Selective inflammatory stimulations enhance release of microglial response factor (MRF)-1 from cultured microglia

The mrf-1 gene has been isolated from microglia exposed to cultured cerebellar granule neurons undergoing apoptosis. We have shown that mrf-1 is upregulated in response to neuronal death and degeneration both in vitro and in vivo. However, the exact role of MRF-1 remains unknown. Here we show that MRF-1 is released from cultured rat microglia, and its release is greatly enhanced under inflammatory conditions. When microglia were treated with ATP, the amount of MRF-1 that was released increased 10-fold compared to the basal level of release. Enhanced MRF-1 release was induced within 10 min and peaked within 1 h; after approximately 4 h, the MRF-1 release had returned to normal. MRF-1 release was stimulated by 2-methyl-thio-ATP (five-fold) and a P2X(7) selective agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (ten-fold). Moreover, the ATP-stimulated MRF-1 release was inhibited by a P2X(7) selective antagonist, oxidized ATP (oATP), and also under a Ca(2+)-free condition. These results indicate that the effects of ATP are dependent on Ca(2+) influx through P2X(7) receptors. MRF-1 release was enhanced by Ca(2+)-ionophore A23187 (sixfold), thapsigargin (threefold); however, it was not enhanced by glutamate or lipopolysaccharide. Moreover, a platelet-activating factor enhanced microglial MRF-1 release in a dose-dependent manner. We also showed that a conditioned medium from cerebellar granule neurons undergoing apoptosis markedly increased MRF-1 release from microglia; that effect was significantly inhibited by oATP. These results indicate that selective inflammatory stimulations, including ATP and PAF, enhance MRF-1 release from microglia through a Ca(2+)-dependent mechanism and suggest that MRF-1 may play a role in cell-cell interactions under inflammatory conditions.     

Histamine H1 receptors in UC-11MG astrocytes and their regulation of cytoplasmic Ca

Experiments were carried out on UC-11MG human astrocytoma cells, a continuous cell line that expresses a broad range of the biochemical and electrophysiological properties found in well-differentiated astrocytes. Because of a number of recent reports that astrocytes may express receptors for a variety of neuro-active substances, we measured the effects of 12 different neurotransmitters on intracellular free Ca²⁺ (Ca_i²⁺) in UC-11MG cells. Of these neurotransmitters only histamine was found to have a significant effect. Further characterization of the nature of the histamine response showed that UC-11MG cells express mepyramine-sensitive H₁ receptors the activation of which causes both mobilization of Ca²⁺ from intracellular stores and entry of Ca²⁺ from the extracellular solution. No evidence was found for the presence of H₂ receptors. The Ca_i²⁺ response was maximal at 300 μM histamine and was attenuated by increasing cell density. We suggest that this neurotransmitter may play a role in astrocytic function in the human CNS.     

Extracellular Ca regulates the stimulus-elicited ATP release from urothelium

Accumulating evidence shows that the epithelial cells in urinary bladder (urothelium) serve as a sensory organ in micturition and/or in nociception pathway by releasing ATP in response to mechanical and/or chemical stimuli. Here, we compared the effects of capsaicin, acetylcholine, and prostaglandin E₂ receptor EP1 agonist (ONO-DI-004) on the urothelial ATP release in primary cultured mouse urothelial cells in low Ca²⁺ medium. All of these chemicals induced a gradual ATP release from urothelium, implying that the downstream Ca²⁺ release from endoplasmic reticulum could trigger the ATP release. Consistent with this suggestion, blockade of inositol 1,4,5-triphosphate receptor reduced the distention-induced ATP release from urothelial tissues. The distention-induced ATP release was not affected by tetrodotoxin. However, an increase in extracellular Ca²⁺ diminished both chemical- and distention-induced ATP release from urothelium. Thus raising the extracellular Ca²⁺ concentration was found to inhibit stimulation-evoked ATP urothelial release.     

