Fig 1: Adiponectin’s (ADPN) effect on trans-junctional currents (Ij) in smooth muscle cells (SMCs) of the gastric fundus. (a,b) Representative original trans-junctional currents tracings, Ij (in pA), recorded in response to a bipolar pulse protocol applied to a gastric SMC before (CTRL) and after an ADPN addition to the bath solution (final concentration 20 nM). (c) Voltage dependence of the trans-junctional instantaneous current (Ij,inst in pA) recorded from the cell analyzed in (a,b) in the absence (open squares, CTRL) or presence of ADPN (filled squares). (d) Voltage dependence of the trans-junctional steady state current (Ij,ss in pA) recorded from the same cell (open circles, CTRL; filled circles, ADPN). (e,f) Representative original trans-junctional currents tracings, Ij (in pA) recorded from a different gastric SMC, before (CTRL) and after ADPN addition in the concomitant presence of guanylate cyclase inhibitor ODQ (ODQ + ADPN) to the bath solution (final concentrations: ODQ 1 µM and ADPN 20 nM). (g) Voltage dependence of the trans-junctional instantaneous current (Ij,inst in pA) recorded from the cell analyzed in (e,f) in CTRL (open squares) or in the presence of ODQ + ADPN (filled triangles). (h) Voltage dependence of the trans-junctional steady state current (Ij,ss in pA) recorded from the same cell (open circles, CTRL; filled diamonds, ODQ + ADPN). (i,j) Mean trans-junctional current values normalized for Cm (in pA/pF), recorded from all of the SMCs analyzed (n = 4 for CTRL and ADPN; n = 3 for ODQ and ODQ + ADPN), were plotted vs. the trans-junctional voltage, Vj. Both the overall Ij,inst–Vj and the Ij,ss–Vj plots are not statistically different from each other in the different conditions (p > 0.05, one-way ANOVA). Data are expressed as mean ± SD. (k–o) Representative confocal immunofluorescence images of Connexin (Cx)43 expression (green) in the four indicated experimental conditions. Nuclei are counterstained in red with PI. (o) Quantitative analyses of Cx43 fluorescence intensity (a.u.) performed on digitized images. * p < 0.05 vs. CTRL; # p < 0.05 vs. ADPN (one-way ANOVA with Bonferroni’s post hoc test).
Fig 2: The effects of adiponectin (ADPN) on resting plasma membrane (RMP) potential and membrane capacitance (Cm) of gastric fundus smooth muscle cells (SMCs) involve guanylate cyclase (GC) activity. (a) RMP evaluation (in mV) in the untreated specimen (CTRL, n = 5), ADPN (20 nM)-treated (ADPN, n = 6), selective GC inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ, 1 µ?)-treated (data related to 5 min ODQ exposure; ODQ, n = 4), and ADPN added in the concomitant presence of ODQ and stimulation repeated after 15 min of exposure (ODQ + ADPN, n = 4). (b) Evaluation of Cm (in pF) as an index of the cell surface. Control SMCs (CTRL, n = 21), ADPN-treated (ADPN, n = 6), ODQ-treated (ODQ, n = 4), and ADPN-treated in the presence of ODQ (ODQ + ADPN, n = 5). Data are expressed as mean ± SD. n is the number of SMCs. * p < 0.05 vs. CTRL; # p < 0.05 vs. ADPN (one-way ANOVA with Bonferroni’s correction).
Fig 3: The effects of adiponectin (ADPN) on outward K+ currents evoked in the smooth muscle cell (SMCs) from the gastric fundus involve guanylate cyclase (GC) activity. Representative family of outward K+ currents evoked by voltage steps from -80 to +50 mV (holding potential, HP = -60 mV) in 10 µM-nifedipine- and 1 µM-tetrodotoxin-containing solution in the untreated sample (CTRL) (a), and in the presence of 20 nM ADPN (b). The representative (c) I–V plot related to the two conditions is shown in (a,b) (open triangles, CTRL; open squares, ADPN). (d) Representative K+ current traces were obtained from a different untreated SMC (CTRL) in the presence of guanylate cyclase inhibitor ODQ 1 µM (e) and in the presence of ODQ + ADPN (f). The representative (g) I–V plot related to the three conditions is shown in (e–f) (open triangles, CTRL; open circles, ODQ; filled squares, ODQ + ADPN). All of the current values are normalized to cell capacitance (Cm) and are indicated in pA/pF. (h) I–V plots related to all of the experiments done in the four different conditions are here reported together for a better comparison: CTRL (open up triangles, n = 6); ADPN (open squares, n = 5); ODQ (open circles, n = 4); ADPN in the presence of ODQ (ODQ + ADPN, filled squares, n = 4). Data are expressed as mean ± SD. * p < 0.05 vs. CTRL; # p < 0.05 vs. ADPN. No statistically significant differences were observed between CTRL, ODQ, and ODQ + ADPN (one-way ANOVA with Bonferroni’s correction; p > 0.05).
Fig 4: Morphological analyses. (a–d) Representative light microscopy images of sections of formalin-fixed and paraffin-embedded full-thickness strips from untreated control (CTRL) and adiponectin (ADPN)-treated murine gastric fundus stained with hematoxylin and eosin (cml = circular muscle layer, ksse = keratinized stratified squamous epithelium, lml = longitudinal muscle layer, m = mucosa, mm = muscularis mucosa, and sm = submucosa). Representative (e,g,i,k) differential interference contrast images (DIC, grey) and (f,h,j,l) relative confocal immunofluorescence images showing the expressions and distribution of a-smooth muscle actin (sma) (green). Nuclei are counterstained in red with propidium iodide (PI). Note that immunostaining of a-sma is along the muscularis mucosa, in the blood vessel wall in the submucosa (f,j), and in the circular and longitudinal muscle layers (h,l). (m–o) Representative ultrastructural transmission electron microscopy (TEM) images of the peripheral cytoplasm of longitudinally sectioned smooth muscle cells (SMCs) of the gastric fundus muscle layer from the CTRL, ADPN-treated strip, and strip treated with carbachol (CCh) to stimulate contraction (positive control). (m) A CTRL cell shows several bundles of irregularly arranged contractile filaments intermingled with dense bodies (arrowheads). (n) An ADPN-treated cell shows a slightly looser network of contractile filaments as compared with the CTRL. (o) A cell treated with CCh shows a denser network of contractile filaments as compared with the control. No substantial differences in the amount and morphology of plasma membrane caveolae (c) or sarcoplasmic reticulum profiles (sr) were observed among the different experimental samples (×60,000).
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