Adenylyl cyclases (ACs) are a family of critically important signaling molecules that are regulat... more Adenylyl cyclases (ACs) are a family of critically important signaling molecules that are regulated by multiple pathways. Adenylyl cyclase 8 (AC8) is a Ca(2+) stimulated isoform that displays a selective regulation by capacitative Ca(2+) entry (CCE), the process whereby the entry of Ca(2+) into cells is triggered by the emptying of intracellular stores. This selectivity was believed to be achieved through the localization of AC8 in lipid raft microdomains, along with components of the CCE apparatus. In the present study, we show that an intact leucine zipper motif is required for the efficient N-linked glycosylation of AC8, and that this N-linked glycosylation is important to target AC8 into lipid rafts. Disruption of the leucine zipper by site-directed mutagenesis results in the elimination of N-glycosylated forms and their exclusion from lipid rafts. Mutants of AC8 that cannot be N-glycosylated are not demonstrably associated with rafts, although they can still be regulated by CCE; however, raft integrity is required for the regulation of these mutants. These findings suggest that raft localized proteins in addition to AC8 are needed to mediate its regulation by CCE.
Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transport... more Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application.
Ca(2+)-sensitive adenylyl cyclases (ACs) orchestrate dynamic interplay between Ca(2+) and cAMP th... more Ca(2+)-sensitive adenylyl cyclases (ACs) orchestrate dynamic interplay between Ca(2+) and cAMP that is a crucial feature of cellular homeostasis. Significantly, these ACs are highly selective for capacitative Ca(2+) entry (CCE) over other modes of Ca(2+) increase. To directly address the possibility that these ACs reside in discrete Ca(2+) microdomains, we tethered a Ca(2+) sensor, GCaMP2, to the N-terminus of Ca(2+)-stimulated AC8. GCaMP2-AC8 measurements were compared with global, plasma membrane (PM)-targeted or Ca(2+)-insensitive AC2-targeted GCaMP2. In intact cells, GCaMP2-AC8 responded rapidly to CCE, but was largely unresponsive to other types of Ca(2+) rise. The global GCaMP2, PM-targeted GCaMP2 and GCaMP2-AC2 sensors reported large Ca(2+) fluxes during Ca(2+) mobilization and non-specific Ca(2+) entry, but were less responsive to CCE than GCaMP2-AC8. Our data reveal that different AC isoforms localize to distinct Ca(2+)-microdomains within the plasma membrane. AC2, which is regulated via protein kinase C, resides in a microdomain that is exposed to a range of widespread Ca(2+) signals seen throughout the cytosol. By contrast, a unique Ca(2+) microdomain surrounds AC8 that promotes selectivity for Ca(2+) signals arising from CCE, and optimizes CCE-mediated cAMP synthesis. This direct demonstration of discrete compartmentalized Ca(2+) signals associated with specific signalling proteins provides a remarkable insight into the functional organization of signalling microdomains.
Regulation of adenylyl cyclases (ACs) by Ca2+ requires capacitative Ca2+ entry (CCE) (Cooper, D. ... more Regulation of adenylyl cyclases (ACs) by Ca2+ requires capacitative Ca2+ entry (CCE) (Cooper, D. M. F. (2003) Biochem. J. 375, 517-529), but whether Ca2+-sensitive phosphodiesterases (PDEs) are similarly discriminating has never been addressed. In the present study, a variety of conditions were devised to manipulate [Ca2+]i so that we could ask whether PDE1 selectively responds to different modes of elevating [Ca2+]i, viz. Ca2+ released from intracellular stores and various modes of Ca2+ entry. In 1321N1 human astrocytoma cells, the endogenous PDE1 (identified as PDE1A by reverse transcriptase-PCR) was largely insensitive to Ca2+ released from carbachol-sensitive stores but was robustly stimulated by a similar rise in [Ca2+]i due to carbachol-induced Ca2+ influx. Gd3+, which effectively blocked thapsigargin-induced CCE and its effect on PDE1A, also inhibited the activation of PDE1A by carbachol-induced Ca2+ entry. However, non-selective ionomycin-mediated Ca2+ entry also activated PDE1A, so that, unlike Ca2+-sensitive ACs, PDE1A cannot discriminate between the different sources of Ca2+ entry. Fractionation of the cells revealed that the Ca2+-calmodulin-stimulated PDE activity was not present at the plasma membrane but was associated with the cytosol and the organellar compartments of the cell. Therefore, the apparent disparity between PDE1A and ACs is likely to be the consequence of their differential subcellular localization. Nevertheless, in a physiological context, where artificial modes of elevating [Ca2+]i are not available, as with ACs, a dependence on CCE would be evident, and it would be the duration of this influx of Ca2+ that would determine how long PDE1A was activated.
