Agonist-evoked Changes Research Articles

Cholesterol Enrichment Impairs Capacitative Calcium Entry, eNOS Phosphorylation & Shear Stress-Induced NO Production.

Endothelial dysfunction, characterized by decreased production or availability of nitric oxide (NO), is widely believed to be the hallmark of early-stage atherosclerosis. In addition, hypercholesterolemia is considered a major risk factor for development of atherosclerosis and is associated with impaired flow-induced dilation. However, the mechanism by which elevated cholesterol levels leads to decreased production of NO is unclear. NO is released in response to shear stress and agonist-evoked changes in intracellular calcium. Although calcium signaling is complex, we have previously shown that NO production by endothelial nitric oxide synthase (eNOS) is preferentially activated by calcium influx via store-operated channels. We hypothesized that cholesterol enrichment altered this signaling pathway (known as capacitive calcium entry; CCE) ultimately leading to decreased NO. Our results show that cholesterol enrichment abolished ATP-induced eNOS phosphorylation and attenuated the calcium response by the preferential inhibition of CCE. Furthermore, cholesterol enrichment also inhibited shear stress-induced NO production and eNOS phosporylation, consistent with our previous results showing a significant role for ATP autocrine stimulation and subsequent activation of CCE in the endothelial flow response.

A role for nitric oxide-mediated glandular hypofunction in a non-apoptotic model for Sjogren's syndrome

To investigate a role for the inflammatory mediator, nitric oxide (NO) in SS, an autoimmune condition characterized by salivary and lacrimal gland hypofunction resulting from failure of acinar cells to secrete. FURA-2 microfluorimetry was used to measure agonist-evoked changes of [Ca(2+)](i) in isolated mouse and human salivary acinar cells following exposure to NO donors. NO had a biphasic effect on salivary acinar function. Acute exposure to NO (2 min) caused a cyclic guanosine monophosphate (GMP)-dependent, 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-sensitive increase in the Ca(2+) signal elicited in response to acetylcholine (ACh) stimulation, consistent with stimulation of ryanodine receptors by cyclic adenosine diphosphate ribose. Prolonged exposure to NO (>40 min) significantly reduced the ACh-evoked Ca(2+) signal by a mechanism independent of cyclic GMP. We found no differences between the responses of human and mouse acinar cells. Our data show that chronic exposure to NO, which is known to be elevated in SS, could have a role in salivary gland hypofunction. We note a similarity in the response to stimulation of salivary acinar exposed to NO and that which we have previously reported in salivary acinar cells isolated from patients with SS. We speculate that NO-mediated nitrosylation of one or more elements of the signal transduction pathway could underlie down-regulation of salivary function in SS.

Kinetic Analysis of the Interaction of the C1 Domain of Protein Kinase C with Lipid Membranes by Stopped-flow Spectroscopy

The diacylglycerol (DG)/phorbol ester-dependent translocation of conventional protein kinase C (PKC) isozymes is mediated by the C1 domain, a membrane-targeting module that also selectively binds phosphatidylserine (PS). Using stopped-flow spectroscopy, we dissect the contribution of DG/phorbol esters (C1 ligand) and PS in driving the association and dissociation of the C1 domain from membranes. Specifically, we examine the binding to membranes of the C1B domain of PKCbeta with a substituted Trp (Y123W) whose fluorescence is quenched upon binding to membranes. Binding of this construct (C1Bbeta-Y123W) to phospholipid vesicles is cooperative with respect to PS content and dependent on C1 ligand, as previously characterized. Stopped-flow analysis reveals that the apparent association rate (k(on)(app)), but not the apparent dissociation rate (k(off)(app)), is highly sensitive to PS content: the 60-fold increase in membrane affinity for vesicles containing no PS compared with 40 mol % PS results primarily from a robust (30-fold) increase in k(on)(app) with little effect (2-fold) on k(off)(app). Membrane affinity is also controlled by the content and structure of the C1 ligand. In contrast to PS, these ligands markedly alter k(off)(app) with smaller effects on k(on)(app). We also show that the affinity for phorbol ester-containing membranes is 2 orders of magnitude higher than that for DG-containing membranes primarily resulting from differences in k(off)(app). Our data are consistent with a model in which the C1 domain is recruited to the membrane via an initial weak electrostatic interaction with PS, followed by a rapid two-dimensional search for ligand, the binding of which retains the domain at the membrane. Thus, PS drives the initial encounter, and DG/phorbol esters retain the domain on membranes. The decreased effectiveness of DG compared with phorbol esters in retaining the C1 domain on membranes contributes to the molecular dichotomy of the rapid, transient nature of DG-dependent PKC signaling versus the chronic hyperactivity of phorbol ester-activated PKC.

