Ecto-nucleotide pyrophosphatase/phosphodiesterase as part of a multiple system for nucleotide hydrolysis by platelets from rats: Kinetic characterization and biochemical properties

In this study, we describe an ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activity in rat platelets. Using p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate for E-NPP, we demonstrate an enzyme activity that shares the major biochemical properties described for E-NPPs: alkaline pH dependence, divalent cation dependence and blockade of activity by metal ion chelator. K_m and V_max values for p-Nph-5′-TMP hydrolysis were found to be 106?±?18?µM and 3.44?±?0.18?nmol p-nitrophenol/min/mg (mean?±?SD,?n?=?5). We hypothesize that an E-NPP is co-localized with an ecto-nucleoside triphosphate diphosphohydrolase and an ecto-5′-nucleotidase on the platelet surface, as part of a multiple system for nucleotide hydrolysis, since they can act under distinct physiological conditions and can be differently regulated. Thus, 0.25?mM suramin inhibited p-Nph-5′-TMP, ATP and ADP hydrolysis, while 0.5?mM AMP decreased only p-Nph-5′-TMP hydrolysis. Besides, 5.0, 10 and 20?mM sodium azide just inhibited ATP and ADP hydrolysis. Angiotensin II (5.0 and 10 nM) affected only ADP hydrolysis. Gadolinium chloride (0.2 and 0.5?mM) strongly inhibited the ATP and ADP hydrolysis. The E-NPP described here represents a novel insight into the control of platelet purinergic signaling.     

Ectonucleotidase activities are altered in serum and platelets of L-NAME-treated rats

It is well known that hypertension is closely associated to the development of vascular diseases and that the inhibition of nitric oxide biosynthesis by administration of Nomega-Nitro-L-arginine methyl ester hydrochloride(L-NAME) leads to arterial hypertension. In the vascular system, extracellular purines mediate several effects;thus, ADP is the most important platelet agonist and recruiting ag ent, while adenosine, an end product of nucleotide metabolism, is a vasodilator and inhibitor of platelet activation and recruitment. Members of several families of enzymes, known as ectonucleotidases, including E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolase), E-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase) and 5'-nucleotidase are able to hydrolyze extracellular nucleotides until their respective nucleosides. We investigated the ectonucleotidase activities of serum and platelets from rats made hypertensive by oral administration of L-NAME (30 mg/kg/day for 14 days or 30 mg/kg/day for 14 days plus 7 days of L-NAME washout, in the drinking water) in comparison to normotensive control rats. L-NAME promoted a significant rise in systolic blood pressure from 112 +/- 9.8 to 158 +/- 23 mmHg. The left ventricle weight index (LVWI) was increased in rats treated with L-NAME for 14 days when compared to control animals. In serum samples, ATP, ADP and AMP hydrolysis were reduced by about 27%, 36% and 27%, respectively. In platelets, the decrease in ATP, ADP and AMP hydrolysis was approximately 27%, 24% and 32%, respectively. All parameters recovered after 7 days of L-NAME washout. HPLC demonstrated a reduction in ADP, AMP and hypoxanthine levels by about 64%, 69% and 87%,respectively. In this study, we showed that ectonucleotidase activities are decreased in serum and platelets from L-NAME-treated rats, which should represent an additional risk for the development of hypertension. The modulation of ectonucleotidase activities may represent an approach to antihypertensive therapy via inhibition of spontaneous platelet activation and recruitment, as well as thrombus formation.     

The influence of bromelain on platelet count and platelet activity in vitro

Bromelain is a general name for a family of sulfhydryl-containing, proteolytic enzymes from the pineapple plant. The aim of the present study was to investigate the influence of bromelain on platelet count, platelet aggregation and platelet activity in vitro. Blood samples were taken from the antecubital vein of 10 healthy male non-smokers. Platelet count decreased after incubation with 2.5 and 5 mg bromelain/ml from 277 +/- 17 platelets/nl before to 256 +/- 21 and 247 +/- 19 platelets/nl after the treatment. The ADP and TRAP-6 induced platelet aggregation led to a significant decrease after the incubation with 2.5 mg (ADP: 48.6 +/- 25.7%; TRAP-6: 49.6 +/- 28.9%) or 5 mg (ADP: 5.0 +/- 4.6%; TRAP-6: 9.0 +/- 4.9%) bromelain/ml in comparison to control (ADP: 81.4 +/- 5.0%; TRAP-6: 77.4 +/- 10.4%). The percentage of unstimulated CD62P positive platelets which were investigated by flow cytometry was minimally higher after incubation with 5 mg bromelain/ml (0.57 +/- 0.48% PC) in comparison to control (0.22 +/- 0.11% PC), but after TRAP-6 stimulation the incubation with 5 mg bromelain/ml led to a remarkable decrease in comparison to the untreated control (50.4 +/- 20.2 to 0.9 +/- 0.8% PC). The changes of CD62P (TRAP-stimulated) and the results of platelet aggregation after incubation with bromelain in vitro may demonstrate the potential of bromelain as a substance for platelet inhibition.     

ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) in human blood platelets

Human platelets contain an ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) that is Ca(2+) dependent, hydrolyses ATP and ADP and also GTP, ITP, CTP, GDP, IDP, CDP. The enzyme does not hydrolyse AMP, p-nitrophenylphosphate, inorganic phosphate or glucose-6-phosphate. Contaminant activities were ruled out because the enzyme was not inhibited by 2 μg/d ouabain, 1.0 μM levamisole, 10 μM ApSA or 1.0 mM azide. The enzyme was sensitive to 100 μM orthovanadate, 100μMApSA and 10 mM azide, reagents that have been described as inhibitors of some other apyrases. A strong inhibition by 1.0 mM NEM was observed, indicating that sulphydryl groups are involved in the enzyme activity. The parallel behaviour of ATPase and ADPase activities and the competition plot presented suggest that ATP and ADP hydrolysis occurs at the same active site. ATP diphosphohydrolase from human platelets may be involved in the modulation of nucleotide concentration in the circulation and thus in vascular tonus.     

Synergistically Stimulated (Na+,K+)-Adenosine Triphosphatase from Plasma Membrane of a Marine Diatom

An ATP-hydrolyzing activity with the properties of a Mg(2+)-dependent (Na(+),K(+))-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from a 20-fold purified plasma membrane fraction of the marine diatom, Nitzschia alba is described.The basal activity requires Mg(2+) and further stimulation by Na(+) or Na(+) plus K(+) is dependent on the presence of Mg(2+); Mn(2+) or Co(2+) can partially substitute for the divalent cation requirement but Ca(2+) equimolar with Mg(2+) inhibits the activity by 54%. ATP is the preferred substrate for the Na(+) plus K(+) stimulated activity, while CTP, UTP, and ADP are only slightly hydrolyzed. The apparent K(m) is 8 x 10(-4) M ATP.The ATP hydrolysis-rate is dependent on the relative concentrations of Na(+) and K(+); the K(0.5) for Na(+) and K(+) are 2 mM and 50 mM, respectively. Basal activity is synergistically stimulated by Na(+) plus K(+) only at certain ion concentrations and shows a strong specificity for both cations.In the presence of Na(+) at 5 mM and K(+) at 350 mM, the ATPase is completely inhibited by p-chloromercuric benzoic acid 10(-4) M, N-ethyl maleimide 10(-3) M, and iodoacetamide 10(-2) M, but is insensitive to ouabain at 10(-7) to 10(-3) M.This study demonstrates for the first time that algal plasma membrane contains an ATPase that is synergistically stimulated by Na(+) and K(+).     

Identification of high-affinity (Kd 0.35 mumol/L) and low-affinity (Kd 7.9 mumol/L) platelet binding sites for ADP and competition by ADP analogues

Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2-3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1-0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3- aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2',3'-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5'-p- fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2',3'-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism

The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis-Menten equation. In the absence of products, the K(M) (the [ATP] required for half-maximal speed) was 28 +/- 1 microM, and the maximum speed was 904 nm/s. P(i) behaved as a competitive inhibitor with K(I) = 9 +/- 1 mM. ADP behaved approximately as a competitive inhibitor with K(I) = 35 +/- 2 microM. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and/or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P(i) is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P(i) before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P(i) release from the other.     

Skeletal muscle proteasome can degrade proteins in an ATP-dependent process that does not require ubiquitin.

