Grass carp somatolactin: I. Evidence for PACAP induction of somatolactin-α and -β gene expression via activation of pituitary PAC-I receptors

2008 ◽  
Vol 295 (2) ◽  
pp. E463-E476 ◽  
Author(s):  
Quan Jiang ◽  
Wendy K. W. Ko ◽  
Ethan A. Lerner ◽  
K. M. Chan ◽  
Anderson O. L. Wong
Keyword(s):  
Gene Expression ◽  
Grass Carp ◽  
Primary Cultures ◽  
Single Copy ◽  
Nerve Fibers ◽  
Pars Intermedia ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Rapid Amplification

Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. At present, SL can be identified only in fish but not in tetrapods and its regulation at the pituitary level has not been fully characterized. Using grass carp as a model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL secretion and synthesis at the pituitary cell level. As a first step, the structural identity of grass carp SL, SLα and SLβ, was established by 5′/3′-rapid amplification of cDNA ends. These two SL isoforms are single-copy genes and are expressed in two separate populations of pituitary cells located in the pars intermedia. In the carp pituitary, PACAP nerve fibers were detected in the nerve tracts of the neurohypophysis and extended into the vicinity of pituitary cells forming the pars intermedia. In primary cultures of grass carp pituitary cells, PACAP was effective in stimulating SL release, cellular SL content, and total SL production. The increase in SL production also occurred with parallel rises in SLα and SLβ mRNA levels. With the use of a combination of molecular and pharmacological approaches, PACAP-induced SL release and SL gene expression were shown to be mediated by pituitary PAC-I receptors. These findings, as a whole, suggest that PACAP may serve as a hypophysiotropic factor in fish stimulating SL secretion and synthesis at the pituitary level. Apparently, PACAP-induced SL production is mediated by upregulation of SLα and SLβ gene expression through activation of PAC-I receptors.

Grass carp somatolactin: II. Pharmacological study on postreceptor signaling mechanisms for PACAP-induced somatolactin-α and -β gene expression

2008 ◽  
Vol 295 (2) ◽  
pp. E477-E490 ◽  
Author(s):  
Quan Jiang ◽  
Mulan He ◽  
Xinyan Wang ◽  
Anderson O. L. Wong
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Grass Carp ◽  
Pharmacological Study ◽  
Primary Cultures ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Fish Model ◽  
Subsequent Activation

Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. However, the signal transduction mechanisms responsible for SL expression are still largely unknown. Using grass carp as an animal model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL gene expression at the pituitary level. In primary cultures of grass carp pituitary cells, SLα and SLβ mRNA levels could be elevated by PACAP via activation of PAC-I receptors. With the use of a pharmacological approach, the AC/cAMP/PKA and PLC/inositol 1,4,5-trisphosphate (IP3)/PKC pathways and subsequent activation of the Ca2+/calmodulin (CaM)/CaMK-II cascades were shown to be involved in PACAP-induced SLα mRNA expression. Apparently, the downstream Ca2+/CaM-dependent cascades were triggered by extracellular Ca2+ ([Ca2+]e) entry via L-type voltage-sensitive Ca2+ channels (VSCC) and Ca2+ release from IP3-sensitive intracellular Ca2+ stores. In addition, the VSCC component could be activated by cAMP/PKA- and PLC/PKC-dependent mechanisms. Similar postreceptor signaling cascades were also observed for PACAP-induced SLβ mRNA expression, except that [Ca2+]e entry through VSCC, PKC coupling to PLC, and subsequent activation of CaMK-II were not involved. These findings, taken together, provide evidence for the first time that PACAP can induce SLα and SLβ gene expression in fish model via PAC-I receptors through differential coupling to overlapping and yet distinct signaling pathways.

