Membrane cycling after the excess retrieval mode of rapid endocytosis in mouse chromaffin cells.

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.

Lithium and KB-R7943 effects on mechanics and energetics of rat heart muscle.

The role of calcium influx on energy expenditure during cardiac contraction was studied. For this purpose, the described ability of lithium and KB-R 7943 (KBR) to diminish Ca entry through Na-Ca exchanger (Ponce-Hornos & Langer, J Mol Cell Cardiol 1980, 12, 1367, Satoh et al., Circulation 2000, 101, 1441) were used. In isolated contractions (contractions elicited after at least 5 min of rest) LiCl 45 mmol L(-1) decreased pressure developed and pressure-time integral from 42.3 +/- 2.7 and 14.5 +/- 1.2 to 32.1 +/- 3.4 mN mm(-2) and 8.3 +/- 0.9 mN mm(-2) s, respectively. A similar effect was observed in regular contractions (at 0.16 Hz stimulation). The presence of KBR (5 micromol L(-1)) in the perfusate induced a slight but not significant decrease in pressure developed and pressure-time integral in steady-state contractions. As it was previously described, the heat involved in a heart muscle contraction can be decomposed into several components (H1, H2, H3 and H4), but only one (H3) was associated with force generation. While H3 decreased with lithium in both types of contractions, H3/PtI ratio remained unaltered, indicating that the economy for pressure maintenance was unaffected. To further investigate the role of Ca entry on force development, a condition in which the contraction is mainly dependent on extracellular calcium was studied. An 'extra' stimulus applied 200 ms after the regular one in a muscle stimulated at 0.16 Hz induces a contraction with this characteristic (Marengo et al., Am J Physiol 1999, 276, H309). Lithium induced a strong decrease in pressure-time integral and H3 associated with this contraction (43 and 45%, respectively) with no change in H3/PtI ratio. Lithium also reduced (53%) an energy component (H2) associated with Ca cycling. The use of KBR showed qualitatively similar results [i.e. a 33% reduction in pressure-time integral associated with the extrasystole (ES) with no changes in H3/PtI ratio and a 30% reduction in the H2 component]. Li and KBR effects appear to be additive and in the presence of 45 mmol L(-1) Li and 5 micromol L(-1) KBR the extrasystole was abolished in 77%. Lithium and KBR effects particularly for the extrasystole can be explained through the inhibition of Ca entry via Na-Ca exchange giving support to the participation of the Na-Ca exchanger in the Ca influx from the extracellular space. In addition, the results also suggest the possibility of an effect of Li on an additional Ca sensitive locus (different than the Na-Ca exchanger). In this connection, in isolated contractions lithium decreased the energy release fraction related to mitochondrial processes (H4) increasing the economy of the overall cardiac contraction.

The heart extrasystole: an energetic approach.

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 (H'2) and one pressure-dependent (H'3) 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 H'2 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.

Tension-dependent and tension-independent energy components of heart 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.

Mechanisms underlying post-rest potentiation in isolated left rat atria.

In the isolated rat left atria the influence of rest intervals on the force developed by the first post rest beat (PRB) was studied. Under control conditions the force developed by the PRB increased (respect to previous steady state at 0.5 Hz) with the increase of the rest interval until 20 sec of pause and decreased with longer intervals. In the presence of caffeine (1 or 4 mM plus high [Ca]0) there was a monotonous fall of the PRB as a function of the rest interval. When extracellular calcium was replaced by Sr the tension developed by PRB vs. rest interval curve rose with a slope lower than the control one and reached the peak at 60 sec. At saturation levels of [Ca]0 the PRB tension development did not vary up to 20 sec pause but the decreasing phase observed after 20 sec of rest interval was still present. At 0.5 mM [Ca]0 the response was similar to control curve. The results in the presence of caffeine and strontium suggest that, in rat atria, the rest potentiation appears to be dependent on the release of calcium from intracellular stores (sarcoplasmic reticulum). This is consistent with the hypothesis proposing that longer resting periods provide a longer interval for the transfer of Ca from uptake to release sites in the sarcoplasmic reticulum.

Mechanisms underlying post-rest potentiation in isolated left rat atria.

In the isolated rat left atria the influence of rest intervals on the force developed by the first post rest beat (PRB) was studied. Under control conditions the force developed by the PRB increased (respect to previous steady state at 0.5 Hz) with the increase of the rest interval until 20 sec of pause and decreased with longer intervals. In the presence of caffeine (1 or 4 mM plus high [Ca]0) there was a monotonous fall of the PRB as a function of the rest interval. When extracellular calcium was replaced by Sr the tension developed by PRB vs. rest interval curve rose with a slope lower than the control one and reached the peak at 60 sec. At saturation levels of [Ca]0 the PRB tension development did not vary up to 20 sec pause but the decreasing phase observed after 20 sec of rest interval was still present. At 0.5 mM [Ca]0 the response was similar to control curve. The results in the presence of caffeine and strontium suggest that, in rat atria, the rest potentiation appears to be dependent on the release of calcium from intracellular stores (sarcoplasmic reticulum). This is consistent with the hypothesis proposing that longer resting periods provide a longer interval for the transfer of Ca from uptake to release sites in the sarcoplasmic reticulum.

[Clinicopathological analysis of fatal cerebellar hematomas]. / Análisis clinicopatológico de hematomas cerebelosos fatales.

An analysis is made of the clinical observations and pathological findings in 16 fatal cerebellar hematomas admitted at the Instituto de Neurología in Montevideo, over a period of 23 years. The absence of definitive lesions of the brainstem in the majority of cases, in spite of the rich symptomatology of brainstem disfunction (horizontal gaze palsies, vegetative syndrome decerebrate movements) is stressed, as well as the rapid evolution of those cases with intraventricular haemorrhage. Missed clinical diagnosis was also frequent in severely ill patients. Discussion is carried out around the importance of early diagnosis in the correct surgical management. Ventriculostomy alone, not followed by immediate craniectomy, may be highly dangerous. Mere evacuation of the cerebellar, and even of the ventricular clots can result insufficient if not followed by ventricular shunting in cases with ventricular inundation.