Mark T. Hori

Long-term fructose feeding impairs vascular relaxation in rat mesenteric arteries

To investigate the long-term influence of insulin resistance and hyperinsulinemia on vascular reactivity, both muscarinic and alpha2-receptor-mediated relaxations and the contribution of nitric oxide to these mechanisms were studied in the fructose-fed rat. Male Sprague-Dawley rats were fed either fructose-rich chow (FFR, n = 6) or normal chow (CNT, n = 6) for 40 weeks. Systolic blood pressure was measured by tail-cuff method. A 3-mm segment of mesenteric artery was excised, cannulated and pressurized, pretreated with prazosin (10(-6) mol/L) and propranolol (3 x 10(-6) mol/L), then precontracted with serotonin (10(-6) mol/L). Endothelium dependent relaxation was induced by addition of acetylcholine (10(-9) to 10(-4) mol/L), or a selective alpha2-agonist B-HT 920 (10(-9) to 10(-5) mol/L), with or without the nitric oxide synthase inhibitor L-NAME (10(-4) mol/L). Systolic blood pressure was significantly higher in FFR at the early period; however, there was no difference at the end of 40 weeks compared to CNT. Fasting plasma insulin was much higher in FFR than in CNT (110+/-62 v 41+/-11 microU/mL, P < .05), whereas plasma glucose was not different. Maximum relaxation to acetylcholine was attained at 10(-6) mol/L in FFR but at 3 x 10(-7) mol/L in CNT. The degree of maximum relaxation attained with acetylcholine was similar in FFR and CNT (89+/-9 and 94+/-4% of precontraction), although attenuated (P < .01) by the addition of L-NAME only in FFR (to 34+/-22%, P < .05) but not in CNT (to 82+/-25%). The half-maximal relaxation dose of acetylcholine was greater in FFR (P < .01) compared with CNT and was significantly increased (P < .05) by L-NAME in both groups. B-HT 920 at 10(-5) mol/L induced a greater relaxation in CNT (36+/-10% of serotonin constriction) than in FFR (19+/-14%, P < .05). These responses were significantly blunted by L-NAME. Thus, muscarinic receptor-mediated vascular relaxation is less sensitive and more nitric oxide dependent in FFR versus CNT. Alpha2-adrenergic-mediated relaxation, predominantly mediated by nitric oxide, is also impaired in FFR. It is possible that prolonged insulin resistance and hyperinsulinemia in FFR could alter endothelial-dependent vasodilatory mechanisms, thereby contributing to the increase in blood pressure seen in this model.

Elevated 12-lipoxygenase activity in the spontaneously hypertensive rat.

We have previously demonstrated that administration of inhibitors of the lipoxygenase (LO) pathway of arachidonic acid metabolism lowers blood pressure in hypertensive rats. In addition, we have shown that LO inhibition attenuates pressor agonist-induced vascular reactivity in vitro and calcium mobilization in cultured vascular smooth muscle cells (VSMC). To further elucidate the relationship between elevated LO activity and hypertension, 4, 8, and 12 week old hypertensive SHR were compared with age-matched Wistar-Kyoto (WKY) rats for plasma 12(S)-hydroxyeicosatetraenoic acid (12-HETE) concentration. 12-HETE levels were significantly elevated in the SHR compared to the WKY (SHR elevated by 154%, 159%, and 272% compared to WKY at 4, 8, and 12 weeks, respectively, P < .01 for all ages). There were no differences in plasma potassium levels between SHR and WKY at any of the ages tested. Plasma aldosterone levels and plasma renin activity were in the normal range at the three ages. At 12 weeks of age, both serum (4.72 +/- 0.23 v 2.18 +/- 0.33 microg/mL, P < .01), and aortic smooth muscle 12-HETE levels (0.94 +/- 0.09 v 0.66 +/- 0.08 microg/mg protein, P < .05) were elevated in SHR compared with WKY. The 12 week old SHR were given a bolus of the LO inhibitor 5,8,11-eicosatriynoic acid (ETI, 7 mg/kg, intravenously) and blood pressure measured after 20 min. ETI reduced mean systolic blood pressure from 175.8 +/- 4.2 to 141.6 +/- 5.9 mm Hg (P < .05). To investigate these effects of HETEs, cultured vascular smooth muscle cells were pretreated for 1 min with 12(S)HETE and then challenged with angiotensin II (AngII). The addition of 12(S)HETE increased AngII-induced intracellular calcium levels in normal cultured rat vascular smooth muscle cells by 78% compared to vehicle (P < .05). Thus, LO products, which are high in SHR, may contribute to vascular tone through alterations in the intracellular calcium signal by potentiating calcium responses to pressors such as Ang II.

