Evidence for leukotriene C4 transport mediated by an ATP-dependent glutathione S-conjugate carrier in rat heart and liver plasma membranes

Using rat heart sarcolemma and liver plasma membrane vesicles, it has been verified that the transport of leukotriene C4 (LTC4) across membranes is an ATP-dependent process; the apparent Km for LTC4 was 150 nM (heart sarcolemma) or 250 nM (liver plasma membrane). S-(2,4-dinitrophenyl)-glutathione (DNP-SG) inhibited LTC4 uptake into the vesicles dose-dependently (I50 = 25 microM for both heart sarcolemma and liver plasma membrane vesicles). Mutual inhibition between LTC4 and DNP-SG in uptake into the vesicles demonstrates that transport of LTC4 is mediated by an ATP-dependent glutathione S-conjugate carrier.     

Leukotriene C4 inhibits ATP-dependent transport of glutathione S-conjugate across rat heart sarcolemma. Implication for leukotriene C4 translocation mediated by glutathione S-conjugate carrier

Sarcolemmal vesicles prepared from rat heart exhibited ATP-dependent uptake of S-(2,4-dinitrophenyl)glutathione (DNP-SG), which obeyed Michaelis-Menten kinetics with an apparent Km of 21 microM for DNP-SG and a Vmax of 0.27 nmol.10 min-1.mg protein-1. Several model glutathione S-conjugates inhibited DNP-SG uptake, but leukotriene C4 inhibited uptake much more significantly even at lower concentrations (competitive inhibition, Ki = 1.5 microM). However, leukotrienes D4 and E4, which lack the gamma-glutamyl moiety, were less effective. The results suggest that the ATP-dependent transport system has a high affinity for leukotriene C4, and may be responsible for the translocation of this compound.     

ATP-dependent transport for glucuronides in canalicular plasma membrane vesicles

Using rat liver canalicular plasma membrane vesicles, it has been verified that the transport of p-nitrophenyl glucuronide (NPG) across membranes is an ATP-dependent process; the apparent Km for NPG was 20 microM. S-(2,4-dinitrophenyl)-glutathione (DNP-SG) inhibited NPG uptake dose-dependently, and NPG or testosterone glucuronide did ATP-dependent DNP-SG uptake similarly. These results suggest that transport of glucuronide is mediated by an ATP-dependent glutathione S-conjugate carrier.     

TRANSPORT OF GLUTATHIONE S-CONJUGATES IN THE YEASTSSACCHAROMYCES CEREVISIAE

Transport of 2,4-dinitrophenyl-S-glutathione (DNP-SG) and a fluorescent glutathione S-conjugate, bimane-S-glutathione (B-SG) was studied in the baker's yeasts (S. cerevisiae). Both conjugates were exported from the cells; the transport was inhibited by fluoride and vanadate like in mammalian cells. B-SG was also found to be accumulated in the vacuoles. The transport rate of DNP-SG outside the cell was higher in a vacuolar-deficient strain. A significant ATP-dependent uptake of (³H)-DNP-SG by vacuoles was found. These results indicate thatS. cerevisiaetransport glutathione S-conjugates both outside the cells and into the vacuoles.     

ATP-dependent primary active transport of cysteinyl leukotrienes across liver canalicular membrane. Role of the ATP-dependent transport system for glutathione S-conjugates

