Inhibition of synaptic transmission by morphine and its antagonism by Ca++ in the isolated sympathetic ganglion of frog and rat

Synaptic transmission is inhibited by morphine in isolated sympathetic ganglia of frog and rat. This action of morphine can competitively be antagonized by naloxone, nalorphine as well as by increasing the Ca++ concentration of the medium. The morphine antagonistic action of Ca++ can be suspended by Mg++ applied in an equimolar concentration. These results show that morphine inhibits synaptic transmission by shifting the ionic balance necessary for transmitter release.     

Effect of mepacrine on gastric motility in the rat

Mepacrine given orally to rats inhibits gastric motility; its blocking effect is comparable to that of chloroquine, papaverine, and drotaverine, but less expressed than that of bencyclane. Similarly as the delayed gastric emptying induced by chloroquine, the effect of mepacrine is antagonized by acetyl-beta-methylcholine in a dose-related fashion, while that of papaverine, drotaverine, and bencyclane remains unchanged after treatment with the cholinomimetic drug.     

Effects of bencyclane on normal and cevadine-modified Na channels in frog skeletal muscle

The effect of 10(-5) mol/l bencyclane on the repetitive electrical activity of muscle membrane was studied with the conventional microelectrode technique. Electrical activity was induced by repetitive stimulation in normal Ringer solution (train) or by a single depolarizing current pulse in the presence of 10(-6) mol/l cevadine (volley). Bencyclane decreased, in a use-dependent manner, the maximum rates of depolarization and repolarization (Vmax+ and Vmax-, resp.) of the action potentials both of the train and the volley. The inhibition of Vmax+ and Vmax- was proportional; however, it was stronger for the volleys than for the trains. The cycle length (mean interspike interval) of the volley was increased by bencyclane; the prolongation was progressive during consecutive cycles. The dissociation of bencyclane from the Na channel was studied by applying trains of different durations with equal pulse numbers. Bencyclane at a higher concentration (5 x 10(-5) mol/l) caused a reversible tonic block: the overshoot potentials, Vmax+ and Vmax- were markedly reduced. The reduction of Vmax- was slightly stronger than that of Vmax+. Slow membrane potential oscillation (SMPO) was evoked by treating the muscle with 10(-4) mol/l of cevadine. The administration of 5 x 10(-6) mol/l bencyclane decreased the frequency of SMPO, while 10(-5) mol/l bencyclane terminated the slow oscillation activity without changing its baseline potential. The present results indicate that bencyclane induces use-dependent inhibition of Na channels in muscle, similarly as do class 1 antiarrhytnmic drugs. Inhibition was observed with both normal and cevadine-modified Na channels.     

The effect of leucine-enkephalin on the peristaltic reflex of the isolated guinea-pig ileum

Leucine (leu)-enkephalin depresses or inhibits the peristaltic reflex of the isolated guinea-pig ileum. Opiate antagonists (naloxone and nalorphine), choline esters (acetylcholine, methacholine and carbachol), cholinomimetics (muscarine and arecoline) and polypeptides which stimulate peristalsis (eledoisin and angiotensin) antagonize the peristaltic block caused by leu-enkephalin. On the other hand, nicotinic ganglionic stimulants (nicotine and dimethylphenylpiperazine) as well as muscarinic ganglionic stimulants (McN-A-343 and AHR-602) do not restore the peristaltic reflex abolished by leu-enkephalin. Thus the inhibitory effect of leu-enkephalin is due mainly to an action on myenteric ganglia as well as on axon terminals of the myenteric plexus subserving the peristaltic reflex. The inhibitory action of leu-enkephalin may be ascribed to the opiate as well as to the cholinoceptive sites in the nervous elements in the myenteric plexus. The blocking action of leu-enkephalin is not associated with ganglionic muscarinic M-1 receptors as well as with ganglionic nicotinic receptors in the myenteric plexus of the guinea-pig isolated ileum.     

