Gap-junction-mediated communication in human periodontal ligament cells.

Periodontal tissue homeostasis depends on a complex cellular network that conveys cell-cell communication. Gap junctions (GJs), one of the intercellular communication systems, are found between adjacent human periodontal ligament (hPDL) cells; however, the functional GJ coupling between hPDL cells has not yet been elucidated. In this study, we investigated functional gap-junction-mediated intercellular communication in isolated primary hPDL cells. SEM images indicated that the cells were in contact with each other via dendritic processes, and also showed high anti-connexin43 (Cx43) immunoreactivity on these processes. Gap-junctional intercellular communication (GJIC) among hPDL cells was assessed by fluorescence recovery after a photobleaching (FRAP) analysis, which exhibited dye coupling between hPDL cells, and was remarkably down-regulated when the cells were treated with a GJ blocker. Additionally, we examined GJs under hypoxic stress. The fluorescence recovery and expression levels of Cx43 decreased time-dependently under the hypoxic condition. Exposure to GJ inhibitor or hypoxia increased RANKL expression, and decreased OPG expression. This study shows that GJIC is responsible for hPDL cells and that its activity is reduced under hypoxia. This is consistent with the possible role of hPDL cells in regulating the biochemical reactions in response to changes in the hypoxic environment.

SSEA-4 is a marker of human deciduous periodontal ligament stem cells.

Although human deciduous teeth are an ideal source of adult stem cells, no method for identifying deciduous periodontal ligament (D-PDL) stem cells has so far been developed. In the present study, we investigated whether stage-specific embryonic antigen (SSEA)-4 is a marker that could be used to isolate D-PDL stem cells. The isolated D-PDL cells met the minimum criteria for mesenchymal stem cells (MSCs): They showed plastic adherence, specific-surface antigen expression, and multipotent differentiation potential. SSEA-4+ D-PDL cells were detected in vitro and in vivo. A flow cytometric analysis demonstrated that 22.7% of the D-PDL cells were positive for SSEA-4. SSEA-4+ clonal D-PDL cells displayed multilineage differentiation potential: They were able to differentiate into adipocytes, osteoblasts, and chondrocytes in vitro. A clonal assay demonstrated that 61.5% of the SSEA-4+ D-PDL cells had adipogenic, osteogenic, and chondrogenic potential. Our present study demonstrated that SSEA-4+ D-PDL cells are a subset of multipotent stem cells. Hence, SSEA-4 is a specific marker that can be used to identify D-PDL stem cells.

A case of granulosa cell tumor of the ovary detected from metastatic foci.

The authors report a case of granulosa cell tumor of the ovary that followed a rare clinical course, where the primary focus did not appear as a mass, and disseminated foci grew in the abdominal cavity. In 2008, a 70-year-old patient, gravida 6 and para 3, was diagnosed with a perihepatic mass, peritoneal dissemination, and an abdominal wall mass as confirmed by computed tomography (CT) scanning. There was no mass lesion in the pelvis. The pathological diagnosis based on the resected mass in the abdominal wall was malignant mesothelioma. During follow-up, abdominal bloating developed from April 2009. CT scans indicated growth of the intraperitoneal lesions. Therefore, the patient received two cycles of combination therapy with cisplatin and pemetrexed. The treatment was discontinued due to lack of efficacy. The intraperitoneal lesions grew but the clinical course was slow and inconsistent with that of malignant mesothelioma. Central pathological review was requested in April 2011, and a granulosa cell tumor was diagnosed. The patient was referred to the department for detailed examination and treatment. The patient underwent incision of the intraperitoneal tumors, simple total hysterectomy, bilateral salpingo-oophorectomy, and omentectomy. The final pathological diagnosis was normal-size adult-type granulosa cell tumor originating from the left ovary. It was a case of granulosa cell tumor without ovarian enlargement where growth of the metastatic foci was the major observation. As complete surgical resection was achieved and no additional therapy was given, the subject was followed on an outpatient basis and no recurrence was identified.

Input-output relation of FitzHugh-Nagumo elements arranged in a trifurcated structure.

