Smooth muscle contraction is essential for lower urinary tract and vascular functions and occupies a central position in pathophysiology and treatment of lower urinary tract symptoms (LUTS) and cardiovascular diseases, both belonging to the most common non-malignant diseases. LUTS consist of both voiding and storage symptoms (Oelke et al., 2009; Gravas et al., 2021). Urethral obstruction is most commonly caused as direct consequence of benign prostatic hyperplasia (BPH), leading to voiding symptoms by prostatic enlargement and increased smooth muscle tone in the hyperplastic prostate (Lepor, 2004). Storage symptoms are caused by spontaneous contractions of the detrusor muscle, referred to as overactive bladder (OAB) (Patel and Chapple, 2008; Chapple, 2011). LUTS commonly lead to considerable loss of quality of life, but also to social withdrawal and depression, while severe complications include urge incontinence, urinary retention, and infections (Chapple, 2011; Oelke et al., 2013). Storage and voiding disorders affect large parts of the population. Estimations for the year 2018 amounted the worldwide number of patients with storage symptoms to 2.7 billion, and those with voiding symptoms to 1.1 billion (Lepor, 2004). While storage symptoms predominantly include nocturia, urge incontinence, and urinary frequency, voiding symptoms most commonly consist of urinary hesitancy, intermittency, and the inability to completely empty the urinary bladder (Chapple, 2011). A considerable proportion of patients with LUTS suggestive of BPH also suffer from OAB-related symptoms, subsumed in the term “mixed LUTS” (Gravas et al., 2021). In both BPH and OAB, exaggerated smooth muscle tone and cell proliferation are important targets for medical therapy (Lepor, 2004; Oelke et al., 2013; Nambiar et al., 2018).

While activation of α₁-adrenoceptors causes smooth muscle contraction in the prostate stroma, hyperplastic prostate stromal cell growth is facilitated through dihydrotestosterone (Lepor, 2004; Hennenberg et al., 2014). Reduction of testosterone is catalyzed by 5α-reductase (5-AR) into its biologically more active metabolite dihydrotestosterone. As α₁-adrenoceptor antagonists (α₁-blockers) are used for the immediate relief of LUTS by inhibiting adrenergic smooth muscle contraction, they are often administered in combination with 5α-reductase inhibitors (5-ARI) for concomitant reduction of prostate size (Oelke et al., 2013). However, α₁-adrenoceptor antagonists improve prostate symptom scores (IPSS) and urinary flow rates (Q_max) by no more than 50 %, and 5-ARI reduce prostate size only up to 25% after long-term use, leading to high discontinuation rates and need for definitive surgery (Michel et al., 1998; Naslund and Miner, 2007; Magistro and Stief, 2020; Tamalunas et al., 2021). Multiple studies have repeatedly suggested that adrenoceptors may promote and regulate cell proliferation and prostate growth (Kyprianou et al., 1998; Kyprianou et al., 2000; Glassman et al., 2001; Chagas-Silva et al., 2014; Nascimento-Viana et al., 2019). Based on findings from cell culture studies, animal models, and analyses of human tissues from patients being treated with α₁-adrenoceptor antagonists, it has been suggested that α₁-adrenoceptors may induce proliferation and suppress apoptosis (Kyprianou et al., 1998; Kyprianou et al., 2000). However, clinical studies fail to confirm the potential inhibitory effect of α₁-blockers on prostate growth, and the European Association of Urology (EAU) guideline’s consensus clearly acknowledges, that α₁-adrenoceptor antagonists do not reduce prostate volume (Roehrborn, 2006; Roehrborn et al., 2008; Roehrborn et al., 2010; Oelke et al., 2013).

