Muscarinic ACh receptor activation causes transmitter release from isolated frog vestibular hair cells

In the frog, vestibular efferent fibers innervate only type-II vestibular hair cells. Through this direct contact with hair cells, efferent neurons are capable of modifying transmitter release from hair cells onto primary vestibular afferents. The major efferent transmitter, acetylcholine (ACh), is known to produce distinct pharmacological actions involving several ACh receptors. Previous studies have implicated the presence of muscarinic ACh receptors on vestibular hair cells, although, surprisingly, a muscarinic-mediated electrical response has not been demonstrated in solitary vestibular hair cells. This study demonstrates that muscarinic receptors can evoke transmitter release from vestibular hair cells. Detection of this release was obtained through patch-clamp recordings from catfish cone horizontal cells, serving as glutamate detectors after pairing them with isolated frog semicircular canal hair cells in a two-cell preparation. Although horizontal cells alone failed to respond to carbachol, application of 20 microM carbachol to the two-cell preparation resulted in a horizontal cell response that could be mimicked by exogenous application of glutamate. All of the horizontal cells in the two-cell preparation responded to 20 microM CCh. Furthermore, this presumed transmitter release persisted in the presence of d-tubocurarine at concentrations that block all known hair cell nicotinic ACh receptors. The effect on the detector cell, imparted by the carbachol application to the hair cell-horizontal cell preparation, was blocked both by 2-amino-5-phosphonopentanoic acid, a selective N-methyl-D-aspartate antagonist, and the muscarinic antagonist, atropine. Thus vestibular hair cells from the frog semicircular canal can be stimulated to release transmitter by activating their muscarinic receptors.     

Opioid receptors mediate a postsynaptic facilitation and a presynaptic inhibition at the afferent synapse of axolotl vestibular hair cells

This study was designed to determine the effects of opiate drugs on the electrical activity of afferent neurons and on the ionic currents of hair cells from semicircular canals. Experiments were done on larval axolotls (Ambystoma tigrinum). The multiunit spike activity of afferent neurons was recorded in the isolated inner ear under both resting conditions and mechanical stimulation. Ionic currents were recorded using voltage clamp of hair cells isolated from the semicircular canal. In the isolated inner-ear preparation, microperfusion of either non-specific opioid receptor antagonist naloxone (10 nM to 1 mM), mu receptor agonist [D-Ala(2), N-Me-Phe(4),Gly(5)-ol]-enkephalin (1 pM to 10 microM), or kappa receptor antagonist nor-binaltorphimine (10 nM to 100 microM) elicited a dose-dependent long-lasting (>5 min) increase of the electrical discharge of afferent neurons. The mu receptor agonist funaltrexamine (1 nM to 100 microM) and the kappa receptor agonist U-50488 (1 nM to 10 microM) diminished the basal spike discharge of vestibular afferents. The delta receptor agonist D-Pen(2)-D-Pen(5)-enkephalin (1 nM to 10 mM) and the antagonist naltrindole (1 nM to 10 mM) were without a significant effect. The only drug that displayed a significant action on hair-cell ionic currents was trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl) benzeneacetamide methanesulfonate (U-50488) that reduced the Ca(2+) current in a dose-dependent fashion. On its own, mu receptor agonist [D-Ala(2), N-Me-Phe(4),Gly(5)-ol]-enkephalin (0.01 and 10 microM) significantly potentiated the response of afferent neurons to the excitatory amino acid agonist (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (0.1 microM), while synaptic transmission was blocked by the use of high-Mg(2+), low-Ca(2+) solutions. Our data indicate that the activity of vestibular afferent neurons may be regulated in a complex fashion by opioid receptors: mu opioid receptors mediating an excitatory, postsynaptic modulatory input to afferent neurons, and kappa receptors mediating an inhibitory, presynaptic input to hair cells.     

Changes in the biochemical and morphological indices of structural disturbance in the peri-infarct area of the myocardium as affected by opioid peptides in an experiment

The effect of opiate peptides (leu-enkephalin, met-enkephalin and beta-endorphin) as well as of enkephalin synthetic analogs (two tetrapeptides and dalargin hexapeptide) on the activity of blood enzymes CPK and LDG1, the scope of myocardial infarction and ultrastructure of periinfarction cardiomyocytes has been studied on 200 white outbred rats with simulated myocardial infarction. The endogenous opiate peptides and dalargin hexapeptide were found to significantly decrease CPK and LDG1 activity and to diminish the size of the infarction. The effect is related to the drugs influence on the survival of periinfarction cardiomyocytes that can be proved electron-microscopically and using colloid lanthanum test.     

