Angiotensin II type 1 receptor blockade partially attenuates hypoxia-induced pulmonary hypertension in newborn piglets: relationship with the nitrergic system

The objective of this study was to observe possible interactions between the renin-angiotensin and nitrergic systems in chronic hypoxia-induced pulmonary hypertension in newborn piglets. Thirteen chronically instrumented newborn piglets (6.3 +/- 0.9 days; 2369 +/- 491 g) were randomly assigned to receive saline (placebo, P) or the AT(1) receptor (AT(1)-R) blocker L-158,809 (L) during 6 days of hypoxia (FiO(2) = 0.12). During hypoxia, pulmonary arterial pressure (Ppa; P < 0.0001), pulmonary vascular resistance (PVR; P < 0.02) and the pulmonary to systemic vascular resistance ratio (PVR/SVR; P < 0.05) were significantly attenuated in the L (N = 7) group compared to the P group (N = 6). Western blot analysis of lung proteins showed a significant decrease of endothelial NOS (eNOS) in both P and L animals, and of AT(1)-R in P animals during hypoxia compared to normoxic animals (C group, N = 5; P < 0.01 for all groups). AT(1)-R tended to decrease in L animals. Inducible NOS (iNOS) did not differ among P, L, and C animals and iNOS immunohistochemical staining in macrophages was significantly more intense in L than in P animals (P < 0.01). The vascular endothelium showed moderate or strong eNOS and AT(1)-R staining. Macrophages and pneumocytes showed moderate or strong iNOS and AT(1)-R staining, but C animals showed weak iNOS and AT(1)-R staining. Macrophages of L and P animals showed moderate and weak AT(2)-R staining, respectively, but the endothelium of all groups only showed weak staining. In conclusion, pulmonary hypertension induced by chronic hypoxia in newborn piglets is partially attenuated by AT(1)-R blockade. We suggest that AT(1)-R blockade might act through AT(2)-R and/or Mas receptors and the nitrergic system in the lungs of hypoxemic newborn piglets.

Angiotensin-(1-7) decreases LPS-induced inflammatory response in macrophages

It has been previously shown that besides its classical role in blood pressure control the reninangiotensin system, mainly by action of angiotensin II on the AT1 receptor, exerts pro-inflammatory effects such as by inducing the production of cytokines. More recently, alternative pathways to this system were described, such as binding of angiotensin-(17) to receptor Mas, which was shown to counteract some of the effects evoked by activation of the angiotensin IIAT1 receptor axis. Here, by means of different molecular approaches we investigated the role of angiotensin-(17) in modulating inflammatory responses triggered in mouse peritoneal macrophages. Our results show that receptor Mas transcripts were up-regulated by eightfold in LPS-induced macrophages. Interestingly, macrophage stimulation with angiotensin-(17), following to LPS exposure, evoked an attenuation in expression of TNF-a and IL-6 pro-inflammatory cytokines; where this event was abolished when the receptor Mas selective antagonist A779 was also included. We then used heterologous expression of the receptor Mas in HEK293T cells to search for the molecular mechanisms underlying the angiotensin-(17)-mediated anti-inflammatory responses by a kinase array; what suggested the involvement of the Src kinase family. In LPS-induced macrophages, this finding was corroborated using the PP2 compound, a specific Src kinase inhibitor; and also by Western blotting when we observed that Ang-(17) attenuated the phosphorylation levels of Lyn, a member of the Src kinase family. Our findings bring evidence for an anti-inflammatory role for angiotensin-(17) at the cellular level, as well as show that its probable mechanism of action includes the modulation of Src kinases activities. J. Cell. Physiol. 227: 21172122, 2012. (C) 2011 Wiley Periodicals, Inc.

