Clinical and Experimental Evaluation of Diagnostic Significance of Alpha-Fetoprotein and Osteopontin at the Early Stage of Hepatocellular Cancer.

We evaluated the possibility of using an experimental model of hepatocellular carcinoma to study oncomarkers of primary liver cancer and compared the diagnostic efficacy of alpha-fetoprotein and osteopontin in the experiment and in clinical practice. Experimental studies were performed on a model of hepatocellular carcinoma induced by administration of diethyl nitrosamine to Fisher-344 rats. In addition, the levels of α-fetoprotein and osteopontin were determined in 35 patients with hepatocellular carcinoma detected at stages I-II according to TNM classification. The proposed model of liver cancer in rats reflects the sequence of stages characteristic of hepatocellular carcinoma in humans: liver fibrosis-cirrhosis-cancer. This model is applicable for the study of tumor markers at the early stage of tumor development. Osteopontin was found to have a more powerful diagnostic potential then alpha-fetoprotein.

[Application of alpha-fetoprotein and osteopontin combination for early diagnosis of hepatocellular carcinoma associated with hepatitis C.]

Liver cirrhosis in the outcome of hepatitis C is the leading cause of hepatocellular carcinoma (HCC) in the world. Early diagnosis and timely treatment of HCC are important for reducing mortality and increasing life expectancy of patients with hepatocellular carcinoma. To assess the risk of HCC, the definition of alpha-fetoprotein (AFP) in the blood is most widely used, but low sensitivity limits its diagnostic value. In 2012, a new HCC biomarker - osteopontin (OPN), which is a secreted phosphoprotein that has a high affinity for integrins was proposed. The level of acute renal failure begins to rise in the early stages of malignancy, before the period of HCC detection by imaging methods, and has significantly better sensitivity than AFP. The purpose of this study is to evaluate the diagnostic efficacy of the combined determination of alpha-fetoprotein and osteopontin in prospective monitoring of patients with chronic hepatitis C in the advanced phase of liver fibrosis. Monitoring of 588 patients with hepatitis C was carried out from February 2013 to February 2019. HCC was detected in 55 of them (2.6% per year). The combination of 2 biomarkers showed better diagnostic efficacy than alpha-fetoprotein and osteopontin separately: AUC 0.85 (95% CI 0.80-0.90) versus AUC 0.63 (95% CI 0.57-0, 70) and AUC 0.82 (95% CI 0.77-0.88), respectively. This combination showed a sensitivity of 85.5% and made it possible to diagnose HCC with a prognostic level of a positive result of 72.3% at 19,4±0,8 weeks before the diagnosis was confirmed by instrumental imaging methods (ultrasound, MRI, CT). In the combined variant, ARF made the greatest contribution to the increase in diagnostic efficacy (AUC). At an early and very early stage of HCC development, isolated HCC elevations were found in only 5.4% of patients. Conclusion: the combined use of alphafetoprotein and osteopontin as a diagnostic panel can be recommended for monitoring patients with liver cirrhosis in the outcome of hepatitis C and predicting HCC at an early stage of development.

Developmental regulation of p53-dependent radiation-induced thymocyte apoptosis in mice.

The production of T cell receptor αß(+) (TCRαß(+) ) T lymphocytes in the thymus is a tightly regulated process that can be monitored by the regulated expression of several surface molecules, including CD4, CD8, cKit, CD25 and the TCR itself, after TCR genes have been assembled from discrete V, D (for TCR-ß) and J gene segments by a site-directed genetic recombination. Thymocyte differentiation is the result of a delicate balance between cell death and survival: developing thymocytes die unless they receive a positive signal to proceed to the next stage. This equilibrium is altered in response to various physiological or physical stresses such as ionizing radiation, which induces a massive p53-dependent apoptosis of CD4(+) CD8(+) double-positive (DP) thymocytes. Interestingly, these cells are actively rearranging their TCR-α chain genes. To unravel an eventual link between V(D)J recombination activity and thymocyte radio-sensitivity, we analysed the dynamics of thymocyte apoptosis and regeneration following exposure of wild-type and p53-deficient mice to different doses of γ-radiation. p53-dependent radio-sensitivity was already found to be high in immature CD4(-) CD8(-) (double-negative, DN) cKit(+) CD25(+) thymocytes, where TCR-ß gene rearrangement is initiated. However, TCR-αß(-) CD8(+) immature single-positive thymocytes, an actively cycling intermediate population between the DN and DP stages, are the most radio-sensitive cells in the thymus, even though their apoptosis is only partially p53-dependent. Within the DP population, TCR-αß(+) thymocytes that completed TCR-α gene recombination are more radio-resistant than their TCR-αß(-) progenitors. Finally, we found no correlation between p53 activation and thymocyte sensitivity to radiation-induced apoptosis.

