Intracellular pH recovery from lactic acidosis of single skeletal muscle fibers

Intracellular pH (pHi), measured with H+-selective microelectrodes, in quiescent frog sartorius muscle fibres was 7.29 +/- 0.09 (n = 13). Frog muscle fibres were superfused with a modified Ringer solution containing 30 mM HEPES buffer, at extracellular pH (pHo) 7.35. Intracellular pH decreased to 6.45 +/- 0.14 (n = 13) following replacement of 30 mM NaCl with sodium lactate (30 mM MES, pHo 6.20). Intracellular pH recovery, upon removal of external lactic acid, depended on the buffer concentration of the modified Ringer solution. The measured values of the pHi recovery rates was 0.06 +/- 0.01 delta pHi/min (n = 5) in 3 mM HEPES and was 0.18 +/- 0.06 delta pHi/min (n = 13) in 30 mM HEPES, pHo 7.35. The Na+-H+ exchange inhibitor amiloride (2 mM) slightly reduced pHi recovery rate. The results indicate that the net proton efflux from lactic acidotic frog skeletal muscle is mainly by lactic acid efflux and is limited by the transmembrane pH gradient which, in turn, depends on the extracellular buffer capacity in the diffusion limited space around the muscle fibres.     

Adenosine transport in bovine chromaffin cells in culture

Bovine adrenal chromaffin cells in culture have a high capacity and affinity for adenosine uptake with Vmax = 14 +/- 2.4 pmol/10(6) cells/min (133 pmol/mg of protein/min) and Km = 1 +/- 0.2 microM. Transport studies, at short time periods, in recently isolated chromaffin cells have Vmax = 15 pmol/10(6) cells/min and Km = 1.1 microM in ATP-depleted cells. Endogenous levels of the various purine nucleosides and bases were determined by high pressure liquid chromatography, with adenosine (3 +/- 1 nmol/10(6) cells), inosine (5.3 +/- 1.2 nmol/10(6) cells), and hypoxanthine (2.1 +/- 0.8 nmol/10(6) cells) being the purine metabolites found in the highest concentration. Taking into account the intracellular water, endogenous levels of 2.1, 3.8, and 1.5 mM, respectively, were obtained. Radioactively labeled adenosine inside the cell underwent enzymatic transformations, producing inosine, hypoxanthine, xanthine, and nucleotides, with their appearance and distribution being a function of the incubation time. When nicotine was used as a secretagogue, the adenosine transformed into the nucleotide pool was released, reaching 18 +/- 8% of the total adenosine found in the nucleotides. Dipyridamole, extensively used clinically, was a strong inhibitor for the adenosine uptake into these cells, with Ki = 5 +/- 0.5 nM and noncompetitive kinetically.     

Bombesin microinjected into the dorsal vagal complex inhibits vagally stimulated gastric acid secretion in the rat

Medullary sites of action for bombesin-induced inhibition of gastric acid secretion were investigated in urethane-anesthetized rats with gastric fistula. Unilateral microinjection of bombesin or vehicle into the dorsal vagal complex was performed using a glass micropipet and pressure ejection of 100 nl volume; gastric acid output was measured every 10 min by flushing the stomach. Microinjection of vehicle into the dorsal vagal complex did not alter gastric acid secretion (1.9 +/- mumol/10) from preinjection levels (2.9 +/- 0.8 mumol/10 min). Microinjection of the stable thyrotropin-releasing hormone (TRH) analog, RX 77368, at a 77 pmol dose into the dorsal vagal complex stimulated gastric acid secretion for 100 min with a peak response at 40 min (24.1 +/- 3.2 mumol/10 min). Concomitant microinjection of RX 77368 (77 pmol) with bombesin (0.6-6.2 pmol) into the dorsal vagal complex dose dependently inhibited by 35-86% the gastric acid response to the TRH analog. Bombesin (6.2 pmol) microinjected into the dorsal vagal complex inhibited by 17% pentagastrin infusion-induced stimulation of gastric acid secretion (13.2 +/- 0.8 mumol/10 min) whereas intracisternal injection induced a 69% inhibition of the pentagastrin response. These results demonstrate that the dorsal motor complex is a sensitive site of action for bombesin-induced inhibition of vagally stimulated gastric secretion. However, other medullary sites must be involved in mediating the inhibitory effect of intracisternal bombesin on pentagastrin-stimulated gastric acid secretion.     

