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N Dascal

Publications and source records attributed to N Dascal.

70 records · Page 4Linked to original sources
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↗
PubMed1986

Expression and modulation of voltage-gated calcium channels after RNA injection in Xenopus oocytes.

Calcium ions flow into cells through several distinct classes of voltage-dependent calcium-selective channels. Such fluxes play important roles in electrical signaling at the cell membrane and in chemical signaling within cells. Further information about calcium channels was obtained by injecting RNA isolated from rat brain, heart and skeletal muscle into Xenopus oocytes. Macroscopic currents through voltage-operated calcium channels were resolved when the endogenous calcium-dependent chloride current was blocked by replacing external calcium with barium and chloride with methanesulfonate. The resulting barium current was insensitive to tetrodotoxin but was completely blocked by cadmium or cobalt. With both heart and brain RNA at least two distinct types of calcium ion conductance were found, distinguishable by their time course and inactivation properties. In oocytes injected with heart RNA, the slowly inactivating component was selectively blocked by the calcium-channel antagonist nifedipine. Barium ion currents induced by heart RNA were modulated by isoproterenol, cyclic adenosine monophosphate, and acetylcholine.

Animals↗
PubMed1986

ATP-evoked membrane responses in Xenopus oocytes.

Voltage-clamp technique and intracellular injections of drugs were used to study the adenosine triphosphate (ATP)-evoked depolarizing current response in the Xenopus laevis oocytes. The depolarizing current was comprised of a fast transient component (D1) followed by a late long-lasting component (D2). It was carried mainly by Cl- ions. The depolarizing current was better elicited by ATP and ADP than by AMP or adenosine and was not blocked either by theophylline (0.2 mM) or by quinidine sulphate (1 mM). The D2 current was sometimes masked by an ATP-evoked K+ hyperpolarizing current which was blocked by theophylline and mediated via P1 purinoceptors. This study suggests that the oocyte's membrane embodies at least two different purinoceptor's types, each of these types subserves a different set of ionic channels.

Adenosine Triphosphate↗
PubMed1986

Involvement of a GTP-binding protein in mediation of serotonin and acetylcholine responses in Xenopus oocytes injected with rat brain messenger RNA.

Injection of poly(A)+ RNA from rat brain into Xenopus oocytes caused the appearance of Cl currents in response to serotonin (5-HT) and acetylcholine (ACh). Both neurotransmitters evoked two-component currents similar in their time course to the oocyte's endogenous cholinergic muscarinic response, which was shown in previous studies to be mediated by IP3 synthesis leading to Ca release from intracellular stores. The responses to ACh and 5-HT exhibited self- and cross-desensitization, i.e., application of either ACh or 5-HT inhibited the subsequent response to either one of the two transmitters. Intracellular injection of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) mimicked the 5-HT and ACh response, and also completely suppressed the response to the subsequent application of either ACh or 5-HT. Treatment of the oocytes with pertussis toxin (PTX) caused a 50% attenuation of ACh and 5-HT responses. In the membranes of both control and mRNA-injected oocytes, PTX catalyzed the ADP-ribosylation of a single Mr = approximately 40,000 protein. Injection of the purified beta gamma-subunits of transducin enhanced the 5-HT response. The 5-HT and GTP-gamma-S responses were inhibited by intracellular injection of the Ca2+ chelator, EGTA, as previously shown for the ACh response. These data suggest that ACh and 5-HT receptors, synthesized in the oocytes on the template of brain mRNA, act through a common pathway that involves (a) a guanine nucleotide binding protein and (b) IP3 production leading to Ca mobilization.

Acetylcholine↗
PubMed1985

Acetylcholine and phorbol esters inhibit potassium currents evoked by adenosine and cAMP in Xenopus oocytes.

In Xenopus laevis oocytes, adenosine and other purinergic agonists induce a K+-conductance increase that is fully mimicked by intracellular application of cAMP. Acetylcholine suppresses the K+-conductance increase caused by adenosine, by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, or by intracellular injection of cAMP. This effect of acetylcholine is not mimicked by intracellular injection of Ca2+ or of the Ca-mobilizing agent inositol 1,4,5-trisphosphate. However, adenosine and cAMP responses are inhibited by 4 beta-phorbol 12,13-dibutyrate and 4 beta-phorbol 12-myristate 13-acetate. These results suggest that, in Xenopus oocytes, the muscarinic inhibition of purinergic and cAMP responses is mediated through the activation of the phospholipid-dependent, Ca-activated protein kinase (protein kinase C).

Acetylcholine↗
PubMed1985

Role of calcium mobilization in mediation of acetylcholine-evoked chloride currents in Xenopus laevis oocytes.

