The role of cell death in the midgut epithelium in Filientomon takanawanum (Protura)

Midgut epithelium in Filientomon takanawanum is composed of epithelial cells and single, sporadic regenerative cells. In 80% of analyzed specimens midgut epithelial cells, as fat body and gonads, are infected with rickettsia-like microorganism. In non-infected specimens young and completely differentiated epithelial cells are distinguished among epithelial cells. Characteristic for midgut epithelial cells regionalization in organelles distribution is not observed. Autophagy is the sporadic process, but if the cytoplasm of epithelium cells possesses numerous spherites and sporadic autophagosomes, the apoptosis begins. Necrosis is observed sporadically.In the midgut epithelium cells of about 80% of analyzed specimens rickettsia-like microorganisms are observed. The more rickettsia-like microorganisms occur in the cytoplasm, the more autophagosomes are formed, and the process of apoptosis proceeds intensively.     

Ultracytochemical localization of Ca++-ATPase activity in the paraphyseal epithelial cells of the frog,Rana esculenta

Summary Ca⁺⁺-ATPase activity was studied ultracytochemically (cf. Ando et al. 1981) in the paraphysis cerebri of the frog. An intense reaction was demonstrated on the plasmalemma of the microvilli at the apical pole of paraphyseal cells; in contrast, the basolateral plasmalemma showed only a slight staining. In addition, mitochondria, gap junctions, cilia, and cytoplasmic elements (e.g., microfilaments) displayed Ca⁺⁺-ATPase activity. Variation of the Ca⁺⁺-concentration in the incubation medium from 0.1 mM to 100 mM altered the Ca⁺⁺-ATPase activity of the cell organelles. The substitution of Ca-by Mg-ions resulted in a conspicuous decrease in the enzyme activity, especially on the apical plasmalemma. Ca⁺⁺-ATPase activity is claimed to be involved in a number of extra-and intracellular functions. In comparison to the epithelium of the adjacent choroid plexus the paraphyseal epithelial cell is thought to be a principal Ca-ion regulator of the cerebrospinal fluid in frogs.Fellow of the Alexander von Humboldt Foundation     

The ultrastructure of the midgut epithelium in millipedes (Myriapoda,Diplopoda)

The midgut epithelia of the millipedes Polyxenus lagurus, Archispirostreptus gigas and Julus scandinavius were analyzed under light and transmission electron microscopies. In order to detect the proliferation of regenerative cells, labeling with BrdU and antibodies against phosphohistone H3 were employed. A tube-shaped midgut of three millipedes examined spreads along the entire length of the middle region of the body. The epithelium is composed of digestive, secretory and regenerative cells. The digestive cells are responsible for the accumulation of metals and the reserve material as well as the synthesis of substances, which are then secreted into the midgut lumen. The secretions are of three types – merocrine, apocrine and microapocrine. The oval or pear-like shaped secretory cells do not come into contact with the midgut lumen and represent the closed type of secretory cells. They possess many electron-dense granules (J. scandinavius) or electron-dense granules and electron-lucent vesicles (A. gigas, P. lagurus), which are accompanied by cisterns of the rough endoplasmic reticulum. The regenerative cells are distributed individually among the basal regions of the digestive cells. The proliferation and differentiation of regenerative cells into the digestive cells occurred in J. scandinavius and A. gigas, while these processes were not observed in P. lagurus. As a result of the mitotic division of regenerative cells, one of the newly formed cells fulfills the role of a regenerative cell, while the second one differentiates into a digestive cell. We concluded that regenerative cells play the role of unipotent midgut stem cells.     

