<Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>: cytochemical localization of a NTPDase1 and an ecto-5′-nucleotidase and effects of adenine nucleotides on cellular viability

Nucleoside triphosphate diphosphohydrolase 1 (NTPDase1), which hydrolyzes extracellular ATP and ADP, and ecto-5-nucleotidase, which hydrolyzes AMP, are characterized for Trichomonas vaginalis. Ultrastructural cytochemical microscopy showed NTPDase1 and ecto-5-nucleotidase activities on the surface of the parasites. High levels of extracellular adenine nucleotides and adenosine did not exert cytolytic effects in intact cells of T. vaginalis. Our results suggest that these enzymes are relevant for the survival of the parasite during exposure to extracellular nucleotides. Since the ecto-localization of these enzymes is essential for the maintenance of adenosine extracellular levels, this nucleoside could be important for the purine salvage pathway in the parasite.     

The cortical stab injury induces beading of fibers expressing ecto-nucleoside triphosphate diphosphohydrolase 3

The ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), an enzyme involved in degradation of extracellular adenosine triphosphate (ATP), is expressed on nerve fibers in different brain regions, including cortex. Here we studied the expression and role of this enzyme after unilateral cortical stab injury in rats. In cortical sections of control rats, NTPDase3 immunoreactivity was associated with two types of fibers: thin processes, occasionally with small mushroom-like protrusions and slightly thicker fibers with more pronounced and more frequent varicosities, whereas immunopositive neuronal perycaria were never observed. Although NTPDase3–positive thin processes and thicker fibers, by general appearance, size and shape, could be dendrites and axons, respectively, they were never immunopositive for microtubule associated protein-2 or neurofilament H subunit. Cortical stab injury induced rapid (within 4 hours) focal varicose swelling that evolved over time to prominent beading of NTPDase3–positive fibers. The NTPDase3–positive fibers in all experimental groups also abundantly express NTPDase1, ecto-5′-nucleotidase and P2X2 receptor channels. Because the brain injury causes a massive ATP release, it is reasonable to conclude that purinoreceptors and ectonucleotidases play an important role in the process of neuritic beading.     

Effect of diadenosine polyphosphates on Ca2+ ATPase activity

 Diadenosine tri-, tetra-, penta-, and hexaphosphate (Ap₃A, Ap₄A, Ap₅A and Ap₆A) have been described as having various effects on vascular tone depending on the number of phosphate groups. This study examined the effect of diadenosine polyphosphates on Ca²⁺ ATPase activity. The activity of the enzyme was measured spectrophotometrically as the difference in hydrolysis of ATP in the presence and absence of Ca²⁺ with various concentrations of ATP and diadenosine polyphosphates. The diadenosine polyphosphates increased the activity of the Ca²⁺ ATPase. The effect tended to be stronger with Ap₅A and Ap₆A than with Ap₃A and Ap₄A in the order of potency: Ap₃A≈AP₄A<Ap₅A≈AP₆A. The stimulatory effect of diadenosine polyphosphates was not competitive with that of ATP, suggesting an allosteric activation of Ca²⁺ ATPase by diadenosine polyphosphates. This effect may be physiologically relevant for limiting the increase in cytosolic free Ca²⁺ concentration elicited by diadenosine polyphosphates by receptor activation and modulating Ca²⁺ ATPase function under resting conditions. Received: 24 January 1997 / Accepted: 9 May 1997     

Evidence that acute taurine treatment alters extracellular AMP hydrolysis and adenosine deaminase activity in zebrafish brain membranes

