L-type Voltage-sensitive Ca2+ Channels Research Articles

Grass Carp Prolactin Gene: Structural Characterization and Signal Transduction for PACAP-induced Prolactin Promoter Activity

In this study, structural analysis of grass carp prolactin (PRL) gene was performed and the signaling mechanisms for pituitary adenylate cyclase-activating peptide (PACAP) regulation of PRL promoter activity were investigated. In αT3-1 cells, PRL promoter activity could be induced by oPACAP38 which was blocked by PACAP antagonist but not the VIP antagonist. The stimulatory effect of oPACAP38 was mimicked by activation of AC/cAMP and voltage-sensitive Ca2+ channel (VSCC) signaling, or induction of Ca2+ entry. In parallel, PACAP-induced PRL promoter activity was negated or inhibited by suppressing cAMP production, inhibiting PKA activity, removal of extracellular Ca2+, VSCC blockade, calmodulin (CaM) antagonism, and inactivation of CaM kinase II. Similar sensitivity to L-type VSCC, CaM and CaM kinase II inhibition were also observed by substituting cAMP analog for oPACAP38 as the stimulant for PRL promoter activity. Moreover, PACAP-induced PRL promoter activity was also blocked by inhibition of PLC signaling, attenuation of [Ca2+]i immobilization via IP3 receptors, and blockade of PI3K/P70S6K pathway. The PACAP-induced PRL promoter activation may involve transactivation of the transcription factor CREB. These results suggest that PACAP can stimulate PRL promoter activation by PAC1 mediated functional coupling of the Ca2+/CaM/CaM kinase II cascades with the AC/cAMP/PKA pathway. Apparently, other signaling pathways, including PLC/IP3 and PI3K/P70S6K cascades, may also be involved in PACAP induction of PRL gene transcription.

PKC-dependent regulation of Kv7.5 channels by the bronchoconstrictor histamine in human airway smooth muscle cells.

Kv7 potassium channels have recently been found to be expressed and functionally important for relaxation of airway smooth muscle. Previous research suggests that native Kv7 currents are inhibited following treatment of freshly isolated airway smooth muscle cells with bronchoconstrictor agonists, and in intact airways inhibition of Kv7 channels is sufficient to induce bronchiolar constriction. However, the mechanism by which Kv7 currents are inhibited by bronchoconstrictor agonists has yet to be elucidated. In the present study, native Kv7 currents in cultured human trachealis smooth muscle cells (HTSMCs) were observed to be inhibited upon treatment with histamine; inhibition of Kv7 currents was associated with membrane depolarization and an increase in cytosolic Ca2+ ([Ca2+]cyt). The latter response was inhibited by verapamil, a blocker of L-type voltage-sensitive Ca2+ channels (VSCCs). Protein kinase C (PKC) has been implicated as a mediator of bronchoconstrictor actions, although the targets of PKC are not clearly established. We found that histamine treatment significantly and dose-dependently suppressed currents through overexpressed wild-type human Kv7.5 (hKv7.5) channels in cultured HTSMCs, and this effect was inhibited by the PKC inhibitor Ro-31-8220 (3 µM). The PKC-dependent suppression of hKv7.5 currents corresponded with a PKC-dependent increase in hKv7.5 channel phosphorylation. Knocking down or inhibiting PKCα, or mutating hKv7.5 serine 441 to alanine, abolished the inhibitory effects of histamine on hKv7.5 currents. These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca2+ influx via L-type VSCCs as a mechanism for histamine-induced bronchoconstriction.

Fluid shear stress induces calcium transients in osteoblasts through depolarization of osteoblastic membrane

Intracellular calcium transient ([Ca2+]i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca2+]i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca2+]i transient in osteoblasts. Fluorescent dyes DiBAC4(3) and fura-2AM were respectively used to detect membrane potential and [Ca2+]i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca2+]i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca2+]i transients. Replacing extracellular Na+ with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca2+]i transients. However, voltage-activated K+ channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca2+ or blocking of L-type voltage-sensitive Ca2+ channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca2+]i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was −43.3mV and the depolarization induced by FSS was about 44mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca2+ mobilization wherein FSS activated SACs to promote Na+ entry to depolarize membrane that, in turn, activated L-VSCCs and Ca2+ influx though L-VSCCs switched on [Ca2+]i response in osteoblasts.

