Isolation,identification and synthesis of locustamyotropin II,an additional neuropeptide of Locusta migratoria: Member of the cephalomyotropic peptide family

An eight residue neuropeptide (Glu-Gly-Asp-Phe-Thr-Pro-Arg-Leu-NH₂) has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata-suboesophageal ganglion complexes of Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae. The peptide designated as locustamyotropin II, or Lom-MT II according to Raina and Gäde (Insect Biochem.18, 785–787, 1988), has a Phe-X-Pro-Arg-Leu-NH₂ carboxyl-terminal in common with the previously identified locustamyotropin I. Locustamyotropin II is also related to leucopyrokinin (Lem-PK), a blocked myotropic neuropeptide isolated from cockroach heads. Both peptides have identical carboxyterminal pentapeptide sequences. The constituent amino acids of this C-terminal are important for biological activity on the Leucophaea hindgut. Lom-MT II differs from Lem-PK in the first three aminoterminal residues. In contrast to Lem-PK and like Lom-MT I, the novel locust peptide is not N-terminally blocked. Lom-MT II has a stimulatory effect on the motility of the oviduct of Locusta but not on the hindgut.     

Cross-Epithelial Hydrogen Transfer from the Midgut Compartment Drives Methanogenesis in the Hindgut of Cockroaches

In the intestinal tracts of animals, methanogenesis from CO₂ and other C₁ compounds strictly depends on the supply of electron donors by fermenting bacteria, but sources and sinks of reducing equivalents may be spatially separated. Microsensor measurements in the intestinal tract of the omnivorous cockroach Blaberus sp. showed that molecular hydrogen strongly accumulated in the midgut (H₂ partial pressures of 3 to 26 kPa), whereas it was not detectable (<0.1 kPa) in the posterior hindgut. Moreover, living cockroaches emitted large quantities of CH₄ [105 ± 49 nmol (g of cockroach)⁻¹ h⁻¹] but only traces of H₂. In vitro incubation of isolated gut compartments, however, revealed that the midguts produced considerable amounts of H₂, whereas hindguts emitted only CH₄ [106 ± 58 and 71 ± 50 nmol (g of cockroach)⁻¹ h⁻¹, respectively]. When ligated midgut and hindgut segments were incubated in the same vials, methane emission increased by 28% over that of isolated hindguts, whereas only traces of H₂ accumulated in the headspace. Radial hydrogen profiles obtained under air enriched with H₂ (20 kPa) identified the hindgut as an efficient sink for externally supplied H₂. A cross-epithelial transfer of hydrogen from the midgut to the hindgut compartment was clearly evidenced by the steep H₂ concentration gradients which developed when ligated fragments of midgut and hindgut were placed on top of each other—a configuration that simulates the situation in vivo. These findings emphasize that it is essential to analyze the compartmentalization of the gut and the spatial organization of its microbiota in order to understand the functional interactions among different microbial populations during digestion.     

Isolation,identification and synthesis of locustapyrokinin II from Locusta migratoria,another member of the FXPRL-amide peptide family

1. A blocked decapeptide was isolated from brain corpora cardiaca-corpora allata sub-oesophageal ganglion extracts of the locust, Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae.2. The primary structure of this myotropic peptide was established as: pGlu-Ser-Val-Pro-Thr-Phe-Thr-Pro-Arg-Leu-NH₂.3. The Chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis.4. This decapeptide is the sixth natural analog of a series of locust peptides with a Phe-X-Pro-Arg-Leu-NH₂ carboxyterminus. This carboxyl terminal sequence is also found in other peptides identified in other insects and it is the biological active core sequence for diverse biological activities: muscle contraction, pheromone production, pigment synthesis and diapauze.5. Like the locustamyotropins and locustapyrokinin I, locustapyrokinin II stimulates contractions of the oviduct in Locusta.     

