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At least 19 recordsLinked to original sources
PubMed2004

Effects of polyamines on the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle myocytes.

The effects of extracellular and intracellular polyamines (PAs), spermine and putrescine, on the cation current (mI(CAT)) evoked either by activating muscarinic receptors with carbachol or by intracellularly applied GTPgammaS (in the absence of carbachol) were studied using patch-clamp recording techniques in single guinea-pig ileal myocytes. Extracellular spermine and putrescine rapidly and reversibly inhibited mI(CAT) in a concentration- and voltage-dependent manner with the IC(50) values at -40 mV of about 1 and 5 mM, respectively. Membrane depolarization relieved the blocking action of PAs although cation conductance activation curve remained N-shaped. The inhibition was similar for both carbachol- and GTPgammaS-evoked currents, suggesting that the cation channel rather than the muscarinic receptor was the primary site of the PA action. In outside-out membrane patches, both cation channel unitary conductance and open probability were reduced. In perforated-patch experiments used to retain cytoplasmic PAs sustained 100 microM carbachol-induced mI(CAT) was significantly smaller (478 +/- 76 pA, n = 7) compared to that recorded using conventional whole-cell configuration with nominally PA-free pipette solution (1314 +/- 76 pA, n = 12), but comparable in size to mI(CAT) with 0.3 mM spermine in the pipette solution (509 +/- 41 pA, n = 19). Intracellular putrescine inhibited mI(CAT) less potently compared to spermine. In conclusion, these results show a novel role of intestinal PAs in mI(CAT) inhibition, which can contribute to their well-known suppressing effect on the gastrointestinal smooth muscle excitability and contractility.

Animals↗
PubMed2003

Three-dimensional culture of human uterine smooth muscle myocytes on a resorbable scaffolding.

The objective of this study was to develop a three-dimensional culture system for the study of human myometrial physiology. Primary cell lines were initiated from human myometrium obtained at the time of term cesarean delivery. After several passages, cells were seeded onto a polyglactin-910 (Vicryl) mesh and maintained in culture. After several days in culture, each mesh was transferred to another culture dish and suspended to avoid contact with the plastic of the dish. Time-lapse videomicroscopy was used to observe cell proliferation and three-dimensional (3-D) fill of the pores of the mesh. Membrane potentials of the cells of this 3-D tissue were measured with a conventional microelectrode. Confocal microscopy was used to assess 3-D morphology. In some experiments, cells were seeded onto two layers of mesh and then cultured as described above. In this two-mesh experiment, force was measured by anchoring one mesh and pulling on the other, using a micrometer-driven strain gauge. In the single-mesh experiment, cells grew into and filled the pores of the mesh by repetitive proliferation, retraction, and proliferation. A confluent, 3-D tissue was obtained within 10 to 14 days of the initial seeding of the mesh. The average membrane potential of the cells within the single mesh was -35 +/- 6 mV. Confocal microscopy demonstrated tissue thickness of 9 to 40 microm (one to eight cells) within the pores of the mesh. In the two-mesh experiment, 2 to 3 weeks in culture yielded confluent 3-D tissues, in which myocytes not only filled the pores of each mesh, but also bridged between the two meshes. The bridging myocytes were able to maintain a tension of 5 g/cm(2) before separation of the two meshes, and coordinated contractions of 40 to 200 cells were observed. We conclude that cultured human myocytes proliferate and form 3-D tissues when supported by Vicryl scaffolding. Tissue grown in 3-D may provide a model system that is sufficient to probe the physiology of cell-to-cell interactions in myometrium.

Biocompatible Materials↗
PubMed1991

A mechanism for testosterone modulation of alpha-1 adrenergic receptor expression in the DDT1 MF-2 smooth muscle myocyte.

