Bovine serum albumin and lysophosphatidic acid stimulate calcium mobilization and reversal of cAMP-induced stellation in rat spinal cord astrocytes

We report that lysophosphatidic acid (LPA) stimulates dynamic calcium (Ca²⁺) fluctuations and morphological rearrangements in astrocytes derived from neonatal rat spinal cord. Addition of 10 μM LPA elicited single Ca²⁺ transients, or biphasic oscillations and sustained increases in intracellular Ca²⁺ ([Ca²⁺]_i). The biphasic Ca²⁺ response consisted of an initial release from intracellular stores, often followed by a sustained elevation or continued oscillations that required Ca²⁺ flux across the cell membrane. The type of Ca²⁺ response, but not the overall magnitude, was dependent on LPA concentration. Higher concentrations (>10 μM) often elicited sustained increases in [Ca²⁺]_i, while lower concentrations stimulated oscillations or single Ca²⁺ transients. It has previously been established that agents that elevate cyclic adenosine monophosphate (cAMP) induce flat astrocytes to adopt a more stellate morphology. LPA can completely reverse this morphological change at a half-maximal concentration of 215 nM. Inhibiting LPA-induced [Ca²⁺]_i fluctuations using BAPTA-AM to buffer [Ca²⁺]_i and EGTA in the bath to prevent transmembrane flux had little effect on the ability of LPA to reverse stellation. LPA is found bound to serum albumin, in which crude preparations have been shown to induce various physiological responses in a number of cell types. Many of the activities have been attributed to albumin-associated lipid factors including LPA. We show that lipid factors associated with BSA can mimic the effect of LPA in both Ca²⁺ mobilization and reversal of cAMP-induced stellation. GLIA 20:163–172, 1997. © 1997 Wiley-Liss Inc.     

P2U nucleotide receptor activation in rat glial cell line induces [Ca2+]i oscillations which depend on cytosolic pH

In single rat glioma cells, the signal transduction process activated by the UTP sensitive purinergic nucleotide receptor was studied by determining [Ca²⁺]_i by Fura-2 fluorescence and measuring pH by BCECF fluorescence to elucidate the control of [Ca²⁺]_i oscillations by intracellular pH. Addition of UTP for long time periods (some min) causes a [Ca²⁺]_i response composed of i) an initial large peak and a following sustained increase (160 s duration), and ii) subsequent regular [Ca²⁺]_i oscillations (amplitude 107 nM, frequency 1.5 oscillations per min). The maintenance of the [Ca²⁺]_i oscillations depends on the continued presence of agonist. The oscillations are abolished by reducing extracellular Ca²⁺ concentration. The interaction of UTP receptors and bradykinin receptors during the [Ca²⁺]_i oscillations was investigated because previous studies have already shown that the peptide causes comparable [Ca²⁺]_i oscillations. During [Ca²⁺]_i oscillations induced by UTP or bradykinin, long-term admission of both hormones (400–500 s) causes a large initial response superimposed on regular [Ca²⁺]_i oscillations. Short pulses (12 s) of the second agonist given in any phase of the oscillations induce large [Ca²⁺]_i peaks. In both cases, the following oscillations are not disturbed. The influence of cytosolic pH was studied by alkalinizing pH_i by application of NH₄Cl. [Ca²⁺]_i oscillations stop after addition of NH₄Cl. Recovery of NH₄Cl-induced alkalinization is reduced by furosemide. To the same degree, the interruption of [Ca²⁺]_i oscillations is significantly prolonged in the presence of furosemide. Thus cytosolic alkalinization suppresses hormone-induced [Ca²⁺]_i oscillations in rat glioma cells. The understanding of the molecular mechanism of this interference of pH should provide an important contribution for unravelling the function of cytosolic pH in cellular signal transduction. © 1996 Wiley-Liss, Inc.     

