Differential regulation of atrial and brain natriuretic peptides and its implications for the management of left ventricular volume overload.

BACKGROUND: In this study, we investigated the possibility that the atrial and brain natriuretic peptide expression in left ventricular volume overload (VOL) is transcriptionally regulated. We further evaluated the diagnostic and/or prognostic potential of this expression for the management of patients with this disorder. DESIGN: We compared the myocardial mRNA expression and plasma levels of the two peptides in VOL patients using donor hearts and in healthy blood donors as controls. RESULTS: The atrial natriuretic peptide (ANP) mRNA was elevated by 38% (P < 0.03) in the right atrium and by 53% (P < 0.003) in the left atrium, but was unchanged in the ventricular chambers of the patient group (n = 19) compared with controls (n = 8). Plasma ANP concentration was elevated by 62% (P < 0.001) compared with blood donor controls (n = 79). It increased slightly (by 36%) 2 h following surgery, and remained at 64% higher (P < 0.03 vs. presurgery) for the 5 days following surgery. The brain natriuretic peptide (BNP) mRNA was elevated by approximately one-fold in both the left ventricle (P < 0.02) and right atrium (P < 0.05), by 94% (P < 0.02) in the right ventricle and by 89% (P < 0.05) in the left atrium. Its plasma level in the patients was 3.4-fold (P < 0.00003) higher than in control subjects. It increased significantly by 1.2-fold (P < 0.01) 2 h following surgery, but dropped significantly (P < 0.05 vs. 2 h post surgery) to presurgical levels 5 days following surgery. CONCLUSION: The results show chamber-specific elevation in both atrial and brain natriuretic peptide expression and differences in their circulating levels in VOL, suggesting that BNP is a potential prognostic indicator in the postsurgical management of these patients.

Evidence for the binding of beta-adrenoceptor blockers to microsomal Na+/K+-ATPase in guinea pig heart preparations.

We reported in a previous study that beta-adrenoceptor blockers inhibit the Mg2+-dependent ATP-hydrolytic function of Na+/K+-ATPase. To determine if this action is a result of binding of beta-blockers to the receptor sites that bind the digitalis glycosides, we performed displacement binding assays of eight beta-blockers with [3H]-ouabain (OUA) in guinea pig myocardial microsomal preparations. In the first series of experiments, 10-200 microM of the beta-blockers were displaced with 250 nM OUA. In the second set of experiments, 10-500 nM of OUA was displaced using 200 microM of the beta-blockers. The drugs showed concentration-dependent receptor occupancy at the different OUA levels. Propranolol (PPN), metoprolol (MTP), and sotalol (STL) showed the strongest binding; nadolol (NDL), indenolol (IDN), and atenolol (ATN) had intermediate binding; carazolol (CRZ) and celiprolol (CLP) had the weakest binding properties. The results suggest that beta-blockers may compete for the same binding sites with ouabain in their inhibition of the Na+/K+-ATPase. These actions may contribute to the mechanism for some of their cardiac effects, especially their proarrhythmic and arrhythmogenic actions.

Angiotensin-converting enzyme polymorphism and the risk of coronary heart disease in the Saudi male population.

OBJECTIVE: To determine the relevance of angiotensin I-converting enzyme (ACE) gene polymorphism for coronary artery disease (CAD) in the Saudi population. METHODS AND RESULTS: DNA of 84 male Saudi patients with established CAD, 36 male controls who underwent angiography, and 327 healthy Saudi male blood donors was amplified by polymerase chain reaction, using oligonucleotide primers flanking the insertion (I)/deletion (D) sites in the polymorphic region of intron 16 of the ACE gene. Polymerase chain reaction amplification resulted in 490-bp (II), 190-bp (DD), or 490- and 190-bp (ID) fragments. The genotype II distribution was 16.7% in the control group, 7.3% in the blood donor group, and 7.2% in the patients with CAD, and the distribution for DD was 58.3%, 47.1%, and 41.0%, respectively. Notably, 61.9% (P <.0001) of CAD patients presented with angina on admission, and 52.4% had diabetes mellitus. CONCLUSIONS: The results show no increased risk of CAD in association with either the II or DD genotypes in the Saudi population. However, further investigation of genotype II as a predictor for atherosclerosis rather than increased risk of coronary heart disease may be indicated.

