Inactivation of depolarization-induced calcium uptake in rat brain synaptosomes

The inactivation of depolarization-induced Ca uptake into rat brain synaptosomes was demonstrated biochemically by comparing⁴⁵Ca fluxes after various intervals of predepolarization achieved by abruptly increasing {K⁺}₀. The chemical composition of the medium was maintained throughout the predepolarization and Ca uptake steps. Under these conditions, inactivation was dependent on depolarization, i.e., basal unstimulated Ca uptake in the presence of 5 mM {K⁺}₀ did not inactivate. Inactivation of stimulated Ca uptake was dependent on the predepolarization interval, moderately dependent on {Ca}₀ and relatively independent of membrane potential, i.e., {K⁺}₀ and ions such as Ni²⁺ and Co²⁺ that blocked Ca uptake. Both cinnarizine and lidoflazine blocked stimulated Ca uptake in a concentration-dependent manner without affecting the % inactivation. Although the amount of stimulated uptake increased greatly between 10 and 30°C, the % inactivation was unaffected by temperature. These findings suggest that inactivation of the presynaptic Ca uptake is an intrinsic property of the channel independent of calcium uptake.     

Requirement of Glycogenolysis for Uptake of Increased Extracellular K+ in Astrocytes: Potential Implications for K+ Homeostasis and Glycogen Usage in Brain

The importance of astrocytic K⁺ uptake for extracellular K⁺ ([K⁺]_e) clearance during neuronal stimulation or pathophysiological conditions is increasingly acknowledged. It occurs by preferential stimulation of the astrocytic Na⁺,K⁺-ATPase, which has higher K_m and V_max values than its neuronal counterpart, at more highly increased [K⁺]_e with additional support of the cotransporter NKCC1. Triggered by a recent DiNuzzo et al. paper, we used administration of the glycogenolysis inhibitor DAB to primary cultures of mouse astrocytes to determine whether K⁺ uptake required K⁺-stimulated glycogenolysis. KCl was increased by either 5 mM (stimulating only the Na⁺,K⁺-ATPase) or 10 mM (stimulating both transporters) in glucose-containing saline media prepared to become iso-osmotic after the addition. DAB completely inhibited both uptakes, the Na⁺,K⁺-ATPase-mediated by preventing Na⁺ uptake for stimulation of its intracellular Na⁺-activated site, and the NKCC1-mediated uptake by inhibition of depolarization- and L-channel-mediated Ca²⁺ uptake. Drugs inhibiting the signaling pathways involved in either of these processes also abolished K⁺ uptake. Assuming similar in vivo characteristics, partly supported by literature data, K⁺-stimulated astrocytic K⁺ uptake must discontinue after normalization of extracellular K⁺. This will allow Kir1.4-mediated release and reuptake by the less powerful neuronal Na⁺,K⁺-ATPase.     

GABA and glutamate fluxes in developing nerve endings

Radiolabeled GABA and glutamate transport into 7 day, 14 day and adult cortical nerve ending preparations was examined. Transport was measured at several Na⁺ concentrations, 19, 27, 43 and 121 mM, and at two temperatures, 15 and 30°C. K_m and V_max values were calculated for all experimental conditions by means of Wilkinson (1961) analysis. A comparison of the day 14 and adult data shows higher K_m values at all Na⁺ concentrations on day 14 for both GABA and glutamate transport. In addition, the temperature dependence of transport was attenuated in the day 14 preparation. Finally, the specificity of GABA transport, as measured by the use of the transport inhibitors β-alanine and 2,4-diaminobutyric acid, was not different between the day 14 and adult preparations. Overall, it is concluded that both GABA and glutamate transport into day 14 nerve endings behave as if “adult” transporter molecules were existing in a more fluid lipid environment, which is the situation found in synaptic membranes prepared from day 14 nerve endings (Hitzemann and Johnson, 1983).Glutamate and GABA transport into 7 day nerve endings is complex and shows marked differences from the day 14 and adult data. Day 7 GABA transport was significantly more sensitive to β-alanine inhibition. Day 7 transport was more sensitive to Na⁺ manipulation and the temperature dependent kinetics show unique Na⁺ effects not seen in the day 14 or adult preparations. For example, at 19 mM Na⁺, 7 day glutamate transport was more temperature dependent than adult transport but as the Na⁺ concentration was increased the reverse was true. The opposite situation for temperature-Na⁺ effects was seen for GABA transport. Finally, no Ca⁺²-dependent component of GABA release could be found in 7 day nerve endings while a significant component was found at day 14. Overall, it is concluded that both glutamate and GABA fluxes in 7 day nerve endings differ both qualitatively from that seen in both day 14 and adult nerve endings.     

