A comparison of d-inositol 1:2-cyclic phosphate 2-phosphohydrolase with other phosphodiesterases of kidney

1. The ability to hydrolyse various phosphodiesterase substrates was examined in subcellular fractions of rat kidney and in serial slices of the kidneys of mouse, rat, guinea pig and ox cut from the cortex perimeter inwards. 2. d-Inositol 1:2-cyclic phosphate 2-phosphohydrolase could be clearly distinguished from phosphodiesterases which hydrolyse 2':3'- and 3':5'-cyclic AMP and p-nitrophenyl thymidine 5'-phosphate (phosphodiesterase I). The hydrolysis of sn-glycero-3-phosphorylcholine showed a distribution identical with that of particle-bound d-inositol 1:2-cyclic phosphate 2-phosphodiesterase, but there was a 30-fold difference in the ratio of enzyme activities between the rat and guinea pig. 3. In rat and mouse kidney, d-inositol 1:2-cyclic phosphate 2-phosphohydrolase is virtually all membrane bound and in the outer cortex, whereas in guinea-pig kidney the enzyme is almost entirely soluble and located throughout the kidney tissue. Some properties of the soluble enzyme are described. 4. Distribution and histochemical studies indicated that in the rat and mouse, phosphodiesterase I is associated with the brush borders of the straight portion (pars recta) of the proximal tubule, whereas inositol 1:2-cyclic phosphate 2-phosphohydrolase and probably glycerylphosphorylcholine diesterase are associated with the brush borders of the convoluted part of the tubule (pars convoluta).     

Potentiometric and colorimetric methods for the assay of 2',3'-cyclic nucleotide 3'-phosphohydrolase

Abstract— A potentiometric titration method for the assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase is presented. Progress curves of the reaction were recorded automatically by pH-stat. 2-Mercaptoethanol was added to the reaction mixture to maintain a linear rate of reaction. The method is suitable for obtaining kinetic parameters and can be used for the rapid assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in nervous tissues. An improved colorimetric method for estimation of 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity at the optimum pH is described. This method employs the two-step procedure in which decyclization by 2′,3′-cyclic nucleotide 3′-phosphohydrolase and dephosphorylation by Escherichia coli alkaline phosphatase (EC 3.1.3.1) are carried out separately under the optimum conditions for each enzyme. The method is sensitive and most convenient for routine assays.     

Improved rapidity and precision in the determination of brain 2',3'-cyclic nucleotide 3'-phosphohydrolase

A rapid and precise method for the determination of brain 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) activity has been developed. Total brain homogenates were treated with deoxycholate, and CNP activity was measured as inorganic phosphate (phosphomolybdic acid, 410 nm) released from the product, 2′-AMP, by alkaline phosphatase. Measurements were carried out under optimal conditions of temperature (30°C) and pH (6.2) using the whole brain of the rat, chicken, and quaking mouse. The entire assay was applicable to multiple samples and could be completed in less than 1 hr.     

Cyclic hydrolase-transfected 3T3 cells have low levels of inositol 1,2-cyclic phosphate and reach confluence at low density.

The cDNA that encodes inositol-1,2-cyclic phosphate 2-phosphohydrolase (cyclic hydrolase), an enzyme that converts inositol 1,2-cyclic phosphate (cIns(1,2)P) to inositol 1-phosphate, was expressed in 3T3 cells to investigate the function of inositol cyclic phosphates. Cells with increased cyclic hydrolase activity had lower levels of cIns(1,2)P and grew to a lower density at confluence than control cells. This relationship was strengthened by the demonstration that several cell types with differences in cyclic hydrolase activity had levels of cIns(1,2)P and saturation densities that also correlated inversely with cyclic hydrolase activity. In addition, cyclic hydrolase activity is higher in cells at confluence compared to subconfluence. These results suggest that cellular cIns(1,2)P levels are determined by cyclic hydrolase activity and play a role in the control of cell proliferation.     

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.     

