Cellular pharmacology of the anti-hepatitis B virus agent beta-L-2',3'-didehydro-2',3'-dideoxy-N4-hydroxycytidine: relevance for activation in HepG2 cells.

Beta-l-2',3'-didehydro-2',3'-dideoxy-N(4)-hydroxycytidine (l-Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED(50)] = 0.03 microM; 50% cytotoxic dose [CD(50)] = 2,500 microM). In the present study, we investigated the intracellular pharmacology of tritiated l-Hyd4C in HepG2 cells. l-[(3)H]Hyd4C was shown to be phosphorylated extensively and rapidly to the 5'-mono-, 5'-di-, and 5'-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of l-Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ss-d-N(4)-hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5'-diphosphocholine derivative of l-Hyd4C, as described for some l-deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 microM l-[(3)H]Hyd4C for 24 h, the 5'-triphosphate accumulated to 19.4 +/- 2.7 pmol/10(6) cells. The predominant peak belonged to 5-diphosphate, with 43.5 +/- 4.3 pmol/10(6) cells. The intracellular half-life of the 5'-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5'-phosphorylated l-deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5'-diphosphate during a drug-free incubation. The high 5'-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of l-Hyd4C.

Two hydrophobic protein fractions of ovine pulmonary surfactant: isolation, characterization, and biophysical activity.

Pulmonary surfactant contains two extremely hydrophobic proteins, SP-B and SP-C. We present a novel HPLC method for the preparation of these hydrophobic proteins. It is based on size-exclusion chromatography using the apolar stationary-phase butyl silica gel and isocratic elution with acidified chloroform/methanol. Samples for HPLC were prepared from sheep lung lavage fluid by centrifugation and extraction with chloroform/methanol. Amino acid analyses of the two protein fractions revealed sequences that are consistent with SP-B and SP-C, respectively. MALDI-TOF-MS analyses of the SP-B fraction showed one major peak of dimeric SP-B with m/z 17,361, and additional peaks of monomeric and oligomeric forms, which are predominantly even numbered. The SP-C fraction showed a peak at m/z 4200, consistent with the theoretical mass of the dipalmitoylated form of this protein. The biophysical activity of pure sheep SP-B and SP-C was evaluated by measuring the surface tension using axisymmetric drop shape analysis for captive bubbles. We found distinct surface pressure versus surface area isotherms of SP-B and SP-C indicating different biophysical activities for these surfactant proteins. The new preparative HPLC method is able to replace the established, time-consuming low-pressure liquid chromatography method for the isolation of SP-B and SP-C from lipids.

Quantitative analysis of hydrophobic pulmonary surfactant proteins by high-performance liquid chromatography with light-scattering detection.

A new method for the separation and quantification of two hydrophobic lung surfactant proteins (SPs) is described. It is based on size-exclusion chromatography using the apolar stationary phase butyl silicagel with a pore size of 30 nm and isocratic elution with chloroform, methanol and trifluoroacetic acid. The samples were prepared from sheep lung lavage fluid by centrifugation and fractional extraction with butanol and chloroform-methanol. The chromatograms show three peaks in the elution order SP-B, SP-C and lipids. A small peak ahead of SP-B, which disappeared after reduction with 2-mercaptoethanol, was oligomeric SP-B. The response of the evaporative light-scattering detector was non-linear. For preparative high-performance liquid chromatography ultraviolet detection at 279 nm is recommended.

Renal nerves are not involved in sodium and water retention during mechanical ventilation in awake dogs.

