Physiological Concentrations Of Mg2 Research Articles

Metal ion-promoted hydrolysis of the antioxidant cardioprotective agent dexrazoxane (ICRF-187) and its one-ring open hydrolysis products to its metal-chelating active form

The metal ion-promoted hydrolysis of the doxorubicin cardioprotective agent dexrazoxane (ICRF-187) has been studied by HPLC and spectrophotometrically. Dexrazoxane is thought to be cardioprotective through the ability of its rings-opened hydrolysis product (D) to chelate loosely bound iron, thus preventing iron-based oxygen free radical damage. While neither Mg(II) nor Ca(II) promoted the hydrolysis of the opening of the first dexrazoxane ring, Zn(II) strongly promoted (60-fold) hydrolysis. However, physiological concentrations of Mg(II) and Ca(II) promoted hydrolysis of the one-ring open dexrazoxane hydrolysis intermediates (B and C) by up to 18-fold. Thus, Mg(II) and Ca(II) may be, in part, responsible for mediating the formation of the active metal ion-chelating form of the drug in vivo. A variety of other metal ions (Mn(II), Co(II), Ni(II), Cu(II) and Zn(II)) strongly promoted hydrolysis of B and C by factors ranging from 25 for Mn(II), to greater than 50,000 for Zn(II) and Cu(II). The Zn(II)-promoted hydrolysis of B and C showed a strong pH dependence, which may be due either to a hydroxide bound to Zn(II) or free hydroxide in solution acting as a nucleophile. Titration of the Fe(III)-B and Fe(III)-D complex with azide resulted in spectral changes consistent with formation of ternary complexes, indicating that these complexes have open or loosely bound coordination sites.

Blocking GABA(A) inhibition reveals AMPA- and NMDA-receptor-mediated polysynaptic responses in the CA1 region of the rat hippocampus.

We have investigated the conditions required to evoke polysynaptic responses in the isolated CA1 region of hippocampal slices from Wistar adult rats. Experiments were performed with extracellular and whole cell recording techniques. In the presence of bicuculline (10 microM), 6-cyano-7-nitroquinoxaline-2-3-dione (10 microM), glycine (10 microM), and a low external concentration of Mg2+ (0.3 mM), electrical stimulation of the Schaffer collaterals/commissural pathway evoked graded N-methyl-D-aspartate (NMDA)-receptor-mediated late field potentials in the stratum radiatum of the CA1 region. These responses were generated via polysynaptic connections because their latency varied strongly and inversely with the stimulation intensity and they were abolished by a high concentration of divalent cations (7 mM Ca2+). These responses likely were driven by local collateral branches of CA1 pyramidal cell axons because focal application of tetrodotoxin (30 microM) in the stratum oriens strongly reduced the late synaptic component and antidromic stimulation of CA1 pyramidal cells could evoke the polysynaptic response. Current-source density analysis suggested that the polysynaptic response was generated along the proximal part of the apical dendrites of CA1 pyramidal cells (50-150 microm below the pyramidal cell layer in the stratum radiatum). In physiological concentration of Mg2+ (1.3 mM), the pharmacologically isolated NMDA-receptor-mediated polysynaptic response was abolished. In control artificial cerebrospinal fluid (with physiological concentration of Mg2+), bicuculline ( 10 microM) generated a graded polysynaptic response. Under these conditions, this response was mediated both by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/NMDA receptors. In the presence of D-2-amino-5-phosphonovalerate (50 microM), the polysynaptic response could be mediated by AMPA receptors, although less efficiently. In conclusion, suppression of gamma-aminobutyric acid-A inhibition reveals glutamate receptor-mediated network-driven events in the isolated CA1 region. These polysynaptic responses are mediated by AMPA and/or NMDA receptors depending on the pharmacological conditions and the external concentration of Mg2+ used. We suggest that these responses are driven by local recurrent collaterals of CA1 pyramidal cells.

Glutamate receptor-mediated synaptic excitation in axons of the lamprey.

