Botulinum toxin type A normalizes alterations in urothelial ATP and NO release induced by chronic spinal cord injury

The purpose of this paper was to simultaneously examine changes in urothelial ATP and NO release in normal and spinal cord injured animals as well as in spinal cord injured animals treated with botulinum toxin type A (BoNT-A). Furthermore we correlated changes in transmitter release with functional changes in bladder contraction frequency, and determined the effects of BoNT-A on bladder efferent nerve function. Normal and spinal cord injured rat bladders were injected on day 0 with either vehicle (saline containing bovine serum albumin) or BoNT-A. On day 2, in vitro neurotransmitter release and bladder strip contractility studies as well as in vivo cystometrographic studies were conducted. Resting ATP release was significantly enhanced following spinal cord injury (i.e. 57% increase, p<0.05) and was unaffected by BoNT-A treatment. SCI increased hypoosmotic evoked urothelial ATP release by 377% (p<0.05). BoNT-A treatment reduced evoked ATP release in SCI bladders by 83% (p<0.05). In contrast, hypoosmotic stimulation induced NO release was significantly inhibited following SCI (i.e. 50%, p<0.05) but recovered in SCI rats treated with BoNT-A (i.e. 195% increase in NO release in SCI-BTX-treated rats compared to SCI controls, p<0.01). Changes in urothelial transmitter release coincided with a significant decrease in non-voiding bladder contraction frequency (i.e. 71%, p<0.05) in SCI-BTX rats compared to SCI rats. While no difference was measured between neurally evoked contractile amplitude between SCI and SCI-BTX animals, atropine (1 microM) inhibited contractile amplitude to a greater extent (i.e. 76%, p<0.05) in the SCI-BTX group compared to the SCI group. We hypothesize that alterations in the ratio of excitatory (i.e. ATP) and inhibitory (i.e. NO) urothelial transmitters promote bladder hyperactivity in rat bladders following SCI that can be reversed, to a large extent, by treatment with BoNT-A.     

Botulinum toxin A inhibits ATP release from bladder urothelium after chronic spinal cord injury

The effects of mechanoreceptor stimulation and subsequent ATP release in spinal cord injured and normal bladders was examined to demonstrate if spinal cord injury (SCI) modulates the basal or evoked release of ATP from bladder urothelium and whether intravesical botulinum toxin A (BTX-A) administration inhibits urothelial ATP release, a measure of sensory nerve activation. A Ussing chamber was used to isolate and separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Following spinal cord injury, resting urothelial release of ATP was ninefold higher than that of normal rats. Botulinum toxin A instillation did not significantly affect the resting release of ATP after spinal cord injury. Evoked ATP release following hypoosmotic stimulation was significantly higher in chronic spinal cord injured compared to normal rat bladders. However, botulinum toxin A treatment markedly reduced ATP release in spinal cord injured bladders by 53% suggesting that ATP release by mechanoreceptor stimulation, as opposed to basal release, occurs by exocytotic mechanisms. In contrast, there was no significant difference in basal or evoked ATP release from bladder serosa following spinal cord injury. Moreover, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side after spinal cord injury, suggesting that its effects were confined to the urothelial side of the bladder preparation. In summary: (1) increased release of ATP from the urothelium of spinal cord injured bladders may contribute to the development of bladder hyperactivity and, (2) mechanoreceptor stimulated vesicular ATP release, as opposed to basal non-vesicular release of ATP, is significantly inhibited in spinal cord injured bladders by intravesical instillation of botulinum toxin A. These results may have important relevance in our understanding of the mechanisms underlying plasticity of bladder afferent pathways following SCI.     

Increased expression of spinal cord Fos protein induced by bladder stimulation after spinal cord injury

These studies examined Fos protein expression in spinal cord neurons synaptically activated by stimulation of bladder afferent pathways after spinal cord injury (SCI). In urethan-anesthetized Wistar rats after SCI for 6 wk, intravesical saline distension significantly (P 相似文献   

Neurochemical plasticity of nitric oxide synthase isoforms in neurogenic detrusor overactivity after spinal cord injury

Nitric oxide (NO) participates in the neural pathways controlling the lower urinary tract (LUT). Expression of NO synthase (NOS) can be upregulated after spinal cord injury (SCI), and altered NOS activity may participate in resulting LUT dysfunction. To investigate distribution of NOS-immunoreactivity (NOS-IR) in neurons of rats following SCI and the possible effects of NOS inhibitors. Expression of neuronal and inducible NOS-IR in lumbosacral spinal cord was assessed in rats. Cystometry was performed to examine effects of intrathecal injection of NOS inhibitor. There was increased expression of neuronal NOS-IR after trauma. Maximum bladder capacity was increased by neuronal NOS (nNOS) inhibitors. Upregulation of nNOS may facilitate emergence of the spinal micturition reflex following SCI; nNOS inhibitor suppressed SCI-induced urinary incontinence by increasing bladder capacity. Our results indicate manipulation of NO production could help treat LUT dysfunction after SCI.     

