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The chick is a widely used system for study of the actions of muscarinic acetylcholine receptors in the cardiovascular, visual, and nervous systems. We report the isolation and functional analysis of the gene encoding the chick M5 muscarinic receptor. RT-PCR analysis indicates that the M5 receptor is expressed at low levels in embryonic chick brain and heart. When expressed in stably transfected Chinese hamster ovary cells, the M5 receptor exhibits high-affinity binding to muscarinic antagonists and mediates robust activation of phospholipase C activity.

The regulation of expression and function of the muscarinic acetylcholine receptor has been studied using several different systems. The role of glycosylation of the m2 receptor was examined by removal of glycosylation sites using site-directed mutagenesis followed by expression in stably transfected cells. The results demonstrated that glycosylation was not required for the synthesis and appearance of the receptors on the cell surface or for the coupling of the receptors to inhibition of adenylyl cyclase activity. Site-directed mutagenesis also was used to demonstrate that the single cysteine in the carboxy terminal domain of the m2 receptor was not required for receptor function, thus rendering unlikely a model suggesting a requirement for palmitoylation of this cysteine in receptor function. The muscarinic receptors expressed in embryonic chick heart were identified by molecular cloning. Two genes were initially identified which are expressed in chick heart and correspond to the chick m2 and m4 receptors. Experiments using the polymerase chain reaction to identify low abundance mRNAs indicate that at least one addition receptor gene is expressed in chick heart. In cell culture, activation of the muscarinic receptors decreases the levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription due to both mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. The elucidation of the factors which regulate the expression and function of muscarinic acetylcholine receptors (mAChR) is of obvious importance in understanding the mechanisms underlying cholinergic transmission. In this chapter, we will describe studies on the expression and function of wild type and mutant muscarinic receptors, the molecular characterization of mAChR expressed in chick heart, and the regulation of mAChR gene expression in response to muscarinic receptor activation.

Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors which are highly conserved across mammalian species. Chick cardiac mAChR, however, have been shown to be pharmacologically, immunologically, and biochemically distinct from m2 mAChR expressed in mammalian heart. We previously reported the isolation and characterization of a novel chicken mAChR, cm4, which is expressed in chick heart and brain. We report here the isolation of an additional chicken mAChR gene whose deduced amino acid sequence is most homologous to the mammalian m2 receptor. Northern blot analysis demonstrated that this chicken m2 gene is also expressed in chick heart and brain. When stably transfected into Chinese hamster ovary (CHO) cells and Y1 adrenal carcinoma cells, the chicken m2 gene expresses a receptor protein which exhibits high affinity binding for the muscarinic antagonist quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the chick m2 receptor has pharmacological properties that are similar to the chick m4 receptor as well as those reported for endogenous mAChR in chick cardiac cells. Consistent with the properties of the chick m4, as well as mammalian m2 and m4 receptors, the chick m2 receptor was able to functionally couple to both the inhibition of adenylate cyclase and the stimulation of phosphoinositide metabolism when expressed in CHO cells, but only the inhibition of adenylate cyclase when expressed in Y1 cells. We conclude from this study that the embryonic chick heart expresses multiple subtypes of mAChR which are highly conserved with their mammalian counterparts. Furthermore, the high degree of conservation between the mammalian m2 and the chick m2 muscarinic receptors suggests that the pharmacological differences that exist between these receptors are due to a relatively small number of specific amino acid changes rather than larger changes in receptor sequence or structure.

Previous studies have demonstrated that muscarinic acetylcholine receptors (mAChR) expressed in chick heart are pharmacologically, immunologically, and biochemically distinct from mAChR expressed in mammalian heart. A chicken genomic clone encoding a mAChR whose deduced amino acid sequence is most homologous to the mammalian m4 receptor has been isolated. Northern blot analysis demonstrated that this gene is expressed in both chick heart and brain. The receptor encoded by this gene was expressed in stably transfected Chinese hamster ovary (CHO) and Y1 adrenal carcinoma cells in order to examine its ligand binding and functional properties. The receptor expressed in CHO and Y1 cells exhibits high affinity binding for the muscarinic antagonists quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the cloned chick m4 receptor exhibits pharmacological properties similar to those previously reported for the chick cardiac receptor. This m4 receptor was able to mediate both agonist-dependent inhibition of forskolin-stimulated cAMP accumulation and agonist-dependent stimulation of phosphoinositide metabolism when expressed in CHO cells. In contrast, when expressed in Y1 cells, the chick m4 receptor mediated agonist-dependent inhibition of forskolin-stimulated cAMP accumulation, but not stimulation of phosphoinositide metabolism. Thus, as with the mammalian cardiac (m2) receptor, the functional specificity of the chick cardiac receptor appears to be dependent on the cell type in which it is expressed.

