Achieving the ratiometric imaging of steroid sulfatase in living cells and tissues with a two-photon fluorescent probe.

Herein, a novel two-photon ratiometric fluorescence assay was proposed for monitoring endogenous steroid sulfatase (STS) activity, which could be applied for the ratiometric imaging of STS activity in the endoplasmic reticulum of living cells and tissues and also could be used to distinguish estrogen-dependent tumor cells from other types of cells.

Is enzymatic hydrolysis a reliable analytical strategy to quantify glucuronidated and sulfated polyphenol metabolites in human fluids?

Phenolic compounds are present in human fluids (plasma and urine) mainly as glucuronidated and sulfated metabolites. Up to now, due to the unavailability of standards, enzymatic hydrolysis has been the method of choice in analytical chemistry to quantify these phase II phenolic metabolites. Enzymatic hydrolysis procedures vary in enzyme concentration, pH and temperature; however, there is a lack of knowledge about the stability of polyphenols in their free form during the process. In this study, we evaluated the stability of 7 phenolic acids, 2 flavonoids and 3 prenylflavanoids in urine during enzymatic hydrolysis to assess the suitability of this analytical procedure, using three different concentrations of ß-glucuronidase/sulfatase enzymes from Helix pomatia. The results indicate that enzymatic hydrolysis negatively affected the recovery of the precursor and free-form polyphenols present in the sample. Thus, enzymatic hydrolysis does not seem an ideal analytical strategy to quantify glucuronidated and sulfated polyphenol metabolites.

Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an "in-house" epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of 3H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using ß-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because ß-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used ß-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that ß-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.

A high-throughput robotic sample preparation system and HPLC-MS/MS for measuring urinary anatabine, anabasine, nicotine and major nicotine metabolites.

BACKGROUND: Most sample preparation methods characteristically involve intensive and repetitive labor, which is inefficient when preparing large numbers of samples from population-scale studies. METHODS: This study presents a robotic system designed to meet the sampling requirements for large population-scale studies. Using this robotic system, we developed and validated a method to simultaneously measure urinary anatabine, anabasine, nicotine and seven major nicotine metabolites: 4-Hydroxy-4-(3-pyridyl)butanoic acid, cotinine-N-oxide, nicotine-N-oxide, trans-3'-hydroxycotinine, norcotinine, cotinine and nornicotine. We analyzed robotically prepared samples using high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry in positive electrospray ionization mode using scheduled multiple reaction monitoring (sMRM) with a total runtime of 8.5 min. RESULTS: The optimized procedure was able to deliver linear analyte responses over a broad range of concentrations. Responses of urine-based calibrators delivered coefficients of determination (R(2)) of >0.995. Sample preparation recovery was generally higher than 80%. The robotic system was able to prepare four 96-well plate (384 urine samples) per day, and the overall method afforded an accuracy range of 92-115%, and an imprecision of <15.0% on average. CONCLUSIONS: The validation results demonstrate that the method is accurate, precise, sensitive, robust, and most significantly labor-saving for sample preparation, making it efficient and practical for routine measurements in large population-scale studies such as the National Health and Nutrition Examination Survey (NHANES) and the Population Assessment of Tobacco and Health (PATH) study.

Contamination of deconjugation enzymes derived from Helix pomatia with the plant bioactive compounds 3,3'-diindolylmethane, 5-methoxypsoralen, and 8-methoxypsoralen.

Bioactive compounds from plant foods are intensely investigated for effects on disease prevention. ß-Glucuronidase/arylsulfatase from Helix pomatia (snail) is commonly used when quantifying exposure to metabolized dietary components. However, we describe here the contamination of multiple formulations of this enzyme preparation with 3,3'-diindolylmethane (DIM), 8-methoxypsoralen (8-MOP), and 5-methoxypsoralen (5-MOP), bioactives from cruciferous and apiaceous vegetables under investigation as putative cancer chemopreventive agents. We identified an Escherichia coli preparation of ß-glucuronidase as free from contamination with any of the compounds tested. These results demonstrate the importance of selecting appropriate enzyme preparations when quantifying naturally occurring, trace level compounds in biological fluids.

