Voltage-gated Ca entry in isolated bovine capillary endothelial cells: evidence of a new type of BAY K 8644-sensitive channel

Isolated bovine capillary endothelial cells have been examined for voltage-dependent Ca entry. All cells displayed a low threshold activity, with the main characteristics of a T-type transient current, when examined using whole-cell recording for activation and inactivation and cell-attached conditions or inside-out patches for the elementary conductance (8 pS). 25% of the cells displayed an additional sustained current in 5 mM CaCl₂ above –40 mV, which was enhanced by application of BAY K 8644, but almost insensitive to superfusion with nicardipine. Two types of channels (2.8 and 21 pS, in 110 mM BaCl₂) were shown to have a BAY K 8644 sensitivity. The large conductance channels were L-type channels. The smaller events were elicited at more hyperpolarized potentials (by some 30 mV). Their mean open time was 16 ms in control conditions. In presence of BAY K 8644, additional long open times were observed (up to 100 ms as compared to 7.8 ms for the time constants of the slow mode of the L-type channel). We refer to these channels as SB channels: of small conductance and sensitive to BAY K 8644. In the presence of nicardipine, SB channels are not noticeably modified, in contrast to the L-type openings which are abolished. Also, SB open times are close to control values when nicardipine is added after a BAY K 8644 application. We suggest that, at physiological concentrations of divalent ions, an SB-type activity is elicited above –40 mV which generates the low threshold sustained current.     

Sustained stimulation shifts the mechanism of endocytosis from dynamin-1-dependent rapid endocytosis to clathrin- and dynamin-2-mediated slow endocytosis in chromaffin cells

Transient stimulation of secretion in calf chromaffin cells is invariably followed by rapid endocytosis (RE), a clathrin- and K(+)-independent process with a half time of several seconds. Here we show that when exocytosis is triggered in a more sustained manner, a much slower form of endocytosis (SE) replaces RE. SE is complete within 10 min and is abolished when anticlathrin antibodies are introduced into the cell or when intracellular K(+) is removed. RE, but not SE, is blocked by intracellular administration of antidynamin-1 antibodies; the inverse specificity was found for antidynamin-2 antibodies. Replacement of extracellular Ca(2+) by Ba(2+) or Sr(2+) completely blocked RE but had little effect on SE. Thus chromaffin cells exhibit two kinetically and mechanistically distinct forms of endocytosis that are coupled to different extents of exocytosis and are mediated by different isoforms of dynamin. We surmise that RE is associated with the transient fusion ("kiss-and-run") mechanism of transmitter release and is the prevalent means of vesicle recapture and recycling under normal physiological conditions, whereas the clathrin-based SE mechanism comes into play only at higher levels of stimulation and may be associated with complete fusion of vesicles with the plasma membrane.     

ADP exerts a protective effect against rundown of the Ca2+ current in bovine chromaffin cells

In isolated chromaffin cells, the high-voltage-activated Ca²⁺ current, recorded using 5 mM Ca²⁺ as the divalent charge carrier, exhibits rundown within 10 min, which is delayed for 1 h at least by the addition of 1 mM adenosine 5-triphosphate (ATP) to the pipette medium. The mechanism of this stabilizing action of ATP has been examined. ATP action is dose dependent; the rundown process, which was delayed at concentrations below 0.4 mM, was totally abolished at higher concentrations. The requirement for ATP was shown to be quite strict: 2 mM inosine 5-triphosphate (ITP) could not replace ATP, whereas guanosine 5-triphosphate (GTP) could, but at higher concentrations. This effect of ATP was shown to require the presence of MgCl₂ and the liberation of a phosphate group since the ATP analogue 5-adenylyl-imidodiphosphate (AMP-PNP) could not act as a substitute for ATP, suggesting an action through either adenosine 5-diphosphate (ADP) or a phosphorylation step. ADP, in the presence of Mg²⁺ only, could replace ATP in the same concentration range. This effect was shown to be specific to ADP; it was maintained after blocking the pathways which convert ADP into ATP, and could not be mimicked by guanosine 5-diphosphate (GDP). Similarly, ATP and ADP effects were abolished at an increased internal Ca²⁺ concentration (pCa 6 instead of pCa 7.7, where pCa = –log₁₀[Ca²⁺]). Nevertheless, the presence of 1 mM Mg-ADP in the bathing solution did not prevent the rundown of the Ca²⁺ channels when going to the inside-out patch recording configuration. In conclusion, the stabilizing effect of ATP may be interpreted by a Mg²⁺-ADP binding site present on high-voltage-activated Ca²⁺ channels. A localization of such an ADP regulatory site on the L-type Ca²⁺ channel itself cannot be excluded, though with an additional requirement since Mg-ADP alone is not able to maintain the corresponding activity on excised patches.     

Ageing changes the cellular basis of the "fight-or-flight" response in human adrenal chromaffin cells.

Stress-induced increases in plasma epinephrine in man have been reported to decrease with age. To investigate the possible cellular basis for this decline we determined the characteristics of calcium currents and their relationship to catecholamine secretion in isolated human adrenal chromaffin (AC) cells. Cells derived from young individuals displayed prominent prepulse facilitation of L-type Ca channels but this property was absent in cells from older subjects. Robust quantal secretion in young cells as determined by amperometry was strongly coupled to the activation of these channels with an average delay of only approximately 3 msec. N- and P-type Ca channels also contributed to secretion but were more weakly coupled to catecholamine release sites. Cells from older subjects secreted much less efficiently and showed only weak coupling between Ca channels and secretion. These studies suggest that the magnitude and timing of adrenal secretion changes with age and that the facilitation Ca channel is key to rapid activation of the fight-or-flight response in young individuals.     

ATP and G proteins affect the runup of the Ca2+ current in bovine chromaffin cells

The Ca²⁺ current recorded by the whole-cell technique in chromaffin cells shows, before the often described rundown, a transient facilitation or runup. Initial current amplitude was 570±165 pA and then it increased by 49±23% (n=19, SD) over 2±1 min in the absence of adenosine 5-triphosphate (ATP). In the presence of ATP, this process occurred with the same magnitude but it was slowed in a dose-dependent manner, lasting 17±2 min with 2 mM ATP (n=8). Since adenosine 5-diphosphate (ADP) does not reproduce this ATP effect, a complex series of phosphorylations is likely to intervene and we show that, at least, a cAMP-dependent i.e., cyclic adenosine monophosphate) phosphorylation occurs. Pertussis toxin (PTX) pretreatment yielded an already maximal Ca²⁺ current (around 1000 pA) at the time of the patch rupture, which only slightly increased thereafter (10%, n=11). Also, guanosine 5-diphosphate (GDP) and guanosine 5-O-(2-thiodiphosphate) (GDP[s]), induced a fast runup, which was absent in the presence of GTP. Furthermore, we show that facilitation does not occur in the presence of dihydrophyridine (DHP) antagonists. Globally, our data suggest that an ATP-dependent phosphorylation stabilizes the inhibitory control exerted by a PTX-sensitive G protein and, as a result, slows down the facilitation of L-type Ca²⁺ channels. The recruitment of L-type channels can also be facilitated by the application of a DHP agonist or a depolarizing prepulse protocol. We show that these processes are only effective over a period which parallels the runup and are not additive to it. This suggests that a single process may underlie these various types of facilitation.