Urinary Bladder Detrusor Research Articles

Involvement of Prostaglandin E2 in the P2Y1 Receptor‐mediated Tonic Contraction of the Rabbit Urinary Bladder

Previously we have provided evidence that excitatory ADP activated P2Y1 receptors contribute for the rabbit urinary bladder contractility. To determine the mechanisms involved in P2Y1-mediated signaling, we have carried out experiments with the isolated rabbit urinary bladder detrusor smooth muscle strips. The contractile responses evoked by ADP or the specific P2Y1 receptor agonist ADPβS were antagonized by the non-selective cyclooxygenase inhibitors indomethacin and meclofenamic acid, as well as by AACOCF3 (10μM), a specific inhibitor of the receptor activated cytosolic Phospholipase A2 (cPLA2). The contractile responses of the detrusor smooth muscle evoked by prostaglandin E2 (PgE2) (EC50, 0.3 μM) but not by PgF2α resembled that evoked by ADPβS (EC50, 0.6 μM). Pretreatment with SC19220 (30 μM), a selective antagonist of the EP1 subtype of the PgE2 receptors abolished contractile responses evoked by ADPβS, while AL-8810, a selective FP (PgF2α) receptor antagonist had no effect. RT-PCR experiments demonstrated that the rabbit detrusor smooth muscle expresses mRNA for the microsomal prostaglandin E2 synthase-2 (mPGES-2). This evidence suggests that activation of P2Y1 receptors may lead to the synthesis of PgE2, which acts in a paracrine manner to evoke and possible amplify the contractile response of the detrusor smooth muscle of the urinary bladder. Supported by NIH HL38126 and the Foundation for Research.

β-NAD is a novel nucleotide released on stimulation of nerve terminals in human urinary bladder detrusor muscle

Endogenous nucleotides with extracellular functions may be involved in the complex neural control of human urinary bladder (HUB). Using HPLC techniques with fluorescence detection, we observed that in addition to ATP and its metabolites ADP, AMP and adenosine, electrical field stimulation (EFS; 4-16 Hz, 0.1 ms, 15 V, 60 s) of HUB detrusor smooth muscle coreleases novel nucleotide factors, which produce etheno-1N(6)-ADP-ribose (eADPR) on etheno-derivatization at high temperature. A detailed HPLC fraction analysis determined that nicotinamide adenine dinucleotide (beta-NAD+; 7.0 +/- 0.7 fmol/mg tissue) is the primary nucleotide that contributes to the formation of eADPR. The tissue superfusates collected during EFS also contained the beta-NAD+ metabolite ADPR (0.35 +/- 0.2 fmol/mg tissue) but not cyclic ADPR (cADPR). HUB failed to degrade nicotinamide guanine dinucleotide (NGD+), a specific substrate of ADP ribosyl cyclase, suggesting that the activity of this enzyme in the HUB is negligible. The EFS-evoked release of beta-NAD+ was frequency dependent and is reduced in the presence of tetrodotoxin (TTX; 0.3 micromol/l), omega-conotoxin GVIA (50 nmol/l), and botulinum neurotoxin A (BoNT/A; 100 nmol/l), but remained unchanged in the presence of guanethidine (3 micromol/l), omega-agatoxin IVA (50 nmol/l), or charbachol (1 micromol/l). Capsaicin (10 micromol/l) increased both the resting and EFS-evoked overflow of beta-NAD+. Exogenous beta-NAD+ (1 micromol/l) reduced both the frequency and amplitude of spontaneous contractions. In conclusion, we detected nerve-evoked overflow of beta-NAD+ and ADPR in HUB. The beta-NAD(+)/ADPR system may constitute a novel inhibitory extracellular nucleotide mechanism of neural control of the human bladder.

EFFECTS OF THE SELECTIVE ACETYLCHOLINESTERASE INHIBITOR TAK-802 ON THE VOIDING BEHAVIOR AND BLADDER MASS INCREASE IN RATS WITH PARTIAL BLADDER OUTLET OBSTRUCTION