Vascular smooth muscle contraction, in turn, is a main cause and important target for drug treatment in wide-spread cardiovascular diseases, including arterial hypertension, diabetic nephropathy, coronary artery disease and others. Worldwide numbers of annual deaths have recently been extrapolated to 7.7–10.4 million for elevated systolic blood pressure, and to 18.6 million in the context of any cardiovascular disease, including ischemic heart disease (Roth et al., 2020; Zhou et al., 2021). Diabetic nephropathy is a major complication of diabetes, affecting up to 35% of diabetic patients and accounting for 50% of patients needing dialysis or kidney transplantation in the United States (DeFronzo et al., 2021; Lin et al., 2021). Reduced renal function in diabetic nephropathy is caused by increased glomerular capillary pressure and elevated smooth muscle tone in efferent glomerular arterioles, which again are related to systemic blood pressure and hypertension (Khavandi et al., 2009; DeFronzo et al., 2021). Consequently, treatment of diabetic nephropathy includes drugs to reduce intrarenal vascular resistance and antihypertensive medications, in addition to glucose-lowering drugs (Cosentino et al., 2020). Thus, while drugs to inhibit vascular smooth muscle contraction are gold standard options in medical treatment of cardiovascular diseases (Brouwers et al., 2021), hypotensive side effects are limiting in medical treatment of voiding symptoms in BPH, where application of α₁-blockers is a mainstay (Oelke et al., 2013).

Agonist-induced smooth muscle contractions are induced by activation of 7-transmembrane G-protein coupled receptors (GPCR) (Somlyo and Somlyo, 2000; Hennenberg et al., 2014) (Figure 1). Receptor-coupled G proteins are each composed of an α, β and γ subunit. Dissociation of Gα from β/γ subunits and receptors accounts for subsequent activation of intracellular post-receptor signaling, finally leading to contraction. Contractile receptors include α₁-adrenoceptors in prostate and vascular smooth muscle, muscarinic receptors in bladder smooth muscle and coronary arteries, and thromboxane A₂ and endothelin-1 receptors in prostate, bladder and vascular smooth muscle. All of them may be coupled to Gα_q or Gα_12/13 subunits, or both (Somlyo and Somlyo, 2000; Hennenberg et al., 2014). However, only little is known about details, e.g., which receptor prefers which Gα, or whether Gα_q is coupled to all of them or about differences in G protein-coupling of the same receptor smooth muscle types of different organs. With YM-254890, the first small molecule inhibitor with presumed specificity for Gα_q has recently become available (Peng et al., 2021). To gain insight into tissue- and receptor-dependent differences in the role of Gα_q for agonist-induced smooth muscle contraction, and for the relevance of Gα_q in growth-related functions of prostate stromal cells, we examined the effect of YM-254890 on smooth muscle contractions of human prostate and detrusor tissues, on vasocontraction of porcine renal and coronary arteries, and on cellular functions of prostate stromal cells.

Contractile smooth muscle receptors may be coupled to Gα_q or Gα_12/13 subunits, or both, but only little is known about the specificities, as to which receptor prefers which Gα, or whether Gα_q is coupled to all of them, and about differences in G protein coupling of the same receptor in different types of smooth muscle (Somlyo and Somlyo, 2000; Hennenberg et al., 2014). With YM-254890, a small molecule inhibitor with presumed specificity for Gα_q has become available (Peng et al., 2021). Our present findings suggest ubiquitous susceptibility to YM-254890 of agonist-induced smooth muscle contractions in four different tissues, including α₁-adrenergic contractions in prostate tissues and renal arteries, cholinergic contractions in detrusor tissues and coronary arteries, and thromboxane- and endothelin-1-induced contractions in all four tissues. Even though comparisons across different series of experiments underlie limitations, some differences between different tissues are conspicuous. Differences became essentially obvious between prostate smooth muscle tissues, where contractions by all agonists were nearly completely inhibited by YM-254890, and detrusor smooth muscle tissues, where contractions were apparently only partly inhibited. In WPMY-1 cells, YM-254890 showed effects on growth-related functions, which were clearly detectable, but clearly limited as well, and probably too weak to translate to effects on prostate growth in vivo. Together, our findings may be discussed in the context of basic smooth muscle physiology but may also add to understanding of LUTS in urology, where experimental findings previously suggested promotion of growth by α₁-adrenoceptors in prostate cells, while α₁-blockers did not reduce prostate volume in patients with BPH (Kyprianou et al., 1998; Kyprianou et al., 2000; Glassman et al., 2001; Chagas-Silva et al., 2014; Nascimento-Viana et al., 2019; Gravas et al., 2021).