Insulin-Mediated Growth in Aortic Smooth Muscle and the Vascular Renin-Angiotensin System

Insulin has been shown to directly affect blood vessel tone and to promote vascular hypertrophy, but the mechanism of these actions remains uncertain. Because angiotensin I (Ang I)-converting enzyme inhibitors have been shown to improve insulin action and to impede the progression of vascular hypertrophy in hypertensive animal models, it is possible that the vascular properties of insulin may be mediated through the tissue renin-angiotensin system (RAS). To evaluate this relationship, we first investigated the effect of insulin on components of the RAS using cultured rat vascular smooth muscle cells (VSMCs). Insulin treatment (1000 microU/mL) markedly increased angiotensinogen mRNA expression and angiotensinogen production. We next investigated the role of the RAS in insulin-mediated cell proliferation, using [3H]thymidine uptake. Studies were done both with insulin alone and in the presence of captopril (1x10(-7) to 10(-5) mol/L) and losartan (1x10(-9) to 10(-7) mol/L). [3H]Thymidine uptake was increased significantly by 1000 microU/mL insulin, and this stimulation was reduced by 1x10(-6) mol/L captopril (-38.8%, P<0.05) and by 1x10(-8) mol/L losartan (-37. 5%, P<0.05). Further studies showed that the degree of insulin-mediated [3H]thymidine uptake in VSMCs could be duplicated by 4x10(-10) mol/L Ang II. Losartan reduced the effects of both Ang II and insulin on [3H]thymidine uptake by about 40% to 45% of baseline (P<0.05). Captopril reduced insulin-mediated [3H]thymidine uptake but did not affect Ang II-mediated [3H]thymidine uptake. In summary, insulin induced significant stimulation of angiotensinogen expression and production and stimulated growth similar to that seen with Ang II in cultured rat VSMCs. Inhibition of Ang II production or its binding to the Ang II type 1 (AT1) receptor inhibited insulin-mediated growth in a fashion similar to that seen with inhibition of Ang II-mediated growth. Thus, insulin can modulate the vascular RAS, and the effect of insulin on vascular growth may be via direct effects on angiotensinogen expression and translation operative through both the AT1 receptor and the conversion of Ang I to Ang II.

Insulin and insulin-like growth factor-I promotes angiotensinogen production and growth in vascular smooth muscle cells.

Background Circulating insulin and insulin-like growth factor-I (IGF-I) levels are increased in patients with hypertension and insulin resistance. Since both hormones are known to have cell growth-promoting effects, they may contribute to the progression of vascular hypertrophy in patients with insulin resistance. Insulin-mediated activation of the vascular renin–angiotensin system (RAS) stimulates growth in cultured rat vascular smooth muscle cells (VSMC). Objective In order to evaluate the role of IGF-I-mediated activation of components of the tissue RAS, we examined the effect of IGF-I receptor stimulation on cell proliferation, and production of angiotensinogen in cultured VSMC. Study Design Aortic VSMC were derived from male Sprague-Dawley rats. IGF-I and insulin-mediated DNA synthesis were estimated by 3 H-thymidine uptake (3H-TdR) with or without the angiotensin I converting enzyme inhibitor, captopril. Moreover, angiotensinogen released by the cells to the culture medium was determined by radioimmunoassay with or without the anti-IGF-I receptor antibody αIR3 or captopril. Results Both IGF-I and insulin increased 3H-TdR uptake by cultured rat VSMC (P < 0.05). Captopril blocked IGF-I and insulin-mediated 3H-TdR uptake (−34.4 ± 1.9% and −32.7 ± 1.8%, P < 0.05, respectively). IGF-I increased the angiotensinogen level in the medium by 30.6 ± 2.9% (P < 0.01). Insulin also stimulated angiotensinogen synthesis by 26.3 ± 2.2% (P < 0.01). Captopril and αIR3 significantly suppressed angiotensinogen production stimulated by both IGF-I and insulin. Conclusions These results indicate that IGF-I as well as insulin stimulates angiotensinogen production and growth in VSMC. Thus, both hormones may independently play a role in progression of the vascular hypertrophy and atherosclerosis in patients with hypertension and insulin resistance through activation of the tissue RAS.

Eicosapentaenoic acid inhibits Ca2+ mobilization and PKC activityin vascular smooth muscle cells*

BACKGROUND Eicosapentaenoic acid is a fish oil fatty acid that has been shown to decrease blood pressure (BP) in humans. The mechanism by which this fatty acid produces this effect is unknown. Angiotensin II increases BP by inducing vasoconstriction of vascular smooth muscle cells, an event that is mediated by an increase of intracellular calcium and an increase of protein kinase C activity. METHODS We determined the effects of eicosapentaenoic acid on angiotensin II-induced calcium signaling, and protein kinase C activity in cultured rat aortic smooth muscle cells. Incorporation of eicosapentaenoic acid into cell phospholipids was determined by gas chromatography/mass spectrometry. Intracellular calcium concentration was determined using fura-2, and protein kinase C activity was assessed by an ELISA assay using a phospho-specific antiserum for protein kinase C substrates. RESULTS We found that eicosapentaenoic acid was incorporated into cell phospholipids within 20 min. Eicosapentaenoic acid (10 or 25 micromol/L) did not alter basal intracellular calcium concentration, but decreased the peak response to 100 nmol/L angiotensin II. Eicosapentaenoic acid also decreased the amount of calcium released by thapsigargin, a drug that releases calcium from the sarcoplasmic reticulum, and decreased cation influx after angiotensin II stimulation. Angiotensin II stimulated phosphorylation of protein kinase C substrates. Preincubation of cells with 10 or 25 micromol/L eicosapentaenoic acid significantly inhibited this phosphorylation. CONCLUSIONS Our results demonstrate that acute incorporation of eicosapentaenoic acid into vascular smooth muscle cell phospholipids inhibits intracellular calcium mobilization and protein kinase C activation. These are potential mechanisms by which eicosapentaenoic acid reduces vasoconstriction.

