Seasonal changes in hepatocyte ultrastructure correlated with the cyclic synthesis of secretory proteins in the winter flounder (Pleuronectes americanus)

Liver tissue was sampled from flounder (Pleuronectes americanus) throughout the year with the intention of documenting changes in the ultrastructure coincident with the production and secretion of antifreeze proteins. In the winter, hepatocytes are dedicated to the production of these proteins and, in the female, also reproductive proteins. In both sexes, liver cells in the summer contain abundant lipid and glycogen stores. In the female, there is a conspicuous hepatocyte transformation from a fat-filled cell in the summer to one with well-developed rough endoplasmic reticulum in the winter. Large amounts of rough endoplasmic reticulum (11.2 mg/gm) were recovered after subcellular fractionation of female wintertime liver. The increased appearance of secretory organelles and the high number of nucleolar profiles observed in winter animals is consistent with the elevated demand for protein secretion and synthesis in both sexes. The fractional volumes occupied by lipid droplets and mitochondria were different when comparisons were made between sex and season. Females contained a greater volume of lipid than did males, and summer animals contained more lipid than those in winter.     

The protein content and morphogenesis of zymogen granules

When zymogen granules, the secretion granules of pancreatic acinar cells, fill, secretory product is accumulated in immature granules, condensing vacuoles. Mature granules are formed when this product (protein) condenses into an osmotically inactive aggregate and, bulk water is expelled. This hypothesis for granule morphogenesis has two elements. The first is that immature granules are precursors to mature granules. The second is that a particular maturational event, condensation, which involves the aggregation of protein, takes place. These hypotheses lead to two straightforward predictions. One, that condensing vacuoles on average, should contain less protein than filled or mature granules. And two, that, due to condensation, mature granules should contain protein at a common concentration. In the current work, both of these predictions were tested using measurements of the protein content of individual granules acquired by X-ray microscopy. Neither prediction was affirmed by the experimental results. First, there was no distinguishable difference in the distribution of protein between immature and mature granules. Second, the protein concentration of mature granules varied widely between preparations, although granules from the same preparation had similar concentrations. From the data we conclude that: 1) mature granules and condensing vacuoles are different, though not necessarily unrelated, types of secretory vesicle, and not two forms of the same object; 2) as such, condensing vacuoles are not precursors to mature granules; 3) all granules do not contain protein at one particular concentration when full, or mature; 4) granule maturation does not involve a condensation step; 5) concentration is not determined by such physical limits as the space available for protein packing or condensation; and 6) the amount of protein contained is physiologically regulated.     

Preparation and discharge of secretion in the subcommissural organ of the rat

Summary The secretion of the subcommissural organ (SCO) of the rat was studied by means of immunocytochemistry at the electron-microscopic level with the use of (1) the polar embedding medium Lowicryl K4M at -30° C, (2) the protein A-gold technique, and (3) a rabbit antiserum against bovine Reissner's fiber (see Sterba et al. 1981).Two different substructures of the ependymal and the hypendymal SCO-cells display a positive immunocytochemical reaction: (1) sacs containing flocculent secretion, which originate from the granular endoplasmic reticulum, and (2) vacuoles filled with fine granular secretion, which are pinched off from the Golgi apparatus. The secretory material of the sacs and the vacuoles is discharged both (i) apically into the cerebrospinal fluid and (ii) basally into intercellular spaces of the SCO-hypendyma. The apically released secretion is condensed to a lamina-like formation, which more caudally assumes the form of Reissner's fiber. The route of the basally released secretion remains, however, vague. The periodically striated bodies, which were thought to be morphological mediators of the discharge of the secretion into the capillaries, are never labeled by gold particles.Supported by grants from the Ministry for Science and Technology of the German Democratic RepublicThe expert technical assistance of Mrs. B. Wolff, Mrs. S. Mehnert, Mrs. E. Siebert, Mrs. Ch. Schneider, and Mrs. I. Seifert is gratefully acknowledged     

Innervation of the pineal gland in the Mongolian gerbil (Meriones unguiculatus)