The involvement of Ca ions in the mediation of muscarinic Cl- current responses in Xenopus oocytes was studied using the voltage-clamp technique and direct measurements of 45Ca efflux. The injection of Ca into the oocytes produced a dose-dependent transient inward (depolarizing) current carried by Cl. This current was occasionally followed by a second, long-lasting inward current. The muscarinic response was evoked by the application of acetylcholine (ACh). It consisted of a transient inward current response, and a long-lasting inward current response, both inward currents carried by Cl ions. Both responses were inhibited by intracellular injection of ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid (EGTA), the long-lasting response being inhibited faster than the transient response. The calmodulin inhibitor, trifluoperazine, inhibited both the Cl-current responses to ACh and to Ca injection. ACh (10 microM) evoked a release of 45Ca from pre-loaded oocytes. This effect was inhibited by atropine (1 microM). In the absence of external Ca, the muscarinic transient and long-lasting responses were partially inhibited. The long-lasting response was more sensitive to the external Ca depletion than the transient response. Repetitive applications of ACh in the absence of external Ca resulted in a progressive decrease in the response amplitudes. Under these conditions, a temporary exposure to normal Ca solution ('Ca window') resulted in a partial recovery of the response amplitudes. The muscarinic inward current responses were not inhibited by nifedipine (20 microM). In the presence of a high external concentration of Mn ions ([Mn]o = 18 mM), the transient response was potentiated. Subsequent applications of ACh in high [Mn]o resulted in progressively decreasing responses. It is concluded that the muscarinic Cl responses in Xenopus oocytes are mediated by an increase in the intracellular free Ca activity, aiCa. Ca ions involved in the mediation of the muscarinic Cl current responses are released from cellular Ca stores. It is also proposed that the transient and long-lasting responses result from the release of Ca from two different stores.

Acetylcholine↗
PubMed1985

Adenosine-induced K+ current in Xenopus oocyte and the role of adenosine 3',5'-monophosphate.

Voltage clamp technique was used in Xenopus laevis oocytes in order to study and compare membrane currents evoked by extracellularly applied adenosine (0.1-10 microM) and intracellularly injected cyclic AMP (0.15-10 microM). The adenosine response is a late long-lasting outward K+ current ("H" current), mediated by the Ra purine receptor subtype. The H current amplitude is directly proportional to (occupancy)3; the KD for adenosine is 3.34 microM. The H current is inhibited by the intracellular injection of protein kinase inhibitors, types II and III (5-450 ng/oocyte) and is usually potentiated by intracellular injection of theophylline (100-300 microM), though extracellular application of theophylline (1-100 microM) reversibly blocks the receptor. Occasionally, the H current is contaminated by a small Cl- current. The cyclic AMP current is also a long-lasting K+ outward current which is potentiated by extracellular theophylline (2 mM). Injection of cyclic AMP inhibits the membrane response to subsequent application of adenosine. The converse inhibition of a cyclic AMP response by an earlier adenosine response is also observed but at very high concentrations of adenosine (greater than 0.6 mM). It was shown by radioimmunoassay that extracellular adenosine increases the level of the intracellular cAMP within a few seconds by about 30%. Intracellular injection of a comparable amount of cAMP was shown to evoke a measurable K+ current. It is proposed that the adenosine-evoked K+ outward current is mediated by a rise in intracellular cAMP.

Adenosine↗
PubMed1984

Acetylcholine promotes progesterone-induced maturation of Xenopus oocytes.

Progesterone-induced maturation of follicle-enclosed and denuded Xenopus laevis oocytes was significantly shortened by a concomitant exposure to acetylcholine. The promotion of maturation by acetylcholine was blocked by the specific muscarinic antagonist atropine. The action of acetylcholine was dose dependent, and the neurotransmitter was effective at very low concentrations. Progesterone progressively reduced the electrophysiological responses of X. laevis oocytes to acetylcholine, which completely disappeared close to the time of germinal vesicle breakdown. Progesterone alone did not elicit any electrophysiological responses. The in vitro effect of acetylcholine on oocyte maturation might reflect a physiological influence of the cholinergic system on an in vivo maturation process.

Acetylcholine↗
PubMed1984

Xenopus oocyte resting potential, muscarinic responses and the role of calcium and guanosine 3',5'-cyclic monophosphate.

