Kazuo Tsunoda

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.

Circadian blood pressure variation in patients with renovascular hypertension or primary aldosteronism.

Circadian blood pressure (BP) variation were studied in patients with renovascular hypertension (RVH) and primary aldosteronism (PA). Ambulatory BP (ABP) was monitored every 5 min for 24 hrs in a ward setting in 23 patients with PA and 17 patients with RVH (13 patients with unilateral renal arterial stenosis and 4 with bilateral stenosis). In patients with RVH, ABP was monitored before and after treatment with a converting enzyme inhibitor or percutaneous transluminal angioplasty. Plasma renin activity (PRA) was high before percutaneous transluminal angioplasty in almost all patients with RVH and low in those with PA. Ordinary circadian BP variation, i.e. nocturnal fall and diurnal rise in BP, was confirmed in the patients with unilateral or bilateral renal artery stenosis. Percutaneous transluminal angioplasty successfully normalized both BP and PRA in those with RVH. Normal circadian BP variation was observed in those with RVH before the treatment with a converting enzyme inhibitor or percutaneous transluminal angioplasty as well as during treatment with the former and after treatment with the latter. Circadian BP variation in the patients with RVH was affected by the pathogenesis of renal artery stenosis alone, i.e, fibromuscular hyperplasia and atherosclerosis; with fibromuscular hyperplasia normal circadian BP variation was observed, while with atherosclerosis, nocturnal BP fall was restricted or eliminated. Circadian BP variation in those with PA before and after excision of adrenal adenoma was essentially similar to that in normal subjects and essential hypertensive patients. From these it seems that in patients with RVH or PA, circadian BP variation is not affected by hypertension per se or by pathogenesis of hypertension.

Interaction of vasopressin and prostaglandins through calcium ion in the renal circulation.

To determine whether the effects of arginine vasopressin (AVP) on the renal and systemic vessels are modulated by prostaglandins (PGs), AVP (10, 20, and 50 mU/kg/min) was infused into the renal artery before and after treatment with indomethacin (8 mg/kg) in anesthetized rabbits. Arginine vasopressin elicited a dose-dependent increase in systemic arterial pressure and renal vasoconstriction. However, after cessation of the infusion, significant renal vasodilation was observed. Indomethacin potentiated the systemic and renal vasoconstrictor actions and attenuated the renal vasodilator reaction induced by AVP. These results suggest that endogenously produced PGs buffer the vasoconstrictor action of AVP, and the renal vasodilator reaction induced by AVP could be mediated through PGs. Further, to investigate whether the effects of AVP on the systemic and renal vessels are mediated by calcium ion (Ca++), the Ca++ entry blocker nifedipine was used. Intravenous administration of nifedipine (50 micrograms/kg) attenuated the systemic and renal vasoconstrictor action of AVP. The renal vasodilator reaction induced by AVP was also diminished after treatment with nifedipine. These results indicate that the systemic and renal vasoconstrictor actions of AVP are mediated through Ca++ influx into the vascular smooth muscle cells. The present study suggests that Ca++ participates in the AVP-induced vasodilator reaction, itself probably mediated by PGs.

DECREASED CONVERSION OF BIG ENDOTHELIN‐1 TO ENDOTHELIN‐1 IN PATIENTS WITH DIABETES MELLITUS

Endothelial cell damage is a primary feature of diabetes mellitus. Endothelin (ET)-1 synthesized by endothelial cells induces vasoconstriction and stimulates endothelial cell growth (Kimura et al. 1988). It is thought that ET-I may be involved in endothelial cell damage in diabetes mellitus. However, some researchers have shown that the plasma-immunoreactive ET concentration in diabetes mellitus is remarkably increased (Takahashi et al. 1990), while others have observed no change in this parameter (Predel et al. 1990). In this study, a highly sensitive and specific sandwich-enzyme immunoassay (EIA) for human ET-1 [l-211 and human big-ET-1 [l-381 was used (Suzuki et al. 1990) to measure simultaneously the plasma concentrations of ET-1 and big-ET-1 in 10 patients aged 32-55 years (43 k 3, x k s.e.m.) with diabetes mellitus type I1 (NIDDM). Their mean blood pressure, haemogloblin A1 and serum creatine concentration were 101.8k3.5 mmHg, 8.1 f 0 . 4 % and 0.75 k 0.03 mg/dL, respectively. Eight age-matched normotensive healthy subjects served as controls. All of the subjects were studied at Tohoku University Hospital. After 30 min of bed rest, blood samples were drawn from the cubital vein into polypropylene tubes containing aprotinin and EDTA-2Na. Plasma immunoreactive (ir)-ET-1 and ir-big-ET-I were extracted with Sep-pak C-18 cartridges and were subjected to sandwich EIA for ir-ET-1 and ir-big-ET-1. These assays consisted of two sets of antibodies that separately recognize the N-terminal and C-terminal portions of ET-1 and big-ET-1. A mouse monoclonal antibody (AwETN40) used as an immobilized antibody in the EIA recognizes the N-terminal loop domain of ET-I and big-ET-1 but does not react with the ET-1 Cterminal heptapeptide [ 15-21]. The Fab’ fragment of rabbit antibodies to C-terminal portions of ET-1 [15-211 or that of big-ET-1 [22-381 was used as the enzyme-labelled antibody after being coupled to horseradish peroxidase. The EIA for ET-1 did not crossreact with big-ET-1 and the EIA for big-ET-1 did not crossreact with ET-1, indicating that each ET-1 can be quantified separately by the EIA. The detection limits of the EIA for ir-ET-1 and ir-big-ET-1 were 0.2 pg per well for both assays. Plasma ir-ET-1 concentration was similar in the patients with diabetes mellitus and the healthy subjects (1.24k0.13 vs 1.45+0.17pg/mL, X+s.e.m., Fig. 1). However, plasma ir-big-ET-1 concentration was markedly elevated in the patients with diabetes mellitus compared with that of the healthy subjects (4.33 & 0.24 vs 3.31 k0.21 pg/mL, P<O.Ol evaluated by Student’s t-test, Fig. 1). Thus, the ratio of plasma ET-1 to plasma big-ET-1 was significantly decreased in the diabetic patients compared with that of the healthy subjects (0.29 + 0.02 vs 0.44 k 0.04, P<O.Ol, Fig. 1).

