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Cannabinoids are the active chemical components of Cannabis sativa (marijuana). The medical use of cannabis goes back over 5,000 years. Cannabinoids produce a very wide array of central and peripheral effects, some of which may have beneficial clinical applications. The discovery of cannabinoid receptors has spawned great interest within the pharmaceutical industry with the hopes of capitalizing on the beneficial effects of cannabis without the unwanted psychotropic effects on the central and peripheral nervous system. This chapter presents an overview of the pharmacology of cannabinoids and their derivatives. It reviews the current literature on central and peripheral cannabinoid receptors as related to effects on the lower urinary tract and the role of these receptors in normal and abnormal urinary tract function. An objective evaluation of the published results of clinical trials of cannabis extracts for the treatment of bladder dysfunction resulting from multiple sclerosis is also presented. It is clear that cannabinoid receptors are present in the lower urinary tract as well as spinal and higher centers involved in lower urinary tract control. Systemic cannabinoids have effects on the lower urinary tract that may be able to become clinically useful; however, a much greater understanding of the mechanisms of cannabinoid receptors in control of the human lower urinary tract is necessary to facilitate development of novel cannabinoid drugs for treatment of pelvic disorders.

Cannabinoids are the active chemical components of Cannabis sativa (marijuana). The medical use of cannabis goes back over 5,000 years. Cannabinoids produce a very wide array of central and peripheral effects, some of which may have beneficial clinical applications. The discovery of cannabinoid receptors has spawned great interest within the pharmaceutical industry with the hopes of capitalizing on the beneficial effects of cannabis without the unwanted psychotropic effects on the central and peripheral nervous system. This chapter presents an overview of the pharmacology of cannabinoids and their derivatives. It reviews the current literature on central and peripheral cannabinoid receptors as related to effects on the lower urinary tract and the role of these receptors in normal and abnormal urinary tract function. An objective evaluation of the published results of clinical trials of cannabis extracts for the treatment of bladder dysfunction resulting from multiple sclerosis is also presented. It is clear that cannabinoid receptors are present in the lower urinary tract as well as spinal and higher centers involved in lower urinary tract control. Systemic cannabinoids have effects on the lower urinary tract that may be able to become clinically useful; however, a much greater understanding of the mechanisms of cannabinoid receptors in control of the human lower urinary tract is necessary to facilitate development of novel cannabinoid drugs for treatment of pelvic disorders.

One theory for the etiology of interstitial cystitis (IC) proposes toxic substances in the urine. This hypothesis was tested in our laboratory by infusing urine into the bladders of rabbits twice weekly for six weeks. For the first study rabbits were treated by one-hour biweekly intravesical exposure to urine from a symptomatic interstitial cystitis patient, a normal volunteer, or physiologic saline. For the second study, animals were exposed to both a high and a low molecular weight fraction of urine pooled from 7 interstitial cystitis patients, 7 normal female volunteers, and physiologic saline. At the end of six weeks the animals were cystoscoped and the bladder was removed the following day for histologic and contractile studies. Post-distention glomerulations were observed in 3 of the 4 whole IC urine-treated animals and an ulcer identical to the classic "Hunner's ulcer" was seen in one of these animals. Post-distention petechial hemorrhages were also noted in all 5 of the high molecular weight IC urine-treated animals but in none of the others, suggesting a difference between IC and normal urine. These IC urine-treated groups also showed the greatest degree of histologic changes including edema and plasma cell infiltrates in the lamina propria, submucosa and perivascular tissue. However, there was no statistically significant difference in bladder capacity, micturition patterns, or contractile response of bladder strips. These results indicate that there are substances with nominal molecular weight greater than 10 kD in interstitial cystitis urine that induce changes in the rabbit bladder that resemble bladders of interstitial cystitis patients.

This is a continuation of studies examining the effectiveness of root repairs and nerve transfers for bladder reinnervation. Our previous retrograde fluorogold tracing studies from the bladder to the spinal cord found regrowth of axons from the spinal cord through the nerve repair site to the bladder which was confirmed electrophysiologically [Ruggieri et al. J Neurotrauma 25:214–24, 2006]. The current study determines whether the pattern of axonal regrowth from the repaired nerves or roots to the bladder is different between the surgical reanastomosis methods.The canine bladder was denervated by transection of all nerve roots from the sacral spinal cord mediating bladder contraction. Reinnervation surgeries included end-on-end repair of transected sacral ventral roots, transfer of coccygeal to sacral ventral roots(CGNT),or transfer of genitofemoral to pelvic nerves(GFNT).Postmortem dialkylcarbocyaninedye tracing with Neurotrace DiI from the distal pelvic nerve to the bladder wall, combined with PGP9.5 neuronal immunohistochemistry, demonstrated innervation by DiI-labeled axons of only parasympathetic postganglionic intramural ganglia in normal controls and sham operated controls, but reinnervation of both intramural ganglia and detrusor muscle directly after repair of sacral ventral roots. GF NT and CG NT also resulted in reinnervation of both intramural ganglia and detrusor muscle, although to a lesser extent than repaired roots.Bladder reinnervation with either the same nerve (orthotopic reinnervation) or with either a primarily somatic nerve (coccygeal) or a primarily sensory nerve (genitofemoral) results in reinnervation of both intramural ganglia as well as direct innervation of detrusor muscle.

