Elimination Of Extracellular Ca2 Research Articles

ZnT1 is a neuronal Zn2+/Ca2+ exchanger

Zinc transporter 1 (ZnT1; SLC30A1) is present in the neuronal plasma membrane, critically modulating NMDA receptor function and Zn2+ neurotoxicity. The mechanism mediating Zn2+ transport by ZnT1, however, has remained elusive. Here, we investigated ZnT1-dependent Zn2+ transport by measuring intracellular changes of this ion using the fluorescent indicator FluoZin-3. In primary mouse cortical neurons, which express ZnT1, transient addition of extracellular Zn2+ triggered a rise in cytosolic Zn2+, followed by its removal. Knockdown of ZnT1 by adeno associated viral (AAV)-short hairpin RNA (shZnT1) markedly increased rates of Zn2+ rise, and decreased rates of its removal, suggesting that ZnT1 is a primary route for Zn2+ efflux in neurons. Although Zn2+ transport by other members of the SLC30A family is dependent on pH gradients across cellular membranes, altered H+ gradients were not coupled to ZnT1-dependent transport. Removal of cytoplasmic Zn2+, against a large inward gradient during the initial loading phase, suggests that Zn2+ efflux requires a large driving force. We therefore asked if Ca2+ gradients across the membrane can facilitate Zn2+ efflux. Elimination of extracellular Ca2+ abolished Zn2+ efflux, while increased extracellular Ca2+ levels enhanced Zn2+ efflux. Intracellular Ca2+ rises, measured in GCaMP6 expressing neurons, closely paralleled cytoplasmic Zn2+ removal. Taken together, these results strongly suggest that ZnT1 functions as a Zn2+/Ca2+ exchanger, thereby regulating the transport of two ions of fundamental importance in neuronal signaling.

Activation of alpha-1 adrenergic receptors increases cytosolic calcium in neurones of the paraventricular nucleus of the hypothalamus.

Norepinephrine (NE) activates adrenergic receptors (ARs) in the hypothalamic paraventricular nucleus (PVN) to increase excitatory currents, depolarise neurones and, ultimately, augment neuro-sympathetic and endocrine output. Such cellular events are known to potentiate intracellular calcium ([Ca2+ ]i ); however, the role of NE with respect to modulating [Ca2+ ]i in PVN neurones and the mechanisms by which this may occur remain unclear. We evaluated the effects of NE on [Ca2+ ]i of acutely isolated PVN neurones using Fura-2 imaging. NE induced a slow increase in [Ca2+ ]i compared to artificial cerebrospinal fluid vehicle. NE-induced Ca2+ elevations were mimicked by the α1 -AR agonist phenylephrine (PE) but not by α2 -AR agonist clonidine (CLON). NE and PE but not CLON also increased the overall number of neurones that increase [Ca2+ ]i (ie, responders). Elimination of extracellular Ca2+ or intracellular endoplasmic reticulum Ca2+ stores abolished the increase in [Ca2+ ]i and reduced responders. Blockade of voltage-dependent Ca2+ channels abolished the α1 -AR induced increase in [Ca2+ ]i and number of responders, as did inhibition of phospholipase C inhibitor, protein kinase C and inositol triphosphate receptors. Spontaneous phasic Ca2+ events, however, were not altered by NE, PE or CLON. Repeated K+ -induced membrane depolarisation produced repetitive [Ca2+ ]i elevations. NE and PE increased baseline Ca2+ , whereas NE decreased the peak amplitude. CLON also decreased peak amplitude but did not affect baseline [Ca2+ ]i . Taken together, these data suggest receptor-specific influence of α1 and α2 receptors on the various modes of calcium entry in PVN neurones. They further suggest Ca2+ increase via α1 -ARs is co-dependent on extracellular Ca2+ influx and intracellular Ca2+ release, possibly via a phospholipase C inhibitor-mediated signalling cascade.

TRPM7-mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562.

Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real‐time RT‐PCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+]i) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2‐APB, Gd3+, and FTY720 dose dependently decreased basal [Ca2+]i. A spontaneously active inward current (I spont) contributive to basal [Ca2+]i was identified by the nystatin‐perforated whole‐cell recording. I spont permeated Ca2+ comparably to Na+, and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA‐mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin‐induced γ‐globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.

Hypoxia-induced contraction of chicken embryo mesenteric arteries: mechanisms and developmental changes.

The fetal cardiovascular responses to acute hypoxia include a redistribution of the cardiac output toward the heart and the brain at the expense of other organs, such as the intestine. We hypothesized that hypoxia exerts a direct effect on the mesenteric artery (MA) that may contribute to this response. Using wire myography, we investigated the response to hypoxia (Po2 ~2.5 kPa for 20 min) of isolated MAs from 15- to 21-day chicken embryos (E15, E19, E21), and 1- to 45-day-old chickens (P1, P3, P14, P45). Agonist-induced pretone or an intact endothelium were not required to obtain a consistent and reproducible response to hypoxia, which showed a pattern of initial rapid phasic contraction followed by a sustained tonic contraction. Phasic contraction was reduced by elimination of extracellular Ca2+ or by presence of the neurotoxin tetrodotoxin, the α1-adrenoceptor antagonist prazosin, or inhibitors of L-type voltage-gated Ca2+ channels (nifedipine), mitochondrial electron transport chain (rotenone and antimycin A), and NADPH oxidase (VAS2870). The Rho-kinase inhibitor Y27632 impaired both phasic and tonic contraction and, when combined with elimination of extracellular Ca2+, hypoxia-induced contraction was virtually abolished. Hypoxic MA contraction was absent at E15 but present from E19 and increased toward the first days posthatching. It then decreased during the first weeks of life and P45 MAs were unable to sustain hypoxia-induced contraction over time. In conclusion, the results of the present study demonstrate that hypoxic vasoconstriction is an intrinsic feature of chicken MA vascular smooth muscle cells during late embryogenesis and the perinatal period.

Nicotine-Upregulation of Alpha 7 nAChR in Xenopus Oocytes

The alpha 7 (α7) subtype of nAChR appears to play critical roles in learning, cognition, and various neuropathologies including nicotine addiction. Nicotine-upregulation of α7 Rs may play a significant role in the latter phenomenon. But whether nicotine-upregulation of α7 Rs reliably occurs, and its underlying mechanism(s), are largely unknown. Previous studies of α7 nAChRs heterologously expressed in Xenopus oocytes failed to observe nicotine-upregulation. These failures might have been due to intracellular accumulation of nicotine and its subsequent slow release from oocytes, resulting in desensitization of α7 Rs during functional assays. In our experiments, 12-14 hr exposure to nicotine (100 μM) 4-5 days post cRNA-injection (PI), followed by extensive washout, yielded statistically-significant ∼2-fold increases in peak α7 currents and net charge as determined by TEVC and α7-protein (by Western blot). Less-extensive washout, as well as 100 nM nicotine incubation, failed to produce upregulation. Instead, inactivated/desensitized currents were observed. GC/MS measurement of nicotine in the washout fluid confirmed that nicotine was continually released from oocytes. The concentration was ∼20 nM at 3-4 hr following washout onset, well in excess of the ∼ 3 nM IC50 value. Exposure of α7-expressing oocytes to cumulative washout fluid produced suppressed α7 currents (∼ 8% of controls). Similar to nicotine, methyllycaconitine, a cell-permeable competitive antagonist of α7 Rs, and carbachol, a stable membrane-impermeable agonist, also produced ∼ 2X-upregulation. However, ACh incubation (which can be hydrolyzed by the oocytes) failed to produce upregulation. We also found that chelation of intracellular Ca2+ by BAPTA-AM completely blocked nicotine upregulation. However, elimination of extracellular Ca2+ had no effect. These results suggest that persistent ligand-binding to α7 Rs (but not necessarily channel gating), leading to increases in [Ca2+]i, was critical for α7 R-upregulation. Several Ca2+-dependent signaling pathways were implicated in nicotine-upregulation of α7.

