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Background Proper function of the gastro-esophageal high pressure zone is essential for the integrity of the antireflux barrier. Mechanisms include tonic contractions and the decreased tone during transient lower esophageal sphincter relaxations. Methods We characterized the pharmacology of nicotinic receptors mediating relaxations of the human upper gastric sphincter (clasp and sling fibers) using currently available subtype selective nicotinic antagonists in tissue from organ transplant donors. Donors with either a history of gastro-esophageal reflux disease or histologic evidence of Barrett's esophagus were excluded. Clasp and sling muscle fiber strips were used for one of three paradigms. For paradigm 1, each strip was exposed to carbachol, washed, exposed to nicotinic antagonists then re-exposed to carbachol. In paradigm 2, strips were exposed to a near maximally effective bethanechol concentration then nicotine was added. Strips then were washed, exposed to nicotinic antagonists then re-exposed to bethanechol followed by nicotine. In paradigm 3, strips were exposed to bethanechol then choline or cytisine. Key Results 100 μM methyllycaconitine has no inhibitory effects on relaxations, eliminating homomeric α7 subtypes. Subtypes composed of α4β2 subunits are also eliminated because choline acts as an agonist and dihydro-beta-erythroidine is ineffective. Conclusions & Inferences Because mecamylamine blocks the relaxations and both choline and cytisine act as agonists in both clasp and sling fibers, the nicotinic receptor subtypes responsible for these relaxations could be composed of α3β4β2, α2β4, or α4β4 subunits.

The following discussion of the esophagogastric junctions includes commentaries on the three component structures of the sphincteric segment between the stomach and the esophagus; the pressure contributions from the three sphincteric components in normal subjects and in gastroesophageal reflux (GERD) patients; the mechanism of action of endoscopic plication to determine the underlying pathophysiology of GERD; and in vitro muscle strip studies of defects within the gastroesophageal sphincteric segment potentially leading to GERD.

Smooth muscle contains multiple muscarinic receptor subtypes, including M2 and M3. M2 receptors outnumber M3 receptors. Based on the potency of subtype selective anticholinergics, contraction is mediated by the M3 subtype. However, results from knockout (KO) mice show that the M2 receptor mediates approximately 45% of the contractile response produced by the M3 receptor. The traditional theory of one receptor mediating a response does not allow assessment of interactions between receptors when more than one receptor participates in a response. Our study was performed using a novel analysis method based on dual receptor occupancy to determine how M2 and M3 receptor subtypes interact to mediate contraction in mouse stomach. Cumulative carbachol concentration contractile responses were determined for wild-type, M2-KO, and M3-KO stomach body smooth muscle. Using affinity constants for carbachol at M2 and M3 cholinergic receptors, the concentration values were converted to fractional receptor occupation. The resulting occupation-effect relations showed maximum effects for the M2 and M3 subtypes, respectively. These occupation-effect relations allow determination of the additive (expected) isobole based on this dual occupancy, thereby providing a curve (mathematically derived) for comparison against the experimentally derived value in wild type. The actual values determined experimentally in the wild type were not statistically significantly different from that predicted by the isobole. This confirms that the interaction between these mutually occupied receptors is additive. The new method of analysis also expands the traditional Schild theory that was based on a single receptor type to which the agonist and antagonist bind.