Adenosine A1 receptors inhibit Ca channels coupled to the release of ACh, but not of GABA, in cultured retina cells

We investigated the effect of adenosine A₁ receptors on the release of acetylcholine (ACh) and GABA, and on the intracellular calcium concentration ([Ca²⁺]_i) response in cultured chick amacrine-like neurons, stimulated by KCl depolarization. The KCl-induced release of [³H]ACh, but not the release of [¹⁴C]GABA, was potentiated when adenosine A₁ receptor activation was prevented by perfusing the cells with adenosine deaminase (ADA) or with 1,3-dipropyl-8-cycloentylxanthine (DPCPX). The changes in the [Ca²⁺]_i induced by KCl depolarization, measured in neurite segments of single cultured cells, were also modulated by endogenous adenosine, acting on adenosine A₁ receptors. Our results show that adenosine A₁ receptors inhibit Ca²⁺ entry coupled to ACh release, but not to the release of GABA, suggesting that the synaptic vesicles containing each neurotransmitter are located in different zones of the neurites, containing different VSCC and/or different densities of adenosine A₁ receptors.     

Domoic acid neurotoxicity in cultured cerebellar granule neurons is controlled preferentially by the NMDA receptor Ca influx pathway

We have monitored real-time alterations in [Ca(2+)](i) in fluo-3-loaded cerebellar granule neurons exposed to domoate, and ascertained the influence of pharmacological blockers of various Ca(2+) entry pathways on intracellular Ca(2+) accumulation, excitatory amino acid (EAA) release and neuronal death. Domoate produced a rapid and concentration-dependent increase in [Ca(2+)](i), the magnitude of which correlated closely with the severity of neuron loss. The increase in [Ca(2+)](i) was derived from activation of NMDA receptors, L-type voltage-sensitive calcium channels (VSCC) and the reversed mode of operation of the Na(+)/Ca(2+) exchanger. When the level of neuroprotection conferred by pharmacological manipulation of these calcium entry pathways was regressed with the corresponding reductions in [Ca(2+)](i) load, it was observed that neuronal vulnerability is controlled preferentially by NMDA receptors. This observation is consistent with our previous study of brevetoxin-induced autocrine excitotoxicity and with the source specificity hypothesis of others [J. Neurochem. 71 (1998) 2349], which suggests that elevation of [Ca(2+)](i) in the vicinity of the NMDA receptor ion channel activates processes leading to neuronal death.     

Prostaglandin E1 inhibits calcium-dependent potentials in mammalian sympathetic neurons

Prostaglandin E1 (PGE1, 10-500 nM) reversibly depressed 3 types of calcium-dependent potentials associated with the spike potential of rabbit superior cervical ganglion cells, namely, the spike after-hyperpolarization, the post-tetanic hyperpolarization, and the Ca2+ spike evoked in a Na+-free/high Ca2+ solution. The results suggest that PGE1 reduces Ca conductance and that this action may underlie its inhibitory action on transmitter release at adrenergic and cholinergic nerve terminals.     

Effect of the removal of extracellular Ca on the response of cytosolic concentrations of Ca to ouabain in carotid body glomus cells of adult rabbits

Effect of the removal of extracellular Ca²⁺ on the response of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to ouabain, an Na⁺/K⁺ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca²⁺]_i (mean±S.E.M.; 38±5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca²⁺]_i increase was abolished by the removal of extracellular Na⁺. D600 (50 μM), an L-type voltage-gated Ca²⁺ channel antagonist, inhibited the [Ca²⁺]_i increase by 57±7% (n=4). Removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i increase, but subsequent washing out of ouabain in Ca²⁺-free solution produced a rise in [Ca²⁺]_i (62±8% increase, n=6, P<0.05), referred to as a [Ca²⁺]_i rise after Ca²⁺-free/ouabain. The magnitude of the [Ca²⁺]_i rise was larger than that of ouabain-induced [Ca²⁺]_i increase. D600 (5 μM) inhibited the [Ca²⁺]_i rise after Ca²⁺-free/ouabain by 83±10% (n=4). These results suggest that ouabain-induced [Ca²⁺]_i increase was due to Ca²⁺ entry involving L-type Ca²⁺ channels which could be activated by cytosolic Na⁺ accumulation. Ca²⁺ removal might modify the [Ca²⁺]_i response, resulting in the occurrence of a rise in [Ca²⁺]_i after Ca²⁺-free/ouabain which mostly involved L-type Ca²⁺ channels.     

Activation of ATP receptor increases the cytosolic Ca(2+) concentration in ventral tegmental area neurons of rat brain

ATP increased the cytosolic Ca(2+) concentration ([Ca](i)) in neurons of ventral tegmental area acutely dissociated from rat brain. The ATP response was dependent on external Ca(2+) and Na(+), and was blocked by voltage-dependent Ca(2+) channel blockers. The results suggest that the ATP-induced depolarization increases Ca(2+) influx via voltage-gated Ca(2+) channels resulting in the increase in [Ca](i).     

