Increased matrix-metalloproteinase-2 and matrix-metalloproteinase-9 expression in the brain of dystrophic mdx mouse

Brain edema and severe alterations of the glial and endothelial cells have recently been demonstrated in the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy, and an increase in microvessel density in patients affected by Duchenne muscular dystrophy has also been shown. In order to further elucidate the mechanisms underlying the angiogenetic processes occurring in Duchenne muscular dystrophy, in this study we analyzed matrix-metalloproteinase-2 and -9 expression in the brain of 20-month-old mdx and control mice by means of immunohistochemistry, in situ hybridization, immunoblotting and gelatin zymography. Moreover, we studied vascular endothelial growth factor expression by means of Western blot and immunohistochemistry, and by dual immunofluorescence using anti-vascular endothelial growth factor and anti matrix-metalloproteinase-2 and-9 antibodies. Ultrastructural features of the brain choroidal plexuses were evaluated by electron microscopy. Spatial relationships between endothelium and astrocyte processes were studied by confocal laser microscopy, using an anti-CD31 antibody as a marker of endothelial cells, and anti-glial fibrillary acidic protein (GFAP) as a marker of glial cells. The results demonstrate that high expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 protein content occurs in mdx brain and in choroidal plexuses where, by in situ hybridization, matrix-metalloproteinase-2 and matrix-metalloproteinase-9 mRNA was localized in the epithelial cells. Moreover, matrix-metalloproteinase-2 mRNA was found in both mdx perivascular astrocytes and blood vessels, while matrix-metalloproteinase-9 mRNA was localized in mdx vessels. Through zymography, increased expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in mdx brain compared with the controls. These enhanced matrix-metalloproteinase levels in mdx mice were found to be associated with increased vascular endothelial growth factor expression, as determined by immunoblotting and immunocytochemistry and with ultrastructural alterations of the mdx choroidal epithelial cells and brain vessels, as previously reported [Nico B, Frigeri A, Nicchia GP, Corsi P, Ribatti D, Quondamatteo F, Herken R, Girolamo F, Marzullo A, Svelto M, Roncali L (2003) Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice. Glia 42:235-251]. Indeed, in the mdx epithelial cells of the plexuses, the apical microvilli were located on the lateral membranes, whereas in the controls they were uniformly distributed over the free ventricular surface. Moreover, by dual immunofluorescence, a colocalization of vascular endothelial growth factor and matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in the ependymal and epithelial cells of plexuses in mdx mice and, under confocal laser microscopy, mdx CD-31 positive vessels were enveloped by less GFAP-positive astrocyte processes than the controls. Overall, these data point to a specific pathogenetic role of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 in neurological dysfunctions associated with Duchenne muscular dystrophy.     

Plasticity in rat uterine sympathetic nerves: the role of TrkA and p75 nerve growth factor receptors

Uterine sympathetic innervation undergoes profound remodelling in response to physiological and experimental changes in the circulating levels of sex hormones. It is not known, however, whether this plasticity results from changes in the innervating neurons, the neuritogenic properties of the target tissue or both. Using densitometric immunohistochemistry, we analysed the effects of prepubertal chronic oestrogen treatment (three subcutaneous injections of 20 microg of beta-oestradiol 17-cypionate on days 25, 27 and 29 after birth), natural peripubertal transition and late pregnancy (19-20 days post coitum) on the levels of TrkA and p75 nerve growth factor receptors in uterine-projecting sympathetic neurons of the thoraco-lumbar paravertebral sympathetic chain (T7-L2) identified using the retrograde tracer Fluorogold. For comparative purposes, levels of TrkA and p75 were assessed in the superior cervical ganglion (SCG) following prepubertal chronic oestrogen treatment. These studies showed that the vast majority of uterine-projecting neurons expressed both TrkA and p75. Both prepubertal chronic oestrogen treatment and the peripubertal transition increased the ratio p75 to TrkA in uterine-projecting neurons, whereas pregnancy elicited the opposite effect. Prepubertal chronic oestrogen treatment had no effects on levels of TrkA or p75 in sympathetic neurons of the SCG. Taken together, our data suggest that neurotrophin receptor-mediated events may contribute to regulate sex hormone-induced plasticity in uterine sympathetic nerves, and are in line with the idea that, in vivo, plasticity in uterine nerves involves changes in both the target and the innervating neurons.     

