GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of G��i���subunits

GAIP (G Alpha Interacting Protein) is a member of the recently described RGS (Regulators of G-protein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein G��i3 and was recently shown to be a GTPase-activating protein (GAP). In AtT-20 cells stably expressing GAIP, we found that GAIP is membrane-anchored and faces the cytoplasm, because it was not released by sodium carbonate treatment but was digested by proteinase K. When Cos cells were transiently transfected with GAIP and metabolically labeled with [35S]methionine, two pools of GAIP���a soluble and a membrane-anchored pool���were found. Since the N terminus of GAIP contains a cysteine string motif and cysteine string proteins are heavily palmitoylated, we investigated the possibility that membrane-anchored GAIP might be palmitoylated. We found that after labeling with [3H]palmitic acid, the membrane-anchored pool but not the soluble pool was palmitoylated. In the yeast two-hybrid system, GAIP was found to interact specifically with members of the G��i subfamily, G��i1, G��i2, G��i3, G��z, and G��o, but not with members of other G�� subfamilies, G��s, G��q, and G��12/13. The C terminus of G��i3 is important for binding because a 10-aa C-terminal truncation and a point mutant of G��i3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of G��i3, which is in keeping with its GAP activity. We conclude that GAIP is a membrane-anchored GAP with a cysteine string motif. This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, suggests GAIP���s possible involvement in membrane trafficking.

A novel heat-activated current in nociceptive neurons and its sensitization by���bradykinin

Pain differs from other sensations in many respects. Primary pain-sensitive neurons respond to a wide variety of noxious stimuli, in contrast to the relatively specific responses characteristic of other sensory systems, and the response is often observed to sensitize on repeated presentation of a painful stimulus, while adaptation is typically observed in other sensory systems. In most cases the cellular mechanisms of transduction and sensitization in response to painful stimuli are not understood. We report here that application of pulses of noxious heat to a subpopulation of isolated primary sensory neurons rapidly activates an inward current. The ion channel activated by heat discriminates poorly among alkali cations. Calcium ions both carry current and partially suppress the current carried by other ions. The current is markedly increased by bradykinin, a potent algogenic nonapeptide that is known to be released in vivo by tissue damage. Phosphatase inhibitors prolong the sensitization caused by bradykinin, and a similar sensitization is caused by activators of protein kinase C. We conclude that bradykinin sensitizes the response to heat by activating protein kinase C.

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.

Preferential Zn2+ influx through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production

Synaptically released Zn2+ can enter and cause injury to postsynaptic neurons. Microfluorimetric studies using the Zn2+-sensitive probe, Newport green, examined levels of [Zn2+]i attained in cultured cortical neurons on exposure to N-methyl-d-asparte, kainate, or high K+ (to activate voltage-sensitive Ca2+ channels) in the presence of 300 ��M Zn2+. Indicating particularly high permeability through Ca2+-permeable ��-amino3-hydroxy-5-methyl-4-isoxazolepropionic-acid/kainate (Ca-A/K) channels, micromolar [Zn2+]i rises were observed only after kainate exposures and only in neurons expressing these channels [Ca-A/K(+) neurons]. Further studies using the oxidation-sensitive dye, hydroethidine, revealed Zn2+-dependent reactive oxygen species (ROS) generation that paralleled the [Zn2+]i rises, with rapid oxidation observed only in the case of Zn2+ entry through Ca-A/K channels. Indicating a mitochondrial source of this ROS generation, hydroethidine oxidation was inhibited by the mitochondrial electron transport blocker, rotenone. Additional evidence for a direct interaction between Zn2+ and mitochondria was provided by the observation that the Zn2+ entry through Ca-A/K channels triggered rapid mitochondrial depolarization, as assessed by using the potential-sensitive dye tetramethylrhodamine ethylester. Whereas Ca2+ influx through Ca-A/K channels also triggers ROS production, the [Zn2+]i rises and subsequent ROS production are of more prolonged duration.

Inositol hexakisphosphate is a physiological signal regulating the K+-inward rectifying conductance in guard cells

