CHOLINESTERASE ACTIVITY OF NODAL AND INTERNODAL REGIONS OF MYELINATED NERVE FIBERS OF FROG

The distribution of cholinesterase (Ch-esterase) in isolated myelinated fibers of the frog has been investigated. Quantitative microgasometric measurements have confirmed the previous histochemical observations. Both approaches indicate that in frog nerve fibers acetylcholinesterase (ACh-esterase) is the only or the predominant enzyme when selective inhibitors and different substrates are used: acetylcholine (ACh), butyrylcholine, and acetyl-B-methylcholine (Mecholyl). By means of the microgasometric technique, a significant difference in ACh-esterase activity between axons isolated from ventral (37.2 ± 1.7 µmole x 10^-5 ACh/mm²/hr) and dorsal roots (2.0 ± 0.9 µmole x 10^-5 ACh/mm²/hr) was found. In the region of the node of Ranvier the enzyme activity (50.4 ± 4.4 µmole x 10^-5 ACh/mm²/hr) appears to be considerably higher than in the internodal area (36.6 ± 2.1 µmole x 10^-5 ACh/mm²/hr). The findings are discussed in relation to the theory of saltatory conduction and the ACh system.     

Conformational Requirements of Acetylcholine Analogues and Local Anaesthetics

ATTEMPTS have been made to view the roles of acetylcholine and of cholinergic agonists as triggering permeability changes in excitable membranes through attachment to acetylcholine receptor biopolymers^1,2. Similarly, it has been proposed that local anaesthetics might block nerve conduction through attachment to axonal acetylcholine receptors^3,4. These considerations raise a number of questions. Are certain specific conformations essential for molecules related to acetylcholine either to “trigger” or to “block” depolarization of excitable membranes ? Are the conformations of such molecules identical in the crystal and in solution ? What are the rotational barriers to conformational alteration of such molecules ?     

The rotational diffusion of the acetylcholine receptor in Torpeda marmorata membrane fragments studied with a spin-labelled alpha-toxin: importance of the 43 000 protein(s)

The rotational diffusion of the acetylcholine (ACh) receptor in subsynaptic membrane fragments from Torpedo marmorata electric organ was investigated with a spin-labelled alpha-bungarotoxin. A toxin with two spin labels was first synthesized; the conventional electron spin resonance spectrum (e.s.r.) of this toxin bound to the receptor indicated: (1) a complete immobilization of the probes; and (2) a strong spin-spin interaction that was not, or barely, seen in solution. The modification of the degree of spin-spin interaction is taken as an indication of a toxin conformational change accompanying its binding to the ACh-receptor. To avoid spin-spin interaction a single-labelled toxin was made and used to follow the rotational diffusion of the receptor by saturation transfer e.s.r. (ST-e.s.r.). With native membranes a high immobilization of the ACh-receptor was noticed. Reduction of the membranes by dithiothreitol had little effect on this motion. Only extraction of the 43 000 protein(s) by pH 11 treatment was able to enhance the rotational diffusion of the ACh-receptor protein (rotational correlation time by ST-e.s.r. in the 0.5 - 1 X 10(-4) s range) and to allow its lateral diffusion in the plane of the membrane fragments (observed by electron microscopy after freeze-etching or negative staining).     

Biochemical investigations of ionic channels in excitable membranes

Summary Ion channels of excitable membranes are composed of a gating device and a selectivity filter. Two strategies are discussed in this review for the biochemical isolation and characterization of these two functional subunits of channels: Membrane molecules involved in ion translocation can be identified in vitro by their pharmacological properties, i.e. by binding assays with radioactive drugs known to selectively affect a special channel in vivo. More desirable is an assay of their true biological function, i.e. translocation of ions through a membrane. Ion flux measurements with natural and reconstituted membrane systems in vitro are recently available.This article summarizes our present knowledge of electrically excitable sodium and potassium channels of nerve membranes and of the chemically excitable sodium/potassium channel of cholinergic synapses, the acetylcholine receptor complex (AChR). Because of the availability of a great variety of drugs binding with high affinity to axonal sodium channels its investigation is more advanced than that of the axonal potassium channel. The lack of high affinity labels for the latter can be possibly overcome by photoaffinity labels which label components of the channel in situ. Initial success is reported with a photoafinity label derived from the potassium channel blocker TEA.Most advanced is the biochemical investigation of the acetylcholine receptor (AChR) which has been purified in milligram quantities. It represents a protein complex composed of different polypeptide chains with different functions regulating the sodium/potassium permeability of cholinergic postsynaptic membranes. Experiments are described to elucidate the quaternary structure, the site of binding of cholinergic ligands and neurotoxins and to prove dynamic conformation changes of the protein which may be the cause for permeability changes of the membrane. The gating device and the ion translocation system (selectivity filter, ionophor) appear to be present in the receptor complex though located possible in different subunits. This is evidenced by reconstitution of excitable membranes from purified AChR and exogenous lipids by a novel and reproducible method.An invited review article.     

