Expression And Clinical Significance Of M_1-5 Receptor Subtype Of Bladder Mucosa In Patients With OAB

Background:Overactive bladder (OAB) is defined as a symptom complex comprising urinary urgency, with or without urgency incontinence,usually with frequency and nocturia, and absence of other local or metabolic factors that would account for those symptoms1,2.The OAB symptoms is the most usual lower urinary tract symptoms, and have great impact on the daily life of the patients.OAB for the health care system is also a heavy burden for its social and economic costs which equal to diabetes or dementia roughly3.Nearly 50% of the patients complain OAB symptoms affected their daily lives; postmenopausal women with urge incontinence easily fractured due to falls; patients spent less time in social activities, easily lonely, easily vulnerable to depression; Nocturia disrupted sleep and affected the physical and mental health of patients4.OAB for the health care system is also a heavy burden, total cost of OAB was＄120 million in the United States in 2000, while the figure for 2007 rosed to$65.9 billion in total and＄1,925 per capita; The total social cost between female patients older than 65 years with OAB and with osteoporosis were 14 billion to 21 billion:5. With the population aging process, such a high prevalence (prevalence rate of people over 65 years more than 30% 5) will seriously increased the economic and social burden.The main drug of treatment of OAB is the M receptor (m muscarinic receptor) antagonist. However, the M receptor antagonist existenced is poor in tissue selective, and have unsatisfactory efficacy, easy to relapse after withdrawal, other shortcomings are difficult to long-term adherence, and dry mouth, constipation and other common side effects6.Irrigated non-selective M receptor antagonist atropine does not affect the physiological urination in normal mice, even high-dose infusion of atropine is only reduced bladder contraction slightly7. A similar experiment take by Fader M et al8 also found that intravesical instillation of atropine and oral oxybutynin have equal effects and fewer side effects, the bladder capacity and quality of life in atropine group improved more significantly.The traditional theory can not explain those phenomenon mentioned above, indicating that perhaps there are other mechanisms have not yet knowen.As a result,it is necessary to further research on the clinical use of M receptor antagonists to provide evidence and guidance OAB ---- such as using new medicines target to M receptor of bladder mucosa, superior subtype selective drugs to enhance the efficacy of bladder and reduce side effects.M receptor has five subtypes:M1 to M5. all of the M receptor subtype mRNA expressed in bladder mucosa.In the human detrusor is mainly M2, M3 receptor, The M3 receptors plays a major role in the direct detrusor contraction 9.In the past,man consider that the major neurotransmitters evoked bladder contraction is Ach which released from cholinergic efferent of detrusor. OAB symptoms are due no inhibition of detrusor contraction, The target of M receptor antagonists is M receptors on the detrusor in disease such as OAB. Now researchers suggests that increased release of Ach by the bladder urothelium, changed M receptor in urothelium and subepithelial myofibroblast, increased bladder afferent nerve activity, may be the cause of the symptoms of OAB16.Bschleipfer T10 found in the urothelium of normal female bladder mucosa present in all subtypes of M receptor (M1R-M5R), RT-PCR expression intensity were: M2R> M3R= M5R> M4R= M1R. Immunofluorescence found that M1R in the basal cell mainly, M2R in almost all umbrella cells, M3R, M4R were uniform distributed in mucosa, M5R reduced from the suburothelium surface to the base gradually.Although studies have found that the M2, M3 receptor mRNA and protein expression of the bladder urothelium and detrusor were increased in diabetic rats; The M2, M3 receptor of bladder urothelium were upregulated in DO (detrusor overactivity) mouse caused by bladder partial outlet obstruction, only M3 increased in detrusor 11.Howwever,There are still few reports about M muscarinic receptor expression in human bladder mucosa in pathological state and the results existence of contradictory12,13.Mukerji G 14 perfromed PUF questionnaire,urgency, frequent urination symptom scores before bladder biopsies in 12 patients with IDO (idiopathic detrusor overactivity),The study illustrated that M2, M3 receptors expressed in the bladder urothelium, nerve fibers in