Activation of endothelial transient receptor potential C3 channel is required for small conductance calcium-activated potassium channel activation and sustained endothelial hyperpolarization and vasodilation of cerebral artery

BACKGROUND: Transient receptor potential C3 (TRPC3) has been demonstrated to be involved in the regulation of vascular tone through endothelial cell (EC) hyperpolarization and endothelium-dependent hyperpolarization-mediated vasodilation. However, the mechanism by which TRPC3 regulates these processes remains unresolved. We tested the hypothesis that endothelial receptor stimulation triggers rapid TRPC3 trafficking to the plasma membrane, where it provides the source of Ca(2+) influx for small conductance calcium-activated K(+) (SKCa) channel activation and sustained EC hyperpolarization. METHODS AND RESULTS: Pressurized artery studies were performed with isolated mouse posterior cerebral artery. Treatment with a selective TRPC3 blocker (Pyr3) produced significant attenuation of endothelium-dependent hyperpolarization-mediated vasodilation and endothelial Ca(2+) response (EC-specific Ca(2+) biosensor) to intraluminal ATP. Pyr3 treatment also resulted in a reduced ATP-stimulated global Ca(2+) and Ca(2+) influx in primary cultures of cerebral endothelial cells. Patch-clamp studies with freshly isolated cerebral ECs demonstrated 2 components of EC hyperpolarization and K(+) current activation in response to ATP. The early phase was dependent on intermediate conductance calcium-activated K(+) channel activation, whereas the later sustained phase relied on SKC a channel activation. The SKC a channel-dependent phase was completely blocked with TRPC3 channel inhibition or in ECs of TRPC3 knockout mice and correlated with increased trafficking of TRPC3 (but not SKC a channel) to the plasma membrane. CONCLUSIONS: We propose that TRPC3 dynamically regulates SKC a channel activation through receptor-dependent trafficking to the plasma membrane, where it provides the source of Ca(2+) influx for sustained SKC a channel activation, EC hyperpolarization, and endothelium-dependent hyperpolarization-mediated vasodilation.Fil: Kochukov, Mikhail Y.. Baylor College of Medicine; Estados UnidosFil: Balasubramanian, Adithya. Baylor College of Medicine; Estados UnidosFil: Abramowitz, Joel. National Institute of Environmental Health Sciences Research; Estados UnidosFil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences Research; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marrelli, Sean P.. Baylor College of Medicine; Estados Unido

Activation of endothelial transient receptor potential C3 channel is required for small conductance calcium-activated potassium channel activation and sustained endothelial hyperpolarization and vasodilation of cerebral artery

BACKGROUND: Transient receptor potential C3 (TRPC3) has been demonstrated to be involved in the regulation of vascular tone through endothelial cell (EC) hyperpolarization and endothelium-dependent hyperpolarization-mediated vasodilation. However, the mechanism by which TRPC3 regulates these processes remains unresolved. We tested the hypothesis that endothelial receptor stimulation triggers rapid TRPC3 trafficking to the plasma membrane, where it provides the source of Ca(2+) influx for small conductance calcium-activated K(+) (SKCa) channel activation and sustained EC hyperpolarization. METHODS AND RESULTS: Pressurized artery studies were performed with isolated mouse posterior cerebral artery. Treatment with a selective TRPC3 blocker (Pyr3) produced significant attenuation of endothelium-dependent hyperpolarization-mediated vasodilation and endothelial Ca(2+) response (EC-specific Ca(2+) biosensor) to intraluminal ATP. Pyr3 treatment also resulted in a reduced ATP-stimulated global Ca(2+) and Ca(2+) influx in primary cultures of cerebral endothelial cells. Patch-clamp studies with freshly isolated cerebral ECs demonstrated 2 components of EC hyperpolarization and K(+) current activation in response to ATP. The early phase was dependent on intermediate conductance calcium-activated K(+) channel activation, whereas the later sustained phase relied on SKC a channel activation. The SKC a channel-dependent phase was completely blocked with TRPC3 channel inhibition or in ECs of TRPC3 knockout mice and correlated with increased trafficking of TRPC3 (but not SKC a channel) to the plasma membrane. CONCLUSIONS: We propose that TRPC3 dynamically regulates SKC a channel activation through receptor-dependent trafficking to the plasma membrane, where it provides the source of Ca(2+) influx for sustained SKC a channel activation, EC hyperpolarization, and endothelium-dependent hyperpolarization-mediated vasodilation.Fil: Kochukov, Mikhail Y.. Baylor College of Medicine; Estados UnidosFil: Balasubramanian, Adithya. Baylor College of Medicine; Estados UnidosFil: Abramowitz, Joel. National Institute of Environmental Health Sciences Research; Estados UnidosFil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences Research; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marrelli, Sean P.. Baylor College of Medicine; Estados Unido

TRPC3 determines osmosensitive [Ca2+]i signaling in the collecting duct and contributes to urinary concentration

