Morphological and transcriptional evaluation of multiple facial cutaneous hyperpigmented spots.

Background: Morphological characteristics of major facial hyperpigmented spots have been well documented. However, detailed alterations of respective transcriptional profile for each spot and in-depth comparisons across multiple spot types have not been reported. Objectives: To comprehensively assess and compare multiple facial hyperpigmented spot types at the morphological and molecular levels by utilising transcriptional expression profiling with correlation to quantified histological features. Methods: Multiple types of facial spot biopsies were collected from Chinese women and compared to additional biopsies taken from adjacent healthy skin. The types of spots included Solar Lentigos with both elongated dermal-epidermal junction (DEJ) (SL[E]) and flat DEJ (SL[F]), Seborrhoeic Keratosis (SK), Melasma, Freckles, Post-inflammatory hyperpigmentation of resolving acne (PIH[A]) and other stimuli (PIH[O]). Combined histomorphometry, immunohistology, and transcriptome analysis for suprabasal-epidermis, basal-epidermis, and dermal compartments dissected by Laser Capture Microdissection (LCM) were conducted and compared across different spot types. Results: Each spot type was confirmed to have the unique histological pathology already documented elsewhere. Most of the spot types except Melasma and PIH (A) revealed similar melanocyte density to adjacent skin. All spots exhibited increased melanin synthesis, melanosome transportation, as well as enhanced melanocyte dendricity, however, each spot revealed a distinct transcriptome regulation pattern in pigmentation pathways. Upregulation of pigmentation genes was also observed in the dermis of SL(F), SL(E), SK and PIH(O), associated with significant modulation of DEJ related genes in basal-epidermis and/or dermal compartments, suggesting potential melanocyte infiltration into the dermis due to impaired DEJ quality. Beyond upregulated pigmentation, for most spots, gene expression in the suprabasal-epidermis regulating keratinisation was significantly upregulated in conjunction with thickened stratum corneum. Furthermore, downregulation of tight junction related genes represented by claudin-1 was observed in majority of spot types, suggesting compromised barrier function could be a similarity across spots. Additionally, Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) was upregulated in all types of spots, indicating involvement of cell senescence as a common theme. Conclusion: This comprehensive and comparative study based on the histological and transcriptional analysis of three skin compartments provided unique insights into specific causations as well as differences and similarities across multiple hyperpigmented spot types.

A regulator of ubiquitin-proteasome activity, 2-hexyldecanol, suppresses melanin synthesis and the appearance of facial hyperpigmented spots.

BACKGROUND: 2-Hexyldecanol has long been used in skin-care products, but has not previously been reported as an active ingredient for skin benefits. OBJECTIVES: To evaluate 2-hexyldecanol in in vitro and ex vivo systems and, if found to be active, progress it to topical clinical testing to determine effects on pigmentation in skin. METHODS: 2-Hexyldecanol was tested in melanocyte cell culture systems (B16 mouse melanoma cells and normal human melanocytes) for its effect on proteolytic activity and melanin production, in the absence and presence of the proteasome-specific inhibitor, MG132. It was further tested in a human skin explant model for its effect on melanin production. Lastly, topically applied 2-hexyldecanol was evaluated for its effect on the appearance of facial pigmentation in an 8-week, randomized, double-blind, vehicle-controlled, split-face incomplete block design study in Chinese women. RESULTS: In submerged cell culture, 2-hexyldecanol upregulated proteolytic activity and decreased melanin synthesis. These effects were antagonized by the proteasome-specific inhibitor MG132. MG132, tested in the absence of 2-hexyldecanol, increased melanin production. In a human skin explant model, topical 2-hexyldecanol suppressed the production of melanin vs. a vehicle control. In a human clinical study in Chinese women (n = 110 observations per test material), a 2-hexyldecanol-containing formulation significantly reduced the appearance of facial hyperpigmented spots vs. its control. CONCLUSIONS: These data indicate that regulation of proteasome activity is a viable target for control of melanin production, that 2-hexyldecanol upregulates proteasomal activity in melanocytes, and that topical 2-hexyldecanol reduces the appearance of hyperpigmentation.

