Capsaicin-sensitive innervation of the guinea-pig taenia caeci

1. Stimulation (2--50 Hz) of the mesenteric nerves of the guinea-pig taenia caeci gave rise to contraction of the muscle obtained from animals pretreated with the adrenergic neuron-blocking agent guanethidine. Contraction was the response to stimulation at low frequencies (2--5 Hz) in about half of the untreated preparations as well. 2. The response was abolished by hyocine (4.5 x 10(-7) M), but was unaffected by the ganglionic blocking agent mecamylamine (4.9 x 10(-5) M). Physostigmine (2.4 x 10(-8) M) enhanced the contractions. 3. Capsaicin (9.8 x10(-6) M) elicited a contraction of the taenia caeci followed by a long-lasting tachyphylaxis. Contraction in response to stimulation of the mesenteric nerves was absent after this pretreatment. 4. Neither the response to direct excitation of the cholinergic neural elements of the myenteric plexus, nor the relaxation caused by stimulation of adrenergic fibres were influenced by capsaicin. "Purinergic" relaxation produced by field stimulation (0.5--10 Hz) remained also unchanged. 5. No functional evidence has been found for the presence of parasympathetic preganglionic fibres among the perivascular nerves applying the taenia. 6. It is concluded that capsaicin-sensitive nerves excite cholinergic neurons of the myenteric plexus.     

Relaxation of guinea-pig fundic strip by adenosine, adenosine triphosphate and electrical stimulation: lack of antagonism by theophylline or ATP treatment.

1 Theophylline relaxed isolated strips of guinea-pig stomach fundus in a dose-dependent manner; above 50 to 100 microM responses showed no fade for up to 90 min. 2 Relaxant responses to adenosine, adenosine triphosphate (ATP), noradrenaline, and to electric field stimulation of non-adrenergic inhibitory nerves were not affected in a significant manner in the presence of 50 microM theophylline. 3 In tissues which showed complete fade of initial responses in the continued presence of 50 microM ATP, the effects of stimulation of non-adrenergic inhibitory nerves remained unaltered, suggesting that the ATP receptor has no function in non-adrenergic inhibitory transmission in this tissue. 4 These findings are opposite to those of Okwuasaba, Hamilton & Cook (1977), who claimed that 50 microM theophylline almost fully inhibited relaxation induced by adenosine ATP and nerve stimulation and that ATP-induced fade also abolished sensitivity to inhibitory nerve stimulation.     

Suramin and the inhibitory junction potential in taenia caeci of the guinea-pig

The effect of surinam on the inhibitory junction potential evoked in smooth muscle cells of guinea-pig taenia caeci by stimulation of intramural nerves at 22 degrees C was investigated. The amplitude of the inhibitory junction potential was reduced concentration dependently by suramin. The suppression of this response by suramin became less pronounced when the number of stimuli increased (pulse rate: 20/s). These results indicate that suramin reduces the inhibitory junction response by interacting with P2-purinoceptors.     

Blockade by WB 4101 of alpha-adrenoceptors in the rat vas deferens and guinea-pig taenia caeci.

The effectiveness of WB 4101, a recently described alpha-adrenoceptor antagonist, in blocking an excitatory and two inhibitory responses to alpha-receptor activation was studied. One of the inhibitory responses was the reduction by the selective alpha-agonist amidephrine of carbachol contractures of isolated guinea-pig taenia caeci. WB 4101 antagonised this inhibition with a Schild plot slope of 0.99 and a pA2 of 8.9. The same pA2 value was obtained for blockade of the contractile effect of amidephrine and noradrenaline on the rat vas deferens. WB 4101 was, however, several hundred times less active in antagonising the inhibitory effect of clonidine on the twitch response of the vas deferens to field stimulation. Incidental observations were that the twitch was increased by low concentrations of amidephrine, and by relatively high concentrations of WB 4101. Because of its potency and postsynaptic selectivity, WB 4101 should be useful for adrenoceptor classification.     

Antagonism of calcium by zinc in guinea-pig isolated taenia caeci and trachealis muscle.

