Role of thyroid hormone receptors in timing oligodendrocyte differentiation.

The timing of oligodendrocyte differentiation is thought to depend on both intracellular mechanisms and extracellular signals. Thyroid hormone (TH) helps control this timing both in vitro and in vivo, but it is still uncertain how it does so. TH acts through nuclear receptors that are encoded by two genes, TRalpha and TRbeta. Previous studies suggested that TRbeta receptors may mediate the effect of TH on oligodendrocyte precursor cells (OPCs). Consistent with this possibility, we show here that overexpression of TRbeta1 promotes precocious oligodendrocyte differentiation, whereas expression of two dominant-negative forms of TRbeta1 greatly delays differentiation. Surprisingly, however, we find that postnatal TRbeta-/- mice have a normal number of oligodendrocytes in their optic nerves and that TRbeta-/- OPCs stop dividing and differentiate normally in response to TH in vitro. Moreover, we find that OPCs do not express TRbeta1 or TRbeta2 mRNAs, whereas they do express TRalpha1 and TRalpha2 mRNAs. These findings suggest that alpha receptors mediate the effect of TH on the timing of oligodendrocyte differentiation. We also show that TRalpha2 mRNA, which encodes a dominant-negative form of TRalpha, decreases as OPCs proliferate in vitro and in vivo. This decrease may help control when oligodendrocyte precursors differentiate.

Cooperation of Sp1 and p300 in the induction of the CDK inhibitor p21WAF1/CIP1 during NGF-mediated neuronal differentiation.

Addition of nerve growth factor (NGF) to PC12 cells promotes neuronal differentiation while inhibiting cell proliferation. In order to understand how NGF exerts its antimitogenic effect during differentiation, we have studied the mechanism by which this factor activates the promoter of the CDK inhibitor p21W4F1/CIP1. The minimal region of the p21 promoter required for the NGF-induction was mapped to a contiguous stretch of 10 bp located 83 bases upstream of the transcription initiation site. This GC-rich region was shown to interact specifically with the transcription factor Sp1 and the related protein Sp3, in either exponentially-growing or NGF-treated PC12 cells. The addition of NGF resulted in an accumulation of the transcriptional co-activator p300 in complexes associated with the NGF-responsive region. Transcriptional activity of Sp1, Sp3 and p300 was specifically induced by NGF in a Gal4-fusion assay, indicating that induction of p21 during neuronal differentiation may involve regulation of the activity of these factors by NGF. Furthermore, p300 was able to act as a co-activator for Sp1-mediated transcriptional activation in PC12 cells, suggesting that p300 and Sp1 may cooperate in activating p21 transcription during the withdrawal of neuronal precursors from the cell cycle. This hypothesis is supported by experiments showing that p300 and Sp1 form complexes in PC12 cells.

The CDK inhibitor p21WAF1/Cip1 is induced through a p300-dependent mechanism during NGF-mediated neuronal differentiation of PC12 cells.

The block of cell proliferation elicited by the addition of nerve growth factor (NGF) to exponentially-growing PC12 cells results, in part, from the inhibition of cyclin D1-associated kinase activity by p21WAF1/CIP1. NGF treatment of PC12 cells provokes the accumulation of p21 mRNA, due to transcriptional activation of the p21 promoter in a p53-independent manner. Transient expression of a mutated form of the adenovirus E1A protein (E1A dCR2), which retains its capacity to bind the transcriptional co-activator p300, completely abolishes the NGF-mediated stimulation of p21 promoter activity. This phenomenon can be reversed by ectopic expression of p300, suggesting that p300 is necessary for the induction of p21 by NGF. In addition, stable expression of E1A dCR2 in PC12 cells results in the inhibition of the NGF response, i.e. it prevents activation of the p21 promoter, cell cycle arrest, and neuronal differentiation. The signalling pathway from the TrkA receptor via the MAP kinase pathway is not altered in these cells. Together, these data indicate that p300 could play a pivotal role in the triggering of the anti-mitogenic effect of NGF and of neuronal differentiation.

Effect of nerve growth factor on the expression of cell cycle regulatory proteins in PC12 cells: dissection of the neurotrophic response from the anti-mitogenic response.

PC12 cells treated with nerve growth factor (NGF) undergo a G1 block and differentiate. Expression of selected cell cycle regulatory proteins was studied under culture conditions which permit observation of a differentiation response independently from a mitogenic or anti-mitogenic response. The expression of all cell cycle regulatory proteins studied is modulated by NGF addition to exponentially-growing cultures in the presence of serum. While levels of most of these proteins decrease, accumulation of cyclin D1 and the cyclin-dependent kinase inhibitor p21 Cip1/WAF1 is observed. Cyclin D1 associated kinase activity is inhibited, correlating with an increase in p21 protein. PC12 cells, synchronized by serum starvation, undergo morphological and functional differentiation in the presence of NGF. Neither cyclin D1 nor p21 are present in such cultures, nor is their expression upregulated by NGF, indicating that they are not required for this process. Removal of serum from differentiated PC12 cells results in loss of these proteins, but has no effect on differentiation or the nonproliferative state in presence of NGF. Together, the results indicate that cyclin D1 and p21 are not necessary for differentiation per se, nor are they required for maintenance of the differentiated state in the absence of serum.

