[First-line treatment with pemetrexed in association with cisplatin in patients with non-operable malignant pleural mesothelioma]. / Association pemetrexed-cisplatine en première ligne dans le mésothéliome pleural malin non opérable.

Malignant pleural mesothelioma (MPM) is an aggressive disease with a poor prognosis. The optimal treatment of MPM was not clearly defined, until the publication of the multicentre, controlled and randomized phase III trial by Vogelzang et al. in 2003, which made the pemetrexed-cisplatin association the gold standard for the non-operable stages. Eleven patients with histologically proven pleural mesothelioma, not candidates for curative surgery, were assessed for eligibility and treated in our hospital. The response rate was similar to the reference study and the toxicity was acceptable. The median survival time was 12.7 months with an objective response rate of 45.5%. The median time to progression was 7.7 months. Neutropenia (all grades included) was the most common haematological toxicity (42.1%) although only one grade 3/4 was noted. Grade 3/4 anaemia and thrombocytopenia were not reported. Nausea and vomiting were the most commonly reported clinical toxicities with 81.8% reported (all grades included). One cutaneous allergic reaction was reported. The combination of pemetrexed and cisplatin chemotherapy provided the best objectives responses, but new therapeutic regimens are still warranted for these patients with a poor prognosis. The results were similar to those obtained in the Vogelzang et al.'s trial despite a selection bias because they correspond to 36.7% of the total recruitment in the unit.

Unimolecular reactions of vibrationally excited CF2ClCHFCH3 and CF2ClCHFCD3: evidence for the 1,2-FCl interchange pathway.

Chemically activated CF2ClCHFCH3 and CF2ClCHFCD3 molecules were prepared with 94 kcal mol-1 of vibrational energy by the recombination of CF2ClCHF and CH3(CD3) radicals at room temperature. The unimolecular reaction pathways were 2,3-FH(FD) elimination, 1,2-ClF interchange and 1,2-ClH elimination; the interchange produces CF3CHClCH3(CF3CHClCD3) with 105 kcal mol-1 of vibrational energy. Rate constants for CF2ClCHFCH3 [CF2ClCHFCD3] were (3.1+/-0.4)x10(6) s-1 [(1.0+/-0.1)x10(6) s-1] for 2,3-FH [FD] loss, (1.5+/-0.2)x10(6) s-1 [(8.3+/-0.9)x10(5) s-1] for 1,2-ClF interchange, and (8.2+/-1.0)x10(5) s-1 [(5.3+/-0.6)x10(5) s-1] for 1,2-ClH [DCl] loss. These correspond to branching fractions of 0.55+/-0.06 [0.43+/-0.04] for 2,3-FH [FD] loss, 0.29+/-0.03 [0.35+/-0.04] for 1,2-ClF interchange, and 0.16+/-0.02 [0.22+/-0.02] for 1,2-ClH [ClD] loss. Kinetic-isotope effects were 3.0+/-0.6 for 2,3-FH [FD] loss, 1.6+/-0.3 for 1,2-ClH loss, and 1.8+/-0.4 for 1,2-ClF interchange. The CF3CHClCH3 (CF3CHClCD3) molecules formed by 1,2-FCl interchange react by loss of HCl [DCl] with rate constants of (5.6+/-0.9)x10(7) s-1 [(2.1+/-0.4)x10(7)] s-1 for an isotope effect of 2.7+/-0.4. Density functional theory was employed to calculate vibrational frequencies and moments of inertia for the molecules and for the transition-state structures. These results were used with RRKM theory to assign threshold energies from comparison of computed and experimental unimolecular rate constants. The threshold energy for ClF interchange is 57.5 kcal mol-1, and those for HF and HCl channels are 2-5 kcal mol-1 higher. Experiments with vibrationally excited CF2ClCF2CF3, CF2ClCF2CF2Cl, and CF2ClCF2Cl, which did not show evidence for ClF interchange, also are reported.

Rate constants and kinetic isotope effects for unimolecular 1,2-HX or DX (X = F or Cl) elimination from chemically activated CF3CFClCH3-d0, -d1, -d2, and -d3.

