3-O-Laurylglyceryl ascorbate reinforces skin barrier function through not only the reduction of oxidative stress but also the activation of ceramide synthesis.

OBJECTIVE: A higher trans-epidermal water loss (TEWL) occurs in rough skin, in elder skin and also in atopic dermatitis. An impaired skin barrier function is considered to be caused by an incomplete construction of the intercellular lamellar structure due to the quantitative reduction of ceramides. Since these symptoms coexist with oxidative stress, we hypothesized that impairment of the skin barrier function is accelerated by oxidative stress. Thus, the purpose of this study was to clarify the effect of oxidative stress on ceramide synthesis and to characterize whether antioxidants can improve skin barrier function. 3-O-Laurylglyceryl ascorbate (VC-3LG), which is a newly amphipathic derivative of ascorbic acid, was evaluated as a candidate antioxidant. METHODS: We characterized the mRNA expression levels of serine palmitoyltransferase (SPT) in normal human epidermal keratinocytes (NHEKs) treated with H2 O2 using real-time PCR analysis. In order to evaluate the effect of VC-3LG on skin barrier function, we used several assays with reconstructed human epidermis equivalents (RHEEs). RESULTS: Ceramide synthesis was down-regulated in NHEKs by oxidative stress. Treatment with VC-3LG abrogated the down-regulation of SPT mRNA in NHEKs caused by oxidative stress, and stimulated SPT mRNA expression levels. In experiments characterizing the antioxidative properties of VC-3LG, VC-3LG reduced oxidative stress in NHEKs by up-regulating catalase mRNA expression. In addition, VC-3LG stimulated the skin barrier function in RHEEs, which had lower TEWL values compared with untreated RHEEs. Furthermore, VC-3LG increased the quantity of ceramide in RHEEs. CONCLUSION: Taken together, we conclude that VC-3LG reinforces the skin barrier function due to its reduction of oxidative stress and its promotion of ceramide synthesis.

Differences in susceptibility to oxidative stress in the skin of Japanese and French subjects and physiological characteristics of their skin.

BACKGROUND: Many researchers have studied differences in conditions of ethnic skin using biophysical measurements. However, few studies to date have focused on the antioxidative capacity of the skin. METHODS: We measured two parameters of oxidative stress in the stratum corneum, catalase activity and protein carbonylation of the stratum corneum (SCCP), in two ethnic groups, Japanese and French subjects, to characterize the susceptibility to oxidative stress. We also measured several physiological parameters at three different skin sites, two sun-exposed sites (cheek and dorsal aspect of the hand) and a sun-protected site (inner upper arm), in both ethnic groups. RESULTS: Transepidermal water loss (TEWL), the size of corneocytes and skin color showed differences between sun-exposed and sun-protected sites regardless of ethnicity. Regarding ethnic differences, catalase activities and parameters of skin hydration and barrier function of Japanese subjects were higher than those of French subjects. However, SCCP values showed a trend contrary to catalase activity. The difference in the b* value indicated that the melanin content of Japanese skin was higher than that of French skin. Pearson's correlation analyses showed that catalase activity and SCCP values had weak relationships with water content, TEWL and skin color in both ethnic groups. CONCLUSION: Differences in susceptibility to oxidative stress, namely melanin content and catalase activity in the skin, induce the better skin condition of Japanese compared with French subjects.

Lysophospholipids improve skin moisturization by modulating of calcium-dependent cell differentiation pathway.

Recent studies have demonstrated that lysophospholipids (LPL) play critical roles in several biological signal transduction pathways to maintain the homoeostasis of cells, tissues and organs. Among them, lysophosphatidic acid (LPA) has been identified as a lipid mediator that induces morphological improvement in the epidermis in mice. In this study, we examined the effects of LPL (soybean-derived phospholipids modified with phospholipase A2 and C) compared with LPA. We initially examined the effects of LPA on normal human epidermal keratinocytes (NHEK) focusing on the expression of profilaggrin and serine palmitoyltransferase (SPT) mRNAs. LPA enhanced the expression of profilaggrin and SPT mRNAs via the modulation of Ca(2+) influx. Based on those results, the influence of LPL on NHEK was examined and was expanded to analyse the expression of two tight junction-related proteins, occludin and claudin-1. LPL had similar effects to increase profilaggrin and SPT mRNA expression and also stimulated the expression of occludin and claudin-1 at the mRNA and protein levels. In accordance with these results, LPL elicited significant improvements in surface water content in human skin. These findings indicate that LPL has the potential to strengthen the skin moisturizing capability by up-regulating the expression of mRNAs encoding components important to skin barrier function and skin hydration.

