Adenosine reuptake inhibition reduces diabetes-induced glomerular hyperfiltration via the adenosine A2a receptor.

Diabetes-induced glomerular hyperfiltration is an early alteration in kidney function in diabetes. Previous studies have shown that reduced adenosine A2a receptor signaling contributes to diabetes-induced glomerular hyperfiltration. The present study investigated the effects of enhanced interstitial adenosine concentration by inhibition of cellular adenosine reuptake, thereby promoting endogenous adenosine signaling. Insulinopenic diabetes was induced by streptozotocin in adult male Sprague-Dawley rats. Two weeks after diabetes induction, kidney function in terms of glomerular filtration rate, and total, cortical, and medullary renal blood flows were evaluated under thiobutabarbital anesthesia during baseline and after renal artery infusion of two doses of the adenosine reuptake inhibitor dilazep. Dilazep did not affect mean arterial pressure indicating that the effects of the interventions were intrarenal. Diabetics had increased glomerular filtration rate compared with controls and dilazep dose-dependently decreased glomerular filtration rate in diabetics, whereas it had no significant effect in controls. Dilazep increased cortical renal blood flows in controls, whereas medullary blood flow was not significantly changed. Dilazep did not affect total renal blood flow in any of the groups but decreased cortical blood flow in diabetics, resulting in decreased filtration fraction by dilazep in diabetics. Pretreatment with the adenosine A2a antagonist ZM241385 prevented intrarenal dilazep-mediated effects on glomerular filtration rate and filtration fraction in diabetics. In conclusion, enhancing intrarenal adenosine signaling by dilazep normalizes diabetes-induced glomerular hyperfiltration at least in part by activation of adenosine A2a receptors.

Affinity, binding kinetics and functional characterization of draflazine analogues for human equilibrative nucleoside transporter 1 (SLC29A1).

In the last decade it has been recapitulated that receptor-ligand binding kinetics is a relevant additional parameter in drug discovery to improve in vivo drug efficacy and safety. The equilibrative nucleoside transporter-1 (ENT1, SLC29A1) is an important drug target, as transporter inhibition is a potential treatment of ischemic heart disease, stroke, and cancer. Currently, two non-selective ENT1 inhibitors (dilazep and dipyridamole) are on the market as vasodilators. However, their binding kinetics are unknown; moreover, novel, more effective and selective inhibitors are still needed. Hence, this study focused on the incorporation of binding kinetics for finding new and improved ENT1 inhibitors. We developed a radioligand competition association assay to determine the binding kinetics of ENT1 inhibitors with four chemical scaffolds (including dilazep and dipyridamole). The kinetic parameters were compared to the affinities obtained from a radioligand displacement assay. Three of the scaffolds presented high affinities with relatively fast dissociation kinetics, yielding short to moderate residence times (RTs) at the protein (1-44 min). While compounds from the fourth scaffold, i.e. draflazine analogues, also had high affinity, they displayed significantly longer RTs, with one analogue (4) having a RT of over 10 h. Finally, a label-free assay was used to evaluate the impact of divergent ENT1 inhibitor binding kinetics in a functional assay. It was shown that the potency of compound 4 increased with longer incubation times, which was not observed for draflazine, supporting the importance of long RT for increased target-occupancy and effect. In conclusion, our research shows that high affinity ENT1 inhibitors show a large variation in residence times at this transport protein. As a consequence, incorporation of binding kinetic parameters adds to the design criteria and may thus result in a different lead compound selection. Taken together, this kinetic approach could inspire future drug discovery in the field of ENT1 and membrane transport proteins in general.

Mechanisms of gemcitabine oral absorption as determined by in situ intestinal perfusions in mice.