1. Spontaneous and evoked synaptic inputs were recorded in vitro in the axons of lamprey reticulospinal neurones. After isolation of the axon from its somata, synaptic inputs were recorded using microelectrode and whole-cell patch clamp recording techniques. 2. Single stimuli applied to the spinal cord elicited Ca(2+)-dependent synaptic potentials with short latencies in reticulospinal axons. These synaptic inputs are capable of summation and generate sufficient depolarization to raise the membrane potential beyond threshold to initiate action potentials. Action potential initiation in the absence of the cell body indicates that these axons show synaptic integration. 3. Both evoked and spontaneous responses comprise at least two components of synaptic drive: a slow component (rise time of 9.6 +/- 2.1 ms) with a reversal potential of -53 +/- 19 mV and a fast component (rise time as fast as 0.85 ms) with a reversal potential of 0.3 +/- 9.1 mV. The responses are Ca2+ dependent, and are blocked by the substitution of Ba2+ for Ca2+ in the saline solution. 4. The slow component of synaptic input was blocked by the gamma-aminobutyric acidA (GABAA) receptor antagonist bicuculline (5 microM). The fast component was blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroqinoxaline-2,3-dione (CNQX; 10 microM) in Ringer solution containing physiological concentrations of Mg2+. Following removal of Mg2+ from the superfusate a further excitatory component was identified that was blocked by application of the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonopentanoate (AP5; 100 microM). 5. Comparison of the kinetic properties and the voltage sensitivity of the isolated components of evoked and spontaneous synaptic activity indicate that these responses are mediated by similar synaptic inputs. 6. These results suggest that axons and presynaptic terminals receive excitatory and inhibitory ionotropic receptor-mediated inputs. Summation of these inputs is possible indicating that the axons act as sites of synaptic integration similar to the role previously attributed only to neuronal dendrites and somata.

Magnesium ion augmentation of inhibitory effects of adenosine on dopamine release in the rat striatum.

The effects of adenosine and magnesium ion (Mg2+) on striatal dopamine release were studied in awake rats by in vivo microdialysis. The mean striatal basal levels of dopamine release at Mg2+ free perfusate were 56.95 +/- 5.30 fmol/sample (for 20 min). By varying the Mg2+ levels in perfusate from 0 mmol/L to 1, 10 or 40 mmol/L, the dopamine release was inhibited by Mg2+ in a level-dependent manner. Perfusion with modified Ringer's solution containing zero Mg2+ and from 5 to 50 mumol/L adenosine, non-selective adenosine agonist, as well as 0.1 mumol/L 2-chloro-N6-cyclopentyladenosine (CCPA), selective adenosine A1 agonist, showed no effect on dopamine release. However, from 5 to 50 mumol/L adenosine and from 0.1 to 1 mumol/L CCPA plus Mg2+ (1 and 40 mumol/L) perfusion decreased the dopamine release. This inhibitory effect of adenosine and CCPA on striatal dopamine release was enhanced by an increase in extracellular Mg2+ levels. Levels of 50 mumol/L of 8-cyclopentyl-1,3-dimethylxanthine (CPT), a selective adenosine A1 receptor antagonist, in perfusate increased the dopamine release under conditions both with and without Mg2+. This stimulatory effect of CPT on striatal dopamine release was reduced by an increase in extracellular Mg2+ levels. As a result, CPT antagonized the inhibitory effects of adenosine and CCPA on dopamine release under conditions of the presence and absence of Mg2+. These results suggest that the inhibition of striatal dopamine release by adenosine was mediated by adenosine A1 receptor. This inhibition was intensified by Mg2+. This study also revealed that the concentrations of Mg2+, which ranged from physiological to supraphysiological, reduced the striatal dopamine release; furthermore it was found that the physiological concentration of Mg2+ potentiated the effects of adenosine agonists, but inhibited adenosine antagonist. Thus, the present study, using in vivo microdialysis preparations, suggests Mg2+ inhibits the calcium ion channels and enhances the adenosinergic function in the central nervous system.

