Effects of Forskolin, Dibutyryl Cyclic AMP, and 5''N-Ethylcarboxamide Adenosine on 22Na Uptake by Rat Brain Synaptosomes Stimulated by Veratridine

The effects of forskolin, dibutyryl cyclic AMP, and 5'-N-ethylcarboxamide adenosine on specific 22Na uptake by synaptosomes stimulated by veratridine were investigated. All substances inhibited 22Na uptake, with forskolin more potent than 5'-N-ethylcarboxamide and this latter one more potent than dibutyryl cyclic AMP. In the absence of preincubation with forskolin, this substance caused little or no effect on 22Na uptake by synaptosomes. In the presence of the adenosine antagonist dipropylsulfophenylxanthine, the inhibitory effect of 5'-N-ethylcarboxamide adenosine on 22Na uptake was consistently antagonized. The results were interpreted as forskolin and 5'-N-ethylcarboxamide adenosine increasing cyclic AMP accumulation, and dibutyryl cyclic AMP mimicking it, and by these mechanisms decreasing sodium uptake through the sodium channels.     

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.     

Ionizing Radiation Alters the Properties of Sodium Channels in Rat Brain Synaptosomes

The effect of ionizing radiation on neuronal membrane function was assessed by measurement of neurotoxin-stimulated 22Na+ uptake by rat brain synaptosomes. High-energy electrons and gamma photons were equally effective in reducing the maximal uptake of 22Na+ with no significant change in the affinity of veratridine for its binding site in the channel. Ionizing radiation reduced the veratridine-stimulated uptake at the earliest times measured (3 and 5 s), when the rate of uptake was greatest. Batrachotoxin-stimulated 22Na+ uptake was less sensitive to inhibition by radiation. The binding of [3H]saxitoxin to its receptor in the sodium channel was unaffected by exposure to ionizing radiation. The effect of ionizing radiation on the lipid order of rat brain synaptic plasma membranes was measured by the fluorescence polarization of the molecular probes 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene. A dose of radiation that reduced the veratridine-stimulated uptake of 22Na+ had no effect on the fluorescence polarization of either probe. These results demonstrate an inhibitory effect of ionizing radiation on the voltage-sensitive sodium channels in rat brain synaptosomes. This effect of radiation is not dependent on changes in the order of membrane lipids.     

Mouse brain synaptosomal sodium channels: activation by aconitine, batrachotoxin, and veratridine, and inhibition by tetrodotoxin

Batrachotoxin, veratridine and aconitine, activators of the voltage-dependent sodium channel in excitable cell membranes, increase the rate of 22Na+ uptake by mouse brain synaptosomes. Batrachotoxin was both the most potent (K0.5, 0.49 microM) and most effective activator of specific 22Na+ uptake. Veratridine (K0.5, 34.5 microM) and aconitine (K0.5, 19.6 microM) produced maximal stimulations of 22Na+ uptake that were 73% and 46%, respectively, of that produced by batrachotoxin. Activation of 22Na+ uptake by veratridine was completely inhibited by tetrodotoxin (I50, 6 nM ), a specific blocker of nerve membrane sodium channels. These results identify appropriate conditions for measuring sodium channel-dependent 22Na+ flux in mouse brain synaptosomes. The pharmacological properties of mouse brain synaptosomal sodium channels described here are distinct from those previously described for sodium channels in rat brain synaptosomes and mouse neuroblastoma cells.     

The Rapid Uptake and Release of [3H]Adenosine by Rat Cerebral Cortical Synaptosomes