Blockade of K+ contractures in skeletal muscle by opioid drugs: a nonstereospecific effect

The effect of several opioid drugs was tested on the K+ contractures in frog's skeletal muscle. These contractures are produced by the entrance of extracellular Ca2+ ions via the voltage-dependent, slow Ca2+ channels located in the T tubules. Morphine and other opioid agonists in concentrations ranging from 10(-10) to 10(-5) M inhibited K+ contractures. The stereoisomers, dextrorphan and levorphanol, were found to have identical potency in inhibiting high K+ contractures, suggesting that this was a nonstereospecific blockade of voltage-dependent calcium channels by the opioid drugs despite the low effective drug concentrations. In agreement with this conclusion it was found that the inhibition of K+ contractures by the opioids was not antagonized by naloxone. It also was observed using a sucrose gap apparatus that these opioid drugs in concentrations used to block the high K+ contractures did not reduce the K+-induced membrane depolarization. Raising the bathing solution Ca2+ concentration from 1.08 to 5 mM produced a reversal of the opioid-induced block of K+ contractures. Finally it was shown that while opioids completely blocked K+ contractures, they did not produce any effect on caffeine contractures showing that opioids do not deplete intracellular Ca2+ stores or inhibit the release of Ca2+ from intracellular sarcoplasmic reticulum stores. It was concluded that several opioid drugs in very low concentrations block K+ contractures in frog's skeletal muscle by a nonstereospecific block of voltage-dependent slow calcium channels.     

Differential contribution of extracellular and intracellular calcium sources to basal transmission and long-term potentiation in the sympathetic ganglion of the rat

Calcium involved in basal ganglionic transmission and long-term potentiation (LTP) can arise either by influx from the extracellular medium or release from intracellular stores. No attempts have yet been made to concurrently explore the contributions of extracellular and intracellular Ca2+ to basal ganglionic transmission or LTP. Here, we investigate this subject using the superior cervical ganglion of the rat. To explore the extracellular Ca2+ contribution, we evaluated basal transmission and LTP at different extracellular Ca2+ concentrations. To assess intracellular Ca2+ release, we explored the contribution of the calcium-induced calcium release process by overactivation or blockade of ryanodine-sensitive Ca2+ receptor channel with caffeine, and also by blocking either IP3R with Xestospongin C or the sarco(endo)plasmic reticulum Ca2+-ATPase pump with thapsigargin. Extracellular Ca2+ affected ganglionic basal transmission and LTP to different extents. While 25% of the physiological Ca2+ concentration supported 80% of basal transmission, 50% of normal Ca2+ was required to achieve 80% of LTP. Notably, disruption of intracellular Ca2+ release by all the drugs tested apparently did not affect basal ganglionic transmission but impaired LTP. We conclude that basal transmission requires only a small level of Ca2+ entry, while LTP expression not only requires more Ca2+ entry but is also dependent on Ca2+ release from intracellular stores.     

Dependence of batrachotoxin block of axoplasmic transport on sodium

Batrachotoxin (BTX) in the low concentration range of 19-190 nM blocks axoplasmic transport in the desheathed cat peroneal nerve in vitro. When the level of Na+ in the incubation medium was reduced to 10 mM, the blocking effect of BTX was much diminished, and in an Na+-free medium BTX had no effect on transport at all. The blocking action of BTX with Na+ present was inhibited by increasing the concentration of Ca2+ in the experimental medium. Relatively small increases were effective with a maximum protection seen when the Ca2+ concentrations were 7-10 mM. The results support the view that an increase in axonal Na+ is inhibitory to the transport mechanism. The results are discussed on the basis of the recently developed transport filament model of axoplasmic transport which takes into account an obligatory role for Ca2+ in transport and its axonal regulation. The possible relation of intraaxonal Na+ concentration to the Ca2+ level is also discussed.     

II - Prostaglandin hyperalgesia: the peripheral analgesic activity of morphine, enkephalins and opioid antagonists.

