Glycated bovine serum albumin for use in feeding trials with animal models - In vitro methodology and characterization of a glycated substrate for modifying feed pellets.

This study investigated different methods to produce Nε-carboxymethyl-lysine (CML)-enriched bovine serum albumin (BSA) as alternatives to the classical approach using glyoxylic acid (GA) and sodium cyanoborohydride (NaBH3CN) which results in toxic hydrogen cyanide (HCN). The reaction of GA (6 mmol/L) and NaBH3CN (21 mmol/L) to produce CML remained the most effective with CML yields of 24-35%, followed by 13-24% using 300 mmol/L glyoxal (GO). GA promoted specific modification of lysine to CML, and fewer structural modifications of the BSA molecule compared with GO, as evidenced by fluorescence and proteomic analyses. GO promoted greater arginine modification compared with GA (76 vs 23%). Despite structural changes to BSA with GO, murine fecal clearance of CML was similar to literature values. Hence, BSA glycation with 300 mmol/L glyoxal is a suitable alternative to GA and NaBH3CN for generating CML-enriched protein free of HCN, but a CML-only fortification model remains to be described.

Busulfan-mediated germ cell depletion does not alter gonad differentiation in the urodele amphibian Pleurodeles waltl.

It is not known in urodele amphibians whether germ cells (GCs) are indispensable for gonadal differentiation. In order to address this question in the newt Pleurodeles waltl, we first cloned the ortholog of VASA which is known as a GC marker in many species. Male (ZZ) and female (ZW) larvae were then exposed to the alkylating agent busulfan (25 µg/ml for 3 days) just after hatching (stage 36). In the main body of busulfan-treated larvae, PwVASA mRNA expression decreased before gonad differentiation in both sexes (at stage 50). This suggested GC depletion which was confirmed by histology, with a complete absence of GCs observed slightly earlier in females (stage 54) than in males (stage 54 + 60 days). In busulfan-treated ZW larvae, the presence of the typical central cavity and expression of a high level of aromatase mRNA confirmed the ovarian phenotype. In busulfan-treated ZZ larvae, the presence of a medulla surrounded by a thin cortex and a low level of aromatase mRNA confirmed the testis phenotype. At the juvenile stage, efferent ducts and lobules were present in the first testis lobe. Taken together, these data suggest that GC depletion does not alter gonad differentiation in P. waltl.

Sexual development of the urodele amphibian Pleurodeles waltl.

Pleurodeles waltl is a urodele amphibian that displays a ZZ/ZW genetic mode of sex determination involving a putative W-borne dominant determinant. This determining pathway can be environmentally inhibited since heat treated ZW larvae undergo a functional female to male sex reversal. Moreover, both genetic sexes can be reversed by treatment of larvae with steroid hormones suggesting they are the major players in the differentiation process. Indeed we demonstrated that i) aromatase expression and activity increase just before ovarian differentiation, ii) aromatase inhibitors induce a female to male sex reversal, iii) estrogens induce male to female sex reversal whereas the opposite is obtained with non-aromatizable androgens, iv) steroidogenic factor 1 and estrogen receptor alpha both display a female-enriched expression following the increase in aromatase activity. The role of endogenous hormones was investigated in a parabiosis model. Surprisingly, in ZW/ZZ associations, the ZW gonad could not differentiate suggesting that the ZZ parabiont produces an inhibiting factor, prior to ovarian differentiation. The role of AMH in this process is discussed, keeping in mind that Mullerian ducts are maintained in males. The development of antibodies and new molecular tools in the near future should help us to better understand the sexual development of this vertebrate.

Female-enriched and thermosensitive expression of steroidogenic factor-1 during gonadal differentiation in Pleurodeles waltl.

