Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K⁺ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated E_k of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward I_ca and outward I_k simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated I_Ca but not the isoprenaline-stimulated I_k. The antibody- or carbachol-induced outward K⁺ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated I_ca were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.     

Platelet calcium pump activity in essential hypertensives and their first-degree relatives

Intracellular free Ca²⁺ concentration has been shown to be elevated in platelets of patients with essential hypertension. This study was designed to characterize Ca²⁺-pump activity of the platelet membranes (surface and intracellular) in these patients. A double-blind study was carried out. Untreated and treated (on R-blockers) essential hypertensives were studied in comparison with normotensive control subjects. First degree blood relatives of essential hypertensives were also studied. The Ca²⁺-activation kinetics of the enzyme showed a significant decrease in the V_max. (for the plasma- and intracellular membranes) and Km (for the intracellular membranes) in the essential hypertensive patients. Increased platelet membrane cholesterol content was observed in these patients. Lowered Ca²⁺-efflux by Ca²⁺-ATPase may lead to elevated intracellular free Ca²⁺-levels in platelets of essential hypertensives. A lowered Ca²⁺-ATPase activity may emerge as a marker for essential hypertension.     

Oxidative damage to sarcoplasmic reticulum Ca2+-pump induced by Fe2+/H2O2/ascorbate is not mediated by lipid peroxidation or thiol oxidation and leads to protein fragmentation

The major protein in the sarcoplasmic reticulum (SR) membrane is the Ca²⁺ transporting ATPase which carries out active Ca²⁺ pumping at the expense of ATP hydrolysis. The aim of this work was to elucidate the mechanisms by which oxidative stress induced by Fenton's reaction (Fe²⁺ + H₂O₂ HO· + OH^–+ Fe³⁺) alters the function of SR. ATP hydrolysis by both SR vesicles (SRV) and purified ATPase was inhibited in a dose-dependent manner in the presence of 0–1.5 MM H₂O₂ plus 50 M Fe²⁺ and 6 mM ascorbate. Ca²⁺ uptake carried out by the Ca²⁺-ATPase in SRV was also inhibited in parallel. The inhibition of hydrolysis and Ca²⁺ uptake was not prevented by butylhydroxytoluene (BHT) at concentrations which significantly blocked formation of thiobarbituric acid-reactive substances (TBARS), suggesting that inhibition of the ATPase was not due to lipid peroxidation of the SR membrane. In addition, dithiothreitol (DTT) did not prevent inhibition of either ATPase activity or Ca²⁺ uptake, suggesting that inhibition was not related to oxidation of ATPase thiols. The passive efflux of ⁴⁵Ca²⁺ from pre-loaded SR vesicles was greatly increased by oxidative stress and this effect could be only partially prevented (ca 20%) by addition of BHT or DTT. Trifluoperazine (which specifically binds to the Ca²⁺-ATPase, causing conformational changes in the enzyme) fully protected the ATPase activity against oxidative damage. These results suggest that the alterations in function observed upon oxidation of SRV are mainly due to direct effects on the Ca²⁺-ATPase. Electrophoretic analysis of oxidized Ca²⁺-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca²⁺-ATPase band and the appearance of low molecular weight peptides (MW < 100 kDa) and high molecular weight protein aggregates. Presence of DTT during oxidation prevented the appearance of protein aggregates and caused a simultaneous increase in the amount of low molecular weight peptides. We propose that impairment of function of the Ca²⁺-pump may be related to aminoacid oxidation and fragmentation of the protein.Abbreviations AcP acetylphosphate - BHT butylhydroxytoluene - DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - SR sarcoplasmic reticulum - SRV sarcoplasmic reticulum vesicles - TBA thiobarbituric acid - TBARS thiobarbituric acid-reactive substances - TFP trifluoperazine     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both ₁-adrenoceptors (₁AR) and ₂-adrenoceptors (₂AR) mediate positive inotropic effects but that only ₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both ₁ AR and ₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of ₁AR and 1/3 of ₂AR, whether or not ₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate ₂AR, we used the ₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the ₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for ₂AR than for - 1 AR; the binding to ₂AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both ₁AR and ₂AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both ₁AR and ₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