Chemically activated CF(3)CFClCH(3), CF(3)CFClCD(3), CF(3)CFClCH(2)D, and CF(3)CFClCHD(2) molecules with 94 kcal mol(-1) of internal energy were formed by the combination of CF(3)CFCl radicals with CH(3), CD(3), CH(2)D, and CHD(2) radicals, which were generated from UV photolysis of CF(3)CFClI and CH(3)I, CD(3)I, CH(2)DI, or CHD(2)I. The total (HF + HCl) elimination rate constants for CF(3)CFClCH(3) and CF(3)CFClCD(3) were 5.3 x 10(6) and 1.7 x 10(6) s(-1) with product branching ratios of 8.7 +/- 0.6 in favor of HCl (or DCl). The intermolecular kinetic isotope effects were 3.22 and 3.18 for the HCl and HF channels, respectively. The product branching ratios were 10.3 +/- 1.9 and 11.8 +/- 1.8 (10.8 +/- 3.8 and 11.6 +/- 1.7) for HCl/HF and DCl/DF, respectively, from CF(3)CFClCH(2)D (CF(3)CFClCHD(2)). The intramolecular kinetic-isotope effects (without correction for reaction path degeneracy) for HCl/DCl and HF/DF elimination from CF(3)CFClCH(2)D (CF(3)CFClCHD(2)) were 2.78 +/- 0.16 and 2.98 +/- 0.12 (0.82 +/- 0.04 and 0.91 +/- 0.03), respectively. Density function theory at the B3PW91/6-311+G(2d,p) and B3PW91/6-31G(d',p') levels was investigated, and the latter was chosen to calculate frequencies and moments of inertia for the molecules and transition states. Rate constants, branching ratios and kinetic-isotope effects then were calculated using RRKM theory with torsional motions treated as hindered internal rotations. Threshold energies for HF and HCl elimination from CF(3)CFClCH(3) were assigned as 61.3 +/- 1.5 and 58.5 +/- 1.5 kcal mol(-1), respectively. The threshold energy for Cl-F interchange was estimated as 67 kcal mol(-1). The difference between the transition states for HCl and HF elimination is discussed.

Age of people and aging problem.

To apply technology to problems of age and aging, we need to better understand these problems and to develop operational technique for their solving. Creation of foundations for such a technique by means of a multifaceted model of age is the main goal of this paper. The suggested approach is based on the system theory of time, multilayer model of evaluation, and on differentiation of three functional subsystems in the human organism: biological, physiological, and psychological.

[Embryos and embryo-like entities: problem of definition in the draft of the Swiss embryonic research law]. / Embryonen und embryo-ähnliche Organismen--Definitionsprobleme im Entwurf zum Embryonenforschungsgesetz.

At the end of May 2002, the draft of the Swiss "Federal Act on Research on Surplus Embryos and Embryonic Stem Cells" (EFG, Embryonic Research Act) reached the pre-legislative consultation stage. Under certain conditions, it would allow research on "surplus" embryos from in-vitro fertilization, and the derivation of embryonic stem cells from surplus embryos for research purposes. The EFG draft defines an embryo as "the developing organism from the point of nuclear fusion until the completion of organ development". New technological developments show that embryo-like entities can also be created without nuclear fusion having taken place. It remains unclear how to treat embryonic entities that don't fall under the draft's narrow definition of an embryo. Expanding this definition would be a welcome improvement.

Antagonistic action of low-fluence and high-irradiance modes of response of phytochrome on germination and beta-mannanase activity in Datura ferox seeds.

