Plasma adenosine and cardiovascular responses to dipyridamole in fetal sheep.

The effects of dipyridamole infusion on fetal arterial plasma adenosine level, [ADO], and the systemic cardiovascular system were studied in 10 fetal sheep at 130-135 days gestational age. Dipyridamole (0.25 mg/kg) was infused into the fetuses intravenously during normoxia and hypoxia. Plasma [ADO] was measured using high-performance liquid chromatography, (HPLC), and fetal heart rate and arterial blood pressure were monitored throughout the study. These studies were performed in the absence and presence of theophylline, an adenosine receptor antagonist. During normoxia (PO2, 23.8 +/- 2.0 Torr), dipyridamole infusion increased fetal plasma [ADO] from 0.82 +/- 0.10 microM to 1.41 +/- 0.16 microM within 1 min (P < 0.01) and fetal heart rate from 157 +/- 6 bpm to 174 +/- 7 bpm (P < 0.01), but did not change mean blood pressure. Fetal plasma [ADO] and fetal heart rate returned to basal levels quickly. Treatment with theophylline did not alter the elevation of plasma [ADO] after dipyridamole infusion, but abolished responses of fetal heart rate to dipyridamole infusion. After 15 min of hypoxia with an average arterial PO2 of 15.4 +/- 1.1 Torr, fetal plasma [ADO] increased to 1.15 +/- 0.14 microM (P < 0.01). Dipyridamole infusion then further raised fetal plasma [ADO] to 1.67 +/- 0.27 microM (P < 0.01). The duration of the increase of fetal plasma [ADO] after dipyridamole infusion was no longer in hypoxia than in normoxia, however there was no significant change in the pattern of transient fetal bradycardia and persistent hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inositol (1,4,5)-trisphosphate activates a calcium channel in isolated sarcoplasmic reticulum membranes.

Sarcoplasmic reticulum membrane vesicles isolated from frog skeletal muscle display high conductance calcium channels when fused into phospholipid bilayers. The channels are selective for calcium and barium over Tris. The fractional open time was voltage-independent (-40 to +25 mV), but was steeply dependent on the free cis [Ca2+] (P0 = 0.02 at 10 microM cis Ca2+ and 0.77 at 150 microM Ca2+; estimated Hill coefficient: 1.6). Addition of ATP (1 mM; cis) further increased P0 from 0.77 to 0.94. Calcium activation was reversed by addition of EGTA to the cis compartment. Magnesium (2 mM) increased the frequency of rapid closures and 8 mM magnesium decreased the current amplitude from 3.4 to 1.2 pA at 0 mV, suggesting a reversible fast blockade. Addition of increasing concentrations of inositol (1, 4, 5)-triphosphate (cis), increased P0 from 0.10 +/- 0.01 (mean +/- SEM) in the control to 0.85 +/- 0.02 at 50 microM in an approximately sigmoidal fashion, with an apparent half-maximal activation at 15 microM inositol (1, 4, 5)-trisphosphate in the presence of 40 microM cis Ca2+. Lower concentrations of this agonist were required to produce a significant increase in P0 when 10 microM or less cis Ca2+ were used. The channel was blocked by the addition to the cis compartment of either 0.5 mM lanthanum, 0.5 microM ruthenium red, or 200 nM ryanodine, all known inhibitors of Ca2+ release from sarcoplasmic reticulum vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in plasma adenosine during simulated birth of fetal sheep

