AIM: To investigate the heterogeneity of basal intracellular free calcium concentration( i) in peritoneal macrophages(PM) and whether it is relative to the reactivity of PM at the single cell level. METHODS: i implicated stimulated were measured by fluorescent microscopic imaging system after loading with fluorescent probe fura-2/AM. Superoxide(O _2)produced by single PM was determined by modified NBT test. RESULTS: The values of basal i determined in 392 PMs of 7 mice showed normal distribution [(54?24) nmol/L, n=392] with wide range(less than 20 nmol/L to more than 100 nmol/L), among which about 50% were in the range of 40-60 nmol/L. When stimulated with PMA or fMLP, i was increased, the peak values were positively correlated with the basal i in one mouse(PMA stimulated cells: r=0.52, P

The binding capacity (R_o) and apparant dissociation constant (K_d) of glucocrticoid binding sites in hepatic cytosol of normal and scalded rats were measured by pseudoscatchard analysis. Compared with control group, the levels of Ro of glucocorticoid receptor was significantly decreased 1 h after scalding, partialy recovered at 12h after scalding and fully recovered at 24 h after scalding. The levels of low-affinity glucocorticoid binding sites were also decreased 12 h after scalding, but had no statistical significance. The possible significance of these results has been discussed.

The effect of glucocorticoids on the down-regulation of glucocorticoid receptor (GR) mRNA was studied in intact rats. GR mRNA was characterized by Northern blot hybridization and quantitated by dot blot hybridization using a human GR cDNA fragment as a probe. Administration of hydrocortisone in polyvinyl alcohol resulted in a rapid increase in plasma glucocorticoids which maintained at stress levels (20 to 40 ?g/dl) for about 3 d. Hepatic GR mRNA decreased significantly to 73.5?63% of control values 6 h following hydrocortisone treatment, after which the decline of GR mRNA was gradual, reaching a minimum of 44.0?5.0% of control levels 3 d after the treatment The effect of hydrocortisone on the down-regulation of hepatic GR mRNA lasted up to 11 d. In contrast, hydrocortisone treatment had no effect on GR mRNA in rat brain. These results are consistent with the changes in GR in rats as reported previously, except that even though the hepatic cytosol GR decreased markedly, no significant changes in hepatic GR mRNA were found 1 h after hydrocortisone treatment, strongly suggesting that the down-regulation of GR by its ligands in vivo occurs at both transcriptional and posttranscriptional levels and is of tissue-specific fashion.

The present study was undertaken by radioligand binding assay to compare the binding properties of U72099E to cytosol and synaptic plasma membrane (SPM)preparations of rat brain. The results showed that the binding of [3H]Glucocorticoids ([3H]GC) was not displaced by U72099E in cytosol but was displaced in SPM with a Ki of 32.2?1.7?mol/L-1,indicating the existence of binding sites for U72099E in plasma membrane (most probably being glucocorticoid membrane receptors, GCMR). The Ki of U72099E is close to the concentrations needed to mimic the actions of it to protect against brain injury, suggesting that GCMR may mediate the beneficial effect of U72099E.

The induction of hsp70 mRNA in human osteosarcoma cells (HOS-8603) and intacl rats under heat stress was studied using hsp70 cDNA labeled with a-32P dATP. The results showed that the induction of hsp70 mRNA was evident in liver, lung, spleen and the most evident was found in brain when the core body temperature of rats was brought to 42℃ for 15 min. The induction of hsp70 mRNA in HOS-8603 was also significant when cells were cultured at 42℃ for 30 min. These results indicated that hsp70 mRNA could be induced both in vitro and in vivo under heat stress condition and the induction of hsp70 mRNA in intact rats was of tissue-specific fashion.

