MAB1522 Sigma-AldrichAnti-Actin Antibody, smooth muscle γ & α actin, clone CGA7

The mechanisms that control phasic and tonic contractions of lymphatic vessels are poorly understood. We hypothesized that rho kinase ROCK, previously shown to increase calcium (Ca2+) sensitivity in vascular smooth muscle, enhances lymphatic contractile activity in a similar fashion. Contractions of isolated rat mesenteric lymphatic vessels were observed at a luminal pressure of 2 cm H2O in a 37°C bath. The expression of ROCK in isolated rat mesenteric lymphatic vessels was assessed by Western blotting and confocal microscopy. The role of ROCK in contractile function was tested using two specific yet structurally distinct inhibitors: H1152 (0.1-10 μM) and Y-27632 (0.5-50 μM). In addition, lymphatics were transfected with constitutively active (ca)-ROCK protein (2 μg/ml) to assess gain of contractile function. Vessel diameter and the concentration of intracellular free Ca2+ ([Ca2+]i) were simultaneously measured in a subset of isolated lymphatics loaded with the Ca2+-sensing dye fura-2. The results show expression of both the ROCK1 and ROCK2 isoforms in lymphatic vessels. Inhibition of ROCK increased lymphatic end diastolic diameter and end systolic diameter in a concentration-dependent manner. Significant reductions in lymphatic tone and contraction amplitude were observed after treatment 1-10 μM H1152 or 25-50 μM Y-27632. H1152 (10 μM) also significantly reduced contraction frequency. Transient increases in [Ca2+]i preceded each phasic contraction, however this pattern was disrupted by either 10 μM H1152 or 50 μM Y-27632 in the majority of lymphatics studied. The significant decrease in tone caused by H1152 or Y-27632 was not associated with a significant change in the basal [Ca2+]i between transients. Transfection with ca-ROCK protein enhanced lymphatic tone, but was not associated with a significant change in basal [Ca2+]i. Our data suggest that ROCK mediates normal tonic constriction and influences phasic contractions in lymphatics. We propose that ROCK modulates Ca2+ sensitivity of contractile proteins in lymphatics.

Dopamine receptors are G protein-coupled receptors that are divided into two subgroups, "D(1)-like" receptors (D(1) and D(5)) that couple to the G(s) protein and "D(2)-like" receptors (D(2), D(3), and D(4)) that couple to G(i). Although inhaled dopamine has been reported to induce bronchodilation in patients with asthma, functional expression of dopamine receptor subtypes has never been described on airway smooth muscle (ASM) cells. Acute activation of G(i)-coupled receptors inhibits adenylyl cyclase activity and cAMP synthesis, which classically impairs ASM relaxation. In contrast, chronic activation of G(i)-coupled receptors produces a paradoxical enhancement of adenylyl cyclase activity referred to as heterologous sensitization. We questioned whether the dopamine D(2)-like receptor is expressed on ASM, whether it exhibits classical G(i)-coupling, and whether it modulates ASM function. We detected the mRNA encoding the dopamine D(2) receptor in total RNA isolated from native human ASM and from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the dopamine D(2) receptor protein in both native human and guinea pig ASM and cultured HASM cells. The dopamine D(2) receptor protein was immunohistochemically localized to both human and guinea pig ASM. Acute activation of the dopamine D(2) receptor by quinpirole inhibited forskolin-stimulated adenylyl cyclase activity in HASM cells, which was blocked by the dopamine D(2) receptor antagonist L-741626. In contrast, the chronic pretreatment (1 h) with quinpirole potentiated forskolin-stimulated adenylyl cyclase activity, which was inhibited by L-741626, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor GF109203X. Quinpirole also stimulated inositol phosphate synthesis, which was inhibited by L-741626 or U73122. Chronic pretreatment (1 h) of the guinea pig tracheal rings with quinpirole significantly potentiated forskolin-induced airway relaxation, which was inhibited by L-741626. These results demonstrate that functional dopamine D(2) receptors are expressed on ASM and could be a novel therapeutic target for the relaxation of ASM.

