Muscarinic acetylcholine receptors of the chick amnion

The presence of muscarinic (M) acetylcholine receptors in the noninnervated chick amnion makes it possible to analyze their functioning with presynaptic effects excluded. The M receptors of the amnion mediating its contraction were identified by testing with selective antagonists: pirenzepine for M₁, methoctramine for M₂, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) for M₃, and tropicamide for M₄ receptor subtype. All antagonists acted as competitive inhibitors of M-acetylcholine receptors. With respect to cholinolytic activity estimated from the response to carbacholine (CBC) (-logIC₅₀), the antagonists could be arranged in the following series: 4-DAMP (8.29) > tropicamide (6.97) > pirenzepine (5.85) > methoctramine (5.63). In addition, the effect of forskolin (5 μM), activator of adenylate cyclase (AC), was unidirectional with ?-adrenergic agonists; it blocked CBC-induced contractile activity of the amnion, whereas phospholipase C (1.25 U/ml) stimulated this activity. These data suggest that CBC-or acetylcholine (ACh)-induced contractile activity of the amnion is mediated by M₃ acetylcholine receptors. Evaluation of contractile response to ACh by the tonic component usually revealed one pool of M₃ acetylcholine receptors. One pool was also revealed after treatment with 4-DAMP, with the Hill coefficient being increased (ACh, n = 1.07; ACh against the 4-DAMP background, n = 1.48). It is possible to detect two pools of M₃-acetylcholine receptors on the basis of either phase-frequency or tonic response, i.e., independently of the test parameter.     

Muscarinic acetylcholine receptors of rat lymphocytes

The muscarinic acetylcholine receptors on rat lymphocytes were determined by [3H]quinuclidinyl benzilate binding studies. Binding of [3H]quinuclidinyl benzilate is rapid (half saturation occurred within 120 s) and highly specific. Muscarinic receptors reveal high lability. The number of receptors on plasma membrane depends on time of incubation as well as on composition of incubation medium. Lymphocytes incubated in nutrient-deficient media lose their surface receptors; enrichment of the medium causes reappearance of the receptors. Appearance of [3H]quinuclidinyl benzilate-binding sites in the incubation medium was under conditions in which binding to lymphocytes was decreased. It is concluded that the number of plasma membrane receptors on rat lymphocytes represents the dynamic steady state in which newly synthesized and degraded receptors are balanced.     

Muscarinic acetylcholine responses in the early embryonic chic retina

The action of acetylcholine on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in early embryonic chick retinae. Whole neural retinae were isolated from embryonic day 3 (E3) chicks and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). Increases in [Ca²⁺]_i were evoked by the puff application of acetylcholine at concentration than 0.1 μM. The Ca²⁺ response became larger in dose–dependant manner up to 10 μM of acetylcholine applied. The rise in [Ca²⁺]_i was not due to the influx of Ca⁺² through calcium channels, but to the release of Ca²⁺ from internal stores. A calcium channel antagonist, nifedipine, which completely blocks the Ca²⁺ rise caused by depolarization with 100 mM K⁺, had no effects on the acetylcholine response and the Ca²⁺ response to acetylcholine occurred even in a Ca²⁺-free medium. The Ca²⁺ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolished the response to 10 μM acetylcholine, whereas d-tubocurarine of 100 μM had no effects. Two muscarinic agonists, muscarine and carbamylcholine (100 μM each), evoked comparable responses with that to 10 μM acetylcholine. The developmental change of the muscarinic response was examined from E3 to E13. The Ca2+ response to 100 μM carbamylcholine was intense at E3-E5, then rapidly declined until E8. The muscarinic Ca²⁺ mobilization we found in the early embryonic chick retina may be regarded as a part of the “embryonic muscarinic system” proposed by Drew's group, which appears transiently and ubiquitously at early embryonic stages in relation to organogenesis. 1994 John Wiley & Sons, Inc.     

Control of adenovirus early gene expression: A class of immediate early products

We have identified the viral mRNAs present in cells in which protein synthesis has been stringently inhibited prior to infection with adenovirus type 2. These species presumably represent the subset of viral mRNAs that are “immediate early” products, requiring only host cell genes for their expression, and they do not include any of the conventionally recognized early mRNAs. Treatment of cells with 100 μM anisomycin inhibits 99.6% of protein synthesis and substantially depresses (by 20–200 fold) the levels of the conventional early mRNAs from regions E1 A, E1B, E2, E3 and E4. Also depressed are species encoding an 87K protein (11.6–31.5 map units) and a 13.6K protein (encoded a short distance to the right of 21.5 map units). The only mRNAs not depressed by this treatment are an mRNA for a 13.5K protein encoded between 17.0 and 21.5 map units, and the mRNA for the late 52,55K protein encoded between 29 and 34 map units, which is also present in small amounts at early times. Further proof that production of the mRNA for the immediate early 13.5K protein is independent of EIA gene function is provided by the observation that it can be detected in cells infected with the EIA deletion mutant d1312.     