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.

Small adrenocortical tumors without apparent clinical endocrine abnormalities. Immunolocalization of steroidogenic enzymes.

Immunohistochemical analysis of steroidogenic enzymes (P-450 side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase, P-450 C21-hydroxylase, P-450 17 alpha-hydroxylase and P-450 11 beta-hydroxylase) was performed on fifteen cases of small adrenocortical adenomas, which were detected incidentally in hormonally asymptomatic patients (non-functioning adrenocortical tumor), in order to study steroidogenesis in these tumors. Immunolocalization revealed that all cases examined expressed all the enzymes in the adrenocortical steroidogenic pathway to various degrees, and in twelve cases abnormalities of precursor hormones and steroid metabolites were clinically observed. Attached non-neoplastic adrenals were present in twelve cases. Among these twelve cases, eight showed cortical atrophy, especially in the zona fasciculata. These atrophied adrenals expressed little immunoreactivity of the enzymes examined. These results strongly indicate that most of small non-functioning adrenocortical tumors have the capacity to produce biologically active steroids including cortisol, although not necessarily associated with hypercorticism. Especially in the cases with cortical atrophy in attached non-neoplastic adrenals, it is considered that autonomous neoplastic production and secretion of cortisol may be insufficient to cause clinical and routine laboratory abnormalities but sufficient to subtly alter the hypothalamic-pituitary-adrenal axis by suppressing ACTH and/or CRF secretion and to result in adrenocortical atrophy. It should be recognized that corticosteroidogenesis does take place in the majority of clinically small non-functioning adrenocortical tumors incidentally detected in hormonally asymptomatic patients when managing these patients.

Chronic synergistic effect of endothelin-1 and angiotensin II on blood pressure in conscious rats.

We assessed whether there is an interaction between angiotensin II (Ang II) and endothelin-1 (ET-1) in the regulation of blood pressure and sodium and water metabolism in rats. Male Sprague-Dawley rats were divided into four groups. Group I rats received Ang II at a subpressor dose (400 micrograms/kg/day i.p.) for up to 6 days. Group II rats received ET-1 at a subpressor dose (3 micrograms/kg/day i.v.) for up to 6 days. Group III rats received both the subpressor dose of Ang II and the subpressor dose of ET-1. Group IV rats received vehicle only. There was no significant difference in systolic blood pressure (SBP) among groups I, II, and IV during the study. On day 6, SBP in groups I, II, and IV was 148.0 +/- 3.0, 142.7 +/- 2.9, and 143.5 +/- 3.0 mm Hg, respectively. On the other hand, SBP in group III was higher than those of the other groups on day 2 and remained elevated thereafter. On day 6, SBP in group III rats was 189.0 +/- 12.5 mm Hg. There were no significant differences in body weight, fluid intake, urine volume, urinary sodium excretion, or urinary potassium excretion among the four groups. The present results suggest that Ang II and ET-1 exert their pressor effects synergistically and might play a role in controlling blood pressure. They also suggest the possibility of the existence of a common pathway in the hypertension-producing mechanism of these two peptides.

Effects of sodium and angiotensin II on urinary active and inactive kallikrein in rats

To assess possible relationships between sodium balance, angiotensin II (ANG II), and renal active and inactive kallikrein, we studied the effects of sodium loading with 1% NaCl and chronic ANG II infusion (900 micrograms/kg per day) on the urinary excretion of total and active kallikrein for 6 days in conscious rats. We determined urinary total, active and inactive kallikrein by measuring kallikrein activity using a kininogenase assay before and after treatment with trypsin (200 micrograms/ml). Sodium loading produced a sustained increase in urinary total, active and inactive kallikrein excretion. Chronic infusion of ANG II induced a sustained increase in urinary total, active and inactive kallikrein excretion in rats on a regular diet. In rats loaded with sodium, however, ANG II did not induce any further changes in urinary kallikrein excretion. Thus, the present study suggests that both sodium loading and ANG II infusion might stimulate the synthesis of renal kallikrein. In addition, it is suggested that ANG II infusion might stimulate the synthesis of kallikrein, at least partly, via the same mechanism as sodium loading does.