Extracellular glucose and dysfunctional insulin receptor signaling independently upregulate arterial smooth muscle TMEM16A expression.

Diabetes alters the function of ion channels responsible for regulating arterial smooth muscle membrane potential, resulting in vasoconstriction. Our prior research demonstrated an elevation of TMEM16A in diabetic arteries. Here, we explored the mechanisms involved in Transmembrane protein 16A (TMEM16A) gene expression. Our data indicate that a Snail-mediated repressor complex regulates arterial TMEM16A gene transcription. Snail expression was reduced in diabetic arteries while TMEM16A expression was upregulated. The TMEM16A promoter contained three canonical E-box sites. Electrophoretic mobility and super shift assays revealed that the -154 nt E-box was the binding site of the Snail repressor complex and binding of the repressor complex decreased in diabetic arteries. High glucose induced a biphasic contractile response in pressurized nondiabetic mouse hindlimb arteries incubated ex vivo. Hindlimb arteries incubated in high glucose also showed decreased phospho-protein kinase D1 and TMEM16A expression. In hindlimb arteries from nondiabetic mice, administration of a bolus dose of glucose activated protein kinase D1 signaling to induce Snail degradation. In both in vivo and ex vivo conditions, Snail expression exhibited an inverse relationship with the expression of protein kinase D1 and TMEM16A. In diabetic mouse arteries, phospho-protein kinase D1 increased while Akt2 and pGSK3ß levels declined. These results indicate that in nondiabetic mice, high glucose triggers a transient deactivation of the Snail repressor complex to increase arterial TMEM16A expression independently of insulin signaling. Conversely, insulin resistance activates GSK3ß signaling and enhances arterial TMEM16A channel expression. These data have uncovered the Snail-mediated regulation of arterial TMEM16A expression and its dysfunction during diabetes.NEW & NOTEWORTHY The calcium-activated chloride channel, TMEM16A, is upregulated in the diabetic vasculature to cause increased vasoconstriction. In this paper, we have uncovered that the TMEM16A gene expression is controlled by a Snail-mediated repressor complex that uncouples with both insulin-dependent and -independent pathways to allow for upregulated arterial protein expression thereby causing vasoconstriction. The paper highlights the effect of short- and long-term glucose-induced dysfunction of an ion channel expression as a causative factor in diabetic vascular disease.

Vasodilators activate the anion channel TMEM16A in endothelial cells to reduce blood pressure.

Systemic blood pressure is acutely controlled by total peripheral resistance as determined by the diameter of small arteries and arterioles, the contractility of which is regulated by endothelial cells lining the lumen of blood vessels. We investigated the physiological functions of the chloride (Cl-) channel TMEM16A in endothelial cells. TMEM16A channels generated calcium (Ca2+)-activated Cl- currents in endothelial cells from control (TMEM16Afl/fl) mice that were absent in those from mice with tamoxifen-inducible, endothelial cell-specific knockout of TMEM16A (TMEM16A ecKO). TMEM16A currents in endothelial cells were activated by the muscarinic receptor agonist acetylcholine and an agonist of the Ca2+ channel TRPV4, which localized in nanoscale proximity with TMEM16A as assessed by single-molecule localization imaging of endothelial cells. Acetylcholine stimulated TMEM16A currents by activating Ca2+ influx through surface TRPV4 channels without altering the nanoscale properties of TMEM16A and TRPV4 surface clusters or their colocalization. In pressurized arteries, activation of TMEM16A channels in endothelial cells induced by acetylcholine; TRPV4 channel stimulation; or intraluminal ATP, another vasodilator, produced hyperpolarization and dilation. Furthermore, deficiency of TMEM16A channels in endothelial cells resulted in increased systemic blood pressure in conscious mice. These data indicate that vasodilators stimulate TRPV4 channels, leading to Ca2+-dependent activation of nearby TMEM16A channels in endothelial cells to produce arterial hyperpolarization, vasodilation, and reduced blood pressure. Thus, TMEM16A is an anion channel in endothelial cells that regulates arterial contractility and blood pressure.

Vasodilators activate TMEM16A channels in endothelial cells to reduce blood pressure.

