AN ATROPINE-LIKE INHIBITORY EFFECT OF DMPP ON RAT ISOLATED URINARY BLADDER

• 1 DMPP inhibits the nerve-mediated contractions of the rat isolated bladder, its effect being greater in preparations from newborn (2 day old) than adult animals. This effect of DMPP was unaffected by hexamethonium. In preparations from adult animals the effect of DMPP increased with frequency of stimulation and was fully prevented by the presence of atropine.
• 2 In bladders from newborn rats low concentrations of furthrethonium (FHR) (10 nM) activated a series of rhythmic contractions which were unaffected by tetrodotoxin and abolished by DMPP through an hexamethonium-insensitive action. On the other hand DMPP did not affect rhythmic contractions produced by a low concentration of eledoisin (60 nM).
• 3 In bladders from adult rats FHR (10 μM) and KCl (30 mM) produced contractures of comparable magnitude. DMPP inhibited, in concentration-related manner the FHR-induced tonic contraction but had little effect on that produced by KCl.
• 4 These findings indicate that in the rat bladder, DMPP antagonizes selectivity cholinergically-mediated contractions through a mechanism which is unaffected by hexamethonium or tetrodotoxin. An ‘atropine-like’ activity of DMPP should be considered.

     

Effects of cooling on cholinergically-mediated contractions of tracheal muscle taken from immature and mature guinea pigs]

The tracheal strip-chain preparations taken from immature (less than 2 weeks old) and mature (greater than 16 weeks old) guinea pigs were used to study the cooling effects of bathing temperature on cholinergically-mediated contractions. In the tracheal muscles from immature animals, cooling of the bathing temperature from 37 degrees C to 20 degrees C augmented both the contractions induced by electrical stimulation (0.05-2 Hz, 0.6 msec, 150 mA) of intramural cholinergic nerves and contractions in response to acetylcholine (0.01-3 microM), while the cooling inhibited the carbachol (0.01-3 microM)-induced contractions. The pretreatment of the tissue with physostigmine (0.05 microM) also augmented both contractions induced by cholinergic nerve stimulation and those in response to acetylcholine (0.01-3 microM), but the cooling did not produce further augmentation of the responses. On the other hand, the tracheal muscles from mature animals showed a reduced contraction in response to cholinergic nerve stimulation by cooling, although the response was potentiated by the physostigmine pretreatment. Histochemical distributions of acetylcholinesterase activities were more marked in the tracheal muscles of immature guinea pigs than in mature ones. From these results, we conclude that increased responsiveness by cooling of the isolated immature guinea pig tracheal muscle to cholinergic nerve stimulation or exogenous acetylcholine may involve the decrease of intramural acetylcholinesterase activities which might be decreased age-dependently.     

The effect of omega conotoxin GVIA,a peptide modulator of the N-type voltage sensitive calcium channels,on motor responses produced by activation of efferent and sensory nerves in mammalian smooth muscle

1. The effect of omega-conotoxin (CTX) GVIA, a peptide which blocks neuronal calcium channels, were investigated on nerve-mediated motor responses in a variety of isolated smooth muscle preparations from rats and guinea-pigs. 2. In the rat or guinea-pig isolated vas deferens CTX (1 nM-1 microM) produced a concentration and time-related inhibition of the response to field stimulation, while the responses to KCl, noradrenaline or adenosine triphosphate were unaffected. In the presence of CTX a series of tetrodotoxin-resistant contractions could be elicited by field stimulation by increasing pulse width and/or voltage. 3. In the rat or guinea-pig isolated urinary bladder, CTX produced a concentration and time-dependent inhibition of twitch responses to field stimulation without affecting the response to exogenous acetylcholine. In the rat bladder the maximal effect did not exceed 25% inhibition while a much larger fraction of the response (about 70%) was inhibited in the guinea-pig bladder. The CTX-resistant response was abolished, in both tissues, by tetrodotoxin. 4. The effects of CTX in the rat bladder were also studied with a whole range of frequencies of field stimulation (0.1-50 Hz). Maximal inhibition was observed toward contractions elicited at frequencies of 2-5 Hz. At low frequencies the inhibitory effects of CTX and atropine were almost additive while at high frequencies of stimulation a large component of the atropine-sensitive response was CTX-resistant. 5. In the rat isolated proximal duodenum, field stimulation in the presence of atropine and guanethidine produced a primary relaxation followed by a rebound contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different effect of antiulcer agents on rat cysteamine-induced duodenal ulcer after sialoadenectomy, but not gastrectomy