The diffusional and osmotic water permeability of the hindgut cuticle in larval Sarcophaga bullata

The diffusional water permeability (P_D) and the pressure filtration coefficient (L_P) of isolated larval hindgut cuticle of the fleshfly, Sarcophaga bullata, were measured using tracer techniques coupled with a simple mathematical model system based on equations of non-equilibrium thermodynamics. Data obtained from the model system were matched to experimental tracer data by means of a mathematical optimization scheme. The following parameter values were obtained: P_D for tritiated water = 1.02 × 10^?6 cm-sec^?1, and L_P = 9.18 × 10^?11 cm³-dyn^?1-sec^?1. These results are now being used to determine reflection coefficients (σ's) and solute mobilities (ω's) for the cuticle system in an attempt to gain an understanding of the mechanisms controlling solute and water movements across the hindgut wall.     

Apparent differences in tryptophan hydroxylation by cockroach (periplaneta americana) nervous tissue and rat brain

Tryptophan hydroxylation in cockroach (Periplaneta americana) nervous tissue was measured and compared to the hydroxylation of tryptophan in rat brain. Tryptophan hydroxylation in both tissues requires a pterine cofactor, and is inhibited by p-chlorophenylalanine. The molecular weight of the protein responsible for hydroxylation of tryptophan in cockroach nervous tissue obtained from gel filtration was estimated to be 54,000.The pH optima and enzyme kinetics differed greatly between the two hydroxylases. Hydroxylation of tryptophan by the enzyme obtained from cockroach tissues incubated with dimethyltetrahydropterine had a pH optimum of about 5.8–5.9 and a K_m in crude enzyme preparations of 2.6 × 10⁻⁶ M and is activity was substrate inhibited above 10⁻⁴ M tryptophan. Hydroxylation of tryptophan by the enzyme obtained from rat brain incubated with dimethyltetrahydropterine had a pH optimum of about 6.5–7.0, a K_m of about 6.7 × 10⁻⁴ M and exhibited no substrate inhibition at tryptophan concentrations up to 2 × 10⁻³ M.When incubated with biopterin, the presumed natural cofactor, the hydroxylase from cockroach tissues had a K_m of about 6.8 × 10⁻⁵ M and no substrate inhibition occurred at tryptophan concentrations up to 2 × 10⁻³ M. Under the same conditions rat hydroxylase had a K_m of 1.1 × 10⁻⁵M and substrate inhibition occurred above 10⁻⁴ M tryptophan.Unlike the mammalian situation, administration of tryptophan peripherally did not change the 5-hydroxytryptamine concentration in cockroach nervous tissue, but did increase tryptophan levels. The low V_max values of the cockroach hydroxylase and the inability of administered tryptophan to elevate 5-hydroxytryptamine levels suggest that in the cockroach hydroxylation of tryptophan itself may be the limiting factor in the biosynthesis of 5-hydroxytryptamine.     

Isolation,primary structure and synthesis of leucomyosuppressin,an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut

1. A neuropeptide that inhibits spontaneous contractions of the isolated cockroach hindgut was purified from head extracts of the cockroach, Leucophaea maderae.2. The inability of aminopeptidase M to degrade the peptide and the presence of glutamic acid in the hydrolysate suggested N-terminal blocking by pyroglutamic acid. The N-terminal pGlu was removed enzymatically and the unblocked fragment was sequenced with an automated peptide sequencer.3. The structure determined (pGlu-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH₂) was synthesized and shown to be both chemically and biologically identical with the natural product.4. Leucomyosuppressin is the first inhibitory neuropeptide isolated and structurally identified from an insect source.     

Isolation and primary structure of two sulfakinin-like peptides from the fleshfly,Neobellieria bullata

1. Two novel insect myotropic peptides termed neosulfakinin-I (Neb-SK-I) and neosulfakininII (Neb-SK-II) were isolated from the heads of 42 thousand fleshflies, Neobellieria bullata (Diplera, Sarcophagidae).2. A series of four, high-performance liquid Chromatographic (HPLC), fractionations performed on columns with different characteristic features yielded two purified biologically active, hindgut motility stimulating fractions, suitable for amino acid sequence analysis.3. The proposed sequences for the two peptides are: Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH₂), (Neb-SK-I) and X-X-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH₂), (Neb-SK-II).4. These sulfakinins exhibit very high homology to putative drosulfakinin sequences which, however, have not yet been isolated, but were deduced from a cloned Drosophila gene encoding these peptides.5. Here we provide the first evidence for the expression of such peptides present in Dipterans.6. Insect sulfakinins show structural identities with the hormonally-active portion of vertebrate gastrin II-, cholecystokinin- and caerulin-related peptides and they share common carboxy terminal sequences with invertebrate/vertebrate peptides of the FMRFamide peptide family.     