We examined the effects of the selective acetylcholinesterase (AChE) inhibitor TAK-802 on voiding behavior and residual urine volume in rats with partial bladder outlet obstruction (BOO) vs rats treated with the nonselective AChE inhibitor distigmine and the muscarinic agonist bethanechol. In addition, the effect of repeat doses of TAK-802 on the bladder mass increase associated with BOO was also examined. Male rats with BOO were used. Six to 8 days after obstruction voiding behavior was observed in a metabolic cage. The animals were then treated orally with 1 drug, and voiding frequency and urine volume at each void were measured for 3 hours. Subsequently the volume of urine retained in the bladder (residual urine) was measured. In another experiment bladder weight in rats with BOO was measured after early repeat doses of TAK-802. BOO increased voiding frequency and decreased average voided volume. TAK-802 and distigmine increased average voided volume, while not causing any change in voiding frequency. On the other hand, bethanechol increased voiding frequency without affecting average voided volume. While all 3 drugs significantly decreased residual urine volume, TAK-802 was most efficacious. In addition, bladder weight in the control BOO group was greater (approximately 2.2-fold) than that in the sham operated group and early repeat administration of TAK-802 prevented the bladder mass increase. AChE inhibitors decreased residual urine volume by restoring voiding function in rats with BOO, although only the effect of TAK-802 was dose dependent. Bethanechol also decreased residual urine volume in a dose dependent manner but by increasing voiding frequency. The prevention of a bladder mass increase by TAK-802 treatment may be attributable to its effect on restoring voiding.

Experimental study of excitability and autorhthmicity in urinary bladder detrusor of diabetes rats

The changes in excitability and autorhthmicity of bladder detrusor in experimental non-insulin dependent diabetes mellitus (NIDDM) rats were observed. Sixty-nine NIDDM rats as NIDDM group and 69 normal rats as control group were enrolled into this experimental study. At 6th, 10th, 14th, 18th, 22nd and 26th week after the rats were injected last time, the changes in the excitability and autorhthmicity of detrusor strips in vitro were observed. The results showed that the threshold of the tension which made the detrusor strips contract was significantly higher in NIDDM group (0.716 +/- 0.325 g) than in control group (0.323 +/- 0.177 g) (F = 59.63, P < 0.001). At different stages, the threshold of the tension resulting the contract of the detrusor strips in NIDDM group was also higher than in control group. At 18th week after STZ injection, the frequency of spontaneous contract of the detrusor strips in NIDDM was significantly higher than in control group (P < 0.05), whereas at 22nd week, that in NIDDM group was significantly lower than in control group (P < 0.05). It was concluded that the decreased excitability of the bladder detrusor was the earliest and most obvious changes in bladder function in diabetes rats and the autorhthmicity had also changed at the early stage of diabetic bladder.

Postjunctional modulation by muscarinic M2 receptors of responses to electrical field stimulation of rat detrusor muscle preparations

1. The aim of the present study was to examine the modulator influence of muscarinic M(2) receptors on responses of rat urinary bladder detrusor muscle evoked by endogenous stimuli, i.e. by stimulation of the bladder innervation. 2. Responses were evoked by electrical field stimulation (EFS; 2-20 Hz, 0.8 ms, 60 V) of isolated strip preparations mounted in organ baths. The tension of the muscle strips was recorded digitally. EFS was performed by applying stimulation with either a short duration (5 s) or a longer duration (to reach peak response; approximately 20 s). 3. Effects of muscarinic receptor antagonists (muscarinic M(1)/M(3) receptor selective: 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP); muscarinic M(2) receptor selective: methoctramine), a beta-adrenergic antagonist (propranolol) and an adenosine receptor antagonist (8-p-sulfophenyltheophylline) were assessed on contractile activity and on poststimulatory relaxations. 4. Low concentrations of methoctramine (10(-8) m) reduced or tended to reduce the EFS-induced contraction, e.g. at 2 Hz by 12% while methoctramine at 10(-7) m had no significant effect. In addition, in the presence of 4-DAMP (10(-9) m), which tended to inhibit contractions at all frequencies (2-20 Hz; -17 to -25%), methoctramine at 10(-8) and 10(-7) m induced a further reduction of the contractile responses (-5 to -10%; 2-20 Hz). 5. The beta-adrenergic receptor antagonist propranolol (10(-6) m) and the adenosine receptor antagonist 8-p-sulfophenyltheophylline (10(-6) m) both increased contractile responses by 9-21% (2-10 Hz, long duration; P < 0.05-0.001) as a consequence of antagonizing relaxatory stimuli. Neither antagonist affected the contractile responses to EFS with the short duration stimulation. Poststimulatory relaxations were reduced by 30-60% (P < 0.05) by propranolol and by 40-60% (P < 0.001) by 8-p-sulfophenyltheophylline, but for 8-p-sulfophenyltheophylline only after stimulation with the short duration. 6. In the presence of methoctramine (10(-7) m), the 8-p-sulfophenyltheophylline-induced increases of the contractile response to long duration EFS were significantly enhanced at 10 Hz (+12 +/- 4%; P < 0.05), whereas no such enhancement of the propranolol inhibitory effect occurred in the presence of methoctramine. However, poststimulatory beta-adrenoceptor-evoked relaxations after short duration EFS were increased by about 35% in the presence of methoctramine, but not those after long duration. 7. Thus, muscarinic M(2) receptor activation inhibits adenosine receptor- and beta-adrenoceptor-evoked relaxations of the rat detrusor muscle. The inhibition occurs via a transient postjunctional mechanism that mainly affects responses with a short latency.

