Effects of Manganese, Calcium, Magnesium and Lithium on the Ouabain-Induced Seizure

The effects of the intra ventricularly administered cations (Mn²⁺, Ca²⁺, Mg²+ and Li+) against the seizure induced by ouabain (3 μg) were investigated. Mn²⁺, Ca²⁺ and Mg²⁺ caused definite sedation and decreased locomotor activity. But Li⁺ was without significant behavioral effect at the doses applied. Among the cations used, Mn²⁺, Ca²⁺ and Mg²⁺ showed significant anticonvulsive effect on the ouabain-induced seizure. In comparison, on the dose and molarto-molar basis, the potency of anticonvulsive action was in the following order: Mn²⁺ > Ca²⁺ > Mg²⁺. On the contrary, the higher dose of Li⁺ potentiated the ouabaininduced seizure. The importance of the increased Ca²⁺ level in the extracellular space or the inhibition of Ca²⁺ uptake as the anticonvulsive effect of Ca²⁺, Mn²⁺ and Mg²⁺ was discussed.     

Effects of divalent cations on the frequency of spontaneous action potentials from the lateral line organ of Xenopus laevis

The effect of superfusion of the internal surface of the skin of Xenopus laevis with saline containing Co2+, Ca2+, Mg2+, or Ba2+, on the frequency of spontaneous action potentials of the lateral line nerve, was studied to investigate the role of extracellular Ca2+ in spontaneous neural activity. Addition of divalent cations to frog saline, either singly or as a mixture of two different ions, produced concentration-dependent suppression of spontaneous rate. The rank order of potency for suppression by each ion, perfused alone, was Co2+ greater than Ca2+ greater than Mg2+ greater than Ba2+. Suppression by combinations of Mg2+ and Co2+, or of Ca2+ and Co2+, was approximated by the sum of the suppressive effects of each cation. Ca2+ was more suppressive than Mg2+ when each of these ions was paired with the same amount of Co2+, while Ca2+ was approximately as suppressive as Co2+ when similarly paired with Mg2+. One interpretation of the suppression by Ca2+ invokes the hypothesis that divalent cations suppress spontaneous activity by charge screening of voltage-sensitive Na+ channels on afferent dendrites and that release of neurotransmitter by the influx of extracellular Ca2+ through voltage-sensitive Ca2+ channels of hair cells may not be the sole mechanism for generation of spontaneous activity in the lateral line. These results quantify the relative suppressive potency of common divalent cations in the lateral line, and serve as a caveat to investigators who interpret a blockade of action potentials by high concentrations of Co2+ or Mg2+ as sufficient evidence for dependence of neurotransmission upon extracellular Ca2+, particularly in acousticolateralis systems.     

Regulation of endogenous calcium and magnesium levels by delta opioid receptors in the rat brain.

The effects of the potent agonist (D-Pen2, D-Pen5) enkephalin (DPDPE), a conformationally restricted ligand which is highly selective for delta opioid receptors, were studied on the endogenous levels and regional distributions of selected mono- and divalent cations in rat brain and thoracic spinal cord by means of atomic absorption spectrophotometry. In general, lower Na+, K+, Ca2+, Mg2+ and Mn2+ levels were characteristic of the medulla and spinal cord compared to that of cortices, striatum and hippocampus, while the highest metal ion levels were detected in the olfactory bulb. Sixty minutes after a single dose of 0.2 microgram DPDPE, administered intracerebroventricularly, transient decreases of endogenous Ca2+ and Mg2+ contents in the parietal cortex, hippocampus and striatum were found with no changes observed in the levels of monovalent cations or Mn2+. A time-dependent down-regulation in Ca2+ and Mg2+ content was also demonstrated, with Ca2+ being faster to respond to DPDPE treatment. The action of DPDPE was dose-dependent (0.2-1.0 micrograms) and could be antagonized by a 30 min naloxone pretreatment. Naloxone alone had no effect on the endogenous cation levels. It is concluded that delta opioid receptors may specifically be involved in the regulation of endogenous ion levels and their movements in the central nervous system of rat.     