1 In guinea-pig isolated taenia caeci and trachealis bathed in a K+-rich, Ca2+-free medium, CaCl2 (0.01-10 mM) produced a concentration-dependent contraction. Zn2+ (0.01-1 mM), Cd2+ (0.01-1 mM), verapamil (0.01-100 microM) and trifluoperazine (1-100 microM) were effective antagonists of CaCl2-induced responses. 2 Zn2+ and Cd2+ in concentrations from 0.01 to 1 mM were without effect on the tone of taenia and trachea in normal Tris solution. Conversely, Zn2+ and Cd2+, in concentrations of 1 mM, caused contraction of these tissues in a K+-rich, Ca2+-free medium. Zn2+ (1 mM)-induced contractions of taenia and trachea were completely inhibited by verapamil (10 microM). 3 In taenia and trachea skinned of their plasma membranes, tension development induced by Ca2+ (10 microM or 1 microM, respectively) was unaffected by verapamil (100 microM), whereas trifluoperazine (100 microM) depressed the maximal tension produced by Ca2+. Segments of skinned preparations contracted in response to low concentrations of Zn2+ (10 microM) or Cd2+ (10 microM). 4 It is concluded that Zn2+ may suppress Ca2+-induced spasm by a direct action on the binding sites of the Ca2+ channel.     

Nitric oxide and ATP co-mediate the NANC relaxant response in the guinea-pig taenia caeci

The effect of the nitric oxide synthase inhibitor N ^G-nitro-l-arginine (l-NOARG; 100 μM) and the P₂ purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS; 50 μM) was investigated on the non-adrenergic, non-cholinergic (NANC) relaxant response of the guinea-pig isolated taenia caeci to electrical field stimulation at 1 or 10 Hz, under isotonic recording conditions. Either drug alone caused an about 50% inhibition, while combining the two drugs nearly abolished the response at both frequencies. The inhibitory effect of l-NOARG (100 μM) was partly reversed by l-arginine (30 mM). PPADS, but not l-NOARG, inhibited the relaxant effect of exogenous ATP, but not that of the nitric oxide donor sodium nitroprusside. It is concluded that both nitric oxide and ATP are involved in the mediation of NANC relaxation in the taenia caeci, in an apparently additive manner. Received: 23 June 1998 / Accepted: 3 July 1998     

Desensitization of guinea-pig taenia caeci smooth muscle induced by a low concentration of carbachol

1. In guinea-pig taenia caeci smooth muscle we have found that 10(-4) mol/L carbachol-induced desensitization to muscarinic agonists develops within 15-30 s, followed by transient resensitization at 1 min, whereas the desensitization to depolarizing high K(+) develops with maximal desensitization at 1 min followed by sustained resensitization up to 30 min. In both cases, Ca(2+)-dependent processes play a crucial role in determining the development of desensitization. 2. To elucidate whether these peculiar processes of desensitization/resensitization may be induced by a lower concentration of carbachol, we examined the development of desensitization induced by 10(-6) mol/L carbachol, because at this concentration carbachol is known to induce biphasic changes in intracellular Ca(2+) concentrations, with a smaller transient increase followed by a larger sustained increase than seen with 10(-4) mol/L carbachol. 3. Contractile responses to muscarinic agonists (carbachol or AHR-602) and high K(+) were desensitized by pretreatment with 10(-6) mol/L carbachol for 30 min in a manner dependent on the presence of extracellular Ca(2+). 4. The development of 10(-6) mol/L carbachol-induced desensitization to these muscarinic agonists in the presence of extracellular Ca(2+) showed three successive phases: fast desensitization within 30 s, followed by transient resensitization at 1 min and the subsequent development of desensitization up to 30 min. In contrast, desensitization to high K(+) did not develop up to 10 min and significant desensitization occurred at 30 min, with no apparent resensitization phase. 5. These results suggest that the characteristics of the Ca(2+)-dependent development of desensitization to muscarinic agonists, but not to high K(+), are well maintained in desensitization induced by a lower concentration of carbachol.     

Calcium and the activation of the alpha 1-adrenoceptors in the guinea-pig taenia caeci.

1. The actions of phenylephrine (0.1-100 mumol l-1) and methoxamine (0.1-100 mumol l-1) were compared with that of adrenaline (0.01-10 mumol l-1) using the single sucrose gap method and mechanical recording in the guinea-pig taenia caeci. Drugs were applied for variable periods of time. 2. The characteristics of the inhibitory effects of alpha-adrenoceptor agonists were the same when exposure time did not exceed 5 min. When the exposure was prolonged, in contrast to the sustained effects of adrenaline (0.1-3 mumol l-1), phenylephrine and methoxamine (1-10 mumol l-1) produced a transient inhibitory action. 3. During the delayed recovery phase of phenylephrine, adrenaline preserved its ability to suppress the spontaneous electrical and mechanical activities of the taenia both when phenylephrine was replaced by adrenaline or when adrenaline was applied in addition to phenylephrine. All the above effects were found in untreated preparations, as well as during blockade of muscarinic cholinoceptors by atropine (1.4 mumol l-1), beta-adrenoceptors by propranolol (3 mumol l-1) and release of endogenous catecholamines by guanethidine (2.5 mumol l-1). 4. In the presence of phorbol 12,13-dibutyrate adrenaline ceased to be effective, while the inhibitory action of phenylephrine was converted to a contraction. 5. In calcium-free conditions in the presence of EGTA (0.4 mmol l-1) the initial hyperpolarization induced by adrenaline and phenylephrine was significantly reduced and with repeated applications of the agonists the inhibitory response disappeared. Similar results were obtained using tissues treated with nifedipine (1 and 10 mumol l-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of adenosine 5’-triphosphate, uridine 5’-triphosphate, adenosine 5’-tetraphosphate and diadenosine polyphosphates in guinea-pig taenia caeci and rat colon muscularis mucosae