Liberation of cyclic AMP and catecholamine from the heart during left stellate stimulation in the anesthetized dog

In open-chest pentothal-chloralose anesthetized dogs, plasma catecholamine and cyclic AMP levels were evaluated in the aortic and coronary sinus blood, during stimulations of the left ansa subclavia (1, 2, and 4 Hz). Basal aortic and coronary sinus catecholamine levels were respectively 0.373 +/- 0.090 and 0.259 +/- 0.048 ng/mL and cyclic AMP levels averaged 21.4 +/- 1.4 and 20.9 +/- 1.6 pmol/mL. Statistically significant increases in cyclic AMP levels were induced by sympathetic stimulations at 1 Hz (2.0 +/- 0.6 pmol/mL, 2 Hz (2.5 +/- 1.2 pmol/mL) and 4 Hz (6.5 +/- 1.5 pmol/mL), concomitantly with elevations of coronary sinus catecholamine levels. Sotalol (5 mg/kg) abolished the increases in coronary sinus cyclic AMP levels induced in coronary sinus cyclic AMP output averaged 282 +/- 30 pmol/min (1 Hz), 662 +/- 160 pmol/min (2 Hz), and 1679 +/- 242 pmol/min (4 Hz). Sympathetically induced cyclic AMP output (4Hz) was blunted by sotalol (-81 +/- 14 pmol/min). Aortic cyclic AMP levels were not significantly influenced by stellate stimulation. Intense correlations were found between increased in coronary sinus plasma catecholamines and cyclic AMP concentration levels (r = 0.81, slope - 1.45, ordinate = -1.42, n = 15) as well as between delta cyclic AMP output versus delta catecholamine output values in the coronary sinus (r = 0.93. slope output levels. Intracoronary infusion of phenylephrine (10 micrograms/min) or nitroprusside (200 micrograms/min) had no influence on cyclic AMP plasma levels whereas aortic and coronary sinus levels were respectively increased 5.5 +/- 1.9 and 7.3 +/- 1.4 pmol/mL during the administration of isoproterenol (5 micrograms/min). These data suggested that plasma cyclic AMP constitutes a sensitive index of cardiac beta-adrenergic activity elicited by the release of endogenous catecholamine during stellate stimulations.     

Effect of human milk folate binding protein on folate intestinal transport

The present study examined the effect of human milk folate binding protein (FBP) on the intestinal transport of 5-methyltetrahydrofolate (5-CH3H4PteGlu). This was performed by examining the transport of radiolabeled 5-CH3H4PteGlu bound to FBP using everted sacs of rat intestine. In the jejunum at pH 6, transport of 27 nM bound 5-CH3H4PteGlu was linear with time for 30 min of incubation. Transport of 13 nM bound 5-CH3H4PteGlu was higher in the jejunum than in the ileum at both pH 6 (2.1 +/- 0.3 and 0.36 +/- 0.03 pmol/g wet wt/25 min, respectively) and pH 8 (1.9 +/- 0.3 and 0.32 +/- 0.02 pmol/g wet wt/25 min, respectively). In the jejunum, transport of 13 nM bound 5-CH3H4PteGlu at pH 6 was less than transport of an equimolar concentration of free 5-CH3H4PteGlu (2.1 +/- 0.3 and 5.1 +/- 0.5 pmol/g wet wt/25 min, respectively) but was similar at pH 8 (1.9 +/- 0.3 and 2.47 +/- 0.3 pmol/g wet wt/25 min, respectively). In the ileum transport of bound and free 5-CH3H4PteGlu was similar at pH 6 (0.36 +/- 0.03) and 0.41 +/- 0.06 pmol/g wet wt/25 min, respectively) and pH 8 (0.32 +/- 0.02 and 0.43 +/- 0.1 pmol/g wet wt/25 min, respectively). The transport process of bound 5-CH3H4PteGlu in the jejunum was energy, temperature, and Na+ dependent, but not pH dependent, and was competitively inhibited by sulfasalazine. Ninety-two percent of the transport substrate that appeared in the serosal compartment following incubation with bound 5-CH3H4PteGlu was found to be free (unbound) 5-CH3H4PteGlu. These results show that human milk FBP decreases the rate of transport of 5-CH3H4PteGlu in the jejunum and suggest that FBP-bound 5-CH3H4PteGlu may utilize the same transport system as free 5-CH3H4PteGlu. The results also suggest a role for human milk FBP in regulating the nutritional bioavailability of folate.     

