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A L Bayer

Publications and source records attributed to A L Bayer.

15 recordsLinked to original sources
PubMed2001

Differential activation of mitogen-activated protein kinase cascades and apoptosis by protein kinase C epsilon and delta in neonatal rat ventricular myocytes.

Protein kinase C (PKC) epsilon and PKCdelta translocation in neonatal rat ventricular myocytes (NRVMs) is accompanied by subsequent activation of the ERK, JNK, and p38(MAPK) cascades; however, it is not known if either or both novel PKCs are necessary for their downstream activation. Use of PKC inhibitors to answer this question is complicated by a lack of isoenzyme specificity, and the fact that many PKC inhibitors stimulate JNK and p38(MAPK) activity. Therefore, replication-defective adenoviruses (Advs) encoding constitutively active (ca) mutants of PKCepsilon and PKCdelta were used to test if either or both of these PKCs are sufficient to activate ERKs, JNKs, and/or p38(MAPK) in NRVMs. Adv-caPKCepsilon infection (1 to 25 multiplicities of viral infection (MOI); 4 to 48 hours) increased total PKCepsilon levels in a time- and dose-dependent manner, with maximal expression observed 8 hours after Adv infection. Adv-caPKCepsilon induced a time- and dose-dependent increase in phosphorylated p42 and p44 ERKs, as compared with a control Adv encoding beta-galactosidase (Adv-nebetagal). Maximal ERK phosphorylation occurred 8 hours after Adv infection. In contrast, JNK was only minimally activated, and p38(MAPK) was relatively unaffected. Adv-caPKCdelta infection (1 to 25 MOI, 4 to 48 hours) increased total PKCdelta levels in a similar fashion. Adv-caPKCdelta (5 MOI) induced a 29-fold increase in phosphorylated p54 JNK, and a 15-fold increase in phosphorylated p38(MAPK) 24 hours after Adv infection. In contrast, p42 and p44 ERK were only minimally activated. Whereas neither Adv induced NRVM hypertrophy, Adv-caPKCdelta, but not Adv-caPKCepsilon, induced NRVM apoptosis. We conclude that the novel PKCs differentially regulate MAPK cascades and apoptosis in an isoenzyme-specific and time-dependent manner.

Adenoviridae↗
PubMed2001

Pyk2 expression and phosphorylation in neonatal and adult cardiomyocytes.

A. L. Bayer, A. G. Ferguson, P. A. Lucchesi and A. M. Samarel. PYK2 Expression and Phosphorylation in Neonatal and Adult Cardiomyocytes. Journal of Molecular and Cellular Cardiology (2001) 33, 1017-1030. Proline-rich tyrosine kinase (PYK2) is a Ca(2+)-dependent, non-receptor protein tyrosine kinase involved in growth factor signaling. Although PYK2 is expressed in a variety of tissues, it has not yet been identified in cardiac muscle. Therefore, immunocytochemical and Western blotting techniques were used to examine PYK2 expression and phosphorylation in neonatal and adult rat ventricular cardiomyocytes (NRVM and ARVM, respectively). PYK2 concentration was much greater in neonatal, than in adult ventricular tissue and cardiomyocytes. In cultured cells, PYK2 expression was highly dependent on [Ca(2+)](i)transients and contractile activity. Non-contracting, low-density NRVM in serum-free culture expressed very low levels of PYK2, while high-density, spontaneously contracting NRVM showed a approximately 12-fold increase in PYK2 expression. Conversely, high-density NRVM treated with nifedipine (10 microM, 48 h) to block spontaneous [Ca(2+)](i)transients and contractile activity resulted in a 2.6-fold decrease in PYK2 levels. Similarly, overnight culture of quiescent ARVM markedly reduced PYK2 levels. Chronic treatment (48 h) of cultured NRVM with the hypertrophic agonist endothelin-1 (ET) (10-300 n M) did not significantly increase PYK2 levels, but strongly shifted the ratio of phosphorylated to total PYK2, indicating that PYK2 phosphorylation accompanies cardiomyocyte hypertrophy. Endothelin-1 also acutely activated PYK2 in both cultured NRVM, and in freshly isolated ARVM. These results suggest that PYK2 is involved in the generation of certain aspects of cardiomyocyte hypertrophy.

