Regulatory effects of senescence marker protein 30 on the proliferation of hepatocytes.

Senescence marker protein 30 (SMP 30) is preferentially expressed in the liver. One of its remarkable functions is the protection of cells against various injuries by enhancement of membrane calcium-pump activity. We analyzed the role of SMP 30 in hepatocyte proliferation. SMP 30 expression was decreased initially, then increased along with hepatic regeneration, after carbon tetrachloride (CCl4) administration. SMP 30 expression was decreased in the necrotic phase and then gradually increased. Its increase was slightly delayed just after the mitotic phase. These results lead us to speculate that mitoses of hepatic cells induce enhanced SMP 30 expression. In contrast, administration of lead nitrate (LN) as a hepatic mitogen induced a more stable increase of SMP 30 expression. To estimate the effect of SMP 30 on cell proliferation, we evaluated hepatic mitosis in wild-type and SMP 30-deficient knockout (KO) mice after CCl4 administration. We found an increase in mitotic numbers in hepatocytes of KO mice. This result suggests that SMP 30 has a suppressive effect on cell proliferation. Suppressive activity of SMP 30 cDNA was shown in cultured hepatoblastic cells. Our results suggest that SMP 30 performs a regulatory function in liver regeneration.

Effects of ouabain on the growth and DNA synthesis of PC12 cells.

We investigated the effects of ouabain and serum from salt-loaded Dahl salt-sensitive (S) rats, which contain abundant ouabain-like compounds, on the growth and DNA synthesis of rat pheochromocytoma PC12 cells. Ouabain decreased the growth of PC12 cells, as evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, in a concentration-dependent fashion. A moderate concentration (10(-7) M) of ouabain increased DNA synthesis, as measured by 5-bromo-2'-deoxyuridine incorporation, and induced transcription of the proto-oncogenes c-myc and c-fos. Serum from salt-loaded Dahl S rats also enhanced DNA synthesis, but serum from Dahl salt-resistant rats did not. Thus ouabain-like compounds may modify the growth or differentiation of neural tissues. This effect may contribute to the development of salt-induced hypertension in Dahl S rats.

Adrenomedullin amidation enzyme activities in hypertensive patients.

Adrenomedullin (AM) is a potent vasodilating peptide secreted from the vasculature of various organs. It is biologically active when its C-terminus is amidated. Recently, an RIA method was developed for measurement of the active form of AM, or mature AM. We here employed this method to investigate the significance of amidation of AM in controlling cardiovascular function. Thirty-six patients under hemodialysis were recruited and divided into hypertensive (n = 25; 157/86 mmHg) and normotensive (n= 11; 116/66 mmHg) groups. Mature AM, immature AM and blood pressure were monitored during hemodialysis in all patients. There was a significant reduction in blood pressure during hemodialysis in both groups, although after hemodialysis blood pressure was still higher in hypertensives than in normotensives (139 +/-14.8/76 +/- 2.5 mmHg vs. 110 +/- 5.1/66.7 +/- 3.1 mmHg). Mature AM before hemodialysis were lower in hypertensives than normotensives and it decreased in both groups. Although mature AM decreased more in normotensives than in hypertensives (-27 +/- 8% vs. -17 +/- 5%), at the end point, its level was still higher in normotensives. The ratio of mature AM/immature AM decreased only in normotensives (-11.4 8.7%), whereas it remained stable in hypertensives (0.2 +/- 5.6%). Both groups showed similar changes in ANP, endothelin, catecholamines, cGMP, and NOx. The low level in mature AM level in hypertensives may have contributed to the higher blood pressure in this group. The attenuation of AM amidation in normotensives indicates that an unspecified amidative enzyme of AM was regulated in order to normalize blood pressure.

Hyperglycemia increases vascular adrenomedullin expression.

