Koichi Miyagawa

Increased oxidative stress impairs endothelial modulation of contractions in arteries from spontaneously hypertensive rats

Objectives The endothelium modulates vascular contractions. We investigated the effects of oxidative stress on endothelial modulation of contractions in hypertension. Methods Changes in isometric tension of femoral arterial rings from spontaneously hypertensive (SHR) and Wistar–Kyoto (WKY) rats were recorded. Results The contractile response to norepinephrine of arteries with endothelium was greater in SHR than in WKY rats (P < 0.0001). Endothelium removal augmented the norepinephrine-induced contraction (P < 0.05). The augmentation was more pronounced in WKY than in SHR, which resulted in comparable contraction of arteries without endothelium in both strains. Nω-nitro-L-arginine methyl ester (100 μmol/l) mimicked the effect of endothelium removal. Production of nitric oxide (NO, assessed by measuring nitrite/nitrate concentrations) during the contraction was not different between SHR and WKY. Vitamin C suppressed the contraction of arteries with endothelium from SHR but not from WKY (P < 0.05). Diphenyleneiodonium and apocynin, inhibitors of nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase, attenuated the contraction of arteries with endothelium from SHR (P < 0.001) but not WKY, but did not affect contractions induced by serotonin. Superoxide generated by xanthine oxidase/hypoxanthine enhanced the norepinephrine-induced contraction of arteries with endothelium from WKY (P < 0.0001), and this effect was reversed by vitamin C. Conclusions In rat femoral arteries, NO released from the endothelium modulates vascular contraction. In SHR, production of superoxide by NADH/NADPH oxidase, which may be activated by norepinephrine, is enhanced, resulting in the inactivation of NO and impairment of endothelial modulation of vascular contractions. Vascular oxidative stress may contribute to the altered circulation in hypertension by impairing endothelial modulation of vascular contractions.

In vitro modulation of primate coronary vascular muscle cell reactivity by ovarian steroid hormones

Susceptibility to drug‐induced coronary vasospasm in rhesus monkeys increases after removal of the ovaries and can be normalized by adding back physiological levels of estradiol‐17β (E2) and/or natural progesterone (P) in vivo as reported recently by our group. Furthermore, the reactivity status (Ca2+ and protein kinase C responses) of freshly isolated and primary culture coronary artery vascular muscle cells (VMC) mimic the intact coronary artery responses to 5‐HT + U46619. Since coronary reactivity is maintained in the isolated VMC, we hypothesized that the reactivity state inherent in the VMC was modulated directly by ovarian steroids in vitro as in the whole animal. To test this hypothesis, we treated hyperreactive VMC from ovariectomized (ovx) monkeys in vitro with E2 or P and measured VMC reactivity to combined stimulation with 5‐HT and U46619, as determined by the amplitude and especially the duration of intracellular Ca2+ signals, as well as protein kinase C (PKC) activation/translocation. VMC were treated for 12–96 h with 3–100 pg/ml E2 (10–365 pM) and/or 0.3–3 ng/ml P (0.95–9.5 nM). Hyperreactive responses to the combination of 5‐HT and U46619 in untreated VMC were significantly and dose‐dependently reduced by treatment in vitro with physiological levels of either E2 or P for at least 24 h. Both the early transient and late sustained increases in intracellular Ca2+ and PKC translocation were blunted, and the effects of 0.2 nM E2 and 3.2 nM P were specifically antagonized by the receptor blockers ICI 182,780 (200 nM) and RU486 (15 nM), respectively. Antibodies to the estrogen receptor and progesterone receptor labeled nuclei in VMC, which were also positively labeled by a smooth muscle myosin heavy chain monoclonal antibody. These data indicate that natural ovarian steroids directly reduce hyperreactive 5‐HT and thromboxane A2‐stimulated Ca2+ and PKC responses of coronary artery VMC from surgically menopausal rhesus macaques. We hypothesize that vascular hyperreactivity, which may be a critical factor involved in the increased incidence of coronary artery vasospasm and ischemic heart disease in postmenopausal women, can be normalized by E2 and/or P through direct actions on coronary artery vascular muscle cells.—Minshall, R. D., Miyagawa, K., Chadwick, C. C., Novy,M. J., Hermsmeyer, K. In vitro modulation of primate coronary vascular muscle cell reactivity by ovarian steroid hormones. FASEB J. 12, 1419–1429 (1998)

Physiologic and pathophysiologic relevance of T-type calcium-ion channels: potential indications for T-type calcium antagonists.