Effects of S21403 (mitiglinide) on postprandial generation of oxidative stress and inflammation in type 2 diabetic patients.

AIM/HYPOTHESIS: Evidence suggests that postprandial hyperglycaemia may be a cardiovascular risk factor in diabetes. Oxidative stress and inflammation are involved in the pathogenesis of diabetic complications and previous studies have shown increased oxidative stress and inflammation in the postprandial phase in diabetic patients. The aim of the present study was to evaluate whether controlling postprandial hyperglycaemia with S21403 (mitiglinide) is accompanied by a reduced generation of oxidative stress and inflammation. SUBJECTS AND METHODS: Forty type 2 diabetic patients participated in the study. Two different breakfast-tests were performed in each patient, with placebo or S21403. Plasma nitrotyrosine, plasma malondialdehyde (MDA), oxidised LDL (oxLDL), plasma total radical-trapping antioxidant parameter (TRAP), IL-6, IL-18, TNF-alpha, plasma glucose and insulin were measured. RESULTS: After the administration of S21403, 40 mg, a rapid stimulation of insulin secretion was observed, accompanied by a reduction of postprandial hyperglycaemia. With S21403, a significant decrease of either nitrotyrosine, MDA and oxLDL levels, and a preservation of plasma TRAP compared with placebo was found. Significant decreases of IL-6, IL-18 and TNF-alpha were also observed with S21403 compared with placebo. CONCLUSIONS/INTERPRETATION: This study shows that controlling postprandial hyperglycaemia with S21403 significantly improves the cluster of oxidative stress and inflammation markers that are increased in the postprandial state in diabetic patients.

The post-prandial state in Type 2 diabetes and endothelial dysfunction: effects of insulin aspart.

OBJECTIVE: Recently, much attention has been focused on the possibility that the post-prandial state may be a cardiovascular risk factor in diabetes. The aim of the present study was to evaluate whether the post-prandial state is associated with endothelial dysfunction in patients with diabetes and to explore the effect on this aspect of managing post-prandial hyperglycaemia by insulin aspart. RESEARCH DESIGN AND METHODS: Twenty-three patients with Type 2 diabetes and 10 normal controls were recruited. In the diabetic patients two different tests were performed in each subject: a standard meal preceded by subcutaneous injection of soluble insulin (0.15 U/kg body weight) or of short-acting insulin aspart (0.15 U/kg body weight). These tests were designed to achieve different levels of post-prandial hyperglycaemia. Controls received a single standard meal test. Immediately before, and 1, 2, 4 and 6 h after each meal, blood glucose, triglycerides, free fatty acids and flow-mediated vasodilation were measured. RESULTS: Compared with regular insulin, insulin aspart significantly reduced the area under the curve for post-prandial hyperglycaemia (58.3 +/- 17.6 vs. 68.1 +/- 17.7; P<0.04), and preserved flow-mediated vasodilation, which was decreased in the post-prandial state (39.4 +/- 2.9 vs. 34.1 +/- 2.2; P<0.01). Triglyceride and free fatty acid levels were not differentially affected by the treatment. In normal controls the meal did not affect flow-mediated vasodilation. CONCLUSION: This study shows that the post-prandial state is accompanied by endothelial dysfunction in Type 2 diabetic patients and that insulin aspart improved endothelial function.

Concomitant primary hyperparathyroidism, Graves' disease and vitamin D deficiency.

The simultaneous occurrence of hyperthyroidism and primary hyperparathyroidism is quite rare. A case is reported here of Graves' disease and primary hyperparathyroidism. Treatment of these concomitant diseases consists in combined thyroidectomy and parathyroidectomy. Histological examination confirmed Graves' disease and revealed the presence of a parathyroid adenoma in one gland and parathyroid hyperplasia in another one. After surgical treatment, secondary hyperparathyroidism due to 25-OH Vitamin D deficiency appeared. To our knowledge, this is the first case in which a Vitamin D absorption deficit was found in presence of hyperthyroidism and hyperparathyroidism without other intestinal absorption deficits. Diagnostic and follow-up difficulties related to these three concomitant pathological conditions are discussed.