Seed germination is often induced by a pulse of red light perceived by phytochrome and cancelled by a subsequent pulse of far-red light. When the pulse of red light is followed by several hours of darkness, a pulse of far-red light is no longer effective and prolonged far-red is necessary to block germination. The aim was to investigate whether the red light pulse and prolonged far-red light act on the same or different processes during germination of Datura ferox seeds. Forty-five hours after the inductive red light pulse, germination could not be blocked by one pulse or six hourly pulses of far-red light, but was significantly reduced by 6 h of continuous far-red light. The pulse of red light increased embryo growth potential and the activities of beta-mannanase and beta-mannosidase extracted from the micropylar region of the endosperm. Continuous far-red light had no effect on embryo growth potential or beta-mannosidase activity, but severely reduced the activity of beta-mannanase. The effect of far-red light had the features of a high-irradiance response of phytochrome. Both germination and beta-mannanase activity were restored by a pulse of red light given after the end of the continuous far-red treatment. It is concluded that the low-fluence response and the high-irradiance response modes of phytochrome have antagonistic effects on seed germination and that the control of beta-mannanase activity is one process where this antagonism is established.

Production and purification of novel secreted human proteins.

Our objective during the last year was to produce and purify 50-80 novel, secreted human proteins identified via high throughput cDNA sequencing and computer analysis. We chose the baculovirus expression vector system in order to obtain secreted, correctly folded, bioactive proteins. Recombinant (re-)baculoviruses (BV) were plaque purified, and pulse-labeling was used to verify the synthesis and secretion of the re-proteins. N-terminal microsequencing was performed to simultaneously confirm the identity of the protein(s) as well as the signal peptide (SP) cleavage site(s). Following sequence confirmation, the proteins were purified to homogeneity and functional assays carried out to determine potential therapeutic applications. We identified proteins with antiviral activity, several novel growth factors, proteins influencing the differentiation of specific cell types, novel proteases and protease inhibitors among others. Certain proteins were expressed both in insect cells and in CHO stable cell lines. In the cases analyzed, we found that the same SP cleavage site was utilized in the two expression systems. Significant differences were observed in the carbohydrate moieties attached to the proteins, though no effects on the biological activity due to these differences have been demonstrated. The BV system has served as a viable alternative for the high throughput, high fidelity expression of many novel secreted human genes. To date, more than 75 new genes have been expressed, and the re-proteins purified. This expression system combines many favorable traits including relative speed, moderate cost but perhaps most importantly, the production of biologically active proteins.

MRI appearances of Listeria rhombencephalitis.

Listeria monocytogenes is a rare cause of central nervous system infection in the non-immunocomprised patient and 10% of these patients develop a rhombencephalitis. We present such a case and discuss the clinical, pathological and radiological features.

A continuous high-resolution physical map spanning 17 megabases of the q12, q13.1, and q13.2 cytogenetic bands of human chromosome 19.

We report the construction of a high-resolution physical map of a 17-Mb region that encompasses the entire q12, q13.1, and q13.2 bands of human chromosome 19. The continuous map extends from a region approximately 400 kb centromeric of the D19S7 marker to the excision repair cross-complementing rodent repair deficiency complementation group 1 (ERCC1) locus. The ordered clone map has been obtained starting from a foundation of cosmid contigs assembled by automated fingerprinting and localized to the cytogenetic map by fluorescence in situ hybridization (FISH). Clonal continuity of the map has been achieved by binning and linking the premapped cosmid contigs by means of yeast artificial chromosomes (YACs). The map consists of a single contig composed of 169 YAC members (minimal spanning path of 18 YACs) linking 165 cosmid contigs. Eighty percent, or about 13.2 Mb of the entire region spanned by the map, has been resolved to the EcoRI restriction map level. Twenty-nine sequence-tagged sites associated with genetic markers or derived from FISH-mapped cosmids have been placed on the map. In addition to the ERCC1 gene area, the map includes the location of the creatine kinase muscle locus (CKM), imidazoledipetidase (PEPD), glucophosphate isomerase (GPI), myelin-associated glycoprotein (MAG), the apolipoprotein E and C (APOE and APOC) genes, and the ryanodine receptor (RYR1) gene. This type of map provides a source of continuously overlapping DNA segments at a level of resolution two orders of magnitude higher than that obtained using YACs alone. In addition, it provides ready-to-use reagents for detailed analyses at the gene level, FISH studies of chromosomal aberrations, and DNA sequencing.

