Fenofibrate improves insulin sensitivity in connection with intramuscular lipid content, muscle fatty acid-binding protein, and beta-oxidation in skeletal muscle.

We investigated the effect of fenofibrate, a peroxisome proliferator-activated receptor-alpha agonist, on insulin sensitivity including lipid metabolism in skeletal muscle. Six-week-old male Sprague-Dawley rats were divided into two groups: those fed a standard chow (control) or a fructose-rich chow (fructose-fed rats (FFRs)) for 6 weeks. FFRs were treated either with a vehicle or with 30 mg/kg per day of fenofibrate for the last 2 weeks. Insulin sensitivity (M-value) was estimated by the euglycemic hyperinsulinemic glucose clamp method. Fatty acid-binding protein (FABP) in skeletal muscle was measured by ELISA, and the expression of FABP mRNA was analyzed by semi-quantitative RT-PCR. The serum and muscle triglyceride (sTG and mTG) levels and the activity of 3-hydroxyacyl-CoA dehydrogenase (HADH), a beta-oxidation enzyme, in muscle were also determined. FFRs showed a lower M-value and higher blood pressure, sTG and mTG than did the control group. The mTG was correlated positively with sTG and negatively with the M-value. Fenofibrate treatment for 2 weeks did not change blood pressure but significantly improved the M-value, sTG and mTG. FABP content and mRNA in the soleus muscle were significantly elevated in FFRs compared with those in the control group. Fenofibrate treatment further increased FABP. The HADH activity was comparable between the control group and FFRs, but significantly increased by fenofibrate treatment. These results suggest that fenofibrate improves insulin sensitivity not only by lowering serum lipids and subsequent influx of fatty acids into muscles but also by reducing intramuscular lipid content via further induction of FABP and stimulation of beta-oxidation in muscles.

Effects of cilnidipine on muscle fiber composition, capillary density and muscle blood flow in fructose-fed rats.

The aim of this study was to examine the roles of muscle fiber composition, capillary density and muscle blood flow in insulin resistance (IR) and the effect of cilnidipine, a calcium channel blocker in fructose-fed rats (FFR). Six-week-old male Sprague-Dawley rats were fed either normal rat chow or fructose-rich chow for 6 weeks. For the last 2 weeks, the rats were treated by gavage with a vehicle (Control and FFR groups) or with cilnidipine (FFR+Cil group). Blood pressure (BP) and insulin sensitivity were assessed in the sixth week. Muscle fiber composition, capillary density and blood flow in the soleus muscle were evaluated. BP of FFR was significantly higher than that of the controls. Cilnidipine significantly lowered BP in FFR. Insulin sensitivity was significantly lower in FFR than in the controls. Cilnidipine significantly improved IR in FFR. The composite ratio of type I fibers in the soleus muscle was significantly lower in FFR than in the controls, but that of type II fibers was significantly higher in FFR. Treatment with cilnidipine resulted in recovery of this ratio to that of the controls. Insulin sensitivity was found to be significantly correlated with the composite ratio of either type I fibers or type II fibers. There were no intergroup differences in capillary density. Muscle blood flow in the FFR+Cil group was higher than that in the Control or FFR groups. These results suggest that muscle fiber composition is linked to IR and that cilnidipine may improve IR in FFR either by modulating muscle fiber composition or by increasing muscle blood flow.

Effects of the Chinese medicine Jiang-Tang-Ke-Li on insulin resistance in fructose-fed rats.

