Potencies and selectivities of inhibitors of acetylcholinesterase and its molecular forms in normal and Alzheimer's disease brain

Eight inhibitors of acetylcholinesterase (AChE), tacrine, bis-tacrine, donepezil, rivastigmine, galantamine, heptyl-physostigmine, TAK-147 and metrifonate, were compared with regard to their effects on AChE and butyrylcholinesterase (BuChE) in normal human brain cortex. Additionally, the IC50 values of different molecular forms of AChE (monomeric, G1, and tetrameric, G4) were determined in the cerebral cortex in both normal and Alzheimer's human brains. The most selective AChE inhibitors, in decreasing sequence, were in order: TAK-147, donepezil and galantamine. For BuChE, the most specific was rivastigmine. However, none of these inhibitors was absolutely specific for AChE or BuChE. Among these inhibitors, tacrine, bis-tacrine, TAK-147, metrifonate and galantamine inhibited both the G1 and G4 AChE forms equally well. Interestingly, the AChE molecular forms in Alzheimer samples were more sensitive to some of the inhibitors as compared with the normal samples. Only one inhibitor, rivastigmine, displayed preferential inhibition for the G1 form of AChE. We conclude that a molecular form-specific inhibitor may have therapeutic applications in inhibiting the G1 form, which is relatively unchanged in Alzheimer's brain.     

Novel acetylcholinesterase inhibitor as increasing agent on rhythmic bladder contractions: SAR of 8-{3-[1-(3-fluorobenzyl)piperidin-4-yl]propanoyl}-1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]quinolin-4-one (TAK-802) and related compounds

As part of an on-going investigation to develop an increasing agent on rhythmic bladder contractions, 1-aryl-3-(1-benzylpiperidin-4-yl)propanones were synthesized and examined as noncarbamate acetylcholinesterase (AChE) inhibitors. Among compounds with various aryl groups, 1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]quinolin-4-one derivative 9c was found to possess a potent AChE inhibition activity with an IC(50) value of 1.3nM. The compound 9c increased rhythmic bladder contractions in Guinea pigs and rats without affecting the basal intravesical pressure, which suggests that 9c may be useful for the treatment of voiding dysfunction caused by detrusor underactivity.     

Effects of lidocaine, mepivacaine and prilocaine on the detrusor muscle of the rat urinary bladder

Lidocaine, mepivacaine and prilocaine cause in rats an increase of the frequency of the spontaneous contractions and an increase of the tone on the detrusor muscle of the urinary bladder. These effects are not antagonized by atropine and by fentola mine. On the contrary verapamil and papaverine antagonize these above effects.     

Preparation and in vitro screening of symmetrical bispyridinium cholinesterase inhibitors bearing different connecting linkage—initial study for Myasthenia gravis implications

Reversible inhibitors (e.g., pyridostigmine bromide, neostigmine bromide) of carbamate origin are used in the early treatment of Myasthenia gravis (MG) to block acetylcholinesterase (AChE) native function and conserve efficient amount of acetylcholine for decreasing number of nicotinic receptors. Carbamate inhibitors are known for many undesirable side effects related to the reversible inhibition of AChE. In contrast, this paper describes 20 newly prepared bispyridinium inhibitors of potential concern for MG. Although some compounds from this series have been known before, they were not assayed for cholinesterase inhibition yet.The newly prepared compounds were evaluated in vitro on human erythrocyte AChE and human plasmatic butyrylcholinesterase (BChE). Their inhibitory ability was expressed as IC₅₀ and compared to standard carbamate drugs. Three compounds presented promising inhibition (in μM range) of both enzymes in vitro similar to the used standards. The novel inhibitors did not present selectivity between AChE and BChE. Two newly prepared compounds were chosen for docking studies and confirmed apparent π–π or π–cationic interactions aside enzyme’s catalytic sites. The kinetics assay confirmed non-competitive inhibition of AChE by two best newly prepared compounds.     

