Calcium homeostasis in rat septal neurons in tissue culture

Septal neutons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca²⁺ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca²⁺]_i) in the neurons was low (50–100 nM). Depolarization of the neurons with 50 mM K⁺ resulted in rapid elevation of [Ca²⁺]_i to 500–1,000 nM showing recovery to baseline [Ca²⁺]_i over several minutes. The increases in [Ca²⁺]_i caused by K⁺ depolarization were completely abolished by the removal of extracellular [Ca²⁺], and were reduced by 80% by the ‘L-type’ Ca²⁺ channel blocker, nimodipine (1 μM). [Ca²⁺]_i was also increased by the excitatory amino andl-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNOX and DNOX. In the absence of extracellular Mg²⁺, large fluctuations in [Ca²⁺]_i were observed that were blocked by removal of extracellular Ca²⁺, by tetrodotoxin (TTX), or by antagonists ofN-methyld-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg²⁺ and TTX, NMDA caused dose-dependent increases in [Ca²⁺]_i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca²⁺]_i in the absence of extracellular Ca²⁺, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca²⁺ stores. Thapsigargin (2 μM) had little effect on [Ca²⁺]_i, or on the recovery from K⁺ depolarization. Removal of extracellular Na⁺ had little effect on basal [Ca²⁺]_i or on responses to high K⁺, suggesting that Na⁺/Ca²⁺ exchange mechanisms do not play a significant role in the short-term control of [Ca²⁺]_i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca²⁺]_i; however, [Ca²⁺]_i recovered as normal from high K⁺ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca²⁺]_i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca²⁺]_i associated with action potential activity was measured. The amplitude of the [Ca²⁺]_i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca²⁺]_i from the modest Ca²⁺ loads imposed on the neuron by action potential trains follows a simple exponential decay (τ = 3–5s).     

Inability to restore resting intracellular calcium levels as an early indicator of delayed neuronal cell death

The hippocampus is especially vulnerable to excitotoxicity and delayed neuronal cell death. Chronic elevations in free intracellular calcium concentration ([Ca²⁺]_i) following glutamate-induced excitotoxicity have been implicated in contributing to delayed neuronal cell death. However, no direct correlation between delayed cell death and prolonged increases in [Ca²⁺]_i has been determined in mature hippocampal neurons in culture. This investigation was initiated to determine the statistical relationship between delayed neuronal cell death and prolonged increases in [Ca²⁺]_i in mature hippocampal neurons in culture. Using indo-1 confocal fluorescence microscopy, we observed that glutamate induced a rapid increase in [Ca²⁺]_i that persisted after the removal of glutamate. Following excitotoxic glutamate exposure, neurons exhibited prolonged increases in [Ca²⁺]_i, and significant delayed neuronal cell death was observed. The N-methyl-D-aspartate (NMDA) channel antagonist MK-801 blocked the prolonged increases in [Ca²⁺]_i and cell death. Depolarization of neurons with potassium chloride (KCl) resulted in increases in [Ca²⁺]_i, but these increases were buffered immediately upon removal of the KCl, and no cell death occurred. Linear regression analysis revealed a strong correlation (R = 0.973) between glutamate-induced prolonged increases in [Ca²⁺]_i and delayed cell death. These data suggest that excitotoxic glutamate exposure results in an NMDA-induced inability to restore resting [Ca²⁺]_i (IRRC) that is a statistically significant indicator of delayed neuronal cell death.     

