Reversal of insulin resistance by ATP in hemorrhagic shock.

Hemorrhagic shock was produced by bleeding rats to a mean arterial pressure of 40 mm Hg (1 mm Hg = 133 N/m2), which was maintained for 2 h. Muscles from these animals ('shock' muscles) showed resistance to the stimulation of glucose uptake by insulin. Addition of 1 mM ATP-MgCl2 to the medium had no effect on basal glucose uptake in either group of muscles, but it permitted insulin to exert its stimulatory effect in 'shock' muscles. An optimal insulin effect on glucose uptake in 'shock' muscles incubated without ATP was observed at an insulin concentration of 0.2 Unit/ml. When 1 mM ATP-MgCl2 was added to the medium, optimal insulin effect in 'shock' muscles was observed at an insulin concentration of 0.007 Unit/ml. Increasing the concentration of ATP-MgCl2 to 2.5 mM in the medium resulted in an optimal insulin effect at an insulin concentration of ATP-MgCl2 to 2.5 mM in the medium resulted in an optimal insulin effect at an insulin concentration of 0.001 Unit/ml in 'shock' muscles. Following 1 h cubation in Krebs-HCO3 medium, intracellular ATP contents of 'shock' muscles were approximately 50% lower than in control muscles. Addition of 1 mM ATP-MgCl2 to the incubation medium had no effect on the intracellular ATP contents of either group of muscles following incubation; however, 2.5 mM ATP-MgCl2 elevated intracellular ATP contents of 'shock' muscles but had no effect in control muscles. Possible mechanisms for this reversal of insulin resistance by ATP-MgCl2 in shock are discussed.     

Metabolism and excretion of exogenous adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate. Studies in the isolated perfused rat kidney and in the intact rat.

Isolated rat kidneys were perfused with a recirculating medium containing exogenous adenosine 3':5'-monophosphate (cyclic AMP) or guanosine 3':5'-monophosphate (cyclic GMP) at an initial concentration of 0.1 mM. Both cyclic nucleotides were rapidly removed from the perfusate. Urinary excretion accounted for about 20% and 40% of the respective cyclic AMP and cyclic GMP lost from the perfusate. The metabolism of the cyclic nucleotides was studied by 14C-labeled cyclic nucleotides in the perfusate. During 60 min, 30% of added cyclic [14C]AMP was metabolized to renal [14C]adenine nucleotides (ATP, ADP, and AMP) and 30% to perfusate [14C]uric acid. Similarly, 20% of cyclic[14C]GMP was metabolized to renal [14C]guanine nucleotides (GTP, GDP, and GMP) and 30% to perfusate [14C]uric acid. Urine contained principally unchanged 14C-labeled cyclic nucleotide. Addition of 0.1 mM cyclic AMP to the perfusate elevated the renal ATP and ADP contents 2-fold. Addition of 0.1 mM of either cyclic AMP or cyclic GMP to the perfusate also elevated the renal production of uric acid 2- to 3-fold. The production and distribution of metabolites of exogenous cyclic nucleotides were also studied in the intact rat. Within 60 min after injection, 3.3 mumol of either 14C-labeled cyclic AMP or cyclic GMP was cleared from the plasma. Kidney cortex and liver were the principal tissues for 14C accumulation. Urinary excretion accounted for about 20 and 45% of the cyclic [14C]AMP and cyclic [14C]GMP lost from the plasma, respectively. The 14C found in the kidney and liver was present almost entirely as the respective purine mono-, di-, and trinucleotides. The other principal metabolite was [14C]allantoin, found in the urine and, to a lesser extent, the liver. The urine contained mostly unchanged 14C-labeled cyclic nucleotide. Unlike the findings with the perfused kidney, [14C]uric acid was not a significant metabolite of the 14C-labeled cyclic nucleotides in these in vivo experiments.     

