Effects of fluoride and caffeine on the metabolism and motility of ejaculated bovine spermatozoa

Treatment of washed, ejaculated bovine sperm with 30 mM sodium fluoride immobilized the cells in a characteristically rigid form. In cells metabolizing endogenous substrates, fluoride decreased respiration by about 60%, but did not inhibit the cells' ability to produce adenosine-5'-triphosphate (ATP) via oxidative phosphorylation and did not block access to endogenous substrates. Fluoride-immobilized sperm maintained maximal ATP titers for at least 60 min, but oligomycin treatment rapidly depleted ATP, indicating that ATP synthesis and metabolism was occurring in immobilized sperm. The putative phosphodiesterase inhibitor caffeine (2.5 mM) restored motility and increased respiration in fluoride-treated sperm, but 8-bromo-adenosine-3',5'-monophosphate (8-bromo-cAMP) did not, even though 8-bromo-cAMP stimulated respiration in control (untreated) sperm. Carboxyfluorescein analysis of the intracellular pH of untreated sperm indicated a normal pH of 6.3. Fluoride addition decreased the apparent intracellular pH slightly, but this effect was attributable to dilution. Caffeine did not change internal pH in untreated or fluoride-immobilized sperm. Fluoride did not appear to affect cAMP metabolism, but caffeine increased intracellular cAMP titers by about 35% in both untreated and fluoride-inhibited sperm. However, caffeine treatment did not mimic 8-bromo-cAMP, as analyzed by electrophoresis and autoradiography of sperm proteins labeled with 32P from endogenously generated [32P]ATP. Clearly, caffeine is not stimulating motility in fluoride-treated sperm by affecting the cyclic AMP system. Fluoride also inhibited motility in digitonin-permeabilized sperm by a mechanism that may have involved magnesium depletion, but caffeine had no stimulatory effect on either untreated or fluoride-immobilized, permeabilized sperm.     

Antagonistic effect of methadone on steroidogenesis and the adenylate cyclase system in isolated rat adrenocortical cells

Methadone exhibits an antagonistic effect toward steroidogenesis which lies prior to progesterone in the biosynthetic pathway in isolated rat adrenal cells. Levels of adenosine cyclic 3′–5′ monophosphate are depressed in a dose dependent fashion in ACTH stimulated cells as is steroidogenesis in cells stimulated with N⁶O²-dibutyryl adenosine cyclic 3′–5′ monophosphate. Stimulation produced by the ACTH analog, O-nitrophenyl sulfenyl ACTH, is also inhibited by methadone. The participation of adenosine cyclic 3′–5′ monophosphate as an obligatory messenger in ACTH stimulated steroidogenesis is discussed with respect to the pharmacological properties of methadone in this system.     

Alkaline pH shifts Ca2+ sparks to Ca2+ waves in smooth muscle cells of pressurized cerebral arteries

The effects of external pH (7.0-8.0) on intracellular Ca(2+) signals (Ca(2+) sparks and Ca(2+) waves) were examined in smooth muscle cells from intact pressurized arteries from rats. Elevating the external pH from 7.4 to 7.5 increased the frequency of local, Ca(2+) transients, or "Ca(2+) sparks," and, at pH 7.6, significantly increased the frequency of Ca(2+) waves. Alkaline pH-induced Ca(2+) waves were inhibited by blocking Ca(2+) release from ryanodine receptors but were not prevented by inhibitors of voltage-dependent Ca(2+) channels, phospholipase C, or inositol 1,4,5-trisphosphate receptors. Activating ryanodine receptors with caffeine (5 mM) at pH 7.4 also induced repetitive Ca(2+) waves. Alkalization from pH 7.4 to pH 7.8-8.0 induced a rapid and large vasoconstriction. Approximately 82% of the alkaline pH-induced vasoconstriction was reversed by inhibitors of voltage-dependent Ca(2+) channels. The remaining constriction was reversed by inhibition of ryanodine receptors. These findings indicate that alkaline pH-induced Ca(2+) waves originate from ryanodine receptors and make a minor, direct contribution to alkaline pH-induced vasoconstriction.     