Muscarinic M1 acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism

Nitric oxide (NO), previously demonstrated to participate in the regulation of the resting membrane potential in skeletal muscles via muscarinic receptors, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh release was estimated by the amplitude of endplate hyperpolarization (H-effect) following a blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine. The muscarinic agonists oxotremorine and muscarine lowered the H-effect and the M1 antagonist pirenzepine prevented this effect occurring at all. Another muscarinic agonist arecaidine but-2-ynyl ester tosylate (ABET), which is more selective for M2 receptors than for M1 receptors and 1,1-dimethyl-4-diphenylacetoxypiperidinium (DAMP), a specific antagonist of M3 cholinergic receptors had no significant effect on the H-effect. The oxotremorine-induced decrease in the H-effect was calcium and calmodulin-dependent. The decrease was negated when either NO synthase was inhibited by N(G)-nitro-L-arginine methyl ester or soluble guanylyl cyclase was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. The target of muscle-derived NO is apparently nerve terminal guanylyl cyclase, because exogenous hemoglobin, acting as an NO scavenger, prevented the oxotremorine-induced drop in the H-effect. These results suggest that oxotremorine (and probably also non-quantal ACh) selectively inhibit the non-quantal secretion of ACh from motor nerve terminals acting on post-synaptic M1 receptors coupled to Ca(2+) channels in the sarcolemma to induce sarcoplasmic Ca(2+)-dependent synthesis and the release of NO. It seems that a substantial part of the H-effect can be physiologically regulated by this negative feedback loop, i.e., by NO from muscle fiber; there is apparently also Ca(2+)- and calmodulin-dependent regulation of ACh non-quantal release in the nerve terminal itself, as calmidazolium inhibition of the calmodulin led to a doubling of the resting H-effect.     

Muscarinic acetylcholine receptors: structural basis of ligand binding and G protein coupling

Muscarinic acetylcholine receptors (m1-m5) were studied by a combined molecular genetic/pharmacologic approach to elucidate the molecular characteristics of the ligand binding site and of the receptor domains involved in G protein coupling. Site-directed mutagenesis studies of the rat m3 muscarinic receptor suggest that the acetylcholine binding domain is formed by a series of hydrophilic amino acids located in the "upper" half of transmembrane domains (TM) III, V, VI, and VII. Moreover, we showed that mutational modification of a TM VI Asn residue (Asn507 in the rat m3 receptor sequence) which is characteristic for the muscarinic receptor family has little effect on high-affinity acetylcholine binding and receptor activation, but results in dramatic reductions in binding affinities for certain subclasses of muscarinic antagonists. The N-terminal portion of the third intracellular loop (i3) of muscarinic and other G protein-coupled receptors has been shown to play a central role in determining the G protein coupling profile of a given receptor subtype. Insertion mutagenesis studies with the rat m3 muscarinic receptor suggest that this region forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents an important G protein recognition surface. Further mutational analysis of this receptor segment showed that Tyr254 located at the N-terminus of the i3 loop of the m3 muscarinic receptor plays a key role in muscarinic receptor-induced Gq activation. The studies described here, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.     

Heparanase modulation of early growth response gene expression

Heparan sulfate (HS), a polysaccharide ubiquitously expressed in animals, is essential for development and homeostasis. Degradation of HS by heparanase, an endoglucuronidase, may affect pathophysiological function. Expression of the heparanase gene has been found elevated in a number of pathological conditions. The goal of this work was to investigate the impact of heparanase on expression of other genes. DNA microarray analysis revealed that 1, 042 genes in the cortex and 1,039 genes in the thalamus are up- or down-regulated more than 2-fold in mouse brain overexpresssing human heparanase. Of these genes, two of the early growth response genes, Egr1 and Egr2, are substantially upregulated in the cortex, but essentially unchanged in the thalamus. RT-PCR analysis demonstrated a significant increase of Egr2, but a minor increase of Egr1, in human embryonic kidney cells stably overexpressing heparanase. The upregulated expression of Egr genes is also observed in hepatoma cells with upregulated expression of heparanase. Earlier studies reported that Egr1 induced heparanase expression; our findings suggest a possible reciprocal regulation of Egr and heparanase expression. Furthermore, overexpression of heparanase influenced expression of most genes involved in heparan sulfate proteoglycan biosynthesis, albeit to a different degree in the cortex and thalamus of the transgenic mice.     

