Differential absorption and esterification of dietary long-chain fatty acids by larvae of the dragonfly, Aeshna cyanea.

In order to evaluate whether dietary long-chain fatty acids were differentially absorbed, Aeshna cyanea larvae received 5 microliters oral doses containing combinations of two radiolabeled fatty acids at nearly equal radioactive and nmolar concentrations: (1) 3H-oleic and 14C-palmitic acids; (2) 3H-oleic and 14C-stearic acids; and (3) 3H-palmitic and 14C-stearic acids. After 3 h or 1 day, hemolymph samples, midgut tissue, midgut contents and fat body tissue were collected and assayed for labeled fatty acids. The 3H/14C ratios indicated that there was a preference for absorption of the monounsaturated oleic acid over both saturated palmitic and stearic acids and that the shorter palmitic acid was absorbed at a higher rate than the longer stearic acid. There were also differences in the 3H/14C ratios of the various lipid classes of the midgut wall, hemolymph, and fat body that reflected differential esterifications and transport of these fatty acids.

Digestion of phosphatidylcholines, absorption, and esterification of lipolytic products by Aeshna cyanea larvae as studied in vivo and in vitro.

Digestion and absorption of phosphatidylcholine by Aeshna cyanea larvae were studied in vivo and in vitro with the isolated digestive juice and isolated midgut. The experiments were performed with stable ether analogues (1-alkyl-2-acyl-,1,2-dialkyl phosphatidylcholine, and 1-monoalkyl-lysophosphatidylcholine), with radioactive 1,2-diacylphosphatidylcholine alternatively labelled in the acyl- and choline moieties, and with several phosphatidylcholine derivatives (1- [1-14C]acyl- and 1-[3H] alkyl-lysophosphatidylcholine, [1-14C]oleic acid, [2-14C]glycerol, phosphoryl[methyl-14C]choline, and [methyl-14C]choline). Chromatographic analyses of the digestion products revealed that phosphatidylcholine was degraded via two interconnected hydrolytic pathways involving phospholipase C, phospholipase A2, lipase, and alkaline phosphatase. Complete hydrolysis by these pathways yielded the same four end products: free fatty acid, glycerol, choline, and Pi, which were absorbed by the midgut enterocytes. Of the intermediate hydrolysates, lysophosphatidylcholine, monoacylglycerol, and possibly phosphorylcholine were also absorbed. Radiolabelled oleic acid, glycerol, lysophosphatidylcholine and monoacylglycerol (as judged from monoacylglycerol absorption) were incorporated into phospholipids and acylglycerols of the midgut enterocytes and were released into the haemolymph primarily in the form of diacylglycerols. In the case of glycerol ingestion, a small fraction of haemolymph radioactivity was associated with free glycerol and glycerolphosphate. After absorption by the enterocytes, radiolabelled choline was partly oxidized to betaine, partly phosphorylated, and partly incorporated into lyso- and phosphatidylcholine. It was recovered from the haemolymph predominantly as free choline, phosphorylcholine, and betaine.

Is monoacylglycerol as an intermediate of triacylglycerol digestion absorbed by Aeshna cyanea larvae?

The isolated digestive juice of Aeshna cyanea larvae hydrolysed trioleoylglycerol preferentially at the terminal 1 and 3 positions, yielding 1, 2-dioleoylglycerol as first intermediate. Hydrolysis continued to 1- and 2-monooleoylglycerol as second intermediate. Separate incubation of monooleoylglycerol revealed that hydrolysis could proceed to completion. Inadequate inhibition of mono[1-14C]oleoylglycerol hydrolysis in the cold and in the presence of the lipase inhibitor tetrahydrolipstatin provided no information on whether monooleoylglycerol was absorbed in addition to free oleic acid. On the other hand, the analogue oleylglyceryl ethers were resistant towards hydrolysis by the digestive juice. Both monoethers and both diethers were esterified with [1-14C]palmitic acid by the homogenate of the midgut wall, whereas esterification in vivo occurred only with the monoethers. These were recovered from the haemolymph after saponification of the joint diacylglycerol and acyl-0-alkylglycerol fraction, indicating that the monoethers had been absorbed and transported into the haemolymph. Ingestion of mono-1-0-[3H]octadecylglycerol showed that the ether was absorbed unchanged by the midgut epithelium, where the major part of the alkyl moiety was oxidized to free fatty acid and incorporated into phospholipids, acylglycerols and acyl-0-alkylglycerols. It is concluded from the absorption of the analogous monoalkylglyceryl ethers that monoacylglyceryl esters are also absorbed by Aeshna larvae.

