Contact-activated migration of melanoma B16 and sarcoma XC cells.

During migration, tumour cells interact with neighbouring neoplastic and normal host cells, and such interaction may influence their motile activity. We investigated the effect of homotypic collisions on the motile activity of two tumour cell lines, mouse melanoma B16 and rat sarcoma XC, and nontransformed human skin fibroblasts. It was found that the tumour cells show only limited motile activity when moving as single cells without contact with neighbours. At a higher density of the culture (and also at a greater number of cell to cell contacts) the activation of motility of investigated tumour cells was observed. On the other hand, the normal human skin fibroblasts showed a typical reaction of density-dependent inhibition of motility. The motile activity of tumour cells was not affected by conditioned media and was visibly dependent on a direct physical contact among colliding cells. The activation of cell movement was observed about 40-50 min after the initial contact between tumour cells. Contact-activated migration of neoplastic cells was inhibited by 50 microM verapamil (a selective voltage-gated calcium channel inhibitor) and 10 microM gadolinium chloride (a nonspecific blocker of mechanosensitive ion channels) but not by pertussis toxin. The observation that homotypic collisions among tumour cells strongly increase their motile activity suggests that contact-activated migration may play a significant role in tumour invasion and metastasis.     

Contact guidance of Walker carcinosarcoma cells by the underlying normal fibroblasts is inhibited by RGD-containing synthetic peptides

The metastatic spread of malignant neoplasms is associated with active migration of cancer cells. The migration of neoplastic cells during the metastatic process may be affected by various extracellular factors, including chemoattractants, haptotactic signals, electric fields, substrate anisotropy, and cell-to-cell contacts. We examined the effect of homotypic collisions and heterotypic interactions with normal human skin fibroblasts on the motile activity of Walker carcinosarcoma cells. It was found that Walker carcinosarcoma cells moving in a dense population neither show contact inhibition of movement when colliding with one another nor increase their motile activity as a result of contact stimulation of motility. On the other hand, when plated onto the surface of aligned fibroblasts, Walker carcinosarcoma cells migrated mainly along the long axes of underlying fibroblasts as a result of contact guidance. The directional character of movement (but not the speed of migration) of Walker carcinosarcoma cells on the surface of aligned fibroblasts was completely effaced by RGD-containing synthetic peptide at a concentration of 1 mg/ml but not by 5 microM verapamil (selective voltage-gated calcium channel inhibitor) or 10 microM gadolinium chloride (non-specific blocker of mechanosensitive ion channels). The suppression of directional character of migration of tumour cells by RGD-containing peptide was associated with the decrease in the amount of fibronectin macromolecules attached to fibroblasts. This suggests that alignment and anisotropic distribution of fibronectin macromolecules may be responsible for contact guidance of tumour cells moving on the surface of fibroblasts.     

Role of Ca2+ in t-PA production by human embryonic lung diploid fibroblast, IMR-90 cells, stimulated by proteose peptone

Proteose peptone (p.peptone) remarkably induced tissue plasminogen activator (t-PA) activity in the conditioned medium of confluently cultured human embryonic lung diploid fibroblast, IMR-90 cells, in a dose-dependent manner. t-PA activity correlated well with the amount of t-PA antigen found in the conditioned medium of IMR-90 cells stimulated by p.peptone. t-PA production by IMR-90 cells stimulated by p.peptone was dependent on extracellular Ca2+ concentration and maximum t-PA production required approximately 3.6 mM extracellular Ca2+. Conversely, elimination of Ca2+ from the culture medium by EGTA, Ca2+ chelate agent, strongly inhibited t-PA production induced by p.peptone. t-PA production induced by p.peptone was inhibited in a dose-dependent manner by Verapamil, which inhibits Ca2+ uptake through the slow channels and also by W-7, an inhibitor of calmodulin. These results suggested that influx of extracellular Ca2+ into IMR-90 cells was caused by p.peptone and induced t-PA production by the cells.     

Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration

Expression of voltage-gated K(+) (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated ([Ca2+](cyt)) following activation of K(+) channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced whole cell K+ currents [I(K(v))] through Kv channels and caused membrane depolarization, which was associated with decreases in ([Ca2+](cyt)), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I(K(v)), E(m), ([Ca2+](cyt)), and RhoA protein and restored cell migration to normal. Elevation of ([Ca2+](cyt)) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C(3) transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.     

Detection of ligand-activated conductive Ca2+ channels in human B lymphocytes

It has been assumed that uptake of extracellular Ca2+ occurs through ligand-activated Ca2+ channels in anti-IgM stimulated human B cells. If so, then uptake should be associated with a depolarizing inward current. Instead, a hyperpolarization due to Ca2+-sensitive K+ conductance is observed. To demonstrate conductive Ca2+ channels in human B lymphocytes, we loaded the cells with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetate (BAPTA), an intracellular Ca2+ chelating agent. This increased the magnitude of the Ca2+ current and delayed the Ca2+-dependent K+ conductance. In BAPTA-loaded B cells suspended in Ca2+-free medium and activated with anti-IgM, reintroduction of Ca2+ resulted in a depolarization that was inhibited by high (microM) concentrations of verapamil and was observed when Ca2+ was replaced by Ba2+ but not Mg2+. These data demonstrate the opening of selective, Ca2+ conductive channels in human B cells following cross-linking of surface immunoglobulins.     

Activity-dependent formation and functions of chondroitin sulfate-rich extracellular matrix of perineuronal nets

Extracellular matrix molecules--including chondroitin sulfate proteoglycans, hyaluronan, and tenascin-R--are enriched in perineuronal nets (PNs) associated with subsets of neurons in the brain and spinal cord. In the present study, we show that similar cell type-dependent extracellular matrix aggregates are formed in dissociated cell cultures prepared from early postnatal mouse hippocampus. Starting from the 5th day in culture, accumulations of lattice-like extracellular structures labeled with Wisteria floribunda agglutinin were detected at the cell surface of parvalbumin-expressing interneurons, which developed after 2-3 weeks into conspicuous PNs localized around synaptic contacts at somata and proximal dendrites, as well as around axon initial segments. Physiological recording and intracellular labeling of PN-expressing neurons revealed that these are large fast-spiking interneurons with morphological characteristics of basket cells. To study mechanisms of activity-dependent formation of PNs, we performed pharmacological analysis and found that blockade of action potentials, transmitter release, Ca2+ permeable AMPA subtype of glutamate receptors or L-type Ca2+ voltage-gated channels strongly decreased the extracellular accumulation of PN components in cultured neurons. Thus, we suggest that Ca2+ influx via AMPA receptors and L-type channels is necessary for activity-dependent formation of PNs. To study functions of chondroitin sulfate-rich PNs, we treated cultures with chondroitinase ABC that resulted in a prominent reduction of several major PN components. Removal of PNs did not affect the number and distribution of perisomatic GABAergic contacts but increased the excitability of interneurons in cultures, implicating the extracellular matrix of PNs in regulation of interneuronal activity.     

The small leucine-rich proteoglycan lumican inhibits melanoma progression

Lumican is a member of the small leucine-rich proteoglycan (SLRP) family. It contributes to the organisation of the collagen network and plays an important role in cell migration and tissue repair. The present study aimed to determine the influence of lumican expression on adhesion, anchorage-dependent and -independent growth, migration, in vitro invasion and in vivo melanoma growth. For that purpose, B16F1 mouse melanoma cells were stably transfected with an expression plasmid containing the complete lumican cDNA. Lumican expression by tumor cells did not change the proliferative activity of mouse melanoma cells in monolayer culture and did not influence either cell adhesion to extracellular matrix gel or type I collagen or cell spreading on these substrates. In contrast, lumican-transfected cells were characterized by a strong reduction of their anchorage-independent proliferation in agarose gel and capacity to invade extracellular matrix gel. After subcutaneous injections of transfected B16F1 cells in syngenic mice, lumican expression significantly decreased subcutaneous tumor formation in vivo, with a concomitant decrease of cyclin D1 expression. Lumican induced and/or increased the apoptosis of B16F1 cells. The results suggest that lumican is involved in the control of melanoma growth and invasion and may be considered, like decorin, as an anti-tumor factor from the extracellular matrix.     

