Somatostatin Signaling Research Articles

Differential expression of preprosomatostatin- and somatostatin receptor-encoding mRNAs in association with the growth hormone-insulin-like growth factor system during embryonic development of rainbow trout ( Oncorhynchus mykiss)

Rainbow trout were used to evaluate the relationship between the somatostatin (SS) signaling and the growth hormone (GH)-insulin-like growth factor (IGF) systems during pre-hatch and post-hatch embryonic development. The expression of preprosomatostatins (PPSS), SS receptors (SSTR), GH receptors (GHR), IGF-1, IGF-2, and IGF type 1 receptors (IGFR1) was examined in various regions at the eyed-egg (29 days post-fertilization, dpf;), post-hatch (53 dpf), swim-up (68 dpf), and complete yolk-absorbed (90 dpf) stages. In head, PPSSI mRNA abundance increased during development while that of PPSSII′ decreased and that of PPSSII″ remained unchanged. In body and tail, mRNA abundance of all PPSSs remained unchanged except that of PPSSII″ which declined in the tail. SSTR expression increased as development progressed in all regions with the exception of SSTR1A mRNA which remained unchanged. mRNA levels of GHR1 declined in all regions of post-hatch embryos, whereas those of GHR2 remained unchanged. Expression of IGF-1 and IGF-2 in head and tail regions increased immediately after hatching, and then declined, whereas the expression of neither IGF changed during development in the body. The expression of IGFR1 mRNAs declined in all regions, reaching their lowest levels at 90 dpf, with the exception of IGFR1A mRNA in the body which remained unchanged. The general decline in the expression of GH-IGF system components during development appears inversely related to a general increase in the expression of SS system elements, and suggests that these two systems interact to regulate the tissue expansion and tissue regression of embryogenesis.

Meningiomas

Meningiomas, the most common intracranial tumors, are dura-based tumors that occur in middle-aged or older adults and have a predilection for women and African Americans. They are almost always benign, but atypical and malignant forms exist. The optimal treatment for symptomatic tumors is complete surgical resection when feasible. Tumors in poor locations can have a higher rate of morbidity and often can be only partially removed. Radiation therapy should be used to treat lesions that are partially removed and lesions of higher grade, despite complete removal. Small lesions may be amenable to radiosurgery, which may obviate surgery in high-risk patients. For lesions that recur or grow despite surgery and radiation, the only option is systemic chemotherapy. No agent is optimal, but hydroxyurea is used most often. Newer approaches such as the use of inhibitors of somatostatin receptors or signal transduction pathways are under investigation.

The Effect of Transforming Growth Factor β on Human Neuroendocrine Tumor BON Cell Proliferation and Differentiation Is Mediated through Somatostatin Signaling

The dual effect of the ubiquitous inflammatory cytokine transforming growth factor beta1 (TGF beta) on cellular proliferation and tumor metastasis is intriguing but complex. In epithelial cell- and neural cell-derived tumors, TGF beta serves as a growth inhibitor at the beginning of tumor development but later becomes a growth accelerator for transformed tumors. The somatostatin (SST) signaling pathway is a well-established antiproliferation signal, and in this report, we explore the interplay between the SST and TGF beta signaling pathways in the human neuroendocrine tumor cell line BON. We defined the SST signaling pathway as a determinant for neuroendocrine tumor BON cells in responding to TGF beta as a growth inhibitor. We also determined that TGF beta induces the production of SST and potentially activates the negative growth autocrine loop of SST, which leads to the downstream induction of multiple growth inhibitory effectors: protein tyrosine phosphatases (i.e., SHPTP1 and SHPTP2), p21(Waf1/Cip1), and p27(Kip1). Concurrently, TGF beta down-regulates the growth accelerator c-Myc protein and, collectively, they establish a firm antiproliferation effect on BON cells. Additionally, any disruption in the activation of either the TGF beta or SST signaling pathway in BON leads to "reversible" neuroendocrine-mesenchymal transition, which is characterized by the loss of neuroendocrine markers (i.e., chromogranin A and PGP 9.5), as well as the altered expression of mesenchymal proteins (i.e., elevated vimentin and Twist and decreased E-cadherin), which has previously been associated with elevated metastatic potential. In summary, TGF beta-dependent growth inhibition and differentiation is mediated by the SST signaling pathway. Therefore, any disruption of this TGF beta-SST connection allows BON cells to respond to TGF beta as a growth accelerator instead of a growth suppressor. This model can potentially apply to other cell types that exhibit a similar interaction of these pathways.

