Yukiko Tokumitsu

Desensitization of β-Adrenergic Receptor-Coupled Adenylate Cyclase in Cerebral Cortex After In Vivo Treatment of Rats with Desipramine

Abstract: Continuous treatment (1–10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the β‐adrenergic receptor‐coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol‐stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of β‐adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl‐5′‐yl‐β‐γ‐imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p‐induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug‐treated rats. The lag was shortened by the addition of isoproterenol, indicating that β–receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of β‐adrenergic receptor‐mediated adenylate cyclase in the cerebral cortex during desipramine therapy.

Troglitazone suppresses cell growth of KU812 cells independently of PPARγ

We examined the effects of troglitazone, one of thiazolidinedione derivatives on human basophilic leukemia cell line KU812. Troglitazone caused the suppression of cell growth, which was suggested to result from the decrease in cyclin E and the hyperphosphorylated form of retinoblastoma tumor suppressor gene product (pRb). In addition, troglitazone caused a decrease in histamine secretion due to the reduced expression of histidine decarboxylase mRNA. Peroxisome proliferator-activated receptor (PPAR)-gamma mRNA was undetectable by reverse transcription-polymerase chain reaction (RT-PCR) in KU812 cells. These findings suggested that troglitazone suppressed cell growth and histamine synthesis independently of PPARgamma.

Inositol-1, 4, 5-trisphosphate accumulation induced by urinary pheromones in female rat vomeronasal epithelium

The mechanisms involved in pheromone-induced responses in the vomeronasal neurons, especially in mammals, are still unclear. In the present study, we examined the effects of rat urine samples containing various types of pheromones regulating gonadal functions on the accumulation of cAMP and inositol 1,4,5-trisphosphate (IP3) in a vomeronasal membrane preparation from the female Wistar rat. Stimulation of the preparation with forskolin induced cAMP accumulation, but stimulation with urine samples excreted from the male Wistar rat, the female Wistar rat, and the male Donryu rat did not change cAMP levels. These results were consistent with the electrophysiological results showing that dialysis of a high concentration of cAMP into the vomeronasal neuron does not induce currents. Stimulation with the three urine samples induced the accumulation of IP3 in the membrane preparation. These results are consistent with previous electrophysiological results [K. Inamura, M. Kashiwayanagi, K. Kurihara, Inositol-1,4,5-trisphosphate induces responses in receptor neurons in rat vomeronasal sensory slices, Chem. Senses 22 (1997) 93-103; K. Inamura, M. Kashiwayanagi, K. Kurihara, Blockage of urinary responses by inhibitors for IP3-mediated pathway in rat vomeronasal sensory neurons, Neurosci. Lett. 233 (1997) 129-132]. After the treatment with Pertussis toxin (PTX), the male Wistar urine did not induce IP3 accumulation significantly. Application of the male Wistar urine decreased ADP-ribosylation of Gi with PTX, while that of the male Donryu urine decreased ADP-ribosylation of Go. Thus, the present results support a mechanism by which the responses of the rat vomeronasal neurons to urinary pheromones are mediated by IP3, Gi and/or Go.

Reduced concentrations of the α-subunit of GTP-binding protein Go in schizophrenic brain

Concentrations of the α-subunits of GTP-binding protein, Go (Goα) and of Gi 2 (Gi 2α) in 6 areas (the hippocampus, parahippocampus, putamen, caudate head, orbital frontal cortex, and lateral temporal cortex) of control and schizophrenic postmortem brains were investigated using the highly sensitive enzyme immunoassay method. There was a significant decrease in Goα in the hippocampus and caudate head of the right hemisphere in schizophrenic patients compared to controls; the ANOVA (a general linear model; SAS Type II) demonstrated a significant diagnosis × side interaction only in the hippocampus. In other areas of the brain, analysis by grouping under diagnosis, side, age, gender, and postmortem delay showed no significant deviations in Goα between controls and schizophrenics. The concentrations of Gi 2α did not differ significantly in any area. These findings contrasted with the results yielded by ADP-ribosylation, which showed decreased pertussis toxin ADP-ribosylated amounts in the hippocampus and putamen of the contralateral (left) hemisphere. Some abnormal receptor — Go or Gi 1 signalling in hippocampus, putamen or caudate head may be involved in the pathogenesis of schizophrenia.