Voltage-dependent -L-type Ca2+ channels participate in regulating neural crest migration and differentiation.

General models of cell activation implicate Ca2+ conductance as pivotal in conveying transmembrane signals. During embryonic development, both cell migration and differentiation are influenced by changes in Ca2+; and, as a consequence, the modulation of Ca2+ is important in the control of many morphogenetic processes. Because Ca2+ conductance may be regulated at voltage-dependent Ca2+ channels (VD-CCs), we investigated whether neural crest cells develop VDCCs and, if so, whether they function in regulating migration and establishing cytomorphology. Autoradiography indicates that neural crest cells in vitro develop -L-type Ca2+ channels during migration and differentiation. Blockage of these channels by verapamil, both in vivo and in vitro, leads to a dramatic and reversible inhibition of neural crest migration. Alterations are manifest in vitro in cell-to-cell and cell-to-substratum contact and in the organization of the actin cytoskeleton. In whole embryos, verapamil or nifedipine inhibits pigment pattern formation. Moreover, blockage of the -L-type Ca2+ channels in whole embryos or cultures, after cells have already migrated and differentiated, results in a significant change in individual cell shape and in the overall pigment cell pattern, suggesting further that maintenance of the differentiated state also requires regulation at the -L-type Ca2+ channel. Since certain aspects of neural crest adhesion and cytoskeletal function are dependent on Ca2+, it is suggested that interactions that regulate the availability of Ca2+ through the VDCC may provide coordinate control of motile and adhesive interactions at the cell-substratum interface.     

Acidic extracellular pH increases calcium influx-triggered phospholipase D activity along with acidic sphingomyelinase activation to induce matrix metalloproteinase-9 expression in mouse metastatic melanoma

Acidic extracellular pH is a common feature of tumor tissues. We have reported that culturing cells at acidic pH (5.4-6.5) induced matrix metalloproteinase-9 expression through phospholipase D, extracellular signal regulated kinase 1/2 and p38 mitogen-activated protein kinases and nuclear factor-kappaB. Here, we show that acidic extracellular pH signaling involves both pathways of phospholipase D triggered by Ca2+ influx and acidic sphingomyelinase in mouse B16 melanoma cells. We found that BAPTA-AM [1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester], a chelator of intracellular free calcium, and the voltage dependent Ca2+ channel blockers, mibefradil (for T-type) and nimodipine (for L-type), dose-dependently inhibited acidic extracellular pH-induced matrix metalloproteinase-9 expression. Intracellular free calcium concentration ([Ca2+]i) was transiently elevated by acidic extracellular pH, and this [Ca2+]i elevation was repressed by EGTA and the voltage dependent Ca2+ channel blockers but not by phospholipase C inhibitor, suggesting that acidic extracellular pH increased [Ca2+]i through voltage dependent Ca2+ channel. In contrast, SR33557, an L-type voltage dependent Ca2+ channel blocker and acidic sphingomyelinase inhibitor, attenuated matrix metalloproteinase-9 induction but did not affect calcium influx. We found that acidic sphingomyelinase activity was induced by acidic extracellular pH and that the specific acidic sphingomyelinase inhibitors (perhexiline and desipramine) and siRNA targeting aSMase/smpd1 could inhibit acidic extracellular pH-induced matrix metalloproteinase-9 expression. BAPTA-AM reduced acidic extracellular pH-induced phospholipase D but not acidic sphingomyelinase acitivity. The acidic sphingomyelinase inhibitors did not affect the phosphorylation of extracellular signal regulated kinase 1/2 and p38, but they suppressed nuclear factor-kappaB activity. These data suggest that the calcium influx-triggered phospholipase D and acidic sphingomyelinase pathways of acidic extracellular pH induced matrix metalloproteinase-9 expression, at least in part, through nuclear factor-kappaB activation.     