Assembly of high-resolution bacterial artificial chromosome, P1-derived artificial chromosome, and cosmid contigs.

The generation of contiguous physical maps is often complicated by a variety of factors including the type of cloning system used. Here we describe procedures for the isolation, rapid characterization, and physical mapping of large-insert recombinant bacterial clones from total human genomic BAC (bacterial artificial chromosome) and PAC (P1-derived artificial chromosome) libraries containing clones with an average insert size of 150 kbp. After initial isolation, the clones were subjected to a variety of fingerprinting procedures including inter-Alu PCR, semiautomated fluorescent finger-printing, and EcoRI restriction fragment mapping. Individual BAC and PAC clones were also used as probes to interrogate arrayed chromosome 19-specific cosmid libraries. The combination of analyses facilitated the identification of chromosome-specific large-insert clones as well as the construction of a large (1.2 Mb) high-resolution BAC, PAC, and cosmid contig in 19q13.2, spanning the region from the carcinoembryonic antigen gene family to the X-ray repair cross complementing 1 DNA repair gene. This type of approach directly demonstrates the utility of large-insert recombinant bacterial clones for the construction of contiguous physical maps of entire chromosomes.

Tacaribe virus gene expression in cytopathic and non-cytopathic infections.

Tacaribe virus (TV) replication was compared in Vero cells infected under conditions leading either to cell death (c.p.e.(+) infection) or to the establishment of persistence (c.p.e.(-) infection). To this end, two virus preparations were employed: one containing a ratio of standard (plaque-forming) viruses to interfering particles (IP) that would induce a distinct lytic response in Vero cells infected at multiplicities giving synchronous infection and another virus stock enriched in IP that would block the cell-killing potential of the cytolytic virus stock. The following results were obtained: (1) No qualitative differences were observed in the species of intracellular viral RNAs in the lytic infection in comparison with infections leading to persistence or during the early stages of persistence. (2) Levels of viral RNAs were severely reduced when the cells were infected with IP in addition to standard viruses, the RNA accumulation being inversely proportional to the ratio of IP to standard viruses used in the infections. (3) Accumulation of the three measurable mRNAs (those corresponding to the glycoprotein precursor [GPC], to the nucleoprotein [N], and to the p11Z protein) ended earlier in the c.p.e.(-) infections (around 18 hr p.i.) than in the c.p.e.(+) infection (45-68 hr p.i.). (4) The rates of synthesis of the GPC, N, and p11Z proteins were largely determined in both the c.p.e.(+) and c.p.e.(-) infections by the amounts of their corresponding mRNAs. (5) The kinetics of accumulation of the S genomes and also the ratios of the S genome to S antigenome were similar in the different infections (accumulation ending at 45-68 hr p.i.). (6) L genome accumulation proceeded for longer time (until 92 hr p.i.) in the c.p.e.(+) infection than in the c.p.e.(-) infections. In the latter accumulation ended at around 45 hr p.i. Until this time ratios of L genome to L antigenome were similar in the different infections. It is concluded that IP affect virus mRNA synthesis early after infection reducing in this way the rate of viral protein synthesis. Low levels of viral proteins might then limit virus replication. In addition, the results support the idea that in TV infections transcription and replication are independently regulated. The implications of these results with regards to the nature and mode of action of TV IP are discussed.

Molecular mimicry of trifluoroacetylated human liver protein adducts by constitutive proteins and immunochemical evidence for its impairment in halothane hepatitis.

