Nitric Oxide Modulates a Late Step of Exocytosis
2000; Elsevier BV; Volume: 275; Issue: 27 Linguagem: Inglês
10.1074/jbc.m000930200
ISSN1083-351X
AutoresJosé David Machado, Fernando Segura, Miguel A. Brioso, Ricardo Borges,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoThe effects of nitric oxide (NO) on the late phase of exocytosis have been studied, by amperometry, on Ba2+-stimulated chromaffin cells. Acute incubation with NO or NO donors (sodium nitroprusside, spermine-NO,S-nitrosoglutathione) produced a drastic slowdown of the granule emptying. Conversely, cell treatment with Nω-nitro-l-arginine methyl ester (a NO synthase inhibitor) or with NO scavengers (methylene blue, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium) accelerated the extrusion of catecholamines from chromaffin granules, suggesting the presence of a NO modulatory tone. The incubation with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine or zaprinast) or with the cell-permeant cGMP analog 8-bromo-cGMP, mimicked the effects of NO, suggesting the involvement of the guanylate cyclase cascade. NO effects were not related to changes in intracellular Ba2+. NO did not modify the duration of feet. Effects were evident even on pre-fusioned granules, observed under hypertonic conditions, suggesting that the fusion pore is not the target for NO, which probably acts by modifying the affinity of catecholamines for the intragranular matrix. NO could modify the synaptic transmitter efficacy through a novel mechanism, which involves the regulation of the emptying of secretory vesicles. The effects of nitric oxide (NO) on the late phase of exocytosis have been studied, by amperometry, on Ba2+-stimulated chromaffin cells. Acute incubation with NO or NO donors (sodium nitroprusside, spermine-NO,S-nitrosoglutathione) produced a drastic slowdown of the granule emptying. Conversely, cell treatment with Nω-nitro-l-arginine methyl ester (a NO synthase inhibitor) or with NO scavengers (methylene blue, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium) accelerated the extrusion of catecholamines from chromaffin granules, suggesting the presence of a NO modulatory tone. The incubation with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine or zaprinast) or with the cell-permeant cGMP analog 8-bromo-cGMP, mimicked the effects of NO, suggesting the involvement of the guanylate cyclase cascade. NO effects were not related to changes in intracellular Ba2+. NO did not modify the duration of feet. Effects were evident even on pre-fusioned granules, observed under hypertonic conditions, suggesting that the fusion pore is not the target for NO, which probably acts by modifying the affinity of catecholamines for the intragranular matrix. NO could modify the synaptic transmitter efficacy through a novel mechanism, which involves the regulation of the emptying of secretory vesicles. catecholamine cytosolic barium concentration 8-bromo-cGMP cytosolic calcium concentration 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium chromogranin A 3-isobutyl-1-methylxanthine Nω-nitro-l-arginine methyl ester nitric-oxide synthase cGMP-dependent protein kinase sodium nitroprusside NO is a short-lived, highly reactive radical involved in several physiological functions such as vasodilatation, macrophage mobility, cytotoxicity, or gene transcription (see Ref. 1.Schmidt H.H.H.W. Walter U. Cell. 1994; 78 (295): 919Abstract Full Text PDF PubMed Scopus (1495) Google Scholar for a review). In addition, NO is a modulator of neurotransmitter-mediated responses in the central nervous system (2.Haley J.E. Wilcox G.L. Chapman P.F. Neuron. 1992; 8: 211-216Abstract Full Text PDF PubMed Scopus (445) Google Scholar). In the adrenal gland, NO could be secreted from the chromaffin cell itself (3.Schwartz P.M. Rodrı́guez-Pascual F. Koesling D. Torres M. Fostermann U. Neuroscience. 1998; 82: 255-265Crossref PubMed Scopus (55) Google Scholar), or paracrine, being secreted from contiguous endothelium (4.Torres M. Ceballos G. Rubio R. J. Neurochem. 