Changes in the expression and current of the Na+/K+ pump in the snail nervous system after exposure to a static magnetic field.

Compelling evidence supports the use of a moderate static magnetic field (SMF) for therapeutic purposes. In order to provide insight into the mechanisms underlying SMF treatment, it is essential to examine the cellular responses elicited by therapeutically applied SMF, especially in the nervous system. The Na(+)/K(+) pump, by creating and maintaining the gradient of Na(+) and K(+) ions across the plasma membrane, regulates the physiological properties of neurons. In this study, we examined the expression of the Na(+)/K(+) pump in the isolated brain-subesophageal ganglion complex of the garden snail Helix pomatia, along with the immunoreactivity and current of the Na(+)/K(+) pump in isolated snail neurons after 15 min exposure to a moderate (10 mT) SMF. Western blot and immunofluorescence analysis revealed that 10 mT SMF did not significantly change the expression of the Na(+)/K(+) pump α-subunit in the snail brain and the neuronal cell body. However, our immunofluorescence data showed that SMF treatment induced a significant increase in the Na(+)/K(+) pump α-subunit expression in the neuronal plasma membrane area. This change in Na(+)/K(+) pump expression was reflected in pump activity as demonstrated by the pump current measurements. Whole-cell patch-clamp recordings from isolated snail neurons revealed that Na(+)/K(+) pump current density was significantly increased after the 10 mT SMF treatment. The SMF-induced increase was different in the two groups of control snail neurons, as defined by the pump current level. The results obtained could represent a physiologically important response of neurons to 10 mT SMF comparable in strength to therapeutic applications.

Antioxidant and molecular chaperone defences during estivation and arousal in the South American apple snail Pomacea canaliculata.

The invasive Pomacea canaliculata estivates during periods of drought and should cope with harmful effects of reoxygenation during arousal. We studied thiobarbituric acid reactive substances (TBARS), enzymatic (superoxide dismutase, SOD and catalase, CAT) and non-enzymatic antioxidants (uric acid and reduced glutathione), and heat shock protein expression (Hsc70, Hsp70 and Hsp90) in (1) active control snails, (2) snails after 45 days of estivation, and (3) aroused snails 20 min and (4) 24 h after water exposure, in midgut gland, kidney and foot. Both kidney and foot (but not the midgut gland) showed a TBARS increase during estivation and a decrease after arousal. Tissue SOD and CAT did not change in any experimental groups. Uric acid increased during estivation in all tissues, and it decreased after arousal in the kidney. Allantoin, the oxidation product of uric acid, remained constant in the midgut gland but it decreased in the kidney until 20 min after arousal; however, allantoin levels rose in both kidney and foot 24 h after arousal. Reduced glutathione decreased during estivation and arousal, in both midgut gland and kidney, and it remained constant in the foot. Hsc70 and Hsp70 kidney levels were stable during the activity-estivation cycle and Hsp90 expression decreases during estivation and recovers in the early arousal. In foot, the expression of Hsp70 and Hsp90 was high during activity and estivation periods and disminished after arousal. Results indicate that a panoply of antioxidant and molecular chaperone defences may be involved during the activity-estivation cycle in this freshwater gastropod.

Using the enzymatic and non-enzymatic antioxidant defense system of the land snail Eobania vermiculata as biomarkers of terrestrial heavy metal pollution.

The present study aimed to investigate the efficacy of antioxidant defense system of the land snail Eobania vermiculata as biomarkers for terrestrial heavy metal pollution. For this reason, a set of biomarkers was performed on land snails E. vermiculata collected from polluted and non-polluted areas in the field. The biomarkers used were lactate dehydrogenase activity, level of lipid peroxidation, activities of catalase, glutathione peroxidase, glutathione-S-transferase, gamma-glutamyl transferase and content of glutathione, metalothionines, total proteins and total lipid in the digestive gland tissue. The correlation between the bioaccumulation of heavy metal in gland tissue and all the biomarkers in the digestive gland was determined. The results revealed appreciable alterations in the biomarker values in field, accompanied by significant correlations among the biomarkers. Therefore, this study suggests E. vermiculata is a suitable sentinel organism and the selected biomarkers show efficacy for terrestrial heavy metal biomonitoring.

