PACAP activates PKA, PKC and Ca(2+) signaling cascades in rat neuroepithelial cells.

Several studies have reported that the PAC(1) receptor (PAC1-R), the specific receptor for PACAP, is expressed at early developmental stages. Here, we describe that the cytosolic Ca(2+) concentration ([Ca(2+)](i)) was increased by PACAP, but not VIP, in a concentration range from 10(-12) to 10(-8) M via the PAC(1)-R in isolated single cells from the rat neural fold. This activation of the cells by PACAP was mimicked by agonists and inhibited by antagonists of the cAMP/PKA and PLC/PKC cascades. These data indicate that PACAP/PAC(1)-R is linked to [Ca(2+)](i) signaling via two G-protein-coupled protein kinase pathways and may thereby play an important role in early neurodevelopment.     

Simvastatin triggers mitochondria-induced Ca2+ signaling alteration in skeletal muscle

Statin drugs represent the major improvement in the treatment of hypercholesterolemia that constitutes the main origin of atherosclerosis, leading to coronary heart disease. Besides tremendous beneficial effects of statins, various forms of muscular toxicity (myalgia, cramp, exercise intolerance, and fatigability) occur frequently. We hypothesized that the iatrogenic effects of statins could result from alterations in Ca²⁺ homeostasis. Acute applications of simvastatin on human skeletal muscle fibers triggered a Ca²⁺ wave of intra-cellular Ca²⁺ that mostly originates from sarcoplasmic reticulum (SR) Ca²⁺-release. In addition, simvastatin increased mitochondrial NADH content and induced mitochondrial membrane depolarization (EC₅₀ = 1.96 μM) suggesting an altered mitochondrial function. Consequently on simvastatin application, a weak mitochondrial Ca²⁺ efflux (EC₅₀ = 7.8μM) through permeability transient pore and Na⁺/Ca²⁺ exchanger was triggered, preceding the large SR-Ca²⁺ release. Increased SR Ca²⁺ content after acute application of statin is also suggested by the increased Ca²⁺ spark amplitude and by the effect of cyclopiazonic acid. We thus conclude that simvastatin induced alterations in mitochondrial function which lead to an increase in cytoplasmic Ca²⁺, SR-Ca²⁺ overload, and Ca²⁺ waves. Taken together, these statin-induced muscle dysregulations may contribute to myotoxicity.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Calmodulin Regulates Ca2+-sensing Receptor-mediated Ca2+ Signaling and Its Cell Surface Expression

The Ca²⁺-sensing receptor (CaSR) is a member of family C of the GPCRs responsible for sensing extracellular Ca²⁺ ([Ca²⁺]_o) levels, maintaining extracellular Ca²⁺ homeostasis, and transducing Ca²⁺ signaling from the extracellular milieu to the intracellular environment. In the present study, we have demonstrated a Ca²⁺-dependent, stoichiometric interaction between CaM and a CaM-binding domain (CaMBD) located within the C terminus of CaSR (residues 871–898). Our studies suggest a wrapping around 1–14-like mode of interaction that involves global conformational changes in both lobes of CaM with concomitant formation of a helical structure in the CaMBD. More importantly, the Ca²⁺-dependent association between CaM and the C terminus of CaSR is critical for maintaining proper responsiveness of intracellular Ca²⁺ responses to changes in extracellular Ca²⁺ and regulating cell surface expression of the receptor.     

Activation of liver cytosol phosphoenolpyruvate carboxykinase by Ca2+ through intracellular redistribution of Mn2+

Calcium has no known direct effect on phosphoenolpyruvate carboxykinase from rat liver cytosol. However, addition of calcium salts to liver postnuclear supernatant led to an increase in assayable enzyme activity in cytosols. This indicates that mitochondria and microsomes present in postnuclear supernatant can participate in observed enzyme activation. The stimulation of phosphoenolpyruvate carboxykinase was prevented by the manganese complexion 1-(2-pyridylazo)-2-naphthol, was not additive with activation by MnCl2 and was inhibited by La3+, Sr2+ and ruthenium red. These data indicate that manganese and mitochondrial or microsomal calcium carriers participate in the mechanism of indirect calcium effect. Measuring of manganese content in cytosols directly, by atomic absorption spectrometry, has provided evidence that there is a pool of manganese associated with mitochondrial and microsomal fraction of rat liver that can be mobilized to the cytosol by calcium ions. The direct addition of this pool of manganese to the cytosol caused the stimulation of phosphoenolpyruvate carboxykinase activity to the same levels as did calcium ions in the postnuclear supernatant. It is postulated that calcium can effect enzyme activity indirectly by releasing manganese from specific cellular compartments into the cytosol.     

