Effect of papaverine on synaptic transmission in the guinea-pig ileum

1. The effect of papaverine, a well known smooth muscle relaxant, was investigated on neural transmission within the enteric nervous system. Segments of guinea-pig ileum were placed in a partitioned bath to enable drugs, including papaverine, to be applied to enteric nerve pathways without interfering with the recording of the smooth muscle contraction. Ascending excitatory enteric nerve pathways were activated by electrical field stimulation in the anal compartment (10 Hz for 2 s, 45 mA, 0.5 ms pulse duration) and the resulting contraction of the intestinal circular muscle in the oral compartment was recorded isotonically.
2. Tetrodotoxin (0.6 μM) and hexamethonium (100 μM) both abolished, or greatly reduced, the contractions when applied to either compartment indicating that nicotinic synapses are involved in this pathway.
3. Papaverine (0.3–30 μM) applied independently to each compartment depressed in a concentration-dependent manner, the nerve-mediated contractions. The IC₅₀ of this inhibitory effect was 3.53 μM for the oral and 4.76 μM for the anal compartments, respectively. Two other phosphodiesterase (PDE) inhibitors, 3-isobutyl-l-methylxanthine (IBMX 10–300 μM) and theophylline (30–1000 μM) added to the anal compartment also inhibited the nerve mediated contractions. Papaverine applied to the anal bath, after IBMX 100 μM (or theophylline 300 μM) further inhibited the nerve-mediated contractions, but was less effective than when applied alone.
4. Phentolamine (1 μM), an α-adrenoceptor antagonist, reduced the inhibitory effect of papaverine, but not that of IBMX (100 μM) or theophylline (300 μM). A combination of phentolamine and IBMX (or theophylline) prevented the inhibitory effect of papaverine.
5. Tetrodotoxin, but not papaverine or hexamethonium, inhibited the contraction elicited by electrical stimulation just anal to the partition indicating that papaverine did not affect the generation or conduction of nerve action potentials.
6. Verapamil (1 μM) and nifedipine (1 μM), two smooth muscle relaxants which act by blocking L-type calcium channels, only inhibited the contractions when applied directly to the recording (oral) compartment. This indicates that L-type Ca²⁺ channels are probably not involved in synaptic transmission in these ascending pathways and thus that the PDE inhibitors do not inhibit synaptic transmission by acting on these channels. ω-Conotoxin GVIA (10 nM), a potent inhibitor of the N-type Ca²⁺ channels, blocked the nerve-mediated contractions applied to either compartment. Whether the PDE inhibitors exert their inhibitory actions via these channels remains to be established.
7. The results indicate that the PDE inhibitors, papaverine, IBMX and theophylline inhibit excitatory enteric neural pathways by depressing synaptic transmission. The inhibitory effect of papaverine (but not IMBX or theophylline) involves, at least in part, the release of noradrenaline from sympathetic nerves acting on α-adrenoceptors on enteric neurones.

     

Substance P-induced autodesensitization inhibits atropine-resistant, neurotensin-stimulated contractions in guinea-pig ileum

The sites of action of γ-aminobutyric acid (GABA) were examined in preparations of the distal colon and ileum of guinea pigs. GABA caused transient relaxations of the longitudinal and circular muscle of the colon and transient constractions followed by relaxation of the muscle of the ileum. The responses of both parts of the intestine were antagonized by tetrodotoxin and by bicuculline. Nerve-free preparations of the longitudinal muscle of the ileum were not affected by GABA, even in concentrations up to 10^?4 g/ml. There was a marked tachyphylaxis of the responses to GABA. Relaxations in response to GABA were not affected by pentolinium or by a combination of phetolamine and propranolol. Contractions in response to GABA were blocked by hyoscine. Neither the ascending excitatory or descending inhibitory components of the peristaltic reflex in the distal colon were affected by bicuculline or by making the intestine tachyphylactic to GABA. The results indicate that GABA stimulates receptor mediating excitation of cholinergic excitatory and enteric inhibitory neurons whereas it has littel or no direct effect on the muscle of the ileum or distal colon.     

Effect of some phosphodiesterase inhibitors on central dopamine mechanisms.