Possible Involvement of Pertussis Toxin Substrates (Gi, Go) in Desipramine-Induced Refractoriness of Adenylate Cyclase in Cerebral Cortices of Rats

To evaluate the efficiency of coupling between β‐receptor and adenylate cyclase catalyst via a GTP‐binding protein, Gs, in the brain membrane two parameters were employed: a β‐agonist‐induced increase in the membrane GTP‐dependent adenylate cyclase activity and a β‐agonist‐induced shortening of the lag time preceding the onset of the steady‐state activation by guanyl‐5′‐yl‐β‐γ‐imidodiphosphate [Gpp(NH)p] of the membrane cyclase. Both parameters showed lower values in membranes from desipramine‐treated rats compared with untreated rats. Thus, coupling of β‐adrenergic receptors to adenylate cyclase in the brain membrane was impaired by the desipramine treatment. Rats once injected intraventricularly with isletactivating protein (IAP), pertussis toxin, were subjected to desipramine treatment, for the purpose of studying effects of another kind of the GTP‐binding protein (Gi), which loses its function as a signal transducer on being ADP‐ribosylated selectively by the toxin. IAP treatment did not impair the β‐receptor coupling by itself, since neither of the above two parameters for the coupling were reduced by IAP treatment. Moreover, the first parameter was normalized. though the second one was not, by superimposition of the IAP treatment upon the desipramine‐treated rats. It seems likely, therefore, that Gi interacts with a Gs‐adenylate cyclase coupling in an inhibitory fashion in brain membranes. The desensitization might be overcome when the inhibitory interaction of Gi on the subsequent process is attenuated by IAP treatment.

Pertussis Toxin Attenuates 5‐Hydroxytryptamine1A Receptor‐Mediated Inhibition of Forskolin‐Stimulated Adenylate Cyclase Activity in Rat Hippocampal Membranes

Abstract: The inhibition of forskolin‐stimulated adenylate cyclase activity by 5‐hydroxytryptamine (5‐HT) receptor agonists was measured in rat hippocampal membranes isolated from animals treated with vehicle or islet‐activating protein (IAP; pertussis toxin). In vehicle‐treated animals, 5‐HT, 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, buspirone, and gepirone were potent in inhibiting forskolin‐stimulated adenylate cyclase activity with EC50 values of 60, 76, 376, and 530 nM, respectively. IAP treatment reduced by 30–55% the 5‐HT1A agonist inhibition of adenylate cyclase activity via 5‐HT1A receptors. The data indicate that the inhibitory guanine nucleotide‐binding protein or Go (a similar GTP‐binding protein of unknown function purified from brain) mediates the 5‐HT1A agonist inhibition of hippocampal adenylate cyclase.

The incorporation of 32Pi into intramitochondrial ADP fraction dependent on the substrate-level phosphorylation

1. A significant amount of 32Pi is incorporated into ADP fraction if mitochondrial phosphorylation is allowed to proceed solely dependent on the endogenous adenine nucleotides even in the absence of uncouplers or inhibitors of oxidative phosphorylation. This formation of [32P]ADP is accompanied by a significant labelling of the GTP fraction as well as by a decrease in mitochondrial AMP. 2. A good correlation, highly significant on a statistical basis, is obtained between the incorporation of 32Pi into ADP on the one hand and the oxidation of [1-14C]glutamate to 14CO2 on the other, under a wide variety of conditions of respiration, suggesting that the substrate-level phosphorylation linked to the oxidation of 2-oxoglutarate leads to the phosphorylation of AMP in rat liver mitochondria. 3. Since intramitochondrial GTP is not directly labelled by the [32P]ATP added, it is concluded that neither nucleoside diphosphokinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) nor adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) is functioning in such an EDTA-containing medium as employed in the present study because of lack of the enzymes inside the inner membrane. This not only indicates that ATP never serves as a phosphate donor for the observed phosphorylation of AMP, but also, along with several other lines of evidence, lends strong support to the view that [32P]GTP generated as a result of the substrate-level phosphorylation is a direct precursor of [32P]ADP through the mediation of GTP:AMP phosphotransferase, which has been verified to be located inside the inner membrane by the significant labelling of GTP by [32P]ADP.

