Evidence for postsynaptic modulation of muscle contraction by a Drosophila neuropeptide

DPKQDFMRFamide, the most abundant FMRFamide-like peptide in Drosophila melanogaster, has been shown previously to enhance contractions of larval body wall muscles elicited by nerve stimulation and to increase excitatory junction potentials (EJPs). The present work investigated the possibility that this peptide can also stimulate muscle contraction by a direct action on muscle fibers. DPKQDFMRFamide induced slow contractions and increased tonus in body wall muscles of Drosophila larvae from which the central nervous system had been removed. The threshold for this effect was approximately 10(-8)M. The increase in tonus persisted in the presence of 7x10(-3)M glutamate, which desensitized postsynaptic glutamate receptors. Thus, the effect on tonus could not be explained by enhanced release of glutamate from synaptic terminals and, thus, may represent a postsynaptic effect. The effect on tonus was abolished in calcium-free saline and by treatment with L-type calcium channel blockers, nifedipine and nicardipine, but not by T-type blockers, amiloride and flunarizine. The present results provide evidence that this Drosophila peptide can act postsynaptically in addition to its apparent presynaptic effects, and that the postsynaptic effect requires influx through L-type calcium channels.     

Action of two corpus cardiacal peptides on muscles of the hindgut of Periplaneta americana L. (Orthoptera: Blattidae)

1. (1) Two small, basic peptides (Ic and II) which stimulate muscles of the hindgut, but not the heart, have been separated from extracts of the corpora cardiaca by ion exchange chromatography and gel filtration.

2. (2) Results obtained with the 5-HT antagonist mianserin hydrochloride suggest that both peptides stimulate contractions of the hindgut by direct action without mediation by an indoleamine such as 5-HT.

3. (3) Extracellular calcium ions are required for rapid contractions of the hindgut stimulated by peptides Ic and II. However, the tonus response to these peptides does not appear to depend on the same inward transmembrane transport of extracellular Ca²⁺ as rapid contractions.

4. (4) Theophylline (5 mM) potentiated the action of both peptides on hindgut muscle, however, stimulation of hindgut muscle by cardiacal extract did not cause a significant increase in the level of cyclic AMP as compared with controls.

5. (5) These results more clearly define the number, character and possible action of cardiacal peptides stimulating hindgut muscle in P. americana.

     

Differential effects of neuropeptides on circular and longitudinal muscles of the crayfish hindgut

Proctolin (Arg-Tyr-Leu-Pro-Thr-OH) and crayfish peptide "DF(2)" (Asp-Arg-Asn-Phe-Leu-Arg-Phe-NH(2)) enhance spontaneous contractions of isolated crayfish hindguts. Both peptides increase the frequency and amplitude of spontaneous, rapid contractions. Proctolin induces a slow contraction, which gives the appearance of an increase in overall tonus. DF(2) has no such effect. To determine whether the peptides affect both longitudinal and circular muscles, hindguts were cut into longitudinal strips and into rings, and contractions were recorded from each. The longitudinal strips generated only rapid contractions, and both peptides increased the frequency and amplitude of such contractions without significantly altering tonus. Rapid contractions were observed in only 1 of 14 preparations of rings. Proctolin induced slow contractions in the rings, and DF(2) had no such effect. The results indicate that neuropeptides have different effects on circular and longitudinal muscles of hindgut.     

The action of iontophoretically applied L-glutamate on an insect visceral muscle

1) lontophoretic application of L-glutamate was employed to study the distribution of glutamate receptors in the superior longitudinal (SL) muscles of the locust (Locusta migratoria) hindgut, in which spontaneous activity was inhibited using normal saline containing 5 mM MgCl_2. 2) Junctional glutamate potentials with a rise time of 50–100 ms (peak) and a decay time of 250–400 ms were recorded at localized sites using ejection pulses in the range 5–10 nC. Most active sites were found in interfiber clefts and were spaced at about 250–300 μm intervals. 3) Desensitization of glutamate receptors occurred using ejection frequencies > 0.2 Hz. Desensitization could be irreversibly blocked using the lectin concanavalin A. 4) Depolarizing (D-) and biphasic depolarizing/hyperpofarizing (DH -) extrajunctional glutamate potentials were observed using ejection pulses > 15 nC. 5) δ-Philanthotoxin (δ-PTX) at concentrations > 0.3 Uml^?1 inhibited junctional glutamate potentials in a dose-dependent manner, 50% inhibition was achieved using 0.45 Uml^?1 δ-PTX. 6) Subthreshold concentrations of proctolin (up to 5 × 10^?10M) had no visible effect on glutamate potentials, suggesting that proctolin possibly does not act by modulating glutamate activity. 7) It is proposed that glutamate plays a transmitter role in SL muscles, while the role of proctolin is still unclear.     