In the urodele amphibian Pleurodeles waltl, sex differentiation is genetically controlled, that is, ZZ male vs ZW female, but may be influenced by temperature, which induces a female-to-male sex reversal. We investigated whether steroidogenic factor 1 (SF-1) could be involved in Pleurodeles sex differentiation or in temperature-dependent sex reversal by cloning a Pleurodeles SF-1 cDNA and examining its developmental expression. The 468-amino-acid deduced protein is highly conserved in comparison with other species. In ZZ and ZW control larvae, SF-1 mRNA is detected at the first stage of the thermosensitive period (TSP) in the gonad-mesonephros-interrenal complex (GMI). By the end of TSP at stage 55, SF-1 is expressed in the gonad (Gd) and in the mesonephros-interrenal (MI) both in ZZ and ZW larvae. During this stage, a transient, ZW-specific increase of SF-1 transcription occurs not only in Gd but also in MI, this increase starting earlier in Gd than in MI. Therefore, in P. waltl, an SF-1 upregulation occurs after the onset of the ovarian-specific increase of aromatase mRNA expression. At the end of metamorphosis, the SF-1 transcription level in Gd and MI is nearly the same in both ZZ and ZW larvae. Besides, after long-term heat treatment leading to sex reversal, SF-1 mRNA upregulation is not observed in ZW larvae, in either Gd or MI. However, SF-1 expression is not decreased after a 48-h heat shock applied at the end of the TSP, suggesting that temperature has no inhibitory effect by itself in long-term heat treatment. Estradiol benzoate treatments show that, at the end of the TSP, SF-1 gene transcription could be controlled by the estrogen level. This is in accordance with the female-enriched SF-1 expression and the decreased SF-1 expression following long-term, sex-reversing heat treatment, which is known to decrease aromatase expression and activity. Thus, it is unlikely that SF-1 is directly involved in Pleurodeles temperature-dependent sex reversal.

Intracellular acidification delays hormonal G2/M transition and inhibits G2/M transition triggered by thiophosphorylated MAPK in Xenopus oocytes.

Xenopus oocyte maturation is analogous to G2/M transition and characterized by germinal vesicle breakdown (GVBD), spindle formation, activation of MPF and Mos-Xp42(Mpk1) pathways. It is accompanied prior to GVBD by a transient increase in intracellular pH. We determined that a well known acidifying compound, NH(4)Cl, delayed progesterone-induced GVBD in a dose-dependent manner. GVBD(50) was delayed up to 2.3-fold by 10 mM NH(4)Cl. Cyclin B2 phosphorylation, Cdk1 Tyr15 dephosphorylation as well as p39(Mos) accumulation, Xp42(Mpk1) and p90(Rsk) phosphorylation induced by progesterone were also delayed by incubation of oocyte in NH(4)Cl. The delay induced by NH(4)Cl was prevented by injection of MOPS buffer pH 7.7. In contrast to acidifying medium, alkalyzing treatment such as Tris buffer pH 9 injections, accelerated GVBD, MPF and Xp42(Mpk1) activation, indicating that pHi changes control early steps of G2/M dynamics. When injected in an immature recipient oocyte, egg cytoplasm triggers GVBD through MPF auto-amplification, independently of protein synthesis. In these conditions, GVBD and Xp42(Mpk1) activation were delayed by high concentration of NH(4)Cl, which never prevented or delayed MPF activation. Strickingly, NH(4)Cl strongly inhibited thiophosphorylated active MAPK-induced GVBD and MPF activation. Nevertheless, Tris pH 9 did not have any effects on egg cytoplasm- or active MAPK-induced GVBD. Taken together, our results suggest that dynamic of early events driving Xp42(Mpk1) and MPF activation induced by progesterone may be negatively or positively regulated by pH(i) changes. However Xp42(Mpk1) pathway was inhibited by acidification alone. Finally, MPF auto-amplification loop was not sensitive to pH(i) changes.

Differential roles of p39Mos-Xp42Mpk1 cascade proteins on Raf1 phosphorylation and spindle morphogenesis in Xenopus oocytes.