Endothelial cells (ECs) regulate vascular contractility to control regional organ blood flow and systemic blood pressure. Several cation channels are expressed in ECs which regulate arterial contractility. In contrast, the molecular identity and physiological functions of anion channels in ECs is unclear. Here, we generated tamoxifen-inducible, EC-specific TMEM16A knockout ( TMEM16A ecKO) mice to investigate the functional significance of this chloride (Cl - ) channel in the resistance vasculature. Our data demonstrate that TMEM16A channels generate calcium-activated Cl - currents in ECs of control ( TMEM16A fl/fl ) mice that are absent in ECs of TMEM16A ecKO mice. Acetylcholine (ACh), a muscarinic receptor agonist, and GSK101, a TRPV4 agonist, activate TMEM16A currents in ECs. Single molecule localization microscopy data indicate that surface TMEM16A and TRPV4 clusters locate in very close nanoscale proximity, with ∼18% exhibiting overlap in ECs. ACh stimulates TMEM16A currents by activating Ca 2+ influx through surface TRPV4 channels without altering the size or density of TMEM16A or TRPV4 surface clusters, their spatial proximity or colocalization. ACh-induced activation of TMEM16A channels in ECs produces hyperpolarization in pressurized arteries. ACh, GSK101 and intraluminal ATP, another vasodilator, all dilate pressurized arteries through TMEM16A channel activation in ECs. Furthermore, EC-specific knockout of TMEM16A channels elevates systemic blood pressure in conscious mice. In summary, these data indicate that vasodilators stimulate TRPV4 channels, leading to Ca 2+ -dependent activation of nearby TMEM16A channels in ECs to produce arterial hyperpolarization, vasodilation and a reduction in blood pressure. We identify TMEM16A as an anion channel present in ECs that regulates arterial contractility and blood pressure. One sentence summary: Vasodilators stimulate TRPV4 channels, leading to calcium-dependent activation of nearby TMEM16A channels in ECs to produce arterial hyperpolarization, vasodilation and a reduction in blood pressure.

Caffeine Consumption plus Physical Exercise Improves Behavioral Impairments and Stimulates Neuroplasticity in Spontaneously Hypertensive Rats (SHR): an Animal Model of Attention Deficit Hyperactivity Disorder.

Attention deficit hyperactivity disorder (ADHD) is a prevalent and disabling disorder, mainly characterized by hyperactivity, inattention, and impulsivity, but also by olfactory and memory impairments that frequently persist throughout lifetime. The pathophysiology of ADHD is complex, involving several brain regions and neural pathways including alterations in adenosine neuromodulation. The administration of caffeine (a non-selective adenosine receptor antagonist) and physical exercise have been independently pointed as effective approaches for the management of ADHD symptoms. Here, we evaluated the effects of caffeine consumption (0.3 mg/mL in drinking water) plus physical exercise in running wheels during 6 weeks-starting during either adolescence (30 days old) or adulthood (4-5 months old)-on behavioral performance (including olfactory discrimination, open field, object recognition, and water maze tests) on the brain levels of monoamines (by high-performance liquid chromatography), on proteins related to synaptic plasticity and on brain-derived neurotrophic factor signaling (by Western blot analysis) in spontaneously hypertensive rats (SHRs), a validated animal model of ADHD. SHRs displayed persistent impairments of olfactory and short-term recognition memory from adolescence to adulthood, which were accompanied by lower levels of synaptosomal-associated protein 25 (SNAP-25) in the prefrontal cortex and hippocampus. The association of caffeine plus physical exercise during adolescence or adulthood restored the olfactory discrimination ability and, in an independent manner, improved short-term recognition memory of SHRs. These benefits were not associated to alterations in locomotor activity or in the hypertensive phenotype. The association of caffeine consumption plus physical exercise during adolescence increased the levels of SNAP-25, syntaxin, and serotonin in the hippocampus and prefrontal cortex, and striatal dopamine levels in SHRs. These results provide new evidence of the potential of caffeine and physical exercise, starting at adolescence or adult life, to improve behavioral impairments and stimulate neuroplasticity in ADHD.

Vasoplegia in sepsis depends on the vascular system, vasopressor, and time-point: a comparative evaluation in vessels from rats subjected to the cecal ligation puncture model.

We evaluated the effects of phenylephrine, norepinephrine, angiotensin II, and vasopressin in mesenteric, renal, carotid, and tail arteries, and in perfused mesenteric vascular bed from rats subjected to the cecal ligation and puncture (CLP) model of sepsis. Phenylephrine and angiotensin II were less efficacious in mesenteric arteries from the CLP 6 h and CLP 18 h groups than in preparations from non-septic animals, but no differences were found for norepinephrine and vasopressin between the preparations. In renal arteries, none of the vasoconstrictors had impaired activity in the CLP groups. Nonetheless, carotid arteries from the CLP 18 h group presented reduced reactivity to all vasoconstrictors tested, but only phenylephrine and norepinephrine had their effects reduced in carotid arteries from the CLP 6 h group. Despite the reduced responsiveness to phenylephrine, tail arteries from septic rats were hyperreactive to vasopressin and norepinephrine at 6 h and 18 h after the CLP surgery, respectively. The mesenteric vascular bed from CLP groups was hyporeactive to phenylephrine, norepinephrine, and angiotensin II, but not to vasopressin. The vascular contractility in sepsis varies from the well-described refractoriness, to unaltered or even hyperresponsiveness to vasoconstrictors, depending on the vessel, the vasoactive agent, and the time period evaluated.