Abstract: Adenosine, a putative inhibitory transmitter or modulator in the brain, is rapidly transported by rat cerebral cortical synaptosomes. The uptake may represent a facilitated diffusion process, which is saturable and temperature-dependent. In this study, the uptake process was very rapid, reaching completion within 60 s of incubation at 37°C, and had an apparent K_m value of 0.9μM and a V_max value of 5.26 pmol/mg protein/ 30 s. Over 70% of the adenosine taken up remained unchanged, whereas 14% was metabolized to inosine. Twelve percent of the adenosine was converted to nucleotides. Rapid uptake of adenosine into rat cerebral cortical synaptosomes was partially inhibited by replacing Na⁺ with choline chloride in the medium. Ca²⁺ ion is important for the uptake process, as inhibition of adenosine uptake occurs in the presence of either Co²- or EGTA. Rapid uptake of adenosine is apparently mediated by a nucleoside carrier, a conclusion based on its inhibition by a variety of purine and pyrimidine nucleosides. Uptake was inhibited by dipyridamole, hexobendine, papaverine, flurazepam, and morphine. Over 60% of the adenosine taken up by the rapid uptake system (30 s) was released by depolarizing agents. In contrast, only 30% of the adenosine taken up during a 15-min incubation period was released under the same conditions. [³H]Adenosine was the predominant purine released in the presence or absence of depolarizing agents. The basal and KCl-evoked release mechanisms were found to be at least partially Ca²⁺-dependent, however, the release of adenosine by veratridine was increased in the presence of EGTA. This finding is in agreement with the reported Ca²⁺-independent release of ATP from brain synaptosomes. The present findings suggest that there are at least two functional pools of adenosine in synaptosomes. Adenosine taken up by different uptake systems may be destined for different uses (metabolism or release) in the neuron.     

Alkaloid neurotoxins-dependent sodium transport in insect synaptic nerve-ending particles

1. Sodium uptake associated with the activation of voltage-sensitive sodium channels by alkaloid activators, batrachotoxin, veratridine, and aconitine in presynaptic nerve terminals isolated from the central nervous system of cockroach (Periplaneta americana) was investigated. 2. Batrachotoxin (K0.5, 0.2 microM) was full agonist as for most effective activator of Na+ uptake; veratridine (K0.5, 2.5 microM) and aconitine (K0.5, 7.6 microM) produced a maximal stimulation of 22Na+ uptake that were 71% and 43% respectively of that produced by batrachotoxin. 3. Veratridine-dependent 22Na+ uptake was completely inhibited by tetrodotoxin (I0.5, 11 nM), a specific inhibitor of the nerve membrane sodium channels. 4. The present study describes appropriate conditions for measuring neurotoxins--stimulated sodium transport in insect central nervous system synaptosomes. The data show that voltage-sensitive sodium channels as defined by specific activation by the alkaloid neurotoxins are qualitatively distinct in insect synaptosomes than those previously described for vertebrate brain synaptosomes, cultured neuronal cell, nerve membrane vesicles and neuroblastoma cells.     

Activation of the Voltage-Sensitive Sodium Channel by a β-Scorpion Toxin in Rat Brain Nerve-Ending Particles

Neurotoxins purified from scorpion venoms previously had been divided into two classes according to their binding properties in rat brain synaptosomes. However, the pharmacological action of beta-scorpion toxin (beta-ScTx) on this preparation has not yet been described. In this report we show that a beta-ScTx induced an increase in 22Na+ uptake through synaptosomal voltage-sensitive sodium channels since this stimulation was abolished by tetrodotoxin (TTX). The increase was smaller than with veratridine and no synergy was observed between beta-ScTx and veratridine, as is the case for alpha-scorpion toxin (alpha-ScTx) and veratridine. The effects of alpha- and beta-ScTx were additive and the concentration-effect curves for each type of toxin were not modified by the other, suggesting that these two types of toxins act through distinct and noninteracting receptor sites. This was confirmed by the absence of mutual modification of the equilibrium and kinetic binding properties. beta-ScTx was shown to inhibit the uptake and to stimulate the release of [3H]gamma-aminobutyric acid. These effects were blocked by TTX, and no synergy was observed with veratridine. It was concluded that all these effects are mediated by the activation of voltage-sensitive sodium channels induced by the binding of beta-ScTx to a receptor site (site 4) distinct from those for other neurotoxins acting on sodium channels.     

Involvement of adenosine A1 receptors in antitussive effect in mice

The effects of N⁶-cyclohexyladenosine, a selective adenosine A₁ receptor agonist, on the capsaicin-induced cough reflex in mice were examined. I.c.v. administration of N⁶-cyclohexyladenosine in doses that ranged from 0.03 to 0.3 nmol decreased the number of coughs in a dose-dependent manner. Pretreatment with 8-cyclopentyl-1,3-theophylline, a selective adenosine A₁ receptor antagonist, significantly reduced the antitussive effect of N⁶-cyclohexyladenosine. On the other hand, CGS21680 (0.3 and 1 nmol, i.c.v.), a selective adenosine A₂ receptor agonist, had no significant effect on the number of capsaicin-induced coughs. These data suggest that adenosine A₁ agonist may have a marked antitussive effect in mice.     