It is well-established that the kidney collecting duct (CD) plays a central role in regulation of systemic water homeostasis. Aquaporin 2 (AQP2)-dependent water reabsorption in the CD critically depends on the arginine vasopressin (AVP) antidiuretic input and the presence of a favorable osmotic gradient at the apical plasma membrane with tubular lumen being hypotonic compared to the cytosol. This osmotic difference creates a mechanical force leading to an increase in [Ca2+]i in CD cells. The significance of the osmosensitive [Ca2+]i signaling for renal water transport and urinary concentration remain unknown. To examine molecular mechanism and physiological relevance of osmosensitivity in the CD, we implemented simultaneous direct measurements of [Ca2+]i dynamics and the rate of cell swelling as a readout of the AQP2-dependent water reabsorption in freshly isolated split-opened CDs of wild type and genetically manipulated animals and combined this with immunofluorescent detection of AVP-induced AQP2 trafficking and assessment of systemic water balance. We identified the critical role of the Ca2+-permeable TRPC3 channel in osmosensitivity and water permeability in the CD. We further demonstrated that TRPC3 -/- mice exhibit impaired urinary concentration, larger urinary volume and a greater weight loss in response to water deprivation despite increased AVP levels and AQP2 abundance. TRPC3 deletion interfered with AQP2 translocation to the plasma membrane in response to water deprivation. In summary, we provide compelling multicomponent evidence in support of a critical contribution of TRPC3 in the CD for osmosensitivity and renal water handling.Fil: Tomilin, Viktor N.. University of Texas; Estados UnidosFil: Mamenko, Mykola. Augusta University; Estados UnidosFil: Zaika, Oleg. University of Texas; Estados UnidosFil: Ren, Guohui. University of Texas; Estados UnidosFil: Marrelli, Sean P.. University of Texas; Estados UnidosFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Pochynyuk, Oleh. University of Texas; Estados Unido

Systemic Administration of the TRPV3 Ion Channel Agonist Carvacrol Induces Hypothermia in Conscious Rodents.

Therapeutic hypothermia is a promising new strategy for neuroprotection. However, the methods for safe and effective hypothermia induction in conscious patients are lacking. The current study explored the Transient Receptor Potential Vanilloid 3 (TRPV3) channel activation by the agonist carvacrol as a potential hypothermic strategy. It was found that carvacrol lowers core temperature after intraperitoneal and intravenous administration in mice and rats. However, the hypothermic effect at safe doses was modest, while higher intravenous doses of carvacrol induced a pronounced drop in blood pressure and substantial toxicity. Experiments on the mechanism of the hypothermic effect in mice revealed that it was associated with a decrease in whole-body heat generation, but not with a change in cold-seeking behaviors. In addition, the hypothermic effect was lost at cold ambient temperature. Our findings suggest that although TRPV3 agonism induces hypothermia in rodents, it may have a limited potential as a novel pharmacological method for induction of hypothermia in conscious patients due to suboptimal effectiveness and high toxicity

Carvacrol-induced hypothermia is associated with a decrease in whole-body energy production.

<p>Whole-body heat production was calculated from oxygen consumption (indirect calorimetry) measured in a metabolic chamber. Mice were injected intraperitoneally with vehicle or carvacrol at 31.6 mg/kg at time 0. N = 6 mice per group in a within-subject design. (A) and (C) Time course of core temperature (A) and heat production measurements (C). * P<0.05 at respective time points, 2-way Repeated Measures ANOVA/Holm-Sidak post-hoc test. (B) and (D) Change in core temperature (B) and heat production (D) from 0 min pre-injection to 30 min post-injection. * P<0.05, t-test.</p

Carvacrol delivered intravenously induces an acute drop in blood pressure in anesthetized mice.

<p>(A) Representative time course graphs showing the effects of a range of doses of carvacrol delivered intraperitoneally (IP) and intravenously (IV) on blood pressure of anesthetized CD-1 mice with stably maintained normal core temperature; PBS: phosphate-buffered saline (IV), used a negative control; PE: phenylephrine, 50 μg/kg IV, used as a validation control for reliable measurements of changes in blood pressure; Carv: carvacrol. (B) The relative change between pre-treatment (calculated as an average over 1 min prior to injection) and post-treatment (an average over 3 min after injection) blood pressure levels after respective treatments. Sol.: Solutol. n = 5 mice in a within-subject design. * P<0.05, one-sample t-test with a hypothesized mean of zero as the null hypothesis (i.e. no change in blood pressure); § P<0.05 vs PBS, t test; # P<0.05 vs Solutol IP, one-way ANOVA/Student-Newman-Keuls test.</p

Induction of cold-seeking behaviors by carvacrol was not detected in the two-plate temperature assay.

<p>(A) Schematic of the experiment. CD-1 mice were intravenously injected with vehicle or carvacrol at 50 mg/kg. 10 min after injection, mice were introduced for 3 min into the temperature-preference apparatus, where they were allowed to move freely and select between two plates set to 30°C (left plate) and 10°C right plate). (B) The percentage of time spent on each plate was measured. This time reveals the preference for the temperature of the respective plate. N = 8 mice per group. P>0.05, t-test. (C) Core temperature was measured during the described protocol every 5 min by wireless temperature transmitters. Carvacrol injection, but not vehicle injection, induced hypothermia. * P<0.05, 2-way Repeated measures ANOVA followed by Student-Neuman-Keuls post-hoc test.</p

The hypothermic effect of carvacrol is lost at low ambient temperature of 10°C.

<p>(A) Adult male CD-1 mice were injected intravenously with vehicle or carvacrol at 50 mg/kg at time 0 and transferred to the temperature-controlled chamber with an ambient temperature set to 10°C. Core temperature of mice was measured by wireless temperature transmitters every 15 min for 1 hour. (B) The drop in core temperature between 0 min pre-injection and 30 min post-injection was calculated from the experiment shown in panel A. n = 6 mice per group. n.s.: P>0.05, t-test.</p