Activation of a P2Y4-like purinoceptor triggers an increase in cytosolic [Ca2+] in the red blood cells of the lizard Ameiva ameiva (Squamata, Teiidae)

An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs) of the lizard Ameiva ameiva store Ca2+ in the endoplasmic reticulum (ER) and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP3 to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgammaS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, alpha, ��-ATP, and ADP failed to do so in a 1- to 200-��m con- centration. The EC50 obtained for the compounds tested was 41.77 ��M for UTP, 48.11 ��M for GTP, 53.11 ��M for UDP, and 30.78 ��M for ATPgammaS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y4-like receptor due to its pharmacological similarity to the mammalian P2Y4 receptor.

Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

Background: Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. Methods: Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. Results: The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 mu M) and PPADS (50 mu M) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 mu M), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 mu M) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 mu M), TNP-ATP (50 mu M) or the purinergic blockers KN-62 (10 mu M) and Ip5I (10 mu M). Incubating P. berghei infected cells with KN-62 (200 mu M) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 mu M) led to an increase in rings forms (82% +/- 4, n = 11) and a decrease in trophozoite forms (18% +/- 4, n = 11). Conclusions: The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.

Stimulation of the blue light phototropic receptor NPH1 causes a transient increase in cytosolic Ca2+

Blue light regulates plant growth and development, and three photoreceptors, CRY1, CRY2, and NPH1, have been identified. The transduction pathways of these receptors are poorly understood. Transgenic plants containing aequorin have been used to dissect the involvement of these three receptors in the regulation of intracellular Ca2+. Pulses of blue light induce cytosolic Ca2+ transients lasting about 80 s in Arabidopsis and tobacco seedlings. Use of organelle-targeted aequorins shows that Ca2+ increases are limited to the cytoplasm. Blue light treatment of cry1, cry2, and nph1 mutants showed that NPH1, which regulates phototropism, is largely responsible for the Ca2+ transient. The spectral response of the Ca2+ transient is similar to that of phototropism, supporting NPH1 involvement. Furthermore, known interactions between red and blue light and between successive blue light pulses on phototropic sensitivity are mirrored in the blue light control of cytosolic Ca2+ in these seedlings. Our observations raise the possibility that physiological responses regulated by NPH1, such as phototropism, may be transduced through cytosolic Ca2+.

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K+ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K+ currents, an inactivating outward-rectifying K+ current was characterized (plant A-type current: IAP). IAP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. IAP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to ���100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward IAP was mainly carried by K+ ions. In contrast to the sustained outward-rectifying K+ currents, cytosolic alkaline pH was found to inhibit IAP and extracellular K+ was required for IAP activity. Furthermore, increasing cytosolic free Ca2+ in the physiological range strongly inhibited IAP activity with a half inhibitory concentration of ��� 94 nM. We present a detailed characterization of an inactivating K+ current in a higher plant cell. Regulation of IAP by diverse factors including membrane potential, cytosolic Ca2+ and pH, and extracellular K+ and Ca2+ implies that the inactivating IAP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.

Mecanismo de interacci��n, calcio dependiente, entre Calcineurina y PERK, involucrado en el Estr��s de Ret��culo Endopl��smico. Ca2+ -dependent mechanism of interaction between Calcineurin and PERK involved in Endoplasmic Reticulum Stress.

