Inhibitory Effect Of Dipyridamole Research Articles

Dipyridamole inhibits TGF-β–induced collagen gene expression in human peritoneal mesothelial cells

Peritoneal matrix accumulation is characteristic of peritoneal fibrosis (PF). Continuous ambulatory peritoneal dialysis (CAPD) patients who had persistent transforming growth factor-beta (TGF-beta) in their drained effluent had an increased risk of PF. We previously reported that TGF-beta stimulates the expression of types I and III collagen mRNA in cultured human peritoneal mesangial cells (HPMCs), which may predispose them to develop PF. Pharmacological interventions to attenuate TGF-beta-stimulated matrix accumulation in HPMC may have therapeutic potential for the treatment of PF. The SMAD family and the extracellular signal-regulated protein kinase (ERK1/2, p44/p42) pathways have been shown to participate in TGF-beta signaling. Our current study identified these signal pathways in HPMCs and investigated the molecular mechanisms involved in the inhibitory effects of dipyridamole on TGF-beta-induced collagen gene expression in HPMCs. HPMCs were cultured from human omentum by an enzyme digestion Expression of collagen alpha1(I) mRNA was determined by Northern blotting. The SMAD proteins and the ERK1/2 activity were determined by Western blotting. TGF-beta-stimulated collagen alpha1(I) mRNA expression of HPMC was inhibited by dipyridamole in a dose-dependent manner. Smad2 and ERK1/2 were activated in response to TGF-beta; however, TGF-beta had little effect on the protein expression of Smad4. The addition of PD98059, which blocked activation of ERK1/2, suppressed TGF-beta-induced collagen alpha1(I) mRNA expression in a dose-dependent manner. At a concentration that inhibited collagen gene expression (17 microg/mL), dipyridamole suppressed ERK1/2 activation by TGF-beta. In contrast, the same concentration of dipyridamole had no effect on TGF-beta-induced activation of Smad2. Dipyridamole inhibits TGF-beta-induced collagen gene expression in HPMC through modulation of the ERK pathway. Our study of dipyridamole may provide therapeutic basis for clinical applications in the prevention of PF.

Estimation of anti-platelet drugs on human platelet aggregation with a novel whole blood aggregometer by a screen filtration pressure method.

1. The effects of anti-platelet drugs on human whole blood aggregation were evaluated using a novel whole blood aggregometer by a screen filtration pressure (SFP) method. 2. The SFP whole blood aggregometer was found to successfully detect whole blood aggregation induced by ADP, collagen and TRAP by measuring the SFP of blood samples. The platelet aggregation threshold index (PATI), the concentration of agonist required with an inducing pressure rate of 50%, varied time-dependently after collection of blood. High values for ADP and collagen were noted immediately after blood collection, suggesting low aggregation activity of platelets, and gradually increase thereafter. 3. Cilostazol (phosphodiesterase 3 inhibitor), dipyridamole, aspirin and tirofiban all inhibited whole blood aggregation in vitro. Inhibitory effects of cilostazol and dipyridamole, but not tirofiban, were markedly enhanced 6 or 7 fold by long pre-incubation (60 min), compared with short pre-incubation (2 min). Such enhancement was only observed with ADP- and not collagen-induced whole blood aggregation. A similar phenomenon was also observed for aggregation with platelet rich plasma (PRP). Cilostazol inhibition of ADP-induced platelet aggregation was more potent with PRP than whole blood (PATI(200)=3.80+/-0.95 microM for whole blood; 2.04+/-0.61 microM for PRP). Inhibitory effects of dipyridamole were attenuated in PRP without erythrocytes. 4. These results demonstrate that the SFP aggregometer can sensitively detect anti-platelet aggregatory effects of various kinds of drugs. So that it is a useful tool for evaluation of anti-platelet drugs.

Dipyridamole and dilazep suppress oxygen radicals in puromycin aminonucleoside nephrosis rats.

