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The role of S100A1 in skeletal muscle is just beginning to be elucidated. We have previously shown that skeletal muscle fibers from S100A1 knockout (KO) mice exhibit decreased action potential (AP)-evoked [Ca.sup.2+] transients, and that S100A1 binds competitively with calmodulin to a canonical S100 binding sequence within the calmodulin-binding domain of the skeletal muscle ryanodine receptor. Using voltage clamped fibers, we found that [Ca.sup.2+] release was suppressed at all test membrane potentials in [S100A1.sup.-/-] fibers. Here we examine the role of S 100A1 during physiological AP-induced muscle activity, using an integrative approach spanning AP propagation to muscle force production. With the voltage-sensitive indicator di-8-aminonaphthylethenylpyridinium, we first demonstrate that the AP waveform is not altered in flexor digitorum brevis muscle fibers isolated from S100A1 KO mice. We then use a model for myoplasmic [Ca.sup.2+] binding and transport processes to calculate sarcoplasmic reticulum [Ca.sup.2+] release flux initiated by APs and demonstrate decreased release flux and greater inactivation of flux in KO fibers. Using in vivo stimulation of tibialis anterior muscles in anesthetized mice, we show that the maximal isometric force response to twitch and tetanic stimulation is decreased in [S100A1.sup.-/-] muscles. KO muscles also fatigue more rapidly upon repetitive stimulation than those of wild-type counterparts. We additionally show that fiber diameter, type, and expression of key excitation-contraction coupling proteins are unchanged in S100A1 KO muscle. We conclude that the absence of S100A1 suppresses physiological AP-induced [Ca.sup.2+] release flux, resulting in impaired contractile activation and force production in skeletal muscle. S100; excitation-contraction coupling; calcium signaling; muscle 10.1152/ajpcell.00180.2010.

Strait KA, Stricklett PK, Chapman M, Kohan DE. Characterization of vasopressin-responsive collecting duct adenylyl cyclases in the mouse. Am J Physiol Renal Physiol 298: F859-F867, 2010. First published December 2, 2009; doi: 10.1152/ajprenal.00109.2009.--Little is known about collecting duct adenylyl cyclase (AC) isoforms or regulation in the mouse. We performed RT-PCR for AC isoforms 1-9 in microdissected cortical (CCD) and outer medullary (OMCD) and acutely isolated inner medullary (IMCD) collecting duct. Ali collecting duct regions contained AC3, AC4, and AC6 mRNA, while CCD and OMCD, but not IMCD, also contained AC5 mRNA. Acutely isolated IMCD expressed AC3, AC4, and AC6 proteins by Western blot analysis. The mIMCD3 cell line expressed AC2, AC3, AC4, AC5, and AC6 mRNA; M-I CCD cells expressed AC2, 3, 4, and 6, while mpkCCD cell lines contained AC3, AC4, and AC6 mRNA. AVP stimulated cAMP accumulation in acutely isolated mouse IMCD; this was reduced by chelation of extracellular calcium (EGTA) and almost completely abolished by blockade of calmodulin (W-7). Blockade of calmodulin kinase with KN-93 or endoplasmic reticulum calcium ATPase (thapsigargin) also reduced the AVP response. A similar inhibitory effect of W-7, KN-93, and thapsigargin was seen on forskolin-stimulated cAMP content in acutely isolated mouse IMCD. These three agents had the same pattern of blockade of AVP- or forskolin-stimulated AC activity in acutely isolated rat IMCD. AVP responsiveness in primary cultures of mouse IMCD was also reduced by W-7, KN-93, and thapsigargin. Small interfering RNA (siRNA) designed to knock down AC3 or AC6 in primary cultured mouse IMCD significantly reduced AVP-stimulated cAMP accumulation. Together, these data are consistent with a role of AC3 and AC6 in the activation of mouse collecting duct by AVP. calcium; cAMP doj: 10.1152/ajprenal.00109.2009.

