Quick-freeze,deep-etch visualization of exocytosis in anterior pituitary secretory cells: localization and possible roles of actin and annexin II

The exocytotic process in the anterior pituitary secretory cells was studied using quick-freeze deep-etch electron microscopy, fluorescein-isothiocyanate-phalloidin staining, heavy meromyosin decoration, and immuno-electron microscopy. The subcortical actin filaments are distributed unevenly in the peripheral cytoplasm. Few secretory granules are seen beneath the plasma membrane in the region where the peripheral cytoplasm is occupied by numerous subcortical actin filaments. On the contrary, in the region free of the subcortical actin filaments, many secretory granules lie in contact with the plasma membrane. Thus, the subcortical actin filaments may control the approach of the secretory granules to the plasma membrane in these cells. The granule and plasma membranes that lie in close proximity are linked by intervening strands. Unfused portions of both membranes remain linked by these strands during membrane fusion and opening. These strands may be involved in membrane contact, fusion and opening during exocytosis. Annexin II (calpactin I) has been demonstrated immunocytochemically to be localized at the contact sites between the granule and plasma membranes, and is therefore a possible component of the intervening strands. Membrane fusion starts within focal regions of both membranes less than 50 nm in diameter. The plasma membrane shows inward depressions toward the underlying granules immediately before fusion. The disappearance of intramembranous particles from the exocytotic site of the membrane has not been observed.     

Gonadotropin-releasing hormone stimulates annexin 5 messenger ribonucleic acid expression in the anterior pituitary cells

We previously reported that annexin 5 is found specifically in gonadotropes and that the expression is dramatically enhanced after ovariectomy. In the present study, the expression of annexin 5 was examined in the primary culture of rat anterior pituitary cells using semiquantitative RT-PCR to determine if it is under the direct control of gonadotropin-releasing hormone (GnRH). Continuous administration of GnRH analog for 1 h enhanced the expression of both FSH beta subunit and annexin 5 mRNA. The expression of annexin 5 mRNA was also augmented by phorbol 12-myristate 13-acetate but not by forskolin. Administration of recombinant rat annexin 5 to the culture increased LH beta mRNA expression. These data clearly demonstrate that the expression of annexin 5 mRNA is directly controlled by GnRH and suggest that annexin 5 is involved in mediating GnRH action in the pituitary gland.     

Association of annexin V with prolactin in the rat anterior pituitary gland.

When pituitary extracts were subjected to non denaturing polyacrylamide gel electrophoresis, an unknown protein was found to associate with a proportion of the prolactin. This protein was dissociated from prolactin by sodium dodecyl sulfate. The protein was purified and sequenced. As the amino terminus was blocked, the amino acid sequences of three peptide fragments were determined. The obtained sequences of 41 amino acids were identical to partial sequences of a known protein, rat Annexin V. The molecular mass, 36 kDa, was also the same as the molecular weight of Annexin V. The existence of Annexin V mRNA in rat pituitary glands was also confirmed by polymerase chain reaction. These results show that Annexin V, a member of the calcium-dependent phospholipid binding proteins, is synthesized in the rat pituitary gland, and suggest its association with prolatin in the gland.     

Targeting of membrane proteins to the regulated secretory pathway in anterior pituitary endocrine cells

Unlike the neuroendocrine cell lines widely used to study trafficking of soluble and membrane proteins to secretory granules, the endocrine cells of the anterior pituitary are highly specialized for the production of mature secretory granules. Therefore, we investigated the trafficking of three membrane proteins in primary anterior pituitary endocrine cells. Peptidylglycine alpha-amidating monooxygenase (PAM), an integral membrane protein essential to the production of many bioactive peptides, is cleaved and enters the regulated secretory pathway even when expressed at levels 40-fold higher than endogenous levels. Myc-TMD/CD, a membrane protein lacking the lumenal, catalytic domains of PAM, is still stored in granules. Secretory granules are not the default pathway for all membrane proteins, because Tac accumulates on the surface of pituitary endocrine cells. Overexpression of PAM is accompanied by a diminution in its endoproteolytic cleavage and in its BaCl(2)-stimulated release from mature granules. Because internalized PAM/PAM-antibody complexes are returned to secretory granules, the endocytic machinery of the pituitary endocrine cells is not saturated. As in corticotrope tumor cells, expression of PAM or Myc-TMD/CD alters the organization of the actin cytoskeleton. PAM-mediated alterations in the cytoskeleton may limit maturation of PAM and storage in mature granules.     

