hc-067047 and Disease-Models--Animal

Vasogenic edema results from blood-brain barrier (BBB) disruption after traumatic brain injury (TBI), and although it can be fatal, no promising therapeutic drugs have been developed as yet. Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable channel that is sensitive to temperature and osmotic pressure. As TRPV4 is known to be responsible for various pathological conditions following brain injury, we investigated the effects of pharmacological TRPV4 antagonists on TBI-induced vasogenic edema in this study. A TBI model was established by inflicting fluid percussion injury (FPI) in the mouse cerebrum and cultured astrocytes. Vasogenic brain edema and BBB disruption were assessed based on brain water content and Evans blue (EB) extravasation into brain tissue, respectively. After FPI, brain water content and EB extravasation increased. Repeated intracerebroventricular administration of the specific TRPV4 antagonists HC-067047 and RN-1734 dose-dependently reduced brain water content and alleviated EB extravasation in FPI mice. Additionally, real-time PCR analysis indicated that administration of HC-067047 and RN-1734 reversed the FPI-induced increase in mRNA levels of endogenous causal factors for BBB disruption, including matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor-A (VEGF-A), and endothelin-1 (ET-1). In astrocytes, TRPV4 level was observed to be higher than that in brain microvascular endothelial cells. Treatment with HC-067047 and RN-1734 inhibited the increase in mRNA levels of MMP-9, VEGF-A, and ET-1 in cultured astrocytes subjected to in vitro FPI. These results suggest that pharmacological inhibition of TRPV4 is expected to be a promising therapeutic strategy for treating TBI-induced vasogenic edema.

Topics: Animals; Astrocytes; Blood-Brain Barrier; Brain Edema; Brain Injuries, Traumatic; Disease Models, Animal; Male; Matrix Metalloproteinase 9; Mice; Morpholines; Pyrroles; Real-Time Polymerase Chain Reaction; Sulfonamides; TRPV Cation Channels; Vascular Endothelial Growth Factor A

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Hydrocephalus is a serious condition that impacts patients of all ages. The standards of care are surgical options to divert, or inhibit production of, cerebrospinal fluid; to date, there are no effective pharmaceutical treatments, to our knowledge. The causes vary widely, but one commonality of this condition is aberrations in salt and fluid balance. We have used a genetic model of hydrocephalus to show that ventriculomegaly can be alleviated by inhibition of the transient receptor potential vanilloid 4, a channel that is activated by changes in osmotic balance, temperature, pressure and inflammatory mediators. The TRPV4 antagonists do not appear to have adverse effects on the overall health of the WT or hydrocephalic animals.

Topics: Animals; Cerebral Cortex; Disease Models, Animal; Hydrocephalus; Morpholines; Nervous System Malformations; Pyrroles; Rats; TRPV Cation Channels

Cardiomyocyte Ca2+ homeostasis is altered with aging via poorly-understood mechanisms. The Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel is an osmotically-activated Ca2+ channel, and there is limited information on the role of TRPV4 in cardiomyocytes. Our data show that TRPV4 protein expression increases in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in cardiomyocyte Ca2+ homeostasis following hypoosmotic stress and to assess the contribution of TRPV4 to cardiac contractility and tissue damage following ischaemia-reperfusion (I/R), a pathological condition associated with cardiomyocyte osmotic stress.. TRPV4 protein expression increased in cardiomyocytes of Aged (24-27 months) mice compared with Young (3-6 months) mice. Immunohistochemistry revealed TRPV4 localization to microtubules and the t-tubule network of cardiomyocytes of Aged mice, as well as in left ventricular myocardium of elderly patients undergoing surgical aortic valve replacement for aortic stenosis. Following hypoosmotic stress, cardiomyocytes of Aged, but not Young exhibited an increase in action-potential induced Ca2+ transients. This effect was mediated via increased sarcoplasmic reticulum Ca2+ content and facilitation of Ryanodine Receptor Ca2+ release and was prevented by TRPV4 antagonism (1 μmol/L HC067047). A similar hypoosmotic stress-induced facilitation of Ca2+ transients was observed in Young transgenic mice with inducible TRPV4 expression in cardiomyocytes. Following I/R, isolated hearts of Young mice with transgenic TRPV4 expression exhibited enhanced contractility vs. hearts of Young control mice. Similarly, hearts of Aged mice exhibited enhanced contractility vs. hearts of Aged TRPV4 knock-out (TRPV4-/-) mice. In Aged, pharmacological inhibition of TRPV4 (1 μmol/L, HC067047) prevented hypoosmotic stress-induced cardiomyocyte death and I/R-induced cardiac damage.. Our findings provide a new mechanism for hypoosmotic stress-induced cardiomyocyte Ca2+ entry and cell damage in the aged heart. These finding have potential implications in treatment of elderly populations at increased risk of myocardial infarction and I/R injury.

