Mohammad Asghar

Oxidative Stress: A Potential Recipe For Anxiety, Hypertension and Insulin Resistance

We recently reported involvement of oxidative stress in anxiety-like behavior of rats. Others in separate studies have demonstrated a link between oxidative stress and hypertension as well as with type 2 diabetes/insulin resistance. In the present study, we have tested a putative role of oxidative stress in anxiety-like behavior, hypertension and insulin resistance using a rat model of oxidative stress. Oxidative stress in rats was produced by xanthine (0.1%; drinking water) and xanthine oxidase (5 U/kg; i.p.). X+XO-treated rats had increased plasma and urinary 8-isoprostane levels (a marker of oxidative stress) and increased malondialdehyde (MDA) levels in the hippocampus and amygdala as compared to control rats. Serum corticosterone (a systemic marker of stress and anxiety) levels also increased with X+XO treatment. Moreover, anxiety-like behavior measured via open-field and light-dark exploration behavior tests significantly increased in X+XO-treated rats. Mean arterial blood pressure measured in anesthetized rats increased in X+XO-treated compared to control rats. Furthermore, plasma insulin but not glucose levels together with homeostasis model assessment (HOMA), an index of insulin resistance, were higher in X+XO-treated rats. Our studies suggest that oxidative stress is a common factor that link anxiety-like behavior, hypertension and insulin resistance in rats.

Potential Contribution of Oxidative Stress and Inflammation to Anxiety and Hypertension

Previously, we have published that pharmacological induction of oxidative stress causes anxiety-like behavior in rats and also is associated with hypertension in these animals. Here, we report that sub-chronic induction of oxidative stress via pharmacological induction leads to i) reduction in glyoxalase (GLO)-1 and glutathione reductase (GSR)-1 expression; ii) calpain mediated reduction of brain derived neurotrophic factor (BDNF) levels; iii) NFκB mediated upregulation of proinflammatory factors interleukin (IL)-6 and tumor necrosis factor (TNF)-α and elevated angiotensin (AT)-1 receptor levels in hippocampus, amygdala and locus coeruleus regions of the brain. Acute oxidative stress has opposite effects. We speculate that regulation of GLO1, GSR1, BDNF, NFκB and AT-1 receptor may contribute to anxiety-like behavior and hypertension in rats.

Role of the angiotensin II AT2 receptor in inflammation and oxidative stress: opposing effects in lean and obese Zucker rats.

Inflammation and oxidative stress are believed to contribute to hypertension in obesity/diabetes. Recently, we reported a role for the AT(2) receptor in blood pressure control in obese Zucker rats. However, the role of AT(2) receptors in inflammation and oxidative stress in obesity is not known. Therefore, in the present study, we tested the effects of the AT(2) receptor agonist CGP-42112A on inflammation and oxidative stress in obese Zucker rats and compared them in their lean counterparts. Rats were systemically treated with either vehicle (control) or CGP-42112A (1 μg·kg(-1)·min(-1); osmotic pump) for 2 wk. Markers of inflammation (CRP, MCP-1, TNF-α, and IL-6) and oxidative stress (HO-1, gp-91(phox)) as well as an antioxidant (SOD) were determined. Control obese rats had higher plasma levels of CRP, MCP-1, TNF-α, IL-6, and HO-1 compared with control lean rats. Conversely, plasma SOD activity was lower in control obese than in control lean rats. Furthermore, the protein levels of TNF-α and gp-91(phox) were higher in the kidney cortex of control obese rats. Interestingly, CGP-42112A treatment in obese rats reduced the plasma and kidney cortex inflammatory (TNF-α, IL-6) and oxidative stress (gp-91(phox)) markers and increased plasma SOD activity to the levels seen in lean control rats. However, CGP-42112A treatment in lean rats increased inflammatory (TNF-α, IL-6) and oxidative stress (gp-91(phox)) markers in the plasma and kidney cortex. Our present studies suggest anti-inflammatory and antioxidative functions of AT(2) receptor in obese Zucker rats but proinflammatory and prooxidative functions in lean Zucker rats.

