Non-adrenergic Non-cholinergic Contraction Research Articles

Nicotine interferes with purinergic signaling in smooth muscle cells isolated from urinary bladders of patients with lower urinary tract symptoms

In patients with outlet obstruction, the contraction of the base is reduced compared to that of healthy individuals, while the contraction of the dome is not affected. Here, we investigated the cellular mechanisms that might be responsible for cholinergic effects blocking non-adrenergic non-cholinergic contractions in the base of the urinary bladder. Smooth muscle cells either from the base or from the dome of human urinary bladders were cultured to determine the contribution of cholinergic and purinergic mechanisms to their Ca2+ homeostasis. While ATP evoked Ca2+ transients in all the cells, nicotine and carbachol induced Ca2+ transients only in 56% and 44% of the cells, respectively. When ATP was administered together with nicotine or carbachol, the amplitudes of the Ca2+ transients recorded from cells prepared from the base of bladders were significantly smaller (42 ± 6% with nicotine and 56 ± 9% with carbachol) than those evoked by ATP alone. This inhibition was much less apparent in the dome of bladders. The inhibition between the cholinergic and purinergic signaling pathways reported in this work may decrease the strength of the contraction of the base of the urinary bladder in patients with outlet obstruction during voiding.

Diabetic plasticity of non-adrenergic non-cholinergic and P2X-mediated rat bladder contractions

We investigated the plasticity effects of diabetes mellitus and diuresis on the non-adrenergic non-cholinergic (NANC) and purinergic (P2X-type) contractile responses in longitudinal rat bladder strips. Female Sprague-Dawley rats received streptozotocin to induce diabetes, or sucrose in water to induce diuresis as a control condition for polyuria. Experiments were carried out at four weeks after treatments, using bladders from non-treated rats as control. Urinary bladder strips were electrically stimulated throughout the experiments to generate neurally evoked contractions (NEC). In all cases, P2X-mediated purinergic contractions were evaluated at the beginning and end of the stimulations with α,β-methylene-adenosine triphosphate (α,βMeATP). The NANC responses were assessed by using two independent protocols. First, cholinergic receptors were activated with carbachol (CCh), followed by inhibition of the muscarinic component with atropine. In the second protocol, the application order for CCh and atropine was reversed. The NANC response, unmasked with the application of atropine, and the P2X purinergic contractions were analyzed. NANC contractions in diabetic bladder strips are more resistant to the desensitizing effects caused by activation of cholinergic receptors. In early stages of experimental diabetes, NANC responses in diabetic strips are less sensitive to functional inhibition mediated by the cholinergic activation. However, P2X-mediated purinergic contractions are more sensitive to desensitization in diabetic or diuretic bladders. For instance preventing muscarinic receptor activation with atropine does not counteract the desensitization of purinergic contractions in either diabetic or diuretic strips. We suggest that diabetes may induce a plasticity of the NANC and P2X-mediated bladder contractile responses. The first one may be associated with diabetic neuropathic damage to bladder nerves, while impaired P2X purinergic contractions might be associated with detrusor hypertrophy observed in diabetic and diuretic strips.

D-serine modulates non-adrenergic non-cholinergic contraction of lower esophageal sphincter in rats

Endogenous d-serine is known to modulate glutamatergic transmission via interaction with the glycine site of N-methyl-d-aspartate (NMDA) receptors. d-serine is synthesized by racemization of l-serine using an enzymatic reaction catalyzed by serine racemase. Although much attention has been focused on the role of d-serine within the central nervous system, the physiological role of d-serine in enteric nervous system has not been investigated. Lower esophageal sphincter (LES) function is known to be modulated by NMDA-dependent mechanisms. The present study was aimed to study the expression of enzymes involved in d-serine metabolism and the function of d-serine in lower esophageal sphincter in rats. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting showed the expression of serine racemase in isolated rat LES. Electrical field stimulation was used to induce non-adrenergic non-cholinergic (NANC) contraction/relaxation of isolated rat LES in an organ bath using an isometric force transducer. The organ bath studies on isolated rat LES showed that incubation with d-serine (100μM) is associated with a significant increase in the NANC contraction of isolated LES. This effect of exogenous d-serine was inhibited by NMDA receptor antagonists (MK-801), suggesting that NMDA receptors are involved in the effects of d-serine on NANC contraction of LES. Incubation with d-serine did not show a significant effect on NANC relaxation within our experimental setting. The results of this study suggest that serine racemase is expressed in LES and d-serine modulates contraction of the lower esophageal sphincter in rats.

