The role of protein kinase C isoenzymes in the pathogenesis of human autoimmune diseases.

The physiological role of protein kinase C (PKC) enzymes in the immune system is presented briefly. From earlier publications of others data were collected how the defects of one/two isoenzymes of PKC system suggested their involvement in the pathogenesis of human autoimmune diseases. Our observations on the defects of seven PKC isoenzymes in the peripheral blood mononuclear cells (PBMC) demonstrate that these molecular impairments are not prerequisits of the pathogenesis of systemic lupus erythematosus (SLE), mixed connective tissue disease and Sjögren's syndrome. However, these defects can modulate the disease activity and symptoms especially in SLE by several pathways. The role of PKC system in other forms of autoimmune diseases is also very small. It was of note that we detected decreased expression of PKC isoenzymes in PBMC of a European white family with an X-linked genetic background showing seasonal undulations in the lupus patient and also in her healthy mother.

Season Dependent Changes in the Expression of Protein Kinase C Isoenzymes in a Female Patient with Systemic Lupus Erythematosus.

We aimed to answer the question whether the decreased expression of protein kinase C (PKC) isoenzymes in the peripheral blood mononuclear cells (PBMC) of patients with systemic lupus erythematosus (SLE) is inherited or not. For this reason we examined the expression of PKC isoenzymes in a European white girl with acute SLE and in her healthy mother and father simultaneously in summer and winter during one year using western blotting and densitometry. We found that in the father the expression of PKC isoenzymes did not differ from that of eight healthy controls included women and men. However, in the "SLE-free" mother and in the patient arrived in July with acute symptoms of lupus, the expression of PKC isoenzymes showed a season dependent undulation in parallel. Namely, in summer the expression values were significantly lower, in winter they were significantly higher than those in the controls. Thus, the decreased expression of PKC isoenzymes in the PBMC of SLE patient is not a disease specific marker; it appears also in her lupus free mother. This phenomenon may be due to a season dependent female genetic background. However, the low PKC levels in summer can still decrease further the low production of IL-2 in T cells of lupus patients augmenting the existing AP-1 defects. This is the first report on the season and female dependent inherited changing of PKC expression in a European white patient with SLE and her mother. Further studies are needed to confirm these findings in other populations.

Protein kinase Cδ promotes proliferation and induces malignant transformation in skeletal muscle.

In this paper, we investigated the isoform-specific roles of certain protein kinase C (PKC) isoforms in the regulation of skeletal muscle growth. Here, we provide the first intriguing functional evidence that nPKCδ (originally described as an inhibitor of proliferation in various cells types) is a key player in promoting both in vitro and in vivo skeletal muscle growth. Recombinant overexpression of a constitutively active nPKCδ in C2C12 myoblast increased proliferation and inhibited differentiation. Conversely, overexpression of kinase-negative mutant of nPKCδ (DN-nPKCδ) markedly inhibited cell growth. Moreover, overexpression of nPKCδ also stimulated in vivo tumour growth and induced malignant transformation in immunodeficient (SCID) mice whereas that of DN-nPKCδ suppressed tumour formation. The role of nPKCδ in the formation of rhabdomyosarcoma was also investigated where recombinant overexpression of nPKCδ in human rhabdomyosarcoma RD cells also increased cell proliferation and enhanced tumour formation in mouse xenografts. The other isoforms investigated (PKCα, ß, ε) exerted only minor (mostly growth-inhibitory) effects in skeletal muscle cells. Collectively, our data introduce nPKCδ as a novel growth-promoting molecule in skeletal muscles and invite further trials to exploit its therapeutic potential in the treatment of skeletal muscle malignancies.

Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes.