An attempt has been made to pharmacologically isolate cholinergic, P(2) purinoceptor-mediated and peptidergic (capsaicin-sensitive, tachykinin-mediated) contraction of the guanethidine-treated rat bladder detrusor preparation, in vitro. The effect of experimental diabetes was assessed on these types of contraction. Responses were evoked by electrical field stimulation (single shocks or 1 Hz for 30 s or 10 Hz for 40 s). Single shocks and 1-Hz stimulation were applied in the presence of (a). atropine (1 microM) or (b). P(2) purinoceptor antagonists (50 microM pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid) [PPADS] plus 100 microM suramin. Long-term electrical field stimulation (10 Hz for 40 s) (c). was applied with both atropine and the P(2) purinoceptor antagonists present in the organ bath. The effects of capsaicin (d). and ATP (e). were also studied. Three groups of experimental animals were used: streptozotocin-treated (50 mg.kg(-1) i.p., 8 weeks before the experiment), parallel solvent-treated and untreated rats. (a). Responses to electrical field stimulation in the presence of atropine were reduced by half by PPADS plus suramin, but were resistant to capsaicin tachyphylaxis. They were enhanced in preparations taken from diabetic rats. (b). Contractions to electrical field stimulation in the presence of PPADS plus suramin were reduced by 2/3 by atropine, but were left unchanged by capsaicin or diabetes. (c). Contractions to long-term stimulation had a quick and a sustained phase. Especially the latter was inhibited by capsaicin tachypyhlaxis; it was also strongly reduced in preparations taken from diabetic rats. (d). Contractions to capsaicin (30 nM and 1 microM) were resistant to tetrodotoxin, strongly reduced by a combination of tachykinin NK(1) and NK(2) receptor antagonists, and slightly reduced in preparations from diabetic animals. Capsaicin (1 microM) had no acute inhibitory action on cholinergic or purinergic responses, nor did it cause relaxation in precontracted preparations treated with tachykinin receptor antagonists. (e) ATP-induced contractions were strongly reduced by PPADS plus suramin (50 plus 100 microM) and to a similar degree by 100 plus 200 microM, respectively. It is concluded that experimental diabetes selectively impairs peptidergic, capsaicin-sensitive responses (especially those that involve impulse conduction) in the rat detrusor preparation. The contractile response to electrical field stimulation that remains after atropine plus the P(2) purinoceptor antagonists has a yet unknown transmitter background.     

Visceromotor responses to calcitonin gene-related peptide (CGRP) in the rat lower urinary tract: evidence for a transmitter role in the capsaicin-sensitive nerves of the ureter

Either intra-arterial or topical administration of calcitonin gene-related peptide (CGRP) had little effect on motility of the urinary bladder in urethane-anaesthetized rats. Only a high concentration (50 microM) of topical CGRP activated the micturition reflex and potentiated the response to exogenous substance P (SP). In the isolated rat bladder CGRP had inconsistent effects on spontaneous or field-stimulated contractions. CGRP neither produced any significant plasma extravasation (Evans blue leakage) in the rat lower urinary tract, nor potentiated the response to exogenous SP. CGRP inhibited motility in the rat isolated proximal urethra and ureters and counteracted the contractile response to neurokinins. An inhibitory effect of capsaicin on stimulated motility of the urethra was observed in all preparations and a small contractile response was evident in about 40% of cases. Lack of desensitization to the action of CGRP prevented the study of its interaction with capsaicin. The inhibitory effect of CGRP in the ureter exhibited a specific desensitization: if the preparations were pre-exposed to exogenous CGRP, the inhibition of motility produced by antidromic activation of the capsaicin-sensitive nerve terminals (field stimulation) as well as the response to capsaicin (1 microM) was prevented but the inhibitory response to isoprenaline was unaffected. These findings indicate that CGRP is able to influence markedly the motility of the rat lower urinary tract, but exhibits marked regional differences in its action. Endogenous CGRP could be the inhibitory transmitter which, when released from capsaicin-sensitive fibers, participate in the control of ureteral motility.     