Reduction in neuronal L-type calcium channel activity in a double knock-in mouse model of Alzheimer's disease

Increased function of neuronal L-type voltage-sensitive Ca2+ channels (L-VSCCs) is strongly linked to impaired memory and altered hippocampal synaptic plasticity in aged rats. However, no studies have directly assessed L-VSCC function in any of the common mouse models of Alzheimer's disease where neurologic deficits are typically more robust. Here, we used cell-attached patch-clamp recording techniques to measure L-VSCC activity in CA1 pyramidal neurons of partially dissociated hippocampal “zipper” slices prepared from 14-month-old wild-type mice and memory-impaired APP/PS1 double knock-in mice. Surprisingly, the functional channel density of L-VSCCs was significantly reduced in the APP/PS1 group. No differences in voltage dependency and unitary conductance of L-VSCCs were observed. The results suggest that mechanisms for Ca2+ dysregulation can differ substantially between animal models of normal aging and models of pathological aging.

Fibroblast growth factor 2 induces apoptosis in the early primary culture of rat cortical neurons

In the central nervous system, fibroblast growth factor 2 (FGF2) is known to have important functions in cell survival and differentiation. In addition to its roles as a neurotrophic factor, we found that FGF2 caused cell death in the early primary culture of cortical neurons. FGF2-induced neuronal cell death showed apoptotic characters, e.g., chromatin condensation and DNA fragmentation. The ultrastructural morphology of FGF2-treated neurons indicated apoptotic features such as progressive cell shrinkage, blebbing of the plasma membrane, loss of cytosolic organelles, clumping of chromatin, and fragmentation of DNA. Tyrosine kinase inhibitors significantly rescued neurons from FGF2-induced apoptosis. FGF2 potentiated a marked influx of Ca 2+ into neurons before apoptosis. Both a calcium chelator and L-type voltage-sensitive Ca 2+ channel (L-VSCC) blockers attenuated FGF2-induced apoptosis, whereas other blockers of VSCCs such as N-type and P/Q-types did not. Blockers of L-VSCCs significantly suppressed FGF2-enhanced Ca 2+ influx into neurons. Moreover, FGF2 also generated reactive oxygen species (ROS) before apoptosis. Radical scavengers reduced not only the FGF2-generated ROS, but also the FGF2-induced Ca 2+ influx and apoptosis. In conclusion, we demonstrated that FGF2 caused apoptosis via L-VSCCs in the early neuronal culture.

Ghrelin stimulation of gonadotropin (LH) release from goldfish pituitary cells: Presence of the growth hormone secretagogue receptor (GHS-R1a) and involvement of voltage-sensitive Ca 2+ channels

Ghrelin (GRLN) stimulates maturational gonadotropin (LH) secretion in goldfish. This study identified GRLN receptors (GHS-Rs) in goldfish tissues and examined the involvement of voltage-sensitive Ca 2+ channels (VSCCs) in ghrelin action. A partial goldfish GHS-R1a sequence was obtained and expression observed in brain, pituitary, spleen, kidney, heart, gill, ovary, testis, and intestine. Synthetic goldfish GRLN (gGRLN 19) stimulated LH secretion from dispersed goldfish pituitary cells in column perifusion and increased intracellular Ca 2+ ([Ca 2+] i) in identified goldfish gonadotropes. gGRLN 19 did not stimulate LH secretion either in the presence of Ca 2+-free media, or the L-type VSCC inhibitors nifedipine and verapamil. Similarly, gGRLN 19-elicited increases in [Ca 2+] i were attenuated by Ca 2+-free media and nifedipine. Furthermore, when LH release and [Ca 2+] i were elevated by Bay K8644, gGRLN 19 had no further effect. These results indicate that GHS-R1a is present in goldfish pituitary and Ca 2+ entry through VSCC mediates direct gGRLN 19 action on LH release in goldfish pituitary cells.

Dynamic interactions between L-type voltage-sensitive calcium channel Cav1.2 subunits and ahnak in osteoblastic cells.