Cholesterol modulates the membrane binding and intracellular distribution of annexin 6

Annexins are Ca(2+)- and phospholipid-binding proteins that are widely expressed in mammalian tissues and that bind to different cellular membranes. In recent years its role in membrane traffic has emerged as one of its predominant functions, but the regulation of its intracellular distribution still remains unclear. We demonstrated that annexin 6 translocates to the late endocytic compartment in low density lipoprotein-loaded CHO cells. This prompted us to investigate whether cholesterol, one of the major constituents of low density lipoprotein, could influence the membrane binding affinity and intracellular distribution of annexin 6. Treatment of crude membranes or early and late endosomal fractions with digitonin, a cholesterol-sequestering agent, displayed a strong reduction in the binding affinity of a novel EDTA-resistant and cholesterol-sensitive pool of annexin 6 proteins. In addition, U18666A-induced accumulation of cholesterol in the late endosomal compartment resulted in a significant increase of annexin 6 in these vesicles in vivo. This translocation/recruitment correlates with an increased membrane binding affinity of GST-annexin 6 to late endosomes of U18666A-treated cells in vitro. In conclusion, the present study shows that changes in the intracellular distribution and concentration of cholesterol in different subcellular compartments participate in the reorganization of intracellular pools of Ca(2+)-dependent and -independent annexin 6.     

Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells

The pituitary cell line, AtT-20, synthesizes adrenocorticotropic hormone (ACTH) as a glycoprotein precursor that is cleaved into mature hormones during packaging into secretory granules. The cells also produce an endogenous leukemia virus (MuLV) that is glycosylated after translation similar to the glycosylation of the ACTH precursor. Our evidence suggests that the envelope glycoprotein and some precursor ACTH get to the cell surface in a vesicle different from the mature ACTH secretory granule. Viral glycoproteins and ACTH precursor are released from the cells much sooner after synthesis than mature ACTH. Isolated secretory granules do not contain significant amounts of the envelope glycoprotein or ACTH precursor. Exposing cells to 8Br-cAMP stimulates release of mature ACTH four to five fold, but has little effect on the release of the ACTH precursor or the viral glycoproteins. We propose that the viral glycoproteins and some of the ACTH precursor are transported by a constitutive pathway, while mature ACTH is stored in secretory granules where its release is enhanced by stimulation.     

Intergranular bridges in the anterior pituitary cell and their possible involvement in Ca2+-induced granule-granule fusion

Quick-freeze deep-etch electron microscopy showed the presence of bridge-like structures between adjacent secretory granules in rat anterior pituitary secretory cells. These intergranular bridges were variable in length and thickness. The finest bridges were 7–8 nm in length, while the longest ones were as long as 80 nm. Annexin II, one of the Ca²⁺-dependent phospholipid-binding proteins, is known to interlink between two membranes and induce aggregation of liposomes and chromaffin granules under the presence of Ca²⁺. In anterior pituitary cells, annexin II was detected by immunoelectron microscopy at the contact sites of secretory granules with other granules. The anterior pituitary cells treated under the presence of extracellular Ca²⁺ with Clostridium perfringens enterotoxin which induces Ca²⁺ influx showed multigranular exocytosis, i.e., multiple fusions of secretory granules with each other and with the plasma membrane. The granule-granule fusion in progress could be captured by the quick-freeze deep-etch technique. The membranes of adjacent secretory granules were partially fused at their contact sites where intergranular strands were no longer seen, while there existed intergranular strands between unfused portions of the granule membranes. From these results, we consider that the intergranular bridges, some of which may be composed of annexin II, are involved in Ca²⁺-induced granule-granule fusion in anterior pituitary cells.     

Interleukin 6 modulation of second messenger systems in anterior pituitary cells.