The functional effects of adenosine 5’-triphosphate (ATP), uridine 5’-triphosphate (UTP), adenosine 5’-tetraphosphate (AP₄) and the diadenosine polyphosphates P₁,P₃-diadenosine triphosphate (Ap₃A), P₁,P₄-diadenosine tetraphosphate (Ap₄A) and P₁,P₅-diadenosine pentaphosphate (Ap₅A) were studied in two isolated smooth muscle preparations thought to contain P_2Y (P2Y₁) receptors, the guinea-pig taenia caeci (which relaxes to ATP) and the rat colon muscularis mucosae (which contracts to ATP). In addition, the breakdown of these compounds by the rat colon muscularis mucosae was investigated by high pressure liquid chromatography. In the guinea-pig taenia caeci all the purine nucleotides caused relaxation with a potency order of Ap₃A=Ap₄A> ATP>AP₄=Ap₅A, and these relaxations were antagonised by suramin with apparent pA₂ values in the region of 5, consistent with activation of a P2Y₁ receptor. In the rat colon muscularis mucosae the nucleotides caused contraction with a potency order of Ap₃A = Ap₄A>ATP=AP₄ =Ap₅A >UTP. However, while suramin (100 μM) inhibited responses to ATP and UTP at all concentrations of agonist, it only inhibited contractions induced by the higher concentrations of AP₄, Ap₃A and Ap₄A and had little effect on contractions induced by Ap₅A. A higher concentration of suramin (1 mM) enhanced contractions induced by ATP but greatly inhibited those induced by UTP and had no effect on responses to the other agonists. The A₁ adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM) had no effect on responses to ATP or UTP but inhibited responses to Ap₃A, Ap₄A, Ap₅A and AP₄. A combination of suramin (1 mM) and DPCPX (10 nM) almost abolished responses to all the agonists. ATP and UTP were rapidly degraded by the rat colon muscularis mucosae while AP₄, Ap₃A, Ap₄A and Ap₅A were degraded more slowly, and the major product detected after breakdown of the purine nucleotides was inosine rather than adenosine. The breakdown of all the nucleotides was inhibited by suramin (1 mM), although this inhibition did not achieve statistical significance in the case of ATP. These results show that while the diadenosine polyphosphates appear to act as P2 agonists in the taenia caeci, in the rat colon muscularis mucosae their major action is via adenosine A₁ receptors rather than via P2 receptors. In addition, although they are more stable than ATP or UTP, their action in this tissue is clearly affected by their degradation which complicates the effects of suramin. Received: 23 March 1998 / Accepted: 29 June 1998     

Changes in membrane potential and phosphoinositides during alpha 1-adrenoceptor stimulation in smooth muscle cells of guinea-pig taenia caeci

The effect of a submaximal concentration of adrenaline (3-5 microM) was studied in taenia caeci smooth muscle cells. Membrane potential hyperpolarization was observed in intact muscle preparations and this response could be separated into two phases, depending on the state of a membrane-bound calcium compartment. The effect of alpha 1-adrenergic stimulation was also measured by [3H]inositol incorporation into phospholipid and inositol phosphate fractions of taenia cell suspensions both in the absence and presence of 2.5 mM extracellular calcium. In the absence of extracellular calcium the inositol phospholipids increased within 15 s after stimulation, followed by enhanced inositol phosphates. With calcium present there was a biphasic increase in the phosphatidylinositol 4,5-bisphosphate (PIP2) fraction with a simultaneous release of inositol phosphates. Lithium ions affected the incorporation of label into the lipids but not into the inositol phosphate fractions. These findings suggest that, in taenia caeci cells, alpha 1-adrenergic-induced membrane hyperpolarization resulting in muscle relaxation is associated with changes in the PIP2 content.     