The effects of short-chain fatty acids on the neuronal membrane functions ofHelix pomatia. II. cholinoreceptive properties

We have examined the effects of short-chain fatty acids on acetylcholine (ACh)-induced transmembrane currents using internally dialyzed neurons of Helix. Decenoic acid, which increased the fluidity of excitable membranes, caused dramatic changes in the voltage sensitivity of ACh currents consisting of an ACh-induced increase in membrane permeability for K+ and Na+ ions and a shift of the Erev of these ACh responses to more positive potentials. Valeric acid, which did not change the membrane fluidity, had no effect on this type of ACh response. Changes of the ENa and ECl had no effect on the size of the decenoic acid-induced shift of the Erev. But the influence of decenoic acid on the voltage sensitivity of ACh-induced currents almost disappeared after the change of the EK by the reduction of the internal K concentration. Decenoic acid had no effect on ACh responses in which K+ ions were not involved in the generation of ACh-induced currents. The results suggest that decenoic acid-induced changes in membrane fluidity modulate cholinoreceptive properties of the neuronal membrane by the inhibition of the K+ carrier involved in the generation of ACh responses.     

Cholinesterase activity per unit surface area of conducting membranes

According to theory, the action of acetylcholine (ACh) and ACh-esterase is essential for the permeability changes of excitable membranes during activity. It is, therefore, pertinent to know the activity of ACh-esterase per unit axonal surface area instead of per gram nerve, as it has been measured in the past. Such information has now been obtained with the newly developed microgasometric technique using a magnetic diver. (1) The cholinesterase (Ch-esterase) activity per mm² surface of sensory axons of the walking leg of lobster is 1.2 x 10^-3 µM/hr. (σ = ± 0.3 x 10^-3; SE = 0.17 x 10^-3); the corresponding value for the motor axons isslightly higher: 1.93 x 10^-3 µM/hr. (σ = ± 0.41 x 10^-3; SE = ± 0.14 x 10^-3). Referred to gram nerve, the Ch-esterase activity of the sensory axons is much higher than that of the motor axons: 741 µM/hr. (σ = ± 73.5; SE = ± 32.6) versus 111.6 µM/hr. (σ = ± 28.3; SE = ± 10). (2) The enzyme activity in the small fibers of the stellar nerve of squid is 3.2 x 10^-4 µM/mm²/hr. (σ = ± 0.96 x 10^-4; SE = ± 0.4 x 10^-4). (3) The Ch-esterase activity per mm² surface of squid giant axon is 9.5 x 10^-5 µM/hr. (σ = ± 1.55 x 10^-5; SE = ± 0.38 x 10^-5). The value was obtained with small pieces of carefully cleaned axons after removal of the axoplasm and exposure to sonic disintegration. Without the latter treatment the figurewas 3.85 x 10^-5 µM/mm²/hr. (σ = ± 3.24 x 10^-5; SE = ± 0.93 x 10^-5). The experiments indicate the existence of permeability barriers in the cell wall surrounding part of the enzyme, since the substrate cannot reach all the enzyme even when small fragments of the cell wall are used without disintegration. (4) On the basis of the data obtained, some tentative approximations are made of the ratio of ACh released to Na ions entering the squid giant axon per cm² per impulse.     

Allethrin interactions with the nicotinic acetylcholine receptor channel

Interactions of the synthetic pyrethroid allethrin with the nicotinic acetylcholine (ACh) receptor/channel were studied in membranes from Torpedo electric organ. Allethrin did not inhibit binding of [3H]ACh to the receptor sites, but inhibited noncompetitively binding of [3H]perhydrohistrionicotoxin ([3H]H12-HTX) to the ionic channel sites in a dose-dependent manner. The inhibition constant (Ki) of [3H]H12-HTX binding in absence of receptor agonist was 30 micro M, while in presence of 100 micro M carbamylcholine it was 4 micro M. This inhibitory effect of allethrin had a negative temperature coefficient. The high affinity binding of allethrin to the channel sites of the nicotinic ACh-receptor may be indicative of a postsynaptic site of action for pyrethroids, in addition to their known action on the sodium channel.     