Gelsolin consists of six homologous domains (G1-G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here, we present the structures of Ca-free human gelsolin and of Ca-bound human G1-G3 in a complex with actin. These structures closely resemble those determined previously for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1-G3/actin structure, whereas it is vacant in the equine version. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding [Ca.sup.2+] and responsible for opening the G2-G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1-G3/actin reveals that the [Ca.sup.2+] locks the G2-G3 interface. Thermal denaturation studies of G2-G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca binding by G2 prolongs the lifetime of partially activated, intermediate conformations in which the protease cleavage site is exposed. actin | calcium activated | calcium dependent | TIRF

Previously, we reported that renal active calcium-transporting genes are highly expressed in female mice and suggested that renal calcium-processing genes play a critical role in normal calcium reabsorption in females (Lee GS, Lee KY, Choi KC, Ryu YH, Paik SG, Oh GT, Jeung EB. J Bone Miner Res 22:1968-1978, 2007). In the current study, we evaluated the differential expression of renal calcium-processing genes in male and female mice. Using microarray analysis, we identified [K.sup.+]-dependent [Na.sup.+]/[Ca.sup.2+] exchanger 3 (NCKX3) as a gene that was differentially expressed in the kidneys of female and male mice. The expression levels of renal NCKX3 mRNA and protein were higher in female than in male mice, whereas there was no difference between the genders in the levels of NCKX3 expression in the brain. Renal NCKX3 localized to the basolateral layer of distal convoluted tubules, indicating that this protein participates in renal calcium reabsorption. To identify putative regulators in the gender-specific expression of NCKX3, several hormones were injected into mature female and male mice. Although any hormones did not alter NCKX3 expression, adrenal gland-secreted hormones aldosterone and hydrocortisone did downregulate renal NCKX3 mRNA expression in female mice, but they did not change its protein levels. Taken together, the results in this study suggest that a high level of renal NCKX3 expression maintain in distal convoluted tubules may play a role in active calcium transport in the kidneys of female mice. calcium homeostasis

Intermittent high-altitude (IHA) hypoxia-induced cardioprotection against ischemia-reperfusion (I/R) injury is associated with the preservation of sarcoplasmic reticulum (SR) function. Although [Ca.sup.2+]/calmodulin (CaM)-dependent protein kinase II (CaMKII) and phosphatase are known to modulate the function of cardiac SR under physiological conditions, the status of SR CaMKII and phosphatase during I/R in the hearts from IHA hypoxic rats is unknown. In the present study, we determined SR and cytosolic CaMKII activity during preischemia and I/R (30 min/30 min) in perfused hearts from normoxic and IHA hypoxic rats. The left ventricular contractile recovery, SR CaMKII activity as well as phosphorylation of phospholamban at [Thr.sup.17], and [Ca.sup.2+]/CaM-dependent SR [Ca.sup.2+]-uptake activity were depressed in the I/R hearts from normoxic rats, whereas these changes were prevented in the hearts from IHA hypoxic rats. Such beneficial effects of IHA hypoxia were lost by treating the hearts with a specific CaMKII inhibitor, KN-93. I/R also depressed cytosolic CaMKII and SR phosphatase activity, but these alterations remained unchanged in IHA hypoxic group. Furthermore, we found that the autophosphorylation at [Thr.sup.287], which confers [Ca.sup.2+]/CaM-independent activity, was not altered by I/R in both groups. These findings indicate that preservation of SR CaMKII activity plays an important role in the IHA hypoxia-induced cardio-protection against I/R injury via maintaining SR [Ca.sup.2+]-uptake activity. cardiac sarcoplasmic reticulum; phospholamban phosphorylation residue; sarcoplasmic reticulum calcium pump adenosinetriphosphatase; phosphatase; cardiac contractile function