Resolution of skeletal muscle inflammation in mdx dystrophic mouse is accompanied by increased immunoglobulin and interferon-gamma production

Mdx mouse, the animal model of Duchenne muscular dystrophy, develops an X-linked recessive inflammatory myopathy with an apparent sustained capacity for muscle regeneration. We analysed whether changes in the skeletal muscle during myonecrosis and regeneration would correlate with functional alterations in peripheral lymphoid tissues. Here we show that during the height of myonecrosis, mdx mice display marked atrophy of peripheral lymph nodes and extensive muscle inflammation. In contrast, enlargement of draining lymph nodes with accumulation of CD4+ CD44+, CD4+ CD25+, CD8+ CD44+ T lymphocytes and type-2 B cells was consistently observed during amelioration of the muscle lesion. In addition, regeneration of the muscular tissue was accompanied by concomitant increase of immunoglobulin-secreting cells in regional lymph nodes and bone marrow. Double immunolabelling analysis revealed intense B cell proliferation and formation of germinal centre in the follicles of dystrophic regional lymph nodes. Furthermore, lymph node cells produced large amounts of IFN-gamma but not IL-4, IL-6 or IL-10 after in vitro mitogen stimulation with Concanavalin A. As these alterations occurred mainly during the recovery period, we suggested that local activation of the immune system could be an influence which mitigates the myonecrosis of muscular tissue in the mdx dystrophic mouse.     

Expression and localization of placenta growth factor and PlGF receptors in human meningiomas

It has previously been suggested that in human brain tumours, endothelial cell proliferation during angiogenesis is regulated by a paracrine mechanism involving vascular endothelial growth factor (VEGF) and its receptors (VEGF receptor 1 and VEGF receptor 2). The mechanism of growth factor up-regulation is based on hypoxic activation of mRNA expression and mRNA stabilization and genetic events, leading to an increase of growth factor gene expression. The role of the other newly discovered VEGF family members with a high specificity for endothelial cells in the pathogenesis of glial neoplasms is unknown. To investigate which other members of the VEGF family are overexpressed in human brain tumours, the mRNA levels of placenta growth factor (PlGF), VEGF-A, and VEGF-B genes were determined by northern blot analysis in surgically obtained human meningiomas. In the 16 meningiomas examined, the mRNA for PlGF was highly expressed in four tumours and VEGF-A mRNA was highly abundant in three tumour samples. There was no close correlation between PlGF mRNA levels and VEGF-A expression levels. VEGF-B mRNA was abundantly expressed in all tumour samples at uniform levels. In a PlGF-positive tumour sample, immunoreactive VEGFR-1 and VEGFR-2 were detected in endothelial cells of the blood vessels. PlGF protein was detectable in most but not all capillaries of the tumour. PlGF is thus highly up-regulated in a subset of human meningiomas and may therefore have functions, in some tumour vessels, connected to endothelial cell maturation and tube formation. These findings suggest that PlGF, in addition to VEGF-A, may be another positive factor in tumour angiogenesis in human meningiomas. Copyright © 1999 John Wiley & Sons, Ltd.     

Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice

 It is commonly accepted, that regenerative capacity of striated muscle is confined to skeletal muscle by activation of satellite cells that normally reside quiescent between the plasmalemma and the basement membrane of muscle fibers. Muscular dystrophies are characterized by repetitive cycles of de- and regeneration of skeletal muscle fibers and by the frequent involvement of the cardiac muscle. Since during the longstanding course of muscular dystrophies there is a permanent demand of myogenic progenitors we hypothesized that this may necessitate a recruitment of additional myogenic precursors from an undifferentiated, permanently renewed cell pool, such as bone marrow (BM) cells. To this end normal and dystrophic (mdx) female mice received bone marrow transplantation (BMT) from normal congenic male donor mice. After 70 days, histological sections of skeletal and cardiac muscle from BMT mice were probed for the donor-derived Y chromosomes. In normal BMT recipients, no Y chromosome-containing myonuclei were detected, either in skeletal or in cardiac muscle. However, in all samples from dystrophic mdx skeletal muscles Y chromosome-specific signals were detected within muscle fiber nuclei, which additionally were found to express the myoregulatory proteins myogenin and myf-5. Moreover, in the hearts of BMT-mdx mice single cardiomyocytes with donor derived nuclei were identified, indicating, that even cardiac muscle cells are able to regenerate by recruitment of circulating BM-derived progenitors. Our findings suggest that further characterization and identification of the BM cells capable of undergoing myogenic differentiation may have an outstanding impact on therapeutic strategies for diseases of skeletal and cardiac muscle. Accepted: 27 October 1998     

