Non-cyclic AMP-dependent, positive inotropic cyclodepsipeptides with negative chronotropy.

The effects of natural cyclodepsipeptides (CDPs) on isolated rat cardiac tissue preparations were examined in vitro. Destruxin A, destruxin B (DB), roseotoxin B (RB), and roseocardin (RC), a novel CDP, each caused a concentration-dependent increase in the contraction force of the right atrium and the papillary and trabecular muscles of the right ventricle at 0.6 to 600 microM. RB, destruxin A, and DB did not affect the half-decay time of relaxation of the papillary muscles, but RC slightly prolonged it, although to a much lesser extent than BA 41899, a calcium sensitizer. This inotropic effect is accompanied by a prolongation of the automatic atrial contraction intervals. The RB-induced increase in the contraction force of papillary muscle was not affected by phentolamine, propranolol, pyrilamine, or cimetidine. RB- and RC-induced increases in the contraction force of papillary muscles were not affected by 3-isobutyl-1-methylxanthine or carbachol. Neither peptide changed the cyclic AMP levels in trabecular muscles. Neither RB nor RC affected the activity of Na(+),K(+)-ATPase from rat kidney. Neither RB, RC, nor DB affected the resting membrane potential or the apparent input resistance of papillary muscles. These results suggest that these CDPs produce both non-cyclic AMP-dependent positive inotropic and negative chronotropic effects.

Roseocardin, a novel cardiotonic cyclodepsipeptide from Trichothecium roseum TT103.

A new cyclodepsipeptide, designated roseocardin, was isolated from the culture broth of Trichothecium roseum TT103. Roseotoxin B and destruxins A and B were also isolated during the same procedure. The structure of roseocardin was determined by EI-MS, NMR and X-ray crystallographic analysis. Roseocardin as well as the other cyclodepsipeptides were shown to produce positive inotropic effects on rat heart muscles.

Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism in the mouse.

Mice were given the extract of cultured Cordyceps sinensis (Cs) (200 mg/kg daily, p.o.) for 3 weeks. In vivo phosphorus-31 nuclear magnetic resonance (NMR) spectra of the liver were acquired at weekly intervals using a surface coil. From 1 to 3 weeks, a consistent increase in the ATP/inorganic phosphate ratio, which represents the high energy state, was observed in the Cs extract-treated mice. The intracellular pH of the Cs extract-treated mice was not significantly different from that of the control mice. No steatosis, necrosis, inflammation or fibrosis were observed in the liver specimens from Cs extract-treated mice.

Differential inhibition of transient and long-lasting calcium channel currents by benzodiazepines in neuroblastoma cells.

The effects of diazepam, nitrazepam, clonazepam, and Ro5-4864 on transient (type I) and long-lasting (type II) calcium channels associated with low-affinity benzodiazepine receptors were investigated using the whole-cell patch-clamp technique. Clonazepam (100 microM), a specific agonist for the central-type benzodiazepine receptor, reduced transient currents through the type I calcium channel by 40% without affecting long-lasting currents through the type II calcium channel. Diazepam and nitrazepam (100 microM), non-specific agonists for both the central- and peripheral-type benzodiazepine receptors, reduced both transient and long-lasting currents equally by 25-30%. A similar non-selective inhibition was observed by Ro5-4864 (1-10 microM), a specific agonist for the peripheral-type benzodiazepine receptor. It is concluded that the two calcium channel types are regulated differentially by two different kinds of benzodiazepines; central-type for type I channel and peripheral-type for both type I and type II channels.

Neurally evoked potentiation of tonic contractions in the guinea-pig vas deferens involves adenosine receptors.