We have reported that plasma adrenomedullin (AM) in hyperglycemic patients was significantly increased compared with normal volunteers. In this report we examined the effects of hyperglycemia on AM expression in the vasculature, the main site of AM production. AM mRNA level in the aorta was higher in the diabetic rats than in the control rats. AM mRNA level and protein kinase C (PKC) activity in cultured vascular smooth muscle cells (VSMC) increased as the glucose concentration in the medium changed from 100mg/dl to 450mg/dl. PKC inhibitors blocked this increase of AM mRNA. Similar osmotic change with mannitol had no effect on AM expression. We conclude that (1) hyperglycemia increases vascular AM expression through PKC-dependent pathway, and (2) the elevated plasma AM in hyperglycemic patients originates from the glucose induced vascular AM expression. We propose the possible role of AM in the pathogenesis of diabetic vascular complications.

Senescence marker protein-30 (SMP30) rescues cell death by enhancing plasma membrane Ca(2+)-pumping activity in Hep G2 cells.

Senescence marker protein-30 (SMP30) has been reported to decrease with aging in the rat liver. SMP30 has been also suggested to play a role as a Ca(2+)-binding protein localized in cytosol of hepatocytes. To elucidate the functional significance of SMP30, we have generated Hep G2 cell lines that stably express large amounts of SMP30 by transfection with human SMP30 cDNA. Using these cell lines, in view of the intracellular Ca2+ homeostasis, we then investigated cytosolic free Ca2+ concentration ([Ca2+]i) and Na(+)-independent Ca2+ efflux from the cells after extracellular ATP stimulation. Although stimulation of cells with ATP caused transient [Ca2+]i increase in both SMP30 and mock transfectants, rate of decrease after peak in [Ca2+]i was enhanced 2-fold by transfection of SMP30. Correspondingly, Ca2+ efflux was significantly increased in SMP30 transfectants compared with mock transfectants. In addition, more SMP30 transfectants survived than mock transfectants when cell death was induced by Ca2+ ionophore treatment. These results suggest that SMP30 regulates [Ca2+]i by modulating plasma membrane Ca(2+)-pumping activity, and thus down-regulation of SMP30 during aging may contribute to deterioration of cellular functions.

A newly identified peptide, proadrenomedullin N-terminal 20 peptide, induces hypotensive action via pertussis toxin-sensitive mechanisms.

Proadrenomedullin N-terminal 20 peptide (PAMP) and adrenomedullin (AM) are novel hypotensive peptides. Although they are derived from the same gene product, proadrenomedullin, their hypotensive mechanisms are different; PAMP inhibits the release of norepinephrine from the peripheral sympathetic nerve endings, whereas AM fosters vasodilation by elevating intracellular cAMP, possibly via activation of cholera toxin-sensitive G proteins. In PC12 cells, PAMP inhibited N-type calcium channel via activation of pertussis toxin-sensitive mechanisms. To clarify the relationship between the hypotensive effect of PAMP and pertussis toxin-sensitive mechanisms, we administered pertussis vaccine intraperitoneally into rats for 3 consecutive days. By using mesenteric artery preparation, we showed that PAMP's ability to decrease norepinephrine overflow was significantly attenuated in pertussis toxin-treated rat (-18.5 +/- 6.9%; P<.05 versus control rats). In electrically stimulated pithed rat, PAMP (20 and 40 nmol/kg) showed a hypotensive effect (-13 +/- 5 and -18 +/- 7 mm Hg, respectively; P<.05, P<.01), whereas in pertussis vaccine-treated rat it did not (-2 +/- 3 and -8 +/- 9 mm Hg, respectively; P=NS). Also, in pithed rat, plasma norepinephrine level was significantly elevated by electrical stimulation in both control (0.323 +/- 0.035 ng/mL) and pertussis vaccine-treated groups (0.355 +/- 0.079 ng/mL). After injection of PAMP (40 nmol/kg), plasma norepinephrine level significantly decreased in the control group (0.225 +/- 0.044 ng/mL; P<.01) but not in the pertussis vaccine-treated group (0.392 +/- 0.021 ng/mL; P=NS). Moreover, in conscious rats, intravenous administration of PAMP (40 nmol/kg) did not evoke hypotension after pertussis vaccine treatment, although untreated controls had significantly decreased arterial pressure (-5 +/- 2 versus -20 +/- 3 mm Hg; P<.01). In contrast to PAMP, the administration of AM (1 nmol/kg) significantly reduced the blood pressure of pertussis vaccine-treated as well as control rats (-20 +/- 5 versus -18 +/- 7 mm Hg; P=NS). These results demonstrate that the ability of PAMP to inhibit norepinephrine release from peripheral sympathetic nerve endings and to decrease blood pressure is pertussis toxin sensitive. Our findings thus suggest that despite being derived from the same gene, PAMP and AM apparently produce hypotension by activating different signaling pathways.