The Ca2+-sensitive adenylyl cyclases (ACs) are exclusively regulated by capacitative Ca2+ entry (... more The Ca2+-sensitive adenylyl cyclases (ACs) are exclusively regulated by capacitative Ca2+ entry (CCE) in nonexcitable cells. The present study investigates whether this Ca2+-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. Ca2+ 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 Ca2+ sensitivity of both ACs, whereas alkaline pH (pH 7.85) enhanced the responsiveness of the enzymes to Ca2+, 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 Ca2+-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 Ca2+-sensitive ACs and other caveolar proteins from local acid shifts.
Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1... more Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are stimulated by Ca(2+)/calmodulin (CaM), have discrete neuronal functions. Although the Ca(2+) sensitivity of AC1 is higher than that of AC8, precisely how these two ACs are regulated by Ca(2+)/CaM remains elusive, and the basis for their diverse physiological roles is quite unknown. Distinct localization of the CaM binding domains within the two enzymes may be essential to differential regulation of the ACs by Ca(2+)/CaM. In this study we compare in detail the regulation of AC1 and AC8 by Ca(2+)/CaM both in vivo and in vitro and explore the different role of each Ca(2+)-binding lobe of CaM in regulating the two enzymes. We also assess the relative dependence of AC1 and AC8 on capacitative Ca(2+) entry. Finally, in real-time fluorescence resonance energy transfer-based imaging experiments, we examine the effects of dynamic Ca(2+) events on the production of cAMP in cells expressing AC1 and AC8. Our data demonstrate distinct patterns of regulation and Ca(2+) dependence of AC1 and AC8, which seems to emanate from their mode of regulation by CaM. Such distinctive properties may contribute significantly to the divergent physiological roles in which these ACs have been implicated.
Lipid rafts are specialized, cholesterol-rich domains of the plasma membrane that are enriched in... more Lipid rafts are specialized, cholesterol-rich domains of the plasma membrane that are enriched in certain signaling proteins, including Ca(2+)-sensitive adenylyl cyclases. This restrictive localization plays a key role in the regulation of the Ca(2+)-stimulable AC8 and the Ca(2+)-inhibitable AC6 by capacitative calcium entry. Interestingly, AC7, a Ca(2+)-insensitive AC, is found in the plasma membrane but is excluded from lipid rafts (Smith, K. E., Gu, C., Fagan, K. A., Hu, B., and Cooper, D. M. F. (2002) J. Biol. Chem. 277, 6025-6031). The mechanisms governing the specific membrane targeting of adenylyl cyclase isoforms remain unknown. To address this issue, a series of chimeras were produced between the raft-targeted AC5 and the non-raft-targeted AC7, involving switching of their major domains. The AC5-AC7 chimeras were expressed in HEK 293 cells and lipid rafts were isolated from the bulk plasma membrane by either detergent-based or non-detergent-based fractionation methods. Additionally, confocal imaging was used to investigate the precise cellular targeting of the chimeras. Surprisingly, the two tandem six-transmembrane domains of AC5 were not required for localization to lipid rafts. Rather, AC5 localization depended on the complete cytoplasmic loops (C1 and C2); constructs with mixed domains were either retained in the endoplasmic reticulum or degraded. Similar conclusions are drawn for the lipid raft localization of the Ca(2+)/calmodulin-stimulable AC8; again, the C1 and C2 domains are critical. Thus, protein-protein interactions may be more important than protein-lipid interactions in targeting these calcium-sensitive enzymes to lipid rafts.
Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular sign... more Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular signaling complexes that serve to localize the activities of cAMP. Evidence is accumulating of direct associations between AKAPs and specific adenylyl cyclase (AC) isoforms to facilitate the actions of protein kinase A on cAMP production. It happens that some of the AC isoforms (AC1 and AC5/6) that bind specific AKAPs are regulated by submicromolar shifts in intracellular Ca(2+). However, whether AKAPs play a role in the control of AC activity by Ca(2+) is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca(2+)-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca(2+) events. This functional interaction between AKAP79/150 and AC8 was observed in HEK293 cells overexpressing the two signaling molecules. Similar findings were made in pancreatic insulin-secreting cells and cultured hippocampal neurons that endogenously express AKAP79/150 and AC8, which suggests important physiological implications for this protein-protein interaction with respect to Ca(2+)-stimulated cAMP production.
The ability of aldosterone to stimulate Na+ transport in a range of epithelial tissues has been k... more The ability of aldosterone to stimulate Na+ transport in a range of epithelial tissues has been known for many years. Early work suggested that aldosterone had a delayed action operating by transcriptional up-regulation of proteins such as the epithelial Na+ channel. However more recent data has suggested that the hormone has a short-term non-genomic action. In this paper we investigate short and long-term actions of aldosterone on Na+ transport in the rabbit urinary bladder. We have shown that aldosterone stimulates epithelial Na+ channel activity, as measured by the amiloride-sensitive short-circuit current over a 3.75 h period and that this action is potentiated by cAMP. Using reverse transcriptase-polymerase chain reaction we have shown that aldosterone and forskolin in combination up-regulate mRNA synthesis for the beta- and gamma-subunits of the epithelial Na+ channel. Using Western blotting we have shown in the case of the beta-subunit that a corresponding increase in channel protein occurs. We have also demonstrated that aldosterone in the presence of inhibitors of phosphodiesterase can stimulate the short-circuit current across rabbit bladder epithelium over a 20 min period. An explanation for the synergistic interaction between aldosterone and cAMP is provided. We have shown that aldosterone can increase cAMP levels within urothelial cells over a 4 min period. We propose that this represents a non-genomic action of the steroid hormone.
Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracel... more Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracellular cAMP signals. Ca2+-sensitive ACs have received far greater attention than the Ca2+/calmodulin-dependent PDE (PDE1) family in governing intracellular cAMP dynamics in response to changes in the cytosolic Ca2+ concentration ([Ca2+]i). Here, we have stably expressed two isoforms of PDE1, PDE1A2 and PDE1C4, in HEK-293 cells to determine whether they exert different impacts on cellular cAMP. Fractionation and imaging showed that both PDEs occurred mainly in the cytosol. However, PDE1A2 and PDE1C4 differed considerably in their ability to hydrolyze cAMP and in their susceptibility to inhibition by the non-selective PDE inhibitor, IBMX and the PDE1-selective inhibitor, MMX. PDE1A2 had an approximately 30-fold greater Km for cAMP than PDE1C4 and yet was more susceptible to inhibition by IBMX and MMX than was PDE1C4. These differences were mirrored in intact cells when thapsigargin-induced capacitative Ca2+ entry (CCE) activated the PDEs. Mirroring their kinetic properties, PDE1C4 was active at near basal cAMP levels, whereas PDE1A2 required agonist-triggered levels of cAMP, produced in response to stimulation of ACs. The effectiveness of IBMX and MMX to inhibit PDE1A2 and PDE1C4 in functional studies was inversely related to their respective affinities for cAMP. To assess the impact of the two isoforms on cAMP dynamics, real-time cAMP measurements were performed in single cells expressing the two PDE isoforms and a fluorescent Epac-1 cAMP biosensor, in response to CCE. These measurements showed that prostaglandin E1-mediated cAMP production was markedly attenuated in PDE1C4-expressing cells upon induction of CCE and cAMP hydrolysis occurred at a faster rate than in cells expressing PDE1A2 under similar conditions. These results prove that the kinetic properties of PDE isoforms play a major role in determining intracellular cAMP signals in response to physiological elevation of [Ca2+]i and thereby provide a rationale for the utility of diverse PDE1 species.