In this study, the propagation of an action potential in a network of excitable elements is studied numerically. The network we consider consists of excitable elements arranged in the shape of a trifurcated structure having three cables. The system has a branch point, a Y junction, at which the three cables are joined. Two types of external stimulations are considered: a single impulsive stimulation at one of the cable terminals, and a pair of stimuli applied to two different terminals. We have found three basic phases depending on the excitability of the elements for a single external stimulus as follows: (1) signal distributor--as the excitability gets higher, the pulse generated by a stimulus splits into two at the branch point, and two pulses are transmitted to the opposite terminals, (2) propagation block--the pulse in the lower excitable chain is blocked at the branch point, and (3) transient propagation--as the excitability is decreased further, we see that the pulse vanishes before reaching the branch point. By the interaction between the pulses that originate from different sources, signal transmission is recovered if the pulses arrive at the branch point nearly synchronously or after a specific delay time. The effects of the repetition of these two types of stimulation are also investigated. Complex spatiotemporal patterns occur due to pulse-pulse interaction and collisions at the branch point. The input-output relationship, which depends crucially on the repetition period and the time lag between the pair of stimuli, is characterized by the stimulus-response ratio and the interspike interval. We also show the effects of noise on the distribution of the interspike interval.

Pair of excitable FitzHugh-Nagumo elements: synchronization, multistability, and chaos.

We analyze a pair of excitable FitzHugh-Nagumo elements, each of which is coupled repulsively. While the rest state for each element is globally stable for a phase-attractive coupling, various firing patterns, including cyclic and chaotic firing patterns, exist in an phase-repulsive coupling region. Although the rest state becomes linearly unstable via a Hopf bifurcation, periodic solutions associated to the firing patterns is not connected to the Hopf bifurcation. This means that the solution branch emanating from the Hopf bifurcation is subcritical and unstable for any coupling strength. Various types of cyclic firing patterns emerge suddenly through saddle-node bifurcations. The parameter region in which different periodic solutions coexist is also found.

Signal propagation and failure in one-dimensional FitzHugh-Nagumo equations with periodic stimuli.

We analyze the effect of additive periodic stimuli in one-dimensional FitzHugh-Nagumo equations in an excitable regime. With a suitable stimulus interval, the suppression of the pulse propagation occurs in some parameter regime. This propagation failure comes from the formation of the "death spot" where successive pulses annihilate. In the parameter regime where the solitary pulse cannot propagate in space stably, however, periodic stimuli cause a propagation of envelope of a traveling pulse under a "resonance" condition, i.e., the pulse at the leading edge disappears successively, however, an envelope is formed and propagates with keeping its shape.

Expression of adrenomedullin and proadrenomedullin N-terminal 20 peptide in PC12 cells after exposure to nerve growth factor.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are multi-functional peptides derived from the same precursor, proadrenomedullin. We have studied the regulatory mechanism of expression of these peptides during neuronal differentiation of rat pheochromocytoma PC12 cells by nerve growth factor (NGF). The cellular levels of the peptides increased slightly, and then progressively decreased below the control by NGF. Immunoreactive (ir)-AM in the medium was transiently increased by NGF. Cytochemical staining showed that ir-AM and ir-PAMP were abundantly present in cytoplasm in the undifferentiated cells, and were decreased during culture with NGF. There was no preferential localization of ir-AM or ir-PAMP in neurites in comparison with in cytoplasm in the differentiated cells. Northern blot analysis showed that mRNA encoding these peptides, as detected as a band of 1.6 kb, increased more than three-fold at 1 h after the addition of NGF and then progressively decreased to one fifth of the control during 72 h. Degradation rate of the mRNA was slowed by NGF even when mRNA level is decreased after 72 h of NGF treatment. The transcription rate of their gene increased transiently and then decreased by the long-term treatment with NGF. These results demonstrate that expression of AM and PAMP is regulated by NGF along with time-dependent differentiation: AM gene transcription is transiently activated by NGF, whereas it was suppressed during neuronal differentiation of the cells.

Induced HMGA1a expression causes aberrant splicing of Presenilin-2 pre-mRNA in sporadic Alzheimer's disease.