Removal of Ca(2+) following depolarization-evoked cytoplasmic Ca(2+) transients in freshly dissociated pyramidal neurones of the rat dorsal cochlear nucleus

Cytoplasmic [Ca²⁺] ([Ca²⁺]_i) was measured using Fura-2 in pyramidal neurones isolated from the rat dorsal cochlear nucleus (DCN). The kinetic properties of Ca²⁺ removal following K⁺ depolarization-induced Ca²⁺ transients were characterized by fitting exponential functions to the decay phase. The removal after small transients (<82 nM peak [Ca²⁺]_i) had monophasic time course (time constant of 6.43±0.48 s). In the cases of higher Ca²⁺ transients biphasic decay was found. The early time constant decreased (from 3.09±0.26 to 1.46±0.11 s) as the peak intracellular [Ca²⁺] increased. The value of the late time constant was 18.15±1.60 s at the smallest transients, and showed less dependence on [Ca²⁺]_i. Blockers of Ca²⁺ uptake into intracellular stores (thapsigargin and cyclopiazonic acid) decreased the amplitude of the Ca²⁺ transients and slowed their decay. La³⁺ (3 mM) applied extracellularly during the declining phase dramatically changed the time course of the Ca²⁺ transients as a plateau developed and persisted until the La³⁺ was present. When the other Ca²⁺ removal mechanisms were available, reduction of the external [Na⁺] to inhibit the Na⁺/Ca²⁺ exchange resulted in a moderate increase of the time constants. It is concluded that in the isolated pyramidal neurones of the DCN the removal of Ca²⁺ depends mainly on the activity of Ca²⁺ pump mechanisms.     

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca²⁺]_i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca²⁺]_i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca²⁺]_i studies. The kinetic profile for [Ca²⁺]_i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca²⁺]_i in guinea pigs, had no effect in rat, and increased [Ca²⁺]_i in all other species. Staurosporine inhibited SFLLRN-induced [Ca²⁺]_i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca²⁺]_i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.     

Protein phosphatase inhibitors prolong Ca-transients and divalent cation-dependent action potentials in leech Retzius cells

Elevation of [K⁺]_o for 30 s from 4 to 120 mM produced a fast and reversible depolarization and transient increase in [Ca²⁺]_i in fura-2 loaded Retzius cells of the leech. The protein phosphatase inhibitor, okadaic acid, significantly slowed the return of [Ca²⁺]_i toward baseline without affecting the amplitude of depolarization or rate of repolarization. Furthermore, okadaic acid and another phosphatase inhibitor, calyculin A, prolonged Ba²⁺-dependent action potentials. These results suggest that the kinetics of Ca²⁺ influx may be regulated by the activity of phosphatases PP-1 and/or PP-2A.     

Restoration of cholinomimetic activity by clonidine in cholinergic plus noradrenergic lesioned rats

Lactate production (J_lac), oxygen consumption rate (Q_O2), plasma membrane potentials (E_m) and cytosolic free calcium levels [Ca²⁺]_i were studied on symaptosomes isolated from rat brains, incubated in presence of high doses of nicardipine (90 μM), diltiazem (0.5 mM) and verapamil (0.25 mM), and submitted to depolarizing stimulation or inhibition of mitochondrial respiration. Nicardipine was able to completely prevent the veratridine-induced stimulation ofJ_lac, Q_O2andE_m depolarization, whereas diltiazem and verapamil were less effective, although the concentrations used were 5 and 3 times higher, respectively, than nicardipine. Diltiazem, verapamil and nicardipine (9 μM) also prevented the veratridine-induced increase in [Ca²⁺]_i, this effect being much less pronounced if the drugs were added after veratridine. Monensin (20 μM) was also able to increase [Ca²⁺]_i but this effect was not affected by verapamil. Synaptosomes were also submitted to an inhibition of respiration of intrasynaptic mitochondria by incubation with rotenone (5 μM); in this condition of mimicked hypoxiaE_m was more positive of about 11 mV; none of the drugs utilized modified this situation. The rotenone-induced 3-fold increase inJ_lac was barely modified by diltiazem and verapamil but it was completely abolished by nicardipine. The possible mechanism of the counteracting action of the drugs towards veratridine stimulation and rotenone inhibition and the involvement of Na⁺/Ca²⁺ exchanger in affecting [Ca²⁺]_i are discussed.     