The effect of five phosphodiesterase (PDE) inhibitors (papaverine, IBMX, theophyllamine, dipyridamol and M & B 22,948) was studied on adenylate cyclase and on cyclic nucleotide phosphodiesterase activities in extracts of rat caudate nucleus. For comparison the effect on DA turnover and on turning behaviour in rats with unilateral lesions of the nigro-neostriatal DA nerurons was studied. Cyclic AMP PDE was inhibited by papaverine, dipyridamol, IBMX, M & B 22,948 and theophyllamine in that order of potency. Cylcic GMP PDE was inhibited by IBMX, papaverine, M & B 22,948 and theophyllamine, but not by dipyridamol. Basal adenylate cyclase washigher if assayed in the presence of papaverine or dipyridamol than if theophyllamine or IBMX was present. The degree of stimulation caused by DA was not significantly influenced by the PDE inhibitors. Papaverine and dipyridamol enhanced DA disappearance in the caudate nucleus and the tuberculum accumbens, but not in the median eminence. Caffeine had no significant effect. Papaverine (1-28 mg/kg) had no signigicant effect on L-dopa (5 mg/kg)-induced turning, and actually inhibited turning induced by the combination of L-dopa (10 mg/kg) and atropine (5 mg/kg). The other four PDE inhibitors all potentiated L-dopa-induced turning. Theophyllamine (20 mg/kg) and IBMX (5 mg/kg) even caused turning when given alone. The data are compatible with the opinion that PDE inhibition leads to an enhanced effect of DA in the caudate nucleus. However, the results also demonstrate that several of the PDE inhibitors have effects on central DA mechanisms that are difficult to explain solely on the basis of PED inhibition.     

Substance P enteric neurons mediate non-colinergic transmission to the circular muscle of the guinea-pig intestine

Summary The sites of action and possible roles of substance P in contracting the circular muscle of the guinea-pig ileum were studied using two analogues of substance P that act as antagonists of some of its actions. These ared-Arg¹,d-Pro²,d-Trp^7,9, Leu¹¹-substance P andd-Pro²,d-Trp^7,9-substance P, referred to by the single letter amino acid codes for the substituting amino acids as (RPWWL)-SP and (PWW)-SP, respectively.Records of circular muscle activity were taken from strips of intestine free of mucosa and submucosa and from rings with all layers of intestine intact. Substance P was equally effective in contracting the circular muscle strips as it was in contracting the longitudinal muscle. The contractions of strips were not blocked by hyoscine (2×10^–6 M) or tetrodotoxin (6×10^–7 M), but were substantially reduced by (RPWWL)-SP (6.7×10^–6 M) or (PWW)-SP (2×10^–5 M). In contrast, contractions of the circular muscle of whole rings of intestine elicited by low concentrations of substance P (4×10^–7M) were blocked by hyoscine or tetrodotoxin but notreduced by the substance P antagonists in the concentrations referred to above. These observations indicate that the antagonists are effective at receptors for substance P on the muscle, but not at substance P receptors on enteric cholinergic nerves.Transmural stimulation of strips of circular muscle or of intestinal rings in the presence of hyoscine evoked contractions that were blocked by tetrodotoxin. These hyoscineresistant, nerve-mediated contractions could be elicited by single pulses in the strips. The contractions were reduced to less than 20% of original amplitude by (RPWWL)-SP (6.7×10^–6M).Reflex contractions of the circular muscle recorded on the oral side of a distension stimulus had a low-threshold, hyoscine-sensitive and a high-threshold, hyoscine-insensitive, component. The low threshold component was unaffected by the substance P antagonists whereas the high threshold component was depressed.It is concluded that substance P nerves are effective in transmitting to the circular muscle, that they are final nerves in non-cholinergic excitatory reflexes, and that the substance P antagonist analogues can be used to distinguish actions of substance P at neural and muscle receptors.     

