A concentration-dependent localization of octopamine-sensitive adenylate cyclase activity in locust skeletal muscle

Summary A concentration-dependent localization of octopamine-sensitive adenylate cyclase activity has been demonstrated in skeletal muscle of the locust, Schistocerca gregaria, using an histochemical technique. In the intermediate speed contracting muscle fibres from the fan region of the extensor-tibiae muscle of the locust hindleg, low concentrations of dl-octopamine (10^–8 M) induce reaction product preferentially in the sarcoplasmic reticular component of the dyads. At slightly higher concentrations (10^–7 and 10^–6 M) lower amounts of diffuse reaction product are also found in the non-dyad sarcoplasmic reticulum and at the sarcolemmal membrane, with occassional amounts of a less diffuse, punctate product in the transverse tubule (T-tubule) component of the dyads. At higher concentrations (10^–5 and 10^–3 M) the predominant product is the dense, plaque-like accumulations of reaction product in the T-tubule component of the dyads. The results are discussed in terms of the likely physiological significance of the accumulation of reaction product in these different locations.     

Histochemical localization of octopamine- and proctolin-sensitive adenylate cyclase activity in a locust skeletal muscle

A histochemical technique for the localization of adenylate cyclase activity has been applied to the extensor-tibiae muscle of the hindleg of the locust, Schistocerca gregaria to localise the sites of action of the modulatory compounds octopamine and proctolin. Octopamine-sensitive adenylate cyclase activity can be demonstrated in fast and intermediate type muscle fibres but not in the limited number of purely slow muscle fibres (3-6) in the fan region at the proximal end of the muscle. In contrast the latter fibres are the only ones in the muscle to exhibit proctolin-sensitive adenylate cyclase activity. In both cases the bulk of the reaction product is localised in the sarcoplasmic reticulum component of the dyads, with lesser amounts occurring beneath the sarcolemmal membrane, in the non-dyad sarcoplasmic reticulum and in the T-tubule system. The results are consistent with physiological data suggesting that proctolin, but not octopamine, mediates its effects on the myogenic rhythm of contraction and relaxation in this muscle by changing the levels of cyclic AMP in the small group of slow muscle fibres which act as the pacemaker for this rhythm.     

Histochemical localization of octopamine- and proctolin-sensitive adenylate cyclase activity in a locust skeletal muscle

Summary A histochemical technique for the localization of adenylate cyclase activity has been applied to the extensortibiae muscle of the hindleg of the locust, Schistocerca gregaria to localise the sites of action of the modulatory compounds octopamine and proctolin. Octopamine-sensitive adenylate cyclase activity can be demonstrated in fast and intermediate type muscle fibres but not in the limited number of purely slow muscle fibres (3–6) in the fan region at the proximal end of the muscle. In contrast the latter fibres are the only ones in the muscle to exhibit proctolinsensitive adenylate cyclase activity. In both cases the bulk of the reaction product is localised in the sarcoplasmic reticulum component of the dyads, with lesser amounts occurring beneath the sarcolemmal membrane, in the non-dyad sarcoplasmic reticulum and in the T-tubule system. The results are consistent with physiological data suggesting that proctolin, but not octopamine, mediates its effects on the myogenic rhythm of contraction and relaxation in this muscle by changing the levels of cyclic AMP in the small group of slow muscle fibres which act as the pacemaker for this rhythm.     

Acetylcholinesterase in membrane fractions derived from sarcotubular system of skeletal muscle: presence of monomeric acetylcholinesterase in sarcoplasmic reticulum and transverse tubule membranes

