Menthol, a secondary alcohol produced by the peppermint herb, Mentha piperita, is widely used in the food and pharmaceutical industries as a cooling/soothing compound and odorant. It induces Ca 2؉ influx in a subset of sensory neurons from dorsal root and trigeminal ganglia, due to activation of TRPM8, a Ca 2؉ -permeable, cold-activated member of the TRP superfamily of cation channels. Menthol also induces Ca 2؉ release from intracellular stores in several TRPM8-expressing cell types, which has led to the suggestion that TRPM8 can function as an intracellular Ca 2؉ -release channel. Here we show that menthol induces Ca 2؉ release from intracellular stores in four widely used cell lines (HEK293, lymph node carcinoma of the prostate (LNCaP), Chinese hamster ovary (CHO), and COS), and provide several lines of evidence indicating that this release pathway is TRPM8-independent: 1) menthol-induced Ca 2؉ release was potentiated at higher temperatures, which contrasts to the cold activation of TRPM8; 2) overexpression of TRPM8 did not enhance the menthol-induced Ca 2؉ release; 3) menthol-induced Ca 2؉ release was mimicked by geraniol and linalool, which are structurally related to menthol, but not by the more potent TRPM8 agonists icilin or eucalyptol; and 4) TRPM8 expression in HEK293 cells was undetectable at the protein and mRNA levels. Moreover, using a novel TRPM8-specific antibody we demonstrate that both heterologously expressed TRPM8 (in HEK293 cells) and endogenous TRPM8 (in LNCaP cells) are mainly localized in the plasma membrane, which contrast to previous localization studies using commercial anti-TRPM8 antibodies. Finally, aequorin-based measurements demonstrate that the TRPM8-independent menthol-induced Ca 2؉ release originates from both endoplasmic reticulum and Golgi compartments.Menthol is a naturally occurring compound responsible for the minty flavor and smell of the mint plant (Mentha piperita). Whereas the first descriptions of the use of menthol as a cooling/soothing compound and odorant date back to ϳ2000 years ago, menthol is still extensively used as an additive in a wide variety of products ranging from ointments and candies to cigarettes. The soothing, refreshing and invigorating feature of the oil made from the peppermint herb is useful in massage for muscle fatigue. It is also used in the treatment of asthma, colic, exhaustion, fever, flatulence, headache, nausea, scabies, sinusitis, and vertigo (1). Currently the best described molecular target of menthol is TRPM8, 2 a member of the melastatin branch of the TRP superfamily of cation channels. TRPM8 was initially identified in a screening procedure aimed at identifying mRNAs that are up-regulated in prostate cancer (2). Subsequently, TRPM8 expression was also demonstrated to be strongly up-regulated in several other primary tumor types including breast, colon, lung, and skin (2). The following studies identified TRPM8 in a subset of dorsal root and trigeminal neurons (3), and found it to function as a plasma membrane Ca 2ϩ -permeable cation ...

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Background information-The IP 3 R (inositol 1,4,5-trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca 2+ release in virtually all cell types. We have previously demonstrated that caspase-3-mediated cleavage of IP 3 R1 during cell death generates a C-terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP 3 R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel.

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Plasma membrane chloride (Cl(-)) pathways play an important role in neuronal physiology. Here, we investigated the role of NKCC1 cotransporters (a secondary active Cl(-) uptake mechanism) in Cl(-) handling in cultured rat dorsal root ganglion neurons (DRGNs) and motor neurons (MNs) derived from fetal stage embryonic day 14. Gramicidin-perforated patch-clamp recordings revealed that DRGNs accumulate intracellular Cl(-) through a bumetanide- and Na(+)-sensitive mechanism, indicative of the functional expression of NKCC1. Western blotting confirmed the expression of NKCC1 in both DRGNs and MNs, but immunocytochemistry experiments showed a restricted expression in dendrites of MNs, which contrasts with a homogeneous expression in DRGNs. Both MNs and DRGNs could be readily loaded with or depleted of Cl(-) during GABA(A) receptor activation at depolarizing or hyperpolarizing membrane potentials. After loading, the rate of recovery to the resting Cl(-) concentration (i.e., [Cl(-)](i) decrease) was similar in both cell types and was unaffected by lowering the extracellular Na(+) concentration. In contrast, the recovery on depletion (i.e., [Cl(-)](i) increase) was significantly faster in DRGNs in control conditions but not in low extracellular Na(+). The experimental observations could be reproduced by a mathematical model for intracellular Cl(-) kinetics, in which DRGNs show higher NKCC1 activity and smaller Cl(-)-handling volume than MNs. On the basis of these results, we conclude that embryonic DRGNs show a higher somatic functional expression of NKCC1 than embryonic MNs. The high NKCC1 activity in DRGNs is important for maintaining high [Cl(-)](i), whereas lower NKCC1 activity in MNs allows large [Cl(-)](i) variations during neuronal activity.