Insulin and the insulin-like growth factors I and II are mitogenic to cultured rat sciatic nerve segments and stimulate [3H]thymidine incorporation through their respective receptors

The factors that control proliferation of Schwann cells during peripheral nerve regeneration are not yet known. In this study we investigated the effects of insulin, insulin-like growth factor I and II (IGF-I and IGF-II), IGF-I analogues, and factors that interfere with their respective receptors, on [³H]thymidine incorporation into cultured nerve segments from the rat sciatic nerve. Segments cultured in nM (0.1–1.7 nM) concentrations of insulin, truncated IGF-I (tIGF-I), long R³IGF-I, or IGF-II exhibited an increase in [³H]thymidine incorporation compared with control segments. IGF-II was most potent. JB1, an IGF-I antagonist, counteracted the effects of tIGF-I and insulin. The results suggest that non-neuronal cells in the nerve segment, probably Schwann cells, possess distinct receptors for insulin, IGF-I, and IGF-II and that these receptors may be involved in the control of Schwann cell proliferation during peripheral nerve regeneration. © 1996 Wiley-Liss, Inc.     

Estrogen and progesterone stimulate Schwann cell proliferation in a sex- and age-dependent manner.

The effects of estrogen and progesterone on Schwann cell proliferation were studied in cultured segments of the rat sciatic nerve from adult male, female, and newborn rats, by measurement of [3H thymidine incorporation or bromo-deoxy-uridine- (BrdU)-labelling and immunocytochemistry. Estrogen (100 nM-500 nM) enhanced [3H] thymidine incorporation in segments from male and newborn rats, while it had no effect on segments from female rats. Progesterone stimulated thymidine incorporation in segments from female and newborn rats (100 nM-500 nM), but caused only a small proliferative response in Schwann cells from male rats at high concentrations. The proliferative effects of estrogen and progesterone were blocked when the segments were cultured in the presence of inhibitors of their respective receptors, ICI 128 780 and zk 112994. The data suggest that Schwann cells possess distinct receptors for estrogen and progesterone and that these receptors may be involved in the control of Schwann cell proliferation. It also shows that the response of Schwann cells to sex hormones varies with sex and perhaps also with age.     

Neuronal stimulation of [3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells

A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl-³H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [³H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [³H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [³H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [³H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [³H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.     

Time-dependent effects of insulin on Schwann cell proliferation in the in vitro regenerating adult frog sciatic nerve

The present study showed that insulin (0.01 μg/ml, ≈? 2 nM) inhibited [³H]-thymidine incorporation in support cells, most likely Schwann cells, of the cultured frog sciatic nerve. A 25–35% inhibition took place in regenerating nerve preparations as well as in preparations devoid of neuronal protein synthesis, i.e., in isolated 5 mm nerve segments and in gangliectomized nerves, suggesting that the effect was direct and not mediated via the neuronal cells. The inhibition by insulin was time-dependent in that an effect was seen after 4 days but not at shorter or at longer periods of culturing. In separate experiments biotinylated insulin was shown to be taken up by Schwann cells in the regenerating nerve. Addition of serum increased the [³H]-thymidine incorporation severalfold and abolished the inhibitory action of insulin. Our results suggest that insulin, at a certain stage of the regeneration programme, exerts a direct, inhibitory effect on the proliferation of the Schwann cells in the cultured frog sciatic nerve. © 1993 Wiley-Liss, Inc.     

Antipsychotics with inverse agonist activity at the dopamine D3 receptor

Summary In NG 108-15 cells expressing the recombinant human D₃ receptor, dopamine agonists enhance [³H]thymidine incorporation and decrease cAMP accumulation. In these cells, but not in wild type cells, haloperidol, fluphenazine, and various other antipsychotics inhibited basal [³H]thymidine incorporation in a concentration-dependent manner. In contrast, other dopamine antagonists such as nafadotride or (+)AJ 76, two D₃-preferring antagonists, were without effect. The concentration-response curve of haloperidol was shifted to the right in presence of nafadotride, with a potency compatible with its nanomolar apparent affinity as neutral antagonist. Pertussis toxin treatment abolished or markedly reduced the responses to haloperidol or fluphenazine. In contrast, no significant enhancement of cAMP accumulation could be observed, under the influence of haloperidol or eticlopride. These data indicate that some dopamine antagonists behave as inverse agonists, and thus appear to inhibit an agonist-independent activity of the D₃ receptor on [³H]thymidine incorporation pathway, but not on the cAMP pathway.     

