Biomedical subjects

I Parker

Publications and source records attributed to I Parker.

At least 19 recordsLinked to original sources

PubMed1992

Messenger RNAs coding for receptors and channels in the cerebral cortex of adult and aged rats.

Poly(A)+ mRNAs from the cerebral cortex of aged (24 months) and young adult (3 months) rats were isolated and injected into Xenopus oocytes to express functional neurotransmitter receptors and voltage-operated channels. Electrophysiological recordings of induced membrane currents were used as a measure of the relative amounts of mRNA encoding different receptors and channels, and to study their functional properties. There were no large differences apparent between mRNAs from aged and adult rats, in marked contrast to the dramatic (1000-fold) changes in mRNA expression that occur during embryonic and postnatal development. The membrane currents induced by glutamate or acetylcholine (ACh) application were roughly one third smaller in oocytes injected with mRNA from aged cerebral cortex than in oocytes injected with mRNA from adult cerebral cortex, whereas currents induced by gamma-aminobutyric acid (GABA), kainate or serotonin (5-HT) application, and by activation of voltage-operated Na+ and Ca2+ channels were not significantly different. We did not observe any age-related differences in the properties of the receptors and channels studied.

Acetylcholine↗

PubMed1992

Effects of alcohols on responses evoked by inositol trisphosphate in Xenopus oocytes.

1. The effects of ethanol and other alcohols on inositol 1,4,5-trisphosphate (InsP3) signalling were studied in Xenopus oocytes by the use of flash photolysis of caged InsP3. Calcium liberation induced by InsP3 was monitored by voltage-clamp recording of Ca(2+)-activated membrane currents, and by fluorescence of the Ca2+ indicator Fluo-3. 2. Membrane current and fluorescence Ca2+ signals evoked by light flashes giving small responses were initially potentiated by bath application of ethanol (80-400 mM). However, the responses subsequently declined while ethanol was present and were strongly reduced or suppressed when it was removed. 3. These effects did not arise artifactually from changes in photolysis of caged InsP3, as similar results were seen with responses evoked by intracellular injections of InsP3. Also, the effects on the membrane current did not arise primarily through actions on the Ca(2+)-dependent Cl- channels, since currents evoked by intracellular injections of Ca2+ were little changed by ethanol. 4. Ethanol reduced the threshold level of InsP3 required to cause Ca2+ liberation. Thus, potentiation was most prominent with small responses evoked by brief light flashes, whereas the predominant effect on larger responses was inhibitory. 5. The facilitatory and inhibitory actions of ethanol persisted after removing extracellular Ca2+. 6. Intracellular injections of ethanol produced an initial inhibition of InsP3 responses, followed, in some oocytes, by a potentiation. 7. Methanol had little effect on InsP3 responses, whereas butanol and other long-chain alcohols produced strong inhibition, but little or no potentiation. 8. We conclude that extracellular application of ethanol produces a rapid potentiation of InsP3-mediated Ca2+ liberation, and a more slowly developing inhibition. The potentiation may arise through stimulation of InsP3 formation at the plasma membrane, whereas the inhibition occurs more deeply in the cell. Both actions were evident at relatively low concentrations (a few tens of millimoles per litre), and might thus be important in the behavioural effects of ethanol intoxication.

Animals↗

PubMed1992

Potentiation of inositol trisphosphate-induced Ca2+ mobilization in Xenopus oocytes by cytosolic Ca2+.