Voltage-sensitive Ca(2+) channels (VSCCs) mediate Ca(2+) permeability in osteoblasts. Association between VSCC alpha(1)- and beta-subunits targets channel complexes to the plasma membrane and modulates function. In mechanosensitive tissues, a 700-kDa ahnak protein anchors VSCCs to the actin cytoskeleton via the beta(2)-subunit of the L-type Ca(v)1.2 (alpha(1C)) VSCC complex. Ca(v)1.2 is the major alpha(1)-subunit in osteoblasts, but the cytoskeletal complex and subunit composition are unknown. Among the four beta-subtypes, the beta(2)-subunit and, to a lesser extent, the beta(3)-subunit coimmunoprecipitated with the Ca(v)1.2 subunit in MC3T3-E1 preosteoblasts. Fluorescence resonance energy transfer revealed a complex between Ca(v)1.2 and beta(2)-subunits and demonstrated their association in the plasma membrane and secretory pathway. Western blot and immunohistochemistry showed ahnak association with the channel complex in the plasma membrane via the beta(2)-subunit. Cytochalasin D exposure disrupted the actin cytoskeleton but did not disassemble or disrupt the function of the complex of L-type VSCC Ca(v)1.2 and beta(2)-subunits and ahnak. Similarly, small interfering RNA knockdown of ahnak did not disrupt the actin cytoskeleton but significantly impaired Ca(2+) influx. Collectively, we showed that Ca(v)1.2 and beta(2)-subunits and ahnak form a stable complex in osteoblastic cells that permits Ca(2+) signaling independently of association with the actin cytoskeleton.

Effects of zonisamide on neurotransmitter release associated with inositol triphosphate receptors

To clarify the antiepileptic mechanisms of zonisamide (ZNS), we determined the interaction between ZNS and inositol-1,4,5-triphosphate receptor (IP3R) on exocytosis of GABA and glutamate in rat frontal cortex using microdialysis. ZNS increased basal GABA release, but not glutamate, concentration-dependently, and reduced concentration-dependently K +-evoked GABA and glutamate releases. Inhibition and activation of IP3R reduced and enhanced basal and K +-evoked GABA releases, respectively. The K +-evoked glutamate release was reduced and enhanced by IP3R antagonist and agonist, respectively, whereas basal glutamate release was increased by IP3R agonist but not affected by IP3R antagonist. Under extracellular Ca 2+ depletion, IP3R agonist increased basal GABA and glutamate releases. The latter effects of IP3R agonist were weakly enhanced by ZNS, but such stimulatory action of ZNS was abolished by extracellular Ca 2+ depletion. In contrast, ZNS inhibited the stimulatory effect of IP3R agonist on K +-evoked release. The stimulatory effect of IP3R agonist on basal release was regulated by N-type voltage-sensitive Ca 2+ channel (VSCC) rather than P- and L-type VSCCs, whereas the stimulatory effect of IP3R agonist on K +-evoked release was regulated by P- and L-type VSCCs rather than N-type VSCC. These results suggest that ZNS-activated N-type VSCC enhances IP3R-associated neurotransmitter release during resting stage, whereas ZNS-induced suppression of P- and L-type VSCCs possibly attenuates IP3R-associated neurotransmitter release during neuronal hyperexcitability. Therefore, the combination of both of these two actions of ZNS on IP3R-associated neurotransmitter release mechanism seems to be involved, at least in part, in the mechanisms of antiepileptic and neuroprotective actions of ZNS.

Ghrelin-induced growth hormone release from goldfish pituitary cells involves voltage-sensitive calcium channels

