Chemical-defined and albumin-free generation of human atrial and ventricular myocytes from human pluripotent stem cells.

Most existing culture media for cardiac differentiation of human pluripotent stem cells (hPSCs) contain significant amounts of albumin. For clinical transplantation applications of hPSC-derived cardiomyocytes (hPSC-CMs), culturing cells in an albumin containing environment raises the concern of pathogen contamination and immunogenicity to the recipient patients. In addition, batch-to-batch variation of albumin may cause the inconsistent of hPSC cardiac differentiation. Here, we demonstrated that antioxidants l-ascorbic acid, trolox, N-acetyl-l-cysteine (NAC) and sodium pyruvate could functionally substitute albumin in the culture medium, and formulated an albumin-free, chemical-defined medium (S12 medium). We showed that S12 medium could support efficient hPSC cardiac differentiation with significantly improved reproducibility, and maintained long-term culture of hPSC-CMs. Furthermore, under chemical-defined and albumin-free conditions, human-induced pluripotent stem cells (hiPSCs) were established, and differentiated into highly homogenous atrial and ventricular myocytes in a scalable fashion with normal electrophysiological properties. Finally, we characterized the activity of three typical cardiac ion channels of those cells, and demonstrated that hPSC-derived ventricular cardiomyocytes (hPSC-vCMs) were suitable for drug cardiac safety evaluation. In summary, this simplified, chemical-defined and albumin-free culture medium supports efficient generation and maintaining of hPSC-CMs and facilitates both research and clinical applications of these cells.

Depression of neuronal excitability and epileptic activities by group II metabotropic glutamate receptors in the medial entorhinal cortex.

Whereas the ionotropic glutamate receptors are the major mediator in glutamatergic transmission, the metabotropic glutamate receptors (mGluRs) usually play a modulatory role. Whereas the entorhinal cortex (EC) is an essential structure involved in the generation and propagation of epilepsy, the roles and mechanisms of mGluRs in epilepsy in the EC have not been determined. Here, we studied the effects of activation of group II metabotropic glutamate receptors (mGluRs II) on epileptiform activity induced by picrotoxin or deprivation of extracellular Mg2+ and neuronal excitability in the medial EC. We found that activation of mGluRs II by application of the selective agonist, LY354740, exerted robust inhibition on epileptiform activity. LY354740 hyperpolarized entorhinal neurons via activation of a K+ conductance and inhibition of a Na+ -permeable channel. LY354740-induced hyperpolarization was G protein-dependent, but independent of adenylyl cyclase and protein kinase A. However, the function of Gßγ was involved in mGluRs II-mediated depression of both neuronal excitability and epileptiform activity. Our results provide a novel cellular mechanism to explain the antiepileptic effects of mGluRs II in the treatment of epilepsy.

Activation of neurotensin receptor 1 facilitates neuronal excitability and spatial learning and memory in the entorhinal cortex: beneficial actions in an Alzheimer's disease model.

Neurotensin (NT) is a tridecapeptide distributed in the CNS, including the entorhinal cortex (EC), a structure that is crucial for learning and memory and undergoes the earliest pathological alterations in Alzheimer's disease (AD). Whereas NT has been implicated in modulating cognition, the cellular and molecular mechanisms by which NT modifies cognitive processes and the potential therapeutic roles of NT in AD have not been determined. Here we examined the effects of NT on neuronal excitability and spatial learning in the EC, which expresses high density of NT receptors. Brief application of NT induced persistent increases in action potential firing frequency, which could last for at least 1 h. NT-induced facilitation of neuronal excitability was mediated by downregulation of TREK-2 K(+) channels and required the functions of NTS1, phospholipase C, and protein kinase C. Microinjection of NT or NTS1 agonist, PD149163, into the EC increased spatial learning as assessed by the Barnes Maze Test. Activation of NTS1 receptors also induced persistent increases in action potential firing frequency and significantly improved the memory status in APP/PS1 mice, an animal model of AD. Our study identifies a cellular substrate underlying learning and memory and suggests that NTS1 agonists may exert beneficial actions in an animal model of AD.

Corticotropin-releasing factor facilitates epileptiform activity in the entorhinal cortex: roles of CRF2 receptors and PKA pathway.