(RS)-2-cis, 4-trans-abscisic acid (ABA), a naturally occurring plant stress hormone, elicited rapid agonist-specific changes in myo-inositol hexakisphosphate (InsP6) measured in intact guard cells of Solanum tuberosum (n = 5); these changes were not reproduced by (RS)-2-trans, 4-trans-abscisic acid, an inactive stereoisomer of ABA (n = 4). The electrophysiological effects of InsP6 were assessed on both S. tuberosum (n = 14) and Vicia faba (n = 6) guard cell protoplasts. In both species, submicromolar concentrations of InsP6, delivered through the patch electrode, mimicked the inhibitory effects of ABA and internal calcium (Cai2+) on the inward rectifying K+ current, IK,in, in a dose-dependent manner. Steady state block of IK,in by InsP6 was reached much more quickly in Vicia (3 min at ���1 ��M) than Solanum (20���30 min). The effects of InsP6 on IK,in were specific to the myo-inositol isomer and were not elicited by other conformers of InsP6 (e.g., scyllo- or neo-). Chelation of Ca2+ by inclusion of 1,2-bis(2-aminophenoxy)ethane-N,N,N���,N���-tetraacetic acid or EGTA in the patch pipette together with InsP6 prevented the inhibition of IK,in, suggesting that the effect is Ca2+ dependent. InsP6 was ���100-fold more potent than Ins(1,4,5)P3 in modulating IK,in. Thus ABA increases InsP6 in guard cells, and InsP6 is a potent Ca2+-dependent inhibitor of IK,in. Taken together, these results suggest that InsP6 may play a major role in the physiological response of guard cells to ABA.

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.

Does the colonic H,K-ATPase also act as an Na,K-ATPase?

We previously have demonstrated that the colonic P-ATPase �� subunit cDNA encodes an H,K-ATPase when expressed in Xenopus laevis oocytes. Besides its high level of amino acid homology (75%) with the Na,K-ATPase, the colonic H,K-ATPase also shares a common pharmacological profile with Na,K-ATPase, because both are ouabain-sensitive and Sch 28080-insensitive. These features raise the possibility that an unrecognized property of the colonic H,K-ATPase would be Na+ translocation. To test this hypothesis, ion-selective microelectrodes were used to measure the intracellular Na+ activity of X. laevis oocytes expressing various combinations of P-ATPase subunits. The results show that expression in oocytes of the colonic H,K-ATPase affects intracellular Na+ homeostasis in a way similar to the expression of the Bufo marinus Na,K-ATPase; intracellular Na+ activity is lower in oocytes expressing the colonic H,K-ATPase or the B. marinus Na,K-ATPase than in oocytes expressing the gastric H,K-ATPase or a �� subunit alone. In oocytes expressing the colonic H,K-ATPase, the decrease in intracellular Na+ activity persists when diffusive Na+ influx is enhanced by functional expression of the amiloride-sensitive epithelial Na+ channel, suggesting that the decrease is related to increased active Na+ efflux. The Na+ decrease depends on the presence of K+ in the external medium and is inhibited by 2 mM ouabain, a concentration that inhibits the colonic H,K-ATPase. These data are consistent with the hypothesis that the colonic H,K-ATPase may transport Na+, acting as an (Na,H),K-ATPase. Despite its molecular and functional characterization, the physiological role of the colonic (Na,H),K-ATPase in colonic and renal ion homeostasis remains to be elucidated.

Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities

Regulation of nonspecific cation channels often underlies neuronal bursting and other prolonged changes in neuronal activity. In bag cell neurons of Aplysia, it recently has been suggested that an intracellular messenger-induced increase in the activity of a nonspecific cation channel may underlie the onset of a 30-min period of spontaneous action potentials referred to as the ���afterdischarge.��� In patch clamp studies of the channel, we show that the open probability of the channel can be increased by an average of 10.7-fold by application of ATP to the cytoplasmic side of patches. Duration histograms indicate that the increase is primarily a result of a reduction in the duration and percentage of channel closures described by the slowest time constant. The increase in open probability was not observed using 5���-adenylylimidodiphosphate, a nonhydrolyzable ATP analog, and was blocked in the presence of H7 or the more specific calcium/phospholipid-dependent protein kinase C (PKC) inhibitor peptide(19���36). Because the increase in activity observed in response to ATP occurred without application of protein kinase, our results indicate that a kinase endogenous to excised patches mediates the effect. The effect of ATP could be reversed by exogenously applied protein phosphatase 1 or by a microcystin-sensitive phosphatase also endogenous to excised patches. These results, together with work demonstrating the presence of a protein tyrosine phosphatase in these patches, suggest that the cation channel is part of a regulatory complex including at least three enzymes. This complex may act as a molecular switch to activate the cation channel and, thereby, trigger the afterdischarge.

