This investigation evaluated the antileukemia properties of a zerumbone (ZER)-loaded nanostructured lipid carrier (NLC) prepared by hot high-pressure homogenization techniques in an acute human lymphoblastic leukemia (Jurkat) cell line in vitro. The apoptogenic effect of the ZER-NLC on Jurkat cells was determined by fluorescent and electron microscopy, Annexin V-fluorescein isothiocyanate, Tdt-mediated dUTP nick-end labeling assay, cell cycle analysis, and caspase activity. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay showed that ZER-NLC did not have adverse effects on normal human peripheral blood mononuclear cells. ZER-NLC arrested the Jurkat cells at G2/M phase with inactivation of cyclin B1 protein. The study also showed that the antiproliferative effect of ZER-NLC on Jurkat cells is through the intrinsic apoptotic pathway via activation of caspase-3 and caspase-9, release of cytochrome c from the mitochondria into the cytosol, and subsequent cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP). These findings show that the ZER-NLC is a potentially useful treatment for acute lymphoblastic leukemia in humans.

Platelets play a central role in inflammation through their direct interaction with other cell types, such as leucocytes and endothelial cells, and by the release of many factors, that is, lipids [such as thromboxane (TX)A2 ] and proteins (a wide number of angiogenic and growth factors) stored in α-granules, and adenosine diphosphate (ADP), stored in dense granules. These platelet actions trigger autocrine and paracrine activation processes that lead to leucocyte recruitment into different tissues and phenotypic changes in stromal cells which contribute to the development of different disease states, such as atherosclerosis and atherothrombosis, intestinal inflammation and cancer. The signals induced by platelets may cause pro-inflammatory and malignant phenotypes in other cells through the persistent induction of aberrant expression of cyclooxygenase (COX)-2 and increased generation of prostanoids, mainly prostaglandin (PG)E2 . In addition to cardiovascular disease, enhanced platelet activation has been detected in inflammatory disease and intestinal tumourigenesis. Moreover, the results of clinical studies have shown that the antiplatelet drug aspirin reduces the incidence of vascular events and colorectal cancer. All these pieces of evidence support the notion that colorectal cancer and atherothrombosis may share a common mechanism of disease, that is, platelet activation in response to epithelial (in tumourigenesis) and endothelial (in tumourigenesis and atherothrombosis) injury. Extensive translational medicine research is necessary to obtain a definitive mechanistic demonstration of the platelet-mediated hypothesis of colon tumourigenesis. The results of these studies will be fundamental to support the clinical decision to recommend the use of low-dose aspirin, and possibly other antiplatelet agents, in primary prevention, that is, even for individuals at low cardiovascular risk.

We have previously shown that platelets express 2 receptor tyrosine kinases, EphA4 and EphB1, and the Eph kinase ligand, ephrinB1, and proposed that transcellular Eph/ephrin interactions made possible by the onset of platelet aggregation promote the further growth and stability of the hemostatic plug. The present study examines how this might occur. The results show that clustering of either ephrinB1 or EphA4 causes platelets to adhere to immobilized fibrinogen via alpha(IIb)beta(3). Adhesion occurs more slowly than with adenosine diphosphate (ADP) and requires phosphatidylinositol 3 (PI3)-kinase and protein kinase C activity but not ephrinB1 phosphorylation. By itself, Eph and ephrin signaling is insufficient to cause aggregation or the binding of soluble fibrinogen, but it can potentiate aggregation initiated by a Ca(++) ionophore or by agonists for thrombin and thromboxane receptors. It also enhances Rap1 activation without requiring ADP secretion, ephrinB1 phosphorylation, or the activation of PI3-kinase and Src. From this we conclude that (1) Eph/ephrin signaling enhances the ability of platelet agonists to cause aggregation provided that those agonists can increase cytosolic Ca(++); (2) this is accomplished in part by activating Rap1; and (3) these effects require oligomerization of ephrinB1 but not phosphotyrosine-based interactions with the ephrinB1 cytoplasmic domain.