The normal physiological contraction of the urinary bladder, which is required for voiding, is predominantly mediated by muscarinic receptors, primarily the M-3 subtype, with the M-2 subtype providing a secondary backup role. Bladder relaxation, which is required for urine storage, is mediated by beta-adrenoceptors, in most species involving a strong beta(3)-component. An excessive stimulation of contraction or a reduced relaxation of the detrusor smooth muscle during the storage phase of the micturition cycle may contribute to bladder dysfunction known as the overactive bladder. Therefore, interference with the signal transduction of these receptors may be a viable approach to develop drugs for the treatment of overactive bladder. The prototypical signaling pathway of M-3 receptors is activation of phospholipase C (PLC), and this pathway is also activated in the bladder. Nevertheless, PLC apparently contributes only in a very minor way to bladder contraction. Rather, muscarinic-receptor-mediated bladder contraction involves voltage-operated Ca2+ channels and Rho kinase. The prototypical signaling pathway of beta-adrenoceptors is an activation of adenylyl cyclase with the subsequent formation of cAMP. Nevertheless, cAMP apparently contributes in a minor way only to beta-adrenoceptor-mediated bladder relaxation. BKCa channels may play a greater role in beta-adrenoceptor-mediated bladder relaxation. We conclude that apart from muscarinic receptor antagonists and beta-adrenoceptor agonists, inhibitors of Rho kinase and activators of BKCa channels may have potential to treat an overactive bladder

Muscarinic acetylcholine receptors, particularly M 3 receptors, are physiologically the most important mechanism to induced urinary bladder smooth muscle contraction. Their prototypical signaling response is a stimulation of phospholipase C (PLC), and this also has been shown in the urinary bladder. Nevertheless, it has remained controversial whether PLC signaling mediates bladder contraction induced by muscarinic receptor agonists. Studies in favor and against a role for PLC differed in their experimental protocol (single versus repeated concentration-response curves within a single preparation) and in the PLC inhibitors that have been used. We have now tested whether previous differential conclusions regarding a role for PLC are related to inhibitors and/or experimental protocols. In a single curve protocol, U-73,122 [1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1 H -pyrrole-2,5-dione] did not attenuate carbachol responses. In a repeated curve protocol, ET-18-OCH 3 (1- O -octadecyl-2- O -methyl- sn -glycero-3-phosphorylcholine) lacked significant inhibition relative to vehicle time controls. In contrast, D609 ( O -tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt) depressed maximal carbachol effects but also nonspecifically inhibited contraction induced by KCl. Neomycin did not affect the carbachol-induced rat urinary bladder contraction. We conclude that previously reported differences relate to the use of inhibitors rather than experimental protocols and that the overall data do not support a role for PLC in M 3 muscarinic receptor-mediated rat bladder contraction.

Although the 'purinergic nerve hypothesis' proposed by Burnstock in the early 1970s was met with considerable skepticism, it is now accepted that certain neurons use a purine nucleotide or nucleoside such as ATP or adenosine as a neurotransmitter. Likewise, early studies indicated that the human bladder is devoid of purinergic nerves mediating contraction; however, later studies demonstrated that purinergic nerve-mediated bladder contraction is increased in pathologic conditions such as interstitial cystitis. Cloning and sequencing studies have revealed four subtypes of adenosine receptors and eight subtypes of P2Y receptors, all of which are G-protein-coupled receptors. There are no reports of the cellular location of these receptors in the human bladder. P2X receptors are ligand-gated ion channels, and seven subunits have been cloned and sequenced. Immunohistochemical studies have determined that P2X(1,2,4) subunits are on detrusor-muscle cells, P2X(1-3,5) subunits are on bladder nerves and P2X(2,3,5) subunits are on bladder urothelial cells. Development of purinergic antagonist drugs with selectivity for P2X(1) receptors on detrusor muscle cells might be useful for treatment of detrusor overactivity. Antagonists with selectivity for P2X(3) receptors on bladder sensory nerves might be clinically beneficial for treatment of urinary urgency, and perhaps chronic pelvic pain.

Introduction : This study examines hypotheses that BDL induces increased guinea pig gallbladder smooth muscle PGE 2 release by up-regulation of COX-2. Methods : BDL, Sham and Control Hartley guinea pig gallbladders were placed in cell culture, grown to confluence and underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacylin Synthase, actin, caldesmon, vinculin, meta-vinculin and tropomyosin and were assayed for basal release of 6-keto- PGF 1 α , PGE 2 and TxB 2 by EIA. Results : BDL did not alter content of smooth muscle cytoskeletal proteins. BDL for 48 h increased smooth muscle cell release of PGE 2 and 6-keto- PGF 1 α by 3-fold or more when compared to the Control and Sham groups. Western Blot analysis showed increased content of COX-2 in the BDL group. Conclusions : BDL for 48 h markedly increased endogenous guinea pig smooth muscle cell PG release, which was due to increased COX-2 synthesis.