Contractile mechanisms coupled to TRPA1 receptor activation in rat urinary bladder

TRPA1 is a member of the transient receptor potential (TRP) channel family present in sensory neurons. Here we show that vanilloid receptor (TRPV1) stimulation with capsaicin and activation of TRPA1 with allyl isothiocyanate or cinnamaldehyde cause a graded contraction of the rat urinary bladder in vitro. Repeated applications of maximal concentrations of the agonists produce desensitization to their contractile effects. Moreover, contraction caused by TRPA1 agonists generates cross-desensitization with capsaicin. The TRP receptor antagonist ruthenium red (10-100 microM) inhibits capsaicin (0.03 microM), allyl isothiocyanate (100 microM) and cinnamaldehyde (300 microM)-induced contractions in the rat urinary bladder. The selective TRPV1 receptor antagonist SB 366791 (10 microM) blocks capsaicin-induced contraction, but partially reduces allyl isothiocyanate- or cinnamaldehyde-mediated contraction. However, allyl isothiocyanate and cinnamaldehyde (10-1000 microM) completely fail to interfere with the specific binding sites for the TRPV1 agonist [(3)H]-resiniferatoxin. Allyl isothiocyanate or cinnamaldehyde-mediated contractions of rat urinary bladder, which rely on external Ca(2+) influx, are significantly inhibited by tachykinin receptor antagonists as well as by tetrodotoxin (1 microM) or indomethacin (1 microM). Allyl isothiocyanate-induced contraction is not changed by atropine (1 microM) or suramin (300 microM). The exposure of urinary bladders to allyl isothiocyanate (100 microM) causes an increase in the prostaglandin E(2) and substance P levels. Taken together, these results indicate that TRPA1 agonists contract rat urinary bladder through sensory fibre stimulation, depending on extracellular Ca(2+) influx and release of tachykinins and cyclooxygenase metabolites, probably prostaglandin E(2). Thus, TRPA1 appears to exert an important role in urinary bladder function.     

Neuropeptide Y in rat detrusor and its effect on nerve-mediated and acetylcholine-evoked contractions.

1. Immunohistochemical and isolated organ bath techniques were used to detect the presence of neuropeptide Y (NPY) in the rat urinary bladder and to determine its effect on tone, spontaneous activity and contractile responses of the detrusor muscle to electrical field stimulation, acetylcholine and alpha,beta-methylene ATP (alpha,beta-MeATP). 2. A very rich presence of NPY-immunoreactive nerve fibres was found mainly within the bundles of detrusor muscle cells. Chronic treatment with 6-hydroxydopamine did not affect the density of NPY-positive nerve fibres. 3. NPY (> 1 nM) enhanced the force and frequency of spontaneous contractions and generated a rise in the resting tone of the detrusor. These effects of NPY on the tone and the spontaneous activity remained unaffected by atropine (3 microM), indomethacin (10 microM) and aspirin (100 microM) but were abolished by Ca(2+)-withdrawal from the bathing medium. 4. The enhancing effects of NPY on the spontaneous contractions and the resting tone were not prevented by the induction of purinoceptor desensitization. 5. NPY (1-250 nM) potentiated electrical field stimulation (EFS, 1-64 Hz, 0.1 ms pulses duration, 10s train duration)-evoked, tetrodotoxin (0.5 microM)-sensitive contractions. The atropine (3 microM)-resistant component of EFS-evoked contractions was also potentiated by NPY. By contrast, the nifedipine (1 microM)-resistant but atropine-sensitive component of EFS-evoked contraction was inhibited by NPY. 6. NPY (250 nM) did not affect acetylcholine-evoked contractions, but potentiated alpha,beta-MeATP-evoked contractions. 7. It is concluded that NPY-innervation of rat urinary bladder is largely confined to the detrusor muscle and is abundant and mainly non-adrenergic.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of topical capsaicin on rat urinary bladder motility in vivo