The liver is the major organ which eliminates leukotriene C4 (LTC4) and other cysteinyl leukotrienes from the blood circulation into bile. Transport of LTC4 was studied using inside-out vesicles enriched in canalicular and sinusoidal membranes from rat liver. The incubation of canalicular membrane vesicles with [3H]LTC4 in the presence of ATP resulted in an uptake of LTC4 into vesicles. The initial rate of ATP-stimulated LTC4 uptake was about 40-fold higher in canalicular than in sinusoidal membrane vesicles. When liver plasma membrane vesicles were incubated in the absence of ATP, an apparent transient uptake of LTC4 was observed which was temperature-dependent and not affected by the osmolarity. This indicates that LTC4 was bound to proteins on the surface of plasma membrane vesicles. Two proteins with relative molecular weights of 17,000 and 25,000 were detected by direct photoaffinity labeling as major LTC4-binding proteins. One protein (Mr 25,000) was ascribed to subunit 1 (Ya) of glutathione S-transferase which was associated with the membrane. LTD4, LTE4, N-acetyl-LTE4, and omega-carboxy-N-acetyl-LTE4 were also transported into liver plasma membrane vesicles in an ATP-dependent manner with initial rates relative to LTC4 (1.0) of 0.46, 0.11, 0.35, and 0.22, respectively. Mutual competition between the cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione for uptake indicated that they are transported by a common carrier. Apparent Km values of the transport system for LTC4, LTD4, and N-acetyl-LTE4 were 0.25, 1.5, and 5.2 microM, respectively. The ATP-dependent transport of LTC4 into vesicles was not inhibited by doxorubicin, daunorubicin, or verapamil, or by the monoclonal antibody C219, suggesting that the transport system differs from P-glycoprotein. Liver plasma membrane vesicles prepared from mutant rats deficient in the hepatobiliary excretion of cysteinyl leukotrienes lacked the ATP-dependent transport of cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione. These results demonstrate that the ATP-dependent carrier system is responsible for the transport of cysteinyl leukotrienes and glutathione S-conjugates from the hepatocytes into bile.     

ATP stimulates the uptake of S-dinitrophenylglutathione by rat liver plasma membrane vesicles

Incubation of inverted plasma membrane vesicles from rat liver with micromolar concentrations of S-dinitrophenylglutathione (DNP-SG) in the presence of ATP resulted in the uptake of DNP-SG into the vesicles. ATP-dependent DNP-SG accumulation was half-maximal with 9 μM DNP-SG, while the K_m for ATP was 320 μM. Glutathione disulfide (GSSG), but not reduced glutathione, inhibited the ATP-dependent accumulation of DNP-SG by the vesicles, suggesting that the same, ATP-dependent transport system is responsible for the extrusion of glutathione conjugates and GSSG from liver cells.     

Identification of P2Y purinoceptors associated with voltage-activated cation channels in cardiac ventricular myocytes of the rat

Extracellular ATP has vasodilatory and inotropic effects in the heart. We have demonstrated that extracellular ATP, in a concentration-dependent manner (10 nM-0.1 mM), increased [Ca2+]i in suspensions of isolated fura-2-loaded rat cardiac ventricular myocytes (maximum 96 +/- 10% increase over basal levels, SEM, n = 12, P less than 0.01). The increase in [Ca2+]i was often biphasic, with an initial fast phase (less than 1 s) of low amplitude, followed by a slower phase of higher amplitude. A second application of ATP had little effect, and ATP abolished the effect of subsequent electrical stimulations, even through the cells were still able to respond with an increase in [Ca2+]i to KCl-induced depolarization or stimulation by caffeine. Pretreatment of cells with nifedipine, verapamil, caffeine, ryanodine, or 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride attenuated the effect of extracellular ATP on [Ca2+]i, and binding of extracellular free calcium by excess EGTA completely abolished the effects of extracellular ATP and electrical stimulation. Extracellular ATP increased bisoxonol fluorescence in ventricular myocytes, indicating depolarization of the sarcolemma. Pretreatment of the myocytes with tetrodotoxin (50 microM), or replacement of NaCl in the incubation buffer with the impermeant cation N-methyl-D-glucamine, suppressed the extracellular ATP effect on [Ca2+]i. ADP and AMP had smaller effects on [Ca2+]i than ATP; adenosine had no effect. ATP analogues showed the following rank order of potency in increasing [Ca2+]i or bisoxonol fluorescence: ATP greater than or equal to 2-methylthioATP much greater than adenosine 5'-O-[3-thio]triphosphate greater than adenosine 5'-[alpha, beta-methylene]triphosphate approximately adenosine 5'-[beta, gamma-methylene]triphosphate approximately adenosine 5'-[beta, gamma-imino]triphosphate greater than adenosine. These data are consistent with the presence of purinoceptors (P2Y subtype) on the sarcolemma of cardiac ventricular myocytes of the rat, which upon activation lead to depolarization and activation of cation channels of the sarcolemma and flux of extracellular Ca2+ into the cells. This may result in further flux of Ca2+ into the cytosol from intracellular stores. The effects of extracellular ATP on [Ca2+]i in rat cardiac ventricular myocytes may, in part, explain the direct inotropic effects of extracellular ATP on the mammalian heart.     