Chemiluminescent simultaneous determination of phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide in the liver and brain of the rat.

The quantification of phospholipid hydroperoxides in biological tissues is important in order to know the degree of peroxidative damage of membrane lipids. For this purpose, optimal conditions for the chemiluminescent simultaneous assay of phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH) in rat liver and brain were determined. A chemiluminescence detection-high performance liquid chromatography (CL-HPLC) method that incorporates cytochrome c and luminol as a post-column hydroperoxide-specific luminescent reagent was used (Miyazawa et al. 1987. Anal. Lett. 20: 915-925; Miyazawa. 1989. Free Radical Biol. Med. 7: 209-217). An n-propylamine-bound silica column with hexane-2-propanol-methanol-water 5:7:2:1 (v/v/v/v) (flow rate 1.0 ml/min) as eluant was used to determine both PCOOH and PEOOH, which were separated from each other and from other lipids and lipid-soluble antioxidants. High reproducibility and sensitivity as low as 10 pmol hydroperoxide-O2 were observed with a mixture of 10 micrograms/ml cytochrome c and 2 micrograms/ml luminol in 50 mM borate buffer (pH 10.0, flow rate 1.1 ml/min) as luminescent reagent and a post-column mixing joint temperature of 40 degrees C. Using the established analytical conditions, it was confirmed that both PCOOH (1324 +/- 122 pmol/g liver, 114 +/- 18 pmol/g brain, mean +/- SD) and PEOOH (728 +/- 89 pmol/g liver, 349 +/- 60 pmol/g brain, mean +/- SD) are present in the liver and brain of Sprague-Dawley rats bred on a slightly modified AIN-76A semisynthetic diet for 3 months. The phospholipid hydroperoxide content in the rat liver was shown to be affected by dietary oils, but not significantly affected in the brain.     

Respiratory and cardiac responses to exercise-simulating peripheral perfusion in endurance trained and untrained rats. I. Reflex responses and changes in perfusion outflow

Ventilatory and circulatory drives elicited by exercise-simulating perfusion of the circulatory isolated hindleg were examined in 10 trained (TR) and untrained (UTR) rats. TR were submitted to endurance training on a motordriven treadmill (30.min-1 at a grade of 10%, 5 days a week for 30 min). Exercise was simulated by perfusion with modified tyrode solutions: I.) hypoxic, enriched with lactic acid (15 mmol.l-1), II.) normoxic, enriched with lactic acid. III.) hypoxic without lactic acid. Perfusion was performed in anaesthetized animals through cannulae in the femoral artery and vein; the hindled was connected to the rest of the body only by nerve and bone. 10 min of control perfusion (normoxic tyrode solution) was followed by a 20 min test period and another 10 min control perfusion. Apart from heart rate (HR), respiratory rate (RR) and several outflow parameters were measured ([K+], [Na+], [lactate], pH, PO2, PCO2). During control period HR was slightly higher in UTR than in TR (375.5 +/- 3.9 (SE) vs. 364.1 +/- 5.5 beats/min-1, p less than 0.6 n.s.), and RR in UTR was significantly higher than those in TR (61.5 +/- 0.4 bpm vs. 55.5 +/- 3.9 breaths.min-1, p less than 0.001). During the test periods both HR and RR in UTR increased significantly while in TR they did not (e.g. in series I mean HR and RR in UTR increased by 8.9 +/- 1.2 beats.min-1 and 1.4 +/- 0.1 breaths.min-1 respectively, whereas in TR the changes were - 2.9 +/- 1.5 beats/min-1 and -0.8 +/- 0.2 breaths.min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutathione and adenosine triphosphate content of in vivo and in vitro matured porcine oocytes