Adenosine is known to inhibit nonshivering thermogenesis in adult brown fat. These experiments were undertaken to test whether fetal adenosine, normally present in high concentrations, suppresses lipolysis in utero and then falls after birth, permitting thermogenesis to begin. To test this hypothesis, we measured fetal plasma adenosine concentration [ADO] using high-performance liquid chromatography in 11 fetal sheep at 135-140 days gestation during simulated birth. During an initial control period, fetal [ADO] averaged 1.9 +/- 0.3 microM, about four times maternal [ADO] (0.4 +/- 0.1 microM, P less than 0.001). The fetus was then cooled by circulating cold water through a plastic coil encircled about the fetal torso. One hour later, when fetal core temperature had decreased 2.3 degrees C, fetal [ADO] averaged 2.8 +/- 0.5 microM, a 50% increase (P less than 0.05), while thermogenesis remained inactive. Next the fetal lungs were ventilated with O2 to raise arterial Po2 to greater than or equal to 150 Torr. Fetal [ADO] decreased only slightly, and thermogenic responses were modest. Finally, the umbilical cord was occluded. Fetal [ADO] decreased rapidly and 60 min later averaged 1.1 +/- 0.2 microM, 40% below initial control (P less than 0.05) and 57% below the previous period (P less than 0.001). As [ADO] fell, strong thermogenic responses became apparent, as indicated by seven- to eightfold increases in plasma glycerol (P less than 0.001) and a doubling in fetal O2 consumption (P less than 0.001). These results are consistent with the hypothesis that high fetal [ADO] inhibits thermogenesis before birth but then decreases after cord occlusion, allowing thermogenesis to begin.(ABSTRACT TRUNCATED AT 250 WORDS)     

High levels of cytosolic free calcium inhibit dephosphorylation of insulin receptor and glycogen synthase.

Treatment of adipocytes with depolarizing concentrations of K+ (40 mM) for 60 min increased [Ca2+]i from 158 +/- 28 nM to 328 +/- 38 nM. This significantly reduced (up to 80% inhibition) dephosphorylation of insulin receptor (IR), EGF receptor (EGF-R) and glycogen synthase (GS). The calcium channel blocker, nitrendipine (30 microM), or Ca2+ free medium completely prevented K(+)-induced inhibition of phosphoprotein phosphatase (PPTase). This effect of high [Ca2+]i was completely reversible when the cells were returned into the non-depolarizing medium. Trypsin treatment (4 micrograms/ml) of the membrane fraction containing inhibited PPTase activity, restored dephosphorylation activity to normal suggesting that elevated [Ca2+]i may inhibit PPTase by promoting its association with the inhibitors. These observations indicate that dephosphorylation of IR and GS can be regulated by [Ca2+]i.     

The Cholinergic Regulation of Intracellular Calcium in the Human Neuroblastoma, SH-SY5Y

The regulation of intracellular calcium by cholinergic agonists was investigated in the human neuroblastoma SH-SY5Y, loaded with fura-2. The resting free Ca2+ concentration in this cell line was 199 +/- 14 nM (mean +/- SEM, n = 19). At 1 mM extracellular Ca2+, high concentrations of carbachol and acetylcholine evoked a biphasic change in intracellular Ca2+ concentration, consisting of a transient initial peak followed by a decline to a plateau that was significantly higher than the basal level. Carbachol (0.5 mM) and acetylcholine (10 microM) caused a maximal increase in the intracellular Ca2+ concentration, reaching a peak of 465 +/- 52 (mean +/- SEM, n = 12) and 422 +/- 48 nM (mean +/- SEM, n = 7), respectively, in less than 4 s. This initial calcium transient declined to a plateau of 268 +/- 36 and 240 +/- 27 nM for carbachol and acetylcholine, respectively, in approximately 40 s. The plateau persisted until the agonist was displaced by the addition of antagonist. Atropine, hexahydrosiladifenidol (HHSD), pirenzepine, and methoctramine inhibited the carbachol-evoked initial calcium transient with Ki values of 0.85 +/- 0.05, 8.3 +/- 1.6, 411 +/- 36, and 240 +/- 46 nM (mean +/- SEM, n = 3), respectively, and the acetylcholine-induced initial calcium transient with Ki values of 0.48 +/- 0.18, 13.5 +/- 8.5, 192 +/- 32, and 414 +/- 25 nM (mean +/- SEM of two experiments), respectively, results suggesting that an M3 muscarinic receptor was predominantly mediating these effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the mechanism of fibrin-specific plasminogen activation by staphylokinase