Intracellular calcium changes associated with cholinergic nicotinic receptor activation in cultured myenteric plexus neurones

Intracellular free calcium concentration ([Ca²⁺]_i) was measured in cultured explants of myenteric plexus neurones by using the fluorescent calcium indicator Indol in combination with patch-clamp techniques. The basal [Ca²⁺]_i was 94 nM and spontaneous oscillations in the internal free calcium concentration were recorded. These oscillations were associated with bursts of action potentials triggered by spontaneous nicotinic excitatory synaptic potentials. Under voltage clamp conditions, application of the selective nicotinic agonist m-hydroxyphenylpropyltri-methylammonium iodide (10 μM) induced an inward current and increased the intracellular free calcium concentration. We conclude that cholinergic synaptic excitatory activity provide a regular calcium entry in myenteric neurone and suggest that the nicotinic channel might be significantly permeable to calcium.     

Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin

Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca²⁺]_i) that was communicated to surrounding cells. Following propagation of the Ca²⁺ wave, many cells showed asynchronous oscillations in [Ca²⁺]_i. Dantrolene sodium (10 μM) inhibited the increase in [Ca²⁺]_i associated with this Ca²⁺ wave by 60-80%, and prevented subsequent Ca²⁺ oscillations. Despite the markedly decreased magnitude of the increase in [Ca²⁺]_i, the rate of propagation and the extent of communication of the Ca²⁺ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 μM) induced an initial increase in [Ca²⁺]_i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca²⁺]_i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of a Ca²⁺ wave from the stimulated cell to adjacent cells. Glutamate (50 μM) induced an initial increase in [Ca²⁺]_i in most cells that was followed by sustained oscillations in [Ca²⁺]_i in some cells. Dantrolene (10 μM) inhibited this initial [Ca²⁺]_i increase caused by glutamate by 65-90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 μm) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca²⁺ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca²⁺ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP₃) mediates the propagation of Ca²⁺ waves whereas Ca²⁺ -induced Ca²⁺ release amplifies Ca²⁺ waves and generates subsequent Ca²⁺ oscillations.     

An Interaction of Oxytocin Receptors with Metabotropic Glutamate Receptors in Hypothalamic Astrocytes

Hypothalamic astrocytes play a critical role in the regulation and support of many different neuroendocrine events, and are affected by oestradiol. Both nuclear and membrane oestrogen receptors (ERs) are expressed in astrocytes. Upon oestradiol activation, membrane‐associated ER signals through the type 1a metabotropic glutamate receptor (mGluR1a) to induce an increase of free cytoplasmic calcium concentration ([Ca²⁺]_i). Because the expression of oxytocin receptors (OTRs) is modulated by oestradiol, we tested whether oestradiol also influences oxytocin signalling. Oxytocin at 1, 10, and 100 nm induced a [Ca²⁺]_i flux measured as a change in relative fluorescence [ΔF Ca²⁺ = 330 ± 17 relative fluorescent units (RFU), ΔF Ca²⁺ = 331 ± 22 RFU, and ΔF Ca²⁺ = 347 ± 13 RFU, respectively] in primary cultures of female post‐pubertal hypothalamic astrocytes. Interestingly, OTRs interacted with mGluRs. The mGluR1a antagonist, LY 367385 (20 nm ), blocked the oxytocin (1 nm )‐induced [Ca²⁺]_i flux (ΔF Ca²⁺ = 344 ± 19 versus 127 ± 11 RFU, P < 0.001). Conversely, the mGluR1a receptor agonist, (RS)‐3,5‐dihydroxyphenyl‐glycine (100 nm ), increased the oxytocin (1 nm )‐induced [Ca²⁺]_i response (ΔF Ca²⁺ = 670 ± 31 RFU) compared to either compound alone (P < 0.001). Because both oxytocin and oestradiol rapidly signal through the mGluR1a, we treated hypothalamic astrocytes sequentially with oxytocin and oestradiol to determine whether stimulation with one hormone affected the subsequent [Ca²⁺]_i response to the second hormone. Oestradiol treatment did not change the subsequent [Ca²⁺]_i flux to oxytocin (P > 0.05) and previous oxytocin exposure did not affect the [Ca²⁺]_i response to oestradiol (P > 0.05). Furthermore, simultaneous oestradiol and oxytocin stimulation failed to yield a synergistic [Ca²⁺]_i response. These results suggest that the OTR signals through the mGluR1a to release Ca²⁺ from intracellular stores and rapid, nongenomic oestradiol stimulation does not influence OTR signalling in astrocytes.     