The endocannabinoid system (ECS) regulates multiple physiological processes, including cutaneous cell growth and differentiation. Here, we explored the effects of the major nonpsychotropic phytocannabinoid of Cannabis sativa, (-)-cannabidiol (CBD), on human sebaceous gland function and determined that CBD behaves as a highly effective sebostatic agent. Administration of CBD to cultured human sebocytes and human skin organ culture inhibited the lipogenic actions of various compounds, including arachidonic acid and a combination of linoleic acid and testosterone, and suppressed sebocyte proliferation via the activation of transient receptor potential vanilloid-4 (TRPV4) ion channels. Activation of TRPV4 interfered with the prolipogenic ERK1/2 MAPK pathway and resulted in the downregulation of nuclear receptor interacting protein-1 (NRIP1), which influences glucose and lipid metabolism, thereby inhibiting sebocyte lipogenesis. CBD also exerted complex antiinflammatory actions that were coupled to A2a adenosine receptor-dependent upregulation of tribbles homolog 3 (TRIB3) and inhibition of the NF-κB signaling. Collectively, our findings suggest that, due to the combined lipostatic, antiproliferative, and antiinflammatory effects, CBD has potential as a promising therapeutic agent for the treatment of acne vulgaris.

The endocannabinoid 2-AG controls skeletal muscle cell differentiation via CB1 receptor-dependent inhibition of Kv7 channels.

Little is known of the involvement of endocannabinoids and cannabinoid receptors in skeletal muscle cell differentiation. We report that, due to changes in the expression of genes involved in its metabolism, the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are decreased both during myotube formation in vitro from murine C2C12 myoblasts and during mouse muscle growth in vivo. The endocannabinoid, as well as the CB1 agonist arachidonoyl-2-chloroethylamide, prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which, per se, instead stimulate differentiation. Importantly, 2-AG also inhibits differentiation of primary human satellite cells. Muscle fascicles from CB1 knockout embryos contain more muscle fibers, and postnatal mice show muscle fibers of an increased diameter relative to wild-type littermates. Inhibition of Kv7.4 channel activity, which plays a permissive role in myogenesis and depends on phosphatidylinositol 4,5-bisphosphate (PIP2), underlies the effects of 2-AG. We find that CB1 stimulation reduces both total and Kv7.4-bound PIP2 levels in C2C12 cells and inhibits Kv7.4 currents in transfected CHO cells. We suggest that 2-AG is an endogenous repressor of myoblast differentiation via CB1-mediated inhibition of Kv7.4 channels.

Function of RasGRP3 in the formation and progression of human breast cancer.

INTRODUCTION: Ras guanine nucleotide exchange factors (RasGEFs) mediate the activation of the Ras signaling pathway that is over activated in many human cancers. The RasGRP3, an activator of H-Ras and R-Ras protein exerts oncogenic effects and the overexpression of the protein is observed in numerous malignant cancer types. Here, we investigated the putative alteration of expression and potential function of RasGRP3 in the formation and progression of human breast cancer. METHODS: The RasGRP3 and phosphoRasGRP3 expressions were examined in human invasive ductal adenocarcinoma derived samples and cell lines (BT-474, JIMT-1, MCF7, SK-BR-3, MDA-MB-453, T-47D) both in mRNA (Q-PCR) and protein (Western blot; immunohistochemistry) levels. To explore the biological function of the protein, RasGRP3 knockdown cultures were established. To assess the role of RasGRP3 in the viability of cells, annexin-V/PI staining and MitoProbe™ DilC1 (5) assay were performed. To clarify the function of the protein in cell proliferation and in the development of chemotherapeutic resistance, CyQuant assay was performed. To observe the RasGRP3 function in tumor formation, the Severe combined immunodeficiency (SCID) mouse model was used. To investigate the role of the protein in Ras-related signaling Q-PCR and Western blot experiments were performed. RESULTS: RasGRP3 expression was elevated in human breast tumor tissue samples as well as in multiple human breast cancer cell lines. Down-regulation of RasGRP3 expression in breast cancer cells decreased cell proliferation, induced apoptosis in MCF7 cells, and sensitized T-47D cells to the action of drugs Tamoxifen and trastuzumab (Herceptin). Gene silencing of RasGRP3 reduced tumor formation in mouse xenografts as well. Inhibition of RasGRP3 expression also reduced Akt, ERK1/2 and estrogen receptor alpha phosphorylation downstream from IGF-I insulin like growth factor-I (IGF-I) or epidermal growth factor (EGF) stimulation confirming the functional role of RasGRP3 in the altered behavior of these cells. CONCLUSIONS: Taken together, our results suggest that the Ras activator RasGRP3 may have a role in the pathological behavior of breast cancer cells and may constitute a therapeutic target for human breast cancer.