1. In the pelvic plexus-vas deferens preparation of the guinea-pig, conditioning stimulation of the pelvic nerves depressed the phasic component of biphasic contractions evoked by test stimulation of the hypogastric nerves, but potentiated the tonic component. 2. Similarly, in the deganglionated vas deferens preparation, conditioning stimulation applied directly to postganglionic nerves issuing from the pelvic nerve side of the pelvic plexus depressed the phasic component of biphasic contractions evoked by test stimulation of the nerves issuing from the plexus on the side of the hypogastric nerve, but potentiated the tonic component. 3. In the deganglionated preparation in the presence of alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-mATP) by which the phasic component was removed, test contractions were markedly reduced by phentolamine and prazosin, and potentiated in a manner dependent on the magnitude of conditioning stimulation. 4. In the deganglionated preparations that were persistently exposed to alpha,beta-mATP (5-10 microM), attempts were made to identify the substance(s) mediating the potentiation of the prazosin-sensitive contractions and their sites of action. 5. Noradrenaline and neuropeptide Y depressed the test contractions. 6. ATP, adenosine 5'-diphosphate, adenosine 5'-monophosphate and adenosine potentiated the contractions. Inosine and adenine were without effect. The effect of ATP was antagonized by 8-phenyltheophylline. N6-Cyclohexyladenosine (CHA) potentiated the contractions in a dose-dependent and 8-phenyltheophylline-sensitive manner. 7. The conditioning stimulation-induced potentiation of test contractions was antagonized by 8-phenyltheophylline, but was further increased by dipyridamole. 8. Methoxamine-evoked contractions were potentiated by conditioning stimulation of the nerves in a manner antagonized by 8-phenyltheophylline, and also by CHA. 9. Adenosine and CHA inhibited in an 8-phenyltheophylline-sensitive manner field stimulation-induced release of 3H-activity from deganglionated vas deferens preloaded with [3H]noradrenaline. 10. In the deganglionated preparation that was not exposed to alpha, beta-mATP, ATP-evoked contractions were potentiated by conditioning stimulation and by CHA. 11. It is indicated that conditioning stimulation and adenosine exert opposite actions on phasic and on tonic contractions in spite of the results showing that each of them postjunctionally potentiates the responses to both ATP and noradrenaline. 12. These results suggest that conditioning stimulation releases adenosine and that adenosine potentiates tonic contractions by increasing the responsiveness of the muscle to noradrenaline through the postjunctional adenosine receptors.

Parasympathetic depression of vas deferens contraction in the guinea-pig involves adenosine receptors.

1. In the guinea-pig pelvic plexus-vas deferens preparation, stimulation of the parasympathetic pelvic nerves contracted the vas deferens then depressed the contractile responses to stimulation of the sympathetic hypogastric nerves. 2. The contraction caused by stimulation of the pelvic nerves was initially phasic then tonic. The contractions were almost abolished by application of hexamethonium to the plexus. The phasic contraction was abolished by alpha,beta-methylene adenosine triphosphate applied to the vas deferens. 3. Conditioning stimulation of the pelvic nerves preferentially depressed the phasic component of test contractions evoked by hypogastric nerve stimulation but did not affect the compound action potentials in postganglionic nerves evoked by test stimulation. 4. When the pelvic plexus was divided into two parts, one with the pelvic nerves and the other with the hypogastric nerves, conditioning stimulation of the pelvic nerves still depressed test contractions evoked by hypogastric nerve stimulation. 5. In the de-ganglionated vas deferens preparation, conditioning stimulation of some postganglionic nerves also depressed contractions evoked by test stimulation of the other postganglionic nerves. 6. 8-Phenyltheophylline (5-20 microM) applied to the vas deferens antagonized the conditioning stimulation-induced depression in both the pelvic plexus-vas deferens and the de-ganglionated preparations. 7. N6-Cyclohexyladenosine (CHA) and N6-(L-2-phenylisopropyl)-adenosine at 0.5 microM preferentially inhibited phasic contractions evoked by the postganglionic nerve stimulation. The effect of CHA was antagonized by 8-phenyltheophylline (10 microM). 8. The results indicate that the mechanism underlying the conditioning stimulation-induced depression of phasic contractions operates not in the ganglia, but through activation of adenosine receptors in the vas deferens.