Major pelvic ganglion electrocautery (MPGE) and spinal cord injury in the rat induce bladder hypertrophy and a change in muscarinic receptor subtypes mediating bladder contraction from predominantly M3 to a combination of M2 and M3. To determine whether this is a result of bladder hypertrophy or denervation, we studied the following groups: sham-operated controls, urinary diversion (DIV), MPGE together with urinary diversion (DIV-DEN), bilateral MPGE (DEN), bladder outlet obstruction (BOO), and MPG decentralization (MPGDEC). The degree of bladder denervation was determined by the maximal carbachol response normalized to the response to electric field stimulation. Receptor subtype density was determined by immunoprecipitation. The affinity of subtype-selective muscarinic antagonists for inhibition of carbachol-induced contractions was used to determine the subtype-mediating contraction. DEN, MPG-DEC, and BOO bladders were hypertrophic whereas DIV bladders were atrophic compared with sham operated. Bladder contraction in sham-operated, DIV, and DIV-DEN was mediated by the M3 receptor subtype, whereas the M2 subtype participated in contraction in the DEN, MPG-DEC, and BOO groups. The hypertrophied bladders had an increase in total and M2 receptor density while all experimental groups showed a reduction in M3 receptor density. Thus bladder hypertrophy, independent from bladder denervation, causes a shift in the muscarinic receptor subtype mediating bladder contraction from M3 toward M2.

Purpose: To characterize a guinea pig behavior model of bladder pain due to intravesical antigen infusion and to determine the role of neurokinin receptor subtypes in mediating this behavior. Materials and Methods: The influence of subtype-selective neurokinin receptor antagonists on increased abdominal licking behavior in response to intravesical antigen infusion in guinea pigs immunized with ovalbumin (OA) was determined. Results: Intravesical OA infusion for 30 minutes induced a significantly greater frequency (about 3-fold) of abdominal licking behavior than during either the 30 minutes pre-challenge or post challenge saline infusions. Treatment with IP capsaicin 7 to 10 days before OA challenge abolished the intravesical antigen-induced behavior. IP injection of the NK1 receptor antagonist CP 99994 (10 mg./kg. or 30 mg./kg.), 30 minutes pretreatment, inhibited the increase in the average number of abdominal licks during antigen infusion. The 30 mg./kg., but not the 10 mg./kg. dose increased the percent of animals showing antinociceptive activity (defined as 4 or less abdominal licks during the antigen infusion). The NK2 receptor antagonist SR 48968 reduced the antigen-induced abdominal licking behavior at IP doses of 3 and 10 mg./kg. but was ineffective at 1 mg./kg. The NK3 receptor antagonist SB 235375 (30 mg./kg., IP) did not reduce this behavior. Conclusions: These results suggest a role for activation of NK1 and NK2, but not NK3 receptors, by tachykinins released from capsaicin-sensitive nerves, in the increased abdominal licking behavior response of guinea pigs to intravesical antigen infusion.

Continuous measurements were made of bladder blood flow by laser Doppler flowmetry in anesthetized dogs during bladder filling and emptying. In both mucosa and muscle, perfusion was inversely proportional to intravesical pressure. There was significantly greater perfusion in the bladder mucosa of males than females at baseline and up to 10 cm water filling pressure but not in the muscle. Intra-arterial infusion of the nitric oxide synthase inhibitor N G-nitro-l-arginine produced a significant decrease in resting bladder perfusion in the mucosa only, with no differences seen in the response to intravesical pressure. Intra-arterial infusion ofl-arginine produced a significant increase in the level of perfusion in the mucosa seen immediately after the bladder was drained. No changes were observed in muscle perfusion afterl-arginine. These results suggest that the perfusion of the bladder mucosa differs by gender and is regulated differently than the bladder muscle, possibly related to the different function of the two layers.