Gemcitabine is a widely used chemotherapeutic drug that is administered via intravenous infusion due to a low oral bioavailability of only 10%. This low oral bioavailability is believed to be the result of gemcitabine's low intestinal permeability and oral absorption, followed by significant presystemic metabolism. In the present study, we sought to define the mechanisms of gemcitabine intestinal permeability, the potential for saturation of intestinal uptake, and the transporter(s) responsible for mediating the oral absorption of drug using in situ single-pass intestinal perfusions in mice. Concentration-dependent studies were performed for gemcitabine over 0.5-2000 µM, along with studies of 5 µM gemcitabine in a sodium-containing buffer ±â€¯thymidine (which can inhibit concentrative (i.e., CNT1 and CNT3) and equilibrative (i.e., ENT1 and ENT2) nucleoside transporters) or dilazep (which can inhibit ENT1 and ENT2), or in a sodium-free buffer (which can inhibit CNT1 and CNT3). Our findings demonstrated that gemcitabine was, in fact, a high-permeability drug in the intestine at low concentrations, that jejunal uptake of gemcitabine was saturable and mediated almost exclusively by nucleoside transporters, and that jejunal flux was mediated by both high-affinity, low-capacity (Km = 27.4 µM, Vmax = 3.6 pmol/cm2/s) and low-affinity, high-capacity (Km = 700 µM, Vmax = 35.9 pmol/cm2/s) transport systems. Thus, CNTs and ENTs at the apical membrane allow for gemcitabine uptake from the lumen to enterocyte, whereas ENTs at the basolateral membrane allow for gemcitabine efflux from the enterocyte to portal venous blood.

Structures of human ENT1 in complex with adenosine reuptake inhibitors.

The human equilibrative nucleoside transporter 1 (hENT1), a member of the SLC29 family, plays crucial roles in adenosine signaling, cellular uptake of nucleoside for DNA and RNA synthesis, and nucleoside-derived anticancer and antiviral drug transport in humans. Because of its central role in adenosine signaling, it is the target of adenosine reuptake inhibitors (AdoRI), several of which are used clinically. Despite its importance in human physiology and pharmacology, the molecular basis of hENT1-mediated adenosine transport and its inhibition by AdoRIs are limited, owing to the absence of structural information on hENT1. Here, we present crystal structures of hENT1 in complex with two chemically distinct AdoRIs: dilazep and S-(4-nitrobenzyl)-6-thioinosine (NBMPR). Combined with mutagenesis study, our structural analyses elucidate two distinct inhibitory mechanisms exhibited on hENT1 and provide insight into adenosine recognition and transport. Our studies provide a platform for improved pharmacological intervention of adenosine and nucleoside analog drug transport by hENT1.

Dilazep analogues for the study of equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2).

As ENT inhibitors including dilazep have shown efficacy improving oHSV1 targeted oncolytic cancer therapy, a series of dilazep analogues was synthesized and biologically evaluated to examine both ENT1 and ENT2 inhibition. The central diamine core, alkyl chains, ester linkage and substituents on the phenyl ring were all varied. Compounds were screened against ENT1 and ENT2 using a radio-ligand cell-based assay. Dilazep and analogues with minor structural changes are potent and selective ENT1 inhibitors. No selective ENT2 inhibitors were found, although some analogues were more potent against ENT2 than the parent dilazep.

Dilazep synergistically reactivates latent HIV-1 in latently infected cells.

The long-lived latently infected cells persist in spite of prolonged highly active anti-retroviral therapy and present a major barrier to a cure of human immunodeficiency virus type 1 (HIV-1) infection. Elimination of this reservoir requires reactivation of the latent virus. None of the current agents can safely and effectively reactivate latent HIV-1 reservoirs. Dilazep, a nucleoside transport inhibitor, is used to treat ischemic dysfunction. However, little is known about the effect of dilazep in inducing HIV expression in latently infected cells. Using the Jurkat T cell model of HIV-1 latency, we found that dilazep effectively reactivates latent HIV-1 gene expression in a dose manner. We observed that dilazep synergistically reactivated latent HIV-1 transcription with valproic acid. We also found that dilazep activates viral latency without inducing cell surface activation markers CD25 and CD69 activation. In summary, dilazep, alone or in combination with VPA, could be useful in future eradication strategies.