Morphine, enkephalins, nalorphine, naloxone and pentazocine are shown to have a peripheral analgesic effect. In our modification of the Randall-Selitto test these substances were 50--100 times more potent than a standard local anaesthetic, lidocaine. At this peripheral site, naloxone did not antagonize the effect of morphine. Morphine had a marked analgesic effect on the hyperalgesia induced by PGE2 and PGI2, BaCl2, Ca2+ ionophore A23187, isoprenaline but not on that induced by dibutyryl cyclic AMP. It was suggested that the peripheral analgesic effect of morphine is due to an inhibition of adenylate-cyclase activity.     

Naloxone inhibits superoxide release from human neutrophils

Using the superoxide dismutase inhibitable reduction of cytochrome c assay, we studied, the effect of (-) naloxone on N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated superoxide (O2-) release from human neutrophils. Neutrophils were pre-incubated with the range of concentrations of (-) naloxone that is administered in models of experimental sepsis (10(-6) - 10(-4.5) M). (-) Naloxone inhibited O2- release in a dose dependent manner. 02- produced by a cell-free xanthine-xanthine oxidase system was not inhibited by (-) naloxone, indicating that (-) naloxone was not scavanging O2-. There was no difference between the effect of (-) and (+) naloxone suggesting that the inhibition of O2- was not specific for an opiate receptor. Another opiate antagonist, nalorphine, as well as the opiate agonist, morphine, also inhibited O2- release in the same concentration range. There was no difference between the effect of naloxone and morphine.     

Effects of beta-endorphin on spontaneous uterine contractions. Prostaglandins production and 45Ca2+ uptake in uterine strips from ovariectomized rats.

The effects of beta-endorphin, Met-enkephalin, dynorphin and SKF 10047 on the constancy of the isometric developed tension (IDT) of the spontaneous contractions of uterine strips isolated from ovariectomized rats were explored. beta-endorphin (10(-6) M) was the only opioid that depressed significantly uterine constancy of IDT in a concentration dependent fashion. Naloxone, neither at 10(-8) M nor at 10(-6) M, altered the negative inotropic influence of beta-endorphin. Moreover, the basal synthesis and outputs of some prostaglandins (PGE1, PGE2 and PGF2 alpha) from rat uteri and the effect of beta-endorphin (10(-6) M), were determined. It was found that the basal synthesis and release of PGs in uteri were significantly inhibited by this endogenous opioid. The effects of beta-endorphin (10(-8), 10(-6) and 10(-5) M) on the basal; and oxytocin or A23187, induced 45Ca2+ uptake, as well as the influence of naloxone were also studied. beta-endorphin at three of the concentrations tested decreased basal uterine 45Ca2+ uptake and this action was not prevented by naloxone (10(-8) M). The presence of oxytocin and of A23187 augmented significantly 45Ca2+ uptake, an effect that was antagonized by beta-endorphin (10(-6) M). The possible role of beta-endorphin in uterine functioning via the modulation of uterine PG synthesis and Ca2+ uptake is discussed.     

Effects of morphine on arachidonic acid metabolism, on Ca2(+)-uptake and on cAMP synthesis in uterine strips from spayed rats

The effects of morphine on arachidonic acid metabolism, on cAMP levels and on basal and induced 45Ca2(+)-uptake, in uterine strips isolated from ovariectomized rats as well as the influence of naloxone, were explored. The presence of morphine (10(-6) M) did not change significantly 14C-arachidonic acid metabolism, basal cAMP levels, or cAMP increment induced by PGE2 or by PGE1. On the other hand morphine (10(-6) M) decreased basal uterine 45Ca2(+)-uptake as much as verapamil (10(-6) M) did, and this action was not prevented by naloxone (10(-8) M). The presence of oxytocin (50 mU.ml-1) augmented 45Ca2(+)-uptake, an effect which was antagonized by morphine (10(-6) M). This inhibitory action of morphine on oxytocin-induced 45Ca2(+)-uptake was not prevented by naloxone (10(-8) M). Furthermore, PGE1 (10(-8) M and (10(-6) M) but not PGE2 (10(-8) and 10(-6) M), stimulated the incorporation of 45Ca2+ into uterine strips, and this action was not altered by morphine. The inhibitory influence of morphine on uterine spontaneous motility and on prostaglandin synthesis and release, previously described by us, is now explained in terms of an inhibition of tissue Ca2(+)-uptake.     