Glutathione (GSH) content in mature porcine oocytes is correlated with subsequent fertilization and developmental success. Adenosine triphosphate (ATP) is an important energy source for maintaining cellular activities and protein synthesis. The objective of this study was to compare GSH and ATP concentrations of in vivo and in vitro matured porcine oocytes. Ovulated, in vivo matured oocytes were frozen at -80 degrees C in groups of 10-20 (GSH) or 5-10 (ATP). In vitro oocytes were matured in either tissue culture medium-199 (TCM199) supplemented with polyvinyl alcohol (PVA) or hyaluronic acid (MAP5), or North Carolina State University-23 (NCSU23) supplemented with porcine follicular fluid (pFF) and frozen as described, or fertilized and cultured. GSH content was determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. ATP content was determined by using the Bioluminescent Somatic Cell Assay Kit. Oocytes matured in vitro in defined TCM199 with PVA or hyaluronic acid, or NCSU23 with pFF had significantly lower concentrations (P < 0.05) of GSH (n = 207, 9.82 +/- 0.71 pmol/oocyte; n = 104, 9.73 +/- 0.81 pmol/oocyte; n = 108, 7.89 +/- 0.66 pmol/oocyte, respectively) compared to in vivo matured oocytes (n = 217, 36.26 +/- 11.00 pmol/oocyte). Concentrations of ATP were not different between treatments (in vivo, n = 70, 0.97 +/- 0.07 pmol/oocyte; TCM-PVA, n = 117, 0.81 +/- 0.13 pmol/oocyte; TCM-MAP, n = 107, 1.02 +/- 0.18 pmol/oocyte; NCSU-pFF, n = 134, 0.71 +/- 0.08 pmol/oocyte). Intracellular ATP content does not appear to be related to developmental potential in porcine oocytes. Low intracellular GSH may be responsible, in part, for lower developmental competence observed in in vitro matured porcine oocytes.     

Human muscle fatigue after glycogen depletion: a 31P magnetic resonance study.

To differentiate the effects of high energy phosphates, pH, and [H2PO4-] on skeletal muscle fatigue, intracellular acidosis during handgrip exercise was attenuated by prolonged submaximal exercise. Healthy human subjects (n = 6) performed 5-min bouts of maximal rhythmic handgrip (RHG) before (CONTROL) and after prolonged (60-min) handgrip exercise (ATTEN-EX) designed to attenuate lactic acidosis in active muscle by partially depleting muscle glycogen. Concentrations of free intracellular phosphocreatine ([PCr]), adenosine triphosphate ([ATP]), and orthophosphate ([P(i)]) and pH were measured by 31P nuclear magnetic resonance spectroscopy and used to calculate adenosine diphosphate [ADP], [H2PO4-], and [HPO4(2-)]. Handgrip force output was measured with a dynamometer, and fatigue was determined by loss of maximal contractile force. After ATTEN-EX, the normal exercise-induced muscle acidosis was reduced. At peak CONTROL RHG, pH fell to 6.3 +/- 0.1 (SE) and muscle fatigue was correlated with [PCr] (r = 0.83), [P(i)] (r = 0.82), and [H2PO4-] (r = 0.81); [ADP] was 22.0 +/- 5.7 mumol/kg. At peak RHG after ATTEN-EX, pH was 6.9 +/- 0.1 and [ADP] was 116.1 +/- 18.2 mumol/kg, although [PCr] and [P(i)] were not different from CONTROL RHG (P greater than 0.05). After ATTEN-EX, fatigue correlated most closely with [ADP] (r = 0.84). The data indicate that skeletal muscle fatigue 1) is multifactorial, 2) can occur without decreased pH or increased [H2PO4-], and 3) is correlated with [ADP] after exercise-induced glycogen depletion.     

Ontogeny of immunoreactive CCK and VIP in pig brain and gut

The concentrations and hormonal forms of CCK and VIP have been determined in extracts of the brain and duodenum of the developing and adult pig. In methanol extracts of the brain cortex, the single hormone form, CCK8, increased from 130 +/- 20 (Mean +/- SEM) pmol/g at birth to an adult level of 300 +/- 50 pmol/g. In acid extracts of brain, the predominant immunoreactive form had N-terminal immunoreactivity and increased from 240 +/- 20 pmol/g at birth to an adult level 490 +/- 30 pmol/g; the C-terminal immunoreactivity was about 10-fold lower. The concentrations and hormonal forms of immunoreactive CCK in duodenal extracts did not appear to be age-related. C-terminal immunoreactivity in methanol extracts averaged 140 +/- 20 pmol/g and in acid extracts 240 +/- 60 pmol/g. The concentration of N-terminal immunoreactivity in acid extracts averaged 490 +/- 70 pmol/g. The VIP concentrations in acid extracts of the brain cortex was 13.5 +/- 2 pmol/g at birth and rose gradually to 30 +/- 9 pmol/g in the adult; in duodenal extracts it was 240 +/- 18 pmol/g at birth and 195 +/- 38 pmol/g in the adult. These results are in marked contrast with the ontogeny of these hormones in the rat in which brain concentrations of CCK and VIP in the neonate are less than 10% of adult levels and in which there are age-related changes in the content of these hormones in the duodenum as well.     

