Evidence for anterograde transport of secretory granules in processes of gastric paracrine (somatostatin) cells

Summary Certain disseminated endocrine-like cells have reviously been found to give off long cytoplasmic processes which end with small bulbous expansions on the membranes of other cell types. It is believed that the process-carrying cells control the functions of the receiving cells by local and directed (paracrine) secretion of messenger molecules (peptides, biogenic monoamines) through their processes. Following injections of amine precursors paracrine cells take up and convert these to the corresponding amines, which can be cytochemically visualized by the Falck-Hillarp formaldehyde-induced fluorescence technique. As the amines are stored in the cytoplasmic (secretory) granules of the cells, they form useful markers for studies of granule turnover and transport. By injecting, at different time intervals, two different precursors (L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine), resulting in amines giving different fluorescence colours in the Falck-Hillarp procedure, we have been able to separately label old and new secretory granule fractions in different fluorescence colours. Examination of such double-labelled paracrine cells (mostly gastric somatostatin cells) indicates that their secretory granules are transported in a proximo-distal direction in the paracrine cell processes (paraxons). This finding strongly supports the concept that paracrine cells control the functions of the cells they contact by way of directed, process-mediated delivery of their secretory products.This work was reported in part at the 29th Congress of the International Union of Physiological Sciences, Sydney, Australia, August 28–September 3, 1983     

Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells

We have studied by electron microscopy and immunocytochemistry the formation of secretory granules containing adrenocorticotropic hormone (ACTH) in murine pituitary cells of the AtT20 line. The first compartment in which condensed secretory protein appears is a complex reticular network at the extreme trans side of the Golgi stacks beyond the TPPase-positive cisternae. Condensed secretory protein accumulates in dilated regions of this trans Golgi network. Examination of en face and serial sections revealed that "condensing vacuoles" are in fact dilations of the trans Golgi network and not detached vacuoles. Only after presumptive secretory granules have reached an advanced stage of morphological maturation do they detach from the trans Golgi network. Frequently both the dilations of the trans Golgi network containing condensing secretory protein and the detached immature granules in the peri-Golgi region have surface coats which were identified as clathrin by immunocytochemistry. Moreover both are the site of budding (or fusion) of coated vesicles, some of which contain condensed secretory protein. The mature granules below the plasma membrane do not, however, have surface coats. Immunoperoxidase labeling with an antiserum specific for ACTH and its precursor polypeptide confirmed that many of the coated vesicles associated with the trans Golgi network contain ACTH. The involvement of the trans Golgi network and coated vesicles in the formation of secretory granules is discussed.     

Syntabulin-mediated anterograde transport of mitochondria along neuronal processes

In neurons, proper distribution of mitochondria in axons and at synapses is critical for neurotransmission, synaptic plasticity, and axonal outgrowth. However, mechanisms underlying mitochondrial trafficking throughout the long neuronal processes have remained elusive. Here, we report that syntabulin plays a critical role in mitochondrial trafficking in neurons. Syntabulin is a peripheral membrane-associated protein that targets to mitochondria through its carboxyl-terminal tail. Using real-time imaging in living cultured neurons, we demonstrate that a significant fraction of syntabulin colocalizes and co-migrates with mitochondria along neuronal processes. Knockdown of syntabulin expression with targeted small interfering RNA or interference with the syntabulin-kinesin-1 heavy chain interaction reduces mitochondrial density within axonal processes by impairing anterograde movement of mitochondria. These findings collectively suggest that syntabulin acts as a linker molecule that is capable of attaching mitochondrial organelles to the microtubule-based motor kinesin-1, and in turn, contributes to anterograde trafficking of mitochondria to neuronal processes.     

Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells

In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion.     

5-Hydroxytryptamine transport in cells and secretory granules from a transplantable rat insulinoma.