Bromocriptine inhibits incorporation of [H]thymidine into rat pituitary tumor cells

The effects of bromocriptine on GH₃ pituitary tumor cell [³H]thymidine incorporation were studied. Cells were grown in the presence of bromocriptine, then exposed to a short-term pulse of [³H]thymidine in serum-free medium containing deoxycytidine (10 μM) to prevent deoxythymidine triphosphate (dTTP) pooling. After 48 h exposure to bromocriptine, basal prolactin (PRL)-secretion during 45 min was inhibited by 50% by 10 μM bromocriptine and thyroid releasing hormone-induced PRL stimulation was suppressed. Incorporation of radiolabelled thymidine into acid-precipitable DNA increased progressively from 15 to 60 min and was abolished by simultaneous incubation with excess unlabelled thymidine (100 μM). Bromocriptine (10 μM) inhibited incorporation of 5–50 μM [³H]thymidine, but this was not reversed by simultaneous incubation with metoclopramide (10 μM). Aminopterin, an inhibitor of endogenous de novo DNA synthesis, stimulated [³H]thymidine incorporation twofold and this increased DNA salvage pathway activity was also blocked by bromocriptine. As incorporation of [³H]thymidine into acid-soluble cell neucleotides was also inhibited by bromocriptine, the data suggest that in these cells the drug inhibits thymidine kinase activity, a salvage pathway of DNA synthesis.     

Differences in the sensitivity to purinergic stimulation of myelinating and non-myelinating Shwann cells in peripheral human and rat nerve

Schwann cells of the peripheral nervous system are distinguished by morphological and functional criteria in myelinating and non-myelinating subtypes. We and others have previously reported that Schwann cells in isolated peripheral human and rat nerve respond to extracellular application of ATP with a rise in the intracellular free calcium concentration [Ca²⁺]_i. In the present study, the receptors mediating these Ca²⁺ transients have been investigated in myelinating and non-myelinating Schwann cells of intact fascicles of isolated human sural nerves, rat ventral roots, and rat vagus nerves. Microfluorometry and confocal laser scanning was used on preparations stained with the Ca²⁺-sensitive dyes Calcium Green-1 and Fura Red. In myelinating Schwann cells of human and rat nerves, the ATP-induced rise of [Ca²⁺]_i resulted from the activation of a P2Y₂ purinoceptor subtype (rank order of potency: UTP ≥ ATP ≫ 2-MeSATP = ADP). In contrast, in non-myelinating Schwann cells, Ca²⁺ transients were produced by activation of a P2Y₁ purinoceptor subtype (rank order of potency: 2-MeSATP > ATP > ADP ≫ UTP). The P1 agonist adenosine and α,β-meATP did not evoke Ca²⁺ signals. Ca²⁺ transients in both types of Schwann cells were found to be due to Ca²⁺ release from cyclopiazonic acid-sensitive intracellular stores. However, inhibition by suramin was only found in non-myelinating Schwann cells. These findings indicate that mammalian Schwann cells express phenotype-specific P2Y receptor subtypes. GLIA 23:374–382, 1998. © 1998 Wiley-Liss, Inc.     

Heparin inhibits the growth of astrocytes in vitro

Heparin at a concentration of 100 μg/ml, inhibits neonatal rat astrocyte proliferation by 45%, and [³H]thymidine incorporation into DNA by 55% when they are stimulated with 2% fetal bovine serum (FBS) in culture. Higher serum concentrations up to 10% FBS decreased this inhibition to 20% and 29% respectively. Inhibition of [³H]thymidine incorporation by heparin is dose-dependent with maximal inhibition at 100 μg/ml, but 39% inhibition is still seen at 1 μg/ml on stimulation by 5% FBS. Heparin or heparin-like molecules in the extracellular matrix of brain capillary walls may be important in the regulation of astrocyte growth in vivo.     

Uptake and release of neurotransmitter candidates, [3H]serotonin, [3H]glutamate,and [3H]γ-aminobutyric acid,in taste buds of the mudpuppy,Necturus maculosus

