Barbara Tolloczko

Mesenteric vascular smooth muscle cells from spontaneously hypertensive rats display increased calcium responses to angiotensin II but not to endothelin-1

Objective To determine the differential calcium responses to two vasoconstrictor peptides, angiotensin II (Ang II) and endothelin-1, in vascular smooth muscle cells derived from mesenteric arteries from young and adult normotensive and hypertensive rats. Methods Effects of Ang II and endothelin-1 on cytosolic free calcium concentration in primary cultured unpassaged single vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto (WKY), Wistar and spontaneously hypertensive rats (SHR) aged 3, 9 and 17 weeks were examined microphotometrically using fura-2 methodology. Results Basal cytosolic free calcium concentration was significantly increased in cells from SHR aged 9 and 17 weeks compared with cells from age-matched WKY and Wistar rats. Ang II and endothelin-1 significantly increased cell cytosolic free calcium in all rat groups at all ages. Responses to low concentrations of Ang II (1 nmol/l) were significantly higher in cells from SHR aged 9 and 17 weeks than in age-matched controls. This was confirmed in cells from rats aged 17 weeks with full concentration-response curves, which also showed that the pD2 for Ang II for WKY rats was significantly different from that of SHR. In cells from SHR at all ages Ang II-stimulated cytosolic free calcium remained persistently high, whereas in cells from WKY and Wistar rats basal levels were reached within 100s after the maximal response. Low concentrations of endothelin-1 elicited significantly lower cytosolic free calcium responses in cells from SHR aged 17 weeks compared with age-matched controls. The time course of cytosolic free calcium responses to endothelin-1 were similar in the groups. Conclusions In primary cultured unpassaged mesenteric vascular smooth muscle cells from adult SHR, cytosolic free calcium concentration responses to Angll are enhanced, whereas responses to low concentrations of endothelin-1 are slightly reduced. The differential effects of these two vasoconstrictor peptides may contribute to their relative roles in modulating vascular smooth muscle cell cytosolic free calcium in SHR.

Airway smooth muscle remodeling is a dynamic process in severe long-standing asthma

BACKGROUND The origin of the excess airway smooth muscle in asthma and when in the course of the disease it is acquired are uncertain. OBJECTIVES We examined the relative sensitivities of 2 markers of proliferation, proliferating cell nuclear antigen (PCNA) and Ki 67, in airway smooth muscle in vivo and in vitro. We then studied whether muscle remodeling is a dynamic process in asthma by quantifying proliferation rate and area. Finally we examined heparin-binding epidermal growth factor as a biomarker of remodeling. METHODS We obtained bronchoscopic biopsies from subjects with moderate or severe asthma and healthy controls (n = 9/group). For in vitro studies, airway smooth muscle cells were cultured from tracheas of transplant donors. The proliferation rate was quantified from PCNA and Ki 67, co-localized to smooth muscle-specific alpha-actin cells in vivo. Muscle area was assessed morphometrically. We examined the expression of heparin-binding epidermal growth factor on tissues by in situ hybridization and by immunohistochemistry and in cells in culture by RT-PCR. RESULTS Proliferating cell nuclear antigen and Ki 67 were highly correlated, but PCNA was a significantly more sensitive marker both in vivo and in vitro. Muscle area was 3.4-fold greater and the fraction of PCNA(+) nuclei in muscle was 5-fold greater in severe asthma than in healthy subjects. Heparin-binding epidermal growth factor was upregulated in proliferating muscle cells in culture and in airway smooth muscle in severe asthmatic tissues. CONCLUSION Proliferating cell nuclear antigen is a highly sensitive marker of proliferation and heparin-binding epidermal growth factor is a potential biomarker during active remodeling of ASM in severe asthma.