Minocycline provides protection against β-amyloid(25-35)-induced alterations of the somatostatin signaling pathway in the rat temporal cortex

Minocycline is a semi-synthetic second-generation tetracycline known to improve cognition in amyloid precursor protein transgenic mice. Whether it can protect the somatostatin (SRIF) receptor-effector system, also involved in learning and memory, from alterations induced by chronic i.c.v. infusion of β-amyloid peptide (Aβ)(25-35) is presently unknown. Hence, in the present study, we tested the effects of minocycline on the SRIF signaling pathway in the rat temporal cortex. To this end, male Wistar rats were injected with minocycline (45 mg/kg body weight) i.p. twice on the first day of treatment. On the following day and during 14 days, Aβ(25-35) was administered i.c.v. via an osmotic minipump connected to a cannula implanted in the left lateral ventricle (300 pmol/day). Minocycline (22.5 mg/kg, i.p.) was injected once again the last 2 days of the Aβ(25-35) infusion. The animals were killed by decapitation 24 h after the last drug injection. Our results show that minocycline prevents the decrease in SRIF receptor density and somatostatin receptor (sst) 2 expression and the attenuated capacity of SRIF to inhibit adenylyl cyclase (AC) activity, alterations present in the temporal cortex of Aβ(25-35)-treated rats. Furthermore, minocycline blocks the Aβ(25-35)-induced decrease in phosphorylated cyclic AMP (cAMP) response element binding protein (p-CREB) content and G-protein-coupled receptor kinase 2 (GRK) protein expression in this brain area. Altogether, the present data demonstrate that minocycline in vivo provides protection against Aβ-induced impairment of the SRIF signal transduction pathway in the rat temporal cortex and suggest that it may have a potential as a therapeutic agent in human Alzheimer's disease, although further studies are warranted.

Somatostatin signaling and the regulation of growth and metabolism in fish

The study of the somatostatins (SS) signaling system in fish has provided important information about the structure, function, and evolution of SSs and their receptors. The SS signaling system elicits widespread biological actions via multiple hormone variants, numerous receptor subtypes, and a variety of signal transduction pathways. SSs alter growth via both direct and indirect actions, including inhibiting growth hormone release at the pituitary, decreasing hepatic GH sensitivity, and lowering plasma IGF-I levels. Metabolism also is significantly influenced by SSs. SSs stimulate the breakdown of energy stores and influences digestion, food intake, nutrient absorption, and food conversion both directly and through the modulation of other hormonal systems. The study of fish, which display a diversity of habitat types and life history forms, reveals that the SS signaling system helps regulate energy partitioning and integrate metabolism with growth and other biological processes.

Selective Regulation of Somatostatin Receptor Subtype Signaling: Evidence for Constitutive Receptor Activation

