The ultrastructural localization of membrane ATPase in rat thin limbs of the loop of Henle

Summary Intraacinar distribution of succinate dehydrogenase (SDH), malate dehydrogenase (MDH), NADP-dependent isocitrate dehydrogenase (IDH), glutamate dehydrogenase (GluDH), lactate dehydrogenase (LDH) and NADH-tetrazolium dehydrogenase (TR) was studied in rat liver cryostat sections by multipositional microphotometric activity determinations. By statistical evaluation, activity of individual enzymes could be related to the acinar topography. Activity was evaluated with regard to distance of measuring position either from afferent (portal) or efferent (hepatic) vessels. Two independent distribution curves were obtained for each enzyme. Acinar distribution of all the enzymes studied followed sigmoid courses with maximal activity of SDH, MDH and LDH in zone 1 (periportal) and GluDH, IDH, TR in zone 3 (pericentral). For all enzymes, maximum activity gradients were confined to zone 2 of the acinus. Data were also evaluated as ratios of activities in zone 1 and zone 3. The following ratios zone 1/zone 3 were obtained: SDH=1.9, MDH=1.7, IDH=0.5, GluDH=0.5, LDH=1.3 and TR=0.6.     

Histotopography and ultrastructure of the thin limbs of the loop of Henle in the hamster

Summary In the kidney of the Syrian hamster the descending thin limbs of both the short and long loops of Henle are not spatially separated from each other and descend between the vascular bundles.Ultrastructurally, five different epithelial types are distinguished in the thin limbs of the short and long loops of Henle. Short loops possess only a descending thin limb with a simply organized epithelium (type 1). Long loops comprise an upper and a lower part of the descending thin limb and the ascending thin limb. The upper part of the long descending thin limb is equipped with a complex and highly interdigitating epithelium with shallow junctions (type 2), which gradually transforms into the simple noninterdigitating type-3 epithelium of the lower part. In a minor portion of long descending thin limbs, however, the upper part begins with an even more complexly organized epithelium (type 2a) than type 2. Type-2a epithelium is conspicuously thicker and possesses a more elaborate mode of cellular interdigitation. Along the descent of this tubular part through the inner stripe of the outer medulla, type-2a epithelium transforms into type-2 epithelium. It is suggested that the long descending thin limbs, which start with type-2a epithelium, belong to the longest loops. The type-4 epithelium of the ascending thin limbs is characterized by flat and extensively interdigitating cells with shallow junctions.The unique pattern of the type-2 a epithelium favors the assumption that solute secretion essentially contributes to the increase in concentration of tubular fluid in long descending thin limbs.This investigation was supported by the Deutsche Forschungsgemeinschaft; project Kr 546 Henlesche Schleife     

The thin limbs of Henle's loop in the rabbit

Summary The thin limbs of short and long loops of Henle of the rabbit kidney were studied by freeze fracture techniques. According to TEM studies of thin sections four segments are discernible: descending thin limbs of short loops, descending thin limbs of long loops, subdivided into an upper and a lower part, and ascending thin limbs (Kaissling and Kriz 1979). This division is supported by findings obtained with the freeze fracture technique and based on differences in the organization of the junctional complexes as well as on differences in the internal morphology of the cell membranes. The descending thin limbs of short loops have junctional complexes established by several closely arranged junctional strands and numerous desmosomes. The upper parts of the long descending thin limbs have tight junctions consisting of a variable number of strands; their outstanding characteristic after freeze fracture is a high density of intramembrane particles in both luminal and baso-lateral membranes. The tight junctions of the lower part of the long descending thin limbs consist of several anastomosing junctional strands, which are, in contrast, loosely arranged; the cell membranes contain only a sparse population of intramembrane particles. The ascending thin limbs are characterized by shallow tight junctions (frequently consisting of only one single junctional strand). Moreover, the epithelial cells of this segment are heavily interdigitated; thereby the tight junctions are correspondingly lengthened.In addition, this study presents further evidence that remarkable species differences occur among thin limb epithelia. The junctional complexes of the long descending thin limbs of the rabbit are organized quite differently from those of small rodents (e.g., rat, Psammomys).The data of this study support the concept that the tight junctions are the main determinant of ionic conductances of the paracellular pathway. However, with reference to recent findings from microperfusion studies, it becomes obvious that a correlation of the junctional morphology with the transepithelial water permeability is lacking, at least for the thin limbs.This investigation was supported by the Deutsche Forschungsgemeinschaft; project Kr 546 Henlesche Schleife     

A possible correlation between ultrastructure and function in the thin descending and ascending limbs of the loop of Henle of rabbit kidney

