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Our understanding of the ways in which peptides are used for communication in the nervous and endocrine systems began with the identification of oxytocin, vasopressin and insulin, each of which is stored in electron dense granules, ready for release in response to an appropriate stimulus. For each of these peptides, entry of its newly synthesized precursor into the endoplasmic reticulum lumen is followed by transport through the secretory pathway, exposing the precursor to a sequence of environments and enzymes that produce the bioactive products stored in mature granules. A final step in the biosynthesis of many peptides is C-terminal amidation by Peptidylglycine α-Amidating Monooxygenase (PAM), an ascorbate- and copper-dependent membrane enzyme that enters secretory granules along with its soluble substrates. Biochemical and cell biological studies elucidated the highly conserved mechanism for amidated peptide production and raised many questions about PAM trafficking and the effects of PAM on cytoskeletal organization and gene expression. Phylogenetic studies and the discovery of active PAM in the ciliary membranes of Chlamydomonas reinhardtii, a green alga lacking secretory granules, suggested that a PAM-like enzyme was present in the last eukaryotic common ancestor. While the catalytic features of human and C. reinhardtii PAM are strikingly similar, the trafficking of PAM in C. reinhardtii and neuroendocrine cells and secretion of its amidated products differ. A comparison of PAM function in neuroendocrine cells, atrial myocytes and C. reinhardtii reveals multiple ways in which altered trafficking allows PAM to accomplish different tasks in different species and cell-types.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is Peptidylglycine α-Amidating Monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adultPamMyh6-cKO/cKOatria revealed a 20-fold drop in the volume fraction of secretory granules and a decrease in peripherally localized Golgi complexes. When primary cultures ofPam0-Cre-cKO/cKOatrial myocytes (PAM floxed, no Cre recombinase) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in proANP levels. Expression of exogenous PAM inPamMyh6-cKO/cKOatrial myocytes produced a dose-dependent increase in proANP content. Strikingly, rescue of proANP content did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact and its basal secretion is stimulated by drugs that inhibit Golgi-localized Arf activators. Increased basal secretion of proANP was a major contributor to its reduced levels inPamMyh6-cKO/cKOmyocytes; the inability of these drugs to inhibit basal proANP secretion byPamMyh6-cKO/cKOmyocytes revealed a role for COPI-mediated recycling of PAM to the endoplasmic reticulum. Analysis of atrial coated vesicles and the ability PAM to make fluorescently-tagged proANP accumulate in thecis-Golgi region of cells lacking secretory granules revealed a non-catalytic role for PAM in soluble cargo trafficking early in the secretory pathway.SignificanceTransmission electron microscopy of atrial cardiomyocytes revealed dense granules resembling those in endocrine cells and neurons, leading to the discovery of the natriuretic peptides stored in these granules. Subsequent studies revealed features unique to atrial granules, including high level expression of Peptidylglycine α-Amidating Monooxygenase (PAM), an enzyme required for the synthesis of many neuropeptides, but not for the synthesis of natriuretic peptides. The discovery that atrial myocytes lacking PAM are unable to produce granules and that PAM lacking its monooxygenase activity can rescue granule formation provides new information about the proANP secretory pathway. A better understanding of the unique features of atrial cell biology should provide insight into atrial fibrillation, the most common cardiac arrhythmia, atrial amyloidosis and heart failure.

Peptidylglycine alpha-amidating monooxygenase (PAM) is highly expressed in neurons and endocrine cells, where it catalyzes one of the final steps in the biosynthesis of bioactive peptides. PAM is also expressed in unicellular organisms such as Chlamydomonas reinhardtii, which do not store peptides in secretory granules. As for other granule membrane proteins, PAM is retrieved from the cell surface and returned to the trans-Golgi network. This pathway involves regulated entry of PAM into multivesicular body intralumenal vesicles (ILVs). The aim of this study was defining the endocytic pathways utilized by PAM in cells that do not store secretory products in granules. Using stably transfected HEK293 cells, endocytic trafficking of PAM was compared to that of the mannose 6-phosphate (MPR) and EGF (EGFR) receptors, established markers for the endosome to trans-Golgi network and degradative pathways, respectively. As in neuroendocrine cells, PAM internalized by HEK293 cells accumulated in the trans-Golgi network. Based on surface biotinylation, >70% of the PAM on the cell surface was recovered intact after a 4 h chase and soluble, bifunctional PAM was produced. Endosomes containing PAM generally contained both EGFR and MPR and ultrastructural analysis confirmed that all three cargos accumulated in ILVs. PAM containing multivesicular bodies made frequent dynamic tubular contacts with younger and older multivesicular bodies. Frequent dynamic contacts were observed between lysosomes and PAM containing early endosomes and multivesicular bodies. The ancient ability of PAM to localize to ciliary membranes, which release bioactive ectosomes, may be related to its ability to accumulate in ILVs and exosomes. (C) 2017 Elsevier GmbH. All rights reserved.

Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1-5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects. ispartof: BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH vol:1866 issue:9 pages:1475-1486 ispartof: location:Netherlands status: published

Peptidylglycine α-amidating monooxygenase (PAM) (EC 1.14.17.3) catalyzes peptide amidation, a crucial post-translational modification, through the sequential actions of its monooxygenase (peptidylglycine α-hydroxylating monooxygenase) and lyase (peptidyl-α-hydroxyglycine α-amidating lyase (PAL)) domains. Alternative splicing generates two different regions that connect the protease-resistant catalytic domains. Inclusion of exon 16 introduces a pair of Lys residues, providing a site for controlled endoproteolytic cleavage of PAM and the separation of soluble peptidylglycine α-hydroxylating monooxygenase from membrane-associated PAL. Exon 16 also includes two O-glycosylation sites. PAM-1 lacking both glycosylation sites (PAM-1/OSX; where OSX is O-glycan-depleted mutant of PAM-1) was stably expressed in AtT-20 corticotrope tumor cells. In PAM-1/OSX, a cleavage site for furin-like convertases was exposed, generating a shorter form of membrane-associated PAL. The endocytic trafficking of PAM-1/OSX differed dramatically from that of PAM-1. A soluble fragment of the cytosolic domain of PAM-1 was produced in the endocytic pathway and entered the nucleus; very little soluble fragment of the cytosolic domain was produced from PAM-1/OSX. Internalized PAM-1/OSX was rapidly degraded; unlike PAM-1, very little internalized PAM-1/OSX was detected in multivesicular bodies. Blue native PAGE analysis identified high molecular weight complexes containing PAM-1; the ability of PAM-1/OSX to form similar complexes was markedly diminished. By promoting the formation of high molecular weight complexes, O-glycans may facilitate the recycling of PAM-1 through the endocytic compartment.

Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is a four-membrane spanning ceramide interacting protein that regulates mTORC1 signaling. Here, we show that LAPTM4B is sorted into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) and released in small extracellular vesicles (sEVs) into conditioned cell culture medium and human urine. Efficient sorting of LAPTM4B into ILV membranes depends on its third transmembrane domain containing a sphingolipid interaction motif (SLim). Unbiased lipidomic analysis reveals a strong enrichment of glycosphingolipids in sEVs secreted from LAPTM4B knockout cells and from cells expressing a SLim-deficient LAPTM4B mutant. The altered sphingolipid profile is accompanied by a distinct SLim-dependent co-modulation of ether lipid species. The changes in the lipid composition of sEVs derived from LAPTM4B knockout cells is reflected by an increased stability of membrane nanodomains of sEVs. These results identify LAPTM4B as a determinant of the glycosphingolipid profile and membrane properties of sEVs.

The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised.

Key features for progression to pancreatic β-cell failure and disease are loss of glucose responsiveness and an increased ratio of secreted proinsulin to insulin. Proinsulin and insulin are stored in secretory granules (SGs) and the fine-tuning of hormone output requires signal mediated recruitment of select SG populations according to intracellular location and age. The GTPase Rac1 coordinates multiple signaling pathways that specify SG release and Rac1 activity is controlled in part by GDP/GTP exchange factors (GEFs). To explore the function of two large multidomain GEFs, Kalirin and Trio in β-cells, we manipulated their Rac1-specific GEF1 domain activity by using small molecule inhibitors and by genetically ablating Kalirin. We examined age related secretory granule behavior employing radiolabeling protocols. Loss of Kalirin/Trio function attenuated radioactive proinsulin release by reducing constitutive-like secretion and exocytosis of 2-hour old granules. At later chase times or at steady state, Kalirin/Trio manipulations decreased glucose stimulated insulin output. Finally, use of a Rac1 FRET biosensor with cultured β-cell lines, demonstrated that Kalirin/Trio GEF1 activity was required for normal rearrangement of Rac1 to the plasma membrane in response to glucose. Rac1 activation can be evoked by both glucose metabolism and signaling through the incretin glucagon-like peptide 1 (GLP-1) receptor. GLP-1 addition restored Rac1 localization/activity and insulin secretion in the absence of Kalirin, thereby assigning Kalirin's participation to stimulatory glucose signaling.

The adaptor protein 1A complex (AP-1A) transports cargo between the trans-Golgi network (TGN) and endosomes. In professional secretory cells, AP-1A also retrieves material from immature secretory granules (SGs). The role of AP-1A in SG biogenesis was explored using AtT-20 corticotrope tumor cells expressing reduced levels of the AP-1A μ1A subunit. A two-fold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non-condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue-stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α-amidating monooxygenase-1 (PAM-1), integral membrane enzymes that enter immature SGs. The non-condensing SGs contained POMC products and PAM-1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM-1 into PHM was unaltered, but PHM basal secretion was increased in sh-μ1A PAM-1 cells. Despite lacking a canonical AP-1A binding motif, yeast two-hybrid studies demonstrated an interaction between the PAM-1 cytosolic domain and AP-1A. Co-immunoprecipitation experiments with PAM-1 mutants revealed an influence of the luminal domains of PAM-1 on this interaction. Thus, AP-1A is crucial for normal SG biogenesis, function and composition.

Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.

Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.

The recycling of secretory granule membrane proteins that reach the plasma membrane following exocytosis is poorly understood. As a model, peptidylglycine alpha-amidating monooxygenase (PAM), a granule membrane protein that catalyzes a final step in peptide processing was examined. Ultrastructural analysis of antibody internalized by PAM and surface biotinylation showed efficient return of plasma membrane PAM to secretory granules. Electron microscopy revealed the rapid movement of PAM from early endosomes to the limiting membranes of multivesicular bodies and then into intralumenal vesicles. Wheat germ agglutinin and PAM antibody internalized simultaneously were largely segregated when they reached multivesicular bodies. Mutation of basally phosphorylated residues (Thr(946), Ser(949)) in the cytoplasmic domain of PAM to Asp (TS/DD) substantially slowed its entry into intralumenal vesicles. Mutation of the same sites to Ala (TS/AA) facilitated the entry of internalized PAM into intralumenal vesicles and its subsequent return to secretory granules. Entry of PAM into intralumenal vesicles is also associated with a juxtamembrane endoproteolytic cleavage that releases a 100-kDa soluble PAM fragment that can be returned to secretory granules. Controlled entry into the intralumenal vesicles of multivesicular bodies plays a key role in the recycling of secretory granule membrane proteins.