Sarcalumenin (SAR), a [Ca.sup.2+]-binding protein located in the longitudinal sarcoplasmic reticulum (SR), regulates [Ca.sup.2+] reuptake into the SR by interacting with cardiac sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase 2a (SERCA2a). We have previously demonstrated that SAR deficiency induced progressive heart failure in response to pressure overload, despite mild cardiac dysfunction in sham-operated SAR knockout (SARKO) mice (26). Since responses to physiological stresses often differ from those to pathological stresses, we examined the effects of endurance exercise on cardiac function in SARKO mice. Wild-type (WT) and SARKO mice were subjected to endurance treadmill exercise training (~65% of maximal exercise ability for 60 min/day) for 12 wk. After exercise training, maximal exercise ability was significantly increased by 5% in WT mice (n = 6), whereas it was significantly decreased by 37% in SARKO mice (n = 5). Cardiac function assessed by echocardiographic examination was significantly decreased in accordance with upregulation of biomarkers of cardiac stress in SARKO mice after training. After training, expression levels of SERCA2a protein were significantly downregulated by 30% in SARKO hearts, whereas they were significantly upregulated by 59% in WT hearts. Consequently, SERCA2 activity was significantly decreased in SARKO hearts after training. Furthermore, the expression levels of other [Ca.sup.2+]-handling proteins, including phospholamban, ryanodine receptor 2, calsequestrin 2, and sodium/calcium exchanger 1, were significantly decreased in SARKO hearts after training. These results indicate that SAR plays a critical role in maintaining cardiac function under physiological stresses, such as endurance exercise, by regulating [Ca.sup.2+] transport activity into the SR. SAR may be a primary target for exercise-related adaptation of the [Ca.sup.2+] storage system in the SR to preserve cardiac function. treadmill; calcium uptake; heart failure; excitation-contraction coupling

Calponin contributes to the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated Mg-ATPase activity of phosphorylated myosin. Previous studies have shown that the contractile agonist acetylcholine induced a direct association of translocated calponin and PKC-[alpha] in the membrane. In the present study, we have determined the domain of PKC-[alpha] involved in direct association with calponin. In vitro binding assay was carried out by incubating glutathione S-transferase-calponin aa 92-229 with His-tagged proteins of individual domains and different combinations of domains of PKC-[alpha]. Calponin was found to bind directly to the full-length PKC-[alpha]. Calponin bound to C2 and C4 domains but not to C1 and C3 domains of PKC-[alpha]. When incubated with proteins of different combination of domains, calponin bound to C2-C3, C3-C4, and C2-C3-C4 but not to C1-C2 or C1-C2-C3. To determine whether these in vitro bindings mimic the in vivo associations, and in vivo binding assay was performed by transfecting colonic smooth muscle cells with Histagged proteins of individual domains and different combinations of domains of PKC-[alpha]. Coimmunoprecipitation of calponin with Histagged truncated forms of PKC-[alpha] showed that C1-C2, C1-C2-C3, C2-C3, and C3-C4 did not associate with calponin. Calponin associated only with full-length PKC-[alpha] and with C2-C3-C4 in cells in the resting state, and this association increased upon stimulation with acetylcholine. These data suggest that calponin bound to fragments that may mimic the active form of PKC-[alpha] and that the functional association of PKC-[alpha] with calponin requires both C2 and C4 domains during contraction of colonic smooth muscle cells. tropomyosin; smooth muscle; contraction; cytoskeleton; signaling

[Ca.sup.2+] was reported to regulate spore germination and aerial hypha formation in streptomycetes; the underlying mechanism of this regulation is not known, cabC, a gene encoding an EF-hand calcium-binding protein, was disrupted or overexpressed in Streptomyces coelicolor M145. On R5- agar, the disruption of cabC resulted in denser aerial hyphae with more short branches, swollen hyphal tips, and early-germinating spores on the spore chain, while cabC overexpression significantly delayed development. Manipulation of the [Ca.sup.2+] concentration in R5- agar could reverse the phenotypes of cabC disruption or overexpression mutants and mimic mutant phenotypes with M145, suggesting that the mutant phenotypes were due to changes in the intracellular [Ca.sup.2+] concentration. CabC expression was strongly activated in aerial hyphae, as determined by Western blotting against CabC and confocal laser scanning microscopy detection of CabC::enhanced green fluorescent protein (EGFP). CabC::EGFP fusion proteins were evenly distributed in substrate mycelia, aerial mycelia, and spores. Taken together, these results demonstrate that CabC is involved in [Ca.sup.2+]-mediated regulation of spore germination and aerial hypha formation in S. coelicolor. CabC most likely acts as a [Ca.sup.2+] buffer and exerts its regulatory effects by controlling the intracellular [Ca.sup.2+] concentration.

The conformation, stability and [Ca.sup.2} binding of two paralogues of [beta][gamma]-crystallins (caulollins) is reported which differ in the length of N-terminal extension. These betagamma-crystallin domains are partially unstructured and gain structure upon binding [Ca.sup.2}.