Calcineurin-mediated dephosphorylation of the human placental membrane receptor for epidermal growth factor urogastrone

The findings of our work were 2-fold: (1) calcineurin (from bovine brain) can catalyze the complete dephosphorylation of the phosphotyrosine and phosphoserine residues in the human placental receptor for epidermal growth factor urogastrone (EGF-URO), and (2) the major calmodulin-binding protein of human placental membranes is a calcineurin-related protein. In terms of its metal ion dependence (Ni2+ greater than Mn2+ greater than Co2+), its calmodulin dependence, and its sensitivity to inhibitors (Zn2+, fluoride, orthovanadate), the phosphotyrosyl protein phosphatase activity of calcineurin, using the EGF-URO receptor as substrate, paralleled the enzyme activity measured with p-nitrophenyl phosphate (PNPP) as a substrate. These characteristics distinguish calcineurin from other classes of protein phosphotyrosyl phosphatases. Calcineurin purified from placental membranes was similar to, if not identical with, bovine brain calcineurin in terms of enzymatic specific activity toward PNPP, subunit electrophoretic mobilities, and immunological cross-reactivity. The enzymatic properties and comparative abundance of calcineurin in the placenta membranes suggest that this enzyme may play an important role in regulating the phosphorylation state of those receptors (e.g., for EGF-URO or insulin) also known to be present in the membranes.     

Evidence against Hydrogen–Calcium Competition Model for Activation of Electrically Excitable Membranes

TO explain the voltage-dependent sodium permeability of excitable membranes, Stephens¹ proposed a model in which sodium-selective channels are normally blocked by calcium ions bound to negatively charged sites located near the outer end of the channels. The calcium ions can be displaced competitively by hydrogen ions, opening the channels to sodium. According to this model, depolarization of an excitable membrane causes an outward flow of hydrogen ions across the membrane. The consequent transient increase in hydrogen ion concentration at the outer surface of the membrane displaces calcium and opens the sodium channels. This model is particularly interesting because it is sufficiently specific to allow direct tests. Stephens shows that it is in general agreement with a variety of experimental data. To test the model further, we have determined the effect of variation in the internal and external concentration of hydrogen ions on sodium currents.     

Excitable membranes--object for evaluating the effect of heavy metal pollution

In acute experiments the interaction of heavy metals (CdCl2 and HgCl2) with neurotransmitters (ACh and 5HT) was studied on the excitable membrane of the identified neurons in the central nervous system of Helix pomatia L. (Gastropoda, Mollusca). It was shown that cadmium and mercury ions exert different influence on both resting and action potentials as well as on the responsiveness of the neural membranes to ACh and 5HT. The selective blocking effect of cadmium and mercury ions can be interpreted on the basis of specificity of postsynaptic receptor structures responsible for the transmitter action and of ion-channels involved in the excitatory processes. The heavy metal effect was not uniform for the different types of neurons, suggesting that pollutants can modify various functions to a different degree. The results show that testing on nerve cell membranes can serve as a useful method and model in investigating the effect of sublethal environmental contamination, as they may cause a profound modulation on the elements of the neural circuitry responsible for the regulation of the animal's behavior.     

Phosphorylation of the GABAa/Benzodiazepine Receptor α Subunit by a Receptor-Associated Protein Kinase