Somatostatin-28 inhibitory action on somatolactin-α and -β gene expression in goldfish

2014 ◽  
Vol 307 (6) ◽  
pp. R755-R768 ◽  
Author(s):  
Quan Jiang ◽  
Anderson O. L. Wong
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Primary Cultures ◽  
Inhibitory Effect ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Structural Identity ◽  
Rapid Amplification ◽  
Differential Coupling

Somatostain (SS) is known to inhibit growth hormone (GH) and prolactin (PRL) secretion. Somatolactin (SL) is a member of the GH/PRL family, but its regulation by goldfish brain somatostatin-28 (gbSS-28) has not been examined. To this end, the structural identity of goldfish SLα was established by 5′/3′-rapid amplification of cDNA ends. As revealed by in situ hybridization and immunohistochemical staining, the expression of SL isoforms was detected in pituitary cells located in the neurointermediate lobe (NIL). The transcripts of goldfish SS receptor 5a (Sst5a) but not Sst1b, Sst2, or Sst3a were detected in the goldfish NIL cells by RT-PCR. In goldfish pituitary cells, gbSS-28 not only had an inhibitory effect on basal SLα and SLβ mRNA levels but also could abolish insulin-like growth factor-stimulated SL gene expression. In primary cultures of goldfish NIL cells, gbSS-28 reduced forskolin-stimulated total cAMP production. With the use of a pharmacological approach, the adenylate cyclase (AC)/cAMP and phospholipase C (PLC)/inositol trisphosphate (IP3)/protein kinase C (PKC) cascades were shown to be involved in gbSS-28-inhibited SLα mRNA expression. Similar postreceptor signaling cascades were also observed for gbSS-28-reduced SLβ mRNA expression, except that PKC coupling to PLC was not involved. These results provide evidence that gbSS-28 can inhibit SLα and SLβ gene expression at the goldfish pituitary level via Sst5 through differential coupling of AC/cAMP and PLC/IP3/PKC cascades.

scholarly journals Signal Transduction Mechanisms for Glucagon-Induced Somatolactin Secretion and Gene Expression in Nile Tilapia (Oreochromis niloticus) Pituitary Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Chaoyi Zhang ◽  
Anji Lian ◽  
Yue Xu ◽  
Quan Jiang
Keyword(s):  
Gene Expression ◽  
Oreochromis Niloticus ◽  
Nile Tilapia ◽  
Primary Cultures ◽  
Hormone Secretion ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Mrna Levels

Glucagon (GCG) plays a stimulatory role in pituitary hormone regulation, although previous studies have not defined the molecular mechanism whereby GCG affects pituitary hormone secretion. To this end, we identified two distinct proglucagons, Gcga and Gcgb, as well as GCG receptors, Gcgra and Gcgrb, in Nile tilapia (Oreochromis niloticus). Using the cAMP response element (CRE)-luciferase reporter system, tilapia GCGa and GCGb could reciprocally activate the two GCG receptors expressed in human embryonic kidney 293 (HEK293) cells. Quantitative real-time PCR analysis revealed that differential expression of the Gcga and Gcgb and their cognate receptors Gcgra and Gcgrb was found in the various tissues of tilapia. In particular, the Gcgrb is abundantly expressed in the neurointermediate lobe (NIL) of the pituitary gland. In primary cultures of tilapia NIL cells, GCGb effectively stimulated SL release, with parallel rises in the mRNA levels, and co-incubation with the GCG antagonist prevented GCGb-stimulated SL release. In parallel experiments, GCGb treatment dose-dependently enhanced intracellular cyclic adenosine monophosphate (cAMP) accumulation with increasing inositol 1,4,5-trisphosphate (IP3) concentration and the resulting in transient increases of Ca2+ signals in the primary NIL cell culture. Using selective pharmacological approaches, the adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) and phospholipase C (PLC)/IP3/Ca2+/calmodulin (CaM)/CaMK-II pathways were shown to be involved in GCGb-induced SL release and mRNA expression. Together, these results provide evidence for the first time that GCGb can act at the pituitary level to stimulate SL release and gene expression via GCGRb through the activation of the AC/cAMP/PKA and PLC/IP3/Ca2+/CaM/CaMK-II cascades.

scholarly journals Modulation of Calmodulin Gene Expression as a Novel Mechanism for Growth Hormone Feedback Control by Insulin-like Growth Factor in Grass Carp Pituitary Cells

