Isovolumetric regulation mechanisms in cultured cerebellar granule neurons

Cultured cerebellar granule neurons exposed to gradual reductions in osmolarity (-1.8 mOsm/min) maintained constant volume up to -50% external osmolarity (pi(o)), showing the occurrence of isovolumetric regulation (IVR). Amino acids, Cl-, and K+ contributed at different phases of IVR, with early efflux threshold for [3H]taurine, D-[3H]aspartate (as marker for glutamate) of pi(o) -2% and -19%, respectively, and more delayed thresholds of -30% for [3H]glycine and -25% and -29%, respectively, for Cl- (125I) and K+ (86Rb). Taurine seems preferentially involved in IVR, showing the lowest threshold, the highest efflux rate (five-fold over other amino acids) and the largest cell content decrease. Taurine and Cl- efflux were abolished by niflumic acid and 86Rb by 15 mM Ba2+. Niflumic acid essentially prevented IVR in all ranges of pi(o). Cl--free medium impaired IVR when pi(o) decreased to -24% and Ba2+ blocked it only at a late phase of -30% pi(o). These results indicate that in cerebellar granule neurons: (i) IVR is an active process of volume regulation accomplished by efflux of intracellular osmolytes; (ii) the volume regulation operating at small changes of pi(o) is fully accounted for by mechanisms sensitive to niflumic acid, with contributions of both Cl- and amino acids, particularly taurine; (iii) Cl- contribution to IVR is delayed with respect to other niflumic acid-sensitive osmolyte fluxes (osmolarity threshold of -25% pi(o)); and (iv), K+ fluxes do not contribute to IVR until a late phase (< -30% pi(o)).     

Volume changes and whole cell membrane currents activated during gradual osmolarity decrease in C6 glioma cells: contribution of two types of K+ channels

Volume changes and whole cell ionic currents activated by gradual osmolarity reductions (GOR) of 1.8 mosM/min were characterized in C6 glioma cells. Cells swell less in GOR than after sudden osmolarity reductions (SOR), the extent of swelling being partly Ca²⁺ dependent. In nominally Ca²⁺-free conditions, GOR activated predominantly whole cell outward currents. Cells depolarized from the initial –79 mV to a steady state of –54 mV reached at 18% osmolarity reduction [hyposmolarity of –18% (H-18%)]. Recordings of Cl^– and K⁺ currents showed activation at H-3% of an outwardly rectifying Cl^– current, with conductance of 1.6 nS, sensitive to niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, followed at H-18% by an outwardly rectifying K⁺ current with conductance of 4.1 nS, inhibited by clofilium but insensitive to the typical K⁺ channel blockers. With 200 nM Ca²⁺ in the patch pipette, whole cell currents activated at H-3% and at H-13% cells depolarized from –77 to –63 mV. A K⁺ current activated at H-1%, showing a rapid increase in conductance, suppressed by charybdotoxin and insensitive to clofilium. These results show the operation of two different K⁺ channels in response to GOR in the same cell type, activated by Ca²⁺ and osmolarity and with different osmolarity activation thresholds. Taurine and glutamate efflux, monitored by labeled tracers, showed delayed osmolarity thresholds of H-39 and H-33%, respectively. This observation clearly separates the Cl^– and amino acid osmosensitive pathways. The delayed amino acid efflux may contribute to counteract swelling at more stringent osmolarity reductions. volume regulation; taurine; hyposmolarity; isovolumetric regulation; regulatory volume decrease     

Expression of<Emphasis Type="Italic"> cefD2</Emphasis> and the conversion of isopenicillin N into penicillin N by the two-component epimerase system are rate-limiting steps in cephalosporin biosynthesis