Nerve growth factor in the adult brain of a teleostean model for aging research: Nothobranchius furzeri

Nerve growth factor (NGF) acts on central nervous system neurons, regulating naturally occurring cell death, synaptic connectivity, fiber guidance and dendritic morphology. The dynamically regulated production of NGF beginning in development, extends throughout adult life and aging, exerting numerous roles through a surprising variety of neurons and glial cells.This study analyzes the localization of NGF in the brain of the teleost fish Nothobranchius furzeri, an emerging model for aging research due to its short lifespan. Immunochemical and immunohistochemical experiments were performed by employing an antibody mapping at the N-terminus of the mature chain human origin NGF. Western blot analysis revealed an intense and well defined band of 20 kDa, which corresponds to proNGF of N. furzeri. Immunohistochemistry revealed NGF immunoreactivity (IR) diffused throughout all regions of telencephalon, diencephalon, mesencephalon and rhomboencephalon. It was detected in neurons and in glial cells, the latter mostly lining the mesencephalic and rhomboencephalic ventricles. Particularly in neurons, NGF IR was localized in perikarya and, to a less extent, in fibers.The widespread distribution of proNGF suggests that it might modulate numerous physiological functions in the adult brain of N. furzeri. The present survey constitutes a baseline study to enhance the understanding of the mechanisms underlying the role of NGF during aging processes.     

Reciprocal changes in expression of mRNA for nerve growth factor and its receptors TrkA and LNGFR in brain of aged rats in relation to maze learning deficits

 Quantitative in situ hybridization was used to examine the expression of mRNA for nerve growth factor (NGF) and its receptors, p140Trk (TrkA) and p75LNGFR (LNGFR), in different brain regions of adult (3-month-old) and aged (27-month-old) Wistar rats. The brain regions studied were hippocampus (dentate gyrus, CA3 region), basal forebrain (medial septum, diagonal band) and caudate-putamen. Prior to hybridization histochemistry behaviorally impaired as well as severely impaired animals were selected from a large group of old rats according to their performance in the Morris water maze. The impaired rats showed longer escape latencies and, thus, implicitly impaired performance in the place version of the task, but did not differ from adult controls on the platform crossing measure registered during the spatial probe trial. The severely impaired rats were significantly impaired on both measures, both in comparison with the adult animals and in comparison with the impaired aged rats. Inspection of the hippocampus revealed no age- or performance-related changes in NGF mRNA levels. The overall expression of TrkA mRNA in basal forebrain and caudate was found to be decreased in the impaired (–20%) as well as the severely impaired aged rats (–17%). A significant increase in p75LNGFR mRNA was found in the basal forebrain of the impaired rats in comparison with the severely impaired aged rats (+35%) and adult animals (+33%). These findings show that age-related maze performance deficits are accompanied by a decrease in basal forebrain and striatal TrkA mRNA expression. The increase in basal forebrain LNGFR mRNA levels observed in impaired, but not severely impaired, aged rats may reflect an early manifestation of processes underlying age-related cognitive deficits and may constitute a restorative and/or compensatory mechanism, since these rats displayed fewer deficits in navigation of the maze. Received: 21 February 1996 / Accepted: 15 October 1996     

Nerve growth factor and its functional receptor TrkA are up-regulated in murine decidual tissue of stress-triggered and substance P-mediated abortion