To compare the gastroesophageal junction of the human with the pig, M2and M3receptor densities and the potencies of M2and M3muscarinic receptor subtype selective antagonists were determined in gastric clasp and sling smooth muscle fibers. Total muscarinic and M2receptors are higher in pig than human clasp and sling fibers. M3receptors are higher in human compared with pig sling fibers but lower in human compared with pig clasp fibers. Clasp fibers have fewer M3receptors than sling fibers in both humans and pigs. Similar to human clasp fibers, pig clasp fibers contract significantly less than pig sling fibers. Analysis of the methoctramine Schild plot suggests that M2receptors are involved in mediating contraction in pig clasp and sling fibers. Darifenacin potency suggests that M3receptors mediate contraction in pig sling fibers and that M2and M3receptors mediate contraction in pig clasp fibers. Taken together, the data suggest that both M2and M3muscarinic receptors mediate the contraction in both pig clasp and sling fibers similar to human clasp and sling fibers.

We reported previously that following ileocystoplasty the structure and pharmacologic response of the implanted ileum changes towards that of the bladder. Specifically, the relaxation response to alpha adrenergic (methoxamine) and purinergic (ATP) stimulation reverses to a contractile response one month after the ileal segment is surgically made part of the urinary bladder. The present study was designed to investigate possible signals for this change and also to determine whether bladder responses would mimic the ileum if surgically interposed into the ileal stream. Rabbits in group 1 underwent bladder interposition into the functioning terminal ileum, rabbits in group 2 underwent tubularized ileocystoplasty and rabbits in group 3 underwent detubularized ileocystoplasty with urinary diversion. Twelve rabbits survived and were available for evaluation; five in group 1, three in group 2 and four in group 3. Analysis was done six weeks after surgery. In group 1 animals, the interposed bladder showed epithelial changes towards ileum and also a change in its in-vitro contractile responses towards that of ileum. In group 2 animals the tubular cystoplastic ileum showed minimal functional and morphologic changes. In group 3 animals, the defunctionalized, detubular cystoplastic ileum showed alpha adrenergic and purinergic response changes towards bladder. These results indicate that detubularization with interruption in the arrangement of smooth muscle fibers as well as the breach in the integrity of neuronal connections is likely to be the primary signal for the change in the ileum towards bladder induced by cystoplasty. The results can not rule out reinnervation of the intestinal segment by bladder nerves. In addition these data demonstrate that the pharmacologic response of the bladder changes towards the ileum within six weeks after the bladder is surgically made part of the ileum.

Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M2 and M3 muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M2 receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M3 density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M2 and M3 receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M2 and M3 receptors to contraction is presented. Using this new method of analysis, it was found that the M2 muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M3 receptor predominantly mediates contraction.

Treatment of the neurogenic bladder in canine models of spinal cord injury presents challenges in ensuring bladder drainage. While vesicostomy is routine for humans, the procedure is not common in canines. Our study of bladder reinnervation involved transection of the nerve roots that mediate bladder contraction in 34 canines. An abdominal vesicostomy was created by fixing the everted mucosa to the skin incision. After euthanasia, we assessed the contractility of in vitro bladder muscle strips in response to muscarinic receptor stimulation. There were a total of 11 complications in 9 of the 34 animals. In two animals, the vesicostomy narrowed such that it could not be catheterized and in two other animals the vesicostomy closed to between 5 and 10 mm diameter. Another animal removed the stitches prior to complete healing, requiring further surgical procedures. In fi ve animals, partial prolapse of the bladder through the vesicostomy required surgical repair, and in one animal the bladder became infected and required antibiotic treatment. In the few animals in which irritation resulted from the constant contact of urine with the skin, daily topical application of petrolatum ointment alleviated this symptom. Gross inspection of the bladder at euthanasia and in vitro contractility of bladder muscle strips from these animals revealed no evidence of changes associated with bladder hypertrophy. This study demonstrated that permanent cutaneous vesicostomy is an optimal refinement method for management of the neurogenic bladder in canines. The procedure avoids the distress as well as potential bladder hypertrophy induced by multiple daily interventions to empty the bladder by either catheterization or manual compression.

We reviewed the current literature on mechanisms involved in the pathogenesis of prostatitis/chronic pelvic pain syndrome (CPPS).A literature review for the years 1966 to 2003 was performed using the MEDLINE database of the United States National Library of Medicine.National Institutes of Health categories I and II prostatitis result from identifiable prostatic infections, whereas patients with category IV are asymptomatic. The majority of symptomatic cases are category III or chronic prostatitis (CP)/CPPS. The etiology of CP/CPPS is unknown. The traditional marker of inflammation, namely white blood cells in prostatic fluids, does not correlate with the predominant symptom of pelvic pain. An imbalance toward increased proinflammatory and decreased anti-inflammatory cytokines has been implicated and a few studies have shown some correlation of this with pelvic pain. The imbalance in some men may result from polymorphisms at the cytokine loci. An autoimmune process may be involved and experimental evidence indicates that this can be under hormonal influence. Recent findings include possible defects in the androgen receptor. The prostate may not even be the source of the symptoms. Pelvic pain also correlates with the neurotrophin nerve growth factor implicated in neurogenic inflammation and central sensitization. Finally, psychological stress may produce measurable biochemical changes and influence the other processes. The role of normal prostatic bacterial flora in inciting the inflammatory response has also been reconsidered.The symptoms of CP/CPPS appear to result from an interplay between psychological factors and dysfunction in the immune, neurological and endocrine systems.