Glutathione reductase of hemolyzates from clinically normal subjects is activated by the addition of flavin-adenine dinucleotide. One-half maximum stimulation could be achieved by approximately 0.02 micromolar flavin-adenine dinucleotide; prior addition of adenosine triphosphate, adenosine diphosphate, or adenosine mnonophosphate prevented activation. Stimulation of glutathione reductase activity of red cells of normal subjects occurred when they were given 5 milligrams of riboflavin daily for 8 days. The degree of stimulation in vitro by flavin-adenine dinucleotide and in vivo by riboflavin was inversely proportional to dietary intake of riboflavin. The variety of clinical disorders which have been associated with glutathione reductase deficiency may have, as a common denominator, abnormalities in flavin-adenine dinucleotide formation.

Glycogen synthase (GS) catalyses the incorporation of uridine diphosphate-glucose into glycogen in skeletal muscle. In concert with the glucose transport step, GS activity is thought to be rate-limiting in the disposal of glucose as muscle glycogen. Glycogen synthase is regulated by both allosteric factors (primarily glucose 6-phosphate) and covalent modification by reversible phosphorylation and dephosphorylation leading to inactivation and activation of GS, respectively. Exercise activates both stimulatory and inhibitory regulators of GS and it is thought that the resultant activity of GS during exercise depends on the relative strength of opposing signals. However, the mechanisms by which exercise regulates GS activity are not fully understood. Glycogen breakdown, the GM-protein phosphatase 1 complex and possibly cellular relocalization of GS may be considered important factors involved in the stimulation of GS activity during exercise, while adenosine monophosphate-activated protein kinase and plasma adrenaline (via protein kinase A) can be considered as essential for the exercise-induced inhibitory signals to GS.

After subarachnoid hemorrhage (SAH), large cerebral arteries are prone to vasospasm. Using a rat model of SAH, we examined whether cortical microvessels demonstrate vasomotor changes that may make them prone to spasm and whether endothelial dysfunction may account for any observed changes. Two days after percutaneous catheterization into the cisterna magna, 0.3 mL of autologous blood was injected into the subarachnoid space. The brain tissue was harvested 20 min later, and microvessels were dissected from the parietal cortex. Vasomotor responses to the thromboxane analog U46619, the protein kinase C agonist phorbol acetate, endothelin-1, adenosine diphosphate, nitroprusside, and isoproterenol were examined in vitroin cerebral arterioles from the control, sham-operated, and SAH animals. Endothelial nitric oxide synthase (NOS3) messenger RNA and protein concentration was measured by northern and western blotting, respectively. Arterioles from the SAH animals demonstrated attenuated dilation to the endothelium-dependent dilator adenosine diphosphate and accentuated constriction to endothelin-1, while responses to the other agents tested were unchanged. NOS3 protein concentration was decreased, but NOS3 messenger RNA was increased after SAH. After SAH, cortical arterioles demonstrate endothelial dysfunction, which may be the basis for microvascular spasm. This is in part related to decreased NOS3, which occurs despite an increase in its transcription.

CONCLUSIONS

Acute microvascular endothelial dysfunction may occur after subarachnoid hemorrhage and contribute to microvascular spasm.

BACKGROUND

Amorphous silica nanoparticles (SiNP) can be used in medical technologies and other industries leading to human exposure. However, an increased number of studies indicate that this exposure may result in cardiovascular inflammation and damage. A high ratio of nitric oxide to peroxynitrite concentrations ([NO]/[ONOO(-)]) is crucial for cardiovascular homeostasis and platelet hemostasis. Therefore, we studied the influence of SiNP on the platelet [NO]/[ONOO(-)] balance and platelet aggregation.

METHODS

Nanoparticle-platelet interaction was examined using transmission electron microscopy. Electrochemical nanosensors were used to measure the levels of NO and ONOO(-) released by platelets upon nanoparticle stimulation. Platelet aggregation was studied using light aggregometry, flow cytometry, and phase contrast microscopy.