Resting potential (r.p.) and muscarinic response mechanisms were studied in Xenopus laevis oocytes using the voltage-clamp technique. Insertion of micro-electrodes into the oocyte produced a 'shunt' membrane conductance which partially sealed after a few minutes. The oocyte resting potential (measured with a single intracellular electrode) ranged from -40 to -60 mV. Ouabain and low K+ solution depolarized both follicles and denuded oocytes. The electrogenic Na+-K+ pump was more active in the latter. In the presence of ouabain, the r.p. agreed with the constant field theory. alpha (PNa+/PK+) was 0.12 in follicles and 0.24 in denuded oocytes. beta (PCl-/PK+) was 0.4 in both. At [Na+]o lower than 70 mM, the r.p. deviated considerably from the constant field predictions. The relatively large value of alpha indicated the major role of Na+ in oocyte r.p. determination. The oocyte muscarinic response was separated into four distinct components: the fast depolarizing Cl- current, 'D1'; the slow depolarizing Cl- current, 'D2'; the slow hyperpolarizing K+ current, 'H'; and the large membrane Cl- current fluctuation, 'F'. The H response reversal potential showed a Nernst relationship to [K+] and was selectively blocked by intracellular injection of tetraethylammonium (TEA). The D1 and D2 reversal potential showed a Nernst relationship to [Cl-]. In Ca2+-deficient, EGTA-containing medium, D2 and F were abolished and D1 and H were reduced. Verapamil inhibited all responses. Increasing [Ca2+]o caused a significant increase in D1, D2 and F response amplitudes. Intracellular injection of 0.6-10 pmol guanosine 3',5'-cyclic monophosphate, induced a large outward K+ current, similar to the muscarinic H response.

Acetylcholine↗
PubMed1982

Adenosine-induced slow ionic currents in the Xenopus oocyte.

Adenosine and its 5'-phosphorylated congeners evoke specific membrane-mediated responses in excitable tissues. Available data suggest that inhibition of the target cell occurs due to hyperpolarization, and in some preparations a compound effect of ATP (excitation and inhibition) has been found. However, the ionic mechanism of the purinergic-mediated response has not been studied by standard intracellular voltage-clamping techniques. Recently, we have discovered purinergic receptors in the Xenopus oocyte, a well defined giant cell amenable to rigorous electrophysiological and biochemical studies. We report here that in these cells, adenosine-induced slow membrane responses consisted of an early depolarizing (D) transient current carried by Cl ions, followed by a steady hyperpolarizing (H) current involving K+ ions. The relative potency sequence for the D current was ATP congruent to ADP greater than AMP congruent to adenosine; this order was reversed for the H current.

Adenosine↗
PubMed1982

Cyclic GMP mimics the muscarinic response in Xenopus oocytes: identity of ionic mechanisms.

Acetylcholine (AcCho) elicits four distinct membrane responses in Xenopus oocytes; the responses can be studied by using the voltage clamp technique. The fastest of the responses, a transient inward current (D1 response), is muscarinic, being evoked by oxotremorine and blocked by atropine but not by curare or hexamethonium. The action of AcCho is cooperative, three transmitter-receptor complexes being required to cause a membrane conductance change, and the dose-response curve in most cases can be fitted by an equation assuming the existence of two binding sites with an affinity ratio of about 11. Guanosine 3',5'-cyclic monophosphate and the 8-bromo and dibutyryl derivatives cause a response similar to D1 in both its time course and the underlying ionic mechanism. The nucleotide-generated response has a smaller amplitude than the AcCho-generated D1.

Acetylcholine↗
PubMed1981

Divalent cations and transmitter release at low concentration of tetrodotoxin.

Transmitter release from frog motor terminals was studied in the presence of very low concentrations of tetrodotoxin (TTX, 4.10(-10)--6.10(-9) g/ml). TTX reversibly reduced the amplitude of the end-plate potential (epp), while leaving the amplitude distribution to follow Poisson's law. The effects of a number of divalent cations were studied in the presence of TTX. It was found that after the addition of TTX there was an increase in the constant of dissociation of calcium and strontium from a hypothetical membrane "release site," while the dissociation constants of magnesium and manganese remained unaltered. It is concluded that the release site is probably intracellular and that a reduced presynaptic spike amplitude, as well as magnesium and manganese ions, decrease the access of calcium and strontium to the site.

Animals↗
PubMedDate not supplied

Inositol 1,4,5-trisphosphate mimics muscarinic response in Xenopus oocytes.

The enhanced metabolism of phosphoinositides, which is associated with a wide variety of stimuli and physiological responses, has been studied intensively. Berridge and his collaborators demonstrated that the first measurable reaction following cell membrane receptor activation is a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and that the product of this reaction, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), could cause a release of non-mitochondrial calcium. These findings have been verified in other systems. Although the relationship between the hydrolysis of PtdIns(4,5)P2 and the mobilization of intracellular calcium was clearly demonstrated, the direct link between Ins(1,4,5)P3 production and the physiological response was only implied. We have investigated the possibility that the intracellular release of Ins(1,4,5)P3 mediates the muscarinic-cholinergic response is Xenopus oocytes, and we show here that intracellularly injected Ins(1,4,5)P3 mimics the muscarinic depolarizing chloride current in Xenopus oocytes. This is the first demonstration of a direct link between phosphoinositides metabolism and a neuro-transmitter-induced physiological response.

Animals↗