The expression and developmental regulation of the alpha and beta subunits of the guanine nucleotide binding regulatory proteins, Gi and Go, were examined in rat atria and ventricles. Protein levels were determined by quantitative immunoblot analysis using affinity purified monospecific antibodies. Northern blot and dot blot analyses were used to characterize and quantitate relative amounts of mRNA encoding these G protein subunits. The concentrations of Go alpha, Gi alpha, and beta subunit protein were found to be greater in adult atrial than in adult ventricular membranes (5.2-, 1.5-, and 2.8-fold, respectively). A corresponding 3.4-fold difference in Go alpha mRNA level was also observed, as well as a 1.3-fold difference in Gi alpha-3 mRNA level. No difference was seen between the amount of beta, Gi alpha-1, Gi alpha-2 mRNA in adult atria and adult ventricles. Comparison of neonatal and adult tissues revealed a developmental decrease in ventricular Gi alpha protein and Gi alpha-2 mRNA levels (70 and 47%, respectively). Developmental decreases were also observed in the amount of mRNA encoding beta and Go alpha in ventricles (47 and 61%, respectively), and beta and Gi alpha-2 in atria (40 and 36%, respectively), while a developmental increase in atrial Gi alpha-3 mRNA levels was observed (57%). These results demonstrate differences in the expression of G protein subunits in rat atria and ventricles, as well as regulation of the levels of these subunits during cardiac development.

The regulation of the number and function of the muscarinic receptors has been investigated in cultured chick cardiac cells and in cells expressing cloned genes encoding mammalian, Drosophila, and chick muscarinic receptors. A serum-free defined medium for the culture of chick embryonic heart cells has been used to study the regulation of mAChR number and function by serum lipoproteins. Addition of rooster high density lipoprotein to the culture medium results in an attenuation of muscarinic receptor-mediated inhibition of cAMP accumulation without a change in the number of receptors or inhibitory G proteins. Clones encoding the mouse m1 receptor and a homologous receptor from Drosophila have been isolated. When expressed in Y1 adrenal cells, both receptors stimulate phosphoinositide hydrolysis but do not inhibit cAMP accumulation. Deletion of 123 out of the 156 amino acids in the third cytoplasmic loop of the mouse m1 receptor does not impair its ability to stimulate phosphoinositide hydrolysis. A genomic clone encoding a muscarinic receptor expressed in chick heart has been isolated. When expressed in Y1 cells, it causes inhibition of cAMP accumulation but does not stimulate phosphoinositide hydrolysis.

Muscarinic acetylcholine receptors belong to the G protein-coupled receptor superfamily and are widely known to mediate numerous functions within the central and peripheral nervous system. Thus, they have become attractive therapeutic targets for various disorders. It has long been known that the parasympathetic system, governed by acetylcholine, plays an essential role in regulating cardiovascular function. Unfortunately, due to the lack of pharmacologic selectivity for any one muscarinic receptor, there was a minimal understanding of their distribution and function within this region. However, in recent years, advancements in research have led to the generation of knockout animal models, better antibodies, and more selective ligands enabling a more thorough understanding of the unique role muscarinic receptors play in the cardiovascular system. These advances have shown muscarinic receptor 2 is no longer the only functional subtype found within the heart and muscarinic receptors 1 and 3 mediate both dilation and constriction in the vasculature. Although muscarinic receptors 4 and 5 are still not well characterized in the cardiovascular system, the recent generation of knockout animal models will hopefully generate a better understanding of their function. This mini review aims to summarize recent findings and advances of muscarinic involvement in the cardiovascular system. [ABSTRACT FROM AUTHOR]

