Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: Effects on prolactin and thyrotropic cells

Summary The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human--thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thryrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.This work has been supported by grants no. 2184-83 and PB 86-0095 from the Comisión Interministerial para la Ciencia y Tecnología, Spain     

Ultrastructural immunolocalization of basic fibroblast growth factor to lipid bodies and secretory granules in human mast cells

Isolated human lung mast cells were used to identify subcellular sites of basic fibroblast growth factor using a postembedding immunogold method. The factor was present in quantity in secretory granules and cytoplasmic lipid bodies. Cisterns of smooth endoplasmic reticulum and ribosome clusters, closely associated with lipid bodies, contained the factor as did the nuclear matrix. Factor-positive lipid bodies were adjacent to nuclear pores and often indented perinuclear cisternae. Altered secretory granules with reduced density, characteristic of secretion by piecemeal degranulation in mast cells, showed reduced gold label for basic fibroblast growth factor; small, electron-lucent (80–100nm) transport vesicles near altered granules were labelled for the factor. Since these mature mast cells do not display extensive arrays of classical secretory organelles, such as rough endoplasmic reticulum and Golgi structures, these new subcellular localizations for basic fibroblast growth factor suggest several possible alternative release routes for a cytokine devoid of a signal sequence characteristic of regulated secretory proteins.     

Combined effects of glucose and oxygen concentrations upon the ultrastructure of cultured islet cells and insulin accumulation in culture media

Rat pancreatic islets cultured for 6 days at 100 or 500 mg/dl glucose and 20 or 7% O2 were examined electron-microscopically, and insulin accumulation in the culture media was assayed immunologically. In the islets cultured at 500 mg/dl glucose and 20% O2, B cells exhibited hypertrophy of granular endoplasmic reticulum and Golgi apparatus, an abundance of free ribosomes, degranulation and the margination of secretory granules. In islets cultured at 500 mg/dl glucose and 7% O2, B cells exhibited dilatation of endoplasmic reticulum cisternae and dominance of Golgi vesicles in addition to the above-mentioned changes. These changes, together with the correlated data on insulin accumulation, are discussed with special reference to the effects of glucose and oxygen upon the synthesis and release of insulin in B cells.     

Serotoninergic control of MSH secretion in the eel. Ultrastructural study after 5-hydroxytryptophan treatment.

Injection of 5-HTP induces a melanodisperson ; MSH cells are markedly stimulated :hormone synthesis (development of Golgi area and endoplasmic reticulum) and release (reduction of secretory granules) are observed. This stimulatory serotoninergic pathway seems antagonistic to the dopaminergic system that inhibits MSH secretion in the eel.     

Concentration of amylase along its secretory pathway in the pancreatic acinar cell as revealed by high resolution immunocytochemistry

Summary The modified protein A-gold immunocytochemical technique was applied to the localization of amylase in rat pancreatic acinar cells. Due to the good ultrastructural preservation of the cellular organelles obtained on glutaraldehyde-fixed, osmium tetroxide-postfixed tissue, the labelling was detected with high resolution over the cisternae of the rough endoplasmic reticulum (RER), the Golgi apparatus, the condensing vacuoles, the immature pre-zymogen granules, and the mature zymogen granules. Over the Golgi area, the labelling was present over the transitional elements of the endoplasmic reticulum, some of the smooth vesicular structures at thecis- andtrans-faces and all the different Golgi cisternae. The acid phosphatase-positive rigidtrans-cisternae as well as the coated vesicles were either negative or weakly labelled. Quantitative evaluations of the degree of labelling demonstrated an increasing intensity which progresses from the RER, through the Golgi, to the zymogen granules and have identified the sites where protein concentration occurs. The results obtained have thus demonstrated that amylase is processed through the conventional RER-Golgi-granule secretory pathway in the pancreatic acinar cells. In addition a concomitance has been found between some sites where protein concentration occurs: thetrans-most Golgi cisternae, the condensing vacuoles, the pre- and the mature zymogen granules, and the presence of actin at the level of the limiting membranes of these same organelles as reported previously (Bendayan, 1983). This suggests that beside their possible role in transport and release of secretory products, contractile proteins may also be involved in the process of protein concentration.     