Ghrelin (GRL) is a stimulator of growth hormone (GH) release in many organisms, including goldfish. As a first study to examine the signalling mechanisms mediating GRL action on GH release in goldfish, we tested the hypothesis that GLR induces GH release from goldfish pituitary cells by enhancing Ca 2+ entry through L-type voltage-sensitive Ca 2+ channels (LVSCCs) using perifusion GH release and fura-2/AM Ca 2+-imaging experiments. Goldfish (g)GRL 19 at 1 nM elicited reversible and repeatable GH responses from dispersed goldfish mixed pituitary cultures. However, the lack of a dose-response relationship in sequential treatments with decreasing concentrations of gGRL 19 (ranging from 10 to 0.01 nM) implicated rapid desensitization of the GH response. Sequential applications of gGRL 19 (1 nM) and salmon GnRH (100 nM), a known Ca 2+-dependent stimulator of GH release, increased intracellular free Ca 2+ levels ([Ca 2+] i) from the same identified somatotropes, suggesting co-expression of GRL and GnRH receptors on single cells. In contrast, 1 nM gGRL 19 failed to elicit GH release and elevation in [Ca 2+] i when the cells are incubated with nominally Ca 2+-free media. When GH release and [Ca 2+] i increases were already stimulated by the LVSCC agonist Bay K8644 (10 μM), addition of 1 nM gGRL 19 did not further elevate these responses. Finally, the LVSCC inhibitors nifedipine (1 μM) and verapamil (1 μM) abolished 1 nM gGRL 19-induced GH release responses while nifedipine eliminated gGRL 19-induced [Ca 2+] i increase. Taken together, the results of this study provide evidence that entry of extracellular Ca 2+ through LVSCCs is a key component of the GRL signalling pathway leading to GH release in the goldfish pituitary.

Cocaine Regulates MEF2 to Control Synaptic and Behavioral Plasticity

Repeated exposure to cocaine causes sensitized behavioral responses and increased dendritic spines on medium spiny neurons of the nucleus accumbens (NAc). We find that cocaine regulates myocyte enhancer factor 2 (MEF2) transcription factors to control these two processes in vivo. Cocaine suppresses striatal MEF2 activity in part through a mechanism involving cAMP, the regulator of calmodulin signaling (RCS), and calcineurin. We show that reducing MEF2 activity in the NAc in vivo is required for the cocaine-induced increases in dendritic spine density. Surprisingly, we find that increasing MEF2 activity in the NAc, which blocks the cocaine-induced increase in dendritic spine density, enhances sensitized behavioral responses to cocaine. Together, our findings implicate MEF2 as a key regulator of structural synapse plasticity and sensitized responses to cocaine and suggest that reducing MEF2 activity (and increasing spine density) in NAc may be a compensatory mechanism to limit long-lasting maladaptive behavioral responses to cocaine.

Hippocampal ‘zipper’ slice studies reveal a necessary role for calcineurin in the increased activity of L-type Ca 2+ channels with aging

Previous studies have shown that inhibition of the Ca 2+-/calmodulin-dependent protein phosphatase calcineurin (CN) blocks L-type voltage sensitive Ca 2+ channel (L-VSCC) activity in cultured hippocampal neurons. However, it is not known whether CN contributes to the increase in hippocampal L-VSCC activity that occurs with aging in at least some mammalian species. It is also unclear whether CN's necessary role in VSCC activity is simply permissive or is directly enhancing. To resolve these questions, we used partially dissociated hippocampal “zipper” slices to conduct cell-attached patch recording and RT-PCR on largely intact single neurons from young-adult, mid-aged, and aged rats. Further, we tested for direct CN enhancement of L-VSCCs using virally mediated infection of cultured neurons with an activated form of CN. Similar to previous work, L-VSCC activity was elevated in CA1 neurons of mid-aged and aged rats relative to young adults. The CN inhibitor, FK-506 (5 μM) completely blocked the aging-related increase in VSCC activity, reducing the activity level in aged rat neurons to that in younger rat neurons. However, aging was not associated with an increase in neuronal CN mRNA expression, nor was CN expression correlated with VSCC activity. Delivery of activated CN to primary hippocampal cultures induced an increase in neuronal L-VSCC activity but did not elevate L-VSCC protein levels. Together, the results provide the first evidence that CN activity, but not increased expression, plays a selective and necessary role in the aging-related increase in available L-VSCCs, possibly by direct activation. Thus, these studies point to altered CN function as a novel and potentially key factor in aging-dependent neuronal Ca 2+ dysregulation.

