Gianluigi D'Agostino

Prejunctional muscarinic inhibitory control of acetylcholine release in the human isolated detrusor: involvement of the M4 receptor subtype.

Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response. In detrusor strips preincubated with [3H]‐choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]‐ACh (76%) and [3H]‐choline (24%). Electrically‐evoked [3H]‐ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release. The effects of various subtype‐preferring muscarinic receptor antagonists were evaluated on [3H]‐ACh release and muscle contraction. The rank potency (−log EC50) orders at pre‐ and postjunctional level were: atropine 4‐diphenyl‐acetoxy‐N‐piperidine (4‐DAMP)>mamba toxin 3 (MT‐3)>tripitramine>para‐fluorohexahydrosiladiphenidol (pF‐HHSiD)methoctraminepirenzepine>tripinamide, and atropine4‐DAMP>pF‐HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT‐3, respectively. The comparison of pre‐ and post‐junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.

Characterization of prejunctional muscarinic autoreceptors in the guinea‐pig trachea

1 The effects of ten muscarinic antagonists on electrically evoked [3H]‐acetylcholine release and muscle contraction were compared in an epithelium‐free preparation of the guinea‐pig trachea that had been preincubated with [3H]‐choline. 2 The M3‐selective antagonists UH‐AH 37, 4‐diphenyl‐acetoxy‐N‐piperidine methobromide and para‐fluorohexahydrosiladiphenidol were more potent in reducing the contractile response than in facilitating the evoked [3H]‐acetylcholine release. Hexahydrosiladiphenidol did not discriminate between pre‐ and postjunctional effects. The rank order of the postjunctional potencies of the ten antagonists as well as the postjunctional pA2 values obtained for hexahydrosiladiphenidol (7.95) and AQ‐RA 741 (7.08) identified the muscular receptor as an M3 subtype. 3 The M2‐selective antagonists methoctramine, AF‐DX 116 and AQ‐RA 741 were more potent in facilitating the evoked [3H]‐acetylcholine release than in inhibiting the contractile response. The increase in release by low concentrations of methoctramine, AF‐DX 116 and AQ‐RA 741 was paralleled by an enhancement of the stimulation‐evoked contractions. 4 Comparison of the pre‐ and postjunctional potencies of the M1‐, M2‐ and M3‐selective antagonists suggests that autoinhibition of acetylcholine release is mediated via an ‘M2‐like’ receptor which differs from the cardiac type M2 receptor in its relatively high affinity for hexahydrosiladiphenidol.

Muscarinic inhibition of acetylcholine release from a novel in vitro preparation of the guinea-pig trachea

SummaryAn isolated preparation of the guinea-pig trachea is described which allows the simultaneous measurement of acetylcholine release and smooth muscle contraction. Incubation of the epithelium-free preparation with [3H]choline resulted in the formation of [3H]acetylcholine. Electrical stimulation caused the release of [3H]acetylcholine and a contractile response. Tetrodotoxin and omission of calcium from the medium abolished both the evoked release and contractions.The muscarinic agonists oxotremorine, carbachol and pilocarpine concentration-dependently inhibited the electrically evoked acetylcholine release and contracted the tracheal smooth muscle. Pre- and postsynaptic EC50 values for a given agonist were not different. Atropine (100 nmol/l) significantly faciliated the evoked acetylcholine release. A concentration of 10 nmol/l atropine did not change the evoked release but antagonized the inhibitory effect of oxotremorine. It is concluded that presynaptic muscarine autoreceptors inhibit the release of acetylcholine from parasympathetic nerves of the guinea-pig trachea.

