Joseph A. Cameron

Gender Differences in the Renal Nitric Oxide (NO) System ☆: Dissociation Between Expression of Endothelial NO Synthase and Renal Hemodynamic Response to NO Synthase Inhibition

Many studies have shown that nitric oxide (NO) production is higher in the systemic vasculature of females than males and is stimulated during pregnancy, a high estrogen state. The present study was performed in rats to determine whether females had a greater expression of endothelial NO synthase (eNOS) in kidneys than did males; whether there were gender differences in the excretion of NO metabolites, nitrate/nitrite; and whether there were gender differences in the renal hemodynamic response to NO synthase inhibition. The renal levels of eNOS mRNA (as measured by ribonuclease protection assays) and protein (as measured by Western blot) were 80% higher in kidneys from females than from males (P < .001). Urinary excretion of NO metabolites, nitrate/nitrite, were not different between males and females. To inhibit eNOS, rats were treated with nitro-L-arginine methyl ester (L-NAME, 3 to 4 mg/kg/day) for 2 weeks. Although there were no differences in basal renal hemodynamics between males and females, when factored for kidney weight, chronic L-NAME increased renal vascular resistance by 130% in males but by only 60% in females, and decreased renal plasma flow by 40% in males but had no effect in females. These data show that although the renal levels of eNOS mRNA and protein are higher in females than in males, the renal vasculature of males is more responsive to NO synthase inhibition. The data suggest that the renal vasculature of males may be more dependent on NO than is the renal vasculature in females.

Original CommunicationsGender Differences in the Renal Nitric Oxide (NO) System: Dissociation Between Expression of Endothelial NO Synthase and Renal Hemodynamic Response to NO Synthase Inhibition☆

Many studies have shown that nitric oxide (NO) production is higher in the systemic vasculature of females than males and is stimulated during pregnancy, a high estrogen state. The present study was performed in rats to determine whether females had a greater expression of endothelial NO synthase (eNOS) in kidneys than did males; whether there were gender differences in the excretion of NO metabolites, nitrate/nitrite; and whether there were gender differences in the renal hemodynamic response to NO synthase inhibition. The renal levels of eNOS mRNA (as measured by ribonuclease protection assays) and protein (as measured by Western blot) were 80% higher in kidneys from females than from males (P < .001). Urinary excretion of NO metabolites, nitrate/nitrite, were not different between males and females. To inhibit eNOS, rats were treated with nitro-L-arginine methyl ester (L-NAME, 3 to 4 mg/kg/day) for 2 weeks. Although there were no differences in basal renal hemodynamics between males and females, when factored for kidney weight, chronic L-NAME increased renal vascular resistance by 130% in males but by only 60% in females, and decreased renal plasma flow by 40% in males but had no effect in females. These data show that although the renal levels of eNOS mRNA and protein are higher in females than in males, the renal vasculature of males is more responsive to NO synthase inhibition. The data suggest that the renal vasculature of males may be more dependent on NO than is the renal vasculature in females.

Inhibition of Ca2+ transport associated with cAMP-dependent protein phosphorylation in rat cardiac sarcoplasmic reticulum by triorganotins

Organotin compounds have been shown to interfere with cardiovascular system. We have studied the in vitro and in vivo effects of tributyltin bromide (TBT), triethyltin bromide (TET) and trimethyltin chloride (TMT) on the cardiac SR Ca2+ pump, as well as on protein phosphorylation of SR proteins, in order to understand the relative potency of these tin compounds. All the three tin compounds inhibited cardiac SR45Ca uptake and Ca2+-ATPase in vitro in a concentration-dependent manner. The order of potency for Ca2+-ATPase as determined by IC50, is TBT (2 μM) > TET (63 μM) > TMT (280 μM). For45Ca uptake, it followed the same order i.e., TBT (0.35 μM) > TET (10 μM) > TMT (440 μM). In agreement with the in vitro results, both SR Ca2+-ATPase and45Ca uptake were significantly inhibited in rats treated with these tin compounds, indicating that these tin compounds inhibit cardiac SR Ca2+ transport. cAMP significantly elevated (70–80%) the32P-binding to SR proteins in vitro in the absence of any organotin. In the presence of organotins, cAMP-stimulated32P-binding to proteins was significantly reduced, but the decrease was concentration dependent only at lower concentrations. The order of potency is TBT > TET > TMT. In agreement with in vitro studies, cAMP-dependent32P bound to proteins was significantly reduced in rats treated with TBT, TET and TMT. SDS-polyacrylamide gel electrophoresis of the cardiac SR revealed at least 30 Coomassie blue stainable bands ranging from 9 to 120 kDa. Autoradiographs from samples incubated in the presence of cAMP indicated32P incorporation in seven bands. Of these, the band corresponding to about 24 kDa molecular weight protein decreased in its intensity with the treatment of organotins. These results suggest that triorganotins may be affecting Ca2+ pumping mechanisms through the alteration of phosphorylation of specific proteins in rat cardiac SR.