Chloride Conductance Is Required for the Protein Kinase A and Rac1-dependent Phosphorylation of Moesin at Thr-558 by KCl in PC12 Cells

Moesin is a member of the ERM family, a family of cross-linkers between the plasma membrane and the actin cytoskeleton, which are known to be activated by phosphorylation. Previously, we reported the RhoA and Rho kinase-dependent phosphorylation of moesin at Thr-558 in hippocampal neuronal cells by glutamate. Here we studied the induction of moesin phosphorylation by KCl (60 mm) in PC12 cells. Moesin phosphorylation at Thr-558 was increased after 2 min of KCl treatment, peaked at 5 min, and returned to the basal level by 60 min. KCl also activated Rac1, but not RhoA, in PC12 cells, and KCl-induced moesin phosphorylation was suppressed in dominant negative Rac1 (N17 Rac1)-expressed cells. The inhibition of protein kinase A (PKA), known as an upstream kinase of Rac1, abolished Rac1 activation and moesin phosphorylation by KCl. Interestingly, the phosphorylation of moesin by KCl was independent of KCl-induced membrane depolarization and calcium influx but was dependent on KCl-induced chloride conductance. 60 mm KCl induced chloride conductance in PC12 cells, and pretreatment with Cl- channel blocker abolished Rac1 activation and moesin phosphorylation by KCl. These results suggest that the phosphorylation of moesin at Thr-558 in PC12 cells by KCl treatment is PKA- and Rac1-dependent and that KCl-induced chloride conductance is involved in the activation of this signaling system.

Acidic ATP activates lymphocyte outwardly rectifying chloride channels via a novel pathway.

Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl- for gluconate(-) shifted the reversal potential, while Cl- channel blockers, 4,4'-diisothiocyanostibene-2,2'-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPgammaS (a poorly hydrolyzable analog of ATP), 2',3'-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 microM suramin (a P2 receptor antagonist), and was partially blocked by 100 microM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca2+ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca(2+)-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors.

Regulation of c-Jun N-terminal kinase activation in hydrogen peroxide induced neurotoxicity

C-Jun N-terminal kinase 1 and 2 (JNK1/2) have been shown to be transiently activated and involved in neurotoxicity. We searched for possible upstream molecules, which are responsible for the regulation of hydrogen peroxide-(H2O2) induced JNK1/2 activation and JNK1/2-mediated apoptotic-like cell death in cultured rat cortical neurons. The results showed that JNK1/2 activation (monitored by anti-diphosphorylated JNK1/2 antibody) was largely prevented by elimination of extracellular Ca2+ or blockage of NMDA-receptors (NMDA-R), and was weakly but significantly decreased by blockage of L-type voltage-gated calcium channel (L-VGCC); furthermore, JNK1/2 activation was largely prevented by inhibition of Ca2+/calmodulin-dependent protein kinase-II (CaMKII) and protein-tyrosine kinases (PTK). We also found that H2O2-induced apoptotic-like cell death was partially prevented by elimination of extracellular Ca2+, or by inhibition of NMDA-R, L-VGCC, PTK and CaMKII, respectively. The above results suggest that in H2O2-induced neurotoxicity, JNK1/2 activation is mainly mediated by NMDA-R and L-VGCC. Consequently, PTK and CaMKII are critical intermediaries in JNK1/2 activation and are mainly responsible for JNK1/2-mediated apoptotic-like cell death.

Characterization of alphaT3-1 cells stably transfected with luteininzing hormone beta-subunit complementary deoxyribonucleic acid.

