Platelet-rich preparations to improve healing. Part I: workable options for every size practice.

Numerous studies have demonstrated that platelet-rich preparations applied to surgical sites, injuries, or wounds are a safe and effective way to promote soft tissue healing and bone growth. Various protocols have been developed for preparing platelet-rich preparations, with subtle but important differences between them. Unfortunately, only a minority of clinicians use platelet-rich preparations, such as platelet-rich plasma and platelet-rich fibrin, in their practice, possibly due to confusion about the different methods and their advantages and disadvantages. Therefore, the different types of preparations are described to help guide the selection of the best method for any size practice. Classic methods generally require large volumes of blood and can be expensive, complicated, and time-intensive. Simpler protocols have been developed recently, which require relatively inexpensive equipment and small blood volumes and, thus, may be more applicable for small clinical practices. Platelet-rich preparations accelerate healing at earlier time points to reduce discomfort and the potential for adverse outcomes, including infection, poor wound closure, and delays in forming strong bone for subsequent procedures (such as implants). However, platelet-rich preparations may also improve long-term outcomes in patients expected to have impaired healing, such as with lifestyle choices (eg, smoking), medications (eg, steroids), diseases (eg, diabetes, osteoporosis, atherosclerosis), and aging, by supplementing the deficient wound environment to restore proper healing. Therefore, both large and small clinical practices would benefit from utilizing platelet-rich preparations to enhance healing in their patients.

Platelet-rich preparations to improve healing. Part II: platelet activation and enrichment, leukocyte inclusion, and other selection criteria.

Multiple platelet-rich preparations have been reported to improve wound and bone healing, such as platelet-rich plasma (PRP) and platelet rich fibrin (PRF). The different methods employed during their preparation are important, as they influence the quality of the product applied to a wound or surgical site. Besides the general protocol for preparing the platelet-rich product (discussed in Part 1 of this review), multiple choices need to be considered during its preparation. For example, activation of the platelets is required for the release and enmeshment of growth factors, but the method of activation may influence the resulting matrix, growth factor availability, and healing. Additionally, some methods enrich leukocytes as well as platelets, but others are designed to be leukocyte-poor. Leukocytes have many important roles in healing and their inclusion in PRP results in increased platelet concentrations. Platelet and growth factor enrichment reported for the different types of platelet-rich preparations are also compared. Generally, TGF-ß1 and PDGF levels were higher in preparations that contain leukocytes compared to leukocyte-poor PRP. However, platelet concentration may be the most reliable criterion for comparing different preparations. These and other criteria are described to help guide dental and medical professionals, in large and small practices, in selecting the best procedures for their patients. The healing benefits of platelet-rich preparations along with the low risk and availability of simple preparation procedures should encourage more clinicians to incorporate platelet-rich products in their practice to accelerate healing, reduce adverse events, and improve patient outcomes.

Determination of the minimal melatonin exposure required to induce osteoblast differentiation from human mesenchymal stem cells and these effects on downstream signaling pathways.

The purpose of this study was to determine the critical time periods of melatonin treatment required to induce human mesenchymal stem cells (hAMSCs) into osteoblasts and to determine which osteogenic genes are involved in the process. The study design consisted of adding melatonin for different times (2, 5, 10, 14 or 21 days) toward the end of a 21-day treatment containing osteogenic (OS+) medium or at the beginning of the 21-day treatment and then withdrawn. The results show that a 21-day continuous melatonin treatment was required to induce both alkaline phosphatase (ALP) activity and calcium deposition and these effects were mediated through MT2Rs. Functional analysis revealed that peak ALP levels induced by melatonin were accompanied by attenuation of melatonin-mediated inhibition of forskolin-induced cAMP accumulation. Immunoprecipitation and western blot analyses, respectively, showed that MT2R/ß-arrestin scaffolds complexed to Gi, MEK1/2 and ERK1/2 formed in these differentiated hAMSCs (i.e., when ALP levels were highest) where ERK1/2 resided primarily in the cytosol. It is hypothesized that these complexes form to modulate the subcellular localization of ERK1/2 to affect osteogenic gene expression. Using real-time RT-PCR, chronic melatonin exposure induced the expression of osteogenic genes RUNX-2, osteocalcin and BMP-2, through MT2Rs. No melatonin-mediated changes in the mRNA expression of ALP, BMP-6 or in the oxidative enzymes MtTFA, PGC-1α, Polγ, NRF-1, PDH, PDK and LDH occurred. These data show that a continuous 21-day melatonin exposure is required to induce osteoblast differentiation from hAMSCs through the formation of MT2R/Gi/ß-arrestin/MEK/ERK1/2 complexes to induce osteogenesis.