Animals↗
PubMed1999

Enhanced allograft survival via simultaneous blockade of transferrin receptor and interleukin-2 receptor.

BACKGROUND: Transferrin receptor (TfR) expression follows the induction of interleukin 2 receptor (IL-2R) expression in a sequence that is necessary to initiate cell proliferation in quiescent T lymphocytes. Therefore, we tested the hypothesis that simultaneous blockade of TfR and IL-2R would be more effective in prolonging allograft survival and suppressing T-cell responses to alloantigen than single receptor blockade by modifying T-cell effectors to alloantigen. METHODS: Neonatal C57BL/6 hearts were transplanted to CBA/J recipients in a heterotopic, nonvascularized cardiac allograft model. Anti-TfR and/or anti-IL-2R or isotype-matched control monoclonal antibodies (mAbs) were administered at 100 microg intravenously on days 0 and 1 of transplantation. RESULTS: Anti-TfR mAb (25.7+/-0.9 days) significantly (P<0.01) prolonged cardiac allograft survival compared with anti-IL-2R mAb (12.5+/-0.9 days) or the isotype control (15.7+/-1.2 days, P<0.01, Wilcoxon rank-sum). Anti-TfR plus anti-IL-2R mAbs significantly (P<0.01) prolonged cardiac allograft survival to 50.7+/-2.0 days compared with the isotype control or either agent alone. These agents in combination down-regulated the intragraft T helper (Th)-1 cytokines, IL-2, interferon-gamma, and IL-15, while up-regulating the Th2 cytokine, IL-4, and completely abrogating the antigen-presenting cell IL-12p40 mRNA expression. CONCLUSIONS: Anti-TfR and anti-IL-2R mAbs are potent immunosuppressants. Combined blockade of TfR and IL-2R at the time of antigen presentation seems to be the most effective by shifting the intragraft Th cytokine paradigm.

Animals↗
PubMed1999

Differential effects of transferrin receptor blockade on the cellular mechanisms involved in graft rejection.

Since transferrin receptor (TfR) appears on activated T cells following the interaction of the antigen-major histocompatibility complex (MHC) with the T cell receptor (TCR) and the appearance of interleukin (IL)-2R, we therefore hypothesize that in vivo blockade of TfR prolongs allograft survival by altering the cellular mechanisms involved in graft rejection. Previous results in our laboratory have demonstrated that anti-TfR monoclonal antibody (mAb) at 100 microg on days 0 and 1 of transplantation significantly prolonged allograft survival to 25.7 +/- 0.9 days in a murine heterotopic, nonvascularized cardiac allograft model. In the current studies, administration of anti-TfR mAb at the time of maximal TfR expression, on days 2 and 3 post-transplantation, failed to prolong allograft survival (13.0 +/- 0.0 days) compared to the isotype controls (10.5 +/- 0.5 and 10.7 +/- 0.4 days) (p < 0.01, Wilcoxon rank sum). A 4-day course of anti-TfR mAb significantly prolonged allograft survival compared to the isotype controls, but was no more effective than a 2-day course of the mAb. Anti-TfR mAb suppressed the mixed lymphocyte response to donor-specific and third-party alloantigen by 78.7% (p < 0.05) and 80.8% (p < 0.05), respectively, while stimulating the CTL response to donor-specific (16.3%, p < 0.05) and third party (49.3%, p < 0.01) alloantigen. Anti-TfR mAb suppressed IL-15 and increased IL-4 intragraft mRNA expression when compared to the isotype controls. Examination of cell surface receptors important during T cell activation revealed alterations in expression following anti-TfR mAb treatment. Anti-TfR mAb is an effective immunosuppressant prolonging allograft survival by altering cell-mediated immune responses and the intragraft cytokine micro-environment.

Animals↗
PubMed1998

T-cell alterations in cardiac allograft recipients after B7 (CD80 and CD86) blockade.