Previous reports have confirmed that steroid hormones modulate the expression of adrenergic receptors on the surface of smooth muscle myocytes. The present study was undertaken to evaluate the mechanism by which testosterone modulates alpha-1 adrenergic receptor expression in the DDT1 MF-2 transformed smooth muscle cell. Utilizing 3H-prazosin radioligand binding studies, alpha-1 adrenergic receptors were noted to increase more than 2 fold in response to incubation with 10(-8)M testosterone for 96 hours. Dihydrotestosterone similarly stimulated a significant increase in alpha-1 receptors; whereas, estradiol and hydrocortisone appeared to suppress the expression of this receptor in DDT myocytes. The testosterone effect was dose related with a maximal response observed in response to 10(-7)M testosterone at both 48 and 96 hours. Kinetic experiments utilizing 10(-8)M testosterone demonstrated a peak effect on alpha-1 receptor expression at 96 hours, and maintenance of the effect for at least 168 hours (7 days). The testosterone effect was completely prevented at both 48 and 96 hours by inhibition of transcription with actinomycin-D, or inhibition of translation with cycloheximide. Consistent with the receptor binding studies, RNA blotting studies have demonstrated maximal alpha-1 receptor mRNA levels at 48-96 hours of testosterone stimulation. In conclusion, these in vitro experiments have confirmed the physiologic concentrations of testosterone stimulate the increased expression of alpha-1 receptors in the DDT1 MF-2 myocyte after a delay of 48-96 hours; and that this effect appears to be mediated by transcription, translation, and synthesis of new proteins in these genital tract myocytes.

Animals↗
PubMed1991

Responses of subcultured rat aortic smooth muscle myocytes to vasoactive agents and KCl-induced depolarization.

We have developed a culture system in which a single-mass primary culture can be used for as long as 6 wk as a source of subcultured smooth muscle myocytes for the study of the changes of their shape upon addition of vasoactive agents (angiotensin, vasopressin, norepinephrine, and serotonin) and KCl depolarization. Responses of subcultivated myocytes were shown to be reproducible with time in primary culture before subculture and consistent with responses of thoracic aorta to the same agents. Effect of KCl depolarization could be blocked with calcium antagonist PN 200-110. Consistently, the presence of calcium L-channels was shown using whole cell patch-clamp recordings. A comparative study of the responses of myocytes derived from two different segments of the thoracic aorta showed that these cells displayed responses with different maximal amplitudes and the same potencies according to their topological origin in the vessel.

Angiotensin II↗
PubMed1990

Calcium currents in isolated rabbit coronary arterial smooth muscle myocytes.

1. Calcium inward currents were recorded from relaxed enzymatically isolated smooth muscle cells from the rabbit epicardial left descending coronary artery using a single-pipette voltage-clamp technique. Outward K+ currents were blocked with CsCl-tetraethylammonium-filled pipette solutions. 2. Relaxed coronary smooth muscle cells had a maximum diameter of 8.6 +/- 0.6 microns and a cell length of 96.7 +/- 3.3 microns when bathed in 2.5 mM [Ca2+]o. The average resting membrane potential at room temperature was -32 +/- 10 mV. The mean cell capacitance was 18.5 +/- 1.7 pF and the input resistance was 3.79 +/- 0.58 G omega. 3. Depolarizing voltage-clamp steps from a holding potential of -80 mV elicited a single time- and voltage-dependent inward current which was dependent upon extracellular [Ca2+]. In 2.5 mM [Ca2+]o, the inward current was activated at a potential of -40 mV and peaked at +10 mV. This current was inhibited by 0.5 mM-CdCl2 and 1 microM-nifedipine and was enhanced with 1 microM-Bay K 8644. No detectable low-threshold, rapidly inactivating T-type calcium current was observed. 4. The apparent reversal potential of this inward current in 2.5 mM [Ca2+]o was +70 mV and shifted by 33.0 mV per tenfold increase in [Ca2+]o. This channel was also more permeable to barium and strontium ions than to calcium ions. 5. Single calcium channel recordings with 110 mM-Ba2+ as the charge carrier revealed a mean slope conductance of 20.7 +/- 0.8 pS. 6. This calcium current (ICa) exhibited a strong voltage-dependent inactivation process. However, the steady-state inactivation curve (f infinity) displayed a slight nonmonotonic, U-shaped dependence upon membrane potential. The potential at which half of the channels were inactivated was -27.9 mV with a slope factor of 6.9 mV. The steady-state activation curve (d infinity) was also well-described by a Boltzmann distribution with a half-activation potential at -4.4 mV and a slope factor of -63 mV. ICa was fully activated at approximately +20 mV. 7. The rate of inactivation was dependent upon the species of ion carrying the current. Both Sr2+ and Ba2+ decreased the rate as well as the degree of inactivation. The tau f (fitted time constant of inactivation) curve displayed a U-shaped relationship in 2.5 mM [Ca2+]o. The reactivation process was voltage dependent and could be described by a single exponential. 8. The current amplitude and the inactivation kinetics were temperature dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2004

Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3-/- mice.