Stimulation of ATP synthesis in hypothermically perfused dog kidneys by adenosine and PO4

During continuous hypothermic perfusion of dog kidneys there occurs a gradual decrease in ATP from about 1.4 to 0.6 μmol/g wet wt after 5 days of preservation. The loss of ATP can be prevented by including both adenosine (10 mM) and PO₄ (25 mM) in the perfusate. Under these conditions kidney cortex ATP levels were more than double control values — 3.5 μmol/g wet wt. Both adenosine and PO₄ were necessary since omission of one substance resulted in no net synthesis of ATP. Furthermore, these high levels of ATP were obtained only if adequate concentrations of adenosine were maintained during perfusion. Following 3 days of perfusion the adenosine level in the perfusate decreased to about 1 mM and under this condition ATP levels were low. Adenosine levels were maintained in the perfusate by two methods: (1) addition of fresh perfusate or (2) pretreatment of the kidney with the adenosine deaminase inhibitor—deoxycoformycin. The increased levels of ATP appear directly related to the availability of nucleotide precursors and the presence of inhibitors of the enzymes involved in the catabolism of nucleotides and nucleosides (PO₄ and deoxycoformycin). Mitochondrial activity was similar in kidneys with high or low ATP levels following 5 days of preservation.     

Effects of differences in substrate supply on the energy metabolism of hypothermically perfused canine kidneys

Experiments were performed on the effects of differences in substrate supply on canine kidneys. Following 2 min of ischemia and flush perfusion for 5 min the kidneys were continuously perfused at 6 °C using albumin perfusate containing free fatty acids and glucose or Haemaccel perfusate without substrates.During 120 hr of perfusion neither potassium nor LDH nor GOT accumulation differed between the two perfusates and up to the 48th hr the tissue contents of adenine nucleotides as well as the energy charge potential were almost identical. The results show that in canine kidneys glucose or FFA supply during hypothermic continuous perfusion does not influence the overall cellular integrity and energetic capacity of the renal cortex at least up to the 48th hr of preservations.     

P2Y(2) receptor-stimulated phosphoinositide hydrolysis and Ca(2+) mobilization in tracheal epithelial cells

Extracellular nucleotides have been implicated in the regulation of secretory function through the activation of P2 receptors in the epithelial tissues, including tracheal epithelial cells (TECs). In this study, experiments were conducted to characterize the P2 receptor subtype on canine TECs responsible for stimulating inositol phosphate (InsP(x)) accumulation and Ca(2+) mobilization using a range of nucleotides. The nucleotides ATP and UTP caused a concentration-dependent increase in [(3)H]InsP(x) accumulation and Ca(2+) mobilization with comparable kinetics and similar potency. The selective agonists for P1, P2X, and P2Y(1) receptors, N(6)-cyclopentyladenosine and AMP, alpha,beta-methylene-ATP and beta, gamma-methylene-ATP, and 2-methylthio-ATP, respectively, had little effect on these responses. Stimulation of TECs with maximally effective concentrations of ATP and UTP showed no additive effect on [(3)H]InsP(x) accumulation. The response of a maximally effective concentration of either ATP or UTP was additive to the response evoked by bradykinin. Furthermore, ATP and UTP induced a cross-desensitization in [(3)H]InsP(x) accumulation and Ca(2+) mobilization. These results suggest that ATP and UTP directly stimulate phospholipase C-mediated [(3)H]InsP(x) accumulation and Ca(2+) mobilization in canine TECs. P2Y(2) receptors may be predominantly mediating [(3)H]InsP(x) accumulation, and, subsequently, inositol 1,4,5-trisphosphate-induced Ca(2+) mobilization may function as the transducing mechanism for ATP-modulated secretory function of tracheal epithelium.     

Rickettsial permeability. An ADP-ATP transport system.

The obligate intracellular parasitic bacterium, Rickettsia prowazeki, has a carrier-mediated transport system for ADP and ATP. The transport of nucleotides was measured by membrane filtration assays; the assay was shown not to harm the relatively labile rickettsiae. The nucleotide transport system was shown to reside in the rickettsiae, not in the contaminating yolk sac mitochondria of the preparation. The influx of nucleotide had an activation energy of 12 to 13 kcal above 22 deg-rees (an apparent transition temperature), and 30 kcal below this value. The uptake of nucleotide was independent of the Mg2+ concentration, but was markedly stimulated by the phosphate concentration. The pH optimum of the influx of nucleotide was pH 7. The specificity of the transport system was remarkable in that it required a specific moiety in each portion of the nucleotide, i.e. an adenine base, a ribose sugar, and two or three, but not one, phosphates. Of the wide variety of compounds tested, the system could transport only ADP, ATP, and (beta, gamma-methylene) adenosine 5'-triphosphate. The influx of nucleotide was a saturable process; half-maximum velocity was achieved at a nucleotide concentration of about 75 muM. ADP and ATP were competitive inhibitors of each other's transport. Although at least 95% of the labeled intracellular nucleotide was exchangeable, efflux of labeled nucleotide was observed only in the presence of unlabeled nucleotide in the medium. Half-maximum efflux was achieved at a concentration of about 75 muM. A large intracellular to extracellular concentration gradient of labeled nucleotide was maintained in the presence of metabolic inhibitors and uncouplers, which completely abolished rickettsial hemolysis. While having no effect on the steady state, KCN and DNP accelerated both influx and efflux. Measurements of the endogenous pool of adenine nucleotides in isolated rickettsiae show that is was large (5 mM), and that these unlabeled nucleotides exchanged, on approximately a 1/1 basis, with exogenously added nucleotide. These studies support the proposal that rickettsiae are not "leaky" to adenine nucleotides or to small molecules in general, and that they have a carrier-mediated transport system which allows an exchange of host and parasite ADP and ATP.     

