Potent,extra-channel influence of several calcium-channel modulators on striatal binding of [3H]tyramine

A number of Ca²⁺-, K⁺-, and Na⁺-channel modulators has been tested with respect to their effects on [³H]tyramine (TY) binding, as a putative marker for the vesicular dopamine (DA) transporter in striatal membrane preparations containing vesicle ghosts. Among organic Ca²⁺-channel modulators, the diphenylalkylamines tested consistently inhibited TY binding: the order of potency was prenylamine>lidoflazine>flunarizine>cinnarizine, with K_i values of 0.1, 0.2, 0.5 and 1.2 M, respectively. Low (up to 100 nM) concentrations of prenylamine did competitively inhibit TY binding, and higher concentrations provoked a mixed-type inhibition. Furthermore, LIGAND-analysis of competition curves revealed a high- and a low-affinity binding site for prenylamine and flunarizine. The TY binding process was also sensitive to selected K⁺- and Na⁺-channel modulators. Since several Ca²⁺-antagonists are known to affect H⁺-ATPase and the bioenergetics of catecholamine storage vesicles in chromaffin granules, thus affecting monoamine storage, the energy requirements for the formation of the TY/carrier complex were here assessed, assuming similarity between chromaffin granules and synaptic vesicles. TY binding, though not reflecting endovesicle-sequestered TY, was indeed strongly sensitive (with K_i coefficients in the fM or low nM range) to the dissipation of the vesicular transmembrane proton concentration ( pH), electrical ( ), and proton electrochemical ( H⁺) gradients, provoked by a number of specifically targeted agents. It is concluded that Ca²⁺-channel agents of the diphenylalkylamine group may directly affect striatal TY binding due to an extrachannel-regulated competition with TY for the vesicular carrier of DA, as well indirectly, by disruption of the transmembrane energization of the reserpine-sensitive carrier.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

Responsiveness of cardiac Na+ channels to antiarrhythmic drugs: The role of inactivation

Summary Elementary Na⁺ currents were recorded at 9°C in inside-out patches from cultured neonatal rat heart myocytes. In characterizing the sensitivity of cooled, slowly inactivating cardiac Na⁺ channels to several antiarrhythmic drugs including propafenone, lidocaine and quinidine, the study aimed to define the role of Na⁺ inactivation for open channel blockade.In concentrations (1–10 mol/liter) effective to depressNP _o significantly, propafenone completely failed to influence the open state of slowly inactivating Na⁺ channels. With 1 mol/liter, _open changed insignificantly to 96±7% of the control. Even a small number of ultralong openings of 6 msec or longer exceeding _open of the whole ensemble several-fold and attaining _open (at –45 mV) in cooled, (-)-DPI-modified, noninactivating Na⁺ channels proved to be drug resistant and could not be flicker-blocked by 10 mol/liter propafenone. The same drug concentration induced in(-)-DPI-modified Na⁺ channels a discrete block with association and dissociation rate constants of 16.1 ± 5.3 × 10⁶ mol^–1 sec^–1 and 675 ± 25 sec^–1, respectively. Quinidine, known to have a considerable affinity for activated Na⁺ channels, in lower concentrations (5 mol/liter) left _open unchanged or reduced, in higher concentrations (10 mol/liter) _open only slightly to 81% of the predrug value whereasNP _o declined to 30%, but repetitive blocking events during the conducting state could never be observed. Basically the same drug resistance of the open state was seen in cardiac Na⁺ channels whose open-state kinetics had been modulated by the cytoplasmic presence of F^– ions. But in this case, propafenone reduced reopening and selectively abolished a long-lasting open state. This drug action is unlikely related to the inhibitory effect onNP _o since hyperpolarization and the accompanying block attenuation did not restore the channel kinetics. It is concluded that cardiac Na⁺ channels cannot be flicker-blocked by antiarrhythmic drugs unless Na⁺ inactivation is removed.     

K+-evoked taurine efflux from cerebellar astrocytes: On the roles of Ca2+ and Na+

The ionic requirements for K⁺-evoked efflux of endogenous taurine from primary cerebellar astrocyte cultures were studied. The Ca²⁺ ionophore A23187 evoked taurine efflux in a dose-dependent fashion with a time-course identical to that of K⁺-induced efflux. The Ca²⁺-channel antagonist nifedipine had no effect upon efflux induced by 10 or 50 mM K⁺. In addition, verapamil did not antagonize 50 mM K⁺-evoked efflux except at high, non-pharmacological concentrations (>100 M), and preincubation with 2 M -conotoxin had no effect on 50 mM K⁺-evoked efflux. Similarly, preincubation with 1 mM ouabain had no effect on the amount of taurine released by K⁺ stimulation, but did accelerate the onset of efflux by 2–4 min. Although 2 M tetrodotoxin had no effect on K⁺-evoked release, replacing Na⁺ with choline abolished the taurine efflux seen in response to K⁺ stimulation. Together, these findings suggest that neuronal N- and L-type Ca²⁺- and voltage-dependent Na⁺-channels are not involved in the influx of Ca²⁺ which appears to be necessary for K⁺-evoked taurine efflux, and that in addition to Ca²⁺, extracellular Na⁺ is also required.     

Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

Summary A vanadate-sensitive H⁺-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H⁺-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H⁺ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H⁺ transport activity was inhibited half-maximally between 1 and 5 M vanadate (Na₃VO₄) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aK _i for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na₃VO₄). ATPase activity of the reconstituted vesicles showed aK _i of 2.6 M for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.     

Modulation of Na+-channels by neurotoxins produces different effects on [3H]ACh release with mobilization of distinct Ca2+-channels

1. Voltage-gated Na⁺ channels are responsible for initiation and conduction of action potentials. The arrival of an action potential at nerve terminal increases intracellular Na⁺ and Ca²⁺ concentrations. Calcium entry into neurons through voltage-dependent calcium channels is associated with a variety of intracellular processes. Scorpion neurotoxins have been used as tools to investigate mechanisms involved in neurotransmitter release. Tityustoxin (TsTX) is an -type toxin that delays Na⁺-channel inactivation. Toxin- (TiTX-) is a -type toxin that induces Na⁺-channel activation at resting potentials.2. In the present work, we describe the effects of both toxins on [³H]acetylcholine ([³H]ACh) release from rat cerebrocortical synaptosomes, in the presence or absence of the calcium channels blockers: -conotoxin-GVIA (-CgTx), 1 M; -agatoxin-IVA (-Aga), 30 nM; -conotoxin-MVIIC (-MVIIC), 1 M; or verapamil, 1M.3. TsTX evokes [³H]ACh release in a concentration-dependent manner with a gradual increase up to saturation at concentrations of 500 nM. However, release of ACh evoked by TiTX- was not linear regarding the toxin concentration. The [³H]-ACh release evoked by TsTX or TiTX- was partially inhibited by -CgTx or -Aga, and blocked with -MVIIC. Verapamil (1 M) had no effect. Tetrodotoxin blocked [³H]ACh release evoked by both toxins.4. These results show that different actions on Na⁺-channels produce different effects on [³H]ACh release with involvement of distinct presynaptic Ca²⁺-channels, which supports the idea that sodium channels may modulate neurotransmitter release.     

Single cardiac outwardly rectifying K+ channels modulated by protein kinase A and a G-protein

Elementary K⁺ currents were recorded at 19 °C in cell-attached and in inside-out patches excised from neonatal rat heart myocytes. An outwardly rectifying K⁺ channel which prevented Na⁺ ions from permeating could be detected in about 10% of the patches attaining (at 5 mmol/l external K⁺ and between – 20 mV and + 20 mV) a unitary conductance of 66 +- 3.9 pS. K _{(outw.-rect.)} ⁺ channels have one open and at least two closed states. Open probability and _open rose steeply on shifting the membrane potential in the positive direction, thereby tending to saturate. Open probability (at –7 mV) was as low as 3 ± 1% but increased several-fold on exposing the cytoplasmic surface to Mg-ATP (100 mol/l) without a concomitant change of _open. No channel activation occurred in response to ATP in the absence of cytoplasmic Mg^–+. The cytoplasmic administration of the catalytic subunit of protein kinase A (120–150 /ml) or GTP--S (100 mol/l) caused a similar channel activation. GDP--S (100 mol/l) was also tested and found to be ineffective in this respect. This suggests that cardiac K _{(outw.-rect.)} ⁺ channels are metabolically modulated by both cAMP-dependent phosphorylation and a G-protein. Offprint requests to: M. Kohlhardt     

Opposite effects of angiotensin II and the protein kinase C activator OAG on cardiac Na+ channels

