Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit ...

University of South Florida

Rho(1) receptor-channels (rho(1)Rs) are GABA-gated chloride channels that exhibit slow kinetics, little desensitization, and inert pharmacology to most anesthetics, except for neuroactive steroids (NSs). NSs differentially modulate rho(1)Rs dependent on the steric arrangement of the hydrogen atom at the fifth carbon position. In particular, the NS allotetrahydrodeoxycorticosterone (5alpha-THDOC) potentiates, whereas 5beta-pregnane-3alpha-ol-20-one (pregnanolone) and 5beta-dihydroprogesterone (5beta-DHP) inhibit rho(1) GABA currents. Here, we used Xenopus laevis oocytes expressing rho(1)Rs as a model system to study the mechanism of NS modulation. The second transmembrane residue, Ile307, was mutated to 16 amino acids. Subsequent testing of these mutants with 5alpha- and 5beta-NSs, at equivalent GABA activity, showed the following paradigm. For 5beta-DHP, Ile307 mutation either altered the degree of inhibition or entirely reversed the direction of modulation, rendering 5beta-DHP a potentiator. Dependent on the mutation, pregnanolone remained an inhibitor, transformed into a potentiator, or converted to inhibitor and potentiator based on concentration. The extent of mode reversal for both 5beta compounds showed a correlation with the side-chain hydrophilicity of the 307 residue. In contrast, Ile307 substitutions did not alter the direction of modulation for 5alpha-THDOC but caused a significant increase in the level of potentiation. Paradoxical to their impact on the mode and/or the degree of modulation, none of the mutations altered the concentration range producing the response significantly for any of the above NSs. Moreover, preincubation of Ile307 mutants with 5alpha or 5beta alone produced an equivalent effect on the activation time course. Based on the above data, a universal model is presented wherein anesthetic compounds like NSs can potentiate or inhibit the activity of ligand-gated ion channels distinct from interaction with alternative binding sites.

Insight into the mechanism of action of neuroactive steroids.

Fast synaptic transmission in mammalian autonomic ganglia is mediated primarily by nicotinic receptors, and one of the most abundant nicotinic acetylcholine receptor subtypes in these neurons contains the alpha7 subunit (alpha7-nAChRs). Unlike alpha7-nAChRs expressed in other cells, the predominant alpha7-nAChR subtype found in rat intracardiac and superior cervical ganglion neurons exhibits a slow rate of desensitization and is reversibly blocked by alpha-bungarotoxin (alphaBgt). We report here the identification of an alpha7 subunit sequence variant in rat autonomic neurons that incorporates a novel 87-base pair cassette exon in the N terminus of the receptor and preserves the reading frame of the transcript. This alpha7 isoform was detected using reverse transcriptase-polymerase chain reaction techniques in neonatal rat brain and intracardiac and superior cervical ganglion neurons. Immunoblot experiments using a polyclonal antibody directed against the deduced amino acid sequence of the alpha7-2 insert showed a pattern of expression consistent with alpha7-2 subunit mRNA distribution. Moreover, the alpha7-2 subunit could be immunodepleted from protein extracts by solid-phase immunoprecipitation techniques using the anti-alpha7 monoclonal antibody 319. The alpha7-2 subunit was shown to form functional homomeric ion channels that were activated by acetylcholine and blocked by alpha-bungarotoxin when expressed in Xenopus laevis oocytes. This alpha7 isoform exhibited a slow rate of desensitization, and inhibition of these channels by alphaBgt reversed rapidly after washout. Taken together, these data indicate that the alpha7-2 subunit is capable of forming functional alphaBgt-sensitive acetylcholine receptors that resemble the alpha7-nAChRs previously identified in rat autonomic neurons. Furthermore, the distribution of the alpha7-2 isoform is not limited to peripheral neurons.

The alpha7 nicotinic acetylcholine receptor subunit exists in two isoforms that contribute to functional ligand-gated ion channels.

The onset of motor learning in rats coincides with exclusive expression of GABAA receptors containing alpha6 and delta subunits in the granule neurons of the cerebellum. This development temporally correlates with the presence of a spontaneously active chloride current through alpha6-containing GABAA receptors, known as tonic inhibition. Here we report that the coexpression of alpha6, beta2, and delta subunits produced receptor-channels which possessed two distinct and separable states of agonist affinity, one exhibiting micromolar and the other nanomolar affinities for GABA. The high-affinity state was associated with a significant level of spontaneous channel activity. Increasing the level of expression or the ratio of beta2 to alpha6 and delta subunits increased the prevalence of the high-affinity state. Comparative studies of alpha6beta2delta, alpha1beta2delta, alpha6beta2gamma2, alpha1beta2gamma2 and alpha4beta2delta receptors under equivalent levels of expression demonstrated that the significant level of spontaneous channel activity is uniquely attributable to alpha6beta2delta receptors. The pharmacology of spontaneous channel activity arising from alpha6beta2delta receptor expression corresponded to that of tonic inhibition. For example, GABAA receptor antagonists, including furosemide, blocked the spontaneous current. Further, the neuroactive steroid 5alpha-THDOC and classical glycine receptor agonists beta-alanine and taurine directly activated alpha6beta2delta receptors with high potency. Specific mutation within the GABA-dependent activation domain (betaY157F) impaired both low- and high-affinity components of GABA agonist activity in alpha6betaY157Fdelta receptors, but did not attenuate the spontaneous current. In comparison, a mutation located between the second and third transmembrane segments of the delta subunit (deltaR287M) significantly diminished the nanomolar component and the spontaneous activity. The possibility that the high affinity state of the alpha6beta2delta receptor modulates the granule neuron activity as well as potential mechanisms affecting its expression are discussed.

