Biomedical subjects

E M Landau

Publications and source records attributed to E M Landau.

At least 19 recordsLinked to original sources

PubMed1991

Activated alpha subunit of Go protein induces oocyte maturation.

The capability of various activated guanine nucleotide binding regulatory protein (G protein) alpha subunits to induce meiotic maturation was studied. Activated Go protein alpha subunit (alpha o*) but not the three inhibitory G protein alpha subunits triggered meiotic maturation in Xenopus oocytes. The effect was concentration dependent with a half-maximal effect in the 100-200 pM range. Injection of alpha o* stimulated protein kinase C activity. Coinjection of the peptide containing residues 19-36 of protein kinase C [PKC-(19-36)], a specific protein kinase C inhibitor, blocked the alpha o*- but not progesterone-induced maturation. Cycloheximide and the injection of antisense oligonucleotides specific to the c-mos transcript blocked alpha o-induced maturation. Immunoprecipitation with a mos protein-specific monoclonal antibody showed that alpha o-injected oocytes had phosphorylated mos protein. When PKC-(19-36) was coinjected with alpha o*, phosphorylated mos protein was not observed. These observations indicate that alpha o*, through protein kinase C and the translation of c-mos, can trigger meiotic division of Xenopus oocytes. Our results raise the possibility that persistently activated G proteins through cellular protooncogenes may regulate cell-cycle resumption.

Animals↗

PubMed1991

Two forms of the bovine brain Go that stimulate the inositol trisphosphate-mediated Cl- currents in Xenopus oocytes. Distinct guanine nucleotide binding properties.

Heterotrimeric GTP-binding proteins from bovine brain were resolved by fast protein liquid chromatography chromatography using Mono Q columns. Two distinct forms of the protein Go were identified. Both forms had stochiometric amounts of alpha- and beta gamma-subunits. The a-subunits of both forms were recognized by an alpha o-specific antiserum, but not by any of the alpha i-specific antisera. The two forms showed distinct migration patterns on 9% sodium dodecyl sulfate-polyacrylamide gels containing 4-8 M urea gradients. Neither form comigrated with the recombinant alpha o1. Both the recombinant alpha o1 and the most abundant form of Go were recognized by an antiserum, H-660, against a peptide encoding amino acids 3-17 of alpha i2. H-660 has been shown previously to recognize alpha o and alpha i (Mumby, S. M., Pang, I. K., Gilman, A. G., and Sternweis, P. C. (1988) J. Biol. Chem. 263, 2020-2026). This more abundant form is called Go A most likely corresponds to the cloned alpha o1. The less abundant form, Go B, was not recognized by H-660. However, both forms of bovine brain Go were recognized by GC/2, an antiserum against the N-terminal region of alpha o1. Hence alpha oA and alpha oB may be different in their N terminus regions. Neither form of bovine brain Go was recognized by an antisera made to a peptide encoding the unique regions of the cloned alpha o2 from HIT cells (Hsu W. H., Rudolph, U., Sanford, J., Bertrand, P., Olate, J., Nelson, C., Moss, L.E., Boyd, A. E., III, Codina, J., and Birnbaumer, L. (1990) J. Biol. Chem. 265, 11220-11226). Go A and Go B have similar guanine nucleotide binding and release properties. Both release GDP within 1 min in the absence of added Mg2+. Both bind guanosine (GTP gamma S) rapidly as well. However Go A binds GTP gamma S about 2.5-fold faster than Go B, in the absence of added Mg2+ ion. Both forms of Go as well as the recombinant alpha o (alpha o1) can increase muscarinic stimulation of inositol trisphosphate-mediated Cl- current in Xenopus oocytes. These data indicate that we have identified two structurally distinct forms of Go that have different guanine nucleotide binding properties and are capable of functioning in the receptor-regulated phospholipase C pathway in Xenopus oocytes.

Animals↗

PubMed1991

Nifedipine blocks calcium-dependent cholinergic depolarization in the guinea pig hippocampus.

