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N Dascal

Publications and source records attributed to N Dascal.

At least 55 records · Page 3Linked to original sources
PubMed1991

Activation of protein kinase C alters voltage dependence of a Na+ channel.

Phorbol esters and purified protein kinase C (PKC) have been shown to down-modulate the voltage-dependent Na+ channels expressed in Xenopus oocytes injected with chick brain RNA. We used the two-electrode voltage-clamp technique to demonstrate that a Na+ channel expressed in oocytes injected with RNA coding for the alpha subunit of the channel alone (VA200, a variant of rat brain type IIA) is also inhibited by PKC activation. The inhibition of Na+ currents, expressed in oocytes injected with either alpha subunit RNA (rat) or total brain RNA (chick), is voltage-dependent, being stronger at negative potentials. It appears to result mainly from a shift in the activation curve to the right and possibly a decrease in the steepness of the voltage dependence of activation. There is little effect on the inactivation process and maximal Na+ conductance. Thus, PKC modulates the Na+ channel by a mechanism involving changes in voltage-dependent properties of its main, channel-forming alpha subunit.

Animals↗
PubMed1990

Interaction between injected Ca2+ and intracellular Ca2+ stores in Xenopus oocytes.

Upon two repetitive deep injections of Ca2+ into Xenopus oocyte (200-300 microns under the membrane), the amplitude of the transient Cl- current induced by the second injection is several-fold higher than that of the first one. This 'potentiation' persists even at 60-90 min intervals between injections. However, in oocytes permeabilized to Ca2+ by the ionophore A23187 in a Ca2(+)-free solution, the potentiation completely disappears after 30 min. It is proposed that the injected Ca2+ is largely taken up by the stores, whereas following the second injection, a higher proportion of Ca2+ reaches the membrane, since the stores are already loaded. In ionophore-treated oocytes, the stores lose the accumulated Ca2+ over several minutes and are then ready to take up Ca2+ again, hindering its arrival at the membrane.

Animals↗
PubMed1990

Modulation of vertebrate brain Na+ and K+ channels by subtypes of protein kinase C.

Effects of purified subtypes I, II and III of protein kinase C (PKC) on voltage-dependent transient K+ (A) and Na+ channels were studied in Xenopus oocytes injected with chick brain RNA. The experiments were performed in the constant presence of 10 nM beta-phorbol 12-myristate-13-acetate (PMA). Intracellular injection of subtype I (tau) reduced the A-current (IA), with no effect on Na+ current (INa). PKC subtype II (beta 1 + beta 2) and III (alpha) reduced both currents. PKC did not affect the response to kainate. Inactivated (heated) or unactivated (injected in the absence of PMA) enzyme and vehicle alone had no effect. Our results strongly suggest that INa and IA in vertebrate neurons are modulated by PKC; all PKC subtypes exert a similar effect on the A-channel while only subtypes II and III modulate the Na+ channel.

Animals↗
PubMed1990

Inactivation of calcium-activated chloride conductance in Xenopus oocytes: roles of calcium and protein kinase C.

Inactivation of Ca2(+)-induced Cl- currents was studied in Xenopus oocytes using the two-electrode voltage-clamp technique. In oocytes permeabilized to Ca2+ by treatment with the ionophore A23187, Ca2+ influx caused by the addition of 2.5-5 mM Ca2+ to the extracellular solution elicited Cl- currents consisting of two components: a fast, transient one (Ifast) and a slow one (Islow). In response to a subsequent application of the same dose of Ca2+, Ifast and Islow were reduced (inactivation phenomenon). The inactivation did not depend on the direction of current flow, but did depend on the duration of the first exposure to Ca2+. The extent of inactivation of Ifast was more significant than that to Islow. Both Ifast and Islow fully recovered from inactivation in less than 30 min. Intracellular injections of 100-400 pmol CaCl2 evoked large inward currents but did not reduce the amplitude of currents evoked by Ca2+ influx. The activator of protein kinase C, beta-phorbol dibutyrate, caused full inhibition of Ifast without any change in Islow. H-7 (1,5-isoquinolinesulfonyl-1,2 methylpiperazine), an inhibitor of protein kinases, strongly reduced the extent of inactivation. Our results suggest that elevation of intracellular Ca2+ by Ca2+ influx through the plasma membrane causes inactivation of the Ca2(+)-dependent Cl- conductance via activation of a Ca2(+)-dependent protein kinase, possibly protein kinase C, whereas Ca2+ arriving at the membrane from inside the cell does not initiate the processes leading to inactivation.

