Antisense oligonucleotide intralesional therapy for human PC-3 prostate tumors carried in athymic nude mice

To address the question whether calretinin (CR) may protect cells against Ca2+ overload or trophic factor deprivation, PC12 cells were transfected with plasmids containing a CR coding region under control of a cytomegalovirus promoter. Nerve growth factor (NGF) treatment induced differentiation, increased transfection efficiency (at least 10-fold) and activated the CR gene (as found by RNase protection method and immunohistochemistry). Exogenous CR expression was identified either in living cells by fluorescence of green fluorescent protein (when the CR coding region was fused to this protein) or in fixed cells by CR immunoreactivity. Undifferentiated and NGF-differentiated populations of transfected cells were incubated in the presence of a Ca(2+)-ionophore or in media deprived of serum or NGF. Expression of exogenous CR in undifferentiated or NGF-treated cells (due to transfection) or endogenous CR (due to gene activation by NGF) did not render PC12 cells more resistant to insults such as Ca(2+)-overload and trophic factor deprivation.     

Reduced expression of E-cadherin in oral squamous cell carcinoma: relationship with DNA methylation of 5'' CpG island

The caffeine-evoked effects on the intracellular Ca2+ concentration ([Ca2+]i) and on the release of dopamine by PC12 cells were investigated. Stimulation by caffeine resulted in a transient Ca2+ release which was followed by a sustained phase of Ca2+ entry through a non-voltage dependent pathway. Treatment with cyclopiazonic acid (CPA) or thapsigargin, inhibitors of the Ca2+ATPase pump of the endoplasmic reticulum, resulted in only a sustained rise in [Ca2+]i in the presence of extracellular Ca2+. Pretreatment of cells with CPA or thapsigargin abolished the subsequent Ca2+ responses to caffeine. Caffeine also evoked the release of dopamine from the cells only in the presence of extracellular Ca2+, which was mimicked by CPA. These results suggest that store-dependent Ca2+ entry evoked by caffeine has an indispensable role in the secretory response in an excitable cell line, PC12 cells.     

Functional coupling of secretion and capacitative calcium entry in PC12 cells

The caffeine-evoked effects on the intracellular Ca2+ concentration ([Ca2+]i) and on the release of dopamine by PC12 cells were investigated. Stimulation by caffeine resulted in a transient Ca2+ release which was followed by a sustained phase of Ca2+ entry through a non-voltage dependent pathway. Treatment with cyclopiazonic acid (CPA) or thapsigargin, inhibitors of the Ca2+ ATPase pump of the endoplasmic reticulum, resulted in only a sustained rise in [Ca2+]i in the presence of extracellular Ca2+. Pretreatment of cells with CPA or thapsigargin abolished the subsequent Ca2+ responses to caffeine. Caffeine also evoked the release of dopamine from the cells only in the presence of extracellular Ca2+, which was mimicked by CPA. These results suggest that store-dependent Ca2+ entry evoked by caffeine has an indispensable role in the secretory response in an excitable cell line, PC12 cells.     

Inositol trisphosphate and cyclic ADP-ribose-mediated release of Ca2+ from single isolated pancreatic zymogen granules

In pancreatic acinar cells low (physiological) agonist concentrations evoke cytosolic Ca2+ spikes specifically in the apical secretory pole that contains a high density of secretory (zymogen) granules (ZGs). Inositol 1,4,5-trisphosphate (IP3) is believed to release Ca2+ from the endoplasmic reticulum, but we have now tested whether the Ca(2+)-releasing messengers IP3 and cyclic ADP-ribose (cADPr) can liberate Ca2+ from AGs. In experiments on single isolated ZGs, we show using confocal microscopy that IP3 and cADPr evoke a marked decrease in the free intragranular Ca2+ concentration. Using a novel high resolution method, we have measured changes in the Ca2+ concentration in the vicinity of an isolated AG and show that IP3 and cADPr cause rapid Ca2+ release from the granule, explaining the agonist-evoked cytosolic Ca2+ rise in the secretory pole.     