Neuronal nitric oxide synthase activity in rat urinary bladder detrusor: participation in M3 and M4 muscarinic receptor function

1. The aim of this paper was to determine the different signalling cascades involved in contraction of the rat urinary bladder detrusor muscle mediated via muscarinic acetylcholine receptors (muscarinic AChR). Contractile responses, phosphoinositides (IPs) accumulation, nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) production were measured to determine the reactions associated with the effect of cholinergic agonist carbachol. The specific muscarinic AChR subtype antagonists and different inhibitors of the enzymatic pathways involved in muscarinic receptor-dependent activation of NOS and cGMP were tested. 2. Carbachol stimulation of M(3) and M(4) muscarinic AChR increased contractility, IPs accumulation, NOS activity and cGMP production. All of these effects were selectively blunted by 4-DAMP and tropicamide, M(3) and M(4) antagonists respectively. 3. The inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), neuronal NOS (nNOS) and soluble guanylate cyclase, but not of protein kinase C and endothelial NOS (eNOS), inhibited the carbachol action on detrusor contractility. These inhibitors also attenuated the muscarinic receptor-dependent increase in cGMP and activation of NOS. 4. In addition, sodium nitroprusside and 8-bromo-cGMP, induced negative relaxant effect. 5. The results obtained suggest that carbachol activation of M(3) and M(4) muscarinic AChRs, exerts a contractile effect on rat detrusor that is accompanied by an increased production of cGMP and nNOS activity. The mechanism appears to occur secondarily to stimulation of IPs turnover via PLC activation. This in turn, triggers cascade reactions involving CaM, leading to activation of nNOS and soluble guanylate cyclase. They, in turn, exert a modulator inhibitory cGMP-mediated mechanism limiting the effect of muscarinic AChR stimulation of the bladder.

Poster 12: Comparative Effects of 1- and 5-Week Ovariectomy on Contractile Function of the Rabbit

OBJECTIVE: The urinary bladder can be considered a target organ for the actions of estrogen. Decreases in circulating estrogen postmenopause have been associated with urinary bladder dysfunctions, including incontinence and detrusor instability. Recent studies indicate that these changes can occur quite quickly when circulating estrogen decreases after ovariectomy. The current study was designed to compare the effects of ovariectomy at 1 and 5 weeks on contractile responses of the rabbit detrusor. METHODS: Female New Zealand white rabbits were ovariectomized and evaluated for the in vitro effects on contractile function (field stimulation [2, 8, and 32 Hz], carbachol [10 uM], ATP [1 mM], and KCl [120 mM]) at 1 and 5 weeks after ovariectomy (n=4–6 rabbits for each group). A control group of 4 rabbits was evaluated simultaneously. RESULTS: 1) Ovariectomy resulted in no significant change in bladder weight at either 1 or 5 weeks. 2) Both 1- and 5-week ovariectomy resulted in decreased maximal contractile responses to 8 and 32 Hz FS. No significant decreases were noted for the maximal responses to ATP, carbachol, or KCl. 3) Both 1- and 5-week ovariectomy resulted in significant decreases in the rate of tension generation for 32 Hz. 4) One-week ovariectomy resulted in a significant decrease in the rate of tension generation to ATP, whereas 5-week ovariectomy resulted in significant and substantial decreases in the rate of tension generation for ATP, carbachol, and KCl. CONCLUSIONS: These findings demonstrate that bladder contractile function is significantly affected by ovariectomy. In previous studies, we have demonstrated that the first sign of contractile dysfunction is a decrease in the rate of tension generation rather than decreases in the maximal contractile responses. These studies indicate that the longer the rabbit is ovariectomized, the greater the level of contractile dysfunctions. Decreased contractile function of the urethra especially may relate to the increased incidence of incontinence postmenopause.