Rat atrial natriuretic factor: Isolation,structure and biological activities of four major peptides

Four peptides possessing both natriuretic activity and smooth muscle relaxant activity were isolated from rat atrium and their amino acid sequences determined. The four peptides designated ANF-I, ANF-II, ANF-III and ANF-IV containing 35, 31, 30 and 25 amino acid residues, respectively, were obtained in a molar ratio of 4:60:20:16. The predominant species ANF-II, which may represent the native form of ANF, has the following sequence: (H₂N)-G-P-R-S-L-R-R-S-S-C-F-G-G-R-I-D-R-I-G-A-Q-S-G-L-G-C-N-S-F-R-Y-(COOH) in which Cys-10 and Cys-26 are disulfide linked. Cleavage of the aspartyl linkage at position 16 by staphylococcal protease caused complete inactivation, indicating that the ring conformation is essential. The dose-response relationships determined for the four pepdides in relaxing norepinephrine-induced contraction of rabbit thoracic aorta showed half-maximum relaxation at concentrations ranging from 1.5 × 10^?9 to 2.5 × 10^?9 M. Comparable dose-response relationships were observed in relaxation of carbacol-induced contraction of chick rectum strips as tested with ANF-II and ANF-IV.     

Levels of aflatoxin B1, bacteria and fungi in feed and food in 1971 — 1987

Totally 39% out of 8371 feed and their component samples were contaminated by aflatoxin B₁. Mean contamination was 36μg/kg with maximum yield 10100 μg/kg. Contamination of samples by total count of organisms, mean contamination and maximum yield, respectively was: 1) bacteria 99%, 2.2×10⁶, 2.4×10⁸; 2) proteolytic bacteria 94%, 1.2×10⁵, 3.0×10⁶;3) moulds 98%, 1.3×10⁵, 9.0×10⁶; 4) yeasts 44 %, 3.3×10⁴, 3.6×10⁶. The samples were contaminated in 92 % byAspergillus spp, in 71% byAspergillus flavus, in 83% byPenicillium spp, and in 20% byFusarium spp with mean contamination 8.3×104, 1.1×10³, 4.2×10⁴, 5.0×10³ , and maximum yield 6.8×10⁶, 1.0×10⁵, 5.0×10⁶, 1.5×10⁶, respectively. Totally 8.5% of strains were aflatoxinogenic and 4.4% of the strains were isolated from feed and 21 % of the strains from grain/nut.     

Calcium and cyclic AMP as possible mediators of neurohormone action in the hindgut of the cockroach, Leucophaea maderae

Adenosine 3′,5′-monophosphate (cyclic AMP) (10⁻⁵ g/ml) often caused a gradual increase in spotaneous contractile activity of the hindgut of the cockroach, Leucophaea maderae, and on rare occasions it would evoke a hormone-like response. However, aminophylline (2·5 × 10⁻⁴ g/ml) was capable of mimicking the neurohormone, and a concentration of 2·5 × 10⁻⁵ g/ml potentiated the contractile response evoked by the neurohormone: these responses were blocked by either the presence of 1 mM manganous ion or in a high potassium solution (162 mM). Propranolol (10⁻⁶ g/ml) and dopamine (10⁻⁴ g/ml) suppressed both spontaneous contractile events and neurohormone action. Dopamine (5 × 10⁻⁶ g/ml) also blocked action potential generation as did propranolol at 10⁻⁴ g/ml.These results lead us to suppose that cyclic AMP might serve as a mediator of neurohormone action by increasing calcium transport across the surface membrane of muscle fibres. Caffeine (2·5 × 10⁻⁴ g/ml), like aminophylline, caused a hormone-like response in normal hindguts. Even when the visceral muscles of the hindgut were depolarized in 162 mM potassium solution (without calcium), caffeine was still capable of inducing a phasic response. However, the addition of 2 mM calcium to such potassium-depolarized preparations caused a gradual increase in muscle tonus and substantially potentiated the response to caffeine.Such findings clearly implicate calcium as the mediator of excitation-contraction coupling in visceral muscle. While the interactions between the neurohormone, cyclic AMP, and calcium seem to be largely associated with the surface membrane and action potential generation.     

