Differences between cellular mechanisms of ATP-and noradrenaline-induced inhibition of visceral smooth muscles under conditions of selective and combined activation of M<Subscript>2</Subscript> or M<Subscript>3</Subscript> cholinoreceptors

Our studies of the role of phospholipase C in inhibitory synaptic action upon visceral smooth muscles demonstrated that, under conditions of carbachol (CCh)-induced pre-activation of cholinoreceptors, ATP-or noradrenaline (NA)-evoked relaxation of these muscles is mediated by the phospholipase C-independent pathway, while the phospholipase C-dependent pathway does not manifest itself as a mechanism that determines the inhibitory effect of the above transmitters. Under conditions of pre-activation of muscarinic cholinoreceptors, ATP-and NA-induced relaxation is continued due to activation of inositol trisphosphate (InsP₃)-sensitive receptors despite the fact that the pathway of inhibition is phospholipase C-independent. This is confirmed by complete depression of the inhibitory effects of ATP and NA against the background of CCh-induced contraction after pre-incubation of the studied preparations together with 100 μM 2-APB, a blocker of InsP₃ receptors. Selective blockings of either M₂ or M₃ cholinoreceptors are accompanied by a complete loss of the ability of the above blocker of InsP₃ receptors (2-APB) to suppress ATP-and NA-induced contraction of smooth muscles in the state of CCh-induced contraction. It can be hypothesized that, under conditions of selective pre-activation of M₂ or M₃ cholinoreceptors, the mechanisms of intracellular signalling mediating the inhibition events are modified. The InsP₃-dependent pathway that determines both adrenergic and purinergic inhibition of smooth muscles is switched off, and the inhibitory action of neurotransmitters is realized under such conditions through the InsP₃-independent pathway. Therefore, in our study we first found differences between cellular mechanisms underlying ATP-and NA-induced inhibition of smooth muscles under conditions of selective activation of either M₂ or M₃ cholinoreceptors and the mechanisms underlying the relaxing action of inhibitory neurotransmitters under conditions of combined synergistic activation of cholinoreceptors of both the above-mentioned subtypes. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 22–31, January–February, 2007.     

Cytophotometric study of the DNA content in the cells of the preoptic area of the hypothalamus in hibernating common frogs

The DNA content in the squashed Feulgen stained cells of the hypothalamic preoptic region in wintering adult and juvenile frogs was measured cytophotometrically. The vast majority of hypothalamic cells studied showed diploid DNA contents. Only 0.9% of tetraploid cells (4c and 2c X 2 cells) was found in this region, both in adult and juvenile frogs. The incidence of tetraploid hypothalamic cells varied among the individuals studied. In some adult frogs as well as in some juveniles no tetraploid cells were detected. The data obtained do not confirm the age related increase in the mean DNA content in frog's hypothalamic cells population.     

The properties of the extraocular muscles of the frog. II. Pharmacological properties of the isolated superior oblique and superior rectus muscles.

The pharmacological properties of the superior oblique and the superior rectus muscles of the frog's eye were investigated in comparison with those of a skeletal muscle (iliofibularis muscle) of the same animal. Acetylcholine causes sustained contractures of the extraocular muscles; this effect is increased by physostigmine and decreased or abolished by d-tubocurarine. Also the applications of succinylcholine, choline or caffeine are able to evoke contractures. There are no striking differences in pharmacological properties between extraocular and skeletal muscles of the frog. The time-course of the contractures and the sensitivity of the muscle preparations to the drugs which evoke contractures are identical in extraocular and iliofibularis muscles. In comparison with skeletal muscles there is no higher sensitivity of the extraocular muscles against curare-like drugs. The existence of adrenergic receptors could not be found neither in extraocular nor in skeletal muscles of the frog. It is concluded that in frogs no pharmacological differences exist between the muscle fibre types which compose the extraocular and the skeletal muscles.     

Non-adrenergic mechanisms of inhibition of the contractile activity of the cat small intestine by histamine, bradykinin, and met-enkephalin

I. a. histamine and bradykinin caused both an activation and an inhibition of jejunal contractile activity. The inhibitory effect was preserved after blockade of muscarinic cholinoreceptors, alpha- and beta-adrenoceptors, and abolished with the nicotinic cholinoceptor blockade. Metenkephalin inhibited the jejunal contractile activity after first activating it. The inhibitory effect of the peptide was preserved after blockade of alpha- and beta-adrenoceptors as well as the nicotinic cholinoceptors. The data obtained suggest that the non-adrenergic inhibitory effect of metenkephalin on intestinal contractions was the result of its depressing action on motor cholinergic neurons, whereas the inhibition of intestinal contractile activity with histamine and bradykinin resulted from their activating action on cholinergic interneurons which activate non-adrenergic inhibitory neurons through nicotinic cholinoceptors.     