Rationale:The synthetic sphingosine analog FTY720 is undergoing clinical trials as an immunomodulatory compound, acting primarily via sphingosine 1-phosphate receptor activation. Sphingolipid and cholesterol homeostasis are closely connected but whether FTY720 affects atherogenesis in humans is not known.Objective:We examined the effects of FTY720 on the processing of scavenged lipoprotein cholesterol in human primary monocyte-derived macrophages.Methods and Results:FTY720 did not affect cholesterol uptake but inhibited its delivery to the endoplasmic reticulum, reducing cellular free cholesterol cytotoxicity. This was accompanied by increased levels of Niemann–Pick C1 protein (NPC1) and ATP-binding cassette transporter (ABC)A1 proteins and increased efflux of endosomal cholesterol to apolipoprotein A-I. These effects were not dependent on sphingosine 1-phosphate receptor activation. Instead, FTY720 stimulated the production of 27-hydroxycholesterol, an endogenous ligand of the liver X receptor, leading to liver X receptor–induced upregulation of ABCA1. Fluorescently labeled FTY720 was targeted to late endosomes, and the FTY720-induced upregulation of ABCA1 was NPC1-dependent, but the endosomal exit of FTY720 itself was not.Conclusions:We conclude that FTY720 decreases cholesterol toxicity in primary human macrophages by reducing the delivery of scavenged lipoprotein cholesterol to the endoplasmic reticulum and facilitating its release to physiological extracellular acceptors. Furthermore, FTY720 stimulates 27-hydroxycholesterol production, providing an explanation for the atheroprotective effects and identifying a novel mechanism by which FTY720 modulates signaling.

Oxysterol binding protein-related protein 2 (ORP2) is a member of the oxysterol binding protein family, previously shown to bind 25-hydroxycholesterol and implicated in cellular cholesterol metabolism. We show here that ORP2 also binds 22(R)-hydroxycholesterol [22(R)OHC], 7-ketocholesterol, and cholesterol, with 22(R)OHC being the highest affinity ligand of ORP2 (K(d) 1.4 x 10(-8) M). We report the localization of ORP2 on cytoplasmic lipid droplets (LDs) and its function in neutral lipid metabolism using the human A431 cell line as a model. The ORP2 LD association depends on sterol binding: Treatment with 5 microM 22(R)OHC inhibits the LD association, while a mutant defective in sterol binding is constitutively LD bound. Silencing of ORP2 using RNA interference slows down cellular triglyceride hydrolysis. Furthermore, ORP2 silencing increases the amount of [(14)C]cholesteryl esters but only under conditions in which lipogenesis and LD formation are enhanced by treatment with oleic acid. The results identify ORP2 as a sterol receptor present on LD and provide evidence for its role in the regulation of neutral lipid metabolism, possibly as a factor that integrates the cellular metabolism of triglycerides with that of cholesterol.

Secretory granules carrying fluorescent cargo proteins are widely used to study granule biogenesis, maturation, and regulated exocytosis. We fused the soluble secretory protein peptidylglycine α-hydroxylating monooxygenase (PHM) to green fluorescent protein (GFP) to study granule formation. When expressed in AtT-20 or GH3 cells, the PHM-GFP fusion protein partitioned from endogenous hormone (adrenocorticotropic hormone, growth hormone) into separate secretory granule pools. Both exogenous and endogenous granule proteins were stored and released in response to secretagogue. Importantly, we found that segregation of content proteins is not an artifact of overexpression nor peculiar to GFP-tagged proteins. Neither luminal acidification nor cholesterol-rich membrane microdomains play essential roles in soluble content protein segregation. Our data suggest that intrinsic biophysical properties of cargo proteins govern their differential sorting, with segregation occurring during the process of granule maturation. Proteins that can self-aggregate are likely to partition into separate granules, which can accommodate only a few thousand copies of any content protein; proteins that lack tertiary structure are more likely to distribute homogeneously into secretory granules. Therefore, a simple “self-aggregation default” theory may explain the little acknowledged, but commonly observed, tendency for both naturally occurring and exogenous content proteins to segregate from each other into distinct secretory granules.

Hormone secretion by pituitary cells is decreased by roscovitine, an inhibitor of cyclin-dependent kinase 5 (Cdk5). Roscovitine treatment reorganizes cortical actin and ultrastructural analysis demonstrates that roscovitine limits the ability of secretory granules to approach the plasma membrane or one another. Trio, a multifunctional RhoGEF expressed in pituitary cells, interacts with peptidylglycine α-amidating monooxygenase, a secretory granule membrane protein known to affect the actin cytoskeleton. Roscovitine inhibits the ability of Trio to activate Rac, and peptides corresponding to the Cdk5 consensus sites in Trio are phosphorylated by Cdk5. Together, these data suggest that control of the cortical actin cytoskeleton, long known to modulate hormone exocytosis and subsequent endocytosis, involves Cdk5-mediated activation of Trio.

A role for heterotrimeric G proteins in the regulation of Golgi function and formation of secretory granules is generally accepted. We set out to study the effect of activation of heterotrimeric G proteins by aluminum fluoride on secretory granule formation in AtT-20 corticotropic tumor cells and in melanotrophs from the rat pituitary. In AtT-20 cells, treatment with aluminum fluoride or fluoride alone for 60 min induced complete dispersal of Golgi, ER-Golgi intermediate compartment and Golgi matrix markers, while betaCOP immunoreactiviy retained a juxtanuclear position and TGN38 was unaffected. Electron microscopy showed compression of Golgi cisternae followed by conversion of the Golgi stacks into clusters of tubular and vesicular elements. In the melanotroph of the rat pituitary a similar compression of Golgi cisternae was observed, followed by a progressive loss of cisternae from the stacks. As shown in other cells, brefeldin A induced redistribution of the Golgi matrix protein GM130 to punctate structures in the cytoplasm in AtT-20 cells, while mannosidase II immunoreactivity was completely dispersed. Fluoride induced a complete dispersal of mannosidase II and GM130 immunoreactivity. The effect of fluoride was fully reversible with reestablishment of normal mannosidase II and GM130 immunoreactivity within 2 h. After 1 h of recovery, showing varying stages of reassembly, the patterns of mannosidase II and GM130 immunoreactivity were identical in individual cells, indicating that Golgi matrix and cisternae reassemble with similar kinetics during recovery from fluoride treatment. Instead of a specific aluminum fluoride effect on secretory granule formation in the trans-Golgi network, we thus observe a unique form of Golgi dispersal induced by fluoride alone, possibly via its action as a phosphatase inhibitor.

