Inhibition of neurogenic and thromboxane A₂‐induced human prostate smooth muscle contraction by the integrin α2β1 inhibitor BTT‐3033 and the integrin‐linked kinase inhibitor Cpd22

Abstract

IntroductionProstate smooth muscle contraction is critical for etiology and treatment of lower urinary tract symptoms in benign prostatic hyperplasia (BPH). Integrins connect the cytoskeleton to membranes and cells to extracellular matrix, what is essential for force generation in smooth muscle contraction. Integrins are composed of different subunits and may cooperate with integrin‐linked kinase (ILK). Here, we examined effects of inhibitors for different integrin heterodimers and ILK on contraction of human prostate tissues.MethodsProstate tissues were obtained from radical prostatectomy. Integrins and ILK were detected by Western blot, real‐time polymerase chain reaction (RT‐PCR), and double fluorescence staining. Smooth muscle contractions of prostate strips were studied in an organ bath. Contractions were compared after application of solvent (controls), the ILK inhibitor Cpd22 (N‐methyl‐3‐(1‐(4‐(piperazin‐1‐yl)phenyl)‐5‐(4'‐(trifluoromethyl)‐[1,1'‐biphenyl]‐4‐yl)‐1H‐pyrazol‐3‐yl)propanamide), the integrin α2β1 inhibitor BTT‐3033 (1‐(4‐fluorophenyl)‐N‐methyl‐N‐[4[[(phenylamino)carbonyl]amino]phenyl]‐1H‐pyrazole‐4‐sulfonamide), or the integrin α4β1/α9β1 inhibitor BOP (N‐(benzenesulfonyl)‐ l‐prolyl‐ l‐O‐(1‐pyrrolidinylcarbonyl)tyrosine sodium salt).ResultsWestern blot analyses of prostate tissues using antibodies raised against integrins α2b, α4, α9, β1, and ILK revealed bands matching the expected sizes of corresponding antigens. Expression of integrins and ILK was confirmed by RT‐PCR. Individual variations of expression levels occurred independently from divergent degree of BPH, reflected by different contents of prostate‐specific antigen. Double fluorescence staining of prostate sections using antibodies raised against integrins α2 and β1, or against ILK resulted in immunoreactivity colocalizing with calponin, suggesting localization in prostate smooth muscle cells. Electric field stimulation (EFS) induced frequency‐dependent contractions, which were inhibited by Cpd22 (3 µM) and BTT‐3033 (1 µM) (inhibition around 37% by Cpd22 and 46% by BTT‐3033 at 32 Hz). The thromboxane A2 analog U46619‐induced concentration‐dependent contractions, which were inhibited by Cpd22 and BTT‐3033 (around 67% by Cpd22 and 39% by BTT‐3033 at 30 µM U46619). Endothelin‐1 induced concentration‐dependent contractions, which were not affected by Cpd22 or BTT‐3033. Noradrenaline and the α1‐adrenergic agonists methoxamine and phenylephrine‐induced concentration‐dependent contractions, which were not or very slightly inhibited by Cpd22 and BTT‐3033. BOP did not change EFS‐ or agonist‐induced contraction.ConclusionsIntegrin α2β1 and ILK inhibitors inhibit neurogenic and thromboxane A2‐induced prostate smooth muscle contraction in human BPH. A role for these targets for prostate smooth muscle contraction may appear possible.

Concentration-dependent alpha1-Adrenoceptor Antagonism and Inhibition of Neurogenic Smooth Muscle Contraction by Mirabegron in the Human Prostate

“…Application of EFS simulates action potentials, resulting in contractions of human prostate tissues by the release of endogenous neurotransmitters, including noradrenaline. Accordingly, tetrodotoxin inhibited EFS-induced contractions under our and similar conditions, as previously reported ( Yu et al, 1994 ; Angulo et al, 2012 ; Li et al, 2020 ; Spek et al, 2021 ). For EFS, tissue strips were placed between two parallel platinum electrodes connected to a CS4 stimulator (Danish Myotechnology, Denmark).…”

Purinergic smooth muscle contractions in the human prostate: estimation of relevance and characterization of different agonists

“…Thus, if contractions in our samples were susceptible to the applied antagonists, this should be seen under our conditions. TTX is known to inhibit EFS-induced prostate smooth muscle contractions by inhibition of endogenous neurotransmission (Angulo et al 2012 ; Li et al 2020b ). In turn, tamsulosin is an α 1 -adrenoceptor antagonist, which is commonly applied for treatment of LUTS suggestive of BPH, and inhibits EFS-induced contractions by preventing the activation of α 1 -adrenoceptors located on prostate smooth muscle cells by neurogenically released noradrenaline (Hennenberg et al 2014b ; Oelke et al 2013 ).…”

“…Stock solutions (10 mM) of α,β-methylene-ATP, β,γ-methylene-ATP, 2-methylthio-ATP, ATPγS, NF023, and PPADS were prepared with water, and stored at − 20 °C until used and before further dilution with water. Tetrodotoxin (TTX) is a sodium channel inhibitor, which inhibits neurotransmitter release and subsequent neurogenic contractions in smooth muscle preparations, including human prostate tissues (Angulo et al 2012 ; Li et al 2020b ). Stock solutions (1 mM) were prepared with water, and stored at − 20 °C until used.…”

Phosphoproteomics identifies potential downstream targets of the integrin α2β1 inhibitor BTT-3033 in prostate stromal cells

“…In our previous study, we showed that 1μM of BTT-3033 (an integrin α2β1 inhibitor) inhibited neurogenic and thromboxane A 2 -induced contractions of human prostate strips, but did not affect endothelin-1-induced contraction and limited the inhibition of contractions by adrenergic agonists (4). In that study, the cytotoxicity tests suggested that the effect of 1 μM of BTT-3033 was reversible after its removal.…”

“…Integrin α2β1, a collagen-I-specific receptor that attaches stromal cells to the ECM, mediates multiple cell functions (6)(7)(8). The cytoplasmic part of integrin α2β1 is the center for focal adhesion (FA) (9) assembly, with the assembling partners including the Src family kinase (SFK), focal adhesion kinase (FAK) and integrin-linked kinase (ILK) of which the modifications may affect biological processes (4,10,11). We conducted a preliminary test (data not shown) that suggested that SFK, FAK, and ILK may not be the downstream targets of BTT-3033 at 1μM in the context of contraction inhibition.…”