Excitatory Amino Acids Stimulate Inositol Phospholipid Hydrolysis and Reduce Proliferation in Cultured Astrocytes

Excitatory amino acids stimulated inositol phospholipid hydrolysis in primary cultures of astrocytes, as reflected by an increased formation of [3H]inositol monophosphate [( 3H]InsP) in the presence of 10 mM Li+. Quisqualate was the most potent activator of inositol phospholipid hydrolysis, followed by glutamate and ibotenate. Kainate exhibited low activity, whereas N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) were inactive. The increase in [3H]InsP formation induced by glutamate was potentiated after 12-h exposure to the proliferative agent epidermal growth factor (EGF), suggesting that activation of the mitotic cycle leads to an enhanced coupling of glutamate recognition sites with phospholipase C. To study how glutamate receptors are involved in regulating cell proliferation, we have measured [methyl-3H]thymidine incorporation in cultured astrocytes. Excitatory amino acids reduced thymidine incorporation with a pharmacological profile similar to that observed for the stimulation of inositol phospholipid hydrolysis. Quisqualate acted as a potent antiproliferative agent, both under basal conditions and in cells stimulated to proliferate by addition of EGF or phorbol 12-tetradecanoate 13-acetate. Glutamate and ibotenate reduced [methyl-3H]thymidine incorporation at high concentrations, whereas kainate, AMPA, and NMDA were virtually inactive. The action of quisqualate on both inositol phospholipid hydrolysis and thymidine incorporation was attenuated by 2-amino-4-phosphonobutyrate, which acted as a weak agonist/competitive antagonist. Other excitatory amino acid receptor antagonists were not effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative pharmacological actions of leucokinins V-VIII on the visceral muscles of Leucophaea maderae

1. Leucokinins V-VIII (Lem-K-V to VIII) did not activate visceral muscles of the cockroach Leucophaea maderae uniformly as a group but rather showed a selective action on the muscles of the hindgut. This organ showed a contractile response to all of the leucokinins at 3 x 10(-10) M that was 2-20% above the mean level of spontaneous activity. The maximum response for each peptide was recorded at 2.1 x 10(-7) M. 2. Both the foregut and the oviduct were 100- to 1000-fold less sensitive than the hindgut, and each of the former required more than 10(-8) M to elicit a detectable excitation. The heart, by comparison, did not respond to any of these peptides. 3. The leucokinins caused a protracted excitation of contractile events in the hindgut that lasted for more than 60 min. Moreover, all four peptides evoked contractions from hindguts after membrane depolarization with 158 mM potassium. These results suggest that nonsynaptic receptors for the peptides exist in visceral muscle.     

In vivo modulation of extracellular hippocampal glutamate and GABA levels and limbic seizures by group I and II metabotropic glutamate receptor ligands

The effects of several metabotropic receptor (mGluR) ligands on baseline hippocampal glutamate and GABA overflow in conscious rats and the modulation of limbic seizure activity by these ligands were investigated. Intrahippocampal mGluR group I agonist perfusion via a microdialysis probe [1 mm (R,S)-3,5-dihydroxyphenylglycine] induced seizures and concomitant augmentations in amino acid dialysate levels. The mGlu1a receptor antagonist LY367385 (1 mm) decreased baseline glutamate but not GABA concentrations, suggesting that mGlu1a receptors, which regulate hippocampal glutamate levels, are tonically activated by endogenous glutamate. This decrease in glutamate may contribute to the reported LY367385-mediated anticonvulsant effect. The mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (50 mg/kg) also clearly abolished pilocarpine-induced seizures. Agonist-mediated actions at mGlu2/3 receptors by LY379268 (100 microm, 10 mg/kg intraperitoneally) decreased basal hippocampal GABA but not glutamate levels. This may partly explain the increased excitation following systemic LY379268 administration and the lack of complete anticonvulsant protection within our epilepsy model with the mGlu2/3 receptor agonist. Group II selective mGluR receptor blockade with LY341495 (1-10 microm) did not alter the rats' behaviour or hippocampal amino acid levels. These data provide a neurochemical basis for the full anticonvulsant effects of mGlu1a and mGlu5 antagonists and the partial effects observed with mGlu2/3 agonists in vivo.     

