Dickkopf3 (Dkk3) is required for maintaining the integrity of secretory vesicles in the mouse adrenal medulla.

REIC (reduced expression in immortalized cells) has been identified as a gene whose expression was reduced in immortalized cultured cells. The REIC gene is identical to Dickkopf-3 (Dkk3), which encodes a secreted glycoprotein belonging to the Dkk family. Previously, we showed that Dkk3 protein is present in the mouse adrenal medulla. However, its role in this tissue has not been elucidated. To explore it, we performed electron microscopic (EM) studies and RNA-sequencing (RNA-seq) analysis on Dkk3-null adrenal glands. EM studies showed that the number of dense core secretory vesicles were significantly reduced and empty vesicles were increased in the medulla endocrine cells. Quantitative PCR (qPCR) analysis showed relative expression levels of chromogranin A (Chga) and neuropeptide Y (Npy) were slightly but significantly reduced in the Dkk3-null adrenal glands. From the result of RNA-seq analysis as a parallel study, we selected three of the downregulated genes, uncoupled protein-1 (Ucp1), growth arrest and DNA-damage-inducible 45 gamma (Gadd45g), and Junb with regard to the estimated expression levels. In situ hybridization confirmed that these genes were regionally expressed in the adrenal gland. However, expression levels of these three genes were not consistent as revealed by qPCR. Thus, Dkk3 maintains the integrity of secreting vesicles in mouse adrenal medulla by regulating the expression of Chga and Npy.

Oncocytoma of the adrenal gland medulla.

We report an unprecedented case of an oncocytoma of the adrenal gland medulla in a 61-year-old woman. The patient presented with right flank pain and hematuria. Computed tomographic studies revealed a right adrenal gland mass that measured 2 cm, which was subsequently excised laparoscopically. Grossly, the tumor in the medulla measured 1.9 × 1.2 cm, weighed 5 g, and had a solid tan-brown cut surface. Histologically, it consisted of large tumor cells containing eosinophilic granular cytoplasm arranged in trabecular and nodular patterns. Electron microscopy revealed closely packed mitochondria in the cytoplasm of almost all tumor cells. The tumor cells were immunohistochemically positive for vimentin. The patient resumed usual activities 2 weeks after surgery, and at 6-month follow-up, she is doing well.

Neural-endocrine mechanisms of respiratory syncytial virus-associated asthma in a rat model.

We examined the underlying neural-endocrine mechanisms of asthma associated with respiratory syncytial virus infection. Thirty Sprague-Dawley rats were randomly divided into control group, respiratory syncytial virus (RSV) group, and anti-nerve growth factor (NGF) IgG group. An RSV infection model was established by nasal drip once a week. In the anti-NGF antibody intervention group, each rat was given an intraperitoneal injection of anti-NGF IgG 3 h before RSV infection. Optical microscopy and transmission electron microscopy were used to observe the structural changes in adrenal medulla cells. Changes in adrenaline and norepinephrine in serum were detected by ELISA. NGF expression was assayed by immunohistochemistry. Expression differences in synaptophysin mRNA were detected by RT-PCR. Transmission electron microscopy displayed widened adrenal medulla intercellular spaces, reduced chromaffin particle concentration, and increased mitochondria in the RSV infection group. At the same time, NGF expression was increased in the RSV infection group significantly. In addition, the adrenaline concentration was significantly decreased compared with the control and anti-NGF antibody groups. Synaptophysin mRNA expression was significantly increased in the RSV infection and anti-NGF antibody groups. However, compared with the RSV infection group, synaptophysin mRNA expression was significantly decreased in the anti-NGF antibody group. We conclude that RSV infection could induce adrenal medulla cell differentiation to nerve cells by over-expression of NGF, resulting in the decreased endocrine function found in asthma progression.

SV2 modulates the size of the readily releasable pool of secretory vesicles.

