Enhanced Hyperthermic Responses to Lipopolysaccharide in Mice Exposed to Repeated Cold Stress.

Lipopolysaccharide (LPS) induces hyperthermia accompanied by various other systemic inflammatory symptoms. The rodents exposed to repeated cold (RC) stress according to a specific schedule are useful as experimental models for autonomic imbalance or fibromyalgia. It is now proven that RC-stressed mice exhibit tolerance to LPS, we examined thermal responses to LPS challenge in RC-stressed mice by monitoring core temperature using the telemetry system. Systemic administration of LPS caused bimodal hyperthermic responses in RC-stressed and unstressed mice. The magnitude of the LPS-induced hyperthermia was greater in RC-stressed mice than in unstressed mice. The RC stress-induced enhancement of hyperthermic responses to LPS was abolished by pretreatment with diclofenac, which is a cyclooxygenase (COX) inhibitor. LPS did not significantly increase COX-2 protein levels in the lung or hypothalamus of RC-stressed or unstressed mice. RC stress did not alter baseline serum corticosterone levels or their increases in response to LPS challenge. These results suggest that RC stress enhances the susceptibility of mice to LPS challenge, leading to greater prostanoid-dependent hyperthermia, which might contribute to tolerance to LPS in RC-stressed mice.

Repeated Cold Stress Enhances the Acute Restraint Stress-Induced Hyperthermia in Mice.

The rodents exposed to repeated cold stress according to a specific schedule, known as specific alternation of rhythm in temperature (SART), exhibit autonomic imbalance, and is now used as an experimental model of fibromyalgia. To explore the susceptibility of SART-stressed animals to novel acute stress, we tested whether exposure of mice to SART stress for 1 week alters the extent of acute restraint stress-induced hyperthermia. Mice were subjected to 7-d SART stress sessions; i.e., the mice were alternately exposed to 24 and 4°C at 1-h intervals during the daytime (09:00-16:00) and kept at 4°C overnight (16:00-09:00). SART-stressed and unstressed mice were exposed to acute restraint stress for 20-60 min, during which rectal temperature was monitored. Serum corticosterone levels were measured before and after 60-min exposure to restraint stress. SART stress itself did not alter the body temperature or serum corticosterone levels in mice. Acute restraint stress increased the body temperature and serum corticosterone levels, both responses being greater in SART-stressed mice than unstressed mice. The enhanced hyperthermic responses to acute restraint stress in SART-stressed mice were significantly attenuated by SR59230A, a ß3 adrenoceptor antagonist, but unaffected by diazepam, an anxiolytic, mifepristone, a glucocorticoid receptor antagonist, or indomethacin, a cyclooxygenase inhibitor. These results suggest that SART stress enhances the susceptibility of mice to acute restraint stress, characterized by increased hyperthermia and corticosterone secretion, and that the increased hyperthermic responses to acute stress might involve accelerated activation of sympathetic ß3 adrenoceptors, known to regulate non-shivering thermogenesis in the brown adipose tissue.

PKC/MEK inhibitors suppress oxaliplatin-induced neuropathy and potentiate the antitumor effects.

Oxaliplatin is a key drug commonly used in colorectal cancer treatment. Despite high clinical efficacy, its therapeutic application is limited by common, dose-limiting occurrence of neuropathy. As usual symptomatic neuropathy treatments fail to improve the patients' condition, there is an urgent need to advance our understanding of the pathogenesis of neuropathy to propose effective therapy and ensure adequate pain management. Oxaliplatin-induced neuropathy was recently reported to be associated with protein kinase C (PKC) activation. It is unclear, however, whether PKC inhibition can prevent neuropathy. In our current studies, we found that a PKC inhibitor, tamoxifen, inhibited oxaliplatin-induced neuropathy via the PKC/extracellular signal-regulated kinase (ERK)/c-Fos pathway in lumbar spinal cords (lumbar segments 4-6). Additionally, tamoxifen was shown to act in synergy with oxaliplatin to inhibit growth in tumor cells-implanted mice. Moreover, mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, PD0325901, suppressed oxaliplatin-induced neuropathy and enhanced oxaliplatin efficacy. Our results indicate that oxaliplatin-induced neuropathy is associated with PKC/ERK/c-Fos pathway in lumbar spinal cord. Additionally, we demonstrate that disruption of this pathway by PKC and MEK inhibitors suppresses oxaliplatin-induced neuropathy, thereby suggesting that PKC and MEK inhibitors may be therapeutically useful in preventing oxaliplatin-induced neuropathy and could aid in combination antitumor pharmacotherapy.

