Elevated sympathetic nervous activity in mice deficient in alphaCGRP.

alpha-Calcitonin gene-related peptide (alphaCGRP) is a pleiotropic neuropeptide implicated in a variety of physiological processes. To better understand the biological functions of alphaCGRP, we developed an alphaCGRP-null mouse model using a gene targeting approach. Recordings of mean arterial pressure (MAP) and heart rate (HR) showed that basal MAP and HR were significantly higher in both anesthetized and conscious, unrestrained alphaCGRP-null mice than in corresponding wild-type mice. The elevated MAP in alphaCGRP-null mice was shown to be the result of elevated peripheral vascular resistance by alpha-adrenergic blockade with prazosin and by transthoracic echocardiogram, which revealed no significant differences between alphaCGRP-null and wild-type mice in the stroke volume, fractional shortening, and ejection fraction. Moreover, evaluation of autonomic nervous activity by measuring HR after pretreatment of atropine and/or atenolol and by analyzing arterial baroreceptor reflexes showed sympathetic nervous activity to be significantly elevated in alphaCGRP-null mice; elevated levels of urinary catecholamine metabolites and decreased HR variability in mutant mice were also consistent with that finding. These findings suggest that alphaCGRP contributes to the regulation of cardiovascular function through inhibitory modulation of sympathetic nervous activity.

Diet-induced hyperhomocysteinemia exacerbates neointima formation in rat carotid arteries after balloon injury.

BACKGROUND: Increasing evidence indicates that elevated plasma homocysteine levels are associated with an increased risk of atherosclerosis and endothelial dysfunction, although little specific information on the mechanisms responsible for the atherogenic effects of homocysteine or on the in vivo contribution made by hyperhomocysteinemia to atherosclerosis is currently available. Because homocysteine is known to exert a direct inhibitory effect on endothelial cell growth in vitro, we hypothesized that this effect contributes to the progression of atherosclerotic lesions initiated by endothelial damage caused by mechanical injury. METHODS AND RESULTS: We prepared diet-induced hyperhomocysteinemic rats in which neointima formation after balloon injury to the common carotid artery was assessed. Moderate hyperhomocysteinemia (plasma homocysteine levels 3- to 4-fold higher than control) significantly exacerbated neointima formation. Oral administration of folate, which had a homocysteine-lowering effect, diminished neointima formation induced by moderate hyperhomocysteinemia. Furthermore, the attenuation of reendothelialization was shown in diet-induced hyperhomocysteinemic rats with Evans blue staining. CONCLUSIONS: Diet-induced hyperhomocysteinemia, even mild to moderate, exacerbates neointima formation after denuding injury, making hyperhomocysteinemia a likely risk factor for postangioplasty restenosis. It may be mediated through an inhibitory effect of homocysteine on reendothelialization. Homocysteine lowering with folate supplementation can effectively ameliorate the detrimental effects of moderate hyperhomocysteinemia. Clinical trials would seem to be warranted.

Hypotension and resistance to lipopolysaccharide-induced shock in transgenic mice overexpressing adrenomedullin in their vasculature.

BACKGROUND: Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. To determine the extent to which chronic AM overproduction affects circulatory physiology under normal and pathological conditions, we used a preproendothelin-1 promoter to establish transgenic mouse lines overexpressing AM in their vasculature. METHODS AND RESULTS: Transgenic mice overexpressing AM mainly in vascular endothelial and smooth muscle cells exhibited significantly lower blood pressure (BP) and higher plasma cGMP levels than their wild-type littermates. Blockade of NO synthase with N(G)-monomethyl-L-arginine elevated BP to a greater degree in AM transgenic mice, offsetting the BP difference between the 2 groups. Despite their lower basal BP, administration of bacterial lipopolysaccharide elicited smaller declines in BP and less severe organ damage in AM transgenic mice than in wild-type mice. Furthermore, the 24-hour survival rate after induction of lipopolysaccharide shock was significantly higher in the transgenic mice. CONCLUSIONS: A chronic increase in vascular AM production reduces BP at least in part via an NO-dependent pathway. In addition, smaller responses to LPS in transgenic mice suggest that AM is protective against the circulatory collapse, organ damage, and mortality characteristic of endotoxic shock.

ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function.

A disintegrin and metalloproteinase (ADAM) represents a protein family possessing both metalloproteinase and disintegrin domains. ADAMTS-1, an ADAM family member cloned from cachexigenic colon adenocarcinoma, is unusual in that it contains thrombospondin type I motifs and anchors to the extracellular matrix. To elucidate the biological role of ADAMTS-1, we developed ADAMTS-1-null mice by gene targeting. Targeted disruption of the mouse ADAMTS-1 gene resulted in growth retardation with adipose tissue malformation. Impaired female fertilization accompanied by histological changes in the uterus and ovaries also resulted. Furthermore, ADAMTS-1(-/-) mice demonstrated enlarged renal calices with fibrotic changes from the ureteropelvic junction through the ureter, and abnormal adrenal medullary architecture without capillary formation. ADAMTS-1 thus appears necessary for normal growth, fertility, and organ morphology and function. Moreover, the resemblance of the renal phenotype to human ureteropelvic junction obstruction may provide a clue to the pathogenesis of this common congenital disease.

Evidence for association between paraoxonase gene polymorphisms and atherosclerotic diseases.

Paraoxonase 1 (PON1) is proposed to have an anti-atherogenic action. Two polymorphisms at the PON1 (M/L55 and Q/R192) have been shown to be associated with coronary artery disease (CAD). This conclusion is not drawn universally, however, and specific ethnic characteristics may be important determinants in this association. Recently two homologues of PON1 - PON2 and PON3 - were identified and Sanghera et al. demonstrated C/S311 polymorphism at PON2 was associated with the risk of CAD. Within that context, we investigated the association between the aforementioned three polymorphisms and CAD and ischemic stroke in a Japanese population. The study population included 431 control subjects, 210 CAD patients, and 235 ischemic stroke patients. Genotype distributions and allele frequencies of M/L55 and C/S311 were similar among the control and patient groups, whereas the R192 allele frequency was significantly higher (P<0.001) in CAD (75%) and ischemic stroke (76%) patients than in control subjects (65%). When confounding influences of other risk factors were controlled for by multivariate analysis, R192 remained an independent risk determinant (additive model: OR (95% CI), P value CAD: 2.01 (1.45-2.79), 0.0001; ischemic stroke: 1.84 (1.34-2.52), 0.0002 (three genotypes into calculation)). Taken together, our data indicate that the Q/R192 is principally associated with both CAD and ischemic stroke in Japanese.

Responses of blood pressure and catecholamine metabolism to high salt loading in endothelin-1 knockout mice.

The molecular mechanism responsible for salt sensitivity is poorly understood. Mice heterozygous for the null mutation of the endothelin-1 (ET-1) gene, Edn1, may be a potential tool for studying this mechanism, because they have elevated blood pressure and disturbances in central sympathetic nerve regulation. In the present study, we used this mouse model to examine the degree to which ET-1 contributes to the responses of blood pressure and catecholamine metabolism to high salt loading. Male Edn1+/- heterozygous mice and Edn1+/+ wild-type littermates were given either a high salt (8%) or a normal salt (0.7%) diet for 4 wk. During the normal diet, renal ET-1 levels in Edn1+/- mice were approximately 50% lower than ET-1 levels in wild-type mice, whereas the high salt diet decreased renal ET-1 levels by about 50% in both Edn1+/- and wild-type mice. The high salt diet significantly increased urinary sodium excretion and fractional excretion of sodium (FENa) but did not affect circulating plasma volume, serum electrolytes, creatinine clearance, or systemic blood pressure. In addition, urinary norepinephrine and normetanephrine excretion were significantly increased, indicating that salt loading can increase sympathetic nerve activity in normal mice. These responses to salt loading did not differ between Edn1+/- mice and their wild-type littermates. We conclude that physiological changes in ET-1 production do not affect the responses of blood pressure and catecholamine metabolism to salt loading, although the renal ET-1 content is decreased by salt loading.