The aim of this study was to determine the effect of Jiang-Tang-Ke-Li (JTKL), a Chinese medicine used to treat diabetes mellitus, on insulin resistance and hypertension in fructose-fed rats (FFR). Six-week-old male Sprague-Dawley rats were fed either normal rat chow (control) or a fructose-rich chow (FFR) for 6 weeks. For the last 2 weeks of the 6-week period of either diet, the rats were treated by gavage with gum arabic solution as a vehicle (control or FFR) or JTKL (3.24 g/kg/day; FFR+JT), and then an euglycemic hyperinsulinemic glucose clamp technique was performed to estimate insulin sensitivity. Systolic blood pressure was measured each week of the 6-week period. At the end of the glucose clamp, the soleus and extensor digitorum longus (EDL) muscles were dissected out for determination of the role of tumor necrosis factor (TNF)-alpha by an ELISA assay. Systolic blood pressures in the FFR groups were significantly higher than that in the control group, although there was no effect on systolic blood pressure for the last 2 weeks of treatment with JTKL. The average rate of glucose infusion during the glucose clamp, as an index of insulin sensitivity (M value), was significantly lower in the FFR than in the control rats, and treatment with JTKL for 2 weeks significantly increased the M value to that of control. TNF-alpha levels were significantly higher in the soleus and EDL muscles of the FFR (480+/-46 and 570+/-45 pg/g wet tissue in the soleus and EDL muscles, respectively) than in those of the control rats (177+/-34 and 206+/-33 pg/g wet tissue in the soleus and EDL muscles, respectively; p<0.01). Treatment with JTKL for 2 weeks significantly lowered TNF-alpha levels to the control levels (189+/-22 and 239+/-92 pg/g wet tissue in the soleus and EDL muscles, respectively). The results suggest that the Chinese medicine JTKL improves insulin resistance and modulates TNF-alpha in the soleus and EDL muscles in hypertensive and insulin-resistant fructose-fed rats.

Gender difference in the relationships among hyperleptinemia, hyperinsulinemia, and hypertension.

Studies reveals that plasma leptin levels (LEP) in females are higher than those in males, and that LEP in hypertensive subjects are higher than those in BMI-matched normotensive subjects. To investigate the relationships among LEP, blood pressure (BP) and insulin sensitivity, we studied these relationships in 133 Japanese males and 263 females. LEP were positively correlated with BP, body mass index, body fat mass (FM) and homeostasis model assessment (HOMA). Regression analysis in which age and FM were adjusted showed LEP were associated with BP and HOMA. Even with adjustment by age, FM and HOMA, LEP were still positively correlated BP in males. LEP in insulin-resistant hypertensives was significantly higher than those in insulin-sensitive hypertensives, in insulin-sensitive normotensives and in insulin-resistant normotensives in males. However, in females, a significantly higher LEP was observed in insulin-resistant subjects than in insulin-sensitive subjects regardless of hypertension. These data suggest that it would be sexual difference in the relationships among hyperleptinemia, hyperinsulinemia and hypertension.

The contribution of skeletal muscle tumor necrosis factor-alpha to insulin resistance and hypertension in fructose-fed rats.

OBJECTIVE: The aim of this study was to determine the role of tumor necrosis factor-alpha (TNF-alpha) in skeletal muscle tissue in insulin resistance and hypertension and the effect of anti-hypertensive medicine on skeletal muscle TNF-alpha in fructose-induced insulin-resistant and hypertensive rats (fructose-fed rats: FFR). DESIGN AND METHODS: Six-week-old male Sprague-Dawley rats were fed either normal rat chow or fructose-rich chow. For the last 2 weeks of a 6-week period of either diet, the rats were treated with a vehicle (control or FFR); temocapril, an angiotensin converting enzyme inhibitor (ACEI); or CS-866, an angiotensin II type 1 receptor blocker (ARB). The euglycemic hyperinsulinemic glucose clamp technique was performed to evaluate insulin sensitivity (M value). TNF-alpha levels in soleus and extensor digitorum longus (EDL) muscles and epididymal fat pads were measured. We also measured the TNF-alpha concentration in an incubated medium secreted from soleus muscle strips with or without angiotensin II. RESULTS: TNF-alpha levels were significantly higher in the soleus and EDL muscles, but not in the epididymal fat, in the FFRs compared with the control rats. Temocapril and CS-866 lowered systolic blood pressure, improved insulin resistance, and reduced TNF-alpha in both skeletal muscles. There were significant negative correlations between M values and TNF-alpha levels in both soleus and EDL muscles. Also, the soleus muscle strip incubation with 10(-7) mol/l angiotensin II increased TNF-alpha secreted into the incubation medium compared to the incubation without angiotensin II. These results suggest that skeletal muscle TNF-alpha is linked to insulin resistance and hypertension and that angiotensin II may be one of the factors that regulate skeletal muscle TNF-alpha.

The effects of an angiotensin-converting enzyme inhibitor and an angiotensin II receptor antagonist on insulin resistance in fructose-fed rats.

