Antioxidants prevent depletion of [Mg]i induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke

Low serum concentrations of Mg²⁺ ions have been reported, recently, in patients with coronary disease, atherosclerosis, and stroke as well as in patients with cerebral hemorrhage. The aim of the present study was to determine whether potent antioxidants [α-tocopherol and pyrrolidine dithiocarbamate (PDTC)] can prevent or ameliorate intracellular Mg²⁺ ([Mg²⁺]_i) depletion associated with cerebral vascular injury induced by alcohol. Exposure of cultured canine cerebral vascular smooth muscle cells to alcohol (10–100 mM) for 24 h induced marked depletion in [Mg²⁺]_i (i.e., 30–65%, depending upon alcohol concentration). Treatment of the cultured cells with either PDTC (0.1 μM) or α-tocopherol (15 μM) for 24 h, alone, failed to interfere with basal [Mg²⁺]_i levels. However, preincubation of the cells with either α-tocopherol or PDTC for 24 h completely inhibited the depletion of [Mg²⁺]_i induced by exposure to 10–100 mM ethanol. These results indicate that α-tocopherol and PDTC prevent decreases in [Mg²⁺]_i produced by ethanol. Moreover, these new results suggest that such protective effects of α-tocopherol and PDTC on cerebral vascular cells might be useful therapeutic tools in prevention and amelioration of cerebral vascular injury and stroke in alcoholics.     

Ethanol inhibition of NMDA mediated depolarizations is increased in the presence of Mg

The inhibitory potency of ethanol upon excitatory amino acid induced depolarizations of rat hippocampal CA1 pyramidal cells was assessed in the presence and absence of magnesium (Mg²⁺) using the grease-gap technique. Ethanol shifted theN-methyl-d-aspartate (NMDA) dose-response curves to the right in a non-parallel manner. In the presence of Mg²⁺, ethanol appeared to be a more effective NMDA antagonist (IC₅₀ 47 mM) than in the absence of Mg²⁺ (IC₅₀ 107 mM). The IC₅₀ for ethanol upon non-NMDA mediated CA1 pyramidal cell depolarizations was in excess of 170 mM. These results strongly suggest a preferential inhibitory action of ethanol against NMDA, rather than non-NMDA, mediated responses. Experiments in which ethanol and Mg²⁺ were covaried indicated that these substances act by two distinct mechanisms to antagonize the action of NMDA. These effects of ethanol, at concentrations which elicit intoxication(< 50mM) but not anesthesia, suggest that the NMDA receptor complex may play an important role in the acute effects of ethanol.     

Effects of extracellular calcium and magnesium on central respiratory control in the brainstem–spinal cord of neonatal rat

The influence of extracellular Ca²⁺ and Mg²⁺ concentrations ([Ca²⁺]_ECF and [Mg²⁺]_ECF, respectively) on central respiratory control was analyzed using the isolated brainstem–spinal cord of the neonatal rat. Central respiratory activity was recorded from the C4 ventral roots. The depth profile of [Ca²⁺]_ECF below the ventral medullary surface was measured with ion-sensitive electrodes. The gradient in [Ca²⁺]_ECF disappeared about 1 h after changing superfusate Ca²⁺ ([Ca²⁺]_CSF) from 2 to 0.5 mM, but not even in 2 h after switching to Ca²⁺-free superfusate. High [Ca²⁺]_CSF (4 mM) or high [Mg²⁺]_CSF (4, 8 mM) decreased respiratory frequency (f_R), whereas low [Ca²⁺]_CSF (0.5 mM) increased f_R and augmented the respiratory CO₂ responsiveness. High [Ca²⁺]_CSF as well as low [Mg²⁺]_CSF (0.5 mM) disturbed respiratory rhythm and pattern, which were markedly restored by high CO₂. The depressing effect of high [Ca²⁺]_ECF and the stimulating effect of low [Ca²⁺]_ECF on the medullary neuronal activity were confirmed by perforated patch recordings. These results suggest that [Ca²⁺]_ECF and [Mg²⁺]_ECF determine the excitability of the respiratory neuron network by modulating the neuronal surface potential, transmembrane Ca²⁺ influx, Ca²⁺-sensitive cation channel gating, and synaptic transmission. Furthermore, some of these actions appear to be antagonized by CO₂/H⁺.     

