Human SNF2L Gene Is Regulated Constitutively and Inducibly in Neural Cells via a cAMP-Response Element

PURPOSE: SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells. MATERIALS AND METHODS: To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR. RESULTS: Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons. CONCLUSION: These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.

Role of cGMP and cAMP in the hemodynamic response to intrathecal sildenafil administration

INTRODUCTION: Results from our laboratory have demonstrated that intracerebroventricular administration of sildenafil to conscious rats promoted a noticeable increase in both lumbar sympathetic activity and heart rate, with no change in the mean arterial pressure. The intracerebroventricular administration of sildenafil may have produced the hemodynamic effects by activating sympathetic preganglionic neurons in the supraspinal regions and spinal cord. It is well documented that sildenafil increases intracellular cGMP levels by inhibiting phosphodiesterase type 5 and increases cAMP levels by inhibiting other phosphodiesterases. OBJECTIVE: To examine and compare, in conscious rats, the hemodynamic response following the intrathecal administration of sildenafil, 8-bromo-cGMP (an analog of cGMP), forskolin (an activator of adenylate cyclase), or dibutyryl-cAMP (an analog of cAMP) in order to elucidate the possible role of the sympathetic preganglionic neurons in the observed hemodynamic response. RESULTS: The hemodynamic responses observed following intrathecal administration of the studied drugs demonstrated the following: 1) sildenafil increased the mean arterial pressure and heart rate in a dose-dependent manner, 2) increasing doses of 8-bromo-cGMP did not alter the mean arterial pressure and heart rate, 3) forskolin did not affect the mean arterial pressure but did increase the heart rate and 4) dibutyryl-cAMP increased the mean arterial pressure and heart rate, similar to the effect observed following the intrathecal injection of the highest dose of sildenafil. CONCLUSION: Overall, the findings of the current study suggest that the cardiovascular response following the intrathecal administration of sildenafil to conscious rats involves the inhibition of phosphodiesterases other than phosphodiesterase type 5 that increase the cAMP level and the activation of sympathetic preganglionic neurons.

Slow Ca2+ channels neither contribute to upstroke of action potential nor to pacemaker potential in spontaneously active freshly isolated three day embryonic chick ventricle.

Contribution of slow Ca2+ channels to the upstroke of action potential (AP) and pacemaker potential was studied by observing the effects of Ca2+ channel activators- high [Ca2+]0, Bay-K-8644, isoproterenol, forskolin and dibutyryl-cAMP on spontaneous AP of freshly isolated 3 day embryonic chick ventricle (3 day ECV). The spontaneous APs showed maximal upstroke velocity (+Vmax), maximum diastolic potential (MDP), overshoot (Eov) and AP duration at -20 mv (APD20) of 42.60 +/- 2.40 V/sec, -59.05 +/- 0.95 my, 16.30 +/- 0.53 mv and 70.32 +/- 4.60 msec, respectively (an average value of 35 preparations). Bay-K-8644 (0.1-0.8 microM), isoproterenol (5-10 pM) and forskolin (0.1-2.0 microM) induced a concentration-dependent increase in APD20 and Eov without affecting +Vmax. Dibutyryl-cAMP (1 microM) also enhanced the APD20 and Eov and had no effect on +Vmax. Elevation of [Ca2+]0 from 0.6 mM to 9.6 mM caused a concentration-dependent increase in APD20 and Eov leaving +Vmax unaltered. Elevated [Ca2+] and the other Ca2+ channel activators had no significant effect on MDP in above concentration range. Increase in APD20 and Eov could be explained at least by activation of slow Ca2+ channels but the lack of any change in +Vmax clearly suggests that the slow Ca2+ channels do not contribute to the upstroke of AP. All these interventions reduced the rate of spontaneous firing without any noticeable effect on MDP. This finding shows that the slow Ca2+ channels also do not contribute directly to the generation of pacemaker potential in spontaneously active freshly isolated 3 day ECV.

Phosphorylation of phospholipase D1 and the modulation of its interaction with RhoA by cAMP-dependent protein kinase

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.

