Inhibition of Transient Receptor Potential Melastatin 3 ion channels by G-protein ßγ subunits.

Transient receptor potential melastatin 3 (TRPM3) channels are activated by heat, and chemical ligands such as pregnenolone sulphate (PregS) and CIM0216. Here, we show that activation of receptors coupled to heterotrimeric Gi/o proteins inhibits TRPM3 channels. This inhibition was alleviated by co-expression of proteins that bind the ßγ subunits of heterotrimeric G-proteins (Gßγ). Co-expression of Gßγ, but not constitutively active Gαi or Gαo, inhibited TRPM3 currents. TRPM3 co-immunoprecipitated with Gß, and purified Gßγ proteins applied to excised inside-out patches inhibited TRPM3 currents, indicating a direct effect. Baclofen and somatostatin, agonists of Gi-coupled receptors, inhibited Ca2+ signals induced by PregS and CIM0216 in mouse dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited inward currents induced by CIM0216 in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in mice. Our data uncover a novel signaling mechanism regulating TRPM3 channels.

A molecular determinant of phosphoinositide affinity in mammalian TRPV channels.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca(2+)-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6.

Transient receptor potential melastatin 3 is a phosphoinositide-dependent ion channel.

Phosphoinositides are emerging as general regulators of the functionally diverse transient receptor potential (TRP) ion channel family. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been reported to positively regulate many TRP channels, but in several cases phosphoinositide regulation is controversial. TRP melastatin 3 (TRPM3) is a heat-activated ion channel that is also stimulated by chemical agonists, such as pregnenolone sulfate. Here, we used a wide array of approaches to determine the effects of phosphoinositides on TRPM3. We found that channel activity in excised inside-out patches decreased over time (rundown), an attribute of PI(4,5)P2-dependent ion channels. Channel activity could be restored by application of either synthetic dioctanoyl (diC8) or natural arachidonyl stearyl (AASt) PI(4,5)P2. The PI(4,5)P2 precursor phosphatidylinositol 4-phosphate (PI(4)P) was less effective at restoring channel activity. TRPM3 currents were also restored by MgATP, an effect which was inhibited by two different phosphatidylinositol 4-kinase inhibitors, or by pretreatment with a phosphatidylinositol-specific phospholipase C (PI-PLC) enzyme, indicating that MgATP acted by generating phosphoinositides. In intact cells, reduction of PI(4,5)P2 levels by chemically inducible phosphoinositide phosphatases or a voltage-sensitive 5'-phosphatase inhibited channel activity. Activation of PLC via muscarinic receptors also inhibited TRPM3 channel activity. Overall, our data indicate that TRPM3 is a phosphoinositide-dependent ion channel and that decreasing PI(4,5)P2 abundance limits its activity. As all other members of the TRPM family have also been shown to require PI(4,5)P2 for activity, our data establish PI(4,5)P2 as a general positive cofactor of this ion channel subfamily.

Activation of TRPV1 channels inhibits mechanosensitive Piezo channel activity by depleting membrane phosphoinositides.

Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca(2+)-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCß indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 and PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin.

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.

Interplay between calmodulin and phosphatidylinositol 4,5-bisphosphate in Ca2+-induced inactivation of transient receptor potential vanilloid 6 channels.

The epithelial Ca(2+) channel transient receptor potential vanilloid 6 (TRPV6) undergoes Ca(2+)-induced inactivation that protects the cell from toxic Ca(2+) overload and may also limit intestinal Ca(2+) transport. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca(2+), calmodulin (CaM), and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in excised inside-out patches. The activity of TRPV6 strictly depended on the presence of PI(4,5)P(2), and Ca(2+)-CaM inhibited the channel at physiologically relevant concentrations. Ca(2+) alone also inhibited TRPV6 at high concentrations (IC(50) = ∼20 µM). A double mutation in the distal C-terminal CaM-binding site of TRPV6 (W695A/R699E) essentially eliminated inhibition by CaM in excised patches. In whole cell patch clamp experiments, this mutation reduced but did not eliminate Ca(2+)-induced inactivation. Providing excess PI(4,5)P(2) reduced the inhibition by CaM in excised patches and in planar lipid bilayers, but PI(4,5)P(2) did not inhibit binding of CaM to the C terminus of the channel. Overall, our data show a complex interplay between CaM and PI(4,5)P(2) and show that Ca(2+), CaM, and the depletion of PI(4,5)P(2) all contribute to inactivation of TRPV6.

Composition and characterization of in situ usable light cured dental drug delivery hydrogel system.

Biodegradable polymers are compatible, permeable and nontoxic, thus they can provide a useful tool for drug delivery or tissue engineering. These polymers can form hydrogels, which are suitable vehicles for different types of materials e.g. drugs, bioactive molecules or cells. In the case of dentistry, photopolymerization is an obvious method to obtain in situ useable devices which can provide a more efficient way of tailoring drug release. A hydrogel system was developed based on poly-gamma-glutamic acid that was modified with methacryloyl groups to achieve this purpose. The resulting new reactive structure was proved by NMR spectroscopy. The swelling ratio of this type of hydrogel has been found remarkable, over 300 % after 24 h, and it can release 5 ng/mm(2) metronidazole. The prepared hydrogels were nontoxic as viability, cytotoxicity tests and cell morphology investigations proved it. These results render this model system an excellent candidate for use as an in situ curing local drug delivery device. The new photoactive system can be utilized in the treatment of periodontal diseases or raising the effectiveness of drugs used only in the minimal effective dose.