[A search for the risk factors for hiccups and evaluation of antiemetic therapy in CDDP-based chemotherapy, using cluster analysis].

Hiccups are often observed in patients treated with cisplatin(CDDP)-based chemotherapy. It has been reported that gender and specific dosages of CDDP and antiemetic drugs(e.g., dexamethasone and 5-HT3 receptor antagonist)using standard therapy are major risk factors in the onset of hiccups. Recently, aprepitant has been added to the antiemetic therapy in CDDP-based chemotherapy. However, it is not known how the onset of hiccups takes place in antiemetic therapy including aprepitant according to the guideline. In this study, we used cluster analysis to classify 229 patients treated with CDDP-based chemotherapy, to investigate the effect of antiemetic therapy on the onset of hiccups and chemotherapy-induced nausea and vomiting(CINV). Our analysis indicated that aprepitant was not a major risk factor for the onset of hiccups in the high CDDP dose group(≥70 mg/m(2)). However, an effect of antiemesis was confirmed in the standard therapy with aprepitant. In conclusion, we suggest that aprepitant is effective for CINV, without causing the onset of hiccups in patients treated with high-dose CDDP-based chemotherapy.

Differential expression of the 5-HT(3A) and 5-HT(3B) receptor in differentiated NG108-15 cells.

Previous work from this laboratory has shown that the serotonin (5-HT) induced response is significantly augmented in differentiated NG108-15 (NG) cells treated with dibutyryl cAMP (Bt(2)cAMP) due to qualitative and quantitative changes in the expression of the 5-HT(3) receptor as demonstrated by specific [(3)H] LY-278584 (a selective 5HT(3) receptor antagonist) binding. In this study, we investigated whether there is any change in the relative expression of the 5-HT(3A) and 5-HT(3B) subunits in NG cells differentiated following Bt(2)cAMP treatment cells. The major findings of this study were that the relative amount of 5-HT(3B) subunit mRNA in Bt(2)cAMP-treated NG cells 5 days following Bt(2)cAMP-treatment was greater than that in the untreated cells. In contrast, the relative expression of the 5-HT(3B) subunit protein in the Bt(2)cAMP-treated NG cells was much less than in the untreated cells, but the relative expression of the 5-HT(3A) subunit in the Bt(2)cAMP-treated NG cells was similar to the untreated cells. Therefore, no relationship between mRNA and protein expression for 5-HT(3A) and 5-HT(3B) subunits in Bt(2)cAMP treated and untreated NG cells were observed. It was also found that fluorescent intensity for the 5-HT(3B) subunit in the cell body of the Bt(2)cAMP treated and untreated NG cells gradually decreased from the day 1-5 after Bt(2)cAMP treatment. However, in specific areas such as the varicosity and nerve endings of the Bt(2)cAMP treated cells, staining intensity for the 5-HT(3B) subunits was stronger than in the untreated cells at the all time points, peaking at day 5 post-treatment. These results suggest that the augmented response induced by 5-HT acting via 5-HT(3) receptors in differentiated NG cells may be due to changes in the relative amount of the 5-HT(3B) subunit, particularly the ratio and distribution of the 5-HT(3A) to (3B) subunits.

Effects of the α1-adrenoceptor agonist phenylephrine on SART stress-induced orthostatic hypotension in rats.

