Free cyclic AMP increases in PC12 cells on depolarization

Agnihotri, Naveen; Kisaalita, William S.; Keith, Charles H.

doi:10.1002/(sici)1097-4547(19970301)47:5<555::aid-jnr11>3.0.co;2-x

Cited by 9 publications

(4 citation statements)

References 32 publications

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“…This agrees with other studies demonstrating that high K ϩ , applied either as a single-step increase or in pulsatile fashion, induces cAMP accumulation in PC12 cells and Xenopus neurons (17,26). In MIN6 cells, the depolarization-induced accumulation of cAMP was significantly inhibited by DDA, suggesting that secretagogues stimulate membrane-bound AC activity.…”

Section: Discussionsupporting

confidence: 92%

Interplay of Ca2+ and cAMP Signaling in the Insulin-secreting MIN6 β-Cell Line

Landa

Harbeck

Kaihara

et al. 2005

Journal of Biological Chemistry

190

216

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Ca2؉ and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca 2؉ and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca 2؉ -regulated cAMP production in living cells is incomplete. Genetically encoded fluorescence resonance energy transfer-based biosensors have made possible real-time imaging of spatial and temporal gradients of intracellular cAMP concentration in single living cells. Here, we used confocal microscopy, fluorescence resonance energy transfer, and insulin-secreting MIN6 cells expressing Epac1-camps, a biosynthetic unimolecular cAMP indicator, to better understand the role of intracellular Ca 2؉ in cAMP production. We report that depolarization with high external K ؉ , tolbutamide, or glucose caused a rapid increase in cAMP that was dependent on extracellular Ca 2؉ and inhibited by nitrendipine, a Ca 2؉ channel blocker, or 2,5-dideoxyadenosine, a P-site antagonist of transmembrane adenylate cyclases. Stimulation of MIN6 cells with glucose in the presence of tetraethylammonium chloride generated concomitant Ca 2؉ and cAMP oscillations that were abolished in the absence of extracellular Ca 2؉ and blocked by 2,5-dideoxyadenosine or 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca 2؉ and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca 2؉ and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca 2؉ and cAMP signaling in electrically excitable endocrine cells and suggest that Ca 2؉ -dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase.

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Section: Discussionsupporting

confidence: 92%

Interplay of Ca2+ and cAMP Signaling in the Insulin-secreting MIN6 β-Cell Line

Landa

Harbeck

Kaihara

et al. 2005

Journal of Biological Chemistry

190

216

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“…Studies in both cell lines and primary neurons that have demonstrated that calcium influx can activate calcium/calmodulin‐dependent adenylyl cyclases to generate cAMP (Agnihotri et al. 1997; Xia and Storm 1997), provide another potential mechanism to activate Ras.…”

Section: Discussionmentioning

confidence: 99%

“…Studies in both cell lines and primary neurons that have demonstrated that calcium influx can activate calcium/calmodulin-dependent adenylyl cyclases to generate cAMP (Agnihotri et al 1997;Xia and Storm 1997), provide another potential mechanism to activate Ras. cAMP activation of Ras has been demonstrated in neuronal cells (Ambrosini et al 2000) and endocrine cells (Busca et al 2000;Tsygankova et al 2000).…”

Section: Discussionmentioning

confidence: 99%

The requirement of Ras and Rap1 for the activation of ERKs by cAMP, PACAP, and KCl in cerebellar granule cells

Obara¹,

Horgan²,

Stork³

2006

Journal of Neurochemistry

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In cerebellar granule cells, the mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) cascade mediates multiple functions, including proliferation, differentiation, and survival. In these cells, ERKs are activated by diverse stimuli, including cyclic adenosine monophosphate (cAMP), pituitary adenylate cyclase activating protein (PACAP), depolarization induced by elevated extracellular potassium (KCl), and the neurotrophin brain-derived neurotrophic factor. Extensive studies in neuronal cell lines have implicated the small G proteins Ras and Rap1 in the activation of ERKs by cAMP, PACAP, and KCl. However, the requirement of Ras and Rap1 in these pathways in cerebellar granule cells has not been addressed. In this study, we utilize multiple biochemical assays to determine the mechanisms of action and requirement of Ras and Rap1 in cultured cerebellar granule cells. We show that both Ras and Rap1 can be activated by cAMP or PACAP via protein kinase (PKA)-dependent mechanisms. KCl activation of Ras also required PKA. Using both adenoviral and transgenic approaches, we show that Ras plays a major role in ERK activation by cAMP, PACAP, and KCl, while Rap1 also mediates activation of a selective membrane-associated pool of ERKs. Furthermore, Rap1, but not Ras, activation by PKA appears to require the action of Src family kinases.

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“…Due to the small working volume, the flow cell allowed rapid displacement of medium, and thus enabled stimulation of the cultured cells with pulses. The results for cAMP imaging have been reported elsewhere (3). We used the flow cell to image fluorescein isothiocyanate (FITC) dextran and Fura-2 in mouse neuroblastoma N1E-115 and human bone osteosarcoma TE-85 cells.…”

Section: Flow Cell Characterization and Usementioning

confidence: 95%

Micro-Perfusion Flow Cell for Imaging Cultured Cells

1999

Self Cite

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We present a unique design for a flow cell with a small working volume that allows rapid displacement of media viewed under high power and short working distance objectives. The flow cell has a small internal depth (ca. 0.033 cm) and volume (ca. 0.05 mL) and is easy to handle. Made of Delrin, the flow cell is biologically inert. We have used the flow cell for fluorescence imaging of PC12 cells loaded with tetramethylrhodamine dextran (TMRD) and other dyes.

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Free cyclic AMP increases in PC12 cells on depolarization

Cited by 9 publications

References 32 publications

Interplay of Ca2+ and cAMP Signaling in the Insulin-secreting MIN6 β-Cell Line

Interplay of Ca2+ and cAMP Signaling in the Insulin-secreting MIN6 β-Cell Line

The requirement of Ras and Rap1 for the activation of ERKs by cAMP, PACAP, and KCl in cerebellar granule cells

Micro-Perfusion Flow Cell for Imaging Cultured Cells

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