Anterior pituitary hormone secretion is under tonic suppression by hypothalamic somatostatin signaling through somatostatin receptor subtypes (SSTs). Because some hormonal axes are known to be abnormally regulated by ligand-independent constitutively active G protein-coupled receptors, we tested pituitary SSTs for selective constitutive signaling. We therefore differentially silenced endogenous SST2, SST3, and SST5 in somatostatin-sensitive ACTH-secreting mouse AtT-20 pituitary corticotroph cells using small inhibitory RNA (siRNA) and analyzed downstream SSTs-regulated pathways. Transfection with siRNA reduced specific receptor subtype mRNA expression up to 82%. Specificity of receptor silencing was validated against negative controls with different gene-selective siRNAs, concordance of mRNA and cAMP changes, reduced potency of receptor-selective agonists, and phenotype rescue by overexpression of the silenced receptor. Mouse SST3 > SST5 > SST2 knockdown increased basal cAMP accumulation (up to 200%) and ACTH secretion (up to 60%). SST2- and SST5-selective agonist potencies were reduced by SST3- and SST5-silencing, respectively. SST5 > SST2 = SST3 silencing also increased basal levels of ERK1/2 phosphorylation. SST3- and SST5-knockdown increased cAMP was only partially blocked by pertussis toxin. The results show that SST2, SST3, and SST5 exhibit constitutive activity in mouse pituitary corticotroph cells, restraining adenylate cyclase and MAPK activation and ACTH secretion. SST3 mainly inhibits cAMP accumulation and ACTH secretion, whereas SST5 predominantly suppresses MAPK pathway activation. Therefore, SST receptor subtypes control pituitary cell function not only through somatostatin binding to variably expressed cell membrane receptor subtypes, but also by differential ligand-independent receptor-selective constitutive action.

A Novel Variant of Ionotropic Glutamate Receptor Regulates Somatostatin Secretion From δ-Cells of Islets of Langerhans

Many metabolic factors affect the secretion of insulin from beta-cells and glucagon from alpha-cells of the islets of Langerhans to regulate blood glucose. Somatostatin from delta-cells, considered a local inhibitor of islet function, reduces insulin and glucagon secretion by activating somatostatin receptors in islet cells. Somatostatin secretion from delta-cells is increased by high glucose via glucose metabolism in a similar way to insulin secretion from beta-cells. However, it is unknown how low glucose triggers somatostatin secretion. Because L-glutamate is cosecreted with glucagon from alpha-cells under low-glucose conditions and acts as a primary intercellular messenger, we hypothesized that glutamate signaling triggers the secretion of somatostatin. In this study, we showed that delta-cells express GluR4c-flip, a newly identified splicing variant of GluR4, an (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptor of rat. After treatment with L-glutamate, AMPA, or kainate, secretion of somatostatin from isolated islets was significantly stimulated under low-glucose conditions. The glutamate-dependent somatostatin secretion was Ca(2+) dependent and blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. Somatostatin in turn inhibited the secretion of L-glutamate and glucagon from alpha-cells. These results indicate that L-glutamate triggers somatostatin secretion from delta-cells by way of the GluR4c-flip receptor under low-glucose conditions. The released somatostatin may complete the feedback inhibition of alpha-cells. Thus, alpha- and delta-cells may communicate with each other through L-glutamate and somatostatin signaling.

Leptin-target neurones of the rat hypothalamus express somatostatin receptors.

Hypothalamic leptinoceptive neurones can be visualized by histochemical demonstration of leptin-induced nuclear translocation of the signalling molecule STAT3. We investigated the relationship of the leptinoceptive neurones to the somatostatin signalling system. With double-labelling immunohistochemistry, we studied the colocalization of leptin-activated transcription factor, STAT3, with somatostatin receptor subtypes, sst1, sst2A, sst2B, sst3 and sst4, or the neuropeptide itself, in the rat hypothalamus. Immunoreactivity for all the entities was widely distributed throughout the entire hypothalamus. Despite the wide distribution, only few cases of colocalization of somatostatin with leptin-activated STAT3 were detected in the paraventricular, arcuate and dorsomedial nuclei. A moderate to high degree of colocalization of nuclear STAT3 and all investigated subtypes of somatostatin receptors was found in the lateral and dorsal hypothalamic areas and in the dorsomedial hypothalamic nucleus. Immunoreactivity for sst1, sst2B and sst4 was present in STAT3-containing nuclei of the paraventricular, periventricular, arcuate and ventromedial hypothalamic neurones, as well as in the retrochiasmatic and posterior hypothalamic areas. Despite the wide distribution of sst2A in the rat hypothalamus, few events of colocalization with leptin-activated STAT3 were observed in the dorsomedial nucleus and in the lateral and dorsal hypothalamic areas only. Many leptin-responsive neurones of the dorsal, lateral, periarcuate, perifornical and posterior hypothalamic areas, as well as in the ventromedial and dorsomedial hypothalamic nuclei, displayed sst3 immunoreactivity at their neuronal cilia. These results provide strong anatomical evidence for the direct interaction of leptin and the somatostatin systems in neuroendocrine control loops such as the energy homeostasis, growth or stress response.