Alterations in spinal cord Fos protein expression induced by bladder stimulation following cystitis

These studies examined Fos protein expression in spinal cord neurons synaptically activated by stimulation of bladder afferent pathways after cyclophosphamide (CYP)-induced bladder inflammation. In urethan-anesthetized Wistar rats with cystitis, intravesical saline distension significantly (P 相似文献   

Increased spinal expression of c-Fos following stimulation of the lower urinary tract in chronic spinal cord-injured rats

c-Fos expression was studied in the lumbar and sacral spinal cord regions involved in processing afferent input from the lower urinary tract and a comparison was made between spinal cord-injured (SCI) animals and control animals with intact neuraxes. Afferent pathways from the lower urinary tract were activated either by insertion of a catheter through the urethra into the urinary bladder or by catheterisation plus induction of reflex micturition contractions by intravesical saline infusion. Placement of a catheter alone elicited Fos expression in a similar number of neurones in SCI and control rats mainly in the medial dorsal horn (MDH) and dorsal commissure (DCM) in the segments L1–2 and L5–S1 with a maximum in L5. Additional saline infusion induced low-frequency, high-amplitude, rhythmic bladder contractions of long duration in the rats with intact spinal cords, whereas in SCI rats, bladder distension elicited reflex contractions at a higher frequency, smaller amplitude and shorter duration. However, the basal and mean bladder pressure, as well as the total contraction time relative to the whole recording time, was not significantly different. Distension-induced bladder contractions markedly increased Fos expression primarily in the spinal segments L5–S1 in the control rats, where the majority of bladder and urethral afferent fibres terminates. Fos-positive cells were located in the MDH, lateral dorsal horn (LDH), DCM and the lateral aspect of laminae V–VII. Compared to controls, Fos expression after spinal cord injury (SCI) occurred in a significantly greater number of neurones throughout the segments L3–S1 following induction of bladder reflexes. The greatest proportional increase in the number of Fos-positive cells occurred in L3–5 which normally receive only little afferent input from the urinary bladder. Cell numbers predominantly increased in the LDH and lateral lamina V–VII. The data are consistent with the concept of a neuroplastic reorganisation of spinal pathways after SCI. Unmasking of silent synapses or formation of new connections by afferent axonal sprouting caudal to the lesion, as evident from the increased numbers of cells expressing Fos after bladder distension, could be factors underlying the emergence of reflexogenic micturition in chronic SCI rats. Accepted: 27 May 1999     

脑桥上中枢损伤与腰骶段脊髓损伤患者的膀胱功能障碍及尿动力特点研究

目的:比较脑桥以上中枢损伤与腰骶段脊髓损伤患者的膀胱功能障碍及尿动力学特点。方法:回顾性分析2011年3月至2014年5月我院收治的78例中枢神经损伤患者的临床资料,包括临床表现、诊断、排尿方式、残余尿、尿动力学检查结果。其中,脑桥以上中枢损伤组43例,腰骶段脊髓损伤组35例,分析和比较两组患者的自由尿流率参数和完全膀胱测压参数。结果:两组间的最大尿流率、排尿量比较差异无统计学意义(P0.05),脑桥上中枢损伤组的残余尿量明显低于腰骶段脊髓损伤组,差异有统计学意义(P0.05)。与腰骶段脊髓损伤组比较,脑桥以上损伤组的膀胱容量明显减少,最大尿流率时的压力、逼尿肌的最大压力及平均压力明显增加,差异均有统计学意义(P0.05)。两组膀胱的顺应性、逼尿肌稳定性比较差异有统计学意义(P0.05),脑桥以上中枢损伤患者的多数表现为低顺应性膀胱(27/43),胸腰段脊髓损伤患者主要表现为高顺应性膀胱(21/35);脑桥以上损伤组多表现为逼尿肌的过度活动(29/43),而腰骶段脊髓损伤组更多表现为逼尿肌的无反射和弱反射(20/35)。结论:脑桥以上损伤患者主要表现为逼尿肌过度活动和膀胱容量的显著降低,以低顺应性膀胱为主;腰骶段脊髓损伤患者的逼尿肌多为无反射和弱反射,以高顺应性膀胱为主。     