Endothelin 1 Stimulates Na+,K+-ATPase and Na+-K+-Cl− Cotransport Through ETA Receptors and Protein Kinase C-Dependent Pathway in Cerebral Capillary Endothelium

Abstract: The effect of endothelins (ET-1 and ET-3) on ⁸⁶Rb⁺ uptake as a measure of K⁺ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K⁺ uptake (EC₅₀ = 0.60 ± 0.15 and 21.5 ± 4.1 nM, respectively), which was inhibited by the selective ET_A receptor antagonist BQ 123 (cyclo-d -Trp-d -Asp-Pro-d -Val-Leu). Neither the selective ET_B agonists IRL 1620 [N-succinyl-(Glu⁹,-Ala^11,15)-ET-1] and sarafotoxin S6c, nor the ET_B receptor antagonist IRL 1038 [(Cys¹¹,Cys¹⁵)-ET-1] had any effect on K⁺ uptake. Ouabain (inhibitor of Na⁺,K⁺-ATPase) and bumetanide (inhibitor of Na⁺-K⁺-Cl^? cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K⁺ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport. ET-1-but not PMA-stimulated K⁺ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na⁺/H⁺ exchange system), suggesting a linkage of Na⁺/H⁺ exchange with ET-1-stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity that is not mediated by PKC.     

The K+- and HCO3−-stimulated phosphatase activities in renal microsomes of rats

The K⁺-stimulated phosphatase activity of microsomes from rat kidney was not inhibited by l-phenylalanine, but the HCO₃^?-stimulated phosphatase activity was markedly inhibited by l-phenylalanine. Valinomycin enhanced the HCO₃^?-stimulated phosphatase activity, but did not enhance the K⁺-stimulated phosphatase activity. Ouabain did not inhibit the HCO₃^?-stimulated phosphatase activity, but inhibited the K⁺-stimulated phosphatase activity.The renal K⁺-stimulated phosphatase activity was suppressed to 40% of the control values by adrenalectomy, but the renal HCO₃^?-stimulated phosphatase activity was little suppressed by adrenalectomy. The renal K⁺-stimulated phosphatase activity in intact and adrenalectomized rats was found to be significantly elevated, in a manner similar to the elevation of the renal (Na⁺ + K⁺)-ATPase activity by aldosterone treatment (P < 0.02).     

Bilayer orientation of membrane-bound NH2 groups across microsomal vesicles and their role in the function of gastric K+-stimulated adenosine triphosphatase

The transversal distribution of the free NH₂ groups associated with phosphatidyl ethanolamine and the intrinsic membrane proteins of the purified pig gastric microsomes was quantitated and their relations to the function of the gastric K⁺-stimulated ATPase was investigated. Three different chemical probes such as 2,4,6-trinitrobenzene sulfonic acid (TNBS), 1-fluoro-2,4-dinitrobenzene (FDNB), and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) were used for the study. The structure-function relationship of the membrane NH₂ groups was studied after modification with the probes under various conditions and relating the inhibition of the K⁺-stimulated ATPase to the ATPase-dependent H⁺ accumulation by the gastric microsomal vesicles. TNBS (2 mm) inhibits nearly completely the K⁺-stimulated ATPase and the vesicular dye accumulation, both in presence and absence of valinomycin plus K⁺. Both the K⁺-ATPase and dye uptake were largely (about 50%) protected against TNBS inhibition if the treatment with TNBS was carried out in presence of 2 mm ATP. TNBS and FDNB labeled 70% of the total microsomal PE; the intra- and extravesicular orientation being 48 and 22%, respectively. The presence or absence of ATP did not have any effect on the TNBS labeling of microsomal PE. ATP, however, significantly (P < 0.05) reduced the labeling of protein-bound NH₂ groups of gastric microsomes by TNBS. The intra- and extravesicular orientation of the protein NH₂ groups were 60 and 40%, respectively. Eighteen percent of the total protein-NH₂ appeared to be associated with the K⁺-stimulated ATPase; the rest being associated with non-ATPase proteins of the microsomes. About half (50%) of the total free NH₂ groups of the K⁺-stimulated ATPase were exposed to the vesicle exterior and were found to play critical roles in gastric ATPase function. The generation of florescence after MDPF conjugation of gastric microsomes was largely (50%) inhibited by ATP. ATP also protected completely the MDPF inhibition of gastric K⁺-stimulated ATPase and dye uptake.     

