Elevation of phospholipase C-ß1 expression by amyloid-ß facilitates calcium overload in neuronal cells.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia. Amyloid-ß (Aß) has long been considered a key cause of neurodegeneration in the AD brain. Although the mechanisms underlying Aß-induced neurodegeneration are not fully understood, a number of recent studies have suggested that intracellular calcium overload mediates this process. In this study, we focused on the cellular function of phospholipase C-ß1 (PLCB1), which regulates calcium signaling by mediating hydrolysis of phosphatidylinositol 4,5-bisphosphate through G-protein coupled receptor pathways. First, we confirmed that acetylcholine-induced calcium release from intracellular stores of SH-SY5Y cells was significantly increased with Aß42 oligomer treatment. We further found that PLCB1 expression was upregulated in Aß42-treated cells, and PLCB1 overexpression in SH-SY5Y cells elicited the calcium overload observed in Aß-treated cells. In addition, Aß42 oligomer-induced calcium overload in SH-SY5Y cells was alleviated by knockdown of PLCB1, indicating that PLCB1 plays an essential role in the neurotoxic process initiated by Aß. The elevation of PLCB1 expression was confirmed in the brain tissues from the 5× familial AD (5×FAD) model mice. These findings suggest that PLCB1 may represent a potential therapeutic target for protecting neuronal cells against excitotoxicity in AD progression.

Effect of Bitter Compounds on the Expression of Bitter Taste Receptor T2R7 Downstream Signaling Effectors in cT2R7/pDisplay-Gα16/gust44/pcDNA3.1 (+) Cells.

Bitterness is an important taste sensation for chickens, which provides useful sensory information for acquisition and selection of diet, and warns them against ingestion of potentially harmful and noxious substances in nature. Bitter taste receptors (T2Rs) mediate the recognition of bitter compounds belonging to a family of proteins known as G-protein coupled receptors. The aim of this study was to identify and evaluate the expression of T2R7 in chicken tongue tissue and construct cT2R7-1 and cT2R7-2-expressing HEK-293T cells to access the expression of PLCß2 and ITPR3 after exposure with different concentrations of the bitter compounds. Using real-time PCR, we show that the relative expression level of T2R7 mRNA in 5, 1, 0.1, and 10-3 mM of camphor and erythromycin solutions and 5 mM of chlorpheniramine maleate solutions was significantly higher than that in 50 mM KCL solutions. We confirmed that the bitter taste receptor T2R7 and downstream signaling effectors are sensitive to different concentrations of bitter compounds. Moreover, T2R7-1 (corresponding to the unique haplotype of the Tibetan chicken) had higher sensitivity to bitter compounds compared with that of T2R7-2 (corresponding to the unique haplotype of the Jiuyuan black-chicken). These results provide great significance of taste response on dietary intake to improve chicken feeding efficiency in poultry production and have certain reference value for future taste research in other bird species.

Phospholipase Cß1 regulates proliferation of neuronal cells.

Cells have developed lineage-specific mechanisms to control proliferation and drive morphologic changes upon differentiation. A hallmark of differentiation is the assembly of signaling molecules that transduce extracellular signals, such as the production of the G protein-regulated enzyme phospholipase Cß (PLCß), which generates calcium signals from sensory stimuli. We found that in most cancerous cell lines there is positive correlation between PLCß1 levels and cell proliferation. In cells of neuronal lineage, however, reducing PLCß1 levels increases the rate of proliferation. Using a combination of biochemical and biophysical methods, we find that, in the G1 phase, a cytosolic population of PLCß1 associates with cyclin-dependent kinase 16 (CDK16), a neuron-specific enzyme that is activated by cyclin Y to inactivate the antioncogenic protein p27Kip1. Binding of PLCß1 directly inhibits CDK16 activity and in turn reduces the ability of cells to enter the S phase. Activation of Gαq by carbachol causes movement of PLCß from the cytosol to the plasma membrane, reducing its association with CDK16. Similarly, the overexpression of activated Gαq moves PLCß1 to the membrane, reverses G1 arrest, and promotes proliferation, thereby connecting external stimuli with cell proliferation. Our results present a model in which the transient high expression of PLCß1 that occurs at the onset of differentiation arrests cells in the G1 phase through its association with CDK16 and allows CDK16 to transition to its postmitotic function of neurite outgrowth and trafficking of synaptic vesicles. The novel role of PLCß1 in neuronal cell proliferation offers a unique interaction that can be manipulated to guide cells into a neuronal phenotype or to develop therapies for neuroblastomas.-Garwain, O., Valla, K., Scarlata, S. Phospholipase Cß1 regulates proliferation of neuronal cells.

