Impact of COVID-19 vaccination in post-COVID cardiac complications.

BACKGROUND: After the acute infection, COVID-19 can produce cardiac complications as well as long-COVID persistent symptoms. Although vaccination against COVID-19 represented a clear reduction in both mortality and ICU admissions, there is very little information on whether this was accompanied by a decrease in the prevalence of post-COVID cardiac complications. The aim of this study was to analyze the relationship between COVID-19 vaccination and the prevalence of post-COVID cardiac injury assessed by echocardiogram, and long-COVID persistent cardiac symptoms. METHODS: All patients who consulted for post-COVID evaluation 14 days after discharge from acute illness were included. Patients with heart disease were excluded. The relationship between complete vaccination scheme (at least two doses applied with 14 days or more since the last dose) and pathological echocardiographic findings, as well as the relationship of vaccination with persistent long-COVID symptoms, were evaluated by multivariate analysis, adjusting for age, sex and clinical variables that would have shown significant differences in univariate analysis. RESULTS: From 1883 patients, 1070 patients (56.8%) suffered acute COVID-19 without a complete vaccination scheme. Vaccination was associated with lower prevalence of cardiac injury (1.35% versus 4.11%, adjusted OR 0.33; 95% CI 0.17-0.65, p=0.01). In addition, vaccinated group had a lower prevalence of persistent long-COVID symptoms compared to unvaccinated patients (10.7% versus 18.3%, adjusted OR 0.52; 95% CI 0.40-0.69, p<0.001). CONCLUSION: Vaccination against COVID-19 was associated with lower post-COVID cardiac complications and symptoms, reinforcing the importance of fully vaccinating the population.

Injuria cardíaca post- COVID19 y su relación con la gravedad del cuadro agudo, los síntomas persistentes y la vacunación / Cardiac Injury after COVID-19 and its Association with the Severity of the Acute Presentation, Persistent Symptoms and Vaccination

RESUMEN Introducción: La infección por COVID-19 se asocia a compromiso cardiovascular en su etapa aguda. La información sobre el compromiso cardíaco post-COVID es muy heterogénea, y la indicación de realizar estudios de imágenes cardíacas de forma rutinaria es aún controvertida. Asimismo, no existe información actualizada sobre el efecto que produjo la vacunación masiva en la incidencia de la injuria cardíaca post-COVID. Objetivos: Analizar la prevalencia de injuria cardíaca mediante ecocardiograma luego de la infección por COVID-19 y su asociación con la gravedad del cuadro agudo y con los síntomas persistentes post-COVID. Como objetivo secundario se exploró la relación de la prevalencia de injuria cardíaca con el inicio de la campaña de vacunación contra COVID-19 en la República Argentina. Material y métodos: Estudio analítico, observacional, prospectivo y unicéntrico. Se incluyeron todos los pacientes consecutivos que consultaron para realizar evaluación post-COVID. Se realizó ecocardiograma transtorácico en todos los pacientes. Se consideró la fecha de inicio de la campaña de vacunación (29/12/2020) para el análisis de los subgrupos pre y post vacunación. Resultados: Se incluyeron los primeros 1000 pacientes que consultaron al centro desde el 01/09/2020 al 01/09/2021. Treinta y nueve (3,9%) presentaron hallazgos patológicos en el ecocardiograma compatibles con injuria post-COVID, incluyendo disfunción ventricular izquierda (2,8%), derrame pericárdico (0,5%) y trastorno de motilidad parietal (0,6%), no conocidos previamente. Los pacientes que padecieron cuadros de COVID-19 agudo moderados o graves presentaron mayor prevalencia de trastornos de motilidad parietal (2,9% versus 0,3%, p = 0,001) y derrame pericárdico (2,9% versus 0,14%, p = 0,001) en comparación con aquellos con cuadros asintomáticos o leves, y esta asociación se mantuvo al ajustar por factores de riesgo cardiovascular y edad (OR 6,7; IC 95% 1,05-42,2, p = 0,04 y OR 25,1; IC 95% 2,1-304,9, p = 0,01 respectivamente). El 19,3% de los pacientes referían síntomas persistentes en la consulta post-COVID; en estos pacientes se observó mayor evidencia de disfunción ventricular izquierda nueva (8,3% vs 2,4%, p<0,005); no obstante, dicha asociación perdió significancia en el análisis multivariado. Respecto a la relación de la injuria cardíaca con el inicio de la vacunación, los 330 pacientes que se realizaron estudios de control post-COVID previamente al inicio de la campaña presentaron mayor prevalencia de injuria que los 670 pacientes luego de esta fecha (6,3% vs 2,7%, p = 0,006). Esta relación se mantuvo en el análisis multivariado (OR 0,35; IC95% 0,17-0,69). Conclusión: La prevalencia de injuria cardíaca evaluada mediante ecocardiograma luego de la infección por COVID-19 fue de 3,9%. Se observó una asociación significativa e independiente entre cuadros iniciales de mayor gravedad y hallazgos patológicos en el ecocardiograma en la etapa post-COVID, no así con los síntomas persistentes. Los pacientes que consultaron luego del inicio de la campaña de vacunación en Argentina presentaron menos prevalencia de injuria cardíaca en comparación con los pacientes de la primera ola.