Alzheimer's disease (AD) is the most prevalent form of age-related dementia, and its effect on society increases exponentially as the population ages. Accumulating evidence suggests that neuroinflammation, mediated by the brain's innate immune system, contributes to AD neuropathology and exacerbates the course of the disease. However, there is no experimental evidence for a causal link between systemic inflammation or neuroinflammation and the onset of the disease.The viral mimic, polyriboinosinic-polyribocytidilic acid (PolyI:C) was used to stimulate the immune system of experimental animals. Wild-type (WT) and transgenic mice were exposed to this cytokine inducer prenatally (gestation day (GD)17) and/or in adulthood. Behavioral, immunological, immunohistochemical, and biochemical analyses of AD-associated neuropathologic changes were performed during aging.We found that a systemic immune challenge during late gestation predisposes WT mice to develop AD-like neuropathology during the course of aging. They display chronic elevation of inflammatory cytokines, an increase in the levels of hippocampal amyloid precursor protein (APP) and its proteolytic fragments, altered Tau phosphorylation, and mis-sorting to somatodendritic compartments, and significant impairments in working memory in old age. If this prenatal infection is followed by a second immune challenge in adulthood, the phenotype is strongly exacerbated, and mimics AD-like neuropathologic changes. These include deposition of APP and its proteolytic fragments, along with Tau aggregation, microglia activation and reactive gliosis. Whereas Aβ peptides were not significantly enriched in extracellular deposits of double immune-challenged WT mice at 15 months, they dramatically increased in age-matched immune-challenged transgenic AD mice, precisely around the inflammation-induced accumulations of APP and its proteolytic fragments, in striking similarity to the post-mortem findings in human patients with AD.Chronic inflammatory conditions induce age-associated development of an AD-like phenotype in WT mice, including the induction of APP accumulations, which represent a seed for deposition of aggregation-prone peptides. The PolyI:C mouse model therefore provides a unique tool to investigate the molecular mechanisms underlying the earliest pathophysiological changes preceding fibrillary Aβ plaque deposition and neurofibrillary tangle formations in a physiological context of aging. Based on the similarity between the changes in immune-challenged mice and the development of AD in humans, we suggest that systemic infections represent a major risk factor for the development of AD.

The extracellular signaling protein Reelin, indispensable for proper neuronal migration and cortical layering during development, is also expressed in the adult brain where it modulates synaptic functions. It has been shown that proteolytic processing of Reelin decreases its signaling activity and promotes Reelin aggregation in vitro, and that proteolytic processing is affected in various neurological disorders, including Alzheimer's disease (AD). However, neither the pathophysiological significance of dysregulated Reelin cleavage, nor the involved proteases and their modulators are known. Here we identified the serine protease tissue plasminogen activator (tPA) and two matrix metalloproteinases, ADAMTS-4 and ADAMTS-5, as Reelin cleaving enzymes. Moreover, we assessed the influence of several endogenous protease inhibitors, including tissue inhibitors of metalloproteinases (TIMPs), α-2-Macroglobulin, and multiple serpins, as well as matrix metalloproteinase 9 (MMP-9) on Reelin cleavage, and described their complex interplay in the regulation of this process. Finally, we could demonstrate that in the murine hippocampus, the expression levels and localization of Reelin proteases largely overlap with that of Reelin. While this pattern remained stable during normal aging, changes in their protein levels coincided with accelerated Reelin aggregation in a mouse model of AD.

BACKGROUND: Emerging evidence indicates that an endogenous autocrine/paracrine system involving gamma-aminobutyric acid (GABA) is present in airways. GABAA channels, GABAB receptors, and the enzyme that synthesizes GABA have been identified in airway epithelium and smooth muscle. However, the endogenous ligand itself, GABA, has not been measured in airway tissues. The authors sought to demonstrate that GABA is released in response to contractile agonists and tonically contributes a prorelaxant component to contracted airway smooth muscle. METHODS: The amount and cellular localization of GABA in upper guinea pig airways under resting and contracted tone was determined by high pressure liquid chromatography and immunohistochemistry, respectively. The contribution that endogenous GABA imparts on the maintenance of airway smooth muscle acetylcholine-induced contraction was assessed in intact guinea pig airway tracheal rings using selective GABAA antagonism (gabazine) under resting or acetylcholine-contracted conditions. The ability of an allosteric agent (propofol) to relax a substance P-induced relaxation in an endogenous GABA-dependent manner was assessed. RESULTS: GABA levels increased and localized to airway smooth muscle after contractile stimuli in guinea pig upper airways. Acetylcholine-contracted guinea pig tracheal rings exhibited an increase in contracted force upon addition of the GABAA antagonist gabazine that was subsequently reversed by the addition of the GABAA agonist muscimol. Propofol dose-dependently relaxed a substance P contraction that was blocked by gabazine. CONCLUSION: These studies demonstrate that GABA is endogenously present and increases after contractile stimuli in guinea pig upper airways and that endogenous GABA contributes a tonic prorelaxant component in the maintenance of airway smooth muscle tone.,

