Atherosclerosis affects calcium signalling in endothelial cells from apolipoprotein E knockout mice before plaque formation

AbstractLittle is known about how hypercholesterolaemia affects Ca2+ signalling in the vasculature of ApoE−/− mice, a model of atherosclerosis. Our objectives were therefore to determine (i) if hypercholesterolaemia alters Ca2+ signalling in aortic endothelial cells before overt atherosclerotic lesions occur, (ii) how Ca2+ signals are affected in older plaque-containing mice, and (iii) whether Ca2+ signalling changes were translated into contractility differences. Using confocal microscopy we found agonist-specific Ca2+ changes in endothelial cells. ATP responses were unchanged in ApoE−/− cells and methyl-β-cyclodextrin, which lowers cholesterol, was without effect. In contrast, Ca2+ signals to carbachol were significantly increased in ApoE−/− cells, an effect methyl-β-cyclodextrin reversed. Ca2+ signals were more oscillatory and store-operated Ca2+ entry decreased as mice aged and plaques formed. Despite clearly increased Ca2+ signals, aortic rings pre-contracted with phenylephrine had impaired relaxation to carbachol. This functional deficit increased with age, was not related to ROS generation, and could be partially rescued by methyl-β-cyclodextrin. In conclusion, carbachol-induced calcium signalling and handling are significantly altered in endothelial cells of ApoE−/− mice before plaque development. We speculate that reduction in store-operated Ca2+ entry may result in less efficient activation of eNOS and thus explain the reduced relaxatory response to CCh, despite the enhanced Ca2+ response

Atherosclerosis differentially affects calcium signalling in endothelial cells from aortic arch and thoracic aorta in Apolipoprotein E knockout mice

Apolipoprotein-E knockout (ApoE-/-) mice develop hypercholesterolemia and are a useful model of atherosclerosis. Hypercholesterolemia alters intracellular Ca2+ signalling in vascular endothelial cells but our understanding of these changes, especially in the early stages of the disease process, is limited. We therefore determined whether carbachol-mediated endothelial Ca2+ signals differ in plaque-prone aortic arch compared to plaque-resistant thoracic aorta, of wild-type and ApoE-/- mice, and how this is affected by age and the presence of hypercholesterolemia. The extent of plaque development was determined using en-face staining with Sudan IV. Tissues were obtained from wild-type and ApoE-/- mice at 10 weeks (pre-plaques) and 24 weeks (established plaques). We found that even before development of plaques, significantly increased Ca2+ responses were observed in arch endothelial cells. Even with aging and plaque formation, ApoE-/- thoracic responses were little changed, however a significantly enhanced Ca2+ response was observed in arch, both adjacent to and away from lesions. In wild-type mice of any age, 1-2% of cells had oscillatory Ca2+ responses. In young ApoE-/- and plaque-free regions of older ApoE-/-, this is unchanged. However a significant increase in oscillations (~13-15%) occurred in thoracic and arch cells adjacent to lesions in older mice. Our data suggest that Ca2+ signals in endothelial cells show specific changes both before and with plaque formation, that these changes are greatest in plaque-prone aortic arch cells, and that these changes will contribute to the reported deterioration of endothelium in atherosclerosis