Isolation of D-myo-inositol 1:2-cyclic phosphate 2-inositolphosphohydrolase from human placenta

We have isolated D-myo-inositol 1:2-cyclic phosphate 2-inositolphosphohydrolase (EC 3.1.4.36) from human placenta. This enzyme catalyzes the conversion of inositol 1:2-cyclic phosphate to inositol 1-phosphate. The enzyme was purified 1300-fold to apparent homogeneity from the soluble fraction of human placenta. The enzyme requires Mn2+ or Mg2+ ions for activity, has an apparent Km for inositol 1:2-cyclic phosphate of 0.15 mM and forms 2.2 mumol of inositol 1-phosphate/min/mg protein. The enzyme does not utilize the cyclic esters of inositol polyphosphates as substrates. The molecular weight determined by gel filtration chromatography is approximately 55,000. Upon electrophoresis in polyacrylamide gels in sodium dodecyl sulfate, the molecular weight was found to be 29,000 both in the presence and absence of beta-mercaptoethanol. The enzyme was inhibited by inositol 2-phosphate (IC50 = 4 microM) and to a lesser degree by inositol 1-phosphate (IC50 = 2 mM) and inositol (IC50 = 4 mM). Zn2+ is a potent inhibitor of enzyme activity (IC50 = 10 microM). Neither Li+ nor Ca2+ had any effect on enzyme activity. This enzyme may serve to generate inositol from inositol cyclic phosphate metabolites produced by the phosphoinositide signaling pathway in cells.     

Studies on the pathophysiology of acute renal failure. II. A histochemical study of the proximal tubule of the rat following administration of mercuric chloride.

Acute renal failure was induced in male rats by the subcutaneous injectioon of 4 mg HgC12 per kg body weight. Enzyme activities of the proximal tubule were studied histochemically at six time intervals from 15 min to 24 h. The enzyme studied were alkaline phosphatase, 5'-nucleotidase, acid phosphatase, alpha-glycerophosphate dehydrogenase (NAD-independent), malic dehydrogenase, succinic dehydrogenase, latic dehydrogenase, glucose-6-phosphate dehydrogenase and glucose-6-phosphatase. Decreases in activity were observed for alkaline phosphatase and 5'-nucleotidase after 15 min. Acid phosphatase was decreased after 30 min. These three enzymes returned to control levels after 3 h, but malic dehydrogenase and alpha-glycerophosphate dehydrogenase were decreased at this time interval. Succinic dehydrogenase was first decreased after 6 h. The earliest morphological changes detectable by light microscopy were observed in pars recta tubules in the medullary rays after 6 h, a time when all enzymes studied showed widespread decreased activity throughout the proximal tubule. After 24 h, the pars convoluta appeared morphologically normal but the pars recta was necrotic and exhibited calcification, whereas enzyme activity was decreased (absent in some cases) in both pars convoluta and pars recta. These results support the hypothesis that Hg++, when given in a sublethal dose, is associated with early histochemical changes in the brush border of the proximal tubule, which may be related to early changes in sodium reabsorption and to the subsequent development of acute renal failure. The observation that changes in plasma membrane-associated enzymes occur early and prior to alterations in enzymes of mitochondria and the endoplasmic reticulum suggests that Hg++ interacts initially with the plasma membrane.     

Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.

The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.     

Quantification of nephrotoxicity in rabbits by automated morphometry of alkaline phosphatase stained kidney sections

Summary Male and female rabbits were injected intravenously with a single dose of either cefroxadine or cefsulodin or cephaloridine. Quantitative determinations of the activity of two brush border membrane enzymes, aminopeptidase and alaline phosphatase, were made in homogenates of cortical kidney tissue, in the urine and morphometrically in proximal tubules of cryostat sections. Morphometry was done by classification and enumeration of proximal tubule sections with the same level of enzyme reaction product using a microscopic television analysis system. By comparison with the control values, no changes were detectable 24 h after the injection of up to 1.2 g cefroxadine or cefsulodin per kg body weight. By contrast, after 300 mg/kg cephaloridine, the concentrations of the two enzymes were decreased in a large number of proximal tubules, i.e. the bursh border membranes, and concomitantly cell degeneration and necrosis took place. Alkaline phosphatase activity in sections and tissue homogenates was reduced to a greater extent than aminopeptidase activity. A corresponding, significant increase in enzymic activity in the urine was only demonstrable in respect of aminopeptidase. The classification of proximal tubules in tissue sections by television analysis on the basis of alkaline phosphatase reaction product concentration appears to be a reliable measure for detecting and quantifying toxic effects on proximal tubules of kidney.Dedicated to Prof. Dr. Gerhard Pfleiderer on the occasion of his 60th birthday     