Blocking of the receptor-stimulated calcium entry into human platelets by verapamil and nicardipine

Verapamil (ED₅₀=3×10⁻⁶ M) and nicardipine (ED₅₀=10⁻⁶ M) inhibited the platelet activating factor (PAF)-induced increase of free cytosolic calcium concentration ([Ca²⁺]_i) in quin2-loaded human platelets. In a Ca-free medium containing 5 mM BaCl₂, PAF stimulated the inflow of Ba²⁺ ions which is completely abolished by verapamil and nicardipine. Simultaneous determination of quin2 fluorescence and ⁴⁵Ca absorption showed that the action of verapamil is accounted for by blocking of the Ca²⁺ entry. Nicardipine suppresses also Ca²⁺ mobilization from intracellular stores. The effects of verapamil and nicardipine are not competitive with respect to PAF.The blockers reduce the [Ca²⁺]_i increase induced by ADP, vasopressin, and PGH₂ analogue U46619.     

Effects of opioids on glutamate induced calcium intracellular increase in individually perfused rat hippocampal neurons in vitro

1. 1. Superfusion of cultured hippocampal cells with glutamate (Glu) 0.5 mM for 5 min induces an increase of [Ca²⁺_i] that is quickly followed by a recovery to control level. Addition of dynorphin or D-pen²-D-pen⁵-enkephalin (DPDPE) induces a persistence of the elevated [Ca²⁺_i], while [D-Ala²,N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO) does not influence it.

2. 2. Superfusion with Glu for 10 min induces a persistent increase of [Ca²⁺_i], that is partially reverted by DAMGO, but not affected by dynorphin or DPDPE.

3. 3. The author suggests a differential influence of selective opioids on the Glu-induced [Ca²⁺_i] increase.

     

μ-Opioid receptor-induced Ca mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca-dependent mechanism

Morphine, a preferential μ-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Although morphine affects cellular differentiation through a Ca²⁺-dependent mechanism, few studies have examined whether Ca²⁺ mediates the effect of opioids on cell proliferation, or whether a particular Ca²⁺ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types mediates the developmental effects of opioids. To address these questions, the present study examined the role of μ-opioid receptors and Ca²⁺ mobilization in morphine-induced astrocyte development. Morphine (1 gmM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca²⁺-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca²⁺ ([Ca²⁺]_o), or in unmodified media containing Ca²⁺ ionophore (A23187), nifedipine (1 μM), dantrolene (10 μM), thapsigargin (100 nM), or l-glutamate (100 μM) for 0-72 h. μ-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca²⁺ ([Ca²⁺]ⁱ) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating μ-opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphine's actions were mimicked by the selective μ, agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca²⁺]_i in developing astroglia. At normal [Ca²⁺]_o, morphine attenuated DNA synthesis by increasing [Ca²⁺]_i; low [Ca²⁺]_o (0.3 mM) blocked this effect, while treatment with Ca²⁺ ionophore or glutamate mimicked morphine's actions. At extremely low [Ca²⁺]_o (< 0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase [Ca²⁺]_i in astrocytes through several pathways, not all affect DNA synthesis or cellular morphology. Nifedipine (which blocks L-type Ca²⁺ channels) did not prevent morphine-induced reductions in BrdU incorporation or cellular differentiation, while thapsigargin (which depletes IP₃-sensitive Ca²⁺ stores) severely affected inhibited DNA synthesis and cellular differentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca²⁺-dependent Ca²⁺ release) selectively blocked the effects of morphine. Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca²⁺-dependent Ca²⁺ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.     

