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Lyso-PS (lyso-phosphatidylserine) has been shown to activate the Gi/o-protein-coupled receptor GPR34. Since in vitro and in vivo studies provided controversial results in assigning lyso-PS as the endogenous agonist for GPR34, we investigated the evolutionary conservation of agonist specificity in more detail. Except for some fish GPR34 subtypes, lyso-PS has no or very weak agonistic activity at most vertebrate GPR34 orthologues investigated. Using chimaeras we identified single positions in the second extracellular loop and the transmembrane helix 5 of carp subtype 2a that, if transferred to the human orthologue, enabled lyso-PS to activate the human GPR34. Significant improvement of agonist efficacy by changing only a few positions strongly argues against the hypothesis that nature optimized GPR34 as the receptor for lyso-PS. Phylogenetic analysis revealed several positions in some fish GPR34 orthologues which are under positive selection. These structural changes may indicate functional specification of these orthologues which can explain the species- and subtype-specific pharmacology of lyso-PS. Furthermore, we identified aminoethyl-carbamoyl ATP as an antagonist of carp GPR34, indicating ligand promiscuity with non-lipid compounds. The results of the present study suggest that lyso-PS has only a random agonistic activity at some GPR34 orthologues and the search for the endogenous agonist should consider additional chemical entities.

To identify amino acids that might be involved in discriminating guanosine-3′,5′-cyclic phosphate (cGMP) towards adenosine-3′,5′-cyclic phosphate (cAMP) binding in the cAMP-specific phosphodiesterases, alignments of different human cyclic nucleotide phosphodiesterases (PDEs) were performed. Eight amino acid residues that are highly conserved in the cAMP-hydrolysing phosphodiesterases (PDE1, PDE3, PDE4, PDE7, PDE8) and that did not show any homologies to the cGMP-specific phosphodiesterases (PDE5, PDE6, PDE9) were selected from these alignments. Using the technique of site-directed mutagenesis, derivatives of PDE4A carrying single mutations at these conserved residues (amino acid positions are given according to the human PDE4A isoform HSPDE4A4B; accession number L20965) were generated and expressed in COS1 cells. The expression products were characterised with regard to cAMP and cGMP hydrolysis and sensitivity towards type-specific inhibitors. The mutation of Phe484 toward Tyr, Ala590 toward Cys, Leu391 and Val501 towards Ala had no significant influence on substrate affinity or specificity. However, the exchange of Trp375 and Trp605 for aliphatic residues abolished catalytic activity and the exchange of Pro595 for Ile led to sevenfold decrease of substrate affinity and an 14-fold decrease of the affinity towards the PDE4-specific inhibitor 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram). Both effects may provide evidence for a structural importance of Trp375, Trp605 and Pro595 for PDE function. By exchanging the aspartate residue for asparagine or alanine at position 440 of the human PDE4A4B isoform, the substrate specificity was altered from the highly specific cAMP hydrolysis to an equally efficient cAMP and cGMP binding and hydrolysis. In addition, the IC50 values for common PDE4-specific inhibitors like rolipram, N-(3,5-dichlorpyrid-4-yl)-3-cyclopentyl-oxy-4-methoxy-benzamide (RPR-73401) and 8-methoxy-5-N-propyl-3-methyl-1-ethyl-imidazo[1,5-a]-pyrido[3,2-e]-pyrazinone (D-22888) were dramatically increased. These results demonstrate an important role of the aspartate at position 440 in determining substrate specificity and inhibitor susceptibility of PDE4A. The strong conservation of this residue suggests that Asp440 may play a similar role in other cAMP-PDEs.

Primary cultures of rat hepatocytes were used to study the combined effects of insulin and dexamethasone on cyclic AMP phosphodiesterase 3 (PDE 3) and glycogen metabolism. PDE activity was measured in extracts obtained by hypotonic shock treatment of the particulate fraction from cultured hepatocytes. PDE 3 was identified by inhibition with ICI 118233, Western blotting, immunoprecipitation of the activity with the use of a new PDE 3B-specific anti-peptide antibody and stimulation of the activity after adding insulin, glucagon and okadaic acid to the culture medium. Specific PDE inhibitors were always used to identify the measured PDE activities. Hypotonic extracts contained 30% PDE 3 and 50% PDE 4. Both PDE types show a nearly constant level during cultivation up to 48 h. Long-term exposure of dexamethasone alone has no effect on PDE 3 activity, whereas, in combination with insulin, the insulin stimulation of PDE 3 activity was found to be increased between 48 and 72 h of cultivation. Additionally, db-cAMP was able to stimulate PDE 3. A possible effect of insulin or db-cAMP on PDE 3B expression could not be found. On the other hand, activation of PDE 3B after 48 h of culturing decreased rapidly after removal of insulin or db-cAMP from the culture medium. Insulin-stimulated incorporation of 14C-glucose into glycogen was inhibited by PDE 3- and PDE 4-specific inhibitors as well as by the unspecific PDE inhibitor IMBX. Inhibitions by PDE 3- and PDE 4-specific inhibitors were found to be additive and reached the same extent as with IMBX. Summarising our results, we can conclude that PDE 3 and PDE 4 effectively control the hepatic glycogen metabolism. Insulin effects on PDE activity and glycogen metabolism require the presence of dexamethasone. Insulin-stimulated PDE seems to play an important role in realising insulin effects on hepatic glycogen metabolism.

We report the presence of KIT variants in granulosa and thecal cells of the follicle and endothelial and steroidogenic cells of the corpus luteum. Transcripts of both full-length splice variants, KIT and KITA, were ubiquitously detected in all cell types, in contrast to transcripts for truncated KIT. RT-PCR with exon-intron-specific primers suggested that KIT transcripts retained intron sequences. We used domain-specific KIT antibodies to identify truncated KIT proteins in cell conditioned media and lysates. These proteins represented soluble KIT and a so far disregarded intracellular KIT fragment, and were ubiquitously present. In contrast, glycosylated variants of full-length KIT were predominantly detected in thecal and endothelial cells. All KIT variants were encountered again in COS-7 cells transfected with a vector containing KITA. Phorbol 12-myristate-13-acetate treatment induced levels of truncated KITs, and this effect was repressed by the metalloproteinase inhibitor TAPI-1. Our findings show that ectodomain cleavage of full-length KIT generates an intracellular KIT. Our experiments suggest that replenishing full-length KIT differs among various ovarian cell types.

Intercellular communication between smooth muscle cells is crucial for contractile behaviour in normal and pathologically altered urinary bladder. Since the study of coupling is difficult in situ, we established cell cultures of bladder smooth muscle cells to analyse coupling mechanisms. Microinjection of Lucifer yellow demonstrated syncytia composed of only a few to several dozen cells. Electron-microscopic examination of freeze-fracture specimens and ultrathin sections revealed that the dye-coupling was based on typical gap junction formation between the cultured smooth muscle cells. Furthermore, we were able to demonstrate gap junctions within the tissue fragments from which the primary cultures were grown. By Western blotting, we found connexin-43-positive protein bands both in native tissue probes from the guinea-pig urinary bladder and in smooth muscle cell cultures. Extracellular electrical stimulation of single cells evoked calcium transients, as visualized by fura-2 ratiofluorimetry. Calcium waves propagated throughout the syncytia with a declining amplitude, showing that the calcium signal was not regenerative. Therefore, the calcium signal was probably transmitted by a diffusible factor. These findings correlated well with the dye-coupling that we found between detrusor smooth muscle cells in situ. The use of smooth muscle cell cultures therefore seems to be a feasible approach for studying coupling behaviour in vitro.

67% of total cAMP phosphodiesterase activity (PDE) in cultured rat hepatocytes could be detected in the cytosol, 15% in plasma membrane, 15% in 'dense vesicle,' and 3% in endoplasmatic reticulum fractions. Up to 84% of the PDE activity of the cytosol is represented by the rolipram-sensitive PDE 4. ICI 118233-inhibited PDE 3 was found predominantly in membranes. We were able to show that dexamethasone acts on the PDE 4 in cytosolic and plasma membrane fractions whereas glucagon effected the PDE 4 of the cytosol and the PDE 3 in 'dense vesicle' membranes. Primary culture of hepatocytes was used to study long-term effects of dexamethasone and glucagon on PDE 4 activity. Addition of dexamethasone (0.1 microM) at the beginning of cultivation leads to a decrease of total PDE 4 activity whereas after 24 h precultivation no dexamethasone effect could be observed. Glucagon effects on PDE 4 were investigated in 20 h precultured hepatocytes. Maximal stimulation was achieved after 2 h of exposure. PDE 4 subtypes A, B , D and, to a lesser degree, subtype C could be detected by RT-PCR analysis. The results of semiquantitative RT-PCR show that the presence of dexamethasone during the first 24 h of cultivation reduced selectively the transcription of PDE 4D, whereas glucagon was without any effect. Also the translation of PDE 4D was reduced as shown in the Western blot. We would like to discuss the way that dexamethasone influences PDE 4D expression-most likely in combination with other factors such as cytokines--during the time of cell plating, whereas glucagon actions are part of metabolic regulations via phosphorylation reactions.