Decoding complex Ca2+ signals through the modulation of Ras signaling

Calcium (Ca(2+)) is a universal regulator of a wide variety of cellular processes. For such control to be achieved, information is encoded within spatial and temporal components of the underlying Ca(2+) signal. One pathway through which Ca(2+) signals are decoded is the Ras binary switch. Here I describe some recent advances that have shed light on how cells can decode the spatial and temporal aspects of Ca(2+) signals through the regulation of this important signalling switch.     

Receptor-mediated activation of electropermeabilized neutrophils. Evidence for a Ca2+- and protein kinase C-independent signaling pathway

Electrically permeabilized neutrophils were used to study the mechanism of activation of the respiratory burst by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (fMLP). Permeabilization was assessed by flow cytometry, radioisotope trapping, and by the requirement for exogenous NADPH for oxygen consumption. A respiratory burst could be elicited by fMLP, phorbol ester, or diacylglycerol in permeabilized cells suspended in EGTA-buffered medium with 100 nM free Ca2+. The fMLP response persisted even in cells depleted of intracellular Ca2+ stores by pretreatment with ionomycin. Therefore, a change in cytosolic free Ca2+ ([Ca2+]i) is not required for receptor-mediated stimulation of the respiratory burst. The responses induced by phorbol ester and diacylglycerol were largely inhibited by H7, a protein kinase C antagonist. In contrast, the stimulation of oxygen consumption by fMLP was unaffected by H7. These results suggest that a third signaling pathway, distinct from changes in [Ca2+]i and activation of protein kinase C, is involved in the response of neutrophils to chemoattractants.     

Thapsigargin decreases the Na+- Ca2 + exchanger mediated Ca2 + entry in pig coronary artery smooth muscle

Na⁺- Ca^2 + exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca^2 + pool along with the SER Ca^2 + pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca^2 + depletion on NCX–SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na⁺-loaded and then placed in either a Na⁺-containing or in a Na⁺-substituted solution. Subsequently, the difference in Ca^2 + entry between the two groups was examined and defined as the NCX mediated Ca^2 + entry. The NCX mediated Ca^2 + entry in the smooth muscle cells was monitored using two methods: Ca^2 +sensitive fluorescence dye Fluo-4 and radioactive Ca^2 +. Ca^2 +-entry was greater in the Na⁺-substituted cells than in the Na⁺-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca^2 + entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na⁺-substituted solution with or without thapsigargin. SER Ca^2 + depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca^2 + entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca^2 + entry may protect the cells against Ca^2 +-overload during ischemia–reperfusion when SERCA2 is known to be damaged.     

Mitochondrial dynamics and Ca2+ signaling

Recent data shed light on two novel aspects of the mitochondria-Ca2+ liaison. First, it was extensively investigated how Ca2+ handling is controlled by mitochondrial shape, and positioning; a playground also of cell death and survival regulation. On the other hand, significant progress has been made to explore how intra- and near-mitochondrial Ca2+ signals modify mitochondrial morphology and cellular distribution. Here, we shortly summarize these advances and provide a model of Ca2+-mitochondria interactions.     

Ca2+ signaling and spinocerebellar ataxia

Spinocerebellar ataxia (SCA) is a neural disorder, which is caused by degenerative changes in the cerebellum. SCA is primarily characterized by gait ataxia, and additional clinical features include nystagmus, dysarthria, tremors and cerebellar atrophy. Forty-four hereditary SCAs have been identified to date, along with >35 SCA-associated genes. Despite the great diversity and distinct functionalities of the SCA-related genes, accumulating evidence supports the occurrence of a common pathophysiological event among several hereditary SCAs. Altered calcium (Ca²⁺) homeostasis in the Purkinje cells (PCs) of the cerebellum has been proposed as a possible pathological SCA trigger. In support of this, signaling events that are initiated from or lead to aberrant Ca²⁺ release from the type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1), which is highly expressed in cerebellar PCs, seem to be closely associated with the pathogenesis of several SCA types. In this review, we summarize the current research on pathological hereditary SCA events, which involve altered Ca²⁺ homeostasis in PCs, through IP₃R1 signaling.     