El Estr��s de Ret��culo Endopl��smico (RE) es inducido por la acumulaci��n de prote��nas sin plegar en el lumen de la organela. Esto se puede observar en diversas situaciones fisio-patol��gicas como durante una infecci��n viral o en proceso isqu��mico. Adem��s, contribuye a la base molecular de numerosas enfermedades ya sea ��ndole metab��lico (Fibrosis qu��stica o Diabetes Miellitus) o neurodegenerativas como mal de Alzheimer o Parkinson (Mutat Res, 2005, 569). Para restablecer la homeostasis en la organela se activa una se��al de transducci��n (UPR), cuya respuesta inmediata es la atenuaci��n de la s��ntesis de prote��na debido a la fosforilaci��n de subunidad alpha del factor eucari��tico de iniciaci��n de translaci��n (eIF2��) v��a PERK. Esta es una prote��na de membrana de RE que detecta estr��s. Bajo condiciones normales, PERK est�� inactiva debido a la asociaci��n de su dominio luminar con la chaperona BIP (Nat Cell Biol, 2000, 2: 326). Frente a una situaci��n de estr��s, la chaperona se disocia causando desinhibici��n. Recientemente, (Plos One 5: e11925) se observ��, bajo condiciones de estr��s, un aumento de Ca2+ citos��lico y un r��pido incremento de la expresi��n de calcineurina (CN), una fosfatasa citos��lica dependiente de calcio, heterodim��rica formada por una subunidad catal��tica (CN-A) y una regulatoria (CN-B). Adem��s, CN interacciona, sin intermediarios, con el dominio citos��lico de PERK favoreciendo su trans-autofosforilaci��n. Resultados preliminares indican que, astrocitos CNA��-/- exhibieron, en condiciones basales, un mayor n��mero de c��lulas muertas y de niveles de eIF2�� fosforilado que los astrocitos CNA��-/-. Hip��tesis: CNA��/B interacciona con PERK cuando el Ca2+ citos��lico esta incrementado luego de haberse inducido Estr��s de RE, lo cual promueve dimerizaci��n y auto-fosforilaci��n de la quinasa, acentu��ndose as�� la fosforilaci��n de eIF2�� e inhibici��n de la s��ntesis de prote��nas. Esta activaci��n citos��lica de PERK colaborar��a con la ya descrita, desinhibici��n luminal llevada cabo por BIP. Cuando el Ca2+ citos��lico retorna a los niveles basales, PERK fosforila a CN, reduciendo su afinidad de uni��n y disoci��ndose el complejo CN/PERK. Objetivo general: Definir las condiciones por las cuales CN interacciona con PERK y regula la fosforilaci��n de eIF2�� e inhibici��n de la s��ntesis de prote��na. Objetivos espec��ficos: I-Estudiar la diferencia de afinidades y dependencia de Ca2+, de las dos isoformas de CN (�� y ��) en su asociaci��n con PERK. Adem��s verificar la posible participaci��n de la subunidad B de CN en esta interacci��n. II-Determinar si la auto-fosforilaci��n de PERK es diferencialmente regulada por las dos isoformas de CN. III-Discernir la relaci��n del estado de fosforilaci��n de CN con su uni��n a PERK. IV-Determinar efectos fisiol��gicos de la interacci��n de CN-PERK durante la respuesta de Estr��s de RE. Para llevar a cabo este proyecto se realizar��n experimentos de biolog��a molecular, interacci��n prote��na-prote��na, ensayos de fosforilaci��n in vitro y un perfil de polisoma con astrocitos CNA��-/- , CNA���-/- y astrocitos controles. Se espera encontrar una mayor afinidad de uni��n a PERK de la isoforma �� de CN y en condiciones donde la concentraci��n de Ca2+ sea del orden micromolar e imite niveles del i��n durante un estr��s. Con respecto al estado de fosforilaci��n de CN, debido a los resultados preliminares, donde solo se la encontr�� fosforilada en condiciones basales, se piensa que CN podr��a interactuar con mayor afinidad con PERK cuando CN se encuentre desfosforilada. Por ��ltimo, se espera encontrar un aumento de eIF2�� fosforilado y una acentuaci��n de la atenuaci��n de la s��ntesis de prote��na como consecuencia de la mayor activaci��n de PERK por su asociaci��n con la isoforma �� de CN en astrocitos donde el Estr��s de RE se indujo por privaci��n de oxigeno y glucosa. Estos experimentos permitir��n avanzar en el estudio de una nueva funci��n citoprotectora de CN recientemente descrita por nuestro grupo de trabajo y sus implicancias en un modelo de isquemia. The accumulation of unfolded proteins into the Endoplasmic Reticulum (ER) activates a signal transduction cascade called Unfolding Protein Response (UPR), which attempts to restore homeostasis in the organelle. (PKR)-like-ER kinase (PERK) is an early stress response transmembrane protein that is generally inactive due to its association with the chaperone BIP. During ER stress, BIP is tritrated by the unfolded protein, leading PERK activation and phosphorylation of eukaryotic initiation factor-2 alpha (eIF2alpha), which attenuates protein s��ntesis. If ER damage is too great and homeostasis is not restored within a certain period of time, an apoptotic response is elicited. We recently demonstrated a cytosolic Ca2+ increase in Xenopus oocytes after induce ER stress. Moreover, calcineurin A/B, a an heterotrimeric Ca2+ dependent phosphatases (CN-A/B), associates with PERK increasing its auto-phosphorylation and significantly enhancing cell viability. Preliminary results suggest that, CN-A��-/- knockout astrocytes exhibit a significant higher eIF2�� phosphorylated level compared to CN-A��-/- astrocytes. Our working hypothesis establishes that: CN binds to PERK when cytosolic Ca2+ is initially increased by ER stress, promoting dimerization and autophosphorylation, which leads to phosphorylation of elF2�� and subsequently attenuation of protein translation. When cytosolic Ca2+ returns to resting levels, PERK phosphorylates CN, reducing its binding affinity so that the CN/PERK complex dissociates. The goal of this project is to determine the conditions by which CN binding to PERK attenuates protein translation during the ER stress response and subsequently, to determine how the interaction of CN with PERK is terminated when stress is removed. To perform this project is planed to do molecular biology experiments, pull down assays, in vitro phosphorylations and assess overall mRNA translation efficiency doing a polisome profile.