Octopamine action on the spontaneous contractions of the isolated nerve cord of Lumbricus terrestris

Octopamine and synephrine were observed to effect the spontaneous rhythmic contractions displayed by the isolated ventral nerve cord of the earthworm, Lumbricus terrestris. octopamine and synephrine produced dose-dependent significant changes in the frequency, amplitude and basal tonus of the spontaneous contractions. Application of adrenergic receptor antagonists suggested the octopamine receptors to have some similarity to vertebrate alpha 1-adrenergic receptors. The spontaneous contractions were not abolished by tetrodotoxin (TTX) which suggested a myogenic origin for the contraction of the ventral nerve cord sheath muscles. Octopamine, in the presence of TTX, increased the basal tonus and maximum force of the spontaneous contractions.     

Pharmacological actions of a new class of neuropeptides, the leucokinins I-IV, on the visceral muscles of Leucophaea maderae

1. Leucokinins I-IV did not activate visceral muscles uniformly as a class but rather showed a selective action on the muscles of the hindgut. This organ showed a contractile response to all of the leucokinins at 3 x 10(-10) M that was 5-10% above the mean level of spontaneous activity. The maximum response for each peptide was recorded at 2.1 x 10(-7) M. 2. Both the foregut and the oviduct were 100-1000 fold less sensitive than the hindgut, and each of the former organs required more than 10(-8) M to elicit a detectable excitation. The heart, by comparison, failed to give consistent responses with any of the peptides. 3. The leucokinins caused a protracted excitation of contractile events in the hindgut that lasted for more than 60 min. Moreover, all four peptides evoked contractions from hindguts after membrane depolarization with 158 mM potassium. 4. This result shows that nonsynaptic receptors for the peptides exist in visceral muscle. The leucokinins showed no evidence of facilitating the reentry of calcium into calcium depleted hindgut preparations.     

Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu>quisqualate (quis)=ibotenate (ibo)>(1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg²⁺). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1_S,3_R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg²⁺, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg²⁺-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms). Thus, in cerebellar granule cells EAAs elicit PPI hydrolysis by acting at two distinct receptor types: (i) metabotropic Glu receptors (mGluR), with pharmacological characteristics suggesting the expression of a unique mGluR receptor that shows certain similarities to those observed for the mGluR1 subtype (Aramori and Nakanishi, 1992) and (ii) NMDA receptors. The physiological agonist, Glu, is able to stimulate both receptor classes.Abbreviations ACPD (1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - AP₃ L-2-amino-3-phosphono-propionate - AP₅ D-2-amino-5-phosphonopentenoate - BMAA -methyl-amino-L-alanine - DIV days in vitro - DNOX 6,7-dinitroouinoxoline-2,3-dione - EAA excitatory amino acids - Glu glutamate - InsP inositol monophosphate - mGluR metabotropic glutamate receptors - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohept-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate - PPI polyphosphoinositide - quis quisqualate     

GABA and glutamate receptors are involved in modulating pacemaker activity in hydra

The effects of gamma-amino butyric acid (GABA) and glutamate, their ionotropic agonists and antagonists on hydra's ectodermal and endodermal pacemaker systems were studied. GABA decreased ectodermal body contraction bursts (CBs) and the number of pulses in a burst (P/CB) and endodermal rhythmic potentials (RPs); tentacle pulses (TPs) were not affected. The GABA(A) agonist, muscimol, and the benzodiazepine receptor agonist, diazepam, mimicked the effects of GABA on the endodermal system. The GABA(A) antagonist bicuculline counteracted GABA's effects. Low concentrations of glutamate increased CBs and RPs. Higher concentrations required concanavalin A (Con A) to produce the same effect on CBs and P/CB. TPs were increased by high concentrations of glutamate and kainate. The ionotropic glutamate agonist, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) also required Con A to increase CBs and RPs. The effects of AMPA were antagonized by 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), which, per se, decreased CBs. The results indicate that GABA and glutamate, acting on their ionotropic receptors, modify the impulses of hydra's pacemaker systems. On the whole GABA decreased the outputs of both ectodermal and endodermal impulse generating systems, while glutamate increased them.     