Features of the modulation of afferent reactions by agonists and antagonists of opiate receptors and competitive stimulation of the skin under conditions of changing barometric pressure

Afferent body reactions were studied under conditions of changes of atmospheric pressure in electrostimulation of skin zones and use of various agonists and antagonists of opiate receptors. The results suggest maintenance of a sufficiently high therapeutic efficacy of transcutaneous electrostimulation at levels of up to 5.5 km above sea level and emphasize the leading role of endogenous ligands of opiate receptors in realization of the analgesic effects of competitive skin stimulation. The results of the study also provide evidence of a high analgesic activity of dalargin, a synthetic analogue of leu-enkephalin, which persisted at a height of up to 7.5 km above sea level. Increasing the resistance of the experimental animals to acute high-altitude hypoxia, this agent facilitates the development of analgesic reactions in electrostimulation of the skin.     

The excitation of frog motoneurones in vitro by the glutamate analogue, DI-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), and the effect of amino acid antagonists

Intracellular recordings were made from motoneurones of the frog spinal cord in vitro and the excitatory effects of the glutamate analogue DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) examined. AMPA (25-100 microM) depolarized motoneurones with an increase in input conductance and an increase in spontaneous cell firing. The time-course of the AMPA response was prolonged compared to L-glutamate. D-Aminophosphonovalerate (1 microM) and D-aminoadipate (100 microM) did not antagonize AMPA-induced excitations while depressing L-glutamate responses. Glutamate diethylester up to 1 mM was not found to be a useful amino acid antagonist since it did not significantly reduce the depolarizations to quisqualate, L-glutamate and AMPA. The results are compatible with an action of AMPA on receptors distinct from those to N-methyl-D-aspartate and shared by quisqualate.     

The effect of dalargin on systemic hemodynamics and microcirculation in experimental traumatic shock

Experiments were conducted on rats and hamsters to study the effect of the synthetic analogue of leu-enkephalin dalargin injected in the early phase of traumatic shock. In experiments on hamsters, short-term increase in arterial pressure (AP) and improvement of microcirculation occurred in the first minutes after the injection. The AP did not increase in rats. This was followed by reduction of AP, deceleration of the blood flow in the microvessels, and increase in the diameter of arterial vessels. These processes developed more rapidly than in the controls. Survival decreased significantly among rats and had a tendency to reduce among hamsters. It is assumed that the inhibiting effect of dalargin on the higher regulating centres of vegetative functions, which is characteristic of opioid peptides, contributed to aggravation of the pathological processes developing in shock and leading to the death of the animals.     

Opioid peptides modulate secretion of the basic determinants of bile secretion

The effects of the synthetic opioid peptide dalargin and the antagonist of opiate receptors naloxone hydrochloride on the rate of bile secretion and concentration of bile acids and sodium ions is studied in rats. It is shown that the substances with opioid activity insignificantly raise the rate of bile secretion and concentration of the studied compounds. It is suggested that the secretion of the carbonate ion and glutathione increase under these conditions. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 5, pp. 498–500, May, 1997     

Hippocampal mossy fiber leu-enkephalin immunoreactivity in female rats is significantly altered following both acute and chronic stress

Research indicates that responses to stress are sexually dimorphic, particularly in regard to learning and memory processes: while males display impaired cognitive performance and hippocampal CA3 pyramidal cell dendritic remodeling following chronic stress, females exhibit enhanced performance and no remodeling. Leu-enkephalin, an endogenous opioid peptide found in the hippocampal mossy fiber pathway, plays a critical role in mediating synaptic plasticity at the mossy fiber-CA3 pyramidal cell synapse. Estrogen is known to influence the expression of leu-enkephalin in the mossy fibers of females, with leu-enkephalin levels being highest at proestrus and estrus, when estrogen levels are elevated. Since stress is also known to alter the expression of leu-enkephalin in various brain regions, this study was designed to determine whether acute or chronic stress had an effect on mossy fiber leu-enkephalin levels in females or males, through the application of correlated quantitative light and electron microscopic immunocytochemistry. Both acute and chronic stress eliminated the estrogen-dependence of leu-enkephalin levels across the estrous cycle in females, but had no effect on male levels. However, following acute stress leu-enkephalin levels in females were consistently lowered to values comparable to the lowest control values, while following chronic stress they were consistently elevated to values comparable to the highest control values. Ultrastructural changes in leu-enkephalin labeled dense core vesicles paralleled light microscopic observations, with acute stress inducing a decrease in leu-enkephalin labeled dense core vesicles, and chronic stress inducing an increase in leu-enkephalin labeled dense-core vesicles in females. These findings suggest that alterations in leu-enkephalin levels following stress could play an important role in the sex-specific responses that females display in learning processes, including those important in addiction.     

