Biosynthesis and secretion of pituitary hormones: dynamics and regulation

Production and secretion of hormones by the pituitary involve highly orchestrated intracellular transport and sorting steps. Hormone precursors are routed through a series of compartments before being packaged in secretory granules. These highly dynamic carriers play crucial roles in both prohormone processing and peptide exocytosis. We have employed the ACTH-secreting AtT-20 cell line to study the membrane sorting events that confer functionality (prohormone activation and regulated exocytosis) to these secretory carriers. The unique ability of granules to promote prohormone processing is attributed to their acidic interior. Using a novel avidin-targeted fluorescence ratio imaging technique, we have found that the trans-Golgi of live AtT-20 cells maintains a mildly acidic (approximately pH 6.2) interior. Budding of secretory granules causes the lumen to acidify to 相似文献   

Accessory subunit Ac45 controls the V-ATPase in the regulated secretory pathway

The vacuolar (H(+))-ATPase (V-ATPase) is crucial for multiple processes within the eukaryotic cell, including membrane transport and neurotransmitter secretion. How the V-ATPase is regulated, e.g. by an accessory subunit, remains elusive. Here we explored the role of the neuroendocrine V-ATPase accessory subunit Ac45 via its transgenic expression specifically in the Xenopus intermediate pituitary melanotrope cell model. The Ac45-transgene product did not affect the levels of the prohormone proopiomelanocortin nor of V-ATPase subunits, but rather caused an accumulation of the V-ATPase at the plasma membrane. Furthermore, a higher abundance of secretory granules, protrusions of the plasma membrane and an increased Ca(2+)-dependent secretion efficiency were observed in the Ac45-transgenic cells. We conclude that in neuroendocrine cells Ac45 guides the V-ATPase through the secretory pathway, thereby regulating the V-ATPase-mediated process of Ca(2+)-dependent peptide secretion.     

Ca(2+) and H+ homeostasis in fission yeast: a role of Ca(2+)/H+ exchange and distinct V-H+-ATPases of the secretory pathway organelles

We determined the H+ and Ca(2+) uptake by fission yeast membranes separated on sucrose gradient and found that (i) Ca(2+) sequestering is due to Ca(2+)/H+ antiporter(s) localized to secretory pathway organelles while Ca(2+)-ATPase activity is not detectable in their membranes; (ii) immunochemically distinct V-H+-ATPases acidify the lumen of the secretory pathway organelles. The data indicate that the endoplasmic reticulum, Golgi and vacuole form a network of Ca(2+) and H+ stores in the single cell, providing favorable conditions for such key processes as protein folding/sorting, membrane fusion, ion homeostasis and Ca(2+) signaling in a differential and local manner.     

Localization of three types of the inositol 1,4,5-trisphosphate receptor/Ca(2+) channel in the secretory granules and coupling with the Ca(2+) storage proteins chromogranins A and B

Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular Ca(2+) store and the presence of the IP(3) receptor (IP(3)R)/Ca(2+) channel on the secretory granule membrane have been established, the identity of the IP(3)R types present in the secretory granules is not known. We have therefore investigated the presence of different types of IP(3)R in the secretory granules of bovine adrenal medullary chromaffin cells using immunogold electron microscopy and found the existence of all three types of IP(3)R in the secretory granules. To determine whether these IP(3)Rs interact with CGA and CGB, each IP(3)R isoform was co-transfected with CGA or CGB into NIH3T3 or COS-7 cells, and the expressed IP(3)R isoform and CGA or CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP(3)R form complexes with CGA and CGB in the cells. To further confirm whether the IP(3)R isoforms and CGA and CGB form a complex in the secretory granules the potential interaction between all three isoforms of IP(3)R and CGA and CGB was tested by co-immunoprecipitation experiments of the mixture of secretory granule lysates and the granule membrane proteins. The three isoforms of IP(3)R were shown to form complexes with CGA and CGB, indicating the complex formation between the three isoforms of IP(3)R and CGA and CGB in the secretory granules. Moreover, the pH-dependent Ca(2+) binding property of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol of Ca(2+)/mol with a dissociation constant (K(d)) of 1.5 mm at pH 5.5 but virtually no Ca(2+) at pH 7.5. The high capacity, low affinity Ca(2+)-binding property of CGB at pH 5.5 is comparable with that of CGA and is in line with its role as a Ca(2+) storage protein in the secretory granules.     

Calcium dynamics in the secretory granules of neuroendocrine cells

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

New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells.

