Immunofluorescent patterns of clathrin and dopamine beta-hydroxylase in chromaffin cells in culture

Summary Bovine chromaffin cells maintained in culture for eight days were loaded with [³H]noradrenaline and then stimulated by a depolarizing concentration (56 mM) of K⁺. Control and stimulated cells were fixed in 3.7% formaldehyde, treated with acetone or Triton X-100, and then exposed to antibodies raised against dopamine beta-hydroxylase (a secretory granule marker) and clathrin, and purified by affinity chromatography. The cellular distribution of the correspondent antigens was investigated by indirect immunofluorescence. Cells treated with anti-dopamine beta-hydroxylase exhibited a granular pattern of fluorescence in the cytosol of the cell body, neurites, and terminal cones. Chromaffin cells exposed to anti-clathrin also showed a punctate pattern of fluorescence staining. However, in this case, the fluorescent dots were smaller than those observed with anti-dopamine beta-hydroxylase, and they were differently distributed. The speckled anti-clathrin fluorescence was preferentially condensed in the juxtanuclear region of the cell bodies, suggesting the possibility that clathrin was concentrated at the level of the Golgi apparatus.The stimulation of cultured chromaffin cells by 10 pulses of 56 mM K⁺ produced 91±2% (n = 5) depletion in the [³H]noradrenaline cell content and a concomitant displacement of the dopamine beta-hydroxylase fluorescence to the periphery of the cells. Four days after cell stimulation the dopamine beta-hydroxylase fluorescence was similar to that observed in control cells. Under the same conditions of stimulation, the distribution of the clathrin fluorescence was unaltered suggesting either that K⁺induced stimulation of the chromaffin cells does not change the cellular distribution of clathrin, or that the changes in the distribution of clathrin are of such low magnitude that they escape detection by fluorescence microscopy.     

Cholinergic stimulation of chromaffin cells induces rapid coating of the plasma membrane

Stimulation of primary bovine adrenal chromaffin cells by carbachol produced a 6-fold increase in cell surface coated pits within 30 s. This coat appeared not to be recruited from a preformed pool at the plasma membrane, but from some pool transparent to electron microscopy. The number of coated pits appeared to decrease rapidly after 1 to 2 min stimulation, but processing for electron microscopy using tannic acid to enhance contrast indicated that both coated pits and closed coated vesicles were increased relative to unstimulated cells for up to 30 min. Analyses of purified adrenal medulla coated vesicles showed a lipid composition close to that expected for cell surface membrane, but there were only trace levels of plasma membrane marker enzymes. Coated vesicles contained significant amounts of both membrane and content proteins characteristic of the chromaffin granule, suggesting that medulla coated vesicles preferentially carry secretion granule proteins. The kinetics of stimulus-dependent formation of coated membrane in the cortical zone of chromaffin cells is closely similar to that observed for secretion granule membrane retrieval.     

Membrane recycling after exocytosis: An ultrastructural study of cultured chromaffin cells

When exocytosis of granule contents is induced by nicotine stimulation, glycoprotein III (a chromaffin granule membrane constituent) is exposed on the surface of cultured chromaffin cells, where it may be labeled with an immunocytochemical tracer. The subsequent fate of this glycoprotein after endocytosis was followed at the ultrastructural level using immunogold methods and was analyzed by morphometry. After stimulation exocytosis membranes newly inserted into the plasma membrane labeled with gold particles for glycoprotein III were found to be endocytosed via coated vesicles and finally found in organelles devoid of chromogranin A, the major secretory granule protein. At intervals between 30 min and 24 h after cell stimulation and immunolabeling, most labeled structures were identified by two different morphological approaches as prelysosomes and lysosomes. In contrast with results obtained on freshly isolated chromaffin cells, it is thus concluded that in cultured cells granule membrane recycling into new granules does not occur. It is suggested that the fate of granule membrane endocytosed after cell stimulation may be influenced by the external conditions to which cells are previously exposed.     

