Regulation Of Intracellular Ca2 Research Articles

Isoflurane Neuroprotection in Rat Hippocampal Slices Decreases with Aging

Most in vitro neuroprotection studies with isoflurane have involved cells obtained during the embryonic or early postnatal period. However, in mature rodents, isoflurane neuroprotection does not persist. The authors determined whether neuroprotection of hippocampal slices with isoflurane decreases with aging and is due to decreased intracellular Ca regulation and survival protein phosphorylation. Hippocampal slices from 5-day-old, 1-month-old, and 19- to 23-month-old rats were deprived of oxygen and glucose for 5-30 min in media bubbled with 1% isoflurane. Cell death was assessed in the CA1, CA3, and dentate regions, and intracellular Ca concentration was measured in CA1 neurons. N-methyl-d-aspartate receptor (NMDAR)-dependent Ca influx was measured and the phosphorylation of NMDARs, and the survival proteins Akt and mitogen-activated protein kinase p42/44 were quantified. Twenty minutes of oxygen and glucose deprivation killed approximately 40-60% of neurons in CA3 and dentate in all age groups. Isoflurane, 1%, reduced death of CA1, CA3, and dentate neurons in slices from 5-day-old rats but not those from 23-month-old rats. In 5-day slices, isoflurane attenuated NMDAR-mediated Ca influx, whereas in aging slices, Ca influx was increased protein kinase C. In aging slices, isoflurane did not increase the phosphorylation of Akt and p42/44. Isoflurane neuroprotection of hippocampal slices during oxygen and glucose deprivation decreases with age. Isoflurane does not prevent large increases in intracellular Ca concentration during oxygen and glucose deprivation and does not induce the phosphorylation of the prosurvival proteins in aging slices. A protein kinase C-mediated increase in NMDAR activity may result in increased excitotoxicity and decreased neuroprotection by volatile anesthetics in the aging brain.

Proton pump inhibitors omeprazole and lansoprazole induce relaxation of isolated human arteries

Vasorelaxant effects of H +/K +-ATPase were previously demonstrated in artery rings isolated from experimental animals. We examined the effects of clinically used H +/K +-ATPase inhibitors on isolated human internal mammary ( n = 19) and radial ( n = 5) arteries. Omeprazole and lansoprazole (30–300 μM) both induced concentration-dependent, reversible and reproducible relaxations of arteries which were precontracted with phenylephrine (5 μM), histamine (15 μM), high K + (80 mM), ouabain (1 μM) and K + free solution. Relaxant responses were similar in both arteries. Presence of N ω-Nitro- l-arginine methyl ester (30 μM) had no effect on lansoprazole-induced responses, thus relaxations are independent from nitric oxide. Relaxation in the K + free medium implies that this action could not be due to the inhibition of H +/K +-ATPase. Lansoprazole (300 μM) inhibited Ca 2+-induced contractions in high K +-Ca 2+ free medium. Omeprazole and lansoprazole may act on a common mechanism which plays a crucial role in regulating human vascular tone and that mechanism appeared to be involved in the regulation of intracellular Ca 2+.

Identification by Mutational Analysis of Amino Acid Residues Essential in the Chaperone Function of Calreticulin

Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.

Activating of ATP-Dependent K+ Channels Comprised of Kir 6.2 and SUR 2B by PGE2 Through EP2 Receptor in Cultured Interstitial Cells of Cajal from Murine Small Intestine

