Angiotensin II receptors in Xenopus oocytes.

Electrical recordings were used to study the sensitivity of native Xenopus oocytes to the octapeptide angiotensin II (AII). AII elicited oscillatory currents associated with an increase in membrane conductance to Cl-. Responsiveness to AII varied greatly between oocytes taken from different frogs, and to a lesser extent between oocytes from the same ovary. Oocytes from frogs showing high sensitivity had response thresholds between 0.5-1.0 nM AII, and at a holding potential of -60 mV, responded to 1 microM AII with currents greater than 3 microA. In contrast, oocytes from some frogs gave no response, even to 10 microM AII. A total of 618 oocytes from 79 frogs were tested for sensitivity to AII, and oocytes from 85% of frogs gave detectable electrical responses. Oscillatory Cl- currents elicited by AII were largely independent of extracellular Ca2+, were abolished by chelation of intracellular Ca2+ using EGTA and were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3). In addition to oscillatory Cl- currents, AII also evoked an influx of extracellular Ca2+, giving rise to a transient inward Cl- current on membrane hyperpolarizing steps. These experiments all suggested that AII responses were elicited through activation of an intracellular messenger pathway triggered by hydrolysis of inositolphospholipids, mobilization of intracellular Ca2+ by inositol polyphosphates, and activation of Ca(2+)-gated Cl- channels. The effect of manual or enzymic defolliculation on AII responses was studied in nine separate experiments recording from 70 defolliculated oocytes. Efficacy of defolliculation procedures was assayed using scanning electron microscopy, which confirmed removal of 90 to greater than 98% of follicular cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracts from plants used in Mexican traditional medicine activate Ca(2+)-dependent chloride channels in Xenopus laevis oocytes.

The two-electrode voltage-clamp technique was employed to investigate the effects of chloroform-methanol (1:1) extracts derived from five medicinal plants on Xenopus laevis oocytes. When evaluated at concentrations of 1 to 500 microg/ml, the extracts prepared from the aerial parts of Baccharis heterophylla H.B.K (Asteraceae), Chenopodium murale L. (Chenopodiaceae), Desmodium grahami Gray (Leguminosae) and Solanum rostratum Dun (Solanaceae) produced concentration-dependent oscillatory inward currents in the oocytes, while the extract of Gentiana spathacea did not induce any response. The reversal potential of the currents elicited by the active extracts was -17 +/- 2 mV and was similar to the chloride equilibrium potential in oocytes. These ionic responses were independent of extracellular calcium. However, they were eliminated by overnight incubation with BAPTA-AM (10 microM), suggesting that the currents were dependent on intracellular Ca2+ increase. Thus the plant extracts activate the typical oscillatory Ca(2+)-dependent Cl- currents generated in the Xenopus oocyte membrane more probably via a mechanism that involves release of Ca2+ from intracellular reservoirs. These observations suggest that Xenopus oocyte electrophysiological recording constitutes a suitable assay for the study of the mechanisms of action of herbal medicines.     

E-series prostaglandins activate cAMP-mediated potassium currents in follicle-enclosed Xenopus oocyte

Receptor-mediated responses to prostaglandins E1 and E2 are shown by electrophysiological methods in follicle-enclosed oocytes of Xenopus laevis. In voltage-clamped oocytes, prostaglandins E1 and E2 elicited an outward hyperpolarizing current. This outward membrane current was caused by an increase in K+ conductance. The prostaglandin-induced current was augmented by adenylate cyclase activator, forskolin, and by phosphodiesterase inhibitor, theophylline, indicating that adenosine 3', 5'-cyclic monophosphate (cAMP) is involved in activating the K+ current. The prostaglandin responses were either abolished or greatly reduced by removing follicular cells with collagenase, suggesting that prostaglandin receptors reside in the follicular cells.     

Changes in intracellular calcium and in membrane currents evoked by injection of inositol trisphosphate into Xenopus oocytes

Intracellular calcium was monitored by the use of aequorin in voltage-clamped oocytes of Xenopus laevis. Injection of inositol trisphosphate (IP3) into oocytes elicited slowly rising and decaying aequorin/calcium signals and produced oscillatory chloride membrane currents. These responses did not depend upon extracellular calcium, since they could be elicited in calcium-free solution and after addition of cobalt or lanthanum to block calcium channels in the surface membrane. We conclude that IP3 causes the release of calcium from intracellular stores in the oocyte. Injections of calcium gave aequorin and membrane current responses that were more transient than those seen with IP3.     