Intestinal absorption of cholesterol, transport in the haemolymph, and incorporation into the fat body and Malpighian tubules of the larval dragonfly Aeshna cyanea.

Doses of 1.5 mumol cholesteryl oleate ingested by Aeshna cyanea larvae were partially hydrolysed in the intestinal lumen and the liberated oleic acid absorbed, while free cholesterol and unhydrolysed cholesteryl oleate were eliminated in the faeces. Ingestion of [4-14C]cholesterol dissolved in olive oil revealed that the larvae also absorbed free cholesterol, the minor part of which (up to 12%) was esterified in the midgut epithelium. Absorption and esterification were markedly enhanced when the same dose of labelled cholesterol was administered in 20% aqueous bovine serum albumin. Radioactivity was rather slowly released into the haemolymph where it was nearly exclusively associated with free cholesterol apart from traces of labelled cholesteryl ester which maximally amounted to 2.9% after 15 days. In the fat body labelled cholesteryl ester maximally amounted to 65% after 15 days, while in the triacylglycerol-storing Malpighian tubules it remained below 1% at all time periods investigated.

The cell junctions of the notochord of Xenopus laevis tadpoles.

The cell junctions of the notochord of Xenopus laevis tadpoles were examined with the electron microscope using thin sections, lanthanum tracer experiments, and freeze-fracture replicas. Both the peripheral and vacuolated cells of the notochord are connected by numerous spot desmosomes characterized by an intercellular desmogloea and intermediate filaments on the cytoplasmic sides. The peripheral cells also display numerous hemidesmosomes facing the underlying basal lamina. Staining with rhodamine-phalloidin for F-actin yielded negative results and suggested that adhaerens-type junctions are absent. Tracer experiments with lanthanum and freeze-fracture replicas clearly revealed the presence of gap junctions between both cell types but no indications of tight junctions were found and no intercellular barrier existed for tracer infiltration of the notochord.

Peroxisomes of the midgut epithelium, malpighian tubules and fat body of larvae of the dragonfly, Aeshna cyanea.

The enterocytes of the midgut epithelium of Aeshna cyanea larvae are rich in peroxisomes while the nidal regenerative and endocrine cells contain only a few. Most of the enterocytic peroxisomes are microperoxisomes lacking a crystalloid nucleoid, but peroxisomes with well developed nucleoid are also present. The peroxisomes are usually concentrated in the basal region of the cells but may also spread into the apical region and closely intermingle with absorptive lipid droplets. They significantly increase in number, when the larvae are regularly fed lipid-rich natural food or long-chain monounsaturated fatty acids that are unusual dietary components of these animals. This observation seems to indicate that the enterocytic peroxisomes are involved in chain shortening and degradation of fatty acids absorbed from the gut lumen. Numerous microperoxisomes are also present in the lipid-storing cells of the Malpighian tubules and fat body.

Lipid transport through the enterocytes of larval Aeshna cyanea (Insecta, Odonata).

The larval enterocytes of A. cyanea absorb lipid after luminal lipolysis in morphologically invisible form by direct membrane transport, presumably molecular diffusion. The lipolytic products are utilized for resynthesis of di- and triglyceride which become visible in the form of lipid droplets in the groundplasm. The putative site of lipid synthesis is the apical ER which locally forms highly ordered complexes. Lipid transport occurs in the form of matrix lipid so that the enterocytes of dragonfly larvae resemble in this respect the lipid-secreting mammocytes rather than the lipid-absorbing mammalian enterocytes. Lipid release involves partial lipolysis and direct membrane transport, possibly including membrane delamination, again in contrast to exocytosis in mammalian enterocytes and apocrine extrusion in mammocytes, which are both indirect membrane transport mechanisms.