The aberrant splicing isoform (PS2V), generated by exon 5 skipping of the Presenilin-2 (PS2) gene transcript, is a diagnostic feature of sporadic Alzheimer's disease (AD). We found PS2V is hypoxia-inducible in human neuroblastoma SK-N-SH cells. We purified a responsible trans-acting factor based on its binding to an exon 5 fragment. The factor was identified as the high mobility group A1a protein (HMGA1a; formerly HMG-I). HMGA1a bound to a specific sequence on exon 5, located upstream of the 5' splice site. HMGA1a expression was induced by hypoxia and the protein was accumulated in the nuclear speckles with the endogenous splicing factor SC35. Overexpression of HMGA1a generated PS2V, but PS2V was repressed by cotransfection with the U1 snRNP 70K protein that has a strong affinity to HMGA1a. HMGA1a could interfere with U1 snRNP binding to the 5' splice site and caused exon 5 skipping. HMGA1a levels were significantly increased in the brain tissue from sporadic AD patients. We propose a novel mechanism of sporadic AD that involves HMGA1a-induced aberrant splicing of PS2 pre-mRNA in the absence of any mutations.

Cerebral endothelial cells are a major source of adrenomedullin.

Adrenomedullin is a peptide hormone with multifunctional biological properties. Its most characteristic effects are the regulation of circulation and the control of fluid and electrolyte homeostasis through peripheral and central nervous system actions. Although adrenomedullin is a vasodilator of cerebral vasculature, and it may be implicated in the pathomechanism of cerebrovascular diseases, the source of adrenomedullin in the cerebral circulation has not been investigated thus far. We measured the secretion of adrenomedullin by radioimmunoassay and detected adrenomedullin mRNA expression by Northern blot analysis in primary cultures of rat cerebral endothelial cells (RCECs), pericytes and astrocytes. We also investigated the expression of specific adrenomedullin receptor components by reverse transcriptase-polymerase chain reaction and intracellular cAMP concentrations in RCECs and pericytes. RCECs had approximately one magnitude higher adrenomedullin production (135 +/- 13 fmol/10(5) cells per 12 h; mean +/- SD, n = 10) compared to that previously reported for other cell types. RCECs secreted adrenomedullin mostly at their luminal cell membrane. Adrenomedullin production was not increased by thrombin, lipopolysaccharide or cytokines, which are known inducers of adrenomedullin release in peripheral endothelial cells, although it was stimulated by astrocyte-derived factors. Pericytes had moderate, while astrocytes had very low basal adrenomedullin secretion. In vivo experiments showed that adrenomedullin plasma concentration in the jugular vein of rats was approximately 50% higher than that in the carotid artery or in the vena cava. Both RCECs and pericytes, which are potential targets of adrenomedullin in cerebral microcirculation, expressed adrenomedullin receptor components, and exhibited a dose-dependent increase in intracellular cAMP concentrations after exogenous adrenomedullin administration. Antisense oligonucleotide treatment significantly reduced adrenomedullin production by RCECs and tended to decrease intraendothelial cAMP concentrations. These findings may suggest an important autocrine and paracrine role for adrenomedullin in the regulation of cerebral circulation and blood-brain barrier functions. Cerebral endothelial cells are a potential source of adrenomedullin in the central nervous system, where adrenomedullin can also be involved in the regulation of neuroendocrine functions.

Adrenomedullin regulates blood-brain barrier functions in vitro.

Adrenomedullin (AM) is an important vasodilator in cerebral circulation, and cerebral endothelial cells are a major source of AM. This in vitro study aimed to determine the AM-induced changes in blood-brain barrier (BBB) functions. AM administration increased, whereas AM antisense oligonucleotide treatment decreased transendothelial electrical resistance. AM incubation decreased BBB permeability for sodium fluorescein (mol. wt 376 Da) but not for Evan's blue albumin (mol. wt 67 kDa), and it also attenuated fluid-phase endocytosis. AM treatment resulted in functional activation of P-glycoprotein efflux pump in vitro. Our results indicate that AM as an autocrine mediator plays an important role in the regulation of BBB properties of the cerebral endothelial cells.

