Poly(ADP-ribosyl)ation basally activated by DNA strand breaks reflects glutamate���nitric oxide neurotransmission

Poly(ADP-ribose) polymerase (PARP) transfers ADP ribose groups from NAD+ to nuclear proteins after activation by DNA strand breaks. PARP overactivation by massive DNA damage causes cell death via NAD+ and ATP depletion. Heretofore, PARP has been thought to be inactive under basal physiologic conditions. We now report high basal levels of PARP activity and DNA strand breaks in discrete neuronal populations of the brain, in ventricular ependymal and subependymal cells and in peripheral tissues. In some peripheral tissues, such as skeletal muscle, spleen, heart, and kidney, PARP activity is reduced only partially in mice with PARP-1 gene deletion (PARP-1���/���), implicating activity of alternative forms of PARP. Glutamate neurotransmission involving N-methyl-d-aspartate (NMDA) receptors and neuronal nitric oxide synthase (nNOS) activity in part mediates neuronal DNA strand breaks and PARP activity, which are diminished by NMDA antagonists and NOS inhibitors and also diminished in mice with targeted deletion of nNOS gene (nNOS���/���). An increase in NAD+ levels after treatment with NMDA antagonists or NOS inhibitors, as well as in nNOS���/��� mice, indicates that basal glutamate-PARP activity regulates neuronal energy dynamics.

Mouse trp2, the homologue of the human trpc2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+ entry channel

Capacitative Ca2+ entry (CCE) is Ca2+ entering after stimulation of inositol 1,4,5-trisphosphate (IP3) formation and initiation of Ca2+ store depletion. One hallmark of CCE is that it can also be triggered merely by store depletion, as occurs after inhibition of internal Ca2+ pumps with thapsigargin. Evidence has accumulated in support of a role of transient receptor potential (Trp) proteins as structural subunits of a class of Ca2+-permeable cation channels activated by agonists that stimulate IP3 formation���very likely through a direct interaction between the IP3 receptor and a Trp subunit of the Ca2+ entry channel. The role of Trp���s in Ca2+ entry triggered by store depletion alone is less clear. Only a few of the cloned Trp���s appear to enhance this type of Ca2+ entry, and when they do, the effect requires special conditions to be observed, which native CCE does not. Here we report the full-length cDNA of mouse trp2, the homologue of the human trp2 pseudogene. Mouse Trp2 is shown to be readily activated not only after stimulation with an agonist but also by store depletion in the absence of an agonist. In contrast to other Trp proteins, Trp2-mediated Ca2+ entry activated by store depletion is seen under the same conditions that reveal endogenous store depletion-activated Ca2+ entry, i.e., classical CCE. The findings support the general hypothesis that Trp proteins are subunits of store- and receptor-operated Ca2+ channels.

��-Aminobutyric acid type B receptors are expressed and functional in mammalian cardiomyocytes

��-Hydroxybutyrate (GHB), an anesthetic adjuvant analog of ��-aminobutyrate (GABA), depresses cell excitability in hippocampal neurons by inducing hyperpolarization through the activation of a prominent inwardly rectifying K+ (Kir3) conductance. These GABA type B (GABAB)-like effects are clearly shown at high concentrations of GHB corresponding to blood levels usually reached during anesthesia and are mimicked by the GABAB agonist baclofen. Recent studies of native GABAB receptors (GABABRs) have favored the concept that GHB is also a selective agonist. Furthermore, cloning has demonstrated that GABABRs assemble heteromeric complexes from the GABABR1 and GABABR2 subtypes and that these assemblies are activated by GHB. The surprisingly high tissue content, together with anti-ischemic and protective effects of GHB in the heart, raises the question of a possible influence of GABAB agonists on excitable cardiac cells. In the present study, we provide electrophysiological evidence that GHB activates an inwardly rectifying K+ current in rat ventricular myocytes. This effect is mimicked by baclofen, reversibly inhibited by GABAB antagonists, and prevented by pertussis toxin pretreatment. Both GABABR1 and GABABR2 are detected in cardiomyocytes by Western blotting and are shown to coimmunoprecipitate. Laser scanning confocal microscopy discloses an even distribution of the two receptors in the sarcolemma and along the transverse tubular system. Hence, we conclude that GABABRs are distributed not only in neuronal tissues but also in the heart, where they can be activated and induce electrophysiological alterations through G-protein-coupled inward rectifier potassium channels.