Modulation of TRPV1-dependent contractility of normal and diabetic bladder smooth muscle by analgesic toxins from sea anemone Heteractis crispa

AimsTRPV1-expressing, capsaicin (CAP)-sensitive afferent fibers innervating bladder in addition to sensory function also exhibit “efferent” features consisting in TRPV1-dependent release of tachykinins (TAC) affecting detrusor smooth muscle (DSM) contractions. Our aim was to investigate the effects of two novel polypeptide inhibitors of TRPV1 from the venom of tropical sea anemone Heteractis crispa, APHC1 and APHC3, on the contractions of DSM from bladders of normal and diabetic rats. Main methodsExperiments were conducted on urothelium-devoid DSM strips from normal rats and rats 8weeks after streptozotocin-induced diabetes by means of contraction force measurements. Key findingsPre-exposure of DSM strips to APHC1 or APHC3 (200nM) specifically inhibited CAP-induced, TRPV1-dependent contractions. Both peptides also transiently enhanced basal tone and spontaneous contractions of DSM strips followed by delayed suppression of electric field stimulation (EFS)-evoked nonadrenergic–noncholinergic (NANC) contractions. The decrease of the amplitude of EFS-evoked NANC contractions by АРНС1 or АРНС3 reached 38.5±3.4% and 25.1±1.6%, respectively, in normal DSM strips and 46.3±3.3% and 43.9±1.8%, respectively, in diabetic ones. APHC-peptide-induced transient enhancement of basal tone could be mimicked by serine protease inhibitor, 4-(2-aminoethyl)bezenesulfonyl fluoride (300μM). SignificanceOur results demonstrate that АРНС1 and АРНС3 may be considered as effective inhibitors of bladder contractility especially during diabetic cystopathy. Modality of action of APHC-polypeptides via the mechanisms involving decreased TRPV1-dependent release of TAC from bladder afferents and suppression of TAC degradation due to their activity as endogenous proteases inhibitors is proposed.

A possible role of the cholinergic and purinergic receptor interaction in the regulation of the rat urinary bladder function

The contractile activation of the upper (dome) and lower (base) parts of the urinary bladder show some differences. Cellular mechanisms that might be responsible for cholinergic effects blocking non-adrenergic non-cholinergic contractions in the base of the rat urinary bladder were investigated. Smooth muscle cells were thus freshly isolated or cultured both from the dome and the base of the rat urinary bladder and the contribution from cholinergic and purinergic pathways to their Ca(2+) homeostasis was examined. The expression of nicotinic acetylcholine (nAChR) and P2X2 purinergic receptors on the cultured cells and on tissue sections was investigated. The ATP-evoked Ca(2+) transients in rat smooth muscle cells did not show any desensitization. However, when ATP was administered together with carbamylcholine (CCh), the latter essentially prevented ATP from evoking Ca(2+) transients in smooth muscle cells from the base (suppression to 12 ± 2.5% of control, n = 57; p < 0.01), but not from the dome (99 ± 5% of control, n = 52; p > 0.05) of the rat urinary bladder. While atropine was unable to modify (6 ± 3% of control, n = 14; p < 0.05), α-bungarotoxin (118 ± 12% of control, n = 20; p > 0.05) blocked the inhibitory effects of CCh. Additionally, α7 subunits of nAChR and P2X2 purinergic receptors were identified using immunocytochemistry, immunohistochemistry, and Western blot in cultured urinary bladder smooth muscle cells, in urinary bladder sections, and in urinary bladder muscle strips, respectively, suggesting that the activation of nAChR modifies the action of ATP.

Plasticity of non-adrenergic non-cholinergic bladder contractions in rats after chronic spinal cord injury

The purpose of this study was to examine the pharmacologic plasticity of cholinergic, non-adrenergic non-cholinergic (NANC), and purinergic contractions in neurogenic bladder strips from spinal cord injured (SCI) rats. Bladder strips were harvested from female rats three to four weeks after T9–T10 spinal cord transection. The strips were electrically stimulated using two experimental protocols to compare the contribution of muscarinic and NANC/purinergic contractions in the presence and the absence of carbachol or muscarine. The endpoints of the study were: (1) percent NANC contraction that was unmasked by the muscarinic antagonist 4-DAMP, and (2) P2X purinergic contraction that was evoked by α,β-methylene ATP. NANC contraction accounted for 78.5% of the neurally evoked contraction in SCI bladders. When SCI bladder strips were treated with carbachol (10μM) prior to 4-DAMP (500nM), the percent NANC contraction decreased dramatically to only 13.1% of the neurally evoked contraction (P=0.041). This was accompanied by a substantial decrease in α,β-methylene ATP evoked P2X contraction, and desensitization of purinergic receptors (the ratio of subsequent over initial P2X contraction decreased from 97.2% to 42.1%, P=0.0017). Sequential activation of the cholinergic receptors with carbachol (or with muscarine in neurally intact bladders) and unmasking of the NANC response with 4-DAMP switched the neurally evoked bladder contraction from predominantly NANC to predominantly cholinergic. We conclude that activation of muscarinic receptors (with carbachol or muscarine) blocks NANC and purinergic contractions in neurally intact or in SCI rat bladders. The carbachol-induced inhibition of the NANC contraction is expressed more in SCI bladders compared to neurally intact bladders. Along with receptor plasticity, this change in bladder function may involve P2X-independent mechanisms.