Class I antiarrhythmic drug effects on ouabain binding to guinea pig cardiac Na+ -K+ ATPase.

The notion that the inhibition of the Mg2+ -dependent ATP-hydrolytic function of the myocardial Na+ -K+ ATPase by class I antiarrhythmic agents occurs as a result of their binding to the same receptor sites as the digitalis glycosides was tested by performing competitive binding assays of [3H]ouabain (OUA) with eight drugs: disopyramide, encainide, lidocaine, lorcainide, phenytoin, procainamide, quinidine, and tocainide in guinea pig heart microsomal preparations. In the first set of experiments, 10-200 microM concentrations of the drugs were preincubated with the enzyme and displacement assays performed with 250 nM OUA. The drugs showed receptor occupancy of 19-32% at 50 microM, 25-44% at 100 microM, and 37-56% at 200 microM. Then, 10-500 nM concentrations of OUA were preincubated with the enzyme, and competitive assays were performed using 200 microM concentrations of the drugs. OUA occupied 39-51% of the receptor sites at 100 nM, 44-67% at 250 nM, and 62-82% at 500 nM, displacing the drugs in a concentration-dependent fashion. The results show that antiarrhythmic drugs interact with the same or similar receptor sites as ouabain on the Na+ -K+ ATPase, pointing to a possible contribution of these interactions to the mechanism for their inhibitory actions on the enzyme, and perhaps their arrhythmogenic effects.

Effects of amiodarone on ouabain binding to microsomal Na+, K(+)-ATPase in guinea-pig heart preparations.

1. The influence of amiodarone on [3H]ouabain (OUA) binding to myocardial Na+, K(+)-ATPase was studied at various KCl concentrations in guinea pig heart microsomal preparations to test the hypothesis that the drug acts on the same receptor sites as cardiac glycosides. 2. First, a series of assays for OUA binding to Na+, K(+)-ATPase were performed in the range of 64-800 nM at 2.5, 5.0 and 10.0 mM K+ concentration. The drug exhibited increasing binding tendency with increasing concentrations and elevation in K+ levels. 3. Competitive binding assays were then performed at 256, 512 and 800 nM OUA in the presence of 50, 100 and 200 microM amiodarone at 5.0 mM KCl, respectively. At each OUA concentration, a concentration-dependent left-to-right shift was observed in the binding affinity with increasing amiodarone concentration. similar assays at 2.5 and 10.0 mM K+ showed the same trends. These effects were significant for 200 mM amiodarone at all K+ levels and OUA concentrations. 4. Furthermore, different OUA concentrations were also shown to displace amiodarone in a concentration-dependent fashion. 5. These results indicate that amiodarone competes with OUA for specific binding sites on myocardial microsomal Na+, K(+)-ATPase. They lead to the conclusion that myocardial Na+, K(+)-ATPase is a possible receptor for some of the cardiac actions of amiodarone, such as its proarrhythmic effects.

HLA-DR types in rheumatic, congenital and degenerative cardiac valve diseases in Saudi patients.

Subjects of Saudi origin were DNA typed for HLA-DR2, DR4 and DRw53 by amplification fragment-length polymorphism and amplification by sequence-specific primer techniques based on polymerase chain reaction. Of these subjects, 125 had sporadic heart valve disease (96 with rheumatic heart disease, 18 with degenerate and 11 with congenital degenerate valve disease) and 77 were healthy Saudi blood donors. While the frequency of individuals typed DR4 was about the same in the rheumatic heart disease as in the control category (30% versus 23%, respectively), it was found to be higher (55%; P<0.02), but below the level of marginal significance after correcting for the number of DR types, in the congenital degenerate valve category. No preferential association of any DR4 subtype could be detected. The incidence of DR2 was lower in the congenital cases compared to that in the controls (9% versus 21%) and remained about the same in the rheumatic heart disease patients as in the controls. The frequency of DRw53 in the degenerate valve categories was slightly lower than that in the controls, but the difference was not significant. The study failed to corroborate the association between HLA-DR4 and rheumatic heart disease shown in previous studies using the serotyping approach.