K+ current stimulation by Cl- in the midgut epithelium of tobacco hornworm (Manduca sexta)

Summary Chloride-stimulated K⁺ secretion by Manduca sexta midgut (5th-instar larvae) was measured as K⁺-carried short-circuit current of the tissue mounted in an Ussing chamber. Microscopic parameters, such as single-channel current and channel density for the rate-determining passive transport step across the basolateral goblet cell membrane (i.e. K⁺ channels), were estimated by means of current-fluctuation analysis of the K⁺ channel blockade by haemolymph-side Ba²⁺ ions. Ba²⁺ was equally effective with Cl^- or gluconate (Glu^-) as the principal ambient anion. The Ba²⁺-induced K⁺ channel conduction noise is reflected by a Lorentzian, or relaxation, noise component in the power spectrum of the K⁺ current fluctuations. A reduced Lorentzian plateau value, but an unchanged corner frequency, were observed when Cl^- was replaced by Glu^-. The results from the analysis of a two-state model of K⁺ channel block by Ba²⁺, with respect to the anion-replacement effects, suggest that the observed changes in K⁺ current and Lorentzian plateau value mirror a complex change of the underlying parameters: Cl^- omission reduces single channel current but increases channel density so that the product of single channel current and channel density is smaller in Glu^- than in Cl^-. It seems likely that basolateral K⁺ channels (1) are subject to anionic gating ligands, and (2) depend on anions with respect to the rate of K⁺ transfer through and open K⁺ channel.Abbreviations a.c. alternating current - single-channel conductance - E _K K⁺ Nernst potential - f frequency contained in current noise - f _c corner frequency - Glu^- gluconate - G _t transepithelial conductance - I _sc short-circuit current - I _K K⁺ current - I _K(max) maximal K⁺ current - i single-channel current - K _Ba barium inhibition constant - K _m Michaelis constant of saturating K⁺ current - k ₀₁ and k ₁₀ barium association and dissociation rate constant, respectively - M K⁺ channel density - S _f power density - S _o Lorentzian plateau value - P _o channel-open probability - P _K K⁺ permeability - V _sc cellular potential at short-circuit These results have already been presented in part, at the 1989 joint meeting of the German and Israel Physiological Societies in Jerusalem (Zeiske et al. 1990).     

Technical considerations for assessing alterations in skeletal muscle sarcoplasmic reticulum Ca++-sequestration functionin vitro

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca⁺⁺-ATPase activity and Ca⁺⁺-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca⁺⁺-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca⁺⁺-activated Ca⁺⁺-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca⁺⁺-uptake was measured at a range of physiological free [Ca⁺⁺] using the Ca⁺⁺ fluorescent dye Indo-1. Maximum Ca⁺⁺-uptake rates when corrected for initial [Ca⁺⁺]_f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca⁺⁺ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca⁺⁺-uptake and Ca⁺⁺-ATPase activity measured in the presence of the Ca⁺⁺ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca⁺⁺-uptake (r=+0.44, P<0.05) and between HOM and CM Ca⁺⁺-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca⁺⁺-uptake function and maximal Ca⁺⁺-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994)     

Isolation of third, fourth, and fifth instar larval midgut epithelia of the moth, Trichoplusia ni

A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population.     

Sugar reception in the blowfly: a possible Ca(++) involvement

The present study investigates the effects of W-7 (a calmodulin antagonist involved in the Ca⁺⁺ cascade) on the response of the ‘sugar’ and ‘water’ cells of labellar chemosensilla in the blowfly Protophormia terraenovae to stimulation with sucrose or fructose. In order to ascertain whether Ca⁺⁺ conductance is involved, the effects of EGTA, one of the most used Ca⁺⁺ chelating agent, and of SK&F-96365, an inhibitor of receptor mediated calcium influx, were also studied. Our electrophysiological data indicate that W-7 addition strongly depresses the ‘sugar’ chemoreceptor response to both sugars and in the case of sucrose stimulation also influences adaptation rate. The Ca⁺⁺ chelator has no significant effects on the response of the ‘sugar’ cell following stimulation with sucrose, but lowers fructose stimulating effectiveness. In the presence of SK&F-96365 both sucrose and fructose responses are inhibited. A possible transduction mechanism for sugar reception is discussed.     