Evidence for two types of 5-hydroxytryptamine receptor on secretomotor neurons of the guinea-pig ileum

Summary Flat sheet preparations of the mucosa plus submucosa from the guinea-pig ileum were placed in Ussing chambers so that short circuit currrent (I _sc), an index of electrolyte movement across the mucosa, could be measured. In these preparations, 5-hydroxytryptamine (5-HT) increasesI _sc indirectly by stimulating both cholinergic and non-cholinergic secretomotor neurons. The 5-HT₃ receptor antagonist, ICS 205–930 (10^–13–10^–5 M), substantially depressed the secretory response due to 5-HT (10^–6 M), but not that produced by direct activation of muscarinic receptors on the mucosal epithelium with carbachol (10^–6 M), or by stimulation of secretomotor neurons with substance P (10^–8 M) or 1,1-dimethyl-4-phenylpiperazinium (10^–5 M). The residual response to 5-HT, after the addition of a maximally effective concentration of ICS 205–930 (10^–6 M), was further reduced by hyoscine (10^–7M). When that part of the 5-HT response attributable to the release of acetylcholine was blocked by hyoscine (10^–7M), ICS 205–930 did not further modify the response to 5-HT. The hyoscine-resistant component was, however, sustantially depressed by tetrodotoxin (3.5 × 10^–7 M). The response remaining after ICS 205–930 and hyoscine was not affected by methysergide (2 × 10^{– 5} M) or cyproheptadine (10^–7 M). We conclude that there are ICS 205–930 sensitive 5-HT receptors on cholinergic secretomotor neurons, and ICS 205–930, methysergide, and cyproheptadine insensitive 5-HT receptors on non-cholinergic secretomotor neurons.     

Effects of some inhibitors of phosphodiesterase on neuromuscular transmission

Summary Phosphodiesterase inhibitors antagonized the d-tubocurarine-induced paralysis of the indirectly stimulated rat phrenic nerve-diaphragm preparation. Ro 7-2956, papaverine and hydrochlorothiazide were active in 1/5 to 1/100 the concentrations of theophylline. This antagonism may be caused by the intracellular accumulation of c-AMP, which in turn may facilitate the release of acetylcholine from motor nerve endings.Some of these results have been presented at the Sixth Annual Meeting of Finnish Medical Societies, Helsinki, March 23–25, 1972.     

Enhancement of opioid inhibition of GABAergic synaptic transmission by cyclo-oxygenase inhibitors in rat periaqueductal grey neurones

Cyclo-oxygenase (COX) inhibitors potentiate opioid inhibition of GABAergic synaptic transmission in rat periaqueductal grey (PAG) (Vaughan et al., 1997). In the present study, the relative contribution of cyclo-oxygenase-1 (COX-1) and COX-2 inhibition to this phenomenon was examined by use of whole-cell patch clamp recordings in brain slices. The μ-receptor partial agonist morphine (10 μM) had little effect on GABAergic synaptic transmission. Morphine reduced the frequency of spontaneous miniature inhibitory postsynaptic currents (m.i.p.s.cs) by 13%. The nonselective COX inhibitor, indomethacin, produced a dose-dependent potentiation of the morpine inhibition of m.i.p.s.c. frequency (maximum inhibition 42%, IC₅₀=6 nM). More selective COX-2 inhibitors produced a similar potentiation of the morphine inhibition of m.i.p.s.c. frequency; however, at greater concentrations (IC₅₀=57 nM piroxicam, 1.7 μM DFU). Maintaining slices in the protein synthesis inhibitor cycloheximide (1 μM), to prevent COX-2 induction, had no effect on the potentiation action of DFU (10 μM). These results demonstrate that the potentiation of opioid inhibition of GABAergic synaptic transmission in PAG is largely a result of inhibition of COX-1 activity. These findings suggest that COX-1, rather than COX-2 inhibition, mediates the synergistic analgesic actions of opioids and non-steroidal anti-inflammatory drugs (NSAIDs) in the midbrain PAG.     

Effects of noradrenaline and somatostatin on basal and stimulated mucosal ion transport in the guinea-pig small intestine