Postsynaptic potentiation of miniature endplate currents in rat diaphragm muscles. Effects of acetylcholinesterase inhibition,temperature, and curare

Miniature endplate potentials (MEPC) were recorded from rat diaphragm muscle fiber. A positive correlation was found in controls between half-decay time and amplitude of individual MEPC, an effect enhanced by acetylcholinesterase (AChE) inhibition (correlation coefficients: 0.29 and 0.49 respectively at a temperature of 28°C). Adding curare following AChE inhibition produced a reduction in the amplitude and duration of MEPC without influencing the correlation relationship between the above-mentioned parameters. This relationship declined significantly with a temperature reduction to 18°C in both the control and cases of AChE inhibition. The increase in MEPC half-decay time following AChE inhibition was greater at 28° than at 18°C; Q₁₀ equalled about two for duration of rising time as compared with around three for MEPC half-decay time. Factors determining the time course of MEPC are discussed. The findings obtained are explained by postsynaptic potential (and cooperative binding of agonists to cholinoreceptors lies at the root of this) and by the pattern of ACh diffusion at the synaptic cleft.A. A. Ukhtomskii Institute of Physiology, A. A. Zhdanov State University, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 4, pp. 504–512, July–August, 1987.     

Effects of antipsychotic drugs on action potential production in skeletal muscle. II. Haloperidol: nonspecific and opiate drug receptor mediated effects.

The effects of haloperidol, an antipsychotic butyrophenone, on excitability and action potential production in frog's sartorius muscle fibers were studied. This drug produced a local-anestheticlike effect which developed slowly over 1 to 5 h with lower concentrations (2.7 to 5.3 X 10(-6 M) but was completely reversed by exposing the muscles to a drug-free solution. In studies with intracellular microelectrodes, evidence was obtained showing that haloperidol decreased excitability and depressed action potential production by inhibiting the specific increase in sodium conductance (gNa) which normally follows an adequate stimulus. Evidence also was obtained showing an inhibition of the secondary increase in potassium conductance (gK). Haloperidol is structurally related to meperidine and it was found that the inhibition of gNa produced by haloperidol is partially antagonized by low concentrations of naloxone (2.8 X 10(-8) and 2.8 X 10(-7) M); as was previously shown for meperidine. Thus haloperidol, like meperidine, suppresses action potential production by two mechanisms of action: one, a nonspecific local-anaestheticlike effect; and the other, a specific inhibition of gNa mediated by means of an opiate drug receptor associated with the muscle fiber membrane. Naloxone did not antagonize the effects of chlorpromazine on gNa.     

Pharmacologic properties of cholinoreceptor antibodies

The study of pharmacological properties of cholinoreceptor antibodies is of definite interest due to the possibility of simulation of some myasthenic conditions. In 1986 the authors obtained antibodies by means of immunization of rabbits with antigenic material from skeletal muscles of the extremities of Balb/c mice. It was shown that incubation of the muscle with cholinoreceptor antibodies decreased its contraction under the effect of various concentrations of acetylcholine.     

Cholinoreceptor sensitivity of the mollusk neuron and frog skeletal muscle to acetylcholine and tetramethylammonium analogs

Studies have been made on the sensitivity of cholinoreceptors in identified isolated neuron from the pedal ganglion of the snail Planorbarius corneus and cholinoreceptors of m. rectus abdominis of the frog Rana temporaria to drugs which differ from acetylcholine by the structure either in cationic group, methylene chain, or ester group. Snail cholinoreceptors were found to be less sensitive to changes in the structure of cationic group and more sensitive to the increase in methylene chain from 3 to 4 groups, as compared to frog cholinoreceptors. The sensitivity of both preparations to changes in ester group, as well as to tetramethylammonium was found to be practically the same. Therefore, the sensitivity of neuronal cholinoreceptors in the snail to the effect of acetylcholine and tetramethylammonium analogues does not significantly differ from the sensitivity of cholinoreceptors in the abdominal muscle of the frog.     