Synaptic glomeruli in the olfactory system of a snail, Achatina fulica

SummaryThe presence of synaptic glomeruli in the olfactory afferent pathway of vertebrates and arthropods is a striking example of neuroanatomical convergence. To test the generality of this analogy, the olfactory receptors in a terrestrial snail, Achatina fulica, were labeled and traced by application of horseradish peroxidase to the epithelial surface at the tip of the posterior tentacles. All afferent fibers enter the digitary extensions of the tentacular ganglion. The majority of fibers travel through the digits to terminate either in the tentacle ganglion per se or in the cerebral ganglion. About 10% of the afferent axons terminate in glomeruli within the digits. The glomeruli are characterized by dense neuropils, numerous synaptic contacts, and enclosure by glia cells and glial processes. Periglomerular interneurons lie in close proximity to the glomeruli. There are about 20 glomeruli per tentacle. They have irregular shapes with a mean individual volume of 38 × 10-5 mm3.

Interleukin-13 inhibits proliferation and enhances contractility of human airway smooth muscle cells without change in contractile phenotype

IL-13 is an important mediator of allergen-induced airway hyperresponsiveness. This Th2 cytokine, produced by activated T cells, mast cells, and basophils, has been described to mediate a part of its effects independently of inflammation through a direct modulation of the airway smooth muscle (ASM). Previous studies demonstrated that IL-13 induces hyperresponsiveness in vivo and enhances calcium signaling in response to contractile agonists in vitro. We hypothesized that IL-13 drives human ASM cells (ASMC) to a procontractile phenotype. We evaluated ASM phenotype through the ability of the cell to proliferate, to contract, and to express contractile protein in response to IL-13. We found that IL-13 inhibits human ASMC proliferation (expression of Ki67 and bromodeoxyuridine incorporation) in response to serum, increasing the number of cells in G0/G1 phase and decreasing the number of cells in G2/M phases of the cell cycle. IL-13-induced inhibition of proliferation was not dependent on signal transducer and activator of transcription-6 but was IL-13Rα2 receptor dependent and associated with a decrease of Kruppel-like factor 5 expression. In parallel, IL-13 increased calcium signaling and the stiffening of human ASMC in response to 1 μM histamine, whereas the stiffening response to 30 mM KCl was unchanged. However, Western blot analysis showed unchanged levels of calponin, smooth muscle α-actin, vinculin, and myosin. We conclude that IL-13 inhibits proliferation via the IL-13Rα2 receptor and induces hypercontractility of human ASMC without change of the phenotypic markers of contractility.

MAP kinases mediate interleukin-13 effects on calcium signaling in human airway smooth muscle cells

Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of allergic asthma through animal models that have shown that IL-13 is both necessary and sufficient to cause airway hyperresponsiveness (AHR). Airway smooth muscle (ASM) is a primary effector of AHR, and IL-13 increases the responsiveness of ASM, by increasing Ca(2+) release intracellularly, to bronchoconstrictors such as histamine. The mechanisms and signaling pathways mediating this effect are incompletely understood. We have investigated the pathways through which IL-13 regulates the Ca(2+) response to histamine in primary human ASM cell cultures. Functional IL-13 receptors were demonstrated by IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mitogen-activated protein kinases (MAPKs). IL-13 increased Ca(2+) responses to histamine. The augmentation of Ca(2+) signaling was not affected by inhibition of STAT6 or p38 MAPK signaling but was prevented by concurrent inhibition of c-jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) MAPKs. This inhibition did not affect the IL-13-induced increase in histamine receptors. We conclude that IL-13 induces potentiation of Ca(2+) responses to contractile agonists by affecting mechanisms downstream of receptors. JNK and ERK MAPKs modulate these mechanisms.

Expression and Regulation of CCR1 by Airway Smooth Muscle Cells in Asthma

C-C chemokines such as CCL11, CCL5, and CCL3 are central mediators in the pathogenesis of asthma. They are mainly associated with the recruitment and the activation of specific inflammatory cells, such as eosinophils, lymphocytes, and neutrophils. It has recently been shown that they can also activate structural cells, such as airway smooth muscle and epithelial cells. The aims of this study were to examine the expression of the CCL3 receptor, CCR1, on human airway smooth muscle cells (ASMC) and to document the regulation of this receptor by cytokines involved in asthma pathogenesis. We first demonstrated that CCR1 mRNA is increased in the airways of asthmatic vs control subjects and showed for the first time that ASMC express CCR1 mRNA and protein, both in vitro and in vivo. Calcium mobilization by CCR1 ligands confirmed its functionality on ASMC. Stimulation of ASMC with TNF-α and, to a lesser extent, IFN-γ resulted in an up-regulation of CCR1 expression, which was totally suppressed by both dexamethasone or mithramycin. Taken together, our data suggest that CCR1 might be involved in the pathogenesis of asthma, through the activation of ASMC by its ligands.

