Ectonucleotidases and purinergic receptors in mouse prostate gland

Abstract

Objectives: Extracellular ATP/ADP and its metabolite adenosine play crucial roles in cellular signaling by interacting with P2 and P1/adenosine receptors. These signaling molecules are regulated by ectonucleotidases, which convert ATP/ADP into adenosine. While recent studies suggest impaired ATP hydrolysis in the aging prostate, the expression and function of ectonucleotidases and purinergic receptors in the prostate gland remain unclear. This study aims to characterize the expression patterns of purinergic enzymes and receptors in the mouse prostate and investigate their functional implications.

Methods: Mouse prostate glands were isolated and analyzed using immunofluorescent staining and microscopy imaging with specific antibodies to detect purinergic enzymes and receptors. Functional studies were conducted to assess prostate smooth muscle contraction in response to purinergic agonists, particularly α,β-meATP and ATPγS.

Results: Our analysis revealed distinct expression patterns of purinergic enzymes and receptors in the prostate: Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) and P2X1 receptors were predominantly localized in prostate smooth muscle cells, ENTPD2 and ecto-5'-nucleotidase (NT5E) in prostate interstitial cells, and alkaline phosphatase (ALPL) in prostate epithelial cells. Notably, ENTPD1 was identified as a key ectonucleotidase expressed in mouse prostate smooth muscle cells. Functionally, P2X1-mediated smooth muscle contraction was triggered by α,β-meATP. However, ATPγS induced contraction even after P2X1 desensitization, suggesting the involvement of additional P2Y receptors. Further analysis confirmed the presence of P2Y1, P2Y2, and P2Y11 receptors in mouse prostate smooth muscle, likely mediating the ATPγS-induced contraction.

Conclusions: This study provides a comprehensive characterization of purinergic signaling components in the mouse prostate. The identification of ENTPD1 in smooth muscle cells and the functional role of multiple P2Y receptors in smooth muscle contraction highlight potential regulatory mechanisms of prostate function. These findings lay the groundwork for future research on purinergic signaling in prostate physiology and its potential implications in age-related dysfunction, both in rodents and humans.

Keywords: P2Y receptor; Purinergic signaling; alkaline phosphatase; mouse prostate; smooth muscle contractility.

Figure 1

Expression of Ectonucleoside triphosphate diphosphohydrolases 1 (ENTPD1) and P2X1 receptors in the smooth muscle of mouse prostate. Mouse prostate tissue was labeled with specific anti-ENTPD1 and anti-P2X1 (green in D) antibodies. (A-C) ENTPD1 Expression: (A) Green fluorescence indicates ENTPD1 localization. (B) Red fluorescence marks Actin (smooth muscle), while blue fluorescence (DAPI) labels nuclei. (C) Merge: ENTPD1 appears primarily in smooth muscle regions, overlapping with actin signals. (D-F) P2X1 Expression: (D) Green fluorescence represents P2X1 receptor localization. (E) Red fluorescence for Actin, and blue for nuclei (DAPI). (F) Merge: P2X1 is predominantly expressed in the smooth muscle layer, colocalizing with actin. Scale bar, 50 µm. These representative images were obtained from a minimum of three mice.

Figure 2

Expression of ENTPD2 in mouse prostate interstitial cells. Mouse prostate tissue was labeled with specific anti-ENTPD2 (green in A and D) antibody. (A-C) ENTPD2 and Actin Expression: (A) Green fluorescence shows ENTPD2 expression. (B) Red fluorescence marks Actin, highlighting smooth muscle structures, while blue (DAPI) labels nuclei. (C) Merge: ENTPD2 appears primarily in interstitial regions surrounding the smooth muscle layer, with minimal colocalization with actin. (D-F) ENTPD2 and CD34 Expression: (D) Green fluorescence represents ENTPD2 localization. (E) Red fluorescence marks CD34, a marker for interstitial cells, while blue (DAPI) stains nuclei. (F) Merge: ENTPD2 partially colocalizes with CD34-positive interstitial cells. Scale bar: 50 µm.

Figure 3

Absence of ENTPD3 in mouse prostate gland. Mouse prostate (A-C) and bladder (D-F) tissues were labeled with specific anti-ENTPD3 antibody (green in A and D). (A) ENTPD3 was not observed in prostate epithelial cells. (B) Red fluorescence marks Actin, primarily labeling smooth muscle structures, while blue fluorescence (DAPI) highlights nuclei. (C) Merged image of ENTPD3 and Actin. (D) ENTPD3 expression in the bladder tissue, localized on the basolateral membrane of bladder urothelial cells (serving as a positive control). (E) Red fluorescence marks Actin. (F) Merged image of ENTPD3 and Actin. Scale bar: 50 µm.

Figure 4

Expression of NT5E (ecto-5’-nucleotidase) and ALPL (alkaline phosphatase) in the prostate gland. A-C. NT5E Expression in Mouse Prostate. A. Green fluorescence represents NT5E expression. B. Red fluorescence labels Actin, primarily marking smooth muscle cells, while blue fluorescence (DAPI) highlights nuclei. C. Merge: NT5E signaling did not colocalize with prostate smooth muscle but mimicked the ENTPD2 expression pattern, indicating localization in interstitial cells. D-F. ALPL Expression in Mouse Prostate. D. Green fluorescence shows ALPL expression. E. Red fluorescence labels Actin. F. Merge: ALPL is highly expressed in the prostate epithelial cell membrane, particularly the apical membrane facing the lumen. Scale bar: 50 µm.

Figure 5

Purinergic contraction forces in mouse prostate tissue. (A-D) Representative traces of prostate smooth muscle contraction from male mice (n=8-10 tissue preparations) in response to various stimuli: KCl depolarization (A), electrical field stimulation (EFS) (B), P2X receptor agonist α,β-meATP (C), and P2 receptor agonist ATPγS (D). (E) Summary data of purinergic contraction forces presented as boxes-and-whiskers. The centerline represents the median, the box encompasses the interquartile range (IQR, 25th to 75th percentile), and the whiskers extend from the minimum to the maximum values. Data were analyzed using the Student’s t-test. *P<0.05.

Figure 6

Expression of ATP-sensitive P2Y receptors in mouse prostate smooth muscle. (A, D, G): Mouse prostate tissues labeled with specific antibodies against P2Y1 (green in A), P2Y2 (green in D), and P2Y11 (green in G) receptors. (B, E, H): Prostate smooth muscle labeled with a red marker. (C, F, I): Merged images showing colocalization of P2Y receptors (green) with prostate smooth muscle (red), resulting in yellow signals. Nuclei are stained blue with DAPI. Scale bar: 50 µm.