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The importance of the immediately releasable pool (IRP) of vesicles was proposed to reside in the maintenance of chromaffin cell secretion during the firing of action potentials at basal physiological frequencies. To accomplish this duty, IRP should be replenished as a function of time. We have previously reported that an action potential-like stimulus (APls) triggers the release of ~50% IRP, followed by a fast dynamin-dependent endocytosis and an associated rapid replenishment process. In this work, we investigated the endocytosis and IRP replenishment produced after the exocytosis of variable IRP fractions in mice primary chromaffin cell cultures. Exocytosis and endocytosis were estimated by membrane capacitance measurements obtained in patch-clamped cells. In addition to the dynamin-dependent fast endocytosis activated after the application of APls or 5 ms squared depolarizations, we found that depolarizations lasting 25-50 ms, which release80% of IRP, are related with a fast dynamin-independent, Ca

The importance of the immediately releasable pool (IRP) of vesicles was proposed to reside in the maintenance of chromaffin cell secretion during the firing of action potentials at basal physiological frequencies. To accomplish this duty, IRP should be replenished as a function of time. We have previously reported that an action potential-like stimulus (APls) triggers the release of ∽50% IRP, followed by a fast dynamin-dependent endocytosis and an associated rapid replenishment process. In this work we investigated the endocytosis and IRP replenishment produced after the exocytosis of variable IRP fractions in mice primary chromaffin cell cultures. Exocytosis and endocytosis were estimated by membrane capacitance measurements obtained in patch-clamped cells. In addition to the dynamin-dependent fast endocytosis activated after the application of APls or 5 ms squared depolarizations, we found that depolarizations lasting 25-50 ms, which release >80% of IRP, are related with a fast dynamin-independent, Ca2+- and protein kinase C (PKC)-dependent endocytosis (time constant < 1 s). PKC inhibitors, such as staurosporine, bisindolylmaleimide XI and prolonged treatments with high concentrations of phorbol esters, reduced and decelerated this endocytosis. Additionally, we found that the inhibition of PKC also abolished a slow component of replenishment (time constant ∽8 s) observed after total IRP exocytosis. Therefore, our results suggest that PKC contributes to the coordination of membrane retrieval and vesicle replenishment mechanisms that occur after the complete exocytosis of IRP.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as reporter of the GLUT4 vesicle-trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 μM ionomycin, indicating that in addition to impair exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 μM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

The maintenance of the secretory response requires a continuous replenishment of releasable vesicles. It was proposed that the immediately releasable pool (IRP) is important in chromaffin cell secretion during action potentials applied at basal physiological frequencies, because of the proximity of IRP vesicles to voltage-dependent Ca2+ channels. However, previous reports showed that IRP replenishment after depletion is too slow to manage such a situation. In this work, we used patch-clamp measurements of membrane capacitance, confocal imaging of F-actin distribution, and cytosolic Ca2+ measurements with Fura-2 to re-analyze this problem in primary cultures of mouse chromaffin cells. We provide evidence that IRP replenishment has one slow (time constant between 5 and 10 s) and one rapid component (time constant between 0.5 and 1.5 s) linked to a dynamin-dependent fast endocytosis. Both, the fast endocytosis and the rapid replenishment component were eliminated when 500 nM Ca2+ was added to the internal solution during patch-clamp experiments, but they became dominant and accelerated when the cytosolic Ca2+ buffer capacity was increased. In addition, both rapid replenishment and fast endocytosis were retarded when cortical F-actin cytoskeleton was disrupted with cytochalasin D. Finally, in permeabilized chromaffin cells stained with rhodamine-phalloidin, the cortical F-actin density was reduced when the Ca2+ concentration was increased in a range of 10–1000 nM. We conclude that low cytosolic Ca2+ concentrations, which favor cortical F-actin stabilization, allow the activation of a fast endocytosis mechanism linked to a rapid replenishment component of IRP. (Figure presented.). Fil: Montenegro, Mauricio Norman. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Bayonés, Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Moya Diaz, José Abelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Borassi, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Toscani, Andrés Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Gallo, Luciana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Marengo, Fernando Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