The in vitro treatment with vitamin D3 is ineffective on the expression of PKC isoenzymes, but decreases further the impaired production of IL-2 in the T lymphocytes of SLE patients.

The objective of the study was to investigate the possibility whether the in vitro treatment with vitamin D3 can restore the impaired expression of protein kinase C (PKC) isoenzymes and IL-2 production in the lymphocytes of patients with systemic lupus erythematosus (SLE). Purified T lymphocytes from 14 patients with SLE and 13 healthy controls were cultured for 48 h in the presence and absence of 1 and 100 nM doses of vitamin D3. The expressions of various PKC isoenzymes were tested by Western blot analysis, and the amounts of various cytokines were detected by ELISA in the culture supernatants. Neither the low (1 nM) nor the high (100 nM) doses of vitamin D3 (1α,-25-dihydroxyvitamin) applied in vitro for 48 h were able to restore the decreased expression of PKC isoenzymes in the T cells of SLE patients. However, 100 nM of vitamin D3 significantly increased the release of IL-10, but suppressed the production of IL-2, IL-6, interferon γ and TNF α in the culture supernatants of both groups. As the low production of IL-2 is one of the main pathologic features of SLE, we recommend to avoid the use of high doses of vitamin D3 for treatment of lupus patients with vitamin D3 deficiency.

Transient receptor potential vanilloid-2 mediates the effects of transient heat shock on endocytosis of human monocyte-derived dendritic cells.

Our goal was to investigate the effect of heat shock on human monocyte-derived dendritic cells (DCs) and to dissect the role of thermosensitive transient receptor potential (TRP) channels in the process. We provide evidence that a short heat shock challenge (43 °C) decreased the endocytotic activity of the DCs and that this effect could be alleviated by the RNAi-mediated knockdown of TRPV2 but, importantly, not by the pharmacological (antagonists) or molecular (RNAi) suppression of TRPV1 and TRPV4 activities/levels. Likewise, the heat shock-induced robust membrane currents were selectively and markedly inhibited by TRPV2 "silencing" whereas modulation of TRPV1 and TRPV4 activities, again, had no effect. These intriguing data introduce TRPV2-coupled signaling as a key player in mediating the cellular actions of heat shock on DCs.

Protein kinase C isoforms have differential roles in the regulation of human sebocyte biology.

Protein kinase C (PKC) isoforms have crucial roles in cutaneous signaling. Interestingly, we lack information about their involvement in human sebaceous gland biology. Therefore, in this current study, we investigated the functions of the PKC system in human immortalized SZ95 sebocytes. Using molecular biological approaches, imaging, and functional assays, we report that SZ95 sebocytes express the conventional cPKCα; the novel nPKCδ, ɛ, and η; and the atypical aPKCζ. Activation of the PKC system by phorbol 12-myristate 13-acetate (PMA) stimulated lipid synthesis (a hallmark of differentiation) and resulted in translocation and then downregulation of cPKCα and nPKCδ. In good accord with these findings, the effect of PMA was effectively abrogated by inhibitors and short interfering RNA-mediated "silencing" of cPKCα and nPKCδ. Of further importance, molecular or pharmacological inhibition of nPKCδ also prevented the lipogenic and apoptosis-promoting action of arachidonic acid. Finally, we also found that "knockdown" of the endogenous aPKCζ activity markedly increased basal lipid synthesis and apoptosis, suggesting its constitutive activity in suppressing these processes. Collectively, our findings strongly argue for the fact that certain PKCs have pivotal, isoform-specific, differential, and antagonistic roles in the regulation of human sebaceous gland-derived sebocyte biology.