The exocytosis of neurotransmitters is regulated by calcium and is plastic - features that suggest specialized regulation of the basic membrane trafficking process. Here we show that Synaptic Vesicle Protein 2 (SV2), a protein specific to neurons and endocrine cells, is required to maintain a pool of vesicles available for calcium-stimulated exocytosis. Direct measures of exocytosis in adrenal chromaffin cells showed that the calcium-induced exocytotic burst, which operationally defines the readily releasable pool of vesicles, was significantly reduced in mice lacking SV2A. Burst kinetics were normal in cells from SV2A knockout animals, however, indicating that SV2 functions before the final events of fusion. Analyses of SDS-resistant SNARE (soluble NSF (N-ethylmaleimide-sensitive fusion) attachment protein receptor) complexes in brain tissue showed that loss of SV2A was associated with fewer SDS-resistant complexes. Our observations indicate that SV2 may modulate the formation of protein complexes required for fusion and therefore the progression of vesicles to a fusion-competent state.

Functional remodeling of gap junction-mediated electrical communication between adrenal chromaffin cells in stressed rats.

An increase in circulating catecholamine levels represents one of the mechanisms whereby organisms cope with stress. In the periphery, catecholamines mainly originate from the sympathoadrenal system. As we reported, in addition to the central control through cholinergic innervation, a local gap junction-delineated route between adrenal chromaffin cells contributes to catecholamine exocytosis. Here, we investigated whether this intercellular communication is modified when the hormonal demand is increased as observed during cold stress. Our results show that in cold exposed rats, gap-junctional communication undergoes a functional plasticity, as evidenced by an increased number of dye-coupled cells. Of a physiological interest is that this upregulation of gap-junctional coupling results in the appearance of a robust electrical coupling between chromaffin cells that allows the transmission of action potentials between coupled cells. This enhancement of gap-junctional communication parallels an increase in expression levels of connexin36 (Cx36) and connexin43 (Cx43) proteins. Both transcriptional and posttranslational mechanisms are involved because Cx36 transcripts are increased in stressed rats and the expression of the scaffolding protein zonula occludens-1, known to interact with both Cx36 and Cx43, is also upregulated. Consistent with an upregulated coupling extent in stressed rats, the cytosolic Ca(2+) concentration rises triggered in a single cell by an iontophoretic application of nicotine occur simultaneously in several neighboring cells. These results describe for the first time a functional plasticity of junctional coupling between adult chromaffin cells that should be crucial for adaptation to stress or sensitization to subsequent stressors.

p60c-src is complexed with a cellular protein in subcellular compartments involved in exocytosis.

We found high levels of the c-src gene product in neuroendocrine tissues from adult animals. To understand the role of this proto-oncogene product, the subcellular localization of p60c-src was studied in neuroendocrine tissue from adrenal medulla. The results indicate that p60c-src was highly enriched in chromaffin granule membranes, in stable association with a protein of 38 kD. The complex with the 38-kD protein was also detected in brain, a tissue known to carry high levels of p60c-src. The 38-kD protein is not calpactin I, II, or synaptophysin. Comparison of its peptide map showed a high degree of conservation among the different species and tissues examined. The interaction between p60c-src and the 38-kD protein involves disulphide bonds that are stable even when the cell fractionation is performed in the presence of a reducing agent. Since the presence of disulphide bonds among cytoplasmic proteins is very unlikely, the possibility of a noncovalent association between p60c-src and the 38-kD protein in vivo is discussed. The 38-kD protein may be involved in a function of p60c-src related to secretory organelles.

Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells.

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH-terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.

Functional and morphological characterization of isolated bovine adrenal medullary cells.

Single bovine adrenal medullary cells have been obtained by retrograde perfusion of adrenal medullae with a solution of 0.05% collagenase in Ca++-free Krebs Henseleit buffer. Chromaffin cells were obtained in high yield (5 X 10(6) cells/g medulla), and more than 95% of these were viable as shown by exclusion of trypan blue. The isolated cells were capable of respiring at a linear rate for a minimum of 120 min. Ultrastructural examination revealed that the cells were morphologically intact, and two distinct types of adrenal medullary cells were identified, on the basis of the morphology of their electron-dense vesicles, as (a) adrenaline-containing and (b) noradrenaline-containing cells. Biochemical analysis showed that the cells contained catecholamines and dopamine-beta-hydroxylase (DBH). The cells released catecholamines and DBH in response to acetylcholine (ACh), and this release was accompanied by changes in the vesicular and surface membranes observed at the ultrastructural level. The time-course of ACh-stimulated catecholamine and DBH release, and the dependence of this release on the concentration of ACh and extracellular Ca++ have been investigated. The isolated cells were pharmacologically sensitive to the action of the cholinergic blocking agents, atropine and hexamethonium.