CD99 type II is a determining factor for the differentiation of primitive neuroectodermal cells

CD99 is a 32-kDa cell surface molecule present on thymocytes, peripheral T cells, many other hematopoietic stem cells and somatic cells were implicated in cell-cell adhesion and cell-activation phenomena. Two major subtypes have been identified so far, designated CD99 type I and type II. We have investigated the correlation between the degree of neural differentiation and the expression of CD99 subtypes in three differentially differentiated cell lines such as CADO-ES1, RD-ES, and SH-N-SY5Y, in order of differentiation. In addition, we induced differentiation of the RD-ES cell line by N(6),2'-dibutyryl-cAMP (db-cAMP). Six days after treatment with db-cAMP, RD-ES cell line has changed its morphology from uniform round cells to cells with neurites, and initially CD99 type II-overexpressed RD-ES cells showed significant down-regulation of CD99 type II, whereas CD99 type I expression remained constant. When RD- ES cells were transfected with the cDNA encoding for CD99 type I-green fluorescence protein (GFP) and type II-GFP, CD99 type II transfected RD-ES cell line remained unchanged with morphology of undifferentiated form. Our data suggest that CD99 type II acts as a negative regulator in the neural differentiation of precursor cells that might occur during nerve system development.

Time sequence of changes in the responsiveness of glycogen breakdown to adrenergic agonists in perfused liver of rats with insulin-induced hypoglycemia

The time-course changes of the responsiveness of glycogen breakdown to a- and Beta-adrenergic agonists during insulin-induced hypoglycemia (IIH) were investigated. Blood glucose levels were decreased prior to the alteration in the hepatic responsiveness to adrenergic agonists. The activation of hepatic glucose production and glycogenolysis by phenylephrine (2 µM) and isoproterenol (20 µM) was decreased in IIH. The changes in the responsiveness of glycogen catabolism were first observed for isoproterenol and later for phenylephrine. Hepatic ß-adrenergic receptors showed a higher degree of adrenergic desensitization than did a-receptors. Liver glycogen synthase activity, glycogen content and the catabolic effect of dibutyryl cyclic AMP (the Beta-receptor second messenger) were not affected by IIH.

Immunocytochemical and morphological effects of short-term stimulation of cultured astrocytes

Both image analysis at light microscopy level and ultrastructural characterization by transmission electron microscopy were employed to evaluate the differentiation stage in young cultured mouse astrocytes after 1-day exposure to dBcAMP, a chemical compound known to induce cell activation. The aim was to validate an experimental model of stimuled astrocytes preserving the properties of recently seeded cells, thus avoiding the overlapping effects of in vitro aging. Differentiated astrocytes, as evidenced by GFAP labeling by streptavidin-perioxidase, doubled their number in treated cultures (45 per cent) versus controls (23 per cent). In addition, a significant increase in processing-bearing astrocytes (elongated forms) to the detriment of immature polygonal astrocytes, was recorded. No noticeable changes were found in cell perimeter, but cell area displayed a significant reduction in labeled surface of astrocytes undergoing morphological differentiation. Concomitantly, electron microscopy showed that radially organized bundles of numerous intermediate filaments compatible with GFAP replaced the few scattered structures observed in control cultures. However methodological caution is advisable as regards the relevance of the in vitro counterpart in situ reactive astrocytes, since cell plasicity is recognized to depend on culture conditions. At any rate, present quantitative results demonstrate that GFAP-positive cell percentage and cell area measurement are adequate parameters of early immunocytochemical and morphological differentiation, respectively, and thus contribute to a better histometric characterization of an easily available substrate to discriminate the wide variety of factors involved in CNS response to injury.

Cholera toxin mediated regulation of the expression of Gq alpha and G11 alpha GTP binding proteins

Previously it has been shown that persistent activation of the stimulatory adenylyl cyclase pathway with cholera toxin (CT) downregulates the Gs alpha polypeptide (80%) in a cAMP-independent manner in C6 glioma cells (Shah, 1997). This study was conducted to examine the short and long term effects of CT on the regulation of pertussis toxin-sensitive and -insensitive G proteins and their transcripts in C6 glioma cells. Treatment of C6 cells with CT (100 ng/ml) up to 16 h had no effect on either Gi or Gq/11 alpha proteins. However, prolonged exposure (24-48 h) caused increased expression of Gi (20-30%) and Gq/11 alpha proteins (40%). Urea gradient gels, which can separate Gq alpha and G11 alpha proteins, revealed that prolonged CT treatment increased the expression of both of these G proteins. The CT-mediated enhanced expression of Gq alpha and G11 alpha proteins was accompanied by increased mRNA levels of these proteins as determined by RT/PCR. Cyclic-AMP elevating agents like forskolin (10 microM) and db-cAMP (1 mM) mimicked the effect of CT on Gi but not Gq/11 alpha proteins. These studies show long term cAMP-dependent regulation of Gi and cAMP-independent expression of Gq/11 alpha proteins in C6 glioma cells.