Background Translational medicine is attracting much attention worldwide and many translational research organizations (TROs) have been established. In China, translational medicine has developed rapidly, but faces many challenges. This study was aimed at exploring these challenges faced by emerging TROs in China. Method A qualitative, multiple case study approach was used to assess the challenges faced by TROs in China. Data were collected between May and August 2012. Results Eight cases were identified. Overall, four themes that characterized TROs in China emerged from analyses: 1. objectives, organizer, and funding resources, 2. participating partners and research teams, 3. management, and 4. achievements. All TROs had objectives related to translating basic discovery to clinic treatment and cultivating translational researchers. In terms of organizer and funding resources, 7 out of 8 TROs were launched only by universities and/or hospitals, and funded mostly through research grants. As for participating partners and multidisciplinary research teams, all but one of the TROs only involved biomedical research institutions who were interested in translational research, and characterized as clinical research centers; 7 out of 8 TROs involved only researchers from biomedicine and clinical disciplines and none involved disciplines related to education, ethnicity, and sociology, or engaged the community. Current management of the TROs were generally nested within the traditional research management paradigms, and failed to adapt to the tenets of translational research. Half of the TROs were at developmental stages defined as infrastructure construction and recruitment of translational researchers. Conclusions TROs in China face the challenge of attracting sustainable funding sources, widening multidisciplinary cooperation, cultivating multi-disciplinary translational researchers and adapting current research management to translational research. Greater emphasis should be placed on increasing multidisciplinary cooperation, and innovating in education programs to cultivate of translational researchers. Efforts should be made to reform research management in TROs, and establish sustainable funding resources. [ABSTRACT FROM AUTHOR]

In the following point-counterpoint article, internationally-acclaimed myopia researchers were challenged to defend the two opposing sides of the topic defined by the title; their contributions, which appear in the order, Point followed by Counterpoint, were peer-reviewed by both the editorial team and an external reviewer. Independently of the invited authors, the named member of the editorial team provided an Introduction and Summary, both of which were reviewed by the other members of the editorial team. By their nature, views expressed in each section of the Point-Counterpoint article are those of the author concerned and may not reflect the views of all of the authors. [ABSTRACT FROM AUTHOR]

Citation information: McBrien NA, Arumugam B, Gentle A, Chow A & Sahebjada S. The M4 muscarinic antagonist MT-3 inhibits myopia in chick: evidence for site of action. Ophthalmic Physiol Opt 2011, 31, 529-539. doi:10.1111/j.1475-1313.2011.00841.x Abstract Purpose: It is well established that the broad-band muscarinic antagonist, atropine is effective at inhibiting the progression of myopia and does so by preventing the elongation of the vitreous chamber of the eye. However, uncertainty remains as to whether this effect occurs through a receptoral mechanism and, if so, which muscarinic receptor subtype mediates this effect. Previous work, in avian and mammalian models of myopia, implicates the M1 and M4 receptors as potential targets. The current study used physiologically relevant concentrations of highly selective muscarinic antagonists (MT-3 and MT-7) to further characterise the role of the M4 receptor in the control of myopia in the chick model of refractive development. Methods: Nine groups of week-old chicks underwent 5 days of monocular deprivation, with a translucent occluder, to induce myopia. These animals had either no injection, scleral puncture with a needle, or daily intravitreal injections of MT-3 (M4-selective), MT-7 (M1-selective) or vehicle. Three concentrations of each antagonist were delivered (250 n m, 2.5 μ m and 10 μ m). After the treatment period, keratometry, retinoscopy and A-Scan ultrasound were used to assess ocular biometry. Results: MT-3 treatment produced a significant dose-dependent reduction in relative myopia (treated−control eye) compared to vehicle treatment (vehicle −10.1 ± 1.1 D vs 10 μ m MT-3 −4.0 ± 1.5 D, p < 0.01). The majority of this effect was due to reduced relative vitreous chamber elongation in drug treated eyes (vehicle +0.26 ± 0.04 mm, 10 μ m MT-3 +0.08 ± 0.07 mm, p < 0.05). In contrast, MT-7 had no significant effect on the development of myopia (MT-7 10 μ m: myopia, −12.1 ± 0.8 D and vitreous chamber depth, +0.23 ± 0.07 mm). Calculations indicate that the experimentally achieved concentrations of MT-3 at intraocular receptors necessary to inhibit 50% of myopia development (between 5 and 50 n m) were consistent with published in vitro affinity constants for the M4 receptor and below those for the M1 receptor. Histology demonstrated that MT-3 at the doses used had no gross effects on the retina, indicating a non-toxic mode of action. Conclusions: In the chick, which lacks a homologue of the mammalian M1 receptor, the above findings represent compelling evidence that muscarinic antagonists prevent myopia progression through an M4-receptor mediated mechanism, most likely located in the retina. [ABSTRACT FROM AUTHOR]