Magnetically localized and wash-free fluorescence immunoassay (MLFIA): proof of concept and clinical applications.

Immunoassays are used for many applications in various markets, from clinical diagnostics to the food industry, generally relying on gold-standard ELISAs that are sensitive, robust, and cheap but also time-consuming and labour intensive. As an alternative, we propose here the magnetically localized and wash-free fluorescence immunoassay (MLFIA): a no-wash assay to directly measure a biomolecule concentration, without mixing nor washing steps. To do so, a fluorescence no-wash measurement is performed to generate a detectable signal. It consists of a differential measurement between the fluorescence of fluorophores bound to magnetic nanoparticles specifically captured by micro-magnets against the residual background fluorescence of unbound fluorophores. Targeted biomolecules (antibodies or antigens) are locally concentrated on micro-magnet lines, with the number of captured biomolecules quantitatively measured without any washing step. The performance of the MLFIA platform is assessed and its use is demonstrated with several biological models as well as clinical blood samples for HIV, HCV and HBV detection, with benchmarking to standard analyzers of healthcare laboratories. Thus, we demonstrated for the first time the versatility of the innovative MLFIA platform. We highlighted promising performances with the successful quantitative detection of various targets (antigens and antibodies), in different biological samples (serum and plasma), for different clinical tests (HCV, HBV, HIV).

Search for Effective Serum Tumor Markers for Early Diagnosis of Hepatocellular Carcinoma Associated with Hepatitis C.

The aim of the study was to identify the most effective serum tumor markers for early diagnosis of hepatocellular carcinoma based on the combination of diagnostic characteristics and correlations. Materials and Methods: There were observed 55 patients with chronic hepatitis C in the stage of liver cirrhosis with a verified diagnosis of hepatocellular carcinoma. The control group consisted of 55 patients with chronic hepatitis C at the stage of liver cirrhosis without hepatocellular carcinoma, comparable to the experimental group in terms of basic clinical profile. The following tumor markers were estimated in both groups: alpha-fetoprotein (AFP), alpha-fetoprotein-L3 (AFP-L3), annexin A2 (ANXA2), heparin-binding growth factor Midkine (MDK), glypican-3 (GPC3), des-gamma-carboxyprothrombin (DCP, PIVKA-II), dickkopf-related protein 1 (DKK-1), osteopontin (OPN), and Golgi protein 73 (GP73). There were also evaluated such indices as diagnostic sensitivity, specificity, positive predictive value, negative predictive value, likelihood ratio of a positive test, the possible correlation between alpha-fetoprotein and other tumor markers. The area under the ROC curve (AUC) was calculated at the 95% confidence interval. Results: The greatest sensitivity was revealed when using heparin-binding growth factor, annexin A2, osteopontin. Alpha-fetoprotein, alpha-fetoprotein-L3, glypican-3, des-gamma-carboxyprothrombin, dickkopf-related protein 1 had the best specificity. AUC>0.75 was found in annexin A2, heparin-binding growth factor, glypican-3, des-gamma-carboxyprothrombin, osteopontin, Golgi protein 73. The likelihood ratio of a positive test result was the highest for glypican-3. A significant correlation was found between alpha-fetoprotein and alpha-fetoprotein-L3, annexin A2, des-gamma-carboxyprothrombin. Conclusion: According to the aggregate indicators of diagnostic efficiency, heparin-binding growth factor, glypican-3, and osteopontin are the most promising tumor markers of those studied. When they are used, integral AUC values are above the average, the level of these tumor markers in the blood of patients with hepatocellular cancer does not correlate with alpha-fetoprotein. They are applicable for diagnosing liver cancer in AFP-negative patients. The combined use of AFP + GPC3, AFP + OPN has already shown their advantages. However, the efficacy of the combination of AFP + MDK, GPC3 + OPN has not been determined yet; therefore, significance of the combined use of these tumor markers in the diagnosis of liver cancer should be investigated in the near future.

