Comparison of LR White and Unicryl as embedding media for light and electron immunomicroscopy of chromaffin cells.

LR White and Unicryl are members of the same family of acrylic embedding resins and are very suitable for "on grid" postembedding immunogold labeling. We studied the ultrastructure of LR White- and Unicryl-embedded cultured chromaffin cells and the immunolocalization of three chromaffin cell proteins, the enzymes dopamine-beta-hydroxylase (DbetaH) and tyrosine hydroxylase (TH), and the membrane fusion and Ca2+ channel protein synexin (annexin VII). We report here that Unicryl is preferable to LR White as an embedding medium for electron microscopy when osmium tetroxide fixation is omitted. The basis for this distinction is better ultrastructural preservation and improved immunodetection efficiency.

Detection of fragmented DNA in apoptotic cells embedded in LR white: A combined histochemical (LM) and ultrastructural (EM) study.

We developed an improved method for the detection of double-strand DNA breaks in apoptotic cells at both the light (LM) and electron microscopic (EM) levels using a modification of the TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labeling (TUNEL) technique. Cultured rat cerebellar granule cells were exposed to low potassium conditions to induce apoptosis. Twenty-four hr after treatment, one group of cells was fixed in situ with 4% paraformaldehyde and labeled for DNA fragmentation characteristic of apoptosis. Apoptotic cells were visualized with diaminobenzidine (DAB) and viewed by LM. The second group of cells was detached from the culture dish, pelleted, fixed with a 4% paraformaldehyde and 0. 2% glutaraldehyde mixture, and embedded in LR White. For LM, the modified TUNEL technique was performed on 1.5-microm LR White sections and apoptotic cells were visualized using an enzymatic reaction to generate a blue precipitate. For EM, thin sections (94 nm) were processed and DNA fragmentation was identified using modified TUNEL with streptavidin-conjugated gold in conjunction with in-depth ultrastructural detail. Alternate sections of cells embedded in LR White can therefore be used for LM and EM TUNEL-based detection of apoptosis. The present findings suggest that the modified TUNEL technique on LR White semithin and consecutive thin sections has useful application for studying the fundamental mechanism of cell death. (J Histochem Cytochem 47:561-568, 1999)

Flat embedding and immunolabelling of SW 1116 colon carcinoma cells in LR white: an improved technique in light and electron microscopy.

Human SW 1116 colon carcinoma cells were grown on matrix-covered coverslips and flat embedded in specially prepared gelatin capsules in the hydrophylic resin LR White. Dehydration and polymerization were carried out so as to maximize preservation of antigenicity. Sections were cut perpendicular to the substratum. To visualize mucin, semithin sections of SW 1116 cells were stained with periodic acid Schiff (PAS) reagent for light microscopy, and ultrathin sections were labelled with a monoclonal mucin antibody (Mab 19-9) and immunogold for electron microscopy. Immunofluorescence was carried out on whole cultured cells using Mab 19-9. The morphological preservation of SW 1116 cells embedded in LR White was comparable to that of Epon-embedded cells. Mucin was localized on the microvillar surface of the apical plasma membrane and occasionally in intercellular spaces between adjacent cells. Mucin was also present in vesicles in the apical and lateral part, and to a lesser extent in the basal part of the cells. We conclude that this new technology significantly improves the morphological preservation of cells and tissues in LR White, while also serving to sustain the antigenicity of cellular antigens.

Gliotoxin-induced cytotoxicity proceeds via apoptosis and is mediated by caspases and reactive oxygen species in LLC-PK1 cells.