Detection of nitrotyrosine in the diabetic plasma: evidence of oxidative stress.

AIMS/HYPOTHESIS: Oxidative stress plays an important role in diabetic vascular complications. It has been shown that an imbalance in the ratio of nitric oxide: superoxide anion, because of a prevalence of superoxide anion, leads to an alteration in vascular reactivity. In this condition peroxynitrite production, resulting from the reaction between nitric oxide and superoxide, could increase. Peroxynitrite is responsible for nitration of tyrosine residues in proteins. Therefore, the presence of nitrotyrosine in plasma proteins is considered indirect evidence of peroxynitrite production. The aim of this study was to demonstrate the presence of nitrotyrosine in the plasma of patients with Type II (non-insulin-dependent) diabetes mellitus and to correlate its concentrations with the plasma concentrations of glucose and antioxidant defenses. METHODS: A total of 40 Type II diabetic patients and 35 healthy subjects were enrolled, and glycaemia, plasma nitrotyrosine, total antioxidant parameter and glycated haemoglobin were measured. Nitrotyrosine was detected by ELISA with a detection limit of 10 nmol/l. RESULTS: Nitrotyrosine was found in the plasma of all diabetic patients (means +/- SD = 0.251 +/- 0.141 micromol/l), whereas it was not detectable in the plasma of healthy control subjects. Nitrotyrosine plasma values were correlated with plasma glucose concentrations (r = 0.38, p < 0.02) but not with total antioxidant parameter or glycated haemoglobin. Total antioxidant parameter was reduced in diabetic patients (p < 0.01). CONCLUSIONS: The presence of nitrotyrosine in the plasma of diabetic patients indicates that peroxynitrite is generated in diabetes, suggesting a possible involvement of peroxynitrite in the development of diabetic complications.

Red wine protects diabetic patients from meal-induced oxidative stress and thrombosis activation: a pleasant approach to the prevention of cardiovascular disease in diabetes.

BACKGROUND: Oxidative stress and thrombosis have been reported to be increased in diabetic patients and involved in the pathogenesis of cardiovascular complications. It has been demonstrated in diabetic patients that consumption of a meal is accompanied by the generation of an oxidative stress and of a hypercoagulable state. It is well recognized that red wine shows antithrombotic activity and that its ingestion increases plasma antioxidant capacity in man. In this study the possibility that red wine consumption may reduce the oxidative stress and thrombosis produced postprandially in diabetic patients has been evaluated. SUBJECTS AND METHODS: Twenty type 2 diabetic patients were studied during fasting consumption of 300 mL of red wine, or during a meal accompanied, or not, by red wine ingestion. RESULTS: Plasma glucose, insulin, triglycerides, total plasma radical-trapping capacity, activated factor VII and prothrombin fragments 1 + 2 were measured in basal state and at 60, 120 and 180 min after the start of each experiment. Low-density lipoprotein (LDL) oxidation was also evaluated at baseline and after 120 min Plasma glucose, insulin, triglycerides and LDL oxidation significantly increased, while the total plasma radical-trapping parameter significantly decreased during the meal test. Consumption of red wine in the fasting state significantly increased total plasma radical-trapping parameter activity, while wine ingestion with a meal counterbalanced the decrease of total plasma radical-trapping parameter and the increase of LDL oxidation. Meal consumption induced an increase in plasma prothrombin fragments 1 + 2 and activated factor VII in diabetic patients. Wine ingestion with the meal significantly reduced the production of both prothrombin fragments 1 + 2 and activated factor VII. Fasting consumption of red wine alone did not show effects on coagulation or LDL oxidation. CONCLUSION: This finding confirms that in the absorptive phase free radicals are produced in diabetic patients, which reduce serum antioxidant defences, increase LDL oxidation and activate the coagulation system. Red wine consumption during a meal significantly preserves plasma antioxidant defences and reduces both LDL oxidation and thrombotic activation. The consumption of a moderate amount of red wine during meals may have a beneficial effect in the prevention of cardiovascular disease in diabetic patients.

alpha 2A/alpha 2C-adrenergic receptor third loop chimera show that agonist interaction with receptor subtype backbone establishes G protein-coupled receptor kinase phosphorylation.