Localization and characterization of carbohydrates in adrenal medullary cells.

The localization and characterization of carbohydrates in adrenal medullary cells were studied by histochemical and cytochemical methods. Adrenaline (A)-and noradrenaline (N)-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. A small amount of glycogen in the form of single beta-particles as well as lysosomes were, however, visualized by this technique. The entire core of the A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded medullae were stained with phosphotungstic acid (PTA) at low pH (0.3). The N granules, in contrast, were mostly unreactive. In the A cells, PTA stained a large part of the Golgi complex, whereas in the N cells the Golgi complex was mostly unstained. In both cell types, the cell coat, lysosomes, and multivesticular bodies reacted to PTA. The periodic acid-Schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation, and greatly diminished by sulfation. In ultrathin sections of GMA- or Araldite-embedded medullae incubated with colloidal iron according to various techniques, the cell coat and lysosomes of both cell types were stained, unlike all the other cytoplasmic organelles. These results indicate that A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins which are probably not acidic.

Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry.

Calpactin I complex, a calcium-dependent phospholipid-binding protein, promotes aggregation of chromaffin vesicles at physiological micromolar calcium ion levels. Calpactin I complex was found to be a globular molecule with a diameter of 10.7 +/- 1.7 (SD) nm on mica. When liposomes were aggregated by calpactin, quick-freeze, deep-etching revealed fine thin strands (6.5 +/- 1.9 [SD] nm long) cross-linking opposing membranes in addition to the globules on the surface of liposomes. Similar fine strands were also observed between aggregated chromaffin vesicles when they were mixed with calpactin in the presence of Ca2+ ion. In cultured chromaffin cells, similar cross-linking short strands (6-10 nm) were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structures approximately 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy on frozen ultrathin sections showed that calpactin I was closely associated with the inner face of the plasma membranes and was especially conspicuous between plasma membranes and adjacent vesicles in chromaffin cells. These in vivo and in vitro data strongly suggest that calpactin I complex changes its conformation to cross-link vesicles and the plasma membrane after stimulation of cultured chromaffin cells.

Calcium-induced displacement of membrane-associated particles upon aggregation of chromaffin granules.

Isolated chromaffin granules incubated in 10 millimolar calcium chloride aggregated, forming contact sites with a pentalaminar membrane structure. These circular attachment sites were free of membrane-associated particles, which accumulated at the periphery. Incubation in 20 millimolar ethylenediaminetetraacetic acid reversed these changes, which are regarded as initial events in the membrane fusion reaction.

Induction of tyrosine 3-monooxygenase in adrenal medulla: role of protein kinase activation and translocation.

The transsynaptic induction of tyrosine 3-monooxygenase (TH) in rat adrenal medulla is preceded by an early increase in the ratio of cyclic adenosine monophosphate (AMP) to cyclic guanosine monophosphate, an activation of cytosol cyclic AMP-dependent protein kinase, and a subsequent translocation of protein kinase catalytic subunits from cytosol to subcellular particles. As a result of this translocation, nuclear protein kinase activity increases during the induction of TH. Transection of splanchnic nerve reverts these events and prevents the induction of TH. Thus, adrenal medulla activation and translocation of cyclic AMP-dependent protein kinase may act as a long-range messenger for the genetic regulation of TH synthesis.

Endopin serpin protease inhibitors localize with neuropeptides in secretory vesicles and neuroendocrine tissues.