The characteristics of the cholinoreceptors on the identified TAN neuron of the giant African snail Achatina fulica]

Acetylcholine, nicotine, a selective agonist of N-cholinoreceptors suberildicholine dibromide, as well as a selective agonist of M-cholinoreceptors 5-methylfurmethide inhibited spike discharges in a dose-dependent manner up to a complete ceasing of the firing in cholinoreceptors situated on the identified neurone TAN of African giant snail Achatina fulica. M-cholinoblocker metamizylum completely prevented the inhibitory effect of methylfurmethide. Central cholinoblocker aetherophen completely prevented the inhibitory effect of suberildicholine dibromide. Metamizylum or aetherophen used alone were only able to decrease the inhibitory effect of acetylcholine, whereas a mixture of these agents suppressed completely the acetylcholine-induced inhibition. The findings suggest that, on the TAN membrane, nicotinic and muscarinic cholinoreceptors co-exist and function in one and the same direction.     

Some observations on cardiac automatism in certain animals

Certain aspects of the acetylcholine hypothesis of cardiac automaticity have been tested in vitro with spontaneously beating cardiac tissue from rabbits, rats, dams, and hagfish. The beat of atria from rabbits and rats may be depressed or excited by acetylcholine, depending upon the state of the tissue. Proguanil and cocaine inhibition of the beat in the rat may be antagonized by acetylcholine so that reversal of the depression occurs. The action of acetylcholine on the hearts of clams was found to be strictly inhibitory. Proguanil and cocaine, in contrast to their action on mammalian atria, exert a stimulatory effect on the heart of the molluscs studied. In fact, cocaine stimulated these hearts when they were inhibited by acetylcholine. Studies on the non-innervated hagfish heart revealed that this tissue is completely insensitive to the action of acetylcholine. Extracts prepared from beating hearts of this species will accelerate hypodynamic hearts of the hagfish as well as of the mussel. An extract of the neurogenic lobster heart was without effect on the hagfish heart. Proguanil was likewise ineffective in concentrations which produced inhibition and excitation in rat and clam hearts respectively. It was concluded that acetylcholine does not play a role in the myogenic automatism of all species, and that another mechanism is responsible is suggested on the basis of results obtained in the hagfish hearts.     

Antigenic properties of T cell antigen-specific receptors isolated from the surface of rabbit and mouse spleen and lymph node cells

The presence ofa allotypic determinants was tested in fractions obtained by gel filtration of antigen-specific receptors isolated by immunoadsorption from lymphoid cells of antigen-stimulateda3-3 rabbits. This technique, as well as the inhibition of the reaction of isolated receptors with anti-T cell receptor antisera (anti R) by anti-a3 antibodies failed to demonstrate the presence of a allotypie determinants. The inhibitory effect of antigen-specific receptors isolated from the lymphoid cells of stimulated A/J mice on the cytotoxic effect of anti-Ia antibodies on mouse spleen cells in the presence of rabbit complement was tested. All preparations inhibited the cytotoxic reaction with the average effectivity of 60%. In order to confirm the presence of Ia determinants on the rabbit and mouse T cell receptor molecules it was shown that the reactions of three anti-R antisera with 12 different receptor preparations were inhibited by anti-la antibodies. SDS-PAGE analyses of¹²⁵I-labelled mouse specific receptors and the precipitate obtained by anti-R antisera showed that T cell receptors were present in fractions with molar mass 100 and 85 kg/mol. The molar mass of the former fraction after reduction and alkylation was 45 kg/mol.     

Do frogs use retinal elevation to measure the distance of a barrier?

Grass frogs, Rana pipiens, will detour around a barrier to reach prey on the other side. However, if the distance between prey and barrier is short, frogs attempt to push through the barrier and reach the prey directly. The relationship between the probability of detouring and the distance between prey and barrier is the same whether the frog's starting position is 4 cm or 8 cm from the barrier. This suggests that frogs measure the absolute separation between the two objects. To discover whether the retinal elevation of the bottom of the barrier contributes to measuring this distance, the relationship between the frequency of detouring and barrier-prey distance was examined in several experiments in which the retinal position of the bottom of the barrier was manipulated. No evidence was obtained that the barrier's retinal elevation helps in gauging distance.On the other hand, retinal elevation influences strongly how far a frog lunges to reach its prey. It is suggested that different cues to distance are applied to the two classes of object because, under natural circumstances, it is difficult to judge where a barrier emerges from the ground. A barrier may be hard to detect below the horizon because of the low contrast between it and the ground, or because vegetation and ground litter mask where the barrier meets the ground. In contrast, the prey's movements make it easily detectable against a stationary background and the prey's short height means that partial occlusion will have little effect on its apparent vertical position in the visual field.     