Prolactin (PRL) has been shown to stimulate intestinal calcium absorption but the mechanism was still unknown. This study aimed to investigate the mechanism and signaling pathway by which PRL enhanced calcium transport in the rat duodenum and Caco-2 monolayer. Both epithelia strongly expressed mRNAs and proteins of PRL receptors. Ussing chamber technique showed that the duodenal active calcium fluxes were increased by PRL in a dose-response manner with the maximal effective dose of 800 ng/ml. This response diminished after exposure to LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Caco-2 monolayer gave similar response to PRL with the maximal effective dose of 600 ng/ml. By nullifying the transepithelial potential difference, we showed that the voltage-dependent paracellular calcium transport did not contribute to the PRL-enhanced flux in Caco-2 monolayer. In contrast, the calcium gradient-dependent paracellular transport and calcium permeability were increased by PRL. Effects of PRL on Caco-2 monolayer were abolished by PI3K inhibitors (LY-294002 and wortmannin), but not by inhibitors of MEK (U-0126) or JAK2 (AG-490). To investigate whether the PRL-enhanced paracellular transport was linked to changes in the epithelial charge selectivity, the permeability ratio of sodium and chloride ([P.sub.Na]/[P.sub.Cl]) was determined. We found that PRL elevated the [P.sub.Na]/[P.sub.Cl] in both epithelia, and the effects were blocked by P13K inhibitors. In conclusion, PRL directly and rapidly stimulated the active and passive calcium transport in the rat duodenum and Caco-2 monolayer via the nongenomic P13K-signaling pathway. This PRL-enhanced paracellular calcium transport could have resulted from altered charge selectivity. charge selectivity; dilution potential; paracellular transport; prolactin receptor; tight junction; transcellular transport doi:10.1152/ajpendo.00142.2007

Aphids, which constitute one of the most important groups of agricultural pests, ingest nutrients from sieve tubes, the photoassimilate transport conduits in plants. Aphids are able to successfully puncture sieve tubes with their piercing mouthparts (stylets) and ingest phloem sap without eliciting the sieve tubes' normal occlusion response to injury. Occlusion mechanisms are calcium-triggered and may be prevented by chemical constituents in aphid saliva injected into sieve tubes before and during feeding. We recorded aphid feeding behavior with the electrical penetration graph (EPG) technique and then experimentally induced sieve tube plugging. Initiation of sieve tube occlusion caused a change in aphid behavior from phloem sap ingestion to secretion of watery saliva. Direct proof of 'unplugging' properties of aphid saliva was provided by the effect of aphid saliva on forisomes. Forisomes are proteinaceous inclusions in sieve tubes of legumes that show calcium-regulated changes in conformation between a contracted state (below calcium threshold) that does not occlude the sieve tubes and a dispersed state (above calcium threshold) that occludes the sieve tubes. We demonstrated in vitro that aphid saliva induces dispersed forisomes to revert back to the nonplugging contracted state. Labeling Western-blotted saliva proteins with [sup.45][Ca.sup.2+] or ruthenium red inferred the presence of calcium-binding domains. These results demonstrate that aphid saliva has the ability to prevent sieve tube plugging by molecular interactions between salivary proteins and calcium. This provides aphids with access to a continuous flow of phloem sap and is a critical adaptation instrumental in the evolutionary success of aphids. calcium-binding | plant-aphid interaction | plugging | saliva proteins | sieve element