Topics: Age Factors; Animals; Calcium; Calcium Signaling; Disease Models, Animal; Humans; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Myocardial Contraction; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Osmotic Pressure; Pyrroles; Sarcoplasmic Reticulum; TRPV Cation Channels

Transient receptor potential vanilloid 4 (TRPV4) receptor modulates pain, and this has been noted in several animal models. However, the involvement of TRPV4 in osteoarthritic (OA) pain remains poorly understood. This study assessed the functional changes in TRPV4 and the expression of its endogenous ligand 5,6-epoxyeicosatrienoic acid (5,6-EET) in a rat monoiodoacetate (MIA)-induced OA pain model (MIA rats). Monoiodoacetate-treated rats showed reduced grip strength as compared to sham-treated rats, and this loss in function could be recovered by the intraarticular administration of a TRPV4 antagonist (HC067047 or GSK2193874). By contrast, the intraarticular administration of the TRPV4 agonist, GSK1016790A, increased the pain-related behaviors in MIA rats but not in sham rats. TRPV4 expression was not increased in knee joints of MIA rats; however, the levels of phosphorylated TRPV4 at Ser824 were increased in dorsal root ganglion neurons. In addition, 5,6-EET was increased in lavage fluids from the knee joints of MIA rats and in meniscectomy-induced OA pain model rats. 5,6-EET and its metabolite were also detected in synovial fluids from patients with OA. In conclusion, TRPV4 was sensitized in the knee joints of MIA rats through phosphorylation in dorsal root ganglion neurons, along with an increase in the levels of its endogenous ligand 5,6-EET. The analgesic effects of the TRPV4 antagonist in the OA pain model rats suggest that TRPV4 may be a potent target for OA pain relief.

Topics: Animals; Arthritis, Experimental; Disease Models, Animal; Ganglia, Spinal; Hand Strength; Iodoacetic Acid; Leucine; Male; Morpholines; Neurons; Osteoarthritis; Pain; Pain Measurement; Phosphorylation; Pyrroles; Rats; Rats, Sprague-Dawley; Sulfonamides; TRPV Cation Channels

Therapeutic target transient receptor potential vanilloid-4 (TRPV-4) is frequently applied in endotoxemia research. It has been reported that HC067047, an inhibitor of TRPV-4, mitigated LPS-induced injury. However, the inhibition of TRPV-4 with HC06047 did not attenuate LPS-induced symptoms and exaggerated pathology. This study was carried with a view to unravelling the reason(s) behind these conflicting results. Different doses of the inhibitor were used in the same degree of sepsis, and their effects were determined through assays for sepsis-related physiological indicators such as endothelial injury markers, coagulation index, organ damage indicators, inflammatory factor levels, and cell apoptosis. The results showed that high or low inhibitor levels had no significant effect on sepsis-related physiological indicators. These findings suggest that proper activation of TRPV-4 in sepsis is important for maintaining normal physiological function. Thus, the degree of TRPV-4 activation should match the severity of sepsis.

Topics: Animals; Apoptosis; Biomarkers; Blood Coagulation; Caspase 3; Cytoprotection; Disease Models, Animal; Endothelial Cells; Interleukin-6; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Morpholines; Pyrroles; Sepsis; TRPV Cation Channels; Tumor Necrosis Factor-alpha

Topics: Animals; Apoptosis; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression; Gene Knockout Techniques; Glycogen Synthase Kinase 3 beta; Heart Function Tests; Mice; Molecular Targeted Therapy; Morpholines; Myocardial Reperfusion Injury; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyrroles; Signal Transduction; TRPV Cation Channels