RESULTS

Amorphous SiNP induced NO release from platelets followed by a massive stimulation of ONOO(-) leading to an unfavorably low [NO]/[ONOO(-)] ratio. In addition, SiNP induced an upregulation of selectin P expression and glycoprotein IIb/IIIa activation on the platelet surface membrane, and led to platelet aggregation via adenosine diphosphate and matrix metalloproteinase 2-dependent mechanisms. Importantly, all the effects on platelet aggregation were inversely proportional to nanoparticle size.

CONCLUSIONS

The exposure of platelets to amorphous SiNP induces a critically low [NO]/[ONOO(-)] ratio leading to platelet aggregation. These findings provide new insights into the pharmacological profile of SiNP in platelets.

Activation of G(i)-coupled receptors in neutrophils stimulates class IB phosphoinositide 3-kinase (PI3K) (also known as PI3Kgamma) through the combined actions of Gbetagamma subunits and the small guanosine triphosphatase (GTPase) Ras, resulting in the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] in the plasma membrane. In most cases, the effectors of this pathway possess a pleckstrin homology (PH) domain that mediates the interaction with and regulation by these two lipid messengers. These direct effectors sit within a complex regulatory network that includes several other signaling pathways and that is responsible for the control of important neutrophil functions, including adhesion, chemotaxis, secretion, and the "respiratory burst" [activation of the nicotinamide adenosine diphosphate (NADPH) oxidase]. Although the molecular details that link the direct effectors of class IB PI3K to these complex cell responses are still largely unknown, these responses involve complex regulation of small GTPases of the Rac, Rho, and Arf families.

Cancer is a chronic disease and its pathogenesis is well correlated with infection and inflammation. Adenosine is a purine nucleoside, which is produced under metabolic stress like hypoxic conditions. Acute or chronic inflammatory conditions lead to the release of precursor adenine nucleotides (adenosine triphosphate (ATP), adenosien diphosphate (ADP) and adenosine monophosphate (AMP)) from cells, which are extracellularly catabolized into adenosine by extracellular ectonucleotidases, i.e., CD39 or nucleoside triphosphate dephosphorylase (NTPD) and CD73 or 5'-ectonucleotidase. It is now well-known that adenosine is secreted by cancer as well as immune cells during tumor pathogenesis under metabolic stress or hypoxia. Once adenosine is released into the extracellular environment, it exerts various immunomodulatory effects via adenosine receptors (A1, A2A, A2B, and A3) expressed on various immune cells (i.e., macrophages, myeloid-derived suppressor cells (MDSCs), natural killer (NK) cells, dendritic cells (DCs), T cells, regulatory T cell (Tregs), etc.), which play very important roles in the pathogenesis of cancer. This review is intended to summarize the role of inflammation and adenosine in the immunopathogenesis of tumor along with regulation of tumor-specific immune response and its modulation as an adjunct approach to tumor immunotherapy.

OBJECTIVE

To investigate the potential effect of atorvastatin 80 mg/day on the pharmacokinetics and pharmacodynamics of the thienopyridines prasugrel and clopidogrel.

METHODS

Open-label, randomized, crossover, two-arm, parallel-group study.

METHODS

Single clinical research center in the United Kingdom.

METHODS

Sixty-nine healthy men aged 18-60 years. Intervention. Subjects received either a loading dose of prasugrel 60 mg followed by a maintenance dose of 10 mg/day or a loading dose of clopidogrel 300 mg followed by 75 mg/day. The drug was given as monotherapy for 10 days, and after a 6-day run-in period with atorvastatin 80 mg/day, the same dosage of atorvastatin was continued with the respective thienopyridine for 10 days. A 14-day washout period separated the treatment regimens.