Regional distribution of a novel calcium/calmodulin-dependent protein kinase mRNA in the rat brain

The regional distribution of a novel Ca(2+)/calmodulin-dependent protein kinase (CaMK-VI) was examined in the adult rat brain by in situ hybridization. High levels of CaMK-VI mRNA were detected in the hippocampus, piriform cortex and habenula, moderate levels in different thalamic nuclei and cerebral cortex, and low levels in the frontal and parietal cortex. This discrete distribution pattern suggests an important role for CaMK-VI in limbic brain regions.     

Cd and Co at micromolar concentrations mobilize intracellular Ca via the generation of inositol 1,4,5-triphosphate in bovine chromaffin cells

To understand the mechanisms underlying the Cd²⁺- and Co²⁺-induced intracellular Ca²⁺ mobilization, we measured the levels of inositol phosphates using bovine chromaffin cells. Studies using HPLC indicated that Cd²⁺, Co²⁺ and methacholine significantly increased the generation of 1,4,5-IP₃. The results suggest that Cd²⁺ and Co²⁺ mobilize Ca²⁺ from IP₃-sensitive Ca²⁺ stores, possibly through the presumptive Cd²⁺ receptor.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

Neuroprotective effect of ginkgolide K on glutamate-induced cytotoxicity in PC 12 cells via inhibition of ROS generation and Ca(2+) influx

Glutamate is considered to be responsible for the pathogenesis of cerebral ischemia disease. [Ca²⁺]_i influx and reactive oxygen species (ROS) production are considered to be involved in glutamate-induced apoptosis process. In this study, we investigated the neuroprotective effects of ginkgolide K in the glutamate-induced rat's adrenal pheochromocytoma cell line (PC 12 cells) and the possible mechanism. Glutamate cytotoxicity in PC 12 cells was accompanied by an increment of malondialdehyde (MDA) content and lactate dehydrogenase (LDH) release, as well as Ca²⁺ influx, bax/bcl-2 ratio, cytochrome c release, caspase-3 protein and ROS generation, and reduction of cell viability and mitochondrial membrane potential (MMP). Moreover, treatment with glutamate alone resulted in decrease activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity. However, pretreatment with ginkgolide K significantly reduced MDA content, LDH release, as well as Ca²⁺ influx, cytochrome c release, bax/bcl-2 ratio, caspase-3 protein and ROS production, and attenuated the decrease of cells viability and MMP. In addition, ginkgolide K remarkedly up-regulated SOD and GSH-PX activities. All these findings indicated that ginkgolide K protected PC12 cells against glutamate-induced apoptosis by inhibiting Ca²⁺ influx and ROS production. Therefore, the present study supports the notion that ginkgolide K may be a promising neuroprotective agent for the treatment of cerebral ischemia disease.     

ATP modulation of large conductance Ca-activated K channels via a functionally associated protein kinase A in CA1 pyramidal neurons from rat hippocampus

Using inside-out configuration of patch clamp techniques, ATP modulation of BK(Ca) channels was studied in hippocampal CA1 pyramidal neurons of adult rat. Intracellular ATP application markedly increased BK(Ca) channel activity, and this ATP-produced increase in BK(Ca) channel activity was characterized by a higher opening frequency with no changes in channel open times. In the presence of specific inhibitor against protein kinase A, H-89, ATP did not induce any increase in the channel activity. Furthermore, adding H-89 after addition of ATP reversed the modulation produced by ATP. In contrast, protein kinase C inhibitor chelerythrine exerted no apparent effects on ATP-induced channel activation. The present study suggests that BK(Ca) channels from hippocampal CA1 pyramidal neurons could be modulated by ATP via a functionally associated protein kinase A-like protein.     