This study was performed to determine whether nerve transfer immediately after spinal root transection would lead to bladder reinnervation in a canine model. In one animal, the left T12 intercostal nerve was mobilized, cut and attached to the severed ends of sacral roots inducing bladder contraction using a graft from the T11 intercostal nerve. On the right side and bilaterally in two other dogs, coccygeal roots innervating tail musculature were cut and attached to the severed bladder sacral roots (coccygeal nerve transfer [CG NT]). In four other dogs, bladder sacral roots were transected in the vertebral column, and the genitofemoral nerve was transferred within the abdomen to the pelvic nerve (genitofemoral nerve transfer [GF NT]). After 14 months for CG NT and 4.5 months for GF NT, electrical stimulation of the pelvic nerve induced bladder pressure and urethral fluid flow on the intercostal nerve transfer side, in each of the five CG NT sites and bilaterally in three of the four GF NT animals. Reinnervation was further shown by retrograde labeling of spinal cord neurons following fluorogold injections into the bladder wall and by histological examination of the root/nerve suture sites. In all CG NT animals, labeled neuronal cell bodies were located in ventral horns in lamina IX of coccygeal cord segments. In the three GF NT animals in which pelvic nerve stimulation induced bladder contraction, abundant labeled cell bodies were observed in lamina IX and lateral zona intermedia of upper lumbar cord. These results clearly demonstrate that bladder reinnervation can be accomplished by immediate nerve transfer of intercostal nerves or coccygeal spinal roots to severed bladder sacral roots, or by transfer of peripheral genitofemoral nerves (L1,2 origin) to pelvic nerves.

In rabbits the contractile response of the urinary bladder is only partially due to cholinergic innervation since atropine does not completely block neuron ally mediated contractions. In the human bladder this atropine resistance is controversial with some reporting atropine resistance in­ vitro while others have stated that the atropine resistance is also tetrodotoxin resistant. Results of the present investigation demonstrate that an atropine resistant, tetrodotoxin sensitive contraction can be evoked in some, but not all human bladder strips. Evidence accumulated over the past few decades indicates that this atropine resistant contraction may be mediated by ATP or a related purine compound. Studies presented herein are designed to develop a radioligand assay for this purinergic receptor. Initial studies indicated that the hydrolysis resistant ATP analog (3,1' methylene ATP offers several advantages over ATP as a potential radio ligand. It is only slowly hydrolyzed by endogenous ATPase and does not inhibit the hydrolysis of ATP indicating that it probably does not bind to the active sites of endogenous ATP hydrolyzing enzymes. In addition (3,1' methylene ATP is 10-100 fold more potent than ATP itself in stimulating contractions of the urinary bladder in-vitro. The radio ligand binding assay herein described can be used to quantitate the density of purinergic receptors, an essential step for determining the role of this system in urinary bladder function and dysfunction. Application of this assay could form the foundation for development of a new class of therapeutic agents for the treatment of urinary bladder dysfunction based on modulation ofthe purinergic nervous system. (J. Ural., 144: 176-181, 1990) The postganglionic nerve evoked contractile response of the urinary bladder of nearly all vertebrate species including man is only partially blocked by antimuscarinic agents such as atropine and scopolamine ' and is relatively unaffected by alpha and beta adrenergic or nicotinic antagonists.! The most likely explanation for this response is that in addition to acetylcho­ line, released by cholinergic nerves, another neurotransmitter released by non-adrenergic, non-cholinergic nerves contributes to the contraction. The presence of these neurons has been alluded to in the early literature by Langley2 and their existence has been suggested throughout the gastrointestinal tract as well as other organs including lung, trachea, esophagus, cardiovas­ cular system, and seminal vesicles.! In the human female blad­ der these nerves have been estimated to be responsible for 50% of the contractile response. 3 Evidence accumulated over the past few decades suggests that the neurotransmitter utilized by these neurons is most likely a purine nucleotide or analog and these nerves have therefore been coined "purinergic" by Burns­ tock" Functionally these neurons are those that produce an effect on the innevated tissue in the presence of both atropine and guanethidine. Although other explanations for the atropine resistant re­ sponse have been put forth including the presence of a barrier to the penetration of atropine, there is little evidence for them. Many substances other than ATP have been explored as can­ didates for mediating the atropine resistant contraction. These include catecholamines, 5-hydroxytryptamine, cyclic AMP, his­ tamine, prostaglandins, alanine, arginine, glycine, glutamic acid, gamma amino butyric acid, bradykinin and substance P.! These substances have been rejected as candidates by most workers since they a) were either inactive or did not mimic the nerve mediated response, b) specific antagonists for these sub

We reviewed clinical and histological findings in 55 patients with interstitial cystitis and 21 with voiding dysfunction secondary to other pathological conditions. Of our interstitial cystitis patients 36% would fail to meet the research definition proposed at a recent National Institutes of Health workshop. Detrusor mastocytosis was present in 64% of our interstitial cystitis patients compared to 80% of the noninterstitial cystitis group. There was no statistically significant difference in mean detrusor mast cell counts between interstitial cystitis and noninterstitial cystitis patients. Biopsies of 12 patients who did meet the proposed National Institutes of Health research definition were evaluated by immunohistochemical techniques. Early results are inconclusive.These studies indicate that interstitial cystitis is a complex disease whose diagnosis presently still must be made from a symptom complex rather than from objective histological criteria, including mastocytosis or the presence of an...

Defects in the urinary bladder urothelium may be an etiological factor in conditions such as recurrent urinary tract infection and interstitial cystitis. Initial studies have demonstrated that the colloidal dye trypan blue can be used as a marker for defects in the integrity of the mucosal epithelium. Male white New Zealand rabbits were separated into 4 groups: Control, in vivo overdistension (via saline infusion), intravesical acetone (20 ml 50% for 1 minute), and intravesical 50% DMSO (20 ml “RIMSO-50” for 30 minutes). Both intravesical instillations were followed by 3–20 ml saline washes. Immediately following acetone or DMSO, 20 ml 1% trypan blue in saline was instilled and left in the bladder for up to 1 hour. After overdistension, 1% trypan blue in saline was infused into the bladder at the same rate until the volume reached that previously defined as the overdistension volume. Control bladders were rinsed with 20 ml saline, twice, before being filled with 20 ml of the trypan blue solution. There was no evidence of trypan blue's staining of tissues in any of the control bladders. After DMSO treatment, there was usually only slight staining of the bladder surface urothelium and subjacent connective tissues. After acetone treatment, microscopic examination of bladder wall showed that nuclei were stained in all tissues of the wall (e.g., urothelium, connective tissue and smooth muscle). After acute frank overdistension, the bladder wall was deep blue throughout, very similar to the acetone-treated bladder. Microscopic analysis demonstrated that the smooth muscle elements were the most heavily stained. In summary 1) trypan blue does not penetrate the normal bladder mucosa, nor is any dye absorbed into the blood stream. 2) Frank overdistension of the bladder defeats the physical integrity of the urothelium as evidenced by penetration of the dye into the submucosal tissue, the peritoneal cavity, and bloodstream. 3) Where DMSO penetrates the urothelium itself, it appears to leave no permanent route by which other molecules can traverse the urothelium after it is removed. 4) Only after the integrity of the urothelial surface is disturbed are the cells of the urothelium susceptible to staining.