The prostaglandin response in guinea-pig taenia caeci and trachea smooth muscle of different ages

The concentration-response curves for the carbachol-induced contraction of smooth muscle cells of guinea-pig taenia caeci and trachea were not dependent on tissue age. The prostaglandin E2 and F2 alpha responses increased with age in taenia caeci in contrast with the PGE2 response evoked in trachea. The high-potassium responses evoked in taenia caeci and trachea both increased at higher tissue age. Methoxyverapamil only inhibited the age-dependent responses. The results suggest that it is mainly the voltage-dependent calcium channels that are involved in the age-dependent prostaglandin response.     

The influence of alpha, beta-methylene ATP on alpha 1-receptor-operated channels in guinea-pig taenia caeci

The influence of the ATP analog alpha, beta-methylene ATP on the action of adrenaline on alpha 1-receptors of smooth muscle cells of guinea-pig taenia caeci was studied by measuring potential changes. The preparation was superfused (1 ml/min) with Krebs solution or calcium-free solution containing atropine (10(-6) M) and propranolol (10(-6) M) at 22 degrees C, using the sucrose-gap method. The ATP analog (10(-5) to 4 X 10(-4) M) and adrenaline (10(-5) M) both caused a transient hyperpolarization in the absence of external calcium (20 min). The response (area under the 'curve') evoked under calcium-free conditions (20 min) increased with the concentration of the ATP analog. The response was diminished when preceded by the adrenaline response (10(-5) M) or when evoked after repeated addition of the analog to the superfusate (35 min). The adrenaline response was also diminished when preceded by the ATP analog. The responses to the ATP analog or adrenaline in the presence of apamin (3 X 10(-7) M) in the absence of external calcium were characterized by depolarization of the muscle cells. Repeated addition of the ATP analog or adrenaline to the preparation under these conditions did not cause any effect. The results suggest strongly that adrenaline and alpha, beta-methylene ATP both activate the same calcium-dependent process, producing calcium mobilization and the opening of apamin-sensitive potassium channels. Besides this action the ATP analog also activates apamin-sensitive potassium channels and this activation is independent of the availability of calcium in the adrenaline-sensitive pool.     

(3H)-adenine nucleotide and (3H)-noradrenaline release evoked by electrical field stimulation, perivascular nerve stimulation and nicotine from the taenia of the guinea-pig caecum

1. The release of [(3)H]-noradrenaline and adenine nucleotide evoked by electrical field stimulation (20-60 V, 30 Hz), perivascular nerve stimulation (20-80 V, 60 Hz) and nicotine (10, 100 muM) was studied in the taenia of the guinea-pig caecum under various conditions.2. Electrical stimulation at high intensity (60 V) caused the release of [(3)H]-adenine nucleotide; however, the inhibitory action of electrical stimulation was proportional to [(3)H]-noradrenaline release.3. The intensity of the inhibitory effect of stimulation of the perivascular nerves was directly related to [(3)H]-noradrenaline release and not associated with the release of [(3)H]-nucleotide.4. Cold storage for more than 8 days, cooling (19 degrees C) or tetrodotoxin treatment (1 mug/ml) abolished the inhibitory responses to electrical stimulation and to nicotine. After these treatments, nicotine and electrical stimulation elicited only contractions; the release of [(3)H]-noradrenaline, but not that of [(3)H]-adenine nucleotide, was inhibited.5. The dissociation of the inhibitory effects of electrical stimulation and nicotine from [(3)H]-nucleotide release does not support the hypothesis that ATP or a related nucleotide is the humoral transmitter of the non-adrenergic inhibition in the taenia of the guinea-pig caecum.     

Thiol reagents and nitric oxide modulate the gating of BKCa channels from the guinea-pig taenia caeci

1. The site of the direct modulation of the gating of BKCa channels by the nitric oxide donor s-nitroso-l-cysteine (NOCys) was examined in excised membrane patches of the guinea-pig taenia caeci by the use of various thiol (sulphydryl)-specific reagents, including N-ethylmaleimide (NEM) and three charged methanethiosulphonate (MTS) reagents, namely positively charged 2-aminoethyl MTS hydrobromide (MTSEA) and [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium (2-sulphonatoethyl) MTS (MTSES), which all specifically convert sulphydryls to a disulphide. 2. At 10 micro mol/L, NOCys transiently increased the probability of opening (N.Po) of the BKCa channels (at 0 mV) after a delay of 1-2 min. 3. Disulphide-reducing agents, such as dithiothreitol (10 micro mol/L), increased N.Po in a manner that was reversed by the sulphide-oxidizing agent thimerosal (10 micro mol/L). Both positively charged MTSET (2.5 mmol/L) and negatively charged MTSES (2.5 mmol/L) rapidly increased N.Po. However, only the MTSES-evoked increase in N.Po remained after a prolonged washout period. 4. The specific alkylating agent of cysteine thiols NEM (1 mmol/L) and the positively charged, but membrane permeable, MTSEA (2.5 mmol/L) decreased N.Po (at 0 mV). 5. Pre-exposure of excised membrane patches to NEM or MTSES prevented the excitatory actions of NOCys (10 micro mol/L). 6. We conclude that MTSES and NOCys must modify thiols on cysteine residues within basic regions of the channel protein that would electrostatically exclude MTSEA and MTSET. A consensus sequence of various mammalian alpha-subunits of the BKCa channel reveals two pairs of cysteine residues surrounded by basic amino acids that could be the site of action for NOCys and MTSES.     