Mechanisms of transport of 5-hydroxytryptamine in the pancreatic B-cell were investigated by using cell suspensions and secretory granules prepared from a transplantable rat insulinoma. (1) Cells incubated with 5-hydroxy[G-3H]tryptamine at concentrations ranging from 0.1 microM to 5 mM accumulated the radioisotope principally by a simple diffusion process. The incorporated radioactivity was recovered principally as the parent molecule and was recovered predominantly in soluble protein and secretory-granule fractions prepared from the tissue. (2) Isolated granules incubated in buffered iso-osmotic medium without ATP accumulated the amine to concentrations up to 38-fold that of the medium. This process was insensitive to reserpine and occurred over a wide range of 5-hydroxytryptamine concentrations (0.075 microM-25 mM). Above 5 mM, 5-hydroxytryptamine accumulation decreased in parallel with the breakdown of the delta pH across the granule membrane. Uptake was favoured by alkaline media and was reduced by the addition of (NH4)2SO4. In both cases a close correlation was observed between uptake and the transmembrane delta pH, a finding that suggested that 5-hydroxytryptamine permeated the membrane as the free base and equilibrated across the membrane with the delta pH. Binding of 5-hydroxytryptamine to granule constituents also played a part in this process. ATP caused a further doubling of granule 5-hydroxytryptamine uptake by a process that was sensitive to reserpine (0.5 microM). Inhibitor studies suggested that amine transport in this instance was linked to the activity of the granule membrane proton-translocating ATPase. (3) It was concluded that the uptake of amines driven by proton gradients across the insulin-granule membrane could account for the accumulation in vivo of amines in the B-cell.     

Topology of chromogranins in secretory granules of endocrine cells

Summary Chromogranins A and B are glycoproteins originally detected in the adrenal medulla. These proteins are also present in a variety of neuroendocrine cells. The subcellular distribution of the chromogranins, and particularly their intra-granular topology are of special interest with respect to their putative functions.Endocrine cells of the guinea pig adrenal medulla, pancreas and gastric mucosa were investigated immunoelectron microscopically for the subcellular distribution of both chromogranins. Out of 13 established endocrine cell types in all locations, only two endocrine cell types showed immunoreactivity for both chromogranin A and B, and eight endocrine cell types showed immunoreactivities only for chromogranin A. These immunoreactivities varied inter-cellularly. Three endocrine cell types were unreactive for the chromogranins. Moreover, some hormonally non-identified endocrine cells in the pancreas and the gastric mucosa also contained chromogranin A immunoreactivities.Subcellularly, chromogranin A or B were confined to secretory granules. In most endocrine cells, the secretory granules showed chromogranin immunoreactivities of varying densities. Furthermore, the intra-granular topology of chromogranin A or B in the secretory granules varied considerably: in some endocrine cell types, i.e. chromaffin-, gastrin- and enterochromaffin-like-cells, chromogranin A immunoreactivity was localized in the perigranular and/or dense core region of the secretory granules; in others, i.e. insulin-, pancreatic polypeptide-and bovine adrenal medulla dodecapeptide-cells, it was present preferentially in the electron-opaque centre of the secretory granules; chromogranin B immunoreactivity was localized preferentially in the perigranular region of the secretory granules of chromaffin cells and gastrin-cells. The inter-cellular and inter-granular variations of chromogranin A and B immunoreactivities point to differences in biosynthesis or processing of the chromogranins among endocrine cells and their secretory granules.     

Evidence for two conductance/exchange pathways for chloride in rat parotid secretory granules

Electron probe x-ray microanalysis was used to determine that bromide is localized to rat parotid secretory granules at early stages of an in situ Cl/Br washout experiment. Chloride efflux and bromide influx across the secretory granule membrane occurred with a time order of minutes. Since the Cl washout data indicated minimal Cl binding within the granule, and therefore minimal Br binding, the Br localization results suggest the presence of two or more anion conductance/exchange pathways in the granule membrane for the Cl (Br) ion.     

Ca increase in secretory granules of stimulated mast cells

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray micro-analysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous granules. A significant increase was also observed for Na and Cl. These changes were not observed (for Ca) or weaker (for Na and Cl) if the cells had been challenged in the absence of nominal extracellular Ca; in this case, there was also a significant decrease in the sulphur content, suggesting a partial dispersion of the organic matrix components. In exocytosed granule matrices, in the presence but not in the absence of extracellular Ca, a slow and long-lasting increase of intragranular free Ca was monitored by changes in the fluorescence of the Ca-sensitive probes Fluo-3 and Calcium Green-5N, accumulated within rat mast-cell secretory granules. These findings are discussed along two lines: It is proposed that the calcium uptake by the exocytosed mast-cell granule matrices can have a physiological relevance for the surrounding tissue. Mast-cell granules do not disperse after exocytosis. The major uptake of Ca which is seen after opening of the exocytotic pore could be responsible for the exceptional stability of the externalized matrices.     