Taurine is one of the most abundant free amino acids in excitable tissues. In the brain, extracellular taurine may act as an inhibitory neurotransmitter, neuromodulator, and neuroprotector. Nucleotides are ubiquitous signaling molecules that play crucial roles for brain function. The inactivation of nucleotide-mediated signaling is controlled by ectonucleotidases, which include the nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto-5′-nucleotidase. These enzymes hydrolyze ATP/GTP to adenosine/guanosine, which exert a modulatory role controlling several neurotransmitter systems. The nucleoside adenosine can be inactivated in extracellular or intracellular milieu by adenosine deaminase (ADA). In this report, we tested whether acute taurine treatment at supra-physiological concentrations alters NTPDase, ecto-5′-nucleotidase, and ADA activities in zebrafish brain. Fish were treated with 42, 150, and 400 mg L⁻¹ taurine for 1 h, the brains were dissected and the enzyme assays were performed. Although the NTPDase activities were not altered, 150 and 400 mg L⁻¹ taurine increased AMP hydrolysis (128 and 153%, respectively) in zebrafish brain membranes and significantly decreased ecto-ADA activity (29 and 38%, respectively). In vitro assays demonstrated that taurine did not change AMP hydrolysis, whereas it promoted a significant decrease in ecto-ADA activity at 150 and 400 mg L⁻¹ (24 and 26%, respectively). Altogether, our data provide the first evidence that taurine exposure modulates the ecto-enzymes responsible for controlling extracellular adenosine levels in zebrafish brain. These findings could be relevant to evaluate potential beneficial effects promoted by acute taurine treatment in the central nervous system (CNS) of this species.     

A Mg-dependent ecto-ATPase is increased in the infective stages of<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis>

In this work, we describe the ability of living epimastigotes of Trypanosoma cruzi to hydrolyze extracellular ATP. In these intact parasites, there was a low level of ATP hydrolysis in the absence of any divalent metal (2.42±0.31 nmol Pi/h×10⁸ cells). ATP hydrolysis was stimulated by MgCl₂, and the Mg-dependent ecto-ATPase activity was 27.15±2.91 nmol Pi/h×10⁸ cells. The addition of MgCl₂ to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. This stimulatory activity was also observed when MgCl₂ was replaced by MnCl₂, but not by CaCl₂ or SrCl₂. The apparent K_m for Mg-ATP^2– was 0.61 mM, and free Mg²⁺ did not increase the ecto-ATPase activity. This ecto-ATPase activity was insensitive to the inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4.diisothiocyanostylbene 2-2-disulfonic acid) as well as suramin, an antagonist of P₂ purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg²⁺-dependent ATPase activity in a dose-dependent manner. A comparison among the Mg²⁺-ecto-ATPase activities of the three forms of T. cruzi showed that the noninfective epimastigotes were less efficient at hydrolyzing ATP than the infective trypomastigote and amastigote stages.     

Effects of diadenosine polyphosphates,ATP and angiotensin II on cytosolic Ca2+ activity and contraction of rat mesangial cells

Diadenosine polyphosphates (Ap _n A) are known to influence cellular Ca²⁺ activity ([Ca²⁺]_i) in several cells. Their vasoactive potency has been described in various systems including the kidney. We examined the effects of diadenosine polyphosphates, adenosine 5-triphosphate (ATP) and angiotensin II (Ang II) on cytosolic Ca²⁺ activity of mesangial cells (MC) in culture obtained from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. [Ca²⁺]_i was measured as a fluorescence ratio F ₃₄₀/F ₃₈₀ with the fura-2 technique using three excitation wavelengths (340 nm, 360 nm and 380 nm) and a photon counting tube. Resting [Ca²⁺]_i was not significantly different in MC from WKY and SHR rats and was measured as 132±9 nmol/l (n=65) and 114±12 nmol/l (n=36), respectively. Diadenosine polyphosphates (Ap₃A–Ap₆A) increased [Ca²⁺]_i transiently with an initial peak and a secondary plateau phase comparable to the effects of ATP or Ang II. Increases in [Ca²⁺]_i induced by all these agonists were not significantly different between MC of WKY and SHR rats. ATP, Ap₃A, Ap₄A, Ap₅A, Ap₆A (each 5 mol/l) increased the fura-2 fluorescence ratio initially by 0.66±0.09 (n=33), 0.52±0.08 (n=18), 0.25±0.05 (n=16), 0.09±0.06 (n=7), 0.09±0.04 (n=11), respectively. A half-maximal initial increase in the fura-2 fluorescence ratio was reached at 22 nmol/l, 0.9 mol/l, 2.0 mol/l and 4.0 mol/l with Ang II, Ap₃A, ATP and Ap₄A, respectively. Ap₄A (100 mol/l, n=18) led to a reversible contraction of MC. Diadenosine polyphosphates increase [Ca²⁺]_i in rat MC, in a similar manner to ATP or Ang II and lead to a contraction of MC, suggesting that these nucleotides are also involved in the control of glomerular haemodynamics.     