Comparative actions of the neuropeptides leucopyrokinin and periplanetin CCI on the visceral muscle systems of the cockroaches Leucophaea maderae and Periplaneta americana

1. The effects of two structurally-related peptides, leucopyrokinin (LPK) and periplanetin CC-I (CCI), on contractile activities of visceral muscle systems were compared in the two cockroaches from which these peptides were originally isolated.2. LPK elicited consistent proctolin-like responses on the hindgut, foregut, oviduct and heart of the Madeira cockroach, Leucophaea maderae, with increases in both amplitude and frequency of contraction. CCI, on the other hand, elicited a mostly tonic response on these tissues.3. For the American cockroach, Periplaneta americana, the responses elicited by LPK and CCI were tonic in nature.4. With the exception of the response of the L. maderae hindgut and heart to LPK, threshold levels for either LPK or CCI on all other tissues of both roaches were considerably higher (10–100 times greater) than those for proctolin on the same tissues.5. The maximum response to any concentration of LPK or CCI on the foregut and oviduct of L. maderae and that on the foregut and hindgut of P. americana never reached more than 60% of the maximum contraction achieved with proctolin.     

Isolation of proctolin,a myotropic peptide,from Periplaneta americana

The isolation of a myotropic substance, proctolin, associated with the innervation of the viscera of the cockroach, Periplaneta americana, is reported. Proctolin, previously described as a neurotransmitter or neurohormone, is a small basic peptide with an estimated molecular weight of 500 to 700. The proctodeal (hindgut) muscles of P. americana contract at threshold levels of about 0·5 ng/ml, equivalent to about 10⁻⁹ M proctolin. Approximately 180 μg proctolin were isolated from 125 kg of whole cockroaches. The isolated peptide was homogeneous on paper and thin-layer chromatography and on high-voltage paper electrophoresis at pH 3·5 and 6·4.     

Leucokinins, a new family of ion transport stimulators and inhibitors in insect Malpighian tubules

Leucokinins are octapeptides isolated from heads of the cockroach Leucophaea maderae. In the cockroach they increase motility of the isolated hindgut. Surprisingly, synthetic leucokinins have biological activity in a different insect and in a different tissue. In isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti, leucokinins depolarize the transepithelial voltage. This effect on voltage is dependent on extracellular Cl. One leucokinin, LK-8, the effects of which were studied further in isolated Malpighian tubules, was found to inhibit transepithelial fluid secretion at low concentrations (10(-11) M threshold), and to stimulate fluid secretion at high concentrations (3.5 x 10(-9) M threshold). Together, the depolarizing effects on voltage and the stimulation of fluid secretion suggest that leucokinins increase the Cl permeability of the tubule wall thereby increasing the availability of Cl for secretion with Na, K and water. Structure-function comparisons of the seven leucokinins studied suggest that the active region of the octapeptide is segregated to the C-terminal pentapeptide. In view of the known effects of leucokinins on hindgut motility in the cockroach, our finding of effects in mosquito Malpighian tubules suggests that leucokinins may be widely distributed in insects where they may have diverse functions in a variety of organs.     

Effects of leucomyosuppressin on the excitation-concentration coupling of insect Leucophaea maderae visceral muscle

1. Leucomyosuppressin (LMS) inhibited neurally evoked contractions of the hindgut of the cockroach Leucophaea maderae. The threshold for this inhibition of LMS was in the range of 1 × 10⁻¹⁰ M.2. LMS caused a sharp reduction in both l-glutamate and proctolin induced contractions. Dose-response profiles of the effect of LMS (held constant at 10⁻⁸M) on variable amounts of proctolin showed an inhibitory effect at 10⁻⁹ M proctolin and below, but at 5 × 10⁻⁹ M proctolin and above, LMS caused no inhibition.3. Potassium (158 mM) depolarized hindguts treated with LMS (10⁻⁸ M) showed a marked reduction (76% ± 2.1) in the proctolin (10⁻⁸ M) response.4. When calcium depleted preparations were returned to normal calcium levels (2 mM) in the presence of proctolin (10 ⁻⁸ M) a contraction occurred that was 45% ± 4 of the maximum in normal saline solution. However, LMS (10⁻⁸ M) reduced this response to only 28% ± 2 of the maximum.5. Proctolin (10⁻⁸ M) induced contractions in the presence of the manganous ions (2mM) fell to 63% ± 4 of the maximum but on the addition of LMS (10⁻⁸M), such responses fell to only 16% ± 5 of the maximum.6. These results offer evidence for a non-synaptic site of action for LMS and a perturbation of key calcium dependent events in the excitation-contraction coupling sequence of visceral muscle by this peptide.     

