Inhibition of smooth muscle contraction and ARF6 activity by the inhibitor for cytohesin GEFs, secinH3, in the human prostate

Abstract

Prostate smooth muscle contraction is critical for etiology and treatment of male lower urinary tract symptoms (LUTS) and is promoted by small monomeric GTPases (RhoA and Rac). GTPases may be activated by guanosine nucleotide exchange factors (GEFs). GEFs of the cytohesin family may indirectly activate Rac, or ADP ribosylation factor (ARF) GTPases directly. Here we investigated the expression of cytohesin family GEFs and effects of the cytohesin inhibitor Sec7 inhibitor H3 (secinH3) on smooth muscle contraction and GTPase activities in human prostate tissues. Of all four cytohesin isoforms, cytohesin-1 and -2 showed the highest expression in real-time PCR. Western blot and fluorescence staining suggested that cytohesin-2 may be the predominant isoform in prostate smooth muscle cells. Contractions induced by norepinephrine, the α-adrenoceptor agonist phenylephrine, the thromboxane A analog U-46619 , and endothelin-1 and -3, as well as neurogenic contractions induced by electric field stimulation (EFS), were reduced by secinH3 (30 µM). Inhibition of EFS-induced contractions appeared to have efficacy similar to that of inhibition by the α-adrenoceptor antagonist tamsulosin (300 nM). Combined application of secinH3 plus tamsulosin caused larger inhibition of EFS-induced contractions than tamsulosin alone. Pull-down assays demonstrated inhibition of the small monomeric GTPase ARF6 by secinH3, but no inhibition of RhoA or Rac1. In conclusion, we suggest that a cytohesin-ARF6 pathway takes part in smooth muscle contraction. This may open attractive new possibilities in medical treatment of male LUTS.

A NAV2729-sensitive mechanism promotes adrenergic smooth muscle contraction and growth of stromal cells in the human prostate

“…7). Similar to NAV2729 and similar to secinH3 in prostate tissues (22), secinH3 (30 M, 2 h) inhibited ARF6 activity in WPMY-1 cells (Fig. 7).…”

“…Recently, the cytohesin inhibitor secinH3 inhibited prostate smooth muscle contraction, which was paralleled by inhibition of ARF6 but not of RhoA or Rac (22). Therefore, we speculated that NAV2729 may inhibit contractions of human prostate tissues.…”

“…Recently, it has been speculated that ARF6 may promote smooth muscle contraction in the human prostate (22). This was based on the observation that the cytohesin inhibitor secinH3 inhibited ARF6, but not Rac1 or RhoA, activity in prostate tissues, which was paralleled by inhibition of prostate smooth muscle contraction and went along with actin breakdown in prostate stromal cells (22). Consequently, a role of ARF6 in smooth muscle contraction was supposed for the first time, but this remains to be proven (22).…”

“…This was based on the observation that the cytohesin inhibitor secinH3 inhibited ARF6, but not Rac1 or RhoA, activity in prostate tissues, which was paralleled by inhibition of prostate smooth muscle contraction and went along with actin breakdown in prostate stromal cells (22). Consequently, a role of ARF6 in smooth muscle contraction was supposed for the first time, but this remains to be proven (22). NAV2729, a small molecule inhibitor with assumed specificity for ARF6, has recently become available (21,23,24).…”

Inhibition of Prostate Smooth Muscle Contraction by Inhibitors of Polo-Like Kinases

“…This is in line with recent evidence, which suggested that smooth muscle contraction of the human prostate is insufficiently understood. In fact, recent studies reported inhibition of prostate smooth muscle contraction by inhibitors for several kinases or GTPases (including c-Jun N-terminal kinase, focal adhesion kinase, src family kinases, LIM kinases, Rac GTPases) ( Strittmatter et al, 2012 ; Kunit et al, 2014 ; Wang et al, 2016a , b ; Herlemann et al, 2018 ; Yu et al, 2018 ), although their involvement of prostate smooth muscle contraction was not known before. This demonstrates that this process is probably more complex than previously assumed, what may be important from a clinical point of view.…”