Stimulation of beta-amyloid precursor protein alpha-processing by phorbol ester involves calcium and calpain activation

Normal processing of Alzheimer's beta-amyloid precursor protein (APP) is markedly stimulated by phorbol esters, but the underlying mechanisms have yet to be fully understood. In this study, we observed that: (a) Phorbol 12,13-dibutyrate (PDBu)-stimulated APP secretion in cultured SH-SY5Y neuroblastoma and fibroblast cells was blocked by EGTA and calpain inhibitors in a concentration-dependent manner, but not by other protease inhibitors. (b) Secretion of fibronectin, another secretory protein tested for comparison, was enhanced by PDBu, but insensitive to calpain inhibitors. (c) PDBu stimulated intracellular calpain activity as measured by the hydrolysis of a fluorogenic calpain substrate. (d) PDBu also induced rapid proteolysis of two endogenous substrates of calpains, i.e., tau and microtubule-associated protein-2 (MAP-2) and the proteolysis was blocked by EGTA and calpain inhibitors. Taken together, these results suggest that stimulation of APP alpha-processing by PDBu is through a mechanism that involves the activation of Ca(2+) and, most notably, calpain. The implications of the findings are discussed in relation to the regulatory mechanism of APP alpha-processing.     

Real-time visualization of cytoplasmic calpain activation and calcium deregulation in acute glutamate excitotoxicity

Although calpain (EC 3.4.22) protease activation was suggested to contribute to excitotoxic delayed calcium deregulation (DCD) via proteolysis of Na⁺/Ca²⁺ exchanger 3 (NCX3), cytoplasmic calpain activation in relation to DCD has never been visualized in real-time. We employed a calpain fluorescence resonance energy transfer substrate to simultaneously image calpain activation and calcium deregulation in live cortical neurons. A calpain inhibitor-sensitive decline in fluorescence resonance energy transfer was observed at 39 ± 5 min after the occurrence of DCD in neurons exposed to continuous glutamate (100 μM). Inhibition of calpain by calpeptin did not delay the onset of DCD, recovery from DCD-like reversible calcium elevations, or cell death despite inhibiting α-spectrin processing by > 90%. NCXs reversed during glutamate exposure, the NCX antagonist KB-R7943 prolonged the time to DCD, and significant NCX3 cleavage following 90 min of glutamate exposure was not observed. Our findings suggest that robust calpain activation associated with acute glutamate toxicity occurs only after a sustained loss in calcium homeostasis. Processing of NCX3 or other calpain substrates is unlikely to be the primary cause of acute excitotoxicity in cortical neurons. However, a role for calpain as a contributing factor or in response to milder glutamate insults is not excluded.     

Severe hypertriglyceridemia caused by Gpihbp1 deficiency facilitates vascular remodeling through increasing endothelial activation and oxidative stress

Hypertriglyceridemia (HTG) is an independent risk factor for atherosclerosis. However, its impact on non-atherosclerotic cardiovascular diseases remains largely unknown. Glycosylphosphatidylinositol anchored high-density lipoprotein binding protein 1 (GPIHBP1) is essential for the hydrolysis of circulating triglycerides and loss of functional GPIHBP1 causes severe HTG. In this study, we used Gpihbp1 knockout (GKO) mice to investigate the potential effects of HTG on non-atherosclerotic vascular remodeling. We compared the aortic morphology and gene expressions between three-month-old and ten-month-old GKO mice and their age-matched wild-type controls. We also conducted similar comparisons between GKO mice and wild-type controls in an angiotensin II (AngII)-induced vascular remodeling model. Our data showed that the intima-media wall of ten-month-old GKO mice but not three-month-olds was significantly thickened compared to wild-type controls. Moreover, ten-month-old GKO mice but not three-month-olds had increased aortic macrophage infiltration and perivascular fibrosis, along with increased endothelial activation and oxidative stress. Similarly, the AngII-induced vascular remodeling, as well as endothelial activation and oxidative stress, were also exacerbated in the GKO mice compared to wild-type controls. In conclusion, we demonstrated that severe HTG caused by Gpihbp1 deficiency could facilitate the onset and progression of non-atherosclerotic vascular remodeling through endothelial activation and oxidative stress in mice.     