Enhanced Ca(2+) influx with mossy fiber stimulation in hippocampal CA3 neurons of spontaneously epileptic rats

This study was performed to determine whether the intracellular Ca²⁺ concentration ([Ca²⁺]_i) is increased in hippocampal CA3 neurons of spontaneously epileptic rats (SER) which show both absence-like and convulsive seizures using hippocampal slices loaded with Calcium Green-1 when a weak single stimulation is given to the mossy fiber. [Ca²⁺]_i in the CA3 area was significantly increased after a single stimulus to mossy fibers in SER, while no changes were detected in normal Wistar rats. These findings suggest the existence of an abnormality in the Ca²⁺ channel in the SER CA3 region and that this is probably responsible for epileptic seizures.     

NT-3 and BDNF protect CNS neurons against metabolic/excitotoxic insults

Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) were recently shown to have biological activity in central neurons. In the present study, NT-3 and BDNF attenuated glucose deprivation-induced neuronal damage dose-dependently in rat hippocampal, septal and cortical cultures. Direct measurements of intraneuronal free calcium levels ([Ca²⁺]_i) and manipulations of calcium inlux demonstrated that NT-3 and BDNF each prevented the elevation of [Ca²⁺]_i that mediated glucose deprivation-induced injury. Studies in cultures depleted of glia indicateda direct action of NT-3 and BDNF on neurons. Neurons pretreated with NT-3 or BDNF for 24 hr were more resistant to glutamate neurotoxicity, and showed attenuated [Ca²⁺]_i responses to glutamate. TrkB (BDNF receptor) and trkC (NT-3 receptor) proteins were present in hippocampal, cortical and septal cultures where they were localied to neuronal cell bodies and neurites. The data demonstrate that NT-3 and BDNF can protect neurons against metabolic and excitotoxic insults, and suggest that these neurotrophins may serve [Ca²⁺]_i-stabilizing and neuroprotective functions in the brain.     

A novel monoclonal antibody against carbohydrates of L1 cell adhesion molecule causes an influx of calcium in cultured cortical neurons

We have studied the function of carbohydrates of the L1 molecule, a member of the immunoglobulin superfamily of adhesion molecules, using a novel monoclonal antibody, mAb-L1(2E12), against L1 molecule. This antibody was specific for the 200 kDa component of mouse L1 molecule and its epitope was N-linked for complex-type oligosaccharides. The mAb-L1(2E12) was found to induce a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cultured mouse embryonic cortical neurons. The rise in [Ca²⁺]_i was dependent on the concentrations of mAb-L1(2E12). The rise seemed to be due to an influx of extracellular Ca²⁺ as EGTA treatment abolished it. Both cadmium and nifedipine blocked the effect of mAb-L1(2E12), suggesting the Ca²⁺ influx was through voltage-operated Ca²⁺ channels, particularly L-type Ca²⁺ channels. These results provide an important insight for understanding the mechanisms by which oligosaccharides of the L1 molecule influence various functions of neural cells.     

Analysis of potential shifts associated with recurrent spreading depression and prolonged unstable spreading depression induced by microdialysis of elevated K in hippocampus of anesthetized rats