Platelet alpha-adrenoceptor density in patients with left ventricular volume overload.

We have evaluated the possibility that, in patients with left ventricular heart disease, the effects of aortic and mitral regurgitation on platelet alpha 2-adrenoceptors may depend on the origin and severity of the overload. Receptor density and binding affinities were estimated by their specific binding to [3H]-yohimbine. In blood donor controls (CON), the receptor density was 4.72 +/- 0.41 fmol/10(6) platelets (n = 31). In the volume overloaded patients (n = 35), the total density was elevated by 57% (p < 0.05) accompanied by a 69% (p < 0.05) increase in plasma epinephrine. Compared with CON, patients with pure aortic regurgitation (AVR, n = 12) showed a 91% (p < 0.0001), pure mitral regurgitation (MVR, n = 15) 43% (p < 0.05) and mixed mitral and aortic valve regurgitation (MOL, n = 8) 23% increase in receptor density. Furthermore, the elevation of the density in the aortic disease was significantly greater (p < 0.05) than in the mixed overload group. There was a weak positive correlation between the increase in receptor density and the ejection fractions (r = 0.27), suggesting that the former may be dependent on the severity of the volume overload. The results show that in left heart valvular disease, aortic regurgitation leads to a highly significant increase in alpha-adrenoceptor density, while the effects of mitral valve disease exhibit borderline significance. These findings probably point to the differences in the extent of influence of the origin and severity of the two forms of left ventricular volume overload (LVO), as well as the ensuing hemodynamic changes on the cardiac contractile apparatus.

Characterization of nucleoside uptake and transport in Entamoeba histolytica.

The uptake and transportation of purine and pyrimidine based nucleosides by trophozoites of axenically grown Entamoeba histolytica (HMI-IMSS) were studied. The trophozoites transported adenosine and its analog tubercidin (1 microM) at a significant rate but poor transportation was observed in case of uridine (about 10% relative rate), inosine (3%), thymidine (2%) and formycin B (1%). The Km for adenosine was 160 +/- 42 microM. Unlabeled nucleosides (100 microM) inhibited adenosine and tubercidin transport. Adenosine related compounds 5'-deoxyadenosine and nebularin inhibited adenosine and tubercidine transport by 50% or more. However, inosine related compounds guanosine, 3'-deoxyinosine and formycin B were less inhibitory. The pyrimidine nucleosides uridine, thymidine and cytidine were poorly inhibitory. 6-[(4 nitrobenzyl)-mercapto] purine ribonucleoside, an inhibitor of mammalian nucleoside transporter, inhibited adenosine or tubercidin transport in E. histolytica variably between 0-30% at 10 microM, but dilazep, a known inhibitor, was inactive upto 10 microM. Increase in temperature from 22 degrees C to 33 degrees C enhanced the rate of transport of adenosine 4.5 fold, tubercidin 7.3 fold and of inosine 4 fold. These findings along with the structure activity figures suggested that transport was mediated and not passive.

Effects of different left ventricular load conditions on myocardial beta-adrenoceptor density in patients with rheumatic heart valvular disease.

1. The possibility that different left ventricular load conditions may influence myocardial beta-adrenoceptor function in various ways was evaluated by determining the receptor density in all four chambers of 69 patients with rheumatic heart valvular disease. 2. The left ventricular beta-adrenoceptor density was reduced by 44% in patients with left ventricular pressure overload (LVP), 66% in left ventricular volume overload (LVV), 56% in mixed volume and pressure overload (MOL), and 60% in those with no left ventricular pressure overload (NOL). Similarly, the right ventricular receptor density decreased significantly by 46%, 54%, 43%, and 46%, left atrial by 15%, 29%, 14%, and 21%; and right atrial by 27%, 30%, 28%, and 12% in LVP, LVV, MOL, and NOL, respectively. Thus, the general trend in the decrease in receptor density was LVV > MOL = NOL > LVP. 3. Furthermore, the LVV patients with the largest decrease in receptor density in all four chambers, similarly exhibited the largest ejection fractions (EF) and left ventricular internal diastolic and systolic diameters. 4. The results show that left ventricular volume overload is a major cause of attenuation in myocardial beta-adrenoceptor density, compared to other forms of ventricular overload in heart valvular disease. 5. Since elevated EF in volume overload patients is an indication of the severity of the disease, the decrease in their myocardial receptor density may be a reflection of the degree of influence of the disease on their sympathetic activity.

