Flow cytometric studies of nucleoside transport regulation in single chromaffin cells.

The present paper reveals that a fluorescent derivative of nitrobenzylthioinosine, 5-(SAENTA-x8)-fluorescein, is a highly specific inhibitor of the neural NBTI-sensitive nucleoside transporter. 5-(SAENTA-x8)-fluorescein inhibited adenosine transport and [3H]NBTI binding with a Ki of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated that 5-(SAENTA-x8)-fluorescein specifically interacted with the NBTI-sensitive nucleoside transporters with high affinity (K[D] = 6 nM). Activation of protein kinases A and C with forskolin or nicotinic receptor agonists, respectively, resulted in 50% inhibition of the fluorescence bound to the cells. Flow cytometry will allow studying nucleoside transport in single cells from heterogeneous neural cell populations.

Differential modulation of nucleoside transport types in neuroblastoma cells by protein kinase activation.

Nucleoside transport regulation in undifferentiated Neuro-2A cells has been studied and found to include Na+-dependent adenosine transport and facilitated diffusion adenosine transport. The latter corresponded to nitrobenzylthioinosine-sensitive nucleoside transport. Short-term treatment of Neuro-2A cells with physiologically relevant signals only modulated the facilitated diffusion component. The stimulation of undifferentiated cells with forskolin or other activators of the protein kinase A pathway, decreased NBTI-sensitive adenosine transport. Treatment of cells with an inactive analogue of forskolin, 1,9-dideoxi-forskolin, had no effect on NBTI-sensitive nucleoside transport. Therefore, the inhibition of protein kinase A activity by pre-incubation with H-89 or the cAMP antagonist, Rp-8-Br-cAMPS, completely prevented the inhibitory effect of forskolin. Similarly, the activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore A-23187 decreased NBTI-sensitive adenosine transport. The effect of PDBu was reversed by pre-incubation of cells with staurosporine. Maximal transport inhibition was obtained by the simultaneous stimulation of cells with a phorbol ester and A-23187 or a phorbol ester and forskolin. The modulation of NBTI-sensitive nucleoside transport corresponded to changes in specific [3H]NBTI binding to Neuro-2A cells. Maximal inhibition correlated well with a maximal enhancement of cAMP production. However, the Na+-dependent adenosine transport in Neuro-2A cells was not modulated by any of these signals.

Fiberoptic bronchoscopy for refractory cough.

Fiberoptic bronchoscopy (FB) has a low yield in the diagnosis of chronic cough (greater than 3 weeks) in unselected patients. We assessed the yield of FB for cough during a four-year period in patients with nonlocalizing chest roentgenograms who were refractory to diagnostic efforts and empiric bronchodilator or antitussive therapy. Seven (28 percent) of 25 patients undergoing FB for cough (of greater than 1,500 bronchoscopies) had diagnostic findings (broncholithiasis, two; tracheobronchopathia osteochondroplastica, two; and tuberculous bronchostenosis, laryngeal dyskinesia, and arytenoid polyp, one each). No tracheobronchial neoplasms were detected. Age greater than 50 years and female sex independently predicted positive results (p = 0.02 Fisher's exact test), while duration of cough (two to 240 months), airflow, and smoking status did not. When patients with prior pulmonary or extrathoracic neoplasms were excluded, seven (35 percent) of 20 studies were diagnostic. Diagnoses potentially could have been made by thoracic computed tomographic scanning in four patients and indirect laryngoscopy in two. Fiberoptic bronchoscopy has a respectable yield for diagnosis of refractory chronic cough and is a reasonable procedure in carefully selected patients.

Pulmonary complications of combination therapy with cyclophosphamide and prednisone.