The interstitial cells of Cajal (ICC) are pacemaker cells in gastrointestinal tract and generate an electrical rhythm in gastrointestinal muscles. We investigated the possibility that PGE₂ might affect the electrical properties of cultured ICC by activating ATPdependent K⁺ channels and, the EP receptor subtypes and the subunits of ATP-dependent K⁺ channels involved in these activities were identified. In addition, the regulation of intracellular Ca²⁺ ([Ca²⁺]i) mobilization may be involved the action of PGE₂ on ICC. Treatments of ICC with PGE₂ inhibited electrical pacemaker activities in the same manner as pinacidil, an ATPdependent K⁺ channel opener and PGE₂ had only a dose-dependent effect. Using RT-PCR technique, we found that ATP-dependent K⁺ channels exist in ICC and that these are composed of K_ir 6.2 and SUR 2B subunits. To characterize the specific membrane EP receptor subtypes in ICC, EP receptor agonists and RT-PCR were used: Butaprost (an EP₂ receptor agonist) showed the actions on pacemaker currents in the same manner as PGE₂. However sulprostone (a mixed EP₁ and EP₃ agonist) had no effects. In addition, RT-PCR results indicated the presence of the EP₂ receptor in ICC. To investigate cAMP involvement in the effects of PGE₂ on ICCs, SQ-22536 (an inhibitor of adenylate cyclase) and cAMP assays were used. SQ-22536 did not affect the effect of PGE₂ on pacemaker currents, and PGE₂ did not stimulate cAMP production. Also, we found PGE₂ inhibited the spontaneous [Ca²⁺]i oscillations in cultured ICC. These observations indicate that PGE₂ alters pacemaker currents by activating the ATP-dependent K⁺ channels comprised of K_ir 6.2-SUR 2B in ICC and this action of PGE₂ are through EP₂ receptor subtype and also the activation of ATP-dependent K⁺ channels involves intracellular Ca²⁺ mobilization.

Differences in mitochondrial movement and morphology in young and mature primary cortical neurons in culture

Mitochondria have many roles critical to the function of neurons including the generation of ATP and regulation of intracellular Ca 2+. Mitochondrial movement is highly dynamic in neurons and is thought to direct mitochondria to specific cellular regions of increased need and to transport damaged or old mitochondria to autophagosomes. Morphology also varies between individual mitochondria and is modulated by fusion and fission proteins such as mitofusin-1 and dynamin-related protein-1, respectively. Although mitochondrial movement and morphology are thought to be modulated to best meet cellular demands, few regulatory signals have been identified. In this study, we examined how the different cellular environments of synaptically immature and mature rat cortical neurons affect mitochondrial movement, morphology, distribution and function. In younger cells, mitochondria were more mobile, were shorter, occupied a smaller percentage of neuronal processes, and expressed greater mitofusin-1 and lower dynamin-related protein-1 protein levels compared with older cells. However, the number of mitochondria per μm of neuronal process, mitochondrial membrane potential and the amount of basally sequestered mitochondrial Ca 2+ were similar. Our results suggest that while mitochondria in young neurons are functionally similar to mature neurons, their enhanced motility may permit faster energy dispersal for cellular demands, such as synaptogenesis. As cells mature, mitochondria in the processes may then elongate and reduce their motility for long-term support of synaptic structures.

Topics on the Na+/Ca2+ Exchanger: Responses of Na+/Ca2+ Exchanger to Interferon-γ and Nitric Oxide in Cultured Microglia

The Na+/Ca2+ exchanger (NCX) plays a role in regulation of intracellular Ca2+ levels, but little is known about the functional role of NCX in microglia. To clarify the role of NCX in microglia, we studied the responses of NCX to pathological conditions such as interferon-γ or nitric oxide (NO) exposure. Treatment with interferon-γ caused a biphasic increase in NCX activity. The delayed increase in NCX activity was accompanied by increases in the mRNA and protein levels. Pharmacological studies show that protein kinase C and tyrosine kinase are involved in the transient and delayed increases in NCX activity, and the extracellular signal-regulated protein kinase is involved in the delayed increase in NCX activity. On the other hand, NO causes apoptotic cell death in cultured microglia. We observed, using the specific NCX inhibitor SEA0400, that NO activates NCX activity and NCX is involved in NO-induced depletion of Ca2+ in the endoplasmic reticulum (ER), leading to ER stress. These results suggest that NCX is involved in the regulation of Ca2+ levels in the ER. The responses of NCX to interferon-γ and NO implies that NCX plays a key role in microglial function.