Production of cAMP by adrenomedullin in human oligodendroglial cell line KG1C: comparison with calcitonin gene-related peptide and amylin.

The actions and the presence of adrenomedullin (AM) were investigated in cultured human oligodendroglial cell line KG1C. AM and AM mRNA were detected in KG1C cells by immunohistochemistry and RT-PCR. mRNAs for calcitonin receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) 1, 2 and 3 but not for calcitonin receptors were detected in the cells, while mRNAs for CRLR, calcitonin receptors and all RAMPs were detected in the human cerebellum. Application of AM resulted in time- and concentration-dependent increases in the cAMP level of KG1C cells. Calcitonin gene-related peptide (CGRP) and amylin, peptides structurally related to AM, also increased cAMP. The potencies for the cAMP production of the three peptides were CGRP > or =AM >> amylin with EC(50) of 8, 18, 90 nM, respectively. The responses induced by AM were strongly inhibited by the CGRP(1) receptor antagonist human CGRP(8-37), and inhibited also by the AM receptor antagonist human AM(22-52). In contrast, the responses induced by CGRP or amylin were inhibited only by CGRP(8-37) and not by AM(22-52). The responses induced by all three peptides were unaffected by the amylin receptor antagonist human amylin(8-37). The CGRP(2) receptor agonist human [Cys(Acm)(2,7)]CGRP significantly increased the cAMP level but the increase was smaller than that caused by CGRP. This increase in cAMP was unaffected by CGRP(8-37), AM(22-52) or by amylin(8-37). These results suggest that in KG1C cells, AM increases cAMP through AM and CGRP(1) receptors, whereas CGRP does so through CGRP(1) and CGRP(2) receptors, and amylin exerts its effects through CGRP(1) receptors. Collectively, these findings imply that AM released from oligodendroglial cells may play a role in the regulation of oligodendrocytes via autocrine/paracrine through AM receptors and CGRP(1) receptors.

Effect of hesperetin, a citrus flavonoid, on the liver triacylglycerol content and phosphatidate phosphohydrolase activity in orotic acid-fed rats.

The effect of dietary hesperetin on the hepatic lipid content and the enzyme activities involved in triacylglycerol (TG) synthesis in rats fed diets with or without 1% orotic acid (OA) was studied. Hepatic TG content was raised by approximately 5-fold after administration of OA for 10 days. The OA-feeding significantly increased the activity of hepatic microsomal phosphatidate phosphohydrolase (PAP), which is the rate-limiting enzyme for TG synthesis. Hepatic glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme activities were also increased. An addition of 1% hesperetin to the OA-supplemented diet resulted in the decrease of the hepatic TG content by 44% and of microsomal PAP activity. Dietary hesperetin alone neither affected liver TG content nor PAP activity significantly. OA-feeding caused an increased liver cholesterol level, whereas simultaneous addition of hesperetin and OA reduced its content to the control level. A slight reduction of hepatic cholesterol by hesperetin was also observed in the OA-free dietary group. The present study demonstrated that dietary hesperetin can reduce the hepatic TG accumulation induced by OA, and this was associated with the reduced activity of TG synthetic enzyme, PAP.

Selective expression of aquaporin 1, 4 and 5 in the rat middle ear.

The middle ear cavity is an air-filled space that must be maintained for effective sound transmission to the inner ear. To examine the mechanisms of water homeostasis in the middle ear, we investigated whether aquaporins (AQPs), a family of water-permeable channels, were expressed in the middle ear. Reverse transcription-polymerase chain reaction and immunoblot analyses revealed that mRNAs encoding AQP1, 4 and 5 (but not 2 or 3) subtypes were expressed in rat middle ear epithelium; AQP1, 4 and 5 were detected as 28-, 30- and 30-kDa proteins, respectively. Immunohistochemical analysis showed that AQP1 was localized at capillary endothelial cells and fibroblasts in lamina propria mucosae; AQP4 was present solely at the basolateral membrane of ciliated cells, whereas AQP5 was on the apical surface of ciliated cells as well as of flat and columnar epithelial cells. The characteristic different localizations of AQP1, 4 and 5 subtypes in the middle ear suggest that middle ear water homeostasis requires the coordinated operation of these AQPs.

