Amplitude Of Excitatory Junction Potentials Research Articles

Modulation of the buccal muscle contraction by identified serotonergic and peptidergic neurons in the snail Achatina fulica

Serotonergic and peptidergic modulation of buccal muscle contraction at an identified neuromuscular synapse in the African giant snail Achatina fulica was examined. A pair of excitatory motoneurons of the radula protractor was identified in the buccal ganglia and these were named B10 neurons. Pharmacological experiments revealed the B10s to be cholinergic. The serotonergic cerebral neuron v-CDN enhanced B10-evoked contraction of the radula protractor, and it is suggested that this effect is mediated postsynaptically by serotonin released from the v-CDN terminals. Immunohistochemical analyses revealed that the B10 motoneurons contained an Achatina cardioexcitatory peptide-1 (ACEP-1)-like substance in their cell body, axon and terminals in the radula protractor. From electron microscopic observation, the ACEP-1-like immunoreactive substance was found to be localized in dense-cored vesicles but not in clear vesicles in the nerve terminals. ACEP-1 applied to the radula protractor markedly enhanced B10-evoked contraction of the muscle by increasing the amplitude of excitatory junction potentials (EJPs). The increase of EJP amplitude in the presence of the peptide was probably due to the increased release of acetylcholine from the terminals of B10. It is suggested that the cholinergic motoneuron B10 uses ACEP-1 as an excitatory cotransmitter.

Physiological effects of two FMRFamide-related peptides from the crayfish Procambarus clarkii

The present study examined the effects of two recently identified neuropeptides on crayfish hearts and on neuromuscular junctions of the crayfish deep abdominal extensor muscles. The two peptides, referred to as NF1 (Asn-Arg-Asn-Phe-Leu-Arg-Phe-NH2) and DF2 (Asp-Arg-Asn-Phe-Leu-Arg-Phe-NH2), increased the rate and amplitude of spontaneous cardiac contractions and increased the amplitude of excitatory junctional potentials (EJPs) in the deep extensors. Both effects were dose-dependent, but threshold and EC50 values for the cardiac effects were at least 10 times lower than for the deep extensor effects. The heart responded equally well to three sequential applications of peptide in any given preparation, but the responses of the deep extensors appeared to decline with successive peptide applications. The results support the hypothesis that these two neuropeptides act as neurohormones to modulate the cardiac and neuromuscular systems in crayfish. Quantal synaptic current recordings from the deep extensor muscles indicate that both peptides increase the number of quanta of transmitter released from synaptic terminals. Neither peptide elicited a measurable change in the size of quantal synaptic currents. NF1 caused a small increase in muscle cell input resistance, while DF2 did not alter input resistance. These data suggest that DF2 increases EJP amplitudes primarily by increasing transmitter release, while the increase elicited by NF1 appears to involve presynaptic and postsynaptic mechanisms.

Neural and myogenic effects of cyclooxygenase products on canine bronchial smooth muscle

In canine bronchi bathed in 10(-6) M indomethacin (IDM), prostaglandin (PG) E2 inhibited electrical field stimulation (EFS)- and acetylcholine (ACh)-mediated contractions and excitatory junction potentials (EJP) in a concentration-dependent manner without altering the resting membrane potential. EFS-induced EJPs were abolished at 10(-7) M PGE2, which shifted responses to ACh 10-fold rightward. Thus PGE2 predominantly inhibited the release of ACh and secondarily decreased smooth muscle response to ACh. U-46619, an analogue of thromboxane A2 (TxA2), initiated tetrodotoxin- and atropine-insensitive contractions in a concentration-dependent manner. U-46619 (10(-9) M) did not alter significantly EFS- or ACh-stimulated contractions and potentiated EFS amplitude of EJPs without depolarizing muscle cells. Either prejunctional activation of ACh release by TxA2 or postjunctional potentiation of the response to ACh can explain these findings. U-46619 (> or = 10(-8) M) depolarized the membrane potential, initiating oscillations accompanied by a large contraction. Addition of 10(-8) M nitrendipine, but not tetraethylammonium (25 mM), blocked the oscillations selectively. Other prostanoids (PGD2, PGI2, and PGF2 alpha) had no significant effects on canine bronchi. In the absence of IDM, PGE2 accumulated, EFS contractions decreased with time, and EJPs disappeared. We conclude that in canine bronchi PGE2 predominantly inhibits ACh release and endogenous PGE2 acts similarly, whereas TxA2 excites, probably at postjunctional sites.

Biochemical machinery involved in the release of ATP from sympathetic nerve terminals.

