Ion and water balance in Gryllus crickets during the first twelve hours of cold exposure

Insects lose ion and water balance during chilling, but the mechanisms underlying this phenomenon are based on patterns of ion and water balance observed in the later stages of cold exposure (12 or more hours). Here we quantified the distribution of ions and water in the hemolymph, muscle, and gut in adult Gryllus field crickets during the first 12 h of cold exposure to test mechanistic hypotheses about why homeostasis is lost in the cold, and how chill-tolerant insects might maintain homeostasis to lower temperatures. Unlike in later chill coma, hemolymph [Na⁺] and Na⁺ content in the first few hours of chilling actually increased. Patterns of Na⁺ balance suggest that Na⁺ migrates from the tissues to the gut lumen via the hemolymph. Imbalance of [K⁺] progressed gradually over 12 h and could not explain chill coma onset (a finding consistent with recent studies), nor did it predict survival or injury following 48 h of chilling. Gryllus veletis avoided shifts in muscle and hemolymph ion content better than Gryllus pennsylvanicus (which is less chill-tolerant), however neither species defended water, [Na⁺], or [K⁺] balance during the first 12 h of chilling. Gryllus veletis better maintained balance of Na⁺ content and may therefore have greater tissue resistance to ion leak during cold exposure, which could partially explain faster chill coma recovery for that species.     

Sexual and seasonal variations in osmoregulation and ionoregulation in the estuarine crab Chasmagnathus granulatus (Crustacea, Decapoda)

The estuarine crab Chasmagnathus granulatus (Crustacea, Decapoda, Brachyura) inhabits salt marshes along the South Atlantic coast from Rio de Janeiro (Brazil) to Patagonia (Argentina). In the present study, salinity tolerance (0-45‰; 16-1325 mOsm/kg H₂O) and hemolymph osmotic and ionic (Na⁺, Cl⁻, and K⁺) regulation in both female and male C. granulatus were analyzed in summer and winter. Results showed that both female and male C. granulatus are euryhaline. Mortality was only observed in extremely low salinity (0‰; 16 mOsm/kg H₂O) for both sexes. For females, the LT₅₀ at 0‰ salinity was similar in summer (20.1 h) and winter (17.4 h). Males were more tolerant to salinity than females in both seasons, and mortality was observed only in summer (LT₅₀ = 50.9 h). Results from freshly collected crabs or long-term (16-day) osmotic and ionic regulation experiments in the laboratory showed that male C. granulatus is a better hyper-osmoregulator than female in summer and winter. However, a hypo-osmoregulatory ability was only observed in females experimentally subjected to salinity 40‰ (1176 ± 11 mOsm/kg H₂O) in both seasons. In both sexes, hyper-osmotic regulation was achieved by hyper-regulating hemolymph Na⁺, Cl⁻, and K⁺ concentration. In females, hypo-osmotic regulation was achieved by hypo-regulating hemolymph Na⁺ and Cl⁻ concentration. Long-term (16-day) osmotic and ionic regulations in different salinities were similar in males or females collected and tested in summer and winter. Despite this lack of a seasonal effect on hemolymph osmoregulatory and ionoregulatory patterns in males or females, a marked seasonal difference in the dynamics of these processes was observed for both sexes. In the first 2 days after hypo-osmotic shock (20‰→5‰; 636→185 mOsm/kg H₂O), variations in female osmolality and ion (Na⁺ and Cl⁻) concentration were larger and faster in winter than in summer, while in males the opposite was observed. Furthermore, a seasonal effect on the crab response to hyper-osmotic shock (20‰→40‰; 636→1176 mOsm/kg H₂O) was only observed in males. A new osmolality and ion (Na⁺ and Cl⁻) concentration steady state was faster achieved in winter than in summer. Regarding sexual differences, females showed a better capacity to hypo-regulate the hemolymph osmolality and Na⁺ concentration than males, even after a sudden increase in salinity (hyper-osmotic shock) in both seasons. On the other hand, males showed a better capacity to hyper-regulate the hemolymph osmolality and Na⁺ concentration than females, even after a sudden decrease in salinity (hypo-osmotic shock), especially in winter. Taken together, results reported in the present study suggest the need to consider both sex and collection season as important factors in future osmotic and ionic regulation studies in estuarine crabs.     