Neurobiological mechanisms of nicotine craving.

Nicotine induces craving, but the degree of craving is believed to be milder than that with other abused drugs. In this article, the neurobiological mechanisms of craving for nicotine and other drugs are reviewed, focusing especially on three factors that can be involved in the development of craving. The first factor is the affective symptoms of withdrawal, the neural basis of which may involve neuroadaptations (desensitization) within the reward systems. Affective symptoms experienced during withdrawal from nicotine are milder than those experienced in withdrawal from other drugs, probably because of its mode of action on the reward systems, which is similar to that of natural rewards. The second factor is the conditioning process, in which environmental stimuli can gain properties of a secondary reinforcer. Nicotine has weak but reliable conditioning effects, and the brain region mediating those effects of nicotine involves the ventral tegmental area. The third factor is a cognitive (memory) process, but little is known about this area.

Nicotine, alcohol, methamphetamine, and inhalant dependence: a comparison of clinical features with the use of a new clinical evaluation form.

The purpose of the present study was to develop a new clinical evaluation form to compare the clinical features of nicotine dependence with those associated with alcohol, methamphetamine, and inhalant dependence. The clinical evaluation form consisted of six scoring items: subjective effects, tolerance, liking (of drug), social disturbance, withdrawal syndrome, and acute psychic and acute physical disorders. A preliminary clinical investigation was performed to test the validity of the evaluation form. Study subjects were those showing dependence on nicotine (n = 25), alcohol (n = 36), methamphetamine (n = 11), and inhalants (n = 6). All subjects met the Diagnostic and Statistical Manual of Mental Disorders (4th ed.) diagnostic criteria for drug dependence, as defined by the Work Group for the chapter "Substance-Related Disorders": M. A. Schuckit, J. E. Helzer, L. B. Cottler, T. Crowley, P. E. Nathan, & G. E. Woody. Nicotine produced subjective effects, tolerance, liking, and psychic withdrawal symptoms, all of which were mild in degree. However, nicotine did not produce social disturbance, physical withdrawal symptoms, or acute psychic or acute physical disorders. With alcohol, acute psychic and acute physical disorders were prominent, and alcohol also produced a moderate degree of influence on various other items that were evaluated. Methamphetamine produced the most serious acute psychic and acute physical disorders with intensive subjective effects. Inhalants were characterized by an intensive degree of acute psychic disorders and subjective effects with mild withdrawal syndrome. Our study findings revealed that the clinical features of drug dependence could be evaluated by using the new clinical evaluation form. Further study is required to clarify the clinical features of nicotine dependence compared with those of other drugs of dependence.

Possible physiological roles of proteolytic products of actin in neutrophils of patients with Behçet's disease.

A truncated actin with an N-terminus of Met-44 is known to be selectively increased in neutrophils of patients with Behçet's disease and to be generated proteolytically by PMN-elastase (Yamashita S. et al., Biol. Pharm. Bull., 23, 519-522 (2000); Biol. Pharm. Bull., 24, 119-122 (2001)). In this study, the functions of the N-terminal peptide consisting of Asp-2 to Val-43 of beta-actin (42-merP) and the truncated actin with an N-terminus of Met-44 were examined. We first confirmed that the 42-merP existed in the patient plasma. The motility of human peripheral blood neutrophils and neutrophilic granulocytes differentiated from HL-60 cells was suppressed by the 42-merP. Furthermore, when neutrophil-like cells from HL-60 cells were preincubated with 10 nm 42-merP, migration of the cells induced by chemotactic factors such as fMLP and IL-8 was suppressed. The release of PMN-elastase, which is a neutrophil granular enzyme that is responsible for the production of the 42-merP and truncated actin, was suppressed by pretreating the neutrophils with 42-merP before fMLP-stimulation. The truncated actin was unable to polymerize in 0.1 M KCl, suggesting that the increase of truncated actin damages the reconstitution capacity of actin in neutrophils of the patients. These results suggest that the increase of 42-merP and truncated actin in patients with Behçet's disease changes functions of neutrophils

Effects of conjugated linoleic acid on serum leptin concentration, body-fat accumulation, and beta-oxidation of fatty acid in OLETF rats.