Possible evidence of contamination by catechins in deconjugation enzymes from Helix pomatia and Abalone entrails.

ß-Glucuronidase and sulfatase are the major deconjugating enzymes used in the cleavage of the glucuronate and sulfate moieties, respectively, from certain conjugated food factors including polyphenols. In the present study, we found that compounds having the same molecular weights as catechins were present in Helix pomatia- and/or Abalone entrails-derived ß-glucuronidase and sulfatase by liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring methods. On the other hand, the same molecular weights as catechins were undetectable in Escherichia coli-derived ß-glucuronidase and Aerobacter aerogenes-derived sulfatase. By high performance liquid chromatography, enzyme-derived catechins were not detected because of approximately 1,000-fold lower sensitivity as compared to LC-MS/MS. These results suggest that the catechins in these enzymes might be attributed to the diets of the organisms as the enzyme sources.

Bioassay based screening of steroid derivatives in animal feed and supplements.

Receptor binding transcription activation bioassays are valuable tools for the screening of steroid hormones in animal feed and supplements. However, steroid derivatives often lack affinity for their cognate receptor and do not show any direct hormonal activity by themselves. These compounds are thus not detected by these kinds of bioassays and need a bioactivation step in order to become active, both in vivo and in vitro. In this study a comparison was made between different in vitro activation methods for hormone esters and hormone glycosides. Testosterone acetate and testosterone decanoate were chosen as model compounds for the hormone esters, representing the broad range of steroid esters of varying polarities, while genistin was used as a substitute model for the steroid-glycosides. Concerning bioactivation of the steroids esters, the efficiency for alkaline hydrolysis was 90-100% and much better as compared to enzymatic deconjugation by esterase. As a result 1 µg testosterone ester per gram of animal feed could easily be detected by a yeast androgen bioassay. When comparing different enzyme fractions for deglycosilation, genistin was shown to be deconjugated most efficiently by ß-glucuronidase/aryl sulfatase from Helix pomatia, resulting in a significant increase of estrogenic activity as determined by a yeast estrogen bioassay. In conclusion, chemical and enzymatic deconjugation procedures for ester and glycoside conjugates respectively, resulted in a significant increase in hormonal activity as shown by the bioassay readouts and allowed effective screening of these derivatives in animal feed and feed supplements.

Phenoloxidase activity of intact and chemically modified functional unit RvH1: a from molluscan Rapana venosa hemocyanin.

o-Diphenol oxidase activities (o-diPO) of chemically modified functional unit RvH1-a of molluscan hemocyanin Rapana venosa were studied using L-Dopa and dopamine as substrates. With L-Dopa as substrate the native FU RvH1-a did not show any o-diPO activity. Therefore the native FU RvH1-a was converted to enzymatic active form, after treatment with SDS, trypsin, urea and different values of pH when its o-diPO activity was studied. The highest artificial induction of o-diPO activity was observed after incubation of FU with 3.0mM SDS, and RvH1-a shows both, dopamine (K(M)=6.53mM, k(cat)/K(M)=1.29) and L-Dopa (K(M)=2.0mM, k(cat)/K(M)=2.1) activity due to a more open active site of the enzyme and better access of the substrates. It was determined that the K(M) value of SDS-activated RvH1-a against dopamine is higher compared to those of hemocyanins from Helix vulgaris, Helix pomatia and native tyrosinase from Ipomoea batatas but much lower than that from Illex argentinus (ST94) tyrosinase and arthropodan hemocyanin from Carcinus aestuarii. The Km value of SDS-activated RvH1-a against L-Dopa is higher than those of hemocyanins from H. vulgaris and Cancer magister, but lower than that of the tyrosinase from Streptomyces albus.

Acetylcholine inhibits nitric oxide (NO) synthesis in the gastropod nervous system.