The GLP-1 receptor is a Class B heptahelical G-protein-coupled receptor that stimulates cAMP production in pancreatic beta-cells. GLP-1 utilizes this receptor to activate two distinct classes of cAMP-binding proteins: protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). Actions of GLP-1 mediated by PKA and Epac include the recruitment and priming of secretory granules, thereby increasing the number of granules available for Ca(2+)-dependent exocytosis. Simultaneously, GLP-1 promotes Ca(2+) influx and mobilizes an intracellular source of Ca(2+). GLP-1 sensitizes intracellular Ca(2+) release channels (ryanodine and IP (3) receptors) to stimulatory effects of Ca(2+), thereby promoting Ca(2+)-induced Ca(2+) release (CICR). In the model presented here, CICR activates mitochondrial dehydrogenases, thereby upregulating glucose-dependent production of ATP. The resultant increase in cytosolic [ATP]/[ADP] concentration ratio leads to closure of ATP-sensitive K(+) channels (K-ATP), membrane depolarization, and influx of Ca(2+) through voltage-dependent Ca(2+) channels (VDCCs). Ca(2+) influx stimulates exocytosis of secretory granules by promoting their fusion with the plasma membrane. Under conditions where Ca(2+) release channels are sensitized by GLP-1, Ca(2+) influx also stimulates CICR, generating an additional round of ATP production and K-ATP channel closure. In the absence of glucose, no "fuel" is available to support ATP production, and GLP-1 fails to stimulate insulin secretion. This new "feed-forward" hypothesis of beta-cell stimulus-secretion coupling may provide a mechanistic explanation as to how GLP-1 exerts a beneficial blood glucose-lowering effect in type 2 diabetic subjects.     

Processing of pro-islet amyloid polypeptide in the constitutive and regulated secretory pathways of beta cells

Islet amyloid is a pathologic characteristic of the pancreas in type 2 diabetes comprised mainly of the beta-cell peptide islet amyloid polypeptide (IAPP; amylin). We used a pulse-chase approach to investigate the kinetics of processing and secretion of the IAPP precursor, proIAPP, in beta cells. By only 20 min after synthesis, a COOH-terminally processed proIAPP intermediate (approximately 6 kDa) was already present in beta cells. Formation of this NH2-terminally extended intermediate was not prevented by arresting secretory pathway transport at the trans-Golgi network (TGN) by either brefeldin A or temperature blockade, suggesting that this initial cleavage step occurs in the TGN before entry of (pro)IAPP into granules. Mature IAPP (approximately 4 kDa) was not detected until 60 min of chase, suggesting that NH2-terminal cleavage occurs in granules. Cells chased in low glucose without Ca2+ or with diazoxide, to block regulated release, secreted both proIAPP (approximately 8 kDa) and a partially processed form (approximately 6 kDa) via the constitutive secretory pathway. Stimulation of regulated secretion resulted in secretion primarily of mature IAPP as well as low levels of both unprocessed (approximately 8 kDa) and partially processed (approximately 6 kDa) proIAPP. We conclude that normal processing of proIAPP is a two-step process initiated by cleavage at its COOH terminus (likely by prohormone convertase 1/3 in the TGN) followed by cleavage at its NH2 terminus (by prohormone convertase 2 in granules) to form IAPP. Both proIAPP and its NH2-terminally extended intermediate appear to be normal secretory products of the beta cell that can be released via either the regulated or constitutive secretory pathways.     

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.     

Ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in AtT-20 cells.

Newly synthesized prohormones and their processing enzymes transit through the same compartments before being packaged into regulated secretory granules. Despite this coordinated intracellular transport, prohormone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 cell line, conversion of pro-opiomelanocortin (POMC) to mature adrenocorticotropic hormone involves the prohormone convertase PC1. The mechanism by which this proteolytic processing is restricted to late secretory compartments is unknown; PC1 activity could be regulated by compartment-specific activators/inhibitors, or through changes in the ionic milieu that influence its activity. By arresting transport in a semi-intact cell system, we have addressed whether metabolically labeled POMC trapped in early secretory compartments can be induced to undergo conversion if the ionic milieu in these compartments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi network did not result in processing, thereby supporting the theory that processing is normally a post-Golgi/trans-Golgi network event. However, acidification of these compartments allowed effective processing of POMC to the intermediate and mature forms. The observed processing increased sharply at a pH below 6.0 and required millimolar calcium, regardless of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is predominantly restricted to acidifying secretory granules, and that a change in pH within these granules is both necessary and sufficient to activate POMC processing.     

