cAMP inhibits IP(3)-dependent Ca(2+) release by preferential activation of cGMP-primed PKG

The singular effects and interplay of cAMP- and cGMP-dependent protein kinase (PKA and PKG) on Ca(2+) mobilization were examined in dispersed smooth muscle cells. In permeabilized muscle cells, exogenous cAMP and cGMP inhibited inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of cAMP and cGMP caused synergistic inhibition that was exclusively mediated by PKG and attenuated by PKA. In intact muscle cells, low concentrations (10 nM) of isoproterenol and sodium nitroprusside (SNP) inhibited agonist-induced, IP(3)-dependent Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of isoproterenol and SNP increased PKA and PKG activities: the increase in PKA activity reflected inhibition of phosphodiesterase 3 activity by cGMP, whereas the increase in PKG activity reflected activation of cGMP-primed PKG by cAMP. Inhibition of Ca(2+) release and muscle contraction by the combination of isoproterenol and SNP was preferentially mediated by PKG. In light of studies showing that PKG phosphorylates the IP(3) receptor in intact and permeabilized muscle cells, whereas PKA phosphorylates the receptor in permeabilized cells only, the results imply that inhibition of IP(3)-induced Ca(2+) release is mediated exclusively by PKG. The effect of PKA on agonist-induced Ca(2+) release probably reflects inhibition of IP(3) formation.     

The alpha2-adrenergic receptor agonist UK 14,304 inhibits secretin-stimulated ductal secretion by downregulation of the cAMP system in bile duct-ligated rats

Secretin stimulates ductal secretion by activation of cAMP PKA CFTR Cl^–/HCO₃^– exchanger in cholangiocytes. We evaluated the expression of _2A-, _2B-, and _2C-adrenergic receptors in cholangiocytes and the effects of the selective ₂-adrenergic agonist UK 14,304, on basal and secretin-stimulated ductal secretion. In normal rats, we evaluated the effect of UK 14,304 on bile and bicarbonate secretion. In bile duct-ligated (BDL) rats, we evaluated the effect of UK 14,304 on basal and secretin-stimulated 1) bile and bicarbonate secretion; 2) duct secretion in intrahepatic bile duct units (IBDU) in the absence or presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na⁺/H⁺ exchanger isoform NHE3; and 3) cAMP levels, PKA activity, Cl^– efflux, and Cl^–/HCO₃^– exchanger activity in purified cholangiocytes. ₂-Adrenergic receptors were expressed by all cholangiocytes in normal and BDL liver sections. UK 14,304 did not change bile and bicarbonate secretion of normal rats. In BDL rats, UK 14,304 inhibited secretin-stimulated 1) bile and bicarbonate secretion, 2) expansion of IBDU luminal spaces, and 3) cAMP levels, PKA activity, Cl^– efflux, and Cl^–/HCO₃^– exchanger activity in cholangiocytes. There was decreased lumen size after removal of secretin in IBDU pretreated with UK 14,304. In IBDU pretreated with EIPA, there was no significant decrease in luminal space after removal of secretin in either the absence or presence of UK 14,304. The inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3. ₂-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases. bicarbonate secretion; chloride efflux; gastrointestinal hormones; intrahepatic biliary epithelium; protein kinase A     

Ca(2+)-sensor region of IP(3) receptor controls intracellular Ca(2+) signaling

Many important cell functions are controlled by Ca(2+) release from intracellular stores via the inositol 1,4,5-trisphosphate receptor (IP(3)R), which requires both IP(3) and Ca(2+) for its activity. Due to the Ca(2+) requirement, the IP(3)R and the cytoplasmic Ca(2+) concentration form a positive feedback loop, which has been assumed to confer regenerativity on the IP(3)-induced Ca(2+) release and to play an important role in the generation of spatiotemporal patterns of Ca(2+) signals such as Ca(2+) waves and oscillations. Here we show that glutamate 2100 of rat type 1 IP(3)R (IP(3)R1) is a key residue for the Ca(2+) requirement. Substitution of this residue by aspartate (E2100D) results in a 10-fold decrease in the Ca(2+) sensitivity without other effects on the properties of the IP(3)R1. Agonist-induced Ca(2+) responses are greatly diminished in cells expressing the E2100D mutant IP(3)R1, particularly the rate of rise of initial Ca(2+) spike is markedly reduced and the subsequent Ca(2+) oscillations are abolished. These results demonstrate that the Ca(2+) sensitivity of the IP(3)R is functionally indispensable for the determination of Ca(2+) signaling patterns.     