Isolation of chromaffin cell plasma membranes on polycationic beads

We have tried to define which proteins of chromaffin cell plasma membranes are facing the cytoplasm by surface labelling a selectively oriented membrane preparation.Viable chromaffin cells were isolated by collagenase treatment of bovine adrenals. Plasma membranes from these cells were isolated on polycationic beads by the method of Jacobson and Branton (Jacobson, B.S. and Branton, D. (1977) Science 195, 302–304). The purity and orientation of the membranes were defined by biochemical and morphological criteria. The membranes, with their external side apposed to the bead surface, were enriched about 10-fold with respect to a whole cell homogenate, and contained only small amounts of contaminating organelles. Surface specific iodination of membranes on beads with 1,3,4,6-tetrachloro-3α, 6α-diphenylglycoluril (Iodogen), followed by polyacrylamide gel electrophoresis, allowed the identification of cytoplasmically exposed proteins. A different pattern was observed when intact cells were labelled prior to membrane isolation. The advantages and possible uses of this immobilized membrane preparation are discussed.     

Phosphorylation of a chromaffin granule-binding protein in stimulated chromaffin cells

A procedure was devised to determine whether in the stimulated chromaffin cell phosphate is incorporated into specific proteins ("chromobindins") that bind to chromaffin granule membranes in a Ca2+-dependent manner. Cells were preincubated with 32P-labeled orthophosphate, then challenged with secretory stimuli. A postmicrosomal supernatant fraction was prepared from the cells and incubated with unlabeled chromaffin granule membranes in the presence of 5 mM Ca2+. Proteins that bound to the membranes were isolated by centrifugation and examined for 32P content by electrophoresis and autoradiography. Stimulation by carbamylcholine, nicotine, 56 mM K+, or 2 mM Ba2+ led to the incorporation of 32P into a 37-kDa protein that had previously been characterized as a substrate for protein kinase C in vitro (chromobindin 9, or CB9; Summers, T. A., and Creutz, C. E. (1985) J. Biol. Chem. 260, 2437-2443). Incorporation of 32P into this protein was dependent on extracellular Ca2+ and followed a time course that paralleled secretion of catecholamines, returning to base-line levels after 30 min, when secretion terminated. 32P was also incorporated into a 58-kDa protein that may be tyrosine hydroxylase and into an unidentified 28-kDa protein in response to cell stimulation, but neither of these proteins bound to granule membranes in a Ca2+-dependent manner. Treatment of cells with phorbol 12,13-dibutyrate, an activator of protein kinase C, led to 32P incorporation into the 37-kDa protein that was only 30% of the level obtained with nicotinic stimulation, suggesting that additional kinases may be involved in phosphorylating this protein in the stimulated cell.     

Retrieving vesicles in secretion-induced rat chromaffin cells contain fodrin

We examined the distribution of fodrin and cytochrome b561 in secretion-induced rat chromaffin cells (epinephrine cells) by immunofluorescence and immunoelectron microscopy. Fasted rats injected with a large dose of insulin were perfusion-fixed and frozen sections of the adrenal medulla were immunolabeled. Fodrin, a peripheral membrane protein, was distributed only in the cell periphery in control cells, but was observed in the cell interior after the insulin treatment; many of the markers were found around small vesicles, 50-200 nm in diameter, and large vacuoles, more than 500 nm in diameter. On the other hand, cytochrome b561, an integral membrane protein, was seen only in the chromaffin granules in control cells, and appeared in small vesicles in the stimulated cells but not in large vacuoles. By double immunolabeling it was shown that cytochrome b561 coexisted with fodrin in the small vesicles. The coexistence of the two proteins was confirmed by the labeling of subcellular particles immunoadsorbed from the insulin-treated adrenal medulla homogenate; vesicles immunoisolated with anti-fodrin antibody on polyacrylamide beads were positively immunolabeled with anti-cytochrome b561 antibody. The present results show that during massive secretion fodrin is taken into the cell interior by vesicles, which may be a mechanism that retrieves the secretory granule membrane from the cell surface.     

Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.     

Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling

The exocytotic exposure and retrieval of an antigen of chromaffin granule membranes were studied with chromaffin cells isolated from bovine adrenal medulla. Cells were incubated with an antiserum against glycoprotein III followed by fluorescein- or gold-labeled anti-IgG. Immunofluorescence on the cell surface was present in a patchy distribution irrespective of whether bivalent antibodies or Fab fragments were used. During subsequent incubation these fluorescent membrane patches were internalized within 45 min. At the ultrastructural level immunogold-labeled patches were present on the surface of stimulated cells. During incubation (5 min to 6 h) these immunolabeled membrane patches became coated, giving rise to coated vesicles and finally to smooth vesicles. These latter vesicles were found spread throughout the cytoplasm including the Golgi region, but Golgi stacks did not become labeled. Part of the immunolabel was transferred to multivesicular bodies, which probably represent a lysosomal pathway. 30 min after incubation immunolabel was also found in electron-dense vesicles apparently representing newly formed chromaffin granules. After 6 h of incubation immunolabel was found in vesicles indistinguishable from mature chromaffin granules. These results provide direct evidence that after exocytosis membranes of chromaffin granules are selectively retrieved from the plasma membrane and are partly recycled to newly formed chromaffin granules, providing a shuttle service from the Golgi region to the plasma membrane.     

Calcium signalling in isolated single chromaffin cells of the rainbow trout (Oncorhynchus mykiss)

Chromaffin cells were isolated from the posterior cardinal vein of rainbow trout (Oncorhynchus mykiss) to assess their suitability as a model system for studying mechanisms of catecholamine secretion in fish and to evaluate intracellular calcium changes associated with cholinoreceptor stimulation. Immunocytochemistry in concert with fluorescence microscopy was employed to identify characteristic chromaffin cell proteins and thus to confirm the presence of these specific cells in suspensions and cultures. Dopamine-β-hydroxylase, an enzyme of the catecholamine-synthesising Blaschko pathway, was identified in cytoplasmic vesicles of the isolated chromaffin cells. The actin filament-severing protein, scinderin, was co-localized with actin in the sub-plasmalemmal membrane of these chromaffin cells. Intracellular calcium [Ca²⁺]_i was measured in single chromaffin cells by microspectrofluorometry using the fluorescent dye Fura-2. Significant increases in [Ca²⁺]_i were observed in chromaffin cells in response to depolarisation of the cell membrane by high concentrations of K⁺ or by the stimulation of the cell by the cholinergic receptor agonists, nicotine, acetylcholine or carbachol. The response to the reversible agonist, nicotine, was attenuated following addition of the nicotinic receptor blocker hexamethonium. Such attenuation, however, did not occur when hexamethonium was added after stimulation with the non-specific irreversible cholinergic agonist, carbachol. These results demonstrate the presence of functional cholinoreceptors, linked to intracellular calcium signalling, on isolated trout chromaffin cells and reveal the potential of these cells as a model system for studying aspects of catecholamine secretion in fish.     

Dynamic changes in chromaffin cell cytoskeleton as prelude to exocytosis

Earlier work by us as well as others has demonstrated that filamentous actin is mainly localized in the cortical surface of chromaffin cell. This F-actin network acts as a barrier to the chromaffin granules, impeding their contact with the plasma membrane. Chromaffin granules contain α-actinin, an anchorage protein that mediates F-actin association with these vesicles. Consequently, chromaffin granules crosslink and stabilize F-actin networks. Stimulation of chromaffin cell produces disassembly of F-actin and removal of the barrier. This interpretation is based on: (1) Cytochemical experiments with rhodamine-labeled phalloidin indicated that in resting chromaffin cells, the F-actin network is visualized as a strong cortical fluorescent ring; (2) Nicotinic receptor stimulation produced fragmentation of this fluorescent ring, leaving chromaffin cell cortical areas devoid of fluorescence; and (3) These changes are accompanied by a decrease in F-actin, a concomitant increase in G-actin, and a decrease in the F-actin associated with the chromaffin cell cytoskeleton (DNAse I assay). We also have demonstrated the presence in chromaffin cells of gelsolin and scinderin, two Ca²⁺-dependent actin filament-severing proteins, and suggested that chromaffin cell stimulation activates scinderin with the consequent disruption of F-actin networks. Scinderin, a protein recently isolated in our laboratory, is restricted to secretory cells and is present mainly in the cortical chromaffin cell cytoplasm. Scinderin, which is structurally different from gelsolin (different pI_s, amino acid composition, peptide maps, and so on), decreases the viscosity of actin gels as a result of its F-actin-severing properties, as demonstrated by electron microscopy. Stimulation of chromaffin cells either by nicotine (10 μM) or high K+ (56 mM) produces a redistribution of subplasmalemmal scinderin and actin disassembly, which preceded exocytosis. The redistribution of scinderin and exocytosis is Ca²⁺-dependent and is not mediated by muscarinic receptors. Furthermore, our cytochemical experiments demonstrate that chromaffin cell stimulation produces a concomitant and similar redistribution of scinderin (fluorescein-labeled antibody) and F-actin (rhodamine phalloidin fluorescence), suggesting a functional interaction between these two proteins. Stimulation-induced redistribution of scinderin and F-actin disassembly would produce subplasmalemmal areas of decreased cytoplasmic viscosity and increased mobility for chromaffin granules. Exocytosis sites, evaluated by antidopamine-β-hydroxylase (anti-DβH) surface staining, are preferentially localized in plasma membrane areas devoid of F-actin.     