Acetylcholine (ACh) is one of the main signals regulating nitric oxide synthase (NOS) expression and nitric oxide (NO) biosynthesis in mammals. However, few comparative studies have been performed on the role of ACh on NOS activity in non-mammalian animals. We have therefore studied the cholinergic control of NOS in the snail Helix pomatia and compared the effects of ACh on NO synthesis in the enteric nervous system of the snail and rat. Analyses by the NADPH-diaphorase reaction, immunocytochemistry, purification with ion-exchange chromatography, Western-blot, and quantitative polymerase chain reaction have revealed the expression of neuronal NOS in the rat intestine and of a 60-kDa subunit of NOS in the enteric nerve plexus of H. pomatia. In H. pomatia, quantification of the NO-derived nitrite ions has established that NO formation is confined to the NOS-containing midintestine. Nitrite production can be elevated by L-arginine but inhibited by N(omega)-nitro-L-arginine. In rats, ACh moderately elevates nitrite production, whereas ACh, the nicotinic receptor agonists (nicotine, acetyl thiocholine iodide, metacholine) and the cholinesterase inhibitor eserine reduce enteric nitrite formation in snails. The nicotinic receptor antagonist tubocurarine also provokes nitrite liberation, whereas the muscarinic receptor agonists or antagonists have no significant effect in snails. In the presence of EDTA or tetrodotoxin, ACh fails to inhibit nitrite production. In pharmacological studies, we have found that ACh contracts the midintestinal muscles and, in snails, simultaneously reduces the antagonistic muscle relaxant effect of L-arginine. Our experiments provide the first evidence for an inhibitory regulation of neuronal NO synthesis by ACh in an invertebrate species.

Pro-oxidant effects of extremely low frequency electromagnetic fields in the land snail Helix aspersa.

Pro-oxidant effects of extremely low frequency (ELF) 50-Hz magnetic fields were investigated in the land snail Helix aspersa exposed both in short-term laboratory treatments and under field conditions by maintaining the organisms in the proximity of a power line for up to 2 months. Oxidative perturbations were investigated as individual antioxidants (catalase, glutathione reductase, glutathione S-transferases, and total glutathione) and total scavenging capacity toward peroxyl radicals and hydroxyl radicals. Accumulation of lipid peroxidation products, destabilization of lysosomal membranes, and loss of DNA integrity were also evaluated as markers of cell damage. The overall results indicated an oxidative challenge caused by ELF magnetic fields with particularly prompt and sensitive responses for catalase, glutathione reductase, and the overall capability to neutralize peroxyl radicals. Cell injuries occurred to different extents according to duration and intensity of electromagnetic exposure and confirmed complex cause-effect relationships between pro-oxidant factors, efficiency of antioxidant defenses, and the onset of oxidative toxicity. This study highlights the importance of a multimarker approach for detecting a wide panel of biological responses, the necessity of investigating the long-term effects of early oxidative responses, and the role of ELF in enhancing susceptibility to other forms of pathologies or diseases.

Nucleotidase activities in membrane preparations of nervous ganglia and digestive gland of the snail Helix aspersa.

Nucleotide-metabolizing enzymes play an important role in the regulation of nucleotide levels. In the present report, we demonstrated an enzyme activity with different kinetic properties in membrane preparations of the nervous ganglia and digestive gland from Helix aspersa. ATPase and ADPase activities were dependent on Ca2+ and Mg2+ with pH optima approximately 7.2 and between 6.0 and 8.0 in digestive gland and nervous ganglia, respectively. The enzyme activities present in membrane preparations of these tissues preferentially hydrolyzed triphosphate nucleotides. In nervous ganglia, the enzyme was insensitive to the classical ATPases inhibitors. In contrast, in digestive gland, N-ethylmaleimide (NEM) produced 45% inhibition of Ca(2+)-ATP hydrolysis. Sodium azide, at 100 microM and 20 mM, inhibited Mg(2+)-ATP hydrolysis by 36% and 55% in digestive gland, respectively. The presence of nucleotide-metabolizing enzymes in these tissues may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in these species.

Some new aspects of 17alpha-estradiol metabolism in man.