Comparative aspects of intracellular proteolytic processing of peptide hormone precursors: studies of proopiomelanocortin processing

In this review, the mechanisms underlying the intracellular processing of peptide hormone precursors, with a focus on proopiomelanocortin (POMC), were discussed on the basis of recent information. POMC as well as other prohormones is processed to active peptides through proteolytic cleavage by prohormone convertases PC1 and/or PC2. However, the cleavage-specificity of PC1 and PC2 in mammals is somewhat different from that in amphibians. From the comparative endocrinological point of view, expression and tissue distribution of PC1 and PC2 were discussed here. In mammals, proteolytic processing of POMC occurs coordinately with the maturation of secretory granules. Studies using immunoelectron microscopy with DAMP (3-[2,4-dinitroanilino]-3'-amino-N-methyldipropylamine) as a pH probe revealed that the acidic pH in the secretory granules, generated by vacular type-H+-ATPase, provides a favorable environment for activating PC1 in AtT-20 cells, a mouse corticotrope tumor cell line. Recent data indicate that the 7B2 protein serves as a chaperone in the regulation of PC2 activation and to control the timing for activating the convertase. Together, secretory granules in endocrine and neuroendocrine cells provide proper sites for biosynthesizing hormones in addition to serving as storage sites and vehicles for the transport of peptide hormones.     

Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells

Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin (proSRIF) (Stoller, T. J., and D. Shields. J. Cell Biol. 1988. 107:2087- 2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37 degrees C. After the 20 degrees C block, the cells were mechanically permeabilized and pro-SRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by preincubating the permeabilized cells with low concentrations of N- ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps. These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occurred at 20 degrees C; in contrast, at 37 degrees C, the budding of secretory vesicles was approximately 40% efficient and was dependent on ATP, GTP, and cytosolic factors. Vesicle formation was inhibited by GTP gamma S suggesting a role for GTP- binding proteins in this process. Vesicle budding was dependent on cytosolic factors that were tightly membrane associated and could be removed only by treating the permeabilized cells with high salt. After high salt treatment, vesicle formation was dependent on added cytosol or the dialyzed salt extract. The formation of nascent secretory vesicles contrasts with prosomatostatin processing which required only ATP for efficient cleavage. Our results demonstrate that prohormone cleavage which is initiated in the TGN, precedes vesicle formation and that processing can be uncoupled from the generation of nascent secretory vesicles.     

Effects of chromogranin expression on inositol 1,4,5-trisphosphate-induced intracellular Ca2+ mobilization

We show here that expression of chromogranins in non-neuroendocrine NIH3T3 cells significantly increased the amount of IP(3)-mediated intracellular Ca(2+) mobilization in these cells, whereas suppression of them in neuroendocrine PC12 cells decreased the amount of mobilized Ca(2+). We have therefore investigated the relationship between the IP(3)-induced intracellular Ca(2+) mobilization and secretory granules. The level of IP(3)-mediated Ca(2+) release in CGA-expressing NIH3T3 cells was 40% higher than in the control cells, while that of CGB-expressing cells was 134% higher, reflecting the number of secretory granules formed. Suppression of CGA and CGB expression in PC12 cells resulted in 41 and 78% reductions in the number of secretory granules, respectively, while the extents of IP(3)-induced Ca(2+) release in these cells were reduced 40 and 69%, respectively. The newly formed secretory granules of NIH3T3 cells contained all three isoforms of the IP(3)Rs. Comparison of the concentrations of the IP(3)R isoforms expressed in the ER and nucleus of chromogranin-expressing and nonexpressing NIH3T3 cells did not show significant differences, indicating that chromogranin expression did not affect the expression of endogenous IP(3)Rs. Nonetheless, the IP(3)R concentrations in secretory granules of chromogranin-expressing NIH3T3 cells were 3.5-4.7-fold higher than those of the ER, similar to the levels found in secretory granules of neuroendocrine chromaffin cells, thus suggesting that the IP(3)Rs targeted to the newly formed secretory granules are newly induced by chromogranins without affecting the expression of intrinsic IP(3)Rs. These results strongly suggest that the extent of IP(3)-induced intracellular Ca(2+) mobilization in secretory cells is closely related to the number of secretory granules.     

Inhibition of the vacuolar H+-ATPase perturbs the transport, sorting, processing and release of regulated secretory proteins.