Metabolic heterogeneity of the proximal and distal kidney tubules

Proximal and distal tubule suspensions were prepared from kidneys of Sprague-Dawley rats by an isolation procedure on a Percoll^R gradient. The marker enzymes alkaline phosphatase (brush border) and hexokinase (cytoplasmic) as well as p-aminohippurate transport capacity, gluconeogenic activity and electron microscopy were used to characterize the two kidney tubule suspensions. The results of this study indicate that cytochrome P-450 is localized to the proximal tubular cells and that the O-deethylation of 7- ethoxycoumarin was higher in the proximal than distal fraction. Both proximal and distal tubules showed glucuronidation and deacetylation capacities and a relatively equal distribution of non-protein sulfhydryls. These studies demonstrate metabolic heterogeneity of the nephron, the proximal tubule being the main site of renal xenobiotic metabolism. Understanding of metabolic heterogeneity of proximal and distal kidney tubules should provide important information regarding cell specific mechanisms of nephrotoxicity.     

Immunolocalization of tissue non-specific alkaline phosphatase in mice

 Immunolocalization of tissue non-specific alkaline phosphatase (TNAP) was examined in murine tissues, employing a specific antiserum to TNAP on frozen sections, 50-μm tissue slices, and paraffin sections. TNAP was detected at high levels in hard tissues including bone, cartilage, and tooth. In bone tissue, the TNAP immunoreactivity was localized on the entire cell surface of preosteoblasts, as well as the basolateral cell membrane of osteoblasts. It was also localized on some resting chondrocytes and most of the proliferative and hypertrophic cells in cartilage. In the incisor, cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts showed particularly strong immunoreactivity. Immunoreactivity was observed in other soft tissues, such as the brush borders of proximal renal tubules in kidney, on cell membrane of the biliary canalicula in liver and in trophoblasts in the placenta. These immunolocalizations were quite similar to enzyme histochemical localizations. However, neither the submandibular gland nor the intestine, which both exhibited alkaline phosphatase activity by enzyme histochemistry, revealed immunoreactivity for TNAP. Therefore, immunocytohistochemical studies for TNAP enabled us to localize the TNAP isozyme, thus distinguishing it from other isozymes. Accepted: 18 October 1996     

Restricted growth of rat kidney proximal tubule cells cultured in serum-supplemented and defined media

Proximal tubules suitable for in vitro culture were prepared from rat kidney cortex by a Ficoll-gradient centrifugation technique which yielded greater than 94% purity. The tubules were seeded into culture dishes, and cell growth was monitored in both Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and in a defined medium consisting of 50:50 Ham's F12 and Dulbecco's supplemented with insulin, transferrin, and hydrocortisone. Growth in serum-containing medium was continuous; however, the specific activity of the brush border enzyme alkaline phosphatase decreased rapidly with time, and the culture morphology became fibroblastic by 6 days. Neither collagen-coating of the dishes nor addition of the differentiation inducer hexamethylene-bisacetamide had any significant effect on growth or enzyme activity of the cultured cells. Theophylline, another inducer of differentiation, proved cytotoxic. Growth of proximal tubule cells in defined medium proceeded for 4 days before irreversible growth arrest occurred. Alkaline phosphatase activity and epithelial morphology remained relatively constant throughout the culture period. Additions of the growth factors triiodothyronine, prostaglandin E2, and epidermal growth factor were unable to unblock the growth arrest. If cells cultured in defined medium for 3 days were switched to serum-supplemented medium, continuous growth occurred, but both alkaline phosphatase activity and epithelial morphology were rapidly lost. As a test of the culture method, rabbit proximal tubule cells were cultured under similar conditions in defined medium. Growth was prolific and continuous for up to, but not exceeding, 30 days, and differentiated properties were retained. It was concluded that both rat and rabbit proximal tubule cells have a limited proliferative capacity in vitro but that the capacity of the rat cell to divide is much reduced relative to the rabbit cell.     

