Enhanced morphine analgesia in mice lacking beta-arrestin 2.

The ability of morphine to alleviate pain is mediated through a heterotrimeric guanine nucleotide binding protein (G protein)-coupled heptahelical receptor (GPCR), the mu opioid receptor (muOR). The efficiency of GPCR signaling is tightly regulated and ultimately limited by the coordinated phosphorylation of the receptors by specific GPCR kinases and the subsequent interaction of the phosphorylated receptors with beta-arrestin 1 and beta-arrestin 2. Functional deletion of the beta-arrestin 2 gene in mice resulted in remarkable potentiation and prolongation of the analgesic effect of morphine, suggesting that muOR desensitization was impaired. These results provide evidence in vivo for the physiological importance of beta-arrestin 2 in regulating the function of a specific GPCR, the muOR. Moreover, they suggest that inhibition of beta-arrestin 2 function might lead to enhanced analgesic effectiveness of morphine and provide potential new avenues for the study and treatment of pain, narcotic tolerance, and dependence.

Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3.

beta-Arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. We identified c-Jun amino-terminal kinase 3 (JNK3) as a binding partner of beta-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with beta-arrestin 2. Cellular transfection of beta-arrestin 2 caused cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activated JNK3 and triggered the colocalization of beta-arrestin 2 and active JNK3 to intracellular vesicles. Thus, beta-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR.

Bone response to intermittent parathyroid hormone is altered in mice null for {beta}-Arrestin2.

Intermittent PTH administration increases bone turnover, resulting in net anabolic effects on bone. These effects are primarily mediated by intracellular cAMP signaling. However, the molecular mechanisms that regulate PTH activity in bone remain incompletely understood. beta-Arrestin2, a G protein-coupled receptor regulatory protein, inhibits PTH-stimulated cAMP accumulation in vitro. Using beta-arrestin2(-/-) (KO) and wild-type (WT) mice, we investigated the response to PTH in primary osteoblasts (POB) and the effects of intermittent PTH administration on bone mass and microarchitecture in vivo. Compared with that in WT mice, PTH-stimulated intracellular cAMP was increased and sustained in KO POB. Intermittent exposure of POB to PTH significantly decreased the ratio of osteoprotegerin (OPG) receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA expression in KO POB, whereas it increased this ratio in WT POB. Total body bone mass and cortical and trabecular bone parameters were 5-10% lower in male KO mice compared with WT, and these differences were magnified upon in vivo administration of intermittent PTH (80 mug/kg.d) for 1 month. Thus, PTH significantly increased total body bone mineral content as well as vertebral trabecular bone volume and thickness in WT, but not KO mice. The anabolic response to PTH in cortical bone was also slightly more pronounced in WT than KO mice. Histomorphometry indicated that PTH prominently stimulated indexes of bone formation in both WT and KO mice, whereas it significantly increased indexes of bone resorption (i.e. osteoclast number and surface) in KO mice only. In conclusion, these results suggest that beta-arrestins may specify the activity of intermittent PTH on the skeleton by limiting PTH-induced osteoclastogenesis.

Synthesis and biochemical studies of estrone sulfatase inhibitors.

The synthesis and biochemical evaluation of estrone sulfatase inhibitors are described. Inhibitors were designed through modifications of the substrate estrone sulfate. An in vitro assay using the microsomal fraction isolated from human term placenta was used to evaluate sulfatase inhibitory activity. All the inhibitors (except sulfonyl chloride analog) exhibited low inhibitory activities in the screening assay. Sulfonyl chloride analog is a strong inhibitor, which caused 91.5% inhibition of the enzymatic activity at 300 microM.

Reducing lead exposure by surveillance system: the Taiwan experience.