ABSTRACT Gut eingepasst: Wechselwirkungen zwischen A‐Kinase‐Ankerproteinen (AKAP) und der Protein... more ABSTRACT Gut eingepasst: Wechselwirkungen zwischen A‐Kinase‐Ankerproteinen (AKAP) und der Proteinkinase A (PKA) spielen eine entscheidende Rolle bei vielen physiologisch relevanten Prozessen, deren Fehlregulation Krankheiten verursacht oder mit ihnen in Zusammenhang steht (z. B. Herzinsuffizienz). Terpyridine wurden als α‐Helixmimetika zur Hemmung solcher Wechselwirkungen entwickelt, die als erste nichtpeptidische Verbindungen die AKAP‐Bindungsstelle der PKA blockieren können.
ABSTRACT A good fit: Interactions between A-kinase anchoring proteins (AKAPs) and protein kinase ... more ABSTRACT A good fit: Interactions between A-kinase anchoring proteins (AKAPs) and protein kinase A (PKA) play key roles in a plethora of physiologically relevant processes whose dysregulation causes or is associated with diseases such as heart failure. Terpyridines have been developed as α-helix mimetics for the inhibition of such interactions and are the first biologically active, nonpeptidic compounds that block the AKAP binding site of PKA.
American journal of physiology. Lung cellular and molecular physiology, 2003
Activation of store-operated Ca(2+) entry inhibits type 6 adenylyl cyclase (EC; AC(6); Yoshimura ... more Activation of store-operated Ca(2+) entry inhibits type 6 adenylyl cyclase (EC; AC(6); Yoshimura M and Cooper DM. Proc Natl Acad Sci USA 89: 6712-6720, 1992) activity in pulmonary artery endothelial cells. However, in lung microvascular endothelial cells (PMVEC), which express AC(6) and turn over cAMP at a rapid rate, inhibition of global (whole cell) cAMP is not resolved after direct activation of store-operated Ca(2+) entry using thapsigargin. Present studies sought to determine whether the high constitutive phosphodiesterase activity in PMVECs rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve. Direct stimulation of adenylyl cyclase using forskolin and inhibition of type 4 phosphodiesterases using rolipram increased cAMP and revealed Ca(2+) inhibition of AC(6). Enzyme activity was assessed using PMVEC membranes, where Ca(2+) and cAMP concentrations were independently controlled. Endogenous AC(6) activity exhibited high- and low-affinity Ca(2+) inhi...
Adenylyl cyclases (ACs) are a family of critically important signaling molecules that are regulat... more Adenylyl cyclases (ACs) are a family of critically important signaling molecules that are regulated by multiple pathways. Adenylyl cyclase 8 (AC8) is a Ca(2+) stimulated isoform that displays a selective regulation by capacitative Ca(2+) entry (CCE), the process whereby the entry of Ca(2+) into cells is triggered by the emptying of intracellular stores. This selectivity was believed to be achieved through the localization of AC8 in lipid raft microdomains, along with components of the CCE apparatus. In the present study, we show that an intact leucine zipper motif is required for the efficient N-linked glycosylation of AC8, and that this N-linked glycosylation is important to target AC8 into lipid rafts. Disruption of the leucine zipper by site-directed mutagenesis results in the elimination of N-glycosylated forms and their exclusion from lipid rafts. Mutants of AC8 that cannot be N-glycosylated are not demonstrably associated with rafts, although they can still be regulated by CCE; however, raft integrity is required for the regulation of these mutants. These findings suggest that raft localized proteins in addition to AC8 are needed to mediate its regulation by CCE.
Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transport... more Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application.
Ca(2+)-sensitive adenylyl cyclases (ACs) orchestrate dynamic interplay between Ca(2+) and cAMP th... more Ca(2+)-sensitive adenylyl cyclases (ACs) orchestrate dynamic interplay between Ca(2+) and cAMP that is a crucial feature of cellular homeostasis. Significantly, these ACs are highly selective for capacitative Ca(2+) entry (CCE) over other modes of Ca(2+) increase. To directly address the possibility that these ACs reside in discrete Ca(2+) microdomains, we tethered a Ca(2+) sensor, GCaMP2, to the N-terminus of Ca(2+)-stimulated AC8. GCaMP2-AC8 measurements were compared with global, plasma membrane (PM)-targeted or Ca(2+)-insensitive AC2-targeted GCaMP2. In intact cells, GCaMP2-AC8 responded rapidly to CCE, but was largely unresponsive to other types of Ca(2+) rise. The global GCaMP2, PM-targeted GCaMP2 and GCaMP2-AC2 sensors reported large Ca(2+) fluxes during Ca(2+) mobilization and non-specific Ca(2+) entry, but were less responsive to CCE than GCaMP2-AC8. Our data reveal that different AC isoforms localize to distinct Ca(2+)-microdomains within the plasma membrane. AC2, which is regulated via protein kinase C, resides in a microdomain that is exposed to a range of widespread Ca(2+) signals seen throughout the cytosol. By contrast, a unique Ca(2+) microdomain surrounds AC8 that promotes selectivity for Ca(2+) signals arising from CCE, and optimizes CCE-mediated cAMP synthesis. This direct demonstration of discrete compartmentalized Ca(2+) signals associated with specific signalling proteins provides a remarkable insight into the functional organization of signalling microdomains.