Renal failure associated with aspergillosis is caused by pathogenic fungi. Gliotoxin is a toxic epipolythiodioxopiperazine metabolite produced by the pathogens. The present study investigated the cytotoxicity and underlying mechanisms induced by gliotoxin in LLC-PK1 cells, a porcine renal proximal tubular cell line. Gliotoxin at 100 ng/ml did not show a cytotoxic effect, but unmasked a dose-dependent cell death induced by TNF-alpha. TNF-alpha-induced cell death in the presence of gliotoxin was associated with hypodiploid nuclei and activation of caspase-3-like proteases. Blockade of caspases by boc-aspartyl (OMe)-fluoromethylketone and z-DEVD.fmk inhibited TNF-alpha-induced cell death. As the concentrations of gliotoxin were increased, gliotoxin killed the cells directly in a dose-dependent manner. Further analyses of DNA fragmentation, hypodiploid nuclei, mitochondrial membrane potential, and plasma membrane integrity revealed that cell death proceeded via apoptosis. Gliotoxin-induced apoptosis was associated with dose-dependent and time-dependent activation of caspase-3-like proteases. Boc-aspartyl (OMe)-fluoromethylketone attenuated the killing effect. Gliotoxin also increased the intracellular levels of reactive oxygen species as measured by flow cytometry. N-acetylcysteine, a well-known antioxidant, completely abolished the gliotoxin-induced caspase-3-like activity, cytotoxicity, and reactive oxygen species. In conclusion, (1) gliotoxin at 100 ng/ml unmasks the ability of TNF-alpha-induced apoptosis, and the effect of TNF-alpha is mediated by caspase-3-like proteases; and (2) at higher concentrations gliotoxin itself induces cell death, which is via apoptosis and dependent on caspase-3-like activity and reactive oxygen species.

Preservation of GABAergic perikarya and boutons after transient ischemia in the gerbil hippocampal CA1 field.

Using an antibody directed against the gamma-aminobutyric acid (GABA)-synthetizing enzyme glutamate decarboxylase (GAD) the fate of the GABAergic innervation was investigated in the hippocampal field CA1 of gerbils up to 14 days after a bilateral transient 5-min occlusion of carotid arteries. As described previously, the CA1 pyramidal cells were subject to the ischemia-induced delayed neuronal death, the first signs of which were detectable after 2 days and which was fully developed after 4 days. Local GAD-immunoreactive neurons and boutons, however, exhibited no changes in their distribution and morphology over the whole 14-day period investigated, as studied both at the light and electron microscopic level. Thus, it can be assumed that the increased excitation observed during the development of delayed neuronal death, is not due to a loss of GABAergic neuronal profiles. The resistance of the GABAergic neurons to the ischemic insult is discussed in relation to the presence of Ca2+-binding proteins in this class of neurons, and the long persistence of innervation in an area nearly devoid of postsynaptic targets is considered.

Gliosis in the LP-BM5 murine leukemia virus-infected mouse: an animal model of retrovirus-induced dementia.

Mice infected with the LP-BM5 murine leukemia virus (MuLV) mixture develop severe immunosuppression, neurotransmitter abnormalities and cognitive impairments in the absence of significant viral or macrophage invasion of the CNS. The time-course of the changes in glial activation have been characterized in an effort to understand the cellular basis of the neurobehavioral abnormalities observed in these mice. Glial activation was determined by measuring the relative changes in F4/80 protein and GFAP immunoreactivity using immunoblots. Augmented F4/80 expression preceded that of GFAP, with global elevations of 4-6-fold at 3 weeks, sustained for up to 12 weeks after inoculation. GFAP immunoreactivity increased 2-fold only in the cerebral cortex and striatum 5 weeks postinfection, declining to control levels by 12 weeks. Immunohistochemistry revealed significant increases in microglial size and staining intensity in the cortex, corpus callosum and striatum, with the development of a unique population of highly ramified, intensely stained microglia and microglial nodules in the corpus callosum and striatum. No evidence of ameboid microglia was found. Astrocyte size and degree of ramification was increased in the hippocampus, cortex, striatum and corpus callosum. Thus, microgliosis is an early event in LP-BM5 infection, preceding astrocytosis, neurotransmitter loss, and development of cognitive deficits. Activated microglia may secrete neurotoxins leading to the neurochemical alterations and cognitive deficits observed in these mice. Because gliosis and microglial nodule formation are hallmarks of HIV-1 encephalopathy, LP-BM5 MuLV-infected C57/B16 mice may afford insights into the mechanisms contributing to the early stages of this syndrome.