Stimulation of glycogenolysis by adenine nucleotides in the perfused rat liver.

Infusion of adenine nucleotides and adenosine into perfused rat livers resulted in stimulation of hepatic glycogenolysis, transient increases in the effluent perfusate [3-hydroxybutyrate]/[acetoacetate] ratio, and increased portal vein pressure. In livers perfused with buffer containing 50 microM-Ca2+, transient efflux of Ca2+ was seen on stimulation of the liver with adenine nucleotides or adenosine. ADP was the most potent of the nucleotides, stimulating glucose output at concentrations as low as 0.15 microM, with half-maximal stimulation at approx. 1 microM, and ATP was slightly less potent, half-maximal stimulation requiring 4 microM-ATP. AMP and adenosine were much less effective, doses giving half-maximal stimulation being 40 and 20 microM respectively. Non-hydrolysed ATP analogues were much less effective than ATP in promoting changes in hepatic metabolism. ITP, GTP and GDP caused similar changes in hepatic metabolism to ATP, but were 10-20 times less potent than ATP. In livers perfused at low (7 microM) Ca2+, infusion of phenylephrine before ATP desensitized hepatic responses to ATP. Repeated infusions of ATP in such low-Ca2+-perfused livers caused homologous desensitization of ATP responses, and also desensitized subsequent Ca2+-dependent responses to phenylephrine. A short infusion of Ca2+ (1.25 mM) after phenylephrine infusion restored subsequent responses to ATP, indicating that, during perfusion with buffer containing 7 microM-Ca2+, ATP and phenylephrine deplete the same pool of intracellular Ca2+, which can be rapidly replenished in the presence of extracellular Ca2+. Measurement of cyclic AMP in freeze-clamped liver tissue demonstrated that adenosine (150 microM) significantly increased hepatic cyclic AMP, whereas ATP (15 microM) was without effect. It is concluded that ATP and ADP stimulate hepatic glycogenolysis via P2-purinergic receptors, through a Ca2+-dependent mechanism similar to that in alpha-adrenergic stimulation of hepatic tissue. However, adenosine stimulates glycogenolysis via P1-purinoreceptors and/or uptake into the cell, at least partially through a mechanism involving increase in cyclic AMP. Further, the hepatic response to adenine nucleotides may be significant in regulating hepatic glucose output in physiological and pathophysiological states.     

[3H]Arachidonic acid metabolism in rat brain minces: Effects of nucleotide triphosphates,CDPcholine and CMP

Rat brain minces were used to investigate the effects of nucleotides on the metabolism of arachidonic acid in nerve tissue. Brain free fatty acids, neutral lipids and phospholipids, were radiolabeled in vivo following intracerebral injection of [³H]arachidonic acid. Minces were prepared from the radiolabeled cerebra and were incubated in a modified Krebs-Ringer buffer with and without various nucleotides. The incubation-induced accumulation of unesterified [³H]arachidonate was reduced in the presence of CDPcholine, ATP, CTP, GTP, and UTP. These nucleotides inhibited choline and inositol glycerophospholipid hydrolysis. They also reduced the amount of labeled diglycerides. However, CDPethanolamine had no effect on arachidonic acid metabolism in the mince preparation and CMP appeared to stimulate further hydrolysis of choline glycerophospholipids, resulting in increased accumulation of [³H]arachidonic acid and labeled diglycerides. We suggest that the production of unesterified [³H]arachidonate and labeled diglycerides is due to the involvement of more than one catabolic reaction, since the high energy nucleotides had similar effects on fatty acid accumulation, but different effects on phospholipid labeling.     

