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R Miledi

Publications and source records attributed to R Miledi.

At least 55 records · Page 3Linked to original sources
PubMed1997

Efficient coupling of 5-HT1a receptors to the phospholipase C pathway in Xenopus oocytes.

To investigate the receptor-channel coupling pathway, the coding region of the 5-HT1a receptor was subcloned into two plasmid vectors pSP64(polyA+) and pSP64T. Compared to the original 5-HT1a receptor construct G-21, both new constructs increased greatly the expression of functional 5-HT1a receptors in Xenopus oocytes, which developed large inward current responses to 5-HT. These responses were dose-dependent (EC50 approximately 150 nM), and could be elicited also by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The 5-HT1a receptor mediated current had an oscillatory time course, and a reversal potential close to the equilibrium potential for Cl- (ca. -25 mV). Moreover, during and for some minutes following the application of 5-HT, these oocytes acquired the property of generating a transient inward current when their membrane was hyperpolarized. These features are characteristic of responses mediated by other receptors (e.g. muscarinic, angiotensin, serum receptors, etc.) that are known to couple to the endogenous PLC/PI second messenger pathway in Xenopus oocytes. In particular, the 5-HT1a receptor mediated current was very similar to the current induced by 5-HT-stimulation of heterogenic 5-HT2c receptors. Our results show further that the 5-HT1a receptor couples to the endogenous PLC/PI pathway much less efficiently than the 5-HT2c receptor. These results demonstrate clearly that the human 5-HT1a receptor can couple efficiently to the Xenopus oocyte endogenous PLC/PI pathway, and provide additional evidence for cell-specific signal transduction.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1996

Threonine-for-leucine mutation within domain M2 of the neuronal alpha(7) nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist.

A study was made of the effects of 5-hydroxytryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken alpha(7) subunit. Acetylcholine (AcCho) elicited large currents (IAcCho) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC50) of 56 microM and a Hill coefficient (nH) of 1.2. The inhibition of IAcCho by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism. 5HT alone did not elicit membrane currents in oocytes injected with the wild-type alpha(7) subunit cDNA. In contrast, 5HT elicited membrane currents (I5HT) in oocytes injected with cDNA encoding an alpha(7) mutant subunit with a threonine-for-leucine-247 substitution (L247T alpha(7)). I5HT was inhibited by the potent nicotinic receptor blockers alpha-bungarotoxin (100 nM) and methyllycaconitine (1 microM). Furthermore, the characteristics of I5HT, including its voltage dependence, were similar to those of IAcCho. The 5HT dose-I5HT response gave an apparent dissociation constant EC50 of 23.5 microM and a Hill coefficient nH of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T alpha(7) for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal alpha(7) nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.

Animals↗
PubMed1996

Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.

Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds.

Animals↗
PubMed1996

Thrombin-induced membrane currents in native Xenopus follicles.

The presence and properties of thrombin receptors have been investigated in Xenopus Laevis follicles. In follicles, voltage-clamped at -60 mV or -80 mV, sub-micromolar concentrations of thrombin (Thr) induce smooth inward currents carried by chloride ions, which desensitize over tens of minutes. This desensitization is prevented by PKC inhibitors (staurosporine and H7). Responses are inhibited by hirudin. Inconsistent responses can be observed in defolliculated oocytes. Thr responses, readily recorded in hypo-osmotic solution are abolished by perfusion of normal frog "Ringer" or by the addition of 50 mM sucrose. Thus Thr acts on receptors present in the follicular cells triggering the opening of chloride channels by acting on receptors, likely to be located on follicular cells, via a PKC-regulated pathway.

Animals↗
PubMed1996

Lysophosphatidic acid-induced neurite retraction in PC12 cells: control by phosphoinositide-Ca2+ signaling and Rho.