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic (GABA(A)/GABA(C)) and metabotropic (GABA(B)) receptors (R). In addition to their location on neurons, GABA and functional GABA(B) receptors have been detected in nonneuronal cells in peripheral tissue. Although the GABA(B)R has been shown to function as a prejunctional inhibitory receptor on parasympathetic nerves in the lung, the expression and functional coupling of GABA(B) receptors to G(i) in airway smooth muscle itself have never been described. We detected the mRNA encoding multiple-splice variants of the GABA(B)R1 and GABA(B)R2 in total RNA isolated from native human and guinea pig airway smooth muscle and from RNA isolated from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the GABA(B)R1 and GABA(B)R2 proteins in human native and cultured airway smooth muscle. The GABA(B)R1 protein was immunohistochemically localized to airway smooth muscle in guinea pig tracheal rings. Baclofen, a GABA(B)R agonist, elicited a concentration-dependent stimulation of [(35)S]GTPgammaS binding in HASM homogenates that was abrogated by the GABA(B)R antagonist CGP-35348. Baclofen also inhibited adenylyl cyclase activity and induced ERK phosphorylation in HASM. Another GABA(B)R agonist, SKF-97541, mimicked while pertussis toxin blocked baclofen's effect on ERK phosphorylation, implicating G(i) protein coupling. Functional GABA(B) receptors are expressed in HASM. GABA may modulate an uncharacterized signaling cascade via GABA(B) receptors coupled to the G(i) protein in airway smooth muscle.

Prostate smooth muscle cells predominantly express alpha1-adrenoceptors (alpha1-AR). alpha1-AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca(2+) entry pathways associated with the activation of alpha1-AR in the prostate have yet to be elucidated. In many cell types, mammalian homologues of transient receptor potential (TRP) genes, first identified in Drosophila, encode TRPC (canonical TRP) proteins. They function as receptor-operated channels (ROCs) which are involved in various physiological processes such as contraction, proliferation, apoptosis, and differentiation. To date, the expression and function of TRPC channels have not been studied in prostate smooth muscle. In fura-2 loaded PS1 (a prostate smooth muscle cell line) which express endogenous alpha1A-ARs, alpha-agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca(2+) influx which depended on the extracellular Ca(2+) and PLC activation but was independent of PKC activation. Thus, we have tested two membrane-permeable analogues of diacylglycerol (DAG), oleoyl-acyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG). They initiated Ca(2+) influx whose properties were similar to those induced by the alpha-agonists. Sensitivity to 2-aminoethyl diphenylborate (2-APB), SKF-96365 and flufenamate implies that Ca(2+)-permeable channels mediated both alpha-agonist- and OAG-evoked Ca(2+) influx. Following the sarcoplasmic reticulum (SR) Ca(2+) store depletion by thapsigargin (Tg), a SERCA inhibitor, OAG and PHE were both still able to activate Ca(2+) influx. However, OAG failed to enhance Ca(2+) influx when added in the presence of an alpha-agonist. RT-PCR and Western blotting performed on PS1 cells revealed the presence of mRNAs and the corresponding TRPC3 and TRPC6 proteins. Experiments using an antisense strategy showed that both alpha-agonist- and OAG-induced Ca(2+) influx required TRPC3 and TRPC6, whereas the Tg-activated (capacitative) Ca(2+) entry involved only TRPC3 encoded protein. It may be thus concluded that PS1 cells express TRPC3 and TRPC6 proteins which function as receptor- and store-operated Ca(2+) entry pathways.