Luteinizing hormone (LH) consists of alpha- and beta-subunits, and synthesis and secretion of LH are regulated by gonadotropin-releasing hormone (GnRH). In order to examine the molecular mechanisms by which GnRH regulates LH secretion, we transfected alphaT3-1 cells with rat LHbeta-subunit cDNA under the control of a constitutive promoter and established a stable cell line of LH2 cells which secreted LH in response to GnRH. Pulsatile and continuous GnRH pretreatments increased gene expression of the alpha-subunit and synthesis of LH, and enhanced the LH secretion by brief treatments with GnRH and 56 mM KCl. The LH secretions were partially blocked by elimination of extracellular Ca2+. GnRH-induced LH secretion was completely inhibited by calphostin C (a protein kinase C inhibitor) and 1 microM wortmannin. In contrast to the GnRH induction, high K+-induced LH secretion was inhibited by KN93, a Ca2+/calmodulin-dependent protein kinase II inhibitor, as well as by 1 microM wortmannin. We also confirmed that activation of cAMP-pathway induced LH secretion, but activation of mitogen-activated protein (MAP) kinase pathway was not involved in LH secretion. These results suggest that GnRH directly regulates LH secretion as well as LHbeta-subunit synthesis, and that LH2 cells are a useful model for the study of LH secretion induced by several secretagogues.

Effect of APGW-Amide on [Ca2+]i in Rat Pheochromocytoma PC12 Cells

In order to determine whether Ala-Pro-Gly-Try-NH2 (APGW-amide) could affect mammalian excitable cells, we investigated the effect of APGW-amide in PC12 cells. APGW-amide caused a rapid [Ca2+]i elevation, which was completely prevented by elimination of extracellular Ca2+ with EGTA and inhibited by two L-type Ca2+ channel blockers. [Ca2+]i elevation was also blocked by a specific PKC inhibitor and prolonged pretreatment of cells with PMA. These results indicate that APGW-amide elevates [Ca2+]i in PC12 cells, possibly by Ca2+ influx via L-type Ca2+ channel activated by PKC.

Characterization of distinct heat shock- and thapsigargin-induced cytoprotective proteins in FRTL-5 thyroid cells.

Heat shock induces the expression of proteins with molecular weights of 70-72 kd and 90 kd, whereas thapsigargin induces the expression of a glucose-regulated protein 78 kd (GRP-78) in certain cells. In this study we examined the induction and cytoprotective effects of heat shock- and thapsigargin-induced proteins in FRTL-5 rat thyroid cells. New protein synthesis was assessed in [35S]methionine-labeled cells and quantitated densitometrically. The expression of specific stress proteins was identified using Western blots, whereas cytoprotection provided by these proteins was evaluated by trypan blue exclusion. Exposure to heat shock (45 degrees C, 15 minutes) induced the expression of proteins with molecular weights at the range of low 70 kD and low 90 kD that peaked between 2-6 hours and returned to baseline within 24 hours. Treatment of cells with thapsigargin (200 nM, 15 minutes) induced the expression of different molecular weight proteins, most likely GRP-78 and -94, that peaked at 4-6 hours and lasted for 24 hours. Neither the removal of growth factors (thyroid-stimulating hormone and insulin) for 5 days nor the elimination of extracellular Ca2+ with EGTA or clamping of the intracellular Ca2+ with BAPTA for 15 minutes affected expression of the heat shock- and the thapsigargin-induced stress proteins. In contrast, protein kinase C inhibitors H7 and GF109203X abolished the expression of all three groups of stress proteins. Both heat shock- and thapsigargin-inuced proteins completely protected cells from subsequent thermal injury (47 degrees C, 35 minutes). The induction of cytoprotective proteins by heat shock and thapsigargin requires the presence of protein kinase C but is Ca(2+)- and growth factor-independent.