Regulation of fibronectin gene expression by cyclic AMP and phorbol myristate acetate in HT-1080 human fibrosarcoma cells

We studied the regulation of fibronectin (FN) gene expression by cAMP and phorbol-12-myristate-13-acetate (PMA) in HT-1080 human fibrosarcoma cells. Dibutyryl cAMP increased FN synthesis and mRNA levels, while PMA inhibited the cAMP-induced FN synthesis. In transient transfection assays, cAMP increased FN promoter activity, while PMA paradoxically enhanced the cAMP-induced promoter activity. Stable transfection experiments, however, showed that neither cAMP or PMA alone nor together affected FN promoter activity. These results suggest that PMA antagonizes the cAMP-induced FN gene expression and that both the action of cAMP and the inhibition of its action by PMA may occur at the posttranscriptional level in HT-1080 cells.

Age-dependent morphological changes in dBcAMP-treated astrocytes

Since changes in cell morphology are conspicuous features of astrocyte reaction, we resorted to an histometric approach to evaluate age influence on such morphological response to activating stimuli. To this end, first subculture of rat brain astrocytes at 1, 9 or 21 days in vitro (DIV) were treated during 2 hs with l mM of dBcAMP, a chemical compound known to induce cell differentiation. Following treatment, immunoperoxidase labeling of GFAP, specific marker of astrocyte activation, was carried out. Although total count of GFAP-positive cell foci was greater in treated samples in all times tested, when such cell foci were evaluated by image analysis, differences between perimeter/area ratios of such foci were only statistifically significant at l DIV. It may be concluded that while dBcAMP effect is maintained despite astrocyte aging, the morphological pattern of response varies markedly along the observation period.

Calcium-activated potassium channels ar involved in the response of mouse Leydig cells to human chorionic gonadotropin

The patch-clamp technique was used to investigate the involvement of ion channels in the response of Leydig cells to gonadotropic hormones (viz.hCG). Recordings in the cell-attached configuration (pipette containing 140 mM KCl) showed unitary events with conductance of 187.9 + or - 5.2 pS(N = 24 patches) in about 70 percent of the cells. These channels were potassium selective and the open channel probability (Po) was always about 1 percent for displacemtne of potential from the resting value in the range of -20 to +60 mV. Treatment of the cells with hCG (2 ng/ml) led to a large increase in the frequency of openings, concomitant with a reduction in the mean closed time and there was essentially no effect on the mean open time of the channels. Dibutyryl cAMP (100 µM) produced an effect similar to that of hCG and both required external calcium for their action. No direct effect of either dibutyryl cAMP or hCG were observed in inside-out patches. Reversal potential measurements on excised inside-out patches demonstrated that the channels were highly potassium selective with unitary conductance of about 206.8 + or - 6.36 pS(mean + or - SEM of 6 measurements), and an estimated permeability of 3.6 x 10-13 + or - 0.2 x 10--13 cm3/s (mean + or - SEM for 6 measurements), in symmetrical 140 mM KCl. The activity of the channel in excised paches was very sensitive to the free-calcium concentration on the intracellular surface of the free-calcium concentration on the intracellular surface of the channel. Po evaluated at + 60mV increased from 3 percent at 10 nM to 47 percent at 100 nM free calcium. The Hill coefficient under these conditions was 1.1. These results demonstrate that Leydig cells have a Ca2+ -activated K+ channel of large unitary conductance, which can be activated upon the binding of hCG to receptors in the cell membrane

Adhesion to laminin is down-regulated upon retinoic acid-induced F9 cell differentiation: a role for alpha6/beta1 integrin

F9 mouse teratocarcinoma cells have a high capacity to adhere to laminin and we identified alpha6/beta1 integrin as the principal laminin-binding protein present in these cells. F9 cells differentiated into parietal endoderm when monolayer cultures were treated with retinoic acid and dibutyryl cyclic AMP. In this process a decreased adherence to laminin was observed due to a lower expression of alpha6/beta1 integrin on the cell surface