The aim of this study was to compare the effects of an angiotensin-converting enzyme (ACE) inhibitor and an angiotensin II receptor (AT) antagonist on insulin resistance, especially on muscle fiber composition in fructose-induced insulin-resistant and hypertensive rats. Six-week-old male Sprague-Dawley rats were fed either normal rat chow (control) or a fructose-rich diet (FFR). For the last two weeks of a six-week period of either diet, the rats were treated with gum arabic solution as a vehicle (control or FFR), angiotensin-converting enzyme inhibitor (FFR+ACE), temocapril (1 mg/kg/ day) or an angiotensin II receptor antagonist (FFR+AT), CS-866 (0.3 mg/kg/day), by gavage, and then the euglycemic hyperinsulinemic glucose clamp technique was performed to evaluate insulin sensitivity. At the end of the glucose clamp, the soleus muscle was dissected for determination of the muscle fiber composition by ATPase methods. Blood pressure at the glucose clamp in the FFR group was significantly higher than that of the control group, and both temocapril and CS-866 significantly lowered the blood pressure of the FFR group. The average rate of glucose infusion during the glucose clamp, as a measure of insulin sensitivity (M value), was significantly lower in the FFR rats compared to the controls (15.4 +/- 0.4, 10.9 +/- 0.6 mg/kg/min, for control and FFR, respectively, P < .01). Both temocapril and CS-866 partially improved the M values compared to FFR (13.2 +/- 0.7, 12.8 +/- 0.5 mg/kg/min, for FFR+ACE, FFR+AT, respectively, P < .01 compared with FFR, P < .05 compared with control). The composite ratio of type I fibers of the soleus muscle was decreased significantly in the FFR rats compared with the controls (82% +/- 2%, 75% +/- 2%, for control and FFR, respectively, P < .01), and both temocapril and CS-866 restored a composite ratio of type I fibers to the same level as that of the controls (81% +/- 1%, 80% +/- 1% for FFR+ACE and FFR+AT, respectively). The M value was significantly correlated with the composition of type I and type II fibers. These results suggest that the fiber composition of skeletal muscle is correlated to insulin resistance, and that both ACE inhibitors and AT antagonists may modulate the muscle fiber composition in a hypertensive and insulin-resistant animal model, fructose-fed rats, to the same extent.

Effects of the Chinese medicine, TSJN on insulin resistance and hypertension in fructose-fed rats.

The aim of this study was to determine the effect of Tang-Shen-Jiao-Nang (TSJN), a Chinese medicine used to treat diabetes mellitus, on insulin resistance and hypertension in fructose-fed rats (FFR). Six-week-old male Sprague-Dawley rats were fed either normal rat chow (control) or a fructose-rich chow (FFR) for 6 wk. For the last 2 or 4 wk of a 6-wk period of either diet, the rats were treated by gavage with gum arabic solution as a vehicle (control or FFR) or TSJN (800 mg/kg/d; FFR+TS), and then we performed the euglycemic hyperinsulinemic glucose clamp technique to estimate insulin sensitivity. Systolic blood pressure was measured weekly for 6 wk. At the end of the glucose clamp, the soleus muscle was dissected out for determination of muscle fiber composition by ATPase methods. Systolic blood pressure was elevated at 2 wk after the start of the fructose-rich chow feeding and persisted thereafter throughout the study. Systolic blood pressure during the glucose clamp in the FFR group was significantly higher than that in the control group. Although there was no effect on systolic blood pressure in rats treated with TSJN for the last 2 wk of their 6-wk diet, those treated with TSJN for the last 4 wk of their 6-wk diet had lower systolic blood pressure than did the rats in the FFR group. The average rate of glucose infusion during the glucose clamp, as a measure of insulin sensitivity (M value), was significantly lower in the FFR than in the controls (10.9 +/- 0.6 and 15.4 +/- 0.4, mg/kg/min, for FFR and controls, respectively; p< 0.01). Treatment with TSJN for 2 wk significantly improved the M value compared to that of the control level (15.1 +/- 0.5 mg/kg/min). The composite ratio of type I fibers in the soleus muscle was significantly decreased in the FFR compared to controls (75.0 +/- 1.7 and 81.7 +/- 1.5%, for FFR and controls, respectively; p< 0.01), and treatment with TSJN for 2 wk led to a recovery composite ratio of type I fiber to the same level as that of the control group (78.7 +/- 1.7% in FFR + TS). The M value was significantly correlated with the compositions of type I and type II fibers (for type I fibers, r= 0.45, p < 0.01, for type II fibers, r= -0.44, p< 0.05). These results suggest that the Chinese medicine TSJN may improve insulin resistance, lower the systolic blood pressure, and modulate muscle fiber composition in hypertensive and insulin-resistant fructose-fed rats.