Identification of a T cell receptor beta chain variable region, V beta 20, that is differentially expressed in various strains of mice.

A cDNA library of TCR beta chain transcripts from BALB/c thymocytes was constructed using anchored polymerase chain reaction (PCR). Screening of this library led to the identification of a V beta gene segment, V beta 20, structurally related to V beta 3 and V beta 17. Genomic analysis of mice displaying deletions in their V beta loci, together with mapping of cosmid clones, situated V beta 20 2.5 kb beside V beta 17. The expression of V beta 20 was estimated by PCR in mice of different H-2 and Mls types. Peripheral T cells from H-2k and H-2d mice did not express V beta 20, whereas in I-E-negative mice (C57Bl/6 and SJL), V beta 20 transcripts were detected. The lack of V beta 20 transcripts in (C57Bl/6 x CBA/J)F1, (C57Bl/6 x BALB/c)F1, and in congenic B6.H-2k mice suggests that the differential use of V beta 20 is due to an I-E-mediated clonal deletion process. The involvement of the Mls super antigens was excluded by analysis of all Mls type combinations. The nature of the V beta 20-deleting element(s) is discussed in the context of the I-E/superantigen systems controlling the expression of V beta 11 and V beta 17.

Neonatal deletion and selective expansion of mouse T cells by exposure to rabies virus nucleocapsid superantigen.

The nucleocapsid (NC) of the rabies virus behaves as an exogenous superantigen (SAg) in humans. In the present report, we analyzed whether it is also a SAg in mice by studying the effect of NC on T cell receptor (TCR) V beta expression in BALB/c mice. Repeated injection of NC in newborn BALB/c mice led to a marked reduction by two- to sixfold of V beta 6 expressing CD4+ T cells in spleen and in peripheral blood. Decrease of V beta 6-expressing CD3+ mature T cells was also observed in thymus. Single NC injection in footpad resulted in a three- to sixfold expansion of V beta 6 CD4+ T cells, but not of CD8+ T cells, in the draining lymph nodes of BALB/c mice. The intensity of the stimulation was dose dependent and was maximal 3 d after the NC injection. The clonal deletion of T cells bearing a particular V beta demonstrates that NC is a SAg in mice. T cells, especially CD4+ T cells, are an essential factor in host resistance to rabies virus and also in the pathophysiology of paralysis; thus, we postulate that a rabies virus component, which stimulates T cells, such as a SAg, may increase virus immunopathogenicity. To evaluate this hypothesis, we compared the course of rabies in adult BALB/c lacking V beta 6, 7, 8.1, and 9 T cells and in normal BALB/c. Immune-related paralysis was decreased in BALB/c missing the NC target V beta T cells. Transfer of V beta 6 but not of V beta 8.1-3 T cells into recipient mice lacking V beta 6, 7, 8.1, and 9 allowed the immune-related paralysis to evolve. Taken together, these results strongly support the hypothesis that T cells expressing rabies SAg-specific V beta 6 T cells, are involved in the genesis of the immunopathology that is characteristic of paralytic rabies.

Neurovascular microcirculatory vasodilation mediated by C-fibers and Transient receptor potential vanilloid-type-1 channels (TRPV 1) is impaired in type 1 diabetes.

Microvascular dysfunction may have an early onset in type 1 diabetes (T1D) and can precede major complications. Our objectives were to assess the endothelial-dependent (acetylcholine, ACh; and post-occlusive hyperemia, PORH), non-endothelial-dependent (sodium nitroprusside, SNP) and neurovascular-dependent (local heating, LH and current induced vasodilation, CIV) microcirculatory vasodilation in T1D patients compared with matched control subjects using a laser speckle contrast imager. Seventeen T1D patients - matched with 17 subjects according to age, gender, Body-Mass-Index, and smoking status - underwent macro- and microvascular investigations. The LH early peak assessed the transient receptor potential vanilloid type 1 channels (TRPV1) mediated vasodilation, whereas the plateau assessed the Nitirc-Oxyde (NO) and endothelium-derived hyperpolarizing factor (EDHF) pathways. PORH explored sensory nerves and (EDHF), while CIV assessed sensory nerves (C-fibers) and prostaglandin-mediated vasodilation. Using neurological investigations, we observed that C-fiber and A-delta fiber functions in T1D patients were similar to control subjects. PORH, CIV, LH peak and plateau vasodilations were significantly decreased in T1D patients compared to controls, whereas there was no difference between the two groups for ACh and SNP vasodilations. Neurovascular microcirculatory vasodilations (C-fibers and TRPV 1-mediated vasodilations) are impaired in TD1 patients whereas no abnormalities were found using clinical neurological investigations. Clinicaltrials: No. NCT02538120.

