Mary F. Walsh

Salt sensitivity in blacks. Salt intake and natriuretic substances.

Accumulating evidence suggests that hypertension in blacks is manifested in part by impaired renal excretion of salt. Consequently, this study was performed to determine if hypertensive and normotensive black subjects differ in their ability to generate known natriuretic substances. Fourteen normotensive and 11 hypertensive blacks were maintained on constant metabolic diets containing either 40 or 180 mmol of salt per day for 14 days each. During the last 4 days of each salt intake period, urine was collected for measurement of sodium, dopamine, and norepinephrine. On the last day of each 14-day dietary period, blood pressures were measured, blood was collected for measurement of plasma atrial natriuretic factor (ANF) and aldosterone, and urine was collected over 2 hours for measurement of prostaglandin E2 (PGE2). Both the normotensive and the hypertensive groups manifested salt sensitivity; then- mean arterial pressure rose by 7 ± 0.2 and 6 ± 0.2%, respectively, when salt intake was increased from 40 to 180 mmoJ/day. The hypertensive group exhibited decreased (p < 0.05) dopamme excretion as compared with the normotensive group for both dietary salt intakes. Plasma ANF levels increased (p < 0.05) in the hypertensive group, but not in the normotensive group, with increasing dietary salt. Plasma aldosterone and urinary norepinephrine and PGE2 were comparable in the two groups for both dietary salt Intakes. These data suggest that salt sensitivity is not unique to hypertensive blacks but occurs in normotensive blacks as well. Decreased renal production of dopamme may be a pathogenic factor in the development and maintenance of hypertension in blacks.

Altered platelet calcium metabolism as an early predictor of increased peripheral vascular resistance and preeclampsia in urban black women.

BACKGROUND Although preeclampsia is an important and relatively common medical problem, its pathophysiology remains unresolved and the search for a biochemical marker that precedes the hemodynamic abnormalities of preeclampsia continues. We designed a study to investigate the hemodynamic changes that characterize preeclampsia and to evaluate the metabolism of platelet intracellular calcium as a possible predictor of the development of preeclampsia. METHODS Hemodynamic measurements and spectrofluorometric determinations of the levels of intracellular calcium in platelets in the basal state and after stimulation with an agonist were performed in 48 nulliparous black women during each trimester of pregnancy. The data on the 14 women (29 percent) in whom preeclampsia developed were then compared with the data on the other 34, who served as normotensive controls. RESULTS There was no significant difference between the two groups in the basal levels of intracellular calcium at any time. In contrast, the levels measured after arginine vasopressin was administered during the first trimester indicated an exaggerated response in the group with preeclampsia as compared with the control group (1494 +/- 388 [+/- SEM] percent vs. 545 +/- 55 percent of base line; P less than 0.0002), which was sustained through the second and third trimesters. All but three of the women with preeclampsia had responses higher than the highest response among the controls. Platelet intracellular calcium responses to arginine vasopressin during the first trimester were a sensitive predictor of the subsequent development of preeclampsia (P less than 0.00009). Although vascular resistance was similar in the two groups during the first trimester, it subsequently decreased in the control group (P less than 0.02) but not in the group with preeclampsia. CONCLUSIONS Our findings indicate that preeclampsia is characterized by the absence of the normal pregnancy-related decrease in vascular resistance, which is preceded in most instances by an exaggerated response of platelet intracellular calcium to arginine vasopressin early in pregnancy. We therefore propose that an increase in the sensitivity of platelet calcium to arginine vasopressin can be used as an early predictor of subsequent preeclampsia.

Troglitazone Reduces Contraction by Inhibition of Vascular Smooth Muscle Cell Ca2+ Currents and Not Endothelial Nitric Oxide Production

The insulin-sensitizing compound troglitazone has evolved into a promising therapeutic agent for type II diabetes. It improves insulin sensitivity and lipoprotein metabolic profiles and lowers blood pressure in humans and rodents. Because troglitazone has insulinlike effects on a number of tissues, we hypothesized that it may reduce vascular tone through stimulation of endothelial-derived nitric oxide (NO) production or by diminution of vascular smooth muscle cell (VSMC) intracellular calcium ([Ca2+]i). Our results show that troglitazone decreases norepinephrine-induced contractile responses in the rat tail artery, an effect not reversed by the NO inhibitor L-nitroarginine methyl ester (L-NAME). In contrast, troglitazone significantly inhibited L-type Ca2+ currents in freshly dissociated rat tail artery and aortic VSMCs and in cultured VSMCs. The data suggest that troglitazone attenuates vascular contractility via a mechanism involving VSMC [Ca2+]i but independent from endothelial generation of NO. Because insulin has been shown to affect vascular tone by both of these mechanisms, troglitazone only partially mimics insulin action in this tissue.

Insulin like growth factor 1 increases vascular smooth muscle nitric oxide production.