Transient receptor potential vanilloid 4 (TRPV4) is widely expressed in the central nervous system and can be activated by multiple stimuli during cerebral ischemia. Recently, we reported that intracerebroventricular (icv.) injection of HC-067047, a specific TRPV4 antagonist, reduced brain infarction following 60-min of middle cerebral artery occlusion (MCAO). This study was undertaken to investigate the molecular mechanisms underlying TRPV4-mediated neuronal injury in cerebral ischemia. We demonstrated that TRPV4 expression was upregulated in the ipsilateral hippocampus at 4 to 48 h post-MCAO, peaking at 18 h post-MCAO. Treatment with TRPV4 antagonists (HC-067047 and ruthenium red) dose-dependently reduced brain infarction at 24 h post-MCAO. Phosphorylation of protein kinase B (p-Akt) was downregulated and that of extracellular signal-related kinase (p-ERK) was upregulated at 8 to 24 h post-MCAO, which was markedly blocked by treatment with HC-067047. Icv. injection of GSK1016790A (a TRPV4 agonist), dose-dependently induced hippocampal neuronal death, accompanied by an increase in phosphorylation of the NR2B subunit of the N-methyl-D-aspartate receptor (NMDAR). In addition, the level of p-Akt was decreased and that of p-ERK was increased by GSK1016790A-injection, which was sensitive to an NR2B antagonist. The neuronal toxicity of GSK1016790A was blocked by treatment with an NR2B antagonist and a phosphatidylinositol-3-kinase (PI3K) agonist but not by administration of a MAPK/ERK kinase antagonist. We conclude that the activation of TRPV4 is upregulated and involved in neuronal injury during cerebral ischemia and that the neurotoxicity associated with TRPV4-activation is mediated through NR2B-NMDAR and the related downregulation of the Akt signaling pathway.

Topics: Animals; Disease Models, Animal; Hippocampus; Infarction, Middle Cerebral Artery; Male; Mice; Morpholines; Phosphatidylinositol 3-Kinases; Pyrroles; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; TRPV Cation Channels

The cation channel transient receptor potential vanilloid (TRPV) 4 is expressed in endothelial and immune cells; however, its role in acute lung injury (ALI) is unclear. The functional relevance of TRPV4 was assessed in vivo, in isolated murine lungs, and in isolated neutrophils. Genetic deficiency of TRPV4 attenuated the functional, histological, and inflammatory hallmarks of acid-induced ALI. Similar protection was obtained with prophylactic administration of the TRPV4 inhibitor, GSK2193874; however, therapeutic administration of the TRPV4 inhibitor, HC-067047, after ALI induction had no beneficial effect. In isolated lungs, platelet-activating factor (PAF) increased vascular permeability in lungs perfused with trpv4(+/+) more than with trpv4(-/-) blood, independent of lung genotype, suggesting a contribution of TRPV4 on blood cells to lung vascular barrier failure. In neutrophils, TRPV4 inhibition or deficiency attenuated the PAF-induced increase in intracellular calcium. PAF induced formation of epoxyeicosatrienoic acids by neutrophils, which, in turn, stimulated TRPV4-dependent Ca(2+) signaling, whereas inhibition of epoxyeicosatrienoic acid formation inhibited the Ca(2+) response to PAF. TRPV4 deficiency prevented neutrophil responses to proinflammatory stimuli, including the formation of reactive oxygen species, neutrophil adhesion, and chemotaxis, putatively due to reduced activation of Rac. In chimeric mice, however, the majority of protective effects in acid-induced ALI were attributable to genetic deficiency of TRPV4 in parenchymal tissue, whereas TRPV4 deficiency in circulating blood cells primarily reduced lung myeloperoxidase activity. Our findings identify TRPV4 as novel regulator of neutrophil activation and suggest contributions of both parenchymal and neutrophilic TRPV4 in the pathophysiology of ALI.

Topics: Acute Lung Injury; Animals; Bone Marrow Transplantation; Calcium Signaling; Capillary Permeability; Disease Models, Animal; Humans; Hydrochloric Acid; Lung; Male; Mice, Knockout; Morpholines; Neutrophil Activation; Neutrophils; Pneumonia; Pulmonary Edema; Pyrroles; TRPV Cation Channels

Neurovascular coupling (NVC) allows increased blood flow to metabolically active neurons and involves the Ca²⁺ -dependent release of vasodilator influences by astrocyte endfeet that encase parenchymal arterioles. We previously reported inversion of NVC from dilation to constriction in brain slices from subarachnoid hemorrhage (SAH) model rats. Corresponding to NVC inversion, there was a marked increase in the amplitude of spontaneous Ca²⁺ oscillations in astrocyte endfeet. Calcium-permeable transient receptor potential vanilloid (TRPV)-4 channels have been reported in astrocyte endfeet, and activators of these channels enhance Ca²⁺ oscillations in healthy animals. Here, we examined the role of TRPV4 channels in the development of high-amplitude spontaneous Ca²⁺ oscillations in astrocyte endfeet and the inversion of neurovascular coupling after SAH. Treatment of brain slices with the TRPV4 channel antagonist, HC-067047 (10 μM), did not alter the amplitude of spontaneous Ca²⁺ oscillations after SAH. In addition, HC-067047 did not inhibit or change SAH-induced inversion of neurovascular coupling. In summary, TRPV4 channels do not appear to be involved in the inversion of neurovascular coupling after SAH. Further studies examining the impact of SAH on additional Ca²⁺ signaling pathways in astrocytes are likely to reveal valuable insights into new therapeutic strategies to advance SAH treatments.

