anandamide and Hypoxia

The two-pore domain potassium channel KCNK3 (TASK-1) is expressed in rat and human pulmonary artery smooth muscle cells. There, it is associated with hypoxia-induced signalling, and its dysfunction is linked to pathogenesis of human pulmonary hypertension. We here aimed to determine its role in hypoxic pulmonary vasoconstriction (HPV) in the mouse, and hence the suitability of this model for further mechanistic investigations, using appropriate inhibitors and TASK-1 knockout (KO) mice. RT-PCR revealed expression of TASK-1 mRNA in murine lungs and pre-acinar pulmonary arteries. Protein localization by immunohistochemistry and western blot was unreliable since all antibodies produced labelling also in TASK-1 KO organs/tissues. HPV was investigated by videomorphometric analysis of intra- (inner diameter: 25-40 μm) and pre-acinar pulmonary arteries (inner diameter: 41-60 μm). HPV persisted in TASK-1 KO intra-acinar arteries. Pre-acinar arteries developed initial HPV, but the response faded earlier (after 30 min) in KO vessels. This HPV pattern was grossly mimicked by the TASK-1 inhibitor anandamide in wild-type vessels. Hypoxia-provoked rise in pulmonary arterial pressure (PAP) in isolated ventilated lungs was affected neither by TASK-1 gene deficiency nor by the TASK-1 inhibitor A293. TASK-1 is dispensable for initiating HPV of murine intra-pulmonary arteries, but participates in sustained HPV specifically in pre-acinar arteries. This does not translate into abnormal rise in PAP. While there is compelling evidence that TASK-1 is involved in the pathogenesis of pulmonary arterial hypertension in humans, the mouse does not appear to serve as a suitable model to study the underlying molecular mechanisms.

Topics: Animals; Arachidonic Acids; Endocannabinoids; Female; Hypoxia; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Polyunsaturated Alkamides; Potassium Channels, Tandem Pore Domain; Pulmonary Artery; RNA, Messenger; Vasoconstriction

Oligodendrocytes are the myelinating cells of the CNS and, like neurons, are highly sensitive to ischemic damage. However, the mechanisms underlying cytotoxicity in oligodendrocytes during hypoxic/ischemic episodes are not fully understood. TASK-1 is a K(+) leak channel that mediates hypoxic depolarisation in neurons. The expression and function of TASK-1 in oligodendrocytes had not previously been addressed. In this study, we investigate the expression of TASK-1 in oligodendrocytes and its role in white matter ischemic damage. Expression of TASK-1 in oligodendrocytes was investigated in the mouse brain using immunostaining. TASK-1 channel function was identified by established pharmacological and electrophysiological strategies, using the whole-cell patch clamp technique in cell cultures of oligodendrocytes from the optic nerve, a typical white matter tract. The role of TASK-1 in hypoxia was examined in isolated intact optic nerves subjected to oxygen glucose deprivation (OGD). Oligodendrocytes are strongly immunopositive for TASK-1 throughout the brain. Patch-clamp identified functional TASK-1-like leak currents in oligodendrocytes using two recognised means of inhibiting TASK-1, decreasing extracellular pH to 6.4 and exposure to the TASK-1 selective inhibitor anandamide. Incubation of optic nerves with methanandamide, a non-hydrolysable form of anandamide, significantly protected oligodendrocytes against hypoxic disruption and death in OGD. Our data demonstrate for the first time that oligodendrocytes express functional TASK-1 channels and provide compelling evidence they contribute to oligodendrocyte damage in hypoxia. Since oligodendrocyte damage is a key factor in ischemic episodes, TASK-1 may provide a potential therapeutic target in stroke and white matter disease.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Biophysical Phenomena; Brain; Calcium Channel Blockers; Electric Stimulation; Endocannabinoids; Gene Expression Regulation; Glucose; Hypoxia; Ischemia; Luminescent Proteins; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin Proteolipid Protein; Nerve Tissue Proteins; Neurons; Oligodendroglia; Optic Nerve; Organ Culture Techniques; Patch-Clamp Techniques; Polyunsaturated Alkamides; Potassium Channels, Tandem Pore Domain; Protons

Endocannabinoids are important regulators of organ homeostasis. Although their role in systemic vasculature has been extensively studied, their impact on pulmonary vessels remains less clear. Herein, we show that the endocannabinoid anandamide (AEA) is a key mediator of hypoxic pulmonary vasoconstriction (HPV) via fatty acid amide hydrolase (FAAH)-dependent metabolites. This is underscored by the prominent vasoconstrictive effect of AEA on pulmonary arteries and strongly reduced HPV in FAAH(-/-) mice and wild-type mice upon pharmacological treatment with FAAH inhibitor URB597. In addition, mass spectrometry measurements revealed a clear increase of AEA and the FAAH-dependent metabolite arachidonic acid in hypoxic lungs of wild-type mice. We have identified pulmonary vascular smooth muscle cells as the source responsible for hypoxia-induced AEA generation. Moreover, either FAAH(-/-) mice or wild-type mice treated with FAAH inhibitor URB597 are protected against hypoxia-induced pulmonary hypertension and the concomitant vascular remodeling in the lung. Thus, the AEA/FAAH pathway is an important mediator of HPV and is involved in the generation of pulmonary hypertension.

Topics: Amidohydrolases; Analysis of Variance; Animals; Arachidonic Acids; Benzamides; Blotting, Western; Carbamates; Chromatography, Liquid; DNA Primers; Endocannabinoids; Hypertension, Pulmonary; Hypoxia; Immunohistochemistry; Lung; Mice; Mice, Knockout; Myocytes, Smooth Muscle; Polyunsaturated Alkamides; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Vasoconstriction

The possible roles of different potassium channels in oxygen sensing mechanisms of the carotid body are still not well defined. It has been suggested that leak potassium (K(+)) channels, voltage gated K(+) channels (Kv) and Ca(2+) dependent large conductance K(+) channels (BK) play important roles in the peripheral chemo-sensing mechanisms of the carotid body. In the present study, we have made an attempt to clarify the extent to which, these channels are involved in such mechanisms using in vitro model of isolated rabbit carotid body. Selective TASK-1 channel blocker, anandamide (3 microM) induced stimulation of the carotid sinus nerve (CSN) activity, in a very similar pattern to normal hypoxic responses, with peak discharge rates of the CSN up to 63 +/- 21% of the hypoxic responses (n = 33), and no additional increase in the CSN activity was observed during the hypoxic stimulation with the co-application of anandamide (n = 6). On the other hand, inhibition of BK channels by TEA (5 mM, n = 6), was sub-maximum and typical hypoxic responses were preserved during the increased CSN activity induced by TEA (n = 7). Maximal stimulation of the CSN activity was obtained by blocking Kv channels with 4AP (2.5 mM, n = 15), which was refractory to the hypoxic response. However the hypoxic response reappeared during hyperpolarization (n = 12). We have found that the leak K(+) channels (TASK-1) seem to be importantly involved in the initiation of the oxygen sensing mechanisms of the rabbit carotid body.

Topics: Administration, Inhalation; Anesthetics; Animals; Arachidonic Acids; Arteries; Carotid Body; Chemoreceptor Cells; Endocannabinoids; Halothane; Hypoxia; Male; Oxygen; Polyunsaturated Alkamides; Potassium Channel Blockers; Potassium Channels, Tandem Pore Domain; Potassium Channels, Voltage-Gated; Rabbits