Abstract: Partially purified preparations of GABA_a/benzodiazepine receptor from rat brain were found to contain high levels of a protein kinase activity that phosphorylated a small number of proteins in the receptor preparations, including a 50-kilodalton (kD) phosphoprotein that comigrated on two-dimensional electrophoresis with purified, immunolabeled, and photolabeled receptor α subunit. Further evidence that the comigrating 50-kD phosphoprotein was, in fact, the receptor α subunit was obtained by peptide mapping analysis: the 50-kD phosphoprotein yielded one-dimensional peptide maps identical to those obtained from iodinated, purified α subunit. Phosphoamino acid analysis revealed that the receptor α subunit is phosphorylated on serine residues by the protein kinase activity present in receptor preparations. Preliminary characterization of the receptor-associated protein kinase activity suggested that it may be a second messenger-independent protein kinase. Protein kinase activity was unaltered by cyclic AMP, cyclic GMP, calcium plus calmodulin, calcium plus phosphatidylserine, and various inhibitors of these protein kinases. Examination of the substrate specificity of the receptor-associated protein kinase indicated that the enzyme preferred basic proteins as substrates. Endogenous phosphorylation experiments indicated that the receptor α subunit may also be phosphorylated in crude membranes by a protein kinase activity present in those membranes. As with phosphorylation of the receptor in purified preparations, its phosphorylation in crude membranes also appeared to be unaffected by activators and inhibitors of second messenger-dependent protein kinases. These findings raise the possibility that the phosphorylation of the α subunit of the GABA_a/ benzodiazepine receptor by a receptor-associated protein kinase plays a role in modulating the physiological activity of the receptor in vivo.     

Acetylcholine in plants: Presence,metabolism and mechanism of action

Acetylcholine (ACh) has been detected in representatives of many taxonomic groups throughout the plant kingdom. The site of its synthesis in plants is probably young leaves. In some plant species choline acetyltransferase (ChAT) activity has been found. This enzyme showing properties similar to animal ChAT, probably participates in ACh synthesis from its precursors, choline and acetyl-Coenzyme A. Acetylcholinesterase (AChE) activity has also been found in many plant tissues. This enzyme decomposes ACh and exhibits properties similar to animal AChE. The presence of both ChAT and AChE in plant tissues suggests that ACh undergoes similar metabolism in plants as it does in animals. Exogenous ACh affects phytochrome-controlled plant growth and development. Mimicking red light (R), ACh stimulates adhesion of root tips to a glass surface and influences leaf movement and membrane permeability to ions. It also affects seed germination and plant growth. Moreover, ACh can modify some enzyme activity and the course of some metabolic processes in plants. Acetylcholine in the presence of calcium ions (Ca²⁺), like R stimulates swelling of protoplast isolated from etiolated wheat leaves. It is proposed that the primary mechanism of action of ACh in plant cells is via the regulation of membrane permeability to protons (H⁺), potassium ions (K⁺), sodium ions (Na⁺) and Ca²⁺.     

ACTH and brain membrane phosphorylation: a model for modulation by neuropeptides

It has been hypothesized that changes in the phosphorylation of synaptic membrane constituents (proteins and lipids) may affect transmission in certain types of synapses. In this paper some of the recent evidence that neuropeptides like ACTH may bring about their behavioral activity by influencing brain protein and lipid phosphorylation is reviewed. An ACTH-sensitive, cAMP-independent protein kinase was isolated from rat brain synaptosomal plasma membranes. This enzyme was partially characterized and it was observed that its activity greatly depended on the presence of calcium ions. One of its substrate proteins B-50 (MW 48,000; IEP 4.5) may play a key role in the turnover of a special class of membrane phospholipids i.e. the (poly)phosphoinositides. Evidence was obtained to suggest that the degree of phosphorylation of the B-50 protein determines the conversion of diphosphoinositol to triphosphoinositol. A model which links the protein phosphorylation to lipid phosphorylation and which points to a functional role for peptides in the regulation of the permeability of brain membranes for calcium ions will be discussed. As the structure-activity relationship for the peptide effects on grooming behavior closely resembles that on phosphorylation, it is assumed that this neurochemical event may indeed be of relevance to the biological activity of the peptide. As the ion permeability may be altered by the peptide it can be suggested that this may lead to modulation of transynaptic information processing in the brain.     