Release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells

Summary The release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells was studied by electron microscopy. The treatment of the cells with SEB at the concentration of 20 μg/ml caused marked increase of the chromaffin granules that either bound to the plasma membrane by the characteristic rods, measuring 15 to 20 nm in length and showing a tubular structure, or budded off at the free cell surface, surrounded by a layer of rod-containing cytoplasm and enclosed by the plasma membrane. The binding between the granular and plasma membranes by the rods did not lead to membrane fusion and exocytosis of the granular content. Many of the bound granules showed vesiculation with loss of the electron-dense core material; at the same time, some of the binding rods contained intraluminal electron-dense material similar to the granular core material. These findings suggested that the electron-dense material (i.e., norepinephrine) of the bound granules was released extracellularly through channels within the rods. Although the granules were bound to the plasma membrane with equal frequency at the free and contiguous cell surfaces, the granular budding occurred only at the free cell surface, indicating that it occurred incidentally to some granules bound at the free cell surfaces. On the basis of the morphological observations, it is postulated that the electron-dense material of the bound granule is selectively released extracellularly through the rods, leaving the vesiculated granules behind in the cytoplasm. The same mode of release of the granular content was observed, though less frequently, in the untreated control cells. No morphological evidence that indicated that the granular content was released extracellularly by exocytosis was found in the treated and control cells. The present observations indicated that the SEB treatment of PC12 cells stimulated the binding of chromaffin granules to the plasma membrane by the rods and the budding of the bound granules at the free cell surface.     

The distribution of Concanavalin A receptor sites on the membrane of chromaffin granules.

The distribution of concanavalin A (con A) receptor sites on the membranes of chromaffin granules has been investigated by binding studies using 125I-labelled con A and by electron-microscope studies using ferritin-labelled con A. In both experiments con A was observed to bind to chromaffin granule membranes but not to intact granules. The ferritin-con A particles bind to only one of the two possible surfaces of the chromaffin granule membranes. These results are in agreement with previous observations concerning the asymmetric distribution of saccharide residues on the surfaces of a number of different plasma membranes. They suggest that for the intracellular membrane of the chromaffin granule the saccharide sites, like those in plasma membranes, are not exposed to the cell cytoplasm. Further work is necessary to establish whether these sites are on the inner surface of the membrane or whether they are unmasked during the conversion of granules to membrane ghosts.     

Characterization of adrenal medullary chromaffin cells by flow cytometry.

BACKGROUND: Adrenomedullary chromaffin cells are neural crest derivatives widely used as a model system to study neurosecretory mechanisms. Morphological, immunohistochemical, and functional data indicate that chromaffin cells are heterogeneous and support the distinction between adrenaline (A)- and noradrenaline (NA)-producing and secreting cells. The aim of this study was to characterize by flow cytometry the two main chromaffin cell subtypes in suspensions of cultured bovine chromaffin cells. METHODS: An indirect immunofluorescence method was used for the specific labeling of two intracellular enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), involved in the synthesis of NA and A, respectively. Flow cytometry analysis of fluorescence labeling was performed in two chromaffin cell fractions differentially enriched in A-containing cells by centrifugation through density gradients. PNMT and DBH-related fluorescence was also correlated with the A and NA content of the cells assayed by HPLC measurements. RESULTS: No significant differences were found in forward-side scatter plots between the two cell fractions (A-enriched cells and mixed cells); however, the degree of labeling of the enzymes and the corresponding PNMT/DBH-related fluorescence ratio was significantly greater in the A-enriched cell fraction. The existence of changes in DBH and PNMT content of chromaffin cells over time (1 week) in culture was also examined. No significant variation in enzyme related fluorescence values was detected in any of the two cell fractions, and this result correlated well with HPLC determinations of the catecholamine content (A and NA) of the cells. CONCLUSIONS: Flow cytometry appears to be a useful technique to characterize chromaffin cell subtypes and to follow their phenotypic changes in response to growth factors.     