Proadrenomedullin N-terminal 20 peptide (PAMP) inhibits proliferation of human neuroblastoma TGW cells.

We investigated the effects of proadrenomedullin N-terminal 20 peptide (PAMP) and adrenomedullin (AM) on the growth of human neuroblastoma TGW cells. Both PAMP and AM inhibited growth and DNA synthesis in neuroblastoma cells. Calcitonin gene-related peptide (CGRP)(8-37), an antagonist to CGRP, abolished the inhibitory effect of AM on growth and DNA synthesis of neuroblastoma cells but did not affect that of PAMP. AM(22-52), an antagonist to AM, also reversed the effect of AM. On the other hand, pertussis toxin (PTX) and omega-conotoxin GIVA blocked the effect of PAMP alone. Thus, PAMP inhibits the growth of neuroblastoma cells by inhibiting N-type Ca2+ channels through PTX-sensitive G protein-coupled receptors, which is different mechanism of AM-induced inhibition of the cell growth.

Effect of insulin on norepinephrine overflow at peripheral sympathetic nerve endings in young spontaneously hypertensive rats.

To determine how the effect of insulin is related to the development of salt-induced hypertension, and whether a hyporesponse to insulin exists in the peripheral sympathetic nerves of a hypertensive model rat, we measured norepinephrine overflow from the periarterial nerve of isolated mesenteric arteries exposed to insulin in spontaneously hypertensive rats (SHR) as well as Wistar-Kyoto rats (WKY) fed diets that were high and low in salt. Salt loading (diet containing 8% salt for 4 weeks) accelerated the development of hypertension in young, spontaneously hypertensive rats (SHR) (157 +/- 5 mm Hg v 198 +/- 4 mm Hg, P < .01) but did not affect the blood pressure of Wistar-Kyoto rats (WKY) (102 +/- 7 mm Hg v 104 +/- 6 mm Hg, P = NS). Basal norepinephrine overflow did not differ in the SHR and WKY rats, but the overflow of norepinephrine after periarterial electrical stimulation (8 Hz 1 min.) was significantly greater in SHR (0.806 +/- 0.079 ng/g) than in WKY (0.723 +/- 0.022 ng/g; P < .01). Although insulin reduced the norepinephrine overflow by periarterial nerve stimulation in both WKY and SHR, the decrease with insulin was significantly greater in the SHR than in WKY (-18.4% +/- 4.0% v -32.0% +/- 4.6%, P < .05). The inhibitory effect of insulin on norepinephrine overflow was reduced by salt loading in SHR (-8.8% +/- 4.0%, P < .05), but not in WKY (-32.5% +/- 4.7%, P = NS). Cocaine and ouabain completely blocked the effect of insulin in all four groups. In contrast to insulin, direct stimulation of Na(+)-K+ ATPase with a high-potassium buffer (12 mmol/L) reduced NE overflow to the same extent among the four groups. These findings show that SHR have a blunted response to the suppression by insulin of norepinephrine overflow. Salt loading reduced the insulin response at peripheral sympathetic nerves of young SHR, but did not affect that of age-matched WKY. Thus, hyporeactivity to insulin may play a role in the development of salt-induced hypertension in young SHR, possibly through a reduced suppression of norepinephrine overflow from sympathetic nerve endings.

Hypotensive effect of a newly identified peptide, proadrenomedullin N-terminal 20 peptide.