Ischemia-reperfusion-induced calpain activation and SERCA2a degradation are attenuated by exercise training and calpain inhibition

The Ca2+-activated protease calpain has been shown to play a deleterious role in the heart during ischemia-reperfusion (I/R). We tested the hypothesis that exercise training would minimize I/R-induced calpain activation and provide cardioprotection against I/R-induced injury. Hearts from adult male rats were isolated in a working heart preparation, and myocardial injury was induced with 25 min of global ischemia followed by 45 min of reperfusion. In sedentary control rats, I/R significantly increased calpain activity and impaired cardiac performance (cardiac work during reperfusion = 24% of baseline). Compared with sedentary animals, exercise training prevented the I/R-induced rise in calpain activity and improved cardiac work (recovery = 80% of baseline). Similar to exercise, pharmacological inhibition of calpain activity resulted in comparable cardioprotection against I/R injury (recovery = 86% of baseline). The exercise-induced protection against I/R-induced calpain activation was not due to altered myocardial protein levels of calpain or calpastatin. However, exercise training was associated with increased myocardial antioxidant enzyme activity (Mn-SOD, catalase) and a reduction in oxidative stress. Importantly, exercise training also prevented the I/R-induced degradation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a. These findings suggest that increases in endogenous antioxidants may diminish the free radical-mediated damage and/or degradation of Ca2+ handling proteins (such as SERCA2a) typically observed after I/R. In conclusion, these results support the concept that calpain activation is an important component of I/R-induced injury and that exercise training provides cardioprotection against I/R injury, at least in part, by attenuating I/R-induced calpain activation.     

Spatial restriction of PDK1 activation cascades by anchoring to mAKAPalpha

The muscle A-kinase anchoring protein (mAKAP) tethers cAMP-dependent enzymes to perinuclear membranes of cardiomyocytes. We now demonstrate that two alternatively spliced forms of mAKAP are expressed: mAKAPalpha and mAKAPbeta. The longer form, mAKAPalpha, is preferentially expressed in the brain. mAKAPbeta is a shorter form of the anchoring protein that lacks the first 244 amino acids and is preferentially expressed in the heart. The unique amino terminus of mAKAPalpha can spatially restrict the activity of 3-phosphoinositide-dependent kinase-1 (PDK1). Biochemical and genetic analyses demonstrate that simultaneous recruitment of PDK1 and ERK onto mAKAPalpha facilitates activation and release of the downstream target p90RSK. The assembly of tissue-specific signaling complexes provides an efficient mechanism to integrate and relay lipid-mediated and mitogenic activated signals to the nucleus.     

Alpha 1-adrenoceptor activation elevates cytosolic calcium in rat pinealocytes by increasing net influx

The regulation of [Ca2+]i in rat pinealocytes was studied using the fluorescent indicator quin2. Pinealocyte resting [Ca2+]i was approximately 100 nM; this rapidly decreased in low Ca2+ medium (approximately 10 microM), indicating there was a high turnover of [Ca2+]i in these cells. Norepinephrine (NE, 10(-6) M) increased [Ca2+]i to approximately 350 nM within 1 min; [Ca2+]i then remained elevated for 30 min. The relative potency of adrenergic agonists was NE greater than phenylephrine much greater than isoproterenol. Phentolamine (10(-6) M) and prazosin (10(-8) M) blocked the effects of adrenergic agonists; in contrast, propranolol (10(-6) M) or yohimbine (10(-6) M) had little or no effect. These observations indicate NE acts via alpha 1-adrenoceptors to elevate [Ca2+]i. The [Ca2+]i response to NE did not occur when [Ca2+]e was reduced to approximately 10 microM by adding EGTA 5s before NE, indicating an increase in net Ca2+ influx is involved rather than mobilization of Ca2+ from intracellular stores. The effect of NE was not blocked by nifedipine (10(-6) M), which did block a K+-induced increase in [Ca2+]i, presumably involving voltage-sensitive channels. Ouabain (10(-5) M) caused a gradual increase in [Ca2+]i; this increase was not blocked by nifedipine. Together these data indicate that pinealocyte [Ca2+]i may be influenced by mechanisms regulated by alpha 1-adrenoceptors, voltage-dependent Ca2+ channels, and perhaps a Na+/Ca2+ exchange mechanism stimulated by ouabain. These studies indicate that the pinealocyte is an interesting model to use to study the adrenergic regulation of [Ca2+]i because of the rapid and prolonged changes in [Ca2+]i produced by alpha 1-adrenoceptor activation.     

Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells

L-type Ca²⁺ channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba²⁺ influx in a depolarizing medium. In the absence of extracellular Ca²⁺, activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba²⁺ influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca²⁺-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in a Ca²⁺-free medium from 30 to 80 nM. This [Ca²⁺]_i increase declined slowly, giving rise to a modest elevation of [Ca²⁺]_i that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca²⁺-free medium. However, when the Ca²⁺ ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca²⁺ channel activity, suggesting that a lower amplitude and prolonged release of Ca²⁺ stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 μM) and ascomycin (5 μM), inhibitors of the Ca²⁺/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1 mM CaCl₂ to the Tg-treated cells increased [Ca²⁺]_i to 165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca²⁺]_i increase elicited by passive depletion of internal Ca²⁺ stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca²⁺]_i via store-dependent Ca²⁺ entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca²⁺ channels in a myocyte model involves a balance between the differential Ca²⁺ sensitivities and opposing actions of PKC and calcineurin.     

Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.     

Chloroquine,quinine and quinidine inhibit calcium release from macrophage intracellular stores by blocking inositol 1,4,5-trisphosphate binding to its receptor

The binding of many ligands to cellular receptors induces a signaling cascade which generates inositol 1,4,5-trisphosphate (IP₃). IP₃ binding to its receptors in various internal compartments causes a rapid Ca²⁺ efflux into the cytosol. We now demonstrate that chloroquine blocks ligand-induced Ca²⁺ mobilization without affecting IP₃ synthesis. The effect is independent of the ligand employed and occurred with five unrelated ligands; namely, α₂-macroglobulin-methylamine, angiotensin II, bradykinin, carbachol, and epidermal growth factor. Chloroquine, quinidine, and quinine, however, block binding of [³H]IP₃ to its receptors by 90%, 88%, and 71%, respectively. These observations suggest a previously undetected mechanism by which these agents may in part function as antimalarials. J. Cell. Biochem. 64:225–232. © 1997 Wiley-Liss, Inc.     

Dynamics of calcium release and uptake by the internal calcium stores in rat sensory neurons

Calcium dynamics in the endoplasmic reticulum of dorsal root ganglion neurons of rats during Ca²⁺ release induced by caffeine and subsequent Ca²⁺ uptake were studied. Calcium release is shown to include two (a short transient and a prolonged slow) phases. We suggest that the transient phase reflects release of free Ca from the calcium store, while the slow phase reflects transition of Ca from a bound form to a free one. The process of Ca²⁺ uptake is characterized by exponential recovery of the calcium level in the store due to the SERCA activity. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 361–363, July–August, 2006.     

Ceramide increase cytoplasmic Ca2+ concentration in Jurkat T cells by liberation of calcium from intracellular stores and activation of a store-operated calcium channel

The effect of ceramide on the cytoplasmic Ca2+ concentration ([Ca2+]i) varies depending on the cell type. We have found that in Jurkat human T cells ceramide increases the [Ca2+]i from a thapsigargin-sensitive calcium pool and the subsequent activation of a capacitative Ca2+ entry. This effect occurs both in the presence and in the absence of extracellular calcium. Addition of ceramine, a non-hydrolysable analogue of ceramide, reproduced its effect on the [Ca2+]i ruling out that this is due to the conversion of ceramide to sphingosine. The effect of ceramide was additive to that obtained by sphingosine, but not to the Jurkat T cells specific antibody OKT3. However, different to the latter, ceramide do not induced an elevation of InsP3. The opening of a store operated Ca2+ channel by ceramide was corroborated by experiments of Fura-2 quenching, using Mn2+ as a surrogate for Ca2+ and confirmed by whole-cell recording patch clamp techniques.     