IFN-γ, IL-4 and IL-13 modulate responsiveness of human airway smooth muscle cells to IL-13

BackgroundIL-13 is a critical mediator of allergic asthma and associated airway hyperresponsiveness. IL-13 acts through a receptor complex comprised of IL-13Rα1 and IL-4Rα subunits with subsequent activation of signal transducer and activator of transcription 6 (STAT6). The IL-13Rα2 receptor may act as a decoy receptor. In human airway smooth muscle (HASM) cells, IL-13 enhances cellular proliferation, calcium responses to agonists and induces eotaxin production. We investigated the effects of pre-treatment with IL-4, IL-13 and IFN-γ on the responses of HASM cells to IL-13.MethodsCultured HASM were examined for expression of IL-13 receptor subunits using polymerase chain reaction, immunofluorescence microscopy and flow cytometry. Effects of cytokine pre-treatment on IL-13-induced cell responses were assessed by looking at STAT6 phosphorylation using Western blot, eotaxin secretion and calcium responses to histamine.ResultsIL-13Rα1, IL-4Rα and IL-13Rα2 subunits were expressed on HASM cells. IL-13 induced phosphorylation of STAT6 which reached a maximum by 30 minutes. Pre-treatment with IL-4, IL-13 and, to a lesser degree, IFN-γ reduced peak STAT6 phosphorylation in response to IL-13. IL-13, but not IFN-γ, pre-treatment abrogated IL-13-induced eotaxin secretion. Pre-treatment with IL-4 or IL-13 abrogated IL-13-induced augmentation of the calcium transient evoked by histamine. Cytokine pre-treatment did not affect expression of IL-13Rα1 and IL-4Rα but increased expression of IL-13Rα2. An anti-IL-13Rα2 neutralizing antibody did not prevent the cytokine pre-treatment effects on STAT6 phosphorylation. Cytokine pre-treatment increased SOCS-1, but not SOCS-3, mRNA expression which was not associated with significant increases in protein expression.ConclusionPre-treatment with IL-4 and IL-13, but not IFN-γ, induced desensitization of the HASM cells to IL-13 as measured by eotaxin secretion and calcium transients to histamine. The mechanism of IL-4 and IL-13 induced desensitization does not appear to involve either downregulation of receptor expression or induction of the IL-13Rα2 or the SOCS proteins.

Secretory glands of the snail tentacle and their relation to the olfactory organ (Mollusca, Gastropoda)

SummaryThe epidermis of the posterior tentacles of the terrestrial snail Achatina fulica was examined by histological and histochemial methods. There are seven types of unicellular glands in the tentacle skin: three mucocytes containing either acid mucopolysaccharides or neutral mucopolysaccharides, or both; two mucocytes containing glycoproteins; a lipid gland; and a protein gland. The mucocytes are considerably more abundant along the shaft of the tentacle than at the tip, where the olfactory organ is situated. Conversely, the lipid glands and the protein glands are found almost exclusively in the olfactory organ. With minor exceptions, none of the foregoing cell types is present in the skin of the head or the foot. These observations indicate a high degree of local specificity in secretory products, consistent with a ubiquitous and generous endowment of glands in the molluscan skin. Collar cells, described by previous authors in closely related species, were not observed.