A monospecific antibody (anti-CF3CO antibody) was obtained by affinity chromatography on a N epsilon-trifluoroacetyl-L-lysine (CF3CO-Lys) matrix of a rabbit polyclonal antiserum, directed against trifluoroacetylated protein adducts (CF3CO-proteins). The anti-CF3CO antibody recognized distinct CF3CO-proteins on immunoblots of a liver biopsy obtained from a human individual 10 h after halothane anaesthesia. Cross-reactive proteins of 52 kDa and 64 kDa were recognized on immunoblots of livers obtained from human individuals not exposed to halothane. Recognition of both CF3CO-proteins and the 52-kDa and 64-kDa cross-reactive proteins was abolished in the presence of 1 mM CF3CO-Lys. Anti-CF3CO antibody, affinity-adsorbed to the 52-kDa or the 64-kDa cross-reactive proteins of human liver, recognized the majority of target CF3CO-proteins on immunoblots of the human liver biopsy of an individual exposed to halothane. Liver biopsies of 5 out of 7 (71%) patients with halothane hepatitis exhibited an absence or low amounts of immunorecognizable 52-kDa and/or 64-kDa cross-reactive proteins. In contrast, of 22 control human individuals tested, all liver tissue samples were positive for the 52-kDa and/or the 64-kDa cross-reactive proteins. These data indicate that epitopes on the cross-reactive proteins of 52 kDa and 64 kDa of human liver bear strong immunochemical resemblance to epitopes on human liver CF3CO-proteins. Low-level expression of the cross-reactive proteins of 52 kDa and 64 kDa is discussed as one possible factor in human susceptibility to halothane hepatitis.

Serum IgG and IgM levels in new and regular long-term plasmapheresis donors.

This Australian study monitored the effects of monthly plasmapheresis on donor serum IgG and IgM levels in 127 new and 124 established plasma donors who donated 1014 units over a five-month period. Of the 251 donors, 3% had reduced total serum protein (TSP) levels, 7% had low IgG levels and 12% had low IgM levels prior to donation on at least one occasion over the study period. Statistical analysis showed that the TSP, IgG and IgM levels of new donors who had donated plasma on less than 10 occasions were no more likely to fall below normal than those of old donors. However, new and old donors whose IgG or IgM levels fell below normal at any time during the study had significantly lower levels of the relevant parameter on entry to the study. Followed longitudinally, IgG and IgM levels in old and new donors tended to fall, although levels fluctuated throughout the study. Statistical analysis failed to show any correlation between TSP levels and IgG or IgM levels. These parameters did not correlate significantly with the number of previous plasmaphereses, donor weight, volume collected or history of infection. This study highlighted the need for regular, specific quantitation of IgG and IgM levels as well as TSP in regular plasmapheresis donors. The frequency of testing is yet to be determined, in view of the high materials and labour costs of such a programme.

Halothane metabolism: immunochemical evidence for molecular mimicry of trifluoroacetylated liver protein adducts by constitutive polypeptides.

A monoform antibody [anti-TFA antibody] against TFA-protein adducts (TFA-adducts) was obtained by affinity purification of a polyclonal antiserum, raised in rabbits against TFA-rabbit serum albumin, on a N-epsilon-TFA-L-lysine matrix coupled to Affi-Gel 102. The anti-TFA antibody did recognize TFA-adducts of distinct molecular mass on Western blots of hepatocyte homogenates or microsomal membranes obtained from rats pretreated with halothane. The anti-TFA antibody also recognized cross-reactive polypeptides with apparent molecular masses of 52 kDa and 64 kDa on Western blots of hepatocyte homogenates obtained from rats not treated with halothane or metabolites thereof. The 52-kDa and 64-kDa cross-reactive polypeptides were localized in the 3,000 x g particulate fraction of liver homogenates. Recognition, on Western blots, of TFA-adducts and both the 52-kDa and 64-kDa cross-reactive polypeptides by anti-TFA antibody was sensitive to competition by N-epsilon-TFA-L-lysine (IC50 less than 100 microM) and N-epsilon-acetyl-L-lysine (IC50 approximately 10 mM). Treatment with piperidine (1 M) did abolish the recognition of TFA-adducts but not that of the 52-kDa and the 64-kDa cross-reactive polypeptides by anti-TFA antibody on Western blots. In antibody-exchange experiments, anti-TFA antibody was affinity-adsorbed on Western blots to the 52-kDa or the 64-kDa cross-reactive polypeptides of the rat heart, followed by spontaneous transfer to target TFA-adducts present on Western blots of rat liver microsomal membranes. The majority of these target TFA-adducts were recognized by anti-TFA antibody transferring from the source 52-kDa or 64-kDa cross-reactive polypeptides. When examined up to 10 days after exposure of rats to a single dose of halothane, no influence on the constitutive level of expression, in the liver, of either cross-reactive polypeptide was observed. In contrast, TFA-adducts were persistent for greater than 90 hr but less than 10 days. In addition to the liver, the 52-kDa and the 64-kDa cross-reactive polypeptides were prominently expressed in the heart and the kidney and, to a much lesser degree, in the spleen, the thymus, the lung, and skeletal muscle of the rat. Considerable variation in the level of expression of the 52-kDa and the 64-kDa cross-reactive polypeptides was recognized in livers of the six human individuals tested so far.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of 5-hydroperoxyeicosatetraenoic acid into dihydroxy- and cysteinyl-leukotrienes by rat hepatocytes: effects of glutathione.