Platelet rich plasma to facilitate wound healing following tooth extraction.

Following tooth removal bone formation normally takes 16 weeks and may result in less than adequate volume for the necessary reconstruction. Platelet rich plasma (PRP) has been promoted as an effective method for improving bone formation. Its use is often expensive, time consuming, or not clinically convenient for the patient and/or clinician. This study examines a simple method for obtaining a "Buffy Coat"-PRP (BC-PRP) and its effect on bone healing following the removal of bilateral mandibular 3rd molars. Subtraction digital radiography and CT scan analysis were used to track changes in radiographic density at PRP treated sites in comparison to ipsilateral non-PRP treated sites. PRP treated sites demonstrated early and significant increased radiographic density over baseline measurements following tooth removal. The greatest benefit of PRP is during the initial 2-week postoperative healing time period (P < .001). During weeks 3 though 12, BC-PRP treatment resulted in significant (P < .0001) increases in bone density compared to control, but there was no significant interaction between time and treatment (P > .05). For the entire time period (0-25 weeks) PRP treatment was significant (P < .0001) and time was significant (P < .0001) but there was no significant interaction (P > .05) between the effect of PRP treatment and time. It required 6 weeks for control extraction sites to reach comparable bone density that PRP treated sites achieved at week 1. Postoperative pain, bleeding, and numbness were not significantly affected by BC-PRP application. Results suggest that this simple technique may be of value to clinicians performing oral surgery by facilitating bone regeneration following tooth extraction.

Microtubules modulate melatonin receptors involved in phase-shifting circadian activity rhythms: in vitro and in vivo evidence.

MT1 melatonin receptors expressed in Chinese hamster ovary (CHO) cells remain sensitive to a melatonin re-challenge even following chronic melatonin exposure when microtubules are depolymerized in the cell, an exposure that normally results in MT1 receptor desensitization. We extended our findings to MT2 melatonin receptors using both in vitro and in vivo approaches. Using CHO cells expressing human MT2 melatonin receptors, microtubule depolymerization prevents the loss in the number of high potency states of the receptor when compared to melatonin-treated cells. In addition, microtubule depolymerization increases melatonin-induced PKC activity but not PI hydrolysis via Gi proteins similar to that shown for MT1Rs. Furthermore, microtubule depolymerization in MT2-CHO cells enhances the exchange of GTP on Gi-proteins using a photoaffinity analog of GTP. To test whether microtubules are capable of modulating melatonin-induced phase-shifts, microtubules are depolymerized specifically within the suprachiasmatic nucleus of the hypothalamus (SCN) of the Long Evans rat and the efficacy of melatonin to phase shift their circadian activity rhythms was assessed and compared to animals with intact SCN microtubules. We find that microtubule depolymerization in the SCN using either Colcemid or nocodazole enhances the efficacy of 10 pm melatonin to phase-shift the activity rhythms of the Long Evans rat. No enhancement occurs in the presence of beta-lumicolchicine, the inactive analog of Colcemid. Taken together, these data suggest that microtubule dynamics can modulate melatonin-induced phase shifts of circadian activity rhythms which may explain, in part, why circadian disturbances occur in individuals afflicted with diseases associated with microtubule disturbances.

Transcriptional response of rat frontal cortex following acute in vivo exposure to the pyrethroid insecticides permethrin and deltamethrin.