Isotopic labeling of synaptosomes that accumulate choline and the effect of narcotic drugs

Subcellular studies of choline uptake of rat striatum indicated a correspondence between the Na⁺-dependent uptake and choline acetyltransferase (ChAc), whereas there was a lack of correspondence between the Na⁺-independent uptake and ChAc. Subcellular studies also showed a correspondence between the Na⁺-dependent uptake and hemicholinium-3 inhibition, and more important, particles that accumulate choline were shown to consist of at least two subcellular populations. A comparison was made of kinetic data from three areas of the rat brain: corpus striatum, cerebral cortex, and hypothalamus. Taken together, our data on choline uptake give added support to the idea that the Na⁺-dependent choline transport is concentrated in the striatum and specifically related to cholinergic nerve endings. Morphine and methadone in vitro inhibited the Na⁺-dependent choline uptake. In vivo morphine induced a significant lowering of theV _max in the rat cerebral cortex, but not in the striatum. This finding is consistent with the known action of morphine on acetylcholine turnover.Preliminary reports of this work were presented at the Fifth Meeting of the American Society for Neurochemistry in New Orleans, March 1974, and the Fall ASPET Meeting in Montreal, August 1974 (1,2).     

The localization of adenosine triphosphatases in morphologically characterized subcellular fractions of guinea-pig brain

1. The distribution of adenosine triphosphatase was studied in morphologically characterized subcellular fractions of guinea-pig brain. The conditions of homogenization were selected so as to favour the survival of nerve endings as organized structures. 2. A fraction consisting mainly of the external membranes of nerve endings was rich in a ouabain-sensitive Na⁺–K⁺-stimulated adenosine triphosphatase which closely resembled that present in the classical microsomal fraction studied by other workers, but which showed a higher specific activity. 3. A dinitrophenol-stimulated adenosine triphosphatase was located in the nerve-ending mitochondria. 4. The synaptic-vesicle fraction contained a small amount of adenosine triphosphatase that differed in its response to several ions and other compounds from the membrane, myelin and mitochondrial fractions, indicating freedom from contamination by these elements.     

Reconstitution and characterization of a sodium-stimulated active aminoisobutyric acid transport system derived from partially purified plasma membranes from mouse fibroblasts transformed by simian virus 40: comparison of reconstituted vesicles with native membrane vesicles

A Na⁺-specific and Na⁺-stimulated active α-aminoisobutyric acid transport system was reconstituted from plasma membranes isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40. The plasma membranes were treated with dimethylmaleic anhydride and then extracted with 2% cholate. The cholate-solubilized supernatant proteins were combined with exogenous phospholipids and eluted through a Sephadex G-50 column. This yielded reconstituted vesicles which in the presence of Na⁺ could actively transport α-aminoisobutyric acid as shown by the transient accumulation above the equilibrium level (overshoot). The overshoot was not obtained with other monovalent cations such as K⁺, Li⁺, and choline⁺. The electrochemical effect of the lipophilic anion, SCN^?, led to greater α-aminoisobutyric acid uptake as compared to that observed with Cl^? or SO₄^2?. The Na⁺-stimulated transport of a-aminoisobutyric acid was a saturable process with an apparent K_m of 2 mm. Studies of the inhibition of α-aminoisobutyric acid transport by other amino acids showed that methylaminoisobutyric acid [specifically transported by A system (alanine preferring)]had a pronounced inhibitory effect on a-aminoisobutyric acid uptake in contrast to the slight inhibitory effect produced by phenylalanine [primarily transported by L system (leucine preferring)]. The results show that the reconstituted vesicles, prepared from partially purified membrane proteins and exogenous phospholipids, regained the same important transport properties of native membrane vesicles, i.e., Na⁺-specific and Na⁺-stimulated concentrative α-aminoisobutyric acid uptake.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