Peptidylglycine-α-amidating monooxygenase (PAM) is a bi-functional enzyme known to catalyze the post-translational bioactivation of signaling peptides. Although PAM is highly concentrated within the cardiac atrium, this tissue does not produce appreciable amounts of α-amidated peptides and thus, the function of PAM in atrium remains largely unknown. In this study, we demonstrate that PAM co-localizes in atrial secretory granules with the storage form of atrial natriuretic peptide (pro-ANP, amino acids 1–126), a hormone involved in the maintenance of blood pressure and fluid homeostasis. ANP is not amidated by PAM, but rather is processed to its active form (amino acids 99–126) by the proteolytic cleavage of pro-ANP. We demonstrate here by subcellular fractionation and biochemical analyses that PAM co-localizes with pro-ANP in secretory granules, where together they constitute the two most abundant membrane-associated proteins, accounting for approximately 95% of the total granular membrane protein. Respectively, light and electron microscopic immunohistochemistry show intense staining for PAM in atrial cardiomyocyctes and subcellular localization of PAM to secretory granules. Additionally, we demonstrate that while pro-ANP is readily found in the soluble contents of the granule lumen, significant amounts remain tightly associated with the membranes even after vigorous washing and estimate the molar ratio of pro-ANP to PAM to be approximately 30:1 in the membrane fraction. We postulate here that the primary function of PAM in the atrium is structural rather than enzymatic. In this regard, PAM may contribute to the packaging of pro-ANP within the secretory granule and possibly function in the presentation of pro-ANP for proteolytic processing.

Lysosome-associated protein transmembrane-4b (LAPTM4B) associates with poor prognosis in several cancers, but its physiological function is not well understood. Here we use novel ceramide probes to provide evidence that LAPTM4B interacts with ceramide and facilitates its removal from late endosomal organelles (LEs). This lowers LE ceramide in parallel with and independent of acid ceramidase-dependent catabolism. In LAPTM4B-silenced cells, LE sphingolipid accumulation is accompanied by lysosomal membrane destabilization. However, these cells resist ceramide-driven caspase-3 activation and apoptosis induced by chemotherapeutic agents or gene silencing. Conversely, LAPTM4B overexpression reduces LE ceramide and stabilizes lysosomes but sensitizes to drug-induced caspase-3 activation. Together, these data uncover a cellular ceramide export route from LEs and identify LAPTM4B as its regulator. By compartmentalizing ceramide, LAPTM4B controls key sphingolipid-mediated cell death mechanisms and emerges as a candidate for sphingolipid-targeting cancer therapies.

The luminal domains of membrane peptidylglycine α-amidating monooxygenase (PAM) are essential for peptide α-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.

N-myc downstream-regulated gene 1 (NDRG1) mutations cause Charcot-Marie-Tooth disease type 4D (CMT4D). However, the cellular function of NDRG1 and how it causes CMT4D are poorly understood. We report that NDRG1 silencing in epithelial cells results in decreased uptake of low-density lipoprotein (LDL) due to reduced LDL receptor (LDLR) abundance at the plasma membrane. This is accompanied by the accumulation of LDLR in enlarged EEA1-positive endosomes that contain numerous intraluminal vesicles and sequester ceramide. Concomitantly, LDLR ubiquitylation is increased but its degradation is reduced and ESCRT (endosomal sorting complex required for transport) proteins are downregulated. Co-depletion of IDOL (inducible degrader of the LDLR), which ubiquitylates the LDLR and promotes its degradation, rescues plasma membrane LDLR levels and LDL uptake. In murine oligodendrocytes, Ndrg1 silencing not only results in reduced LDL uptake but also in downregulation of the oligodendrocyte differentiation factor Olig2. Both phenotypes are rescued by co-silencing of Idol, suggesting that ligand uptake through LDLR family members controls oligodendrocyte differentiation. These findings identify NDRG1 as a novel regulator of multivesicular body formation and endosomal LDLR trafficking. The deficiency of functional NDRG1 in CMT4D might impair lipid processing and differentiation of myelinating cells.

This article reviews the neuroanatomical studies on the distribution of nitric oxide synthase (NOS) in neurons and nerve fibers innervating the submandibular gland. Specificity of NADPH-diaphorase activity as a histochemical marker of neuronal NOS is discussed in light of corresponding NOS immunoreactivity. Anatomical data suggest that nitric oxide may affect neural regulation of the submandibular gland through both sympathetic, parasympathetic and sensory divisions of the autonomic nervous system. NOS-containing nerve terminals in the gland parenchyme are mainly vascular and either parasympathetic and/or sensory in nature, while sympathetic terminals lack NOS. Most postganglionic parasympathetic neurons are intensely NOS-immunoreactive. Some of the preganglionic parasympathetic neurons show vague reactivity, while their terminals in the submandibular ganglia stain heavily. The postganglionic sympathetic neurons normally show only barely visible reactivity, while manipulations interrupting axonal continuity increase neuronal NOS content. A subpopulation of the preganglionic sympathetic neurons and their terminals are intensely reactive. The observations summarized here suggest that nitric oxide participates in the control of blood flow through the gland, while direct effect on secretion is unlikely.

The effect of monensin on the Golgi complex, formation of secretory granules and basal beta-endorphin secretion in cultured melanotrophs from the rat pituitary was studied. Earlier studies on the effect of monensin on regulated secretion have generally showed only minor effects on secretory granules. The initial (within 5 min) effect of monensin on the melanotroph was the appearance of large vacuoles at the trans-side of the thiamine pyrophosphatase-positive trans-most Golgi cisternae. This was associated with a dose-dependent inhibition of the condensation of electron-dense secretory products. After 1 h of treatment with 1 microM monensin the Golgi stack was completely vacuolized. At the same time mature secretory granules were enlarged to severalfold their original size, and after 4 h of treatment secretory granules were no longer observed. Despite the marked effects on granule formation and mature secretory granules monensin did not affect the basal release of beta-endorphin-immunoreactive material during continued incubation for up to 4 h, indicating that basal peptide secretion can bypass the monensin block.