Ca2+ depletion from granules inhibits exocytosis. A study with insulin-secreting cells

The secretory compartment is characterized by low luminal pH and high Ca2+ content. Previous studies in several cell types have shown that the size of the acidic Ca2+ pool, of which secretory granules represent a major portion, could be estimated by applying first a Ca2+ ionophore followed by agents that collapse acidic pH gradients. In the present study we have employed this protocol in the insulin-secreting cell line Ins-1 to determine whether the Ca2+ trapped in the secretory granules plays a role in exocytosis. The results demonstrate that a high proportion of ionophore-mobilizable Ca2+ in Ins-1 cells resides in the acidic compartment. The latter pool, however, does not significantly contribute to the [Ca2+]i changes elicited by thapsigargin and the inositol trisphosphate-producing agonist carbachol. By monitoring membrane capacitance at the single cell level or by measuring insulin release in cell populations, we show that Ca2+ mobilization from nonacidic Ca2+ pools causes a profound and long lasting increase in depolarization-induced secretion, whereas breakdown of granule pH had no significant effect. In contrast, releasing Ca2+ from the acidic pool markedly reduces secretion. It is suggested that a high Ca2+ concentration in the secretory compartment is needed to sustain optimal exocytosis.     

Neuronal Ca2+ sensor 1, the mammalian homologue of frequenin, is expressed in chromaffin and PC12 cells and regulates neurosecretion from dense-core granules

Neuronal Ca2+ sensor 1 (NCS-1) is the mammalian homologue of the Ca2+-binding protein frequenin previously implicated in regulation of neurotransmission in Drosophila (Pongs, O., Lindemeier, J., Zhu, X. R., Theil, T., Endelkamp, D., Krah-Jentgens, I., Lambrecht, H.-G., Koch, K. W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J. , Canal, I., Barbas, J. A., and Ferrus, A. (1993) Neuron 11, 15-28). NCS-1 has been considered to be expressed only in neurons, but we show that NCS-1 expression can be detected in bovine adrenal chromaffin and PC12 cells, two widely studied model neuroendocrine cells. NCS-1 was present in both cytosolic and membrane fractions including purified chromaffin granules, and in immunofluorescence, its distribution overlapped with peripheral punctate staining seen with the synaptic-like microvesicle marker synaptophysin in PC12 cells. The possible functional role of NCS-1 in exocytosis of dense-core granules was tested using transient transfection in PC12 cells and assay of co-transfected growth hormone (GH) release. Overexpression of NCS-1 increased evoked GH release in intact cells in response to ATP. No effect of overexpression was seen on GH release because of Ca2+ in permeabilized cells suggesting that NCS-1 may have a regulatory but not direct role in neurosecretion.     

Transport via the regulated secretory pathway in semi-intact PC12 cells: role of intra-cisternal calcium and pH in the transport and sorting of secretogranin II

To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi-intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 microM [Ca2+]free to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 microM GTP gamma S, 50 microM AlF3-5 or 5 micrograms/ml BFA. When added after immature granules had formed, GTP gamma S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SgII via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SgII into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.     

Examination of the role of ADP-ribosylation factor and phospholipase D activation in regulated exocytosis in chromaffin and PC12 cells

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.     

Exocytosis of secretory granules in the juxtaglomerular granular cells of kidneys

BACKGROUND: There is little agreement as to the secretory process of renin granules in juxtaglomerular granular cells (JG cells) of kidneys, although a large number of studies of the regulation of renin secretion have been reported. METHODS: The structural correlation between the stimuli and the secretory process was examined in mouse JG cells on renal cortical slices incubated with the beta-adrenergic agonist, isoproterenol; the loop diuretic, furocemide; the Ca2+ chelator, EGTA; and the actin filament-disrupting agent, cytochalasin B. RESULTS AND CONCLUSIONS: Treatment with isoproterenol (10(-5)-10(-3) M) or furocemide (10(-3) M) in Ca(2+)-containing medium did not significantly affect the ultrastructure of JG cells. In slices incubated with isoproterenol or furocemide in the Ca(2+)-free medium, JG cells occasionally contained a few electron-lucent granules at the cell periphery in addition to the electron-dense mature granules observed in the control slices. On rare occasions, the JG cells displayed omega-shaped cavities with electron-lucent matrices, a feature similar to the contents of electron-lucent granules. Cytochalasin B markedly promoted the effects of these stimulants in Ca(2+)-free medium. These findings suggest that participation of actin filament disassembly in the exocytotic process of the mature granules in JG cells.     