YM-254890 is a cyclic depsipeptide compound, which was originally isolated from Chromobacterium sp. and claimed as a specific Gα_q inhibitor (Taniguchi et al., 2003; Takasaki et al., 2004). YM-254890 inhibits signaling mediated by Gα_q-coupled receptors, with a high potency and an IC₅₀ around 30 nM, by blocking guanosine triphosphate (GTP) exchange from guanosine diphosphate (GDP), thereby preventing Gα_q activation (Nishimura et al., 2010). Based on previous findings confirming that 30 nM YM-254890 abolished UTP-activated P2Y₂ receptor-mediated Ca²⁺ signaling and ERK1/2 phosphorylation in human coronary endothelial cells, we applied YM-254890 in a concentration of 30 nM in organ bath experiments, and concentrations ranging from 10 to 100 nM in cell culture experiments (Peng et al., 2021). However, YM-254890 was also shown to inhibit G_s protein-mediated cyclic adenosine monophosphate (cAMP) production in the same study with a calculated IC₅₀ of 30 nM (Peng et al., 2021).

Agonist-induced smooth muscle contractions are caused by activation of 7-transmembrane receptors, coupled to heterotrimeric G proteins each including a Gα_q and/or Gα_12/13 subunit (Somlyo and Somlyo, 2000; Hennenberg et al., 2014). Receptor activation results in subsequent activation of intracellular post-receptor signaling by Gα subunits, ultimately leading to contraction by accumulation of phosphorylated myosin light chains (MLC) through activation of MLC kinase or inactivation of MLC phosphatase (Somlyo and Somlyo, 2000; Hennenberg et al., 2014). In fact, contractile receptors in all smooth muscle types share three prototypic intracellular signaling pathways, which are believed to be activated in parallel, including inositol 1,4,5-trisphosphate (IP₃)/calcium-dependent mechanisms of MLC kinase activation, and MLC phosphatase inactivation by diacylglycerol- (DAG)-mediated protein kinase C activation and by RhoA/Rho kinase (Somlyo and Somlyo, 2000; Hennenberg et al., 2014). IP₃ and DAG are generated by phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C (PLC), which is typically activated by Gα_q in smooth muscle cells (Frazier et al., 2008; Black et al., 2016). Based on exemplary findings, activation of RhoA has been attributed/related to Gα_12/13 (Gohla et al., 2000; Black et al., 2016). However, whether proposed concepts of an Gα_q/PLC axis and Gα_12/13 axis can be in fact generalized to all contractile receptors and to all types of smooth muscle cells, remains uncertain and to be proven (Somlyo and Somlyo, 2000; Black et al., 2016). In fact, the extent of RhoA activation was found to differ with contractile agonists (Sakurada et al., 2001). While thromboxane receptors were supposed to couple preferentially to Gα_12/13, endothelin-1 was presumed to use Gα_q and Gα_12/13 for signal transduction (Offermanns et al., 1994). On the other hand, Gα_q may activate RhoA as well, together reflecting the highly anecdotic and exemplary character of available findings (Momotani et al., 2011). Thus, the preference of different contractile receptors, or of different smooth muscle types for either Gα_q or Gα_12/13 still represents a central question in smooth muscle physiology. Here, we examined effects of YM-254890 on agonist-induced and neurogenic contractions in four smooth muscle-rich tissues, including human prostate and detrusor tissues, and porcine renal and coronary arteries.