The effects of topical capsaicin on urinary bladder motility were investigated following saline-induced distension of the bladder wall in urethane-anaesthetized rats and compared to the effects of topical substance P and acetylcholine. Capsaicin and substance P produced similar excitatory effects in both quiescent and rhythmically contracting bladders, i.e., a TTX resistant tonic contraction followed by a series of rhythmic, TTX sensitive, phasic contractions. Acetylcholine, in doses equieffective in producing TTX resistant contractions was less effective than capsaicin or substance P in triggering neurogenic rhythmic contractions of bladder muscle. Atropine pretreatment prevented the neurogenic component of the excitatory effect of both capsaicin and substance P. Repeated applications of capsaicin but not of substance P led to desensitization. Bladders of animals pretreated (4 days before) with a large dose of s.c. capsaicin developed insensitivity to topical capsaicin and a larger volume of saline was required to trigger neurogenic rhythmic contractions of the detrusor muscle. These results suggest that capsaicin acts by interfering with the mechanism(s) regulating the threshold for the micturition reflex to occur.     

Involvement of bradykinin in trypsin-induced urinary bladder contraction in cyclophosphamide-treated rats

Protease-activated receptor-2 (PAR-2) is activated by serine proteases, such as trypsin and mast cell tryptase. Previous studies have demonstrated that both trypsin and PAR-2 activating peptide contract isolated rat urinary bladder preparations, however, the mechanisms are not fully understood. In the present study, we examined the role of bradykinin in contractions induced by trypsin and the PAR-2 agonist 2-furoyl-LIGRL-NH(2) in urinary bladders isolated from control or cyclophosphamide (CYP)-induced cystitis rats. The contractile effects of trypsin were significantly greater in the preparations obtained from CYP-treated rats than in those from controls. The bradykinin B2 receptor antagonist Hoe 140 did not affect trypsin-induced contractions in control rat bladders, whereas it significantly reduced the contractile effects of trypsin on bladders from CYP-treated rats. On the other hand, Hoe 140 failed to affect contractions induced by the PAR-2 agonist 2-furoyl-LIGRL-NH(2). These results suggest that the actions of trypsin on urinary bladders in cystitis rats are partly exerted through stimulation of bradykinin B2 receptor in a PAR-2-independent manner. This mechanism seems to be involved in the enhancement of trypsin-induced bladder contractions observed after induction of cystitis with CYP in rats.     

Four motor effects of capsaicin on guinea-pig distal colon.

The motor effects of capsaicin on the guinea-pig distal colon have been investigated in vivo and in vitro. Capsaicin (0.1-10 micrograms kg-1 i.v.) produced a transient relaxation which was reduced by pretreatment with capsaicin itself, atropine, hexamethonium, phentolamine or guanethidine and almost abolished by tetrodotoxin (TTX). Topically applied capsaicin produced a transient inhibition of tone and spontaneous activity prevented by topically applied TTX. In isolated preparations of distal colon, capsaicin produced a transient, TTX- and atropine-sensitive contraction which was followed by a depression of the contractile activity. The depressant effect was unaffected by atropine plus guanethidine but was greatly reduced by TTX, indicating activation of intramural non-adrenergic, non-cholinergic (NANC) mechanisms. The depressant effect on the first exposure to capsaicin (1 microM) was greater than that produced by a second, third or fourth exposure. In preparations excised from capsaicin-pretreated animals, capsaicin (1 microM) only produced an inhibitory effect on spontaneous contractions. Desensitization did not occur to this inhibitory effect. In preparations pre-exposed to capsaicin (1 microM, 1 h before), capsaicin (1-30 microM) produced a concentration-related inhibition of spontaneous contractions (IC50 = 19 microM) and of the high K+-induced tonic contraction (IC50 = 23 microM). A similar effect on spontaneous motility was produced by capsaicin in colonic segments excised from capsaicin-pretreated guinea-pigs (IC50 = 16 microM) or guinea-pigs treated with TTX (IC50 = 20 microM). It is concluded that, in vivo, capsaicin activates inhibitory reflexes, presumably due to stimulation of primary afferent fibres. This effect involves, at least in part, activation of sympathetic nerves to this organ. The contractile effect of capsaicin on the isolated colon involves activation of intramural cholinergic neurones, whereas the TTX-sensitive component of the inhibitory effect involves either release of an inhibitory transmitter through an axon reflex arrangement or activation of NANC neurones. In addition, at high concentrations capsaicin produces a direct depression of smooth muscle contraction.     