Neurotensin elevates hepatic bile acid secretion in chickens by a mechanism requiring an intact enterohepatic circulation

Neurotensin (NT), given intravenously at 10-50 pmol/kg per min to anesthetized female chickens equipped with a bile duct fistula, dose-dependently elevated hepatic bile flow and bile acid output but only when the enterohepatic circulation was maintained by returning the bile to the intestinal lumen. Infusion of NT at 10 and 50 pmol/kg per min increased the average hepatic bile acid output over a 30-min period to 138 +/- 11 and 188 +/- 13% of control, respectively. During infusion of NT, plasma levels of immunoreactive NT (iNT) increased in time from the basal level (14 +/- 1.3 pM) to reach steady state at 30 min. There was a near linear relationship between the dose of NT infused and the increment in plasma iNT. In addition, infusion of NT at 40 pmol/kg min gave a plasma level of iNT (approximately/= 88 pM) which was within the range of those observed during duodenal perfusion with lipid (54-300 pM) and near to that measured in hepatic portal blood from fed animals (52 +/- 5 pM). Perfusion of duodenum with lipid released endogenous NT and increased the rate of hepatic bile flow. When NT antagonist SR48692 was given, bile flow rate decreased to the basal level. These results suggest that intestinal NT, released by lipid, may participate in the regulation of hepatic bile acid output by a mechanism requiring an intact enterohepatic circulation.     

Properties of adenylate cyclase from plasma membranes of the rabbit myometrium in different functional states

Adenylate cyclase of plasma membranes from the nonpregnant rabbit myometrium shows the maximum activity at pH 7.7-7.9, is characterized by apparent Km for ATP amounting to 0.38 +/- 0.09 mM, V--125 +/- 34.4 pmol min/mg protein, is activated at most by 15-20 mM Mg2+ and F-. Adenylate cyclase of plasma membranes from the pregnant rabbit myometrium is characterized by apparent Km for ATP amounting to 0.74 +/- 0.06 mM, V--77.3 +/- 6.0 pmol/min/mg protein, is activated at most by 5-10 mM Mg2+ and 10-15 mM F-; the pH optimum for the adenylate cyclase in this functional state is 7.3. Adenylate cyclase in the state of labour is characterized by apparent Km for ATP amounting to 0.46 +/- 0.11 mM, V--34.8 +/- 4.6 pmol/min/mg protein, is activated at most by 10-15 mM Mg2+ and F-, shows the same activity at pH 7.3-8.5. Adenylate cyclase of myometrium in three investigated states is activated by 2 mM EGTA; 10(-7) M Ca2+ decreases activation caused by EGTA; higher concentrations of Ca2+ decrease the basal activity of the enzyme.     

A 31P-NMR study on the recovery of intracellular pH in LLC-PK1/Cl4 cells from intracellular alkalinization

The regulation of intracellular pH (pHi) in a renal epithelial cell line, LLC-PK1/Cl4, during re-acidification from an alkaline load was studied by 31P-NMR. Intracellular alkalinization was induced by 10 mM ammonium glucuronate or by preloading with and subsequent removal of 20% CO2; the rate of re-acidification was found to be 0.047 pH units/min and 0.053 pH units/min, respectively. This rate of re-acidification was inhibited by 83% if Cl- was removed from the extracellular medium. A similar inhibition was found in the presence of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) (76% inhibition) and 1 mM bumetanide (81% inhibition). No change in recovery was found after removing sodium from the extracellular medium, indicating that LLC-PK1/Cl4 cells recover from an intracellular alkaline load by a Cl-/HCO3- exchanger, which is SITS- and bumetanide-sensitive and has no requirement for sodium. In addition, the steady-state pHi in Cl4 cells was monitored by 31P-NMR. Removal of Cl- from the extracellular medium introduced an increase in pHi by 0.33 pH units, whereas 1 mM SITS and 1 mM bumetanide caused an increase in pHi by 0.14 or 0.13 pH units. In the presence of 1 mM amiloride, an inhibitor of the Na+/H+ exchanger, the steady-state pHi did not change significantly. These results indicate that at pHo 7.4 the steady-state intracellular pH of LLC-PK1/Cl4 cells strongly depends on the activity of the Cl-/HCO3- exchanger. Under the same conditions the activity of the Na+/H+ exchanger seems to be negligible.     