The possibility that cholinergic stimulation might directly activate a receptor-operated Ca2+ channel was investigated in the CA1 region of guinea pig hippocampus using intracellular recording techniques. Two cholinergic responses were studied: (1) the plateau depolarization evoked by cholinergic stimulation in the presence of Ba2+; and (2) the Ca2(+)-dependent component of membrane depolarization. Both of these responses were blocked by 1-5 microM of nifedipine, a blocker of voltage-dependent L-type Ca2+ channels. In addition, the plateau response was mimicked by direct postsynaptic depolarization in the presence of Ba2+. We conclude that cholinergic stimulation does not directly activate a Ca2+ conductance in these neurons, but rather leads to the indirect activation of L channels which may be located both pre- and postsynaptically.

Animals↗

PubMed1990

Long-term potentiation in rat hippocampus is inhibited by low concentrations of ethanol.

Acute ethanol ingestion impairs memory in humans at concentrations associated with mild intoxication. A possible neurophysiological correlate of this effect is the suppression by ethanol of long-tem potentiation (LTP), a persistent increase in synaptic efficiency which has been proposed as a substrate for memory. However, in previous studies ethanol has been shown to impair LTP only at very high concentrations, near the lethal level in humans. We now report that ethanol can significantly reduce LTP in rat hippocampus at concentrations as low as 5 mM, a level attainable following ingestion of a single alcoholic drink. We also demonstrate that the potency of ethanol in depressing LTP correlates well with its potency in inhibiting the response to N-methyl-D-aspartate, an agonist at the glutamate receptors implicated in LTP induction. The influence of low ethanol concentrations on LTP may contribute to the memory impairment associated with its use in humans.

Animals↗

PubMed1990

Cholinergic stimulation enhances long-term potentiation in the CA1 region of rat hippocampus.

The effect of the cholinergic agonist carbachol on a putative substrate for memory (long-term potentiation; LTP) was investigated in slices of rat hippocampus (CA1 region). Carbachol (5 microM) increased LTP when the presynaptic depression of the EPSP was controlled. The results indicate that carbachol enhances the effectiveness of the tetanus, probably through postsynaptic mechanisms. This effect may have implications for the role of acetylcholine in memory and the use of cholinergics in memory disorders.

Animals↗

PubMed1990

Go protein as signal transducer in the pertussis toxin-sensitive phosphatidylinositol pathway.

Receptors stimulating phospholipase C do so through heterotrimeric GTP-binding proteins to produce two second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol. In spite of the detailed understanding of phospholipase C structure and phosphatidyl inositol signalling, the identity of the GTP-binding protein involved is so far unknown. To address this issue, we have used the Xenopus oocyte in which muscarinic receptors couple to phospholipase C through a pertussis toxin-sensitive GTP-binding protein. In this cell, InsP3 mobilizes intracellular Ca2+ to evoke a Cl- current. The magnitude of this Cl- current is proportional to the amount of InsP3 in the cell, and therefore can be used as an assay for InsP3 production. We report here that the activated alpha-subunit of the GTP-binding protein GO, when directly injected into oocytes, evokes a Cl- current by mobilizing Ca2+ from intracellular InsP3-sensitive stores. We also show that holo-GO, when injected into oocytes, can specifically enhance the muscarinic receptor-stimulated Cl- current. These data indicate that GO can serve as the signal transducer of the receptor-regulated phospholipase C in Xenopus oocytes.

Animals↗

PubMed1989

Coupling of exogenous receptors to phospholipase C in Xenopus oocytes through pertussis toxin-sensitive and -insensitive pathways. Cross-talk through heterotrimeric G-proteins.

