Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The modulation of the rat cortical m1 muscarinic receptor mRNA was studied with a method of quantitation using the polymerase chain reaction after conversion to complementary DNA (cDNA) with AMV reverse transcriptase (RT/PCR). Primers specific to the C3 region of the m1 mRNA were employed. The y-intercepts from the linear regions of semilogarithmic plots of PCR product versus cycle number were used as measures of the levels of m1 muscarinic mRNA-measured relative to that of glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA (the 'ratio' method). Alternatively, m1 mRNA in total cortical RNA samples was quantitated from the increase in product elicited by adding a known amount of exogenous m1 muscarinic cDNA sequence (the 'spiking' method). This allowed calculation of absolute level of m1 mRNA, which was 4.1 pg/micrograms total RNA. We measured the level of the rat cortical m1 mRNA after 1 week of chronic receptor blockade with atropine, showing upregulation of 154% by the GAPDH/m1 ratio method and 145% by the spiking method. That this transcriptional alteration was specific was indicated by the finding that the level of GAP-43 mRNA was not affected by atropine treatment.
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PMID:Chronic atropine administration up-regulates rat cortical muscarinic m1 receptor mRNA molecules: assessment with the RT/PCR. 137 72

Five muscarinic receptor genes (m1-m5) that encode distinct muscarinic receptor subtypes have been cloned. Because of their structural homology and pharmacological similarity, ligand binding probes currently available do not clearly distinguish among the subtypes. To obtain a clear distribution within the CNS of molecularly defined muscarinic receptor subtypes, seven brain regions were examined for the expression of the respective mRNAs. The most sensitive method for detecting mRNA is through amplification of the respective cDNAs. Brain regions were obtained from male Wistar rats, and total RNA was isolated. The isolates were extensively treated with RNase-free DNase to remove any residual genomic DNA. Total RNA (1 microgram) was reverse-transcribed using random primers and reverse transcriptase. The resulting cDNA was amplified using a thermal cycler, and the polymerase chain reaction (PCR)-amplified products were analyzed by gel electrophoresis containing ethidium bromide and visualized with fluorescent illumination. PCR-amplified samples were also injected directly onto an HPLC anion exchange column and quantified by UV detection. Each of the five muscarinic subtypes was found in every brain region examined. The m1 subtype was most abundant in cortex and gradually declined in content caudally to the spinal cord. The m2 subtype was most abundant in thalamus-hypothalamus and ponsmedulla. The m4 subtype was found in greatest amount in the striatum, whereas m3 and m5 were expressed consistently throughout the CNS. The combination of RT-PCR and HPLC provides a rapid and sensitive method for quantifying the expression of mRNA coding for all five muscarinic receptor subtypes derived from the CNS.
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PMID:m1-m5 muscarinic receptor distribution in rat CNS by RT-PCR and HPLC. 751 60

The aim was to explore whether biliary epithelial cells show muscarinic acetylcholine receptors and to investigate their role in ductular bile formation. In both, isolated rat biliary epithelial cells and Mz-Cha-1 cells, a biliary epithelial cell line, binding of [3H]N-methyl-scopolamine occurred with 0.718 +/- 0.08 and 0.482 +/- 0.05 fmol per 10(6) cells, respectively. To characterize the involved second messenger, intracellular Ca2+ levels were monitored by confocal microscopy. Stimulation of biliary epithelial cells with carbachol produced an increase in free cytosolic Ca2+ levels that declined to baseline values describing a sinusoidal oscillation curve. Increasing concentrations of the agonist decreased latency of the response and increased oscillation frequency. Similar results were obtained in Mz-Cha-1 cells. The intracellular Ca2+ originated from IP3 sensitive intracellular stores and from the extracellular medium. The Ca2+ response could partially be blocked by atropine and completely by pirenzepine, a specific muscarinic receptor-type M1 antagonist. The presence of M1 receptor messenger RNA (mRNA) in biliary epithelial cells was confirmed by reverse transcriptase polymerase chain reaction. In the isolated perfused guinea pig liver, a model with high ductular bile flow, carbachol induced a dose dependent decrease of bile flow by 79.6% +/- 9.8% at 50 mumol/L carbachol (P < .001), without affecting perfusion pressure or biliary electrolyte concentrations. It is concluded that biliary epithelial cells express muscarinic acetylcholine receptors. Stimulation of this receptor leads to cholestasis. This could be because of changes in peribiliary permeability and/or inhibition of biliary epithelial cell secretory function.
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PMID:Muscarinic acetylcholine receptor stimulation of biliary epithelial cells and its effect on bile secretion in the isolated perfused liver [corrected]. 909 80