ABSTRACT Background: COVID-19 is associated with cardiovascular involvement in the acute phase. The information about cardiac involvement after COVID-19 is heterogeneous, and the indication to routinely perform cardiac imaging tests is still controversial. There is no updated information on the effect mass vaccination has on the incidence of cardiac injury after COVID-19. Objectives: The primary objective of this study was to evaluate the prevalence of cardiac injury after COVID-19 by transthoracic echocardiography and its association with the severity of the acute phase and with persistent symptoms after recovery. The secondary objective was to explore the association of the prevalence of cardiac injury with the beginning of the vaccination campaign against COVID-19 in Argentina. Methods: We conducted an observational, single-center, and retrospective study. All the consecutive patients who consulted for post-COVID-19 evaluation were included. All the patients underwent transthoracic echocardiography. The date the vaccination campaign started (12/29/2020) was considered the cut-off point for the analysis of the pre-vaccination and postvaccination subgroups. Results: The first 1000 patients who consulted in our center between 09/01/2020 and 09/01/2021 were included. Thirty-nine patients (3.9%) had new abnormal echocardiographic findings suggestive of cardiac injury after COVID-19, including left ventricular dysfunction (2.8%), pericardial effusion (0.5%), and wall motion abnormalities (0.6%). Patients with moderate or severe acute COVID-19 presented a higher prevalence of wall motion abnormalities (2.9% versus 0.3%, p= 0.001) and pericardial effusion (2.9% versus 0.14%, p = 0.001) compared to those with asymptomatic or mild COVID-19 and this association remained after adjusting for cardiovascular risk factors and age (OR 6.7, 95% CI 1.05-4.2, p = 0.04, and OR 25.1, 95% 2.1-304.9, p = 0.01 respectively). The percentage of patients who reported persistent symptoms during consultation after COVID-19 was 19.3%, and they had higher evidence of new left ventricular dysfunction (8.3% vs. 2.4%, p < 0.005); however, this association lost significance on multivariate analysis. When the association of cardiac injury with the start of vaccination was considered, the 330 patients who underwent post-COVID assessment before the vaccination campaign started had a higher prevalence of injury than the 670 patients evaluated after this date (6.3% vs. 2.7%, p = 0.006). and this association persisted on multivariate analysis (OR 0.35; 95%CI 0.17-0.69). Conclusion: The prevalence of cardiac injury assessed by echocardiography after COVID-19 was 3.9%. There was a significant and independent association between the severe initial presentations and the abnormal echocardiographic findings after COVID-19, but not with persistent symptoms. Patients who consulted after the vaccination campaign started in Argentina had a lower prevalence of cardiac injury compared with those patients in the first wave.