Membrane currents elicited by porcine vasoactive intestinal peptide (VIP) in follicle-enclosed Xenopus oocytes

Membrane currents were recorded from voltage-clamped Xenopus laevis oocytes, surrounded by their enveloping follicular and epithelial cells. Porcine vasoactive intestinal peptide (VIP) generated a membrane current due to an increase in membrane conductance to K+. The VIP current was mimicked by the adenylate cyclase activator forskolin and was potentiated by phosphodiesterase inhibitors, suggesting that adenosine 3',5'-cyclic monophosphate (cyclic AMP) plays a role in mediating the response. Though resembling the follicle's responses to catecholamines and adenosine in ionic basis and apparent mechanism, the response to VIP was not blocked by catecholaminergic or purinergic antagonists, indicating the presence of a specific VIP receptor in the follicle. Among the VIP related peptides, PHM-27 generated similar but smaller K+ currents and porcine secretin and glucagon neither elicited a response nor blocked that to VIP. After treating follicles with collagenase to remove the epithelial and follicular cells the responses to VIP were either substantially reduced or abolished, suggesting that the VIP receptors and K+ channels are both located in the follicular cells.     

Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C

We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.     

Phosphatidic acid and lysophosphatidic acid stimulate receptor-regulated membrane currents in the Xenopus laevis oocyte.

External application of dioleoyl-phosphatidic acid and oleoyl-lysophosphatidic acid stimulated Ca(2+)-dependent chloride currents in voltage-clamped Xenopus laevis oocytes. The responses were observed in oocytes from which follicular cells had been removed, indicating they were intrinsic to the oocyte itself. The lipid-induced Ca(2+)-dependent chloride currents were observed in the absence of extracellular calcium, were blocked by intracellular injection of the calcium chelator, bis(O-aminophenoxy)-ethane N,N,N'N'-tetraacetic acid, and could not be elicited by direct intracellular injection of the active lipids. The thresholds for dose-dependent current responses to dioleoyl-phosphatidic acid (100 nM) and for oleoyl-lysophosphatidic acid (10 nM) indicated that the lipid activities on oocytes were potent. With repeated or prolonged administration of either active lipid, responses exhibited desensitization. These results demonstrate that the Xenopus oocyte expresses endogenous functional responses for the mitogenic lipids phosphatidic acid and lysophosphatidic acid and thus provides a powerful model for characterization of the pharmacology and transduction pathways of these responses.     

Effects of ethanol on three endogenous membrane conductances present in Xenopus laevis oocytes

The Xenopus oocyte expression and recording system has allowed a detailed analysis of the physiology and pharmacology of neuronal ion channels including their sensitivity to ethanol. It is important however, to ascertain the effects of a particular drug on the channels inherently expressed by oocytes to ensure that drug effects ascribed to the expressed recombinant receptors are manifested solely through those receptors. In this study, the effects of ethanol were determined on three endogenous currents that can be elicited in oocytes and other cells by various manipulations. The inward cation current, IC, was activated by perfusing naive oocytes with a divalent-free recording solution. Ethanol (25-100 mM) modestly inhibited IC with 100 mM ethanol producing a 7-8% inhibition of steady state currents. The store-operated or capacitative calcium current (I(SOC)) was activated in thapsigargin-treated oocytes by switching from a calcium-free solution to one containing 10 mM calcium. In thapsigargin-treated oocytes also injected with EGTA to block calcium-activated chloride currents, ethanol (100 mM) had no effect on the store-operated calcium current. In contrast, ethanol (10-100 mM) dose-dependently inhibited the calcium-dependent chloride current (I(Cl(Ca)) in thapsigargin-treated oocytes. A voltage-jump protocol was used to separate the two components of I(Cl(Ca)), I(Cl-1) and I(Cl-2). Under these conditions, ethanol (100 mM) inhibited I(Cl-1) currents to a greater extent (38%) than it did I(Cl-2) currents (14%). These results show that Xenopus oocytes express endogenous ion channels that are differentially sensitive to ethanol.     

Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.     

Acetylcholine receptors in monkey and rabbit oocytes

Membrane potential responses to acetylcholine (ACh, 10(-7)-10(-3 M) were investigated in monkey and rabbit ovarian oocytes. In monkey oocytes ACh most commonly elicited a short-latency hyperpolarization concomitant with a decreased membrane input resistance (Rin). Under voltage-clamp short-latency ACh currents had an equilibrium potential of approximately -40 mV. In rabbit oocytes responses to ACh consisted of an increase in Rin or of a depolarization with an equilibrium potential of approximately -15 mV. Curare, hexamethonium, and atropine (10(-5)-10(-3) M) did not block these ACh responses. Thus, the oocyte membrane in the rabbit contains ACh receptors that cannot be classified as either muscarinic or nicotinic.     

The sensitivity of G protein-activated K+ channels toward halothane is essentially determined by the C terminus

G protein-activated K(+) channels (GIRKs or Kir3.x) are targets for the volatile anesthetic, halothane. When coexpressed with the m(2) acetylcholine (ACh) receptor in Xenopus oocytes, agonist-activated GIRK1(F137S)- and GIRK2-mediated currents are inhibited by halothane, whereas in the absence of ACh, high concentrations of halothane induce GIRK1(F137S)-mediated currents. To elucidate the molecular mechanism of halothane action on GIRK currents of different subunit compositions, we constructed deletion mutants of GIRK1(F137S) (GIRK1(Delta363*)) and GIRK2 (GIRK2(Delta356)) lacking the C-terminal ends, as well as chimeric GIRK channels. Mutated GIRK channels showed normal currents when activated by ACh but exhibited different pharmacological properties toward halothane. GIRK2(Delta356) showed no sensitivity against the inhibitory action of halothane but was activated by halothane in the absence of an agonist. GIRK1(Delta363*) was activated by halothane more efficiently. Currents mediated by chimeric channels were inhibited by anesthetic concentrations that were at least 30-fold lower than those necessary to decrease GIRK2 wild type currents. Glutathione S-transferase pulldown experiments did not show displacement of bound Gbetagamma by halothane, indicating that halothane does not interfere with Gbetagamma binding. Single channel experiments revealed an influence of halothane on the gating of the channels: The agonist-induced currents of GIRK1 and GIRK2, carried mainly by brief openings, were inhibited, whereas higher concentrations of the anesthetic promoted long openings of GIRK1 channels. Because the C terminus is crucial for these effects, an interaction of halothane with the channel seems to be involved in the mechanism of current modulation.     

Agonist-activated Ca2+ influx and Ca2+ -dependent Cl- channels in Xenopus ovarian follicular cells: functional heterogeneity within the cell monolayer

Xenopus follicles are endowed with specific receptors for ATP, ACh, and AII, transmitters proposed as follicular modulators of gamete growth and maturation in several species. Here, we studied ion‐current responses elicited by stimulation of these receptors and their activation mechanisms using the voltage‐clamp technique. All agonists elicited Cl^? currents that depended on coupling between oocyte and follicular cells and on an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), but they differed in their activation mechanisms and in the localization of the molecules involved. Both ATP and ACh generated fast Cl^? (F_Cl) currents, while AII activated an oscillatory response; a robust Ca²⁺ influx linked specifically to F_Cl activation elicited an inward current (I_iw,Ca) which was carried mainly by Cl^? ions, through channels with a sequence of permeability of SCN^? > I^? > Br^? > Cl^?. Like F_Cl, I_iw,Ca was not dependent on oocyte [Ca²⁺]_i; instead both were eliminated by preventing [Ca²⁺]_i increase in the follicular cells, and also by U73122 and 2‐APB, drugs that inhibit the phospolipase C (PLC) pathway. The results indicated that F_Cl and I_iw,Ca were produced by the expected, PLC‐stimulated Ca²⁺‐release and Ca²⁺‐influx, respectively, and by the opening of I_Cl(Ca) channels located in the follicular cells. Given their pharmacological characteristics and behavior in conditions of divalent cation deprivation, Ca²⁺‐influx appeared to be driven through store‐operated, calcium‐like channels. The AII response, which is also known to require PLC activation, did not activate I_iw,Ca and was strictly dependent on oocyte [Ca²⁺]_i increase; thus, ATP and ACh receptors seem to be expressed in a population of follicular cells different from that expressing AII receptors, which were coupled to the oocyte through distinct gap‐junction channels. J. Cell. Physiol. 227: 3457–3470, 2012. © 2011 Wiley Periodicals, Inc.     