The somatostatin analogue TT-232 induces apoptosis in A431 cells: Sustained activation of stress-activated kinases and inhibition of signalling to extracellular signal-regulated kinases

TT-232 is a somatostatin analogue containing a five-residue ring structure. The present report describes TT-232-induced signalling events in A431 cells, where a 4-h preincubation with the peptide irreversibly induced a cell death program, which involves DNA-laddering and the appearance of shrunken nuclei, but is unrelated to somatostatin signalling. Early intracellular signals of TT-232 include a transient two-fold activation of the extracellular signal-regulated kinase (ERK2) and a strong and sustained activation of the stress-activated protein kinases c-Jun NH 2-terminal kinase (JNK)/SAPK and p38MAPK. Blocking the signalling to ERK or p38MAPK activation had no effect on the TT-232-induced cell killing. At the commitment time for inducing cell death, TT-232 decreased EGFR-tyrosine phosphorylation and prevented epidermial growth factor (EGF)-induced events like cRaf-1 and ERK2 activation. Signalling to ERK activation by FCS, phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor (PDGF) was similarly blocked. Our data suggest that TT-232 triggers an apoptotic type of cell death, concomitant with a strong activation of JNK and a blockade of cellular ERK2 activation pathways.

Calcium responses to thyrotropin-releasing hormone, gonadotropin-releasing hormone and somatostatin in phospholipase css3 knockout mice.

These studies examined the importance of phospholipase Cbeta (PLCbeta) in the calcium responses of pituitary cells using PLCbeta3 knockout mice. Pituitary tissue from wild-type mice contained PLCbeta1 and PLCbeta3 but not PLCbeta2 or PLCbeta4. Both Galphaq/11 and Gbetagamma can activate PLCbeta3, whereas only Galphaq/11 activates PLCss1 effectively. In knockout mice, PLCbeta3 was absent, PLCbeta1 was not up-regulated, and PLCbeta2 and PLCbeta4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCbeta3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCbeta3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCbeta3 knockout animals These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCbeta3 through Gbetagamma, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCbeta1 and PLCbeta3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nM and 1 microM TRH and GnRH were identical in pituitary cells from wild-type and PLCbeta3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCbeta1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCbeta3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.

Structure-Function Relationships of the Signaling System for the Somatostatin Peptide Hormone Family

Somatostatins are a diverse family of peptide hormones that regulate a vast array of biological processes in vertebrates, including the modulation of growth, development, and metabolism. The multi-functional nature of the somatostatin family arises from the an elaborate, multi-faceted signaling system consisting of somatostatin signaling molecules, G-protein-coupled receptors, and cellular effector pathways. A striking aspect of this signaling system is the substantial diversity at every level. The signal molecules themselves display considerable structural heterogeneity. This molecular heterogeneity results from tissue-specific differential processing of a single large precursor protein (preprosomatostatin) as well as from the existence of multiple somatostatin genes, each giving rise to different precursors. In addition, numerous SS receptor subtypes have been characterized (five in mammals), some of which exhibit preferential binding to one ligand form over another. Propagation of the signal results from linkage of the receptors via numerous types of G-proteins to several different cellular effector pathways, including adenylyl cyclase, various protein kinases, numerous ion channels, and phospholipase C/inositol-3-phosphate. Ultimately, a particular response in a given target cell may be determined by structural interactions between and among the various elements of the signaling system.