TRP channels and Ca2+ signaling

There is a rapidly growing interest in the family of transient receptor potential (TRP) channels because TRP channels are not only important for many sensory systems, but they are crucial components of the function of neurons, epithelial, blood and smooth muscle cells. These facts make TRP channels important targets for treatment of diseases arising from the malfunction of these channels in the above cells and for treatment of inflammatory pain. TRP channels are also important for a growing number of genetic diseases arising from mutations in various types of TRP channels. The Minerva-Gentner Symposium on TRP channels and Ca(2+) signaling, which took place in Eilat, Israel (February 24-28, 2006) has clearly demonstrated that the study of TRP channels is a newly emerging field of biomedicine with prime importance. In the Eilat symposium, investigators who have contributed seminal publications and insight into the TRP field presented their most recent, and in many cases still unpublished, studies. The excellent presentations and excitement generated by them demonstrated that much progress has been achieved. Nevertheless, it was also evident that the field of TRP channels is still in its infancy in comparison to other fields of ion channels, and even the fundamental knowledge of the gating mechanism of TRP channels is still unsolved. The beautiful location of the symposium, together with informal intensive discussions among the participants, contributed to the success of this meeting.     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

PKA induces Ca2+ release and enhances ciliary beat frequency in a Ca2+-dependent and -independent manner

The intent of this work was to evaluate the role of cAMP inregulation of ciliary activity in frog mucociliary epithelium and toexamine the possibility of cross talk between the cAMP- andCa²⁺-dependent pathways in thatregulation. Forskolin and dibutyryl cAMP induced strong transientintracellular Ca²⁺ concentration([Ca²⁺]_i)elevation and strong ciliary beat frequency enhancement with prolongedstabilization at an elevated plateau. The response was not affected byreduction of extracellular Ca²⁺concentration. The elevation in[Ca²⁺]_iwas canceled by pretreatment with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, thapsigargin, and a phospholipase C inhibitor, U-73122. Underthose experimental conditions, forskolin raised the beat frequency to amoderately elevated plateau, whereas the initial strong rise infrequency was completely abolished. All effects were canceled by H-89,a selective protein kinase A (PKA) inhibitor. The results suggest adual role for PKA in ciliary regulation. PKA releasesCa²⁺ from intracellular stores,strongly activating ciliary beating, and, concurrently, producesmoderate prolonged enhancement of the beat frequency by aCa²⁺-independent mechanism.

     

Lipid phosphate phosphatase-1 and Ca2+ control lysophosphatidate signaling through EDG-2 receptors

The serum-derived phospholipid growth factor, lysophosphatidate (LPA), activates cells through the EDG family of G protein-coupled receptors. The present study investigated mechanisms by which dephosphorylation of exogenous LPA by lipid phosphate phosphatase-1 (LPP-1) controls cell signaling. Overexpressing LPP-1 decreased the net specific cell association of LPA with Rat2 fibroblasts by approximately 50% at 37 degrees C when less than 10% of LPA was dephosphorylated. This attenuated cell activation as indicated by diminished responses, including cAMP, Ca(2+), activation of phospholipase D and ERK, DNA synthesis, and cell division. Conversely, decreasing LPP-1 expression increased net LPA association, ERK stimulation, and DNA synthesis. Whereas changing LPP-1 expression did not alter the apparent K(d) and B(max) for LPA binding at 4 degrees C, increasing Ca(2+) from 0 to 50 micrometer increased the K(d) from 40 to 900 nm. Decreasing extracellular Ca(2+) from 1.8 mm to 10 micrometer increased LPA binding by 20-fold, shifting the threshold for ERK activation to the nanomolar range. Hence the Ca(2+) dependence of the apparent K(d) values explains the long-standing discrepancy of why micromolar LPA is often needed to activate cells at physiological Ca(2+) levels. In addition, the work demonstrates that LPP-1 can regulate specific LPA association with cells without significantly depleting bulk LPA concentrations in the extracellular medium. This identifies a novel mechanism for controlling EDG-2 receptor activation.     

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC

It has been suggested that L-type Ca²⁺ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca²⁺ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca²⁺ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba²⁺ currents (I_Ba) through L-type Ca²⁺ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca²⁺ channel activity but did not alter the voltage-dependent characteristics of Ca²⁺ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31–8425 or Go-6983, prevented I_Ba enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I_Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I_Ba when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca²⁺ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current