The mechanism of calcium channel facilitation in bovine chromaffin cells.

Abstract

1. This study was planned to clarify the mechanism of Ca2+ channel facilitation by depolarizing prepulses given to voltage-clamped bovine chromaffin cells. The hypothesis for an autocrine modulation of such channels was tested by studying the effects of a soluble vesicle lysate (SVL) on whole-cell Ba2+ currents (IBa).2. SVL was prepared from a bovine adrenal medullary homogenate. The ATP content in this concentrated SVL amounted to 3a 18 + 0a12 mm (n = 4). The concentration of noradrenaline and adrenaline present in the SVL was 11 2 + 0 97 and 15X2 + 2 mm, respectively (n = 5). A 1:1000 dilution of SVL in the external solution halved the magnitude of IBa and produced a 7-fold slowing of its activation kinetics. The blocking effects of SVL were concentration dependent and quickly reversed upon washout. 3. Inhibition and slowing of the kinetics of IBa by SVL could be partially reversed by strong depolarizing prepulses (+90 mV, 45 ms). This reversal of inhibition, called Ca2P channel facilitation, persisted in the presence of 3 uM nifedipine.4. Intracellular dialysis of GDP-/l-S (0 5 mM) or pretreatment of the cells with pertussis toxin (100 ng ml-' for 18-24 h) prevented the reduction in peak current caused by a 1 :100 dilution of SVL; no prepulse facilitation could be observed under these conditions. 5. The receptor blockers naloxone (10 uM) or suramin (100 uM) and PPADS (100 uM) largely antagonized the effects of SVL. Treatment of SVL with alkaline phosphatase or dialysis against a saline buffer to remove low molecular mass materials (<1O kDa) considerably reduced the activity of SVL.6. Stopping the flow of the external solution (10 mm Ba2+) gradually reduced the size, and slowed down the activation phase, of the current. Prepulse facilitation of IBa was absent or weak in a superfused cell, but was massive upon flow-stop conditions in the presence or absence of 3 uM nifedipine. 7. Our experiments suggest that facilitation by prepulses of whole-cell current through Ca2+ channels is due to the suppression of an autoinhibitory autocrine loop present in bovine chromaffin cells. By acting at least on purinergic and opiate receptors, the exocytotic release of ATP and opiates will cause a tonic inhibition of the current through a G-proteinmediated mechanism. Such a mechanism will be removed by strong depolarizing prepulses, and will involve preferentially non-L-type channels. In the light of these and other recent results, previously held views on the selective recruitment by prepulses of dihydropyridinesensitive Ca2+ channels are not tenable.t To whom correspondence should be addressed.

Neuronal Ca2+ Sensor-1/Frequenin Functions in an Autocrine Pathway Regulating Ca2+ Channels in Bovine Adrenal Chromaffin Cells

“…The inhibited pool consists of a complex mixture of all non-Ltype channels in chromaffin cells including P/Q-and N-type. This regulatory pathway operates by pertussis toxin-sensitive, G-protein-dependent mechanisms (31,41,42) likely to involve, at least in part, direct interaction of G-protein ␤␥-subunits with Ca 2ϩ channels (43,44). We revealed this inhibited current by addition of the purinergic and opioid antagonists suramin and naloxone as described previously (28,31,32).…”

“…They can, however, be recruited into a functional pool under various conditions (28,31,32,41,42). The inhibited pool consists of a complex mixture of all non-Ltype channels in chromaffin cells including P/Q-and N-type.…”

“…The Ca 2ϩ currents were found to be predominantly non-L-type as 3 M nifedipine resulted in only between 0 and 18% inhibition of currents whereas these were abolished by addition of 250 M cadmium (n ϭ 4 cells). Under the conditions used here, non-L-type Ca 2ϩ currents are under tonic autocrine inhibition (31). This is due to endogenous release of ATP and opioids from chromaffin cells in the dish.…”

“…This is due to endogenous release of ATP and opioids from chromaffin cells in the dish. This inhibition can be removed by addition of P 2y purinergic and opioid receptor antagonists (28,31,32). In order to examine the relationship between the additional Ca 2ϩ current in NCS1-(E120Q)-expressing cells and the recruitment of this pool of quiet Ca 2ϩ channels, we examined the effect of addition of a combination of 100 M suramin and 10 M naloxone to block purinergic and opioid receptors, respectively.…”

Old and emerging concepts on adrenal chromaffin cell stimulus-secretion coupling

“…Most of them are effectively activated by the neurotransmitter molecules released by CCs (ATP, opioids, A, NA) and induce a marked depression of Ca 2+ currents. Here also, the Madrid group was determinant in demonstrating that the purified content of secretory vesicles (soluble vesicle lysate) when applied on bovine CCs had a potent voltage-dependent depressive action on N-and P/Q-type Ca 2+ currents (Cav2.1, Cav2.2) that was prevented by mixtures of broad spectrum opioidergic and purinergic antagonists [2,33]. L-type channels (Cav1.2 and Cav1.3) are also autocrinally modulated by β1-and β2-AR but the action is voltage independent and can be either depressive or potentiating [18].…”