BACKGROUND: Pyrethroids are neurotoxic pesticides that interact with membrane bound ion channels in neurons and disrupt nerve function. The purpose of this study was to characterize and explore changes in gene expression that occur in the rat frontal cortex, an area of CNS affected by pyrethroids, following an acute low-dose exposure. RESULTS: Rats were acutely exposed to either deltamethrin (0.3 - 3 mg/kg) or permethrin (1 - 100 mg/kg) followed by collection of cortical tissue at 6 hours. The doses used range from those that cause minimal signs of intoxication at the behavioral level to doses well below apparent no effect levels in the whole animal. A statistical framework based on parallel linear (SAM) and isotonic regression (PIR) methods identified 95 and 53 probe sets as dose-responsive. The PIR analysis was most sensitive for detecting transcripts with changes in expression at the NOAEL dose. A sub-set of genes (Camk1g, Ddc, Gpd3, c-fos and Egr1) was then confirmed by qRT-PCR and examined in a time course study. Changes in mRNA levels were typically less than 3-fold in magnitude across all components of the study. The responses observed are consistent with pyrethroids producing increased neuronal excitation in the cortex following a low-dose in vivo exposure. In addition, Significance Analysis of Function and Expression (SAFE) identified significantly enriched gene categories common for both pyrethroids, including some relating to branching morphogenesis. Exposure of primary cortical cell cultures to both compounds resulted in an increase (approximately 25%) in the number of neurite branch points, supporting the results of the SAFE analysis. CONCLUSION: In the present study, pyrethroids induced changes in gene expression in the frontal cortex near the threshold for decreases in ambulatory motor activity in vivo. The penalized regression methods performed similarly in detecting dose-dependent changes in gene transcription. Finally, SAFE analysis of gene expression data identified branching morphogenesis as a biological process sensitive to pyrethroids and subsequent in vitro experiments confirmed this predicted effect. The novel findings regarding pyrethroid effects on branching morphogenesis indicate these compounds may act as developmental neurotoxicants that affect normal neuronal morphology.

Protein biomarkers associated with growth and synaptogenesis in a cell culture model of neuronal development.

Cerebellar granule cells (CGC) provide a homogenous population of cells which can be used as an in vitro model for studying the cellular processes involved in the normal development of the CNS. They may also be useful for hazard identification as in vitro screens for developmental neurotoxicity. The present study examined morphologic and biochemical markers of CGC neurite outgrowth and synaptogenesis in vitro using both qualitative and quantitative approaches. CGC exhibit a rapid outgrowth of neurites over 14 days in vitro, concomitant with the expression of the synaptic protein Synapsin 1 that was observed as puncta associated with cell bodies and neurites. The expression of neurotypic proteins associated with the cytoskeleton (NF68, MAP2), growth cones (GAP-43) and the synapse (Synapsin I) present an ontogeny that reflects the morphological growth of CGC. The utility of these neurotypic proteins as biomarkers was examined by inhibiting CGC growth using pharmacologic inhibitors of PKC activity and the MAP kinase pathway. Quantitative analysis of neurite outgrowth was performed using an automated image acquisition and analysis system. Treatment of CGC with the MAP kinase pathway inhibitor U0126 significantly decreased total neurite outgrowth, while the inhibitor of classic PKC isoforms Bis I had no effect on this measure. The ontogenetic expression of neurotypic proteins was reduced after treatment with both inhibitors. In particular, Synapsin 1 and GAP-43 expression were both significantly reduced by chemical treatment. These data demonstrate that neurotypic proteins can be used as biomarkers of neuronal development in vitro, and in some cases, may detect changes that are not apparent using morphologic measures.

Developmental neurotoxicity testing in vitro: models for assessing chemical effects on neurite outgrowth.

In vitro models may be useful for the rapid toxicological screening of large numbers of chemicals for their potential to produce toxicity. Such screening could facilitate prioritization of resources needed for in vivo toxicity testing towards those chemicals most likely to result in adverse health effects. Cell cultures derived from nervous system tissue have proven to be powerful tools for elucidating cellular and molecular mechanisms of nervous system development and function, and have been used to understand the mechanism of action of neurotoxic chemicals. Recently, it has been suggested that in vitro models could be used to screen for chemical effects on critical cellular events of neurodevelopment, including differentiation and neurite growth. This review examines the use of neuronal cell cultures as an in vitro model of neurite outgrowth. Examples of the cell culture systems that are commonly used to examine the effects of chemicals on neurite outgrowth are provided, along with a description of the methods used to quantify this neurodevelopmental process in vitro. Issues relating to the relevance of the methods and models currently used to assess neurite outgrowth are discussed in the context of hazard identification and chemical screening. To demonstrate the utility of in vitro models of neurite outgrowth for the evaluation of large numbers of chemicals, efforts should be made to: (1) develop a set of reference chemicals that can be used as positive and negative controls for comparing neurite outgrowth between model systems, (2) focus on cell cultures of human origin, with emphasis on the emerging area of neural progenitor cells, and (3) use high-throughput methods to quantify endpoints of neurite outgrowth.