The paracellular pathway in the lepidopteran larval midgut: modulation by intracellular mediators

The features of the paracellular pathway, an important route for the transfer of ions and molecules in epithelia, are in insects still poorly investigated and it has not yet been elucidated how the septate junction (SJ) acts as a transepithelial barrier. In this study, some properties of the paracellular pathway of Bombyx mori larval midgut, isolated in Ussing chambers, were determined and the modulation of SJ permeability by intracellular events disclosed. Diffusion potentials evoked by transepithelial gradients of different salts indicated that the junction bore weak negative charges and that the paracellular pathway was selective with respect to ion charge and size. In standard conditions, the transepithelial resistance was 28.2+/-2.1 Omega cm(2), a value indicating that the midgut is a low resistance epithelium. The modulation of midgut SJ by typical enhancers of mammalian tight junction permeability known to act on the cytoskeleton was studied by measuring the shunt resistance and the lumen-to-haemolymph flux of sucrose. An increase of the intracellular level of cAMP and Ca(2+) caused a significant decrease of the shunt resistance and an increase of SJ permeability. The attenuation of Ca(2+) effect in the presence of the calcium channel blocker nifedipine indicated that the influx of external Ca(2+) into the cytoplasm was important for the opening of the SJ, as well as the release of Ca(2+) from the intracellular stores.     

Voltage clamping with single microelectrodes: Comparison of the discontinuous mode and continuous mode using the Axoclamp 2A amplifier

Summary The voltage clamp technique is a powerful method for studying the physiology of excitable membrane. This technique has made possible the determination of ionic responses generated by activation of either receptor-mediated or voltage-dependent processes. The development of the whole-cell, tight-seal voltage clamp method has allowed the analysis and examination of membrane physiology at the single cell level. The method allows the characterization of voltage-dependent ionic conductances both at the macroscopic (whole-cell) and at the microscopic (unitary conductance or single channel) level in cells less than 10 µm in diameter, a feat difficult to achieve with conventional fine-tipped micropipettes.In this paper, several methologies used for culturing neuronal and non-neuronal cells in the laboratory are described. A comparison between the two modes of voltage clamp using blunt-tipped patch-microelectrodes, the switching (discontinuous) and the non-switching (continuous) modes, of the Axoclamp-2A amplifier is made. Some results on membrane currents obtained from neuronal and non-neuronal cells using the single electrode whole-cell tight-seal voltage clamp is illustrated. The possible existence of two inactivating K⁺ currents, one dependent on Ca⁺⁺ the other is not, is discussed.     

Barium modifies the concentration dependence of active potassium transport by insect midgut

Summary The rate of active K⁺ transport by the isolated lepidopteran midgut shows a rectangular hyperbolic relation to [K⁺] over the range 20 to 70mm K⁺ in the absence of any divalent cation. Addition of Ba⁺⁺ to the hemolymph (K⁺ uptake) side introduces a linear component to the concentration dependence, such that active K^– transport is decreased at [K⁺] of 55mm or less, but increased transiently at higher [K⁺]. As [Ba⁺⁺] is increased over the range 2 to 8mm the linear component increases and the saturating component decreases; in 8mm Ba⁺⁺ the concentration dependence is dominated by the linear component. The effect of Ba⁺⁺ cannot easily be accounted for by simple competition with K⁺ for basal membrane uptake sites. Similar effects might be exercised by other alkali earth cations, since the concentration dependence of active K⁺ transport possesses a substantial linear component in solutions containing 5mm Ca⁺⁺ and 5mm Mg⁺⁺ (the alkali earth metal concentrations of standard lepidopteran saline).     

The fine structure of the midgut epithelium in a centipede,Scolopendra cingulata (Chilopoda,Scolopendridae), with the special emphasis on epithelial regeneration

Scolopendra cingulata has a tube-shaped digestive system that is divided into three distinct regions: fore-, mid- and hindgut. The midgut is lined with a pseudostratified columnar epithelium which is composed of digestive, secretory and regenerative cells. Hemocytes also appear between the digestive cells of the midgut epithelium. The ultrastructure of three types of epithelial cells and hemocytes of the midgut has been described with the special emphasis on the role of regenerative cells in the protection of midgut epithelium. The process of midgut epithelium regeneration proceeds due to the ability of regenerative cells to proliferate and differentiate according to a circadian rhythm. The regenerative cells serve as unipotent stem cells that divide in an asymmetric manner.Additionally, two types of hemocytes have been distinguished among midgut epithelial cells. They enter the midgut epithelium from the body cavity. Because of the fact that numerous microorganisms occur in the cytoplasm of midgut epithelial cells, we discuss the role of hemocytes in elimination of pathogens from the midgut epithelium. The studies were conducted with the use of transmission electron microscope and immunofluorescent methods.     