Isolation and characterization of the inositol cyclic phosphate products of phosphoinositide cleavage by phospholipase C. Metabolism in cell-free extracts

The phosphoinositides are metabolized by phospholipase C in response to hormone or agonist stimulation in many cell types to produce diglyceride and water-soluble inositol phosphates. We have recently shown that the phospholipase C reaction products include cyclic phosphate esters of inositol. One of these, inositol 1, 2-cyclic 4,5-trisphosphate, is active in promoting Ca2+ mobilization in platelets and in inducing changes in conductance in Limulus photoreceptors similar to those produced by light (Wilson, D. B., Connolly, T. M., Bross, T. E., Majerus, P. W., Sherman, W. R., Tyler, A., Rubin, L. J., and Brown, J. E. (1985) J. Biol. Chem. 260, 13496-13501. In the current study, we have examined the metabolism of the inositol phosphates. We find that both cyclic and non-cyclic inositol trisphosphates are metabolized by inositol 1,4,5-trisphosphate 5-phosphomonoesterase, to inositol 1,2-cyclic bisphosphate and inositol 1,4-bisphosphate, respectively. However, the apparent Km of the enzyme for the cyclic substrate is approximately 10-fold higher than for the non-cyclic substrate. These inositol bisphosphates are more slowly degraded to inositol 1,2-cyclic phosphate and inositol 1-phosphate, respectively. Inositol 1,2-cyclic phosphate is then hydrolyzed to inositol 1-phosphate, which in turn is degraded to inositol and inorganic phosphate by inositol 1-phosphate phosphatase. The human platelet inositol 1,2-cyclic phosphate hydrolase enzyme and a similar rat kidney hydrolase do not utilize the cyclic polyphosphate esters of inositol as substrates. These results suggest that the inositol cyclic phosphates and the non-cyclic inositol phosphates are metabolized separately by phosphatases to cyclic and non-cyclic inositol monophosphates. The cyclic monophosphate is then converted to inositol 1-phosphate by a cyclic hydrolase. We suggest that the enzymes that metabolize the inositol phosphates may serve to regulate cellular responses to these compounds.     

Subcellular localization of rat kidney phosphate independent glutaminase

The phosphate independent glutaminase is contained in the brush border membrane of the rat kidney proximal tubule cells. This glutaminase activity cofractionates with the brush border membrane marker activities, alkaline phosphatase and γ-glutamyltranspeptidase, during differential centrifugation. About 30% of these activities are recovered with the mitochondrial fraction, the remainder is pelleted in the heavy microsomal fraction. The phosphate independent glutaminase in both fractions bands, during isopycnic centrifugation, with a mean density of 1.16–1.17 and is coincident with both brush border membrane marker activities. The isolation of intact, individual kidney cells was accomplished by initial perfusion of the kidneys in situ with a collagenase-papain solution followed by a brief incubation in the same enzyme solution. Incubation of isolated cells with a higher concentration of papain results in selective release of the phosphate independent glutaminase. The fact that this occurs without appreciable release of a cytoplasmic marker activity, lactate dehydrogenase, suggests that the phosphate independent glutaminase may be localized on the external surface of the kidney cells.     

Identification of the microvillar 110-kDa calmodulin complex (myosin-1) in kidney.

The epithelial layer lining the proximal convoluted tubule of mammalian kidney contains a brush border of numerous microvilli. These microvilli appear in structure to be very similar to the microvilli on epithelial cells of the small intestine. Microvilli found in both the small intestine and the proximal convoluted tubules in kidney have a core bundle of actin filaments bundled by the accessory proteins villin and fimbrin. Along the length of intestinal microvilli, lateral links can be observed to connect the core bundle of actin filaments to the membrane. These cross-bridges are comprised of a 110-kDa calmodulin complex which belongs to a class of single-headed myosin molecules, collectively referred to as myosin-1. We now report that an analogous calmodulin-binding polypeptide of 105 kDa has been identified in rat kidney cortex. The 105-kDa polypeptide is preferentially found in purified kidney brush borders, can be extracted with ATP, and co-elutes with calmodulin on gel filtration and anion exchange chromatography. Fractions containing the 105-kDa polypeptide exhibit a modest ATPase activity in buffer containing CaCl2. The partially purified 105-kDa polypeptide will bind iodinated calmodulin and will sediment with F-actin in buffer containing ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or Ca2+. The addition of ATP partially reverses this association with F-actin. These results indicate that myosin-1, in addition to its presence in intestinal brush borders, is present in the brush border of kidney. We also provide preliminary evidence to indicate that the 105-kDa polypeptide is not restricted to tissues possessing a brush border.     