Adenosine and adenine nucleotides are independently released from both the nerve terminals and the muscle fibres upon electrical stimulation of the innervated skeletal muscle of the frog

The independent release of adenosine and adenine nucleotides upon electrical stimulation was studied in the innervated sartorius muscle of the frog after blockade of the extracellular catabolism of adenosine monophosphate (AMP) through exo-AMP deaminase and ecto-5-nucleotidase. Nerve stimulation (30 min, 0.2Hz) induced the release of both adenosine (19±3 pmol) and adenine nucleotides (101±7 pmol). Experiments performed in the presence of tubocurarine (5 M) to prevent purine release due to nerve-evoked muscle twitching, or under direct stimulation of the muscle in low calcium solutions to prevent pre-synaptic release of purines, showed that there was an evoked release of adenosine and adenine nucleotides both from the nerve endings and from the twitching muscle fibres. Removal of ecto-5-nucleotidase inhibition shows that the catabolism of adenine nucleotides released during stimulation contributes in about 50% to the amount of endogenous extracellular adenosine. When only one of the enzymes catabolizing AMP (ecto-5-nucleotidase or exo-AMP deaminase) was inhibited, the evoked release of adenine nucleotides was undetectable, suggesting that each enzyme is able to catabolize all the AMP formed from adenine nucleotides released upon stimulation. It is concluded that the concentration of endogenous extracellular adenosine is under the control of the relative activities of exo-AMP deaminase and ecto-5-nucleotidase.Brief accounts of some of the results in this study have been published previously (refs. [6, 7]).     

Mutagen-mediated enhancement of HIV-1 replication in persistently infected cells

Lethal mutagenesis, a new antiviral strategy to extinguish virus through elevated mutation rates, was explored in H61-D cells an HIV-1 persistently infected lymphoid cell line. Three mutagenic agents: 5-hydroxy-2^′-deoxycytidine (5-OHdC), 5-fluorouracil (5-FU) and 2,2^′-difluoro-2^′-deoxycytidine (gemcitabine) were used. After 54 passages, treatments with 5-FU and gemcitabine reduced virus infectivity, p24 and RT activity. Treatment with the pyrimidine analog 5-OHdC resulted in increases of p24 production, RT activity and infectivity. Rise in viral replication by 5-OHdC during HIV-1 persistence is in contrast with its inhibitory effect in acute infections. Viral replication enhancement by 5-OHdC was associated with an increase in intracellular HIV-1 RNA mutations. Mechanisms of HIV-1 replication enhancement by 5-OHdC are unknown but some potential factors are discussed. Increase of HIV-1 replication by 5-OHdC cautions against the use, without previous analyses, of mutagenic nucleoside analogs for AIDS treatment.     

Intracellular ADP activates ATP-sensitive K+ channels in vascular smooth muscle cells of the guinea pig portal vein

Vasodilatation following tissue ischemia is assumed to partially result from activation of ATP-dependent K⁺ channels (K_ATP). To assess the effect of cytosolic adenosine nucleotides, the balance of which depends on tissue pO₂, on K_ATP, we have measured steady state outward currents (SSC) by the whole-cell clamp technique in smooth muscle cells of the guinea pig portal vein at different concentrations of ATP and ADP in the pipette solution. Glibenclamide, a selective inhibitor of K_ATP, was used as a pharmacological tool. — With no nucleotides in the pipette solution (Ca²⁺-free), the SSC determined at +20 mV was unaffected by glibenclamide, while with 0.1 mM ATP or with 0.1 mM ADP, the SSC exhibited a glibenclamide-sensitive component indicating activation of K_ATP. At 5 mM ATP and no ADP, hardly any effect of glibenclamide on the SSC was detected, suggesting inhibition of K_ATP by this high concentration of ATP. With 0.1 mM ADP at 5 mM ATP however, activation of K_ATP was achieved. — At 10^–7 M Ca²⁺ in the pipette solution, an increased SSC was measured, but the responses to the nucleotides and/or glibenclamide were not modified. — These findings suggest that in vivo, ADP may be involved in the regulation of vascular K_ATP, linking tissue pO₂ with vascular tone and tissue perfusion.     