Reactive oxygen species (ROS) are involved in the pathophysiology of puromycin aminonucleoside (PAN) nephrosis. To elucidate further the role of radicals in PAN nephrosis and the to determine the particular radical species scavenged by dipyridamole (DPM) and dilazep (DZ), we applied chemiluminescence and electron spin resonance (ESR) techniques. Chemiluminescence of glomeruli, which were isolated on day 7 from rats injected with 100 mg kg-1 PAN, was measured with or without scavengers. The inhibitory effects of DPM and DZ on hydroxyl radical adduct formation in the Fenton's reaction were evaluated using ESR. Chemiluminescence was greater in glomeruli from rats with PAN nephrosis than in the the glomeruli of control rats. This increase was suppressed by superoxide dismutase, catalase, dimethylthiourea and also by DPM and DZ. ESR indicated that DPM and DZ inhibited hydroxyl radical adduct formation with a second-order rate constant of 2.9 x 10(10) and 1.6 x 10(10) (mol L(-1) s(-1) respectively, similar to that of dimethylthiourea. DPM and DZ scavenge hydroxyl radicals, thereby alleviating PAN nephrosis.

Inhibitory Effect of Dipyridamole and its Derivatives on Lipid Peroxidation in Mitochondria

Dipyridamole (DIP), 2,6-bis(diethanolamino)-4,8-dipiperidino-[5,4-d]pyrimidine, is a coronary vasodilator widely used in clinics. It has also been reported to have coactivator activity for a number of antitumour drugs and antioxidant activity in membrane systems. In recent years we have been studying the spectroscopic properties of this drug and several of its derivatives as well as their interaction with charged micelles and phospholipid monolayers. A strong interaction of DIP and DIP derivatives with these model membrane systems and a dependence of the strength of the interaction upon the chemical structure of the DIP derivative was observed. Here, the antioxidant effect of DIP and the derivatives, RA14, RA47, and RA25, was compared. We observed that although it strongly inhibits the iron-induced lipoperoxidation on mitochondria (IC 50 = 1 μM), it shows no protection against an organic oxidant, cumene hydroperoxide. The order of hydrophobicity of the DIP derivatives, DIP > RA14 > RA47 > RA25, correlates very well with both the values of the association constants of these derivatives to micelles, their localization in the micelles, and phospholipid films and their antioxidant effect on mitochondria. So, a very good correlation of the structure of the drug in regarded to the nature of its substituents with the biological activity is observed. Essentially the same result was observed either measuring the lipid peroxidation or the membrane fluidity by ESR, suggesting that the effect of DIP and DIP derivatives is probably associated to their binding to the lipid bilayer and not to interaction with membrane proteins.

Smooth Muscle Cell–Derived Adenosine Inhibits Cell Growth

Several endogenous factors generated within the vessel wall have been implicated in contributing to the vascular remodeling process associated with hypertension and atherosclerosis. Furthermore, substances generated by smooth muscle cells (SMCs) are known to regulate SMC proliferation in an autocrine fashion. Adenosine is a vasodilator synthesized by SMCs, and exogenous adenosine inhibits SMC proliferation. However, whether adenosine produced endogenously has antimitogenic effects is not known. Hence, we evaluated the effects of SMC-derived adenosine on 2.5% fetal calf serum-induced proliferation of rat aortic SMCs. SMC proliferation was assayed by measurement of DNA synthesis ([3H]thymidine incorporation) and cell counting. To determine the effects of endogenous adenosine on SMC proliferation, we stimulated growth-arrested SMCs with 2.5% fetal calf serum in the presence and absence of modulators of adenosine levels, including (1) erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA; inhibits adenosine deaminase), (2) dipyridamole (blocks adenosine transport and inhibits phosphodiesterase), (3) dipyridamole plus EHNA, and (4) adenosine with or without EHNA. [3H]Thymidine incorporation and cell number were measured after 24 and 96 hours, respectively. EHNA and dipyridamole inhibited both FCS-induced DNA synthesis and cell proliferation in a concentration-dependent manner. Furthermore, extracellular (in medium) adenosine levels were significantly increased when cultured cells were treated with EHNA, and the inhibitory effects of dipyridamole as well as exogenous adenosine were enhanced in the presence of EHNA. Additionally, the inhibitory effects of dipyridamole and EHNA on DNA synthesis were significantly reduced in the presence of KF17837, an A2 adenosine receptor antagonist. These results indicate that SMC-derived adenosine can inhibit SMC proliferation. Hence, it is possible that a defect in localized adenosine synthesis within the vessel wall could contribute to vascular thickening and neointima formation.