Turnover and Storage of Newly Synthesized Adenine Nucleotides in Bovine Adrenal Medullary Cell Cultures

The adenine nucleotide stores of cultured adrenal medullary cells were radiolabeled by incubating the cells with 32Pi and [3H]adenosine and the turnover, subcellular distribution, and secretion of the nucleotides were examined. ATP represented 84-88% of the labeled adenine nucleotides, ADP 11-13%, and AMP 1-3%. The turnover of 32P-adenine nucleotides and 3H-nucleotides was biphasic and virtually identical; there was an initial fast phase with a t1/2 of 3.5-4.5 h and a slow phase with a half-life varying from 7 to 17 days, depending upon the particular cell preparation. The t1/2 of the slow phase for labeled adenine nucleotides was the same as that for the turnover of labeled catecholamines. The subcellular distribution of labeled adenine nucleotides provides evidence that there are at least two pools of adenine nucleotides which make up the component with the long half-life. One pool, which contains the bulk of endogenous nucleotides (75% of the total), is present within the chromaffin vesicles; the subcellular localization of the second pool has not been identified. The studies also show that [3H]ATP and [32P]ATP are distributed differently within the cell; 3 days after labeling 75% of the [32P]ATP was present in chromaffin vesicles while only 35% of the [3H]ATP was present in chromaffin vesicles. Evidence for two pools of ATP with long half-lives and for the differential distribution of [32P]ATP and [3H]ATP was also obtained from secretion studies. Stimulation of cell cultures with nicotine or scorpion venom 24 h after labeling with [3H]adenosine and 32Pi released relatively twice as much catecholamine as 32P-labeled compounds and relatively three times as much catecholamine as 3H-labeled compounds.     

Effect of extracellular ATP level on flow-induced Ca++ response in cultured vascular endothelial cells

Cultured vascular endothelial cells loaded with the highly fluorescent Ca(++)-sensitive dye Fura-2 were exposed to the flow of a fluid containing various concentrations of ATP (0, 0.5, 1, 5 microM) in an apparatus designed on the basis of fluid dynamics, and simultaneous changes in intracellular free Ca++ concentration were monitored by photometric fluorescence microscopy. The flow rate of the perfusate was altered from 0 to 6.3 to 22.8 to 39.0 cm/sec, inducing shear stress on the cell surface of 0, 2.9, 10.4, and 17.9 dynes/cm2, respectively. Although no significant change in intracellular Ca++ level was observed at ATP levels below 100 nM, at an ATP level of 500 nM, the intracellular Ca++ level increased together with an increase in the flow rate of the perfusate. At this level of ATP, the intracellular Ca++ levels at flow rates of 0, 6.3, 22.8, and 39.0 cm/sec were 44.8 +/- 7.3, 60.3 +/- 10.7, 74.0 +/- 5.8 and 89.4 +/- 6.4 nM (mean +/- SD; n = 8), respectively. At ATP levels over 1 microM, the flow-rate dependency of Ca++ response became less clear than that observed at the ATP level of 500 nM. These Ca++ responses to changes in flow rate disappeared when extracellular Ca++ was chelated by adding 2 mM of EGTA to the perfusate. These results suggest that the vascular endothelial cell has a mechanism that elevates the intracellular Ca++ level in accord with the flow rate at appropriate ATP concentrations, and that changes in intracellular Ca++ level under this mechanism seem to be chiefly caused by the influx of extracellular Ca++ into cells.     

Extracellular ATP perturbs transmembrane ion fluxes, elevates cytosolic [Ca2+], and inhibits phagocytosis in mouse macrophages

In addition to its important role in intracellular metabolic pathways, ATP appears to function as a neurotransmitter in mammalian neurones. The extracellular effects of ATP are not restricted to neurones. We describe the effects of ATP on transmembrane fluxes of monovalent and divalent cations and on phagocytosis in the J774 mouse macrophage cell line and in mouse macrophages elicited by intraperitoneal injection of thioglycollate broth. Of all nucleotides tested, only ATP is capable of depolarizing the macrophage plasma membrane potential, promoting Na+ influx and K+ efflux, effecting an increase in intracellular free Ca2+, and inhibiting phagocytosis. Nonhydrolyzable ATP analogs had no effect on membrane permeability or phagocytosis. The effect mediated by ATP is not accompanied by an increase in membrane permeability to nucleotides, indicating that the action of ATP is restricted to the external surface of macrophages.     