Neurotransmitters in vertebrate taste buds have not yet been identified with confidence. Serotonin, glutamate, and γ-aminobutyric acid (GABA) have been postulated, but the evidence is incomplete. We undertook an autoradiographic study of [³H]serotonin, [³H]glutamate, and [³H]GABA uptake in lingual epithelium from the amphibian, Necturus maculosus, to determine whether taste bud cells would accumulate and release these substances. Lingual epithelium containing taste buds was incubated in low concentrations (0.4–6 μM) of these tritiated transmitter candidates and the tissue was processed for light microscopic autoradiography. Merkel-like basal taste cells accumulated [³H]serotonin. When the tissue was treated with 40 mM K⁺ after incubating the tissue in [³H]serotonin, cells released the radiolabelled transmitter. Furthermore, depolarization (KCl)-induced release of [³H]serotonin was Ca-dependent: if Ca²⁺ was reduced to 0.4 mM and 20 mM Mg²⁺ added to the high K⁺ bathing solution, Merkel-like basal cells did not release [³H]serotonin. In contrast, [³H]glutamate was taken up by several cell types, including non-sensory epithelial cells, Schwann cells, and some taste bud cells. [³H]glutamate was not released by depolarizing the tissue with 40 mM K⁺. [³H]GABA uptake was also widespread, but did not occur in taste bud cells. [³H]GABA accumulated in non-sensory epithelial cells and Schwann cells. These data support the hypothesis that serotonin is a neurotransmitter or neuromodulator released by Merkel-like basal cells in Necturus taste buds. The data do not support (nor rule out) a neurotransmitter role for glutamate or GABA in taste buds. J. Comp. Neurol. 392:199–208, 1998. © 1998 Wiley-Liss, Inc.     

The mechanism of calcium-independent catecholamine depleting action of monensin from clonal rat pheochromocytoma cells

Monensin, a monovalent cation ionophore, induced profound release of radiolabeled materials from clonal rat pheochromocytoma cells (PC12h) preloaded with [³H]norepinephrine (NE). The release was suppressed in the absence of external Na⁺, but was not affected at all in the absence of external Ca²⁺. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), that was monitored by means of a fluorescent Ca²⁺ indicator, Quin 2, was temporarily increased upon a depolarizing stimulus of high-K⁺, which induced the Ca²⁺-dependent release of [³H]NE from PC12h cells. On the other hand, monensin induced only a slight increase in [Ca²⁺]_i. The radiolabeled materials released by high-K⁺ treatment were mainly [³H]NE, whereas those by monensin were mainly the metabolites of [³H]NE. Pargyline, a monoamine oxidase inhibitor, suppressed both the degradation of [³H]NE,stored in PC12h cells and the monensin-induced release of radiolabeled compounds from them. Monensin decreased the content of [³H]NE in storage granules of pargyline-treated cells. Thus, it is likely that monensin expels NE from the storage vesicles to cytosol and then its metabolites by monoamine oxidase are released in a non-exocytotic manner.     

Ca mobilization and capacitative Ca entry regulate DNA synthesis in cultured chick retinal neuroepithelial cells

Release of Ca²⁺ from intracellular Ca²⁺ stores (Ca²⁺ mobilization) and capacitative Ca²⁺ entry have been shown to be inducible in neuroepithelial cells of the early embryonic chick retina. Both types of Ca²⁺ responses decline parallel with retinal progenitor cell proliferation. To investigate their potential role in the regulation of neuroepithelial cell proliferation, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the Ca²⁺ pump of intracellular Ca²⁺ stores, and of SK&F 96365, an inhibitor of capacitative Ca²⁺ entry, on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chicks and in dissociated cultures from E7 and E9 chick retinae. We demonstrate that both antagonists inhibit [³H]-thymidine incorporation in a dose-dependent manner without affecting cell viability or morphology. The inhibition of [³H]-thymidine incorporation by SK&F 96365 occurred in the same concentration range (IC₅₀: 4 μM) as the blockade of capacitative Ca²⁺ entry in the E3 retinal organ culture. At a concentration of 5 μM SK&F 96365, DNA synthesis was reduced by 71, 40 and 32% in the E3, E7 and E9 cultures, respectively. Application of DBHQ at concentrations which led to depletion of intracellular Ca²⁺ stores also inhibited [³H]-thymidine incorporation with IC₅₀ values of 20–30 μM in the different cultures. Our results suggest the involvement of Ca²⁺ mobilization and capacitative Ca²⁺ entry in the regulation of DNA synthesis in the developing neural retina.     

Extracellular matrix upregulates synthesis of glucosylceramide-based glycosphingolipids in primary Schwann cells