RESULTS

Blood samples were collected before and at various time points after dosing on days 1 and 11 for determination of plasma concentrations of metabolites and for measurement of platelet aggregation induced by adenosine 5'-diphosphate 20 microM and vasodilator-stimulated phosphoprotein (VASP). Coadministration of atorvastatin did not alter exposure to active metabolites of prasugrel or clopidogrel after the loading dose and thus did not alter inhibition of platelet aggregation (IPA). During maintenance dosing, atorvastatin administration resulted in 17% and 28% increases in the area under the plasma concentration-time curve (AUC) values of prasugrel's and clopidogrel's active metabolites, respectively. These small changes in AUC did not result in a significant change in IPA response to prasugrel but did result in a significant increase in IPA during clopidogrel maintenance dosing at some, but not all, of the time points on day 11. Coadministration of atorvastatin with either prasugrel or clopidogrel had no effect on VASP phosphorylation relative to the thienopyridine alone after the loading dose.

CONCLUSIONS

Coadministration of atorvastatin 80 mg/day with prasugrel or clopidogrel did not negatively affect the antiplatelet response to either drug after a loading dose or during maintenance dosing. The lack of a clinically meaningful effect of high-dose atorvastatin on the pharmacodynamic response to prasugrel after the loading or maintenance dose indicates that no dosage adjustment should be necessary in patients receiving these drugs concomitantly.

We have evaluated the contributions of nitric oxide (NO) and prostacyclin (PGI2) in the in vivo antiplatelet effects of clinically useful nitrovasodilators. In rats, intravenous infusion of three NO donors, glyceryl trinitrate, sodium nitroprusside, or 3'-morpholinosydnonimine, the stable metabolite of molsidomine, released 6-keto PGF1alpha (the stable metabolite of PGI2) and inhibited ex vivo human platelet aggregation to adenosine diphosphate by at least 80%. In in vitro studies, glyceryl trinitrate, sodium nitroprusside, and 3'-morpholinosydnonimine, at clinically attainable concentrations, increased cyclooxygenase activity in endothelial cells (EC), which resulted in a four- to sixfold release of 6-keto PGF1alpha. Pretreatment of the EC with hemoglobin which binds to and inactivates the biological actions of NO, but not by methylene blue (MelB), attenuated the NO-mediated PGI2 from the EC by at least 70%. Release of 6-keto PGF1alpha by the NO donors increased the ability of these compounds to inhibit thrombin-induced human platelet aggregation by at least 10 times; this potentiation was inhibited by hemoglobin but not by MeB. MeB blocked the direct anti-platelet effect of the NO donors in the absence of EC. In summary, we have demonstrated that NO, directly as well as together with an NO-driven cyclooxygenase activation (and hence PGI2), release contributes to the marked anti-platelet effects observed after the in vivo administration of clinically used nitrovasodilators.

A large variability in the antiplatelet response to clopidogrel has been consistently reported. Recently, a P2Y12 haplotype was shown to be associated with enhanced adenosine diphosphate (ADP)-induced platelet aggregation in healthy volunteers. The aim of this study was to test in patients (n = 416) scheduled for coronary artery stenting whether P2Y12 haplotype H2 carriage is associated with increased ADP-induced platelet aggregation after administration of a 600 mg loading dose of clopidogrel. Blood was drawn from the arterial sheath at least 2 h after administration of 100 mg aspirin and 600 mg clopidogrel. ADP-induced platelet aggregation was assessed in platelet-rich plasma with light-transmission aggregometry. P2Y12 haplotypes (H1/H2) and P2Y12 C32T genotypes were determined with TaqMan assays. Haplotype combinations and genotypes were not associated with parameters of ADP-induced platelet aggregation after administration of a 600 mg loading dose of clopidogrel. Maximal ADP (5 mumol/l)-induced platelet aggregation was similar in patients carrying haplotype H2 and homozygous carriers of haplotype H1 (43.9 +/- 21.4 versus 43.2 +/- 21.1%, respectively; P = 0.77). Carriage of P2Y12 H2 haplotype does not seem to affect the platelet response to a 600 mg loading dose of clopidogrel in coronary artery disease patients prior to stenting.