Traditional approaches for risk assessment of ischemic heart disease (IHD) are based on the physiological consequences of an epicardial coronary stenosis. Of note, normal coronary arteries or nonobstructive coronary artery disease (CAD) is a common finding in women with signs and symptoms of ischemia. Therefore, assessment of risk based on a coronary stenosis approach may fail in women. Positron emission tomography (PET) quantifies absolute myocardial blood flow (MBF) which may help to elucidate other mechanisms involved such as endothelial dysfunction and alterations in the smooth muscle cell relaxation responsible for IHD in women. The objective of the present review is to describe the current state of the art of PET imaging in assessing IHD in women with nonobstructive CAD. Fil: Campisi, Roxana. Instituto Argentino de Diagnostico y Tratamiento; Argentina. Diagnostico Maipu; Argentina Fil: Marengo, Fernando Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

AIM It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca2+ entry through voltage-dependent Ca2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca2+ current and intracellular Ca2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells. METHODS Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca2+ was estimated using epifluorescence microscopy and fluo-8 (salt form). RESULTS Cells stimulated by brief depolatizations in absence of extracellular Ca+2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca+2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant

It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by CaExocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic CaCells stimulated by brief depolatizations in absence of extracellular CaWe demonstrated that Ca

To review the value of myocardial blood flow and coronary flow reserve (CFR) measurements, as assessed by positron emission tomography (PET), in women with suspected ischemic heart disease. CFR is a noninvasive measure of coronary vasomotor function that integrates the hemodynamic effects of epicardial coronary stenosis, diffuse atherosclerosis, and microvascular dysfunction on myocardial tissue perfusion and has emerged as an imaging marker of cardiovascular risk, independently of the degree of obstructive coronary artery disease (CAD). Normal coronary arteries or nonobstructive CAD is a common finding in women with ischemia. Thus, assessment of risk based on a coronary stenosis approach may fail in women. PET is able to quantify absolute myocardial blood flow and CFR which may help to elucidate other mechanisms involved such as microvascular dysfunction and diffuse epicardial CAD, responsible for the disease in women.

The extent and type of hormones and active peptides secreted by the chromaffin cells of the adrenal medulla have to be adjusted to physiological requirements. The chromaffin cell secretory activity is controlled by the splanchnic nerve firing frequency, which goes from approximately 0.5 Hz in basal conditions to more than 15 Hz in stress. Thus, these neuroendocrine cells maintain a tonic release of catecholamines under resting conditions, massively discharge intravesicular transmitters in response to stress, or adequately respond to moderate stimuli. In order to adjust the secretory response to the stimulus, the adrenal chromaffin cells have an appropriate organization of Ca2+ channels, secretory granules pools, and sets of proteins dedicated to selectively control different steps of the secretion process, such as the traffic, docking, priming and fusion of the chromaffin granules. Among the molecules implicated in such events are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Ca2+ sensors like Munc13 and synaptotagmin-1, chaperon proteins such as Munc18, and the actomyosin complex. In the present review, we discuss how these different actors contribute to the extent and maintenance of the stimulus-dependent exocytosis in the adrenal chromaffin cells. Fil: Marengo, Fernando Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Cárdenas, Ana M.. Universidad de Valparaíso; Chile