Mechanisms of Disease: ryanodine receptor defects in heart failure and fatal arrhythmia

Abnormal regulation of intracellular Ca(2+) by sarcoplasmic reticulum plays a part in the mechanism underlying contractile and relaxation dysfunction in heart failure (HF). The protein-kinase-A-mediated hyperphosphorylation of ryanodine receptors in the sarcoplasmic reticulum has been shown to cause the dissociation of FKBP12.6 (also known as calstabin-2) from ryanodine receptors in HF. In addition, several disease-linked mutations in the ryanodine receptors have been reported in patients with catecholaminergic polymorphic ventricular tachycardia or arrhythmogenic right ventricular cardiomyopathy type 2. The unique distribution of these mutation sites has led to the concept that the interaction among the putative regulatory domains within the ryanodine receptors has a key role in regulating channel opening. The knowledge gained from various studies of ryanodine receptors under pathologic conditions might lead to the development of new pharmacological or genetic strategies for the treatment of HF or cardiac arrhythmia. In this review, we focus on the role of the Ca(2+)-release channel, the ryanodine receptor, in the pathogenesis of HF and fatal arrhythmia, and the possibility of developing new therapeutic strategies for targeting this receptor.

Fibrocystin interacts with CAML, a protein involved in Ca 2+ signaling

The predicted structure of the autosomal recessive polycystic kidney disease protein, fibrocystin, suggests that it may function as a receptor, but its function remains unknown. To understand its function, we searched for proteins that interact with the intracellular C-terminus of fibrocystin using the yeast two-hybrid system. From the screening, we found calcium modulating cyclophilin ligand (CAML), a protein involved in Ca 2+ signaling. Immunofluorescent analysis showed that both proteins are co-localized in the apical membrane, primary cilia, and the basal body of cells derived from the distal nephron Epitope-tagged expression constructs of both proteins were co-immunoprecipitated from COS7 cells. The intracellular C-terminus of fibrocystin interacts with CAML, a protein with an intracellular distribution that is similar to that of PKD2. Fibrocystin may participate in regulation of intracellular Ca 2+ in the distal nephron in a manner similar to PKD1 and PKD2 that are involved in autosomal dominant polycystic kidney disease.

Regulation of intracellular calcium levels by polyunsaturated fatty acids, arachidonic acid and docosahexaenoic acid, in astrocytes: possible involvement of phospholipase A2

Pathological conditions in the brain, such as ischemia, trauma and seizure are accompanied by increased levels of free n-6 and n-3 polyunsaturated fatty acids (PUFA), mainly arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3). A neuroprotective role has been suggested for PUFA. For investigation of the potential molecular mechanisms involved in neuroprotection by PUFA, we studied the regulation of the concentration of intracellular Ca2+ ([Ca2+]i) in rat brain astrocytes. We evaluated the presence of extracellular PUFA and the release of intracellular PUFA. Interestingly, only the constitutive brain PUFA AA and DHA, but not eicosapentaenoic acid (EPA) had prominent effects on intracellular Ca2+. AA and DHA suppressed [Ca2+]i oscillation, inhibited store-operated Ca2+ entry, and reduced the amplitudes of Ca2+ responses evoked by agonists of G protein-coupled receptors. Moreover, prolonged exposure of astrocytes to AA and DHA brought the cells to a new steady state of a moderately elevated [Ca2+]i level, where the cells became virtually insensitive to external stimuli. This new steady state can be considered as a mechanism of self-protection. It isolates disturbed parts of the brain, because AA and DHA reduce pathological overstimulation in the tissue surrounding the damaged area. In inflammation-related events, frequently AA and DHA exhibit opposite effects. However, in astrocytes AA and DHA exerted comparable effects on [Ca2+]i. Extracellularly added AA and DHA, but not EPA, were also able to induce the release of [3H]AA from prelabeled astrocytes. Therefore, we also suggest the involvement of phospholipase A2 activation and lysophospholipid generation in the regulation of intracellular Ca2+ in astrocytes.