Prostacyclin-mediated activation of peroxisome proliferator-activated receptor �� in colorectal cancer

There is evidence from both genetic and pharmacologic studies to suggest that the cyclooxygenase-2 (COX-2) enzyme plays a causal role in the development of colorectal cancer. However, little is known about the identity or role of the eicosanoid receptor pathways activated by COX-derived prostaglandins (PG). We previously have reported that COX-2-derived prostacyclin promotes embryo implantation in the mouse uterus via activation of the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) ��. In light of the recent finding that PPAR�� is a target of ��-catenin transactivation, it is important to determine whether this signaling pathway is operative during the development of colorectal cancer. Analysis of PPAR�� mRNA in matched normal and tumor samples revealed that expression of PPAR��, similar to COX-2, is up-regulated in colorectal carcinomas. In situ hybridization studies demonstrate that PPAR�� is expressed in normal colon and localized to the epithelial cells at the very tips of the mucosal glands. In contrast, expression of PPAR�� mRNA in colorectal tumors was more widespread with increased levels in transformed epithelial cells. Analysis of PPAR�� and COX-2 mRNA in serial sections suggested they were colocalized to the same region within a tumor. Finally, transient transfection assays established that endogenously synthesized prostacyclin (PGI2) could serve as a ligand for PPAR��. In addition, the stable PGI2 analog, carbaprostacyclin, and a synthetic PPAR�� agonist induced transactivation of endogenous PPAR�� in human colon carcinoma cells. We conclude from these observations that PPAR��, similar to COX-2, is aberrantly expressed in colorectal tumors and that endogenous PPAR�� is transcriptionally responsive to PGI2. However, the functional consequence of PPAR�� activation in colon carcinogenesis still needs to be determined.

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K+ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K+ currents, an inactivating outward-rectifying K+ current was characterized (plant A-type current: IAP). IAP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. IAP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to ���100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward IAP was mainly carried by K+ ions. In contrast to the sustained outward-rectifying K+ currents, cytosolic alkaline pH was found to inhibit IAP and extracellular K+ was required for IAP activity. Furthermore, increasing cytosolic free Ca2+ in the physiological range strongly inhibited IAP activity with a half inhibitory concentration of ��� 94 nM. We present a detailed characterization of an inactivating K+ current in a higher plant cell. Regulation of IAP by diverse factors including membrane potential, cytosolic Ca2+ and pH, and extracellular K+ and Ca2+ implies that the inactivating IAP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.

Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia

Many pathogens causing diarrhea do so by modulating ion transport in the gut. Respiratory pathogens are similarly associated with disturbances of fluid balance in the respiratory tract, although it is not known whether they too act by altering epithelial ion transport. Here we show that influenza virus A/PR/8/34 inhibits the amiloride-sensitive Na+ current across mouse tracheal epithelium with a half-time of about 60 min. We further show that the inhibitory effect of the influenza virus is caused by the binding of viral hemagglutinin to a cell-surface receptor, which then activates phospholipase C and protein kinase C. Given the importance of epithelial Na+ channels in controlling the amount of fluid in the respiratory tract, we suggest that down-regulation of Na+ channels induced by influenza virus may play a role in the fluid transport abnormalities that are associated with influenza infections.

Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosis-associated mutations in copper-zinc superoxide dismutase

The mechanism by which mutations in the superoxide dismutase (SOD1) gene cause motor neuron degeneration in familial amyotrophic lateral sclerosis (ALS) is unknown. Recent reports that neuronal death in SOD1-familial ALS is apoptotic have not documented activation of cell death genes. We present evidence that the enzyme caspase-1 is activated in neurons expressing mutant SOD1 protein. Proteolytic processing characteristic of caspase-1 activation is seen both in spinal cords of transgenic ALS mice and neurally differentiated neuroblastoma (line N2a) cells with SOD1 mutations. This activation of caspase-1 is enhanced by oxidative challenge (xanthine/xanthine oxidase), which triggers cleavage and secretion of the interleukin 1�� converting enzyme substrate, pro-interleukin 1��, and induces apoptosis. This N2a culture system should be an instructive in vitro model for further investigation of the proapoptotic properties of mutant SOD1.

Evidence for the existence of a sulfonylurea-receptor-like protein in plants: Modulation of stomatal movements and guard cell potassium channels by sulfonylureas and potassium���channel���openers

Limitation of water loss and control of gas exchange is accomplished in plant leaves via stomatal guard cells. Stomata open in response to light when an increase in guard cell turgor is triggered by ions and water influx across the plasma membrane. Recent evidence demonstrating the existence of ATP-binding cassette proteins in plants led us to analyze the effect of compounds known for their ability to modulate ATP-sensitive potassium channels (K-ATP) in animal cells. By using epidermal strip bioassays and whole-cell patch-clamp experiments with Vicia faba guard cell protoplasts, we describe a pharmacological profile that is specific for the outward K+ channel and very similar to the one described for ATP-sensitive potassium channels in mammalian cells. Tolbutamide and glibenclamide induced stomatal opening in bioassays and in patch-clamp experiments, a specific inhibition of the outward K+ channel by these compounds was observed. Conversely, application of potassium channel openers such as cromakalim or RP49356 triggered stomatal closure. An apparent competition between sulfonylureas and potassium channel openers occurred in bioassays, and outward potassium currents, previously inhibited by glibenclamide, were partially recovered after application of cromakalim. By using an expressed sequence tag clone from an Arabidopsis thaliana homologue of the sulfonylurea receptor, a 7-kb transcript was detected by Northern blot analysis in guard cells and other tissues. Beside the molecular evidence recently obtained for the expression of ATP-binding cassette protein transcripts in plants, these results give pharmacological support to the presence of a sulfonylurea-receptor-like protein in the guard-cell plasma membrane tightly involved in the outward potassium channel regulation during stomatal movements.

Uncoupling proteins 2 and 3 are highly active H+ transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone)

Based on the discovery of coenzyme Q (CoQ) as an obligatory cofactor for H+ transport by uncoupling protein 1 (UCP1) [Echtay, K. S., Winkler, E. & Klingenberg, M. (2000) Nature (London) 408, 609���613] we show here that UCP2 and UCP3 are also highly active H+ transporters and require CoQ and fatty acid for H+ transport, which is inhibited by low concentrations of nucleotides. CoQ is proposed to facilitate injection of H+ from fatty acid into UCP. Human UCP2 and 3 expressed in Escherichia coli inclusion bodies are solubilized, and by exchange of sarcosyl against digitonin, nucleotide binding as measured with 2���-O-[5-(dimethylamino)naphthalene-1-sulfonyl]-GTP can be restored. After reconstitution into vesicles, Cl��� but no H+ are transported. The addition of CoQ initiates H+ transport in conjunction with fatty acids. This increase is fully sensitive to nucleotides. The rates are as high as with reconstituted UCP1 from mitochondria. Maximum activity is at a molar ratio of 1:300 of CoQ:phospholipid. In UCP2 as in UCP1, ATP is a stronger inhibitor than ADP, but in UCP3 ADP inhibits more strongly than ATP. Thus UCP2 and UCP3 are regulated differently by nucleotides, in line with their different physiological contexts. These results confirm the regulation of UCP2 and UCP3 by the same factors CoQ, fatty acids, and nucleotides as UCP1. They supersede reports that UCP2 and UCP3 may not be H+ transporters.