Phencyclidine block of Ca2+ ATPase in rat heart sarcoplasmic reticulum

Phencyclidine hydrochloride (PCP) also known as Angel Dust is a very potent psychotomimetic drug of abuse. Besides its central nervous system (CNS) effects PCP produces a number of adverse effects in a variety of tissues including the cardiovascular system. Since PCP is known to alter the cellular calcium homeostasis the present studies were initiated to determine the changes in cardiac Ca2+ ATPase activity in rats treated with PCP. For in vitro studies the cardiac sarcoplasmic reticulum (SR) fractions prepared from normal rats were incubated with 25, 50 and 100 microM PCP and the enzyme activities were estimated. Whereas, for in vivo studies the cardiac SR fractions prepared from rats treated with PCP (10 mg/kg body wt. single dose, intra-peritoneally (i.p.)) and sacrificed at different time intervals were used. PCP reduced the Ca2+ ATPase activity significantly both in vitro and in vivo. A 50% inhibition of the enzyme activity was obtained with 100 microM PCP in vitro. A significant reduction of SR Ca2+ ATPase was also evident as early as 1 h after treatment of rats with PCP. The reduction of Ca2+ ATPase activity in SR was irreversible even at 12 h after treatment. The in vitro kinetic studies revealed that PCP was found to be a competitive inhibitor of Ca2+ ATPase with respect to the substrate, ATP, and non-competitive with respect to Ca2+ activation. These results indicate that PCP alters the myocardial Ca2+ homeostasis by inhibiting the Ca2+ ATPase in cardiac SR in rats. Inhibition of SR Ca2+ ATPase may result in the impairment of contraction and relaxation coupling processes in the myocardium.

Dietary calcium supplementation restores pressure natriuresis responses in Dahl-S rats

Calcium supplementation prevents hypertension in Dahl S (DS) rats. Because abnormal pressure natriuresis may contribute to the development of hypertension, we examined the effect of calcium on pressure natriuresis. DS and Dahl R (DR) rats maintained on a 4% sodium diet containing either 0.5% or 2% calcium for 4 weeks were anesthetized; sodium excretion, renal blood flow, and inulin clearance were determined at perfusion pressures of 100, 125, and 156 mm Hg. Inulin clearance and renal blood flow were not different between groups. Sodium excretion increased with increasing renal perfusion pressure in all groups. The slope of the line relating renal perfusion pressure to sodium excretion was greater (P < .05) in DR rats than in DS rats on normal calcium intakes. High calcium intake normalized the slope of the line relating renal perfusion pressure to sodium excretion in DS rats, but had no effect on DR rats. Thus, dietary calcium supplementation normalizes the blunted pressure natriuresis response in the DS rat and may contribute to the prevention of hypertension.

Inhibition of calcium ATPase by phencyclidine in rat brain.

Phencyclidine (PCP) is a potent psychotomimetic drug of abuse and has profound effect on the functioning of the central nervous system (CNS). Many of the CNS functions are known to be mediated by calcium (Ca2+). In the present study we have investigated the effects of PCP on Ca2+ ATPase activity in rat brain both in vitro and in vivo. For in vitro studies, synaptic membrane fractions prepared from normal rat brain were incubated with PCP at different concentrations (25-100 μM) before the addition of substrate. For n vivo studies, rats were treated with a single moderate dose of PCP (10 mg/kg, IP) and animals were sacrificed at 1,2, 6 and 12 h after treatment. Ca2+ ATPase activity in synaptic membrane fractions was assayed by estimation of inorganic phosphate. PCP inhibited the Ca2+ ATPase in vitro in a concentration dependent manner with significant effect at 50 and 100 μM. A significant time-dependent reduction of the Ca2+ ATPase activity was evident in vivo. As early as 2 h after the treatment of rats with PCP the ATPase activity was significantly reduced. The reduction of Ca2+ ATPase observed even at 12 h after treatment suggesting a prolonged presence of the drug in the brain tissue. Further, kinetic studies in vitro indicated PCP to be a competitive inhibitor of Ca2+ ATPase with respect to the substrate, ATP. The present findings indicate that PCP inhibits synaptic membrane Ca2+ ATPase thus altering cellular Ca2+ homeostasis in CNS which may partially explain the pharmacological effects of the drug and/or its neurotoxicity.