Carbamazepine prevents breakdown of neurotransmitter release induced by hyperactivation of ryanodine receptor

To clarify the mechanisms of the pharmacological action of carbamazepine (CBZ), we determined the effect of CBZ on GABA and glutamate release associated with the ryanodine receptor (Ryr)-sensitive Ca 2+-induced Ca 2+-releasing system (CICR) in the rat hippocampus using microdialysis. The therapeutically relevant concentration of CBZ increased basal GABA release without affecting basal glutamate release; however, K +-evoked releases were concentration-dependently reduced by CBZ. Lower-concentration ryanodine increased basal and K +-evoked releases of GABA and glutamate in a concentration dependent manner, whereas higher-concentration ryanodine reduced them. These inflection points in the concentration-response curves of ryanodine for neurotransmitter release (critical concentrations) were shifted to the left by K +-evoked stimulation. The critical concentration of ryanodine in GABA release was lower than that in glutamate release. During the resting stage, the critical concentrations of ryanodine were unaffected by inhibition of L-type, N-type and P-type voltage-sensitive Ca 2+ channels (VSCCs) but were prevented by CBZ; however, during the neuronal hyperexcitable stage, the critical concentration was increased by CBZ, L-type and P-type VSCC inhibitors but not the N-type VSCC inhibitor. Therefore, a therapeutically relevant concentration of CBZ protects against the breakdown of the neurotransmitter release mechanism induced by hyperactivation of Ryr via inhibition of L-type and P-type VSCCs as well as inhibition of Ryr-sensitive CICR. These actions of CBZ appear to be involved, at least partially, in its anti-seizure mechanisms.

Signaling mechanisms for α2-adrenergic inhibition of PACAP-induced growth hormone secretion and gene expression grass carp pituitary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent growth hormone (GH)-releasing factor in lower vertebrates. However, its functional interactions with other GH regulators have not been fully characterized. In fish models, norepinephrine (NE) inhibits GH release at the pituitary cell level, but its effects on GH synthesis have yet to be determined. We examined adrenergic inhibition of PACAP-induced GH secretion and GH gene expression using grass carp pituitary cells as a cell model. Through activation of pituitary alpha2-adrenoreceptors, NE or the alpha2-agonist clonidine reduced both basal and PACAP-induced GH release and GH mRNA expression. In carp pituitary cells, clonidine also suppressed cAMP production and intracellular Ca2+ levels and blocked PACAP induction of these two second messenger signals. In GH3 cells transfected with a reporter carrying the grass carp GH promoter, PACAP stimulation increased GH promoter activity, and this stimulatory effect could be abolished by NE treatment. In parallel experiments, clonidine reduced GH primary transcript and GH promoter activity without affecting GH mRNA stability, and these inhibitory actions were mimicked by inhibiting adenylate cyclase (AC), blocking protein kinase A (PKA), removing extracellular Ca2+ in the culture medium, or inactivating L-type voltage-sensitive Ca2+ channels (VSCC). Since our recent studies have shown that PACAP can induce GH secretion in carp pituitary cells through cAMP/PKA- and Ca2+/calmodulin-dependent mechanisms, these results, taken together, suggest that alpha2-adrenergic stimulation in the carp pituitary may inhibit PACAP-induced GH release and GH gene transcription by blocking the AC/cAMP/PKA pathway and Ca2+ entry through L-type VSCC.

PTH-Induced Actin Depolymerization Increases Mechanosensitive Channel Activity to Enhance Mechanically Stimulated 2+ Signaling in Osteoblasts*

Disruption of the actin cytoskeleton with cytochalasin D enhanced the mechanically induced increase in intracellular Ca(2+) ([Ca(2+)](i)) in osteoblasts in a manner similar to that of PTH. Stabilization of actin with phalloidin prevented the PTH enhanced [Ca(2+)](i) response to shear. Patch-clamp analyses show that the MSCC is directly influenced by alterations in actin integrity. PTH significantly enhances the fluid shear-induced increase in [Ca(2+)](i) in osteoblasts, in part, through increased activation of both the mechanosensitive, cation-selective channel (MSCC) and L-type voltage-sensitive Ca(2+) channel (L-VSCC). Both stimuli have been shown to produce dynamic changes in the organization of the actin cytoskeleton. In this study, we examined the effects of alterations in actin polymerization on [Ca(2+)](i) and MSCC activity in MC3T3-E1 and UMR-106.01 osteoblasts in response to shear +/- PTH pretreatment. MC3T3-E1 or UMR-106.01 cells were plated onto type I collagen-coated quartz slides, allowed to proliferate to 60% confluency, and mounted on a modified parallel plate chamber and subjected to 12 dynes/cm(2). For patch-clamp studies, cells were plated on collagen-coated glass coverslips, mounted on the patch chamber, and subjected to pipette suction. Modulators of actin cytoskeleton polymerization were added 30 minutes before the experiments, whereas channel inhibitors were added 10 minutes before mechanical stimulation. All drugs were maintained in the flow medium for the duration of the experiment. Depolymerization of actin with 1-5 microM cytochalasin D (cyto D) augmented the peak [Ca(2+)](i) response and increased the number of cells responding to shear, similar to the increased responses induced by pretreatment with 50 nM PTH. Stabilization of actin with phalloidin prevented the PTH enhanced [Ca(2+)](i) response to shear. Inhibition of the MSCC with Gd(3+) significantly blocked both the peak Ca(2+) response and the number of cells responding to shear in cells pretreated with either PTH or cyto D. Inhibition of the L-VSCC reduced the peak [Ca(2+)](i) response to shear in cells pretreated with PTH, but not with cyto D. Patch-clamp analyses found that addition of PTH or cyto D significantly increased the MSCC open probability in response to mechanical stimulation, whereas phalloidin significantly attenuated the PTH-enhanced MSCC activation. These data indicate that actin reorganization increases MSCC activity in a manner similar to PTH and may be one mechanism through which PTH may reduce the mechanical threshold of osteoblasts.