Separation and determination of 14C-labelled intermediates of the citric acid cycle and related compounds.

Abstract A new systematic procedure is presented which permits a complete chromatographic resolution of the intermediates of the citric acid cycle as well as the related amino and keto acids. The adsorption onto ion-exchange columns followed by the subsequent elution therefrom forms the first step of the present procedure. Meanwhile, unstable keto acids are enzymatically converted into the respective amino acids. This preliminary process is very effective in desalting the biological specimen and also results in a broad division of a large number of intermediates into three subgroups, i.e., amino, keto and other organic acid fractions. Each of these subgroups is then readily resolved into individual acids by means of one-dimensional thinlayer chromatography. 14 C-Compounds added to the reaction mixture of rat liver mitochondria were recovered with a good reproducibility throughout the entire course of the present procedure.

Protein kinase A-dependent IL-6 production induced by calcitonin in human glioblastoma A172 cells.

In human glioblastoma A172 cells, interleukin-6 (IL-6) production was induced by interleukin-1 beta (IL-1 beta) and dibutyryl cyclic AMP. These cells have been shown to induce IL-6 production via a cAMP-protein kinase A system. Since calcitonin (CT) and calcitonin gene-related peptide (CGRP) are known to increase cAMP accumulation in murine and rat astrocytes, we examined whether these neuropeptides induced IL-6 production in A172 cells. Human CT and human CGRP increased IL-6 production and cAMP accumulation in a dose-dependent manner. A specific protein kinase A inhibitor, H-89, inhibited both CT- and CGRP-induced IL-6 production. CT and CGRP have been shown to cross-react with each other. To exclude the possibility of this cross-reactivity, we studied the additive effects of CT and CGRP and the inhibitory effects of specific inhibitors. When 100 nM CT was added, cAMP accumulation stimulated by 10 nM CGRP (the maximal dose) was increased. CGRP (8-37), a specific CGRP receptor inhibitor, inhibited cAMP accumulation and IL-6 production induced by CGRP, but did not inhibit these effects when they were induced by CT. Salmon CT (8-32), a specific inhibitor of the CT receptor, inhibited cAMP accumulation induced by CT, but did not inhibit the effect induced by CGRP. These results demonstrated that CT can induce IL-6 production via cAMP accumulation and the effects of CT are mediated via its own receptors.

Suppression of insulin-stimulated phosphatidylinositol 3-kinase activity by the β3-adrenoceptor agonist CL316243 in rat adipocytes

Insulin increased 2‐deoxyglucose (2‐DG) uptake via the translocation of glucose transporter (GLUT) 4 to the plasma membrane fraction in rat adipocytes. The stimulatory actions of insulin were accompanied by both an increase in the immunoreactive p85 subunit of phosphatidylinositol (PI) 3‐kinase in the plasma membrane fractions and PI 3‐kinase activation by tyrosine phosphorylation of the p85 subunit. The β3‐adrenoceptor agonist CL316243 (CL) suppressed all the insulin actions in adenosine deaminase (ADA)‐treated cells, but was without effect in non‐ADA‐treated cells. The inhibitory effects of CL on GLUT 4 translocation and PI 3‐kinase activation were abolished by the addition of N6‐phenylisopropyl adenosine. Cholera toxin treatment, which markedly increased intracellular cAMP levels, suppressed increases in the levels of GLUT 4 and PI 3‐kinase in the plasma membrane fractions in response to insulin. In addition, dibutyryl (Bt2) cAMP also impaired the activation of PI 3‐kinase by insulin. These results indicated that CL suppressed insulin‐stimulated glucose transport under conditions where cAMP levels were markedly increased (∼12‐fold). The inhibitory actions of PI 3‐kinase activation by insulin were exerted even when cAMP, 8‐bromo‐cAMP, or Bt2 cAMP was added to immunoprecipitates of the p85 subunit of PI 3‐kinase, after treating the cells with insulin. These results suggest that CL suppressed insulin‐stimulated PI 3‐kinase activity via a cAMP‐dependent mechanism, at least in part, direct cAMP action in ADA‐treated adipocytes, by which PI 3‐kinase activation was inhibited, resulting in the decrease in GLUT 4 translocation and subsequent 2‐DG uptake in response to insulin.