Unique properties of muscularis mucosae smooth muscle in guinea pig urinary bladder

The muscularis mucosae, a type of smooth muscle located between the urothelium and the urinary bladder detrusor, has been described, although its properties and role in bladder function have not been characterized. Here, using mucosal tissue strips isolated from guinea pig urinary bladders, we identified spontaneous phasic contractions (SPCs) that appear to originate in the muscularis mucosae. This smooth muscle layer exhibited Ca(2+) waves and flashes, but localized Ca(2+) events (Ca(2+) sparks, purinergic receptor-mediated transients) were not detected. Ca(2+) flashes, often in bursts, occurred with a frequency (～5.7/min) similar to that of SPCs (～4/min), suggesting that SPCs are triggered by bursts of Ca(2+) flashes. The force generated by a single mucosal SPC represented the maximal force of the strip, whereas a single detrusor SPC was ～3% of maximal force of the detrusor strip. Electrical field stimulation (0.5-50 Hz) evoked force transients in isolated detrusor and mucosal strips. Inhibition of cholinergic receptors significantly decreased force in detrusor and mucosal strips (at higher frequencies). Concurrent inhibition of purinergic and cholinergic receptors nearly abolished evoked responses in detrusor and mucosae. Mucosal SPCs were unaffected by blocking small-conductance Ca(2+)-activated K(+) (SK) channels with apamin and were unchanged by blocking large-conductance Ca(2+)-activated K(+) (BK) channels with iberiotoxin (IbTX), indicating that SK and BK channels play a much smaller role in regulating muscularis mucosae SPCs than they do in regulating detrusor SPCs. Consistent with this, BK channel current density in myocytes from muscularis mucosae was ～20% of that in detrusor myocytes. These findings indicate that the muscularis mucosae in guinea pig represents a second smooth muscle compartment that is physiologically and pharmacologically distinct from the detrusor and may contribute to the overall contractile properties of the urinary bladder.     

Cardiac responses of Pacific oyster Crassostrea gigas to agents modulating cholinergic function

To examine the functional effects of cholinergic modulation compounds in oyster hearts and to explore their possible use in monitoring intoxication with acetylcholine-esterase (AChE) inhibitors such as organophosphates, tests were performed with in situ oyster heart preparations. The endogenous cholinergic agonist acetylcholine (ACh), AChE-resistant synthetic agonist carbachol, and the reversible carbamate type of AChE inhibitor physostigmine, all potently depressed spontaneous cardiac contractility. The depression was reversed by extensive washout, or prevented by muscarinic cholinergic antagonist atropine. The irreversible organophosphate type AChE inhibitor parathion or its active metabolite paraoxon at concentrations up to 100 microM failed to depress cardiac contractility. While other reversible AChE inhibitors such neostigmine and pyridostigmine also depressed the contractility, organophosphate AChE inhibitors malathion, diazinon, or phenthoate did not. Despite the differential effect in depressing cardiac function between the reversible and irreversible inhibitors, both of these inhibitors effectively inhibited cardiac AChE activity. The results suggest that the activation of muscarinic cholinergic receptors is coupled to inhibitory cardiac modulation, and organophosphate AChE inhibitors may inhibit only an AChE isozyme located at sites that are not important for control of cardiac activity in oysters.     

Studies on new,centrally active and reversible acetylcholinesterase inhibitors

We have synthesized the tertiary amines of pyridostigmine and neostigmine, 3-pyridinol dimethylcarbamate (norpyridostigmine) and 3-dimethylaminophenol dimethylcarbamate (norneostigmine) respectively, and we have tested their abilities to cross the blood-brain barrier and inhibit mouse brainAChE activity. The in vivo inhibition of AChE activity by norpyridostigmine reaches 72% at 10 minutes which is comparable to that seen with physostigmine (73% at 10 minutes). Inhibition by norneostigmine is less effective (50% at 10 minutes) and approaches that obtained with tetrahydroaminoacridine (57% at 10 minutes). These data show that both norpyridostigmine and norneostigmine cross the blood-brain barrier and that they are effective inhibitors of mouse brain AChE activity. These drugs could be useful in the treatment of memory, impairment associated with Alzheimer's disease, and other memory disorders.     

Suppression of human detrusor smooth muscle excitability and contractility via pharmacological activation of large conductance Ca2+-activated K+ channels