Calcium-dependent neutrophil secretion: characterization and regulation by annexins

To gain direct access to the secretory machinery and study the regulation, mechanisms, and effectors of Ca2+-dependent neutrophil secretion, we developed an efficient and reproducible method of plasma membrane permeabilization using streptolysin O. We confirmed previous studies that permeabilized neutrophils secrete in response to calcium alone, but we also found that the Ca2+ dose-response is biphasic. Secretion is detectable at <1.0 microM Ca2+ and reaches a plateau between 1.0 and 60 to 80 microM. When stimulated with >80 microM Ca2+, secretion is two- to threefold greater than at lower [Ca2+], suggesting that two distinct mechanisms of Ca2+-dependent secretion that differ in their affinity for Ca2+ exist in neutrophils. Although permeabilization allows 100% leak of lactate dehydrogenase, maximum secretion from permeabilized cells is 80% that of f-met-leu-phe-stimulated intact cells, indicating that the essential components of the Ca2+-dependent secretory apparatus are predominantly, if not entirely, membrane bound. Permeabilization causes leakage of 100% of annexins V and VI, but 41% of annexin I and 12% of annexin III are retained. Immunofluorescence microscopy revealed that retained annexins I and III are associated with granule membranes. Addition of soluble annexins I and III to permeabilized cells increased Ca2+-induced secretion up to 15% and 90%, respectively, implying that both annexins participate in this secretory pathway. While annexin V is not required for secretion, it inhibits the low Ca2+-affinity mechanism of secretion.     

Video-microscopy for analysis of molecular dynamics in cells

Real-time analysis of molecular dynamics in living cells was studied by developed video-microscopes. Two new detective methods were reported, one is for analysis of ciliary movement and the other is the qualitative analysis of exocytosis of insulin-containing granules with a video-enhanced light/fluorescent microscope. For analysis of ciliary movement, glass beads were migrated in the flow. The migration speed parallel to the flow produced by ciliary beating was used as an index of the beating activity. When tracheal epithelium isolated from mouse was incubated with ambroxol, and expectorant known to activate ciliary beat frequency, the floating speeds of glass beads were changed with 1 min of incubation. The results suggest that the present method is useful not only for screening of expectorants but also for the study of molecular mechanisms underlying ciliary beat of tracheal epithelium. Visualization of the moment of the release of contents from insulin-containing granules was achieved using video-enhanced fluorescent microscopy in MIN6 cells of mouse insulinoma cell line. A fluorescent amino acridine dye, quinacrine, was found to be incorporated into low-pH secretory granules, including insulin, in the cells. The granules which incorporated quinacrine emitted a slightly blue-green fluorescence. Upon stimulation with glucose, release of the quinacrine fluorescence from granules were observed. The present method would be useful for quantitative analysis of secretion of insulin from MIN6 cells as well as pancreatic beta-cells.     

Gonadotropin response to naloxone in the mare: effect of time of year and reproductive status

Effects of a novel dihydropyridine type of antihypertensive drug, cilnidipine, on the regulation of the catecholamine secretion closely linked to the intracellular Ca2+ were examined using nerve growth factor (NGF)-differentiated rat pheochromocytoma PC12 cells. By measuring catecholamine secretion with high-performance liquid chromatography coupled with an electrochemical detector, we showed that high K+ stimulation evoked dopamine release from PC12 cells both before and after NGF treatments. Cilnidipine depressed dopamine release both from NGF-treated and untreated PC12 cells in a concentration-dependent manner. In contrast, inhibition by nifedipine was markedly decreased in the differentiated PC12 cells. With intracellular Ca2+ concentration ([Ca2+]i) measurements using fura 2, the elevation of high K+-evoked [Ca2+]i was separated into nifedipine-sensitive and -resistant components. The nifedipine-resistant [Ca2+]i increase was also blocked by cilnidipine, as well as omega-conotoxin-GVIA. By the use of the conventional whole-cell patch-clamp technique, the compositions of the high-voltage-activated Ca2+ channel currents in the NGF-treated PC12 cells were divided into types: L-type, N-type, and residual current components. It was also estimated that cilnidipine at 1 and 3 micromol/L strongly blocked the N-type current without affecting the residual current. These results suggest that cilnidipine inhibits catecholamine secretion from differentiated PC12 cells by blocking Ca2+ influx through the N-type Ca2+ channel, in addition to its well-known action on the L-type Ca2+ channel.     

Activation of protein kinase C by intracellular free calcium in the motoneuron cell line NSC-19