Hypobaric Preconditioning Modifies Group I mGluRs Signaling in Brain Cortex.

The study assessed involvement of Ca(2+) signaling mediated by the metabotropic glutamate receptors mGluR1/5 in brain tolerance induced by hypoxic preconditioning. Acute slices of rat piriform cortex were tested 1 day after exposure of adult rats to mild hypobaric hypoxia for 2 h at a pressure of 480 hPa once a day for three consecutive days. We detected 44.1 ± 11.6 % suppression of in vitro anoxia-induced increases of intracellular Ca(2+) levels and a fivefold increase in Ca(2+) transients evoked by selective mGluR1/5 agonist, DHPG. Western blot analysis of cortical homogenates demonstrated a 11 ± 4 % decrease in mGluR1 immunoreactivity (IR), and in the nuclei-enriched fraction a 12 ± 3 % increase in IR of phospholipase Cß1 (PLCß1), which is a major mediator of mGluR1/5 signaling. Immunocytochemical analysis of the cortex revealed increase in the mGluR1/5 and PLCß1 IR in perikarya, and a decrease in IR of the neuronal inositol trisphosphate receptors (IP3Rs). We suggest that enhanced expression of mGluR5 and PLCß1 and potentiation of Ca(2+) signaling may represent pro-survival upregulation of Ca(2+)-dependent genomic processes, while decrease in mGluR1 and IP3R IR may be attributed to a feedback mechanism preventing excessive intracellular Ca(2+) release.

An increased expression of PI-PLCß1 is associated with myeloid differentiation and a longer response to azacitidine in myelodysplastic syndromes.

This study tested the hypothesis that PI-PLCß1 is associated with myeloid differentiation and that its expression could be useful for predicting the response of MDS patients to azacitidine, as the clinical effect of epigenetic treatments is often detectable only after several cycles of therapy. To this end, PI-PLCß1 was quantified on 70 MDS patients (IPSS risk: 13 Low, 20 Int-1, 31 Int-2, 6 High) at baseline and during the first 3 cycles of azacitidine. Results were then compared with the hematologic response, as assessed after the sixth cycle of azacitidine therapy. Overall, 60 patients completed 6 cycles of azacitidine, and for them, a clinical and molecular evaluation was possible: 37 of these patients (62%) showed a specific increase of PI-PLCß1 mRNA within the first 3 cycles, which was associated with a longer duration of response and with an increased myeloid differentiation, as evidenced by PI-PLCγ2 induction and the recruitment of specific myeloid-associated transcription factors to the PI-PLCß1 promoter during azacitidine response. Moreover, the increase of cyclin D3 gene expression throughout all of the therapy showed that PI-PLCß1-dependent signaling is indeed activated in azacitidine responder patients. Taken together, our results show that PI-PLCß1 quantification in MDS predicts the response to azacitidine and is associated with an increased myeloid differentiation.

PLCß1a and PLCß1b selective regulation and cyclin D3 modulation reduced by kinamycin F during k562 cell differentiation.

Here we report that both PLCß1a and PLCß1b are relevant regulators of erythropoiesis in that kinamycin F, a potent inducer of γ-globin production in K562 cells, caused a selectively reduction of both PLCß1 isozymes even though the results point out that the effect of the drug is mainly directed toward the expression of the PLCß1a isoform. We have identified a different role for the two isozymes as regulators of K562 differentiation process induced by kinamycin F. The overexpression of PLCß1b induced an increase in γ-globin expression even in the absence of kinamycin F. Moreover during K562 differentiation, cyclin D3 level is regulated by PLCß1 signaling pathway. Namely the amplification of the expression of the PLCß1a, but not of PLCß1b, is able to maintain high levels of expression of cyclin D3 even after treatment with kinamycin F. This could be due to their different distribution in the cell compartments since the amount of PLCß1b is mainly present in the nucleus in respect to PLCß1a. Our data indicate that the amplification of PLCß1a expression, following treatment with kinamycin F, confers a real advantage to K562 cells viability and protects cells themselves from apoptosis.

