Tissue transglutaminase overexpression in the brain potentiates calcium-induced hippocampal damage.

Tissue transglutaminase (tTG) post-translationally modifies proteins in a calcium-dependent manner by incorporation of polyamines, deamination or crosslinking. Moreover, tTG can also bind and hydrolyze GTP. tTG is the major transglutaminase in the mammalian nervous system, localizing predominantly in neurons. Although tTG has been clearly demonstrated to be elevated in neurodegenerative diseases and in response to acute CNS injury, its role in these pathogenic processes remains unclear. Transgenic mice that overexpress human tTG (htTG) primarily in CNS neurons were generated to explore the role of tTG in the nervous system and its contribution to neuropathological processes. tTG transgenic mice were phenotypically normal and were born with the expected Mendelian frequency. However, when challenged systemically with kainic acid, tTG transgenic mice, in comparison to wild-type (WT) mice, developed more extensive hippocampal neuronal damage. This was evidenced by a decreased number of healthy neurons, and increased terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) labeling as an indicator of neuronal cell death in the kainic acid-treated transgenic mice. Moreover, the duration and severity of seizures developed by htTG transgenics in response to kainic acid administration were significantly more pronounced than those observed in WT mice. These data indicate for the first time that tTG may play an active role in excitatory amino acid-induced neuronal cell death, which has been postulated to be an important component of acute CNS injury and chronic CNS neurodegenerative conditions.

2-[4-(3,4-Dimethylphenyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393), a selective dopamine D4 receptor antagonist.

2-[4-(3,4-Dimethylphenlyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393) was identified as a potent dopamine D4 receptor antagonist with excellent receptor selectivity. [3H]-spiperone competition binding assays showed that A-381393 potently bound to membrane from cells expressing recombinant human dopamine D4.4 receptor (Ki=1.5 nM), which was 20-fold higher than that of clozapine (Ki=30.4 nM). A-381393 exhibited highly selective binding for the dopamine D4.4 receptor (>2700-fold) when compared to D1, D2, D3 and D5 dopamine receptors. Furthermore, in comparison to clozapine and L-745870, A-381393 exhibits better receptor selectivity, showing no affinity up to 10 microM for a panel of more than 70 receptors and channels, with the exception of moderate affinity for 5-HT2A (Ki=370 nM). A-381393 potently inhibited the functional activity of agonist-induced GTP-gamma-S binding assay and 1 microM dopamine induced-Ca2+ flux in human dopamine D4.4 receptor expressing cells, but not in human dopamine D2L or D3 receptor cells. In contrast to L-745870, A-381393 did not exhibit any significant intrinsic activity in a D4.4 receptor. In vivo, A-381393 has good brain penetration after subcutaneous administration. A-381393 inhibited penile erection induced by the selective D4 agonist PD168077 in conscious rats. Thus, A-381393 is a novel selective D4 antagonist that will enhance the ability to study dopamine D4 receptors both in vitro and in vivo.

Enhanced G-protein activation by a mixture of Abeta(25-35), Abeta(1-40/42) and zinc.

Beta-amyloid peptides (Abetas) bind to several G-protein coupled receptor proteins and stimulate GTPase activity in neurons. In this study we determined the effects of Abeta(1-42), Abeta(1-40), Abeta(25-35) and their mixtures on [(35)S]GTP binding in rat brain cortical membranes in the absence and presence of zinc. We found that the Abetas alone induced a concentration-dependent activation of G-proteins (IC50 approximately 10(-6) m), while aggregated Abeta fibrils only affected GTP binding at concentrations above 10(-5) m. Mixing Abeta(25-35) with Abeta(1-42) or Abeta(1-40) induced a several-fold increase in GTP-binding. This potentiation followed a bell shaped curve with a maximum at 50 : 50 ratios. No potentiating effect could be seen by mixing Abeta(1-40) and Abeta(1-42) or highly aggregated Abetas. Zinc had no effect on Abeta(1-40/42) but strongly potentiated the Abeta(25-35) or the mixed peptides-induced GTP-binding. Changes in secondary structure accompanied the mixed peptides or the peptide/zinc complexes induced potentiation, revealing that structural alterations are behind the increased biological action. These concentration dependent potentiating effects of zinc and the peptide mixtures could be physiologically important at brain regions where peptide fragments and/or zinc are present at elevated concentrations.

Chronic intrathecal morphine administration produces homologous mu receptor/G-protein desensitization specifically in spinal cord.