Extrinsic origin of the capsaicin-sensitive innervation of rat duodenum: possible involvement of calcitonin gene-related peptide (CGRP) in the capsaicin-induced activation of intramural non-adrenergic non-cholinergic neurons

Capsaicin produces a concentration-related relaxation of the longitudinal muscle of the rat isolated duodenum in the presence of atropine (3 microM) plus guanethidine (3 microM). This effect of capsaicin is partly (about 40%) antagonized by tetrodotoxin (1.0 microM) suggesting the involvement of intramural non-adrenergic non-cholinergic (NANC) neurons. The capsaicin-induced relaxations are unaffected by previous bilateral vagotomy or removal of the inferior mesenteric ganglion but are completely prevented by removal of the coeliac ganglia plus the superior mesenteric ganglion (72 h before). Acute duodenal denervation did not modify the response to capsaicin. Unlike various neuropeptides (substance P, kassinin, neurokinin A, cholecystokinin octapeptide, somatostatin, vasoactive intestinal polypeptide) only the calcitonin gene-related peptide (CGRP) closely mimicked, both qualitatively and quantitatively, the capsaicin-induced relaxations. The CGRP-induced relaxations were unaffected by hexamethonium and partly reduced (about 40%) by tetrodotoxin. In preparations desensitized to adenosine-triphosphate (ATP) a putative NANC inhibitory neurotransmitter of the rat duodenum, the effects of CGRP were reduced (about 30%) as compared to controls. After ATP-desensitization tetrodotoxin did not produce any further reduction of the CGRP-induced relaxations suggesting the involvement of endogenous ATP in the neuronal (tetrodotoxin-sensitive) component of the CGRP-induced relaxations. Either ATP- or CGRP-desensitization reduced (about 50 and 65% respectively) the amplitude of the capsaicin-induced relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of the capsaicin-sensitive innervation of the rat urinary bladder in the activation of micturition reflex

Capsaicin applied on the serosal surface of the urinary bladder in urethane-anaesthetized rats produces two distinct types of motor effects: a tetrodotoxin-, hexamethonium- and lidocaine-insensitive 'tonic' contraction and a series of tetrodotoxin-, hexamethonium- and lidocaine-sensitive rhythmic contractions. Both 'tonic' and rhythmic contractions are abolished by bladder denervation indicating their neurogenic origin. The rhythmic but not the 'tonic' component of the contractile effect of capsaicin is abolished by spinal cord transection indicating activation of a supraspinal micturition reflex. The motor effects of topical capsaicin are unaffected by pretreatment with indomethacin or diphenhydramine plus cimetidine. Pretreatment with a large dose of subcutaneous (SC) capsaicin increases both volume and pressure threshold for micturition while amplitude of micturition contraction is unaffected. Moreover the spinal somatovesical reflex elicited by pinching of the perineal skin is unaffected by capsaicin-desensitization. The intracerebroventricular (ICV) administration of capsaicin reproduces the effects of SC capsaicin on the bladder response to saline filling. Rats pretreated with ICV capsaicin are as sensitive as controls in reacting to noxious heat (hot plate test) while the wiping response to instillation of capsaicin into one eye was abolished. These findings provide functional evidence for the presence in the rat urinary bladder of a capsaicin-sensitive innervation which subserves a sensory function in relaying volume/pressure information from detrusor muscle to central nervous system. Information carried through these capsaicin-sensitive fibers appears to be relevant for initiation of a supraspinal vesico-vesical micturition reflex. Functional evidence indicates that these fibers may terminate at supraspinal level.     

The role of sensory neuropeptides in motor innervation of the hamster isolated urinary bladder