The biochemical mechanisms underlying the process of facilitation in sympathetic nerve terminals of the guinea-pig vas deferens have been investigated by intracellular and focal extracellular recording techniques. Activation of protein kinase C by the phorbol ester, phorbol 12,13-dibutyrate (PDBu) greatly increased the amplitude of excitatory junction potentials (e.j.ps) from the first stimulus in a train and altered the pattern of facilitation. The configuration of the extracellularly recorded nerve terminal impulse and the sensitivity of the postjunctional membrane to released ATP were unaffected. The specific protein kinase C inhibitor, Ro-31,8220, abolished facilitation and antagonized the effects of PDBu. These results suggest that protein kinase C plays a fundamental role in ATP release from sympathetic nerves and in particular in the mechanisms underlying facilitation.

Neural and myogenic effects of leukotrienes C4, D4, and E4 on canine bronchial smooth muscle

The leukotrienes (LTs), referred to as the slow-reacting substance of anaphylaxis (SRS-A), are reported to have little or no activity in the canine airway. The objective of this study was to determine whether LTC4, LTD4, and LTE4 (10(-10)-10(-7) M) play a role in neuromuscular control of third- to fifth-order canine bronchi. In the presence of 1 microM indomethacin (Indo), canine bronchial smooth muscle contracted and was depolarized in a concentration-dependent manner by LTC4 or LTD4 but not by LTE4. LTC4 and LTD4 concentration-response curves were not significantly affected when conducted in the presence of any of the following: 10(-7) M propranolol (beta-adrenoceptor antagonist), 10(-6) M chlorpheniramine (H1-receptor antagonist), 10(-6) M ketanserin (nonselective 5-hydroxytryptamine receptor antagonist), 10(-7) M atropine (muscarinic receptor antagonist), and 10(-6) M tetrodotoxin (sodium channel blocker). LTC4 and LTD4 also potentiated electrical field-stimulated (EFS) excitatory junction potentials (EJPs), suggesting a possible prejunctional enhancement of acetylcholine release. In the absence of Indo, no postjunctional responses to LTC4 and LTD4 occurred. Endogenous prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha (a stable metabolite of PGI2) levels from canine bronchi were significantly reduced by Indo. In the presence of Indo, addition of > or = 10(-8) M of PGE2 suppressed contractions to LTC4 and LTD4. These data suggest that the decrease in PGE2 and PGI2 production by Indo is sufficient to unmask the excitatory postjunctional actions of LTC4 and LTD4 on bronchial smooth muscle. Serine borate (45 mM; an inhibitor of gamma-glutamyl transpeptidase, which prevents the conversion of LTC4 to LTD4) increased selectively the contractile activity of LTC4. L-Cysteine (3 mM; an inhibitor of an aminopeptidase, which prevents the conversion of LTD4 to LTE4) enhanced the contractile responses to LTD4. Serine borate increased the amplitude and duration of EFS contractions and potentiated the amplitude of EFS EJPs; the last effects were prevented by nordihydroguaiaretic acid. These and other studies suggest that LTs are synthesized by canine bronchi and have receptors on canine bronchial smooth muscle but that contractions to LTC4 and LTD4 in the canine airway are usually not observed because of the presence of inhibitory prostanoids (PGE2 and PGI2). We suggest that decreases in PGE2 and PGI2 in models of airway disease in combination with increases in LTC4, LTD4, and thromboxane A2 may contribute to airway hyperresponsiveness in vitro.

Effects of epithelial cell supernatant on membrane potential and contraction of dog airway smooth muscles.

We investigated the effects of cultured epithelial cells and supernatants on resting membrane potential and excitatory neuroeffector transmission in smooth muscle cells of dog trachea and bronchioles. The mean resting membrane potential of the mucosa-free tracheal smooth muscle cells was -59.5 +/- 1.5 mV (+/- SD). Application of cultured epithelial cells (> 2.5 x 10(5) cells/ml) hyperpolarized the membrane, resulting in a potential of -64.5 +/- 1.7 mV. The supernatant of the cultured epithelial cells also increased the resting membrane potential of the mucosa-free tracheal smooth muscle cells by 4 to 9 mV. These hyperpolarizing actions were not modified by indomethacin (10(-5) M), l-NG-nitroarginine (10(-5) M), or oxyhemoglobin (10(-5) M), but were inhibited by glibenclamide (10(-6) M). The supernatants of the cultured epithelial cells completely or partially suppressed the contractile response of epithelium-denuded bronchioles to electrical field stimulations and suppressed the amplitude of excitatory junction potentials of the trachealis evoked by electrical field stimulations. Indomethacin prevented the inhibitory effect of supernatants on the amplitude of twitch contractions and excitatory junction potentials and markedly suppressed supernatant-associated inhibition of the excitatory neuroeffector transmission. Furthermore, indomethacin with AA861, a lipoxygenase inhibitor, completely suppressed this effect. Our findings suggest that cultured airway epithelial cells spontaneously release at least two factors. One factor selectively modulates the resting membrane potential, and the other inhibits the excitatory neuroeffector transmission.