Analysis of a rapid, simple, and inexpensive technique used to obtain platelet-rich plasma for use in clinical practice.

The use of platelet-rich plasma (PRP) has become more generally accepted, and implant dentists are using PRP more frequently to promote the healing of oral surgical and/or periodontal wounds. Critical elements of PRP are thought to be growth factors contained within the concentrated platelets. These growth factors are known to promote soft-tissue healing, angiogenesis and osteogenesis. We present a rapid, simple, and inexpensive methodology for preparing PRP using the Cliniseal centrifuge method. This study demonstrates that platelets are concentrated approximately 6-fold without altering platelet morphology. Further we demonstrate that key growth factors, platelet-derived growth factor BB (PDGF-BB), transforming growth factor B (TGF-B1), vasculature endothelial growth factor (VEGF), and epidermal growth factor (EGF) are present in comparable or higher concentrations than those reported with the use of other techniques. Prolonged bench set time (>3 hours) after centrifugation resulted in decreased concentration of TGF-B1 but not decreased concentration of PDGF-BB, VEGF, or EGF. This study confirms the molecular aspects of PRP obtained using this inexpensive and efficient methodology.

Expanding the test set: Chemicals with potential to disrupt mammalian brain development.

High-throughput test methods including molecular, cellular, and alternative species-based assays that examine critical events of normal brain development are being developed for detection of developmental neurotoxicants. As new assays are developed, a "training set" of chemicals is used to evaluate the relevance of individual assays for specific endpoints. Different training sets are necessary for each assay that would comprise a developmental neurotoxicity test battery. In contrast, evaluation of the predictive ability of a comprehensive test battery requires a set of chemicals that have been shown to alter brain development after in vivo exposure ("test set"). Because only a small number of substances have been well documented to alter human neurodevelopment, we have proposed an expanded test set that includes chemicals demonstrated to adversely affect neurodevelopment in animals. To compile a list of potential developmental neurotoxicants, a literature review of compounds that have been examined for effects on the developing nervous system was conducted. The search was limited to mammalian studies published in the peer-reviewed literature and regulatory studies submitted to the U.S. EPA. The definition of developmental neurotoxicity encompassed changes in behavior, brain morphology, and neurochemistry after gestational or lactational exposure. Reports that indicated developmental neurotoxicity was observed only at doses that resulted in significant maternal toxicity or were lethal to the fetus or offspring were not considered. As a basic indication of reproducibility, we only included a chemical if data on its developmental neurotoxicity were available from more than one laboratory (defined as studies originating from laboratories with a different senior investigator). Evidence from human studies was included when available. Approximately 100 developmental neurotoxicity test set chemicals were identified, with 22% having evidence in humans.

Neurite outgrowth assessment using high content analysis methodology.

High content analysis of neurite outgrowth enables the rapid and comprehensive phenotypic assessment of individual neurons in a multiwell format amenable to high throughput assays. The resulting data are considered "high content" because multiple measurements of neuronal outgrowth and morphometric data are calculated from hundreds of individual cells within each image. This approach has been widely adopted by the pharmaceutical industry to accelerate neurological drug discovery and in vitro safety assessment. High content technology utilizes automated fluorescent and/or brightfield microscopy for image acquisition. The acquired images are then quantified using mathematical algorithms to measure pertinent neurobiological morphometric information, including neurite length, count, and extent of branching for each cell within the images. Furthermore, evaluation of the individual cell-level measurements enables the detection of subpopulations of cellular responders not apparent when examining well-level averages. Using this technology, neurite outgrowth can be quantified in each well, derived from hundreds of cell measurements in a 96-well microplate in approximately 30 min.

Neuronal models for evaluation of proliferation in vitro using high content screening.

