Yoshiaki Masuyama

Bone mineral density in women with essential hypertension

Recent studies have reported the abnormalities in calcium metabolism at the systemic level in human hypertension as well as in experimental hypertension. Because bone is the largest store of calcium in the body, the bone calcium content and mineralization may represent the entire calcium balance. The present study was undertaken to investigate the bone mineral density (BMD) in women with essential hypertension by means of the dual-energy X-ray absorptiometric (DXA) method. The DXA analysis showed a significant decrease in BMD in female hypertensive subjects compared with normotensive subjects. In addition, the BMD was inversely correlated with systolic blood pressure in women. The 24-h urinary calcium excretion was significantly greater in female hypertensive subjects than in female normotensive subjects. Furthermore, the greater the urinary calcium excretion, the lower the BMD in women. The values of serum total calcium, total magnesium, ionized calcium, and 1, 25(OH)2 vitamin D were not different between hypertensive and normotensive subjects. The results of the present study demonstrated that DXA provided an index of whole calcium balance, and suggest that high blood pressure might be associated with reduced BMD in female hypertension.

Effects of galanin on dopamine release in the central nervous system of normotensive and spontaneously hypertensive rats

Galanin is a 29-amino acid peptide and widely distributed in the brain, although its significance in the control of neural activities is undefined. In the present study, we describe the effects of galanin on the electrically evoked release of dopamine in the rat central nervous system. In addition, to elucidate a possible role of galanin in the regulation of dopaminergic transmission in hypertension, we examined whether the effect of galanin on dopamine release might be altered in the central nervous system of spontaneously hypertensive rats (SHR). Galanin (1 x 10(-8) to 1 x 10(-7) mol/L) inhibited the stimulation (1 Hz)-evoked [3H]dopamine release by a comparable amount in striatal slices of Sprague-Dawley rats, although the basal release of dopamine was not affected by the peptide. In the striatum of SHR, the electrical stimulation (1 Hz)-evoked [3H]dopamine release was significantly smaller than in the striatum of Wistar-Kyoto (WKY) rats. However, the inhibitory effect of galanin on the stimulation-evoked [3H]dopamine release was significantly more pronounced in SHR than in WKY rats. These results show that galanin significantly reduced the release of dopamine in rat striatum. Furthermore, the greater inhibitory effect of galanin on dopamine release in SHR suggests that galanin might actively participate in the regulation of dopaminergic nerve activity in hypertension.

Modulation of norepinephrine release by galanin in rat medulla oblongata.

Galanin, a 29-amino acid peptide, is widely distributed in both the central and peripheral nervous systems and is colocalized with catecholamines, although its physiological significance remains to be elucidated. In the present study we investigated the regulatory mechanisms of galanin on norepinephrine release in rat medulla oblongata. In slices of medulla oblongata of Sprague-Dawley rats, galanin inhibited the stimulation-evoked [3H]norepinephrine release in a concentration-dependent manner (fractional release ratio during electrical stimulation: control 0.937 +/- 0.043, mean +/- SEM, n = 6; galanin 1 x 10(-7) M 0.501 +/- 0.037, n = 6, p less than 0.05; and galanin 1 x 10(-6) M 0.299 +/- 0.018 n = 6, p less than 0.05). Galanin potentiated inhibition of [3H]norepinephrine release by the alpha 2-agonists (UK 14,304 and clonidine). The blockade of alpha 2-adrenergic receptors by RX 781094 diminished the inhibition of norepinephrine release by galanin. Pretreatment of pertussis toxin, which interferes with the coupling of inhibitory guanosine triphosphate-binding proteins to adenylate cyclase, significantly attenuated the suppressive effects of galanin on norepinephrine release. In slices of medulla oblongata obtained from spontaneously hypertensive rats (SHR), the inhibitory effect of galanin on norepinephrine release was significantly less than in those from age-matched Wistar-Kyoto rats. These results show that galanin might inhibit the stimulation-evoked norepinephrine release in rat medulla oblongata, at least partially mediated by alpha 2-adrenergic receptors and the pertussis toxin-sensitive guanosine triphosphate-binding proteins. Moreover, less suppression of norepinephrine release by galanin in SHR suggests that galanin might be involved in the regulation of central sympathetic nervous activity in hypertension.