Insulin like growth factor 1 (IGF-1) vasodilates, increases blood flow and lowers blood pressure; nitric oxide (NO) has been suggested to mediate some of these effects. We studied the role of IGF-1 in the regulation of NO production in vascular smooth muscle cells (VSMC). IGF-1 induced a concentration and time-dependent increase in NO release from endothelium-denuded aortic rings. Pre-incubation with cycloheximide or aminoguanidine blocked IGF-1-stimulated NO release. In addition, a six-fold increase in NO production by VSMC was seen upon incubation with IGF-1. These results suggest that IGF-1 induces NO release in VSMC through a process that involves new protein synthesis and the inducible isoform of nitric oxide synthase.

Impaired calcium metabolism associated with hypertension in Zucker obese rats

Recent data from our laboratory indicate that reduced membrane Ca-adenosine triphosphatase (ATPase) activity in non-insulin-dependent diabetics may be responsible for increases in intracellular calcium and, consequently, for elevated vascular resistance. Since obesity is frequently associated with hypertension, even before the development of overt diabetes, we evaluated blood pressure and erythrocyte cation levels and membrane Na/K-ATPase and Ca-ATPase in Zucker obese rats and their lean controls (n = 10 per group). Intra-arterial blood pressure, determined via a femoral cannula, demonstrated elevated systolic and diastolic pressure in the obese rats (P less than .05). There were no significant differences in Na/K-ATPase between groups, but there was a decrease in Ca-ATPase (P less than .01) in the obese rats and an increase in tissue and cellular calcium content (P less than .05). These data demonstrate a specific impairment in membrane Ca-ATPase activity in obese rats they may have caused the observed increase in cellular calcium and, consequently, increased blood pressure. These phenomena may result from impaired insulin activation of membrane Ca-ATPase in these insulin-resistant animals.

Calcium and Protein Kinase C Mediate High-Glucose-Induced Inhibition of Inducible Nitric Oxide Synthase in Vascular Smooth Muscle Cells

Abnormal vascular smooth muscle (VSMC) proliferation is a key feature in diabetes-associated atherosclerotic disease. Since nitric oxide inhibits VSMC tone, migration, adhesion, and proliferation, we examined the effects of high glucose on IL-1beta-induced NO release from VSMCs in culture. Confluent smooth muscle cells, preincubated with either 5 mmol/L (mM) or 20 mmol/L (mM) glucose for 48 hours, were stimulated with IL-1beta. Nitrite was measured in the culture medium after 24 hours. IL-1beta-induced a 15-fold increase in NO production in normal glucose medium. Glucose (10 to 30 mmol/L (mM)) significantly reduced the response to IL-1beta. High glucose (20 mmol/L (mM)) inhibited IL-1beta-evoked NO production by approximately 50%. IL-1beta-stimulated [3H] citrulline-forming activity of the nitric oxide synthase (NOS) was also significantly lower in high-glucose-exposed cells, and this was reflected in diminished cellular levels of NOS protein. To assess the role of protein kinase C (PKC), membrane PKC activity was measured, and glucose (20 mmol/L (mM)) significantly increased it. Immunoblotting of the membranes revealed a glucose-induced increase in the PKC betaII isoform. 1,2-Dioctanoyl-glycerol, a PKC activator, mimicked the high-glucose effect on IL-1beta-induced NO release, while staurosporine, a PKC inhibitor, reversed it. The role of calcium in the glucose-mediated inhibition of cytokine-induced NO release was determined by treatment with BAPTA, an intracellular chelator of calcium. BAPTA partially reversed the inhibitory effects of glucose. Increasing intracellular calcium by A23187, an ionophore or thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, significantly decreased IL-1beta-induced NO release and NOS expression. These results indicate that glucose-induced inhibition of IL-1beta-stimulated NO release and NOS expression may be mediated by PKC activation and increased intracellular calcium.

Absence of Adhesion Triggers Differential FAK and SAPKp38 Signals in SW620 Human Colon Cancer Cells that may Inhibit Adhesiveness and Lead to Cell Death

Background: Upon adhesion, anchorage-dependent cells transmit survival signals from the matrix into the cell. Loss of anchorage leads to anoikis. Resistance to anoikis may influence tumor progression and metastasis. To better understand the pathways that regulate the choice between adhesion and cell death, we examined FAK, c-Src andMAPKinase activities in SW620 human colon cancer cells. Methods: SW620 cell suspensions were first allowed to adhere to collagen I for 30 minutes and adherent cells were subsequently counted. FAK, p38, c-Src and ERK1/2 phosphorylation were assessed by Western blot in adherent cells and in cells prevented from adhesion by plating unto BSA-pacificated dishes. p38 and FAK were inhibited by SB203580 (20 µM) or by specific FAK antisense nucleotides or FAK siRNA, respectively, and adhesion quantitated. Apoptosis (anoikis) after lack of adhesion was measured colorimetrically in control cells and in cells treated with SB203580. Results: Adhesion to collagen I nearly doubled FAK phosphorylation at Y397, the autophosphorylation site, and decreased p38 activation by 60% (p<0.001) but did not affect FAK phosphorylation at Y576, the c-Src dependent site. Lowering FAK expression with FAK antisense decreased adhesion to collagen I; the larger decrease in FAK expression obtained with the siRNA (43 ± 2%) resulted in significantly greater inhibition of adhesion not only to collagen I but also to collagen IV and fibronectin. The p38 inhibitor diminished anoikis and enhanced adhesion. Interestingly, the SB compound also significantly inhibited FAK phosphorylation at Y397 (23 ± 3% in adherent, 30 ± 4% in non-adherent cells at 30 minutes and 35 ± 4% in adherent, 46 ± 14% in non-adherent cells after 6 hours, p<0.05 for each) and greatly enhanced phosphorylation of ERK1/2, a putative anti-apoptotic component of the MAPK cascade. Conclusions: In the absence of adhesion, SW620 cells exhibit increased p38 but decreased FAK activation, signals that may promote cell death. Our observations with the p38 inhibitor SB203580 indicate that inside-out signals, from p38 to FAK, may regulate both adhesion and anoikis in SW620 cells. In addition, the data suggest the presence of cross-talk between the pro-apoptotic p38 and anti-apoptotic ERK1/2 pathways.