Phosphorylation promotes the desensitization of the opioid-induced Ca increase in NG108-15 cells

Using the fluorescent Ca²⁺ indicator fura-2, we demonstrated that, in a single NG108-15 cell, acute repetitive challenge with leucine-enkephalin (EK) results in a gradual reduction of the increase of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) at agonist exposure times of 90 s or less; increasing the EK exposure time of each challenge from 30 to 90 s results in greater desensitization, with complete desensitization occurring at 90 s exposure. Similar results are seen with ATP. In opioid-desensitized cells, bradykinin can still induce a marked [Ca²⁺]_i increase, while exposure of desensitized cell to 50 mM K⁺ restores the response EK-induced, suggesting a role of intracellular Ca²⁺ stores in the desensitization process. Pretreatment of cells with certain protein kinase inhibitors, including N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) and staurosporine, prevented desensitization, while others, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and {1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl- -tyrosyl]-4-phenyl-piperazine (KN-62), had no effect. In contrast, activation of protein kinase C by phorbol 12-myristate 13-acetate promoted desensitization. Thus, the desensitization is dependent on protein phosphorylation. HA1004 alone did not alter EK- or bradykinin-induced inositol 1,4,5-trisphosphate (IP₃) generation; however, the inhibitory effect of calyculin A on EK- or bradykinin-induced IP₃ generation was reversed by HA1004. In addition, in the presence of HA1004, the blockade of Ca²⁺ influx by either verapamil or removal of extracellular Ca²⁺ or the depletion of Ca²⁺ pools by thapsigargin still led to desensitization, suggesting that phosphorylation does not alter the activity of the Ca²⁺ transporters involved in Ca²⁺ influx and Ca²⁺ release. Our results imply that emptying of intracellular Ca²⁺ stores and protein phosphorylation in the phospholipase C signaling pathway play roles in the process of desensitization.     

Effect of nilvadipine on the voltage-dependent Ca channels in rat hippocampal CA1 pyramidal neurons

Effects of nilvadipine on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) were compared with other organic Ca²⁺ antagonists in acutely dissociated rat hippocampal CA1 pyramidal neurons. The inhibitory effects of nilvadipine, amlodipine and flunarizine on LVA I_Ca were concentration- and use-dependent. The apparent half-maximum inhibitory concentrations (IC₅₀s) at every 1- and 30-s stimulation were 6.3×10⁻⁷ M and 1.8×10⁻⁶ M for flunarizine, 1.9×10⁻⁶ M and 7.6×10⁻⁶ M for nilvadipine, and 4.0×10⁻⁶ M and 8.0×10⁻⁶ M for amlodipine, respectively. Thus, the strength of the use-dependence was in the sequence of nilvadipine>flunarizine>amlodipine. Nilvadipine also inhibited the HVA I_Ca in a concentration-dependent manner with an IC₅₀ of 1.5×10⁻⁷ M. The hippocampal CA1 neurons were observed to have five pharmacologically distinct HVA Ca²⁺ channel subtypes consisting of L-, N-, P-, Q- and R-types. Nilvadipine selectively inhibited the L-type Ca²⁺ channel current which comprised 34% of the total HVA I_Ca. On the other hand, amlodipine non-selectively inhibited the HVA Ca²⁺ channel subtypes. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca²⁺ influx through both LVA and HVA L-type Ca²⁺ channels, in combination with the cerebral vasodilatory action, may prevent neuronal damage during ischemia.     

Localization of mRNA for Ca/calmodulin-dependent protein kinase I in the brain of developing and mature rats

The gene expression of Ca²⁺/calmodulin-dependent protein kinase I (CaM kinase I) in the brain of developing and adult rats was examined by in situ hybridization histochemistry. During the development, CaM kinase I showed two chronological expression patterns; the persistent and relative high expression as observed in the olfactory bulb and cerebellar cortex, and the gradual decrease in the expression during the postnatal development as observed in most other brain regions. The gene expression was not detected in the germinal ventricular zone and cerebellar external granular layer. In the mature brain, CaM kinase I mRNA was expressed widely, though weakly in general, in almost all neurons, except for the olfactory bulb, cerebellum and hippocampus expressing at high intensity. These findings suggest that CaM kinase I may play a variety of neuronal Ca²⁺/calmodulin-mediated signaling processes in the developing and mature brains.     