The mechanisms of insulin sensitivity improving effects of angiotensin converting enzyme inhibitor.

It is well-known that angiotensin converting enzyme (ACE) inhibitor not only decreases blood pressure (BP) but also improves insulin sensitivity. To elucidate the mechanisms of these actions of ACE inhibitor, we evaluated its effect on both BP and insulin sensitivity (M-value) as estimated by the glucose clamp technique in essential hypertensives in comparison with the effect of angiotensin receptor (AT) antagonist. We also evaluated the effect of ACE inhibitor on BP, M-value and muscle fiber composition in fructose-fed rats (FFR) as an insulin-resistant hypertensive model with or without treatment with Hoe 140 (kinin receptor antagonist). In essential hypertensives, both ACE inhibitor and AT antagonist decreased BP and improved insulin sensitivity to the same extent. In FFR, ACE inhibitor also decreased BP and improved insulin sensitivity. Moreover, Hoe 140 showed no effect on these actions of ACE inhibitor. The composite ratio of type I fiber of soleus muscle was decreased significantly in FFR compared to control and ACE inhibitor produced a recovery of the composite ratio of type I fiber to the same as control. These results suggested that muscle fiber composition of skeletal muscle is linked to insulin resistance, and that ACE inhibitor may modulate muscle fiber composition through its vasodilative effect in hypertension. These results also suggest that for vasodilation, it is more important to inhibit angiotensin II than to block degradation of kinins or to improve insulin sensitivity by ACE inhibitor.

The contribution of nitric oxide to diuretic and natriuretic effects of renal kinins in normotensive rats.

We have reported that diuresis and natriuresis due to increase in renal kinins induced by the neutral endopeptidase 24.11 (NEP) inhibitor were attenuated by nitric oxide (NO) synthase inhibitor. To further clarify the water-sodium excretory mechanism of renal kinins, we estimated NO2+NO3 (NOx) and cGMP in plasma and urine with and without a specific NEP inhibitor, thiorphan. P-aminohippuric acid (PAH) and inulin were injected into male Sprague-Dawley rats. Vehicle (n = 8) or thiorphan (30 mg/kg, n = 10) was injected after the control period. Mean blood pressure (MBP), plasma and urinary PAH, inulin, NOx and cGMP, urinary volume (UV) and urinary sodium excretion (UNaV) were measured before and after injection of the reagents. MBP, renal plasma flow and glomerular filtration rate were not affected by thiorphan. Plasma NOx and cGMP with thiorphan did not differ from the vehicle, while urinary NOx and cGMP increased. None of the variables were affected by vehicle. UV and UNaV were higher with thiorphan than with vehicle. Positive correlation was found between urinary deltaNOx and deltacGMP. Each urinary deltaNOx and deltacGMP was significantly correlated to both deltaUV and deltaUNaV. Urinary NOx and cGMP were increased while maintaining correlations to UV and UNaV, but plasma NOx and cGMP were not affected by thiorphan. This implies that the mechanism of water-sodium excretion induced by NEP inhibitor is mediated by renal NO. Therefore, renal NO may contribute to the diuretic and natriuretic effects of renal kinins.

Alteration of muscle fiber composition linking to insulin resistance and hypertension in fructose-fed rats.