Modulatory effects of aluminum, calcium, lithium, magnesium, and zinc ions on [3H]MK-801 binding in human cerebral cortex

The independent and combined effects of Ca2+, Mg2+, Zn2+, Al3+ and Li+ on [3H]MK-801 binding in human cerebral cortical membranes were studied to further characterize the modulatory effects of metal ions on the N-methyl-D-aspartate (NMDA) receptor-ionophore. Glycine, in the presence of glutamate, significantly intensified the Mg2+ inhibition of [3H]MK-801 binding whereas it masked the Ca2+ enhancement and slightly diminished the Zn2+ inhibition. Both Ca2+ and Mg2+ reduced the Zn2+ inhibitory potency. Aluminum demonstrated a potent, relatively glycine-insensitive inhibition of [3H]MK-801 binding as an amorphous Al(OH)3 polymer rather than as the free ion. Cationic modulation of the NMDA receptor-ionophore appears to be regulated at multiple sites which have significant allosteric interactions.     

Selective antagonism of frog cerebellar synaptic transmission by manganese and cobalt ions.

Parallel fiber-Purkinje cell synaptic transmission of the frog cerebellum can be selectively blocked by Mn2+ and Co2+. The interaction of these cations with Ca2+ indicates that they act, as has been found for other chemical synapses, at the presynaptic terminals where Ca2+ is required for release of neurotransmitters. Climbing fiber-Purkinje cell synaptic transmission was similarly blocked by Mg2+, Mn2+ and Co2+.     

The effects of excitatory amino acids on intracellular calcium in single mouse striatal neurons in vitro

Using microspectrofluorimetry and the calcium-sensitive dye fura-2, we examined the effect of excitatory amino acids on [Ca2+]i in single striatal neurons in vitro. N-methyl-D-aspartic acid (NMDA) produced rapid increases in [Ca2+]i. These were blocked by DL-2-amino-5-phosphonovaleric acid (AP5), by Mg2+, by phencyclidine, and by MK801. The block produced by Mg2+ and MK801 could be relieved by depolarizing cells with veratridine. When external Ca2+ was removed, NMDA no longer increased [Ca2+]i. Furthermore, the effects of NMDA were not blocked by concentrations of La3+ that blocked depolarization induced rises in [Ca2+]i. Substitution of Na+o by Li+ did not block the effects of NMDA. Concentrations of L-glutamate greater than or equal to 10(-6) M also increased [Ca2+]i. The effects of moderate concentrations of glutamate were blocked by AP5 but not by La3+ or by substitution of Na+ by Li+. The effects of glutamate were blocked by removal of external Ca2+ but were not blocked by concentrations of Mg2+ or MK801 that completely blocked the effects of NMDA. The glutamate analogs kainic acid (KA) and quisqualic acid also increased [Ca2+]i. The effects of KA were blocked by removal of external Ca2+ but not by La3+, Mg2+, MK801, or replacement of Na+ by Li+. Although AP5 was able to block the effects of KA partially, very high concentrations were required. These results may be explained by considering the properties of glutamate-receptor-linked ionophores. Excitatory amino acid induced increases in [Ca2+]i are consistent with the possibility that Ca2+ mediates excitatory amino acid induced neuronal degeneration.     

Role of cations in olfactory reception

The effects of changed ionic environments on the olfactory responses in the carp, the rainbow trout, and the bullfrog were examined by recording the stimulant-induced waves from the olfactory bulb. (a) Application of stimulants (various species of amino acids and beta-ionone) dissolved in deionized water to the EDTA-treated olfactory epithelium of the carp did not induce any response. The addition of various species of salts to the stimulating solution reversibly restored the response. (b) The responses of the carp to L-alanine in the presence of MgCl2 and MgSO4 and those in the presence of KCl and K4Fe(CN)6 are described by single curves, respectively, as a function of concentration of the cations, suggesting that the cations support olfactory reception. (c) All the inorganic cations examined (Li+, NH3+, K+, Ca2+, Mg2+, Co2+, Mn2+, Cd2+) and organic cations (Tris+, choline+, bis-Tris propane2+) were effective to support the response of the carp, whereas TEA+, TBA+, triethanolamine+, and bis-Tris+ were ineffective. (d) The olfactory responses of the rainbow trout and the bullfrog were also reduced by removal of ions from the surface of the epithelia and recovered by addition of ions. (e) It is suggested that the cations do not act as current carriers across the apical olfactory cell membrane for the generation of the receptor potential.     