1994; 63: 988-996Crossref PubMed Scopus (59) Google Scholar). In addition, NO could also be released by afferent nerves (5.Marley P.D. McLeod J. Anderson C. Thomson K.A. J. Autonom. Nerv. Sys. 1995; 54: 184-194Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 6.Afework M. Ralevic V. Burnstock G. Neurosci. Lett. 1995; 190: 109-112Crossref PubMed Scopus (14) Google Scholar). To date, many in vitro studies have been carried out to elucidate the role of NO/cGMP on the secretory processes of chromaffin cells. Results are still controversial; O'Sullivan and Burgoyne (7.O'Sullivan A.J. Burgoyne R.D. J. Neurochem. 1990; 54: 1805-1808Crossref PubMed Scopus (69) Google Scholar) reported a potentiation of CA1 release induced by various NO-releasing agents, whereas others have found a dose-dependent inhibition of secretion (8.Rodrı́guez-Pascual F. Miras-Portugal M.T. Torres M. Mol. Pharmacol. 1996; 49: 1058-1070PubMed Google Scholar, 9.Oset-Gasque M.J. Parramón M. Hortelano S. Boscá L. González M.P. J. Neurochem. 1994; 63: 1693-1700Crossref PubMed Scopus (62) Google Scholar), or no changes at all (10.Shono M. Houchi H. Oka M. Nakaya Y. J. Cardiovasc. Pharmacol. 1997; 30: 419-423Crossref PubMed Scopus (7) Google Scholar, 11.Kumai T. Tanaka M. Tateishi T. Asoh M. Kobayashi S. Jpn. J. Pharmacol. 1998; 77: 205-210Crossref PubMed Scopus (4) Google Scholar). NO is also reported to increase basal secretion (3.Schwartz P.M. Rodrı́guez-Pascual F. Koesling D. Torres M. Fostermann U. Neuroscience. 1998; 82: 255-265Crossref PubMed Scopus (55) Google Scholar, 9.Oset-Gasque M.J. Parramón M. Hortelano S. Boscá L. González M.P. J. Neurochem. 1994; 63: 1693-1700Crossref PubMed Scopus (62) Google Scholar). NO induces CA synthesis through tyrosine hydroxylase activation (11.Kumai T. Tanaka M. Tateishi T. Asoh M. Kobayashi S. Jpn. J. Pharmacol. 1998; 77: 205-210Crossref PubMed Scopus (4) Google Scholar). The present view is that the main role of NO is the control of adrenal blood flow, whereas its modulation on the bulk of CA release seems to be small (12.Moro M.A. Michelena P. Sánchez-Garcı́a P. Palmer R. Moncada S. Garcı́a A.G. Eur. J. Pharmacol. 1993; 246: 213-218Crossref PubMed Scopus (27) Google Scholar). Catecholamines and other soluble components are stored within chromaffin granules at very high concentrations: 0.5–1m (13.Finnegan J.M. Pihel K. Cahill P.S. Huang L. Zerby S.E. Edwing A.G. Kennedy R.T. Wightman R.M. J. Neurochem. 1996; 66: 1914-1923Crossref PubMed Scopus (121) Google Scholar, 14.Albillos A. Dernick G. Horstmann H. Almers W. Alvarez de Toledo G. Lindau M. Nature. 1997; 398: 509-512Crossref Scopus (480) Google Scholar, 15.Kopell W.N. Westhead E.W. J. Biol. Chem. 1982; 257: 5707-5710Abstract Full Text PDF PubMed Google Scholar, 16.Sen R. Sharp R.R. Biochim. Biophys. Acta. 1982; 721: 70-82Crossref PubMed Scopus (12) Google Scholar), thus creating a high intragranular osmotic pressure. Complexation of intragranular substances will reduce the osmotic forces, thereby preventing granule lysis (15.Kopell W.N. Westhead E.W. J. Biol. Chem. 1982; 257: 5707-5710Abstract Full Text PDF PubMed Google Scholar, 16.Sen R. Sharp R.R. Biochim. Biophys. Acta. 1982; 721: 70-82Crossref PubMed Scopus (12) Google Scholar, 17.Helle K.B. Reed R.K. Ehrhart M. Aunis D. Angeletti R.H. Acta Physiol. Scand. 1990; 138: 565-574Crossref PubMed Scopus (34) Google Scholar, 18.Sharp R.R. Sen R. Biophys. J. 1982; 40: 17-25Abstract Full Text PDF PubMed Scopus (5) Google Scholar). No mechanisms are known at present that regulate this intragranular matrix complex. Amperometric techniques allow the direct observation of time-course kinetics of single secretory events and have been successfully used to study the late phase of exocytosis (19.Schroeder T.J. Borges R. Finnegan J.M. Pihel K. Amatore C. Wightman R.M. Biophys. J. 1996; 70: 1061-1068Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 20.Leszczyszyn D.J. Jankowski J.A. Viveros O.H. Diliberto Jr., E.J. Near J.A. Wightman R.M. J. Biol. Chem. 1990; 265: 14736-14737Abstract Full Text PDF PubMed Google Scholar, 21.Pihel K. Travis E.R. Borges R. Wightman R.M. Biophys. J. 1996; 71: 1633-1640Abstract Full Text PDF PubMed Scopus (67) Google Scholar, 22.Borges R. Travis E.R. Hoechstetler S.E. Wightman R.M. J. Biol. Chem. 1997; 272: 8325-8331Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Here, we show conclusively that NO, acting on the guanylate cyclase cascade, produces dramatic changes on quantal