Regulation of adenylyl cyclases (ACs) by Ca2+ requires capacitative Ca2+ entry (CCE) (Cooper, D. ... more Regulation of adenylyl cyclases (ACs) by Ca2+ requires capacitative Ca2+ entry (CCE) (Cooper, D. M. F. (2003) Biochem. J. 375, 517-529), but whether Ca2+-sensitive phosphodiesterases (PDEs) are similarly discriminating has never been addressed. In the present study, a variety of conditions were devised to manipulate [Ca2+]i so that we could ask whether PDE1 selectively responds to different modes of elevating [Ca2+]i, viz. Ca2+ released from intracellular stores and various modes of Ca2+ entry. In 1321N1 human astrocytoma cells, the endogenous PDE1 (identified as PDE1A by reverse transcriptase-PCR) was largely insensitive to Ca2+ released from carbachol-sensitive stores but was robustly stimulated by a similar rise in [Ca2+]i due to carbachol-induced Ca2+ influx. Gd3+, which effectively blocked thapsigargin-induced CCE and its effect on PDE1A, also inhibited the activation of PDE1A by carbachol-induced Ca2+ entry. However, non-selective ionomycin-mediated Ca2+ entry also activated PDE1A, so that, unlike Ca2+-sensitive ACs, PDE1A cannot discriminate between the different sources of Ca2+ entry. Fractionation of the cells revealed that the Ca2+-calmodulin-stimulated PDE activity was not present at the plasma membrane but was associated with the cytosol and the organellar compartments of the cell. Therefore, the apparent disparity between PDE1A and ACs is likely to be the consequence of their differential subcellular localization. Nevertheless, in a physiological context, where artificial modes of elevating [Ca2+]i are not available, as with ACs, a dependence on CCE would be evident, and it would be the duration of this influx of Ca2+ that would determine how long PDE1A was activated.
The Ca2+-sensitive adenylyl cyclases (ACs) are exclusively regulated by capacitative Ca2+ entry (... more The Ca2+-sensitive adenylyl cyclases (ACs) are exclusively regulated by capacitative Ca2+ entry (CCE) in nonexcitable cells. The present study investigates whether this Ca2+-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. Ca2+ 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 Ca2+ sensitivity of both ACs, whereas alkaline pH (pH 7.85) enhanced the responsiveness of the enzymes to Ca2+, 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 Ca2+-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 Ca2+-sensitive ACs and other caveolar proteins from local acid shifts.
Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1... more Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are stimulated by Ca(2+)/calmodulin (CaM), have discrete neuronal functions. Although the Ca(2+) sensitivity of AC1 is higher than that of AC8, precisely how these two ACs are regulated by Ca(2+)/CaM remains elusive, and the basis for their diverse physiological roles is quite unknown. Distinct localization of the CaM binding domains within the two enzymes may be essential to differential regulation of the ACs by Ca(2+)/CaM. In this study we compare in detail the regulation of AC1 and AC8 by Ca(2+)/CaM both in vivo and in vitro and explore the different role of each Ca(2+)-binding lobe of CaM in regulating the two enzymes. We also assess the relative dependence of AC1 and AC8 on capacitative Ca(2+) entry. Finally, in real-time fluorescence resonance energy transfer-based imaging experiments, we examine the effects of dynamic Ca(2+) events on the production of cAMP in cells expressing AC1 and AC8. Our data demonstrate distinct patterns of regulation and Ca(2+) dependence of AC1 and AC8, which seems to emanate from their mode of regulation by CaM. Such distinctive properties may contribute significantly to the divergent physiological roles in which these ACs have been implicated.