ClC-3, a member of the large superfamily of ClC voltage-dependent Cl(-) channels, has been proposed as a molecular candidate responsible for volume-sensitive osmolyte and anion channels (VSOACs) in some cells, including heart and vascular smooth muscle. However, the reported presence of native VSOACs in at least two cell types from transgenic ClC-3 disrupted (Clcn3(-/-)) mice casts considerable doubt on this proposed role for ClC-3. We compared several properties of native VSOACs and examined mRNA transcripts and membrane protein expression profiles in cardiac and pulmonary arterial smooth muscle cells from Clcn3(+/+) and Clcn3(-/-) mice to: (1) test the hypothesis that native VSOACs are unaltered in cells from Clcn3(-/-) mice, and (2) test the possibility that targeted inactivation of the Clcn3 gene using a conventional murine global knock-out approach may result in compensatory changes in expression of other membrane proteins. Our experiments demonstrate that VSOAC currents in myocytes from Clcn3(+/+) and Clcn3(-/-) mice are remarkably similar in terms of activation and inactivation kinetics, steady-state current densities, rectification, anion selectivity (I(-) > Cl(-)>> Asp(-)) and sensitivity to block by glibenclamide, niflumic acid, DIDS and extracellular ATP. However, additional experiments revealed several significant differences in other fundamental properties of native VSOACs recorded from atrial and smooth muscle cells from Clcn3(-/-) mice, including: differences in regulation by endogenous protein kinase C, differential sensitivity to block by anti-ClC-3 antibodies, and differential sensitivities to [ATP](i) and free [Mg(2+)](i). These results suggest that in response to Clcn3 gene deletion, there may be compensatory changes in expression of other proteins that alter VSOAC channel subunit composition or associated regulatory subunits that give rise to VSOACs with different properties. Consistent with this hypothesis, in atria from Clcn3(-/-) mice compared to Clcn3(+/+) mice, quantitative analysis of ClC mRNA expression levels revealed significant increases in transcripts for ClC-1, ClC-2, and ClC-3, and protein expression profiles obtained using two-dimensional polyacrylamide gel electrophoresis revealed complex changes in at least 35 different unidentified membrane proteins in cells from Clcn3(-/-) mice. These findings emphasize that caution needs to be exercised in simple attempts to interpret the phenotypic consequences of conventional global Clcn3 gene inactivation.

Adenosine Triphosphate↗
PubMed1992

Membrane actions of calcitonin gene-related peptide in cardiac and smooth muscle myocytes.

Calcitonin gene-related peptide is a 37-amino acid neuropeptide acting as a transmitter of nonadrenergic, noncholinergic nerves in the heart. Binding sites of high affinity have been reported in coronary arteries, in atria, and, of minor density, in ventricular myocardium. These sites are likely linked to G-proteins mediating modifications of ion channel opening probability and duration and to stimulation of adenylate cyclase activity and cAMP-mediated alterations of ion channel activities. In isolated and perfused guinea pig hearts, low concentrations of CGRP (1-3 nM) exerted no chronotropic effect, but increased coronary flow slightly. Atrioventricular conduction duration and effective refractory period of atrioventricular conduction were prolonged by 3 nM of CGRP. The higher concentration of 10 nM increased the sinus rate, and the effects on the atrioventricular node were counterbalanced. HV and QRS duration of the ECG remained essentially unchanged, but persistent ventricular fibrillation was inducible by burst stimulation in all CGRP-treated hearts. Results in human myometrial myocytes indicate that CGRP exerted direct G protein-mediated activation of potassium channels, leading to hyperpolarization and smooth muscle relaxation. Activation of potassium channels, most prominent in smooth muscle relaxation, is likely an additional factor in the cardiostimulatory profile of CGRP.

Action Potentials↗
PubMed2006

Inactivation of calcium channels in vascular smooth muscle myocytes.