BACKGROUND/AIMS: The endopin serpin protease inhibitors have been identified by molecular studies as components of secretory vesicles that produce neuropeptides. Endopin 1 inhibits trypsin-like serine proteases, and endopin 2 inhibits cathepsin L that produces neuropeptides in secretory vesicles. To assess the secretory vesicle and neuroendocrine tissue distribution of these endopins, the goal of this study was to define specific antisera for each endopin isoform and to examine their localization with neuropeptides and in neuroendocrine tissues. METHODS: This study utilized methods consisting of Western blots, immunoelectron microscopy, and immunofluorescence microscopy for evaluation of the localization of endopin protease inhibitors in neuroendocrine tissues. RESULTS: Immunoelectron microscopy with these selective antisera demonstrated the localization of endopins 1 and 2 within secretory vesicles of adrenal medulla (bovine). Cellular immunofluorescence confocal microscopy illustrated the high level of colocalization of endopins 1 and 2 with enkephalin and NPY neuropeptides that are present in secretory vesicles of adrenal medullary chromaffin cells in primary culture. Tissue distribution studies (by Western blots) showed the expression of endopins 1 and 2 in bovine brain, pituitary, adrenal medulla, and other neuroendocrine tissues. CONCLUSIONS: These results implicate endopins 1 and 2 as endogenous protease inhibitors in neuropeptide-containing secretory vesicles and neuroendocrine tissues.

Cathepsin L participates in the production of neuropeptide Y in secretory vesicles, demonstrated by protease gene knockout and expression.

Neuropeptide Y (NPY) functions as a peptide neurotransmitter and as a neuroendocrine hormone. The active NPY peptide is generated in secretory vesicles by proteolytic processing of proNPY. Novel findings from this study show that cathepsin L participates as a key proteolytic enzyme for NPY production in secretory vesicles. Notably, NPY levels in cathepsin L knockout (KO) mice were substantially reduced in brain and adrenal medulla by 80% and 90%, respectively. Participation of cathepsin L in producing NPY predicts their colocalization in secretory vesicles, a primary site of NPY production. Indeed, cathepsin L was colocalized with NPY in brain cortical neurons and in chromaffin cells of adrenal medulla, demonstrated by immunofluorescence confocal microscopy. Immunoelectron microscopy confirmed the localization of cathepsin L with NPY in regulated secretory vesicles of chromaffin cells. Functional studies showed that coexpression of proNPY with cathepsin L in neuroendocrine PC12 cells resulted in increased production of NPY. Furthermore, in vitro processing indicated cathepsin L processing of proNPY at paired basic residues. These findings demonstrate a role for cathepsin L in the production of NPY from its proNPY precursor. These studies illustrate the novel biological role of cathepsin L in the production of NPY, a peptide neurotransmitter, and neuroendocrine hormone.

Immunomorphometric variations of sustentacular cells of the male viscacha adrenal medulla during the annual reproductive cycle. Effects of androgens and melatonin.

The adrenal medulla is crucial for the survival of species facing significant environmental changes. The parenchyma is composed mainly of chromaffin cells, ganglion cells and sustentacular cells (SC). The male viscacha exhibits seasonal variations of gonadal activity and other metabolic functions. The aim of this work was to investigate the influence of the reproductive conditions on the morphology of SC of this rodent. In addition, the effects of testosterone and melatonin on these cells were studied. Immunoexpression of S100 protein, GFAP and vimentin were analyzed. Furthermore, the distribution of adrenergic and noradrenergic chromaffin cells subpopulations was studied for the first time in this species. SC present long cytoplasmic processes in contact with chromaffin cells, probably generating an intraglandular communication network. Significant differences (p < 0.05) in the %IA (percentage of immunopositive area) for the S100 protein were observed according to winter (4.21 ±â€¯0.34) and summer (3.51 ±â€¯0.15) values. In castrated animals, the %IA (6.05 ±â€¯0.35) was significantly higher in relation to intact animals (3.95 ±â€¯0.40). In melatonin-treated animals the %IA (3.62 ±â€¯0.23) was significantly higher compared to control animals (2.65 ±â€¯0.26). GFAP immunoexpression was negative and no noradrenergic chromaffin cells were detected suggesting an adrenergic phenotype predominance. Vimentin was observed in SC, endothelial cells and connective tissue. Results indicate that SC exhibit variations along the annual reproductive cycle, along with castration and the melatonin administration. Our results suggest that in this rodent SC are not only support elements, but also participate in the modulation of the activity of the adrenal medulla; probably through paracrine effects.