Color discrimination with broadband photoreceptors.

BACKGROUND: Color vision is commonly assumed to rely on photoreceptors tuned to narrow spectral ranges. In the ommatidium of Drosophila, the four types of so-called inner photoreceptors express different narrow-band opsins. In contrast, the outer photoreceptors have a broadband spectral sensitivity and were thought to exclusively mediate achromatic vision. RESULTS: Using computational models and behavioral experiments, we demonstrate that the broadband outer photoreceptors contribute to color vision in Drosophila. The model of opponent processing that includes the opsin of the outer photoreceptors scored the best fit to wavelength discrimination data. To experimentally uncover the contribution of individual photoreceptor types, we restored phototransduction of targeted photoreceptor combinations in a blind mutant. Dichromatic flies with only broadband photoreceptors and one additional receptor type can discriminate different colors, indicating the existence of a specific output comparison of the outer and inner photoreceptors. Furthermore, blocking interneurons postsynaptic to the outer photoreceptors specifically impaired color but not intensity discrimination. CONCLUSIONS: Our findings show that receptors with a complex and broad spectral sensitivity can contribute to color vision and reveal that chromatic and achromatic circuits in the fly share common photoreceptors.

Transient decrease in nociceptor GRK2 expression produces long-term enhancement in inflammatory pain.

In heterozygous mice, attenuation of G-protein-coupled receptor kinase 2 (GRK2) level in nociceptors is associated with enhanced and prolonged inflammatory hyperalgesia. To further elucidate the role of GRK2 in nociceptor function we reversibly decreased GRK2 expression using intrathecal antisense oligodeoxynucleotide (AS-ODN). GRK2 AS-ODN administration led to an enhanced and prolonged hyperalgesia induced by prostaglandin E(2), epinephrine and carrageenan. Moreover, this effect persisted unattenuated 2weeks after the last dose of antisense, well after GRK2 protein recovered, suggesting that transient attenuation of GRK2 produced neuroplastic changes in nociceptor function. Unlike hyperalgesic priming induced by transient activation of protein kinase C epsilon (PKCε), (Aley et al., 2000; Parada et al., 2003b), the enhanced and prolonged hyperalgesia following attenuation of GRK2 is PKCε- and cytoplasmic polyadenylation element binding protein (CPEB)-independent and is protein kinase A (PKA)- and Src tyrosine kinase (Src)-dependent. Finally, rats treated with GRK2 AS-ODN exhibited enhanced and prolonged hyperalgesia induced by direct activation of second messengers, adenyl cyclase, Epac or PKA, suggesting changes downstream of G-protein-coupled receptors. Because inflammation can produce a decrease in GRK2, such a mechanism could help explain a predilection to develop chronic pain, after resolution of acute inflammation.

Phospholipase C beta 1 expression in the dorsolateral prefrontal cortex from patients with schizophrenia at different stages of illness.

OBJECTIVE: Our recent microarray study detected decreases in the expression of phospholipase C beta 1 mRNA in the dorsolateral prefrontal cortex from subjects with schizophrenia at different stages of illness. Thus we aimed to validate and extend these findings. METHOD: We measured levels of mRNA and protein for phospholipase C beta 1 variant a and b using real-time PCR and western blot analysis, respectively, in the dorsolateral prefrontal cortex from subjects with schizophrenia, who had a short (< 7 years) or long (> 22 years) duration of illness. RESULTS: Compared to age/sex matched controls, levels of phospholipase C beta 1 variant a and b mRNAs were decreased (-33% and -50%, respectively) in short duration schizophrenia. By contrast, only variant a mRNA was decreased (-24%) in long duration schizophrenia. There was no significant difference in the protein levels of either phospholipase C beta 1 variant in schizophrenia, irrespective of duration of illness (variant a; P = 0.84, variant b; P = 0.73). CONCLUSION: Our data confirm that phospholipase C beta 1 transcript levels are decreased in the dorsolateral prefrontal cortex from subjects with schizophrenia. However, the changes in levels of mRNA do not translate into a change at the level of protein. It is possible protein expression is regulated independently of mRNA and it remains to be determined whether there is a functional consequence of this change in mRNA relating to the pathophysiology of schizophrenia.

