Yoshinobu Takata

Adrenoceptors in SHR: Alterations in binding characteristics and intracellular signal transduction pathways

There is much data on altered adrenoceptor function in the heart, blood vessel and kidney from spontaneously hypertensive rats (SHR). The enhancement of vascular and renal alpha-adrenoceptor function, i.e. vasoconstriction and retention of water and sodium, may contribute to the development and maintenance of the hypertension, whereas cardiac alpha1-adrenoceptor may be of minor physiological significance. Alpha1-adrenoceptor-mediated signal transduction as a whole is increased in SHR vascular tissues, but the intracellular signaling per receptor in the kidney seems to be decreased despite increased alpha1-adrenoceptor density. On the other hand, cardiac and vascular beta-adrenoceptor responsiveness is attenuated in SHR. Reduced vasorelaxation mediated by beta-adrenoceptors may also contribute to high blood pressure. The impaired cardiovascular beta-adrenoceptor function in SHR does not appear to be necessarily explained by alterations observed at receptor levels. Alterations in signal transduction should be also considered. Limited data on renal beta-adrenoceptor density and its signaling suggest decreased or unaltered cyclic AMP formation per receptor in SHR. We will review alterations in both binding characteristics and each component of intracellular signal transduction pathways in cardiovascular and renal adrenoceptors of SHR.

Differential Effects of Verapamil, Nicardipine and Diltiazem on Ca2+-Dependent and Ca2+-Independent Noradrenaline Release and Contraction in Isolated Canine Saphenous Veins

We reported that verapamil, nicardipine and diltiazem inhibited both 3H overflow and contraction induced by transmural nerve stimulation (TNS) in the canine saphenous vein preloaded with 3H-noradrenaline. In the present study, the effects of these Ca channel antagonists in the presence of tetraethylammonium (TEA) were investigated to evaluate whether the antagonists act by inhibiting Ca2+ influx into the nerve endings. The effects of the drugs on both responses to tyramine were also studied. In the 1 mmol/l-TEA-pretreated veins, verapamil and nicardipine inhibited only the contraction. Diltiazem inhibited the two parameters, the effects being nearly equipotent to those in the absence of TEA. The L-cis isomer of diltiazem inhibited the TNS-evoked responses to a similar degree as diltiazem, which was unaffected by 1 mmol/l TEA. Verapamil, nicardipine and diltiazem inhibited the TNS-evoked 3H overflow in 0.25 mmol/l Ca2+ and 20 mmol/l TEA medium, the inhibitions by the two former antagonists being greater than those seen in normal Krebs solution. TEA (1 mmol/l) added after the Ca channel antagonists, overcame the inhibition of the evoked overflow by diltiazem more poorly than that by verapamil or nicardipine. Only verapamil inhibited both responses to tyramine. These data together with previous results suggest that unlike verapamil and nicardipine, diltiazem may inhibit the TNS-evoked neurotransmitter release via a mechanism being unrelated to its Ca2+ influx-inhibiting action, resulting in an inhibition of the contraction, and that only verapamil inhibits the tyramine-evoked neurotransmitter release.

Antihypertensive activity and pharmacokinetics of KD3-671, a nonpeptide AT1-Receptor antagonist, in renal hypertensive dogs

The antihypertensive activity and pharmacokinetics of KD3-671 (previously named KT3-671), a nonpeptide AT1-receptor antagonist, were investigated in renal hypertensive dogs with normal or high plasma renin activity (PRA). A single administration of KD3-671 at 3 and 10 mg/kg, p.o., to the hypertensive dogs with high PRA dose-dependently reduced mean blood pressure (MBP), which was not correlated with plasma KD3-671 concentration. Significant increases in PRA and plasma angiotensin (Ang) II occurred 2 h after KD3-671 dosing. Enalapril at 3 mg/kg, p.o., also reduced MBP. Neither KD3-671 nor enalapril affected heart rate. When given orally once a day for 29 days to the hypertensive dogs with normal PRA, KD3-671 at 3 and 10 mg/kg/day dose-dependently reduced MBP, which was smaller than that in the dogs with high PRA. This was the case for enalapril. The hypotension induced by the first dose of KD3-671 or enalapril was consistently observed after doses 8, 15, 22, and 29. After cessation of repeated dosing, no rebound phenomenon in MBP was observed. Pharmacokinetic parameters of KD3-671 were not influenced by repeated dosing. KD3-671 markedly increased both PRA and plasma Ang II concentration at 2 h after dosing. These results suggest that KD3-671 may be useful for the treatment of hypertension.