Praziquantel has no direct effect on (Na++K+)-ATPases and (Ca2+-Mg2+)ATPases of Schistosoma mansoni

Therapeutic concentrations of praziquantel produce a rapid and intense contraction of the human flatworm Schistosoma mansoni. As an action on ATPases responsible for calcium homeostasis arises as a possible explanation for the molecular mechanism of this effect, we tested here the effect of praziquantel on different preparations from male adult worms that were previously characterized for their content in (Na⁺+K⁺)-ATPase and (Ca²⁺-Mg²⁺)ATPase activities from different origins. Concentrations as high as 100 μM praziquantel did not inhibit (Na⁺+K⁺)-ATPase from tegument and carcass nor (Ca²⁺-Mg ²⁺)ATPase from heterogeneous (P₁) and microsomal (P₄) fractions. As 100 μM praziquantel was also without effect on calcium permeability of microsomal vesicles actively loaded with ⁴⁵Ca²⁺, the present results discard three hypotheses recently raised for the mechanism of praziquantel-induced contraction of S. mansoni.     

Chronic exposure to soil salinity in terrestrial species: Does plasticity and underlying physiology differ among specialized ground-dwelling spiders?

In salt marshes, the alternation of low and high tides entails rapid shifts of submersion and aerial exposure for terrestrial communities. In these intertidal environments, terrestrial species have to deal with an osmotic loss in body water content and an increase in sodium chloride concentration when salt load increases. In salt marshes, spiders represent an abundant arthropod group, whose physiological ecology in response to variations of soil salinity must be further investigated. In this study, we compared the effect of salinity on the survival and physiology of three species of Lycosidae; two salt marsh species (Arctosa fulvolineata and Pardosa purbeckensis) and one forest species (P. saltans). Spiders were individually exposed at three salinity conditions (0‰, 35‰ and 70‰) and survival, changes in body water content, hemolymph ions (Na⁺, Ca²⁺, Mg²⁺, K⁺; ICP-MS technique) and metabolites (mainly amino acids, polyols, sugars; LC and GC techniques) were assessed. The survival of the forest species P. saltans was very quickly hampered at moderate and high salinities. In this spider, variations of hemolymph ions and metabolites revealed a quick loss of physiological homeostasis and a rapid salt-induced dehydration of the specimens. Conversely, high survival durations were measured in the two salt-marsh spiders, and more particularly in A. fulvolineata. In both P. purbeckensis and A. fulvolineata, the proportion of Na⁺, Ca²⁺, Mg²⁺, K⁺ remained constant at the three experimental conditions. Accumulation of hemolymph Na⁺ and amino acids (mainly glutamine and proline) demonstrated stronger osmoregulatory capacities in these salt-marsh resident spiders. To conclude, even if phylogenetically close (belonging to the same, monophyletic, family), we found different physiological capacities to cope with salt load among the three tested spider species. Nevertheless, physiological responses to salinity were highly consistent with the realized ecological niches of the spiders.     

Chill-tolerant Gryllus crickets maintain ion balance at low temperatures

Insect cold tolerance is both phenotypically-plastic and evolutionarily labile, but the mechanisms underlying this variation are uncertain. Chill-susceptible insects lose ion and water homeostasis in the cold, which contributes to the development of injuries and eventually death. We thus hypothesized that more cold-tolerant insects will better maintain ion and water balance at low temperatures. We used rapid cold-hardening (RCH) and cold acclimation to improve cold tolerance of male Gryllus pennsylvanicus, and also compared this species to its cold-tolerant relative (Gryllus veletis). Cold acclimation and RCH decreased the critical thermal minimum (CT_min) and chill coma recovery time (CCR) in G. pennsylvanicus, but while cold acclimation improved survival of 0 °C, RCH did not; G. veletis was consistently more cold-tolerant (and had lower CCR and CT_min) than G. pennsylvanicus. During cold exposure, hemolymph water and Na⁺ migrated to the gut of warm-acclimated G. pennsylvanicus, which increased hemolymph [K⁺] and decreased muscle K⁺ equilibrium potentials. By contrast, cold-acclimated G. pennsylvanicus suffered a smaller loss of ion and water homeostasis during cold exposure, and this redistribution did not occur at all in cold-exposed G. veletis. The loss of ion and water balance was similar between RCH and warm-acclimated G. pennsylvanicus, suggesting that different mechanisms underlie decreased CCR and CT_min compared to increased survival at 0 °C. We conclude that increased tolerance of chilling is associated with improved maintenance of ion and water homeostasis in the cold, and that this is consistent for both phenotypic plasticity and evolved cold tolerance.     