release of CA by single chromaffin cells, probably acting on the intragranular matrix. To our knowledge, this is the first experimental report suggesting that the kinetic of vesicular release can be modulated by drugs or second messengers. In addition, we have found evidence indicating that the interaction of intragranular components can be modulated under physiological conditions. If this effect of NO were extended to dense core vesicles of sympathetic neurons, it would result in significant changes on synaptic efficacy, even releasing the same amount of noradrenaline quanta. Noradrenaline, SNP, IBMX, zaprinast, methylene blue, 8-Br-cGMP, cultured media, sera, and collagenase type IA were purchased from Sigma-Aldrich (Madrid, Spain). Fura-2/AM, Pluronic acid, and S-nitrosoglutathione were obtained from Molecular Probes (Eugene, OR). Spermine-NO, l-NAME, and C-PTIO were purchased from RBI (Natick, MA). NO gas (N30) was purchased from Air Liquide (Tenerife, Spain). Urografin® was obtained from Schering España (Madrid, Spain). Culture plates were from Corning (Cambridge, MA). All salts used for buffer preparation were reagent grade. Bovine adrenal chromaffin cells, enriched in adrenaline, were prepared as described elsewhere (23.Moro M.A. López M.G. Gandı́a L. Michelena P. Garcı́a A.G. Anal. Biochem. 1990; 185: 243-248Crossref PubMed Scopus (187) Google Scholar). Cells were planted on 12-mm diameter glass coverslips at an approximate density of 5 × 105 cells/coverslip. Cells were maintained at 37 °C in a 5% CO2 environment and used at room temperature between 1 and 4 days of culture. Carbon fiber microelectrodes were prepared as described (24.Kawagoe K.T. Zimmerman J.B. Wightman R.M. J. Neurosci. Methods. 1993; 48: 225-240Crossref PubMed Scopus (343) Google Scholar). Carbon fibers (5 μm radius; Thornel P-55, Amoco Corp., Greenville, SC) were the kind gift of Prof. R. M. Wightman (University of North Carolina at Chapel Hill, NC). Electrochemical recordings were performed using an Axopatch 200B (Axon Instruments, Foster City, CA). A fixed potential of +650 mV was maintained between the carbon fiber electrode versus an Ag/AgCl pellet reference electrode. Electrodes were backfilled with 3m KCl to connect to the headstage. Electrodes were tested with a flow-injection system with noradrenaline standard solutions using an EI-400 potentiostat (Ensman Inst. Bloomington, IN) (24.Kawagoe K.T. Zimmerman J.B. Wightman R.M. J. Neurosci. Methods. 1993; 48: 225-240Crossref PubMed Scopus (343) Google Scholar). Glass coverslips with adhering adrenal cells were washed in Krebs-HEPES buffer solution containing (in mm): NaCl (140), KCl (5.Marley P.D. McLeod J. Anderson C. Thomson K.A. J. Autonom. Nerv. Sys. 1995; 54: 184-194Abstract Full Text PDF PubMed Scopus (36) Google Scholar), MgCl2 (1.2), CaCl2 (2.Haley J.E. Wilcox G.L. Chapman P.F. Neuron. 1992; 8: 211-216Abstract Full Text PDF PubMed Scopus (445) Google Scholar), glucose (11.Kumai T. Tanaka M. Tateishi T. Asoh M. Kobayashi S. Jpn. J. Pharmacol. 1998; 77: 205-210Crossref PubMed Scopus (4) Google Scholar), and HEPES (10.Shono M. Houchi H. Oka M. Nakaya Y. J. Cardiovasc. Pharmacol. 1997; 30: 419-423Crossref PubMed Scopus (7) Google Scholar), brought to pH 7.35 with NaOH. Cells were placed in a perfusion chamber positioned on the stage of an inverted microscope (Leica DM-IRB, Wetzlar, Germany). Amperometric measurements were performed with the carbon fiber microelectrode gently touching the cell membrane. Cell release was stimulated by 5-s pressure ejection of 5 mm Ba2+ from a micropipette placed 40 μm away from the cell. Ba2+ was used as a secretagogue because it does not require receptor activation or membrane depolarization and because it produces a low frequency of secretory event, so that during spike analysis the initial and final points of each wave can be easily distinguished. Experiments using hypertonic solutions were performed as described previously (22.Borges R. Travis E.R. Hoechstetler S.E. Wightman R.M. J. Biol. Chem. 1997; 272: 8325-8331Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Briefly, cells were incubated in hypertonic Krebs (750 mosm, obtained by adding NaCl) solution for 5 min, in the presence or in the absence of 10 μm SNP. Under