Many of the structural domains involved in Ca2+ channel (CACN) inactivation are also involved in determining their sensitivity to antagonist inhibition. We hypothesize that differences in inactivation properties and their structural determinants may suggest candidate domains as targets for the development of novel, selective antagonists. The characteristics of Ca2+ current (ICa) inactivation, steady-state inactivation (SSIN), and recovery from inactivation were studied in freshly dispersed smooth muscle cells from rabbit portal vein (RPV) using whole-cell, voltage-clamp methods. The time course of inactivation could be represented by two time constants. Increasing ICa by increasing [Ca2+]o or with more negative holding potentials decreased both time constants. With Sr2+, Ba2+, or Na+ as the charge carrier, ICa inactivation was also represented by two time constants, both of which were larger than those found with Ca2+. With Ca2+, Sr2+, or Ba2+ as the charge carrier, both time constants had minimum values near the voltage associated with maximum current. When Na+ (140 mM) was the charge carrier, voltages for Imax (-20 mV) or taumin (0 mV) did not correspond. SSIN of ICa had a half-maximum voltage of -32 +/- 4 mV for Ca2+, -43 mV +/- 5 mV for Sr2+, -41 +/- 5 mV for Ba2+, and -68 +/- 6 mV for Na+. The slope factor for SSIN per e-fold voltage change was 6.5 +/- 0.2 mV for Ca2+, 6.8 +/- 0.3 for Sr2+, and 6.6 +/- 0.2 for Ba2+, representing four equivalent charges. When Na+ or Li+ was the charge carrier, the slope factor was 13.5 +/- 0.7 mV, representing two equivalent charges. For ICa in rat left ventricular (rLV) myocytes, there was no difference in the slope factor of SSIN for Ca2+ and Na+. The rate of recovery of ICa from inactivation varied inversely with recovery voltage and was independent of the charge carrier. These results suggest that inactivation of ICa in PV myocytes possess an intrinsic voltage dependence that is modified by Ca2+. For RPV but not rLV ICa, the charge of the permeating ion confers the voltage-dependency of SSIN.

Animals↗
PubMed2001

Identification of a novel isoform of the cyclic-nucleotide phosphodiesterase PDE3A expressed in vascular smooth-muscle myocytes.

We have identified a new cyclic-nucleotide phosphodiesterase isoform, PDE3A, and cloned its cDNA from cultured aortic myocytes. The nucleotide sequence of its coding region is similar to that of the previously cloned myocardial isoform except for the absence of the initial 300-400 nt that are present in the latter, as confirmed by reverse-transcriptase-mediated PCR, 5' rapid amplification of cDNA ends and a ribonuclease protection assay. Expression in Spodoptera frugiperda (Sf9) cells yields a protein with catalytic activity and inhibitor sensitivity typical of the PDE3 family. The recombinant protein's molecular mass of approx. 131 kDa is compatible with translation from an ATG sequence corresponding to nt 436-438 of the myocardial PDE3A coding region. Antibodies against residues 424-460 (nt 1270-1380) and 1125-1141 (nt 3373-3423) of the myocardial isoform react with an approx. 118 kDa band in Western blots of homogenates of human aortic myocytes, whereas antibodies against residues 29-42 (nt 85-126) do not react with any bands in these homogenates. Our results suggest that a vascular smooth-muscle isoform ('PDE3A2') is a product of the same gene as the longer myocardial ('PDE3A1') and the shorter placental ('PDE3A3') isoforms and is generated pre-translationally in a manner that results in the absence of the 145 N-terminal amino acids of PDE3A1.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Adrenergic stimulation of diacylglycerol production in genital tract smooth muscle myocytes.

Diacylglycerol (DAG) production has not been reported in previous studies that have characterized inositol phosphate production during alpha-1 adrenergic receptor signal transduction in the DDT1 MF-2 genital tract myocytes. The current study sought to measure norepinephrine (NE)-stimulated DAG production in these transformed myocytes utilizing thin layer chromatography. DAG production was characterized as an alpha-1 adrenergic mediated event utilizing subtype specific adrenergic agonist and antagonists. DAG production occurred in response to physiologic concentration of NE, was apparent by 30 s and was significantly increased by 2 min. Maximal DAG production was unaffected by pretreatment of the myocytes for 96 h with testosterone, which has previously been shown to induce a doubling of alpha-1 adrenergic receptors in these cells. In contrast, testosterone pretreatment did result in a shift of the dose-response curve resulting in a significantly lower EC50 for NE in the treated cells compared to control myocytes. In conclusion, these studies have confirmed that DAG production occurs as a component of alpha-1 adrenergic signal transduction in the DDT1 MF-2 myocytes; transduction events that were modulated by testosterone resulting in increased agonist sensitivity.

Animals↗
PubMed2000

Altered ion channel activity in murine colonic smooth muscle myocytes in an experimental colitis model.