Relationship between synaptosomal uptake and release of [14C]gaba, [14C]diaminobutyric acid and [14C]beta-alanine.

[¹⁴C]GABA is taken up by rat brain synaptosomes via a high affinity, Na⁺-dependent process. Subsequent addition of depolarizing levels of potassium (56.2 MM) or veratridine (100 μM) stimulates the release of synaptosomal [¹⁴C]GABA by a process which is sensitive to the external concentration of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺. However, the relatively smaller amount of [¹⁴C]GABA taken up by synaptosomes in the absence of Na⁺ is not released from synaptosomes by Ca²⁺ -dependent, K ⁺-stimulation. [¹⁴C]DABA, a competitive inhibitor of synaptosomal uptake of GABA (Iversen & Johnson , 1971) is also taken up by synaptosomal fractions via a Na ⁺ -dependent process; and is subsequently released by Ca²⁺ -dependent, K⁺-stimulation. On the other hand, [¹⁴C]β-alanine, a purported blocker of glial uptake systems for GABA (Schon & Kelly , 1974) is a poor competitor of GABA uptake into synaptosomes. Comparatively small amounts of [¹⁴C] β-alanine are taken up by synaptosomes and no significant amount is released by Ca²⁺ -dependent, K⁺-stimulation. These data suggest that entry of [¹⁴C]GABA into a releasable pool requires external Na⁺ ions and maximal evoked release of [¹⁴C]GABA from the synaptosomal pool requires external Ca²⁺ ions. The GABA analogue, DABA, is apparently successful in entering the same or similar synaptosomal pool. The GABA analogue, β-alanine, is not. None of the compounds or conditions studied were found to simultaneously affect both uptake and release processes. Compounds which stimulated release (veratridine) or inhibited release (magnesium) were found to have minimal effect on synaptosomal uptake. Likewise compounds (DABA) or conditions (Na⁺-free medium) which inhibited uptake, had little effect on release.     

RAPID EFFECTS OF VERATRIDINE, TETRODOTOXIN, GRAMICIDIN D, VALINOMYCIN AND NaCN ON THE NA+, K+ AND ATP CONTENTS OF SYNAPTOSOMES

Abstract— The effects of brief exposures of a number of depolarizing agents on ²⁴Na⁺ influx and on the Na⁺, K⁺ and ATP contents of synaptosomes were studied using a Millipore filtration technique to terminate the reaction. When synaptosomes were incubated in normal medium, there was a rapid influx of ²⁴Na⁺ and a gain in Na’contents; neither the ²⁴Na⁺ influx nor the Na⁺ gain were blocked by tetrodotoxin suggesting that this Na⁺ entry did not involve Na⁺-channels. Veratridine markedly increased the rate of ²⁴Na⁺ influx into synaptosomes and also increased the Na⁺ content and decreased the K⁺ content of synaptosomes within the first 10s of exposure. The normal ion contents were reversed by 1 min. The effects of veratridine on Na⁺ influx and on synaptosomal ion contents were prevented by tetrodotoxin and required Na⁺ in the medium. The ionophores gramicidin D and valinomycin also rapidly reversed the Na⁺ and K⁺ contents of synaptosomes, but these effects could not be blocked by tetrodotoxin. The reducing effect of gramicidin D on synaptosomal K⁺ content required Na’in the medium, whereas valinomycin caused a fall in the K⁺ content of synaptosomes in a Na⁺-free medium. Veratridine and gramicidin D, at concentrations known to reverse the synaptosomal ion contents, did not affect synaptosomal ATP levels. In contrast, valinomycin and NaCN caused an abrupt fall in synaptosomal ATP levels. The above findings suggest that veratridine quickly alters synaptosomal Na⁺ and K⁺ contents by opening Na ⁺-channels in the presynaptic membrane, and provide direct evidence for the existence of Na⁺-channels in synaptosomes. In contrast, gramicidin D and valinomycin appear to act independently of Na ⁺-channels, possibly by their ionophoric effects and, in the case of valinomycin, by diminishing synaptosomal ATP contents and hence diminishing Na⁺-pump activity. The rapid reversals of Na⁺ and K⁺ contents by these drugs could affect the resting membrane potentials, Na⁺-Ca²⁺ exchange across the synaptosomal membrane, and the release, synthesis and uptake of neurotransmitters by synaptosomes.     