GABA and glutamate fluxes in developing nerve endings

Radiolabeled GABA and glutamate transport into 7 day, 14 day and adult cortical nerve ending preparations was examined. Transport was measured at several Na⁺ concentrations, 19, 27, 43 and 121 mM, and at two temperatures, 15 and 30°C. K_m and V_max values were calculated for all experimental conditions by means of Wilkinson (1961) analysis. A comparison of the day 14 and adult data shows higher K_m values at all Na⁺ concentrations on day 14 for both GABA and glutamate transport. In addition, the temperature dependence of transport was attenuated in the day 14 preparation. Finally, the specificity of GABA transport, as measured by the use of the transport inhibitors β-alanine and 2,4-diaminobutyric acid, was not different between the day 14 and adult preparations. Overall, it is concluded that both GABA and glutamate transport into day 14 nerve endings behave as if “adult” transporter molecules were existing in a more fluid lipid environment, which is the situation found in synaptic membranes prepared from day 14 nerve endings (Hitzemann and Johnson, 1983).Glutamate and GABA transport into 7 day nerve endings is complex and shows marked differences from the day 14 and adult data. Day 7 GABA transport was significantly more sensitive to β-alanine inhibition. Day 7 transport was more sensitive to Na⁺ manipulation and the temperature dependent kinetics show unique Na⁺ effects not seen in the day 14 or adult preparations. For example, at 19 mM Na⁺, 7 day glutamate transport was more temperature dependent than adult transport but as the Na⁺ concentration was increased the reverse was true. The opposite situation for temperature-Na⁺ effects was seen for GABA transport. Finally, no Ca⁺²-dependent component of GABA release could be found in 7 day nerve endings while a significant component was found at day 14. Overall, it is concluded that both glutamate and GABA fluxes in 7 day nerve endings differ both qualitatively from that seen in both day 14 and adult nerve endings.     

Hemolymph ion composition and volume changes in the supralittoral isopod Ligia pallasii Brandt, during molt

We analyzed ion composition and volume of the hemolymph of Ligia pallasii in four different stages of the molt cycle using capillary electrophoresis and ³H-inulin. The main ions in the hemolymph were Na⁺, K⁺, Mg²⁺, Ca²⁺, and Cl⁻. The Ca²⁺concentration increased significantly during the molt by 47% from intermolt to intramolt and by 37% from intermolt to postmolt, probably due to resorption of Ca²⁺ from the cuticle and sternal CaCO₃ deposits. The K⁺ concentration increased significantly by 20% during molt. The hemolymph volume normalized to the dry mass of the animals decreased by 36% from intermolt to late premolt. This was due to a reduction in the hemolymph volume and to an increase in dry mass of the animals during premolt. A sudden increase in the hemolymph volume occurring between late premolt and intramolt served to expand the cuticle. Since the Na⁺, K⁺, Mg²⁺, and Cl⁻ concentrations did not change significantly from late premolt to intramolt, the increase in hemolymph volume suggests an uptake of seawater rather than freshwater. Accepted: 7 March 2000     

Effects of azadirachtin on six inorganic cation distributions in Ostrinia furnacalis (G.)