Relationship between alterations in lymphocyte and myocardial beta-adrenoceptor density in patients with left heart valvular disease.

1. We have evaluated the possibility that alterations in lymphocyte beta-adrenoceptor density may be related to changes in the myocardial population in left heart valvular diseases. Receptor density and their binding affinities were estimated using [125I]-iodocyanopindolol. 2. The lymphocyte (LC) beta-adrenoceptor density was 43.4 +/- 5.6 fmol/mg protein in the controls (n = 35) and 81% lower in heart valvular patients (n = 86). In myocardial controls (n = 18), the left ventricular (LV) receptor density was 167.2 +/- 29.8 fmol/mg protein, right ventricular (RV) density was 123.1 +/- 14.6 fmol/mg, left atrial (LA) density was 81.6 +/- 10.5 fmol/mg and right atrial (RA) 108.1 +/- 14.5 fmol/mg. Compared with this group, the receptor density of the study patients (n = 47) decreased by 67, 43, 24 and 32% in the LV, RV, LA and RA, respectively. The decrease in LC was twice that of the average total myocardial receptor density. 3. When patients were classified according to their left ventricular load conditions as having either left ventricular pressure overload (LVP), left ventricular volume overload, mixed lesions (MOL) and no left ventricular overload (mitral stenosis; NOL), the attenuation in LC receptor density reached statistical significance for all four groups, without showing significant difference between the individual groups. In contrast, the decrease in all chambers was predominantly due to volume overload. MOL and NOL exerted intermediate effects that were significant in the LV, while LVP did not contribute to the changes in the LA. 4. Accordingly, the reduction in peripheral beta-adrenoceptor density may reflect the extent to which particularly the volume overload exerts its influence on myocardial beta-adrenoceptors in left heart valvular patients.

Sympathetic activity in children undergoing balloon valvuloplasty of pulmonary stenosis.

We studied the influence of balloon valvuloplasty on alpha- and beta-adrenoceptor densities, plasma catecholamine, and cAMP levels in children and infants with pulmonary stenosis before and 10 min after balloon dilatation, employing as controls children undergoing transcatheter occlusion of patent ductus arteriosus (PDA) with Qp/Qs ratio < 1.5. In the PDA group, the alpha-adrenoceptor density (Bmax) was 3.75 +/- 0.72 fmol/10(7) cells (n = 15) before occlusion and remained unchanged at 3.35 +/- 0.47 fmol 10 min thereafter. In the pulmonary stenosis patients (n = 31), the receptor density was 59% higher (p < 0.05) before, and decreased to PDA levels 10 min after, the procedure. The control beta-adrenoceptor density was 64.8 +/- 11.0 fmol/10(6) cells before, and 71.2 +/- 13.2 fmol 10 min after, occlusion. In the study group, the density was 23% lower (p < 0.07) and increased to the PDA levels 10 min after the dilatation. Compared with the PDA, pre- and postdilatation plasma norepinephrine levels were not significantly changed; epinephrine was slightly elevated before, but increased by 73% after, dilatation; dopamine was 80% (p < 0.05); and cAMP was 37% higher before, and remained elevated at 70 and 23% above the PDA values after, the procedure. Accordingly, alpha-adrenoceptor density is significantly elevated in children with pulmonary stenosis and decreases significantly immediately after balloon valvuloplasty. On the other hand, beta-adrenoceptor density is attenuated and increases toward normal levels after the procedure. The immediate reversal of the receptor levels after balloon valvuloplasty suggests that this procedure exerts acute effects on the sympathetic functional level in this disease.

Effect of left ventricular pressure and volume overload on alpha-adrenoceptor activity in patients with rheumatic heart valvular disease.