Activation of cholinergic receptors blocks non-adrenergic non-cholinergic contractions in the rat urinary bladder

In the present study, the plasticity of the non-adrenergic non-cholinergic (NANC) response was investigated. Isolated rat bladder strips were electrically stimulated and the evoked contractions were isometrically recorded. The NANC part of the contractions were unmasked by applying 500 nM 4-DAMP, a potent muscarinic antagonist. Treatment of the bladder strips with 10 μM carbachol (a cholinergic agonist) increased the muscle tone but did not alter the neurally evoked contractions. However, carbachol decreased: (1) the NANC response from 74.6% to 33.3% of control and (2) the purinergic contractile response to α,β-methylene ATP (α,β-mATP) (10 μM) from 97.0% to 43.4% ( p < 0.05). Treatment with the cholinesterase inhibitor eserine (10 μM) also significantly decreased the NANC response to 21.1% ( p < 0.0001). The purinergic receptor antagonist suramin (100 μM) did not affect the neurally evoked contractions, however; subsequent addition of 4-DAMP decreased the contractions to 31%. Activation of the smooth muscle cholinergic receptors (with carbachol or eserine) and purinergic receptors (with α,β-mATP) decreased the NANC contractions and the direct contractile response to α,β-mATP. When the electrically evoked contractions were facilitated by the L-type Ca 2+ channel activator, Bay-K 8644 the subsequent application of 4-DAMP did not unmask inhibited NANC contractions. We conclude that activation of muscarinic receptors by cholinergic agonist, carbachol or by endogenous acetylcholine (ACh) induce a cascade of events that leads to diminished purinergic response and consequently an inhibition of the bladder NANC response.

Role for NK 1 and NK 2 receptors in the motor activity in mouse colon

The present study examined the effects induced by endogenous and exogenous activation of NK 1 and NK 2 receptors on the mechanical activity of mouse proximal colon. Experiments were performed in vitro recording the changes in intraluminal pressure from isolated colonic segments. Electrical field stimulation in the presence of atropine and guanethidine produced a small relaxation, followed by nonadrenergic noncholinergic (NANC) contraction. SR140333, NK 1 receptor antagonist, or SR48968, NK 2 receptor antagonist, significantly reduced the contraction, although SR48968 appeared more efficacious. The co-administration of SR140333 and SR48968 virtually abolished the NANC contraction. [Sar 9, Met(O 2) 11]-substance P, selective NK 1 receptor agonist, induced a concentration-dependent biphasic effect, contraction followed by reduction of the mechanical spontaneous activity. Both effects were antagonized by SR140333, but not by SR48968. [β-Ala 8]-neurokinin A (4-10), selective NK 2 receptor agonist, evoked concentration-dependent contraction, which was antagonized by SR48968, but not by SR140333. The contraction induced by [Sar 9, Met(O 2) 11]-substance P, but not by [β-Ala 8]-neurokinin A (4-10), was reduced by tetrodotoxin or atropine, and increased by N ω-nitro-L-arginine methyl ester (L-NAME), inhibitor of nitric oxide synthase. The inhibitory effects induced by [Sar 9, Met(O 2) 11]-substance P were abolished by tetrodotoxin or L-NAME. The results of the present study suggest that in mouse colon both NK 1 and NK 2 receptors are junctionally activated by endogenous tachykinins to cause an additive response. NK 1 receptors appear to be located on cholinergic and on nitrergic neurons as well as on smooth muscle cells, whereas NK 2 receptors seem to be present exclusively on smooth muscle cells.