Anion transport in red blood cells and arginine-specific reagents. Interaction between the substrate-binding site and the binding site of arginine-specific reagents

Phenylglyoxal is found to be a potent inhibitor of sulfate equilibrium exchange across the red blood cell membrane at both pH 7.4 and 8.0. The inactivation exhibits pseudo-first-order kinetics with a reaction order close to one at both pH 7.4 and 8. The rate constant of inactivation at 37 degrees C was found to be 0.12 min-1 at pH 7.4 and 0.19 min-1 at pH 8.0. Saturation kinetics are observed if the pseudo-first order rate constant of inhibition is measured as a function of phenylglyoxal concentration. Sulfate ions as well as chloride ions markedly decrease the rate of inactivation by phenylglyoxal at pH 7.4, suggesting that the modification occurs at or near to the binding site for chloride and sulfate. The decrease of the rate of inactivation produced at pH 8.0 by chloride ions is much higher than that produced by sulfate ions. Kinetic analysis of the protection experiments showed that the loaded transport site is unable to react with phenylglyoxal. From the data it is concluded that the modified amino acid(s) residues, presumably arginine, is (are) important for the binding of the substrate anion.     

Cyclic 3',5'-AMP phosphodiesterase of Neurospora crassa

Cyclic 3′,5′-AMP (cAMP) phosphodiesterase activity can be demonstrated in extracts of $\text{Neurospora}\text{crassa}$. The activity is particulate, has a pH optimum of 7.4, and consists of two forms that have different cAMP binding constants. Methylxanthines, inorganic phosphate, and EDTA are inhibitors of the diesterase as are ATP, ADP, and 8-bromo-cAMP. The enzymatic activity is stimulated by histamine and imidazole. These properties suggest that the $\text{Neurospora}$ enzyme is more closely related to the mammalian than to bacterial cAMP phosphodiesterases.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

Protease inhibitors block hormonal activation of adenylate cyclase

To investigate the mechanism of serine protease stimulation of rat ovarian adenylate cyclase, a variety of synthetic protease inhibitors were used. These inhibitors blocked trypsin, chymotrypsin and hCG stimulation of adenylate cyclase in nearly the same manner. The inhibition of hormone stimulated adnylate cyclase could not be explained by a loss of [¹²⁵I]hCG binding. Cholera toxin and epinephrine stimulation of adenylate cyclase were similarly inhibited, whereas basal and fluoride-stimulated activities were only affected by higher doses of the inhibitors. The results suggest that adenylate cyclase in the ovary may be regulated by membrane protease activity.     

Reaction of bovine and equine growth hormones with tetranitromethane.

Bovine and equine growth hormones were chemically modified with tetranitromethane, at pH 7.4 during 5 h and at pH 8.0 in the presence of 8 M urea during 1 h. a) Both hormones have very similar but not identical reactivities. b) The nitration of the reactive tyrosines and tryptophan residues at pH 7.4 produces no detectable changes in their immunological or somatotrophic activities. C) The nitration of all tyrosine residues in both hormones gives rise to a complete loss of somatographic activity with no alteration of the immunological activity.     

Augmentation of keratinocyte differentiation by the epidermal mitogen, 8-bromo-cAMP

The effect of the epidermal mitogen, 8-bromo-cAMP, on keratinocyte differentiation was studied. A 3 X 10(-4) M dose of 8-bromo-cAMP was added to primary neonatal mouse epidermal keratinocyte cultures that slowly proliferate, stratify and differentiate over 2-3 weeks time. [3H]Thymidine autoradiography coupled with an NH4Cl plus reducing agent technic which separates basal and differentiating keratinocytes was used to determine the target cell for the 8-bromo-cAMP mitogenic effect. A histologic stain and a four buffer protein extraction protocol, in conjunction with PAGE and fluorographic technics, were used to assess the differentiation of the cultures. The data indicated that 8-bromo-cAMP primarily stimulated the proliferation of the basal cell monolayer. Simultaneous with the mitogenic effect was an increase in the production of keratohyalin granule, keratin and cell envelope proteins, which are specific markers of epidermal differentiation. The results indicate that keratinocytes stimulated by the epidermal mitogen 8-bromo-cAMP simultaneously express differentiation-related processes.     