Diadenosine polyphosphate-induced inhibition of cardiac KATP channels: Operative state-dependent regulation by a nucleoside diphosphate

It has been proposed that the regulatory action of mononucleotides, such as ATP and UDP, on cardiac ATP-sensitive K⁺ (K_ATP) channels is determined by the state of the channel. Recently, dinucleotides, such as diadenosine tetraphosphate (Ap₄A) and diadenosine pentaphosphate (Ap₅A), have been recognized as novel intracellular ligands of cardiac K_ATP channels. However, it is not known whether the state of K_ATP channels also determines the response of the channel to dinucleotides. Therefore, we examined the action of diadenosine polyphosphates on K_ATP channel activity during different operative channel states, using the inside-out patch clamp technique applied to patches excised from guinea-pig ventricular myocytes. Spontaneous openings of K_ATP channels (or operative condition 1) were inhibited by Ap₄A and Ap₅A. Addition of UDP, which on its own did not affect spontaneous openings of K_ATP channels, prevented Ap₄A and Ap₅A to inhibit spontaneous K_ATP channel activity. In contrast, following run-down of spontaneous channel activity, UDP induced K_ATP channel openings (or operative condition 2), but could no longer antagonize the inhibitory effect of Ap₄A and Ap₅A. We conclude that the inhibitory action of diadenosine polyphosphates on K_ATP channels can be blocked by UDP only when K_ATP channels are in operative condition 1, but not in operative condition 2. Thus, the operative condition of KATp channels determines the UDP-mediated regulation of the diadenosine polyphosphate-dependent inhibitory channel gating. This finding further suggests that the operative state of the cardiac K_ATP channel protein is a critical determinant of the regulatory action of an intracellular ligand.     

Nucleotide hydrolysis by solubilized membrane-bound enzymes of the brush border plasma membrane of Hymenolepis diminuta

Pyrophosphate, p-nitrophenyl phosphate and a variety of pyrimidine and purine nucleotides are hydrolyzed by the solubilized membrane-bound enzymes of the brush border plasma membrane of Hymenolepis diminuta. The pH optima (or ranges) for hydrolysis of substrates are 8.0 (pyrophosphate), 8.8 (p-nitrophenyl phosphate), 8.4-8.9 (nucleoside monophosphates), and 7.1-8.1 (nucleoside triphosphates); all substrates, with the exception of nucleoside triphosphates, have a higher affinity for the solubilized enzyme at pH 7.4 than at their optimal pH for hydrolysis. ATP is degraded completely by the enzyme preparation to adenosine and inorganic phosphate, but since neither ADP nor ATP accumulate in the incubation medium it is not known whether ATP hydrolysis involves the sequential hydrolysis of terminal phosphate groups. Isoelectric focusing and various chromatographic procedures (gel permeation, ion-exchange and hydrophobic interaction chromatography) fail to separate the alkaline phosphatase, phosphodiesterase, 5'-nucleotidase, adenosine triphosphatase and ribonuclease activities associated with the solubilized membrane preparation. Additionally, inhibitor studies indicate that only a single enzyme with low substrate specificity is involved in the hydrolysis of nucleotides, p-nitrophenyl phosphate, pyrophosphate and hexose phosphate esters. Purines and pyrimidines and their nucleosides interact with the active site, and in some instances activity of the enzyme is stimulated by an unknown mechanism.     