Fully-grown G2-arrested Xenopus oocytes resume meiosis upon hormonal stimulation. Resumption of meiosis is characterized by germinal vesicle breakdown, chromosome condensation, and organization of a bipolar spindle. These cytological events are accompanied by activation of MPF and the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathways. The latter cascade is activated upon p39(Mos) accumulation. Using U0126, a MEK1 inhibitor, and p39(Mos) antisense morpholino and phosphorothioate oligonucleotides, we have investigated the role of the members of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) in spindle morphogenesis. First, we have observed at a molecular level that prevention of p39(Mos) accumulation always led to MEK1 phosphorylation defects, even when meiosis was stimulated through the insulin Ras-dependent pathway. Moreover, we have observed that Raf1 phosphorylation that occurs during meiosis resumption was dependent upon the activity of MEK1 or Xp42(Mpk1) but not p90(Rsk). Second, inhibition of either p39(Mos) accumulation or MEK1 inhibition led to the formation of a cytoplasmic aster-like structure that was associated with condensed chromosomes. Spindle morphogenesis rescue experiments using constitutively active Rsk and purified murine Mos protein suggested that p39(Mos) or p90(Rsk) alone failed to promote meiotic spindle organization. Our results indicate that activation of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathway is required for bipolar organization of the meiotic spindle at the cortex.

Steroids, aromatase and sex differentiation of the newt Pleurodeles waltl.

In the newt Pleurodeles waltl, genetic sex determination obeys female heterogamety (female ZW, male ZZ). In this species as in most of non-mammalian vertebrates, steroid hormones play a key role in sexual differentiation of gonads. In that context, male to female sex reversal can be obtained by treatment of ZZ larvae with estradiol. Male to female sex reversal has also been observed following treatment of ZZ larvae with testosterone, a phenomenon that was called the "paradoxical effect". Female to male sex reversal occurs when ZW larvae are reared at 32 degrees C during a thermosensitive period (TSP) that takes place from stage 42 to stage 54 of development. Since steroids play an important part in sex differentiation, we focussed our studies on the estrogen-producing enzyme aromatase during normal sex differentiation as well as in experimentally induced sex reversal situations. Our results based on treatment with non-aromatizable androgens, aromatase activity measurements and aromatase expression studies demonstrate that aromatase (i) is differentially active in ZZ and ZW larvae, (ii) is involved in the paradoxical effect and (iii) might be a target of temperature. Thus, the gene encoding aromatase might be one of the master genes in the process leading to the differentiation of the gonad in Pleurodeles waltl.

Effect of cadmium on gonadogenesis and metamorphosis in Pleurodeles waltl (urodele amphibian).

In the amphibian Pleurodeles waltl, steroid hormones play a key role in sex differentiation. Since cadmium has been reported to block receptors of sex steroid hormones, we analyzed the effects of this heavy metal on Pleurodeles larvae gonadogenesis. At stage 42, larvae die in the presence of 10.9 microM Cd in the rearing tap water, with TL(50) of 46.3 h, but the concentration of 5.5 microM is tolerated for more than 60 days. When used at 5.5 microM cadmium accumulation measured by atomic absorption spectrophotometry (AAS) in total homogenates of larvae at stage 54 (after 77 days of exposure to the heavy metal) reached 58.1 microg/g of dry weight. At stage 54, we did not detect inhibitory effects on gonadogenesis in larvae reared in the presence of 5.5 microM Cd since stage 42. When the exposure to 5.5 microM Cd was lengthened after stage 54, metamorphosis was delayed and could not be completed. When larvae were exposed to 10.9 microM Cd from stage 54, metamorphosis did not occur and gonad development was stopped. Our study demonstrates a lack of a direct effect of cadmium on sex determination-differentiation but a strong inhibitory effect on metamorphosis, which impairs further gonadal development.

Functional analysis of the structural basis of homophilic cadherin adhesion.

