Deprivation Of Extracellular Ca2 Research Articles

Ca2+ -dependent down-regulation of human histamine H1 receptors in Chinese hamster ovary cells.

Gq/11 protein-coupled human histamine H1 receptors in Chinese hamster ovary cells stimulated with histamine undergo clathrin-dependent endocytosis followed by proteasome/lysosome-mediated down-regulation. In this study, we evaluated the effects of a sustained increase in intracellular Ca2+ concentrations induced by a receptor-bypassed stimulation with ionomycin, a Ca2+ ionophore, on the endocytosis and down-regulation of H1 receptors in Chinese hamster ovary cells. All cellular and cell-surface H1 receptors were detected by the binding of [3 H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H1 receptor ligands, mepyramine and pirdonium, respectively. The pretreatment of cells with ionomycin markedly reduced the mepyramine- and pirdonium-sensitive binding sites of [3 H]mepyramine, which were completely abrogated by the deprivation of extracellular Ca2+ and partially by a ubiquitin-activating enzyme inhibitor (UBEI-41), but were not affected by inhibitors of calmodulin (W-7 or calmidazolium) and protein kinase C (chelerythrine or GF109203X). These ionomycin-induced changes were also not affected by inhibitors of receptor endocytosis via clathrin (hypertonic sucrose) and caveolae/lipid rafts (filipin or nystatin) or by inhibitors of lysosomes (E-64, leupeptin, chloroquine, or NH4 Cl), proteasomes (lactacystin or MG-132), and a Ca2+ -dependent non-lysosomal cysteine protease (calpain) (MDL28170). Since H1 receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H1 receptors, even after the ionomycin treatment, H1 receptors appeared to exist in a form to which [3 H]mepyramine was unable to bind. These results suggest that H1 receptors are apparently down-regulated by a sustained increase in intracellular Ca2+ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.

Dual effects of nobiletin, a citrus polymethoxy flavone, on catecholamine secretion in cultured bovine adrenal medullary cells

Nobiletin, a compound of polymethoxy flavones found in citrus fruits, possesses a wide range of pharmacological activities. Here we report the effects of nobiletin on catecholamine secretion in cultured bovine adrenal medullary cells. Nobiletin (1.0-100 microM) concentration-dependently stimulated catecholamine secretion and (45)Ca(2+) influx. Its stimulatory effect of nobiletin on catecholamine secretion was abolished by deprivation of extracellular Ca(2+) and partially inhibited by specific inhibitors of voltage-dependent Ca(2+) channels and Na(+)/Ca(2+) exchangers. On the other hand, nobiletin suppressed catecholamine secretion and (22)Na(+) and (45)Ca(2+) influx induced by acetylcholine, an agonist of nicotinic acetylcholine receptors, in a concentration-dependent manner. It also inhibited catecholamine secretion, (22)Na(+) influx and/or (45)Ca(2+) influx induced by veratridine, an activator of voltage-dependent Na(+) channels, and 56 mM K(+), an activator of voltage-dependent Ca(2+) channels. In Xenopus oocytes expressing alpha3beta4 neuronal acetylcholine receptors, nobiletin directly inhibited the current evoked by acetylcholine in a concentration-dependent manner similar to that observed in catecholamine secretion. The present findings suggest that nobiletin, by itself, stimulates catecholamine secretion via activation of voltage-dependent Ca(2+) channels or Na(+)/Ca(2+) exchangers, whereas it inhibits catecholamine secretion induced by acetylcholine through the suppression of Na(+) influx and Ca(2+) influx in cultured bovine adrenal medullary cells.

Regulation of Cl- conductance in delayed shortening and shrinkage of outer hair cells.

Tetanic electrical field stimulation induces slow and delayed shortening of isolated outer hair cells (OHCs) from guinea pigs concomitant with a decrease in cell volume and intracellular Cl- concentration ([Cl-]i). These responses are dependent on the entry of Ca2+ and the activation of both Ca(2+)-activated K+ channels and furosemide-sensitive Cl- channels. In this study, the regulatory mechanisms of the Cl- channels of OHCs were investigated. Tetanic stimulation induced shrinkage and a decrease in [Cl-]i, the latter being unrelated to the activity of the Na+/K+/2Cl- cotransporter. These responses were inhibited by tetraethylammonium, and the inhibition was overcome by an increase in the K+ conductance by valinomycin, though valinomycin alone did not affect the resting cell volume and [Cl-]i. The deprivation of extracellular Ca2+ inhibited the stimulation-induced changes in volume and [Cl-]i even in the presence of valinomycin. These results suggest that electrical stimulation-induced delayed shortening and shrinkage of OHCs are induced by Cl- efflux, which is electrically coupled to K+ efflux and independently activated following an increase in intracellular Ca2+ concentration.