A Requirement for K+-Channel Activity in Growth Factor–Mediated Extracellular Signal-Regulated Kinase Activation in Human Myeloblastic Leukemia ML-1 Cells

Voltage-gated K+ channels have been shown to be required for proliferation of various types of cells. Much evidence indicates that K+-channel activity is required for G1 progression of the cell cycle in different cell backgrounds, suggesting that K+-channel activity is required for early-stage cell proliferation in these cells. However, little is known about the molecular mechanisms that underlie this phenomenon. We have shown in human myeloblastic leukemia ML-1 cells that K+ channels are activated by epidermal growth factor (EGF), whereas serum starvation deprivation suppressed their activity. In addition, voltage-gated K+ channels are required for G1/S-phase transition of the cell cycle. We report here that suppression of K+ channels prevented the activation of extracellular signal-regulated protein kinase 2 (ERK-2) in response to EGF and serum. However, blockade of K+ channels did not prevent ERK-2 activation induced by 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Elimination of extracellular Ca2+ did not alter either ERK-2 activation or the effect of K+-channel blockade on ERK-2 activation. Our data demonstrate that the K+ channel is a part of the EGF-mediated mitogenic signal-transduction process and is required for initiation of the EGF-mediated mitogen-activated protein kinase (MAPK) pathways. Our findings may thus explain why an increase in K+-channel activity is associated with cell proliferation in many types of cells, including ML-1 cells.

An Ultraviolet-activated K+ Channel Mediates Apoptosis Of Myeloblastic Leukemia Cells

Exposure of mammalian cells to UV light causes initial changes in the cell membrane, induces phosphorylation and clustering of growth factor/cytokine receptors, and activates the Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathway leading to programmed cell death (apoptosis). In this study, we found that an early event in the cell membrane of myeloblastic leukemia (ML-1) cells was the vigorous activation of the voltage-gated K+ channel by UV irradiation. The strong enhancement by UV irradiation of K+ channel activity in the cell membrane subsequently activated the JNK/SAPK signaling pathway and resulted in myeloblastic leukemia cell apoptosis. Suppression of UV-induced K+ channel activation with specific channel blockers prevented UV-induced apoptosis through inhibition of UV-induced activation of the proteins SEK (SPAK kinase) and JNK. However, suppression of K+ channel activity could not protect cells from etoposide-induced apoptosis, which bypasses the membrane event. Elimination of extracellular Ca2+ had no effect on the UV-induced and K+ channel-mediated JNK/SAPK activation. Thus, we have identified a novel mechanism in which activation of K+ channels by UV-irradiation upstream of SEK and SAPK/JNK mediates UV-induced myeloblastic cell apoptosis.

Constitutively Active Gα12, Gα13, and Gαq Induce Rho-dependent Neurite Retraction through Different Signaling Pathways

In neuronal cells, activation of a certain heterotrimeric G protein-coupled receptor causes neurite retraction and cell rounding via the small GTPase Rho. However, the specific heterotrimeric G proteins that mediate Rho-dependent neurite retraction and cell rounding have not yet been identified. Here we investigated the effects of expression of constitutively active Galpha subunits on the morphology of differentiated PC12 cells. Expression of GTPase-deficient Galpha12, Galpha13, and Galphaq, but not Galphai2, caused neurite retraction and cell rounding in differentiated PC12 cells. These morphological changes induced by Galpha12, Galpha13, and Galphaq were completely inhibited by C3 exoenzyme, which specifically ADP-ribosylates and inactivates Rho. The tyrosine kinase inhibitor tyrphostin A25 blocked the neurite retraction and cell rounding induced by Galpha13 and Galphaq. However, tyrphostin A25 failed to inhibit the Galpha12-induced neuronal morphological changes. On the other hand, inhibition of protein kinase C or elimination of extracellular Ca2+ blocked the neurite retraction and cell rounding induced by Galphaq, whereas the morphological effects of Galpha12 and Galpha13 did not require activation of protein kinase C and extracellular Ca2+. These results demonstrate that activation of Galpha12, Galpha13, and Galphaq induces Rho-dependent morphological changes in PC12 cells through different signaling pathways.