PDGF-induced signaling in proliferating and differentiated vascular smooth muscle: Effects of altered intracellular Ca regulation

Platelet-derived growth factor-BB (PDGF)-induced intracellular signaling is involved in phenotypic modulation of vascular smooth muscle (VSM). This study has examined the PDGF-induced Ca2+ increase and the resultant effect on signaling pathways in proliferative compared with fully differentiated VSM. PDGF-induced changes in Ca2+ were measured in portal vein (PV) myocytes from 2-4-day-old (proliferating), compared to 6-week-old (differentiated), Sprague Dawley rats. Phospholipase C (PLC)gamma expression and activation of extracellular signal-regulated kinase (ERK) 1/2 was determined by immunoblotting or confocal immunolabelling. Activation of the Ca(2+)-dependent transcription factor, nuclear factor of activated T-cells (NFATc), was assessed by electromobility shift assay. PDGF increased the intracellular Ca2+ concentration in differentiated, but not in proliferating, PV myocytes. This is probably due to very low expression of PLC(gamma) in proliferating PV. In 6-week-old PV, PDGF stimulation induced nuclear translocation and activation of NFATc. PDGF did not induce NFATc activation in neonatal PV. PDGF-induced ERK1/2 activation was observed in both 2-4-day-old and 6-week-old PV. In 6-week-old PV, ERK1/2 activation was Ca(2+)-dependent and protein kinase C-dependent. However in 2-4-day-old PV, PDGF-induced ERK1/2 activation was via a Ca(2+)-independent, atypical protein kinase C. PLC(gamma) expression was also decreased in the neointima, compared to media, of balloon-injured rabbit subclavian arteries. The regulation of PDGF-induced Ca2+ increases by PLC(gamma) expression in VSM may provide a mechanism for coordinating different signaling pathways leading to activation of specific transcription factors. This may play an important role in the phenotypic modulation of VSM.

Differential Effects of Bupivacaine and Ropivacaine Enantiomers on Intracellular Ca2+Regulation in Murine Skeletal Muscle Fibers

Increased intracellular Ca concentrations are considered to be a major pathomechanism in local anesthetic myotoxicity. Racemic bupivacaine and S-ropivacaine cause Ca release from the sarcoplasmic reticulum of skeletal muscle fibers and simultaneously inhibit Ca reuptake. Examining the optical isomers of both agents, the authors investigated stereoselective effects on muscular Ca regulation to get a closer insight in subcellular mechanisms of local anesthetic myotoxicity. R- and S-enantiomers as well as racemic mixtures of both agents were tested in concentrations of 1, 5, 10, and 15 mm. Saponin-skinned muscle fibers from the extensor digitorum longus muscle of BALB/c mice were examined according to a standardized procedure. For the assessment of effects on Ca uptake and release from the sarcoplasmic reticulum, agents were added to the loading solution and the release solution, respectively, and force and Ca transients were monitored. The effects of S-enantiomers on both Ca release and reuptake were significantly more pronounced than those of racemic mixtures and R-enantiomers, respectively. In addition, the effects of racemates were markedly stronger than those of R-enantiomers. With regard to Ca release, the effects of bupivacaine isomers were more pronounced than the isomers of ropivacaine. These data show that stereoselectivity is involved in alterations of intracellular Ca regulation by bupivacaine and ropivacaine. S-enantiomers seem to be more potent than R-enantiomers, with intermediate effects of racemic mixtures. In addition, lipophilicity also seems to determine the extent of Ca release by local anesthetics.

Regulation of intracellular Ca2+ and calcineurin by NO/PKG in proliferation of vascular smooth muscle cells

