Isolated Light Chain of Tetanus Toxin Inhibits Exocytosis: Studies in Digitonin-Permeabilized Cells

Previous work indicates that the heavy chain of tetanus toxin is responsible for the binding of the toxin to the neuronal membrane and its subsequent internalization. In the present study, the light chain of tetanus toxin mimicked the holotoxin in inhibiting Ca2+-dependent secretion of [3H]norepinephrine from digitonin-permeabilized adrenal chromaffin cells. Preincubation of tetanus toxin with monoclonal antibodies to the light chain prevented the inhibition by tetanus toxin. Preincubation of tetanus toxin with nonimmune ascites fluid or with monoclonal antibodies directed against the C fragment (the C-terminal of the heavy chain) or the heavy-chain portion of the B fragment did not prevent inhibition by tetanus toxin. The data indicate that the light chain is responsible for the intracellular blockade of exocytosis.     

Requirement of ATP for Exocytotic Release of Catecholamines from Digitonin-Permeabilized Adrenal Chromaffin Cells

Cultured chromaffin cells were preincubated with digitonin to deplete endogenous ATP from the cell cytoplasm. Catecholamine release from these digitonin-pretreated cells was then studied in the presence and absence of exogenous ATP to elucidate a possible involvement of the cytoplasmic ATP in the exocytotic process. The preincubation of the cells with digitonin in the ATP-free permeabilizing medium resulted in a marked decline of the releasing response to a calcium challenge. Furthermore, the declined activity of catecholamine release caused by digitonin pretreatment was restored by the presence of ATP, but not by other adenine nucleotides, and this recovery was observed in a manner dependent on the concentration of ATP. These findings, therefore, seem to indicate that a decrease in the releasing activity of the digitonin-pretreated cells may be due to the removal of endogenous ATP from the cytoplasmic space of the cells, thus suggesting that the cytoplasmic ATP may be involved in the exocytotic mechanism of catecholamine secretion.     

Stimulatory Effect of Ascorbic Acid on Norepinephrine Biosynthesis in Digitonin-Permeabilized Adrenal Medullary Chromaffin Cells

The regulatory role of ascorbic acid in norepinephrine biosynthesis was studied using digitonin-permeabilized chromaffin cells. When permeabilized chromaffin cells were incubated with [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine) in calcium-free medium, the amounts of radioactive dopamine and norepinephrine measured in the cell fraction were increased as a function of incubation time and dopamine concentration. Both the accumulation of dopamine and the formation of norepinephrine were shown to require the presence of Mg-ATP in the medium. These results indicate that the permeabilization of chromaffin cells by digitonin treatment does not disrupt the functions of chromaffin granules, including dopamine uptake, norepinephrine formation, and storage of these amines. Using this permeabilized cell system, the effect of ascorbic acid on the rates of dopamine uptake and hydroxylation was investigated. The formation of norepinephrine was stimulated by ascorbic acid at concentrations of 0.5-2 mM in the presence of Mg-ATP. By contrast, dopamine uptake was not affected by the presence or absence of ascorbic acid in the medium. These findings provide evidence that ascorbic acid may stimulate the conversion of dopamine to norepinephrine by increasing dopamine beta monooxygenase activity rather than by increasing the substrate supply of dopamine. These observations also suggest that the rate of norepinephrine biosynthesis in adrenal medullary cells may be regulated by the concentration of ascorbic acid within the cell cytoplasm.     

Examination of the Role of ADP-Ribosylation Factor and Phospholipase D Activation in Regulated Exocytosis in Chromaffin and PC12 Cells

Abstract: The possible role of ADP-ribosylation factor (ARF)-activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin-permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF-stimulated PLD activity in these cell types. Exocytosis from cytosol-depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol-depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca²⁺ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA-sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.     