IP(3)-dependent intracellular Ca(2+) release is required for cAMP-induced c-fos expression in hippocampal neurons

Endothelial argininosuccinate synthetase 1 (ASS1) regulates the provision of l-arginine to nitric oxide synthase 3 (NOS3). Previous studies demonstrated that endothelial ASS1 expression was induced by laminar shear stress (LSS) and that this enzyme plays a role in maintaining anti-inflammatory microenvironments through enhancing NO production. However, differently from the case of NOS3, the regulatory mechanism for the endothelial ASS1 expression in response to LSS is not well understood. This study addressed a specific issue whether endothelial ASS1 expression is regulated by Kruppel-like factors (KLFs) that are presumed to coordinate endothelial gene expressions in response to LSS. The cDNA microarray data indicated that LSS stimulated the expression of numerous KLFs in human umbilical vein endothelial cells. KLF4 showed the highest fold increase and LSS-dependent increases of KLF4 and most other KLFs were similar in young versus senescent endothelial cells. LSS-induced KLF4 expression was verified by RT-PCR and Western blotting. LSS-induced ASS1 expression and NO production were suppressed by a small interfering RNA for KLF4. The ectopic expression of KLF4 led to the increase of ASS1 expression and NO production. The present study demonstrated a key regulatory role of KLF4 in the endothelial ASS1 expression and NO production in response to LSS.     

Cytosolic Ca(2+) controls the loading dependence of IP(3)-induced Ca(2+) release.

It is still debated whether inositol 1,4, 5-trisphosphate(IP(3))-induced Ca(2+) release is loading-dependent. We now report that stimulation of the IP(3) receptor by luminal Ca(2+) depends on the cytosolic [Ca(2+)] in permeabilized A7r5 cells. The EC(50) and maximal extent of Ca(2+) release were loading-dependent in the presence of 5 mM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid: the EC(50) increased 1.9-fold and the maximal release decreased from 88 to 52% when the stores contained 73% less Ca(2+). In the presence of 0.3 microM free Ca(2+), the EC(50) for filled and less filled stores differed, however, only 1.2-fold and the maximal Ca(2+) release was respectively 96 and 87% of the total releasable Ca(2+). At 1 microM free Ca(2+), the difference in EC(50) between filled and less filled stores again became larger (2.2-fold) and the maximal Ca(2+) release decreased from 93 to 87% when the stores contained less Ca(2+).     

The small chemical vacuolin-1 inhibits Ca(2+)-dependent lysosomal exocytosis but not cell resealing

Resealing after wounding, the process of repair following plasma membrane damage, requires exocytosis. Vacuolins are molecules that induce rapid formation of large, swollen structures derived from endosomes and lysosomes by homotypic fusion combined with uncontrolled fusion of the inner and limiting membranes of these organelles. Vacuolin-1, the most potent compound, blocks the Ca(2+)-dependent exocytosis of lysosomes induced by ionomycin or plasma membrane wounding, without affecting the process of resealing. In contrast, other cell structures and membrane trafficking functions including exocytosis of enlargeosomes are unaffected. Because cells heal normally in the presence of vacuolin-1, we suggest that lysosomes are dispensable for resealing.     

FAM3D inhibits glucagon secretion via MKP1-dependent suppression of ERK1/2 signaling

Dysregulated glucagon secretion is a hallmark of type 2 diabetes (T2D). To date, few effective therapeutic agents target on deranged glucagon secretion. Family with sequence similarity 3 member D (FAM3D) is a novel gut-derived cytokine-like protein, and its secretion timing is contrary to that of glucagon. However, the roles of FAM3D in metabolic disorder and its biological functions are largely unknown. In the present study, we investigated whether FAM3D modulates glucagon production in mouse pancreatic alpha TC1 clone 6 (αTC1-6) cells. Glucagon secretion, prohormone convertase 2 (PC2) activity, and mitogen-activated protein kinase (MAPK) pathway were assessed. Exogenous FAM3D inhibited glucagon secretion, PC2 activity, as well as extracellular-regulated protein kinase 1/2 (ERK1/2) signaling and induced MAPK phosphatase 1 (MKP1) expression. Moreover, knockdown of MKP1 and inhibition of ERK1/2 abolished and potentiated the inhibitory effect of FAM3D on glucagon secretion, respectively. Taken together, FAM3D inhibits glucagon secretion via MKP1-dependent suppression of ERK1/2 signaling. These results provide rationale for developing the therapeutic potential of FAM3D for dysregulated glucagon secretion and T2D.     