Enhanced turnover of phosphatidylcholine in platelets of hypertensive rats. Possible involvement of a phosphatidylcholine-specific phospholipase C.

In an attempt to determine the mechanism involved in the hyperreactivity of platelets in primary hypertension, the dynamic behavior of phospholipids was investigated in quiescent platelets of spontaneously hypertensive rats (SHR) compared to normotensive controls. By using 32Pi, [methyl-3H]choline or [3H]glycerol as the radioactive precursors, the labeling of phosphatidylcholine (PC) was shown to be markedly enhanced (10-20-times) in SHR compared to controls. This difference between SHR and controls could not be ascribed to differences either in the actual amount of PC or in the uptake of various labels, suggesting that PC turnover was markedly enhanced in platelets of SHR. The [methyl-3H]choline labeling of phosphocholine and of CDP-choline was twice as high in SHR as in controls; chase experiments showed that when the label disappeared from phosphocholine, it was rapidly converted to PC. The results indicated that in rat platelets, PC synthesis occurred mainly via the CDP-choline pathway, and suggested that CTP:phosphocholine cytidylyltransferase was the rate-limiting step; they also indicated that the activity of this enzyme and that of choline kinase might be enhanced in SHR platelets compared to Wistar-Kyoto rat (WKY) platelets, and may thus be responsible for the enhanced PC synthesis. From these results, the existence of a PC-specific phospholipase C activity involved in PC turnover in SHR platelets can be envisaged.

Metabolism of phosphoinositides in the rat erythrocyte membrane. A reappraisal of the effect of magnesium on the 32P incorporation into polyphosphoinositides.

The metabolism of phosphoinositides was investigated in the red blood cell membrane of the rat by measuring 32P-incorporation into phospholipids after incubation of membranes with [gamma-32P]ATP in a medium containing magnesium. A new chromatographic procedure has been developed which facilitates the separation of triphosphoinositide, diphosphoinositide and phosphatidylinositol from the phospholipids present in lipid extracts of incubated 'ghost' under our experimental conditions only two phospholipids, diphosphoinositide and triphosphoinositide, were 32P-labelled. Furthermore, the results indicate that either di-or triphosphoinositide could be labelled preferentially, depending upon the magnesium concentration of the incubation medium. This clarifies some apparent discrepancies reported in the literature between the 32 P labelling of polyphosphoinositides observed in intact erythrocytes and that observed with 'ghost' membranes. In addition, the enzymatic pathways involved in the phosphoinositide metabolism are discussed.

Novel vascular biology of third-generation L-type calcium channel antagonists: ancillary actions of amlodipine.

Calcium channel blockers (CCBs) were developed as vasodilators, and their use in cardiovascular disease treatment remains largely based on that mechanism of action. More recently, with the evolution of second- and third-generation CCBs, pleiotropic effects have been observed, and at least some of CCBs' benefit is attributable to these mechanisms. Understanding these effects has contributed greatly to elucidating disease mechanisms and the rationale for CCB use. Furthermore, this knowledge might clarify why drugs are useful in some disease states, such as atherosclerosis, but not in others, such as heart failure. Although numerous drugs used in the treatment of vascular disease, including statins and angiotensin-converting-enzyme inhibitors, have well-described pleiotropic effects universally accepted to contribute to their benefit, little attention has been paid to CCBs' potentially similar effects. Accumulating evidence that at least 1 CCB, amlodipine, has pharmacologic actions distinct from L-type calcium channel blockade prompted us to investigate the pleiotropic actions of amlodipine and CCBs in general. There are several areas of research; foci here are (1) the physicochemical properties of amlodipine and its interaction with cholesterol and oxidants; (2) the mechanism by which amlodipine regulates NO production and implications; and (3) amlodipine's role in controlling smooth muscle cell proliferation and matrix formation.