The Influence of the Intracellular Potential on Potassium Uptake by Beetroot Tissue

Intracellular potentials were measured in beetroot tissue during the steady-state uptake of K⁺ from various solutions. In solutions containing bicarbonate, the membrane potential becomes up to 70 mv more negative than the estimated equilibrium potential for K⁺. The uptake of K⁺ from such solutions is correlated with variations in the potential, both when the bicarbonate concentration is changed and also when the metabolic activity of the tissue is changed by washing in water for various periods. However, the estimated permeability to K⁺ varies from 0.4 x 10^-7 to 1.5 x 10^-7 cm·sec^-1. It is postulated that the change of potential arises from the metabolic transport of HCO₃^- into the cell or H⁺ outwards, and that the associated uptake of K⁺ is partly or entirely by passive diffusion across the cell membrane. In contrast, K⁺ uptake from KCl solutions is not accompanied by any significant change in the membrane potential, which remains relatively close to the K⁺ equilibrium potential. In solutions containing both KHCO₃ and KCl, it appears that an amount of K⁺ equal to the influx of Cl^- is taken up independently of the potential, while the component of K⁺ uptake which is not balanced by Cl^- uptake is related to the potential in the manner described. These results suggest that K⁺ uptake is linked to Cl^- uptake in an electrically neutral active transport process.     

UPTAKE OF ACETYLCHOLINE AND CHOLINE INTO RAT BRAIN CORTICAL SLICES AND SYNAPTOSOMES AS RELATED TO 32Pi, INCORPORATION INTO THEIR PHOSPHOLIPIDS

—The role of ACh-stimulated ³²P_i incorporation into the phospholipids of rat cerebral cortex slices and isolated nerve endings (synaptosomes) has been studied. ACh stimulation is not connected with any carrier-mediated uptake of ACh. Such uptake may occur in slices in the presence of the anticholinesterase Sarin but barely in the presence of eserine. Regardless of the nature of the anticholinesterase used, rat cerebral cortex synaptosomes that respire and show high and low affinity choline uptake do not accumulate ACh against a concentration gradient. At exogenous ACh concentrations of 10^–5m and above, some ACh enters the synaptosomes by diffusion and significantly stimulates ³²P_i incorporation into phosphatidic acid. It is discussed whether, in isolated nerve endings, an increase in cytoplasmic ACh concentration due to diffusion may induce vesicle turnover to keep a balance between ‘free’ and bound ACh or if a presynaptic ACh receptor is responsible for the observed changes in phosphatidic acid. The distribution of accumulated radioactivity derived from exogenous choline and ACh respectively between ACh, choline, phosphorylcholine and betaine has been studied in slices and isolated nerve endings.     

Four types of potassium currents in motor nerve terminals of snake

The experiments were perfomed on transvcrsus abdominis muscle of Elaphe dione by subendothelial recording. The results indicate that in snake motor nerve endings there exist four types of K* channels, i.e. voltage-dependent fast and slow K channels, Ca2 -activated K channel and ATP-sensitive K channel, (i) The typical wave form of snake terminal current was the double-peaked negativity in standard solution. The first peak was at-tributed to Na influx (INa) in nodes of Ranvier. The second one was blocked by 3, 4-aminopyridine (3, 4-DAP) or te-traethylammonium (TEA), which corresponded to fast K outward current (IKF) through the fast K* channels in terminal part, (ii) After IKF as well as the slow K current (IKS) were blocked by 3, 4-DAP, the TEA-sensitive Ca2 -dependent K current (IK(Ca)) passing through Ca2 -activated K channel was revealed, whose amplitude depended on [K ]and [Ca2 ] It was blocked by Ba2 , Cd2 or Co2 . (iii) IK.F and IK(Ca) were blocked by TEA, while IK.S was retained. It     

Cadmium alteration of root physiology and potassium ion fluxes

Segments of oat (Avena sativa L.) roots which had been exposed to 1 millimolar CdSO₄ in quarter-strength Hoagland No. 1 solution exhibited decreased respiratory rates, ATP levels, membrane-bound ATPase activity, and reduced K⁺ fluxes. Respiration and ATP levels were decreased after a 2-hour treatment with 1 millimolar CdSO₄ to 65 and 75%, respectively, of control rates. A membrane-bound, Mg²⁺-dependent, K⁺-stimulated acid ATPase was rapidly inhibited to 12% of control activity in the presence of 1 millimolar CdSO₄. Potassium uptake into root segments was inhibited to 80% of control values after 30 minutes in the presence of CdSO₄. A 2-hour pretreatment of root segments with CdSO₄ inhibited K⁺ uptake to 15% of control values. Cytoplasmic K⁺ efflux was inhibited with 1 millimolar CdSO₄.