Expression of muscarinic receptor subtypes in rat gastric smooth muscle was examined with reverse transcriptase-polymerase chain reaction (RT-PCR). Under the condition for detecting the messages of m1-m4 subtypes in brain, atrium, and gastric mucosa, only the fragments of m2 and m3 subtypes were amplified with RNA prepared from rat gastric smooth muscles. Furthermore, the amplified fragments were digested by restriction enzymes, reconfirming that the predicted size products of m2 and m3 contain the partial DNA sequences of m2 and m3 subtypes, respectively. We measured gastric motility in rats with a pressure transducer system under the continuous venous infusion of the muscarinic antagonists atropine and butylscopolamine (nonselective), AF-DX 116 (M2), zamifenacine (M3), and glucagon. Heart rate was monitored simultaneously in the tail. Gastric motility was inhibited in the presence of glucagon and zamifenacine without alteration of heart rate, whereas there was no inhibition in the presence of AF-DX 116 even after the augmentation of heart rate was observed. Gastric emptying was also suppressed in the presence of zamifenacine, which had an effect comparable with that of atropine, butylscopolamine, and glucagon. These results indicate that the activation of the M3 subtype in gastric smooth muscle causes its contraction, and the M3 selective antagonist could be a potentially useful drug without an adverse effect on the heart for radiological and endoscopic examination in the upper gastrointestinal tract.
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PMID:Expression of muscarinic receptor subtypes in rat gastric smooth muscle: effect of M3 selective antagonist on gastric motility and emptying. 914 41

A significant body of evidence suggests that the development and maintenance of elevated blood pressure in the spontaneously hypertensive rat (SHR), a genetic model for essential hypertension, is due at least partly to a central hyper-cholinergic state. For example, this strain responds with an exaggerated pressor response to pharmacological stimulation of central muscarinic receptors in certain brain regions compared to normotensive Wistar Kyoto rats (WKY). At least part of the enhanced response to central muscarinic receptor stimulation in SHR is due to the altered expression of post-synaptic receptors. In the present study, the reverse transcriptase-polymerase chain reaction and autoradiographic techniques were used to estimate the relative levels of mRNA and density of receptor binding sites for the five subtypes of muscarinic receptors within the rostral ventrolateral medulla (RVL) of SHR and WKY. Adult (12-week-old) SHR exhibited an increase in the levels of both M2 muscarinic mRNA, and M2 receptor binding sites in RVL compared to age-matched normotensive WKY. Similarly, 4-week-old pre-hypertensive SHR exhibited increased levels of M2 mRNA in whole medulla oblongata, and an increase in the number of binding sites for M2 receptors in the RVL. Since the RVL is known to integrate tonic cholinergic sympathoexcitatory input, these results suggest that the increased expression of M2 muscarinic receptors in this region represents one neurochemical correlate for the maintenance of excessive central efferent sympathetic nervous activity in the SHR. Since the neurochemical change precedes the development of hypertension, the altered medullary M2 receptor expression may play a role as an initiating or predisposing factor for the development of hypertension in SHR.
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PMID:Increased expression of M2 muscarinic receptor mRNA and binding sites in the rostral ventrolateral medulla of spontaneously hypertensive rats. 918 22

1. We have previously shown that nitric oxide (NO) production is essential for cholinergic inhibition of the beta-adrenergic stimulated L-type calcium current (ICa-L) in rabbit pacemaker (sino-atrial node (SAN)) cells. The present experiments demonstrate the presence of constitutive nitric oxide synthase (cNOS) in SAN cells, and characterize the NO-mediated cholinergic response. 2. Immunohistochemical staining, using an antibody prepared against endothelial cNOS, demonstrated that this enzyme was present in single myocytes obtained from the SAN. 3. The activation of cNOS is known to be Ca2+ and calmodulin dependent. Strongly buffering intracellular Ca2+ with the membrane-permeable chelator BAPTA-AM (10 microM) significantly reduced (and in some cases abolished) the attenuation of ICa-L by the muscarinic agonist carbamylcholine (CCh). In contrast, the CCh-induced activation of an outward K+ current, IK,ACh, was unaffected by buffering of [Ca2+]i. The calmodulin inhibitor 48/80 (20 microM) also abolished the attenuation of ICa-L by CCh, with no change in the activation of IK,ACh. 4. Neither thapsigargin nor ryanodine (5-10 microM), agents which deplete intracellular Ca2+ stores, significantly changed the attenuation of ICa-L by CCh. 5. Pertussis toxin (PTX) completely abolished both the inhibitory action of CCh on ICa-L and the activation of IK,ACh. This establishes that a PTX-sensitive GTP-binding protein links the muscarinic receptor to NO synthase activation in SAN cells. 6. Our hypothesis is that NO leads to activation of a cyclic GMP (cGMP)-activated phosphodiesterase (PDE II) as a mechanism for enhanced cyclic AMP breakdown and ICa-L attenuation. This was supported by showing that a specific inhibitor of PDE II, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), blocks the effect of CCh on ICa-L, but not on IK,ACh. Using reverse transcriptase-polymerase chain reaction techniques, we have established that PDE II is the dominant cyclic nucleotide phosphodiesterase isoform in SAN cells.
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PMID:Characteristics of nitric oxide-mediated cholinergic modulation of calcium current in rabbit sino-atrial node. 959 96