Norepinephrine release and neuropeptide Y in medulla oblongata of spontaneously hypertensive rats.

Neuropeptide Y is colocalized with norepinephrine in both central and peripheral noradrenergic neurons. In this study, we examined the regulatory mechanisms of neuropeptide Y on norepinephrine release in the medulla oblongata of rats. Neuropeptide Y inhibited the stimulation-evoked [3H] norepinephrine release in a dose-dependent manner in slices of medulla oblongata of Sprague-Dawley rats (1 Hz, S2/S1 ratio, control, 0.946±0.040 [±SEM], n=6; neuropeptide Y 1×10* M, 0.676 ±0.022, n=6, p < 0.05; neuropeptide Y lxlO″7 M, 0.589±0.014, n=6, p < 0.05). Neuropeptide Y potentiated inhibition of [3H]norepinephrine release by the a⁁-agonists UK 14,304 and clonidine. The blockade of ≪,-adrenergic receptors by RX 781,094 diminished inhibitory effects of neuropeptide Y on norepinephrine release. Pretreatment of pertussis toxin (a toxin that interferes with the coupling of inhibitory receptors to adenylate cyclase) attenuated the suppression of norepinephrine release by neuropeptide Y. In spontaneously hypertensive rats, the inhibitory effect of UK 14,304 and neuropeptide Y on norepinephrine release from the medulla oblongata was significantly less than in age-matchedWistar-Kyoto rats. These results show that neuropeptide Y inhibits norepinephrine release partially mediated by o⁁-adrenergic receptors and the pertussis toxin-sensitive guanosine triphosphate-binding proteins in rat medulla oblongata. Furthermore, less suppression of norepinephrine release by UK 14,304 and neuropeptide Y in spontaneously hypertensive rats suggests that a,-adrenergic receptors and neuropeptide Y might be involved in the regulation of central sympathetic tone in hypertension.

Enhanced DNA synthesis of cultured vascular smooth muscle cells from spontaneously hypertensive rats Difference of response to growth factor, intracellular free calcium concentration and DNA synthesizing cell cycle

It is widely reported that cultured vascular smooth muscle cells (CVSMCs) from spontaneously hypertensive rats (SHR) show enhanced proliferation compared with cells from Wistar-Kyoto rats (WKY). The present studies were designed to find out whether this exaggerated proliferation in SHR is determined genetically and, if so, to evaluate the mechanism on the cell cycle. (1) Incorporation of [3H]thymidine into DNA was enhanced in CVSMCs from 3- and 12-week-old SHR compared with WKY but not in CVSMCs from DOCA-salt hypertensive rats compared with the cells from sham-operated rats. (2) DNA synthesis in SHR cells was enhanced further by addition of insulin (which is considered to be a progression factor) but not by arginine-vasopressin (AVP; considered to be a competence factor) or by angiotensin II (AII). On the other hand, insulin, AVP and AII significantly augmented DNA synthesis in WKY cells. (3) Intracellular free calcium concentration was slightly, but significantly, higher in SHR cells. (4) An increase in the population of DNA-synthesizing S-phase cells and decrease in (G2 + M)-phase cells in SHR were observed by flowcytometry. These data suggest (1) that enhanced DNA synthesis in CVSMCs from SHR is determined genetically, (2) that enhanced DNA synthesis in CVSMCs from SHR is largely dependent on an increased proportion of S-phase cells and (3) that this increase in S-phase cells in CVSMCs from SHR could be due to enhanced competence gene expression in SHR cells. (4) The increased intracellular free calcium concentration is compatible with an activation of the inositol-trisphosphate pathway.