Obviously, contractions of prostate and detrusor tissues showed divergent susceptibilities to YM-254890, as contractions by all examined agonists were only partly inhibited in detrusor tissues, but virtually completely in prostate tissues. In the light of this observation, it may be speculated that contractile receptors are coupled to Gα_q/11 in human prostate smooth muscle cells, but to lesser extent (e.g., only to parts of receptor populations) in human bladder smooth muscle. In fact, this is in line with findings suggesting that human bladder smooth muscle contraction by muscarinic receptors does not (or not necessarily) involve activation of PLC (Frazier et al., 2008). Activation of PLC by contractile receptors is imparted by Gα_q/11, which is considered a prototypical pathway of agonist-induced smooth muscle contraction, with the exception of M3 receptors in bladder smooth muscle (Frazier et al., 2008). As inhibitions of non-cholinergic contractions in our detrusor tissues remained incomplete, it may be concluded that this phenomenon is not limited to M3 receptors but includes other contractile receptors in bladder smooth muscle as well. However, the previously noted special role of bladder M3 receptors is apparently not bladder-specific, as we observed full inhibition of cholinergic contractions in coronary arteries. Thus, contractile receptors seem to be universally coupled to Gα_q/11 in the human prostate, while the contribution of Gα_q to agonist-induced contractions in the human detrusor is lower.

In fact, it has been suggested that contractions by cholinergic agonists may differ from those by other agonists. In bladder and airway smooth muscle tissues, β-adrenoceptor agonists exhibited lower efficacy as relaxing compounds if tissues were precontracted by muscarinic agonists, compared to precontraction with other agents, including high-molar KCl (Dale et al., 2014). At a clinical level, this stands in unison with the Symphony trial (NCT01340027), which showed a positive impact on patients treated for OAB with combinations of antimuscarinic agents and the β₃-agonist mirabegron, decidedly advocating the combination use of different drug classes (Abrams et al., 2015). While precise mechanisms underlying the unique characteristics of muscarinic contractions remain unclear, PLC- and thus, Gα_q-independent components of bladder smooth muscle contraction are well known and may include voltage-operated calcium channels and Rho kinase (Frazier et al., 2008). This is consistent with the findings by Phelps and colleagues, who used porcine urothelium and lamina propria, and observed partial reduction of GPCR-mediated contraction by nifedipine, which was maintained in a Ca²⁺-free environment, suggesting internal stores of Ca²⁺, activated by G_q/11 receptor proteins in those tissues (Phelps et al., 2022). Notably for YM-254890, inhibitory effects on such a wide range of contractile agonists through the specific Gα_q/11 inhibitor are novel and may indeed be due to shared post-receptor signaling in smooth muscle contractile receptors.

Regarding renal and coronary arteries, certain differences exist as well: while α₁-adrenergic contractions are known from most vessel types but do not occur in coronary arteries, cholinergic contractions typically do not occur in renal interlobar arteries (Moreland and Bohr, 1984; Li et al., 2021). Accordingly, we examined cholinergic contractions only in coronary arteries. α₁-Adrenergic contractions are shared by vascular and prostate smooth muscle, but showed slightly divergent reactions to YM-254890. Contractions by all three α₁-agonists appeared to be inhibited stronger in prostate tissues than in interlobar arteries, where they were partly or incompletely inhibited. The same applied to U46619-induced contractions. Whether this reflects differences in smooth muscle tissue types, or involvement of G_s protein-coupled post receptor signaling pathways (prostate vs. vascular, e.g., α_1A vs. α_1D subtypes of α₁-adrenoceptors), or species-dependent differences between smooth muscle tissues from humans and pigs, cannot be clarified on the basis of our data.

However, the inhibitory effect of YM-254890 in our study eems fairly Gα_q-specific, while an involvement of Gα_s inhibition appears highly unlikely. While YM-254890 was recently also reported as a Gα_s inhibitor, the effects we observed may not be due to inhibition of Gα_s-mediated cAMP production (Peng et al., 2021). Thus, if YM-254890 inhibited Gα_s in our tissues, we would not expect inhibition of contraction, but increased contraction, considering that Gα_s causes cAMP production, finally resulting in smooth muscle relaxation. In line, we observed that YM-254890 was not able to inhibit KCl-induced contractions in any of the examined tissues, which should occur, if the compound inhibited Gα_s and subsequent cAMP production Thus, it is fairly possible to support specificity of YM-254890 to Gα_q.