The mechanism of plasminogen activation by recombinant staphylokinase was studied both in the absence and in the presence of fibrin, in purified systems, and in human plasma. Staphylokinase, like streptokinase, forms a stoichiometric complex with plasminogen that activates plasminogen following Michaelis-Menten kinetics with Km = 7.0 microM and k2 = 1.5 s-1. In purified systems, alpha 2-antiplasmin inhibits the plasminogen-staphylokinase complex with k1(app) = 2.7 +/- 0.30 x 10(6) M-1 s-1 (mean +/- S.D., n = 12), but not the plasminogen-streptokinase complex. Addition of 6-aminohexanoic acid induces a concentration-dependent reduction of k1(app) to 2.0 +/- 0.17 x 10(4) M-1 s-1 (mean +/- S.D., n = 5) at concentrations greater than or equal to 30 mM, with a 50% reduction at a 6-aminohexanoic acid concentration of 60 microM. Staphylokinase does not bind to fibrin, and fibrin stimulates the initial rate of plasminogen activation by staphylokinase only 4-fold. Staphylokinase induces a dose-dependent lysis of a 0.12-ml 125I-fibrin-labeled human plasma clot submersed in 0.5 ml of citrated human plasma; 50% lysis in 2 h is obtained with 17 nM staphylokinase and is associated with only 5% plasma fibrinogen degradation. Corresponding values for streptokinase are 68 nM and more than 90% fibrinogen degradation. In the absence of a fibrin clot, 50% fibrinogen degradation in human plasma in 2 h requires 790 nM staphylokinase, but only 4.4 nM streptokinase. These results suggest the following mechanism for relatively fibrin-specific clot lysis with staphylokinase in a plasma milieu. In plasma in the absence of fibrin, the plasminogen-staphylokinase complex is rapidly neutralized by alpha 2-antiplasmin, thus preventing systemic plasminogen activation. In the presence of fibrin, the lysine-binding sites of the plasminogen-staphylokinase complex are occupied and inhibition by alpha 2-antiplasmin is retarded, thus allowing preferential plasminogen activation at the fibrin surface.     

Elevated interstitial adenosine concentrations do not activate the muscle reflex

The purpose of the present study was to examine the effects of adenosine perfusion of the isolated triceps surae muscle group in the decerebrate cat on interstitial adenosine concentrations as well as heart rate and blood pressure responses. In six male cats (6.0 +/- 0.21 kg), the triceps surae muscle group of both legs was perfused with an artificial blood solution containing no additives (control) and then with blood containing 20 mM or 100 microM adenosine for 10 min. An intact muscle reflex was confirmed by bolus injections of 50 mM phosphate and/or saturated KCl administered into the triceps surae muscle via the cannulated popliteal artery before and after adenosine blood perfusion. Microdialysis of the triceps surae muscle group during muscle perfusion revealed that interstitial adenosine was elevated (P < 0.05) from 0.9 +/- 0.3 microM during control blood perfusion to 2,421 +/- 547 microM during 20 mM adenosine perfusion. In addition, interstitial adenosine levels were increased (P < 0.05) from 1.1 +/- 0.3 microM during control blood perfusion to 4.1 +/- 1.2 microM during perfusion with 100 microM adenosine. Despite the large increases in interstitial adenosine levels, perfusion of the triceps surae muscle group with the two blood adenosine solutions resulted in no significant increases in heart rate or blood pressure. These data strongly suggest that elevated interstitial adenosine concentrations do not play a role in activating the muscle reflex and confirm our previous in vivo human findings (J Appl Physiol 83: 1045-1053, 1997).     