Non-opioid dynorphin binding site on secretory vesicles of a pituitary-derived cell line

Accumulating evidence indicates that the endogenous opioid peptides dynorphinA-(1-17) and dynorphinA-(1-13) interact not only with opioid but also with yet poorly characterized non-opioid receptors. The latter have been implicated in a number of the effects of dynorphins including induction of ACTH release in sheep and in AtT 20 cells, a pituitary-derived mouse cell line. AtT 20 cells do not express opioid receptors and therefore are particularly suitable for search of non-opioid dynorphin receptors. We report here that ³H-dynorphinA-(1-13)-NH₂ associates specifically with AtT 20 cells, apparently through an uptake process and a binding site. Within the cell, it binds preferentially to fractions containing secretory vesicles, with a K_d of about 100 nM. DynorphinA-(1-17), and several non-opioid fragments of dynorphin, including A-(2-17), A-(2-16) and A-(2-13), compete with ³H-dynorphinA-(1-13)-NH₂ for that site with IC₅₀s ranging from 200 nM to 2 μM. ACTH(1-39) also competes with ³H-dynorphinA-(1-13)-NH₂ for the site with an IC₅₀ of about 300 nM. DynorphinA-(2-17) at μM concentrations stimulates release of ACTH from the isolated vesicles. The results indicate the presence of a non-opioid dynorphin binding site on the secretory vesicle fractions of AtT20 cells that might be involved in ACTH release. The ability of ACTH itself to compete for the binding sites associated with the vesicles suggest that those sites may be involved in an autocrine loop.     

Spermine is neuroprotective against anoxia and N-methyl- -aspartate in hippocampal slices

Polyamines were implicated as either neurotoxic or neuroprotective in several models of stroke. Spermine augments the excitotoxicity mediated by the N-methyl- -aspartate (NMDA) receptor because this receptor is activated at micromolar spermine concentrations. However, at higher concentrations, spermine could be neuroprotective because it blocks the NMDA receptor and voltage-activated Ca²⁺ channels. In this work, acute hippocampal slices were exposed to 1 mM spermine and either 10 min of anoxia or 0.5 mM NMDA. The percent recovery of population spikes was the measure of neuroprotection. One millimolar spermine was robustly neuroprotective; however, 0.1 mM spermine and 1 mM putrescine were not. The neuroprotective concentration of spermine was higher than the physiological concentration of free spermine. However, during an excitotoxic episode, extracellular Ca²⁺ is decreased, enabling the inhibitory activity of lower spermine concentration. In addition, several noxious stimuli trigger the release of intracellular spermine and could raise local levels of spermine. Therefore, it is possible that spermine has a neuroprotective role in vivo.     

Myelin contains neutral sphingomyelinase activity that is stimulated by tumor necrosis factor-α