Nerve growth factor-induced neurite outgrowth of rat pheochromocytoma PC 12 cells: dependence on extracellular Mg2+ and Ca2+

Dependence of neurite outgrowth on extracellular Mg2+ and Ca2+ was studied in nerve growth factor-responsive pheochromocytoma PC 12 cells under assay conditions in which neurite formation was independent of both RNA synthesis and protein synthesis. NGF-induced neurite formation occurred maximally in the presence of extracellular Mg2+ at concentrations greater than 1.0 mM. However, extracellular Ca2+ alone did not stimulate the neurite formation, and inhibited this process at higher concentrations (greater than 10 mM). These data are consistent with the fact that NGF-mediated neurite extension occurred in assay medium containing either 1.0 mM EGTA or 0.5 mM LaCl3. Other divalent cations so far tested proved to be negative, suggesting that this phenomenon appears to be specific to Mg2+. Moreover, quantitative analysis revealed that the length and thickness of neurites formed were controlled by the presence of extracellular Ca2+. Thus, neurites formed at lower concentrations of Ca2+ in the presence of 1.0 mM Mg2+ and NGF were found to be thinner and longer than those formed at higher concentrations of Ca2+, suggesting that Ca2+ and Mg2+ have separate regulatory functions in the formation of neurites of PC 12 cells.     

Differential ontogeny of divalent cation effects on rat brain delta-, mu-, and kappa-opioid receptor binding

The effect of the divalent cations, Mn2+, Mg2+ and Ca2+ on rat forebrain delta-, mu- and kappa-receptor binding was examined during postnatal development. It was found that delta-receptor binding, assessed with [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) (+ 10 nM D-Ala2- MePhe4-Gly-ol5-enkephalin (DAMGE)), was stimulated by the 3 cations in a dose- and developmental time-dependent manner. delta-Binding was most sensitive to the cations during the first week postnatal, prior to the appearance of high-affinity delta-binding. In contrast, inhibition of mu-receptor binding ([3H]DAMGE) by divalent cations appeared early in development and remained constant throughout the postnatal period. Divalent cation inhibition of kappa-binding ([3H]ethylketocyclazocine ([3H]EKC) + 100 nM DAMGE and 100 nM DADLE) appeared after the second week postnatal. These results demonstrate that the characteristics and postnatal development of divalent cation modulation of mu-, delta- and kappa-binding is distinctly different. Thus, the neonate may be a good model system to examine the binding properties and functions of delta- and kappa-receptor subtypes.     

Divalent cations and fast axonal transport in chemically desheathed (triton X-treated) frog sciatic nerve

We have studied the ability of divalent cations to restore to normal axonal transport (AXT) which was inhibited by deprivation of Ca2+ and/or Mg2+ ions. The epi- and perineurium of the frog sciatic nerve were damaged by a 30-s wash in Triton X-100 containing frog Ringer's. This treatment did not affect either AXT or nerve levels of Ca2+ and Mg2+, but made the ions more easily extractable with a Ca2+- and Mg2+-free Ringer's solution (CMFR). Inhibition of AXT was achieved by incubating Triton X-100-treated nerves in CMFR + EGTA for 5 h, followed by an additional incubation for 12 h in CMFR or Ringer's devoid of only Ca2+ (CFR). These treatments reduced Ca2+ and Mg2+ contents by 77% and 38% respectively. Addition of Ca2+ (1.1 mM) during the 12-h period stimulated AXT, measured as accumulation of 3H-labelled components in front of a ligature, several fold. Mg2+ could not substitute for Ca2+ but potentiated the stimulating effect of Ca2+. Addition of other divalent cations did not affect AXT (Sr2+ and Ba2+) or potentiated the inhibition caused by Ca2+-deprived medium (Mn2+ and Co2+). ATP and creatine phosphate contents were similar in nerves incubated in Ca2+-deprived medium and in Ca2+-containing Ringer's. Thus, inhibition of AXT in the former situation was not due to a decreased availability of high energy phosphates. Two calcium antagonists, D-600 and nifedipin, which are potent smooth muscle relaxants, effectively blocked AXT. The present results suggest that Ca2+ is specifically required to maintain AXT and that an analogy exists between Ca2+ regulation during smooth muscle contraction and AXT.     