BACKGROUND: The role of renal nerves during positive end-expiratory pressure ventilation (PEEP) has only been investigated in surgically stressed, anesthetized, unilaterally denervated dogs. Anesthesia, sedation, and surgical stress, however, decrease urine volume and sodium excretion and increase renal sympathetic nerve activity independent of PEEP. This study investigated in awake dogs the participation of renal nerves in mediating volume and water retention during PEEP. METHODS: Eight tracheotomized, trained, awake dogs were used. The protocol consisted of 60 min of spontaneous breathing at a continuous positive airway pressure of 4 cm H2O, followed by 120 min of controlled mechanical ventilation with a mean PEEP of 15-17 cm H2O (PEEP), and 60 min of continuous positive airway pressure. Two protocols were performed on intact dogs, in which volume expansion had (hypervolemic; electrolyte solution, 0.5 ml x kg(-1) x min(-1)) and had not (normovolemic) been instituted. This was repeated on the same dogs 2 or 3 weeks after bilateral renal denervation. RESULTS: Hypervolemic dogs excreted more sodium and water than did normovolemic dogs. There was no difference between intact and renal-denervated dogs. Arterial pressure did not decrease when continuous positive airway pressure was switched to PEEP. Plasma renin activity, aldosterone, and antidiuretic hormone concentrations were greater in normovolemic dogs. The PEEP increased aldosterone and antidiuretic hormone concentrations only in normovolemic dogs. CONCLUSIONS: In conscious dogs, renal nerves have no appreciable contribution to sodium and water retention during PEEP. Retention in normovolemic dogs seems to be primarily caused by an activation of the renin-angiotensin system and an increase in the antidiuretic hormone. Excretion rates depended on the volume status of the dogs.

Vasopressin and renin-angiotensin maintain arterial pressure during PEEP in nonexpanded, conscious dogs.

Increases of plasma arginine vasopressin (AVP) and plasma renin activity (PRA) during controlled mechanical ventilation (CMV) with positive end-expiratory pressure (PEEP) induce positive fluid balances by decreasing renal excretion. We investigated whether elevated levels of AVP and/or PRA maintain mean arterial pressure (MAP) during PEEP under conditions where plasma volume is not expanded. Six conscious chronically tracheotomized beagle dogs, kept under standardized conditions, were investigated in four protocols. They were 1) control: 1 h spontaneous breathing with a continuous positive airway pressure of 4 cmH2O (CPAP 4) followed by 2 h CMV with PEEP, resulting in a mean airway pressure of approximately 20 cmH2O (CMV 20 referred to as "PEEP"); 2) vasopressin blockade: 1 h CPAP 4, 2 h PEEP after intravenous application of an AVP V1-receptor antagonist (AVPA); 3) converting enzyme inhibition: 1 h CPAP 4, 2 h PEEP plus angiotensin-converting enzyme inhibition (ACEI); and 4) combined blockade: 1 h CPAP 4, 2 h PEEP plus AVPA + ACEI. In AVPA + ACEI, MAP decreased during PEEP from 101 +/- 4 to 75 +/- 10 mmHg, glomerular filtration rate (GFR) decreased from 3.6 +/- 0.3 to 1.7 +/- 0.7 ml.min-1.kg body wt-1, heart rate increased from 95 +/- 10 to 122 +/- 7 beats/min, plasma aldosterone increased from 62 +/- 26 to 353 +/- 63 pg/ml, plasma epinephrine increased from 81 +/- 15 to 352 +/- 89 pg/ml (all changes P < 0.05), and plasma norepinephrine did not change. Neither MAP nor GFR changed during PEEP in control experiments in which both PRA and AVP increased, in AVPA experiments in which PRA increased, or in ACEI experiments in which AVP increased. We conclude that both AVP and angiotensin II contribute to the maintenance of MAP and GFR during PEEP. When both hormones are inhibited, no immediate compensation exists to prevent an acute fall in MAP and GFR.

Quantitative analysis of pulmonary surfactant phospholipids by high-performance liquid chromatography and light-scattering detection.

An improved high-performance liquid chromatographic method for the separation and quantitation of nine phospholipid classes is described. It is based on normal-phase chromatography with silica gel as stationary phase and a binary gradient with mixtures of chloroform, methanol and water as mobile phase. The response of the evaporative light-scattering detector was non-linear. Peak areas were proportional to the power 1.7 of the masses. Phospholipids in lung lavage samples were enriched by liquid extraction prior to HPLC analysis. The described method is a rapid and accurate procedure for the quantitative analysis of phospholipid classes in biological samples.