Microsomes were isolated from white rabbit muscle and separated into several fractions by centrifugation in a discontinuous sucrose density gradient. Four membrane fractions were obtained namely surface membrane, light, intermediate and heavy sarcoplasmic reticulum. The origin of these microsomal vesicles was investigated by studying biochemical markers of sarcoplasmic reticulum and surface and T-tubular membranes. The transverse tubule derived membranes were further purified by using a discontinuous sucrose density gradient after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. All membrane preparations displayed acetylcholinesterase activity (AChE, EC 3.1.1.7), this being relatively more concentrated in T-tubule membranes than in those derived from sarcoplasmic reticulum. The membrane-bound AChE of unfractioned microsomes notably increased its activity by aging, treatment with detergents and low trypsin concentrations indicating that the enzyme is probably attached to the membrane in an occluded form, the unconstrained enzyme displaying higher activity than the vesicular acetylcholinesterase.Sedimentation analysis of Triton-solubilized AChE from different membrane fractions revealed enzymic multiple forms of 13.5S, 9–10S and 4.5–4.8S, the lightest form being the predominant one in all membrane preparations. Therefore, in both sarcoplasmic reticulum and T-tubule membrane the major component of AChE appears to be a membrane-bound component, probably a G₁ form.     

Monoclonal antibody specific for the T-tubule of skeletal muscle

Monoclonal antibodies were raised against a triad-enriched (sarcoplasmic reticulum-T-tubule complex) microsomal membrane fraction of rabbit skeletal muscle. The avidin-biotin complex (ABC) immunoperoxidase staining method was used to screen hybrid colonies. Positive antibodies exhibited a granular doublet pattern at the A-I junction, consistent with the location of triads in rabbit muscle. One monoclonal antibody, M171, was further characterized by ultrastructural and immunoadsorption techniques. Postembedding electron immunocytochemistry was performed on tissue sections embedded in Lowicryl K4M. Goat anti-mouse immunoglobulin absorbed to 10 nm colloidal gold particles was used as an ultrastructural label. In these studies, M171 recognized an epitope at the triads and at periodic openings along the plasmalemma. Immunoadsorption on protein transfers of isolated sarcoplasmic reticulum, surface membrane (plasmalemma and T-tubule), and triad-enriched fractions showed that M171 reacts with a surface membrane component. Taken together, these studies suggest that M171 recognizes an epitope associated with the T-tubule at the triad and at the "mouth" of the T-system at the plasmalemma.     

NUCLEOSIDE PHOSPHATASE ACTIVITIES IN RAT CARDIAC MUSCLE

Localizations of aldehyde-resistant nucleoside phosphatase activities in frozen sections of rat cardiac muscle have been studied by electron microscopy. Activities are higher after fixation with formaldehyde than with glutaraldehyde. After incubation with adenosine triphosphate or inosine diphosphate at pH 7.2, reaction product is found in the "terminal cisternae" or "transverse sacs" of the sarcoplasmic reticulum, which, together with the "intermediary vesicles" (T system), constitute the "dyads" or "triads". Reaction product is also present at the membranes of micropinocytotic vacuoles which apparently form from the plasma membrane of capillary endothelial cells and from the sarcolemma. In certain regions of the intercalated discs, reaction product is found within the narrow spaces between sarcolemmas of adjacent cells and within micropinocytotic vacuoles that seem to form from the sarcolemma. With inosine diphosphate, reaction product is also found in other parts of the sarcoplasmic reticulum. After incubation with cytidine monophosphate at pH 5, reaction product is present in the transverse sacs of sarcoplasmic reticulum, in micropinocytotic vacuoles in capillary endothelium, and in lysosomes of muscle fibers and capillaries. The possible significance of the sarcoplasmic reticulum phosphatases is discussed in relation to the role the reticulum probably plays in moving calcium ions and thereby controlling contraction and relaxation of the muscle fiber.     

Calcium modulation of phosphoinositide kinases in transverse tubule vesicles from frog skeletal muscle