Our previous data indicate that M3 muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by selective alkylation of M3 receptors with 4-DAMP mustard and suggest that the M2 receptor subtype may be involved in inhibition of beta-adrenergic receptor induced relaxation, therefore, allowing recontraction. Alkylation resulted in 85% of M3 receptors and 65% of M2 receptors unable to bind radioligand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and direct carbachol contractions were inhibited by antagonists with affinities consistent with M3 receptor mediated contraction. In contrast, the affinities of antagonists for inhibition of carbachol induced recontractions following isoproterenol stimulated relaxation in the presence of 90 mM KCl, indicated a contractile function for the M2 receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role for the M2 subtype in bladder smooth muscle contraction.

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.

The purpose of this study was to characterize the muscarinic receptor subtypes in the individual lobes of the rat prostate. Immunoprecipitation was performed on homogenates of these 3 lobes using antibodies to the m1-m4 muscarinic receptor subtypes. Reverse transcriptase polymerase chain reaction assays (RT-PCR) were also performed using primers specific for each of the five muscarinic receptor subtypes (m1-m5). The susceptibility of the receptors to degradation by endogenous prostate proteases was assessed by mixing rat ventral prostate with rat heart (m2) and rat parotid (m3) prior to immunoprecipitation. In the ventral lobe, transcripts for the m1-m4 subtypes were amplified whereas in the dorsal and lateral lobes only the m2 and m3 sets of primers amplified PCR products of the predicted size. Immunoprecipitation of the ventral lobe resulted in predominantly m3 receptors, while the majority of receptors immunoprecipitated from lateral and dorsal lobes were the m2 subtype. The m3 muscarinic subtype was apparently susceptible to degradation by prostate proteases whereas the m2 subtype was not. These results demonstrate a regional distribution in the subtypes of muscarinic receptors in the rat prostate, and a greater susceptibility of the m3 receptor to degradation during immunoprecipitation than the m2 subtype.

Background Increased nicotinic receptor mediated relaxation in the gastroesophageal antireflux barrier may be involved in the pathophysiology of reflux. This study is designed to determine whether the defects we previously identified in gastroesophageal reflux disease patients in- vivo are due to abnormalities of the gastric sling, gastric clasp, or lower esophageal circular (LEC) muscle fibers. Methods Muscle strips from whole stomachs and esophagi were obtained from 16 normal donors and 15 donors with histologically proven Barrett's esophagus. Contractile and relaxant responses of gastric sling, gastric clasp, or LEC fibers were determined to increasing concentrations of carbachol and to nicotine after inducing maximal contraction to bethanechol. Muscarinic receptor density was measured using subtype selective immunoprecipitation. Key Results Barrett's esophagus gastric sling and LEC fibers have decreased carbachol-induced contractions. Barrett's esophagus sling fibers have decreased M2-muscarinic receptors and LEC fibers have decreased M3 receptors. Relaxations of all three fiber types are greater in Barrett's esophagus specimens to both high carbachol concentrations and to nicotine following bethanechol precontraction. The maximal response to bethanechol is greater in Barrett esophagus sling and LEC fibers. Conclusions & Inferences The increased contractile response to bethanechol in Barrett's specimens indicates that the defect is likely not due to the smooth muscle itself. The enhanced nicotinic receptor mediated response may be involved in greater relaxation of the muscles within the high-pressure zone of the gastroesophageal junction during transient lower esophageal sphincter relaxations and during deglutitive inhibition and may be involved in the pathophysiology of gastroesophageal reflux disease.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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)

