Comparison of contractile responses of the guinea pig ileum longitudinal muscle to ethanol and GABAA agonists

The guinea pig ileum myenteric plexus contains GABAA receptors linked to chloride ion channels which are pharmacologically similar to those in the central nervous system. The present study examined the reported ability of acute ethanol treatment to directly activate GABAA receptors or to increase GABAA agonist-mediated activation of the GABAA receptor in the myenteric plexus. Direct addition of ethanol to preparations of the guinea pig ileum longitudinal muscle had two effects. Immediately after ethanol (10-300 mM) was added to the tissue bath a concentration-related contractile response was observed which became maximal within 10 sec and then decayed over the next 60 sec. Contractile responses to higher concentrations of ethanol (greater than 100 mM) also were followed by a sustained reduction of longitudinal muscle tone. Contractions evoked by gamma-aminobutyric acid (GABA) and GABAA agonists, 3-aminopropane sulfonic acid (APSA) (3-100 microM) or muscimol (0.3-30 microM) developed maximally and decayed within 20 sec. Acetylcholine (0.01-10 microM) induced contractions were sustained over several minutes. Preincubation of tissue strips in ethanol (30 mM) for 1 min did not alter concentration relationships for GABA, muscimol or APSA contractile responses. Furthermore, addition of ethanol (10-100 mM) simultaneously with APSA, or 0.5, 2 or 5 min before the addition of APSA, also failed to consistently enhance contractile responses. Ethanol (30 mM) also did not alter desensitization-induced reductions in contractile responses to muscimol (3 microM) caused by preincubation of tissues with muscimol (1 microM). Finally, contractile responses to ethanol and APSA were completely blocked by atropine (0.1 microM) and tetrodotoxin (0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential inhibition of cholinergic and noncholinergic neurogenic contractions by 5-hydroxytryptamine1A receptor agonists in guinea pig ileum.

Previous studies have shown that 5-hydroxytryptamine1A (5-HT1A) receptors are localized only to a subset of myenteric neurons in guinea pig ileum; the purpose of this study was to determine the possible functional significance of this differential receptor localization. Nerve-mediated contractions of the longitudinal muscle, myenteric plexus of guinea pig ileum were studied in vitro. Contractions were evoked by single transmural electrical stimuli (0.5-msec duration, at 0.1 Hz; cholinergic) or trains of stimuli (10 or 20 Hz for 1 sec; noncholinergic; scopolamine, 1 microM present). The 5-HT1A agonist, 8-hydroxy-2-(n-dipropylamino)tetralin (DPAT), reduced cholinergic contractions by no more than 14% in the concentration range of 0.001 to 0.3 microM. At high concentrations (1 to 100 microM), DPAT inhibited longitudinal muscle, myenteric plexus contractions; the EC50 was 6 microM. 5-Hydroxytryptamine (5-HT) and 5-carboxamidotryptamine (5-CT) reduced longitudinal muscle, myenteric plexus contractions by a maximum of 35% and 24% at 100 and 30 microM, respectively. Spiperone and 1-(2-methoxyphenyl)-4-[4-(2-phthalimidobutyl]piperazine (NAN-190), two 5-HT1A receptor antagonists, did not block DPAT-induced inhibition of cholinergic contractions. DPAT (3, 10 and 30 microM) shifted histamine concentration-response curves to the right in an apparently competitive manner; Schild analysis yielded a pA2 of 5.2. DPAT (3, 10, 30 and 100 microM) also shifted bethanechol concentration-response curves to the right in an apparently competitive manner; Schild analysis yielded a pA2 of 5.5. These data indicate that DPAT blocks histamine and muscarinic receptors on longitudinal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual effects of endothelins -1, -2 and -3 on guinea pig field-stimulated ileum: possible mediation by two receptors coupled to pertussis toxin-insensitive mechanisms.

