Residence of Adenylyl Cyclase Type 8 in Caveolae Is Necessary but Not Sufficient for Regulation by Capacitative Ca2+Entry

Smith, Karen E.; Gu, Chen; Fagan, Kent A.; Hu, Biao; Cooper, Dermot M.F.

doi:10.1074/jbc.m109615200

Cited by 86 publications

(115 citation statements)

References 42 publications

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“…8, Western blotting of HEK 293 cell membrane fractions using an NHE1-specific monoclonal antibody showed high expression of the exchanger in fractions 4 and 5, alongside caveolin (the marker of lipid rafts) and transiently expressed AC8. The latter is consistent with previous studies in our laboratory showing that the AC8 protein is enriched in these caveolin-containing fractions (24). A small amount of NHE1 was found in fractions 7-9, which corresponded to the bulk plasma membrane regions, as identified by the marker ␤-adaptin (37).…”

Section: Nhe1 Activity Protects Ac8 From Local Acid Shifts In Hek 293supporting

confidence: 81%

“…Cariporide was a gift from Dr. Juergen Puenter at Avetis Pharma (Frankfurt, Germany). Polyclonal AC8 antibody was a gift from Dr. J. J. Cali, as previously described (24). Polyclonal caveolin antibody was obtained from BD Transduction Laboratories (Erembodegem, Belgium), and polyclonal ␤-adaptin antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).…”

Section: Methodsmentioning

confidence: 99%

“…In the intact cell, the Ca 2ϩ -sensitive ACs are regulated exclusively by capacitative Ca 2ϩ entry (CCE) or Ca 2ϩ entry through voltage-gated Ca 2ϩ channels (19 -22). The selectivity of the cyclases for discrete modes of Ca 2ϩ entry rather than other sources of cytosolic Ca 2ϩ increase alludes to the co-localization of the ACs with preferred Ca 2ϩ entry sites, a quality that is at least partly dependent on the selective localization of Ca 2ϩ -sensitive ACs in cholesterolrich domains of the plasma membrane, known as lipid rafts or caveolae (23,24). It is conceivable then that, during periods of cellular activity, the overall degree of cAMP production might reflect local changes in both [Ca 2ϩ ] and pH i .…”

Section: Intracellular Ph (Ph I )mentioning

confidence: 99%

“…Cells-Although we have eliminated some possible mechanisms in explaining the discrepancies between in vitro and in vivo effects of pH, a final hypothesis considered the possibility that Ca 2ϩ -regulated ACs were protected from intracellular acid shifts due to their localization in lipid rafts or caveolae (24,37). We hypothesized that pH is tightly regulated in these cellular microdomains, such that application of a weak acid will produce a sustained pH i decrease in the bulk cell cytosol, but localized acid extrusion within the caveolar domain could minimize local pH i changes.…”

Section: Nhe1 Activity Protects Ac8 From Local Acid Shifts In Hek 293mentioning

confidence: 99%

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Localized Na+/H+ Exchanger 1 Expression Protects Ca2+-regulated Adenylyl Cyclases from Changes in Intracellular pH

Willoughby

Masada

Crossthwaite

et al. 2005

Journal of Biological Chemistry

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The Ca 2؉ -sensitive adenylyl cyclases (ACs) are exclusively regulated by capacitative Ca 2؉ entry (CCE) in nonexcitable cells. The present study investigates whether this Ca 2؉ -dependent modulation of AC activity is further regulated by local pH changes that can arise beneath the plasma membrane as a consequence of cellular activity. Ca 2؉ stimulation of AC8 expressed in HEK 293 cells and inhibition of endogenous AC6 in C6-2B glioma cells exhibited clear sensitivity to modest pH changes in vitro. Acid pH (pH 7.14) reduced the Ca 2؉ sensitivity of both ACs, whereas alkaline pH (pH 7.85) enhanced the responsiveness of the enzymes to Ca 2؉ , compared with controls (pH 7.50). Surprisingly, in the intact cell, the response of AC8 and AC6 to CCE was largely unperturbed by similar changes in intracellular pH (pH i ), imposed using a weak acid (propionate) or weak base (trimethylamine). A range of hypotheses were tested to identify the mechanism(s) that could underlie this lack of pH effect in the intact cell. The pH sensitivity of CCE in HEK 293 cells is likely to dampen the effects of pH i on Ca 2؉ -regulated ACs and may partly explain the discrepancy between in vitro and in vivo data. However, we have found that the Na ؉ /H ؉ exchanger (NHE), NHE1, is functionally active in these cells, and like AC8 (and AC6) it resides in lipid rafts or caveolae, which may create cellular microdomains where pH i is tightly regulated. An abundance of NHE1 in these cellular subdomains may generate a privileged environment that protects the Ca 2؉ -sensitive ACs and other caveolar proteins from local acid shifts. Intracellular pH (pH i )1 is a fundamental determinant of cell function that, until recently, was considered to be tightly regulated due to rapid H ϩ diffusion, buffering, and ion transport mechanisms. However, it is now recognized that modest fluctuations in pH i can arise during "physiological" cellular activity (1-9). Furthermore, recent studies have provided evidence for pH i microdomains just beneath the plasma membrane, which can exhibit transient pH shifts on the order of several tenths of a pH unit, as a consequence of the local activity of plasma membrane transporters (10 -12). Fluctuations of [H ϩ ] within these microdomains could potentially exert profound effects on the numerous cellular proteins residing within or near the plasma membrane, including the cAMP-producing adenylyl cyclase (AC). Early in vitro studies of plasma membrane preparations demonstrated that the catalytic activity of AC is steeply pH-dependent, over the range of pH 6.0 -8.5, where acidification or alkalinization, respectively, decreases or increase cAMP synthesis (13-15).In addition to the pH dependence of AC catalytic activity, the Ca 2ϩ sensitivity of the Ca 2ϩ -regulated ACs is also likely to be highly pH-sensitive. Four of the nine AC isoforms cloned to date are regulated by submicromolar concentrations of Ca 2ϩ . AC1 and AC8 are stimulated by Ca 2ϩ acting via calmodulin, whereas AC5 and AC6 are inhibited, independently of calmodulin (16)...