Determination of cadmium in blood, plasma, and urine by electrothermal atomic absorption spectrophotometry after isolation by anion-exchange chromatography

The determination of cadmium in whole blood, urine, or plasma by atomic absorption using electrothermal atomization is described. In preparation for atomic absorption analysis, cadmium was concentrated on an anion-exchange column, significantly lowering the limit of detection and allowing for the first time the accurate and precise determination of plasma cadmium concentrations in persons/animals with low-level cadmium exposures. Recovery of 109Cd from spiked whole blood, plasma, and urine into supernatants of nitric acid-deproteinated samples averaged 99, 100, and 95%, respectively. Anion-exchange isolation of the anionic chlorocadmium complex removed 99.8% of the major elements associated with a deproteinated whole blood sample. The recovery of 109Cd from the anion-exchange column was 92.2 +/- 0.9% (mean +/- SE, N = 35). The separation of cadmium from constituents in blood, urine, or plasma in this manner allowed comparison of unknown samples to aqueous standards with a defined acid matrix using commercially available acids. The mean intra-assay coefficient of variation (CV) was 12 +/- 3% (mean +/- SE, N = 6) for blood, plasma, and urine samples having cadmium concentrations of 0.1-0.8 microgram/liter. The interassay CV was 13% (N = 7) for a blood sample containing 0.6 microgram Cd/liter. The recovery of known amounts of cadmium added to blood, plasma, and urine in the range of 0.2 to 5.0 micrograms Cd/liter was 97 +/- 6% (mean +/- SE, N = 4).     

Colorimetric determination of chloride in biological samples by using mercuric nitrate and diphenylcarbazone

A colorimetic method is outlined for the determination of the chloride ion in biological samples (blood serum, plasma, and urine). The present method is based on the quantitative reduction of free mercuric ions by chloride ions. Chloride ions form an indissociable complex with mercuric ions. The remaining free mercuric ions form a purple complex with diphenylcarbazone with an absorption maximum at 550 nm. The reduction of color intensity at 550 nm is directly proportional to chloride concentration in the sample. The linear concentration range in the final reaction mixture was 0–100 μM with a correlation coefficient of −0.9997. The coefficient of variation for the 50 μM chloride ion in the final reaction mixture was 0.9% (n=6). The analyzed value of chloride concentration in the human control serum Accutrol™ Normal (Sigma) was 101±4 mM (mean±SD, n=12). The certified value of chloride in Accutrol Normal by Sigma is 102 mM, with a mean in the range 91–113 mM. This method was applied to the measurement of urinary chloride excretion in experimental rats. During 16-h urine collection, no food was given and rats had free access to purified water. The urinary excretion rate of chloride was 23.6±9.3 μmol/h (mean±SD, n=8) and 126.2±28.0 μmol/h (n=8) for rats fed a normal diet (2.6 g NaCl/kg diet) and a high-salt diet (82.6 g NaCl/kg diet) for 70 d prior to urine collection, respectively. This method is appropriate for low concentrations of chloride in samples or when sample volume is limiting, as in many animal studies such as metabolic urine collection from rats. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of the products that may also be suitable.     

Electrochemical determination of azidothymidine in human whole blood.

An electrochemical method based on differential pulse voltammetry is presented for the determination of AZT in whole blood of fasted subjects. A protein-free supernatant of whole blood is prepared using HClO4 precipitation followed by neutralization with phosphate buffer. The AZT is reduced at a hanging mercury drop electrode. The linear dynamic range of standards in buffer is from the detection limit of 4.1 nM to 206.5 microM (1.1 to 55,200 ng/ml). However, in spiked blood samples the linear dynamic range is from 0.029 to 0.29 microM (7.75 to 77.5 ng/ml). The whole blood assay yields a recovery of 92.30 +/- 5.92% compared to the standard solution assay. After a 30-min preparation time, each sample can be analyzed in 10 min by a manual procedure.     