Clotrimazole (CLT), a member of the antifungal imidazole family of compounds, has been found to inhibit both calcium (Ca2+)-activated 86Rb and potassium (K) fluxes of human red cells and to inhibit red cell binding of 125I-charybdotoxin (ChTX) [11]. We have now used patch-clamp techniques to demonstrate reversible inhibition of whole cell KCa2+ currents in murine erythroleukemia (MEL) cells by submicromolar concentrations of CLT. Inhibition was equivalent whether currents were elicited by bath application of the Ca2+ ionophore A23187 or by dialyzing cells with a pipette solution containing micromolar concentrations of free Ca2+. The extent of inhibition of whole cell MEL KCa2+ currents was voltage-dependent, decreasing with increasing test potential. We also determined the single channel basis of the CLT inhibition in MEL cells by demonstrating the inhibition of a calcium-activated, ChTX-sensitive K channel by CLT in outside-out patches. The channel was also blocked by the des-imidazolyl metabolite of CLT, 2-chlorophenyl-bisphenyl-methanol (MET II) [15], thus demonstrating that the imidazole ring is not required for the inhibitory action of CLT. Single KCa2+ channels were also evident in inside-out patches of MEL cells. Block of K current by CLT was not unique to MEL cells. CLT also inhibited a component of the whole cell K current in PC12 cells. Channel specificity of block by CLT was determined by examining its effects on other types of voltage-sensitive currents. CLT block showed the following rank order of potency: K currents in PC12 cells > Ca2+ currents in PC12 cells > Na currents in sympathetic neurons. These results demonstrate that direct inhibition of single KCa2+ by CLT can be dissociated from inhibition of cytochrome P-450 in MEL cells.     

Double effect of ethanol on intracellular Ca2+ levels in undifferentiated PC12 cells

In PC12, a cellular line derived from a rat pheochromocytoma, ethanol (EtOH) induces a different effect depending on the concentration used. When resting cells are incubated with an alcohol concentration less than or equal to 120 mM, the [Ca2+]i increased with a double phase pattern. If the alcohol concentration was increased over 120-160 mM, EtOH reversed its effect and the [Ca2+]i decreased. This decrease was strongly inhibited if KCl-depolarized cells were used and was completely abolished if the substrate constituted EtOH-chronically treated cells. The Ca2+ increase is the consequence of an activation of L-type voltage-activated channels, while the other voltage-dependent channels (N-type), the receptor-operated channels and the Ca2+ extrusion pump present in these cells are not involved in EtOH action. These findings indicate that EtOH can induce (by different mechanisms) both potentiating and inhibiting effects on [Ca2+]i in PC12 cells in relation to the alcohol dose effectively present in the suspension medium.     

Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin-secreting cells

Insulin-secreting cells express four GTPases of the Rab3 family. After separation of extracts of INS-1 cells on a sucrose density gradient, the bulk of the A, B, and C isoforms was recovered in the fractions enriched in insulin-containing secretory granules. Rab3D was also mainly associated with secretory granules, but a fraction of this isoform was localized on lighter organelles. Analyses by confocal microscopy of immunostained HIT-T15 cells transfected with epitope-tagged constructs confirmed the distribution of the Rab3 isoforms. Transfection of HIT-T15 cells with GTPase-deficient mutants of the Rab3 isoforms decreased nutrient-induced insulin release to different degrees (D>B>A>C), while overexpression of Rab3 wild types had minor or no effects. Expression of the same Rab3 mutants in PC12 cells provoked an inhibition of K+-stimulated secretion of dense core vesicles, indicating that, in beta-cells and neuroendocrine cells, the four Rab3 isoforms play a similar role in exocytosis. A Rab3A/C chimera in which the carboxyterminal domain of A was replaced with the corresponding region of C inhibited insulin secretion as Rab3A. In contrast, a Rab3C/A chimera containing the amino-terminal domain of C was less potent and reduced exocytosis as Rab3C. This suggests that the degree of inhibition obtained after transfection of the Rab3 isoforms is determined by differences in the variable amino-terminal region.     

Etiology of the cribra orbitalia: effect of amino acid profile in bone collagen and the iron content of bone minerals]

The total calcium content of secretory granules, Cag, was evaluated in isolated neurohypophysial nerve endings. The Cag in the resting state, as measured by X-ray microanalysis, is relatively high with an average of 7.4 +/- 0.6 mmol/kg wet weight. Following a depolarizing potassium challenge, a subpopulation of granules with even higher Cag could be detected, dispersed over a wider range of concentrations (up to 70 mmol/kg wet weight). After subsequent rinsing in physiological saline, Cag decreased to control values. This could have resulted from Ca2+ extrusion, or from preferential secretion of calcium-enriched granules. Our data can be interpreted in favor of the second explanation since no decrease in Cag was observed when secretion was blocked by a hyperosmotic saline. The effect of hyperosmotic conditions on isolated nerve endings was further studied by monitoring free cytoplasmic Ca2+ with the calcium-sensitive dye Fura-2 and by conventional electron microscopy. It was demonstrated that hyperosmotic treatment alone did not increase basal cytosolic Ca2+ concentrations but did significantly reduce the potassium-induced cytosolic rise in Ca2+. Electron microscopy of nerve endings in hyperosmotic conditions showed numerous exocytotic figures at various stages. The observed changes in Cag are in accord with a published hypothesis which proposes that intragranular calcium is a significant variable in regulated secretion.     