The endogenous phospholipid mediator lysophosphatidic acid (LPA) caused growth cone collapse, neurite retraction, and cell flattening in differentiated PC12 cells. Neurite retraction was blocked by cytochalasin B and ADP-ribosylation of the small-molecular-weight G protein Rho by the Clostridium botulinum C-3 toxin. LPA induced a transient rise in the level of inositol 1,4,5-trisphosphate, and retraction was blocked by inhibitors of phospholipase beta. Repeated application of LPA elicited homologous desensitization of the Ca2+ mobilization response. The activation of the phosphoinositide (PIP)-Ca2+ second messenger system played a permissive role in the morphoregulatory response. Blockers of protein kinase C--chelerythrine, a myristoylated pseudosubstrate peptide, staurosporine, and depletion of protein kinase C from the cells by long-term phorbol ester treatment--all diminished neurite retraction by interfering with LPA-induced Ca2+ mobilization, which was required for the withdrawal of neurites. A brief 15-min treatment with 4 beta-phorbol 12-myristate 13-acetate also blocked retraction and Ca2+ mobilization, by inactivating the LPA receptor. Inhibition of protein tyrosine phosphorylation by herbimycin diminished retraction. Although activation of the PIP-Ca2+ second messenger system appears necessary for the Rho-mediated rearrangements of the actin cytoskeleton, bradykinin, which activates similar signaling events, failed to cause retraction, indicating that a yet unidentified novel mechanism is also involved in the LPA-induced morphoregulatory response.

Actins↗
PubMed1996

Lysophosphatidic acid-induced neurite retraction in PC12 cells: neurite-protective effects of cyclic AMP signaling.

Effects of the cyclic AMP second messenger system were studied on the retraction of neurites elicited by the phospholipid mediator lysophosphatidic acid (LPA) in PC12 cells. LPA stimulation inhibited adenylyl cyclase, indicating that the LPA receptor couples to the heterotrimeric Gi proteins. However, pertussis toxin or expression of dominant negative Ras did not prevent neurite retraction. In contrast, cholera toxin, forskolin, and application of dibutyryl-cyclic AMP prevented neurite retraction. The neurite-protective effect of forskolin was blocked by Rp-adenosine 3',5'-phosphorothioate. Forskolin and dibutyryl-cyclic AMP both failed to protect neurites in A126-1B2 and 123.7 cells, which lack cyclic AMP-activated protein kinase. Data indicate that elevation of cyclic AMP levels triggers a cyclic AMP-activated protein kinase-dependent mechanism that opposes the functioning of the morphoregulatory signaling activated by LPA. ADP-ribosylation of Rho by the Clostridium botulinum C-3 toxin in 123.7 cells caused neuronal differentiation, indicated by neurite extension, and blocked LPA-induced neurite retraction. LPA activates Gq- and Gi-linked signaling in parallel; therefore, a morphoregulatory signaling network hypothesis is proposed versus the simplistic approach of a signaling pathway. The signaling network integrates the receptor-activated individual, sequential, and parallel signaling events into an interactive network whose individual components may fulfill required and permissive functions encoding the cellular response.

ADP Ribose Transferases↗
PubMed1996

Design and in vitro pharmacology of a selective gamma-aminobutyric acidC receptor antagonist.

In mammals, receptors for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) are divided into three pharmacological classes, which are denoted GABAA, GABAB, and GABAC. GABAC receptors are defined by their insensitivity to the GABAA receptor antagonist bicuculline and the GABAB receptor agonist (-)-baclofen. GABAC receptors probably are a heterogeneous group of proteins. The most extensively studied mammalian GABAC receptors are those found in neurons of the outer retina. These receptors are GABA-gated Cl- channels comprised of p subunits, of which there are two subtypes. The physiological functions served by GABAC receptors are largely unknown; to determine the functions, it would be useful to have GABAC-selective ligands. In a previous study, we found that isoguvacine, a GABAA-selective agonist, and 3-aminopropyl-(methyl)phosphinic acid (3-APMPA), a GABAB-selective agonist, show affinity for retinal GABAC receptors. In particular, 3-APMPA is an antagonist with low micromolar potency (Kb approximately 1 microM). Here, we report the synthesis and pharmacological characterization of (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA), a hybrid of isoguvacine and 3-APMPA designed to retain affinity for GABAC receptors but not to interact with GABAA or GABAB receptors. Electrical assays show that TPMPA is a competitive antagonist of cloned human mu 1 GABAC receptors expressed in Xenopus laevis oocytes (Kb approximately 2 microM). TPMPA is > 100-fold weaker as an inhibitor of rat brain GABAA receptors expressed in oocytes (Kb approximately 320 microM) and has only weak agonist activity on GABAB receptors assayed in rat hippocampal slices (EC50 approximately 500 microM). TPMPA should be a useful pharmacological probe with which to investigate GABAC receptor function in the outer retina and in any other areas of the nervous system in which these types of receptor are present.