Intrinsic fluorescence as a probe of structure-function relationships in Ca2+-transport ATPases

Applications of intrinsic fluorescence measurements in the study of Ca²⁺-transport ATPases are reviewed. Since the initial reports showing that the fluorescence emission was sensitive to Ca²⁺ binding, a substantial amount of work has focused on the use of both steady-state and time-resolved fluorescence spectroscopy to investigate structure-function relationships in sarcoplasmic reticulum and plasma membrane Ca²⁺-ATPases. These studies have revealed ligand-induced conformational changes, as well as provided information on protein-protein, protein-solvent and/or protein-lipid interactions in different functional states of these proteins. The main results of these studies, as well as possible future prospects are discussed.     

Effects of salinity on turgor pressure and fertility in Tolypella (Characeae)

We present the first experimental results on salinity tolerance and regulation mechanisms in the genus Tolypella. The two species investigated, T. nidifica and T. glomerata, regulate turgor pressure with almost complete effectiveness by adjustment of K+ and CT concentrations. Sucrose is also involved. The mechanism is basically identical to the mechanism of turgor pressure regulation previously identified in representatives of the genera Chara and Lamprothamnium. Since Chara and Lamprothamnium on the one hand and Tolypella on the other belong to different phylogenetic branches that separated early in the geological history of the Characeae, the K+ regulation mechanism can be assumed to represent an ancient pattern derived from a salt-tolerant common ancestor. Furthermore, our experiments provide evidence that salinity is a limiting factor for fertility in both T. nidifica and T. glomerata. Although the onset of gametangia covers the whole range of salinities tested here (0–29 psu), 12psu was the inhibitory level for the formation of mature oospores. Fertilization is probably disturbed by an increase in salinity. An inability to reproduce sexually under euryhaline conditions could explain why the distribution of the two species is restricted to oligo- and mesohaline environments, despite the wide range of salinity tolerance of their vegetative apparatus.     

Comparative study of methodologies for obtaining β-glucosidase immobilized on dextran-modified silica

In this study, two procedures for the immobilization of β-glucosidase on silica are compared. The first approach comprises a preliminary stabilization of β-glucosidase by coupling with dextran dialdehyde and subsequent immobilization of the obtained β-glucosidase dextran dialdehyde with aminopropylsilica. In the second approach, β-glucosidase is immobilized on silica modified with a dextran-dialdehyde coating. Enzyme immobilized via coupling with dextran dialdehyde and subsequent attachment with aminopropylsilica show a remarkably enhanced thermostability. Enzyme immobilized by the alternative approach demonstrated an inferior thermoresistance. The difference in behavior of the immobilized enzyme obtained via these two methods can be explained considering the number of links between the enzyme and carrier. Enzyme immobilized on dextran dialdehydecoated silica is fixed via a limited number of links. On the other hand, with soluble β-glucosidase-dextran conjugates, the enzyme configuration is already stabilized via a high number of links with the dextran backbone. It is clear from this study that the sequence of reactions in immobilizing enzymes on silica support via a dextran-dialdehyde linker has a significant effect on the final properties.     