H(2)O(2)-induced Ca(2+) overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway

Generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload are key mechanisms involved in ischemia-reperfusion (I/R)-induced myocardial injury. The relationship between I/R injury and Ca(2+) overload has not been fully characterized. The increase in Na(+)/H(+) exchanger (NHE-1) activity observed during I/R injury is an attractive candidate to link increased ROS production with Ca(2+) overload. We have shown that low doses of H(2)O(2) increase NHE-1 activity in an extracellular signal-regulated kinase (ERK)-dependent manner. In this study, we examined the effect of low doses of H(2)O(2) on intracellular Ca(2+) in fura 2-loaded, spontaneously contracting neonatal rat ventricular myocytes. H(2)O(2) induced a time- and concentration-dependent increase in diastolic intracellular Ca(2+) concentration that was blocked by inhibition of ERK1/2 activation with 5 microM U-0126 (88%) or inhibition of NHE-1 with 5 microM HOE-642 (50%). Increased NHE activity was associated with phosphorylation of the NHE-1 carboxyl tail that was blocked by U-0126. These results suggest that H(2)O(2) induced Ca(2+) overload is partially mediated by NHE-1 activation secondary to phosphorylation of NHE-1 by the ERK1/2 MAP kinase pathway.     

Activation of the human intermediate-conductance Ca(2+)-activated K(+) channel by 1-ethyl-2-benzimidazolinone is strongly Ca(2+)-dependent.

Modulation of the cloned human intermediate-conductance Ca(2+)-activated K(+) channel (hIK) by the compound 1-ethyl-2-benzimidazolinone (EBIO) was studied by patch-clamp technique using human embryonic kidney cells (HEK 293) stably expressing the hIK channels. In whole-cell studies, intracellular concentrations of free Ca(2+) were systematically varied, by buffering the pipette solutions. In voltage-clamp, the hIK specific currents increased gradually from 0 to approximately 300 pA/pF without reaching saturation even at the highest Ca(2+) concentration tested (300 nM). In the presence of EBIO (100 microM), the Ca(2+)-activation curve was shifted leftwards, and maximal currents were attained at 100 nM Ca(2+). In current-clamp, steeply Ca(2+)-dependent membrane potentials were recorded and the cells gradually hyperpolarised from -20 to -85 mV when Ca(2+) was augmented from 0 to 300 nM. EBIO strongly hyperpolarised cells buffered at intermediate Ca(2+) concentrations. In contrast, no effects were detected either below 10 nM (no basic channel activation) or at 300 nM Ca(2+) (V(m) close to E(K)). Without Ca(2+), EBIO-induced hyperpolarisations were not obtainable, indicating an obligatory Ca(2+)-dependent mechanism of action. When applied to inside-out patches, EBIO exerted a Ca(2+)-dependent increase in the single-channel open-state probability, showing that the compound modulates hIK channels by a direct action on the alpha-subunit or on a closely associated protein. In conclusion, EBIO activates hIK channels in whole-cell and inside-out patches by a direct mechanism, which requires the presence of internal Ca(2+).     

A CaMKII/calcineurin switch controls the direction of Ca(2+)-dependent growth cone guidance

Axon pathfinding depends on attractive and repulsive turning of growth cones to extracellular cues. Localized cytosolic Ca2+ signals are known to mediate the bidirectional responses, but downstream mechanisms remain elusive. Here, we report that calcium-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) phosphatase provide a switch-like mechanism to control the direction of Ca(2+)-dependent growth cone turning. A relatively large local Ca2+ elevation preferentially activates CaMKII to induce attraction, while a modest local Ca2+ signal predominantly acts through CaN and phosphatase-1 (PP1) to produce repulsion. The resting level of intracellular Ca2+ concentrations also affects CaMKII/CaN operation: a normal baseline allows distinct turning responses to different local Ca2+ signals, while a low baseline favors CaN-PP1 activation for repulsion. Moreover, the cAMP pathway negatively regulates CaN-PP1 signaling to inhibit repulsion. Finally, CaMKII/CaN-PP1 also mediates netrin-1 guidance. Together, these findings establish a complex Ca2+ mechanism that targets the balance of CaMKII/CaN-PP1 activation to control distinct growth cone responses.     