Animals↗
PubMed1995

Functional role of follicular cells in the generation of osmolarity-dependent Cl- currents in Xenopus follicles.

1. Osmolarity-dependent (osmo-dependent) ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using the two-microelectrode voltage-clamp technique, combined with intra-oocyte pressure injection of sucrose or polyethylene glycols (PEGs). 2. Intra-oocyte injections of sucrose or PEG (3-25 nmol) generated inward membrane currents (follicles held at -60 mV) associated with an increase in membrane conductance. These currents were carried mainly by chloride ions (ICl(osm)), and were strongly attenuated by increasing the tonicity of the external medium, or by external application of La3+ (0.1-1 mM). 3. The ability to generate ICl(osm) depended on the molecular weight of the injected PEG. Injections of PEG 200 or 300 generated ICl(osm) in 95% of the follicles tested, PEG 600 generated comparable currents in only 20% of the follicles, while similar injections of PEG 1000 did not elicit ICl(osm). 4. Octanol (1-1.5 mM), a gap junction channel blocker, reversibly inhibited 50-90% of the ICl(osm) generated by injections of sucrose or PEG 300. Moreover, sucrose or PEG injections did not elicit ICl(osm) in defolliculated oocytes. 5. It is concluded that an increase in the internal osmolarity of the follicular cells activates a mechanism, probably involving cellular swelling, which leads to the opening of ICl(osm) channels most probably located in the follicular cell membrane.

1-Octanol↗
PubMed1995

Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane.

Xenopus oocytes are a valuable aid for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Their use has recently been extended by the demonstration that oocytes can incorporate foreign membranes carrying preassembled receptors and channels. Here we show that when reconstituted in an artificial lipid matrix and injected into Xenopus oocytes, purified nicotinic acetylcholine receptors are efficiently inserted into the plasma membrane, where they form "clusters" of receptors that retain their native properties. This constitutes an innovative approach that, besides allowing the analyses of membrane fusion processes, is also a powerful technique for studying the characteristics and regulation of many membrane proteins (with their native stoichiometry and configuration) upon reinsertion into the membrane of a very convenient host cell system.

Animals↗
PubMed1995

Activation of GABA rho 1 receptors by glycine and beta-alanine.

Neurons of the vertebrate retina possess receptors for many neurotransmitters. Particularly interesting is a new type of GABA receptor (GABA rho) that, in contrast to GABAA and GABAB receptors, shows very little desensitization, is not blocked by bicuculline, and is not activated by baclofen. Homomeric human GABA rho 1 receptors were expressed in Xenopus oocytes. In addition to GABA, and related agonists, GABA rho 1 receptors respond to glycine (Gly) and beta-alanine (beta-Ala) by generating Cl- currents that do not desensitize and are resistant to bicuculline. The half-maximal concentrations for Gly and beta-Ala currents were 14.2 +/- 1.3 mM and 0.66 +/- 0.11 mM respectively. The current responses to Gly and beta-Ala were blocked by picrotoxin and TBPS. The cross-sensitivity of GABA rho 1 receptors to Gly and beta-Ala may play a role in retinal physiology.

Animals↗
PubMed1995

Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.

A method was developed to transplant assembled nicotinic acetylcholine receptors (AcChoRs) and Cl- channels from the electric organ of Torpedo to the membrane of Xenopus oocytes. Membrane vesicles from Torpedo electroplaques were injected into the oocytes and, within a few hours, the oocyte membrane acquired AcChoRs and Cl- channels. The mechanism of expression of these receptors and channels is very different from that which follows the injection of mRNA, since the appearance of receptors after membrane injection does not require de novo protein synthesis or N-glycosylation. This, and other controls, indicate that the foreign receptor-bearing membranes fuse with the oocyte membrane and cause the appearance of functional receptors and channels. All this makes the Xenopus oocyte an even more powerful tool for studies of the structure and function of membrane proteins.

Acetylcholine↗
PubMed1995

A monovalent cationic conductance that is blocked by extracellular divalent cations in Xenopus oocytes.

1. Native Xenopus oocytes were voltage clamped and exposed to Ringer solutions containing low concentrations of divalent cations. Oocytes, held at -60 mV, developed a reversible non-inactivating smooth inward current (Ic) associated with an increase in membrane conductance. 2. Ic was selectively carried by cations (Na+, K+), indicating that the current was not the result of a non-specific membrane breakdown, but was due instead to removal of a blocking effect of divalent cations on a specific population of endogenous ionic channels located in the oocyte membrane. 3. The blocking effects of Ca2+ and Mg2+ were voltage dependent, implying action at a binding site within the pore of the cationic channel. For example, the half-maximal inhibition (IC50) of Ic by Ca2+ was 61 microM in oocytes held at -60 mV and 212 microM in oocytes held at 0 mV. 4. The Ic channels could be unblocked by depolarization of the membrane even in the presence of physiological concentrations of Ca2+ or Mg2+. The unblocking of the channels was observed as a slowly developing outward current. 5. The novel cationic current was substantially reduced following in vitro maturation of oocytes by treatment with progesterone (10 microM, 4-5 h). 6. The physiological role of Ic channels remains to be elucidated. Nonetheless, their characteristics explain the ionic basis of the sensitivity of oocytes to reductions in extracellular divalent cations and raise the possibility that the channels play a role in calcium homeostasis.

Animals↗
PubMed1995

Two forms of acetylcholine receptor gamma subunit in mouse muscle.

Nicotinic acetylcholine receptors (nAcChoRs) of skeletal muscle are heterosubunit ligand-gated channels that mediate signal transmission from motor nerves to muscle. While cloning murine nAcChoR subunits, to gain an insight into the receptor diversity across species, we detected two forms of gamma subunits in the myogenic C2C12 cell line. Both forms are functional when expressed in Xenopus oocytes. One gamma subunit [long gamma (gamma 1)] was almost identical to that previously cloned in the murine BC3H-1 tumor cell line. The second form of gamma subunit [short gamma (gamma s)] lacked 156 bp (52 amino acids) in the extracellular N terminus, adjoining the hydrophobic segment M1, which corresponds to the fifth exon of the gamma-subunit gene. The two forms of gamma subunit coexist during myogenesis in vitro and in 17-day embryonic and denervated adult muscle fibers in vivo. However, the gamma s variant was the only form of gamma subunit in newborn muscle. In dissociated muscle fibers of newborn mice, AcCho-evoked channel openings were more prolonged when compared with C2C12 myotubes or denervated adult muscle fibers. The gamma s subunit may, thus, contribute to the structural and functional diversity of nAcChoRs in muscle cells.

Acetylcholine↗
PubMed1995

Effects of serotonergic agents on neuronal nicotinic acetylcholine receptors.

In Xenopus oocytes expressing neuronal nicotinic acetylcholine receptors (nAcChoRs), made up of alpha 2 and beta 4 subunits, acetylcholine (AcCho) elicited ionic membrane currents (AcCho currents) that were modulated by serotonergic agents. Both agonists and antagonists specific for various serotonin (5-hydroxytryptamine, 5HT) receptor subtypes interacted directly with alpha 2 beta 4 nAcChoRs: 5HT, (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin, methysergide, spiperone, and ketanserin reversibly reduced the amplitude of AcCho currents and accelerated their decay. The AcCho-current time course decayed with two exponential functions. In the presence of 5HT, the fast time constant of current decay (tau f) was not greatly modified, but the slow time constant (tau s) was reduced. With AcCho and 5HT both at 100 microM, tau s was reduced from 140 s to 85 s. The order of potency for inhibition of AcCho current amplitudes was (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin > methysergide > spiperone > ketanserin > 5HT. The inhibition was voltage-dependent but the magnitude of the voltage dependence for the different blockers did not correspond to their blocking potency: e.g., the block with spiperone was stronger than with 5HT, but it was less voltage-dependent. Our results suggest that serotonergic agents block neuronal nAcChoRs in a noncompetitive manner, similar to the block of muscle nAcChoR by curare and other substances. These results show that neuronal nAcChoR channels that have been activated by their specific neurotransmitter may be modulated by nonspecific neurotransmitters and their antagonists. These effects may help to better understand brain functions as well as the mode of action of the many serotonergic agents that are used in medical practice.

Animals↗
PubMed1995

Xenopus Gq alpha subunit activates the phosphatidylinositol pathway in Xenopus oocytes but does not consistently induce oocyte maturation.

