Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B2 bradykinin and M1 muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP3 receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP3 to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca2+]i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca2+]i were blocked by depletion of Ca2+ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca2+ from IP3-sensitive Ca2+ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.     

Inositol 1,4,5-trisphosphate [correction of tris-phosphate] activation of inositol trisphosphate [correction of tris-phosphate] receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-trisphosphate (IP3) [corrected] binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at approximately 300 nM-1 microM, the open probability remained elevated (approximately 0.8) in the presence of saturating levels (10 microM) of IP3, even as [Ca2+]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+]i (Kact) of 210 nM and Hill coefficient (Hact) approximately 2; and inhibitory sites with half-maximal inhibitory [Ca2+]i (Kinh) of 54 microM and Hill coefficient (Hinh) approximately 4. Lowering IP3 concentration was without effect on Ca2+ activation parameters or Hinh, but decreased Kinh with a functional half-maximal activating IP3 concentration (KIP3) of 50 nM and Hill coefficient (HIP3) of 4 for IP3. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.     

A new role for IP3 receptors: Ca2+ release during nuclear vesicle fusion

During nuclear assembly, vesicles derived from the mitotic disassembly of the nuclear membranes reform the nuclear envelope. The vesicles first bind to chromosomes, specifically recognize other nuclear vesicles and then fuse to enclose the chromosomes. The proteins that mediate these events are largely unknown. Using reconstituted extracts of Xenopus eggs, we found that nuclear vesicle fusion required elevated (microM) concentrations of free Ca2+ [Sullivan KMC. Busa WB. Wilson KL. (1993) Cell, 73, 1411-1422]. Our data suggest that Ca2+ is released from the vesicle lumen by the activation of IP3 receptors (ligand-gated Ca2+ channels). We propose that the role of IP3 receptors during nuclear assembly may be analogous to that of voltage-gated Ca2+ channels during regulated secretion: to provide a microdomain of high cytosolic Ca2+ that triggers fusion. In this article, we will briefly describe current ideas about nuclear assembly and disassembly, and summarize the evidence that IP3 receptors are required for nuclear vesicle fusion. We will discuss parallels between our results and the role of voltage-gated Ca2+ channels, and Ca2+, in regulated exocytosis. Finally, we will address the question of how IP3 receptors are activated during nuclear vesicle fusion: is there a signal that stimulates IP3 production, or is the channel activated directly?     

Calcium-sensitive calcium influx in photoreceptor inner segments

The effect of external calcium concentration ([Ca2+]o) on membrane potential-dependent calcium signals in isolated tiger salamander rod and cone photoreceptor inner segments was investigated with patch-clamp and calcium imaging techniques. Mild depolarizations led to increases in intracellular Ca2+ levels ([Ca2+]i) that were smaller when [Ca2+]o was elevated to 10 mM than when it was 3 mM, even though maximum Ca2+ conductance increased 30% with the increase in [Ca2+]o. When external calcium was lowered to 1 mM [Ca2+]o, maximum Ca2+ conductance was reduced, as expected, but the mild depolarization-induced increase in [Ca2+]i was larger than in 3 mM [Ca2+]o. In contrast, when photoreceptors were strongly depolarized, the increase in [Ca2+]i was less when [Ca2+]o was reduced. An explanation for these observations comes from an assessment of Ca2+ channel gating in voltage-clamped photoreceptors under changing conditions of [Ca2+]o. Although Ca2+ conductance increased with increasing [Ca2+]o, surface charge effects dictated large shifts in the voltage dependence of Ca2+ channel gating. Relative to the control condition (3 mM [Ca2+]o), 10 mM [Ca2+]o shifted Ca2+ channel activation 8 mV positive, reducing channel open probability over a broad range of potentials. Reducing [Ca2+]o to 1 mM reduced Ca2+ conductance but shifted Ca2+ channel activation negative by 6 mV. Thus the intracellular calcium signals reflect a balance between competing changes in gating and permeation of Ca2+ channels mediated by [Ca2+]o. In mildly depolarized cells, the [Ca2+]o-induced changes in Ca2+ channel activation proved stronger than the [Ca2+]o-induced changes in conductance. In response to the larger depolarizations caused by 80 mM [K+]o, the opposite is true, with conductance changes dominating the effects on channel activation.     