Residue Ile89 in human plasma membrane monoamine transporter influences its organic cation transport activity and sensitivity to inhibition by dilazep.

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter belonging to the equilibrative nucleoside transporter (ENT) family. Despite its distinct substrate specificity from the classic nucleoside transporters ENT1 and 2, PMAT appears to share similar protein architecture with ENT1/2 and retains low affinity binding to classic ENT inhibitors such as nitrobenzylmercaptopurine riboside (NBMPR) and the coronary vasodilators dilazep and dipyridamole. Here we investigated the role of residue Ile89, a position known to be important for ENT interaction with dilazep, dipyridamole, and nucleoside substrates, in PMAT transport function and its interaction with classic ENT inhibitors using Madin-Darby canine kidney (MDCK) cells stably expressing human PMAT. Substitution of Ile89 in PMAT with Met, the counterpart residue in ENT1, resulted in normal plasma membrane localization and protein expression. Transport kinetic analysis revealed that I89M mutant had a 2.7-fold reduction in maximal transport velocity (V(max)) with no significant change in apparent binding affinity (K(m)) towards the prototype PMAT substrate 1-methyl-4-phenylpyridinium (MPP+), suggesting that I89 is an important determinant for the catalytic activity of PMAT. Dose-dependent inhibition studies further showed that the I89M mutation significantly increased PMAT's sensitivity to dilazep by 2.5-fold without affecting its sensitivity to dipyridamole and NBMPR. Located at the extracellular end of transmembrane domain 1 of PMAT, I89 may occupy an important position close to the substrate permeation pathway and may be involved in direct interaction with the vasodilator dilazep.

Dilazep decreases lipopolysaccharide-induced nitric oxide and TNF-alpha synthesis in RAW 264 cells.

Dilazep dihydrochloride (dilazep) is used to treat ischemic dysfunction, although the mechanisms underlying the anti-inflammatory effects of the drug have not yet been elucidated. The present study evaluated the anti-inflammatory effect of dilazep. Dilazep suppressed the production of nitric oxide (NO) and the expression of TNF-alpha mRNA by lipopolysaccharide (LPS) in RAW 264 cells. However, 1400W, an inducible NO synthase inhibitor, suppressed the production of NO but did not suppress the expression of TNF-alpha mRNA following treatment with LPS. Caffeine, an adenosine antagonist, restored LPS-stimulated NO synthesis, which is suppressed by dilazep. Therefore, these observations may suggest that the suppression of NO synthesis after dilazep treatment in RAW 264 cells is caused by the inhibition of TNF-alpha expression via adenosine receptors.

Identification of the ENT1 antagonists dipyridamole and dilazep as amplifiers of oncolytic herpes simplex virus-1 replication.

Oncolytic herpes simplex virus-1 (oHSV) vectors selectively replicate in tumor cells, where they kill through oncolysis while sparing normal cells. One of the drawbacks of oHSV vectors is their limited replication and spread to neighboring cancer cells. Here, we report the outcome of a high-throughput chemical library screen to identify small-molecule compounds that augment the replication of oHSV G47Delta. Of the 2,640-screened bioactives, 6 compounds were identified and subsequently validated for enhanced G47Delta replication. Two of these compounds, dipyridamole and dilazep, interfered with nucleotide metabolism by potently and directly inhibiting the equilibrative nucleoside transporter-1 (ENT1). Replicative amplification promoted by dipyridamole and dilazep were dependent on HSV mutations in ICP6, the large subunit of ribonucleotide reductase. Our results indicate that ENT1 antagonists augment oHSV replication in tumor cells by increasing cellular ribonucleoside activity.

Estimation of endogenous adenosine activity at adenosine receptors in guinea-pig ileum using a new pharmacological method.