Structure-Function Relationships of the Signaling System for the Somatostatin Peptide Hormone Family1

Somatostatins are a diverse family of peptide hormones that regulate a vast array of biological processes in vertebrates, including the modulation of growth, development, and metabolism. The multi-functional nature of the somatostatin family arises from the an elaborate, multi-faceted signaling system consisting of somatostatin signaling molecules, G-protein-coupled receptors, and cellular effector pathways. A striking aspect of this signaling system is the substantial diversity at every level. The signal molecules themselves display considerable structural heterogeneity. This molecular heterogeneity results from tissue-specific differential processing of a single large precursor protein (preprosomatostatin) as well as from the existence of multiple somatostatin genes, each giving rise to different precursors. In addition, numerous SS receptor subtypes have been characterized (five in mammals), some of which exhibit preferential binding to one ligand form over another. Propagation of the signal results from linkage of the receptors via numerous types of G-proteins to several different cellular effector pathways, including adenylyl cyclase, various protein kinases, numerous ion channels, and phospholipase C/inositol-3-phosphate. Ultimately, a particular response in a given target cell may be determined by structural interactions between and among the various elements of the signaling system.

Somatostatin analogs stimulate DNA-dependent protein kinase activity in human gastric tumoral cell-line HGT1

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylates, in the presence of double-stranded DNA, several transcription-, replication- and repair -factors. Its interaction with the DNA-binding regulatory component Ku (p86-/p70-Ku) is required for stabilization and activity. We have previously shown that p86-Ku behaves as a specific receptor for the growth inhibitory tetradecapeptide, somatostatin. In this work, we investigate a possible regulation by somatostatin analogs, of DNA-PK activity in the human gastric tumoral HGT1/clone6 cell-line. We demonstrate that a 48 h-preincubation of cells with octreotide or RC-160, stimulates DNA-PK activity by 8 and 10 fold with ED 50S of 1 and 0.1 nM, respectively. These stimulations appearing only after 3 h were inhibited by cycloheximide. They were not observed in a cell clone which was transfected by a cDNA encoding p86-Ku antisense. This study demonstrates the existence of a new somatostatin signaling pathway involving the stimulation of DNA-PK activity.

Can GHRH or GH secretagogues re-initiate pituitary GH pulsatility?

Can GHRH or GH secretagogues re-initiate pituitary GH pulsatility?

Sst2 Somatostatin Receptor Mediates Cell Cycle Arrest and Induction of p27 Kip1: EVIDENCE FOR THE ROLE OF SHP-1

Activation of the somatostatin receptor sst2 inhibits cell proliferation by a mechanism involving the stimulation of the protein-tyrosine phosphatase SHP-1. The cell cycle regulatory events leading to sst2-mediated growth arrest are not known. Here, we report that treatment of Chinese hamster ovary cells expressing sst2 with the somatostatin analogue, RC-160, led to G1 cell cycle arrest and inhibition of insulin-induced S-phase entry through induction of the cyclin-dependent kinase inhibitor p27(Kip1). Consequently, a decrease of p27(Kip1)-cdk2 association, an inhibition of insulin-induced cyclin E-cdk2 kinase activity, and an accumulation of hypophosphorylated retinoblastoma gene product (Rb) were observed. However, RC-160 had no effect on the p21(Waf1/Cip1). When sst2 was coexpressed with a catalytically inactive mutant SHP-1 in Chinese hamster ovary cells, mutant SHP-1 induced entry into cell cycle and down-regulation of p27(Kip1) and prevented modulation by insulin and RC-160 of p27(Kip1) expression, p27(Kip1)-cdk2 association, cyclin E-cdk2 kinase activity, and the phosphorylation state of Rb. In mouse pancreatic acini, RC-160 reverted down-regulation of p27(Kip1) induced by a mitogen, and this effect did not occur in acini from viable motheaten (mev/mev) mice expressing a mutant SHP-1 with markedly deficient enzymes. These findings provide the first evidence that sst2 induces cell cycle arrest through the up-regulation of p27(Kip1) and demonstrate that SHP-1 is required for maintaining high inhibitory levels of p27(Kip1) and is a critical target of the insulin, and somatostatin signaling cascade, leading to the modulation of p27(Kip1).

Arachidonate 5-lipoxygenase and its activating protein: prominent hippocampal expression and role in somatostatin signaling.