Under basal conditions the action potential firing rate of adrenal chromaffin cells is lower than 0.5 Hz. The maintenance of the secretory response at such frequencies requires a continuous replenishment of releasable vesicles. However, the mechanism that allows such vesicle replenishment remains unclear. Here, using membrane capacitance measurements on mouse chromaffin cells, we studied the mechanism of replenishment of a group of vesicles released by a single action potential-like stimulus (APls). The exocytosis triggered by APls (ETAP) represents a fraction (40%) of the immediately releasable pool, a group of vesicles highly coupled to voltage dependent calcium channels. ETAP was replenished with a time constant of 0.73 � 0.11 s, fast enough to maintain synchronous exocytosis at 0.2-0.5 Hz stimulation. Regarding the mechanism involved in rapid ETAP replenishment, we found that it depends on the ready releasable pool; indeed depletion of this vesicle pool significantly delays ETAP replenishment. On the other hand, ETAP replenishment also correlates with a dynamin-dependent fast endocytosis process (τ = 0.53 � 0.01 s). In this regard, disruption of dynamin function markedly inhibits the fast endocytosis and delays ETAP replenishment, but also significantly decreases the synchronous exocytosis during repetitive APls stimulation at low frequencies (0.2 and 0.5 Hz). Considering these findings, we propose a model in where both the transfer of vesicles from ready releasable pool and fast endocytosis allow rapid ETAP replenishment during low stimulation frequencies. Fil: Moya Diaz, José Abelino. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Alvarez, Yanina Daniela. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Montenegro, Mauricio Norman. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina Fil: Bayonés, Lucas. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina Fil: Belingheri, Ana Verónica. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: González-Jamett, Arlek M.. Universidad de Valparaiso; Chile Fil: Cárdenas, Ana M.. Universidad de Valparaiso; Chile Fil: Marengo, Fernando Diego. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina

Aim: After exocytosis, neuroendocrine cells and neurones keep constant the plasma membrane and the releasable vesicle pools by performing endocytosis and vesicular cycling. Patch-clamp capacitance measurements on chromaffin cells showed that strong Ca +2 entry activates excess retrieval: a rapid endocytosis process that retrieves more membrane than the one fused by preceding exocytosis. The main purpose of the present experiments was to study the recycling pathway that follows excess retrieval, which is unknown. Methods: Membrane recycling after exocytosis‐endocytosis can be studied by fluorescence imaging assays with FM1-43 (Perez Bay et al. Am J Physiol Cell Physiol 2007; 293, C1509). In this work, we used this assay in combination with fluorescent dextrans and specific organelle-targeted antibodies to study the membrane recycling after excess retrieval in mouse chromaffin cells. Results: Excess retrieval was observed after the application of high-K + or cholinergic agonists during 15 or 30 s in the presence of FM1-43. We found that the excess retrieval membrane pool (defined as endocytosis–exocytosis) was associated with the generation of a non-releasable fraction of membrane (up to 30% of plasma membrane surface) colocalizing with the lysosomal compartment. The excess retrieval membrane pool followed a saturable cytosolic Ca 2+ dependency, and it was suppressed by inhibitors of L-type Ca 2+ channels, endoplasmic reticulum Ca 2+ release and PKC. Conclusion: Excess retrieval is not associated with the cycling of releasable vesicles, but it is related to the formation of non-releasable endosomes. This process is activated by a concerted contribution of Ca 2+ entry through L-channels and Ca 2+ release from endoplasmic reticulum.

J. Neurochem. (2011) 116, 155–163. Abstract In neuroendocrine cells, such as adrenal chromaffin cells, the exocytosis of hormone-filled vesicles is triggered by a localized Ca2+ increase that develops after the activation of voltage-dependent Ca2+ channels. To reach the fusion competent state, vesicles have to go through a series of maturation steps that involve the detachment from cytoskeletal proteins, docking and priming. However, the fusion readiness of vesicles will also depend on their proximity to the calcium source. The immediately releasable pool is a small group of ready-to-fuse vesicles, whose fusion is tightly coupled to Ca2+ entry through channels. Recent work indicates that such coupling is not produced by a random distribution between vesicles and channels, but would be the result of a specific interaction of immediately releasable vesicles with particular Ca2+ channel subtypes. The immediately releasable pool is able to sustain, with high efficiency, the secretion triggered by the small and localized Ca2+ gradients produced by brief depolarizations at low frequencies, like action potentials at basal conditions in adrenal chromaffin cells.