Modulation of [3H]dopamine release by neuropeptide Y in rat striatal slices

Neuropeptide Y, a 36-amino-acid peptide, has a wide and specific distribution in the central nervous system. In this study we examined the regulatory mechanisms of neuropeptide Y on dopamine release in the rat central nervous system. The effects of neuropeptide Y on the electrically stimulated [3H]dopamine release were investigated in superfused striatal slices of Sprague-Dawley rats, spontaneously hypertensive rats and Wistar-Kyoto rats. Neuropeptide Y (1 x 10(-8) - 1 x 10(-7) mol/1) reduced the stimulation (1 Hz)-induced [3H]dopamine release by a comparable amount in Sprague-Dawley rats. The blockade of dopamine D2 receptors by the dopamine D2 receptor antagonist, sulpiride, diminished the inhibitory effects of neuropeptide Y on the stimulation-evoked [3H]dopamine release. Pretreatment of slices with pertussis toxin (a potent inhibitor of G1-proteins) attenuated the suppression of the stimulation-evoked [3H]dopamine release by neuropeptide Y. Unlabelled dopamine itself reduced the stimulation-evoked [3H]dopamine release, and the inhibitory effect was also attenuated in the pertussis toxin-pretreated slices. In spontaneously hypertensive rats, the inhibitory effect of neuropeptide Y on the stimulation-evoked [3H]dopamine release was more pronounced than that in Wistar-Kyoto rats. The results of the present study showed that neuropeptide Y inhibited the stimulation-evoked dopamine release partially mediated by dopamine D2 receptors and the pertussis toxin-sensitive G1-proteins in rat striatum. Furthermore, the greater effect of neuropeptide Y on dopamine release in spontaneously hypertensive rats suggests a possible involvement of the peptide in regulating the central dopaminergic nerve activity in hypertension.

Production of nerve growth factor by cultured vascular smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto rats.

Objective: Nerve growth factor (NGF) is a neurotrophic protein which acts on peripheral sympathetic nerves. Elevated NGF in vascular tissues of young spontaneously hypertensive rats (SHR) has been reported. The aim of the present study was to compare the amount of NGF secreted from cultured vascular smooth muscle cells (VSMC) and mesenteric artery and thoracic aorta segments from SHR and Wistar—Kyoto (WKY) rats. Methods: VSMC prepared by the enzyme digestion method from the thoracic aortic media of 14-week-old SHR and age-matched WKY rats were subcultured in Dulbeccos modified Eagles medium containing 10% fetal calf serum. Segments of mesenteric artery and thoracic aorta from 4-week-old SHR and age-matched WKY rats were similarly cultured. The NGF content in conditioned medium was measured using an enzyme immunoassay. The protein content of VSMC was measured by the Lowry method. Results: Total NGF content in the cell culture medium was increased during an exponential growth phase and then gradually decreased during a quiescent phase in both rat strains. There were no significant differences in the levels of NGF secreted from mesenteric artery and thoracic aorta segments between the SHR and WKY rats. The differences in cellular protein content between SHR and WKY rats were very small. Conclusions: In contrast to the reports of increased NGF in SHR tissues, our data demonstrate that NGF secretion was lower in VSMC from SHR, and was equivalent in mesenteric artery and thoracic aorta segments from SHR and WKY rats. We have no clear explanation for these observations, but the present results indicate that upregulation of NGF in SHR tissues is not responsible for a simple enhancement of NGF synthesis in VSMC, and suggest a breakdown of the regulatory mechanism or mechanisms of NGF gene expression in SHR tissues.

Hyperinsulinemia is a determinant of membrane fluidity of erythrocytes in essential hypertension.