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) can be categorized into molecularly divergent groups by their differential sensitivity to pertussis toxin. Receptors specifically use either pertussis toxin-sensitive or-insensitive G-proteins to couple to specific effectors. Receptor stimulation of phospholipase C, however, is pertussis toxin sensitive in some systems and pertussis toxin insensitive in others. We studied the coupling of receptors to phospholipase C by expressing receptors from both systems into a single cell, the Xenopus oocyte. [Arg8]Vassopressin (AVP) receptors from liver and cholecystokinin-8(sulfated) (CCK) receptors from brain were expressed in oocytes by intracellular injection of RNA. Both receptors stimulated a Ca2+-dependent Cl- current which can also be evoked by intracellular injection of inositol 1,4,5-tris-phosphate. Hence, receptor stimulation of phospholipase C was measured as the evoked Ca2+-dependent Cl- current. The liver AVP receptor, which is known to stimulate phospholipase C in a pertussis toxin-insensitive manner (Lynch, C. J., Prpic, V., Blackmore, P. F., and Exton, J. H. (1986) Mol. Pharmacol. 29, 196-203), was found to stimulate phospholipase C through a pertussis toxin-sensitive pathway in the Xenopus oocyte. The CCK receptor from brain stimulated phospholipase C through a pertussis toxin-insensitive pathway. Both AVP and CCK stimulation of phospholipase C were attenuated by the intracellular injection of excess G-protein beta gamma subunits. Neither pertussis toxin treatment nor intracellular injection of beta gamma subunits affected any steps subsequent to inositol 1,4,5-tris-phosphate production. From these data we conclude that both the pertussis toxin-sensitive and -insensitive pathways for receptor coupling to phospholipase C are transduced by heterotrimeric G-proteins. We also find that there is a lack of coupling fidelity of receptors to G-proteins in stimulation of phospholipase C which can be influenced by the membrane environment.

Animals↗

PubMed1989

Ethanol suppresses hippocampal cell firing through a calcium and cyclic AMP-sensitive mechanism.

The effects of ethanol were studied intracellularly in hippocampal pyramidal cells in vitro. Ethanol, 50-100 mM, produced a marked suppression of neuronal firing. This effect was blocked by treating the cell with cyclic 3', 5'-adenosine monophosphate (cAMP) or cadmium ions. Ethanol had no effect on the after-hyperpolarizing current. It is concluded that the ethanol-induced reduction of firing rate is due to a calcium-dependent process, and modulated by cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1989

Functional muscarinic supersensitivity in denervated rat hippocampus.

The effects of carbachol (CCh), a cholinergic agonist, were compared in voltage-clamped hippocampal pyramidal neurons in vitro, obtained from normal and fimbria-fornix-lesioned rats. A substantial increase in sensitivity to the effects of CCh was seen in denervated neurons. The supersensitivity was demonstrated on both the inward leak current and the calcium-dependent potassium current, IAHP. These findings provide convincing evidence for cholinergic denervation supersensitivity in the hippocampus.

Acetylcholinesterase↗

PubMed1989

A novel calcium-dependent chloride current in Xenopus oocytes injected with brain messenger RNA.

1. Membrane currents were studied in voltage-clamped Xenopus laevis oocytes which had been injected with total rat brain RNA. 2. When the membrane potential was stepped from -100 to +10 mV, two components of outward current were observed which were named Tout1 and Tout2. 3. Both Tout1 and Tout2 were eliminated in chloride-free or calcium-free media and blocked by 9-anthroic acid, indicating that they represented calcium-dependent chloride currents. 4. Both currents were dependent on extracellular calcium (1.8-10 mM), with Tout1 showing a greater sensitivity to changes in calcium concentration. 5. Tout2 but not Tout1 was blocked by intracellular injection of 300-600 pmol, BaCl2 (final concentration in the oocyte: 0.3-0.6 mM). Injection of KCl had no effect on either Tout1 or Tout2. 6. Tout2 but not Tout1 was enhanced by low concentrations of serotonin (0.5-2 nM). This effect was blocked by 0.1 microM-mianserin. Higher concentrations (above 10 nM) of serotonin decreased the amplitude of Tout2. The effect of serotonin was blocked by the protein kinase inhibitor, H-7 (25 microM). 7. Tout2 but not Tout1 was enhanced by 10 nM-phorbol myristate acetate. Higher concentrations of the phorbol ester decreased the amplitude of Tout2. 8. It is concluded that in oocytes injected with RNA there is an induction of a novel component of the calcium-induced chloride current (Tout2). This current reflects a second process of chloride channel opening which can be enhanced by serotonin via activation of protein kinase C.