Direct actions of organophosphate anticholinesterases on nicotinic and muscarinic acetylcholine receptors

Four nerve agents and one therapeutic organophosphate (OP) anticholinesterase (anti-ChE) bind to acetylcholine (ACh) receptors, inhibit or modulate binding of radioactive ligands to these receptors, and modify events regulated by them. The affinity of nicotinic (n) ACh receptors of Torpedo electric organs and most muscarinic (m) ACh receptors of rat brain and N1E-115 neuroblastoma cultures for the OP compounds was usually two to three orders of magnitude lower than concentrations required to inhibit 50% (IC-50) of ACh-esterase activity. However, a small population of m-ACh receptors had an affinity as high as that of ACh-esterase for the OP compound. This population is identified by its high-affinity [³H]-cis-methyldioxolane ([³H]-CD) binding. Although sarin, soman, and tabun had no effect, (O-ethyl S[2-(diisopropylamino)ethyl)] methyl phosphonothionate (VX) and echothiophate inhibited competitivel the binding of receptors. However, VX was more potent than echothiophate in inhibiting this binding and 50-fold more potent in inhibiting carbamylcholine (carb)-stimulated [³H]-cGMP synthesis in N1E-115 neuroblastoma cells—both acting as m receptor antagonist. All five OPs inhibited [³H]-CD binding, with IC-50s of 3, 10, 40, 100, and 800 nM for VX, soman, sarin, echothiophate, and tabun, respectively. The OP anticholinesterases also bound to allosteric sites on the n-ACh receptor (identified by inhibition of [³H]-phencyclidine binding), but some bound as well to the receptor's recognition site (identified by inhibition of [¹²⁵I]-α-bungarotoxin binding). Soman and echothiophate in micromolar concentrations acted as partial agonists of the n-ACh receptor and induced receptor desensitization. On the other hand, VX acted as an open channel blocker of the activated receptor and also enhanced receptor desensitization. It is suggested that the toxicity of OP anticholinesterases may include their action on n-ACh as well as m-ACh receptors if their concentrations in circulation rise above micromolar levels. At nanomolar concentrations their toxicity is due mainly to their inhibition of ACh-esterase. However, at these low concentrations, many OP anticholinesterases (eg, VX and soman) may affect a small population of m-ACh receptors, which have a high affinity for CD. Such effects on m-ACh receptors may play an important role in the toxicity of certain OP compounds.     

Definition of physical integrity of synaptosomes and myelosomes by enzyme osmometry

Isotonic requirements for synaptosomes were shown to vary with the concentration of sucrose or mannitol in the isolation medium, as well as with their differential permeability to polyols and ions. The technique of enzyme osmometry, which permits quantitation of the osmotic integrity in a heterogeneous population, was used to defined the osmotic requirements for synaptosomes and myelosomes in a variety of ionic and non-electrolyte media. Important differences, observed in the rank order of permeability of synaptosomal and myelosomal membranes to electrolyte media, were consistent with the known channel density/electrical activity of the corresponding plasma membranes.     

Functional analysis of transmembrane domain 2 of the M1 muscarinic acetylcholine receptor

Ala substitution scanning mutagenesis has been used to probe the functional role of amino acids in transmembrane (TM) domain 2 of the M1 muscarinic acetylcholine receptor, and of the highly conserved Asn43 in TM1. The mutation of Asn43, Asn61, and Leu64 caused an enhanced ACh affinity phenotype. Interpreted using a rhodopsin-based homology model, these results suggest the presence of a network of specific contacts between this group of residues and Pro415 and Tyr418 in the highly conserved NPXXY motif in TM7 that exhibit a similar mutagenic phenotype. These contacts may be rearranged or broken when ACh binds. D71A, like N414A, was devoid of signaling activity. We suggest that formation of a direct hydrogen bond between the highly conserved side chains of Asp71 and Asn414 may be a critical feature stabilizing the activated state of the M1 receptor. Mutation of Leu67, Ala70, and Ile74 also reduced the signaling efficacy of the ACh-receptor complex. The side chains of these residues are modeled as an extended surface that may help to orient and insulate the proposed hydrogen bond between Asp71 and Asn414. Mutation of Leu72, Gly75, and Met79 in the outer half of TM2 primarily reduced the expression of functional receptor binding sites. These residues may mediate contacts with TM1 and TM7 that are preserved throughout the receptor activation cycle. Thermal inactivation measurements confirmed that a reduction in structural stability followed the mutation of Met79 as well as Asp71.     