Ultrastructural studies of the secretion of calcium carbonate by the serpulid polychaete worm,Pomatoceros caeruleus

Summary Pomatoceros caeruleus possesses a pair of simple acinar calcium-secreting glands lying in the ventral peristomium. Each gland has a single large secretory acinus containing columnar secretory cells with basal nuclei. Golgi complexes and flattened cisternae of the rough endoplasmic reticulum are abundant in the midregion and secretory vacuoles fill the apical cytoplasm. Elongate microvilli extend from the apices of the cells into the gland lumen. An organelle-free zone, the intracellular channel, extends from near the base almost to the apex of the cells. It is bordered on one side by the lateral cell membranes and is separated from the organelle compartment by elongate profiles of the rough endoplasmic reticulum.The secretory products of the calcium-secreting glands have the form of cubic or rhombohedral granules with average dimensions of 150–200 m on a side. The granules are composed of a fibrous organic matrix in which needle-like calcite crystals are deposited. The possible mode of synthesis of the calcified secretory granules is discussed.Part of this work represents a portion of a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Duke University. I wish to express my thanks to Dr. Karl M. Wilbur and Dr. Norimitsu Watabe for their advice and encouragement during this study. This study was supported by Public Health Service Grants 5TI DE 92-05 and DE 02668 from the National Institutes of Health.     

Hypothesis: one rate-limiting step controls the magnitude of both phases of glucose-stimulated insulin secretion

The biphasic secretory response of pancreatic -cells to abrupt and sustained exposure to glucose is well documented. Some of the ATP-sensitive K⁺ (K_ATP) channel-dependent mechanisms underlying the first phase of insulin release are known; the mechanisms underlying the second phase are less well known. The hypothesis we propose is that one rate-limiting step, controlling the conversion of granules in a readily releasable (RR) docked granule pool to an immediately releasable (IR) pool, is responsible for the magnitude of both phases of release. Furthermore, we propose that the K_ATP channel-independent signaling pathway regulates this rate-limiting step. The size of the IR pool of granules that constitutes the first phase is determined under resting conditions by the forward and reverse rates of conversion of granules in the RR and IR pools. The resulting equilibrium position determines the maximum number of -cell granules available for release during the first phase upon exposure to glucose. At the nadir between the two phases, the IR pool has been depleted so that the rate of granule release is equal to the low forward rate for the conversion of RR to IR granules. After the nadir, the forward rate is accelerated during the rising portion of the second phase until it reaches a maximum rate at the plateau. glucose signaling pathways; granule pools     

Multiple rough endoplasmic cisternae in “C” cells

Summary Multiple rough endoplasmic cisternae were found in C cells of the adult rat, at interphase. They are considered to be normal constituents of C cells. Their morphological relation to rough endoplasmic reticulum and their close proximity to mitochondria, Golgi dictyosomes and secretory granules suggest that they may have a role in the secretory activity of this endocrine cell.     

Immuno-electron microscopy of formation,degradation and exocytosis of the ovulation neurohormone in Lymnaea stagnalis

Summary The cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis are involved in the control of egg laying and associated behaviour by releasing various peptides. One of these is the ovulation hormone (CDCH). The cellular dynamics of this peptide have been studied using an antiserum raised to a synthetic portion of CDCH comprising the 20–36 amino acid sequence. With the secondary antibody-immunogold technique, specific immunoreactivity was found in all CDC. Rough endoplasmic reticulum and Golgi apparatus showed very little reactivity as did secretory granules that were in the process of being budded off from the Golgi apparatus. However, secretory granules that were being discharged from the Golgi apparatus, were strongly reactive. Secretory granules within lysosomal structures revealed various degrees of immunoreactivity, indicating their graded breakdown. Large electrondense granules, formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were only slightly reactive. In the axon terminals secretory granules released their contents into the haemolymph by the process of exocytosis. The exteriorized contents were in most cases clearly immunopositive.The possibility has been discussed that CDCH is cleaved from its polypeptide precursor within secretory granules during granule discharge from the Golgi apparatus; subsequently, the mature secretory granules would be transported towards the neurohaemal axon terminals where they release CDCH into the haemolymph. Superfluous secretory material would be degraded by the lysosomal system including the large electron-dense granules.     