The formation of basement membrane around Schwann cells that are in contact with axons is necessary for Schwann cell differentiation and myelin formation in the peripheral nervous system. However, primary Schwann cells grown on basement membrane in the absence of neuronal influence show increased proliferation rather than differentiation, which implies that the signals generated by Schwann cell-basement membrane interactions are multipotential. We examined the effect of matrigel, an exogenous basement membrane preparation, and other extracellular matrix growth surfaces on primary Schwann cells to determine if the resulting interactions play a role in the control of glycosphingolipid synthesis. Isolated primary Schwann cells grown on a thin layer of matrigel rapidly adhered to the surface and exhibited a greater degree of cell spreading when compared to cells grown on the nonspecific substrate polylysine. Labeling of the cells with [³H]galactose between 24 and 48 hr after plating revealed that the incorporation of [³H]galactose into glucosylceramide-based glycosphingolipids increased from 1.5-3-fold on matrigel in comparison to cells grown on polylysine. The major labeled glycolipids under both conditions were GM3 ganglioside and two neutral glycolipids that comigrated with GbOse₄Cer (GalNAcβ1-3Galα1-4Galβ1-1Cer) and GbOse₅Cer (GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) standards. There was little or no increase in the incorporation of [³H]leucine, [³H]galactose, or [³H]glucosamine into proteins or [³H]palmitic acid into phospholipids, free ceramides, or sphingomyelin, suggesting that the matrigel-induced increase in the synthesis of the glycolipids was selective. In the absence of serum, there was little or no difference in the levels of glycolipid labeling between cells grown on the two substrata, demonstrating that serum factors were required for matrigel to have this effect. When cells were grown on surfaces coated with individual extracellular matrix components, those cells grown on laminin and collagen IV showed an increase in glycolipid labeling similar to that produced by matrigel, while labeling increased to a lesser degree for the other components tested. Thus, the signals generated by interactions between Schwann cells and basement membrane, particularly the laminin and collagen IV constituents, contribute to the regulation of glycolipid synthesis which in turn may affect cell morphology and proliferation. © 1996 Wiley-Liss, Inc.     

Glial DNA synthesis and cell proliferation in the lesioned frontal cortex of the rat

Proliferation of rat neuroglial cells was quantified following a lesion of the frontal cortex, with the rate of incorporation of intraventricularly administered [³H]thymidine ([³H]TdR) into cortical DNA serving as an index of glial proliferation. Incorporation of [³H]uridine into the corresponding RNA fractions did not serve this purpose. The intraventricular route of administration of thymidine greatly reduced the amount of [³H]TdR needed to label DNA relative to systemic injection. The rate of incorporation of [³H]TdR into DNA was linear for 75 min post-injection. Significantly more [³H]TdR was incorporated into DNA of the lesioned frontal cortex than that of the contralateral control cortex, during the first 4 days post-trauma. The majority of the acid-insoluble radioactivity (from [³H]TdR) was localized in the nuclear subcellular fraction of the cortex. Experiments indicated that the enhanced incorporation of [³H]TdR was not the result of altered metabolism or pool sizes of TdR in the lesioned cortex. Histological analysis indicated that there was a significant increase in the number of glial cells in the lesioned cortex by day 4 post-lesion, which corresponded to the increase in DNA synthetic activity.It was concluded that mechanical trauma to the frontal cortex of the rat results in an increase in the number of glial cells at and near the lesion which is accompanied by an increase in incorporation of [³H]TdR into cortical DNA. This method of measuring posttraumatic DNA synthesis has several advantages over autoradiography.     

Stimulation of DNA synthesis in Balbc 3T3 cells by peripheral nerve degenerating in vitro

The release of mitogenic substances from degenerating peripheral nerves was detected and characterized in vitro. Cultures of serum-starved, subconfluent $\text{Balb}\text{c}$ 3T3 cells were exposed 24 h to myelinated peripheral nerve fascicles, with [³H]thymidine added during the last 3 h. Cells exposed to peripheral nerves incorporated twice as much [³H]thymidine as control cultures without nerves (P < 0.005). Autoradiography showed a graded decrease in labeling index with increasing distance from nerves. The mitogenic response varied in a dose-dependent manner with increasing nerve length. Also, the response varied according to the degree of myelination. Myelinated sciatic nerve fascicles caused greater incorporation of [³H]thymidine (P < 0.005) than unmyelinated abdominal vagus nerves of similar size, suggesting myelin-derived growth factor activity. Evidence from other laboratories has led to the hypothesis that during peripheral nerve injury, myelin proteins are degraded by lysosome-derived acid proteinases yielding mitogenic polypeptide fragments. We report that the addition of the acid proteinase inhibitor, pepstatin, to the culture media caused a small but significant decrease (P < 0.05) in the mitogenic effect of peripheral nerves. The work supports the concept that the cell proliferation accompanying Wallerian degeneration is stimulated by mitogens released by the injured nerve.     