In the present study, to determine a possible role of insulin in the regulation of membrane functions, we have examined the relationship between plasma insulin level and membrane fluidity of erythrocytes in patients with essential hypertension and normotensive subjects. Membrane fluidity of erythrocytes obtained from hypertensive and normotensive subjects were evaluated by means of an electron paramagnetic resonance and spin-labeling method. The order parameter (S for 5-nitroxide stearate) and the peak height ratio (ho/h(-1) for 16-nitroxide stearate) obtained from electron paramagnetic resonance spectra of erythrocyte membranes were significantly higher in patients with essential hypertension than in normotensive subjects. The finding indicated that the erythrocyte membrane fluidity was lower in essential hypertension than in normotensive controls. The plasma concentration of insulin while fasting was also significantly greater in hypertensive patients than in normotensive subjects. In addition, the plasma insulin level was significantly correlated with the values of the order parameter (S) and the peak height ratio (ho/h(-1)), which showed that the higher plasma insulin was associated with the lower membrane fluidity of erythrocytes. These results support the hypothesis that insulin may actively participate in the regulation of membrane fluidity of erythrocytes in essential hypertension.

Adrenomedullin and membrane fluidity of erythrocytes in mild essential hypertension

OBJECTIVE Adrenomedullin is a newly discovered 52 amino acid peptide that has a potent vasodilating action. The present study was undertaken to investigate the role of adrenomedullin in the regulation of membrane fluidity of erythrocytes in patients with essential hypertension. METHODS AND RESULTS We used an electron paramagnetic resonance and spin-labeling method. Adrenomedullin significantly decreased the order parameter for 5-nitroxide stearate and peak height ratio for 16-nitroxide stearate obtained from electron paramagnetic resonance spectra of erythrocyte membranes in normotensive volunteers (mean +/- SEM order parameter value: control, 0.718 +/- 0.003, n = 16; adrenomedullin at 10(-9) mol/l, 0.692 +/- 0.004, n = 16, P < 0.05; adrenomedullin at 10(-8) mol/l, 0.690 +/- 0.004, n = 16, P < 0.05; adrenomedullin at 10(-7) mol/l, 0.683 +/- 0.004, n = 16, P < 0.05). The findings showed that adrenomedullin increased the membrane fluidity of erythrocytes. In addition, the effect of adrenomedullin was significantly potentiated by prostaglandin E1 and dibutyryl cyclic AMP. In contrast, the calcium ionophore A23187 counteracted the actions of adrenomedullin. In patients with essential hypertension, who had higher order parameter values, the membrane fluidity of erythrocytes was significantly lower than in the normotensive control subjects (order parameter: 0.728 +/- 0.004 in hypertensives, n = 20; 0.692 +/- 0.002 in normotensives, n = 36, P < 0.01). The effect of adrenomedullin on membrane fluidity was more pronounced in the erythrocytes of essential hypertensive than in the erythrocytes of normotensive subjects (change in the order parameter with adrenomedullin at 10(-9) mol/l: -4.2 +/- 0.3% in hypertensives, n = 20; -1.8 +/- 0.2% in normotensives, n = 20, P < 0.05; adrenomedullin at 10(-8) mol/l: -4.5 +/- 0.3% in hypertensives, n = 20; -1.8 +/- 0.2% in normotensives, n = 36, P < 0.05). CONCLUSIONS The results of the present study demonstrate that adrenomedullin significantly increased the membrane fluidity of erythrocytes. The mechanisms were partially mediated by a prostaglandin E1- and cyclic AMP-dependent pathway which might be linked to changes in intracellular calcium kinetics. The greater effect of adrenomedullin in patients with essential hypertension suggests that the peptide might actively participate in the regulation of membrane functions in hypertension.

Effects of neuropeptide Y on norepinephrine release in hypothalamic slices of spontaneously hypertensive rats

We describe the effects of neuropeptide Y (NPY) on [3H]norepinephrine (NE) release from hypothalamic slices of spontaneously hypertensive rats (SHR). The electrical stimulation (1 Hz)-evoked [3H]NE release was significantly greater in hypothalamic slices of SHR than in those of Wistar Kyoto rats (WKY). NPY inhibited the stimulation-evoked [3H]NE release in a dose-dependent manner. The inhibitory effect of NPY was significantly attenuated in SHR compared with WKY. The results may indicate that the less inhibitory effect of NPY on NE release induces increased sympathetic nerve activity in the hypothalamus of SHR.