Modulation of P2Z/P2X(7) receptor activity in macrophages infected with Chlamydia psittaci

Given the role that extracellular ATP (ATP(o))-mediated apoptosis may play in inflammatory responses and in controlling mycobacterial growth in macrophages, we investigated whether ATP(o) has any effect on the viability of chlamydiae in macrophages and, conversely, whether the infection has any effect on susceptibility to ATP(o)-induced killing via P2Z/P2X(7) purinergic receptors. Apoptosis of J774 macrophages could be selectively triggered by ATP(o), because other purine/pyrimidine nucleotides were ineffective, and it was inhibited by oxidized ATP, which irreversibly inhibits P2Z/P2X(7) purinergic receptors. Incubation with ATP(o) but not other extracellular nucleotides inhibits the growth of intracellular chlamydiae, consistent with previous observations on ATP(o) effects on growth of intracellular mycobacteria. However, chlamydial infection for 1 day also inhibits ATP(o)-mediated apoptosis, which may be a mechanism to partially protect infected cells against the immune response. Infection by Chlamydia appears to protect cells by decreasing the ability of ATP(o) to permeabilize macrophages to small molecules and by abrogating a sustained Ca(2+) influx previously associated with ATP(o)-induced apoptosis.     

Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms

We have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only.     

Internalized insulin-receptor complexes are unidirectionally translocated to chloroquine-sensitive degradative sites. Dependence on metabolic energy

Insulin receptors on the surface of isolated rat adipocytes were photoaffinity labeled at 12 degrees C with the iodinated photoreactive insulin analogue, 125I-B2 (2-nitro-4-azidophenylacetyl)-des-PheB1-insulin, and the pathways in the intracellular processing of the labeled receptors were studied at 37 degrees C. During 37 degrees C incubations, the labeled 440-kDa insulin receptors were continuously internalized (as assessed by trypsin inaccessibility) and degraded such that up to 50% of the initially labeled receptors were lost by 120 min. Metabolic poisons (0.125-0.75 mM 2,4-dinitrophenol (DNP) and 1-10 mM NaF), which led to dose-dependent depletion of adipocyte ATP pools, inhibited receptor loss, and caused up to 3-fold increase in intracellular receptor accumulation. This effect was due to inhibition of intracellular receptor degradation, and there was no apparent effect of the metabolic poisons on initial internalization of the receptors. Following maximal intracellular accumulation of labeled insulin receptors in the presence of NaF or DNP, removal of these agents resulted in a subsequent, time-dependent degradation of the accumulated receptors. However, when the lysosomotropic agent, chloroquine (0.2 mM), was added immediately following removal of the metabolic poisons, further degradation of the intracellularly accumulated receptors was prevented, suggesting that the chloroquine-sensitive degradation of insulin receptors occurs distal to the site of inhibition by NaF or DNP. To confirm this, maximal intracellular accumulation of labeled receptors was first allowed to occur in the presence of chloroquine and the cells were then washed and reincubated in chloroquine-free media in the absence or presence of NaF or DNP. Under these conditions, degradation of the intracellularly accumulated receptors continued to occur, and NaF or DNP failed to block the degradation. In summary, these results indicate that the loss of cell surface insulin receptors in adipocytes involves: 1) initial internalization of the receptors to a nondegradative intracellular compartment by a process that is relatively insensitive to ATP depletion, followed by 2) a highly energy-dependent unidirectional translocation of the receptors from this compartment to chloroquine-sensitive site(s) of degradation.     