Summary An ultrastructural study of the thin loops of Henle has been made in the renal papilla of the rabbit. Animals in different states of water balance were used but no morphological difference was observed in the loops obtained from animals in different experimental groupings. The cytoplasm of the squamous cells lining the limbs was characterised by a paucity of organelles. Descending and ascending limbs were distinguishable. A distinct morphological difference was seen in the junctional regions of cell processes of the descending and ascending thin limbs of the loop. The ascending limb processes were joined by continuous tight junctions whereas the descending limb junctional regions invariably showed a space of at least 70 Å between adjacent processes. It is suggested that there may be a correlation between the structure of these junctional regions and the different permeability characteristics of the two limbs. The thin ascending limb must, on physiological evidence, be relatively impermeable with reference to the thin descending limb.The author wishes to thank Professor F. R. Johnson for his advice and assistance, and Mr. R. F. Birchenough, Mr. P. L. Hyam and Mr. J. Manston for valuable technical assistance.     

Polarized expression of different monocarboxylate transporters in rat medullary thick limbs of Henle.

Extracellular lactic acid is a major fuel for the mammalian medullary thick ascending limb (MTAL), whereas under anoxic conditions, this nephron segment generates a large amount of lactic acid, which needs to be excreted. We therefore evaluated, at both the functional and molecular levels, the possible presence of monocarboxylate transporters in basolateral (BLMVs) and luminal (LMVs) membrane vesicles isolated from rat MTALs. Imposing an inward H(+) gradient induced the transient uphill accumulation of L-[(14)C]lactate in both types of vesicles. However, whereas the pH gradient-stimulated uptake of L-[(14)C]lactate in BLMVs was inhibited by anion transport blockers such as alpha-cyano-4-hydroxycinnamate, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), and furosemide, it was unaffected by these agents in LMVs, indicating the presence of a L-lactate/H(+) cotransporter in BLMVs, but not in LMVs. Under non-pH gradient conditions, however, the uptake of L-[(14)C]lactate in LMVs was transstimulated 100% by L-lactate, but by only 30% by D-lactate. Furthermore, this L-lactate self-exchange was markedly inhibited by alpha-cyano-4-hydroxycinnamate and DIDS and almost completely by 1 mM furosemide, findings consistent with the existence of a stereospecific carrier-mediated lactate transport system in LMVs. Using immunofluorescence confocal microscopy and immunoblotting, the monocarboxylate transporter (MCT)-2 isoform was shown to be specifically expressed on the basolateral domain of the rat MTAL, whereas the MCT1 isoform could not be detected in this nephron segment. This study thus demonstrates the presence of different monocarboxylate transporters in rat MTALs; the basolateral H(+)/L-lactate cotransporter (MCT2) and the luminal H(+)-independent organic anion exchanger are adapted to play distinct roles in the transport of monocarboxylates in MTALs.     

The ultrastructural localization of two basement membrane components: entactin and laminin in rat tissues

The localization of two noncollagenous components of basement membranes, laminin and entactin, was determined in rat kidney, muscle, and small intestine using electron immunohistochemistry. In the renal glomerulus anti-laminin antibodies reacted with the basement membrane of peripheral capillary loops and with mesangial matrix. In the peripheral capillary loop laminin was preferentially distributed in both laminae rarae. This was in contrast to anti-entactin that localized in peripheral capillary loops but not in mesangial matrix. Even in the peripheral capillary loops it had a different distribution than laminin. Entactin was found predominantly in the lamina rara interna. In renal tubular basement membranes both antibodies localized throughout the full thickness of the basement membranes, with laminin having a preferential distribution in the lamina rara, whereas entactin was more evenly distributed. In the basement membrane of the duodenal mucosa entactin localized in the lamina densa, whereas laminin was present in both laminae. In skeletal muscle both antibodies had similar localization in all basement membranes. These results demonstrate that entactin is an intrinsic component of basement membranes. They also demonstrate that basement membranes from different tissues have subtle variations in content and/or assembly of the different components. It is likely that these variations may be reflected in different functional properties.     