Purified myelin from mouse brain was found to contain two forms of neutral sphingomyelinase, one Mg²⁺ dependent and the other Mg²⁺ independent. The former had a pH optimum of 7.5 and K_m of 0.35 mM, whereas the corresponding values for the latter were pH 8.0 and K_m 3.03 mM. Specific activity of the Mg²⁺-dependent enzyme showed a rostral-caudal gradient, ranging from 75 nmol/mg protein/hr in myelin from cerebral hemispheres to 21 nmol/mg protein/hr in myelin from spinal cord. Relative specific activity was approximately 20% that of brain stem or cerebral hemisphere homogenate. Treatment of myelin with taurocholate or high salt concentration did not significantly reduce activity of the Mg²⁺-dependent enzyme. The activity of that enzyme did not change with time or in the presence or absence of protease inhibitors; by contrast, that of the Mg²⁺-independent enzyme decreased sharply in the absence of protease inhibitors but rose in their presence. To test for the effect of tumor necrosis factor-α (TNFα) on myelin sphingomyelinase, mouse brain myelin was labeled in vivo by intracerebral injection of [³H]acetate into 18–20-day-old mice. After 40 hr, brain stems were removed, minced, and treated with TNFα in Krebs-Ringer solution, after which myelin was immediately isolated. Separation and counting of individual lipids revealed TNFα treatment to cause increased labeling of myelin ceramide and cholesterol ester with concomitant decrease in myelin sphingomyelin. Western blotting of myelin proteins using antibodies to the two TNFα receptors as probes revealed the presence of the p75 receptor. Implications of these findings in relation to possible mechanisms of autoimmune demyelination are discussed. J. Neurosci. Res. 50:466–476, 1997. © 1997 Wiley-Liss, Inc.     

Extracellular magnesium and calcium reduce myotonia in ClC-1 inhibited rat muscle

Loss-of-function mutations in the ClC-1 Cl^? channel trigger skeletal muscle hyperexcitability in myotonia congenita. For reasons that remain unclear, the severity of the myotonic symptoms can vary markedly even among patients with identical ClC-1 mutations, and may become exacerbated during pregnancy and with diuretic treatment. Since both these conditions are associated with hypomagnesemia and hypocalcemia, we explored whether extracellular Mg²⁺ and Ca²⁺ ([Mg²⁺]_o and [Ca²⁺]_o) can affect myotonia. Experimental myotonia was induced in isolated rat muscles by ClC-1 inhibition and effects of [Mg²⁺]_o or [Ca²⁺]_o on myotonic contractions were determined. Both cations dampened myotonia within their physiological concentration ranges. Thus, myotonic contractile activity was 6-fold larger at 0.3 than at 1.2 mM [Mg²⁺]_o and 82-fold larger at 0.3 than at 1.27 mM [Ca²⁺]_o. In intracellular recordings of action potentials, the threshold for action potential excitation was raised by 4–6 mV when [Mg²⁺]_o was elevated from 0.6 to 3 mM, compatible with an increase in the depolarization of the membrane potential necessary to activate the Na⁺ channels. Supporting this notion, mathematical simulations showed that myotonia went from appearing with normal Cl^? channel function to disappearing in the absence of Cl^? channel function when Na⁺ channel activation was depolarized by 6 mV. In conclusion, variation in serum Mg²⁺ and Ca²⁺ may contribute to phenotypic variation in myotonia congenita patients.     

Spermine increases paired-pulse facilitation in area CA1 of hippocampus in a calcium-dependent manner

The effect of spermine on neurotransmission was studied in area CA1 of the hippocampal slice preparation. Paired-pulse stimulation (20 ms interpulse interval) was delivered to stratum radiatum; the evoked field potential responses were recorded simultaneously from stratum radiatum and from stratum pyramidale. At mM and sub-mM concentrations, spermine decreased the slope of pEPSP in stratum radiatum and the area of the conditioning population spike in stratum pyramidale. Short-latency paired-pulse inhibition of the population spike was converted to facilitation by spermine. These effects of spermine resembled those observed at low calcium concentration. In addition, dose-response and input-output curves determined at various Ca²⁺ concentrations demonstrated that the depressant effects of spermine were larger at low Ca²⁺ levels. The results support the notion that spermine competitively blocks presynaptic voltage-sensitive Ca²⁺ channels, thus causing a decreased release of neurotransmitter. Since spermine is present in brain, it is likely that it is a natural modulator of Ca²⁺ channels.     