Role of galanin receptor 1 in peristaltic activity in the guinea pig ileum

Galanin effects are mediated by distinct receptors, galanin receptor 1 (GAL-R1), GAL-R2 and GAL-R3. Here, we analyzed 1) the role of GAL-R1 in cholinergic transmission and peristalsis in the guinea-pig ileum using longitudinal muscle-myenteric plexus preparations and intact segments of the ileum in organ bath, and 2) the distribution of GAL-R1 immunoreactivity in the myenteric plexus with immunohistochemistry and confocal microscopy. Galanin inhibited electrically stimulated contractions of longitudinal muscle-myenteric plexus preparations with a biphasic curve. Desensitization with 1 microM galanin suppressed the high potency phase of the curve, whereas the GAL-R1 antagonist, RWJ-57408 (1 microM), inhibited the low potency phase. Galanin (3 microM) reduced the longitudinal muscle contraction and the peak pressure, and decreased the compliance of the circular muscle. All these effects were antagonized by RWJ-57408 (1 or 10 microM). RWJ-57408 (10 microM) per se did not affect peristalsis parameters in normal conditions, nor when peristalsis efficiency was reduced by partial nicotinic transmission blockade with hexamethonium. In the myenteric plexus, GAL-R1 immunoreactivity was localized to neurons and to fibers projecting within the plexus and to the muscle. GAL-R1 was expressed mostly by cholinergic neurons and by some neurons containing vasoactive intestinal polypeptide or nitric oxide synthase. This study indicates that galanin inhibits cholinergic transmission to the longitudinal muscle via two separate receptors; GAL-R1 mediates the low potency phase. The reduced peristalsis efficiency could be explained by inhibition of the cholinergic drive, whereas the decreased compliance is probably due to inhibition of descending neurons and/or to the activation of an excitatory muscular receptor. Endogenous galanin does not appear to affect neuronal pathways subserving peristalsis in physiologic conditions via GAL-R1.

Comparative studies of the postjunctional activities of some very potent muscarinic agonists

SummaryThis study was undertaken to determine the potenties of seven muscarinic agonists (methylfurtrethonium, dioxolane, oxathiolane, carbachol, muscarine, muscarone and oxotremorine) on the postjunctional muscarinic receptors of seven isolated preparations (guinea pig taenia-coli, ileum, jejunum, trachea and atria and rat jejunum and urinary bladder).The results indicate that the rank order of sensitivity of the preparations varies independently of the potency of the agonist used and it is almost the same for all the compounds with the exception of oxotremorine.Muscarone was the most potent compound in all the tissues. Intergroup comparisons in each preparation and the evaluation of the equieffective molar ratios relative to muscarone revealed that carbachol possesses a certain degree of cardioselectivity and oxathiolane, on the other hand, is much less active on the cardiac tissue than on the others.Oxotremorine is a peculiar compound endowed with cardioselectivity.

Tachykinin NK2 receptors facilitate acetylcholine release from guinea-pig isolated trachea

The release of newly synthesised [3H]acetylcholine was evoked by electrical field stimulation (5 Hz, 600 pulses) of epithelium-deprived guinea-pig trachea strips after sensory neuropeptides depletion with 3 microM capsaicin. The selective tachykinin NK(2) receptor agonist [betaAla(8)]neurokinin A-(4-10) increased in a concentration-dependent manner the electrically-induced release of [3H]acetylcholine. The facilitatory effect was antagonised by the selective non-peptide tachykinin NK(2) receptor antagonist, SR 48968 (apparent pK(B) 8.9). The tachykinin NK(1) and NK(3) receptor agonists substance P methyl ester and senktide (both 10 and 100 nM), respectively, did not affect the evoked release of [3H]acetylcholine. It is concluded that the cholinergic nerves of guinea-pig trachea are endowed with prejunctional facilitatory tachykinin receptors of the NK(2) subtype.