Increases in vascular α1-adrenoceptor affinity and PI turnover in DOCA-salt hypertensive rats

The pD2 value for norepinephrine and the pA2 value for prazosin in mesenteric larger resistance vessels from deoxycorticosterone acetate (DOCA)-salt hypertensive rats were significantly higher than the values of normotensive rats, suggesting that alpha 1-adrenoceptors have a higher affinity in hypertensive rats. The accumulation of [3H]inositol monophosphate induced by norepinephrine was significantly greater in the mesenteric artery from hypertensive rats than in the mesenteric artery from normotensive rats. These results suggest that the enhanced phosphatidylinositol turnover seen in DOCA-salt hypertensive rats may contribute, at least partly, to the higher affinity of alpha 1-adrenoceptors.

KT3-671, an angiotensin AT1 receptor antagonist, attenuates vascular but not cardiac responses to sympathetic nerve stimulation in pithed rats.

Effects of KT3-671 on vascular and cardiac sympathetic neurotransmission were investigated in pithed rats. The pressor response to spinal stimulation (5 Hz) of the pithed rat without the adrenals was approximately 75% of that with the adrenals. Guanethidine (8 mg/kg, i.v.) decreased by about 76% the pressor response to sympathetic stimulation in the pithed rat with intact adrenals and the guanethidine-resistant response was almost completely abolished by bilateral adrenalectomy. Therefore, the following experiments were done using the pithed rat without the adrenals. KT3-671 (1–10 mg/kg, i.v.) as well as losartan (1–10 mg/kg, i.v.) inhibited dose-dependently the pressor response to sympathetic stimulation. KT3-671 was approximately four times more potent than losartan in inhibiting the pressor response. The two angiotensin II subtype 1 receptor antagonists (10 mg/kg, i.v.) did not affect the pressor response to exogenously administered norepinephrine. Neither KT3-671 nor losartan influenced the tachycardia induced by spinal stimulation and isoprenaline. Intravenous infusion of angiotensin II (100 ng/kg/min) did not affect both pressor and tachycardic responses to sympathetic stimulation. In conclusion, KT3-671 as well as losartan inhibits vascular but not cardiac sympathetic neurotransmission of the pithed rats, which may contribute to its overall antihypertensive efficacy.

Differential effects of Ca antagonists on the noradrenaline release and contraction evoked by nerve stimulation in the presence of 4-aminopyridine

1 We previously reported that verapamil, nicardipine and diltiazem inhibited both neurotransmitter release and contraction evoked by transmural nerve stimulation (TNS) in the canine saphenous vein. 2 To evaluate whether the three Ca antagonists act on the nerve endings by inhibiting Ca2+ influx, the effects of the three antagonists were studied in the presence of 4‐aminopyridine (4‐AP) 3 × 10–4 m on the TNS‐evoked tritium overflow and contraction of canine saphenous veins preloaded with [3H]‐noradrenaline. 3 4‐AP increased both tritium overflow and contraction evoked by TNS, but did not enhance the contraction induced by exogenous noradrenaline (10 nmol). In the veins pretreated with 4‐AP, verapamil (3 × 10–5 m) and nicardipine (10–5 m and 3 × 10–5 M) caused no significant effects on the TNS‐evoked tritium overflow, but they still inhibited the contraction. Diltiazem (10–5 m and 3 × 10–5 m) significantly inhibited both responses to TNS in the veins pretreated with 4‐AP, the effects being nearly equipotent to those in the absence of 4‐AP. The (–)‐cis isomer of diltiazem (10−5 m and 3 × 10−5 M), which is about 100 times less potent than diltiazem in inhibiting Ca2+ influx, inhibited both responses to TNS in the presence of 4‐AP to almost the same degree as diltiazem. 4 When 4‐AP was added after the Ca antagonists, it reversed the TNS‐evoked tritium overflow inhibiting actions of verapamil (3 × 10−5 m) and nicardipine (3 × 10−5 m) much more effectively than that of diltiazem (3 × 10−5 m). 5 Tetracaine (4 × 10−6 M) significantly inhibited the TNS‐evoked tritium overflow and contraction, which were unaffected by 4‐AP. 6 Sodium salicylate (10−2 M) failed to modify the inhibition of TNS‐evoked tritium overflow following diltiazem (3 × 10−5 M), but it enhanced that of tetracaine (4 × 10−6 M). 7 Verapamil but not diltiazem and nicardipine significantly increased the spontaneous tritium overflow from veins pretreated with 4‐AP. 8 The present study together with previous results suggests that diltiazem but not verapamil and nicardipine may inhibit the TNS‐evoked neurotransmitter release through an action other than inhibition of Ca2+ influx into the adrenergic nerve endings, allowing an inhibition of the resulting contraction.