Identification and phylogenetic classification of eleven putative P-type calcium transport ATPase genes in the yeastsSaccharomyces cerevisiae andSchizosaccharomyces pombe

Calcium is an essential second messenger in yeast metabolism and physiology. So far, only four genes coding for calcium translocating ATPases had been discovered in yeast. The recent completion of the yeastSaccharomyces cerevisiae genome allowed us to identify six new putative Ca⁺⁺-ATPases encoding genes. Protein sequence homology analysis and phylogenetic classification of all putative Ca⁺⁺-ATPase gene products from the yeastsSaccharomyces cerevisiae andSchizosacchraomyces pombe reveal three clusters of homologous proteins. Two of them comprises seven proteins which might belong to a new class of P-type ATPases of unknown subcellular location and of unknown physiological function.     

Metabolic pathways for diacylglycerol biosynthesis and release in the midgut of larval Manduca sexta

The pathway for the synthesis of diacylglycerol in larval Manduca sexta midgut was studied. Fifth instar larvae were fed with [9,10–³H]–oleic acid–labeled triolein and the incorporation of the label into lipid intermediates was analyzed as a function of time. The results showed that the triacylglycerol was hydrolyzed to fatty acids and glycerol in the midgut lumen. In midgut tissue, the labeled fatty acids were rapidly incorporated into phosphatidic acid, diacylglycerol and triacylglycerol, but no significant labeling of monoacylglycerol was observed. Dual-labeling experiments were performed in order to characterize the kinetics of diacylglycerol biosynthesis in the midgut, its incorporation into hemolymph lipophorin and its clearance from hemolymph. The results were best described by a model in which the rate-limiting step in diacylglycerol biosynthesis was the uptake of fatty acid from the lumen of the midgut. Once in the cell the fatty acid was rapidly incorporated in phosphatidic acid and diacylglycerol. Diacylglycerol was converted to triacylglycerol or exported into hemolymph. The interconversion of diacylglycerol and triacylglycerol was fairly rapid, suggesting that triacylglycerol serves as a reservoir from which diacylglycerol can be produced. This mechanism permits the cell to maintain a low steady-state concentration of diacylglycerol and yet efficiently absorb fatty acids from the lumen of the midgut.     

A novel arrangement of midgut epithelium and hepatic cells implies a novel regulation of the insulin signaling pathway in long-lived millipedes

Nutrients absorbed by the epithelial cells of the millipede midgut are channeled to a contiguous population of hepatic cells where sugars are stored as glycogen. In insects and other arthropods, however, nutrients absorbed by midgut epithelia are first passed across the epithelial basal surface to the hemolymph before storage in fat body. The inter-digitation of cellular processes at the interface of hepatic and midgut epithelial cells offers a vast surface area for exchange of nutrients. At this interface, numerous small vesicles with the dimensions of exosomes (∼30 nm) may represent the mediators of nutrient exchange. Longevity and the developmental arrest of diapause are associated with reduced insulin signaling. The long lifespans for which millipedes are known may be attributable to a novel pathway with reduced insulin signaling represented by the novel arrangement of hepatic storage cells and midgut epithelial absorbing cells.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called "regenerative" cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.     