To determine whether Ca2+/calcineurin mediated the inhibitory effects of nitric oxide /cGMP-dependent protein kinase (NO/PKG) on the proliferation of vascular smooth muscle cells (VSMC). Proliferation and viability of primary VSMC from rat aorta were measured using [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay and acridine orange and ethidium bromide staining, respectively. Cytosolic Ca2+ was determined by Fluo-3/AM. Calcineurin protein and its activity were assayed using immunoblotting and free inorganic phosphate analysis, respectively. (+/-)-S-nitroso-N-acetyl-penicillamine (SNAP) and Sp-8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Sp-8-pCPT-cGMPS) decreased phenylephrine (PE)-induced proliferation of VSMC by 27.3% and 36.6%, respectively, but Rp-8-[(4-chlorophenyl)thio]-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS) increased PE-induced proliferation of VSMC. SNAP, Sp-8-pCPT-cGMPS, and Rp-8-pCPT-cGMPS did not affect the viability of VSMC. Calcineurin protein was decreased by 63.1% and its activity was decreased by 59.7% in smooth muscle cells (SMC) pretreated with verapamil (Ver) and then stimulated by PE. In SMC pretreated with Ver, the absorbance of cells stimulated by PE decreased by 22.0% and was further inhibited by the additional treatment of SNAP and Sp-8-pCPT-cGMPS. In SMC pretreated with cyclosporin A (CsA), the absorbance of cells stimulated by PE decreased by 36.7%, but could not be further altered by the additional treatment of SNAP, Sp-8-pCPT-cGMPS, and Rp-8-pCPT-cGMPS. In addition, Ver inhibited PE-induced intracellular Ca2+ variations, which could be further inhibited by SNAP and Sp-8-pCPT-cGMPS, but not by Rp-8-pCPT-cGMPS. Moreover, the increase in calcineurin activity induced by PE was inhibited by SNAP and Sp-8-pCPT-cGMPS, but was promoted by Rp-8-pCPT-cGMPS. NO/PKG regulates calcineurin activity via the modulation of intracellular Ca2+ concentration, and thus partially inhibits the proliferation of VSMC without affecting their viability.

Functional Proteins Involved in Regulation of Intracellular Ca2+ for Drug Development: Pharmacology of SEA0400, a Specific Inhibitor of the Na+-Ca2+ Exchanger

The Na+-Ca2+ exchanger (NCX) is involved in regulation of intracellular Ca2+ concentration. A specific inhibitor of NCX has been required for clarification of the physiological and pathological roles of NCX. We have developed 2-[4-[(2,5-difluorophenyl)methoxy] phenoxy]-5-ethoxyaniline (SEA0400), a highly potent and selective inhibitor of NCX. SEA0400 in the concentration range that inhibits NCX exhibits negligible affinities for the Ca2+ channels, Na+ channels, K+ channels, noradrenaline transporter, and 14 receptors; and it does not affect the activities of the store-operated Ca2+ channel, Na+-H+ exchanger, and several enzymes including Na+,K+-ATPase and Ca2+-ATPase. Furthermore, recent studies show that SEA0400 attenuates ischemia-reperfusion injury in the brain, heart, and kidney and radiofrequency lesion-induced edema in rat brain. These findings suggest that NCX plays a key role in ischemia-reperfusion injury and may be a target molecule for treatment of reperfusion injury-related diseases.

Somatic Ca 2+ dynamics in response to choline-mediated excitation in histaminergic tuberomammillary neurons

Histaminergic tuberomammillary (TM) neurons of the posterior hypothalamus have been implicated in cognition, alertness and sleep-wakefulness cycles. Spontaneous firing of TM neurons has been associated with histamine release and wakefulness. The expression of α7 nicotinic acetylcholine receptors (nAChRs) in TM neurons suggests a role for endogenous choline and for nicotinic drugs in the regulation of intracellular Ca 2+ metabolism, normal TM neuronal activity and histamine release. First, we established the link between TM neuronal spontaneous firing frequency and cytosolic free Ca 2+ concentration ([Ca 2+] i). A strong correlation was observed: an onset of spontaneous firing (3–4Hz) was accompanied by a 20-fold increase in [Ca 2+] i from 56±18nM to 1.0±0.6μM. The same range of firing frequencies has been observed in TM neurons in vivo and is associated with wakefulness. Secondly, choline-induced activation of α7 nAChRs did not elevate [Ca 2+] i directly, i.e. in the absence of high-threshold voltage-gated Ca 2+ channel (HVGCC) activation. Cd 2+ (200μM) completely blocked all Ca 2+ signals, but inhibited only 37±16% of α7 nAChR-mediated currents. Thirdly, the responsiveness of [Ca 2+] i to choline-mediated excitation was inhibited by hyperpolarization and enhanced by depolarization, sensitizing [Ca 2+] i at membrane voltages associated with normal TM neuronal activity. These properties of [Ca 2+] i define the ability of TM neurons to translate cholinergic stimuli of identical strengths into different cytosolic Ca 2+ effects, providing the physiological substrate for state-specific modulation of incoming cholinergic information and would be expected to play a very important role in determining activity profiles of TM neurons exposed to elevated concentrations of cholinergic agents, such as choline and nicotine.