Timescales of IP(3)-evoked Ca(2+) spikes emerge from Ca(2+) puffs only at the cellular level

The behavior of biological systems is determined by the properties of their component molecules, but the interactions are usually too complex to understand fully how molecular behavior generates cellular behavior. Ca(2+) signaling by inositol trisphosphate receptors (IP(3)R) offers an opportunity to understand this relationship because the cellular behavior is defined largely by Ca(2+)-mediated interactions between IP(3)R. Ca(2+) released by a cluster of IP(3)R (giving a local Ca(2+) puff) diffuses and ignites the behavior of neighboring clusters (to give repetitive global Ca(2+) spikes). We use total internal reflection fluorescence microscopy of two mammalian cell lines to define the temporal relationships between Ca(2+) puffs (interpuff intervals, IPI) and Ca(2+) spikes (interspike intervals) evoked by flash photolysis of caged IP(3). We find that IPI are much shorter than interspike intervals, that puff activity is stochastic with a recovery time that is much shorter than the refractory period of the cell, and that IPI are not periodic. We conclude that Ca(2+) spikes do not arise from oscillatory dynamics of IP(3)R clusters, but that repetitive Ca(2+) spiking with its longer timescales is an emergent property of the dynamics of the whole cluster array.     

WNK1 activates ERK5 by an MEKK2/3-dependent mechanism

WNK1 belongs to a unique protein kinase family that lacks the catalytic lysine in its normal position. Mutations in human WNK1 and WNK4 have been implicated in causing a familial form of hypertension. Here we report that overexpression of WNK1 led to increased activity of cotransfected ERK5 in HEK293 cells. ERK5 activation was blocked by the MEK5 inhibitor U0126 and expression of a dominant negative MEK5 mutant. Expression of dominant negative mutants of MEKK2 and MEKK3 also blocked activation of ERK5 by WNK1. Moreover, both MEKK2 and MEKK3 coimmunoprecipitated with endogenous WNK1 from cell lysates. WNK1 phosphorylated both MEKK2 and -3 in vitro, and MEKK3 was activated by WNK1 in 293 cells. Finally, ERK5 activation by epidermal growth factor was attenuated by suppression of WNK1 expression using small interfering RNA. Taken together, these results place WNK1 in the ERK5 MAP kinase pathway upstream of MEKK2/3.     

Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells

The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca(2+)-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca(2+) homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression increases the Ca(2+) leak (while leaving Ca(2+) accumulation unaffected), hence reducing the steady-state [Ca(2+)] levels. As a direct consequence, the [Ca(2+)] increases caused by inositol 1,4,5 trisphosphate (IP3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria. Bcl-2 overexpression also reduced the rate of Ca(2+) influx activated by Ca(2+) store depletion, possibly by an adaptive downregulation of this pathway. By interfering with Ca(2+)-dependent events at multiple intracellular sites, these effects of Bcl-2 on intracellular Ca(2+) homeostasis may contribute to the protective role of this oncogene against programmed cell death.     

Caveolin-3 inhibits growth signal in cardiac myoblasts in a Ca2+-dependent manner

Caveolin, a major protein component of caveolae, directly interacts with multiple signaling molecules, such as Ras and growth factor receptors, and inhibits their function. However, the role of the second messenger system in mediating this inhibition by caveolin remains poorly understood. We examined the role of Ca2+-dependent signal in caveolin- mediated growth inhibition using a rat cardiac myoblast cell line (H9C2), in which the expression of caveolin- 3, the muscle specific subtype, can be induced using the LacSwitch system. Upon induction with IPTG and serum-starvation, the expression of caveolin-3 was increased by 3.3-fold relative to that of mock-induced cells. The recombinant caveolin-3 was localized to the same subcellular fraction as endogenous caveolin-3 after sucrose gradient purification. Angiotensin II enhanced ERK phosphorylation, but this enhancement was significantly decreased in caveolin-3-induced cells in comparison to that in mock-induced cells. Similarly, when cells were stimulated with fetal calf serum, DNA synthesis, as determined by [3H]-thymidine incorporation, was significantly decreased in caveolin- 3-induced cells. When cells were treated with Ca2+ chelator (BAPTA and EGTA), however, this attenuation was blunted. Calphostin (PKC inhibitor), but not cyclosporine A treatment (calcineurin inhibitor), blunted this attenuation in caveolin-3 induced cells. Our findings suggest that caveolin exhibits growth inhibition in a Ca2+-dependent manner, most likely through PKC, in cardiac myoblasts.     

Initiation of IP(3)-mediated Ca(2+) waves in Xenopus oocytes.