BACKGROUND: Specific alternation of rhythm in temperature (SART)-stressed rats, an animal model of autonomic imbalance, exhibit low blood pressure and tachycardia during consciousness and under anesthesia. In addition, these rats easily develop orthostatic hypotension (OH) as a response to postural manipulation. Hence, we studied the influence of the adrenalin α1-receptor agonist phenylephrine on stress-induced OH in SART-stressed rats and unstressed rats. METHODS: Male Wistar rats weighing 250-300 g were used. Rats were fixed in the supine position under urethane anesthesia. Blood pressure was directly measured from the left common carotid artery and ECG was recorded simultaneously. RESULTS: The maximum decrease in blood pressure and the area under the blood pressure-time curve were both large, while the %reflex was small in the SART-stressed rats compared with unstressed rats. In the SART-stressed rats, prolonged intravenous administration of phenylephrine reduced OH at a dose that barely affected unstressed rats. CONCLUSION: The results suggested that sympathetic dysfunction is a factor underlying SART stress-induced OH.

Specific alternation of rhythm in temperature (SART) stress-induced irritable bowel syndrome-like changes in mice and effects of drugs.

Stress is closely associated with the manifestation and progress of irritable bowel syndrome (IBS). For the purpose of establishing experimentally the relationship between IBS and stress, the transportation capacity of the small intestine in specific alternation of rhythm in temperature (SART)-stressed animals was studied using charcoal transportation method. The charcoal suspension was administered orally into the stomach of fasting mice. Mice were sacrificed after a certain time and %charcoal transit (%CT) of the small intestine was measured. The %CTs in SART-stressed mice were greater than those in unstressed or continuously cold-stressed mice. This increase in %CT remained for 1 week after discontinuation of SART stress loading. Cholinergic blockers decreased %CTs in SART-stressed mice. Increases in %CT by a cholinesterase inhibitor were less in SART-stressed mice than in unstressed mice. Increases of %CT in SART-stressed mice were suppressed by Neurotropine. These results suggested that the parasympathetic hypertonicity, not just cold, played a role in the increases in the transportation capacity in SART-stressed mice and that these animals can be a useful tool for elucidation of the mechanism of IBS.

Changes in characteristics of the specific binding of [3H]LY-278584, a 5-HT3-receptor antagonist, on differentiated NG108-15 cells.

We have reported previously that the concentration of intracellular Ca2+ evoked by serotonin (5-HT) was significantly augmented in differentiated NG108-15 (NG) cells treated with dibutyryl cAMP and the enhanced response occurred via 5-HT3 receptors. We investigated changes in the characteristics for specific binding of [(3)H]LY-278584 (a specific antagonist of the 5-HT3 receptor) on membranes from differentiated NG cells. The results indicated that the K(d) and B(max) values for the specific binding to differentiated NG cells were significantly smaller and larger, respectively, than those for undifferentiated NG cells. The binding was significantly inhibited by 10 nM tropisetron, a specific 5-HT3-receptor antagonist, but not by any other types of 5-HT-receptor antagonists. These results suggested that the enhanced response by 5-HT in differentiated NG cells was due to both qualitative and quantitative changes in the 5-HT3 receptor.

Induction of metallothionein in mouse cerebellum and cerebrum with low-dose thimerosal injection.