Summary Noradrenaline (NA) and somatostatin (SOM) stimulate intestinal water and ion absorption and are found in mucosal nerve fibres and nerve terminals in submucous ganglia of the guinea-pig small intestine. As the main projection of submucous neurons is to the mucosa, NA and SOM might alter mucosal transport either by a direct effect on the epithelium or indirectly, by affecting submucous neurons. In this study these two possible sites of action of NA and SOM have been investigated in mucosa-submucosa preparations of guinea-pig ileum. In addition, the actions of NA and SOM on the secretory responses caused by stimulation of different populations of submucous neurons have been studied. The stimulants of secretion used were a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 10^–5 M), 5-hydroxytryptamine (5-HT, 10^–7 M) and electrical field stimulation (EFS), which activate cholinergic, noncholinergic and mixed populations of submucous secretomotor neurons, respectively.Segments of intestine were dissected free of external muscle and myenteric plexus and mounted in Ussing chambers. Short-circuit current (I _sc) was measured as an indication of net active ion transport across the tissue. NA (10^–8 M) and SOM (>10^–10 M) each caused a decrease in I _sc, indicating a net increase in ion absorption. The NA response was abolished and the magnitude of the SOM response was reduced to 20% by tetrodotoxin (10^–7 M). DMPP, 5-HT and EFS each stimulated nerves that increased I _sc and each of these responses was significantly diminished by NA and SOM; for both NA and SOM the decrease in the DMPP response was significantly greater than the decrease observed in the response to carbachol (10^–6 M). Phentolamine (10^–6 M) abolished all of the effects of NA but caused no change in the SOM effects. These studies have shown that NA and SOM cause similar changes in net ion transport, that their actions are primarily on submucous secretomotor neurons and that NA and SOM can diminish the responses to stimulation of both cholinergic and noncholinergic submucous neurons.In this tissue it is also known that SOM coexists with NA in noradrenergic nerve terminals in the submucosa. However, when applied together, NA and SOM caused no greater decrement in the carbachol and 5-HT responses than would be predicted by adding the separate effects of NA and SOM. Hence there was no obvious interaction between NA and SOM effects on mucosal transport.     

Neurogenic regulation of dendritic cells in the intestine

Antigen presenting cells like dendritic cells (DC) are responsible for the initiation of adaptive immune responses via the T helper cells they activate. The type of T cell responses DC induce is dependant on the local immunological environment where antigen has been taken up. In the gut, resident DC are phenotypically and functionally shaped by epithelial and stromal cell derived signals, the cytokine microenvironment, and neuronal products. These factors can control the activation state of DC thereby inducing tolerance for food and commensal organisms or immunity against pathogenic microbes. The enteric nervous system (ENS) is increasingly recognized as an important regulatory factor in intestinal immune cell control. Neurotransmitters and neuropeptides like acetylcholine (ACh), norepinephrine (NE) and vasoactive intestinal peptide (VIP) are released by neurons of the ENS and can affect the function of DC and subsequent immune responses. The critical balance between tolerance and protective immunity is disrupted in inflammatory bowel disease, which results in an exaggerated immune response against commensal bacteria. In this review we discuss the effects of ACh, VIP, and NE on DC function. DC express various receptors for these neuron derived products and can alter DC co-stimulatory molecule expression, cytokine release and subsequent T cell activation in an anti-inflammatory fashion. Knowledge about these interactions will help find new drug targets and may facilitate the development of specific therapies for diseases like inflammatory bowel disease (IBD).     

Effects of imidazoline derivatives on cholinergic motility in guinea-pig ileum: involvement of presynaptic α2-adrenoceptors or imidazoline receptors?