Highly purified transverse tubule membranes isolated from frog skeletal muscle phosphorylate phosphatidylinositol to phosphatidylinositol 4-phosphate and phosphatidylinositol (4,5)-bisphosphate. The two phosphorylation reactions have different calcium requirements. Phosphorylation of phosphatidylinositol to phosphatidylinositol 4-phosphate, which takes place in both isolated transverse tubules and sarcoplasmic reticulum membrane, is independent of calcium in a range of concentrations from 10(-9) to 10(-6) M, and is progressively inhibited to 10% of the maximal values by increasing calcium to 10(-4) M or higher (K0.5 = 5 X 10(-6) M). In contrast, phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol (4,5)-bisphosphate, a reaction exclusively present in transverse tubule membranes, is maximal at calcium concentrations higher than 2 X 10(-6) M and decreases to 30% of maximal values at calcium concentrations of 2 X 10(-7) M or lower (K0.5 = 10(-6) M). Unlike frog membranes, transverse tubules from rabbit muscle need exogenous phosphatidylinositol 4-phosphate in order to produce the bisphosphate derivative in the same range of calcium concentrations. Inositol (1,4,5)-trisphosphate has been proposed recently as a chemical messenger in excitation-contraction coupling in skeletal muscle. Calcium regulation of the synthesis of phosphatidylinositol (4,5)-bisphosphate, the membrane-bound precursor of inositol (1,4,5)-trisphosphate, might have physiological implications regarding modulation of excitation-contraction coupling by intracellular calcium levels.     

Effects of calcium ions and adenosine diphosphate on the activities of NAD+-linked isocitrate dehydrogenase from the radular muscles of the whelk and flight muscles of insects.

1. The activity of NAD+-linked isocitrate dehydrogenase from the radular muscle of the whelk is higher than those in many vertebrate muscles and only slightly lower than in the flight muscles of insects. The enzyme activity from the whelk (Buccinum undatum) is stable for several hours after homogenization of the radular muscle, whereas that from insect flight muscle is very unstable. Consequently, the enzyme from the whelk muscle is suitable for a systematic investigation of the effects of Ca2+ and ADP. 2. The sigmoid response of the enzyme activity to isocitrate concentration is markedly increased by raising the Ca2+ concentration from 0.001 to 10 muM, but it is decreased by ADP. The inhibitory effect of Ca2+ is most pronounced at pH7.1; it is not observed at pH 6.5. Similar effects are observed for the enzyme from the flight muscle of the locust (Schistocerca gregaria) and the water bug (Lethocerus cordofanus). The percentage activation by ADP of the enzyme from either the whelk or the insects is greater at 10 muM-Ca2+, and 50% of the maximum activation is obtained at 0.10 and 0.16 mM-ADP for the enzyme from whelk and locust respectively at this Ca2+ concentration. At 10 muM-Ca2+ in the absence of added ADP, the apparent Km for isocitrate is markedly higher than in other conditions. Ca2+ concentrations of 0.01, 0.1 and 0.2 muM cause 50% inhibition of maximum activity of the enzyme from the muscles of the whelk, locust and water bug respectively. 3. Recent work has indicated that mitochondria may play a complementary role to the sarcoplasmic reticulum in the control of the distribution of Ca2+ in muscle. The opposite effects of Ca2+ on the activities of isocitrate dehydrogenase and mitochondrial glycerol phosphate dehydrogenase from muscle tissue are consistent with the hypothesis that changes in the intracellular distribution of Ca2+ control the activities of these two enzymes in order to stimulate energy production for the contraction process in the muscle. Although both enzymes are mitochondrial, glycerol phosphate dehydrogenase resides on the outer surface of the inner membrane and responds to sarcoplasmic changes in Ca2+ concentration (i.e. an increase during contraction), whereas the isocitrate dehydrogenase resides in the matrix of the mitochondria and responds to intramitochondrial concentrations of Ca2+ (i.e. a decrease during contraction). It is suggested that changes in intramitochondrial Ca2+ concentrations are primarily responsible for regulation of the activity of NAD+-isocitrate dehydrogenase in order to control energy formation for the contractile process. However, when the muscle is at rest, changes in intramitochondrial concentrations of ADP may regulate energy formation for non-contractile processes.     