Regional differences between the immunohistochemical distribution of Ca/calmodulin-dependent protein kinase II α and β isoforms in the brainstem of the rat

The distribution of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) α and β isoforms in the brainstem of adult rats was investigated using an immunohistochemical method with two monoclonal antibodies which specifically recognize the α and β isoform, respectively. We found that these isoforms were differentially expressed by neurons in the substantia nigra, red nucleus, dorsal cochlear nucleus, pontine nuclei and inferior olivary nucleus. Neurons in the inferior olivary nucleus express the α isoform, but not the β isoform. In contrast, neurons in the substantia nigra, red nucleus and pontine nuclei were immunostained with the β antibody, but not with the α antibody. In the dorsal cochlear nucleus, neurons in layers I and II were α-immunopositive, whereas neurons in layers III and IV were β-immunopositive. Therefore, the distribution of the CaM kinase II α-immunopositive neurons is completely different from that of CaM kinase II β-immunopositive neurons. Next we examined the possible coexistence of CaM kinase II α isoform and glutamate or that of CaM kinase II β isoform and glutamic acid decarboxylase (GAD) in the single neuron by double immunofluorescence labelling using a pair of anti-α and anti-glutamate antibodies, or a pair of anti-β and anti-GAD antibodies. The results indicated that neurons expressing anti-α immunoreactivity were also immunopositive against anti-glutamate antibody, and neurons expressing β isoform were also immunopositive against anti-GAD antibody, suggesting that α-immunopositive neurons are classified as excitatory-type neurons, and on the contrary, β-immunopositive neurons are classified as inhibitory-type neurons. In conclusion, the present study confirmed that α- and β-isoforms of CaM kinase II are differentially expressed in the nuclei of the brainstem and have different roles.     

Neuron-specific expression of reporter gene in transgenic mice carrying the 5′-upstream region of mouse P/Q-type Ca channel α1A subunit gene fused to E. coli lacZ reporter gene

To dissect the molecular mechanisms underlying the neuron-specific expression of the P/Q type calcium channel α_1A subunit gene, transgenic mice carrying a 0.5-kb, 1.5-kb, 3.0-kb or 6.3-kb 5′-upstream region of the gene fused to Escherichia coli lacZ reporter gene were produced. In transgenic mice carrying the 1.5-kb, 3.0-kb or 6.3-kb 5′-upstream region, the reporter gene was exclusively expressed in the nervous system, although those with the 0.5-kb 5′-upstream region failed to show reporter expression. Histological examinations showed that the three 5′-upstream regions induced distinct expression patterns of the reporter gene in the CNS and adrenal medulla. The 1.5-kb 5′-upstream region drove reporter gene expression in the olfactory bulb, dorsal cortex and hippocampus, while the regulatory element for the expression in the amygdaloid nucleus, septum, habenula medial nucleus, choroid plexus, substantia nigra, inferior colliculus, pontine nucleus and cerebellum was located in the 5′-upstream sequence between 1.5 kb and 6.3 kb. In the cerebellum, the expression of the reporter gene was induced by the 3.0-kb region in granule cells, whereas it was induced by the 6.3-kb region in Purkinje cells. The expression of the reporter gene in chromaffin cells in the adrenal medulla was induced only by the 6.3-kb 5′-upstream region. These results suggest that the expression of the mouse P/Q-type Ca²⁺ channel α_1A subunit gene is regulated in a complex fashion by both positive and negative cis-regulatory elements.     

The putative OP4 antagonist, [Nphe]nociceptin(1-13)NH2, prevents the effects of nociceptin in neuropathic rats

Nociceptin or orphanin FQ (N/OFQ) is the natural ligand of the opioid receptor-like 1 receptor (ORL-1), which has been also classified as the fourth member of the opioid family of receptors and named OP₄. Elucidation of the biological role of N/OFQ has been hampered by the lack of compounds that selectively block the OP₄ receptor. Recently, a N/OFQ derivative, [Nphe¹]N/OFQ(1-13)NH₂, has been found to possess OP₄ antagonistic properties both in vitro and in vivo models. We investigated its spinal effect in the chronic constriction injury of the sciatic nerve in the rat, a model relevant to neuropathic pain in humans. Intrathecal (i.t.) administration of N/OFQ (0.2–20 nmoles) dose-dependently reversed mechanical allodynic-like behavior, while [Nphe¹]N/OFQ(1-13)NH₂ (20–120 nmoles, i.t.) was ineffective on its own. [Nphe¹]N/OFQ(1-13)NH₂ (60–120 nmoles, i.t.) antagonized N/OFQ (about 80% of reduction) but did not modify the activity of morphine (20 nmoles, i.t.). These results further support, for the first time in a chronic model of pain, the specific antagonistic profile of [Nphe¹]N/OFQ(1-13)NH₂vs the OP₄ receptor. This pseudopeptide is an interesting pharmacological tool to better clarify the role of N/OFQ in pathophysiology.