The conversion of isopenicillin N into penicillin N in Acremonium chrysogenum is catalyzed by an epimerization system that involves an isopenicillin N-CoA synthethase and isopenicillin N-CoA epimerase, encoded by the genes cefD1 and cefD2. Several transformants containing two to seven additional copies of both genes were obtained. Four of these transformants (TMCD26, TMCD53, TMCD242 and TMCD474) showed two-fold higher IPN epimerase activity than the untransformed A. chrysogenum C10, and produced 80 to 100% more cephalosporin C and deacetylcephalosporin C than the parental strain. A second class of transformants, including TMCD2, TMCD32 and TMCD39, in contrast, showed a drastic reduction in cephalosporin biosynthesis relative to the untransformed control. These transformants had no detectable IPN epimerase activity and did not produce cephalosporin C or deacetylcephalosporin C. They also expressed both endogenous and exogenous cefD2 genes only after long periods (72–96 h) of incubation, as shown by Northern analysis, and were impaired in mycelial branching in liquid cultures. The negative effect of amplification of the cefD1 - cefD2 gene cluster in this second class of transformants is not correlated with high gene dosage, but appears to be due to exogenous DNA integration into a specific locus, which results in a pleiotropic effect on growth and cefD2 expression. Communicated by P. J. Punt     

Study of antigen-processing steps reveals preferences explaining differential biological outcomes of two HLA-A2-restricted immunodominant epitopes from human immunodeficiency virus type 1

Cytotoxic T-lymphocyte (CTL) responses directed to different human immunodeficiency virus (HIV) epitopes vary in their protective efficacy. In particular, HIV-infected cells are much more sensitive to lysis by anti-Gag/p17(77-85)/HLA-A2 than to that by anti-polymerase/RT(476-484)/HLA-A2 CTL, because of a higher density of p17(77-85) complexes. This report describes multiple processing steps favoring the generation of p17(77-85) complexes: (i) the exact COOH-terminal cleavage of epitopes by cellular proteases occurred faster and more frequently for p17(77-85) than for RT(476-484), and (ii) the binding efficiency of the transporter associated with antigen processing was greater for p17(77-85) precursors than for the RT(476-484) epitope. Surprisingly, these peptides, which differed markedly in their antigenicity, displayed qualitatively and quantitatively similar immunogenicity, suggesting differences in the mechanisms governing these phenomena. Here, we discuss the mechanisms responsible for such differences.     

Regulation of the human bradykinin B2 receptor expressed in sf21 insect cells: a possible role for tyrosine kinases

The functional regulation of the human bradykinin B2 receptor expressed in sf21 cells was studied. Human bradykinin B2 receptors were immunodetected as a band of 75-80 kDa in membranes from recombinant baculovirus-infected cells and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 receptors, detected in membranes by [(3)H-bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfection. In these cells, bradykinin induced a transient increase of intracellular calcium ([Ca(2+)](i)) in fura 2-AM loaded sf21 cells, and promoted [(35)S]-GTP(gamma)S binding to membranes. The effects of bradykinin were dose dependent (with an EC(50) of 50 nmol/L for calcium mobilization) and were inhibited by N-alpha-adamantaneacetyl-D-Arg-[Hyp(3),Thi(5,8),D-phe(7)]-Bk, a specific B2 receptor antagonist. When the B2 antagonist was applied at the top of the calcium transient, it accelerated the decline of the peak, suggesting that calcium mobilization at this point was still influenced by receptor occupation. No calcium mobilization was elicited by 1 micromol/L (Des-Arg(9))-Bk, a B1 receptor agonist that did not inhibit the subsequent action of 100 nmol/L bradykinin. No effect of bradykinin was detected in uninfected cells or cells infected with the wild-type baculovirus. Bradykinin-induced [Ca(2+)](i) mobilization was increased by genistein and tyrphostin A51. These tyrosine kinase inhibitors did not modify basal levels of [Ca(2+)](i). Homologous desensitization of the B2 receptor was observed after repeated applications of bradykinin, which resulted in attenuated changes in intracellular calcium. In addition, genistein promoted an increased response to a third exposure to the agonist when applied after washing the cells that had been previously challenged with two increasing doses of bradykinin. Genistein did not affect the calcium mobilization induced by activation of the endogenous octopamine G protein-coupled receptor or by thapsigargin. The B2 receptor, detected by confocal microscopy in unpermeabilized cells, remained constant at the surface of cells stimulated with bradykinin for 10 min, in the presence or absence of genistein. Agonist-promoted phosphorylation of the B2 receptor was markedly accentuated by genistein treatment. Phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by bradykinin, could act in a step previous to receptor phosphorylation. Interestingly, genistein prevented agonist-induced G protein uncoupling from B2 receptors, determined by in vitro bradykinin-stimulated [(35)S]-GTP(gamma)S binding, in membranes from bradykinin pretreated cells. Our results suggest that tyrosine kinase(s) regulate the activity of the human B2 receptor in sf21 cells by affecting its coupling to G proteins and its phosphorylation.     