Functional characterisation of tachykinin receptors in the circular muscle layer of the mouse ileum

Objectives Tachykinins are important mediators in neuromuscular signalling but have not been thoroughly characterised in the mouse gut. We investigated the participation of tachykinin receptors in contractility of circular muscle strips of the mouse ileum. Results Electrical field stimulation (EFS) of excitatory nonadrenergic noncholinergic (NANC) nerves induced frequency-dependent contractions which were mimicked by substance P (SP). Desensitisation of SP and NK 1, NK 2 or NK 3 receptors significantly reduced contractions to EFS. The NK 1 receptor blocker RP67580 significantly inhibited NANC contractions to EFS. The NK 2 and NK 3 receptor blockers nepadutant and SR142801 did not affect NANC contractions per se but increased the RP67580-induced inhibition of NANC contractions to EFS. Contractions to SP were significantly reduced by RP67580 but not affected by nepadutant or SR142801. The NK 1 and NK 2 receptor agonists, septide and [β-ala 8]-NKA 4-10 (β-A-NKA), respectively, but not the NK 3 receptor agonist senktide-induced dose-dependent contractions. Atropine inhibited and l-NNA augmented contractions to septide. Contractions to β-A-NKA were insensitive to atropine but augmented by l-NNA. Conclusions Tachykinins mediate NANC contractions to EFS in the mouse small intestine. Endogenously released tachykinins activate mainly NK 1 receptors, located on cholinergic nerves and smooth muscle cells and, to a lesser degree, NK 2 and NK 3 receptors, most likely located presynaptically.

Interactions of volatile anesthetics with cholinergic, tachykinin, and leukotriene mechanisms in isolated Guinea pig bronchial smooth muscle.

We studied relaxation of airway smooth muscle by sevoflurane, desflurane, and halothane in isolated guinea pig bronchi. Ring preparations were mounted in tissue baths filled with physiological salt solution and continuously aerated with 5% CO(2) in oxygen. Electrical field stimulation induced contractions sensitive to tetrodotoxin, indicating nerve-mediated responses. These consisted of an atropine-sensitive cholinergic phase and a nonadrenergic noncholinergic (NANC) phase sensitive to SR48968, a neurokinin-2 receptor antagonist. Anesthetics were added to the gas aerating the tissue baths. Sevoflurane and desflurane at 1.0 minimum alveolar anesthetic concentration and halothane at 1.0-2.0 minimum alveolar anesthetic concentrations inhibited both cholinergic and NANC contractions to electrical field stimulation. None of the anesthetics affected responses to exogenously applied neurokinin A, a likely mediator of NANC contractions, suggesting prejunctional inhibition of NANC neurotransmission. The anesthetics did not affect the initiation of contractile responses to leukotriene C(4) (LTC(4)), a mediator of asthmatic bronchoconstriction. However, sevoflurane and desflurane both relaxed bronchi in a steady-state contraction achieved by LTC(4). Surprisingly, halothane did not relax LTC(4) contractions. Concerning LTC(4)-elicited bronchoconstriction, sevoflurane and desflurane were more potent airway smooth muscle relaxants in vitro. Halothane, sevoflurane, and desflurane attenuated airway smooth muscle tone via inhibition of cholinergic and nonadrenergic noncholinergic neurotransmission. Sevoflurane and desflurane reduced leukotriene C(4)-induced bronchoconstriction, whereas halothane did not. This indicates a beneficial role for sevoflurane and desflurane in asthmatics.

TACHYKININERGIC EXCITATORY NEUROTRANSMISSION IN THE PIG INTRAVESICAL URETER

The present investigation was designed to study the role played by neurokinin A (NKA) in the non adrenergic non cholinergic (NANC) neurotransmission of the pig intravesical ureter. We used immunohistochemical techniques to evidence the distribution of NKA-immunoreactive (NKA-IR) fibers in the pig intravesical ureter. We have also performed isolated organ bath experiments to release endogenous tachykinins from ureteral nerves and to characterize the functionally active receptor through which endogenous ligands evoke contraction, and to show the effect of exogenous tachykinins on intravesical ureteral smooth muscle. NKA-IR fibers were found penetrating through ureteral adventitia and distributed in the subepithelial and muscular layers. NKA-IR fibers were not found around small arteries supplying the ureter or in the associated intramural ganglia. Electrical field stimulation (EFS, 1 ms duration, 2 to 16 Hz, 20 s trains) performed in NANC conditions evoked frequency-dependent contractions which were reduced by capsaicin (10-5 M) and GR 94800 (3 x 10-8 M), sensory neurotoxin and NK2 receptor antagonist, respectively. Contractions to EFS were abolished by tetrodotoxin (10-6 M). Exogenous NKA and substance P (SP) induced dose-dependent contractions, characterized by an increase of the ureteral basal tone, NKA being more potent than SP. These results suggest that tachykinins, especially NKA, released from capsaicin-sensitive primary afferents, are involved in the NANC excitatory neurotransmission, contracting the smooth muscle via NK2 receptors activation, in the pig intravesical ureter. NKA at this level does not seem to participate in the regulation of local blood flow, plasmatic extravasation or ganglionar transmission.