Objectifs La paraplegie est une pathologie pourvoyeuse des handicaps chroniques et severes incluant une dysfonction mictionnelle complexe. Les techniques de re-innervation vesicale par chirurgie de transposition radiculaire lombo-sacree pourraient permettre de recouvrer une fonction mictionnelle en cas de traumatisme medullaire complet. Evaluer l’efficacite d’une technique chirurgicale de re-innervation vesicale par anastomose radiculaire L7-S1 unilaterale dans un modele de chat paraplegique en T9-T10. Methodes Etude pilote chez 8 chats paraplegiques ayant subi une section medullaire complete en T9-T10. Une anastomose radiculaire unilaterale entre les racines ventrales motrices de L7 et S1 a ete realisee 1 mois plus tard pour 6 chats. Les 2 autres animaux representant le groupe temoin. Evaluation bilaterale realisee a 3, 5, 7 et 9 mois par stimulation cutanee electrique et manuelle du membre inferieur (Dermatome L7 ipsilateral), associee a une evaluation urodynamique couplee a un enregistrement electromyographique du sphincter uretral. Resultats A 9 mois de suivi, 33,3 % (n = 2) des chats ayant beneficie d’une chirurgie de re-innervation vesicale ont presente un flux urinaire declenche par une stimulation cutanee ipsilaterale. 66,7 % des chats (n = 4) ont aussi presente une augmentation de pression detrusorienne provoquee par stimulation cutanee. Aucun flux urinaire ou augmentation de pression detrusorienne n’a ete mis en evidence dans le groupe temoin ou apres stimulation du membre inferieur controlateral. Tous les chats etaient vivants a l’issu du suivi. Conclusion Une chirurgie de re-innervation vesicale par transposition radiculaire en infra-lesionnel est possible et peut induire un flux mictionnel apres stimulation cutanee. Ces resultats sont a confirmer par des etudes presentant un echantillon plus important, un suivi plus long et une experimentation d’autres type de techniques chirurgicales (bilaterale, anastomose supra-lesionnelle) avant d’envisager un transfert de cette technique en pratique clinique.

Aims Our previous studies showed that bladder hypertrophy shifts the muscarinic receptor subtype mediating contraction from M3 towards M2 along with increased M2 and decreased M3 protein concentration. We quantified mRNA for M1 through M5 receptors to determine whether the changes in M2 and M3 protein levels was due to changes in transcription. Methods Bladder hypertrophy was induced by bladder outlet obstruction (BOO), major pelvic ganglion electrocautery (DEN), and major pelvic ganglion decentralization (DEC). Bladder atrophy was induced by ureteral diversion (DIV). Additional groups included denervated and diverted (DEN-DIV), sham operated (SHAM), and normal (NOR) controls. Transcripts were quantified using a multiplex ribonuclease protection assay (RPA) and receptor protein density was determined by immunoprecipitation. Receptor transcripts were expressed per unit total RNA. Results Although all five receptor subtype transcripts were detected in all experimental groups, the densities of M1, M4, and M5 were much lower than for the M2 and M3 subtype. There were more M2 receptor transcripts than all the others, consistent with M2 protein determinations. M2 transcripts were significantly increased in DEN and BOO bladders. Surprisingly, M3 transcripts were also significantly increased in BOO. There was a significant correlation (r = 0.98, P

The muscarinic receptor subtype-activated signal transduction mechanisms mediating rat urinary bladder contraction are incompletely understood. M(3) mediates normal rat bladder contractions; however, the M(2) receptor subtype has a more dominant role in contractions of the hypertrophied bladder. Normal bladder muscle strips were exposed to inhibitors of enzymes thought to be involved in signal transduction in vitro followed by a single cumulative concentration-response curve to the muscarinic receptor agonist carbachol. The outcome measures were the maximal contraction, the potency of carbachol, and the affinity of the M(3) -selective antimuscarinic agent darifenacin for inhibition of contraction. Inhibition of phosphoinositide-specific phospholipase C (PI-PLC) with 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH(3)) reduces carbachol potency and reduces darifenacin affinity, whereas inhibition of phosphatidyl choline-specific phospholipase C (PC-PLC) with O-tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt (D609) attenuates the carbachol maximal contraction. Inhibition of rho kinase with (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632) reduces carbachol potency and increases darifenacin affinity. Inhibition of rho kinase, protein kinase A (PKA), and protein kinase G (PKG) with 1-(5-isoquinolinesulfonyl)-homopiperazine.HCl (HA-1077) reduces the carbachol maximal contraction, carbachol potency, and darifenacin affinity. Inhibition of protein kinase C (PKC) with chelerythrine increases darifenacin affinity, whereas inhibition of rho kinase, PKA, PKG, and PKC with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine.2HCl (H7) reduces the carbachol maximum and carbachol potency while increasing darifenacin affinity. Inhibition of rho kinase, PKA, and PKG with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H89) reduces carbachol maximum and carbachol potency. Both the M(2) and the M(3) receptor subtype are involved in normal rat bladder contractions. The M(3)subtype seems to mediate contraction by activation of PI-PLC, PC-PLC, and PKA, whereas the M(2) signal transduction cascade may include activation of rho kinase, PKC, and an additional contractile signal transduction mechanism independent of rho kinase or PKC.