these hypertonic conditions, secretion was elicited by pulse injection of isotonic Krebs solution. In order to reduce NO degradation, free O2 was reduced from the stock solution. Krebs solution was bubbled with pure N2 into a sealed bottle for about 60 min. Five milliliters of the above solution were transferred into 7-ml sealed vials and bubbled again for another 10 min, keeping a pure N2 atmosphere in the empty space. This degassing procedure reduced the pO2 to 15–20 mmHg (ABL-2, Radiometer, Copenhagen, Denmark), equivalent to 20–40 μm free O2. In a fume hood, NO gas was on-line bubbled through a sealed bottle containing 5 n NaOH, to get rid of acid-generated material and then to an empty 7-ml vial for 1 min at 0.5 bar, maintaining the flow for 5 min. Two milliliters of the deoxygenated solution were injected into the vial containing pure NO using a gas-tight syringe through a rubber stopper. The vial was kept at 4 °C and used within 2 h. NO concentration in the solution, measured by Griess method, was 2.8 mm. Amperometric signals were low-pass filtered at 1 KHz and sampled at 4 KHz and collected using a locally written software (Labview for Macintosh, National Instruments, Austin, TX). Data analysis was carried out using locally written macros for IGOR (Wavemetrics, Lake Oswego, OR). These macros allow the automatic digital filtering, secretory spike identification, and build histograms for spike classification. All the above macros are free shareware. Fig. 1 describes the parameters measured from each secretory spike. Once the beginning and the end points were found, the computer obtained the maximum amplitude of the oxidation current (I max), which was expressed in pA. The ascending slope (m) was determined from the linear part of the trace located between 25% and 75% of theI max; hence, this parameter is not affected by the presence of the pre-spike phenomenon (foot), m being expressed in nA/s. The time to peak (t P) was determined between the point at which the back-extrapolation of the slope line crossed the base line and the point ofI max. This parameter partially shows the slow dissociation of adrenaline from intragranular proteic matrix. Total granule release (Q) was obtained by integration of the curve, which indicates the amount of oxidizing substances released and is expressed in pC. Q was normalized as the cubic root (Q 1/3) and two spike fade constants (τ =I max− I max/e) and (τ′ = I max/e) taken from the adjusted exponential decay. Because of day-to-day variations in electrode sensitivity and cell responsiveness, significant differences were currently observed between untreated cells, used as controls, from different days. For this reason, effects of drugs on secretory spikes were compared with control experiments carried out under the same conditions. Statistical analysis was carried out by the non-parametric Mann-Whitney Utest. Glass coverslips with adhering adrenal cells were washed twice in Krebs buffer solution and incubated with 2 μm fura-2/AM (stock solution dissolved in 20% Pluronic F-127 in Me2SO) and 0.1% fetal calf serum for 45 min. Cells were then washed twice to remove extracellular dye and placed in the perfusion chamber, as described above. Intracellular Ba2+ was measured using a computer-operated monochromator (TILL Photonics, Munich, Germany) controlled by Labview software. Fluorescence signals were low-pass filtered at 510 nm and detected by a photomultiplier mounted to a viewfinder (TILL Photonics) that defined the area of interest over which the fluorescence intensity was integrated. Data of [Ba2+]c time courses were collected at 10 Hz and expressed as fluorescence ratios (F 360) and (F 380). The direct application of NO produced drastic effects on the time course of secretory spikes, which are summarized on TableI and II. These effects were reproduced with all of the NO donors tested (TableII). Spermine-NO was particularly potent promoting a fall in granules emptying kinetics. Incubation withS-nitrosoglutathione and direct NO application produced similar concentrationdependent changes, which relate closely to their described abilities for producing free NO (25.Ferrero R. Rodrı́guez-Pascual F. Miras-Portugal M.T. Torres M. Br. J. Pharmacol. 