Control of cAMP signalling in the cellular migration of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterised by a very high mortality rate and is the 4th most common cause of cancer death (Siegel et al., 2012). The disease initially develops asymptomatically, and at the time of diagnosis patients usually have multiple metastases (Rhim et al., 2012). It would therefore be highly desirable to develop treatments which specifically impede the ability of PDAC cells to metastasise by interfering with the cellular processes responsible for efficient cellular migration. Intracellular signalling cascades, which utilise various signalling proteins, ultimately lead to the appropriate cell coordination and enable efficient cellular motility. One such signalling pathway that participates in the regulation of migration is controlled by the second messenger cyclic adenosine monophosphate (cAMP) (Howe, 2004). Several effectors of cAMP have been found which include protein kinase A (PKA) (Tasken & Aandahl, 2004), exchange factors activated by cAMP (EPAC) (Bos, 2006), and cyclic nucleotide-regulated cation channels (Biel, 2009). PKA has been intimately linked with several cellular processes which contribute towards cell motility. In most cases, the various specific effects of PKA signalling require selective targeting of the kinase into microdomains through interaction with A-kinase-anchoring proteins (AKAPs) (Pidoux & Tasken, 2010). Other cAMP effectors such as EPAC have defined roles in controlling various aspects of migration, such as cellular adhesion to the extracellular matrix (Bos, 2005). The effect of modulating cAMP signalling on the rate of migration has been investigated in several cancer types. Interestingly the results obtained were rather varied; both inhibition and stimulation of migration was observed (Chen et al., 2008; Baljinnyam et al., 2009; Grandoch et al., 2009; Shaikh et al., 2012). However, the effect of cAMP, and its effectors, on the rate of migration has not been investigated in PDAC; this was the main aim of this study. Classical cAMP elevating agents such as forskolin and 3-Isobutyl-1-methylxanthine (IBMX), as well as the cAMP analogue 8-Bromoadenosine 3’5’-cyclic monophosphate (8Br-cAMP), were found to inhibit migration of the PANC-1 cells. The role of cAMP signalling was further supported by the results of experiments utilising cAMP FRET sensors, which were imaged in live single cells. Further characterisation of cAMP effects in 4 other diverse PDAC cell lines yielded similar results, indicating that the mechanism of inhibition was common to all PDAC cell types tested. PANC-1 cell invasion was also inhibited by cAMP elevation. I went on to investigate events such as cell ruffling and focal adhesion assembly, which are processes closely associated with cellular motility. Dual transfection with a cAMP sensor and GFP tagged paxillin revealed a relationship between cAMP elevation and the loss of paxillin from focal adhesions, which was quickly reversible upon cAMP returning back to basal levels. Using a similar approach, peripheral cell ruffling was found to be inhibited by intracellular cAMP elevation. These results indicated that the inhibition of migration upon cAMP elevation was likely to occur as a result of immediate signalling events (and not due to cAMP-dependent changes in gene expression). The final part of the project concentrated on the individual contribution of the downstream effectors of cAMP, with particular emphasis on selective PKA and EPAC modulation. Utilising both PKA and EPAC sensors, I determined the appropriate concentrations of N6-benzoyl-cAMP (6Bnz) and 8-pCPT-2’OMe-cAMP (8pCPT) required to achieve selective PKA and EPAC activation respectively. Interestingly, I found that the two effectors had opposing actions; EPAC activation was found to induce migration, while PKA was found to suppress migration. Further investigation utilised a potent and selective PKA inhibitor peptide (PKI), which upon expression was found to prevent inhibition of ruffling, paxillin loss from focal adhesions, and inhibition of migration in response to cAMP elevation. Furthermore, it was found that suppression of basal PKA activity had a tendency to induce migration. I also utilised a cell permeable peptide (st-Ht31) which inhibits PKA interaction with AKAPs, thus effectively reducing its function by uncoupling the kinase from its specific signalling microdomains. The resulting effect was found to be a large potentiation of PANC-1 migration, which further highlighted the importance of PKA activity in the control of migration

KATP channels in the nodose ganglia mediate the orexigenic actions of ghrelin

Ghrelin is the only known hunger signal derived from the peripheral tissues. Ghrelin overcomes the satiety signals evoked by anorexigenic molecules, such as cholecystokinin (CCK) and leptin, to stimulate feeding. The mechanisms by which ghrelin reduces the sensory signals evoked by anorexigenic hormones, which act via the vagus nerve to stimulate feeding, are unknown. Patch clamp recordings of isolated rat vagal neurons show that ghrelin hyperpolarizes neurons by activating K+ conductance. Administering a KATP channel antagonist or silencing Kir6.2, a major subunit of the KATP channel, abolished ghrelin inhibition in vitro and in vivo. Patch clamp studies show that ghrelin inhibits currents evoked by leptin and CCK‐8, which operate through independent ionic channels. The inhibitory actions of ghrelin were abolished by treating the vagal ganglia neurons with pertussis toxin, as well as phosphatidylinositol 3‐kinase (PI3K) or extracellular signal‐regulated kinase 1 and 2 (Erk1/2) small interfering RNA. In vivo gene silencing of PI3K and Erk1/2 in the nodose ganglia prevented ghrelin inhibition of leptin‐ or CCK‐8‐evoked vagal firing. Feeding experiments showed that silencing Kir6.2 in the vagal ganglia abolished the orexigenic actions of ghrelin. These data indicate that ghrelin modulates vagal ganglia neuron excitability by activating KATP conductance via the growth hormone secretagogue receptor subtype 1a–Gαi–PI3K–Erk1/2–KATP pathway. The resulting hyperpolarization renders the neurons less responsive to signals evoked by anorexigenic hormones. This provides a mechanism to explain the actions of ghrelin with respect to overcoming anorexigenic signals that act via the vagal afferent pathways.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113677/1/tjp6781.pd