Chloride channels mediate the response to gonadotropin-releasing hormone (GnRH) in Xenopus oocytes injected with rat anterior pituitary mRNA

Functional expression of receptors for GnRH was studied using Xenopus laevis oocytes injected with poly(A)+ mRNA extracted from rat anterior pituitary glands. Whole-cell currents were monitored using two-electrode voltage-clamp techniques. In oocytes which responded to both GnRH and TRH, the GnRH response showed a longer latency and time-to-peak than the TRH response. The response to GnRH or an agonist of GnRH receptors, buserelin (1 nM-1 microM) consisted of current fluctuations and occurred in a dose-dependent manner. This GnRH response was blocked by the Cl- channel blockers 9-AC (9-anthracene carboxylic acid; 1 mM), 4,4'-diisothiocyanastilbene-2,2'-disulfonic acid (0.1 mM), and diphenylamine-2-carboxylic acid (0.1 mM). The reversal potential for the GnRH-induced current fluctuations was -25 mV, comparable with the reported Cl- equilibrium potential in Xenopus oocytes, and its shift, when the external concentration of Cl- was changed, was reasonably described by the Nernst equation. These results indicate that the GnRH-induced response was dependent on the activity of Cl- channels. Ca2+ also plays a role, as the GnRH-induced response was reversibly suppressed by a calmodulin inhibitor, chlordiazepoxide (0.2 microM), and by a blocker of intracellular Ca2+ release, TMB-8 (8-(N.N-diethylamino) octyl-3,4,5-trimethoxybenzoate; 0.1-0.2 mM). It is concluded that GnRH (and TRH) receptors, expressed in Xenopus oocytes by injecting exogenous mRNA from rat anterior pituitary glands, operate via activation of Ca2+-dependent Cl- channels.     

Native ion current coupled to purinergic activation via basal and mechanically induced ATP release in Xenopus follicles

Xenopus follicle-enclosed oocytes are endowed with purinergic receptors located in the follicular cell membrane; their stimulation by ATP elicits an electrical response that includes generation of a fast inward current (F(Cl)) carried by Cl(-). Here, it was found that mechanical stimulation of the follicle provoked a native electrical response named I(mec). This was dependent on coupling between oocyte and follicular cells, because I(mec) was eliminated by enzymatic defolliculation or application of uncoupling drugs, such as heptanol or carbenoxolone. Moreover, the characteristics of I(mec) suggested that it corresponded with opening of the Cl(-) channel involved in F(Cl). For example, I(mec) showed cross-talk with the membrane mechanism that activates the F(Cl) response and anionic selectivity similar to that displayed by F(Cl). Also like F(Cl), I(mec) was independent of extracellular or intracellular Ca(2+). Furthermore, I(mec) was inhibited by superfusion with a purinergic antagonist, suramin, or by an enzyme that rapidly hydrolyzes ATP, apyrase. The response to mechanical stimulation was reconstituted in defolliculated oocytes expressing P2X channels as an ATP sensor. Recently, it has been shown that ATP release from the Xenopus oocyte is triggered by mechanical stimulation. Together, these observations seemed to indicate that I(mec) is activated through a mechanism that involves oocyte release of ATP that diffuses and activates purinergic receptors in follicular cells, with subsequent opening of F(Cl) channels. Thus, I(mec) generation disclosed a paracrine communication system via ATP between the oocyte and its companion follicular cells that might be of physiological importance during the growth and development of the gamete.     