Altered non-adrenergic non-cholinergic neurotransmission in gastric fundus from streptozotocin-diabetic rats

The influence of streptozotocin-induced diabetes has been investigated on responses to non-adrenergic, non-cholinergic (NANC) nerve stimulation in rat gastric fundus. NANC relaxations in precontracted muscle strips from diabetic rats were smaller than those from control rats. In addition, the relaxations in diabetic but not control rats were followed by rapidly-developing frequency-dependent contractions. In the presence of α-chymotrypsin and N g -nitro- l-arginine methyl ester ( l-NAME), the NANC contractions were markedly enhanced in diabetic rats. Treatment with the aldose reductase inhibitor, sorbinil, did not affect NANC relaxations or contractions in tissues from diabetic rats, and responses remained significantly different from those from control rats. The findings suggest that diabetes impairs relaxations to NANC nerve stimulation in the rat gastric fundus, and that a contractile NANC neurotransmitter(s) is released in diabetic rats. The results also suggest that diabetes-induced alterations in the NANC nerve response are not caused by increased activity of the aldose reductase pathway.

Nonadrenergic, noncholinergic responses of the rabbit detrusor smooth muscle and the role of L-arginine/nitric oxide pathway in these responses.

In the present study, nonadrenergic, noncholinergic (NANC) responses of rabbit detrusor smooth muscle and the possible involvement of the L-arginine/nitric oxide (L-ARG/NO) pathway was investigated. In the presence of atropine (10(-6) M) and guanethidine (10(-5) M), frequency-response curves were obtained by stimulating tissue with 10-sec trains at increasing frequencies (1-10 Hz) with 3-min intervals between stimulations. Electrical field stimulation (EFS) evoked a biphasic response in rabbit detrusor smooth muscle, consisting of an initial contraction followed by relaxation. ATP desensitization significantly inhibited contractions. L-NAME (10(-5) M) increased the contractions by a maximum of 33 +/- 5% at 1 Hz, 37 +/- 5% at 2 Hz, 18 +/- 4% at 4 Hz, 20 +/- 3% at 8 Hz and 15 +/- 4% at 10 Hz. In detrusor preparations, exposure to L-NAME (NG-nitro-L-arginine methyl ester, 10(-5) M) significantly reduced the maximal relaxation to electrical stimulation to 7 +/- 3% of the control. In the presence of L-ARG (10(-4) M), contractions induced by electrical field stimulation (EFS) at 1, 2, 4, 8 and 10 Hz were reduced by 20 +/- 4, 24 +/- 3, 25 +/- 3, 20 +/- 3 and 30 +/- 5%, respectively. Exposure to L-ARG (10(-4) M) significantly increased the maximal relaxation to electrical stimulation to 52 +/- 5% of the control. Exogenously applied ATP (10(-5)-10(-2) M) to rabbit detrusor muscle resulted in contractions while sodium nitroprusside (10(-7)-10(-3) M) caused concentration-dependent relaxation. These results suggest that nitric oxide (NO) acts as an inhibitory NANC neurotransmitter in the rabbit detrusor smooth muscle. Additionally, NANC contractions mediated by ATP released from NANC nerves, may be masked by the L-ARG/NO pathway in this tissue.

Excitatory transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of cannabinoid CB1 receptors.

1. The effect of cannabinoid drugs has been investigated on cholinergic and non-adrenergic non-cholinergic (NANC) contractile responses to the circular smooth muscle of guinea-pig ileum elicited by electrical field stimulation (EFS). 2. The cannabinoid receptor agonist WIN 55,212-2 (1-1000 nM) and the putative endogenous ligand anandamide (0.1-100 microM) both produced a concentration-dependent inhibition of the cholinergic (9-57% and 1-51% inhibition) and NANC (9 55% and 2-57% inhibition) contractile responses. WIN 55,212-2 and anandamide did not modify the contractions produced by exogenous acetylcholine or substance P. 3. Apamin (30 nM), a blocker of Ca2+-activated K+ channels, reduced the inhibitory effect of WIN 55,212-2 on cholinergic, but not NANC, contractile response. NG-nitro-L-arginine methyl ester (100 microM), an inhibitor of nitric oxide synthase, or naloxone (1 microM), an opioid receptors antagonist, did not modify the inhibitory effect of WIN 55,212-2 on both cholinergic and NANC contractions. 4. The inhibitory effects of WIN 55,212-2 and anandamide on both cholinergic and NANC contractile response was competitively antagonized by the cannabinoid CB1 receptor antagonist SR 141716A (10-1000 nM). 5. In absence of other drugs, SR 141716A (1-1000 nM) enhanced cholinergic (1-45% increase) and NANC (2-38% increase) contractile responses elicited by electrical stimulation, but did not modify the contractions produced by acetylcholine or substance P. 6. It is concluded that activation of prejunctional cannabinoid CB1 receptors produces inhibition of cholinergic and NANC excitatory responses in the guinea-pig circular muscle. The inhibition of cholinergic (but not NANC) transmission involves activation of apamin-sensitive K+ channels. In addition, an endogenous cannabinoid ligand could inhibit cholinergic and NANC transmission in the guinea-pig ileal circular muscle.