Rat alpha6beta2delta GABAA receptors exhibit two distinct and separable agonist affinities.

Phencyclidine (PCP) and ketamine are dissociative anesthetics capable of inducing analgesia, psychomimetic behavior, and a catatonic state of unconsciousness. Despite broad similarities, there are notable differences between the clinical actions of ketamine and PCP. Ketamine has a lower incidence of adverse effects and generally produces greater CNS depression than PCP. Both noncompetitively inhibit NMDA receptors, yet there is little evidence that these drugs affect GABA(A) receptors, the primary target of most anesthetics. alpha6beta2/3delta receptors are subtypes of the GABA(A) receptor family and are abundantly expressed in granular neurons within the adult cerebellum. Here, using an oocyte expression system, we show that at anesthetically relevant concentrations, ketamine, but not PCP, modulates alpha6beta2delta and alpha6beta3delta receptors. Additionally, at higher concentrations, ketamine directly activates these GABA(A) receptors. Comparatively, dizocilpine (MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]), a potent noncompetitive antagonist of NMDA receptors that is structurally unrelated to PCP, did not produce any effect on alpha6beta2delta receptors. Of the recombinant GABA(A) receptor subtypes examined (alpha1beta2, alpha1beta2gamma2, alpha1beta2delta, alpha4beta2gamma2, alpha4beta2delta, alpha6beta2gamma2, alpha6beta2delta, and alpha6beta3delta), the actions of ketamine were unique to alpha6beta2delta and alpha6beta3delta receptors. In dissociated granule neurons and cerebellar slice recordings, ketamine potentiated the GABAergic conductance arising from alpha6-containing GABA(A) receptors, whereas PCP showed no effect. Furthermore, ketamine potentiation was absent in cerebellar granule neurons from transgenic functionally null alpha6(-/-) and delta(-/-)mice. These findings suggest that the higher CNS depressant level achieved by ketamine may be the result of its selective actions on alpha6beta2/3delta receptors.

Ketamine, but not phencyclidine, selectively modulates cerebellar GABA(A) receptors containing alpha6 and delta subunits.

Bartonella henselae (Bh) is an emerging zoonotic pathogen that has been associated with a variety of human diseases, including bacillary angiomatosis that is characterized by vasoproliferative tumor-like lesions on the skin of some immunosuppressed individuals. The study of Bh pathogenesis has been limited to in vitro cell culture systems due to the lack of an animal model. Therefore, we wanted to investigate whether the zebrafish embryo could be used to model human infection with Bh. Our data showed that Tg(fli1:egfp)(y1) zebrafish embryos supported a sustained Bh infection for 7 days with >10-fold bacterial replication when inoculated in the yolk sac. We showed that Bh recruited phagocytes to the site of infection in the Tg(mpx:GFP)uwm1 embryos. Infected embryos showed evidence of a Bh-induced angiogenic phenotype and an increase in the expression of genes encoding pro-inflammatory factors and pro-angiogenic markers. However, infection of zebrafish embryos with a deletion mutant in the major adhesin (BadA) resulted in little or no bacterial replication and a diminished host response, providing the first evidence that BadA is critical for in vivo infection. Thus, the zebrafish embryo provides the first practical model of Bh infection that will facilitate efforts to identify virulence factors and define molecular mechanisms of Bh pathogenesis.

Zebrafish embryo model of Bartonella henselae infection.

Anaesthetic molecules act on synaptic transmission via the allosteric modulation of ligand-gated chloride channels, such as hetero-oligomeric αβγ GABA receptors. To elucidate the overall activation paradigm via allosteric versus orthosteric sites, we used highly homologous, but homo-oligomeric, ρ receptors that are contrastingly insensitive to anaesthetics and respond partially to several full GABA αβγ receptor agonists. Here, we coexpressed varying ratios of RNAs encoding the wild-type and the mutated ρ subunits, which are anaesthetic-sensitive and respond with full efficacy to partial GABA agonists, to generate distinct ensembles of receptors containing five, four, three, two, one, or zero mutated subunits. Using these experiments, we then demonstrate that, in the pentamer, three anaesthetic-sensitive ρ subunits are needed to impart full efficacy to the partial GABA agonists. By contrast, five anaesthetic-sensitive subunits are required for direct activation by anaesthetics alone, and only one anaesthetic-sensitive subunit is sufficient to confer the anaesthetic-dependent potentiation to the GABA current. In conclusion, our data indicate that GABA and anaesthetics holistically activate the GABA ρ receptor through distinct subunit level rearrangements and suggest that in contrast to the global impact of GABA via orthosteric sites, the force of anaesthetics through allosteric sites may not propagate to the neighbouring subunits and, thus, may have only a local and limited effect on the ρ GABA receptor model system.

Jahanshah Amin

Department of Molecular Pharmacology and Physiology,
Morsani College of Medicine,
Department of Molecular Pharmacology and Physiology, Morsani College of Medicine

University of South Florida

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Jahanshah Amin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular Pharmacology and Physiology,Morsani College of Medicine,Department of Molecular Pharmacology and Physiology, Morsani College of Medicine

University of South Florida

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Department of Molecular Pharmacology and Physiology,
Morsani College of Medicine,
Department of Molecular Pharmacology and Physiology, Morsani College of Medicine