Activation and inhibition of Na/K-ATPase by filipin-cholesterol complexation. A correlative biochemical and ultrastructural study on the microsomal and purified enzyme of the avian salt gland.

The Na/K-ATPase-rich microsomal fraction and purified Na/K-ATPase membranes of the salt-stressed avian salt gland were studied at defined filipin/cholesterol molar ratios (F/C) using enzyme assay and electron microscopy including negative staining, thin sectioning and freeze fracturing. Comparative examinations of detergent-treated microsomal fractions and the use of electron microscopic tracers revealed that F/C up to 2 activated latent Na/K-ATPase in sealed right-side-out vesicles by increasing membrane permeability without disrupting the vesicular membrane. Therefore, filipin offers an alternative to the detergents for the activation of latent vectorial membrane enzymes and a possible tool to examine their subcellular localization and sidedness in the membrane. The same F/C had no stimulatory effect on the microsomal anion-ATPase suggesting that the 2 ATPases are not located in the same membrane. Increasing F/C applied to the unfixed Na/K-ATPase membranes caused an increase in the number of structural F-C-complexes and a progressive lateral displacement of the enzyme particles which finally led to a separation of the areal distribution of these structures at F/C = 10. Such displacements did not occur in unfixed microsomes and were prevented by glutaraldehyde fixation of the purified membranes. F/C exceeding 2 progressively and temperature-dependently inhibited the Na/K-ATPase in its membrane-bound states, whereas the solubilized enzyme was rather insensitive. The structural and biochemical data suggest that inhibition results from the perturbation of the lipidic microenvironment of the enzyme caused by filipin-cholesterol complexation.

Plasma membranes, cell junctions and cuticle of the rectal chloride epithelia of the larval dragonfly Aeshna cyanea.

The cell membranes and cell junctions of the rectal chloride epithelia of the larval dragonfly Aeshna cyanea were examined in thin sections and by freeze-fracture. These epithelia function in active ion absorption and maintain a high concentration gradient between the haemolymph and the fresh-water environment. Freeze-fracturing reveals fine-structural differences in the intramembraneous particles of the luminal and contraluminal plasma membranes of these epithelia, reflecting the functional diversity of the two membranes, which are separated by the junctional complex. The particle frequency of the basolateral plasma membranes is reduced after transfer of the larvae into high concentrations of environmental salinity. The junctional complex is located in the apical region and composed of three types of cell junctions: the zonula adhaerens, seen in freeze-fracture as a nearly particle-free zone; the extended and highly convoluted pleated septate junction and randomly interspersed gap junctions of the inverted type. Gap junctions also occur between the basolateral plasma membranes. They provide short-cuts in the diffusion pathway for direct and rapid co-ordination of the interconnected cell processes. Colloidal and ionic lanthanum tracer solutions applied in vivo from the luminal side penetrate through the cuticle via epicuticular depressions, but invade only the apical portion of the junctional complex. This indicates that the pleated septate junction constitutes a structural control of the paracellular pathway across the chloride epithelia, which are devoid of tight junctions. The structure of the pleated septate junctions is interpreted as a device for the extension of the diffusion distance, which is inversely related to the net diffusion. A conservative estimate of the total length of the junction, and the number and extension of septa reveals that the paracellular route exceeds the transcellular route by a factor of 50.

Ultrastructure of the purified and reconstituted Na/K-ATPase of the avian salt gland.