We endeavored to use a basic and well-controlled experimental system to characterize the extent and time sequence of sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA) involvement in the development of cardiac hypertrophy, including transcription, protein expression, [Ca.sup.2+] transport, and cytoplasmic [Ca.sup.2+] signaling. To this end, hypertrophy of neonatal rat cardiac myocytes in culture was obtained after adrenergic activation with phenylephrine (PE). Micrographic assessment of myocyte size, rise of [[sup.14]C]phenylalanine incorporation and total protein expression, and increased transcription of atrial natriuretic factor demonstrated unambiguously the occurrence of hypertrophy. An early and prominent feature of hypertrophy was a reduction of the SERCA2 transcript, as determined by RT-PCR with reference to a stable marker such as glyceraldehyde-3-phosphate dehydrogenase. Reduction of [Ca.sup.2+]-ATPase protein levels and [Ca.sup.2+] transport activity to ~50% of control values followed with some delay, evidently as a consequence of a primary effect on transcription. Cytosolic [Ca.sup.2+] signaling kinetics, measured with a [Ca.sup.2+]-sensitive dye after electrical stimuli, were significantly altered in hypertrophic myocytes. However, the effect of PE hypertrophy on cytosolic [Ca.sup.2+] signaling kinetics was less prominent than observed in myocytes subjected to drastic SERCA2 downregulation with small interfering RNA or inhibition with thapsigargin (10 nM). We conclude that SERCA2 undergoes significant down-regulation after hypertrophic stimuli, possibly due to lack of SERCA gene involvement by the hypertrophy transcriptional program. The consequence of SERCA2 downregulation on [Ca.sup.2+] signaling is partially compensated by alternate [Ca.sup.2+] transport mechanisms. These alterations may contribute to a gradual onset of functional failure in long-term hypertrophy. calcium adenosinetriphosphatase; calcium transport doi:10.1152/ajpcell.00441.2006

[Ca.sup.2+] enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a [Ca.sup.2+] pulse. The G293S and G283S mutations delayed the dissipation of [Ca.sup.2+] transients induced in CHO cells by [InsP.sub.3]. In organotypic cultures, [Ca.sup.2+] imaging of vestibular hair cells showed that the dissipation of stereociliary [Ca.sup.2+] transients induced by [Ca.sup.2+] uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells. hereditary hearing loss | mutations | calcium homeostasis | calcium transport

Aging and glycolytic inhibition (GI) are known to alter intracellular calcium ion ([Ca.sup.2+.sub.i]) handling in cardiac myocytes, causing early afterpotentials (EADs) and delayed afterpotentials. We hypothesized that aging and GI interact synergistically in intact hearts to generate EADs and triggered activity leading to atrial fibrillation (AF). We studied isolated and Langendorff-perfused hearts of young (age 3-5 mo, N = 8) and old (age 27-29 mo, N = 14) rats subjected to GI (0 glucose + 10 mmol/l pyruvate). Epicardial atrial activation maps were constructed using optical action potentials, while simultaneously monitoring [Ca.sup.2+.sub.i] by means of dual-voltage and calcium-sensitive fluorescent dyes. During GI, spontaneous AF occurred in 13 of 14 old but in no young rats. AF was initiated by EAD-induced triggered activity at the left atrial pulmonary vein junction (LA-PVJ). The triggered activity initially propagated as single wave front, but within 1 s degenerated into multiple wavelets. The EADs and triggered activity in the old atria were associated with significantly elevated diastolic [Ca.sup.2+.sub.i] levels at the LA-PVJ, where the time constant [tau] of the [Ca.sup.2+.sub.i] transient decline and action potential duration were significantly (P < 0.01) prolonged compared with atrial sites 5 mm away from LA-PVJ. During GI and rapid atrial pacing, spatially discordant APD and [Ca.sup.2+.sub.i] transient alternans developed in the old hut not young atria, leading to AF. Atria in old rats had significantly more fibrotic tissue than atria in young rats. We conclude that GI interacts with the aged and fibrotic atria to amplify [Ca.sup.2+.sub.i] handling abnormalities that facilitate EAD-mediated triggered activity and AF. action potential; early after depolarization; calcium transient; interstitial fibrosis; atrial fibrillation