Stimulatory and inhibitory effects of adrenaline and 8-bromo-cAMP on insulin-sensitive 2-deoxyglucose transport in rat adipocytes

The effects of adrenaline and 8-bromo-cAMP on 2-deoxyglucose uptake in isolated rat adipocytes were investigated under conditions of unidirectional flux, in which the transport process is rate limiting. Adrenaline showed a dualistic effect in the absence of insulin. At concentrations below 1 microM adrenaline stimulated 2-deoxyglucose uptake; at higher concentrations adrenaline inhibited the uptake. In the presence of insulin at the maximum effective concentration, addition of adrenaline further increased the 2-deoxyglucose uptake by about 50%. In the presence of insulin 8-bromo-cAMP had the same effect as adrenaline. In the absence of insulin, 8-bromo-cAMP only inhibited 2-deoxyglucose uptake.     

Nitrate-Sensitive ATPase Activity and Proton Pumping in Guard Cell Protoplasts of Commelina

ATPase activity was measured in crude homogenates of guard cellprotoplasts of Commelina communis L. using a linked enzyme assay.A low level of azide-sensitive ATPase activity was detectedwith a pH optimum of 6.8. This activity was stimulated by 0.01%(v/v) Triton X-100, and the pH optimum shifted to pH 7.4. Nitrate-sensitiveATPase activity was measured in the presence of azide and showeda pH optimum around pH 8.0. Proton pumping activity in a mixedpopulation of vesicles from GCP was monitored using fluorescencequenching of quinacrine. Mg-ATP dependent proton pumping wasobserved at pH 8.0, but not at pH 6.6. The activity at pH 8.0was inhibited by nitrate and DCCD but not vanadate. These dataindicate that activity of the tonoplast proton pump was beingmeasured. There was, however, no evidence for a tonoplast cation(K⁺)/proton antiporter under these assay conditions as potassiumdid not reduce the initial rate of pH gradient formation orincrease the rate of collapse of a pre-formed gradient afterinhibition of the pump. Key words: Tonoplast ATPase, proton pump, guard cell protoplasts, Commelina     

Inhibition of cyclic GMP formation and aggregation in Dictyostelium by the intracellular Ca2+ antagonist TMB-8

Aggregation in Dictyostelium discoideum was shown in previous studies employing EGTA to require Ca2+, but the intra- or extracellular site of action of this ion and its role in chemotaxis were not determined [1]. In this investigation we show that the intracellular Ca2+ immobilising agent TMB-8 does not affect binding of the signalling nucleotide, cAMP, to the cell surface receptors but abolishes the rapid accumulation of intracellular cGMP and subsequent chemotactic aggregation. We infer that movement of Ca2+ from membrane-bound stores is triggered by binding of cAMP to the cell-surface receptor and that this plays a primary role in stimulating cGMP formation and chemotaxis.     

Formation of 8-S-l-cysteinylguanosine from 8-bromoguanosine and cysteine

When 8-bromoguanosine was incubated with cysteine at pH 7.4 and 37 °C, a previously unidentified product was formed as a major product in addition to guanosine. The product was identified as a cysteine substitution derivative of guanosine at the 8 position, 8-S-l-cysteinylguanosine. The reaction was accelerated under mildly basic conditions. The cysteine adduct of guanosine was fairly stable and decomposed with a half-life of 193 h at pH 7.4 and 37 °C. Similar results were observed for incubation of 8-bromo-2′-deoxyguanosine with cysteine. The results suggest that 8-bromoguanine in nucleosides, nucleotides, RNA, and DNA can react with thiols resulting in stable adducts.     

Hymenolepis diminuta: Partial characterization of membrane-bound nucleotidase activities (ATPase and 5′-nucleotidase) in the isolated brush border membrane

Adenosine triphosphatase (ATPase; EC 3.6.1.3) and 5′-nucleotidase (5′-NTase; EC 3.1.3.5) activities of the isolated brush border membrane of Hymenolepis diminuta have been studied. The pH optimum for ATPase activity is 7.4, and divalent cations are necessary for maximum activity; no Na⁺-K⁺ activated ATPase is present in the isolated brush border membrane. ATPase activity is inhibited by molybdate and phosphorylated monosaccharides, but not by N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB), or fluoride. The pH optimum for 5′-NTase activity is 9.6–10.2, and divalent cations are necessary for maximum activity. 5′-NTase activity is inhibited by molybdate at pH 9.6 and 7.4, and activated by NEM and pCMB and pH 9.6 and 7.4, respectively; fluoride has no effect on 5′-NTase activity. Solubilization of the brush border membrane fraction in 1% sodium dodecyl sulfate has no inhibitory action on either enzyme activity.     

