Melatonin-induced suppression of PC12 cell growth is mediated by its Gi coupled transmembrane receptors

The effects of pertussis toxin, an uncoupler of Gi protein from adenylate cyclase, and luzindole, a competitive inhibitor of melatonin receptor binding, were examined for their ability to inhibit melatonin-induced suppression of PC12 cell growth. Both agents inhibited the melatonin response suggesting that melatonin may be acting through one of its Gi coupled cell surface receptors. This is confirmed by Western blots demonstrating the presence of MT1 receptors in PC12 cells. Coupling of the Gi protein to these receptors is demonstrated by failure of melatonin to suppress cell growth in PKA deficient A126-1B2-1 mutant PC12 cells. Similarly, melatonin failed to prevent cell proliferation when cells were incubated in the presence of the PKA inhibitor, Rp-cAMP. Retinoic acid and dexamethasone, agents known to effect PC12 cell growth and/or differentiation, displayed differential effects on the actions of melatonin. In the presence of melatonin and low concentrations of retinoic acid (100 nM), PC12 cell proliferation was stimulated compared to that seen with either agent alone, whereas no increase in cell proliferation was observed when higher concentrations of retinoic acid (100 microM) were used. The effects of dexamethasone on suppression of PC12 cell growth were additive with that of melatonin whereas, 1,25-dihydroxyvitamin D(3) (IC(50)=10 nM), which by itself had no effect on PC12 cell growth, was found to inhibit the melatonin response. This study demonstrates that inhibition of PC12 cell growth, at physiological concentrations of melatonin, is mediated by cAMP-dependent cell surface receptors and this response is altered by other growth factors known to effect PC12 cell proliferation and differentiation.     

Antioxidant Effect of Melatonin in Human Retinal Neuron Cultures

This study investigates whether the neurohormone melatonin can prevent the retinal neuronal injury caused by reactive oxygen species (ROS) in cultured human retinal neuronal cells. Cultures of human retinal neuronal cells established from a variety of donors were grown to 14 days and then subjected to experimental hypoxanthine/xanthine oxidase (HX/XO)-induced injury. Intracellular production of ROS by administration of HX/XO was confirmed by flow cytometry; the ROS resulted in both apoptotic and necrotic pattern of cell death in the retinal neuron cultures. The efficacy of melatonin against ROS injury was quantitated by MTT assay, enzyme immunoassay, and immunocytochemistry for neurofilament protein. The antioxidative effect of melatonin was compared with that of α-tocopherol. Retinal neuronal injury significantly reduced in a dose–response manner by a treatment of 1.0–8.0 mM α-tocopherol. Melatonin, in concentrations of more than 2.0 mM, also significantly reduced the injury. About 70% of cells are rescued by pretreatment with 1.0 mM α-tocopherol and 8.0 mM melatonin in the MTT assay. Our observations suggest that melatonin can rescue retinal neurons from ROS injury in human retinal cell cultures.     

Melatonin alters the metabolism of the β-amyloid precursor protein in the neuroendocrine cell line PC12

The deposition of amyloid plaques in brain parenchyma is one of the major pathological hallmarks of Alzheimer’s disease (AD). The amyloid in senile plaques is composed of the amyloid β-peptide (Aβ) of 39–43 amino acid residues derived from a larger β-amyloid precursor protein (βAPP). Soluble derivatives of βAPP (sAPP) lacking the cytoplasmic tail, transmembrane domain, and a small portion of the extracellular domain are generated proteolytically by “secretases.” Using cell cultures, the authors analyzed the level of sAPP in neuroblastoma and pheochromocytoma (PC12) cells by immunoblotting samples from conditioned media and cell lysates. Normal levels of secretion of sAPP into conditioned media were severely inhibited by treating cells with melatonin (3–4 mM). The inhibitory effect of melatonin on the secretion of sAPP can be reversed. When the cells that were pretreated with melatonin for 10 h were washed, the normal level of secretion of sAPP was restored. Northern blot analyses indicated that the treatment of PC12 cells with melatonin resulted in a significant decrease in the level of mRNA encoding βAPP, β-actin, and glyceraldehyde-3-phosphate dehydrogenase, and that the treatment of a human neuroblastoma cell line with melatonin resulted in no change in levels of these messages. The secretion of sAPP into the conditioned medium was substantially reduced in the differentiated cells similar to reductions observed in melatonin-treated undifferentiated PC12 cells. Melatonin was found to potentiate the nerve growth factor-mediated differentiation in PC12 cells at 24 h. Taken together, these data suggest that melatonin regulates the metabolism of βAPP and other housekeeping genes in a cell-type specific manner, and that melatonin accelerates the early process of neuronal differentiation.     