Azadirachtin (Az), as a botanical insecticide, is relatively safe and biodegradable. It affects a wide vaariety of biological processes, including the reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using ion chromatography, we analyzed changes in six inorganic cation (Li⁺, Na⁺, NH₄ ⁺, K⁺, Mg²⁺, and Ca²⁺) distributions of the whole body and hemolymph in Ostrinia furnacalis (G.) after exposure to sublethal doses of Az. The results showed that Az dramatically interfered with Na⁺, NH₄ ⁺, K⁺, Mg²⁺, and Ca²⁺ distributions in hemolymph of O. furnacalis (G.) and concentrations of these five cations dramatically increased. However, in the whole body, the levels of K⁺, Mg²⁺, and Ca²⁺ significantly, decreased after exposure to Az, except that Na⁺ and NH₄ ⁺ remained constant. Li⁺ was undetected in both the control and treated groups in the whole body and hemolymph. It is suggested that Az exerts its insecticidal effects on O. furnacalis (G.) by interfering with the inorganic cation distributions related to ion channels.     

Voltage-dependent Inwardly Rectifying Potassium Conductance in the Outer Membrane of Neuronal Mitochondria

Potassium fluxes integrate mitochondria into cellular activities, controlling their volume homeostasis and structural integrity in many pathophysiological mechanisms. The outer mitochondrial membrane (OMM) is thought to play a passive role in this process because K⁺ is believed to equilibrate freely between the cytosol and mitochondrial intermembrane space. By patch clamping mitochondria isolated from the central nervous systems of adult mitoCFP transgenic mice, we discovered the existence of I_OMMKi, a novel voltage-dependent inwardly rectifying K⁺ conductance located in the OMM. I_OMMKi is regulated by osmolarity, potentiated by cAMP, and activated at physiological negative potentials, allowing K⁺ to enter the mitochondrial intermembrane space in a controlled regulated fashion. The identification of I_OMMKi in the OMM supports the notion that a membrane potential could exist across this membrane in vivo and suggests that the OMM possesses regulated pathways for K⁺ uptake.     

A mosaic analysis of the apterous mutation in Drosophila melanogaster

An analysis of three phenotypes expressed by the apterous-four (ap⁴) mutant of Drosophila melanogaster has been carried out in $\text{ap}{}^4\text{ap}{}^{\mathrm{+}}$ mosaic adults. The wing and haltere deficiency phenotype was found to be autonomous for entirely mutant structures. However, small patches of mutant anterior and posterior wing margin cells can exist in mosaic wings. Examples of duplicated and triplicated lengths of bristle rows were often found associated with the existing mutant patches. About 20% of the mosaics expressed the phenotype of precocious adult death, dying 30–40 hr after eclosion. The focus for this phenotype was located in the posterior region of the abdomen, and a strong correlation was found between expression of this phenotype and the presence of mutant Malpighian tubules. The focus for juvenile hormone deficiency in ap⁴ adults was located near that for precocious adult death; in fact, the two foci may be identical. It is suggested that defective functioning of ap⁴ adult Malpighian tubules results in abnormal hemolymph leading to early death. Inadequate juvenile hormone secretion could result indirectly from impaired glandular functioning attributable to abnormal adult hemolymph.     

Mode of action of Bacillus thuringiensis δ-endotoxin: General characteristics of intoxicated Bombyx larvae

The general pathology induced by δ-endotoxin in terms of larval behavior and hemolymph chemistry has been widely studied in the so-called Type I insect, Bombyx mori. The succession of symptoms is divided into four arbitrary stages: Stage 0, appearance and locomotion normal, no feeding; Stage 1, slightly sluggish; Stage 2, extremely sluggish; and Stage 3, complete paralysis. The action of δ-endotoxin is highly specific to the midgut since contractile movement of both foregut and hindgut continues long after all locomotor activity and heartbeat have stopped. Immediately after the silkworm stops feeding and blood pH sharply rises, there is an associated abrupt rise in the K⁺ concentration of hemolymph. Thereafter, the rise in K⁺ is linear while the rise in pH is not. In vivo measurements have not yielded the same simple linear dependence of pH on K⁺ concentrations that is found in in vitro mixtures of hemolymph and midgut juice. Ligation experiments showed that the same pathological sequence (rise of pH and K⁺ concentration, and general paralysis) follows whether the toxin has unrestricted access to the entire midgut or only part of it (anterior or posterior). From the results of injections of midgut juice or various salt solutions into hemocoel, we came to the conclusions that the blood pH and the symptoms are not necessarily parallel and the intact midgut and Malpighian tubules have strong functions for ion regulation.     