Cross tolerance between nitroglycerin and neural relaxation of the rabbit sphincter of Oddi

We studied whether non-adrenergic, non-cholinergic (NANC) relaxation of the rabbit sphincter of Oddi was influenced by tolerance to nitroglycerin (NG)in vitro. Sphincter of Oddi (SO) muscle rings precontracted with EC50concentrations of cholecystokinin octapeptide (CCK8) were exposed to cumulative increases in NG concentrations and tested for relaxation by measurement of isometric tension. A separate group of six rings was subjected to a preceding exposure to 275 μmnitroglycerin over 60 min to inducein vitrotolerance to nitroglycerin. The rings (both tolerant and non-tolerant) were subjected to electrical field stimulation (FS: 50 V, 0.1 ms, 20 Hz, 3 and 10 stimuli). The rings were then preincubated with NANC solution: phentolamine, oxprenolol and atropine (all 1 μm) for 20 min and FS was applied again. FS was repeated after additional incubation withNG-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthase (30 μm) and after a successive incubation with 3 mml-arginine (20 min). Maximum contractions produced by CCK8 in `tolerant' and ‘non-tolerant’ sphincters were 29.9±5.8 and 28.3±5.2 mN, respectively. The sensitivity to CCK8 also was not different between the two groups with EC50(−log M) values of 8.5±0.2 and 8.3±0.1, respectively. FS evoked twitchlike contraction followed by relaxation in the ampullary SO in both `tolerant' and `non-tolerant' preparations. Incubation in NANC solution resulted in monophasic relaxations in response to FS in non-tolerant sphincters but not in tolerant ones.l-NAME (30 μm) reversed NANC relaxation in non-tolerant muscle rings whereas it failed to modify NANC contractions in the tolerant preparations.l-arginine (3 mm) reversed the inhibitory effect ofl-NAME on NANC relaxation in the `non-tolerant' rings and it was without effect on FS-induced contractions in the `tolerant' SO. As measured by radioimmunoassay, tolerance to NG was without any significant effect on tissue content of both cyclic adenosine 3′:5′ monophosphate (cAMP) and cyclic guanosine 3′:5′ monophosphate (cGMP). FS significantly increased tissue cAMP and cGMP content in ‘non-tolerant’ preparations. FS failed to increase the level of either cyclic nucleotide in `tolerant' tissue. We conclude that NANC relaxation of the ampullary part of the rabbit SO is significantly impaired in the state of tolerance to NG`in vitro'.

EVIDENCE OF ADENOSINE 5′-TRIPHOSPHATE RELEASE FROM NERVE AND P2X-PURINOCEPTOR MEDIATED CONTRACTION DURING ELECTRICAL STIMULATION OF RAT URINARY BLADDER SMOOTH MUSCLE

Purpose We provided direct evidence for the existence of purinergic innervation in the rat urinary bladder. Materials and Methods The non-adrenergic non-cholinergic (NANC) innervation was studied in 4-month-old Wistar rats. Electric-field stimulation (EFS) of the detrusor muscle strips in the presence of four autonomic blockers (atropine 10 sup −6 M, guanethidine 10 sup −6 M, phentolamine 10 sup −6 M and propranolol 10 sup −6 M) showed NANC contractions accounted for about 50% of the maximum contractile response. The adenyl purines released from nerves by EFS were detected by HPLC after conversion to ethenopurines. The amount of total purine released was frequency-dependent and could be totally suppressed by tetradotoxin (10 sup −6 M). The amount of ATP released was significantly greater than those for ADP, AMP and adenosine (p <0.05, n = 4). Desensitization induced by alpha, beta-MeATP (10 sup −6 to 10 sup −4 M), a P2x receptor agonist, reduced the NANC contraction. In addition, the NANC contraction was also abolished by P2 receptor blocker suramin (10 sup −4 to 10 sup −3 M) and P2x receptor blocker PPADS (10 sup −5 to 10 sup −4 M.). Conclusion The results of the present study give evidence to support purinergic nerve-mediated bladder smooth muscle contractions in the rat. Among the purine nucleotides, ATP is the dominant purinergic neurotransmitter released and P2x receptor activation is responsible for the NANC contractile response.