the detrusor layer. M2, M3 of subepithelial myofibroblast-like cell increased in patients with IDO, and correlated with urgency, frequent urination symptom score; M2, M3 receptors in urothelium and detrusor were unchanged.Kate H Moore et al 15 have observed M2, M3 expression in 20 cases female patients with IDO and found M2 was no change in bladder mucosa, M3 was decreased. Datta S et al 16also compared bladder suburothelium M1-3 receptor in 24 cases of patients with IDO,and found M1, M3 receptor were downregulated on the bladder urothelium.Recently, Soumendra N Datta et al 17 found that M1, M3 receptor of suburothelium were downregulation in 36 patients with DO using immunohistochemical methods,and the downregulation is negatively correlated to urgency and frequency of 24 hours;M1, M3 was decreased in urothelium.The expressed levels of M1 to M5 mRNA of bladder mucosa in patients with DO/IDO in bladder recommended above has a contradiction, and all have a common characteristic:that is, patients are derived from DO.OAB is a clinical symptom diagnosis, distinct from the diagnosis of detrusor overactivity (DO) which is made by urodynamic assessment.The overlap between urodynamically-defined DO and subjectively-reported OAB is substantial. DO refers to patients with no inhibition of detrusor contraction in the process the urinary bladder pressure-volume determination test. Thus, DO can not be equated to OAB18 19-21. That is, to date, there is not a real research of M receptor of bladder mucosa in patients with OAB has been reported.The five subtypes of M receptors exist in multiple organs, and also play an important role, such as salivary glands (M1, M3), gastrointestinal smooth muscle (M2, M3), eyes (M3, M5), heart (M2), the brain (M1, M3, M4, M5) 16, which is related to common side effects of M-receptor antagonists observed clinically, such as dry mouth, constipation, blurred vision, tachycardia, mental symptoms. To avoid these side effects,people need to change the route of administration or development of new subtype-selective, tissue selective drug,and also need to study of the changes in M receptor expression of bladder mucosa and its clinical significance,which will contribute to further clear its mechanism, clinical use of more subtype-selective drugs or with a more appropriate route of administration.In summary, the observation the distribution of bladder mucosa M receptor in the patients with OAB and expression changes associated clinical symptoms, which will help to further explore the pathogenesis of OAB and the pharmacol mechanism of M receptor antagonists; may change the route administration of M receptor antagonist, increase efficacy, reduce side effects; help to identify potential therapeutic targets, provid a reference tissue to the development of more subtype-selective, more selective drugs.Objective:The overactive bladder is common and distressing bladder hypersensory syndromes that has a significant impact on the quality of life of many people worldwide.The etiology and pathogenesis of OAB not yet clear, which makes it difficult to treat. The primary treatment of OAB is drug therapy, while the most commonly used drug is an oral M receptor (m muscarinic cholinergic receptor) antagonist.For the pathogenesis of OAB, people usually considered that detrusor overactivity leads to urgency, urge incontinence and other symptoms of OAB. Therefore, it is generally believed that M receptor antagonist blockaded M receptor on the detrusor, then reduced the bladder efferent activity, thereby inhibiting the detrusor contraction.However, oral recommended dose of M-receptor antagonist can improve symptoms such as urinary frequency, urgency, urge incontinence and increase bladder capacity, and rarely cause difficulty urinating or urinary retention in patients with OAB; In addition, the bladder M receptor antagonist atropine can significantly improve symptoms in patients with detrusor overactivity8. This means, M receptor antagonists may block M receptors in the bladder mucosa by a mechanism which inhibited the bladder sensory pathways (afferent pathway) in patients with OAB.Although the bladder mucosa has become one of hot discussed target in the researchs of OAB. But the function of M receptor on the bladder is unclear, at present only the distribution of M receptor subtypes in a small number of patients with bladder detrusor overactivity has reported, but their findings existed contradiction12,13. So far, There not have detection of M receptor subtypes on the bladder mucosa in patients with OAB