In this study we have characterized the role of sensory fibers and of the sensory peptides, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP), on the contractile responses evoked by single pulse electrical field stimulation (EFS) in the hamster urinary bladder. EFS of the hamster isolated urinary bladder produced twitch contractions which were unaffected by atropine but abolished by tetrodotoxin. The P2 purinoreceptor antagonist PPADS (30 microM) inhibited twitches by 66+/-4% on its own and by 78+/-3% in the presence of atropine. The selective tachykinin NK2 receptor antagonist nepadutant produced a slight but consistent reduction of twitch amplitude (-21+/-3%) at 1 microM. Addition of nepadutant to atropine and PPADS did not further increase their inhibitory effect. The application of hCGRP (10-300 nM) produced a concentration-dependent inhibition of twitches (Emax -38+/-3%, EC50=12 nM) and a small reduction of tone (0.5+/-0.09 mN). Similar effects were obtained with capsaicin (0.1-10 microM) which inhibited EFS-evoked contractions with an EC50 of 100.0 nM and a maximal effect of 34+/-4% inhibition at 1 microM. Under submaximal parameters of stimulation NKA (10 nM) increased the amplitude of twitches by 45+/-6% and produced a concentration-dependent tonic contraction (EC50=55.9 nM). The CGRP1 receptor subtype antagonist, hCGRP(8-37), increased by 29+/-8% the EFS-evoked contractions and significantly reduced the response to 0.1 microM CGRP. Capsaicin (10 microM) increased both CGRP-LI and NKA-LI release from superfused slices of hamster urinary bladder by about sixfold and by about 70%, over baseline, respectively. A second application of capsaicin was ineffective, indicating a complete desensitization of sensory nerve efferent function. In the hamster urinary bladder the sensory neuropeptides NKA and CGRP are co-released by sensory fibers after stimulation either by EFS or capsaicin. However, the role of CGRP appears functionally predominant.     

Prejunctional facilitatory alpha 1-adrenoceptors in the rat urinary bladder.

1. The effect of activation of alpha 1-adrenoceptors on acetylcholine (ACh) release and neurally evoked contractile responses induced by electrical field stimulation was investigated in smooth muscle strips from the rat urinary bladder. 2. Neurogenic contractions were facilitated by the alpha 1-adrenoceptor agonists, phenylephrine (PE) (2-128 microM) and methoxamine (2-128 microM) in a dose-dependent manner. These agents also increased small amplitude spontaneous contractions of bladder strips and in 10% of strips increased basal tone. However, contractions elicited by exogenous ACh (1-10 microM) were not affected by alpha 1-agonists. 3. The magnitude of the PE facilitation was higher at lower frequencies (1-5 Hz) or at submaximal intensities of stimulation and at lower Ca2+ concentrations (0.5-1 mM). The selective alpha 1-adrenoceptor antagonist, terazosin (TRZ) (0.05-1 microM), competitively inhibited (pA2 value: 8.6) the PE facilitation of the neurally evoked contractions but not the PE induced increase of spontaneous contractions. 4. [3H]-noradrenaline (NA) and [14C]-ACh release evoked by electrical field stimulation were increased (140% and 173%, respectively) by 2 microM PE. TRZ (0.05-0.1 microM) blocked the PE facilitation of ACh release but not the facilitation of NA release. TRZ alone did not alter the release of ACh or NA nor the amplitude of the neurogenic contractions. 5. PE (2 microM) did not alter the basal release of ACh but did increase (by 180%) the basal release of NA. Desipramine (2 microM) blocked this effect of PE and also the PE-facilitation of evoked ACh and NA release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurogenically mediated contractions of dog basilar artery involve the release of a thromboxane-like substance

Electrical field stimulation of dog isolated basilar artery produced neurogenically mediated contractions which were unaffected by phentolamine (1 microM), atropine (1 microM), ketanserin (1 microM) or methiothepin (0.1 microM). Responses were abolished by GR32191 (1-10 nM), BM 13.177 (0.1-10 microM) or flurbiprofen (0.5 microM) and markedly attenuated by dazoxiben (1-10 microM). Removal of the endothelium by Triton X-100-perfusion did not modify the magnitude of contractions to electrical stimulation and GR32191 still abolished the responses. GR32191 (1-10 nM) did not modify neurogenically mediated contraction of rabbit ear artery or potassium chloride-induced contraction of dog basilar artery. The results suggest that electrical field stimulation of dog basilar artery causes contractions which are mediated via a cyclo-oxygenase product with characteristics similar to thromboxane. This thromboxane-like substance is not endothelial in origin, nor released by contraction of the cerebrovascular smooth muscle per se and is therefore derived from a subendothelial, possibly neuronal, source.     