Protein kinase C isozymes that mediate enhancement of neurite outgrowth by ethanol and phorbol esters in PC12 cells

Using PC12 cells to study ethanol's effects on growth of neural processes, we found that ethanol enhances NGF- and basic FGF-induced neurite outgrowth. Chronic ethanol exposure selectively up-regulates δ and ε protein kinase C (PKC) and increases PKC-mediated phosphorylation in PC12 cells. Since PKC regulates differentiation, we investigated the role of PKC in enhancement of neurite outgrowth by ethanol. Like ethanol, 0.3–10 nM phorbol 12-myristate, 13-acetate (PMA) increased NGF-induced neurite outgrowth. However, higher concentrations did not, and immunoblot analysis demonstrated that 100 nM PMA markedly depleted cells of β, δ and ε PKC. PMA (100 nM) also down-regulated β, δ and ε PKC in ethanol-treated cells and completely prevented enhancement of neurite outgrowth by ethanol. In contrast, the cAMP analogue 8-bromoadenosine cAMP did not completely mimic the effectsof ethanol on neurite outgrowth, and ethanol was able to enhance neurite formation in mutant PC12 cells deficient in protein kinase A (PKA). These findings implicate β, δ or εPKC, but not PKA, in the neurite-promoting effects of ethanol and PMA. Since chronic ethanol exposure up-regulates δ and ε, but not βPKC, these findings suggest that δ or εPKC regulate neurite outgrowth.     

Ultrastructural confirmation of neuronal protection by melatonin against the neurotoxin 6-hydroxydopamine cell damage

6-Hydroxydopamine (6-OHDA) is a neurotoxin used in the induction of experimental Parkinson's disease in both animals and cultured neuronal cells. Biochemical and molecular approaches showed previously that low doses of 6-OHDA induced apoptosis in PC12 cells, while high doses of this neurotoxin induced necrosis. Melatonin has been shown to protect against the neuronal programmed cell death induced by 6-OHDA, although it was not able to prevent the massive necrotic cellular death occurring after the addition of high doses of the neurotoxin. In the present work, we demonstrate by ultrastructural analysis that although low doses of 6-OHDA induced apoptosis in PC12 cells, it also damaged the non-apoptotic cells, morphologically corresponding this damage to incipient and reversible necrotic lesions. When the doses of the neurotoxin increase, there are still apoptotic cells, although most of the cells show necrotic irreversible lesions. We also found that melatonin partially prevents the incipient necrotic lesions caused by low doses of 6-OHDA. The fact that melatonin was shown in previous work to prevent apoptosis caused by low doses of 6-OHDA, but not necrosis induced by high doses of the neurotoxin, seemed to indicate that this agent is only able to protect against apoptosis. However, our present results, melatonin preventing also the incipient necrotic neuronal lesions, suggest that this hormone may provide a general protection against cell death, suggesting that higher doses should be tried in order to prevent the necrotic cell death induced by high doses of the neurotoxin.     