The proteasome (the multicatalytic endoproteinase complex) in mammalian tissues hydrolyzes proteins and several types of peptides. When this structure was isolated rapidly from rabbit skeletal muscle in the presence of glycerol, its various peptidase and protease activities showed a large reversible activation by physiological concentrations of ATP (Ka = 0.3-0.5 mM). Hydrolysis of succinyl-Leu-Leu-Val-Tyr-(4-methylcoumaryl-7-amide) was stimulated up to 12-fold by ATP, whereas degradation of casein and bovine serum albumin increased 4- to 7-fold. Neither ADP nor AMP had any effect. CTP, GTP, UTP, and the nonhydrolyzable analogs adenosine 5'-[beta,gamma-imino]triphosphate (AMPP[NH]P) and adenosine 5'-[alpha,beta-methylene]triphosphate (AMP[CH2]PP) increased peptide hydrolysis as well as ATP did. However, only ATP stimulated casein breakdown and only in the presence of Mg2+. Thus, nucleotide binding allows activation of the peptidase functions, but ATP hydrolysis seems necessary for enhanced degradation of proteins. The ATP effect on proteolysis was reversible and did not require ubiquitin. Sensitivity to ATP was labile, and with storage at 4 degrees C the enzyme became fully active in the absence of ATP or Mg2+. The ATP-activated form closely resembles the proteasome complex described previously, which did not show ATP dependence: both have molecular masses of 650 kDa, contain the same 8-10 subunits, and are precipitated by the same antibodies. A similar ATP-activated form was found in rabbit liver but not in rabbit reticulocytes. The proteasome seems to represent a ubiquitin-independent, ATP-stimulated proteolytic activity within nucleated mammalian cells.     

ATP diphosphohydrolase in human platelets from patients with coronary arteries heart disease

ATP diphosphohydrolase is an enzyme described in platelets and may be related to the control of ADP-dependent platelet aggregation. Platelet aggregation in atherosclerotic coronary arteries, and the release of platelet-derived factors, play an important role in coronary artery disease syndromes. In this study, we determined the activity of ATP diphosphohydrolase in platelets from patients with chronic and acute coronary artery disease syndromes and healthy persons. The following groups were studied: healthy persons (group I), patients with chronic heart disease (group II) and acute heart disease (group III). Results did not demonstrate differences between the groups studied. The control group demonstrated a lower range of enzyme activity. The patients from groups II and III had ingested drugs with actions upon the cardiovascular system and the effect, in vitro, of these drugs upon the ATP diphosphohydrolase activity in human platelets was also investigated. The in vitro experiments demonstrated that 2.0 mM acetylsalicylic acid inhibited ATP hydrolysis by human platelets by approximately 55%. Significant correlation was observed between ADP hydrolysis and glucose blood levels in the control group and between ATP hydrolysis and triglycerides in the group II. These results contribute to our understanding of a possible relationship between ATP diphosphohydrolase and thrombogenesis.     

ATP diphosphohydrolase in human platelets from patients with coronary arteries heart disease

ATP diphosphohydrolase is an enzyme described in platelets and may be related to the control of ADP-dependent platelet aggregation. Platelet aggregation in atherosclerotic coronary arteries, and the release of platelet-derived factors, play an important role in coronary artery disease syndromes. In this study, we determined the activity of ATP diphosphohydrolase in platelets from patients with chronic and acute coronary artery disease syndromes and healthy persons. The following groups were studied: healthy persons (group I), patients with chronic heart disease (group II) and acute heart disease (group III). Results did not demonstrate differences between the groups studied. The control group demonstrated a lower range of enzyme activity. The patients from groups II and III had ingested drugs with actions upon the cardiovascular system and the effect, in vitro , of these drugs upon the ATP diphosphohydrolase activity in human platelets was also investigated. The in vitro experiments demonstrated that 2.0 mM acetylsalicylic acid inhibited ATP hydrolysis by human platelets by approximately 55%. Significant correlation was observed between ADP hydrolysis and glucose blood levels in the control group and between ATP hydrolysis and triglycerides in the group II. These results contribute to our understanding of a possible relationship between ATP diphosphohydrolase and thrombogenesis.     