Inhibition of [Ca2+]i Transients in Rat Adrenal Chromaffin Cells by Neuropeptide Y Role for a cGMP-dependent Protein Kinase-activated K+ Conductance

The effects of neuropeptide Y on the intracellular level of Ca²⁺ ([Ca²⁺]_i) were studied in cultured rat adrenal chromaffin cells loaded with fura-2. A proportion (16%) of cells exhibited spontaneous rhythmic [Ca²⁺]_i oscillations. In silent cells, oscillations could be induced by forskolin and 1,9–dideoxyforskolin. This action of forskolin was not modified by H-89, an inhibitor of protein kinase A. Spontaneous [Ca²⁺_i fluctuations and [Ca²⁺]_i fluctuations induced by forskolin- and 1,9-dideoxyforskolin were inhibited by neuropeptide Y. Increases in [Ca²⁺]_i induced by 10 and 20 mM KCI but not by 50 mM KCI were diminished by neuropeptide Y. However, neuropeptide Y had no effect on [Ca²⁺]_i increases evoked by (-)BAY K8644 and the inhibitory effect of neuropeptide Y on responses induced by 20 mM KCI was not modified by o-conotoxin GVIA, consistent with neither L- nor N-type voltage-sensitive Ca²⁺ channels being affected by neuropeptide Y. Rises in [Ca²⁺]_i provoked by 10 mM tetraethylammonium were not decreased by neuropeptide Y, suggesting that K⁺ channel blockade reduces the effect of neuropeptide Y. However, [Ca²⁺]_i transients induced by 1 mM tetraethylammonium and charybdotoxin were still inhibited by neuropeptide Y, as were those to 20 mM KCI in the presence of apamin. The actions of neuropeptide Y on [Ca²⁺]_i transients provoked by 20 and 50 mM KCI, 1 mM tetraethylammonium, (-)BAY K8644 and charybdotoxin were mimicked by 8–bromo-cGMP. In contrast, 8–bromo-CAMP did not modify responses to 20 mM KCI or 1 mM tetraethylammonium. The inhibitory effects of neuropeptide Y and 8–bromo-cGMP on increases in [Ca²⁺]_i induced by 1 mM tetraethylammonium were abolished by the Rp-8–pCPT-cGMPS, an inhibitor of protein kinase G, but not by H-89. A rapid, transient increase in cGMP level was found in rat adrenal medullary tissues stimulated with 1 μM neuropeptide Y. Rises in [Ca²⁺]_i produced by DMPP, a nicotinic agonist, but not by muscarine, were decreased by neuropeptide Y. Our data suggest that neuropeptide Y activates a K⁺ conductance via a protein kinase G-dependent pathway, thereby opposing the depolarizing action of K⁺ channel blocking agents and the associated rise in [Ca²⁺]_i.     

Synaptic transmission and paired-pulse behaviour of CA1 pyramidal cells in hippocampal slices from a hibernator at low temperature: importance of ionic environment

To investigate the effects of ionic changes possibly associated with hibernation, hippocampal slices prepared from golden hamsters were studied in artificial cerebrospinal fluid (ACSF) of variable composition (K⁺ 3–5 mM, Ca²⁺ 2–4 mM, Mg²⁺ 2–4 mM, pH 7.0–7.7) at temperatures of 15–20°C, just above the temperature below which synaptic transmission is blocked. Population action potentials (population spikes, PSs) of CA1 pyramidal cells were evoked by stimulation of the Schaffer collaterals/commissural fibers with paired pulses (interpulse interval 50 ms, interval pairs 30 s). The responses evoked at given temperatures were investigated as a function of extracellular ion concentrations. In ACSF containing 3 mM K⁺, 2 mM Ca²⁺ and 2 mM Mg²⁺, PSs could be evoked at temperatures of > ∼ 16°C whereas at lower temperatures synaptic transmission was blocked. The threshold temperature was slightly higher for the first (PS1) than for the second PS (PS2) evoked by paired-pulse stimulation. The slices displayed paired-pulse facilitation (PPF) at all temperatures. Elevation of [K⁺]₀ from 3 to 5 mM depressed the amplitudes of both PS1 and PS2, with a stronger effect on PS2. PPF was reduced and, at near-threshold temperatures, turned into paired-pulse depression (PPD). Elevation of [Ca²⁺]₀ from 2 to 4 mM increased the amplitude of PS1. The amplitude of PS2, in contrast, was reduced at near-threshold temperatures. PPF turned into PPD. Elevation of [Mg²⁺]₀ from 2 to 4 mM reduced the amplitudes of both PS1 and PS2, with a stronger effect on PS1. Accordingly, PPF was increased. Acidification by 0.3 pH units strongly depressed the amplitudes of PS1 as well as PS2 and increased PPF. Alkalization by 0.4 pH units had only weak effects in the opposite direction. Changes in the ionic composition comparable to those investigated in the present study presumably occur in the brain interstitium of hamsters during entrance into hibernation. According to our results, such changes depress synaptic transmission at low temperatures in the hamster hippocampus in vitro. This modulation may be important for the regulation of neuronal activity during entrance into hibernation.     