Overactive bladder syndrome is frequently associated with increased detrusor smooth muscle (DSM) contractility. We tested the hypothesis that pharmacological activation of the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel with NS-1619, a selective BK channel opener, reduces the excitability and contractility of human DSM. We used the amphotericin-perforated whole cell patch-clamp technique on freshly isolated human DSM cells, live-cell Ca(2+) imaging, and isometric DSM tension recordings of human DSM strips obtained from open bladder surgeries. NS-1619 (30 μM) significantly increased the amplitude of the voltage step-induced whole cell BK currents, and this effect was abolished by pretreatment with 200 nM iberiotoxin (IBTX), a selective BK channel inhibitor. In current-clamp mode, NS-1619 (30 μM) significantly hyperpolarized the resting membrane potential, and the hyperpolarization was reversed by IBTX (200 nM). NS-1619 (30 μM) significantly decreased the intracellular Ca(2+) level in isolated human DSM cells. BK channel activation with NS-1619 (30 μM) significantly inhibited the amplitude, muscle force, frequency, duration, and tone of the spontaneous phasic and pharmacologically induced DSM contractions from human DSM isolated strips. IBTX (200 nM) suppressed the inhibitory effects of NS-1619 on spontaneous contractions. The amplitude of electrical field stimulation (0.5-50 Hz)-induced contractions was significantly reduced by NS-1619 (30 μM). Our data suggest that pharmacological activation of BK channels could represent a novel treatment option to control bladder dysfunction in humans.     

Phasic Contractions in Urinary Bladder from Juvenile versus Adult Pigs

Aims

Alterations in properties of the bladder with maturation are relevant physiologically and pathophysiologically. The aim of this study was to investigate alterations in bladder properties with maturation in juvenile vs. adult pig, focussing on differences between layers of the bladder wall (mucosa vs. detrusor) and the presence and functional contribution of interstitial cells (ICs).

Methods

Basal and cholinergic-induced phasic contractions (PCs) in mucosal and denuded-detrusor strips from juvenile and adult pigs were assessed. Expression of c-kit, a marker of ICs, was investigated in the mucosa and the detrusor layers of the pig bladder. The functional role of ICs in mediating PCs was examined using imatinib.

Results

Mucosal strips from juvenile and adult pig bladders demonstrated basal PCs whilst denuded-detrusor strips did not. PCs of mucosal strips from juvenile pigs were significantly greater than those from adult bladders. Immunoreactivity for c-kit was detected in mucosa and detrusor layers of pig bladder. Histological studies demonstrated a distinct layer of smooth muscle between the urothelium and bladder detrusor, termed the muscularis mucosa. Imatinib was only effective in inhibiting PCs in mucosal strips from juvenile pigs. Imatinib inhibited the carbachol-induced PCs of both juvenile and adult denuded-detrusor strips, although strips from juvenile bladders demonstrated a trend towards being more sensitive to this inhibition.

Conclusions

We confirm the presence of c-kit positive ICs in pig urinary bladder. The enhanced PCs of mucosal strips from juvenile animals could be due to altered properties of ICs or the muscularis mucosa in the bladders of these animals.     

Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca²⁺-activated K⁺ channel

Detrusor smooth muscle (DSM) exhibits increased spontaneous phasic contractions under pathophysiological conditions such as detrusor overactivity (DO). Our previous studies showed that activation of cAMP signaling pathways reduces DSM contractility by increasing the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel activity. Here, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig DSM excitability and contractility by increasing BK channel activity. Utilizing isometric tension recordings of DSM isolated strips and the perforated patch-clamp technique on freshly isolated DSM cells, we examined the mechanism of DSM relaxation induced by PDE inhibition. Inhibition of PDEs by 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor, significantly reduced DSM spontaneous and carbachol-induced contraction amplitude, frequency, duration, muscle force integral, and tone in a concentration-dependent manner. IBMX significantly reduced electrical field stimulation-induced contractions of DSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on DSM contractility, indicating a role for BK channels in DSM relaxation mediated by PDE inhibition. IBMX increased the transient BK currents (TBKCs) frequency by ～3-fold without affecting the TBKCs amplitude. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ～2-fold and hyperpolarized the DSM cell resting membrane potential by ～6 mV. Blocking the BK channels with paxilline abolished the IBMX hyperpolarizing effects. Under conditions of blocked Ca(2+) sources for BK channel activation, IBMX did not affect the depolarization-induced steady-state whole cell BK currents. Our data reveal that PDE inhibition with IBMX relaxes guinea pig DSM via TBKCs activation and subsequent DSM cell membrane hyperpolarization.     