In vivo study of the role of muscarinic receptors in the parasympathetic control of rabbit colonic motility

The aim of the present study was to elucidate the role of the non-M 1 muscarinic receptors, in the extrinsic and intrinsic nerve control of in vivo colonic motility. Experiments were performed on the proximal colon of anaesthetized rabbits. In this species, the parasympathetic innervation of the proximal colon originates from the vagus nerves. The action of methoctramine and 4-diphenylacetoxy- N-methylpiperidine methobromide (4-DAMP) was studied on excitatory junction potentials (EJPs), and on inhibitory junction potentials (IJPs) elicited in smooth muscle cells by stimulating parasympathetic efferents. The effects of the same drugs on spontaneous spiking activity were also investigated. The EJPs either decreased or disappeared after intra-arterial (i.a.) administration of 4-DAMP (45 pg to 450 ng). In the presence of 4-DAMP, further intravenous (i.v.) administration of pirenzepine (0.1 mg · kg −1) had facilitatory effects on the inhibitory pathway, i.e., after abolition of the EJPs, vagal stimulation elicited IJPs. With the highest dose of 4-DAMP, vagal stimulation immediately elicited IJPs the amplitude of which still increased after pirenzepine. In the presence of 4-DAMP, the spontaneous spike discharge was not noticeably altered. Methoctramine (0.37 to 75 μg, i.a. or 50 μg to 0.2 mg · kg −1, i.v.) increased the amplitude of the EJPs, whereas it decreased that of the IJPs. In addition, at the same doses, it either initiated or increased spike discharges that were not altered by pirenzepine up to 0.2 mg · kg −1, i.v. The so-called rebound excitation occurring after IJPs was not affected by methoctramine. No change in the EJP or IJP amplitude was observed with gallamine at sufficiently high doses to paralyse striated muscles (up to 3 mg · kg −1 · h −1). It is concluded that the parasympathetic excitatory pathway to smooth muscle is blocked by 4-DAMP, whereas it is facilitated by methoctramine. 4-DAMP has no effect on the inhibitory pathway which is strongly depressed by methoctramine; however, the fact that these two drugs have opposite effects indicates that 4-DAMP and methoctramine may act on different muscarinic receptor subtypes. In addition, the facilitatory effects of pirenzepine on IJPs observed in animals pre-treated with 4-DAMP, indicates that the latter drug may act on non-M 1 and non-M 2 (presumably M 3) muscarinic receptors. Methoctramine acts on non-M 1 and non-M 3 (presumably M 2) receptors. The spike discharge induced by methoctramine is presumably due to an increased release of acetylcholine, and possibly also of a non-cholinergic transmitter which has excitatory effects on smooth muscle, the identification of which requires further investigations. The inhibitory effects of methoctramine on IJPs indicate that non-adrenergic non-cholinergic (NANC) neurones which are inhibitory of smooth muscle are controlled by inhibitory intramural neurones, the activity of which is expressed through a muscarinic mechanism involving non-M 1 and non-M 3, presumably M 2 receptors.

Facilitation and depression at different branches of the same motor axon: evidence for presynaptic differences in release

This study provides evidence that a neuron can exhibit differences in activity-dependent transmitter release at two synaptic sites due to variations in the properties of its presynaptic terminals. Two muscles in the stomatogastric system of the lobster Homarus americanus are innervated by a single motor neuron but respond differently to that motor neuron's input, resulting in two different movements evoked by one motor neuron. During continued motor neuron stimulation, the gm8 muscle contracts slowly and maintains contraction, while the gm9 muscle contracts rapidly and then relaxes. These different muscle responses can be accounted for, in large part, by the properties of the respective neuromuscular synapses: the excitatory junctional potentials recorded in gm8 are initially small but summate and facilitate with repeated stimulation, while those in gm9 are initially large but depress with repeated stimulation. Presynaptic differences in neurotransmitter release contribute strongly to the divergent responses; reduction of the excitatory junction potential amplitude by partial postsynaptic receptor blockade or by desensitization does not change the amount of depression at gm9. However, reduction of neurotransmitter release with low-Ca2+, high-Mg2+ saline removes gm9 synaptic depression and reveals that both neuromuscular junctions exhibit frequency-dependent homosynaptic facilitation. Postsynaptic differences in muscle input resistance and muscle composition may enhance the effects of the divergent release properties, but are not responsible for the activity-dependent changes. Ultrastructural features of the nerve terminals on the two muscles are consistent with the differential output of the terminals; the synapses on gm9 are larger and have more presynaptic dense bars than their counterparts on gm8. These data suggest that the basis for the differences in transmitter release between the two muscles may be a higher density of release sites in the gm9 synapses that leads to a higher output of neurotransmitter, rapid depletion of transmitter stores, and synaptic depression.

Calcium released by photolysis of DM-nitrophen triggers transmitter release at the crayfish neuromuscular junction.