Inositol trisphosphate receptors: Ca2+-modulated intracellular Ca2+ channels

The three subtypes of inositol trisphosphate (InsP3) receptor expressed in mammalian cells are each capable of forming intracellular Ca2+ channels that are regulated by both InsP3 and cytosolic Ca2+. The InsP3 receptors of many, though perhaps not all, tissues are biphasically regulated by cytosolic Ca2+: a rapid stimulation of the receptors by modest increases in Ca2+ concentration is followed by a slower inhibition at higher Ca2+ concentrations. Despite the widespread occurrence of this form of regulation and the belief that it is an important element of the mechanisms responsible for the complex Ca2+ signals evoked by physiological stimuli, the underlying mechanisms are not understood. Both accessory proteins and Ca2+-binding sites on InsP3 receptors themselves have been proposed to mediate the effects of cytosolic Ca2+ on InsP3 receptor function, but the evidence is equivocal. The effects of cytosolic Ca2+ on InsP3 binding and channel opening, and the possible means whereby the effects are mediated are discussed in this review.     

The effect of mersalyl on inositol trisphosphate receptor binding and ion channel function

A number of thiol-reactive agents induce repetitive Ca2+ spiking in cells by a mechanism thought to involve sensitization of the inositol 1,4,5-trisphosphate receptor (IP3R). To further define the basis of this interaction, we have studied the effect of several thiol-reactive agents on [3H]IP3 binding, IP3-gated channel activity, and conformation of the IP3R in membranes from hepatocytes, cultured WB rat liver epithelial cells, and cerebellum microsomes. At 4 degrees C, the organomercurial thiol-reactive agent mersalyl markedly stimulates (3-4fold) [3H]IP3 binding to permeabilized hepatocytes. The closely related molecule, thimerosal, has only a small stimulatory effect under these conditions, and GSSG or N-ethylmaleimide are without effect. The stimulatory effect of mersalyl was associated with a decrease in Kd of the IP3R with no change in Bmax. Mersalyl was without effect on detergent-solubilized hepatocyte binding sites or on the [3H]IP3 binding activity of cerebellum microsomes. In contrast to thimerosal, which potentiates IP3-mediated Ca2+ release, mersalyl blocked IP3-gated Ca2+ channels. Mersalyl pretreatment of WB membranes altered the pattern of immunoreactive receptor fragments generated upon subsequent cleavage of the receptor with proteinase K. This effect was not reproduced by thimerosal and was also not observed in experiments on cerebellum microsomes. We conclude that the WB cell and brain IP3 receptors are differently regulated by modification of thiol groups. Reaction of the WB cell IP3 receptor with mersalyl alters its conformation and modifies the accessibility of sites on the protein that are cleaved by proteinase K. In the presence of mersalyl, the receptor has high affinity for IP3 but is inactive as a Ca2+ channel. This contrasts with the high affinity receptor/active Ca2+ channel induced by thimerosal, suggesting that even closely related thiol agents may interact at different thiol groups.     

The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct from those of the endoplasmic reticulum

In the past few years, intracellular organelles, such as the endoplasmic reticulum, the nucleus and the mitochondria, have emerged as key determinants in the generation and transduction of Ca2+ signals of high spatio-temporal complexity. Little is known about the Golgi apparatus, despite the fact that Ca2+ within its lumen controls essential processes, such as protein processing and sorting. We report the direct monitoring of the [Ca2+] in the Golgi lumen ([Ca2+]Golgi) of living HeLa cells, using a specifically targeted Ca2+-sensitive photoprotein. With this probe, we show that, in resting cells, [Ca2+]Golgi is approximately 0.3 mM and that Ca2+ accumulation by the Golgi has properties distinct from those of the endoplasmic reticulum (as inferred by the sensitivity to specific inhibitors). Upon stimulation with histamine, an agonist coupled to the generation of inositol 1,4,5-trisphosphate (IP3), a large, rapid decrease in [Ca2+]Golgi is observed. The Golgi apparatus can thus be regarded as a bona fide IP3-sensitive intracellular Ca2+ store, a notion with major implications for the control of organelle function, as well as for the generation of local cytosolic Ca2+ signals.     