Gonadotropin releasing hormone modulates γ-aminobutyric acid-evoked intracellular calcium increase in immortalized hypothalamic gonadotropin releasing hormone neurons

To examine the functional role of calcium signaling in the interactive modulation of gonadotropin releasing hormone (GnRH) neurons by γ-aminobutyric acid (GABA) and GnRH itself, we analyzed the intracellular calcium level ([Ca²⁺]_i), using fura-2AM fluorescent dye in immortalized hypothalamic GT1-1 cells. GT1-1 cells showed spontaneous [Ca²⁺]_i oscillations, which were dependent on extracellular Ca²⁺ level, L-type Ca²⁺ channel and SK-type K⁺ channel. When GABA or a specific GABA_A type receptor agonist, muscimol was applied to the media, [Ca²⁺]_i rapidly increased through L-type Ca²⁺ channel in a dose-dependent manner, and subsequently decreased below the basal level without any oscillation. However, a specific GABA_B type receptor agonist, baclofen showed no effect. On the other hand, application of GnRH or its potent agonist buserelin, rapidly abolished the spontaneous [Ca²⁺]_i oscillations. Interestingly, a prior treatment with buserelin abolished GABA-evoked increase in [Ca²⁺]_i in a noncompetitive manner. Since buserelin also blocked K⁺-evoked increase in [Ca²⁺]_i, we suggest that GnRH may block spontaneous [Ca²⁺]_i oscillation through modulating the L-type [Ca²⁺]_i channel activity. These results show that GABAergic agents may exert both stimulatory and inhibitory controls over the GnRH neuronal activity, and GnRH can block the stimulatory effect of GABA, implicating the possible existence of an ultrashort feedback circuit.     

Calcium oscillations in a subpopulation of S-antigen-immunoreactive pinealocytes of the rainbow trout (Oncorhynchus mykiss)

By means of the fura-2 technique and image analysis the intracellular concentration of free calcium ions [Ca²⁺]_i was examined in isolated rainbow trout pinealocytes identified by S-antigen immunocytochemistry. Approximately 30% of the pinealocytes exhibited spontaneous [Ca²⁺]_i oscillations whose frequency differed from cell to cell. Neither illumination with bright light nor dark adaptation of the cells had an apparent effect on the oscillations. Removal of extracellular Ca²⁺ or application of 10 μM nifedipine caused a reversible breakdown of the [Ca²⁺]_i oscillations. Application of 60 mM KCl elevated [Ca²⁺]_i in 90% of the oscillating and 50% of the non-oscillating pinealocytes. The effect of KCl was blocked by 50 μM nifedipine. These results suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca²⁺]_i in trout pinealocytes. Experiments with thapsigargin (2 μM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role for regulation of [Ca²⁺]_i remains elusive. Treatment with norepinephrine (100 pM–50 μM), previously shown to induce calcium release from intracellular calcium stores in rat pinealocytes, had no apparent effect on [Ca²⁺]_i in any trout pinealocyte. This finding conforms to the concept that noradrenergic mechanisms are not involved in signal transduction in the directly light-sensitive pineal organ of anamniotic vertebrates.     