Cytosolic calcium levels and stress induced y-amino butyrate synthesis in asparagus mesophyll cells

Numerous investigations have demonstrated large increases in y-amino butyrate (GABA) levels in response to a variety of stresses such as touch or cold shock (Wallace et ale 1984) Circumstantial evidence indicating a role of Ca2 + in these increases includes elevated Ca2+ levels in response to touch and cold shock (Knight et ale 1991), and the demonstration of a calmodulin binding domain on glutamate decarboxylase (GAD), the enzyme responsible for GABA synthesis (Baum et al 1993) In the present study the possible role of Ca2+ and calmodulin in stimulation of GAD and subsequent GABA accumulation was examined using asparagus mesophyll cells. Images of cells loaded with the Ca2+ indicator Fluo-3 revealed a rapid and transient increase in cytosolic Ca2+ in response to cold shock. GABA levels increased by 106% within 15 min. of cold shock. This increase was inhibited 70% by the calmodulin antagonist W7, and 42% by the Ca2+ channel blocker La3+.. Artificial elevation of intracellular Ca2+ by the Ca2+ionophore A23187 resulted in an 61% increase in GABA levels. Stimulation of GABA synthesis by ABA resulted in an 83% increase in GABA levels which was inhibited 55% by W7. These results support the hypothesis that cold shock stimulates Ca2+ entry into the cytosol of the cells which results in Ca2+/calmodulin mediated activation of GAD and consequent GABA synthesis.

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca2+ - and K+ -permeable conductance in root cells

Plant cell growth and stress signaling require Ca2+ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH_. In root cells, extracellular OH_ activates a plasma membrane Ca2+-permeable conductance that permits Ca2+ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca2+-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH_-activated Ca2+- and K+-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca2+ in response to OH_. An OH_-activated Ca2+ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca2+-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca2+ in plants.

Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: Redistribution of calmodulin as a possible means of regulating Ca2+���mobilization

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly bound to multiple sites on InsP3 receptors and Ca2+ increased the binding by 190% �� 10%; the half-maximal effect occurred when the Ca2+ concentration was 184 �� 14 nM. In the absence of Ca2+, calmodulin caused a reversible, concentration-dependent (IC50 = 3.1 �� 0.2 ��M) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect was similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 ��M) inhibited the Ca2+ release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways.

Mitochondrial Ca2+-induced Ca2+ Release Mediated by the Ca2+ Uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca2+ during cell stimulation. The present study focuses on the pathways for mitochondrial Ca2+ efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca2+ uptake and increased the cytosolic [Ca2+] ([Ca2+]c) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca2+]c kinetics and inhibited Ca2+ release from Ca2+-loaded mitochondria. This effect was due to inhibition of mitochondrial Na+/Ca2+ exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca2+]c triggered fast Ca2+ release from these depolarized Ca2+-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca2+-induced Ca2+ release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca2+ uniporter. This novel kind of mitochondrial Ca2+-induced Ca2+ release might contribute to Ca2+ clearance from mitochondria that become depolarized during Ca2+ overload.

Mitochondrial Contribution to the Anoxic Ca2+ Signal in Maize Suspension-Cultured Cells1

Anoxia induces a rapid elevation of the cytosolic Ca2+ concentration ([Ca2+]cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca2+ release is important for gene activation and survival in O2-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca2+ to the anoxic [Ca2+]cyt elevation in maize cells. Imaging of intramitochondrial Ca2+ levels showed that a majority of mitochondria released their Ca2+ in response to anoxia and took up Ca2+ upon reoxygenation. We also investigated whether the mitochondrial Ca2+ release contributed to the increase in [Ca2+]cyt under anoxia. Analysis of the spatial association between anoxic [Ca2+]cyt changes and the distribution of mitochondrial and other intracellular Ca2+ stores revealed that the largest [Ca2+]cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca2+]cyt under aerobic conditions but prevented a [Ca2+]cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca2+]cyt and participate in the signaling of O2 deprivation.