AIM: Adenosine modulates neurotransmission and in the intestine adenosine is continuously released both from nerves and from smooth muscle. The main effect is modulation of contractile activity by inhibition of neurotransmitter release and by direct smooth muscle relaxation. Estimation of adenosine concentration at the receptors is difficult due to metabolic inactivation. We hypothesized that endogenous adenosine concentrations can be calculated by using adenosine receptor antagonist and agonist and dose ratio (DR) equations. METHODS: Plexus-containing guinea-pig ileum longitudinal smooth muscle preparations were made to contract intermittently by electrical field stimulation in organ baths. Schild plot regressions were constructed with 2-chloroadenosine (agonist) and 8-(p-sulfophenyl)theophylline (8-PST; antagonist). In separate experiments the reversing or enhancing effect of 8-PST and the inhibiting effect of 2-chloroadenosine (CADO) were analysed in the absence or presence of an adenosine uptake inhibitor (dilazep), and nucleoside overflow was measured by HPLC. RESULTS: Using the obtained DR, baseline adenosine concentration was calculated to 28 nm expressed as CADO activity, which increased dose dependently after addition of 10(-6) m dilazep to 150 nm (P < 0.05). HPLC measurements yielded a lower fractional increment (80%) in adenosine during dilazep, than found in the pharmacological determination (440%). CONCLUSION: Endogenous adenosine is an important modulator of intestinal neuro-effector activity, operating in the linear part of the dose-response curve. Other adenosine-like agonists might contribute to neuromodulation and the derived formulas can be used to calculate endogenous agonist activity, which is markedly affected by nucleoside uptake inhibition. The method described should be suitable for other endogenous signalling molecules in many biological systems.

Mutation of Trp29 of human equilibrative nucleoside transporter 1 alters affinity for coronary vasodilator drugs and nucleoside selectivity.

hENT1 (human equilibrative nucleoside transporter 1) is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and NBMPR (nitrobenzylmercaptopurine ribonucleoside). When a library of randomly mutated hENT1 cDNAs was screened using a yeast-based functional complementation assay for resistance to dilazep, a clone containing the W29G mutation was identified. Multiple sequence alignments revealed that this residue was highly conserved. Mutations at Trp29 were generated and tested for adenosine transport activity and inhibitor sensitivity. Trp29 mutations significantly reduced the apparent V(max) and/or increased the apparent K(m) values for adenosine transport. Trp29 mutations increased the IC50 values for hENT1 inhibition by dipyridamole, dilazep, NBMPR, soluflazine and draflazine. NBMPR and soluflazine displayed remarkably similar trends, with large aromatic substitutions at residue 29 resulting in the lowest IC50 values, suggesting that both drugs could interact via ring-stacking interactions with Trp29. The W29T mutant displayed a selective loss of pyrimidine nucleoside transport activity, which contrasts with the previously identified L442I mutant that displayed a selective loss of purine nucleoside transport. W29T, L442I and the double mutant W29T/L442I were characterized kinetically for nucleoside transport activity. A helical wheel projection of TM (transmembrane segment) 1 suggests that Trp29 is positioned close to Met33, implicated previously in nucleoside and inhibitor recognition, and that both residues line the permeant translocation pathway. The data also suggest that Trp29 forms part of, or lies close to, the binding sites for dipyridamole, dilazep, NBMPR, soluflazine and draflazine.

Visualisation of the effects of dilazep on rat afferent and efferent arterioles in vivo.