Role of vasoactive peptides in high glucose-induced increased expression of Gαq/11 proteins and associated signaling in vascular smooth muscle cells.

We have recently shown that A10 vascular smooth muscle cells (VSMCs) exposed to high glucose exhibited enhanced expression of G(alpha)q and PLCbeta proteins. Since high glucose has been reported to increase the levels of vasoactive peptides and oxidative stress, the present study was undertaken to investigate the implication of angiotensin II (Ang II), endothelin (ET)-1, and oxidative stress in the high glucose-induced enhanced expression of G(alpha)q/11 and PLCbeta proteins and associated signaling in A10 VSMCs. The levels of G(alpha)q, G(alpha)11, PLCbeta-1, and PLCbeta-2 proteins, as determined by Western blotting, were significantly higher in A10 VSMCs exposed to high glucose than in control cells. The elevated levels were restored to control values by the antioxidant diphenyleneiodonium (DPI), as well as by the antagonist of Ang II AT1 receptor losartan and the antagonists of ETA and ETB receptors BQ123 and BQ788, respectively. In addition, ET-1-stimulated production of inositol trisphosphate (IP3), which was enhanced by high glucose, was also restored toward control levels by DPI. Furthermore, the enhanced production of superoxide anion (O2-), increased NADPH oxidase activity, and enhanced expression of p22phox and p47phox proteins induced by high glucose were restored to control levels by losartan, BQ123, and BQ788. These results suggest that through increased oxidative stress, high glucose-induced enhanced levels of endogenous Ang II and ET-1 may contribute to the increased levels of G(alpha)q/11 and mediated signaling in A10 VSMCs.

Co-expression of nAChRs and molecules of the bitter taste transduction pathway by epithelial cells of intrapulmonary airways.

AIMS: The ability to sense the bitter taste of nicotine is an important component of addiction to, and withdrawal from, cigarette smoking. alpha-Gustducin and phospholipase C-beta2 (PLC-beta2), molecules involved in the taste transduction pathway, have been identified in airway epithelial solitary chemosensory cells (SCCs). Airway epithelial cells also express multiple nicotinic acetylcholine receptors (nAChRs). However, the relationship between nAChRs and molecules of taste transduction in response to nicotine is not known. This study was designed to determine whether nAChRs and the taste transduction molecules alpha-gustducin, PLC-beta2 and bitter taste receptors (T2R38) reside at sites of the intrapulmonary airways where interaction with the nicotine components of cigarette smoke is likely. MAIN METHODS: We used the reverse transcription-polymerase chain reaction (RT-PCR) to detect alpha-gustducin, PLC-beta2 and T2R38 mRNA and immunohistochemistry to localize expression of these proteins by nAChR expressing cells of the airway. KEY FINDINGS: RT-PCR demonstrated the presence of mRNA for alpha-gustducin, PLC-beta2 and T2R38. Immunohistochemistry showed the expression of alpha-gustducin, PLC-beta2 and T2R38 by subsets of epithelial cells at all levels of the intrapulmonary airways including bronchi, terminal and respiratory bronchioles. Double labeling demonstrated the co-expression of alpha-gustducin with alpha3, alpha4, alpha5, alpha7 and beta2, as well as, PLC-beta2 and T2R38 with alpha4, alpha5 and beta2 nAChR subunits. SIGNIFICANCE: These findings provide morphological evidence for the presence of molecules of the bitter taste transduction pathway in nAChR expressing SCCs of the intrapulmonary airways. These SCCs may, thus, constitute a peripheral component of the bitter taste signal transduction pathway for nicotine.

Phosphatidic acid potentiates G(alpha)q stimulation of phospholipase C-beta1 signaling.