Evaluation of renal function and fluid homeostasis during recovery from exercise-induced hyponatremia

Renal function including fluid and electrolyte balance was studied during recovery in eight subjects who developed symptomatic hyponatremia (HN; plasma sodium concentration less than 130 mM) during an 88-km ultramarathon footrace and compared with results for normonatremic runners [NN; n = 18, mean postrace plasma sodium concentration, 138.2 +/- 1.2 (SE) mM]. Estimated fluid intake during the race for HN was 12.5 +/- 1.6 (SE) liters over 9 h 41 min (+/- 28 min). HN excreted a net fluid excess of 2.95 +/- 0.56 (range 1.2-5.9) liters compared with a fluid deficit of 2.7 +/- 0.3% body weight in NN. The sodium deficit was 153 +/- 35 mmol in HN and 187 +/- 37 mmol in NN. Despite the fluid overload, plasma volume was decreased by 24.1 +/- 5.0% in HN compared with 8.2 +/- 2.6% in NN. Serum renin activity (5.1 +/- 2.0 ng.ml-1.h-1), aldosterone concentrations (410 +/- 34 ng/l), creatinine clearances (174.8 +/- 28.2 ml/min), and urine output (6.4 +/- 1.0 ml/min) were markedly elevated in HN during recovery. Thus the hyponatremia of exercise results from fluid retention in subjects who ingest abnormally large fluid volumes during prolonged exercise.     

Zinc and calcium ions cooperatively modulate ADAMTS13 activity

ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) multimers. The metal ion dependence of ADAMTS13 activity was examined with multimeric VWF and a fluorescent peptide substrate based on Asp(1596)-Arg(1668) of the VWF A2 domain, FRETS-VWF73. ADAMTS13 activity in citrate-anticoagulated plasma was enhanced approximately 2-fold by zinc ions, approximately 3-fold by calcium ions, and approximately 6-fold by both ions, suggesting cooperative activation. Cleavage of VWF by recombinant ADAMTS13 was activated up to approximately 200-fold by zinc ions (K(D) (app) approximately 0.5 microM), calcium ions (K(D) (app) approximately 4.8 microM), and barium ions (K(D) (app) approximately 1.7 mM). Barium ions stimulated ADAMTS13 activity in citrated plasma but not in citrate-free plasma. Therefore, the stimulation by barium ions of ADAMTS13 in citrated plasma appears to reflect the release of chelated calcium and zinc ions from complexes with citrate. At optimal zinc and calcium concentrations, ADAMTS13 cleaved VWF with a K(m) (app) of 3.7 +/- 1.4 microg/ml (approximately 15 nM for VWF subunits), which is comparable with the plasma VWF concentration of 5-10 microg/ml. ADAMTS13 could cleave approximately 14% of VWF pretreated with guanidine HCl, suggesting that this substrate is heterogeneous in susceptibility to proteolysis. ADAMTS13 cleaved FRETS-VWF73 with a K(m) (app) of 3.2 +/- 1.1 microM, consistent with an approximately 200-fold decrease in affinity compared with VWF. ADAMTS13 cleaved VWF and FRETS-VWF73 with roughly comparable catalytic efficiency of 55 microM(-1) min(-1) and 18 microM(-1) min(-1), respectively. The striking preference of ADAMTS13 for VWF suggests that substrate recognition depends on structural features or exosites on multimeric VWF that are missing from FRETS-VWF73.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Determination of procarbazine in human plasma by liquid chromatography with electrospray ionization mass spectrometry