Purpose: We provide an overview of the medical literature supporting the combined use of muscarinic and α-adrenergic antagonist therapy for the treatment of voiding dysfunction. Materials and Methods: The MEDLINE database (1966 to 2004) of the United States National Library of Medicine was searched for pertinent studies. Results: Although the mechanism of action of α-adrenergic antagonist therapy for voiding dysfunction has traditionally been assumed to be relaxation of the periurethral, prostatic and bladder neck smooth muscle, substantial evidence supports action at extraprostatic sites involved in micturition, including the bladder dome smooth muscle, peripheral ganglia, spinal cord and brain. Likewise the mechanism of action of anticholinergic therapy has been traditionally assumed to be inhibition of the M 3 muscarinic receptor subtypes that mediate normal bladder contractions. However, M 2 receptor mediates hypertrophied bladder contractions and there is evidence for an M 2 component to the suprasacral control of voiding. Conclusions: Based on the physiology of α-adrenergic and muscarinic receptors the inhibition of each one would be expected to be more beneficial than that of either alone because they would work on 2 components of detrusor function. Patients who would likely benefit from this combination therapy are men with lower urinary tract symptoms, women with urgency/frequency syndrome (overactive bladder), patients with uninhibited bladder contractions due to neurogenic bladder, and patients with pelvic pain and voiding symptoms, ie interstitial cystitis and chronic prostatitis/chronic pelvic pain syndrome.

OBJECTIVES Several reports have demonstrated the effects of obesity on prostate cancer. Also several reports have linked expression of vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (FGF-2) to prostate cancer aggressiveness. The objective of this study was to determine whether a difference exists between lean and obese Zucker rat sera on proliferation prostate cancer cell lines, as well as to examine the differences in FGF-2 and VEGF concentrations. METHODS Ten-week-old female obese and lean Zucker rat sera were subjected to charcoal stripping and tested for the proliferation of human LNCaP and rat AT3B-1 prostate cancer cells. An acetonitrile extract of the charcoal used to strip the sera was also tested for mitogenicity. VEGF and FGF-2 concentrations were determined by enzyme-linked immunosorbent assay. RESULTS Both unstripped and charcoal-stripped obese rat sera had a greater mitogenic effect than did the lean sera on the LNCaP cell line. Charcoal stripping of both obese and lean sera reduced the mitogenic effect on the AT3B-1 cell line. The acetonitrile extract of the charcoal used to strip the sera was unable to recover this proliferative effect. The concentration of VEGF was greater in the obese serum than in the lean serum, and charcoal stripping reduced the concentrations of both FGF-2 and VEGF. CONCLUSIONS The finding of greater VEGF in obese rat sera, as well as greater mitogenic responses on human prostate cancer cells in vitro, suggests this as one of the many possible mechanisms involved in obesity-related prostate cancer biology.

Bladder muscle specimens from seven patients with neurogenic bladder dysfunction were analyzed to determine whether the muscarinic receptor subtype mediating contraction shifts from M3 to the M2 subtype as found in the denervated, hypertrophied rat bladder. Seven bladder specimens were analyzed from six female and one male patients. Six of the patients had traumatic cervical spinal cord injuries (C4-C7), and the other patient had an L1 congenital myelomeningocele. This was compared with results from bladder specimens obtained from eight organ transplant donors. The affinities of three subtype-selective muscarinic receptor antagonists for inhibition of carbachol-induced contractions were determined. The affinity of the M3 selective antagonists darifenacin or p-fluoro-hexahydrosiladifenadol (p-F-HHSiD) was determined in six of the seven spinal injury patient specimens. The affinity was consistent with M2-mediated contractions in four of these six specimens, intermediate between M2 and M3 in one specimen, and within the M3 range in one specimen. The other specimen, tested only with the M2 selective antagonist methoctramine, showed an M3 affinity. In the organ donors, the affinity of p-F-HHSiD was within the M2 range for six of seven specimens, whereas the affinity of darifenacin was within the M3 range for five of six and intermediate between M2 and M3 for the other specimen tested. The affinity of methoctramine in both organ donor specimens tested was within the M3 range. Whereas normal detrusor contractions are mediated by the M3 receptor subtype, in patients with neurogenic bladder dysfunction as well as certain organ transplant donors, contractions can be mediated by the M2 muscarinic receptor subtype.

M3 muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M2 receptors participate in contraction because M3-selective antagonists [ para-fluoro-hexahydro-sila-diphenidol ( p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M2-selective antagonist methoctramine in the denervated bladder is consistent with M3 receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M2 receptor and one by the M3 receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine: p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M2 and M3 receptors can induce contraction. In the denervated bladder, the M2 and the M3 receptors interact in a facilitatory manner to mediate contraction.

The following discussion on the physiology of the esophagus includes commentaries on the function of the muscularis mucosa and submucosa as a mechanical antireflux barrier in the esophagus; the different mechanisms of neurological control in the esophageal striated and smooth muscle; new insights from animal models into the neurotransmitters mediating lower esophageal sphincter (LES) relaxation, peristalsis in the esophageal body (EB), and motility of esophageal smooth muscle; differentiation between in vitro properties of the lower esophageal circular muscle, clasp muscle, and sling fibers; alterations in the relationship between pharyngeal contraction and relaxation of the upper esophageal sphincter (UES) in patients with dysphagia; the mechanical relationships between anterior hyoid movement, the extent of upper esophageal opening, and aspiration; the application of fluoroscopy and manometry with biomechanics to define the stages of UES opening; and nonpharmacological approaches to alter the gastroesophageal junction (GEJ).