Summary Elementary Na⁺ currents were recorded at 19°C in cell-attached and inside-out patch-clamp experiments to study the influence of the vasoactive peptide angiotensin II (A II) and of the diacylglycerol analogue OAG (1-oleoyl-2-acetyl-snglycerol) on open probability and gating properties of single cardiac Na⁺ channels from cultured neonatal rat cardiocytes. Treating the cardiocytes with A II caused Na⁺ channel activation: reconstructed peak I_Na increased to 137 ± 17.5% of control at 3 mol/liters and to 176 ± 42% at 30 mol/liter. This NP_o increase developed without major changes in open state and burst activity, even at 30 mol/liter. OAG (6 mol/liter) did not mimic this A II action. By contrast, OAG treatment of the cardiocytes had the opposite effect on NP_o and diminished reconstructed peak I_Na to 67 ± 4.9% of the control. The putative protein kinase C inhibitor staurosporine (0.2 mol/liter) abolished this I_Na depression and led to a normalization of NP_o. OAG had the same effect on isolated Na⁺ channels. Exposure of the cytoplasmic surface of inside-out patches to 1 mol/liter OAG reversibly depressed, in the simultaneous presence of 50 mol/liter Mg-ATP, the reconstructed peak I_Na to 40 ± 9.7% of the control but left i _unit, _open and burst activity unaffected. No NP_o depression was obtained in the absence of Mg-ATP indicating that Mg-ATP may serve as phosphate donor. Obviously, after phosphorylation by protein kinase C, cardiac Na⁺ channels attain a reduced open probability but appear to preserve their kinetic properties. It is also concluded that activation of protein kinase C is not the mechanism underlying the A II induced channel activation.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ko 778/2-3), Bonn.     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Veratridine blocks voltage-gated potassium current in human T lymphocytes and in mouse neuroblastoma cells

(i) Effects of veratridine on ionic conductances of human peripheral blood T lymphocytes have been investigated using the whole-cell patch-clamp technique, (ii) Veratridine reduces the net outward current evoked by membrane depolarizations. The reduction originates from block of a 4-aminopyridine-sensitive, voltage-gated K⁺ current, (iii) Human T lymphocytes do not appear to express voltage-gated Na⁺ channels, since inward currents are observed neither in control nor in veratridine- and bretylium-exposed lymphocytes. (iv) The effect of veratridine consists of an increase in the rate of decay of the voltage-gated K⁺ current and a reduction of the peak current amplitude. Both effects depend on veratridine concentration. Halfmaximum block occurs at 97 m and the time constant of decay is reduced by 50% at 54 m of veratridine. (v) Possible mechanisms of veratridine action are discussed. The increased rate of K⁺ current decay is most likely due to open channel block. The decrease of current amplitude may involve an additional mechanism. (vi) In cultured mouse neuroblastoma N1E-115 cells, veratridine blocks a component of voltage-gated K⁺ current, in addition to its effect on voltage-gated Na⁺ current. This result shows that the novel effect of veratridine is not confined to lymphocytes.We thank Jacobien Künzel of the Wilhelmina Hospital for Children, Utrecht, for providing the blood samples and Aart de Groot for technical assistance. The research was supported by a fellowship of the Royal Netherlands Academy of Arts and Sciences to M. Oortgiesen.     

Significance of the β-Subunit in the Biogenesis of Na+/K+-ATPase

This review summarizes our experiments on the significance of the -subunit in the functional expression of Na⁺/K⁺-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na⁺/K⁺-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH₂-terminal domain to the extracellular COOH-terminus: the NH₂-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na⁺/K⁺-ATPase by Na⁺/K⁺- and H⁺/K⁺-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na⁺/K⁺-ATPase -subunit and from Ile89 to the COOH–terminus of the H⁺/K⁺-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na⁺/K⁺-ATPase.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Endogenous Adenosine Modulation of 22Na Uptake by Rat Brain Synaptosomes

To evaluate if endogenous extracellular adenosine influences sodium channel activity in nerve terminals, we investigated how manipulations of extracellular adenosine levels influence ²²Na uptake by rat brain synaptosomes stimulated with veratridine (VT). To decrease extracellular adenosine levels, adenosine deaminase (ADA) that converts adenosine into an inactive metabolite was used. To increase extracellular adenosine levels, we used the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA), as well as the inhibitor of adenosine transport, nitrobenzylthioinosine (NBTI). ADA (0.1–5U/ml) caused an excitatory effect on ²²Na uptake stimulated by veratridine, which was abolished in the presence of the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 25M). Both the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1–10M) and the adenosine deaminase inhibitor EHNA (10–25M) inhibited ²²Na uptake by rat brain synaptosomes. It is suggested that adenosine is tonically inhibiting sodium uptake by rat brain synaptosomes.     

Active transport in phototrophic bacteria

Phototrophic bacteria utilize light-driven, cyclic electron flow to pump protons out of their cytoplasm, creating an electrochemical proton gradient, _H+, outside acid and positive. These bacteria exchange external protons for internal cations (Na⁺, K⁺ and Ca⁺²), allowing the cells to maintain a nearly constant internal pH while maintaining the electrical component of _H+. Na⁺/H⁺ exchange also establishes an electrochemical Na⁺ gradient. Phototrophic bacteria are able to utilize these electrochemical gradients as energy sources for the uptake of a wide variety of metabolites (e.g., sugars, organic acids and amino acids) via metabolite/cation symports.     