Animals↗

PubMed1988

Functional expression of brain cholecystokinin and bombesin receptors in Xenopus oocytes.

Total RNA was extracted from 15-day-old whole rat brains. Microinjection of the RNA into Xenopus laevis oocytes induced electrophysiological responsiveness to cholecystokinin-8 (CCK) and bombesin (BBS) but not to corticotropin-releasing factor (CRF) or somatostatin. The responses to CCK and BBS were similar in shape, time course, and reversal potential to that induced by receptor mediated phospholipid breakdown and that which is induced by intracellular injection of IP3. These responses were not blocked by atropine or by mianserin, did not require extracellular Ca2+ and were completely suppressed by intracellular injection of EGTA.

Animals↗

PubMed1988

Activation of ionic currents in Xenopus oocytes by corticotropin-releasing peptides.

Oocytes of the African frog Xenopus laevis are shown by electrophysiological methods to possess receptors for corticotropin-releasing factor (CRF), arginine-vasopressin (AVP) and cholecystokinin (CCK). Oocytes surrounded by their follicular cell envelope responded to CRF or AVP with an outward hyperpolarizing current. This current was mediated by an increased conductance of K+ ions. Pretreatment with the adenylate cyclase activator forskolin or with the cAMP phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) potentiated the responses to these peptides indicating that the cAMP second messenger system may mediate the responses. Oocytes stripped of the follicular envelope, which cannot generate cAMP-dependent K+ currents, did not respond to either CRF or AVP. Oocytes exposed to CCK responded with an inward depolarizing current. This current was carried by an increased conductance to Cl-ions. Removal of the follicular cell layer did not affect the response to CCK. The shape, time course, and reversal potential of the Cl-current suggest that CCK acts through the phosphatidylinositol pathway.

Animals↗

PubMed1988

Beta gamma subunits of GTP-binding proteins inhibit muscarinic receptor stimulation of phospholipase C.

This study examines the mechanism of guanine nucleotide-binding protein (G protein) coupling of receptors to phospholipase C. The Xenopus oocyte has a muscarinic receptor-activated Cl- current that is mediated by inositol 1,4,5-trisphosphate. Modulation of the muscarinic receptor-evoked Cl- current was examined under voltage clamp in oocytes injected with resolved G-protein subunits. The presence of an alpha subunit of G proteins in oocytes was shown by pertussis toxin-labeling of a 41-kDa band in oocyte membranes. The presence of the beta subunit of G proteins was demonstrated by immunoblotting experiments with an antiserum (U-49) that is specific for the beta subunit. Pertussis toxin treatment of oocytes resulted in the uncoupling of muscarinic receptors from activation of the Cl- current. Cells microinjected with 1.5 ng of human erythrocyte beta gamma-subunit complex or 1.0 ng of bovine brain beta gamma-subunit complex showed approximately a 95% reduction in the evoked Cl- current. Cells injected with equal volumes of protein storage vehicle showed no change in response. Cells injected with boiled beta gamma subunits, bovine serum albumin, or resolved alpha subunits also showed no reduction in response. Cells injected with various concentrations of beta gamma subunits showed a concentration dependence with half-maximal inhibition of the muscarinic activated Cl- current at about 10 nM. Cells injected with 1.0 ng of bovine brain beta gamma subunits could not respond to bath-applied agonist but could generate the Cl- current on intracellular injection of inositol 1,4,5-trisphosphate. These observations suggest that there is a G protein responsible for muscarinic receptor-mediated signal transduction through phospholipase C and that it is an alpha beta gamma heterotrimer. It appears that the mode of action of the G protein in the phospholipase C system may be similar to that of the hormone-activated adenylyl cyclase.

Animals↗

PubMed1988

An analysis of the depolarization produced in guinea-pig hippocampus by cholinergic receptor stimulation.