Changes in galanin immunoreactivity in rat lumbosacral spinal cord and dorsal root ganglia after spinal cord injury.

Alterations in the expression of the neuropeptide galanin were examined in micturition reflex pathways 6 weeks after complete spinal cord transection (T8). In control animals, galanin expression was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (4) the lateral collateral pathway in lumbosacral spinal segments. Densitometry analysis demonstrated significant increases (P < or = 0.001) in galanin immunoreactivity (IR) in these regions of the S1 spinal cord after spinal cord injury (SCI). Changes in galanin-IR were not observed at the L4-L6 segments except for an increase in galanin-IR in the dorsal commissure in the L4 segment. In contrast, decreases in galanin-IR were observed in the L1 segment. The number of galanin-IR cells increased (P < or = 0.001) in the L1 and S1 dorsal root ganglia (DRG) after SCI. In all DRG examined (L1, L2, L6, and S1), the percentage of bladder afferent cells expressing galanin-IR significantly increased (4-19-fold) after chronic SCI. In contrast, galanin expression in nerve fibers in the urinary bladder detrusor and urothelium was decreased or eliminated after SCI. Expression of the neurotrophic factors nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) was altered in the spinal cord after SCI. A significant increase in BDNF expression was present in spinal cord segments after SCI. In contrast, NGF expression was only increased in the spinal segments adjacent and rostral to the transection site (T7-T8), whereas spinal segments (T13-L1; L6-S1), distal to the transection site exhibited decreased NGF expression. Changes in galanin expression in micturition pathways after SCI may be mediated by changing neurotrophic factor expression, particularly BDNF. These changes may contribute to urinary bladder dysfunction after SCI.

Cooperative attachment of cross bridges predicts regulation of smooth muscle force by myosin phosphorylation

Serine 19 phosphorylation of the myosin regulatory light chain (MRLC) appears to be the primary determinant of smooth muscle force development. The relationship between MRLC phosphorylation and force is nonlinear, showing that phosphorylation is not a simple switch regulating the number of cycling cross bridges. We reexamined the MRLC phosphorylation-force relationship in slow, tonic swine carotid media; fast, phasic rabbit urinary bladder detrusor; and very fast, tonic rat anococcygeus. We found a sigmoidal dependence of force on MRLC phosphorylation in all three tissues with a threshold for force development of approximately 0.15 mol P(i)/mol MRLC. This behavior suggests that force is regulated in a highly cooperative manner. We then determined whether a model that employs both the latch-bridge hypothesis and cooperative activation could reproduce the relationship between Ser(19)-MRLC phosphorylation and force without the need for a second regulatory system. We based this model on skeletal muscle in which attached cross bridges cooperatively activate thin filaments to facilitate cross-bridge attachment. We found that such a model describes both the steady-state and time-course relationship between Ser(19)-MRLC phosphorylation and force. The model required both cooperative activation and latch-bridge formation to predict force. The best fit of the model occurred when binding of a cross bridge cooperatively activated seven myosin binding sites on the thin filament. This result suggests cooperative mechanisms analogous to skeletal muscle that will require testing.

Molecular biological researches of the lower urinary tract function

Adrenergic alpha1 and beta receptors are present in the target organs of sympathetic nerve and they participate in the signal transduction mechanism of the lower urinary tract. Adrenergic alpha1 receptors are present in urethral and prostatic smooth muscles, and contract these muscles. Among these receptor subtypes, the alpha1-A receptor has the most important role, and mRNA expression of the corresponding alpha1-a subtype is predominant. In the human urinary bladder detrusor smooth muscle, the expression of adrenergic beta3 receptor subtype mRNA is predominant, and relaxation of detrusor smooth muscle is mediated mainly via beta3 receptor. Afferent nerve with lower threshold can easily transmit bladder sensation and takes an important role in the pathophysiology of urge urinary incontinence. Successful molecular cloning of vanilloid receptors, which are present in these afferent nerves, revealed that vanilloid receptors are ion-channels, sensitive for heat and pH, and termed VR1 and VRL1. Among purinergic receptors, ion channel type P2X3 receptor is found in afferent nerve fibers and plays some roles in the signal transduction of bladder sensation. In the near future, agonist for the adrenergic beta3 receptor and selective antagonists for VR1, VRL1, or P2X3 will possibly become drugs for pollakisuria and urge urinary incontinence.