Effect of MPTP on dopaminergic neurons in the goldfish brain: a light and electron microscope study.

The neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes a Parkinsonian syndrome in the goldfish (Carassius auratus), characterized by transient bradykinesia, the accumulation of MPP+ in the brain, and a decrease in the forebrain and midbrain content of catecholamines (Pollard et al., FASEB J., 6 (1992) 3108-3116). Using light and electron microscopy, we studied the effect of MPTP on the distribution and ultrastructure of tyrosine hydroxylase (TH)-immunoreactive, dopaminergic neurons, and on the ultrastructure of other selected areas of the goldfish brain. Goldfish were treated with MPTP (50 mg/kg) in the absence or presence of L-deprenyl (10 mg/kg) or clorgyline (10 mg/kg). In the medial part of the central telencephalon, the nucleus telencephali, pars medialis, MPTP caused a decrease in the number of TH-immunoreactive neurons and distortions in their labelling pattern. Electron microscopic observations showed that MPTP caused swelling of cell processes, changes in neuronal nuclear profiles, dilation of endoplasmic reticulum, intracellular vacuolization and membrane distortions, and degeneration of neuronal fibers in this brain area. MPTP also caused a small reduction and some diffuseness in the labelling of dopaminergic neurons in several diencephalic periventricular nuclei. Moreover, MPTP induced cell swelling and degeneration in the subependymal cell layers along the forebrain ventricles. In all areas, L-deprenyl appeared to partially prevent the MPTP-induced degenerative changes. We conclude that in the goldfish MPTP causes marked histochemical changes in selected dopaminergic brain systems coincident with the Parkinson-like locomotor and neurochemical deficits.

Identification and localization of a kainate binding protein in the frog inner ear by electron microscopy immunocytochemistry.

A kainate binding protein (KBP) was studied in Rana pipiens inner ear using monoclonal and polyclonal antibodies against affinity purified KBP from frog brain. The KBP identified and analyzed in inner ear tissue homogenates, with one- and two-dimensional immunoblots, was similar to the affinity purified KBP and to the antibody-identified frog brain KBP. As brain KBP, inner ear KBP had 5 main components in the molecular weight dimension, centered at Mr = 48,000; however, inner ear KBP had a greater abundance of the higher molecular weight components. Light and electron microscopy observations showed KBP immunostaining at two locations: (1) in the dendrites of the eight nerve afferent fibers contacting sensory hair cells, with the postsynaptic density being more intensely stained; and (2) on the cytoplasmic membrane of fibroblasts present in the inner ear connective tissue which displayed intense immunostaining. The presence of kainate (KA) binding sites in the inner ear was assessed using in vitro receptor autoradiography. [3H]KA binding sites were found in connective tissue areas confirming the immunocytochemistry results. The postsynaptic localization of the KBP in afferent endings, strongly supports it as being a component of the KA receptor complex. However, its presence on fibroblasts situated in the inner ear connective tissue makes its function hypothetical. The dual presence of the KBP on non-neuronal cells as well as at postsynaptic membrane sites suggests the existence of a family of proteins involved in KA binding and KA receptors with a complex organization.

Identification and ultrastructural localization of a calretinin-like calcium-binding protein (protein 10) in the guinea pig and rat inner ear.

Calretinin has been identified as a brain specific calcium-binding protein which appears as a prominent protein in the cochlear nucleus. We identified and localized calretinin in the guinea pig and rat inner ear using polyclonal antibodies. Immunoblot analyses of guinea pig and rat auditory nerve homogenates revealed an immunoreactive band migrating with the same molecular weight as the purified protein, at Mr = 29 k. Immunocytochemistry was carried out at the light and electron microscope levels. In the guinea pig cochlea, inner hair cells, Deiters' cells, Hensen's cells and interdental cells of the spiral limbus were stained. Most of the cochlear ganglion cells were immunostained. In the guinea pig vestibular organs, the staining was exclusively neuronal and localized in large nerve fibers and nerve calices of the apex of the cristae. Only some vestibular ganglion cells were stained. In the rat cochlea, inner hair cells and most of the ganglion neurons were immunoreactive. In the rat vestibule, large nerve fibers and calices were stained as were some type II hairs cells. Only some vestibular ganglion cells were reactive. Electron microscopic observations of immunostained guinea pig cochlea and vestibule showed that the staining was cytosolic. In addition, specific sub-localization was also found in the apical portion of the nerve calices in association with microvesicles. These results describe the discrete localization of calretinin in the cochlea and in the vestibular receptors and suggest a function associated with biochemical regulations at the level of microvesicles in vestibular afferent neurons.