1999; 127: 779-787Crossref PubMed Scopus (62) Google Scholar). Spike decay was also affected; τ′ increased with 200 nm NO from 12.9 ms to 20.3, whereas τ changed from 27.1 to 42.3 ms, indicating that NO strongly slowed down the last phase of exocytosis. The observed spike shape changes were not caused by a decrease in electrode sensitivity, as SNP did not modify the oxidation curves observed in the flow stream system used for electrode calibration (data not shown).Table IThe effects of NO on secretory spike parametersI maxQt 12mt PnnpApCmsnA/smsspikescellsControl41.9 ± 1.81.4 ± 0.0624.8 ± 0.811.6 ± 0.620 ± 117617NO (20 nm)20.1 ± 0.91.0 ± 0.0533.1 ± 1.14.2 ± 0.233 ± 2 7168NO (200 nm)19.0 ± 1.11.1 ± 0.0741.0 ± 1.62.7 ± 0.251 ± 4 3326The effects of NO incubation are shown together with their own control cells (see "Results"). Data are expressed in the units indicated. See Fig. 1 for explanation of each parameter. Open table in a new tab Table IIThe effects of various NO/guanylate cyclase activators and blockers on secretory spike parameters (normalized data)I maxQt 12mt Pnsans, number of spikes; nc, number of cells.nc ans, number of spikes; nc, number of cells.NO (20 nm) 48* 71*133* 36*165* 716 8NO (200 nm) 45* 79165* 23*255* 332 6Puffed SNP (10 μm) 74*100136* 61*155*105914SNP 1 μM 64* 94154* 33*247*131011SNP 10 μM 36* 62*161* 24*371* 87121SPER-NO 100 μM 27* 55*177* 18*150* 52313GSNO 20 μM 54* 73*140* 31*360*1446 78-Br-cGMP 36* 63*137* 34*110 31911IBMX 5 mM 73*104128* 54*135*128615IBMX + SNP 64* 88125* 50*115* 94615Zaprinast 10 μM 63*110166* 37*195* 57212Zaprinast + SNP 60*124*217* 26*248* 52311L-NAME 10 μM 98** 87 85*117* 63*153613L-NAME 100 μM 78* 56* 69* 96 63* 83713C-PTIO 10 nM223*111 64*331*104 78510Methylene blue142* 62* 52*290* 58* 36413Data were normalized as percentages of their own control group. Statistical analysis (Mann-Whitney U test) was performed on original data. Significant differences, p < 0.01 (**) or p < 0.001 (*). SNP was incubated at 10 μm.a ns, number of spikes; nc, number of cells. Open table in a new tab The effects of NO incubation are shown together with their own control cells (see "Results"). Data are expressed in the units indicated. See Fig. 1 for explanation of each parameter. Data were normalized as percentages of their own control group. Statistical analysis (Mann-Whitney U test) was performed on original data. Significant differences, p < 0.01 (**) or p < 0.001 (*). SNP was incubated at 10 μm. Fig. 2 shows histograms from secretory spikes obtained in the absence or in the presence of 10 μm of the NO donor SNP incubated for 10–20 min. SNP caused a dramatic reduction in the spike I max, averaging a fall to a 36% of control that was accompanied by at 1/2 average increase of 161% (Table II). Virtually, no spikes over 60 pA were found upon SNP treatment. Conversely, the number of events with a t 1/2 of over 40 ms was greatly increased. The releasing speed decayed as the ascending slopes of spikes were drastically reduced. The histogram in Fig. 2 shows that the number of secretory events with at P over 10 ms in duration was largely increased. NO effects were even more pronounced with 100 μm SNP, but a dramatic reduction in the number of spikes prevented us from using these data. Total granule release remained unaltered at low concentration of the drug, whereas a reduction was observed when SNP was raised to 10 μm. In order to rule out SNP effects caused by NO metabolites accumulated along drug incubation, 10 μm SNP was also applied for 10 s in the vicinity of a cell. The effects of this brief application, although less pronounced, were qualitatively similar (Table II); I max dropped from 45 to 34 pA, andt 1/2 rose 36%. Fig. 3 describes how NO affected the time course of spikes. Incubation with 100 μm spermine-NO for 10 min produced a drastic change in spike shape, which included a reduction in theI max and in the m (ascending slope), accompanied with an increase in the t P, τ, τ′, and t 1/2. The effect of NO on exocytotic kinetics occurs in few seconds (Fig. 4).Figure 4Time course of NO effects on quantal release. Typical amperometric trace (from five) of a secretory response from a cell stimulated for 5 s with 5 mmBa2+ (dot); 30 s later NO solution was added to the cell chamber to get