κ Receptor Activation Attenuates l-trans-Pyrrolidine-2,4-Dicarboxylic Acid-Evoked Glutamate Levels in the Striatum

Abstract: The effects of local κ receptor activation and blockade on extracellular striatal glutamate levels evoked by reverse microdialysis of l - trans -pyrrolidine-2,4-dicarboxylic acid ( l - trans -PDC) were investigated. l - trans -PDC elevates extracellular glutamate levels in vivo by acting as a competitive substrate for plasma membrane excitatory amino acid transporters. The selective κ-opioid receptor agonist U-69593 (1-100 n M ) significantly attenuated l - trans -PDC-stimulated glutamate levels in a concentration-dependent manner. The selective κ receptor antagonist nor -binaltorphimine (1-100 n M ) reversed the U-69593-induced decrease in l - trans -PDC-evoked glutamate levels also in a concentration-dependent manner, indicating that the U-69593-induced reduction was mediated by κ receptor activation. In addition, nor -binaltorphimine significantly elevated basal extracellular glutamate levels, implying that κ receptors tonically regulate glutamate efflux in the striatum. Previous data from this laboratory have shown that l - trans -PDC-evoked extracellular glutamate levels are partially calcium-sensitive. The present study demonstrated that the inhibition of l - trans -PDC-evoked glutamate levels by reduced calcium perfusion was not altered by U-69593. Therefore, κ receptors regulate the calcium-dependent component of l - trans -PDC-evoked extracellular glutamate levels in the striatum.     

Proctolin: a peptide transmitter candidate in insects.

The slow, striated muscles of the proctodeum (hindgut) of the cockroach, $\text{Periplaneta americana}$ (L.), were examined pharmacologically with reference to the responses evoked by nerve stimulation, glutamate, 5-HT, and proctolin, a myotropic peptide from $\text{Periplaneta}$ recently isolated and identified. The graded contractions evoked by repetitive nerve stimulation were simulated by 5-HT and proctolin at threshold concentrations of about 10⁻⁷ and 10⁻⁹ M respectively; responses to glutamate (∼10⁻⁴ M) were not similarly graded. The 5-HT receptors are distinct from other receptors, including the post-synaptic receptors, since they were specifically blocked by bromolysergic acid diethylamide. Proctolin was fully active on TTX-treated or surgically denervated muscle indicating that the proctolin receptors are located on the muscle fibre membrane. Tyramine, at threshold levels 5×10⁻⁸ M, reversibly antagonized the responses evoked by proctolin and by nerve stimulation but was without effect on the 5-HT and glutamate responses. Neurally evoked responses were potentiated by subthreshold concentrations of proctolin but not by glutamate. Pharmacologically, the proctolin and post-synaptic receptors appear to be identical and distinct from the glutamate and 5-HT receptors. Since proctolin is known to be a constituent of an efferent pathway of the proctodeal nerves, the evidence suggests that it may function as an excitatory transmitter substance. Peptidergic transmission is discussed in relation to the ultrastructural organization of the proctodeal nerve terminals which contain neurosectory granules in addition to electron-lucent, synaptic vesicles.     

Glutamate stimulates the phosphorylation of glial fibrillary acidic protein in slices of immature rat hippocampus via a metabotropic receptor

Phosphorylation of the astrocyte cell marker glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats (10–16 days postnatal) was strongly stimulated by glutamate in the presence of Ca²⁺. This effect apparently occurred via a metabotropic receptor since the specific agonist of metabotropic glutamate receptors, 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), stimulated GFAP phosphorylation by 173% whilst the mixed agonists, ibotenate and quisqualate, stimulated to a lesser extent. Ionotropic agonists were mainly ineffective. The action of 1S,3R-ACPD was blocked by (+)-2-amino-3-phosphonopropionic acid ( -AP₃) a specific antagonist of the metabotropic glutamate receptor coupled to the hydrolysis of phosphoinositides and was reduced by 70% by preincubation of the slices with pertussis toxin. In contrast to these results with immature animals glutamate had little or no effect on the phosphorylation of GFAP in hippocampal slices from adult rats.     

Role of Group III Metabotropic Glutamate Receptors in Excitotoxin-Induced Cerebellar Granule Cell Death