The alpha(2A)-adrenergic receptor (AR) undergoes rapid agonist-promoted desensitization due to phosphorylation by G protein-coupled receptor kinases (GRKs) 2 and 3 at serines in the third intracellular loop of the receptor. In contrast, the alpha(2C)AR fails to display such desensitization or phosphorylation, which has been presumed to be due to this receptor lacking GRK phosphorylation sites. However, the alpha(2C)AR has multiple serines and threonines in putative favorable motifs within its third intracellular loop. We considered that the conformation of the third intracellular loop imposed by agonists binding to the transmembrane-spanning domains could be the basis of this subtype-specific property, rather than the presence or absence of phosphoacceptors per se. To address this, alpha(2A)/alpha(2C) third loop chimeric receptors were constructed. In whole cell phosphorylation studies, the alpha(2A) with the alpha(2C) third loop receptor underwent agonist-promoted phosphorylation while the alpha(2C) with the alpha(2A) third loop receptor did not, indicating that the agonist interaction with the parent receptor backbone establishes the phosphorylation phenotype. We postulated then that agonists with diverse structures that distinctly interact with alpha(2)AR should display different degrees of phosphorylation independent of receptor activation. Indeed, several full and partial agonists were identified, which evoked phosphorylation that was not related to intrinsic activity as established by [(35)S]guanosine 5'-3-O-(thio)triphosphate binding. Taken together, it appears that phosphorylation of the alpha(2)AR evoked by agonist is highly sensitive to the conformation of the third intracellular loop induced/stabilized by agonist to such an extent that these properties dictate the extent of phosphorylation of the loop when phosphoacceptors are present, and are the basis for subtype-specific phosphorylation.

Meal-induced oxidative stress and low-density lipoprotein oxidation in diabetes: the possible role of hyperglycemia.

Oxidative stress and its contribution to low-density lipoprotein (LDL) oxidation have been implicated in the pathogenesis of vascular diabetic complications. However, the relationship between hyperglycemia, hyperinsulinemia, hyperlipidemia, and oxidative stress is still debated. If plasma glucose and/or insulin and/or lipid are some of the most important determinants of oxidative stress in diabetes, then their typical postprandial elevations in diabetes would be expected to favor oxidative stress and LDL oxidation. To test this hypothesis, in type 2 diabetic patients, we evaluated the effects of two different standard meals designed to produce different levels of postprandial hyperglycemia on the plasma oxidative status and LDL oxidation. The meals were administered in randomized order to each of 10 type 2 diabetic patients. Blood samples were collected at baseline and 60 and 120 minutes after the meals. In every sample, plasma levels of glucose, insulin, cholesterol, triglycerides, nonesterified fatty acids (NEFAs), malondialdehyde (MDA), and the total radical-trapping antioxidant parameter (TRAP) were measured. LDL susceptibility to oxidation was evaluated at baseline and after 120 minutes. Plasma glucose, insulin, triglycerides, and MDA increased and NEFAs and TRAP significantly decreased after either meal. The variations in plasma glucose, MDA, and TRAP were significantly greater and LDL was more susceptible to oxidation after the meal that produced a significantly higher degree of hyperglycemia. These results suggest that postprandial hyperglycemia may contribute to oxidative stress in diabetic patients, providing a mechanistic link between hyperglycemia and diabetic vascular disease.