Calcium oscillations in single cultured Chinese hamster ovary cells stably transfected with a cloned human cholecystokinin (CCK)B receptor.

In Chinese hamster ovary cells stably expressing the cloned human cholecystokinin (CCK)B/ gastrin receptor, cholecystokinin octapeptide (CCK-8) evoked increases in [Ca2+]i monitored by digitized video imaging of fura-2 fluorescence ratios. At concentrations around 10 pM, CCK-8 elicited [Ca2+]i oscillations, which were blocked by elimination of extracellular Ca2+, by a phospholipase C inhibitor, U-73122, by a protein kinase C inhibitor, H7, as well as by phospholipase A2 (PLA2) inhibitors, ONO-RS-082 and aristolochic acid. At higher concentrations, CCK-8 induced a single biphasic [Ca2+]i rise consisting of a large peak followed by a lower sustained plateau, while the response turned into [Ca2+]i oscillation when the extracellular Ca2+ was eliminated or a PLA2 inhibitor was included. CCK-8 stimulated the release of arachidonic acid, and this was inhibited by aristolochic acid. Arachidonic acid caused an increase in [Ca2+]i which was dependent upon extracellular Ca2+. These results suggest that the activation of PLA2 might be involved, at least in part, in the Ca2+ influx that maintains the sustained plateau phase of [Ca2+]i as well as the [Ca2+]i oscillation when CCKB receptors are stimulated.

Characterization of voltage-sensitive Na+ and K+ currents recorded from acutely dissociated pelvic ganglion neurons of the adult rat.

1. Electrophysiological properties of acutely dissociated neurons from the major pelvic ganglion (MPG) of the adult male rat were studied with whole cell patch-clamp recording techniques. The MPG neurons innervating the urinary bladder were labeled by retrograde axonal tracing methods with the use of a fluorescent dye, Fast Blue (FB) injected into the bladder wall and identified with a fluorescent microscope. 2. Passive and active membrane properties such as resting membrane potential, input resistance, duration of action potentials, thresholds for spike activation, or duration of afterhyperpolarization in unidentified MPG neurons were comparable with those of FB-labeled neurons innervating the urinary bladder. The action potential in both unidentified and bladder efferent MPG neurons was reversibly abolished by tetrodotoxin (TTX, 1 microM). The afterhyperpolarization of the TTX-sensitive action potential in both groups was reduced by application of Cd2+ (0.1 mM) and further suppressed by tetraethylammonium (TEA, 10 mM). Extracellularly applied TEA increased the duration of the action potential, and 4-aminopyridine (4-AP, 1 or 2 mM) also reduced the spike afterhyperpolarization and increased the spike duration. The duration of the action potential was decreased and the rate of spike repolarization was increased by approximately 2.5-fold with negative shift of membrane potential from -40 to -80 mV. 3. The isolated Na+ current was reversibly blocked by 1 microM TTX and had a mean peak amplitude of 127.3 pA/pF when activated from a holding potential of -70 mV in the external solution containing 100 mM Na+. The Na+ conductance reached half-maximal activation at a membrane potential of -21.5 mV with a slope factor of 4.9 mV. The steady-state inactivation of Na+ conductance occurred at membrane potentials more depolarized than -90 mV, and the half-maximal inactivation was obtained at -57.5 mV with a slope factor of 8.8 mV. 4. The fast-transient A-type K+ current (IA) was activated at membrane potentials more depolarized than -60 mV from a holding membrane potential of -100 mV, reached a peak amplitude within 10 ms after the onset of depolarizing voltage steps, and decayed within 20-30 ms at membrane potential depolarizations to +20 to +30 mV. The IA current activated by a voltage step to +20 mV from a holding potential of -100 mV averaged 102.1 pA/pF. The half-maximal activation of the IA conductance was obtained at a membrane potential of -21.2 mV with a slope factor of 9.9 mV. In steady-state inactivation of IA current, the half-maximal inactivation occurred at -76.5 mV and the slope factor was 8.0 mV. 5. The delayed K+ current was reduced by 25-35% by bath application of Cd2+ or the elimination of extracellular Ca2+ ions. The bath application of 4-AP (2 mM) suppressed the IA current by 75% and the delayed K+ current by 60%. Extracellularly applied TEA (10 mM) suppressed the delayed K+ current by 90%, but suppressed the IA current by only 16%. 6. These results indicate that bladder neurons and unidentified neurons in the MPG have similar properties including a TTX-sensitive Na+ current and three distinct types of voltage-sensitive K+ currents-IA current, Ca(2+)-activating K+ current, and delayed rectifier K+ current-that contribute to the repolarization phase of the action potential. These electrical properties of the MPG neurons resemble those of sympathetic neurons in the superior cervical and inferior mesenteric ganglia.

