Dataset Information

Synergistic Signaling by Light and Acetylcholine in Mouse Iris Sphincter Muscle.

ABSTRACT: The mammalian pupillary light reflex (PLR) involves a bilateral brain circuit whereby afferent light signals in the optic nerve ultimately drive iris-sphincter-muscle contraction via excitatory cholinergic parasympathetic innervation [1, 2]. Additionally, the PLR in nocturnal and crepuscular sub-primate mammals has a "local" component in the isolated sphincter muscle [3-5], as in amphibians, fish, and bird [6-10]. In mouse, this local PLR requires the pigment melanopsin [5], originally found in intrinsically photosensitive retinal ganglion cells (ipRGCs) [11-19]. However, melanopsin's presence and effector pathway locally in the iris remain uncertain. The sphincter muscle itself may express melanopsin [5], or its cholinergic parasympathetic innervation may be modulated by suggested intraocular axonal collaterals of ipRGCs traveling to the eye's ciliary body or even to the iris [20-22]. Here, we show that the muscarinic receptor antagonist, atropine, eliminated the effect of acetylcholine (ACh), but not of light, on isolated mouse sphincter muscle. Conversely, selective genetic deletion of melanopsin in smooth muscle mostly removed the light-induced, but not the ACh-triggered, increase in isolated sphincter muscle's tension and largely suppressed the local PLR in vivo. Thus, sphincter muscle cells are bona fide, albeit unconventional, photoreceptors. We found melanopsin expression in a small subset of mouse iris sphincter muscle cells, with the light-induced contractile signal apparently spreading through gap junctions into neighboring muscle cells. Light and ACh share a common signaling pathway in sphincter muscle. In summary, our experiments have provided details of a photosignaling process in the eye occurring entirely outside the retina.

SUBMITTER: Wang Q

PROVIDER: S-EPMC8577559 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Two forms (I and II) of phosphoinositide-specific phospholipase C (PLC) were purified from the cytosol of bovine iris sphincter by sequential chromatography on DEAE-Sepharose, EAH-Sepharose, heparin-Sepharose, Sephacryl S-200 gel filtration and Mono Q HR columns. The final step resulted in specific activities of PLC-I and PLC-II of 4.3 and 5.9 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein, which represented up to 295-fold purification compared with that of the starting supernatant. The purified enzymes were further investigated for the presence of isoenzymes and characterized for molecular mass, substrate specificity, pH, Ca2+ requirements and kinetic parameters. Using monoclonal antibodies, PLC-I was identified as PLC-delta 1. The apparent molecular mass of PLC-I as determined by SDS/PAGE and gel filtration was 85 kDa. PLC-II contained an apparently invisible protein band that reacted with the antibody against PLC-gamma 1, and a major 109 kDa protein band that was not recognized by any of the PLC monoclonal antibodies. Further purification of PLC-II by size-exclusion h.p.l.c. resulted in elution of the enzyme activity as a single peak which corresponded to 109 kDa position. Again, this PLC activity was not recognized by any of the PLC monoclonal antibodies. However, the 109 kDa protein activity was recognized by a polyclonal antibody raised against a rat PLC-gamma 1 fragment (amino acids 1272-1287), thus suggesting that this protein is a proteolytic product of PLC-gamma 1. PLC-delta 1 and PLC-gamma 1 were identified in the supernatant fraction and PLC-beta 1 in the membrane fraction of the iris sphincter. Although immunologically different, the catalytic properties of PLC-I and PLC-II were quite similar. The Vmax and Km values for phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis were three to five times greater than those for PI hydrolysis. Both forms preferred PIP and PIP2 over PI and both were inactive against phosphatidylcholine. With PIP2 as substrate, the optimal pH values for PLC-I and PLC-II were 6.5 and 7.5 respectively. Unlike PIP2, PI hydrolysis by both forms was dependent on the presence of free Ca2+. The maximal hydrolysis of PI and PIP2 by both forms occurred at 200 and 5 microM Ca2+ respectively. Incubation of the purified enzymes with the catalytic subunit of protein kinase A (PKA) and [gamma-32P]ATP resulted in increased phosphorylation of PLC-I and PLC-II, but it had no inhibitory effect on their enzyme activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Project description:1. Paired iris smooth muscles from rabbits were incubated for 30 min at 37 degrees C in an iso-osmotic salt medium containg glucose, inositol, cytidine and [32P]phosphate. 2. One of the pair was then incubated at 37 degrees C for 10 min in unlabelled medium containing 10mM-2-deoxyglucose and the other was incubated in the presence of acetylcholine plus eserine (0.05mM each). 2-Deoxyglucose, which was included in the incubation medium to minimize the biosynthesis of triphosphoinositide from ATP and diphosphoinositide, decreased the amount of labelled ATP by 71% and inhibited further 32P incorporation from ATP into triphosphoinositide by almost 30%. 3. Acetylcholine (0.05mM) increased significantly the loss of 32P from triphosphoinositide (the 'triphosphoinositide effect') in 32P-labelled iris muscle. This effect was measured both chemically and radiochemically. It was also observed when 32Pi was replaced by myo-[3H]inositol in the incubation medium. 4. The triphosphoinositide effect was blocked by atropine but not by D-tubocurarine. Further, muscarinic but not nicotinic agonists were found to provoke this effect. 5. Acetylcholine decreased by 28% the 32P incorporation into triphosphoinositide, presumably by stimulating its breakdown. This decrement in triphosphoinositide was blocked by atropine, but not by D-tubocurarine. 6. The triphosphoinositide effect was accompanied by a significant increase in 32P labelling, but not tissue concentration, of phosphatidylinositol and phosphatidic acid. The possible relationship between the loss of 32P label from triphosphoinositide in response to acetylcholine and the concomitant increase in that of phosphatidylinositol and phosphatidic acid is discussed. 7. The presence of triphosphoinositide phosphomonoesterase, the enzyme that might be stimulated in the iris smooth muscle by the neurotransmitter, was demonstrated, and, under our methods of homogenization and assay, more than 80% of its activity was localized in the particulate fraction.