Topography of cat medullary ventral surface hypoxic acidification.

The topographic relationship between previously identified medullary ventral surface respiratory chemosensitive regions and brain surface extracellular fluid (ECF) acid production during acute hypoxia was explored in anesthetized, paralyzed, and artificially ventilated cats. Glass pH electrodes (0.8-mm diam, sheathed in stainless steel tubing) were mounted in mechanical contact with surfaces of medullary surface or adjacent pyramids, pons, spinal cord, or parietal cortex. Isocapnic hypoxia of 5 min [at arterial O2 saturation (SaO2) = 48 +/- 10%] reduced pH over rostral (Mitchell) and caudal (Loeschcke) areas by 0.12 +/- 0.09 and 0.07 +/- 0.04, respectively (n = 10, P < 0.05). Change in pH (delta pH) was proportional to desaturation with slopes 100 delta pH/delta SaO2 of 0.45 (rostral) and 0.20 (caudal) (R = 0.91 and 0.88, respectively). pH drop usually began within 3 min of hypoxia, became stable between 5 and 15 min, began to rise within 2 min of reoxygenation, and returned to control within 10 min. During equally hypoxic tests, intermediate area (Schl?fke), pons, and spinal cord surfaces showed no significant acid shift. Parietal cortex ECF pH dropped more slowly but steadily by 0.079 +/- 0.034 during 20 min at SaO2 = 50% after a small but significant initial alkaline shift, and acidification of cortical surface continued for > 5 min after reoxygenation. We conclude that medullary ventral chemosensitive regions produce more lactic acid during hypoxia than neighboring brain surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Acute Ethanol on Release of Endogenous Adenosine from Rat Cerebellar Synaptosomes

The effects of pharmacologically relevant concentrations of ethanol on the release of endogenous adenosine from rat cerebellar synaptosomes were investigated. Release was conducted for 5, 10, 30, or 60 s after which time the incubation medium (containing the released adenosine) was rapidly separated from the synaptosomal membranes by vacuum filtration. The adenosine content of the filtrate was measured by HPLC-fluorescence detection. Both basal and KCl-stimulated adenosine release consisted of an initial rapid phase, for the first 10 s, that was followed by a relatively slower phase. Basal endogenous adenosine release was estimated as 199 +/- 14 pmol/mg protein/5 s. Potassium (chloride) increased adenosine release from the basal level to 433 +/- 83 pmol/mg protein/5 s. Ethanol caused a dose-dependent increase of adenosine release. The interaction between dilazep and ethanol indicates that ethanol-stimulated release does not involve the dilazep-sensitive transport system. The results support previous findings that indicate that cerebellar adenosine is involved in the mediation of ethanol-induced motor disturbances in the rat.     

Measurement and analysis of intracellular ATP levels in metabolically engineered Zymomonas mobilis fermenting glucose and xylose mixtures

Intracellular adenosine-5'-triphosphate (ATP) levels were measured in a metabolically engineered Zymomonas mobilis over the course of batch fermentations of glucose and xylose mixtures. Fermentations were conducted over a range of pH (5-6) in the presence of varying initial amounts of acetic acid (0-8 g/L) using a 10% (w/v) total sugar concentration (glucose only, xylose only, or 5% glucose/5% xylose mixture). Over the design space investigated, ethanol process yields varied between 56.6% and 92.3% +/- 1.3% of theoretical, depending upon the test conditions. The large variation in process yields reflects the strong effect pH plays in modulating the inhibitory effect of acetic acid on fermentation performance. A corresponding effect was observed on maximum cellular specific growth rates, with the rates varying between a low of 0.15 h(-1) observed at pH 5 in the presence of 8 g/L acetic acid to a high of 0.32 +/- 0.02 h(-1) obtained at pH 5 or 6 when no acetic acid was initially present. While substantial differences were observed in intracellular specific ATP concentration profiles depending upon fermentation conditions, maximum intracellular ATP accumulation levels varied within a relatively narrow range (1.5-3.8 mg ATP/g dry cell mass). Xylose fermentations produced and accumulated ATP at much slower rates than mixed sugar fermentations (5% glucose, 5% xylose), and the ATP production and accumulation rates in the mixed sugar fermentations were slightly slower than in glucose fermentations. Results demonstrate that higher levels of acetic acid delay the onset and influence the extent of intracellular ATP accumulation. ATP production and accumulation rates were most sensitive to acetic acid at lower values of pH.     