Rapid structural and functional alterations have been noted in several models of partial outlet obstruction. To better characterize the rapid progression of alterations, the partially obstructed urinary bladders of mature NZW male rabbits were studied at 1, 3, 5, 7 and 14 days of outlet obstruction with respect to muscarinic receptor density, DNA, RNA, lipid and hydroxyproline content. Functional characteristics were assessed by measuring the in vitro response of the whole bladder to cholinergic and field stimulation. Wet weight increased eight-fold by day 7, decreasing to four-fold at day 14. Receptor density decreased by 50% by day 1 and remained low throughout. Although DNA concentration varied only slightly from controls, RNA increased four-fold by day 7. Hydroxyproline concentration per mg. tissue decreased in the obstructed bladder, yet total hydroxyproline content of the obstructed bladder significantly increased. Total lipids increased significantly during day 3 through 7 and decreased by day 14. Cystometry revealed a large capacity low pressure system at day 1 which rapidly changed to a low compliance system of lesser volume by day 14. Bladder emptying was significantly impaired in all obstructed specimens. Additionally, electrical field stimulation was significantly less effective than cholinergic stimulation in effecting bladder emptying. The above findings suggest that rapid changes in biochemical parameters occur during the early stage of acute obstruction which may in part be secondary to metabolic or inflammatory alterations in the detrusor. It additionally suggests that the myogenic alterations in partial outlet obstruction are rapid and partially adaptive, while neurogenic alterations appear degenerative and display a lesser degree of short term adaptation.

There are several methodologies available for determining the contractile effects of specific agents on isolated smooth muscle. The standard isolated bath technique utilizes strips mounted in a physiological buffer. The agent under study is added to the buffer and the response is recorded on a polygraph. The superfused strip technique utilizes strips in which buffer is pumped over the strip at relatively low flow rates. The agent under investigation is either infused into the stream of buffer bathing the tissue strip or administered directly onto the tissue. We have compared the response of these two methodologies using isolated strips of rabbit urinary bladder body. Isolated strips of rabbit urinary bladder were mounted in standard isolated baths containing 30 ml Tyrode's solution. The response of these strips to the autonomic agonists bethanechol, ATP, and isoproterenol were determined via cumulative addition at 5-minute intervals. Similar strips were mounted as above, after equilibration, the Tyrode's was drained, and fresh oxygenated Tyrode's was pumped over the strip at a rate of 4 ml per minute. Flow was stopped and drugs were administered directly onto the tissue in 100-μl aliquots. After either maximal response or 2 minutes, flow was resumed. Drugs were added at 10-minute intervals. The results can be summarized as follows: 1) The superfused bladder strips responded to 100-fold lower amounts of autonomic agonist than the strips in the isolated bath. 2) The maximum response to ATP (an agent that produces rapid desensitization) was threefold greater in the superfused model than in the bladder bath, and the rate of response of the tissues in the superfused system was significantly greater than in the isolated baths. In summary, the superfused isolated bladder preparation is an excellent methodology to use in specific investigations.