Animals↗
PubMed1990

Short- and long-term desensitization of serotonergic response in Xenopus oocytes injected with brain RNA: roles for inositol 1,4,5-trisphosphate and protein kinase C.

In Xenopus oocytes injected with rat brain RNA, serotonin (5HT) and acetylcholine (ACh) evoke membrane responses through a common biochemical cascade that includes activation of phospholipase C, production of inositol 1,4,5-trisphosphate (Ins1,4,5-P3), release of Ca2+ from intracellular stores, and opening of Ca-dependent Cl- channels. The response is a Cl- current composed of a transient component (5HT1 or ACh1) and a slow, long-lasting component (5HT2 or ACh2). Here we show that only the fast, but not the slow, component of the response is subject to desensitization that follows a previous application of the transmitter. The recovery of 5HT1 from desensitization is biphasic, suggesting the existence of two types of desensitization: short-term desensitization (STD), which lasts for less than 0.5 h; and long-term desensitization (LTD) lasting for up to 4 h. The desensitization between 5HT and ACh is heterologous and long-lasting. We searched for (a) the molecular target and (b) the cause of desensitization. (a) Pre-exposure to 5HT does not reduce the response evoked by intracellular injection of Ca2+ and by Ca2+ influx. Cl- current evoked by intracellular injection of Ins1,4,5-P3 was reduced shortly after application of 5HT, but fully recovered 30 min later. Thus, the Cl- channel is not a target for desensitization. Neither Ins1,4,5-P3 receptor nor the Ca2+ store is a target of LTD but they may be the targets of STD. (b) Ca2+ injection did not inhibit the 5HT response, suggesting that Ca2+ is not a sole cause of STD or LTD.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1989

Modulation of the voltage-dependent sodium channel by agents affecting G-proteins: a study in Xenopus oocytes injected with brain RNA.

The effects of agents known to affect G-proteins on voltage-dependent, tetrodotoxin-sensitive Na+ channels were studied in Xenopus oocytes injected with rat brain RNA, using two-electrode voltage-clamp technique. The non-hydrolysable analogue of GTP, GTP-gamma-S, known to activate G-proteins, inhibited the Na+ current (INa). The decrease in the amplitude of INa was not accompanied by changes in activation or inactivation characteristics of the channel. The non-hydrolysable analogue of GDP, GDP-beta-S, had no effect on INa. The responses to gamma-aminobutyric acid and kainate in the same oocytes were also attenuated by GTP-gamma-S. Pertussis toxin, which inactivates some G-proteins by catalyzing their ADP-ribosylation, enhanced INa, but did not prevent the inhibition of INa by GTP-gamma-S. We conclude that the Na+ channel, and possibly the GABA and kainate receptors and/or channels, are coupled to a G-protein. The activation of the G-protein modulates the channels either directly, or via activation of biochemical cascade possibly involving production of second messengers and channel phosphorylation.

Animals↗
PubMed1989

Evidence for the existence of a cardiac specific isoform of the alpha 1 subunit of the voltage dependent calcium channel.

