Aruna Natarajan

Lipid Rafts Keep NADPH Oxidase in the Inactive State in Human Renal Proximal Tubule Cells

Recent studies have indicated the importance of cholesterol-rich membrane lipid rafts (LRs) in oxidative stress-induced signal transduction. Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases, the major sources of reactive oxygen species, are implicated in cardiovascular diseases, including hypertension. We tested the hypothesis that NADPH oxidase subunits and activity are regulated by LRs in human renal proximal tubule cells. We report that a high proportion of p22phox and the small GTPase Rac1 are expressed in LRs in human renal proximal tubule cells. The D1-like receptor agonist, fenoldopam (1 &mgr;mol/L per 20 minutes) dispersed Nox subunits within LRs and non-LRs and decreased oxidase activity (30.7±3.3%). In contrast, cholesterol depletion (2% methyl-β-cyclodextrin [βCD]) translocated NADPH oxidase subunits out of LRs and increased oxidase activity (154.0±10.5% versus control, 103.1±3.4%), which was reversed by cholesterol repletion (118.9±9.9%). Moreover, NADPH oxidase activation by βCD (145.5±9.0%; control: 98.6±1.6%) was also abrogated by the NADPH oxidase inhibitors apocynin (100.4±3.2%) and diphenylene iodonium (9.5±3.3%). Furthermore, βCD-induced reactive oxygen species production was reversed by knocking down either Nox2 (81.0±5.1% versus βCD: 162.0±2.0%) or Nox4 (108.0±10.8% versus βCD: 152.0±9.8%). We have demonstrated for the first time that disruption of LRs results in NADPH oxidase activation that is abolished by antioxidants and silencing of Nox2 or Nox4. Therefore, in human renal proximal tubule cells, LRs maintain NADPH oxidase in an inactive state.

The D5 Dopamine Receptor Mediates Large-Conductance, Calcium- and Voltage-Activated Potassium Channel Activation in Human Coronary Artery Smooth Muscle Cells

Large-conductance, calcium- and voltage-activated potassium (BKCa) channels hyperpolarize coronary artery smooth muscle cells, causing vasorelaxation. Dopamine activates BKCa channels by stimulating D1-like receptor-mediated increases in cAMP in porcine coronary artery myocytes. There are two D1-like receptors (R), D1R and D5R. We hypothesize that the specific D1-like receptor involved in BKCa channel activation in human coronary artery smooth muscle cells (HCASMCs) is the D5R and that activation occurs via cAMP cross-activation of cGMP-dependent protein kinase (PKG), rather than cAMP-dependent protein kinase (PKA). The effects of D1-like receptor agonists and antagonists on BKCa channel opening in HCASMCs were examined in the presence and absence of PKG/PKA inhibition by cell-attached patch clamp. In the absence of commercially available ligands specific for D1R or D5R, D1R or D5R protein was down-regulated by transfecting HCASMCs with human D1R or D5R antisense oligonucleotides, respectively: cells transfected with scrambled oligonucleotides and nontransfected HCASMCs served as controls. The predominant ion channel conducting outward currents in nontransfected HCASMCs was identified as the large-conductance, calcium- and voltage-activated potassium (BKCa) channel, which was activated by D1-like receptor agonists despite PKA inhibition with (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid (KT 5720) (300 nM), but was abolished by inhibiting PKG with 9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b-11a-triazadibenzo(a,g) cycloocta(cde)-trinden-1-one (KT 5823) (300 nM). D1-like receptor agonists activated BKCa channels in all transfected cells except those transfected with D5R antisense oligonucleotides. Thus, the dopamine (D1-like) receptor mediates activation of BKCa channels in HCASMCs by D5R, not D1R, and via PKG, not PKA. This is the first report of differential D1-like receptor regulation of vascular smooth muscle function in human cells.

The Renin-Angiotensin and Renal Dopaminergic Systems Interact in Normotensive Humans

The renin-angiotensin-aldosterone (RAAS) and renal dopaminergic systems interact to maintain sodium balance. High NaCl intake increases renal synthesis of dopamine and dopaminergic receptor activity, decreasing epithelial sodium transport, whereas sodium deficit activates the RAAS, increasing epithelial sodium transport. We tested the hypothesis that attenuation of the natriuretic effect of dopamine D1-like receptors during salt restriction results in part from increased RAAS activity in seven salt-resistant normotensive adults using a double-blind placebo-controlled balanced crossover design. All subjects attained sodium balance on low (50 mmol Na(+)/day) and high (300 mmol Na(+)/day) NaCl diets, administered 4 weeks apart. Sodium, potassium, lithium, para-aminohippurate, and creatinine clearances were measured before, during, and after a 3-hour infusion of fenoldopam, a D1-like receptor agonist, with and without pretreatment with enalapril, an angiotensin converting enzyme inhibitor. On the high NaCl diet, fenoldopam-induced natriuresis was associated with the inhibition of renal proximal and distal tubule sodium transport. On the low NaCl diet, fenoldopam decreased renal distal tubule sodium transport but did not cause natriuresis. The addition of enalapril to fenoldopam restored the natriuretic effect of fenoldopam and its inhibitory effect on proximal tubule sodium transport. Thus, on a high NaCl diet fenoldopam causes natriuresis by inhibiting renal proximal and distal tubule transport, but on a low NaCl diet the increased RAAS activity prevents the D1-like receptor from inhibiting renal proximal tubule sodium transport, neutralizing the natriuretic effect of fenoldopam. These results demonstrate an interaction between the renin-angiotensin and renal dopaminergic systems in humans and highlight the influence of dietary NaCl on these interactions.