Calcium dynamics in the secretory granules of neuroendocrine cells

Cellular Ca(2+)signaling results from a complex interplay among a variety of Ca(2+) fluxes going across the plasma membrane and across the membranes of several organelles, together with the buffering effect of large numbers of Ca(2+)-binding sites distributed along the cell architecture. Endoplasmic and sarcoplasmic reticulum, mitochondria and even nucleus have all been involved in cellular Ca(2+) signaling, and the mechanisms for Ca(2+) uptake and release from these organelles are well known. In neuroendocrine cells, the secretory granules also constitute a very important Ca(2+)-storing organelle, and the possible role of the stored Ca(2+) as a trigger for secretion has attracted considerable attention. However, this possibility is frequently overlooked, and the main reason for that is that there is still considerable uncertainty on the main questions related with granular Ca(2+) dynamics, e.g., the free granular [Ca(2+)], the physical state of the stored Ca(2+) or the mechanisms for Ca(2+) accumulation and release from the granules. This review will give a critical overview of the present state of knowledge and the main conflicting points on secretory granule Ca(2+) homeostasis in neuroendocrine cells.     

Studies on the transport of secretory granules in the magnocellular hypothalamic neurons

Summary The axonal flow of neurosecretory elementary granules has been studied in the paraventricular neurons of the rat (PVN), with the help of three techniques: light microscopy, radioautography after labelling with ³⁵S-L-cysteine, and electron microscopy.Colchicine treatment does not alter the uptake of ³⁵S cysteine in the PVN but the flow of labelled neurosecretory material towards the neurohypophysis is interrupted. Interruption of the axonal flow is also evidenced by the stagnation of neurosecretory granules at the periphery of the neuronal cytoplasm and by the presence of numerous axonal swellings, heavily loaded with neurosecretory granules and often containing abnormal elongated granules, surrounded by a single membrane, oriented more or less parallely to the long axis of the axons. Other cell organelles and neurotubules are not altered. The present experiments bring further evidence of the arrest by colchicine of the axonal flow of secretory granules without apparent changes of the neurotubules.This work was supported by a grant (1970/1971) from the Belgian National Fund for Scientific Research (J. F.-D), and by grant No 1120 from the Belgian National Fund for Medical Research (P. D).The authors wish to thank Mrs. A.-M. Hunninck-Couck for her devoted and skillful technical assistance, and are endebted to Dr. J. C. Heuson for kindly supplying the rats.     

Regulation of chloride transport in parotid secretory granules by membrane fluidity.

Zymogen granule membranes contain Cl- conductance and Cl/anion exchange activities that become important for primary fluid production after fusion with the apical plasma membrane of the acinar cell. We have used steady-state fluorescence anisotropy of diphenylhexatriene derivatives and measurements of Cl- transport in isolated secretory granules to determine the contribution of membrane fluidity to the regulation of transport across the granule membrane. Secretory granules from several unstimulated glands (rat pancreas and parotid, rabbit gastric glands) were shown to have low membrane fluidity compared to plasma membranes. In addition, Cl- transport activity in different granule preparations showed a strong correlation to the membrane fluidity when measured with 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), but not with 3-[p-(6-phenyl)-1,3,5-hexatrienyl)-phenyl]propionic acid (PA-DPH). These data suggest that TMA-DPH preferentially partitions into a specific lipid environment associated with, or which exerts an influence on, the Cl- transport proteins and that increases in the fluidity of this environment are associated with higher transport rates. Data from other types of plasma membranes indicate that TMA-DPH partitions much more than PA-DPH into the cytoplasmic leaflet, suggesting that this part of the granule membrane is involved in the observed fluidity changes. Furthermore, increasing the bulk membrane fluidity with the local anesthetics benzyl alcohol and n-alkanols increased the Cl- transport rates up to 10-fold. This increase was apparently through specific transporters as anion selectivity was maintained in spite of the higher absolute rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Topology of chromogranins in secretory granules of endocrine cells.

Chromogranins A and B are glycoproteins originally detected in the adrenal medulla. These proteins are also present in a variety of neuroendocrine cells. The subcellular distribution of the chromogranins, and particularly their intra-granular topology are of special interest with respect to their putative functions. Endocrine cells of the guinea pig adrenal medulla, pancreas and gastric mucosa were investigated immunoelectron microscopically for the subcellular distribution of both chromogranins. Out of 13 established endocrine cell types in all locations, only two endocrine cell types showed immunoreactivity for both chromogranin A and B, and eight endocrine cell types showed immunoreactivities only for chromogranin A. These immunoreactivities varied inter-cellularly. Three endocrine cell types were unreactive for the chromogranins. Moreover, some hormonally non-identified endocrine cells in the pancreas and the gastric mucosa also contained chromogranin A immunoreactivities. Subcellularly, chromogranin A or B were confined to secretory granules. In most endocrine cells, the secretory granules showed chromogranin immunoreactivities of varying densities. Furthermore, the intra-granular topology of chromogranin A or B in the secretory granules varied considerably: in some endocrine cell types, i.e. chromaffin-, gastrin- and enterochromaffin-like-cells, chromogranin A immunoreactivity was localized in the perigranular and/or dense core region of the secretory granules; in others, i.e. insulin-, pancreatic polypeptide- and bovine adrenal medulla dodecapeptide-cells, it was present preferentially in the electron-opaque centre of the secretory granules; chromogranin B immunoreactivity was localized preferentially in the perigranular region of the secretory granules of chromaffin cells and gastrin-cells. The inter-cellular and inter-granular variations of chromogranin A and B immunoreactivities point to differences in biosynthesis or processing of the chromogranins among endocrine cells and their secretory granules.     