Tachykinin NK 1 and NK 2 receptors mediate non-adrenergic noncholinergic excitatory neuromuscular transmission in the human ileum

Tachykinin NK 1 and NK 2 receptor selective antagonists and agonists were used to study excitatory nonadrenergic non-cholinergic (NANC) transmission in circular muscle strips from human ileum by the sucrose-gap method. In the presence of atropine (1 μM), guanethidine (3 μM), indomethacin (3 μM), apamin (0.1 μM) and N ω-nitro-L-arginine (L-NOARG, 30 μM), electrical field simulation (EFS) produced a NANC inhibitory junction potential (i.j.p.) followed by NANC excitatory junction potential (e.j.p.) with superimposed action potentials and contraction of the circular muscle of human ileum. The selective tachykinin NK 1 receptor antagonist, GR 82334 (0.1–3 μM) produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. (IC 50 = 0.21 μM) and contraction (IC 50 = 0.21 μM). The selective tachykinin NK 2 receptor antagonist, MEN 10627 (0.01–1 μM), likewise produced a concentration-dependent inhibition of the EFS-evoked NANC e.j.p. (IC 50 = 0.07 μM) and contraction (IC 50 = 0.03 μM). Either antagonist was more effective in inhibiting the mechanical than the electrical response to EFS. Neither GR 82334 nor MEN 10627 had any effect on the apamin- and L-NOARG-resistant NANC i.j.p. Activation of the NK 1 or NK 2 receptors by the selective receptor agonists, [Sar 9]substance P (SP) sulfone and [βAla 8]neurokinin A (NKA) (4–10), respectively (0.3 μM for 20 s each), produced depolarization with superimposed action potentials and contractions. GR 82334 selectively inhibited the responses to [Sar 9]]SP sulfone, without affecting the responses to [βAla 8]NKA (4–10). MEN 10627 inhibited the responses to [βAla 8]NKA (4–10), without affecting the responses to [Sar 9]SP sulfone. We conclude that both tachykinin NK 1 and NK 2 receptors co-operate in producing NANC excitation and contraction of the circular muscle in human ileum.

CGRP potentiates excitatory transmission to the circular muscle of guinea-pig colon

We aimed to assess whether calcitonin gene-related peptide (CGRP) can modulate the release of tachykinins which are the main nonadrenergic noncholinergic (NANC) excitatory transmitters to the circular muscle of the guinea-pig proximal colon. In organ bath experiments, electrical field stimulation (EFS) in the presence of atropine (1 μM) and guanethidine (3 μM) evoked twitch phasic NANC contractions which were abolished by the combined administration of tachykinin NK 1 and NK 2 receptor antagonists. Human αCGRP (CGRP, 1–100 nM) produced a concentration-dependent potentiation of the amplitude of the NANC contractions induced by EFS while salmon calcitonin (up to 1 μM) had no effect. The potentiating effect of CGRP was unaffected by in vitro capsaicin pretreatment (10 μM for 15 min), peptidase inhibitors (captopril, bestatin and thiorphan, 1 μM each), apamin (0.3 μM) plus l-nitroarginine ( l-NOARG, 100 μM) and by the CGRP 1 receptor antagonist, the C-terminal fragment CGRP(8–37) (1 μM). The NK 2 receptor antagonist MEN 10627 which, when administered alone, had only a partial inhibitory effect on the amplitude of NANC twitches, concentration-dependently (10 nM–1 μM) inhibited the potentiating effect of CGRP. CGRP (1–100 nM) produced a concentration-dependent potentiation of the atropine-sensitive cholinergic contractions evoked by EFS in the presence of guanethidine and of tachykinin NK 1 and NK 2 receptor antagonists. Similar to the effect of CGRP, application of capsaicin (0.1–1 μM) potentiated the amplitude of the NANC contraction to EFS, an effect undergoing complete desensitization upon a second application of the drug. CGRP (0.1 μM) did not affect the contractile action of a submaximally effective concentration of neurokinin A (2 nM) while it inhibited that induced by substance P (2 nM). In sucrose gap, single pulse EFS in the presence of atropine (1 μM) and guanethidine (3 μM) induced an inhibitory junction potential (i.j.p.) and a small excitatory junction potential (e.j.p.). CGRP (0.1 μM) produced membrane hyperpolarization and relaxation without affecting i.j.p. amplitude but concomitantly increased the e.j.p. amplitude to induce a contraction in correspondence to each electrical pulse. In the presence of the NK 1 receptor antagonist, GR 82334 (3 μM), the membrane hyperpolarization and relaxation produced by CGRP and the EFS-evoked i.j.p. were unaffected, while the potentiating effect of CGRP on the EFS-evoked NANC e.j.p. and the corresponding contraction were abolished. We conclude that, in addition to the previously characterized direct smooth muscle relaxant action via CGRP 1 receptors (Maggi et al. Regulatory Peptides 61, 27–36, 1996), CGRP also induces a remarkable potentiation of excitatory neurotransmission to the circular muscle of the guinea-pig colon via CGRP 2 receptors. The latter effect, documented in this study, is evidenced on both the atropine-sensitive and the atropine-resistant (tachykinin-mediated) components of excitatory transmission: this effect does not involve mediator(s) release from capsaicin-sensitive primary afferent nerves, nor inhibition of peptide degradation or modulation of NANC inhibitory transmission.