We investigated the efficacy of a 4-wk supplementation of conjugated linoleic acid (CLA) as free fatty acid (FFA) or triacylglycerol (TG) on serum leptin concentration, body-fat accumulation, and mitochondrial beta-oxidation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. A significant reduction of serum leptin concentration (42%) and a decrease in the wet weights of perirenal, epididymal, and omental/visceral-adipose tissue in TG-CLA and FFA-CLA groups were found in comparison with the OLETF control group. Both forms of CLA supplementation produced a 5.2% decrease in body weight compared with the control even though food intake was similar in the OLETF groups. Moreover, both forms of CLA enhanced carnitine-palmitoyltransferase activity in brown adipose tissue, perirenal adipose tissue, red gastrocnemius muscle, and liver in comparison with the OLETF control group. Serum concentrations of non-esterified fatty acid and TG also were reduced in rats fed diets supplemented with TG-CLA and FFA-CLA.

Down-regulation of cell surface insulin receptors by sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor in adrenal chromaffin cells.

Long-term (> or =12 h) treatment of cultured bovine adrenal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), caused a time (t(1/2)=16.3 h)- and concentration (IC50=37.8 nM)-dependent decrease of cell surface 125I-insulin binding by 35%, but did not change the Kd value. TG caused a sustained increase of cytoplasmic concentration of Ca2+ ([Ca2+]c) in a biphasic manner, and the effect of TG on 125I-insulin binding was abolished by BAPTA-AM. Western blot analysis showed that TG lowered insulin receptor (IR) beta-subunit level in membrane, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network (TGN), was not changed by TG. These results suggest that inhibition of SERCA by TG and the subsequent increase of [Ca2+]c down-regulates cell surface IR by retarding externalization of IR from the TGN.

Up-regulation of cell surface sodium channels by cyclosporin A, FK506, and rapamycin in adrenal chromaffin cells.

Treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by 56% in a time (t(1/2) = 15.2 h)- and concentration (EC(50) = 2.9 microM)-dependent manner but did not change the K(d) value. In CsA-treated cells, veratridine-induced (22)Na(+) influx was augmented with no change in the EC(50) of veratridine; also, alpha- and beta-scorpion venom and Ptychodiscus brevis toxin-3 enhanced veratridine-induced (22)Na(+) influx in a more than additive manner, as in nontreated cells. CsA treatment for 1 to 24 h inhibited calcineurin activity, measured by the in vitro assay, with the IC(50) of 0.6 microM but did not alter cellular level of calcineurin. FK506 or rapamycin elevated [(3)H]STX binding by 36 or 25%, whereas GPI-1046, an immunophilin ligand incapable to inhibit calcineurin, or okadaic acid, an inhibitor of protein phosphatases 1 and 2A, had no increasing effect. The rise of [(3)H]STX binding by CsA was attenuated by the coincident treatment with brefeldin A (BFA), an inhibitor of vesicular exit from the trans-Golgi network. The internalization rate of cell surface Na(+) channels, as determined in the presence of BFA, was decreased in CsA (but not rapamycin)-treated cells (t(1/2) = 20.3 h), compared with nontreated cells (t(1/2) = 13.7 h). CsA treatment, however, did not elevate cellular levels of Na(+) channel alpha-subunit and Na(+) channel alpha- and beta(1)-subunit mRNAs. In CsA-treated cells, veratridine-induced (45)Ca(2+) influx via voltage-dependent Ca(2+) channels and catecholamine secretion were enhanced, whereas high K(+)-induced (45)Ca(+) influx was not. Thus, the inhibition of calcineurin or rapamycin-binding protein causes up-regulation of cell surface functional Na(+) channels via modulating externalization and internalization of Na(+) channels, thus enhancing Ca(2+) channel gating and catecholamine secretion.

Heterogeneous increases of cytoplasmic calcium: distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels.

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.