Phosphatidic acid (PA) is interactive with G(alpha)q-linked agonists to stimulate GPCR signaling via phospholipase C-beta(1) (PLC-beta(1)). Phorbol 12-myristate 13-acetate (PMA) increases cellular levels of PA and phospholipase D activity (PLD). This study evaluated whether PMA can stimulate PLC-beta(1) activity via PA, independent of GPCR input in transfected COS 7 cells. PMA alone had little effect on PLC activity in cells co-transfected with PLC-beta(1) and G(alpha)q. Activated G(alpha)q, induced by co-transfecting muscarinic cholinergic receptor (m1R), was necessary for stimulation of PLC-beta(1) activity by PMA. Stimulation by PMA was dependent on the PA-regulatory motif of PLC-beta(1) implicating PA in this mechanism. PLD1 knockdown by antisense decreased responsiveness of PLC-beta(1) to both PMA and carbachol. PA alone thus has little effect on PLC-beta(1) activity, but PA and PLD1 synergize with activated G(alpha)q to stimulate PLC-beta(1) signaling. Coordinate interaction with activated G(alpha)q may serve as an important mechanism to fine tune response to ligands while preventing spurious initiation of PLC-beta signaling by PA in cells.

Sour ageusia in two individuals implicates ion channels of the ASIC and PKD families in human sour taste perception at the anterior tongue.

BACKGROUND: The perception of sour taste in humans is incompletely understood at the receptor cell level. We report here on two patients with an acquired sour ageusia. Each patient was unresponsive to sour stimuli, but both showed normal responses to bitter, sweet, and salty stimuli. METHODS AND FINDINGS: Lingual fungiform papillae, containing taste cells, were obtained by biopsy from the two patients, and from three sour-normal individuals, and analyzed by RT-PCR. The following transcripts were undetectable in the patients, even after 50 cycles of amplification, but readily detectable in the sour-normal subjects: acid sensing ion channels (ASICs) 1a, 1beta, 2a, 2b, and 3; and polycystic kidney disease (PKD) channels PKD1L3 and PKD2L1. Patients and sour-normals expressed the taste-related phospholipase C-beta2, the delta-subunit of epithelial sodium channel (ENaC) and the bitter receptor T2R14, as well as beta-actin. Genomic analysis of one patient, using buccal tissue, did not show absence of the genes for ASIC1a and PKD2L1. Immunohistochemistry of fungiform papillae from sour-normal subjects revealed labeling of taste bud cells by antibodies to ASICs 1a and 1beta, PKD2L1, phospholipase C-beta2, and delta-ENaC. An antibody to PKD1L3 labeled tissue outside taste bud cells. CONCLUSIONS: These data suggest a role for ASICs and PKDs in human sour perception. This is the first report of sour ageusia in humans, and the very existence of such individuals ("natural knockouts") suggests a cell lineage for sour that is independent of the other taste modalities.

Gq-initiated cardiomyocyte hypertrophy is mediated by phospholipase Cbeta1b.

Activation of the heterotrimeric G protein Gq causes cardiomyocyte hypertrophy in vivo and in cell culture models. Hypertrophic responses induced by pressure or volume overload are exacerbated by increased Gq activity and ameliorated by Gq inhibition. Gq activates phospholipase Cbeta (PLCbeta) subtypes, resulting in generation of the intracellular messengers inositol(1,4,5)tris-phosphate [Ins(1,4,5)P(3)] and sn-1,2-diacylglycerol (DAG), which regulate intracellular Ca(2+) and conventional protein kinase C subtypes, respectively. Gq can also signal independently of PLCbeta, and the involvement of either Ins(1,4,5)P(3) or DAG in cardiomyocyte hypertrophy has not been unequivocally established. Overexpression of one splice variant of PLCbeta1, specifically PLCbeta1b, in neonatal rat cardiomyocytes causes increased cell size, elevated protein/DNA ratio, and heightened expression of the hypertrophy-related marker gene, atrial natriuretic peptide. The other splice variant, PLCbeta1a, had no effect. Expression of a 32-aa C-terminal PLCbeta1b peptide, which competes with PLCbeta1b for sarcolemmal association, prevented PLC activation and eliminated hypertrophic responses initiated by Gq or Gq-coupled alpha(1)-adrenergic receptors. In contrast, a PLCbeta1a C-terminal peptide altered neither PLC activity nor cellular hypertrophy. We conclude that hypertrophic responses initiated by Gq are mediated specifically by PLCbeta1b. Preventing PLCbeta1b association with the sarcolemma may provide a useful therapeutic target to limit hypertrophy.

Compartmentation of the cerebellar nuclei of the mouse.