We have investigated the activity of calcium and potassium channels in a murine model of experimental colitis. Colonic myocytes from dextran sulphate sodium (DSS)-treated mice were examined by whole cell patch clamp techniques. Myeloperoxidase activity was enhanced 3. 5-fold in DSS-treated mouse colon. In whole cell voltage clamp, depolarization predominantly evoked net transient outward currents in DSS-treated mice and inward Ca(2+) currents in control myocytes. Voltage-dependent L-type Ca(2+) currents were studied using intracellular Cs(+) in the patch pipette. Inward Ca(2+) currents were markedly suppressed in inflamed colon. The peak currents at +10 mV depolarization were -3.93 +/- 0.88 pA/pF in control (n = 12) and -1.14 +/- 0.19 (n = 10) in DSS mice. In contrast there was no change in the amplitude, kinetics, or steady-state inactivation properties of the transient outward currents in control or DSS-treated colonic myocytes. Inflammation significantly enhanced activation of the ATP-sensitive K(+) channel. At a holding potential of -50 mV, the K(ATP) channel opener lemakalim induced an inward current of 2.02 +/- 0.5 pA/pF in control (n = 20) and 4.19 +/- 1.17 pA/pF in DSS-treated colon. These currents were abolished by glibenclamide. The present results suggest that inflammation of the colon results in selective changes in ion channel activity of smooth muscle cells.

Animals↗
PubMed2006

LIM-only protein, CRP2, switched on smooth muscle gene activity in adult cardiac myocytes.

Smooth muscle alpha-actin gene activity appears in promyocardial cells well before cardiac myocyte differentiation and is down-regulated during the onset of rhythmic contractility and cardiac morphogenesis. The levels of LIM-only CRP2 correlated well with smooth muscle gene activity. Cardiomyocyte-specific expression of CRP2 in transgenic mice showed robust expression of smooth muscle cell-specific transcripts and protein filaments in the adult heart. Protein transduction of a recombinant CRP2 protein, fused to the protein transduction domain of HIV, into neonatal heart cells induced de novo synthesis of smooth muscle cell-specific transcripts and proteins. The LIM zinc fingers in CRP2 were found to collaborate with Brg1 of the SNF/SWI complexes, recruited serum response factor, and remodeled smooth muscle target gene chromatin through histone acetylation. CRP2 may have a cytoskeletal role, but as a nuclear protein, CRP2 acted as a potent transcription coadaptor that remodeled silent cardiac myocyte chromatin and directed serum response factor-dependent smooth muscle gene activity.

Actins↗
PubMed1989

Adrenergic stimulation of inositol-phosphate production in a genital tract smooth muscle cell line.

Catecholamines are important in the modulation of smooth muscle contractile activity; this study was undertaken to evaluate adrenoceptor stimulation of intracellular inositol-phosphate production in a genital tract smooth muscle myocyte. DDT1 MF-2 smooth muscle myocytes, derived from a hamster ductus deferens leiomyosarcoma, were loaded with 3H-inositol, incubated in 10 mM LiCl, then stimulated with adrenergic agonists with and without antagonists. Subsequently, the inositol phosphates were isolated by anion-exchange chromatography. In the presence of norepinephrine (NE), inositol trisphosphate (IP3) was produced by 30 s and peaked at 2 min; inositol 1-phosphate was also apparent by 30 s, and continued to increase over 15 min. Clonidine (an alpha-2 agonist), isoproterenol, and NE in the presence of phentolamine or prazosin (an alpha-1 antagonist) failed to increase IP3. In contrast, NE in the presence of yohimbine (an alpha-2 antagonist) or propranolol stimulated IP3 production to levels comparable to that stimulated by NE alone. These studies provide evidence that inositol phosphate production is involved in alpha-1 adrenergic signal transduction in DDT1 MF-2 myocyte.

Adrenergic Agonists↗
PubMed2005

Electrophysiological and molecular identification of voltage-gated sodium channels in murine vascular myocytes.

A voltage-gated Na+ current was characterised in freshly dissociated mouse portal vein (PV) smooth muscle myocytes. The current was found superimposed upon the relatively slow L-type Ca2+ current and was resistant to conventional Ca2+ channel blockers but was abolished by external Na+ replacement and tetrodotoxin (TTX, 1 microM). The molecular identity of the channel responsible for this conductance was determined by RT-PCR where only the transcripts for Na+ channel genes SCN7a, 8a and 9a were detected. The presence of the protein counterparts to the SCN8a and 9a genes (NaV1.6 and NaV1.7, respectively) on the individual smooth muscle myocytes were confirmed in immunocytochemistry, which showed diffuse staining around a predominantly plasmalemmal location. TTX inhibited the action potential in individual myocytes generated in the current clamp mode but isometric tissue tension experiments revealed that TTX (1 and 5 microM) had no effect on the inherent mouse PV rhythmicity. However, the Na+ channel opener veratridine (10 and 50 microM) significantly increased the length of contraction and the interval between contractions. This effect was not influenced by pre-incubation with atropine, prazosin and propranolol, but was reversed by TTX (1 microM) and completely abolished by nicardipine (1 microM). Furthermore, preincubation with the reverse-mode Na+-Ca2+ exchange blocker KB-R7943 (10 microM) also inhibited the veratridine response. We have established for the first time the molecular identity of the voltage-gated Na+ channel in freshly dispersed smooth muscle cells and have shown that these channels can modulate contractility through a novel mechanism of action possibly involving reverse mode Na+-Ca2+ exchange.