1. The ability of cytosolic Ca2+ ions to modulate inositol 1,4,5-trisphosphate (Insp3)-induced Ca2+ liberation from intracellular stores was studied in Xenopus oocytes using light flash photolysis of caged InsP3. Changes in cytosolic free Ca2+ level were effected by inducing Ca2+ entry through ionophore and voltage-gated plasma membrane channels and by injection of Ca2+ through a micropipette. Their effects on Ca2+ liberation were monitored by video imaging of Fluo-3 fluorescence and by voltage clamp recording of Ca(2+)-activated membrane Cl- currents. 2. Treatment of oocytes with the Ca2+ ionophores A23187 and ionomycin caused a transient elevation of cytosolic Ca2+ level when cells were bathed in Ca(2+)-free solution, which probably arose because of release of Ca2+ from intracellular stores. 3. Membrane current and Fluo-3 Ca2+ signals evoked by photoreleased InsP3 in ionophore-treated oocytes were potentiated when the intracellular Ca2+ level was elevated by raising the Ca2+ level in the bathing solution. 4. Responses to photoreleased InsP3 were similarly potentiated following activation of Ca2+ entry through voltage-gated Ca2+ channels expressed in the plasma membrane. 5. Ca(2+)-activated membrane currents evoked by depolarization developed a delayed 'hump' component during sustained photorelease of InsP3, probably because Ca2+ ions entering through the membrane channels triggered liberation of Ca2+ from intracellular stores. 6. Ba2+ and Sr2+ ions were able to substitute for Ca2+ in potentiating InsP3-mediated Ca2+ liberation. 7. Gradual photorelease of InsP3 by weak photolysis light evoked Ca2+ liberation that began at particular foci and then propagated throughout, but not beyond that area of the oocyte exposed to the light. Local elevations of intracellular Ca2+ produced by microinjection of Ca2+ acted as new foci for the initiation of Ca2+ liberation by InsP3. 8. In resting oocytes, intracellular injections of Ca2+ resulted only in localized elevation of intracellular Ca2+, and did not evoke propagating waves. 9. The results show that cytosolic Ca2+ ions potentiate the ability of InsP3 to liberate Ca2+ from intracellular stores. This process may be important for the positive feedback mechanism underlying the generation of Ca2+ spikes and waves, and for interactions between the InsP3 pathway and Ca2+ ions entering cells through voltage- and ligand-gated channels.

Animals↗

PubMed1992

Characteristics of membrane currents evoked by photoreleased inositol trisphosphate in Xenopus oocytes.

Photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor was used to study characteristics of Ca(2+)-activated Cl- currents activated in Xenopus oocytes by the InsP3-Ca2+ signaling pathway. Photolysis flashes shorter than a threshold duration evoked no response, but the current amplitude then grew about linearly as the flash duration was further lengthened. Currents directly evoked by photorelease of Ca2+ from a caged precursor grew linearly with increasing flash duration and showed a small threshold before they were activated. However, the major part of the threshold of InsP3-evoked responses appears to arise because a certain concentration of InsP3 (estimated to be approximately 60 nM) is required to evoke Ca2+ liberation. Subthreshold conditioning flashes potentiated responses to subsequent flashes, and the potentiation increased linearly with increasing conditioning flash duration before abruptly declining. The potentiation decayed exponentially with a time constant of approximately 17 s with increasing interflash interval. Currents evoked by photoreleased InsP3 began after a latency that shortened from 10 s or longer to 100 ms as the photolysis intensity was increased. This dose dependence of the latency could be quantitatively explained by the time required for the InsP3 concentration to rise above threshold. Intracellular injection of heparin (a competitive antagonist at the InsP3 receptor) increased the threshold for InsP3 action, as did increased temperature. We conclude that several characteristics of InsP3-evoked responses, including their dose dependence, latency, and facilitation with paired stimuli, arise because a distinct threshold level of InsP3 is required to evoke release of Ca2+ from intracellular stores.

Animals↗

PubMed1991

Regenerative release of calcium from functionally discrete subcellular stores by inositol trisphosphate.

Fluorescence imaging was used to determine the spatial and temporal patterns of subcellular calcium (Ca2+) liberation induced in Xenopus oocytes by photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor. Increasing levels of InsP3 evoked Ca2+ release that began in a graded manner but, at varying threshold levels of InsP3, localized sites then showed transient and asynchronous 'puffs' of Ca2+ release. With higher levels of InsP3, Ca2+ from adjacent sites formed a focus for initiation of a propagating Ca2+ wave. The results show that InsP3-sensitive Ca2+ stores are arranged as distinct and functionally independent units, and that Ca2+ is released in both graded and regenerative fashions.

Animals↗

PubMed1991

Receptors of the serotonin 1C subtype expressed from cloned DNA mediate the closing of K+ membrane channels encoded by brain mRNA.

The modulation of K+ channels by serotonin (5-HT) receptors was studied by coinjecting Xenopus oocytes with mRNA transcribed in vitro from a cloned 5-HT 1C subtype (5-HT1C) receptor gene, together with size-fractionated mRNA isolated from rat cerebral cortex that expresses K+ channels. After intracellular loading with EGTA to block Ca2(+)-dependent chloride currents, these oocytes responded to 5-HT with an inward current associated with a decrease in membrane conductance. Membrane current responses were small or absent in oocytes injected with either mRNA alone. We conclude that 5-HT1C receptors are able to cause the closing of a class of K+ channels expressed by cortex mRNA in a Ca2(+)-independent manner. The coupling between the receptors and channels appears to be mediated by the inositol phospholipid second messenger pathway, since activation of this pathway by application of serum evoked a similar closing current.