5-Lipoxygenase-activating protein (FLAP) is an 18-kDa integral membrane protein required, in peripheral cells, for the activation of 5-lipoxygenase (5-LO) and for the resulting synthesis of leukotrienes from arachidonic acid. In the brain, the leukotrienes have been implicated in several pathophysiological events and in the electrophysiological effect of somatostatin, yet the cellular origin and role of these messenger molecules are still poorly understood. In the present study, we used reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry to demonstrate that 5-LO and FLAP are expressed in various regions of the rat brain, including hippocampus, cerebellum, primary olfactory cortex, superficial neocortex, thalamus, hypothalamus, and brainstem. Highest levels of expression were observed in cerebellum and hippocampus. In the latter we demonstrate the colocalization of 5-LO and FLAP in CA1 pyramidal neurons. Moreover, electrophysiological experiments show that selective inhibition of FLAP with the compound MK-886 (0.25-1 microM) prevents the somatostatin-induced augmentation of the hippocampal K+ M-current. Our results provide necessary evidence for the presence and signaling role of 5-LO and FLAP in central neurons and strongly support their proposed participation in somatostatin-receptor transmembrane signaling.

Intracellular degradation of prohormone-chloramphenicol-acetyl-transferase chimeras in a pre-lysosomal compartment.

Small peptide hormones (less than 50 amino acids) are synthesized as larger inactive precursors. Work from several laboratories, including our own, has implicated the propeptide of various precursors in mediating intracellular transport and targeting to secretory granules. We previously demonstrated that the proregion of prosomatostatin, one of the simplest peptide hormone precursors, when fused to alpha-globin, enabled the globin polypeptide to be transported to the regulated secretory pathway. To identify sorting motifs in this propeptide, we have now constructed a chimera comprising the somatostatin signal peptide and proregion fused to chloramphenicol acetyl transferase (CAT) and a control protein consisting of the signal peptide fused to CAT, both of which were expressed in rat anterior-pituitary GH3 cells. Both molecules were translocated into the endoplasmic reticulum (ER) efficiently and core-glycosylated on the single cryptic N-linked glycosylation site present in CAT. Surprisingly, the glycosylated propeptide-CAT and signal without CAT were degraded intracellularly with half-lives of 30 min and 90 min, respectively. Based on the kinetics of degradation, temperature sensitivity, and resistance to lysosomotrophic agents, we suggest that degradation occurred in the ER. Our data imply that the pro-region is not an a priori universal sorter, but only directs heterologous peptides to the secretory pathway when the passenger peptide assumes a secretion-competent conformation.

The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells.

We have investigated the role of the somatostatin propeptide in mediating intracellular transport and sorting to the regulated secretory pathway. Using a retroviral expression vector, two fusion proteins were expressed in rat pituitary (GH3) cells: a control protein consisting of the beta-lactamase signal peptide fused to chimpanzee alpha-globin (142 amino acids); and a chimera of the somatostatin signal peptide and proregion (82 amino acids) fused to alpha-globin. Control globin was translocated into the endoplasmic reticulum as determined by accurate cleavage of its signal peptide; however, alpha-globin was not secreted but was rapidly and quantitatively degraded intracellularly with a t 1/2 of 4-5 min. Globin degradation was insensitive to chloroquine, a drug which inhibits lysosomal proteases, but was inhibited at 16 degrees C suggesting proteolysis occurred during transport to the cis-Golgi apparatus. In contrast to the control globin, approximately 30% of the somatostatin propeptide-globin fusion protein was transported to the distal elements of the Golgi apparatus where it was endoproteolytically processed. Processing of the chimera occurred in an acidic intracellular compartment since cleavage was inhibited by 25 microM chloroquine. 60% of the transported chimera was cleaved at the Arg-Lys processing site in native prosomatostatin yielding "mature" alpha-globin. Most significantly, approximately 50% of processed alpha-globin was sorted to the regulated pathway and secreted in response to 8-Br-cAMP. We conclude that the somatostatin propeptide mediated transport of alpha-globin from the endoplasmic reticulum to the trans-Golgi network by protecting molecules from degradation and in addition, facilitated packaging of alpha-globin into vesicles whose secretion was stimulated by cAMP.