TRPM8 (melastatine-related transient receptor potential member 8), a member of the transient receptor potential (TRP) superfamily of cation channels, has been shown to be a calcium-channel protein. TRPM8 mRNA has also been shown to be overexpressed in prostate cancer and is considered to play an important role in prostate physiology. This study was designed to determine the androgen-regulation mechanisms for TRPM8 mRNA expression and to identify the phenotype of TRPM8-expressing cells in the human prostate. Our findings show that trpm8 gene expression requires a functional androgen receptor. Furthermore, this article argues strongly in favour of the fact that the trpm8 gene is a primary androgen-responsive gene. Single-cell reverse transcriptase PCR and immunohistochemical experiments also showed that the trpm8 gene was mainly expressed in the apical secretory epithelial cells of the human prostate and trpm8 down-regulation occurred during the loss of the apical differentiated phenotype of the primary cultured human prostate epithelial cells. The androgen-regulated trpm8 expression mechanisms are important in understanding the progression of prostate cancer to androgen-independence. These findings may contribute to design a strategy to predict prostate cancer status from the TRPM8 mRNA level. Furthermore, as the TRPM8 channel is localized in human prostate cells, it will be interesting to understand its physiological function in the normal prostate and its potential role in prostate cancer development.

We examined the expression of smooth muscle cytoskeleton in spindle-shaped cells in the capsule of hepatocellular carcinoma (HCC) and the septa of liver cirrhosis (LC). Serial sections of livers resected from 11 patients were stained with monoclonal antibodies against vimentin, desmin, smooth muscle actin (1A4, HHF35, CGA7) and smooth muscle myosin heavy chain isoforms (SM1, SM2). Capsular spindle-shaped cells exhibited a cytoskeletal feature indicative of intermediately differentiated smooth muscle cells. Computer-assisted morphometry revealed that the proportions of 1A4-, HHF35-, CGA7- and SM1- positive areas to vimentin-positive area were 88.0+/-11.0%, 50.8+/-17.4%, 25.3+/-16.4% and 19.4+/-12.4% (n=11) in main tumours and 86.6+/-9.4%, 50.9+/-18.7%, 21.1+/-12.3% and 17.6+/-9.7% (n=12) in daughter tumours, indicating that spindle-shaped cells are heterogeneous in cytoskeletal expression. Septal spindle-shaped cells in LC lacked the cytoskeletal proteins specific to differentiated smooth muscle cells (CGA7, SM1, SM2 and desmin). Electron microscopically, capsular spindle-shaped cells contained more microfilaments and less rough endoplasmic reticulum than do septal cells. Intermediately differentiated smooth muscle cells are induced in the capsule of HCC but not in the septa of LC, suggesting a role for stromal interaction by tumour cells in the induction of smooth muscle cells.

Cytodifferentiation of smooth muscle cells has been analyzed immunocytochemically during rat intestinal development and in chimaeric intestines by using monoclonal antibodies reacting specifically with smooth muscle actin species (CGA7 [10] and anti-alpha SM-1 [40]). As development proceeds, the various intestinal muscle layers differentiate in the following order: (1) cells expressing smooth muscle actin appear within the mesenchyme of the 15-day fetal rat intestine, in the circular muscle-forming area, the differentiation of cells in the presumptive longitudinal muscle layer starting with a 48-h delay; (2) smooth muscle fibers appear within the connective tissue core of the villi shortly after birth, in parallel with a progressive formation of the muscularis mucosae, which becomes clear-cut only in the course of the 2nd week after birth; (3) a distinct cell layer in the innermost part of the circular muscle layer arises during the perinatal period. Thereafter, the fluorescence pattern remains unchanged until the adult stage. Chimaeric intestines were constructed by the association of 14-day fetal intestinal epithelium and cultured fetal rat or human skin fibroblasts. These fibroblastic cells did not express actin at the time at which they were associated. The immunocytochemical analysis of smooth muscle actin in the hybrid intestines, which had developed as intracoelomic grafts for 12 days, revealed that the skin fibroblastic cells had been induced by the intestinal epithelial cells to differentiate into smooth muscle cells. Such a result was also obtained with allantoic endoderm. It was not obvious in cocultures of intestinal epithelium with skin fibroblastic cells. However, when intestinal epithelial cells were cocultured with intestinal mesenchymal cells, actin expression was stimulated in the latter cell population.