Project description:We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid from membrane phospholipids. The ability of the activated endothelin-A receptor, which is coupled to both Gq- and Gi-proteins, to recruit and activate this complex signal transduction pathway remains to be elucidated. Further studies on the mechanism of these relationships could provide important information about the functions of p38 MAP kinase in smooth muscle.

Project description:1. The mechanism of acetylcholine-stimulated breakdown of phosphatidyl-myo-inositol 4,5-bisphosphate and its dependence on extracellular Ca(2+) was investigated in the rabbit iris smooth muscle. 2. Acetylcholine (50mum) increased the breakdown of phosphatidylinositol bisphosphate in [(3)H]inositol-labelled muscle by 28% and the labelling of phosphatidylinositol by 24% of that of the control. Under the same experimental conditions there was a 33 and 48% increase in the production of (3)H-labelled inositol trisphosphate and inositol monophosphate respectively. Similarly carbamoylcholine and ionophore A23187 increased the production of these water-soluble inositol phosphates. Little change was observed in the (3)H radioactivity of inositol bisphosphate. 3. Both inositol trisphosphatase and inositol monophosphatase were demonstrated in subcellular fractions of this tissue and the specific activity of the former was severalfold higher than that of the latter. 4. The acetylcholine-stimulated production of inositol trisphosphate and inositol monophosphate was inhibited by atropine (20mum), but not tubocurarine (100mum); and it was abolished by depletion of extracellular Ca(2+) with EGTA, but restored on addition of low concentrations of Ca(2+) (20mum). 5. Calcium-antagonistic agents, such as verapamil (20mum), dibenamine (20mum) or La(3+) (2mm), also abolished the production of the water-soluble inositol phosphates in response to acetylcholine. 6. Release of inositol trisphosphate from exogenous phosphatidylinositol bisphosphate by iris muscle microsomal fraction (;microsomes') was stimulated by 43% in the presence of 50mum-Ca(2+). 7. The results indicate that increased Ca(2+) influx into the iris smooth muscle by acetylcholine and ionophore A23187 markedly activates phosphatidylinositol bisphosphate phosphodiesterase and subsequently increases the production of inositol trisphosphate and its hydrolytic product inositol monophosphate. The marked increase observed in the production of inositol monophosphate could also result from Ca(2+) activation of phosphatidylinositol phosphodiesterase. However, there was no concomitant decrease in the (3)H radioactivity of this phospholipid.

Project description:BACKGROUND:Degeneration of smooth muscles in sphincters can cause debilitating diseases such as fecal incontinence. Skeletal muscle-derived cells have been effectively used in clinics for the regeneration of the skeletal muscle sphincters, such as the external anal or urinary sphincter. However, little is known about the in vitro smooth muscle differentiation potential and in vivo regenerative potential of skeletal muscle-derived cells. METHODS:Myogenic progenitor cells (MPC) were isolated from the skeletal muscle and analyzed by flow cytometry and in vitro differentiation assays. The differentiation of MPC to smooth muscle cells (MPC-SMC) was evaluated by immunofluorescence, flow cytometry, patch-clamp, collagen contraction, and microarray gene expression analysis. In vivo engraftment of MPC-SMC was monitored by transplanting reporter protein-expressing cells into the pyloric sphincter of immunodeficient mice. RESULTS:MPC derived from human skeletal muscle expressed mesenchymal surface markers and exhibit skeletal myogenic differentiation potential in vitro. In contrast, they lack hematopoietic surface marker, as well as adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Cultivation of MPC in smooth muscle differentiation medium significantly increases the fraction of alpha smooth muscle actin (aSMA) and smoothelin-positive cells, while leaving the number of desmin-positive cells unchanged. Smooth muscle-differentiated MPC (MPC-SMC) exhibit increased expression of smooth muscle-related genes, significantly enhanced numbers of CD146- and CD49a-positive cells, and in vitro contractility and express functional Cav and Kv channels. MPC to MPC-SMC differentiation was also accompanied by a reduction in their skeletal muscle differentiation potential. Upon removal of the smooth muscle differentiation medium, a major fraction of MPC-SMC remained positive for aSMA, suggesting the definitive acquisition of their phenotype. Transplantation of murine MPC-SMC into the mouse pyloric sphincter revealed engraftment of MPC-SMC based on aSMA protein expression within the host smooth muscle tissue. CONCLUSIONS:Our work confirms the ability of MPC to give rise to smooth muscle cells (MPC-SMC) with a well-defined and stable phenotype. Moreover, the engraftment of in vitro-differentiated murine MPC-SMC into the pyloric sphincter in vivo underscores the potential of this cell population as a novel cell therapeutic treatment for smooth muscle regeneration of sphincters.

Project description:PurposeThe α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse.MethodsTo examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes.ResultsIn the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while β3 and β4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), α2, α9, β4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), β2, β4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of α3-α5 and α9-containing nAChR subunits and m1-m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of α9 and α10 nAChR subunit expressions in the retina of any species.ConclusionsRather than a simple upregulation of a single AChR subunit or subtype, the absence of the α7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.

Dataset Information

Synergistic Signaling by Light and Acetylcholine in Mouse Iris Sphincter Muscle.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets