Search

Dietary calcium is believed to reduce colon cancer risk, but the mechanism by which this occurs is poorly understood. Employing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we previously showed that a high-calcium diet (hCa) significantly abrogated hyperplasia in the distal colons of NIH-Swiss mice. Here, we explored the mechanism of dietary protection by hCa by analyzing the expression of genes involved in the regulation of Ca uptake/flux in the intestinal epithelium, including the Ca-sensing receptor, vitamin D receptor, Ca binding protein, and transient receptor potential cation channels, subfamily V, members 5 and 6 (TRPV5/6). Interestingly, while TRPV6 expression increased significantly during TMCH, the expression of the other gene products was unchanged. This elevated TRPV6 expression was significantly abrogated by a hCa diet. Immunofluorescence revealed apical membrane localization of TRPV6 in the normal colon, whereas during TMCH we observed intense apical pole and cytoplasmic staining along the entire longitudinal crypt axis, including the expanded proliferating zone. The hCa diet reversed this effect. In humans, overexpression of TRPV6 was associated with early-stage colon cancer, and in colon carcinoma cells, inhibition of TRPV6 expression by small interfering RNA inhibited their proliferation and induced apoptosis. TRPV6 small interfering RNA also diminished the transcriptional activity of the calcium-dependent nuclear factors in activated T cells. Thus the aberrant overexpression of TRPV6 contributes to colonic crypt hyperplasia in mice and to colon cancer cell proliferation in humans. Therefore, it is likely that suppression of TRPV6 by a hCa diet is required for its protective effects in the colon. crypt hyperplasia; TRPV6; cancer prevention doi: 10.1152/ajpgi.00193.2010.

Bicarbonate secretion in the intestine (duodenum) of marine fish has been suggested to play a major role in regulation of calcium availability for uptake. However, while the end process may lead to carbonate precipitation, regulation of transport of calcium and/or bicarbonate may actually result in fine-tuning of calcium availability for transport. To test this hypothesis, sea bream (Sparus auratus) duodenal preparations were mounted in Ussing-type chambers and the effect of parathyroid hormone-related protein (PTHrP) and stanniocalcin 1 (STC 1) on the control of intestinal bicarbonate secretion and calcium transport was analyzed. As expected, PTHrP increased net calcium uptake, as a result of an increase of calcium uptake without changes in calcium efflux. In contrast, purified sea bream STC 1 caused a minor decrease of calcium uptake and a two- to threefold increase in calcium efflux. As a result, STC 1 was able to invert the calcium flux from net calcium uptake to net calcium loss, which is in keeping with its known actions as a hypocalcemic factor. Furthermore, both PTHrP and STC 1 regulate intestinal bicarbonate secretion. PTHrP increased calcium uptake and simultaneously reduced the single factor that induces calcium precipitation, bicarbonate secretion. In contrast, STC 1, while reversing the calcium net flux to make it secretory, promoted intestinal bicarbonate secretion, both actions directed to decrease the calcium gradient across the epithelium and promote immobilization in the form of bicarbonate in the intestinal lumen. Together our results provide robust evidence to support an antagonistic action of PTHrP and STC 1 in the fine control of movements of both calcium and bicarbonate in the intestine of seawater fish. ion regulation; sea water; sea bream doi: 10.1152/ajpregu.00378.2009.

Endurance impact exercise, e.g., running, is known to enhance the intestinal calcium absorption. However, nonimpact exercise, e.g., swimming, is more appropriate for osteoporotic patients with cardiovascular diseases or disorders of bone and joint, but the effect of swimming on the intestinal calcium transport was unknown. This study, therefore, aimed to investigate the transepithelial calcium transport and the expression of related genes in the intestine of rats trained to swim nonstop 1 h/day, 5 days/wk for 2 wk. We found that endurance swimming stimulated calcium transport in the duodenum, proximal jejunum, and cecum, while decreasing that in the proximal colon. Swimming affected neither the transepithelial potential difference nor resistance. As demonstrated by real-time PCR, the small intestine, especially the duodenum, responded to swimming by upregulating a number of genes related to the transcellular calcium transport, i.e., TRPV5, TRPV6, calbindin-[D.sub.9k], PMC[A.sub.1b], and NCX1, and the paracellular calcium transport, i.e., ZO-1, ZO-2, ZO-3, cingulin, occludin, and claudins, as well as nuclear receptor of 1,25[(OH).sub.2][D.sub.3]. In contrast, swimming downregulated those genes in the colon. Microarray analysis showed that swimming also altered the expression of duodenal genes related to the transport of several ions and nutrients, e.g., [Na.sup.+], [K.sup.+], [C1.sup.-], glucose, and amino acids. In conclusion, endurance swimming enhanced intestinal calcium absorption, in part, by upregulating the calcium transporter genes. The present microarray study also provided relevant information for further investigations into the intestinal nutrient and electrolyte transport during nonimpact exercise. gene ontology; microarray; nonimpact exercise; real-time polymerase chain reaction; Ussing chamber

There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on [beta]-cell function and survival. In the present study, we sought to determine the mechanism by which palmitate affects intracellular [Ca.sup.2+], and in particular the role of the endoplasmic reticulum (ER). In human [beta]-cells and MIN6 cells, palmitate rapidly increased cytosolic [Ca.sup.2+] through a combination of [Ca.sup.2+] store release and extracellular [Ca.sup.2+] influx. Palmitate caused a reversible lowering of ER [Ca.sup.2+], measured directly with the fluorescent protein-based ER [Ca.sup.2+] sensor D1ER. Using another genetically encoded indicator, we observed long-lasting oscillations of cytosolic [Ca.sup.2+] in palmitate-treated cells. In keeping with this observed ER [Ca.sup.2+] depletion, palmitate induced rapid phosphorylation of the ER [Ca.sup.2+] sensor protein kinase R-like ER kinase (PERK) and subsequently ER stress and [beta]-cell death. We detected little palmitate-induced insulin secretion, suggesting that these [Ca.sup.2+] signals are poorly coupled to exocytosis. In summary, we have characterized [Ca.sup.2+]-dependent mechanisms involved in altered [beta]-cell function and survival induced by the free fatty acid palmitate. We present the first direct evidence that free fatty acids reduce ER [Ca.sup.2+] and shed light on pathways involved in lipotoxicity and the pathogenesis of type 2 diabetes. diabetes; free fatty acids; fluorescence resonance energy transfer; calcium homeostasis; endoplasmic reticulum

Phospholemman (PLM) belongs to the FXYD family of small ion transport regulators. When phosphorylated at [Ser.sup.68], PLM inhibits cardiac [Na.sup.+]/[Ca.sup.2+] exchanger (NCX1). We previously demonstrated that the cytoplasmic tail of PLM interacts with the proximal intracellular loop (residues 218-358), but not the transmembrane (residues 1-217 and 765-938) or [Ca.sup.2+]-binding (residues 371-508) domains, of NCX1. In this study, we used intact [Na.sup.+]/[Ca.sup.2+] exchanger with various deletions in the intracellular loop to map the interaction sites with PLM. We first demonstrated by Western blotting and confocal immunofluorescence microscopy that wild-type (WT) NCX1 and its deletion mutants were expressed in transfected HEK-293 cells. Cotransfection with PLM and NCX1 (or its deletion mutants) in HEK-293 cells did not decrease expression of NCX1 (or its deletion mutants). Coexpression of PLM with WT NCX1 inhibited NCX1 current ([I.sub.NaCa]). Deletion of residues 240-679, 265-373, 250-300, or 300-373 from WT NCX1 resulted in loss of inhibition of [I.sub.NaCa] by PLM. Inhibition of [I.sub.NaCa] by PLM was preserved when residues 229-237, 270-300, 328-330, or 330-373 were deleted from the intracellular loop of NCX1. These results suggest that PLM mediated inhibition of [I.sub.NaCa] by interacting with two distinct regions (residues 238-270 and 300-328) of NCX1. Indeed, [I.sub.NaCa] measured in mutants lacking residues 238-270, 300-328, or 238270 + 300-328 was not affected by PLM. Ghitathione S-transferase pull-down assays confirmed that PLM bound to fragments corresponding to residues 218-371, 218-320, 218-270, 238-371, and 300-373, but not to fragments encompassing residues 250-300 and 371-508 of NCX1, indicating that residues 218-270 and 300-373 physically associated with PLM. Finally, acute regulation of [I.sub.NaCa] by PLM phosphorylation observed with WT NCX1 was absent in 250300 deletion mutant but preserved in 229-237 deletion mutant. We conclude that PLM mediates its inhibition of NCX1 by interacting with residues 238-270 and 300-328. FXYD1; ion transport; sodium-calcium exchange

When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production by NO synthase (NOS), matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extraeellular matrix, and presentation of HGF to the c-met receptor as demonstrated by a primary culture and in vivo assays. We now add evidence that calcium-calmodulin is involved in the satellite cell activation cascade in vitro. Conditioned medium from cultures that were treated with a calcium ionophore (A23187, ionomycin) for 2 h activated cultured satellite cells and contained active HGF, similar to the effect of mechanical stretch or NO donor treatments. The response was abolished by addition of calmodulin inhibitors (calmidazolium, W-13, W-12) or a NOS inhibitor [N.sup.G]-nitro-L-arginine methyl ester hydrochloride but not by its less inactive enantiomer [N.sup.G]-nitro-Darginine methyl ester hydrochloride. Satellite cells were also shown to express functional calmodulin protein having a calcium-binding activity at 12 h postplating, which is the time at which the calcium ionophore was added in this study and the stretch treatment was applied in our previous experiments. Therefore, results from these experiments provide an additional insight that calcium-calmodulin mediates HGF release from the matrix and that this step in the activation pathway is upstream from NO synthesis. muscle regeneration; stretch-activation

It has been suggested that store-operated [Ca.sup.2+] entry (SOC) facilitates catecholamine secretion and synthesis in bovine adrenal medullary (AM) cells. However, there has been no experimental result clearly showing that cation channel activity is enhanced by store [Ca.sup.2+] depletion. Thus the present experiments were undertaken to address the issue of whether rat AM cells have SOC channels. Inhibition of the sarco(endo)plasmic reticulum [Ca.sup.2+] (SERCA) pump resulted in a sustained increase in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in rat AM cells. This increase was completely suppressed by 2 mM [Ni.sup.2+] but not by 100 [micro]M D600. A bath application of [Ni.sup.2+], but not D600, produced an outward current at -60 mV in rat AM cells, whereas exposure to a SERCA pump inhibitor did not affect either the whole cell current level or the [Ni.sup.2+]-induced outward current. The refilling of intracellular store sites was suppressed by the addition of [Ni.sup.2+] to the perfusate. RT-PCR revealed that transcripts for transient receptor potential channels 1 (TRPC1) and 5 (TRPC5) were present in rat adrenal medullas. Immunocytochemistry showed that TRPC1 channels, which have been implicated in SOC in certain types of cells, were mainly localized in the endoplasmic reticulum (ER) and not in the plasma membrane, and that STIM1, a [Ca.sup.2+] sensor in the ER, was not expressed in rat AM cells. On the basis of these results, we conclude that rat AM cells lack the SOC mechanism. calcium store sites; transient receptor potential channel; endoplasmic reticulum

Relatively little is known about the contribution of [Ca.sup.2+]-dependent and -independent mechanisms in the contractility of neonatal gastrointestinal smooth muscle. We therefore studied [Ca.sup.2+] homeostasis and [Ca.sup.2+] sensitization mechanisms in 10-day-old and adult guinea pig gallbladder smooth muscle to elucidate developmental changes in these processes. Gallbladder contractility was evaluated by isometrical tension recordings from strips, intracellular [Ca.sup.2+] concentration was estimated by epifluorescence microscopy of fura-2-loaded isolated cells, and protein expression and phosphorylation were assessed by Western blot analysis. The neonatal gallbladder contracted significantly less to CCK than adult tissue, but this correlated with an increased [Ca.sup.2+] mobilization, suggesting immaturity of [Ca.sup.2+] sensitization mechanisms. The enhanced [Ca.sup.2+] release in the newborn gallbladder was the result of the increase in the size of the releasable [Ca.sup.2+] pool. Moreover, in neonatal smooth muscle cells, neither the plasma membrane [Ca.sup.2+] pump nor the [Na.sup.+]/[Ca.sup.2+] exchanger collaborate in the extrusion of [Ca.sup.2+]. In contrast, in these cells, there is an increase in phospholamban phosphorylation, which could drive to an overactivity of the sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase pump. The reduced [Ca.sup.2+] sensitivity in neonatal tissues was demonstrated by the lack of effect to Y-27362, an inhibitor of Rho kinase (ROCK), and GF-109203X, an inhibitor of PKC, on agonist-induced contraction. In addition, the neonatal gallbladder showed lower levels of RhoA, ROCK, PKC, and two effectors [C-kinase-dependent inhibitor of 17 kDa (CPI-17) and myosin phosphatase targetting 1 (MYPT1)] as well as an absence of CPI-17 and MYPT1 phosphorylation in response to agonists. In conclusion, our results indicate that the main mechanisms involved in smooth muscle contractility are under developmental regulation. [Ca.sup.2+]-dependent contraction; [Ca.sup.2+] sensitization; gastrointestinal smooth muscle

We hypothesized that testosterone at physiological levels enhances cardiac contractile responses to stimulation of both [[alpha].sub.1]- and [[beta].sub.1]-adrenoceptors by increasing [Ca.sup.2+] release from the sarcoplasmic reticulum (SR) and speedier removal of [Ca.sup.2+] from cytosol via [Ca.sup.2+]-regulatory proteins. We first determined the left ventricular developed pressure, velocity of contraction and relaxation, and heart rate in perfused hearts isolated from control rats, orchiectomized rats, and orchiectomized rats without and with testosterone replacement (200 [micro]g/100 g body wt) in the presence of norepinephrine ([10.sup.-7] M), the [[alpha].sub.1]-adrenoceptor agonist phenylephrine ([10.sup.-6] M), or the nonselective [beta]-adrenoceptor agonist isoprenaline ([10.sup.-7] M) in the presence of 5 x [10.sup.-7] M ICI-118,551, a [[beta].sub.2]-adrenoceptor antagonist. Next, we determined the amplitudes of intracellular [Ca.sup.2+] concentration transients induced by electrical stimulation or caffeine, which represent, respectively, [Ca.sup.2+] release via the ryanodine receptor (RyR) or releasable [Ca.sup.2+] in the SR, in ventricular myocytes isolated from the three groups of rats. We also measured [sup.45][Ca.sup.2+] release via the RyR. We then determined the time to 50% decay of both transients, which represents, respectively, [Ca.sup.2+] reuptake by sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA) and removal via the sarcolemmal [Na.sup.+]/[Ca.sup.2+] exchanger (NCX). We correlated [Ca.sup.2+] removal from the cytosol with activities of SERCA and its regulator phospholamban as well as NCX. The results showed that testosterone at physiological levels enhanced positive inotropic and lusitropic responses to stimulation of [[alpha].sub.1]- and [[beta].sub.1]-adrenoceptors via the androgen receptor. The increased contractility and speedier relaxation were associated with increased [Ca.sup.2+] release via the RyR and faster [Ca.sup.2+] removal out of the cytosol via SERCA and NCX. orchiectomy; androgen receptor; left ventricular developed pressure; velocity of contraction and relaxation; electrical-induced intracellular [Ca.sup.2+] concentration transients; caffeine-induced intracellular [Ca.sup.2+] concentration transients; ryanodine receptor; sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase; [Na.sup.+]/[Ca.sup.2+] exchanger

PDGF B chain or PDGF receptor (PDGFR)-[beta]-deficient (-/-) mice lack mesangial cells. To study responses of [alpha]- and [beta]-receptor activation to PDGF ligands, metanephric mesenchymal cells (MMCs) were established from embryonic day E11.5 wild-type (+/+) and -/- mouse embryos. PDGF BB stimulated cell migration in +/+ cells, whereas PDGF AA did not. Conversely, PDGF AA was chemotactic for -/- MMCs. The mechanism by which PDGFR-[beta] inhibited AA-induced migration was investigated. PDGF BB, but not PDGF AA, increased intracellular [Ca.sup.2+] and the production of reactive oxygen species (ROS) in +/+ cells. Transfection of -/- MMCs with the wild-type [beta]-receptor restored cell migration and ROS generation in response to PDGF BB and inhibited AA-induced migration. Inhibition of [Ca.sup.2+] signaling facilitated PDGF AA-induced chemotaxis in the wild-type cells. The antioxidant N-acetyl-L-cysteine (NAC) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) abolished the BB-induced increase in intracellular [Ca.sup.2+] concentration, suggesting that ROS act as upstream mediators of [Ca.sup.2+] in suppressing PDGF AA-induced migration. These data indicate that ROS and [Ca.sup.2+] generated by active PDGFR-[beta] play an essential role in suppressing PDGF AA-induced migration in +/+ MMCs During kidney development, PDGFR [beta]-mediated ROS generation and [Ca.sup.2+] influx suppress PDGF AA-induced chemotaxis in metanephric mesenchyme. reactive oxygen species; calcium

To gain insight into the mechanisms that underlie angiotensin II (ANG II)-induced cytoplasmic [Ca.sup.2+] concentration ([[Ca].sub.cyt]) oscillations in medullary pericytes, we expanded a prior model of ion fluxes. ANG II stimulation was simulated by doubling maximal inositol trisphosphate (I[P.sub.3]) production and imposing a 90% blockade of [K.sup.+] channels. We investigated two configurations, one in which ryanodine receptors (RyR) and I[P.sub.3] receptors (I[P.sub.3]R) occupy a common store and a second in which they reside on separate stores. Our results suggest that [Ca.sup.2+] release from stores and import from the extracellular space are key determinants of oscillations because both raise [Ca] in subplasmalemmal spaces near RyR. When the [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR) threshold of RyR is exceeded, the ensuing [Ca.sup.2+] release is limited by [Ca.sup.2+] reuptake into stores and export across the plasmalemma. If sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA) pumps do not remain saturated and sarcoplasmic reticulum [Ca.sup.2+] stores are replenished, that phase is followed by a resumption of leak from internal stores that leads either to [[Ca].sub.cyt] elevation below the CICR threshold (no oscillations) or to elevation above it (oscillations). Our model predicts that oscillations are more prone to occur when I[P.sub.3]R and RyR stores are separate because, in that case, [Ca.sup.2+] released by RyR during CICR can enhance filling of adjacent I[P.sub.3] stores to favor a high subsequent leak that generates further CICR events. Moreover, the existence or absence of oscillations depends on the set points of several parameters, so that biological variation might well explain the presence or absence of oscillations in individual pericytes. mathematical model; sarcoplasmic reticulum stores; ryanodine receptors; inositol trisphosphate receptors; medullary pericytes

Defecation in the nematode worm Caenorhabditis elegans is a highly rhythmic behavior that is regulated by a [Ca.sup.2+] wave generated in the 20 epithelial cells of the intestine, in part through activation of the inositol 1,4,5-trisphosphate receptor. Execution of the defecation motor program (DMP) can be modified by external cues such as nutrient availability or mechanical stimulation. To address the likelihood that environmental regulation of the DMP requires integrating distinct cellular and organismal processes, we have developed a method for studying coordinate [Ca.sup.2+] oscillations and defecation behavior in intact, freely behaving animals. We tested this technique by examining how mutations in genes known to alter [Ca.sup.2+] handling [including egl-8/phospholipase C (PLC)-[beta], kqt-3/KCNQ1, sca-1/sarco (endo)plasmic reticulum [Ca.sup.2+] ATPase, and unc-43/[Ca.sup.2+]-CaMKII] contribute to shaping the [Ca.sup.2+] wave and asked how [Ca.sup.2+] wave dynamics in the mutant backgrounds altered execution of the DMP. Notably, we find that [Ca.sup.2+] waves in the absence of PLC[beta] initiate ectopically, often traveling in reverse, and fail to trigger a complete DMP. These results suggest that the normal supremacy of the posterior intestinal cells is not obligatory for [Ca.sup.2+] wave occurrence but instead helps to coordinate the DMP. Furthermore, we present evidence suggesting that an underlying pacemaker appears to oscillate at a faster frequency than the defecation cycle and that arrhythmia may result from uncoupling the pacemaker from the DMP rather than from disrupting the pacemaker itself. We also show that chronic elevations in [Ca.sup.2+] have limited influence on the defecation period but instead alter the interval between successive steps of the DMP. Finally, our results demonstrate that it is possible to assess [Ca.sup.2+] dynamics and muscular contractions in a completely unrestrained model organism. calcium; oscillation; Caenorhabditis elegans; biosensor

Cardiac P2X purinergic receptors can mediate an increase in myocyte contractility and a potentially important role in the heart. The P2[X.sub.4] receptor (P2[X.sub.4]R) is an important subunit of native cardiac P2X receptors. With transgenic mice with cardiac-specific overexpression of P2[X.sub.4]R (Tg) used as a model, the objectives here were to characterize the P2X receptor-mediated cellular contractile and [Ca.sup.2+] transient effects and to determine the mechanism underlying the receptor-induced increase in myocyte contractility. In response to the agonist 2-methylthioATP (2-meSATP), Tg myocytes showed an increased intracellular [Ca.sup.2+] transient, as defined by fura 2 fluorescence ratio, and an enhanced contraction shortening that were unaccompanied by cAMP accumulation or L-type [Ca.sup.2+] channel activation. The increased [Ca.sup.2+] transient was not associated with any alteration in action potential duration, resting membrane potential, or diastolic fluorescence ratio or rates of rise and decline of the [Ca.sup.2+] transient. Simultaneous [Ca.sup.2+] transient and contraction measurements did not show any agonist-mediated change in myofilament [Ca.sup.2+] sensitivity. However, activation of the overexpressed P2[X.sub.4] receptor caused an enhanced SR [Ca.sup.2+] loading, as evidenced by a 2-meSATP-evoked increase in the caffeine-induced inward current and [Ca.sup.2+] transient. Similar data were obtained in wild-type mouse ventricular myocytes. Thus an increased SR [Ca.sup.2+] content, occurring in the absence of cAMP accumulation or L-type [Ca.sup.2+] channel activation, is the principal mechanism by which cardiac P2X receptor mediates a stimulatory effect on cardiac myocyte contractility. purines; calcium; sarcoplasmic reticulum

Throughout the world many people use artificial sweeteners (AS) for the purpose of reducing caloric intake. The most prominently used of these molecules include saccharin, aspartame (Nutrasweet), acesulfame-K, and cyclamate. Despite the caloric advantage they provide, one key concern in their use is their aversive aftertaste that has been characterized on a sensory level as bitter and/or metallic. Recently, it has been shown that the activation of particular T2R bitter taste receptors is partially involved with the bitter aftertaste sensation of saccharin and acesulfame-K. To more fully understand the biology behind these phenomena we have addressed the question of whether AS could stimulate transient receptor potential vanilloid-1 (TRPV1) receptors, as these receptors are activated by a large range of structurally different chemicals. Moreover, TRPV1 receptors and/or their variants are found in taste receptor cells and in nerve terminals throughout the oral cavity. Hence, TRPV1 activation could be involved in the AS aftertaste or even contribute to the poorly understood metallic taste sensation. Using [Ca.sup.2+] imaging on TRPV1 receptors heterologously expressed in the human embryonic kidney (HEK) 293 cells and on dissociated primary sensory neurons, we find that in both systems, AS activate TRPV1 receptors, and, moreover, they sensitize these channels to acid and heat. We also found that TRPV1 receptors are activated by CuS[O.sub.4], ZnS[O.sub.4], and FeS[O.sub.4], three salts known to produce a metallic taste sensation. In summary, our results identify a novel group of compounds that activate TRPV1 and, consequently, provide a molecular mechanism that may account for off tastes of sweeteners and metallic tasting salts. multisensory taste; pain; calcium imaging

Mitochondria are an important source of reactive oxygen species (ROS) formed as a side product of oxidative phosphorylation. The main sites of oxidant production are complex I and complex III, where electrons flowing from reduced substrates are occasionally transferred to oxygen to form superoxide anion and derived products. These highly reactive compounds have a well-known role in pathological states and in some cellular responses. However, although their link with [Ca.sup.2+] is well studied in cell death, it has been hardly investigated in normal cytosolic calcium concentration ([[[Ca.sup.2+]].sub.i]) signals. Several [Ca.sup.2+] transport systems are modulated by oxidation. Oxidation increases the activity of inositol 1,4,5-trisphosphate and ryanodine receptors, the main channels releasing [Ca.sup.2+] from intracellular stores in response to cellular stimulation. On the other hand, mitochondria are known to control [[[Ca.sup.2+]].sub.i] signals by [Ca.sup.2+] uptake and release during cytosolic calcium mobilization, specially in mitochondria situated close to [Ca.sup.2+] release channels. Mitochondrial inhibitors modify calcium signals in numerous cell types, including oscillations evoked by physiological stimulus. Although these inhibitors reduce mitochondrial [Ca.sup.2+] uptake, they also impair ROS production in several systems. In keeping with this effect, recent reports show that antioxidants or oxidant scavengers also inhibit physiological calcium signals. Furthermore, there is evidence that mitochondria generate ROS in response to cell stimulation, an effect suppressed by mitochondrial inhibitors that simultaneously block [[[Ca.sup.2+]].sub.i] signals. Together, the data reviewed here indicate that [Ca.sup.2+]-mobilizing stimulus generates mitochondrial ROS, which, in turn, facilitate [[[Ca.sup.2+]].sub.i] signals, a new aspect in the biology of mitochondria. Finally, the potential implications for biological modeling are discussed. mitochondria; calcium

Intracellular ANG II induces biological effects in nonrenal cells, but it is not known whether it plays a physiological role in renal proximal tubule cells (PTCs). PTCs express angiotensinogen, renin, and angiotensin-converting enzyme mRNAs, suggesting the presence of high levels of intracellular ANG II. We determined if microinjection of ANG II directly in single PTCs increases intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) and, if so, elucidated the cellular mechanisms involved. Changes in [[Ca.sup.2+]]i responses were studied by fluorescence imaging using the [Ca.sup.2+] indicator fluo 3. ANG II (1 nM) was microinjected directly in the cells, whereas cell-surface angiotensin type 1 (A[T.sub.1]) receptors were blocked by losartan (10 [micro]M). When ANG II (1 nM) was added to the perfusate, there was a marked increase in [[[Ca.sup.2+]].sub.i] that was blocked by extracellular losartan. With losartan in the perfusate, intracellular microinjection of ANG II elicited a robust increase in cytoplasmic [[[Ca.sup.2+]].sub.i] that peaked at 30 s (basal: 2.2 [+ or -] 0.3 vs. ANG II: 14.9 [+ or -] 0.4 relative fluorescence units; P < 0.01). Chelation of extracellular [Ca.sup.2+] with EGTA (2 mM) did not alter microinjected ANG II-induced [[[Ca.sup.2+].sub.i] responses ([Ca.sup.2+] free + ANG II: 12.3 [+ or -] 2.6 relative fluorescence units, not significant vs. ANG II); however, pretreatment with thapsigargin to deplete intracellular [Ca.sup.2+] stores or with U-73122 to inhibit phospholipase C (1 [micro]M each) markedly attenuated microinjected ANG II-induced [[[Ca.sup.2+].sub.i] responses. Combined microinjection of ANG II and losartan abolished [[[Ca.sup.2+]].sub.i] responses, whereas a combination of ANG II and PD-123319 had no effect. These data demonstrate for the first time that direct microinjection of ANG II in single PTCs increases [[[Ca.sup.2+]].sub.i] by stimulating intracellular A[T.sub.1] receptors and releases [Ca.sup.2+] from intracellular stores, suggesting that intracellular ANG II may play a physiological role in PTC function. intracellular calcium; kidney: microinjection; proximal tubules; receptor-mediated endocytosis

Recent genetic analysis has identified a pivotal role of primary cilia in the pathogenesis of polycystic kidney disease (PKD). However, little is known regarding how cilia loss/dysfunction contributes to cyst development. In epithelial cells, changes in apical fluid flow induce cilia-mediated [Ca.sup.2+] entry via polycystin-2 (PC2), a cation channel. The Oak Ridge Polycystic Kidney (orpk) mouse contains a mutated Tg737 gene that disrupts expression of polaris, a protein required for ciliogenesis. These studies examine the effect of cilia malformation on [Ca.sup.2+] entry in orpk cilia(-) collecting duct principal cells, and in orpk cells in which wild-type Tg737 was reintroduced, orpk cilia(+). [[[Ca.sup.2+]].sub.i] was monitored in confluent cell monolayers using fluorescence microscopy. Intrinsic apical [Ca.sup.2+] entry was measured by [Mn.sup.2+] quenching and [Ca.sup.2+] depletion/readdition under flow conditions below the threshold for stimulation. We found that unstimulated apical [Ca.sup.2+] entry was markedly increased in cilia(-) cells and was sensitive to [Gd.sup.3+], an inhibitor of PC2. Electrophysiological measurements demonstrate increased abundance of an apical channel, consistent with PC2, in cilia(-) cells. Immunofluorescence studies revealed that PC2, normally expressed on and at the base of cilia in orpk cilia(+) cells, was observed throughout the apical membrane in cilia(-) cells. Furthermore, cilia(-) cells displayed elevated subapical [Ca.sup.2+] levels measured with the near-membrane [Ca.sup.2+] indicator FFP-18. We propose that cilia exert a tonic regulatory influence on apical [Ca.sup.2+] entry, and absence of cilia results in loss of spatial organization of PC2, causing unregulated [Ca.sup.2+] entry and elevations in subapical [[Ca.sup.2+]], a factor which may contribute to cyst formation. Oak Ridge Polycystic Kidney; autosomal recessive polycystic kidney disease; polycystin-2; [Ca.sup.2+] permeability

TRPV4, a renally expressed nonselective cation channel of the transient receptor potential (TRP) family, is gated by hypotonicity. Kinases of the WNK family influence expression and function of the thiazide-sensitive [Na.sup.+]-[Cl.sup.-] cotransporter, and monogenic human hypertension has been linked to mutations in the gene coding for WNK4. Along with TRPV4, WNK isoforms are highly expressed in the distal nephron. We show here that coexpression of WNK4 downregulates TRPV4 function in human embryonic kidney (HEK-293) cells and that this effect is mediated via decreased cell surface expression of TRPV4; total abundance of TRPV4 in whole cell lysates is unaffected. The effect of the related kinase WNK1 on TRPV4 function and surface expression was similar to that of WNK4. Disease-causing point mutations in WNK4 abrogate, but do not eliminate, the inhibitory effect on TRPV4 function. In contrast to wild-type WNK4, a kinase-dead WNK4 point mutant failed to influence TRPV4 trafficking; however, deletion of the entire WNK4 kinase domain did not blunt the effect of WNK4 on localization of TRPV4. Deletion of the extreme COOH-terminal putative coiled-coil domain of WNK4 abolished its effect. In immunoprecipitation experiments, we were unable to detect direct interaction between TRPV4 and either WNK kinase. In aggregate, these data indicate that TRPV4 is functionally regulated by WNK family kinases at the level of cell surface expression. Because TRPV4 and WNK kinases are coexpressed in the distal nephron in vivo and because there is a tendency toward hypercalcemia in [TRPV4.sup.-/-] mice, we speculate that this pathway may impact systemic [Ca.sup.2+] balance. In addition, because WNK kinases and TRPV4 are activated by anisotonicity, they may comprise elements of an osmosensing or osmotically responsive signal transduction cascade in the distal nephron. hypertension; calcium balance; hypotonicity; osmoregulation

The hormone leptin and the gut peptide CCK synergistically interact to enhance the process of satiation. Although this interaction may occur at several levels of the neuroaxis, our previous results indicate that leptin can specifically enhance the satiation effect of CCK by acting on subdiaphragmatic vagal afferent neurons. Because of this localized action, we hypothesized that a high proportion of vagal afferent neurons innervating the stomach or duodenum would be responsive to leptin and/or CCK. To test this hypothesis, we measured changes in cytosolic calcium levels induced by leptin and CCK in cultured nodose ganglion neurons labeled with a retrograde neuronal tracer injected into either the stomach or the duodenum. In the neurons labeled from the stomach, CCK activated 74% (39 of 53) compared with only 35% (34 of 97) of nonlableled cells. Of the CCK-responsive neurons 60% (18 of 30) were capsaicin-sensitive. Leptin activated 42% (22 of 53) of the stomach innervating neurons compared with 26% of nonlabeled neurons. All of the leptin-sensitive neurons labeled from the stomach also responded to CCK. In the neurons labeled from the duodenum, CCK activated 71% (20 of 28). Of these CCK-responsive neurons 80% (12 of 15) were capsaicin sensitive. Leptin activated 46% (13 of 28) of these duodenal innervating neurons, of which 89% (8 of 9) were capsaicin-sensitive. Among neurons labeled from the duodenum 43% (12 of 28) were responsive to both leptin and CCK, compared with only 15% (15 of 97) of unlabeled neurons. Our results support the hypothesis that vagal afferent sensitivity to CCK and leptin is concentrated in neurons that innervate the stomach and duodenum. These specific visceral afferent populations are likely to comprise a substrate through which acute leptin/CCK interactions enhance satiation. nodose; calcium imaging; satiation; cholecystokinin