Previous studies have shown that chronic i.v. treatment with morphine or heroin decreased mu opioid receptor activation of G-proteins in specific brain regions. The present study examined the effect of intrathecal (i.t.) morphine administration on receptor/G-protein coupling in the spinal cord. In spinal cord membranes, [35S]GTP gamma S binding was stimulated by agonists of several G-protein-coupled receptors, including mu opioid (DAMGO), delta opioid (DPDPE), GABA(B) (baclofen), cannabinoid CB(1) (WIN 55,212-2), muscarinic cholinergic (carbachol) and adenosine A(1) (PIA). [35S]GTP gamma S autoradiography revealed that most of this agonist activation of G-proteins was localized to laminae I and II of dorsal horn. To determine the effects of chronic morphine on these receptor activities, rats were treated for 7 days with 0.11 mg/kg/day i.t. morphine, and receptor activation of G-proteins was determined by [35S]GTP gamma S autoradiography of brain and spinal cord. In spinal cord sections, chronic morphine treatment decreased DAMGO-stimulated [35S]GTP gamma S binding in laminae I and II at all levels of spinal cord examined. There were no effects of morphine treatment on [35S]GTP gamma S stimulation in spinal cord by other receptor systems examined (Adenosine A(1) and GABA(B)), and no significant effects of chronic i.t. morphine treatment were observed in brain sections. These data show that homologous desensitization of mu receptor/G-protein coupling occurs specifically in spinal cord following chronic morphine administration.

Guanine nucleotide transport by atractyloside-sensitive and -insensitive carriers in isolated heart mitochondria.

In previous work (McKee EE, Bentley AT, Smith RM Jr, and Ciaccio CE, Biochem Biophys Res Commun 257: 466-472, 1999), the transport of guanine nucleotides into the matrix of intact isolated heart mitochondria was demonstrated. In this study, the time course and mechanisms of guanine nucleotide transport are characterized. Two distinct mechanisms of transport were found to be capable of moving guanine nucleotides across the inner membrane. The first carrier was saturable, displayed temperature dependence, preferred GDP to GTP, and did not transport GMP or IMP. When incubated in the absence of exogenous ATP, this carrier had a V(max) of 946 +/- 53 pmol. mg(-1). min(-1) with a K(m) of 2.9 +/- 0.3 mM for GDP. However, transport of GTP and GDP on this carrier was completely inhibited by physiological concentrations of ATP, suggesting that this carrier was not involved with guanine nucleotide transport in vivo. Because transport on this carrier was also inhibited by atractyloside, this carrier was consistent with the well-characterized ATP/ADP translocase. The second mechanism of guanine nucleotide uptake was insensitive to atractyloside, displayed temperature dependence, and was capable of transporting GMP, GDP, and GTP at approximately equal rates but did not transport IMP, guanine, or guanosine. GTP transport via this mechanism was slow, with a V(max) of 48.7 +/- 1.4 pmol. mg(-1). min(-1) and a K(m) = 4.4 +/- 0.4 mM. However, because the requirement for guanine nucleotide transport is low in nondividing tissues such as the heart, this transport process is nevertheless sufficient to account for the matrix uptake of guanine nucleotides and may represent the physiological mechanism of transport.

Pharmacological and biochemical strategies to increase the accumulation of arabinofuranosylguanine triphosphatein primary human leukemia cells.

Purine nucleoside phosphorylase deficiency leads to a dGTP-mediated T-lymphopenia, suggesting that an analogue of deoxyguanosine would be selectively effective in T-cell disease. 9-beta-D-Arabinofuranosylguanine (ara-G) is relatively resistant to hydrolysis by purine nucleoside phosphorylase and selectively toxic to T cells, but its low solubility has prevented its use in the clinic. 2-Amino-6-methoxy-arabinofuranosylpurine (GW506U) serves as the water-soluble prodrug for ara-G. A Phase I trial in patients with refractory hematological malignancies demonstrated that the clinical responses to this agent were directly related to the peak levels of ara-G 5'-triphosphate (ara-GTP) in target cells. The aim of the present study was to develop and test strategies to increase intracellular accumulation of ara-GTP in primary human leukemia cells of myeloid and B-lymphoid origin. Three strategies were tested. First, incubations with 100 microM ara-G for 4 h produced a linear median accumulation rate of 19 microM/h (range, 2-45 microM/h; n = 15) in lymphoid leukemia cells and 16 microM/h (range, 0.5-41 microM/h; n = 11) in myeloid leukemia cells. Saturation of ara-GTP accumulation was achieved only after 6-8 h exposure in both lymphoid and myeloid leukemia cells, suggesting a rationale for prolonged infusion. Second, a dose-dependent increase in ara-GTP accumulation was observed with incubations of 10-300 microM ara-G for 3 h. Hence, dosing regimens that achieve high plasma levels of ara-G during therapy may increase cellular levels of ara-GTP. Finally, a biochemical modulation approach using in vitro incubation of leukemia cells with 10 microM 9-beta-D-arabinofuranosyl-2-fluoroadenine for 3 h, followed by either 50 or 100 microM ara-G for 4 h, resulted in a statistically significant median 1.3-fold (range, 1.1-9.0-fold; P = 0.034) and 1. 8-fold (range, 0.9-10.6 fold; P = 0.018) increase in ara-GTP compared to cells incubated with ara-G alone. Extension of these studies to ex vivo incubations confirmed our in vitro findings. These strategies will be used in the design of clinical protocols to increase ara-GTP accumulation in leukemia cells during therapy.