Although the effects of dilazep hydrochloride (dilazep), a nucleoside transport inhibitor, have been examined, there have been no visualisation studies on the physiological effects of dilazep on the glomerular arterioles. The purpose of this study was to visualise and evaluate the effects of dilazep and consequently the effects of adenosine, which dilazep augments by measuring glomelurar diameters, renal blood flow and resistance in rats in vivo. We time-sequentially examined afferent and efferent arteriolar diameter changes using an intravital videomicroscope and renal blood flow. We administered dilazep at a dose of 300 microg/kg intravenously. To further investigate the effects of dilazep, rats were pre-treated with 8-p-sulfophenyl theophylline (a nonselective adenosine receptor antagonist), 8-cyclopentyl-1,3-dipropylxanthine (an A1 receptor antagonist), or 3,7-dimethyl-1-propargylxanthine (an A2 receptor antagonist). Dilazep constricted the afferent and efferent arterioles at the early phase and dilated them at the later phase, with the same degree of vasoconstrictive and vasodilatory effect on both arterioles. A1 blockade abolished vasoconstriction and augmented vasodilatation at the later phase and A2 blockade abolished vasodilatation and augmented vasoconstriction at the early phase. Non-selective blockade abolished both early vasoconstriction and later vasodilatation. In conclusion, adenosine augmented by dilazep constricted the afferent and efferent arterioles of the cortical nephrons at the early phase and dilated both arterioles at the later phase via A1 and A2 adenosine receptor activation, respectively. That the ratio of afferent to efferent arteriolar diameter was fairly constant suggests that intraglomerular pressure is maintained in the acute phase by adenosine despite the biphasic flow change.

Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter.

Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V(max) of 3.4 +/- 1 pmol.microl(-1).s(-1), with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs (K(m) = 96 +/- 37 microM; V(max) = 1.6 +/- 0.3 pmol.microl(-1).s(-1)) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine (K(i) = 19 +/- 7 microM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na(+) or H(+). PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.

Residues 334 and 338 in transmembrane segment 8 of human equilibrative nucleoside transporter 1 are important determinants of inhibitor sensitivity, protein folding, and catalytic turnover.

Equilibrative nucleoside transporters (ENTs) are important for the metabolic salvage of nucleosides and the cellular uptake of antineoplastic and antiviral nucleoside analogs. Human equilibrative nucleoside transporter 1 (hENT1) is inhibited by nanomolar concentrations of structurally diverse compounds, including dipyridamole, dilazep, nitrobenzylmercaptopurine ribonucleoside (NBMPR), draflazine, and soluflazine. Random mutagenesis and screening by functional complementation for inhibitor-resistant mutants in yeast revealed mutations at Phe-334 and Asn-338. Both residues are predicted to lie in transmembrane segment 8 (TM 8), which contains residues that are highly conserved in the ENT family. F334Y displayed increased V(max) values that were attributed to increased rates of catalytic turnover, and N338Q and N338C displayed altered membrane distributions that appeared to be because of protein folding defects. Mutations of Phe-334 or Asn-338 impaired interactions with dilazep and dipyridamole, whereas mutations of Asn-338 impaired interactions with draflazine and soluflazine. A helical wheel projection of TM 8 predicted that Phe-334 and Asn-338 lie in close proximity to other highly conserved and/or hydrophilic residues, suggesting that they form part of a structurally important region that influences interactions with inhibitors, protein folding, and rates of conformational change during the transport cycle.

Cardiac mechanical dysfunction induced by ischemia-reperfusion in perfused heart isolated from stroke-prone spontaneously hypertensive rats.