Gating and permeation properties of two types of calcium channels in neuroblastoma cells.

The gating and permeation properties of two types of calcium channels were studied in the neuroblastoma cell line N1E-115. Calcium channel currents as carried by Ba2+ (50 mM) were recorded using the whole-cell variation of the patch electrode voltage-clamp technique. The two types of calcium channels showed similar membrane potential dependence with respect to the steady-state activation and inactivation gating properties. However, the properties of the long-lasting type II channels were shifted approximately 30 mV in the depolarizing direction compared with those of the transient type I channels. Activation of type I channels developed with a sigmoidal time course which was described by m2 kinetics, whereas the activation of type II channels was described by a single exponential function. Tail current upon repolarization followed an exponential decay in either type of calcium channels. In comparison to type I channels, the activation process of type II channels was shifted approximately 30 mV in the positive direction, while the deactivation process showed a 60 mV shift in the positive direction. The rate constants of activation obtained from the activation and deactivation processes indicated that under comparable membrane potential conditions, type II channels close 2.4 times faster than type I channels upon repolarization. When external 50 mM Ba2+ was replaced with Ca2+ or Sr2+ on the equimolar basis, the amplitudes of transient and long-lasting currents were altered without a significant change in their time courses. The ion permeability ratios determined from the maximum amplitude of the inward current were as follows: Ba2+ (1.0) = Sr2+ (1.0) greater than Ca2+ (0.7) for type I channels, and Ba2+ (1.0) greater than Sr2+ (0.7) greater than Ca2+ (0.3) for type II channels. Replacement of Ba2+ with Ca2+ caused a 10-12 mV positive shift in the current-voltage relation for type II channels. However, the shift for type I channels was much less. This suggests that negative surface charges are present around type II channels. After correction for the surface charge effect on the ion permeation, there was no significant difference between the permeability ratios of these cations for the two channel types. It was concluded that the two types of calcium channels have many common properties in their gating and permeation mechanisms despite their differential voltage sensitivity and ion selectivity.

Maitotoxin-induced membrane current in neuroblastoma cells.

Maitotoxin (MTX) is a potent marine toxin isolated from the toxic dinoflagellate, Gambierdiscus toxicus. We have examined the possibility of MTX activating calcium channels using cultured neuroblastoma cells (N1E-115). MTX (10 ng/ml) produced a depolarization of the membrane, which was prevented by the removal of Ca2+ from the external medium. Under voltage clamp conditions, membrane currents were recorded with 50 mM Ba2+ as a charge carrier through calcium channels. After application of MTX (1 ng/ml), an inward current necessary to hold the membrane at -90 mV increased progressively. This was followed by a gradual decrease of the transient inward Ba2+ current through type I calcium channels recorded at -30 mV which was eventually abolished. A similar tendency was observed in the long-lasting inward Ba2+ current through type II calcium channels, which was recorded at +10 mV. The MTX action was antagonized by calcium channel blockers such as verapamil (100 microM) and La3+ (1 mM). A high concentration of verapamil (500 microM) blocked both types of calcium channels persistently. After washout of verapamil but while the calcium channels were still blocked, MTX (1 ng/ml) induced a steady-state current. The MTX-induced current showed an inward-rectifying property with a reversal potential of approximately -30 mV. The results suggest that the MTX-induced current does not flow through calcium channels. Thus, MTX may create a pore in the membrane with pharmacological properties similar to those of calcium channels.

Cyclic nucleotide potentiation of muscarinic responses in neuroblastoma cells.