Inhibitors of arachidonic acid metabolism have variable effects on calcium signaling pathways

The metabolic pathways of arachidonic acid (AA) have been shown to be important in the regulation of cellular function. Several studies have demonstrated both direct and indirect effects of products of these pathways in the regulation of vascular actions, and in particular on signaling pathways. Because intracellular calcium concentration is a significant mediator of stimulus-coupled signal transduction, we investigated the effects of AA pathway inhibitors on angiotensin II (Ang II)-induced calcium mobilization in cultured rat vascular smooth muscle cells (VSMC). Thus, specific calcium pools were examined for differential effects resulting from inhibitors of the three major pathways of AA metabolism. Inhibition of lipoxygenase (LO) with 2.5 micromol/L of 5,8,11 eicosatriynoic acid (ETI), cyclooxygenase (CO) with 2 micromol/L of ibuprofen (IBU), and cytochrome P-450 (P450) with 1 micromol/L of 7-ethoxyresorufin (7ER) all reduced total Ang II-induced intracellular calcium transients ([Ca2+]i) in fura 2-loaded cultured rat VSMC. However, the sites of action affected were unique for each inhibitor. Pretreatment of VSMC with either ETI or IBU inhibited thapsigargin (TG) (1 micromol/L)-sensitive calcium increments (control; 118.0 +/- 33.1 nmol/L, n = 9, ETI; 34.7 +/- 4.8 nmol/L, n = 9, IBU; 40.3 +/- 8.8 nmol/L, n = 8, P < .05 v control). Both caffeine (CAF) and ryanodine (RY) treatment attenuated Ang II-induced [Ca2+]i; however, pretreatment with ETI, IBU, or 7ER did not alter this effect. In other studies, the LO inhibitor ETI attenuated Ang II-induced Ca2+ influx, whereas inhibitors of CO and P450 pathways had no effect. These data show that 1) E

Decreased erythrocyte insulin binding in hypertensive subjects with hyperinsulinemia

BACKGROUND This study investigates erythrocyte insulin receptor binding and affinity in subjects with hypertension and hyperinsulinemia. Insulin receptor-binding function has not been extensively studied in hypertensive subjects. METHODS Insulin receptor density, binding affinity, and protein tyrosine kinase activity were measured in erythrocytes from 18 hypertensive and 16 normotensive subjects. Insulin sensitivity was measured by the fasting plasma insulin/glucose ratio and the homeostatic assessment model algorithm (HOMA) index. Erythrocyte insulin binding was determined by a competitive binding assay and protein tyrosine kinase activity was measured by an enzyme-linked immunoabsorbent assay technique. RESULTS Fasting plasma insulin/glucose ratio and the insulin resistance index (HOMA) were significantly higher in the hypertensive versus normotensive subjects. Receptor saturation of the high affinity binding sites (Bmax) was reduced in the hypertensive versus control subjects. The Kd values were lower in the erythrocytes from hypertensive than control subjects. Insulin-induced protein tyrosine kinase activity was decreased in erythrocytes from hypertensive versus control subjects. CONCLUSIONS A reduced erythrocyte insulin receptor density and tyrosine protein kinase activity may reflect insulin receptor dysfunction in hypertensive individuals who have insulin resistance and hyperinsulinemia. More information is needed examining insulin receptor function in other target tissues such as fat or skeletal muscle cells before defects in the insulin receptor can be firmly proposed as a cause of the metabolic syndrome.

Arachidonic Acid Metabolites in the Vasculature

Early evidence that prostaglandins (PGs) or other arachidonic acid (AA) metabolites might play a role in blood vessels and blood pressure regulation focused on the kidney (1). Hypertension accompanying the removal of the kidneys could not be attributed to alterations in fluid and electrolyte function alone, for ureteral implantation into the vena cava created the same degree of renal failure but substantially less elevation of blood pressure (1). Subsequent identification of the PGs as vasodilatory lipids present in the medullary interstitial cells suggested that these compounds might be the renal vasodilatory hormones that prevented renoprival hypertension. The first PGs identified in the kidney, PGE2 and PGA2, were vasodilatory and promoted sodium excretion when they were infused into the renal artery. However, PGE2 was metabolized by one pass through the pulmonary circulation in animals (2), and in humans, a high degree of extraction of PGE2 by the lung was demonstrated (3). Although PGA2 escaped pulmonary degradation it was rapidly cleared from the circulation (3). Subsequent studies indicated that PGs of the A family probably do not exist in vivo and most likely are dehydration products of PGE produced in extraction procedures. Within the kidney, PGE2 has been shown to be the principal PG and may have significant effects on renal blood flow (especially in reduced flow states), glomerular function, renal renin release, and water reabsorption (4).