate on effluent radioactivity from pancreatic islets labelled with [(32)P]phosphate and myo-[2-(3)H]inositol

The anomers of both D-glucose pentaacetate and L-glucose pentaacetate were recently found to display insulinotropic potential. In order to progress in understanding the mode of action of these esters in islet cells, we have now investigated whether they mimic the effect of nutrient secretagogues to cause a phosphate flush and activation of phospholipase C in isolated islets. For this purpose, rat pancreatic islets were prelabelled with either [(32)P]orthophosphate or myo-[2-(3)H]inositol and placed in a perifusion chamber. In the absence of any other exogenous nutrient, the administration of alpha-D-glucose pentaacetate (1.7 mM) from 46 to 70 min of perifusion increased, after an initial transient fall, both 32P and 3H fractional outflow rates and stimulated insulin release from the perifused islets. No secondary rise in either (32)P or (3)H outflow and no sizeable stimulation of insulin release was observed, however, in response to Beta-L-glucose pentaacetate (also 1.7 mM). These findings are consistent with the view that the insulinotropic action of alpha-D-glucose pentaacetate entails a nutrient-like component leading to the occurrence of both a phosphate flush and hydrolysis of phosphoinositides. This is not the case, however, for Beta-L-glucose pentaacetate. The latter ester might act directly on a yet unidentified receptor, the early secretory response to alpha-D-glucose pentaacetate also apparently involving such a direct effect of the ester itself.     

Covalent interaction of [3H]-dopamine with rat brain proteins in vivo and with the dopamine-reuptake site of nerve endings in vitro

Isolated nerve endings have been demonstrated to undergo saturable, covalent interactions with [3H]-dopamine under physiological conditions; and the reaction is greatly accelerated by flash photolysis with ultraviolet light. With intact nerve endings, under conditions where dopamine reuptake occurs, benztropine and cocaine (inhibitors of dopamine reuptake), but not atropine or haloperidol (a postsynaptic antagonist), prevent the reaction. The reaction also occurs in vivo following the intraventricular administration of [3H]-dopamine, the reaction being greatest with mitochondria, followed by the nerve ending and myelin. With the use of sodium dodecylsulfate-gel electrophoresis, a number of proteins of varying molecular weight were labeled, and the pattern of labeling was similar in vitro and in vivo. One protein, with a MW of about 60,000 was labeled to an exceptionally high degree. A number of protein bands showed decreased radiolabeling in the presence of benztropine, a finding which suggests that they may be associated with the reuptake site. Both the addition of ascorbic acid and unlabeled dopamine inhibited the reactivity of [3H]-dopamine, and the effects were concentration dependent. In the absence of photolysis, the reaction (3H]-dopamine to synaptic membranes attained saturation within 10 min, but with photolysis and reaction continued at a constant rate even after 20 min. The results are discussed in relation to the use of [3H]-dopamine as a photoaffinity label of the dopamine reuptake site and in relation to the nature of the reactions with and without photolysis.     

The distribution of [3H]synthanecine A bis-N-ethylcarbamate and its metabolites in the rat

Using long exposures of stripping film autoradiographs before processing, mixtures of weakly and strongly labelled nuclei were seen in different areas of the mouse spleen. Previous results (Harris et al., 1973) led to the conclusion that many cells, not in division cycle, were labelling with (3H) thymidine and that this process was important for the development of specific antibody-producing cells following stimulation with an antigen such as sheep red cells (SRC). The present data are an analysis of the (3H) thymidine labelling kinetics in the spleens of mice reared in conventional or germ-free conditions. The labelling seen in the 24 h following an injection of (3H) thymidine could best be interpreted on the basis of synthesis of unstable DNA. The changes in the pattern, and distribution of labelled nuclei as well as the intensity of their labelling was not compatible with cell division only, but was also the result of movement of labelled material between the lymphoid cells of the organ. Germ-free mice were followed for 24 days following a single injection of (3H) thymidine. The rate of uptake of label into the spleen was much slower than has been found previously in mice reared in conventional conditions. When SRC were injected 2 h after giving (3H) thymidine the labelling of lymphoid cells in the spleen and blood was quite different to controls given (3H) thymidine alone. Detailed analysis indicated that turnover of labelled material, presumably DNA, as well as cells was involved. This turnover of DNA could be considered to be metabolic in the sense that renewal, increase in amount, loss, and transfer to other cells were involved. These, and other studies, in vivo (Harris & Olsen, 1973) and in vitro (Harris et al. 1975) indicate that such processes, involving DNA, are highly relevant to the development of antibody-producing capacity by cells responding to antigenic challenge.     

