Morphometrical features of left atrial appendage in the AF patients subjected to left atrial appendage closure

Background: This study aimed to evaluate the morphometrical features of left atrial appendage (LAA) in patients with atrial fibrillation, subjected to left atrial appendage percutaneous closure (LARIAT) for stroke prevention. Materials and methods: Computed tomography (CT) scans of 51 patients with atrial fibrillation subjected to LARIAT procedure were comparatively evaluated with 50 patients with sinus rhythm (control group). Three-dimensional (3D) reconstructions were created using volume-rendering for evaluation. Results: No differences were found in LAA types of distribution (cauliflower: 25.5 vs. 34.0%, chicken wing: 45.1 vs. 46.0%, windsock: 29.4 vs. 20.0%, all p>0.05) between groups. However, the study group was characterized by LAAs with a lower number of lobes. The LAA orifice anteroposterior and transverse diameters (19.3±4.12 vs. 17.2±4.0mm, p=0.01 and 25.1±5.1 vs. 20.5±4.4mm, p=0.001), orifice area (387.2±133.9 vs. 327.1±128.3mm2, p=0.02) and orifice perimeter (70.2±12.5 vs. 61.2±11.6mm, p=0.04) was significantly larger in atrial fibrillation patients. More oval LAA orifices was found in atrial fibrillation group (94.0 vs. 70.4%, p=0.001). No statistically significant differences were found in LAA body length (47.4±15.4 vs. 43.7±10.9mm, p=0.17), body width (24.7±5.6 vs. 24.4±5.8mm, p=0.81), and chamber depth (17.7±3.5 vs. 16.5±3.8mm, p=0.11). Calculated LAA ejection fraction was significantly lower in study group compared to healthy patients (16.4±14.9 vs. 48.2±12.9%, p=0.001). Conclusions: Important morphometrical differences in LAA orifice have been found, which was significantly larger and more oval in patients with atrial fibrillation compared to healthy controls. Although no difference in LAA body type and size was observed; the LAA ejection fraction was significantly lower in atrial fibrillation rhythm patients

Inactivation and tachyphylaxis of heat-evoked inward currents in nociceptive primary sensory neurones of rats

Membrane currents evoked by repeated noxious heat stimuli (43–47 °C) of 3 s duration were investigated in acutely dissociated dorsal root ganglion (DRG) neurones of adult rats. The heat stimuli generated by a fast solution exchanger had a rise time of 114 ± 6 ms and a fall time of 146 ± 13 ms.When heat stimuli were applied to heat-sensitive small (≤ 32·5 μm) DRG neurones, an inward membrane current (Iheat) with a mean peak of 2430 ± 550 pA was observed (n = 19). This current started to activate and deactivate with no significant latency with respect to the heat stimulus. The peak of Iheat was reached with a rise time of 625 ± 115 ms. When the heat stimulus was switched off Iheat deactivated with a fall time of 263 ± 17 ms.During constant heat stimulation Iheat decreased with time constants of 4–5 s (inactivation). At the end of a 3 s heat stimulus the peak current was reduced by 44 ± 5 % (n = 19).Current-voltage curves revealed outward rectifying properties of Iheat and a reversal potential of −6·3 ± 2·2 mV (n = 6). Inactivation was observed at all membrane potentials investigated (−80 to 60 mV); however, inactivation was more pronounced for inward currents (37 ± 5 %) than for outward currents (23 ± 6 %, P < 0·05).When neurones were investigated with repeated heat stimuli (3 to 5 times) of the same temperature, the peak current relative to the first Iheat declined by 48 ± 6 % at the 3rd stimulus (n = 19) and by 54 ± 18 % at the 5th stimulus (n = 4; tachyphylaxis).In the absence of extracellular Ca2+ (buffered with 10 mm EGTA) inactivation (by 53 ± 6 %) and tachyphylaxis (by 42 ± 7 % across three stimuli) were still observed (n = 8). The same was true when intracellular Ca2+ was buffered by 10 mm BAPTA (inactivation by 49 ± 4 %, tachyphylaxis by 52 ± 7 % across three stimuli; n = 13). Thus, inactivation and tachyphylaxis were mainly independent of intra- and extracellular Ca2+.These results indicate that inactivation and tachyphylaxis of heat-evoked inward currents can be observed in vitro, similar to adaptation and suppression of action potential discharges elicited by comparably fast heat stimuli in vivo. Whereas the voltage dependence of Iheat resembles that of capsaicin-induced membrane currents (ICaps), the independence of inactivation and tachyphylaxis of Iheat from calcium is in clear contrast to ICaps. A similar difference in calcium dependence of inactivation has been reported between heat-evoked and capsaicin-induced currents through the cloned capsaicin receptor channel VR1. Thus, the properties of Iheat and of VR1 largely account for the adaptation and suppression of heat-evoked nociceptor discharges

Capacitative Ca2+ entry is graded with degree of intracellular Ca2+ store depletion in bovine vascular endothelial cells

