Molecular analysis of the regulation of muscarinic receptor expression and function.

We have investigated the molecular mechanisms involved in the regulation of muscarinic acetylcholine receptor gene expression and localization and generated knockout mice to study the role of the M1 muscarinic receptor in vivo. We have used the MDCK cell system to demonstrate that different subtypes of mAChR can be targeted to different regions of polarized cells. We have also examined the developmental regulation of mAChR expression in the chick retina. Early in development, the M4 receptor is the predominant mAChR while the levels of the M2 and M3 receptors increase later in development. The level of M2 receptor is also initially very low in retinal cultures and undergoes a dramatic increase over several days in vitro. The level of M2 receptor can be increased by a potentially novel, developmentally regulated, secreted factor produced by retinal cells. The promoter for the chick M2 receptor gene has been isolated and shown to contain a site for GATA-family transcription factors which is required for high level cardiac expression. The M2 promoter also contains sites which mediate induction of transcription in neural cells by neurally active cytokines. We have generated knockout mice lacking the M1 receptor and shown that these mice do not exhibit pilocarpine-induced seizures and muscarinic agonist-induced suppression of the M-current potassium channel in sympathetic neurons.

Tyrosines 905 and 915 of gp130 are required for maximum induction of m2 muscarinic acetylcholine receptor and VIP gene transcription by cytokines in neuronal cells.

1. Leukemia inhibitory factor action is mediated by a heterodimeric receptor consisting of two subunits, gp130 and the low-affinity leukemia inhibitory factor receptor (LIFR). 2. We used chimeric receptors containing the intracellular domain of either the LIFR or gp130 to identify regions of the receptors required for induction of the m2 muscarinic acetylcholine receptor gene in IMR-32 and SN56 neuronal cells. 3. While chimeric receptors containing the intracellular domain of gp130 were able to induce transcription from both the m2 and the vasoactive intestinal peptide (VIP) gene promoters, chimeric receptors containing the intracellular domain of the LIFR were incapable of mediating induction of the m2 gene despite being able to induce VIP transcription. 4. Deletion and mutagenesis studies identified two tyrosines, Y905 and Y915, which were required for maximal induction of the m2 and VIP genes. 5. Because Y905 and Y915 are reported to be the only tyrosine residues in gp130 that bind Stat1, these results suggest that this transcription factor plays a key role in the induction of transcription of both the m2 and the VIP genes.

Disruption of a neuropeptide gene, flp-1, causes multiple behavioral defects in Caenorhabditis elegans.

Neuropeptides serve as important signaling molecules in the nervous system. The FMRFamide (Phe-Met-Arg-Phe-amide)-related neuropeptide gene family in the nematode Caenorhabditis elegans is composed of at least 18 genes that may encode 53 distinct FMRFamide-related peptides. Disruption of one of these genes, flp-1, causes numerous behavioral defects, including uncoordination, hyperactivity, and insensitivity to high osmolarity. Conversely, overexpression of flp-1 results in the reciprocal phenotypes. On the basis of epistasis analysis, flp-1 gene products appear to signal upstream of a G protein-coupled second messenger system. These results demonstrate that varying the levels of FLP-1 neuropeptides can profoundly affect behavior and that members of this large neuropeptide gene family are not functionally redundant in C. elegans.

GATA factor-dependent regulation of cardiac m2 muscarinic acetylcholine gene transcription.

The m2 subtype is the predominant muscarinic acetylcholine receptor subtype expressed in heart and regulates the rate and force of cardiac contraction. We have previously reported the isolation of the promoter region for the chick m2 receptor gene and defined a region of the chick m2 promoter sufficient for high level expression in cardiac primary cultures. In this manuscript we demonstrate transactivation of cm2 promoter by the GATA family of transcription factors. In addition, we define the GATA-responsive element in the chick m2 promoter and demonstrate that this element is required for expression in cardiac primary cultures. Finally, we demonstrate specific binding of both a chick heart nuclear protein and of cloned chick GATA-4, -5, and -6 to the GATA-responsive element.

Regulation of muscarinic receptor expression and function in cultured cells and in knock-out mice.