Chromium(III)ATP inactivating (Na+ + K+)-ATPase supports Na+-Na+ and Rb+-Rb+ exchanges in everted red blood cells but not Na+,K+ transport

The chromium(III) complex of ATP, an MgATP complex analogue, inactivates (Na+ + K+)-ATPase by forming a stable chromo-phosphointermediate. The rate constant k2 of inactivation at 37 degrees C of the beta, gamma-bidentate of CrATP is enhanced by Na+ (K0.5 = 1.08 mM), imidazole (K0.5 = 15 mM) and Mg2+ (K0.5 = 0.7 mM). These cations did not affect the dissociation constant of the enzyme-chromium-ATP complex. The inactive chromophosphoenzyme is reactivated slowly by high concentrations of Na+ at 37 degrees C. The half-maximal effect on the reactivation was reached at 40 mM NaCl, when the maximally observable reactivation was studied. However, 126 mM NaCl was necessary to see the half-maximal effect on the apparent reactivation velocity constant. K+ ions hindered the reactivation with a Ki of 70 microM. Formation of the chromophosphoenzyme led to a reduction of the Rb+ binding sites and of the capacity to occlude Rb+. The beta, gamma-bidentate of chromium(III)ATP (Kd = 8 microM) had a higher than the alpha, beta, gamma-tridentate of chromium(III)ATP (Kd = 44 microM) or the cobalt tetramine complex of ATP (Kd = 500 microM). The beta, gamma-bidentate of the chromium(III) complex of adenosine 5'-[beta, gamma-methylene]triphosphate also inactivated (Na+ + K+)ATPase. Although CrATP could not support Na+, K+ exchange in everted vesicles prepared from human red blood cells, it supported the Na+-Na+ and Rb+-Rb+ exchange. It is concluded that CrATP opens up Na+ and K+ channels by forming a relatively stable modified enzyme-CrATP complex. This stable complex is also formed in the presence of the chromium complex of adenosine 5'-[beta, gamma-methylene]triphosphate. Because the beta, gamma-bidentate of chromium ATP is recognized better than the alpha, beta, gamma-tridentate, it is concluded that the triphosphate site recognizes MgATP with a straight polyphosphate chain and that the Mg2+ resides between the beta- and the gamma-phosphorus. The enhancement of inactivation by Mg2+ and Na+ may be caused by conformational changes at the triphosphate site.     

ATP-dependent Cl- uptake by plasma membrane vesicles from the rat brain

Uptake of Cl- by plasma membrane vesicles from the rat brain was stimulated by ATP at 37 degrees C, but not by beta, gamma-methylene ATP or at 0 degrees C. The addition of Triton X-100 or sucrose to the incubation medium diminished the ATP-stimulated Cl- uptake, suggesting that Cl- was transported across the membranes into the intravesicular space. This ATP-stimulated Cl- uptake was not affected by 1 mM ouabain. 1 microM oligomycin, 0.1 mM gamma-aminobutyric acid or 0.1 mM picrotoxin. Thus, non-mitochondrial ATP-driven Cl- transport through a system other than Na, K-ATPase or Cl- channels occurs in neuronal plasma membrane vesicles.     

Evidence for the presence of P2-purinoceptors at the surface of human articular chondrocytes in monolayer culture

Extracellular purines can act at purinoceptors to influence metabolic processes. Nucleotide-metabolizing ectoenzymes may modulate such purinergic effects, and their occurrence in a tissue may suggest the presence of purinoceptors. Thus, following the identification of ecto-nucleoside triphosphate pyrophosphatase in cultured human articular chondrocytes, we have studied whether these cells express P2-type purinoceptors. Release of prostaglandin E (PGE) was monitored, since articular chondrocytes synthesize and secrete PGE, and activation of P2-purinoceptors frequently results in enhanced prostaglandin production. Extracellular ATP and ADP stimulated PGE production, whereas AMP and adenosine had only limited effects. ATP concentrations as low as 5 microM were effective, and maximal responses were achieved at 50-100 microM ATP. GTP, UTP and ITP also elicited responses, but tended to be less effective than ATP at equivalent concentrations. Of the analogues of ATP that were tested, only adenosine 5'-(beta,gamma-methylene)triphosphate stimulated PGE production. The response to extracellular ATP was virtually abolished by indomethacin. Treatment of the cells with the P1-purinoceptor antagonist, 8-phenyltheophylline, or with pertussis toxin reduced both basal and ATP-stimulated PGE production, but did not substantially decrease the ratio of ATP-stimulated to basal PGE production. These results indicate the presence of P2-purinoceptors in cultured human articular chondrocytes, and suggest that extracellular ATP may have physiological and pathological effects in human articular cartilage.     