Fine ultrastructure and biochemistry of PC12 cells: a comparative approach to understand neurotoxicity.

The PC12 cell line is commonly used as a tool to understand the biochemical mechanisms underlying the physiology and degeneration of central dopamine neurons. Despite the broad use of this cell line, there are a number of points differing between PC12 cells and dopamine neurons in vivo which are missed out when translating in vitro data into in vivo systems. This led us to compare the PC12 cells with central dopamine neurons, aiming at those features which are predictors of in vivo physiology and degeneration of central dopamine neurons. We carried out this comparison, either in baseline conditions, following releasing or neurotoxic stimuli (i.e. acute or chronic methamphetamine), to end up with therapeutic agents which are suspected to produce neurotoxicity (l-DOPA). Although the neurotransmitter pattern of PC12 cells is close to dopamine neurons, ultrastructural morphometry demonstrates that, in baseline conditions, PC12 cells possess very low vesicles density, which parallels low catecholamine levels. Again, compartmentalization of secretory elements in PC12 cells is already pronounced in baseline conditions, while it is only slightly affected following catecholamine-releasing stimuli. This low flexibility is caused by the low ability of PC12 cells to compensate for sustained catecholamine release, due both to non-sufficient dopamine synthesis and poor dopamine storage mechanisms. This contrasts markedly with dopamine-containing neurons in vivo lending substance to opposite findings between these compartments concerning the sensitivity to a number of neurotoxins.

Morphology and ultrastructure of the adrenal gland in Bactrian camels (Camelus bactrianus).

In the present study, we examined the morphological features of the adrenal gland in Bactrian camel by means of digital anatomy, light and electron microscopy. Our findings testified that the gland was divided into three parts, capsule, cortex and medulla from outside to inside as other mammals, and the cortex itself was further distinguished into four zones: zona glomerulosa, zona intermedia, zona fasciculate and zona reticularis. Notably, the zona intermedia could be seen clearly in the glands from females and castrated males, whereas it was not morphologically clear in male. There was a great deal of lipid droplets in the zona fasciculate, while it was fewer in the zona glomerulosa and zona reticularis. The cytoplasm of adrenocortical cell contained rich mitochondria and endoplasmic reticulum. The adrenal medulla was well-developed with two separations of external and internal zones. The most obvious histological property of adrenal medulla cells were that they contained a huge number of electron-dense granules enveloped by the membrane, and so medulla cells could be divided into norepinephrine cells and epinephrine cells. Moreover, the cortical cuffs were frequently present in adrenal gland. Results of this study provides a theoretical basis necessary for ongoing investigations on Bactrian camels and their good adaptability in arid and semi-arid circumstances.

Effects of colocynth alkaloids and glycosides on Wistar rats fed high-fat diet. A biochemical and morphological study.

INTRODUCTION: In traditional medicine, Citrullus colocynthis is used to treat diabetes, hyperlipidemia, cardiovascular diseases, inflammation, and oxidative stress, all of which can appear when a diet rich in vegetable fats, such as palm oil, is continuously consumed. Such high-fat diets are chronic stressors of the hypothalamic-pituitary-adrenal axis. The objective of our study was to analyze and evaluate the effects of colocynth total alkaloids and glycosides on metabolic, hormonal, and structural disorders of the adrenal medulla in Wistar rats fed a high-fat diet. MATERIAL AND METHODS: Twenty six Wistar rats were distributed as follows: six control animals received a standard laboratory diet; twenty experimental rats received the standard laboratory diet supplemented with palm oil - the high-fat diet (HFD). After seven months of this diet, the HFD group was subdivided into rats treated for the next 2 months with either alkaloid extract (HFD-ALk group) or ethanol extract of glycosides (HFD-GLc) or animals on HFD only. Plasma metabolites and ACTH concentrations were measured by standard methods. Sections of adrenal medulla were stained by Heidenhain-Azan method and Sudan Black. RESULTS: The adrenal medulla of the HFD rats showed prominent structural changes, such as hypertrophy of chromaffin and ganglion cells, vacuolation, inflammatory foci, and fibrosis. The biochemical and hormonal parameters were significantly improved in the HFD rats treated with alkaloid and glycoside extracts of Citrullus colocynthis. Moreover, the morphological changes of the adrenal medulla were attenuated in HFD-ALk and HFD-Glc rats. CONCLUSIONS: The results of the study indicate that phytotherapy using Citrullus colocynthis alkaloids may correct metabolic and hormonal perturbations as well as adrenal medulla structure of rats maintained on HFD.