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.

Gene targeting and Calcium handling efficiencies in mouse embryonic stem cell lines.

AIM: To compare gene targeting efficiencies, expression profiles, and Ca(2+) handling potentials in two widely used mouse embryonic stem cell lines. METHODS: The two widely used mouse embryonic stem cell lines, R1 and HM-1, were cultured and maintained on Mitomycin C treated mouse embryonic fibroblast feeder cell layers, following standard culture procedures. Cells were incubated with primary and secondary antibodies before fluorescence activated cell sorting analysis to compare known pluripotency markers. Moreover, cells were harvested by trypsinization and transfected with a kinase-inactive murine Tyk2 targeting construct, following the BioRad and Amaxa transfection procedures. Subsequently, the cells were cultured and neomycin-resistant cells were picked after 13 d of selection. Surviving clones were screened twice by polymerase chain reaction (PCR) and finally confirmed by Southern blot analysis before comparison. Global gene expression profiles of more than 20 400 probes were also compared and significantly regulated genes were confirmed by real time PCR analysis. Calcium handling potentials of these cell lines were also compared using various agonists. RESULTS: We found significant differences in transfection efficiencies of the two cell lines (91% ± 6.1% vs 75% ± 4.2%, P = 0.01). Differences in the targeting efficiencies were also significant whether the Amaxa or BioRad platforms were used for comparison. We did not observe significant differences in the levels of many known pluripotency markers. However, our genome-wide expression analysis using more than 20 400 spotted cDNA arrays identified 55 differentially regulated transcripts (P < 0.05) implicated in various important biological processes, including binding molecular functions (particularly Ca(2+) binding roles). Subsequently, we measured Ca(2+) signals in these cell lines in response to various calcium agonists, both in high and low Ca(2+) solutions, and found significant differences (P < 0.05) in the regulation of Ca(2+) homeostasis between the investigated cell lines. Then we further compared the detection and expression of various membrane and intracellular Ca(2+) receptors and similarly found significant (P < 0.05) variations in a number of calcium receptors between these cell lines. CONCLUSION: Results of this study emphasize the importance of considering intrinsic cellular variations, during selection of cell lines for experiments and interpretations of experimental results.

Human female hair follicles are a direct, nonclassical target for thyroid-stimulating hormone.

Pituitary thyroid-stimulating hormone (TSH) regulates thyroid hormone synthesis via receptors (TSH-R) expressed on thyroid epithelial cells. As the hair follicle (HF) is uniquely hormone-sensitive and, hypothyroidism with its associated, increased TSH serum levels clinically can lead to hair loss, we asked whether human HFs are a direct target for TSH. Here, we report that normal human scalp skin and microdissected human HFs express TSH-R mRNA. TSH-R-like immunoreactivity is limited to the mesenchymal skin compartments in situ. TSH may alter HF mesenchymal functions, as it upregulates alpha-smooth muscle actin expression in HF fibroblasts. TSH-R stimulation by its natural ligand in organ culture changes the expression of several genes of human scalp HFs (for example keratin K5), upregulates the transcription of classical TSH target genes and enhances cAMP production. Although the functional role of TSH in human HF biology awaits further dissection, these findings document that intracutaneous TSH-Rs are fully functional in situ and that HFs of female individuals are direct targets for nonclassical, extrathyroidal TSH bioregulation. This suggests that organ-cultured scalp HFs provide an instructive and physiologically relevant human model for exploring nonclassical functions of TSH, in and beyond the skin.

Inhibition of human hair follicle growth by endo- and exocannabinoids.

Recent studies strongly suggest that the cannabinoid system is a key player in cell growth control. Since the organ-culture of human hair follicles (HF) offers an excellent, clinically relevant model for complex tissue interaction systems, we have asked whether the cannabinoid system plays a role in hair growth control. Here, we show that human scalp HF, intriguingly, are both targets and sources of endocannabinoids. Namely, the endocannabinoid N-arachidonoylethanolamide (anandamide, AEA) as well as the exocannabinnoid delta (9) -tetrahydrocannabinol dose-dependently inhibited hair shaft elongation and the proliferation of hair matrix keratinocytes, and induced intraepithelial apoptosis and premature HF regression (catagen). These effects were inhibited by a selective antagonist of cannabinoid receptor-1 (CB1). In contrast to CB2, CB1 was expressed in a hair cycle-dependent manner in the human HF epithelium. Since we successfully identified the presence of endocannabinoids in human HF, our data strongly suggest that human HF exploit a CB1-mediated endocannabinoid signaling system for negatively regulating their own growth. Clinically, CB1 agonists may therefore help to manage unwanted hair growth, while CB1 antagonists might counteract hair loss. Finally, human HF organ culture offers an instructive, physiologically relevant new research tool for dissecting "nonclassical" effects of endocannabinoids and their receptor-mediated signaling in general.