A monoclonal antibody directed against a synthetic peptide reacts with a cell surface rabbit class I MHC molecule.

Monoclonal antibodies were prepared against synthetic peptides synthetized on the basis of nucleotide sequence from cDNA and genomic clones encoding a class I antigen expressed by the rabbit RL-5 cell line. Using a peptide corresponding to positions 61-73 of the N domain, we were able to obtain an hybridoma producing monoclonal antibody which recognized the peptide as well as the native class I antigen. This hybridoma, designated anti-61, reacted with cell surface molecules on RL-5 cells and on human HeLa cells transfected with the 19-1 gene encoding RL-5 class I antigen. No reactivity with HeLa cells prior to transfection could be detected by radioimmunoassay or by fluorescent activated cell sorter analyses, although these cells were strongly positive in both assays with anti-human class I reagents. Antibody reaction to positive cells could be inhibited by the homologous peptide but not by unrelated peptides. Anti-61 antibody precipitated a 41,000 mol. wt molecule from RL-5 cells with an N-terminal amino acid sequence corresponding to the RL-5 class I antigen.

Physicochemical studies on the antigen-antibody complexes of two monoclonal antibodies against rabbit thymocytes.

We have characterized two monoclonal antibodies directed against rabbit thymocyte antigens. Using internally labelled MD3 cells (a transformed T-cell line), ART-F immunoprecipitated a membrane glycoprotein of 95,000 mol. wt, while ART-A immunoprecipitated two major glycoproteins, one of 95,000 mol. wt, the other of 105,000 mol. wt as analysed on reduced SDS-PAGE. ART-A recognizes 650,000 sites both on rabbit thymocytes and MD3 cells with an association constant of 1.5 X 10(5)/M. ART-F shows a biphasic binding curve with a high affinity constant of 3.3 and 0.6 X 10(8)/M and a low affinity constant of 2.2 and 6.9 X 10(5)/M for thymocytes and MD3 cells respectively. The numbers of high affinity and low affinity antigens are 200,000 for the former and 800,000 for the latter. On the basis of the kinetic data, the difference between low and high affinity is attributed to the difference in association-rate constants. The affinity constants calculated from the reaction-rate constants are in good agreement with those obtained from equilibrium measurements. While ART-F prevents the binding of ART-A, ART-A does not inhibit the affinity binding of ART-F. The analysis of the equilibrium, inhibition and kinetic data allow us to present a model for the structural relation of the epitopes recognized on the T-cells by the two monoclonal antibodies.

Evidence for antigen driven selection in two monoclonal auto-antibodies derived from nonobese diabetic mice.

The nonobese diabetic (NOD) mouse is a model of human type I diabetes. This diabetes is due to massive infiltration of the pancreatic beta cell of islets by autoreactive T cells (insulitis) followed by the destruction of insulin-producing cells. Circulating autoantibodies are also detected, notably against glutamic acid decarboxylase, peripherin and insulin. Two monoclonal autoantibodies directed against insulin and peripherin were obtained by fusing NOD spleen and myeloma cells. We report here the nucleotide sequence of the genes encoding for the V regions of these two antibodies. Somatic mutations were identified by comparing the light chain nucleotide sequence of one of these autoantibodies with its germline counterpart precursor established from NOD mice after PCR gene amplification. The other one displays N additions on both sides of the D region. These results strongly suggest that both autoantibodies have undergone diversification, either N additions or somatic mutations, and therefore present structural features of antibodies derived from animals immunized against exogenous antigens.

Pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 superantigen.

Superantigens (SAg) are proteins of bacterial or viral origin able to activate T cells by forming a trimolecular complex with both MHC class II molecules and the T cell receptor (TCR), leading to clonal deletion of reactive T cells in the thymus. SAg interact with the TCR through the beta chain variable region (Vbeta), but the TCR alpha chain has been shown to have an influence on the T cell reactivity. We have investigated here the role of the TCR alpha chain in the modulation of T cell reactivity to Mtv-7 SAg by comparing the peripheral usage of Valpha2 in Vbeta6(+) (SAg-reactive) and Vbeta8.2(+) (SAg non-reactive) T cells, in either BALB/D2 (Mtv-7(+)) or BALB/c (Mtv-7(-)) mice. The results show, first, that pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 SAg. Second, there is a strikingly different distribution of the Valpha2 family members in CD4 and CD8 populations of Vbeta6 but not of Vbeta8.2 T cells, irrespective of the presence of Mtv-7 SAg. Third, the alpha chain may play a role in the overall stability of the TCR/SAg/MHC complex. Taken together, these results suggest that the Valpha domain contributes to the selective process by its role in the TCR reactivity to SAg/MHC class II complexes, most likely by influencing the orientation of the Vbeta domain in the TCR alphabeta heterodimer.