Experimental traumatic brain injury (TBI) produces cholinergic neurotransmission deficits that may contribute to chronic spatial memory deficits. Cholinergic neurotransmission deficits may result from presynaptic alterations in the storage and release of acetylcholine (ACh) or from changes in the receptors for ACh. The vesicular ACh transporter (VAChT) mediates accumulation of ACh into secretory vesicles, and the M2 muscarinic receptor subtype can modulate cholinergic neurotransmission via a presynaptic inhibitory feedback mechanism. We examined the effects of controlled cortical impact (CCI) injury on hippocampal VAChT and M2 muscarinic receptor subtype protein and medial septal mRNA levels at 4 weeks following injury. Rats were anesthetized and surgically prepared for CCI injury (4 m/sec, 2.5 to 2.9 mm in depth) and sham surgery. Animals were sacrificed, and coronal sections (35 microm thick) were cut through the dorsal hippocampus for VAChT and M2 immunohistochemistry. Semiquantitative measurements of VAChT and M2 protein in hippocampal homogenates from injured and sham rats were assessed with Western blot analysis. Changes in VAChT and M2 mRNA levels were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). At 4 weeks after injury, both immunohistochemical and Western blot methods demonstrated an increase in hippocampal VAChT protein. An increase in VAChT mRNA was also observed. Immunohistochemistry demonstrated a loss of M2; however, there was no significant change in M2 mRNA levels in comparison with sham controls. These changes may represent a compensatory response of cholinergic neurons to increase the efficiency of ACh neurotransmission chronically after TBI through differential transcriptional regulation.
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PMID:Differential effects of traumatic brain injury on vesicular acetylcholine transporter and M2 muscarinic receptor mRNA and protein in rat. 1044 68

Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.
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PMID:Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation. 1076 52

Expression of muscarinic receptors in rat islets, RINm5F cells, and INS-1 cells was established by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantified by RNase protection. Both methods indicated that m3 and m1 receptors were expressed approximately equally in the various cellular preparations and to a much greater extent than the m5 subtype. However, the cell lines, especially RINm5F cells, expressed less of a given receptor subtype than did islets. Immunohistochemistry indicated that m3 receptors were expressed throughout the islet core. Binding studies using the radiolabeled muscarinic receptor antagonist QNB demonstrated a maximal binding capacity of INS-1 cells of 23.0+/-2.9 fmol/mg protein. Functional analyses were undertaken using INS-1 cells stably transfected with either m1 or m3 receptor cDNAs. Overexpression of either receptor did not affect basal responses but markedly enhanced maximal responses to the muscarinic receptor agonist carbachol. Although maximal hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP2) was twofold greater in m1-transfectants as compared with m3-transfectants, cell lines overexpressing either receptor gave essentially equivalent secretory responses to a full range of carbachol doses. The results demonstrate that both m1 and m3 muscarinic receptors are well expressed in pancreatic beta-cells, functionally linked to signaling pathways, and capable of initiating insulin secretion with equal potencies.
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PMID:Quantitative and functional characterization of muscarinic receptor subtypes in insulin-secreting cell lines and rat pancreatic islets. 1086 60

Acetylcholine (ACh) produces pain when applied to human skin and excites cutaneous mechanoreceptors and nerve terminals. These effects are partially mediated by activation of muscarinic receptors. The expression of muscarinic receptor subtype M2 has been shown in sensory neurons of rat dorsal root ganglia using reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and immunohistochemistry. The purpose of the present study was to determine whether these M2 receptors are targeted to the peripheral endings of sensory neurons in the rat skin. Double-staining histochemical procedures were employed using a specific antiserum to M2 receptors combined with either of the following neuronal markers: an antiserum to the neuropeptide substance P, an antiserum to the protein gene product 9.5, which is a marker for peripheral nerve fibres, and the histochemical marker of a subpopulation of unmyelinated C-fibre afferents, I-B4, the Bandeira simplicifolia-derived isolectin. The M2 receptor subtype was found on different populations of nerve fibres. In the nerve plexus at the epidermal-dermal junction, M2 receptors are mainly present on I-B4-positive axons but are absent on fibres with substance P immunoreactivity. Sweat glands receive M2-receptor-immunoreactive fibres that express neither I-B4 binding nor substance P immunoreactivity, whereas blood vessels of the deeper dermis are innervated by I-B4-positive nerve fibres that are immunoreactive for M2 receptors and substance P. In addition to axon profiles, keratinocytes and endothelial cells also exhibit M2 receptor immunoreactivity. The results show the presence of M2 receptors in neuronal and non-neuronal cells, suggesting multiple effects of acetylcholine in the skin.
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PMID:Immunohistochemical localization of muscarinic receptors (M2) in the rat skin. 1092 69


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