Temporal Characteristics of Potassium-Stimulated Acetylcholine Release and Inactivation of Calcium Influx in Rat Brain Synaptosomes

Abstract: The time course of Ca²⁺-dependent [³H]acetylcholine ([³H]ACh) release and inactivation of ⁴⁵Ca²⁺ entry were examined in rat brain synaptosomes depolarized by 45 m M [K⁺]_o. Under conditions where the intrasynaptosomal stores of releasable [³H]ACh were neither exhausted nor replenished in the course of stimulation, the K⁺-evoked release consisted of a major (40% of the releasable [³H]ACh pool), rapidly terminating phase ( t _1/2 = 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K⁺-stimulated Ca²⁺ entry suggests the presence of fast-inactivating, slow-inactivating, and noninactivating, or very slowly inactivating, components. The fast-inactivating component of the K⁺-stimulated Ca²⁺ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K⁺ stimulus. The noninactivating Ca²⁺ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K⁺]_o the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage-dependent Ca channels.     

Uptake and Release of N-Methyl-d-Aspartate by Rat Brain Slices

Abstract: The excitant amino acid, N -methyl- d -aspartate, was actively taken up by slices of rat cerebral cortex. This uptake was Na⁺ - and temperature-dependent, but was relatively inefficient (K_m 3 MM, V_max 0.07 μmol/g/min) compared with that of other acidic amino acids. The uptake of N -methyl- d -aspartate does not appear to have a rate-limiting influence on the time course of N -methyl- d -aspartate-induced excitation since potent uptake inhibitors, such as threo-3-hydroxy- l -aspartate, do not influence the excitant action of N -methyl- d -aspartate. The relatively prolonged excitant action of this acidic amino acid may be the result of relatively slow dissociation of the activated receptor complex. Reloaded N -methyl- d -aspartate can be released from rat brain slices by stimulation with K⁺ ions. Such K⁺-stimulated release appeared to be Ca²⁺-independent, unlike the K⁺-stimulated release of preloaded d -aspartate. These findings suggest that N -methyl- d -aspartate may be a weak but selective substrate for a glial acidic amino acid uptake system.     

Dopaminergic Properties of Cultured Rat Carotid Body Chemoreceptors Grown in Normoxic and Hypoxic Environments

Abstract: Using dissociated carotid body (CB) cultures prepared from neonatal (postnatal days 5–7; P7) or juvenile (postnatal day 19–20; P20) rats, we compared catecholaminergic properties and mechanisms of O₂ sensing in glomus cells grown in normoxic (Nox; 20% O₂) and chronically hypoxic (CHox; 6% O₂) environments for up to 2 weeks. In Nox cultures, basal dopamine (DA) release, determined by HPLC and normalized to the number of tyrosine hydroxylase-positive glomus cells present, was similar for P7 and P20 cultures (∼0.3 pmol/1,000 cells/15 min) and was unaffected by culture duration (2 vs. 12 days). Acute hypoxia (5 and 10% O₂) caused a dose-dependent stimulation (6× and 3× basal, respectively) in DA release, that was inhibited by nifedipine (10 µ M ). DA release was also stimulated by high extracellular K⁺ (30 m M ) and iberiotoxin (200 n M ), a selective blocker of P o ₂-regulated, Ca-dependent K⁺ channel in glomus cells. The stimulatory effect of iberiotoxin was similar to 5% O₂ in P20 cultures, but substantially less (about one-half) in P7 cultures. In contrast, in CHox cultures, basal DA release was substantially elevated, ∼8× Nox levels, although this did not correlate with significant differences in stores. Further, whereas acute hypoxia (5% O₂) and high K⁺ also stimulated DA release in CHox cultures (∼2× and ∼3× basal), iberiotoxin (200 n M ) did not. Thus, after chronic hypoxia in vitro, there is an enhanced basal catecholamine release and an apparent down-regulation of functional Ca-dependent K⁺ channels in CB chemoreceptors. These cellular adaptations may relate to changes in CB chemosensitivity during chronic hypoxemia.     