Modulation of spontaneous activity in the overactive bladder: the role of P2Y agonists

Spinal cord transection (SCT) leads to an increase in spontaneous contractile activity in the isolated bladder that is reminiscent of an overactive bladder syndrome in patients with similar damage to the central nervous system. An increase in interstitial cell number in the suburothelial space between the urothelium and detrusor smooth muscle layer occurs in SCT bladders, and these cells elicit excitatory responses to purines and pyrimidines such as ATP, ADP, and UTP. We have investigated the hypothesis that these agents underlie the increase in spontaneous activity. Rats underwent lower thoracic spinal cord transection, and their bladder sheets or strips, with intact mucosa except where specified, were used for experiments. Isometric tension was recorded and propagating Ca(2+) and membrane potential (E(m)) waves were recorded by fluorescence imaging using photodiode arrays. SCT bladders were associated with regular spontaneous contractions (2.9 ± 0.4/min); ADP, UTP, and UDP augmented the amplitude but not their frequency. With strips from such bladders, a P2Y(6)-selective agonist (PSB0474) exerted similar effects. Fluorescence imaging of bladder sheets showed that ADP or UTP increased the conduction velocity of Ca(2+)/E(m) waves that were confined to regions of the bladder wall with an intact mucosa. When transverse bladder sections were used, Ca(2+)/E(m) waves originated in the suburothelial space and propagated to the detrusor and urothelium. Analysis of wave propagation showed that the suburothelial space exhibited properties of an electrical syncitium. These experiments are consistent with the hypothesis that P2Y-receptor agonists increase spontaneous contractile activity by augmenting functional activity of the cellular syncitium in the suburothelial space.     

Role of Potassium Channels in the Effects of Hydrogen Sulfide on Contractility of Gastric Smooth Muscle Cells in Rats

The effect of sodium hydrosulfide (NaHS), a hydrogen sulfide (H₂S) donor, on spontaneous contractile activity of rat gastric smooth muscle cells was analyzed. Experiments were conducted on gastric stripes under conditions of isometric contraction. It was shown that NaHS has a biphasic effect on spontaneous contractile activity, increasing tonic tension and the amplitude of phasic contractions within the first minutes since application. This initial phase is followed by a decrease in amplitude, basal tone, and frequency of spontaneous contractions. The inhibitory effect of NaHS was dose-dependent at concentrations from 10 to 600 μM. Preliminary application of tetraethylammonium and 4-aminopirydine, inhibitors of voltage-gated and calciumactivated potassium channels, prevented a NaHS-induced initial increase in basal tone and phasic contraction amplitude. Activation of ATP-dependent potassium channels (KATP-channels) by diazoxide prevented in part a NaHS-induced decrease in basal tone and amplitude of spontaneous contractions. Glibenclamide, an inhibitor of K_ATP-channels, decreased the inhibitory effect of NaHS on amplitude, basal tone and frequency of spontaneous contractions. It was concluded that in rat gastric smooth muscles the excitatory effect of H₂S is mediated by the inhibition of voltagegated and calcium-activated potassium channels, while its inhibitory effect involves the activation of K_ATP-channels.     

Some new carbacylamidophosphates as inhibitors of acetylcholinesterase and butyrylcholinesterase

The differences in the inhibition activity of organophosphorus agents are a manifestation of different molecular properties of the inhibitors involved in the interaction with the active site of enzyme. We were interested in comparing the inhibition potency of four known synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl2, constituting organophosphorus compounds, where R = CCl3 (1), CHCl2 (2), CH2Cl (3) and CF3 (4), and four new ones with the general formula RC(O)NHP(O)(R')2, where R' = morpholine and R = CCl3 (5), CHCl2 (6), CH2Cl (7), CF3 (8), on AChE and BuChE activities. In addition, in vitro activities of all eight compounds on BuChE were determined. Besides, in vivo inhibition potency of compounds 2 and 6, which had the highest inhibition potency among the tested compounds, was studied. The data demonstrated that compound 2 from the compound series 1 to 4 and compound 6 from the compound series 5 to 8 are the most sensitive as AChE and BuChE inhibitors, respectively. Comparing the IC50 values of these compounds, it was clear that the inhibition potency of these compounds for AChE are 2- to 100-fold greater than for BuChE inhibition. Comparison of the kinetics (IC50, Ki, kp, KA and KD) of AChE and BuChE inactivation by these compounds resulted in no significant difference for the measured variables except for compounds 2 and 6, which appeared to be more sensitive to AChE and BuChE by significantly higher kp and Ki values and a lower IC50 value in comparison with the other compounds. The LD50 value of compounds 2 and 6, after oral administration, and the changes of erythrocyte AChE and plasma BuChE activities in albino mice were studied. The in vivo experiments, similar to the in vitro results, showed that compound 2 is a stronger AChE and BuChE inhibitor than the other synthesized carbacylamidophosphates. Furthermore, in this study, the importance of electropositivity of the phosphorus atom, steric hindrance and leaving group specificity were reinforced as important determinants of inhibition activity.     

