Angiotensin II (Ang II) has been implicated in the pathogenesis of the vascular injury associated with hypertension and diabetes mellitus. Increased vascular permeability is an important early manifestation of endothelial dysfunction and the pathogenesis of atherosclerosis. How Ang II contributes to endothelial dysfunction and promotes an increase in vascular permeability is unknown but is classically attributed to its pressor actions. We demonstrate that human vascular smooth muscle cells express abundant mRNA for vascular permeability/endothelial growth factor. Vascular permeability factor is a 34- to 42-kD glycoprotein that markedly increases vascular endothelial permeability and is a potent endothelial mitogen. Ang II potently induced a concentration-dependent (maximal, 10(-7) mol/L) and time-dependent increase in vascular permeability factor mRNA expression by human vascular smooth muscle cells that was maximal after 3 hours and diminished by 24 hours. Ang II-induced vascular permeability factor mRNA expression by human vascular smooth muscle cells was inhibited by the specific Ang II receptor antagonist losartan (DuP 753), confirming that this is an Ang II receptor subtype 1-mediated event. These results describe a new action of Ang II on human vascular smooth muscle, notably the induction of vascular permeability factor mRNA expression. The wide spectrum and potent activity of vascular permeability factor suggest a novel mechanism whereby Ang II could locally and directly influence the permeability, growth, and function of the vascular endothelium independent of changes in hemodynamics.

Objective-The platelet ADP receptors P2Y1 and P2Y12 play a pivotal role in platelet aggregation. There is marked interindividual variation in platelet response to ADP. We studied whether genetic variants in the P2Y1 or P2Y12 genes affect platelet response to ADP. Methods and Results-The P2Y1 and P2Y12 genes were screened for polymorphisms. Associations between selected polymorphisms and the platelet response to ADP (0.1, 1.0, and 10 mol/L), assessed by whole blood flow cytometric measurement of fibrinogen binding to activated glycoprotein IIb-IIIa, were then determined in 200 subjects. Five polymorphisms were found in the P2Y1 gene and 11 in the P2Y12 gene. All polymorphisms were silent. A P2Y1 gene dimorphism, 1622A͘G, was associated with a significant (Pϭ0.007) effect on platelet ADP response, with a greater response in carriers of the G allele (frequency 0.15). The effect was seen at all concentrations of ADP but greatest at 0.1 mol/L ADP, where the response in GG homozygotes was on average 130% higher than that seen in AA homozygotes (Pϭ0.006). Key Words: platelets Ⅲ thrombosis Ⅲ genes Ⅲ receptors Ⅲ adenosine diphosphate P latelet activation and thrombus formation play an integral role in the hemostatic mechanism after vascular injury. After disruption of atherosclerotic plaques, platelet aggregation also plays an important role in development of myocardial infarction and other acute coronary syndromes. 1 Binding of platelets to von Willebrand factor and collagen is the initiating event in platelet activation. This leads to platelet degranulation and release of ADP. ADP, acting via specific receptors, causes further platelet activation and platelet aggregation. Therefore, ADP plays a key direct role in platelet activation. ADP-mediated activation also partly explains the platelet response to other agonists. 2 Two platelet ADP receptors, P2Y1 and P2Y12, have been shown to initiate platelet activation when stimulated in concert. 3 Both are heterotrimeric G-protein-coupled receptors: P2Y1 to Gq and P2Y12 to Gi. Stimulation at P2Y1 leads to intracellular calcium mobilization and platelet shape change, 4 whereas stimulation at P2Y12 leads to inhibition of adenylyl cyclase 5 and activation of phosphoinositide-3 kinase. 6 The end effect is affinity modulation of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor for fibrinogen, resulting in fibrinogen binding and platelet aggregation. 7 It is well recognized that there is substantial interindividual variation in platelet response to ADP. 8,9 The reasons for the interindividual variation are poorly defined, but the observation that the response is stable over time, in a given individual, 9 raises the possibility that at least in part, it may be genetically controlled because of variation in the P2Y1 and/or P2Y12 genes. Conclusions-P2Y1 and P2Y12 genes are located on chromosome 3. The P2Y1 gene spans Ϸ4 kb 10 and is made up of a single exon of 3122 base pairs encoding a 372-aa protein. 11 The P2Y12 gene spans 47 kb and is made up of 3 exons and 2 introns. 12 There are ...

The role of the kidney in initiating hypertension has been much debated. Here we demonstrate that a recently identified gene of yet unknown function, termed SA, which is differentially expressed in the kidney of the spontaneously hypertensive rat, cosegregates with an increase in blood pressure in F2 rats derived from a cross of the spontaneously hypertensive rat with normotensive Wistar-Kyoto rats, accounting for 28 and 21% of the genetic variability in systolic and diastolic blood pressures, respectively. Further, the genotype at this locus appears to determine the level of expression of the gene in the kidney. The findings provide strong evidence for a primary genetic involvement of the kidney in hypertension. (J. Clin. Invest. 1993.