In Vitro and In Vivo Inhibition of Rat Brain Nitric Oxide Synthase Activity by Phencyclidine

Phencyclidine (PCP) is a widely abused psychoactive drug that perturbs many neurotransmitter systems studied to date. Nitric oxide (NO) has been established as a neuronal messenger and its rapid diffusibility across cell membranes makes NO an extensive and versatile messenger in brain development and functioning. The present study was initiated to investigate the effect of PCP on rat brain nitric oxide synthase (NOS) activity both in vitro and in vivo. Brain cytosolic fractions from normal rats were used for in vitro and in vivo studies. The rats were treated with a single dose of PCP (10 mg/kg, intraperitoneally); the brains were removed at 0, 1, 2, 6, and 12 hours after PCP treatment and the cytosolic fractions were prepared by homogenization and centrifugation. NOS activity was assessed by quantifying the release of [3H]-citrulline from [3H]-arginine. PCP significantly inhibited rat brain NOS in vitro in a concentration (0.05–2 mM)-dependent manner. The kinetic evaluation of arginine, NADPH, and Ca2+ activation of NOS revealed that PCP (0.5 mM) inhibited NOS activity competitively with respect to arginine and NADPH and noncompetitively inhibited with respect to Ca2+. PCP also caused a time-dependent reduction of brain NOS activity in vivo as early as 1 hour after treatment. Even after 12 hours of PCP treatment, NOS activity did not reverse to its normal level as compared to the control group, suggesting sequestration and persistence of the drug in the central nervous system. These results suggest that inhibition of brain NOS by PCP might be one of the mechanisms through which PCP causes neurotoxicity.

The Effect of Glucocorticoids and LPS on the Functional Activity of RAW Cell Line

It is well documented that glucocorticoids are potent anti-inflammatory and immunosuppressive agents that are known to affect cell mediated inflammation by the inhibition of cellular proliferation and cytokine production. The literature is lacking knowledge in elucidating the mode of action of such agents on the transformed like cells in culture. Therefore, RAW (macrophage like) cells was selected as a model to determine the effects of cortisol administration or cortisol in the presence of LPS on the cells metabolic functions. Cells were treated with physiological concentrations of cortisol or cortisol + LPS for periods of 24, 48 and 72 hours. After each phase, cell numbers, cellular damage and cellular morphology were determined. The results indicated cortisol and cortisol + LPS treated cells inhibited cellular proliferation as well as increased cellular MDA levels as early as 24 hours. Overall, the results indicate that cortisol has a remarkable effect on RAW cellular proliferation similar to the reduction seen in our previous findings using HEP-2 cells. In addition to reduction in cellular number the cells ability to adjust to a bacterial challenge may be directly altered. These results provided important information for patients who are immunosuppressed or chronically exposed to stressful conditions.

Abstract 3369: Differential biotherapeutic advantages of honey in targeting the Warburg effect and survival of MRC-5 and A549 cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Lung cancer is a one of the most prevalent and deadly cancers in United States. Experimental evidence support that cancer cells do exhibit higher glycolytic rates than normal cells (Warburg effect). To exploit this unique cancer-dependent ATP generation phenomenon, we hypothesize that exposure of cancer cells to organic inhibitors of glycolysis would negatively impact their survival and alter their growth and viability resulting from the vast decrease in their essential glycolytic ATP production; no negative consequences will be seen on normal lung cells. The human lung fibroblast cell line MRC-5 and the human alveolar epithelial cell line A549 were used as models for normal lung and lung cancer in vitro in this study. Using standard methods, both cell lines were maintained and exposed to honey and D-glucose reagents at concentration levels ranging from 31.3-2,000 µg/ml in 96 well plates in quadruplets and experiments repeated at least three times using MTT, and cell counting (T4 Cellometer) assays as well as phase-contrast photo-imaging. Our results indicate that exposure of both cell lines to these natural nutraceutic organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Honey and D-glucose showed statistically significant (p<0.05) differential negative effects on the A549 line in comparison to its unexposed control as well as to their effects on the MRC-5 cell line. Results show a promising role of honey and D-glucose as metabolites of interest for selective management of cancerous cells. Citation Format: Ibrahim O. Farah, Veshell L. Lewis, Wellington K. Ayensu, Joseph A. Cameron. Differential biotherapeutic advantages of honey in targeting the Warburg effect and survival of MRC-5 and A549 cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3369. doi:10.1158/1538-7445.AM2014-3369