Ventral otic cell lines as developmental models of auditory epithelial and neural precursors.

Conditionally immortal cell lines were established from the ventral otocyst of the Immortomouse at embryonic day 10.5 and selected to represent precursors of auditory sensory neural and epithelial cells. Selection was based upon dissection, tissue-specific markers, and expression of the transcription factor GATA3. Two cell lines expressed GATA3 but possessed intrinsically different genetic programs under differentiating conditions. US/VOT-E36 represented epithelial progenitors with potential to differentiate into sensory and nonsensory epithelial cells. US/VOT-N33 represented migrating neuroblasts. Under differentiating conditions in vitro the cell lines expressed very different gene expression profiles. Expression of several cell- and tissue-specific markers, including the transcription factors Pax2, GATA3, and NeuroD, differed between the cell lines in a pattern consistent with that observed between their counterparts in vivo. We suggest that these and other conditionally immortal cell lines can be used to study transient events in development against different backgrounds of cell competence.

Characterization of a metabotropic glutamate receptor type 5-green fluorescent protein chimera (mGluR5-GFP): pharmacology, surface expression, and differential effects of Homer-1a and Homer-1c.

Metabotropic glutamate receptor 5 (mGluR5) can modulate synaptic transmission by increasing intracellular Ca2+ and it plays a role in several forms of synaptic plasticity. We have constructed a fusion of human mGluR5 and green fluorescent protein (mGluR5-GFP). Expression of mGluR5-GFP in clonal cell lines yielded a functional fluorescent receptor with pharmacological profiles similar to wild-type mGluR5. mGluR5-GFP coimmunoprecipitated with Homer-1c, indicating that addition of GFP to the C-terminal did not prevent Homer binding. Coexpression of wild-type mGluR5 or mGluR5-GFP with Homer 1c, but not Homer-1a, resulted in reduced receptor surface localization and the formation of intracellular clusters. Neither Homer-1a nor Homer-1c had any effect on mGluR1 or mGluR1-GFP distribution. mGluR5-GFP expressed alone or in combination with Homer-1a formed dimers in HEK cells. Coexpression with Homer-1c, however, prevented mGluR5-GFP dimerization. Neither Homer altered the agonist profiles of mGluR5 or mGluR5-GFP. These data indicate that the functional expression of mGluR5 is regulated by Homer-1c and demonstrate that mGluR5-GFP provides a useful tool to study the molecular pharmacology and cell biology of mGluRs in real-time.

Effect of divalent metal ions on the binding of tissue factor and activated factor VII.

Surface plasmon resonance technology was used to study the binding of activated factor VII to tissue factor, in the presence of divalent metal ions. The binding was calcium-dependent, with the optimum calcium concentration at 5 mM. Only very minimal binding was observed in the absence of added calcium ions and presence of 10 mM EGTA. The effect of the calcium concentration on the apparent dissociation constants for the TF/VIIa complex formation was also investigated. In the absence of calcium ions but in the presence of either magnesium or zinc ions, no significant binding of activated factor VII to tissue factor was detected. However some binding was observed with manganese ions, indicating that manganese ions could partially replace calcium ions to support the formation of the TF/VIIa complex. These results are consistent with studies of the effect of divalent metal ions on the amidolytic activity of the TF/VIIa complex.

The C-terminal domain of the mGluR1 metabotropic glutamate receptor affects sensitivity to agonists.

The metabotropic glutamate receptor (mGluR) subtype 1 exists as at least three variants (-1a, -1b, and -1c) generated by alternative splicing at the C-terminal domain. Fluorometric Ca2+ measurements were used to compare the concentration dependency of agonist-induced rises in intracellular free Ca2+ concentration ([Ca2+]i) in human embryonic HEK 293 cells transiently expressing rat mGluR1a, mGluR1b, or mGluR1c. The rank order of agonist potencies was quisqualate >> (2S,1'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] and did not differ among the splice variants. However, agonists were consistently more potent at mGluR1a than at mGluR1c and mGluR1b. In the same system, we characterized the agonist pharmacology of two chimeric rat mGluR3/1 receptors where the first and/or the second intracellular loop(s) and the C-terminal domain were exchanged with the corresponding mGluR1a or mGluR1c sequences and that were previously shown to mediate elevations in [Ca2+]i in response to agonists. The potency of agonists was higher at the chimera having the C-terminus of mGluR1a as compared with those having the mGluR1c C-terminus. Both chimeric mGluR3/1 receptors had the same rank order of agonist potencies: L-CCG-I >> (1S,3R)-ACPD approximately quisqualate. These data support the hypothesis that the C-terminal domain of mGluRs plays a role in determining the potency of agonists for inducing mGluR-mediated functional responses.

The agonist selectivity of a class III metabotropic glutamate receptor, human mGluR4a, is determined by the N-terminal extracellular domain.