We investigated the difference in mechanical function after ischemia and reperfusion between Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) or stroke-prone SHR (SHRSP) using the isolated working heart model, in order to examine postischemic mechanical dysfunction in the severely hypertrophied heart. Systolic blood pressure of SHRSP was higher than that of SHR and WKY, and the left ventricular wall in SHRSP was thicker than in WKY. Mechanical dysfunction of the heart during reperfusion following ischemia (11 min) in SHRSP was severer than that in SHR and WKY, and recovery of the cardiac energy charge potential (ECP) level in SHRSP was lower than that in SHR and WKY. Twenty-five, 12 and 11 min-ischemia in WKY, SHR and SHRSP, respectively, caused a similar level of cardiac mechanical damage. Also, the ECP levels were almost equivalent among them at the end of 20 min reperfusion following each time of ischemia. Under each ischemic condition, a Ca2+-channel blocker, diltiazem, and an adenosine potentiator, dilazep, produced a beneficial effect on the post-ischemic dysfunction in SHR and WKY. However, neither cardioprotective drug led to recovery of the mechanical dysfunction of the heart during reperfusion following ischemia in SHRSP. Thus, the severely hypertrophied heart such as that in SHRSP was more susceptible to cardiac reperfusion dysfunction, than the moderately hypertrophied heart such as that in SHR. These results suggest that the cardioprotective effects of drugs may be deteriorated in severe hypertrophied hearts.

Dilazep and dipyridamole inhibit tissue factor expression on monocytes induced by IgG from patients with antiphospholipid syndrome.

AIM: To investigate whether antiplatelet agents, dilazep and dipyridamole, inhibit tissue factor (TF) expression on monocytes induced by IgG from patients with antiphospholipid syndrome (APS). METHODS: Freshly isolated peripheral blood monocytes were allowed to adhere on plastic and then cultured in media containing patient or control antibodies and/or other agonists with or without dilazep or dipyridamole. The TF activity on monocytes was investigated by measuring factor VIIa-dependent generation of factor Xa, using a chromogenic substrate and the TF mRNA expression was examined by real-time PCR (TaqMan PCR). RESULTS: The TF activity on monocytes induced by APS IgG (250 mg/L) was inhibited by dilazep (0.15-150 micromol/L) and dipyridamole (0.2-200 micromol/L) in a dose-dependent fashion. But, the TF mRNA expression induced by APS IgG was not inhibited. Theophylline (500 micromol/L), an adenosine receptor antagonist, could counteract the inhibitory effect of dilazep and dipyridamole on TF activity. CONCLUSION: Antiplatelet agents, dilazep and dipyridamole, block APS IgG-induced monocytes TF expression at a post-transcriptional level, partly by adenosine receptor pathway. Pharmacological agents that block monocytes TF activity, such as dilazep and dipyridamole, are a novel therapeutic approach in APS.

Nucleoside transporter subtype expression and function in rat skeletal muscle microvascular endothelial cells.

1. Microvascular endothelial cells (MVECs) form a barrier between circulating metabolites, such as adenosine, and the surrounding tissue. We hypothesize that MVECs have a high capacity for the accumulation of nucleosides, such that inhibition of the endothelial nucleoside transporters (NT) would profoundly affect the actions of adenosine in the microvasculature. 2. We assessed the binding of [(3)H]nitrobenzylmercaptopurine riboside (NBMPR), a specific probe for the inhibitor-sensitive subtype of equilibrative NT (es), and the uptake of [(3)H]formycin B (FB), by MVECs isolated from rat skeletal muscle. The cellular expression of equilibrative (ENT1, ENT2, ENT3) and concentrative (CNT1, CNT2, CNT3) NT subtypes was also determined using both qualitative and quantitative polymerase chain reaction techniques. 3. In the absence of Na(+), MVECs accumulated [(3)H]FB with a V(max) of 21+/-1 pmol microl(-1) s(-1). This uptake was mediated equally by es (K(m) 260+/-70 microm) and ei (equilibrative inhibitor-insensitive; K(m) 130+/-20 microm) NTs. 4. A minor component of Na(+)-dependent cif (concentrative inhibitor-insensitive FB transporter)/CNT2-mediated [(3)H]FB uptake (V(i) 0.008+/-0.005 pmol microl(-1) s(-1) at 10 microm) was also observed at room temperature upon inhibition of ENTs with dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]pyrimidine)/NBMPR. 5. MVECs had 122,000 high-affinity (K(d) 0.10 nm) [(3)H]NBMPR binding sites (representing es transporters) per cell. A lower-affinity [(3)H]NBMPR binding component (K(d) 4.8 nm) was also observed that may be related to intracellular es-like proteins. 6. Rat skeletal muscle MVECs express es/ENT1, ei/ENT2, and cif/CNT2 transporters with characteristics typical of rat tissues. This primary cell culture model will enable future studies on factors influencing NT subtype expression, and the consequent effect on adenosine bioactivity, in the microvasculature.