NMDA receptor-mediated calcium entry in the absence of AMPA receptor activation in rat dorsal horn neurons

This study tested quantitatively the degree to which selective activation of NMDA receptors increases the intracellular concentration of free Ca 2+ ions ([Ca 2+] i) in neurons isolated from the rat spinal cord dorsal horn and grown in culture. The results show that activation of NMDA receptors, without additional depolarizing stimuli, produce [Ca 2+] i increases in the neuronal cell bodies in the presence of physiological concentrations of Mg 2+. Furthermore, this phenomenon occurs during synaptic activation of NMDA receptors. Thus, it cannot be assumed that in physiological extracellular [Mg 2+], there is an absolute requirement for additional membrane depolarization before NMDA receptor activation stimulates significant increases in [Ca 2+] 1.

Postsynaptic modulation of NMDA synaptic currents in rat hippocampal microcultures by paired-pulse stimulation.

1. Paired-pulse synaptic stimulation of hippocampal neurons in microcultures resulted in depression of synaptic currents mediated by both NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. However, NMDA EPSCs were more severely depressed than AMPA EPSCs. 2. Partial NMDA receptor blockade reduced paired-pulse depression of NMDA but not of AMPA synaptic currents while partial AMPA receptor blockade had no effect on paired-pulse depression of AMPA EPSCs. These results suggest that ion flux through NMDA receptors is important in paired-pulse depression of NMDA responses but has no effect on AMPA responses. 3. Low extracellular Ca2+ concentrations or positive postsynaptic holding potentials reduced paired-pulse depression of NMDA EPSCs to near that of AMPA responses. 4. Brief paired applications of exogenous glutamate to neurons produced Ca(2+)-dependent depression similar to the depression of NMDA synaptic responses and synaptic stimulation depressed responses to exogenously applied NMDA. 5. Physiological concentrations of Mg2+ prevented expression of the postsynaptic modulation of NMDA EPSCs at -70 mV, but partial relief of Mg2+ block of the NMDA channel with depolarization increased paired-pulse depression of NMDA EPSCs.

Kainate-induced elevations of intracellular Ca2+ and extracellular glutamate are partially decreased by NMDA receptor antagonists in cultured cerebellar granule neurons

Several lines of evidence indicate that physiological activity of N-methyl-D-aspartate (NMDA) receptor was blocked by physiological concentration of Mg2+ (1.2 mM). However, the activity of NMDA receptor may not be blocked totally with this concentration of Mg2+ under elevated membrane potential by kainate. Here, we described the effect of Mg2+ on NMDA receptor and how much of NMDA receptor functions could be activated by kainate. Effects of NMDA receptor antagonist on kainate-induced elevation of intracellular Ca2+ levels ([Ca2+]i) and extracellular glutamate level were examined in cultured rat cerebellar granule neurons. Kainate-induced elevation of [Ca2+]i was not affected by physiological concentration of Mg2+ though NMDA-induced elevation was blocked by the same concentration of Mg2+. Kainate-induced elevation of [Ca2+]i was decreased by 32% in the presence of NMDA antagonists, MK-801 and CPP (3-[2-carboxypiperazine-4-yl]propyl-1-phosphonic acid), in Mg2+ free buffer. Kainate receptor-activated glutamate release was also decreased (30%) by MK-801 or CPP. These results show that certain extent of elevations of intracellular Ca2+ and extracellular glutamate by kainate is due to coactivation of NMDA receptors.

Modulation of N-methyl- d-aspartate receptor activity by oxidative stress conditions in chick retinal cells