Reactivity of urinary bladder smooth muscle in guinea pigs to acetylcholine and carbachol--participation of acetylcholinesterase

The authors examined the influence of acetylcholinesterase inhibitor (neostigmine) on the in vitro reactivity of urinary bladder smooth muscle (UBSM) in guinea pigs. The aim of the present study was to determine the participation of pharmacokinetic properties of acetylcholine and carbachol in different UBSM reactivity to these mediators. In vitro method of organ baths was used and reactivity of UBSM strips to cumulative doses of acetylcholine and carbachol was tested before and after the incubation with neostigmine (10(-4) mol.l(-1)). Neostigmine caused a significant increase of UBSM reactivity to acetylcholine. The UBSM reactivity to acetylcholine was significantly higher at concentrations of 10(-5) and 10(-4) mol.l(-1) compared to carbachol at the same concentrations. These findings indicate that in addition to different mediator affinity to muscarinic receptors and to their different intrinsic activity, the pharmacokinetic properties of acetylcholine and carbachol also participate in UBSM reactivity.     

Synaptic acetylcholinesterase of chicken muscle changes during development from a hybrid to a homogeneous enzyme.

The asymmetric (20S) form of acetylcholinesterase (AChE) in 1-day-old chick muscle is a hybrid enzyme containing both AChE (110 kd) and butyrylcholinesterase (BuChE, 72 kd) catalytic subunits. However, we now report that the asymmetric AChE extracted or immunopurified from older adult chicken muscles, where it is the endplate form, shows a progressive developmental loss of the BuChE subunit and its activities, centred around 4 weeks of age, while the AChE and collagenous subunits remain. In confirmation, using differential labelling and co-sedimentation it was shown that the hybrid 20S AChE/BuChE form of 1-day chick muscle is gradually and completely replaced during muscle maturation by a 21.3S form, also collagen-tailed but otherwise homogeneous in AChE catalytic subunits. Two other changes occur concomitantly. Firstly, the AChE catalytic subunit of the adult form has a lower apparent mol. wt in gel electrophoresis, by 5 kd, than the same subunit in the 1-day hybrid enzyme; this difference does not reside in the carbohydrate attachments. Secondly, the collagen tail changes, in that some conformation-dependent epitopes on it disappear in the same period. Hence, a major reorganization of the asymmetric AChE, involving all three types of subunit, occurs in the course of muscle development.     

Design, synthesis and evaluation of isaindigotone derivatives as dual inhibitors for acetylcholinesterase and amyloid beta aggregation

A series of isaindigotone derivatives and analogues were designed, synthesized and evaluated as dual inhibitors of cholinesterases (ChEs) and self-induced β-amyloid (Aβ) aggregation. The synthetic compounds had IC(50) values at micro or nano molar range for cholinesterase inhibition, and some compounds exhibited strong inhibitory activity for AChE and high selectivity for AChE over BuChE, which were much better than the isaindigotone derivatives previously reported by our group. Most of these compounds showed higher self-induced Aβ aggregation inhibitory activity than a reference compound curcumin. The structure-activity relationship studies revealed that the derivatives with higher inhibition activity on AChE also showed higher selectivity for AChE over BuChE. Compound 6c exhibiting excellent inhibition for both AChE and self-induced Aβ aggregation was further studied using CD, EM, molecular docking and kinetics.     

Length-dependent regulation of basal myosin phosphorylation and force in detrusor smooth muscle

Urinary bladder (detrusor) smooth muscle is active in the absence of an external stimulus. Tone occurs even "at rest" during the filling phase, and it is elevated in patients with overactive bladder. This study examined the role of muscle length on tone and the level of basal myosin light chain phosphorylation (MLC(20P)). MLC(20P) was 23.9 +/- 1% (n = 58) at short lengths (zero preload; L(z)). An increase in length from L(z) to the optimal length for contraction (L(o)) caused a reduction in MLC(20P) to 15.8 +/- 1% (n = 49). Whereas 10 microM staurosporine reduced MLC(20P) at L(z), 1 microM staurosporine, a Ca(2+)-free solution, and inhibitors of MLC kinase, protein kinase C (PKC) and RhoA kinase (ROK) did not. However, 1 microM staurosporine and inhibitors of ROK inhibited MLC(20P) and tone at L(o). These data support the hypothesis that a Ca(2+)-independent kinase, possibly ZIP-like kinase, regulates MLC(20P) at L(z), whereas in detrusor stretched to L(o), additional kinases, such as ROK, participate.     