Inhibition of lactate export by quercetin acidifies rat glial cells in vitro

The relationship between glial lactate release and glial intracellular pH (pH_i) regulation is studied using C6 glioma cells and rat astrocytes in vitro, and the lactate transport inhibitors quercetin and α-cyano-4-hydroxycinnamate (CHC). pH_i is measured using 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The results show that lactate release is mediated partly by a specific lactate transport system inhibitable by quercetin (50 μM), but not by CHC (5 mM). Inhibition by quercetin results in a significant 3–4-fold increase of intracellular lactate and a decrease of intracellular pH to 6.9. A participation of quercetin-inhibitable lactate transport in glial pH_i-regulation is suggested by the observation that pH_i-homeostasis after acidification by diffusion of undissociated lactic acid into the cell is inhibited by quercetin. The existence of a system controlling lactate release in glial cells may also reflect a function of astrocytes to supply neurons with lactate.     

Uptake and release of adenosine in isolated rat fat cells

Radioactively labelled adenosine and adenine were rapidly taken up by isolated rat fat cells, and incorporated into nucleotides, of which ATP dominated. The overall process had an apparent K_m of 1–5 μM. During incubation, especially in the presence of lipolytic agents, there was a reduction in labelled ATP with a compensatory increase in ADP, AMP, cAMP and nucleosides. The build-up of adenosine during incubation was inhibited by theophylline, which inhibits 5′-nucleotidase. Radioactivity released from perifused fat cells consisted mainly of nucleoside material, of which adenosine predominated. Lipolytic stimulation caused no significant increase in nucleoside outflow from perifused cells, whereas oxygenation was capable of reducing this outflow. It is concluded that adenosine is formed by fat cells as a consequence of ATP breakdown. Stimulation of lipolysis during activation of the sympathetic nerves leads to reversible ATP breakdown and adenosine release. Adenosine might therefore act as a modulator of lipolysis in vivo under these conditions, even though it does not serve as a feed back regulator in the proper sense.     

The 5′CL-PCBP RNP complex, 3′ poly(A) tail and 2A are required for optimal translation of poliovirus RNA

In this study, we showed that the 5′CL-PCBP complex, 3′ poly(A) tail and viral protein 2A^pro are all required for optimal translation of PV RNA. The 2A^pro-mediated stimulation of translation was observed in the presence or absence of both the 5′CL and the 3′ poly(A) tail. Using protein-RNA tethering, we established that the 5′CL-PCBP complex is required for optimal viral RNA translation and identified the KH3 domain of PCBP2 as the functional region. We also showed that the 5′CL-PCBP complex and the 3′ poly(A) tail stimulate translation independent of each other. In addition to the independent function of each element, the 5′CL and the 3′ poly(A) tail function synergistically to stimulate and prolong translation. These results are consistent with a model in which the 5′CL-PCBP complex interacts with the 3′ poly(A)-PABP complex to form a 5′-3′ circular complex that facilitates ribosome reloading and stimulates PV RNA translation.     