Thimerosal, an ethyl mercury compound, is used worldwide as a vaccine preservative. We previously observed that the mercury concentration in mouse brains did not increase with the clinical dose of thimerosal injection, but the concentration increased in the brain after the injection of thimerosal with lipopolysaccharide, even if a low dose of thimerosal was administered. Thimerosal may penetrate the brain, but is undetectable when a clinical dose of thimerosal is injected; therefore, the induction of metallothionein (MT) messenger RNA (mRNA) and protein was observed in the cerebellum and cerebrum of mice after thimerosal injection, as MT is an inducible protein. MT-1 mRNA was expressed at 6 and 9 h in both the cerebrum and cerebellum, but MT-1 mRNA expression in the cerebellum was three times higher than that in the cerebrum after the injection of 12 microg/kg thimerosal. MT-2 mRNA was not expressed until 24 h in both organs. MT-3 mRNA was expressed in the cerebellum from 6 to 15 h after the injection, but not in the cerebrum until 24 h. MT-1 and MT-3 mRNAs were expressed in the cerebellum in a dose-dependent manner. Furthermore, MT-1 protein was detected from 6 to 72 h in the cerebellum after 12 microg/kg of thimerosal was injected and peaked at 10 h. MT-2 was detected in the cerebellum only at 10 h. In the cerebrum, little MT-1 protein was detected at 10 and 24 h, and there were no peaks of MT-2 protein in the cerebrum. In conclusion, MT-1 and MT-3 mRNAs but not MT-2 mRNA are easily expressed in the cerebellum rather than in the cerebrum by the injection of low-dose thimerosal. It is thought that the cerebellum is a sensitive organ against thimerosal. As a result of the present findings, in combination with the brain pathology observed in patients diagnosed with autism, the present study helps to support the possible biological plausibility for how low-dose exposure to mercury from thimerosal-containing vaccines may be associated with autism.

Expression of metallothionein mRNAs on mouse cerebellum microglia cells by thimerosal and its metabolites.

Effects of thimerosal and its metabolites, ethyl mercury and thiosalicylate, on the expression of metallothionein (MT) mRNAs in mouse cerebellum microglia cell line, C8-B4 cells, were studied. The level of MT-1 mRNA significantly decreased at early hours and recovered time-dependently 24h after thimerosal was added to the C8-B4 cells. However, MT-2 and MT-3 mRNA expressions did not change from the control group. In contrast, the expression of MT-1 mRNA increased in a mouse neuroblastoma cell line 6h after incubation with thimerosal. In addition, the level of MT-1 mRNA decreased in C8-B4 cells 6h after the addition of thiosalicylate, but ethyl mercury induced MT-1 mRNA expression. When cell viability was compared with thimerosal, thiosalicylate, and ethyl mercury, the viability of C8-B4 cells decreased dose-dependently 24h after either thimerosal or ethyl mercury was added; however, the viability increased dose-dependently until 15 microM thiosalicylate was added. From the present results, it is concluded that the expression of MT-1 mRNA may be mediated by different factors than the expression of MT-2 mRNA in C8-B4 cells. The reduction of MT-1 mRNA level by thiosalicylate may affect the proliferation of C8-B4 cells.

Hydrogen sulfide evokes neurite outgrowth and expression of high-voltage-activated Ca2+ currents in NG108-15 cells: involvement of T-type Ca2+ channels.

We investigated if stimulation of T-type Ca(2+) channels with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H(2)S), could cause neuronal differentiation of NG108-15 cells. Like dibutyryl cyclic AMP (db-cAMP), treatment with NaHS at 1.5-13.5 mM for 16 h enhanced neurite outgrowth in a concentration-dependent manner. Synergistic neuritogenic effect was obtained in the cells stimulated with NaHS in combination with db-cAMP at subeffective concentrations. Exposure to NaHS or db-cAMP for 2 days resulted in enhancement of expression of high-voltage-activated currents consisting of N-, P/Q-, L- and also other types, but not of T-type currents. Mibefradil, a pan-T-type channel blocker, abolished the neuritogenesis induced by NaHS, but not by db-cAMP. The NaHS-evoked neuritogenesis was also completely blocked by pretreatment with BAPTA/AM, a chelator of intracellular Ca(2+), and by zinc chloride at a concentration known to selectively inhibit Ca(v)3.2 isoform of T-type Ca(2+) channels, but not Ca(v)3.1 or Ca(v)3.3. Further, L-ascorbate, recently proven to selectively inhibit Ca(v)3.2, abolished the neuritogenic effect of NaHS, but not db-cAMP. Our data thus demonstrate that NaHS/H(2)S is a novel inducer of neuronal differentiation in NG108-15 cells, as characterized by neuritogenesis and expression of high-voltage-activated currents, and suggest the involvement of T-type Ca(2+) channels, especially Ca(v)3.2.

