Cytofluorometric quantitation of 5-hydroxytryptamine and heparin in individual mast cell granules.

Cytofluorometric quantitation of 5-hydroxytryptamine (5-HT) and heparin in individual mast cell granules is described. The technique is based on micromanipulation of intact mast cells reacted with formaldehyde or stained with Berberine sulfate and the use of a cytofluorometer equipped with a sensitive peak detecting device. The quantities of 5-HT and heparin contained in mast cell granules which are of the order of 10(-16) and 10(-13) g, respectively were expressed as relative fluorescence guanta. The results of measurements on representative samples of mast cell granules indicate that all granules contain heparin as well as 5-HT, and that there are large variations in both 5-HT and heparin content within the granule populations of individual cells. A dose dependent increase in 5-HT content in both cells and individual mast cell granules occurred 24 hr after the injection of 10--50 mg L-5-hydroxytryptophan/kg intraperitoneally. There was no evidence for an increase in the heparin content of granules or cells, indicating that a new synthesis of granular macromolecules is not required for the 5-HT uptake. The results further suggest that 5-HT may be stored initially in a cytoplasmic extragranular pool and then taken up in the mast cell granules.     

Intracellular localization of serotonin in mast cells of the colon in normal and colitis rats

Intracellular localization of serotonin (5-HT) in the mast cells of two phenotypes in normal rat colon and dextran sodium sulphate-induced colitis was studied by immunoelectron microscopy with a quantitative analysis of the distribution of immunogold labelling. Mucosal mast cells in normal rats contained round shape secretory granules with varying electron density. Immunogold labelling for 5-HT was concentrated over the secretory granules. In mucosal mast cells from colitis rats, vacuolated granules without 5-HT labelling were frequently observed and immunogold labelling over the secretory granules was significantly increased compared to controls. On the other hand, connective tissue mast cells in normal rats contained oval shape secretory granules with homogeneous electron density. Their immunogold labelling was diffusely scattered over the secretory granules as well as over the cytoplasm. In connective tissue mast cells from colitis rats, secretory granules with high electron density were increased and the immunogold labelling over the secretory granules was much higher than that in controls. The present results suggest that intracellular localization of 5-HT is different in two phenotypes of mast cells and they may release 5-HT in a different manner. Mucosal mast cells may release 5-HT by a degranulation or exocytosis, while connective tissue mast cells may release 5-HT by a diacrine manner of secretion.     

Small GTPase Rab4 regulates Ca2+-induced alpha-granule secretion in platelets

Upon activation, platelets release many active substances stored in alpha- and dense-core granules. However, the molecular mechanisms governing regulated exocytosis are not yet fully understood. Here, we have established an assay system using permeabilized platelets to analyze the Ca(2+)-induced exocytosis of both types of granules, focusing on RabGTPases. Incubation with Rab GDP dissociation inhibitor, an inhibitory regulator of RabGTPases, reduced membrane-bound RabGTPases extensively, and caused strong inhibition of the Ca(2+)-induced secretion of von Willebrand factor (vWF) stored in alpha-granules, but not that of [(3)H]5-hydroxytryptamine (5-HT) in dense-core granules. Specifically, Rab4 co-fractionated with vWF and P-selectin (an alpha-granule marker) upon separation of platelet organelles by density gradient centrifugation. Incubation of the permeabilized platelets with cell extracts expressing the dominant negative mutant of His-tagged Rab4S22N, but not with those of similar mutant His-Rab3BT36N, inhibited the vWF secretion, whereas neither of the cell extracts affected the [(3)H]5-HT secretion. Importantly, the inhibition of vWF secretion was rescued by depleting the cell extracts of the His-Rab4S22N with nickel beads. Thus, in platelets, the regulatory mechanisms governing alpha- and dense-core granule secretions are distinct, and Rab4 is an essential regulator of the Ca(2+)-induced exocytosis of alpha-granules.     