Neuronal differentiation of Ca channel by nerve growth factor

The inhibitory effect of nicardipine, a potent Ca²⁺ channel blocker in muscular cells, on the Ca²⁺ channel of clonal rat pheochromocytoma cells (PC12h) and cultured rat adrenal medullary cells was studied during the neuronal differentiation mediated by nerve growth factor (NGF). Nicardipine at nM-order concentrations suppressed the high-K⁺-evoked, Ca²-dependent release of preloaded [³H]norepinephrine from PC12h cells and adrenal medullary cells, whereas it scarcely inhibited the release from the cultured rat brainstem cells. The inhibitory actions of nicardipine on both PC12h and newborn rat adrenal medullary cells were significantly decreased after these cells were cultured in the presence of NGF. These results suggest that the changes in Ca²⁺ channel are accompanied by the neuronal differentiation mediated by NGF.     

Transmural compression-induced proliferation and DNA synthesis through activation of a tyrosine kinase pathway in rat astrocytoma RCR-1 cells

Gliosis results from abnormal proliferation of glial cells and often occurs in response to brain or spinal cord injury. There are many factors that trigger gliosis associated with such injuries, including ischemia, humoral factors produced by the injured tissue, and possibly mechanical compression itself. In the present study, the effects of mechanical compression on cell proliferation and DNA synthesis were examined in vitro with the rat astrocyte cell line RCR-1. Pressure was applied to cells by instilling compressed helium into sealed plates or flasks in which the partial pressure of oxygen were maintained constant. Compression resulted in time- and intensity-dependent increases in cell number and [³H]thymidine incorporation, with maximum effects apparent at 10 min and 120 mmHg. Compression-induced cell proliferation and DNA synthesis were not inhibited by gadolinium (Gd³⁺), a blocker of stretch-activated ion channels, or by inhibitors of protein kinase A, protein kinase C, or Ca²⁺/calmodulin-dependent protein kinases. However, the tyrosine kinase inhibitor genistein inhibited these effects of compression in a concentration-dependent manner. Conditioned medium from compressed cells also induced cell proliferation and DNA synthesis at atmospheric pressure in a genistein-sensitive manner. These results suggest that transmural compression triggers the release of a factor (or factors) that induces cell proliferation and DNA synthesis through a tyrosine kinase pathway in RCR-1 cells.     

Different proliferative responses of Gi/o-protein-coupled receptors in human myometrial smooth muscle cells

The majority of studies investigating the proliferative effect of G_i/o-protein-coupled receptor agonists are performed in recombinant receptor systems or cell lines. In these systems the relative stoichiometry of receptors compared to other cell components might be changed, which may lead to anomalies in cellular responses in contrast to natural occurring systems. In the present study, we have used primary cultures of smooth muscle cells (SMCs) isolated from human myometrium to characterize the proliferative effects of agonists binding to two different G protein-coupled receptors. Treatment of quiescent SMCs with lysophosphatidic acid (LPA) and noradrenaline resulted in significant increases in [³H]thymidine incorporation. However, LPA was almost four times more effective than noradrenaline in this respect. The proliferative effects of the agonists could be completely blocked by pertussis toxin, indicating that the response are mediated through G_i/o-proteins. The selective α₂-adrenergic receptor (α₂-AR) antagonist yohimbine dose-dependently reduced the effect of noradrenaline suggesting that the proliferative response was mediated through α₂-ARs. The proliferative effects induced by LPA and noradrenaline was markedly reduced in SMCs treated with the tyrosine kinase inhibitor genistein and the cAMP elevating compound forskolin. However, LPA but not noradrenaline induced rapid rises in the cytosolic free Ca²⁺ concentration [Ca²⁺]_i. The ability to increase Ca²⁺ might be one explanation why LPA produce a more pronounced proliferative response than noradrenaline in primary cultures of human myometrial SMCs.     

Influence of non-neuronal cells on regeneration of the rat sciatic nerve

The ability of the rat sciatic nerve to regenerate into a previously frozen distal nerve segment was studied and compared to regeneration after a crush lesion. The regeneration rate in the frozen segment was 1.9 mm/day, which was approximately half of that observed after a crush lesion (3.3 mm/day). If an unfrozen nerve segment was left intact beyond the frozen section, the rate of regeneration increased to 3.2 mm/day. However, a fresh nerve segment sutured along the frozen segment did not significantly affect the rate of regeneration. Incorporation of [3H]thymidine in the regenerating nerve, analyzed after 1, 3 and 6 days, showed an increased labelling in the frozen segment. This increase spread from the proximal nerve segment into the frozen section. In nerves where a segment was left intact beyond the frozen section, [3H]thymidine incorporation was seen to enter the frozen section from both sides. The spreading of [3H]thymidine incorporation appeared to correlate with the rate of regeneration. However, the same pattern of incorporation could be observed in nerves where regeneration was detained by a transection. The results suggest that Schwann and/or other cells which invade the frozen nerve segment affect the rate of axonal elongation, and that the migration of these cells occurs independently of regenerating fibers.