Extracellular magnesium and calcium reduce myotonia in isolated ClC‐1 chloride channel‐inhibited human muscle

Introduction: Experimental myotonia induced in rat muscle by ClC‐1 chloride channel‐inhibited has been shown to be related inversely to extracellular concentrations of Mg²⁺ and Ca²⁺ ([Mg²⁺]_o and [Ca²⁺]_o) within physiological ranges. Because this implicates a role for [Mg²⁺]_o and [Ca²⁺]_o in the variability of symptoms among myotonia congenita patients, we searched for similar effects of [Mg²⁺]_o and [Ca²⁺]_o on myotonia in human muscle. Methods: Bundles of muscle fibers were isolated from abdominal rectus in patients undergoing abdominal surgery. Myotonia was induced by ClC‐1 inhibition using 9‐anthracene carboxylic acid (9‐AC) and was assessed from integrals of force induced by 5‐Hz stimulation for 2 seconds. Results: Myotonia disappeared gradually when [Mg²⁺]_o or [Ca²⁺]_o were elevated throughout their physiological ranges. These effects of [Mg²⁺]_o and [Ca²⁺]_o were additive and interchangeable. Conclusions: These findings suggest that variations in symptoms in myotonia congenita patients may arise from physiological variations in serum Mg²⁺ and Ca²⁺. Muscle Nerve 51 : 65–71, 2015     

Li+/Mg2+ competition at therapeutic intracellular Li+ levels in human neuroblastoma SH‐SY5Y cells

Objectives: One proposed mechanism of lithium action in the treatment of bipolar disorder is that Li⁺ competes with Mg²⁺ for Mg²⁺ binding sites within the cell. In this study, we investigated this competition at therapeutic intracellular Li⁺ levels in human neuroblastoma SH‐SY5Y cells.

Methods: We used fluorescence spectroscopy and a Mg²⁺ indicator, furaptra, to investigate this competition in human neuroblastoma SH‐SY5Y cells. Atomic absorption spectrophotometry was used for determination of the intracellular Li⁺ levels.

Results: The neuroblastoma cells, incubated in 15 mM or 30 mM Li⁺‐containing buffer, showed a significant increase in free intracellular Mg²⁺ levels [using a positive linear within‐groups contrast t‐test, the 15 mM condition produced t(2)=5.0, one‐tailed p<0.02, and the 30 mM Li⁺‐incubation conditions gave t(2)=9.2, one‐tailed p<0.006] but did not significantly increase over time in the Li⁺‐free condition [t(2)=0.1, one‐tailed p>0.96]. At the earlier times during the incubation (1 or 10 min for the 15 mM or 30 mM Li⁺‐containing buffers), the intracellular Li⁺ concentrations were 0.6–2.5 mM, values which are comparable to those reached in the brain of Li⁺‐treated patients.

Conclusion: We demonstrated that competition between Li⁺ and Mg²⁺ can occur at therapeutic intracellular Li⁺ levels.     