Recent advances in receptor technology have demonstrated that subtypes of each autonomic receptor exist. Using both direct radio-ligand studies and the inhibition of receptor binding by subtype-selective pharmacological antagonists, we have studied the distribution of subtypes of alpha and beta adrenergic receptors and muscarinic cholinergic receptors in the urinary bladder of the rabbit and man. Alpha adrenergic receptors were quantified by direct binding of tritiated prazosin (alpha-1), yohimbine (alpha-2), and the non-selective alpha adrenergic ligand dihydroergocriptine (DHE). These studies demonstrated that the distribution of alpha receptor subtypes in the bladder base (for both rabbit and human) is approximately 80% alpha-1 and 20% alpha-2. Beta receptor subtypes were identified by the inhibition of the non-selective ligand 3H-dihydroalprenalol (DHA) by the beta-1 selective inhibitor ICI-89 and the beta-2 selective inhibitor ICI-118. Initial studies demonstrated that the beta adrenergic density of the bladder body was 92 fmol per mg. protein for the rabbit and 32 fmol per mg. protein for human bladder body. Inhibition of DHA binding by ICI-118 demonstrated a single class of receptor with an IC50 of approximately 0.013 microM for both rabbit and human. Inhibition of DHA binding by ICI-89 also demonstrated one class of receptors with an IC50 of approximately 9.0 microM for both species. These results indicate that there are primarily beta-2 receptors in the rabbit and human bladder body. Although the number of muscarinic subtypes in existence is currently being re-evaluated, there are at least two which can be identified by the selective muscarinic agent pirenzepine (PZP). The brain has been shown to contain both high and low affinity PZP sites. Using both direct PZP binding to the bladder body, and the inhibition by PZP of the non-selective radio-ligand quinuclidinyl benzylate (QNB), we have demonstrated that both the rabbit and human bladder body have no observable high affinity PZP-selective binding and the inhibition of 3H-QNB by PZP demonstrated that there was only the low-affinity PZP binding site. Although receptor subtypes in the bladder have been the subject of numerous investigations, this is the first study describing the distribution of both adrenergic and cholinergic receptor subtypes in both the rabbit and human.

The function of the urinary bladder is to store and expell urine. Micturition is accomplished via a coordinated, well-sustained contraction of sufficient force to expel the entire contents of the urinary bladder. Studies in several labs have demonstrated that a functional bladder contraction consists of two phases: the first phase is represented by a rapid rise in intravesical pressure (isometric contraction) to a peak, followed by a prolonged period of increased (sustained) intravesical pressure (tension). Using isolated strip techniques, the in vitro whole bladder model, and several metabolic methodologies, we have studied contractile, functional, and biochemical parameters of these two phases of contraction. The results of our studies were as follows: 1) There was a small but significant decrease in net ATP at the peak of contraction, but not during the plateau phase. Creatine phosphate (CP) was significantly reduced at both times. 2) There was a rapid decrease in NADH fluorescence during the peak phase of contraction, which indicates an increase in oxidative metabolisvm. 3) The ED50 for the fluorescence response to bethanechol was significantly lower than the ED50 for contraction. 4) Whereas anoxia produced a gradual decline in both intracellular ATP and peak contraction, there was an immediate loss in the ability to maintain a contraction (ability of the bladder to empty). The results of these studies indicate that whereas preformed ATP mediates the initial contractile response to bethanechol, the plateau phase of bladder contraction (and the ability of the bladder to empty) may be mediated by high-energy intermediates of oxidative phosphorylation (aerobic metabolism).

Although adenyl cyclase stimulation has been associated with beta-receptor activation in the heart and some smooth muscle systems, it has been shown not to correlate with beta receptor activation in several other smooth muscle preparations. The present study investigates the relationship between cyclic AMP formation and beta-adrenergic stimulation in the rabbit urinary bladder. The intracellular concentration of cyclic AMP was determined in fresh rabbit bladder and isolated strips incubated under a variety of conditions. The results can be summarized as follows: (1) there was no difference between the cyclic AMP concentration in the bladder base and the bladder body, (2) incubation with theophylline significantly increased the concentration of cyclic AMP in all bladder strips, (3) isoproterenol significantly increased cyclic AMP in the bladder dome but not in the base, and (4) propranolol specifically inhibited the cyclic AMP increase produced by isoproterenol. Incubation with methoxamine, bethanechol, ATP, or diltiazem had no effect on the cyclic AMP concentration. The difference in the response to isoproterenol between bladder base and body correlates very well with both the distribution of beta receptors and the contractile response of these sections to isoproterenol. These studies demonstrate that the bladder body contains an isoproterenol-sensitive adenyl cyclase system.