Novel Ca 2+ signaling pathway for AVP mediated vasoconstriction

[Arg8]-vasopressin (AVP) is a peptide hormone which exerts vasoconstrictor effects by binding to V1a vasopressin receptors on vascular smooth muscle cells (VSMC). This effect is attributed to activation of phosphoinositidase C (PIC) and consequent release of Ca2+ from the sarcoplasmic reticulum. Interestingly, half-maximal activation of PIC requires nanomolar AVP concentrations, whereas circulating AVP concentrations rarely exceed 100pM. Using cultured VSMC, we previously identified a novel Ca2+ signaling pathway activated by 10–100pM AVP. This Ca2+ signaling pathway is distinguished from the PIC pathway by its dependence upon protein kinase C (PKC) activation and L-type voltage-sensitive Ca2+ channels (VSCC). In the present study, we tested the hypothesis that picomolar AVP concentrations would induce PKC- and VSCC-dependent vasoconstriction in isolated pressurized rat mesenteric arteries. AVP (10−14 to 10−6 M) induced a concentration-dependent constriction of arteries that was reversible with a V1a vasopressin receptor antagonist. 30pM AVP induced significant vasoconstriction (~45% of maximum), which was prevented by PKC inhibition with calphostin-C (250nM) or Ro-31–8220 (1μM) as well as by blockade of VSCC with verapamil (10μM). In contrast, maximal vasoconstriction induced by 10nM AVP was insensitive to either PKC inhibition or blockade of VSCC. These results suggest that two dose-dependent signaling pathways exist for AVP-induced vasoconstriction. Accordingly, we conclude that the vasoconstrictor actions of picomolar AVP within the systemic circulation may be dependent upon the novel Ca2+ signaling pathway involving PKC activation and L-type VSCC. NIH RO1-HL070670 and Falk Fellowship

Fluid Shear-Induced ATP Secretion Mediates Prostaglandin Release in MC3T3-E1 Osteoblasts

ATP is rapidly released from osteoblasts in response to mechanical load. We examined the mechanisms involved in this release and established that shear-induced ATP release was mediated through vesicular fusion and was dependent on Ca2+ entry into the cell through L-type voltage-sensitive Ca2+ channels. Degradation of secreted ATP by apyrase prevented shear-induced PGE2 release. Fluid shear induces a rapid rise in intracellular calcium ([Ca2+]i) in osteoblasts that mediates many of the cellular responses associated with mechanotransduction in bone. A potential mechanism for this increase in [Ca2+]i is the activation of purinergic (P2) receptors resulting from shear-induced extracellular release of ATP. This study was designed to determine the effects of fluid shear on ATP release and the possible mechanisms associated with this release. MC3T3-E1 preosteoblasts were plated on type I collagen, allowed to proliferate to 90% confluency, and subjected to 12 dynes/cm2 laminar fluid flow using a parallel plate flow chamber. ATP release into the flow media was measured using a luciferin/luciferase assay. Inhibitors of channels, gap junctional intercellular communication (GJIC), and vesicular formation were added before shear and maintained in the flow medium for the duration of the experiment. Fluid shear produced a transient increase in ATP release compared with static MC3T3-E1 cells (59.8 +/- 15.7 versus 6.2 +/- 1.8 nM, respectively), peaking within 1 minute of onset. Inhibition of calcium entry through the L-type voltage-sensitive Ca2+ channel (L-VSCC) with nifedipine or verapamil significantly attenuated shear-induced ATP release. Channel inhibition had no effect on basal ATP release in static cells. Ca(2+)-dependent ATP release in response to shear seemed to result from vesicular release and not through gap hemichannels. Vesicle disruption with N-ethylmaleimide, brefeldin A, or monensin prevented increases in flow-induced ATP release, whereas inhibition of gap hemichannels with either 18alpha-glycyrrhetinic acid or 18beta-glycyrrhetinic acid did not. Degradation of extracellular ATP with apyrase prevented shear-induced increases in prostaglandin E2 (PGE2) release. These data suggest a time line of mechanotransduction wherein fluid shear activates L-VSCCs to promote Ca2+ entry that, in turn, stimulates vesicular ATP release. Furthermore, these data suggest that P2 receptor activation by secreted ATP mediates flow-induced prostaglandin release.