Polymorphism of the methionine synthase gene : association with homocysteine metabolism and late-onset vascular diseases in the Japanese population.

Methionine synthase and 5,10-methylenetetrahydrofolate reductase (MTHFR) sequentially catalyze the remethylation of homocysteine to methionine. A point mutation in the encoding region of the methionine synthase gene, which results in substitution of an aspartic acid for a glycine residue (D919G), has been identified in patients of the cblG genetic complementation group; these patients exhibit significantly decreased methionine synthase activity. Nevertheless, the D919G mutation has also been reported to be common in the general population. In this study, we analyzed the distribution of methionine synthase D/G polymorphism in the Japanese population and examined the extent to which it is associated with altered homocysteine metabolism and late-onset vascular diseases. We studied 215 patients with coronary artery disease, 251 patients with histories of ischemic stroke, and 257 control subjects. The methionine synthase genotype was analyzed by polymerase chain reaction followed by HaeIII digestion; allele frequencies for the D919G variant of the enzyme proved to be similar in all 3 subject groups (control subjects, 0.17; coronary artery disease patients, 0. 17; and ischemic stroke patients, 0.19). Furthermore, in patients with ischemic stroke, plasma levels of homocyst(e)ine and folate were similar, irrespective of methionine synthase genotype. Thus, the methionine synthase D919G mutation was found to be common in the Japanese general population, and it appears unlikely that this polymorphism has a major effect on homocysteine metabolism and/or the onset of vascular diseases.

Methylenetetrahydrofolate reductase gene polymorphism and ischemic stroke in Japanese.

Hyperhomocyst(e)inemia has been identified as an independent risk factor for atherosclerotic and thromboembolic diseases such as coronary artery disease, cerebral artery disease, and venous thrombosis. Recently, the alanine/valine (A/V) gene polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR), one of the key enzymes that catalyzes the remethylation of homocysteine, was reported. The VV genotype is correlated with increased plasma homocyst(e)ine levels as a result of the reduced activity and increased thermolability of this enzyme. In this study, we examined the association between the V allele of the MTHFR gene and ischemic stroke in an elderly Japanese population. The diagnosis of cerebral infarction of all study patients was confirmed by CT of the brain. The MTHFR genotype was analyzed by polymerase chain reaction followed by HinfI digestion. In 256 stroke patients and 325 control subjects, the frequencies of the V allele were 0.45 and 0.32, respectively. The odds ratios and 95% confidence intervals adjusted for the other risk factors were, respectively, 1.51 (1.02 to 2.23) for the AV genotype and 3.35 (1.94 to 5.77) for the VV genotype compared with the AA genotype. Both of these effects were statistically significant (P=0.041 and P<0.001, respectively). In patients with multiple infarcts in particular, the allele frequency of the V mutation was 0.56, and the association between the V allele and stroke was highly significant. Plasma homocyst(e)ine levels were significantly higher in patients with the VV genotype than in patients with the AA or AV genotype, especially those with low plasma folate levels. The V allele of the MTHFR gene was significantly associated with cerebral infarction in an elderly Japanese population in a codominant manner. The VV genotype may contribute to risk for ischemic stroke through a predisposition to increased plasma homocyst(e)ine levels, and dietary folate supplementation may be of benefit, particularly to patients with this genotype.

Effects of cilostazol on late lumen loss after Palmaz-Schatz stent implantation.