Functional Proteins Involved in Regulation of Intracellular Ca2+ for Drug Development: Desensitization of N-Methyl-D-aspartate Receptor Channels

N-Methyl-D-aspartate (NMDA) receptors are a principal subtype of excitatory ligand-gated ion channels with crucial roles in a variety of physiological and pathological processes in the mammalian central nervous system (CNS). In contrast to synaptic non-NMDA receptors that rapidly internalize, synaptic NMDA receptors have been shown to be rather static. However, recent accumulating evidence gives rise to the possibility that NMDA receptors may also undergo internalization. In our studies, repeated but not sustained stimulation by an agonist indeed induced desensitization of NMDA-receptor channels, which may be due to a decrease in the number of NMDA receptors expressed on cellular surfaces by internalization in a manner similar to the clathrin-induced endocytosis. Desensitization of NMDA-receptor channels provides a clue for the elucidation of fundamental mechanisms underlying dynamic regulation of the number of NMDA receptors at synapses, which is undoubtedly critical for the maintenance of both integrity and functionality in the CNS.

Functional Proteins Involved in Regulation of Intracellular Ca2+ for Drug Development: The Extracellular Calcium Receptor and an Innovative Medical Approach to Control Secondary Hyperparathyroidism by Calcimimetics

Circulating levels of calcium ion (Ca2+) are maintained within a narrow physiological range mainly by the action of parathyroid hormone (PTH) secreted from parathyroid cells. Parathyroid cells can sense small fluctuations in plasma Ca2+ levels by virtue of a cell surface Ca2+ receptor (CaR) that belongs to the superfamily of G-protein-coupled receptors. Calcimimetics are positive allosteric modulators that activate the CaR on parathyroid cells and thereby immediately suppress PTH secretion. Pre-clinical studies with NPS R-568, a first generation calcimimetic compound, have demonstrated that daily oral administration inhibits the elevation of plasma PTH levels and parathyroid gland hyperplasia and ameliorates impaired bone qualities in rats with chronic renal insufficiency. The results of clinical trials with cinacalcet hydrochloride, a second generation calcimimetic compound, have shown that calcimimetics possess lowering effects not only on serum PTH levels but also on serum calcium × phosphorus product levels, a hallmark of an increased risk for cardiovascular death in dialysis patients with end-stage renal disease (ESRD). Thus, calcimimetics have considerable potential as an innovative medical approach to manage secondary hyperparathyroidism associated with ESRD. Indeed, cinacalcet hydrochloride has been approved in several countries and is the first positive allosteric modulator of any G protein-coupled receptor to reach the market.

Functional Proteins Involved in Regulation of Intracellular Ca2+ for Drug Development: Role of Calcium/Calmodulin-Dependent Protein Kinases in Ischemic Neuronal Death

Excessive elevation of intracellular calcium level seems to be a trigger of ischemic neuronal injury. Calcium/calmodulin (CaM)-dependent protein kinase kinase (CaM-KK) is an upstream kinase for CaM kinase IV (CaM-KIV) that was reported to prevent apoptosis through phosphorylation of CREB (cyclic AMP responsive element-binding protein). We here observed that CaM-KK could directly activate Akt, thereby preventing apoptosis in cultured cells. Then we examined changes in Akt and CaM-KIV activities in gerbil forebrain ischemia. In 5-minischemia-caused delayed neuronal death in hippocampal CA1 neurons, Akt and CaM-KIV activities were decreased after reperfusion. On the other hand, during induction of ischemic tolerance, Akt activity gradually and persistently increased in the CA1 neurons with transient increase in CREB phosphorylation. Inhibition of Akt activity with wortmannin or CREB-DNA binding with CRE-decoy injection resulted in failure of generation of ischemic tolerance. These results indicated activation of Akt and CaM-KIV play important roles in induction of the ischemic tolerance. Activation of CaM-KK may provide a new strategy for overcoming the ischemic stress.