Compromiso cardíaco y su relación con la gravedad del cuadro agudo y los síntomas persistentes en la convalecencia de infección por COVID-19 / Cardiovascular Involvement and its Relationship with the Severity of the Acute Phase and Persistent Symptoms during Recovery from COVID-19 Infection

RESUMEN Introducción: La infección por COVID-19 se asocia a compromiso cardiovascular en su etapa aguda. La información sobre el compromiso cardíaco en la etapa de convalecencia de la enfermedad tanto en pacientes con y sin síntomas persistentes es limitada. Objetivos: 1. Analizar el compromiso cardíaco mediante ecocardiograma en la etapa de convalecencia de la enfermedad por COVID-19; 2. Explorar su asociación con: a) gravedad del cuadro agudo y b) persistencia de síntomas. Métodos: Estudio analítico, observacional, prospectivo y unicéntrico. Se incluyeron pacientes consecutivos que consultaron al centro para realizar evaluación post-COVID. Se realizó ecocardiograma Doppler color transtorácico en busca de hallazgos patológicos. Resultados: Se incluyeron 600 pacientes desde el 01/09/2020 al 01/05/2021. Veintinueve (4,8%) presentaron hallazgos patológicos en el ecocardiograma. Los pacientes con cuadros iniciales moderados o graves presentaron mayor prevalencia de trastornos de motilidad parietal (4,3% versus 0,5%, p = 0,02) y derrame pericárdico (4,3% versus 0,24%, p = 0,01) en comparación con aquellos con cuadros asintomáticos o leves. En el ajuste multivariado esta asociación no alcanzó significación estadística. El 28,6% de los pacientes referían síntomas persistentes, no observándose una asociación entre la presencia de los mismos y los hallazgos ecocardiográficos patológicos. Conclusión: La prevalencia de compromiso cardíaco evaluado mediante ecocardiograma en la etapa de convalecencia de la enfermedad por COVID-19 fue de 4,8%. Los pacientes con cuadros iniciales más graves presentaron más hallazgos patológicos. La significancia no se sostuvo en el análisis multivariado. Los síntomas persistentes no se asociaron a mayor compromiso cardíaco.

ABSTRACT Background: The acute phase of COVID-19 infection is associated with cardiovascular involvement, but there is limited information regarding this relationship in the recovery phase from this disease both in patients with or without persistent symptoms. Objectives: The aims of this study were: 1. To analyze cardiovascular involvement by echocardiography in the recovery phase from COVID-19 disease, and 2. To explore its association with: a) the severity of the acute phase and b) the presence of persistent symptoms. Methods: An analytical, observational, prospective and single-center study was carried out, including consecutive patients attending the center for post-COVID-19 evaluation who underwent a transthoracic color Doppler echocardiogram looking for pathological outcomes. Results: A total of 600 patients were included from September 1, 2020 to May 1, 2021, and 29 of these patients (4.8%) presented pathological findings in the echocardiogram. Patients with moderate or severe acute phase COVID-19 infection had a higher prevalence of wall motion disorders (4.3% vs. 0.5%, p=0.02) and pericardial effusion (4.3% vs. 0.24%, p=0.01) compared with those with asymptomatic or mild symptoms; however, after multivariate adjustment, this association did not reach statistical significance. In 28.6% of cases, patients reported persistent symptoms, with no evident association between their presence and pathological echocardiographic results. Conclusion: The prevalence of cardiovascular involvement evaluated by echocardiography was 4.8% in the recovery phase from COVID-19 disease. Patients with more severe initial clinical presentation exhibited more pathological findings, but the significance was not sustained in the multivariate analysis. Persistent symptoms were not associated with greater cardiovascular involvement.

HERC1 Ubiquitin Ligase Is Required for Hippocampal Learning and Memory.