In the presence of glutathione (GSH 400 microM), rat hepatocyte homogenates converted 5-hydroperoxyeicosatetraenoic acid (5-HPETE), via the intermediate leukotriene A4, into leukotriene C4 (LTC4) and leukotriene B4 (LTB4); 5-hydroxyeicosatetraenoic acid (5-HETE) was also a prominent product. During a 5-min incubation with 100 microM (13.4 microgram) 5-HPETE, 0.24 ng of LTC4, 15.4 ng of all-trans-LTB4, 4.3 ng of LTB4, and 12.4 micrograms of 5-HETE were formed/mg of protein. In incubations devoid of GSH, 38.6 ng of all-trans-LTB4, 8.8 ng of LTB4, and 2.2 micrograms of 5-HETE were formed/mg of protein, and 3.3 micrograms of intact 5-HPETE could be recovered. The presence of GSH induced a time-dependent rapid depletion of 5-HPETE, paralleled by large increases in the formation of 5-HETE; formation of LTC4 was detected in the presence but not in the absence of GSH. Addition of thiomalic acid (0.1 mM) or penicillamine (0.2 mM), both inhibitors of selenium-dependent GSH peroxidases, increased formation rates of LTC4 by factors of 3 and 2, respectively, whereas the suppressive effects of GSH on the formation of LTB4 were partially reversed. These results suggest that hepatocytes are capable of the simultaneous synthesis of cysteinyl- and dihydroxy-leukotrienes as well as 5-HETE; the availability of the precursor 5-HPETE and the profile of leukotrienes formed are dependent on the GSH concentration and the extent of GSH peroxidase activity.

Halothane metabolism: Kupffer cells carry and partially process trifluoroacetylated protein adducts.

Kupffer cells, prepared 18 h after pretreatment of rats with a single dose of halothane, did carry TFA-adducts which were recognized on Western blots by a anti-TFA-antibody. Based on apparent molecular weight, the pattern of the major TFA-adducts within Kupffer cells was similar to that observed in hepatocytes. When kept in primary culture, Kupffer cells processed TFA-adducts of apparent molecular weight of 220 kD, 110 kD and 74 kD within 24 or 48 h; in contrast, other TFA-adducts were persistent for at least 48 h in Kupffer cells. The data suggest a role for Kupffer cells in processing of chemically altered proteins in the liver.

Extracellular matrix: differential influence on growth and biosynthesis patterns of vascular smooth muscle cells from SHR and WKY rats.