Endocrinology ◽  
2005 ◽  
Vol 146 (9) ◽  
pp. 3821-3835 ◽  
Author(s):  
Longfei Huo ◽  
Guodong Fu ◽  
Xinyan Wang ◽  
Wendy K. W. Ko ◽  
Anderson O. L. Wong
Keyword(s):  
Gene Expression ◽  
Feedback Control ◽  
Grass Carp ◽  
Cell Model ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Hormone Synthesis ◽  
Igf I ◽  
Gh Gene

Abstract Calmodulin (CaM), the Ca2+ sensor in living cells, is essential for biological functions mediated by Ca2+-dependent mechanisms. However, modulation of CaM gene expression at the pituitary level as a means to regulate pituitary hormone synthesis has not been characterized. In this study we examined the functional role of CaM in the feedback control of GH by IGF using grass carp pituitary cells as a cell model. To establish the structural identity of CaM expressed in the grass carp, a CaM cDNA, CaM-L, was isolated from the carp pituitary using 3′/5′ rapid amplification of cDNA ends. The open reading frame of this cDNA encodes a 149-amino acid protein sharing the same primary structure with CaMs reported in mammals, birds, and amphibians. This CaM cDNA is phylogenetically related to the CaM I gene family, and its transcripts are ubiquitously expressed in the grass carp. In carp pituitary cells, IGF-I and IGF-II induced CaM mRNA expression with a concurrent drop in GH transcript levels. These stimulatory effects on CaM mRNA levels were not mimicked by insulin and appeared to be a direct consequence of IGF activation of CaM gene transcription without altering CaM transcript stability. CaM antagonism and inactivation of calcineurin blocked the inhibitory effects of IGF-I and IGF-II on GH gene expression, and CaM overexpression also suppressed the 5′ promoter activity of the grass carp GH gene. These results, as a whole, provide evidence for the first time that IGF feedback on GH gene expression is mediated by activation of CaM gene expression at the pituitary level.

PKC and ERK are differentially involved in gonadotropin-releasing hormone-induced growth hormone gene expression in the goldfish pituitary

2005 ◽  
Vol 289 (6) ◽  
pp. R1625-R1633 ◽  
Author(s):  
Christian Klausen ◽  
Takeshi Tsuchiya ◽  
John P. Chang ◽  
Hamid R. Habibi
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Primary Cultures ◽  
Gonadotropin Releasing Hormone ◽  
Growth Hormone Gene ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Releasing Hormone ◽  
Gh Secretion ◽  
Gh Gene

Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus and stimulates the synthesis and secretion of gonadotropin hormones. In addition, GnRH also stimulates the production and secretion of growth hormone (GH) in some fish species and in humans with certain clinical disorders. In the goldfish pituitary, GH secretion and gene expression are regulated by two endogenous forms of GnRH known as salmon GnRH and chicken GnRH-II. It is well established that PKC mediates GnRH-stimulated GH secretion in the goldfish pituitary. In contrast, the signal transduction of GnRH-induced GH gene expression has not been elucidated in any model system. In this study, we demonstrate, for the first time, the presence of novel and atypical PKC isoforms in the pituitary of a fish. Moreover, our results indicate that conventional PKCα is present selectively in GH-producing cells. Treatment of primary cultures of dispersed goldfish pituitary cells with PKC activators (phorbol ester or diacylglycerol analog) did not affect basal or GnRH-induced GH mRNA levels, and two different inhibitors of PKC (calphostin C and GF109203X) did not reduce the effects of GnRH on GH gene expression. Together, these results suggest that, in contrast to secretion, conventional and novel PKCs are not involved in GnRH-stimulated increases in GH mRNA levels in the goldfish pituitary. Instead, PD98059 inhibited GnRH-induced GH gene expression, suggesting that the ERK signaling pathway is involved. The results presented here provide novel insights into the functional specificity of GnRH-induced signaling and the regulation of GH gene expression.