To evaluate the performance of a lead-surveillance program in reducing blood lead levels of workers in Taiwan, the authors conducted prospective and cross-sectional studies. A total of 6 905 workers, whose job titles indicated a direct exposure to lead in 1995, were included in this surveillance system. In this study, the authors compared the mean blood lead levels in 1994 (i.e., year of onset of surveillance) with that in 1995 in workers of major industries. Lead-exposed workers had a statistically significant decrease (i.e., average of 1.8 microg/dl) in blood lead levels during this 1-y period. The decrease was particularly obvious in individuals who worked in chemical products manufacturing, ship building/repairing, and plastic products manufacturing. The significant decreases in blood lead levels in these workers indicated that this surveillance system was effective. Surveillance, combined with control measures, might be an important means by which occupational lead exposure can be reduced.

[Preclinical pharmacologic evaluation of bleomycin A6].

Bleomycin A6 is an antineoplastic antibiotic. Toxicologic study showed that the LD50 values of bleomycin A6 in mice were 92 +/- 7.4 mg/kg (IV), 72 +/- 5.6 mg/kg (IM), 82 +/- 4.5 mg/kg (SC), 102 +/- 0.6 mg/kg (IP) and 520 +/- 0.1 mg/kg (PO), respectively. Wistar rats were divided into 6 groups. Doses of 20, 10, 5, 2 and 0.5 mg/kg/day were given intraperitoneally every other day for 60 days. Renal damage to various degrees was found in rats with doses higher than 2 mg/kg. However, no pathologic changes were observed in the liver, heart, lungs, spleen, stomach, intestines, pancreas or brain. Three groups of dogs (3 in each group) were injected i.v. every other day for 60 days with a dose of 1.0, 0.5 and 0.1 mg/kg, respectively. The three dogs receiving 1.0 mg/kg died between the 4th and 7th week. Dogs treated with 1.0 and 0.5 mg/kg had renal damage and skin ulceration. With a dose of 0.1 mg/kg, dogs showed good tolerance and no renal damage was found. Multiple doses of bleomycin A6 injected into rabbit muscles caused no obvious local changes. After IV injection into rabbits, the maximal serum concentration of bleomycin A6 was 18.5 micrograms/ml, having a half life of 2.8 hours and 7.7% of the drug were excreted in the urine within 12 hours.

The protective effect of the large-scale use of PHKC rabies vaccine in humans in China.

Reported are the results obtained with different immunization schedules of adjuvant or freeze-dried concentrated (FDC) primary hamster kidney cell (PHKC) rabies vaccine on volunteers. The FDC vaccine (potency, 4.5 IU), which was inoculated in six doses, on days 0, 3, 7, 14, 30 and 90, and the adjuvant vaccine (potency 2.5 IU), which was inoculated in five doses, on days 0 and 7 (double dose), 14, 30 and 90, induced earlier, higher, and more persistent neutralizing antibody titres than the adjuvant vaccine which was inoculated in five doses on days 0, 3, 7, 14 and 30. The persistence of the neutralizing antibody titres induced by three intradermal doses of vaccine administered on days 0 (4 sites), 7 (2 sites), and 28 (1 site) was lower than that induced by six intramuscular doses administered on days 0, 3, 7, 14, 30, and 90. A cell-mediated immunity (CMI) was also induced in vaccinees who received the adjuvant vaccine. The protective effect of the adjuvant vaccine was better than that of the previously used Semple vaccine and has had a positive effect on the epidemiology of human rabies in China.

CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program.

Previous studies showed that CCAAT/enhancer binding protein alpha (C/EBP alpha) is required for differentiation of 3T3-L1 preadipocytes induced by exogenous hormonal agents. It was not possible to ascertain, however, whether C/EBP alpha alone is sufficient to induce differentiation because its antimitogenic activity precluded propagating 3T3-L1 cell lines that constitutively express C/EBP alpha at high levels. This problem was circumvented by using 3T3-L1 preadipocytes stably transfected with an isopropyl beta-D-thiogalactoside (IPTG)-inducible p42 C/EBP alpha expression vector system. IPTG-induced expression of the 42-kDa isoform of C/EBP alpha in preadipocytes caused expression of several endogenous adipocyte-specific genes (genes encoding the 422 adipose P2 protein, glucose transporter 4, and C/EBP alpha) and the accumulation of cytoplasmic triglyceride. Thus, C/EBP alpha is not only necessary but also is sufficient to trigger differentiation of growth-arrested 3T3-L1 preadipocytes without use of exogenous hormonal agents.