延髓头端腹外侧区血管紧张素Ⅱ对脊髓投射神经元氨基酸递质释放的调节

用脊髓(T8)中间外侧柱(IML)微透析方法结合高效液相色谱(HPLC)技术,研究延髓头端腹外侧区(RVLM)微量注射血管紧张素Ⅱ(ANGⅡ,100pmol,n=11)后脊髓IML氨基酸递质释放的变化.在RVLM区微量注射ANGⅡ(100pmol,n=11),能显著增加(P<0.01)脊髓(T8)内天门冬氨酸(ASP,从4.75±1.01升至8.90±2.28pmol/20μl)和谷氨酸(GLU,从18.99±8.64升至73.88±29.26pmol/20μl)的释放.在同一RVLM部位注射losartan(10nmol,n=8)可以显著抑制注射ANGⅡ引起的GLU释放升高反应(P<0.05).免疫荧光双标记结合共聚焦显微镜观察到RVLM内62%～91%的谷氨酸能神经元呈AT1受体免疫阳性.此结果提示ANGⅡ诱发的脊髓内谷氨酸释放可能来源于RVLM内AT1受体免疫阳性的谷氨酸能脊髓投射神经元.     

Early molecular-level changes in rat bladder wall tissue following spinal cord injury

Previously, we demonstrated using a rat model of spinal cord injury (SCI) that bladder wall tissue compliance significantly increased within the first 2 weeks following injury. In order to explore the potential molecular-level mechanisms of this event, the present study quantified molecules pertinent to bladder tissue remodeling and changes in mechanical properties. An initial gene array analysis followed by real-time qPCR revealed that the message levels for tropoelastin and lysyl oxidase were as high as 8-fold in SCI rats compared to normal. Furthermore, both the message and protein levels of TGF-beta1 and IGF-1, known stimulators of elastin synthesis, in SCI rat bladders were significantly higher compared to those of normal rats. Taken together, it can be speculated that functional changes of the bladder associated with SCI induce release of select growth factors, which, in turn, stimulate elastogenesis that lead to alteration of biomechanical properties of the wall tissue.     

Effect of naftopidil,an alpha1D/A-adrenoceptor antagonist,on the urinary bladder in rats with spinal cord injury

AimsAlpha_1D-adrenoceptors (α_1D-ARs) located in the spinal cord are involved in the control of lower urinary tract function. In order to clarify the effect of α_1D-ARs on storage function in the spinal cord, we examined the effect of oral administration and intrathecal injection of the α_1D/A-AR antagonist, naftopidil, on bladder activity, as well as the effect of naftopidil on bladder wall histology, in female rats with spinal cord injury (SCI).Main methodsAdult female Sprague–Dawley rats with Th9–10 spinal cord transection were used. In SCI rats with or without 5 mg/day of naftopidil for 4 weeks, bladder activity was examined via continuous cystometry. In other SCI rats, bladder activity was examined before and after intrathecal injection of naftopidil. In addition, bladder wall histology was compared between SCI rats with or without oral administration of naftopidil for 4 weeks.Key findingsOral administration of naftopidil decreased the number of non-voiding contractions (NVCs). Intrathecal injection of naftopidil prolonged the interval between voiding contractions, decreased the maximum voiding contraction pressure and the number of NVCs, and increased bladder capacity without affecting the residual urine volume. Oral administration of naftopidil also decreased bladder wall fibrosis.SignificanceThe α_1D/A-AR antagonist naftopidil might act on the bladder and spinal cord to improve detrusor hyperreflexia in the storage state in SCI female rats. Naftopidil also suppressed bladder wall fibrosis, suggesting that it may be effective for the treatment of neurogenic lower urinary tract dysfunction after SCI.     