Contractile effects of cysteamine on the guinea-pig ileum

Cysteamine (beta-mercaptoethylamine HCl) (1.0-40.0 mM) induced a concentration-dependent increase in tonic and phasic contractions of segments of guinea-pig ileum in vitro. Myenteric plexus-longitudinal muscle (MPLM) preparations also responded with an increase in tonic contractions but phasic contractions were either greatly reduced or absent, indicating that these were a response of the circular muscle. Atropine (5 microM) inhibited the cysteamine-induced contractions, whereas hexamethonium and guanethidine had no effect, suggesting that cysteamine was acting at least partly via a cholinergic mechanism involving muscarinic receptors. Tetrodotoxin increased the phasic contractions of ileal segments, but had no effect on the tonic component. Treatment of MPLM preparations with morphine (1 microM) resulted in a small reduction in responsiveness to cysteamine, and blocked electrically-induced contractions by at least 90%. Since morphine acts by inhibiting acetylcholine release via hyperpolarization of intrinsic neurones, a small but significant part of the cysteamine-induced contractions probably resulted from stimulation of acetylcholine release from intrinsic neurones. Following a response to high cysteamine concentrations (greater than 15 mM) tissues were refractory to subsequent cysteamine administration. Cross-desensitization between cysteamine and acetylcholine also occurred, as short-term (1-3 min) incubation of MPLM preparations with high concentrations of either compound (1-10 microM acetylcholine or 20 mM cysteamine) resulted in a reduced responsiveness to both. A reduced sensitivity to acetylcholine or cysteamine was obtained following long-term (45 min) incubation with acetylcholine (1 microM). Removal of Na+ from the incubation medium negated this effect. In contrast, the refractoriness to acetylcholine or cysteamine following long-term (45 min) incubation with cysteamine (20 mM) was accentuated in low Na+ medium. It is suggested that cysteamine induces a contraction of both the circular and longitudinal muscle of the guinea-pig ileum by stimulating the release of acetylcholine from intrinsic neurones, by an action at the level of the smooth muscle muscarinic receptor, and possibly by a non-cholinergic mechanism. However, the mechanisms by which acetylcholine and cysteamine induce tissue refractoriness probably differ.     

Regional differences in the effects of capsaicin and tachykinins on motor activity and vascular permeability of the rat lower urinary tract

Summary 1. The effects of capsaicin, substance P (SP) and neurokinin A (NKA) on motor activity and vascular permeability was investigated in the rat lower urinary tract (bladder dome and neck, proximal urethra and ureters). 2. Capsaicin produced contractions of the rat bladder dome and neck and of the proximal urethra in vitro, which were unaffected by tetrodotoxin and abolished by ganglionectomy. SP and NKA were almost equipotent in producing a contraction of the rat isolated bladder dome or neck and urethra. However, the maximal response to NKA was about twice that of SP on the urethra and bladder neck. 3. Capsaicin did not affect motility of the unstimulated rat isolated ureter, while NKA or SP activated rhythmic contractions, NKA being about 850 times more potent than SP. Either capsaicin or field stimulation produced a transient inhibition of the NKA-activated rhythmic contractions of the rat isolated ureter which was prevented by capsaicin-desensitization. 4. The capsaicin-(1 M) or field stimulation-induced inhibition of NKA-activated rhythmic contractions of the rat isolated ureter were unaffected by removal of pelvic ganglia but abolished by cold storage (72 h at 4°C). 5. Intravenous capsaicin induced an inflammatory response (Evans blue leakage) in the bladder, proximal urethra and ureters in vivo. Plasma extravasation was greater in the ureters, urethra and bladder neck than in the dome. SP, NKA and histamine produced a dose-dependent dye leakage in all segments of the rat urinary tract, the response being slightly greater in the bladder neck than in the dome. 6. The capsaicin-induced inflammatory response was abolished by systemic capsaicin-desensitization and reduced, to a variable extent, by pelvic ganglionectomy, in the various tissues examined. Topical application of tetrodotoxin on the bladder dome failed to affect the capsaicin-induced plasma extravasation in the urinary bladder. 7. These findings indicate that chemoceptive, capsaicin-sensitive nerves are present throughout the whole rat lower urinary tract and their activation determines a variety of visceromotor responses and an increase of vascular permeability. In various instances the response to capsaicin may be explained by the action of tachykinins but some effects may involve other sensory neuropeptides. Send offprint requests to C. A. Maggi at the above address     

The effects of Bay K 8644 and nifedipine on the responses of rat urinary bladder to electrical field stimulation, beta,gamma-methylene ATP and acetylcholine.