In the rabbit bladder, pregnancy and prolonged bladder contractions decrease both muscarinic receptor density and contractile response, whereas newborns show enhanced muscarinic contractile response. Although the M(2) receptor predominates in rabbit bladder, we and others have shown that the affinity of a series of subtype selective muscarinic antagonists for inhibition of muscarinic agonist-induced contractions is most consistent with the pharmacologically defined M(3) receptor directly mediating smooth muscle contraction. Bladders from fetal rabbits, gravid rabbits, and male rabbits exposed to 4 hr of induced spontaneous contractions were used to determine whether changes in receptor density and contractility are due to a selective decrease in either the M(2) or M(3) muscarinic receptor subtype. To determine organ specificity, the heart and uterus were also studied. Gravid rabbits of 3 weeks' gestation and their fetal rabbits were studied. In male rabbits, bladder contractions were induced for 4 hr by ligating the catheterized penis at its base. Muscarinic receptor density and subtype distribution were determined by radioligand binding and immunoprecipitation. Receptor density was 24% lower in gravid bladder body, unchanged in gravid bladder base, 54% lower in gravid uterus, 115% higher in fetal bladders, and 34% lower after induced bladder contractions. Immunoprecipitation showed greater M(2) receptors than M(3) in all tissues studied, whereas M(l) and M(4) receptors were undetectable. There was no difference from control in the ratio of M(2) to M(3) receptor in any tissues except that a greater proportion of M(3) receptors was found in male vs. female bladders. Changes in contractile response to cholinergic stimulation in the gravid, fetal, and experimental detrusor instability model are associated with changes in total receptor density and not solely with changes in the M(3) receptor subtype that mediates bladder smooth muscle contraction. Neurourol. Urodynam. 18:511-520, 1999.

In vitro bladder contractions in response to cumulative carbachol doses were measured in the presence of selective muscarinic antagonists from rats that had their major pelvic ganglion bilaterally removed. Denervation induced both hypertrophy and a supersensitivity of the bladders to agonist. The affinities in control bladders for antagonism of carbachol-induced contractions were consistent with M3-mediated contractions. Affinities in denervated bladders for 4-diphenlacetoxy- N-methylpiperidine methiodide (8.5) and p-fluoro hexahydrosilodifenidol (6.6) were consistent with M2-mediated contractions, although the methoctramine affinity (6.5) was consistent with M3-mediated contractions. Subtype-selective immunoprecipitation of muscarinic receptors revealed a 50% increase in total and a 60% increase in M2 receptor density with no change in M3 receptor density in denervated bladders compared with normal or sham-operated controls. 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 mediates smooth muscle contraction in the denervated bladder.

Subtype-selective muscarinic antagonists effects on carbachol-induced and electric field-stimulated contractility of rat bladder were compared in vitro. Schild plot analysis of cumulative carbachol dose-response curves in the presence of antagonists was consistent with M3-mediated bladder contractions. However, nerve-evoked contractions were inhibited 15% at 30 Hz ( P < 0.01) by 10 nM pirenzepine (M1-selective antagonist), whereas 10 nM methoctramine (M2-selective antagonist) increased these contractions by 17% at 30 Hz ( P < 0.01). Identical doses had no effect on carbachol-induced contractions, indicating prejunctional M1 facilitory and M2 inhibitory receptors. m1 Receptors could not be identified by subtype-selective antibodies, nor could the m1 transcript be identified by Northern hybridization. However, m1, m2, m3, and m4 transcripts were identified in rat bladder using the reverse transcriptase-polymerase chain reaction, providing support for the existence of the m1 subtype. In conclusion, strong evidence is provided for the existence of prejunctional M1 facilitory and M2 inhibitory and postjunctional M3 receptors modulating contractility in the rat urinary bladder.

The prostate gland from several animal species contains variable levels of muscarinic subtypes, but only the human prostate expresses significant levels of the m1 subtype. We studied muscarinic receptor activity in human benign prostatic hypertrophy (BPH) as well as several cell lines derived from prostate cancer. The BPH we studied expresses approximately 75% of the m1 receptor and undetectable levels of the other receptor subtypes whereas PC3 cells express only the m3 receptor subtype. DU145 and LnCaP cells express approximately equal levels of m1 and m3 receptor subtypes. Only the PC3 cells responded to carbachol with an increase in turnover of polyphosphoinositides, and none of the cell lines responded with effects on cAMP metabolism. Co-precipitation of receptors with heterotrimeric guanine nucleotide-binding regulatory proteins demonstrated interactions of the m1 receptors with Gi, Gq and G16 in BPH tissue and of the m1 and m3 receptors with Gi, Gq and G12 in PC3 and DU145 cells. Mitogen activated protein kinase (ERK) activity was seen in response to carbachol in PC3 and DU145 but not LnCaP cells. Finally, carbachol promoted cell proliferation in all three cell lines. Thus, there appears to be no consistent relationship between ERK activity, cell proliferation, and the subtype mediating the proliferative response, amongst these prostate cancer cell lines.