We have investigated the possibility that the various left ventricular load conditions may exert different effects on the sympathetic function by comparing the influence of volume (VOL) and pressure (POL) overload on platelet alpha-adrenoceptor activity, plasma catecholamines and cAMP in 44 patients with rheumatic heart valvular disease. Receptor activity was determined by radioligand binding methods, catecholamines by HPLC using an electrochemical detector, and cAMP by radioimmunoassay. The mean alpha-adrenoceptor density (Bmax) of the control group (n = 29) was 4.71 +/- 0.41 fmol per 10(7) platelets and the corresponding dissociation constant (Kd) was 2.47 +/- 0.15 nM. In VOL patients, the density was elevated by 70% (P < 0.0001), but it remained unchanged in the POL patients. In contrast to the Bmax, the Kd of the VOL group was not changed, and it increased by 34% (P < 0.01) in the POL group. Norepinephrine was elevated by 91% (P < 0.05) in POL, and epinephrine increased by 65% (P < 0.05) in POL and 71% (P < 0.05) in VOL. These results suggest that the sympathetic nervous system responds to left ventricular volume overload by increasing alpha-adrenoceptor density with no apparent change in receptor affinity toward [3H]-yohimbine binding, and to left ventricular pressure overload by decreasing their binding affinity without a parallel decrease in receptor density. The increase in receptor density in VOL is accompanied by an increase in plasma epinephrine, and the decrease in binding affinity in POL is associated with increased plasma norepinephrine and epinephrine levels.

Comparison of the effect of left ventricular volume and pressure overload on beta-adrenoceptor density in left heart valvular disease.

The sympathetic responses in left heart valvular disease may depend on the ventricular load conditions. We proposed to evaluate this possibility by comparing the effects of left ventricular pressure (LVP) and volume (LVV) overload on beta-adrenoceptor density and ligand binding affinity in lymphocytes and in the four myocardial chambers in rheumatic heart valvular patients. Receptor activity was determined by radioligand binding using [125I]iodocyanopindolol. In the lymphocytes (n = 45), the beta-adrenoceptor density was reduced by 88% (P < 0.001) in LVP patients (n = 15) and 79% (P < 0.001) in LVV patients (n = 30) compared with 23 controls. In the myocardium, the receptor density of the LVP (n = 12) was attenuated by 55% (P < 0.05) in the left ventricle, 42% in the right ventricle, 13% in the left atrium, and 37% in the right atrium, while in LVV patients (n = 22) it decreased by 73% (P < 0.01) in the left ventricle, 62% (P < 0.05) in the right ventricle, 30% in the left atrium, and 34% in the right atrium compared with 15 controls. Thus, the reduction in density was greatest in lymphocytes and least in the atria in both groups. The decrease in ventricular density of the LVV group was similar to the reduction in the lymphocytes and two-fold higher than in the atrial density. These alterations were significantly greater for the LVV than for the LVP group. The Kd for the myocardial receptor binding to [125I]iodocyanopindolol was not significantly influenced in either group, but was lower in the lymphocytes. These findings suggest that in patients with left heart valvular disease, there is a significant attenuation in both peripheral and myocardial beta-adrenoceptor density. The decrease in receptor density is significantly greater in the left ventricular volume overload than in the left ventricular pressure overload patients.

Beta adrenoceptor density in patients with left-sided valvular regurgitation.