The effect of oxidative stress, induced by ascorbate (1.5 mM)/Fe 2+ (7.5 μM), on the cellular responses to N-methyl- d-aspartate (NMDA) receptor activation was evaluated by measuring the release of [ 3H]GABA induced by NMDA from cultured retina cells. In retina cells submitted to oxidative stress the [ 3H]GABA release evoked by NMDA, in a medium containing physiological concentrations of Mg 2+ (1.6 mM) and K + (4 mM), was significantly higher than in control cells. The [ 3H]GABA release evoked by NMDA was potentiated by glycine and was abolished by MK-801, suggesting that the [ 3H]GABA release was due to NMDA receptor activation. The increased effect of NMDA in peroxidized cells was significantly reduced by TTX, suggesting that the higher cellular responses to the activation of NMDA receptors are due to a hyperexcitability of retina cells submitted to oxidative stress. No significant differences were found between the average resting membrane potential of control and peroxidized cells. However, membrane potential is more tightly regulated by K +-channels sensitive to 4-aminopyridine (100 μM), α-dendrotoxin (100 nM) and γ-dendrotoxin (100 nM) under oxidative stress.

Hexokinase and glucokinase binding in permeabilized guinea-pig hepatocytes

The release of glucokinase (hexokinase IV) from digitonin-permeabilized hepatocytes from rat, guinea pig or mouse liver is inhibited by physiological concentrations of Mg2+ (> 0.25 mM). Preincubation of hepatocytes with fructose increases glucokinase release during permeabilization in the presence of Mg2+ but decreases glucokinase release in the absence of Mg2+, suggesting that fructose causes translocation of glucokinase from the Mg(2+)-dependent site. Glucose (25 mM) and sorbitol (1 mM) also induce translocation of glucokinase from the Mg(2+)-dependent site in guinea-pig, as in rat hepatocytes, but glucose is less effective than fructose or sorbitol, and the concentrations of fructose and sorbitol that cause half-maximal activation (A50) are 3-fold and 20-fold higher, respectively, in guinea-pig than in rat hepatocytes (170 microM and 257 microM, compared with 61 microM and 13 microM). Dihydroxyacetone and glycerol have no effect on fructose-induced or sorbitol-induced translocation in guinea-pig hepatocytes, in contrast with the potentiation and inhibition, respectively, by these substrates in rat hepatocytes. Some, but not all, of the differences between rat and guinea-pig hepatocytes could be due to the more reduced cytoplasmic NADH/NAD+ redox state in guinea-pig cells. The activity of low-Km hexokinases accounts for 30% of total hexokinase activity (low-Km hexokinases + glucokinase) in guinea-pig hepatocytes. Of the low-Km hexokinase activity, approx. 30% is released in the presence of Mg2+, 9% shows Mg(2+)-dependent binding and 60% shows Mg(2+)-independent binding. There was no substrate-induced translocation of low-Km hexokinase activity, indicating that translocation is specific for hexokinase IV.

Effects of Ethanol on NMDA Receptors in Brain: Possibilities for Mg2+ ‐Ethanol Interactions

The major excitatory neurotransmitter in the CNS is L-glutamate, and one of the subtypes of L-glutamate receptors, the N-methyl-D-aspartate (NMDA) subtype, has been found to be quite sensitive to inhibition by low concentrations of ethanol (5-50 mM). The NMDA receptor-ion channels are unique in that they exhibit a voltage-dependent blockade by physiological concentrations of Mg2+, a blockade that is relieved as the cell membrane is depolarized. Several lines of evidence also suggest that the activity of this receptor-channel complex may be regulated through a high-affinity Mg2+ site, which is distinct from the channel-blocking site and could even be located on the extracellular domain of the protein. This high-affinity Mg2+ site has been shown to increase the binding of N-[1-(2-thienyl) cyclohexyl]piperidine within the ion channel, as well as the binding of competitive antagonist such as 3-(+/-)-carboxypiperazine-4-yl)-[1,2]-propyl-1-phosphonic acid and the receptor coactivator glycine. The relationship between the acute effects of ethanol on receptor activation and the regulatory properties of Mg2+ is not yet known, although the hypomagnesemia that occurs in chronic alcoholism could certainly have implications for receptor function. A significant amount of molecular characterization of the multiple isoforms of the NMDA receptor-ion channel will be required before the role of Mg2+ can be clarified and any relationship between Mg2+ regulation and ethanol inhibition established.