Rab18 inhibits secretory activity in neuroendocrine cells by interacting with secretory granules

Rab proteins comprise a complex family of small GTPases involved in the regulation of intracellular membrane trafficking and reorganization. In this study, we identified Rab18 as a new inhibitory player of the secretory pathway in neuroendocrine cells. In adrenal chromaffin PC12 cells and pituitary AtT20 cells, Rab18 is located at the cytosol but associates with a subpopulation of secretory granules after stimulation of the regulated secretory pathway, strongly suggesting that induction of secretion provokes Rab18 activation and recruitment to these organelles. In support of this, a dominant-inactive Rab18 mutant was found to distribute diffusely in the cytosol, whereas a dominant-active Rab18 mutant was predominantly associated to secretory granules. Furthermore, interaction of Rab18 with secretory granules was associated to an inhibition in the secretory activity of PC12 and AtT20 cells in response to stimulatory challenges. Association of Rab18 with secretory granules was also observed by immunoelectron microscopy in normal, non-tumoral endocrine cells (pituitary melanotropes), wherein Rab18 protein content is inversely correlated to the level of secretory activity of cells. Taken together, these findings suggest that, in neuroendocrine cells, Rab18 acts as a negative regulator of secretory activity, likely by impairing secretory granule transport.     

Electron microscope identification of endocrine secretory granules in endothelial cells

Electron microscopy has revealed endocrine secretory granules in the endothelial cells of the rat liver, spleen and bone marrow capillaries. The granules were responsible for serotonin, melatonin, catecholamines and insulin synthesis. Local mechanisms of hormonal control of hemostasis are discussed.     

Evidence for the involvement of KIF4 in the anterograde transport of L1-containing vesicles

In this study we present evidence about the cellular functions of KIF4. Using subcellular fractionation techniques and immunoisolation, we have now identified a type of vesicle that associates with KIF4, an NH(2)-terminal globular motor domain kinesin-like protein. This vesicle is highly concentrated in growth cones and contains L1, a cell adhesion molecule implicated in axonal elongation. It lacks synaptic vesicle markers, receptors for neurotrophins, and membrane proteins involved in growth cone guidance. In cultured neurons, KIF4 and L1 predominantly localize to the axonal shaft and its growth cone. Suppression of KIF4 with antisense oligonucleotides results in the accumulation of L1 within the cell body and in its complete disappearance from axonal tips. In addition, KIF4 suppression prevents L1-enhanced axonal elongation. Taken collectively, our results suggest an important role for KIF4 during neuronal development, a phenomenon which may be related to the anterograde transport of L1-containing vesicles.     

Zinc-containing 7S-NGF complex. Evidence from zinc histochemistry for localization in salivary secretory granules

7S-NGF is a pro-protein containing a neurotrophic subunit, beta-NGF, which has been localized by immunocytochemistry to the granules of granular convoluted tubule (GCT) cells in certain murine salivary glands [Watson et al., Anat Rec (1985) 213:365]. The 7S-NGF pro-protein contains zinc and is stabilized by zinc ions [Pattison and Dunn, Biochemistry (1976) 15:3696]. In the present work, dithizone, toluene sulfonamide quinoline (TSQ), and neo-Timm's methods for zinc were used to determine whether zinc histochemistry could be used to visualize the zinc associated with the 7S-NGF complex and, if so, whether zinc histochemistry might corroborate the reported localization of the 7S-NGF complex in GCT secretory granules. The results indicate that intensity of zinc staining varies with the reported variations in NGF levels in different salivary glands, and that the zinc is selectively concentrated in the GCT secretory granules. We suggest that zinc histochemistry may be a useful marker for the presence of the zinc-stabilized 7S-NGF pro-protein.