Procarbazine is a cytotoxic chemotherapeutic agent used in the treatment of lymphomas and brain tumors. Its pharmacokinetic behavior remains poorly understood even though more than 30 years have elapsed since the drug was approved for clinical use. To characterize the pharmacokinetics of procarbazine in brain cancer patients during a phase I trial, a method for determining the drug in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) was developed and thoroughly validated. Plasma samples were prepared for analysis by precipitating proteins with trichloroacetic acid and washing the protein-free supernatant with methyl tert-butyl ether to remove excess acid. The solution was separated on a Luna C-18 analytical column using methanol-25 mM ammonium acetate buffer, pH 5.1 (22:78, v/v) as the mobile phase at 1.0 ml/min. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H](+) ions at m/z 222.2 for procarbazine and at m/z 192.1 for the internal standard (3-dimethylamino-2-methylpropiophenone). Procarbazine and the internal standard eluted as sharp, symmetrical peaks with retention times (mean+/-S.D.) of 6.3+/-0.1 and 9.9+/-0.3 min, respectively. Calibration curves of procarbazine hydrochloride in human plasma at concentrations ranging from 0.5 to 50 ng/ml exhibited excellent linearity. The mean absolute recovery of the drug from plasma was 102.9+/-1.0%. Using a sample volume of 150 microl, procarbazine was determined at the 0.5 ng/ml (1.9 nM) lower limit of quantitation with a mean accuracy of 105.2% and an interday precision of 3.60% R.S.D. on 11 different days over 5 weeks. During this same time interval, the between-day accuracy for determining quality control solutions of the drug in plasma at concentrations of 2.0, 15 and 40 ng/ml ranged from 97.5 to 98.2% (mean+/-S.D., 97.9+/-0.4%) and the precision was 3.8-6.2% (mean+/-S.D., 5.1+/-1.2%). Stability characteristics of the drug were thoroughly evaluated to establish appropriate conditions to process, store and prepare clinical specimens for chromatographic analysis without inducing significant chemical degradation. The sensitivity achieved with this assay permitted the plasma concentration-time profile of the parent drug to be accurately defined following oral administration of standard doses to brain cancer patients.     

Regional and whole-body markers of nitric oxide production following hyperemic stimuli

The measurement of nitric oxide (NO) bioavailability is of great clinical interest in the assessment of vascular health. However, NO is rapidly oxidized to form nitrite and nitrate and thus its direct detection in biological systems is difficult. Venous plasma nitrite (nM concentrations) has been shown to be a marker of forearm NO production following pharmacological stimulation of the endothelium utilizing acetylcholine (Ach). In the present study, we demonstrate, within 15 apparently healthy subjects (34.1 +/- 7.3 years), that reactive hyperemia of the forearm, a physiological endothelial stimulus, results in a 52.5% increase in mean plasma nitrite concentrations (415 +/- 64.0 to 634 +/- 57.1 nM, P = 0.015). However, plasma nitrite is readily oxidized to nitrate within plasma, and thus its utility as a marker of NO production within the clinical setting may be limited. Alternatively, NOx (predominantly nitrate) is relatively stable in plasma (microM concentrations), but is produced by sources other than the vasculature and has been shown to be unsuitable as a measure of localized NO production. We reasoned that the principle source of NOx generation during exercise is NO production and thus have examined the change in NOx following treadmill exercise stress. In this study, 12 apparently healthy subjects showed an increase (from baseline) in venous NOx at peak effort and during recovery (12 +/- 9.1 and 17 +/- 15.3 microM respectively, P < 0.05). In contrast, 10 subjects with cardiovascular disease showed no significant increases. Additionally, a correlation between VO(2peak) and the change in circulating NOx (r(2) = 0.4585, P < or = 0.01) indicated the subjects who could exercise hardest also produced the most NO.     

Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin, alpha 1-acid glycoprotein and albumin

The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37 degrees C. At 1.7 nM L-[3H]-NE binding was approximately 25%. The binding was rapid and was not influenced by the incubation time. [3H]-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37 degrees C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles.mg-1 protein, and low-affinity sites with a Kd of 2.7 microM and a Bmax of 3.3 pmoles.mg-1 protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. NE binding to pure human plasma proteins was also studied using ultrafiltration. Scatchard analyses revealed a single class of very high-affinity binding sites for prealbumin (Kd 4.9 nM), a single class of binding sites for alpha 1-acid glycoprotein (Kd 54 microM) and two classes of binding sites for albumin with high (Kd 1.7 microM) and low (Kd 0.8 mM) affinities respectively. The main results obtained in this study - a) reversibility of NE binding, b) stability of free and bound NE in plasma, c) involvement of the prealbumin as a specific binding protein - point out to a specific transport for NE in human blood plasma.     