Iontophoresis of bases, nucleosides, and nucleotides.

PURPOSE: To investigate whether transdermal iontophoresis may be potentially useful for delivery of oligonucleotide drugs, the electrotransport of representative bases (uracil and adenine), nucleosides (uridine and adenosine) and nucleotides (AMP, ATP, GTP and imido-GTP) across mammalian skin in vitro has been considered. METHODS: While the passive permeability of all compounds investigated (from 1 mM solutions at pH 7.4) was very low, the application of constant current iontophoresis (0.55 mA/cm2) significantly enhanced the transport of both charged and uncharged species. RESULTS: The efficiency of delivery depended only weakly upon lipophilicity, varied quite linearly with concentration (for AMP and ATP), was inversely sensitive to molecular weight, and was strongly influenced by charge. Neutral solutes were delivered better from the anode than the cathode, as expected; post-iontophoresis, passive permeabilities were greater than those of the untreated controls, suggesting that iontophoretically-induced changes in barrier function cannot be completely repaired in in vitro model systems. The triphosphate nucleotides, ATP and GTP, were essentially completely metabolized (presumably to their corresponding mono-phosphates) during their iontophoretic delivery, while imido-GTP was apparently resistant to enzymatic attack; however, comparison of the transport data from AMP and ATP suggested that ATP metabolism occurred primarily after the rate-limiting step of iontophoresis. CONCLUSIONS: The results obtained are consistent with the general patterns of behavior previously observed in investigations of amino acid and peptide electrotransport. It remains to be seen whether extension of the research described here to larger oligonucleotide species is a feasible long-term objective.

Characterization of M1- and M2-muscarinic receptors in calf retina membranes.

Muscarinic receptors are identified in bovine retina membranes by the specific binding of 1-[benzilic-4,4'-3H]-quinuclidinyl benzilate [3H]-QNB. Binding occurs to one population of non-cooperative binding sites: KD = 0.11 +/- 0.02 nM and Bmax = 0.61 +/- 0.07 pmol/mg protein. Competition binding curves of the M1-selective antagonist pirenzepine are shallow. Computer-analysis reveals the presence of 45 +/- 1% M1-receptors (high affinity sites for pirenzepine, Ki = 31 +/- 10 nM). The remaining low affinity sites (Ki = 1.0 +/- 0.3 microM) are denoted as M2-receptors. Competition binding curves with the agonist carbachol are shallow as well. 1 mM GTP causes a rightward shift and a steepening of the carbachol curve, whereas 1 mM N-ethylmaleimide (NEM) provokes a leftward shift and also a steepening of the curve. The GTP effect is abolished by NEM. Binding of the antagonists [3H]-QNB, atropine or pirenzepine is not modulated by GTP nor by NEM.

GTP-mediated Ca2+ release in rough endoplasmic reticulum. Correlation with a GTP-sensitive increase in membrane permeability.

Guanine nucleotides have been reported to stimulate reticular Ca2+ release. By using the structure-linked latency of microsomal mannose-6-phosphate phosphatase as an index of microsomal permeability [Arion, Ballas, Lange & Wallin (1976) J. Biol. Chem. 251, 4901-4907], the effects of GTP on Ca2+ release and membrane permeability were compared in liver microsomes. In a stripped rough-microsome preparation, GTP caused a dose-dependent increase in mannose 6-phosphate permeability. Half-maximal and maximal effects were observed at 3 microM- and 10 microM-GTP respectively. The time course of the change in membrane permeability coincided with the time course of GTP-dependent Ca2+ release. This increase in microsomal permeability displayed positive to-operativity with respect to GTP (Hill coefficient = 1.8). By analogy to the GTP-dependent Ca2+ release process, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]-triphosphate inhibited the ability of GTP to alter microsomal permeability, but were without effect when added alone. In the presence of 50 microM-GTP, complete inhibition of the GTP-dependent increase in microsomal permeability was achieved with 10 microM-guanosine 5'-[gamma-thio]triphosphate, whereas a 25% inhibition was observed with 10 microM-guanosine 5'-[beta gamma-imido]triphosphate. In contrast with previous observations in crude microsomal preparations, GTP-dependent Ca2+ release in the stripped rough-microsome preparation did not require the addition of poly(ethylene glycol), although the latter did stimulate the rate of Ca2+ release. The ability of GTP to alter microsomal permeability was blocked by prior treatment with the thiol reagent p-hydroxymercuribenzoate; complete inhibition was observed after a 10 min exposure to 50 microM. Inhibition was reversed by subsequent treatment with dithiothreitol. The marked similarities between the two GTP-sensitive processes indicate that they may function via the same mechanism.