Oral cyclophosphamide and prednisone are standard treatment for some neoplasms and necrotizing systemic vasculitis and are advocated with increasing frequency for idiopathic interstitial lung disease. During a 15-month period, we observed four cases of acute respiratory failure from Pneumocystis carinii pneumonia (PCP) in patients treated with oral cyclophosphamide and prednisone. One patient each had polyarteritis nodosa, Wegener's granulomatosis, bronchiolitis obliterans with organizing pneumonia, and chronic lymphocytic leukemia with red blood cell aplasia. Hypoalbuminemia (serum albumin level less than 3.0 g/dl) and daily therapy were associated with increased risk for development of PCP (p less than 0.05). None of the patients had leukopenia (less than 3,500/cu mm) or neutropenia (less than 1,000/cumm) at diagnosis. All were negative for the human immunodeficiency virus. Patients receiving oral cyclophosphamide and prednisone may be at higher or increasing risk for PCP. A high index of suspicion and aggressive evaluation for opportunistic infection are needed in these patients; consideration for trimethoprim-sulfamethoxazole prophylaxis and development of more quantitative measures of immunosuppression are needed.

Effect of forskolin and cyclic AMP analog on adenosine transport in cultured chromaffin cells.

The adenosine transport in cultured chromaffin cells was inhibited by the presence of the adenylate cyclase activator, forskolin, and a cAMP analog. The V(max) values of this transport obtained for control and in the presence of 8-(-4-chlorophenylthio)adenosine-3?:5?-monophosphate cyclic (ClPhcAMP, 100 ?M) or forskolin (0.5 ?M) were 85 +/- 5; 45 +/- 1.5 and 38 +/- 3 pmol/10(6) cells/min, respectively. The K(m) values were not significantly modified. The number of adenosine transporters in cultured chromaffin cells, measured by nitrobenzylthioinosine (NBTI) binding, were decreased by the above mentioned effectors. The values of binding sites per cell were 30,000 +/- 3200; 12,000 +/- 1000 and 21,300 +/- 2000 for control, ClPhcAMP and forskolin, respectively; without changing the dissociation constant. When the binding studies were conducted with cellular homogenates, a significant decrease in the maximal binding capacity for nitrobenzylthioinosine was obtained. The values were as follows: 0.087 +/- 0.01 pmol/mg protein for control, 0.044 +/- 0.02 pmol/mg protein for ClPhcAMP; and 0.032 +/- 0.01 pmol/mg protein for forskolin. In this neural tissue, the adenosine transport system seems to be inhibited by stimulation of the adenylate cyclase or by the cyclic AMP analogue that enters the cells. These results suggest that this inhibition could be mediated by a molecular modification of adenosine transporters, the binding with NBTI is therefore a possible parameter of this modification.

Hemoptysis: a manifestation of pulmonary disease confidently managed by military physicians.

Military physicians can confidently manage hemoptysis with a systematic approach and optimal timing of consultation. Begin with a thorough history, physical examination, and chest x-ray. In our series of 177 cases, a cause for hemoptysis was found in 78% of those with abnormal chest x-rays but in only 21% of those with normal chest x-rays. All 36 cases of bronchogenic carcinoma were associated with an abnormal chest x-ray. A normal chest x-ray was associated with no cause found for the hemoptysis (44 cases) or bronchitis (25 cases), with no carcinomas developing upon a 2-year follow-up. Hospitalization is indicated with excessive bleeding or to allay patient or physician) anxiety. Diagnostic bronchoscopy is usually indicated, especially to localize the bleeding in massive hemoptysis (greater than 600 cc per 24 hours) when surgery may be indicated. Prompt referral should be the rule with bleeding from a mycetoma, diffuse bronchiectasis, or with recurrent significant hemorrhage (greater than 200 cc). In an active-duty population, these instances are fortunately rare, and conservative management and elective referral are the norm.

Nitrobenzylthioinosine binding cooperativity in chromaffin tissue membranes.

The nucleoside transporter present in chromaffin tissue membranes has been studied by [3H]nitrobenzylthioinosine (NBTI) binding. This ligand presents a high affinity, with a Kd value of 2.1 +/- 0.2 nM and a Bmax of 1.7 +/- 0.2 pmol/mg protein. From the Scatchard and the semilogarithmic graphical representations a positive cooperativity was deduced, with a Hill coefficient of 1.7 +/- 0.4. In displacement studies of NBTI by the non labelled compound, the Hill coefficient was also higher than 1 (1.44 +/- 0.11) in the presence of ATP. This nucleotide seems necessary to maintain the number of high affinity binding sites.