Antisense CCAAT/enhancer-binding protein RNA suppresses coordinate gene expression and triglyceride accumulation during differentiation of 3T3-L1 preadipocytes.

Previous studies suggest that the CCAAT/enhancer-binding protein (C/EBP) functions in the coordinate expression of adipocyte genes during differentiation of 3T3-L1 preadipocytes. We sought to block expression of C/EBP selectively using a bovine papilloma virus (BPV) vector to direct transcription of a approximately 0.4-kb segment of C/EBP cDNA (in antisense orientation) containing translated sequence 5' to that encoding the basic and leucine zipper regions of the protein. Vector-directed expression of antisense C/EBP RNA in 3T3-L1 preadipocytes inhibited expression of C/EBP mRNA and protein, as well as several adipose-specific mRNAs, and also prevented cytoplasmic triglyceride accumulation. Rescue of the "adipocyte phenotype" was accomplished by transfection of cells expressing antisense RNA with a modified BPV vector that directs transcription of the complementary sense C/EBP RNA.

A comparison of seven visual fatigue assessment techniques in three data-acquisition VDT tasks.

We compared 7 methods of measuring visual fatigue--accommodation power, visual acuity, pupil diameter, critical fusion frequency (CFF), eye movement velocity, subjective rating of visual fatigue, and task performance--for their sensitivity to visual load. In the experiment, 10 participants performed a monitoring task at 2 viewing distances, read articles under 2 levels of screen contrast, and tracked visual targets at 2 different speeds. The same measurement techniques, excluding pupil diameter and eye movement velocity, were compared by extending the task time from 20 to 60 min with the same VDT tasks to test for possible improvement in sensitivity. The results indicated that sensitivities of accommodation power, visual acuity, and CFF were greatly improved by a longer task period, but these 3 measurement techniques did not distinguish among tasks. Pupil diameter, eye movement velocity, and subjective rating of visual fatigue were sensitive in differentiating tracking from reading and monitoring tasks. Eye movement velocity and subjective rating were sensitive to the changes in target velocity of the tracking task. Although task performance was not directly comparable to other measurement techniques, it helped to ensure that participants maintained the same performance level by devoting more resources to the high-load conditions. Actual or potential applications of this research include using some of these assessment techniques for the design of adaptive displays.

beta-arrestins regulate mitogenic signaling and clathrin-mediated endocytosis of the insulin-like growth factor I receptor.

beta-Arrestins mediate agonist-dependent desensitization of G protein-coupled receptors and target the receptors to clathrin-coated pits for internalization. Here we report an expanded role of beta-arrestins in promoting clathrin-mediated endocytosis of a tyrosine kinase growth factor receptor, i.e. the insulin-like growth factor I (IGF-1) receptor. beta-Arrestins bind to the ligand-occupied IGF-1 receptors, promote their endocytosis, and enhance IGF-1-dependent mitogen-activated protein kinase phosphorylation and DNA synthesis. Our results suggest a role for beta-arrestins in regulating mitogenic signaling and clathrin-mediated endocytosis of receptors not classically coupled to G proteins.

Selective induction of E2F1 in response to DNA damage, mediated by ATM-dependent phosphorylation.

