Functional membrane vesicles from the nervous system of insects (original) (raw)
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Density of sodium channels in insect synaptic nerve endings
Neurochemistry International, 1990
Specific binding of [ll-3H]saxitoxin (STX) and activity of ouabain sensitive adenosinetriphosphatase (Na ÷, K+-ATPase) were determined in neuronal membrane fractions using a subcellular preparation from the central nervous system of the cockroach Periplaneta americana. The nerve ending fractions (synaptosomes) contained 90-95% of the total specific activity of ouabain sensitive Na +, K +-ATPase, and 60-70% of specific STX binding of the crude nerve homogenate. Sodium influx induced by veratridine in synaptosomes was inhibited by saxitoxin at a half-maximal concentration of 4 nM, and kinetics were consistent with reversible binding of one molecule of saxitoxin to each sodium channel receptor site, with an equilibrium dissociation constant (Ko) of approx. 3 nM. The density of saturable binding sites was 2 pmol/mg protein which was estimated to correspond to about 95 binding sites per/tm 2 of synaptic membrane. The results of transport and binding data show that insect synaptosomes possess the capability to conduct inward sodium currents at least comparable to those found in other neuronal membranes, and thus provide a physiologically viable preparation to assess the effect of cation fluxes on the synaptic transmitter release process.
The role of the synaptic vesicles in transmission at the insect nerve-muscle junction
Life Sciences, 1973
The uhraslrueture of nerve-muscle synapses on red and white fibres of locust (Schistocerca gregarta) rett'actor un~.is ~nusele Hbres was examined before and after stimulation. At a stim~latlon~equelley of 15t(z, the sy'n~psos on the white fit:a'es fatigued completely; those on the"red fi6res also fatibmed, bul; during prolonged stimulat.ion they showed intermittent ~riods of reoo~ery. Synaptie ~'esieles at both types of fatigued synapses were reduced in vo~umtt c6~paved with controls and at ffttigued synapses on white fibres the vesicles had irregular outlines. Aggregation of vesicIcs were observed at fatigued synapses on red fibres anti fhe synaptic cleft width at these synapses was less than at centre! syrtapses on red fibl~-',s. These results are discussed in the light of the ve~icle hypothesis for.,synaptic transmission, According to the vesicle hypothesL~ t:or t4-a~l~itter ~tot,age in, and release from axon terminals (1, 2), synaptio fatigue o~ght b# o, xl~eted to l~ad to changes in distribution, population density and, possibly, l~otume of s~napti¢ ve~ieles. To test this, we have examined the ultrastrueturo of t~o J, ypo~ of i~s, eQt norvo-musele synapse which have been fatigued by high-frequency s~mola/,ioa. Those two types of synapse are found on the red and white fibres of the raotathor~¢ r~*tructor tmguis mu,s:-'le of the locust, Schistocerca gregaria (3).
Insect Biochemistry
Aima'act-High yields of relatively pure, morphologically well-preserved, functionally competent synaptosomes were prepared from brains of moths of Mamestra configurata using a modified microscale Ficoll flotation technique. Typical preparations yielded I0 mg of synaptosomal protein per gram of moth brains. The moth brain synaptosomes were virtually free of endoplasmic reticulurn and mitochondrial contaminants as judged from marker enzyme studies and electron microscopy. Voltage-dependent Ca 2+ ion transport was studied using the moth brain synaptosome preparations. Synaptosomes took up radioactive 45Ca 2+ from the incubation medium. The rate of uptake was increased up to threefold when the synaptosomes were incubated in a depolarizing, high [K +] medium. Time course studies indicated that voltage-dependent Ca 2+ uptake was composed of an early (<2 sec) fast phase and a late (> 10 sec) slow phase. ATP-dependent Ca 2+ ion transport was studied in moth brain synaptosome membrane vesicles prepared from synaptosomes by osmotic shock and purified on a second Ficoll gradient. The inside-out synaptosome membrane vesicles contained an ATP-dependent calcium ion pump which transported 4SCa2+ from the incuation medium into the interior of the vesicle in the presence of ATP. The calcium ionophore A23187 rapidly released accumulated 45Ca2+ from the vesicles. The maximal rate of ATPdependent Ca 2+ transport occurred at a [Ca 2+ free] of 0. I to 0.2 nM, indicating that the transport process has a very high affinity for Ca 2+ ions.