Activation of alpha-2 adrenergic receptors stimulates GABA release by astrocytes

Norepinephrine is one of the key neurotransmitters in the hippocampus, but its role in the functioning of the neuroglial networks remains unclear. Here we show that norepinephrine suppresses NH₄Cl-induced oscillations of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in hippocampal neurons. We found that the inhibitory effect of norepinephrine against ammonium-induced [Ca²⁺]_i oscillations is mediated by activation of alpha-2 adrenergic receptors. Furthermore, UK 14,304, an agonist of alpha-2 adrenergic receptors, evokes a biphasic [Ca²⁺]_i elevation in a minor population of astrocytes. This elevation consists of an initial fast, peak-shaped [Ca²⁺]_i rise, mediated by G_iβγ subunit and subsequent PLC-induced mobilization of Ca²⁺ from internal stores, and a plateau phase, mediated by a Ca²⁺ influx from the extracellular medium through store-operated and TRPC3 channels. We show the correlation between the Ca²⁺ response in astrocytes and suppression of [Ca²⁺]_i oscillations in neurons. The inhibitory effect of UK 14,304 is abolished in the presence of gallein, an inhibitor of G_βγ-signaling. In turn, application of the agonist in the presence of the PLC inhibitor decreases the frequency and amplitude of [Ca²⁺]_i oscillations in neurons but does not suppress them. The same effect is observed in the presence of bicuculline, a GABA(A) receptor antagonist. We demonstrate that UK 14,304 application increases the frequency and amplitude of slow outward chloride currents in neurons, indicating the release of GABA by astrocytes. Thus, our findings indicate that the activation of astrocytic alpha-2 adrenergic receptors stimulates GABA release from astrocytes via G_iβγ subunit-associated signaling pathway, contributing to the suppression of neuronal activity.     

Ca2+ influx into leech glial cells and neurones caused by pharmacologically distinct glutamate receptors

The effect of glutamatergic agonists on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of neuropile glial cells and Retzius neurones in intact segmental ganglia of the medicinal leech Hirudo medicinalis was investigated by using iontophoretically injected fura-2. In physiological Ringer solution the [Ca²⁺]_i levels of both cell types were almost the ssame (glial cells: 58 ± 30 nM, n = 51; Retzius neurones: 61 ± 27 nM, n = 64). In both cell types glutamate, kainate, and quisqualate induced an increase in [Ca²⁺]_i which was inhibited by 6,7-dinitroquinoxaline-2,3-dione (DNQX). This increase was caused by a Ca²⁺ influx from the extracellular space because the response was greatly diminished upon removal of extracellular Ca²⁺. The glutamate receptors of neuropile glial cells and Retzius neurones differed with respect to the relative effectiveness of the agonists used, as well as with regard to the inhibitory strenght of DNQX. In Retzius neurones the agonist-induced [Ca²⁺]_i increase was abolished after replacing extracellular Na⁺ by organic cations or by mM amounts of Ni²⁺, whereas in glial cells the [Ca²⁺]_i increase was largely preserved under both conditions. It is concluded that in Retzius neurones the Ca²⁺ influx is predominantly mediated by voltage-dependent Ca²⁺ channels, whereas in neuropile glial cells the major influx occurs via the ion channels that are associated with the glutamate receptors.     

Identification of neuropeptide Y receptors in cultured astrocytes from neonatal rat brain

Specific binding sites for neuropeptide Y could be demonstrated in primary cultures of astrocytes from neonatal rat brain. Neuropeptide Y binding was saturable, reversible, and temperature dependent as revealed by saturation studies and kinetic experiments. Scatchard analysis of equilibrium binding data indicated a single population of high-affinity binding sites with respective K_D and B_max values of 0.43 nM and 6.9 fmol/2.7 × 10⁵ cells. Physiological responses induced by neuropeptide Y could be detected in a distinct subpopulation of cultured astrocytes on the basis of two criteria: (1) electrophysiological responses and (2) single cell measurements of changes in [Ca²⁺]_i. In that fraction of cells responding (20–70%, varying among cultures from different preparations), brief application of neuropeptide Y led to a membrane potential depolarization, lasting several minutes. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, neuropeptide Y induced an inward current with a similar time course as the neuropeptide Y-induced membrane depolarization. As detected by single cell microfluorimetric (fura-2) measurements neuropeptide Y induced an increase of [Ca²⁺]_i which was caused by the entry of extracellular Ca²⁺. Both the [Ca²⁺]_i increase and the electrophysiological responses were unaffected by pretreatment of the astrocytes with pertussis toxin. © 1993 Wiley-Liss, Inc.     

