Differential Expression Of Cholinergic Muscarinic Receptor Subtypes In Normal Human Prostate

Background and Objective:The human prostate is densely innervated by hypogastric and pelvic nerves. It contains cholinergic neurons and noradrenergic nerves. It is widely believed that the prostatic epithelium receives a cholinergic innervation while the stroma receives a predominantly noradrenergic innervation. The acetylcholine plays an important role in regulating the growth and function of the gland. The cholinoceptors contain nicotinic (N) receptors and muscarinic (M) receptors. The various responses to cholinomimetics affecting contractility of the prostate appear to involve M receptors rather than N receptors. And five subtypes of muscarinic receptor (M_～M₅) were recognized. The different subtypes of the muscarinic receptors have different functions and locations in the gland. Subtyping of the muscarinic receptors in the human prostate especially the normal gland has not been extensively studied, and different approaches have sometimes yielded conflicting findings.In our study, immunohistochemistry was used to study the density and localization of M₁,M₂, and M₃ and real-time RT-PCR was employed to analyse the gene expression of M₁～M₅ of muscarinic receptors the in order to get the location and distribution of subtypes of the muscarinic receptors in the normal prostate.Materials and methods:Seventeen fresh cadaver prostate glands stored in liquid nitrogen were used.Patient mean age was 32.6(23-48) years old. Group A consists of 10 patients for immunohistochemistry test. Group B has 7 patients for immunohistochemistry and real-time RT-PCR test. The prostate samples were classified as transitional zone, central zone, peripheral zone, bladder neck, urethra, and pre-fibromuscular stroma. RNA extracted from each part was tested by real-time RT-PCR, and tissue slides stained with M1M2, and M3 antibodies in immunohistochemistry.Results:Immunohistochemistry1. In Groups A and B, Mi ^M3 shows positive in all the areas of the prostate.2. The three M receptors (Mi -Mi) located both in epithelium and stroma in all parts of prostate gland.3. Mi receptor has highest density in central zone (p<0.05), while M3 has lowest in bladder neck (p<0.05).4. Mi receptor shows highest density in central zone and peripheral zone, and much higher than M2 and M3 (p<0.05).5. The subtypes of the muscarinic receptors (Mi ^M3) have stronger staining in epithelium than in stroma (p<0.05).Real-time RT-PCR:1. Subtypes of the muscarinic receptors (Mi -Ms) were expressed variously in all parts of prostate gland. Transitional zone, pre-fibromuscular stroma, central zone and peripheral zone had higher expression, whereas urethra and bladder neck lower.2. M2 receptor expressed highest, M4and M5 lowest, Mj andM3 in the middle in the all six parts. M2 receptor had higher expression in transitional zone, peripheral zone, bladder neck, and pre-fibromuscular stroma than other areas. M2 receptor expressed 2-fold higher in pre-fibromuscular stroma, transitional zone and peripheral zone than Mi receptor.3. Mi receptor expressed much higher in central zone than in other areasConclusion:1. Muscarinic (M) receptors distributed widely in normal human prostate gland;2. The five subtypes of M receptor (Mi -Ms) were found in all parts of the gland;3. All subtypes of cholinoceptors expressed mainly in epithelium but weakly in stroma of the gland;4. M2 receptor is expressed strongest, M4 and Ms weakest, while Mi and M3 moderate;5. Mi receptor expressed much higher in the central zone than in other zones.