IL-4 inhibits osteoclast formation through a direct action on osteoclast precursors via peroxisome proliferator-activated receptor ��1

IL-4 is a pleiotropic immune cytokine secreted by activated TH2 cells that inhibits bone resorption both in vitro and in vivo. The cellular targets of IL-4 action as well as its intracellular mechanism of action remain to be determined. We show here that IL-4 inhibits receptor activator of NF-��B ligand-induced osteoclast differentiation through an action on osteoclast precursors that is independent of stromal cells. Interestingly, this inhibitory effect can be mimicked by both natural as well as synthetic peroxisome proliferator-activated receptor ��1 (PPAR��1) ligands and can be blocked by the irreversible PPAR�� antagonist GW 9662. These findings suggest that the actions of IL-4 on osteoclast differentiation are mediated by PPAR��1, an interpretation strengthened by the observation that IL-4 can activate a PPAR��1-sensitive luciferase reporter gene in RAW264.7 cells. We also show that inhibitors of enzymes such as 12/15-lipoxygenase and the cyclooxygenases that produce known PPAR��1 ligands do not abrogate the IL-4 effect. These findings, together with the observation that bone marrow cells from 12/15-lipoxygenase-deficient mice retain sensitivity to IL-4, suggest that the cytokine may induce novel PPAR��1 ligands. Our results reveal that PPAR��1 plays an important role in the suppression of osteoclast formation by IL-4 and may explain the beneficial effects of the thiazolidinedione class of PPAR��1 ligands on bone loss in diabetic patients.

Flexibility of the Kir6.2 inward rectifier K+ channel pore

Interactions of sulfhydryl reagents with introduced cysteines in the pore-forming (Kir6.2) subunits of the KATP channel were examined. 2-Aminoethyl methanethiosulfonate (MTSEA+) failed to modify Cd2+-insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels. Although a single Cd2+ ion is coordinated by L164C, four MTSEA+ ���hits��� can occur, each sequentially reducing the single-channel current. A dimeric fusion of control-Kir6.2 and L164C subunits generates Cd2+-insensitive channels, confirming that at least three cysteines are required for coordination, but MTSEA+ modification of the dimer occurs in two hits. L164C channels were not modified by bromotrimethyl ammoniumbimane (qBBr+), even though qBBr+ caused voltage-dependent block (as opposed to modification) that was comparable to that of MTSEA+ or 3-(triethylammonium)propyl methanethiosulfonate (MTSPTrEA+), implying that qBBr+ can also enter the inner cavity but does not modify L164C residues. The Kir channel pore structure was modeled by homology with the KcsA crystal structure. A stable conformation optimally places the four L164C side chains for coordination of a single Cd2+ ion. Modification of these cysteines by up to four MTSEA+ (or three MTSPTrEA+, or two qBBr+) does not require widening of the cavity to accommodate the derivatives within it. However, like the KcsA crystal structure, the energy-minimized model shows a narrowing at the inner entrance, and in the Kir6.2 model this narrowing excludes all ions. To allow entry of ions as large as MTSPTrEA+ or qBBr+, the entrance must widen to >8 ���, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement.

Allosteric modulation of Ca2+ channels by G proteins, voltage-dependent facilitation, protein kinase C, and Cav�� subunits

N-type and P/Q-type Ca2+ channels are inhibited by neurotransmitters acting through G protein-coupled receptors in a membrane-delimited pathway involving G���� subunits. Inhibition is caused by a shift from an easily activated ���willing��� (W) state to a more-difficult-to-activate ���reluctant��� (R) state. This inhibition can be reversed by strong depolarization, resulting in prepulse facilitation, or by protein kinase C (PKC) phosphorylation. Comparison of regulation of N-type Ca2+ channels containing Cav2.2a ��1 subunits and P/Q-type Ca2+ channels containing Cav2.1 ��1 subunits revealed substantial differences. In the absence of G protein modulation, Cav2.1 channels containing Cav�� subunits were tonically in the W state, whereas Cav2.1 channels without �� subunits and Cav2.2a channels with �� subunits were tonically in the R state. Both Cav2.1 and Cav2.2a channels could be shifted back toward the W state by strong depolarization or PKC phosphorylation. Our results show that the R state and its modulation by prepulse facilitation, PKC phosphorylation, and Cav�� subunits are intrinsic properties of the Ca2+ channel itself in the absence of G protein modulation. A common allosteric model of G protein modulation of Ca2+-channel activity incorporating an intrinsic equilibrium between the W and R states of the ��1 subunits and modulation of that equilibrium by G proteins, Cav�� subunits, membrane depolarization, and phosphorylation by PKC accommodates our findings. Such regulation will modulate transmission at synapses that use N-type and P/Q-type Ca2+ channels to initiate neurotransmitter release.

Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels

The cystic fibrosis transmembrane conductance regulator (CFTR) protein has the ability to function as both a chloride channel and a channel regulator. The loss of these functions explains many of the manifestations of the cystic fibrosis disease (CF), including lung and pancreatic failure, meconium ileus, and male infertility. CFTR has previously been implicated in the cell regulatory volume decrease (RVD) response after hypotonic shocks in murine small intestine crypts, an effect associated to the dysfunction of an unknown swelling-activated potassium conductance. In the present study, we investigated the RVD response in human tracheal CF epithelium and the nature of the volume-sensitive potassium channel affected. Neither the human tracheal cell line CFT1, expressing the mutant CFTR-��F508 gene, nor the isogenic vector control line CFT1-LC3, engineered to express the ��gal gene, showed RVD. On the other hand, the cell line CFT1-LCFSN, engineered to express the wild-type CFTR gene, presented a full RVD. Patch-clamp studies of swelling-activated potassium currents in the three cell lines revealed that all of them possess a potassium current with the biophysical and pharmacological fingerprints of the intermediate conductance Ca2+-dependent potassium channel (IK, also known as KCNN4). However, only CFT1-LCFSN cells showed an increase in IK currents in response to hypotonic challenges. Although the identification of the molecular mechanism relating CFTR to the hIK channel remains to be solved, these data offer new evidence on the complex integration of CFTR in the cells where it is expressed.

DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature

Several DEG/ENaC cation channel subunits are expressed in the tongue and in cutaneous sensory neurons, where they are postulated to function as receptors for salt and sour taste and for touch. Because these tissues are exposed to large temperature variations, we examined how temperature affects DEG/ENaC channel function. We found that cold temperature markedly increased the constitutively active Na+ currents generated by epithelial Na+ channels (ENaC). Half-maximal stimulation occurred at 25��C. Cold temperature did not induce current from other DEG/ENaC family members (BNC1, ASIC, and DRASIC). However, when these channels were activated by acid, cold temperature potentiated the currents by slowing the rate of desensitization. Potentiation was abolished by a ���Deg��� mutation that alters channel gating. Temperature changes in the physiologic range had prominent effects on current in cells heterologously expressing acid-gated DEG/ENaC channels, as well as in dorsal root ganglion sensory neurons. The finding that cold temperature modulates DEG/ENaC channel function may provide a molecular explanation for the widely recognized ability of temperature to modify taste sensation and mechanosensation.

A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain

Alterations in sodium channel expression and function have been suggested as a key molecular event underlying the abnormal processing of pain after peripheral nerve or tissue injury. Although the relative contribution of individual sodium channel subtypes to this process is unclear, the biophysical properties of the tetrodotoxin-resistant current, mediated, at least in part, by the sodium channel PN3 (SNS), suggests that it may play a specialized, pathophysiological role in the sustained, repetitive firing of the peripheral neuron after injury. Moreover, this hypothesis is supported by evidence demonstrating that selective ���knock-down��� of PN3 protein in the dorsal root ganglion with specific antisense oligodeoxynucleotides prevents hyperalgesia and allodynia caused by either chronic nerve or tissue injury. In contrast, knock-down of NaN/SNS2 protein, a sodium channel that may be a second possible candidate for the tetrodotoxin-resistant current, appears to have no effect on nerve injury-induced behavioral responses. These data suggest that relief from chronic inflammatory or neuropathic pain might be achieved by selective blockade or inhibition of PN3 expression. In light of the restricted distribution of PN3 to sensory neurons, such an approach might offer effective pain relief without a significant side-effect liability.