Summary Parasympathetic stimulation of parotid glands has been studied in vivo, a) in normal resting glands, b) 72 h after post-ganglionic sympathectomy and c) after adrenergic degranulation of the acinar cells.Morphological results in each gland were compared with a similarly pretreated, but not parasympathetically stimulated, contralateral gland from the same animal.On parasympathetic stimulation of glands with densely granulated acinar cells (groups a- and b-) a variable, but usually relatively small, tendency for vacuole formation occurred in some cells. After prior degranulation of the cells (group c-) the tendency for vacuole formation was greatly accentuated. This indicates that the pre-existing metabolic state of the cells can influence the responses to stimulation of a single nerve. Dilatation of rough endoplasmic reticulum and nuclear changes were also more prominent after parasympathetic stimulation of previously degranulated acinar cells, and this suggests that parasympathetic impulses may have strong activating effects on resynthesis under these conditions. It is also likely that parasympathetic stimulation induced some, albeit small, degree of degranulation and, since this occurred in the absence of sympathetic nerves (group b-) it was probably the consequence of a direct cholinergic effect. The present results therefore indicate that the concept of an absolute dichotomy between parasympathetic and sympathetic responses is not tenable in this tissue.Analyses of saliva for amylase and peroxidase gave complex results but indicate that the two enzymes are not necessarily secreted in parallel. The morphological results support the idea that some enzyme molecules may have entered the saliva without being prepackaged into secretory granules, but could have passed directly from dilated cisterns of rough endoplasmic reticulum into intra-cellular vacuoles, and this tendency was most apparent after para-sympathetic stimulation of previously degranulated cells.Travel grants from the Wellcome Trust to J.R. Garrett are gratefully acknowledged. This work has been helped by the technical assistance of Mr. P.S.A. RowleyM.R.C. Research Assistant     

The effects of 11-methyl 16,16 dimethyl prostaglandin E2 on gastric acid secretion in man

11-Methyl 16,16 Dimethyl Prostaglandin E₂ (TM-PGE) was administered orally to man in dosages of 2.5, 5, 7.5 and 10 μg/kg. Maximal inhibition of basal secretion was 52 and 78% and submaximal histamine-stimulated secretion 45 and 70% for volume and acid output, respectively. Secretory inhibition was observed for approximately two hours after ingestion of the drug. No effect was observed on serum gastrin levels. Side effects occurred with equal frequency in the placebo and drug groups. TM-PGE is well tolerated and inhibits both basal and submaximal histamine-stimulated acid secretion in man. Further evaluation may prove it to be helpful in the clinical treatment of acid hypersecretory states and peptic ulcer disease.     

Restitution of granule stores in the rabbit parotid gland after isoprenaline-induced secretion

Summary A detailed stereological analysis has been made of the organelle content of rabbit acinar cells during the restoration of granule stores following extensive degranulation with isoprenaline (IPR). Rabbits were sacrificed 2, 4, 8, 12 and 16 h after IPR administration and the volumes and proportions of intracellular organelles were compared with those of untreated glands.At 2 h only 5% of cell volume was occupied by secretion granules, but there was already evidence of nascent granule formation. The volume per cell of secretion granules increased in sigmoid fashion and by 16 h amounted to 350 m³/cell, 36% of cell volume, which are values similar to those of the control replete glands. IPR treatment caused some initial swelling of the cells, and there were transient increases in the volumes of several compartments. However, the volume of mitochondrial and lysosomal compartments had returned to control levels by 4 h and that of the nucleus by 8 h. The greatest increase was in the volume of the rough endoplasmic reticulum which had increased by nearly 70% by 2 h and remained enlarged throughout the period of restitution. However, neither the volume nor the proportion of the smooth membraned compartment varied throughout the period of analysis.The results are analysed in the light of the overall response of the cells to IPR and the interaction of the organelles during the synthetic phase of the secretory cycle. They are presented as a basis for ensuing studies of the granule populations and the membrane composition of the cells during the restoration of granule stores.     

