The Effects of Age on Prostatic Responses to Oxytocin and the Effects of Antagonists

Abstract

Benign prostatic hyperplasia (BPH) is an age-related enlargement of the prostate with urethral obstruction that predominantly affects the middle-aged and older male population, resulting in disruptive lower urinary tract symptoms (LUTS), thus creating a profound impact on an individual's quality of life. The development of LUTS may be linked to overexpression of oxytocin receptors (OXTR), resulting in increased baseline myogenic tone within the prostate. Thus, it is hypothesised that targeting OXTR using oxytocin receptor antagonists (atosiban, cligosiban, and β-Mercapto-β,β-cyclopentamethylenepropionyl1, O-Me-Tyr2, Orn8]-Oxytocin (ßMßßC)), may attenuate myogenic tone within the prostate. Organ bath and immunohistochemistry techniques were conducted on prostate tissue from young and older rats. Our contractility studies demonstrated that atosiban significantly decreased the frequency of spontaneous contractions within the prostate of young rats (**** p < 0.0001), and cligosiban (* p < 0.05), and ßMßßC (**** p < 0.0001) in older rats. Additionally, immunohistochemistry findings revealed that nuclear-specific OXTR was predominantly expressed within the epithelium of the prostate of both young (*** p < 0.001) and older rats (**** p < 0.0001). In conclusion, our findings indicate that oxytocin is a key modulator of prostate contractility, and targeting OXTR is a promising avenue in the development of novel BPH drugs.

Keywords: BPH; LUTS; oxytocin; oxytocin receptor antagonists.

Figure 1

Effects of atosiban on the frequency of prostate movement. Application of atosiban (n = 5) (1 µM) significantly downregulated the % change in baseline frequency of spontaneous contractions in the prostate of young (a) but not older (b) rats (unpaired t-test, **** p < 0.0001) (**** denotes significant while ns shows non-significant effects).

Figure 2

Effects of atosiban (1 µM) on prostate (spontaneous) contractions. Atosiban (1 µM) showed a significant effect on the baseline integral of spontaneous contractions in young (a) but not older (c) rats. However, some effect was observed on the baseline maximum parameters of spontaneous contractions in both young (b) and older (d) rats, calculated relative to the percentage of maximum concentration of potassium chloride (KCl; 20 mM) (unpaired t-test, n = 5, p ≥ 0.05) (* denotes significant and ns shows non-significant effects).

Figure 2

Effects of atosiban (1 µM) on prostate (spontaneous) contractions. Atosiban (1 µM) showed a significant effect on the baseline integral of spontaneous contractions in young (a) but not older (c) rats. However, some effect was observed on the baseline maximum parameters of spontaneous contractions in both young (b) and older (d) rats, calculated relative to the percentage of maximum concentration of potassium chloride (KCl; 20 mM) (unpaired t-test, n = 5, p ≥ 0.05) (* denotes significant and ns shows non-significant effects).

Figure 3

Administration of exogenous cligosiban (1 µM) significantly attenuated the % change in baseline integral (a), maximum value (b), and frequency (c) of spontaneous contractile activity within the prostate of older (7–9 months) rats, averaged relative to the percentage of maximum concentration of potassium chloride (KCl; 20 mM) (n = 5, unpaired t-test, * p < 0.05) (* denotes significant effect).

Figure 4

Effects of ßMßßC (1 µM) on smooth muscle (prostate) contractions. ßMßßC showed a significant effect in decreasing the % change in baseline integral (a) and frequency (c) of spontaneous contractile activity generated within the prostate of older (7–9 months) rats. However, some change (non-significant) was detected in the baseline maximum value (b) of spontaneous contractions (n = 5, unpaired t-test, * p < 0.05 and **** p < 0.0001) (* and **** denote significant while ns shows non-significant effects).

Figure 5

Immunofluorescence of a young rat prostate. Representative of double immunofluorescence (IF) staining for α-SMA and OXTR of negative control of a rat prostate (Ai), positive control (rat testis) (Aii). IF images (Ba) DAPI reveal nuclear staining, (Bb) highlight OXTR staining, (Bc) show actin staining and (Bd) merge image displays colocalisation of epithelial cells with OXTR within the prostate (scale bar = 100 µm). Graph (C) demonstrates differences in the intensity of OXTR staining between the nuclear and cytoplasmic regions of epithelial and smooth muscle cells (One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001) [NC-MERGE = Negative Control; PC-TESTIS = Positive Control Testis; DAPI = 4′,6-diamidino-2-phenylindole; OXTR = Oxytocin Receptor; α-SMA = Alpha Smooth Muscle Actin; MERGE = Merged Image; Nuc-Epi = Nucleus Epithelium; Nuc-SM = Nucleus Smooth Muscle; Cyt-Epi = Cytoplasm Epithelium; Cyt-SM = Cytoplasm Smooth Muscle] (*, *** and **** denotes significant while ns shows non-significant effects).