Lipid rafts are specialized, cholesterol-rich domains of the plasma membrane that are enriched in... more Lipid rafts are specialized, cholesterol-rich domains of the plasma membrane that are enriched in certain signaling proteins, including Ca(2+)-sensitive adenylyl cyclases. This restrictive localization plays a key role in the regulation of the Ca(2+)-stimulable AC8 and the Ca(2+)-inhibitable AC6 by capacitative calcium entry. Interestingly, AC7, a Ca(2+)-insensitive AC, is found in the plasma membrane but is excluded from lipid rafts (Smith, K. E., Gu, C., Fagan, K. A., Hu, B., and Cooper, D. M. F. (2002) J. Biol. Chem. 277, 6025-6031). The mechanisms governing the specific membrane targeting of adenylyl cyclase isoforms remain unknown. To address this issue, a series of chimeras were produced between the raft-targeted AC5 and the non-raft-targeted AC7, involving switching of their major domains. The AC5-AC7 chimeras were expressed in HEK 293 cells and lipid rafts were isolated from the bulk plasma membrane by either detergent-based or non-detergent-based fractionation methods. Additionally, confocal imaging was used to investigate the precise cellular targeting of the chimeras. Surprisingly, the two tandem six-transmembrane domains of AC5 were not required for localization to lipid rafts. Rather, AC5 localization depended on the complete cytoplasmic loops (C1 and C2); constructs with mixed domains were either retained in the endoplasmic reticulum or degraded. Similar conclusions are drawn for the lipid raft localization of the Ca(2+)/calmodulin-stimulable AC8; again, the C1 and C2 domains are critical. Thus, protein-protein interactions may be more important than protein-lipid interactions in targeting these calcium-sensitive enzymes to lipid rafts.
Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular sign... more Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular signaling complexes that serve to localize the activities of cAMP. Evidence is accumulating of direct associations between AKAPs and specific adenylyl cyclase (AC) isoforms to facilitate the actions of protein kinase A on cAMP production. It happens that some of the AC isoforms (AC1 and AC5/6) that bind specific AKAPs are regulated by submicromolar shifts in intracellular Ca(2+). However, whether AKAPs play a role in the control of AC activity by Ca(2+) is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca(2+)-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca(2+) events. This functional interaction between AKAP79/150 and AC8 was observed in HEK293 cells overexpressing the two signaling molecules. Similar findings were made in pancreatic insulin-secreting cells and cultured hippocampal neurons that endogenously express AKAP79/150 and AC8, which suggests important physiological implications for this protein-protein interaction with respect to Ca(2+)-stimulated cAMP production.
The ability of aldosterone to stimulate Na+ transport in a range of epithelial tissues has been k... more The ability of aldosterone to stimulate Na+ transport in a range of epithelial tissues has been known for many years. Early work suggested that aldosterone had a delayed action operating by transcriptional up-regulation of proteins such as the epithelial Na+ channel. However more recent data has suggested that the hormone has a short-term non-genomic action. In this paper we investigate short and long-term actions of aldosterone on Na+ transport in the rabbit urinary bladder. We have shown that aldosterone stimulates epithelial Na+ channel activity, as measured by the amiloride-sensitive short-circuit current over a 3.75 h period and that this action is potentiated by cAMP. Using reverse transcriptase-polymerase chain reaction we have shown that aldosterone and forskolin in combination up-regulate mRNA synthesis for the beta- and gamma-subunits of the epithelial Na+ channel. Using Western blotting we have shown in the case of the beta-subunit that a corresponding increase in channel protein occurs. We have also demonstrated that aldosterone in the presence of inhibitors of phosphodiesterase can stimulate the short-circuit current across rabbit bladder epithelium over a 20 min period. An explanation for the synergistic interaction between aldosterone and cAMP is provided. We have shown that aldosterone can increase cAMP levels within urothelial cells over a 4 min period. We propose that this represents a non-genomic action of the steroid hormone.
Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracel... more Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracellular cAMP signals. Ca2+-sensitive ACs have received far greater attention than the Ca2+/calmodulin-dependent PDE (PDE1) family in governing intracellular cAMP dynamics in response to changes in the cytosolic Ca2+ concentration ([Ca2+]i). Here, we have stably expressed two isoforms of PDE1, PDE1A2 and PDE1C4, in HEK-293 cells to determine whether they exert different impacts on cellular cAMP. Fractionation and imaging showed that both PDEs occurred mainly in the cytosol. However, PDE1A2 and PDE1C4 differed considerably in their ability to hydrolyze cAMP and in their susceptibility to inhibition by the non-selective PDE inhibitor, IBMX and the PDE1-selective inhibitor, MMX. PDE1A2 had an approximately 30-fold greater Km for cAMP than PDE1C4 and yet was more susceptible to inhibition by IBMX and MMX than was PDE1C4. These differences were mirrored in intact cells when thapsigargin-induced capacitative Ca2+ entry (CCE) activated the PDEs. Mirroring their kinetic properties, PDE1C4 was active at near basal cAMP levels, whereas PDE1A2 required agonist-triggered levels of cAMP, produced in response to stimulation of ACs. The effectiveness of IBMX and MMX to inhibit PDE1A2 and PDE1C4 in functional studies was inversely related to their respective affinities for cAMP. To assess the impact of the two isoforms on cAMP dynamics, real-time cAMP measurements were performed in single cells expressing the two PDE isoforms and a fluorescent Epac-1 cAMP biosensor, in response to CCE. These measurements showed that prostaglandin E1-mediated cAMP production was markedly attenuated in PDE1C4-expressing cells upon induction of CCE and cAMP hydrolysis occurred at a faster rate than in cells expressing PDE1A2 under similar conditions. These results prove that the kinetic properties of PDE isoforms play a major role in determining intracellular cAMP signals in response to physiological elevation of [Ca2+]i and thereby provide a rationale for the utility of diverse PDE1 species.
ABSTRACT Gut eingepasst: Wechselwirkungen zwischen A‐Kinase‐Ankerproteinen (AKAP) und der Protein... more ABSTRACT Gut eingepasst: Wechselwirkungen zwischen A‐Kinase‐Ankerproteinen (AKAP) und der Proteinkinase A (PKA) spielen eine entscheidende Rolle bei vielen physiologisch relevanten Prozessen, deren Fehlregulation Krankheiten verursacht oder mit ihnen in Zusammenhang steht (z. B. Herzinsuffizienz). Terpyridine wurden als α‐Helixmimetika zur Hemmung solcher Wechselwirkungen entwickelt, die als erste nichtpeptidische Verbindungen die AKAP‐Bindungsstelle der PKA blockieren können.
ABSTRACT A good fit: Interactions between A-kinase anchoring proteins (AKAPs) and protein kinase ... more ABSTRACT A good fit: Interactions between A-kinase anchoring proteins (AKAPs) and protein kinase A (PKA) play key roles in a plethora of physiologically relevant processes whose dysregulation causes or is associated with diseases such as heart failure. Terpyridines have been developed as α-helix mimetics for the inhibition of such interactions and are the first biologically active, nonpeptidic compounds that block the AKAP binding site of PKA.
American journal of physiology. Lung cellular and molecular physiology, 2003
Activation of store-operated Ca(2+) entry inhibits type 6 adenylyl cyclase (EC; AC(6); Yoshimura ... more Activation of store-operated Ca(2+) entry inhibits type 6 adenylyl cyclase (EC; AC(6); Yoshimura M and Cooper DM. Proc Natl Acad Sci USA 89: 6712-6720, 1992) activity in pulmonary artery endothelial cells. However, in lung microvascular endothelial cells (PMVEC), which express AC(6) and turn over cAMP at a rapid rate, inhibition of global (whole cell) cAMP is not resolved after direct activation of store-operated Ca(2+) entry using thapsigargin. Present studies sought to determine whether the high constitutive phosphodiesterase activity in PMVECs rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve. Direct stimulation of adenylyl cyclase using forskolin and inhibition of type 4 phosphodiesterases using rolipram increased cAMP and revealed Ca(2+) inhibition of AC(6). Enzyme activity was assessed using PMVEC membranes, where Ca(2+) and cAMP concentrations were independently controlled. Endogenous AC(6) activity exhibited high- and low-affinity Ca(2+) inhi...
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