Suppression of detrusor-sphincter dysynergia by GABA-receptor activation in the lumbosacral spinal cord in spinal cord-injured rats

We investigated the effects of intrathecal application of GABAA- or GABAB-receptor agonists on detrusor-sphincter dyssynergia (DSD) in spinal cord transection (SCT) rats. Adult female Sprague-Dawley rats were used. At 4 wk after Th9-10 SCT, simultaneous recordings of intravesical pressure and urethral pressure were performed under an awake condition to examine the effect of intrathecal application of GABAA and GABAB agonists (muscimol and baclofen, respectively) or GABAA and GABAB antagonists (bicuculline and saclofen, respectively) at the level of L6-S1 spinal cord. In spinal-intact rats, the effects of bicuculline and saclofen on bladder and urethral activity were also examined. During urethral pressure measurements, DSD characterized by urethral pressure increases during isovolumetric bladder contractions were observed in 95% of SCT rats. However, after intrathecal application of muscimol or baclofen, urethral pressure showed urethral relaxation during isovolumetric bladder contractions. The effective dose to induce inhibition of urethral activity was lower compared with the dose that inhibited bladder contractions. The effect of muscimol and baclofen was antagonized by intrathecal bicuculline and saclofen, respectively. In spinal-intact rats, intrathecal application of bicuculline induced DSD-like changes. These results indicate that GABAA- and GABAB-receptor activation in the spinal cord exerts the inhibitory effects on DSD after SCT. Decreased activation of GABAA receptors due to hypofunction of GABAergic mechanisms in the spinal cord might be responsible, at least in part, for the development of DSD after SCT.     

Distribution and subtype determination ofβ-receptors in the spinal cord of the adult rat

1. We determined the number of beta-receptors in the whole spinal cord of the adult rat and in the cervical, thoracal, and lumbal/sacral parts. 2. The undivided spinal cord contains 47 +/- 10 fmol/mg beta-receptors (KD = 2066 +/- 982 pmol/liter), and the cervical part of the spinal cord contains 53 +/- 8 fmol/mg protein (KD = 3224 +/- 1775 pmol/liter). The thoracal part shows 40 +/- 1 fmol/mg protein (KD = 3229 +/- 104 pmol/liter), and the lumbal/sacral spinal cord contains 48 +/- 8 fmol/mg protein (KD = 3610 +/- 1610 pmol/liter). 3. Competitive inhibition studies with l-practolol, dl-atenolol, and ICI 118,551 were performed and we calculated by a computer program in the whole spinal cord the following ratio of beta-receptor subtypes: 80 +/- 5% Beta 1-receptors and 20 +/- 5% beta 2-receptors. 4. The basal and (-)-isoproterenol- and NaF-stimulated activity of adenylate cyclase was highest in the cervical part of the spinal cord and equally distributed between the thoracal and the lumbal/sacral parts. 5. The whole synaptosomal protein of the cervical part of the spinal cord contained 132 +/- 20 fmol, the thoracal part 117 +/- 3 fmol, and the lumbal/sacral part 133 +/- 22 fmol.     

The effect of supplementation wit n-3 fatty acids on the physical performance in subjects with spinal cord injury

A global physical evaluation was performed in 21 males with spinal cord injury (SCI), at the beginning and at three and six months of omega-3 fatty acid (FA) supplementation. A significant increase in the proportion of eicosapentaenoic acid and docosahexanoic acid in plasma was observed in response to the supplementation (p<0.05). After six months of FA supplementation, strength endurance time increased from 127.7+/-19.0 s to 215.2+/-45.6 s in the right arm, and from 139+/-27.6 s to 237.7+/-48.7 s, in the left arm. The time to perform 20 repetitions of 70% maximum workload showed a reduction of 41% between the first and the third test. The time taken to cover a 90 meter long track, with a 6% slope, was reduced from 66.9+/-8.0 s to 59.3+/-6.7 s, at the end of the study (p<0.05). In conclusion, omega-3 FA supplementation could contribute to improve the functional capabilities in SCI subjects.     

三七总皂苷对脊髓损伤大鼠运动功能恢复的作用

目的：探讨三七总皂苷对大鼠脊髓损伤（SCI）后运动功能恢复的作用。方法：正常SD大鼠随机分为5组（n=8）：正常对照组（Normal）、假手术组（Sham）、脊髓损伤（SCI）和脊髓损伤+三七总皂苷组（PNS）（n=8）。所有大鼠分别在造模前及造模后第1、3、7、14、21和28天接受运动功能评分（BBB）和运动诱发电位（MEP）检查,观察大鼠后肢运动功能的恢复情况。结果：造模后,Sham组、PNS组、SCI组BBB评分低于正常;MEP波幅低于正常;潜伏期较正常延长。PNS组与同期SCI组比较,第7、14、21、28天的BBB评分差异有统计学意义（P<0.05）;第7天、14天、21天、28天,MEP检查波幅（Amp）和潜伏期（Lat）组内有显著差异,并且与同期SCI组比较差异有统计学意义（P<0.05）。结论：三七总皂苷可促进大鼠SCI后运动功能的恢复。     