Transport ATPase cytochemistry: ultrastructural localization of potassium-dependent and potassium-independent phosphatase activities in rat kidney cortex

A cytochemical method for the light and electron microscope localization of the K- and Mg-dependent phosphatase component of the Na-K-ATPase complex was applied to rat kidney cortex, utilizing p-nitrophenylphosphate (NPP) as substrate. Localization of K-N-ATPase activity in kidneys fixed by perfusion with 1% paraformaldehyde -0.25% glutaraldehyde demonstrated that distal tubules are the major cortical site for this sodium transport enzyme. Cortical collecting tubules were moderately reactive, whereas activity in proximal tubules was resolved only after short fixation times and long incubations. In all cases, K-NPPase activity was restricted to the cytoplasmic side of the basolateral plasma membranes, which are characterized in these neplron segments by elaborate folding of the cell surface. Although the rat K-NPPase appeared almost completely insensitive to ouabain with this cytochemical medium, parallel studies with the more glycoside-sensitive rabbit kidney indicated that K-NPPase activity in these nephron segments is sensitive to this inhibitor. In addition to K-NPPase, nonspecific alkaline phosphatase also hydrolyzed NPP. The latter could be differentiated cytochemically from the specific phosphatase, since alkaline phosphatase was K-independent, insensitive to ouabain, and specifically inhibited by cysteine. Unlike K-NPPPase, alkaline phosphatase was localized primarily to the extracellular side of the microvillar border of proximal tubules. A small amount of cysteine-sensitive activity was resolved along peritubular surfaces of proximal tubules. Distal tubules were unreactive. In comparative studies, Mg-ATPase activity was localized along the extracellular side of the luminal and basolateral surfaces of proximal and distal tubules and the basolateral membranes of collecting tubules.     

The ultrastructural localization of metallothionein in cadmium exposed rat liver

Summary The ultrastructural localization of metallothionein (MT) was investigated in the liver of male Wistar rats by a cryo-immunocytochemical technique. The liver parenchymal and sinusoidal cells were studied in both cadmium-exposed (3 × 1.2 mg kg⁻¹ as cadmium chloride) and non-treated animals. Treatment with cadmium induced the synthesis of MT yet differences in the distribution were evident amongst the various types of liver cell. MT was found most abundantly in the parenchymal and endothelial cells, yet was absent in the stellate cell and sparsely distributed in the Kupffer cell. In the cells where MT gene expression was induced, the metalloprotein was distributed within both the nuclear and cytoplasmic compartments. The significance of the nuclear localization of MT is discussed.     

Mg2+- ATPase in the fish brain and its ultrastructural localization

Effects of γ-aminobutyric acid (GABA) and ethanol on Mg²⁺-ATPase from mitochondrial and microsomal fractions of the fish brain were studied. GABA (10^-8–10^-4 M) activates microsomal Mg²⁺-ATPase, but has no effect on the mitochondrial enzyme activity. This effect of GABA on the microsomal Mg²⁺-ATPase was absent in the presence of 8% ethanol. Ethanol at 1– 10% concentrations inhibits the basal microsomal Mg²⁺-ATPase and has no effect on the mitochondria enzyme. Using cytochemical technique, Mg²⁺-ATPase was revealed both in neurons and in glial cells. The ethanol-sensitive Mg²⁺-ATPase is located in the area of synaptic junctions and is bound to plasma, vesicular, and smooth endoplasmic reticulum membranes.     

Further studies on the ultrastructural localization of a magnesium dependant neutral ATPase in arteries

Summary A further analysis of the ultrastructural localization of a Mg⁺⁺ dependant neutral ATPase in arteries (thoracic aorta and basilar artery) has been performed in light of recent findings concerning the use of differential fixation and pitfalls in the standard Wachstein-Meisel (W-M) technique. The localization of reaction product was documented following fixation in 5% and 10% formaldehyde and 5% glutaraldehyde, and following incubation in the standard W-M media with ATP, AMP, -glycerophosphate as substrates. These results were compared to the localization using a modified W-M medium with ATP in which the lead ion concentration was reduced to 1.8 mM. Using the standard W-M procedure, formalin fixation gave a more intense but also a more diffuse (both intra- and extracellular) precipitate of reaction product than glutaraldehyde. The localization to cell structure remained the same in both cases, namely to the outer cell membrane, within its invaginations and in pinocytotic vesicles of both endothelial and smooth muscle cells. Following incubation in a medium with lower lead ion concentration, less extracellular precipitate was found and following glutaraldehyde fixation, very sparse precipitate of reaction product was localized to the cell membrane and its invaginations, often on the cytoplasmic side. The reduction of extracellular precipitate following pre-incubation in 5 mM cystein was believed to be due to inhibition of an unspecific alkaline phosphatase and phosphomonoesterase which had diffused out of the cell following fixation. Cysteine had no effect on the ATPase of the vascular wall. The significance of these results was discussed in light of previous studies on blood vessels and newer insights into this technique.     

Membranous localization and properties of ATPase of rat liver lysosomes.