Inactivation effects of UV irradiation and ozone treatment on the yeast and the mold in mineral water.

In recent years, bottled mineral water has undergone inactivation by methods other than the traditional heat treatment during the production process; there are fewer reports of the effectiveness of these inactivation methods on yeasts and molds in mineral water than on bacteria and protozoan oocysts. In this study, we evaluated the effects of UV irradiation and ozone treatment compared with heat treatment at 85 degrees C on yeast cells and mold spores inoculated into mineral water. A 5-log reduction occurred at a UV radiation dose of 31,433 microJ/cm2 for Saccharomyces cerevisiae and at 588,285 microJ/cm2 for Penicillium pinophilum. The treatment time for 5-log reduction estimated for UV irradiation was about 0.6 min for S. cerevisiae and about 10.7 min for P. pinophilum; at an ozone concentration of 0.1 ppm, it was 1.75 min for S. cerevisiae and 2.70 min for P. pinophilum, and at a concentration of 0.6 ppm, it was 0.32 min for S. cerevisiae and 0.57 min for P. pinophilum. Comparison of the inactivation effects among the three methods showed that UV irradiation and ozone treatment were less effective than heat treatment at 85 degrees C. Thus, when UV irradiation and ozone treatment are used for inactivation of mineral water, it seems that they need to be combined with heat treatment to achieve a definite effect. Yeast cells are more sensitive to all three inactivation methods than are mold spores, and the sensitivity of yeast cells and mold spores to these inactivation methods may vary among genera.

A cytotoxic ribonuclease targeting specific transfer RNA anticodons.

The carboxyl-terminal domain of colicin E5 was shown to inhibit protein synthesis of Escherichia coli. Its target, as revealed through in vivo and in vitro experiments, was not ribosomes as in the case of E3, but the transfer RNAs (tRNAs) for Tyr, His, Asn, and Asp, which contain a modified base, queuine, at the wobble position of each anticodon. The E5 carboxyl-terminal domain hydrolyzed these tRNAs just on the 3' side of this nucleotide. Tight correlation was observed between the toxicity of E5 and the cleavage of intracellular tRNAs of this group, implying that these tRNAs are the primary targets of colicin E5.

A Zn(II)-glycine complex suppresses UVB-induced melanin production by stimulating metallothionein expression.

Oxidative stress caused by ultraviolet (UV) radiation generates reactive oxygen species (ROS) in the skin, induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyper-pigmentation. Thus, increasing the anti-oxidative ability of skin cells is expected to be a good strategy for skin-lightening cosmetics. Metallothionein (MT) is one of the stress-induced proteins and is known to exhibit a strong anti-oxidative property. We previously reported that a zinc(II) complex with glycine (Zn(II)(Gly)(2)) effectively induces MT expression in cultured human keratinocytes. To determine its potential as a new skin lightening active, we examined whether Zn(II)(Gly)(2) regulates the release of melanocyte-activating factors from UVB-irradiated keratinocytes and affects melanin production in a reconstructed human epidermal equivalent. Conditioned medium from UVB-irradiated keratinocytes accelerated melanocyte proliferation to 110%, and that increase could be prevented by pre-treatment with Zn(II)(Gly)(2). In addition, Zn(II)(Gly)(2) significantly reduced both the production of prostaglandin E(2) and proopiomelanocortin expression in UVB-irradiated keratinocytes. Zn(II)(Gly)(2) also decreased melanin production in a reconstructed human epidermal equivalent. These results indicate that MT-induction in the epidermis effectively up-regulates tolerance against oxidative stress and inhibits the secretion of melanocyte growth and activating factors from keratinocytes. Thus, Zn(II)(Gly)(2) is a good candidate as a new skin-lightening active.

cAMP responsive element-mediated regulation of the gene transcription of the alpha 1B adrenergic receptor by thyrotropin.