The distribution of neurofilament proteins and peripherin in the pituitary gland of the rat was studied with a panel of monoclonal and polyclonal antibodies recognizing different neurofilament subunits. In the posterior lobe, a dense plexus of neurofilament- and peripherin-immunoreactive fibers was seen. In the intermediate lobe neurofilament- and peripherin-immunoreactivity was seen only in nerve fibers in the connective tissue septa, while no immunoreactivity was seen in parenchymal nerve fibers. Bilateral sympathetic ganglionectomy did not affect peripherin-immunoreactivity, indicating that the peripherin-immunoreactive fibers are of central origin. In the anterior lobe, a few solitary neurofilament- and peripherin-immunoreactive fibers were observed. Western blotting confirmed the presence of 150 kD and 200 kD neurofilament proteins in the posterior lobe. No neurofilament protein was detected in the intermediate and anterior lobes. Abundant intermediate filaments were seen with electron microscopy in the nerve fibers of the connective tissue septa in the intermediate lobe. In the parenchymal nerve fibers only microtubules were seen, indicating that the lack of neurofilament immunoreactivity is due to absence of neurofilaments.

The effect of vagotomy and pyloroplasty on the density of nerve fibers containing bombesin/gastrin-releasing peptide (GRP), calcitonin gene-related peptide (CGRP), and galanin as well as histamine-, 5-hydroxytryptamine (5-HT)-, and somatostatin-containing cells in the oxyntic mucosa of the rat stomach was studied. Ten days after vagotomy and pyloroplasty the density of histamine-containing cells in the oxyntic mucosa was increased by 70% (P

We characterize here ORP11, a member of the oxysterol-binding protein family. ORP11 is present at highest levels in human ovary, testis, kidney, liver, stomach, brain, and adipose tissue. Immunohistochemistry demonstrates abundant ORP11 in the epithelial cells of kidney tubules, testicular tubules, caecum, and skin. ORP11 in HEK293 cells resides on Golgi complex and LE, co-localizing with GFP-Rab9, TGN46, GFP-Rab7, and a fluorescent medial-trans-Golgi marker. Under electron microscopic observation, cells overexpressing ORP11 displayed lamellar lipid bodies associated with vacuolar structures or the Golgi complex, indicating a disturbance of lipid trafficking. N-terminal fragment of ORP11 (aa 1-292) localized partially to Golgi, but displayed enhanced localization on Rab7- and Rab9-positive LE, while the C-terminal ligand-binding domain (aa 273-747) was cytosolic, demonstrating that the membrane targeting determinants are N-terminal. Yeast two-hybrid screen revealed interaction of ORP11 with the related ORP9. The interacting region was delineated within aa 98-372 of ORP9 and aa 154-292 of ORP11. Overexpressed ORP9 was able to recruit EGFP-ORP11 to membranes, and ORP9 silencing inhibited ORP11 Golgi association. The results identify ORP11 as an OSBP homologue distributing at the Golgi-LE interface and define the ORP9-ORP11 dimer as a functional unit that may act as an intracellular lipid sensor or transporter.

The vacuolar H+-ATPase (V-ATPase) establishes pH gradients along secretory and endocytic pathways. Progressive acidification is essential for proteolytic processing of prohormones and aggregation of soluble content proteins. The V-ATPase V0 subunit is thought to have a separate role in budding and fusion events. Prolonged treatment of professional secretory cells with selective V-ATPase inhibitors (bafilomycin A1, concanamycin A) was used to investigate its role in secretory-granule biogenesis. As expected, these inhibitors eliminated regulated secretion and blocked prohormone processing. Drug treatment caused the formation of large, mixed organelles, with components of immature granules and lysosomes and some markers of autophagy. Markers of the trans-Golgi network and earlier secretory pathway were unaffected. Ammonium chloride and methylamine treatment blocked acidification to a similar extent as the V-ATPase inhibitors without producing mixed organelles. Newly synthesized granule content proteins appeared in mixed organelles, whereas mature secretory granules were spared. Following concanamycin treatment, selected membrane proteins enter tubulovesicular structures budding into the interior of mixed organelles. shRNA-mediated knockdown of the proteolipid subunit of V0 also caused vesiculation of immature granules. Thus, V-ATPase has a role in protein sorting in immature granules that is distinct from its role in acidification.

The effect of a 4-min secretory stimulation with 10(-6) M isoproterenol on the peripheral cytoplasmic zone of intermediate lobe cells of the rat pituitary incubated in vitro was studied. Two widths of the peripheral zone, a 450 nm zone and a 250 nm zone, were analyzed with electron-microscopical morphometry. Isoproterenol stimulation did not induce any significant effect on the number of electron-dense secretory granules contained within the 250 or the 450 nm zone. A 50% decrease was, however, noted in the number of electron-lucent granules situated in immediate association with the plasma membrane. The majority of the transection diameters of electron-dense granules in the peripheral zone of control cells fell within a range of 135-230 nm. A relative increase in the number of transections over 230 nm was noted after isoproterenol stimulation. In conclusion, isoproterenol stimulation induced a preferential release of electron-lucent granules from the peripheral cytoplasmic zone of the intermediate lobe cells, indicating that this subpopulation of granules represents the immediately releasable secretory pool of these cells. In addition, large electron-dense granules appeared in the peripheral zone during isoproterenol stimulation.