Enteroendocrine cells and 5-HT availability are altered in mucosa of guinea pigs with TNBS ileitis

Enteroendocrine cells act as sensory transducers, releasing 5-HT and numerous peptides that are involved in regulating motility, secretion, and gut sensation. The action of mucosal 5-HT is terminated by a 5-HT reuptake transporter (SERT). In this study, we examined the hypothesis that ileitis leads to changes in enteroendocrine cell populations and mucosal 5-HT availability. Ileitis was induced in guinea pigs by intraluminal injection of 2,4,6-trinitrobenzenesulfonic acid and experiments were conducted 3, 7, and 14 days after treatment. The number of somatostatin, neurotensin, and 5-HT-immunoreactive cells increased at 3 and 7 days of ileitis, respectively, whereas no significant changes in the numbers of cholecystokinin, glucagon-like peptide-2, glucose-dependent insulinotropic peptide, and peptide YY-immunoreactive cells were observed. Chemical stimulation of the inflamed mucosa with sodium deoxycholic acid significantly increased 5-HT release compared with basal release. Mechanical stimulation of the mucosa potentiated the effect of the chemical stimuli at day 7. Epithelial SERT immunoreactivity was significantly reduced during the time course of inflammation. Thus changes in enteroendocrine cell populations and 5-HT availability could contribute to the altered motility and secretion associated with intestinal inflammation by disrupting mucosal signaling to enteric nerves involved in peristaltic and secretory reflexes.     

The role of Na+/H+ exchanger in serotonin secretion from porcine blood platelets

This study was undertaken to evaluate whether a link exists between the activation of protein kinase C (PKC), operation of Na(+)/H(+) exchanger (NHE), cell swelling and serotonin (5-HT) secretion in porcine platelets. Activation of platelets by thrombin or phorbol 12-myristate 13-acetate (PMA), a PKC activator, initiated a rapid rise in the activity of Na(+)/H(+) exchanger and secretion of 5-HT. Both thrombin- and PMA-evoked activation of Na(+)/H(+) exchanger was less pronounced in the presence of ethyl-isopropyl-amiloride (EIPA), an NHE inhibitor, and by GF 109203X, a PKC inhibitor. Monensin (simulating the action of NHE) caused a dose-dependent release of 5-HT that was not abolished by GF 109203X or EGTA. Lack of Na(+) in the suspending medium reduced thrombin-, PMA-, and monensin-evoked 5-HT secretion. GF 109203X nearly completely inhibited 5-HT release induced by PMA-, partly that induced by thrombin, and had no effect on 5-HT release induced by monensin. EIPA partly inhibited 5-HT release induced by thrombin and nearly totally that evoked by PMA. Electronic cell sizing measurements showed an increase in mean platelet volume upon treatment of cells with monensin, PMA or thrombin. The PMA- and thrombin-evoked rise in mean platelet volume was strongly reduced in the presence of EIPA. As judged by optical swelling assay monensin and PMA produced a rapid rise in platelet volume. The swelling elicited by PMA was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Hypoosmotically evoked platelet swelling did not affect platelet aggregation but significantly potentiated thrombin-evoked release of 5-HT and ATP. Taken together, these results show that in porcine platelets PKC may promote 5-HT secretion through the activation of NHE. It is hypothesized that enhanced Na(+)/H(+) antiport may result in a rise in cell membrane tension (due to cell swelling) which in turn facilitates fusion of secretory granules with the plasma membrane leading to 5-HT secretion.     

Toxoplasma gondii ADP-ribosylation factor 1 mediates enhanced release of constitutively secreted dense granule proteins

Toxoplasma gondii dense granules are morphologically similar to dense matrix granules in specialized secretory cells, yet are secreted in a constitutive, calcium-independent fashion. We previously demonstrated that secretion of dense granule proteins in permeabilized parasites was augmented by the non-hydrolyzable GTP analogue guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (Chaturvedi, S., Qi, H., Coleman, D. L., Hanson, P., Rodriguez, A., and Joiner, K. A. (1998) J. Biol. Chem. 274, 2424-2431). As now demonstrated by pharmacological and electron microscopic approaches, GTPgammaS enhanced release of dense granule proteins in the permeabilized cell system. To investigate the role of ADP-ribosylation factor 1 (ARF1) in this process, a cDNA encoding T. gondii ARF1 (TgARF1) was isolated. Endogenous and transgenic TgARF1 localized to the Golgi of T. gondii, but not to dense granules. An epitope-tagged mutant of TgARF1 predicted to be impaired in GTP hydrolysis (Q71L) partially dispersed the Golgi signal, with localization to scattered vesicles, whereas a mutant impaired in nucleotide binding (T31N) was cytosolic in location. Both mutants caused partial dispersion of a Golgi/trans-Golgi network marker. TgARF1 mutants inhibited delivery of the secretory reporter, Escherichia coli alkaline phosphatase, to dense granules, precluding an in vivo assessment of the role of TgARF1 in release of intact dense granules. To circumvent this limitation, recombinant TgARF1 was purified using two separate approaches, and used in the permeabilized cell assay. TgARF1 protein purified on a Cibacron G3 column and able to bind GTP stimulated dense granule secretion in the permeabilized cell secretion assay. These results are the first to show that ARF1 can augment release of constitutively secreted vesicles at the target membrane.     