Arginine vasopressin triggers intracellular calcium release, a calcium-activated potassium current and exocytosis in identified rat corticotropes

Arginine vasopressin (AVP) stimulates the secretion of ACTH from pituitary corticotropes. We investigated the action of AVP in single corticotropes of male rats. Corticotropes were identified with the reverse hemolytic plaque assay using antibodies against ACTH. Using the whole-cell recording technique in conjunction with the fluorescent Ca2+ indicator, indo-1 to measure the concentration of cytosolic free Ca2+ ([Ca2+]i), we show that AVP triggers a transient and plateau pattern of Ca2+ signal. The [Ca2+]i elevation activates the apamin-sensitive Ca2+-activated K+ current, which, in turn, causes membrane hyperpolarization. The Ca2+ signal can be elicited in the absence of extracellular Ca2+ and is mimicked by intracellular inositol 1,4,5-trisphosphate (IP3). Both GDP-beta-S and heparin inhibit the AVP response. Thus, AVP triggers intracellular Ca2+ release from the (IP3)-sensitive store via a GTP binding protein-coupled phosphoinositide pathway. Using the high temporal resolution capacitance measurement to detect exocytosis in single corticotropes, we show that a burst of exocytosis is evoked during the AVP-triggered [Ca2+]i elevation. Exocytosis can also be triggered when Ca2+ is released directly from the IP3-sensitive store via flash photolysis of caged IP3. We conclude that AVP-stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release from the IP3-sensitive store.     

Inositol 1,4,5-trisphosphate-induced Ca2+ release is regulated by cytosolic Ca2+ in intact skeletal muscle

Microinjection of inositol 1,4,5-trisphosphate (InsP3) into intact skeletal muscle fibers isolated from frogs (Rana temporaria) increased resting cytosolic Ca2+ concentration ([Ca2+]i) as measured by double-barreled Ca2+-selective microelectrodes. In contrast, microinjection of inositol 1-phosphate, inositol 1,4-biphosphate, and inositol 1,4,5,6-tetrakisphosphate did not induce changes in [Ca2+]i. Incubation in low-Ca2+ solution, or in the presence of L-type Ca2+ channel blockers did not affect InsP3-induced release of cytosolic Ca2+. Neither ruthenium red, a blocker of ryanodine receptor Ca2+-release channels, nor cytosolic Mg2+, a known inhibitor of the Ca2+-induced Ca2+-release process, modified the InsP3-induced release of cytosolic Ca2+. However, heparin, a blocker of InsP3 receptors, inhibited InsP3-induced release of cytosolic Ca2+. Also, pretreatment with dantrolene or azumulene, two inhibitors of cytosolic Ca2+ release, reduced [Ca2+]i, and prevented InsP3 from inducing release of cytosolic Ca2+. Incubation in caffeine or lengthening of the muscle increased [Ca2+]i and enhanced the ability of InsP3 to induce release of cytosolic Ca2+. These results indicate that InsP3, at physiological concentrations, induces Ca2+ release in intact muscle fibers, and suggest that the InsP3-induced Ca2+ release is regulated by [Ca2+]i. A Ca2+-dependent effect of InsP3 on cytosolic Ca2+ release could be of importance under physiological or pathophysiological conditions associated with alterations in cytosolic Ca2+ homeostasis.     

Analytical steady-state solution to the rapid buffering approximation near an open Ca2+ channel

We derive an analytical steady-state solution for the Ca2+ profile near an open Ca2+ channel based on a transport equation which describes the buffered diffusion of Ca2+ in the presence of rapid stationary and mobile Ca2+ buffers (Wagner and Keizer, 1994). This steady-state rapid buffering approximation gives an upper bound on local Ca2+ elevations such as Ca2+ puffs or sparks when conditions for the validity of the rapid buffering approximation are met and is an alternative to approximations that assume that mobile buffers are unsaturable. This result also provides an analytical estimate of the cytosolic Ca2+ domain concentration ([Ca2+]d) near a channel pore and shows the dependence of [Ca2+]d on moderate concentrations of endogenous mobile buffer, Ca2+ indicator dye, and bulk cytosolic Ca2+. Assuming a simple relationship between [Ca2+]d and the lumenal depletion domain of an intracellular Ca2+ channel, lumenal and cytosolic Ca2+ profiles are matched to give an implicit analytical expression for the effect of bulk lumenal Ca2+ on [Ca2+]d.     