The family of voltage-gated calcium-ion (Ca2+) channels is critical in the role of transmembrane signaling of excitable cells throughout the body. Within the cardiovascular system, two types of Ca2+ channels have been identified: the L-type channel and the T-type channel. These two types of Ca2+ channels have distinct electrophysiologic identities, and although the roles of the T-type Ca2+ channels have not been firmly established, there are many reasons for believing that the roles of the T-type and L-type Ca2+ channels are distinct. T-type Ca2+ channels have the appropriate characteristics to generate pacemaker activity in the sinoatrial node. In vascular smooth muscle, they appear to be involved in maintenance of coronary and peripheral vasomotor tone and control of vascular growth and remodeling. Characterization of the T-type Ca2+ channels will be facilitated by the availability of mibefradil, a novel calcium antagonist that selectively blocks T-type Ca2+ channels. Mibefradil is associated with a reduction in heart rate but not with negative inotropic effects or neurohormonal stimulation. It is thought that the unique pharmacologic effects of mibefradil are related to blockade of T-type Ca2+ channels, and it is hypothesized that this action will have a positive impact on cardiovascular morbidity and mortality via cardioprotective and renoprotective effects. However, much work needs to be done to fully test this hypothesis.

Protein kinase C mechanism enhances vascular muscle relaxation by the Ca2+ antagonist, Ro 40-5967.

Actions of the novel Ca2+ antagonist, Ro 40-5967, which displays unusual efficacy against endothelin (ET)-induced contractions, were studied in isolated vascular muscle cells (VMCs) using the fluorescent protein kinase C (PKC) indicator, BODIPY 12 alpha-phorbol ester 13 beta-acetate (PBA-BODIPY). High-sensitivity (photon-counting) digital-imaging microscopy quantified PKC distribution within VMCs and showed translocation from the cytosol to the cell surface membrane on stimulation with ET. ET (1 nM) stimulated translocation of PBA-BODIPY fluorescence that peaked at 4 min, increasing from 19 +/- 2% to 29 +/- 2% surface membrane (edge) distribution (n=44, p<0.05). Increases in membrane-associated PKC due to translocation began within 2 min and persisted for about 10 min, after which a gradual decline to control levels occurred. Upon exposure to Ro 40-5967 (10 microM), there was an inhibition of fluorescence intensity throughout the cell. Average fluorescence intensity decreased to 84 +/- 4% (n=20, p<0.05) of that in prestimulus controls. Cell/membrane was also reduced to below unstimulated control levels. Amlodipine failed to decrease PKC fluorescence intensity or translocation to the surface membrane. These data suggest that there is an important direct PKC inhibitory action of Ro 40-5967 that would at least partially explain relaxation of ET-induced contractions.

Reactivity-based coronary vasospasm independent of atherosclerosis in rhesus monkeys

OBJECTIVES We studied the hypothesis that in the absence of vascular pathology, coronary artery vasospasm occurs as a result of local regions of vascular muscle hyperreactivity. We aimed to explore the basis for a functional etiology of those vasospasms not explained on a structural basis. BACKGROUND Ovariectomized rhesus monkeys (Macaca mulatta) without injury or significant vascular disease were stimulated with platelet release products, and angiograms were compared with those from vasospasms induced in human patients. METHODS We used intracoronary (IC) injections of serotonin, thromboxane A2 (U46619), endothelin 1 or angiotensin II in concentrations 3 to 10 times that which reduced coronary artery diameter by 50%. RESULTS Although no agent alone caused vasospasm, the combination of pathophysiologic concentrations of serotonin and the stable thromboxane A2 mimetic, U46619, injected through an IC catheter, synergistically caused coronary vasospasm on the second or third challenge in five of seven monkeys. These drug-induced vasospasms were similar to vasospasms induced by mechanical injury followed by serotonin, and to those stimulated in human IC diagnostic tests, as judged by onset, appearance, kinetics and vasodilator reversal. CONCLUSIONS These studies in ovariectomized monkeys revealed that coronary vasospasm can be stimulated without preexisting vascular pathology, endothelial denudation or injury. Reproducible vasospasm of primate coronary arteries in response to these two endogenous pathophysiologic vasoconstrictors, which are thought to be precipitating stimuli in the etiology of vasospasm, suggests that structure-independent epicardial vasospasm can be an important element in serious cardiac ischemic events, particularly the focal, persistent vasospasms that occur without plaques or injury.

