Molecular cloning and expression of HRLRRP, a novel heart-restricted leucine-rich repeat protein.

We isolated a novel leucine-rich repeat protein (LRRP) cDNA from E13 mouse embryos by the in silico approach. The cDNA encoded a protein of 274 amino acids having 7 leucine-rich repeat motifs at the center of the protein. An in vitro transcription/translation study showed that the cDNA coded for a peptide of approximately 31kDa. Northern blot analysis suggested that the mRNA of this novel LRRP was expressed only in the heart, although RT-PCR indicated slight expression in skeletal muscle as well. The transcripts of this gene and Nkx-2.5/Csx were detected in the early stage of cardiac differentiation of P19CL6 embryonal carcinoma cells treated with 1% dimethyl sulfoxide. The fusion protein made between it and GFP was detected at a high level in mitochondria and a low level in the nuclei of COS7 cells. The nuclei of the adult mouse heart were strongly stained with the antibody raised against the synthetic peptide of the protein. Therefore, we designated the gene as heart-restricted leucine-rich repeat protein (HRLRRP) and assume that mouse HRLRRP may play important roles in cardiac development and/or cardiac function.

Relationship between ligand binding and YIPP motif in the C-terminal region of human AT1 receptor.

The YIPP (tyrosine-isoleucine-proline-proline, amino acids 319-322) motif within the C-terminal part of the human AT(1) receptor is associated with angiotensin II (AII)-induced activation of the Jak-STAT pathway and phospholipase Cgamma1 phosphorylation. We report here that mutations of the YIPP motif strongly affect ligand-binding to the receptor. We analysed AT(1) receptors of the wild type (WT) and 11 mutants with a FLAG-epitope-tag within their C-terminal portion. Mutations of the "P-P" amino acid sequence of this motif decreased both AII binding and the AII-induced intracellular Ca(2+) transients. Mutant and WT receptors were expressed equally in the cell membrane and were localized within the plasma membrane. These results suggest that the "P-P" amino acid sequence within the YIPP motif is important for AII binding to the AT(1) receptor.

Heterogeneous distribution of beta-adrenoceptors and muscarinic receptors in the sinoatrial node and right atrium of the dog.

1. The pacemaker site is known to shift away from the sinoatrial (SA) node in response to autonomic stimuli. 2. To test whether the shifting of the impulse-initiation site that occurs during autonomic nerve stimulation can be explained by the spatial distributions of beta-adrenoceptor and muscarinic receptors, we obtained densities (B(max)) and dissociation constants (K((d)) for these receptors in three regions along the sulcus terminalis (the SA node itself and regions superior and inferior to it) and a region of the right atrial appendage in the dog. 3. The Bmax values for [(125)I]-iodocyanopindolol binding to beta-adrenoceptors were not different among the four regions. The Bmax value for [(3)H]-quinuclidinyl benzilate binding to muscarinic receptors was significantly higher in the SA node area than in any other region (P < 0.01), although there was no difference among the other three regions. 4. For each receptor, K(d) values were similar among the four regions. 5. These results suggest that spatial variations in the densities of beta-adrenoceptors and muscarinic receptors are not important for shifting the impulse-initiation site and that other factors, such as non-uniform innervation by autonomic nerve fibres, may be mainly responsible for the pacemaker shift induced by autonomic nerve stimulation.

KDR-5169, a new gastrointestinal prokinetic agent, enhances gastric contractile and emptying activities in dogs and rats.

KDR-5169, 4-amino-5-chloro-N-[1-(3-fluoro-4-methoxybenzyl)piperidin-4-yl]-2-(2-hydroxyethoxy)benzamide hydrochloride dihydrate, is a new prokinetic with a dual action, i.e., stimulation of the 5-HT4 receptor and antagonism of the dopamine D2 receptor. In this study, we determined in vitro activities of KDR-5169 towards both receptors and demonstrated the effect of the compound on gastrointestinal motor activity in conscious dogs and rats. In dogs, intravenous KDR-5169 stimulated upper gastrointestinal motility in the fasting state and also eliminated the depressive effect of 3,4-dihydroxyphenylalanine (L-DOPA) on this motility in the postprandial state. The effect of KDR-5169 on gastric emptying was further characterized by the use of three rat gastroparesis models (dopamine D2 receptor agonist (quinpirol)-, abdominal surgery-, or combined-situation-induced). Domperidone (a dopamine D2 receptor antagonist) was effective in the quinpirol-delay and combination-delay models, and cisapride and mosapride (5-HT4 receptor agonists) were effective in the surgery-delay model. Only KDR-5169 eliminated the delay of gastric emptying in all three models. In addition, KDR-5169 accelerated emptying to above the normal level in the combination-delay model. These results suggest that KDR-5169 would be effective in various types of gastric ileus caused by different mechanisms.