To elucidate the molecular mechanism of the stimulatory effect of thyrotropin on the gene regulation of alpha 1B adrenergic receptor in functioning rat thyroid (FRTL-5) cells, we established a competitive reverse-transcriptase (RT) polymerase chain reaction (PCR) and nuclear run-off assay to quantify changes in mRNA levels and transcription rates. A binding assay showed that FRTL-5 cells predominantly expressed alpha 1B adrenergic receptor and that thyrotropin increased its expression sevenfold. By means of RT-PCR, we found that thyrotropin induced an 11-fold increase in alpha 1B receptor mRNA abundance. The nuclear run-off assay demonstrated that thyrotropin caused a ninefold increase at the gene transcriptional level, which occurred in the presence of the protein synthesis inhibitor cycloheximide. The half-life of the alpha 1B receptor mRNA in cells incubated with thyrotropin for 1 h increased 1.5-fold but returned to the original value after 12 h. Dibutyryl cAMP and forskolin mimicked the stimulatory effects of thyrotropin on the gene transcriptional level. The 5'-flanking region of the rat alpha 1B receptor gene contained a putative cAMP responsive element (CRE) at nucleotide -438 relative to the translation start site. The promoter analysis using the reporter gene indicated that the CRE motif confers the cAMP sensitivity to the transcription of the rat alpha 1B receptor gene. These results demonstrated that a CRE-mediated mechanism is involved in the transcriptional regulation of the alpha 1B receptor gene by thyrotropin without requiring new protein synthesis.

Cardiac-specific overexpression of angiotensin II AT2 receptor causes attenuated response to AT1 receptor-mediated pressor and chronotropic effects.

Angiotensin (Ang) II has two major receptor isoforms, AT1 and AT2. Currently, AT1 antagonists are undergoing clinical trials in patients with cardiovascular diseases. Treatment with AT1 antagonists causes elevation of plasma Ang II which selectively binds to AT2 and exerts as yet undefined effects. Cardiac AT2 level is low in adult hearts, whereas its distribution ratio is increased during cardiac remodeling and its action is enhanced by application of AT1 antagonists. Although in AT2 knock-out mice sensitivity to the pressor action of Ang II was increased, underlying mechanisms remain undefined. Here, we report the unexpected finding that cardiac-specific overexpression of the AT2 gene using alpha-myosin heavy chain promoter resulted in decreased sensitivity to AT1-mediated pressor and chronotropic actions. AT2 protein undetectable in the hearts of wild-type mice was overexpressed in atria and ventricles of the AT2 transgenic (TG) mice and the proportions of AT2 relative to AT1 were 41% in atria and 45% in ventricles. No obvious morphological change was observed in the myocardium and there was no significant difference in cardiac development or heart to body weight ratio between wild-type and TG mice. Infusion of Ang II to AT2 TG mice caused a significantly attenuated increase in blood pressure response and the change was completely blocked by pretreatment with AT2 antagonist. This decreased sensitivity to Ang II-induced pressor action was mainly due to the AT2-mediated strong negative chronotropic effect and exerted by circulating Ang II in a physiological range that did not stimulate catecholamine release. Isolated hearts of AT2 transgenic mice perfused using a Langendorff apparatus also showed decreased chronotropic responses to Ang II with no effects on left ventricular dp/dt max values, and Ang II-induced activity of mitogen-activated protein kinase was inhibited in left ventricles in the transgenic mice. Although transient outward K+ current recorded in cardiomyocytes from AT2 TG mice was not influenced by AT2 activation, this study suggested that overexpression of AT2 decreases the sensitivity of pacemaker cells to Ang II. Our results demonstrate that stimulation of cardia AT2 exerts a novel antipressor action by inhibiting AT1-mediated chronotropic effects, and that application of AT1 antagonists to patients with cardiovascular diseases has beneficial pharmacotherapeutic effects of stimulating cardiac AT2.