Grape Powder Supplementation Prevents Oxidative Stress–Induced Anxiety-Like Behavior, Memory Impairment, and High Blood Pressure in Rats

We examined whether or not grape powder treatment ameliorates oxidative stress-induced anxiety-like behavior, memory impairment, and hypertension in rats. Oxidative stress in Sprague-Dawley rats was produced by using L-buthionine-(S,R)-sulfoximine (BSO). Four groups of rats were used: 1) control (C; injected with vehicle and provided with tap water), 2) grape powder-treated (GP; injected with vehicle and provided for 3 wk with 15 g/L grape powder dissolved in tap water), 3) BSO-treated [injected with BSO (300 mg/kg body weight), i.p. for 7 d and provided with tap water], and 4) BSO plus grape powder-treated (GP+BSO; injected with BSO and provided with grape powder-treated tap water). Anxiety-like behavior was significantly greater in BSO rats compared with C or GP rats (P < 0.05). Grape powder attenuated BSO-induced anxiety-like behavior in GP+BSO rats. BSO rats made significantly more errors in both short- and long-term memory tests compared with C or GP rats (P < 0.05), which was prevented in GP+BSO rats. Systolic and diastolic blood pressure was significantly greater in BSO rats compared with C or GP rats (P < 0.05), whereas grape powder prevented high blood pressure in GP+BSO rats. Furthermore, brain extracellular signal-regulated kinase-1/2 (ERK-1/2) was activated (P < 0.05), whereas levels of glyoxalase-1 (GLO-1), glutathione reductase-1 (GSR-1), calcium/calmodulin-dependent protein kinase type IV (CAMK-IV), cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) were significantly less (P < 0.05) in BSO but not in GP+BSO rats compared with C or GP rats. We suggest that by regulating brain ERK-1/2, GLO-1, GSR-1, CAMK-IV, CREB, and BDNF levels, grape powder prevents oxidative stress-induced anxiety, memory impairment, and hypertension in rats.

Grape Powder Intake Prevents Ovariectomy-Induced Anxiety-Like Behavior, Memory Impairment and High Blood Pressure in Female Wistar Rats

Diminished estrogen influence at menopause is reported to be associated with cognitive decline, heightened anxiety and hypertension. While estrogen therapy is often prescribed to overcome these behavioral and physiological deficits, antioxidants which have been shown beneficial are gaining nutritional intervention and popularity. Therefore, in the present study, utilizing the antioxidant properties of grapes, we have examined effect of 3 weeks of grape powder (GP; 15 g/L dissolved in tap water) treatment on anxiety-like behavior, learning-memory impairment and high blood pressure in ovariectomized (OVX) rats. Four groups of female Wistar rats were used; sham control, sham-GP treated, OVX and OVX+GP treated. We observed a significant increase in systolic and diastolic blood pressure in OVX rats as compared to sham-controls. Furthermore, ovariectomy increased anxiety-like behavior and caused learning and memory impairment in rats as compared to sham-controls. Interestingly, providing grape powder treated water to OVX rats restored both systolic and diastolic blood pressure, decreased anxiety-like behavior and improved memory function. Moreover, OVX rats exhibited an impaired long term potentiation which was restored with grape powder treatment. Furthermore, ovariectomy increased oxidative stress in the brain, serum and urine, selectively decreasing antioxidant enzyme, glyoxalase-1 protein expression in the hippocampus but not in the cortex and amygdala of OVX rats, while grape powder treatment reversed these effects. Other antioxidant enzyme levels, including manganese superoxide dismutase (SOD) and Cu/Zn SOD remained unchanged. We suggest that grape powder by regulating oxidative stress mechanisms exerts its protective effect on blood pressure, learning-memory and anxiety-like behavior. Our study is the first to examine behavioral, biochemical, physiological and electrophysiological outcome of estrogen depletion in rats and to test protective role of grape powder, all in the same study.