Metal Selectivity of Exocytosis in α-Toxin-Permeabilized Bovine Chromaffin Cells

Abstract: Metal selectivity of exocytosis was analyzed by comparing the effects of polyvalent metal cations Ca²⁺, Ba²⁺, Sr²⁺, Pb²⁺, La³⁺, Cd²⁺, Co²⁺, Tb³⁺, Mn²⁺, and Zn²⁺ on the release of norepinephrine (NE) from staphylococcal α-toxin-permeabilized bovine chromaffin cells. Pb²⁺, La³⁺, Cd²⁺, Sr²⁺, and Ba²⁺ activated NE secretion accompanied by the release of intragranular dopamine β-hydroxylase but not cytosolic lactate dehydrogenase, indicating the activation of the mechanism of exocytosis. The release triggered by saturating concentrations of Pb²⁺, La³⁺, Cd²⁺, and Sr²⁺ was nonadditive with Ca²⁺, indicating a common site of action. In contrast, the Ba²⁺-evoked NE release was additive with Ca²⁺ and the Ca²⁺ agonists Pb²⁺, La³⁺, Cd²⁺, and Sr²⁺, suggesting that Ba²⁺ activates secretion at a site distinct from the Ca²⁺ receptor. In distinction to the NE release evoked by Pb²⁺, La³⁺, Cd²⁺, and Ba²⁺, the Sr²⁺-evoked NE release was associated with a significant elevation of Ca²⁺ concentration in the medium and abolished by Ca²⁺ chelation. This indicates that the secretagogue effect of Sr²⁺ was indirect and secondary to the displacement of bound Ca²⁺. Co²⁺ and Mn²⁺ inhibited the NE release evoked by Ca²⁺, Sr²⁺, Pb²⁺, La³⁺, and Cd²⁺ but had no effect on the Ba²⁺-dependent secretion. Tb³⁺ and Zn²⁺ were without effect on exocytosis.     

Calcium Is Released by Exocytosis Together with Catecholamines from Bovine Adrenal Medullary Cells

We have tested the hypothesis that exocytosis is a possible export route for calcium from bovine adrenal medullary cells. After prelabelling cells in primary tissue culture with 45Ca, evoked 45Ca export and catecholamine secretion show the same time course, a similar fraction of the total pool of 45Ca and catecholamine is released, and the same concentrations of carbamylcholine or KCl are required for half-maximal triggered release. Increasing the osmolarity of the extracellular medium or treating the cells with botulinum toxin type D inhibits both evoked catecholamine secretion and 45Ca export to the same extent without inhibiting 45Ca influx. Incorporation of 45Ca into chromaffin granules is very slow, however, and incorporated 45Ca is not immediately releasable. 45Ca entering the cell during short-term stimulation is not found in the releasable pool during a second period of triggered secretion. Our data suggest that chromaffin granules are the largest pool of intracellular calcium in bovine adrenal medullary cells and that most of the calcium in chromaffin granules does not rapidly exchange with cytoplasmic Ca, but can be released directly by exocytosis. Exocytosis does not appear to play a major role in exporting Ca that enters the cell during short-term stimulation.     

Loss and Ca2+-Dependent Retention of Scinderin in Digitonin-Permeabilized Chromaffin Cells: Correlation with Ca2+-Evoked Catecholamine Release

Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca(2+)-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca(2+)-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca(2+)-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca(2+)-dependent catecholamine release observed in permeabilized chromaffin cells.     