Angiotensin II type 2 receptor overexpression activates the vascular kinin system and causes vasodilation.

Angiotensin II (Ang II) is a potent vasopressor peptide that interacts with 2 major receptor isoforms - AT1 and AT2. Although blood pressure is increased in AT2 knockout mice, the underlying mechanisms remain undefined because of the low levels of expression of AT2 in the vasculature. Here we overexpressed AT2 in vascular smooth muscle (VSM) cells in transgenic (TG) mice. Aortic AT1 was not affected by overexpression of AT2. Chronic infusion of Ang II into AT2-TG mice completely abolished the AT1-mediated pressor effect, which was blocked by inhibitors of bradykinin type 2 receptor (icatibant) and nitric oxide (NO) synthase (L-NAME). Aortic explants from TG mice showed greatly increased cGMP production and diminished Ang II-induced vascular constriction. Removal of endothelium or treatment with icatibant and L-NAME abolished these AT2-mediated effects. AT2 blocked the amiloride-sensitive Na(+)/H(+) exchanger, promoting intracellular acidosis in VSM cells and activating kininogenases. The resulting enhancement of aortic kinin formation in TG mice was not affected by removal of endothelium. Our results suggest that AT2 in aortic VSM cells stimulates the production of bradykinin, which stimulates the NO/cGMP system in a paracrine manner to promote vasodilation. Selective stimulation of AT2 in the presence of AT1 antagonists is predicted to have a beneficial clinical effect in controlling blood pressure.

Angiotensin AT(1) and AT(2) receptors differentially regulate angiopoietin-2 and vascular endothelial growth factor expression and angiogenesis by modulating heparin binding-epidermal growth factor (EGF)-mediated EGF receptor transactivation.

Angiotensin II (Ang II)-mediated signals are transmitted via heparin binding epidermal growth factor (EGF)-like growth factor (HB-EGF) release followed by transactivation of EGF receptor (EGFR). Although Ang II and HB-EGF induce angiogenesis, their link to the angiopoietin (Ang)-Tie2 system remains undefined. We tested the effects of Ang II on Ang1, Ang2, or Tie2 expression in cardiac microvascular endothelial cells expressing the Ang II receptors AT(1) and AT(2). Ang II significantly induced Ang2 mRNA accumulations without affecting Ang1 or Tie2 expression, which was inhibited by protein kinase C inhibitors and by intracellular Ca(2+) chelating agents. Ang II transactivated EGFR via AT(1), and inhibition of EGFR abolished the induction of Ang2. Ang II caused processing of pro-HB-EGF in a metalloproteinase-dependent manner to stimulate maturation and release of HB-EGF. Neutralizing anti-HB-EGF antibody blocked EGFR phosphorylation by Ang II. Ang II also upregulated vascular endothelial growth factor (VEGF) expression in an HB-EGF/EGFR-dependent manner. AT(2) inhibited AT(1)-mediated Ang2 expression and phosphorylation of EGFR. In an in vivo corneal assay, AT(1) induced angiogenesis in an HB-EGF-dependent manner and enhanced the angiogenic activity of VEGF. Although neither Ang2 nor Ang1 alone induced angiogenesis, soluble Tie2-Fc that binds to angiopoietins attenuated AT(1)-mediated angiogenesis. These findings suggested that (1) Ang II induces Ang2 and VEGF expression without affecting Ang1 or Tie2 and (2) AT(1) stimulates processing of pro-HB-EGF by metalloproteinases, and the released HB-EGF transactivates EGFR to induce angiogenesis via the combined effect of Ang2 and VEGF, whereas AT(2) attenuates them by blocking EGFR phosphorylation. Thus, Ang II is involved in the VEGF-Ang-Tie2 system via HB-EGF-mediated EGFR transactivation, and this link should be considerable in pathological conditions in which collateral blood flow is required.

Generation of active oxygen species from advanced glycation end-products (AGEs) during ultraviolet light A (UVA) irradiation and a possible mechanism for cell damaging.