Involvement of amyloid precursor protein in functional synapse formation in cultured hippocampal neurons

Amyloid precursor protein (APP) is known to be widely expressed in neuronal cells, and enriched in the central and peripheral synaptic sites. Although it has been proposed that APP functions in synaptogenesis, no direct evidence has yet been reported. In this study we investigated the involvement of APP in functional synapse formation by monitoring spontaneous oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cultured hippocampal neurons. As more and more neurons form synapses with each other during the culture period, increasing numbers of neuronal cells show synchronized spontaneous oscillations of [Ca²⁺]_i. The number of neurons that showed synchronized spontaneous oscillations of [Ca²⁺]_i was significantly lower when cultured in the presence of monoclonal antibody 22C11 against the N-terminal portion of APP. Moreover, incubation with excess amounts of the secretory form of APP or the N-terminal fragment of APP also inhibited the increase in number of neurons with synchronized spontaneous oscillations of [Ca²⁺]_i. The addition of monoclonal antibody 22C11 or secretory form of APP did not, however, affect MAP-2-positive neurite outgrowth. These findings suggest that APP play a role in functional synapse formation during CNS development. J. Neurosci. Res. 51:185–195, 1998. © 1998 Wiley-Liss, Inc.     

Characteristics of thrombin-induced calcium signals in rat astrocytes

The protease thrombin seems to play a central role in events following neural injury, whereby the enzyme can act, in concert with other molecules as a hormone or as a growth factor. In cells derived from the nervous system, thrombin induces changes in morphology and proliferation. The signalling mechanisms involved in these thrombin-activated processes are still unclear. In the present study we investigated Ca²⁺ signals in fura-2 loaded rat astrocytes in primary culture. Brief stimulation of astrocytes with thrombin induced a dose-dependent transient elevation of [Ca²⁺]_i, best fitted by a double-sigmoidal curve giving two EC₅₀ values of 3 pM and 150 pM. Continuous superfusion of cells with thrombin induced Ca²⁺ responses with three different types of kinetics. In 48% of the cells tested a single transient rise superimposed with fast fluctuations of [Ca²⁺]_i was seen. The following complex long-term changes of [Ca²⁺]_i, dependent on the presence of the agonist thrombin, were observed: i) a biphasic [Ca²⁺]_i elevation, characterized by an initial peak followed by a sustained plateau phase (in 43% of the cells) and ii) oscillations of [Ca²⁺]_i (in 9% of the cells). The observed Ca²⁺ responses were inhibited by the phospholipase C (PLC) inhibitor U-73122 and the thrombin inhibitor protease nexin-1/glia-derived nexin. The synthetic thrombin receptor activating peptide could mimic the thrombin-induced changes of [Ca²⁺]_i. In astrocytes in Ca²⁺-free medium, thrombin induced a sharp single transient Ca²⁺ rise, without superimposed fluctuations. After depletion of intracellular Ca²⁺ stores with thapsigargin the Ca²⁺ response to thrombin was diminished or completely suppressed indicating that thrombin induces the release of Ca²⁺ from intracellular stores. During long-term Ca²⁺ responses, omission of extracellular Ca²⁺ resulted in a reversible interruption of the signal. In conclusion our results demonstrate that thrombin by activation of its plasma membrane receptor induces through activation of PLC different types of Ca²⁺ responses. The complex Ca²⁺ signals are generated by an interplay of InsP₃-mediated Ca²⁺ release from intracellular stores and Ca²⁺ entry across the plasma membrane. GLIA 21:361–369, 1997. © 1997 Wiley-Liss, Inc.     

GABAB receptors increase intracellular calcium concentrations in chromaffin cells through two different pathways: Their role in catecholamine secretion