It has been shown that inhaled cigarette smoke activates vagal pulmonary C fibers and rapidly adapting receptors (RARs) in the airways and that nicotine contained in the smoke is primarily responsible. This study was carried out to determine whether nicotine alone can activate pulmonary sensory neurons isolated from rat vagal ganglia; the response of these neurons was determined by fura-2-based ratiometric C[A.sub.2]+ imaging. The results showed: 1) Nicotine ([10.sup.-4] M, 20 s) evoked a transient increase in intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]) in 175 of the 522 neurons tested ([DELTA][[Ca.sup.2+].sub.i] = 142.2 [+ or -] 12.3 nM); the response was reproducible, with a small reduction in peak amplitude in the same neurons when the challenge was repeated 20 rain later. 2) A majority (59.7%) of these nicotine-sensitive neurons were also activated by capsaicin ([10.sup.-7] M). 3) 1,1-Dimethyl-4-phenylpiperazinium iodide (DMPP; [10.sup.-4] M, 20 s), a selective agonist of the neuronal nicotinic acetylcholine receptors (NnAChRs), evoked a pattern of response similar to that of nicotine. 4) The responses to nicotine and DMPP were either totally abrogated or markedly attenuated by hexamethonium ([10.sup.-4] M). 5) In anesthetized rats, right atrial bolus injection of nicotine (75-200 [micro]g/kg) evoked an immediate (latency lung afferents; C fibers; airway irritation; cigarette smoke; 1,1-dimethyl4-phenylpiperazinium iodide

Glucocorticoids, such as prednisolone, are often used in clinic because of their anti-inflammatory and immunosuppressive properties. However, glucocorticoids reduce bone mineral density (BMD) as a side effect. Malabsorption of [Ca.sup.2+] in the intestine is supposed to play an important role in the etiology of low BMD. To elucidate the mechanism of glucocorticoid-induced [Ca.sup.2+] malabsorption, the present study investigated the effect of prednisolone on the expression and activity of proteins responsible for active intestinal [Ca.sup.2+] absorption including the epithelial [Ca.sup.2+] channel TRPV6, calbindin-[D.sub.9K], and the plasma membrane ATPase PMCA1b. Therefore, C57BL/6 mice received 10 mg/kg body wt prednisolone daily by oral gavage for 7 days and were compared with control mice receiving vehicle only. An in vivo [sup.45][Ca.sup.2+] absorption assay indicated that intestinal [Ca.sup.2+] absorption was diminished after prednisolone treatment. We showed decreased duodenal TRPV6 and calbindin-[D.sub.9K] mRNA and protein abundance in prednisolone-treated compared with control mice, whereas PMCAlb mRNA levels were not altered. Importantly, detailed expression studies demonstrated that in mice these [Ca.sup.2+] transport proteins are predominantly localized in the first 2 cm of the duodenum. Furthermore, serum [Ca.sup.2+] and 1,25-dihydroxyvitamin [D.sub.3] [1,25[(OH).sub.2][D.sub.3]] concentrations remained unchanged by prednisolone treatment. In conclusion, these data suggest that prednisolone reduces the intestinal [Ca.sup.2+] absorption capacity through diminished duodenal expression of the active [Ca.sup.2+] transporters TRPV6 and calbindin-[D.sub.9K] independent of systemic 1,25[(OH).sub.2][D.sub.3]. 1,25-dihydroxyvitamin [D.sub.3]; duodenum; epithelial calcium channel 2; calcium transporter 1

The TRP family of ion channels transduce an extensive range of chemical and physical signals. TRPC6 is a receptor-activated nonselective cation channel expressed widely in vascular smooth muscle and other cell types. We report here that TRPC6 is also a sensor of mechanically and osmotically induced membrane stretch. Pressure-induced activation of TRPC6 was independent of phospholipase C. The stretch responses were blocked by the tarantula peptide, GsMTx-4, known to specifically inhibit mechanosensitive channels by modifying the external lipid-channel boundary. The GsMTx-4 peptide also blocked the activation of TRPC6 channels by either receptor-induced PLC activation or by direct application of diacylglycerol. The effects of the peptide on both stretch- and diacylglycerol-mediated TRPC6 activation indicate that the mechanical and chemical lipid sensing by the channel has a common molecular mechanism that may involve lateral-lipid tension. The mechanosensing properties of TRPC6 channels highly expressed in smooth muscle cells are likely to play a key role in regulating myogenic tone in vascular tissue. GsMTx-4 peptide | mechanosensitivity | tarantula venom | myogenic tone | calcium signals

