Embryonic Kidney Cell Line HEK293 Research Articles

Regulation of natriuretic peptide (urodilatin) release in a human kidney cell line

To identify the molecular mechanisms underlying the release of a renal natriuretic peptide (NP) we selected a human kidney cell line (HEK 293) that displays several characteristics of distal tubular cells. Cells were exposed to different extracellular and intracellular stimuli, and the effect on NP release was measured with a specific urodilatin radioimmunoassay, as well as with an atrial NP (ANP) radioimmunoassay. In the absence of stimuli, HEK 293 cells showed a basal release of urodilatin immunoreactivity and ANP immunoreactivity. Raising the osmolality of the secretion medium with sodium chloride and various other osmolytes rapidly increased cellular NP secretion. Elevation of intracellular cAMP levels by forskolin plus 3-isobutyl-1-methylxanthine and administration of phorbol-12-myristate-13-acetate together with the calcium-ionophore A23187 also resulted in respective increases in the amount of secreted peptide. HEK 293 cells exhibit the endogenous expression of both particulate and soluble guanylyl cyclases. In the presence of 8-Br-cGMP, cell cultures showed the enhanced secretion of an ANP immunoreactive peptide only, indicating that guanylyl cyclase activation provoked the secretion of ANP immunoreactivity but not of urodilatin immunoreactivity. The human embryonic kidney cell line HEK 293 represents a renal cellular model system in which we have identified a rapid and regulated release of NPs in response to the osmotic effect of increased extracellular sodium chloride and various intracellular stimuli.

Genomic organisation, alternative splicing and primary structure of human matrilin-4

We have recently cloned a cDNA for mouse matrilin-4. By sequence comparison we identified the 12 kb long human matrilin-4 gene as a part of a high-throughput genomic sequence (HS453C12) in the databases. Additionally we found a human matrilin-4 expressed sequence tag (H54037) in the database that had been mapped to chromosome 20q13.1–2. The gene contains 10 exons and, like the matrilin-1 gene, the human matrilin-4 gene contains an AT-AC intron between the two exons en coding the coiled-coil domain. The cDNA sequence of human matrilin-4 was determined by sequencing of RT-PCR products obtained from mRNA of the human embryonic kidney cell line HEK 293. At the amino acid level it showed an overall sequence identity to the mature mouse matrilin-4 of 91% with a maximum of 97% in the second vWFA-like module. Alternative splicing leads to three different mRNAs. They all encode the putative signal peptide, the two vWFA-like domains and the potential coiled-coil α-helical oligomerisation domain but differ in that either one, two or three EGF-like domains are retained in the mature mRNA. Due to a G to A mutation at the splice donor site of intron C, the third exon encodes an untranslated pseudo-exon specifying the first EGF-like domain when compared to mouse matrilin-4.

Identification of Mel 1a melatonin receptors in the human embryonic kidney cell line HEK293: evidence of G protein-coupled melatonin receptors which do not mediate the inhibition of stimulated cyclic AMP levels

Binding assays using 2-[ 125I]iodomelatonin revealed high-affinity, guanosine 5′- O-(3-thiotriphosphate) sensitive, melatonin binding sites ( B max 1.1 fmol/mg protein) in the human embryonic kidney cell line HEK293. Competition studies using the selective melatonin receptor antagonist luzindole and RT-PCR techniques identified these sites as human Mel 1a melatonin receptors. Challenge of HEK293 cells with 1 μM melatonin had no effect on forskolin stimulated cyclic AMP levels, whereas in HEK293 cells engineered to stably over-express the human Mel 1a melatonin receptor ( B max>400 fmol/mg protein) melatonin dose-dependently inhibited stimulated cyclic AMP levels (IC 50 7.7 pM). These data may indicate that certain tissues, expressing low levels of G protein-coupled melatonin receptors, do not display melatonin mediated inhibition of cAMP.