We previously showed that ablation of caveolin-1 (Cav-1) gene expression in mice promotes neointimal hyperplasia in vivo, a phenomenon normally characterized by smooth muscle cell (SMC) migration and proliferation. Whether these defects are cell autonomous, i.e., due to loss of Cav-1 within SMCs or loss of Cav-1 expression in other adjacent cell types in vivo, remains unknown. Cav-1 has been shown to associate with receptors for many vasoactive factors on the SMC surface. Therefore, Cav-1 might be an important regulator of SMC proliferation, migration, and signal transduction. To mechanistically dissect the role of Cav-1 in SMC signaling, we isolated SMCs from the aortas (AoSMCs) of Cav-1-deficient ([Cav-1.sup.-/-]) mice and characterized these cells with respect to their proliferation, migration, and [Ca.sup.2+] response to an important vasoactive factor, endothelin-1 (ET-1). 5-Bromo-2'-deoxyuridine incorporation and a wound-healing assay showed an increase in proliferation and migration rates in [Cav-1.sup.-/-] compared with wild-type ([Cav-1.sup.-/-]) AoSMCs. AoSMCs demonstrated upregulation of phosphorylated ERK1/2, cyclin D1, and proliferating cell nuclear antigen and reduced expression of the cyclin-dependent kinase inhibitor [p27.sup.Kip1]. The [Ca.sup.2+] response was examined in the presence of ET-1 and assessed by confocal microscopy with the [Ca.sup.2+]-sensitive fluorescent probe fluo 3. When treated with ET-1, Cav-[1.sup.-/-] AoSMCs exhibited a faster and larger increase in free intracellular [Ca.sup.2+] than [Cav-1.sup.+/+] cells. The ET-1-induced response in [Cav-1.sup.-/-] cells was mediated by the ETB receptor, as shown using the ETB receptor antagonist BQ-788 and the ETA receptor antagonist BQ-123. In [Cav-1.sup.-/-] cells, ETA receptor expression was reduced and E[T.sub.B] receptor expression was upregulated. Therefore, Cav-1 ablation increased the ET-1-induced [Ca.sup.2+] response in SMCs by altering the type and expression level of the ET receptor (i.e., receptor isoform switching). These data suggest a novel regulatory role for Cav-1 in SMCs with respect to their proliferation, migration, and [Ca.sup.2+]-mediated signaling. vascular disease; calcium response; endothelin receptors; neointimal hyperplasia; caveolae; caveolin

Transient receptor potential melastatin- (TRPM) and vanilloid-related (TRPV) channels are nonselective cation channels pertinent to diverse physiological functions. Multiple TRPM and TRPV channel subtypes have been identified and cloned in different tissues. However, their information in vascular tissue is scant. In this study, we sought to identify TRPM and TRPV channel subtypes expressed in rat deendothelialized intralobar pulmonary arteries (PAs) and aorta. With RT-PCR, mRNA of TRPM2, TRPM3, TRPM4, TRPM7, and TRPM8 of TRPM family and TRPV1, TRPV2, TRPV3, and TRPV4 of TRPV family were detected in both PAs and aorta. Quantitative real-time RT-PCR showed that TRPM8 and TRPV4 were the most abundantly expressed TRPM and TRPV subtypes, respectively. Moreover, Western blot analysis verified expression of TRPM2, TRPM8, TRPV1, and TRPV4 proteins in both types of vascular tissue. To examine the functional activities of these channels, we monitored intracellular [Ca.sup.2+] transients ([[[Ca.sup.2+]].sub.i]) in pulmonary arterial smooth muscle cells (PASMCs) and aortic smooth muscle cells (ASMCs). The TRPM8 agonist menthol (300 [micro]M) and the TRPV4 agonist 4[alpha]-phorbol 12,13-didecanoate (1 [micro]M) evoked significant increases in [[[Ca.sup.2+]].sub.i] in PASMCs and ASMCs. These [Ca.sup.2+] responses were abolished in the absence of extracellular [Ca.sup.2+] or the presence of 300 [micro]M [Ni.sup.2+] but were unaffected by 1 [micro]M nifedipine, suggesting [Ca.sup.2+] influx via nonselective cation channels. Hence, for the first time, our results indicate that multiple functional TRPM and TRPV channels are coexpressed in rat intralobar PAs and aorta. These novel [Ca.sup.2+] entry pathways may play important roles in the regulation of pulmonary and systemic circulation. transient receptor potential channels; calcium signaling; nonselective cation channels