Animals↗

PubMed1991

Inositol 1,3,4,6-tetrakisphosphate mobilizes calcium in Xenopus oocytes with high potency.

Injection of Ins(1,3,4,6)P4 into Xenopus oocytes evoked Ca2(+)-dependent membrane currents with a potency 5-10 times less than Ins(1,4,5)P3, whereas Ins(1,3,4)P3 and Ins(1,3,4,5,6)P5 were almost ineffective. Responses to Ins(1,3,4,6)P4 arose through liberation of intracellular Ca2+ and through entry of extracellular Ca2+. These results, together with the observation that Ins(1,3,4,6)P4 facilitated responses to Ins(1,4,5)P3, suggests that both of these compounds may act on the same intracellular receptors.

Animals↗

PubMed1991

Microinjection into Xenopus oocytes: a precise semi-automatic instrument and optimal parameters for injection of mRNAs.

A new apparatus for the injection of Xenopus oocytes is described which provides semi-automatic cell handling together with highly accurate and reproducible volume delivery. Using the system requires very little skill, yet it gives 6.3% average reproducibility in the 5 to 70 nl volume range. The instrument uses a fixed injector system driven by an Inchworm piezoelectric positioner or, in a low-cost version, by a Rainin EDP-2 battery-operated motorized pipette. A movable, vacuum-operated oocyte holder minimizes lateral movement of the oocyte during injection. Oocytes injected with the system show better survival and enhanced expression of mRNA compared with those injected with a widely used type of manual injector (Coleman, 1984).

Animals↗

PubMed1991

Inositol tetrakisphosphate liberates stored Ca2+ in Xenopus oocytes and facilitates responses to inositol trisphosphate.

1. The actions of the putative second messenger inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) were studied by injecting it into voltage-clamped oocytes while recording Ca(2+)-dependent chloride membrane currents and, in some experiments, fluorescence signals from Ca2+ indicators. 2. Ins(1,3,4,5)P4 evoked a rise in intracellular Ca2+ and associated chloride current in oocytes bathed in normal or Ca(2+)-free Ringer solutions. The fluorescence Ca2+ signal showed a prolonged rise with superimposed oscillations, whereas the current reflected only the oscillatory component. 3. Injections of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) evoked currents showing an initial transient, followed by oscillations. Ins(1,3,4,5)P4 evoked similar oscillations, but the transient component was usually small or absent. Ins(1,3,4,5)P4 was about 20-fold less potent than Ins(1,4,5)P3, as measured by comparing doses required to elicit currents with the same integral. The most sensitive oocytes responded to about 1 fmol Ins(1,3,4,5)P4 and 0.1 fmol Ins(1,4,5)P3. 4. Injections of Ins(2,4,5)P4 evoked oscillatory currents, with a potency about three times greater than Ins(1,4,5)P3. Ins(1,3,4)P4 was ineffective in some oocytes even at doses of several picomoles, but in other oocytes evoked small transient and oscillatory currents with a potency 100 times or more less than Ins(1,3,4,5)P4. 5. Injections of Ins(1,3,4,5)P4 made into the animal hemisphere of the oocyte evoked larger currents than injections into the vegetal hemisphere. 6. Photo-release of Ins(1,4,5)P3 from caged Ins(1,4,5)P4 loaded into the oocyte was used to examine interactions between Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Injection of low (ca 1 fmol) doses of Ins(1,3,4,5)P4 shortly before a light flash greatly facilitated currents evoked by photo-release of near-threshold amounts of Ins(1,4,5)P3. This facilitation was unaffected by removal of extracellular Ca2+ and arose because Ins(1,3,4,5)P4 reduced the threshold amount of Ins(1,4,5)P3 required to evoke a response. 7. Larger amounts (several femtomoles) of Ins(1,3,4,5)P4 depressed responses evoked by photo-release of Ins(1,4,5)P3. This may arise because Ca2+ liberated by Ins(1,3,4,5)P4 inhibits the ability of Ins(1,4,5)P3 to release further Ca2+. 8. We conclude that Ins(1,3,4,5)P4 liberates intracellular Ca2+ in the oocyte in a manner similar to that of Ins(1,4,5)P3, and suggest that a physiological role for Ins(1,3,4,5)P4 may be to facilitate responses to Ins(1,4,5)P3.