Control of exocytosis from adrenal chromaffin cells

1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and secretion induced by nicotinic stimulation or depolarization in intact cells were closely correlated. The results provide strong support for Ca2+ entry being the trigger for exocytosis. 3. Experiments in which the H+ electrochemical gradient across the intracellular secretory granule (chromaffin granule) membrane was altered indicated that the gradient does not play an important role in exocytosis. 4. Ca2+ entry into the cells is associated with activation of phospholiphase C and a rapid translocation of protein kinase C to membranes. 5. The plasma membrane of chromaffin cells was rendered permeable to Ca2+, ATP, and proteins by the detergent digitonin without disruption of the intracellular secretory granules. In this system in which the intracellular milieu can be controlled, micromolar Ca2+ directly stimulated catecholamine secretion. 6. Treatment of the cells with phorbol esters and diglyceride, which activate protein kinase C, enhanced phosphorylation and subsequent Ca2+-dependent secretion in digitonin-treated cells. 7. Phorbol ester-induced secretion could be specifically inhibited by trypsin. The experiments indicate that protein kinase C modulates but is not necessary for Ca2+-dependent secretion.     

Visualization of the exocytosis/endocytosis secretory cycle in cultured adrenal chromaffin cells

Cultured bovine adrenal medullary chromaffin cells were stimulated to secrete catecholamines by addition of veratridine or nicotine. The formation of an exocytotic pit exposes a major secretory granule membrane antigen, the enzyme dopamine beta-hydroxylase, to the external medium. By including antiserum to this enzyme in the medium, we were able to visualize sites of exocytosis by decoration of bound antibody using a fluorescent second antibody. Internalization of this antibody- antigen complex was then followed in chase experiments: approximately half the surface complex was internalized in 15-30 min. In other experiments, secretion was triggered in the absence of antiserum, and surface enzyme was revealed by binding antibodies at various times after secretion had been halted by an antagonist. Surface patches of antigen remained discrete from the bulk of the plasma membrane for at least 30 min, although a substantial proportion of the antigen was internalized within this time. Cell surface concanavalin A receptors were internalized at a roughly similar rate, suggesting that mechanisms may be similar. After internalization, chromaffin granule membranes fused to larger structures, possibly lysosomes, and were transported over a few hours to the perinuclear region of the cell.     

Calmodulin-binding proteins in granule and plasma membranes from bovine chromaffin cells

Calmodulin-binding proteins in chromaffin granule membrane and chromaffin cell plasma membranes have been investigated and compared. Chromaffin granules were purified by centrifugation over a 1.7 M sucrose layer. Plasma membranes were obtained in a highly purified form by differential and isopycnic centrifugation. Enzymatic determinations of 5'-nucleotidase, a generally accepted plasma membrane marker, showed a 40-50-fold enrichment as compared to the cell homogenate. Marker enzyme studies demonstrated only minimal contamination by other subcellular organelles. After solubilization with Triton X-100, calmodulin-binding proteins were isolated from chromaffin granule membranes and plasma membranes by affinity chromatography on a calmodulin/Sepharose 4B column. On two-dimensional polyacrylamide gelelectrophoresis a prominent protein (Mr = 65,000, pI ranging from 5.1 to 6) consisting of multiple spots, was present in the calmodulin-binding fraction from chromaffin granule membranes as well as from plasma membranes. Besides this 65 kDa protein both fractions had at least four groups of proteins in common. Also, proteins typical for either preparation were observed. In the calmodulin-binding protein preparations from chromaffin granule membranes a prominent spot with Mr = 80,000 and a pH ranging from 5.0 to 5.7 was present. This protein was enzymatically and immunologically identified as dopamine-beta-monooxygenase.     