17Alpha-estradiol (1,3,5(10)-estratriene-3,17alpha-diol) together with a tracer dose of the tritium-labeled compound was administered orally and sublingually to male volunteers. The serum concentrations of 17alpha-estradiol (free and liberated by enzymatic hydrolysis) were quantified by GC/MS, and the serum total radioactivity and urinary radioactivity excretion were determined. After oral administration, 17alpha-estradiol was rapidly and intensively conjugated; only tiny quantities of the free steroid (<1% of total) appeared in serum. Sublingual administration resulted in temporary (up to 3 h p.a.) higher serum levels of the free compound. The metabolite patterns obtained by TLC of extracts from serum and urine demonstrated that 17alpha-estradiol is the subject of a poor phase I metabolism in man. A great discrepancy was found in the serum concentrations of 17alpha-estradiol (free + conjugated) determined by GC/MS and the serum radioactivity expressed in 17alpha-estradiol equivalents. By TLC analysis of the steroid conjugates extracted from serum, various 17alpha-estradiol conjugate peaks were found. By enzymatic hydrolysis with beta-glucuronidase/aryl sulfatase from Helix pomatia they were only partially cleaved. Thus, the difference between the serum radioactivity and the 17alpha-estradiol levels determined by GC/MS had to be attributed to an incomplete conjugate hydrolysis. It has been shown with the synthesized 17alpha-estradiol sulfate conjugates that only the 3-sulfate is cleaved by enzymatic hydrolysis, whereas the 17-sulfate group resists enzymatic hydrolysis. The methanolysis procedure (acetyl chloride in MeOH) has proved to be an efficient method for cleaving both the 3-sulfate group and the 17-sulfate group. In contrast to the 17alpha-estradiol conjugates in serum, the urinary conjugates were intensively split by the enzyme preparation. From this, it has to be concluded that the serum conjugates were deconjugated and newly reconjugated before urinary excretion.

Influence of Helix pomatia enzyme preparations on the oxidative conversion of some clostebol acetate metabolites in urine.

Clostebol acetate (4-chloro-testosterone acetate) is an anabolic steroid used for fattening purposes in cattle breeding. To safeguard public health, its use has been prohibited by the European Commission since 1986. Screening for its urinary metabolites is therefore an important tool for the control of possible violations. Because those metabolites appear conjugated to glucuronic acid or sulfate, deconjugation prior to analysis is necessary. This work describes the variability in results seen with the use of various commercial preparations of Helix pomatia (SHP) for enzymatic hydrolysis of the conjugates. A simultaneous oxidative side reaction was observed, converting metabolites with a 3-OH-4-ene structure into a 3-oxo-4-ene structure. This was not observed when samples were incubated without enzyme or in the presence of heat-inactivated SHP. GC-MS analysis revealed oxidation of some metabolites of clostebol acetate, 4-chloro-4-androsten-3alpha-ol-17-one and 4-chloro-4-androsten-3alpha,17beta-diol, changing them into other metabolites, 4-chloro-4-androsten-3,1 7-dione and clostebol (4-chloro-testosterone), respectively. Based on the difference in cross-reactivities of the antibodies for these metabolites, comparative analysis in enzyme immunoassay, following enzymatic hydrolysis, confirmed this transformation. This oxidative conversion phenomenon could be of great importance when considering the choice or target analytes for screening bovine urine.

The effect of calcium pump inhibitors on the response of intracellular calcium to caffeine in snail neurones.

We have measured intracellular free calcium ([Ca(2+)]i) using Fura-2 or Ca(2+)-sensitive microelectrodes in voltage-clamped neurones of the snail, Helix aspersa. Caffeine-induced transient increases in [Ca(2+)]i were normally followed by a brief fall of [Ca(2+)]i below its pre-caffeine level. We investigated the cause of this undershoot by raising [Ca(2+)]i; and by inhibiting the plasma membrane or endoplasmic reticulum Ca ATPases (PMCA or SERCA respectively). When the cell membrane potential was decreased from -60 to -25mV, steady-state [Ca(2+)]i increased. The caffeine-induced transients were smaller while the undershoots were larger than in control conditions. When the PMCA was inhibited by high pH the steady-state [Ca(2+)]i increased by 100-400nM. The caffeine-induced [Ca(2+)]i increase and the subsequent undershoot both became larger. Injection of orthovanadate, which inhibits the PMCA and increases [Ca(2+)]i, did not block either effect of caffeine. But when the SERCA was inhibited by cyclopiazonic acid the undershoot disappeared. The phosphodiesterase inhibitor IBMX did not influence the undershoot. These results suggest that the undershoot is generated by the Ca(2+)] ATPase of the stores rather than that of the plasma membrane. Since the undershoot increased as [Ca(2+)]i increased, we conclude that at higher levels of [Ca(2+)]i the stores refill more rapidly.