Analysis of the nucleotide sequence variation of the antigen-binding domain of DR alpha and DQ alpha molecules as related to the evolution of papillomavirus-induced warts in rabbits.

We previously found that regression of skin warts induced by the Shope cottontail rabbit papillomavirus in New Zealand White rabbits, as well as malignant conversion of persistent warts, are linked to a restriction fragment length polymorphism of the major histocompatibility complex class II DR alpha and DQ alpha genes. To find out whether this immunogenetic control could be connected with the antigen binding and presentation function of the alpha 1 domain of class II molecules, we have sequenced the exon 2 of the four DR alpha EcoRI and six of the seven DQ alpha PvuII restriction fragment length polymorphism alleles identified, and deduced the encoded amino acid sequences. We found no amino acid polymorphism among DR alpha alleles, indicating that the alpha 1 domain of the DR alpha chain does not condition wart regression or cancer development. In contrast, 27 of the 82 amino acids of the DQ alpha 1 domain were found variable, defining five amino acid sequence alleles. The restriction fragment length polymorphism allele linked to regression and another allele not linked to regression share the same alpha 1 domain, indicating that wart regression is rather conditioned by a closely linked gene. The most divergent DQ alpha 1 allele, however, was that associated with a higher risk of cancer. Alignment of rabbit and human DQ alpha exon 2 alleles disclosed that amino acid charge variations occur at positions assumed to be important for peptide binding in humans. By modulating the affinity for tumor-specific antigenic peptides, such transitions could affect immune surveillance and, thus, condition the risk for progression to carcinoma of papillomavirus-associated lesions.

Vitamin D3 metabolite injections to thyroparathyroidectomized pregnant rats: effects on calcium-binding proteins of maternal duodenum and of fetoplacental unit.

Fetomaternal relationships with respect to vitamin D metabolism were investigated in thyroparathyroidectomized (TPTX) pregnant rats, with or without treatment with different vitamin D3 metabolites. Calcium-binding protein (CaBP) in maternal duodenum was used as an index of 1,25-(OH)2D3 status of the mother. Pregnant rats were TPTX on day 12.5 and CaBP was measured on 21.5 days of gestation by RIA in maternal duodenal mucosa and in the fetoplacental unit (placenta, fetal membranes, and fetal intestine). In the duodenum of TPTX mothers, the CaBP concentration was reduced by 50%. This fall was associated with a decrease of 1,25-(OH)2D in maternal plasma. CaBP in maternal duodenum increased by the administration of 1,25-(OH)2D3 or 1,24,25-(OH)3D3. In contrast, 24,25-(OH)2D3 injections to TPTX mothers were ineffective. In both placenta and fetal membranes, CaBPs decreased by 20% in TPTX mothers and were normalized only in 1.25-(OH)2D3-treated TPTX mothers. In the fetal intestine, CaBP variations paralleled those of maternal duodenal CaBP. The data indicate that plasma levels of 1,25-(OH)2D in TPTX pregnant rats are partly under the control of maternal parathyroid glands, and they support that even in pregnancy, the CaBP concentration in maternal duodenum may well reflect the 1,25-(OH)2D status of the mother. The CaBP synthesis in placenta and fetal membranes are vitamin D-dependent, and their regulation differs from that of intestinal CaBP. It app]ears that 1 alpha-hydroxylase activities of the fetoplacental unit (placenta and fetal kidney) are blunted in TPTX animals and that CaBP synthesis in the fetus depends on the presence of 1 alpha-hydroxylated vitamin D3 metabolites in the mother.

Hypersensitivity of phospholipase C in platelets of spontaneously hypertensive rats.