DIPYRIDAMOLE AS A MODULATOR OF MULTIDRUG-RESISTANCE IN TUMOR-CELLS IN-VITRO

The aim of the present study was to investigate in vitro the effect on LoVo cells (LoVo-S, doxorubicin-sensitive, and LoVo-R, doxorubicin-resistant) on the association of dipyridamole and certain antiblastic drugs (doxorubicin, idarubicin, mitoxantrone, teniposide) with different mechanisms of action. The cells were treated for 48 hours with the drugs, or were left for 24 hours without treatment (recovery period). The results demonstrate that dipyridamole possesses an inhibitory effect on cell growth, and that it potentiates the cytotoxic effect of the tested antiblastic drugs, particularly on LoVo-R cells. In the recovery period the inhibitory effect of dipyridamole on LoVo-S and LoVo-R cells was reversible, while the effect of the antitumour drugs, either alone or in association with dipyridamole, persisted. We suggest that dipyridamole has a synergistic effect on LoVo cell growth when associated with antiblastics; the increased effect produced on LoVo-R cells is indicative for a reversion of multidrug resistance, probably via a mechanism related to transmembrane transport system.

Novel nucleoside transport inhibitors of natural origin.

The salvage pathway of nucleotide biosynthesis is one of the attractive targets for cancer chemotherapy. Dipyridamole, a nucleoside transport inhibitor, may be used for blocking the salvage pathways. Studies have demonstrated that dipyridamole can reduce the rescue effect of exogenous nucleosides and potentiated the cytotoxicity of acivicin, an antimetabolite of de novo pathways of nucleotide biosynthesis, in hepatoma cells1, 2. The inhibitory effect of dipyridamole was cell growth phase -dependent. Cultured hepatoma cells in lag and log phase were highly sensitive to dipyridamole; by contrast, cells in stationary phase were insensitive3, 4. The combination of acivicn and dipyridamole administered in rats bearing s.c. transplanted hepatoma yielded a summation effect, decreasing NTP and dNTP pools5. The synergistic effect of dipyridamole and acivicin was also demonstrated in colon cancer cells6. There have been reports that dipyridamole potentiated the cytotoxicity of several antimetabolites including methotrexate, 5-fluorouracil and N-phosphonacetyl-L-aspartate (PALA)7, 8, 9. In addition to potentiating antimetabolites, dipyridamole was found to enhance the cytotoxicity of doxorubicin, etoposide, vincristine and mitoxantrone10, 11, 12. These synergistic effects of dipyridamole and drugs which are not antimetabolites were due to, at least in part, increased intracellular concentrations of anticancer drugs. Drug sensitivity of multidrug resistant cells may be modulated by dipyridamole13. The use of dipyridamole in cancer chemotherapy has drawn much attention and clinical trials have been underway14, 15. The study of dipyridamole indicates that blocking nucleoside salvage pathways by inhibition of nucleoside transport may be one of the effective ways in chemotherapy. By examination of a series of compounds of natural origin, we have found that green tea polyphenols (GP), antibiotics C3368-A (CA) and C3368-B (CB) are highly active in blocking nucleoside transport in cancer cells. Investigations have demonstrated that GP, CA or CB show synergistic effects with anticancer drugs.