Characteristics for enhanced response of serotonin-evoked ion dynamics in differentiated NG108-15 cells.

Characteristics for the up-regulated response in the concentration of intracellular calcium ion ([Ca(2+)]( i )) and in the sodium ion (Na(+)) current by serotonin (5-HT) were investigated in differentiated neuroblastoma x glioma hybrid NG108-15 (NG) cells. The results for the changes in [Ca(2+)]( i ) by 5-HT were as follows, (1) The 5-HT-induced Ca(2+) response was inhibited by 3 x 10(-9) M tropisetron (a 5-HT(3) receptor blocker), but not by other types of 5-HT receptor blockers; (2) The 5-HT-induced Ca(2+) response was mainly inhibited by calciseptine (a L-type Ca(2+) blocker), but not by other types of Ca(2+) channel blockers or 10(-7) M TTX (a voltage-sensitive Na(+) channel blocker); (3) When the extracellular Na(+) was removed by exchange with choline chloride or N-methyl-D-glucamine, the 5-HT-induced Ca(2+) response was extremely inhibited. The results for the 5-HT-induced Na(+) current by the whole cell patch-clamp technique were as follows, (1) The 5-HT-induced Na(+) current in differentiated cells was significantly larger than that in undifferentiated cells; (2) The ED(50) value for 5-HT-induced Na(+) current in undifferentiated and differentiated cells was almost the same, about 4 x 10(-6) M each other; (3) The 5-HT-induced Na(+) current was completely blocked by 3 x 10(-9) M tropisetron, but not by other 5-HT receptor antagonists and 10(-7) M TTX. These results suggested that 5-HT-induced Ca(2+) response in differentiated NG cells was mainly due to L-type voltage-gated Ca(2+) channels allowing extracellular Na(+) to enter via 5-HT(3) receptors, but not through voltage-gated Na(+) channels.

Enhancement of sodium current in NG108-15 cells during neural differentiation is mainly due to an increase in NaV1.7 expression.

It is well known that morphological and functional changes during neural differentiation sometimes accompany the expression of various voltage-gated ion channels. In this work, we investigated whether the enhancement of sodium current in differentiated neuroblastoma x glioma NG108-15 cells treated with dibutyryl cAMP is related to the expression of voltage-gated sodium channels. The results were as follows. (1) Sodium current density on peak voltage in differentiated cells was significantly enhanced compared with that in undifferentiated cells, as detected by the whole-cell patch clamp method. The steady-state inactivation curve in differentiated cells was similar to that for undifferentiated cells, but a hyperpolarized shift in the activation curve for differentiated cells was observed. The sodium currents of differentiated and undifferentiated cells were completely inhibited by 10(-7) M tetrodotoxin (TTX). (2) The only Na(V) mRNA with an increased expression level during neuronal differentiation was that for NaV1.7, as observed by real-time PCR analysis. (3) The increase in the level of NaV1.7 alpha subunit expression during neuronal differentiation was also observed by immunocytochemistry; in particular, the localization of NaV1.7 alpha subunits on the soma, varicosities and growth cone was significant. These results suggest that the enhancement of TTX-sensitive sodium current density in differentiated NG108-15 cells is mainly due to the increase in the expression of the TTX-sensitive voltage-gated Na+ channel, NaV1.7.

Hydrogen sulfide as a novel nociceptive messenger.