Vagal control of heart rate (HR) is mediated by direct and indirect actions of ACh. Direct action of ACh activates the muscarinic K+ (KACh) channels, whereas indirect action inhibits adenylyl cyclase. The role of the KACh channels in the overall picture of vagal HR control remains to be elucidated. We examined the role of the KACh channels in the transfer characteristics of the HR response to vagal stimulation. In nine anesthetized sino-aortic-denerved and vagotomized rabbits, the vagal nerve was stimulated with a binary white-noise signal (0-10 Hz) for examination of the dynamic characteristic and in a step-wise manner (5, 10, 15, and 20 Hz/mm) for examination of the static characteristic. The dynamic transfer function from vagal stimulation to HR approximated a first- order, low-pass filter with a lag time. Tertiapin, a selective KACh channel blocker (30 nmol/kg iv), significantly decreased the dynamic gain from 5.0 ± 1.2 to 2.0 ± 0.6 (mean ± SD) beats·min-1·Hz-1 (P < 0.01) and the corner frequency from 0.25 ± 0.03 to 0.06 ± 0.01 Hz (P < 0.01) without changing the lag time (0.37 ± 0.04 vs. 0.39 ± 0.05 s). Moreover, tertiapin significantly attenuated the vagal stimulation-induced HR decrease by 46 ± 21, 58 ± 18, 65 ± 15, and 68 ± 11% at stimulus frequencies of 5, 10, 15, and 20 Hz, respectively. We conclude that KACh channels contribute to a rapid HR change and to a larger decrease in the steady-state HR in response to more potent tonic vagal stimulation. [ABSTRACT FROM AUTHOR]

Background: In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. Results: The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4- DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Conclusions: Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion. [ABSTRACT FROM AUTHOR]

Glucocorticoids affect the expression and density of neurotransmitter receptors in many tissues but data concerning the heart are contradictory and incomplete. We injected rats with hydrocortisone for 1–12 days and measured the densities of cardiac muscarinic receptors, α1-, β1- and β2-adrenoceptors and propranolol-resistant binding sites (formerly assumed to be the putative β4-adrenoceptor). Some aspects of intracellular signalling were also evaluated: we measured adenylyl cyclase activity (basal, isoprenaline- and forskolin-stimulated and carbachol-inhibited), the coupling between muscarinic receptors and G proteins and basal and isoprenaline-stimulated heart rate. The density of cardiac muscarinic receptors increased (in both the atria and the ventricles). The density of β1-adrenoceptors increased in the atria and was little changed in the ventricles. The density of β2-adrenoceptors increased in both the atria and the ventricles. The number of α1-adrenoceptors decreased initially, followed by a transient increase in the atria and did not change in the ventricles. The density of propranolol-resistant binding sites first increased and then diminished in the atria and did not change in the ventricles. Although there were noticeable changes in receptor densities, the stimulatory and inhibitory effects on adenylyl cyclase, basal and isoprenaline-stimulated heart rate and the coupling between muscarinic receptors and G proteins were not significantly altered. This may indicate that changes in receptor densities might be one of the mechanisms maintaining stable functional output. [ABSTRACT FROM AUTHOR]

1 A zebrafish M[sub 2] muscarinic acetylcholine receptor (mAChR) gene was cloned. It encodes 495 amino acids in a single exon. The derived amino acid sequence is 73.5% identical to its human homologue. 2 Competitive binding studies of the zebrafish M[sub 2] receptor and [³H]-NMS gave negative log dissociation constants (pK[sub i]) for each antagonist as follows: atropine (9.16)>himbacine (8.05)≥4DAMP (7.83)>AF-DX 116 (7.26)≥pirenzepine (7.18)≥tropicamide (6.97)≥methoctramine (6.82)≥p-F-HHSiD (6.67)>carbachol (5.20). The antagonist affinity profile correlated with the profile of the human M[sub 2] receptor, except for pirenzepine. 3 Reverse transcription polymerase chain reaction and Southern blotting analysis demonstrated that the M[sub 2] mAChR mRNA levels increased during the segmentation period (12 h post-fertilization; h.p.f.) in zebrafish. By whole-mount in situ hybridization, the M[sub 2] mAChR was first detectable in the heart, vagus motor ganglion, and vagus sensory ganglion at 30, 48 and 60 h.p.f., respectively. 4 The muscarinic receptor that mediates carbachol (CCh)-induced bradycardia was functionally mature at 72 h.p.f. The effect of CCh-induced bradycardia was antagonized by several muscarinic receptor antagonists with the order of potency (pIC[sub 50] values): atropine (6.76)>methoctramine (6.47)>himbacine (6.10)>4-DAMP (5.72)>AF-DX 116 (4.77), however, not by pirenzepine, p-FHHSiD, or tropicamide (< 10 µM). 5 The effect of CCh-induced bradycardia was abolished completely before 56 h.p.f, by M[sub 2] RNA interference, and the bradycardia effect gradually recovered after 72 h.p.f. The basal heart rate was increased in embryos injected with M[sub 2] mAChR morpholino antisense oligonucleotide (M[sub 2] MO) and the effect of CCh-induced bradycardia was abolished by M[sub 2] MO in a dose-dependent manner. In conclusion, the results suggest that the M[sub 2] mAChR inhibit basal heart rate in zebrafish embryo and the M[sub 2]... [ABSTRACT FROM AUTHOR]