Cuticular control of diffusional water permeability

The diffusional water permeability (P_d) in various gut structures of the fleshfly, Sarcophaga bullata, was measured using tritiated water. Water P_d in the larval hindgut was 3.91 × 10^?6 cm/sec, whereas in the adult hindgut it was 4.4 × 10^?4 cm/sec. The presence of cuticle in various parts of the gut apparently controls the water permeabilities of these structures. Furthermore, the water permeability of the cuticle may be correlated with the mechanism for the production of a hyperosmotic excretion in the hindgut.     

Some characteristics of a new isolate of Helicosporidium and its effect upon mosquitoes

A Helicosporidium sp. was isolated by feeding spores concentrated by continuous flow centrifugation from ditch water to starved Heliothis zea larvae. This Helicosporidium sp was infectious to Anopheles quadrimaculatus, Culex salinarius, and Aedes aegypti with IC₅₀'s of 4.4 × 10², 2.6 × 10⁴, and 2.4 × 10⁴ spores/ml, respectively. Larval mortality was dosage dependent with LC₅₀ values 72 hr postexposure of 6.8 × 10⁴ for An. quadrimaculatus, 9.4 × 10³ for Cx. salinarius, and 1.5 × 10⁵ for Ae. aegypti. The spores of this Helicosporidium were also tolerant of freezing and desiccation. Because of these traits and the melanization response they provoked in host tissues, this is probably not naturally a mosquito pathogen and is most likely from a terrestrial insect.     

Effects of soil-treatment with fungal biopesticides on pupation behaviors,emergence success and fitness of tea geometrid,Ectropis grisescens (Lepidoptera: Geometridae)

Ectropisgrisescens Warren (Lepidoptera: Geometridae) is one of the most severe defoliating pests of tea plants. Synthetic pesticides have been widely applied to control this pest in tea plantations, but pesticide residues may decrease the quality and safety of tea products. In the present study, we hypothesized that soil treatment with the two commercial biopesticides (Shuiguxin^®) based on Metarhizium anisopliae (Metchnikoff) Sorokin and Beauveria bassiana (Balsamo) could reduce the survivorship and fitness of E. grisescens. Wandering larvae of E. grisescens were allowed to pupate in soil treated with each biopesticide, and the concentrations of M. anisopliae (Shuiguxin^®) and B. bassiana (Shuiguxin^®) that produced the 50% mortality values (LC₅₀) were 2.9 × 10⁶ and 1.6 × 10⁷ conidia/g soil, respectively. Artificial burying the pupae using soil treated with M. anisopliae (Shuiguxin^®) and B. bassiana (Shuiguxin^®) (1 × 10⁸ or 1 × 10⁹ conidia/g soil for both biopesticides) also significantly reduced emergence success of E. grisescens. In addition, choice tests showed that soil treated with the high concentration of M. anisopliae (Shuiguxin^®) or B. bassiana (Shuiguxin^®) had repellent effects on pupating E. grisescens. However, sublethal concentrations (LC₂₅ and LC₅₀) of both biopesticides did not significantly affect fecundity, fertility and longevity of post-emerged adults. Our study showed that soil treatment with the two commercial biopesticides caused direct mortality of pupating E. grisescens, but may not effectively suppress E. grisescens populations at sublethal concentrations. The realistic application of the fungal dosage in fields should be determined in future studies     

The susceptibility of the pea moth, Cydia nigricana, to infection by the granulosis virus of the codling moth, Cydia pomonella

Laboratory studies demonstrated that neonate larvae of the pea moth, Cydia nigricana, are susceptible to infection with a granulosis virus (CpGV) isolated from the codling moth, Cydia pomonella. Comparative LC₅₀ values for C. nigricana and C. pomonella are 1.90 × 10⁵ and 1.54 × 10⁴ capsules/ml of diet, respectively. The virus extracted from CpGV-infected pea moth larvae is serologically related, and probably identical, to CpGV.     