The anti-anginal drug fendiline elevates cytosolic Ca(2+) in rabbit corneal epithelial cells

The effect of the anti-anginal drug fendiline on intracellular free Ca(2+) levels ([Ca(2+)](i)) in a rabbit corneal epithelial cell line (SIRC) was explored using fura-2 as a fluorescent Ca(2+) indicator. At a concentration above 1 microM, fendiline increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 7 microM. The [Ca(2+)](i) response consisted of an immediate rise and an elevated phase. Extracellular Ca(2+) removal decreased half of the [Ca(2+)](i )signal. Fendiline induced quench of fura-2 fluorescence by Mn(2+) (50 microM), suggesting the presence of Ca(2+) influx across the plasma membrane. This Ca(2+) influx was abolished by La(3+) (50 microM), but was insensitive to dihydropyridines, verapamil and diltiazem. Fendiline (10 microM)-induced store Ca(2+) release was largely reduced by pretreatment with thapsigargin (1 microM) (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+). Conversely, pretreatment with 10 microM fendiline abolished thapsigargin-induced Ca(2+) release. Fendiline (10 microM)-induced Ca(2+) release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Cumulatively, this study shows that fendiline induced concentration-dependent [Ca(2+)](i )increases in corneal epithelial cells by releasing the endoplasmic reticulum Ca(2+) in a phospholipase C-independent manner, and by causing Ca(2+) influx.     

A novel regulatory mechanism for amino acid absorption in lepidopteran larval midgut

A number of methyl and ethyl esters of naturally occurring amino acids exert a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids along the midgut of the silkworm Bombyx mori. L-Leucine methyl ester (Leu-OMe), one of the most effective activators, induces a large increase of the initial rate of leucine uptake in midgut brush border membrane vesicles (BBMV) from the anterior-middle (AM) region, and a small effect in BBMV from the posterior (P) region. Nonetheless, the methyl ester causes in both regions a relevant K(+)-, Deltapsi- and pH-independent increase of the intravesicular accumulation of the amino acid. The activation by Leu-OMe proves that amino acid absorption can be modulated all along the B. mori larval midgut and that the AM region, where the ability to transport and concentrate the substrate is very low, is more susceptible than the P region. Leucine uptake in AM-BMMV can be activated by amino acid methyl esters with definite structural requisites, with the following order of potency: L-leucine>L-phenylglycine>L-methionine>L-phenylalanine>L-norleucinez.Gt;L-isoleucine. The activation is stereospecific and occurs also with some ethyl esters (e.g. leucine and phenylalanine). No activation was observed with esters of amino acids with short hydrophobic or polar side-chains. The activation mechanism here described plays a fundamental role in larval growth since silkworms reared on artificial diets supplemented with leucine or methionine methyl esters reach maximum body weight 12-18 h before control larvae and spin cocoons with a larger shell weight. This novel regulatory mechanism of an amino acid transport protein appears to be widespread among lepidopteran larvae.     

Differential calcium signaling in dairy cows with specific CXCR1 genotypes potentially related to interleukin-8 receptor functionality

Neutrophil migration and activation are critical components of innate immunity and are mediated by a variety of inflammatory mediators, which include interleukin-8 (IL-8) and epithelial-derived neutrophil activating peptide-78 (ENA-78). Limited knowledge on the expression of receptors for these inflammatory mediators (CXCR1 and CXCR2) in bovine, in addition to the association of a polymorphism (G→C) in position +777 of the CXCR1 gene with impaired neutrophil function, prompted evaluation of CXCR1 and CXCR2 mRNA and protein expression, ligand binding affinity, and intracellular receptor signaling in neutrophils from cows with different CXCR1 genotypes. Initial observations revealed that overall IL-8 receptor numbers appeared to be lower in cows with a CC genotype compared to cows with a GG genotype. However, in the presence of SB225002, a CXCR2 inhibitor, CXCR1 affinity was about fivefold lower in cows with a CC genotype and may have resulted in an underestimation of receptor numbers in cows with this genotype. In addition, intracellular calcium ([Ca⁺⁺]_i) release was lower in cows with a CC genotype when cells were stimulated with IL-8 but not ENA-78. Furthermore, when neutrophils were stimulated with an optimal dose of IL-8 in the presence of SB225002, [Ca⁺⁺]_i release was lower in cows with a CC genotype, suggesting differential CXCR1 signaling among genotypes. These findings offer knowledge of the role that each of these receptors plays in the inflammatory response in the bovine and provide insight into the potential mechanisms that may be affected in neutrophils of cows with different CXCR1 genotypes.