Influence of Potassium Concentration in Microdialysis Perfusate on Basal and Stimulated Striatal Dopamine Release: Effect of Ceruletide, a Cholecystokinin-Related Peptide

Abstract: The in vivo microdialysis method was used to study the effect of the cholecystokinin-related peptide, ceruletide, on extracellular levels of dopamine (DA) in the striatum following perfusion with various K⁺ concentrations. Increasing the K⁺ concentration in the perfusate from 4 to 15 or 17.5 m M did not change basal DA release or release evoked by electrical stimulation of the medial forebrain bundle (MFB). However, when the perfusing solution contained 20 or 30 m M K⁺, dose-dependent reductions of both basal and MFB-stimulated DA release occurred. Subcutaneous administration of ceruletide at 160 μg/kg had no influence on the basal or MFB-stimulated DA release with 4 or 15 m M K⁺ in the perfusate. However, after perfusion with 17.5 m M K⁺, ceruletide significantly attenuated the basal and MFB-stimulated DA release. Carbachol (10 μ M ) locally applied via the dialysis probe also attenuated MFB-stimulated DA release after perfusion with 17.5 m M K⁺. From these results, we conclude that under appropriate depolarization of striatal DA terminals, ceruletide induces further depolarization and inactivation of nigrostriatal DA terminals. The present data suggest that this effect may be mediated via intrinsic cholinergic neurons in the striatum.     

In Vitro Effect of Tetanus Toxin on GAB A Release from Rat Hippocampal Slices

Abstract Ca²⁺-dependent K⁺-stimulated γ-aminobutyric acid release from rat hippocampal slices was reduced about 30% by pre-incubation of the slices with 10⁴ mouse LD₅₀/ml tetanus toxin for 3 h at 37°C.     

A Toxin Fraction (FTX) from the Funnel-Web Spider Poison Inhibits Dihydropyridine-Insensitive Ca2+ Channels Coupled to Catecholamine Release in Bovine Adrenal Chromaffin Cells

Abstract: In adrenal chromaffin cells, depolarization-evoked Ca²⁺ influx and catecholamine release are partially blocked by blockers of L-type voltage-sensitive Ca²⁺ channels. We have now evaluated the sensitivity of the dihydropyridine-resistant components of Ca²⁺ influx and catecholamine release to a toxin fraction (FTX) from the funnel-web spider poison, which is known to block P-type channels in mammalian neurons. FTX (1:4,000 dilution, with respect to the original fraction) inhibited K⁺-depolarization-induced Ca²⁺ influx by 50%, as monitored with fura-2, whereas nitrendipine (0.1–1 μ M ) and FTX (3:3), a synthetic FTX analogue (1 m M ), blocked the [Ca²⁺]_i transients by 35 and 30%, respectively. When tested together, FTX and nitrendipine reduced the [Ca²⁺]_i transients by 70%. FTX or nitrendipine reduced adrenaline and noradrenaline release by ∼80 and 70%, respectively, but both substances together abolished the K⁺-evoked catecholamine release, as measured by HPLC. The ω-conotoxin GVIA (0.5 μ M ) was without effect on K⁺-stimulated ⁴⁵Ca²⁺ uptake. Our results indicate that FTX blocks dihydropyridine- and ω-conotoxin-insensitive Ca²⁺ channels that, together with L-type voltage-sensitive Ca²⁺ channels, are coupled to catecholamine release.     

NMDA-mediated modulation of dopamine release is modified in rat prefrontal cortex and nucleus accumbens after chronic nicotine treatment

In this study, we investigate the effects of chronic administration of (−)nicotine on the function of the NMDA-mediated modulation of [³H]dopamine (DA) release in rat prefrontal cortex (PFC) and nucleus accumbens (NAc). In the PFC synaptosomes NMDA in a concentration-dependent manner evoked [³H]DA release in rats chronically treated with vehicle (14 days) with an EC₅₀ of 13.1 ± 2.0 μM. The NMDA-evoked overflow of the [³H]DA in PFC nerve endings of rats treated with (−)nicotine was significantly lower (−43%) than in vehicle treated rats. The EC₅₀ was 9.0 ± 1.4 μM. Exposure of NAc synaptosomes of rats treated with vehicle to NMDA produced an increase in [³H]DA overflow with an EC₅₀ of 14.5 ± 5.5 μM. This effect was significantly enhanced in chronically treated animals. The EC₅₀ was 10.5 ± 0.5 μM. The K⁺-evoked release of [³H]DA was not modified by the (−)nicotine administration. Both the changes of the NMDA-evoked [³H]DA overflow in the NAc and PFC disappeared after 14 days withdrawal. The results show that chronic (−)nicotine differentially affects the NMDA-mediated [³H]DA release in the PFC and NAc of the rat.     