Acid secretagogues induce Ca 2+ mobilization coupled to K + conductance activation in rat parietal cells in tissue culture

Intracellular recordings from cultured parietal cells of the rat gastric fundus showed that carbachol, pentagastrin, histamine (in the presence of isobutylmethylxanthine; IBMX) and dibutyryl cyclic AMP induced hyperpolarizing responses which were sensitive to a K+ channel blocker, quinine. The Ca2+ ionophore, ionomycin, also induced a quinine-sensitive hyperpolarization. Deprivation of extracellular Ca2+ preferentially inhibited the hyperpolarizing responses to histamine (plus IBMX) and to dibutyryl cyclic AMP. Caffeine, oxalate and dantrolene sodium, which are known to affect Ca2+ transport in the endoplasmic reticulum, selectively inhibited the carbachol response. Mitochondrial inhibitors (KCN and carbonylcyanide p-trifluoromethoxyphenylhydrazone) preferentially suppressed the gastrin response. Cytosolic Ca2+ measurements with fura-2 indicated that significant increases in the intracellular concentration of free Ca2+ were induced not only by Ca2+-mediated acid secretagogues (carbachol and gastrin), but also by a cyclic AMP-mediated secretagogue (histamine plus IBMX). Dibutyryl cyclic AMP also increased cytosolic Ca2+ ions. It is concluded that stimulation of receptors to histamine, carbachol and gastrin gives rise to mobilization of Ca2+ ions into the cytoplasm from the different sources, thereby stimulating Ca2+-activated K+ channels in cultured rat parietal cells.

C1q induces chemotaxis and K+ conductance activation coupled to increased cytosolic Ca2+ in mouse fibroblasts.

Cultured mouse fibroblasts (L cells) respond to whole C with a slow hyperpolarization. Among the C components tested, C1q was found to be most effective. In contrast, the cell did not respond to C1, in which the collagen-like region of the C1q molecule is masked. The C1q-induced hyperpolarizing response was inhibited by collagen or C1q-specific antisera. Human diploid skin fibroblasts (Flow 1,000 cells) also exhibited similar membrane potential changes in response to whole C or C1q. After repeated applications of C1q, the cell membrane became unresponsive (desensitized). The treatment of L cells with pronase E inhibited the C1q-induced response, whereas the response to ATP, which is known to interact to its own receptor, was still preserved. The reversal potential of C responses was close to the K+ equilibrium potential. The hyperpolarizing response was inhibited by a blocker of Ca2+-activated K+ channels in fibroblasts (quinine), by deprivation of extracellular Ca2+ or by a Ca2+ channel blocker (nifedipine). By means of Ca2+-selective microelectrodes, the cytosolic free Ca2+ concentration was found to increase from 126 to 206 nM upon stimulation of L cells with C1q. Using an agarose-well method, L cells were observed to migrate predominantly toward C1q or whole C. It is concluded that the fibroblasts have the C1q receptor sensitive to pronase E and that activation of C1q receptors gives rise to Ca2+ influx, triggering an increase in the cytosolic free Ca2+ ions, which in turn induces a hyperpolarizing response as a result of the stimulation of Ca2+-activated K+ channels and initiates chemotaxis to C1q.

Factors responsible for oscillations of membrane potential recorded with tight-seal-patch electrodes in mouse fibroblasts.

In giant fibroblastic L cells, penetration of a conventional microelectrode brought about marked decreases in the membrane potential and input resistance measured with a patch electrode under tight-seal whole-cell configuration, and repeated hyperpolarizations were often observed upon penetration. Therefore, the question arose whether such leakage artifact is a causal factor for generation of the membrane potential oscillation even in giant L cells. During whole-cell recordings, however, regular potential oscillations were observed in the cells that had not been impaled with a conventional microelectrode, as far as the Ca2+ buffer was not strong in the pipette solution. Oscillatory changes in the intracellular potential were detected by extracellular recordings with a tight-seal patch electrode in the cell-attached configuration. Thus, the potential oscillation occurs even in the absence of penetration-induced leakage or without rupture of the patch membrane. Withdrawal of a micropipette from one cell was often found to induce marked cell damage and elicit oscillatory hyperpolarizations in a neighboring cell with a certain time lag. The longer the distance between the injured and recorded cells, the greater was the time lag. Application of the cell lysate on the cell surface also gave rise to oscillatory hyperpolarizations. After repeated applications of the lysate, the membrane became unresponsive (desensitized), suggesting the involvement of receptors for the lysate factor. The lysates of different cell species (mouse lymphoma L5178Y cells or human epithelial Intestine 407 cells) produced similar effects. The effective component was heat stable and distinct from ATP. Lysate-induced hyperpolarizations were inhibited by deprivation of extracellular Ca2+ and by application of a Ca2+ channel blocker (nifedipine) or a K+ channel blocker (quinine) in the same manner as spontaneous oscillatory hyperpolarizations. It is concluded that the mouse fibroblast exhibits membrane potential oscillations, when the cell was activated, presumably via receptor systems, by some diffusible factors released from damaged cells.