The import of nuclear-encoded proteins into chloroplasts is tightly controlled on both sides of the envelope membranes. Regulatory circuits include redox-control as well as calcium-regulation, with calmodulin being the likely mediator of the latter. Using affinity-chromatography on calmodulin-agarose, we could identify the inner envelope translocon component Tic32 as the predominant calmodulin-binding protein of this membrane. Calmodulin-binding assays corroborate the interaction for heterologously expressed as well as native Tic32. The interaction is calcium-dependent and is mediated by a calmodulin-binding domain between Leu-296 and Leu-314 close to the C-proximal end of the pea Tic32. We furthermore could establish Tic32 as a bona fide NADPH-dependent dehydrogenase. NADPH but not NADH or [NADP.sup.+] affects the interaction of Tic110 with Tic32 as well as Tic62. At the same time, dehydrogenase activity of Tic32 is affected by calmodulin. In particular, binding of NADPH and calmodulin to Tic32 appear to be mutually exclusive. These results suggest that redox modulation and calcium regulation of chloroplast protein import convene at the Tic translocon and that both could be mediated by Tic32. calcium signaling | protein import | Tic translocon | redox-regulation | short-chain dehydrogenases

Chronic metabolic acidosis results in a negative calcium balance as a result of bone resorption and renal calcium loss. However, reports on the changes in intestinal calcium transport have been controversial. The present investigation therefore aimed to study the effects of chronic metabolic acidosis induced by 1.5% N[H.sub.4]Cl administration on the three components of duodenal calcium transport, namely, solvent drag-induced, transcellular active, and passive paracellular components, in rats using an in vitro Ussing chamber technique. The relative mRNA expression of genes related to duodenal calcium transport was also determined. We found that 21-day chronic metabolic acidosis stimulated solvent drag-induced and transcellular active duodenal calcium transport but not passive paracellular calcium transport. Our results further demonstrated that an acute direct exposure to serosal acidic pH, in contrast, decreased solvent drag-induced calcium transport in a pH-dependent fashion but had no effect on transcellular active calcium transport. Neither the transepithelial resistance nor duodenal permeability to [Na.sup.+], [Cl.sup.-], and [Ca.sup.2+] via the passive paracellular pathway were altered by chronic metabolic acidosis, suggesting that widening of the tight junction and changes in the charge-selective property of the tight junction did not occur. Thus the enhanced duodenal calcium transport observed in chronic metabolic acidosis could have resulted from a long-term adaptation, possibly at the molecular level. RT-PCR study revealed that chronic metabolic acidosis significantly increased the relative mRNA expression of duodenal genes associated with solvent drag-induced transport, i.e., the [[beta].sub.1]-subunit of [Na.sup.+]-[K.sup.+]-ATPase, zonula occludens-1, occludin, and claudin-3, and with transcellular active transport, i.e., transient receptor potential vanilloid family [Ca.sup.2+] channels 5 and 6 and plasma membrane [Ca.sup.2+]-ATPase isoform 1b. Total plasma calcium and free ionized calcium and magnesium concentrations were also increased, whereas serum parathyroid hormone and 1[alpha],25-dihydroxyvitamin [D.sub.3] levels were not changed. The results indicated that 21-day chronic metabolic acidosis affected the calcium metabolism in rats partly through enhancing the mRNA expression of crucial duodenal genes involved in calcium absorption, thereby stimulating solvent drag-induced and transcellular active calcium transport in the duodenum. tight junction; dilution potential; N[H.sub.4]Cl; trifluorperazine; Ussing chamber doi:10.1152/ajpgi.00108.2006

Histone arginine methylation is a posttranslational modification linked to the regulation of gene transcription. Unlike other post-translational modifications, methylation has generally been regarded as stable, and enzymes that demethylate histone arginine residues have not been identified. However, it has recently been shown that human peptidylarginine deiminase 4 (PAD4), a [Ca.sup.2+]-dependent enzyme previously known to convert arginine residues to citrulline in histones, can also convert monomethylated arginine residues to citrulline both in vivo and in vitro. Citrullination of histone arginine residues by the enzyme antagonizes methylation by histone arginine methyltransferases and is thus a novel post-translational modification that regulates the level of histone arginine methylation and gene activity. Here we present the crystal structures of a [Ca.sup.2+]-bound PAD4 mutant in complex with three histone N-terminal peptides, each consisting of 10 amino acid residues that include one target arginine residue for the enzyme (H3/Arg-8, H3/Arg-17, and H4/Arg-3). To each histone N-terminal peptide, the enzyme induces a [beta]-turn-like bent conformation composed of five successive residues at the molecular surface near the active site cleft. The remaining five residues are highly disordered. The enzyme recognizes each peptide through backbone atoms of the peptide with a possible consensus recognition motif. The sequence specificity of the peptide recognized by this enzyme is thought to be fairly broad. These observations provide structural insights into target protein recognition by histone modification enzymes and illustrate how PAD4 can target multiple arginine sites in the histone N-terminal tails. calcium binding | histone modification | rheumatoid arthritis | protein deimination/citrullination | x-ray crystal structure