Characterization of Nucleotide Transport into Rat Brain Synaptic Vesicles

ATP transport to synaptic vesicles from rat brain has been studied using the fluorescent substrate analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP). The increase in intravesicular concentration was time dependent for the first 30 min, epsilon-ATP being the most abundant nucleotide. The complexity of the saturation curve indicates the existence of kinetic and allosteric cooperativity in the nucleotide transport, which exhibits various affinity states with K0.5 values of 0.39 +/- 0.06 and 3.8 +/- 0.1 mM with epsilon-ATP as substrate. The Vmax values obtained were 13.5 +/- 1.4 pmol x min(-1) x mg of protein(-1) for the first curve and 28.3 +/- 1.6 pmol x min(-1) x mg of protein(-1) considering both components. This kinetic behavior can be explained on the basis of a mnemonic model. The nonhydrolyzable adenine nucleotide analogues adenosine 5'-O-3-(thiotriphosphate), adenosine 5'-O-2-(thiodiphosphate), and adenosine 5'-(beta,gamma-imino)triphosphate and the diadenosine polyphosphates P1,P3-di(adenosine)triphosphate, P1,P4-di(adenosine)tetraphosphate, and P1,P5-di(adenosine)pentaphosphate inhibited the nucleotide transport. The mitochondrial ATP/ADP exchange inhibitor atractyloside, N-ethylmaleimide, and polysulfonic aromatic compounds such as Evans blue and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid also inhibit epsilon-ATP vesicular transport.     

Interaction between L-arginine: no system and cyclooxygenase metabolic products of arachidonic acid in coronary autoregulation.

Coronary autoregulation (CA) is the intrinsic ability of the heart to maintain its nutritive blood supply constant over a wide range of perfusion pressure. This phenomenon is regulated through several control mechanisms, while metabolic and myogenic control mechanism have dominant effects. In last few years, endothelial control mechanism, which is part of metabolic control, was intensive investigated. Dominant topic of endothelial-investigation was bioregulatory L-arginine: NO system, with his effective product--nitric oxide (NO). On the other hand, cyclooxygenase metabolic pathway products of arachidonic acid plays an important role in the control of vasomotor tone of coronary arteries. For this purpose, the aim of our study was to evaluate role of L-arginine: NO system, cyclooxygenase metabolites of arachidonic acid, as well as, their interactions in the control of CA of the isolated rat heart.. In our study rat hearts autoregulate CF between 50 and 90 cm H2O of CPP. Basal release (at 60 cm H2O) of NO (as nitrite), cAMP, cGMP and HX+X (i.e. adenosine) amounted to 2.85+/-0.25 nmol/min/g wt, 29.45+/-2.22 pmol/min/g wt, 0.43+/-0.08 pmol/min/g wt and 37.50+/-2.89 nmol/min/g wt respectively. Release of NO, cAMP and cGMP were strictly parallel with CPP-CF curve, while release of adenosine (i.e. HX + X) was an inverse function of perfusion pressure. Inhibition of NOS (L-NAME, 30 micromol/l) significantly widened autoregulatory range (40-100 cm H2O), with significant reduction in CF and NO- and cGMP release, while release of cAMP was completely reversed in the presence of L-NAME. However, inhibition of cyclooxygenase didn't influence autoregulatory range, with similar changes of NO- and cAMP-release and completely inversed values of released adenosine. When L-NAME an indomethacin (an nonspecific COX-inhibitor), 3 micromol/l where added together, they exhibit interactions between these two enzymatic systems. Namely, when L-NAME was added first, indomethacin didn't influence hemodynamic effects of NOS-inhibitor. On the other hand, when COX-inhibitor was added first, L-NAME widened autoregulatory range in small manner as after control autoregulatory experiments (40-90 cm H2O). All hemodynamic changes were followed with similar changes in NO-release, what suggest that exist interaction between L-arginine: NO system and COX-metabolites in the regulation of coronary autoregulation.     