1. Bay K 8644 (0.33 nM to 1 microM) greatly increased the contractions of rat urinary bladder detrusor muscle induced by beta, gamma-methylene ATP (beta, gamma-MeATP, 10 microM) and by electrical field stimulation of the purinergic component (the cholinergic response was blocked by atropine). 2. The contractions induced by acetylcholine (ACh, 10 microM) and by electrical field stimulation of the cholinergic component (the purinergic response was blocked following desensitization by alpha, beta-MeATP) were also potentiated by Bay K 8644, although to a lesser extent than the purinergic responses. 3. Nifedipine (1 nM to 3.3 microM) inhibited all the contractions induced by beta, gamma-MeATP, ACh and electrical field stimulation. However, while the responses to beta, gamma-MeATP and electrical field stimulation of the purinergic component were almost abolished, a substantial proportion of the responses to ACh and electrical field stimulation of the cholinergic component were nifedipine resistant. 4. The concentration-effect curves for the potentiation by Bay K 8644 of the responses to beta, gamma-MeATP, ACh and electrical field stimulation were shifted to the right by nifedipine (10 nM). At concentrations greater than 1 microM, Bay K 8644 inhibited contraction. 5. It is concluded that voltage-sensitive calcium channels play an important role in the excitatory mechanical action of P2X-purinoceptor-mediated purinergic responses in the rat urinary bladder, while cholinergic-mediated responses are less dependent on such channels.     

Content and contractile effect of arginine vasopressin in rat urinary bladder

The contractile response of normal male rat urinary bladders to exogenous arginine vasopressin (AVP) and the AVP content of normal and denervated bladders were investigated. In isolated detrusor strips, the maximal response to AVP was about 12% of the contraction elicited by KCl (124 mM), and the EC50 value was 1.03 +/- 0.13 x 10(-8) M. The response to transmural nerve stimulation was not affected by the presence of AVP. Addition of an AVP receptor antagonist strongly reduced the response to exogenous AVP, but did not affect contractions in response to nerve stimulation. In normal bladders, the concentration of immunoreactive (ir) AVP was 29 +/- 6.0 x 10(-15) mol/g. Three days after denervation the bladders had increased 2.4-fold in weight. At this time, the concentration of irAVP was not different from the control value, but the total content had increased significantly. Characterization of bladder irAVP by reverse-phase HPLC revealed that 66.5% of the total immunoreactivity eluted in the position of synthetic AVP. The results suggest a non-neuronal localization of bladder irAVP.     

Non-cholinergic inotropic responses evoked by electric field stimulation in the isolated rat urinary bladder. Possible participation of substance P

The present study explores whether a peptide, such as substance P (SP), has some role subserving the atropine-resistant component of electrically-evoked contractions, in isolated rat urinary bladders. The electric field stimulation (EFS) employed herein, consisted in square wave pulses of 5 Hz, 50 ms duration and supramaximal voltage (40 V), applied for 10 sec, every 3 min and conducted to the tissue via a pair of platinum ring electrodes, surrounding the isolated preparations. In order to assess whether electric stimuli, induced urinary bladder inotropism through the activation of nerve structures, degeneration of intramural nerve elements was attempted by cooling the tissue (48 h at 4-5 degrees C). After such procedure, 80-90% inhibition of responses to EFS, was detected. Moreover, tetrodotoxin, at 10(-6) M, evoked similar effects than cooling. Atropine, at 10(-6) M, failed to produce a significant decrement of contractile responses, whereas at 10(-5) M, the electrically-induced inotropism declined around 40%, in comparison with controls. In another set of experiments, atropinized urinary bladders (atropine at 10(-5) M) were exposed to capsaicin (5 X 10(-6) M) and this coincided with decreased (-43%) responses to EFS. Next, SP, at 10(-9) M, was added to the medium containing capsaicin and complete restoration of full contractile responses to EFS, was observed. Inasmuch as it has been proposed that capsaicin releases SP from sensory nerve fibers and since our experiments show that SP restored the inotropism elicited by electric stimuli on capsaicin-exposed preparations, it is suggested that SP could be involved, at least in part, in the non-cholinergic, EFS-evoked, contractile responses of isolated rat urinary bladders.