The role of the G(i)-coupled platelet P2Y(12) receptor in platelet function has been well established. However, the functional effector or effectors contributing directly to alphaIIbbeta3 activation in human platelets has not been delineated. As the P2Y(12) receptor has been shown to activate G protein-gated, inwardly rectifying potassium (GIRK) channels, we investigated whether GIRK channels mediate any of the functional responses of the platelet P2Y(12) receptor. Western blot analysis revealed that platelets express GIRK1, GIRK2, and GIRK4. In aspirin-treated and washed human platelets, 2 structurally distinct GIRK inhibitors, SCH23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) and U50488H (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate), inhibited adenosine diphosphate (ADP)-, 2-methylthioADP (2-MeSADP)-, U46619-, and low-dose thrombin-mediated platelet aggregation. However, the GIRK channel inhibitors did not affect platelet aggregation induced by high concentrations of thrombin, AYPGKF, or convulxin. Furthermore, the GIRK channel inhibitors reversed SFLLRN-induced platelet aggregation, inhibited the P2Y(12)-mediated potentiation of dense granule secretion and Akt phosphorylation, and did not affect the agonist-induced G(q)-mediated platelet shape change and intracellular calcium mobilization. Unlike AR-C 69931MX, a P2Y(12) receptor-selective antagonist, the GIRK channel blockers did not affect the ADP-induced adenlylyl cyclase inhibition, indicating that they do not directly antagonize the P2Y(12) receptor. We conclude that GIRK channels are important functional effectors of the P2Y(12) receptor in human platelets.

The nature of the inhbitory non-adrenergic non-cholinergic (NANC) neurotransmitter responsible for neurogenic relaxation of rat duodenum was studied with in vitro techniques. Adenosine 5'-triphosphate (ATP)(1 mM), gamma-aminobutyric acid (GABA, 1 mM), dimethylphenylpiperazinium (DMPP, 0.1 mM) and field stimulation (60 V, 2 ms, 0.1 Hz) produced transient relaxation followed by rebound contraction. In contrast vasoactive intestinal polypeptide (VIP) (0.3 microM) and noradrenaline (1 microM) induced relaxation which set in more slowly and lasted longer. Tetrodotoxin (0.85 microM) abolished field stimulation-induced relaxation but not ATP-, VIP- or noradrenaline-induced relaxation. Nucleotide pyrophosphatase (0.25 U/ml), but not the proteolytic enzyme alpha-chymotrypsin (2 U/ml), selectively antagonized NANC relaxation. The rank order of potency of various adenine derivatives for inducing relaxation was adenosine-5'-triphosphate greater than adenosine-5'-diphosphate much greater than adenosine greater than adenosine-5'-monophosphate. ATP-induced relaxation was selectively antagonized by the putative P2 purinoceptor antagonist reactive blue 2, but unaffected by the selective P1 purinoceptor antagonist 8-phenyltheophylline. The duration of ATP- as well as beta-gamma-methylene adenosine-5'-triphosphate (a stable analogue of ATP)-induced relaxation was similar and was unaffected by indomethacin 10 microM (which abolished the rebound contraction). In those preparations whose contractile tone was increased by using a high-K+ medium the ability of ATP to elicit relaxation was markedly reduced, while GABA- and DMPP-induced relaxation was abolished. On the other hand, ATP-, GABA- and DMPP-induced relaxation of the tonic component of 5-hydroxytryptamine (5-HT)(0.1 mM)-induced contraction was similar to that observed in control conditions. These findings add further weight to the proposal that endogenous ATP is involved in determining NANC relaxation of rat duodenum.