Prejunctional effects of muscarinic agonists on 3H-acetylcholine release in the rat urinary bladder strip

SummaryThe inhibitory effects of some muscarinic agonists on tritiated acetylcholine release evoked by field stimulation were investigated in the rat urinary bladder strip. The acetylcholine stores of the preparation were labelled with 3H-choline. Electrical field stimulation caused an outflow of tritium, reflecting the release of 3H-acetylcholine. The release of 3H-acetylcholine was decreased in a concentration-dependent manner by all the agonists tested: oxotremorine, muscarone, muscarine, carbachol and methylfurtrethonium. On the contrary, only muscarine and muscarone enhanced the basal efflux of tritium in a concentration-dependent fashion. Concentration-response curves were determined both at 2 Hz and at 1 Hz by using intermittent administration of the drugs. Maximal depression in release (by 78 – 82%) was observed in experiments at 1 Hz. A similar inhibition was obtained at 2 Hz frequency only when a low concentration of calcium (0.6 mM) in the medium was used. Oxotremorine was the most potent among the tested compounds with the same intrinsic activity as the other drugs. In contrast to the other agonists investigated, oxotremorine showed in about 10-fold greater potency at pre- than at postjunctional muscarine receptors in the rat urinary bladder. This difference might depend either on heterogeneity of muscarine receptors or on different mechanism(s) relating to the transducing properties of receptors at the pre- and postjunctional level. A comparison between the relative prejunctional potencies in the rat urinary bladder and in the guinea pig myenteric plexus (data from the literature) suggests that prejunctional muscarine receptors are similar in these tissues. Furthermore, the findings obtained with a low concentration of calcium in the medium may support the view that intraneuronal availability of calcium plays a significant role in modulating the prejunctional negative feed-back mechanism in the rat urinary bladder.

An appraisal of recently patented compounds for bladder overactivity and urinary incontinence

Bladder overactivity and urinary incontinence are lower urinary tract syndromes for which there is no adequate pharmacological treatment. The latter condition is associated with an involuntary loss of urine causing a hygienic and social concern to the patient. Recently, central and peripheral mechanisms that control bladder storage and voiding processes have been partly addressed and clarified. Novel antimuscarinic agents, as well as newer formulations of available drugs and selective antagonists at α1D-receptors, have been patented, in addition to selective agonists at the β3-adrenoceptor and selective antagonists at 5-hydroxytryptamine, subtype 1A and 7 (5-HT1A and 5-HT7) receptors. Other potential therapeutic options include neurokinin type 1 and 2 (NK1/NK2) tachykinin receptor antagonists and potassium channel openers. The aforementioned agents may contribute to the pharmacological armamentarium in urology treatment, although their therapeutic effectiveness and safety profile remain to be validated in the clinical setting.

Isolated porcine bronchi provide a reliable model for development of bronchodilator anti-muscarinic agents for human use

Background and purpose: In human airways, muscarinic acetylcholine receptors (mAChRs) exert a predominant role in the control of airways resistance and anti‐muscarinic agents are currently included in the pharmacological treatment of chronic obstructive pulmonary disease (COPD). However, the development of more effective mAChR antagonists is hampered by considerable species variability in the ultrastrucural and functional control of airway smooth muscle, making extrapolation of any particular animal model questionable. This study was designed to characterize the mAChRs in a bronchial preparation from pigs, animals considered to provide close models of human biology.

The influence of verapamil and papaverine on the calcium- and epinephrine-induced responses of isolated guinea-pig atria☆

The effects of papaverine and verapamil on Ca2+ - and epinephrine evoked responses were investigated on spontaneously beating atria of the guinea-pig. Papaverine showed a non competitive antagonism against epinephrine but was ineffective against Ca2+; verapamil antagonized both the action of epinephrine and Ca2+ in a different manner but at the same concentrations. The pharmacological prerequisite to classify an anti-Ca2+ substance as a blocker of Ca2+ entry (that is the ability to selectively antagonize the Ca2+ -induced contraction relative to those of norepinephrine in vascular smooth muscle) was not met by the results obtained in the cardiac tissue. The behaviors of verapamil and papaverine with regard to Ca2+ response suggest another criterion for classifying a substance as a Ca2+ entry blocker based on the ability or inability to inhibit the response of the atrial preparation to Ca2+.