The Latency of Exocytosis Varies with the Mechanism of Stimulated Release in PC12 Cells

Abstract: To compare the time course of different mechanisms of chemically stimulated release, amperometric detection of dopamine was carried out at single PC12 cells. The rapid response of carbon fiber microelectrodes allowed the detection of single exocytotic events, thus providing time-resolved information about the dynamics of stimulated release, in particular the latency between the stimulation of a cell and the secretion of catecholamines. On rapid depolarization of the cell membrane caused by application of 105 m M K⁺, almost immediate (6 ± 1 s) release of dopamine was observed. Stimulation with 1 m M nicotine, involving the stimulant binding to a ligand-gated ion channel, resulted in a short (37 ± 5 s) delay between stimulation and secretion. Application of 1 m M muscarine to the cells caused a long (103 ± 11 s) latency before exocytosis was detected. A biphasic response that appeared to be similar to a combination of nicotine- and muscarine-stimulated release was observed when cells were stimulated with 10 m M acetylcholine. Thus, it appears that the dynamics of stimulated release at single PC12 cells is significantly affected by the mechanism leading to exocytosis.     

Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells

Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca²⁺ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca²⁺ sources: (a) Ca²⁺ surrounding single cells was transiently displaced by applying agonist with or without Ca²⁺ from an ejection pipette. (b) Intracellular stores of Ca²⁺ were depleted by soaking cells in Ca²⁺-free plus 1 mM EGTA solution before transient exposure to agonist plus Ca²⁺. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca²⁺]_i and secretion in cells with or without internal Ca²⁺ stores, but only when applied with Ca²⁺ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca²⁺ was included in the pipette, but neither produced responses in cells depleted of internal Ca²⁺ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca²⁺]_i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ～20-s refractory period was found between repeated caffeine-evoked exocytotic bursts even though [Ca²⁺]_i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca²⁺ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca²⁺. These results and conclusions are consistent with our original observations in the perfused adrenal gland.     

Effects of Tetanus Toxin on Catecholamine Release from Intact and Digitonin-Permeabilized Chromaffin Cells

Abstract: Tetanus exotoxin inhibited Ca²⁺-dependent cate-cholamine secretion in a dose-dependent manner in digito-nin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca²⁺. The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca²⁺ concentrations. Tetanus toxin also inhibited catechol-amine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca²⁺-dependent exocytotic pathway to inhibit secretion.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

Regulatory Role of the GTP-Binding Protein, Go, in the Mechanism of Exocytosis in Adrenal Chromaffin Cells

To elucidate the possible involvement of GTP-binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and antibodies against the G proteins (Gi and G(o)) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine-evoked catecholamine release. High K(+)-, histamine-, or gamma-aminobutyric acid-evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10(-4) M acetylcholine was decreased significantly in PTX-treated cells. In digitonin-permeabilized cells, PTX pretreatment resulted in a decrease of the half-maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP-gamma-S (a nonhydrolyzable GTP analogue) inhibited Ca(2+)-dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the alpha subunit of the PTX-sensitive G protein, G(o). Treatment with similar antibodies against the alpha subunit of Gi had no effect. These findings suggest that G(o) directly controls the Ca(2+)-triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.     

Evidence Against an Acute Inhibitory Role of nSec-1 (Munc-18) in Late Steps of Regulated Exocytosis in Chromaffin and PC12 Cells

Abstract: nSec-1 (munc-18) is a mammalian homologue of proteins implicated in constitutive exocytosis in yeast and neurotransmission in Caenorhabditis elegans and Drosophila . Mutant phenotypes in these species suggest that nSec-1 is likely to be required for neurotransmission. Various other data have been interpreted as suggesting that nSec-1 could also be a negative regulator of Ca²⁺-dependent exocytosis. We have tested this possibility by introducing exogenous nSec-1 into permeabilised chromaffin or PC12 cells and examining its effects on Ca²⁺-induced and α-soluble N -ethylmaleimide-sensitive fusion protein attachment protein-stimulated exocytosis. No effects of exogenous nSec-1 were observed in these assays. In addition, the effect of nSec-1 overexpression in transiently transfected PC12 cells on reporter growth hormone (GH) secretion was examined. Overexpression of nSec-1 resulted in a marked increase in GH production, reflected in an increase in both cell-associated and medium GH levels. The relative amounts retained in the cells were unaffected by nSec-1 overexpression, indicating that GH storage was unaffected and that the major effect was on its synthesis. In contrast, nSec-1 overexpression did not affect the proportion of GH that was released following stimulation in intact or permeabilised cells. These results suggest either that nSec-1 is already expressed at sufficient levels and remains so following permeabilisation or that nSec-1 may not be an acute inhibitory regulator of Ca²⁺-dependent exocytosis in chromaffin or PC12 cells.     