This study compared the effects of endothelin-1 (ET-1), ET-2 and ET-3 on the guinea pig field-stimulated ileum. All ETs (0.3-30 nM) caused graded inhibitions of nerve-mediated responses followed by sustained contractions. The rank order of potencies for the twitch depressor effect (IC50S) was ET-3 = ET-1 greater than ET-2, with ET-3 causing greater maximal inhibition than ET-1 or ET-2. The rank order of potencies for contraction (EC50S) was ET-1 = ET-2 greater than ET-3, with ET-1 causing greater maximal contraction than ET-2 or ET-3. Twitch inhibition by ET-1 (3 nM) was unaffected by indomethacin (5.6 microM), cromakalim (10 microM), glibenclamide (3 microM) or nicardipine (0.1 microM). ET-1-induced contraction was unaltered by tetrodotoxin (0.3 microM), atropine (0.3 microM) or glibenclamide, but was reduced by indomethacin. Cromakalim and nicardipine virtually abolished ET-1-induced contraction. ET-1 (up to 30 nM) did not potentiate submaximal contractions induced by acetylcholine, histamine, bradykinin or substance P. ET-3 relaxed ileal segments precontracted with either acetylcholine (0.3 microM) or histamine (1 microM). Pretreatment of guinea pigs with pertussis toxin (50 micrograms/kg i.p., 6 days beforehand) did not influence either effects of ET-1 on the field-stimulated ileum. Our data suggest that the dual effects of ETs on the guinea pig isolated ileum are mediated by distinct receptors and possibly involve different mechanisms of action. The transient inhibition of responses to field stimulation seems unrelated to activation of ATP-sensitive potassium channels and is rather insensitive to L-type Ca++ channel blockade.     

A subtype of the gamma-aminobutyric acid(B) receptor regulates cholinergic twitch response in the guinea pig ileum

The pharmacological profile of the gamma-aminobutyric acid (GABA)(B) receptor regulating cholinergic twitch contraction in the guinea pig ileum myenteric plexus-longitudinal muscle preparation was investigated. GABA and (-)-baclofen inhibited the contraction, exhibiting quite close potencies (pD(2) for GABA = 5.70; pD(2) for (-)-baclofen = 5.33). The compound CGP 47656 also reduced the cholinergic twitch concentration (pD(2) = 5.42), but its efficacy was significantly lower than that of (-)-baclofen or GABA. Added at varying concentrations, CGP 47656 modified the concentration-response curve of (-)-baclofen as expected for a partial agonist. Phaclofen, CGP 36742, CGP 35348, and CGP 52432 behaved as competitive antagonists of (-)-baclofen, exhibiting the following pA(2) values: 3.90, 4.88, 5.02, and 7.82, respectively. The compound CGP 56999 behaved as a potent noncompetitive GABA(B) receptor antagonist. In comparing the pharmacological profile of the ileal receptor with those of the previously characterized pharmacological subtypes of the GABA(B) receptor present in the central nervous system, it can be seen that the GABA(B) receptor inhibiting cholinergic twitch contraction in guinea pig ileum myenteric plexus-longitudinal muscle mostly resembles the receptor located on somatostatin human neocortex nerve terminals.     

Mechanism of contractile response to bombesin of the longitudinal muscle from guinea pig ileum

The mechanism of action of bombesin in the longitudinal muscle with myenteric plexus (LM-MP) preparation from guinea pig ileum was examined. Bombesin (3 x 10(-10) to 3 X 10(-7) M) induced contraction in a concentration-dependent manner. The contractions induced by bombesin at concentrations of 10(-9) M and less were tetrodotoxin- and scopolamine-sensitive, and those induced at concentrations of 10(-8) M and greater were tetrodotoxin-sensitive, and partially scopolamine-sensitive. In association with the mechanical response to bombesin, the peptide induced Ca(++)-dependent and tetrodotoxin-sensitive release of acetylcholine from the LM-MP preparation. Bicuculine inhibited partly both the cholinergic contraction and the release of acetylcholine induced by bombesin, suggesting the involvement of a gamma-aminobutyric acid (GABA)-ergic mechanism. Bombesin also induced Ca(++)-dependent and tetrodotoxin-sensitive release of endogenous and [3H]GABA from the LM-MP preparation. These results indicate that bombesin induces contraction mainly mediated mediated by cholinergic and partially mediated by noncholinergic excitatory neurons. GABAergic neurons may be involved in a part of the response to bombesin mediated by the cholinergic neuron.     