BACKGROUND: T-cell activation requires engagement of the T cell receptor with the antigen-MHC and simultaneous ligation of the coreceptor CD28. CD28 binds both the CD80 (B7-1) and CD86 (B7-2) ligands on antigen-presenting cells. The functional role of these costimulatory pathways in transplantation is not completely understood. We tested the hypothesis that in vivo blockade of the CD28 pathway via the anti-CD80 and anti-CD86 monoclonal antibodies (mAbs) would prolong allograft survival. METHODS: Neonatal C57BL/6J (H2b) hearts were transplanted to CBA/J (H2k) recipients in a heterotopic nonvascularized model, with anti-CD80 and/or anti-CD86 mAbs being administered intravenously at the time of allografting (day 0) and on the following day (day 1). RESULTS: Anti-CD80 mAb (29.8+/-1.5 days) and anti-CD86 mAb (30.8+/-0.5 days) alone significantly prolonged allograft survival compared with the isotype control (10.7+/-0.4 days, P < 0.01, Wilcoxon rank sum). The concurrent (days 0 and 1) and sequential administration of anti-CD86 mAb on days 0 and 1 plus anti-CD80 mAb on days 2 and 3 prolonged allograft survival to >80 days. Simultaneous administration of anti-CD80 and anti-CD86 mAbs significantly suppressed donor-specific cytotoxic T lymphocyte responses to alloantigen. Anti-CD86 mAb suppressed intragraft interleukin (IL)-4, IL-10, IL-12 p40, and IL-15 mRNA expression. CONCLUSIONS: Anti-CD80 and/or anti-CD86 mAbs are potent immunosuppressants in prolonging allograft survival. Combined blockade of the B7 (CD80 and CD86) ligands seems to be the most effective in prolonging allograft survival and suppressing donor-specific allogeneic cytotoxic T lymphocyte responses. In vivo blockade of CD86, in comparison to CD80, had the greatest immunosuppressive effect on day 7 intragraft cytokines, suggesting its role on early allogeneic immune responses.

Animals↗
PubMed1998

Anti-transferrin receptor monoclonal antibody: a novel immunosuppressant.

BACKGROUND: Transferrin receptor is a widely distributed cell surface receptor present on most proliferating and highly specialized quiescent cells. Expression of transferrin receptor on the surface of immune cells is up-regulated during T-cell activation after the interaction of the antigen-MHC with the T cell receptor. The role of transferrin receptor in T-cell activation has not been well-established. Since transferrin receptor is physically associated with the CD3 zeta-chain, blockade of transferrin receptor has the potential to interfere with the T-cell signals important in transplant rejection. METHODS: Anti-transferrin receptor monoclonal antibody (mAb) was administered in vivo and in vitro to determine whether this agent was effective in prolonging allograft survival and altering cell-mediated immunity. RESULTS: Using donor C57BL/6J (H2b) hearts transplanted to CBA/J (H2k) recipients, anti-transferrin receptor mAb at the time of transplantation prolonged cardiac allograft mean survival time to 25.7+/-0.9 days compared with untreated (13.3+/-0.6 days, P < 0.05) or isotype-matched (10.7+/-0.4 days, P < 0.05) controls. Anti-transferrin receptor mAb administered in vivo failed to suppress the subsequent allogeneic responses. However, when added to culture, anti-transferrin receptor mAb suppressed the allogeneic cytotoxic T lymphocyte response by 79-100% but not the mixed lymphocyte response. CONCLUSIONS: These studies are the first to suggest that transferrin receptor is a potential therapeutic target for clinical transplantation. Future studies will determine the most efficacious dose and time for maximal immunosuppression and the mechanisms responsible for the immunosuppression exhibited by antitransferrin receptor mAb.

Adjuvants, Immunologic↗
PubMed1998

Transferrin receptor in T cell activation and transplantation.