We investigated the effect of interleukin-6 (IL-6) on second messenger systems in anterior pituitary (AP) cells. The acute exposition of membranes derived from the pituitary gland to IL-6 did not modify basal and forskolin-stimulated adenylate cyclase (AC) activity, as well as inositol phosphate (IP) production and free [Ca(++)]i. Preincubation of AP cells with IL-6 for 20 min did not affect basal second messengers levels, while completely abolished the stimulation by VIP of AC activity, partially inhibited forskolin-stimulated cAMP formation and reduced TRH-stimulated IP production. Finally, the pretreatment of AP cells for 20 min with IL-6 also reduced the TRH-induced rise in free [Ca(++)]i.     

Angiotensin II phosphorylation of extracellular signal-regulated kinases in rat anterior pituitary cells

We studied the effects of ANG II on extracellular signal-regulated kinase (ERK)1/2 phosphorylation in rat pituitary cells. ANG II increased ERK phosphorylation in a time- and concentration-dependent way. Maximum effect was obtained at 5 min at a concentration of 10-100 nM. The effect of 100 nM ANG II was blocked by the AT1 antagonist DUP-753, by the phospholipase C (PLC) inhibitor U-73122, and by the MAPK kinase (MEK) antagonist PD-98059. The ANG II-induced increase in phosphorylated (p)ERK was insensitive to pertussis toxin blockade and PKC depletion or inhibition. The effect was also abrogated by chelating intracellular calcium with BAPTA-AM or TMB-8 by depleting intracellular calcium stores with a 30-min pretreatment with EGTA and by pretreatment with herbimycin A and PP1, two c-Src tyrosine kinase inhibitors. It was attenuated by AG-1478, an inhibitor of epidermal growth factor receptor (EGFR) activation. Therefore, in the rat pituitary, the increase of pERK is a Gq- and PLC-dependent process, which involves an increase in intracellular calcium and activation of a c-Src tyrosine kinase, transactivation of the EGFR, and the activation of MEK. Finally, the response of ERK activation by ANG II is altered in hyperplastic pituitary cells, in which calcium mobilization evoked by ANG II is also modified.     

Phagosome-lysosome fusion. Characterization of intracellular membrane fusion in mouse macrophages

Several approaches have been used to study the determinants of phagosome-lysosome fusion in intact mouse macrophages. Lysosomes were labeled with the fluorescent vital dye acridine orange and the rate and extent of their fusion with yeast-containing phagosomes was monitored by fluorescence microscopy. Fusion was also assayed by electron microscopy, using horseradish peroxidase or thorium dioxide as a marker for secondary lysosomes. Good agreemen samples with an enzymatic marker, and thorium dioxide-labeled samples evaluated by stereology. The rate of usion as assayed by fluorescence was not affected by the number of particles ingested, serum concentration, or prior uptake of digestible or nondigestible substances. With this assay it was possible to observe the rate of fusion separate from and uninfluenced by the phagocytic rate. Both the rate and extent of fusion were dramatically increased after several days in culture and similar changes were found by use of the EM assays. Fusion was strongly affected by incubation temperature, having a Q10 of 2.5 No detectable fusion occurred below 15 degrees C, and this inhibition was rapidly reversed when cells were returned to 37 degrees C.     

Recovery of surface membrane in anterior pituitary cells. Variations in traffic detected with anionic and cationic ferritin

Cells dissociated from rat anterior pituitaries were incubated with native or cationized ferritin (CF) to trace the fate of surface membrane. Native ferritin, which did not bind to the cell surface, was taken up in small amounts by bulk-phase endocytosis and was found increasingly (over 1-2 h) concentrated in lysosomes. CF at 100-fold less concentrations bound rapidly to the cell membrane, was taken up by endocytosis in far greater amounts, and within 15-60 min was found increasingly within multiple stacked Golgi cisternae, around forming secretion granules, and within elements of GERL, as well as within lysosomes. The findings demonstrate that the fate of the tracer--and presumably also that of the surface membrane--varies with the same molecule differing only in net charge: vesicles carrying anionic ferritin (net negative charge) fuse only with elements of the lysosomal system whereas those carrying CF (net positive charge) can fuse not only with elements of the lysosomal system, but also with elements along the secretory pathway (Golgi cisternae and condensing granules) as well.     