pH-dependent stability of EGX, a multi-functional cellulase from mollusca, Ampullaria crossean

Cellulose and hemicellulose are the most abundant andmajor constituents of plants and the potential rawmaterials for enzyme hydrolysis related biotechnologicalprocesses. The cellulase and hemicellulase have beenconsidered as two of the most important enzymes inbiotechnology market which can hydrolyze cellulosic ma-terials to fermentable sugars for various biotechnologicalpurposes [1,2], and can also be used in textile- and paper-making industry [3]. It is important to use these enzymesunder re…     

The effect of medium pH on rate of growth, neurite formation and acetylcholinesterase activity in mouse neuroblastoma cells in culture.

Cell division, neurite formation and acetylcholinesterase activity were examined in a clone (NBA2) of mouse neuroblastoma cells maintained for up to 120 hours in medium with pH values between 6.6 and 8.0. Growth rate decreased as pH was reduced from 7.8 to 6.6. Generation time at pH 7.4 was 25 hours, while the rate of cell division was negligible at pH 6.6. The total number of cells at stationary phase was less at the lower pH values. Neurite formation was enhanced markedly as the pH was reduced from 7.4 to 6.6. Acetylcholinesterase activity was 5- to 8-fold greater in cells exposed to medium at pH 6.6 than in cells maintained in medium at pH 7.4. The reduction in the rate of cell division and increases in neurite formation and acetylcholinesterase activity at pH 6.6 were reversible upon exposure of the cells to pH 7.4 medium. Cell viability was greater than 90% at all medium pH values over a period of 120 hours. Uncloned T-59 mouse neuroblastoma cells were affected similarly by changes in pH. These results show that manipulation of the environmental pH can reversibly alter growth, neurite formation, and acetylcholinesterase activity of mouse neuroblastoma cells in culture.     

Spatio-temporal dynamic analysis of bid activation and apoptosis induced by alkaline condition in human lung adenocarcinoma cell.

Activation of initiator and effector caspases and Bid cleavage are apoptotic characteristic features. They are associated with cell alkalization or acidification in some models of apoptosis. The alteration of culture conditions such as extracellular pH value and the overexpression of Bid plasmids may induce cell apoptosis. In present report, we used fluorescence confocal imaging and fluorescence resonance energy transfer (FRET) techniques based on green fluorescent proteins (GFPs) to monitor the spatio-temporal dynamics of Bid translocation and caspase-3 activation in real time in living human lung adenocarcinoma (ASTC-a-1) cells under neutral (pH 7.4) and alkaline (pH 8.0) conditions. The cells transfected with Bid-CFP plasmid did not show apoptotic characteristics for 96 hours under an atmosphere of 95% air, 5% CO(2) at pH 7.4 and 37 degrees C, implying that the overexpression of Bid-CFP plasmid does not induce cell apoptosis. However, all the cells underwent apoptosis after being placed in the alkaline culture (pH 8.0). The dynamic results in single living cell showed that the alkaline condition at pH of 8.0 induced Bid cleavage and tBid translocation to mitochondria at about 1.5 hour, and then induced the caspase-3 activation and cell apoptosis. These results show that the alkaline sondition (pH=8.0) induces cell apoptosis by activating caspase-8, which cleaves Bid to tBid, tBid translocation to mitochondria, and then activating the caspase-3 in the ASTC-a-1 cells.     

Stimulation of rat liver adenylate cyclase by halothane.

Rat liver membranes prepared by a modification of the procedure of Neville were exposed to clinical and toxic concentrations of the general anesthetic, halothane, for 10 minutes. Basal, glucagon (5 × 10⁻⁵M) and sodium fluoride (20 mM) stimulated adenylate cyclase activity was assayed. Clinical and toxic concentrations of halothane augmented basal adenylate cyclase activity. Glucagon and sodium fluoride stimulated adenylate cyclase activity was enhanced at greater than clinically useful halothane concentrations only. The study provides direct evidence that halothane stimulates adenylate cyclase, the extent of augmentation of enzyme activity is halothane concentration dependent, and modified by other drugs.