Animals↗

PubMed1991

Caffeine inhibits inositol trisphosphate-mediated liberation of intracellular calcium in Xenopus oocytes.

1. Voltage-clamp recording of Ca(2+)-activated chloride currents in Xenopus oocytes was used to study the effects of caffeine on the liberation of intracellular Ca2+ induced by photo-release of inositol 1,4,5-trisphosphate (InsP3) from caged InsP3. Bath application of caffeine, at concentrations between 0.1 and 10 mM, reduced or abolished the current evoked by photo-release of InsP3 and by microinjection of InsP3. 2. Caffeine did not appreciably reduce currents evoked by injection of Ca2+ into oocytes, whereas measurements using the Ca2+ indicator Rhod-2 showed that it instead inhibited the liberation of Ca2+ by InsP3. 3. Caffeine increased the threshold amount of InsP3 required to evoke a current response and proportionally reduced the currents evoked by suprathreshold levels of InsP3. 4. Theophylline and 3-isobutyl-1-methylxanthine (IBMX) were much less potent than caffeine, and few changes were seen in the InsP3 responses following application of forskolin or intracellular injection of cyclic AMP. Thus, inhibition of InsP3 responses by caffeine does not arise through inhibition of phosphodiesterase enzymes. 5. Even at high (10 mM) concentrations, caffeine did not itself elicit any clear Ca(2+)-activated current. It is therefore unlikely that inhibition of the InsP3 responses arise because caffeine itself liberates Ca2+ from intracellular stores. 6. The site of action of caffeine is intracellular, because injections of caffeine into the oocyte strongly inhibited responses to InsP3, whereas local extracellular applications of similar amounts were almost without effect.

Animals↗

PubMed1990

Localized all-or-none calcium liberation by inositol trisphosphate.

Laser confocal microscopy was used to monitor calcium ion (Ca2+) liberation from highly localized (micrometer) regions of intact Xenopus oocytes in response to photo-released inositol 1,4,5-trisphosphate (InsP3). Local Ca2+ release varied in an all-or-none manner with increasing amount of InsP3, in contrast to signals recorded from larger areas, which grew progressively as the concentration of InsP3 was raised above a threshold. Liberation of Ca2+ was restricted to within a few microns of the site of InsP3 release and, in response to agonist activation, localized regions of the oocyte showed asynchronous oscillations in cytoplasmic Ca2+ release. Results obtained with this technique provided direct evidence that InsP3-induced Ca2+ liberation was quantized and suggest that the InsP3-sensitive Ca2+ pool may be a collection of independent, localized compartments that release Ca2+ in an all-or-none manner.

Animals↗

PubMed1990

A slowly inactivating potassium current in native oocytes of Xenopus laevis.

Membrane currents were recorded in voltage-clamped oocytes of Xenopus laevis in response to voltage steps. We describe results obtained in oocytes obtained from one donor frog, which showed an unusually large outward current upon depolarization. Measurements of reversal potentials of tail currents in solutions of different K+ concentration indicated that this current is carried largely by K+ ions. It was strongly reduced by extracellular application of tetraethylammonium, though not by Ba2+ or 4-aminopyridine. Removal of surrounding follicular cells did not reduce the K+ current, indicating that it arises across the oocyte membrane proper. Activation of the K+ conductance was first detected with depolarization to about -12 mV, increased with a limiting voltage sensitivity of 3 mV for an e-fold change in current, and was half-maximally activated at about +10 mV. The current rose following a single exponential timecourse after depolarization, with a time constant that shortened from about 400 ms at -10 mV to about 15 ms at +80 mV. During prolonged depolarization the current inactivated with a time constant of about 4 s, which did not alter greatly with potential. The K+ current was independent of Ca2+, as it was not altered by addition of 10 mM Mn2+ to the bathing medium, or by intracellular injection of EGTA. Noise analysis of K+ current fluctuations indicated that the current is carried by channels with a unitary conductance of about 20 ps and a mean open lifetime of about 300 ms (at room temperature and potential of +10 to +20 mV).

4-Aminopyridine↗

PubMed1990

Changes in messenger RNAs coding for neurotransmitter receptors and voltage-operated channels in the developing rat cerebral cortex.