Leukotriene C(4) (LTC(4)) does not share a cellular efflux mechanism with cGMP: characterisation of cGMP transport by uptake to inside-out vesicles from human erythrocytes

The transport of cGMP out of cells is energy requiring and has characteristics compatible with an ATP-energised anion pump. In the present study a model with inside-out vesicles from human erythrocytes was employed for further characterisation of the cGMP transporter. The uptake of leukotriene C(4) (LTC(4)), a substrate for multidrug resistance protein (MRP), was concentration-dependently inhibited by the leukotriene antagonist MK571 (IC(50)=110+/-20 nM), but cGMP was unable to inhibit LTC(4) uptake. Oxidised glutathione (GSSG) and glutathione S-conjugates caused a concentration-dependent inhibition of [(3)H]cGMP uptake with IC(50) of 2200+/-700 microM for GSSG, 410+/-210 microM for S-(p-nitrobenzyl)glutathione and 37+/-16 microM for S-decylglutathione, respectively. Antioxidants such as reduced glutathione and dithiothreitol did not influence transport for concentrations up to 100 microM, but both inhibited cGMP uptake with approx. 25% at 1 mM. The cGMP pump was sensitive to temperature without activity below 20 degrees C. The transport of cGMP was dependent on pH with maximal activity between pH 8.0 and 8.5. Calcium caused a concentration-dependent inhibition with IC(50) of 43+/-12 microM. Magnesium gave a marked activation in the range between 1 and 20 mM with maximum effect at 10 mM. The other divalent cations, Mn(2+) and Co(2+), were unable to substitute Mg(2+), but caused some activation at 1 mM. EDTA and EGTA stimulated cGMP transport concentration-dependently with 50% and 100% above control at 100 microM, respectively. The present study shows that the cGMP pump has properties compatible with an organic anion transport ATPase, without affinity for the MRP substrate LTC(4). However, the blockade of the cGMP transporter by glutathione S-conjugates suggests it is one of several GS-X pumps.     

2-Mercaptoacetate administration depresses the beta-oxidation pathway through an inhibition of long-chain acyl-CoA dehydrogenase activity.

In an investigation of the link between Pi transport and alkaline phosphatase in mammalian small intestine, the characteristics of Pi uptake by brush-border membrane vesicles prepared from rat intestine were compared with the properties of the tissue alkaline phosphatase. The NaCl-dependent Pi uptake had a Km of 0.1 mM at pH 7.5 and was inhibited totally by 1 mM-arsenate and by 1 mM-vanadate. These compounds are also potent competitive inhibitors of the alkaline phosphatase activity of the vesicles, with Ki values less than 5 microM at pH 7.5. When the effect on Pi uptake of several other potent inhibitors of alkaline phosphatase, including phosphonates and phosphate analogues, was tested, however, it was found that there was little, if any, inhibition of transport under conditions in which the inhibition of phosphatase activity was total. Incubation of the vesicles for 20 min with oxidized adenosine 5'-[beta gamma-imido]triphosphate followed by rapid gel filtration to remove the inhibitor resulted in an irreversible loss of phosphatase activity, but left Pi transport unimpaired. Conversely, a similar prolonged incubation with adenosine 5'-[beta-thio]diphosphate or adenosine 5'-[gamma-thio]triphosphate had no effect on alkaline phosphatase activity but resulted in a permanent partial loss of transport capability. The failure to demonstrate an inhibition of Pi transport resulting from inhibition of alkaline phosphatase and the different responses of enzymic activity and Pi transport to irreversible inhibition make it very unlikely that the enzyme is directly involved in the transport system.     