Opioid peptides as neuroregulators: Potential areas for the study of genetic-behavioral mechanisms

The opioid peptides have been related to behavior in both animal and human studies. Further investigation can be anticipated which could lead to the elucidation of genetic controls over enzymes which process these peptides and the receptors upon which the peptides act. The enzymes, both synthetic and degradative, can lead to the formation of different forms of the opiate peptides. Differential control of these enzymes or of the multiple forms of opiate receptors could lead to discrete changes in opiate status and subsequent behavioral changes. Conversely, genetically regulated behavioral modification could also lead secondarily to opiate changes.Support of this work was provided by the Office of Naval Research (ONR N00014-79-C-0796; SRO-001), relating to opioid peptides and stress; the National Institute on Drug Abuse (DA-01207), relating to opioid peptides and drug abuse; and the National Institute of Mental Health (MH-23861 and MH-30854), relating to opioid peptides and mental disorders. S.F.M. was a Visiting Professor at the Nancy Pritzker Laboratory in 1980–1981. We would like to thank Rosemary Schmele and Norma Varon for their gracious secretarial assistance.     

Selective presynaptic insectotoxin (alpha-latroinsectotoxin) isolated from black widow spider venom.

A homogenous protein of 120,000 mol. wt isolated from black widow spider (Lactrodectus mactans tredecimguttatus) venom and referred to as alpha-latroinsectotoxin was highly potent (4 nM) in the induction of an increase of the frequency of miniature excitatory postsynaptic potentials in blowfly (Calliphora vicina) larvae neuromuscular preparations. In the frog nerve ending, however, even 50 nM alpha-latroinsectotoxin failed to affect transmitter release. Pretreatment of insect preparations with alpha-latrotoxin or frog preparations with alpha-latroinsectotoxin did not prevent the specific effect of consequent applications of alpha-latroinsectotoxin (insect) and alpha-latrotoxin (frog), respectively. The binding of labelled [125I]alpha-latroinsectotoxin to insect and [125I]alpha-latrotoxin to bovine membrane preparations was saturable and highly specific. The presynaptic effect, but not the binding of alpha-latroinsectotoxin, was dependent on the presence of divalent cations in the external medium. Mg2+ could readily substitute for Ca2+ and increase of transmitter release induced by alpha-latroinsectotoxin also occurred in Ca(2+)-free solutions. Pretreatment of preparations with 300 micrograms/ml concanavalin A completely abolished both the presynaptic effect of alpha-latroinsectotoxin and its binding to insect membrane preparations. Thus, the phenomenology of alpha-latroinsectotoxin action on insects resembles in general that described for the action of alpha-latrotoxin on vertebrates. The selectivity of alpha-latrotoxin and alpha-latroinsectotoxin seems to be due to differences in the structure of neurotoxin receptors in nerve endings of vertebrates and insects, although the mode of presynaptic action has a great deal in common.     

Effect of morphine and leucine-enkephalin on the electrophysiological responses of Pacinian corpuscles of the cat

The effects of morphine and Leu-enkephalin on the electrical activity of Pacinian corpuscles isolated from cat mesentery were studied. These drugs caused a short-term increase and subsequent decrease of the impulse activity, accompanied by a monotonic growth of the threshold for firing of action potentials and amplitudes of generator and action potentials. Naloxone induced an effect similar to that of morphine. The results obtained indicate that morphine and Leu-enkephalin evoke an increase in membrane potential of a nerve ending. These data may form the basis of the modulating effect of opioid peptides on encapsulated mechanoreceptor activity.     

Opioid peptides inhibit the action of oestradiol on human myometrial cells in culture.