Cloning and characterization of two cDNAs encoding sulfatases in the Roman snail, Helix pomatia.

The sulfatase from the snail Heli pomatia is widely used for analytical applications. We have investigated the content of sulfatases in H. pomatia, using a biochemical and a molecular approach. A 112-kDa protein from the intestinal juice of H. pomatia comigrated with sulfatase activity when chromatographed on Sephacryl S300 and concanavalin A-Sepharose. The N-terminal amino acid sequence of the protein was similar to one of three sulfatase motifs defined by sequence alignment of known sulfatases. Degenerate primers designed from the motifs and the N-terminal amino acid sequence obtained were used to generate PCR fragments and to isolate both a full-length and a 3'-truncated cDNA encoding H. pomatia sulfatases, designated SULF1 and SULF2. SULF1 consists of 503 amino acids and shows 53-55% identity to the mammalian arylsulfatase B. The amino acid sequence deduced from the 878-bp SULF2 cDNA fragment is 55% identical with SULF1. Both SULF1 and SULF2 contain the cysteine residue conserved in the active site of many sulfatases, which is known to be posttranslationally modified into formylglycine in eukaryotic sulfatases. However, the SULF1 and SULF2 cDNAs do not code for the protein purified. This indicates the presence of at least three sulfatase genes in H. pomatia.

Conversion of adenosine(5') oligophospho(5') adenosines into inosine(5') oligophospho(5') inosines by non-specific adenylate deaminase from the snail Helix pomatia.

Until now, the catabolism of adenosine(5')triphospho(5')adenosine (Ap3A) and adenosine(5')tetraphospho(5')adenosine (Ap4A) has been thought to commence with either hydrolytic or phosphorolytic cleavage of their oligophosphate chains, depending on the organism. Here, we show that in the extracts from the retractile 'foot' of the snail Helix pomatia deamination predominates; the adenosine moieties of these and other adenosine(5')oligophospho(5')adenosines (ApnAs) undergo successive deamination leading, via an inosine(5')oligophospho(5')adenosine (IpnA), to the corresponding inosine(5')oligophospho(5')inosine (IpnI). The reactions are catalyzed by the non-specific adenylate deaminase described earlier (Stankiewicz, A.J. (1983) Biochem. J. 215, 39-44). We describe TLC and HPLC systems which allow the separation of any of the deaminated derivatives from its parent compound; Ap2A, Ap3A, Ap4A or Ap5A. The Km values for these substrates are 20, 22, 32 and 39 microM, respectively, whereas the Km for 5'-AMP is 12 microM. Relative substrate specificities for these compounds amount to 25, 18, 14, 7 and 100. The enzyme was shown also to deaminate phosphonate and thiophosphate analogues of Ap3A.

Localization and distribution of alkaline phosphatase activity in the hepatopancreas of the snail.

Histocytochemical methods were used to investigate alkaline phosphatase activity in the digestive gland (hepatopancreas) of the common garden snail Helix aspersa. Histochemical findings and light microscopic observations showed that enzymatic activity was confined mainly to the basal connective tissue that enveloped the adenomeres. Transmission electron microscopy showed that enzymatic activity was localized in the plasma membrane, and showed an intercellular distribution along the lateral surfaces and the basal portions of the cells in different adenomeres. Alkaline phosphatase activity was also found in the plasma membrane of fibrocytes of the basal connective tissue enveloping the adenomeres. Enzymatic activity was seen around the fat droplets of glandular cells. The possible involvement of alkaline phosphatase in processes or remodelling of the basal connective tissue that envelopes the gland is discussed.