Intragranular pH rapidly modulates exocytosis in adrenal chromaffin cells Marcial Camacho,* Jose ´ D. Machado,* Mo ´ nica S. Montesinos,* Manuel Criado and Ricardo Borges* *Unidad de Farmacologı ´a, Facultad de Medicina, Universidad de La Laguna, Tenerife, Spain  Instituto de Neurociencias de Alicante, Universidad Miguel Herna ´ ndez-CSIC, Sant Joan d’Alacant, Spain Abstract Several drugs produce rapid changes in the kinetics of exocy- tosis of catecholamines, as measured at the single event level with amperometry. This study is intended to unveil whether the mechanism(s) responsible for these effects involve changes in the intravesicular pH. Cell incubation with bafilomycin A1, a blocker of the vesicular proton pump, caused both a deceler- ation in the kinetics of exocytosis and a reduction in the cate- cholamine content of vesicle. These effects were also observed upon reduction of proton gradient by nigericin or NH 4 Cl. pH measurements using fluorescent probes (acridine orange, quinacrine or enhanced green fluorescent protein–synapto- brevin) showed a strong correlation between vesicular pH and the kinetics of exocytosis. Hence, all maneuvers tested that decelerated exocytosis also alkalinized secretory vesicles and vice versa. On the other hand, calcium entry caused a transient acidification of granules. We therefore propose that the regu- lation of vesicular pH is, at least partially, a necessary step in the modulation of the kinetics of exocytosis and quantal size oper- ated by some cell signals. Keywords: acridine orange, ATP-dependent vesicular proton pump, bafilomycin A1, enhanced green fluorescent protein, quinacrine, secretion. J. Neurochem. (2006) 96, 324–334. Exocytosis constitutes the main cellular mechanism for the secretion of neurotransmitters. This finding gave support to the classical quantal theory of neurotransmission, which maintained that neurotransmitters were released in discrete packages from the nerve terminals onto the postsynaptic cell (Del Castillo and Katz 1954). This implies that the modu- lation of a postsynaptic response can only take place through a change in the number of vesicles that undergo exocytosis at the nerve terminals. However, recent amperometric meas- urements at the single event level show that the concentration reaching the postsynaptic cell can be rapidly modulated without change in the number of released vesicles (Burgoyne and Barclay 2002). Two separate major mechanisms have been observed by which such a modulation is achieved; the first involves changes in the vesicular content (Sulzer and Pothos 2000), whereas the second acts through changes in the kinetics of exocytosis at the single event level (Machado et al. 2000). It is conceivable that either both mechanisms underlay the action of certain drugs or that they are the endpoint of cellular signaling routes. Previous studies by others and our own laboratory have shown that certain drugs alter the vesicular content and others the kinetics of exocytosis (Borges et al. 2002; Burgoyne and Barclay 2002). However, the mechanisms underlying changes such as those caused by activation of PKA (cAMP-dependent protein kinase) or PKG (cGMP- dependent protein kinase) were not tested in this respect. The vesicular pH could play a key role in the control of aggregation of vesicular solutes. The resting luminal pH of chromaffin granules is around 5.5 (Winkler and Westhead 1980), which roughly coincides with the pK a of the major vesicular protein chromogranin A. This protein plays a key role in overcoming the osmotic gradient resulting from the Received January 22, 2005; revised manuscript received August 1, 2005; accepted September 9, 2005. Address correspondence and reprint requests to Ricardo Borges, Unidad de Farmacologı ´a, Facultad de Medicina, Universidad de La Laguna, E-38071-La Laguna, Tenerife, Spain. E-mail: rborges@ull.es Abbreviations used: AO, acridine orange; C-PTIO, 2-(4-carboxy- phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DIDS, 4,4¢-di- isothiocyanostilbene-2,2¢-disulfonic acid; DMPP, dimethylphenyl- piperazinium; EGFP, enhanced green fluorescent protein; NO, nitric oxide; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; QNC, quinacrine; SNP, sodium nitroprusside; V-ATPase, ATP-dependent vesicular proton pump. Journal of Neurochemistry , 2006, 96, 324–334 doi:10.1111/j.1471-4159.2005.03526.x Ó 2005 The Authors 324 Journal Compilation Ó 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 324–334