The present study investigates the possibility that imidazoline receptors mediate modulation of cholinergic motor functions of the guinea-pig ileum. For this purpose, the effects of a series of compounds with known affinity for α₂-adrenoceptors and/or imidazoline recognition sites were examined on the cholinergic twitch contractions evoked by electrical field stimulation (0.1 Hz) of longitudinal muscle-myenteric plexus preparations. Additional experiments were carried out on ileal strips preincubated with [³H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation (1 Hz). The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. α-Methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline and xylazine caused a concentration-dependent inhibition of twitch responses (IC₅₀ from 0.13 to 1.05 μM; E_max from 85.9 to 92.5%). Rilmenidine and agmatine were less potent in reducing the twitch activity, and the latter compound acted also with low intrinsic activity (IC₅₀=44.9 μM; E_max=35.5%). In interaction experiments, the inhibitory action of clonidine on twitch responses was competitively antagonized by RX 821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), idazoxan, rauwolscine, yohimbine and BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)-methyl]-4,5-dihydroimidazoline), whereas prazosin (10 μM), ARC 239 (2-(2,4-(O-methoxy-phenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione; 10 μM) and BRL 41992 (1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazol[1,5-a]azepi-ne; 10 μM) were without effect. Rauwolscine antagonized the inhibitory effects of various agonists on ileal twitch activity in a competitive manner and with similar potency. Agmatine and idazoxan did not significantly modify the twitch contractions when tested in the presence of α₂-adrenoceptor blockade by rauwolscine (3 μM) or RX 821002 (1 μM). Linear regression analysis showed that the affinity values of antagonists correlated with their affinity at the α_2A and α_2D binding sites as well as at previously classified α_2A/D adrenoceptor subtypes, whereas no significant correlation was obtained when comparing the potency estimates of agonists and antagonists with the affinity at I₁ or I₂ binding sites. When tested on the electrically induced outflow of tritium, α-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline, xylazine and rilmenidine yielded inhibitory concentration-response curves which were shifted rightward to a similar extent in the presence of rauwolscine (3 μM). In the absence of further drugs, agmatine significantly reduced the evoked tritium outflow at the highest concentrations tested (10 and 100 μM), whereas idazoxan (up to 100 μM) was without effect. When RX 821002 (1 μM) was added to the superfusion medium, neither agmatine nor idazoxan modified the evoked outflow of radioactivity. The results argue against modulation by imidazoline receptors of acetylcholine release from myenteric plexus nerve terminals. They provide evidence that compounds endowed with imidazoline-like structures affect the cholinergic motor activity of the guinea-pig ileum by interacting with presynaptic α₂-adrenoceptors belonging to the α_2D subtype. Received: 10 October 1997 / Accepted: 14 March 1998     

Modulation of peristalsis by NK3 receptor antagonism in guinea-pig isolated ileum is revealed as intraluminal pressure is raised

1. NK(3) tachykinin receptors mediate slow excitatory transmission in the enteric nervous system and play a role in reflexes induced by the intestinal stretch or mucosal compression. However, there is little evidence to suggest that these receptors are important in peristalsis. We have examined the effects of the NK(3) receptor antagonist, talnetant, on peristalsis in guinea-pig isolated ileum induced by optimal and by supra-maximal distension pressures. 2. At the guinea-pig NK(3) receptor, talnetant was shown to have high affinity (pK(B) 8.8) and selectivity over the guinea-pig NK(1) and NK(2) receptors. 3. Peristaltic waves in the ileum elicited by optimal distension pressures (1-3 cmH(2)O) were unaffected by talnetant at a supra-maximal concentration (250 nm). 4. Distension at a higher pressure (4 cmH(2)O) induced peristalsis in which there was incomplete closure of the lumen during each peristaltic wave and an increase in the periods of inactivity observed between bursts of peristaltic activity. The addition of talnetant (250 nm) increased the number of peristaltic events by reducing these periods of inactivity and thus, increased the productivity of the peristaltic reflex. 5. The data suggest that NK(3) receptors are not involved in the modulation of peristaltic movements by physiological stimuli, but they may have a role in modulation of reflexes in extreme or pathological conditions.     

Evidence that axons containing substance P in the guinea-pig ileum are of intrinsic origin

Summary Acid extracts from both normal and extrinsically denervated ileum contained a compound which was indistinguishable from synthetic substance P; this compound was assayed by examining its contractile effect on the longitudinal muscle of segments of ileum in which receptors for acetylcholine and histamine were blocked. Contractions caused by the compound were markedly and selectively antagonized when the ileum was made insensitive to the action of substance P. The activities in the extract and of synthetic substance P were both destroyed by chymotrypsin but were not affected by trypsin or carboxypeptidase B. The concentrations of substance P-like material in normal and extrinsically denervated segments were not significantly different, being equivalent to 0.48 g of substance P per g of external muscle plus myenteric plexus. A compound with substance P-like activity was liberated by stimulation of intramural nerves, either electrically or by dimethylphenylpiperazinium, in both normal and extrinsically denervated segments of ileum. The release of this compound was prevented by tetrodotoxin and its action on the muscle was blocked when the ileum was made insensitive to the action of substance P. Experiments with transmural stimulation showed that excitatory nerve pathways involving substance P neurons extend for less than 4 cm along the intestine.     