The role of cyclic nucleotides in modulating acetylcholine-induced and dopamine-induced responses was examined with cultured neuroblastoma N1E-115 cells by means of intracellular recording techniques. Acetylcholine-induced muscarinic hyperpolarization and muscarinic depolarization were potentiated by bath application of a dibutyryl analog of adenosine 3',5'-phosphate (cyclic AMP) or phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone. Dibutyryl cyclic AMP did not affect the resting membrane potential and membrane resistance. Acetylcholine-induced nicotinic depolarization was unaffected by dibutyryl cyclic AMP or phosphodiesterase inhibitors. Intracellular pressure injection of cyclic AMP caused a potentiation of muscarinic hyperpolarization and muscarinic depolarization without marked change in the resting membrane potential. Nicotinic depolarization and dopamine depolarization were not affected by cyclic AMP injection. Among the possible metabolites of cyclic AMP, injection of adenosine potentiated muscarinic hyperpolarization, but did not change nicotinic depolarization and dopamine depolarization. Injection of guanosine 3',5'-phosphate (cyclic GMP) potentiated muscarinic hyperpolarization and muscarinic depolarization without effect on nicotinic depolarization and dopamine depolarization. We conclude that cyclic AMP and cyclic GMP enhance muscarinic responses in neuroblastoma cells. It is suggested that synaptic transmission in the nervous system may be modulated postsynaptically by changes in intracellular cyclic nucleotide levels.

Characterization of two types of calcium channels in mouse neuroblastoma cells.

1. Two types of voltage-sensitive calcium channels were identified and studied in the neuroblastoma cell line N1E-115. Calcium channel currents as carried by Ba2+ (50 mM) were recorded using the whole-cell variation of the patch-electrode voltage-clamp technique. 2. A transient (type I) inward Ba2+ current was evoked by a step depolarization from a holding potential of -80 mV to potentials more positive than -50 mV. The current amplitude became maximum around -20 mV. 3. A depolarization to potentials more positive than -20 mV evoked a long-lasting (type II) component of the inward Ba2+ current. This component reached its maximum around +10 mV and did not inactivate during a prolonged depolarizing pulse lasting 400 ms. 4. When preceded by a 5 s conditioning pulse to -30 mV, step depolarization failed to evoke a transient current due to inactivation. However, it induced a long-lasting current. 5. A transient current isolated as the component sensitive to conditioning depolarization became faster in its time course and smaller in its amplitude with membrane depolarization. The current direction was still inward at +60 mV. 6. From the differential voltage sensitivity and the independent channel activity described above, calcium channels responsible for the transient current (type I channel) and those responsible for the long-lasting current (type II channel) were considered to be two different entities. 7. Cd2+ preferentially blocked type II channels, whereas La3+ was a highly potent blocker for both types of calcium channels. 8. The relative potency for block by polyvalent cations was as follows (apparent dissociation constant in microM): La3+, 1.5 much greater than Ni2+, 47 greater than Cd2+, 160 = Co2+, 160 for type I channels, and La3+, 0.9 greater than Cd2+, 7.0 much greater than Ni2+, 280 greater than Co2+, 560 for type II channels. 9. The two types of calcium channels were equally sensitive to the temperature. The current amplitude was reduced by cooling below 30 degrees C. The temperature coefficient (Q10) value was estimated to be 3.0 between 20 and 30 degrees C, and 15.0 below 20 degrees C. Above 30 degrees C, warming reduced the amplitude slightly. 10. External application of dibutyryl adenosine 3',5'-phosphate (dibutyryl cyclic AMP) (1 mM) caused an increase in the amplitude of the type II current by 30-50%, while failing to enhance the type I component.(ABSTRACT TRUNCATED AT 400 WORDS)

Block of calcium channels by enkephalin and somatostatin in neuroblastoma-glioma hybrid NG108-15 cells.