The β-adrenoceptor (β-AR) mediated signal transduction pathway in cardiomyocytes is known to involve β1- and β2-ARs, stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). The activation of β1- and β2-ARs has been shown to increase heart function by increasing Ca2+-movements across the sarcolemmal membrane and sarcoplasmic reticulum through the stimulation of Gs-proteins, activation of AC and PKA enzymes and phosphorylation of the target sites. The activation of PKA has also been reported to increase phosphorylation of some myofibrillar proteins (for promoting cardiac relaxation) and nuclear proteins (for cardiac hypertrophy). The activation of β2-AR has also been shown to affect Gi-proteins, stimulate mitogen activated protein kinase and increase protein synthesis by enhancing gene expression. β1- and β2-ARs as well as AC are considered to be regulated by PKA- and protein kinase C (PKC)-mediated phosphorylations directly; both PKA and PKC also regulate β-AR indirectly through the involvement of β-AR kinase (βARK), β-arrestins and Gβγ-protein subunits. Genetic manipulation of different components and regulators of β-AR signal transduction pathway by employing transgenic and knockout mouse models has provided insight into their functional and regulatory characteristics in cardiomyocytes. The genetic studies have also helped in understanding the pathophysiological role of βARK in heart dysfunction and therapeutic role of βARK inhibitors in the treatment of heart failure. Varying degrees of defects in the β-AR signal transduction system have been identified in different types of heart failure to explain the attenuated response of the failing heart to sympathetic stimulation or catecholamine infusion. A decrease in β1-AR density, an increase in the level of Gi-proteins and overexpression of βARK are usually associated with heart failure; however, these attenuations have been shown to be dependent upon the type and stage of heart failure as well as region of the heart. Both local and circulating renin-angiotensin systems, sympathetic nervous system and endothelial cell function appears to regulate the status of β-AR signal transduction pathway in the failing heart. Thus different components and regulators of the β-AR signal transduction pathway appears to represent important targets for the development of therapeutic interventions for the treatment of heart failure. [ABSTRACT FROM AUTHOR]

The structural basis for the selectivity of the antagonist UH-AH 37 at human muscarinic acetylcholine receptors was investigated by expressing mutant receptors in COS-7 cells. Previous studies have demonstrated that the interaction between UH-AH 37 and [[sup 3] H]N-methylscopolamine in equilibrium assays is competitive and that the high affinity of UH-AH 37 for the M[sub 5] subtype, compared to M[sub 2] , is due to an epitope in the sixth transmembrane domain (TM6) or the third outer loop of the receptor. By mutating each nonconserved residue in this region of M[sub 2] and M[sub 5] to its counterpart in the other receptor, we identified a threonine residue in the middle of TM6 uniquely responsible for the higher affinity of the M[sub 5] receptor (M[sub 1] , M[sub 3] , and M[sub 4] receptors also carry a threonine at that location and also have high affinity for UH-AH 37). The mutant receptor in which the corresponding alanine of the M[sub 2] receptor was replaced by threonine, M[sub 2] [sup 401] ala ⇒ thr, expressed enhanced affinity for pirenzepine as well as for UH-AH 37. The chick M[sub 2] receptor, which expresses anomalously high affinity for pirenzepine, differs from its mammalian counterparts by the presence of a threonine at this position. Affinities of AF-DX 116 and 4-DAMP, as well as the allosteric potency of UH-AH 37, were not sensitive to the M[sub 2] [sup 401] ala ⇒ thr mutation.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The selective α-adrenergic agonist methoxamine (10-10M), in the presence of propranolol (10M), can reduce both the inwardly rectifying K background current ( I) and the muscarinic cholinergic receptor-activated K current ( I) in rabbit atrial myocytes resulting in action potential prolongation during the final phase of repolarization and a depolarization of the resting membrane potential. The reduction of these K current(s) by α-adrenoceptor stimulation was insensitive to pre-treatment of artial myocytes with pertussis toxin (0.15-0.5 μg/ml) and was irreversible following intracellular dialysis with the non-hydrolysable guanosine triphosphate (GTP) analogue, Gpp(NH)p (1−5×10M). Neither the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulphonyl)-2-methylpiperoxine (H-7) (5×10M) and staurosporine (1×10M), nor 'downregulation' of PKC by prolonged phorbol ester exposure (5×10M, for 7-8 h) had an effect on the α-adrenergic modulation of this K current. Under cellattached patch-clamp conditions, bath application of methoxamine reversibly decreased acetylcholine-induced single-channel activity, thus confirming the observed reduction of the ACh-induced current under whole-cell voltage clamp. These results demonstrate that the αadrenoceptor, once activated, can reduce current through two different inwardly rectifying K channels in rabbit atrial myocytes. These current changes are mediated via a pertussis toxin-insensitive GTP-binding protein, and do not appear to involve the activation of PKC. [ABSTRACT FROM AUTHOR]