Kinetics of release of serotonin from isolated secretory granules. I. Amperometric detection of serotonin from electroporated granules.

We developed a method for measuring the efflux of 5-hydroxytryptamine (5-HT, serotonin) from isolated intact granules of the mast cell of the beige mouse. This method combines electroporation of the vesicle membrane with amperometric detection of 5-HT. A single secretory granule is placed between two platinum electrodes (distance approximately 100 microm) and positioned adjacent (<1 microm) to a carbon fiber microelectrode. A short (approximately 30 micros) high-intensity voltage pulse (electric field of approximately 5 kV/cm) is delivered to the electrodes to trigger the mechanical breakdown of the granule membrane, which activates the release of 5-HT. We observed concurrent swelling of the granule matrix with the oxidation of 5-HT at the carbon fiber electrode (overpotential + 650 mV). Similar to the release of secretory products during exocytosis, the oxidation current exhibits a spike-like time course with a noninstantaneous rising phase (time between onset of current and maximum flux, t(max)) with approximately 25% of the molecules released during this period. When the current reaches its maximum, the granule matrix attains its maximum swollen state. We found that the rising phase depends on the initial cross-sectional area of the granule (t(max) approximately 21r2) and reflects the time required for membrane rupture. The average t(1/2)spike of the amperometric spikes was found to be approximately 150 ms, which is 3-7 times faster than the t(1/2) measured during cellular exocytosis.     

A highly Ca2+-sensitive pool of granules is regulated by glucose and protein kinases in insulin-secreting INS-1 cells

We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca(2+) to directly measure the Ca(2+) sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca(2+) sensitivity of release in that a small proportion of granules makes up a highly Ca(2+)-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca(2+). A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca(2+) are not located close to Ca(2+) channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca(2+)-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca(2+), suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca(2+) sensor that is poised to respond to modest, global elevations of [Ca(2+)](i).     

Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine

The parasympathetic control of goblet cell secretion and the membrane events accompanying accelerated mucus release were studied in large intestinal mucosal biopsies maintained in an organ culture system. The secretory response of individual goblet cells to 10(-6) M acetylcholine chloride with 3 x 10(-3) M eserine sulfate (a cholinesterase inhibitor) was assessed by light microscopy and autoradiography, by scanning and transmission electron microscopy, and by freeze-fracture. Goblet cells on the mucosal surface are unaffected by acetylcholine. In crypt goblet cells acetylcholine-eserine induces rapid fusion of apical mucous granule membranes with the luminal plasma membrane (detectable by 2 min), followed by sequential, tandem fission of the pentalaminar, fused areas of adjacent mucous granule membranes. These events first involve the most central apical mucous granules, are then propagated to include peripheral granules, and finally spread toward the most basal granules. By 60 min, most crypt cells are nearly depleted. The apical membrane, although greatly amplified by these events, remains intact, and intracellular mucous granules do not coalesce with each other. During rapid secretion membrane-limited tags of cytoplasm are observed attached to the cavitated apical cell surface. These long, thin extensions of redundant apical membrane are rapidly lost, apparently by being shed into the crypt lumen.     

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.     

Myosin 5a controls insulin granule recruitment during late-phase secretion

We have examined the importance of the actin-based molecular motor myosin 5a for insulin granule transport and insulin secretion. Expression of myosin 5a was downregulated in clonal INS-1E cells using RNAinterference. Stimulated hormone secretion was reduced by 46% and single-cell exocytosis, measured by capacitance recordings, was inhibited by 42% after silencing. Silencing of Slac-2c/MYRIP, which links insulin granules to myosin 5a, resulted in similar inhibition of single-cell exocytosis. Antibody inhibition of the myosin 5a-Slac-2c/MYRIP interaction significantly reduced the recruitment of insulin granules for release. The pool of releasable granules independent of myosin 5a activity was estimated to approximately 550 granules. Total internal reflection microscopy was then applied to directly investigate granule recruitment to the plasma membrane. Silencing of myosin 5a inhibited granule recruitment during late phase of insulin secretion. In conclusion, we propose a model where insulin granules are transported through the actin network via both myosin 5a-mediated transport and via passive diffusion, with the former playing the major role during stimulatory conditions.     