Staurosporine induces the outgrowth of neurites from the dorsal root ganglion of the chick embryo and PC12D cells

Staurosporine, a potent inhibitor of protein kinases, caused the rapid outgrowth of neurites from cultured dorsal root ganglia of chick embryos and from PC12D cells, a subline of PC12 cells. Treatment of dorsal root ganglia with 1 to 20 nM staurosporine resulted in the extensive outgrowth of neurites that were indistinguishable from those induced by NGF, as assessed by phase-contrast microscopy, electron microscopy and cytochemical staining of actin and tubulin. However, neurites generated from the ganglia in response to the higher concentrations of staurosporine (40–100 nM) seemed to have different characteristics, possible as a result of the inhibition of cell migration from ganglia. The sequential changes in morphology of PC12D cells in response to staurosporine and to NGF were revealed by staining of actin. Ruffling membranes emerged at the margins of PC12D cells within 4 min after the addition of staurosporine or of NGF. From 10 min to 24 h after the addition of either compound, the ruffles were transformed into several projections that became growing neurites. The formation of ruffles and the outgrowth of neurites were both apparent at a concentration of staurosporine of 10 nM. The neurites that emerged from PC12D cells in response to staurosporine and in response to NGF were indistinguishable under the phase-contract microscope and after staining of actin and tubulin. However, staurosporine never promoted survival of PC12D cells in serum-free conditions as that promoted by NGF. The observations indicate that staurosporine at nanomolar concentrations may reproduce the neurogenic changes that induced by NGF in primed neuronal cells, although it can not mimic the action of NGF that supports survival of neurons.     

Amyloid β peptide induces necrosis rather than apoptosis

Amyloid β peptide (AβP), a major component of Alzheimer's disease plaques, is toxic to rat pheochromocytoma PC12 cells and to rat cortical neurons. A reduction in cell survival could be detected after 24 h incubation with 0.01 to 20 μM of the 25–35 peptide fragment (β25–35) of AβP. To study the mechanism of cell death induced by AβP, the morphological as well as the biochemical features of neuronal cell death were analyzed. To distinguish between necrosis and apoptosis, PC12 cell death caused by β25–35 was compared to that induced by serum deprivation, a process known to be apoptotic in these cells. The DNA-degradation pattern of AβP treated cells appeared random rather than at distinct internucleosomal sites as with apoptosis. Electron microscopic studies of NGF-treated PC12 cells and cortical primary cultures exposed to 20 μM β25–35 revealed immediate cellular damage such as vacuolization of the cytoplasm, breakdown of Golgi-apparatus and other membrane systems, and neurite disintegration. This was followed by total collapse of the cytoplasm and cell lysis. These data show that AβP toxicity occurs via a necrotic rather than an apoptotic pathway.     

Neurotransmitters, KCl and antioxidants rescue striatal neurons from apoptotic cell death in culture

Striatal neurons grown in low density culture on serum-free media and in the absence of glia die within 3 days of plating. In this study, we sought to determine the mechanism of cell death (e.g., apoptosis) and whether trophic influences, such as, growth factors, neurotransmitters, antioxidants or KCl-mediated depolarization could improve their survival. We found that striatal neurons grown in this manner die via apoptosis unless treated with one of several different rescuing agents. One way to prevent the death of most striatal neurons was continual treatment with 5–20 μM dopamine (DA) or other monoamines. Although the survival effect of DA was mimicked by the specific D1 receptor agonist, SKF38393, no D1 or D2 receptor antagonists blocked the effect. As with DA, chronic depolarization with KCl (12–39 mM) or treatment with antioxidants, such as the vitamin E analog, Trolox (10–10–500 μM), or the hormone, melatonin (10–10–500 μM) also rescued striatal neurons from impending cell death. Surprisingly, growth factors, such as BDNF, bFGF, GDNF, NGF, NT3 and EGF, demonstrated no ability to rescue striatal neurons in this model, suggesting that death was not solely caused by the absence of essential trophic factors. We conclude that a variety of agents, but not growth factors, can prevent the demise of striatal neurons, presumably by neutralizing damage at one or more steps in the death cascade.     

β-Amyloid polypeptide increases calcium-uptake in PC12 cells: a possible mechanism for its cellular toxicity in Alzheimer's disease

Calcium-uptake into PC12 cells was measured by incubation with⁴⁵Ca after the cells were exposed for 24 h to β-amyloid peptide(1–40) at concentrations between 0 and 46 μM. The rate of influx of⁴⁵Ca into PC12 cells was constant for the first 10 min. For 46 μM β-amyloid peptide(1–40), the rate of influx was about 1,300 ions/s/μm² and the number of cells decreased significantly. There was no significant decrease in cell number when cells were exposed to β-amyloid in calcium-free medium. These results indicate that β-amyloid increases calcium uptake into PC12 cells, and suggest that the increased uptake is responsible for the toxicity of β-amyloid in PC12 cells.     