Dipyridamole inhibits red cell-induced platelet activation

We have recently shown that red blood cells can induce spontaneous platelet aggregation (SPA) in whole blood ex vivo, which could be inhibited by dipyridamole. Since this drug, at therapeutic doses is not an effective inhibitor of platelet aggregation in platelet rich plasma, the inhibition of platelet interaction with the red cell was thought to be the mechanism of its action. Values for the percentage fall in the single platelet count due to SPA in whole blood after 3 and 6 min rollermixing were: control 15 ± 2.2 and 42 ± 2.9; 6 μM dipyridamole 6 ± 1.1 ( P < 0.001) and 31 ± 2.6 ( P < 0.01); 12 μM dipyridamole 2 ± 0.9 ( P < 0.0005) and 22 ± 2.3 ( P < 0.0005) (mean ± SEM, n = 10). Electron microscopic observation revealed that the aggregation involves an initial platelet adhesion to the red blood cell; the adherent platelets then become activated and serve as foci for the growing aggregates. Dipyridamole appeared to inhibit the initial platelet adhesion to the red cell (the principal trigger mechanism for SPA) which may mimic the initiation of thrombosis in some situations in vivo. The inhibitory effect of dipyridamole on the platelet-red cell interaction suggests that this drug has antithrombotic potential in situations where red blood cells have a trigger role in platelet activation and may explain why the drug has been more effective in some situations than in others.

Inhibition of insulin-stimulated glucose transport in rat adipocytes by nucleoside transport inhibitors

In isolated rat adipocytes, basal as well as insulin-stimulated 3- O-methylglucose transport was inhibited nearly completely (maximal inhibition: 95%) by the nucleoside transport inhibitors dipyridamole (IC 50 = 5 μM), nitrobenzylthioguanosine (20 μM), nitrobenzylthioinosine (35 μM) and papaverine (130 μM). Transport kinetics in the presence of 10 μM dipyridamole revealed a significant increase in the transport K m value of 3- O-methylglucose (3.45±0.6 vs 2.36±0.29 mM in the controls) as well as a decrease in the V max value (4.84±0.95 vs 9.03±1.19 pmol/s per μl lipid in the controls). Half-maximally inhibiting concentrations of dipyridamole were one order of magnitude higher than those inhibiting nucleoside (thymidine) uptake (0.48 μM). The inhibitory effect of dipyridamole (5 μM) reached its maximum within 30 s. The agent failed to affect insulin's half-maximally stimulating concentration (0.075 nM) indicating that it did not interfere with the mechanism by which insulin stimulates glucose transport. Further, dipyridamole fully suppressed the glucose-inhibitable cytochalasin B binding (IC 50 = 1.65±0.05 μM). The data indicate that nucleoside transport inhibitors reduce glucose transport by a direct interaction with the transporter or a closely related protein. It is suggested that glucose and nucleoside transporters share structural, and possibly functional, features.

Role of plasma adenosine in the antiplatelet action of hl 725, a potent inhibitor of camp phosphodiesterase: Species differences.

The potent inhibitor of platelet cAMP phosphodiesterase (PDE) HL 725 (9,10-Dimethoxy-2-mesitylimino-3-methyl-3, 4,6,7-tetrahydro-2H-pyrimido(6,1-A)-isoquinoline-4-one-hydrochloride), was examined for its effects on human and rat platelet aggregation. Strong inhibitory effects are seen on collagen-induced platelet aggregation both in rat platelet-rich plasma (PRP) (IC 50, 54 ± 12 nM) and whole blood (ICsO, 57 ± 25 nM). Compared to the effects on rat platelets, HL 725 is about two-fold less inhibitory in human PRP ( IC50, 94±29 nM) and whole blood (IC50, 126 ± 50 nM). The inhibitory action of HL 725 can be reversed by washing and resuspension of the platelets, suggesting that HL 725 does not bind tightly to cAMP PDE. If human or rat PRP is pretreated with adenosine deaminase, an enzyme that degrades adenosine or 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, the inhibitory effect of HL 725 is reversed. Similar blockade of the inhibitory actions of several other inhibitors of cAMP PDE such as RA 233, RX-RA 69 (analogs of dipyridamole) and oxagrelate is seen by adenosine deaminase pretreatment. The nucleoside transport inhibitors, dilazep and dipyridamole which are non-inhibitory alone to platelet aggregation, strongly potentiate (about 10-fold) the inhibitory action of HL 725 on collagen-induced platelet aggregation in human whole blood. However, if the whole blood is pretreated with adenosine deaminase, no inhibitory effect of dipyridamole plus HL 725 is seen on platelet aggregation. These studies demonstrate that plasma adenosine plays a crucial role in the antiaggregatory actions of HL 725 and several other inhibitors of cAMP PDE both in human and rat blood.