Adenosine A1 and A2 receptors mediate presynaptic inhibition and postsynaptic excitation in guinea pig submucosal neurons

Intracellular recordings were made from guinea pig submucosal neurons in vitro. Adenosine, 2-[p-(carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine (CGS21680), 2-chloroadenosine (CADO), 5'-N-ethylcarboxamidoadenosine (NECA), R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), N6-cyclohexyladenosine (CHA) and 1-deaza-2-chloro-N6-cyclopentyladenosine (DCCPA) were applied by adding them to the superfusion solution. Adenosine (30 nM to 30 microM) depolarized S-type neurons neurons and this was mimicked by analogs with potency order: CGS21680 = NECA greater than R-PIA greater than CADO greater than adenosine greater than CHA much greater than DCCPA. 8-Cyclopentyltheophylline (CPT) blocked the depolarizing action of CADO or R-PIA; this antagonism was surmountable and the dissociation equilibrium constant (KD) estimated by the Schild method was 295 nM. Synaptic potentials were evoked by focal stimulation of nerve strands running between submucosal ganglia. The nicotinic excitatory postsynaptic potential was reduced by adenosine and analogs with potency order CHA = R-PIA greater than DCCPA = NECA = CADO greater than adenosine much greater than CGS21680; CPT competitively antagonized this effect of CADO or CHA with a KD of 13 nM. The noradrenergic inhibitory postsynaptic potential was also reduced; the potency order was R-PIA greater than CHA = CADO = NECA = DCCPA greater than adenosine much greater than CGS21680; the KD of CPT as an antagonist was 7 nM. It is concluded that adenosine directly depolarizes submucosal neurons by acting at an A2 receptor, and that it inhibits the release of acetylcholine (from intramural nerves) and noradrenaline (from sympathetic nerves) by acting at a presynaptic A1 receptor.     

Suppression by muscarinic M2 stimulation of the contractile mechanism via histamine H1 receptors in the longitudinal muscle of guinea pig ileum

The longitudinal muscle of the guinea pig ileum is well known to contract in response to stimulation of either muscarinic or histamine H1 receptors. However, we found that simultaneous application of agonists to both receptors, methacholine (MeCh) and histamine (Hist), at their maximum concentrations (both at 10(-5) M), induced a contraction of the muscle with a pattern very similar to that observed with MeCh alone. In the muscle contracted by MeCh, a muscarinic antagonist, atropine, caused a transient relaxation, but a histamine H1 blocker, pyrilamine, had no effect on the tension. Experiments using pirenzepine indicated that muscarinic-M1 receptors did not seem to be involved in the mechanism of this phenomenon. The atropine-induced relaxation was not affected by phentolamine (10(-6) M), propranolol (10(-6) M) or tetrodotoxin (10(-6) M). After pretreatment of the muscle with 10(-5) M MeCh for 10 min, the contractile effect of 10(-5) M Hist was suppressed intensely for a certain time period, but that of 10(-5) M MeCh was not diminished. On the other hand, pretreatment of the muscle with 10(-5) M Hist for 10 min did not influence the contraction by either MeCh or Hist. The suppression by MeCh of the Hist action developed dependently on the concentration of MeCh and the time length of exposure to MeCh, and the persistence was temperature-dependent. Furthermore, this suppression was not surmounted by the elevation of Hist concentration. A similar phenomenon was also observed in the muscle which was immersed in the nutrient solution without CaCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct determination of acetylcholine release by radioimmunoassay and presence of presynaptic M1 muscarinic receptors in guinea pig ileum

Radioimmunoassay for acetylcholine (ACh) with a sensitivity of 10 pg/tube was applied to the direct determination of ACh output from the nerve endings in longitudinal muscle strips of guinea pig ileum. The strips were preincubated with an irreversible cholinesterase inhibitor and superfused with Krebs' solution under various experimental conditions. Pirenzepine (0.1-10 microM) and atropine (10-100 nM) produced an increase in electrically evoked ACh output through the inhibition of presynaptic muscarinic receptors. Contractile response to endogenous ACh released by electrical stimulation was enhanced by pirenzepine and atropine at lower concentrations, whereas the highest concentrations of pirenzepine (10 microM) and atropine (100 nM) caused a reduction in the enhanced contractile response and a significantly diminished response, respectively. These results demonstrate that the concentrations of pirenzepine and atropine, effective in inhibiting presynaptic muscarinic receptors, differ from those inhibiting postsynaptic muscarinic receptors and suggest the possibility that presynaptic M1 muscarinic receptors regulating ACh output may be present in the guinea pig ileum.     