The aim of this study was to examine the role of muscle fiber composition in insulin resistance and the effect of a calcium channel antagonist on insulin sensitivity in fructose-induced insulin resistant and hypertensive rats. Six-week-old male Sprague-Dawley rats were fed either normal rat chow (control) or fructose-rich diet (FFR). For the last 2 weeks of a 6-week period of either diet, the rats were treated, by gavage, with gum arabic solution (control or FFR) or a dihydropyridine calcium channel antagonist, benidipine hydrochloride (3 mg/kg/day: FFR + Ca), then the euglycemic hyperinsulinemic glucose clamp technique was performed to evaluate insulin sensitivity. Blood pressure was measured weekly for 6 weeks. At the end of the glucose clamp, the soleus muscle was dissected out for determination of muscle fiber composition by ATPase methods. Blood pressure was elevated at 2 weeks after the start of fructose-rich chow feeding and persisted thereafter throughout the study. Blood pressure at the glucose clamp in the FFR was significantly higher than that in the control group (142 +/- 2 v 155 +/- 2 mm Hg, P < .01) and the calcium antagonist significantly lowered blood pressure of FFR (136 +/- 6 mm Hg for FFR +/- Ca, P < .05). The average rate of glucose infusion during glucose clamp, as a measure of insulin sensitivity (M value), was significantly lower in the FFR than in the control (15.4 +/- 0.4 v 10.9 +/- 0.6 mg/kg/min, P < .01). The calcium channel antagonist partially improved the M value compared to that of FFR (13.4 +/- 0.7 mg/kg/min in FFR +/- Ca, P < .01 compared to FFR, P < .05 compared to control). The composite ratio of type I fiber in soleus muscle was significantly decreased in FFR compared to control (81.7 +/- 1.5% v 75.0 +/- 1.7%, P < .01), and the composite ratio of type I fiber in rats treated with the calcium channel antagonist (FFR +/- Ca) recovered to the control level (79.9 +/- 1.1%, P < .05 compared to FFR). The M value was significantly correlated with the compositions of type I and type II fibers (for type I fibers, r = 0.80, P < .01; for type II fibers, r = -0.81, P < .01). These results suggest that fiber composition of skeletal muscle links insulin resistance and that a calcium channel antagonist may modulate muscle fiber composition in hypertensive animal model, fructose-fed rats.

[The effects of angiotensin II receptor antagonists on insulin resistance].

Many of hypertensive individuals have glucose intolerance and dyslipidemia, and insulin resistance is common disorder on the basis of these diseases. It is important to take care of these metabolic disease for not only the control of hypertension, blood glucose and hyperlipidemia, but also the prevention of atherosclerosis. We reviewed the effects of angiotensin II receptor antagonists on insulin resistant syndrome.

Effect of an angiotensin II receptor antagonist, candesartan, on insulin resistance and pressor mechanisms in essential hypertension.

Candesartan (8 mg/d) was administered for 2 weeks to eight patients with essential hypertension to investigate the effect of an angiotensin II receptor antagonist on insulin sensitivity. The effect of candesartan on sodium-retaining action and the activation of pressor systems by hyperinsulinaemia, which might be related to pressor mechanisms in essential hypertension, was also investigated in this study. In patients with essential hypertension, candesartan restored insulin sensitivity to the level of that in normotensive subjects. Candesartan treatment attenuated the sodium-retaining action of hyperinsulinaemia. These findings suggest that inhibition of angiotensin II action by candesartan can improve insulin sensitivity and inhibit the sodium-retaining action associated with hyperinsulinaemia in essential hypertension.

Effects of aging on the insulin actions for the glucose metabolism and renal function in normotensives and essential hypertensives.

It has been suggested that hyperinsulinemia compensating insulin resistance in glucose metabolism may be a pathogenic factor in essential hypertension. On the other hand, age-associated increases in the prevalence of glucose intolerance and hypertension are also well established. The aim of this study is to clarify the influence of aging on insulin sensitivity in glucose metabolism and on renal sodium handling under hyperinsulinemia, which may relate to high blood pressure in insulin-resistant subjects. Fifty-two normotensive subjects and 61 patients with essential hypertension were evaluated in this study. The subjects of these groups were divided into young (<40 years old) and middle-elderly (> or = 40 years old): young normotensives (Y-NT, n = 22); middle-elderly normotensives (ME- NT, n = 30); young hypertensives (Y-HT, n = 9); and middle-elderly hypertensives (ME-HT, n = 52). Using the euglycemic hyperinsulinemic glucose clamp, insulin sensitivity was assessed as M value. Just before the start and the termination of the glucose clamp, creatinine clearance (Ccr) and urinary excretion of sodium (UNaV) were measured. In addition, renal plasma flow assessed as para-aminohippuric acid clearance was also measured at the same time in several subjects; 8 Y-NT, 8 ME-NT, 3 Y-HT, and 10 ME-HT. The M value was significantly lower in ME-NT, Y-HT, and ME-HT, compared to Y-NT, although blood sugar and immunoreactive insulin levels were similar in all four groups. In normotensive subjects, there was a significant, negative correlation between age and M value. However, this correlation was not observed in hypertensive patients. UNaV decreased in ME-NT, Y-HT, and ME-HT, but not in Y-NT under hyperinsulinemia by the glucose clamp, whereas Ccr showed no significant change in any group. In all subjects, the change of UNaV (deltaUNaV) correlated significantly and positively with the M value. Renal plasma flow significantly increased under hyperinsulinemia by the glucose clamp in only Y-HT, but not in the other groups. There was a significant, positive correlation between deltaUNaV and the change of renal plasma flow under hyperinsulinemia by the glucose clamp. These results suggested that both the impairments of the insulin sensitivity and insulin-induced vasodilation at the renal artery with aging may partially contribute to age-related elevation of blood pressure through renal sodium retention by compensating hyperinsulinemia. On the other hand, it seems reasonable to assume that these abnormalities, which can contribute to high blood pressure in essential hypertension, already may exist at lower ages in essential hypertensive patients.