Blunted attenuation of angiotensin II-mediated Ca2+ transients by insulin in cultured unpassaged vascular smooth muscle cells from spontaneously hypertensive rats

Insulin attenuates vasoconstrictor-stimulated intracellular calcium ([Ca2+]i) responses in cells from normotensive rats. To determine whether these effects may be altered in hypertension, this study assesses the effects of physiologic concentrations of insulin on angiotensin II-stimulated [Ca2+]i in primary unpassaged cultured vascular smooth muscle cells (VSMC) from mesenteric arteries from spontaneously hypertensive rats (SHR), Wistar Kyoto (WKY), and Wistar rats. [Ca2+]i was measured microphotometrically in cells from 3-, 9-, and 17-week-old rats by the Fura 2 methodology. Basal, angiotensin II-stimulated (1 nmol/L), and insulin-attenuated (70 microU/mL) angiotensin II-induced [Ca2+]i did not differ in VSMC obtained from 3-week-old WKY, Wistar, and SHR. Basal and angiotensin II-stimulated [Ca2+]i in VSMC from 9- and 17-week-old SHR was significantly greater (P < .01) than that in cells from age-matched WKY and Wistar rats. Insulin decreased angiotensin II-stimulated [Ca2+]i responses in all groups, but the effect was significantly blunted in cells from 9- and 17-week-old SHR. The magnitude of inhibition of angiotensin II-stimulated [Ca2+]i responses induced by insulin was 63 +/- 12 nmol/l (WKY), 60 +/- 10 nmol/L (Wistar), and 28 +/- 8 nmol/L (SHR), (P < .01 v normotensive) in cells from 9-week-old rats and 70 +/- 15 nmol/L (WKY), 67 +/- 12 nmol/L (Wistar), and 25 +/- 10 nmol/L (SHR) (P < .01 v normotensive) in cells from 17-week-old rats. Insulin increased angiotensin II-stimulated [Ca2+]i recovery rate to basal values in cells from WKY rats, but had no effect on the rate of recovery in VSMC from SHR. (ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide synthesis by tracheal smooth muscle cells by a nitric oxide synthase-independent pathway.

Nitric oxide (NO) is known to be synthesized from L-arginine in a reaction catalyzed by NO synthase. Liver cytochrome P-450 enzymes also catalyze the oxidative cleavage of C==N bonds of compounds containing a -C(NH2)==NOH function, producing NO in vitro. The present study was designed to investigate whether there was evidence of a similar pathway for the production of NO in tracheal smooth muscle cells. Formamidoxime (10(-2) to 10(-4) M), a compound containing -C(NH2)==NOH, relaxed carbachol-contracted tracheal rings and increased intracellular cGMP in cultured tracheal smooth muscle cells, whereas L-arginine had no such effect. NO was detectable in the medium containing cultured tracheal smooth muscle cells when incubated with formamidoxime. Ethoxyresorufin (10(-7) to 10(-4) M), an alternate cytochrome P-450 substrate, inhibited formamidoxime-induced cGMP accumulation as well as tracheal ring relaxation in cultured tracheal smooth muscle cells. The NO synthase inhibitors Nomega-nitro-L-arginine (10(-3) M) and NG-monomethyl-L-arginine (10(-3) M) had no effect on formamidoxime-induced cGMP accumulation. These results suggest that NO can be synthesized from formamidoxime in tracheal smooth muscle cells, presumably by a reaction catalyzed by cytochrome P-450.Nitric oxide (NO) is known to be synthesized from l-arginine in a reaction catalyzed by NO synthase. Liver cytochrome P-450 enzymes also catalyze the oxidative cleavage of CN bonds of compounds containing a -C(NH2)NOH function, producing NO in vitro. The present study was designed to investigate whether there was evidence of a similar pathway for the production of NO in tracheal smooth muscle cells. Formamidoxime (10-2 to 10-4 M), a compound containing -C(NH2)NOH, relaxed carbachol-contracted tracheal rings and increased intracellular cGMP in cultured tracheal smooth muscle cells, whereasl-arginine had no such effect. NO was detectable in the medium containing cultured tracheal smooth muscle cells when incubated with formamidoxime. Ethoxyresorufin (10-7 to 10-4 M), an alternate cytochrome P-450 substrate, inhibited formamidoxime-induced cGMP accumulation as well as tracheal ring relaxation in cultured tracheal smooth muscle cells. The NO synthase inhibitors N ω-nitro-l-arginine (10-3 M) and N G-monomethyl-l-arginine (10-3 M) had no effect on formamidoxime-induced cGMP accumulation. These results suggest that NO can be synthesized from formamidoxime in tracheal smooth muscle cells, presumably by a reaction catalyzed by cytochrome P-450.