1. Spontaneous and evoked transmitter release at the crayfish neuromuscular junction were potentiated in response to photolytic release of calcium from the 'caged' calcium compound DM-nitrophen, which had previously been injected into presynaptic terminals. 2. The amount of calcium released from DM-nitrophen photolysis depends on the concentration of DM-nitrophen, its photoproducts, Ca2+, Mg2+, H+, ATP and the cell's native buffer. Since none of these are known in the crayfish terminal, the study was conducted in a qualitative fashion. 3. Photolytic release of calcium from DM-nitrophen increased excitatory junctional potentials (EJPs) by a range of 2-31 times over control values and the miniature excitatory junctional potential (MEJP) frequency increased from resting values of 1-10 quanta/s to 3000-11,000 quanta/s. 4. Extracellular calcium was not required for the light-evoked asynchronous release of transmitter. Calcium-bound DM-nitrophen previously pressure injected into crayfish presynaptic terminals increased the MEJP frequency from resting values of 1-8 quanta/s to 800-10,000 quanta/s during photolysis in a calcium-free cobalt Ringer solution. 5. Iontophoresis of calcium-free DM-nitrophen into presynaptic terminals released transmitter upon photolysis, but only in a calcium-containing Ringer solution. This suggests that DM-nitrophen is capable of binding calcium once injected into terminals, but this is dependent on the presence of external calcium. 6. Photolysis of DM-nitrophen at lower light intensities produced a slower rate of transmitter release. 7. Brief light exposures, i.e. those which photolysed 5-20% of the DM-nitrophen, resulted in a rapid decay of postsynaptic responses on extinguishing the light, due to rebinding of photolytically released calcium to unphotolysed DM-nitrophen. Longer light exposures which completely photolysed DM-nitrophen, leaving only the low affinity photoproducts, produced a slow decay of transmitter release after the light pulse, presumably due to the active extrusion of calcium from the presynaptic terminals. 8. During photolysis of DM-nitrophen, the time courses of changes in EJP amplitude and MEJP frequency were different, indicating that the two measures of transmitter release were not linearly related. 9. MEJP frequency and EJP amplitudes during DM-nitrophen photolysis were fitted to a 'non-linear summation model' in which photolytically released calcium sums with calcium entering during an action potential to evoke transmitter release with a calcium co-operativity of five.

Responses to sympathetic nerve stimulation of the sinus venosus of the toad.

1. The changes in membrane potential produced by sympathetic nerve stimulation were recorded from sinus venosus preparations of the toad, Bufo marinus, in which beating had been prevented by the dihydropyridine calcium antagonist, nifedipine. 2. Supramaximal sympathetic stimuli initiated long-lasting excitatory junction potentials which started with the same latencies, some 1 to 2 s, as did sympathetic tachycardias recorded from beating preparations. 3. Brief trains of stimuli increased the amplitude of excitatory junction potentials and shortened their latency of onset. Similarly when excitatory junction potentials were facilitated their latency of onset was shortened. 4. The time courses of excitatory junction potentials were prolonged by cooling the preparation but unchanged when the neuronal uptake of catecholamines was inhibited. 5. In arrested preparations, beta-adrenoceptor activation causes a hyperpolarization, as did the inhibition of phosphodiesterases or the activation of adenylate cyclase. This contrasts with the depolarization produced by sympathetic nerve stimulation which could be mimicked by the rapid application of either adrenaline or noradrenaline but not by beta-adrenoceptor activation, phosphodiesterase inhibition or by adenylate cyclase activation. 6. The results are discussed in relation to the idea that neuronally released adrenaline activates a set of adrenoceptors which are linked to a set of channels by a pathway that does not involve cyclic AMP.

Effects of PAF on excitatory neuro-effector transmission in dog airways.