Action Potentials↗
PubMed2003

Vesicle-associated protein-A is differentially expressed during intestinal smooth muscle cell differentiation.

Gastrointestinal (GI) smooth muscle diseases represent a major health concern affecting in excess of 2 million people each year. Little is currently known regarding the molecular mechanisms controlling either normal or pathogenic GI smooth muscle development. In an effort to identify the specific gene products responsible for modulating GI smooth muscle cell (SMC) differentiation, we performed differential display on distinct intestinal SMC (ISMC) phenotypes. This analysis identified over 40 unique transcripts that appeared to be differentially expressed in distinct SMC phenotypes. One such transcript that appeared to be preferentially expressed in immature smooth muscle myocytes was identified as vesicle-associated membrane protein, associated protein A (VAP-A). Northern blot analysis confirmed that VAP-A was expressed threefold higher in immature smooth muscle myocytes when compared with both smooth muscle myoblasts and mature smooth muscle myocytes. VAP-A mRNA was differentially expressed during normal rat development and showed peak levels of expression in the intestine during late embryogenesis and early neonatal development. These observations provide the first evidence that VAP-A-mediated membrane trafficking may play an important role in modulating ISMC differentiation.

Amino Acid Sequence↗
PubMedDate not supplied

Regulation of acetylcholine-induced phosphorylation of PLD1 in porcine tracheal smooth muscle.

The muscarinic agonist, acetylcholine (ACh), stimulates phospholipase D (PLD) activity in tracheal smooth muscle cells. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) also stimulates PLD in this tissue. Activation of ACh-induced PLD was inhibited by the tyrosine kinase inhibitor genistein in a concentration-dependent manner. Presently known isoforms of PLD, PLD1 and PLD2, were identified in tracheal smooth muscle and their activation-induced phosphorylation status studied. Both ACh and PMA increased phosphorylation of PLD1 that was significantly blocked by genistein or the PKC inhibitor calphostin C. PLD2 phosphorylation was not detected in the present experiments. Western blots probed with an anti-phosphotyrosine antibody indicate that PLD1 in this tissue is phosphorylated on tyrosine residues after ACh or PMA stimulation. Tyrosine phosphorylation of PLD1 was blocked by genistein and calphostin C. No tyrosine residues were phosphorylated on PLD2. Taken together, these results demonstrate that porcine tracheal smooth muscle cells express both isoforms PLD1 and PLD2. However, on muscarinic activation only PLD1 in this tissue is phosphorylated by PKC via a tyrosine-kinase-dependent pathway.

Acetylcholine↗
PubMed2005

Deficient innervation characterizes intestinal strictures in a rat model of colitis.

Intestinal strictures are a common complication of Crohn's disease leading to serious consequences. With unknown etiology and cellular composition, strictures can be neither prevented nor reversed by current therapeutic strategies, and research has been limited by the lack of a well-developed animal model. We observed the sporadic occurrence of intestinal strictures at Day 35 in the TNBS rat model of colitis, which persisted beyond Day 90. Strictured tissue showed fusion, thickening, and disorganization of the smooth muscle layers. Immunocytochemistry revealed that all strictures were characterized by deficient innervation with a complete loss of intrinsic neurons, and a 92% loss of total axons per area. The number of alpha-smooth muscle actin-positive smooth muscle cells (SMC) increased in strictures, but immunolabeling showed phenotypic modulation of these cells, with the SMC phenotype (desmin-positive, vimentin-negative) entirely replaced by a myofibroblast phenotype (desmin-negative, vimentin-positive). Although cellular structure still predominated in the strictured regions, histochemistry showed increased extracellular matrix collagen, from 6 +/- 0.9% to 22 +/- 4% of total area. With previous evidence for neural loss in colitis, and in vitro studies showing neural regulation of smooth muscle cell (SMC) growth, we conclude that the regional loss of innervation may initiate tissue re-modeling that is characteristic of stricture formation.

Animals↗