Effects of adenosine triphosphate and alkaline phosphatase on solubilized 3,5,3'-triiodothyronine-binding activity

The T3-binding activity of salt-extractable nuclear proteins from rat liver was affected when ATP (2-10 mM; pH 8.0) was added concomitantly with T3 in the incubation medium. Scatchard analysis revealed that the equilibrium association constant was significantly reduced [5 mM ATP, 0.3 +/- 0.1 (+/- SE) 10(10) M-1; control, 1.1 +/- 0.15 X 10(10) M-1], but the maximum binding capacity remained unchanged. Similar values of inhibition were obtained when unbound receptors were preincubated with ATP. ATP achieved its maximal effect after 45 min of incubation at 30 C. Dilution experiments indicated that the effect of ATP was reversible. The inhibiting potency of nucleoside triphosphates at pH 8.0 was in the following order: ATP = CTP greater than GTP, whereas UTP had no effect. Nonhydrolyzable analogs of ATP were also inhibitory, and HPLC fractionation showed an approximately 98% recovery of ATP after incubation with nuclear extract. The adenine ring with at least two phosphates was essential, since ADP was as potent as ATP, whereas AMP had no effect. When the pH of the incubation medium was lowered to 7.3, the T3-binding activity was inhibited by ATP in the 0.1-1 mM range. Magnesium (3 mM) greatly increases the ATP effect at pH 7.3, but not at pH 8. The T3-binding activity was also drastically reduced when calf intestine alkaline phosphatase was added concomitantly in the incubation medium. Eight micrograms per ml enzyme were necessary to inhibit the T3 specific binding by 50% (30 C for 45 min). Scatchard analysis showed that the receptor affinity for T3 was decreased (control, 1.1 +/- 0.02 x 10(10) M-1; alkaline phosphatase, 0.41 +/- 0.03 x 10(10) M-1; n = 6), whereas the maximum binding capacity remained unchanged. Incubations performed with increasing concentrations of beta-mercaphoethanol (2.5, 5, 10, and 25 mM) revealed that the phosphatase inhibitory effect is thiol dependent. The inhibition was maximal at 2.5 mM and progressively decreased at 5 and 10 mM. No inhibition occurred at 25 mM. When a saturating concentration of T3 was employed, the specific binding was decreased at low thiol concentrations. These observations show that the nuclear T3 receptors may be modulated by ATP/ADP and phosphorylation/dephosphorylation processes. It is proposed that in vitro dephosphorylation leads to rapid oxydation of sulfhydryl groups which are essential for optimum T3 binding.     

Regulation of the nucleotide dependence of the cardiac sarcolemma Ca(2+)-ATPase.

The nucleotide dependence of the Ca(2+)-ATPase purified from cardiac sarcolemma by calmodulin-affinity chromatography was investigated for preparations either in the basal state or activated by three procedures: (i) addition of calmodulin; (ii) addition of phosphatidylserine and (iii) controlled proteolysis. Upon activation, the maximal velocity of ATP hydrolysis increases by a factor of 4-5, while the curves of ATP dependence of ATP hydrolysis change from hyperbolic to biphasic, revealing the presence of two Kmapp for ATP. A tight coupling between Ca2+ and ATP binding sites was also observed. At high ATP concentration, the ATPase activity of the basal state shows a complex dependence on Ca2+ concentration, increasing sharply at millimolar Ca2+. Our results indicate that this increase in ATPase activity is paralleled by the appearance of a second, low affinity Kmapp for ATP. When only the high affinity site for ATP is occupied the ATPase activity of the basal state displays a simple, hyperbolic dependence on the Ca2+ concentration. In addition, increasing Ca2+ concentration appears to decrease the ATP binding at the low affinity site of the enzyme. The effect of ADP on ATP hydrolysis was also examined. The finding that ADP is a potent inhibitor of the purified Ca(2+)-ATPase from heart suggests that the stimulatory action of ADP observed in cardiac sarcolemmal vesicles is not an intrinsic property of the enzyme.     

5'-nucleotidase activity is altered by hypo- and hyperthyroidism in platelets from adult rats

Thyroid disorders are associated to a number of vascular diseases that involve processes such as platelet aggregation and vascular tone control. Since, these processes can be also affected by ATP, ADP and adenosine levels, we investigate the hydrolysis of these nucleotides in platelets from hyperthyroid, hypothyroid, and hypothyroid with hormonal replacement rats. Hyperthyroidism was induced by daily injections of L-thyroxine (T4) 25 microg/100 g body weight for 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) for 14 days. In the hormonal replacement group, hypothyroid rats were injected with T4 (5 microg/100 g body weight, i.p.) for 5 days. The AMP hydrolysis by platelets was increased 49% in hyperthyroid rats and decreased 50% in response to hypothyroidism, while the ATP and ADP hydrolysis was not altered in both groups. Besides, the T4 replacement significantly reversed the inhibition of the AMP hydrolysis observed in hypothyroid rats. Our findings indicate that the thyroid disorders affect the 5'-nucleotidase activity and consequently can alter the adenosine levels in a reversible manner in platelet fraction. Since, adenosine is able to inhibit platelet aggregation and acts as a potent vasodilator, these results can contribute to a better comprehension of the vascular events described in thyroid disorders.     

