Ken Omata

Possible Role of P-450 Metabolite of Arachidonic Acid in Vasodilator Mechanism of Angiotensin II Type 2 Receptor in the Isolated Microperfused Rabbit Afferent Arteriole

Although angiotensin II type 2 (AT2) receptor has recently been cloned, its functional role is not well understood. We tested the hypothesis that selective activation of AT2 receptor causes vasodilation in the preglomerular afferent arteriole (Af-Art), a vascular segment that accounts for most of the preglomerular resistance. We microperfused rabbit Af-Arts at 60 mmHg in vitro, and examined the effect of angiotensin II (Ang II; 10(-11)-10(-8) M) on the luminal diameter in the presence or absence of the Ang II type 1 receptor antagonist CV11974 (CV; 10(-8) M). Ang II was added to both the bath and lumen of preconstricted Af-Arts. Ang II further constricted Af-Arts without CV (by 74+/-7% over the preconstricted level at 10(-8) M; P < 0.01, n = 7). In contrast, in the presence of CV, Ang II caused dose-dependent dilation; Ang II at 10(-8) M increased the diameter by 29+/-2% (n = 7, P < 0.01). This dilation was completely abolished by pretreatment with an AT2 receptor antagonist PD123319 (10(-7) M, n = 6), suggesting that activation of AT2 receptor causes vasodilation in Af-Arts. The dilation was unaffected by inhibiting either nitric oxide synthase (n = 7) or cyclooxygenase (n = 7), however, it was abolished by either disrupting the endothelium (n = 10) or inhibiting the cytochrome P-450 pathway, particularly the synthesis of epoxyeicosatrienoic acids (EETs, n = 7). These results suggest that in the Af-Art activation of the AT2 receptor may cause endothelium-dependent vasodilation via a cytochrome P-450 pathway, possibly by EETs.

Vasodilation mediated by angiotensin II type 2 receptor is impaired in afferent arterioles of young spontaneously hypertensive rats.

Renal vasoconstrictor action of angiotensin II (Ang II) is exaggerated in the spontaneously hypertensive rat (SHR) before development of hypertension. We have recently demonstrated that in the rabbit afferent arteriole (Af-Art) activation of the AT2 receptor causes vasodilation, which modulates the vasoconstrictor action of Ang II mediated by the AT1 receptor. In this study, we tested the hypothesis that vasoconstrictor action of Ang II is exaggerated in SHR Af-Arts due to an impaired function of the AT2 receptor before development of hypertension. Af-Arts were microdissected from the superficial cortex of 4- to 5-week-old SHR or age-matched Wistar Kyoto rats (WKY), and perfused at 60 mm Hg in vitro. Ang II (10–11 to 10–8 M) decreased the luminal diameter of Af-Arts of both strains in a dose-dependent manner. However, the constriction was stronger in SHR; at 10–10 M, the diameter decreased by 34 ± 4% in SHR (n = 6) compared to 18 ± 3% in WKY (n = 6; p < 0.01). Pretreatment with PD123319 (PD), an AT2 receptor antagonist, significantly augmented Ang II-induced constriction in WKY but not SHR Af-Arts; at 10–10 M, the diameter now decreased by 41 ± 5 and 37 ± 1% in SHR (n = 6) and WKY (n = 6), respectively. Thus, blockade of the AT2 receptor abolished the difference in Ang II action on Af-Arts between strains. Moreover, with the AT1 receptor blockade Ang II caused dose-dependent dilation of preconstricted Af-Arts only in WKY (27 ± 5% at 10–8 M, n = 5), and the dilation was abolished by simultaneous treatment with PD. In contrast, no such dilation was observed in SHR Af-Arts. These results suggest that activation of the AT2 receptor modulates AT1 receptor vasoconstriction in WKY Af-Arts, while impaired modulatory function of AT2 receptor may play a role in the exaggerated vasoconstrictor action of Ang II on the Af-Art of prehypertensive SHR.