Ureters of mature New Zealand white male rabbits were studied at 7 and 14 days following partial bladder outlet obstruction. Muscarinic receptor density; intracellular concentrations of DNA, RNA, lipid, and hydroxyproline; and functional characteristics of the whole ureter were assessed as a means of characterizing the ureteral secondary response to partial bladder obstruction. Wet weight increased 2-fold by day 7 and almost to 3-fold by day 14. Ureteral length increased 15% by day 7 and 29% by day 14. DNA concentration varied only slightly from controls while RNA increased by 68% by day 7 but returned to near normal range by day 14. Total hydroxyproline content increased, although hydroxyproline concentration per mg tissue decreased. Total lipids increased by 73% over the first 7 days but subsequently decreased to 27% above control levels by day 14. After 1 week of obstruction, muscarinic receptor density was 35.5% of controls, and after 2 weeks of obstruction, muscarinic density was 52.5% of controls. Although muscarinic receptor density decreased significantly, the number of receptors per ureter did not substantially change. Flow studies demonstrated increased opening pressures at 7 days and decreased opening pressures by day 14. Flow as a function of constant pressure was not significantly changed by the 7th day but increased 24% by the 14th day. Bethanechol (muscarinic agonist) caused similar decreases in flow in control and 1- and 2-week obstructed ureters. These studies indicate that 1 week following partial outlet obstruction, ureters demonstrate increased protein synthesis and lipid deposition resulting in increased weight and length. By the 2nd week, protein synthesis and lipid content begin to return to normal. Weight and length remain significantly increased and the capacity for flow through the ureter improves.

Subtypes of muscarinic receptors have been proposed by several investigators, based on differences in the potency of muscarinic agonists in various tissues. Development of selective muscarinic agonists and antagonists for the urinary bladder would be extremely useful since side effects of the presently used drugs often limit their clinical usefulness. Direct investigation of muscarinic receptor subtypes by radioligand binding techniques has recently become possible. The muscarinic antagonist pirenzepine distinguishes between a high affinity and low affinity binding site in several tissues. Results of the present investigation indicate that only the low affinity pirenzepine binding site is present in both human and rabbit bladder. Muscarinic receptor linked biochemical responses (increased phosphatidyl inositol turnover and inhibition of adenyl cyclase) were also consistent with the presence of only the low affinity pirenzepine binding receptor subtype in both human and rabbit bladder.

Functionally, the urinary bladder can be divided into two sections: the bladder body and bladder base. Pharmacologically, the bladder body responds to a greater extent than the bladder base to cholinergic agonists (contraction), beta-adrenergic agonists (relaxation), and purinergic agonists (contraction), whereas the bladder base responds to a greater extent than the bladder body to alpha-adrenergic agonists (contraction). The autonomic receptor density parallels the pharmacological response; that is, the bladder body contains higher densities of muscarinic cholinergic and beta-adrenergic receptors than the bladder base, whereas the base contains a higher density of alpha-adrenergic receptors. The purpose of this present study was to compare a variety of biochemical factors between the rabbit urinary bladder base and body. Sections of rabbit bladder body and base were rapidly dissected and frozen in liquid nitrogen. Tissue concentrations of DNA, RNA, hydroxyproline, lipids, glycogen, cyclic AMP, CP, ATP, ADP, and AMP were determined. The results demonstrated that the bladder base contained a 34% greater concentration of DNA, whereas the bladder body contained a 127% greater concentration of RNA. The bladder base contained 39.6% and 36% greater concentrations of hydroxyproline and glycogen, respectively. The concentration of lipids, cyclic AMP, and adenine nucleotides was equal in the two areas. The observed differences between bladder body and base may be related to the significantly greater work (energy utilized to actively expel urine) performed by the bladder body.