The ontogenetic development of poly(A)+ mRNAs coding for receptors to several neurotransmitters (kainate, glutamate, acetylcholine, and serotonin) and voltage-operated channels (sodium and calcium) was studied by isolating total poly(A)+ mRNA from the brains of rats at various developmental stages and injecting it into Xenopus oocytes. The oocytes translated the foreign mRNA and incorporated functional receptor/ion channel complexes into the cell membrane. Thus, recording of induced membrane currents in voltage-clamped oocytes gave a measure of the relative amounts of the different messengers. Responses induced by kainate, glutamate, acetylcholine, and serotonin all increased with age and reached a maximum in oocytes injected with mRNA from adult cortex. Messenger RNAs for the earliest ages examined, Embryonic Days 15 and 18, expressed little or no response to kainate, glutamate, or acetylcholine, while 50-70% of the adult response was reached by Postnatal Day 10. In contrast, the serotonin-induced response was already comparatively large (16% of the adult level) in oocytes injected with mRNA from Embryonic Day 15 brain and increased postnatally to adult levels. The expression of voltage-dependent sodium and calcium channels was small in oocytes injected with mRNA from embryonic animals and increased postnatally to reach a maximum in oocytes injected with mRNA from adult animals.

Aging↗

PubMed1990

Serotonin receptors expressed in Xenopus oocytes by mRNA from brain mediate a closing of K+ membrane channels.

Membrane currents evoked by serotonin (5-HT) were studied in Xenopus oocytes injected with rat brain mRNA. Intracellular EGTA was used to abolish the Ca2(+)-dependent oscillatory Cl- current to 5-HT, revealing an underlying smooth inward current. This was associated with a decreased membrane conductance, was antagonized by Ba2+ and Zn2+ (but not TEA), and probably arises through a closing of K+ channels. Half-maximal responses were obtained with 30 nM 5-HT, while 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) was ineffective. Furthermore, methysergide, mianserin and lysergic acid antagonized the K(+)-closing response to 5-HT, consistent with it being mediated through 5-HT1C receptors. The largest K(+)-closing responses were induced by a size fraction of mRNA which also induced a large K+ conductance, suggesting that the response requires expression of both receptors and K+ channels. The K(+)-closing response induced in the oocyte resembles the M- and S-type currents described in, respectively, mammalian and invertebrate neurons.

Animals↗

PubMed1990

Inhibition by Ca2+ of inositol trisphosphate-mediated Ca2+ liberation: a possible mechanism for oscillatory release of Ca2+.

Light-flash photolysis of caged inositol 1,4,5-trisphosphate (InsP3) was used to generate reproducible transients of free InsP3 in Xenopus oocytes, and the resulting liberation of Ca2+ from intracellular stores was monitored by recording Ca2+-activated membrane currents and by use of the fluorescent Ca2+ indicator fluo-3. InsP3-mediated Ca2+ release was inhibited by elevating the intracellular free Ca2+ level, either by microinjecting Ca2+ into the cell or by applying conditioning light flashes to liberate Ca2+. This inhibition followed a slow time course, being maximal after about 2 s and subsequently declining over several seconds. Negative feedback of Ca2+ ions on InsP3-mediated Ca2+ liberation may explain the oscillatory release of Ca2+ seen during activation of inositol phospholipid signaling in the oocyte, and the time course of the inhibition is consistent with the period of the oscillations.

Activity Cycles↗

PubMed1989

Ionic and charge-displacement currents evoked by temperature jumps in Xenopus oocytes.

Membrane currents were recorded in voltage-clamped oocytes of Xenopus laevis. Currents were produced in response to temperature jumps imposed by a heating lamp. Responses were larger when the animal (pigmented) hemisphere of the oocyte was illuminated as compared to the vegetal hemisphere; they arose because of a thermal effect as they were attenuated by removal of infrared wavelengths. The temperature jump responses comprised two distinct components: (i) a slow maintained current, which inverted direction at a membrane potential of about -25 mV and, (ii) a fast transient current, which at all potentials examined (-160 to +30 mV), was inward at the onset of a light flash and outward at the offset. The slow component probably arises through temperature-dependent changes in the 'leakage' current of the oocyte, and measurements of reversal potentials in solutions of different ionic composition indicated that currents carried by Na+ and H+ ions contribute to the response. In contrast, the fast component was not altered by changes in composition of the bathing solution. This observation, together with the finding that the charge movements associated with the on and off transients were of similar magnitude, suggest that the fast current may arise because of the displacement of charges across the plasma membrane.

Animals↗

PubMed1989

Latencies of membrane currents evoked in Xenopus oocytes by receptor activation, inositol trisphosphate and calcium.