Strategy, Management and Health PolicyVenture Capital Enabling TechnologyPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IVThe effects of structural modifications of adenine nucleotides previously shown to enhance either agonist (2-thioether groups) or antagonist (additional phosphate moieties at the 3'- or 2'-position) properties at P2Y(1) receptors were examined at recombinant rat P2X(1), P2X(2), P2X(3), and P2X(4) receptors expressed in Xenopus oocytes. The potency of P2Y(1) agonists HT-AMP (2-(hexylthio)adenosine-5'-monophosphate) and PAPET (2-[2-(4-aminophenyl)ethylthio]adenosine-5'-triphosphate) was examined at P2X receptors. Both nucleotides showed a preference for the Group I (α,β-meATP-sensitive, fast-inactivating) P2X sub-units. HT-AMP was 5-fold more potent than ATP at P2X(3) receptors and a partial agonist at all except P2X(2) receptors, at which it was a full agonist. The efficacy of HT-AMP was as low as 23% at P2X(4) receptors. PAPET was a weak partial agonist at rat P2X(4) receptors and a nearly full agonist at the other subtypes. At rat P2X(3) receptors, PAPET was more potent than any other known agonist (EC(50) = 17 ± 3 nM). MRS 2179 (N(6)-methyl-2'-deoxyadenosine 3', 5-bisphosphate, a potent P2Y(1) receptor antagonist) inhibited ATP-evoked responses at rat P2X(1) receptors with an IC(50) value of 1.15 ± 0.21 μM. MRS 2179 was a weak antagonist at rat P2X(3) receptors, with an IC(50) value of 12.9 ± 0.1 μM, and was inactive at rat P2X(2) and P2X(4) receptors. Thus, MRS 2179 was 11-fold and 130-fold selective for P2Y(1) receptors vs. P2X(1) and P2X(3) receptors, respectively. MRS 2209, the corresponding 3'-deoxy-2'-phosphate isomer, was inactive at rat P2X(1) receptors, thus demonstrating its greater selectivity as a P2Y(1) receptor antagonist. Various adenine bisphosphates in the family of MRS 2179 containing modifications of either the adenine (P2Y(1) antagonists with 2- and 6-substitutions), the phosphate (a 3',5'-cyclic diphosphate, inactive at P2Y(1) receptors), or the ribose moieties (antagonist carbocyclic analogue), were inactive at both rat P2X(1) and P2X(3) receptors. An anhydrohexitol derivative (MRS 2269) and an acyclic derivative (MRS 2286), proved to be selective antagonists at P2Y(1) receptors, since they were inactive as agonist or antagonist at P2X(1) and P2X(3) receptors.

The effects of tetrandrine (6,6', 7,12-tetramethoxy-2, 2'-dimethyl-berbaman) on the mitochondrial function were assessed on oxidative stress, mitochondrial permeability transition (MPT), and bioenergetics of rat liver mitochondria. At concentrations lower than 100 nmol/mg protein, tetrandrine decreased the hydrogen peroxide formation, the extent of lipid peroxidation, the susceptibility to Ca(2+)-induced opening of MPT pore, and inhibited the inner membrane anion channel activity, not significantly affecting the mitochondrial bioenergetics. High tetrandrine concentrations (100-300 nmol/mg protein) stimulated succinate-dependent state 4 respiration, while some inhibition was observed for state 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations. The respiratory control ratio and the transmembrane potential were depressed but the adenosine diphosphate to oxygen (ADP/O) ratio was less affected. A slight increase of the inner mitochondrial membrane permeability to H(+) and K(+) by tetrandrine was also observed. It was concluded that low concentrations of tetrandrine afford protection against liver mitochondria injury promoted by oxidative-stress events, such as hydrogen peroxide production, lipid peroxidation, and induction of MPT. Conversely, high tetrandrine concentrations revealed toxicological effects expressed by interference with mitochondrial bioenergetics, as a consequence of some inner membrane permeability to H(+) and K(+) and inhibition of the electron flux in the respiratory chain. The direct immediate protective role of tetrandrine against mitochondrial oxidative stress may be relevant to clarify the mechanisms responsible for its multiple pharmacological actions.