Mechanical characteristics of isotonic work and high-energy phosphates in frog ventricle after 1-fluoro-2,4-dinitrobenzene]

The blocking of the creatinphosphokinase by 1-fluoro-2,4-dinitrobenzene (FDNB) allows to investigate the relationship between ATP-supply, contractility and relaxability of the frog's myocardium. In isotonically working isolated ventricles of frogs the time of work, systolic and diastolic volume, velocity of contraction and relaxation as well as the levels of CP, ATP, ADP and AMP were measured at different intervals until termination of each experiment. CP shows a small variation, ATP decreases to 60% and ADP + AMP increase for the same amount under FDNB during the development of a slight inhibition of contractility and a continuously growing inhibition and retardation of relaxation until systolic arrest. ATP content and volume of relaxation correlated strictly. The contracture and the diminished contractility are caused by the decrease of ATP, producing a lack of substrate for Ca transport and actin-myosin-ATPase. This models the course of events during an insufficiency like in angina pectoris and in myocardial infarction.     

The atropine-like action, not removed by naloxone, of the opioid dermorphin on the inotropic effects of acetylcholine

Opioid dermorphin induced a negative inotropic effect on the isolated perfused by Straube frog's heart, this effect was blocked by naloxone. On the background of dermorphin negative inotropic effect acetylcholine inhibited the ventricular contractile activity to the same degree as in the control experiments before dermorphin injection. But after the combined infusion of naloxone and dermorphin removed the opioid inotropic effect, the negative inotropic effect of acetylcholine became significantly weaker than in the control. It has been concluded that there are opiate receptors in the frog's ventricular myocardium, their activation leads to the negative inotropic effect. Dermorphin may act like atropine on the inotropic effects of acetylcholine, this action doesn't depend on the opiate receptors activation.     

Substantial species differences in relation to formation and degradation of N-acyl-ethanolamine phospholipids in heart tissue: an enzyme activity study

The formation of N-acyl-ethanolamines (NAEs), including the cannabinoid receptor ligand anandamide, and their precursors N-acyl-ethanolamine phospholipids (NAPEs) are catalyzed by NAPE-hydrolyzing phospholipase D (NAPE-PLD) and N-acyl-transferase, respectively. NAPE and NAE are suggested to have beneficial effects on the heart, but in the literature there are indications of species differences in the activity of these enzymes. We have examined heart microsomes from rats, mice, guinea pigs, rabbits, frogs, cows, dogs, cats, mini pigs and human beings for activities of these two enzymes. N-Acyl-transferase activity was very high in dogs and cats (>13 pmol/min/mg protein) whereas it was very low to barely detectable in the other species (<3 pmol/min/mg protein). NAPE-PLD activity was very high in rats and guinea pigs (>45 pmol/min/mg protein) whereas it was 9 pmol/min/mg protein in frogs and below that in the other species. The ratio of activity between the two enzymes varied from 0.002 to 15 in the investigated species. The activity of the two enzymes in rat hearts as opposed to rat brain did not change during development. These results indicate that there may be substantial species differences in the generation of anandamide and other NAEs as well as NAPEs in heart tissues.     

Mechanisms of serotonin effect on motility of ileocaecal zone in alert rabbits

I.V. administration of serotonin (2 mg kg(-1)) to alert rabbits changed the ileal, caecal, and colon motility including excitatory and inhibitory components. Initial rise of contractile activity was quickly replaced by its diminishing followed by a longer enhancement of the motility, and then by the final, inhibitory, phase. Under blockade of beta1- and beta2-adrenoreceptors with propranolol inhibition of ileal and caecal contractile activity with serotonin was preserved, the effect of circulating catecholamines on beta-adrenoreceptors of smooth muscle cells seems to be excluded as a cause of the serotonin inhibitory effect. In conditions of blockade of pre- and postsynaptic alpha-adrenoreceptors with phentolamine, there was no significant diminishing of the contractile activity in the ileo-caecal zone below the initial level induced by serotonin in control experiments. Intensification of the ileo-caecal zone contractile activity under the effect of serotonin persisted in conditions of blockade of muscarinic cholinoreceptors and was proceeding with participation of non-cholinergic excitatory mechanism.     