The effect of GABA antagonism on propulsive activity of the guinea-pig large intestine

Possible involvement of γ-aminobutyric acid (GABA) in intestinal motility of the guinea-pig has been investigated using the speed of propulsion of faecal pellets along isolated segments of guinea-pig distal colon, and the rate of pellet expulsion from freshly excised colon. GABA antagonism, by bicuculline or by tachyphylaxis to GABA, substantially reduced intestinal motility and generally reduced the amplitude of the reflex contraction associated with pellet propulsion. These results support the view that GABAergic mechanisms may be involved in the propulsive activity of the guinea-pig distal colon.     

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 μM) 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. N^G-nitro-L-arginine methyl ester (100 μM), an inhibitor of nitric oxide synthase, or naloxone (1 μM), 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.

     

Endogenous CCK depresses contractile activity within the ascending myenteric reflex pathway of rat ileum

The ascending excitatory reflex is an important part of the myenteric reflex. In order to study the ascending neural pathways, isolated segments of rat ileum were stimulated by electrical stimulation of the gut wall (20 V, 3 pulses per second, 1 ms) using platinum electrodes. The excitatory contractile response was recorded using perfused manometric side-hole tubing located 2 and 4 cm orally to the stimulation site. The contractile response to electrical stimulation was abolished by atropine (10(-6) M) or hexamethonium (10(-4) M). The excitatory response increased after administration of the cholecystokinin A (CCK(A)) receptor antagonists lorglumide (3x10(-6) M: +44.1%), devazepide (10(-8) M: +19.4%; 10(-7) M: +30.0%) and SR-27897 (10(-10) M: +21.8%, 10(-8) M: +47.0%, P<0.05, n=8). However, the CCK(B) receptor antagonist L-365,260 also caused a significant increase in the oral excitation (10(-6) M: +27.4%). sCCK-8 caused a significant reduction in the ascending response (10(-8) M: -11.5%) and induced spontaneously occurring contractions at doses ranging from 10(-10)-10(-6) M. CCK-9 significantly increased the ascending response (10(-7) M: +10.9%, P<0.05). However, caerulein (10(-10) M: -25.9%, 10(-8) M: -26.8%; P<0.01) and pentagastrin (10(-10) M: -20.2%, P<0.05; 10(-8) M: -23.7%, P<0.01; 10(-6) M: -28.3%, P<0.001) reduced the ascending contractile response significantly. These data, obtained with potent and highly specific CCK receptor antagonists, demonstrate an inhibitory role of endogenously released CCK within the ascending neural pathway. The data further suggest that exogenously applied CCK-related peptides have different effects on the myenteric reflex which might be due to excitation of the different involved neurons (short and long ascending inter- and motorneurons) in an unphysiological order. Thus in experiments investigating more complex neuronal circuits, experiments with antagonists should be regarded as more specific.     

Role of extra- and intracellular calcium in the contractile action of agonists in the guinea-pig ileum

Summary The effects of Ca²⁺-channel blockers (nifedipine and verapamil), EGTA, caffeine or the removal of external Ca²⁺ on the contractile action of different agonists and transmural electrical stimulation were examined in isolated segments of the proximal and terminal part of the guinea-pig ileum. The effects of agonists and nerve stimulation on membrane potential were also studied by means of the sucrose gap method. Acetylcholine-elicited contractions in both parts and noradrenaline-as well as histamine-induced contractions in the terminal part of the ileum were composed of an initial phasic and a sustained tonic component. Single pulse transmural nerve stimulation elicited smooth muscle twitches, whereas addition of CaCl₂ to the tissue bath containing Ca²⁺-free and high-K⁺ medium elicited a sustained contraction. Both verapamil and nifedipine were more potent in inhibiting the tonic phase of the responses to the agonists or CaCl₂ than inhibiting the phasic contractions elicited by transmural nerve stimulation, acetylcholine or noradrenaline. The excitatory junction potentials (e j.p.s.) as well as smooth muscle twitches were reduced only by high nifedipine concentrations. The effects of acetylcholine on membrane potential and input membrane resistance were affected minimally by the omission of extracellular Ca²⁺, while the contractions gradually disappeared on repetitive agonist application in the absence of external Ca²⁺ and were blocked by caffeine preexposure. In Ca²⁺-free solution noradrenaline and histamine partially reduced each other's motor effect, while neither of them changed the contractile action of acetylcholine, yet the contraction induced by noradrenaline was prevented and that of histamine significantly reduced by preexposure to acetylcholine. These results suggest that the potency of acetylcholine to release Ca²⁺ from its caffeine-sensitive intracellular stores is much higher than that of histamine and noradrenaline. Send offprint requests to V. Bauer at the above address     