Cardiovascular and Autonomic Influences on Blood Pressure

The cardiovascular system provides appropriate organ and tissue perfusion at rest and at times of stress by regulation of blood pressure (BP). The arterial pressure level at any given time reflects the composite activities of the heart and the peripheral circulation.

Salt intake influences the interaction between the renin-angiotensin and renal dopaminergic systems in normotensive humans

D discovery is a complex process which involves an interdisciplinary approach to design effective feasible drugs (1). The development of new drugs with potential therapeutic applications is one of the most complex process in the pharmaceutical industry (2). Millions of dollars and man hours are dedicated to the discovery of new therapeutic agents. Rational drug discovery process combat and supersede the conventional process with the advent of proteomic, genomic and structural information (3).U in conjunction with other technologies the light beam becomes a uniquely powerful tool to study cells. Currently light source is used not only to observe cells, but also to stimulate cellular chemistry. This area of biophotonics is called “caged compounds” as synthetic organic chemistry say “Photolabile compounds” is used to make biologically signalling molecules functionally inert. Irradiation with light liberates the caged molecules, thus “switching on” a chosen signaling pathway. Such light-triggered release of molecules from “caged” forms also offers the potential to deliver innocuous agents to cells, tissues, and organisms, where they can be unmasked to their active states. Because light can be readily tuned and focused, it can be spatially and temporally controlled to provide “on-command” drug delivery, unmasking of biochemical agents for enzyme and protein activation, and other biochemical and physiological studies.T effects of artemisinin derivatives, artemether and lumefantrine on the activity of glucose 6-phosphate dehydrogenase (G6PD) and some haematological parameters in rats (Rattus novergicus) were studied. The experimental animals were randomly distributed into four groups: those administered Tween 80 (control), those administered artemether (8 mg/kg body weight), those administered lumefantrine (48 mg/kg body weight) and those co-administered artemether (8 mg/kg body weight) and lumefantrine (48 mg/kg body weight). The drugs were orally administered twice daily for three days to half of the animals while the remaining half were dosed for six days. Animals were subsequently anaesthetized in diethyl ether, blood samples were collected by cardiac puncture and the organs were excised and weighed. The following parameters were assessed in blood and liver homogenate: glucose, G6PDH, packed cell volume (PCV), haemoglobin (HB), white blood cell count (WBC), neutrophil, leukocyte, eosinophil, monocyte and basophil. After 3 days of administration, results showed significant decreases (p 0.05) in plasma and homogenate glucose, G6PD activity and haematological parameters. It could be concluded that the administration of artemether and lumefantrine after 3 days to non-malaria infected rats showed hypoglycaemia and reduced haematological indices, while increasing G6PD activity.T study is to reveal whether artificial hemolyzed blood samples obtained during clinical operation affect the pharmacokinetic (PK) profile and bioequivalence (BE) study results. A validated LC-MS/MS method was applied to analyze both hemolyzed and non-hemolyzed plasma samples derived from healthy volunteers to whom clopidogrel, methylprednisolone and ropinirole were administrated orally in three different pilot BE studies conducted in the clinical research center. During the first period of BE studies, for drug concentrations of hemolyzed and non-hemolyzed plasma samples, clopidogrel were 862.57±860.16 (pg/mL) and 920.61±959.14 (pg/mL) at 1 hour post dosing; methylprednisolone were 155.21±33.60 (pg/mL) and 160.01±29.9 (pg/mL) at 2.5 hours post dosing; ropinirole were 1322.87±392.96 (pg/mL) and 1151.42±299.91 (pg/mL) at 4 hours post dosing. During the second period, the values are 895.61±590.47 (pg/mL) and 941.60±601.91 (pg/mL) for clopidogrel; 160.01±29.99 (pg/mL) and 127.40±41.61 (pg/mL) for methylprednisolone; 1146.30±249.89 (pg/mL) and 1220.01±196.67 (pg/mL) for ropinirole. The drug concentration between hemolyzed and non-hemolyzed plasma samples did not yield a significant difference (p>0.05). In conclusion, although hemolysis may physically change the characteristics of the plasma samples, it doesn’t significantly affet the accuracy of PK profile of clopidogrel, methylprednisolone and ropinirole. However, hemolysis should always be avoided in the practice of clinical studies to get ideal plasma for analysis.Free radical scavenging activity of the Malaysian Leech Saliva extract, Hirudinaria manillensis Abbas Mohammad Ghawi1, Abdualrahman M.Abdualkader2, Ahmed Merzouk3 and Mohamed Alaama2 Basic Medical Science Department, Faculty of Pharmacy, International Islamic University Malaysia, Malaysia. Pharmaceutical Chemistry Department, Faculty of Pharmacy, International Islamic University Malaysia, Malaysia BIOPEP SOLUTIONS INC., Vancouver, BC CanadaTrypanosomatids cause deadly diseases in humans. Of the various biochemical pathways in trypanosomatids, glycolysis, has received special attention because of being sequestered in peroxisome like organelles critical for the survival of the parasites. This study focuses on phosphoglycerate kinase (PGK) from Leishmania spp. which, exists in two isoforms, the cytoplasmic PGKB and glycosomal PGKC differing in their biochemical properties. Computational analysis predicted the likelihood of a transmembrane helix only in the glycosomal isoform PGKC, of approximate length 20 residues in the 62-residue extension, ending at, arginine residues R471 and R472. From experimental studies using circular dichroism and NMR with deuterated sodium dodecyl sulfate, we find that the transmembrane helix spans residues 448 +/- 2 to 476 in Leishmania mexicana PGKC. The significance of this observation is discussed in the context of glycosomal transport and substrate tunneling