The cerebellar nuclei integrate inhibitory input from Purkinje cells with excitatory input from mossy and climbing fiber collaterals and are the sole cerebellar output. Numerous studies have shown that the cerebellar cortex is highly compartmentalized into hundreds of genetically determined, reproducible topographic units--transverse zones and parasagittal stripes--that can be identified through the expression patterns of numerous molecules. The Purkinje cell stripes project to the cerebellar nuclei. However, there is no known commensurate topographic complexity in the cerebellar nuclei. Rather, conventional anatomical descriptions identify four major subdivisions--the medial, anterior and posterior interposed, and lateral nuclei--together with a few intranuclear subdivisions. To begin to address the apparent complexity gap, we have used a panel of antigens and transgenes to reveal a reproducible molecular heterogeneity in the mouse cerebellar nuclei. Based on the differential expression patterns, singly and in combination, a new cerebellar nuclear topographic map has been constructed. This reveals the subdivision of the cerebellar nuclei into at least 12 reproducible expression domains. We hypothesize that such heterogeneity is the counterpart of the zones and stripes of the cerebellar cortex.

Compartmentation of the cerebellar cortex of hummingbirds (Aves: Trochilidae) revealed by the expression of zebrin II and phospholipase C beta 4.

The parasagittal organization of the mammalian cerebellar cortex into zones has been well characterized by immunohistochemical, hodological and physiological studies in recent years. The pattern of these parasagittal bands across the cerebellum is highly conserved across mammals, but whether a similar conservation of immunohistochemically defined parasagittal bands occurs within birds has remained uncertain. Here, we examine the compartmentation of the cerebellar cortex of a group of birds with unique cerebellar morphology-hummingbirds (Trochilidae). Immunohistochemical techniques were used to characterize the expression of zebrin II (aldolase C) and phospholipase C beta 4 (PLC beta 4) in the cerebellar cortex of two hummingbird species. A series of zebrin II immunopositive/immunonegative parasagittal stripes was apparent across most folia representing three major transverse zones: an anterior zone with a central stripe flanked by three lateral stripes on either side; a central zone of high/low immunopositive stripes; and a posterior zone with a central stripe flanked by four to six lateral stripes on either side. In addition, both folia I and X were uniformly immunopositive. The pattern of PLC beta 4 immunoreactivity was largely complementary-PLC beta 4 positive stripes were zebrin II negative and vice versa. The similarity of zebrin II expression between the hummingbirds and the pigeon indicates that the neurochemical compartmentation of the cerebellar cortex in birds is highly conserved, but species differences in the number and width of stripes do occur.

mGluR5-PLCbeta4-PKCbeta2/PKCgamma pathways in hippocampal CA1 pyramidal neurons in pilocarpine model of status epilepticus in mGluR5+/+ mice.

While it is generally accepted that phospholipase C (PLC) and protein kinase C (PKC) are down-stream proteins involved in metabotropic glutamate receptor 5 (mGluR5)-related signal transduction, we still do not know which subtype of PLC or PKC is specifically regulated after mGluR5 activation. In the present study in mGluR5 wild-type (mGluR5+/+) mice, we showed induced PKCbeta2 or PKCgamma expression at the border between the stratum oriens and alveus (O/A border) at 2h during pilocarpine induced status epilepticus (SE), and in the stratum pyramidale in CA1 area at 1 day after pilocarpine induced SE; at 1 day, induced expression of PLCbeta4 in the stratum pyramidale of CA1 area was observed. Furthermore, double labeling revealed the co-localization of induced PKCbeta2 or PKCgamma with mGluR5 or with induced PLCbeta4 in the stratum pyramidale of CA1 area. These induced expression, however, were not found in mGluR5 mutant (mGluR5-/-) mice. It suggests that induced PLCbeta4-PKCbeta2/PKCgamma at 1 day after pilocarpine induced SE in pyramidal neurons or PKCbeta2 or PKCgamma in interneurons at O/A border at 2h during pilocarpine induced SE may be specifically linked to the activation of mGluR5. When compared to mGluR5+/+ mice, significant shorter latency (from pilocarpine injection to the occurrence of status epilepticus) and maintenance period (from beginning to the end of status epilepticus) for status epilepticus in mGluR5-/- mice were also demonstrated. It is possible that mGluR5 may play a negative role in initiation of status epilepticus by interacting with muscarinic acetylcholine receptor in mGluR5+/+ mice.

Corticotropin-releasing hormone stimulates SGK-1 kinase expression in cultured hippocampal neurons via CRH-R1.