1. Effects of PAF on excitatory neuro-effector transmission in smooth muscle cells of mucosa-free trachea and epithelium-intact bronchiole of the dog were investigated, by isometric tension recording, microelectrode and double sucrose-gap methods. 2. PAF (10(-11)-10(-7) M) dose-dependently enhanced the amplitude of contraction evoked by repetitive field stimulations (10 stimuli at 20 Hz) in both tracheal and bronchiolar tissues. At higher concentrations PAF (> 10(-8) M) increased the amplitude of contraction to a greater extent in the bronchiole than in the trachea. 3. In both muscle tissues, in parallel to the amplitude of contraction, PAF markedly enhanced the amplitude of excitatory junction potentials (e.j.ps) evoked by a single field stimulation in a dose-dependent manner, with no change in the resting membrane potential or input membrane resistance of the smooth muscle cells. PAF (5 x 10(-7) M) enhanced the amplitude of e.j.p. to a greater extent in the bronchiole than in the trachealis. In contrast, lyso-PAF (10(-10)-10(-7) M) showed no effect on e.j.p. amplitude in bronchiolar tissues. At a high concentration (10(-7) M) lyso-PAF slightly enhanced the e.j.p. amplitude in tracheal tissue, however the lyso-PAF induced stimulation of e.j.p. amplitude in the trachea was small compared to that of PAF. 4. PAF (10(-7) M) had no effect on the membrane depolarization induced by acetylcholine (ACh, 10(-9)-10(-5) M) and carbachol (10(-9)-10(-5) M) in tracheal smooth muscle cells. 5. The PAF-antagonists CV3988 (5 x i0-7 M) or WEB2086 (5 x 10-7 M) significantly enhanced the e.j.p. amplitude themselves, PAF (5 x 10-8 M) further enhanced the ej.p. amplitude in the presence of WEB2086 (5 x l0-7 M) but not CV3988 (5 x 10-7 M). In contrast, the new PAF-antagonist, E 6123(5 x l0-8 M), did not affect the ej.p. amplitude itself, and completely inhibited the increase in ej.p. amplitude caused by 5 x 10-8 M PAF. On the other hand, in the presence of the Hi-antagonist,mepyramine, PAF (5 X 10-8 M) further enhanced the ej.p. amplitude.6. The leukotriene synthesis inhibitor AA-861 (10-6 M) or leukotriene antagonist ONO1078 (10-7 M)inhibited the increase in ej.p. amplitude caused by 5 X 10-8 M PAF, respectively.7. In the presence of AA-861 (10-6 M), leukotriene B4 (LTB4, 10-' M) or LTD4 (10-8 M) slightly, and LTC4 (10- M) markedly enhanced the ej.p. amplitude. In contrast, LTE4 (10-8 M) significantly suppressed the e.j.p. amplitude.8. PAF (5 x 10-8 M) attenuated the depression phenomena of ej.ps observed during double stimulus experiments at different time intervals (5-10 s), but had no effect on the summation of ej.ps during repetitive field stimulation at a high frequency (20 Hz) in the trachealis.9. These results indicate that PAF potentiates excitatory neuro-effector transmission mainly through stimulating the release of lipoxygenase products, mainly LTC4 in the dog airway smooth muscle tissues.

Role of M1 muscarinic receptors in the parasympathetic control of colonic motility in cats and rabbits.

1. The effects of pirenzepine (a selective blocking agent of M1 muscarinic receptors) were studied on excitatory junction potentials (EJPs) and inhibitory junction potentials (IJPs) elicited on colonic smooth muscle by stimulation of efferent parasympathetic nerve fibres in anaesthetized cats and rabbits. 2. Pirenzepine (25 micrograms kg-1 to 0.2 mg kg-1, i.v.) decreased the amplitude of EJPs or abolished them. In pirenzepine, parasympathetic stimulation elicited IJPs in most cases. 3. In both species, pirenzepine initiated spontaneous IJPs, indicating an increase in the activity of the non-adrenergic, non-cholinergic (NANC) inhibitory neurones. 4. The results suggest that M1 muscarinic receptors are involved in synaptic transmission within intramural plexuses, at interneuronal synapses in the parasympathetic excitatory pathway to colonic smooth muscle, but are not involved in the pathway to the NANC inhibitory neurones. The facilitation of IJPs by pirenzepine suggests that, under normal physiological conditions, NANC neurones are tonically inhibited by an intramural nervous circuit involving M1 muscarinic receptors.

Multiple motor pathways to single smooth muscle cells in the ferret trachea.

1. We investigated the distribution and characteristics of motor pathways to individual smooth muscle cells activated by electrical stimulation of either, single nerves which enter the tracheal plexus (inlet nerves), or a longitudinal nerve trunk (LNT) located near the entrance of an inlet nerve into the plexus. Excitatory junction potentials (EJPs) were recorded using intracellular microelectrodes as an index of smooth muscle cell activation. In all experiments EJPs were completely blocked by tetrodotoxin and by atropine. 2. In smooth muscle fields located in the caudal direction from the point of inlet or LNT nerve stimulation, neural input decreased as a function of distance. There was evidence of a demarcated area innervated by neurons entering the plexus in one inlet nerve. In smooth muscle fields located in the rostral or transverse direction from the site of nerve stimulation, no such demarcated area could be identified. 3. Of the smooth muscle cells located within the innervated fields studied, 83-95% were activated following stimulation of a single inlet nerve or LNT. Evoked EJPs were similar in different innervated cells or units of electrically coupled cells located within the same 1 mm2 'field'. 4. There was overlapping cholinergic motor input to single smooth muscle cells originating from neurons present in different inlet nerves or different neurons present in the same inlet nerve or region of the LNT. Multiple small step increases in the voltage used to stimulate a LNT resulted in three or four step increases in EJP amplitudes. This gives a minimal value for the number of motor pathways that can be activated by neurons in a region of LNT leading to a single smooth muscle cell. 5. Motor pathways to smooth muscle cells located in caudal and rostral fields ran initially in the LNT and exited in proximity to the smooth muscle cell studied. 6. Motor pathways used in transmitting signals to smooth muscle cells to different areas of trachealis muscle varied in their sensitivity to hexamethonium or curare. EJPs evoked in fields located in the caudal direction from the stimulating electrode were abolished by these drugs. Muscle cells located in different rostral fields showed EJPs that were either sensitive or resistant to these drugs. 7. The rostral hexamethonium-resistant pathway ran initially in the LNT but it exited from the LNT several millimetres before reaching the level of the smooth muscle field innervated. This pathway followed stimulation frequencies up to 25 Hz. The final neuron in this pathway released acetylcholine and evoked EJPs were entirely inhibited by atropine.(ABSTRACT TRUNCATED AT 400 WORDS)