The glucocorticoid receptor of hepatic cytosol of rat was determined by the exchange assay in the presence of endogenous corticosterone. The condition of ex-chame was 0℃ , 4.5~5 hours. The dissociation of corticosterone-receptor complex was about 90% under such a condition. The effect of the endogenous corticosterore on the assay was evaluated by the measurement of [3H]-dexamethasone binding after preincubation of the liver cytosol from adrenalectomized rats with different amounts of corticosterone. The results indicated that, at a corticosterone concentration of less than 6 nM the error of the specific binding capacity (Ro) of [3H]-dexa-methasone was no more than 10%,and the apparant dissociation constant (Kd) was essentially unchanged. The Ro and Kd of glucocorticoid receptor of the hepatic cytosol of normal and scalded rats were measured by this method. The Ro was decressed in the scalded rats. The levels of Ro in hepatic cytosol from rats after 1, 12,24, 36 hours and 5 days of scalding were 76.4?15.2%,70.6?22.0%79.7?5.8%, 113.0?24.8% and 102.9 ?17.6% (mean?SD) of the control group respectively. The apparant Kd slightly increased 1 hour after scalding. The possible mechanisms of the above changes were discussed.

The high-affinity glucocorticoid binding sites (HAGS) and the low-affinity glucocorticoid binding sites (LAGS) with steroid specificity were demostrated in cerebral cytosol of rats by using the radioligand binding assay. The Kd of HAGS and LAGS were (178?0.71)?l0-8mol/L and (2.12?1.06)?10-6mol/L respecitively as estimated by Scatchard and Pseudoscatchard analysis. Glucocorticoid receptors (GR) in the traumatized(left) hemisphere cytosol were decreased more significantly than those in both the control (right) hemisphere cytosol at 6h postinjury and normal brain tissue (P

In order to study the physiological action of glucocorticoid receptor (GR) in intact animal, an animal model of GR depletion was developed in the rat by mifepristone (RU486), an antagonist of GR. Mifepristone 50 mg/kg body weight mixed in polyvinyl alcohol (PVA) was injected intramuscularly every 12 h in order to maintain the plasma mifepristone at the concentration of about 10-6mol/L for 72 h. In the meanwhile, the plasma corticosterone (B) was stabilized at about 15 ?g/dl by unilateral adrenalectomy 3 d before. In this model mifepristone occupied 88.1 ?10.7% of the total GR in hepatic, brain cytosol and thymocytes as measured by the exchange assay with [3H]dexamethasone (Dex) as ligand. Some indexes of glucocorticoid response were measured in the control, model and bilateral adrenalectomized (AdxT) rats. The polymorphonuclear eosinophil leukocyte (PME) count in blood and phospholipase A2(PLA2) activity in serum were decreased and tyrosine aminotransferase (TAT) activity of the liver was elevated in the control rats after stress provoked by epinephrine, but these changes were reversed in the model rats as well as in the AdxT rats. Thus it may be concluded that decrease of GR to about 12% may result in adrenoglucocorticoid insufficiency in spite of the normal functioning adrenal gland and high plasma level of B. So far as we know this is the first success to produce experimental endocrine insufficiency in a receptor depletion animal model and to quantitate the occupational threshold of about 12% of the total GR in rats.

The specific glucocorticoid binding of adrenalectomized rats was measured by the radioligand binding assay.The [3H]dexamethasone was used as ligand.The binding capacity (R0) and dissociation constant (Kd) were calculated.The specific binding sites present the following characteristics : 1.Low binding capacity , (R0: 264?50 fmole/mg protein) ; 2.High affinity,(Kd:2.5?0.9?10-8M) ; 3.The specificity of steroid binding ; 4.The presence in the other target organs , such as brain, kidney and thymus;5.The degree of saturation in vivo of the hepatic cytosol binding is closely correlated to the induction of hepatic enzymes, tyrosine aminotransferase.These indicate that the specific binding sites of dexamethasone are functionally significant glucocorticoid receptors of the rat liver cytosol.

Scatchard, pseudoscatchard and competitive analyses showed that there were two glucocorticoid (GC) binding sites in rat hepatic cytosol: high-affinity glucocorticoid binding sites (HAGS) and low-affinity glucocorticoid binding sites (LAGS). Pseudoscatchard analysis showed the dissociation constant (Kd) of LAGS was 4.1+1.4umol/L (n=4). The inactivation of LAGS in vitro was slower" than that of HAGS. LAGS and HAGS could not be separated by DEAE-cellulose chfomatography. HAGS were saturable and had steroid specificity for glucocorticoid just as the classic glucocorticoid receptor. Triamcinolone acetonide, RU486 and RU26988 competed equally well to LAGS while aldosterone, progesterone, estradiol and testosterone did not compete with LAGS. These results suggest that LAGS have steroid specificity for glucocorticoids. The biological actions of LAGS await fur ther study.