Dopamine-Mediated Inhibition of Renal Na,K-ATPase Is Reduced by Insulin

Abstract— Recently we have reported that rosiglitazone treatment of obese Zucker rats reduced plasma insulin and restored the ability of dopamine to inhibit Na,K‐ATPase (NKA) in renal proximal tubules. The present study was performed to test the hypothesis that a chronic increase in levels of insulin causes a decrease in expression of the D1 receptor and its uncoupling from G proteins, which may account for the diminished inhibitory effect of dopamine on NKA in obese Zucker rats. We conducted experiments in primary proximal tubule epithelial cells obtained from Sprague‐Dawley rat kidneys. These cells at 80% to 90% confluence were pretreated with insulin (100 nmol/L for 24 hours) in growth factor–/serum‐free medium. SKF‐38393, a D1 receptor agonist, inhibited NKA activity in untreated cells, but the agonist failed to inhibit enzyme activity in insulin‐pretreated cells. Basal NKA activity was similar in untreated and insulin‐pretreated cells. Measurement of D1 receptors in the plasma membranes revealed that [3H]SCH‐23390 binding, a D1 receptor ligand, as well as D1 receptor protein abundance, was significantly reduced in insulin‐pretreated cells compared with untreated cells. SKF‐38393 (10 &mgr;mol/L) elicited significant stimulation of [35S]GTP[gamma]S binding in the membranes from control cells, suggesting that the D1 receptor–G protein coupling was intact. However, the stimulatory effect of SKF‐38393 was absent in membranes from insulin‐pretreated cells. We suggest that chronic exposure of cells to insulin causes both the reduction in D1 receptor abundance and its uncoupling from G proteins. These phenomena might account for the diminished inhibitory effect of dopamine on NKA activity in hyperinsulinemic rats.

Altered Functioning of Both Renal Dopamine D1 and Angiotensin II Type 1 Receptors Causes Hypertension in Old Rats

Activation of renal dopamine D1 (D1R) and angiotensin II type 1 receptors (AT1Rs) influences the activity of proximal tubular sodium transporter Na,K-ATPase and maintains sodium homeostasis and blood pressure. We reported recently that diminished D1R and exaggerated AT1R functions are associated with hypertension in old Fischer 344 × Brown Norway F1 (FBN) rats, and oxidative stress plays a central role in this phenomenon. Here we studied the mechanisms of age-associated increase in oxidative stress on diminished D1R and exaggerated AT1R functions in the renal proximal tubules of control and antioxidant Tempol-treated adult and old FBN rats. Although D1R numbers and D1R agonist SKF38393-mediated stimulation of [35S]-GTP&ggr;S binding (index of D1R activation) were lower, G protein–coupled receptor kinase 4 (kinase that uncouples D1R) levels were higher in old FBN rats. Tempol treatment restored D1R numbers and G protein coupling and reduced G protein–coupled receptor kinase 4 levels in old FBN rats. Angiotensin II–mediated stimulation of [35S]-GTP&ggr;S binding and Na,K-ATPase activity were higher in old FBN rats, which were also restored with Tempol treatment. We also measured renal AT1R function in adult and old Fischer 344 (F344) rats, which, despite exhibiting an age-related increase in oxidative stress and diminished renal D1R function, are normotensive. We found that diuretic and natriuretic responses to candesartan (indices of AT1R function) were similar in F344 rats, a likely explanation for the absence of age-associated hypertension in these rats. Perhaps, alterations in both D1R (diminished) and AT1R (exaggerated) functions are necessary for the development of age-associated hypertension, as seen in old FBN rats.