Role of Ca2+ in differentiation mediated by nerve growth factor and dibutyryl cyclic AMP in PC12 cells

Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca2+ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca2+ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca2+ influx, and Ca2+ mobilization from intracellular Ca2+ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca2+ accumulation by nonmitochondrial Ca2+ pools or the thapsigargin (TG)-sensitive Ca2+ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca2+ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca2+ . Therefore, NGF and dbcAMP induced different effects on Ca2+ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca2+ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca2+ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.     

Cysteine string protein functions directly in regulated exocytosis

Cysteine string protein (Csp) is essential for neurotransmitter release in Drosophila. It has been suggested that Csp functions by regulating the activity of presynaptic Ca2+ channels, thus controlling exocytosis. We have examined the effect of overexpressing Csp1 in PC12 cells, a neuroendocrine cell line. PC12 cell clones overexpressing Csp1 did not show any changes in morphology, granule number or distribution, or in the levels of other key exocytotic proteins. This overexpression did not affect intracellular Ca2+ signals after depolarization, suggesting that Csp1 has no gross effect on Ca2+ channel activity in PC12 cells. In contrast, we show that Csp1 overexpression enhances the extent of exocytosis from permeabilized cells in response to Ca2+ or GTPgammaS in the absence of Ca2+. Because secretion from permeabilized cells is not influenced by Ca2+ channel activity, this represents the first demonstration that Csp has a direct role in regulated exocytosis.     

Ca2+-induced deprotonation of peptide hormones inside secretory vesicles in preparation for release

The acidic environment inside secretory vesicles ensures that neuropeptides and peptide hormones are packaged in a concentrated condensed form. Although this is optimal for storage, decondensation limits release. Thus, it would be advantageous to alter the physical state of peptides in preparation for exocytosis. Here, we report that depolarization of the plasma membrane rapidly increases enhanced green fluorescent protein (EGFP)-tagged hormone fluorescence inside secretory vesicles. This effect requires Ca2+ influx and persists when exocytosis is inhibited by N-ethylmaleimide. Peptide deprotonation appears to produce this response, because it is not seen when the vesicle pH gradient is collapsed or when a pH-insensitive GFP variant is used. These data demonstrate that Ca2+ evokes alkalinization of the inside of secretory vesicles before exocytosis. Thus, Ca2+ influx into the cytoplasm alters the physical state of intravesicular contents in preparation for release.     

Effects of reserpine on ECL-cell ultrastructure and histamine compartmentalization in the rat stomach

The histamine-storing ECL cells in the stomach play a key role in the control of acid secretion. They contain granules, secretory vesicles and microvesicles, and sustained gastrin stimulation results in the additional formation of vacuoles and lipofuscin bodies. The cells are rich in the vesicle monoamine transporter type-2 (VMAT-2), which can be inhibited by reserpine. The present study examines the effect of reserpine on ECL-cell ultrastructure and histamine compartmentalization. Rats received reserpine and/or gastrin. Reserpine was given twice by the intraperitoneal route (25 mg/kg once daily). Gastrin-17 was given by subcutaneous infusion (5 nmol/kg/h), starting at the time of the first reserpine injection and continuing for 4 days when the rats were killed. At this stage, histamine in the oxyntic mucosa was unaffected by reserpine but elevated by gastrin. Immunocytochemical analysis (confocal microscopy) showed ECL-cell histamine in control and gastrin-treated rats to be localized in cytoplasmic organelles (e.g., secretory vesicles). After treatment with reserpine alone or reserpine+gastrin, ECL-cell histamine occurred mainly in the cytosol. Planimetric analysis (electron microscopy) of ECL cells showed reserpine to increase the number, size and volume density of the granules and to reduce the size and volume density of the secretory vesicles. Gastrin reduced the number and volume density of granules and secretory vesicles, increased the number and volume density of microvesicles and caused vacuoles and lipofuscin bodies to appear. Reserpine+gastrin increased the number, volume density and size of the granules. Reserpine prevented the effects of gastrin on secretory vesicles, vacuoles and microvesicles, but did not prevent the development of lipofuscin. Our findings are in line with the views: (1) that preformed cytosolic histamine is taken up by granules/secretory vesicles via VMAT-2, that histamine is instrumental in the transformation of granules into secretory vesicles and in their consequent enlargement and (2) that vacuoles are formed by the fusion of large secretory vesicles.