Whereas corticotropin-releasing factor (CRF) has been considered as the most potent epileptogenic neuropeptide in the brain, its action site and underlying mechanisms in epilepsy have not been determined. Here, we found that the entorhinal cortex (EC) expresses high level of CRF and CRF2 receptors without expression of CRF1 receptors. Bath application of CRF concentration-dependently increased the frequency of picrotoxin (PTX)-induced epileptiform activity recorded from layer III of the EC in entorhinal slices although CRF alone did not elicit epileptiform activity. CRF facilitated the induction of epileptiform activity in the presence of subthreshold concentration of PTX which normally would not elicit epileptiform activity. Bath application of the inhibitor for CRF-binding proteins, CRF6-33, also increased the frequency of PTX-induced epileptiform activity suggesting that endogenously released CRF is involved in epileptogenesis. CRF-induced facilitation of epileptiform activity was mediated via CRF2 receptors because pharmacological antagonism and knockout of CRF2 receptors blocked the facilitatory effects of CRF on epileptiform activity. Application of the adenylyl cyclase (AC) inhibitors blocked CRF-induced facilitation of epileptiform activity and elevation of intracellular cyclic AMP (cAMP) level by application of the AC activators or phosphodiesterase inhibitor increased the frequency of PTX-induced epileptiform activity, demonstrating that CRF-induced increases in epileptiform activity are mediated by an increase in intracellular cAMP. However, application of selective protein kinase A (PKA) inhibitors reduced, not completely blocked CRF-induced enhancement of epileptiform activity suggesting that PKA is only partially required. Our results provide a novel cellular and molecular mechanism whereby CRF modulates epilepsy.

GABA(B) receptor activation inhibits neuronal excitability and spatial learning in the entorhinal cortex by activating TREK-2 K+ channels.

The entorhinal cortex (EC) is regarded as the gateway to the hippocampus and thus is essential for learning and memory. Whereas the EC expresses a high density of GABA(B) receptors, the functions of these receptors in this region remain unexplored. Here, we examined the effects of GABA(B) receptor activation on neuronal excitability in the EC and spatial learning. Application of baclofen, a specific GABA(B) receptor agonist, inhibited significantly neuronal excitability in the EC. GABA(B) receptor-mediated inhibition in the EC was mediated via activating TREK-2, a type of two-pore domain K(+) channels, and required the functions of inhibitory G proteins and protein kinase A pathway. Depression of neuronal excitability in the EC underlies GABA(B) receptor-mediated inhibition of spatial learning as assessed by Morris water maze. Our study indicates that GABA(B) receptors exert a tight control over spatial learning by modulating neuronal excitability in the EC.

UTP regulation of ion transport in alveolar epithelial cells involves distinct mechanisms.

UTP is known to regulate alveolar fluid clearance. However, the relative contribution of alveolar type I cells and type II cells to this process is unknown. In this study, we investigated the effects of UTP on ion transport in type I-like cell (AEC I) and type II-like cell (AEC II) monolayers. Luminal treatment of cell monolayers with UTP increased short-circuit current (I(sc)) of AEC II but decreased I(sc) of AEC I. The Cl(-) channel blockers NPPB and DIDS inhibited the UTP-induced changes in I(sc) (DeltaIsc) in both types of cells. Amiloride, an inhibitor of epithelial Na(+) channels (ENaC), abolished the UTP-induced DeltaI(sc) in AEC I, but not in AEC II. The general blocker of K(+) channels, BaCl(2), eliminated the UTP-induced DeltaI(sc) in AEC II, but not in AEC I. The intermediate conductance (IK(Ca)) blocker, clofilium, also blocked the UTP effect in AEC II. The signal transduction pathways mediated by UTP were the same in AEC I and AEC II. Furthermore, UTP increased Cl(-) secretion in AEC II and Cl(-) absorption in AEC I. Our results suggest that UTP induces opposite changes in I(sc) in AEC I and AEC II, likely due to the reversed Cl(-) flux and different contributions of ENaC and IK(Ca). Our results further imply a new concept that type II cells contribute to UTP-induced fluid secretion and type I cells contribute to UTP-induced fluid absorption in alveoli.

Modulation of GABAergic transmission by muscarinic receptors in the entorhinal cortex of juvenile rats.

Whereas the entorhinal cortex (EC) receives profuse cholinergic innervations from the basal forebrain and activation of cholinergic receptors has been shown to modulate the activities of the principal neurons and promote the intrinsic oscillations in the EC, the effects of cholinergic receptor activation on GABAergic transmission in this brain region have not been determined. We examined the effects of muscarinic receptor activation on GABA(A) receptor-mediated synaptic transmission in the superficial layers of the EC. Application of muscarine dose-dependently increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from the principal neurons in layer II/III via activation of M(3) muscarinic receptors. Muscarine slightly reduced the frequency but had no effects on the amplitude of miniature IPSCs recorded in the presence of tetrodotoxin. Muscarine reduced the amplitude of IPSCs evoked by extracellular field stimulation and by depolarization of GABAergic interneurons in synaptically connected interneuron and pyramidal neuron pairs. Application of muscarine generated membrane depolarization and increased action potential firing frequency but reduced the amplitude of action potentials in GABAergic interneurons. Muscarine-induced depolarization of GABAergic interneurons was mediated by inhibition of background K(+) channels and independent of phospholipase C, intracellular Ca(2+) release, and protein kinase C. Our results demonstrate that activation of muscarinic receptors exerts diverse effects on GABAergic transmission in the EC.