Proadrenomedullin N-terminal 20 peptide (PAMP) and adrenomedullin (AM), which are both derived from proadrenomedullin, exhibit marked hypotensive effects. We recently reported that PAMP but not AM reduced the release of norepinephrine from peripheral sympathetic nerve endings. Our present objective was to clarify the involvement of the sympathetic nervous system in the hypotensive action of PAMP and AM. Intravenous administration of PAMP (10, 20, and 50 nmol/kg) to conscious rats induced less reflex tachycardia (5 +/- 5, 10 +/- 5, and 14 +/- 6 beats per minute [bpm]) than that of AM in 0.1, 0.5, and 1.0 nmol/kg doses (5 +/- 8, 20 +/- 7, and 38 +/- 5 bpm, P < .01) although both agents showed similar hypotensive effects. We evaluated the effect of PAMP on blood pressure in pithed rats whose sympathetic nervous systems were abolished. In pithed rats, AM (-2 +/- 1, -7 +/- 1, and -10 +/- 3 mm Hg; NS, P < .05, and P < .01, respectively) but not PAMP evoked hypotension. In contrast, administration of PAMP (-3 +/- 1, -11 +/- 2, and -14 +/- 4 mm Hg; P < .05, P < .01, and P < .01, respectively) as well as adrenomedullin (-2 +/- 2, -10 + 3, and -15 +/- 4 mm Hg; NS, P < .01, and P < .01) significantly reduced blood pressure in electrically stimulated, pithed rats, which had reached almost the same levels as in conscious rats. In electrically stimulated, pithed rats, plasma norepinephrine level was reduced by PAMP but not by vehicle or AM. These findings suggest that the hypotensive effect of PAMP is mainly due to inhibition of peripheral sympathetic nerve activity.

Proadrenomedullin NH(2)-terminal 20 peptide, a new product of the adrenomedullin gene, inhibits norepinephrine overflow from nerve endings.

Proadrenomedullin NH(2)-terminal 20 peptide (PAMP) and adrenomedullin, which are derived from proadrenomedullin, exhibit remarkable hypotensive action. We investigated the effect of PAMP and adrenomedullin on peripheral sympathetic neutral transmission. Using perfused rat mesenteric arteries, PAMP (0, 1, 5, and 10 pmol/ml) decreased norepinephrine overflow by periarterial electrical nerve stimulation in a dose-dependent fashion (0.244 +/- 0.043, 0.231 +/- 0.048, 0.195 +/- 0.061 and 0.168 +/- 0.051 ng/gram tissue weigh: NS, P < 0.05, and P < 0.02, respectively). In contrast to PAMP, adrenomedullin (1, 5, and 10 pmol/ml) did not change it. In contrast, vasoconstrictive response of mesenteric arteries to exogenous norepinephrine was significantly attenuated by 10 pmol/ml of adrenomedullin but not by the same dose of PAMP. Calcitonin gene-related peptide (8-37) [CGRP(8-37)], a CGRP receptor antagonist, inhibited the vasodilatory effect of adrenomedullin but could not suppress the sympathoinhibitory effect of PAMP. Neither a nicotinic antagonist, hexamethonium, nor a presynaptic alfa2 antagonist, yohimbine, blocked the sympathoinhibitory effect of PAMP. Thus, it suggests that PAMP and adrenomedullin, which are derived from the same gene, exhibit different hypotensive mechanisms: PAMP inhibits neural transmission at peripheral sympathetic nerve ending, although adrenomedullin directly dilates vascular smooth muscle, possibly through CGRP-like receptor.

Tumor necrosis factor-alpha induces vascular hyporesponsiveness in Sprague-Dawley rats.

The cytokine tumor necrosis factor-alpha (TNF alpha) is one of the major mediators of septic shock. Because vasodilation is a hallmark of sepsis and decreased vascular responsiveness has been implicated in the pathogenesis of septic shock, we studied the effect of TNF alpha on the mean blood pressure in conscious rats and vascular responsiveness to vasoconstrictors ex vivo using the standard organ bath method. Intravenous infusion of TNF alpha (0.006 or 0.06 mg/kg/hr for 10 hours) decreased mean blood pressure in a dose-dependent fashion. Contractile responses to norepinephrine were depressed dose-dependently in the aortic rings both with and without its endothelium. Aortic contractions by potassium depolarization were also depressed. These results suggest that TNF alpha induces non-specific vascular hyporesponsiveness, which is independent of the presence of the endothelium. The TNF alpha-induced vascular hyporesponsiveness might contribute to the hypotensive action of TNF alpha.