20-HETE Requires Increased Vascular Tone to Constrict Rabbit Afferent Arterioles

Renal production of 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450-dependent arachidonate metabolite, increases during development of hypertension in spontaneously hypertensive rats, and inhibition of its production prevents hypertension. Since 20-HETE is a potent vasoconstrictor, these findings suggest that 20-HETE may contribute to the development of hypertension by elevating renal vascular resistance. In this study we examined the direct action of 20-HETE on the afferent arteriole, a vascular segment crucial to the control of renal vascular resistance. Rabbit afferent arterioles were microperfused at 60 mm Hg in vitro, and 20-HETE was added to the lumen. Although 20-HETE (10(-10) to 10(-6) mol/L) had no effect on the diameter of non-treated afferent arterioles (n=6), it caused dose-dependent constriction when vascular tone was increased with norepinephrine (0.3 micromol/L); 20-HETE at 10(-6) mol/L decreased diameter by 43 +/- 4% (n=6, P < .001). This constriction was abolished by disrupting the endothelium (n=5). Moreover, pretreatment with the cyclooxygenase inhibitor indomethacin (50 micromol/L) or the thromboxane/endoperoxide receptor antagonist SQ29548 (1 micromol/L) significantly (P < .03) attenuated 20-HETE-induced constriction: 20-HETE at 10(-6) mol/L constricted norepinephrine-treated afferent arterioles by 28 +/- 3% (n=6) and 25 +/- 4% (n=5), respectively. These results demonstrate that an increase in afferent arteriolar tone is required for the vasoconstrictor action of 20-HETE, which is partly mediated by the endothelial cyclooxygenase pathway. THus, increased production of 20-HETE in the kidney and increase in afferent arteriolar tone, both of which often precede the development of hypertension, may synergistically contribute to the development of hypertension by elevating renal vascular resistance.

Effects of ouabain on blood pressure regulation in rats

In order to test the hypothesis that a circulating inhibitor of the sodium-potassium ATPase pump may cause a concomitant rise in blood pressure and increased sodium excretion, we studied chronic effects of continuous infusion of ouabain, an inhibitor of sodium-potassium ATPase, for up to 6 days on systolic blood pressure and urinary sodium excretion in conscious rats. We also evaluated the effect of this substance in rats with hypertension induced by chronic infusion of norepinephrine. Continuous infusion of ouabain (1.2 mg/kg per day) into the jugular vein by an osmotic minipump did not induce any changes in systolic blood pressure and urinary sodium excretion in intact rats on regular diets. Furthermore it did not cause a change in systolic blood pressure in rats drinking 1% NaCl, and in unilaterally nephrectomized rats drinking 1% NaCl, when compared with vehicle-infused animals. When the same dose of ouabain was administered simultaneously with 1.8 mg/kg per day norepinephrine infused intraperitoneally by another osmotic minipump in conscious rats, systolic blood pressure rose on day 1 to only 129.3 +/- 2.8 mmHg compared with the rist to 145.0 +/- 2.0 mmHg when norepinephrine alone was infused (P less than 0.01). The antihypertensive effect of ouabain was sustained for the entire experimental period lasting for 6 days and was not associated with any changes in urinary sodium excretion. The administration of ouabain to rats made hypertensive by a 3-day infusion of norepinephrine, returned the blood pressure to control levels, and the antihypertensive effect was sustained throughout the experimental period lasting a further 3 days and was not associated with any changes in urinary sodium excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of atrial natriuretic factor on angiotensin II-induced hypertension in rats.

To assess the physiological role of atrial natriuretic factors in blood pressure regulation, we studied the effect of chronic infusion of a synthetic atrial natriuretic factor of 25 amino acid residues (Arg 102-Tyr 126) in rats with angiotensin II-induced hypertension. Rats were studied while on a normal sodium diet or during sodium loading with 1% NaCl solution used as drinking water. Systolic blood pressure decreased slightly during combined infusion of synthetic atrial natriuretic factor, 150 micrograms/kg/day, and angiotensin II, 900 micrograms/kg/day. This effect was sustained for 3 days in rats receiving a regular sodium intake (p less than 0.01) and during sodium loading (p less than 0.01). Administration of synthetic atrial natriuretic factor to rats made hypertensive by a 3-day infusion of angiotensin II reduced blood pressure slightly, but not to control levels, and this effect was sustained for the remaining 3 days of the experiment in both dietary groups. These results indicate that a nonhypotensive dose of synthetic atrial natriuretic factor can modulate the vasopressor effect of angiotensin II. Thus, the attenuating effect may be involved in blood pressure regulation independently of sodium metabolism, although its actual physiological importance remains undetermined.