Bladder contraction is mediated by a number of intracellular biochemical reactions including glycolysis, mitochondrial respiration, phosphocreatine kinase, and various ATPases. In order to understand how alterations in intracellular metabolism can affect bladder function (or how alterations in bladder function can affect intracellular metabolism), we must first understand and describe normal bladder metabolism. Using the rabbit as a model for bladder function, the present study compares the intracellular concentrations of a variety of important compounds between the bladder base and body. The intracellular concentrations of phosphocreatine (PC), creatine, ATP, ADP, and AMP were determined in samples of rabbit urinary bladder body and base. The values for PC and creatine were significantly lower in the bladder base than in the bladder body. Adenine nucleotide concentrations were similar in the two sections. The concentrations of NAD + NADH, NADP, CTP, CDP, CMP, GTP, GDP, GMP, and UTP, UDP, UMP were also measured. Of these compounds, the concentrations of GTP and UTP in the bladder body were nearly double those in the bladder base. The intracellular concentrations of the other tissue constituents were similar in the two bladder sections. These studies demonstrate that the bladder is not biochemically homogeneous but that the bladder base and body differ not only functionally but also metabolically. Experiments in which strips of bladder body were incubated in vitro in oxygenated Tyrode's solution containing glucose showed that the tissue concentrations of PC, ATP, GTP, and UTP decreased substantially following 30 min of incubation, with no further decrease during a subsequent 60-min incubation.

Imipramine is a tricyclic antidepressant that has been demonstrated to be useful in the treatment of certain voiding dysfunctions. Imipramine has a variety of pharmacological effects including direct antimuscarinic activity, inhibition of catecholamine reuptake, direct muscle relaxant, and calcium antagonism. Using the in-vitro whole bladder model we have studied the effect of imipramine on the rate and magnitude of both intravesical pressure generation and bladder emptying in response to field stimulation. The results can be summarized as follows: at concentrations as low as 1 mumol/l imipramine causes a significant inhibition of volume expulsion without significantly affecting pressure generation. Imipramine produced a dose-dependent inhibition of both pressure development and percent volume emptying; however, it was substantially more potent in inhibiting the ability of the bladder to empty than to generate pressure.

Intestinal segments are commonly incorporated in the urinary system as conduits, reservoirs, and as implants in augmentations of bladders. This practice, initiated in 1888 by Tizzone, only became popular in the 1950s. Although both bladder and intestine are composed primarily of smooth muscle, there are significant morphological and functional differences between these tissues. This present study compares the pharmacological properties of intestinal smooth muscle to those of bladder smooth muscle. Ileum was taken as a representative section from small intestine, sigmoid colon from the large bowel, and the bladder body from the urinary bladder. The results can be summarized as follows: Bethanechol (cholinergic agonist) produced a rapid and sustained increase in tension in the bladder, a marked increase in the amplitude and frequency of phasic contractions in the ileum, and a sustained increase in tension in the sigmoid colon. Methoxamine (α-adrenergic agonist) and ATP (purinergic agonist) produced an increase in tension in the bladder, and reduced the tension in both ileum and sigmoid. Isoproterenol (β-adrenergic agonist) produced a relaxation in all three tissues.

Bladder augmentation or substitution with ileal segments is being used to a greater extent in recent years. The goal of the present study is to characterize the physiologic and pharmacologic changes in the ileal segment that occur following ileal cystoplasty. Four weeks following ileocystoplasty in rabbits, the contractile response of sections of normal terminal ileum, bladder body, and the cystoplastic segment of ileum (ileocystoplasty) was studied in isolated muscle baths. Results were as follows: 1) The ileum responded to a maximal dose of bethanechol with an increase in the amplitude and frequency of phasic contractions whereas the bladder displayed a rapid and sustained increase in basal tension. The cystoplastic ileal segment showed a marked increase in basal tension with superimposed phasic contractions. 2) Maximally effective doses of ATP and methoxamine reduced tension in the normal ileal segments but contracted both the bladder and cystoplastic ileal segments. 3) Isoproterenol reduced tension in all three segments. The pharmacological response of the cystoplastic segment of ileum significantly shifted from that of the ileum toward the response of the bladder. This effect is important as a manifestation of the functional plasticity of smooth muscle.