PROBLEM: Stress, elicited by environmental and social conditions, is known to affect the homeostasis of the nervous, endocrine and immune systems. In pregnancy, perceived stress results in a predomination of inflammatory abortion-associated Th1 cytokines over immunosuppressive, pregnancy-protective-associated Th2 cytokines, putatively via neuropeptide substance P (SP). Nerve growth factor (NGF), an important trophic factor for sympathetic neurons, has been implicated in the responsiveness of immune-competent cells through its functional receptor, tropomyosin receptor kinase (TrkA). Thus, the aim of the present study was to identify a cross-talk between distinct neurotrophic and immune mediators in pregnancy maintenance. METHOD OF STUDY: Using immune fluorescence, we evaluated decidual and placental expression of NGF and TrkA on gestation day (gd) 13.5 in the abortion-prone mouse model CBA/J x DBA/2J in (1) CBA/J female control mice; (2) CBA/J mice exposed to stress on gd 5.5; and (3) CBA/J mice injected with SP on gd 5.5 to mimick stress perception. RESULTS: Stress and SP injection significantly increased the abortion rate and up-regulated decidual NGF and TrkA expression compared with the control. Stress, but not SP injection down-regulated placental NGF, whereas no changes in placental TrkA were observed. CONCLUSION: Our data suggest a functional role for NGF in stress-triggered, SP-mediated abortion.     

Immunohistochemical evaluation of the protein expression of nerve growth factor and its TrkA receptor in rat limbic regions following electroshock seizures

Repeated (but not acute) exposure to brief, non-injurious seizures evoked by minimal electroconvulsive shock (ECS) decreases neuronal death in limbic system and increases mRNA levels for nerve growth factor (NGF). Thus, the induction of NGF is a potential mechanism for the neuroprotection evoked by repeated ECS. The neuroprotective action of NGF is mediated by the TrkA receptor. This study determined whether repeated ECS exposure increased TrkA and NGF protein levels. To determine the functional significance of changes in these proteins, we compared the effects of ECS given daily either for 7 days (chronic ECS) or for 1 day (acute ECS). After chronic ECS, upregulation of both NGF and TrkA was found in perirhinal cortex, thalamus, and amygdala. In hippocampus, TrkA was upregulated in CA2, CA3 and CA4. NGF increase in hippocampus was found in CA1 and dentate gyrus. In frontal cortex and substantia innominata, an increase in NGF (but not in TrkA) was found. In most brain regions, TrkA and NGF remained unchanged after acute ECS. Our results demonstrate that repeated exposure to ECS causes an upregulation of TrkA and NGF proteins in several limbic areas in which neuroprotective effects are observed suggesting that NGF contributes to ECS-evoked neuroprotection.     

Effects of increased nerve growth factor plasma levels on the expression of TrkA and p75 in rat testicles

In addition to their well-known roles within the nervous system, the neurotrophins and their receptors regulate some functions in the reproductive system. In this study we used combined morphological and immunohistochemical techniques to investigate the presence and cellular localization in the rat testicle of the two receptors of nerve growth factor (NGF), i.e. TrkA and p75(NTR). Furthermore, to evaluate whether increased plasma levels of NGF affect the ageing process, 4-methylcathechol (4-MC), an inductor of NGF synthesis, was administered. Both TrkA and p75(NTR) were expressed in rat testicles, but the pattern and intensity of immunoreaction were marginally different between them. In adult rats TrkA was expressed in spermatozoa and spermatids, and p75 was expressed in spermatogonia. In newborn rats TrkA immunoreactivity was found in the Leydig cells, whereas p75 was detected in a cellular layer that surrounds the seminiferous tubules. In adult treated animals the immunoreaction for TrkA and p75(NTR) was also localized in the spermatocytes, whereas in newborn treated rats no changes in the pattern of immunoreaction was observed. The present findings suggest a role of the NGF/TrkA/p75 system in the physiology of reproduction, but the practical relevance of this remains to be established.     

Vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF) play a central role in the pathogenesis of digital clubbing

Digital clubbing is associated with many unrelated serious diseases but its pathogenesis remains a clinical enigma. It has been hypothesized that platelet clusters impacting in the distal vasculature mediate the morphological changes of clubbing. Since the multifunctional cytokines vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) are released on platelet aggregation and are hypoxically regulated, the present study has examined their role in clubbing using immunohistochemistry. Basic fibroblast growth factor (bFGF), transforming growth factor-beta 1 (TGF-beta1), microvessel density, carbonic anhydrase IX (CAIX), hypoxia inducible factor (HIF)-1alpha, and HIF-2alpha were also measured. There was a significant increase in VEGF (p = 0.01), pKDR (p = 0.03), PDGF (p = 0.017), and HIF-1alpha and HIF-2alpha (p = 0.004 and p = 0.004, respectively) expression together with a significant increase in microvessel density (p = 0.03) in the stroma in clubbed digits compared with controls. There was no difference in CAIX (p = 0.25), TGF-beta1 (p = 0.66) or bFGF (p = 0.18) between affected and control groups. These findings suggest that VEGF and PDGF are released after platelet impaction and that their expression is hypoxically enhanced in the stroma after capillary occlusion. VEGF may synergize with PDGF in inducing the stromal and vascular changes present in digital clubbing.     

Vascular endothelial growth factor and its type 2 receptor in tumors and serum of patients with renal cancer

It was found that renal cancer tissue is characterized by increased content of VEGF not depending on the parameters of normal tissue and expression of VEGFR2 receptor. At the same time, the content of VEGFR2 in the tumor is determined by the state of the surrounding tissue and increases in low number of patients with tumors invading the renal capsule. Serum concentrations of VEGF and VEGFR2 in patients with renal cancer did not significantly differ from the corresponding values in the control group. No significant correlations between the levels of VEGF and VEGFR2 in the blood and tumor tissue were found. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 145, No. 6, pp. 691–694, June, 2008     

Coexpression of heparanase, basic fibroblast growth factor and vascular endothelial growth factor in human esophageal carcinomas

Heparan sulfate (HS), which is degraded by heparanase, plays an important role in cell adhesion, insolubility of the extracellular matrix (ECM) and as a reservoir for various growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). In the present study, we examined the immunohistochemical expression of heparanase, bFGF and VEGF, and evaluated the correlation between their expression and microvessel density (MVD) in human esophageal carcinomas. Heparanase, bFGF and VEGF were immunolocalized predominantly to the carcinoma cells, but they were also localized to the endothelial cells of microvessels near the carcinoma cell nests. In carcinomas with invasion of the muscular layer or adventitia, heparanase staining was stronger at the invasive areas of carcinomas than the intraepithelial spread. Expression of heparanase and bFGF and the degree of MVD were associated with tumor invasion, lymph node metastasis and pathological stages. Cases with positive staining for heparanase, bFGF or VEGF tended to have a higher MVD than those without staining, and carcinomas with concomitant expression of heparanase, bFGF and VEGF showed the highest MVD. The level of heparanase mRNA expression was directly correlated with the MVD. In addition, heparanase-positive cases had a higher positive ratio of bFGF and VEGF compared with the heparanase-negative cases. These data suggest the possibility that heparanase may contribute to not only cancer cell invasion but also angiogenesis probably through degradation of HS in the ECM and release of bFGF and VEGF from the HS-containing ECM.     

Membrane abnormalities and Ca homeostasis in muscles of the mdx mouse,an animal model of the Duchenne muscular dystrophy: a review

Muscles of the mdx mouse lack dystrophin, a cytoskeletal protein. Mdx fibres exhibit an increased fragility to hypo-osmotic shock and to forced lengthening, an abnormal opening time of stretch-sensitive calcium channels. The question of a chronic elevated [Ca²⁺]_i value is a matter of controversy. We have analysed Ca homeostasis in smooth and skeletal muscles from the adult mdx mouse. The wall of the vas deferens was loaded with the fluorescent Ca indicator Fura-2-AM (cell-diffusible). Resting [Ca²⁺]_i was measured after changes of the electrochemical potential for Ca²⁺ and after KCl or electrical stimulations. In no instance was a difference observed between these and similar muscles from control mice. Single striated fibres were isolated by collagenase treatment of the flexor digitorum brevis muscle and loaded with Fura-2-AM. The value of resting [Ca²⁺]_i was measured using an in situ calibration procedure which took account of Ca buffering by Fura-2. A chronic increase of cytosolic Ca²⁺ was not confirmed. The expression of the intracellular Ca-binding protein, parvalbumin, was measured. It increased by about threefold in fast mdx muscles (tibialis anterior) but remained undetectable in the soleus. It is hypothesized that parvalbumin helps to maintain [Ca²⁺]_i within normal values. This hypothesis will be discussed in connection with dystrophy phenotypes in mutant dogs and in human patients.     