Chromaffin cell exocytosis is triggered by Ca(2+) entry through several voltage-dependent channel subtypes. Because it was postulated that immediately releasable vesicles are closely associated with Ca(2+) channels, we wondered what channel types are specifically coupled to the release of this pool. To study this question, cultured mouse chromaffin cell exocytosis was followed by patch-clamp membrane capacitance measurements. The immediately releasable pool was estimated using paired pulse stimulation, resulting in an upper limit of 31+/-3 fF for control conditions (I(Ca): 25+/-2 pA/pF). The N-type channel blocker omega-conotoxin-GVIA affected neither I(Ca) nor the immediately releasable pool exocytosis; although the L channel blocker nitrendipine decreased current by 50%, it did not reduce this pool significantly; and the R channel inhibitor SNX-482 significantly reduced the current but induced only a moderate decrease in the estimated IRP exocytosis. In contrast, the P/Q channel blocker omega-Agatoxin-IVA decreased I(Ca) by 37% but strongly reduced the immediately releasable pool (upper limit: 6+/-1 fF). We used alpha1A subunit knockout mice to corroborate that P/Q Ca(2+) channels were specifically linked to immediately releasable vesicles, and we found that also in this preparation the exocytosis of this pool was severely decreased (6+/-1 fF). On the other hand, application of a strong stimulus that caused the fusion of most of releasable vesicles (3 min, 50 mM K(+)) induced similar exocytosis for wild type and knockout cells. Finally, whereas application of train stimulation on chromaffin cells derived from wild type mice provoked typical early synchronous and delayed asynchronous exocytosis components, the knockout derived cells presented a strongly depressed early exocytosis but showed a prominent delayed asynchronous component. These results demonstrate that P/Q are the dominant calcium channels associated to the release of immediately releasable pool in mouse chromaffin cells.

Neurons and neuroendocrine cells must retrieve plasma membrane excess and refill vesicle pools depleted by exocytosis. To perform these tasks cells can use different endocytosis/recycling mechanisms whose selection will impact on vesicle recycling time and secretion performance. We used FM1-43 to evaluate in the same experiment exocytosis, endocytosis, and recovery of releasable vesicles on mouse chromaffin cells. Various exocytosis levels were induced by a variety of stimuli, and we discriminated the resultant endocytosis-recycling responses according to their ability to rapidly generate releasable vesicles. Exocytosis of ≤20% of plasma membrane (provoked by nicotine/acetylcholine) was followed by total recovery of releasable vesicles. If a stronger stimulus (50 mM K+and 2 mM Ca2+) provoking intense exocytosis (51 ± 7%) was applied, endocytosis still retrieved all the fused membrane, but only a fraction (19 ± 2%) was releasable by a second stimulus. Using ADVASEP-7 or bromophenol blue to quickly eliminate fluorescence from noninternalized FM1-43, we determined that this fraction became releasable in

Traditionally, it was accepted that long-term hormone replacement therapy (HRT) has a cardiovascular beneficial effect in postmenopausal women with and without coronary artery disease (CAD). However, randomized trials in postmenopausal women have not shown any benefit in either primary or secondary prevention of cardiovascular events. Therefore, these findings have raised the question of whether traditional HRT (i.e., estrogen and progesterone) has a cardioprotective effect in women at risk for or with established CAD. Concerns about the use of conventional HRT have led to a search for alternatives. Tibolone is a synthetic compound with estrogenic, androgenic, and progestogenic properties that relieves climacteric symptoms and prevents postmenopausal bone loss. Tibolone possesses a tissue-selective mechanism of action that differs from that of estrogen and/or progestogen. Unlike these compounds, tibolone's metabolites play a central role in its mode of action. Tibolone is widely used for HRT. However, its clinical impact on cardiovascular disease is still under study. The current review focuses on the effects of tibolone on the cardiovascular system and discusses clinical investigations with this compound in postmenopausal women.

The relationship between the localized Ca 2+ concentration and depolarization-induced exocytosis was studied in patch-clamped adrenal chromaffin cells using pulsed-laser Ca 2+ imaging and membrane capacitance measurements. Short depolarizing voltage steps induced Ca 2+ gradients and small “synchronous” increases in capacitance during the pulses. Longer pulses increased the capacitance changes, which saturated at 16 fF, suggesting the presence of a small immediately releasable pool of fusion-ready vesicles. A Hill plot of the capacitance changes versus the estimated Ca 2+ concentration in a thin (100 nm) shell beneath the membrane gave n = 2.3 and K d = 1.4 μM. Repetitive stimulation elicited a more complex pattern of exocytosis: early pulses induced synchronous capacitance increases, but after five or more pulses there was facilitation of the synchronous responses and gradual increases in capacitance continued between pulses (asynchronous exocytosis) as the steep submembrane Ca 2+ gradients collapsed. Raising the pipette Ca 2+ concentration led to early facilitation of the synchronous response and early appearance of asynchronous exocytosis. We used this data to develop a kinetic model of depolarization-induced exocytosis, where Ca 2+ -dependent fusion of vesicles occurs from a small immediately releasable pool with an affinity of 1–2 μM and vesicles are mobilized to this pool in a Ca 2+ -dependent manner.