Evidence that inhibitory neurotransmission differs between the proximal and distal segments of guinea-pig taenia caeci

The effect of atropine (1 microM) and N(G)-nitro-L-arginine (L-NOARG, 10 microM) on electrical field stimulation induced relaxation in proximal and distal segments of guinea-pig taenia caeci in the presence of guanethidine (4 microM) was studied. The frequency-dependent relaxations were lower in proximal than in distal segments both in the presence and in the absence of atropine. The effect of L-NOARG (an inhibitor of nitric oxide (NO) synthase) on relaxation in the presence of atropine depended on the frequency of electrical stimulation and the segment used; the effect of L-NOARG was greater in proximal segments than in distal segments. In the absence of atropine, the inhibitory effect of L-NOARG was the same in both segments at all frequencies tested. This study demonstrates differences between the opposite extremes of guinea-pig taenia caeci in relaxations induced by electrical stimulation. Our data also show a role of NO that is dependent on the integrity of cholinergic transmission.     

Inhibitory effects of AH 21-132 in guinea-pig isolated ileum and taenia caeci.

1. AH 21-132 is being investigated as a potential chemotherapeutic agent for bronchial asthma. The present experiments were designed to determine whether AH 21-132 shares the activity of theophylline as an antagonist at adenosine A1 receptors and to assess its potency as a relaxant in intestinal smooth muscle. 2. In the transmurally-stimulated guinea-pig ileum, theophylline (1 mM), but not AH 21-132 (1 and 10 microM), antagonized twitch depression induced by adenosine. Higher concentrations (100 microM and 1 mM) of AH 21-132 themselves had a depressant effect. Neither theophylline (1 mM) nor AH 21-132 (1 and 10 microM) antagonized twitch depression induced by noradrenaline. 3. AH 21-132 (100 microM and 1 mM) depressed maximum contractions of ileum induced by both acetylcholine (ACh) and histamine. 4. In ileum treated with hyoscine (1 microM), AH 21-132 (greater than 10 microM) caused a concentration-dependent depression of the log concentration-effect curve for potassium chloride. 5. Simultaneous extracellular electrophysiological and mechanical recording from taenia caeci showed that AH 21-132 (100 microM-1 mM) inhibited spontaneous tension waves and their associated bursts of electrical spike activity. 6. Intracellular electrophysiological recording from taenia caeci showed that the mechano-inhibitory effect of 1 mM AH 21-132 was accompanied by abolition of spontaneous spike activity. Following spike abolition, the membrane potential assumed a value very close to that observed during periods of electrical quiescence prior to drug exposure. 7. AH 21-132 inhibited the activity of cyclic AMP-dependent and cyclic GMP-dependent phosphodiesterases derived from homogenates of ileal smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bradykinin receptors in the guinea-pig taenia caeci are similar to proposed BK3 receptors in the guinea-pig trachea, and are blocked by HOE 140.

1. Bradykinin (BK) receptors of the guinea-pig taenia caeci were compared with those of the guinea-pig trachea, a preparation proposed to possess novel BK3 receptors. 2. Bradykinin-evoked contractile responses were unaffected in both preparations by the selective BK1 receptor antagonist [des-Arg9,Leu8]-BK (1 microM-10 microM). The BK2 receptor antagonists, D-Arg-[Hyp3,D-Phe7]-BK and D-Arg-[Hyp3,Thi5,8,D-Phe7]-BK, both had low affinities (apparent pKB estimates less than 6) which did not differ significantly between the two preparations (P greater than 0.05). In contrast, the novel bradykinin receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (HOE 140) potently antagonized responses to bradykinin with relatively high affinity (apparent pKB = 8.42 +/- 0.15 and 8.94 +/- 0.16 in the taenia caeci, and trachea, respectively). 3. We conclude that the bradykinin receptors in the guinea-pig taenia caeci have similar recognition properties to those present in the guinea-pig trachea, and in this respect the taenia caeci represents a useful preparation for the further study of proposed novel BK3 receptors.