Two-Photon Imaging of Stroke OnsetIn VivoReveals That NMDA-Receptor Independent Ischemic Depolarization Is the Major Cause of Rapid Reversible Damage to Dendrites and Spines

We adapt a mouse global ischemia model to permit rapid induction of ischemia and reperfusion in conjunction with two-photon imaging to monitor the initial ionic, structural, and functional implications of brief interruptions of blood flow (6-8 min) in vivo. After only 2-3 min of global ischemia, a wide spread loss of mouse somatosensory cortex apical dendritic structure is initiated during the passage of a propagating wave (3.3 mm/min) of ischemic depolarization. Increases in intracellular calcium levels occurred during the wave of ischemic depolarization and were coincident with the loss of dendritic structure, but were not triggered by reperfusion. To assess the role of NMDA receptors, we locally applied the antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate] at concentrations sufficient to fully block local NMDA agonist-evoked changes in intracellular calcium levels in vivo. Changes in dendritic structure and intracellular calcium levels were independent of NMDA receptor activation. Local application of the non-NMDA glutamate receptor antagonist CNQX also failed to block ischemic depolarization or rapid changes in dendrite structure. Within 3-5 min of reperfusion, damage ceased and restoration of synaptic structure occurred over 10-60 min. In contrast to a reperfusion promoting damage, over this time scale, the majority of spines and dendrites regained their original structure during reperfusion. Intrinsic optical signal imaging of sensory evoked maps indicated that reversible alteration in dendritic structure during reperfusion was accompanied by restored functional maps. Our results identify glutamate receptor-independent ischemic depolarization as the major ionic event associated with disruption of synaptic structure during the first few minutes of ischemia in vivo.

Functional characterization of mouse α4β2 nicotinic acetylcholine receptors stably expressed in HEK293T cells

Mouse alpha4beta2 nicotinic acetylcholine receptors (nAchRs) were stably expressed in HEK293T cells. The function of this stable cell line, termed mmalpha4beta2, was assessed using an aequorin-based luminescence method that measures agonist-evoked changes in intracellular calcium. Agonist-elicited changes in intracellular calcium were due primarily to direct entry of calcium through the alpha4beta2 channel, although release of calcium from intracellular stores contributed approximately 28% of the agonist-evoked response. Agonist pharmacologies were very similar between the mmalpha4beta2 cells and most cell lines that stably express human alpha4beta2 nAchRs. Based on agonist profiles and sensitivity to the antagonist dihydro-beta-erythroidine (DHbetaE), the predominant alpha4beta2 nAchR expressed in the mmalpha4beta2 cells exhibits a pharmacology that most resembles the DHbetaE-sensitive component of 86Rb+ efflux from mouse brain synaptosomes. However, when evaluated with the aequorin assay, the mmalpha4beta2 nAchR was found to be atypically sensitive to blockade by the presumed alpha7-selective antagonist methyllycaconitine (MLA), exhibiting an IC50 value of 31 +/- 0.1 nm. Similar IC50 values have been reported for the MLA inhibition of nicotine-stimulated dopamine release, a response that is mediated by beta2-subunit-containing nAchRs and not alpha7-subunit-containing nAchRs. Consequently, at low nanomolar concentrations, MLA may not be as selective for alpha7-containing nAchRs as previously thought.

Acetylcholine-evoked calcium mobilization and ion channel activation in human labial gland acinar cells from patients with primary Sjögren's syndrome.