Figure 5

Immunofluorescence of a young rat prostate. Representative of double immunofluorescence (IF) staining for α-SMA and OXTR of negative control of a rat prostate (Ai), positive control (rat testis) (Aii). IF images (Ba) DAPI reveal nuclear staining, (Bb) highlight OXTR staining, (Bc) show actin staining and (Bd) merge image displays colocalisation of epithelial cells with OXTR within the prostate (scale bar = 100 µm). Graph (C) demonstrates differences in the intensity of OXTR staining between the nuclear and cytoplasmic regions of epithelial and smooth muscle cells (One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001) [NC-MERGE = Negative Control; PC-TESTIS = Positive Control Testis; DAPI = 4′,6-diamidino-2-phenylindole; OXTR = Oxytocin Receptor; α-SMA = Alpha Smooth Muscle Actin; MERGE = Merged Image; Nuc-Epi = Nucleus Epithelium; Nuc-SM = Nucleus Smooth Muscle; Cyt-Epi = Cytoplasm Epithelium; Cyt-SM = Cytoplasm Smooth Muscle] (*, *** and **** denotes significant while ns shows non-significant effects).

Figure 6

Measurement of oxytocin receptor (OXTR) within old rat prostate. Immunofluorescence staining (Aa) DAPI for nuclear staining, (Ab) OXTR staining, (Ac) actin staining, and (Ad) merge image reveals colocalisation of OXTR with epithelial cells (scale bar = 100 µm). Graph (B) compares nuclear and cytoplasmic staining of OXTR of epithelium and smooth muscle cells (One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001). [DAPI = 4′,6-diamidino-2-phenylindole; OXTR = Oxytocin Receptor; α-SMA = Alpha Smooth Muscle Actin; MERGE = Merged Image; Nuc-Epi = Nucleus Epithelium; Nuc-SM = Nucleus Smooth Muscle; Cyt-Epi = Cytoplasm Epithelium; Cyt-SM = Cytoplasm Smooth Muscle] **** denotes significant while ns shows non-significant effects).

Figure 6

Measurement of oxytocin receptor (OXTR) within old rat prostate. Immunofluorescence staining (Aa) DAPI for nuclear staining, (Ab) OXTR staining, (Ac) actin staining, and (Ad) merge image reveals colocalisation of OXTR with epithelial cells (scale bar = 100 µm). Graph (B) compares nuclear and cytoplasmic staining of OXTR of epithelium and smooth muscle cells (One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001). [DAPI = 4′,6-diamidino-2-phenylindole; OXTR = Oxytocin Receptor; α-SMA = Alpha Smooth Muscle Actin; MERGE = Merged Image; Nuc-Epi = Nucleus Epithelium; Nuc-SM = Nucleus Smooth Muscle; Cyt-Epi = Cytoplasm Epithelium; Cyt-SM = Cytoplasm Smooth Muscle] **** denotes significant while ns shows non-significant effects).

Figure 7

Immunohistochemistry analysis of rat prostate. (a,b) compare the density of nuclear and cytoplasmic staining of oxytocin receptor (OXTR) within the epithelial compartment, while (c,d) the smooth muscle compartment of young and older rat prostate (unpaired t-test) [Nuc-Epi = Nucleus Epithelium; Cyt-Epi = Cytoplasm Epithelium; Nuc-SM = Nucleus Smooth Muscle; and Cyt-SM = Cytoplasm Smooth Muscle] (** and *** denotes significant while ns shows non-significant effects).

Figure 7

Immunohistochemistry analysis of rat prostate. (a,b) compare the density of nuclear and cytoplasmic staining of oxytocin receptor (OXTR) within the epithelial compartment, while (c,d) the smooth muscle compartment of young and older rat prostate (unpaired t-test) [Nuc-Epi = Nucleus Epithelium; Cyt-Epi = Cytoplasm Epithelium; Nuc-SM = Nucleus Smooth Muscle; and Cyt-SM = Cytoplasm Smooth Muscle] (** and *** denotes significant while ns shows non-significant effects).