In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity (hazard identification). In order to identify potential developmental neurotoxicants, a battery of in vitro tests for neurodevelopmental processes such as cell proliferation, differentiation, growth, and synaptogenesis has been proposed. The development of in vitro approaches for toxicity testing will require choosing a model system that is appropriate to the endpoint of concern. This study compared several cell lines as models for neuronal proliferation. The sensitivities of neuronal cell lines derived from three species (PC12, rat; N1E-115, mouse; SH-SY5Y, human) to chemicals known to affect cell proliferation were assessed using a high content screening system. After optimizing conditions for cell growth in 96-well plates, proliferation was measured as the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA during S phase. BrdU-labeled cells were detected by immunocytochemistry and cell counts were obtained using automated image acquisition and analysis. The three cell lines showed approximately 30-40% of the population in S phase after a 4h pulse of BrdU. Exposure to the DNA polymerase inhibitor aphidicolin for 20 h prior to the 4h pulse of BrdU significantly decreased proliferation in all three cell lines. The sensitivities of the cell lines were compared by exposure to eight chemicals known to affect proliferation (positive controls) and determination of the concentration inhibiting proliferation by 50% of control (I(50)). PC12 cells were the most sensitive to chemicals; 6 out of 8 chemicals (aphidicolin, cadmium, cytosine arabinoside, dexamethasone, 5-fluorouracil, and methylmercury) inhibited proliferation at the concentrations tested. SH-SY5Y cells were somewhat less sensitive to chemical effects, with five out of eight chemicals inhibiting proliferation; dexamethasone had no effect, and cadmium inhibited proliferation only at concentrations that decreased cell viability. Data from the N1E-115 cell line was extremely variable between experiments, and only 4 out of 8 chemicals resulted in inhibition of proliferation. Chemicals that had not been previously shown to alter proliferation (negative controls) did not affect proliferation or cell viability in any cell line. The results show that high content screening can be used to rapidly assess chemical effects on proliferation. Three neuronal cell lines exhibited differential sensitivity to the effect of chemicals on this endpoint, with PC12 cells being the most sensitive to inhibition of proliferation.

Comparison of PC12 and cerebellar granule cell cultures for evaluating neurite outgrowth using high content analysis.

Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process underlying nervous system development that can be quantified using automated microscopy and image analysis (high content analysis). The present study characterized and compared the PC-12 cell line (NS-1) and primary cultures of cerebellar granular cells (CGC), as models for assessing chemical effects on neurite outgrowth. High content analysis of neurite outgrowth was performed using the Cellomics ArrayScan V(Ti) automated epifluorescent imaging system to acquire and analyze images of beta-tubulin immunostained cells in 96-well plates. Cell viability was assessed using the CellTiter-Glo assay. Culture of NS-1 or CGC in nerve growth factor or serum respectively, rapidly induced neurite outgrowth that increased over four days in vitro. Seven compounds previously shown to affect neurite outgrowth in vitro were tested in both models for changes in total neurite length and cell viability. In NS-1 cells, four chemicals (PKC inhibitor Bis-I, MEK inhibitor U0126, trans-Retinoic acid, methylmercury) inhibited neurite outgrowth, while lead, amphetamine and valproic acid had no effect. In CGC, five chemicals inhibited neurite outgrowth (Bis-I, U0126, lead, methylmercury, and amphetamine), while trans-Retinoic acid decreased cell viability but not neurite outgrowth. Valproic acid was without effect. The sensitivity of the two models was chemical specific: NS-1 cells were more sensitive to Bis-I, methylmercury and trans-Retinoic acid, while CGC were more sensitive to U0126, lead, and amphetamine. For every chemical (except trans-Retinoic acid), neurite outgrowth was equal to or more sensitive than cell viability. In comparison, out of seven chemicals without prior evidence for effects on neurite outgrowth, only one decreased neurite outgrowth (diphenhydramine in CGC). These findings demonstrate that the effects of chemicals on neurite outgrowth may be cell type specific.

Development of a high-throughput screening assay for chemical effects on proliferation and viability of immortalized human neural progenitor cells.