The regulation of total creatine content in a myoblast cell line

Total cellular creatine content is an important bioenergetic parameter in skeletal muscle. To understand its regulation we investigated creatine transport and accumulation in the G8 cultured skeletal myoblast line. Like other cell types, these contain a creatine transporter, whose activity, measured using a radiolabelling technique, was saturable (Km = 110 ± 25 M) and largely dependent on extracellular [Na⁺]. To study sustained influences on steady state creatine concentration we measured total cellular creatine content using a fluorimetric method in 48 h incubations. We found that the total cellular creatine content was relatively independent of extracellular creatine concentration, consistent with high affinity sodium-dependent uptake balanced by slow passive efflux. Accordingly, in creatine-free incubations net creatine efflux was slow ( 5 ± 1 % of basal creatine content per day over 6 days), while creatine content in 48 h incubations was reduced by 28 ± 13% of control by the Na⁺,K⁺-ATPase inhibitor ouabain. Creatine accumulation after 48 h was stimulated by treatment with the mixed - and -adrenergic agonist noradrenaline, the -adrenergic agonist isoproterenol, the ₂-agonist clenbuterol and the cAMP analogue N⁶,2-O-dibutyryladenosine 3,5-cyclic monophosphate, but was unaffected by the ₁ adrenergic agonist methoxamine. The noradrenaline enhancement of creatine accumulation at 48 h was inhibited by the mixed - and -antagonist labetalol and by the -antagonist propranolol, but was unaffected by the ₂ antagonist phentolamine; greater inhibition was caused by the ₂ antagonist butoxamine than the ₁ antagonist atenolol. Creatine accumulation at 48 h was increased to 230 ± 6% of control by insulin and by 140 ± 13% by IGF-I (both at 3 nM). Creatine accumulation at 48 h was also increased to 280 ± 40% of control by 3,3,5-triiodothyronine (at 70 M) and to 220 ± 35% of control by amylin (60 nM). As 3,3,5-triiodothyronine, amylin and isoproterenol all stimulate the Na⁺,K⁺-ATPase, we suggest that they stimulate Na⁺-creatine cotransport indirectly by increasing the transmembrane [Na⁺] concentration gradient and membrane potential.Abbreviations IGF-I insulin-like growth factor I - IGF-II insulin-like growth factor II - T₃ 3,3,5-triiodothyronine - CGRP calcitonin gene-related peptide     

Interaction among anion,cation and glucose transport proteins in the human red cell

Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, _DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID₅₀ (0.3±0.1 m) for H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is virtually the same as the ID₅₀ (0.47±0.04 m) for H₂-DIDS inhibition of red cell Cl^– flux, thus relating _DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in _DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID₅₀ for cytochalasin B modulation of _DBDS is 0.1±0.2 m in good agreement withK _D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na⁺,K⁺-ATPase, increases red cell Cl^– exchange flux in red cells by a factor of about two. This interaction indicates that the Na⁺,K⁺-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.     

Influence of isolation media on synaptosomal properties: Intracellular pH,pCa, and Ca2+ uptake

Preparations of synaptosomes isolated in sucrose or in Na⁺-rich media were compared with respect to internal pH (pH₁), internal Ca²⁺ concentration ([Ca²⁺]_i), membrane potential and⁴⁵Ca²⁺ uptake due to K⁺ depolarization and Na⁺/Ca²⁺ exchange. We found that synaptosomes isolated in sucrose media have a pH_i of 6.77±0.04 and a [Ca²⁺]_i of about 260 nM, whereas synaptosomes isolated in Na⁺-rich ionic media have a pH_i of 6.96±0.07 and a [Ca²⁺]_i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca²⁺ only by voltage sensitive calcium channels (VSCC'S) when K⁺-depolarized, while the Na⁺-rich synaptosomes take up⁴⁵Ca²⁺ both by VSCC'S and by Na⁺/Ca²⁺ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca²⁺ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca²⁺ uptake by VSCC'S, but not by Na⁺/Ca²⁺ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca²⁺ flux through channels and through Na⁺/Ca²⁺ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca²⁺ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF 2,7-Biscarboxyethyl-5(6)-carboxyfluorescein - BCECF/AM acetoxymethyl ester of BCECF - [Ca²⁺]_i Internal free calcium ion concentration - CBZ-DMB 5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil - DMB 2, 4-dimethylbenzamil - DMSO dimethyl sulfoxide - Indo-1/AM acetoxymethyl ester of Indo-1 - MES 2-|N-Morpholino|ethanesulfonic acid - NMG N-methyl-D-glucamine - pH_i internal pH - TPP⁺ tetraphenylphosphonium - _p plasma membrane potential     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.