Vacuolar H+-ATPases (V-ATPases) are multisubunit enzymes that acidify various intracellular organelles, including secretory pathway compartments. We have examined the effects of the specific V-ATPase inhibitor bafilomycin A1 (Baf) on the intracellular transport, sorting, processing and release of a number of neuroendocrine secretory proteins in primary Xenopus intermediate pituitary cells. Ultrastructural examination of Baf-treated intermediate pituitary cells revealed a reduction in the amount of small dense-core secretory granules and the appearance of vacuolar structures in the trans-Golgi area. Pulse-chase incubations in combination with immunoprecipitation analysis showed that in treated cells, the proteolytic processing of the newly synthesized prohormone proopiomelanocortin, prohormone convertase PC2 and secretogranin III (SgIII) was inhibited, and an intracellular accumulation of intact precursor forms and intermediate cleavage products became apparent. Moreover, we found that treated cells secreted considerable amounts of a PC2 processing intermediate and unprocessed SgIII in a constitutive fashion. Collectively, these data indicate that in the secretory pathway, V-ATPases play an important role in creating the microenvironment that is essential for proper transport, sorting, processing and release of regulated secretory proteins.     

Contributions of intracellular compartments to calcium dynamics: implicating an acidic store

Many cells show a plateau of elevated cytosolic Ca(2+) after a long depolarization, suggesting delayed Ca(2+) release from intracellular compartments such as mitochondria and endoplasmic reticulum (ER). Mouse pancreatic beta-cells show a thapsigargin-sensitive plateau ('hump') of Ca(2+) after a 30 s depolarization but not after a 10 s depolarization. Surprisingly, this hump depends primarily on compartments other than the mitochondria or ER. It is reduced by only 22% upon blocking mitochondrial Na(+)-Ca(2+) exchange and by only 18% upon blocking ryanodine or IP(3) receptors together. Further, the time course of ER Ca(2+) measured by a targeted cameleon does not depend on the duration of depolarizations. Instead, the hump is reduced 35% by treatments with the dipeptide glycylphenylalanine beta-napthylamide, a tool often used to lyse lysosomes. We show that this dipeptide does not disturb ER functions, but it lyses acidic compartments and releases Ca(2+) into the cytosol. Moreover, it induces leaks in and possibly lyses insulin granules and stops mobilization of secretory granules to the readily releasable pool in beta-cells. We conclude that the dipeptide compromises dense-core secretory granules and that these granules comprise an acidic calcium store in beta-cells whose loading and/or release is sensitive to thapsigargin and which releases Ca(2+) after cytosolic Ca(2+) elevation.     

Evidence for distinct dibasic processing endopeptidases with Lys-Arg and Arg-Arg specificities in neurohypophysial secretory granules.

Two Ca(2+)-dependent endopeptidases endowed with specificities for paired basic residues have been disclosed in rat and ox neurohypophysial secretory granules. Specificities investigated by using synthetic fluorogenic substrates showed the presence of a Lys-Arg endopeptidase with optimum pH close to the granule pH (5.5) and of an Arg-Arg endopeptidase more active at pH 7.0. Granule extracts have virtually no activity towards Lys-Lys-containing substrate or monobasic substrates. Pro-Gly-Lys-Arg-chloromethylketone appears a very efficient inhibitor for the Lys-Arg enzyme. Soluble and membrane-bound forms of both endopeptidases have been detected. pH-dependence of membrane binding and partitioning into Triton X-114 suggest that the membrane-bound form of Lys-Arg endopeptidase is associated through an amphiphilic alpha-helix. It is proposed that the enzyme Lys-Arg cleaves prooxytocin and provasopressin at their signal sequence Gly-Lys-Arg when these precursors arrive in the neurosecretory granules. The processing proceeds in the granules through carboxypeptidase E and alpha-amidating enzyme complex for giving mature pharmacologically active nonapeptide hormones.     

Calcium depletion blocks proteolytic cleavages of plasma protein precursors which occur at the Golgi and/or trans-Golgi network. Possible involvement of Ca(2+)-dependent Golgi endoproteases.