The platelet-related effects of tenecteplase versus alteplase versus reteplase.

Clinical studies have investigated the combination of glycoprotein (GP) IIb/IIIa inhibitors and thrombolytic agents for acute myocardial infarction. However, thrombolytic agents alone may possess direct antiplatelet properties that could affect reperfusion. Blood from 11 patients with coronary disease and five healthy subjects was incubated for 30 min with tenecteplase (4, 12, and 24 microg/ml), alteplase (1, 4, and 10 microg/ml), reteplase (1, 5, and 10 microg/ml) or control buffer. Platelet aggregation induced by 1, 20 and 50 micromol/l adenosine diphosphate (ADP), the stimulated expression of GP IIb/IIIa and P-selectin, and plasma fibrinogen levels were determined. Platelet aggregation in patients was inhibited by medium and high concentrations of alteplase when induced by 1 micromol/l ADP [1.6 +/- 0.5%, P = 0.001 and 0.9 +/- 0.2%, P = 0.002 versus 8.3 +/- 1.6% (control)] and 20 micromol/l ADP [46.9 +/- 3.9%, P = 0.001 and 46.2 +/- 4.8%, P = 0.001 versus 65.7 +/- 2.7% (control)]. High concentration tenecteplase was associated with lower aggregation by 20 micromol/l ADP (58 +/- 2.1% versus control, P = 0.033). There were no changes in GP IIb/IIIa activation or P-selectin expression in patients or healthy subjects. Platelet aggregation (1 micromol/l ADP) in healthy subjects was inhibited only by high doses of alteplase (P = 0.001). Plasma fibrinogen levels were significantly decreased after treatment with reteplase at 1 microg/ml(1.53 +/- 0.21 versus 2.65 +/- 0.31, P = .009) and 5 microg/ml(1.55 +/- 0.16 versus 2.65 +/- 0.31, P = .005). Alteplase inhibits platelet aggregation more than tenecteplase and reteplase. The attenuation of platelet aggregation by alteplase is dissociated from the expression of activated GP IIb/IIIa and P-selectin, and by fibrinogen degradation. These results suggest that alteplase exerts its antiplatelet effect independent of GP IIb/IIIa and P-selectin expressions and fibrinogen degradation. These findings may be directly relevant to the effect of alteplase on reperfusion and to future studies using combined platelet inhibitors and thrombolytic therapy.     

5′-nucleotidase activity is altered by hypo- and hyperthyroidism in platelets from adult rats

Thyroid disorders are associated to a number of vascular diseases that involve processes such as platelet aggregation and vascular tone control. Since, these processes can be also affected by ATP, ADP and adenosine levels, we investigate the hydrolysis of these nucleotides in platelets from hyperthyroid, hypothyroid, and hypothyroid with hormonal replacement rats. Hyperthyroidism was induced by daily injections of L-thyroxine (T4) 25?μg/100?g body weight for 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) for 14 days. In the hormonal replacement group, hypothyroid rats were injected with T4 (5?μg/100?g body weight, i.p.) for 5 days. The AMP hydrolysis by platelets was increased 49% in hyperthyroid rats and decreased 50% in response to hypothyroidism, while the ATP and ADP hydrolysis was not altered in both groups. Besides, the T4 replacement significantly reversed the inhibition of the AMP hydrolysis observed in hypothyroid rats. Our findings indicate that the thyroid disorders affect the 5′-nucleotidase activity and consequently can alter the adenosine levels in a reversible manner in platelet fraction. Since, adenosine is able to inhibit platelet aggregation and acts as a potent vasodilator, these results can contribute to a better comprehension of the vascular events described in thyroid disorders.     