Capsaicin-induced currents with distinct desensitization and Ca2+ dependence in rat trigeminal ganglion cells.

1. Whole cell patch-clamp records from cultured rat trigeminal ganglion cells having soma diameters ranging from 20 to 50 microM revealed that capsaicin activated two inward currents and an outward current. At -60 mV, the inward currents could be distinguished by their different peak times, which were 4.2 +/- 3.1 and 41.4 +/- 16.4 (SD) s. 2. Cells with the smallest soma diameters had the largest current densities. 3. The more rapidly activating current had a linear current-voltage relation and a reversal potential near 0 mV. 4. The more slowly activating current is not a Ca(2+)-activated Cl- current. 5. The peak of the rapid current (Ip)-capsaicin concentration (C) relationship was characterized by Ip/Ipmax = [1 + (C/Kd)n]-1, where n = 1.2 and the dissociation constant (Kd) = 0.68 microM. 6. The rapidly activating current was heterogeneous in regards to both its rate of activation and extent of desensitization. In cells bathed in buffer containing calcium and held at -60 mV, most of the capsaicin-activated currents desensitized. Removal of extracellular Ca2+ could reduce, eliminate, or have no effect on desensitization. 7. At positive holding potentials the currents very slowly desensitized, even in the presence of Ca2+. 8. Repeated 30-s applications of 1 microM capsaicin separated by 0.5, 2.5, and 5.5 min all induced tachyphylaxis. Tachyphylaxis decreased exponentially until the current remained approximately constant. Decreasing the time between capsaicin applications increased the extent of tachyphylaxis, whereas elimination of extracellular Ca2+ markedly reduced tachyphylaxis.

Role of [Ca2+]i in lethal oxidative injury in rat cultured inner medullary collecting duct cells.

Reactive oxygen metabolites have been implicated in the pathogenesis of toxic, ischaemic and immunologically mediated renal injury. An increase in the cytosolic free Ca2+ concentration ([Ca2+]i) has been proposed as a mechanism of oxidative stress-induced cell injury. We used a fluorescence spectrometer and a fluorescence probe to measure the [Ca2+]i and viability of rat primary cultured inner medullary collecting duct (IMCD) cells during oxidative stress induced by 5 mM tert-butyl hydroperoxide (TBHP). Initially, this oxidative stress evoked a small increase in [Ca2+]i which was followed by a slower sustained increase from the resting level of 170.8 +/- 38.8 nM to 1490.5 +/- 301.7 nM after 60 min, and this preceded the loss of plasma membrane integrity, measured by the propidium iodide fluorescence method. The elimination of extracellular Ca2+ from the culture medium prevented the TBHP-induced [Ca2+]i increase and improved cell viability. Restoration of extracellular Ca2+ resulted in an immediate and large increase in [Ca2+]i and extensive cell death. Verapamil, a Ca2+ channel blocker, inhibited the [Ca2+]i increase and afforded significant protection against cellular injury following exposure to TBHP-induced oxidative stress. Extracellular acidosis also prevented the increase in [Ca2+]i and cell death caused by this oxidative stress. These results are consistent with the hypothesis that oxidative stress-induced IMCD cellular injury may be the result of increased [Ca2+]i caused, in part, by activation of voltage-dependent Ca2+ channels.