Abstract: The neuronal effects of the metabotropic glutamate receptor agonist (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid have been studied in cultured rat cerebellar granule cells, and compared with those of the endogenous excitotoxin glutamate, and the dietary excitotoxin β- N -methylamino- l -alanine. Glutamate, β- N -methylamino- l -alanine, and (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid all caused concentration-dependent cerebellar granule cell death over a 24-h exposure period. The metabotropic antagonist ( RS )-α-methyl-4-carboxyphenylglycine reduced glutamate-, β- N -methylamino- l -alanine-, and (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid-induced death by 50, 37, and 90%, respectively. (1 S ,3 R )-Aminocyclopentane-1,3-dicarboxylic acid-induced death was unaffected by the group I antagonist ( RS )-1-aminoindan-1,5-dicarboxylic acid, increased by the group II antagonist ethylglutamic acid, and markedly decreased by the group III antagonist ( RS )-α-methylserine- O -phosphate. Neither (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid nor the group I agonist ( RS )-3,5-dihydroxyphenylglycine caused an increase in intracellular free calcium levels. The group III agonist l -(+)-2-amino-4-phosphonobutyric acid also induced concentration-dependent cerebellar granule cell death, and so it was suggested that the group III metabotropic glutamate receptors were responsible for (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid-induced death. Blocking these receptors with ( RS )-α-methylserine- O -phosphate also prevented a proportion of glutamate- and β- N -methylamino- l -alanine-induced death.     

Excitatory neurotransmitters in the tentacle flexor muscles responsible for space positioning of the snail olfactory organ

Recently, three novel flexor muscles (M1, M2 and M3) in the posterior tentacles of the snail have been described, which are responsible for the patterned movements of the tentacles of the snail, Helix pomatia. In this study, we have demonstrated that the muscles received a complex innervation pattern via the peritentacular and olfactory nerves originating from different clusters of motoneurons of the cerebral ganglia. The innervating axons displayed a number of varicosities and established neuromuscular contacts of different ultrastructural forms. Contractions evoked by nerve stimulation could be mimicked by external acetylcholine (ACh) and glutamate (Glu), suggesting that ACh and Glu are excitatory transmitters at the neuromuscular contacts. Choline acetyltransferase and vesicular glutamate transporter immunolabeled axons innervating flexor muscles were demonstrated by immunohistochemistry and in Western blot experiments. Nerve- and transmitter-evoked contractions were similarly attenuated by cholinergic and glutamatergic antagonists supporting the dual excitatory innervation. Dopamine (DA, 10^?5 M) oppositely modulated thin (M1/M2) and thick (M3) muscle responses evoked by stimulation of the olfactory nerve, decreasing the contractions of the M1/M2 and increasing those of M3. In both cases, the modulation site was presynaptic. Serotonin (5-HT) at high concentration (10^?5 M) increased the amplitude of both the nerve- and the ACh-evoked contractions in all muscles. The relaxation rate was facilitated suggesting pre- and postsynaptic site of action. Our data provided evidence for a DAergic and 5-HTergic modulation of cholinergic nerves innervating flexor muscles of the tentacles as well as the muscles itself. These effects of DA and 5-HT may contribute to the regulation of sophisticated movements of tentacle muscles lacking inhibitory innervation.     

Some pharmacological properties of neuromuscular transmission in the oviduct of the locust,Locusta migratoria

The effects of various pharmacological agents on neurally evoked contractions of the visceral muscles of the oviduct of Locusta migratoria have been examined. The pentapeptide, proctolin, at low concentrations (10^?11 M?10^?10 M), induced an increase in the amplitude of neurally evoked contractions and basal tonus, and induced the appearance and increased the frequency of myogenic contractions. Glutamate, at 10^?4 M, produced a small transient contraction which in some preparations was accompanied by a reduction in amplitude of neurally evoked contractions. Octopamine, at 10^?6 M, reduced the amplitude of neurally evoked contractions and also resulted in a relaxation of the muscles. The octopaminergic effects were inhibited by the α-aminergic antagonist phentolamine. Neurally evoked contractions were unaffected by dopamine, 5-HT or the acetylcholine receptor antagonists atropine and hexamethonium. Acetylcholine increased the amplitude of neurally evoked contractions, but only at the high concentration of 10^?3 M. The possible role of proctolin and glutamate as excitatory neuro-transmitters and the inhibitory action of octopamine is discussed.     