Effect of different calcium modulators on motilin-induced contractions of the rabbit duodenum. Comparison with acetylcholine

Motilin and acetylcholine (ACh) have a direct contractile effect on rabbit small intestinal smooth muscle. To explore the role of calcium influx in these contractions, we studied the effect of extracellular calcium concentration and of calcium antagonists on the response of longitudinal muscle preparations from rabbit duodenum. Motilin- (10(-7) M) and ACh- (10(-4) M)-induced contractions were abolished in Ca2+-depleted medium. ACh (10(-4) M) or motilin (10(-8) and 10(-7) M) increased the contractile response to added Ca2+ to 130 +/- 6%, 129 +/- 10% and 145 +/- 5% of the maximal response to Ca2+ added alone (10 mM in a cumulative concentration response curve). The sensitivity to Ca2+ was greater in the presence of ACh and motilin (EC50 = 1.0 and 1.1 mM Ca2+) than in the absence of any agonist (1.7 mM). In cumulative concentration response (CCR) curves for motilin and ACh, pD2'-values were 7.0 and 6.6 for diltiazem, 8.4 and 7.8 for verapamil (two calcium entry blockers), 5.6 and 5.2 for TMB-8 (an inhibitor of intracellular calcium), 5.3 and 5.2 for TFP (a calmodulin-antagonist). All CCR-curves showed metactoid-like action of the antagonistic drugs. We conclude that ACh and motilin cause calcium to enter the smooth muscle cell. They are probably operating via separate channels, and use a mechanism which differs from K+-induced influx. Intracellular calcium stores appear to play a minor role in these contractions.     

The role of extra- and intracellular calcium in pepsinogen extrusion by isolated gastric glands]

It was found that carbacholine stimulated pepsinogen extrusion by isolated guinea pig stomach glands which were incubated in Ca(2+)-free medium, containing EGTA (0.25 mM). This effect could be imitated by caffeine (10 mM), a specific activator of Ca2+ release from intracellular pools. Extracellular Ca2+ in the concentrations over 0.125 mM increased pepsinogen extrusion which was stimulated by carbacholine. The interdependence between the level of pepsinogen extrusion and Ca2+ concentration in the medium had S-shaped character. La3+ ions (10(-4) mM) inhibited pepsinogen extrusion already in the first minutes after its activation by carbacholine. When testing other cations (Sr2+, Mg2+, Ba2+) it was found that only Sr2+ had some influence on pepsinogen extrusion. Thus, it can be concluded that both intra- and extracellular Ca2+ take part in the activation of pepsinogen extrusion. Obviously the role of extracellular Ca2+ consists in the support of reactivity of stomach glands to the action of stimulators of secretion.     

Glucose-stimulated sequestration of Ca2+ in clonal insulin-releasing cells. Evidence for an opposing effect of muscarinic-receptor activation.

Net fluxes of Ca2+ and acid production were studied in clonal insulin-releasing cells (RINm5F) by using colour indicators and dual-wavelength spectrophotometry. After equilibration with a medium containing 10-20 microM-Ca2+, only minimal amounts of Ca2+ (0.08 mmol/kg of protein) were released from the cells by subsequent additions of the respiratory blocker antimycin A and the Ca2+ ionophore A23187. The presence of 20 mM-glucose resulted in an almost 5-fold increase of the acid production and in a stimulated net uptake of Ca2+. The latter process was independent of the extracellular Ca2+ concentration and reached saturation after 20 +/- 1 min, when it corresponded to 1.18 +/- 0.07 mmol of calcium/kg of protein. Whereas the thiol reagent iodoacetamide suppressed the acid production, interference with mitochondrial function by using antimycin A or the uncoupler carbonyl cyanide m-chlorophenylhydrazone had the opposite effect. The latter two drugs induced a selective release of Ca2+ from a pool containing 35% of that taken up during glucose exposure. Most of the remaining Ca2+ was liberated by A23187 or iodoacetamide. Carbamoylcholine was also selective in mobilizing glucose-stimulated calcium, but this calcium (17%) appeared to originate from the pool insensitive to mitochondrial poisons. The action of carbamoylcholine was blocked by atropine and did not depend on the presence of extracellular Na+. The opposite effects of glucose and muscarinic-receptor activation on a non-mitochondrial calcium pool are consistent with participation of the endoplasmic reticulum in the glucose-induced sequestration of Ca2+ in pancreatic beta-cells.     