Immunocytochemical and morphometrical study of thyrotropic cells of Rana ridibunda

Summary Indirect immunoflorescence and PAP techniques for light microscopy as well as the immunogold complex technique for electron microscopy were used to localize and identify thyrotropic (TSH) producing cells in the pars distalis of Rana ridibunda. A double immunostaining procedure was used to distinguish TSH cells from other glycoprotein hormone producing cells. Rabbit anti-human--TSH was used as the primary antiserum and revealed a basophil, PAS and alcian blue positive cell type in the ventro-central zone of the gland. Under the electron microscope, TSH cells show irregular morphology, polymorphic secretory granules with diameters ranging between 120 and 375 nm and poor development of the endoplasmic reticulum and Golgi complex; they are usually polarized towards capillaries. Ultrastructural morphometry (point-counting method) was used to evaluate stereological parameters of rough endoplasmic reticulum, Golgi complex, secretory granules and mitochondria.This work has been supported by grant 2184-83 from the Comisión Asesora (CAICYT) of Spain     

The effect of reserpine on the pars intermedia of the rat pituitary

Summary Reserpine has a stimulatory effect on the pars intermedia of the rat pituitary, probably mediated by its action on regulatory catecholaminergic nerves. The effect of single intraperitoneal injections of 0.1–20 mg/kg b.w. of reserpine was studied in adult male rats. Reserpine at a dose of 2 mg/kg b.w. induced degranulation, orientation of the secretory granules along the cell membrane and loss of formaldehyde-chloral-induced fluorescence, accompanied by an activation of the granular endoplasmic reticulum and the Golgi apparatus. With higher doses progressive degranulation and loss of fluorescence were observed. The effect was, however, heterogeneous, and with all doses cells displaying normal ultrastructure and normal fluorescence were regularly present.To study the release of granular products (containing a different components of the pro-opiomelanocortin chain) from individual cells, formaldehyde-chloral induced fluorescence and -MSH- and -endorphin immunoreactivies were demonstrated in consecutive sections from pituitaries of rats given 8 mg/kg body weight of reserpine 24 h before sacrifice. The results indicate coordinated release of these granular products at the cellular level after reserpine treatment.This work was supported by Finska Läkaresällskapet     

Immunocytochemical localization of parathyroid hormone in rabbit parathyroid glands

Immunocytochemical localization of parathyroid hormone was examined in the rabbit parathyroid gland by means of protein A-gold technique. Protein A-gold particles were observed on the secretory granules and the large secretory granules thought to be storage granules. No protein A-gold particles were observed on cisternae of the endoplasmic reticulum and the Golgi apparatus.     

Immunocytochemical localization of parathyroid hormone in hamster parathyroid gland

The immunocytochemical localization of parathyroid hormone was examined in the hamster parathyroid gland by using the protein A-gold technique. Protein A-gold particles were concentrated over secretory granules, large secretory granules thought to be storage granules and Golgi vacuoles. No protein A-gold particles were detected over large vacuolar bodies and cisternae of the granular endoplasmic reticulum.     

Involvement of the Golgi apparatus in the secretion of α-amylase from gibberellin-treated barley aleurone cells

Localisation of -amylase (EC 3.2.1.1) in barley aleurone cells treated with gibberellic acid has been achieved using protein A-gold-labelled polyclonal antibodies. Gold particles were located almost exclusively over the lumen of the rough endoplasmic reticulum and cisternae of the Golgi apparatus. The label was most concentrated over the Golgi apparatus. This indicates that the Golgi is involved in the secretion of -amylase protein from aleurone cells.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - PBS phosphate-buffered saline     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The ultrastructure of the intermediate lobe of the hypophysis was studied in Anolis carolinensis with the use of a threefold aldehyde fixative. Lizards with a brown skin were selected. The possibility of two types of secretory cells is discussed; neither cell type is innervated. Type I cells are rarely found and contain dense granules approximately 0.3 m in diameter; Type II cells vary widely in secretory activity. Most of the Type II cells contain a large number of dense secretory granules (up to about 1.3 m in diameter) almost filling the cytoplasm. Rough endoplasmic reticulum (RER), Golgi apparatus and mitochondria are poorly developed. Only some of these cells show signs suggesting a high secretory activity, namely a well developed RER, Golgi apparatus and numerous mitochondria. In these cells the RER sometimes forms large intracisternal droplets (up to 7 m in diameter). Two of the animals exhibited a more uniform, high secretory activity. Large (about 2 m in diameter), pale vacuoles, probably of extracellular character, were found mostly in the vicinity of the perivascular septum. Their role in the release of MSH is discussed. The present data, which are discussed with reference to earlier findings (Forbes, 1972), form the morphological basis for an experimental study on regulation of MSH release (Larsson et al., 1979).Supported by grants from the Swedish Natural Science Research Council (to P. Meurling) and the Royal Physiographic Society of LundThe authors are indebted to Mrs. Ingrid Hallberg, Mrs. Kirsten Thörneby and Mrs. Lena Sandell for valuable technical assistance and to Miss Inger Norling for photographic aid     