Muscle cramps and elevated serum creatine phosphokinase levels induced by β-adrenoceptor blockers

We have assessed the propensity of β-adrenoceptor blockers to cause muscle cramps and to raise the serum creatine phosphokinase (CPK) level in 78 patients with essential hypertension. After a control period, a β-adrenoceptor blocker without intrinsic sympathomimetic activity (ISA; propranolol, metoprolol or arotinolol) was administered for three months. Thereafter, the patients were randomised to receive a β-adrenoceptor blocker with ISA (pindolol or carteolol) for three months or a β-adrenoceptor blocker without ISA for a further three months. This pattern was continued until all β-adrenoceptor blockers had been given. At the end of each period, CPK and CPK-MB levels were measured.Of the 78 subjects, muscle cramps occurred in 27 during treatment with pindolol and 32 during treatment with carteolol. No complaints were made by subjects treated with propranolol and arotinolol, but muscle cramps were reported in 2 treated with metoprolol. While muscle cramps were caused both by pindolol and carteolol in 16 subjects, they were caused by either of these drugs in the remainder of the subjects. Muscle cramp occurred mainly in the calves when the patients were in bed at night. Serum CPK and CPK-MB levels increased significantly during treatment with pindolol (control period vs pindolol, CPK=96 vs 133 IU · ml−1, CPK-MB=14 vs 18 IU · ml−1) or carteolol (CPK=117 IU · ml−1, CPK-MB=18 IU · ml−1) while the levels during treatment with propranolol, arotinolol and metoprolol did not change from those in the control period. The change in serum CPK during treatment with carteolol or pindolol was significantly correlated with the control serum CPK level. No correlation was observed between muscle cramps and serum CPK level. There were individual differences in the severity of muscle cramps, with some subjects complaining frequently of severe muscle cramps.Because muscle cramps are frequently experienced at night, they disturb sleep and lower the quality of life in patients. This problem should be considered during treatment with β-adrenoceptor blockers with ISA.

Vasopressin stimulates Cl- transport in ascending thin limb of Henle's loop in hamster.

The effect of arginine vasopressin (AVP) on NaCl transport was investigated in the isolated microperfused hamster ascending thin limb of Henles loop by measuring transepithelial voltage (Vt) and transmural 22Na+ and 36Cl- fluxes. In the presence of a transmural NaCl concentration gradient (100 mM higher in the lumen), Vt was 8.4 +/- 0.4 mV. Addition of 1 nM AVP to the basolateral solution increased Vt to 9.6 +/- 0.4 mV, which corresponds to an increase in the Cl- to Na+ permselectivity ratio (PCl/PNa) from 2.8 +/- 0.2 to 3.4 +/- 0.2. AVP at physiological concentrations increased Vt in a dose-dependent manner with an ED50 of 5 pM. AVP increased the Cl- efflux coefficient from 99.6 +/- 6.3 to 131.4 +/- 10.6 x 10(-7) cm2/s without affecting the Na+ efflux coefficient. 5-Nitro-2-(3-phenyl-propylamino)-benzoate (0.2 mM), a Cl- channel inhibitor, in the perfusate decreased the basal Cl- efflux coefficient and inhibited the AVP-induced increase in this parameter. The AVP-induced increase in Vt was not affected by [d(CH2)5(1),O-Me-Tyr2,Arg8] vasopressin, a V1 receptor antagonist, but was abolished by [d(CH2)5,D-Ile2,Ile4,Arg8] vasopressin, a V2 receptor antagonist. The selective V2 agonist dDAVP in 1 nM also increased Vt from 8.6 +/- 0.7 to 9.5 +/- 0.6 mV. Dibutyryl cAMP and forskolin both increased Vt, whereas H89, an inhibitor of cAMP-dependent protein kinase, abolished the AVP-induced increase in Vt. These results demonstrate that AVP stimulates Cl- transport in the ascending thin limb of Henles loop by activating Cl- channels via a signal transduction cascade comprising V2 receptors, adenylate cyclase, and cAMP-dependent protein kinase. The ascending thin limb of Henles loop thus participates in the formation of concentrated urine as one of the target renal tubular segments of AVP.

Difference in urinary LTE4 and 11-dehydro-TXB2 excretion in asthmatic patients.