Transferrin receptor (TfR) expression is up-regulated during T cell activation after the interaction of the T cell receptor with the antigen-major histocompatibility complex and the expression of interleukin-2 (IL-2) receptor. We hypothesize that anti-TfR monoclonal antibody (mAb) will prolong allograft survival by altering T cell responses. In a murine heterotopic nonvascularized cardiac allograft model, CBA/J (H-2k) recipients were transplanted with neonatal C57BL/6 (H-2b) donor hearts. Anti-TfR or isotype-matched control mAbs (100 microg) were administered at the time of transplantation and on the following day. Splenocytes from naive CBA/J mice were stimulated in vitro with C57BL/6 alloantigen. Anti-TfR mAb was administered at 5 microg/mL during the initiation of culture. Cytotoxic T lymphocyte (CTL) and mixed lymphocyte responses (MLR) were performed to assess T cell function. After 24 h in culture, cells were harvested, RNA isolated, and semi-quantitative reverse transcriptase-polymerase chain reaction performed. Anti-TfR mAb prolonged allograft survival to 25.7 +/- 0.9 days compared to the isotype control (10.7 +/- 0.4 days, P < 0.01, Wilcoxon rank sum). Anti-TfR mAb completely abrogated the CTL response and suppressed the MLR by 70-86% compared to the isotype controls. Anti-TfR mAb suppressed IL-2, interferon-gamma (IFN-gamma), IL-10, and IL-12 p40 mRNA expression, but had no effect on IL-4, IL-12 p35, and IL-15 mRNA expression. In conclusion, anti-TfR mAb prolongs allograft survival, suppresses T cell function, and alters IL-2, IL-10, IL-12 p40, and IFN-gamma mRNA expression. These data suggest that the down-regulation in IL-12 mRNA by anti-TfR mAb may prevent the development of T helper cells, thereby promoting graft survival and altering cell-mediated immune responses. The partial effect by anti-TfR mAb on cytokine mRNA expression may be due to other contributing factors such as costimulation.

Animals↗
PubMed1993

Cytoplasmic and nuclear signaling pathways of V1-vascular vasopressin receptors.

We studied the cytoplasmic and nuclear signaling pathways of V1-vascular AVP receptors of human platelets, primary cultures of renal glomerular mesangial cells, and established cultures of the A7r5 aortic smooth muscle cell line. The immediate transmembrane signals are triggered by the formation of ligand-receptor complexes as illustrated by binding experiments with [3H]AVP (Kd = 2.50 nM), d(CH2)5Tyr(Me)AVP (Kd = 0.62 nM), the linear V1 antagonist phenylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-[125I]Tyr-NH2 (Kd = 1.42 nM) or by fluorescence experiments with linear antagonists like phenylacetyl-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 coupled to biotin and made fluorescent by labeling with tetramethylrhodamine-avidin. We used several approaches (radioreceptor binding, radioactive labeling, autoradiographic, enzymatic, photoaffinity labeling, and immunoblotting procedures) to identify the guanine nucleotide regulatory protein coupled to V1-vascular vasopressin receptors. AVP-stimulated GTPase activity of human platelet membranes was blocked by pretreatment with antibodies specific for the C-terminal of the newly described Gq alpha protein. In the presence of MgCl2, AVP increased labeling by the photoreactive GTP analog [alpha-32P]azidoanilido GTP of a platelet membrane protein of apparent molecular mass of 42 kDa. AVP effect was reversed by the specific V1-vascular antagonist d(CH2)5Tyr(Me)AVP and labeling was completely abolished by GTP gamma s.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Role of eicosanoids in vasopressin-induced calcium mobilization in A7r5 vascular smooth muscle cells.

The role of arachidonic acid (AA) and its metabolites in vasopressin (AVP)-induced calcium mobilization in A7r5 aortic smooth muscle cells was explored by intracellular calcium monitoring, [14C]AA labeling, and high-performance liquid chromatography (HPLC) techniques. In fura 2-loaded A7r5 cells, AA potentiated AVP-stimulated increase in intracellular free Ca2+ ([Ca2+]i). The cyclooxygenase inhibitor indomethacin reduced both the AA- and AVP-induced influx of extracellular Ca2+. AVP-induced [Ca2+]i transients were not altered by lipoxygenase inhibitors but were reduced in a dose-dependent fashion by ketoconazole, an inhibitor of cytochrome P-450 monooxygenases. Among several epoxygenase metabolites of AA tested, 5,6-epoxyeicosatrienoic acid potentiated AVP-induced [Ca2+]i transients. Reverse-phase HPLC analysis of lipid extracts from A7r5 cells prelabeled with [14C]AA isolated a radioactive peak that did not coelute with established products of cyclooxygenase-, lipoxygenase-, or cytochrome P-450-catalyzed oxidations of AA. This peak was significantly increased after AVP stimulation and was completely blocked by preincubation with ketoconazole. Thus the stimulation of V1-vascular AVP receptors of A7r5 cells triggers several cytoplasmic signaling pathways involving AA metabolite formation through the cyclooxygenase and epoxygenase pathways.