Endothelin-1 gene expression in endothelial cells is potently inhibited by a vasodilator, dilazep.

Endothelin-1 (ET-1) is considered to be involved in various cardiovascular and renal disorders. The objective of this study was to investigate whether a vasodilator and antiplatelet agent, 1,4-bis[3-(3,4,5-trimethoxybenzoyloxy) propyl]perhydro-1,4-diazepine dihydrochloride monohydrate (dilazep, DZ), has an ET-1-inhibiting effect in vitro. Bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) pretreated with fetal calf serum were treated with DZ and preproET-1 (PpET-1) transcription was evaluated by Northern blot analysis. ET-1 peptide release in culture medium was evaluated by radioimmunoassay. The effect of DZ on the ET-1 promoter/enhancer apparatus was evaluated in transfection experiments using -5 kb ET-1 promoter/enhancer constructs. Modest inhibition of PpET-1 gene transcription was detected after 30 min of DZ treatment (0.56+/-0.19 vs. 1 , p<0.01) and more marked inhibition was seen at 24 h (0.04+/-0.04 vs. 1, p<0.0001). ET-1 peptide release was suppressed strongly after 3 h (382.5+/-2.9 vs. 673.5+/-74.5pg/ml, p< 0.001) and 24 h (38.8+/-9.8 vs. 5,075+/-52.0pg/ml, p<0.0001). DZ potently inhibited PpET-1 transcription in a concentration-dependent manner (0.42+/-0.18 vs. 1, p<0.001, at 100micromol/l). DZ suppressed PpET-1 transcription in confluent HUVEC at 3 h (0.41 +/-0.11 vs. 1, p<0.0001). DZ strongly inhibited PpET-1 transcription after 1 h of thrombin (TH) treatment (0.30+/-0.01 vs. 1.51+/-0.03, p<0.0001). Transfection experiments using the 5 kb ET-1 promoter-luciferase plasmid revealed that DZ strongly suppressed ET-1 promoter activity by 99% (p<0.01). DZ potently inhibited ET-1 gene expression at the transcription level in serum- or TH-treated endothelial cells.

Characterization of monocyte tissue factor activity induced by IgG antiphospholipid antibodies and inhibition by dilazep.

Increasing evidence suggests that autoantibodies directly contribute to hypercoagulability in the antiphospholipid syndrome (APS). One proposed mechanism is the antibody-induced expression of tissue factor (TF) by blood monocytes. Dilazep, an antiplatelet agent, is an adenosine uptake inhibitor known to block induction of monocyte TF expression by bacterial lipopolysaccharide. In the current study we characterized the effects of immunoglobulin G (IgG) from patients with APS on monocyte TF activity and investigated whether dilazep is capable of blocking this effect. IgG from 13 of 16 patients with APS significantly increased monocyte TF activity, whereas normal IgG had no effect. Time-course experiments demonstrated that APS IgG-induced monocyte TF mRNA levels were maximal at 2 hours and TF activity on the cell surface was maximal at 6 hours. Dilazep inhibited antibody-induced monocyte TF activity in a dose-dependent fashion but had no effect on TF mRNA expression. The effect of dilazep was blocked by theophylline, a nonspecific adenosine receptor antagonist. In conclusion, IgG from certain patients with APS induce monocyte TF activity. Dilazep inhibits the increased expression of monocyte TF activity at a posttranscriptional level, probably by way of its effect as an adenosine uptake inhibitor. Pharmacologic agents that block monocyte TF activity may be a novel therapeutic approach in APS.