A new approach to insect-pest control—combination of neurotoxins interacting with voltage sensitive sodium channels to increase selectivity and specificity

Voltage-sensitive sodium channels are responsible for the generation of electrical signals in most excitable tissues and serve as specific targets for many neurotoxins. At least seven distinct classes of neurotoxins have been designated on the basis of physiological activity and competitive binding studies. Although the characterization of the neurotoxin receptor sites was predominantly performed using vertebrate excitable preparations, insect neuronal membranes were shown to possess similar receptor sites. We have demonstrated that the two mutually competing antiinsect excitatory and depressant scorpion toxins, previously suggested to occupy the same receptor site, bind to two distinct receptors on insect sodium channels. The latter provides a new approach to their combined use in insect control strategy. Although the sodium channel receptor sites are topologically separated, there are strong allosteric interactions among them. We have shown that the lipid-soluble sodium channel activators, veratridine and brevetoxin, reveal divergent allosteric modulation on scorpion α-toxins binding at homologous receptor sites on mammalian and insect sodium channels. The differences suggest a functionally important structural distinction between these channel subtypes. The differential allosteric modulation may provide a new approach to increase selective activity of pesticides on target organisms by simultaneous application of allosterically interacting drugs, designed on the basis of the selective toxins. Thus, a comparative study of neurotoxin receptor sites on mammalian and invertebrate sodium channels may elucidate the structural features involved in the binding and activity of the various neurotoxins, and may offer new targets and approaches to the development of highly selective pesticides.     

A possible model for receptors in excitable membranes

A model is proposed for receptors in excitable membranes based on the following assumptions. The receptor site and the process it excites in the membrane are located close to each other. The change of the electrostatic potential in the neighbourhood of the receptor site on the adsorption of a molecule (or ion) influences a potential dependent process in the membrane, such as ion permeability, rate of enzymatic reactions, ion binding etc. A comment is also made about the connection between measured physiological activity of a molecule and its ?real” physical activity.     

Plasma membrane-associated protein tyrosine phosphatase activity in hamster spermatozoa

Plasma membranes of caput and cauda epididymal spermatozoa of hamster exhibited protein phosphatase activity. This membrane-associated protein phosphatase was identified as a protein tyrosine phosphatase based on its ability to hydrolyse a substrate specific for PTPase, by inhibition of its activity with a specific inhibitor of PTPase (sodium orthovanadate) and by the inability to inhibit its activity with calyculin, okadaic acid, trifluoperazine, calcium, EGTA, and EDTA, which are specific inhibitors of other protein phosphatases, namely PP-1, PP-2A, PP-2B, and PP-2C respectively. The specific activity of the protein tyrosine phosphatase both in the caput and cauda epididymal sperm plasma membranes was similar, implying that the enzyme may not be solely responsible for the differential phosphorylation of membrane proteins observed during maturation (Uma Devi et al. 1997. Mol Reprod Dev 47:341-350). Thus the significance of the PTPase activity in epididymal maturation still remains to be determined. The membrane-associated PTPase may not be essential for acquisition of motility. However, it appears that the activity is essential for the sustenance of motility since sodium orthovanadate, which specifically inhibits PTPase activity, also inhibits motility of spermatozoa and decreases the overall velocity of the spermatozoa by decreasing the average path velocity, straight line velocity, curvilinear velocity, and amplitude of lateral head displacement of the treated spermatozoa.     

Regulation of atrial contraction in the seawater-adapted eel,Anguilla japonica

The atrium isolated from the seawater-adapted eel beats spontaneously in normal Ringer solution for more than 10 hr. The strength of beating was inhibited by acetylcholine (ACh) and the inhibitory effects were blocked by atropine, a muscarinic ACh-receptor antagonist, indicating existence of muscarinic ACh-receptor on the atrium. The atrial contractility was stimulated by catecholamines and their agonists; the order of potency being isoproterenol > adrenaline (AD) = noradrenaline (NA) > phenylephrine > clonidine. The stimulatory effects of AD was completely blocked by propranolol, a β-adrenoceptor antagonist, but not by phentolamine, an α-adrenoceptor antagonist. These data were consistent with characteristics of β-adrenoceptors. Further characterization of the β-receptor was not attempted. The positive inotropic and chronotropic actions of AD were not completely blocked either by atenolol, a β₁-adrenoceptor antagonist, or by ICI 118551, a β₂-adrenoceptor antagonist. When electrical current with a short duration (0.25 msec) was passed through the atrium, the beating was inhibited initially, then enhanced later. The initial inhibition was inhibited by atropine and the later enhancement was blocked by propranolol. These results indicate that the electrical stimulation releases ACh and catecholamine(s) from the nerve endings. The positive inotropic and chronotropic effects of catecholamines were mimicked by tyramine, a catecholamine releaser from sympathetic nerve endings.