Functional Proteins Involved in Regulation of Intracellular Ca2+ for Drug Development: Chronic Nicotine Treatment Upregulates L-Type High Voltage-Gated Calcium Channels

Neurochemical mechanisms underlying drug dependence and withdrawal syndrome remain unclear. In this review, we discuss how chronic nicotine exposure to neurons affects expression of diazepam binding inhibitor (DBI), an endogenous anxiogenic neuropeptide supposed to be a common substance participating drug dependence, and function of L-type high voltage-gated Ca2+ channels (HVCCs). We also discuss the functional interaction between DBI and L-type HVCCs in nicotine dependence. Both DBI levels and [45Ca2+] influx significantly increased in the brain from mice treated with nicotine for long term, which was further enhanced after abrupt cessation of nicotine and was abolished by nicotinic acetylcholine receptor (nAChR) antagonists. Similar responses of DBI expression and L-type HVCC function were observed in cerebral cortical neurons after sustained exposure to nicotine. In addition, increased DBI expression was inhibited by antagonists of nAChR and L-type HVCCs. Sustained exposure of neurons to nicotine significantly enhanced expression of α1 and α2/δ1 subunits for L-type HVCCs and caused an increase in the Bmax value of [3H]verapamil binding to the particulate fractions. Therefore, it is concluded that the alterations in DBI expression is mediated via increased influx of Ca2+ through upregulated L-type HVCCs and these neurochemical changes have a close relationship with development of nicotine dependence and/or its withdrawal syndrome.

Functional Proteins Involved in Regulation of Intracellular Ca2+ for Drug Development: Preface

Functional Proteins Involved in Regulation of Intracellular Ca2+ for Drug Development: Preface

Polycystin-1 Activates the Calcineurin/NFAT (Nuclear Factor of Activated T-cells) Signaling Pathway

Regulation of intracellular Ca(2+) mobilization has been associated with the functions of polycystin-1 (PC1) and polycystin-2 (PC2), the protein products of the PKD1 and PKD2 genes. We have now demonstrated that PC1 can activate the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway through Galpha(q) -mediated activation of phospholipase C (PLC). Transient transfection of HEK293T cells with an NFAT promoter-luciferase reporter demonstrated that membrane-targeted PC1 constructs containing the membrane proximal region of the C-terminal tail, which includes the heterotrimeric G protein binding and activation domain, can stimulate NFAT luciferase activity. Inhibition of glycogen synthase kinase-3beta by LiCl treatment further increased PC1-mediated NFAT activity. PC1-mediated activation of NFAT was completely inhibited by the calcineurin inhibitor, cyclosporin A. Cotransfection of a construct expressing the Galpha(q) subunit augmented PC1-mediated NFAT activity, whereas the inhibitors of PLC (U73122) and the inositol trisphosphate and ryanodine receptors (xestospongin and 2-aminophenylborate) and a nonspecific Ca(2+) channel blocker (gadolinium) diminished PC1-mediated NFAT activity. PC2 was not able to activate NFAT. An NFAT-green fluorescent protein nuclear localization assay demonstrated that PC1 constructs containing the C-tail only or the entire 11-transmembrane spanning region plus C-tail induced NFAT-green fluorescent protein nuclear translocation. NFAT expression was demonstrated in the M-1 mouse cortical collecting duct cell line and in embryonic and adult mouse kidneys by reverse transcriptase-PCR and immunolocalization. These data suggest a model in which PC1 signaling leads to a sustained elevation of intracellular Ca(2+) mediated by PC1 activation of Galpha(q) followed by PLC activation, release of Ca(2+) from intracellular stores, and activation of store-operated Ca(2+) entry, thus activating calcineurin and NFAT.