Fatty acid kinetics and carbohydrate metabolism during electrical exercise in spinal cord-injured humans

Motor center activity and reflexes from contracting muscle have been shown to be important for mobilization of free fatty acids (FFA) during exercise. We studied FFA metabolism in the absence of these mechanisms: during involuntary, electrically induced leg cycling in individuals with complete spinal cord injury (SCI). Healthy subjects performing voluntary cycling served as controls (C). Ten SCI (level of injury: C5-T7) and six C exercised for 30 min at comparable oxygen uptake rates (approximately 1 l/min), and [1-14C]palmitate was infused continuously to estimate FFA turnover. From femoral arteriovenous differences, blood flow, muscle biopsies, and indirect calorimetry, leg substrate balances as well as concentrations of intramuscular substrates were determined. Leg oxygen uptake was similar in the two groups during exercise. In SCI, but not in C, plasma FFA and FFA appearance rate fell during exercise, and plasma glycerol increased less than in C (P < 0.05). Fractional uptake of FFA across the working legs decreased from rest to exercise in all individuals (P < 0.05) but was always lower in SCI than in C (P < 0.05). From rest to exercise, leg FFA uptake increased less in SCI than in C subjects (14 +/- 3 to 57 +/- 20 vs. 41 +/- 13 to 170 +/- 57 micromol x min(-1) x leg(-1); P < 0.05). Muscle glycogen breakdown, leg glucose uptake, carbohydrate oxidation, and lactate release were higher (P < 0.05) in SCI than in C during exercise. Counterregulatory hormonal changes were more pronounced in SCI vs. C, whereas insulin decreased only in C. In conclusion, FFA mobilization, delivery, and fractional uptake are lower and muscle glycogen breakdown and glucose uptake are higher in SCI patients during electrically induced leg exercise compared with healthy subjects performing voluntary exercise. Apparently, blood-borne mechanisms are not sufficient to elicit a normal increase in fatty acid mobilization during exercise. Furthermore, in exercising muscle, FFA delivery enhances FFA uptake and inhibits carbohydrate metabolism, while carbohydrate metabolism inhibits FFA uptake.     

TRPA1 receptor modulation attenuates bladder overactivity induced by spinal cord injury

The ankyrin-repeat transient receptor potential 1 (TRPA1) has been implicated in pathological conditions of the bladder, but its role in overactive bladder (OAB) following spinal cord injury (SCI) remains unknown. In this study, using a rat SCI model, we assessed the relevance of TRPA1 in OAB induced by SCI. SCI resulted in tissue damage, inflammation, and changes in bladder contractility and in voiding behavior. Moreover, SCI caused upregulation of TRPA1 protein and mRNA levels, in bladder and in dorsal root ganglion (DRG; L6-S1), but not in corresponding segment of spinal cord. Alteration in bladder contractility following SCI was evidenced by enhancement in cinnamaldehyde-, capsaicin-, or carbachol-induced bladder contraction as well as in its spontaneous phasic activity. Of relevance to voiding behavior, SCI induced increase in the number of nonvoiding contractions (NVCs), an important parameter associated with the OAB etiology, besides alterations in other urodynamic parameters. HC-030031 (TRPA1 antagonist) treatment decreased the number and the amplitude of NVCs while the TRPA1 antisense oligodeoxynucleotide (AS-ODN) treatment normalized the spontaneous phasic activity, decreased the cinnamaldehyde-induced bladder contraction and the number of NVCs in SCI rats. In addition, the cinnamaldehyde-induced bladder contraction was reduced by exposure of the bladder preparations to HC-030031. The efficacy of TRPA1 AS-ODN treatment was confirmed by means of the reduction of TRPA1 expression in the DRG, in the corresponding segment of the spinal cord and in the bladder, specifically in detrusor muscle. The present data show that the TRPA1 activation and upregulation seem to exert an important role in OAB following SCI.     