Quinolinic acid protects rat cerebellar granule cells from glutamate-induced apoptosis.

The effects of quinolinic acid (QUIN) on glutamate-induced excitotoxicity were examined in primary cultures of rat cerebellar granule neurons. Exposing these neurons to QUIN (< or =2.5 mM) in the presence of glucose and Mg2+ had no effect on their viability. Although pretreating neurons with QUIN (10 microM) for 6 h did not reduce necrotic death induced by glutamate exposure in the absence of glucose and Mg2+, QUIN pretreatment significantly suppressed glutamate-induced apoptosis by 68% (as indicated by DNA fragmentation) in cultures containing glucose and Mg2+. Furthermore, the N-methyl-D-aspartate (NMDA) receptor antagonist AP-5 reversed QUIN-induced neuroprotection, while the non-NMDA antagonist CNQX had no effect. This study demonstrates that pathophysiologically relevant concentrations of QUIN can protect neurons from apoptosis mediated via the NMDA receptor.

Pathophysiological aspects of blood-brain barrier permeability in epileptic seizures.

Blood-brain barrier (BBB) permeability to macromolecules was assessed during seizures induced by pentylenetetrazole, bicuculline, methoxypyridoxine, methionine sulfoximine, and kainic acid. It was observed that each convulsant induced a specific pattern of regional BBB opening. This was, however, only the case when systemic blood pressure (BP) rose with seizure onset. The analysis of regional cerebral blood flow revealed that a high increase in flow in rabbits with BP rise is related to the normal flow at rest in the single brain region, but not to BBB permeability. In rabbits without BP increase, regional flow increase was low but well modulated and is possibly a better indicator for neuronal activity. The ultrastructural analysis showed that macromolecular transport over the cerebrovascular endothelium is by pinocytosis, an neurotransmitter controlled process. It is suggested that seizure-induced regional BBB opening is determined by two factors: release of neurotransmitters due to the process of auto-regulation during peripheral pressure increase, and change in local neurotransmitter milieu due to the action of the convulsant and/or the seizure activity.

Characterization of a calcium current in a vertebrate cholinergic presynaptic nerve terminal.

Calcium currents were recorded from a cholinergic presynaptic nerve terminal in the chick ciliary ganglion using the whole-cell voltage-clamp technique. The presynaptic element of this synapse is in the form of a calyx that envelops the postsynaptic ciliary neuron. A method was developed to isolate the ciliary neuron, expose the calyx, and apply patch-clamp electrodes under visual control. The presynaptic Ca current activated at +30 mV with a fast time constant of about 1.5 msec and deactivated at -80 mV with a time constant of about 0.5 msec, values that are consistent with a role in action-potential-dependent transmitter release. The calyx Ca current was blocked by 0.1 mM Cd or 2 microM omega-conotoxin and was resistant to voltage-dependent inactivation. The presynaptic Ca channel exhibits similarities to the N-type group but differs from these by the minimal voltage-dependent inactivation. This type of channel, designated CaN-PT (N-like, presynaptic terminal), may play a key role in transmitter release at many vertebrate fast-transmitting synapses.

The blood-brain barrier to horseradish peroxidase at the onset of bicuculline-induced seizures in hypothalamus, pallidum, hippocampus, and other selected regions of the rabbit.