Insulin-like growth factor as a novel stimulator for somatolactin secretion and synthesis in carp pituitary cells via activation of MAPK cascades

2011 ◽  
Vol 301 (6) ◽  
pp. E1208-E1219 ◽  
Author(s):  
Quan Jiang ◽  
Wendy K. W. Ko ◽  
Anderson O. L. Wong
Keyword(s):  
Growth Factor ◽  
P38 Mapk ◽  
Grass Carp ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Insulin Like Growth Factor ◽  
Cell Content ◽  
Mapk Cascades ◽  
Igf I

Somatolactin (SL), a member of the growth hormone/prolactin family, is a pituitary hormone unique to fish models. Although SL is known to have diverse functions in fish, the mechanisms regulating its secretion and synthesis have not been fully characterized. Using grass carp pituitary cells as a model, here we examined the role of insulin-like growth factor (IGF) in SL regulation at the pituitary level. As a first step, the antisera for the two SL isoforms expressed in the carp pituitary, SLα and SLβ, were produced, and their specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the carp pituitary. Western blot using these antisera revealed that grass carp SLα and SLβ could be N-linked glycosylated and their basal secretion and cell content in carp pituitary cells could be elevated by IGF-I and -II treatment. These stimulatory effects occurred with parallel rises in SLα and SLβ mRNA levels, and these SL gene expression responses were not mimicked by insulin but blocked by IGF-I receptor inactivation. In carp pituitary cells, IGF-I and -II could induce rapid phosphorylation of IGF-I receptor, MEK1/2, ERK1/2, MKK3/6, and p38 MAPK; and SLα and SLβ secretion, protein production, and mRNA expression caused by IGF-I and -II stimulation were negated by inactivating MEK1/2 and p38 MAPK. Parallel inhibition of PI3K and Akt, however, were not effective in these regards. These results, taken together, provide evidence that IGF can upregulate SL secretion and synthesis at the pituitary level via stimulation of MAPK- but not PI3K/Akt-dependent pathways.

scholarly journals Paracrine regulation of growth hormone gene expression by gonadotrophin release in grass carp pituitary cells: functional implications, molecular mechanisms and signal transduction

2005 ◽  
Vol 34 (2) ◽  
pp. 415-432 ◽  
Author(s):  
Hong Zhou ◽  
Yonghua Jiang ◽  
Wendy K W Ko ◽  
Wensheng Li ◽  
Anderson O L Wong
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Adenylate Cyclase ◽  
Mrna Expression ◽  
Grass Carp ◽  
Janus Kinase ◽  
Growth Hormone Gene ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Gh Gene

Growth hormone (GH) is known to stimulate luteinizing hormone (LH) release via paracrine interactions between somatotrophs and gonadotrophs. However, it is unclear if LH can exert a reciprocal effect to modulate somatotroph functions. Here we examined the paracrine effects of LH on GH gene expression using grass carp pituitary cells as a cell model. LH receptors were identified in grass carp somatotrophs and their activation by human chorionic gonadotropin (hCG) increased ‘steady-state’ GH mRNA levels. Removal of endogenous LH by immunoneutralization using LH antiserum inhibited GH release and GH mRNA expression. GH secretagogues, including gonadotrophin releasing hormone (GnRH), pituitary adenylate cyclase-activating polypeptide (PACAP) and apomorphine, were effective in elevating GH mRNA levels but these stimulatory actions were blocked by LH antiserum. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was not affected by hCG but was enhanced by LH immunoneutralization. Treatment with LH antiserum also suppressed basal levels of mature GH mRNA and primary transcripts. hCG increased cAMP synthesis in carp pituitary cells and hCG-induced GH mRNA expression was mimicked by forskolin but suppressed by inhibiting adenylate cyclase and protein kinase A. Similarly, the stimulatory actions of hCG and forskolin on GH mRNA expression were blocked by inhibiting Janus kinase 2 (JAK2) and MAP kinase (MAPK), including P42/44MAPK and P38 MAPK. These results suggest that LH is essential for the maintenance of GH release, GH gene expression, and somatotroph responsiveness to GH-releasing factors. The paracrine actions of LH on GH mRNA expression are mediated by a concurrent increase in GH gene transcription and GH mRNA turnover, probably through JAK2/MAPK coupled to the cAMP-dependent pathway.