Exocytosis in adrenal chromaffin cells is strongly influenced by the pattern of stimulation. To understand the dynamic and spatial properties of the underlying Ca(2+) signal, we used pulsed laser Ca(2+) imaging to capture Ca(2+) gradients during stimulation by single and repetitive depolarizing stimuli. Short single pulses (10-100 ms) lead to the development of submembrane Ca(2+) gradients, as previously described (F. D. Marengo and J. R. Monck, 2000, Biophysical Journal, 79:1800-1820). Repetitive stimulation with trains of multiple pulses (50 ms each, 2Hz) produce a pattern of intracellular Ca(2+) increase that progressively changes from the typical Ca(2+) gradient seen after a single pulse to a Ca(2+) increase throughout the cell that peaks at values 3-4 times higher than the maximum values obtained at the end of single pulses. After seven or more pulses, the fluorescence increase was typically larger in the interior of the cell than in the submembrane region. The pattern of Ca(2+) gradient was not modified by inhibitors of Ca(2+)-induced Ca(2+) release (ryanodine), inhibitors of IP(3)-induced Ca(2+) release (xestospongin), or treatments designed to deplete intracellular Ca(2+) stores (thapsigargin). However, we found that the large fluorescence increase in the cell interior spatially colocalized with the nucleus. These results can be simulated using mathematical models of Ca(2+) redistribution in which the nucleus takes up Ca(2+) by active or passive transport mechanisms. These results show that chromaffin cells can respond to depolarizing stimuli with different dynamic Ca(2+) signals in the submembrane space, the cytosol, and the nucleus.

Fil: Bonazzola, Patricia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Cardiologicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiologicas; Argentina

Most accepted single particle tracking methods are able to obtain high-resolution trajectories for relatively short periods of time. In this work we apply a straightforward combination of single-particle tracking microscopy and metallic nanoparticles internalization on mouse chromaffin cells to unveil the intracellular trafficking mechanism of metallic-nanoparticle-loaded vesicles (MNP-V) complexes after clathrin dependent endocytosis. We found that directed transport is the major route of MNP-Vs intracellular trafficking after stimulation (92.6% of the trajectories measured). We then studied the MNP-V speed at each point along the trajectory, and found that the application of a second depolarization stimulus during the tracking provokes an increase in the percentage of low-speed trajectory points in parallel with a decrease in the number of high-speed trajectory points. This result suggests that stimulation may facilitate the compartmentalization of internalized MNPs in a more restricted location such as was already demonstrated in neuronal and neuroendocrine cells (Bronfman et al 2003 J. Neurosci. 23 3209-20). Although further experiments will be required to address the mechanisms underlying this transport dynamics, our studies provide quantitative evidence of the heterogeneous behavior of vesicles mobility after endocytosis in chromaffin cells highlighting the potential of MNPs as alternative labels in optical microscopy to provide new insights into the vesicles dynamics in a wide variety of cellular environments. Fil: Gabriel, Manuela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Moya Diaz, José Abelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Gallo, Luciana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Marengo, Fernando Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Estrada, Laura Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina

The consequences of an extrasystole (ES) on cardiac muscle’s energetics and Ca2+homeostasis were investigated in the beating heart. The fraction of heat release related to pressure development (pressure dependent) and pressure-independent heat release were measured during isovolumic contractions in arterially perfused rat ventricle. The heat release by a contraction showed two pressure-independent components (H1 and H2) of short evolution and a pressure-dependent component (H3). The additional heat released by ES was decomposed into one pressure-independent ([Formula: see text]) and one pressure-dependent ([Formula: see text]) component with time courses similar to those of control components H2 and H3. ES also induced the potentiation of pressure development (P) and heat release during the postextrasystolic (PES) beat. The slope of the linear relationship between pressure-dependent heat and pressure maintenance was similar in control, ES, and PES contractions (0.08 ± 0.01, 0.10 ± 0.02, and 0.08 ± 0.01 mJ ⋅ g−1⋅ mmHg−1⋅ s−1, respectively). The potentiation of H2 (heat component related with Ca2+removal processes) in PES was equal to [Formula: see text] at 0.3, 0.5, 1, and 2 mM Ca2+, suggesting that the extra amount of Ca2+mobilized during ES was recycled in PES. Pretreatment with 1 mM caffeine to deplete sarcoplasmic reticulum Ca2+content inhibited both the mechanical and energetic potentiation of PES. However, the heat released and the pressure developed during ES were not changed by sarcoplasmic reticulum depletion. The results suggest that 1) the source of Ca2+for ES would be entirely extracellular, 2) the Ca2+entered during ES is accumulated in the sarcoplasmic reticulum, and 3) the Ca2+stored by the sarcoplasmic reticulum during ES induces an increased contribution of this organelle during PES compared with the normal contraction.

Heat production and isovolumetric pressure development (P) were measured simultaneously in the arterially perfused rat ventricle. The time course of the calorimetric signal that follows a contraction could be decomposed into four components of energy released. Three of these components (H1, H2, and H4) were pressure independent, only H3 correlated with either P or the pressure-time integral (PtI) (r > 0.78, n = 36, P < 0.01). The dimensionless slope of the regression of H3 on P was 0.24 (an index of muscle economy) and the absence of O2 (N2 replacement) decreased it to 0.178 suggesting that 26% of H3 is related to oxidative metabolism. H4 was the most affected by the lack of O2 in the perfusate. It decreased to 16% in the first beat under N2 without change in P or in H1, H2 or H3, and disappeared (1.6 +/- 1.0 mJ.g-1) in the fourth contraction under N2 (while P, H1, H2 and H3 remained over 64% of their control values). H4 was activated during the first 1-3 beats after a quiescent period and remained active for several seconds (even in the absence of subsequent stimulation) as if the basal metabolism had been increased to a new steady level. H1 and H2 were dependent on the extracellular Ca. The magnitudes of both H1 (1.8 +/- 0.2 mJ.g-1) and H2 (2.7 +/- 0.2 mJ.g-1) were similar to those reported for the fast and slow components of activation heat in skeletal muscle. If twin stimuli are applied (200 ms apart), additional energy is released (+3.0 +/- 0.3 mJ.g-1) that can be decomposed in two components similar to those identified as H2 and H3. The magnitude of H1, its absence in the twin contraction and its Ca dependency suggest an association with Ca-binding processes (mainly Troponin C). The presence of an H2 component during the twin contraction, its magnitude and Ca dependence gives support to a relationship between H2 and Ca removal processes.

It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca(2+) channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca(2+) current. Accordingly, in the present work we found that the Ca(2+) current flowing through P/Q-type Ca(2+) channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels. To investigate the mechanism that underlies the coupling between the immediately releasable pool and P/Q-type channels we transiently expressed in mouse chromaffin cells peptides corresponding to the synaptic protein interaction site of Cav2.2 to competitively uncouple P/Q-type channels from the secretory vesicle release complex. This treatment reduced the efficiency of Ca(2+) current to induce exocytosis to similar values as direct inhibition of P/Q-type channels via ω-agatoxin-IVA. In addition, the same treatment markedly reduced immediately releasable pool exocytosis, but did not affect the exocytosis provoked by sustained electric or high K(+) stimulation. Together, our results indicate that the synaptic protein interaction site is a crucial factor for the establishment of the functional coupling between immediately releasable pool vesicles and P/Q-type Ca(2+) channels. Fil: Alvarez, Yanina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina; Fil: Belingheri, Ana Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina; Fil: Perez Bay, Andrés Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina; Fil: Javis, Scott E.. University of Calgary. Hotchkiss Brain Institute; Canada; Fil: Tedford, H. William. University of Calgary. Hotchkiss Brain Institute; Canada; Fil: Zamponi, Gerald. University of Calgary. Hotchkiss Brain Institute; Canada; Fil: Marengo, Fernando Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