K(+)-channel blockers do not decrease acetylcholine depolarizations in canine trachealis.

Using the double sucrose gap, we have examined the role of K+ channels in the cholinergic depolarizations in response to field stimulation and acetylcholine (Ach) in canine trachealis. Acetylcholine-like depolarization per se decreased electrotonic potentials from hyperpolarizing currents. The net effect of acetylcholine (10(-6) M) depolarization on membrane conductance was a small increase after the depolarization was compensated by current clamp. Reversal potentials for acetylcholine depolarization and for the excitatory junction potential (EJP) were determined by extrapolation to be 20-30 mV positive to the resting potential, previously shown to be approximately -55 mV. They were shifted positively by tetraethylammonium ion (TEA) at 20 mM or Ba2+ at 1 mM. TEA or Ba2+ initially depolarized the membrane and increased membrane resistance. Repolarization of the membrane restored any reductions in EJP amplitudes associated with depolarization. After 15 min, the membrane potential partially repolarized, and acetylcholine-induced depolarization and contractions were then increased by TEA. 4-Aminopyridine depolarized the membrane but decreased membrane resistance. Apamin (10(-6) M), charybdotoxin (10(-7) M), and glybenclamide (10(-5) M) each failed to significantly depolarize membranes, increase membrane resistance, or reduce EJP amplitudes or depolarization to 10(-6) M Ach. Glybenclamide reduced depolarizations to added acetylcholine slightly. TEA occasionally reduced the EJP markedly, but this was shown to be most likely a prejunctional effect mediated by norepinephrine release. TEA alone among K(+)-channel blockers slowed the onset and the time courses of the EJP as well as the acetylcholine-induced depolarization. K(+)-channel closure cannot be a complete explanation of acetylcholine-induced membrane effects on this tissue. Acetylcholine must have increased the conductance of an ion with a reversal potential positive to the resting potential in addition to any effect to close K+ channels.

Effects of vasoactive intestinal polypeptide antagonists on cholinergic neurotransmission in dog and cat trachea.

1. The effects of vasoactive intestinal polypeptide (VIP) antagonists [AC-Tyr1, D-Phe2]-GRF(1-29)-NH2 and [4-Cl-D-Phe6, Leu17]-VIP on excitatory neuroeffector transmission in the dog and cat trachea were investigated by use of microelectrode, double sucrose-gap and tension recording methods. 2. In the dog trachea, repetitive stimuli at high frequency (20 Hz) markedly enhanced the amplitude of contraction, the amplitude of contractions evoked by 50 stimuli at 20 Hz relative to that evoked by 5 stimuli being 14.2 +/- 3.8 times (n = 7, +/- s.d.). In the cat, the summation was much less marked, the amplitude of contractions evoked by 50 stimuli relative to that evoked by 5 stimuli being only 2.1 +/- 0.6 times (n = 5, +/- s.d.). Neither VIP antagonist had any effect on the relationship between the number of stimuli at 20 Hz and the relative amplitude of contraction in the dog trachea, but did enhance the amplitude of contractions to 1.1-1.5 times control in the cat trachea. 3. VIP antagonists dose-dependently enhanced the amplitude of excitatory junction potentials (e.j.ps) evoked by a single stimulus in the cat trachea, without changing the resting membrane potential or input membrane resistance of the smooth muscle cells. However, neither antagonist had any effect on the amplitude of the e.j.p. in the dog trachea. 4. Neither VIP antagonist had any effect on the post-junctional response of smooth muscle cells to exogenously applied acetylcholine (ACh; 10(-9)-10(-5) M) in the dog or cat trachea.5. In the cat trachea, VIP (10-11 M) suppressed the ej.p. amplitude to 0.74 +/- 0.09 times the control value (n = 6). However, after pretreatment of the tissue with the VIP antagonists [Ac-Tyr', D-Phe2]-GRF(1-29)- H2 (10-8M) and [4-Cl-D-Phe6, Leu17]-VIP (10-8M), VIP (10-11 M) did not suppress the ej.p. amplitude, indicating that VIP antagonists block the presynaptic inhibitory action of exogenous VIP.6. In parallel with the enhancement of contraction, ej.ps showed marked summation when repetitive field stimulations were applied at high frequency (20 Hz) in the dog trachea. The relationship between the relative amplitude of the ej.p. and number of stimuli at 20 Hz was linear and the slope was 2.2 +/- 0.3 mV/stimulation. VIP antagonists did not affect this relationship. However, in the cat trachea, summation of ej.ps was not at all marked and a linear relationship was not observed with the double sucrose-gap method. Incubation of the cat tracheal tissue with either of the VIP antagonists (10-8 or 10-7M) markedly enhanced the summation of ej.ps evoked by repetitive field stimulation at 20 Hz, and after the treatment a linear relationship between the number of stimuli and the amplitude of ej.ps was observed, the slopes being 0.6 +/- 0.1 (n = 8) and 0.55 +/- 0.1 mV/stimulation (n = 5), respectively.7. These results indicate that both VIP antagonists, [Ac-Tyr', D-Phe2]-GRF(1-29)-NH2 and [4-Cl-DPhe6, Leu17]-VIP, have a prejunctional action accelerating the excitatory neuroeffector transmission, presumably by enhancing transmitter release from the vagus nerves in the cat, but not in the dog trachea.