The effects of calcium depletion on the proteolytic cleavage and secretion of plasma protein precursors were investigated in primary cultured rat hepatocytes and HepG2 cells. When the cells were incubated with A23187, the calcium-specific ionophore, in a medium lacking CaCl2, precursors of serum albumin and the third and fourth components of complement, C3 and C4, respectively, were found to be released into the medium. The addition of ionomycin or EGTA to the medium inhibited the processing of pro-C3 as well. Blocking the secretory pathway either at the mixed endoplasmic reticulum/Golgi in the presence of brefeldin A or at the endoplasmic reticulum/tubular-vesicular structure at a reduced temperature caused accumulation of pro-C3 within hepatocytes or HepG2 cells, indicating that the cleavage of the precursor occurs at a later stage of the secretory pathway. Once the blockade was released by incubating the cells either in the brefeldin A-free medium or at 37 degrees C, the secretion of plasma proteins resumed, irrespective of the presence of A23187. However, the processing of pro-C3 was almost completely inhibited in the presence of A23187, with only the precursor being released into the medium, implying that a decline in Ca2+ levels within the cell modulates the activity of a Golgi endoprotease responsible for the cleavage of pro-C3. When incubated with isolated Golgi membranes, pro-C3 secreted from Ca(2+)-depleted cells was cleaved in vitro into their subunits in the presence of Ca2+ but not in its absence, pointing to the involvement of a Ca(2+)-dependent Golgi endoprotease in the processing of pro-C3. These results collectively suggest that calcium depletion blocks the proteolytic cleavages of plasma protein precursors presumably by exhausting a Ca2+ pool available to the Ca(2+)-dependent processing enzyme(s) located at the Golgi and/or trans-Golgi network.     

Association of newly synthesized islet prohormones with intracellular membranes

Results from recent studies have indicated that pancreatic islet prohormone converting enzymes are membrane-associated in islet microsomes and secretory granules. This observation, along with the demonstration that proglucagon is topologically segregated to the periphery within alpha cell secretory granules in several species, led us to investigate the possibility that newly synthesized islet prohormones might be associated with intracellular membranes. Anglerfish islets were incubated with [3H]tryptophan and [14C]isoleucine for 3 h, then fractionated by differential and density gradient centrifugation. Microsome (M) and secretory granule (SG) fractions were halved, sedimented, and resuspended in the presence or absence of dissociative reagents. After membrane lysis by repeated freezing and thawing, the membranous and soluble components were separated by centrifugation. Extracts of supernatants and pellets were chromatographed by gel filtration; fractions were collected and counted. A high proportion (77-79%) of the newly synthesized proinsulin and insulin was associated with both M and SG membranes. Most of the newly synthesized proglucagons and prosomatostatins (12,000-mol-wt precursors) were also membrane-associated (86-88%) in M and SG. In contrast, glucagon- and somatostatin-related peptides exhibited much less membrane-association in SG (24-31%). Bacitracin, bovine serum albumin EDTA, RNAse, alpha-methylmannoside, N-acetylglucosamine, and dithiodipyridine had no effect on prohormone association with membranes. However, high salt (1 M KCl) significantly reduced membrane- association of prohormones. Binding of labeled prohormones to SG membranes from unlabeled tissue increased with incubation time and was inhibited by unlabeled prohormones. The pH optimum for prohormone binding to both M and SG membranes was 5.2. It is suggested that association of newly synthesized prohormones with intracellular membranes could be related to the facilitation of proteolytic processing of prohormones and/or transport from their site of synthesis to the secretory granules.     

Inositol 1,4,5-trisphosphate receptor/Ca2+ channel modulatory role of chromogranin A, a Ca2+ storage protein of secretory granules

The secretory granules of neuroendocrine cells, which contain large amounts of Ca(2+) and chromogranins, have been demonstrated to release Ca(2+) in response to inositol 1,4,5-trisphosphate (IP(3)), indicating the IP(3)-sensitive intracellular Ca(2+) store role of secretory granules. In our previous study, chromogranin A (CGA) was shown to interact with several secretory granule membrane proteins, including the IP(3) receptor (IP(3)R), at the intravesicular pH 5.5 (Yoo, S. H. (1994) J. Biol. Chem. 269, 12001-12006). To examine the functional aspect of this coupling, we measured the IP(3)-mediated Ca(2+) release property of the IP(3)R reconstituted into liposomes in the presence and absence of CGA. Presence of CGA in the IP(3)R-reconstituted liposome significantly enhanced the IP(3)-mediated Ca(2+) release from the liposomes. Moreover, the number of IP(3) bound to the reconstituted IP(3)R increased. The fluorescence energy transfer and IP(3)R Trp fluorescence quenching studies indicated that the structure of reconstituted IP(3)R becomes more ordered and exposed in the presence of CGA, suggesting that the coupled CGA in the liposome caused structural changes of the IP(3)R, changing it to a structure that is better suited to IP(3) binding and subsequent Ca(2+) release. These results appear to underscore the physiological significance of IP(3)R-CGA coupling in the secretory granules.     

Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells.

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.