Assessment of the role of TRH in the release of [3H]-dopamine from rat nucleus accumbens-lateral septum slices

We have studied [3H]-dopamine ([3H]-DA) release from rat nucleus accumbens lateral septum slices in response to various paradigms aimed at increasing endogenous or exogenous thyrotropin releasing hormone (TRH) concentrations in the extracellular space. High KCl concentrations significantly enhanced [3H]-DA release by fourfold. TRH (10(-4) or 5 x 10(-4) M) did not affect [3H]-DA release. The release of [3H]-DA was not stimulated by TRH either in the presence of N-1-carboxy-2-phenylethyl (N(im)benzyl)-histidyl-beta naphthylamide, a specific pyroglutamyl peptidase II inhibitor, or that of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptidase I. None of the peptidase inhibitors modified the [3H]-DA release by themselves. These results suggest that the TRH stimulation of [3H]-DA release in vitro observed in previous studies is not due to peptide inactivation but may be due to a nonspecific effect. TRH enhancement of DA release in nucleus accumbens in vivo may not be the result of a direct effect of TRH on DA terminals.     

In vivo and in vitro effects of muscarinic receptor antagonists on contractions and release of [3H]acetylcholine in the rabbit urinary bladder

We show here that hemocytes and leukocytes with phagocytic activity from both invertebrates (Planorbarius corneus, Viviparus ater) and vertebrates (Carassius auratus, Rana esculenta) express pro-opiomelanocortin (POMC) mRNA, as assessed by in situ hybridization with a digoxigenin-labeled human DNA probe. These data are in accord with previous observations from our laboratories on the presence in these cells of POMC-derived peptides and strongly suggest that these molecules--highly conserved throughout evolution--play an important role in cell locomotion and phagocytosis. POMC mRNA was also detected in lymphocytes of R. esculenta, but not of C. auratus, suggesting that from anuran amphibians onwards lymphocytes also express this gene. This phenomenon could be related to the appearance of more than one immunoglobulin isotype in anurans.     

In vivo and in vitro regulation of [3H]glyburide binding to brain sulfonylurea receptors in obesity-prone and resistant rats by glucose

Select brain neurons increase their firing rate when ambient glucose levels rise, possibly via a neuronal ATP-sensitive K+ (KATP) channel and its associated sulfonylurea receptor (SUR). We used receptor autoradiographic binding of 20 nM [3H]glyburide (in the presence or absence of Gpp(NH)p which blocks binding to low-affinity sites) to assess the in vivo and in vitro effects of altering glucose availability upon high- and low-affinity binding to brain SUR. Since the brain's ability to monitor and regulate glucose metabolism is critical to maintenance of energy balance, testing was done in chow-fed male Sprague-Dawley rats which had an underlying predisposition to develop either diet-induced obesity (DIO-prone) or to be diet-resistant (DR-prone) when subsequently fed a high-energy diet. Under control conditions, both in vivo and in vitro studies showed DIO-prone rats to have reduced levels of low-, but not high-affinity [3H]glyburide binding in most forebrain areas. As compared to equiosmolar infusions of mannitol, 60 min unilateral intracarotid glucose infusions at 4 mg/kg/min in awake rats reduced low-affinity [3H]glyburide binding in numerous hypothalamic and amygdalar areas of both DR- and DIO-prone rats with little effect on high-affinity binding. Only in the paraventricular nucleus of DR-prone rats was there a phenotype-specific downregulation of low-affinity binding. Brain sections from other rats were incubated with [3H]glyburide in the presence of 0, 5 or 10 mM glucose. The resultant in vitro effects of glucose were more variable and widespread than intracarotid infusions. Here, glucose often increased low-affinity [3H]glyburide binding, particularly in DR-prone rats at 5 mM. Again, there was little effect on high-affinity binding. Thus, glucose may affect the firing of glucose-responsive neurons by indirectly altering KATP channel function via its effects on low-affinity cell body SUR.     

Further characterization of [3H]-CGS 21680 binding sites in the rat striatum and cortex

1. The putative high affinity binding site for the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N- ethylcarboxamidoadenosine (CGS 21680) in the rat cerebral cortex was characterized by use of a number of selective A1 and A2 adenosine receptor ligands, and compared to the characteristics of the more abundant striatal A2A receptor. 2. The binding of [3H]-CGS 21680 to cortical membranes was performed at pH 5.5, in order to increase the amount of specific binding. 3. Reduction of the pH from 7.4 to 5.5 increased the apparent affinity of the striatal binding side for both agonists and antagonists. The relative order of potencies of both groups of ligands were the same at both pH values, and were consistent with binding to the A2A receptor. There was no observable change in the Bmax, the values being 415 and 446 fmol mg-1 protein at pH 5.5 and 7.4 respectively. 4. The cortical binding site yielded a Bmax value of 117 fmol mg-1 protein. The relative order of potencies of the adenosine receptor ligands observed at this binding site were not the same as those observed in the striatum, exhibiting a profile with both A1 and A2 characteristics. 5. Further characterization of this cortical binding site in the presence of the A1 selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) revealed a more typical A2A profile. This indicated that under the conditions used there were two components of [3H]-CGS 21680 binding, approximately 20% of the A1 receptor and 80% to the A2A receptor. 6. It is concluded that in the cerebral cortex there is a CGS 21680 binding site showing the characteristic properties of the striatal A2A receptor, and no evidence was obtained for the existence of a novelA2A-like binding site.