Effect of sodium rhein on electrically-evoked and agonist-induced contractions of the guinea-pig isolated ileal circular muscle

1. This study examined the effects of sodium rhein (0.03–30 μM) on the contractions of the isolated circular muscle of guinea-pig ileum induced by acetylcholine (100 nM), substance P (3 nM) and electrical stimulation (10 Hz for 0.3 s, 100 mA, 0.5 ms pulse duration). The effect of sodium rhein was also evaluated on the ascending excitatory reflex using a partitioned bath (oral and anal compartments). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 20 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the guinea-pig intestinal circular muscle in the oral compartment was recorded.
2. Sodium rhein (0.3, 3 and 30 μM) significantly potentiated (52±11% at 30 μM) acetylcholine-induced contractions. In the presence of tetrodotoxin (0.6 μM) or ω-conotoxin GVIA (10 nM) sodium rhein (3 and 30 μM) did not enhance, but significantly reduced (49±10% and 44±8%, respectively, at 30 μM) acetylcholine-induced contractions.
3. Sodium rhein (0.3, 3 and 30 μM) significantly increased (65±11% at 30 μM) substance P-induced contractions. In the presence of tetrodotoxin (0.6 μM), ω-conotoxin GVIA (10 nM) or atropine (0.1 μM), sodium rhein (3 and 30 μM) significantly reduced (50±10%, 55±8% and 46±10%, respectively, at 30 μM) substance P-induced contractions.
4. N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) abolished the potentiating effect of sodium rhein on acetylcholine and substance P-induced contractions. At the highest concentration (30 μM), sodium rhein, in presence of L-NAME, reduced the acetylcholine (30±6%)- or substance P (36±6%)-induced contractions.
5. Sodium rhein (30 μM) significantly potentiated (29±9%) the electrically-evoked contractions. L-NAME (100 μM), but not phentolamine, enhanced the effect of sodium rhein. Sodium rhein (30 μM) significantly increased (32±9%) the ascending excitatory reflex when applied in the oral, but not in the anal compartment.
6. These results indicate that sodium rhein (i) activates excitatory cholinergic nerves on circular smooth muscle presumably through a facilitation of Ca²⁺ entry through the N-type Ca²⁺ channel, (ii) has a direct inhibitory effect on circular smooth muscle and (iii) does not affect enteric ascending neuroneural transmission. Nitric oxide could have a modulatory excitatory role on sodium rhein-induced changes of agonist-induced contractions and an inhibitory modulator role on sodium rhein-induced changes of electrically-induced contractions.

     

Mechanisms underlying the intestinal fluid secretion evoked by nociceptive serosal stimulation of the rat

Summary Intestinal net fluid transport was measured in vivo continuously with a gravimetric method. Chemical stimulation of the jejunal serosa with hydrochloric acid (0.1 M), ethanol (20%), cat bile or 7-deoxycholic acid (10 mM) evoked an intestinal fluid secretion. Hexamethonium (10 mg/kg b. wt. i. v.) or serosal application of lidocaine (1% solution) partially blocked this secretory response. Bradykinin and prostaglandin E₁, two important inflammatory mediators, elicited fluid secretion when applied to the serosal surface at a concentration of 10^–4M. This secretion was also partly inhibited by hexamethonium. Furthermore indomethacin (10 mg/kg b. wt. i. v.) or pyrilamine (10 mg/kg b. wt. i. v.), a H₁-receptor blocker, partly inhibited the secretory response caused by chemical stimulation of the serosa while cimetidine (1 mg/kg b. wt. i. v.), a H₂-receptor blocker, had no effect. Freeze sectioned samples from chemically stimulated intestines were examined by fluorescence microscopy. A leakage of i. v. administrated Evans blue labelled albumin into the interstitial space of the serosa and the outer layer of the muscularis was found.It is concluded: (1) The intestinal fluid secretion studied is mainly elicited by nociceptive stimulation of nerves in the serosa or the outer muscularis. (2) The reflex may be activated by the local release of histamine, kinins and prostaglandins. (3) The reflex studied is part of an inflammatory response.     