Inhibition of guinea pig ileum contractions mediated by a class of histamine receptor resembling the H3 subtype

Segments of guinea pig ileum were isolated and placed on coaxial electrodes in organ baths filled with oxygenated Krebs' solution at 37 degrees C. Twitch responses were produced with rectangular-wave stimuli (0.5 msec; 10 Hz; 4.8-12 V) delivered in 1-sec trains every 10 sec. After addition of pyrilamine to block H1-mediated contractions, histamine and N alpha-methylhistamine (NMH) inhibited the twitch responses with EC50 values of 64 and 1.7 nM, respectively. In contrast, there was no difference in potency between histamine and NMH at contractile H1 receptors in ileum longitudinal muscle or at chronotropic H2 receptors in the atria. Impromidine blocked the inhibitory effect of NMH in a competitive manner with a KB value of 26 nM, whereas inhibition caused by gamma-aminobutyric acid or by adenosine remained unaffected by impromidine. At concentrations that completely prevented the electrically induced twitch, NMH did not alter contractions produced by exogenous acetylcholine. Inhibitory responses to NMH were unaffected by cimetidine, hexamethonium, combined alpha and beta adrenergic receptor blockade, naloxone, 8-phenyltheophylline or gamma-aminobutyric acid receptor desensitization. These data support the presence of inhibitory histamine receptors in the ileum that are pharmacologically similar to the presynaptic H3 autoreceptors found in rat cortex. The distribution of H3 receptors, therefore, may not be confined to the central nervous system and their function, as with alpha-2 adrenergic sites, may not be limited to that of an autoreceptor.     

Histamine receptors in the myenteric plexus-longitudinal muscle of the guinea-pig ileum: H1- and H2-receptor-mediated potentiation of the contractile response to electrical stimulation

The effect of histamine on the contractile response to low frequency-electrical field stimulation in the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum was investigated. By blocking the direct increase in smooth muscle tone caused by histamine with low concentrations of pyrilamine (10(-9)--5 x 10(-8) M) a dose-dependent, histamine-induced potentiation of the twitch response to electrical stimulation was observed. Blocking the direct actions of histamine with concentrations of pyrilamine greater than 10(-7) M resulted in a biphasic histamine dose-response curve: lower histamine concentrations produced a dose-dependent decrease of the twitch response; higher concentrations produced a potentiation. The potentiating effect of histamine was inhibited by high concentrations of H2-receptor antagonists. Tiotidine (ICI 125, 211) had a pA2 of 5.25, 100 times greater than its pA2 in isolated guinea-pig atria. Blockade of the actions of the H2-receptor agonists dimaprit and tetrahydrozoline also required greater antagonist concentrations. The selective H1-receptor agonist, 2-(2-thiazolyl)-ethylamine, also enhanced the response to electrical stimulation. The potentiating effect of histamine could be blocked by hexamethonium (10(-7)--10(-5) M) but not by atropine. Atropine (10(-9)--10(-8) M) did prevent the decrease in the contraction amplitude induced by histamine in the presence of 10(-7) M pyrilamine. The purinergic antagonist theophylline, adrenergic antagonists or depletion of endogenous catecholamines were without effect. Tetrodotoxin (10(-6) M) inhibited the augmentation induced by histamine. Our results demonstrate that histamine potentiates the acetylcholine-mediated contractile response to electrical field stimulation of guniea-pig ileum via H1- and what may be an H2-receptor subtype.     

Antigen-induced enhancement of noncholinergic contractile responses to vagus nerve and electrical field stimulation in guinea pig isolated trachea.

Nonadrenergic, noncholinergic contractions were elicited by electrical field stimulation (EFS) (2 Hz, 1 msec, 12 V for 15 sec) of the distal aspect of guinea pig trachea pretreated with atropine (1 microM), propranolol (1 microM) and indomethacin (3 microM). The contractions were abolished by pretreatment with the sensory C-fiber toxin capsaicin or by a combination of the neurokinin (NK)1 receptor antagonist, CP 96,345 (0.1 microM), and the NK2 receptor antagonist, MEN 10376 (3 microM), and were markedly attenuated by tetrodotoxin. In animals actively sensitized to ovalbumin, the addition of threshold concentrations of antigen markedly increased the noncholinergic contractile responses to EFS (approximately 3- to 6-fold). This potentiation was long lasting, persisting virtually unchanged for 60 min, whereas the antigen-induced contractions were shorter lived, usually lasting less than 30 min. The ovalbumin-induced potentiation of the neuronal response was not observed in tissues pretreated with capsaicin or treated with tetrodotoxin. This antigen-induced potentiation of capsaicin-sensitive, EFS-induced contractions was not mimicked by serotonin or prostaglandin D2. However, it was mimicked by histamine. Moreover, the histamine H1 receptor antagonist pyrilamine (0.3 microM) reversed the potentiation elicited by ovalbumin. The effect of ovalbumin challenge was also examined on the distal trachea with the right vagus nerve intact. Noncholinergic contractions to EFS and vagus nerve stimulation were enhanced equally by threshold concentrations of antigen. The results support the hypothesis that antigen challenge releases histamine which acts via H1 receptors to enhance noncholinergic contractions due to the release of tachykinins from capsaicin-sensitive fibers in the guinea pig trachea.     