Effect of Dipyridamole on Spontaneous Platelet Aggregation in Whole Blood Decreases with the Time After Venepuncture: Evidence for the Role of ADP

SummarySpontaneous platelet aggregation (SPA) was studied in human whole blood at 3, 5, 10, 20, 30, 40 and 60 minutes after venepuncture. Using a whole blood platelet counter, SPA was quantified by measuring the fall in single platelet count upon rollermixing aliquots of citrated blood at 37° C. The extent of SPA increased with the time after venepuncture, with a correlation coefficient of 0.819. The inhibitory effect of dipyridamole (Dipy) on SPA was studied: (a) 10 μM at each time interval; (b) 0.5-100 μM at 3 and 30 minutes and (c) 15 μM in combination with 100 μM adenosine, 8 μM 2-chloroadenosine (2ClAd, an ADP receptor blocker) and 50 μM aspirin. There was a rapid decrease in the inhibitory effect of Dipy with the time after venepuncture; the correlation coefficient was -0.533. At all the concentrations studied, Dipy was more effective at 3 minutes than at 30 minutes after venepuncture. A combination of Dipy with adenosine, 2ClAd or aspirin was a more effective inhibitor of SPA than either drug alone. However, when 15 μM Dipy and 10 μM Ad were added together, the inhibitory effect of Dipy was not increased significantly, suggesting that Dipy inhibits platelet aggregation independent of Ad. The increase in SPA with the time after venepuncture was abolished when blood was taken directly into the anticoagulant containing 5 μM 2ClAd. It is suggested that ADP released from the red blood cells is responsible for the increased platelet aggregability with the time after venepuncture and makes a serious contribution to the artifacts of in vitro platelet function studies.

Adenosine and dipyridamole: actions and interactions on the contractile response of guinea-pig ileum to high frequency electrical field stimulation.

The action of adenosine on the myenteric plexus-longitudinal muscle strips from guinea-pig ileum to high frequency electrical field stimulation (10 Hz) was investigated. Electrically induced contractions were reduced markedly by tetrodotoxin (0.2 microM) and atropine (1 microM), and partially by noradrenaline (3 microM) and morphine (3 microM). Adenosine, adenosine 5'-monophosphate (AMP) and adenosine triphosphate (ATP) produced a concentration-dependent inhibition of the high frequency contractions over the range of 0.1-100 microM, the most potent being adenosine. The concentration-response curve for adenosine was significantly shifted to the left by dipyridamole (10 nM), while dipyridamole at higher concentrations (30 nM-10 microM), depressed the contraction markedly by itself. Dipyridamole decreased [3H]-adenosine uptake into strips of ileum in a concentration-dependent manner. There was a significant correlationship between the reduction of adenosine uptake and the inhibition of the contraction induced by dipyridamole (r = 0.970). In strips desensitized to adenosine or treated with adenosine deaminase, the inhibitory effect of dipyridamole was significantly reduced. The present investigation revealed that adenosine depressed responses of guinea-pig ileum to high frequency electrical stimulation and suggested that the inhibitory effect of dipyridamole may be closely associated with the behaviour of endogenous adenosine or related compounds.