We have investigated the role of the protein ubiquitous mitochondrial creatine kinase (uMtCK) in the formation and stabilization of inner and outer membrane contact sites. Using liver mitochondria isolated from transgenic mice, which, unlike control animals, express uMtCK in the liver, we found that the enzyme was associated with the mitochondrial membranes and, in addition, was located in membrane-coated matrix inclusions. In mitochondria isolated from uMtCK transgenic mice, the number of contact sites increased 3-fold compared with that observed in control mitochondria. Furthermore, uMtCK-containing mitochondria were more resistant to detergent-induced lysis than wild-type mitochondria. We conclude that octameric uMtCK induces the formation of mitochondrial contact sites, leading to membrane cross-linking and to an increased stability of the mitochondrial membrane architecture.

The pathogenetic processes underlying schizophrenia are unknown, but the organic changes observed in the brain tissue of schizophrenic patients suggest the possibility of findings also in the cerebrospinal fluid (CSF) of patients suffering from this disorder. In a recently published study micrometer-sized spherical particles in CSF were isolated from significantly more schizophrenic patients compared to controls. The functional importance of these particles is unknown, but if replicated in independent study samples they could function as trait markers and spur further investigations. We analyzed CSF samples from five patients with a diagnosis of schizophrenia, nine patients with chronic pain syndrome and six healthy controls with scanning electron microscopy in accordance with the protocol described in the original report. Similar particles were identified as in the previous study, but they were not associated with disease status (P=1.00). It cannot be excluded that these divergent results are due to methodological differences, but presently the most parsimonious explanation would be that the original finding was a false positive result. More investigation is needed to clarify the source and relevance of these particles.

Monensin has been shown to cause nonexocytotic release of catecholamines from adrenal medullary and PC12 cells. We examined the effect of monensin on peptide secretion with cultured melanotropes from the rat pituitary as a model. 1 µM monensin caused an immediate, transient increase in β-endorphin secretion. The effect was still seen in a calcium-free medium, but was totally abolished in a sodium-free medium. Intracellular calcium concentration was measured with Fura 2: no increase was observed during monensin stimulation. Hypo-osmolar medium mimicked the effect of monensin, causing a 12-fold transient increase in β-endorphin secretion. This effect was not abolished in either calcium-free or sodium-free medium. No increase in the number of exocytotic figures captured by tannic acid incubation was observed during 5 min of incubation with 1 µM monensin or hypo-somolar medium. We thus show that monensin causes β-endorphin secretion from the melanotrope and that this effect is due to sodium influx and resultant cell swelling. The calcium independency and lack of increase of exocytotic figures suggest that swelling-induced secretion is nonexocytotic, possibly via transient exocytotic pore opening.

The production and regulated secretion of bioactive peptides require a series of lumenal enzymes to convert inactive precursors into bioactive peptides plus several cytosolic proteins to govern granule formation, maturation, translocation, and exocytosis. Peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme essential for biosynthesis of many peptides, is an integral membrane protein with trafficking information in both its lumenal and cytosolic domains. Kalirin, a PAM cytosolic domain interactor protein with spectrin-like repeats and GDP/GTP exchange factor activity for Rac1, is expressed with PAM in neurons but is not expressed in the anterior pituitary or AtT-20 corticotrope cells. Expression of Kalirin alters the cytoskeletal organization of Chinese hamster ovary and AtT-20 cells expressing membrane PAM. Expression of membrane PAM also alters cytoskeletal organization, demonstrating the presence of endogenous proteins that can mediate this effect. Significant amounts of both PAM and Kalirin fractionate with cytoskeletal elements. Since cytoskeletal organization is critical for exocytosis, constitutive-like and regulated secretions were evaluated. Whereas the constitutive-like secretion of adrenocorticotropic hormone (ACTH) is increased by expression of membrane PAM, regulated secretion is eliminated. Expression of Kalirin in AtT-20 cells expressing membrane PAM restores stimulated secretion of ACTH. Thus, Kalirin or its homologue may be essential for regulated secretion, and the PAM-Kalirin interaction may coordinate intragranular with cytosolic events.

The amount of colonic substance P and substance P-receptors is increased in ulcerative colitis, which may denote that substance-P is involved as a neurogenic mediator in the inflammatory process of ulcerative colitis. We studied the anatomical distribution of elevated colonic substance P in ulcerative colitis and assessed morphometrically whether the changes in substance P correlate with alterations in colonic innervation. Full-thickness specimens of colonic wall were obtained from normal human colons (N = 9) and the most and least affected regions of ulcerative colitis colons (N = 10) and immunostained for substance P. Substance P immunoreactivity index was calculated by multiplying each intensity value by the number of pixels exhibiting this intensity value. The numbers of substance P-immunoreactive nerve fibers in the lamina propria were markedly increased, and their fluorescence intensity was enhanced in ulcerative colitis. The longitudinal muscle layer contained substance P-immunoreactive nerve fibers in ulcerative colitis, but not in the controls. The substance P-immunoreactive index (= number x intensity of nerve fibers) was 3.42 +/- 1.49 in controls, 21.19 +/- 7.79 in mild ulcerative colitis regions (P0.05), and 29.68 +/- 9.81 in severe ulcerative colitis regions (P0.01). Increase in the number of substance P nerve fibers is in accordance with the hypothesis that substance P contributes to neurogenic mediation of inflammation in ulcerative colitis.

Local production of catecholamines in the stomach of the rat was studied by immunohistochemical demonstration of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), the enzymes catalyzing the formation of dopamine, noradrenaline and adrenaline, respectively. A rich innervation of TH- and DBH-immunoreactive nerve fibers was seen in the muscular layers and the myenteric plexus, in the submucosa and in the walls of submucosal blood vessels and in the lamina propria at the base of the epithelial layer. In addition, TH-, but not DBH-immunoreactive nerve fiber networks surrounding ganglion cells in the myenteric plexus were frequently observed, indicating dopaminergic preganglionic innervation of the myenteric plexus. In the oxyntic epithelium, single TH- and DBH-immunoreactive fibers extended in the strands of lamina propria as far as the middle portion of the gastric glands. A small population of single angulate cells in the oxyntic epithelium showed TH-, but not DBH-immunoreactivity. No specific PNMT immunoreactivity was observed.