Experiments were performed to determine the effect of ouabain on the release of relaxing factor(s) from cultured endothelial cells, and its action on the effect of the relaxing factor(s) on arterial smooth muscle. A column of porcine aortic endothelial cells grown on microcarrier beads in suspension culture was perfused with modified Krebs-Ringer bicarbonate solution. The release of relaxing factor(s) by the endothelial cells was detected under bioassay conditions by measuring the relaxing activity of the perfusate overflowing a ring of canine coronary artery (without endothelium) contracted with prostaglandin F2 alpha. Incubation of the endothelial cells with ouabain did not affect the relaxation of the bioassay ring under basal conditions or upon stimulation of the endothelial cells with ADP but impaired the relaxation induced by bradykinin or the calcium ionophore A23187. Incubation of the bioassay ring with ouabain reduced the relaxation under basal conditions as well as the relaxation induced by ADP but did not affect the relaxation observed upon stimulation with bradykinin and A23187 and the endothelium-independent relaxations induced by nitric oxide. These experiments suggest that cultured porcine aortic endothelial cells release two endothelium-derived relaxing factors; one is released under basal conditions and upon stimulation with adenosine diphosphate and the other (which presumably is nitric oxide) upon stimulation with bradykinin and the calcium ionophore A23187.

1. Single-channel currents activated by extracellular adenosine 5'-triphosphate (ATP-induced currents) were recorded in cultured muscle cells of Xenopus laevis using the cell-attached patch clamp technique. 2. The amplitude histogram of the ATP-induced currents had two distinct peaks, corresponding to 60 pS (high-conductance (gamma) channels currents) and 41 pS (low-gamma channel currents). The peak values of the currents were unaltered during 1-6 days in culture. 3. The mean open time of the two types of ATP-induced currents was 0.93 ms for high-gamma and 0.86 ms for low-gamma channel currents at 50 mV hyperpolarization. The reversal potential of the ATP-induced current, estimated from the I-V relationship, ranged between -5 and -15 mV. 4. The open-state probability of currents induced by 10 microM-ATP decreased in the presence of 20 microM-d-tubocurarine. 5. The frequency of ATP-induced current events depended upon the ATP concentration. The current events were first detected at 0.1 microM-ATP and occurred with increasing frequency up to 10 microM-ATP. At concentrations higher than 10 microM, the frequency of current events decreased. 6. When acetylcholine (ACh, 0.1 nM) was applied together with various concentrations of ATP, the frequency of current events increased 2- to 3-fold at the ATP concentration range between 0.1 and 10 microM. At higher concentrations of ATP the frequency decreased again. When ACh (0.1 nM) was applied without ATP, current events were rarely observed. 7. Two types of ATP-induced currents were also observed with adenylylimido 5'-diphosphate (AMP-PNP) at one-hundred micromolar concentrations. Neither AMP (adenosine 5'-monophosphate) nor ADP (adenosine 5'-diphosphate) (1-500 microM) induced channel events. 8. It is concluded that the nicotinic ACh receptor channels in cultured Xenopus skeletal muscle cells are opened by micromolar concentrations of exogenous ATP. The possible physiological significance is discussed.

BACKGROUND

Recent studies have shown that adenosine triphosphate (ATP) is liberated from macula densa cells in response to increased tubular NaCl in vitro. We tested the hypothesis that increased NaCl in the macula densa stimulates the release of ATP, resulting in extracellular formation of adenosine which is involved in signal transmission of the tubuloglomerular feedback response.

METHODS

Rabbit afferent arterioles and attached macula densas were simultaneously microperfused in vitro. Tubuloglomerular feedback was induced by increasing macula densa Na/Cl from 11/10 to 81/80 mmol/L and was measured before and after treatment.