Cilostazol inhibits intimal hyperplasia after stent implantation into canine iliac arteries. To determine the antiproliferative effect of this agent, cilostazol or aspirin was randomly given for 6 mo to 36 patients treated with Palmaz-Schatz stent implantation. Initial success was obtained in 34 patients. Repeat angiography was performed in 33 patients, and the complete angiographic data were obtained in 22 lesions of the cilostazol group and in 21 lesions of the aspirin group. The reference diameter and minimal luminal diameter were similar in both groups before and immediately after stent implantation. At follow-up, minimal luminal diameters were significantly greater in the cilostazol group than in the aspirin group (P < 0.001). Late loss and loss index were significantly smaller in the cilostazol group than in the aspirin group (P < 0.001). These results suggest that cilostazol reduces angiographic late lumen loss and thereby may reduce the incidence of restenosis after Palmaz-Schatz stent implantation.

[A clinical phase III trial of MR-20 in gynecologic nephrotoxicity of cisplatin--a comparative study in MR-20-treated and control patients on cyclical intermittent cisplatin treatment].

UNLABELLED: MR-20 was administered to 52 gynecological cancer patients who presented with nephrotoxicity from cisplatin (CDDP) treatment over 3 courses at 33 institutions throughout Japan during the period from July 1992 through March 1994, in order to study its suppressive effect on the nephrotoxicity as well as its safety; and the results are reported in this paper. METHODS: The efficacy and usefulness of MR-20 were studied by a MR-20-untreated-controlled, non-double-blind manner. An efficacy rate of 72.0% was achieved in the MR-20 group, and 37.0% in the untreated group: MR-20 was significantly more effective for nephrotoxicity than the MR-20-untreated group. Ccr was prevented from significant variations in the MR-20 group, compared with the untreated group. It was considered that MR-20 is a safe drug, and that it is useful in suppressing the nephrotoxicity of CDDP treatment.

[Ten-year follow-up of percutaneous transluminal coronary angioplasty].

The long-term efficacy of percutaneous transluminal coronary angioplasty (PTCA) was evaluated in 124 patients who underwent PTCA between October 1982 and June 1985. Seventy-six patients had their proximal coronary arteries completely revascularized by PTCA (including repeat PTCA) and follow-up angiography showed the vessels were patent 3 to 6 months after the last intervention (group A). The other 48 patients showed incomplete revascularization (group B). The 10-year event-free survival rates from cardiac death and non-fatal myocardial infarction were 89.7% in group A and 93.5% in group B, respectively. Survival rates free from cardiac death, myocardial infarction, coronary bypass surgery, and repeat angioplasty for new lesions were 82.5% and 62.7%, respectively (p < 0.001). Ninety-nine percent of patients in group A and 79.5% of patients in group B were asymptomatic at the follow-up. Coronary angiography was performed in a total of 22 patients (19 patients in group A, 3 in group B). In group A, myocardial infarction occurred in one patient 8 years after PTCA. The infarct-related lesion seemed to be the same as the previous PTCA lesion. Although the other 20 PTCA lesions were all patent without progression, nine significant and new lesions were found on the coronary angiograms. Successfully revascularized vessels remained patent for over 10 years, but new lesions sometimes occur in segments other than the previously treated segment.

Production of endothelin-1 in vascular endothelial cells is regulated by factors associated with vascular injury.

We studied the effects of factors associated with vascular injury on the production of endothelin-1 (ET-1) in cultured porcine aortic endothelial cells. ET-1 mRNA expression and peptide production in endothelial cells were increased by thrombin, transforming growth factor beta 1 (TGF beta 1), interleukin-1 (IL-1) and tumor necrotizing factor alpha (TNF alpha). The analysis of the enhancer/promoter region of the human ET-1 gene showed that the ET-1 gene transcription is regulated in a cell-specific manner and is activated by thrombin, TGF beta 1 and IL-1. These results suggest that the production of ET-1 in endothelial cells is regulated by factors associated with platelet aggregation, macrophage infiltration and the formation of atherosclerotic lesions.