The rates and the degree of Cd²⁺ inhibition of the parameters listed above suggest that one of the first sites of Cd²⁺ action is the plasmalemma K⁺ carrier (ATPase) in oat roots.

     

Induction of a high-capacity nitrate-uptake mechanism in barley roots prompted by nitrate uptake through a constitutive low-capacity mechanism

Roots of nitrate-starved and nitrate-pretreated seedlings of Hordeum vulgare were used to investigate the induction of a high-capacity uptake mechanism for nitrate. When exposed to 0.2 mmol·l^-1KNO₃, nitrate-starved roots took up nitrate at a rate of approx. 1 mol·(g FW)^-1·h^-1; K⁺ was absorbed at a rate ten-times higher. Nitrate uptake accelerated after a lag of about 1 h, until it matched the rate of K⁺ uptake about 4 h later. p-Fluorophenylalanine (FPA), which prevents the synthesis of functioning proteins, suppressed the development of the high-capacity mechanism. Pretreatment of the roots with 0.2 mmol·l^-1 Ca(NO₃)₂ for 24 h established the high-capacity mechanism. Pretreated roots were able to absorb nitrate at high rates immediately upon exposure to 0.2 mmol·l^-1KNO₃, in the absence or presence of FPA. The high-capacity mechanism, once established, appeared to have a protein turnover as slow as that of the low-capacity mechanism or that of the mechanism involved in the uptake of K⁺. In contrast, the mechanisms for the transport of nitrate and K⁺ into the xylem vessels were completely blocked by FPA within 1 h of application, confirming earlier evidence for a rapid turnover of the transport proteins in the xylem parenchyma.Nitrate reduction proceeded at rates which were roughly one-tenth as large as the rates of the respective nitrate-uptake processes, indicating that nitrate-reductase activity was determined by the rate of nitrate uptake and not vice versa.We conclude that the formation of a high-capacity nitrate-uptake mechanism in barley roots occurs in response to nitrate uptake through a constitutive mechanism of low capacity which appears to function as a sensing mechanism for nitrate in the environment of the roots.Abbreviation FPA p-fluorophenylalanine     

Ionic and Metabolic Requirements for High-Affinity Choline Uptake and Acetylcholine Synthesis in Nerve Terminals at a Neuromuscular Junction

Abstract: We have shown previously that in the chick ciliary nerve-iris muscle preparation Na⁺-dependent high-affinity choline uptake was confined to the nerve terminals. In this paper the sodium-dependent high-affinity choline uptake (SDHACU), which is coupled to acetylcholine (ACh) synthesis, was further characterized by measuring uptake of [³H]choline and its conversion to [³hjach under a variety of ionic and metabolic perturbations. Mannitol equilibration with the extracellular space was found to occur in less than 1 min in this preparation. Na⁺-dependent choline (Ch⁺) uptake was shown to be linear for 16 min and to reach an equilibrium before Na⁺-independent Ch⁺ uptake, which continued to increase for 60 min. Elevated [K⁺]₀ concentrations inhibited Ch⁺ uptake and ACh synthesis. Glycolytic and respiratory inhibitors also reduced both processes, as did ouabain and omission of [K⁺]₀. Incubation conditions that reduce transmitter release had no effect on inhibition by high [K⁺]₀. Reduction of SDHACU and sodium-dependent ACh synthesis by depolarization with high [K⁺]₀ or by inhibition of Na, K-ATPase implies that the electrochemical gradients for Ch⁺ and Na⁺ are important in providing a driving force for high-affinity Ch⁺ uptake. The inhibition by metabolic blockers suggests active transport, but the effects may be indirect, caused by reduced Na, K-ATPase activity and alterations in membrane potential. While most metabolic inhibitors exerted parallel effects on both Ch⁺ uptake and ACh synthesis, in some cases Ch⁺ uptake was more strongly inhibited than ACh synthesis. This occurred in preparations incubated with high [K⁺]₀ and ouabain. Na⁺-dependent Ch⁺ uptake and ACh synthesis were found to be temperature-dependent with a Q₁₀ (20–30°) of 3.6 and 6.6, respectively and a Q₁₀ (30–40°) of 1.3 and 1.0, respectively. Inhibition of acetylcholinesterase by paraoxon increases to 92% the proportion of the Ch⁺ taken up which is converted to ACh. ACh did not reduce Ch⁺ transport when present at 100 μM.     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