The structures of many cell surface adhesion proteins comprise multiple tandem repeats of structurally similar domains. In many cases, the functional significance of this architecture is unknown, and there are several cases in which evidence for individual domain involvement in adhesion has been contradictory. In particular, the extracellular region of the adhesion glycoprotein cadherin consists of five tandemly arranged domains. One proposed mechanism postulated that adhesion involves only trans interactions between the outermost domains. However, subsequent investigations have generated several competing models. Here we describe direct measurements of the distance-dependent interaction potentials between cadherin mutants lacking different domains. By quantifying both the absolute distances at which opposed cadherin fragments bind and the quantized changes in the interaction potentials that result from deletions of individual domains, we demonstrate that two domains participate in homophilic cadherin binding. This finding contrasts with the current view that cadherins bind via a single, unique site on the protein surface. The potentials that result from interactions involving multiple domains generate a novel, modular binding mechanism in which opposed cadherin ectodomains can adhere in any of three antiparallel alignments.

Differential expression of P450 aromatase during gonadal sex differentiation and sex reversal of the newt Pleurodeles waltl.

A better understanding of vertebrate sexual differentiation could be provided by a study of models in which genetic sex determination (GSD) of gonads can be reversed by temperature. In the newt Pleurodeles waltl, a P450 aromatase cDNA was isolated from adult gonads, and the nucleotide or deduced amino acid sequences showed a high level of identity with various vertebrate species. In adults, aromatase expression was found in gonads and brain. In developing gonads, the expression was found to fit with the thermo-sensitive period (TSP) and was detected in both ZZ and ZW larvae, as well as in ZW submitted during the whole TSP to a masculinizing temperature. In the latter individuals, in situ hybridization and semi quantitative RT-PCR showed that, at the end of TSP, aromatase expression was at the same level than in normal ZZ larvae and was significantly lower than in normal ZW ones. Furthermore, temperature-induced down regulation did not occur when heating was performed at the end of TSP. Our results confirm the importance of aromatase regulation in female versus male differentiation and demonstrate that a down regulation of aromatase expression is involved in the process of sex reversal.

Effects of androgens on sex differentiation of the urodele Pleurodeles waltl.

In nonmammalian vertebrates, steroids have been hypothesized to induce somatic sex differentiation, since manipulations of the steroidal environment of gonads have led to various degrees of sex reversal. Whereas the critical role of estrogens in ovarian differentiation is well documented, studies on androgens have produced a perplexing variety of results depending upon species variations and nature of androgens used. In this way, testosterone induces masculinization of females in some species but provokes paradoxical feminization of males in many other species such as the urodelan Pleurodeles waltl. In reptiles this phenomenon could be interpreted by conversion of exogenous testosterone to estradiol by aromatase. Treatments of Pleurodeles larvae with nonaromatizable androgens bring support to this hypothesis and suggest a role of androgens in sex differentiation. Dihydrotestosterone (DHT) could not induce the paradoxical feminization of ZZ larvae. In addition, DHT as well as 11beta-hydroxy-androstenedione could drive a functional male differentiation of ZW larvae. Moreover, other 5alpha reduced androgens also induced sex reversal of female larvae. Yet, the 5alpha reductase inhibitor CGP 53133 and antiandrogens such as flutamide or cyproterone acetate did not exert any effect on male sex differentiation of ZZ larvae. Though the precise role of androgens is still unknown, especially for 11-oxygenated androgens, our results suggest an implication in male sex differentiation. In this way, testosterone could play a pivotal role in being metabolized either into other androgens during testis differentiation or into estradiol during ovarian differentiation.

Modelling Alzheimer-specific abnormal Tau phosphorylation independently of GSK3beta and PKA kinase activities.

In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into paired helical filaments. These Tau variants displayed specific epitopes that are immunoreactive with anti-phospho-Tau antibodies such as AT100. As shown in in vitro experiments, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase A (PKA) may be key kinases in these phosphorylation events. In the present study, Tau was microinjected into Xenopus oocytes. Surprisingly, in this system, AT100 was generated without any GSK3beta and PKA contribution during the progesterone or insulin-induced maturation process. Our results demonstrate that a non-modified physiological process in a cell model can generate the most specific Alzheimer epitope of Tau pathology.

Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH(2)-terminal EC1 domain in the homophilic binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding. Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer-dimer equilibrium with an affinity constant of approximately 64 microm. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity. The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment-based adhesion assay. A protein with only the first two NH(2)-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W. Brieher, N. Lavrik, B. Gumbiner, and D. Leckband. 1999. PROC: Natl. Acad. Sci. USA. 96:11820-11824; Sivasankar, S., B. Gumbiner, and D. Leckband. 2001. Biophys J. 80:1758-68). We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.

c-Mos proteolysis is independent of the CA(2+) rise induced by 6-DMAP in Xenopus oocytes.

In Xenopus oocytes, metaphase II arrest is due to a cytostatic factor (CSF) that involves c-Mos, maintaining a high MPF (cdk1/cyclin B) activity in the cell. At fertilization, a rise in intracellular calcium triggers the proteolysis of both cyclin B and c-Mos. The kinase inhibitor 6-dimethylaminopurine (6-DMAP) is also able to release matured Xenopus oocytes from metaphase II block. This is characterized by c-Mos proteolysis without degradation of cyclin B. We hypothesized that 6-DMAP induced an increase in intracellular calcium. Using the calcium-sensitive fluorescent dye Fura-2, we observed a systematic increase in intracellular calcium following 6-DMAP application. In matured oocytes previously microinjected with the calcium chelator BAPTA, no calcium changes occurred after 6-DMAP addition; however, c-Mos was still proteolysed. In oocytes at the GVBD stage, c-Mos proteolysis occurred in response to 6-DMAP but not to calcium ionophore treatment. We suggest that c-Mos proteolysis is rather controlled by a phosphorylation-dependent process.

Differential effects of 6-DMAP, olomoucine and roscovitine on Xenopus oocytes and eggs.

The effects of the new cyclin-dependent kinase inhibitors, roscovitine and olomoucine, on oocytes and eggs of Xenopus laevis were investigated and compared with those of 6-dimethylamino purine (6-DMAP). The inhibitory properties of 6-DMAP, olomoucine and roscovitine towards p34cdc2-cyclin B isolated from Xenopus eggs revealed K-IC50 values of 300, 40 and 10 microM respectively. The three compounds inhibited progesterone-induced maturation with M-IC50 values of 200, 100 and 20 microM. These values were consistent with the K-IC50 values but the ratio M-IC50/K-IC50 was higher for roscovitine and olomoucine than for 6-DMAP. The disappearance of spindle and condensed chromosomes without pronucleus formation was observed when 1 mM 6-DMAP was applied for 4 h at germinal vesicle breakdown or at metaphase II, whereas no effect was observed using 1 mM olomoucine or 50 microM roscovitine. Changes in the electrophoretic mobility of p34cdc2 and erk2 were observed only in homogenates of matured oocytes or eggs exposed for 4 h to 1 mM 6-DMAP. When the drugs were microinjected into matured oocytes, olomoucine (100 microM) and roscovitine (50 microM) induced pronucleus formation more efficiently than did 6-DMAP (100 microM). Taken together, these results demonstrate that Xenopus oocytes possess a lower permeability to olomoucine and roscovitine and that these new compounds are suitable for in vivo studies after germinal vesicle breakdown provided they are microinjected.

Activation of Xenopus eggs by the kinase inhibitor 6-DMAP suggests a differential regulation of cyclin B and p39(mos) proteolysis.

In Xenopus eggs, metaphase II arrest is due to the cytostatic factor that maintains a high level of MPF activity. Kinases are important in this phenomenon since p39(mos) and MAPK play a part in the cytostatic activity whereas p34(cdc2) is the catalytic subunit of MPF. Fertilization induces a rise in intracellular calcium leading to egg activation that can be mimicked by calcium-increasing agents such as calcium ionophore. We have performed on Xenopus eggs a biochemical comparison of the effects of the kinase inhibitor 6-DMAP and the calcium ionophore. Both drugs were able to induce pronucleus formation but the underlying molecular events were different. The inactivation of MAPK occurred earlier in eggs exposed to 6-DMAP. Cyclins B1 and B2 were stable and p39(mos) was proteolysed in 6-DMAP-treated eggs while the three proteins underwent degradation in A23187-treated ones. These results suggest a differential regulation of ubiquitin-dependent proteolysis of cyclin B and p39(mos).