A selective effect of protein kinase C activators on noradrenaline release compared with subsequent contraction in canine isolated saphenous veins

1 Effects of protein kinase C (PKC) activators and inhibitors on both tritium overflow and subsequent contraction evoked by transmural nerve stimulation (TNS) were investigated in canine saphenous veins prelabelled with [3H]‐noradrenaline. 2 Activation of PKC by stepwise increasing concentrations (0.01 nm–1 μm) of 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), phorbol 12,13‐dibutyrate (PDBu) or mezerein caused a significant and concentration‐dependent enhancement of the tritium overflow evoked by TNS, while the activators failed to affect the corresponding contraction except with the highest concentration of PDBu when the contraction was significantly reduced. Phorbol, which is inactive on PKC, had no effects on the tritium overflow and contraction induced by TNS. 3 PKC inhibitors, polymyxin B (1 and 10 μm) and the isoquinolinesulphonamide, H‐7 (1 μm), inhibited significantly the phorbol ester‐potentiated tritium overflow evoked by TNS with no effects on the contraction. H‐7 and the related inhibitor H‐8 at 10 μm reduced significantly both responses to TNS in the presence of TPA, while they suppressed only the TNS‐induced contraction in the absence of TPA. 4 None of the PKC activators or inhibitors affected the spontaneous tritium overflow. 5 PDBu (0.01 and 0.1 μm) elevated resting tension of the veins more effectively than TPA and mezerein. 6 These results suggest that PKC may modulate electrically stimulated noradrenaline release from adrenergic nerve endings of the canine saphenous veins and the PKC activators may act more selectively on presynaptic than postsynaptic sites, but have no apparent effect on postjunctional noradrenergic mechanisms.

Possible involvement of both N- and L-type voltage-dependent Ca channels in adrenergic neurotransmission of canine saphenous veins in low Ca2+ plus tetraethylammonium medium

SummaryThe involvement of N- and L-type voltage-dependent Ca channels (VDCCs) in adrenergic neurotransmission under the superfusion with 0.25 mM Ca2+ + 20 mM tetraethylammonium (low Ca2+ + TEA) medium has been studied by examining the effects of ω-conotoxin GVIA (ω-CTX) and dihydropyridine antagonists and agonist on transmural nerve stimulation (TNS)-evoked 3H overflow from canine saphenous veins preloaded with [3H]-noradrenaline. Nisoldipine (10 and 30 μM) and nifedipine (30 μM) reduced significantly the TNS-evoked 3H overflow in low Ca2+ + TEA medium, while the two dihydropyridine antagonists failed to suppress it in normal Krebs medium. Bay K 8644 (30 and 100 nM) produced a significant and concentration-dependent enhancement of the TNS-evoked 3H overflow in low Ca2+ + TEA medium. The enhancing effects of Bay K 8644 were antagonized by both 3 μM nisoldipine and 10 μtM nifedipine. ω-CTX inhibited markedly the TNS-evoked 3H overflow in both normal Krebs and low Ca2+ + TEA media, the inhibition by ω-CTX being ten times more potent in low Ca2+ + TEA medium. Nisoldipine (30 μM), when combined with 1 nM ω-CTX, produced a further significant inhibition of the TNS-evoked 3H overflow in low Ca2+ + TEA medium. However, no additional inhibition by 30 μM nisoldipine was observed when ω-CTX concentration was raised to 2 nM. In the veins superfused with normal Krebs medium, nisoldipine (30 μM) did not affect the inhibitory effect of 10 nM ω-CTX on the evoked 3H overflow. The low Ca2+ + TEA medium increased the spontaneous 3H overflow, which was not influenced by ω-CTX and dihydropyridines. These results suggest that in low Ca2+ + TEA medium but not normal Krebs one, Ca2+ entry via both N- and L-type VDCCs may be involved in adrenergic neurotransmission in the canine saphenous veins.