Rat mast cell tryptase

Rat mast cell tryptase is located largely if not totally in the cell's secretory granules. When the active site reagent [3H]diisopropyl fluorophosphate was used to label tryptase and chymase simultaneously, the ratio of tryptase:chymase active sites was determined to be 0.05. In comparison to chymase and tryptase in other species and chymase in the rat, rat tryptase is poorly bound to the granule matrix as evidenced by (1) its release parallel to histamine on induction of secretion and (2) its appearance in the supernatant when isolated granules were stripped of their membranes with hypotonic medium. Tryptase on release from the granule is moderately stable at a pH of 5.0 but unstable at pH 7.5, the pH that the enzyme encounters on secretion from the cell. These several properties indicate that the role of rat mast cell tryptase extracellularly is likely to differ greatly from that of chymase.     

An osmotic mechanism for exocytosis from dissociated chromaffin cells

Dissociated chromaffin cells from bovine adrenal medulla were stimulated to secrete epinephrine and dopamine beta-hydroxylase with a variety of secretagogues in a study designed to test the hypothesis that the chemiosmotic lysis reaction of isolated chromaffin granules might in some way be related to the mechanism of release during exocytosis. Increasing the osmotic strength of the incubation medium with either NaCl or sucrose led to suppression of secretion of epinephrine from the cells regardless of whether secretion was induced with veratridine or acetylcholine. Suppression of secretion was approximately exponential with respect to osmotic strength. Epinephrine secretion occurred only if the medium contained a permeant anion such as chloride, and secretion induced by veratridine was suppressed when Na isethionate replaced NaCl in the medium. In an extensive study with different monovalent anions veratridine supported epinephrine secretion according to the following activity series: Br-, I-, NO3- greater than methylsulfate, SCN- greater than Cl greater than acetate much greater than isethionate. A similar series, except for the potency of NO3-, was observed with A23187 as agonist. In general, the anion series for granule lysis was analogous. However, there was a poor quantitative correlation between the anion dependence of chemiosmotic granule lysis and the anion dependence of cell secretion. Anion transport inhibitors such as probenecid and pyridoxal phosphate also inhibited secretion while the stilbene disulfonates were inactive. The ineffectiveness of the stilbene disulfonates further distinguished chemiosmotic granule lysis from cell secretion. Secretion of catecholamines, induced by veratridine or nicotine, a cholinergic agonist, was suppressed when NaCl in the medium was replaced by isosmotic sucrose and unexpectedly low levels of dopamine beta-hydroxylase were observed in some cases. In sum, these properties of secreting chromaffin cells resembled some properties of isolated chromaffin granules incubated in ATP and Cl-, but were different in a number of instances. We, therefore, have interpreted our data to indicate that while some mechanistic relationships may indeed exist between the release event in exocytosis from chromaffin cells and the chemiosmotic lysis reaction characteristic of isolated chromaffin granules, an understanding of the energetics of exocytosis awaits the discovery of reasons for the quantitative differences between the two systems.     

Regulation of secretory granule pH in insulin-secreting cells

Luminal acidification is important for the maturation of secretory granules, yet little is known regarding the regulation of pH within them. A pH-sensitive green fluorescent protein (EGFP) was targeted to secretory granules in RIN1046-38 insulinoma cells by using a construct in which the EGFP gene was preceded by the nucleotide sequence for human growth hormone. Stimulatory levels of glucose doubled EGFP secretion from cell cultures, and potentiators of glucose-induced insulin secretion enhanced EGFP release. Thus this targeted EGFP is useful for population measurements of secretion. However, less than ~4% of total cell EGFP was released after 1.5 h of stimulation. Consequently, when analyzed in single cells, fluorescence of the targeted EGFP acts as an indicator of pH within secretory granules. Glucose elicited a decrease in granule pH, whereas inhibitors of the V-type H(+)-ATPase increased pH and blocked the glucose effect. Granule pH also was modified by effectors of the protein kinase A pathway, with activation eliciting granule alkalinization, suggesting that potentiation of peptide release by cAMP may involve regulated changes in secretory granule pH.     

Kinetics of release of serotonin from isolated secretory granules. II. Ion exchange determines the diffusivity of serotonin.