Inotropic, electrophysiological and biochemical responses to histamine in rabbit papillary muscles: evidence for coexistence of H1- and H2-receptors

This study was performed to determine the subtypes of histamine receptors that are involved in the electrophysiological, inotropic and biochemical responses to histamine in isolated rabbit papillary muscles. Histamine increased force of contraction and shortened action potential duration (APD) in a concentration-dependent manner. The former was antagonized by chlorpheniramine, a H1-antagonist, whereas the latter was blocked by cimetidine, a H2-antagonist. However, even when H1-receptors were blocked entirely by chlorpheniramine, histamine still produced a positive inotropic effect, an effect which was antagonized by cimetidine. On the other hand, when H2-receptors were eliminated by cimetidine, histamine caused a H1-receptor mediated APD prolongation. Carbachol attenuated the decrease in APD but not the increase in force of contraction caused by histamine. Cyclic AMP and cyclic GMP levels both were elevated significantly by histamine. The increase in cyclic AMP level induced by histamine was abolished by cimetidine, but not altered by chlorpheniramine, whereas the converse was true for the increase in cyclic GMP level. Additionally, histamine produced a significant stimulation of phosphoinositide hydrolysis as measured by [3H]inositol monophosphate accumulation, although its extent was far less than that produced by carbachol. The phosphoinositide response to histamine was blocked by chlorpheniramine. These data suggest that H1- and H2-receptors coexist in rabbit ventricles. Stimulation of H1- and H2-receptors with histamine independently sets off the biochemical responses linked specifically to the respective subtypes of histamine receptors. On the other hand, the inotropic and electrophysiological responses to histamine are governed predominantly by H1- and H2-receptors, respectively, and this results in an apparent restriction of the expression of the responses mediated by another subtype.     

Evidence for a noradrenergic innervation to "atypical" beta adrenoceptors (or putative beta-3 adrenoceptors) in the ileum of guinea pig.

Indirect evidence suggests that beta-1 adrenoceptors in the guinea pig ileum are innervated but it has not been determined whether "atypical" beta adrenoceptors also receive a postganglionic sympathetic innervation. To answer this question, experiments were undertaken using electrical stimulation of para-arterial sympathetic neurons to evoke relaxation in isolated segments of guinea pig ileum. Tension was developed in the ileal segments by either transmural electrical field stimulation to evoke the cholinergic "twitch" response, or by histamine to produce a steady-state contracture. Para-arterial sympathetic nerve stimulation evoked a frequency-dependent inhibition of the twitch response which was blocked by guanethidine and restored by dexamphetamine, indicating typical noradrenergic transmission. In preparations contracted with histamine and pretreated with benextramine to block alpha adrenoceptors, para-arterial sympathetic nerve stimulation evoked frequency-dependent relaxations which were reduced in magnitude but not abolished by the following beta adrenoceptor antagonists: bromoacetylalprenololmenthane (1 microM) or a combination of ICI 118,551 (0.3 microM) and CGP 20712A (0.1 microM). Remaining responses were blocked by compounds exhibiting affinity for atypical beta adrenoceptors, (-)-alprenolol (3 microM) and nadolol (300 microM), as well as the agonist (-)-isoproterenol (10 microM; to saturate the atypical beta adrenoceptor). However, relaxations to papaverine were unaffected by these treatments. Experiments revealed that potential cotransmitters (ATP, neuropeptide Y and somatostatin) do not appear to play a detectable role in relaxations produced by para-arterial sympathetic nerve stimulation. The results demonstrate, for the first time, that atypical beta adrenoceptors in guinea pig ileum receive a noradrenergic innervation.     