Trophic effects of purines in neurons and glial cells

In addition to their well known roles within cells, purine nucleotides such as adenosine 5' triphosphate (ATP) and guanosine 5' triphosphate (GTP), nucleosides such as adenosine and guanosine and bases, such as adenine and guanine and their metabolic products xanthine and hypoxanthine are released into the extracellular space where they act as intercellular signaling molecules. In the nervous system they mediate both immediate effects, such as neurotransmission, and trophic effects which induce changes in cell metabolism, structure and function and therefore have a longer time course. Some trophic effects of purines are mediated via purinergic cell surface receptors, whereas others require uptake of purines by the target cells. Purine nucleosides and nucleotides, especially guanosine, ATP and GTP stimulate incorporation of [3H]thymidine into DNA of astrocytes and microglia and concomitant mitosis in vitro. High concentrations of adenosine also induce apoptosis, through both activation of cell-surface A3 receptors and through a mechanism requiring uptake into the cells. Extracellular purines also stimulate the synthesis and release of protein trophic factors by astrocytes, including bFGF (basic fibroblast growth factor), nerve growth factor (NGF), neurotrophin-3, ciliary neurotrophic factor and S-100beta protein. In vivo infusion into brain of adenosine analogs stimulates reactive gliosis. Purine nucleosides and nucleotides also stimulate the differentiation and process outgrowth from various neurons including primary cultures of hippocampal neurons and pheochromocytoma cells. A tonic release of ATP from neurons, its hydrolysis by ecto-nucleotidases and subsequent re-uptake by axons appears crucial for normal axonal growth. Guanosine and GTP, through apparently different mechanisms, are also potent stimulators of axonal growth in vitro. In vivo the extracellular concentration of purines depends on a balance between the release of purines from cells and their re-uptake and extracellular metabolism. Purine nucleosides and nucleotides are released from neurons by exocytosis and from both neurons and glia by non-exocytotic mechanisms. Nucleosides are principally released through the equilibratory nucleoside transmembrane transporters whereas nucleotides may be transported through the ATP binding cassette family of proteins, including the multidrug resistance protein. The extracellular purine nucleotides are rapidly metabolized by ectonucleotidases. Adenosine is deaminated by adenosine deaminase (ADA) and guanosine is converted to guanine and deaminated by guanase. Nucleosides are also removed from the extracellular space into neurons and glia by transporter systems. Large quantities of purines, particularly guanosine and, to a lesser extent adenosine, are released extracellularly following ischemia or trauma. Thus purines are likely to exert trophic effects in vivo following trauma. The extracellular purine nucleotide GTP enhances the tonic release of adenine nucleotides, whereas the nucleoside guanosine stimulates tonic release of adenosine and its metabolic products. The trophic effects of guanosine and GTP may depend on this process. Guanosine is likely to be an important trophic effector in vivo because high concentrations remain extracellularly for up to a week after focal brain injury. Purine derivatives are now in clinical trials in humans as memory-enhancing agents in Alzheimer's disease. Two of these, propentofylline and AIT-082, are trophic effectors in animals, increasing production of neurotrophic factors in brain and spinal cord. Likely more clinical uses for purine derivatives will be found; purines interact at the level of signal-transduction pathways with other transmitters, for example, glutamate. They can beneficially modify the actions of these other transmitters.     

Effects of diadenosine polyphosphates,ATP and angiotensin II on membrane voltage and membrane conductances of rat mesangial cells

Diadenosine polyphosphates have been shown to influence renal perfusion pressure. As mesangial cells may contribute to these effects we investigated the effects of diadenosine triphosphate (Ap₃A), diadenosine tetraphosphate (Ap₄A), diadenosine pentaphosphate (Ap₅A) and diadenosine hexaphosphate (Ap₆A) on membrane voltage (V _m) and membrane conductance (g _m) in mesangial cells (MC) of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats in primary and long-term culture. We applied the patch-clamp technique in the fast-whole-cell configuration to measure V _m and g _m. To compare the effects of diadenosine polyphosphates with hitherto known agonists we also tested adenosine 5-triphosphate (ATP) and angiotensin II (Ang II). As there was no significant difference in the V _m values in MC of WKY (–42±1 mV, n=70) and SHR rats (–45±2 mV, n=99) as well as in the agonist-induced changes of V _m, all data were pooled. The V _m of all the cells was –44±1 mV (n=169) and g _m was 15.9±1.8 nS (n=141). Ion-exchange experiments showed the presence of a K⁺ and a non-selective cation conductance in resting MC whereas a Cl^– conductance or a Na⁺selective conductance could not be observed. Ap₃A, Ap₄A, Ap₅A, AP₆A and ATP each at a concentration of 5 mol/l, led to a significant depolarization of V _m by 5±2 mV (n=14), 7±1 mV (n=25), 3±1 mV (n=23), 2±1 mV (n=16), and 14±2 mV (n=23), respectively. For Ap₄A, the most potent diadenosine polyphosphate, we determined the half-maximally effective concentration (EC ₅₀) as 6 mol/l (n=5–25), for ATP as 2 mol/l (n=9–37), and for Ang II as 8 nmol/l (n=6–18). Ap₄A 100 mol/l increased g _m significantly by 55±20% (n=16), 100 mol/l ATP by 135±60% (n=18). The diadenosine polyphosphates examined were able to depolarize V _m (Ang II >ATP> Ap₄A>Ap₃A>Ap₅A>Ap₆A) by activation of a Cl^– conductance and a non-selective cation conductance, as do ATP or Ang II.     