Thyrotrophin-Releasing Hormone Analogues Increase Dopamine Release from Slices of Rat Brain

Abstract: Rat brain slices were incubated with a high concentration of K⁺, thyrotrophin-releasing hormone (TRH), or one of two biologically stable TRH analogues (CG 3509 or RX 77368). Basal release of endogenous dopamine, measured by electrochemical detection, was increased by K⁺ (30 m M ) from slices of hypothalamus, septum, nucleus accumbens, and striatum. CG 3509 (10⁵–10⁻³ M ) increased the release of dopamine from slices of nucleus accumbens, septum, and hypothalamus in a dose-dependent fashion, whereas RX 77368 (10⁻⁴ M ) increased the release of dopamine from the septum only. Neither analogue increased the release of striatal dopamine. The results provide further evidence for specific regional interactions between TRH and dopamine in rat brain.     

Turgor- dependent membrane permeability in relation to calcium level

The relationship between the inhibiting effect of Ca²⁺ and of low turgor pressure on K⁺ release from fresh-cut discs of carrot ( Daucus carota var. Nantes) storage tissue was studied. A range of Ca²⁺ concentrations in the tissue was obtained by adding 0.5 m M EDTA or CaSO₄ at different concentrations to the medium. Calcium inhibited K⁺ release in fully turgid cells (2.5 μmol K⁺ g⁻¹ h⁻¹ in 0.5 m M EDTA vs 0.4 μmol K⁺ g⁻¹ h⁻¹ in 10 m M CaSO₄). Less turgid cells, obtained by equilibration with 0.2 M mannitol, released K⁺ at only 30% of the rate of the turgid cells, yet the pattern of K⁺ release as a function of Ca²⁺ level was similar in both turgid and non-turgid cells. Removal of calcium by EDTA occasionally injured cell membranes in the fully turgid discs but never in the less turgid ones. In view of the additive effect of Ca²⁺ and low turgor on K⁺ release regardless of the treatment order, it is suggested that the two factors exert their effect on membrane permeability independently of each other.     

Chronic Dopamine D2 Receptor Activation Does Not Affect Survival and Differentiation of Cultured Dopaminergic Neurons: Morphological and Neurochemical Observations

Abstract: Primary cultures of rat ventral mesencephalon were used to elucidate the role of chronic stimulation of dopamine (DA) D₂ autoreceptors in the development of fetal dopaminergic neurons in vitro. Cultured dopaminergic neurons, as visualized by tyrosine hydroxylase immunocytochemistry, became more differentiated in the course of cultivation time and exhibited specific high-affinity uptake for [³H]DA. In rat striatal tissue, activation of D₂ receptors has been shown to inhibit the release of DA. Previously accumulated [³H]DA was released from the cultures upon depolarization in a Ca²⁺-dependent manner. K⁺-evoked [³H]DA release could be inhibited by the selective D₂ receptor agonists LY 171555 and N0437 in a concentration-dependent manner. The inhibitory effects of LY 171555 and N0437 were antagonized by the selective DA D₂ receptor antagonist sulpiride. These observations are indicative for the expression of functional D₂ receptors in the cultures. Daily treatment of these cultures for 7 days with LY 171555 or sulpiride did not lead to any change in protein content, the number of tyrosine hydroxylase-immunoreactive neurons, or the uptake capacity for [³_H]DA. Our data demonstrate that chronic stimulation of DA D₂ receptors does not impair survival or differentiation of cultured fetal dopaminergic neurons.     

Regulation of Serotonin Release in the Frontal Cortex and Ventral Hippocampus of Homozygous Mice Lacking 5-HT1B Receptors: In Vivo Microdialysis Studies

Abstract: To assess the involvement of the serotonin receptor subtype 5-HT_1B as terminal autoreceptor regulating 5-HT release in mice, we compared basal values and potassium-evoked changes of extracellular 5-HT levels obtained by in vivo microdialysis in two serotoninergic terminal projection areas of conscious wild-type mice with those measured in homozygous mutant mice lacking the gene encoding the 5-HT_1B receptor. In the frontal cortex and ventral hippocampus, basal and K⁺-evoked 5-HT release did not differ between the two strains of mice studied. The infusion via reverse microdialysis of the selective 5-HT_1B receptor agonist CP-93,129 (500 n M ) decreased significantly K⁺-evoked 5-HT release in the frontal cortex (by −44%) and ventral hippocampus (by −32%) of wild-type mice but had no effect in mutants. In a similar manner, the mixed 5-HT_1B-5-HT_1D receptor agonist sumatriptan (800 n M ) decreased significantly K⁺-evoked 5-HT release in the frontal cortex (by −46%) of wild-type mice but had no effect in mutants. These results demonstrated that 5-HT_1B knockout mice are not as sensitive to full (CP-93,129) and mixed (sumatriptan) 5-HT_1B receptor agonists as are wild-type mice. These data provide in vivo evidence that, in mice, 5-HT_1B, but not 5-HT_1D, autoreceptors inhibit 5-HT release at nerve terminals located in the frontal cortex and ventral hippocampus.     