Glutamate and glycine decrease the affinity of [ 3H]MK-801 binding in the presence of Mg 2+

The NMDA receptor is coupled to a cation-selective ion channel, which has been implicated in important brain functions such as long-term potentiation and burst firing, and in neuronal death associated with stroke and epilepsy. We have investigated the binding properties of [ 3H]MK-801, which binds selectively to the open state of the NMDA channel, at physiological concentrations of Mg 2+ in membrane preparations of the rat cerebral cortex. Glutamate and glycine were found to enhance [ 3H]MK-801 binding at low concentrations and inhibit [ 3H]MK-801 binding at high concentrations. The inhibition of [ 3H]MK-801 binding was due to an enhancement of the dissociation rate constant and was reversed by competitive glutamate and glycine antagonists. These findings could be explained by a glutamate- and glycine-induced decrease in the affinity of [ 3H]MK-801 binding sites within activated NMDA channels, in the presence of Mg 2+. This decrease in [ 3H]MK-801 affinity may correspond to a decreased affinity of the site where mg 2+ causes a voltage-dependent block of the NMDA channel.

Hypotonic fragility of outer membrane and activation of external pathway of NADH oxidation in rat liver mitochondria are increased with age

Great importance is attached to structural and functional deterioration of mitochondria as a reason for ageing of an organism; the attention of many scientists has been concentrated on such questions as age changes in the system of oxidative phosphorylation, damage of mitochondrial DNA by free radicals generated in the respiratory chain and inclusion of some fragments of mitochondrial DNA into the nuclear genome. Mitochondrial high amplitude swelling in a cell under some extreme conditions can possibly play a very important role in mechanisms of deterioration of energy transformation function, in activation of lipid peroxidation and mitochondrial DNA damage as a result of outer membrane disruption and release of enzymes from the intermembrane space (e.g. superoxide dismutase and adenylate kinase). In this work the age changes of the hypotonic fragility of the outer membrane of rat liver mitochondria and the activation of the external, rotenone-insensitive pathway of NADH oxidation have been examined. It is shown that the obligatory condition for activation of rotenone-insensitive NADH oxidation is a break in the outer membrane and that the rate of NADH oxidation substantially increases in the presence of physiological concentrations of Mg 2+ which cause a multiple increase in the affinity of the inner membrane to cytochrome c. Research on the rate of rotenone-insensitive NADH oxidation with respect to the osmotic pressure, the ionic strength of the medium, the presence of Mg 2+ ions and cytochrome c in the medium has demonstrated a considerable increase in the hypotonic fragility of the outer membrane of liver mitochondria with age in male rats. In female rats the age changes were insignificant. It is supposed that the damage to the outer membrane of mitochondria in cells can serve as one of the possible explanations of both decrease in the reliability of an aged organism under extreme conditions and sex differences of life-span.

Dissociation of nitric oxide generation and kainate-mediated neuronal degeneration in primary cultures of rat cerebellar granule cells

In the presence of physiological concentrations of Mg 2+ and in glycine-free buffer, the relationship between KA-mediated generation of NO and neurotoxicity in cultures of cerebellar granule cells of the rat was examined. The neuronal damage elicited by KA was not dependent on the presence of l-arginine, a precursor of NO, since neither the potency nor magnitude of KA-mediated cell death was altered in either the absence or presence of exogenously applied l-arginine. Similarly, with the exception of 4-hydroxy-azobenzene-4'-sulfonic acid, disodium salt dihydrate (HBS), the salt associated with N G-monomethyl- l-arginine (di-( p-hydroxyazobenzene- p′-sulfonate) (MA(HBS)), treatment with several different competitive NO synthetase inhibitors did not provide protection against the toxicity of KA. However, the ability of KA to induce neuronal damage was significantly decreased in cerebellar granule cells treated with either HBS or α-tocopherol (VE). On the basis of these results, it is concluded that the generation of free radicals may be involved in the process of KA-elicited neuronal death in cultures of cerebellar granule cells but that this is unrelated to the synthesis of NO. This conclusion agrees with both in vivo and in vitro studies, implicating the involvement of free radicals in non-NMDA mediated neuronal damage.