Pharmacological characterization of endothelin-stimulated phosphoinositide breakdown and cytosolic free Ca2+ rise in rat C6 glioma cells.

Because increasing evidence indicates that glial cells are a target of endothelin, we have characterized endothelin-induced phosphoinositide (PI) turnover and Ca2+ homeostasis in C6 glioma cells. Endothelin-1 (ET) increased formation of 3H-inositol phosphate (IP) from PI and elicited an increase in cytosolic free Ca2+ ([Ca2+]i) in rat C6 glioma. In the presence of Li+, the increase in 3H-inositol trisphosphate formation was rapid, reaching its peak at 5 min after stimulation. ET also elicited a rapid and sustained increase in [Ca2+]i in a dose-dependent manner (1-100 nM). The rank orders of efficacy for ET-related peptides in increasing [Ca2+]i were ET = ET-2 greater than sarafotoxin greater than ET-3. Both ET-mediated stimulation of IP formation and [Ca2+]i increase were largely inhibited in the absence of external Ca2+ but unaffected by the depletion of external Na+ and the presence of dihydropyridine derivatives or verapamil. Inorganic Ca2+ channel blockers Cd2+, La3+, and Mn2+ at 1 mM inhibited both responses induced by ET. Cross-desensitization and nonadditivity were observed for both events among ET-related peptides tested, but not between ET and ATP. Pretreatment of cells with pertussis toxin (PTX) attenuated the PI response to ET, but had no effect on ET-elicited [Ca2+]i increase. ET-induced Ca2+ mobilization (measured in Ca(2+)-free medium) was only transient and was inhibited by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate. Moreover, the intracellular Ca2+ pools mobilized by ET and ATP appeared to overlap, as indicated by their partial heterologous desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chelation of endogenous membrane calcium inhibits gamma-aminobutyric acid uptake in synaptosomes

In a previous work, we have demonstrated that calcium chelators induce the release of gamma-aminobutyric acid (GABA) from synaptosomes in a Na+ -dependent manner and that this release is blocked by cations such as Mg2+, La3+, and ruthenium red. In the present study, we show that treatment of synaptosomes with 0.1 mM EGTA in the absence of both Ca2+ and Mg2+ inhibits the sodium-dependent high-affinity uptake of [3H]GABA by about 50%. This inhibition increased to about 65% with 1.5 mM EGTA, and it was completely prevented by an excess of Ca2+ or by 1.2 mM Mg2+. In contrast, when EDTA was used as a chelator, Mg2+ was unable to reverse the inhibition. The inhibitory effect of 0.1 mM EGTA was also prevented by 250 microM La3+ or by 20 microM ruthenium red. In the absence of chelators and the presence of Ca2+ and Mg2+, 50 microM and 200 microM La3+ inhibited GABA uptake by about 20 and 50%, respectively, whereas 20 microM ruthenium red produced a nonsignificant 25% inhibition and nifedipine was without effect. It is concluded that the membrane-surface negative charges, probably those of the sialic acid molecules that have been implicated in the functioning of the GABA carrier, must be neutralized by endogenous Ca2+ or by another cation in order to permit the adequate function of the transporter. The inhibition by La3+ in the absence of the chelators could be explained by a binding of this cation to the Na+ sites on the GABA carrier.     

Ionomycin, a Ca++ ionophore, increases platelet volume independently of the Na+/H+ exchanger

A Ca++-ionophore, ionomycin, increased the volume of human platelets suspended in a Ca++-containing buffer. This change in cell volume was dependent upon ionomycin and extracellular Ca++ concentrations, suggesting that the volume change occurs when the intracellular Ca++ reaches a certain level (greater than uM as determined by aequorin method). The ionomycin-induced volume increase was suppressed by replacement of extracellular Na+ with membrane-impermeable N-methyl-D-glucamine or Cs+, but not with Li+, K+, or Rb+. Ethylisopropylamiloride, a potent inhibitor of the Na+/H+ exchanger, had only weak inhibitory effect, and the apparent Km for Na+ was approximately 350 mM, which is much larger than that of the Na+/H+ exchanger. It is suggested that certain mechanisms other than the Na+/H+ exchanger are responsible for ionomycin-induced volume increase.     