Hemolymph volume of noninfected and Plasmodium berghei-infected Anopheles stephensi.

The hemolymph volume of Anopheles stephensi adult female mosquitoes was determined by a radioisotope dilution technique. [carboxy-¹⁴C]Inulin was injected into the hemocoels of mosquitoes with a calibrated capillary needle. After sufficient time for thorough mixing, the labeled hemolymph was collected from groups of 50 mosquitoes by a centrifugation technique. Total hemolymph volume was calculated by a conventional formula for radioisotope dilution. The mean hemolymph volume of the newly emerged adult female mosquitoes was 336 nl/mosquito. The ratio of hemolymph volume to body weight was 0.25 μl/mg body wt. By 14 days after emergence, hemolymph volume had dropped to 190 nl/mosquito. Infection of mosquitoes with the rodent malaria parasite, Plasmodium berghei, had no significant effect on hemolymph volume of the mosquito.     

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

Control of Diapause by Acidic pH and Ammonium Accumulation in the Hemolymph of Antarctic Copepods

Life-cycles of polar herbivorous copepods are characterised by seasonal/ontogenetic vertical migrations and diapause to survive periods of food shortage during the long winter season. However, the triggers of vertical migration and diapause are still far from being understood. In this study, we test the hypothesis that acidic pH and the accumulation of ammonium (NH₄ ⁺) in the hemolymph contribute to the control of diapause in certain Antarctic copepod species. In a recent study, it was already hypothesized that the replacement of heavy ions by ammonium is necessary for diapausing copepods to achieve neutral buoyancy at overwintering depth. The current article extends the hypothesis of ammonium-aided buoyancy by highlighting recent findings of low pH values in the hemolymph of diapausing copepods with elevated ammonium concentrations. Since ammonia (NH₃) is toxic to most organisms, a low hemolymph pH is required to maintain ammonium in the less toxic ionized form (NH₄ ⁺). Recognizing that low pH values are a relevant factor reducing metabolic rate in other marine invertebrates, the low pH values found in overwintering copepods might not only be a precondition for ammonium accumulation, but in addition, it may insure metabolic depression throughout diapause.     

Response of the insect immune system to three different immune challenges

Insects rely on an innate immune system to effectively respond to pathogenic challenges. Most studies on the insect immune system describe changes in only one or two immune parameters following a single immune challenge. In addition, a variety of insect models, often at different developmental stages, have been used, making it difficult to compare results across studies. In this study, we used adult male Acheta domesticus crickets to characterize the response of the insect innate immune system to three different immune challenges: injection of bacterial lipopolysaccharides (LPS); injection of live Serratia marcescens bacteria; or insertion of a nylon filament into the abdomen. For each challenge, we measured and compared hemolymph phenoloxidase (PO) and lysozyme-like enzyme activities; the number of circulating hemocytes; and the nodulation responses of challenged and un-challenged crickets. We found that injection of an LD₅₀ dose of LPS from Escherichia coli elicited a more rapid response than an LD₅₀ dose of LPS from S. marcescens. LPS injection could cause a rapid decrease 2 hpi, followed by an increase by 7 dpi, in the number of circulating hemocytes. In contrast, injection of live S. marcescens produced a rapid increase and then decrease in hemocyte number. This was followed by an increase in the number of hemocytes at 7 dpi, similar to that observed following LPS injection. Both LPS and live bacteria decreased hemolymph PO activity, but the timing of this effect was dependent on the challenge. Live bacteria, but not LPS, induced an increase in lysozyme-like activity in the hemolymph. Insertion of a nylon filament induced a decrease in hemolymph PO activity 2 h after insertion of the filament, but had no effect on hemocyte number or lytic activity. Our results indicate that the innate immune system’s response to each type of challenge can vary greatly in both magnitude and timing, so it is important to assess multiple parameters at multiple time points in order to obtain a comprehensive view of such responses.     