A High-Affinity Ca2+ Pump, ECA1, from the Endoplasmic Reticulum Is Inhibited by Cyclopiazonic Acid but Not by Thapsigargin1

To identify and characterize individual Ca2+ pumps, we have expressed an Arabidopsis ECA1 gene encoding an endoplasmic reticulum-type Ca2+-ATPase homolog in the yeast (Saccharomyces cerevisiae) mutant K616. The mutant (pmc1pmr1cnb1) lacks a Golgi and a vacuolar membrane Ca2+ pump and grows very poorly on Ca2+-depleted medium. Membranes isolated from the mutant showed high H+/Ca2+-antiport but no Ca2+-pump activity. Expression of ECA1 in endomembranes increased mutant growth by 10- to 20-fold in Ca2+-depleted medium. 45Ca2+ pumping into vesicles from ECA1 transformants was detected after the H+/Ca2+-antiport activity was eliminated with bafilomycin A1 and gramicidin D. The pump had a high affinity for Ca2+ (Km = 30 nm) and displayed two affinities for ATP (Km of 20 and 235 ��m). Cyclopiazonic acid, a specific blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, inhibited Ca2+ transport (50% inhibition dose = 3 nmol/mg protein), but thapsigargin (3 ��m) did not. Transport was insensitive to calmodulin. These results suggest that this endoplasmic reticulum-type Ca2+-ATPase could support cell growth in plants as in yeast by maintaining submicromolar levels of cytosolic Ca2+ and replenishing Ca2+ in endomembrane compartments. This study demonstrates that the yeast K616 mutant provides a powerful expression system to study the structure/function relationships of Ca2+ pumps from eukaryotes.

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca2+]cyt response. Cyclic-nucleotide-mediated elevations in [Ca2+]cyt involved both internal and external Ca2+ stores. Both cAMP- and cGMP-mediated [Ca2+]cyt elevations could be inhibited by the Ca2+-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca2+]cyt. Addition of the adenylate cyclase inhibitor 2���,5���-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca2+]cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca2+ signaling.

Heat-Shock-Induced Changes in Intracellular Ca2+ Level in Tobacco Seedlings in Relation to Thermotolerance1

Exposure of plants to elevated temperatures results in a complex set of changes in gene expression that induce thermotolerance and improve cellular survival to subsequent stress. Pretreatment of young tobacco (Nicotiana plumbaginifolia) seedlings with Ca2+ or ethylene glycol-bis(��-aminoethylether)-N,N,N���,N���-tetraacetic acid enhanced or diminished subsequent thermotolerance, respectively, compared with untreated seedlings, suggesting a possible involvement of cytosolic Ca2+ in heat-shock (HS) signal transduction. Using tobacco seedlings transformed with the Ca2+-sensitive, luminescent protein aequorin, we observed that HS temperatures induced prolonged but transient increases in cytoplasmic but not chloroplastic Ca2+. A single HS initiated a refractory period in which additional HS signals failed to increase cytosolic Ca2+. However, throughout this refractory period, seedlings responded to mechanical stimulation or cold shock with cytosolic Ca2+ increases similar to untreated controls. These observations suggest that there may be specific pools of cytosolic Ca2+ mobilized by heat treatments or that the refractory period results from a temporary block in HS perception or transduction. Use of inhibitors suggests that HS mobilizes cytosolic Ca2+ from both intracellular and extracellular sources.

Cytosolic Na+ controls and epithelial Na+ channel via the Go guanine nucleotide-binding regulatory protein.

In tight Na+-absorbing epithelial cells, the fate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-beta-S, pertussis toxin, and antibodies against the alpha-subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-.

Intrasarcomere [Ca2+] gradients in ventricular myocytes revealed by high speed digital imaging microscopy.

Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that significant intrasarcomeric [Ca2+] gradients should exist during activation. To directly test for this, we measured [Ca2+] distribution in single cardiac myocytes using fluorescent [Ca2+] indicators and high speed, three-dimensional digital imaging microscopy and image deconvolution techniques. Steep cytosolic [Ca2+] gradients from the t-tubule region to the center of the sarcomere developed during the first 15 ms of systole. The steepness of these [Ca2+] gradients varied with treatments that altered Ca2+ release from internal stores. Electron probe microanalysis revealed a loss of Ca2+ from the junctional SR and an accumulation, principally in the A-band during activation. We propose that the prolonged existence of [Ca2+] gradients within the sarcomere reflects the relatively long period of Ca2+ release from the SR, the localization of Ca2+ binding sites and Ca2+ sinks remote from sites of release, and diffusion limitations within the sarcomere. The large [Ca2+] transient near the t-tubular/ junctional SR membranes is postulated to explain numerous features of excitation-contraction coupling in cardiac muscle.