The activation of GABA_B receptors of adrenal chomaffin cells produces an increase of [Ca²⁺]_i measured by fura-2 AM techniques. GABA_B agonists 3-aminopropylphosphinic acid or (-)baclofen, at concentrations of 0.5 mM, increased basal Ca²⁺, values 332 ± 60.9 and 306 ± 40.5 nM, respectively, in cells suspended in a 2.5 mM Ca²⁺ buffer. The GABA_B-induced increase of [Ca²⁺]_i seemed to have two different components. The first was due to an entry from the extracellular medium mainly through L-type voltage-dependent Ca²⁺ channels as the dihydropiridine nifedipine 50 μM was able to decrease it more than 60%, while ω-conotoxin, which blocks N-type channels, did not produce any change in the GABA_B-evoked Ca²⁺ increment. The second component was due to a release of Ca²⁺ from intracellular pools and was about one-third of the total GABAB-induced increase of [Ca²⁺]_i. GABA_B receptors stimulated inositol 1,4,5-trisphosphate-sensitive and not the caffeine-sensitive Ca²⁺ store. In a low Ca²⁺ buffer after treatment with 2 μM angiotensin II, neither 0.5 mM 3-APPA nor baclofen were able to produce an additional increase of [Ca²⁺]_i, whereas 4 mM caffeine had no effect on GABA_B response. This intracellular Ca²⁺ mobilization could be due to inositol 1,4,5-trisphosphate accumulation produced by the activation of GABA_B receptors. In fact, the specific agonists after 10 minutes incubation produced a dosedependent increase of inositol 1,4,5-trisphosphate. The maximal effect was obtained at 100 μM baclofen and 3-APPA, and it was 3.63 ± 0.75 and 3.2 ± 1.5 times the basal levels (7.3 ± 0.3 pmol/10⁶ cells), respectively. In the absence of extracellular Ca²⁺, GABA_B-evoked catecholamine secretion and cyclic AMP formation were reduced more than 70%, suggesting an important role of extracellular Ca²⁺ in GABA_B mechanisms in adrenal chromaffin cells. © 1995 Wiley-Liss, Inc.     

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.     

Thyrotrophin-Releasing Hormone Raises Cytosolic Free Calcium Concentration in Human Adenomatous Somatotrophs and Corticotrophs; Comparison with in vivo Responsiveness to Thyrotrophin-Releasing Hormone in Patients with Acromegaly or Cushing's Disease

The effect of thyrotrophin-releasing hormone (TRH) on intracellular free Ca²⁺ concentration, [Ca²⁺)i, was investigated with the fluorescent dye fura-2 in cell suspensions obtained from 13 human growth hormone-secreting adenomas and 6 adrenocorticotrophin-secreting adenomas. Preoperatively, 9 out of 13 acromegalic patients showed a positive growth hormone response to TRH administration while none of the 6 patients with Cushing's disease had a plasma adrenocorticotrophin increase after TRH injection. In all the growth hormone-secreting adenomas the addition of TRH (100 nM) caused a significant rise in [Ca²⁺]i (from a resting level of 133±40 (±SD) to a value of 284±119 nM at 100 nM TRH, n = 42; P<0.001). The transient induced by TRH was found to have a dual origin, one due to Ca²⁺ mobilization from intracellular stores which was maintained in presence of EGTA (3mM) and verapamil (10 μM) and a plateau phase due to Ca²⁺ influx from the extracellular media. Somatostatin (0.1 μM) lowered both resting [Ca²⁺]i and TRH-induced transients. The effect of gonadotrophin-releasing hormone on [Ca²⁺]i was evaluated on cell suspensions obtained from 6 growth hormone-secreting adenomas. Gonadotrophin-releasing hormone (100 nM) caused a marked rise in [Ca²⁺]i (from 179±25 to 283±15nM) on the cell suspension obtained from the only in vivo responsive adenoma while it was ineffective in the remaining 5. Although TRH was ineffective in modifying plasma adrenocorticotrophin levels in all patients with Cushing's disease, in 5 out of 6 tumors the addition of 100 nM TRH caused a significant rise in [Ca²⁺]i (from 102.5 ± 36 to 163±66 nM, n = 22; P < 0.005). However, the effect of TRH on [Ca²⁺]i was significantly lower than that caused by arginine vasopressin, a physiological stimulator of adrenocorticotrophin release ([Ca²⁺]i values; 145±78 nM at 100 nM TRH versus 300±140 at 10 nM arginine vasopressin, n = 15; P<0.05). Moreover, the effect of arginine vasopressin on [Ca²⁺]i was detectable at concentrations as low as 0.1 nM while TRH was effective at concentrations higher than 1 nM. By contrast, gonadotrophin-releasing hormone was ineffective in increasing [Ca²]i in all the adrenocorticotrophin-secreting adenomas studied. Collectively, these data indicate that sensitivity to TRH is present in almost all the growth hormone- and adrenocorticotrophin-secreting adenomas independently of the responsiveness of the individual patients to the peptide.     