Oxidative stress alters renal D1 and AT1 receptor functions and increases blood pressure in old rats

Aging is associated with an increase in oxidative stress and blood pressure (BP). Renal dopamine D1 (D1R) and angiotensin II AT1 (AT1R) receptors maintain sodium homeostasis and BP. We hypothesized that age-associated increase in oxidative stress causes altered D1R and AT1R functions and high BP in aging. To test this, adult (3 mo) and old (21 mo) Fischer 344 × Brown Norway F1 rats were supplemented without/with antioxidant tempol followed by determining oxidative stress markers (urinary antioxidant capacity, proximal tubular NADPH-gp91phox, and plasma 8-isoprostane), D1R and AT1R functions, and BP. The D1R and AT1R functions were determined by measuring diuretic and natriuretic responses to D1R agonist (SKF-38393; 1 μg·kg(-1)·min(-1) iv) and AT1R antagonist (candesartan; 10 μg/kg iv), respectively. We found that the total urinary antioxidant capacity was lower in old rats, which increased with tempol treatment. In addition, tempol decreased the elevated NADPH-gp91phox and 8-isoprostane levels in old rats. Systolic, diastolic, and mean arterial BPs were higher in old rats and were reduced by tempol. Although SKF-38393 produced diuresis in both adult and old rats, urinary sodium excretion (UNaV) increased only in adult rats. While candesartan increased diuresis and UNaV in adult and old rats, the magnitude of response was greater in old rats. Tempol treatment in old rats reduced candesartan-induced increase in diuresis and UNaV. Our results demonstrate that diminished renal D1R and exaggerated AT1R functions are associated with high BP in old rats. Furthermore, oxidative stress may cause altered renal D1R and AT1R functions and high BP in old rats.

Activation of dopamine D1-like receptor causes phosphorylation of α1-subunit of Na+,K+-ATPase in rat renal proximal tubules

Abstract Dopamine causes inhibition of Na + ,K + -ATPase activity via activation of dopamine D 1 -like receptors. It is the phosphorylation of Serine 18 of the α 1 -subunit of Na + ,K + -ATPase which results in the inhibition of the enzyme activity; however, such a phosphorylation by dopamine D 1 -like receptor agonist has not been demonstrated in the proximal tubules. We show here by immunoprecipitation and detection with phosphoserine antibody that SKF 38393, a dopamine D 1 -like receptor agonist, causes phosphorylation of the α 1 -subunit of Na + ,K + -ATPase. The effect of (±)-1-phenyl-2,3,4,5-tetrahydro-(1 H )-3-benzazepine-7,8-diol hydrochloride, SKF 38393, is blocked by R (+)-7-choro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1 H -benzazepine hydrochloride, SCH 23390, a dopamine D 1 -like receptor antagonist, and staurosporin, a protein kinase C inhibitor. The phosphorylation is also increased by phorbol 12–13 dibutyrate ester. However, Rp-cAMP triethylamine, an inhibitor of protein kinase A, does not affect the SKF 38393-mediated phosphorylation of Na + ,K + -ATPase. Therefore, these results provide the evidence that dopamine D 1 -like receptor activation causes phosphorylation of the α 1 -subunit of Na + ,K + -ATPase in renal proximal tubules via protein kinase C pathway.

Potential Dopamine-1 Receptor Stimulation in Hypertension Management

The role of dopamine receptors in blood pressure regulation is well established. Genetic ablation of both dopamine D1-like receptor subtypes (D1, D5) and D2-like receptor subtypes (D2, D3, D4) results in a hypertensive phenotype in mice. This review focuses on the dopamine D1-like receptor subtypes D1 and D5 (especially D1 receptors), as they play a major role in regulating sodium homeostasis and blood pressure. Studies mostly describing the role of renal dopamine D1-like receptors are included, as the kidneys play a pivotal role in the maintenance of sodium homeostasis and the long-term regulation of blood pressure. We also attempt to describe the interaction between D1-like receptors and other proteins, especially angiotensin II type 1 and type 2 receptors, which are involved in the maintenance of sodium homeostasis and blood pressure. Finally, we discuss a new concept of renal D1 receptor regulation in hypertension that involves oxidative stress mechanisms.