To test the hypothesis that the determinants for agonist selectivity of class III metabotropic glutamate receptors (mGluRs) are localized in the N-terminal extracellular domain, a chimaeric cDNA was constructed where 519 amino acids of the N-terminal extracellular domain of human mGluR1b were exchanged with the corresponding region of human mGluR4. The pharmacological profile of the chimaera, designated hmGlu(R4)1-519/1b, was analysed by recordings of intracellular calcium concentration ([Ca2+]i) in transiently transfected HEK 293 cells and compared with that of human mGluR1b and human mGluR4a stably expressed in Chinese hamster ovary cells. Application of 100 microM L-2-amino-4-phosphonobutyrate (L-AP4), a class III mGluR-specific agonist, induced a rise in [Ca2+]i in hmGlu(R4)1-519/1b but not in hmGluR1b expressing cells. In contrast, application of quisqualate (100 microM) induced a rise in [Ca2+]i at hmGluR1b but not at hmGlu(R4)1-519/1b. Dose-response analysis with L-AP4 and L-glutamate at hmGlu(R4)1-519/1b revealed a half-maximal effect (EC50) of 16.0 microM and 196 microM, respectively. The EC50 values for quisqualate, glutamate and (1S,3R)-ACPD at hmGluR1b were 10.25 microM, 225 microM and 3060 microM, respectively. The rank order of agonist potency of hmGlu(R4)1-519/1b corresponds to that of hmGluR4 (L-AP4 > L-glutamate > (1S,3R)-ACPD > quisqualate) but is different from that of hmGluR1b (quisqualate > glutamate >> (1S,3R)-ACPD).

Two sulphonated dye compounds which compete for inositol 1,4, 5-trisphosphate binding to rat liver microsomes: effects on 5'-phosphatase activity.

The ability of heparin to interact with the Ins 1,4,5-P3 receptor is dependent on its chain length and degree of sulphation. Here we report results obtained with two sulphonated dye compounds of known structures and molecular weights below 1000, cibacron blue and Patent blue. Both compounds compete for Ins 1,4,5-P3 binding to rat liver microsomes and also inhibit Ins 1,4,5-P3 5'-phosphatase activity in the same preparation. Comparison with the effects of heparin show these to be two separate actions of the compounds.

The effect of heparin on the inositol 1,4,5-trisphosphate receptor in rat liver microsomes. Dependence on sulphate content and chain length.

Heparin is known to inhibit the binding of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) to high-affinity binding sites and to inhibit Ins 1,4,5-P3-induced Ca2+ release from intracellular membrane-bound stores [(1987) J. Biol. Chem. 262, 12132-12136; (1987) FEBS Lett. 228, 57-59]. We have performed studies to clarify the structural requirements for this action of heparin in rat liver microsomes. Both N- and O-linked sulphate groups contribute to binding activity, since de-N-sulphated heparin was without effect on the Ins 1,4,5-P3 receptor whereas a polyxylan bearing only O-linked sulphates (pentosan polysulphate) was as active as heparin. Therefore, the density of negative charge contributed by sulphate groups is important for the binding of heparin. Heparins with high and low affinity for antithrombin III both inhibited Ins 1,4,5-P3 binding. There was a strong dependence on chain length, since binding activity decreased dramatically as the size of the heparin chain was reduced below that of 18-24 monosaccharide units.

Sodium exchange during hypoxia and on reoxygenation in the isolated rabbit heart.

Sodium exchange was examined during and after a period of hypoxic and substrate free perfusion in the isolated but arterially perfused interventricular septum of the rabbit heart. Temperature was 35 degrees C and muscles were stimulated at a mean rate of 90 beats/min. The uptake and washout of isotopes of sodium were followed during hypoxia and on reoxygenation. The extracellular space was estimated from the distribution volume of 51Cr-EDTA. During 45 mins hypoxia the intracellular sodium increased from 9.6 +/- 1.1 to 22.6 +/- 1.9 mmol/kg wet tissue. At this time maximal contracture had developed and recovery of mechanical function on reoxygenation was small. No increased efflux of sodium could be detected on reoxygenation. A net efflux of sodium could be detected when conditions were chosen to stimulate sodium--calcium exchange. These experiments do not support the hypothesis that the previously reported influx of calcium on reoxygenation is directly linked to a net loss of sodium.

Phospholipid turnover during cell-cycle traverse in synchronous Chinese-hamster ovary cells. Mitogenesis without phosphoinositide breakdown.