High plasma immunoreactive leptin level in essential hypertension.

Insulin resistance, the most important factor in metabolic syndrome X, has been considered to raise blood pressure. Recently it was reported that insulin resistance was related to an elevated plasma level of leptin, which is an adipocyte-specific ob gene product and which plays a role in food intake suppression, thermogenesis, and energy expenditure through the activation of the hypothalamus. However there are no reports that deal with the relationship of insulin resistance to plasma leptin and blood pressure. To evaluate the role of leptin in essential hypertensives, two groups of subjects who were carefully matched for body mass index (BMI) were studied; 22 normotensives (NT, age: 46.5 +/- 2.6 years, BMI: 23.9 +/- 0.4 kg/m2, male/female: 14/8) and 45 mild-to-moderate essential hypertensives (EHT, age: 51.9 +/- 2.0 years, BMI: 24.5 +/- 0.4 kg/m2, male/female: 21/24). We applied the euglycemic hyperinsulinemic glucose clamp technique to all subjects and insulin sensitivity was evaluated as the M value. EHT showed a significantly lower M value (160.2 +/- 7.4 v 184.3 +/- 7.3 mg/m2/min, P < .05) and higher basal plasma immunoreactive leptin level (7.6 +/- 0.8 v 5.0 +/- 0.8 ng/mL, P < .05) than NT, despite the fact that there was no significant difference between NT and EHT in age, gender, or BMI. The relationship between mean blood pressure and leptin showed a significant positive correlation in all of the subjects (r = 0.31, P < .05), suggesting that leptin may be related to a pathophysiology of essential hypertension.

Urinary kallikrein in Dahl-Iwai salt-sensitive and -resistant rats.

This study was designed to evaluate the differences between the renal kallikrein in newly established Dahl-Iwai rats under salt loading and that of Sprague-Dawley rats (SD). Urinary kallikrein quantity and activity was markedly lower in Dahl-Iwai rats than in SD even during the control period. Moreover, kallikrein quantity and activity in Dahl-Iwai salt-sensitive rats (SS) were clearly diminished in comparison with salt-resistant rats (SR). The kallikrein activity/ quantity ratio was also lower in SS and SR than in SD during the control period. After salt loading, systolic blood pressure increased only in SS. Kallikrein activity in SS and SR, and kallikrein quantity in SS were increased, whereas those in SD did not change. Although the kallikrein activity/quantity ratio in SR reached the same level in SD after salt loading, that in SS was lower throughout the experiment. These results suggest that Dahl-Iwai rats are less able hereditarily to produce renal kallikrein and that there may exist structurally abnormal kallikrein that may have a lower activity. Different kinetics of renal kallikrein between SS and SR by salt loading might be explained by kallikrein inhibitors or abnormal kallikrein or nonkallikrein kininogenase. These different kinetics of renal kallikrein may play some role on blood pressure elevation in SS.

Gender-related differences in androgen regulation of thromboxane A2 receptors in rat aortic smooth-muscle cells.