We have investigated the molecular and cellular basis for the regulation of expression and function of the muscarinic acetylcholine receptors. Treatment of cultured chick cardiac cells with the agonist carbachol results in decreased levels of mRNA encoding the m2 and m4 receptors. Treatment of chick embryos in ovo with carbachol results in decreased levels of mRNA encoding the potassium channel subunits GIRK1 and GIRK4 as well as the m2 receptor. There are thus multiple pathways for the regulation of mAChR responsiveness by long-term agonist exposure. Immunoblot, immunoprecipitation, and solution hybridization analyses have been used to quantitate the regulation of mAChR expression in chick retina during embryonic development. The m4 receptor is the predominant subtype expressed early in development, while the expression of the m3 and m2 receptors increases later in development. A cAMP-regulated luciferase reporter gene has been used to demonstrate that the m2 and m4 receptors have distinct specificities for coupling to G-protein subtypes to mediate inhibition of adenylyl cyclase. This system has also been used to demonstrate that beta-arrestin1 and beta-adrenergic receptor kinase-1 act synergistically to promote receptor desensitization. We have isolated the promoter region for the chick m2 receptor gene, identified regions of the promoter required to drive high level expression in cardiac and neural cells, and have identified a region which confers sensitivity of gene expression to neurally active cytokines. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we have used the method of targeted gene disruption by homologous recombination to generate mice deficient in the m1 receptor.

Isolation and characterization of the chicken m2 acetylcholine receptor promoter region: induction of gene transcription by leukemia inhibitory factor and ciliary neurotrophic factor.

We have isolated the promoter region and determined the start sites of transcription for the gene encoding the chicken m2 (cm2) muscarinic acetylcholine receptor. Transfection experiments, using cm2-luciferase reporter gene constructs, demonstrated that a 789-bp genomic fragment was sufficient to drive high level expression in chicken heart primary cultures, while an additional 1.2-kb region was required for maximal expression in mouse septal/ neuroblastoma (SN56) cells. Treatment of SN56 cells with the cytokines ciliary neurotrophic factor and leukemia inhibitory factor increases expression of endogenous muscarinic acetylcholine receptors and results in a 4- to 6-fold induction of cm2 promoter driven luciferase expression. We have mapped a region of the cm2 promoter that is necessary for induction by cytokines.

The flp-1 propeptide is processed into multiple, highly similar FMRFamide-like peptides in Caenorhabditis elegans.

Previously, we described a gene, flp-1, that encodes seven FMRFamide-like peptides from two alternatively spliced transcripts in the nematode Caenorhabditis elegans. To determine whether all or a subset of the predicted peptides coded for by flp-1 are produced in vivo, we undertook the isolation of FMRFamide-like peptides from C. elegans. Six FLRFamide-containing peptides, all contained within the putative translation products of the flp-1 gene, were isolated from extracts of mixed stage animals. By quantitative PCR analysis of RNA from mixed stage animals, we found that the shorter transcript of flp-1 has a higher level of expression than the longer transcript.

Alternatively spliced transcripts of the flp-1 gene encode distinct FMRFamide-like peptides in Caenorhabditis elegans.

We have isolated and characterized several cDNAs and the corresponding genomic region of a gene encoding multiple FMRFamide-like neuropeptides from the nematode Caenorhabditis elegans. The gene, named flp-1, consists of six exons of which four encode FMRFamide-like peptides. The cDNA and genomic sequences revealed that two distinct transcripts are generated by the use of an alternative 3' splice acceptor site between exons 3 and 4. This alternative splice results in the substitution of AGSDPNFLRFG for one of the copies of SADPNFLRFG found in the other translation product. Based on PCR analysis of RNA from mixed-stage animals, both transcripts are expressed. This gene is the first example of a distinct FMRFamide-like peptide being derived from alternative splicing, suggesting a unique role for the substituted peptide in the animal.

Effect of thermal history on the glassy state of indapamide.

The effects of thermal history, e.g. cooling rate, annealing, etc., on the thermal behaviour of indapamide glass were determined by differential scanning calorimetry (DSC). The glass was prepared by heating indapamide crystals (m.p. 162 degrees C) to 180 degrees C, and then cooling the melt to room temperature. The glass transition temperature (Tg) of the material was 98 degrees C. An endotherm, due to thermal relaxation of the glass, was observed in the DSC thermogram when indapamide glass was prepared by slow cooling or was annealed isothermally at a temperature below Tg. Such enthalpy relaxation may be observed during ageing of pharmaceutical glasses and might influence their physico-chemical properties.