Amino acids modulate ion transport and fluid secretion by insect Malpighian tubules
Journal of Experimental Biology, 2003
SUMMARYInsect haemolymph typically contains very high levels of free amino acids. This study shows that amino acids can modulate the secretion of ions and water by isolated Malpighian tubules of Rhodnius prolixus and Drosophila melanogaster. Secretion rates of Rhodnius tubules in amino-acid-free saline increase after addition of serotonin to a peak value, then slowly decline to a plateau. Addition of glutamine, glutamate or aspartate to such tubules increases secretion rates dramatically relative to the controls in amino-acid-free saline, and these increases are sustained for 1-2h. Seven other amino acids have more modest stimulatory effects, whereas lysine and arginine are inhibitory. Secreted fluid pH and Na+ concentration increase and K+ concentration decreases in response to glutamine. Pre-incubation of unstimulated tubules in saline solutions containing amino acids followed by stimulation with serotonin in amino-acid-free saline shows that the effects of amino acids far outlast...
Electrogenic behavior of synaptic vesicles from Torpedo californica
Journal of Biological Chemistry, 1978
Electrical potential changes in pure synaptic vesicles from Torpedo californica were monitored with the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide. Vesicles resuspended in variable external sodium ion in the presence of gramicidin established sodium ion membrane diffusion potentials. Vesicles resuspended in choline or acetylcholine chloride became hyperpolarized upon addition of gramicidin. Hyperpolarization was subsequently partially reversed spontaneously by choline or acetylcholine influx, which was confirmed by gel filtration, to yield a new, less negative, stable membrane potential. Thus, acetylcholine and choline are taken up electrogenically by synaptic vesicles. Acetylcholine is stored in synaptic vesicles and is recognized as the mediator of nerve impulses at many types of nerve terminals (l), including the electric organ of Torpedo californica (2). The organ is embryologically derived from muscle but contains up to 1000 times greater innervation than mammalian muscle. Thus, this tissue is a rich source of homogeneously cholinergic synaptic vesicles, which makes it a very desirable model system for characterization of their structure and dynamic function (3). Vesicles from the Torpedo electric organ are membranebounded, 880 A in diameter, and contain approximately 0.08 M ATP and 0.36 M acetylcholine, and an acidic core protein called vesiculin (2). Binding of exogenous acetylcholine to intact Torpedo vesicles (4) and to vesicle ghosts (5) has been demonstrated. Acetylcholine but not ATP spontaneously leaks slowly from isolated vesicles (3). Isolated vesicles have been found to contain significant quantities of Na+, K+, Mg2+, Cat+, and Zn*+ (6). We present here preliminary results on transport properties exhibited by purified Torpedo vesicles for some physiologically important ions. MATERIALS AND METHODS ' J. E. Rothlein and S. M. Parsons, unpublished observation. * The abbreviation used is: Hepes, 4-(2-hydroxyethyll-l-piperazineethanesulfonic acid.
Specific stimulated uptake of acetylcholine by Torpedo electric organ synaptic vesicles
Proceedings of the National Academy of Sciences, 1980
The specificity of acetylcholine uptake by synaptic vesicles isolated from the electric organ of Torpedo californica was studied. In the absence of cofactors, [3H]acetylcholine was taken up identically to[14C]choline in the same solution (passive uptake), and the equilibrium concentration achieved inside the vesicles was equal to the concentration outside. In the presence of MgATP, [3H]acetylcholine and [14C]choline in the same solution were taken up identically, except only about half as much of each was taken up (suppressed uptake). [3H]Acetylcholine uptake was stimulated by MgATP and HCO3- about 4-fold relative to suppressed uptake, for a net concentrative uptake of about 2:1 (stimulated uptake). Uptake of [14C]choline in the same solution remained at the suppressed level. [3H]Acetylcholine taken up under stimulated conditions migrated with vesicles containing [14C]mannitol on analytical glycerol density gradients during centrifugation. Vesicle were treated with nine protein modi...