Two different types of channels are targets for potassium channel openers in Xenopus oocytes

K+ channel openers elicit K+ currents in follicle-enclosed Xenopus oocytes. The most potent activators are the pinacidil derivatives P1075 and P1060. The rank order of potency to activate K+ currents in follicle-enclosed oocytes was: P1075 (K0.5:5 microM) greater than P1060 (K0.5:12 microM) greater than BRL38227 (lemakalim) (K0.5:77 microM) greater than RP61410 (K0.5:100 microM) greater than (-)pinacidil (K0.5:300 microM). Minoxidil sulfate, nicorandil, RP49356 and diazoxide were ineffective. Activation by the K+ channel openers could be abolished by the antidiabetic sulfonylurea glibenclamide. It was not affected by the blocker of the Ca(2+)-activated K+ channels charybdotoxin. The various K+ channel openers failed to activate glibenclamide-sensitive K+ channels in defolliculated oocytes, but BRL derivatives (K0.5 for BRL38226 is 150 microM) and RP61419 inhibited a background current. The channel responsible for this background current is K+ permeable but not fully selective for K+. It is resistant to glibenclamide. It is inhibited by Ba2+, 4-aminopyridine, Co2+, Ni2+ and La3+.     

Hyperpolarization-activated Cl current elicited by pituitary adenylate cyclase activating polypeptide in Xenopus oocytes

We examined the electrophysiological effect of pituitary adenylate cyclase activating polypeptide (PACAP) in isolated Xenopus laevis oocytes in vitro. In conventional two-electrode voltage clamp experiments, PACAP (1–10 μM) activated an inward rectifier current at membrane potentials more negative than −60 mV without causing any significant change in currents at potentials more positive than −60 mV both in the follicle-enclosed oocyte and in the defolliculated oocyte. This current reversed at −22.5 mV, close to the theoretical value of Cl⁻ equilibrium potential and the reversal potential of this current was shifted positively by reducing [Cl⁻]_o. This current was blocked by Cl⁻ channel blocker SITS and Ba²⁺. Furthermore, VIP and adenylate cyclase activator forskolin did not elicit the currents. In conclusion, PACAP elicited the hyperpolarization-activated Cl⁻ current in Xenopus laevis oocytes. This current may modulate the membrane potential of the oocyte, thereby affecting the oocyte physiology.     

Co-expression of calcium-dependent protein kinase with the inward rectified guard cell K+ channel KAT1 alters current parameters in Xenopus laevis oocytes

Increased guard cell cytosolic [Ca2+] is known to be involved in signal transduction pathways leading to stomatal closure, and inhibit the inward rectifying guard cell K+ channel KAT1. Guard cell calcium-dependent protein kinase (CDPK) has been shown to phosphorylate KAT1; such phosphorylation is known to modulate other K+ channels involved in signal transduction cascades. The work reported here focused on demonstrating CDPK-dependent inhibition of KAT1 currents. A cDNA encoding soybean CDPK was generated and it's translation product was shown to be functional; demonstrating Ca2+-dependent autophosphorylation and phosphorylation of a target protein. Ion currents were monitored using voltage clamp techniques upon expression of KAT1 in Xenopus laevis oocytes. Coexpression of recombinant CDPK with KAT1 in oocytes altered the kinetics and magnitude of induced K+ currents; at a given hyperpolarizing command voltage, the magnitude of KAT1 currents was reduced and the half-time for channel activation was increased. This finding supports a model of Ca2+-dependent ABA inhibition of inward K+ currents in guard cells as being mediated by CDPK phosphorylation of KAT1.     

Angiotensin II stimulates an endogenous response in Xenopus laevis ovarian follicles

While responses to angiotensin II have previously been induced in Xenopus laevis oocytes after injection of messenger RNA extracted from mammalian tissue, no endogenous responses of ovarian tissue to this hormone have been reported. Here we describe such an endogenous dose-dependent response to angiotensin II, detected by conventional electrophysiological techniques, in follicular oocytes. The ED50 of the response was estimated to be 0.15 +/- 0.07 microM (S.E.M.). Maximal depolarization, obtained at 1 microM angiotensin II, was 18.3 +/- 1.4 mV (n = 18, three experiments using oocytes from two toads, mean resting membrane potential = -42 +/- 2 mV). The response was absent from collagenase-treated oocytes or follicular oocytes treated with octanol, suggesting that the receptors are predominantly in the follicular layer surrounding the oocytes.