Effects of phosphodiesterase isoenzyme inhibitors on cutaneous inflammation in the guinea-pig.

1. Inflammation is central to the pathophysiology of asthma. The recent findings that different inflammatory cells may express different phosphodiesterase (PDE) isoenzymes have centered attention on inhibitors of these isoenzymes as new drugs for the treatment of asthma. In this study, we investigated the effect of different PDE isoenzyme inhibitors on the accumulation of 111In-labelled eosinophils and local oedema formation at sites of allergic- and mediator-induced inflammation in guinea-pig skin. 2. Systemic treatment with SK&F 94120, a type III PDE inhibitor, or zaprinast, a type V PDE inhibitor, had no effect on the 111In-eosinophil accumulation and oedema formation induced by i.d. injection of zymosan-activated plasma (ZAP), PAF, histamine or in a passive cutaneous anaphylaxis (PCA) reaction. 3. Systemic treatment with rolipram, a type IV PDE inhibitor, effectively inhibited 111In-eosinophil accumulation induced by ZAP, PAF, histamine and in a PCA reaction. However, oedema formation measured in the same sites was not affected. Systemic administration of higher doses of theophylline produced similar results. In contrast, 111In-neutrophil accumulation induced by ZAP or in a PCA reaction was not altered by systemic treatment with rolipram. 4. Locally-injected rolipram had little effect on 111In-eosinophil accumulation and oedema formation induced by histamine, PAF and in a PCA reaction. 5. These data show that systemic, but not local, treatment with rolipram effectively inhibits allergic- and mediator-induced 111In-eosinophil accumulation but not oedema formation or 111In-neutrophil accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of desipramine-induced blockade of neuronal uptake mechanisms on adrenoceptor-mediated responses in the guinea-pig colon

In order to clarify whether adrenoceptors in the guinea-pig distal colon are under sympathetic control, we assessed possible variations in the sensitivity to adrenoceptor agonists after blockade of neuronal catecholamine uptake mechanisms by desipramine (DMI). First, experiments were carried out to investigate the effects of DMI added in the organ bath on propulsion velocity, endogenous and [³H] prelabelled acetylcholine overflow, electrically evoked noradrenaline overflow and longitudinal smooth muscle tone. Secondly, we studied the effects of adrenoceptor agonists on the above parameters in untreated animals and in animals chronically treated with DMI.DMI added in the organ bath at concentrations equal to or higher than 30 nM inhibited all the parameters under study. Thus, when evaluating the effect of DMI on concentration-response curves to adrenoceptor agonists, concentrations which were per se inactive were used. DMI added in the organ bath at concentrations up to 30 nM potentiated the inhibitory effects of exogenous noradrenaline on propulsion velocity and acetylcholine overflow, but it did not affect the concentration-response curve to exogenous noradrenaline on longitudinal smooth muscle tone. Furthermore, 30 nM DMI inhibited propulsion velocity during sympathetic nerve stimulation. In preparations obtained from animals chronically treated with DMI, no significant change of propulsion velocity, endogenous and [³H] prelabelled acetylcholine overflow was found with respect to untreated animals. Nevertheless, in such preparations subsensitivity to isoprenaline (acting mainly on muscular -adrenoceptors) and clonidine (acting on neuronal ₂-adrenoceptors) and supersensitivity to phenylephrine were observed. Electrically evoked noradrenaline overflow was enhanced, in a frequency-dependent way, by yohimbine and inhibited by clonidine.We conclude that in the guinea-pig colon: 1) - and -adrenoceptors are under tonic neuronal control, as indicated by the sensitivity changes to - and -adrenoceptor agonists after chronic DMI treatment; 2) exogenous NA reaching neuronal, but not muscular adrenoceptors, is affected by neuronal uptake mechanisms; 3) NA released by adrenergic terminals undergoes neuronal uptake and is controlled by ₂-autoreceptors. Correspondence to: G. M. Frigo at the above address