Reflex cardiovascular and ventilatory responses to increasing H+ activity in cat hindlimb muscle

Static exercise increases arterial pressure, heart rate, and ventilation, effects which are believed in part to arise reflexly from a metabolic stimulus in the working muscle. In anesthetized cats, we tested the hypothesis that intra-arterial injections of lactic and hydrochloric acid, which created levels of these substances in muscle similar to those seen during contraction, reflexly increased cardiovascular and ventilatory function. Hydrochloric acid (32 and 57 mM; 1 ml) injected into the arterial supply of the triceps surae decreased intramuscular pH from 7.26 +/- 0.05 to 7.17 +/- 0.05 (P less than 0.01) and reflexly increased arterial pressure (23 +/- 7 mmHg; P less than 0.01), heart rate (11 +/- 2 beats/min; P less than 0.001), and ventilation (187 +/- 72 ml/min; P less than 0.05). Static contraction of the triceps surae decreased intramuscular pH from 7.28 +/- 0.06 to 7.13 +/- 0.06 (P less than 0.01). Lactic acid was more potent in causing reflexes than was equimolar HCl. For example, lactic acid containing 4 mM lactate and 0.87 mM H+ reflexly increased arterial pressure, heart rate, and ventilation, whereas 0.87 mM HCl did not. Intra-arterial sodium lactate (13 and 33 mM) at a neutral pH had no effect on these variables. We conclude that contraction-induced accumulation of H+, especially that arising from lactic acid, might provide a metabolic stimulus to evoke reflex autonomic effects.     

Cyclic oxidation-reduction reactions regulate thromboxane A2/prostaglandin H2 receptor number and affinity in human platelet membranes

The radiolabeled thromboxane A2/prostaglandin H2 (TXA2/PGH2) agonist 125I-BOP bound to the TXA2/PGH2 receptor on human platelet membranes. Scatchard analysis showed that pretreatment of platelet membranes with the reducing agent dithiothreitol (DTT) (10 mM) for 10 min decreased maximal 125I-BOP binding (Bmax) from 1.51 +/- 0.11 pmol/mg to 0.51 +/- 0.05 pmol/mg (p = 0.001) and increased the affinity of the remaining binding sites (Kd = 647 +/- 64 pM (untreated), 363 +/- 46 pM (treated), p = 0.006). Prolonged incubation of membranes with DTT (10 mM) for 40 min further reduced the Bmax to 0.23 +/- 0.08 pmol/mg (p = 0.001 from untreated), and the binding affinity remained elevated (Kd = 334 +/- 117 pM, p = 0.035 from untreated). Kinetic analysis of 125I-BOP binding indicated that the apparent increase in binding affinity after DTT treatment was due exclusively to an increase in the rate of ligand-receptor association with no change in dissociation rate. The effects of DTT on 125I-BOP binding were dose-dependent with an EC50 of 8.1 +/- 0.2 mM. DTT inactivation of TXA2/PGH2 receptors was time-dependent with a second order rate constant (k2) of 0.123 M-1 s-1 at 20 degrees C. The platelet membrane 125I-BOP binding site was partially protected from DTT inactivation by prior occupation with the ligand. TXA2/PGH2 receptor protection by I-BOP was dose-dependent and linearly related (r = 0.97, p = 0.002) to the proportion of receptors occupied, but was incomplete since agonist occupation of 89% of the total number of receptors resulted in only a 38% protective effect. Inhibition of 125I-BOP binding after reduction with DTT could be made permanent by addition of the sulfhydryl alkylating agent N-ethylmaleimide (25 mM), but was completely reversed by reoxidation with dithionitrobenzoic acid (DTNB) (5 mM). Oxidation of untreated receptors with DTNB resulted in a 64% increase in 125I-BOP binding sites from 1.65 +/- 0.12 pmol/mg to 2.70 +/- 0.08 pmol/mg (p = 0.013) without affecting binding affinity. DTNB-induced increases in 125I-BOP binding were concentration-dependent with an EC50 of 668 +/- 106 microM and occurred in less than 1 min at 37 degrees C. In the absence of DTT, alkylation of free sulfhydryl groups with N-ethylmaleimide reduced 125I-BOP Bmax in platelet membranes to 0.85 +/- 0.08 pmol/mg (p = 0.003), but did not change the affinity of the remaining receptors. The EC50 for N-ethylmaleimide inactivation of TXA2/PGH2 receptors was 139 +/- 8 mM, and the k2 in time course experiments was 0.067 M-1 s-1 at 20 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)