Hydrogen sulfide (H(2)S), an endogenous gasotransmitter, modulates various biological events such as inflammation in the mammalian body. The present study investigated possible involvement of H(2)S in peripheral nociceptive processing. Intraplantar (i.pl.) administration of NaHS, a H(2)S donor, produced prompt hyperalgesia in rats, accompanied by expression of Fos in the spinal dorsal horn. The H(2)S-evoked hyperalgesia was blocked by 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), an oxidizing agent, or ethosuximide and mibefradil, T-type Ca(2+) channel inhibitors. L-Cysteine, an endogenous source for H(2)S, given i.pl., also elicited hyperalgesia, an effect being abolished by DL-propargylglycine (PPG) and beta-cyanoalanine (BCA), inhibitors of cystathionine-gamma-lyase, a H(2)S synthesizing enzyme. PPG and/or BCA partially inhibited the hyperalgesia induced by i.pl. lipopolysaccharide, an effect being reversed by i.pl. NaHS. In the patch-clamp study using undifferentiated NG108-15 cells that express T-type, but not other types, of Ca(2+) channels, NaHS enhanced the currents through the T-type channels, an effect being blocked by DTNB. Thus, H(2)S appears to function as a novel nociceptive messenger through sensitization of T-type Ca(2+) channels in the peripheral tissues, particularly during inflammation.

Paclitaxel-2'-Ethylcarbonate prodrug can circumvent P-glycoprotein-mediated cellular efflux to increase drug cytotoxicity.

PURPOSE: The aim of the study was to investigate whether 2'-ethylcarbonate-linked paclitaxel (TAX-2'-Et) circumvents P-glycoprotein (P-gp)-mediated cellular efflux and cytotoxicity enhanced by TAX-2'-Et activation within human culture cells transfected with a rabbit liver carboxylesterase (Ra-CES) cDNA. MATERIALS AND METHODS: TAX-2'-Et transport was characterized in a human colon carcinoma cell line (Caco-2) and paclitaxel (TAX)-resistant ovarian carcinoma cells (SKOV3/TAX60). Expression of P-gp, multidrug resistance protein (MRP) 2 and Ra-CES was detected by Western blotting. Cytotoxicity against Ra-CES-expressing cells and cellular amount of TAX produced were determined by MTT assay and using HPLC, respectively. RESULTS: Unlike rhodamine123 and TAX, TAX-2'-Et did not exhibit polarized transport in the Caco-2 cells in the absence or presence of verapamil. P-gp levels were expressed much higher in the SKOV3/ TAX60 cells than in the Caco-2 cells. MRP2 protein was not detectable in the SKOV3/TAX60 cells. Uptake by the SKOV3/TAX60 cells was similar in quantity to the amount internalized by P-gp-negative SKOV3 cells. In the SKOV3/TAX60 cells, cellular uptake of TAX-2'-Et was not altered regardless of the absence or presence of verapamil. The cytotoxicity to the untransfected SKOV3 cells induced by TAX-2'-Et was significantly lower than that induced by TAX. In the Ra-CES-expressing SKOV3 line, the EC50 value of TAX (10.6 nM) was approximately four-fold higher than that of TAX-2'-Et (2.5 nM). Transfection of Ra-CES into another TAX-resistant ovarian carcinoma cells (KOC-7c) conferred a high level of TAX-2'-Et cytotoxicity via prodrug activation. The intracellular levels of TAX produced from TAX-2'-Et in the Ra-CES-positive KOC-7c cells significantly increased compared with the levels seen in exposure of the untransfected KOC-7c cells to TAX. CONCLUSIONS: TAX-2'-Et can circumvent P-gp-associated cellular efflux of TAX. TAX-2'-Et is converted into TAX by the Ra-CES, supporting its potential use as a theoretical GDEPT strategy for cancer cells expressing high levels of P-gp. The TAX-2'-Et prodrug efficiently increased the amount of intracellular TAX, which mediates tumor cell death.

Enhancement of serotonin- and bradykinin-evoked calcium ion dynamics in differentiated NG108-15 cells.

