PubMed · 5159502
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H J Schneider, A Hesse. 1971. [Trace elements in urinary calculi: lithium].. https://pubmed.ncbi.nlm.nih.gov/5159502/
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We have investigated Ca2+-induced exocytosis from human neutrophils using the whole cell patch-clamp capacitance technique. Microperfusion of Ca2+ buffer solutions (<30 nM to 5 mM free Ca2+) through the patch-clamp pipette revealed a biphasic activation of exocytosis by Ca2+. The first phase was characterized by high affinity (1.5-5 microM) and low apparent cooperativity (<=2) for Ca2+, and the second phase by low affinity (approximately 100 microM) and high cooperativity (>6). Only the second phase was accompanied by loss of myeloperoxidase, suggesting that the low-affinity exocytosis reflected release of peroxidase-positive (primary) granules, while the high-affinity exocytosis reflected release of peroxidase-negative (secondary and tertiary) granules. At submaximal Ca2+ concentrations, only a fraction of a given granule population was released. This submaximal release cannot simply be explained by Ca2+ modulation of the rate of exocytosis, and it suggests that the secretory response of individual cells is adjusted to the strength of the stimulus. The Ca2+ dependence of the high- and low-affinity phases of neutrophil exocytosis bears a resemblance to endocrine and neuronal exocytosis, respectively. The occurrence of such high- and low-affinity exocytosis in the same cell is novel, and suggests that the Ca2+ sensitivity of secretion is granule-, rather than cell-specific.
1. Human T lymphocytes express both voltage-gated (K(V)) and Ca2+-activated (K(Ca)) potassium channels. The K(Ca) channel is activated by elevations of intracellular Ca2+ ([Ca2+]i) at concentrations attained during physiological Ca2+ signalling. Whether or not the K(V) channel is affected by [Ca2+]i is a matter of controversy. Here, the interaction between the K+ channels of lymphocytes and [Ca2+]i was studied using cell-attached and whole-cell patch-clamp recordings, while [Ca2+]i of the same cell was monitored simultaneously by fura-2 imaging or from the activity of the K(Ca) channels. 2. The K+ channels in cell-attached patches were measured using a high K+ pipette solution. The K(V) conductance was quantified as the integral of the inward current during voltage ramp stimulation, yielding a measure independent of the cell membrane potential. Whereas the open probability of the K(Ca) channel showed an absolute dependence on [Ca2+]i, the K(V) channel was little affected by [Ca2+]i. The K(V) conductance is not reduced by elevations of [Ca2+]i in the range 0-8 muM. On the contrary, a modest but consistent increase in the K(V) current component in cell-attached currents was observed when [Ca2+]i was elevated. 3. The absence of inhibition of the K(V) current by [Ca2+]i was also apparent from whole-cell measurements with pipette solutions buffered to 1 microM free Ca2+: following break-in to whole-cell configuration, depolarizing voltage ramps were applied at regular intervals to activate the K(V) current while the K(Ca) current was measured from the slope of the ramp current below the activation of the voltage-gated current. During the gradual activation of the K(Ca) current, as the cell interior was perfused with the pipette solution, the K(V) current remained constant in amplitude. 4. In the initial period following break-in to whole-cell configuration, a gradual increase in the rate of K(V) current inactivation was generally observed. However, the time course of this change in kinetics was much slower than the perfusion of the cell interior, as judged from the activation of the K(Ca) conductance, ruling out a direct effect of Ca2+, within the physiological range, on K(V) channel kinetics.
Hypoxia/reoxygenation injury to cultured endothelial cells results in cytoskeletal rearrangement and second messenger activation related to increased monolayer junctional permeability. Cytoskeletal rearrangement by reactive oxygen species may be related to specific activation of the phospholipase D (PLD) pathway. Human umbilical vein endothelial cell monolayers are exposed to H2O2 (100 microM) or metabolites of the PLD pathway for 1-60 min. Changes in cAMP levels, Ca2+ levels, PIP2 production, filamin distribution, and intercellular gap formation are then quantitated. H2O2-induced filamin translocation from the membrane to the cytosol occurs after 1-min H2O2 treatment, while intercellular gap formation significantly increases after 15 min. H2O2 and phosphatidic acid exposure rapidly decrease intracellular cAMP levels, while increasing PIP2 levels in a Ca2+-independent manner. H2O2-induced cAMP decreases are prevented by inhibiting phospholipase D. H2O2-induced cytoskeletal changes are prevented by inhibiting phospholipase D, phosphatidylinositol-4-phosphate kinase, phosphoinositide turnover, or by adding a synthetic peptide that binds PIP2. These data indicate that metabolites produced downstream of H2O2-induced PLD activation may mediate filamin redistribution and F-actin rearrangement.