Bovine adrenal endothelial cells express nucleoside transporters nonregulated by protein kinases A and C.

The present investigation characterizes the nucleoside transporters in bovine adrenomedullary endothelial cells and their possible regulation by the action of protein kinases A and C to establish comparisons with the nucleoside transport system in chromaffin cells. The nucleoside transport proved to be a nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with high affinity for adenosine. These endothelial cells had a high density of nucleoside transporters (660,000 +/- 130,000 transporters/ cell), measured by NBTI binding, and the efficiency was close to 2 adenosine molecules internalized transporter-1.s-1. The stimulation of the cells with bradykinin and P1,P4-di(adenosine-5')tetraphosphate, which raise the intracellular Ca2+ concentration, did not modulate the adenosine transport. When the cells were stimulated with signals coupled to adenosine 3',5'-cyclic monophosphate intracellular production, such as norepinephrine and isoproterenol, the adenosine transport was not modified. Furthermore, the treatment of the cells with direct activators of both protein kinases A and C had no effect on adenosine transport, in contrast to that reported in chromaffin cells.

Effects of phorbol esters and secretagogues on nitrobenzylthioinosine binding to nucleoside transporters and nucleoside uptake in cultured chromaffin cells.

Secretagogues inhibited adenosine uptake in chromaffin cells without causing apparent changes in the uptake affinity. The inhibition caused by carbachol, nicotine and acetylcholine reached 50%. This inhibition was reproduced by the action of protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA; 100 nM), phorbol 12,13-dibutyrate (PDBu; 100 nM), dicaproin (10 micrograms/ml) and tricaprylin (10 micrograms/ml), with inhibitions of Vmax. of 18, 20, 37 and 47% respectively. No changes in the affinity of uptake were observed with these effectors. Down-regulation of protein kinase C by phorbol esters decreased the inhibitory effects of carbachol on adenosine uptake. Binding studies with nitrobenzylthioinosine (NBTI) showed a similar decrease in the number of transporters when chromaffin cells were treated with the same effectors used for the uptake studies. The high-affinity dissociation constants showed minor changes with respect to the control. The ratio between maximal uptake capacity and the transporter number per cell was not significantly modified by the action of secretagogues or direct effectors of protein kinase C. The number of high-affinity binding sites for NBTI was decreased in cellular homogenates by the direct action of protein kinase C activators, with staurosporine able to reverse this action. Protein kinase C from bovine brain in the presence of ATP and effectors, decreased the number of high-affinity NBTI-binding sites in purified chromaffin cell plasma membranes. These data suggest the possibility of a molecular modification at the transporter level.

Effect of P2Y agonists on adenosine transport in cultured chromaffin cells.

Adenosine transport in cultured chromaffin cells was inhibited by purinergic P2y-receptor agonists without significant changes in the affinity constant, the values being between 1 +/- 0.4 and 1.6 +/- 0.6 microM. The Vmax parameter was modified significantly, being 40 +/- 1.0, 26 +/- 5.0, 32 +/- 3.0, and 22 +/- 4.7 pmol/10(6) cells/min for control, adenosine-5'-O-(2-thiodiphosphate), 5'-adenylylimidodiphosphate, and P1,P4-di(adenosine-5'-) tetraphosphate (Ap4A) (100 microM for every effector), respectively. Ap4A, a physiological ligand for P2y receptors in chromaffin cells, showed the highest inhibitory effect (45%). This transport inhibition is explained by an increase in the cytosolic Ca2+ concentration ([Ca2+]i) and the activation of protein kinase C (PKC). Experiments of [Ca2+]i measurement with the fura-2 technique showed that P2y agonists, as well as bradykinin, were able to increase [Ca2+]i, this effect being independent of the presence of extracellular Ca2+. The peptide bradykinin, determined to be coupled to phosphatidylinositol hydrolysis and internal Ca2+ mobilization in chromaffin cells, exhibited a behavior similar to that of P2y agonists in adenosine transport inhibition (39%). P2y agonists and bradykinin increased PKC activity associated with the membrane fraction (about 50% increase in particulate PKC activity with respect to controls). The present studies suggest that adenosine transport is regulated by P2y-purinergic receptors mediated via Ca2+ mobilization and PKC activation.