GFAP-positive hippocampal astrocytes in situ respond to glutamatergic neuroligands with increases in [Ca2+]i

It is becoming increasingly clear that astrocytes play very dynamic and interactive roles that are important for the normal functioning of the central nervous system. In culture, astrocytes express many receptors coupled to increases in intracellular calcium ([Ca²⁺]_i). In vivo, it is likely that these receptors are important for the modulation of astrocytic functions such as the uptake of neurotransmitters and ions. Currently, however, very little is known about the expression or stimulation of such astrocytic receptors in vivo. To address this issue, confocal microscopy and calcium sensitive fluorescent dyes were used to examine the dynamic changes in astrocytic [Ca²⁺]_i, within acutely isolated hippocampal slices. Astrocytes were subsequently identified by immunocytochemistry for glial fibrillary acidic protein. In this paper, we present data indicating that hippocampal astrocytes in situ respond to glutamate, kainate, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), N-methyl-D-aspartate (NMDA), and depolarization with increases in [Ca²⁺]_i. The increases in [Ca²⁺]_i occurred in both the astrocytic cell bodies and the processes. Temporally the changes in [Ca²⁺]_i were very dynamic, and various patterns ranging from sustained elevations to oscillations of [Ca²⁺]_i were observed. Individual astrocytes responded to neuroligands selective for both ionotropic and metabotropic glutamate receptors with increases in [Ca²⁺]_i. These findings indicate that astrocytes in vivo contain glutamatergic receptors coupled to increases in [C²⁺]_i and are able to respond to neuronally released neurotransmitters. (c) 1995 Wiley-Liss, Inc.     

Flow cytometric study of mitochondrial dysfunction after AMPA receptor activation

The effect of AMPA-receptor stimulation on MMP and on the concentration of intracellular calcium ([Ca²⁺]_i) was studied in dissociated CGC from rat pups, by flow cytometry. In the presence of cyclothiazide, AMPA induced a sodium-independent decrease in MMP up to 30.7 ± 2.5%. This effect was antagonized by CNQX and NBQX. Mepacrine and dibucaine reversed the effect of AMPA on MMP, suggesting that it is mediated by a release of arachidonic acid. AMPA alone induced a slight (about 7%) increase in [Ca²⁺]_i. In the presence of cyclothiazide, AMPA induced a concentration-dependent [Ca²⁺]_i increase up to 29.10 ± 2.10% that was not reversed by flunarizine. This increase was similar to that observed in a Na⁺-free medium, and was antagonized by CNQX and NBQX, but not by MK-801. Mitochondria play a key role in the modulation of [Ca²⁺]_i since a significant [Ca²⁺]_i increase was found in the presence of FCCP. On the other hand, the dantrolene-sensitive calcium pools do not participate in the [Ca²⁺]_i increase induced by stimulation of AMPA receptors. It is concluded that when AMPA-receptor desensitization is blocked, a decrease in MMP and an increase in [Ca²⁺]_i occurs, which could be additional events to potentiate neuronal cell death induced by glutamate. J. Neurosci. Res. 52:684–690, 1998. © 1998 Wiley-Liss, Inc.     

Involvement of Na+–Ca2+ Exchanger in Reperfusion-induced Delayed Cell Death of Cultured Rat Astrocytes