Export pumps for glutathione S-conjugates

The release of glutathione S-conjugates from cells is an ATP-dependent process mediated by integral membrane glycoproteins belonging to the recently discovered multidrug-resistance protein (MRP) family. Many lipophilic compounds conjugated with glutathione, glucuronate, or sulfate are substrates for export pumps of the MRP family. In humans six MRP isoforms encoded by different genes have been cloned. Orthologs of MRP have been identified in many species including yeast, plants, and nematodes. Human MRP1 and MRP2 are currently best characterized with respect to substrate specificity by measurements of ATP-dependent transport into inside-out membrane vesicles. High-affinity substrates include the glutathione S-conjugate leukotriene C4, S-(2,4dinitrophenyl)glutathione, bilirubin glucuronosides, and 17beta-glucuronosyl estradiol. In addition, glutathione disulfide is transported by MRP1 and MRP2. Reduced glutathione may be released from cells in a process directly or indirectly mediated by members of the MRP family. Proteins of the MRP family are indispensable for transport of glutathione S-conjugates and glutathione disulfide into the extracellular space and play, therefore, a decisive role in detoxification and defense against oxidative stress.     

Low- and high-Km transport of dinitrophenyl glutathione in inside out vesicles from human erythrocytes.

Kinetic studies on the low- and high-Km transport systems for S-2,4-dinitrophenyl glutathione (DNP-SG) present in erythrocyte membranes were performed using inside-out plasma membrane vesicles. The high-affinity system showed a Km of 3.9 microM a Vmax of 6.3 nmol/mg protein per h, and the low-affinity system a Km of 1.6 mM and a Vmax of 131 nmol/mg protein per h. Both uptake components were inhibited by fluoride, vanadate, p-chloromercuribenzoate (pCMB) and bis(4-nitrophenyl)dithio-3,3'-dicarboxylate (DTNB). The low-Km uptake process was less sensitive to the inhibitory action of DTNB as compared to the high-Km process. N-Ethylmaleimide (1 mM) inhibited the high-Km process only. The high-affinity uptake of DNP-SG was competitively inhibited by GSSG (Ki = 88 microM). Vice versa, DNP-SG inhibited competitively the low-Km component of GSSG uptake (Ki = 3.3 microM). The high-Km DNP-SG uptake system was not inhibited by GSSG. The existence of a common high-affinity transporter for DNP-SG and GSSG in erythrocytes is suggested.     

Vacuolar transport of the glutathione conjugate of trans-cinnamic acid

Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-glutatione were used to study the vacuolar transport of phynylpropanoid-glutathione conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-conjugated [14C]trans-cinnamic acid. Uptake of the conjugate in the presence of MgATP was not dependent on a trans-tonoblast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 microM; 14% inhibition). Uptake of the conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) glutathione (500 microM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated Km and Vmax values for uptake were 142 microM amd 5.95 nmol mg(-1) min(-1), respectively. Based on these results, phenylpropanoid-glutation conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the glutathione S-conjugate pump(s) located in the tonoplast membrane.     

Stimulation of Ca2+ efflux from sarcoplasmic reticulum by preincubation with ATP and inorganic phosphate.

Preincubation of sarcoplasmic reticulum with 1 mM-ATP completely inhibits Ca2+ accumulation and stimulates ATPase activity by over 2-fold. This effect of ATP is obtained only when the preincubation is carried out in the presence of Pi, but not with arsenate, chloride or sulphate. The inhibition by ATP of Ca2+ accumulation is pH-dependent, increasing as the pH is increased above 7.5. Inhibition of Ca2+ accumulation is observed on preincubation with ATP, but not with CTP, UTP, GTP, ADP, adenosine 5'-[beta gamma-methylene]triphosphate or adenosine 5'-[beta gamma-imido]triphosphate. The presence of Ca2+, but not Mg2+, during the preincubation, prevents the effect of ATP + Pi on Ca2+ accumulation. The ATP + Pi inhibition of Ca2+ accumulation is not due to modification of the ATPase catalytic cycle, but rather to stimulation of a rapid Ca2+ efflux from actively or passively loaded vesicles. This Ca2+ efflux is inhibited by dicyclohexylcarbodi-imide. Photoaffinity labelling of sarcoplasmic-reticulum membranes with 8-azido-[alpha-32P]ATP resulted in specific labelling of two proteins, of approx. 160 and 44 kDa. These proteins were labelled in the presence of Pi, but not other anions.     

