Background and Objectives
The aim of our study was to test a platelet‐rich plasma releasate (PRP‐R/SRGF) from CaCl2‐activated platelets as a source of growth factors for the expansion of mesenchymal ...stromal cells (MSCs). PRP‐R/SRGF, obtained with a low‐cost procedure, is characterized by a reduced variability of growth factor release.
Materials and Methods
PRP‐R/SRGF is a clinical‐grade quality solution obtained from CaCl2‐activated platelets. Its activity was evaluated by measuring the proliferation, the phenotype, the differentiation potential and the immunosuppressive properties of MSCs derived from bone marrow (BM) and adipose tissue (AT).
Results
PRP‐R/SRGF was more active than FBS to expand BM‐ and AT‐derived MSCs. PRP‐R/SRGF treatment did not affect the expression of typical MSCs surface markers, neither MSCs differentiation potential nor their capability to inhibit activated T‐cell proliferation.
Conclusions
The clinical‐grade PRP‐R/SRGF may be used in the clinical setting for the expansion of MSCs.
Volatile anesthetics and alcohols enhance transmission mediated by gamma-aminobutyric acid type A receptors (GABA(A)Rs) in the central nervous system, an effect that may underlie some of the ...behavioral actions of these agents. Substituting a critical serine residue within the GABA(A)R alpha(1) subunit at position 270 with the larger residue histidine eliminated receptor modulation by isoflurane, but it also affected receptor gating (increased GABA sensitivity). To correct the shift in GABA sensitivity of this mutant, we mutated a second residue, leucine at position 277 to alanine. The double mutant alpha(1)(S270H,L277A)beta(2)gamma(2S) GABA(A)R was expressed in Xenopus laevis oocytes and human embryonic kidney (HEK)293 cells, and it had near-normal GABA sensitivity. However, rapid application of a brief GABA pulse to receptors expressed in HEK293 cells revealed that the deactivation was faster in double mutant than in wild-type receptors. In all heterologous systems, the enhancing effect of isoflurane and ethanol was greatly decreased in the double mutant receptor. Homozygous knockin mice harboring the double mutation were viable and presented no overt abnormality, except hyperactivity. This knockin mouse line should be useful in determining which behavioral actions of volatile anesthetics and ethanol are mediated by the GABA(A)Rs containing the alpha(1) subunit.
Despite the prevalence of their use, little is currently known of the molecular mechanisms of action of inhaled drugs of abuse. Recent studies have shown effects on NMDA, GABA
A and glycine receptors ...in vitro, suggesting that inhalants may exert at least some of their pharmacological effects on ligand-gated ion channels. Enhancement of serotonin-3 receptor function has been shown to play a role in the reinforcing properties of drugs of abuse. We tested the hypothesis that the commonly abused inhaled agents 1,1,1-trichloroethane, trichloroethylene, and toluene enhance serotonin-3 receptor function. All three inhalants significantly and reversibly potentiated, in a dose-dependent manner, serotonin-activated currents mediated by mouse serotonin-3A receptors expressed in
Xenopus oocytes. Our findings add the serotonin-3 receptor to the growing list of molecular targets commonly affected by both inhalants and classic CNS depressants such as ethanol and the volatile anesthetics.
GABA type A receptors (GABA(A)-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with ...specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABA(A)-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705-714, 2004; Pharmacol Biochem Behav 90:95-104, 2008; J Psychiatr Res 42:184-191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism.
The mechanism by which the inhaled anesthetic isoflurane produces amnesia and immobility is not understood. Isoflurane modulates GABA(A) receptors (GABA(A)-Rs) in a manner that makes them plausible ...targets. We asked whether GABA(A)-R α2 subunits contribute to a site of anesthetic action in vivo. Previous studies demonstrated that Ser270 in the second transmembrane domain is involved in the modulation of GABA(A)-Rs by volatile anesthetics and alcohol, either as a binding site or a critical allosteric residue. We engineered GABA(A)-Rs with two mutations in the α2 subunit, changing Ser270 to His and Leu277 to Ala. Recombinant receptors with these mutations demonstrated normal affinity for GABA, but substantially reduced responses to isoflurane. We then produced mutant (knockin) mice in which this mutated subunit replaced the wild-type α2 subunit. The adult mutant mice were overtly normal, although there was evidence of enhanced neonatal mortality and fear conditioning. Electrophysiological recordings from dentate granule neurons in brain slices confirmed the decreased actions of isoflurane on mutant receptors contributing to inhibitory synaptic currents. The loss of righting reflex EC(50) for isoflurane did not differ between genotypes, but time to regain the righting reflex was increased in N(2) generation knockins. This effect was not observed at the N(4) generation. Isoflurane produced immobility (as measured by tail clamp) and amnesia (as measured by fear conditioning) in both wild-type and mutant mice, and potencies (EC(50)) did not differ between the strains for these actions of isoflurane. Thus, immobility or amnesia does not require isoflurane potentiation of the α2 subunit.
Background and Objectives The aim of our study was to test a platelet-rich plasma releasate (PRP-R/SRGF) from CaCl sub(2)-activated platelets as a source of growth factors for the expansion of ...mesenchymal stromal cells (MSCs). PRP-R/SRGF, obtained with a low-cost procedure, is characterized by a reduced variability of growth factor release. Materials and Methods PRP-R/SRGF is a clinical-grade quality solution obtained from CaCl sub(2)-activated platelets. Its activity was evaluated by measuring the proliferation, the phenotype, the differentiation potential and the immunosuppressive properties of MSCs derived from bone marrow (BM) and adipose tissue (AT). Results PRP-R/SRGF was more active than FBS to expand BM- and AT-derived MSCs. PRP-R/SRGF treatment did not affect the expression of typical MSCs surface markers, neither MSCs differentiation potential nor their capability to inhibit activated T-cell proliferation. Conclusions The clinical-grade PRP-R/SRGF may be used in the clinical setting for the expansion of MSCs.