Depolarization triggers intracellular magnesium surge in cultured dorsal root ganglion neurons

Intracellular magnesium concentration ([Mg²⁺]_i) of cultured dorsal root ganglion (DRG) neurons was measured using the magnesium indicator Mag-Fura-2/AM. [Mg²⁺]_i was 0.48±0.08 mM (mean±SEM, n=23) at rest, and it increased 3-fold by depolarization with a 60-mM K⁺ solution. The [Mg²⁺]_i increase was observed in the absence of extracellular Mg²⁺, but the increase disappeared in the absence of extracellular Ca²⁺. 50 μM cadmium or 100 μM verapamil, a Ca²⁺ channel blocker, also diminished the rise of [Mg²⁺]_i. The additional measurement of an intracellular Ca²⁺ concentration ([Ca²⁺]_i) indicated that the [Mg²⁺]_i rise requires a threshold concentration of [Ca²⁺]_i to be reached; above 60 nM. The present results indicate that depolarization induces a Ca²⁺-influx through voltage dependent Ca channels and this causes the release of Mg²⁺ from intracellular stores into the cytoplasm.     

Carbachol Potentiates Q Current and Activates a Calcium-dependent Non-specific Conductance in Rat Hippocampus In Vitro

Intracellular recordings were made from CA1 neurons in rat hippocampal slices maintained in vitro. When Na⁺ currents were blocked with tetrodotoxin and K⁺ conductances known to be sensitive to suppression by muscarinic agonists were blocked by 2 mM Ba²⁺, CA1 cells were depolarized by carbachol (3 – 10 μM) with an attendant conductance increase, whereas prior to Ba²⁺ the agonist produced a decrease or no change in conductance. Under voltage clamp at ～–60 mV and in the presence of tetrodotoxin and Ba²⁺, carbachol (3 – 10 μM) induced a variable-latency biphasic inward current of up to 380 pA associated with a conductance increase of ～50%. The first phase was associated with an increase (more than 2-fold) of the Cs⁺-sensitive, hyperpolarization-activated cationic current, I_Q. Carbachol also accelerated the kinetics of I_Q at – 100 mV with an average 24% reduction in its activation time constant. The second phase reflected an additional inward current that was Cs⁺-resistant, displayed little apparent voltage sensitivity and had a mean extrapolated reversal potential, determined in the presence of external Cs⁺ (>5 mM), of ～–20 mV. In a small proportion of cells the second phase of inward current was followed (or overlapped) by an outward current, also associated with a conductance increase, which reversed at ～–70 mV. These carbachol actions were prevented by extracellular 300 μM Cd²⁺ and 2 mM Mn²⁺, by high levels (>5 mM) of extracellular Mg²⁺ or Ca²⁺, and by omission of Ca²⁺ or reduction of extracellular Na⁺ to 25 mM by substitution of NaCl with Tris or N-methyl-d -glucamine. Carbachol action was not mimicked by oxotremorine (≤60 μM), but was irreversibly blocked by this drug. Likewise, atropine (100 nM) irreversibly and gallamine (10 μM) reversibly antagonized carbachol's action. The action of carbachol was blocked shortly after prior exposure of slices to 2 – 5 mM caffeine. Chronic or acute incubation of slices with 2 mM Li⁺ potentiated (between 1- and 2-fold) carbachol responses. The data indicate that muscarinic activation increases cationic flux by a calcium-dependent potentiation of I_Q and activation of a non-selective conductance. The probability that inositol phospholipid metabolism is involved in triggering these events is discussed.     