The effect of acetylcholine on inositol lipid metabolism and intracellular calcium concentrations in bovine anterior pituitary cells

Acetylcholine (ACh) increased the intracellular calcium concentration in bovine anterior pituitary cells. In the presence of the calcium channel antagonists verapamil (20 microM) or nitrendepine (1 microM) the increase in calcium was partially inhibited but showed both transient and sustained components. In the presence of EGTA (2.5 mM) only the transient component was observed. ACh also decreased inositol radioactivity in phosphatidylinositides and increased it in inositol phosphates. It is concluded that the increase in calcium caused by acetylcholine requires both the entry of external calcium and mobilisation of internal calcium. Replacement of external sodium by N-methyl-D-glucamine inhibited the rises in calcium and inositol phosphate labelling in response to ACh. Tetrodotoxin (3 microM) or ouabain (50 microM) did not affect either response to ACh. Verapamil did not affect the calcium rise induced by ACh in the absence of external sodium. The phorbol ester PMA (10 nM) caused a transient rise in calcium and inhibited the calcium rise caused by acetylcholine: it did not modify the effect of acetylcholine on inositol phosphates. The dependence of the stimulation of external calcium entry and inositol phosphate production on external sodium ions and protein kinase C is discussed.     

Calcium ionophore and phorbol ester increase membrane binding of annexin a7 in alveolar type II cells

The fusion of lamellar body with plasma membrane, a distal obligatory step in exocytosis of lung surfactant, may be mediated by annexin a7 (anx a7; synexin). To understand the mechanism of anx a7 action, we tested the hypothesis that anx a7 binding to membranes would increase in order to facilitate membrane fusion during stimulation of lung surfactant secretion. Isolated rat alveolar type II cells were treated with established secretagogues of lung surfactant and the membrane and cytosol fractions were analyzed for in vitro binding of anx a7. In cells treated with calcium ionophore (A23187) or phorbol 12-myristate 13-acetate (PMA), anx a7 binding to the membrane fraction was increased by 120%, while that to the cytosol fraction was decreased by 40%, when compared with binding to corresponding fractions from control cells. Protein kinase inhibitors prevented the PMA effects on anx a7 binding. The lamellar body and plasma membrane fractions of A23187-treated cells also showed increased binding of anx a7. The lamellar bodies of A23187-treated cells showed lower K(m) for Ca(2+) and higher maximal binding of anx a7, when compared with those from control cells. Collectively, our findings suggest that these two agents modify membrane proteins to regulate anx a7 binding, which may facilitate increased membrane fusion activity during stimulation of surfactant secretion.     

Sequential analysis of trans-SNARE formation in intracellular membrane fusion

SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Q(a), Q(b), and Q(c)) from target (t)-SNAREs and one (R) from the vesicular (v)-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Q(a) SNARE, leaving behind a Q(bc)R subcomplex. This subcomplex serves as an acceptor for a Q(a) SNARE from the opposite membrane, leading to Q(a)-Q(bc)R trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the Q(bc)R cis-complex and the formation of the Q(a)-Q(bc)R trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex). This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.     

Correlation between electrical activity and intracellular Ca2+ oscillations in GH3 rat anterior pituitary cells

Simultaneous measurements of electrical activity and intracellular Ca(2+) levels were performed in perforated-patch current-clamped individual GH3 cells. Both in cells showing brief (<100 ms) and long action potentials (APs), we found a good correlation between the averaged intracellular Ca2+ concentration ([Ca2+]i) and AP frequency, but not between the mean [Ca2+]i and AP duration. Nevertheless, the magnitude of spontaneous Ca2+ oscillations was highly dependent on the size and duration of the APs. The decay of the Ca2+ transients was not slowed when the size of the oscillations was varied either spontaneously or after elongation of the AP with the K+ channel blocker tetraethyl ammonium. Furthermore, the recovery from Ca2+ loads similar to those induced by the APs was slightly retarded after treatment of the cells with intracellular store Ca2+-ATPase inhibitors. Among previous results showing that caffeine-induced [Ca2+]i increases are secondary to electrical activity enhancements in GH3 cells, these data indicate that the Ca2+ entry triggered via APs is the primary determinant of the [Ca2+]i variations, and that Ca2+-induced Ca2+ release has a minor contribution to Ca2+ oscillations recorded during spontaneous activity. They also point to modulation of electrical activity patterns as a crucial factor regulating spontaneous [Ca2+]i signalling, and hence pituitary cell functions in response to physiological secretagogues.     