Aortic regurgitation differs from mitral regurgitation in that it is a result of combined volume and pressure overload, while the latter represents an almost pure volume overload. In this study, we tested the possibility that these two forms of left ventricular volume overload exert different effects on beta-adrenoceptor density. Lymphocyte (n = 33) and myocardial (n = 22) beta-adrenoceptor densities were evaluated by [125I]-iodocyanopindolol binding in volume-overload patients with left heart valvular disease, compared with 31 healthy donor blood and 15 donor heart controls, made available as a result of failing to get matching recipient. The total lymphocyte (LC) beta-adrenoceptor density decreased from 43.4 +/- 5.5 fmol mg-1 protein in controls to 9.2 +/- 2.7 fmol (P < 0.001) in heart valvular patients. In the myocardial controls, the left ventricular (LV)-receptor density was 126.7 +/- 19.5 fmol; right ventricular (RV), 123.1 +/- 14.6 fmol; left atrial (LA), 81.6 +/- 10.5 fmol; and right atrial (RA), 108.1 +/- 14.5 fmol mg-1 protein. Compared to this group, the total LV-receptor density of the patients decreased by 63%, RV by 54%, LA by 31% and RA by 34%. The decrease in receptor density exhibited a positive correlation with increasing ejection fractions in both the left (r = 0.38) and right (r = 0.44) ventricles, indicating that the former was dependent on the extent of the disease. These changes were accompanied by a 44% increase in plasma epinephrine, 13% in norepinephrine and a 27% decrease in dopamine levels. Based on the predominant left ventricular volume overload classified as aortic regurgitation (AVR), mitral regurgitation (MVR), and mixed aortic and mitral regurgitation (MOL), the attenuation in myocardial-receptor densities showed the following trend: MOL > AVR > AVR. The results show a global reduction in myocardial and LC beta-adrenoceptor density, which depends on the origin and the gravity of the LV volume overload.

Regulation of sympathetic activity in children with various congenital heart diseases.

We studied the alpha- and beta-adrenoceptor activity and catecholamine and cAMP levels in 112 children and infants admitted to the hospital for diagnostic or interventional catheterization of tetralogy of Fallot, ventricular septal defects with or without hypertension, pulmonary stenosis, coarctation of the aorta, and various complex cyanotic congenital cardiac diseases and compared them with 14 children undergoing transcatheter occlusion of patent ductus arteriosus with insignificant left-to-right-shunts. The mean total platelet alpha-adrenoceptor density of the study population was elevated by 73%. Both the increases in acyanotic (p < 0.05) and cyanotic (p < 0.005) patients as well as the difference between the two groups (p < 0.01) were significant. Based on the congenital disease classification, the elevation in receptor density was also significant in all groups of patients, except coarctation of the aorta. On the other hand, the mean lymphocyte beta-adrenoceptor density was attenuated by 27%, showing significant difference between the acyanotic and the patent ductus arteriosus groups, but none between acyanotic and cyanotic or cyanotic and the patent ductus arteriosus groups. Among the congenital groups, only the left-to-right shunts and the pulmonary stenosis group showed significant (p < 0.05) decrease in beta-adrenoceptor density, whereas the affinity of all the groups toward [125I]iodocyanopindolol was hardly influenced. The plasma levels of all three catecholamines, norepinephrine, epinephrine, and dopamine, were elevated, but cAMP remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of lymphocyte beta-adrenoceptor activity and Gs-protein in patients with rheumatic heart valvular disease.

In order to test whether the beta-adrenoceptor activity in rheumatic heart valvular disease depends on the ventricular load conditions, we determined their density and binding affinity to [125I]-iodocyanopindolol in lymphocytes, as well as plasma catecholamine and cAMP levels in 69 patients with regurgitant and stenotic lesions of the aortic and mitral valves. The patients were classified as having left ventricular pressure overload (LVP), left ventricular volume overload (LVV), mixed lesions (MOL) or right ventricular pressure overload (RVP). The beta-adrenoceptor activity was determined by radioligand binding methods, catecholamines by high performance liquid chromatography using an electrochemical detector and cAMP by radioimmunoassay. The mean beta-adrenoceptor density (Bmax) of the control group was 60.1 +/- 9.5 fmol (n = 29) per 10(6) lymphocytes. In the study population, the density was decreased by 83% in LVP, 78% in LVV, 87% in MOL and 86% in RVP. Plasma norepinephrine was elevated by 89% in LVP and 60% in MOL, epinephrine by 43% in LVP, 50% in VOL, 115% in MOL and 20% in RVP, while dopamine was not significantly changed, and cAMP was slightly elevated in all four groups. Screening for activating mutational changes in the Gs alpha-protein gave negative results, possibly dissociating the elevation in plasma cAMP from stimulatory effects of such abnormalities in the Gs-protein signaling.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-dependent nucleoside transport in mouse lymphocytes, human monocytes, and hamster macrophages and peritoneal exudate cells.