Thromboxane A2 (TXA2) has been implicated as an important mediator of cardiovascular diseases. Aortas obtained from male rats are more sensitive to TXA2 mimetics compared with those obtained from females. A similar phenomenon has been reported in canine coronary arteries. To determine whether there is a gender-related difference in the regulation of TXA2 receptors by androgenic steroids, we determined the effect of testosterone and dihydrotestosterone (DHT) on TXA2 receptor density in cultured rat aortic smooth-muscle (RASM) cells and guinea pig coronary artery smooth-muscle (CASM) cells. TXA2 receptor density (B(max)) and dissociation constant (Kd) were determined by radioligand binding studies with (125)I-BOP, a TXA2 receptor agonist. Testosterone significantly (p < 0.05) increased TXA2 receptor density in cultured RASM cells and guinea pig CASM cells. DHT significantly (p < 0.005) increased the B(max) in male RASM cells (62 +/- 2 vs. 40 +/- 3 fmol/mg protein; n = 7; p < 0.005). DHT increased the B(max) values in both male and female RASM cells, but the increase was significantly (p < 0.05) less in female than in male RASM cells (57 +/- 10% increase for male and 31 +/- 5% for female). Androgen-receptor protein was detected in RASM cells by Western blot and was less in the female RASM cells than in the male. The results indicate that RASM cells possess an androgen receptor and that gender-related differences exist in the regulation of expression of TXA2 receptors by androgens.

Inhibition of testosterone 5 alpha-reductase: evidence for tissue-specific regulation of thromboxane A2 receptors.

Testosterone has been implicated as a risk factor for cardiovascular diseases and thromboxane A2 (TXA2) may be an important pathophysiologic mediator for them. Testosterone has been shown to increase TXA2 receptor density in several cell types. Testosterone is reduced at the 5 alpha position to its active metabolite, dihydrotestosterone, by 5 alpha-reductase. We determined the effects of epristeride, a 5 alpha-reductase inhibitor, on the density of TXA2 receptors in rat aortic smooth muscle cells and human erythroleukemia cells, a megakaryocyte-like cell, in vitro, and in rat platelets and aortic membranes in vivo. In rat aortic smooth muscle cells, epristeride significantly (P < .01, n = 5) blocked the effect of testosterone to increase TXA2 receptor density (Bmax: 44 +/- 3, 76 +/- 7, 48 +/- 4 and 46 +/- 4 fmol/mg protein, for control cells, cells treated with testosterone (200 nM), cells treated with testosterone and epristeride (10 nM) and cells treated with epristeride, respectively. Epristeride did not block the effect of testosterone in human erythroleukemia cells. Treatment of male rats with epristeride for 2 weeks significantly (P < .01) decreased TXA2 receptor density in aortic membranes (41 +/- 3 for vehicle, n = 10; 27 +/- 3 fmol/mg protein for epristeride, n = 11) but did not significantly change TXA2 receptor density in platelets. Maximum contractile responses of rat aortas to U46619, a TXA2 mimetic, were significantly (P < .001) lower in epristeride-treated rats than in vehicle-treated rats (4.2 +/- 0.1 for vehicle, n = 16, 3.0 +/- 0.2 g tension for epristeride, n = 15). In conclusion, regulation of expression of TXA2 receptors by testosterone in cells of vascular origin, but not in platelets, appears to be via DHT.

Characterization of the cloned HEL cell thromboxane A2 receptor: evidence that the affinity state can be altered by G alpha 13 and G alpha q.

Thromboxane A2 (TXA2) induces activation of platelets and vascular smooth muscle contraction via cell surface receptors. A platelet type TXA2 receptor from the megakaryocyte-like HEL cell was cloned with a deduced amino acid sequenced identical to that previously reported for the human placental TXA2 receptor. Transient expression of the HEL cell TXA2 receptor cDNA and radioligand binding studies with the agonist 125I-BOP showed a single class of binding sites with an affinity comparable to a low affinity platelet TXA2 receptor. Using a series of 13-azapinane TXA2 analogs, which discriminate between TXA2 receptor subtypes in platelets and vascular smooth muscle, we found that the cloned HEL cell TXA2 receptor is characteristic of a platelet type TXA2 receptor and that its binding characteristics are different from those of vascular smooth muscle cells. The affinity of the HEL cell TXA2 receptor for 125I-BOP was significantly (P < .05) increased upon co-transfection with G alpha 13 alone, or with G alpha q alone and with G alpha 13 and G alpha 12 together (n = 4-6). GTP gamma S significantly (P < .05) decreased the affinity of the receptor for 125I-BOP in COS-7 cell membranes coexpressing HEL-TXR and G alpha 13 to a value comparable to HEL-TXA2 receptor alone. We conclude that 1) the cloned HEL cell TXA2 receptor has pharmacological characteristics of a low affinity platelet type receptor and 2) that the affinity state of this receptor may be influenced by interaction with G alpha 13 and G alpha q.