A Mg(2+)-dependent ecto-phosphatase activity on the external surface of Trypanosoma rangeli modulated by exogenous inorganic phosphate

In this work, we characterized a Mg(2+)-dependent ecto-phosphatase activity present in live Trypanosoma rangeli epimastigotes. This enzyme showed capacity to hydrolyze the artificial substrate for phosphatases, p-nitrophenylphosphate (p-NPP). At saturating concentration of p-NPP, half-maximal p-NPP hydrolysis was obtained with 0.23mM Mg(2+). Ca(2+) had no effect on the basal phosphatase activity, could not substitute Mg(2+) as an activator and in contrast inhibited the p-NPP hydrolysis stimulated by Mg(2+). The dependence on p-NPP concentration showed a normal Michaelis-Menten kinetics for this phosphatase activity with values of V(max) of 8.94+/-0.36 nmol p-NP x h(-1) x 10(-7) cells and apparent K(m) of 1.04+/-0.16 mM p-NPP. Mg(2+)-dependent ecto-phosphatase activity was stimulated by the alkaline pH range. Experiments using inhibitors, such as, sodium fluoride, sodium orthovanadate and ammonium molybdate, inhibited the Mg(2+)-dependent ecto-phosphatase activity. Inorganic phosphate (Pi), a product of phosphatases, inhibited reversibly in 50% this activity. Okadaic acid and microcystin-LR, specific phosphoserine/threonine phosphatase inhibitors, inhibited significantly the Mg(2+)-dependent ecto-phosphatase activity. In addition, this phosphatase activity was able to recognize as substrates only o-phosphoserine and o-phosphothreonine, while o-phosphotyrosine was not a good substrate for this phosphatase. Epimastigote forms of T. rangeli exhibit a typical growth curve, achieving the stationary phase around fifth or sixth day and the Mg(2+)-dependent ecto-phosphatase activity decreased around 10-fold with the cell growth progression. Cells maintained at Pi-deprived medium (2 mM Pi) present Mg(2+)-dependent ecto-phosphatase activity approximately threefold higher than that maintained at Pi-supplemented medium (50 mM Pi).     

Intracellular signaling for vasoconstrictor coupling factor 6: novel function of beta-subunit of ATP synthase as receptor

Coupling factor 6 (CF6), a component of adenosine triphosphate (ATP) synthase, is circulating and functions as an endogenous vasoconstrictor by inhibiting cytosolic phospholipase A2. We showed a high plasma level of CF6 in human hypertension. The present study focused on the identification and characterization of a receptor for CF6 and its post-receptor signaling pathway. Incubation of human umbilical vein endothelial cells (HUVECs) with an excess of free CF6 reduced by 50% the immunoreactivity for the antibody to beta-subunit of ATP synthase at the cell surface, but unaffected that for the alpha-subunit antibody. A significant displacement of radioligand was observed at 3x10(-9) through 10(-7) M unlabeled CF6, and the Kd was 7.6 nM. Adenosine diphosphate (ADP) at 10(-7) M and beta-subunit antibody suppressed the binding of (125)I-CF6 by 81.3+/-9.7% and 32.0+/-2.0%, respectively, whereas the alpha-subunit antibody unaffected it. The hydrolysis activity of ATP to ADP was increased by 1.6-fold by CF6 at 10(-7) M, and efrapeptin at 10(-5) M, an inhibitor of ATP synthase, blocked it. CF6 at 10(-7) M decreased intracellular pH in 2',7'-bis(carboxyethyl-5 (6))-carboxyfluorescein-loaded HUVEC. Amyloride at 10(-4) M augmented the pH decrease in response to CF6, whereas efrapeptin at 10(-5) M blocked it. Arachidonic acid release was suppressed by CF6, and it was reversed by efrapeptin at 10(-5) M or beta-subunit antibody or ADP at 10(-7) M. The beta-subunit antibody suppressed coupling factor 6-induced increase in blood pressure. These indicate that membrane-bound ATP synthase functions as a receptor for CF6 and may have a previously unsuspected role in the genesis of hypertension by modulating the concentration of intracellular hydrogen.     