Limitations of our study certainly include variability of data in organ bath experiments, which was most pronounced in our experiments with neurogenic detrusor contractions. Still, several factors may generally account for high variability between patients. Tissue heterogeneity is particularly high in human tissues, due to individual variations (i.e., larger than in standardized laboratory animals), age (i.e., greater variability and span than in standardized laboratory animals), or even different pathological backgrounds, e.g., BPH, OAB, or mixed LUTS. To reduce limitations, we verified the neurogenic origin of EFS-induced detrusor contractions, by showing they were fully sensitive to TTX, and referred all contractions to KCl.

In parallel to prostate smooth muscle tone, static obstruction as a direct consequence of prostatic stromal cell growth may contribute to peri-urethral compression in BPH as well and presents an important target for drug treatment (Oelke et al., 2013; Hennenberg et al., 2014). 5-ARI decrease prostate size, although by no more than 25% and are applied to prevent disease progression and complications (Fusco et al., 2018; Gravas et al., 2021). Cytokines, growth factors and in particular hormones (e.g., androgens, estrogens) have a decisive influence on prostate growth, and molecular mechanisms at the level of intracellular signaling are an important and clinically relevant focus of interest (Sampson et al., 2007; Timms and Hofkamp, 2011). Based on experimental studies, it has been repeatedly suggested, that α₁-adrenoceptors mediate prostatic growth. After chronic administration of phenylephrine in vivo, rats and mice develop hypertrophic and dysplastic changes in the prostate (Golomb et al., 1998; Marinese et al., 2003). Similarly, findings from sympathectomized rats suggested connections between prostatic growth and sympathetic innervation via α₁-adrenoceptors (McVary et al., 1994). In fact, prostatic α₁-adrenoceptors induce proliferation, suppress apoptosis, and activate growth-promoting kinases in prostate cells, including ERK1/2 and others (Kyprianou et al., 1998; Kyprianou et al., 2000; Glassman et al., 2001; Kanagawa et al., 2003; Maroni et al., 2004; Hennenberg et al., 2014). Together, these findings raised the assumption that treatment with α₁-adrenoceptor antagonists in BPH may prevent prostatic growth. In contrast, there is large consensus from numerous clinical studies and decades of clinical experience, that α₁-blockers do not reduce prostate volume in patients with LUTS/BPH (Roehrborn, 2006; Roehrborn et al., 2008; Roehrborn et al., 2010; Gravas et al., 2021).

This persistent controversy prompted us to examine effects of YM-254890 on growth-related functions of WPMY-1 cells. In previous studies, YM-254890 abolished ERK1/2 activation in exposed tissues, probably by inhibition of Gα_q/11 (Peng et al., 2021). We observed that YM-254890 reduced proliferation rate, growth in colony formation assays and viability in WPMY-1 cells, while the number of apoptotic cells was increased by YM-254890. These effects occurred within a concentration range of 10–100 nM. Data of some series allowed curve fitting, which pointed to IC₅₀ values for YM-254890 in nanomolar ranges, which are in line with previous values for inhibition of Gα_q/11 by YM-254890 (Peng et al., 2021). While the observed effects may be due to anti-proliferative effects facilitated through an ERK1/2 inhibition by YM-254890, we recognize that cellular toxicity of YM-254890 may have contributed as well. However obvious, effects on growth-related functions of stromal cells were clearly limited and small as well, and in vivo translation may not be possible based on these experiments. However, this may explain why previous studies in experimental models suggested α₁-adrenergic growth of prostate cells, but α₁-blockers did not affect prostate size in vivo (Oelke et al., 2013; Hennenberg et al., 2014).