Aberrant 3H in Ehrlich mouse ascites tumor cell nucleotides after in vivo labeling with myo-[2-3H]- and L-myo-[1-3H]inositol: implications for measuring inositol phosphate signaling

After in vivo radiolabeling of Ehrlich cells for 24h with conventional myo-[2-3H]inositol we previously demonstrated an aberrant 3H-labeling of ATP that interfered in the HPLC analysis of inositol trisphosphates. This aberrant 3H-labeling was accounted for by the extensive kidney catabolism of myo-[2-3H] inositol with delivery of 3H-labeled metabolites to extrarenal tissues. As expected, the aberrant labeling of ATP is markedly reduced with the use of 3H-myo-inositol labeled at L-C1 rather than at C2, reflecting that the 3H at L-C1 disappears in the first step of the myo-inositol catabolism: the oxidative conversion to D-glucuronate. In contrast, with the 3H at C2 of myo-inositol, the 3H-C2 passes into the pentose phosphate conversions with resulting labeling of nucleotides. The extent of catabolism to 3H-labeled water, the cellular accumulation of 3H-myo-inositol, the incorporation into cellular inositol phospholipids, and the labeling pattern of cellular phosphoinositides were all found to be similar for the two labeled myo-inositol moieties. With the use of L-myo-[1-3H]inositol an aberrant 3H-labeling at about 25% remained, for which a presumptive mechanism is proposed. L-myo-[1-3H]Inositol appears nevertheless to be a preferable alternative to myo-[2-3H]inositol for tracing the intact myo-inositol molecule after in vivo labeling, with minimized interference from aberrant 3H-labeling of nucleotides.     

Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 Modulates Local ATP-Induced Calcium Signaling in Human HaCaT Keratinocytes

Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP) as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP) accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.     

Diadenosine polyphosphates and the control of cyclic AMP concentrations in isolated rat liver cells.

Extracellular diadenosine polyphosphates (Ap(n)A), through their interactions with appropriate P(2) receptors, influence a diverse range of intracellular activities. In particular, Ap(4)A stimulates alterations in intracellular calcium homeostasis and subsequent activation of glycogen breakdown in isolated liver cells. Here we show that, like ATP, Ap(4)A and other naturally occurring diadenosine polyphosphates attenuate glucagon-stimulated accumulation of cyclic AMP in isolated rat liver cells. The characteristics of Ap(4)A- and ATP-dependent modulation of glucagon-stimulated cyclic AMP accumulation are similar. These results are discussed in the context of the repertoire of intracellular signalling processes modulated by extracellular nucleotides.     

Studies on the energy-linked Ca2+ accumulation in pig heart mitochondria - role of Mg2'ons

Comparative intracellular distribution of Ca2+, Mg2+ and adenine nucleotides has been studied in pig heart by differential centrifugation or fractional extraction and has shown that Mg2+ and ATP are associated mainly with soluble fractions whereas Ca2+ and ADP are more tightly bound to subcellular structures. Ca2+ accumulation and Ca2+ stimulated respiration were studied in pig heart mitochondria under different energetic conditions in the absence or presence of phosphate. Ca2+ concentrations of about 1200 nmoles/mg protein inhibit Ca2+ accumulation, site I substrate oxidation and induce an efflux of mitochondrial Mg2+. These deleterious effects of Ca2+ on respiration occur even in the absence of phosphate or oxidizable substrate; they are completely prevented by ruthenium red only, and partially prevented by the addition of M2+ to the medium. The kinetics of Ca2+ uptake become of the sigmoidal type when Mg2+ is present. This cation strongly inhibits the rate of Ca2+ uptake in the presence of added phosphate and decreases the affinity of Ca2+ for its transport system. In the absence of phosphate, Mg2+ has no effect on Ca2+ uptake. The possible physiological implications of these findings are discussed     

Potentiation of oxidative cell injury in hepatocytes which have accumulated Ca2+

Incubation of freshly isolated rat hepatocytes with exogenous ATP, but not with succinate, resulted in intracellular Ca2+ accumulation which was partly prevented when the inhibitor of mitochondrial Ca2+ sequestration, ruthenium red, was also present in the medium. Although the bulk of the accumulated Ca2+ was sequestered by the mitochondria, formation of surface blebs and stimulation of phosphorylase alpha activity during incubation of the hepatocytes with ATP indicate that this treatment was also associated with an increase in cytosolic free Ca2+ concentration. When hepatocytes loaded with Ca2+ by preincubation with ATP were exposed to either 2-methyl-1,4-naphthoquinone or t-butyl hydroperoxide, the cytotoxicity of both agents was markedly potentiated. Our results suggest that ATP-induced Ca2+ accumulation in hepatocytes is not due to contamination of the cell suspension with damaged cells or free intracellular organelles and that the intracellular Ca2+ concentration can affect the response to toxic agents.