Leucine-enkephalin, methionine-enkephalin, and morphine caused a reversible block of Ca2+ channel currents in neuroblastoma-glioma hybrid cells (NG108-15). The long-lasting (type 2) component of the Ca2+ channel current was blocked by leucine-enkephalin, while the transient (type 1) component was not affected. The enkephalin-induced blocking action was antagonized by naloxone and appears to be mediated by delta-opiate receptors. Two different aspects of the blocking effect were detected, a resting block and a recovery from block during prolonged depolarizing pulses. Recovery from block was more complete, and its time course was more rapid, with depolarization to more positive potentials. The dose dependence of the type 2 channel block at rest indicated a one-to-one binding stoichiometry, with an apparent dissociation constant of 8.8 nM. Somatostatin exerted a similar selective blocking action on the type 2 Ca2+ channel. The time- and voltage-dependent block of type 2 Ca2+ channels may provide a mechanism underlying the enkephalinergic presynaptic inhibition of transmitter release and the somatostatin block of pituitary growth hormone release.

Cyclic AMP-mediated potentiation of muscarinic hyperpolarization in neuroblastoma cells.

Adenosine, 2-chloroadenosine and prostaglandin E1 which are known to increase cyclic AMP in neuroblastoma cells potentiated the acetylcholine-induced muscarinic hyperpolarization of the cells without changing the resting membrane potential. The potentiation caused by 2-chloroadenosine was further augmented by Ro 20-1724, a phosphodiesterase inhibitor. A direct intracellular pressure application of cyclic AMP potentiated the muscarinic hyperpolarization without changing the resting membrane potential. Morphine which inhibits adenylate cyclase antagonized 2-chloroadenosine-induced potentiation of the muscarinic hyperpolarization. These results suggest that changes in cyclic AMP level modulate the muscarinic response of neuroblastoma cells.

Transplantation of extensor carpi radialis longus muscle in normal and dystrophic chicks.

The etiology of avian muscular dystrophy was examined by a cross-transplantation technique. Care was taken for the transplants to regenerate and develop under neural influence, by using the small extensor carpi radialis longus (ECRL) muscle. The ECRL muscles were exchanged between normal and dystrophic chicks 2 to 3 days ex ovo, and the muscle weight, number of muscle fibers, muscle fiber size, and contractile properties of the transplanted muscles were observed 60 to 65 days after operation when the tissue reconstitution was virtually complete. The results obtained for the physiologic, anatomic, and histologic parameters strongly suggested that there exists some failure in the host environment of the dystrophic chicken. The analyses of the histologic parameters suggested that a genetic disorder may also reside in the muscle tissue itself. The myotonic nature of the muscle membrane, however, probably does not contribute significantly to the abnormal behavior of dystrophic chickens. The importance of some neurogenic abnormalities in avian muscular dystrophy is discussed in relation to the results reported by other investigators.

Substance P as an excitatory transmitter of primary afferent neurons in guinea-pig sympathetic ganglia.

Electrophysiological and neurochemical experiments were carried out to examine a possible transmitter role substance P in the prevertebral ganglia of the guinea-pig. When potentials were recorded intracellularly from neurons of the isolated ganglia, stimulation of the pre- or postganglionic nerves elicited a non-cholinergic slow excitatory postsynaptic potential (EPSP). This synaptic potential was compared with the effects of substance P. Brief application of substance P caused a depolarization of the ganglion cells with a similar time course to that of the non-cholinergic slow EPSP. Changes in membrane resistance during the substance P-induced depolarization resembled those associated with the non-cholinergic slow EPSP. During the substance P-induced depolarization the non-cholinergic slow EPSP was markedly depressed. Attempts were made to determine the origin of the fibers eliciting the non-cholinergic slow EPSP. In the inferior mesenteric ganglia isolated together with preganglionic nerves that retained intact connections with spinal nerve roots, dorsal root stimulation evoked a non-cholinergic slow EPSP but not a cholinergic fast EPSP in the ganglion cells, whereas ventral root stimulation caused only cholinergic fast EPSPs. Following the prolonged treatment with capsaicin, the non-cholinergic slow EPSP was greatly depressed or abolished. Radioimmunoassay revealed that after ligation or section of pre- or postganglionic nerves an accumulation of substance P occurred in the proximal stumps of the interrupted nerves. Stimulation with high potassium medium evoked a release of immunoreactive substance P from the prevertebral ganglia and the release was calcium-dependent. The present findings suggests that axon collaterals of certain visceral primary efferents form synapses with principal cells in the prevertebral ganglia and release substance P as a transmitter for the non-cholinergic slow EPSP.