an estimated NO concentration of 200 nm (triangle). Vertical dashed lines indicate periods of 20 s wheret 1/2 values were measured. Numbersbetween lines show average values fort 1/2. The number of secretory spikes computed are in parentheses. Calibration barsare shown on the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Due to the large differences within control data from one day to another, each treatment was compared with its own untreated control cells, from the culture of the same day using the same electrode. TableII shows data normalized with their own control. Although the effects of NO on total CA released by Ba2+ were not analyzed in detail, a discrete reduction in spike firing, of about 15%, was observed. In addition, the average spike charge observed was reduced by 20–40%. The guanylate cyclase PKG is known to be the main cellular transduction system for NO. In order to test if cGMP could mimic the NO effects, cells were treated with 10 μm cGMP-permeable analog 8-Br-cGMP. Results are summarized on Table II. Incubation for 20–30 min caused changes of spike shape qualitatively similar to those found with NO and NO donors. The secretory speed was profoundly slowed, and spikes were indistinguishable from NO-treated cells. Similarly, τ values were affected to the same extend, τ′ increased from 9.47 to 12.7 ms, whereas τ changed from 20.8 to 31.4 ms, indicating that cGMP affected as well the very last phase of exocytosis. Endogenous cellular levels of cGMP can also be increased by inhibiting its degradation. Table II shows the effects of 20 min of incubation with two phosphodiesterase inhibitors, IBMX and the more specific inhibition of cGMP-phosphodiesterase, zaprinast. When applied alone, both substances produced net changes on spike shape similar to those observed with NO donors. In the presence of 10 μm SNP, slight additive effects were observed, suggesting that both agents act through the same mechanism. Zaprinast increased τ′ from 13.4 to 22.5 ms and τ from 30 to 49.3 ms, whereas these values were increased by IBMX from 19 to 24.7 ms and 36.6 to 47.3 ms, respectively; the addition of SNP did not significantly modify the τ values obtained with IBMX. Cells were treated with l-NAME at 37 °C for 30 min and exocytotic spikes recorded in the presence of the drug. Lowl-NAME concentrations (10 μm) promoted significant changes on the spike t 1/2,m, and t P values (Table II). Although data obtained with 100 μml-NAME were qualitatively similar, they should be interpreted with caution because of the total granule release reduction observed (44%). The effects ofl-NAME persisted during incubation but rapidly disappeared upon drug removal, indicating a reversible NOS inhibition. Highl-NAME concentrations (1 mm) resulted in a drastic reduction of the number of secretory spikes, probably due to a nonspecific or toxic effect (data not shown). The presence of NOS within chromaffin cells suggested the existence of a NO basal tone which probably modulates continuously the kinetics of the exocytosis. This basal tone was revealed by NO sequestration using NO scavengers. Table II shows the effects of cell incubation with methylene blue and C-PTIO on Ba2+-evoked secretory spikes. Neither methylene blue nor C-PTIO caused CA release. However, both agents induced a concentration-dependent reduction of the t 1/2, which was accompanied with an increase in m and a shortening oft P. C-PTIO was more potent than methylene blue, probably because of its specificity and ability to serve as NO scavenger; the I max was significantly increased to 223% after only 4–5 min of incubation, revealing the presence of a basal NO activity within cultured cells. An unexpected effect observed with NO scavengers was the changes found on spike charge. Table IIshows that C-PTIO increased Q, whereas methylene blue induced a reduction. However, in all cases, an increase in theI max together with a reduction int 1/2 and τ was observed. Fig. 3 summarizes the effect of 10 min of incubation with a low concentration of C-PTIO (10 nm); spikes became taller and thinner, and the CA concentration reaching electrode was much bigger. Note that NO could account for 10-fold changes in the CA concentration reaching electrode (I max). One possible