Advanced glycation end-products (AGEs) have been reported to be accumulated in dermal skin. However, the role of AGEs in the photoaging of human skin remains unknown, and for this reason, we have examined the interaction between AGEs and ultraviolet A light (UVA) from both the chemical and biological aspects. Previously, we reported that exposing human dermal fibroblasts to UVA in the presence of AGEs that were prepared with bovine serum albumin (BSA) decreased the cell viability due to superoxide anion radical s (.O2(-)) and hydroxyl radicals (.OH) generated by AGEs under UVA irradiation, and active oxygen species are detected with ESR spin-trapping. To identify the active oxygen species in detail and to clarify the cell damaging mechanism, we performed several experiments and the following results were obtained. (1) In ESR spin-trapping, by addition of dimethyl sulfoxide and superoxide dismutase, ESR signals due to .O2(-) -derived DMPO-OOH and .OH-derived DMPO-OH adducts, respectively, were detectable. (2) UVA-irradiated AGEs elevated the lipid peroxide levels in both fibroblasts and liposomes. But the peroxidation in liposomes was inhibited by addition of deferoxamine. (3) Survival of fibroblasts exposed to UVA in the presence of AGEs was elevated by addition of deferoxamine. And finally, (4) survival of fibroblasts was found to be regulated by the level of H2O2. On the basis of these results, we propose a possible mechanism in which AGEs under UVA irradiation generate active oxygen species involving .O2(-), H2O2, and .OH, and the .OH species plays a harmful role in promoting cell damage.

Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines.

BACKGROUND: Bone marrow implantation (BMI) was shown to enhance angiogenesis in a rat ischemic heart model. This preclinical study using a swine model was designed to test the safety and therapeutic effectiveness of BMI. METHODS AND RESULTS: BM-derived mononuclear cells (BM-MNCs) were injected into a zone made ischemic by coronary artery ligation. Three weeks after BMI, regional blood flow and capillary densities were significantly higher (4.6- and 2.8-fold, respectively), and cardiac function was improved. Angiography revealed that there was a marked increase (5.7-fold) in number of visible collateral vessels. Implantation of porcine coronary microvascular endothelial cells (CMECs) did not cause any significant increase in capillary densities. Labeled BM-MNCs were incorporated into approximately 31% of neocapillaries and corresponded to approximately 8.7% of macrophages but did not actively survive as myoblasts or fibroblasts. There was no bone formation by osteoblasts or malignant ventricular arrhythmia. Time-dependent changes in plasma levels for cardiac enzymes (troponin I and creatine kinase-MB) did not differ between the BMI, CMEC, and medium-alone implantation groups. BM-MNCs contained 16% of endothelial-lineage cells and expressed basic fibroblast growth factor>>vascular endothelial growth factor>angiopoietin 1 mRNAs, and their cardiac levels were significantly upregulated by BMI. Cardiac interleukin-1beta and tumor necrosis factor-alpha mRNA expression were also induced by BMI but not by CMEC implantation. BM-MNCs were actively differentiated to endothelial cells in vitro and formed network structure with human umbilical vein endothelial cells. CONCLUSIONS: BMI may constitute a novel safety strategy for achieving optimal therapeutic angiogenesis by the natural ability of the BM cells to secrete potent angiogenic ligands and cytokines as well as to be incorporated into foci of neovascularization.

Iodine-123 metaiodobenzylguanidine images reflect intense myocardial adrenergic nervous activity in congestive heart failure independent of underlying cause.