Contribution of distinct structural elements to activation of calpain by Ca2+ ions

The effect of Ca2+ in calpain activation is mediated via several binding sites in the enzyme molecule. To test the contribution of structural elements suspected to be part of this Ca2+ relay system, we made a site-directed mutagenesis study on calpains, measuring consequential changes in Ca2+ binding and Ca2+ sensitivity of enzyme activity. Evidence is provided for earlier suggestions that an acidic loop in domain III and the transducer region connecting domains III and IV are part of the Ca2+ relay system. Wild-type Drosophila Calpain B domain III binds two to three Ca2+ ions with a K(d) of 3400 microm. Phospholipids lower this value to 220 microm. Ca2+ binding decreases in parallel with the number of mutated loop residues. Deletion of the entire loop abolishes binding of the ion. The Ca2+ dependence of enzyme activity of various acidic-loop mutants of Calpain B and rat m-calpain suggests the importance of the loop in regulating activity. Most conspicuously, the replacement of two adjacent acidic residues in the N-terminal half of the loop evokes a dramatic decrease in the Ca2+ need of both enzymes, lowering half-maximal Ca2+ concentration from 8.6 to 1.3 mm for Calpain B and from 250 to 7 microm for m-calpain. Transducer-region mutations in m-calpain also facilitate Ca2+ activation with the most profound effect seen upon shortening the region by deletion mutagenesis. All of these data along with structural considerations suggest that the acidic loop and the transducer region form an interconnected, extended structural unit that has the capacity to integrate and transduce Ca2+-evoked conformational changes over a long distance. A schematic model of this extended transducer mechanism is presented.     

The antigen receptor on a human T cell line initiates activation by increasing cytoplasmic free calcium

A monoclonal antibody to the antigen-receptor on the T cell line Jurkat induces substantial increases in [Ca++]i. Ca++ ionophores can substitute for this antibody in activation by increasing [Ca++]i to levels comparable with those seen with the antigen-receptor antibody. Stimulation with either the antigen-receptor antibody or a Ca++ ionophore leads to the appearance of the same phosphoproteins. These results suggest that the antigen-receptor initiates T cell activation by increasing [Ca++]i.     

In contrast to sheep, goats adapt to dietary calcium restriction by increasing intestinal absorption of calcium

Many studies aimed at understanding calcium homeostasis in the cow use sheep or goats as ruminant models. However, the comparability of relevant homeostatic processes between ruminant species has not been assessed. Therefore, we investigated whether the mechanisms of maintaining calcium homeostasis are similar in different ruminant species. Dietary calcium of goats was restricted along with treatment with calcitriol in a similar protocol to that in a recent study with sheep. Plasma calcium and phosphate and parameters of bone metabolism were analysed. Gastrointestinal calcium transport was characterised in vitro in Ussing chambers. The expression of apical epithelial calcium channels, calbindin-D(9K), and the basolateral plasma membrane Ca(2+)-ATPase was determined by quantitative RT-PCR and Western blot analysis. In contrast to sheep, the goats were able to compensate for low dietary calcium supply by increasing active calcium absorption in the small intestine, especially in the jejunum. As in sheep, the observed ruminal calcium transport of goats was affected neither by the calcium restricted diet nor by the calcitriol treatment, thus indicating the presence of an alternative, vitamin D-independent mechanism of calcium transport in the forestomachs. These results demonstrate that mechanisms for maintaining calcium homeostasis differ significantly between ruminant species.     

The importance of calcium influx, calpain and calmodulin for the activation of CaCo-2 cell death pathways by Clostridium perfringens enterotoxin

CaCo-2 cells exhibit apoptosis when treated with low doses of Clostridium perfringens enterotoxin (CPE), but develop oncosis when treated with high CPE doses. This study reports that the presence of extracellular Ca(2+) in treatment buffers is important for normal activation of both those cell death pathways in CPE-treated CaCo-2 cells. Normal development of CPE-induced cell death pathway effects, such as morphologic damage, DNA fragmentation, caspase activation, mitochondrial membrane depolarization and cytochrome c release, was strongly inhibited when CaCo-2 cells were CPE-treated in Ca(2+)-free buffers. When treatment buffers contained Ca(2+), CPE caused a rapid increase in CaCo-2 cell Ca(2+) levels, apparently because of increased Ca(2+) influx through a CPE pore. High CPE doses caused massive changes in cellular Ca(2+) levels that appear responsible for activating oncosis, whereas low CPE doses caused less perturbations in cellular Ca(2+) levels that appear responsible for activating apoptosis. Both CPE-induced apoptosis and oncosis were found to be calmodulin- and calpain-dependent processes. As Ca(2+) levels present in the intestinal lumen resemble those of Ca(2+)-containing treatment buffers used in this study, perturbations in cellular Ca(2+) levels and calpain/calmodulin-dependent processes are also probably important for inducing enterocyte cell death during CPE-mediated gastrointestinal disease.     