Spontaneous action potentials and [Ca.sup.2+] transients were investigated in intact gallbladder preparations to determine how electrical events propagate and the cellular mechanisms that modulate these events. Rhythmic phasic contractions were preceded by [Ca.sup.2+] flashes that were either focal (limited to one or a few bundles), multifocal (occurring asynchronously in several bundles), or global (simultaneous flashes throughout the field). [Ca.sup.2+] flashes and action potentials were abolished by inhibiting sarcoplasmic reticulum (SR) [Ca.sup.2+] release via inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] channels with 2-aminoethoxydiphenyl borate and xestospongin C or by inhibiting voltage-dependent [Ca.sup.2+] channels (VDCCs) with nifedipine or diltiazem or nisoldipine. Inhibiting ryanodine channels with ryanodine caused multiple spikes superimposed upon plateaus of action potentials and extended quiescent periods. Depletion of SR [Ca.sup.2+] stores with thapsigargin or cyclopiazonic acid increased the frequency and duration of [Ca.sup.2+] flashes and action potentials. Acetylcholine, carbachol, or cholecystokinin increased synchronized and increased the frequency of [Ca.sup.2+] flashes and action potentials. The phospholipase C (PLC) inhibitor U-73122 did not affect [Ca.sup.2+] flash or action potential activity but inhibited the excitatory effects of acetylcholine on these events. These results indicate that [Ca.sup.2+] flashes correspond to action potentials and that rhythmic excitation in the gallbladder is multifocal among gallbladder smooth muscle bundles and can be synchronized by excitatory agonists. These events do not depend on PLC activation, but agonist stimulation involves activation of PLC. Generation of these events depends on [Ca.sup.2+] entry via VDCCs and on [Ca.sup.2+] mobilization from the SR via Ins(1,4,5)[P.sub.3] channels. calcium transients; gallbladder motility; slow waves; action potentials

Voltage-gated [Ca.sup.2+] channels in arterial myocytes can mediate [Ca.sup.2+] release from the sarcoplasmic reticulum and, thus, induce contraction without the need of extracellular [Ca.sup.2+] influx. This metabotropic action of [Ca.sup.2+] channels (denoted as calcium-channel-induced calcium release or CCICR) involves activation of G proteins and the phospholipase C-inositol 1,4,5-trisphosphate pathway. Here, we show a form of vascular tone regulation by extracellular ATP that depends on the modulation of CCICR. In isolated arterial myocytes, ATP produced facilitation of [Ca.sup.2+]-channel activation and, subsequently, a strong potentiation of CCICR. The facilitation of L-type channel still occurred after full blockade of purinergic receptors and inhibition of G proteins with GDP[beta]S, thus suggesting that ATP directly interacts with [Ca.sup.2+] channels. The effects of ATP appear to be highly selective, because they were not mimicked by other nucleotides (ADP or UTP) or vasoactive agents, such as norepinephrine, acetylcholine, or endothelin-1. We have also shown that CCICR can trigger arterial cerebral vasoconstriction in the absence of extracellular calcium and that this phenomenon is greatly facilitated by extracellular ATP. Although, at low concentrations, ATP does not induce arterial contraction per se, this agent markedly potentiates contractility of partially depolarized or primed arteries. Hence, the metabotropic action of L-type [Ca.sup.2+] channels could have a high impact on vascular pathophysiology, because, even in the absence of Ca.sup.2+] channel opening, it might mediate elevations of cytosolic Ca.sup.2+] and contraction in partially depolarized vascular smooth muscle cells exposed to small concentrations of agonists. L-type [Ca.sup.2+] channel | vascular contractility | excitation-contraction coupling