Profound bradycardia after amyl nitrite in patients with a tendency to vasovagal episodes.

Two patients with mild aortic insufficiency inhaled amyl nitrite during routine echocardiographic examinations. One developed sinus arrest and syncope and the other had pronounced sinus bradycardia. The mechanism of this paradoxical response is unclear. Caution should be exercised when amyl nitrite is administered for diagnostic purposes.

In situ intestinal absorption of a poorly water-soluble drug from mixed micellar solutions of bile salt and lipolysis products in rats.

The role of a bile salt (sodium taurocholate) and lipolysis products (monoglyceride and fatty acid) in the intestinal absorption of a poorly water-soluble drug, diazepam, was investigated. Absorption rates and bioavailabilities were determined with the in situ rat gut technique of Doluisio et al. and analyzing the diazepam concentrations in the luminal solution, intestinal membrane, blood and lymph. The absorption rate constant of diazepam decreased with the increase in bile salt concentration. Thus, although the bile salt increased the solubility of diazepam, the net effect was a decrease in apparent diazepam absorption rate. On the other hand, the lipolysis products incorporated in the bile salt micelles increased the solubility of diazepam without affecting the absorption rate constant, and as a result the apparent absorption rate of diazepam increased. In addition, the solubilized diazepam was absorbed almost uniformly throughout the small intestine. The drug solubilized in mixed micelles of bile salt-lipolysis products was not absorbed through the lymphatic system along with the lipolysis products, rather it was absorbed directly into the blood. The possible mechanism of the effect of dietary fat in the absorption of drug is discussed.

pH-Solubility profile of papaverine hydrochloride and its relationship to the dissolution rate of sustained-release pellets.

The pH-solubility profile of papaverine hydrochloride (I) was determined using the phase-solubility technique and equilibrium solubilities in buffers. The release of I from sustained-release pellets consisting of a shellac-based matrix was determined by the USP basket technique and was found to exhibit zero-order kinetics. Release rates at various pH values of the permeating solvent were compared with the pH-solubility profile and were directly proportional to the solubility below, but not above, the apparent pHmax (3.9). This lack of proportionality was also shown by the intrinsic dissolution rates. The effect was attributed to the self-buffering action of I and the metastability of the papaverine salt-base system in the vicinity of pHmax. It is postulated that the outer layer of polymer and filler on the surface of the pellets forms a barrier which determines the rate of release. The inner matrix serves as a drug reservoir in which the internal pH may not be the same as the bulk pH.

Clinical and hemodynamic correlation in patients with pericardial effusion and swinging heart by echocardiography.

The clinical and hemodynamic findings in 13 consecutive patients with "swinging heart" on M-mode echocardiography were analyzed. In these patients the anterior right ventricular and posterior left ventricular walls and interventricular septum moved almost parallel to each other throughout the cardiac cycle, often with exaggerated excursion. In 10 of 13 patients right heart catheterization revealed the hemodynamic profile of cardiac tamponade, while one additional patient was found to have evidence of cardiac compression at the time of surgery. In the remaining two patients no acute invasive diagnostic procedures were performed. During the same observation period cardiac tamponade was observed in five patients without echocardiographic evidence of a swinging heart, and four of these had large clots in the pericardial space. Thus, the swinging heart pattern appears to be a reliable marker of cardiac tamponade, except in those patients with intrapericardial lesions which mechanically limit cardiac motion.

Pulmonary valve gonococcal endocarditis. A forgotten disease.

Although gonococcal infections of the pulmonary valve were common before the introduction of antibiotics, such infections have rarely been reported since penicillin became available. In an elderly man with gonococcal endocarditis of the pulmonary valve the non-specific signs and symptoms, the late appearance of a pulmonary murmur, and the sterility of early blood cultures made the diagnosis unclear until three weeks after admission. Endocarditis was localised to the pulmonary valve by M-mode and cross-sectional echocardiography. Echocardiography may be useful for diagnosing endocarditis in patients with fever of unknown origin. Gonococcal infection should be suspected in patients with pulmonary vegetations and sterile blood cultures.