Nerve growth factor survival signaling in cultured hippocampal neurons is mediated through TrkA and requires the common neurotrophin receptor P75

The role of the common neurotrophin receptor p75 (p75NTR) in neuronal survival and cell death remains controversial. On the one hand, p75NTR provides a positive modulatory influence on nerve growth factor (NGF) signaling through the high affinity neurotrophin receptor TrkA, and hence increases NGF survival signaling. However, p75NTR may also signal independently of TrkA, causing cell death or cell survival, depending on the cell type and stage of development. Here we demonstrate that TrkA is expressed in primary cultures of hippocampal neurons and is activated by NGF within 10 min of exposure. In primary hippocampal cultures neuroprotection by NGF against glutamate toxicity was mediated by NF-kappaB and accompanied by an increased expression of neuroprotective NF-kappaB target genes Bcl-2 and Bcl-xl. In mouse hippocampal cells lacking p75NTR (p75NTR-/-) activation of TrkA by NGF was not detectable. Moreover, neuroprotection by NGF against glutamate toxicity was abolished in p75NTR-/- neurons, and the expression of bcl-2 and bcl-xl was markedly reduced as compared to wildtype cells. NGF increased TrkA phosphorylation in hippocampal neurons and provided protection that required phosphoinositol-3-phosphate (PI3)-kinase activity and Akt phosphorylation, whereas the mitogen-activated protein kinases (MAPK), extracellular-regulated kinases (Erk) 1/2, were not involved. P75NTR signaling independent of TrkA, such as increased neutral sphingomyelinase (NSMase) activity causing enhanced levels of ceramide, were not detected after exposure of hippocampal neurons to NGF. Interestingly, inhibition of sphingosine-kinase blocked the neuroprotective effect of NGF, suggesting that sphingosine-1-phosphate was also involved in NGF-mediated survival in our cultured hippocampal neurons. Overall, our results indicate an essential role for p75NTR in supporting NGF-triggered TrkA signaling pathways mediating neuronal survival in hippocampal neurons.     

High level of tubulin microheterogeneity in the mouse brain

Using a high resolution isoelectric focusing method, we have demonstrated a high level of tubulin heterogeneity in mouse brain: 21 isotubulins were identified. Determination of the apparent molecular weight and analysis of the peptide map of each isotubulin species allowed us to identify 7 α and 14 β tubulin subunits. Furthermore, variations in this tubulin population during brain development were confirmed; in particular, an increase in the number of β isotubulins was observed at the beginning of the postnatal period.     

Epidermal growth factor and insulin-like growth factor I are localized in different compartments of salivary gland duct cells. lmmunohistochemical evidence

Immunohistochemical methods were used to map EGF (epidermal growth factor) and IGF-I (insulin-like growth factor I; somatomedin C) immunoreactivities in salivary glands of adult rodents. Epidermal growth factor is, as is NGF (nerve growth factor), limited in distribution to the granules in granular duct cells in the submandibular gland. Insulin-like growth factor I is, in contrast, cytoplasmic and has a much more widespread distribution. It is seen in intercalated, striated and granulated duct cells as well as in apical parts of excretory duct cells. The parotid and the palatine salivary glands, lacking EGF immunoreactivity, have their IGF-I immunoreactivity similarly distributed as the submandibular gland. Isoproterenol treatment of adult male rats results in rapid and extensive growth of the submandibular and the parotid glands, which double their weights in just a few days. Isoproterenol causes release of granules from the submandibular granular duct cells and decrease in frequency of EGF immunoreactive cells. However, there is no or only minor concomitant changes in the distribution and intensity of the IGF-I immunoreactivity in these duct cells. Our results indicate that the trophic peptides EGF (and NGF) and IGF-I are localized in different compartments in salivary gland duct cells and that divergent pathways control their release.