Evidence for central α2-adrenergic modulation of rat pineal melatonin synthesis

This study was performed to distinguish central and peripheral α₂-adrenoceptors in the inhibition of rat pineal melatonin synthesis. The rats received lipo- or hydrophilic α₂-adrenoceptor ligand injections at middark; after 1 or 2 h the pineal melatonin contents were measured. The lipophilic agonist medetomidine (100 μg/kg s.c.) suppressed the melatonin contents significantly, while the hydrophilic agonists ST-91 and p-aminoclonidine (10 or 100 μg/kg i.v.) did not. The suppression by medetomidine was counteracted by the lipophilic antagonist yohimbine (0.3–3.0 mg/kg i.p.) but not by the hydrophilic antagonist L-659,066 (1–10 mg/kg i.v.). In conclusion, the suppression of nocturnal melatonin synthesis by α₂-adrenoceptor agonists is mainly of central origin.     

Direct actions of anticholinesterases on the neuronal nicotinic acetylcholine receptor channels

Recent studies have suggested that anticholinesterases including organophosphates and carbamates act directly on the nicotinic acetylcholine receptor (AChR) channel. We performed whole-cell and single-channel patch-clamp experiments to elucidate the mechanism of action of anticholinesterases on the nicotinic AChR in rat clonal phaeochromocytoma (PC12) cells. Neostigmine and carbaryl showed a biphasic effect; enhancement and suppression of carbachol-induced whole-cell currents. The currents induced by 100 μM carbachol was enhanced by the first co-application with 10 or 100 μM neostigmine, and the current was eventually suppressed below the control level during repeated co-applications. The decay phase of current was accelerated by neostigmine. Carbaryl at 0.1 μM greatly potentiated the carbachol-induced current, and at higher concentrations (0.3–3 μM), current was suppressed. In single-channel experiments, these compounds increased the short closures or gaps during channel opening without changing the single-channel conductance. Mean open time and burst duration were decreased in the presence of neostigmine and carbaryl. These results indicate that neostigmine and carbaryl directly block the nicotinic AChR channel.     

Melatonin administration to dogs

Summary Melatonin concentrations in serum and urine were examined following oral administration of melatonin to dogs. Four different doses of melatonin ranging from 10 to 80 mg per kg of body weight were given. Melatonin was rapidly absorbed and reached a maximum serum level after 20–30 min, with a distribution phase of 3.5 hours and elimination half life (t1/2) of 5 hours. The fraction excreted in the urine was 0.25% of the administrated dose during the first 5 hours. These results as well as the diurnal rhythm of serum melatonin in the dog are similar to corresponding data reported in the human.This study was presented September 30, 1980 by L. Wetterberg at the International Symposium on Melatonin in Bremen, Federal Republic of Germany.     

In vitro protection of 3-nitropropionic acid-induced toxicity of astrocytes by basic fibroblast growth factor and thrombin

The protective action of basic fibroblast growth factor (bFGF) and thrombin on cultured cortical astrocytes in vitro to the toxic effects of 3-nitropropionic acid (3-NPA) was examined. 3-NPA produced concentration- and time-dependent astrocyte loss as indicated by decrease in the number of glial fibrillary acidic protein (GFAP) positive cells and increase in lactate dehydrogenase (LDH) level of the culture medium. The 3-NPA-induced cellular loss was apparent within 12 h and was maximal at 24 h. The presence of bFGF (10 ng/ml) by itself increased the number of astrocytes at various time intervals, and attenuated the 3-NPA-induced cell loss significantly at various concentrations (0.017–1.7 mM), in 24 and 48 h of exposure. Lower concentrations of thrombin (up to 1 nM) had no effect on the number of astrocytes but higher concentrations (10–100 nM) produced greater cell loss. Thrombin (1 nM) prevented the 3-NPA-induced decrease in GFAP positive cells at both 24 and 48 h intervals. This was further substantiated by the fact that thrombin as low as 0.01 nM attenuated the 3-NPA-induced increase of LDH activity at 24 and 48 h exposure times. But, with 1 nM of thrombin, the attenuation of the LDH activity was seen only at 24 h. The results indicate that 3-NPA produced acute astrocyte toxicity and was attenuated by bFGF and lower concentrations of thrombin.     