Effects of aerobic exercise training on cardiac renin-angiotensin system in an obese Zucker rat strain

Objective: Obesity and renin angiotensin system (RAS) hyperactivity are profoundly involved in cardiovascular diseases, however aerobic exercise training (EXT) can prevent obesity and cardiac RAS activation. The study hypothesis was to investigate whether obesity and its association with EXT alter the systemic and cardiac RAS components in an obese Zucker rat strain. Methods: The rats were divided into the following groups: Lean Zucker rats (LZR); lean Zucker rats plus EXT (LZR+EXT); obese Zucker rats (OZR) and obese Zucker rats plus EXT (OZR+EXT). EXT consisted of 10 weeks of 60-min swimming sessions, 5 days/week. At the end of the training protocol heart rate (HR), systolic blood pressure (SBP), cardiac hypertrophy (CH) and function, local and systemic components of RAS were evaluated. Also, systemic glucose, triglycerides, total cholesterol and its LDL and HDL fractions were measured. Results: The resting HR decreased (, 12%) for both LZR+EXT and OZR+EXT. However, only the LZR+EXT reached significance (p, 0.05), while a tendency was found for OZR versus OZR+EXT (p = 0.07). In addition, exercise reduced (57%) triglycerides and (61%) LDL in the OZR+EXT. The systemic angiotensin I-converting enzyme (ACE) activity did not differ regardless of obesity and EXT, however, the OZR and OZR+EXT showed (66%) and (42%), respectively, less angiotensin II (Ang II) plasma concentration when compared with LZR. Furthermore, the results showed that EXT in the OZR prevented increase in CH, cardiac ACE activity, Ang II and AT2 receptor caused by obesity. In addition, exercise augmented cardiac ACE2 in both training groups. Conclusion: Despite the unchanged ACE and lower systemic Ang II levels in obesity, the cardiac RAS was increased in OZR and EXT in obese Zucker rats reduced some of the cardiac RAS components and prevented obesity-related CH. These results show that EXT prevented the heart RAS hyperactivity and cardiac maladaptive morphological alterations in obese Zucker rats.

Contribution of the genes of the renin-angiotensin system to interindividual differences in blood pressure levels in Caucasians from Rochester, Minnesota

The Renin-Angiotensin system (RAS) regulates blood pressure through its effects on vascular tone, renal hemodynamics, and renal sodium and fluid balance. The genes encoding the four major components of the RAS, angiotensinogen, renin, angiotensin I-converting enzyme (ACE), and angiotensin II receptor type 1 (AT1), have been investigated as candidate genes in the pathogenesis of essential hypertension. However, studies have primarily focused on small samples of diseased individuals, and, therefore, have provided little information about the determinants of interindividual variation in blood pressure (BP) in the general population.^ Using data from a large population-based sample from Rochester, MN, I have evaluated the contribution of variation in the region of the RAS genes to interindividual variation in systolic, diastolic, and mean arterial pressure in the population-at-large. Marker genotype data from four polymorphisms located within or very near these genes were first collected on 3,974 individuals from 583 randomly ascertained three-generation pedigrees. Haseman-Elston regression and variance component methods of linkage analysis were then carried out to estimate the proportion of interindividual variance in BP attributable to the effects of variation at these four measured loci.^ A significant effect of the ACE locus on interindividual variation in mean arterial pressure (MAP) was detected in a sample of siblings belonging to the youngest generation. After allowing for measured covariates, this effect accounted for 15-25% of the interindividual variance in MAP, and was even greater in a subset with a positive family history of hypertension. When gender-specific analyses were carried out, this effect was significant in males but not in females. Extended pedigree analyses also provided evidence for an effect of the ACE locus on interindividual variation in MAP, but no difference between males and females was observed. Circumstantial evidence suggests that the ACE gene itself may be responsible for the observed effects on BP, although the possibility that other genes in the region may be at play cannot be excluded.^ No definitive evidence for an effect of the renin, angiotensinogen, or AT1 loci on interindividual variation in BP was obtained in this study, suggesting that the impact of these genes on BP may not be great in the Caucasian population-at-large. However, this does not preclude a larger effect of these genes in some subsets of individuals, especially among those with clinically manifest hypertension or coronary heart disease, or in other populations. ^

A new strategy to decrease N-methyl-d-aspartate (NMDA) receptor coactivation: Inhibition of d-serine synthesis by converting serine racemase into an eliminase

Serine racemase is a brain-enriched enzyme that synthesizes d-serine, an endogenous modulator of the glycine site of N-methyl-d-aspartate (NMDA) receptors. We now report that serine racemase catalyzes an elimination reaction toward a nonphysiological substrate that provides a powerful tool to study its neurobiological role and will be useful to develop selective enzyme inhibitors. Serine racemase catalyzes robust elimination of l-serine O-sulfate that is 500 times faster than the physiological racemization reaction, generating sulfate, ammonia, and pyruvate. This reaction provides the most simple and sensitive assay to detect the enzyme activity so far. We establish stable cell lines expressing serine racemase and show that serine racemase can also be converted into a powerful eliminase in cultured cells, while the racemization of l-serine is inhibited. Likewise, l-serine O-sulfate inhibits the synthesis of d-serine in primary astrocyte cultures. We conclude that the synthetic compound l-serine O-sulfate is a better substrate than l-serine as well as an inhibitor of d-serine synthesis. Inhibition of serine racemase provides a new strategy to selectively decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.

Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents.

Apoptosis induced in myeloid leukemic cells by wild-type p53 was suppressed by different cleavage-site directed protease inhibitors, which inhibit interleukin-1 beta-converting enzyme-like, granzyme B and cathepsins B and L proteases. Apoptosis was also suppressed by the serine and cysteine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone (TPCK) [corrected], but not by other serine or cysteine protease inhibitors including N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), E64, pepstatin A, or chymostatin. Protease inhibitors suppressed induction of apoptosis by gamma-irradiation and cycloheximide but not by doxorubicin, vincristine, or withdrawal of interleukin 3 from interleukin 3-dependent 32D non-malignant myeloid cells. Induction of apoptosis in normal thymocytes by gamma-irradiation or dexamethasone was also suppressed by the cleavage-site directed protease inhibitors, but in contrast to the myeloid leukemic cells apoptosis in thymocytes was suppressed by TLCK but not by TPCK. The results indicate that (i) inhibitors of interleukin-1 beta-converting enzyme-like proteases and some other protease inhibitors suppressed induction of apoptosis by wild-type p53 and certain p53-independent pathways of apoptosis; (ii) the protease inhibitors together with the cytokines interleukin 6 and interferon-gamma or the antioxidant butylated hydroxyanisole gave a cooperative protection against apoptosis; (iii) these protease inhibitors did not suppress induction of apoptosis by some cytotoxic agents or by viability-factor withdrawal from 32D cells, whereas these pathways of apoptosis were suppressed by cytokines; (iv) there are cell type differences in the proteases involved in apoptosis; and (v) there are multiple pathways leading to apoptosis that can be selectively induced and suppressed by different agents.

Tonic stimulation of renin gene expression by nitric oxide is counteracted by tonic inhibition through angiotensin II.

This study was designed to examine the possible involvement of prostaglandins and nitric oxide (NO) in the renin stimulatory effect of angiotensin II (AngII) antagonists. To this end, plasma renin activities (PRAs) and renal renin mRNA levels were assayed in rats that were treated with the Ang-converting enzyme inhibitor ramipril or with the AngII AT1-receptor antagonist losartan. Ramipril and losartan increased PRA values from 7.5 +/- 1.6 to 86 +/- 6 and 78 +/- 22 ng of AngI per h per ml and renin mRNA levels from 112 +/- 9% to 391 +/- 20% and 317 +/- 10%, respectively. Inhibition of prostaglandin formation with indomethacin did not influence basal or ramipril-affected PRA. Basal renin mRNA levels also were unchanged by indomethacin, while increases in renin mRNA levels after ramipril treatment were slightly reduced by indomethacin. Inhibition of NO synthase by nitro-L-arginine methyl ester (L-NAME) reduced PRA values to 3.2 +/- 0.9, 34 +/- 13, and 12.1 +/- 2.7 ng of AngI per h per ml in control, ramipril-treated, and losartan-treated animals, respectively. Renin mRNA levels were reduced to 77 +/- 14% under basal conditions and ramipril- and losartan-induced increases in renin mRNA levels were completely blunted after addition of L-NAME. The AngII antagonists, furthermore, induced an upstream recruitment of renin-expressing cells in the renal afferent arterioles, which was also blunted by L-NAME. These findings suggest that renin mRNA levels are tonically increased by NO and that the action of NO is counteracted by AngII.