A calcium dependent de-adhesion mechanism regulates the direction and rate of cell migration: a mathematical model

Cell migration has long been studied by a variety of techniques and many proteins have been implicated in its regulation. Integrins, key proteins that link the cell to the extracellular matrix, are central to adhesion complexes whose turnover defines the rate of cell locomotion. The formation and disassembly of these adhesions is regulated by both intracellular and extracellular factors. In this study we have focused on the Ca2+-dependent protein network (module) that disassembles the adhesion complexes. We have developed a mathematical model that includes the Ca2+-dependent enzymes micro-calpain and phospholipase C (PLC) as well as IP3 receptors and stretch activated Ca2+ channels, all of which have been reported to regulate migration. The model also considers the spatial effects of Ca2+ propagation into lamella. Our model predicts differential activation of calpain at the leading and trailing edges of the cell. Since disassembly of integrin adhesive contacts is proportional to the degree of calpain activation, this leads to cell migration in a preferred direction. We show how the dynamics of Ca2+ spiking affects calpain activation and thus changes the disassembly rate of adhesions. The spiking is controlled by PLC activity and currents through stretch-activated Ca2+ channels. Our model thus combines the effects of various molecular factors and leads to a consistent explanation of the regulation of the rate and direction of cell migration.     

Integrin and Cadherin Synergy Regulates Contact Inhibition of Migration and Motile Activity

Integrin receptors play a central role in cell migration through their roles as adhesive receptors for both other cells and extracellular matrix components. In this study, we demonstrate that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of cell migration. In addition to promoting proliferation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A. Horwitz. 1996. J. Cell Biol. 133:169–184), ectopic expression of the α5 integrin in cultures of primary quail myoblasts promotes a striking contact-mediated inhibition of cell migration. Myoblasts ectopically expressing α5 integrin (α5 myoblasts) move normally when not in contact, but upon contact, they show inhibition of migration and motile activity (i.e., extension and retraction of membrane protrusions). As a consequence, these cells tend to grow in aggregates and do not migrate to close a wound. This phenotype is also seen with ectopic expression of β1 integrin, paxillin, or activated FAK (CD2 FAK) and therefore appears to result from enhanced integrin-mediated signaling. The contact inhibition observed in the α5 myoblasts is mediated by N-cadherin, whose expression is upregulated more than fivefold. Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type and mutant N-cadherins all implicate N-cadherin in the contact inhibition of migration. Ectopic expression of N-cadherin also produces cells that show inhibited migration upon contact; however, they do not show suppressed motile activity, suggesting that integrins and cadherins coordinately regulate motile activity. These observations have potential importance to normal and pathologic processes during embryonic development and tumor metastasis.     

The role of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion

The biochemical and biophysical roles of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion were studied. (1) Various kinds of cell, such as Ehrlich ascites tumor cells, mouse melanoma cells (B16-CW1 cells) and human epidermoid carcinoma cells (KB cells), could fuse in Ca2+-free medium containing a cheletor, glycoletherdiaminetetraacetic acid, in the same way as in Ca2+-containing medium. (2) The ATP content in Ehrlich ascites tumor cells decreased rapidly when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (3) Intracellular adenine nucleotides leaked out into the reaction medium when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (4) On addition of the virus, O2 consumption of Ehrlich ascites tumor cells decreased in Ca2+-free medium, but not in Ca2+-containing medium. (5) HVJ (Sendai virus) did not affect production of lactate by Ehrlich ascites tumor cells in both Ca2+-free medium and Ca2+-containing medium. These observations suggest that the role of extracellular Ca2+ in virus-induced cell fusion is to maintain the ATP and other intracellular metabolite contents at normal levels instead of triggering the fusion reaction itself.     