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.

Effects of testosterone on neuromuscular transmission in rat isolated urinary bladder

Testosterone was examined for its effects on neuromuscular transmission in rat and shrew urinary bladder. In isolated preparations of detrusor muscle from sexually immature male rats (8–10 weeks old) at concentrations of 100–300 μM, it inhibited neuromuscular transmission in a concentration-dependent manner and it also inhibited responses to applied carbachol and diadenosine pentaphosphate (Ap 5A, a P2X receptor agonist). Ethanol (at or above 38 mM), the solvent for testosterone, also caused significant inhibition of neurogenic contractions as well as carbachol- and Ap 5A-induced contractions. In older, sexually mature male rats (over 16 weeks old), testosterone and ethanol had similar effects to those observed in the young male rat, although both were slightly less potent. In young virgin female rats (8–12 weeks old), testosterone and ethanol inhibited neuromuscular transmission; testosterone was approximately 1000 times more potent than in male rats, with a threshold concentration of 30 nM. In the insectivore, Suncus murinus, testosterone (0.1 μM–1 mM) caused inhibition of neurogenic and chemogenic responses, but ethanol had no significant effect. Flutamide (50 μM), a genomic testosterone-receptor antagonist, did not inhibit any of the responses to testosterone. It is concluded that testosterone acts predominantly on a postjunctional nongenomic receptor to inhibit urinary bladder detrusor muscle contraction.

Pharmacological and molecular biological evidence for ETA endothelin receptor subtype mediating mechanical responses in the detrusor smooth muscle of the human urinary bladder

The aim of this study was to characterize endothelin receptor subtypes of the detrusor muscle of the human urinary bladder. The receptor subtypes mediating endothelin (ET)-1-induced activity in the human detrusor smooth muscles have been characterized using isometric contraction and reverse transcription-polymerase chain reaction (RT-PCR). ET-1 (a non-selective ET receptor agonist; 10(-10) M to 10(-6) M) exhibited concentration-dependent contractions in human urinary bladder with a plateau at concentrations above 3x10(-7) M. Neither IRL1620 nor sarafotoxin S6c (both ETB-selective agonists; 10(-10) M to 10(-6) M) elicited contractile activity in the human urinary bladder detrusor smooth muscle. FR139317 (an ETA-selective antagonist; 10(-7) M to 10(-5) M) produced a marked shift to the right of the ET-1 concentration-response curve in human urinary bladder detrusor smooth muscle (from the Schild plot TpA2=7.96; slope=0.95). In contrast, RES701-1 (an ETB-selective antagonist; 10(-7) M to 10(-5) M) had no effect on the ET-1 concentration-response curve. RT-PCR revealed positive amplification of ETA receptor mRNA fragment, but not ETB. These results indicate that the ET-1-induced contractile effects of urinary bladder detrusor smooth muscle seem to be mediated mainly by the ETA receptor, not by the ETB receptor.

Early Ambulation Reduces the Incidence of Urinary Retention after Spinal Anesthesia for Benign Anorectal Surgery

Background : It has been known that bed rest after spinal anesthesia for benign anorectal surgery helps to reduce the incidence of postdural puncture headache, but the bed rest position is thought to have a negative effect on urinary retention, as a result of anxiety, anal distention, bladder distention and pain-induced reflex inhibition of the urinary bladder detrusor muscle. We investigate the effect of early ambulation after spinal anesthesia on postdural puncture headache and on urinary retention compared with bed rest for 24 hours. Methods : One hundred and fifty-three ASA Physical Status Ⅰ patients received spinal anesthesia for scheduled benign anorectal surgery. We applied hyperbaric 0.5% tetracaine 6 mg (1.2 ml) using 25 gauge Quincke needles with the cutting bevel parallel to the dural fibers at the patients' sitting position. All patients were randomly divided into an early ambulation group or bed rest group. We investigated the incidence of urinary retention and headache. The duration of operation, the perioperative intravenous fluid volume, surgical technique and postoperative pain regimen were standardized. Results : The incidence of urinary retention in the early ambulation group (16/75, 21.3%) was lower than that in bed rest group (32/78, 41.0%). There was no difference in any parameters between the two groups. Conclusions : To reduce the incidence of urinary retention, early ambulation after spinal anesthesia for benign anorectal surgery is recommended over bed rest with no increase in the incidence of postdural puncture headache. (Korean J Anesthesiol 1999; 37: 1001∼1006)

Effect of exogenous adenosine and its monophosphate on the contractile response to acetylcholine in the human isolated detrusor muscle strips.