The nerve terminals in the intermediate and posterior lobes of the rat pituitary gland are reported to show colocalization of serotonin and tyrosine hydroxylase. This study examined the extent of this colocalization in the pituitary gland and in the nuclei considered to project to the pituitary. In the intermediate lobe, two types of nerve fibers were encountered, one containing serotonin (5-HT-IR) and tyrosine hydroxylase (TH-IR) immunoreactivities and the other showing 5-HT-IR only. Instead, there was no colocalization in the posterior lobe. In the hypothalamus, colchicine treatment with l -tryptophan and pargyline injections resulted in 5-HT-IR in some neurons in the dorsomedial, periventricular and arcuate nuclei, some of which in the arcuate and periventricular nuclei were also TH-IR. In the raphe nuclei no colocalization of 5-HT-IR and TH-IR was observed. Catecholamine neurotoxin, 6-hydroxydopamine, abolished the 5-HT-IR and dramatically reduced the TH-IR in the intermediate lobe nerve fibers. Both effects were prevented by cocaine, a monoamine uptake inhibitor, but not by fluoxetine, a specific serotonin uptake inhibitor. Serotonin neurotoxin p-chloroamphetamine (PCA) had no effect on intermediate lobe fibers, although it caused complete disappearance of 5-HT-IR from the posterior lobe nerve fibers. This effect was prevented by fluoxetine. Our results indicate, that colocalization of serotonin and TH observed in the intermediate lobe occurs both in the nerve terminals within the lobe and in some nuclei that innervate it. Furthermore, drug treatments suggest that serotonin in the intermediate lobe is localized in catecholaminergic fibers, which do not posses a specific serotonin uptake mechanism.

The formation of secretory granules in chronically hypersecretory melanotrophs in the rat pituitary was studied. Hypersecretion was induced by treatment with the dopamine antagonist haloperidol (1.5 mg/kg daily for 7 days), which releases the normal neural dopaminergic inhibition of secretion from the melanotroph. Morphometric analysis showed a 100% increase in the volume fraction of granular endoplasmic reticulum after haloperidol treatment, while the volume fractions of electron-dense granules, electron-lucent granules and the Golgi apparatus were unaltered. The mean diameter of the mature secretory granules was increased by 10%, indicating a 30% increase in mean granule volume. A similar increase in diameter was observed in condensing granules within the Golgi area. With earlier results on the effect of chronic inhibition the study shows that a main adaptive response of the melanotroph to altered secretory conditions is a change in the volume of the secretory granules, regulated by a mechanism that operates at an early stage of granule formation.

The internalization of the extracellular markers horseradish peroxidase (HRP) and cationized ferritin (CF) by the melanotrophs of the intermediate lobe of the rat pituitary was studied during short-time incubation of mechanically dissociated cells or in cell culture after 5 days. After a 30 min exposure, the tracers were found in electron-lucent granules or vacuoles of approximately the same size as the secretory granules, situated 200–500 nm from the cell membrane. In the cultured cells, which showed a higher rate of tracer uptake, internalization was followed for 1, 2 and 5 min after labelling and during 2 h of exposure. Initially, the label was seen only in coated pits and coated vesicles at the cell membrane. Larger vacuoles were first seen after 2–5 min of incubation. After 2 h of exposure the labelling pattern was distinctly different for the two tracers. CF was found in larger vacuoles of varying morphology, in dilatations at the base of cilia, within Golgi saccules and at the edge of the electron-dense core of forming secretory granules. HRP was found in an extensive array of tubulovesicular structures extending throughout the cytoplasm. The Golgi complex and forming granules were, however, not labelled with HRP. The study identifies part of the electron-lucent granules or vacuoles in the melanotroph as endosomes, and shows that the melanotrophs sort CF and HRP via diverting pathways after internalization, suggesting that granule membrane, and possibly its functional components, can be recycled in these cells.

The role of the preepithelial mucus-HCO-3 layer in protection against intracellular acidosis was investigated in isolated Necturus gastric antral mucosa exposed to luminal acid by simultaneous measurement of intracellular pH (pH(i)) and extracellular surface pH (pHs) in surface epithelium with microelectrode technique. Acidification of the luminal perfusate to pH 2.5 acidified pH(i) in surface epithelial cells from 7.33 +/- 0.02 to 7.20 +/- 0.04, whereas pHs fell from 6.75 +/- 0.21 to 5.20 +/- 0.25 (P < 0.01; n = 9), followed by a steady state for at least 2 h. Inhibition of epithelial HCO-3 secretion and transport by removal of serosal HCO-3 and CO2 (HEPES and O2 substitution) during acid exposure provoked a progressive acidification of pHs from 5.60 +/- 0.41 to 2.74 +/- 0.14 in 30 min (P < 0.01; n = 9), which was accompanied, after a 5- to 10-min delay, by acidification of pH(i) from 7.21 +/- 0.03 to 5.68 +/- 0.26 (P < 0.01). Digestion of the surface mucus gel by pepsin (5% wt/vol) at pH 2.5 caused a slow acidification of pHs from 5.22 +/- 0.59 to 3.60 +/- 0.46 within 2 h. This was followed by a more rapid acidification to 2.53 +/- 0.38 (P < 0.01; n = 7), with concomitant acidification of pH(i) from 7.19 +/- 0.05 to 6.03 +/- 0.33 (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

A phenylethanolamine N-methyltransferase-(PNMT)-immunoreactivity, present without the other catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), has been previously detected in the central nervous system and in endocrine cells of the islets of Langerhans and the pituitary intermediate lobe of the rat. In the present study a similar PNMT-like immunoreactivity is demonstrated in the rat parathyroid gland. The immunoreactivity was distinctly localized to the cell periphery, and present in all glandular cells. The thyroid gland was negative. In the parathyroid TH- and DBH-immunoreactivity was seen only in vascular nerve fibers; no glandular cells were stained. The functional significance of the PNMT-like immunoreactivity is not known. The absence of TH- and DBH-immunoreactivity and the low level of adrenaline detected in the parathyroid, and the peripheral localization of the immunoreactivity may indicate an alternative enzyme function or the detection of an immunologically related protein common to pancreatic, pituitary and parathyroid endocrine cells.