Topics: Animals; Astrocytes; Calcium Signaling; Disease Models, Animal; Hyperemia; Male; Morpholines; Organ Culture Techniques; Pyrroles; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; TRPV Cation Channels

The pathogenesis of pain in chronic pancreatitis is poorly understood, and its treatment can be a major clinical challenge. Surgical and other invasive methods have variable outcomes that can be unsatisfactory. Therefore, there is a great need for further discovery of the pathogenesis of pancreatitis pain and new therapeutic targets. Human and animal studies indicate a critical role for oxidative stress and activation of transient receptor potential (TRP) cation channel subfamily members TRPV1 and TRPA1 on pancreatic nociceptors in sensitization mechanisms that result in pain. However, the in vivo role of transient receptor potential cation channel subfamily V member 4 (TRPV4) in chronic pancreatitis needs further evaluation. The present study characterized a rat alcohol/high fat diet (AHF)-induced chronic pancreatitis model with hypersensitivity, fibrotic pathology, and fat vacuolization consistent with the clinical syndrome. The rats with AHF-induced pancreatitis develop referred visceral pain-like behaviors, i.e. decreased hindpaw mechanical thresholds and shortened abdominal and hindpaw withdrawal latency to heat. In this study, oxidative stress was characterized as well as the role of TRPV4 in chronic visceral hypersensitivity. Lipid peroxidase and oxidative stress were indicated by increased plasma thiobarbituric acid reactive substances (TBARS) and diminished pancreatic manganese superoxide dismutase (MnSOD). The secondary sensitization associated with AHF-induced pancreatitis was effectively alleviated by the TRPV4 antagonist, HC 067047. Similarity of the results to those with the peripherally restricted μ-opiate receptor agonist, loperamide, suggested TRPV4 channel activated peripheral sensitization. This study using a reliable model that provides pre-clinical correlates of human chronic pancreatitis provides further evidence that TRPV4 channel is a potential therapeutic target for treatment of pancreatitis pain.

Topics: Analgesics; Animals; Diet, High-Fat; Disease Models, Animal; Drug Evaluation, Preclinical; Ethanol; Hot Temperature; Loperamide; Male; Morpholines; Oxidative Stress; Pain; Pain Threshold; Pancreatitis, Chronic; Pyrroles; Random Allocation; Rats, Inbred F344; Receptors, Opioid, mu; Touch; TRPV Cation Channels

Individuals with functional lower urinary tract disorders including interstitial cystitis (IC)/bladder pain syndrome (BPS) and overactive bladder (OAB) often report symptom (e.g., urinary frequency) worsening due to stress. One member of the transient receptor potential ion channel vanilloid family, TRPV4, has recently been implicated in urinary bladder dysfunction disorders including OAB and IC/BPS. These studies address the role of TRPV4 in stress-induced bladder dysfunction using an animal model of stress in male rats. To induce stress, rats were exposed to 7 days of repeated variate stress (RVS). Quantitative PCR data demonstrated significant (P ≤ 0.01) increases in TRPV4 transcript levels in urothelium but not detrusor smooth muscle. Western blot analyses of split urinary bladders (i.e., urothelium and detrusor) showed significant (P ≤ 0.01) increases in TRPV4 protein expression levels in urothelial tissues but not detrusor smooth muscle. We previously showed that RVS produces bladder dysfunction characterized by decreased bladder capacity and increased voiding frequency. The functional role of TRPV4 in RVS-induced bladder dysfunction was evaluated using continuous, open outlet intravesical infusion of saline in conjunction with administration of a TRPV4 agonist, GSK1016790A (3 μM), a TRPV4 antagonist, HC067047 (1 μM), or vehicle (0.1% DMSO in saline) in control and RVS-treated rats. Bladder capacity, void volume, and intercontraction interval significantly decreased following intravesical instillation of GSK1016790A in control rats and significantly (P ≤ 0.01) increased following administration of HC067047 in RVS-treated rats. These results demonstrate increased TRPV4 expression in the urothelium following RVS and that TRPV4 blockade ameliorates RVS-induced bladder dysfunction consistent with the role of TRPV4 as a promising target for bladder function disorders.