Regulation of cyclooxygenase-2 pathway by HER2 receptor

In the present work, we characterized the receptor properties and the conductive features of the inositol (1,4,5)-trisphosphate (IP3)-activated Ca2+ channels present in excised plasma-membrane patches obtained from mouse macrophages and A431 cells. We found that the receptor properties of the channels tested were similar to those of the IP3 receptor (IP3R) expressed in the endoplasmic reticulum (ER) membrane. These properties include activation by IP3, inhibition by heparin, time-dependent inactivation by high IP3 concentrations, activation by guanosine 5'o-thiotriphosphate and regulation by arachidonic acid. On the other hand, in terms of conductive properties, the channel closely resembles Ca2+-release-activated Ca2+ channels (Icrac). These conductive properties include extremely low conductance (approximately 1 pS), very high selectivity for Ca2+ over K+ (PCa/PK>1000), inactivation by high intracellular Ca2+ concentration and, importantly, strong inward rectification. Notably, the same channel was activated by: (1) agonists in the cell-attached mode of channel recording, and (2) cytosolic IP3 after patch excision. Although the possibility cannot be completely excluded that a novel type of IP3R is expressed exclusively in the plasma membrane, in their entirety our findings suggest that the plasma membrane of mouse macrophages and A431 cells contains Icrac-like Ca2+ channels coupled to an IP3-responsive protein which displays properties similar to those of the IP3R expressed in the ER membrane.     

Intracellular Ca2+ during metabolic activation of KATP channels in spontaneously active dorsal vagal neurons in medullary slices

Intracellular Ca2+ ([Ca2+]i) and membrane properties were measured in fura-2 dialysed dorsal vagal neurons (DVN) spontaneously active at a frequency of 0.5-5 Hz. [Ca2+]i increased by about 30 nm upon rising spike frequency by more than 200% due to 20-50 pA current pulses or 10 micrometer serotonin. It fell by 30 nm upon block of spiking by current-injection, tetrodotoxin or Ni2+ and also during hyperpolarization due to gamma-aminobutyric acid or opening of adenosine triphosphate (ATP) -sensitive K+ (KATP) channels with diazoxide. KATP channel-mediated hyperpolarizations during anoxia or cyanide produced an initial [Ca2+]i decrease which reversed into a secondary Ca2+ rise by less than 100 nm. Similar moderate rises of [Ca2+]i were observed during block of aerobic metabolism under voltage-clamp as well as in intact cells, loaded with fura-2 AM. The magnitude of the metabolism-related [Ca2+]i transients did not correlate with the amplitude of the KATP channel-mediated outward current. [Ca2+]i did not change during diazoxide-induced or spontaneous activation of KATP outward current observed in 10% of cells after establishing whole-cell recording. Increasing [Ca2+]i with cyclopiazonic acid did not activate KATP channels. [Ca2+]i was not affected upon block of outward current with sulphonylureas, but these KATP channel blockers were effective to reverse inhibition of spike discharge and, thus, the initial [Ca2+]i fall upon spontaneous or diazoxide-, anoxia- and cyanide-induced KATP channel activation. A sulphonylurea-sensitive hyperpolarization and [Ca2+]i fall was also revealed in the early phase of iodoacetate-induced metabolic arrest, whereas after about 20 min, occurrence of a progressive depolarization led to an irreversible rise of [Ca2+]i to more than 1 micrometer. The results indicate that KATP channel activity in DVN is not affected by physiological changes of intracellular Ca2+ and the lack of a major perturbance of Ca2+ homeostasis contributes to their high tolerance to anoxia.     

Different mechanisms for [Ca2+]i oscillations induced by carbachol and high concentrations of [Ca2+]o in the rat ventricular myocyte