Biochemical, pharmacological and electrophysiological evidence implies the existence of tissue specific isoforms of the L-type VDCC. The alpha 1 and alpha 2 subunits of the skeletal muscle calcium channel have been previously cloned and their amino acid sequence deduced. Here we report the isolation and sequencing of a partial cDNA that encodes a heart specific isoform of the alpha 1 subunit. The amino acid sequence deduced from this part cDNA clone shows 64.7% similarity with the skeletal muscle alpha 1 subunit. Northern analysis reveals 2 hybridizing bands, 8.5 and 13 kb, in contrast to one 6.5 kb band in the skeletal muscle. Selective inhibition of mRNA expression in Xenopus oocytes by complementary oligodeoxy-nucleotides derived from the heart clone provides further evidence that the cDNA corresponds to an essential component of the VDCC. These data further support the existence of tissue-specific isoforms of the L-type VDCC.

Amino Acid Sequence↗
PubMed1989

Specific block of calcium channel expression by a fragment of dihydropyridine receptor cDNA.

Although the structure of rabbit skeletal muscle dihydropyridine (DHP) receptor, deduced from cDNA sequence, indicates that this protein is the channel-forming subunit of voltage-dependent calcium channel (VDCC), no functional proof for this prediction has been presented. Two DNA oligonucleotides complementary to DHP-receptor RNA sequences coding for putative membrane-spanning regions of the DHP receptor specifically suppress the expression of the DHP-sensitive VDCC from rabbit and rat heart in Xenopus oocytes. However, these oligonucleotides do not suppress the expression of the DHP-insensitive VDCC and of voltage-dependent sodium and potassium channels. Thus, the gene for DHP receptor of rabbit skeletal muscle is closely related, or identical to, a gene expressed in heart that encodes a component of the DHP-sensitive VDCC. The DHP-sensitive and DHP-insensitive VDCCs are distinct molecular entities.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Protein kinase C modulates neurotransmitter responses in Xenopus oocytes injected with rat brain RNA.

Oocytes of the frog Xenopus laevis express various exogenous neurotransmitter receptors and ion channels when injected with RNA from excitable tissues. The oocytes serve as a convenient model system in which modulation of neurotransmitter responses can be studied. We examined the effects of activators and an inhibitor of protein kinase C (PKC) on responses to serotonin (5-HT), acetylcholine (ACh), kainate, and gamma-aminobutyric acid (GABA) in oocytes injected with RNA from rat brain. The PKC activators beta-phorbol esters 4 beta-phorbol-12-myristate-13-acetate (PMA) and 4 beta-phorbol-12,13-dibutyrate (PDBu), as well as the synthetic diacylglycerol, 1-oleyl-2-acetylglycerol (OAG), significantly inhibited the responses to 5-HT and ACh (both known to be mediated by mobilization of intracellular Ca2+); the first (transient) phase of these responses was affected stronger than the second, slow phase. PKC activators also reduced the response to GABA. The effect of PDBu on the response to kainate was dual; either inhibition or potentiation were observed at different concentrations of PDBu. The inactive analogue of PMA, the alpha-PMA, was without effect on the responses to 5-HT and GABA. The PKC inhibitor 1,5-isoquinolinesulfonyl-2-methylpiperazine (H7) suppressed the inhibitory effect of PDBu on 5-HT response. Amiloride, a blocker of the Na+/H+ exchange (which is known to be activated by PKC in some tissues), did not suppress the effects of PDBu. We concluded that activation of PKC down-regulates the responses to 5-HT, ACh and GABA, and has a dual effect on response to kainate. Possible mechanisms of these effects are discussed.

Acetylcholine↗
PubMed1989

Two calcium-activated chloride conductances in Xenopus laevis oocytes permeabilized with the ionophore A23187.