Dynamic changes in the concentration of intracellular free-calcium ion ([Ca(2+)](i)) by carbachol (CCh) and neurotransmitter candidates was investigated in undifferentiated and differentiated neuroblastomaxglioma hybrid NG108-15 (NG) cells. [Ca(2+)](i) was increased in a dose-dependent manner by bradykinin (BK) and serotonin (5-HT) in differentiated NG cells, and the response to BK and 5-HT was significantly greater than that in undifferentiated NG cells. The EC(50) value of BK was approximately 1.5 x 10(-8)M in both undifferentiated and differentiated NG cells. The EC(50) value of 5-HT in differentiated NG cells was about 5 x 10(-6)M. The response to BK and 5-HT was almost completely inhibited by 10 nM Hoe140 (a BK B2 receptor antagonist) and 3 nM tropisetron (a 5-HT(3) receptor antagonist), respectively. These results suggest that there are some mechanisms by which the response evoked by BK and 5-HT is up-regulated in differentiated NG cells.

Enhancement of veratridine-induced sodium dynamics in NG108-15 cells during differentiation.

Developmental changes in dynamics of Na+ were studied in neuroblastomaxglioma hybrid NG108-15 cells during differentiation which was induced by dibutyryl cAMP (Bt2cAMP). Ratiometric Na+ imaging with a Na+-sensitive fluorescent dye SBFI (sodium-binding benzofuran isophthalate) revealed that the intracellular Na+ concentration ([Na+]i) was not affected by the application of high K+ (60 mM) solution to either control or differentiated cells. When cells were exposed to 50 microM veratridine (Vtd), an agonist of voltage-sensitive sodium channels (VSSCs), a significant increase in [Na+]i was observed in differentiated but not in undifferentiated cells. Calculated mean [Na+]i value increased from the basal 10.4 to 44.1 mM in response to 50 microM Vtd. This Vtd response was reversibly inhibited by tetrodotoxin (TTX), a specific blocker for VSSCs, in a dose-dependent manner (IC50 = 1 nM). It is suggested that VSSCs in NG108-15 cells are sensitive to TTX and Vtd and that the number of VSSCs increases during differentiation.

Increased response to high KCl-induced elevation in the intracellular-Ca(2+) concentration in differentiated NG108-15 cell and the inhibitory effect of the L-type Ca(2+) channel blocker, calciseptine.

Characteristics of the increasing effect for the concentration of intracellular calcium ions ([Ca(2+)](i)) by high-KCl application were investigated in the neuroblastomaxglioma hybrid NG108-15 cell line (NG108-15 cells). The present study confirmed that the increasing effect of [Ca(2+)](i) by high-KCl application in single NG108-15 cells, differentiated with dibutyryl cAMP (Bt(2)cAMP), was significantly enhanced, compared to undifferentiated cells. The following observations were made at first: (1) The response to high-KCl application, in both undifferentiated and differentiated cells, was significantly inhibited by calciseptine (CaS), an L-type Ca(2+) channel blocker, but not by N-, P- and R-type Ca(2+) channel blockers. The IC(50) values for CaS in both undifferentiated and differentiated cell was almost identical. (2) The inhibitory effect of CaS was irreversible. (3) The increasing effect for [Ca(2+)](i) by high-KCl application was completely dependent on the presence of extracellular calcium ions. (4) The increased [Ca(2+)](i) by high-KCl application under a plateau concentration was quickly decreased to basal levels when the high-KCl solution was exchanged for a high-KCl solution containing EGTA (without CaCl(2)). Together, these results suggest that the enhancement of the response effect of [Ca(2+)](i) by high-KCl application in differentiated single NG108-15 cells was mainly due to the quantitative increase of L-type voltage-sensitive calcium channels (VSCCs), which were irreversibly inhibited by CaS.

Effects of calmodulin and Ca2+ channel blockers on omega-conotoxin GVIA binding to crude membranes from alpha1B subunit (Cav2.2) expressed BHK cells and mice brain lacking the alpha1B subunits.