Anterograde axonal transport of peptidylglycine alpha-amidating monooxygenase in rat sciatic nerves.

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.

Interleukin 1 increases the production of endothelin-1 by cultured endothelial cells.

We examined the effect of human recombinant interleukin 1 (IL-1) on the production of endothelin-1 by cultured porcine endothelial cells. The induction of endothelin-1 mRNA began within 1 hr of exposure to IL-1, showed twin peaks at 4 and 24 hr, and declined thereafter. Enzyme-linked immunosorbent assay revealed that the amount of endothelin-1 peptide in conditioned media was also increased by IL-1 in a dose- and time-dependent manner. Our results suggested that IL-1, a macrophage-derived cytokine, may affect the contraction and proliferation of vascular smooth muscle cells by stimulating the production of endothelin by endothelial cells.

The effects of anesthetics on the concentrations of cholecystokinin octapeptide sulfate-like immunoreactivity in rat brain regions.

Cholecystokinin octapeptide sulfate-like immunoreactivity (CCK-8S-LI) was determined by radioimmunoassay in rat brain areas following injections of pentobarbital, halothane and chloral hydrate. Time-dependent changes in the concentrations of CCK-8S-LI were different between pentobarbital and chloral hydrate in all brain regions studied. After pentobarbital injection, CCK-8S-Li peaked at 30-60 min in the frontal cortex, nucleus accumbens, striatum and substantia nigra; after chloral hydrate injection, CCK-8S-LI peaked at 120 min in the hypothalamus, nucleus accumbens and substantia nigra. Both anesthetics induced almost the same sleeping times. Halothane inhalation caused increases in the concentrations of CCK-8S-LI in the amygdala and hippocampus. In addition, following intracardial perfusion of saline for 30 min after pentobarbital anesthesia, the concentrations of CCK-8S-LI increased in the nucleus accumbens, and decreased in the frontal cortex. These results suggest that since different anesthetics cause different changes in CCK levels, anesthetics affect studies of these neurons.

In vivo release of cholecystokinin-like immunoreactivity in rat frontal cortex under freely moving conditions.

Release of cholecystokinin-like immunoreactivity (CCK-LI) was measured in rat medial prefrontal cortex in vivo by brain dialysis and enzyme immunoassay under freely moving condition. Perfusion of 50 mM K+ resulted in the increase of CCK-LI in the dialysate. The data on high-performance liquid chromatography (HPLC) of the dialysate showed that the increase of the CCK-LI was mainly due to the increase of CCK octapeptide sulfate itself. In the preliminary experiments, we applied this brain dialysis method for determination of CCK-LI release with drug treatment. After treatment with sulpiride (i.p.), a D2 dopamine receptor antagonist, a significant increase of CCK-LI was observed, indicating that this brain dialysis technique is applicable to detect change in the level of CCK-LI release after a certain drug treatment.

Temperature- and growth-phase-dependent changes in membrane fatty acid compositions of Brevibacterium ammoniagenes.

Fatty acids newly synthesized by Brevibacterium ammoniagenes grown at different temperatures were analyzed. The assay temperature, not the growth temperature, was found to be the major factor affecting the unsaturated/saturated ratio of newly synthesized fatty acids in logarithmic-phase cells. However, in the stationary-phase cells the growth temperature also affected the product profile significantly; cells grown at 7 degrees C produced relatively more oleate and stearate and less palmitate and hexadecenoate when shifted up to 37 degrees C than did cells grown and assayed at 37 degrees C. The unsaturated/saturated ratio as well as average chain length of fatty acids also varied along with the progress of isothermal growth phase. These changes in fatty acid product profiles observed in vivo could be mimicked in vitro assays of the fatty acid synthetase by changing malonyl-CoA concentrations. Our results suggest that the malonyl-CoA concentration is a factor which, in addition to temperature, determines growth-phase-dependent and growth-temperature-dependent changes in the unsaturated/saturated ratios of fatty acids.