BACKGROUND AND PURPOSEEndostatin (ES) is a c‐terminal proteolytic fragment of collagen XVIII with promising antitumour properties in several tumour models, including human glioblastoma. We hypothesized that this peptide could interact with plasma membrane ion channels and modulate their functions.EXPERIMENTAL APPROACHUsing cell proliferation and migration assays, patch clamp and Western blot analysis, we studied the effects of ES on the proliferation and migration of human glioblastoma U87 cells, mediated by T‐type Ca²⁺channels.KEY RESULTSExtracellular application of ES reversibly inhibited T‐type Ca²⁺channel currents (T‐currents) in U87 cells, whereas L‐type Ca²⁺currents were not affected. This inhibitory effect was associated with a hyperpolarizing shift in the voltage‐dependence of inactivation but was independent of G‐protein and protein tyrosine kinase‐mediated pathways. All three α₁subunits of T‐type Ca²⁺channels (Ca_V3), α_1G(Ca_V3.1), α_1H(Ca_V3.2) and α_1I(Ca_V3.3), were endogenously expressed in U87 cells. Using transfected HEK293 or CHO cells, we showed that only Ca_V3.1 and Ca_V3.2, but not Ca_V3.3 or Ca_V1.2 (L‐type), channel currents were significantly inhibited. More interestingly, ES inhibited the proliferation and migration of U87 cells in a dose‐dependent manner. Pretreatment of the cells with the specific T‐type Ca²⁺channel blocker mibefradil occluded these inhibitory effects of ES.CONCLUSION AND IMPLICATIONSThis study provides the first evidence that the antitumour effects of ES on glioblastoma cells is through direct inhibition of T‐type Ca²⁺channels and gives new insights into the future development of a new class of antiglioblastoma agents that target the proliferation and migration of these cells.LINKED ARTICLEThis article is commented on by Santoniet al., pp. 1244–1246 of this issue. To view this commentary visithttp://dx.doi.org/10.1111/j.1476‐5381.2012.01908.x

Hydrogen sulfide (H(2)S), an endogenous gas molecule synthesized by cystathionine-β-synthetase (CBS), is involved in inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms of CBS-H(2)S signaling in peripheral nociceptive processing remain unknown. We demonstrated that peripheral inflammation induced by intraplantar injection of complete Freund adjuvant significantly up-regulated expression of CBS at both protein and mRNA levels in rat dorsal root ganglia (DRG). The CBS inhibitors hydroxylamine and aminooxyacetic acid attenuated mechanical hyperalgesia in a dose-dependent manner and reversed hyperexcitability of DRG neurons in inflamed rats. Intraplantar administration of NaHS (its addition mimics CBS production of H(2)S) or l-cysteine in healthy rats elicited mechanical hyperalgesia. Application of NaHS in vitro enhanced excitability and tetrodotoxin (TTX)-resistant sodium current of DRG neurons from healthy rats, which was attenuated by pretreatment of protein kinase A inhibitor H89. Methylation-specific PCR and bisulfite sequencing demonstrated that promoter region of cbs gene was less methylated in DRG samples from inflamed rats than that from controls. Peripheral inflammation did not alter expression of DNA methyltransferase 3a and 3b, the 2 major enzymes for DNA methylation, but led to a significant up-regulation of methyl-binding domain protein 4 and growth arrest and DNA damage inducible protein 45α, the enzymes involved in active DNA demethylation. Our findings suggest that epigenetic regulation of CBS expression may contribute to inflammatory hyperalgesia. H(2)S seems to increase TTX-resistant sodium channel current, which may be mediated by protein kinase A pathway, thus identifying a potential therapeutic target for the treatment of chronic pain.

Insulin-like growth factor 1 (IGF-1) is implicated in the nociceptive (pain) sensitivity of primary afferent neurons. We found that the IGF-1 receptor (IGF-1R) functionally stimulated voltage-gated T-type Ca²⁺ (Ca_V3) channels in mouse dorsal root ganglia (DRG) neurons through a mechanism dependent on heterotrimeric G protein (heterotrimeric guanine nucleotide–binding protein) signaling. IGF-1 increased T-type channel currents in small-diameter DRG neurons in a manner dependent on IGF-1 concentration and IGF-1R but independent of phosphatidylinositol 3-kinase (PI3K). The intracellular subunit of IGF-1R coimmunoprecipitated with G_αo. Blocking G protein signaling by the intracellular application of guanosine diphosphate (GDP)-β-S or with pertussis toxin abolished the stimulatory effects of IGF-1. Antagonists of protein kinase Cα (PKCα), but not of PKCβ, abolished the IGF-1–induced T-type channel current increase. Application of IGF-1 increased membrane abundance of PKCα, and PKCα inhibition (either pharmacologically or genetically) abolished the increase in T-type channel currents stimulated by IGF-1. IGF-1 increased action potential firing in DRG neurons and increased the sensitivity of mice to both thermal and mechanical stimuli applied to the hindpaw, both of which were attenuated by intraplantar injection of a T-type channel inhibitor. Furthermore, inhibiting IGF-1R signaling or knocking down Ca_V3.2 or PKCα in DRG neurons abolished the increased mechanical and thermal sensitivity that mice exhibited under conditions modeling chronic hindpaw inflammation. Together, our results showed that IGF-1 enhances T-type channel currents through the activation of IGF-1R that is coupled to a G protein–dependent PKCα pathway, thereby increasing the excitability of DRG neurons and the sensitivity to pain.

The corticotropin-releasing factor (CRF) peptides CRF and uro-cortins 1 to 3 are crucial regulators of mammalian stress and inflammatory responses, and they are also implicated in disorders such as anxiety, depression, and drug addiction. There is considerable interest in the physiological mechanisms by which CRF receptors mediate their widespread effects, and here we report that the native CRF receptor 1 (CRFR1) endogenous to the human embryonic kidney 293 cells can functionally couple to mammalian Ca_V3.2 T-type calcium channels. Activation of CRFR1 by either CRF or urocortin (UCN) 1 reversibly inhibits Ca_V3.2 currents (IC₅₀ of ∼30 nM), but it does not affect Ca_V3.1 or Ca_V3.3 channels. Blockade of CRFR1 by the antagonist astressin abolished the inhibition of Ca_V3.2 channels. The CRFR1-dependent inhibition of Ca_V3.2 channels was independent of the activities of phospholipase C, tyrosine kinases, Ca²⁺/calmodulin-dependent protein kinase II, protein kinase C, and other kinase pathways, but it was dependent upon a cholera toxin-sensitive G protein-mediated mechanism relying upon G protein βγ subunits (Gβγ). The inhibition of Ca_V3.2 channels via the activation of CRFR1 was due to a hyperpolarized shift in their steady-state inactivation, and it was reversible upon washout of the agonists. Given that UCN affect multiple aspects of cardiac and neuronal physiology and that Ca_V3.2 channels are widespread throughout the cardiovascular and nervous systems, the results point to a novel and functionally relevant CRFR1-Ca_V3.2 T-type calcium channel signaling pathway.