Intrinsic differences in the filling response of the guinea pig duodenum and ileum

Transit of intestinal contents differs between the duodenum and ileum in intact animals. To determine whether this relates to intrinsic differences in the capacity, distensibility, or filling response of these segments, we used the isolated duodenum and ileum of 13 guinea pigs to record: (1) increases in the diameter and luminal volume produced by specific pressure heads and (2) the pressure waves and movements of the segments in response to bolus injections. We found that the duodenum and the ileum had similar diameters in the empty state, and when filled with volumes below 0.4 ml both generated baseline pressures below 3 cm H2O. However, the ileum increased its diameter and volume significantly more in response to increases in inflow pressure than the duodenum did. Conversely, injection of bolus volumes led to pressure waves of higher amplitude and longer duration in the duodenum than in the ileum. The pressure waves were produced by sharply defined ring contractions in the ileum; there was bulging of the walls downstream from the contraction site and collapse upstream from it. In the duodenum, the contraction involved a longer segment and was shallow with ill-defined margins. These intrinsic differences in the capacity, distensibility, and contraction patterns of the duodenum and ileum are likely to affect luminal transit and other mechanical functions of these intestinal segments in the intact animal.     

Effects of platelet-activating factor on β- and H2-receptor-mediated increase of myocardial contractile force in isolated perfused guinea pig hearts

Platelet-activating factor (PAF) has been termed an important mediator of cardiovascular shock due to immunological reactions, including anaphylaxis and endotoxic reactions. Previous studies have shown that PAF is a potent cardiodepressive agent inducing a drastic coronary constriction and a sustained impairment of myocardial contractility. In this study, an attempt was made to further characterize the prolonged PAF effects on coronary circulation and myocardial contractile force in the isolated guinea pig heart perfused at constant pressure. An intracoronary PAF bolus (0.18 nmol, related to coronary flow rates of 1 ml/min) induced a precipitous decrease of coronary flow rates, left ventricular pressure, and left ventricular contraction (peak positive dP/dt), which was followed by a slow increase reaching new steady state after 15 min (-48%, -40%, -42% below baseline, respectively). If the specific PAF antagonist WEB 2086 (3.65 nmol/min, related to coronary flow rates of 1 ml/min) was infused 30 min after PAF administration, the prolonged PAF-mediated cardio-depressive effects were rapidly reversed. Several studies indicate that PAF induces a down regulation of beta-adrenoreceptors in different cell types, including human lung tissue. Therefore, a further objective of the study was to evaluate whether PAF selectively impairs the positive inotropic effects of beta-receptor agonists or also inhibits the contractile effects of inotropic drugs, which are known to enhance cardiac contractility independently of beta-receptors. In these experiments, the beta-agonist isoproterenol and the H2-agonist impromidine were administered as intracoronary boluses (0.35 nmol and 0.14 nmol, respectively, related to coronary flow rates of 1 ml/min) prior to PAF injection and 30 min after PAF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic alpha adrenoceptor modulation of neurally mediated cholinergic excitatory and nonadrenergic noncholinergic inhibitory responses in guinea pig trachea

Cholinergic and nonadrenergic noncholinergic (NANC) excitatory nerves in guinea pig trachea are subject to presynaptic alpha-2 adrenoceptor inhibitory control. Although the trachea is also innervated by NANC inhibitory nerves, little is known about their presynaptic regulation. The present study assessed the capacity of alpha-1 and alpha-2 adrenoceptor agonists to modulate NANC inhibitory nerves and for comparison, cholinergic excitatory nerves in guinea pig trachea. To eliminate effects of intrinsic sympathetic nerve stimulation and prostanoid production, tissues were pretreated with guanethidine, propranolol and indomethacin. The alpha-2 adrenoceptor agonist, clonidine (1 microM), induced a 12-fold rightward shift of the frequency-response curve for neurally mediated cholinergic contractions but had no effect on the concentration-response curve for exogenously administered acetylcholine. This action of clonidine was inhibited in a concentration-dependent manner by the alpha-2 adrenoceptor antagonist, yohimbine, and by the alpha-1 adrenoceptor antagonist, prazosin, NANC inhibitory responses were unaffected by clonidine (1 microM). The alpha-1 adrenoceptor agonist, phenylephrine (1 microM), failed to influence responses induced by cholinergic or NANC inhibitory nerve stimulation, acetylcholine or vasoactive intestinal peptide. Furthermore, in tissues treated with propranolol but not subjected to adrenergic neuronal blockade with guanethidine, neurally mediated cholinergic responses were not altered by yohimbine (0.3 microM) treatment. These results indicate that in guinea pig trachea: 1) cholinergic nerves are modulated by presynaptic, prazosin-sensitive inhibitory presynaptic alpha-2 adrenoceptors and 2) NANC inhibitory nerves do not possess presynaptic, modulatory alpha adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Failure of aerosolized endothelin (ET-1) to induce bronchial hyperreactivity in the guinea pig