Integrating rapid responses to 1,25-dihydroxyvitamin D 3 with transcriptional changes in osteoblasts: Ca 2+ regulated pathways to the nucleus

It is well established that 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) treatment of target cells including osteoblasts activates both membrane-initiated rapid Ca 2+ responses linked to influx through voltage sensitive Ca 2+ channels (VSCCs) and longer term nuclear receptor-mediated changes in gene expression. We recently reported use of a cDNA microarray strategy to identify transcriptional changes after 3 and 24 h of treatment with 1,25(OH) 2D 3 and with an analog of 1,25(OH) 2D 3 (25(OH)-16ene-23yne-D 3 [AT]) that activates Ca 2+ influx without binding to the nuclear receptor. Among 5000 different clones on the array filters, we identified families of genes in osteoblasts that were altered two-fold or greater following treatment with 1,25(OH) 2D 3 or analog AT for 3 h. Cluster analysis further revealed complex patterns of changes in gene expression, indicative of multiple pathways to the nucleus. Evidenced by changes in target gene expression, activation of a Ca 2+/CaMK/CREB/CRE pathway clearly occurs and modulates expression of a variety of genes associated with changes in protein secretion including those involved in paracrine regulation of bone resorption, RANKL and osteoprotegerin (OPG). The changes in gene expression can be inhibited by L-type VSCC channel blockers, confirming the role of Ca 2+ entry in pathway activation. These findings provide clear evidence of rapid changes in gene expression associated with Ca 2+ influx after treatment with 1,25(OH) 2D 3, and open the door to novel nuclear receptor-independent signaling pathways that affect gene transcription.

Protective effects of a selective L-type voltage-sensitive calcium channel blocker, S-312- d, on neuronal cell death

Amyloid β protein (Aβ)- and human group IIA secretory phospholipase A 2 (sPLA 2-IIA)-induced neuronal cell death have been established as in vitro models for Alzheimer’s disease (AD) and stroke. Both sPLA 2-IIA and Aβ causes neuronal apoptosis by increasing the influx of Ca 2+ through L-type voltage-sensitive Ca 2+ channel (L-VSCC). In the present study, we evaluated effects of a selective L-VSCC blocker, S-(+)-methyl 4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitro-phenyl)thieno[2,3- b]pyridine-5-carboxylate ( S-312- d), on Aβ- and sPLA 2-IIA-induced neuronal apoptosis in primary cultures of rat cortical neurons. S-312- d significantly rescued cortical neurons from Aβ- and sPLA 2-IIA-induced cell death. Both cell death stimuli caused the appearance of apoptotic features such as plasma membrane blebs, chromatin condensation, and DNA fragmentation. S-312- d completely suppressed these apoptotic features. Before apoptosis, the two death ligands markedly enhanced an influx of Ca 2+ into neurons. S-312- d significantly prevented neurons from sPLA 2-IIA- and Aβ-induced Ca 2+ influx. Furthermore, the neuroprotective effect of S-312- d was more potent than that of another L-VSCC blocker, nimodipine. On the other hand, blockers of other VSCCs such as the N-type and P/Q-type calcium channels had no effect on the neuronal cell death, apoptotic features and Ca 2+ influx. In conclusion, we demonstrated that S-312- d rescues cortical neurons from Aβ- and sPLA 2-IIA-induced apoptosis.