There is considerable public concern that the majority of commercial chemicals have not been evaluated for their potential to cause developmental neurotoxicity. Although several chemicals are assessed annually under the current developmental neurotoxicity guidelines, time, resource, and animal constraints prevent testing of large numbers of chemicals using this approach. Thus, incentive is mounting to develop in vitro methods to screen chemicals for their potential to harm the developing human nervous system. As an initial step toward this end, the present studies evaluated an automated, high-throughput method for screening chemical effects on proliferation and viability using ReNcell CX cells, a human neural progenitor cell (hNPC) line. ReNcell CX cells doubled in approximately 36 h and expressed the neural progenitor markers nestin and SOX2. High-throughput assays for cell proliferation (5-bromo-2'-deoxyuridine incorporation) and viability (propidium iodide exclusion) were optimized and tested using known antiproliferative compounds. The utility of this in vitro screen was evaluated further using a set of compounds containing eight known to cause developmental neurotoxicity and eight presumably nontoxic compounds. Six out of eight developmental neurotoxicants significantly inhibited ReNcell CX cell proliferation and/or viability, whereas two out of eight nontoxic chemicals caused only minimal effects. These results demonstrate that chemical effects on cell proliferation and viability can be assessed via high-throughput methods using hNPCs. Further development of this approach as part of a strategy to screen compounds for potential effects on nervous system development is warranted.

Assessment of chemical effects on neurite outgrowth in PC12 cells using high content screening.

Identification of chemicals that pose a hazard to the developing nervous system is the first step in reducing human exposure and preventing health risks to infants and children. In response to the need for more efficient methods to identify potential developmental neurotoxicants, the present study evaluated the utility of an automated high content screening system to detect chemical effects on neurite outgrowth in Neuroscreen-1 cells (NS-1), a subclone of PC12 cells. Plating 2000 NS-1 cells per well with 100 ng/ml nerve growth factor for 96 h produced optimal neurite growth in a 96-well format. Using this protocol, five chemicals that had been previously shown to inhibit neurite outgrowth in PC12 cells were examined. Inhibition of neurite outgrowth (assessed as total neurite length per cell) was observed for all five chemicals. For three of the chemicals, inhibition was associated with decreased cell viability. To demonstrate the utility of this approach for screening, a further set of chemicals (eight known in vivo developmental neurotoxicants and eight chemicals with little evidence of in vivo neurotoxicity) were tested over a wide concentration range (1 nM-100 microM). Trans-retinoic acid, dexamethasone, cadmium, and methylmercury inhibited neurite outgrowth, although dexamethasone and cadmium only affected neurite outgrowth at concentrations that decreased viability. Amphetamine facilitated neurite outgrowth, whereas valproic acid, diphenylhydantoin, and lead had no effect. Of the chemicals that were not neurotoxic, there were no effects on cell viability, but two (dimethyl phthalate and omeprazole) increased neurite outgrowth at the highest concentration tested. These results demonstrate that a high content screening system can rapidly quantify chemical effects on neurite outgrowth in vitro. Concentration-response data for both neurite outgrowth and cell viability allowed for the determination of the specificity of chemical effects on a neurodevelopmental endpoint. Further studies will examine the utility of other in vitro preparations for cell-based assays of neurite outgrowth.

Melatonin enhances alkaline phosphatase activity in differentiating human adult mesenchymal stem cells grown in osteogenic medium via MT2 melatonin receptors and the MEK/ERK (1/2) signaling cascade.