Ca2+ sensitivity of volume‐regulatory K+ and Cl‐ channels in cultured human epithelial cells.

1. During exposure to a hypotonic solution, cultured human epithelial cells (Intestine 407) exhibited a regulatory volume decrease (RVD) after initial osmotic swelling. 2. The volume readjustment was slowed by elevating the extracellular K+ concentration and facilitated by reducing the extracellular Cl- concentration. Not only putative K+ channel blockers, quinine and Ba2+, but also a stilbene derivative Cl- channel blocker (SITS) inhibited the RVD. 3. The volume recovery of hypoosmotically swollen cells was very much suppressed by the deprivation of extracellular Ca2+ ions or by chelation of cytosolic Ca2+ ions with Quin-2 loaded within the cells. 4. Biphasic membrane potential changes were associated with the RVD process at low extracellular K+ and Cl- concentrations. The initial hyperpolarizing response was inhibited by quinine and Ba2+, whereas the late depolarizing response was inhibited by SITS. The deprivation of extracellular Ca2+ inhibited the initial hyperpolarizing phase but not the late depolarizing phase. 5. Two-microelectrode voltage clamp studies showed that the initial hyperpolarization and late depolarization were associated with quinine-sensitive outward currents and SITS-sensitive inward currents, respectively. The reversal potentials estimated from the current-voltage curves were about -80 mV for the initial response and -27 mV for the late response. Tenfold changes in the K+ and Cl- concentrations shifted these reversal potentials by 50 mV for the initial response and by 42 mV for the late response. 6. Under whole-cell recordings, similar current changes were observed in the cells exposed to a hypotonic solution, when the intracellular Ca2+ ions were moderately buffered with 1 mM-EGTA in the dialysing solution filled in a patch pipette. When most Ca2+ ions were chelated with 10 mM-EGTA in the pipette solution, the initial outward current as well as the corresponding hyperpolarization was suppressed, but the late current associated with the depolarizing phase was preserved. 7. Intracellular Ca2+ injections induced an increase in the quinine-sensitive K+ conductance but failed to activate the Cl- conductance. 8. It is concluded that both K+ and Cl- channels are involved in the regulatory volume decrease, and that the former channel is exclusively activated by elevation of the cytosolic Ca2+ concentration in the epithelial cells.

Atrial natriuretic factor and cyclic guanosine 3',5'-monophosphate in vascular smooth muscle.

To elucidate the molecular mechanism of the vascular action of atrial natriuretic factor (ANF), we investigated the effects of synthetic ANF and sodium nitroprusside on the levels of intracellular cyclic nucleotides and prostacyclin (measured as its stable metabolite 6-keto-prostaglandin F1 alpha) in cultured vascular smooth muscle cells from rat mesenteric artery and, in some experiments, from rat renal artery. Both ANF and sodium nitroprusside increased intracellular cyclic guanosine 3',5'-monophosphate (cGMP) levels in a dose-dependent manner but did not affect cyclic adenosine 3',5'-monophosphate levels or 6-keto-prostaglandin F1 alpha synthesis. The stimulatory effect of ANF and sodium nitroprusside on cGMP levels were additive. Neither the deprivation of extracellular Ca2+ nor calcium entry blockers affected ANF-stimulated cGMP levels. Preincubation of ANF or sodium nitroprusside with kallikrein attenuated only the effect of ANF on cGMP levels. The effect of kallikrein was abolished by serine protease inhibitors. In contrast, the oxidant methylene blue inhibited the effect of sodium nitroprusside on cGMP levels, but not that of ANF. The stimulatory effect of ANF on cGMP levels was greater in cells from renal artery than in those from mesenteric artery. These results in cultured vascular smooth muscle cells further support the hypothesis that cGMP mediates the vasorelaxant action of ANF.