Inhibitory mechanism of dipyridamole on platelet aggregation [formula omitted

The effects of dipyridamole on platelet aggregation ex vivo and in vitro and on platelet cyclic AMP phosphodiesterase (PDE) were studied, and the mechanism of the ex vivo effects was assessed. Both the ADP- and collagen-induced aggregations ex vivo were inhibited dose-responsively by oral administration of dipyridamole. Maximum dipyridamole levels in the plasma were reached at 30 min after the administration. The inhibitory effects of dipyridamole on platelet aggregation ex vivo reached a maximum at between 1 and 2 hrs. On the other hand, the ADP-induced aggregation in vitro and cyclic AMP PDE activity were not inhibited until after 10 min of incubation at a low concentration of dipyridamole. This mode of inhibition of platelet aggregation in vitro and of cyclic AMP PDE activity agreed with the mode of inhibition in the case of platelet aggregation ex vivo . It is suggested therefore that the ex vivo effects, observed with only a low dipyridamole concentration in the plasma, may be due primarily to inhibition by dipyridamole of the cyclic AMP PDE in platelets.

Prevention of platelet aggregation in patients with prostatic cancer during estrogen therapy

In prostatic cancer patients treated with synthetic estrogen, hexestrol, increased levels of platelet aggregation were observed in accordance with a significant decrease of platelet cyclic AMP. Dipyridamole, which is known to inhibit phosphodiesterase activity, was used to prevent this platelet aggregating effect of hexestrol. Inhibitory effect of dipyridamole on platelet aggregation was demonstrated in patients treated with hexestrol during one year of observation.

The effect of Dipyridamole on platelet adhesiveness and aggregation

The effect of dipyridamole on human platelet adhesivenss was studied by the author's modification of Hellem's II method and its effect on platelet aggregation was observed by the turbidmetric method of Born using aggregometer (Bryston).The adhesivenss, in vitro, was inhibited by dipyridamole at 100-500μg/ml blood, and the inhibited degree of the the adhesiveness was slighter than that of ADP-induced aggregation. In vivo, by intravenous injection of dipyridamole, the adhesiveness was inhibited obviously during 5-15 minutes, and recovered after 30 minutes. It was also lowered during the course of oral abministration of dipyridamole (75-250mg/day), but was rised by the nterruption of its administration.ADP-aggregation (0.4μg) was also inhibited by dipyridamole at 50-500μg/ml PRP. Only secondary aggregation was inhibited by its low concentrations, whereas primary aggregation was also inhibited by its high concentrations. Lag phases of adrenalin (1μg)-and collagen (50%)-aggregation were prolonged by this inhibitor at 50-200μg/ml, and such aggregation was completely inhibited at the concentration, over 200μg/ml.The inhibitory effect of dipyridamole upon ADP-induced aggregation was not influenced by the period of its pre-incubation with PRP and by HCl used in these experiments. Platelet aggregation induced by ADP (0.4-40μg/ml of PRP) which had been incubated with 100μg of dipyridamole was similar to that induced by the same concentrations of ADP without the preincubation.Fifty μg of dipyridamole/ml PRP slightly inhibited ADP release induced by Kaolin, and its concentration over 200μg/ml inhibited the release so remarkably that ADP thus released was not enough to aggregate platelets.Bovine fibrinogen-aggregation was not influenved by dipyridamole at the concentration up to 500μg/ml.

Beeinflussung der Monosaccharid-Permeabilit�t des Menschen-Erythrocyten durch eine Pyrimido-Pyrimidin-Verbindung

Results are reported on the influence of dipyridamole on monosaccharide penetration into human red blood cells. As could be shown by means of osmotic methods, dipyridamole (10−4 mol/l) inhibits the uptake of d-glucose, d-fructose, d-ribose, d-mannose, d-galactose, d-xylose, and l-arabinose into human erythrocytes to a different degree. The results are discussed with respect to the inhibitory effects of dipyridamole on the transfer of adenosine, orthophosphate, and monosaccharides across the human red cell membrane.