Cations differentially affect subpopulations of L-glutamate receptors in rat synaptic plasma membranes

Several cations were examined for their ability to specifically affect one of the 3 L-glutamate (L-Glu) binding sites in rat forebrain synaptic plasma membranes (i.e. Na+-dependent, Cl--dependent and Cl--independent). Na+-dependent binding was potently inhibited by K+ and NH4+ ions. Other monovalent cations tested (Cs+, Li+, triethylammonium) had no effect on this binding site. Polyvalent cations (Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Cr3+) also had little effect on the Na+-dependent L-Glu binding site. Cl--dependent L-Glu binding was potently inhibited by Na+ ions but was not affected by other monovalent ions. All of the divalent cations were potent inhibitors of both Cl--dependent and -independent binding. The results show that these binding sites of L-Glu can be distinguished by their response to cations and suggest possible novel modes of regulation in vivo.     

Taste responses to electrolytes in the frog glossopharyngeal nerve: initial process of taste reception

In taste reception, it has been proposed that changes in surface potential on the apical membrane of taste cells bring about activation of taste cells during chemical stimulation. To ascertain whether changes in the surface potential are involved in taste reception of electrolytes, unitary discharges were recorded from single water fibers of the frog glossopharyngeal nerve with a suction electrode. The surface potential is a function of both the charge density of the membrane surface and the ionic strength of the medium. Low concentrations of CaCl2 (less than 1 mM) were very effective stimuli. However, 0.01-1 mM LaCl3 and HCl (pH 3.0-4.5), which alter the surface potential in the positive direction, had no excitatory effect. Transition metal cations, such as Mn2+, Co2 and Ni2+, had an excitatory effect, but the responses to these cations appeared at relatively high concentrations (greater than 5 mM), in spite of the high affinity of the receptor membrane for these cations. The results suggest that the surface charge of the apical membrane is not associated with the excitation caused by electrolytes. MgCl2 (greater than 5 mM) and NaCl (greater than 100 mM) were also effective stimuli, whereas choline Cl (100-1000 mM) had no excitatory effect. An increase in the ionic strength was achieved by the addition of 100-300 mM choline Cl to stimulating solutions of MgCl2 or NaCl. The responses to Mg2+ and Na+ were not affected by the increase in the ionic strength. The results obtained here indicate that changes in the surface potential on the surface of the apical membrane are not involved in taste reception of electrolytes. Alteration of the surface potential of the membrane in the positive direction would bring about a reduction in the local concentration of cations in the vicinity of the membrane. Hence, the presence of divalent cations in the medium may affect the response to monovalent cations. However, addition of 100 mM MgCl2 to the stimulating solution of NaCl did not affect the concentration-response curve for NaCl. This result suggests that the surface charge density of the apical membrane is very low and hence the magnitude of the surface potential is very small. The results also suggest that Mg2+ and Na+ activate the taste cells by two separate, non-interacting processes. The present study suggests that, in the initial process of taste reception, only the binding of each separate cation to its appropriate receptor site (specific receptor site) leads to activation of the receptor.     

丙泊酚可调节缺血再灌注损伤脊髓Ca2+, Mg2+, Cu2+，Zn2+的平衡

检测血清和脊髓匀浆中Ca2+、Mg2+、Cu2+、Zn2+的变化规律，揭示丙泊酚对缺血再灌注损伤脊髓保护作用的可能机制。结果发现，随着缺血再灌注损伤时间的延长，兔血清Ca2+，Cu2+浓度逐渐升高，Mg2+，Zn2+浓度逐渐下降，至脊髓损伤后7d，以上离子变化最明显；缺血再灌注损伤7 d，兔缺血脊髓匀浆中的各离子浓度变化与血清中相一致。给予丙泊酚干预后，缺血再灌注期间兔血清和脊髓匀浆中Ca2+、Mg2+、Cu2+、Zn2+浓度均无显著的波动。提示丙泊酚可通过稳定或恢复脊髓缺血再灌注损伤区金属离子的平衡发挥对脊髓缺血再灌注损伤的保护作用。     

Spontaneous voltage and current fluctuations in tissue cultured mouse dorsal root ganglion cells