T-tubule depolarization-induced SR Ca2+ release is controlled by dihydropyridine receptor- and Ca(2+)-dependent mechanisms in cell homogenates from rabbit skeletal muscle

In vertebrate skeletal muscle, the voltage-dependent mechanism of rapid sarcoplasmic reticulum (SR) Ca2+ release, commonly referred to as excitation-contraction (EC) coupling, is believed to be mediated by physical interaction between the transverse (T)-tubule voltage-sensing dihydropyridine receptor (DHPR) and the SR ryanodine receptor (RyR)/Ca2+ release channel. In this study, differential T-tubule and SR membrane monovalent ion permeabilities were exploited with the use of an ion-replacement protocol to study T-tubule depolarization-induced SR 45Ca2+ release from rabbit skeletal muscle whole-cell homogenates. Specificity of Ca2+ release was ascertained with the use of the DHPR antagonists D888, nifedipine and PN200-110. In the presence of the "slow" complexing Ca2+ buffer EGTA, homogenates exhibited T-tubule depolarization-induced Ca2+ release comprised of an initial rapid phase followed by a slower release phase. During the rapid phase, approximately 20% of the total sequestered Ca2+ (approximately 30 nmol 45Ca2+/mg protein), corresponding to 100% of the caffeine-sensitive Ca2+ pool, was released within 50 ms. Rapid release could be inhibited fourfold by D888. Addition to release media of the "fast" complexing Ca2+ buffer BAPTA, at concentrations > or = 4 mM, nearly abolished rapid Ca2+ release, suggesting that most was Ca2+ dependent. Addition of millimolar concentrations of either Ca2+ or Mg2+ also greatly reduced rapid Ca2+ release. These results show that T-tubule depolarization-induced SR Ca2+ release from rabbit skeletal muscle homogenates is controlled by T-tubule membrane potential- and by Ca(2+)- dependent mechanisms.     

Single glutamate gated channels in insect visceral muscle

Single glutamate-gated ion channels with a conductance of 135 pS are demonstrated in tonic muscle fibres of the locust hindgut. Channel kinetics closely resemble those of glutamatergic channels in locust skeletal muscles. Glutamate concentrations increasing within the range from 5 X 10(-5) to 1 X 10(-3) M result in an increase of the frequency of channel opening and a decrease in channel closed times. Delta-philanthotoxin, a toxin isolated from the venom of the digger wasp Philanthus triangulum, inhibits channel activity by blocking open channels and increasing channel closed times.     

Inhibition of the activity of membrane-bound Ca2+-ATPase in the sarcoplasmic reticulum fragments of rabbit skeletal muscles by the alkaloid sanguinarine

Sanguinarine, a plant DNA-intercalator, is shown to inhibit the enzyme activity of the membrane-bound Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum fragments. This inhibition could be interpreted by the well known ability of this alkaloid to interact with sulphydryl groups of the enzymes. Sanguinarine is a weaker inhibitor of this reaction than a sulphydryl group poison Ag+. The I50 is 3.10(-6) M for Ag+ and 7.10(-5) M for sanguinarine in the reaction medium with NO3- substituted for Cl-. In the standard reaction medium containing Cl-, the I50 for sanguinarine is 1.8.10(-4) M. In this case sanguinarine activates Ca2+-ATPase at low concentrations presumably because of uncoupling ATP hydrolysis from Ca2+ transport through membrane. Other agents studied are: DNA-intercalators--ethidium bromide, acriflavine, acridine orange; DNA-complexing antibiotics--actinomycin D, and olivomycin, alkaloids, quinine, morphine, berberine and an uncoupler of oxidative phosphorylation 2,4-dinitrophenol. These were found not to inhibit Ca2+-ATPase activity up to the concentrations of 10(-3)-10(-4) M.     

Properties of a phosphorylated intermediate of the Ca,Mg-activated ATPase of microsomal vesicles from uterine smooth muscle