Diosgenin-caused changes of the adrenal gland histological parameters in a rat model of the menopause.

Diosgenin, a steroidal sapogenin of natural origin, has demonstrated benefits when it comes to the treatment of malignancies, cardiovascular issues and menopausal symptoms. In this study, we investigated the histological changes of the adrenal gland after diosgenin application in a rat model of the menopause. Middle-aged, acyclic female Wistar rats were divided into control (C; n=6) and diosgenin treated (D; n=6) groups. Diosgenin (100mg/kg b.w./day) was orally administered for four weeks, while C group received the vehicle alone. A histological approach included design-based stereology, histochemistry and immunohistochemistry. The adrenal cortex volume decreased in D females by 15% (p<0.05) while the volume of adrenal medulla increased (p<0.05) by 64%, compared to the same parameters in C group. Volume density of the zona glomerulosa (expressed per absolute adrenal gland volume) in D rats increased (p<0.05) by 22% in comparison with C animals. Diosgenin treatment decreased (p<0.05) the volume density of the zona fasciculata (expressed per volume of adrenal cortex) by 15% when compared to C females. Absolute volume of the zona reticularis in D group decreased (p<0.05) by 38% in comparison with the same parameter in C rats. Also, after diosgenin application, the volume density of the zona reticularis (expressed per volume of adrenal cortex) and the zona reticularis cell volume were decreased by 51% and 20% (p<0.05) respectively, compared to C animals. Our results, reflecting a decrease in many stereological parameters of the adrenal cortex, indicate that diosgenin took over the role of corticosteroid precursors and became incorporated into steroidogenesis.

Chromaffin cells in the adrenal homolog of Aphanius fasciatus (teleost fish) express piecemeal degranulation in response to osmotic stress: a hint for a conservative evolutionary process.

The effect of severe osmotic stress on the ultrastructural morphology of chromaffin cells in the adrenal homolog of Aphanius fasciatus, a small eurhyaline teleost living in saltpans, was evaluated by electron microscopy quantitative analysis. Fishes were transferred from salt water, whose salinity was 3.7%, to dechlorinated tap water and chromaffin cells were studied at resting condition and after 2 and 48 hr from the beginning of the experiment. Ultrastructural examination revealed a series of granule and cytoplasmic changes highly specific for piecemeal degranulation (PMD), a secretory process based on vesicular transport of cargoes from within granules for extracellular release, which was previously described in chromaffin cells of the mouse, rat, and human adrenal medulla. There was indeed a significant trend toward loss of content material from chromaffin granules accompanied by enlargement of granule size. Remarkably, chromaffin granules maintained their individual close structure during the whole releasing process and eventually transformed into large empty containers. A dramatic increase in the density of small, membrane-bound, variably electron-dense vesicles free in the cytoplasm or attached to granules was recognized during the first 2 hr of stress response. These features fell to control levels after 48 hr. A similar time-course pattern was observed concerning the formation of budding projections from the surface of chromaffin granules. This study provides new insight into PMD physiology and suggests that PMD is part of an adaptive secretory response to severe osmotic stress in fishes. From an evolutionary point of view, this study lends support to the concept that PMD is a secretory mechanism highly conserved throughout vertebrate classes.