The relationship between the intensity of combined formaldehyde-chloral vapour-induced fluorescence and the concentration of amino-terminal tryptophyl-peptide in model experiments was found to be non-linear. At a certain concentration the intensity began to increase more slowly than the concentration, and when the concentration further increased the intensity even began to decrease. Based on the studies previously reported and on the above findings it seems that fluorescence induced by combined formaldehyde-chloral vapour, glyoxylic acid vapour and possibly also other combined formaldehyde and carbonyl compounds in the hypophyseal cells containing amino-terminal tryptophyl-peptides is quenched in normal conditions due to the high local concentration. Thus, small to moderate changes in the amounts of amino-terminal tryptophyl-peptides cannot be observed by measuring the fluorescence intensity. In tissue experiments the intensity of combined formaldehyde-chloral vapour-induced fluorescence in the rat pars intermedia was measured after reserpine treatment, which decreases the number of hormone storage granules as demonstrated electron microscopically. The fluorescence intensity measurements were combined with an estimation of the amounts of amino-terminal tryptophyl-peptides extracted from hypophyses and separated in thin-layer chromatography, and subsequently demonstrated by combined formaldehyde-chloral vapour and a protein stain (amido black). Reserpine treatment decreased the fluorescence intensity in the pars intermedia and in thin-layer chromatography, and the staining of the fluorescent band with amido black was also decreased. Amino-terminal tryptophyl-peptides appeared to be depleted from the pars intermedia cells together with endorphins and other hormones of the ACTH/MSH cells containing tryptophan.

The cells of the mammalian adenohypophysis store a substance, probably a tryptophyl-peptide, which exhibits formaldehyde-induced fluorescence (FIF). The fluorescence intensity and the number of cells in different parts of the adenohypophysis of human fetus or rat were studied after the following treatments: formaldehyde vapour alone, acidification of the formaldehyde-treated sections with hydrochloric acid or with glacial acetic acid, acetyl chloride vapour alone, as well as combined formaldehyde and acetyl chloride vapour.

The catecholamine-containing nerve fibers of the rat pituitary were studied by immunohistochemical demonstration of the catecholamine-synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Immunohistochemical demonstration of TH confirms earlier catecholamine fluorescence histochemical studies showing a fine network of varicose fibers in both the intermediate and the neural lobe, with the most dense aggregation of fibers at the border between the lobes. DBH-immunoreactive fibers were much less in number, and confined to the neural lobe, where both vascular and parenchymal fibers were seen. With the antibody to PNMT bright staining was seen in all the glandular cells of the intermediate lobe, while the neural lobe was negative. No immunoreactive structures were observed in the anterior lobe. Functionally the study confirms the presence of an extensive dopaminergic innervation of the neurointermediate lobe, giving an anatomical basis for the tonic inhibitory action of dopamine on the intermediate lobe cells and for recent observations attributing dopamine a local regulatory function also in the neural lobe. In addition to vascular noradrenaline-containing fibers as described earlier the study shows parenchymal DBH-immunoreactive fibers in the neural lobe, suggesting a local role for noradrenaline in this lobe. The nature of the cellular PNMT-immunoreactivity in the intermediate lobe remains to be established. The cellular localization of the PNMT-immunoreactivity was distinctly different than that of the alpha-MSH-immunoreactivity within the intermediate lobe cells and reserpine treatment did not affect the PNMT-immunoreactivity although it induced a heterogeneous depletion of alpha-MSH and related peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

The normal femoral artery and its branches were found to be innervated with a dense network of adrenergic nerves. The nerve plexus around the vein was sparse. Adventitial stripping of the femoral vessels, with or without division and reanastomosis, caused local disappearance of catecholamine fluorescence in the stripped area. The distal adrenergic innervation remained normal if the femoral nerve was left intact. Division of the femoral nerve, alone or in combination with blood vessel division and reanastomosis, caused total disappearance of catecholamine fluorescence from the adrenergic nerves of the entire distal neurovascular tree examined. At the end of the observation period of 36 weeks from the time of division of the nerve, artery and vein with subsequent microvascular anastomosis, numerous adrenergic nerves were observed to have crossed the suture line. The vascular nerve plexus around the femoral vessels was dense in places, but in other places sparse or absent. It seems that the reinnervation occurs not only over the suture line, but also together with other regenerating nerves from the adjacent tissues and by collateral sprouting from adjacent adrenergically normally innervated areas.

The ultrastructure of the incubated intermediate lobe of the rat pituitary and the morphological effect of isoproterenol stimulation on its cells were studied under in vitro conditions. The general structure of isolated neurointermediate lobes maintained for 2-3 h in vitro was well preserved, and the presence of intact nerve terminals establishing synaptic contacts with the glandular cells of the intermediate lobe was confirmed. Removal of the intermediate lobe from central inhibition leads to increased hormonal secretion, which was reflected by large Golgi areas and the appearance of secretory images. However, no obvious degranulation or peripheral migration of the secretory granules after 2-3 h in vitro was seen. The secretory granules varied in electron density; totally electron-lucent granules were regularly observed and exocytotic phenomena were shown. In addition, more extensive invaginations suggesting secretion by compound exocytosis were seen. A three-fold increase in the beta-endorphin secretion during a 4-min stimulation with 10(-6) M isoproterenol did not induce any morphometrically detectable changes in the incubated cells. This indicates that only a minor fraction of the total granule content is mobilized during an acute increase in secretory activity.