Evidence for Non-adrenergic Non-cholinergic Contractile Responses in Bovine and Swine Trachea

Non-adrenergic non-cholinergic (NANC) contraction of airway smooth muscle has been observed in some but not all animal species. The aim of this study was to investigate the NANC-contractile responses in bovine and swine trachea. Proximal and distal bovine and swine trachea were cut in strips and placed in 10 ml organ baths equilibrated in Krebs Henseleit (KH) solution and electrically stimulated (10 sec, 60 V, 2 ms, 4, 10 and 30 Hz). Contractile frequency response curves performed in the presence of the muscarinic antagonist, atropine (100 mM), the angiotensin converting enzyme inhibitor, captopril (1 μM) and the neutral endopeptidase inhibitor, thiorphan (1 μM), added 30 min prior to electrical field stimulation (EFS). In some tissues, incubated with atropine thiorphan and captopril, were also evaluated the effects of a pretreatment with capsaicin (10 μM) or a selective NK1receptor antagonist, SR 14033 (100 nM) added to the baths 30 min prior to EFS. Bovine and swine proximal and distal tracheal preparations contracted in a frequency-dependent manner to EFS (4, 10 and 30 Hz). Some experiments were also performed with substance P (0.1nM to 1 μM) in absence or in presence of SR 14033 (10 nM or 100 nM). At the maximum frequency tested (30 Hz), the contractile response elicited in bovine proximal and distal preparations was 194.5±17.1% and 229.7±24.1%, of ACh (100 μM), respectively. Similarly, the contractile response elicited by EFS (30 Hz) in swine proximal and distal preparations was 187.2±12.1% and 181.6±9.2% of ACh (100 μM), respectively. In tissues incubated with atropine, a significant decrease in smooth muscle sensitivity to EFS was observed (P<0.05). When tissues were pretreated with captopril and thiorphan, a significant increase in the contractile response to EFS (30 Hz) was observed in all tested tissue preparations (bovine, proximal 210.1±14.4%, distal 264.3±16.2%; swine, proximal 199.3±14.9%, distal 206.3±16.2%,P<0.05). In the presence of atropine, captopril and thiorphan a significant increase in the contractile response was observed in bovine and swine distal preparations compared with tissues incubated with atropine only (P<0.05). These effects were antagonized by a pretreatment with a selective NK1receptor antagonist, SR 14033. A pretreatment with capsaicin statistically (P<0.05) enhanced EFS-induced contraction in all tested preparations respect to tissues incubated with atropine, thiorphan and captopril. Substance P induced a concentration dependent contraction of bovine and swine isolated tracheal preparations which was antagonized by a pretreatment with a selective NK1receptor antagonist, SR 14033. No significant difference in the contractile potency (EC50) nor in maximum response (Emax) was observed to exogenously administered substance P between proximal and distal tracheal preparations. These data suggest that NANC contractile responses are present in bovine and swine trachea and are more evident in distal airways.

Activation of large conductance potassium channels inhibits the afferent and efferent function of airway sensory nerves in the guinea pig.

Sensory nerves play an important role in airway disease by mediating central reflexes such as cough, and local axon reflexes resulting in the peripheral release of neuropeptides. We have tested whether the benzimidazolone compound, NS1619, an opener of large conductance calcium-activated potassium (BK Ca) channels, inhibits the activity of sensory fibers, and central and local airway reflexes in guinea pig airways. In in vitro single fiber recording experiments, NS1619 applied to identified receptive fields in the trachea inhibited the firing of A(delta)-fibers evoked by hypertonic saline and distilled water, and bradykinin-evoked firing of C-fibers. Electrically evoked nonadrenergic noncholinergic contractions of isolated bronchi mediated by the release of neurokinin A (NKA) from C-fibers, but not those elicited by exogenous NKA, were inhibited by NS1619. These effects of NS1619 were prevented by iberiotoxin, a selective blocker of BK Ca channels. In conscious guinea pigs, cough evoked by aerosolized citric acid was also inhibited by NS1619. These data show that BK Ca channel activation inhibits sensory nerve activity, resulting in a reduction of both afferent and efferent function. BK Ca channel openers may therefore be of potential benefit in reducing neurogenic inflammation and central reflexes seen during inflammatory conditions of the airways, and may represent a new class of antitussive drug.