Hyperhomocyst(e)inemia is associated with an increased risk of coronary artery disease and myocardial infarction. Both genetic and environmental factors influence the plasma level of homocysteine. One of the metabolic pathways for homocysteine involves the enzyme methylenetetrahydrofolate reductase (MTHFR), which regulates the conversion of homocysteine to methionine. A thermolabile variant of MTHFR is associated with reduced enzyme activity and increased plasma homocysteine levels. Recently, the cause of this variant of MTFHR has been identified as a single base change altering an alanine to a valine residue in the protein. Using a PCR-based assay to distinguish the normal and thermolabile variants of MTHFR in this study, we investigated whether the thermolabile variant is a genetic risk factor for myocardial infarction. In a study of 532 subjects (310 myocardial infarction patients and 222 population-based controls), we found no difference in either MTHFR genotype distribution (p = 0.57) or allele frequencies (p = 0.68) between cases and controls. The allele frequencies of the thermolabile variant were 0.34 and 0.35 in cases and controls, respectively. The age- and sex-stratified odds ratio for risk of myocardial infarction associated with homozygosity for the thermolabile variant was 0.85 (95% CI 0.50-1.50, p = 0.57) and that with carriage of the thermolabile allele was 1.06 (95% CI 0.73-1.52, p = 0.76). The odds ratios remained non-significant when restricted to young subjects (< 60 years) or males, and were not influenced by several other risk factors for myocardial infarction considered either singly or in combination. Interestingly, in both cases and controls, there was a trend toward a higher prevalence of hypertension in subjects carrying the normal allele, although as this is a post-hoc finding it needs to be interpreted with caution. The thermolabile variant of MTHFR is not a major risk factor for myocardial infarction and is unlikely to explain a significant proportion of the reported association of hyperhomocyst(e)inemia with coronary artery disease.

The production of different transcripts (transcript heterogeneity) is a feature of many genes that may result in phenotypic variation. Several mechanisms, that occur at both the DNA and RNA level have been shown to contribute to this transcript heterogeneity in mammals, all of which involve either the rearrangement of sequences within a genome or the use of alternative signals in linear, contiguous DNA or RNA. Here we describe tissue-specific repetition of selective exons in transcripts of a rat gene (SA) with a normal exon-intron organization. We conclude that nonlinear mRNA processing can generate tissue-specific transcripts.

Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein-SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254-1545), rSUR2A-CTB (residues 1254-1403) and rSUR2A-CTC (residues 1294-1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358-1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294-1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294-1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels.

ATP-sensitive potassium (K ATP ) channels are abundantly expressed in the myocardium.Although a definitive role for the channel remains elusive they have been implicated in the phenomenon of cardioprotection, but the precise mechanism is unclear. We set out to test the hypothesis that the channel protects by opening early during ischemia to shorten action potential duration and reduce electrical excitability thus sparing intracellular ATP. This could reduce reperfusion injury by improving calcium homeostasis.Using a combination of contractile function analysis, calcium fluorescence imaging and patch clamp electrophysiology in cardiomyocytes isolated from adult male Wistar rats, we demonstrated that the opening of sarcolemmal K ATP channels was markedly delayed after cardioprotective treatments; ischemic preconditioning, adenosine and PMA. This was due to the preservation of intracellular ATP for longer during simulated ischemia therefore maintaining sarcolemmal K ATP channels in the closed state for longer. As the simulated ischemia progressed, K ATP channels opened to cause contractile, calcium transient and action potential failure, however there was no indication of any channel activity early during simulated ischemia to impart an energy sparing hyperpolarization or action potential shortening.We present compelling evidence to demonstrate that early opening of sarcolemmal K ATP channels during simulated ischemia is not part of the protective mechanism imparted by ischemic preconditioning or other PKC-dependent cardioprotective stimuli. On the contrary, channel opening was actually delayed. We conclude that sarcolemmal K ATP channel opening is a consequence of ATP depletion, not a primary mechanism of ATP preservation in these cells. 3Highlights:• Opening of the SarcoK ATP channel was proposed to be cardioprotective• Channel opening was delayed after cardioprotective stimuli• Ca 2+ & ATP levels were maintained during ischemia independent of SarcoK ATP opening• Mitochondrial function preserved during ischemia, independent of SarcoK ATP opening• Early opening of SarcoK ATP is not involved in PKC-dependent cardioprotection

We have identified and characterised a cDNA encoding a novel gene, designated myocyte stress 1 (ms1), that is up-regulated within 1 h in the left ventricle following the application of pressure overload by aortic banding in the rat. The deduced ms1 protein of 317 amino acids contains several putative functional motifs, including a region that is evolutionarily conserved. Distribution analysis indicates that rat ms1 mRNA expression is predominantly expressed in striated muscle and progressively increases in the left ventricle from embryo to adulthood. These findings suggest that ms1 may be important in striated muscle biology and the development of pressure-induced left ventricular hypertrophy. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.