Uptake and release of possible false transmitter amino acids by rat brain tissue

—The uptake of l [¹⁴C]glutamine by a crude isolated nerve ending fraction of rat brain was found to be linear with time for at least 5 min, profoundly temperature-dependent, apparently half-saturated at a substrate concentration of 0·26 mm , partially inhibited by dinitrophenol and ouabain and elevated [K⁺], weakly Na⁺-dependent, poorly inhibited by drugs which block uptake of biogenic amines and more strongly inhibited by glutamic acid (IC₅₀= 0·5mm ) than by aspartic acid, GABA, glycine or methionine. The [¹⁴C]glutamine taken up appeared to be associated with nerve endings and was released by membrane-disruption; about 20 per cent was associated with free mitochondria. Glutamine, δ-aminolevulinic acid and several other amino acids were poor inhibitors of [³H]GABA-uptake; δ-aminolevulinic acid was a poor inhibitor of [³H]glutamine-uptake, whereas glutamine was a moderately effective competitive inhibitor (K_i= 1 mm ). [¹⁴C]glutamine and [³H]GABA were released from brain slices by electrical stimulation or 50 mm K⁺, while labeled δ-aminolevulinic acid, leucine, urea, amphetamine and tyramine were poorly released. [¹⁴C]glutamine was not released by unlabeled glutamate or several aromatic amines. We conclude that the neuropsychiatric features of porphyria are not likely due to a ‘false transmitter’ role for δ-aminolevulinic acid although such a role for glutamine in hepatic encephalopathy or other neuropsychiatric diseases should be considered.     

Discrimination between Alkali Metal Cations by Yeast : II. Cation interactions in transport

K⁺ is a competitive inhibitor of the uptake of the other alkali metal cations by yeast. Rb⁺ is a competitive inhibitor of K⁺ uptake, but Li⁺, Na⁺, and Cs⁺ act like H⁺. At relatively low concentrations they behave as apparent noncompetitive inhibitors of K⁺ transport, but the inhibition is incomplete. At higher concentrations they inhibit the remaining K⁺ transport competitively. Ca⁺⁺ and Mg⁺⁺ in relatively low concentrations partially inhibit K⁺ transport in an apparently noncompetitive manner although their affinity for the transport site is very low. In each case, in concentrations that produce "noncompetitive" inhibition, very little of the inhibiting cation is transported into the cell. Competitive inhibition is accompanied by appreciable uptake of the inhibiting cation. The apparently noncompetitive effect of other cations is reversed by K⁺ concentrations much higher than those necessary to essentially "saturate" the transport system. A model is proposed which can account for the inhibition kinetics. This model is based on two cation-binding sites for which cations compete, a carrier or transporting site, and a second nontransporting (modifier) site with a different array of affinities for cations. The association of certain cations with the modifier site leads to a reduction in the turnover of the carrier, the degree of reduction depending on the cation bound to the modifier site and on the cation being transported.     

ATP-dependent chloride transport in plasma membrane vesicles from Aplysia intestine

A Cl⁻-stimulated ATPase activity, which is sensitive to both thiocyanate and vanadate, has been localized to the plasma membrane of Aplysia enterocytes. Utilizing plasma membrane vesicles from Aplysia enterocytes, ATP stimulated Cl⁻ uptake to approximately 2.5-times that of control in a Na⁺, K⁺ and HCO₃⁻-free medium. This ATP-dependent Cl⁻ uptake was sensitive to both thiocyanate and vanadate. These results are consistent with the hypothesis that the active Cl⁻ absorptive process in Aplysia intestine could be a Cl⁻-stimulated ATPase found in the enterocyte plasma membrane.