Previous work has established a role for p53 in triggering apoptosis in response to DNA damage; p53 also induces apoptosis in response to deregulation of the Rb cell cycle pathway. The latter event is consistent with a role for the Rb-regulated E2F1 protein as a specific inducer of apoptosis and p53 accumulation. We now show that DNA damage leads to a specific induction of E2F1 accumulation, dependent on ATM kinase activity and that the specificity of E2F1 induction reflects a specificity in the phosphorylation of E2F1 by ATM as well as the related kinase ATR. We identify a site for ATM/ATR phosphorylation in the amino terminus of E2F1 and we show that this site is required for ATM-mediated stabilization of E2F1. Finally, we also show that E2F1 is required for DNA damaged induced apoptosis in mouse thymocytes. We conclude that the cellular response to DNA damage makes use of signals from the Rb/E2F cell cycle pathway.

Control of adipocyte differentiation by CCAAT/enhancer binding protein alpha (C/EBP alpha).

Upon differentiation of 3T3-L1 preadipocytes into adipocytes transcription of many adipose-specific genes is coordinately activated. A differentiation-induced factor, later identified as C/EBP alpha, binds to and transactivates the promoters of these genes. Vector-directed expression of antisense C/EBP alpha RNA in preadipocytes blocked expression of C/EBP alpha, as well as adipose-specific mRNAs, and also prevented cytoplasmic triglyceride accumulation. Rescue of the 'adipocyte phenotype' was accomplished by transfection of the antisense cells with a complementary sense C/EBP alpha RNA expression vector. Using an IPTG-inducible double-vector LacSwitch C/EBP alpha expression system, it was found that differentiation can be induced without exogenous hormone inducers. These findings indicate that C/EBP alpha is not only required, but is sufficient, to trigger differentiation of 3T3-L1 preadipocytes. The C/EBP alpha gene promoter possesses a C/EBP binding site through which C/EBP alpha autoactivates its own expression. A nuclear protein referred to as CUP (C/EBP undifferentiated protein) that binds to a bipartite element in the C/EBP alpha promoter just 5' to the C/EBP binding site has been purified and characterized. During differentiation of preadipocytes, expression of CUP activity decreases as expression of C/EBP alpha increases. Evidence suggests that a CUP-containing protein complex bridges between the CUP (repression) and C/EBP (autoactivation) elements in the promoter and may maintains the C/EBP alpha gene in the repressed state prior to differentiation.

Glucocorticoids reciprocally regulate expression of the CCAAT/enhancer-binding protein alpha and delta genes in 3T3-L1 adipocytes and white adipose tissue.

Glucocorticoid agonists, i.e. dexamethasone or triamcinolone acetonide, rapidly induce expression of CCAAT/enhancer-binding protein (C/EBP) delta and repress expression of C/EBP alpha in fully differentiated 3T3-L1 adipocytes. Within 30 min of glucocorticoid treatment, the cellular level of C/EBP delta rises dramatically, increasing > 100-fold within 6 h. Concurrently, the level of C/EBP alpha decreases, reaching a minimum within 4 h. The dexamethasone concentration dependence and steroid specificity of these responses suggest that both processes are mediated by the glucocorticoid receptor. The reciprocal effects of dexamethasone on the steady-state levels of C/EBP alpha and C/EBP delta can be accounted for kinetically and quantitatively by changes in their mRNA levels and by the transcription rates of their respective genes. The glucocorticoid-induced changes in expression of the C/EBP isoforms are correlated with the transcriptional activation of the SCD1 gene, an adipocyte gene known to be transactivated by C/EBP isoforms. Glucocorticoids also regulate expression of the C/EBP isoforms in vivo. Within 4 h of administration of dexamethasone or triamcinolone acetonide to adult rats, expression of C/EBP delta is induced in white adipose tissue while expression of C/EBP alpha is repressed. Like the response in 3T3-L1 adipocytes, the effects of dexamethasone on C/EBP alpha in white adipose tissue are rapid and transient.

A 30-kDa alternative translation product of the CCAAT/enhancer binding protein alpha message: transcriptional activator lacking antimitotic activity.