Inhibition of protein tyrosine phosphatases blocks calcium-induced activation of metaphase II-arrested oocytes of Xenopus laevis.

We have studied the effect of a protein tyrosine phosphatases (PTP) inhibitor on calcium-induced activation of Xenopus laevis oocytes arrested at metaphase II. Ammonium molybdate microinjection blocked pronucleus formation following A23187 treatment while cortical granules still underwent exocytosis. Pronuclei still occurred in ammonium molybdate-injected oocytes following 6-DMAP addition. Changes that usually occurred following A23187 exposure were inhibited in the presence of ammonium molybdate in the oocyte: MAPK dephosphorylation, p34(cdc2) rephosphorylation and cyclin B2 and p39(mos) proteolysis. These results suggest that a PTP is involved in the activation of the ubiquitin-dependent degradation machinery.

Intra-M phase-promoting factor phosphorylation of cyclin B at the prophase/metaphase transition.

Activation of Cdc2-cyclin B (or M phase-promoting factor (MPF)) at the prophase/metaphase transition proceeds in two steps: dephosphorylation of Cdc2 and phosphorylation of cyclin B. We here investigated the regulation of cyclin B phosphorylation using the starfish oocyte model. Cyclin B phosphorylation is not required for Cdc2 kinase activity; both the prophase complex dephosphorylated on Cdc2 with Cdc25 and the metaphase complex dephosphorylated on cyclin B with protein phosphatase 2A display high kinase activities. An in vitro assay of cyclin B kinase activity closely mimics in vivo phosphorylation as shown by phosphopeptide maps of in vivo and in vitro phosphorylated cyclin B. We demonstrate that Cdc2 itself is the cyclin B kinase; cyclin B phosphorylation requires Cdc2 activity both in vivo (sensitivity to vitamin K3, a Cdc25 inhibitor) and in vitro (copurification with Cdc2-cyclin B, requirement of Cdc2 dephosphorylation, and sensitivity to chemical inhibitors of cyclin-dependent kinases). Furthermore, cyclin B phosphorylation occurs as an intra-M phase-promoting factor reaction as shown by the following: 1) active Cdc2 is unable to phosphorylate cyclin B associated to phosphorylated Cdc2, and 2) cyclin B phosphorylation is insensitive to enzyme/substrate dilution. We conclude that, at the prophase/metaphase transition, cyclin B is mostly phosphorylated by its own associated Cdc2 subunit.

Effect of procaine on membrane potential and intracellular pH in Xenopus laevis oocytes.

The effect of the local anaesthetic procaine on the intracellular pH, pHi, and electrophysiological properties of full-grown Xenopus oocytes was studied. In spite of its interference with both the pH-sensitive microelectrodes and fluorescent probe BCECF, we have shown that procaine induced an intracellular acidification rather than the alkalization commonly observed in most cells. The resting pHi of Xenopus oocytes loaded with BCECF was 7.36 +/- 0.04 (n = 16). Addition of 10 mM procaine to the bath at pH 7.5 caused pHi to decrease to a new steady state value of 6.97 +/- 0.05 (n = 9). A similar behaviour of pHi was observed with microelectrodes. Procaine also promoted a rise in membrane conductance and a membrane depolarization. These changes in membrane potential and conductance were not caused by the decrease in pHi since the addition of sodium propionate at pH 7.5 produced the same decrease of pHi as procaine, but resulted in only a slight depolarization with superimposed oscillations. Current measurements using two-electrode voltage clamp showed that the depolarization was associated with an inward current. No significant effect on this current was observed when replacing Cl, K or Na in the external medium. The absence of effect of Cl and K channel inhibitors argues against the involvement of Cl and K currents during the procaine response.