Actin polymerization and correct filament organization are required for any type of smooth muscle contraction. YM-254890 quantitatively and qualitatively altered actin organization in a concentration-dependent manner. Together with the completely abolished adrenergic contractions in human prostate smooth muscle tissues, we assume that Gα_q/11 and its upstream receptors promote contraction not only by MLC phosphorylation but also by actin-dependent mechanisms, at least in prostate smooth muscle. Considering that anticontractile effects were limited in detrusor tissues, it appears possible that the role and contribution of Gα_q/11-dependent actin organization differs between smooth muscle-rich organs. While YM-254890 seems to marginally increase apoptosis, there was otherwise no increase in undirected cell death. Consequently, decrease in viability observed in CCK-8 assays may not be attributed to cell death due to cytotoxicity, which is why we performed flow cytometry. Thus, disruption of actin organization may be due to Gα_q/11-specific action of YM-254890, as proliferation was disproportionately reduced in a very limited manner only. Divergent incubation periods in the organ bath (30 min) and in cell culture (72 h) certainly pose another limitation of our study, as it remains unclear whether disruption of actin filament organization was due to specific inhibition of G_q protein signaling, or if toxicity may have contributed. Furthermore, we do not know if contractile agonists would have had an effect on WPMY-1 cells in cell culture experiments.

Medical therapy for LUTS suggestive of BPH includes α₁-adrenoceptor antagonists for rapid relieve of voiding symptoms by inhibiting prostate smooth muscle contraction, and 5-ARI for long-term reduction of prostate size, and to prevent disease progression (Fusco et al., 2018; Gravas et al., 2021). Medical therapy for OAB is primarily based on anticholinergic medications for storage symptoms. However, discontinuation rates due to limited efficacy and disproportional side effects range around 80% within 12 months (Sexton et al., 2011). Meanwhile, the limited efficacies have been explained by non-adrenergic prostate smooth muscle contractions and by non-cholinergic detrusor contractions (thromboxane, endothelin), and OAB-induced detrusor contractions are supposed to be non-cholinergic in origin. Nevertheless, non-adrenergic and non-cholinergic contractions in the lower urinary tract have hardly been taken into account in basic research so far. With the β₃-adrenergic agonist mirabegron, the first alternative to anticholinergics was introduced. However, its efficacy does not exceed that of established antimuscarinics, so that medical treatment of storage disorders still represents a significant problem (Dahm et al., 2017). As regulation of prostatic and bladder smooth muscle tone remains a crucial target in LUTS, BPH and OAB, increased understanding of molecular mechanisms may be beneficial for future medical treatment options (McNeal, 1990). Thus, fundamental understanding of underlying mechanisms in smooth muscle contraction, including organ-specific characterization of contractile receptors, may not only be relevant for the lower urinary tract, but for the cardiovascular system and other smooth muscle-rich organs as well.

Our findings suggest shared intracellular signaling by Gα_q-coupled contractile receptors in multiple smooth muscle-rich organs, but also point to possible differences. α₁-Adrenergic contractions in prostate smooth muscle and renal arteries, cholinergic contractions in detrusor tissues and coronary arteries, and thromboxane- and endothelin-induced contractions in each of these tissues showed ubiquitous susceptibility for YM-254890. Differences were most obvious between prostate smooth muscle, where contractions by all agonists were nearly completely inhibited, and detrusor smooth muscle, where contractions by all agonists were inhibited only partly. YM-254890 affected growth-related functions in prostate stromal cells, which were clearly detectable, but clearly limited as well. Our findings significantly add to improved understanding of basic smooth muscle physiology but may also be applicable in the context of LUTS treatment in urology, where previous findings suggested promotion of growth by α₁-adrenoceptors in prostate cells, while α₁-blockers do not reduce prostate volume in patients with BPH. Together this may aid in developing new pharmaceutical targets for LUTS and antihypertensive medication in the future.