Ultrastructural demonstration of exocytosis in intact and saponin-permeabilized cultured bovine chromaffin cells

Exocytosis is the release of intracellular vesicular contents directly to the cell exterior after fusion of the vesicular and plasma membranes. It is generally accepted as the process by which transmitters and hormones are released from neurons and neurosecretory cells. There is overwhelming biochemical evidence that exocytosis is the mechanism by which catecholamines are released from adrenal chromaffin cells. With the exception of the hamster, however, there is little ultrastructural evidence to support such a mechanism. We have used a modified in vitro tannic-acid method to visualize exocytosis by transmission electron microscopy in intact and saponin-permeabilized bovine chromaffin cells. When cells are exposed to tannic-acid-containing medium, the content of vesicles involved in exocytosis is coagulated in situ as the vesicle opens to the exterior. Numerous exocytotic profiles were observed. The exposed vesicle contents appeared more granular than those of vesicles in the cell interior. Tannic acid also made the plasma membrane more distinct. Small holes were apparent in the plasma membrane of saponin-treated cells, with little disruption of underlying cytoplasmic structure. Furthermore, when these cells were stimulated with calcium, exocytosis was evident only at regions of intact plasma membrane, not at the holes. Parallel measurements of secretion showed no secretion in the presence of tannic acid. Pretreatment with tannic acid prevented subsequent secretion by intact cells and markedly reduced that of permeabilized cells, indicating a probable change in the nature of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

Urethral chromaffin cells

Summary The urethral mucosa of the rat, rabbit and guinea-pig was examined with both fluorescence and electron microscopy. Employing the former technique, numerous brightly fluorescing flask-shaped cells were observed amongst the basal cells of the urethral epithelium in all three species. In the electron microscope cells with a similar shape and distribution are distinguished by their content of membrane-limited dense granules, extensive Golgi membranes and bundles of filaments. In favourable planes of section short microvilli extend from the apical region of these cells which are joined to neighbouring urethral epithelial cells by zonulae occludentes. These fluorescent, granule-containing cells are classified as urethral chromaffin cells.Fluorescent nerves were not observed in relation to the urethral epithelium although the electron microscope revealed axons lying singly or in groups both beneath and between the urethral epithelial cells. Many of these axons appear varicose and contain small, agranular vesicles, a few large granulated vesicles and numerous mitochondria. Occasionally a vesicle-containing axon lay adjacent to a urethral chromaffin cell. While a direct autonomic innervation of these cells could not be discounted it is concluded that the majority of nerves probably perform a sensory function.     

Bidirectional transbilayer lipid movement in human platelets as vizualized by the fluorescent membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene

Transbilayer movement of the fluorescent membrane probe TMA-DPH [1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene] in the plasma membrane of human platelets was investigated by measuring fluorescence intensity and fluorescence decay. Labeling of unstimulated platelets by TMA-DPH results in a rapid increase in fluorescence intensity, leveling off within 1 min. Dilution of platelets into buffer without TMA-DPH leads to an almost complete rapid efflux of TMA-DPH, indicating that TMA-DPH labels only the outer leaflet of the plasma membrane. Transbilayer movement of the fluorescent probe in unstimulated platelets could be observed upon prolonged incubation and occurs with a t1/2 of 60-90 min. Stimulation of platelets with thrombin directly after the initial rapid uptake of TMA-DPH results in a fast increase in membrane-bound TMA-DPH, fully explained by the increase in plasma membrane caused by secretion of intracellular storage organelles. No indications for increased transbilayer movement of the probe were found, since dilution of thrombin-stimulated TMA-DPH-labeled platelets into buffer without TMA-DPH indicated no uptake of TMA-DPH by intracellular membranes. In contrast to thrombin, stimulation of TMA-DPH-labeled platelets with the Ca2(+)-ionophore ionomycin results in a much larger increase in fluorescence intensity. This process is accompanied by labeling of intracellular membranes as indicated by incomplete efflux of TMA-DPH after dilution of the stimulated platelets. Thus, stimulation of platelets by ionomycin gives rise to rapid and massive inward movement of TMA-DPH (t1/2 approximately 10-12 s). Prolonged incubation of platelets in the absence of any stimulus allows labeling of the total lipid pool, including intracellular membranes.(ABSTRACT TRUNCATED AT 250 WORDS)