OBJECTIVES: This study was undertaken to assess myocardial adrenergic activity using iodine-123 metaiodobenzylguanidine (MIBG) imaging in patients with heart failure. BACKGROUND: In patients with congestive heart failure, adrenergic nerve activity is accelerated. However, whether myocardial adrenergic nerve activity reflects the severity of heart failure and its relation to the underlying cause have not yet been elucidated. METHODS: Planar MIBG images were obtained from 96 patients with heart failure and compared with images from 9 age-matched healthy subjects. Groups 1 and 2 included 65 patients with heart failure related to impaired myocardial function and whose left ventricular ejection fraction was < 40% (group 1 = 40 patients with dilated cardiomyopathy; group 2 = 25 patients with ischemic cardiomyopathy). Group 3 included 31 patients with heart failure related to a mechanical abnormality and whose left ventricular ejection fraction was > 40% (mitral regurgitation in 16, aortic regurgitation in 9, aortic and mitral regurgitation in 4, ruptured aneurysm of Valsalva in 2). Myocardial uptake of MIBG was calculated as the heart/mediastinal activity ratio. Storage and release of MIBG were calculated as percent myocardial MIBG washout from 15 min to 4 h after isotope injection. RESULTS: The heart/mediastinal activity ratio in the immediate images (15 min) showed a significant decrease only in patients with severe heart failure (groups 1 and 2). The myocardial washout was accelerated in all three heart failure groups. The level of myocardial washout was related to severity of heart failure and correlated well with New York Heart Association functional classification. CONCLUSIONS: In severe heart failure associated with cardiomyopathy, norepinephrine uptake is reduced. In addition, myocardial adrenergic nerve activity is accelerated in proportion to severity of heart failure, independent of the underlying cause.

Colicin E3 and its immunity genes.

A DNA segment of plasmid ColE3-CA38 was cloned into pBR328 and its nucleotide sequence was determined. This segment contains the putative promoter-operator region, the structural genes of protein A (gene A) and protein B (gene B) of colicin E3, and a part of gene H. Just behind the promoter region, there is an inverted repeat structure of two 'SOS boxes', the specific binding site of the lexA protein. This suggests that the expression of colicin E3 is regulated directly by the lexA protein. Genes A and B face the same direction, with an intergenic space of nine nucleotides between them. ColE3-CA38 and ColE1-K30 are homologous in their promoter-operator regions, but hardly any homology was found in their structural genes. On the other hand, ColE3-CA38 is fairly homologous to CloDF13 throughout the regions sequenced, with some exceptions including putative receptor-binding regions. By deletion mapping of the immunity gene and recloning of gene B, it was shown genetically that protein B itself is the actual immunity substance of colicin E3. It was also found that the expression of E3 immunity partially depends on the recA function. Thus, we propose two modes of expression of E3 immunity: in the uninduced state, only a slight amount of protein B is produced constitutively to protect the cell from being attacked by the exogenous colicin; and in the SOS-induced state, a large amount of protein B is produced to protect the protein synthesis system of the host cell from ribosome inactivation by endogenously produced colicin E3.

Detection of minimal residual disease using clonospecific primers for CDRIII in patients with acute B lymphocytic leukemia with or without Philadelphia chromosome: possibility of clinical application as a tool for improving prognosis.

We attempted to identify the minimal residual leukemic clone as related to the clinical course in patients with acute B lymphocytic leukemia (B-ALL). DNA was extracted from stored bone marrow slides, and the third complementarity determining region (CDRIII) was amplified by polymerase chain reaction (PCR) using primers with consensus sequences for VH and JH. After amplification of the CDRIII band, the DNA fragment of CDRIII was inserted into the cloning vector PUC118. After cloning, the DNA sequences for CDRIII were determined. Clonospecific DNA sequences in CDRIII were selected, and clonospecific primers for each patient were synthesized. Using the clonospecific primers, we carried out second-round PCR to detect minimal residual disease (MRD) during several stages of the clinical course. Basically, the sensitivity of detection for MRD was between 10(-4) and 10(-5) cells. Even when leukemic cells were not detected in the morphologic study, with this detection system, the MRD was identified as an amplified CDRIII band stained with ethidium bromide on agarose gel. After bone marrow transplantation (BMT), MRD was detected for at least 4 months. In this article, we discuss the difference in sensitivity of detection for MRD between the BCR-ABL fusion gene and CDRIII in Philadelphia chromosome-positive (Ph+) B-ALL, as well as the possible clinical application of this method to predict relapse and prognosis.

Production of chronic congestive heart failure by rapid ventricular pacing in the rabbit.