Persisting modification of dendritic calcium influx by excitatory amino acid stimulation in isolated CA1 neurons

Spatiotemporal changes of the intracellular calcium ion (Ca 2+) were recorded by digital ratio imaging of fura-2 in pyramidal neurons acutely isolated from the adult guinea-pig hippocampus. Increases in calcium were evoked in tetrodotoxin (2 μM) containing saline either by stimulation with the excitatory arnino acids, glutamate or N-methyl- d-aspartate, or by depolarization with high potassium (50 mM). Local stimulation with excitatory amino acids, applied from a microelectrode with 1-2-s iontophoretic pulses at the dendrite, induced a rapid increase in intracellular Ca 2+ predominantly supported by a Ca 2+ influx at the site of stimulation (primary response). Ca 2+ levels recovered withing 1–2 min in about one-half of the neurons examined. In the remaining neurons the initial exposure to excitatory amino acids induced a non-recovering gradient of Ca 2+, highest at the site of stimulation, that lasted for periods of minutes (secondary response). Within the population that showed recovery from the initial agonist exposure, a second, or in some cases, a third application triggered the sustained, secondary response. Pretreatment of neurons with the protein kinase inhibitor sphingosine (10 μM) blocked development of the secondary response but had no effect on the primary response to the excitatory amino acids. There were no Ca 2+ increases in Ca 2+-free medium with either agonist, and responses to N-methyl- d-aspartate were blocked by 2-amino-4-phosphovaleric acid and significantly reduced at physiological concentrations of Mg 2+ (1.8 mM). The maintained gradient of Ca 2+ was supported by a continuous influx of calcium from outside the cell. In contrast, dendritic gradients of Ca 2+ induced by short exposures to high potassium (50 mM, 5 s) collapsed immediately at the end of the stimulus and could be repeatedly evoked. Minute-long exposures to high K, induced large, repeatable changes in Ca 2+ but there was always rapid recovery in normal saline. K depolarization applied after excitatory amino acid stimulation produced larger Ca 2+ changes than the same K stimulus applied before the cell was stimulated with the excitatory amino acid. Bath application of GABA (10–100 μM) reduced the magnitude of the maintained Ca 2+ gradients. The functional significance of the extended, secondary response cannot be directly established from these measurements on isolated neurons, but its properties could give rise, in part, to mechanisms involved in neural plasticity, in kindling epileptogenesis or in glutamate-induced toxicity.

Effects of monovalent and divalent cations on 3-(+)[125I]iododizocilpine binding to the N-methyl-D-aspartate receptor of rat brain membranes.

We have investigated the binding of 3-[125I]iododizocilpine ([125I]iodo-MK-801) to the N-methyl-D-aspartate (NMDA) receptor in well-washed rat brain membranes. [125I]Iododizocipline binding was displaced by the following: dizocilpine greater than thienylphencyclidine greater than phencyclidine greater than ketamine. Binding of [125I]iododizocilpine was enhanced by glutamate, glycine, and spermidine, whose actions could be reversed by CGS-19755, 7-chlorokynurenate, and arcaine, respectively. [125I]Iododizocilpine binding was also enhanced by a number of divalent cations, including Ba2+, Ca2+, Mg2+, Mn2+, and Sr2+, and several monovalent cations, including Na+ and K+. These cations enhanced [125I]iododizocilpine binding by an action at the polyamine site. In addition, the inhibitory effects associated with high concentrations of these cations was markedly reduced compared to those found in previous studies with [3H]dizocilpine. Analysis of the ability of spermidine, Mg2+, and Sr2+ to alter the inhibition of [125I]iododizocilpine by arcaine gave pA2 values of 5.41, 4.47, and 4.93, corresponding to EC50 concentrations of 3.9, 34.7, and 12.0 microM, respectively, suggesting that physiological concentrations of Mg2+ may occupy the polyamine site. These results demonstrate that [125I]iododizocilpine is a useful probe for the NMDA receptor. Moreover, its high specific activity and relative insensitivity to the inhibitory actions of divalent cations should make [125I]iododizocilpine a valuable ligand for the study of NMDA receptors in intact cellular systems.