target site of NO could be the interference with Ba2+ movements. A series of experiments was done measuring [Ba2+]c in the absence and in the presence of 10 μm SNP. Fig. 5 shows representative traces ofF 360/F 380 ratios obtained with cells loaded with fura-2. Cells treated with SNP showed no changes on the ascending part of the traces. However, a significant increase on [Ba2+]c of 18 ± 2% was observed on the plateau of [Ba2+]c traces (6 cells of each group). In any case, the increased [Ba2+]clevels were maintained in both groups of cells for 6–8 min after the stimulus, the time usually taken for amperometric recording. A series of experiments was carried out in order to elucidate the cellular target site for NO. Foot (pre-spike feature) duration indicates the elapsed time for formation of the fusion pore. If a given substance modifies the fusion pore machinery, the duration of the foot might be altered. However, no differences on foot duration were found between foot produced in control conditions and cells incubated with 10 μm SNP: 17.4 ± 1.1 ms (n = 99) versus 15.5 ± 1.2 ms (n = 84), or 8-Br-cGMP: 14.8 ± 0.8 ms (n = 87) versus 15.6 ± 1.3 ms (n = 64). In amperometric recordings, only the 35% of spikes exhibited foot (13.Finnegan J.M. Pihel K. Cahill P.S. Huang L. Zerby S.E. Edwing A.G. Kennedy R.T. Wightman R.M. J. Neurochem. 1996; 66: 1914-1923Crossref PubMed Scopus (121) Google Scholar, 19.Schroeder T.J. Borges R. Finnegan J.M. Pihel K. Amatore C. Wightman R.M. Biophys. J. 1996; 70: 1061-1068Abstract Full Text PDF PubMed Scopus (141) Google Scholar). In this study, measurements were only performed on spikes where the beginning and finishing points of foot were clearly distinguishable. We have shown that cell stimulation under conditions of high tonicity (i.e. >700 mosm), promoted the partial exocytosis of chromaffin granules (22.Borges R. Travis E.R. Hoechstetler S.E. Wightman R.M. J. Biol. Chem. 1997; 272: 8325-8331Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Ba2+ application caused an increase in the [Ba2+]c, which was not accompanied by secretory spikes. However, exocytotic pore formation had already occurred, as demonstrated by the fact that brief pressure injection of isotonic saline caused many exocytotic events, which lasted throughout the time of application. These secretory spikes had lost 50% of their content, and they did not possess foot because they came from pre-fusioned granules that could only swell in response to isotonic media (Fig. 1S⃞). After Ba2+ stimulation under hypertonic conditions, granules were already opened; changes produced in spike shape must not be caused by an effect on the fusion pore but on another target, probably on the affinity of CA for the intragranular matrix. Data obtained from Fig. 6 (table inset) show significant changes on spike shape obtained from pre-fusioned granules, which mimicked those produced under normal conditions. Moreover, pre-fusioned granules progressively lost CA, indicated by the gradual fall in Q values along the time from Ba2+ stimulation. NO partially prevented this loss. As shown in Fig. 6, there were statistical differences betweenQ values obtained from control and SNP groups, indicating that NO increased the affinity of CA for its intragranular matrix. The results of this study demonstrate that NO, even at low concentrations, produces profound effects on the kinetics of secretory spikes (Figs. 2 and 3). We also show that exocytosis is modulated by a basal NO tone present within the cultured cells. Our data suggest that most of the NO action is carried out through the guanylate cyclase PKG pathway, as incubation with cGMP analog 8-Br-cGMP and phosphodiesterase inhibitors mimicked NO effects. Previous studies have reported changes on spike shape by altering temperature (21.Pihel K. Travis E.R. Borges R. Wightman R.M. Biophys. J. 1996; 71: 1633-1640Abstract Full Text PDF PubMed Scopus (67) Google Scholar), ionic composition (26.Jankowski J.A. Finnegan J.M. Wightman R.M. J. Neurochem. 1994; 63: 1739-1747Crossref PubMed Scopus (64) Google Scholar, 27.Alés E. Tabares L. Poyato J.M. Valero V. Lindau M. Alvarez de Toledo G. Nat. Cell Biol. 1999; 1: 40-44Crossref PubMed
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