Activity-dependent hepatocyte growth factor expression and its role in organogenesis and cancer growth suppression

Studies by Murphy et al. have shown that neuronal stimulation can activate immediate early genes that code for transcription factors. Recent data suggest that Ca 2+ elevation in both neuronal cytoplasmic and nuclear compartments is responsible for the coupling of synaptic excitation to gene expression. Deisseroth et al. suggest that Ca 2+ influx through L-type voltage-sensitive Ca 2+ channels (VSCCs) activates cytoplasmic Ca 2+ targets such as calmodulin (CaM). The Ca 2+–CaM complex then translocates to the nucleus leading to Ca 2+ and cAMP response element-binding protein (CREB) phosphorylation and gene expression. Reports have shown that L-type VSCCs are found on the vagus nerve. Other studies have suggested that activation of L-type VSCCs leads to a Ca 2+ store-dependent elevation of nuclear [Ca 2+] that triggers gene expression by more direct activation of nuclear Ca 2+/CaM-dependent protein kinase (CaMK). Moreover, nuclear transcription factors such as DREAM are themselves Ca 2+-dependent, further supporting the importance of both nuclear and cytoplasmic Ca 2+ elevation in regulating gene expression. Our simulation studies suggest that intense synaptic stimulation in combination with amplification by release from intracellular Ca 2+ stores can produce elevations in nuclear Ca 2+ concentration and CaMK phosphorylation leading to CREB phosphorylation and gene expression. One of the downstream events would be the production of hepatocyte growth factor (HGF). HGF has trophic, repair, therapeutic or mitotic effect on kidney, pancreas, spleen, liver, lung, heart and spinal cord. These organs and systems' regeneration can be achieved by either upregulation of HGF release from the vagus nerve or upregulation of HGF production within the system (spinal cord). Conversely, inhibition of HGF release from the vagus nerve can inhibit cancer growth. Vagus nerve seems to be the nerve that nature intends to regulate organ growth and regeneration, it is very possible that other than HGF and injurin, other growth factors could be found in the vagus nerve. Electrical depolarization and hyperpolarization of the vagus nerve would be the most natural and effective way to induce organ regeneration and suppress cancer growth, respectively. A similar pathway seems to exist for different organs as HGF has trophic, repair, therapeutic or mitotic effect on different vagally innervated organs.

Decoding of synaptic voltage waveforms by specific classes of recombinant high-threshold Ca(2+) channels.

Studies suggest that the preferential role of L-type voltage-sensitive Ca(2+) channels (VSCCs) in coupling strong synaptic stimulation to transcription is due to their selective activation of local chemical events. However, it is possible that selective activation of the L-type channel by specific voltage waveforms also makes a contribution. To address this issue we have examined the response of specific Ca(2+) channel types to simulated complex voltage waveforms resembling those encountered during synaptic plasticity (gamma and theta firing frequency). L-, P/Q- and N-type VSCCs (alpha1C, alpha1A, alpha1B/beta1B/alpha2delta, respectively) were all similarly activated by brief action potential (AP) waveforms or sustained step depolarization. When complex waveforms containing large excitatory postsynaptic potentials (EPSPs), APs and spike accommodation were applied under voltage clamp we found that the integrated L-type VSCC current was approximately three times larger than that produced by the P/Q- or N-type Ca(2+) channels (gamma frequency 1 s stimulation). For P/Q- or N-type channels the complex waveforms led to a smaller current than that expected from the response to a simple 1 s step depolarization to 0 or +20 mV. EPSPs present in the waveforms favoured the inactivation of P/Q- and N-type channels. In contrast, activation of the L-type channel was dependent on both EPSP- and AP-mediated depolarization. Expression of P/Q-type channels with reduced voltage-dependent inactivation (alpha1A/beta2A/alpha2delta) or the use of hyperpolarized intervals between AP stimuli greatly increased their response to complex voltage stimuli. We propose that in response to complex synaptic voltage waveforms P/Q- and N-type channels can undergo selective voltage-dependent inactivation leading to a Ca(2+) current mediated predominantly by L-type channels.