Effect of intensive glycaemic control on fibrinogen plasma concentrations in patients with Type II diabetes mellitus. Relation with beta-fibrinogen genotype.

Recent studies show that in diabetic subjects an increase of plasma fibrinogen concentration is associated with a high risk of cardiovascular complications. Environmental and genetic factors contribute to the plasma fibrinogen concentration. Several studies indicate a relation between the polymorphism in the 5' region of the beta-fibrinogen gene and plasma protein concentrations and in diabetes the possible influence of hyperglycaemia on fibrinogen is still debated. In this study we investigated these relations. Hind III polymorphism was evaluated by a polymerase chain reaction-technique. On the basis of the observed allelic combination of fibrinogen beta-gene polymorphism and the existence of poor metabolic control (glycated haemoglobin > or = 7.5%), 50 Type II diabetic patients were selected. They were divided into three groups according to their beta-gene polymorphism (alpha1alpha1: n = 20, alpha1alpha2: n = 15, alpha2alpha2: n = 15) and then intensive insulin therapy was started. After 3 months of intensive treatment, the improvement in glycaemic control was equivalent, in terms of glycated haemoglobin, in all the three groups. A fibrinogen reduction was observed in alpha1alpha2 and alpha2alpha2 but not in alpha1alpha1 subjects. These results underline a possible relation between fibrinogen genotypes and glycaemic control in determining plasma fibrinogen concentrations in diabetic patients.

Agonist-mediated downregulation of G alpha i via the alpha 2-adrenergic receptor is targeted by receptor-Gi interaction and is independent of receptor signaling and regulation.

One mechanism of long-term agonist-promoted desensitization of alpha2AR function is downregulation of the cellular levels of the alpha subunit of the inhibitory G protein, Gi. In transfected CHO cells expressing the human alpha2AAR, a 40.1 +/- 3.3% downregulation of Galphai2 protein occurred after 24 h of exposure of the cells to epinephrine, which was not accompanied by a decrease in Galphai2 mRNA. The essential step that targets Gi for degradation by agonist occupancy of the receptor was explored using mutated alpha2AAR lacking specific structural or functional elements. These consisted of 5HT1A receptor and beta2AR sequences substituted at residues 113-149 of the second intracellular loop and 218-235 and 355-371 of the N- and C-terminal regions of the third intracellular loop (altered Gi and Gs coupling), deletion of Ser296-299 (absent GRK phosphorylation), and substitution of Cys442 (absent palmitoylation and receptor downregulation). Of these mutants, only those with diminished Gi coupling displayed a loss of agonist-promoted Gi downregulation, thus excluding Gs coupling and receptor downregulation, palmitoylation, and phosphorylation as necessary events. Furthermore, coupling-impaired receptors consisting of mutations in the second or third loops ablated Gi downregulation, suggesting that a discreet structural motif of the receptor is unlikely to represent a key element in the process. While pertussis toxin ablated Gi downregulation, blocking downstream intracellular consequences of alpha2AAR activation or mimicking these pathways by heterologous means failed to implicate cAMP/adenylyl cyclase, phospholipase C, phospholipase D, or MAP kinase pathways in alpha2AAR-mediated Gi downregulation. Taken together, agonist-promoted Gi downregulation requires physical alpha2AAR-Gi interaction which targets Gi for degradation in a manner that is independent of alpha2AAR trafficking, regulation, or second messengers.

Meal-generated oxidative stress in type 2 diabetic patients.

OBJECTIVE: Free radical production has been reported to be increased in diabetic patients and to be involved in the pathogenesis of diabetic complications. In this study, a standardized meal was administered to 10 type 2 diabetic patients and 10 healthy matched normal subjects to evaluate its effects on plasma oxidative stress generation. RESEARCH DESIGN AND METHODS: In diabetic patients, at baseline and after the meal, plasma malondialdehyde (MDA), vitamin C, protein SH groups, uric acid, vitamin E, and total plasma radical-trapping parameter, which evaluates plasma antioxidant capacity due to known and unknown antioxidants present in the plasma as well as their mutual cooperation, were measured. RESULTS: After the meal, plasma MDA and vitamin C increased, while protein SH groups, uric acid, vitamin E, and total plasma radical-trapping parameter decreased more significantly in the diabetic subjects than in control subjects. CONCLUSIONS: This finding shows that in the absorptive phase, free radicals are produced in diabetic patients. Since plasma glucose, but not insulin, rose significantly more in diabetic subjects than in control subjects, hyperglycemia may play an important role in the generation of postprandial oxidative stress in diabetic patients.