1. Adenosine (0.1-1 mM) or its 5'-monophosphate (5'-AMP) induced a concentration-dependent relaxation of tension caused by acetylcholine (0.2 microM) in human urinary bladder detrusor strips. 2. This effect was antagonized concentration dependently by theophylline at an apparent pA2 value of about 5. 3. Maximum relaxation by adenosine or 5'-AMP never exceeded 50% and 80%, respectively, of acetylcholine-induced tension. Relaxation by some beta2-adrenoceptor agonists (0.1-0.2 mM) or norepinephrine was limited to about 50% of maximum. 4. The responses to adenosine and terbutaline were additive, causing full relaxation, and suggesting mobilization of distinct mechanisms underlying muscle relaxation.

Neuromuscular transmission and innervation in the urinary bladder of the insectivore Suncus murinus

In isolated preparations of the urinary bladder detrusor of the house musk shrew Suncus murinus (order: insectivora; family: Soricidae), electrical field stimulation (0.5–32 pulses/s) evoked neurogenic contractile responses that were markedly attenuated by atropine (1 μM). The non-cholinergic component was reduced but not abolished by the P 2-purinoceptor antagonist, suramin (300 μM). Thus, neuromuscular transmission in the suncus urinary bladder is effected by cholinergic and purinergic nerves together with an as-yet unidentified component. Using immunohistochemical methods, the suncus urinary bladder was seen to be supplied by nerves containing neuropeptide Y, tyrosine hydroxylase, vasoactive intestinal polypeptide, galanin, substance P, calcitonin gene-related peptide and type I nitric oxide synthase. The pattern of responses to electrical field stimulation was more similar to that of humans and Old World primates, than to that of rodents or lagomorphs. The pattern of innervation of the bladder wall, in terms of the distribution of populations containing a given neuropeptide, was very similar to that in humans. Hence, Suncus murinus may provide a novel species for modelling the neuropharmacology of the human bladder, and also for studying the evolution of autonomic innervation.

Purinergic Modulation of Rat Urinary Bladder Detrusor Smooth Muscle

1. Rat detrusor muscle was responsive to both ATP and adenosine; ATP elicited an excitatory response, whereas adenosine had an inhibitory effect. 2. ATP and adenosine had an inhibitory modulatory action on responses to acetylcholine, potassium depolarization and field stimulation. 3. Quinidine inhibited the ATP response and blocked the inhibitory effect of ATP on acetylcholine, potassium-depolarization and field-stimulation responses. The effect of adenosine remained unaltered in the presence of quinidine. 4. Caffeine and theophylline blocked the adenosine inhibition of responses to field stimulation. 5. It is concluded that excitatory P 2-type purinoreceptors mediated by ATP and inhibitory P 1-type purinoreceptors mediated by adenosine exist in rat urinary bladder detrusor smooth muscle and that both ATP and adenosine exhibit a modulatory action on detrusor muscle agonist-induced responses.

Effect of several bicyclic peptide and cyclic pseudopeptide tachykinin NK2 receptor antagonists in the human isolated urinary bladder.

1. We have studied several tachykinin NK2 receptor antagonists, bearing a monocyclic pseudopeptide (MEN 10,508, MEN 10,573, MEN 10,581, MEN 10,612, MEN 10,619 and MEN 10,677), or bicyclic peptide (MEN 10,627, MEN 10,692, MEN 10,771, MEN 10,882 and MEN 10,993) structure, on the human isolated urinary bladder detrusor muscle against neurokinin A as an agonist, and compared their affinities in this preparation with those for NK2 receptors expressed in the rabbit isolated pulmonary artery and hamster isolated trachea. 2. In the human bladder, all the antagonists tested produced a concentration-dependent and competitive antagonism of neurokinin A-mediated contractions: among the cyclic pseudopeptides MEN 10,677 (pKB = 8.0) was the most potent antagonist, while among the bicyclic analogues it was MEN 10,993 (pKB = 8.8). 3. In general, the bicyclic peptide antagonists tested were more potent than the monocyclic pseudopeptide compounds, either in the human urinary bladder or in the rabbit pulmonary artery or hamster trachea, showing a nanomolar affinity for the human NK2 receptor. 4. A highly significant correlation was found between the estimated pKB values of all the antagonists tested in the human urinary bladder and rabbit pulmonary artery (r2 = 0.94, n = 12, P < 0.01), whereas no linear correlation was found between pKB values measured in the human urinary bladder and hamster trachea (r2 = 0.52, n = 12, P > 0.05): these observations provide further pharmacological evidence for receptor homology between the human and rabbit NK2 receptor. 5. The present results point out the class of NK2 receptor antagonists bearing a bicyclic peptide structure, like MEN 10,627, as candidates for testing in pathological conditions, such as bladder hyperactivity, for which preclinical evidence indicates that a therapeutic effect could result from the block of the tachykinin NK2 receptor.