Na/K-ATPase of salt-stressed salt glands of the domestic duck (Anas platyrhynchos) was purified in membrane-bound form by incubation of the microsomal fraction with sodium dodecylsulphate and ATP followed by discontinuous sucrose gradient centrifugation. Gel electrophoresis of the purified plasma membrane preparation substantially showed the two polypeptide subunits of the Na/K-ATPase both of which stained with the periodic acid-Schiff reagent. About 99% of the total ATPase activity was ouabain-inhibitable amounting to 1300 mumol Pi/(mg protein X h) of specific activity. The anion-stimulated, ouabain-insensitive ATPase increased parallel to the Na/K-ATPase up to the microsomal fraction until it totally vanished during SDS incubation. Electron microscopy of thin sections revealed that the purified fraction consisted of flat and cup-shaped triple-layered membrane fragments. Particles arranged into clusters and strands were visible as 3 to 5 nm surface particles in negatively stained suspensions and as 8 to 10 nm intramembraneous particles in freeze fracture replicas. The differential distribution of the intramembraneous particles on the fracture faces reflected the structural membrane asymmetry. Solubilization of Na/K-ATPase led to the disappearance of intramembraneous particles. Incorporation of the solubilized enzyme into phosphatidylcholine vesicles again showed 8 to 10 nm particles apparently orientated at random in the artificial membrane. Control liposomes prepared in the absence of solubilized enzyme were devoid of intramembraneous particles. These results clearly demonstrate that the avian salt gland Na/K-ATPase exists as 8 to 10 nm particles in both the purified plasma membrane and the artificial phospholipid membrane.

The loop diuretic furosemide as non-competitive inhibitor of C1-/HCO3-ATPases of vertebrate kidneys and insect rectum.

1. The effect of the diuretic drug furosemide was studied in detail on ouabain-insensitive, SCN- and OCN- -sensitive C1-/HCO-3-ATPase in homogenates from larval dragonfly rectum (Aeshna cyanea), frog (Rana temporaria) and mouse (Mus musculus) kidney. 2. The in vitro inhibition by the drug studied on the HCO-3-activated enzyme is non-competitive with an inhibitor constant of Ki=4.3 mM furosemide in the case of insect rectum and Ki=0.9 mM furosemide in the case of frog and mouse kidney. 3. Furosemide even at 10 mM concentration which completely inhibits the anion-dependent ATPase has only a little inhibitory effect on the Na+/K+-ATPase of the 3 tissues. 4. The data suggest that furosemide may affect an active chloride transport system involving a C1-/HCO-3-ATPase.

[Demonstration and properties of a Cl-/HCO3--ATPase in the avian salt gland (author's transl)]. / Nachweis und Eigenshaften einer Cl-/HCO3--ATPase in der Vogelsalzdrüse.

An anion-stimulated, Mg2+-dependent, ouabain-insensitive ATPase is present in salt gland homogenates of domestic ducks (Anas platyrhynchos). The enzyme is unspecifically stimulated by various inorganic and organic anions including amino and sulfonic acids which are often used as buffer components (e. g. histidine, Bicin, PIPES, MES and HEPES). Therefore, the demonstration of ATPase stimulation by chloride strongly depends on the type and concentration of the buffer used and may also largely interfere with the stimulation caused by other anions present in the incubation medium. Of the inorganic anions tested chloride and bicarbonate appear to be the favorite physiological activators, but the possible role of carbonic acids in the stimulation of the anion-dependent ATPase should not be neglected. Km values are approximately 5.8 mM for Cl- and approximately 8.7 mM for HCO-3-activation. Maximal ATPase stimulation is obtained at 25 mM Cl- and approximately 30 mM HCO-3, respectively. The simultaneous presence of bicarbonate decreases chloride affinity and Vmax, and shifts the chloride optimum to lower concentrations. ATP is the most preferred substrate. Maximal activation by Cl- and HCO-3 occurs at ATP concentrations between 0.5 and 1 mM. ATP affinity increases in the presence of Cl- and HCO-3, respectively. Both chloride and bicarbonate require a Mg2+ to ATP ratio of approximately 0.5 and a pH value of 8.0 to 8.5 for optimal stimulation. Stimulation by Cl- and HCO-3 is inhibited by thiocyanate, cyanate and by the diuretic drugs furosemide, ethacrynic acid and mersalyl. Incubation media adapted for the simultaneous demonstration of both chloride and bicarbonate activation contained 10 to 20 mM histidine-Tris buffer at pH 8.0 to 8.5, 150 mM sucrose, 0.2 mM ouabain, 0.5 mM magnesium acetate, 1 mM ATP, (pH adjusted to 8.0-8.5 with Tris or NaOH), with and without 25 mM sodium chloride or 25 mM sodium bicarbonate.