Ca2+ transport by intact synaptosomes: the voltage-dependent Ca2+ channel and a re-evaluation of the role of sodium/calcium exchange

The verapamil-sensitive Ca2+ channel in the synaptosomal plasma membrane is investigated. Verapamil is without effect on Ca2+ uptake or steady-state content in synaptosomes with a polarized plasma membrane, but completely inhibits the additional Ca2+ uptake following plasma-membrane depolarization by high [K+], by veratridine plus ouabain or by high concentrations of the permeant cation tetraphenylphosphonium. Verapamil-insensitive Ca2+ influx and steady-state content are identical in polarized and depolarized synaptosomes, even though the Na+ electrochemical potential is greatly decreased in the latter, indicating that Na+/Ca2+ exchange is not a significant mechanism for Ca2+ efflux under these conditions. A transient Na+-dependent Ca2+ efflux can only be observed on addition of Na+ to Na+-depleted depolarized synaptosomes. While 0.2 mM verapamil decreases the ate of 86Rb+ efflux and 22Na+ entry during depolarization induced by veratridine plus ouabain, the final steady-state Na+ accumulation is not inhibited. Ca2+ efflux from synaptosomes following mitochondrial depolarization does not occur by a verapamil-sensitive pathway.     

Retention of nerve terminals in dispersed parotid acinar cells.

The effects of veratridine, an agent known to increase Na permeability in excitable tissues, were determined on a dispersed cell preparation from the rat parotid gland. The uptake of ²²Na by these parotid cells was increased in the presence of veratridine but not to as great an extent as with carbachol. The veratridine effect was blocked by both tetrodotoxin (TTX) and a combination of receptor blockers, atropine and phentolamine. TTX had no effect on the increase in ²²Na uptake due to carbachol. Electron microscopic examination revealed the presence of nerve terminals in the dispersed cell preparation, often in very close apposition to individual cells. It is likely that these nerve terminals are the primary sites of actions of veratridine and TTX and not the parotid acinar cells. The possibility of the presence of unmyelinated nerve fibers should be taken into account in the analyses of experimental data obtained with dispersed cell preparations.     

Role of synaptosomal Na-accumulation in transmitter release

The mechanism whereby Na⁺, K⁺-ATPase inhibitors such as ouabain trigger transmitter release in a calcium-independent manner remains obscure. We have examined the possible role of intra-synaptosomal sodium ion accumulation in ouabain-induced acetylcholine (ACh) release by: 1) Measuring²²Na accumulation in cat cortical synaptosomes in the presence of ouabain, A23187, veratridine, or strophanthidin over the same time course in which we previously determined their effects on ACh release; and 2) measuring synaptosomal²²Na accumulation and ACh-release in the presence of ouabain plus tetrodotoxin in normal or calcium-free buffer. Our results indicate that tetrodotoxin-dependent²²Na accumulation is at least partially responsible for ouabain-induced ACh release in normal and calcium-free media, but that this ion-accumulation per se is not sufficient to elicit release with other secretogogues.Dedicated to Henry McIlwain.     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Uptake of [N?Me?3H]-choline by synaptosomes from the central nervous system of Locusta migratoria

The accumulation of ³H-choline by isolated synaptosomes from the central nervous system of locust was studied at concentrations varying from 0.05 to 40μM. Kinetic analysis of the saturable process revealed a high-affinity and a low-affinity system. The high-affinity uptake was competitively inhibited by hemicholinium-3 and was absolutely dependent on external sodium. Elevated potassium concentrations inhibited choline uptake. The choline uptake by insect synaptosomes was found to be remarkably resistant to a variety of metabolic inhibitors. The reduced choline uptake under depolarizing conditions (high potassium concentration or veratridine) in the absence of calcium implies that electrochemical gradients are important for high-affinity choline uptake. Depolarization of preloaded synaptosomes under appropriate conditions resulted in a significant release of newly accumulated choline radioactivity.     