were reported.In summary, the study observed the distribution of bladder mucosa M receptor in the patients with OAB and expression changes associated clinical symptoms, which will help to further explore the pathogenesis of OAB and the pharmacol mechanism of M receptor antagonists; may change the route administration of M receptor antagonist, increase efficacy, reduce side effects; help to identify potential therapeutic targets, provid a reference tissue to the development of more subtype-selective, more selective drugs.Methods:This object were divided into two groups:control group, overactive bladder group. Voiding diary OABSS, King's questionnaire were recorded before the experiment. All biopsy specimens were obtained by cystoscopy.(1) OAB symptoms score and quality of life scoreIncluding the overactive bladder symptom score sheet (OABSS), King's questionnaire score.(2)M receptor subtypes quantitative determinationThe expression of M1-5 receptor in the wall of bladder mucosa in patients and control were determined by fluorescence quantitative PCR.(3)the relationship between M receptor subtypes and bladder symptoms and quality of life in patients with OAB The results of the questionnaires were evaluated, and their correlation between the expression of M receptor subtypes were analyzed.Results:10 cases in OAB group, the average age of 38.70±10.71 years; 11 cases in normal control group the average age of 49.64±13.11 years; age difference between groups was not statistically significant (F= 4.328, P= 0.051).In control group,6 patients were female and 5 males,7 females and 3 males in OAB group, compared gender differences between the groups was not statistically significant (using Fisher exact test, P=0.659).1, The array of M receptor mRNA copies were compared between the control group and OAB group:In the table below, M receptor subtype in patients with OAB mRNA copies in order were:M3> M4> M5> M2> M1. Control group were:M3> M4> M5> M1> M2. From the M receptor subtype mRNA in terms of ranking, there are some differences between the two groups.Table 1 M1-M5 Comparison of copy number of bladder mucosa in patients with OAB (median)Table 1 M1-M5 Comparison of copy number of bladder mucosa in patients with OAB (median)M1 M2 M3 M4 M5 (n=10) (n=9) (n=10) (n=10) (n=10) OAB 0.27(0.21-0.55) 0.70(0.09-4.57) 46.35(11.97-168.83) 36.86(7.45-68.76) 10.13(1.66-20.87)Table 2 M1-M5 Comparison of copy number of bladder mucosa in control group (median)M1 M2 M3 M4 M5 (n2=11) (n2=9) (n2=11) (n2=11) (n2=11) control 0.24(0.11-0.52) 0.09(0.02-1.31) 12.79(3.86-269.35) 10.79(5.03-91.02) 9.47(3.78-28.76)M3, M4, M5 receptor were rich whether in OAB or control group of the bladder mucosa, most of which was M3 receptor. Moreover, M3 receptor was relevant to the scores of first 5,6 part of King's quality of life (r=0.788,0.654; P= 0.035,0.040.)2, M1-M5 receptor mRNA copy number of bladder mucosa between OAB and control groups were compared with Mann-Whitney test,, Z values were-0.63;-1.28;-0.63;-0.85;-0.14. P= 0.557; 0.222; 0.557; 0.426; 0.918. There were not statistically significant differences.3, The correlations between M1-M5 receptor mRNA and the symptoms of OAB patients:Total OABSS in patients with OAB was 7.70±3.16 points; OABSS urgency symptom score 3.50±2.07 points;Total score of King's quality of life was 529.72±208.15 points.M4 receptor was related to scores and urgency symptoms with OABSS, r=0.740,0.723; P=0.014,0.018.M4, M5 receptor was related to Part 5 in King's quality of life scores, r= 0.788,0.768; P= 0.035,0.044.Conclusions:1, M muscarinic receptor mRNA of patients with OAB in order were:M3> M4> M5> M2> M1. The control group were:M3> M4> M5> M1> M2., there are some differences between the two groups.2, Whether in OAB or control group of the bladder mucosa,most of which was M3 receptor. Moreover, M3 receptor was relevant to the scores of first 5,6 part of King's quality of life; indicating that the widely used M3 selective receptor antagonists for treatment of OAB may also have effects on the bladder mucosa.The shudy provides a theoretical basis for use of selective M3 receptor antagonist to improve the quality of life in clinic. Selective M3 receptor antagonist on personal life, emotional improvements should be more apparent.3, M4 were related to urgency symptoms and OABSS score in patients with OAB; M4, M5 receptor expression also related to Part 5 in King's quality of life scores. This shows that M4, M5 also play a role in the pathogenesis of OAB, M4, M5 receptor should be pay attention in future studies.