Smooth muscle cells from spontaneously hypertensive rats (SHR) elaborated extracellular matrix (ECM) material in culture that was more stimulatory to growth of cells from normotensive (WKY) animals than their own matrix. Both cell types elaborated ECMs consisting of glycoconjugate material (proteoglycans, glycopeptides) elastin, and collagens, but there were differences in the relative proportions of the compounds synthesized. Cells from SHR produced an ECM richer in elastin than that synthesized by WKY derived cells (approximately 19% vs. 11%, respectively). However, the latter elaborated ECMs containing more (approximately 45% for WKY vs. 29% for SHR) glycoconjugate material than the former. The lysyloxidase-mediated cross-linking of elastin was more rapid in cultured cells from SHR animals than from their normotensive counterparts and may be as a consequence of increased substate (tropoelastin) availability in ECMs from SHR animals. The relative proportions and sulphate levels of the glycosaminoglycans associated with matrix material elaborated by the two cell types were similar. Radiolabelled glycoconjugate material was degraded by cells (SHR/WKY) when they were plated upon pre-formed ECMs, and their patterns of synthesis of new matrix was markedly altered under such conditions. New matrix material elaborated by cells plated upon ECM-coated dishes consisted predominantly of glycopeptide and proteoglycan matrix components. Epidermal growth factor promoted the incorporation of [3H]-thymidine into DNA by quiescent cells, and this was also markedly stimulated when cells were plated onto ECM-coated plasticware rather than onto plastic substratum.

Enhanced responsiveness to angiotensin II in vascular smooth muscle cells from spontaneously hypertensive rats is not associated with alterations in protein kinase C.

This study compares vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats with respect to protein kinase C and intracellular responses to angiotensin II (Ang II). Ang II-induced degradation of polyphosphoinositides and accumulation of inositol di- and tris-phosphates was enhanced (approximately twofold) in hypertensive-derived cells, without a change (vs. normotensive-derived cells) in half-maximally effective concentrations of Ang II. Intracellular pH (approximately 6.6) was comparable between both cell isolates at quiescence, but alkalinization induced by Ang II, serum, or phorbol ester was greater (delta 0.1-0.2 pH units) for hypertensive-derived cells. For both cell types, the intracellular pH response to these agonists was prevented in the presence of Na+-H+ exchange inhibitors. S6 kinase activation induced by Ang II was enhanced (approximately twofold) in hypertensive-derived cells, whereas activation in response to serum or 12-O-tetradecanoylphorbol 13-acetate did not differ significantly between the two cell types. Quantitation of protein kinase C by immunoblotting and [3H]phorbol dibutyrate binding procedures revealed no differences between the two smooth muscle cell isolates (at quiescence or in the presence of serum) with respect to either total amounts or subcellular distribution. Sensitivity of protein kinase C to phorbol ester was apparently also not different between the two cell types, as assessed from dose-dependent (phorbol ester) S6 kinase activation profiles. Phorbol ester caused a similar subcellular redistribution of [3H]phorbol dibutyrate binding in the two cell isolates, but for both, minimal (10%) translocation occurred in response to Ang II. The data suggest that enhanced agonist responsiveness in vascular smooth muscle cells is unlikely to involve alterations in protein kinase C.

Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats.

Aortic smooth muscle cells from spontaneously hypertensive rats (SHR) exhibit inappropriate proliferation characteristics in culture that suggest a modified response to serum mitogens or growth factors. The present study compares vascular smooth muscle cells from SHR and normotensive Wistar-Kyoto (WKY) rats with respect to their proliferative and functional response to growth factors. Specific attention was focused on the interaction of these vascular smooth muscle cells with epidermal growth factor. An increased growth rate of vascular smooth muscle cells from SHR (vs. WKY rats) was observed when cells were cultured in the presence of serum (10% and 0.5%), but not under serum-free conditions. The additional presence of low serum concentrations (0.5%) was required for epidermal growth factor to elicit a proliferative response, whereupon smooth muscle cells from SHR displayed an increased (vs. WKY rats) growth rate. Saturation binding of [125I]epidermal growth factor to intact smooth muscle cells indicated a twofold increase in receptor density in SHR-derived cells (p less than 0.001 vs. WKY rats) without an alteration in affinity for the growth factor. Cells derived from SHR also exhibited greater functional responsiveness to epidermal growth factor when compared with smooth muscle cells from WKY rats as evidenced by amplifications of both S6 kinase activation, phosphoinositide catabolism, elevation of intracellular pH, and DNA synthesis (nuclear labeling). We conclude that increased responsiveness of SHR-derived smooth muscle cells to epidermal growth factor could contribute to alterations in vascular smooth muscle growth and tone that may be fundamental to the pathogenesis of hypertension and atherosclerosis.