Thesis presents new results about the mechanosensitivity of the smooth muscle
layer (detrusor) of the urinary bladder and the contribution of TREK-1, Piezo1, and
TRPV4 ion channels to the overall mechanosensitivity in the smooth muscle cell
(SMC). Thesis presents new results about the mechanosensitivity of the smooth
muscle layer (detrusor) of the urinary bladder and the contribution of TREK-1,
Piezo1, and TRPV4 ion channels to the overall mechanosensitivity in the smooth
muscle cell (SMC). These channels come from different families, but share a
common sensitivity to mechanical stimulation, such as pressure, shear stress, or
stretch, which are typical stimuli for the urinary bladder. The main mechanoreceptors
of the urinary bladder are afferent neurons and the urothelial layer which covers the
internal cavity. However, the detrusor, and the SMC itself, also have independent
mechanosensitivity, although there are not many works about this. To study the
mechanosensitivity of isolated myocytes, we designed device for creating
hydrodynamic stimulation of the cell membrane with a narrow stream of solution.
This work shows for the first time the existence of three populations of SMC in terms
of sensitivity to mechanical stimulation: the first (55%) is electrophysiologically
insensitive to mechanical stimulation, the second (26%) generates an input current
with Ca2+/Na+ conductance, the third (19%) has an output mechanoactivated current
with K+ conductivity. Using PCR and immunocytochemistry methods, we have
shown that three mechanosensitive ion channels, TREK-1, TRPV4, and Piezo1 are
expressed in the detrusor. We performed experiments with the application of L-
methionine (a TREK-1 inhibitor), which blocked 90% of the mechanoactivated
output current. This proves that the activity from the 3rd group of SMC was caused
precisely by the activity of TREK-1, the only mechanoreceptor in detrusor SMC,
which was shown by functional methods in the literature. In addition, while
investigating the nature of the non-selective mechanosensitive channel from the 2nd
group of SMC, we found that mechanical stimulation of the detrusor SMC generates
an increase in the intracellular concentration of Ca2+ ions ([Ca2+]i). Further studies of
the detrusor SMC showed that the increase in [Ca2+]i could not be related to TRPV4
activity. This conclusion was formed from the insensitivity of SMC to the selective
5
activator of TRPV4 – GSK1016790A, shown by visualization of calcium signals with
the Ca2+-sensitive dye Fluo-4 and patch clamp. In addition, we provided evidence for
the functional presence of Piezo1 in the SMC of detrusor for the first time. Yoda1
(selective Piezo1 agonist) in detrusor SMC and DRG neurons induced a canonical
Piezo1-mediated Ca2+ signal. However, in electrophysiological experiments, Yoda1
inhibited the outward currents, instead of activating the high-conductance inward
currents typical for Piezo1. Further studies showed that the Yoda1-derived effect is
overlaps with glibenclamide-induced inhibition of the ATP-sensitive potassium
channels (KATP). This indicates that Piezo1 may be involved in regulation of the open
state of KATP, inhibiting its activity through Ca2+ mobilization and associated
downstream signaling.
