Abstract 1455 Distribution of TRPV4 Channels in Mouse Lung Microvasculature Under Acidic pH and High Temperature

The lung endothelial barrier serves as an integral part of homeostasis and the disruption of this barrier can lead to several lung pathologies and disorders. A large part of this barrier functioning resides in the superfamily of transient receptor potential called the transient receptor potential vanilloid (TRPV4) channel, which is a non-selective calcium permeable channel that take residence in the endothelium lining of the lung. It is known that TRPV4 channels activate in response to various factors including mechanical stress, reactive oxygen species, temperature, and pH. Although calcium influx through TRPV4 channel is implicated in maintaining homeostasis in the lung microcirculation, it may also play a pivotal role in pathologic calcium signaling under conditions like acute lung infection. However, it is not known how the channel distribution changes in response to conditions that emulate states of infection and disease. The purpose of this experiment was to identify and evaluate the TRPV4 channel distribution when subjected to high temperatures and acidic pH. Lung slices of 500 μm were collected from Cdh5-GCaMP8 mice (transgenic mice with Ca2+-dependent fluorescent indicator expressed specifically within vascular endothelial cells) and mounted on Slygard inserts. For this experiment, four conditions were used to assess the effects of temperature and acidic pH. Two of the slices were subjected to pH 7.1 using an acidic HEPES buffer as the bathing solution while the other two slices were bathed in physiological pH of 7.4. Two of the slices were exposed to a temperature of 40 degrees Celsius while the other two were kept at room temperature. The slices exposed to high temperature were kept in a temperature-controlled chamber for 45 minutes while the room temperature slices were kept for the same amount of time. We performed immunofluorescent staining using TRPV4 specific antibody to capture several planes of tissue using confocal microscopy which allowed for identification of the location and intensity of TRPV4. All the slices showed positive TRPV4 staining throughout the tissue. The control condition showed puncta and isolated spots of TRPV4 signal throughout cells. This indicates normal, homeostatic conditions of most of the channels isolated in vesicles in the cytosol. However, the high temperature+low pH had the brightest intensity of signal and continuous cellular staining all over the tissues with increased protein aggregates. This may suggest there is an increased distribution of channels in the membrane when subjected to these conditions. These findings implicate a possible recruitment effect on the vesicles or increased vesicle exocytosis of TRPV4 to the membrane. This may alter the activity of the channels by increasing calcium signaling during infection, which has been shown by previous studies to cause pathological signaling and effects. Future studies will be performed to examine the tissues with membrane specific antibodies to further asses location of channels. Also, live calcium signaling studies will be useful in determining if the increased activity of channels during infection conditions such as temperature and pH will positively correlate with increased calcium signaling. Different time points will be performed to distinguish when the system achieves a pathological state to understand possible methods of prevention against lung infection.