P2X7 receptor inhibition improves recovery after spinal cord injury

Secondary injury exacerbates the extent of spinal cord insults, yet the mechanistic basis of this phenomenon has largely been unexplored. Here we report that broad regions of the peritraumatic zone are characterized by a sustained process of pathologic, high ATP release. Spinal cord neurons expressed P2X7 purine receptors (P2X7R), and exposure to ATP led to high-frequency spiking, irreversible increases in cytosolic calcium and cell death. To assess the potential effect of P2X7R blockade in ameliorating acute spinal cord injury (SCI), we delivered P2X7R antagonists OxATP or PPADS to rats after acute impact injury. We found that both OxATP and PPADS significantly improved functional recovery and diminished cell death in the peritraumatic zone. These observations demonstrate that SCI is associated with prolonged purinergic receptor activation, which results in excitotoxicity-based neuronal degeneration. P2X7R antagonists inhibit this process, reducing both the histological extent and functional sequelae of acute SCI.     

Change in muscarinic modulation of transmitter release in the rat urinary bladder after spinal cord injury

Muscarinic facilitation of 14C-ACh release from post-ganglionic parasympathetic nerve terminals was studied in bladder strips prepared from spinal intact (SI) and spinal cord transected (SCT) rats. The spinal cord was transected at the lower thoracic spinal segments 3 weeks prior to the experiments. Using non-facilitatory stimulation (2 Hz) the release of ACh in spinal intact rats did not change in the presence of a non-specific muscarinic antagonist, atropine (100 nM), an M(1) specific antagonist (pirenzepine, 50 nM) or an M(1)-M(3) specific antagonist (4-DAMP, 5 nM). However, during a facilitatory stimulation paradigm (10 Hz or 40 Hz, 100 shocks) atropine and pirenzepine, but not 4-DAMP inhibited the release of ACh in bladders from spinal intact rats, indicating an M(1) receptor-mediated facilitation. In spinal cord transected rats, 2 Hz stimulation-induced release was significantly inhibited by atropine or 4-DAMP but not by pirenzepine indicating that a pre-junctional facilitatory mechanism mediated via M(3) muscarinic receptors could be induced by a non-facilitatory stimulation paradigm after spinal injury. In bladders of spinal cord transected rats, 10 Hz stimulation-evoked release of ACh was also inhibited by atropine and 4-DAMP (5 nM) but not by pirenzepine (50 nM). These results indicate that pre-junctional muscarinic receptors at cholinergic nerve endings in the bladder change after chronic spinal cord injury. It appears that low affinity M(1) muscarinic receptors are replaced by high affinity M(3) receptors. This change in modulation of ACh release may partly explain the bladder hyperactivity after chronic spinal cord injury.     

M2 receptors in genito-urinary smooth muscle pathology

In vitro bladder contractions in response to cumulative carbachol doses were measured in the presence of selective muscarinic antagonists from rats which had their major pelvic ganglion bilaterally removed (denervation, DEN) or from rats in which the spinal cord was injured (SCI) via compression. DEN induced both hypertrophy (505+/-51 mg bladder weight) and a supersensitivity of the bladders to carbachol (EC50=0.7+/-0.1 uM). Some of the SCI rats regained the ability to void spontaneously (SPV). The bladders of these animals weighed 184+/-17 mg, significantly less than the bladders of non voiding rats (NV, 644+/-92 mg). The potency of carbachol was greater in bladder strips from NV SCI animals (EC50=0.54+/-0.1 uM) than either bladder strips from SPV SCI (EC50=0.93+/-0.3 microM), DEN or control (EC50=1.2+/-0.1 microM) animals. Antagonist affinities in control bladders for antagonism of carbachol induced contractions were consistent with M3 mediated contractions. Antagonist affinities in DEN bladders for 4-diphenlacetoxy-N-methylpiperidine methiodide (4-DAMP, 8.5) and para fluoro hexahydrosilodifenidol (p-F-HHSiD, 6.6); were consistent with M2 mediated contractions, although the methoctramine affinity (6.5) was consistent with M3 mediated contractions. p-F-HHSiD inhibited carbachol induced contraction with an affinity consistent with M2 receptors in bladders from NV SCI (pKb=6.4) animals and M3 receptors in bladders from SPV SCI animals (pKb=7.9). Subtype selective immunoprecipitation of muscarinic receptors revealed an increase in total and an increase in M2 receptor density with no change in M3 receptor density in bladders from DEN and NV SCI animals compared to normal or sham operated controls. M3 receptor density was lower in bladders from SPV SCI animals while the M2 receptor density was not different from control. This increase in M2 receptor density is consistent with the change in affinity of the antagonists for inhibition of carbachol induced contractions and may indicate that M2 receptors or a combination of M2 and M3 receptors directly mediate smooth muscle contraction in bladders from DEN and NV SCI rats.