Phosphorylation and functional desensitization of the alpha2A-adrenergic receptor by protein kinase C.

We have investigated the potential for protein kinase C (PKC) to phosphorylate and desensitize the alpha2A-adrenergic receptor (alpha2AAR). In whole-cell phosphorylation studies, recombinantly expressed human alpha2AAR displayed an increase in phosphorylation after short-term exposure to 100 nM phorbol 12-myristate-13-acetate (PMA) that was blocked by preincubation with a PKC inhibitor. This increase in receptor phosphorylation over basal amounted to 172 +/- 40% in COS-7 cells and 201 +/- 40% in Chinese hamster ovary cells. In permanently transfected Chinese hamster fibroblast cells, PKC activation by brief exposure of the cells to PMA resulted in a marked desensitization of alpha2AAR function, amounting to a 68 +/- 4% decrease in the maximal agonist (UK14304)-stimulated intracellular calcium release. Such desensitization was blocked by the PKC inhibitor bisindolylmaleimide I and was not evoked by an inactive phorbol ester. The desensitization of this agonist response was not caused by PKC-mediated augmentation of G protein-coupled receptor kinase activity, because PMA-promoted desensitization of a mutated alpha2AAR that lacked G protein-coupled receptor kinase phosphorylation sites was identical to that of wild-type alpha2AAR. To test whether PKC phosphorylation is a mechanism by which alpha2AAR can be regulated by other receptors, the alpha1bAR was co-expressed with the alpha2AAR in Chinese hamster ovary cells. Upon selective activation of alpha1bAR, the function of alpha2AAR underwent a 53 +/- 5% desensitization. Thus, cellular events that result in PKC activation promote phosphorylation of the alpha2AAR and lead to substantial desensitization of receptor function. This heterologous regulation also represents a mechanism by which rapid crosstalk between the alpha2AAR and other receptors can occur.

Antioxidant defences are reduced during the oral glucose tolerance test in normal and non-insulin-dependent diabetic subjects.

BACKGROUND: Free radical production has been reported to be increased in patients with diabetes mellitus, and it has been suggested that hyperglycaemia may directly contribute to the generation of oxidative stress. The aim of the present study was to evaluate the effects of an acute increase in glycaemia on plasma antioxidant defences. RESULTS: During the oral glucose tolerance test (OGTT), plasma concentration of protein-bound sulphydryl (SH) groups, vitamin C, vitamin E and uric acid significantly decreased in normal as well as non-insulin-dependent diabetes mellitus (NIDDM) subjects. Total plasma radical-trapping activity, which evaluates plasma antioxidant capacity due to known and unknown antioxidants present in the plasma as well as their mutual co-operation, was also significantly reduced. CONCLUSION: This finding supports the hypothesis that hyperglycaemia may, even acutely, induce an oxidative stress.

Hyperglycemia-induced circulating ICAM-1 increase in diabetes mellitus: the possible role of oxidative stress.