Multiple transduction mechanisms of dopamine, somatostatin and angiotensin II receptors in anterior pituitary cells

The concept of multifactorial pituitary control is now well established. As in other cell systems, integration of complex messages involves dynamic interactions of receptors and coupling mechanisms. Regulation of adenohypophyseal secretions has been shown to involve cyclic AMP production, the modulation of phosphatidylinositol phosphate breakdown and Ca2+ mobilization. Dopamine, somatostatin and angiotensin II receptors are negatively coupled to adenylate cyclase in anterior pituitary cells. In the case of angiotensin, this effect on adenylate cyclase appears paradoxical since the peptide markedly stimulates prolactin secretion. In fact, angiotensin II also markedly stimulates inositol phosphate production and this effect could account for the stimulated hormone secretion. In addition, dopamine could inhibit inositol phosphate production stimulated by angiotensin II and thyrotropin-releasing hormone. Dopamine and somatostatin also directly modulate voltage-dependent calcium channels, perhaps through a direct coupling with potassium channels. On the other hand, steroids modulate the sensitivity of adenohypophyseal cells to neurohormones by different mechanisms. In the case of somatostatin, it increases the number of specific binding sites, while in the case of dopamine estradiol affects the transduction mechanisms of D2 dopamine receptors. In conclusion, dopamine and somatostatin receptors appear coupled to various transduction mechanisms through pertussis-sensitive G proteins in anterior pituitary cells.     

Transformation of Golgi membrane into the envelope of herpes simplex virus in rat anterior pituitary cells

Envelopment of herpes simplex virus type-1 (HSV-1) was investigated in relation to membrane differentiation in dissociated anterior pituitary cells. The number of cells stained positively with anti-HSV-1 serum was increased from 16 h to 31 h post infection. During this period, electron microscopy revealed that a number of nucleocapsids (unenveloped particles) were accumulated in the Golgi area, where they frequently became surrounded by a double membrane of short Golgi cisternae or by one with a Golgi associated endoplasmic reticulum lysosome (GERL)-like structure. The inner membrane of the cisterna surrounding the nucleocapsids showed regional specialization which was characterized by increased thickness and electron opacity. Acid phosphatase activity, a marker for GERL or trans Golgi cisternae, appeared in the cytoplasmic short cisternae surrounding the nucleocapsids, whereas glucose-6-phosphatase activity, a marker for the nuclear envelope or for endoplasmic reticulum, was not demonstrated in such cisternae. Monoclonal antibody against glycoprotein gD revealed that gD was localized in the trans Golgi membrane as well as in the envelope of the virion. The antibody-binding sites were highly concentrated in the area where Golgi membranes showed increased opacity. Furthermore, nucleocapsids were surrounded exclusively by gD-positive cisternal (Golgi or Golgi-derived) membranes. Thus, our results indicate that the envelope of HSV is derived from trans Golgi cisterna (GERL), and that some viral components, including gD, destined for the envelope may be assembled initially in the Golgi membrane, which is thereby transformed into the envelope of the virus.     

Novel mechanism of intracellular calcium release in pituitary cells

In sea urchin eggs an enzymatic metabolite of beta-NAD+, called cyclic ADP-ribose (cADPR), is as potent and powerful a releaser of sequestered intracellular Ca2+ as is inositol 1,4,5-trisphosphate (IP3). The enzyme that synthesizes cADPR is present in several vertebrate animal tissues, but the Ca(2+)-releasing activity of cADPR has not been described in mammalian cells. We report here that incubation of beta-NAD+ with cell-free extracts of several rat tissues (including pituitary gland) generates a product which releases intracellular Ca2+ stores in permeabilized rat pituitary GH4C1 cells. This product has the biological characteristics of cADPR (it acts after depletion of the IP3 stores and after blockade of the IP3 receptor by heparin). The response is mimicked, in a concentration-dependent manner, by authentic cADPR and is desensitized by prior incubation with cADPR. We conclude that cADPR is not only synthesized by certain mammalian cells but also acts in such cells to release compartmentalized intracellular Ca2+ by a mechanism that differs from that used by IP3. Therefore, cADPR may serve, in addition to IP3, as a second messenger for intracellular Ca2+ mobilization in mammalian cells.