OBJECTIVE: The aim was to produce a model of low output congestive heart failure by rapid pacing in rabbits. METHODS: To perform rapid pacing in rabbits, a custom made pacemaker was developed which is light (about 80 g) and can pace at up to 400 beats.min-1 for more than two weeks. A thoracotomy was done and two electrodes were sutured onto the left ventricle. A central venous pressure line was chronically implanted. With the use of this pacemaker, rabbits were paced at 350-400 beats.min-1 for several weeks. RESULTS: Central venous pressure increased from 1.4(SEM 0.2) to 6.4(0.5) mm Hg (p < 0.01, n = 14). After pacing for 16.1(1.6) d, haemodynamic studies were performed under anaesthesia with thiamylal sodium. Left ventricular end diastolic pressure was higher in the paced rabbits (n = 10) than in the control rabbits which underwent sham operation but were not paced (n = 6), at -0.6(0.6) v 19.3(2.0) mm Hg (p < 0.01). Cardiac output [673(56) v 536(45) ml.min-1, p < 0.10] and +dP/dt [1433(97) v 722(51) mm Hg.s-1, p < 0.01] were lower in the paced rabbits (n = 7-8) than in the control rabbits (n = 6). The paced rabbits had more ascites [1.9(1.0) v 45.9(18.9) ml, p < 0.05] and pleural effusion [0.4(0.3) v 12.9(6.7) ml, p < 0.10] than control rabbits. Plasma noradrenaline was higher in the paced rabbits (n = 11) than in the control rabbits (n = 7), at 1.59(0.43) v 0.60(0.05) ng.ml-1 (p < 0.05). The ratio of wet heart weight or lung weight to body weight was higher (p < 0.01) in the paced rabbits than in the control rabbits. CONCLUSIONS: Chronic biventricular congestive heart failure can be produced in rabbits by rapid pacing.

Thyrotropin and triiodothyronine regulate iodothyronine 5'-deiodinase messenger ribonucleic acid levels in FRTL-5 rat thyroid cells.

We investigated the regulation of type I iodothyronine 5'-deiodinase (5'-D) gene expression by TSH and T3 in FRTL-5 rat thyroid cells. Northern blot analysis revealed that these cells express a 5'-D messenger RNA (mRNA) species of 2.1 kilobases. Readdition of TSH to FRTL-5 cells, precultured in both thyroid hormones and TSH-depleted medium for 4 days, increased 5'-D mRNA levels, reaching a maximum (2.8-fold compared to control) after 12 h of TSH (10 microU/ml) stimulation. Dibutyryl cAMP (DBC) and forskolin mimicked this stimulatory effect of TSH on 5'-D mRNA levels. T3 also increased the 5'-D mRNA levels, reaching a maximum (2-fold compared to control) after 8 h of T3 (10(-9) M) stimulation. Addition of TSH (10 microU/ml) or DBC (1 mM) together with T3 (10(-9) M) further increased 5'-D mRNA levels, reaching a maximum (5-fold compared to control) after 12 h of stimulation. Examination of the rate of disappearance of 5'-D mRNA levels after inhibition of mRNA transcription by actinomycin-D revealed that neither TSH nor T3 significantly affected the rate of disappearance. Cycloheximide, a protein synthesis inhibitor, almost completely blocked the induction of 5'-D mRNA by TSH and DBC, but did not block the induction by T3. These results suggest that both TSH and T3 increase 5'-D mRNA levels probably by increasing transcription rate, and that TSH regulates it, in part via the second messenger cAMP, for which cycloheximide-sensitive de novo protein synthesis is required, whereas T3 does without requiring it.

Synergistic effect of thyroid hormone and thyrotropin on iodothyronine 5'-deiodinase in FRTL-5 rat thyroid cells.