The renin-angiotensin system and male reproduction: new functions for old hormones

The blood-borne renin-angiotensin system (RAS) is known best for its role in the maintenance of blood pressure and electrolyte and fluid homeostasis. However, numerous tissues show intrinsic angiotensin-generating systems that cater for specific local needs through actions that add to, or differ from, the circulating RAS. The male reproductive system has several sites of intrinsic RAS activity. Recent focus on the epididymis, by our laboratories and by others, has contributed important details about the local RAS in this tissue. The RAS components have been localized morphologically and topographically; they have been shown to be responsive to androgens and to hypoxia; and angiotensin has been shown to influence tubular, and consequently, fluid secretion. Components of the RAS have also been found in the testis, vas deferens, prostate and semen. Angiotensin II receptors, type 1 and, to a lesser extent, type 2 are widespread, and angiotensin IV receptors have been localized in the prostate. The roles of the RAS in local processes at these sites are still uncertain and have yet to be fully elucidated, although there is evidence for involvement in tubular contractility, spermatogenesis, sperm maturation, capacitation, acrosomal exocytosis and fertilization. Notwithstanding this evidence for the involvement of the RAS in various important aspects of male reproduction, there has so far been a lack of clinical evidence, demonstrable by changes in fertility, for a crucial role of the RAS in male reproduction. However, it is clear that there are several potential targets for manipulating the activity of the male reproductive system by interfering with the locally generated angiotensin systems.

Independent, marked associations of alleles of the insulin receptor and dipeptidyl carboxypeptidase-I genes with essential hypertension

1. There is evidence to suggest that essential hypertension is a polygenic disorder and that it arises from yet-to-be-identified predisposing variants of certain genes that influence blood pressure. The cloning of various hormone, enzyme, adrenoceptor and hormone receptor genes whose products are involved in blood pressure control and the identification of polymorphisms of these has permitted us to test their genetic association with hypertension. 2. Cross-sectional analyses of a number of candidate gene markers were performed in hypertensive and normotensive subjects who were selected on the basis of both parents being either hypertensive or normotensive, respectively, and the difference in total alleles on all chromosomes for each polymorphism between the hypertensive and normotensive groups was test by χ analysis with one degree of freedom. 3. A marked association was observed between hypertension and insertion alleles of polymorphisms of the insulin receptor gene (INSR) (P<0.0040) and the dipeptidyl carboxypeptidase-1 (angiotensin I-converting enzyme; kininase II) gene (DCP1) (P<0.0018). No association with hypertension was evident, however, for polymorphisms of the growth hormone, low-density lipoprotein receptor, renal kallikrein, α2- and β1-adrenoreceptor, atrial natriuretic factor and insulin genes. 4. All but one of the hypertensive subjects had at least one of the hypertension-associated alleles, and although subjects homozygous for both were three times more frequent in the hypertensive group, examination of the nine possible genotypes suggested that the INSR and DCP1 alleles are independent markers for hypertension. 5. The present results suggest that genetic variant(s) in close linkage disequilibrium with polymorphisms at INSR and DCP1 may be involved in part in the aetiology of essential hypertension.

Histone deacetylase inhibitors target diabetes via chromatin remodeling or as chemical chaperones?

Globally, obesity and diabetes (particularly type 2 diabetes) represents a major challenge to world health. Despite decades of intense research efforts, the genetic basis involved in diabetes pathogenesis & conditions associated with obesity are still poorly understood. Recent advances have led to exciting new developments implicating epigenetics as an important mechanism underpinning diabetes and obesity related disease. One epigenetic mechanism known as the "histone code" describes the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as lysine acetyltransferases or KATs and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. Some of the known inhibitors of HDACs (HDACi) have also been shown to act as "chemical chaperones" to alleviate diabetic symptoms. In this review, we discuss the available evidence concerning the roles of HDACs in regulating chaperone function and how this may have implications in the management of diabetes. © 2009 Bentham Science Publishers Ltd.

Enzymatic reaction of the immobilized enzyme on porous silicon studied by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

Desorption/ionization on silicon mass spectrometry (DIOS-MS) is a matrix-free technique that allows for the direct desorption/ionization of low-molecular-weight compounds with little or no fragmentation of analytes. This technique has a relatively high tolerance for contaminants commonly found in biological samples. DIOS-MS has been applied to determine the activity of immobilized enzymes on the porous silicon surface. Enzyme activities were also monitored with the addition of a competitive inhibitor in the substrate solution. It is demonstrated that this method can be applied to the screening of enzyme inhibitors. Furthermore, a method for peptide mapping analysis by in situ digestion of proteins on the porous silicon surface modified by trypsin, combined with matrix-assisted laser desorption/ionization-time of flight-MS has been developed.