Lysosomes isolated from rat liver were found to have ATPase activity (EC No 3.6.1.3). Subfractionation of the lysosomes revealed a membranous localization of ATPase activity. The enzyme has half maximal activity at 0.2 mM ATP and is inhibited by high concentrations of ATP. The apparent Km for divalent metal is 0.2 mM, and either Ca2+ or Mg2+ give maximal activity. The ATPase activity has latency when lysosomes are isolated from rats treated with Triton WR-1339. This latency may be due to the presence of internalized sucrose because the activity of L fraction lysosomes is much less latent and Triton WR-1339 itself is not inhibitory. The latency of glucosaminidase, a marker enzyme for lysosomes, contrasts with the low latency of the ATPase and points to an ATPase with an exposed active site in intact lysosomes.     

Cytochemical localization of plasma membrane ATPase activity in plant cells

Summary The cytochemical localization of ATPase activity has been investigated in maize root cells using both lead and cerium-based capture methods. With both methods, staining at the plasma membrane was observed in all cells of the root, although the precipitate obtained with cerium was more uniform and granular than that with lead. Controls using no substrate or no magnesium, -glycerophosphate to replace ATP, vanadate or boiled tissue generally showed little or no staining. However, biochemical studies on purified plasma membrane fractions showed that ATPase activity was markedly inhibited by fixation, particularly by glutaraldehyde, and also by lead and cerium ions. Non-enzymic hydrolysis of ATP by cerium was greater than that by lead. The value and limitations of these procedures for the localization of plasma membrane H⁺-ATPase activity are summarized in relation to previous criticisms of these methods.Abbreviations DTT dithiothreitol - EDTA ethylene diaminetetraacetic acid - GP B-glycerophosphate - PCMBS p-chloromercuribenzene sulphonic acid - PMSF phenylmethylsulphonyl fluoride     

The ultrastructural localization of antigen B10 of the hepatocyte plasma membrane in mouse hepatomas

The ultrastructure of the murine hepatocyte plasma membrane antigen (Ag B10) was studied by immunoelectron microscopy in 5 spontaneous and 3 chemical-induced hepatomas. Ag B10 was associated with plasmalemma of bile canaliculi and membrane of microvilli as in normal liver. Sometimes it was connected with plasmalemma of lateral domain of tumor cells. The availability of Ag B10 in the matrix of bile canaliculi and within microvilli was shown.     

The vacuolar-type ATPase from insect plasma membrane: immunocytochemical localization in insect sensilla

Summary This comparative immunocytochemical investigation provides evidence that the electrogenic potassium pump of insect sensilla is a vacuolar-type proton ATPase energizing potassium-proton antiport, as was shown recently for the electrogenic potassium pump in the larval midgut of the sphinx moth Manduca sexta. Antennal sensilla of the saturniid moth Antheraea pernyi were probed with antibodies to the midgut vacuolar-type ATPase. The monoclonal antibodies recognized their epitopes in the native and SDS-denatured state, and bound specifically to the subunit with the relative molecular mass (M_r) of 67000 (antibody 86-3) or to the subunits of M_r 28000 and 16000 (antibody 47-5). Both antibodies labelled the apical region of the auxiliary cells, as was demonstrated by immunofluorescence microscopy. Immunogold-electron microscopy localized the binding sites of the 47-5 antibody in the highly folded apical plasma membranes of the auxiliary cells. Labelling was selective and was detected in all types of examined sensilla (S. trichodea, S. styloconica, S. coeloconica). These findings are in agreement with the current view that an electrogenic potassium pump is situated in the apical plasma membrane of the auxiliary cells and that the pump is involved in driving the receptor current. They support the hypothesis that a proton-motive force generated by a vacuolar-type ATPase provides an alternative to the classical Na⁺/K⁺-ATPase to energize animal plasma membranes.     

Characterization and localization of ouabain-insensitive Na-dependent ATPase activities along the rat nephron.

Single segments of rat nephron contain two distinct ouabain-insensitive, K-independent, Na-dependent ATPase activities: a Na-stimulated ATPase and a Na-inhibited ATPase. Na-inhibited ATPase activity is found in the proximal tubule and the thick ascending limb of Henle's loop but is absent in the collecting tubule whereas Na-stimulated ATPase is exclusively located in the proximal convoluted tubule. Na-inhibited ATPase, but not Na-stimulated ATPase, is totally abolished in the presence of 100 microM Ca2+. Conversely, Na-stimulated ATPase, but not Na-inhibited ATPase, is curtailed when nephron segments are preincubated at pH 7.2 whereas it is activated at pH 7.8. Finally, Na-stimulated ATPase displays an apparent Km for Na+ of approximately 10 mM, and is dose-dependently inhibited by the diuretic triflocin (IC50 approximately 6 x 10(-6) M).