Effects of acetaldehyde on electrical activity during neuroeffector transmission in guinea-pig vas deferens

The effects of acetaldehyde on electrical activity during sympathetic neuroeffector transmission were studied in the guinea-pig vas deferens. Application of 1 mM acetaldehyde produced a slow depolarization of the smooth muscle membrane. The amplitudes of facilitated excitatory junction potentials (EJPs) evoked by nerve stimulation were slightly decreased. A higher concentration of acetaldehyde (5 mM) initially hyperpolarized and later depolarized the membrane. The decrease in EJP amplitudes was more pronounced during hyperpolarization. Acetaldehyde (5 mM) increased the frequency of the spontaneous EJPs and reduced their amplitudes, whereas action potentials in postganglionic nerves were unaffected. Acetaldehyde (1–5 mM) decreased the amplitudes of EJPs in vasa pretreated with reserpine but did not alter the resting membrane potentials. The decrease in the EJP amplitudes together with the hyperpolarization of the membrane could be responsible for the early inhibitory effect of acetaldehyde on neuroeffector transmission. The slow depolarization, which is presumably mediated by endogenous noradrenaline, may cause the late facilitatory effect.

Peptidergic and serotonergic facilitation of a neuromuscular synapse in Aplysia

(1) A buccal muscle motor neuron which synthesizes the neuromodulatory small cardioactive peptides (SCPs) was identified in the buccal ganglion of Aplysia by using a combination of electrophysiological and single cell biochemical experiments. This neuron was designated B38. (2) Exogenous SCP B enhanced B38-induced contractions when perfused over the target muscle, the rostral portion of the buccal I3 muscle. SCP B potentiation of muscle contraction was associated with an increase in the excitatory junction potential (EJP) amplitude recorded from the muscle fibers, increased muscle cyclic AMP (cAMP) content, hyperpolarization of the muscle fibers, and an increase in the muscle fiber membrane conductance. Exogenous SCP B also depolarized the cell body of B38 and increased electrical coupling between the symmetrically paired B38 neurons. (3) These results suggest that the SCPs may be co-released from B38 along with an unidentified conventional neurotransmitter to homosynaptically facilitate B38 synaptic transmission by modulating presynaptic and postsynaptic components. (4) Stimulation of the identified serotonergic metacerebral neuron or perfusion of exogenous serotonin (5-HT) over the I3 muscle also potentiated B38-induced muscle contractions and EJP amplitude. Thus the B38 neuromuscular synapse represents a peripheral site of serotonergic heterosynaptic facilitation in Aplysia. (5) Presynaptic and postsynaptic serotonergic effects were qualitatively similar to those of SCP B. Serotonergic effects on muscle fiber hyperpolarization and increase in muscle fiber membrane conductance were similar in magnitude to those of SCP B but 5-HT induced a much larger increase in the EJP amplitude which was additive with that of SCP B. (6) The effect of 5-HT on the EJP amplitude was associated with inhibition of a slowly decaying component of synaptic facilitation. Concentrations of SCP B that increased the EJP were much less effective at inhibiting the slow component of facilitation. These observations indicate that 5-HT also exerted a presynaptic effect on B38 transmitter release. (7) Both 5-HT and SCP B increased muscle cAMP levels and application of forskolin mimicked many of their effects, suggesting that at least some of the postsynaptic effects were mediated by increased cAMP levels in the I3 muscle.