1. The effects of carbachol on hippocampal pyramidal neurones were studied in tissue slices in vitro with intracellular microelectrodes, employing current clamp and voltage clamp methods. 2. The calcium-dependent potassium current, IAHP, and the voltage-dependent potassium current, IM, were both reversibly blocked by the application of carbachol (5-10 microM). 3. Carbachol (1-10 microM) induced a steady inward current under circumstances in which both IAHP and IM were inactive. This inward current was sometimes difficult to reverse upon carbachol wash-out, an effect possibly related to receptor desensitization. 4. The depolarizing effect of carbachol was reversed by 0.1 microM-atropine, and exhibited an apparent dissociation coefficient of 1.2 microM for carbachol and 18 nM for pirenzepine, indicating that it is mediated by activation of an M1 muscarinic receptor. 5. The depolarizing effect or inward current induced by carbachol was completely blocked by the potassium channel blockers caesium, tetraethylammonium and barium. 6. The slope of the current-voltage (I-V) plots in carbachol was reduced in the majority of cells, and crossed the control I-V plots at a negative membrane potential. The reversal potentials in carbachol shifted in a positive direction when bathing potassium concentration was increased. 7. In a number of cells, the I-V curves in carbachol were parallel to or converged positively with the control I-V curves. 8. The effects of carbachol were compared to those of serotonin, which increases a 'pure' potassium conductance. Serotonin (10 microM) produced an increase in the slope of the I-V curve, with a reversal potential sensitive to changes in bathing potassium concentration. The carbachol reversal potential values were negative to those of serotonin at 5 and 10 mM-potassium. The equilibrium potentials for carbachol and serotonin were equal at 25 mM-potassium. 9. The negative values of the reversal potential at 5 and 10 mM-potassium and the occurrence of non-crossing I-V characteristics in carbachol could be explained by postulating a second effect of carbachol: namely, a non-specific conductance increase in the dendrites. 10. It is concluded that carbachol depolarizes pyramidal cells in the hippocampus by blocking a voltage-insensitive potassium leak channel and does so by activating M1 muscarinic receptors. In addition, carbachol may also activate a second conductance in the dendrites, which could account for the anomalous I-V characteristics sometimes seen in response to carbachol in these cells.

Animals↗

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

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↗

PubMed1981

pH dependence of the acetylcholine receptor channel: a species variation.

The effects of pH changes on the miniature endplate current (mepc) and on endplate current fluctuations (acetylcholine [ACh] noise) were examined at the neuromuscular junction in vitro in two species of frogs. In Rana pipiens the relationship between the decay time constant of the mepc (tau') and pH had a symmetrical bell shape; the value of tau' being largest at pH 7 and decreasing at more acid or more alkaline pH. In acid pH the mepc amplitude (A) decreased relative to its value at pH 7, and in alkaline pH A increased. In Rana ridibunda a narrower and asymmetric bell-shaped dependence of tau' on pH, having a maximum of pH 5.5, was found. The mepc amplitude was again reduced in acid pH but had a peak at pH 5.5. Also, its value at pH 9 was larger than at pH 7. These results were obtained with a number of different buffers and were not found to be sensitive to the nature of the buffer chosen. By performing ACh-noise analysis we found that in Rana pipiens at acid pH (5.5-5.0), the single channel conductance (gamma) and the single channel open time (tau) were significantly reduced relative to their value at pH 7. However, in Rana ridibunda at acid pH (5.4) gamma was unchanged and tau was markedly increased relative to their values at pH 7. The results can be explained quantitatively by electrostatic interaction between two fixed and titratable ionic groups and a mobile charge in the receptor molecule. The model fits the data for groups having pKs approximately 4.8 and approximately 9.8 for Rana pipiens and approximately 4.6 and approximately 6.3 for Rana ridibunda. The groups can be tentatively identified as amino acid residues; glutamic or aspartic and lysine or tyrosine for Rana pipiens; glutamic or aspartic and histidine for Rana ridibunda. The difference in the fitted values of the other model parameters for these two species can be attributed to differences in the spatial configuration of the charged groups.

Acetylcholine↗