Gas chromatography-mass spectrometry of cis-9,10-epoxyoctadecanoic acid (cis-EODA). II. Quantitative determination of cis-EODA in human plasma

Cytochrome P450 dependent epoxidation and non-enzymic lipid peroxidation of oleic acid (cis-9-octadecenoic acid) result in the formation of cis-9,10-epoxyoctadecanoic acid (cis-EODA). This oleic acid oxide has been identified indirectly in blood and urine of humans. Reliable concentrations of circulating cis-EODA have not been reported thus far. In the present article, we report on the first GC-tandem MS method for the accurate quantitative determination in human plasma of authentic cis-EODA as its pentafluorobenzyl (PFB) ester. cis-[9,10-2H2]-EODA (cis-d2-EODA) was synthesized by chemical epoxidation of commercially available cis-[9,10-2H2]-9-octadecenoic acid and used as an internal standard for quantification. Endogenous cis-EODA and externally added cis-[9,10-2H2]-EODA were isolated from acidified plasma samples (1 ml; pH 4.5) by solvent or solid-phase extraction, converted into their PFB esters, isolated by HPLC and quantified by selected reaction monitoring. The parent ions [M-PFB]- at mass-to-charge ratio (m/z) 297 for cis-EODA and m/z 299 for (cis-d2-EODA) were subjected to collisionally-activated dissociation and the corresponding characteristic product ions at m/z 171 and 172 were monitored. In plasma of nine healthy humans (5 females, 4 males), cis-EODA was found to be present at 47.6+/-7.4 nM (mean+/-S.D.). Plasma cis-EODA levels were statistically insignificantly different (P=0.10403, t-test) in females (51.1+/-3.4 nM) and males (43.1+/-2.2 nM). cis-EODA was identified as a considerable contamination in laboratory plastic ware and found to contribute to endogenous cis-EODA by approximately 2 nM. The present GC-tandem MS method should be useful in investigating the physiological role(s) of cis-EODA in humans.     

Interstitial pH, K(+), lactate, and phosphate determined with MSNA during exercise in humans

The purpose of the present study was to use the microdialysis technique to simultaneously measure the interstitial concentrations of several putative stimulators of the exercise pressor reflex during 5 min of intermittent static quadriceps exercise in humans (n = 7). Exercise resulted in approximately a threefold (P < 0.05) increase in muscle sympathetic nerve activity (MSNA) and 13 +/- 3 beats/min (P < 0.05) and 20 +/- 2 mmHg (P < 0.05) increases in heart rate and blood pressure, respectively. During recovery, all reflex responses quickly returned to baseline. Interstitial lactate levels were increased (P < 0.05) from rest (1.1 +/- 0.1 mM) to exercise (1. 6 +/- 0.2 mM) and were further increased (P < 0.05) during recovery (2.0 +/- 0.2 mM). Dialysate phosphate concentrations were 0.55 +/- 0. 04, 0.71 +/- 0.05, and 0.48 +/- 0.03 mM during rest, exercise, and recovery, respectively, and were significantly elevated during exercise. At the onset of exercise, dialysate K(+) levels rose rapidly above resting values (4.2 +/- 0.1 meq/l) and continued to increase during the exercise bout. After 5 min of contractions, dialysate K(+) levels had peaked with an increase (P < 0.05) of 0.6 +/- 0.1 meq/l and subsequently decreased during recovery, not being different from rest after 3 min. In contrast, H(+) concentrations rapidly decreased (P < 0.05) from resting levels (69.4 +/- 3.7 nM) during quadriceps exercise and continued to decrease with a mean decline (P < 0.05) of 16.7 +/- 3.8 nM being achieved after 5 min. During recovery, H(+) concentrations rapidly increased and were not significantly different from baseline after 1 min. This study represents the first time that skeletal muscle interstitial pH, K(+), lactate, and phosphate have been measured in conjunction with MSNA, heart rate, and blood pressure during intermittent static quadriceps exercise in humans. These data suggest that interstitial K(+) and phosphate, but not lactate and H(+), may contribute to the stimulation of the exercise pressor reflex.     