Fetal mouse dorsal root ganglion (DRG) neurons were maintained in primary dissociated cell culture for periods of 7 days to 3 months. Intracellular recordings from these cells revealed the presence of spontaneous subthreshold potentials in 101/177 neurons studied. When measured at the resting membrane potential, these spontaneous voltage events took two forms: (a) high frequency potential fluctuations several millivolts in peak-to-peak amplitude and (b) small, discrete hyperpolarizations. Neurons exhibiting either type of event were designated as 'active' DRG cells. No spontaneous potentials were seen in DRG cells hyperpolarized to membrane voltages more negative than -64 +/- 11.5 mV (n = 5 cells). Under voltage-clamp conditions, the subthreshold potentials of active DRG cells were replaced by fluctuations in outward current. The power spectral density, S(f) of these current fluctuations was approximated by an equation of the form S(f) = (S(o)/[1 + (f/fc) alpha] where 2 less than or equal to a less than or equal to 3 and the half-power frequency fc = 11.3 +/- 3.1 Hz at 23 degrees C (n = 17 cells). The spontaneous voltage fluctuations of active DRG cells were abolished in Ca2+-free saline, and of the divalent metal cations Sr2+, Mg2+, Ba2+, Co2+ and Mn2+, only Sr2+ could substitute for Ca2+ in the maintenance of this activity. Tetraethylammonium ions (1-10 mM) reversibly blocked the spontaneous potentials, while caffeine (10 mM) increased the frequency of these events. The spontaneous voltage fluctuations were not dependent on the presence of spinal cord neurons in the culture plate, and they were also observed in cultured DRG cells derived from adult mice.     

Brain cationic ATPase activities in epileptic (El) mice

Total, Mg2+-, Na+,K+-, and Ca2+-ATPase activities were studied in fresh brain membrane preparations from adult epileptic (El) mice and nonepileptic C57BL/6J (B6) mice. The El mice have an inherited type of temporal lobe epilepsy. No significant differences were observed between the El and B6 mice for any of the ATPase activities in the hippocampus, brain stem, or cerebellum. These findings indicate that seizure susceptibility in El mice is not associated with differences in the activities of these cationic ATPases and that seizure susceptibility in El mice and audiogenic DBA/2 mice may involve different biochemical mechanisms.     

Calcium-activated cationic channel in rat sensory neurons

Ion channels in sensory neurons are molecular sensors that detect external stimuli and transduce them to neuronal signals. Although Ca2+-activated nonselective cation (CAN) channels were found in many cell types, CAN channels in mammalian sensory neurons are not yet identified. In the present study, we describe an ion channel that is activated by intracellular Ca2+ in cultured rat sensory neurons. Half-maximal concentration of Ca2+ in activating the CAN channel was approximately 780 micro m. The current-voltage relationship of this channel was linear with a unit conductance of 28.8 +/- 0.4 pS at -60 mV in symmetrical 140 mm Na+ solution. The CAN channel was permeable to monovalent cations such as Na+, K+, Cs+, and Li+, but poorly permeable to Ca2+. The CAN channel in mammalian sensory neurons was reversibly blocked by intracellular adenine nucleotides, such as ATP, ADP, and AMP. Interestingly, single-channel currents activated by Ca2+ were blocked by fenamates, such as flufenamic acid, a class of nonsteroidal anti-inflammatory drugs. Thus, these results suggest that CAN channels in mammalian sensory neurons would participate in modulating nociceptive neural transmission in response to ever-changing intracellular Ca2+ in the local microenvironment.     

Effects of tetraethylammonium-chloride and divalent cations on the afterhyperpolarization following repetitive firing in leech neurons

In leech Retzius cells, repetitive activity evoked a prolonged Ca2+-dependent after-hyperpolarization (PAH) (30-60 s) accompanied by an increase in input conductance. PAH persisted in Retzius cells, as well as in nociceptive (N) cells, when Sr2+ but not Mg2+ was substituted for Ca2+. In the presence of tetraethylammonium-chloride (TEA) or Ba2+, PAH was replaced by a Ca2+-dependent, Mg2+-blockable depolarization which was present in the order N greater than R. Careful study of the differences in such phenomena in identified cells may improve our understanding of the differential susceptibility of various neurons to hyperexcitability.