Isolation and properties of an actomyosin-like protein from glial cells of bovine brain

An enriched fraction of glial cells was isolated by centrifugation in a Ficollsucrose density gradient from bovine cerebral cortical tissue, and an actomyosinlike protein (AMLP) was obtained from it. The yield of the AMLP was 0.05% relative to glial cell protein. The AMLP was found to contain bound nucleotides and to give reversible association-dissociation reactions characteristic of AMLP under the influence of Mg²⁺ ions and ATP. ATPase of glial AMLP, which is activated by Ca²⁺ ions by a greater degree than Mg²⁺, differs from the AMLP of neuronal origin.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Department of Biochemistry, Tbilisi University. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 3, pp. 280–282, March, 1977.     

Binding of 5'-O-(2-thiodiphosphate) to rat brain membranes is prevented by diadenosine tetraphosphate and correlates with ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) activity

The distribution of binding sites for [(35)S]5'-O-(2-thiodiphosphate) ([(35)S]ADPbetaS), a radioligand of P2Y(1,12,13) receptors, and of ecto-nucleotide pyrophosphatase phosphodiesterase activity were analyzed in the rat forebrain. Binding sites for the radilogand are widespreadly distributed in the rat forebrain, showing the highest density in hypothalamus. K(d) values were in the range 1-2 nM. Diadenosine tetraphosphate (Ap(4)A) and diethenoadenosine tetraphosphate, epsilon-(Ap(4)A), displaced the radioligand, indicating dinucleotide binding to ADPbetaS-recognizing P2Y receptors. Activity ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), able to hydrolyze Ap(4)A and other diadenosine polyphosphates, is also widely distributed through the rat forebrain, with the highest activity in hypothalamus. These results suggests that Ap(4)A signalling mediated by P2Y(1,12,13) receptors and enzymatically regulated by NPP1 activity may be particularly important in hypothalamus and add new support for neurotransmitter/neuromodulatory functions of diadenosine polyphosphates in brain.     

Transmitter-induced changes of the membrane voltage of HT29 cells

The colonic carcinoma cell line HT₂₉ was used to examine the influence of agonists increasing cytosolic cAMP and Ca²⁺ activity on the conductances and the cell membrane voltage (V _m). HT₂₉ cells were grown on glass cover-slips. Cells were impaled by microelectrodes 4–10 days after seeding, when they had formed large plaques. In 181 impalements V _m was –51±1 mV. An increase in bath K⁺ concentration from 3.6 mmol/l to 18.6 mmol/l or 0.5 mmol/l Ba²⁺ depolarized the cells by 10±1 mV (n=49) or by 9±2 mV (n=3), respectively. A decrease of bath Cl^– concentration from 145 to 30 mmol/l depolarized the cells by 11±1 mV (n=24). Agents increasing intracellular cAMP such as isobutylmethylxanthine (0.1 mmol/l), forskolin (10 mol/l) or isoprenaline (10 mol/l) depolarized the cells by 6±1 (n=13), 15±3 (n=5) and 6±2 (n=3) mV, respectively. In hypoosmolar solutions (225 mosmol/l) cells depolarized by 9±1 mV (n=6). Purine and pyrimidine nucleotides depolarized the cells dose-dependently with the following potency sequence: UTP > ATP > ITP > GTP > TIP > CTP = 0. The depolarization by ATP was stronger than that by ADP and adenosine. The muscarinic agonist carbachol led to a sustained depolarization by 27±6 mV (n=5) at 0.1 mmol/l, and to a transient depolarization by 12±4 mV (n=5) at 10 mol/l. Neurotensin depolarized with a half-maximal effect at around 5 nmol/l. The depolarization induced by nucleotides and neurotensin was transient and followed by a hyperpolarization. We confirm that HT₂₉ cells possess Cl^–- and K⁺-conductive pathways. The Cl^– conductance is regulated by intracellular cAMP level, cytosolic Ca²⁺ activity, and cell swelling. The K⁺ conductance in HT₂₉ cells is regulated by intracellular Ca²⁺ activity.Supported by DFG Gre 480/10 and GIF Proj. no. I-86-100.10/ 88