Sodium Transport in Capillaries Isolated from Rat Brain

Abstract: Brain capillary endothelial cells form a bloodbrain barrier (BBB) that appears to play a role in fluid and ion homeostasis in brain. One important transport system that may be involved in this regulatory function is the Na⁺,K⁺-ATPase that was previously demonstrated to be present in isolated brain capillaries. The goal of the present study was to identify additional Na⁺ transport systems in brain capillaries that might contribute to BBB function. Microvessels were isolated from rat brains and ²²Na ⁺ uptake by and efflux from the cells were studied. Total ²²Na ⁺ uptake was increased and the rate of ²²Na ⁺ efflux was decreased by ouabain, confirming the presence of Na⁺,K⁺-ATPase in capillary cells. After inhibition of Na⁺,K⁺-ATPase activity, another saturable Na ⁺ transport mechanism became apparent. Capillary uptake of ²²Na ⁺ was stimulated by an elevated concentration of Na ⁺or H⁺ inside the cells and inhibited by extracellular Na⁺, H⁺, Li⁺, and NH₄⁺. Amiloride inhibited ²²Na ⁺ uptake with a K_i between 10^?5 and 10^?6M but there was no effect of 1 mM furosemide on ²²Na⁺ uptake by the isolated microvessels. These results indicate the presence in brain capillaries of a transport system capable of mediating Na ⁺/ Na ⁺ and Na ⁺/H ⁺ exchange. As a similar transport system does not appear to be present on the luminal membrane of the brain capillary endothelial cell, it is proposed that Na ⁺/H ⁺ exchange occurs primarily across the antiluminal membrane.     

The Characterization of [3H] Adenosine Uptake into Rat Cerebral Cortical Synaptosomes

: Uptake of adenosine, a putative inhibitory transmitter or modulator, was investigated in rat cerebral cortical synaptosomes. The accumulation of [³H]adenosine into synaptosomes, using an adenosine concentration of 10 μ.m , was linear for 30 min at 37°C. The uptake appeared to be mediated by kinetically saturable processes with apparent K_m's of 1 μam (“high-affinity A”) and 5 μm (“high-affinity B”), both of which were partially sensitive to the presence of external sodium and calcium ions. Both uptake processes were partially inhibited by 2,4-dinitrophenol, implying the presence of active uptake and diffusional components. A study of the metabolites of adenosine taken up by the two uptake systems indicates that the major metabolites were adenosine and nucleotides. However, adenosine incorporated by the high-affinity A uptake system is more likely to form deaminated metabolites, such as hypoxanthine and inosine, indicating a possible functional difference between the two uptake processes. A detailed comparison of the inhibitory properties of certain adenosine analogues and other pharmacological agents has revealed differences between the two adenosine uptake systems. Since the glial contamination in synaptosomal preparations is well established, one of the uptake systems we observed in the present study might be of glial origin. This notion is supported by the findings that the K_m values and kinetic properties of papaverine action in the synaptosomal high-affinity A uptake system are similar to those of astrocytes reported in the literature. In conclusion, the uptake processes of synaptosomal preparations show that accumulation of adenosine into neuronal (and possibly glial) elements may play a major role in regulating the extracellular adenosine concentration. Uptake inhibitors, such as diazepam, may exert, at least in part, their pharmacological actions by interfering with the regulation of extracellular adenosine concentrations.     

Influence of membrane potential on the sodium-dependent uptake of gamma-aminobutyric acid by presynaptic nerve terminals: Experimental observations and theoretical considerations