Study of Cardiotonic Effect of Venom of the Green Toad Bufo viridis

In experiments on the isolated heart of frogs, cats, and rats, cardiotonic effect of the green toad Bufo viridis Laur. venom was studied. It has been shown that both the venom and the fraction of bufodienolides isolated from it produce an increase of the strength of cardiac contractions and, to a lesser extent, of the heart rate in cold-blooded and warm-blooded animals. A high selectivity of the venom inotropic effect was seen as an increase of the rate characteristics of elevation or reduction of the pressure in the rat heart left ventricle. The venom and bufadienolides increase the frog atrial trabecula contraction without a rise of the slow incoming (calcium) current. A similarity of mechanisms of cardiotonic effects of the venom and of the plant cardiac glycosides is discussed.     

Reaction of phosphofructokinase 2/fructose 2,6-bisphosphatase with monoclonal antibodies. A proof of the bifunctionality of the enzyme

Monoclonal antibodies were derived from mice immunized against homogeneous chicken liver phosphofructokinase 2/fructose 2,6-bisphosphatase. Of 112 clones, 30 were found to secrete antibodies that specifically reacted with the antigen in enzyme-linked immunoabsorbant assay (ELISA) while 17, which were ELISA-negative, produced antibodies that affected the enzymic activity of the antigen. Four clones were subcloned and used for an extensive investigation of the reaction of the corresponding antibodies with the supposedly bifunctional enzyme. A definite proof of the bifunctionality of the enzyme was obtained from the two following observations. First, the two activities were similarly retained by the four antibodies that had been coupled to Sepharose. Second, one of the antibodies inhibited both activities with the same efficiency. Furthermore, the antigen-antibody reaction led to the formation of aggregates with an apparent molecular mass of several megadaltons, showing that the two subunits of the antigen reacted with the same antibody and were therefore identical. The four monoclonal antibodies affected the activity of phosphofructokinase 2. This effect was seen as an up to 17-fold activation as well as an up to 85% inhibition. Only one of the four antibodies (antibody 10) had inhibitory effects on fructose 2,6-bisphosphatase, an effect which was in part explained by a decrease in the rate of formation of the intermediary phosphoenzyme. All the effects described above were obtained on both the chicken liver and the pigeon muscle enzymes but with lower doses of antibody in the case of the former enzyme. Antibody 10 was also shown to react with mouse liver phosphofructokinase 2/fructose 2,6-bisphosphatase, and with phosphofructokinase 2 from chicken brain, heart and testis and from frog skeletal muscle and liver. None of the four antibodies cross-reacted with phosphofructokinase 2 from Saccharomyces cerevisiae or from spinach leaves.     

RGNTF单抗与多抗的特性和抑制作用（简报）

We have immunized Balb/c mice and rabbits with a minute quantity of a 30 kD neuronotrophic factor which was isolated from the extract of newborn rat tectum (Te) by Phast System gel electrophoresis. Splenic cells from the immunized mice were hybridized with NS-1 mouse myeloma cells. Three clones were selected from 576 wells of hybridomas and were capable of secreting monoclonal antibodies specific to the retinal ganglion neuronotrophic factor (RGNTF-MAbs), namely A1, D3 and E8. Subtyping of the three monoclonal antibodies revealed that A1 and D3 are IgG3 and E8 is IgM. They maintained secreting antibodies even after six months of culturing in vitro. In order to determine the specificities of these antibodies, we have used their ascites fluids containing antibodies at a different dilutions to study their effects on the survival of retinal ganglion cells in vitro. The results indicated that at the dilution ranges of 1:250 to 2000, all three monoclonal antibodies exhibited inhibition on the survival of retinal ganglion cells and the inhibition increased with increases in antibody concentrations; especially at a dilution of 1:250, the E8 monoclonal antibody reaching 70% inhibition and A1 and D3 reaching 66% and 62% inhibition, respectively. Polyclonal antibodies from rabbits exhibited similar but weaker results of inhibition. We can conclude that the monoclonal and polyclonal antibodies can specifically inhibit the activity of the 30 kD retinal ganglion neuronotrophic factor.