Involvement of a guanine-nucleotide-binding protein-mediated mechanism in the enhancement of arachidonic acid liberation by phorbol 12-myristate 13-acetate and Ca2+ in saponin-permeabilized platelets

A mechanism by which protein kinase C potentiates arachidonic acid (AA) liberation in rabbit platelets was examined using [3H]AA-labeled, saponin (7 micrograms/ml)-permeabilized rabbit platelets. Pretreatment of the [3H]AA-labeled platelets with 4 beta-phorbol 12-myristate 13-acetate (PMA, 10-40 nM) or 1,2-dioctanoylglycerol (DOG, 20 microM) enhanced [3H]AA liberation induced by an addition of Ca2+ (1 mM) after cell permeabilization, whereas 4 alpha-phorbol 12,13-didecanoate (80 nM) did not exert such an effect. The potentiating effects of PMA and DOG were inhibited by staurosporine (200 nM). PMA (40 nM) also potentiated [3H]AA liberation induced by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S, 100 microM), 5'-guanylyl imidodiphosphate (200 microM) or NaF (20 mM) plus AlCl3 (10 microM) in the presence of Ca2+ (100 microM). The enhancement by PMA of the GTP gamma S-induced AA liberation was also inhibited by staurosporine (200 nM). Furthermore, guanosine 5'-[beta-thio]diphosphate (GDP beta S, 0.5-2 mM) suppressed the PMA (40 nM)- and DOG (20 microM)-enhanced, Ca2+ (1 mM)-dependent [3H]AA liberation. This inhibitory effect of GDP beta S was reversed by a further addition of GTP gamma S (200 microM). However, pertussis toxin (0.2-1 micrograms/ml) had no effect on the PMA-enhanced [3H]AA liberation. These results indicate a possibility that protein kinase C may potentiate AA liberation through a guanine-nucleotide-binding protein-mediated mechanism in saponin-permeabilized rabbit platelets.     

Increased free calcium in endothelial cells under stimulation with adenine nucleotides

The release of vasodilating substances from the vascular endothelium has been postulated to depend on a rise in the level of intracellular free calcium (Cai++). We measured Cai++ in intact monolayers of calf endothelial cells, grown in culture, that were loaded with the fluorescent calcium indicator quin 2. Fluorescence (excitation wavelength 340 nm, emission wavelength 492 nm) was calibrated by raising Cai++ to a maximum with the calcium ionophore ionomycin (0.1 microM) and by lowering it to a minimum with ionomycin plus manganese (0.4 mM), which quenches quin 2 fluorescence completely. Loss of fluorescent dye from the cells was calculated from fluorescence at the isosbestic excitation wavelength (365 nm). Resting Cai++ was 71 +/- 3 (SEM) nM. ATP (adenosine-5'-triphosphate) raised Cai++ dose-dependently and reversibly to 458 +/- 60 nM at a concentration of 10 microM, and at 0.1 mM to values close to those that occurred under ionomycin. ADP (A-5'-PP) and AMP (A-5'-P) had smaller effects with a maximal Cai++ of 287 +/- 72 nM at 30 microM ADP and 176 +/- 17 nM at 0.1 mM AMP. At these concentrations, ADP and AMP attenuated significantly the increase of Cai++ under ATP (10 microM). Adenosine (0.1 or 0.3 mM) and acetylcholine (0.1 to 30 microM) enhanced Cai++ inconsistently, by a maximum of 50 nM. These effects were abolished by theophylline and atropine, respectively. In the absence of extracellular calcium, ATP still raised Cai++, although endothelial responsiveness declined after repetitive stimulations. We conclude that activation of purinergic receptors increases intracellular free calcium in endothelial cells, and that this increase is probably an essential trigger for synthesis of prostacyclin and the labile endothelium-derived relaxant factor.