In neuroendocrine cells, such as adrenal chromaffin cells, the exocytosis of hormone-filled vesicles is triggered by a localized Ca(2+) increase that develops after the activation of voltage-dependent Ca(2+) channels. To reach the fusion competent state, vesicles have to go through a series of maturation steps that involve the detachment from cytoskeletal proteins, docking and priming. However, the fusion readiness of vesicles will also depend on their proximity to the calcium source. The immediately releasable pool is a small group of ready-to-fuse vesicles, whose fusion is tightly coupled to Ca(2+) entry through channels. Recent work indicates that such coupling is not produced by a random distribution between vesicles and channels, but would be the result of a specific interaction of immediately releasable vesicles with particular Ca(2+) channel subtypes. The immediately releasable pool is able to sustain, with high efficiency, the secretion triggered by the small and localized Ca(2+) gradients produced by brief depolarizations at low frequencies, like action potentials at basal conditions in adrenal chromaffin cells.

Endocytosis is a crucial process for neuroendocrine cells that ensures membrane homeostasis, vesicle recycling, and hormone release reliability. Different endocytic mechanisms have been described in chromaffin cells, such as clathrin-dependent slow endocytosis and clathrin-independent rapid endocytosis. Rapid endocytosis, classically measured in terms of a fast decrease in membrane capacitance, exhibits two different forms, ‘‘rapid compensatory endocytosis’’ and ‘‘excess retrieval.’’ While excess retrieval seems to be associated with formation of long-lasting endosomes, rapid compensatory endocytosis is well correlated with exocytotic activity, and it is regarded as a mechanism associated to rapid vesicle recycling during normal secretory activity. It has been suggested that rapid compensatory endocytosis may be related to the prevalence of a transient fusion mode of exo-endocytosis. In the latter mode, the fusion pore, a nanometric-sized channel formed at the onset of exocytosis, remains open for a few hundred milliseconds and later abruptly closes, releasing a small amount of transmitters. By this mechanism, endocrine cell selectively releases low molecular weight transmitters, and rapidly recycles the secretory vesicles. In this article, we discuss the cellular and molecular mechanisms that define the different forms of exocytosis and endocytosis and their impact on vesicle recycling pathways. Fil: Cárdenas, Ana María. Universidad de Valparaíso. Centro Interdisciplinario de Neurociencia; Chile Fil: Marengo, Fernando Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

Background —The effect of long-term smoking on coronary vasomotion and vasodilator capacity in healthy smokers is unknown. Methods and Results —Myocardial blood flow (MBF) was quantified with [ 13 N]ammonia and positron emission tomography (PET) at rest, during cold pressor testing (endothelium-dependent vasomotion), and during dipyridamole-induced hyperemia in 16 long-term smokers and 17 nonsmokers. MBF at rest did not differ between the 2 groups. Cold induced similar increases in rate-pressure product (RPP) in smokers and nonsmokers. However, MBF increased only in nonsmokers and was, during cold, higher than in smokers (0.91±0.18 versus 0.78±0.14 mL · g −1 · min −1 , P P =0.0006, and 0.99±0.25 versus 0.96±0.27, P =NS). The hyperemic response to dipyridamole and the myocardial flow reserve did not differ between the 2 groups. In a multiple regression model adjusted for age, sex, serum lipid levels, years of smoking, and pack-years, years of smoking was the strongest predictor of the normalized blood flow response to cold ( P Conclusions —The normal hyperemic response to dipyridamole in long-term smokers indicates a preserved endothelium-independent coronary vascular smooth muscle relaxation, whereas the abnormal response to cold suggests a defect in coronary vasomotion likely located at the level of the coronary endothelium. Its severity depends on the total exposure time to smoking.