We measured the efflux of 5-hydroxytryptamine (5-HT, serotonin) from an intact secretory granule extracted from the mast cell of the beige mouse. The efflux was measured with amperometry after rupture of the granule membrane was triggered by electroporation. We determined the diffusivity of 5-HT within the secretory granule to be 2.0 x 10(-8) cm2 s(-1) when the granule is in contact with a physiological saline and found that this diffusivity depends on the valence of the cation in the external electrolyte. There is a fivefold increase in the diffusion coefficient of 5-HT determined in CsCl (150 mM, pH 7.2) at 3.7 x 10(-8) cm2 s(-1) compared to that determined in histamine dihydrochloride (Hi, 100 mM at pH 4.5) at 0.7 x 10(-8) cm2 s(-1). We found that the rate of expansion of the granule matrix observed in physiological medium correlates with the efflux of 5-HT, and that the rate of swelling of the matrix and the efflux depend on the microviscosity within the granule matrix and not the bulk viscosity of the external solution. The low diffusivity of 5-HT (approximately 500-fold less than in the bulk), the observation that the valence of the counterion affects this diffusivity, and the relationship between the volume changes of the matrix and the efflux suggest that 5-HT is released from the granule by ion exchange. We discuss the implications of this result for exocytotic release in mast cells and propose that an ion exchange mechanism could control the rate of release in other secretory systems.     

Similar molecular requirements for antigen receptor-triggered secretion of interferon and granule enzymes by cytolytic T lymphocytes

At least two biologically significant responses are triggered by the crosslinking of the T-cell receptor (TcR) on the surface of cloned cytotoxic T lymphocytes (CTL): synthesis and secretion of macrophage-activating factor(s) (MAF) that can be attributed to interferon-gamma (IFN) and release of preformed cytolytic granules. We directly compared the molecular requirements for synthesis and secretion of IFN and secretion of granule enzymes triggered in the same cell by the same activating ligand (antigen or monoclonal antibody (mAb) to TcR). An increase in the surface density of activating ligand (immobilized anti-TcR mAb) enhanced both secretion of IFN and secretion of granules. Secretion of IFN occurred immediately after synthesis: only low (but detectable) levels of IFN were detected in cell cytosolic or particulate fractions isolated from Percoll gradients of lysed CTL, while very high levels of IFN were found in the stimulated CTL culture fluids. Inhibitors of RNA synthesis and protein synthesis blocked secretion of IFN, but did not inhibit release of preformed cytolytic granules. The requirement for TcR crosslinking in triggering both secretion of granules and secretion of IFN from CTL was pharmacologically reproduced by the synergistic action of PMA, a protein kinase C activator, and the Ca2+ ionophore A23187. Both secretion of IFN and secretion of granules were absolutely dependent upon extracellular Ca2+: EGTA completely blocked both TcR- and PMA/A23187-induced secretion of IFN and exocytosis of granules. These studies suggest that similar molecular mechanisms are involved in secretion of newly synthesized IFN and secretion of preformed cytolytic granules. One notable difference between the molecular requirements for the two secretory events was a much lower concentration requirement for PMA for IFN synthesis and secretion than for granule secretion in the synergistic interactions with A23187. Implications of these studies for the exocytosis model of cell-mediated cytotoxicity are discussed.     

Post-translational processing and secretory pathway of human atriopeptin in rat pheochromocytoma cells.

Atriopeptin (AP) is expressed in several tissues with each tissue capable of specific differences in processing of the prohormone (pro-AP) to mature low molecular forms of the peptide. Since pro-AP has low biological activity, processing into mature AP is a critical activation event. This observation prompted us to study whether granule storage or regulated secretion of AP is essential for cleavage of mature peptide. We examined the processing of AP in adrenal medulla derived cells, using the rat pheochromocytoma cell line (PC12 cell) stably transfected with a genomic human AP DNA in the presence and absence of nerve growth factor (NGF), and also examined the mechanism of AP secretion and compared the results with those obtained using transfected chinese hamster ovary cells (CHO cells). The amount of prohormone was 5-10 fold higher than that of low molecular form of AP in the transfected PC12 cells. This ratio was essentially unchanged in differentiated PC12 cells after NGF treatment of the cells. Potassium depolarization of the transfected PC12 cells caused a 5-fold increase in AP release into the medium primarily as the intact prohormone. On the other hand, transfected CHO cells only exhibited constitutive AP release which is non-response to depolarization. These results suggest that the AP prohormone is sorted into secretory granules as the prohormone in PC12 cells and undergoes regulated release in response to depolarization indicating granule storage or release is not the critical determinant of AP prohormone cleavage.