Adenosine 5'-triphosphate (ATP) is an excitatory cotransmitter with noradrenaline to the smooth muscle of the rat prostate gland

Abstract

1. This study investigated whether adenosine 5'-triphosphate (ATP) is involved in neurotransmission to the rat prostate gland. 2. Fluorescence immunohistochemistry carried out on formaldehyde-fixed and frozen sections of rat prostate showed immunoreactivity for the P2X(1)-receptor in the fibromuscular stroma surrounding the secretory acini but not in the glandular epithelium. P2X(2)-, P2X(3)-, P2X(4)- and P2X(7)-receptors were immunonegative in the rat prostate stroma. Double-staining procedures showed P2X(1)-receptor immunoreactivity to be colocalized with alpha-actin immunoreactivity. 3. Isolated organ bath studies investigated whether drugs, which modify purinergic mechanisms, are able to affect contractility of the rat prostate gland. Suramin (100 micro M) and alphabetamethylene ATP (10 micro M) inhibited contractile responses to trains of electrical-field stimulation (70 V, 0.5 ms, 0.1-2 Hz) in the absence and presence of prazosin (300 nM). Responses to 5-20 Hz were unaffected by suramin or alphabetamethylene ATP. 4. Exogenous application of ATP analogues to unstimulated isolated preparations of rat prostate produced concentration-dependent suramin (100 micro M) sensitive transient contractions with a relative order of potency: alphabetamethylene ATP>betagammamethylene ATP>ATP. 5. Adenosine and adenosine 5'-monophosphate (AMP) did not produce contractile responses. 6. These results suggest that P2X(1)-receptors for ATP, which mediate contractions are present in the fibromuscular stroma of the rat prostate. The relative order of potency of ATP analogues in producing contractions of the rat prostate is consistent with the activation of P2X(1)-receptors. Inhibition by suramin and alphabetamethylene ATP of electrically evoked nerve-mediated contractions of the rat prostate implies that ATP contributes to this contractile response and is therefore a cotransmitter with noradrenaline during low-frequency stimulation.

Figure 1

Representative photomicrographs showing microscopy of the same cross-section of rat prostate (n=6 rats) following double immunolabelling with rabbit polyclonal antibody to P2X₁-receptor (left panel) and mouse monoclonal antibody to actin (right panel). P2X₁-receptor and actin immunostaining (indicated by arrows) is colocalized in the fibromuscular stroma (S) between the glandular acini. The epithelium (E) that lines the lumen of the acini was immunonegative in both cases. Scale bar=50 μM.

Figure 2

Representative traces showing the effects of suramin (100 μM), αβmethylene ATP (10 μM) and prazosin (300 nM) on responses to electrical-field-stimulation (—) (0.5 ms pulse duration, 70 V, 0.1–20 Hz for 10 pulses or 10 s)-induced contractions of isolated preparations of the rat prostate.

Figure 3

Mean contractile responses to electrical-field stimulation (0.1–20 Hz, 0.5 ms, 70 V for 10 pulses or 10 s) following administration of: (open bars) no drug or (closed bars) guanethidine (10 μM) (upper panel) or tetrodotoxin (1 μM) (lower panel). Each column represents the mean±s.e.m. of six experiments. ^*Indicates a significant difference from corresponding control response (^*P<0.05; ANOVA, followed by post hoc Tukey–Kramer correction).

Figure 4

Mean contractile responses to electrical-field stimulation (0.1–20 Hz, 0.5 ms, 70 V for 10 pulses or 10 s) following administration of: (open bars) no drug, (closed bars) suramin (100 μM) (upper panel) or αβmethylene ATP (10 μM) (lower panel), (diagonally striped) prazosin (300 nM), (cross-hatched) suramin (100 μM) and prazosin (300 nM) (upper panel) or αβmethylene ATP (10 μM) and prazosin (300 nM) (lower panel). Each column represents the mean±s.e.m. of six experiments. ^*Indicates a significant difference from control response (^*P<0.05; ANOVA, followed by post hoc Tukey–Kramer correction). ^#Indicates a significant difference from response in the presence of prazosin (^#P<0.05; ANOVA, followed by post hoc Tukey–Kramer correction).

Figure 5

Representative traces showing the effects of βγmethylene ATP (1–100 μM) on unstimulated isolated preparations of rat prostate gland in the absence (upper panel) and presence (lower panel) of the P2-receptor antagonist suramin (100 μM). Arrows indicate administration of each concentration of βγmethylene ATP. W=washout.

Figure 6

Mean log concentration–response curves for the excitatory effects of: ATP, βγmethylene ATP and αβmethylene ATP on unstimulated isolated rat prostatic preparations. Results are expressed as the mean peak force developed to each concentration of agonist. Each point represents the mean±s.e.m. of six experiments.

Figure 7

Mean log concentration–response curves for the excitatory effects of ATP (upper panel), βγmethylene ATP (centre panel) and αβmethylene ATP (lower panel) on unstimulated isolated rat prostatic preparations: in the presence and absence of suramin (100 μM). Results are expressed as the mean peak force developed to each concentration of agonist. Each point represents the mean± s.e.m. of six experiments. P-values are for the concentration × treatment interaction of a repeated-measures ANOVA and represent the difference in the concentration–response curves in the absence and presence of suramin. Asterisks indicate a significant difference (^*P<0.05; ^**P<0.005).