A phosphorylated intermediate of the CaMg-ATPase is demonstrated in microsomal preparations from uterine smooth muscle. Characterization included the use of activators, inhibitors, and sodium dodecyl sulfate (SDS)-gel electrophoresis. The phosphorylation was a function of the ATP and Ca concentrations. The dissociation constant KATP was 2.7 X 10(-6) M and KCa was 1.7 X 10(-6) M. Mg was obligatory for the reaction. Na azide, ouabain, or the substitution of NaCl for KCl did not affect the reaction. Phosphorylation was inhibited by Salyrgan, ADP, or 20 mM calcium. SDS-polyacrylamide gel electrophoresis at pH 2.4 demonstrated phosphorylation of predominantly one protein with a molecular weight of 100,000. Hydroxylamine and, to a lesser extent, neutral and alkaline pH caused dephosphorylation. This indicates the presence of an acylphosphate bond in the phosphoprotein. The above findings are consistent with the phosphorylated intermediate being a Ca,Mg-ATPase. The inhibition by 20 mM calcium indicates that the Ca,Mg-ATPase of smooth muscle differs from that of striated muscle sarcoplasmic reticulum.     

Sequential docking, molecular differentiation, and positioning of T-Tubule/SR junctions in developing mouse skeletal muscle.

Skeletal muscle Ca(2+) release units (CRUs) are junctions of the surface membrane/T-tubule system and the sarcoplasmic reticulum (SR) that function in excitation-contraction coupling. They contain high concentrations of dihydropyridine receptors (DHPRs) in the T-tubules and of ryanodine receptors (RyR) in the SR and they are positioned at specific locations in the sarcomere. In order to characterize the sequence of developmental steps leading to the specific molecular and structural organization of CRUs, we applied a range of imaging techniques that allowed us to follow the differentiation of the membrane compartments and the expression of junctional proteins in developing mouse diaphragm muscle. We find that docking of the two membrane systems precedes the incorporation of the RyRs into the junctions, and that T-tubule/SR junctions are formed and positioned at the I-A interface at a stage when the orientation of T-tubule is predominantly longitudinal. Thus, the sequence of developmental events is first the docking of T-tubules and SR, secondly the incorporation of RyR in the junctions, thirdly the positioning of the junctions in the sarcomere, and only much later the transverse orientation of the T-tubules. These sequential stages suggests an order of inductive processes for the molecular differentiation and structural organization of the CRUs in skeletal muscle development.     

Spectrophotometric and fluorometric assay of superoxide ion using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole

Two highly sensitive spectrophotometric methods are developed and described for the measurement of superoxide ion radical derived from KO2 as well as O2*- generated either from the xanthine-xanthine oxidase reaction or by the addition of nicotinamide adenine dinucleotide (NADH) to skeletal muscle sarcoplasmic reticulum (SR) vesicles. These methods allow quantification of superoxide ion concentration by monitoring its reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), either by recording absorbance of the final reaction product at a wavelength of 470 nm or by measuring its fluorescence emission intensity at 550 nm using an excitation wavelength of 470 nm. The extinction coefficient of the active product was determined to be 4000 M(-1) cm(-1). A lower limit second-order bimolecular rate constant of 1.5+/-0.3x10(5) M(-1) s(-1) was estimated from kinetic stopped-flow analysis for the reaction between NBD-Cl and KO2. A plot of absorbance versus concentration of superoxide was linear over the range 2 to 200 microM KO2, whereas higher sensitivities were obtained from fluorometric measurements down into sub-micromolar concentrations with a limit of detection of 100 nM KO2. This new spectrophotometric assay showed higher specificity when compared with some other commonly used methods for detection of superoxide (e.g., nitroblue tetrazolium). Results presented showed good experimental agreement with rates obtained for the measurement of superoxide ion when compared with other well-known probes such as acetylated ferri cytochrome c and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT). A detailed discussion of the advantages and limitations of this new superoxide ion probe is presented.     

Conditions of spontaneous gelatinization of sarcoplasmic proteins]

Ca2+ ions at low concentration (10(-4)-10(-5) M) when added to ultracentrifugates of sarcoplasmic proteins with minimal content of sarcoplasmic reticulum fragments completely prevent protein gelatinization or gels, formed in these conditions, quickly dilute. Ca2+ ions at the concentration of 10(-3) M prevented in these conditions gelatinization in all the cases studied. Strong gels of sarcoplasmic proteins are formed afterwards in weak acid (pH 6.1-6.75), neutral (pH 7.0) and weak alkaline (pH 7.25-7.45 and higher) media. It is suggested that gelatinization of sarcoplasmic proteins is closely related with the development of plastic tonus, obturatory muscle function and viscous after-effect.     