A型行为核心特征与血管紧张素转换酶基因型相关性研究

In order to study the role of inherited factors and Type A Behavior Pattern (TABP) in the development of CHD, the present study chose the angiotensin I-converting enzyme (ACE) gene as the target gene, and investigated the associations of TABP, the polymorphism of ACE gene with susceptibility to development of CHD in the healthy population and CHD patients from Northern China. 1. Correlation Analysis Between TABP and serum level of ACE in Chinese healthy individuals TABP and serum of ACE were determined in 137 Chinese healthy individuals. The results showed that there was a significant correlation between the scores of CH in TABP invertory and the serum level of ACE. 2 The distribution charicteristics of ACE gene polymorphism frequencies and association with serum level of ACE in Chinese healthy individuals population The polymorphism of ACE gene and serum of ACE were determined in 137 Chinese healthy individuals. The results showed that: the ethnic differences in I/D polymorphism of ACE gene are obvious; deletion polymorphism of the ACE gene is associated with serum ACE level. 3. The relationship between insertion/deletion polymorphism of ACE gene and CHD in a Chinese population I/D polymorphism in intron 16 of the ACE gene was determined by polymerase chain reaction(PCR) in a study of 109 patients with CHD. The results showed: The frequencies of DD genotype(0.39) and D allele(0.63) were higher among the CHD group than among the control subjects(0.12 and 0.42 respectively, P < 0.01). Furthermore, MI and multivessel disease was more strongly associated with (P < 0.01). It is indicated that D allele and DD gentype of ACE might be an important risk factor for CHD, especially for MI or multivessel disease in Chinese population. 4. Correlation Analysis Between Type A Behavior Pattern and the Polymorphism of ACE Gene The polymorphism of ACE gene and type A behavior pattern (TABP) survey were determined in 291 Chinese healthy individuals. The result showed that the higher frequency of rare D allele of an insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene was found in type A behavior individuals compared with type B behavior individuals in 291 healthy individuals; there was a significant correlation between the scores of CH in TABP invertory and DD genotype of the ACE gene. It is suggested that the behavioral attributes of competitiveness, achievement striving, hostility, being irritated easily and impatience may be associated with heredity. 5. Correlation Analysis Between Angiotensin I-Converting Enzyme Gene Polymorphism, Type A Behavior Pattern and Coronary Heart Disease in Chinese The polymorphism of ACE gene and type A behavior pattern (TABP) survey were determined in 109 patients with CHD. The results showed the development of coronary heart disease(CHD) is influenced mainly by the behavioral attributes of competitiveness, achievement striving, hostility, being irritated easily and impatience; the deletion polymorphism of ACE gene may be play a important role in the process of it. 6. Correlation Analysis Between Type A Behavior Pattern Core Components and the Polymorphism of ACE Gene The polymorphism of ACE gene and type A behavior pattern (TABP) survey were determined in1306 Chinese healthy individuals. The results showed that there was a significant correlation between the scores of CH in TABP invertory and DD genotype of the ACE gene. Furthermore, the behavioral attributes of hostility, being irritated easily and impatience may be associated with heredity. At the end of this research, in terms of theory, the research approaches of TABP and the factors influenced the relationship between TABP and CHD were explored and discussed. Furthermore, several new opinions were put forward.

Radiolabeled inhibitors as probes for imaging mutant IDH1 expression in gliomas: Synthesis and preliminary evaluation of labeled butyl-phenyl sulfonamide analogs.