scholarly journals Differential Effects of Gonadotropin-Releasing Hormone (GnRH) Pulse Frequency on Gonadotropin Subunit and GnRH Receptor Messenger Ribonucleic Acid Levels in Vitro*

Endocrinology ◽  
1997 ◽  
Vol 138 (3) ◽  
pp. 1224-1231 ◽  
Author(s):  
Ursula B. Kaiser ◽  
Andrzej Jakubowiak ◽  
Anna Steinberger ◽  
William W. Chin
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Pulse Frequency ◽  
Gnrh Receptor ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Subunit Gene ◽  
Differential Effects ◽  
Messenger Ribonucleic Acid

Abstract The hypothalamic hormone, GnRH, is released and transported to the anterior pituitary in a pulsatile manner, where it binds to specific high-affinity receptors and regulates gonadotropin biosynthesis and secretion. The frequency of GnRH pulses changes under various physiological conditions, and varying GnRH pulse frequencies have been shown to regulate differentially the secretion of LH and FSH and the expression of the gonadotropin α, LHβ, and FSHβ subunit genes in vivo. We demonstrate differential effects of varying GnRH pulse frequency in vitro in superfused primary monolayer cultures of rat pituitary cells. Cells were treated with 10 nm GnRH pulses for 24 h at a frequency of every 0.5, 1, 2, or 4 h. α, LHβ, and FSHβ messenger RNA (mRNA) levels were increased by GnRH at all pulse frequencies. α and LHβ mRNA levels and LH secretion were stimulated to the greatest extent at a GnRH pulse frequency of every 30 min, whereas FSHβ mRNA levels and FSH secretion were stimulated maximally at a lower GnRH pulse frequency, every 2 h. GnRH receptor (GnRHR) mRNA levels also were increased by GnRH at all pulse frequencies and were stimulated maximally at a GnRH pulse frequency of every 30 min. Similar results were obtained when the dose of each pulse of GnRH was adjusted to maintain a constant total cumulative dose of GnRH over 24 h. These data show that gonadotropin subunit gene expression is regulated differentially by varying GnRH pulse frequencies in vitro, suggesting that the differential effects of varying GnRH pulse frequencies on gonadotropin subunit gene expression occur directly at the level of the pituitary. The pattern of regulation of GnRHR mRNA levels correlated with that of α and LHβ but was different from that of FSHβ. This suggests that α and LHβ mRNA levels are maximally stimulated when GnRHR levels are relatively high, whereas FSHβ mRNA levels are maximally stimulated at lower levels of GnRHR expression, and that the mechanism for differential regulation of the gonadotropins by varying pulse frequencies of GnRH may involve levels of GnRHR. Furthermore, these data suggest that the mechanisms whereby varying GnRH pulse frequencies stimulate α, LHβ, and GnRHR gene expression are similar, whereas the stimulation of FSHβ mRNA levels may be different.

scholarly journals In situ hybridization analysis of anterior pituitary hormone gene expression during fetal mouse development.

1994 ◽  
Vol 42 (8) ◽  
pp. 1117-1125 ◽  
Author(s):  
M A Japón ◽  
M Rubinstein ◽  
M J Low
Keyword(s):  
Gene Expression ◽  
Anterior Pituitary ◽  
Beta Subunit ◽  
Pars Intermedia ◽  
Pituitary Hormone ◽  
Pars Distalis ◽  
Mouse Development ◽  
Glycoprotein Hormone ◽  
Anterior Pituitary Hormone ◽  
Stimulating Hormone