RESULTS

We first tested whether hydrolysis of ATP is required for tubuloglomerular feedback. When we enhanced conversion of ATP to adenosine by adding hexokinase or apyrase to the bath and arteriole lumen, the tubuloglomerular feedback response was augmented. During the control period, tubuloglomerular feedback decreased arteriole diameter by 2.2 +/- 0.2 microm. In the presence of hexokinase, tubuloglomerular feedback decreased diameter by 3.4 +/- 0.3 microm (N= 8) (P < 0.05, with vs. without hexokinase). In the apyrase group, tubuloglomerular feedback decreased diameter by 2.7 +/- 0.4 microm during the control period. When apyrase was added, tubuloglomerular feedback decreased diameter by 4.7 +/- 0.4 microm (N= 8) (P < 0.05, with vs. without apyrase). When hydrolysis of adenosine monophosphate (AMP) to adenosine was blocked by supplementing the bath with 100 micromol/L alpha,beta-methylene adenosine 5'-diphosphate (MADP), an inhibitor of 5'-nucleotidase, tubuloglomerular feedback response was blocked and diameter remained unchanged. We next studied whether ATP released from the macula densa binds to P(2) receptors and activates the tubuloglomerular feedback response. The P(2) purinergic receptor inhibitor suramin was added to both arteriole lumen and bath. During the control period, tubuloglomerular feedback decreased diameter by 3.7 +/- 0.5 microm. Suramin (100 micromol/L) did not significantly inhibit tubuloglomerular feedback, since in the presence of suramin diameter decreased by 3.8 +/- 0.3 microm (N= 7). Finally, we added the adenosine A(1) receptor inhibitor FK838 to both bath and lumen and found that it completely blocked high NaCl-induced tubuloglomerular feedback.

CONCLUSIONS

We concluded that ATP released from the macula densa is broken down to form AMP in the extracellular space. AMP in turn is degraded by ecto-5'-nucleotidases to adenosine, which mediates signal transmission of the tubuloglomerular feedback response.

Platelet-activating factor (PAF), a naturally occurring phospholipid, is a potent activator of various biological processes, including platelet aggregation. The mechanisms by which PAF acts are largely unknown, partly because of the lack of specific inhibitors for PAF-elicited responses. It was found that in washed human platelets the psychotropic triazolobenzodiazepine drugs alprazolam and triazolam potently inhibited PAF-induced changes in shape, aggregation, and secretion. The effects were specific for PAF activation, since the responses of human platelets to adenosine diphosphate, thrombin, epinephrine, collagen, arachidonate, and the calcium ionophore A23187 were not inhibited by the triazolobenzodiazepines. These psychotropic drugs should be useful in investigating the possibility that PAF or PAF-like phospholipids play a role in neuronal function and in elucidating biochemical mechanisms activated specifically by PAF in a variety of cells.

1. A detailed laboratory method is described for the labelling of human platelets with [111In]indium oxine. The 45 min method is simple, requires on 26 ml of blood and is suitable for routine clinical use. 2. After the labelling and resuspension of the platelets in plasma, aggregation responses to both adenosine diphosphate and collagen were similar to those of normal platelet-rich plasma. Less than 5% of the [111In]indium oxine was released by secretory function of platelets. 3. Labelling efficiencies of 90.1 +/- 4.29% (n = 28) were achieved in 60 s by normal concentrations of plasma-free platelet suspensions. 4. Platelet survival in vivo in healthy volunteer subjects follows a linear function with a survival time of 8.44 +/- 0.18 days.

The goal of this study was to determine the role of the synthesis and release of nitric oxide in modulating alterations in microvascular permeability of the hamster cheek pouch in response to adenosine 5'-diphosphate and bradykinin. We used intra-vital fluorescent microscopy to examine the permeability of the hamster cheek pouch to agonists before and following application of enzymatic inhibitors of nitric oxide, NG-monomethyl-L-arginine (L-NMMA; 0.01, 0.1, and 1.0 microM) and NW-nitro-L-arginine methyl ester (L-NAME; 0.01, 0.1, and 1.0 microM). Increases in permeability of the hamster cheek pouch were quantitated by the formation of microvascular leaky sites. ADP and bradykinin produced an increase in the number of venular leaky sites, and superfusion of L-NMMA and L-NAME significantly decreased ADP- and bradykinin-induced increases in microvascular permeability. To determine the specificity of nitric oxide blockade on microvascular permeability, we examined changes in permeability in response to adenosine, and examined the effects of D-NMMA on microvascular permeability. Adenosine-induced increases in permeability were not altered by treatment with L-NMMA, and D-NMMA did not inhibit ADP-induced increases in microvascular permeability. Thus, these findings suggest that production of nitric oxide, in response to application of ADP and bradykinin, has a role in modulating macromolecular permeability of the hamster cheek pouch in vivo.