Mutations in the human HERC1 E3 ubiquitin ligase protein develop intellectual disability. The tambaleante (tbl) mouse carries a HERC1 mutation characterized by cerebellar ataxia due of adult cerebellar Purkinje cells death by extensive autophagy. Our previous studies demonstrated that both the neuromuscular junction and the peripheral nerve myelin sheaths are also affected in this mutant. Moreover, there are signs of dysregulated autophagy in the central nervous system in the tbl mouse, affecting spinal cord motor neurons, and pyramidal neurons of the neocortex and the hippocampal CA3 region. The tbl mutation affects associative learning, with absence of short- and long-term potentiation in the lateral amygdala, altered spinogenesis in their neurons, and a dramatic decrease in their glutamatergic input. To assess whether other brain areas engaged in learning processes might be affected by the tbl mutation, we have studied the tbl hippocampus using behavioral tests, ex vivo electrophysiological recordings, immunohistochemistry, the Golgi-Cox method and transmission electron microscopy. The tbl mice performed poorly in the novel-object recognition, T-maze and Morris water maze tests. In addition, there was a decrease in glutamatergic input while the GABAergic one remains unaltered in the hippocampal CA1 region of tbl mice, accompanied by changes in the dendritic spines, and signs of cellular damage. Moreover, the proportions of immature and mature neurons in the dentate gyrus of the tbl hippocampus differ relative to the control mice. Together, these observations demonstrate the important role of HERC1 in regulating synaptic activity during learning.

The HERC1 ubiquitin ligase regulates presynaptic membrane dynamics of central synapses.

HERC1 is a ubiquitin ligase protein, which, when mutated, induces several malformations and intellectual disability in humans. The animal model of HERC1 mutation is the mouse tambaleante characterized by: (1) overproduction of the protein; (2) cerebellar Purkinje cells death by autophagy; (3) dysregulation of autophagy in spinal cord motor neurons, and CA3 and neocortical pyramidal neurons; (4) impairment of associative learning, linked to altered spinogenesis and absence of LTP in the lateral amygdala; and, (5) motor impairment due to delayed action potential transmission, decrease synaptic transmission efficiency and altered myelination in the peripheral nervous system. To investigate the putative role of HERC1 in the presynaptic dynamics we have performed a series of experiments in cultured tambaleante hippocampal neurons by using transmission electron microscopy, FM1-43 destaining and immunocytochemistry. Our results show: (1) a decrease in the number of synaptic vesicles; (2) reduced active zones; (3) less clathrin immunoreactivity and less presynaptic endings over the hippocampal main dendritic trees; which contrast with (4) a greater number of endosomes and autophagosomes in the presynaptic endings of the tambaleante neurons relative to control ones. Altogether these results show an important role of HERC1 in the regulation of presynaptic membrane dynamics.

Proteoglycans involved in bidirectional communication between mast cells and hippocampal neurons.

BACKGROUND: Mast cells (MCs) in the brain can respond to environmental cues and relay signals to neurons that may directly influence neuronal electrical activity, calcium signaling, and neurotransmission. MCs also express receptors for neurotransmitters and consequently can be activated by them. Here, we developed a coculture model of peritoneal MCs, incubated together with dissociated hippocampal neurons for the study of cellular mechanisms involved in the mast cell-neuron interactions. METHODS: Calcium imaging was used to simultaneously record changes in intracellular calcium [Ca2+]i in neurons and MCs. To provide insight into the contribution of MCs on neurotransmitter release in rat hippocampal neurons, we used analysis of FM dye release, evoked by a cocktail of mediators from MCs stimulated by heat. RESULTS: Bidirectional communication is set up between MCs and hippocampal neurons. Neuronal depolarization caused intracellular calcium [Ca2+]i oscillations in MCs that produced a quick response in neurons. Furthermore, activation of MCs with antigen or the secretagogue compound 48/80 also resulted in a neuronal [Ca2+]i response. Moreover, local application onto neurons of the MC mediator cocktail elicited Ca2+ transients and a synaptic release associated with FM dye destaining. Neuronal response was partially blocked by D-APV, a N-methyl-D-aspartate receptor (NMDAR) antagonist, and was inhibited when the cocktail was pre-digested with chondroitinase ABC, which induces enzymatic removal of proteoglycans of chondroitin sulfate (CS). CONCLUSIONS: MC-hippocampal neuron interaction affects neuronal [Ca2+]i and exocytosis signaling through a NMDAR-dependent mechanism.