Endocannabinoids regulate growth and survival of human eccrine sweat gland-derived epithelial cells.

The functional existence of the emerging endocannabinoid system (ECS), one of the new neuroendocrine players in cutaneous biology, is recently described in the human skin. In this study, using human eccrine sweat gland-derived immortalized NCL-SG3 model cells and a wide array of cellular and molecular assays, we investigated the effects of prototypic endocannabinoids (anandamide, 2-arachidonoylglycerol) on cellular functions. We show here that both endocannabinoids dose-dependently suppressed proliferation, induced apoptosis, altered expressions of various cytoskeleton proteins (e.g., cytokeratins), and upregulated lipid synthesis. Interestingly, as revealed by specific agonists and antagonists as well as by RNA interference, neither the metabotropic cannabinoid receptors (CB) nor the "ionotropic" CB transient receptor potential ion channels, expressed by these cells, mediated the cellular actions of the endocannabinoids. However, the endocannabinoids selectively activated the mitogen-activated protein kinase signaling pathway. Finally, other elements of the ECS (i.e., enzymes involved in the synthesis and degradation of endocannabinoids) were also identified on NCL-SG3 cells. These results collectively suggest that cannabinoids exert a profound regulatory role in the biology of the appendage. Therefore, from a therapeutic point of view, upregulation of endocannabinoid levels might help to manage certain sweat gland-derived disorders (e.g., tumors) characterized by unwanted growth.

Activation of transient receptor potential vanilloid-3 inhibits human hair growth.

In the current study, we aimed at identifying the functional role of transient receptor potential vanilloid-3 (TRPV3) ion channel in the regulation of human hair growth. Using human organ-cultured hair follicles (HFs) and cultures of human outer root sheath (ORS) keratinocytes, we provide the first evidence that activation of TRPV3 inhibits human hair growth. TRPV3 immunoreactivity was confined to epithelial compartments of the human HF, mainly to the ORS. In organ culture, TRPV3 activation by plant-derived (e.g., eugenol, 10-1,000 µM) or synthetic (e.g., 2-aminoethoxydiphenyl borate, 1-300 µM) agonists resulted in a dose-dependent inhibition of hair shaft elongation, suppression of proliferation, and induction of apoptosis and premature HF regression (catagen). Human ORS keratinocytes also expressed functional TRPV3, whose stimulation induced membrane currents, elevated intracellular calcium concentration, inhibited proliferation, and induced apoptosis. Of great importance, these effects on ORS keratinocytes were all mediated by TRPV3, as small interfering RNA-mediated silencing of TRPV3 effectively abrogated the cellular actions of the above agonists. These findings collectively support the concept that TRPV3 signaling is a significant player in human hair growth control. Therefore, TRPV3 and the related intracellular signaling mechanism might function as a promising target for pharmacological manipulations of clinically relevant hair growth disorders.

The synthetic bryostatin analog Merle 23 dissects distinct mechanisms of bryostatin activity in the LNCaP human prostate cancer cell line.

Bryostatin 1 has attracted considerable attention both as a cancer chemotherapeutic agent and for its unique activity. Although it functions, like phorbol esters, as a potent protein kinase C (PKC) activator, it paradoxically antagonizes many phorbol ester responses in cells. Because of its complex structure, little is known of its structure-function relations. Merle 23 is a synthetic derivative, differing from bryostatin 1 at only four positions. However, in U-937 human leukemia cells, Merle 23 behaves like a phorbol ester and not like bryostatin 1. Here, we characterize the behavior of Merle 23 in the human prostate cancer cell line LNCaP. In this system, bryostatin 1 and phorbol ester have contrasting activities, with the phorbol ester but not bryostatin 1 blocking cell proliferation or tumor necrosis factor alpha secretion, among other responses. We show that Merle 23 displays a highly complex pattern of activity in this system. Depending on the specific biological response or mechanistic change, it was bryostatin-like, phorbol ester-like, intermediate in its behavior, or more effective than either. The pattern of response, moreover, varied depending on the conditions. We conclude that the newly emerging bryostatin derivatives such as Merle 23 provide powerful tools to dissect subsets of bryostatin mechanism and response.