The preparation, in vitro screening and molecular docking of symmetrical bisquaternary cholinesterase inhibitors containing a but-(2E)-en-1,4-diyl connecting linkage

Carbamate inhibitors (e.g. pyridostigmine bromide) are used as a pre-treatment for the prevention of organophosphorus poisoning. They work by blocking the native function of acetylcholinesterases (AChE) and thus protect AChE against irreversible inhibition by organophosphorus compounds. However, carbamate inhibitors are known for their many undesirable side effects related to the carbamylation of AChE. In this paper, we describe 17 novel bisquaternary compounds and have analysed their effect on AChE inhibition. The newly prepared compounds were evaluated in vitro using both human erythrocyte AChE and human plasmatic butyrylcholinesterase. Their inhibitory ability was expressed as the half maximal inhibitory concentration (IC??) and then compared to the standard carbamate drugs and two AChE reactivators. One of these novel compounds showed promising AChE inhibition in vitro (nM range) and was better than the currently used standards. Additionally, a kinetic assay confirmed the non-competitive inhibition of hAChE by this novel compound. Consequently, the docking results confirmed the apparent π-π or π-cationic interactions with the key amino acid residues of hAChE and the binding of the chosen compound at the enzyme catalytic site.     

Deletion of Dicer in smooth muscle affects voiding pattern and reduces detrusor contractility and neuroeffector transmission

MicroRNAs have emerged as important regulators of smooth muscle phenotype and may play important roles in pathogenesis of various smooth muscle related disease states. The aim of this study was to investigate the role of miRNAs for urinary bladder function. We used an inducible and smooth muscle specific Dicer knockout (KO) mouse which resulted in significantly reduced levels of miRNAs, including miR-145, miR-143, miR-22, miR125b-5p and miR-27a, from detrusor preparations without mucosa. Deletion of Dicer resulted in a disturbed micturition pattern in vivo and reduced depolarization-induced pressure development in the isolated detrusor. Furthermore, electrical field stimulation revealed a decreased cholinergic but maintained purinergic component of neurogenic activation in Dicer KO bladder strips. The ultrastructure of detrusor smooth muscle cells was well maintained, and the density of nerve terminals was similar. Western blotting demonstrated reduced contents of calponin and desmin. Smooth muscle α-actin, SM22α and myocardin were unchanged. Activation of strips with exogenous agonists showed that depolarization-induced contraction was preferentially reduced; ATP- and calyculin A-induced contractions were unchanged. Quantitative real time PCR and western blotting demonstrated reduced expression of Cav1.2 (Cacna1c). It is concluded that smooth muscle miRNAs play an important role for detrusor contractility and voiding pattern of unrestrained mice. This is mediated in part via effects on expression of smooth muscle differentiation markers and L-type Ca(2+) channels in the detrusor.     

Role for pituitary adenylate cyclase activating polypeptide in cystitis-induced plasticity of micturition reflexes

Pituitary adenylate cyclase activating polypeptide (PACAP) peptides are expressed and regulated in sensory afferents of the micturition pathway. Although these studies have implicated PACAP in bladder control, the physiological significance of these observations has not been firmly established. To clarify these issues, the roles of PACAP and PACAP signaling in micturition and cystitis were examined in receptor characterization and physiological assays. PACAP receptors were identified in various tissues of the micturition pathway, including bladder detrusor smooth muscle and urothelium. Bladder smooth muscle expressed heterogeneously PAC(1)null, PAC(1)HOP1, and VPAC(2) receptors; the urothelium was more restricted in expressing preferentially the PAC(1) receptor subtype only. Immunocytochemical studies for PAC(1) receptors were consistent with these tissue distributions. Furthermore, the addition of 50-100 nM PACAP27 or PACAP38 to isolated bladder strips elicited transient contractions and sustained increases in the amplitude of spontaneous phasic contractions. Treatment of the bladder strips with tetrodotoxin (1 muM) did not alter the spontaneous phasic contractions suggesting direct PACAP effects on bladder smooth muscle. PACAP also increased the amplitude of nerve-evoked contractions. By contrast, vasoactive intestinal polypeptide had no direct effects on bladder smooth muscle. In a rat cyclophosphamide (CYP)-induced cystitis paradigm, intrathecal or intravesical administration of PAC(1) receptor antagonist, PACAP6-38, reduced cystitis-induced bladder overactivity. In summary, these studies support roles for PACAP in micturition and suggest that inflammation-induced plasticity in PACAP expression in peripheral and central micturition pathways contribute to bladder dysfunction with cystitis.