Phases of the exocytotic fusion pore.

Membrane fusion and fission are fundamental processes in living organisms. Membrane fusion occurs through the formation of a fusion pore, which is the structure that connects two lipid membranes during their fusion. Fusion pores can form spontaneously, but cells endow themselves with a set of proteins that make the process of fusion faster and regulatable. The fusion pore starts with a narrow diameter and dilates relatively slowly; it may fluctuate in size or can even close completely, producing a transient vesicle fusion (kiss-and-run), or can finally expand abruptly to release all vesicle contents. A set of proteins control the formation, dilation, and eventual closure of the fusion pore and, therefore, the velocity at which the contents of secretory vesicles are released to the extracellular medium. Thus, the regulation of fusion pore expansion or closure is key to regulate the release of neurotransmitters and hormones. Here, we review the phases of the fusion pore and discuss the implications in the modes of exocytosis.

Synaptotagmin-7 controls the size of the reserve and resting pools of synaptic vesicles in hippocampal neurons.

Continuous neurotransmitter release is subjected to synaptic vesicle availability, which in turn depends on vesicle recycling and the traffic of vesicles between pools. We studied the role of Synaptotagmin-7 (Syt-7) in synaptic vesicle accessibility for release in hippocampal neurons in culture. Synaptic boutons from Syt-7 knockout (KO) mice displayed normal basal secretion with no alteration in the RRP size or the probability of release. However, stronger stimuli revealed an increase in the size of the reserve and resting vesicle pools in Syt-7 KO boutons compared with WT. These data suggest that Syt-7 plays a significant role in the vesicle pool homeostasis and, consequently, in the availability of vesicles for synaptic transmission during strong stimulation, probably, by facilitating advancing synaptic vesicles to the readily releasable pool.

[Estimation of the "vascular age" based on body mass index in a population in primary prevention. Association with subclinical carotid atherosclerosis]. / Estimación de la «edad vascular¼ basada en el índice de masa corporal en una población en prevención primaria. Asociación con la aterosclerosis carotídea subclínica.

BACKGROUND AND OBJECTIVES: To know the relationship between "vascular age" (VA) and the diagnosis of subclinical atherosclerosis could improve cardiovascular risk stratification. OBJECTIVES: 1) to know the VA in a primary prevention population, and 2) to determine the relationship between VA and the presence of carotid atherosclerotic plaque (CAP). PATIENTS AND METHODS: We calculated VA based on body mass index (BMI). We obtained the difference between VA and chronological age (Delta). The screening of CAP was done by ultrasound. We analyzed the association between quintiles of VA and Delta with the presence of CAP. ROC analysis was performed. RESULTS: In total, 411 patients were included (age 47 [10] years, 54% men). The VA and Delta were 55 (15) and 7 (9) years respectively. In 75% of the patients VA was higher than the chronological age (50%≥ 6 years). Subjects with CAP had significantly higher VA (66 [11] versus 50 [14] years, P<.0001) and Delta (13 [9] versus 5 [7] years, P<.0001) than subjects without CAP. We observed a positive association of quintiles of VA and Delta with the prevalence of CAP. The area under de curve and the optimal cutoff point of VA for the detection of CAP were 0.813 and 60 years, respectively, and for Delta, 0,771 and 11 years, respectively. CONCLUSIONS: The VA based on BMI could be a simple tool to estimate the presence of CAP and improve cardiovascular risk stratification in patients in primary prevention.

Push-and-pull regulation of the fusion pore by synaptotagmin-7.