"Sebocytes' makeup": novel mechanisms and concepts in the physiology of the human sebaceous glands.

The pilosebaceous unit of the human skin consists of the hair follicle and the sebaceous gland. Within this "mini-organ", the sebaceous gland has been neglected by the researchers of the field for several decades. Actually, it was labeled as a reminiscence of human development ("a living fossil with a past but no future"), and was thought to solely act as a producer of sebum, a lipid-enriched oily substance which protects our skin (and hence the body) against various insults. However, due to emerging research activities of the past two decades, it has now become evident that the sebaceous gland is not only a "passive" cutaneous "relic" to establish the physico-chemical barrier function of the skin against constant environmental challenges, but it rather functions as an "active" neuro-immuno-endocrine cutaneous organ. This review summarizes recent findings of sebaceous gland research by mainly focusing on newly discovered physiological functions, novel regulatory mechanisms, key events in the pathology of the gland, and future directions in both experimental and clinical dermatology.

Histamine and H1 -histamine receptors faster venous circulation.

The study has analysed the action of histamine in the rabbit venous system and evaluated its potential role in contraction during increased venous pressure. We have found that a great variety exists in histamine sensitivity and H(1) -histamine receptor expression in various types of rabbit veins. Veins of the extremities (saphenous vein, femoral vein, axillary vein) and abdomen (common iliac vein, inferior vena cava) responded to histamine by a prominent, concentration-dependent force generation, whereas great thoracic veins (subclavian vein, superior vena cavas, intrathoracic part of inferior vena cava) and a pelvic vein (external iliac vein) exhibited slight sensitivity to exogenous histamine. The lack of reactivity to histamine was not due to increased activity of nitric oxide synthase (NOS) or heme oxygenase-1. H(1) -histamine receptor expression of veins correlated well with the histamine-induced contractions. Voltage-dependent calcium channels mediated mainly the histamine-induced force generation of saphenous vein, whereas it did not act in the inferior vena cava. In contrast, the receptor-operated channels were not involved in this response in either vein. Tyrosine phosphorylation occurred markedly in response to histamine in the saphenous vein, but not in the inferior vena cava. Histamine induced a prominent ρ kinase activation in both vessels. Protein kinase C and mitogen-activated protein kinase (MAPK) were not implicated in the histamine-induced intracellular calcium sensitization. Importantly, transient clamping of the femoral vein in animals caused a short-term constriction, which was inhibited by H(1) -histamine receptor antagonist in vivo. Furthermore, a significantly greater histamine immunopositivity was detected in veins after stretching compared to the resting state. We conclude that histamine receptor density adapts to the actual requirements of the circulation, and histamine liberated by the venous wall during increased venous pressure contributes to the contraction of vessels, providing a force for the venous return.

Endocannabinoids modulate human epidermal keratinocyte proliferation and survival via the sequential engagement of cannabinoid receptor-1 and transient receptor potential vanilloid-1.