Ca(2+)-inactivated K+ current is modulated by endothelin-1 in B-16 murine melanoma cells

It is well established that endothelin-1 (ET-1) plays a role in differentiation and proliferation in a variety of cells such as fibroblasts and human melanoma cells via a receptor-mediated mechanism. However, whether ET-1 modulates ion channel activity in these cell types is still unknown. In this report, we recorded the voltage-dependent outward K+ current in cultured B16 melanoma cells using the patch-clamp technique. Biophysical and pharmacological properties of the K+ current, and the effect of ET-1 on the K+ current were investigated. When cells were loaded with a Ca(2+)-chelating agent (EGTA or BAPTA), the K+ current amplitude gradually increased with time after establishment of the whole cell configuration. Replacement of Ca2+ with Co2+ in the extracellular medium caused no significant modulation of the K+ current amplitude. Addition of BaCl2 or quinidine to the extracellular solution reduced the K+ current amplitude, whereas the K+ current was insensitive to tetraethylammonium. ET-1 (10 nM) reversibly decreased the K+ current amplitude and accelerated the decay of the K+ current. The ET-1-induced inhibitory effect displayed no desensitization following repeated ET-1 application. Pretreatment with pertussis toxin (PTX) or perfusion of cells with the protein kinase C (PKC) inhibitor H-7 abolished the inhibitory effect of ET-1 on the K+ current. We conclude that the outward K+ current recorded in murine B-16 melanoma cells represents a Ca(2+)-inactivated K+ current, and that the inhibitory effect of ET-1 on the K+ current may reveal a novel mechanism to control the differentiation and proliferation of melanoma cells.     

Effect of UV-B irradiation on release of arachidonic acid from B-16 melanoma cells

B-16 melanoma cells in culture were prelabeled with (3H)-arachidonate, and exposed to UV radiation. Immediately after irradiation the cells released labeled materials. This UV-stimulated release was inhibited by mepacrine (20 microM) and calmodulin inhibitor W7 (0.5 microM). To determine the influence of extracellular Ca2+ on the UV-stimulated release, experiments were made with media containing various concentrations of Ca2+. The release decreased significantly at lower Ca2+ concentrations. These results suggest that Ca2+-calmodulin-dependent phospholipase A2 was involved in UV-stimulated release of radiolabeled materials, possibly arachidonic acid and its metabolites, from the cells.     

Epidermal growth factor-stimulated DNA synthesis requires an influx of extracellular calcium

The dependency of normal cell proliferation on adequate extracellular Ca2+ levels was further investigated by determining the role of Ca2+ influx in epidermal growth factor (EGF)-induced rat liver epithelial (T51B) cell DNA synthesis. Fura-2-loaded T51B cells responded with an increase in [Ca2+]i to EGF (5-50 ng/ml) that was blocked by low (25 microM) extracellular Ca2+ or by pretreatment with 50 microM La3+ to inhibit plasma membrane Ca2+ flux. Confluent T51B cells treated for 24 h with EGF (0.1-50 ng/ml) dose-dependently incorporated [3H]-thymidine into cell nuclei. Low extracellular Ca2+ or addition of La3+ prevented the EGF-stimulated rise in labeled nuclei, indicating that a movement of Ca2+ into the cell was required for DNA synthesis. This was supported by our findings that bradykinin, which induced a rise in [Ca2+]i by opening plasma membrane Ca2+ channels in T51B cells (but not A23187, thrombin or ATP, which raise [Ca2+]i primary through mobilization of intracellular Ca2+ stores), potentiated DNA synthesis stimulated by submaximal doses of EGF. Potentiation of the action of EGF by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), indicates that activation of protein kinase C and an influx of Ca2+ share a common mechanism for initiating DNA synthesis.     