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Section: Nhe1 Activity Protects Ac8 From Local Acid Shifts In Hek 293supporting

confidence: 81%

Section: Methodsmentioning

confidence: 99%

Section: Intracellular Ph (Ph I )mentioning

confidence: 99%

Section: Nhe1 Activity Protects Ac8 From Local Acid Shifts In Hek 293mentioning

confidence: 99%

See 2 more Smart Citations

Localized Na+/H+ Exchanger 1 Expression Protects Ca2+-regulated Adenylyl Cyclases from Changes in Intracellular pH

Willoughby

Masada

Crossthwaite

et al. 2005

Journal of Biological Chemistry

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“…It has been shown that expression of specific isoforms, subcellular localization, and differential regulation of ACs greatly contribute to the diverse and tissue-specific regulation of cAMP (33)(34)(35)(36)(37). Here we report a novel regulatory pathway for control of cAMP synthesis in three hematopoietic cell lines.…”

Section: Discussionmentioning

confidence: 99%

Regulation of cAMP Responses by the G12/13 Pathway Converges on Adenylyl Cyclase VII

Jiang

Collins

Davis

et al. 2008

Journal of Biological Chemistry

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Regulation of intracellular cAMP by multiple pathways enables differential function of this ubiquitous second messenger in a context-dependent manner. Modulation of G s -stimulated intracellular cAMP has long been known to be modulated by the G i and G q /Ca 2؉ pathways. Recently, the G 13 pathway was also shown to facilitate cAMP responses in murine macrophage cells. We report here that this synergistic regulation of cAMP synthesis by the G s and G 13 pathways is mediated by a specific isoform of adenylyl cyclase, AC7. Furthermore, this signaling paradigm exists in several hematopoietic lineages and can be recapitulated by exogenous expression of AC7 in HEK 293 cells. Mechanistic characterization of this synergistic interaction indicates that it occurs downstream of receptor activation and it can be mediated by the ␣ subunit of either G 12 or G 13 . Our results demonstrate that AC7 is a specific downstream effector of the G 12/13 pathway.

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Implication of adenylyl cyclase 8 in pathological smooth muscle cell migration occurring in rat and human vascular remodelling

Guéguen¹,

Keuylian²,

Mateo³

et al. 2010

The Journal of Pathology

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Recently, we discovered on primary cell cultures that adenylyl cyclase type 8 (AC8) was involved in the transition of rat vascular smooth muscle cells (VSMCs) to an inflammatory phenotype. Here we demonstrate, in human vessels displaying early or advanced atherosclerotic lesions, that: (a) only intimal VSMCs strongly express AC8; and (b) very few AC8-positive VSMCs were detected in the medial layer, either in atherosclerotic or healthy arteries. Furthermore, over-expressing AC8 in primary rat VSMC cultures triggered the recolonization of a wounded zone and similar results were obtained in the presence of mitomycin, a potent inhibitor of proliferation. This phenomenon was prevented by silencing AC8. Indeed, in IL-1 beta-treated cells, AC8 silencing halted migration and decreased the matrix-metalloproteinases 2/9 secretion, known to be involved in VSMC migration. In vivo, we showed: (a) a pronounced up-regulation of AC8 expression in highly migrating VSMCs of the injured rat carotid artery; (b) an undetectable AC8 labelling in re-endothelized vessels where neo-intimal thickening had stopped. From our data, we conclude that AC8 expression appears closely linked to the properties developed by VSMCs in atherosclerosis and post-angioplasty neo-intima formation leading to restenosis. In addition, it reinforces the idea that VSMC responses to their cell environment greatly depend on the AC isoforms expressed and attributes a new role for AC8 in these pathological vascular processes.

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Residence of Adenylyl Cyclase Type 8 in Caveolae Is Necessary but Not Sufficient for Regulation by Capacitative Ca2+Entry

Cited by 86 publications

References 42 publications

Localized Na+/H+ Exchanger 1 Expression Protects Ca2+-regulated Adenylyl Cyclases from Changes in Intracellular pH

Localized Na+/H+ Exchanger 1 Expression Protects Ca2+-regulated Adenylyl Cyclases from Changes in Intracellular pH

Regulation of cAMP Responses by the G12/13 Pathway Converges on Adenylyl Cyclase VII

Implication of adenylyl cyclase 8 in pathological smooth muscle cell migration occurring in rat and human vascular remodelling

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