Bcl-2 accelerates the neuronal differentiation: new evidence approaching to the biofunction of Bcl-2 in the neuronal system

The proto-oncogene product Bcl-2 is unique in that it inhibits apoptosis rather than promoting cell proliferation. In the present study, we encountered a new possible role of Bcl-2 in the neuronal differentiation. Rat pheochromocytoma PC12 cells have been known as the model of neuronal differentiation by the stimulation of NGF. Bcl-2 transfected PC12 (MB2) cells showed the accelerated neuronal differentiation, as compared with control PC12 (V4) cells. In addition, chemotherapeutic agents Taxol which has been known as neurotoxic compound, induced the acute neuronal cell atrophy and suppressed neuronal differentiation. This neuronal cell atrophy and suppression of neuronal differentiation were not due to apoptotic cell death. Interestingly, Bcl-2 rescued PC12 cells from both neuronal cell atrophy and suppression of neuronal differentiation. Taxol suppressed polymerization between neurofilament light and heavy (NF-L and NF-H), and MB2 cell extract rescued it. We, therefore, suggest the acceleration of polymerization between NF-L and NF-H as the new possible role of Bcl-2.     

Melatonin enhances horizontal cell sensitivity in salamander retina

Intracellular electrophysiological recording techniques were utilized to investigate the possible function of retinal melatonin in the larval tiger salamander. Endogenous retinal melatonin was present and appeared to bind to a membrane-enriched fraction of the salamander retina, as determined by radioimmunoassay and receptor binding studies. Melatonin added through the purfusion bath to flat-mounted retinas resulted in a horizontal cell(HC) hyperpolarization of 10–20 mV. Additionally, the amplitude of HC responses to short test flashes increased in the presence of melatonin. Voltage-intensity plots revealed that application of 500 μM of melatonin caused an increase of the HC light sensitivity and this effect was reversible. These results suggest that melatonin synthesized and released during the dark period of the diurnal cycle may alter the sensitivity of second-order neurons at a time of day when photopic input is at its lowest level.     

Circadian rhythm of melatonin release in pineal gland culture: arg-vasopressin inhibits melatonin release

The mammalian pineal gland is known to receive a noradrenergic sympathetic efferent signal from the suprachiasmatic nucleus (SCN) via the superior cervical ganglion. Arg-vasopressin (AVP) containing neurons in the SCN is one of the output paths of circadian information to the other brain areas. AVP release from the SCN is suppressed by melatonin. In turn, we determined the direct effect of AVP on melatonin release using pineal gland explant culture. AVP (1 μM) suppressed melatonin release. Noradrenaline stimulated melatonin release was attenuated by AVP. In turn, the expression of the melatonin synthesis enzyme arylalkylamine N-acetyltransferase mRNA in the rat SCN was reported. We measured melatonin content in the SCN in rats kept under the light–dark cycle and constant dim light. Melatonin in the SCN was higher during the dark period than that in the light. A similar tendency was also observed in the SCN of animals kept under a constant dim light. It was suggested that the reciprocal regulation of melatonin release and AVP release occurs in the SCN and pineal gland.     