Desensitization of the isolated beta 2-adrenergic receptor by beta-adrenergic receptor kinase, cAMP-dependent protein kinase, and protein kinase C occurs via distinct molecular mechanisms.

Exposure of beta 2-adrenergic receptors (beta 2ARs) to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Phosphorylation of the beta 2AR by several distinct kinases plays an important role in this desensitization phenomenon. In this study, we have utilized purified hamster lung beta 2AR and stimulatory guanine nucleotide binding regulatory protein (Gs), reconstituted in phospholipid vesicles, to investigate the molecular properties of this desensitization response. Purified hamster beta 2AR was phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), or beta AR kinase (beta ARK), and receptor function was determined by measuring the beta 2AR-agonist-promoted Gs-associated GTPase activity. At physiological concentrations of Mg2+ (less than 1 mM), receptor phosphorylation inhibited coupling to Gs by 60% (PKA), 40% (PKC), and 30% (beta ARK). The desensitizing effect of phosphorylation was, however, greatly diminished when assays were performed at concentrations of Mg2+ sufficient to promote receptor-independent activation of Gs (greater than 5 mM). Addition of retinal arrestin, the light transduction component involved in the attenuation of rhodopsin function, did not enhance the uncoupling effect of beta ARK phosphorylation of beta 2AR when assayed in the presence of 0.3 mM free Mg2+. At concentrations of Mg2+ ranging between 0.5 and 5.0 mM, however, significant potentiation of beta ARK-mediated desensitization was observed upon arrestin addition. At a free Mg2+ concentration of 5 mM, arrestin did not potentiate the inhibition of receptor function observed on PKA or PKC phosphorylation. These results suggest that distinct pathways of desensitization exist for the receptor phosphorylated either by PKA or PKC or alternatively by beta ARK.

Rapid Modulation of Spinach Leaf Nitrate Reductase by Photosynthesis

Assimilatory nitrate reductase activity (NRA) in crude spinach leaf (Spinacia oleracea) extracts undergoes rapid changes following fluctuations in photosynthesis brought about by changes in external CO(2) or by water stress (WM Kaiser, E Brendle-Behnisch [1991] Plant Physiol 96:363-367). A modulation of NRA sharing several characteristics (stability, response to Mg(2+) or Ca(2+), kinetic constants) with the in vivo modulation was obtained in vitro by preincubating desalted leaf extracts with physiological concentrations of Mg(2+) and ATP (deactivating) or AMP (activating). When nitrate reductase (NR) was inactivated in vivo by illuminating leaves at the CO(2) compensation point, it could be reactivated in vitro by incubating leaf extracts with AMP. For the in vitro inactivation, ATP could be replaced by GTP or UTP. Nonhydrolyzable ATP analogs (beta, gamma-imido ATP, beta, gamma-methyl-ATP) had no effect on NR, whereas gamma-S-ATP caused an irreversible inactivation. This suggests that NR modulation involves ATP hydrolysis. In contrast to NR in crude leaf extracts, partially purified NR did not respond to ATP or AMP. ATP and AMP levels in whole leaf extracts changed in the way predicted by the modulation of NRA when leaves were transferred from photosynthesizing (low ATP/AMP) to photorespiratory (high ATP/AMP) conditions. Adenine nucleotide levels in leaves could be effectively manipulated by feeding mannose through the leaf petiole. NRA followed these changes as expected from the in vitro results. This suggests that cytosolic ATP/AMP levels are indeed the central link between NRA in the cytosol and photosynthesis in the chloroplast. Phosphorylation/dephosphorylation of NR or of NR-regulating protein factors is discussed as a mechanism for a reversible modulation of NR by ATP and AMP.

Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2

Several components of the phosphoinositide cycle have been found to interact specifically and at physiological concentrations with the plasma membrane-associated clathrin assembly (adaptor) protein AP-2. These include phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, which are present at the plasma membrane, as well as other polyphosphoinositols. ATP and other polyphosphate molecules complete with the polyphosphoinositols, however, they are at least 80-fold less potent. Also, the effect of ATP, unlike the polyphosphoinositols, is blocked by physiological concentrations of Mg2+. Photoaffinity labeling of AP-2 by [alpha-32P]8-azidoadenosine 5'-triphosphate and its competition by polyphosphoinositols has been used to identify the alpha subunit of the AP-2 complex as the site of specific interaction with the polyphosphoinositols and to confirm direct ultrafiltration binding experiments. Proteolytic dissection of the labeled AP-2 demonstrated that binding occurred exclusively on the N-terminal portion of the alpha subunit. Interaction of purified AP-2 with sub-microM concentrations of polyphosphoinositols has inhibitory effects on a novel AP-2 self-association described in the accompanying paper (Beck, K. A., and Keen, J. H., J. Biol. Chem. 266, 4437-4441), and at higher concentrations on the binding of AP-2 to dissociated clathrin trimers as well as AP-2-mediated clathrin coat assembly. Review of the literature shows that several physiological stimuli that are known to result in increased coat pit formation in intact cells correlate with increased phosphoinositide turnover. These in vivo correlations and the in vitro observations reported here suggest that coated membrane and phosphoinositide cycles may be interdependent within cells.

The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1–60 μM and 50–370 μM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca 2+-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca 2+-binding affinity was 1.5–2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca 2+-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca 2+-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca 2+-ATPase activity of permeabilized erythrocytes. At Ca 2+ concentrations below 0.7 μM, ouabain (0.5–1 mM) activated whereas at higher Ca 2+ concentrations it inhibited the Ca 2+-ATPase activity. Taking this observation into account the Na +/K +-ATPase was determined as the difference of between the ATPase activities in the presence of Na + and K + and in the presence of K + alone. At physiological concentration of Mg 2+ (370 μM), the addition of 0.3–1 μM Ca 2+ increased Na +/K +-ATPase activity by 1.5–3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg 2+ concentration (e.g., 50 μM) only Na +/K +-ATPase inhibition by Ca 2+ was seen. It was found that at [NaCl] < 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca 2+-free media. This activating effect of furosemide was enhanced with a diminution of [Na +] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na +/K +-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb + influx into intact erythrocytes.

Development and regulation of excitatory amino acid receptors involved in the release of arachidonic acid in cultured hippocampal neural cells

Release of [ 3H]arachidonic acid mediated by excitatory amino acid (EAA) receptors was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. Treatment with N- methyl- d-aspartate (NMDA), quisqualate (QA) and kainate resulted in age- and dose-dependent stimulation of [ 3H]arachidonic acid release. During development, the maximum response for NMDA was observed relatively earlier (at 7 days) than those for QA and kainate (at 14 days) in the hippocampal neuronal cultures. The half maximal effects were obtained at about 15 μM NMDA at all ages studied and about 0.5 μM QA at 14 and 20 days. At optimum concentrations NMDA- and QA-induced releases were additive. Unlike with neurons, treatment with all the 3 EAA receptor agonists, NMDA, QA and kainate, had no significant effect on [ 3H]arachidonate release in hippocampal astroglial cells. In cultured 14-day-old neurons, the increases in NMDA- and QA-mediated [ 3H]arachidonic acid release were completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and the ionotropic QA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. But the ionotropic QA receptor agonist α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) had no significant effect on [ 3H]arachidonate release, indicating that interaction between ionotropic QA and metabolotropic QA receptors may be essential for optimal QA-mediated arachidonic acid release. At physiological concentrations of Mg 2+ (1.2 mM), AMPA was found to potentiate NMDA-induced release of [ 3H]arachidonic acid; the effect appeared to be related to a removal of Mg 2+ blockade mediated by mild depolarisation. Our findings would suggest a role for associative stimulation of different types of EAA receptors in the effector mechanisms responsible for signal transduction and synaptic plasticity in the hippocampus.