Barium and Calcium Stimulate Secretion from Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells by Similar Pathways

We compared the characteristics of secretion stimulated by EGTA-buffered Ba(2+)- and Ca(2+)-containing solutions in digitonin-permeabilized bovine adrenal chromaffin cells. Half-maximal secretion occurred at approximately 100 microM Ba2+ or 1 microM Ca2+. Ba(2+)-stimulated release was not due to release of sequestered intracellular Ca2+ because at a constant free Ba2+ concentration, increasing unbound EGTA did not diminish the extent of release due to Ba2+. The maximal extents of Ba(2+)- and Ca(2+)-dependent secretion in the absence of MgATP were identical. MgATP enhanced Ba(2+)-induced secretion to a lesser extent than Ca(2+)-induced secretion. Half-maximal concentrations of Ba2+ and Ca2+, when added together to cells, yielded approximately additive amounts of secretion. Maximal concentrations of Ba2+ and Ca2+ when added together to cells for 2 or 15 min were not additive. Tetanus toxin inhibited Ba(2+)- and Ca(2+)-dependent secretion to a similar extent. Ba2+, unlike Ca2+, did not activate polyphosphoinositide-specific phospholipase C. These data indicate that (1) Ba2+ directly stimulates exocytosis, (2) Ba(2+)-induced secretion is stimulated to a lesser extent than Ca(2+)-dependent secretion by MgATP, (3) Ba2+ and Ca2+ use similar pathways to trigger exocytosis, and (4) exocytosis from permeabilized cells does not require activation of polyphosphoinositide-specific phospholipase C.     

Calcium Homeostasis in Digitonin-Permeabilized Bovine Chromaffin Cells

The regulation of cytosolic calcium was studied in digitonin-permeabilized chromaffin cells. Accumulation of ⁴⁵Ca²⁺ by permeabilized cells was measured at various Ca²⁺ concentrations in the incubation solutions. In the absence of ATP, there was a small (10–15% of total uptake) but significant increase in accumulation of Ca²⁺ into both the vesicular and nonvesicular pools. In the presence of ATP, the permeabilized cells accumulated Ca²⁺ into carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-sensitive and -insensitive pools. The CCCP-sensitive pool—mainly mitochondria—was active when the calcium concentration was > 1 μM and was not saturated at 25 μM. The Ca²⁺ sequestered by the CCCP-insensitive pool could be inhibited by vanadate and released by inositol trisphosphate, a combination suggesting that this pool was the endoplasmic reticulum. The CCCP-insensitive pool had a high affinity for calcium, with an EC₅₀ of ～1 μM. When the Ca²⁺ concentration was adjusted to the level in the cytoplasm of resting cells (0.1 μM), the presumed endoplasmic reticulum pool was responsible for ～90% of the ATP-stimulated calcium uptake. At a calcium level similar to the acetylcholine-stimulated level in intact cells (5–10 μM), most of the Ca²⁺ (>95%) went into the CCCP-sensitive pool.     