Corticotropin-releasing hormone (CRH) has been shown to exhibit various functions in hippocampus. In the present study, we examined the effect of CRH on the expression of serum/glucocorticoid-inducible protein kinase-1 (SGK-1), a novel protein kinase, in primary cultured hippocampal neurons. A dose-dependent increase in mRNA and protein levels of SGK-1 as well as frequency of SGK-1-positive neurons occurred upon exposure to CRH (1 pmol/l to 10 nmol/l). These effects can be reversed by the specific CRH-R1 antagonist antalarmin but not by the CRH-R2 antagonist astressin 2B. Blocking adenylate cyclase (AC) activity with SQ22536 and PKA with H89 completely prevented CRH-induced mRNA and protein expression of SGK-1. Blockage of PLC or PKC did not block CRH-induced SGK-1 expression. Our results suggest that CRH act on CRH-R1 to stimulate SGK-1 mRNA and protein expression in cultured hippocampal neurons via a mechanism that is involved in AC/PKA signaling pathways.

Ins(1,4,5)P(3) regulates phospholipase Cbeta1 expression in cardiomyocytes.

The functional significance of the Ca2+-releasing second messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3), IP(3)) in the heart has been controversial. Ins(1,4,5)P(3) is generated from the precursor lipid phosphatidylinositol(4,5)bisphosphate (PIP(2)) along with sn-1,2-diacylglycerol, and both of these are important cardiac effectors. Therefore, to evaluate the functional importance of Ins(1,4,5)P(3) in cardiomyocytes (NRVM), we overexpressed IP(3) 5-phosphatase to increase degradation. Overexpression of IP(3) 5-phosphatase reduced Ins(1,4,5)P(3) responses to alpha(1)-adrenergic receptor agonists acutely, but with longer stimulation, caused an overall increase in phospholipase C (PLC) activity, associated with a selective increase in expression of PLCbeta1, that served to normalise Ins(1,4,5)P(3) content. Similar increases in PLC activity and PLCbeta1 expression were observed when Ins(1,4,5)P(3) was sequestered onto the PH domain of PLCdelta1, a high affinity selective Ins(1,4,5)P(3)-binding motif. These findings suggested that the available level of Ins(1,4,5)P(3) selectively regulates the expression of PLCbeta1. Cardiac responses to Ins(1,4,5)P(3) are mediated by type 2 IP(3)-receptors. Hearts from IP(3)-receptor (type 2) knock-out mice showed heightened PLCbeta1 expression. We conclude that Ins(1,4,5)P(3) and IP(3)-receptor (type 2) regulate PLCbeta1 and thereby maintain levels of Ins(1,4,5)P(3). This implies some functional significance for Ins(1,4,5)P(3) in the heart.

Decreased expression of phospholipase C-beta 1 protein in endoplasmic reticulum stress-loaded neurons.

The endoplasmic reticulum (ER) plays a critical role in the maintenance of intracellular homeostasis and its dysfunction is thought to lead to neuronal death, which results in neurodegenerative disorders. Since phospholipase C (PLC) isozymes are involved in maintenance of the intracellular Ca2+ concentration by regulating Ca2+ release from the ER, their expression might be affected by ER stress. Of these isozymes, PLC-beta 1 and -gamma 1, in particular, are known to protect cells from oxidative stress and thus alteration of their expression profile under ER stress-loaded conditions is interesting. Using primary cultured rat cortical neurons, we here examined whether expression of PLC-beta 1 and -gamma 1 was altered in ER stress-loaded neurons induced by tunicamycin (Tm). In ER stress-loaded neurons treated with Tm in the range of 0.03-3 microg/ml for 20 h, the viability of the neurons was decreased dose-dependently, the decrease being significant with 0.3 or more microg/ml, and expression of the representative ER stress markers, GRP78/BiP, and cleaved caspase-3 and -12, was increased after 24 h postincubation, confirming the induction of ER stress in the neurons. In the ER stress-loaded neurons obtained on Tm treatment, the expression level of PLC-beta 1 decreased dose-dependently. On the other hand, there was no difference in the PLC-gamma 1 protein expression level between control and ER stress-loaded neurons. Overall, we demonstrated that ER stress decreases the expression of PLC-beta 1, but not -gamma 1, in neurons.