ICAM-1 is one of the most important intercellular adhesion molecules involved in atherogenesis. Previous studies reported increased circulating ICAM-1 plasma levels in NIDDM patients with or without vascular complications. It has been suggested that an acute increase of plasma glucose may produce an oxidative stress in man, and in vitro studies have demonstrated that high glucose and free radicals induce cellular expression of ICAM-1. In this study, three different experiments were performed in nine NIDDM patients and in seven matched healthy controls: oral glucose tolerance test, antioxidant glutathione i.v. administration for two h, oral glucose tolerance test plus glutathione i.v. administration. Blood samples were drawn at -15 min and every 30 min from 0 to 180 min. During the oral glucose tolerance test, circulating ICAM-1 plasma levels significantly increased in both diabetic and normal subjects. Glutathione administration during the oral glucose tolerance test abolished this phenomenon. Glutathione administered alone significantly decreased circulating ICAM-1 plasma levels in diabetic patients, while no effect was observed in the normal subjects. These data suggest that hyperglycemia may induce an increase of circulating ICAM-1 plasma levels through an oxidative stress, and that the antioxidant glutathione counterbalances this effect. These data support the hypothesis of a causal relationship linking hyperglycemia, oxidative stress and atherogenesis in diabetes mellitus.

Total plasma antioxidant capacity predicts thrombosis-prone status in NIDDM patients.

OBJECTIVE: To explore the hypothesis that a relationship exists between free radical activity and abnormalities in hemostasis in NIDDM. RESEARCH DESIGN AND METHODS: The use of the total radical-trapping antioxidant parameter (TRAP) has very recently been proposed to explore the antioxidant property of a plasma and their mutual cooperation. In the present study, TRAP, vitamin E, vitamin C, vitamin A, uric acid, protein-bound SH (thiol) groups, fibrinogen, prothrombin fragments F1 + 2, and D-dimer have been evaluated in 46 NIDDM patients and 47 healthy matched control subjects. RESULTS: In NIDDM patients, TRAP, vitamin A, SH groups, and uric acid were significantly reduced, whereas the level of vitamin E was significantly increased. Vitamin C was similar in the two groups. Fibrinogen, prothrombin fragment 1 + 2, and D-dimer were increased in diabetic patients. TRAP, but no single other antioxidant, had a strong inverse association with fibrinogen, prothrombin fragment 1 + 2, and D-dimer. CONCLUSIONS: These findings are consistent with the hypothesis that oxidative stress may condition coagulation activation in diabetics. However, the data suggest that it is the total antioxidant capacity rather than any single plasma antioxidant that is the most relevant parameter.

Role of the amino terminus of the third intracellular loop in agonist-promoted downregulation of the alpha2A-adrenergic receptor.

A prominent feature of long-term regulation of the alpha2A-adrenergic receptor (alpha2AAR) is a loss of cellular receptors over time (downregulation). The molecular determinants of downregulation were sought by targeting regions of the receptor involved in G protein coupling and phosphorylation. Mutated receptors, consisting of chimeric substitutions of analogous beta2-adrenergic receptor (beta2AR) and serotonin 5-hydroxytryptamine1A (5-HT1A) receptor sequence into the second intracellular loop (ICL2) (residues 113-149), the amino terminus (residues 218-235) and carboxy terminus (residues 355-371) of ICL3, and a deletion of the beta-adrenergic receptor kinase (betaARK) phosphorylation sites in the third intracellular loop (ICL3) (residues 293-304), were expressed in Chinese hamster ovary (CHO) cells. Wild-type alpha2AAR underwent 31% +/- 3% downregulation after 24 h of exposure to 100 microM epinephrine. Loss of downregulation was observed with some mutants, but this was not related to functional coupling to inhibitory or stimulatory guanine nucleotide regulatory binding proteins (Gi or GS) or to phosphorylation. Rather, any mutant with a substitution of the amino terminus of ICL3 (regardless of whether the substitution was with beta2AR or 5-HT1A sequence) resulted in upregulation. Studies with an inhibitor of protein synthesis indicated that the primary mechanism of downregulation of the alpha2AAR is agonist-promoted degradation of receptor protein which requires a destabilization sequence in the amino terminus of ICL3. Thus, in contrast to other G protein-coupled receptors, in which G protein coupling or phosphorylation are critical for long-term agonist regulation, the alpha2AAR has a specific structural domain distinct from these other functional regions that serves to direct agonist-promoted downregulation.