Aerosol administration of endothelin (ET-1) has been shown to provoke a potent bronchoconstriction in the guinea pig. We investigated whether or not, aerosolized ET-1 induces a bronchial hyperreactivity in the guinea pig. Aerosolized ET-1 (10 micrograms/ml for 60 min) did not alter the dose-response curve, established by successive aerosol administration of acetylcholine (ACh) 3-4 h and 18-24 h after challenge with the peptide. In a second protocol, aerosolized ET-1 (10 micrograms/ml for 3 min) induced, in anaesthetized guinea pigs a bronchopulmonary response but did not alter the dose-response curve to aerosolized ACh established 30 min after the challenge. These results suggest that ET-1 may participate to the early, but not the late alteration of the bronchopulmonary tone observed during pathophysiological conditions.     

Characterization of high affinity [3H]pirenzepine and (-)-[3H] quinuclidinyl benzilate binding to muscarinic cholinergic receptors in rabbit peripheral lung

We have characterized the binding of the selective muscarinic antagonist [3H]pirenzepine ([3H])PZ) and the classical muscarinic antagonist (-)-[3H]quinuclidinyl benzilate ((-)-[3H]QNB) to muscarinic cholinergic sites in rabbit peripheral lung membranes. For both radioligands, high affinity binding with pharmacologic specificity was demonstrated. The high affinity Kd for [3H]PZ binding determined from saturation isotherms was 4.5 nM and the Kd for (-)-[3H]QNB binding was 6.2 pM. Comparison of the total binding capacity values determined by saturation experiments with [3H] PZ and (-)-[3H]QNB demonstrates that approximately 78% of the total muscarinic binding sites in rabbit peripheral lung bind [3H]PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (-)-[3H]QNB binding by the muscarinic agonist carbachol in peripheral lung membranes. If the pulmonary muscarinic receptor with high affinity for PZ proves to have an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.     

Acetylcholine-induced desensitization of the contractile response to histamine in Guinea pig ileum is prevented by either pertussis toxin treatment or by selective inactivation of muscarinic M(3) receptors

We have studied the role of M(2) and M(3) muscarinic receptors in acetylcholine-mediated desensitization of the contractile response to histamine in the guinea pig ileum. Treatment of the isolated ileum with acetylcholine (30 microM) for 20 min caused a marked desensitization of the contractile response to histamine. When measured 5 min after washout of acetylcholine, the EC(50) value of histamine increased 5.8-fold compared with that estimated before acetylcholine treatment, whereas the maximal response was unaffected. This shift in the EC(50) value of histamine was maximal at the earliest time measured after acetylcholine treatment (5 min), and normal sensitivity recovered in approximately 20 min. Acetylcholine-induced desensitization was prevented by uncoupling of M(2) receptors from G(i) with pertussis toxin or by selective inactivation of M(3) receptors with N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP mustard). The shifts in the EC(50) values of histamine measured 5 min after acetylcholine treatment were only 2.0- and 1.8-fold in pertussis toxin- and 4-DAMP mustard-treated ilea, respectively. Both pertussis toxin- and 4-DAMP mustard-treatment had little or no effect on histamine-induced contractions in control ileum. Measurement of histamine-stimulated inositol phosphate accumulation in the longitudinal muscle of the ileum showed little or no inhibitory effect of prior exposure to acetylcholine, indicating that the majority of the heterologous desensitization occurs downstream from phospholipase Cbeta activation. Collectively, our results suggest that activation of both M(2) and M(3) receptors is required for heterologous desensitization of histamine-mediated contractions in the guinea pig ileum.