The effects of narcotic analgesics and narcotic antagonists on ganglionic transmission in the rat.

The effects of narcotic analgesics, narcotic-antagonist analgesics and narcotic antagonists on ganglionic transmission in the superior cervical ganglia of the rat were studied $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. $\text{In}$$\text{vivo}$ administration of morphine, meperidine, methadone, pentazocine or naltrexone blocked ganglionic transmission. Levorphanol, cyclazocine, nalorphine and naloxone had no effect on ganglionic transmission in this procedure. $\text{In}$$\text{vitro}$ studies confirmed the $\text{in}$$\text{vivo}$ results with the exception of levorphanol, cyclazocine and nalorphine, which were also found to block ganglionic transmission $\text{in}$$\text{vitro}$. In both preparations, naloxone did not antagonize the effect of morphine, suggesting that the effects of morphine and the other opiates were nonspecific. Similar potency of $\text{d}$- and $\text{l}$-isomers of pentazocine and cyclazocine support this conclusion. The observation that naltrexone blocked ganglionic transmission, but the other pure narcotic antagonist, naloxone, was inactive is somewhat unique to this test procedure and possibly significant.     

Modulation of thapsigargin-induced calcium mobilisation by cyclic AMP-elevating agents in human lymphocytes is insensitive to the action of the protein kinase A inhibitor H-89

Ca2+ mobilisation from internal stores and from the extracellular medium is one of the primary events involved in lymphocyte activation and proliferation. Regulation of these processes by adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) was studied in Fura2-loaded human peripheral blood lymphocytes. Cytosolic Ca2+ concentration ([Ca2+]i) was measured in single cells by the use of a ratio imaging fluorescence microscope and Ca2+ mobilisation was achieved by the use of the endoplasmic reticulum (ER) Ca2+ ATPase inhibitor, thapsigargin (Thg). Our results show that both activation and inhibition of PKA, with forskolin (FSK) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H-89), respectively, inhibited the Thg-induced Ca2+ entry. Furthermore, FSK also reduced the ability of Thg to release Ca2+ from internal stores. This reduction was inhibited by the adenylyl cyclase (AC) inhibitor 9-(tetrahydro-2-furanyl)-9-H-purin-6-amine (SQ22,536), but not by the PKA inhibitor H89, indicating that cAMP but not PKA is responsible for this effect. FSK effect was mimicked by dibutyryl cAMP (dbcAMP) and by inhibition of phosphodiesterases (PDEs) with rolipram (ROL) and milrinone (MIL). We also showed that a very high concentration of H-89 (100 microM) releases Ca2+ from an intracellular pool, although this action is probably independent of PKA inhibition. Neither 10 microM H-89 nor other cAMP/PKA-modulating drugs had any effect on the basal [Ca2+]i of human lymphocytes. We conclude that PKA may act as a fine modulator of capacitative Ca2+ entry, while cAMP has a PKA-independent interaction with the Ca2+ stores of human lymphocytes.     

Investigation of beta-endorphin nonopioid reception in preimplantation development of mouse embryo in vitro

The effect of beta-endorphin on 2-, 4- and 8-cell embryo development in vitro was studied. It is shown, that hormone has no effect on 2-cell embryos development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number ofblastocyst formation increases in presence of 0.1 microM beta-endorphin in embryo cultured medium but the number of blastocyst with abnormal structure decreases. The effect of hormone on the change of intracellular concentration of Ca2+ ion in 2-, 4- and 8-cell mouse embryo has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase, and phospholipase activity blockers and opioid blocker naloxone on the change of intracellular concentration of Ca2+ ion in early mouse embryo in the presence of beta-endorphin have been also studied. It is shown that 2-cell embryo has opioid and nonopioid beta-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopoioid beta-endorphin receptors. It is also shown that the effect of beta-endorphin in the early mouse embryo through a nonopioid receptors occurs with the participation of intracellular Ca2+ and adenylate cyclase signaling system.     