Development of new potent agonists able to interact with two postulated subsites of the cholecystokinin CCK-B receptor

Summary Since the biochemical and pharmacological profile of BC 197 and BC 264, two CCK₈-derived agonists with high specificity for CCK-B receptors, suggests their potential interaction with two CCK-B receptor subsites, it appeared essential to design new series of compounds that would be able to discriminate between these two subsites. As CCK₄ is the shortest fragment of CCK which interacts selectively with CCK-B receptors, compounds derived from the C-terminal tetrapeptide domain of BC 264, Boc-Trp-(NMe)Nle-Asp-Phe-NH₂, and of the cyclic compound BC 197, were prepared. While RB 360 (N-(cycloamido)-α-Me(R)Trp-[(2S)-2-amino-9-((cycloamido)carbonyl)nonanoyl]-Asp-Phe-NH₂), like BC 197, has a CCK-B₁ profile with anxiogenic-like effects in the elevated plus-maze test, RB 400 (HOOC-CH₂-CO-Trp-(NMe)Nle-Asp-Phe-NH₂), like BC 264, seems to be a specific CCK-B₂ agonist, able to increase attention and/or memory processes in the Y-maze test.     

Protein Kinase FA/Glycogen Synthase Kinase-3α After Heparin Potentiation Phosphorylates τ on Sites Abnormally Phosphorylated in Alzheimer''s Disease Brain

In this work, we tested the effect of ion channel blockers and of phorbol ester treatments on [³H]dopamine ([³H]DA) release and neurotensin (NT)-induced facilitation of [³H]DA release from cultures of rat fetal mesencephalic cells. The potassium channel blockers tetraethylammonium and 4-aminopyridine increased basal [³H]DA release and decreased K⁺-evoked [³H]DA release, whereas apamin was without effect. K⁺-evoked [³H]DA release was decreased by ω-conotoxin and nifedipine, totally suppressed by cadmium, and unaffected by amiloride. These results show the differential sensitivity of [³H]DA release to blockade of various ion channels and suggest the involvement of N-type, L-type, and non-L-non-N-type, but not T-type, voltage-sensitive calcium channels in K⁺-evoked release. Phorbol 12-myristate 13-acetate increased both spontaneous and K⁺-evoked [³H]DA release, suggesting a modulatory action of protein kinase C on DA release in this system. Unexpectedly, however, the effects of the phorbol ester were not counteracted by the protein kinase C inhibitors H7, staurosporine, or polymyxin B. NT-induced facilitation of K⁺-evoked [³H]DA release was insensitive to most of the ion channel blockers, except cadmium (64% decrease in NT effect), suggesting that the corresponding potassium' and calcium channels were not involved in the effect of NT on [³H]DA release in this system. The NT effect was totally suppressed by phorbol ester treatments, indicating a possible desensitization of the corresponding transduction mechanisms after protein kinase C activation.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.     

Hypoxia Enhances [3H]Noradrenaline Release Evoked by Nicotinic Receptor Activation from the Human Neuroblastoma SH-SY5Y

Abstract: We have used the human sympathetic neuronal line SH-SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K⁺]_o (100 m M ) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [³H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K⁺-evoked release was unaffected by hypoxia (P o ₂≅ 30–38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca²⁺_o with 1 m M EGTA abolished NA release. K⁺-evoked release was also dramatically reduced in the presence of 200 µ M Cd²⁺ to block voltage-gated Ca²⁺ channels, but DMPP-evoked release was less affected. In hypoxia, DMPP-evoked Cd²⁺-resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH-SY5Y cells in response to nAChR activation by increasing Ca²⁺ influx through the nAChR pore, or by activating an unidentified Cd²⁺-resistant Ca²⁺-influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.     