The action of valproate on spontaneous epileptiform activity in the absence of synaptic transmission and on evoked changes in [Ca]0 and [K]0 in the hippocampal slice

The effects of valproate (VPA) on neuronal excitability and on changes in extracellular potassium ([K⁺]₀) and calcium ([Ca²⁺]₀) were investigated with ion selective-reference electrode pairs in area CA₁ of rat hippocampal slices. Field potential responses to single ortho- and antidromic stimuli were unaltered by VPA (1–5 mM). The afferent volley evoked in the Schaffer-commissural fibers was also unaffected. In contrast, VPA (1 mM) depressed frequency potentiation and paired pulse facilitation markedly. Decreases in [Ca²⁺]₀ induced either by repetitive stimulation or by application of the excitatory amino acids N-methyl-d-aspartate and quisqualate were reduced, and the latter results suggest that VPA interferes with postsynaptic Ca²⁺ entry. When synaptic transmission was blocked by lowering [Ca²⁺]₀ (0.2 mM) and elevating [Mg²⁺]₀ (7 mM), prolonged afterdischarges elicited by antidromic stimulation were blocked by VPA. VPA also suppressed the spontaneous epileptiform activity seen when [Ca²⁺]₀ was lowered to 0.2 mM, without elevating [Mg²⁺]₀. The amplitudes of the rises in [K⁺]₀ induced by repetitive orthodromic stimulation were only slightly depressed and those elicited by antidromic stimulation were generally unaltered by VPA, as were laminar profiles of stimulus-evoked [K⁺]₀ signals. These results indicate that VPA has membrane actions in addition to known effects on excitatory and inhibitory transmitter pools.     

Lipopolysaccharide regulates both serotonin- and thrombin-induced intracellular calcium mobilization in rat C6 glioma cells: Possible involvement of nitric oxide synthase-mediated pathway

To investigate the mechanisms by which lipopolysaccharide (LPS) affects Ca²⁺ signaling systems, we studied the effects of LPS on the serotonin (5-HT)- or thrombin-induced intracellular Ca²⁺ ([Ca²⁺]_i) increase in rat C6 glioma cells. Pretreatment of the cells with 1 μg/ml LPS for 24 hr significantly inhibited [Ca²⁺]_i increase induced by 10 μM 5-HT- or 0.5 U/ml thrombin. Its inhibitory effects were both dose- and time-dependent. Treatment with 1 mM dibutyryl cGMP (dbcGMP) for 30 min also significantly inhibited the 5-HT- and thrombin-induced [Ca²⁺]_i increase to approximately 60–70% of control. However, simultaneous pretreatment with LPS and dbcGMP did not show any synergistic inhibition. The simultaneous pretreatment with LPS and the potent cGMP-dependent protein kinase (PKG) inhibitors H-8 and KT5823 for 24 hr significantly antagonized the inhibitory effect of LPS. Pretreatment of the cells with 1 μg/ml LPS for 24 hr significantly enhanced cGMP accumulation, while dexamethasone and NMMA (NOS inhibitors) significantly attenuated the LPS-induced enhancement in cGMP accumulation. In addition, pretreatment of the cells with 100 nM dexamethasone for 24 hr significantly suppressed LPS-induced inducible nitric oxide synthase (iNOS; type II NOS, NOS-II) protein expression. These results indicate that LPS may inhibit both 5-HT- and thrombin-induced [Ca²⁺]_i increase via iNOS expression and PKG activation pathway in rat C6 glioma cells. J. Neurosci. Res. 51:517–525, 1998. © 1998 Wiley-Liss, Inc.     

Membrane-bound enzymes of erythrocytes in human muscular dystrophy: (Na+ + K+-ATPase, Ca2+-ATPase, K+- and Ca2+-p-nitrophenylphosphatase.

Four enzyme activities were studied in erythrocyte membranes from patients with Duchenne and congenital myotonic muscular dystrophy. (Na⁺ + K⁺)-stimulated, Mg²⁺-dependent adenosinetriphosphatase, measured in two different media, showed normal activity and ouabain inhibition, as did K⁺-stimulated p-nitrophenylphosphatase. The specific activity of Ca²⁺-stimulated p-nitrophenylphosphatase was twice normal in Duchenne membranes. Ca²⁺-stimulated, Mg²⁺-dependent adenosine-triphosphatase was augmented in membranes from both Duchenne and congenital myotonic muscular dystrophic patients. The cause of the increased activities may be the necessity for compensating an alteration in the calcium metabolism in the dystrophic erythrocytes.Several kinetic parameters of the two Ca²⁺-stimulated enzyme activities were studied in Duchenne and control membranes. Most were not changed, with the exception of the Na⁺-stimulation of Ca²⁺-ATPase. In Duchenne membranes two affinity sites were present with half maximal activating concentrations of 58 ± 4 and 4 ± 1 mM Na⁺. In control membranes only one affinity site was found with K_a = 26 ± 9 mM Na⁺.     