Effects of a hydroxy-cinnamoyl conjugate of spermidine on arthropod neuromuscular junctions

N1-coumaroyl spermidine is structurally similar to acylpolyamines found in spider and wasp venoms, which are known to block arthropod glutamate receptors. N1-coumaroyl spermidine reduced the amplitude of excitatory postsynaptic potentials recorded in crayfish muscle. This effect was dose dependent, with an IC50 value of 70 micromol l(-1). N1-coumaroyl spermidine reversibly reduced the amplitude of potentials elicited by iontophoretic application of L-glutamate, indicating a direct effect on postsynaptic glutamate receptors. Neither 1 mmol l(-1) spermidine nor 1 mmol l(-1) coumaric acid altered excitatory postsynaptic potential amplitude, indicating that blockage requires the conjugated phenolic polyamine. N1-coumaroyl spermine, a slightly longer phenolic polyamine, reduced excitatory postsynaptic potential amplitude with approximately the same potency as N1-coumaroyl spermidine. Thus, potency of blockage does not appear to be affected in this experimental preparation by small changes in length of the polyamine. N1-coumaroyl spermidine also reduced excitatory postsynaptic potentials in muscles of the insect Drosophila. The ability of N1-coumaroyl spermidine to attenuate synaptic transmission at insect neuromuscular synapses lends support to the notion that plant-derived phenolic polyamines might serve as natural insecticides.     

Lactate metabolism in resting and contracting canine skeletal muscle with elevated lactate concentration.

This study was undertaken to quantitatively account for the metabolic disposal of lactate in skeletal muscle exposed to an elevated lactate concentration during rest and mild-intensity contractions. The gastrocnemius plantaris muscle group (GP) was isolated in situ in seven anesthetized dogs. In two experiments, the muscles were perfused with an artificial perfusate with a blood lactate concentration of ~9 mM while normal blood gas/pH status was maintained with [U-(14)C]lactate included to follow lactate metabolism. Lactate uptake and metabolic disposal were measured during two consecutive 40-min periods, during which the muscles rested or contracted at 1.25 Hz. Oxygen consumption averaged 10.1 +/- 2.0 micromol. 100 g(-1). min(-1) (2.26 +/- 0.45 ml. kg(-1). min(-1)) at rest and 143.3 +/- 16.2 micromol. 100 g(-1). min(-1) (32.1 +/- 3.63 ml. kg(-1). min(-1)) during contractions. Lactate uptake was positive during both conditions, increasing from 10.5 micromol. 100 g(-1). min(-1) at rest to 25.0 micromol. 100 g(-1). min(-1) during contractions. Oxidation and glycogen synthesis represented minor pathways for lactate disposal during rest at only 6 and 15%, respectively, of the [(14)C]lactate removed by the muscle. The majority of the [(14)C]lactate removed by the muscle at rest was recovered in the muscle extracts, suggesting that quiescent muscle serves as a site of passive storage for lactate carbon during high-lactate conditions. During contractions, oxidation was the dominant means for lactate disposal at >80% of the [(14)C]lactate removed by the muscle. These results suggest that oxidation is a limited means for lactate disposal in resting canine GP exposed to elevated lactate concentrations due to the muscle's low resting metabolic rate.     

Suppression of cortical functional hyperemia to vibrissal stimulation in the rat by epoxygenase inhibitors

Application of glutamate to glial cell cultures stimulates the formation and release of epoxyeicosatrienoic acids (EETs) from arachidonic acid by cytochome P-450 epoxygenases. Epoxygenase inhibitors reduce the cerebral vasodilator response to glutamate and N-methyl-D-aspartate. We tested the hypothesis that epoxygenase inhibitors reduce the somatosensory cortical blood flow response to whisker activation. In chloralose-anesthetized rats, percent changes in cortical perfusion over whisker barrel cortex were measured by laser-Doppler flowmetry during whisker stimulation. Two pharmacologically distinct inhibitors were superfused subdurally: 1) N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), an epoxygenase substrate inhibitor; and 2) miconazole, a reversible cytochrome P-450 inhibitor acting on the heme moiety. Superfusion with 5 micromol/l MS-PPOH decreased the hyperemic response to whisker stimulation by 28% (from 25 +/- 9 to 18 +/- 7%, means +/- SD, n = 8). With 20 micromol/l MS-PPOH superfusion, the response was decreased by 69% (from 28 +/- 9% to 9 +/- 4%, n = 8). Superfusion with 20 micromol/l miconazole decreased the flow response by 67% (from 31 +/- 6% to 10 +/- 3%, n = 8). Subsequent superfusion with vehicle restored the response to 26 +/- 11%. Indomethacin did not prevent MS-PPOH inhibition of the flow response, suggesting that EET-related vasodilation was not dependent solely on cyclooxygenase metabolism of 5,6-EET. Neither MS-PPOH nor miconazole changed baseline flow, reduced the blood flow response to an adenosine A(2) agonist, or decreased somatosensory evoked potentials. The marked reduction of the cortical flow response to whisker stimulation with two different types of epoxygenase inhibitors indicates that EETs play an important role in the physiological coupling of blood flow to neural activation.