Summary Sodium, potassium and veratridine were tested for their effects on the uptake of gamma-aminobutyric acid (GABA) by pinched-off presynaptic nerve terminals (synaptosomes). As noted by previous investigators, the uptake from media containing 1 m GABA (high-affinity uptake) is markedly Na-dependent; the uptake averaged 65 pmoles/mg synaptosome protein × min, with [Na]₀=145mm and [K]₀=5mm, and declined by about 90% when the external Na concentration ([Na]₀) was reduced to 13mm (Na replaced by Li). The relationship between [Na]₀ and GABA uptake was sigmoid, suggesting that two or more Na⁺ ions may be required to activate the uptake of one GABA molecule. Thermodynamic considerations indicate that with a Na⁺/GABA stoichiometry of 21, the Na electrochemical gradient, alone, could provide sufficient energy to maintain a maximum steady-state GABA gradient ([GABA] _i /[GABA]₀) of about 10⁴ across the plasma membrane of GABA-nergic terminals.In Ca-free media with constant [Na]₀, GABA uptake was inhibited, without delay, by increasing [K]₀ or by introducing 75 m veratridine; the effect of veratridine was blocked by 200nm tetrodotoxin. The rapid onset (within 10 sec) of the veratridine and elevated-K effects implies that alterations in intra-terminal ion concentrations are not responsible for the inhibition. The uptake of GABA was inversely proportional to log [K]₀. These observations are consistent with the idea that the inhibitory effects of both veratridine and elevated [K]₀ may be a consequence of their depolarizing action. The data are discussed in terms of a barrier model (Hall, J. E., Mead, C.A., Szabo, G. 1973.J. Membrane Biol. 11:75) which relates carrier-mediated ionic flux to membrane potential.     

Effect of Adenosine on Na+ and Cl− Currents in A6 Monolayers. Receptor Localization and Messenger Involvement

The effect of adenosine regulation on sodium and chloride transport was examined in cultured A₆ renal epithelial cells. Adenosine and its analogue N⁶-cyclopentyladenosine (CPA) had different effects on short-circuit current (I _sc) depending on the side of addition. Basolateral CPA addition induced an approximately threefold increase of the I _sc that reached a maximum effect 20 min after addition and was completely inhibited by preincubation with either an A₂ selective antagonist, CSC, or the sodium channel blocker, amiloride. Apical CPA addition induced a biphasic I _sc response characterized by a rapid fourfold transient increase over its baseline followed by a decline and a plateau phase that were amiloride insensitive. The A₁ adenosine antagonist, CPX, completely prevented this response. This I _sc response to apical CPA was also strongly reduced in Cl⁻-free media and was significantly inhibited either by basolateral bumetanide or apical DPC preincubation. Only basolateral CPA addition was able to induce an increase in cAMP level. CPA, added to cells in suspension, caused a rapid rise in [Ca²⁺] _i that was antagonized by CPX, not affected by CSC and prevented by thapsigargin preincubation. These data suggest that basolateral CPA regulates active sodium transport via A₂ adenosine receptors stimulating adenylate cyclase while apical CPA regulates Cl⁻ secretion via A₁ receptor-mediated changes in [Ca²⁺] _i .     

Phosphate uptake in Chara: membrane transport via Na/Pi cotransport

Phosphate uptake in the freshwater charophyte plant Chara corallina was found to be strongly dependent on the presence of Na in the external medium. Based on the reciprocal stimulations of ³²Pi uptake by Na and ²²Na uptake by Pi, the logical mechanism for Pi uptake appears to be a nNa/Pi symport with a half‐maximal stimulation (K_m) for Na of approximately 300 μM and a K_m for Pi of approximately 10 μM . Comparison of the stimulations of ³²Pi and ²²Na influxes at pH 6 gives a stoichiometry of Na : Pi of 5·68. The reduction in Pi influx with increasing pH is consistent with the transported species being the monovalent H₂PO₄^?. In voltage‐clamp experiments, currents elicited by Pi in the presence of Na were equivalent to an influx of positive charge which exceeded the measured influxes of ³²P by a factor of 6·26. Intracellular perfusion was used to examine the dependence of Pi influx on ATP and Na. In perfused cells, Pi influx was low when ATP was absent from the internal medium or Na was absent from the external medium. Addition of ATP alone had little effect whereas addition of Na alone increased the ³²Pi influx slightly. Addition of both ATP and Na together restored Pi influx to rates comparable to those of intact cells. It is suggested that the ATP is required for membrane hyperpolarization which in turn drives the highly electrogenic flux of Pi with up to 6 Na. However, consideration of the electrochemical potential differences for Na and Pi at pH less than 6 shows that nNa/Pi would not be feasible. It is suggested that at low pH, H⁺ may substitute for Na.