The goals of this study were to determine (a) if melatonin enhances human adult mesenchymal stem cell (hAMSC) differentiation into osteoblasts as assessed by measuring alkaline phosphatase (ALP) enzyme activity, and (b) identify potential signal transduction pathways that mediate this process. ALP activity significantly increased in hAMSCs following a 10-day incubation in osteogenic medium, relative to hAMSCs incubated in basal growth medium alone. Melatonin (50 nm), added in combination with the osteogenic medium, significantly increased ALP activity relative to osteogenic medium alone. Co-exposure of hAMSCs to osteogenic medium supplemented with melatonin and either pertussis toxin or the melatonin receptor antagonists, luzindole or 4P-PDOT (MT2 receptor selective), inhibited the melatonin-induced increase in ALP activity, indicating the involvement of melatonin receptors, in particular, MT2 receptors. Assessment of melatonin receptor function following exposure to osteogenic medium containing either vehicle or melatonin produced dichotomous results. That is, if the differentiation of hAMSCs into an osteoblast was induced by osteogenic medium alone, then 2-[125I]-iodomelatonin binding and melatonin receptor function increased. However, examination of melatonin receptor function following chronic melatonin exposure, an exposure that resulted in a 50% enhancement in ALP activity, revealed that these receptors were desensitized. This was reflected by a complete loss in specific 2-[125I]-iodomelatonin binding as well as melatonin efficacy to inhibit forskolin-induced cAMP accumulation. Further characterization of the mechanisms underlying melatonin's effects on these differentiation processes revealed that MEK (1/2) and ERK (1/2), epidermal growth factor receptors, metalloproteinase and clathrin-mediated endocytosis were essential while PKA was not. Our results are consistent with a role for melatonin in osteoblast differentiation. If so, then, the decrease in plasma melatonin levels observed in humans during late adulthood may further enhance susceptibility to osteoporosis.

Therapeutic treatments potentially mediated by melatonin receptors: potential clinical uses in the prevention of osteoporosis, cancer and as an adjuvant therapy.

Melatonin's therapeutic potential is grossly underestimated because its functional roles are diverse and its mechanism(s) of action are complex and varied. Melatonin produces cellular effects via a variety of mechanisms in a receptor independent and dependent manner. In addition, melatonin is a chronobiotic agent secreted from the pineal gland during the hours of darkness. This diurnal release of melatonin impacts the sensitivity of melatonin receptors throughout a 24-hr period. This changing sensitivity probably contributes to the narrow therapeutic window for use of melatonin in treating sleep disorders, that is, at the light-to-dark (dusk) or dark-to-light (dawn) transition states. In addition to the cyclic changes in melatonin receptors, many genes cycle over the 24-hr period, independent or dependent upon the light/dark cycle. Interestingly, many of these genes support a role for melatonin in modulating metabolic and cardiovascular physiology as well as bone metabolism and immune function and detoxification of chemical agents and cancer reduction. Melatonin also enhances the actions of a variety of drugs or hormones; however, the role of melatonin receptors in modulating these processes is not known. The goal of this review is to summarize the evidence related to the utility of melatonin as a therapeutic agent by focusing on its other potential uses besides sleep disorders. In particular, its use in cancer prevention, osteoporosis and, as an adjuvant to other therapies are discussed. Also, the role that melatonin and, particularly, its receptors play in these processes are highlighted.

Modulation of melatonin receptors and G-protein function by microtubules.

Chronic melatonin exposure produces microtubule rearrangements in Chinese hamster ovary (CHO) cells expressing the human MT1 melatonin receptor while at the same time desensitizing MT1 receptors. Because microtubule rearrangements parallel MT1 receptor desensitization, we tested whether microtubules modulate receptor responsiveness. We determined whether depolymerization of microtubules by Colcemid, which prevents melatonin-induced outgrowths in MT1-expressing CHO cells, also prevents MT1 receptor desensitization by affecting G(alpha)-GTP exchange on G-proteins. In this study, we found that depolymerization of microtubules in MT1 receptor expressing cells, prevented melatonin-induced receptor desensitization reflected by an increase in the number of high potency sites when compared with melatonin-treated cells. Further examination of the mechanism(s) underlying this desensitization suggested that these effects occurred at the level of G-proteins. Depolymerization of microtubules during melatonin-induced desensitization, attenuated forskolin-induced cAMP accumulation, the opposite of which usually occurs following melatonin exposure alone. Concomitant to this attenuation in the forskolin response was a reduction in the amount of G(i alpha) protein coupled to MT1 receptors and an increase in [32P] azidoanilido GTP incorporation into G(i) proteins. These data are consistent with the findings that microtubule depolymerization did not affect MT1/G(q) coupling nor did it affect melatonin-induced phosphoinositide hydrolysis following melatonin exposure. However, interestingly, microtubule depolymerization enhanced melatonin-induced protein kinase C activation that was blocked in the presence of pertussis toxin. These data demonstrate that microtubule dynamics can modulate melatonin receptor function through their actions on G(i) proteins and impact on downstream signaling cascades.