Urothelium muscarinic activation phosphorylates CBSSer227 via cGMP/PKG pathway causing human bladder relaxation through H2S production

Abstract

The urothelium modulates detrusor activity through releasing factors whose nature has not been clearly defined. Here we have investigated the involvement of H2S as possible mediator released downstream following muscarinic (M) activation, by using human bladder and urothelial T24 cell line. Carbachol stimulation enhances H2S production and in turn cGMP in human urothelium or in T24 cells. This effect is reversed by cysthationine-β-synthase (CBS) inhibition. The blockade of M1 and M3 receptors reverses the increase in H2S production in human urothelium. In T24 cells, the blockade of M1 receptor significantly reduces carbachol-induced H2S production. In the functional studies, the urothelium removal from human bladder strips leads to an increase in carbachol-induced contraction that is mimicked by CBS inhibition. Instead, the CSE blockade does not significantly affect carbachol-induced contraction. The increase in H2S production and in turn of cGMP is driven by CBS-cGMP/PKG-dependent phosphorylation at Ser227 following carbachol stimulation. The finding of the presence of this crosstalk between the cGMP/PKG and H2S pathway downstream to the M1/M3 receptor in the human urothelium further implies a key role for H2S in bladder physiopathology. Thus, the modulation of the H2S pathway can represent a feasible therapeutic target to develop drugs for bladder disorders.

Possible role of hydrogen sulfide as an endogenous relaxation factor in the rat bladder and prostate

“…In this study, both enzymes were undetectable at the gene or protein level in the bladder of Wistar rats. Even though gene expression levels of both enzymes in the bladder have not been investigated before, some reports showed that protein bands of CBS and CSE were detected in the bladder of humans and Sprague‐Dawley rats and CBS protein bands were detected in a human urothelial cell line (T24 cells) . Immunoreactivity of both enzymes was observed in nerve fibers in the smooth muscle layer of the pig bladder neck, while in the human bladder, mainly CSE was detected in the smooth muscle layer, but not in the urothelium .…”

“…In contrast, in this study, rat BL‐D and BL‐T strips contained these components and enzymes related to H 2 S biosynthesis were expressed not only in the smooth muscle layer but also in the urothelium of both bladder tissues. In addition, human urothelium samples treated with L‐cysteine generated H 2 S in vitro . Therefore, the response of H 2 S to a detrusor might differ with or without an urothelium, which can regulate detrusor smooth muscle contractility as a source of endogenous H 2 S. Even though it is unclear whether there are any species‐specific differences in the response of H 2 S in the bladder among Wistar rats, Sprague‐Dawley rats, guinea pigs, and humans, exogenous H 2 S can induce relaxation of the Wistar rat bladder and prostate.…”

“…A limitation of this study is that effects of H 2 S biosynthesis inhibition or H 2 S scavenging on bladder and prostate contractility were not investigated. Some compounds are reported to inhibit CBS, CSE, or MPST, and in human urothelium, carbachol‐induced enhancement of H 2 S production was attenuated by a “CBS inhibitor.” However, these compounds have limitations, low potency and non‐selectivity . In addition, to our knowledge, there is nor report demonstrating selective H 2 S scavengers.…”

“…Even though gene expression levels of both enzymes in the bladder have not been investigated before, some reports showed that protein bands of CBS and CSE were detected in the bladder of humans and Sprague-Dawley rats 9,20 and CBS protein bands were detected in a human urothelial cell line (T24 cells). 21 Immunoreactivity of both enzymes was observed in nerve fibers in the smooth muscle layer of the pig bladder neck, 8 while in the human bladder, mainly CSE was detected in the smooth muscle layer, but not in the urothelium. 9 Even though the anti-CBS and anti-CSE antibodies used in previous studies 8,9,20,21 were different from those used in this study, species-specific differences may exist in the distribution patterns of both enzymes in the bladder among Wistar rats, Sprague-Dawley rats, pigs, and humans.…”

Role of uL3 in Multidrug Resistance in p53-Mutated Lung Cancer Cells

“…Although it is well established that cystine and glutamate are substrates of system xc - , it has been recently demonstrated that cystathionine is a novel substrate of the cystine/glutamate transporter [39]. Cystathionine-β-synthase (CBS), a key enzyme in the trans-sulfuration metabolic pathway involved in the modulation of H 2 S production [40,41], converts homocysteine to cystathionine, which is converted by CSE to cysteine required for synthesis of the antioxidant glutathione GSH [37]. Silencing CBS severely reduces cellular GSH levels [42].…”