Leucine Transport in Brush Border Membrane Vesicles From Freshwater Insect Larvae
Leucine transport across brush border membrane vesicles prepared from four insect species common to European freshwater streams has been characterized. The species studied were: Ephemera danica (Ephemeroptera: Ephemeridae), Isoperla grammatica (Plecoptera: Perlodidae), Hydropsyche pellucidula (Trichoptera: Hydropsychidae), and Hybomitra bimaculata (Diptera: Taban-idae). The transport differed among the studied taxa for several features, including pH and sodium dependence, substrate affinity and specificity, and efficiency. In H÷ pellucidula and E÷ danica, leucine uptake was higher at pH 7.4 than at more alkaline or acidic pH values, whereas in I÷ grammatica and H÷ bimaculata, the uptake was rather constant when pH varied from 5.0 to 7.4, then strongly decreased at pH 8.8. All but E÷ danica displayed a transient intravescicular leucine accumulation in the presence of sodium, suggesting the existence of a cation-leucine symport mechanism. The sodium dependence ranged according to the following order: H÷ pellucidula > I÷ grammatica > H÷ bimaculata > E÷ danica÷ Moreover, in H÷ pellucidula and I÷ grammatica, the sodium-dependence was stronger at pH 8.8 than at pH 7.4. In E÷ danica, leucine uptake was sodium-independent at all pH values. The highest value of V max (45.3 pmol·s 1 ·mg proteins 1) was in E÷ danica, which, however, displayed the lowest affinity (K m 137 mM) when compared to the kinetic parameters of other taxa. The V max and K m values were: 40 and 52.5, 32.1 and 12.5, and 4.5 and 230 for H÷ bimaculata, H÷ pellucidula, and I÷ grammatica, respectively. The obtained results are discussed within our current knowledge of amino acid transport systems in insects. Arch. Contract grant sponsor: University of Milano-Bicocca (Progetto F.A .R.). Abbreviations used: CAPS = 3-cyclohexylamino-1-propansulfonic acid; HEPES = 4-2-hydroxyethyl)-1-piperazineethansulfonic acid; MES = 4-morpholineethansulfonic acid; TMA = tetramethylammonium.
Journal of Biochemistry, 1998
The phospholipid translocation from the outer to the inner leaflet of synaptic vesicles isolated from the electric organ of the Japanese electric ray, Narke japonica, was measured using fluorescent phospholipid probes. Phosphatidylcholine (PC), phosphatidylethanol amine (PE), or phosphatidylserine (PS) with a fluorescent NBD-labeled short acyl chain at the sn-2 position was mixed with purified synaptic vesicles and the probe in the outer leaflet of the membranes was reduced with dithionite to quench the fluorescence from time to time. The percentage of fluorescence remaining after the dithionite treatment served as an index for the phospholipid translocation. The results obtained indicated that about 30, 13, and 9% of NBD-PE, NBD-PS, and NBD-PC, respectively, were translocated from the outer to the inner leaflet in 3 h. Thus, the translocation activity in synaptic vesicle membranes was much higher for PE than for PS, in contrast to the previous results obtained with plasma membranes, including synaptosomal membranes. The percentages of the phospho lipid in the inner leaflet at equilibrium were estimated to be 41, 31, and 14% for PE, PS, and PC, respectively. The translocation was inhibited by pretreatment with an SH reagent, iodoacetamide, indicating the involvement of a proteinaceous translocator. These data may provide a biochemical basis for elucidating the mechanisms of membrane fusion and exocytosis at nerve endings.