Inositol (1,4,5)-trisphosphate (IP(3)) evokes Ca(2+) liberation in Xenopus oocytes as elementary events (Ca(2+) puffs) that become coupled to propagate Ca(2+) waves with increasing [IP(3)]. To investigate this transition between local and global Ca(2+) signaling, we developed an optical method for evoking rapid subcellular Ca(2+) elevations, while independently photoreleasing IP(3) and simultaneously recording confocal Ca(2+) images. Focal Ca(2+) elevations triggered waves within 100 ms of photoreleasing IP(3), compared with latencies of seconds following photorelease of IP(3) alone. Wave velocity varied with [IP(3)] but was independent of time after photorelease of IP(3), indicating that delayed wave initiation did not involve slow binding of IP(3) to its receptors. The amount of Ca(2+) required to trigger a wave was approximately 10-fold greater than the average size of puffs, and puffs showed no progressive increase in magnitude before waves initiated. Instead, Ca(2+) puffs contributed to a slow rise in basal free [Ca(2+)], which further increased puff frequency and sensitized IP(3) receptors so that individual events then triggered waves. Because the wave threshold is much greater than the size of the elementary puff, cells can employ both local and global signaling mechanisms, and the summation of stochastic behavior of elementary events allows generation of reproducible periodic waves.     

Ca(2+)-dependent ubiquitination of calmodulin in yeast.

Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner by ubiquityl-calmodulin synthetase (uCaM-synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253-256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM-synthetase conjugated ubiquitin Ca(2+)-dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (reticulocyte) uCaM-synthetase. As estimated from autoradiograms the monoubiquitination product (first-order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23-26 kDa and the second-order conjugate an apparent molecular mass of ca. 28-32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.     

Calmodulin regulates Ca(2+)-dependent feedback inhibition of store-operated Ca(2+) influx by interaction with a site in the C terminus of TrpC1

The mechanism involved in [Ca(2+)](i)-dependent feedback inhibition of store-operated Ca(2+) entry (SOCE) is not yet known. Expression of Ca(2+)-insensitive calmodulin (Mut-CaM) but not wild-type CaM increased SOCE and decreased its Ca(2+)-dependent inactivation. Expression of TrpC1 lacking C terminus aa 664-793 (TrpC1DeltaC) also attenuated Ca(2+)-dependent inactivation of SOCE. CaM interacted with endogenous and expressed TrpC1 and with GST-TrpC1 C terminus but not with TrpC1DeltaC. Two CaM binding domains, aa 715-749 and aa 758-793, were identified. Expression of TrpC1Delta758-793 but not TrpC1Delta715-749 mimicked the effects of TrpC1DeltaC and Mut-CaM on SOCE. These data demonstrate that CaM mediates Ca(2+)-dependent feedback inhibition of SOCE via binding to a domain in the C terminus of TrpC1. These findings reveal an integral role for TrpC1 in the regulation of SOCE.     

IP3-independent signalling of OX1 orexin/hypocretin receptors to Ca2+ influx and ERK

OX1 orexin receptors (OX1R) have been shown to activate receptor-operated Ca2+ influx pathways as their primary signalling pathway; however, investigations are hampered by the fact that orexin receptors also couple to phospholipase C, and therewith inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ release. We have here devised a method to block the latter signalling in order to focus on the mechanism of Ca2+ influx activation by OX1R in recombinant systems. Transient expression of the IP3-metabolising enzymes IP3-3-kinase-A (inositol-1,4,5-trisphosphate-->inositol-1,3,4,5-tetrakisphosphate) and type I IP3-5-phosphatase (inositol-1,4,5-trisphosphate-->inositol-1,4-bisphosphate) almost completely attenuated the OX1R-stimulated IP3 elevation and Ca2+ release from intracellular stores. Upon attenuation of the IP3-dependent signalling, the receptor-operated Ca2+ influx pathway became the only source for Ca2+ elevation, enabling mechanistic studies on the receptor-channel coupling. Attenuation of the IP3 elevation did not affect the OX1R-mediated ERK (extracellular signal-regulated kinase) activation in CHO cells, which supports our previous finding of the major importance of receptor-operated Ca2+ influx for this response.     

Mechanism of Ca(2+)-dependent desensitization in TRP channels

The 30+ members of the family of TRP channels are diverse in their physiological roles, yet the mechanisms that regulate their gating may be conserved. In particular, all TRP channels show an activity-dependent inhibition which is mediated by Ca(2+). The mechanism by which Ca(2+) inhibits TRP channels is currently a matter of intense debate, with Ca(2+)-regulated kinases, phosphatases, phospholipases and calmodulin all proposed to be involved. In this review, we will discuss different mechanisms for Ca(2+)-dependent desensitization in TRP channels. We will conclude with a model that focuses on Ca(2+)-dependent activation of phospholipase C and Ca(2+) binding to calmodulin and propose that the phospholipase C and calmodulin pathways are structurally and functionally coupled.