We have recently shown that lipid mediators of the emerging endocannabinoid system (ECS) are key players of growth control of the human pilosebaceous unit. In this study, we asked whether the prototypic endocannabinoid anandamide (N-arachidonoylethanolamine, AEA) has a role in growth and survival of epidermal keratinocytes (KCs). Using human cultured KCs and skin organ-culture models, and by employing combined pharmacological and molecular approaches, we provide early evidence that AEA markedly suppresses KC proliferation and induces cell death, both in vitro and in situ. Moreover, we present that these cellular actions are mediated by a most probably constitutively active signaling mechanism that involves the activation of the metabotropic cannabinoid receptor CB(1) and a sequential engagement of the "ionotropic cannabinoid receptor" transient receptor potential vanilloid-1 (TRPV1). Finally, we demonstrate that the cellular effects of AEA are most probably due to a Ca(2+) influx via the non-selective, highly Ca(2+)-permeable ion channel TRPV1, and the concomitant elevation of intracellular Ca(2+) concentration. The data reported here may encourage one to explore whether the targeted manipulation of the above signaling pathway of the cutaneous ECS could become a useful adjunct treatment strategy for hyperproliferative human dermatoses such as psoriasis or KC-derived skin tumors.

Conformationally constrained analogues of diacylglycerol (DAG). 31. Modulation of the biological properties of diacylgycerol lactones (DAG-lactones) containing rigid-rod acyl groups separated from the core lactone by spacer units of different lengths.

Diacylglycerol lactones built with a rigid 4-[(methylphenyl)ethynyl]phenyl rod that is separated from the exocyclic acylcarbonyl of the DAG-lactone core by a spacer unit of variable length were synthesized and studied. Binding affinities for a panel of classical and novel PKC isozymes in two different phospholipid environments, one corresponding to the plasma membrane of cells, were determined. The kinetics and site of translocation for the PKC isozymes alpha and delta upon treatment with the compounds were also studied as well as the early response of ERK phosphorylation and the late response of induction of apoptosis in the human prostatic carcinoma cell line LNCaP. Finally, the compounds were evaluated in terms of their interaction with biomimetic lipid/polydiacetylene membranes by the associated chromatic response. The different spatial disposition of the rigid structural motif on the DAG-lactones contributes to differential activity.

Increased expressions of cannabinoid receptor-1 and transient receptor potential vanilloid-1 in human prostate carcinoma.

PURPOSE: Recently, functional cannabinoid receptor-1 (CB1) and vanilloid receptor-1 (TRPV1) have been described in human prostate and prostate cancer-derived cell lines where the activation of the receptors resulted in inhibition of cellular growth. We, however, lack the description of the expression of these molecules in human prostate cancer (PCC) and in benign prostate hyperplasia (BPH). METHODS: Therefore, immunohistochemistry, Western blotting, and quantitative "real-time Q-PCR were performed to define the expressions of CB1 and TRPV1 in healthy and diseased prostate tissues. RESULTS: CB1 was identified in epithelial and smooth muscle cells types of the human prostate, whereas TRPV1 was exclusively localized to the mucosal cells. We also found that the expression of CB1 and TRPV1 (both at the protein and mRNA levels) were significantly up-regulated in PCC. However, while the increased expression of TRPV1 showed a proper correlation with increasing PCC tumor grades, such phenomenon was not observed with CB1. In addition, we also measured markedly elevated CB1 levels in BPH tissues whilst the expression of TRPV1 was not altered when compared to healthy control prostate. CONCLUSIONS: Our findings strongly argue for that (1) the CB1 and TRPV1 molecules as well as their ligands may indeed possess a promising future role in the treatment of PCC; (2) TRPV1 may also serve as a prognostic factor in PCC; and (3) CB1 may act as a potential target molecule in the therapeutic management of BPH.

Protein kinase C isoenzymes differentially regulate the differentiation-dependent expression of adhesion molecules in human epidermal keratinocytes.