Effects of azelastine on vagal neuroeffector transmission in canine and human airway smooth muscle

Azelastine is a newly developed antiallergic drug that is reported to antagonize histamine and leukotrienes in addition to its inhibitory action on release of chemical mediators. In the present study, the effects of azelastine on neuroeffector transmission in the airway smooth muscles with double sucrose gap and isometric tension-recording methods were evaluated. Azelastine (10(-8) to 10(-6) mol/L) markedly decreased the contractile response of human bronchial and dog tracheal muscle strips to electrical field stimulation (10 pulses at 20 V, 20 Hz, 800 microseconds) in a dose-dependent manner. In parallel with actions on twitch contractions, azelastine suppressed the amplitude of excitatory junction potentials of dog trachea without changing the resting membrane potential and input resistance of smooth muscle cells. However, azelastine did not alter acetylcholine sensitivity of the smooth muscle cells. These results indicate that azelastine possesses an inhibitory action on the release of acetylcholine by vagal nerve terminals. This inhibitory effect of azelastine may contribute to the treatment of asthma in addition to its antiallergic actions.

A comparison of the actions of cromakalim and nifedipine on rabbit isolated mesenteric artery

This study shows that cromakalim (0.4–10 μM) can concentration-dependently hyperpolarize the smooth muscle cell membrane and increase 86Rb efflux from rabbit mesenteric small arteries at concentrations which inhibit noradrenaline-induced increases in perfusion pressure in this preparation. Hyperpolarisation of the cell membrane by cromakalim was inhibited by prior exposure of the tissue to glibenclamide (1 μM). Noradrenaline (> 1 μM) depolarized the smooth muscle cell membrane and this effect was reduced in the presence of cromakalim. Experiments involving repetitive stimulation of the perivascular nerves in this tissue showed that cromakalim (2–10 μM) reduced excitatory junction potential amplitude and fall time without affecting the facilitation process. The results of this study suggest that in rabbit small mesenteric arteries the vasodilator action of cromakalim is a consequence of the opening of 86Rb-permeable potassium channels. It is unlikely that any component of the vasorelaxant effects of cromakalim is due to a direct effect on voltage-operated calcium channels or a prejunctional effect on neuroeffector transmission.

Distension-evoked ascending and descending reflexes in the circular muscle of guinea-pig ileum: an intracellular study

Reflex responses evoked by distension of the guinea-pig small intestine were recorded from the circular muscle with intracellular microelectrodes. For this purpose a mechanically stable preparation that allowed the intestinal wall to be distended within 9 mm of the recording site was developed. A segment of intestine was opened along the mesenteric border and pinned mucosa uppermost over a balloon set in the base of an organ bath, so that inflation of the balloon could distend the intestinal wall without simultaneously pushing against the mucosa. Compound excitatory junction potentials (EJPs) and compound inhibitory junction potentials (IJPs) were recorded at sites up to 40 mm oral and anal to the distending stimulus, respectively. The compound EJPs recorded orally had amplitudes of up to 24 mV and declined to baseline during distensions that exceeded 10–15 s. Distensions at intervals of less than 20 s evoked successively smaller oral compound EJPs; after four distensions in 30 s the amplitude of the compound EJP had fallen to less than 10%. The amplitude of the oral compound EJP was reduced by hyoscine (1 μM), but the extent of the reduction depended on the degree of distension; responses to mild stimuli were blocked, whereas those to strong stimuli were only slightly reduced. The amplitude of the hyoscine-resistant component of the compound EJP was markedly reduced by antagonists of substance P receptors in the muscle. In the presence of muscarinic and substance P receptor antagonists, a transient compound IJP could be detected on the oral side of the stimulus. The compound IJPs recorded anal to the distension had amplitudes up to 22 mV but the potential returned to baseline during prolonged distension. In the presence of hyoscine (1 μM) some inhibitory activity continued throughout prolonged stimuli. Compound IJP amplitudes were not significantly reduced by repeated distensions separated by more than 6 s. At anal sites a transient depolarization (off-response) was recorded immediately following the termination of a distension in some preparations. The off-response was unaffected by hyoscine and was more readily observed after the further addition of substance P antagonists. The compound IJPs were almost completely blocked by apamin (0.2 μM). The compound EJPs and IJPs recorded orally were blocked by hexamethonium (100 μM), but the amplitudes of compound IJPs recorded anally were significantly reduced by hexamethonium (100–200 μM) only at recording sites greater than 15 mm from the centre of the balloon. The off-response was reduced by hexamethonium at all sites. All responses to distension were blocked by tetrodotoxin (0.5 μM). Oral compound EJPs and anal compound IJPs of normal amplitude could still be evoked after removal of the mucosa and nearly all circular muscle from the site of distension. Such lesions did not prevent transmission of these reflexes along the intestine. Thus, both sensory neurons and the nervous pathways activated by stretch lie within the myenteric plexus.