HVC lesions modify immediate early gene expression in auditory forebrain regions of femalesongbirds

It is well established that auditory forebrain regions of oscine birds are essential for the encoding of species‐typical songs and are, therefore, vital for recognition of song during sociosexual interactions. Regions such as the caudal medial nidopallium (NCM) and the caudal medial mesopallium (CMM) are involved in perceptual processing of song and the formation of auditory memories. There is an additional telencephalic nucleus, however, that has also been implicated in species recognition. This nucleus is HVC, a prominent nucleus that sits at the apex of the song system, and is well known for its critical role in song learning and song production in male songbirds. Here, we explore the functional relationship between auditory forebrain regions (i.e., NCM and CMM) and HVC in female canaries (Serinus canaria). We lesion HVC and examine immediate early gene responses to conspecific song presentation within CMM and NCM to explore whether HVC can modulate auditory responses within these forebrain regions. Our results reveal robust deficits in ZENK‐ir in CMM and NCM of HVC‐lesioned females when compared with control‐ and sham‐lesioned females, indicating that functional connections exists between HVC and NCM/CMM. Although these connected regions have been implicated in song learning and production in males, they likely serve distinct functions in female songbirds that face the task of song recognition rather than song production. Identifying functional connections between HVC and auditory regions involved in song perception is an essential step toward developing a comprehensive understanding of the neural basis of song recognition. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Muscarinic acetylcholine receptors involved in the regulation of neural stem cell proliferation and differentiation in vitro

Neural stem cells (NSCs) are currently considered powerful candidates for cell therapy in neurodegenerative disorders such as Parkinson's disease. However, it is not known when and how NSCs begin to differentiate functionally. Recent reports suggest that classical neurotransmitters such as acetylcholine (Ach) are involved in the proliferation and differentiation of neural progenitor cells, suggesting that neurotransmitters play an important regulatory role in development of the central nervous system (CNS). We have shown by calcium imaging and immunochemistry that proliferation and differentiation are enhanced by M2 muscarinic Ach receptors (mAchR) expressed on the NSC surface and on their neural progeny. Moreover, atropine, an mAchR antagonist, blocks the enhancement and inhibits the subsequent differentiation of NSCs. Further understanding of this neural-nutrition role of Ach might elucidate fetal brain development, the brain's response to injury, and learning and memory.     

Site in the cat-86 regulatory leader that permits amicetin to induce expression of the gene.

Expression of the plasmid gene cat-86 is induced in Bacillus subtilis by two antibiotics, chloramphenicol and the nucleoside antibiotic amicetin. We proposed that induction by either drug causes the destabilization of a stem-loop structure in cat-86 mRNA that sequesters the ribosome-binding site for the cat coding sequence. The destabilization event frees the ribosome-binding site, permitting the initiation of translation of cat-86 mRNA. cat-86 induction is due to the stalling of a ribosome in a leader region of cat-86 mRNA, which is located 5' to the RNA stem-loop structure. A stalled ribosome that is active in cat-86 induction has its aminoacyl site occupied by leader codon 6. To test the hypothesis that a leader site 5' to codon 6 permits a ribosome to stall in the presence of an inducing antibiotic, we inserted an extra codon between leader codons 5 and 6. This insertion blocked induction, which was then restored by the deletion of leader codon 6. Thus, induction seems to require the maintenance of a precise spatial relationship between an upstream leader site(s) and leader codon 6. Mutations in the ribosome-binding site for the cat-86 leader, RBS-2, which decreased its strength of binding to 16S rRNA, prevented induction. In contrast, mutations that significantly altered the sequence of RBS-2 but increased its strength of binding to 16S rRNA did not block induction by either chloramphenicol or amicetin. We therefore suspected that the proposed leader site that permitted drug-mediated stalling was located between RBS-2 and leader codon 6. This region of the cat-86 leader contains an eight-nucleotide sequence (conserved region I) that is largely conserved among all known cat leaders. The codon immediately 5' to conserved region I differs, however, between amicetin-inducible and amicetin-noninducible cat genes. In amicetin-inducible cat genes such as cat-86, the codon 5' to conserved region I is a valine codon, GTG. The same codon in amicetin-noninducible cat genes is a lysine codon, either AAA or AAG. When the GTG codon immediately 5' to conserved region I in cat-86 was changed to AAA, amicetin was no longer active in cat-86 induction, but chloramphenicol induction was unaffected by the mutation. The potential role of the GTG codon in amicetin induction is discussed.