Characterization and autoradiographic localization of [ 3H]α,β-methylene ATP binding sites in cat urinary bladder

1. 1. The characteristics and distribution of [ 3h]α,β-methylene ATP ([ 3H]α,β-MeATP), a radioligand for P 2x-purinoceptors, binding sites in cat urinary bladder detrusor were examined. 2. 2. Saturation analysis revealed that, in cat bladder membrane preparations, only one population of binding sites with high affinity ( K d =1.8 nM) was present, in contrast to other species where both high-and low-affinity binding sites are present. Another feature is that the density of the binding sites in the cat bladder ( B max =21.2 pmol/mg protein) is considerably higher (about 2-fold) than the high-affinity binding component in the rat bladder membrane preparations. 3. 3. Displacement experiments with unlabelled purinoceptor ligands indicate that [ 3H]α,β-MeATP mainly binds to P 2x-purinoceptors. The order of binding displacement activity was: α,β-methylene ATP, β,γ-methylene ATP>2-methylthioATP>ATP>suramin and l-β,γ-methylene ATP>adenosine. 4. 4. Autoradiographic study demonstrated dense specific binding sites of [ 3H]α,β-MeATP on detrusor smooth muscle of cat bladder. 5. 5. The results of this study are consistent with pharmacological studies for the existence of P 2x-purinoceptors in cat bladder.

Age-related differential susceptibility to calcium channel blocker and low calcium medium in rat detrusor muscle: response to field stimulation.

The influences of aging on the response of urinary bladder detrusor smooth muscle in response to neurogenic stimulation (electric field stimulation) were investigated in rats aged 3-4 months (young) and 18-20 months (old). In addition, age-associated changes in calcium homeostasis were evaluated by comparing the basal intracellular free calcium concentration ([Ca2+]i), the change of [Ca2+]i in response to field stimulation, the contractile response at different extracellular calcium concentrations (Ca2+ = 1.8, 0.6, and 0 mM), and the sensitivity to the L-type calcium channel blocker. In both groups, the frequency-response curve in response to field stimulation (1-32 Hz) was similar. The contractile response to field stimulation was biphasic (phasic and tonic). The phasic/tonic ratio at different frequencies was similar between the two groups. The contractile responses were equally inhibited by atropine (1 microM) and preincubation with alpha, beta-methylene ATP (50 microM). Tetrodotoxin (1 microM) virtually abolished the responses to field stimulation in both groups. The biphasic components showed different sensitivities to diltiazem and low calcium. The tonic component was more readily inhibited by diltiazem, whereas the phasic component was more sensitive to low calcium medium. In general, the response to lower frequency stimulations was more sensitive to both diltiazem and low calcium. The aged group was more sensitive to the inhibitory effects of both low calcium and diltiazem. In Ca2(+)-free medium, the contractile response to higher frequency (e.g., 32 Hz) was temporarily preserved in most of the preparations. This response disappeared after repeated stimulation. The magnitude of this Ca2(+)-free response to 32 Hz stimulation was significantly lower in the aged group than in the young group (18.1 +/- 1.4 vs.29.1 +/- 3.2% of the control response in normal Tyrode medium, P < 0.05). The basal [Ca2+]i levels were similar in the two groups (young: 131 +/- 12 nM, n = 5; old: 118 +/- 10 nM, n = 4, P > 0.05). The magnitude and time course of the [Ca2+]i response to field stimulation were also similar in the two groups. It is concluded that aging seems to have little effect on the neuromuscular activities of rat urinary bladder. However, the calcium homeostasis may be altered as evidenced by the increased sensitivity to diltiazem and low extracellular calcium.