1. Currents evoked by elevated intracellular free Ca2+ in Xenopus laevis oocytes were studied using the two-electrode voltage clamp technique. The elevation in Ca2+ concentration was achieved in three ways: by the use of the divalent cation ionophore A23187; by application of Ca2+-mobilizing neurotransmitters serotonin and acetylcholine (ACh); by the entry of Ca2+ through voltage-dependent channels. 2. In most experiments, the membrane was permeabilized to Ca2+ by a 15 min pretreatment with A23187 in a Ca2+-free solution. Exposure of the ionophore-treated oocytes to external Ca2+ elicited an inward current (at holding potentials of -40 to -60 mV). At external Ca2+ concentrations ([Ca2+]) between 0.1 and 1 mM, the current had a time-to-peak of at least 10 s, and slowly decayed over tens of seconds. At [Ca2+] greater than 2 mM, the inward current had two distinct kinetic components, a fast and transient one (Ifast) and a slow one (Islow). 3. The main carrier of the Ca2+-evoked inward current was Cl-. Several data indicate the existence of a tetraethylammonium (TEA)-sensitive K+ conductance. No evidence for a Na+ current was found. 4. The two components of the Ca2+-evoked inward current in ionophore-permeabilized oocytes, and the two components of the current evoked by ACh and serotonin (the latter in oocytes injected with rat brain RNA but untreated with A23187), were blocked by intracellular injection of the Ca2+ chelator, ethyleneglycolbis-(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA). The two components of these currents displayed different sensitivity to Ca2+ buffering; higher doses of EGTA were necessary to inhibit the slow component than the fast one. 5. One to two minutes of treatment with 2 mM-9-anthracene carboxylic acid (9-AC) fully blocked Ca2+-dependent Cl- current evoked by Ca2+ influx through voltage- dependent Ca2+ channels in intact (untreated with A23187) oocytes. In ionophore-treated oocytes, block of Ifast was observed at holding potentials at which the current was outward (i.e. due to Cl- influx); Islow was inhibited only partially. The block of Ca2+-evoked Cl- efflux by 9-AC developed much more slowly and was less potent. to explain these results, the existence of two sites of 9-AC action is proposed. 6. Exposure of the ionophore-permeabilized oocytes to 0.1-0.2 mM [Ca2+] strongly reduced the response to higher concentrations of Ca2+. Ifast displayed stronger Ca2+-dependent inactivation than Islow.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1988

Is a decrease in cyclic AMP a necessary and sufficient signal for maturation of amphibian oocytes?

Acetylcholine rapidly lowered the intracellular levels of cyclic AMP in stage 5 and 6 Xenopus laevis oocytes. Acetylcholine alone did not induce oocyte maturation, though it did accelerate maturation induced by progesterone. The effect of acetylcholine on oocyte maturation was independent of extracellular calcium concentration. Adenosine increased cyclic AMP and abolished the progesterone-induced decrease in cyclic AMP levels in follicles and in denuded oocytes. This effect of adenosine was blocked by the Ra purinergic receptor antagonist, theophylline. Despite those effects, adenosine alone induced maturation in stage 6 oocytes and accelerated progesterone-induced maturation in both stage 5 and 6 cells. Adenosine also induced a significant increase in the rate of 45Ca efflux from oocytes in the presence and the absence of external calcium. We suggest that the activation of cell surface receptors involved in the release of calcium from cellular stores may induce or accelerate oocyte maturation independently of small changes in intracellular cyclic AMP concentration.

Acetylcholine↗
PubMed1987

Further characterization of the slow muscarinic responses in Xenopus oocytes.

In immature follicular oocytes of the frog Xenopus laevis, application of muscarinic agonists evokes a complex response consisting of a fast and a slow Cl currents (the dominant responses), Cl current fluctuations, and a less prominent slow K current. The characteristics of the slow ACh-evoked potassium current were studied using the two-electrode voltage clamp method, and compared to those of the ACh-evoked Cl currents. In experiments designed to study the K current response separately, without the interference of ACh-evoked Cl currents, the holding potential was set close or equal to Cl equilibrium potential (measured as the reversal potential of the ACh-evoked Cl current). The Cl current responses were studied in cells that had negligible K current response. The dose-response curve of the potassium response followed classical Michaelis-Menten kinetics. The dose-response characteristics of the slow ACh-evoked Cl current displayed a positive cooperativity of at least 3. In spite of this difference, kinetic analysis revealed that these two responses, as well as the fast Cl current response that was characterized earlier (Dascal and Landau 1982), had almost identical apparent equilibrium dissociation constants (0.29-0.39 microM), suggesting involvement of a single receptor class. Both K and Cl currents were reduced (to 32-56% of control) by millimolar concentrations of phosphodiesterase (PDE) inhibitors, theophylline and isobutylmethylxanthine. Elevation of extracellular Ca concentration from 1 to 10 mM doubled the K current; depletion of external Ca caused a partial inhibition of this response. The K current was potentiated by 0.1 microM 4-phorbol 12,13-dibutyrate (PDBu).(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1987

Dissociation of acetylcholine- and cyclic GMP-induced currents in Xenopus oocytes.