Animals↗
PubMed1993

Linear V1-vascular vasopressin antagonists suitable for radioiodination, biotinylation, and fluorescent labeling.

We modified several linear V1-vascular arginine vasopressin (AVP) antagonists to obtain compounds suitable for radioiodination, biotinylation, and fluorescent labeling. In binding competition experiments with human platelet V1-vascular AVP receptors, the linear V1 antagonist phenylacetyl-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 (PhaaGln) displayed the greatest affinity [dissociation constant (Kd) = 0.05 +/- 0.01 nM]. The radioiodinated compound phenylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-125I-labeled Tyr-NH2 (125I-labeled TyrPhaa) was characterized by a high affinity (Kd = 1.42 +/- 0.19 nM), a low nonspecific binding, and good stability. PhaaGln coupled to dodecabiotin retained a good affinity for V1-vascular AVP receptors (Kd = 1.41 +/- 0.20 nM). The complex PhaaGln-dodecabiotin-avidin is bifunctional, since an avidin-agarose column specifically bound V1-vascular AVP receptors labeled with 125I-TyrPhaa-dodecabiotin. In A7r5 vascular smooth muscle cells loaded with fura-2, PhaaGln and PhaaGln-dodecabiotin were pure antagonists as they blocked AVP-induced calcium mobilization but did not elicit a calcium signal by themselves. V1-vascular AVP receptors of A7r5 vascular smooth muscle cells were visualized by bound PhaaGln-dodecabiotin made fluorescent by labeling with fluorescein avidin. Thus linear V1-vascular AVP antagonists can be used as high affinity and specificity radioiodinated, biotinylated, and fluorescent probes to explore V1-vascular AVP receptors of human and animal origin.

Amino Acid Sequence↗
PubMed1992

Effects of amiloride analogues on AVP binding and activation of V1-receptor-expressing cells.

We tested the interactions between amiloride analogues and V1 vascular arginine vasopressin (AVP) receptors of human platelets, rat glomerular mesangial cells, and A7r5 smooth muscle cells by using radioligand binding techniques, intracellular calcium monitoring, platelet aggregation, and cell contraction techniques. Amiloride analogues were competitive inhibitors of both the agonist [3H]AVP and the tritiated V1 antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-O-methyl)tyrosine]AVP ([3H]d(CH2)5Tyr(Me)AVP) binding to V1 AVP receptors in the three different cell types used. The order of potency was ethylisopropyl amiloride (EIPA) greater than Benzamil greater than amiloride. AVP mobilization of intracellular calcium was blocked by the V1 antagonist d(CH2)5Tyr(Me)AVP and was reduced by EIPA in a dose-dependent manner. Moreover, EIPA also inhibited prostaglandin F2 alpha mobilization of intracellular calcium. Alkalinization of the intracellular pH with ammonium chloride reversed the inhibitory effect of EIPA but not that of the V1 antagonist on AVP-induced calcium mobilization. Both amiloride and EIPA blocked AVP-induced aggregation of human platelets and contraction of mesangial cells and glomeruli preparations independently of receptor site antagonism. In conclusion, amiloride analogues interfere with activation of V1 vascular receptors by AVP at different levels including binding to the receptor site, mobilization of intracellular calcium, cell contraction or aggregation, and presumably alteration of intracellular ion transports.

Amiloride↗
PubMed1991

Multiple signaling pathways of V1-vascular vasopressin receptors of A7r5 cells.

We explored the nature and time course of the multiple signal transduction pathways for V1-vascular vasopressin (AVP) receptors of A7r5 aortic smooth muscle cells in culture by using radioligand binding techniques, intracellular calcium monitoring, and polyphosphoinositide and phospholipid analyses. V1-vascular AVP receptors of A7r5 cells were characterized by the agonist radioligand [3H]AVP and the antagonist radioligand [3H]d(CH2)5Tyr(Me)AVP. Affinity and capacity of agonist but not antagonist binding were modulated by MgCl2 and aluminum fluoride, suggesting that the receptors are coupled to a guanine nucleotide regulatory protein. In fura-2-loaded A7r5 cells, AVP induced within seconds a dose-dependent increase of free intracellular Ca++ ([Ca++]i) consisting of a rapid transient spike and a sustained increase lasting for 3-5 min. The baseline [Ca++]i was 136 +/- 18 nM, the maximum [Ca++]i response to AVP was 1,582 +/- 297 nM, and AVP ED50 was 1.87 +/- 0.15 nM. Diverse experiments performed with EGTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester, Mn++, ionomycin, terbutylbenzo hydroquinone, and nicardipine suggested that the initial spike resulted from both intracellular Ca++ release from the endoplasmic reticulum and extracellular Ca++ influx, whereas the sustained phase depended on dihydropyridine-insensitive extracellular Ca++ influx. Experiments done with indomethacin and arachidonic acid indicated that AVP-induced extracellular Ca++ influx was in part dependent on phospholipase A2 activation. In [3H]myoinositol and [3H]arachidonate-labeled A7r5 cells, AVP stimulated inositol 1,4,5 trisphosphate and 1,2 diacylglycerol production via activation of phospholipase C. Also, AVP stimulated a transphosphatidylation reaction through activation of phospholipase D in A7r5 cells labeled with [3H]1-O-alkyl lysoglycerophosphocholine. Thus, the stimulation of V1-vascular AVP receptors of A7r5 cells triggers several signaling pathways. The immediate and transient [Ca++]i rise due to mobilization of intracellular and extracellular Ca++ is associated with the activation of phospholipases A2 and C, and the sustained activation of phospholipase D.

Animals↗
PubMed1990

Reconstitution of solubilized V1 vasopressin receptors of human platelets.

We describe the reconstitution of solubilized human platelet arginine vasopressin (AVP) receptors into phospholipid vesicles. Purified platelet plasma membranes enriched in AVP receptors [binding equilibrium dissociation constant (Kd) = 1.87 +/- 0.14 nM, maximum number of binding sites (Bmax) = 261 +/- 10 fmol/mg protein] were solubilized with 20 mM sodium cholate. Phospholipid vesicles made of 10% cholesterol, 20% egg phosphatidylcholine, and 70% egg phosphatidylserine were formed by bath sonication. Solubilized AVP receptors were incorporated into the vesicles while the detergent was removed by filtration through Sephadex G-100. The reconstituted receptors retained a high affinity for [3H]AVP (Kd = 3.19 +/- 0.13 nM, Bmax = 257 +/- 9 fmol/mg). Competition experiments with different AVP analogues confirmed the V1 vascular nature of the reconstituted receptors. Saturation experiments carried out with the agonist [3H]AVP and the V1 antagonist [3H]d(CH2)5Tyr(Me)AVP revealed that agonist binding to the reconstituted receptors was divalent cation dependent, whereas antagonist binding was not. Moreover, the affinity of the agonist [3H]AVP for the reconstituted receptors was modulated by the nonhydrolyzable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), whereas [3H]d(CH2)5Tyr(Me)AVP binding affinity was not. The phospholipid vesicles could be loaded with free fura-2 and displayed an enhanced fluorescence caused by calcium entry after addition of ionomycin. However, stimulation by AVP did not induce an increase of free calcium inside the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine Vasopressin↗
PubMed1980

Computer support for ultrasonic diagnosis.

Differential diagnosis by means of A-scan echography depends to an important extent on the echogram. These characteristics can be evaluated and statistically analysed by computer programs. The analysis is aided by the storage in the computer of a series of echograms in a short period of time (3 seconds). The possible uses of the programs which have been developed are demonstrated.

Computers↗