Characterization of lipophilicity and antiproliferative activity of E-2-arylmethylene-1-tetralones and their heteroanalogues.

A molecular library based on E-2-arylmethylene-1-tetralone has been designed and synthesized. A reversed phase high performance liquid chromatographic (RP-HPLC) method has been developed and applied to separate them and to characterize their lipophilicity. The chromatographic method applied here was suitable to separate the structural (ortho and para) isomers of compounds and was sensitive enough to differentiate their lipophilicities. The measured (k') and computer calculated (CLOGP) lipophilicity values has been compared. Good linear correlation has been found in the case of these structurally related molecules. In vitro biological assay has been performed with Methylene blue dying to investigate the antiproliferative potency of the compounds synthesized in this work. The measured (k') and calculated (CLOGP) lipophilicities of the compounds were compared with the antiproliferative activities and an optimum value of lipophilicity has been found for these compounds.

Kinetic and pharmacological properties of human brain Na(+)/H(+) exchanger isoform 5 stably expressed in Chinese hamster ovary cells.

The recently cloned Na(+)/H(+) exchanger isoform 5 (NHE5) is expressed predominantly in brain, yet little is known about its functional properties. To facilitate its characterization, a full-length cDNA encoding human NHE5 was stably transfected into NHE-deficient Chinese hamster ovary AP-1 cells. Pharmacological analyses revealed that H(+)(i)-activated (22)Na(+) influx mediated by NHE5 was inhibited by several classes of drugs (amiloride compounds, 3-methylsulfonyl-4-piperidinobenzoyl guanidine methanesulfonate, cimetidine, and harmaline) at half-maximal concentrations that were intermediate to those determined for the high affinity NHE1 and the low affinity NHE3 isoforms, but closer to the latter. Kinetic analyses showed that the extracellular Na(+) dependence of NHE5 activity followed a simple hyperbolic relationship with an apparent affinity constant (K(Na)) of 18.6 +/- 1.6 mM. By contrast to other NHE isoforms, NHE5 also exhibited a first-order dependence on the intracellular H(+) concentration, achieving half-maximal activation at pH 6.43 +/- 0.08. Extracellular monovalent cations, such as H(+) and Li(+), but not K(+), acted as effective competitive inhibitors of (22)Na(+) influx by NHE5. In addition, the transport activity of NHE5 was highly dependent on cellular ATP levels. Overall, these functional features distinguish NHE5 from other family members and closely resemble those of an amiloride-resistant NHE isoform identified in hippocampal neurons.

Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain.

To isolate a cDNA encoding Na+/H+ exchanger isoform 5 (NHE5), we screened a human spleen library using exon sequences of the NHE5 gene. Clones spanning 2.9 kilobase pairs were isolated; however, they contained several introns and were missing coding sequences at both the 5' and 3' ends. The missing 5' sequences were obtained by 5'-rapid amplification of cDNA ends and by analysis of an NHE5 genomic clone, and the missing 3' sequences were obtained by 3'-rapid amplification of cDNA ends. Polymerase chain reaction amplification of brain cDNA yielded products in which each of the introns had been correctly excised, whereas the introns were retained in products from spleen and testis, suggesting that the NHE5 transcripts expressed in these organs do not encode a functional transporter. The intron/exon organization of the NHE5 gene was analyzed and found to be very similar to that of the NHE3 gene. The NHE5 cDNA, which encodes an 896-amino acid protein that is most closely related to NHE3, was expressed in Na+/H+ exchanger-deficient fibroblasts and shown to mediate Na+/H+ exchange activity. Northern blot analysis demonstrated that the mRNA encoding NHE5 is expressed in multiple regions of the brain, including hippocampus, consistent with the possibility that it regulates intracellular pH in hippocampal and other neurons.

Endosomal recycling of the Na+/H+ exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway.

The NHE3 isoform of the Na+/H+ exchanger localizes to both the plasmalemmal and endosomal compartments in polarized epithelial and transfected Chinese hamster ovary (AP-1) cells. It is unclear how the distribution of NHE3 between these compartments is regulated. In this study, we examined the potential involvement of phosphatidylinositol 3'-kinase (PI3-K) in regulating the activity and distribution of NHE3, as this lipid kinase has been implicated in modulating vesicular traffic in the endosomal recycling pathway. Wortmannin and LY294002, both potent inhibitors of PI3-K, markedly inhibited NHE3-mediated H+ extrusion across the plasma membrane in a concentration- and time-dependent manner. The subcellular distribution of the antiporters was monitored by transfecting epitope-tagged NHE3 into AP-1 cells. In parallel with the inhibition of transport, PI3-K antagonists induced a pronounced loss of NHE3 from the cell surface and its accumulation in an intracellular compartment, as assessed by immunofluorescence microscopy and enzyme-linked immunosorbent assays. Further analysis using cells transfected with antiporters bearing an external epitope tag revealed that the redistribution reflected primarily a decrease in the rate of recycling of intracellular NHE3 to the cell surface. The wortmannin-induced inhibition and redistribution of NHE3 were prevented when cells were incubated at 4 degreesC, consistent with the known temperature dependence of the endocytic process. These observations demonstrate that NHE3 activity is controlled by dynamic endocytic and recycling events that are modulated by PI3-K.

Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. phosphorylation-dependent and -independent mechanisms.

We recently identified a region within the cytoplasmic C-terminal tail of the Na+/H+ exchanger NHE3 isoform (residues 579 to 684) which is essential for inhibition of transport activity by cAMP-dependent protein kinase (PKA) (Cabado, A. G., Yu, F. H., Kapus, A., Gergely, L., Grinstein, S., and Orlowski, J. (1996) J. Biol. Chem. 271, 3590-3599). To further define determinants of PKA regulation, six serine residues located in potential recognition sequences for PKA within, or adjacent to, this region (positions 552, 605, 634, 661, 690, and 691) were altered either independently or in various combinations using site-directed mutagenesis. Wild type and mutant NHE3s tagged with the influenza virus hemagglutinin epitope were stably expressed in exchanger-deficient Chinese hamster ovary cells (AP-1) for functional studies. Of the individual mutations examined, only substitutions at Ser605 or Ser634 affected sensitivity to forskolin, an activator of adenylate cyclase, although partial inhibition of NHE3 activity by forskolin remained. By contrast, simultaneous mutation of both these serines completely abolished cAMP-mediated inhibition of NHE3 without greatly affecting basal transport activity. Two-dimensional analysis of tryptic digests of immunoprecipitated NHE3 labeled in vivo with [32P]orthophosphate revealed several phosphopeptides under basal conditions. Phosphorylation was increased approximately 3-fold in one of these peptides following forskolin treatment, and this change was eliminated by mutation of residue Ser605. Thus, phosphorylation of Ser605 is essential for cAMP-mediated inhibition of NHE3. In addition, Ser634 is also required for the effect of cAMP, even though this residue does not become phosphorylated upon activation of PKA.

Investigation of the chemical stability of a new growth hormone-releasing hormone (GHRH) analogue by HPLC.

The stability of a new active growth hormone-releasing hormone analogue (D-Ala2,Nle27,(gamma-amino-butyric acid)30-GHRH(1-30)-NH2) was investigated during storage at different temperatures in aqueous solution. Samples stored for various periods of time were analysed by HPLC. It is concluded that in aqueous solution D-Ala2, Nle27,(gamma-amino-butyric acid)30-growth hormone-releasing hormone (1-30)-NH2 is stable: at least for 36 days at 4 degrees C; for 28 days at 25 degrees C; and for 10 days at 37 degrees C.

New Gaba-containing analogues of human growth hormone-releasing hormone (1-30)-amide: I. Synthesis and in vitro biological activity.

Analogues of human growth hormone-releasing hormone (1-30)-amide have been developed. All analogues have been modified in position 27 with Nle and with Gaba in position 30. Additional D-amino-acids have been inserted in the GHRH(1-30)-NH2 sequence: A-1741: Nle27,Gaba30-GH-RH(1-30)-NH2 A-495: D-Ala2,Nle27,Gaba30-GH-RH(1-30)-NH2 A-515: D Ala2,Leu15,Nle27,Gaba30-GH-RH(1-30)-NH2 A-527: D-Ala2,D-Arg11,Leu15,Nle27,Gaba30-GH-RH(1-30)-NH2. Our analogues were synthesized by solid phase peptide synthesis and were tested is two different in vitro systems and in rat pituitary cell cultures. A-495 and A-1741 were found to be the most active in releasing GH, however they showed different activities in the two different test systems. A-495 exhibited higher potency in the superfusion system (1.63 fold potency of the GHRH (1-29)-amide), while A-1741 evoked higher GH release from cultured pituitary cells (1.5-2.5 times higher than the GH-RH(1-44)-amide). The other analogues (A-515 and A-527) were found to be equipotent to the standard molecule. We can conclude that Nle27 and Gaba30 substitutions appeared to be a good modification in in vitro test systems, and Gaba30 substitution served as a good spacer during the synthesis, since it made the coupling of the C-terminal amino acids easier and produced quantitative coupling. In addition to the advantageous properties in the synthesis these modifications with or without D-Ala at the N-terminus increased the in vitro biological activity to 1.5-2.5 fold of the GHRH molecule.(ABSTRACT TRUNCATED AT 250 WORDS)