Full-length (42 kDa) CCAAT/enhancer binding protein alpha (C/EBP alpha) (p42) has been implicated in the transcriptional activation of adipocyte genes including the 422(aP2) and C/EBP alpha genes during differentiation of 3T3-L1 preadipocytes. We have identified a 30-kDa isoform (p30) of C/EBP alpha that is expressed by 3T3-L1 adipocytes, mouse adipose tissue, and rat liver. In vitro translation of wild-type C/EBP alpha mRNA or transient transfection with a wild-type C/EBP alpha vector gave rise to similar levels of p42 and p30. Mutational analysis revealed that p30 is an alternative translation product initiated at the third in-frame methionine codon of the C/EBP alpha message. p30C/EBP alpha binds to the C/EBP sites within and activates reporter gene expression driven by the 422(aP2) and C/EBP alpha gene promoters. Although transfection of 3T3-L1 preadipocytes with a strong p30C/EBP alpha expression vector is insufficient to induce differentiation, this vector advances the differentiation program. Unlike p42C/EBP alpha, which inhibits cell proliferation, p30C/EBP alpha is not antimitotic. Thus, the N-terminal 12-kDa segment of full-length C/EBP alpha contains an amino acid sequence necessary for antimitotic activity. During differentiation of 3T3-L1 preadipocytes and during hepatocyte development, the cellular p42C/EBP alpha/p30C/EBP alpha ratio changes, raising the possibility of a regulatory role.

Feedback regulation of beta-arrestin1 function by extracellular signal-regulated kinases.

The functions of beta-arrestin1 to facilitate clathrin-mediated endocytosis of the beta2-adrenergic receptor and to promote agonist-induced activation of extracellular signal-regulated kinases (ERK) are regulated by its phosphorylation/dephosphorylation at Ser-412. Cytoplasmic beta-arrestin1 is almost stoichiometrically phosphorylated at Ser-412. Dephosphorylation of beta-arrestin1 at the plasma membrane is required for targeting a signaling complex that includes the agonist-occupied receptors to the clathrin-coated pits. Here we demonstrate that beta-arrestin1 phosphorylation and function are modulated by an ERK-dependent negative feedback mechanism. ERK1 and ERK2 phosphorylate beta-arrestin1 at Ser-412 in vitro. Inhibition of ERK activity by a dominant-negative MEK1 mutant significantly attenuates beta-arrestin1 phosphorylation, thereby increasing the concentration of dephosphorylated beta-arrestin1. Under such conditions, beta-arrestin1-mediated beta2-adrenergic receptor internalization is enhanced as is its ability to bind clathrin. In contrast, if ERK-mediated phosphorylation is increased by transfection of a constitutively active MEK1 mutant, receptor internalization is inhibited. Our results suggest that dephosphorylated beta-arrestin1 mediates endocytosis-dependent ERK activation. Following activation, ERKs phosphorylate beta-arrestin1, thereby exerting an inhibitory feedback control of its function.

Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence.

Morphine is a powerful pain reliever, but also a potent inducer of tolerance and dependence. The development of opiate tolerance occurs on continued use of the drug such that the amount of drug required to elicit pain relief must be increased to compensate for diminished responsiveness. In many systems, decreased responsiveness to agonists has been correlated with the desensitization of G-protein-coupled receptors. In vitro evidence indicates that this process involves phosphorylation of G-protein-coupled receptors and subsequent binding of regulatory proteins called beta-arrestins. Using a knockout mouse lacking beta-arrestin-2 (beta arr2-/-), we have assessed the contribution of desensitization of the mu-opioid receptor to the development of morphine antinociceptive tolerance and the subsequent onset of physical dependence. Here we show that in mice lacking beta-arrestin-2, desensitization of the mu-opioid receptor does not occur after chronic morphine treatment, and that these animals fail to develop antinociceptive tolerance. However, the deletion of beta-arrestin-2 does not prevent the chronic morphine-induced up-regulation of adenylyl cyclase activity, a cellular marker of dependence, and the mutant mice still become physically dependent on the drug.