Rabbits were subjected to bicuculline-induced generalized seizures of 15-min duration to elucidate the mechanism by which the macromolecule horseradish peroxidase (HRP) traverses the blood-brain barrier (BBB) in specific brain areas. Transendothelial pinocytosis at the level of arterioles was the main route of passage. In addition, in thalamus and hippocampus pinocytotic vesicles were observed in capillaries. In thalamus, hypothalamus and septum vesicles in the endothelium of venules were also present. Repeatedly, pinocytotic vesicles were ejecting their content into the interendothelial clefts, so that the presence of HRP reaction product between adjacent tight junctions cannot be considered a conclusive evidence for their opening. The HRP, which had reached the neuropil due to the seizure-evoked BBB opening, accumulated in the interstitial spaces and penetrated the synaptic cleft. Uptake of the tracer in vesicular form into presynaptic boutons, presumably excitatory ones as diagnosed by their ultrastructural features, was observed in all brain regions. The uptake was rare in septum, periaqueductal gray, hypothalamus, and cerebellar cortex; frequent in pallidum, hippocampus, and medulla oblongata; and very intense in thalamus. Uptake in postsynaptic dendrites was present mostly in the vicinity of boutons. Incorporation into glial processes was rare and confined to perivascular astrocytes. It is suggested, that HRP traverses the BBB by regionally selective, transmitter-controlled pinocytotic transport and that the neuronal uptake of the foreign protein is at least partially dependent on the involvement of synapses of particular brain regions in the paroxysmal activity during the generalized seizures.

Preparation of affinity-purified, biotinylated tetanus toxin, and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells.

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 1:1 to 20:1 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60-80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using 125I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2-3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these "primed" cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.

Role of extracellular proteins in the dynamics of vasogenic brain edema.

The relationship between extravasation of proteins into extracellular spaces of brain parenchyma and the water content of such regions were evaluated in an experimental model. In this model, a temporary opening of the blood-brain barrier (BBB) to proteins was produced without significant injury to the cellular elements of brain tissue. Rabbits were subjected to bolus injection of their own blood under 360-400 mm Hg pressure via the internal carotid artery. The opening of the barrier and its duration were evaluated with Evans blue (EB), horseradish peroxidase (HRP), and sodium fluorescein (NaFl) tracers. The water content of brain tissue was assessed by specific gravity (SG) measurements in 1-mm-diameter tissue samples. Quantitative evaluation of protein penetration into brain tissue was carried out using 125I bovine serum albumin (BSA). The opening of the BBB to proteins persisted up to 9 h, whereas the barrier remained permeable to small molecular NaFl for 24 h. The SG measurements indicated in the areas of EB extravasation a progressive increment in water content up to 9 h, i.e., the duration of BBB opening to proteins. Following this, there was a progressive clearance of edema in spite of the BBB remaining open for NaFl for 24 h. Quantitative evaluations of 125I-BSA and SG in the same tissue samples, supported by statistical analysis, indicated approximately linear relationship analysis, indicated approximately linear relationship between albumin and water, implying a strong correlation between the development of vasogenic edema and extravasation of proteins into extracellular spaces.

Specific adhesion between pheochromocytoma (PC12) cells and adrenal medullary endothelial cells in co-culture.

Chromaffin cells in the adrenal medulla are found in close proximity to capillary endothelial cells, thereby forming the classical endocrine complex. To examine the possible chemical basis of their interaction in more detail, we have grown bovine adrenal medullary endothelial (BAME) cells in monolayer cultures and added to them pheochromocytoma (PC12) cells, a chromaffin tumor cell line of rats. The PC12 cells were chosen because of the similarities they share with adrenal medullary chromaffin cells. PC12 cells rapidly attached to BAME cells cultures, their rate of adhesion being significantly enhanced over binding of PC12 cells to either uncoated plates or to monolayers of unrelated cell cultures. Consistent with this observation, we noted that the extracellular matrix (ECM) derived from the BAME cells did not enhance PC12 cell adhesion and did not promote neurite sprouting as previously described for ECM derived from corneal endothelial cells. The specific adhesion between PC12 and BAME cells could be abolished by cell surface extracts derived from these two cells but not by extracts derived from unrelated cell types. This activity was heat-labile, sensitive to trypsin and, to a lesser extent, to neuraminidase. We therefore conclude that PC12 cells may interact with BAME cells by specific proteinaceous adhesive factors associated with their plasma membranes. These interactions might represent the formative role of cell-cell contacts in the organization of the developing adrenal gland.