Maturation of Potassium-Stimulated Respiration in Rat Cerebral Cortical Slices

Abstract: Maximal dinitrophenol-stimulated respiration and K⁺-stimulated respiration were measured polarographically in cerebral cortical slices taken from rats aged 2–60 days. Increasing K⁺ concentrations produced an increase in respiration in slices from animals aged 15 days and older, but not in slices from animals aged 10 days and younger. Dinitrophenol-stimulated respiration, or the maximal respiratory capacity of the tissue, showed a similar increase between 10 and 15 days of age. At each age the maximal respiratory capacity was 6–8 ng at 0/mg protein min greater than the maximal K⁺-stimulated respiration.     

Effects of 2-iodobenzanilide on potassium uptake, H+ extrusion and K+-stimulated ATPase of corn roots

The carboxanilide systemic fungicide 2-iodobenzanilide (2-IB) after 2 h pretreatment at 0.25 m M inhibited K⁺ and SO₄^2- uptake by excised corn roots ( Zea mays L., cv. Dekalb 342) up to ca 70 and 40%, respectively. Proton extrusion from corn roots was also reduced by ca 50% after 1 h contact, and the microsomal K⁺-stimulated ATPase activity from corn roots and pea stems ( Pisum sativum L., cv. Alaska) inhibited by 50 and 72%, respectively. In contrast, the Mg²⁺-ATPase activities of microsomes and mitochondria at pH 6.0 and 8.7, respectively, were unaffected. After 2 h of preincubation with 0.25 m M 2-IB, O₂ consumption by corn roots and pea stems was inhibited by 12 and 18%, respectively. ATP content of corn roots was not altered by 2-IB treatment. Therefore, energy availability "in vivo" was unaffected and the primary effect on corn roots is suggested to be at the plasmalemma ATPase which forms the proton gradient.
With isolated pea stem mitochondria, 0.25 m M 2-IB inhibited O₂ consumption by ca 60% when NADH or malate plus pyruvate were added as substrates; when succinate was used O₂ consumption was unaffected. The mode of action on isolated mitochondria was different from that shown for carboxin and also formerly attributed to the whole class of carboxanilide fungicides.     

Differential fading of inhibitory and excitatory B2 bradykinin receptor responses in rat sympathetic neurons: a role for protein kinase C

Through inhibitory and excitatory effects on sympathetic neurons, B₂ bradykinin receptors contribute to protective and noxious cardiovascular mechanisms. Presynaptic inhibition of sympathetic transmitter release involves an inhibition of Ca_V2 channels, neuronal excitation an inhibition of K_V7 channels. To investigate which of these mechanisms prevail over time, the respective currents were determined. The inhibition of Ca²⁺ currents by bradykinin reached a maximum of 50%, started to fade within the first minute, and became attenuated significantly after ≥ 4 min. The inhibition of K⁺ currents reached a maximum of 85%, started to fade after > 3 min, and became attenuated significantly after ≥ 7 min. Blocking Ca²⁺-independent protein kinase C (PKC) enhanced the inhibition of Ca²⁺ currents by bradykinin and delayed its fading, left the inhibition of K⁺ currents and its fading unaltered, and enhanced the reduction of noradrenaline release and slowed its fading. Conversely, direct activation of PKC abolished the inhibition of noradrenaline release and largely attenuated the inhibition of Ca²⁺ currents. These results show that the inhibitory effects of bradykinin in sympathetic neurons are outweighed over time by its excitatory actions because of more rapid, PKC-dependent fading of the inhibitory response.     

Characterization of the plasmalemma ATPase activity from roots of Plantago major ssp. pleiosperma, purified by the two-phase partitioning method

A purified plasmalemma preparation from roots of Plantago major L. ssp. pleiosperma (Pilger) was obtained by the two-phase partitioning method, using 6.5% (w/w) of Dextran T-500 and polyethylene glycol 3350, respectively. The distribution of murker enzymes proved the purity of the plasmalemma fraction. The ATPase activity was characterized by determining its sensitivity to anions, cations and inhibitors. The Mg²⁺-dependent ATPase activity peaked at pH 7.25, K⁺-stimulation at pH 6.75, and the Cl⁻ -stimulation both at pH 6.75 and 7.5 (all in the presence of 3 m M MgSO₄). The plasmalemma preparations hydrolyzed preferentially ATP (in the presence of Mg²⁺), although they were less specific for ATP at pH 7.5 than at pH 6.75. The Cl⁻ - stimulated ATPase is probably associated with and located on the plasmalemma. The question if the Cl⁻ -stimulated activity is due to an ATPase distinct from the classical K⁺-stimulated ATPase is considered.