Evidence that adenine nucleotides modulate nucleoside-transporter function. Characterization of uridine transport in chromaffin cells and plasma membrane vesicles.

Uridine transport was investigated in cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue. In intact cells, the kinetic parameters for uridine uptake were Km 150 +/- 45 microM, and Vmax 414 +/- 17 pmol . 10(6) cells-1 . min-1. This low affinity for uridine and its inhibition by low concentrations of nitrobenzylthioinosine (Ki 3 nM) and dipyridamole (Ki 54 nM) are consistent with a facilitated diffusion nucleoside transport system. The IC50 value for the adenosine transport inhibition by uridine was very high (240 microM), agreeing with the relative affinities of these nucleosides in the chromaffin cell nucleoside transport system, which was 150-fold higher for adenosine than for uridine. Uridine was significantly metabolized in chromaffin cells but not in plasma membrane vesicles. The affinity of uridine transport measured in these membrane vesicles was reproducible and similar to the affinity found for intact cells with a Km value of 185 +/- 11 microM and a Vmax value of 4.24 +/- 0.10 pmol . mg protein-1 . s-1. These membrane preparations were employed to investigate the regulatory action of ATP and other nucleotide analogues on nucleoside transport. ATP increased the Vmax value but the Km value was not significantly modified. Adenosine 5'-[beta,gamma-imino]triphosphate, 1,N6-ethenoadenosine 5'-triphosphate, and adenosine(5')-tetraphospho(5')adenosine(Ap4A) at 100 microM were able to mimic the ATP effect. These results agree with a regulatory role of ATP, and the uridine transport on chromaffin plasma membrane vesicles is a good model for analyzing the nucleoside-transporter function and regulation.

Effect of 5'-(N-ethylcarboxamido)adenosine on adenosine transport in cultured chromaffin cells.

Extracellular adenosine is transported into chromaffin cells by a high-affinity transport system. The action of adenosine receptor ligands was studied in this cellular model. 5'-(N-Ethylcarboxamido)adenosine (NECA), an agonist of A2 receptors, activated adenosine transport. Km values for adenosine were 4.6 +/- 1.0 (n = 5) and 10.2 +/- 3.0 microM (n = 5) for controls and 100 nM NECA, respectively. The Vmax values were 66.7 +/- 23.5 and 170.2 +/- 30 pmol/10(6) cells/min for controls and 100 nM NECA, respectively. The A1 agonist N6-cyclohexyladenosine, the A1 antagonist 8-cyclopentyl-1, 3-dipropylxanthine, and the A1-A2 antagonist 1,3-dipropyl-8-(4-[(2-aminoethyl)amino]-carbonylmethyloxyphenyl)- xanthine did not significantly modify the adenosine transport in this system. Binding studies done with [3H]dipyridamole, a nucleoside transporter ligand, did not show changes in either the number or affinity of transporter sites after NECA treatment. This ligand can enter cells and quantifies the total number of transporters. The binding studies with [3H]-nitrobenzylthioinosine, which quantifies the plasma membrane transporters, showed a Bmax of 19,200 +/- 800 and 23,200 +/- 700 transporters/cell for controls and 100 nM NECA, respectively. No changes in the KD were obtained. The effects of NECA were not mediated through adenylate cyclase activation, because its action was not imitated by forskolin.

MR and CT findings in pulmonary artery sarcoma.

Pulmonary artery sarcomas are a rare and frequently forgotten cause of pulmonary artery occlusion. We present a case report that details magnetic resonance and new CT findings, which may help establish early diagnosis of this infrequently encountered tumor.