1. The purpose of the present study was to explore the different mechanisms of [Ca2+]i oscillations induced by high concentrations of either carbachol (CCh) or extracellular Ca2+ ([Ca2+]o). First, we compared the oscillations induced by CCh at concentrations of 100-300 micromol/L and [Ca2+]o (5 mmol/L) in the single rat ventricular myocyte. Second, we studied CCh- and [Ca2+]o-induced [Ca2+]i oscillations following either interference with the production of inositol trisphosphate (IP3), reductions in cytosolic Ca2+ ([Ca2+]i), inhibition of Ca2+ influx and Na+-Ca2+ exchange or depletion of Ca2+ from its intracellular store. 2. The [Ca2+]i oscillations induced by CCh were frequent and were superimposed on [Ca2+]i transients in electrically stimulated cells, whereas those induced by high [Ca2+]o were occasional and occurred in quiescent cells and between [Ca2+]i transients in electrically stimulated cells. In both cases, [Ca2+]i oscillations were preceded by an increase in resting levels of [Ca2+]i. 3. Carbachol-induced [Ca2+]i oscillations were accompanied by an increase in amplitude and prolongation of the time of decline to 80% of the peak of the [Ca2+]i transient, while high [Ca2+]o-induced [Ca2+]i oscillations were the opposite. 4. A reduction of [Ca2+]o to 0.1 mmol/L and treatment with Ni2+ or ryanodine or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid AM (BAPTA-AM) abolished the [Ca2+]i oscillations induced by both CCh and high [Ca2+]o. 5. The calcium channel blockers verapamil and nifedipine and inhibitors of phospholipase C (neomycin and U-73122) abolished the [Ca2+]i oscillations induced by CCh; Li+ accelerated the onset of the [Ca2+]i oscillations induced by CCh. 6. These observations suggest that the mechanisms responsible for the [Ca2+]i oscillations induced by CCh and high [Ca2+]o are different from each other. Other than an increase in extracellular Ca2+ influx as a mechanism common for both CCh- and high [Ca2+]o-induced [Ca2+]i oscillations, the CCh-induced [Ca2+]i oscillations involve influx of Ca2+ via L-type Ca2+ channels, Na+-Ca2+ exchange, mobilization of intracellular Ca2+ and IP3 production.     

Spontaneous and repetitive calcium transients in C2C12 mouse myotubes during in vitro myogenesis

Fluorescence videomicroscopy was used to monitor changes in the cytosolic free Ca2+ concentration ([Ca2+]i) in the mouse muscle cell line C2Cl2 during in vitro myogenesis. Three different patterns of changes in [Ca2+]i were observed: (i) [Ca2+]i oscillations; (ii) faster Ca2+ events confined to subcellular regions (localized [Ca2+]i spikes) and (iii) [Ca2+]i spikes detectable in the entire myotube (global [Ca2+]i spikes). [Ca2+]i oscillations and localized [Ca2+]i spikes were detectable following the appearance of caffeine-sensitivity in differentiating C2Cl2 cells. Global [Ca2+]i spikes appeared later in the process of myogenesis in cells exhibiting coupling between voltage-operated Ca2+ channels and ryanodine receptors. In contrast to [Ca2+]i oscillations and localized [Ca2+]i spikes, the global events immediately stopped when cells were perfused either with a Ca2+-free solution, or a solution with TTX, TEA and verapamil. To explore further the mechanism of the global [Ca2+]i spikes, membrane currents and fluorescence signals were measured simultaneously. These experiments revealed that global [Ca2+]i spikes were correlated with an inward current. Moreover, while the depletion of the Ca2+ stores blocked [Ca2+]i oscillations and localized [Ca2+]i spikes, it only reduced the amplitude of global [Ca2+]i spikes. It is suggested that, during the earlier stages of the myogenesis, spontaneous and repetitive [Ca2+]i changes may be based on cytosolic oscillatory mechanisms. The coupling between voltage-operated Ca2+ channels and ryanodine receptors seems to be the prerequisite for the appearance of global [Ca2+]i spikes triggered by a membrane oscillatory mechanism, which characterizes the later phases of the myogenic process.     

Depletion of intracellular calcium stores facilitates the influx of extracellular calcium in platelet derived growth factor stimulated A172 glioblastoma cells

Calcium signaling in non-excitable cells is the consequence of calcium release from intracellular stores, at times followed by entry of extracellular calcium through the plasma membrane. To study whether entry of calcium depends upon the level of saturation of intracellular stores, we measured calcium channel opening in the plasma membrane of single confluent A172 glioblastoma cells stimulated with platelet derived growth factor (PDGF) and/or bradykinin (BK). We monitored the entry of extracellular calcium by measuring manganese quenching of Indo-1 fluorescence. PDGF raised intracellular calcium concentration ([Ca2+]i) after a dose-dependent delay (tdel) and then opened calcium channels after a dose-independent delay (tch). At higher doses (> 3 nM), BK increased [Ca2+]i after a tdel approximately 0 s, and tch decreased inversely with both dose and peak [Ca2+]i. Experiments with thapsigargin (TG), BK, and PDGF indicated that BK and PDGF share intracellular Ca2+ pools that are sensitive to TG. When these stores were depleted by treatment with BK and intracellular BAPTA, tdel did not change, but tch fell to almost 0 s in PDGF stimulated cells, indicating that depletion of calcium stores affects calcium channel opening in the plasma membrane. Our data support the capacitative model for calcium channel opening and the steady-state model describing quantal Ca2+ release from intracellular stores.     

Cyclosporin A inhibits apical secretory K+ channels in rabbit cortical collecting tubule principal cells

We used the cell-attached patch clamp configuration to examine the effect of basolateral cyclosporin A (CsA) exposure on low conductance K+ channels found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures. Baseline K+ channel activity, measured as mean NPo (number of channels x open probability), was 2.7 +/- 1.1 (N = 29). NPo fell by 69% (0.84 +/- 0.32; N = 32) in cultures pretreated with 500 ng/ml CsA for 30 minutes prior to patching. Chelation of intracellular [Ca2+]i (10 mM BAPTA/AM; N = 8) or removal of extracellular Ca2+ (N = 9), but not prevention of [Ca2+]i store release (10 microM TMB-8; N = 7), abolished CsA-induced inhibition. This suggested that CsA effects were mediated by an initial rise in [Ca2+]i via Ca2+ influx. Either 25 nM AVP (N = 10) or 0.25 microM thapsigargin (N = 8) (causing IP3-dependent and -independent release of [Ca2+]i stores, respectively) augmented, while 25 pM (N = 6) or 250 pM AVP (N = 8) reversed CSA-induced channel inhibition. Apical membrane protein kinase C (PKC) activation with 0.1 microM phorbol ester, PMA (N = 8) or 10 microM synthetic diacylglycerol, OAG (N = 7), mimicked (mean NPo = 0.99 +/- 0.40) the inhibitory effect of CsA. Apical PKC inhibition by prolonged apical exposure to PMA (N = 10) or 100 microM D-sphingosine (N = 6) blocked CsA's effect. Cyclic AMP increasing maneuvers, 10 microM forskolin (N = 5) or 0.5 mM db-cAMP (N = 8), stimulated basal K+ channel activity in the absence of CsA. In Conclusion: (1) basolateral exposure to CsA inhibits the activity of apical membrane 13 pS channels responsible for physiologic K+ secretion in rabbit CCT principal cells. (2) The inhibition is mediated by changes in intracellular Ca2+ and activation of apical PKC. (3) Pharmacologic AVP (nM) augments CsA-induced inhibition by releasing intracellular Ca2+ stores; more physiologic AVP (pM) attenuates channel inhibition, probably through cAMP generation. (4) Inhibition of apical secretory K+ channels by CsA likely contributes to decreased kaliuresis and clinical hyperkalemia observed in patients on CsA therapy.     

Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol

Eukaryotic cells respond to many hormones and neurotransmitters with increased activity of the enzyme phospholipase C and a subsequent rise in the concentration of intracellular free calcium ([Ca2+]i). The increase in [Ca2+]i occurs as a result of the release of Ca2+ from intracellular stores and an influx of Ca2+ through the plasma membrane; this influx of Ca2+ may or may not be store-dependent. Drosophila transient receptor potential (TRP) proteins and some mammalian homologues (TRPC proteins) are thought to mediate capacitative Ca2+ entry. Here we describe the molecular mechanism of store-depletion-independent activation of a subfamily of mammalian TRPC channels. We find that hTRPC6 is a non-selective cation channel that is activated by diacylglycerol in a membrane-delimited fashion, independently of protein kinases C activated by diacylglycerol. Although hTRPC3, the closest structural relative of hTRPC6, is activated in the same way, TRPCs 1, 4 and 5 and the vanilloid receptor subtype 1 are unresponsive to the lipid mediator. Thus, hTRPC3 and hTRPC6 represent the first members of a new functional family of second-messenger-operated cation channels, which are activated by diacylglycerol.     

Expression of calcium channels in adult cardiac myocytes is regulated by calcium

In addition to playing a significant role in cardiac excitation-contraction coupling, intracellular Ca2+ ([Ca2+]i) can regulate gene expression. While the mechanisms regulating expression of Ca2+ channels are not entirely defined, some evidence exists for Ca2+-dependent regulation. Using an adult ventricular myocyte culture system, we determined the effects of Ca2+ on: (1) abundance of mRNA for L-type Ca2+ channel alpha1 subunit (DHP receptor); (2) amount of DHP receptors; and (3) whole-cell Ca2+ current (ICa). Rat ventricular myocytes were cultured for 1-3 days in serum-free medium containing either normal (1.8 mM) or high (4.8 mM) Ca2+. Exposing myocytes to high Ca2+ rapidly elevated [Ca2+]i as determined by fura-2. Northern blot analysis revealed that culturing cells in high Ca2+ produced 1.5-fold increase in mRNA levels for the DHP receptor. The abundance of DHP receptors, determined by ligand binding, was two-fold greater in myocytes after 3 days in high Ca2+. Moreover, peak ICa was larger in myocytes cultured for 3 days in high Ca2+ (-17.8+/-1.5 pA/pF, n=26) than in control cells (-11.0+/-1.0 pA/pF, n=23). Voltage-dependent activation and inactivation, rates of current decay, as well as percent increases in ICa elicited by Bay K8644 were similar in all groups. Therefore, larger ICa is likely to represent a greater number of functional channels with unchanged kinetics. Our data support the conclusion that transient changes in [Ca2+]i can modulate DHP receptor mRNA and protein abundance, producing a corresponding change in functional Ca2+ channels in adult ventricular myocytes.     

Control of ionic currents in guard cell vacuoles by cytosolic and luminal calcium

Activity of vacuolar ion channels can be regulated by the cytosolic free Ca2+ concentration ([Ca2+]cyt). Using the whole-vacuole mode of patch-clamp with Vicia faba guard cell vacuoles, three distinct cation currents were apparent that were differentially regulated by [Ca2+]cyt. At 'zero' to 100 nM [Ca2+]cyt, instantaneous currents typical of Fast Vacuolar (FV) channels were activated. A 10 fold KCl gradient directed out of the vacuole increased FV currents (up to fivefold) at negative potentials compared with the currents in symmetrical KCl. At [Ca2+]cyt higher than 100 nM, instantaneous currents became smaller and voltage-independent (non-rectifying) and were typical of Vacuolar K(+)-selective (VK) channels. These currents were less sensitive to a KCl gradient than were the FV currents, being stimulated less than twofold at negative potentials. Reversal potentials measured in the presence of a KCl gradient indicated a high K+ permeability of both FV and VK currents. At [Ca2+]cyt higher than 600 nM time-dependent currents elicited by positive potentials were typical of Slow Vacuolar (SV) channel activation. When the Ca2+ mole fraction in the cytosolic or luminal solution was varied the reversal potential of SV currents (determined by tail current analysis) passed through maximum or minimum values. The resultant calculated apparent permeability ratios varied with ionic conditions but indicated high Ca2+ and K+ permeabilities. If a Cl- permeability was assumed then the apparent PCa was lower. However, substitution of Cl- by the larger (impermeant) anion gluconate had no effect on the reversal potential of SV tail currents in the presence of Ca2+ and a K+ gradient, demonstrating that the assumption of Cl- permeability of the SV channel is invalid. Single-channel SV currents also decreased with increasing cytosolic Ca2+ mole fraction. These data indicate that the SV channel is highly cation selective, shows characteristics typical of a multi-ion pore and derives ion selectivity by Ca2+ binding. The SV channel currents could also be Mg(2+)-activated and were demonstrated to be Mg(2+)-permeable in the absence of Ca2+. The apparent permeability ratio (PMg:PK) also varied under different ionic conditions. The results indicate not only that FV, VK and SV channels are all present in a single cell type, but also that each is differentially regulated by [Ca2+]cyt. The respective roles of these channels in vacuolar ion release are discussed, and possible conditions are presented in which these channels could be activated by disparate signalling pathways during stomatal closure.     

Inhibition of bradykinin-induced calcium increase by phosphatase inhibitors in neuroblastoma x glioma hybrid NG108-15 cells

Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by bradykinin by approximately 63%. This inhibition was dependent on the concentration of okadaic acid with an IC50 of 0.15 nM. Okadaic acid treatment only lowered the maximal response of [Ca2+]i increase and had no effect on the EC50 value for bradykinin regardless of the presence of extracellular Ca2+. Neither the capacity of 45Ca2+ accumulation within intracellular nonmitochondrial Ca2+ stores nor the magnitude of [Ca2+]i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP3) generation, the Mn2+ influx, and the capacity of mitochondrial Ca2+ accumulation. Furthermore, the sensitivity of IP3 in the Ca2+ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca2+]i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP3, and the slowed rate of Ca2+ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca2+ signaling cascade in NG108-15 cells.