Light- and electron-microscopic radioautographic study of glycoprotein secretion in the granular duct of the submandibular gland of the male mouse

Summary L-³H-fucose was injected intravenously into adult male mice, after which, at different time intervals, the submandibular glands were removed and processed for light-and electron-microscopic radioautography. This radio active hexose was taken up by newly synthesized glycoproteins in the cells lining the granular ducts which were maximally labeled at 4 h after injection. Between 4 and 72 h the amount of labeled glycoproteins decreased moderately indicating that these macromolecules undergo a slow renewal. The main subcellular site of incorporation of 3 H-fucose into glycoproteins was the Golgi apparatus. From this organelle labeled glycoproteins were transferred to small secretory granules (diameter up to 1.0 m) located not only near the Golgi region but also throughout the apical cytoplasm. At 1 h after injection the concentration of label reached a maximum in the small secretory granules and labeling of medium (diameter between 1.1 and 2.0 m) and large (diameter over 2.0 m) granules was very low. At this postinjection interval the secretion product inside the lumen of the duct was already labeled. Between 1 and 72 h after injection the concentration of radioactivity in the small secretory granules decreased intensely while increasing in the medium and in the large ones. The concentration of fucose label reached a maximum in the medium secretory granules at 24 h and in the large ones at 72 h after injection. Additional experiments using mice previously injected with 4 intraperitoneal doses of ³H-fucose given 3 h apart demonstrated that the large granules undergo a very slow renewal. Some were found to be labeled as long as 28 days after administration of ³H-fucose. Recorded in this latter series of experiments was the labeling pattern of dense bodies that were regularly visualized in the cells lining the granular ducts. Their significance in the secretory process is discussed. In conclusion, newly synthesized glycoproteins are transferred from the Golgi apparatus to small secretory granules which carry a readily releasible pool of these macromolecules to the lumen of the duct. The small secretory granules also transfer newly synthesized glycoproteins to medium and large secretion granules which store a pool that is released very slowly. This characterizes the large secretory granules as the intracellular sites of storage of secretion products. The results of this investigation were correlated with the knowledge about the chemical composition of the different macromolecules that are known to be synthesized by the secretory cells of the granular ducts of the submandibular gland of the mouse.     

Cytological effects of urecholine stimulation on the rat pancreas

Summary Stimulation of the exocrine pancreas by the secretagogue urecholine causes degranulation of the acinar cells. Under in vivo conditions, this degranulation is not uniform throughout the tissue. Indeed some of the acini are almost completely depleted of their granules while others display the appearance of resting acini. A noticeable feature is that all the cells of the same acinus display a comparable degree of degranulation. Moreover, groups of neighbouring acini seem to respond simultaneously suggesting that the secretory stimulus is propagated from one acinus to the other. In vitro stimulation of dispersed acini also showed that some of the acini are more responsive than others indicating that this phenomenon can not be attributed to accessibility of the secretagogue to its receptor. These observations lead us to the concept that the response of the pancreatic acinar cell is controlled at the level of the acinus.     

Inhibition of glycoconjugate secretion by colchicine and cytochalasin B

Summary The effects have been analyzed of cytochalasin B and colchicine on the secretion of glycoconjugates by human bronchial expiants labeled in vitro with radioactive glucosamine. Both cytochalasin B and colchicine had no effect on baseline ¹⁴C-labeled glycoconjugate release but caused a dose-dependent (10^–7–10^–4 M) inhibition of ¹⁴C-glycoconjugate release and discharge of labeled macromolecules from mucous and serous cells induced by 5 · 10^–5 M methacholine.Quantitative autoradiographic analyses showed that neither cytochalasin B nor colchicine inhibited ³H-threonine or ³H-glucosamine incorporation into mucous and serous cells of the submucosal glands or goblet cells of the airway epithelium. Colchicine (10^–5 M) but not cytochalasin B significantly reduced the rate at which labeled macromolecules were transported through mucous, serous and goblet cells but this effect was not observed until 4 h after the addition of colchicine. Neither cytochalasin B nor colchicine affected the basal rate of labeled-macromolecule discharge from mucous, serous or goblet cells. At a concentration of 10^–5 M, both agents completely inhibited the increase in labeled-macromolecule discharge induced in mucous and serous cells by methacholine.Our results suggest that in the submucosal gland of human airways microtubules and microfilaments may be important in secretagogue-induced but not in baseline cellular glycoconjugate discharge, implying that the mechanisms of the two processes differ significantly. Furthermore, a role for microtubules is suggested in the transport of secretory granules through mucous, serous and goblet cells.Supported by National Institutes of Health Research Grant 5R01HL22444. The authors gratefully acknowledge the technical assistance of Mr. Tudor Williams, Mr. Eduardo Quintanilla and Ms. Maureen Hayes