Positive lusitropic effect and diminished myofibrillar sensitivity to calcium produced by cAMP on toad (Bufo Arenarum Hensel) ventricle

En tiritas intactas de ventrículo de sapo, se estudió el efecto relajante o lusitrópico positivo de diferentes intervenciones qu aumentan el AMPc intracelular. El isoproterenol aumentó la tensión desarrollada (DT), la velocidad máxima de contracción (+T), y la velocidad máxima de relajación (-T aumentó proporcionalmente más que +T a concentraciones de isoproterenol desde 10-8 a 10-4M, por lo que la relación +T/-T disminuyó significativamente. Una dosis única de isoproterenol (3x10**-8M) aumentó significativamente los niveles de AMPc desde 0.174 ñ 0.022 a 0.329 ñ pmoles/mg peso húmedo y produjo un aumento en la contractilidad de 69 ñ 13% y una disminución de +T/-T de 18.5 ñ 4.55%. La administración de 10**-3M de dibutiril AMPc(dAMPc) aumentó significativamente DT y +T y disminuyó significativamente la relación +T/-T. Efectos similares produjo la administración de milrinona, un inhibidor específico de la fosfodiesterasa de AMPc. La papaverina, un inhibidor inespecífico de fosfodiesterasas, no produjo aumentos en +T, pero aumentó significativamente -T. En trabéculas desprovistas químicamente de membrana, la sensibilidad al calcio de las proteínas contráctiles aumentó significativamente por la administración de 10**-5M del inhibidor de fosfodiesterasa 3-isobutil-1-metil-xantina (IBMX). La administración de 10**-3M de dAMPc no afectó la sensibilidad al calcio de las trabéculas desprovistas de membrana. Sin embargo la misma concentración de dAMPc produjo una disminución en la sensibilidad al calcio de las proteínas contráctiles cuando se administró en presencia de IBMX o de papavarina. Los resultados indicarían que el efecto relajante del isoproterenol es mediado en el ventrículo de sapo por un aumento en los niveles de AMPc intracelular. Estos resultados sugieren además que la disminución de la sensibilidad al calcio de los miofilamentos podría ser un mecanismo por el que el AMPc produce su efecto relajante

Insulin and IGF-I stimulated RNA synthesis in primary cultures of neuronal cells: involvement of cyclic AMP and protein kinase-c

La insulina y el IGF-I promueven el crecimiento de las células neuronales de rata en cultivo primario. Con el objeto de investigar el mecanismo de transducción de señales hormonales en este sistema biológico, estudiamos el efecto de agonistas de AMP cíclico y un estimulador de la proteína kinasa-C sobre la síntesis de ARN basal e inducida por hormonas. Los agentes que aumentan los níveles de AMP cíclico endógenos (foraskolina, dibutiril-AMP cíclico, toxina colérica) bloquearon los efectos estimuladores de la insulina y el factor de crecimiento; el dibutiril AMP cíclico, sin embargo, no alteró la unión de las hormonas a sus receptores. Aunque a diferencia de los agentes antes mencionados, el ester de forbol elevó significativamente la síntesis de ARN basal; este, no obstante, inhibió la estimulación por la insulina. Este último efecto probablemente fue mediado por un incremento en los niveles de AMP cíclico, como se ha encontrado en otros tipos de células. La estaurosporina, un inhibidor de la proteína kinasa-C, también bloqueó los efectos de la insulina sobre la síntesis de RNA

Further studies on the influence of dibutyryl cAMP, theophylline and prostaglandin E1 on composition and biosynthesis of phospholipids in Microsporum gypseum.

Exogenous supplementation of dibutyryl cAMP and cAMP modulators like theophylline and prostaglandin E1 in the growth medium of Microsporum gypseum lead to increase in the levels of phosphatidylcholine and lysophosphatidylcholine and thereby in total phospholipid content. These observations were further confirmed by the increased incorporation of [32P]orthophosphoric acid into total phospholipid and [14C]choline into phosphatidylcholine and lysophosphatidylcholine. The activity of sn-glycerol-3-phosphate acyltransferase, the enzyme involved in phospholipid synthesis, was stimulated in the presence of dibutyryl cAMP, theophylline and PGE1 supporting the increased synthesis of phospholipids.