The turnover of phospholipids was investigated in quiescent serum-starved Chinese-hamster ovary (CHO-K1) cells stimulated to progress through the cell cycle by the addition of dialysed bovine serum. A variety of radiolabelling techniques were employed to study the rapid effects of serum on phospholipids and later events during G1 and S phases of the cell cycle. Pulse-labelling studies using [32P]Pi revealed that there was a stimulation of the synthesis rate of all phospholipids investigated during the initial few hours after serum addition. The greatest stimulation (20-fold) was observed in phosphatidylcholine, and the smallest in the polyphosphoinositides (PPIs). Mock stimulation with serum-free medium caused a similar increase in PPI turnover, but little or no effect on turnover of other phospholipids. This effect could be accounted for by a stimulation of the turnover of cellular ATP pools increasing [32P]ATP specific radioactivity. Late G1 and S phases were associated with a decrease in the rate of synthesis of all phospholipids. Phosphatidic acid was the only phospholipid whose labelling fell below that in mock-stimulated cells during the period of the cell cycle. Stimulation of serum-starved cells that had been prelabelled with myo-[2-3H]inositol caused no change in the amounts of inositol trisphosphate, but both serum-stimulated and mock-stimulated cells exhibited similar small decreases in both inositol bisphosphate and inositol monophosphate, of approx. 30% after 30 s. When cells were serum-stimulated in the presence of 10 mM-Li+, there was no increase in the size of the total inositol phosphate pool. We conclude that mitogenic stimulation and cell-cycle traverse cause profound and complex effects on phospholipid turnover in CHO-K1 cells, but there is no evidence for a role of inositol lipid turnover in the proliferative response to serum in this cell line.

Elevated phosphatidylinositol kinase activity in Rous sarcoma virus-transformed cells. Lack of evidence for enzyme translocation.

Phosphatidylinositol kinase (E.C. 2.7.1.67) activity of rat fibroblasts transformed by Rous sarcoma virus (RSV) was measured and compared with immunoprecipitated protein tyrosine kinase activity associated with pp60v-src. Both enzyme activities were elevated in the particulate fractions from wild-type RSV-transformed cells and cells transformed by a temperature-sensitive mutant of RSV when grown at the permissive temperature. The presence of the non-ionic detergent Nonidet P-40 in the phosphatidylinositol kinase assays stimulated the soluble and particulate forms of the enzyme to different degrees but did not affect the relative differences between transformed and untransformed cells. Our results indicate that phosphatidylinositol kinase activity is a good correlate of RSV transformation and suggest a functional relationship between pp60v-src and phosphatidylinositol kinase.

Alpha-adrenoceptor stimulation, lysophosphoglycerides, and lipid peroxidation in reoxygenation induced calcium uptake in rabbit myocardium.

Calcium uptake on reoxygenation of hypoxic cardiac muscle is well documented. Alpha-adrenoceptor stimulation by released catecholamines, lysophosphoglycerides and lipid peroxidation have all been suggested as mediators of this effect. We have measured the uptake of Ca2+ on reoxygenation in the isolated arterially perfused interventricular septum of the rabbit heart. The alpha agonist phenylephrine (1 mumol X litre-1) did not alter calcium uptake, and the presence of either prazosin (1 mumol X litre-1) or phentolamine (10 mumol X litre-1) did not alter the reoxygenation induced Ca2+ uptake. Lysophosphatidylcholine caused an increase in Ca2+ uptake above 8 mumol X litre-1 but also produced a simultaneous increase in the distribution volume of 51Cr-EDTA, an extracellular space marker, indicating loss of membrane integrity. Hydrogen peroxide and cumene hydroperoxide both caused an increased Ca2+ uptake, but no disruption of the cell membrane; the effect on Ca2+ uptake could be inhibited by Ni2+ ions. Alpha adrenergic stimulation and lysophosphoglycerides do not appear to be key to Ca2+ uptake on reoxygenation, but lipid peroxidation of the sarcolemma is a possible mechanism.

Mechanical response of rabbit myocardium and coronary arteries to leukotriene D4. Failure to demonstrate a role in the pathophysiology of hypoxia.

We have studied the effect of leukotriene D4 (LTD4) on rabbit and rat myocardial contractility and on rabbit coronary arteries. A concentration of 2 X 10(-7) M caused only a small reduction of myocardial contractility, but caused a contraction of smooth muscle in coronary arteries similar to that obtained with potassium (30 mmoles/liter). LTD4 (2 X 10(-7) M) added to the perfusate 10 min before or at the time of reoxygenation after a period of 30 min of hypoxia did not alter contractility or resting tension. LTD4 is unlikely to be a contributing factor in the initiation of cell necrosis on reoxygenation of hypoxic myocardium.

Calcium out of control.

The accumulation of calcium during myocardial hypoxia or ischaemia followed by reoxygenation or reperfusion is related to the development of cell necrosis and may be an important causal mechanism. Influx of calcium is a late event during hypoxia but occurs abruptly on reoxygenation or reperfusion. On reoxygenation calcium influx is not altered by nifedipine or quiescence but can be prevented by nickel (3 mM), cyanide (5 mM) or FCCP (10(-6) M). The extracellular marker 51Cr-EDTA does not enter the intracellular fluid on reoxygenation but can when the cell membrane is disrupted by a detergent, Brij'35, or the calcium paradox. The results suggest that the uptake of calcium on reoxygenation or reperfusion is related to the reintroduction of oxygen and caused by an increased calcium influx down the concentration gradient. The flux is not through the slow calcium channel and is not due to disruption of the membrane. The effects of CN- and FCCP and the unaltered calcium efflux suggest that the major part of the calcium uptake is stored in intracellular compartments and is not located in the intracellular fluid.