Differential effect of calmodulin antagonists on MG132-induced mitochondrial dysfunction and cell death in PC12 cells

Defects in proteasome function have been suggested to be involved in the pathogenesis of neurodegenerative diseases. We examined the effect of calmodulin antagonists on proteasome inhibitor-induced mitochondrial dysfunction and cell viability loss in undifferentiated PC12 cells. Caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk) and antioxidants attenuated cell death and decrease in GSH contents in PC12 cells treated with 20 microM MG132, a proteasome inhibitor. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) had a differential inhibitory effect on the MG132-induced cell death and GSH depletion depending on concentration with a maximal inhibitory effect at 0.5-1 microM. Addition of trifluoperazine and W-7 reduced the MG132-induced nuclear damage, loss of the mitochondrial transmembrane potential followed by cytochrome c release, formation of reactive oxygen species and elevation of intracellular Ca(2+) levels in PC12 cells. Calmodulin antagonists at 5 microM exhibited a cytotoxic effect on PC12 cells but attenuated the cytotoxicity of MG132. The results suggest that the toxicity of MG132 on PC12 cells is mediated by activation of caspase-8, -9 and -3. Trifluoperazine and W-7 at the concentrations of 0.5-1 microM may attenuate the MG132-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering of the intracellular Ca(2+) levels as well as calmodulin inhibition.     

Stereological estimates of postnatal structural differentiation in a sexually dimorphic hypothalamic nucleus involved in vocal control

Display of a specific, courtship vocalization and other masculine functions in the adult gerbil, is associated with a sexually differentiated hypothalamic nucleus, the Sexually Dimorphic Area pars compacta (SDApc). Total SDApc volume and vocal function differentiate neonatally. Since total volume is a rudimentary measure of brain nucleus differentiation, we examined the more detailed cytoarchitectural parameters behind SDApc development in gerbils, cell number, density per nucleus and individual nuclear (soma) volume. Unbiased stereological estimates were made on thick (20–40 μm) brain sections from postnatal days 1 (D1), 3 (D3), 6 (D6), 16 (D16), 40 (D40) and 60 (D60) animals. Sex differences in stereological parameters were not apparent on D1 but from D3, SDApc growth patterns widely differed between the sexes. Significant differences in (i) cell number, and (ii) nuclear volume were found at D3 and D60, respectively. In males, cell number increased between D1–D6 but subsequently decreased from the D6 value by approximately 80% to reach the value of D16 which remained constant. Cell density paralleled the decrease in cell number between D6–D16 in males, whereas a progressive expansion in nuclear volume occurred between D1–D40. Male total SDApc volume enlarged between D1–D3 and D40–D60. Conversely in females, cell number and density declined between D1–D3 and D1–D40, respectively, and then remained at these low values. Cell volume, however, increased up to D40 and then significantly decreased. The resulting change to female total SDApc volume was a reduction immediately after birth, D1–D3, to a constant low value. We conclude that first, the association between various stereological measures and total SDApc volume was minimal, suggesting independent mechanisms of sexual differentiation for each cytoarchitectonic parameter. Second, the neonatal peak in SDApc cell number indicates cell migration taking place contemporaneously with cell death in males. Third, the effect of changes in cytoarchitectural components between D6–D16 and D40–D60 in males is probably due to SDApc dendritic volume expansion, suggesting that the male SDApc retains plasticity until at least puberty. Fourth, the decrease in the number of cells in females early in neonatal life, suggests programmed cell death.     

Gi and RGS proteins provide biochemical control of androgen receptor nuclear exclusion

Nuclear localization of androgen receptors (ARs) is essential for their activity. Melatonin induces AR nuclear exclusion via increase in cGMP, calcium, and protein kinase C (PKC) activation, presumably through G-protein(s). The effects of regulators of G-protein signaling (RGS) on AR localization were studied in AR-expressing PC3 cells. Gi-specific RGS10 inhibited melatonin but not cGMP-induced AR nuclear exclusion, independent of androgen. No evidence for Gq activation by melatonin was found. However, Gi/Gq-selective RGS4 inhibited AR nuclear exclusion downstream of PKC activation—an effect that was abrogated by constitutively active Gq. RGS10 and RGS4, but not RGS2, ablated the inhibitory effects of melatonin on AR reporter gene activity. For the first time, these data show regulation by Gi and Gi-specific RGS protein-mediated AR nuclear exclusion, which is potentially important in the treatment of AR-dependent cancers and neurodegenerative disorders. They also reveal a role for a Gq protein downstream of PKC activation in AR nuclear localization.