Thrombin-induced aggregation and serotonin release were markedly enhanced in platelets from spontaneously hypertensive rats (SHR) when compared with those from normotensive Wistar-Kyoto rats (WKY). Since phosphoinositides are involved in calcium-mediated platelet responses, the metabolism of these lipids was investigated in SHR and WKY by using 32P-labeled quiescent platelets. In unstimulated cells, both the rate and extent of 32P incorporation into individual inositol-containing phospholipids and phosphatidic acid were identical in SHR and WKY. This finding suggests that the pool size and basal turnover of phosphoinositides did not differ between the two strains. In contrast, early thrombin-induced phosphoinositide metabolism, when monitored as changes in [32P]phosphatidic acid, was significantly higher in SHR than in WKY. For example, a 20-second exposure to thrombin, 0.3 U/ml, induced the formation of 1.6 times more [32P]phosphatidic acid in SHR than in WKY. These results provide evidence for a leftward shift of the dose-response and time-course curves of thrombin-induced [32P]phosphatidic acid formation in SHR. Moreover, the extent of the difference between SHR and WKY was independent of the extracellular calcium concentration. Following thrombin stimulation, [32P]phosphatidic acid formation likely reflects the initial agonist-receptor interaction; therefore, these results suggest that phospholipase C activity is enhanced in platelets of SHR and that the hypersensitivity of phospholipase C in SHR may play a role in the overall alteration of cell calcium handling and, hence, in the platelet responses of SHR.

Platelet phosphatidylcholine turnover in experimental hypertension.

To gain insight into the membrane alteration that could account for the hyperresponsiveness of platelets in hypertension, we have investigated whether, in resting platelets of hypertensive rats, the metabolism of phospholipids was modified. Because preliminary results indicated a specific acceleration of phosphatidylcholine turnover in spontaneously hypertensive rats, the possible relation between such an abnormality and hypertension was investigated by studying phosphorus-32 labeling of phosphatidylcholine (taken as an index of its turnover) in various experimental models of hypertension. The data showed that phosphatidylcholine turnover 1) was considerably increased in platelets from spontaneously hypertensive (even at the prehypertensive stage) and stroke-prone rats compared with Wistar or Wistar-Kyoto control rats, 2) did not differ between deoxycorticosterone-salt-treated hypertensive and control rats, and 3) was increased in Dahl salt-sensitive rats fed a high NaCl diet (hence hypertensive rats), compared with either the rats fed a low NaCl diet or the salt-resistant rats. These results indicate that an increase in phosphatidylcholine turnover is a consequence of neither hypertension nor high salt intake and appears likely to be of genetic origin. These data allow us to suggest the existence, in platelets, of a relation between phosphatidylcholine turnover, free cytoplasmic Ca2+, and responsiveness to stimuli. Because phosphatidylcholine is assumed to participate in signal transduction, an increase in its turnover in platelets might be considered as a primary membrane abnormality that, in primary hypertension, results in platelet hyperresponsiveness.

Altered turnover of polyphosphoinositides in the erythrocyte membrane of the spontaneously hypertensive rat.

The metabolism of inositol phospholipids of the erythrocyte membrane was compared in normotensive Wistar-Kyoto (WKY), spontaneously hypertensive (SHR), and stroke-prone SHR (SHR-SP) rats. This was performed on isolated ghost membranes by measuring the incorporation of 32P from [ gamma-32P ] adenosine triphosphate (ATP) into the diphosphoinositides (DPI) and the triphosphoinositides (TPI) which were the only 32P-labeled phospholipids. 32P-labeling of TPI was altered in adult and 3-week-old SHR as well as in SHR-SP compared to WKY controls; the radioactivity associated with TPI in hypertensive rats was about 30% lower than that associated with TPI in age-matched normotensive controls. By contrast, the radioactivity associated with DPI was similar in both hypertensive and normotensive rats. Measurement of the phosphoinositide distribution in both SHR and WKY indicates that the change observed in 32P-TPI could not be accounted for by a reduced phosphatidylinositol content in SHR membrane. Measurement of the Mg2+-activated TPI-phosphomonoesterase and of the Ca2+-activated polyphosphoinositide phosphodiesterase activities did not show any significant difference between SHR and WKY. It thus appears that the altered phosphoinositide metabolism observed in hypertensive rats was a consequence of some alteration in the activity of kinases which are responsible for the conversion of phosphatidylinositol into DPI and TPI. These results also suggest that the defect in phosphoinositide metabolism observed in genetically hypertensive rats was not a consequence of the blood pressure elevation and could be related to the pathogenesis of hypertension.