In the rabbit bladder, pregnancy has been shown to induce a significant decrease in both muscarinic receptor density and response to muscarinic stimulation. Neonatal rabbit bladders have a high muscarinic receptor density and contractile response to bethanechol stimulation. The bladders from 7 gravid rabbits, 7 age-matched virgin controls, and 32 fetal rabbits of 3 week gestation were studied. Compared to control tissue, filtration binding demonstrated receptor density to be 24.3% lower in gravid bladder dome, 41.2% lower in gravid bladder base, and 114.8% higher in fetal bladders. While total receptor density was not different from control in gravid heart, fetal hearts showed a 2.5 fold increased receptor density. There was also a 61% reduction in muscarinic receptor density in the gravid uterus. Immunoprecipitation assays using muscarinic receptor subtype specific antisera were used to measure the relative levels of m1, m2, m3 and m4 receptors. The m2 receptor was the predominant subtype in the bladder and uterus, and the only subtype detected in rabbit heart. The m3 receptor protein was also present, but in lower levels in the bladder and uterus. The m1 and m4 receptors were not detected in any of the tissues studied. Furthermore, the relative percent of each receptor did not statistically change for the gravid or fetal rabbit bladder, uterus, or heart, when compared to its control. Differences in the contractile response to cholinergic stimulation of the gravid bladder and uterus, and of the fetal bladder then, can be attributed to changes in muscarinic receptor density and not to changes in receptor subtype.

Background We sought to determine how the individual components of the distal esophagus and proximal stomach form the gastroesophageal junction high-pressure zone (GEJHPZ) antireflux barrier. Methods An endoscopic ultrasound/manometry catheter was pulled through the proximal stomach and distal esophagus in 20 normal subjects. The axial length and width of individual structures on endoscopic ultrasound were measured. The anatomic orientation of gastroesophageal junction (GEJ) components was examined in two organ donor specimens using micro-computed tomography (micro-CT). Key Results The three distinct structures identified within the GEJHPZ, from distal to proximal, were as follows: the gastric clasp and sling muscle fiber complex, crural diaphragm, and lower esophageal circular smooth muscle fibers (LEC). The LEC was statistically significantly thicker than adjacent esophageal muscles. These structures were associated with three pressure peaks. The pressure peak produced by the clasp/sling fiber complex often overlapped with the pressure peak from the crural diaphragm. The most proximal peak, associated with the LEC, was significantly greater and bimodal in nine of 20 subjects. This bimodal LEC pressure peak correlated with two areas of thickened muscle observed with ultrasound. Micro-CT of GEJ from organ donors confirmed the two areas of thickened muscle. Conclusions & Inferences Three distinct anatomic structures, the clasp and sling muscle fibers, crural diaphragm, and LEC combine to form the antireflux barrier of the proximal stomach and distal esophagus. The clasp and sling muscle fibers combine with the crural diaphragm to form a distal pressure profile. The more proximal LEC has a bimodal pressure profile in some patients.

ents of the HPZ. From prior anatomic studies using simultaneous high resolution manometry and high frequency ultrasound with pharmacologic manipulation, these HPZ components represent a proximal lower esophageal circular muscle sphincter (LEC), a mid crural diaphragm (CD) and a distal gastric sling and clasp muscle fiber complex. The average pressure in each of these components was 24.7+/10.7mmHg in the LEC, 33.2+/-17.2mmHg in the crural diaphragm and 15.83+/-8.03 in the clasp sling muscle fiber complex. The width of the LEC, crural diaphragm and gastric sling clasp fiber complex was 1.01 +/0.25cm, 1.29 +/0.37cm and 0.76 +/0.3 cm respectively. The total length of the high-pressure zone was 4.08 +/0.61 cm. The distance between the peak pressure within the gastric sling and clasp muscle fiber complex and the crural diaphragm was 1.7 +/0.53cm and the distance between the crural diaphragm and the LEC was 1.4 +/-0.48. CONCLUSIONUsing high spatial resolution manometry within the high-pressure zone of the gastroesophageal junction, it is possible to resolve the high-pressure zone into 3 distinct and separate components. These components represent the gastric sling and clasp fiber muscle complex in the proximal stomach, the crural diaphragm measured in the distal esophagus and the LEC muscle proximal to the CD. The average pressure, width and distance between these components were measured.

The present study used radioligand binding and in vitro contractility experiments to identify and characterize a peripheral-type benzodiazepine receptor PBR in rabbit urinary bladder. [3H]PK11195 bound to bladder membranes with high-affinity and density (Kd = 5.2 nM., Bmax = 268 fmol./mg. protein), indicating the presence of a PBR. [3H]flunitrazepam bound with high-affinity and density (Kd = 1.2 nM., Bmax = 48 fmol./mg. protein). The rank order potency of various benzodiazepines and isoquinoline carboxamides in displacing the binding of [3H]PK11195 was Ro5-4864 > diazepam = flunitrazepam » Rol5-1788 = clonazepam. Ro5-4864 and PK11195 inhibited nerve-evoked contractions in a concentration-dependent manner (IC50 = 42 μΜ. and 56 μΜ., respectively). Carbachol- and KCl-induced contractions were also inhibited by Ro5-4864 and PK11195. KCl-induced contractions were inhibited to a greater extent than carbachol-induced or field-stimulated contractions with all the drugs tested. Both Ro5-4864 and PK11195 significantly increased the ED50 for calcium-induced contractions following a cholinergic stimulus compared with control. These data demonstrate the presence of a PBR in urinary bladder capable of altering contractility in vitro through modulation of calcium activity.

Background: We recently showed that the gastric sling/clasp muscle complex does not contribute to the high pressure zone in GERD patients indicating a role in pathophysiology. Aim: We determined the cholinergic contractile response in clasp, sling and lower esophageal circular (LEC) fibers. Methods: Stomach and esophagi were obtained from 17 human transplant donors: 11 with no GERD history, 4 with probable GERD (proton pump inhibitor use) and 2 with definite GERD (Barrett's esophagitis). The contractile response to increasing carbachol concentrations was determined. Muscarinic receptor density was measured by subtype selective immunoprecipitation of [3]H-QNB binding. Results: Clasp and LEC fibers from definite GERD have decreased maximal contractile response. Contractility is increased in sling fibers of both definite and probable GERD. Concentrations of carbachol higher than 100 μM induce relaxations that are decreased in clasp fibers of probable and definite GERD. Relaxations are greater in sling fibers from definite GERD and higher in LEC from probable and definite GERD. Total and M2 receptor density is statistically lower in the GERD than non-GERD specimens in both clasp and sling fibers and M3 density is statistically lower in GERD than non-GERD clasp fibers. Conclusion: This suggest that a myogenic defect in clasp fibers may be involved in GERD pathophysiology in the organ donors with Barrett's esophagitis. The greater contractile response in the sling fibers from GERD donors suggest a possible compensatory mechanism for the lack of contractility of the clasp muscle fibers.

