Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with spontaneous calcium oscillations and enhanced calcium responses following endothelin-1 stimulation

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P) and thereby regulates cellular transformation. To investigate the role Ins(1,4,5)P-mediated Ca oscillations play in cellular transformation, we studied Ins(1,4,5)P-mediated Ca responses in cells underexpressing the 43 kDa 5-phosphatase. Chronic reduction in 43 kDa 5-phosphatase enzyme activity resulted in a 2.6-fold increase in the resting Ins(1,4,5)P concentration and a 4.1-fold increase in basal intracellular Ca. The increased Ins(1,4,5)P levels resulted in partial emptying (40%) of the Ins(1,4,5)P-sensitive Ca store, however, store-operated Ca influx remained unchanged. In addition, Ins(1,4,5)P receptors were chronically downregulated in unstimulated cells, as shown by a 53% reduction in [H]Ins(1,4,5)P binding to microsomal receptor sites. Agonist stimulation with endothelin-1 resulted in the rapid rise and fall of Ins(1,4,5)P and Ins(1,3,4,5)P levels, with no significant differences in the rates of hydrolysis of these second messengers in antisenseor vector-transfected cells. These studies indicate, in contrast to its predicted action, the 43 kDa 5-phosphatase does not metabolise Ins(1,4,5)P and Ins(1,3,4,5)P post agonist stimulation. Cells with decreased 43 kDa 5-phosphatase activity exhibited spontaneous Ca oscillations in the absence of any agonist stimulation, and increased sensitivity and amplitude of intracellular Ca responses to both high and low dose endothelin-l stimulation. We conclude the 43 kDa 5-phosphatase exerts a profound influence on Ins(1,4,5)P-induced Ca spiking, both in the unstimulated cell and following agonist stimulation. We propose the enhanced Ca oscillations may mediate cellular transformation in cells underexpressing the 43 kDa 5-phosphatase

Multiple Neural Oscillators and Muscle Feedback Are Required for the Intestinal Fed State Motor Program

After a meal, the gastrointestinal tract exhibits a set of behaviours known as the fed state. A major feature of the fed state is a little understood motor pattern known as segmentation, which is essential for digestion and nutrient absorption. Segmentation manifests as rhythmic local constrictions that do not propagate along the intestine. In guinea-pig jejunum in vitro segmentation constrictions occur in short bursts together with other motor patterns in episodes of activity lasting 40–60 s and separated by quiescent episodes lasting 40–200 s. This activity is induced by luminal nutrients and abolished by blocking activity in the enteric nervous system (ENS). We investigated the enteric circuits that regulate segmentation focusing on a central feature of the ENS: a recurrent excitatory network of intrinsic sensory neurons (ISNs) which are characterized by prolonged after-hyperpolarizing potentials (AHPs) following their action potentials. We first examined the effects of depressing AHPs with blockers of the underlying channels (TRAM-34 and clotrimazole) on motor patterns induced in guinea-pig jejunum, in vitro, by luminal decanoic acid. Contractile episode durations increased markedly, but the frequency and number of constrictions within segmenting bursts and quiescent period durations were unaffected. We used these observations to develop a computational model of activity in ISNs, excitatory and inhibitory motor neurons and the muscle. The model predicted that: i) feedback to ISNs from contractions in the circular muscle is required to produce alternating activity and quiescence with the right durations; ii) transmission from ISNs to excitatory motor neurons is via fast excitatory synaptic potentials (EPSPs) and to inhibitory motor neurons via slow EPSPs. We conclude that two rhythm generators regulate segmentation: one drives contractions within segmentation bursts, the other the occurrence of bursts. The latter depends on AHPs in ISNs and feedback to these neurons from contraction of the circular muscle

Diet-Induced Obesity Impairs Endothelium-Derived Hyperpolarization via Altered Potassium Channel Signaling Mechanisms