Two-dimensional confocal images of organization, density, and gating of focal Ca2+ release sites in rat cardiac myocytes

In cardiac myocytes Ca2+ cross-signaling between Ca2+ channels and ryanodine receptors takes place by exchange of Ca2+ signals in microdomains surrounding dyadic junctions, allowing first the activation and then the inactivation of the two Ca2+-transporting proteins. To explore the details of Ca2+ signaling between the two sets of receptors we measured the two-dimensional cellular distribution of Ca2+ at 240 Hz by using a novel confocal imaging technique. Ca2+ channel-triggered Ca2+ transients could be resolved into dynamic ���Ca2+ stripes��� composed of hundreds of discrete focal Ca2+ releases, appearing as bright fluorescence spots (radius ��� 0.5 ��m) at reproducible sites, which often coincided with t-tubules as visualized with fluorescent staining of the cell membrane. Focal Ca2+ releases triggered stochastically by Ca2+ current (ICa) changed little in duration (���7 ms) and size (���100,000 Ca ions) between ���40 and +60 mV, but their frequency of activation and first latency mirrored the kinetics and voltage dependence of ICa. The resolution of 0.95 �� 0.13 reproducible focal Ca2+ release sites per ��m3 in highly Ca2+-buffered cells, where diffusion of Ca2+ is limited to 50 nm, suggests the presence of about one independent, functional Ca2+ release site per half sarcomere. The density and distribution of Ca2+ release sites suggest they correspond to dyadic junctions. The abrupt onset and termination of focal Ca2+ releases indicate that the cluster of ryanodine receptors in individual dyadic junctions may operate in a coordinated fashion.

A model for amplification of hair-bundle motion by cyclical binding of Ca2+ to mechanoelectrical-transduction channels

Amplification of auditory stimuli by hair cells augments the sensitivity of the vertebrate inner ear. Cell-body contractions of outer hair cells are thought to mediate amplification in the mammalian cochlea. In vertebrates that lack these cells, and perhaps in mammals as well, active movements of hair bundles may underlie amplification. We have evaluated a mathematical model in which amplification stems from the activity of mechanoelectrical-transduction channels. The intracellular binding of Ca2+ to channels is posited to promote their closure, which increases the tension in gating springs and exerts a negative force on the hair bundle. By enhancing bundle motion, this force partially compensates for viscous damping by cochlear fluids. Linear stability analysis of a six-state kinetic model reveals Hopf bifurcations for parameter values in the physiological range. These bifurcations signal conditions under which the system���s behavior changes from a damped oscillatory response to spontaneous limit-cycle oscillation. By varying the number of stereocilia in a bundle and the rate constant for Ca2+ binding, we calculate bifurcation frequencies spanning the observed range of auditory sensitivity for a representative receptor organ, the chicken���s cochlea. Simulations using prebifurcation parameter values demonstrate frequency-selective amplification with a striking compressive nonlinearity. Because transduction channels occur universally in hair cells, this active-channel model describes a mechanism of auditory amplification potentially applicable across species and hair-cell types.

Connexins regulate calcium signaling by controlling ATP release

Forced expression of gap junction proteins, connexins, enables gap junction-deficient cell lines to propagate intercellular calcium waves. Here, we show that ATP secretion from the poorly coupled cell lines, C6 glioma, HeLa, and U373 glioblastoma, is potentiated 5- to 15-fold by connexin expression. ATP release required purinergic receptor-activated intracellular Ca2+ mobilization and was inhibited by Cl��� channel blockers. Calcium wave propagation also was reduced by purinergic receptor antagonists and by Cl��� channel blockers but insensitive to gap junction inhibitors. These observations suggest that cell-to-cell signaling associated with connexin expression results from enhanced ATP release and not, as previously believed, from an increase in intercellular coupling.

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.

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.

Bimodal regulation of Na+���Ca2+ exchanger by ��-adrenergic signaling pathway in shark ventricular myocytes

In shark heart, the Na+���Ca2+ exchanger serves as a major pathway for both Ca2+ influx and efflux, as there is only rudimentary sarcoplasmic reticulum in these hearts. The modulation of the exchanger by a ��-adrenergic agonist in whole-cell clamped ventricular myocytes was compared with that of the Na+���Ca2+ exchanger blocker KB-R7943. Application of 5 ��M isoproterenol and 10 ��M KB-R7943 suppressed both the inward and the outward Na+���Ca2+ exchanger current (INa���Ca). The isoproterenol effect was mimicked by 10 ��M forskolin. Isoproterenol and forskolin shifted the reversal potential (Erev) of INa���Ca by approximately ���23 mV and ���30 mV, respectively. An equivalent suppression of outward INa���Ca by KB-R7943 to that by isoproterenol produced a significantly smaller shift in Erev of about ���4 mV. The ratio of inward to outward exchanger currents was also significantly larger in isoproterenol- than in control- and KB-R7943-treated myocytes. Our data suggest that the larger ratio of inward to outward exchanger currents as well as the larger shift in Erev with isoproterenol results from the enhanced efficacy of Ca2+ efflux via the exchanger. The protein kinase A-mediated bimodal regulation of the exchanger in parallel with phosphorylation of the Ca2+ channel and enhancement of its current may have evolved to satisfy the evolutionary needs for accelerated contraction and relaxation in hearts of animals with vestigial sarcoplasmic Ca2+ release stores.

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.