Membrane Cholesterol Regulates Smooth Muscle Phasic Contraction

The regulation of contractile activity in smooth muscle cells involves rapid discrimination and processing of a multitude of simultaneous signals impinging on the membrane before an integrated functional response can be generated. The sarcolemma of smooth muscle cells is segregated into caveolar regions-largely identical with cholesterol-rich membrane rafts—and actin-attachment sites, localized in non-raft, glycerophospholipid regions. Here we demonstrate that selective extraction of cholesterol abolishes membrane segregation and disassembles caveolae. Simultaneous measurements of force and [Ca2+]i in rat ureters demonstrated that extraction of cholesterol resulted in inhibition of both force and intracellular Ca2+ signals. Considering the major structural reorganization of cholesterol-depleted sarcolemma, it is intriguing to note that decreased levels of membrane cholesterol are accompanied by a highly specific inhibition of phasic, but not tonic contractions. This implies that signalling cascades that ultimately lead to either phasic or tonic response may be spatially segregated in the plane of the sarcolemma. Replenishment of cholesterol restores normal contractile behavior. In addition, the tissue function is re-established by inhibiting the large-conductance K+-channel. Sucrose gradient ultracentrifugation in combination with Western blotting analysis demonstrates that its α-subunit is associated with detergent-resistant membranes, suggesting that the channel might be localized within the membrane rafts in vivo. These findings are important in understanding the complex signalling pathways in smooth muscle and conditions such as premature labor and hypertensio

Ion channel mechanisms of rat tail artery contraction-relaxation by menthol involving, respectively, TRPM8 activation and L-type Ca2+ channel inhibition

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 μM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 μM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two other common TRPM8 agonists, WS-12 and icilin, also inhibited L-ICa With respect to L-ICa inhibition, WS-12 is the most selective agonist. It augmented PE-induced contractions, whereas any secondary phase of vasorelaxation (as with menthol) was completely lacking. Thus TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels and largely obscure TRPM8-mediated vasoconstriction. These findings will promote our understanding of the vascular TRPM8 role, especially the well-known hypotensive effect of menthol, and may also have certain translational implications (e.g., in cardiovascular surgery, organ storage, transplantation, and Raynaud's phenomenon)

Leptin Resistance in Vagal Afferent Neurons Inhibits Cholecystokinin Signaling and Satiation in Diet Induced Obese Rats

Background and Aims: The gastrointestinal hormone cholecystokinin (CCK) plays an important role in regulating meal size and duration by activating CCK1 receptors on vagal afferent neurons (VAN). Leptin enhances CCK signaling in VAN via an early growth response 1 (EGR1) dependent pathway thereby increasing their sensitivity to CCK. In response to a chronic ingestion of a high fat diet, VAN develop leptin resistance and the satiating effects of CCK are reduced. We tested the hypothesis that leptin resistance in VAN is responsible for reducing CCK signaling and satiation. Results: Lean Zucker rats sensitive to leptin signaling, significantly reduced their food intake following administration of CCK8S (0.22 nmol/kg, i.p.), while obese Zucker rats, insensitive to leptin, did not. CCK signaling in VAN of obese Zucker rats was reduced, preventing CCK-induced up-regulation of Y2 receptor and down-regulation of melanin concentrating hormone 1 receptor (MCH1R) and cannabinoid receptor (CB1). In VAN from diet-induced obese (DIO) Sprague Dawley rats, previously shown to become leptin resistant, we demonstrated that the reduction in EGR1 expression resulted in decreased sensitivity of VAN to CCK and reduced CCK-induced inhibition of food intake. The lowered sensitivity of VAN to CCK in DIO rats resulted in a decrease in Y2 expression and increased CB1 and MCH1R expression. These effects coincided with the onset of hyperphagia in DIO rats. Conclusions: Leptin signaling in VAN is required for appropriate CCK signaling and satiation. In response to high fat feeding

Escherichia coli-mediated impairment of ureteric contractility is uropathogenic E. coli specific.

BACKGROUND: Ureters are fundamental for keeping kidneys free from uropathogenic Escherichia coli (UPEC), but we have shown that 2 strains (J96 and 536) can subvert this role and reduce ureteric contractility. To determine whether this is (1) a widespread feature of UPEC, (2) exhibited only by UPEC, and (3) dependent upon type 1 fimbriae, we analyzed strains representing epidemiologically important multilocus sequence types ST131, ST73, and ST95 and non-UPEC E. coli. METHODS: Contractility and calcium transients in intact rat ureters were compared between strains. Mannose and fim mutants were used to investigate the role of type 1 fimbriae. RESULTS: Non-UPEC had no significant effect on contractility, with a mean decrease after 8 hours of 8.8%, compared with 8.8% in controls. UPEC effects on contractility were strain specific, with decreases from 9.47% to 96.7%. Mannose inhibited the effects of the most potent strains (CFT073 and UTI89) but had variable effects among other UPEC strains. Mutation and complementation studies showed that the effects of the UTI89 cystitis isolate were fimH dependent. CONCLUSIONS: We find that (1) non-UPEC do not affect ureteric contractility, (2) impairment of contractility is a common feature of UPEC, and (3) the mechanism varies between strains, but for the most potent UPEC type 1 fimbriae are involved