Chromosomal localization of the human annexin III (ANX3) gene

The annexins or lipocortins are a new family of calcium-dependent phospholipid-binding proteins. Annexin III has been previously identified as inositol 1,2-cyclic phosphate 2-phosphohydrolase (EC 3.1.4.36), an enzyme of inositol phosphate metabolism, and also as placental anticoagulant protein III, lipocortin III, calcimedin 35-alpha, and an abundant neutrophil cytoplasmic protein. In this study, the gene (ANX3) encoding annexin III was localized to human chromosome 4 at band q21 (q13-q22) by (1) polymerase chain reaction analysis of a human-rodent hybrid cell panel, confirmed by genomic Southern blot analysis of the same panel with a cDNA probe and (2) in situ hybridization with a cDNA probe.     

Identification of a phosphodiesterase that converts inositol cyclic 1:2-phosphate to inositol 2-phosphate.

Inositol 2-phosphate (Ins(2)P) has been identified in several cell types. The cellular levels of Ins(2)P appear to be directly correlated with the levels of inositol 1:2-cyclic phosphate (cIns(1:2)P) (Ross, T. S., Wang, F. P., and Majerus, P. W. (1992) J. Biol. Chem. 267, 19919-19923). In this study we have detected an enzyme in extracts from CV-1 cells and rat cerebellum that converts cIns(1:2)P to Ins(2)P and inositol 1-phosphate. This enzyme (designated cyclic hydrolase II) is not the same protein previously designated cIns(1:2)P 2-phosphohydrolase (cyclic hydrolase I). The products, heat inactivation curves, pH optima, and metal dependence of these two activities are different, and the two activities were separated by DEAE and gel filtration chromatography. Mixing of cyclic hydrolase I with cyclic hydrolase II does not effect the activity of either. The Km of the CV-1 cyclic hydrolase II for D-cIns(1:2)P is 10 microM. The enzyme is approximately 55 kDa as estimated by gel filtration analysis in the presence of sodium chloride and 120 kDa in its absence.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca2+/Mg2+)-ATPase,and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Intestinal brush border hydrolase topography. Effects of vitamin D-3 and filipin

Intestinal brush borders were isolated from vitamin D-3-treated and vitamin D-deficient chicks, and protein topography in the paired preparations assessed by the enzymatic release of four marker hydrolases. Exposure of the brush borders to the protease bromelain resulted in soluble levels of alkaline phosphatase, leucine aminopeptidase, maltase, and sucrase activities from preparations of vitamin D-3-treated birds that were 42%, 75%, 64%, and 56%, respectively, of corresponding activities released in preparations from rachitic chicks. Analyses for recovery of enzyme activity revealed that bromelain treatment selectively inactivated 43% of the alkaline phosphatase activity of brush borders obtained from vitamin D-3-replete birds, and preferentially diminished recovered sucrase activity in preparations from vitamin D-deficient chicks. In additional experiments, brush borders isolated from rachitic birds were treated in vitro with the polyene antibiotic filipin or an equivalent volume of vehicle. Subsequent exposure of such preparations to bromelain resulted in little or no differences in levels of marker hydrolase specific activities released from filipin- or vehicle-treated brush borders. However, analyses of membrane-bound specific activities after treatment of brush border preparations with a range of filipin concentrations, revealed a biphasic inhibition of approx. 30% for both maltase and sucrase, relative to vehicle controls, and a smaller effect on alkaline phosphatase and leucine aminopeptidase.     

Histological and enzyme histochemical changes in the kidney of male bullhead (Cottus gobio) during the spawning period

Histological and enzyme histochemical studies were carried out on the excretory kidney of the male bullhead ( Cottus gobio ). During the spawning season striking morphofunctional changes were observed in the second proximal segment of the kidney tubule. The tubular epithelium was greatly hypertrophied, strongly basophilic and produced a PAS-positive secretion. The enzyme histochemical pattern also changed conspicuously during this time: the alkaline phosphatase activity in the brush border was greatly reduced; the acid phosphatase and non-specific esterase activity in the cytoplasm was distinctly elevated.