Actin Involvement in Exocytosis from PC12 Cells: Studies on the Influence of Botulinum C2 Toxin on Stimulated Noradrenaline Release

Botulinum C2 toxin is known to ADP-ribosylate actin. The toxin effect was studied on [3H]noradrenaline secretion of PC12 cells. [3H]Noradrenaline release was stimulated five- to 15-fold by carbachol (100 microM) or K+ (50 mM) and 10-30-fold by the ionophore A23187 (5 microM). Pretreatment of PC12 cells with botulinum C2 toxin for 4-8 h at 20 degrees C, increased carbachol-, K+-, and A23187-induced, but not basal, [3H]noradrenaline release maximally 1.5-to three-fold, whereas approximately 75% of the cellular actin pool was ADP-ribosylated. Treatment of PC12 cells with botulinum C2 toxin for up to 1 h at 37 degrees C also increased stimulated [3H]noradrenaline secretion, whereas toxin treatment for greater than 1 h decreased the enhanced [3H]noradrenaline release stimulated by carbachol and K+ but not by A23187. Concomitantly with toxin-induced stimulation of secretion, 20-50% of the cellular actin was ADP-ribosylated, whereas greater than 60% of actin was modified when exocytosis was attenuated. The data indicate that ADP-ribosylation of actin by botulinum C2 toxin largely modulates stimulation of [3H]noradrenaline release. Moreover, the biphasic toxin effects suggest that distinct mechanisms are involved in the role of actin in secretion.     

Lack of Phospholipase D Activity in Chromaffin Cells: Bradykinin-Stimulated Phosphatidic Acid Formation Involves Phospholipase C in Chromaffin Cells but Phospholipase D in PC12 Cells

The role of lipid-bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor-linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase (DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of chromaffin cells prelabelled (24 h) with 32Pi leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [gamma-32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.     

Hydrolysis-Resistant GTP Analogs Stimulate Catecholamine Release from Digitonin-Permeabilized PC12 Cells

Abstract: The effect of the hydrolysis-resistant GTP analogs, guanosine 5'- O -(3-thiotriphosphate) (GTPγS) and guanylyl imidodiphosphate (GMPPNP), on norepinephrine (NE) secretion from digitonin-permeabilized rat pheochromocytoma cells, PC12, was examined. Although secretion in the presence of saturating Ca²⁺ (10 μ M ) was not affected by GTP7S or GMPPNP, secretion in the absence of Ca²⁺ was stimulated by these GTP analogs. Secretion induced by saturating concentrations of GTPγS or GMPPNP was approximately 80% of that induced by 10 μ M Ca²⁺. Half-maximum stimulation was induced by 30 μ M GTPγS or GMPPNP. Both Ca²⁺-stimulated and GTPγS-stimulated secretion were ATP dependent and inhibited by N -ethylmaleimide. The GTPγS-stimulated secretion of NE from permeabilized PC12 cells does not appear to result from either the release of Ca²⁺ or the activation of protein kinase C. Activation of protein kinase C by pretreatment of intact cells with 12- O -tetradecanoyl-phorbol 13-acetate caused a 50% increase in both Ca²⁺-stimulated and GTP7S-stimulated secretion. Cholera and pertussis toxins did not affect Ca²⁺-stimulated or GTPγS-stim-ulated NE secretion. Guanosine 5'- O -(2-thiodiphosphate) (GDPβS) and GTP inhibited GTPγS-stimulated secretion but not Ca²⁺-stimulated secretion. The inability of GDPβS to inhibit Ca²⁺-stimulated secretion indicates that the process affected by GTPγS is not an essential step in the Ca²⁺-stimulated pathway.     

Store-Operated Ca2+ Influx and Voltage-Gated Ca2+ Channels Coupled to Exocytosis in Pheochromocytoma (PC12) Cells

Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+ and caffeine. K+-evoked exocytosis was entirely dependent on Ca2+ influx through voltage-gated Ca2+ channels, and of the subtypes of such channels present in these cells, influx through N-type was primarily responsible for triggering exocytosis. L-type channels played a minor role in mediating K+-evoked secretion, whereas P/Q-type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca2+. Application of caffeine in Ca2+-free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+ was restored to the extracellular solution (in the absence of caffeine), store-operated Ca2+ influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L-type Ca2+ channels, but less than that caused by Ca2+ influx through N-type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+ influx through pathways other than voltage-gated Ca2+ channels.