A mathematical model quantifying GnRH-induced LH secretion from gonadotropes

A mathematical model is developed to investigate the rate of release of luteinizing hormone (LH) from pituitary gonadotropes in response to short pulses of gonadotropin-releasing hormone (GnRH). The model includes binding of the hormone to its receptor, dimerization, interaction with a G protein, production of inositol 1,4, 5-trisphosphate, release of Ca(2+) from the endoplasmic reticulum, entrance of Ca(2+) into the cytosol via voltage-gated membrane channels, pumping of Ca(2+) out of the cytosol via membrane and endoplasmic reticulum pumps, and release of LH. Cytosolic Ca(2+) dynamics are simplified (i.e., oscillations are not included in the model), and it is assumed that there is only one pool of releasable LH. Despite these and other simplifications, the model explains the qualitative features of LH release in response to GnRH pulses of various durations and different concentrations in the presence and absence of external Ca(2+).     

A novel granular cell type of locust Malpighian tubules: ultrastructural and immunocytochemical study

Summary A novel secretory cell type in the initial segment of the Malpighian tubules of the locusts Schistocerca gregaria and Locusta migratoria is described ultrastructurally and studied by means of immunocytochemical techniques. The cells show abundant rough endoplasmic reticulum with interspersed Golgi zones. The richness of the cell secretory machinery and the presence of apical dense plemorphic granules suggest a role in secretion of proteinaceous material to the tubule lumen. The surprising finding of ACTH (1–24)-, -MSH-, and 7B2-like immunoreactivity for this cell is discussed.     

Brefeldin A induces callose formation in onion inner epidermal cells

Summary The antibiotic fungal toxin brefeldin A (BFA) causes synthesis of additional cell wall material in adult differentiated onion inner epidermal cells at concentrations of 5–30 g/ml. This tertiary wall contains callose and is layered on the secondary cellulosic wall in a time- and dose-dependent manner. Initially, callose is found in pit fields in the form of small vesicular patches. With time and dose, depositions grow in size and form large plugs invaginating into the cell, where the adjacent cytoplasm forms bulky accumulations and contains many organelles including endomembranes. Within the cytoplasm, BFA exerts the characteristic morphological effects on the secretory system including changes of the Golgi stacks, formation of large vesicles, and proliferation of dilated cisternae of the endoplasmic reticulum. Higher concentrations of BFA (60 g/ml) lead to disintegration of the Golgi apparatus; they have no effects on the cell wall, no callose synthesis occurs. We conclude from these observations that BFA has two independent targets in onion cells. BFA acts on the plasma membrane, hence operating as an elicitor of plant defense reactions and thus activates callose synthesis. BFA acts also on the membranes of the secretory system and influences budding and fusion of vesicles at the endoplasmic reticulum and at the dictyosomes. These two mechanisms occur in parallel, suggesting that the secretory system still can play its presumed role in callose synthesis. Only when dictyosomes are completely disintegrated, no more callose is formed.Abbreviations BFA Brefeldin A - PM plasma membrane - GA Golgi apparatus - ER endoplasmic reticulum - GS glucan synthetase Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Effects of hypergravity environment on the parathyroid gland of the isoproterenol-treated hamster

The ultrastructure of the parathyroid glands of hamsters subjected to 5 g environment after an intraperitoneal injection of isoproterenol was studied. In the isoproterenol-treated hamsters exposed to hypergravity environment, the Golgi complexes, cisternae of the granular endoplasmic reticulum and lipid droplets were significantly increased and secretory granules were significantly decreased compared with those of the control group. In addition, many chief cells contained numerous prosecretory granules in the Golgi areas and several secretory granules were situated close to the plasma membrane of the chief cells. These results suggest that the synthesis and release of parathyroid hormone may be markedly stimulated in the parathyroid glands of the isoproterenol-treated hamsters exposed to hypergravity environment.