Docosahexaenoic acid block of neuronal voltage-gated K+ channels: subunit selective antagonism by zinc.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid is highly enriched in neuronal membranes, and several studies suggest that DHA is critical for neuronal development. We have investigated the effects of exogenously applied DHA on voltage-gated K+ channels using patch-clamp techniques. DHA produced a concentration-dependent inhibition of the sustained outward current in isolated neocortical neurons. This blocking action was examined in more detail with two cloned neuronal K+ channels (Kv1.2 and Kv3.1a) expressed in mammalian fibroblasts. DHA produced a potent inhibition of depolarization-activated K+ currents from cells expressing these channels (Kd values, 1.8 +/- 0.1 muM and 690 +/- 60 nM, for Kv1.2 and Kv3.1a, respectively, at +40 mV). The DHA block of both channel types was rapidly reversed (approximately 2 sec) by bovine serum albumin, which binds the fatty acid. Micromolar concentrations of extracellular Zn2+ non-competitively antagonized DHA inhibition of Kv1.2 channels, whereas there was little effect on DHA block of Kv3.1a channels. Experiments with membrane patches from Kv1.2 transfected cells demonstrated that the DHA block occurred from the outside, suggesting that the fatty acid interacts directly with an external domain of the ion channel. DHA may serve as a local messenger molecule that selectively modulates the activity of certain voltage-gated K+ channels in a Zn2(+)-dependent fashion.

Anandamide, an endogenous cannabinoid, inhibits Shaker-related voltage-gated K+ channels.

Anandamide has been identified in porcine brain as an endogenous cannabinoid receptor ligand and is believed to be a counterpart to the psychoactive component of marijuana, delta 9-tetrahydrocannabinol (delta 9-THC). Here we report that anandamide directly inhibits (IC50, 2.7 muM) Shaker-related Kv1.2 K+ channels that are found ubiquitously in the mammalian brain. Delta 9-THC also inhibited Kv1.2 channels with comparable potency (IC50, 2.4 muM), as did several N-acyl-ethanolamides with cannabinoid receptor binding activity. Potassium current inhibition occurred through a pertussis toxin-insensitive mechanism and was not prevented by the cannabinoid receptor antagonist SR141716A. Utilizing excised patches of Kv1.2 channel-rich membrane as a rapid and sensitive bioassay, we found that phospholipase D stimulated the release of an endogenous anandamide-like K+ channel blocker from rat brain slices. Structure-activity studies were consistent with the possibility that the released blocker was either anandamide or another N-acyl-ethanolamide.

Human papillomavirus (HPV) status of non-tobacco related squamous cell carcinomas of the lateral tongue.

OBJECTIVES: The human papillomavirus (HPV) is an important cause of some head and neck squamous cell carcinomas (HNSCCs), but its role in cancer of the lateral tongue is debatable. Suspicion of HPV causation is heightened when these lateral tongue carcinomas arise in patients that are young and/or have never smoked. The purpose of this study was to determine the incidence of transcriptionally active high risk HPV in these tumors, with a particular emphasis on non-smoking patients who are often presumed to have HPV-positive tumors. METHODS: We evaluated 78 HNSCCs of the lateral tongue for the presence of HPV using p16 immunohistochemistry and an RNA in situ hybridization assay targeting HPV E6/E7 mRNA. The study population was enriched for patients without traditional risk factors such as smoking and drinking. RESULTS: P16 overexpression was detected in 9 (11.5%) of 78 cases, but HPV E6/E7 mRNA transcripts were detected in only 1 (1.3%) case (positive predictive value of p16 staining for the presence of transcriptionally active HPV=0.12). HPV mRNA transcripts were not detected in any patient under 40 (n=11), or in patients who had never smoked (n=44), had quit smoking (n=15), and/or were only light consumers of alcohol (n=57). CONCLUSIONS: HPV is not detected in the vast majority of lateral tongue carcinomas. In light of the observation that HPV plays little if any role in the development of these cancers, routine HPV testing is unwarranted , even for patients without traditional risk factors. P16 staining is not a reliable marker for the presence of transcriptionally active HPV at this particular anatomic site.

Time- and voltage-dependent block of delayed rectifier potassium channels by docosahexaenoic acid.

Docosahexaenoic acid (22:6n3) acts at an extracellular site to produce a voltage- and time-dependent block of the delayed rectifier current (IK) similar to that classically described for intracellularly applied quaternary ammonia compounds. In dissociated cells from the pineal gland, some long-chain polyunsaturated fatty acids reduced both late sustained (IK) (for 22:6n3, IC50 = 2.5 +/- 0.3 microM) and early transient (IA) (IC50 = 2.0 +/- 0.1 microM) components of potassium current when applied extracellularly, whereas the monounsaturate oleic acid had minimal efficacy. From comparisons of other related fatty acids, it was determined that there is a structural requirement for polyunsaturation to block IK. In contrast, chain-elongated 22-carbon polyunsaturates acted similarly to their precursor 20-carbon fatty acids (arachidonic acid and eicosapentanoic acid). Block of IK by 22:6n3 was accompanied by a dose-dependent acceleration of the current decay in both whole-cell and outside-out membrane patches, and 22:6n3 increased the macroscopic inactivation rate of IA. The combined "eicosanoid" inhibitor eicosatetraenoic acid, when included in the patch pipette, did not antagonize the action of 22:6n3. Instead, eicosatetraenoic acid produced a direct block of IK when applied extracellularly at high concentrations (25 microM). Analyses of voltage- and time-dependent block by 22:6n3 support the hypothesis that certain fatty acids directly interact with and preferentially block the open state of some potassium channels. We also describe an interaction between fatty acid block and zinc; 22:6n3 failed to block either IA or IK in the presence of zinc or cadmium, whereas extracellular calcium did not affect the response. These studies suggest a possible biological function for 22:6n3 in the nervous system, which may underlie its essential role during neural development.