1. Application of serum to Xenopus oocytes elicits an oscillatory chloride membrane current, which begins after a latency of several seconds or minutes, and is mediated through a phosphoinositide-calcium signalling pathway. We studied the characteristics and origin of this latency in voltage-clamped oocytes. 2. Bath application of low doses of serum evoked responses beginning after latencies of 1 min or more. The latency decreased with increasing dose and reached a minimal value of several seconds that did not decrease with further increases in serum concentration. Experiments to study this minimal latency were done by applying brief 'puffs' of serum and other agonists at high concentrations from a local extracellular pipette. 3. The mean latency of the response evoked by local serum application was about 7 s (at 22-24 degrees C), but individual responses showed a wide variation, from 2 s to over 20 s. Diffusion of serum from the pipette tip to the membrane did not contribute appreciably to this delay, since short (less than 100 ms) delays were obtained when KCl was applied in the same way. 4. Currents evoked by acetylcholine and serotonin, in oocytes induced to acquire muscarinic and serotonergic receptors following injection of brain messenger RNA, began following latencies similar to that of the serum response. 5. The response latency was shorter when serum was applied to the vegetal rather than the animal hemisphere of the oocyte, even though smaller currents were obtained. 6. The latency showed a slight dependence upon membrane potential, becoming shorter with depolarization. 7. Cooling to temperatures below about 22 degrees C produced a striking lengthening of the delay, corresponding to a Q10 of about 5. In contrast, above 22 degrees C the temperature dependence was slight, with a Q10 of about 1.25. 8. Intracellular injections of calcium and inositol 1,4,5-trisphosphate (IP3) evoked chloride currents with short (a few tens of milliseconds) latency. Short (100 ms) latency responses were also evoked when intracellularly loaded caged IP3 was photolysed by strong illumination, but weak illumination gave responses with latencies of over 1 min. 9. Measurements of intracellular free calcium, made with Fura-2 and Indo-1, showed an increase following serum application beginning coincident with the onset of the membrane current response.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗

PubMed1989

Membrane currents elicited by divalent cations in Xenopus oocytes.

1. Membrane currents were recorded from voltage-clamped Xenopus oocytes in response to bath application of various divalent cations. 2. In oocytes from 93 of 160 frogs tested, Co2+ ions evoked slow, oscillatory membrane currents. Sensitivity to Co2+ varied greatly between oocytes from different frogs, but was relatively consistent for oocytes taken from the same ovary. Oocytes with high sensitivity had response thresholds of 5-10 microM, and gave currents greater than 1 microA to 1 mM-CoCl2. In contrast, oocytes from some frogs gave no oscillatory response even to 10 mM-CoCl2. With responsive oocytes, Cd2+, Ni2+, Zn2+, Mn2+ and Cr2+ ions (5 microM to 1 mM) also elicited oscillations, whereas Sr2+, Ba2+ and Ca2+ (0.1-10 mM) showed very little activity, and Mg2+ ions, none. 3. Responses to divalent cation were well preserved in defolliculated oocytes, indicating they were generated in the oocyte membrane itself, and were not dependent on the presence of enveloping follicular cells. 4. The oscillatory currents reversed around -20 mV (the chloride equilibrium potential) and rectified strongly at potentials more negative than about -60 mV. The oscillations were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3), were largely preserved after removal of external Ca2+, but were abolished following chelation of intracellular Ca2+ by EGTA. Intraoocyte injection of Co2+ ions failed to generate oscillatory currents. 5. Currents elicited by divalent cations resembled the oocyte's oscillatory responses to acetylcholine and a serum protein. However, the response to divalent cations was not blocked by atropine and furthermore, the relative sensitivities to these agonists varied independently between oocytes from different frogs. 6. We conclude that extracellular Cd2+, Ni2+, Zn2+, Co2+, Mn2+ and Cr2+ interact with the oocyte surface to raise cytosolic levels of inositol phosphates. This causes mobilization of intracellular Ca2+, in turn activating Ca2+-gated Cl- channels in the oocyte membrane. 7. In addition to the large oscillatory currents, divalent cations generated small (5-50 nA), smooth, maintained currents associated with decreases in membrane conductance. The size and ionic basis of these currents varied between oocytes from different frogs. 8. Zinc ions also elicited smooth currents, associated with an increase in membrane conductance, and carried predominantly by K+. This response was specific to Zn2+ and occurred independently of oscillatory Cl- currents. The K+ current was abolished by defolliculation, was potentiated by the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine,and showed facilitation with K+ currents generated by the adenylate cyclase activator forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