Osmotic Swelling-Induced Changes in Cytosolic Calcium Do Not Affect Regulatory Volume Decrease in Rat Cultured Suspended Cerebellar Astrocytes

Abstract: Hyposmotic swelling-induced changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and their influence on regulatory volume decrease (RVD) were examined in rat cultured suspended cerebellar astrocytes. Hyposmotic media (50 or 30%) evoked an immediate rise in [Ca²⁺]_i from 117 nM to a mean peak increase of 386 (50%) and 220 nM (30%), followed by a maintained plateau phase. Ca²⁺ influx through the plasmalemma as well as release from internal stores contributed to this osmosensitive [Ca²⁺]_i elevation. Omission of external Ca²⁺ or addition of Cd²⁺, Mn²⁺, or Gd³⁺ did not reduce RVD, although it was decreased by La³⁺ (0.1–1 mM). Verapamil did not affect either the swelling-evoked [Ca²⁺]_i or RVD. Maneuvers that deplete endoplasmic reticulum (ER) Ca²⁺ stores, such as treatment (in Ca²⁺-free medium) with 0.2 µM thapsigargin (Tg), 10 µM 2,5-di-tert-butylhydroquinone, 1 µM ionomycin, or 100 µM ATP abolished the increase in [Ca²⁺]_i but did not affect RVD. However, prolonged exposure to 1 µM Tg blocked RVD regardless of ER Ca²⁺ content or cytosolic Ca²⁺ levels. Ryanodine (up to 100 µM) and caffeine (10 mM) did not modify [Ca²⁺]_i or RVD. BAPTA-acetoxymethyl ester (20 µM) abolished [Ca²⁺]_i elevation without affecting RVD, but at higher concentrations BAPTA prevented cell swelling and blocked RVD. We conclude that the osmosensitive [Ca²⁺]_i rise occurs as a consequence of increased Ca²⁺ permeability of plasma and organelle membranes, but it appears not relevant as a transduction signal for RVD in rat cultured cerebellar astrocytes.     

Feeding, body weight, and sensitivity to non-ingestive reward stimuli during and after 12-day continuous central infusions of melanocortin receptor ligands

The brain melanocortin system mediates downstream effects of hypothalamic leptin and insulin signaling. Yet, there have been few studies of chronic intracerebroventricular (i.c.v.) melanocortin receptor (MCR) agonist or antagonist infusion. Although there is evidence of interaction between melanocortin and dopamine (DA) systems, effects of chronic MCR ligand infusion on behavioral sensitivity to non-ingestive reward stimuli have not been investigated. The objective of this study was to investigate effects of chronic i.c.v. infusion of the MCR agonist, MTII, and the MCR antagonist, SHU9119, on food intake, body weight, and sensitivity to rewarding lateral hypothalamic electrical stimulation (LHSS) and the reward-potentiating (i.e., threshold-lowering) effect of D-amphetamine. The MCR antagonist, SHU9119 (0.02 microg/h) produced sustained hyperphagia and weight gain during the 12-day infusion period, followed by compensatory hypophagia and an arrest of body weight gain during the 24-day post-infusion period. At no point during the experiment was sensitivity to LHSS or D-amphetamine (0.25mg/kg, i.p.) altered. The MCR agonist, MTII (0.02 microg/h) produced a brief hypophagia (3 days) followed by a return to control levels of daily intake, but with body weight remaining at a reduced level throughout the 12-day infusion period. This was followed by compensatory hyperphagia and weight gain during the 24-day post-infusion period. There was no change in sensitivity to non-ingestive reward stimuli during the infusion of MTII. However, sensitivity to D-amphetamine was increased during the 24-day post-infusion period. It therefore seems that changes in ingestive behavior that occur during chronic MCR ligand infusion may not affect the response to non-ingestive reward stimuli. However, it is possible that the drive to re-feed and restore body weight following MCR agonist treatment includes neuroadaptations that enhance the incentive effects of drug stimuli.