In Xenopus follicular oocytes, activation of muscarinic receptors evokes a slow potassium current (H-response); a similar current is evoked by intracellular injection of cyclic guanosine 3',5'-monophosphate, cGMP (Dascal et al. 1984). We have tested the hypothesis that cGMP may be the second messenger that mediates the opening of K channel by acetylcholine (ACh). ACh elevated the intracellular level of cGMP with a time course similar to that of the development of the muscarinic H-response; maximal increase in cGMP concentration above the control was about 0.2 pmole/oocyte. The amount of injected cGMP that produced a detectable K current ("threshold dose") varied between 0.5 and 3 pmole/oocyte. At low doses of cGMP, the slope of log dose-log response curve was about 2.5, suggesting involvement of a biochemical process with a positive cooperativity of at least 3. Higher doses of cGMP evoked, in addition to the outward current, an irregular, rapidly developing, long-lasting inward current, that never reached amplitudes comparable to those of ACh-evoked Cl currents. The K current elicited by cGMP was insensitive to elevation or depletion of external Ca. It was potentiated by isobutylmethylxanthine (IBMX). ACh strongly inhibited the cGMP-evoked K current when applied at the plateau of the latter. 4-Phorbol 12,13-dibutyrate (PDBu) (1 microM) rapidly and completely inhibited the cGMP response. It is concluded, that most of the results presented in this report contradict the hypothesis that cGMP is the intracellular mediator of ACh-induced changes in membrane conductance in the oocytes.

1-Methyl-3-isobutylxanthine↗
PubMed1987

The use of Xenopus oocytes for the study of ion channels.

Recently, in addition to the "traditional" research on meiotic reinitiation and fertilization mechanisms, the oocytes of the African frog Xenopus laevis have been exploited for the study of numerous aspects of ion channel function and regulation, such as the properties of several endogenous voltage-dependent channels and the involvement of second messengers in mediation of neurotransmitter-evoked membrane responses. In addition, injection of these cells with exogenous messenger RNA results in production and functional expression of foreign membranal proteins, including various voltage- and neurotransmitter-operated ion channels originating from brain, heart, and other excitable tissues. This method provides unique opportunities for the study of the structure, function, and regulation of these channels. A multidisciplinary approach is required, involving molecular biology, electrophysiology, biochemistry, pharmacology, and cytology.

Animals↗
PubMed1987

Rat brain 5-HT1C receptors are encoded by a 5-6 kbase mRNA size class and are functionally expressed in injected Xenopus oocytes.

Injection of rat brain RNA into Xenopus laevis oocytes induces synthesis of receptors that show an electrophysiological response to bath application of serotonin. While there are at least 4 pharmacologically distinct subtypes of 5-HT binding sites in the rat brain, we find that the pharmacological characteristics of the predominant electrophysiologically active receptor synthesized in Xenopus oocytes are most consistent with those of the 5-HT1C subtype. Additional electrophysiologically active 5-HT receptor types could not be detected. Injection of mRNA isolated from a number of rat brain regions shows that the choroid plexus is particularly enriched for 5-HT1C mRNA. Oocytes injected with RNA isolated from this region respond 16 or 8 times more strongly to serotonin than do oocytes injected with RNA isolated from cortex or substantia nigra, respectively. In addition, by fractionation of rat brain mRNA through agarose gels, we have identified a single RNA size class of about 5-6 kbase that encodes this serotonin receptor.

Animals↗