Stimulative effect of glia maturation factor and acetylcholine on active amino acid uptake and Ca, Mg-ATPase activity in nodose ganglia excised from adult rats

During aerobic incubation at 37°C, active uptake of labeled non-metabolizable α-aminoisobutyric acid (AIB) into isolated nodose ganglia (NG) excised from adult rats was accelerated to nearly twice that of the control, by the addition of glia maturation factor (GMF, 5 μg/ml) in a dose-dependent manner. A similar but moderate stimulative effect on ganglionic AIB uptake was caused by the addition of acetylcholine (ACh, 1 mM) plus eserine (0.1 mM). This effect, however, was not antagonized by nicotinic (hexamethonium, C₆, 0.1 mM) or muscarinic (atropine, 0.1 mM) blockers. The GMF-induced amino acid uptake seemed to be inhibited by further addition of ACh. On the other hand, ganglionic Ca²⁺, Mg²⁺-ATPase activity was greatly stimulated by either GMF or ACh. These results suggest that the increase in AIB uptake induced by GMF or ACh is possibly linked to Ca²⁺, Mg²⁺-ATPase activity in NG cell membranes.     

Anions modulate cation-induced responses of single units of the frog glossopharyngeal nerve

Fibers of the frog glossopharyngeal nerve that are sensitive to water stimulation also respond to Ca, Mg and Na salts. During stimulation with a salt, the cation and the anion are applied together and the anion could influence the response to the cation. We examined this interaction using single unit recordings at the level of fungiform papilla. Nerve impulses of large amplitude were recorded in response to the stimulation of adjacent papillae with increasing concentrations of various Ca, Mg and Na salts. For a given cation, the elicited spike frequency depended on the anion. For example, the responses of single fibers to Ca²⁺ concentrations above 0.1 mM were maximal with CaSO₄ and minimal with Ca(SCN)₂. The rank order of efficiency was CaSO₄ > CaCl₂ = CaBr₂ = Ca(NO₃)₂ > Ca(SCN)₂ for Ca²⁺ ions at 5 mM. The effects of these anions were reversed for Mg and Na salts, the rank orders being Mg(SCN)₂ > Mg(NO₃)₂ ≥ MgBr₂ = MgCl₂ > MgSO₄, for Mg²⁺ ions at 200 mM, and NaSCN > NaI ≥ NaNO₃ > NaBr > NaCl NaF = Na₂SO₄, for Na⁺ ions at 500 mM. All these sequences correspond to the lyotropic rank order of the anions. In stimulation by a mixture of Ca and Na salts, which have different rank orders with respect to anions, either the response to Ca²⁺ ions or the response to Na⁺ ions could be eliminated as a result of mutual antagonism between Ca²⁺ and Na⁺ ions. In this case, the rank order of anions was dependent only on the cation that was able to exert a stimulatory effect in the mixture. Threshold concentrations for Ca, Mg and Na salts are influenced by cationic properties, but not by anionic properties. We hypothesize that anions can modulate the efficacy of cation transduction by binding to a membrane element that interacts with each of the three distinct receptors for Ca²⁺, Mg²⁺ and Na⁺ ions without altering the affinities of these receptors for the respective cations. The present results cannot be interpreted in terms of permeability of the apical membrane to anions and changes in surface potential on the apical membrane. The possibility is discussed that an anion-selective paracellular pathway between taste cells is responsible for the effect of anions on the cation-induced response.