Phorbol ester contracts rabbit thoracic aorta by increasing intracellular calcium and by activating calcium influx

The ability of the phorbol ester tumor promoter, PDB, to activate contraction and stimulate calcium influx was investigated in rabbit thoracic aorta. PDB caused a strong, slowly-developing sustained contraction in physiological salt solution which was concentration-related (0.01 to 10.0 microM). PDB-induced contractions (0.1 microM) in calcium-free medium were attenuated but not prevented. PDB (1.0 microM) maximally stimulated Ca influx above basal control, vehicle = 39.2 +/- 2.2; PDB 1.0 microM = 70.7 +/- 6.7 mumoles Ca/kg tissue; N = 16, p less than 0.01). These data suggest that PDB activates rabbit thoracic aorta by a combination of intracellular and extracellular calcium dependent mechanisms.     

Calmodulin modulates the delay period between release of calcium from internal stores and activation of calcium influx via endogenous TRP1 channels

In the present study we have explored the role of calmodulin (CaM) and inositol 1,4,5-trisphosphate receptor (IP(3)R) in the communication process activated after the release of calcium from the endoplasmic reticulum (ER) and the activation of calcium influx via endogenous TRP1 channels from Chinese hamster ovary cells. Experiments using combined rapid confocal calcium and electrophysiology measurements uncovered a consistent delay of around 900 ms between the first detectable calcium released from the ER and the activation of the calcium current. This delay was evident with two different methods used to release calcium from the ER: either the blockade of the microsomal calcium ATPase with thapsigargin or activation of bradykinin receptors linked to the IP(3) cascade. Direct application of IP(3) or a peptide from the NH(2)-terminal region of the IP(3)R activated store operated calcium, reducing the delay period. Introduction of CaM into the cell via the patch pipette increased the delay period from 900 +/- 100 ms to 10 +/- 2.1 s (n = 18). Furthermore, the use of selective CaM antagonists W7 and trifluoperazine maleate resulted in a substantial reduction of the delay period to 200 +/- 100 ms with 5 microm trifluoperazine maleate (n = 16) and 150 +/- 50 ms with 500 nm W7 (n = 22). CaM reduced also the current density activated by thapsigargin or brandykinin to about 60% from control. The CaM antagonists did not affect significantly the current density. The results presented here are consistent with an antagonistic effect of IP(3)R and CaM for the activation of store operated calcium after depletion of the ER. The functional competition between the activating effect of IP(3)R and the inhibiting effect of CaM may modulate the delay period between the release of calcium from the ER and the activation of calcium influx observed in different cells, as well as the amount of current activated after depletion of the ER.     

NADPH oxidase activation increases the sensitivity of intracellular Ca2+ stores to inositol 1,4,5-trisphosphate in human endothelial cells

Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca(2+), but whether NADPH oxidase activation directly affects Ca(2+) signaling is unknown. NADPH stimulated the production of superoxide anion and H(2)O(2) in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rac1, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H(2)O(2) each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP(3)) required to release intracellularly stored Ca(2+) and shifted the InsP(3)-Ca(2+) release dose-response curve to the left. Concentrations of H(2)O(2) as low as 3 microm increased the sensitivity of intracellular Ca(2+) stores to InsP(3) and decreased the InsP(3) EC(50) from 423.2 +/- 54.9 to 276.9 +/- 14. 4 nm. The effect of NADPH on InsP(3)-stimulated Ca(2+) release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H(2)O(2) increases the sensitivity of intracellular Ca(2+) stores to InsP(3) in human endothelial cells. Since Ca(2+)-dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction.