Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles1

The initial rate of Ca2+ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca2+-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca2+-selective minielectrodes to determine pCa values. The initial rate of Ca2+ influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca2+ movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K+. In addition, Ca2+ was shown to move across the membrane vesicles in the presence of a K+ diffusion potential gradient. The potential-stimulated rate of Ca2+ transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl3, verapamil, and nifedipine had little or no effect. These results indicate that Ca2+ transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.

Up-regulation of pressure-activated Ca(2+)-permeable cation channel in intact vascular endothelium of hypertensive rats.

In endothelial cells, stretch-activated cation channels have been proposed to act as mechanosensors for changes in hemodynamic forces. We have identified a novel mechanosensitive pressure-activated channel in intact endothelium from rat aorta and mesenteric artery. The 18-pS cation channel responded with a multifold increase in channel activity when positive pressure was applied to the luminal cell surface with the patch pipette and inactivated at negative pipette pressure. Channel permeability ratio for K+, Na+, and Ca2+ ions was 1:0.98:0.23. Ca2+ influx through the channel was sufficient to activate a neighboring Ca2(+)-dependent K+ channel. Hemodynamic forces are chronically disturbed in arterial hypertension. Endothelial cell dysfunction has been implicated in the pathogenesis of arterial hypertension. In two comparative studies, density of the pressure-activated channel was found to be significantly higher in spontaneously hypertensive rats and renovascular hypertensive rats compared with their respective normotensive controls. Channel activity presumably leads to mechanosensitive Ca2+ influx and induces cell hyperpolarization by K+ channel activity. Both Ca2+ influx and hyperpolarization are known to induce a vasodilatory endothelial response by stimulating endothelial nitric oxide (NO) production. Up-regulation of channel density in hypertension could, therefore, represent a counterregulatory mechanism of vascular endothelium.

SNAP-25 and synaptotagmin involvement in the final Ca(2+)-dependent triggering of neurotransmitter exocytosis.

In neurons, depolarization induces Ca2+ influx leading to fusion of synaptic vesicles docked at the active zone for neurotransmitter release. While a number of proteins have now been identified and postulated to participate in the assembly and subsequent disengagement of a vesicle docking complex for fusion, the mechanism that ultimately triggers neuroexocytosis remains elusive. Using a cell-free, lysed synaptosomal membrane preparation, we show that Ca2+ alone is sufficient to trigger secretion of glutamate and furthermore that Ca(2+)-signaled exocytosis is effectively blocked by antibodies and peptides to SNAP-25, a key constituent of the vesicle docking complex. In addition, Ca2+ inhibits the ability of synaptotagmin, a synaptic vesicle protein proposed as a calcium sensor and triggering device, to associate with this docking complex. These results support a model in which Ca(2+)-dependent triggering of neurotransmission at central synapses acts after ATP-dependent potentiation of the docking-fusion complex for membrane fusion.

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.

N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.

Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells.

Whole-cell patch-clamp recordings and single-cell Ca2+ measurements were used to study the control of Ca2+ entry through the Ca2+ release-activated Ca2+ influx pathway (ICRAC) in rat basophilic leukemia cells. When intracellular inositol 1,4,5-trisphosphate (InsP3)-sensitive stores were depleted by dialyzing cells with high concentrations of InsP3, ICRAC inactivated only slightly in the absence of ATP. Inclusion of ATP accelerated inactivation 2-fold. The inactivation was increased further by the ATP analogue adenosine 5'-[gamma-thio]triphosphate, which is readily used by protein kinases, but not by 5'-adenylyl imidodiphosphate, another ATP analogue that is not used by kinases. Neither cyclic nucleotides nor inhibition of calmodulin or tyrosine kinase prevented the inactivation. Staurosporine and bisindolylmaleimide, protein kinase C inhibitors, reduced inactivation of ICRAC, whereas phorbol ester accelerated inactivation of the current. These results demonstrate that a protein kinase-mediated phosphorylation, probably through protein kinase C, inactivates ICRAC. Activation of the adenosine receptor (A3 type) in RBL cells did not evoke much Ca2+ influx or systematic activation of ICRAC. After protein kinase C was blocked, however, large ICRAC was observed in all cells and this was accompanied by large Ca2+ influx. The ability of a receptor to evoke Ca2+ entry is determined, at least in part, by protein kinase C. Antigen stimulation, which triggers secretion through a process that requires Ca2+ influx, activated ICRAC. The regulation of ICRAC by protein kinase will therefore have important consequences on cell functioning.

The kinetics of quantal transmitter release from retinal amacrine cells.

Exocytosis of transmitter at most synapses is a very fast process triggered by the entry of Ca2+ during an action potential. A reasonable expectation is that the fast step of exocytosis is followed by slow steps readying another vesicle for exocytosis but the identity and kinetics of these steps are presently unclear. By voltage clamping both pre- and postsynaptic neurons in an isolated pair of retinal amacrine cells, we have measured evoked synaptic currents and responses to single vesicles of transmitter (minis). From these currents, we have computed the rate of exocytosis during a sustained presynaptic depolarization. We show here that for these cells, release is consistent with a scheme of "fire and reload." Large Ca2+ influx causes the rapid release of a small number of vesicles, typically approximately 10 per presynaptic neuron, likely corresponding to those vesicles already docked. After this spike of exocytosis whose peak is 150 quanta per release site per s, continued Ca2+ influx sustains release at only 22 quanta per release site per s, probably rate-limited by the docking of fresh vesicles.

Convergent and parallel activation of low-conductance potassium channels by calcium and cAMP-dependent protein kinase.

K+ channels, which have been linked to regulation of electrogenic solute transport as well as Ca2+ influx, represent a locus in hepatocytes for the concerted actions of hormones that employ Ca2+ and cAMP as intracellular messengers. Despite considerable study, the single-channel basis for synergistic effects of Ca2+ and cAMP on hepatocellular K+ conductance is not well understood. To address this question, patch-clamp recording techniques were applied to a model liver cell line, HTC hepatoma cells. Increasing the cytosolic Ca2+ concentration ([Ca2+]i) in HTC cells, either by activation of purinergic receptors with ATP or by inhibition of intracellular Ca2+ sequestration with thapsigargin, activated low-conductance (9-pS) K+ channels. Studies with excised membrane patches suggested that these channels were directly activated by Ca2+. Exposure of HTC cells to a permeant cAMP analog, 8-(4-chlorophenylthio)-cAMP, also activated 9-pS K+ channels but did not change [Ca2+]i. In excised membrane patches, cAMP-dependent protein kinase (the downstream effector of cAMP) activated K+ channels with conductance and selectivity identical to those of channels activated by Ca2+. In addition, cAMP-dependent protein kinase activated a distinct K+ channel type (5 pS). These data represent the differential regulation of low-conductance K+ channels by signaling pathways mediated by Ca2+ and cAMP. Moreover, since low-conductance Ca(2+)-activated K+ channels have been identified in a variety of cell types, these findings suggest that differential regulation of K+ channels by hormones with distinct signaling pathways may provide a mechanism for hormonal control of solute transport and Ca(2+)-dependent cellular functions in the liver as well as other nonexcitable tissues.

The inositol 1,4,5-trisphosphate receptor is essential for T-cell receptor signaling.

Antigen-specific activation of T lymphocytes, via stimulation of the T-cell antigen receptor (TCR) complex, is marked by a rapid and sustained increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). It has been suggested that the second messenger inositol 1,4,5-trisphosphate (IP3) produced after TCR stimulation binds to the IP3 receptor (IP3R), an intracellular Ca(2+)-release channel, and triggers the increase in [Ca2+]i that activates transcription of the gene for T-cell growth factor interleukin 2 (IL-2). However, the role of the IP3R in T-cell signaling and possibly in plasma membrane Ca2+ influx in T cells remains unproven. Stable transfection of T cells (Jurkat) with antisense type 1 IP3R cDNA prevented type 1 IP3R expression, providing a tool for dissecting the role of IP3 signaling during T-cell activation. T cells lacking type 1 IP3R failed to increase [Ca2+]i or produce IL-2 after TCR stimulation. Moreover, depletion of intracellular Ca2+ stores without TCR activation stimulated Ca2+ influx in cells lacking the type 1 IP3R. These results establish that the type 1 IP3R is required for intracellular Ca2+ release that triggers antigen-specific T-cell proliferation but not for plasma membrane Ca2+ influx.

Oxygen-sensitive calcium channels in vascular smooth muscle and their possible role in hypoxic arterial relaxation.

We have investigated the modifications of cytosolic [Ca2+] and the activity of Ca2+ channels in freshly dispersed arterial myocytes to test whether lowering O2 tension (PO2) directly influences Ca2+ homeostasis in these cells. Unclamped cells loaded with fura-2 AM exhibit oscillations of cytosolic Ca2+ whose frequency depends on extracellular Ca2+ influx. Switching from a PO2 of 150 to 20 mmHg leads to a reversible attenuation of the Ca2+ oscillations. In voltage-clamped cells, hypoxia reversibly reduces the influx of Ca2+ through voltage-dependent channels, which can account for the inhibition of the Ca2+ oscillations. Low PO2 selectively inhibits L-type Ca2+ channel activity, whereas the current mediated by T-type channels is unaltered by hypoxia. The effect of low PO2 on the L-type channels is markedly voltage dependent, being more apparent with moderate depolarizations. These findings demonstrate the existence of O2-sensitive, voltage-dependent, Ca2+ channels in vascular smooth muscle that may critically contribute to the local regulation of circulation.

Colocalization of calcium entry and exocytotic release sites in adrenal chromaffin cells.

"Snapshot" images of localized Ca2+ influx into patch-clamped chromaffin cells were captured by using a recently developed pulsed-laser imaging system. Transient opening of voltage-sensitive Ca2+ channels gave rise to localized elevations of Ca2+ that had the appearance of either "hotspots" or partial rings found immediately beneath the plasma membrane. When the Ca2+ imaging technique was employed in conjunction with flame-etched carbon-fiber electrodes to spatially map the release sites of catecholamines, it was observed that the sites of Ca2+ entry and catecholamine release were colocalized. These results provide functional support for the idea that secretion occurs from "active zone"-like structures in neuroendocrine cells.

Modulação da diferenciação neural de células troco embrionárias por transientes de cálcio intracelulares: papéis dos receptores purinérgicos e de canais de cálcio voltagem-dependentes

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal.

Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia, accounting for 60-70% of cases in subjects over 65 years of age. Several postulates have been put forward that relate AD neuropathology to intellectual and functional impairment. These range from free-radical-induced damage, through cholinergic dysfunction, to beta-amyloid-induced toxicity. However, therapeutic strategies aimed at improving the cognitive symptoms of patients via choline supplementation, cholinergic stimulation or beta-amyloid vaccination, have largely failed. A growing body of evidence suggests that perturbations in systems using the excitatory amino acid L-glutamate (L-Glu) may underlie the pathogenic mechanisms of (e.g.) hypoxia-ischemia, epilepsy, and chronic neurodegenerative disorders such as Huntington's disease and AD. Almost all neurons in the CNS carry the N-methyl-D-aspartate (NMDA) subtype of ionotropic L-glutamate receptors, which can mediate post-synaptic Ca2+ influx. Excitotoxicity resulting from excessive activation of NMDA receptors may enhance the localized vulnerability of neurons in a manner consistent with AD neuropathology, as a consequence of an altered regional distribution of NMDA receptor subtypes. This review discusses mechanisms for the involvement of the NMDA receptor complex and its interaction with polyamines in the pathogenesis of AD. NMDA receptor antagonists have potential for the therapeutic amelioration of AD. (C) 2004 Elsevier Ltd. All rights reserved.

A small-molecule lead compound for the treatment of Alzheimer's disease

At autopsy, Alzheimer's disease is characterised by the presence of amyloid plaques and neurofibrillary tangles, made up of two peptide sequences, amyloid-beta(1-40) (A beta 40) and amyloid-beta(1-42) (A beta 42). In Tyrode's solution (2 mM Ca2+), 10 mu M A beta 42 peptide almost immediately aggregates and eventually forms p-sheets. This aggregation can be inhibited with 4,5-dianilinophthalimide (DAPH). Ca2+-permeant AMPA receptors are involved in the neuronal Ca2+ influx (neurotoxicity) induced by the A beta 42 peptide in cultured neuronal cells. The Ca2+ influx observed with pre-incubated A beta 42 peptide was inhibited by DAPH. DAPH also inhibits epidermal growth factor receptor kinase, and this will prevent its development for use in Alzheimer's disease. The potential of DAPH as a small-molecule lead compound for the treatment of Alzheimer's disease next requires the separation of the structural requirements that reverse fibril formation and inhibit epidermal growth factor receptor kinase.

TRPV2 Channels Contribute to Stretch-Activated Cation Currents and Myogenic Constriction in Retinal Arterioles

<p>Purpose: Activation of the transient receptor potential channels, TRPC6, TRPM4, and TRPP1 (PKD2), has been shown to contribute to the myogenic constriction of cerebral arteries. In the present study we sought to determine the potential role of various mechanosensitive TRP channels to myogenic signaling in arterioles of the rat retina.</p><p>Methods: Rat retinal arterioles were isolated for RT-PCR, Fura-2 Ca2+ microfluorimetry, patch-clamp electrophysiology, and pressure myography studies. In some experiments, confocal immunolabeling of wholemount preparations was used to examine the localization of specific mechanosensitive TRP channels in retinal vascular smooth muscle cells (VSMCs).</p><p>Results: Reverse transcription-polymerase chain reaction analysis demonstrated mRNA expression for TRPC1, M7, V1, V2, V4, and P1, but not TRPC6 or M4, in isolated retinal arterioles. Immunolabeling revealed plasma membrane, cytosolic and nuclear expression of TRPC1, M7, V1, V2, V4, and P1 in retinal VSMCs. Hypoosmotic stretch-induced Ca2+ influx in retinal VSMCs was reversed by the TRPV2 inhibitor tranilast and the nonselective TRPP1/V2 antagonist amiloride. Inhibitors of TRPC1, M7, V1, and V4 had no effect. Hypoosmotic stretch-activated cation currents were similar in Na+ and Cs+ containing solutions suggesting no contribution by TRPP1 channels. Direct plasma membrane stretch triggered cation current activity that was blocked by tranilast and specific TRPV2 pore-blocking antibodies and mimicked by the TRPV2 activator, Δ9-tetrahydrocannabinol. Preincubation of retinal arterioles with TRPV2 blocking antibodies prevented the development of myogenic tone.</p><p>Conclusions: Our results suggest that retinal VSMCs express a range of mechanosensitive TRP channels, but only TRPV2 appears to contribute to myogenic signaling in this vascular bed.</p>