We cloned the Xenopus laevis form of Gq alpha subunit to study its effects on oocyte maturation. Injection of Xenopus Gq alpha mRNA into stage 6 oocytes activated the phospholipase C/phosphatidylinositol pathway. The oocyte membrane became permeable to calcium ions and was able to generate transient inward currents (T(in)), due to the opening of Ca(2+)-dependent Cl- channels. The T(in) amplitude developed over several hours and disappeared by 24 hr. Diacylglycerol levels were found to parallel the appearance and disappearance of the T(in). The concurrent decline of T(in) values and diacylglycerol was not due to a failure in the synthesis of Gq alpha protein, which was produced continuously for > 24 hr. After Xenopus Gq alpha mRNA injection, germinal vesicle breakdown (GVBD) was variable (0-100%) in stage 6 oocytes, whereas none of the stage 4 oocytes underwent GVBD. In contrast, stage 6 oocytes injected with mRNA encoding the Go alpha G protein consistently underwent GVBD but did not acquire T(in). Our results show that activation of phospholipase C is not an absolute requisite for the induction of maturation, although in oocytes of some frogs phospholipase C activation can trigger a pathway to GVBD.

Animals↗
PubMed1994

Osmo-dependent Cl- currents activated by cyclic AMP in follicle-enclosed Xenopus oocytes.

The role of adenosine 3',5'-cyclic monophosphate (cAMP) in generating the osmo-dependent slow inward membrane currents (S(in)) elicited by activation of follicle stimulating hormone (FSH) or acetylcholine (ACh) receptors was studied in voltage-clamped, follicle-enclosed oocytes of Xenopus laevis (follicles). Forskolin (FSK) also generated S(in) currents, and in low concentrations it potentiated the S(in) currents elicited by FSH but not those elicited by ACh. Moreover, intra-oocyte injections of cAMP elicited similar slow inward currents (cAMP-S(in)) that: (i) were carried mainly by chloride ions; (ii) were abolished by defolliculating the oocytes; and (iii) were dependent on the osmolarity of the external medium. Compared with the Ca(2+)-dependent chloride channels that are located in the oocyte membrane; the cAMP-activated S(in) channels were less permeable to I- and Br-, and their current-voltage relation did not rectify strongly at negative potentials. Generation of cAMP-S(in) desensitized the FSH-S(in) currents, but did not have effects on both the S(in) and the fast chloride current (F(in)) specifically elicited by ACh. Furthermore, follicular phospholipase C activation through stimulation of angiotensin II (AII) receptors failed to generate the current responses elicited by ACh. We conclude that cAMP acts as a potent second messenger in generating the osmo-dependent Cl- currents elicited by FSH but not those elicited by ACh. The mechanisms underlying the ACh responses remain unknown. The osmo-dependent chloride channels activated by cAMP may play a role in the control of volume of the follicular cells-oocyte complex.

Animals↗
PubMed1994

Cationic modulation of rho 1-type gamma-aminobutyrate receptors expressed in Xenopus oocytes.

A study was made of the effects of di- and trivalent cations on homomeric rho 1-type gamma-aminobutyrate (GABA rho 1) receptors expressed in Xenopus oocytes after injection of mRNA coding for the GABA rho 1 subunit. GABA elicited large currents with a Kd approximately 1 microM. The properties of these GABA rho 1 receptors were similar to those of native bicuculline-resistant GABA receptors expressed by retinal mRNA. GABA rho 1 currents showed very little desensitization, were blocked by picrotoxin but not by bicuculline, and were not modulated by barbiturates, benzodiazepines, or beta-carbolines. Zn2+ reversibly decreased GABA rho 1 responses (IC50 = 22 microM). Other divalent cations were also tested and their rank order of potency was: Zn2+ approximately Ni2+ approximately Cu2+ >> Cd2+, whereas Ba2+, Co2+, Sr2+, Mn2+, Mg2+, and Ca2+ showed little or no effect. In contrast, La3+ reversibly potentiated the GABA currents mediated by homomeric GABA rho 1 receptors, with an EC50 = 135 microM and a maximal potentiation of about 100% (GABA, 1 microM; La3+, 1 mM). Other lanthanides showed similar effects (Lu3+ > Eu3+ > Tb3+ > Gd3+ > Er3% > Nd3+ > La3+ > Ce3+). Thus, GABA rho 1 receptors contain sites for cationic recognition, and in particular, Zn2+ may play a role during synaptic transmission in the retina.

Animals↗