Role of membrane-bound 5''-nucleotidase in nucleotide uptake by the moderate halophile Vibrio costicola

Intact cells of Vibrio costicola hydrolyzed ATP, ADP, and AMP. The membrane-bound 5'-nucleotidase (C. Bengis-Garber and D. J. Kushner, J. Bacteriol. 146:24-32, 1981) was solely responsible for these activities, as shown by experiments with anti-5'-nucleotidase serum and with the ATP analog, adenosine 5'-(beta gamma-imido)-diphosphate. Fresh cell suspensions rapidly accumulated 8-14C-labeled adenine 5'-nucleotides and adenosine. The uptake of ATP, ADP, and AMP (but not the adenosine uptake) was inhibited by adenosine 5'-(beta gamma-imido)-diphosphate similarly to the inhibition of the 5'-nucleotidase. Furthermore, the uptake of nucleotides had Mg2+ requirements similar to those of the 5'-nucleotidase. The uptake of ATP was competitively inhibited by unlabeled adenosine and vice versa; inhibition of the adenosine uptake by ATP occurred only in the presence of Mg2+. These experiments indicated that nucleotides were dephosphorylated to adenosine before uptake. The hydrolysis of [alpha-32P]ATP as well as the uptake of free adenosine followed Michaelis-Menten kinetics. The kinetics of uptake of ATP, ADP, and AMP also each appeared to be a saturable carrier-mediated transport. The kinetic properties of the uptake of ATP were compared with those of the ATP hydrolysis and the uptake of adenosine. It was concluded that the adenosine moiety of ATP was taken up via a specific adenosine transport system after dephosphorylation by the 5'-nucleotidase.     

Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.     

Selective activation of Ca2+ influx by extracellular ATP in a pancreatic beta-cell line (HIT)

The action of exogenous ATP on cytoplasmic free Ca2+ ([Ca2+]i) was studied in insulin secreting cells using fura-2. Stimulation of clonal pancreatic beta-cells (HIT) with ATP (range 2-20 microM) evoked a sustained elevation in [Ca2+]i. ATP selectively promoted Ca2+ influx and not Ca2+ mobilization since (1) the effect required external Ca1+ and (2) was observed in cells in which internal stores were depleted with ionomycin (3) the rate of Mn2+ influx, measured as the quenching of the fura-2 signal, was accelerated by ATP. The action of ATP was unaffected by the voltage-sensitive Ca2+ channel blockers nifedipine and verapamil as well as by a depolarizing concentration of K+. The effect on [Ca2+]i was highly specific for ATP since AMP, ADP, adenosine 5'-[gamma-thio]triphosphate, adenosine 5'-[beta, gamma-methylene]triphosphate, GTP and adenosine were ineffective. In normal pancreatic islet cells, both exogenous ATP (range 0.2-2 microM) and ADP induced a transient Ca2+ elevation that did not require external Ca2+. The nucleotide specificity of the effect on [Ca2+]i suggests that ATP activates P2 gamma purinergic receptors in normal beta-cells. Thus, ATP evokes a Ca2+ signal in clonal HIT cells and normal islet cells by different transducing systems involving distinct purinoreceptors. A novel mechanism for increasing [Ca2+]i by extracellular ATP is reported in HIT cells, since the nucleotide specificity and the selective activation of Ca2+ influx without mobilization of internal Ca2+ stores cannot be explained by mechanisms already described in other cell systems.     

Effect of ATP analogs of DNA synthesis in isolated nuclei

Optimal synthesis of DNA in Ehrlich ascites cell nuclei is shown to be dependent upon the presence of both ATP and ADP. ATP can be replaced only by dATP. An ATP regenerating system is less effective than ATP alone or ATP in combination with ADP. ATP does not stimulate DNA synthesis primarily by maintenance of deoxyribonucleotide triphosphate levels. When the inhibition of DNA synthesis by high ATP levels is taken into account, the ATP analogs adenosine 5'-(alpha,beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)-triphosphate, and adenosine 5'-(beta, gamma-imino)triphosphate can neither substitute for ATP nor inhibit the ATP stimulation of DNA synthesis. Adenosine 5'-(3-thio)triphosphate, however, is a competitive inhibitor of DNA synthesis.