The potential shifts (ΔV_o) associated with spreading depression (SD) were analysed with the help of multiple extracellular recording and ion-selective microelectrodes in the CA1 region of the dorsal hippocampus of anesthetized rats. Recurrent waves of SD were induced by perfusing high K⁺ solution through microdialysis probes. SD-related ΔV_o had a composite wave shape, consisting of an early, rapidly shifting part (phase I) followed by a slower shift to a second negative maximum (phase II). ΔV_o shifts in stratum radiatum usually started earlier, always lasted longer and had lartger amplitude than those recorded in stratum pyramidale. The ΔV_o responses in stratum radiatum had an inverted saddle shape created by a transient relatively positive “hump” interposed between phases I and II. During this “hump”, the potentials in the two layers transiently approached one another. During continuous high K⁺ dialysis, successive ΔV_o waves episodes evolved according to a consistent pattern: while phase I remained unchanged, phase II increased in amplitude and duration with each episode. Eventually, a depressed state developed which lasted for many minutes, termed here prolonged unstable spreading depression. During phase I, ΔV_o and extracellular K ([K⁺]_o) changes were correlated. During phase II, [K⁺]_o decreased even as ΔV_o continued to increase. During SD, [Ca²⁺]_o decreased to <0.01 mM. During phases I and II, both [Ca²⁺]_o and [Na⁺]_o remained low. the recoverries of [Ca²⁺]_o and [Na⁺]_o had an initial fast and a later much slower phase and took several minutes longer than the recoveries of [K⁺]_o and ΔV_o. Depth profiles of ΔV_o and Δ[K⁺]_o revealed strikingly steep gradients early and late during a wave; but voltage and ion gradients were not precisely correlated either in time or in space. We conclude that ΔV_o of phases I and II are generated by different processes. Membrane ion currents cannot fully explain the ΔV_o responses. The possible contributions by ion diffusion and by active ion transport are discussed. The extremely low level to which [Ca²⁺]_o sinks during SD, and its two-phase recovery, indicate intracellular sequestration or binding of substantial amounts of Ca²⁺ ions. The residual deficit of [Ca²⁺_o following recovery of SP shifts may account for the persistent depression of synaptic transmission after repolarization of neurons.     

The action of valproate on spontaneous epileptiform activity in the absence of synaptic transmission and on evoked changes in [Ca2+]o and [K+]o 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.     

The influx of Ca and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca²⁺ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca²⁺]_i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca²⁺ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca²⁺]_i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca²⁺ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca²⁺]_i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca²⁺ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.     

Simultaneous monitoring of three key neuronal functions in primary neuronal cultures

The coupling of Ca²⁺ influx to synaptic vesicle (SV) recycling in nerve terminals is essential for neurotransmitter release and thus neuronal communication. Both of these parameters have been monitored using fluorescent reporter dyes such as fura-2 and FM1-43 in single central nerve terminals. However, their simultaneous monitoring has been hampered by the proximity of their fluorescence spectra, resulting in significant contamination of their signals by bleedthrough. We have developed an assay that simultaneously monitors both SV recycling and changes in intracellular free Ca²⁺ ([Ca²⁺]_i) in cultured neurons using the reporter dyes FM4-64 and fura-2AM. By monitoring both fura-2 and FM4-64 emission in the far red range, we were able to visualize functionally independent readouts of both SV recycling and [Ca²⁺]_i independent of fluorescence bleedthrough. We were also able to incorporate an assay of cell viability without any fluorescence bleedthrough from either fura-2 or FM4-64 signals, using the dye SYTOX Green. We propose that this assay of three key neuronal functions could be simply translated into a high content screening format for studies investigating small molecule inhibitors of these processes.     

Dual action of estrogen on glutamate-induced calcium signaling: mechanisms requiring interaction between estrogen receptors and src/mitogen activated protein kinase pathway

Conjugated equine estrogens (CEE) is the most widely prescribed pharmaceutical estrogen replacement therapy (ERT) for postmenopausal women in the United States and is the ERT of the Women’s Health Initiative. Previous studies from our laboratory have demonstrated that CEE exerts neurotrophic and neuroprotective effects in neurons involved in learning and memory, and which are affected in Alzheimer’s disease. The present work demonstrates that CEE potentiated the rise in intracellular calcium ([Ca²⁺]_i) following exposure to physiological concentrations of glutamate. In contrast, the reverse effect occurred in the presence of excitotoxic levels of glutamate exposure, where CEE attenuated the rise in [Ca²⁺]_i. Potentiation of the glutamate response was mediated by the NMDA receptor, as the NMDA receptor antagonist MK-801 blocked the CEE-induced potentiation, whereas the L-type calcium channel blocker nifedipine did not. Further, the CEE-potentiated glutamate response was mediated by a src tyrosine kinase, as the tyrosine kinase inhibitor PP2 blocked the potentiation induced by CEE and neurons treated with CEE displayed increased phosphorylated tyrosine. The inhibition by CEE of [Ca²⁺]_i rise in the presence of excitotoxic levels of glutamate was mediated by mitogen activated protein kinase (MAPK), as the protective effect of CEE was blocked by inhibiting MAPK activation with PD98059. These data provide potential mechanisms to explain the cognitive enhancing and neuroprotective effects exerted by ERT.     