Role of substance P as a sensory transmitter in spinal cord and sympathetic ganglia.

The hypothesis that substance P (SP) might be a transmitter of primary sensory neurons was first proposed by Lembeck in 1953. A large amount of evidence supporting this hypothesis has recently accumulated, particularly since the elucidation of the chemical structure of SP by Leeman and her colleagues in 1971, which made a number of new approaches possible (e.g. radioimmunoassay for SP, immunohistochemistry and electrophysiological tests of SP action on central and peripheral neurons). SP is concentrated in certain primary afferent terminals in the spinal cord, is released therefrom when the dorsal roots are electrically stimulated, and exerts a powerful excitant action on spinal neurons. It is therefore likely that SP produces excitatory postsynaptic potentials (EPSPs) in spinal neurons, although the characteristics of SP-mediated EPSPs, i.e. their time course, ionic mechanisms, etc., remain to be revealed. Recent electrophysiological and neurochemical studies on the prevertebral ganglia of the guinea-pig strongly suggest that SP is released from axon collaterals of visceral primary afferent neurons in the ganglia and serves as a transmitter that generates non-cholinergic slow EPSPs in principal cells. There is evidence that this SP-mediated synaptic transmission in the sympathetic ganglia is under the influence of enkephalinergic presynaptic inhibition. Some preliminary experiments on the interaction between SP and enkephalins in the spinal cord are described.

Enkephalins presynaptically inhibit cholinergic transmission in sympathetic ganglia.

Recent biochemical and immunohistochemical studies have shown that the opioid peptides, enkephalins, occur in nerve terminals and cell bodies in mammalian sympathetic ganglia1-3. Opiates and enkephalins are thought to inhibit synaptic transmission in the peripheral nervous tissues as well as in the central nervous system4-12. The mechanisms of the opiate actions, however, are not entirely clear; both pre- and postsynaptic sites of action have been proposed7-9,11,12. As acetylcholine is known to be the major neurotransmitter in the autonomic ganglia and as the mechanism of synaptic transmission is well clarified13, analysis of the peptide action could be more easily but equally usefully carried out in the peripheral synapses than in central synapses. We now report that enkephalins presynaptically inhibit cholinergic transmission in sympathetic ganglia.

Substance P: depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurons.

The substance P content, glutamic acid decarboxylase and choline acetyltransferase activities and the level of [3H]diprenorphine binding were measured in various regions of the lumbar spinal cord of rats after unilateral section of the sciatic nerve or after dorsal rhizotomy. Sciatic nerve section produced a 75--80% depletion of substance P in the dorsal horn but did not change the substance P content of the ventral horn. The onset of substance P depletion occurred within 7 days and was maintained for 2 months. The substance P content of the dorsal root ganglia and both the peripheral and central branches of primary sensory neurons was also reduced after sciatic nerve section. Glutamic acid decarboxylase and choline acetyltransferase activity were unchanged; however, a small decrease in opiate receptor binding occurred 1 month after nerve section. Dorsal rhizotomy produced an 80% depletion of substance P in the dorsal horn. In addition, the substance P content of the ventral horn was significantly reduced. Glutamic acid decarboxylase activity in the dorsal horn was unaffected by dorsal rhizotomy whereas opiate receptor binding was reduced by 40%. From these studies it appears that peripheral nerve injury results in the degeneration of primary sensory neurons which contain and release substance P as neurotransmitter.