In chromaffin cells, Ca(2+) binding to synaptotagmin-1 and -7 triggers exocytosis by promoting fusion pore opening and fusion pore expansion. Synaptotagmins contain two C2 domains that both bind Ca(2+) and contribute to exocytosis; however, it remains unknown whether the C2 domains act similarly or differentially to promote opening and expansion of fusion pores. Here, we use patch amperometry measurements in WT and synaptotagmin-7-mutant chromaffin cells to analyze the role of Ca(2+) binding to the two synaptotagmin-7 C2 domains in exocytosis. We show that, surprisingly, Ca(2+) binding to the C2A domain suffices to trigger fusion pore opening but that the resulting fusion pores are unstable and collapse, causing a dramatic increase in kiss-and-run fusion events. Thus, synaptotagmin-7 controls fusion pore dynamics during exocytosis via a push-and-pull mechanism in which Ca(2+) binding to both C2 domains promotes fusion pore opening, but the C2B domain is selectively essential for continuous expansion of an otherwise unstable fusion pore.

Myosin II contributes to fusion pore expansion during exocytosis.

During exocytosis, the fusion pore expands to allow release of neurotransmitters and hormones to the extracellular space. To understand the process of synaptic transmission, it is of outstanding importance to know the properties of the fusion pore and how these properties affect the release process. Many proteins have been implicated in vesicle fusion; however, there is little evidence for proteins involved in fusion pore expansion. Myosin II has been shown to participate in the transport of vesicles and, surprisingly, in the final phases of exocytosis, affecting the kinetics of catecholamine release in adrenal chromaffin cells as measured by amperometry. Here, we have studied single vesicle exocytosis in chromaffin cells overexpressing an unphosphorylatable form (T18AS19A RLC-GFP) of myosin II that produces an inactive protein by patch amperometry. This method allows direct determination of fusion pore expansion by measuring its conductance, whereas the release of catecholamines is recorded simultaneously by amperometry. Here we demonstrated that the fusion pore is of critical importance to control the release of catecholamines during single vesicle secretion in chromaffin cells. We proved that myosin II acts as a molecular motor on the fusion pore expansion by hindering its dilation when it lacks the phosphorylation sites.

Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore.

Release of charged neurotransmitter molecules through a narrow fusion pore requires charge compensation by other ions. It has been proposed that this may occur by ion flow from the cytosol through channels in the vesicle membrane, which would generate a net outward current. This hypothesis was tested in chromaffin cells using cell-attached patch amperometry that simultaneously measured catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that <2% of cations, if any, enter the vesicle through its membrane. Instead, we show that flux of catecholamines through the fusion pore, measured as an amperometric foot signal, decreases when the extracellular cation concentration is reduced. The results reveal that the rate of transmitter release through the fusion pore is coupled to net Na+ influx through the fusion pore, as predicted by electrodiffusion theory applied to fusion-pore permeation, and suggest a prefusion rather than postfusion role for vesicular cation channels.

Monitoring synaptic function at the neuromuscular junction of a mouse expressing synaptopHluorin.

We monitored presynaptic exocytosis and vesicle recycling at neuromuscular junctions of transgenic mice expressing synaptopHluorin (spH), using simultaneous optical and electrophysiological recordings. Synaptic transmission was indistinguishable from that in wild-type controls. Fluorescence rose during and decayed monotonically after stimulus trains to the nerve, with amplitudes and decay times increasing with the amount of stimulation. The relatively large size of synaptic terminals allowed us to examine the spatial profile of fluorescence changes. We identified hot spots of exocytosis, which were stable with repeated trains. Photobleach experiments showed that spH freshly exposed by nerve stimulation was not preferentially retrieved by compensatory endocytosis; instead, most retrieved spH preexisted in the surface membrane. Finally, we compared fluorescence and electrical [summed end-plate potentials (EPPs)] estimates of exocytosis, which diverged during repeated trains, as fluorescence exceeded summed EPPs, although the average amplitude of miniature EPPs was unchanged. This might reflect exocytosis of spH-containing, acetylcholine-free ("empty") vesicles or other organelles during intense stimulation.