Rapid calcium release from the isolated sarcoplasmic reticulum is triggered via the attached transverse tubular system

Rapid replacement of 0.15 M K gluconate with 0.15 M choline Cl led to multiphasic Ca2+ release from a heavy fraction of rabbit skeletal muscle microsomes. Following the initial lag period (0-50 ms), about 15 nmol of Ca2+/mg of protein was rapidly released with first-order rate constants k = 60-140 s-1. Subsequently, a larger amount of Ca2+ (up to 56 nmol/mg) was released at a slower rate (k = 0.8-1.5 s-1). The Ca2+ released in both rapid and slow phases was reaccumulated within 60 s. In agreement with a previous report (Caswell, A. H., Lau, Y. H., Garcia, M., and Brunschwig, J-P. (1979) J. Biol. Chem. 254, 202-208), French press treatment of the tubule/sarcoplasmic reticulum (SR) complex results in dissociation of transverse tubular membrane (T-tubules) from SR. Subsequent incubation with 0.4 M potassium cacodylate results in the reassociation of the complex, as shown by sucrose density-gradient sedimentation. Upon T-tubule dissociation, both rapid and slow Ca2+ release was inhibited. Upon reassociation, the rapid Ca2+ release was completely restored and the slow phase partially restored. The results indicate that the T-tubule associated with SR plays a crucial role in triggering rapid Ca2+ release induced by ionic replacement. Other types of Ca2+ release, e.g. those induced by Ca2+ alone or with drugs such as caffeine and quercetin, are unaffected by T-tubule dissociation, and hence produced by direct stimulation of the SR membrane.     

Interaction between Paranosema locustae and Metarhizium anisopliae var. acridum, two pathogens of the desert locust, Schistocerca gregaria under laboratory conditions

The interaction between two pathogens, the microsporidian Paranosema locustae Canning and the fungus Metarhizium anisopliae var. acridum Driver and Milner was studied under laboratory conditions in an attempt to develop an improved method of microbial control for the desert locust, Schistocerca gregaria Forsk?l. Fifth-instar locust nymphs, reared in the laboratory, were treated with various concentrations of one of the two pathogens or with both pathogens. The numbers of locusts killed were recorded each day and the production of pathogen spores within the dead locusts was assessed at the end (day 21) of each experiment. Locust nymphs treated with both P. locustae and M. anisopliae died sooner than nymphs infected with only one of the pathogens. At the lower concentrations of pathogen tested, the effects of the two pathogens were additive. At the higher concentrations the combined effects were synergistic. In terms of locust mortality, there was no evidence of any antagonistic effects between the two pathogens. However, the production of spores by P. locustae was reduced considerably when the host insects were infected also with M. anisopliae. For example, nymphs treated initially with P. locustae and then treated 3 and 10 days later with M. anisopliae produced 3-20 times and 2.5-8 times fewer spores, respectively, than nymphs treated only with P. locustae. Hence, in areas where M. anisopliae is applied, the natural persistence of P. locustae in the local grasshopper and locust populations may be diminished.     

Synthesis of polyphosphoinositides in transverse tubule and sarcoplasmic reticulum membranes of frog skeletal muscle.