Bronchoconstrictor cysteinyl leukotrienes (LT) and thromboxane (TX) A2 have been implicated in the pathogenesis of asthma. Determination of urinary leukotriene E4 (LTE4) and 11-dehydro-TXB2 levels are often used to assess cysteinyl LT and TXA2 production in humans. To define the potential role in the pathogenesis of asthma, we investigated the urinary LTE4 and 11-dehydro-TXB2 levels. LTE4 and 11-dehydro-TXB2 levels were determined using liquid chromatography/tandem mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS), respectively. Urinary LTE4 levels in asthmatic patients (192 +/- 122 pg/mg creatinine, n = 14) were significantly higher (P < 0.005) than those in healthy volunteers (55 +/- 16 pg/mg creatinine, n = 13), but no significant difference in 11-dehydro-TXB2 levels was observed. A significant inverse correlation (r = -0.821, P < 0.005) was found between urinary LTE4 levels and the forced expiratory volume in 1 s (FEV1) but no significant correlation was observed between urinary 11-dehydro-TXB2 levels and FEV1. The present findings suggest that cysteinyl LTs play a more important role in the pathogenesis of asthma than TXA2.

Sex and age-related differences in the urinary excretion of TXB2 in normal human subjects: a possible pathophysiological role of TXA2 in the aged kidney

Urinary excretion of thromboxane B2 (TXB2), a stable metabolite of TXA2, was measured by radioimmunoassay in 58 normal subjects (17 males and 41 females) aged from 19 to 98 years. The basal level of urinary TXB2 in normal subjects was 100 +/- 6.7 ng/day (mean +/- SEM). In young males, contamination of urine specimens with seminal fluid, which is known to contain large amounts of prostaglandin E, did not change the level of urinary TXB2. Urinary excretion of TXB2 was significantly higher in male subjects than in female (124 +/- 12.6 vs 90 +/- 7.4 ng/day, p less than 0.05). There was no significant age-related difference in 24-h urinary excretion of TXB2 in normal subjects. However, when urinary excretion of TXB2 was expressed as a function of urinary creatinine excretion, it was significantly higher in the elderly (60-93 years old) than in the younger subjects (19-59 years old)(141 +/- 15.7 vs 99 +/- 7.7 ng/g creatinine, p less than 0.02). Renal TXA2 may have a pathophysiological role in the functional impairment of the kidney in elderly people.

Role of thromboxane A2 in the hypotensive effect of captopril in essential hypertension.

We have previously reported that captopril stimulates thromboxane A2 synthesis in patients with essential hypertension. In the present study, the hypotensive effects of captopril and OKY-046, a selective inhibitor of thromboxane A2 synthetase, were studied in nine patients with essential hypertension to determine whether thromboxane A2 is involved in the regulation of blood pressure. A single oral dose of OKY-046 (400 mg) decreased urinary thromboxane B2 (a stable metabolite of thromboxane A2) excretion significantly (from 113 +/- 19.0 to 51.0 +/- 6.1 pg/min; p less than 0.01) and increased urinary sodium excretion significantly (from 73.0 +/- 15.3 to 113.0 +/- 14.4 microEq/min; p less than 0.01), but no change was observed in mean arterial pressure. The administration of OKY-046 (600 mg/day) for 3 days induced a significant and sustained decrease in urinary thromboxane B2 excretion, but it did not affect the mean arterial pressure. Although captopril (50 mg) alone induced a significant increase in urinary thromboxane B2 excretion (from 91.4 +/- 11.0 to 297.3 +/- 30.8 pg/min; p less than 0.001) and a significant decrease in mean arterial pressure (from 97.0 +/- 4.7 to 88.1 +/- 5.1 mm Hg; p less than 0.01), captopril in combination with OKY-046 induced a decrease both in urinary thromboxane B2 excretion (from 70.8 +/- 12.3 to 54.2 +/- 14.7 pg/min; p less than 0.01) and in mean arterial pressure (from 105.1 +/- 3.8 to 84.2 +/- 3.6 mm Hg; p less than 0.01). Thus, the hypotensive effect of captopril was potentiated by OKY-046. OKY-046 did not affect the changes in plasma renin activity and plasma aldosterone concentration and blunted urinary prostaglandin E2 and 6-keto-prostaglandin F1 alpha excretion in response to captopril. These results indicate that thromboxane A2 counteracts the hypotensive effect of captopril in patients with essential hypertension.