Fusion pore regulation of transmitter release.

During the last decade a wealth of new information about the properties of the exocytotic fusion pore is changing our current view of exocytosis. The exocytotic fusion pore, a necessary stage before the full merging of the vesicle membrane with the plasma membrane, is becoming a key cellular structure that might critically control the amount of neurotransmitter released into the synaptic cleft and that can be subjected to control by second messengers and phosphorylated proteins. Fusion pores form, expand to fully merge membranes, or can close leaving an intact and identical synaptic vesicle in place for a new round of exocytosis. Transient formation of fusion pores is the mechanistic representation of the "kiss-and-run" hypothesis of transmitter release and offers new alternatives for synaptic vesicle recycling besides to the classical mechanism mediated by clathrin coat endocytosis. For vesicle recycling transient fusion pores ensures a fast mechanism for maintaining an active pool of synaptic vesicles. The size reached by transient fusion pores and the time spent on the open state can determine the release of subquantal synaptic transmission, which could be a mechanism of synaptic potentiation. In this review we will described the electrophysiological and fluorescence methods that contribute to further explore the biophysical properties of the exocytotic fusion pore and the relevant experiments obtained by these methods.

The fusion pore.

The secretory process requires many different steps and stages. Vesicles must be formed and transported to the target membrane. They must be tethered or docked at the appropriate sites and must be prepared for fusion (priming). As the last step, a fusion pore is formed and the contents are released. Release of neurotransmitter is an extremely rapid event leading to rise times of the postsynaptic response of less than 100 micro s. The release thus occurs during the initial formation of the exocytotic fusion pore. To understand the process of synaptic transmission, it is thus of outstanding importance to understand the molecular structure of the fusion pore, what are the properties of the initial fusion pore, how these properties affect the release process and what other factors may be limiting the kinetics of release. Here we review the techniques currently employed in fusion pore studies and discuss recent data and opinions on exocytotic fusion pore properties.

Secretory vesicles membrane area is regulated in tandem with quantal size in chromaffin cells.

The number of transmitter molecules released in a quantal event can be regulated, and recent studies suggest that the modulation of quantal size is associated with corresponding changes in vesicle volume (Colliver et al., 2000; Pothos et al., 2002). If so, this could occur either by distension of the vesicle membrane or by incorporation and removal of vesicle membrane. We performed simultaneous measurements of vesicle membrane area and catecholamine release in individual quantal events from chromaffin cells using cell-attached patch amperometry. Cells were treated with reserpine, a vesicular monoamine transport blocker that decreases quantal size, or l-dopa, a catecholamine precursor that increases quantal size. We show that decrease and increase in quantal size are associated with a respective decrease and increase in vesicle membrane area. These results point to a novel mechanism of vesicle membrane dynamics by which vesicles physically change their membrane area in response to changes in transmitter content such that the intravesicular concentration of transmitter is maintained.

Exocytosis of single chromaffin granules in cell-free inside-out membrane patches.

In chromaffin cells, exocytosis of single granules and properties of the fusion pore--the first connection between vesicular lumen and extracellular space --can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.

Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules.

Rab3D, a member of the Rab3 subfamily of the Rab/ypt GTPases, is expressed on zymogen granules in the pancreas as well as on secretory vesicles in mast cells and in the parotid gland. To shed light on the function of Rab3D, we have generated Rab3D-deficient mice. These mice are viable and have no obvious phenotypic changes. Secretion of mast cells is normal as revealed by capacitance patch clamping. Furthermore, enzyme content and overall morphology are unchanged in pancreatic and parotid acinar cells of knockout mice. Both the exocrine pancreas and the parotid gland show normal release kinetics in response to secretagogue stimulation, suggesting that Rab3D is not involved in exocytosis. However, the size of secretory granules in both the exocrine pancreas and the parotid gland is significantly increased, with the volume being doubled. We conclude that Rab3D exerts its function during granule maturation, possibly by preventing homotypic fusion of secretory granules.