Comparison of tolerance to intravenous nitroglycerin during nicorandil and intermittent nitroglycerin patch in healthy volunteers.

BACKGROUND: Nitrate tolerance is associated with a loss in the hemodynamic response to nitrate during repeated administration. Nicorandil is a new potassium channel agonist with additional nitrate properties. The aim of this study was to determine whether 7-day nicorandil (10 mg orally twice a day) administration attenuates the response to single-dose intravenous nitroglycerin (0.45 mg over 1 minute) in comparison with 7-day intermittent nitroglycerin patch administration (10 mg for 16 of 24 hours). METHODS: This was an open, randomized crossover study performed in 12 healthy volunteers. Blood pressure, heart rate, and their oscillations were measured with use of a noninvasive device. Low-frequency oscillations (66 to 129 mHz) of blood pressure reflect sympathetic activity. Reflex sympathetic activation was measured as after versus before intravenous nitroglycerin difference in low frequency oscillations of blood pressure and heart rate on day 0 and day 7 of each treatment period. Measurements after single-dose intravenous nitroglycerin included the maximum decrease in systolic blood pressure and maximum increase in heart rate and sympathetic activation. Tolerance in each group was assessed as the difference in each parameter between day 7 and day 0. RESULTS: Attenuation of the intravenous nitroglycerin-induced decrease in systolic blood pressure (day 7 - day 0) was - 10 +/- 10 mm Hg during use of the nitroglycerin patch and -2 +/- 11 mm Hg during nicorandil (p = 0.03). Similarly, the change in low frequency oscillations of systolic blood pressure was -79 +/- 144 mm Hg-Hz-1/2 during nitroglycerin administration and 60 +/- 139 mm Hg-Hz-1/2 during nicorandil administration (p = 0.04). CONCLUSION: These results indicate that 7-day administration of nicorandil does not attenuate single-dose intravenous nitroglycerin-induced hemodynamic changes or sympathetic activation. In healthy volunteers and at this dosage (10 mg twice a day), cross tolerance between nicorandil and nitroglycerin does not occur.

Reproducibility of non-invasive measurement and of short-term variability of blood pressure and heart rate in healthy volunteers.

1. Spectral analyses of blood pressure and heart rate oscillations are increasingly used to assess the influences of diseases and drugs on the autonomic nervous system. Such influences can only be interpreted in view of the spontaneous variability of these oscillations. We therefore studied the reproducibility of power spectral analyses of blood pressure and heart rate fluctuations measured by a non-invasive finger plethysmographic method in 24 healthy volunteers. 2. Intra-observer reproducibility was assessed from measurements obtained on 3 consecutive days and 1 month later in each subject. Inter-observer reproducibility was assessed by comparing measurements made by two observers on one occasion. 3. There was no significant difference in standard haemodynamic and spectral analysis parameters (low frequency: 60-130 mHz and high frequency: respiration rate +/- 30 mHz) measured on 3 consecutive days and 1 month later in each subject. The standard deviation of differences between systolic blood pressure or heart rate oscillations on different occasions was in the 150-200 and 50-100 mm Hg Hz-1/2 or beats min-1 Hz-1/2 range for low frequency and high frequency oscillations respectively. Similar results were found when inter-observer reproducibility was considered. 4. From these results, we derived a sample-size table giving the number of subjects to be included in studies of cross-over or parallel design in order to detect a non-random difference in spectral analysis parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical evaluation of pain during subcutaneous injections of low molecular weight heparins in healthy volunteers.

In order to compare the intensity and the duration of the pain following the subcutaneous injection of low molecular weight heparins, 0.4 ml nadroparin, 0.4 ml enoxaparin and 0.4 ml placebo (NaCl 0.9%) were administered at 1 week intervals to 12 healthy volunteers in a randomised, double-blind, three-period study. Pain was assessed by visual analogue and verbal category scales. Based on visual analogue scale scores, nadroparin resulted in significantly less pain than enoxaprin at 1 and 5 min after injection (6.2 +/- 2.6 mm vs 21.9 +/- 5.9 mm; P < 0.05 and 3.5 + 1.5 mm vs 14.3 +/- 4 mm; P < 0.05, respectively, mean +/- s.e. mean). Similar results were obtained using verbal category scale. Confidence interval testing for difference between groups showed that nadroparin injection resulted in less pain than enoxaparin at 1 min (-15.8: -31.2 to -0.4 mm).