The effect of opioid peptides on cultured, oestradiol-stimulated human myometrial cells was examined. Oestradiol increased cell densities in mixed-cell (smooth muscle cells + stromal fibroblasts) cultures by 40%. This oestradiol-induced stimulation of cell proliferation was decreased to control values by D-met2-pro5-enkephalinamide. The half-effective inhibitory concentration of enkephalinamide was 0.3 nmol/l. The opioid-induced inhibition of cell proliferation was blocked completely by the specific opiate receptor antagonist naloxone, while naloxone did not have any effect on its own. This opioid effect was mediated dominantly by the mu opiate receptor. The optimal concentration for oestradiol to stimulate uterine cell proliferation was 2.2 nM. The basal rate of cell proliferation was not affected by enkephalinamide. In saturation experiments, the parameters of specific [3H]-naloxone binding were: dissociation constant = 1.02 nM, maximal binding capacity = 2910 binding sites/cell, Hill coefficient = 1.029. In human myometrial pure smooth muscle cell cultures, oestradiol decreased the proliferation of cells. Progesterone potentiated these oestradiol effects, but had no effect on its own. Enkephalinamide was also able to block the effects of oestradiol, but naloxone did not antagonize it. In summary, here we present a novel inhibitory role of endogenous opioid peptides in the regulation of cell growth and proliferation in the human uterus.     

The transmitter role of glutamate in nervous systems

Acceptance of L-glutamate as an excitatory transmitter was relatively slow compared to other transmitters, such as GABA. However, the transmitter role of L-glutamate has received more attention recently. In the crustacean neuromuscular junction L-glutamate fulfills most of the criteria for the transmitter identification. Studies on the glutamate receptor antagonist suggest that L-glutamate may also be the transmitter at the squid giant synapse. L-Glutamate may be the excitatory transmitter in many parts of CNS, although the evidence for this hypothesis is still not complete. The role of L-aspartate as the transmitter in CNS is not clear but its contribution may be less than that of L-glutamate. Glutamate receptors have been classified into three groups according to pharmacological properties. However, recent observations suggest that the situation seems more complicated than previously expected and more detailed comparison between synaptic potentials and glutamate action is needed.     

Central representation and opioid modulation of gastric mechanoreceptor activity in the rat

The effects of gastric distension on single-unit activity recorded from the dorsal vagal nucleus in the rat medulla have been studied using microelectrodes in anesthetized animals. Out of 97 units studied, 64 responded to gastric distension in one of four characteristic ways. It was possible to modify these gastric distension responses by iontophoretic application of Leu-enkephalin and naloxone. The predominant effect was diminution of the centrally recorded gastric distension response by Leu-enkephalin. These studies show it is possible to modulate the central representation of gastric mechanoreceptor activity by an opioid peptide that is common to both the gut and the brain. It is suggested these studies provide evidence that opiate effects on the gastrointestinal tract are mediated centrally as well as peripherally.     

Effect of the leu-enkephalin analog dalargin on DNA synthesis in the myocardium and lingual epithelium of rats in early postnatal ontogeny

The DNA synthesis is studied in the myocardium and lingual epithelium of 4–5-day-old rats 24 h after a single administration of the leu-enkephalin analog dalargin in a dose of 10 or 100 μg/kg. At 10 μg/kg, dalargin significantly decreases the number of DNA-synthesizing nuclei in the left atrium, while at 100 μg/kg it significantly decreases the number of DNA-synthesizing nuclei in the left atrium and both ventricles. Dalargin does not change the DNA synthesis in the lingual epithelial cells. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 123, No. 1, pp. 43–45, January, 1997     

Opioid peptides and opiate alkaloids in immunoregulatory processes

Among the various non-neuronal cell types known to express and utilize neuropeptides, those of the immune system have received much attention in recent years. In particular, comparative studies in vertebrates and invertebrates have shown that endogenous opioid peptides are engaged in receptor mediated autoregulatory immune and neuroendocrine processes. The majority of these immune processes are stimulatory, as determined by their effects on conformational changes indicative of immunocyte activation, cellular motility, and phagocytosis. Endogenous opioid peptides form an effective network of messenger molecules in cooperation with cytokines, opiate alkaloids, and certain regulatory enzymes (neutral endopeptidase 24.11). Peptide-mediated immunostimulatory effects observed in this system are operationally counteracted by the inhibitory effects of morphine and related opiates. Opioid/opiate signaling processes are mediated by several types of receptors with different degrees of selectivity. Among them the recently identified, opioid insensitive µ₃ receptor deserves attention on account of its specificity for opiate alkaloids.     