BACKGROUND: The vascular endothelium plays a critical role in the control of blood flow. Altered endothelium-mediated vasodilator and vasoconstrictor mechanisms underlie key aspects of cardiovascular disease, including those in obesity. Whilst the mechanism of nitric oxide (NO)-mediated vasodilation has been extensively studied in obesity, little is known about the impact of obesity on vasodilation to the endothelium-derived hyperpolarization (EDH) mechanism; which predominates in smaller resistance vessels and is characterized in this study. METHODOLOGY/PRINCIPAL FINDINGS: Membrane potential, vessel diameter and luminal pressure were recorded in 4(th) order mesenteric arteries with pressure-induced myogenic tone, in control and diet-induced obese rats. Obesity, reflecting that of human dietary etiology, was induced with a cafeteria-style diet (∼30 kJ, fat) over 16-20 weeks. Age and sexed matched controls received standard chow (∼12 kJ, fat). Channel protein distribution, expression and vessel morphology were determined using immunohistochemistry, Western blotting and ultrastructural techniques. In control and obese rat vessels, acetylcholine-mediated EDH was abolished by small and intermediate conductance calcium-activated potassium channel (SK(Ca)/IK(Ca)) inhibition; with such activity being impaired in obesity. SK(Ca)-IK(Ca) activation with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) and 1-ethyl-2-benzimidazolinone (1-EBIO), respectively, hyperpolarized and relaxed vessels from control and obese rats. IK(Ca)-mediated EDH contribution was increased in obesity, and associated with altered IK(Ca) distribution and elevated expression. In contrast, the SK(Ca)-dependent-EDH component was reduced in obesity. Inward-rectifying potassium channel (K(ir)) and Na(+)/K(+)-ATPase inhibition by barium/ouabain, respectively, attenuated and abolished EDH in arteries from control and obese rats, respectively; reflecting differential K(ir) expression and distribution. Although changes in medial properties occurred, obesity had no effect on myoendothelial gap junction density. CONCLUSION/SIGNIFICANCE: In obese rats, vasodilation to EDH is impaired due to changes in the underlying potassium channel signaling mechanisms. Whilst myoendothelial gap junction density is unchanged in arteries of obese compared to control, increased IK(Ca) and Na(+)/K(+)-ATPase, and decreased K(ir) underlie changes in the EDH mechanism

Remodeling of Purinergic Receptor-Mediated Ca2+ Signaling as a Consequence of EGF-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca 2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR TETRA) and observed significant changes in the potency of ATP (EC 50 0.175 μM (-EGF) versus 1.731 μM (+EGF), P<0.05), and the nature of the ATP-induced Ca 2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca 2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca 2+ homeostasis. To determine whether changes in ATP-mediated Ca 2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X 5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X 5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis

Endothelin-1 and endothelin-3 stimulate calcium mobilization by different mechanisms in vascular smooth muscle

The mechanisms by which endothelin-1 (ET-1) and endothelin-3 (ET-3) stimulate Ca2+ mobilization were investigated in rat aortic smooth muscle cells. Both ET-1 and ET-3 potently stimulated mobilization of Ca2+ from intracellular stores, however only ET-1-stimulated Ca2+ mobilization appeared to occur as a consequence of an elevation in cellular inositol trisphosphate (IP3) concentration. Neomycin, an inhibitor of phospholipase C, inhibited both the increase in [3H]IP3 formation and the mobilization of Ca2+ induced by ET-1, however it did not affect Ca2+ mobilization induced by ET-3. Together these findings indicate that ET-1 stimulates Ca2+ mobilization via an increase in IP3, whereas the effect of ET-3 appears to be mediated by a separate, IP3-independent signalling pathway

Intracellular Ph in Human Arterial Smooth-Muscle - Regulation by Na+/h+ Exchange and a Novel 5-(N-Ethyl-N-Isopropyl)Amiloride-Sensitive Na+-Dependent and Hco3--Dependent Mechanism

We investigated in a physiological salt solution (PSS) containing HCO the intracellular pH (pH(i)) regulating mechanisms in smooth muscle cells cultured from human internal mammary arteries, using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Na influx rates. The recovery of pH(i) from an equivalent intracellular acidosis was more rapid when the cells were incubated in CO/HCO-buffered PSS than in HEPES-buffered PSS. Recovery of pH(i) was dependent on extracellular Na (K(m), 13.1 mM); however, it was not attenuated by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), indicating the absence of SITS-sensitive HCO-dependent mechanisms. Recovery instead apparent mostly dependent on processes sensitive to 5-(N-ethyl-N-isopropyl)amiloride (EIPA), indicating the involvement of Na/H exchange and a previously undescribed EIPA-sensitive Na- and HCO-dependent mechanism. Differentiation between this HCO-dependent mechanism and Na/H exchange was achieved after depletion of cellular ATP. Under these conditions, the NHCl-induced Na influx rate stimulated by intracellular acidosis was markedly attenuated in HEPES-buffered PSS but not in CO/HCO-buffered PSS. EIPA also appeared to inhibit the two mechanisms differentially. In HEPES-buffered PSS containing 20 mM Na, the EIPA inhibition curve for the intracellular acidosis-induced Na influx was monophasic (IC, 39 nM), whereas in an identical CO/HCO-buffered PSS, the inhibition curve exhibited biphasic characteristics (IC, 37.3 nM and 312 μM). Taken together, the results indicate that Na/H exchange and a previously undescribed EIPA-sensitive Na- and HCO-dependent mechanism play an important role in regulating the pH(i) of human vascular smooth muscle. The involvement of the latter mechanism depends on the severity of the intracellular acidosis, varying from approximately 25% in severe intracellular acidosis up to 50% at lesser, more physiological, levels of induced acidosis