Characteristics for the specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to crude membranes from BHKN101 cells expressing the alpha1B subunits of Cav2.2 channels and from mice brain lacking the alpha1B subunits of Cav2.2 channels, particularly, the effects of CaM and various Ca2+ channel blockers on these specific bindings were investigated. Specific binding of 125I-omega-CTX GVIA to the crude membranes from BHKN101 cells was observed, but not from control BHK6 cells. omega-CTX GVIA, omega-CTX MVIIC and omega-CTX SVIB inhibited the specific binding of 125I-omega-CTX GVIA to crude membranes from BHKN101 cells, and the IC50 values for omega-CTXGVIA, omega-CTX MVIIC and omega-CTX SVIB were 0.07, 8.5 and 1.7 nM, respectively. However, omega-agatoxin IVA and calciseptine at concentrations of 10(-9)-10(-6) M did not inhibit specific binding. Specific binding was also about 80% inhibited by 20 microg protein/ml CaM. The amount of 125I-omega-CTX GVIA (30 pM) specifically bound to membranes from brain of knockout mice lacking alpha1B subunits of Cav2.2 channels was about 30% of that to the crude membranes from brain of wild-type. On the other hand, specific binding of 125I-omega-CTX MVIIC (200 pM) was observed on the crude membranes of both BHKN101 and control BHK6 cells. The specific binding of 125I-omega-CTX MVIIC (200 pM) was not inhibited by omega-CTX GVIA and omega-CTX SVIB, and also omega-Aga IVA and calciseptine at concentrations of 10(-9)-10(-7) M, although specific binding was almost completely dose dependently inhibited by non-radiolabeled omega-CTX MVIIC (IC50 value was about 0.1 nM). 20 microg protein/ml CaM did not inhibit specific binding. Therefore, these results suggest that BHKN101 cells have a typical Cav2.2 channels which are also inhibited by CaM and have not specific binding sites for omega-CTX MVIIC, although omega-CTX MVIIC is a blocker for both Cav2.1 (alpha1A; P/Q-type) and Cav2.2 channels.

Characteristics of omega-conotoxin GVI A and MVIIC binding to Cav 2.1 and Cav 2.2 channels captured by anti-Ca2+ channel peptide antibodies.

A New Binding Method (NBM) was used to investigate the characteristics of the specific binding of 125I-omega-conotoxin (omega-CTX) GVIA and 125I-omega-CTX MVIIC to Cav2.1 and Cav2.2 channels captured from chick brain membranes by antibodies against B1Nt (a peptide sequence in Car2.1 and Cav2.2 channels). The results for the NBM were as follows. (1) The ED50 values for specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to Cav2.1 and Cav2.2 channels were about 68 and 60 pM, respectively, and very similar to those (87 and 35 pM, respectively) to crude membranes from chick brain. (2) The specific 125I-omega-CTX GVIA (100 pM) binding was inhibited by omega-CTX GVIA (0.5 nM), dynorphine A (Dyn), gentamicin (Gen), neomycin (Neo) and tobramicin (Tob) (100 microM each), but not by omega-agaconotoxin (Aga) IVA, calciseptine, omega-CTX SVIB, omega-CTX MVIIC (0.5 nM each), PN200-110 (PN), diltiazem (Dil) or verapamil (Ver) (100 microM each). Calmodulin (CaM) inhibited the specific binding in a dose-dependent manner (IC50 value of about 100 microg protein/ml). (3) The specific 125I-omega-CTX MVIIC (60 pM) binding was inhibited by omega-CTX MVIIC, omega-CTX GVIA, omega-CTX SVIB (0.5 nM each), Dyn, Neo and Tob (100 microM, each), but not by omega-Aga IVA, calciseptine (0.5 nM each), PN, Dil, Ver (100 microM each) or 100 microg protein/ml CaM. These results suggested that the characteristics of the specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to Cav2.1 and Cav2.2 channels in the NBM were very similar to those to crude membranes from chick brain, although the IC50 values for CaM and free Ca2+ of CaM were about 33- and 5000-fold higher, respectively, than those for the specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to crude membranes.