Effect of Opiate Peptide Dalargin and Des-Tyr-Dalargin on Cardiac Pump Function during Ischemia-Reperfusion

Experiments on isolated perfused rat heart showed that nonselective micro- and delta-opiate receptor agonist dalargin decreased contractility of the intact heart, but had no effect on pump function of the ischemic myocardium. Dalargin analogue des-Tyr-dalargin not binding to opiate receptors decreased contractility of intact myocardium and isolated heart exposed to 45-min total ischemia. We hypothesize that the influence of dalargin is related to activation of cardiac delta-opiate receptors, while the inotropic effect of des-Tyr-dalargin is mediated by other receptors.     

Effects of naloxone, nalorphine and dalargin on the course of shock due to the combination of mechanical trauma and acute blood loss

Acute experiments on 75 adult rabbits were made to study action of intravenous injections of naloxone (0.1 mg/kg), nalorphine (0.5 and 2.0 mg/kg) and dalargin (0.1 mg/kg) in early and late periods of irreversible shock caused by combination of non-shockogenic mechanical trauma of the hip and safe blood loss from the femoral artery. In early shock antagonists of opiate receptors did not influence the outcomes while injection of dalargin prolonged survival of the animals noticeably. In late shock injection of the drugs had no significant influence on the course of the pathological process while reinfusion did not help the animals to survive. In the late shock injection of the above drugs after blood reinfusion increased the rabbits' survival.     

Modulation of alcohol and nicotine responses through the endogenous opioid system

It has been estimated that more than 80% of alcoholics are also nicotine dependent and that, vice versa, the rate of alcoholism is substantially increased by a factor of 4–10 in the nicotine-dependent population. However, the cause for this very high degree of comorbidity is still largely unknown. At the molecular and cellular level, both drugs have very different mechanisms of action. Nicotine specifically activates ligand-gated ion channels in the brain, which are normally gated by acetylcholine, while alcohol interacts with various neurotransmitter receptors. Despite this diversity, both drugs seem to engage the endogenous opioid system as a modulator of some of its pharmacological effect. An acute exposure to nicotine or alcohol leads to a release of opioid peptides in specific brain regions, thus resulting in an activation of their corresponding receptors. If the brain is exposed repeatedly or chronically to these drugs, adaptive changes in the level and expression of opioid peptides and receptors occur. These adaptive changes are thought to contribute to the homeostatic or allostatic adaptations of the brain, which have been associated with drug dependence. This review summarizes pharmacological and genetic studies in animal models and in humans that have addressed the role of specific opioid peptides and receptors in various stages of the addiction process.     

Endogenic opioid peptides and antiarrhythmic effect of adaptation to stress

Adaptation of rats to repeated short-term immobilization increases cardiac resistance to an arrhythmogenic action of coronary artery occlusion (10 min) and reperfusion (10 min) in rats anesthetized with ketamine and artificially ventilated. We examined the role of opioid receptors and endogenous opioid peptides in the development of this antiarrhythmic effect produced in response to repeated periods of immobilization stress. We found that repeated daily stress during a 15-day period resulted in an increase of leu-enkephalin in blood plasma, in the suprarenal gland and myocardium. Adaptation to stress also resulted in an increase in beta-endorphinl-31 in blood plasma, the hypophysis, hypothalamus and midbrain. Pretreatment with selective mu, delta and cappa opioid receptor (OR) antagonists had no effect on the incidence of occlusion and reperfusion-induced arrhythmias in non-adapted control rats. However, pretreatment with the selective muOR antagonist CTAP (0.5 mg/kg) intravenously completely abrogated the antiarrhythmic effect of adaptation. Selective delta and cappa receptor antagonists did not affect the antiarrhythmic effect of adaptation. Prior administration of the selective muOR agonist DALDA (0.1 mg/kg) decreased the incidence of occlusion and reperfusion-evoked arrhythmias in non-adapted rats. This effect was abolished by pretreatment with the selective muOR antagonist CTAP (0.5 mg/kg). These data suggest that mu opioid receptors and endogenous opioid peptides play an important role in the antiarrhythmic effect of adaptation to stress in rats.