Effect of the cations sodium,potassium and calcium on the interaction of hyaluronate chains: a light scattering and viscometric study

Light scattering and viscometric studies have been carried out on two preparations, A and B, of rooster comb hyaluronate. Sedimentation rate studies have also been performed with A. Light scattering measurements in 0.2 m KCl for preparation A gave a molecular weight of 3.3 × 10⁶ and for B, 1.0 × 10⁶. In (0.1–0.3) M NaCl similar measurements gave a particle weight for A of (4.4–6.4 × 10⁶ and for B (1.7–2.8 × 10⁶. In 0.066 m CaCl₂ molecular weight values of 9.5 × 10⁶ for A and 1.7 × 10⁶ for B were obtained. Thus in the presence of Na⁺ and Ca²⁺ ions aggregates of chains persisted into dilute solution. Measurements by light scattering on A and B in 4 m guanidinium chloride gave values in the same range as those obtained in 0.2 m KCl. Sedimentation rate studies on A gave values of 10.3 Svedbergs in 0.2 m KCl and 12.2 Svedbergs in 0.2 m NaCl and 0.066m CaCl₂. The shear dependence of the viscosity was studied using a conicylindrical viscometer at shear rates between 0.5 and 20 s^?1. Preparation A in 0.2 m KCl and NaCl yielded values for (ν_sp/cc→0 of 5000 and 7100 ml g^?1 respectively in keeping with the tendency to aggregate. The behaviour for preparation B was similar. In 0.066 m CaCl₂ there was a marked dependence of viscosity on shear speed below 10 s^?1 for all concentrations and the value of (ν_sp/c)→0 at 0 s^?1 for preparation A was 7700 ml g^?1 while at a shear rate of 8 s^?1 (ν_sp/c)c→0 ? 5000 ml g ^?1. Similar effects were found for preparation B and the data suggest associations of chains disruptable by weak shear forces. The increase in viscosity with concentration in the presence of 0.066 m CaCl₂ was much less than in the presence of KCl or NaCl, suggesting that the Ca²⁺ had a marked effect on the ”rigidity’ of the molecules in solution. A viscometric titration experiment with Ca^2? showed that a level of 0.02 m CaCl₂ in 0.2 m NaCl was sufficient to produce the change in viscosity presented above and that significant perturbations of the viscosity were present at 0.005?0.01 m CaCl₂.     

Isolated epithelial cells of the toad bladder. Their preparation, oxygen consumption, and electrolyte content

Epithelial cells of the toad bladder were disaggregated with EDTA, trypsin, hyaluronidase, or collagenase and were then scraped free of the underlying connective tissue. In most experiments EDTA was complexed with a divalent cation before the tissue was scraped. Q _OO2, sucrose and inulin spaces, and electrolytes of the isolated cells were measured. Cells disaggregated by collagenase or hyaluronidase consumed O₂ at a rate of 4 µl hr^-1 dry wt^-1. Q _OO2 was increased 50% by ADH (100 U/liter) or by cyclic 3'',5''-AMP (10 mM/liter). Na⁺-free Ringer''s depressed the Q _OO2 by 40%. The Q _OO2 of cells prepared by trypsin treatment or by two EDTA methods was depressed by Na⁺-free Ringer''s but was stimulated relatively little by ADH. Two other EDTA protocols produced cells that did not respond to Na⁺ lack or ADH. The intracellular Na⁺ and K⁺ concentrations of collagenase-disaggregated cells were 32 and 117 mEq/kg cell H₂O, respectively. Cation concentrations of hyaluronidase cells were similar, but cells that did not respond to ADH had higher intracellular Na⁺ concentrations. Cells unresponsive to ADH and Na⁺ lack had high sucrose spaces and low transcellular membrane gradients of Na⁺, K⁺, and Cl^-. The results suggest that trypsin and EDTA disaggregation damage the active Na⁺ transport system of the isolated cell. Certain EDTA techniques may also produce a general increase in permeability. Collagenase and hyaluronidase cells appear to function normally.