Dopamine inhibits calcium flux in the 7315a prolactin-secreting pituitary tumour

Cells of the 7315a prolactin-secreting tumour express biochemically normal cell-surface receptors for dopamine. However, dopamine inhibits prolactin release from these cells only when the basal rate of prolactin release is augmented by increasing the intracellular and/or extracellular calcium concentration of the tumour cells. This suggests that dopaminergic modulation of calcium ion flux could have a central physiological role in these neoplastic cells. In 7315a cells we examined the ability of dopamine to regulate 45Ca2+ influx and fractional 45Ca2+ efflux under conditions of enhanced calcium flux using the calcium channel activator, maitotoxin. It was observed that unidirectional calcium influx stimulated by maitotoxin was significantly inhibited by dopamine. Maitotoxin stimulated fractional efflux and prolactin release from the tumour cells and dopamine simultaneously inhibited both processes by a haloperidol-reversible mechanism. Therefore, in 7315a cells dopamine receptor activation is coupled to inhibition of calcium flux as at least one component in the regulation of prolactin release. These cells may provide further opportunity to study intracellular signalling mechanisms that are modulated by dopamine receptor activity.     

A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration

Cell migration relies on a tight temporal and spatial regulation of the intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in turn depends on Ca2+ influx via channels in the plasma membrane whose molecular nature is still largely unknown for migrating cells. A mechanosensitive component of the Ca2+ influx pathway was suggested. We show here that the capsaicin-sensitive transient receptor potential channel TRPV1, that plays an important role in pain transduction, is one of the Ca2+ influx channels involved in cell migration. Activating TRPV1 channels with capsaicin leads to an acceleration of human hepatoblastoma (HepG2) cells pretreated with hepatocyte growth factor (HGF). The speed rises by up to 50% and the displacement is doubled. Patch clamp experiments revealed the presence of capsaicin and resiniferatoxin (RTX)-sensitive currents. In contrast, HepG2 cells kept in the absence of HGF are not accelerated by capsaicin and express no capsaicin- or RTX-sensitive current. The TRPV1 antagonist capsazepine prevents the stimulation of migration and inhibits capsaicin-sensitive currents. Finally, we compared the contribution of capsaicin-sensitive TRPV1 channels to cell migration with that of mechanosensitive TRPV4 channels that are also expressed in HepG2 cells. A specific TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, does not increase the displacement. In summary, we assigned a novel role to capsaicin-sensitive TRPV1 channels. They are important Ca2+ influx channels required for cell migration.     

Functional expression of voltage-gated calcium channels in human melanoma

The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression.     

Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.     

Inhibition of inositol trisphosphate-stimulated calcium mobilization by calmodulin antagonists in rat liver epithelial cells

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), an intracellular second messenger produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate, interacts with cytoplasmic membrane structures to elicit the release of stored Ca2+. Ins(1,4,5)P3-induced Ca2+ mobilization is mediated through high affinity receptor binding sites; however, the biochemical mechanism coupling receptor occupation with Ca2+ channel opening has not been identified. In studies presented here, we examined the effects of naphthalenesulfonamide calmodulin antagonists, W7 and W13, and a new selective antagonist, CGS 9343B, on Ca2+ mobilization stimulated by Ins(1,4,5)P3 in neoplastic rat liver epithelial (261B) cells. Intact fura-2 loaded cells stimulated by thrombin, a physiological agent that causes phosphatidylinositol 4,5-bisphosphate hydrolysis and Ins (1,4,5)P3 release, responded with a rise in cytoplasmic free Ca2+ levels that was dose dependently inhibited by W7(Ki = 25 microM), W13 (Ki = 45 microM), and CGS 9343B (Ki = 110 microM). Intracellular Ca2+ release stimulated by the addition of Ins(1,4,5)P3 directly to electropermeabilized 261B cells was similarly inhibited by pretreatment with anti-calmodulin agents. W7 and CGS 9343B, which potently blocked Ca2+/calmodulin-dependent protein kinase, had no significant effect on protein kinase A or C in dose range required for complete inhibition of Ca2+ mobilization. Ca2+ release channels and Ca2+-ATPase pump activity were also unaffected by calmodulin antagonist treatment. These results indicate that calmodulin is tightly associated with the intracellular membrane mechanism coupling Ins(1,4,5)P3 receptors to Ca2+ release channels