Topics: Administration, Intravesical; Animals; Disease Models, Animal; Gene Expression Regulation; Leucine; Male; Morpholines; Pyrroles; Rats; Rats, Wistar; RNA, Messenger; Sulfonamides; Time Factors; TRPV Cation Channels; Urinary Bladder; Urinary Bladder, Neurogenic; Urinary Incontinence, Stress; Urodynamics; Urological Agents

Detection of external irritants by head nociceptor neurons has deep evolutionary roots. Irritant-induced aversive behavior is a popular pain model in laboratory animals. It is used widely in the formalin model, where formaldehyde is injected into the rodent paw, eliciting quantifiable nocifensive behavior that has a direct, tissue-injury-evoked phase, and a subsequent tonic phase caused by neural maladaptation. The formalin model has elucidated many antipain compounds and pain-modulating signaling pathways. We have adopted this model to trigeminally innervated territories in mice. In addition, we examined the involvement of TRPV4 channels in formalin-evoked trigeminal pain behavior because TRPV4 is abundantly expressed in trigeminal ganglion (TG) sensory neurons, and because we have recently defined TRPV4's role in response to airborne irritants and in a model for temporomandibular joint pain. We found TRPV4 to be important for trigeminal nocifensive behavior evoked by formalin whisker pad injections. This conclusion is supported by studies with Trpv4(-/-) mice and TRPV4-specific antagonists. Our results imply TRPV4 in MEK-ERK activation in TG sensory neurons. Furthermore, cellular studies in primary TG neurons and in heterologous TRPV4-expressing cells suggest that TRPV4 can be activated directly by formalin to gate Ca(2+). Using TRPA1-blocker and Trpa1(-/-) mice, we found that both TRP channels co-contribute to the formalin trigeminal pain response. These results imply TRPV4 as an important signaling molecule in irritation-evoked trigeminal pain. TRPV4-antagonistic therapies can therefore be envisioned as novel analgesics, possibly for specific targeting of trigeminal pain disorders, such as migraine, headaches, temporomandibular joint, facial, and dental pain, and irritation of trigeminally innervated surface epithelia.

Topics: Animals; Butadienes; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fixatives; Formaldehyde; Keratinocytes; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Morpholines; Neurons; Nitriles; Pain; Pyrroles; Trigeminal Ganglion; TRPV Cation Channels; Ubiquitin Thiolesterase; Vibrissae

Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive channel in pulmonary arterial smooth muscle cells (PASMCs). Its upregulation by chronic hypoxia is associated with enhanced myogenic tone, and genetic deletion of trpv4 suppresses the development of chronic hypoxic pulmonary hypertension (CHPH). Here we further examine the roles of TRPV4 in agonist-induced pulmonary vasoconstriction and in the enhanced vasoreactivity in CHPH. Initial evaluation of TRPV4-selective antagonists HC-067047 and RN-1734 in KCl-contracted pulmonary arteries (PAs) of trpv4(-/-) mice found that submicromolar HC-067047 was devoid of off-target effect on pulmonary vasoconstriction. Inhibition of TRPV4 with 0.5 μM HC-067047 significantly reduced the sensitivity of serotonin (5-HT)-induced contraction in wild-type (WT) PAs but had no effect on endothelin-1 or phenylephrine-activated response. Similar shift in the concentration-response curve of 5-HT was observed in trpv4(-/-) PAs, confirming specific TRPV4 contribution to 5-HT-induced vasoconstriction. 5-HT-induced Ca(2+) response was attenuated by HC-067047 in WT PASMCs but not in trpv4(-/-) PASMCs, suggesting TRPV4 is a major Ca(2+) pathway for 5-HT-induced Ca(2+) mobilization. Nifedipine also attenuated 5-HT-induced Ca(2+) response in WT PASMCs but did not cause further reduction in the presence of HC-067047, suggesting interdependence of TRPV4 and voltage-gated Ca(2+) channels in the 5-HT response. Chronic exposure (3-4 wk) of WT mice to 10% O2 caused significant increase in 5-HT-induced maximal contraction, which was partially reversed by HC-067047. In concordance, the enhancement of 5-HT-induced contraction was significantly reduced in PAs of CH trpv4(-/-) mice and HC-067047 had no further effect on the 5-HT induced response. These results suggest unequivocally that TRPV4 contributes to 5-HT-dependent pharmaco-mechanical coupling and plays a major role in the enhanced pulmonary vasoreactivity to 5-HT in CHPH.

Topics: Animals; Calcium Signaling; Chronic Disease; Disease Models, Animal; Dose-Response Relationship, Drug; Familial Primary Pulmonary Hypertension; Hypertension, Pulmonary; Hypoxia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Pulmonary Artery; Pyrroles; Serotonin; Sulfonamides; Time Factors; TRPV Cation Channels; Vasoconstriction; Vasoconstrictor Agents