The effects of T3 and T4 on the iodothyronine 5'-deiodinase (5'-D) activity in FRTL-5 rat thyroid cells were investigated. T3 and T4 stimulated the 5'-D activity in a dose-dependent manner. Kinetic studies showed that the stimulation of the 5'-D by T3 was associated with an increase in maximum velocity (Vmax) in [11.9 +/- 0.2 (mean +/- SE) and 25.4 +/- 0.9 pmol I-released/mg protein.min, respectively, in control and cultured with 10(-9) M T3 for four days] but without a change in apparent Michaelis-Menten constant (Km) (94.8 +/- 5.3 nM and 105.4 +/- 12.1 nM, respectively). Furthermore, cycloheximide (5 microM) completely abolished the stimulatory effect of T3 on the 5'-D activity. T3 and T4 also enhanced the 5'-D activity stimulated by TSH in a dose-dependent manner. Kinetic studies showed that the stimulatory effect of T3 on the 5'-D stimulated by TSH was again associated with an increase in Vmax (86.0 +/- 4.0 and 166.5 +/- 1.9 pmol I- released/mg protein.min, respectively, cultured with 0.3 U/liter TSH and cultured with TSH plus 10(-9) M T3 for four days) without a change in apparent Km (114.0 +/- 7.4 nM and 111.6 +/- 12.5 nM, respectively). Cycloheximide (5 microM) completely abolished the stimulatory effect of TSH plus T3 on the 5'-D activity. There were no significant differences observed between cells cultured with TSH and with TSH plus T3 in either the intra- or extracellular cAMP contents. Furthermore, T3 enhanced the 5'-D activity stimulated by (Bu)2 cAMP. These results strongly suggest that T3 or T4 was synergistic with TSH in stimulating the 5'-D activity in FRTL-5 cells, and that cAMP production would be an important component of the synergism.

Intra-arterial infusion of insulin attenuates vasoreactivity in human forearm.

Hyperinsulinemia may contribute to the development of hypertension. The aim of the present study was to determine whether hyperinsulinemia modulates vascular reactivity to phenylephrine or angiotensin II. In 10 young, healthy volunteers, the left brachial artery was cannulated for drug infusion and direct measurements of arterial pressure. We measured forearm blood flow by a strain-gauge plethysmograph while infusing phenylephrine (0.2, 0.8, and 2.4 micrograms/min) and angiotensin II (5, 10, and 20 ng/min) locally into the brachial artery before and during simultaneous intra-arterial infusion of insulin (0.15 mU/kg per minute). Forearm vascular resistance was calculated from directly measured arterial pressure and forearm blood flow. Intra-arterial infusion of insulin raised the local plasma insulin level from 10.3 +/- 1.4 to 133.3 +/- 21.1 microU/mL (P < .01) and did not change blood glucose level in the venous effluents of the forearm. Insulin infusion slightly but not significantly increased basal forearm blood flow (4.6 +/- 1.5 to 5.5 +/- 0.9 mL/min per 100 milliliters, NS) and decreased forearm vascular resistance (22.1 +/- 2.1 to 20.3 +/- 2.8 U, NS). Phenylephrine and angiotensin II increased forearm vascular resistance dose dependently before and during simultaneous insulin infusion (P < .01 for both). Intra-arterial infusion of insulin attenuated vascular reactivity to phenylephrine (P < .01) and angiotensin II (P < .01). None of these drugs changed blood pressure or heart rate. Our results suggest that hyperinsulinemia attenuates vascular reactivity in the forearm resistance vessels in healthy humans.

3,3',5'-Triiodothyronine inhibits collagen-induced human platelet aggregation.

To clarify further the activity of rT3, we examined the effect of rT3 on collagen-induced platelet activation as reflected by aggregation, serotonin release, and protein phosphorylation. rT3, T4, T3, and triiodothyroacetic acid inhibited collagen-induced platelet aggregation and serotonin release from platelets in a dose-dependent manner. However, thyronine did not inhibit collagen-induced platelet aggregation. The concentration at which rT3 inhibited by 50% collagen-induced platelet aggregation was 30 +/- 4 (mean +/- SE) mumol/L. rT3, T4, and T3 did not differ significantly in their abilities to inhibit platelet aggregation. Moreover, rT3 inhibited collagen-induced phosphorylation of the 20-kilodalton protein (myosin light chain) in platelets. In contrast, rT3 did not inhibit 12-O-tetradecanoylphorbol 13-acetate (TPA)- or thrombin-induced platelet aggregation and inhibited only minimally TPA-induced 40-kilodalton protein phosphorylation. These results suggest that rT3 inhibits collagen-induced platelet activation by inhibiting the activity of myosin light chain kinase and that it may be interesting to investigate some kinds of activity of rT3.