A novel receptor for diadenosine polyphosphates coupled to calcium increase in rat midbrain synaptosomes

1. Diadenosine polyphosphates, Ap4A and Ap5A, as well as ATP, alpha,beta-MeATP and ADP-beta-S, were able to elicit variable intrasynaptosomal Ca2+ increases in rat midbrain synaptic terminals. The origin of the Ca2+ increment was the extra synaptosomal space since the elimination of extracellular Ca2+ abolished the effect of all the agonists. 2. The P2-purinoceptor antagonist, suramin, did not affect the Ca(2+)-increase evoked by diadenosine polyphosphates but dramatically blocked the Ca2+ entry induced by ATP and its synthetic analogues. 3. The actions of Ap5A and ATP on the intrasynaptosomal Ca2+ increase did not cross-desensitize. 4. Concentration-response studies for diadenosine polyphosphates showed pD2 values of 54.5 +/- 4.2 microM and 55.6 +/- 3.8 microM for Ap4A and Ap5A, respectively. 5. The entry of calcium induced by diadenosine polyphosphates could be separated into two components. The first represented a selective voltage-independent Ca2+ entry; the second, a sustained phase which was voltage-dependent. 6. Studies on the voltage-dependent Ca(2+)-channels involved in the effects of the diadenosine polyphosphates, demonstrated that omega-conotoxin G-VI-A inhibited the sustained Ca(2+)-entry, suggesting the participation of an N-type Ca(2+)-channel. This toxin was unable to abolish the initial cation entry induced by Ap4A or Ap5A. omega-Agatoxin IV-A, tetrodotoxin, or nifedipine did not inhibit the effects of the diadenosine polyphosphates. 7. The effect of ATP on Ca(2+)-entry was abolished by nifedipine and omega-conotoxin G-VI-A, suggesting the participation of L- and N-type Ca(2+)-channels in the response to ATP. 8. These data suggest that Ap4A, Ap5A and ATP activate the same intracellular Ca2+ signal through different receptors and different mechanisms. Ap4A and Ap5A induce a more selective Ca2+-entry in a voltage-independent process. This is the first time that a selective action of diadenosine polyphosphate through receptors other than P1 and P2-purinoceptors has been described.

Fluid flow modulates vascular endothelial cytosolic calcium responses to adenine nucleotides.

To determine whether fluid flow influences the action of soluble vasoactive agonists on vascular endothelium. Confluent monolayers of bovine aortic endothelial cells (BAEC) were cultured on glass coverslips, prelabeled with the Ca(2+)-sensitive dye fura-2, and placed in a parallel-plate flow chamber designed to generate defined laminar fluid flow. Cytosolic free Ca2+ concentration ([Ca2+]i) in individual BAEC was monitored during perfusion with medium containing adenine nucleotide under defined flow conditions. Continuous perfusion with ATP (0.3-3.0 microM) or ADP (0.1-1.0 microM) evoked repetitive oscillations in [Ca2+]i in individual BAEC. The frequency of the [Ca2+]i oscillations was dependent on both nucleotide concentration and levels of applied shear stress; at constant bulk concentration of nucleotide, the frequency increased with shear stress. Stopping flow in the continuous presence of agonists immediately extinguished the oscillatory response. Elimination of extracellular Ca2+ did not inhibit the [Ca2+]i oscillations. In the presence of nonhydrolyzable nucleotide analog, ATP gamma S or ADP beta S, application of flow resulted in similar shear-dependent [Ca2+]i oscillations, suggesting that flow modulation of the [Ca2+]i response was not simply due to depletion of ATP or ADP in the vicinity of BAEC monolayers as a result of hydrolysis of nucleotides by ectonucleotidases. These findings suggest that local hemodynamic conditions may modulate the action of vasoactive agents on the vascular endothelium in vivo.