Impact of Recovery from Desensitization on Acid-sensing Ion Channel-1a (ASIC1a) Current and Response to High Frequency Stimulation

ASIC1a is a neuronal sodium channel activated by external H⁺ ions. To date, all the characterization of ASIC1a has been conducted applying long H⁺ stimuli lasting several seconds. Such experimental protocols weaken and even silence ASIC1a currents to repetitive stimulation. In this work, we examined ASIC1a currents by methods that use rapid application and removal of H⁺. We found that brief H⁺ stimuli, <100 ms, even if applied at high frequency, prevent desensitization thereby generate full and steady peak currents of human ASIC1a. Kinetic analysis of recovery from desensitization of hASIC1a revealed two desensitized states: short- and long-lasting with time constants of τ_Ds ≤0.5 and τ_Dl = 229 s, while in chicken ASIC1a the two desensitized states have similar values τ_D 4.5 s. It is the large difference in stability of the two desensitized states that makes hASIC1a desensitization more pronounced and complex than in cASIC1a. Furthermore, recovery from desensitization was unrelated to cytosolic variations in pH, ATP, PIP₂, or redox state but was dependent on the hydrophobicity of key residues in the first transmembrane segment (TM1). In conclusion, brief H⁺-stimuli maintain steady the magnitude of peak currents thereby the ASIC1a channel is well poised to partake in high frequency signals in the brain.     

Na-Stimulation of Photosynthesis in the Cyanobacterium Synechococcus UTEX 625 Grown on High Levels of Inorganic Carbon

Photosynthesis of washed cells of Synechococcus UTEX 625 grown on 5% CO₂ was markedly stimulated (647 ± 50%) at pH 8.0 by the addition of low concentrations of NaCl (concentration required for half-maximal response, K_½, = 18 micromolar). Studies with KCl and Na₂SO₄ showed that the stimulation was due to Na⁺. Photosynthesis at pH 6.1 was only slightly stimulated by Na⁺. The response of photosynthesis at pH 8.0 to [Na⁺] was strongly sigmoidal for dissolved inorganic carbon ([DIC] ≤ 500 micromolar). Cells grown with high total [DIC], but air-levels of CO₂, at pH 9.6 showed the same response to low [Na⁺]. The absence of Na⁺ could be partially, but not completely overcome, by higher [DIC]. Various methods for examining CO₂ or HCO₃⁻ use (K_½^CO₂ determination; isotopic disequilibrium; and consideration of HCO₃⁻ dehydration rate) were consistent with CO₂ use by the cells, but HCO₃⁻ use could not be ruled out. Isotopic disequilibrium studies showed that CO₂ use was stimulated by Na⁺. Cells grown on 5% CO₂ accumulated DIC against a concentration gradient by a process (or processes) dependent on Na⁺. No evidence for uptake of Na⁺ concomitant with DIC uptake could be found. The lack of O₂ evolution during the initial and most rapid period of DIC accumulation suggested that the required energy was obtained from cyclic photophosphorylation.     

High-performance liquid chromatographic–fluorimetric assay for cathepsin A (lysosomal protective protein) activity

A rapid and sensitive assay for the determination of cathepsin A activity is reported. This method is based on fluorimetric detection of a dansylated peptide, 5-dimethylaminonaphthalene-1-sulfonyl-l-Phe, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-l-Phe-l-Leu, after separation by high-performance liquid chromatography using a C₁₈ reversed-phase column and isocratic elution. This method is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl-l-Phe at concentrations as low as 300 fmol, yields highly reproducible results and requires less than 7.0 min per sample for separation and quantitation. The optimum pH for cathepsin A activity was 4.5–5.0. The K_m and V_max values were respectively 14.9 μM and 27.91 pmol/μg/h with the use of enzyme extract obtained from mouse kidney. Cathepsin A activity was strongly inhibited by Ag⁺, Hg²⁺, diisopropylfluorophosphate and p-chloromercuriphenylsulphonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in kidney. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.