Mouse splenocytes and hamster peritoneal exudate cells (PEC), including macrophages, were shown to contain a predominantly Na(+)-dependent and inhibitor (6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside, NBMPR)-resistant transport system for adenosine and other nucleosides. Adenosine (1 microM) was transported about equally in mouse thymocytes and human monocytes from peripheral blood by a Na(+)-dependent system and the NBMPR-sensitive facilitated diffusion system. Hamster PEC also transported inosine, tubercidin, formycin B, uridine, and thymidine in a NBMPR-insensitive manner. With the exception of formycin B, all nucleosides were phosphorylated intracellularly to varying degree, adenosine being almost fully phosphorylated. During the time course of routine experiments (30 s) formycin B was concentrated twofold over external medium levels (1 microM) without any drop-off in the transport rate. On the basis of metabolic studies it was estimated that uridine and tubercidin were also transported against a concentration gradient. Inosine, guanosine, 2'-deoxyadenosine, tubercidin, formycin B, and the pyrimidines uridine, thymidine, and cytidine (all 100 microM) inhibited transport of adenosine and inosine about 50-100%, while 3'-deoxyinosine showed weak inhibitory action. Transport of thymidine was strongly inhibited by nucleosides except by 3'-deoxyinosine. The Na(+)-dependent, active, and concentration transport system appears to be a feature of many immune-type cells, and its presence offers particular conceptual possibilities for the therapy of infections located in these cells.

In vivo effectiveness of several nucleoside transport inhibitors in mice and hamsters.

The in vivo nucleoside transport inhibitory effects of 6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside (NBMPR), used as its 5'-monophosphate derivative (NBMPR-P), dilazep, mioflazine and its derivatives soluflazine, R57974 and R75231, were investigated in BALB/c mice. The extent and duration of action were followed by assaying adenosine transport in blood cells sampled at time intervals following i.p. administration (ca. 20 mg/kg). Dilazep and R57974 were found to be short-acting inhibitors, while NBMPR-P and R75231 were similar in their action and caused essentially full inhibition of adenosine transport over a 4- to 5-h period. Mioflazine and soluflazine were rather ineffective, causing only partial inhibition. R75231 was also active after oral administration which, when repeated three times in 4-h intervals, resulted in essentially full transport inhibition up to 20 h following the initial dose. Effects of NBMPR-P, R57974 and dilazep on adenosine transport in blood cells were also measured in blood cells of hamsters after i.p. administration of the same doses. All three drugs caused full transport inhibition, but the action of dilazep and R75231 showed reversal within about 30 min and 2 h, respectively, while NBMPR-P caused full inhibition for at least 3-4 h. These results demonstrate the potential of the mioflazine derivative R75231 to be useful in vivo, possibly even after p.o. administration, for host protection against the actions of cytotoxic nucleosides used in experimental antiparasitic therapy or other studies requiring suppression of nucleoside transport.

Sodium-dependent and inhibitor-insensitive uptake of adenosine by mouse peritoneal exudate cells.

[8-3H]Adenosine uptake in mouse peritoneal exudate cells, harvested following i.p. challenge with Complete Freund's Adjuvant from BALB/c mice, was found to be insensitive to common nucleoside transport inhibitors such as dilazep or 6-[(4-nitrobenzyl)mercapto]purine ribonucleoside and to require sodium ion, being inactive when sodium was replaced by lithium or potassium. These findings also applied to the adherent (macrophages) and nonadherent (polymorphonuclear cells) cell fractions prepared from the peritoneal cell mixture. Uptake was inhibited by several nucleosides including deoxyadenosine, inosine, uridine, thymidine and, to a lesser extent, by the adenosine analog tubercidin, while adenine, fructose, glucose and ribose were without effect. Uptake [8-3H]adenosine was fully matched by rapid intracellular phosphorylation to AMP, ADP and ATP. Inosine was a substrate for the transporter, but tubercidin was not. The system clearly is distinct from carrier-mediated, nonconcentrative transport and has similarities to concentrative, sodium-dependent nucleoside transporters described in other cell types.