We have previously shown that a dialyzed, lyophilized saline extract from bovine urothelium can restore the antiadherence activity of the rabbit bladder following mucin removal with 50% acetone. This report describes results of initial purification of antiadherence factor(s) from bovine bladder mucin and describes results of biochemical analysis in an attempt to elucidate possible mechanism of this antiadherence activity. Separation performed by gel filtration (Spectra gel AcA 34, 20-350 KD range) results in three fractions. Only the low molecular weight fraction had a statistically significant inhibitory effect on bacterial adherence to the mucin deficient rabbit bladder. After overnight dialysis against running deionized water and lyophilization, the crude extract contained 60% protein while gel filtration fractions 1-3 contained 35%, 90% and 15% by weight respectively. The first fraction (apparent high molecular weight, greater than 350 kD) did not appear to enter SDS polyacrylamide electrophoresis gels (SDS-PAGE, 3-12%) or agarose gels (0.5%) to any significant extent. In the fractions that displayed antiadherence activity (the crude extract, fractions 2 and 3) SDS-PAGE bands were seen corresponding to an apparent molecular weight of 78 kD in addition to bands co-migrating with bovine serum albumin (BSA). BSA itself slightly increases bacterial adherence in this model. Most of the albumin of the crude extract was found in the second fraction (60%). On the other hand most of the sulfate and sugar of the crude extract was found in the third, low molecular weight fraction. Since sulfated polysaccharides such as heparin and dextran sulfate are very effective antiadherence agents in this rabbit bladder model, it is conceivable that the sulfated sugar content of the third fraction is responsible for its antiadherence effect on the mucin deficient rabbit bladder.

The Bmax for [3H]QNB binding in the bladders of alcohol preferring (AA) rats was only approximately 60% of that in the alcohol non-preferring (ANA) rats. No significant change in Bmax for [3H]QNB binding in bladder was observed between alcohol insensitive (AT) and alcohol sensitive (ANT) rats. No significant change in Kd for [3H]QNB binding in bladder was observed between the four different rat lines studied. Therefore, alcohol preference but not sensitivity is associated with a decrease in muscarinic receptor density in the rat bladder. Because all of the rats used in this study were ethanol-naive, the decrease in muscarinic receptor density in the bladders of alcohol preferring rats is associated with genetic factors inherent to this rat line. Further studies are needed to determine if these observations are tissue specific or specific to the m2 subtype, which predominates in the rat bladder.

This study determined how closely the contractile physiology of the pig gastroesophageal junction follows the human. We obtained human tissue from organ transplant donors and pig tissue from a slaughterhouse. Total, M-2 and M-3 receptor density was determined by subtype specific immunoprecipitation. Total and M-2 are higher in pig than human. M-3 receptors are 2 fold higher in human than pig sling and over 2 fold lower in human than pig clasp fibers. The methoctramine and darifenacin potency to inhibit bethanechol contractions, calculated by classic Schild analysis, indicates that both M-2 and M-3 receptors cause contraction which violates the assumption of one receptor causing the effect. An analysis method relating dual occupation of M-2 andM-3 receptors to the contractile response was developed based on the published Ka values of M-2 and M-3 receptors for bethanechol of 170 μM and 110 μM respectively, and mass-action binding which, at equilibrium, gives receptor occupation = [A][R] / ([A] + Ka), where [A] denotes the agonist concentration, [R] is the receptor concentration and Ka is the agonist dissociation constant (reciprocal of affinity). Three dimensional plots for M-2 and M-3 occupation and contractile response are shown in the figure. Although the M-3 receptor subtype density is different between human and pig, the physiology of the contractile response is similar. This indicates that the pig may be a good model for human gastroesophageal junction physiology. Muscarinic receptor density (fMol/mg solubile protein)

The circular muscle fibers at the end of the esophagus are traditionally considered the lower esophageal sphincter. However, the first barrier to gastric reflux is actually the clasp/sling fiber complex of the stomach. Relaxation mediates transient lower esophageal sphincter relaxation underlying the pathophysiology of gastroesophageal reflux disease (GERD). This study determined the pharmacologic specificity of the nicotinic receptor mediated relaxation of pre-contracted strips of human and porcine clasp muscle fibers. Human specimens were obtained from organ transplant donors. Clasp fiber muscle strips were exposed to increasing carbachol concentrations (human) or acetylcholine with physostigmine to block cholinesterase (pig). At concentrations higher than 30 uM, abrupt relaxations were produced. After 60 minutes of repeated washing, strips were exposed to various ganglionic and neuromuscular nicotinic receptor antagonists for 30 minutes then rechallenged with cholinergic agonists. Results, as a percentage of relaxation in time control strips are shown in the figure below. The neuromuscular blockers d-tubocurarine, decamethonium (human) and pancuronium (pig) inhibited relaxation whereas the ganglionic blocker hexamethonium (human) and the alpha7 selective antagonist methyllycaconitine did not. Other ganglionic blockers such as MG624, NDNI and TMPH blocked these relaxations whereas mecamylamine was only partially effective in human tissue. These results indicate that the pharmacology of the nicotinic receptor mediating relaxation of the gastric clasp fibers may be unique and a potential target for development of selective agents for treatment of GERD.