Synthesis of polyphosphoinositides has been studied in transverse (T-) tubule and sarcoplasmic reticulum (SR) membrane fractions of frog skeletal muscle, following 32P-labeling with [gamma-32P]ATP. Purified SR and T-tubule fractions respectively synthesize 9.4 +/- 0.8 and 71.9 +/- 9.8 pmol PtdInsP/mg per min, indicating nearly 8-fold higher activity of PtdIns kinase in the T-tubules than in the SR. The activity of this enzyme in both membrane systems is maximum at pH 7 and pCa 6. PtdInsP2 is synthesized from the endogenous PtdInsP, only in T-tubule membranes by the action of PtdInsP kinase. This lipid is the most intensely 32P-labeled phosphoinositide (181.7 +/- 9.2 pmol/mg per min) in these membranes. PtdIns kinase in the T-tubule and SR membranes, and PtdInsP kinase in the former are modulated by the free [Mg2+]. Loss of radiolabel from transiently maximal 32P-incorporation in polyphosphoinositides in T-tubule membranes, concomitant with a decrease in the ATP concentration in the incubation buffer, shows the occurrence of phosphoinositidases in these membranes. Under the conditions used, no such activities were evident in SR membranes. Compound 48/80, a mixture of condensation products of N-methyl-p-methoxyphenethylamine with formaldehyde, known to block phosphoinositidase C and phospholipase A2, causes a dose-dependent increase in the 32P-label of PtdInsP, in T-tubule membranes. The synthesis of lyso PtdInsP2, a deacylated form of PtdInsP2 which occurs in nearly equal quantities in both T-tubule and SR membranes, may result from a mechanism independent of phospholipase A2.     

Evidence for a possible neurotransmitter/neuromodulator role of tyramine on the locust oviducts

Visualization of the tyraminergic innervation of the oviducts was demonstrated by immunohistochemistry, and the presence of tyramine was confirmed using high-performance liquid chromatography coupled to electrochemical detection. Oviducts incubated in high-potassium saline released tyramine in a calcium-dependent manner. Stimulation of the oviducal nerves also resulted in tyramine release, suggesting that tyramine might function as a neurotransmitter/neuromodulator at the locust oviducts. Tyramine decreased the basal tension, and also attenuated proctolin-induced contractions in a dose-dependent manner over a range of doses between 10(-7) and 10(-4) M. Low concentrations of tyramine attenuated forskolin-stimulated cyclic AMP levels in a dose-dependent manner. This effect was not blocked by yohimbine. High concentrations of tyramine increased basal cyclic AMP levels of locust oviducts in a dose-dependent manner; however, the increases in cyclic AMP were only evident at the highest concentrations tested, 5 x 10(-5) and 10(-4) M tyramine. The tyramine-induced increase in cyclic AMP shared a similar pharmacological profile with the octopamine-induced increase in cyclic AMP. Tyramine increased the amplitude of excitatory junction potentials at low concentrations while hyperpolarizing the membrane potential by 2-5 mV. A further increase in the amplitude of the excitatory junction potentials and the occurrence of an active response was seen upon washing tyramine from the preparation. These results suggest that tyramine can activate at least three different endogenous receptors on the locust oviducts a putative tyramine receptor at low concentrations, a different tyramine receptor to inhibit muscle contraction, and an octopamine receptor at high concentrations.     

Experimental studies upon a bundle of tonic fibres in the locust extensor tibialis muscle

The bundle of tonic fibres situated at the proximal end of the locust metathoracic extensor tibialis muscle is innervated by the dorsal unpaired median neurone (DUMETi) as well as by the slow excitatory (SETi)) and common inhibitor (CI) neurones. It is not innervated by the fast excitatory neurone (FETi).These fibres contract spontaneously and rhythmically. The myogenic rhythm can be modified by neural stimulation.Spontaneous slow depolarizing potentials resembling the pacemaker potentials of insect cardiac muscle were demonstrated in these fibres.The actions of glutamate on the tonic muscle fibres are not compatible with its being a specific excitatory transmitter. Glutamate can stimulate weak contractions of the muscle, but this action is inhibited when chloride ions are removed from the saline.10^?6 M Octapamine hyperpolarizes the tonic fibre membrane. Octopamine, GABA and glutamate all inhibit the myogenic contractions and reduce the force of the neurally evoked contractions.The tonic muscle is very responsive to proctolin. At 5 × 10^?11 M proctolin enhances the force and increases the frequency of myogenic contractions. At 10^?9 M it depolarizes the muscle membrane potential, and at that and higher concentrations it causes the muscle to contract. At 2 × 10^?7 M proctolin induces contractures which resemble those evoked by sustained high-frequency neural stimulation. Iontophoretic experiments show that proctolin receptors occur at localized sites on the tonic fibre membrane.