In endothelial cells, release of Ca2+ from endoplasmic reticulum (ER) Ca2+ stores activates Ca2+ influx via the capacitative Ca2+ entry (CCE) pathway. In cultured bovine pulmonary artery endothelial cells, we investigated the relationship between intracellular Ca2+ store load and CCE activity, as well as the kinetics of CCE activation and deactivation, by simultaneously measuring changes in [Ca2+]i and unidirectional manganese (Mn2+) entry through the CCE pathway.Submaximal concentrations of ATP caused quantal release of Ca2+ from the ER, resulting in a dose-dependent depletion of Ca2+ stores and acceleration of Mn2+ entry. Mn2+ entry rate, as a measure of CCE activity, was graded with the amount of released Ca2+. Maximal activation of CCE did not require complete store depletion.Slow depletion of the ER by exposure to the ER Ca2+ pump inhibitor cyclopiazonic acid resulted in a delayed activation of CCE, revealing a temporal dissociation between release of Ca2+ from intracellular stores and activation of CCE.During [Ca2+]i oscillations, at frequencies higher than 0·5 spikes min−1, each Ca2+ spike resulted in a progressive acceleration of CCE without leading to oscillations of Ca2+ entry. In contrast, low frequency [Ca2+]i oscillations were paralleled by transient CCE that was activated and deactivated with each Ca2+ spike, resulting in an oscillatory pattern of Ca2+ entry.It is concluded that CCE is a rapidly activating process which is graded with store depletion and becomes fully activated before complete depletion. The duration of CCE activation correlates with the degree of store depletion and the time that is required to refill depleted stores. Overall, a mechanism of graded CCE prevents exhaustion of intracellular Ca2+ reserves and provides an efficient way to respond to variable degrees of intracellular store depletion

Ca2+ signalling and PKCα activate increased endothelial permeability by disassembly of VE—cadherin junctions

The role of intracellular Ca2+ mobilization in the mechanism of increased endothelial permeability was studied. Human umbilical vein endothelial cells (HUVECs) were exposed to thapsigargin or thrombin at concentrations that resulted in similar increases in intracellular Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i in both cases was due to release of Ca2+ from intracellular stores and influx of extracellular Ca2+.Both agents decreased endothelial cell monolayer electrical resistance (a measure of endothelial cell shape change) and increased transendothelial 125I-albumin permeability. Thapsigargin induced activation of PKCα and discontinuities in VE-cadherin junctions without formation of actin stress fibres. Thrombin also induced PKCα activation and similar alterations in VE-cadherin junctions, but in association with actin stress fibre formation.Thapsigargin failed to promote phosphorylation of the 20 kDa myosin light chain (MLC20), whereas thrombin induced MLC20 phosphorylation consistent with formation of actin stress fibres.Calphostin C pretreatment prevented the disruption of VE-cadherin junctions and the decrease in transendothelial electrical resistance caused by both agents. Thus, the increased [Ca2+]i elicited by thapsigargin and thrombin may activate a calphostin C-sensitive PKC pathway that signals VE-cadherin junctional disassembly and increased endothelial permeability.Results suggest a critical role for Ca2+ signalling and activation of PKCα in mediating the disruption of VE-cadherin junctions, and thereby in the mechanism of increased endothelial permeability

Nitric oxide inhibits capacitative Ca2+ entry and enhances endoplasmic reticulum Ca2+ uptake in bovine vascular endothelial cells

In vascular endothelial cells, elevation of cytosolic free calcium concentration ([Ca2+]i) causes activation of nitric oxide synthase (NOS) and release of nitric oxide (NO). The goal of the study was to characterize the interplay between [Ca2+]i and NO production in this cell type. Simultaneous measurements of [Ca2+]i and intracellular NO concentration ([NO]i) in cultured bovine vascular endothelial cells (CPAE cell line) with the fluorescent indicators fura-2 and DAF-2, respectively, revealed that Ca2+ influx following agonist-induced intracellular Ca2+ store depletion (capacitative Ca2+ entry, CCE) represents the preferential Ca2+ source for the activation of the Ca2+-calmodulin-dependent endothelial NOS (eNOS). Exposure to the NO donor sodium nitroprusside (SNP) showed that high NO levels suppressed CCE and had an inhibitory effect on Ca2+ extrusion by the plasmalemmal Ca2+-ATPase. This inhibitory effect on CCE was mimicked by the membrane-permeant cGMP analogue 8-bromo-cGMP, but was reversed by the NO scavenger haemoglobin and prevented by the inhibitor of the NO-sensitive guanylate cyclase ODQ. Brief exposure to SNP reduced the peak of ATP-induced Ca2+ release from the endoplasmic reticulum (ER) and accelerated Ca2+ reuptake into the ER. Prolonged incubation with SNP resulted in enhanced Ca2+ loading of the ER, as revealed by direct measurements of store content with the ER-entrapped low-affinity Ca2+ indicator mag-fura-2. The results suggest that in vascular endothelial cells, NO synthesis is under autoregulatory control that involves NO-dependent [Ca2+]i regulation. Via cGMP-dependent inhibition of CCE and acceleration of Ca2+ sequestration into the ER, NO can lower [Ca2+]i and therefore exert an autoregulatory negative feedback on its own Ca2+-dependent synthesis