Noradrenergic depression of neuronal excitability in the entorhinal cortex via activation of TREK-2 K+ channels.

The entorhinal cortex is closely associated with the consolidation and recall of memories, Alzheimer disease, schizophrenia, and temporal lobe epilepsy. Norepinephrine is a neurotransmitter that plays a significant role in these physiological functions and neurological diseases. Whereas the entorhinal cortex receives profuse noradrenergic innervations from the locus coeruleus of the pons and expresses high densities of adrenergic receptors, the function of norepinephrine in the entorhinal cortex is still elusive. Accordingly, we examined the effects of norepinephrine on neuronal excitability in the entorhinal cortex and explored the underlying cellular and molecular mechanisms. Application of norepinephrine-generated hyperpolarization and decreased the excitability of the neurons in the superficial layers with no effects on neuronal excitability in the deep layers of the entorhinal cortex. Norepinephrine-induced hyperpolarization was mediated by alpha(2A) adrenergic receptors and required the functions of Galpha(i) proteins, adenylyl cyclase, and protein kinase A. Norepinephrine-mediated depression on neuronal excitability was mediated by activation of TREK-2, a type of two-pore domain K(+) channel, and mutation of the protein kinase A phosphorylation site on TREK-2 channels annulled the effects of norepinephrine. Our results indicate a novel action mode in which norepinephrine depresses neuronal excitability in the entorhinal cortex by disinhibiting protein kinase A-mediated tonic inhibition of TREK-2 channels.

Differential effects of thiopental and pentobarbital on spinal GABA(A) receptors.

General anesthetics thiopental and pentobarbital possess very similar chemical structures whereas their clinical potency is quite different. The underlying molecular mechanism is not fully understood. This study was designed to assess the differential effects of thiopental and pentobarbital on GABA(A) receptors of mechanically dissociated rat spinal dorsal horn neurons by using whole-cell patch-clamp technique. Pentobarbital, at a concentration of 30 microM, which markedly enhanced sub-saturated GABA-induced current (I(GABA)), had no effect on thiopental-induced maximal current. Similarly, the pentobarbital-induced maximal current was also not affected by 30 microM thiopental. Moreover, a linear summation of thiopental-induced maximal current and pentobarbital-induced sub-maximal current was observed. In addition, pentobarbital failed to further enhance I(GABA) in the presence of thiopental at a concentration with maximal modulatory effects on I(GABA), and vice versa. Our results thus suggest that thiopental and pentobarbital might exert the GABA mimetic effects independently, but share a common mechanism to produce the GABA modulatory effects.

Modulation of gamma-aminobutyric acid A receptor function by thiopental in the rat spinal dorsal horn neurons.

To assess the actions of thiopental at the spinal dorsal horn level, we examined the effects of thiopental using the whole cell patch-clamp technique on mechanically dissociated rat spinal dorsal horn neurons. Thiopental, at large concentrations, elicited a current (I(Thio)) through activation of chloride conductance, and its threshold concentration was approximately 50 microM. I(Thio) was sensitive to bicuculline, a gamma-aminobutyric acid (GABA)A receptor antagonist, but not to strychnine, a glycine receptor antagonist. At a clinically relevant concentration (30 muM), thiopental markedly enhanced the peak amplitude of a subsaturating GABA-induced current (I(GABA)) but not that of a saturating GABA-induced current. Furthermore, thiopental prolonged the time constants of both desensitization and deactivation of I(GABA). At a large concentration (300 muM), it inhibited the peak amplitude of I(GABA), which may be the result of open-channel blockade. In addition, at 30 microM, thiopental increased the duration and decreased the frequency of GABAergic miniature inhibitory postsynaptic currents. These results indicate that thiopental enhances GABAergic inhibitory transmission and suggest that GABA(A) receptors in the spinal cord are a potential target through which thiopental causes immobility and depresses the response to noxious stimuli.

Thiopental inhibits glycine receptor function in acutely dissociated rat spinal dorsal horn neurons.

Whole-cell patch-clamp was used to assess the modulatory effect of thiopental (Thio) on glycine (Gly) receptor in mechanically dissociated rat spinal dorsal horn neurons. It was found that Thio inhibited the amplitude, accelerated the desensitization and prolonged the deactivation of Gly-induced currents (IGly) in a concentration-dependent manner. In addition, a rebound current occurred after washout of the co-application of Gly and Thio in most neurons tested. Moreover, the inhibitory effect of Thio was not the result of cross-inhibition between Gly and GABAA receptors. Furthermore, taurine-induced currents, a low-affinity agonist for Gly receptors, were also markedly inhibited by Thio in a similar way to IGly. These results indicate that Thio suppresses Gly receptor function and suggest that Thio anesthetic actions might not be mediated by Gly receptors. We speculate that the weak muscle relaxation and the limited analgesic effects observed during Thio anesthesia may attribute to its inhibitory effects on Gly receptors.