The specific removal of negatively-charged sialic acid by neuraminidase produces a large increase in cardiac myocyte Ca uptake (17.3 +/- 1.1 mmol Ca/kg dry weight) and marked cell contracture. Importantly, the insertion of the negatively-charged amphiphile dodecyl sulfate in the sarcolemma eliminates the increased calcium uptake and preserves contractile function. In the present study, we further examine the role of sialic acid-Ca interaction and, specifically, the role of gangliosides (sialic acid-containing glycolipids) in cardiac cells' Ca permeability. Neonatal cell culture and adult ventricular myocytes were used. The major findings of this study are: (1) while dodecyl sulfate inhibits cellular calcium uptake and contracture development induced by sialic acid removal, cationic and neutral amphiphiles are without effect. (2) Ca channel blockers (nifedipine and protamine) and the Na/Ca exchange inhibitor Ni do not modify the effect of sialic acid removal. (3) A non-classical-channel related whole-cell current appears and increases after 21 +/- 2.2 min treatment with 0.02 U/ml neuraminidase (n = 4). Incubation with neuraminidase in the presence of dodecyl sulfate greatly delays the appearance of these currents to 44.4 +/- 6.1 min (n = 4). (4) The use of a specific probe for GM1 ganglioside, the cholera toxin B subunit (3 micrograms/ml), induces a moderate but clear increase in cellular Ca (1.63 +/- 0.3 mmol Ca kg dry weight; n = 8). However, this increase was not modified by treatment with dodecyl sulfate. (5) Finally, 50 mU/ml endoglycoceramidase, an enzyme which specifically cleaves the link between the sialic acid-containing oligosaccharide and ceramide of gangliosides, induced a significant increase in Ca uptake (4.4 +/- 0.9 mmol Ca kg dry weight, n = 4). These results indicate the importance of negatively charged sialic acid-containing gangliosides in the maintenance of cardiac cell physiological Ca permeability. The increase in Ca uptake induced by the removal of sialic acid seems to be mediated by development of a Ca "leak" via other than classical cation channels.

Cardiac myocyte Ca transport systems, such as sarcoplasmic reticulum Ca-ATPase and sarcolemmal Na/Ca exchange (Na/Ca), are critically dependent on temperature. The purpose of this work was to study the effect of temperature on cellular Ca compartmentation and its exchange characteristics in intact functional neonatal cultured myocytes. The Na/Ca mediated Ca exchange (CaNa/Ca)--including its sarcoplasmic reticulum (SR) and sarcolemmal (SL) contributions, a slow exchange component related to mitochondrial Ca and the La displaceable Ca pool were studied combining isotopic and gas-dissection techniques for membrane isolation. The major findings of this study are: (i) The amount of Ca exchanged through CaNa/Ca is clearly dependent on temperature (Q10 approximately 1.6) in the range studied (17-37 degrees C); (ii) the addition of 1 microM nifedipine does not modify the temperature dependence of CaNa/Ca; (iii) the sarcolemmal bound fraction contributing to CaNa/Ca is not changed by temperature; (iv) the increase in CaNa/Ca with temperature is explained by an increment in the contribution of SR-Ca to CaNa/Ca; (v) a fraction of SR which does not exchange via CaNa/Ca at low temperatures can be released and mobilized by caffeine-this caffeine sensitive fraction is reduced as temperature is increased and is no longer measurable as a separate entity at 37 degrees C; (vi) if we consider (iv) and (v) together, SR content would be temperature dependent with a Q10 of approximately 1.5; (vii) a La displaceable pool, which represents over 66% of the total exchangeable Ca, increases in the range of 22-33 degrees C with a Q10 of 1.25 which is consistent with a pool distribution of 70% SL-bound and 30% SR-derived [Post J.A., Langer G.A. Cellular origin of the rapidly exchangeable calcium pool in the cultured neonatal rat heart cell. Cell Calcium 1992; 13: 627-634]; and (viii) the rate constant for the mitochondrial Ca component increases by 60% from 22 degrees C to 37 degrees C, but Ca content in this organelle is not modified over this temperature range.

We used pulsed laser imaging to measure the development and dissipation of Ca(2+) gradients evoked by the activation of voltage-sensitive Ca(2+) channels in adrenal chromaffin cells. Ca(2+) gradients appeared rapidly (