Influx and efflux of inorganic carbon in Synechococcus UTEX625

The CO₂ and HCO₃^? fluxes in air-grown cells of Synechococcus UTEX 625 al pH 8-0 were measured during dark to light and light to dark transitions using a mass spectrometer and sampling of the reaction medium. The kinetic parameters for initial uptake of CO₂ and HCO₃^? were determined during the initial period of illumination. The development of the internal C_i pool was followed up to steady-state photosynthesis, which occurred when the size of the internal inorganic carbon pool remained apparently constant for a limited period. The experimental procedure confirmed that only CO₂ transport occurred with 100mmolm^?3 Na⁺ and that both CO₂ and HCO^?3 transport occurred with 25molm^?3 Na⁺. The K_1/2 values of initial CO₂ and HCO₃ uptake were 0.7 and 17.2 mmolm^?3respectively and agreed closely with the K_1/2 values of net CO₂ and HCO₃^? transport during steady-state photosynthesis, which were 0.66 and 17.1 mmolm^?3 respectively. Maximum rates of CO₂and HCO₃^? transport were 423 and 219mmolh^?1 g^?1 Chl. Maximum CO₂ efflux observed upon darkening was 118mmolh^?1 g^?1 Chl. A permeability coefficient of the cell for CO₂ of 3 × 10^?8 m s^?1 was determined from the dark CO₂ efflux assuming an internal pH of 7.2 in the dark. Following the initial CO₂ uptake in the light, the extracellular [CO₂] steadily declined when only CO₂ transport was allowed, but an increase in the extracellular [CO₂] when HCO₃^? transport was allowed to proceed suggested that an enhanced CO₂ efflux occurred as a result of the larger size of the intracellular C_i pool.     

HCO3− and CO2 leakage from Synechococcus UTEX 625

The leakage of various inorganic carbon species from air-grown cells of Synechococcus UTEX 625 was investigated after a light to dark transition or during a light period using a mass spectrometer under a wide variety of experimental conditions. Total inorganic carbon efflux and CO₂ efflux during the initial period of darkness were measured with or without carbonic anhydrase in the reaction medium respectively. The HCO₃^? efflux after a light to dark transition was estimated by difference. Carbon dioxide efflux in the light was measured by inhibiting CO₂ transport with either Na₂S or COS₃ or quenching the ¹³C inorganic carbon transport by the addition of ¹²C inorganic carbon in excess. In cells in which CO₂ fixation was inhibited, when only the HCO₃^? transport system was fully operative, CO₂ effluxed continuously during the light period at a rate equal to about 25% of that in darkness. When only the CO₂ transport system was operative, HCO₃^? effluxed during the light period. The difference between the light and dark efflux rates was consistent with a 0.6 unit decrease in the intracellular pH upon darkening the cells. The permeabilities of the cell for CO₂ (2.94 ± 0.14 ± 10^?8ms^?1; mean ± SE, n=137) and HCO₃^? (1.4–1.7 ± 10^?9 ms^?1) were calculated.     

Distribution and activity of microsomal NADPH-dependent monooxygenases and amino acid decarboxylases in cruciferous and non-cruciferous plants, and their relationship to foliar glucosinolate content

The NADPH-dependent conversion of amino acids to their aldoximes is an initial step in glucosinolate biosynthesis. A number of microsomal aldoxime-forming monooxygenase activities were detected in leaves from a variety of glucosi-nolate-containing species, whereas barley, bean and tobacco leaves did not contain any such activities. The substrates for these monooxygenases in each species largely correlated with the spectrum of glucosinolates found in that species. No activity was detected that metabolized homomethionine (supposed precursor of 2-propenylglucosinolate [sinigrin]), even in species where sinigrin was the major glucosinolate. In Sinapis species containing hydroxybenzylglucosinolate (sinalbin), activity with L-Tyr was detected, whereas Brassica species containing sinalbin had no such activity. However, these Brassicas did contain an L-Phe monooxygenase activity. Partial characterization of the monooxygenases indicated that in Brassica species, Nasturtium officinalis and Raphanus sativus these resembled the flavin-linked monooxygenases previously found in oilseed rape (Brassica napus) and Chinese cabbage (Brassica campestris). The L-Tyr-dependent activity in Sinapis species, and the L-Phe-dependent activity in Tropacolum majus, had characteristics of cytochrome P450-type enzymes. No similarity was found with any other known amino acid metabolizing enzymes (including decarboxylases, amino acid oxidases and diamine/polyamine oxidases).