We have investigated the signalling properties of the naturally occuring intercellular signalling molecule lysophosphatidic acid (LPA) in primary human skin fibroblasts. LPA stimulated phospholipase C activity resulting in the formation of inositol 1,4,5-trisphosphate (IP) which was accompanied by a concentration-dependent increase in intracelluar calcium concentration ([Ca]). The increase in [Ca] was subject to homologous desensitisation but not to heterologous desensitisation by sphingosine-1-phosphate. The half-maximal effect of LPA on the rise in [Ca] was attained at 7-20 nM. IP formation and Ca mobilisation were highly pertussis toxin (PTX)-sensitive (100% and 75%, respectively). LPA also inhibited forskolin-stimulated formation of cAMP, which was partially reversed (51%) when fibroblasts were pretreated with PTX. To directly test the involvement of guanine nucleotide-binding regulatory proteins (G proteins), LPA-induced binding of the stable GTP analogue GTPγS was measured. LPA induced an increase in GTPγS binding, which was completely inhibited by PTX, implicating the involvement of G-type G proteins in LPA signalling. Furthermore, LPA increased DNA synthesis and cell proliferation. Finally, LPA induced the migration of human skin fibroblasts, which in conjunction with the stimulation of cell growth strengthens the presumed involvement of LPA in wound healing and tissue regeneration. Both effects (cell growth and migration) were almost completely PTX-sensitive. Overall, these investigations in primary cultures of human skin fibroblasts confirm and extend our knowledge about LPA signalling, suggesting a pivotal role of receptor coupled activation of G-type proteins at least in this cell type. [ABSTRACT FROM AUTHOR]

The postnatal alterations of the composition of a subunit isoforms (G, G G, and of G proteins, the adenylyl cyclase activity as well as of cAMP-regulated phosphoproteins e.g. troponin and phospholamban were investigated in the ventricular tissue of 1, 7, 30 days old rats. Quantitative immunodetection revealed a 5.7-fold decrease in G at 30th postnatal day compared with the postnatal day 1 and up to 15-fold at 4 months. The amounts of G and G as well as the G subunits were found to be higher in the earlier life period compared to the adult. In contrast, the content of G was uneffected by the developmental state. Basal adenylyl cyclase activity (pmoles cAMP/min × mg protein) increased from 30.9 ± 5.0, 36.8 ± 5.0 to 63.9 ± 5.9 at 1st, 7th and 30th postnatal day, respectively. Isoprenaline (100 μM) enhanced the activity of adenylyl cyclase from day 1, 7-30 from 46.2 ± 7.0, 79.1 ± 9.2 to 120.5 ± 7.2, respectively. The effects of forskolin and NaF on adenylyl cyclase activity was found to be not influenced within the first postnatal month. Furthermore, a developmentally controlled expression of cardiac troponin I was observed (6-fold from the first to the 28th postnatal day) whereas the level of phospholamban was found to be age-independent. In conclusion, there is an increase in the efficiency of the β-adrenergic signal transfer mainly caused by a reduction of the inhibitiory G proteins and a dominance of the G-linked pathway in the postnatal rat heart. Furthermore the developmentally controlled expression of troponin I might be of functional importance in the cAMP-supported relaxation. Additionally, altered G, G and G pattern of the developing rat ventricle may play a role in the observed change of α-adrenerg-mediated heart contractility as well as in cardiac differentiation and growth processes. [ABSTRACT FROM AUTHOR]