Inhibition of a Cl-/HCO3(-)ATPase in the avian salt gland by furosemide and ethacrynic acid.

An ouabain-insensitive, Mg2+-dependent, chloride- and bicarbonate-stimulated ATPase is present in salt gland homogenates of domestic ducks (Anas platyrhynchos). Furosemide and ethacrynic acid are non-competitive inhibitors of this enzyme in vitro. The inhibitor constant of furosemide is Ki = 2.5 mM and of ethacrynic acid Ki = 1.9 mM. In contrast, under the same conditions the salt gland Na+/K+-ATPase activity is not inhibited by furosemide, whereas ethacrynic acid becomes inhibitory only at higher concentrations (10 mM).

[Biochemical demonstration of HCO3--und Cl--dependent ATPase activities in the rectum of larval dragonflies and inhibition of rectal chloride uptake by thiocyanate (author's transl)]. / Biochemischer Nachweis von CHO3-- und Cl--abhängigen ATPase-Aktivitäten im Rectum von anisopteren Libellenlarven und Hemmung der rectalen Chloridaufnahme durch Thiocyanat.

Hydrogencarbonate and chloride activated, ouabain-insensitive ATPase activities are demonstrated in the salt-absorbing rectum of larval dragonflies. Maximal activation is achieved at approx. 30 mM HCO3- and 20 mM Cl-, respectively. The stimulation of each anion obeys Michaelis-Menten kinetics Km values are 4.65 mM for HCO3-- and 10.25 mM for Cl--activation. The activating anion of one type of ATPase simultaneously exerts an inhibitory effect on the other. Cl--activation is also reduced by Mg.ATP in concentrations above 0.5 mM and by Tris-Hepes buffer exceeding 2.5 mM. Both anion-dependent ATPase activities are found enriched in subcellular membraneous fractions of the rectum. Thiocyanate inhibits both activities and causes a significant decrease in rectal uptake of radioactive chloride from hypo-osmotic external solution. In the case of HCO3- dependent ATPase a competitive inhibition as SCN- was found with an inhibitor constant of Ki=0.5 mM.

Comparative biochemical, histochemical and autoradiographic studies of Na+/k+-ATPase in the rectum of dragonfly larvae (Odonata, Aeshnidae).

Na+/K+-ATPase localization in the rectal wall of larval Aeshna cyanea (Insecta, Odonata) was studied with histochemical precipitation techniques and 3H-ouabain autoradiography in conjunction with biochemical measurements of enzyme activities and radiospectrometry of 3H-ouabain binding, respectively. The NPP-strontium and ATP-lead methods led to complete inhibition of Na+/K+-ATPase in this organ and hence to unreliable histochemical results. The 3H-ouabain binding technique revealed sodium pump sites at the basolateral plasma membranes of the absorptive rectal chloride epithelia.

Chloride cells of larval Notonecta glauca and Naucoris cimicoides (Hemiptera, Hydrocorisae). Fine structure and cell counts at different salinities.

The chloride cells of the larval waterbugs, Notonecta glauca L. and Naucoris cimicoides L., ultrastructurally resemble the caviform chloride cells of other aquatic insects as well as those of teleost fish. The predominant features are abundant mitochondria, basolateral plasma membrane infoldings and an apical cavity possessing numerous microvilli. After histochemical precipitation of chloride, dense deposits of silver chloride are present in the apical region. The cuticular area overlying the chloride cells is extremely reduced in thickness and differentiated into a plug-like structure. The 1st instars of Notonecta glauca raised from the eggs at various hypo-osmotic salt concentrations have identical numbers of thoracal chloride cells regardless of the external salinity. The number of cells progressively increases with each moult. However, in corresponding larval stages, there is a significant decrease in the number of thoracal chloride cells relative to increases in the external salinity. These results are consistent with the presumptive involvement of the chloride cells in osmotic hyperregulation.