Epidermal expression of adhesion molecules such as desmogleins (Dsg) and cadherins is strongly affected by the differentiation status of keratinocytes. We have previously shown that certain protein kinase C (PKC) isoforms differentially alter the growth and differentiation of human epidermal HaCaT keratinocytes. In this paper, using recombinant overexpression and RNA interference, we define the specific roles of the different PKC isoenzymes in modulation of expression of adhesion molecules in HaCaT keratinocytes. The level of Dsg1, a marker of differentiating keratinocytes, was antagonistically regulated by two Ca-independent 'novel' nPKC isoforms; i.e. it increased by the differentiation-promoting nPKCdelta and decreased by the growth-promoting nPKCepsilon. The expression of Dsg3 (highly expressed in proliferating epidermal layers) was conversely regulated by these isoenzymes, and was also inhibited by the differentiation inducer Ca-dependent 'conventional' cPKCalpha. Finally, the expression of P-cadherin (a marker of proliferating keratinocytes) was regulated by all of the examined PKCs, also in an antagonistic manner (inhibited by cPKCalpha/nPKCdelta and stimulated by cPKCbeta/nPKCepsilon). Collectively, the presented results strongly argue for the marked, differential, and in some instances antagonistic roles of individual Ca-dependent and Ca-independent PKC isoforms in the regulation of expression of adhesion molecules of desmosomes and adherent junctions in human epidermal keratinocytes.

The analgesic drug, tramadol, acts as an agonist of the transient receptor potential vanilloid-1.

BACKGROUND: Tramadol is an effective analgesic substance widely used in medical practice. Its therapeutic action have been mainly attributed to the activation of mu-opioid receptors as well as to the inhibition of neurotransmitter reuptake mechanisms and various voltage- and ligand-gated ion channels of the nociceptive system. As transient receptor potential vanilloid-1 (TRPV1, "the capsaicin receptor") has been shown to function as a central integrator molecule of pain sensation, our aim in the current study was to define the involvement of TRPV1 in the complex mechanism of action of tramadol. METHODS: To achieve these goals, we used single-cell Ca-imaging as well as fluorescent image plate reader assays on Chinese hamster ovary (CHO) cells heterologously over-expressing TRPV1. RESULTS: We found that (1) tramadol, similar to the well-known TRPV1 agonist, capsaicin, significantly increased [Ca(2+)](i) of TRPV1-CHO cells in a concentration-dependent fashion; (2) its effect was reversibly prevented by the TRPV1 antagonist capsazepine; (3) repeated application of tramadol resulted in marked tachyphylaxis; and (4) tramadol did not modify [Ca(2+)](i) in control (empty vector expressing) CHO cells. CONCLUSIONS: Collectively, these findings strongly support the intriguing and novel concept that tramadol acts as an agonist of TRPV1. Considering that activation of TRPV1 on sensory neurons is followed by a local release of vasoactive neuropeptides and a marked desensitization of the afferent fibers (hence termination of pain sensation), our findings may equally explain both the desired analgesic as well as the often-seen, yet "unexpected," local side effects (e.g., initiation of burning pain and erythema) of tramadol.

Investigation of micronized titanium dioxide penetration in human skin xenografts and its effect on cellular functions of human skin-derived cells.

Titanium dioxide (TiO2) nanoparticles are ubiquitously used materials in everyday life (e.g. paints,household products and plastic goods). However, despite the wide array of common applications, their pathogenetic role was also suggested under certain conditions (e.g. pulmonary neoplasias and lung fibrosis). From a dermatological point of view, it is also of great importance that TiO2 also serves as a physical photoprotective agent in sunscreens and is widely used in various cosmetic products. However, the effect of TiO2 on human cutaneous functions is still unknown. Therefore, in the current study, we investigated the in vivo penetration of TiO2 via human skin transplanted to immunodeficient mice and,furthermore, we measured the in vitro effects of nanoparticles on various functional properties of numerous epidermal and dermal cells in culture. Hereby, using various nuclear microscopy methods, we provide the first evidence that TiO2nanoparticles in vivo do not penetrate through the intact epidermal barrier. However, we also report that TiO2, when exposed directly to cell cultures in vitro, exerts significant and cell-type dependent effects on such cellular functions as viability, proliferation, apoptosis and differentiation. Therefore, our novel findings will hopefully inspire one to systemically explore in future, clinically oriented trials whether there is indeed a risk from micronized TiO2-containing products on skin with an impaired stratum corneum barrier function.