In some cells, Ca²⁺ depletion induces an increase in intracellular Ca²⁺ ([Ca²⁺]_i) after reperfusion with Ca²⁺-containing solution, but the mechanism for the reperfusion injury is not fully elucidated. Using an antisense strategy we studied the role of the Na⁺-Ca²⁺ exchanger in reperfusion injury in cultured rat astrocytes. When astrocytes were perfused in Ca²⁺-free medium for 15–60 min, a persistent increase in [Ca²⁺]_i was observed immediately after reperfusion with Ca²⁺-containing medium, and the number of surviving cells decreased 3–5 days latter. The increase in [Ca²⁺]_i was enhanced by low extracellular Na⁺ ([Na⁺]_o) during reperfusion and blocked by the inhibitors of the Na⁺-Ca²⁺ exchanger amiloride and 3,4-dichlorobenzamil, but not by the Ca²⁺ channel antagonists nifedipine, Cd²⁺ and Ni²⁺. Treatment of astrocytes with antisense, but not sense, oligodeoxynucleotide to the Na⁺-Ca²⁺ exchanger decreased Na⁺–Ca²⁺ exchanger protein level and exchange activity. The antisense oligomer attenuated reperfusion-induced increase in [Ca²⁺]ⁱ and cell toxicity. The Na⁺-Ca²⁺ exchange inhibitors 3,4-dichlorobenzamil and ascorbic acid protected astrocytes from reperfusion injury partially, while the stimulators sodium nitroprusside and 8-bromo-cyclic GMP and low [Na⁺]_o exacerbated the injury. Pretreatment of astrocytes with ouabain and monensin caused similar delayed glial cell death. These findings suggest that Ca²⁺ entry via the Na⁺–Ca²⁺ exchanger plays an important role in reperfusion-induced delayed glial cell death.     

Cytosolic free [Ca2+] in single T-lymphocytes from depressed patients and healthy controls

Summary Human lymphocytes are widely used as peripheral models for central neurones. Alterations in immune function have been reported in depressed patients, e.g. mitogen-induced proliferation is impaired during depression. One possible causative mechanism could be altered [Ca²⁺]_i regulation. Phytohaemagglutinin (PHA)-induced rise of [Ca²⁺]_i has been found to be diminished in lymphocyte suspensions from depressed patients (Ecker et al., this issue). We measured PHA-induced rise of [Ca²⁺]_i in single Fura-2 AM-loaded T11⁺ lymphocytes of patients with major depression and controls to further analyse [Ca²⁺]_i regulation in depression.The [Ca²⁺]_i of resting lymphocytes was 57±2 nmol/l (mean ± SEM). There was no difference in resting [Ca²⁺]_i of resting lymphocytes of patients and controls. PHA evoked an increase of [Ca²⁺]_i an 7 out of 14 cells from control subjects up to 400–500 nmol/l. In contrast, only 4 out of 13 cells from depressed patients showed an increase of [Ca²⁺]_i up to 200 nmol/l. In a small fraction of cells from both groups the [Ca²⁺]_i signal is oscillating.Our preliminary data confirm alteration of [Ca²⁺]_i regulation in lymphocytes of depressed patients.     

Mobilization of intracellular calcium by substance p in a human astrocytoma cell line (U-373 MG)

Variations in intracellular free calcium concentration (Δ[Ca²⁺]_i) were measured in intact and isolated human astrocytoma cells (U373 MG) loaded with fura-2 acetoxymethylester. Microperfusion of 50 nM substance P (SP), applied for 1 s, increased [Ca²⁺]_i by 351 nM from a stable basal level of [Ca²⁺]_i of 26 nM. The peak Δ[Ca²⁺]_i induced by SP was dose dependent with a threshold of 10_-3 nM, an ED₅₀ of 1.3 nM and a maximal effect for concentrations of SP greater than 100 nM. The NKI receptor agonist, [Sar⁹Met(O₂)¹¹]SP, mimicked the effect of SP, while the NK₂ and NK₃ selective receptor agonists, [N1¹⁰]NKA(4-10) and senktide, respectively, had no effect. The Δ[Ca²⁺]_i induced by SP was unaffected by 100 μM cadmium or by removal of extracellular calcium ions. Caffeine up to 30 mM had no effect on [Ca²⁺]_i. In contrast, thapsigargin increased resting [Ca²⁺]_i by 92 nM and reduced the Δ[Ca²⁺]_i induced by SP. A pertussis treatment (500 ng/ml-24 h) did not modify the Δ[Ca²⁺]_i induced by SP. We conclude that SP, acting on a NK1 receptor, mobilizes cytosolic calcium from an intracellular calcium pool which can be partially depleted by thapsigargin. © 1994 Wiley-Liss, Inc.