INTRODUCTION: Malignant gliomas frequently harbor mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Studies suggest that IDH mutation contributes to tumor pathogenesis through mechanisms that are mediated by the neomorphic metabolite of the mutant IDH1 enzyme, 2-hydroxyglutarate (2-HG). The aim of this work was to synthesize and evaluate radiolabeled compounds that bind to the mutant IDH1 enzyme with the goal of enabling noninvasive imaging of mutant IDH1 expression in gliomas by positron emission tomography (PET). METHODS: A small library of nonradioactive analogs were designed and synthesized based on the chemical structure of reported butyl-phenyl sulfonamide inhibitors of mutant IDH1. Enzyme inhibition assays were conducted using purified mutant IDH1 enzyme, IDH1-R132H, to determine the IC50 and the maximal inhibitory efficiency of the synthesized compounds. Selected compounds, 1 and 4, were labeled with radioiodine ((125)I) and/or (18)F using bromo- and phenol precursors, respectively. In vivo behavior of the labeled inhibitors was studied by conducting tissue distribution studies with [(125)I]1 in normal mice. Cell uptake studies were conducted using an isogenic astrocytoma cell line that carried a native IDH1-R132H mutation to evaluate the potential uptake of the labeled inhibitors in IDH1-mutated tumor cells. RESULTS: Enzyme inhibition assays showed good inhibitory potency for compounds that have iodine or a fluoroethoxy substituent at the ortho position of the phenyl ring in compounds 1 and 4 with IC50 values of 1.7 μM and 2.3 μM, respectively. Compounds 1 and 4 inhibited mutant IDH1 activity and decreased the production of 2-HG in an IDH1-mutated astrocytoma cell line. Radiolabeling of 1 and 4 was achieved with an average radiochemical yield of 56.6 ± 20.1% for [(125)I]1 (n = 4) and 67.5 ± 6.6% for [(18)F]4 (n = 3). [(125)I]1 exhibited favorable biodistribution characteristics in normal mice, with rapid clearance from the blood and elimination via the hepatobiliary system by 4 h after injection. The uptake of [(125)I]1 in tumor cells positive for IDH1-R132H was significantly higher compared to isogenic WT-IDH1 controls, with a maximal uptake ratio of 1.67 at 3 h post injection. Co-incubation of the labeled inhibitors with the corresponding nonradioactive analogs, and decreasing the normal concentrations of FBS (10%) in the incubation media substantially increased the uptake of the labeled inhibitors in both the IDH1-mutant and WT-IDH1 tumor cell lines, suggesting significant non-specific binding of the synthesized labeled butyl-phenyl sulfonamide inhibitors. CONCLUSIONS: These data demonstrate the feasibility of developing radiolabeled probes for the mutant IDH1 enzyme based on enzyme inhibitors. Further optimization of the labeled inhibitors by modifying the chemical structure to decrease the lipophilicity and to increase potency may yield compounds with improved characteristics as probes for imaging mutant IDH1 expression in tumors.

Studies on acyl pyrrolidine inhibitors of HCV RNA-dependent RNA polymerase to identify a molecule with replicon antiviral activity

The SAR development is described for a series of N-acyl pyrrolidine inhibitors of the Hepatitis C virus RNA-dependent RNA polymerase, NS5B, from tractable Delta 21 enzyme inhibitors to an example with antiviral activity in a cellular assay (HCV replicon). (c) 2007 Elsevier Ltd. All rights reserved.

Increased endocytosis in retinal vascular endothelial cells grown in high glucose medium is modulated by inhibitors of nonenzymatic glycosylation

We sought to determine if hyperglycaemia is responsible for increased retinal vascular endothelial-cell (RVEC) endocytosis in diabetes and to assess the role of nonenzymatic glycosylation in mediation of this novel endothelial-cell pathology. RVECs were propagated in media containing either 5 or 25 mmol/l glucose for up to 10 days after which they were exposed to the protein tracer horseradish peroxidase for 30 min. The level of RVEC endocytosis was quantified in intact cell monolayers by electron microscopic stereology, and in cell lysates by a simple spectrophotometric method. The effect of the nonenzymatic glycosylation inhibitors, aminoguanidine and D-lysine, on high-glucose medium induced changes in RVEC endocytosis was tested by inclusion of these agents in the culture medium. RVECs exposed to 25 mmol/l glucose showed a stepwise increase in endocytosis of horseradish peroxidase culminating in a two- to threefold increase after 10 days. Endocytosis returned to normal levels after a further 10 days in 5 mmol/l glucose medium. The increase in RVEC endocytosis was markedly reduced, but not completely normalised, by aminoguanidine and D-lysine. Exposure of cultured RVECs to 25 mmol/l glucose causes an increase in endocytosis of similar magnitude to that experienced by RVEC in early diabetes, and implicates hyperglycaemia in the latter situation. A significant component of the increase in RVEC endocytosis appears to be mediated by nonenzymatic glycosylation.