Effects of the fraction (1-10 kDa) of the brain of a Yakut horse on kinetic parameters of Ca2+ transport system in sarcolemma vesicles of cardiomyocytes]

The effect of the fraction (1-10 kDa) obtained from the brain of cold-adapted animal (Yakut horse) on Ca2+ transport in sarcolemma vesicles of cardiomyocytes was investigated. It was shown that during insertion of Yakut horse brain fraction into incubation medium at the concentration from 10(-9) M to 3.10(-5) M at Ca2+ transport substrate concentration from 0.1 mM to 1.0 mM, the rate of Ca2+ passive penetration into vesicles slightly increased and at Ca2+ transport substrate concentration 3 mM, which is physiologic, a decrease of rate values was established for all concentrations of the fraction (1-10 kDa) of Yakut horse. While studying the kinetics of an active Ca2+ transport for all investigated concentrations of the fraction (1-10 kDa) of Yakut horse brain from 10(-9) M to 3.10(-5) M at Ca2+ concentration in incubation medium from 10(-7) to 3.10(-6) M, calcium accumulation rates by vesicles exceeded control values. So we can suppose that application of brain fraction (1-10 kDa) of genotypically cold-adapted animal, results in a decrease of intracellular Ca2+ concentration.     

III - Prostaglandin hyperalgesia: relevance of the peripheral effect for the analgesic action of opioid-antagonists

Morphine injected into the rat cerebral ventricles had a marked analgesic effect, while no effect was observed with pentazocine and naloxone or nalorphine caused a strong hyperalgesia. Administered systemically (IP) naloxone and nalorphine caused a transitory analgesia followed by a long lasting hyperalgesic effect; morphine and pentazocine showed only an analgesic effect. It was concluded that the site of analgesic action of opioid-antagonists is peripheral rather than central. The peptidase-resistant enkephalin-analog, BW 180c, which does not cross the blood brain barrier, caused a marked analgesia by IP administration to paws made hyperalgesic by PGE2 or carrageenin. It is suggested that agents derived from morphine, morphine-antagonists, enkephalins or cGMP devoid of central effect but having a strong peripheral effect may constitute a new class of safer analgesics.     

Opioid agonist modulation of cytoplasmic free Ca2+ level in concanavalin A-stimulated mouse lymphocytes.

In this study the influence of mu-, delta-, and kappa-selective opioid agonists (DAMGO, DSLET, and dynorphin A (1-13)) on cytoplasmic free Ca2+ ([Ca2+]i) level in normal and concanavalin-A (Con A)-activated mouse lymphocytes was investigated. [Ca2+]i was measured using the fluorescent dye FURA-2AM. The opioid peptides at 10-12-10-7 M induced some increase in [Ca2+]i in non-activated lymphocytes. However, DAMGO and DSLET (10-13-10-7 M) considerably inhibited a Con A-induced increase in [Ca2+]i. The inhibiting effect of both peptides was higher after 20-min preincubation compare to 2-h preincubation. The effect of the kappa-agonist dynorphin A (1-13) was significantly different depending on the duration of cell pretreatment and the concentration of the peptide used. After preincubation for 20 min at low concentrations (10-12-10-11 M) it slightly stimulated, while at higher (10-10-10-7 M) concentrations it inhibited lymphocyte response to Con A. After preincubation for 2 h, pronounced stimulation of mitogen-induced Ca2+ flux was observed at peptide concentration 10-9 M. The effects of opioids were antagonized by naloxone. These data indicate that functionally active opioid receptors expressed on lymphocytes could be involved in early stages of mitogen activation.