Detection and localization of synexin (Annexin VII) in Xenopus adult and embryonic tissues using an antibody to the Xenopus N-terminal PGQM repeat domain.

Synexin (Annexin VII) is a widely distributed member of the annexin gene family which forms calcium channels and drives calcium-dependent membrane fusion. In Xenopus laevis, different synexins contain two to six tandem repeats of the tetra amino acid sequence PGQM in the unique N-terminal, with a distribution specific to adult tissues and embryonic stages. Immunogold studies using the PGQM-specific polyclonal antibody showed that synexin is localized in adult muscle to myosin-rich A-bands, Z-bands, and T-tubules, and in other adult tissues to nuclei and mitochondria and other formed elements. In oocytes, synexin was also found associated with yolk granules. The PGQM tandem repeats could represent interaction sites for other proteins, and we therefore synthesized a synthetic peptide containing the maximum six tandem repeats [NH2-(PGQM)6-Y-COOH] to test this hypothesis. We found that the peptide alone could specifically bind and crosslink to different proteins in a tissue-specific manner. In liver, it bound to a single 35-kDa protein. In muscle, it bound to four proteins (35, 45, 48, and 116 kDa). Therefore, we conclude that the PGQM domain is accessible to specific antibodies and that the PGQM repeat is sufficiently ordered to unambiguously identify specific binding proteins in different Xenopus tissues.

The MPTP-induced parkinsonian syndrome in the goldfish is associated with major cell destruction in the forebrain and subtle changes in the optic tectum.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can induce a parkinsonian syndrome in humans and nonhuman primates, which is susceptible to treatment and prevention by drugs such as L-DOPA and L-deprenyl. Recently, we have reported that MPTP can also cause a parkinsonian syndrome in the common goldfish, which appears to faithfully mirror the neurochemical and behavioral aspects of the action of MPTP in the higher vertebrates. In addition, we recently identified the likely teleost equivalent of the substantia nigra in the goldfish forebrain, the "nucleus pars medialis," on the basis of its destruction by MPTP and selective protection by the MAO-B blocker L-deprenyl. In the present work we substantiate this conclusion by examining tissue destruction the goldfish forebrain at increasing MPTP concentrations, up to the the LD50 of 200 mg/kg. In addition, we show that at the highest MPTP dose subtle changes also occur with low frequency in nondopaminergic cells in the optic tectum, and in ependymal cells lining the midbrain ventricle. The effects on ependymal cells are similar to those previously noted in the forebrain. We conclude that the goldfish model continues to faithfully mimic the histologic pattern of parkinsonian tissue destruction engendered by MPTP in primate models.

Granule swelling in stimulated bovine adrenal chromaffin cells: regulation by internal granule pH.

Adrenal medullary chromaffin cells secrete catecholamines through exocytosis of their intracellular chromaffin granules. Osmotic granule swelling has been implicated to play a role in the generation of membrane stress associated with the fusion of the granule membrane. However, controversy exists as to whether swelling occurs before or after the actual fusion event. Using morphometric methods we have determined the granule diameter distributions in rapidly frozen, freeze-substituted chromaffin cells. Our measurements show that intracellular chromaffin granules increase in size from an average of 234 nm to 274 nm or 277 nm in cells stimulated to secrete with nicotine or high external K+, respectively. Granule swelling occurs before the formation of membrane contact. Ammonium chloride, an agent which inhibits stimulated catecholamine secretion by approximately 50% by altering the intragranular pH, also inhibits granule swelling. In addition, ammonium chloride-treated secreting cells show more granule-plasma membrane contacts than untreated secreting cells. Sodium propionate induces granule swelling in the absence of secretagogue and has been shown to enhance nicotine- and high K(+)- induced catecholamine release. These results indicate that in adrenal chromaffin cells granule swelling is an essential step in exocytosis before fusion pore formation, and is related to the pH of the granule environment.