Activity of purified NAD-specific isocitrate dehydrogenase at modulator and substrate concentrations approximating conditions in mitochondria

The kinetic parameters of NAD-specific isocitrate dehydrogenase from bovine heart were examined at levels of substrates and effectors approximating the concentrations reported for isolated intact heart mitochondria in different respiratory states. The effect of changing ADP/ATP ratios (with total adenine nucleotides constant at 8 mmol/L) on enzyme activity was measured at constant concentrations of the substrates magnesium D-isocitrate (0.10 mmol/L) and NAD+ (3.0 mmol/L), the positive effector magnesium citrate (1.0 mmol/L) and the negative effector NADPH (1.5 mmol/L) at pH 7.4. Enzyme activity increased with increasing ADP/ATP ratios as a result of activation by rising ADP concentrations and not due to decreasing inhibition by falling levels of ATP. Increasing ADP decreased the inhibition by NADPH, and this effect was enhanced by magnesium citrate and by free Ca2+. In incubation media containing all of the above effectors, the S0.5 for enhancement of activity by free Ca2+ was 10 to 20 mumol/L at ratios of total ADP/total ATP between 2.0 and 0.1. This value is in the range of intramitochondrial concentrations of free Ca2+,1 but it is appreciably larger than S0.5 of Ca2+ (0.6 to 1 mumol/L) for the enhancement of ADP activation, which was determined in the absence of other effectors. When both the NAD+/NADH and the ADP/ATP ratios were decreased, a further decline in activity was found. The effect of the decreasing NAD+/NADH ratio was due to inhibition by NADH (apparent I0.5 = 0.23 +/- 0.03 mmol/L) since NAD+ was saturating over the range examined.     

Regulation by cell metabolism and adenine nucleotides of a K channel in insulin-secreting B cells (RIN m5F).

A potassium channel in membranes of the insulin-secreting B-cell line, RIN m5F, was studied using the patch-clamp technique. In cell-attached patches, and for pipette solutions containing 140 mM KCl, the I-V curves exhibited a pronounced rectification, with the conductance being higher when the current flowed from the electrode into the cell (50 pS). Addition of glucose (5-7 mM) to the bath was sufficient to abolish the channel activity, while low doses of the metabolic inhibitor 2,4-dinitrophenyl enhanced it, indicating a link between channel activity and cell metabolism. In excised inside-out patches, the activity of a channel with similar conductive properties was inhibited by ATP in a dose-dependent fashion, the half-maximal concentration being approximately 70 microM. ADP also acted as an inhibitor, but with much lower potency. However, when ADP was present, the blocking effectiveness of ATP was reduced, suggesting a nonadditive interaction of the two nucleotides with the channel. In conclusion, complete closure of the K channel by glucose (5-7 mM) suggests that this channel may be responsible for the early phase of the B-cell depolarization, which is seen in normal cells for glucose increments up to similar values. In addition, since ATP and ADP are both present in the cells, the reduction by ADP of the blocking efficacy of ATP in excised patches may help one to understand why, in intact cells, the channel is still functional for intracellular ATP concentrations that in excised patches would completely suppress its activity.     

Adenosine diphosphate stimulation of cultured hematopoietic cell lines

Adenosine diphosphate (ADP) plays a critical role in platelet activation both by exogenous stimulation and the release of endogenous intracellular stores. As the platelet ADP receptor is not well defined, we have chosen to identify and characterize several cell lines that possess functional receptors for this nucleotide. Rat promegakaryoblasts (RPM), human erythroleukemia cells (HEL), U937, and K562 leukemia cells responded to ADP, as measured by a rapid increase in intracellular calcium. In the case of RPM cells, ADP was the only naturally occurring platelet agonist capable of eliciting this response. Binding studies with [3H]ADP and fixed cells showed 3.99 +/- 1.77 x 10(5) binding sites/cell for RPM cells (apparent dissociation constant [kd] = 7.75 +/- 2.3 x 10(-8) mol/L), 8.19 +/- 3.25 x 10(5) sites/cell for HEL cells (kd = 2.15 +/- 0.84 x 10(-7) mol/L, 1.15 +/- 0.23 x 10(6) sites/cell for U937 cells (kd = 2.20 +/- 0.53 x 10(-7) mol/L) and 5.39 +/- 2.80 x 10(5) sites/cell for K562 cells (kd = 1.37 +/- 0.39 x 10(-7) mol/L), Inhibition studies with unlabeled nucleotides and analogues showed that binding was approximately 85% specific and the inhibitory pattern was similar to that seen with mature platelets. The purine base adenosine resulted in little or no inhibition. These studies indicate that both human and rat hematopoietic cell lines possess intact ADP receptors and may be useful tools in future studies of the structure and function of this important platelet-activation system.