Effects of mibefradil on intracellular Ca2+ release in cultured rat cardiac fibroblasts and human platelets

The Ca2+ antagonist mibefradil at supratherapeutic concentrations induced a sustained increase of cytosolic Ca2+ in cultured rat cardiac fibroblasts and human platelets which lack sensitivity to K+ depolarization and Ca2+ channel block by verapamil or other Ca2+ antagonists. At concentrations above 10 μM, mibefradil elevated substantially cytosolic [Ca2+] without affecting the peak level of agonist-induced Ca2+ transients. These Ca2+-mobilizing actions of 10 or 100 μM mibefradil stand in contrast to the Ca2+ antagonism and relaxation of vascular muscle at 1μM concentrations. Since a substantial part of mibefradil-induced increase in cytosolic Ca2+ was independent of extracellular Ca2+, and in order to define better the mechanism of Ca2+ increase, we exposed permeabilized cultured rat cardiac fibroblasts and human platelets to mibefradil at concentrations sufficiently high to identify covert effects. In permeabilized fibroblasts or platelets mibefradil at concentrations above 10 μM activated dose-dependent Ca2+ release from intracellular Ca2+ stores. Verapamil had no effect at concentrations of up to 100 μM. Mibefradil-induced Ca2+ release was not affected by ryanodine, thapsigargin, removal of ATP or dithioerythreitol, indicating that neither Ca2+- nor disulfide reagent-induced Ca2+ release were involved and that mibefradil did not release Ca2+ by inhibition of the Ca2+-ATPase pump of endoplasmic reticulum. The rate, but not the amplitude, of mibefradil-induced Ca2+ release is increased up to fourfold in the presence of pentosan polysulphate or heparin, two potent inhibitors of inositol 1,4,5-trisphosphate-induced Ca2+ release. Depletion of Ca2+ stores of permeabilized cells using inositol 1,4,5-trisphosphate in the presence of thapsigargin completely blocked mibefradil-induced Ca2+ release, and depletion of Ca2+ stores by mibefradil prevented further Ca 2+ release by inositol 1,4,5-trisphosphate. Mibefradil at supratherapeutic concentrations (≥/10 μM) thus mobilized Ca2+ from an inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in cultured rat cardiac fibroblasts and human platelets.

Renoprotective Effect of Calcium Channel Blockers in Combination with an Angiotensin Receptor Blocker in Elderly Patients with Hypertension. A Randomized Crossover Trial Between Benidipine and Amlodipine

Anti-hypertensive medication with an angiotensin II receptor blocker (ARB) is effective in slowing the progression of chronic kidney disease. The present study was designed to investigate whether calcium channel blockers (CCBs) in combination with an ARB differentially affect kidney function. Elderly hypertensive patients with chronic kidney disease (n = 17, 72 ± 6 years old) were instructed to self-measure blood pressure. They were randomly assigned to receive either benidipine (4–8 mg/day) or amlodipine (5–10 mg/day) combined with olmesartan (10 mg/day). After 3 months, CCBs were switched in each patient and the same protocol was applied for another 3 months. At baseline, significant correlation was obtained between urine albumin (22.8 ± 16.7 (median ± median absolute deviation) mg/g creatinine) and self-measured blood pressure (170 ± 23/87 ± 10 (mean ± SD) mmHg, r = 0.65, p < 0.01). Both regimens reduced blood pressure to a similar extent (139 ± 22/75 ± 11 mmHg and 133 ± 17/72 ± 10 mmHg, respectively; both p < 0.001), while urine albumin decreased only after combination therapy including benidipine (11.7 ± 6.1 mg/g creatinine, p < 0.05). Benidipine, but not amlodipine, in combination with olmesartan, reduced urinary albumin excretion in elderly hypertensive patients with chronic kidney disease. The results suggest the importance of selecting medications used in combination with ARB in hypertensive patients with chronic kidney disease.

Novel concepts in Ca2+ channel selectivity

Properties of mibefradil Mibefradil produces a unique combination of T-type Ca2+ channel block and protein kinase C inhibition, and thus effective vasodilation, without adverse positive chronotropic or negative inotropic cardiac actions. Lack of reflex sympathetic activation In contrast to virtually every other vasodilator, mibefradil avoids the reflex sympathetic activation and increased heart rate that form the practical limit for existing vasodilator action. The freedom from increased heart rate is especially important for maintaining an adequate blood flow in the coronary circulation. Conclusions Mibefradil offers a new concept in Ca2+ channel antagonism, with long-term protection against the pathophysiological consequences of hypertension.