Background and Objectives
The aim of our study was to test a platelet‐rich plasma releasate (PRP‐R/SRGF) from CaCl
2
‐activated platelets as a source of growth factors for the expansion of ...mesenchymal stromal cells (MSCs). PRP‐R/SRGF, obtained with a low‐cost procedure, is characterized by a reduced variability of growth factor release.
Materials and Methods
PRP‐R/SRGF is a clinical‐grade quality solution obtained from CaCl
2
‐activated platelets. Its activity was evaluated by measuring the proliferation, the phenotype, the differentiation potential and the immunosuppressive properties of MSCs derived from bone marrow (BM) and adipose tissue (AT).
Results
PRP‐R/SRGF was more active than FBS to expand BM‐ and AT‐derived MSCs. PRP‐R/SRGF treatment did not affect the expression of typical MSCs surface markers, neither MSCs differentiation potential nor their capability to inhibit activated T‐cell proliferation.
Conclusions
The clinical‐grade PRP‐R/SRGF may be used in the clinical setting for the expansion of MSCs.
Adult female rats, undernourished at perinatal age, were evaluated for anxiolytic action in the plus-maze test after acute and chronic administration of diazepam (DZP) and pentobarbital (PTB). ...Deprived (D) rats chronically treated with vehicle showed an increased anxiety as compared with control (C) animals. A single intraperitoneal (i.p.) administration of DZP (1 mg/kg) or PTB (7.5 mg/kg) produced similar anticonflict effect in both C and D rats. Tolerance to the anxiolytic effect of DZP and PBT developed in C rats after a 15-day administration schedule, whereas no tolerance was observed in D animals. Drug disposition was not altered after chronic treatment either in C or in D rats. γ-aminobutyric acid (GABA)-mediated chloride uptake in microsacs of cerebral cortex of naive D rats was decreased as compared with naive C rats. After chronic DZP administration (1 mg/kg/day i.p. for 15 days), GABA-mediated
36Cl
− influx in brain cortex microsacs of C rats did not change; however, GABA efficacy was increased in microsacs of D animals. In addition, chronic DZP treatment induced GABA-benzodiazepine uncoupling in brain cortex of C rats, but not in D animals, as assessed by chloride uptake in microsacs. Chronic PTB treatment (7.5 or 30 mg/kg/day i.p. for 15 days) did not modify GABA stimulation or GABA-PTB interaction in cortical microsacs of C or D rats.
Background: Volatile anesthetics (VAs) alter the function of key central nervous system proteins but it is not clear which, if any, of these targets mediates the immobility produced by VAs in the ...face of noxious stimulation. A leading candidate is the glycine receptor, a ligand-gated ion channel important for spinal physiology. VAs variously enhance such function, and blockade of spinal glycine receptors with strychnine affects the minimal alveolar concentration (an anesthetic EC50) in proportion to the degree of enhancement. Methods: We produced single amino acid mutations into the glycine receptor alpha 1 subunit that increased (M287L, third transmembrane region) or decreased (Q266I, second transmembrane region) sensitivity to isoflurane in recombinant receptors, and introduced such receptors into mice. The resulting knockin mice presented impaired glycinergic transmission, but heterozygous animals survived to adulthood, and we determined the effect of isoflurane on glycine-evoked responses of brainstem neurons from the knockin mice, and the minimal alveolar concentration for isoflurane and other VAs in the immature and mature knockin mice. Results: Studies of glycine-evoked currents in brainstem neurons from knockin mice confirmed the changes seen with recombinant receptors. No increases in the minimal alveolar concentration were found in knockin mice, but the minimal alveolar concentration for isoflurane and enflurane (but not halothane) decreased in 2-week-old Q266I mice. This change is opposite to the one expected for a mutation that decreases the sensitivity to volatile anesthetics. Conclusion: Taken together, these results indicate that glycine receptors containing the alpha 1 subunit are not likely to be crucial for the action of isoflurane and other VAs.
Amino acids (AAs) in the extracellular portion of the transmembrane domain of several inhibitory ligand‐gated ion channels participate in an alcohol binding site. To extend these studies to neuronal ...nicotinic acetylcholine receptors (nAChRs), we focused on an AA (L262) located in the same region of the second transmembrane domain of the α2 subunit of neuronal nAChRs. Single‐point mutation of α2L262 was carried out, the resulting α2 subunits co‐expressed with wild‐type β4 subunits in Xenopus laevis oocytes, and studied using two‐electrode voltage clamp. Ethanol enhancement of ACh responses was diminished α2(L262F)β4 or abolished α2(L262G)β4, α2(L262S)β4 and α2(L262A)β4. Mutation of the homologous AA in β4 β4(L258A) did not modify the ethanol modulation and the mutation in α2 was dominant, because ethanol did not enhance ACh responses in α2(L262A)β4(L258A) nAChRs. n‐Alcohols (ethanol through octanol) were applied to α2(L262A)β4 nAChRs. As described previously for other nAChRs, short‐chain alcohols enhanced, intermediate‐chain alcohols had no effect and long‐chain alcohols inhibited ACh responses in the wild‐type receptor. For α2(L262A)β4 nAChRs the alcohol enhancing effect was absent, and the alcohol inhibitory action was increased. Although this suggests removal of an alcohol enhancing site through mutagenesis, we cannot rule out the enhancement of action at an alcohol inhibitory site.
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