Transforming Growth Factor-β Stimulates Intestinal Epithelial Focal Adhesion Kinase Synthesis via Smad- and p38-Dependent Mechanisms

Focal adhesion kinase (FAK) regulates cell migration, proliferation, and apoptosis. FAK protein is reduced at the edge of migrating gut epithelial sheets in vitro, but it has not been characterized in restitutive gut mucosa in vivo. Here we show that FAK and activated phospho-FAK (FAK(397)) immunoreactivity was lower in epithelial cells immediately adjacent to human gastric and colonic ulcers in vivo, but dramatically increased in epithelia near the ulcers, possibly reflecting stimulation by growth factors absent in vitro. Transforming growth factor (TGF)-beta, but not fibroblast growth factor, platelet-derived growth factor, or vascular endothelial growth factor, increased FAK levels in Caco-2 and IEC-6 cells. Epithelial immunoreactivity to TGF-beta and phospho-Smad3 was also higher near the ulcers, varying in parallel with FAK. The TGF-beta receptor antagonist SB431542 completely blocked TGF-beta-induced Smad2/3 and p38 activation in IEC-6 cells. SB431542, the p38 antagonist SB203580, and siRNA-mediated reduction of Smad2 and p38alpha prevented TGF-beta stimulation of both FAK transcription and translation (as measured via a FAK promoter-luciferase construct). FAK(397) levels were directly related to total FAK protein expression. Although gut epithelial motility is associated with direct inhibition of FAK protein adjacent to mucosal wounds, TGF-beta may increase FAK protein near but not bordering mucosal ulcers via Smad2/3 and p38 signals. Our results show that regulation of FAK expression may be as important as FAK phosphorylation in critically influencing gut epithelial cell migration after mucosal injury.

Insulin-Like Growth Factor-I Decreases Mean Blood Pressure and Selectively Increases Regional Blood Flow in Normal Rats

Abstract The insulin-like growth factor-I (IGF-I) and its receptors are widely distributed in peripheral vascular tissue, yet their role in the regulation of blood pressure and blood flow remains unknown. This study investigated the effect of IGF-I on blood pressure and selected regional blood flow in normal Wistar rats anesthetized with chloralose/urethane. The femoral artery was cannulated and used to monitor arterial blood pressure. Electromagnetic flow probes were placed around the left common iliac artery, left renal artery, and the superior mesenteric artery, and used to measure blood flow. IGF-I (2.6 μg, 5.1 or 10.3 nmol/animal iv) was injected as a bolus into the femoral vein. Following the injection of IGF-I (10.3 nmol), we observed a significant decrease of plasma glucose (57%) and a significant decrease of mean arterial pressure (MAP) that continued to decline throughout the 60-min experimental period. IGF-I (5.1 nmol) significantly decreased blood glucose by 44% and decreased the MAP by 14% with a nadir at 15 min and recovery after 60 min. A smaller dose of IGF-I (2.6 nmol) did not significantly decrease the blood glucose but resulted in a slight but significant decrease in MAP. The heart rate was increased by 10.3 and 5.1 but not 2.5 nmol of IGF-I. IGF-I (10.3 nmol) was associated with regional vascular responses with a preferential increase in flow of the iliac and superior mesenteric vessels, measured as vascular conductance. IGF-I (5.1 and 2.6 nmol) increased preferentially renal vascular conductance. Preinfusion with L-NAME, a nitric oxide inhibitor, inhibited the effects of IGF-I on flow. We conclude that IGF-I can selectively dilate vascular beds leading to a decrease in blood pressure and that the response to IGF-I is mediated by nitric oxide.

Extracellular pressure stimulates colon cancer cell proliferation via a mechanism requiring PKC and tyrosine kinase signals

Abstract.  Pressure in colonic tumours may increase during constipation, obstruction or peri‐operatively. Pressure enhances colonocyte adhesion by a c‐Src‐ and actin‐cytoskeleton‐dependent PKC‐independent pathway. We hypothesized that pressure activates mitogenic signals.