We used 35S-labeled oligonucleotides and cRNAs (riboprobes) to detect the temporal order and spatial pattern of anterior pituitary hormone gene expression in (B6CBF1 x B6CBF1)F2 fetal mice from embryonic Day 9.5 (E9.5) to postnatal Day 1 (P1). Pro-opiomelanocortin (POMC) mRNA was expressed in the basal diencephalon on Day E10.5, in the ventromedial zone of the pars distalis on Day E12.5, and in the pars intermedia on Day E14.5. The common alpha-glycoprotein subunit (alpha-GSU) mRNA first appeared in the anterior wall of Rathke's pouch on Day E11.5 and extended to the pars tuberalis and ventromedial zone of the pars distalis on Day E12.5. Thyroid-stimulating hormone-beta (TSH beta) subunit mRNA was expressed initially in both the pas tuberalis and ventromedial pars distalis on Day E14.5, with an identical spatial distribution to alpha-GSU at the time. In contrast, luteinizing hormone-beta (LH beta) subunit and follicle-stimulating hormone beta (FSH beta) subunit mRNAs were detected initially only in the ventromedial pars distalis on Days E16.5 and E17.5, respectively, in an identical distribution to each other. POMC-, alpha-GSU-, TSH beta, LH beta-, and FSH beta-positive cells within the pars distalis all increased in number and autoradiographic signal with differing degrees of spatial expansion posteriorly, laterally, and dorsally up to Day P1. POMC expression was typically the most intense and extended circumferentially to include the entire lateral and dorsal surfaces of the pars distalis. The expression of both growth hormone (GH) and prolactin (PRL) started coincidentally on Day E15.5. However PRL cells localized in the ventromedial area similarly to POMC and the glycoprotein hormone subunits, whereas GH cells were found initially in a more lateral and central distribution within the lobes of the pars distalis. Somatotrophs increased dramatically in number and autoradiographic signal, extending throughout the pars distalis except for the most peripheral layer of cells on Day E17.5. Mammotrophs also increased in number but less abundantly than somatotrophs, and PRL expression remained more confined to central-medial and ventrolateral areas of the pars distalis up to Day P1. These data demonstrate distinctive patterns of expression for each of the major anterior pituitary hormone genes during development of the mouse pituitary gland and suggest that different groups of committed cells are the immediate precursors to the terminally differentiated hormone-secreting cell types.

Phorbol 12-myristate 13-acetate alters SR Ca(2+)-ATPase gene expression in cultured neonatal rat heart cells

1996 ◽  
Vol 271 (3) ◽  
pp. H1031-H1039 ◽  
Author(s):  
M. Qi ◽  
J. W. Bassani ◽  
D. M. Bers ◽  
A. M. Samarel
Keyword(s):  
Gene Expression ◽  
Sarcoplasmic Reticulum ◽  
Mrna Stability ◽  
Primary Cultures ◽  
Neonatal Rat ◽  
Response To Treatment ◽  
Similar Degree ◽  
Heart Cells ◽  
Mrna Levels ◽  
Atpase Gene

Primary cultures of neonatal rat ventricular myocytes were used to examine how the cardiac myocyte cytoplasmic Ca2+ ([Ca2+]i) transient and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) gene expression change in response to treatment with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). Exposure of neonatal myocytes to PMA (200 nM, 48-72 h) produced myocyte growth and a 70% prolongation of the half-time for [Ca2+]i decline induced by potassium depolarization in the absence of extracellular Na+ (in which the sarcoplasmic reticulum Ca2+ pump is the main mechanism responsible for [Ca2+]i decline). The reduced rate of [Ca2+]i transient decline corresponded to a 53% reduction in SERCA2 protein levels and a 43% reduction in SERCA2 mRNA levels as compared with control myocytes. Exposure to PMA for as little as 30 min or for as long as 48 h produced a similar degree of SERCA2 mRNA downregulation over time. PMA-induced downregulation of SERCA2 mRNA levels was blocked by either 10 nM staurosporine or 4 microM chelerythrine, whereas treatment with either agent alone increased SERCA2 mRNA levels as compared with control cells. Actinomycin D mRNA stability assays revealed that PMA treatment appeared to markedly destabilize the relatively long-lived SERCA2 mRNA transcript. Taken together, these results indicate that downregulation of SERCA2 gene by PMA in cultured neonatal myocytes occurs at least in part by alterations in mRNA stability and results in functional alterations in [Ca2+]i decline that are similar to that observed in the hypertrophied and failing adult myocardium.

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