Prostaglandin E1 and dibutyryl cyclic AMP enhance platelet resistance to deformation.

The effect of prostaglandin E₁ (PGE₁) on platelets is mediated through the PGE₁ receptor and the consequent maintenance of the platelet's discoid shape. The effects of PGE₁ and dibutyryl cAMP (dbcAMP) on the deformability of human platelets were studied. Deformability tests based upon the micropipette aspiration on the platelets were performed by using pipettes with radii (Rp) of 0.26-0.36 gm. The time course of the extension length (Dp, in μg) of the platelets in response to aspiration with a negative pressure (ΔP) of 5 cm H₂ O (ΔP × Rp = 0.15 dynes/cm) was analyzed. PGE₁ treatment (0.1 μM) resulted in a decrease of platelet deformability as compared with results obtained for apparently non-activated, control platelets. The deformation index, i.e., Dp/Rp (PGE₁ -treated) / Dp/Rp (control), was significantly reduced to 0.90 ± 0.04. DbcAMP treatment also significantly decreased the deformability of platelets and this decrease was dbcAMP dose dependent. In contrast, colchicine- or cytochalasin D-treated platelets increased deformability. PGE₁ -treated platelets had a higher [cAMP]_i than controls. Platelets treated with PGE₁ or dbcAMP showed a reduced [Ca²⁺]i increment induced by thrombin as compared to non-treated controls. These results indicate that PGE₁ and dbcAMP treatment of platelets is accompanied by an enhancement of platelet resistance to deformation. The increased [cAMP]_i and low [Ca²⁺]_i after PGE₁ treatment may limit the rearrangement of cytoskeleton and thus enhance platelet resistance to deformation.     

Enhancement of phagocytosis by dynorphin A in mouse peritoneal macrophages

The effects of the opioid peptide dynorphin A (DynA) on phagocytosis in peritoneal macrophages was examined by flow cytometry (FCM). DynA enhanced phagocytosis in a dose-dependent manner. Leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), β-neo-endorphin (βNeo-End), DynA(9–17) and DynA(13–17) had no such activity, -Neo-endorphin ( Neo-End), dynorphin B (DynB), DynA(l–13) and DynA(6–17) enhanced phagocytosis less effectively than DynA. Naloxone did not inhibit the enhancement of phagocytosis induced by DynA. Unstimulated control phagocytosis was partially suppressed in Ca²⁺-free EGTA-containing solution and even in this solution DynA enhanced phagocytosis. However, the enhancement by DynA was suppressed in EGTA- and BAPTA-AM-containing Ca²⁺-free solution. The present study showed that enhancement of phagocytosis by DynA was independent of extracellular Ca²⁺ ([Ca²⁺]_o) and dependent on intracellular Ca²⁺ ([Ca²⁺]_i). The present results support DynA being one of the mediators from the nervous system that modulates the immune system.     

Tetanus-induced asynchronous GABA release in cultured hippocampal neurons

Asynchronous GABA release was studied in cultured hippocampal neurons using paired whole-cell recordings. Tetanization of the presynaptic GABAergic neuron was accompanied by a train of IPSCs which showed tetanic depression. Asynchronous IPSCs (asIPSCs) also developed during the train and continued for 1.85±0.3 s after the stimulation. The threshold frequency for evoking asIPSCs was 10 Hz, while maximal asynchronous activity was achieved at 40 Hz. Perfusion with EGTA-AM blocked asIPSCs. The elevation of [Ca²⁺]_i that accompanies presynaptic action potential firing triggers asynchronous release of GABA vesicles, thereby counteracting tetanic depression of synchronous IPSCs.