Recent evidence has indicated that the salivary gland dysfunction associated with Sjögren's syndrome (SjS) is not necessarily due to immune-mediated destruction of acinar tissue. SjS sufferers may possess substantial reserves of acinar tissue but nevertheless be incapable of maintaining salivary flow rates in the normal range. We have investigated the ability of isolated labial gland acinar cells from SjS patients to fluid secrete by measuring agonist-evoked changes in intracellular Ca(2+) ([Ca(2+)](i)) using fura-2 microfluorimetry and activation of K(+) and Cl(-) channels using the patch-clamp whole cell technique. We can confirm that stimulation with a super-maximal dose of acetylcholine (ACh) increased [Ca(2+)]i equally in both control acinar cells and those derived from SjS patients. However, at submaximal concentrations, the dose-response curve for ACh was shifted to the right by approximately one order of magnitude in acinar cells from SjS patients compared to control acinar cells. Patch-clamp measurements consistent with the presence of Ca(2+)-activated K(+) and Cl(-) conductances were obtained from both control acinar cells and those obtained from SjS patients. Dose-dependent activation of the ion channels by acetylcholine was also right-shifted in acinar cells from SjS patients compared to control cells. Our data show that labial gland acinar cells from SjS patients were capable of responding to agonist stimulation by mobilizing [Ca(2+)](i) and activating K(+) and Cl(-) channels consistent with the requirements of fluid secretion. However, the persistent loss of sensitivity to ACh observed in from SjS patients may account for the lack of saliva production observed in these patients in vivo.

An investigation of interactions between the immune system and stimulus-secretion coupling in mouse submandibular acinar cells. A possible mechanism to account for reduced salivary flow rates associated with the onset of Sjögren's syndrome.

To determine whether chronic exposure to lymphocyte-derived cytokines could inhibit the fluid secretory mechanism in salivary gland acinar cells and so account for the loss of gland function seen in the early stages of Sjögren's syndrome. Mouse submandibular acinar cells maintained in primary culture were exposed to a profile of cytokines produced by concanavalin A-activated splenic lymphocytes in vitro for periods up to 72 h. Agonist-evoked changes in intracellular Ca(2+) were determined microfluorimetrically in both control and cytokine-treated cells. Acinar cells maintained in primary culture in the presence of cytokines for up to 72 h were able to mobilize intracellular calcium in response to stimulus by acetylcholine in an identical fashion to those maintained in primary culture in the absence of cytokines. Acute application of the conditioned medium produced by the activated lymphocytes had an antisecretory effect on acetylcholine-evoked Ca(2+) mobilization, which was found to be mediated by cholinesterase rather than by cytokines. Neither chronic nor acute exposure to the profile of cytokines released by concanavalin A-activated splenic lymphocytes interfered in any way with the second messenger cascade and fluid and electrolyte secretion in acinar cells. Our data suggest an alternative hypothesis, in which elevated levels of cholinesterase can metabolize acetylcholine released within the salivary glands and thus prevent fluid secretion.

Exposing guinea pigs to ozone for 1 wk enhances responsiveness of rapidly adapting receptors.

Acute exposure to ozone causes changes in breathing pattern and lung function which may be caused in part by stimulation of rapidly adapting receptors (RARs). The consequences of repeated daily ozone exposure on RAR responsiveness are unknown, although ozone-induced changes in pulmonary function diminish with repeated exposure. Accordingly, we investigated whether repeated daily ozone exposure diminishes the general responsiveness of RARs. Guinea pigs (n = 30) were exposed to 0.5 parts/million ozone or filtered air (8 h/day for 7 days). The animals were then anesthetized, and RAR impulse activity, dynamic compliance (Cdyn), and lung resistance were recorded at baseline and in response to four stimuli: substance P, methacholine, hyperinflation, and removal of positive end-expiratory pressure. Repeated daily ozone exposure exaggerated RAR responses to substance P, methacholine, and hyperinflation without causing physiologically relevant effects on baseline or substance P- and methacholine-induced changes in Cdyn and lung resistance. Because agonist-evoked changes in RAR activity preceded Cdyn changes, the data suggest that repeated daily ozone exposure enhances RAR responsiveness via a mechanism other than changes in Cdyn.

Development of a functional assay for Ca 2+ release activity of IP 3R and expression of an IP 3R gene fragment in the baculovirus-insect cell system

Receptor-stimulated phosphoinositide (PI) hydrolysis is an important and ubiquitous mechanism of intracellular signaling. Inositol 1,4,5-trisphosphate (IP 3), generated by phosphoinositide (PI) hydrolysis, binds to and gates an intracellular Ca 2+ channel, the IP 3 receptor (IP 3R), which is therefore a central component of this signaling cascade. Here we describe the development of a baculovirus (BV)/ Sf(S. frugiperda) cell system that can be used to look at IP 3R function. Agonist-evoked changes in intracellular Ca 2+ levels [Ca 2+] i were measured (using Fura2) in Sf cells expressing the gene encoding the muscarinic acetylcholine receptor (vmlAchR). Furthermore, we have constructed a recombinant BV (vlP 3R) , with the core of the IP 3R ligand-binding domain from the Drosophila IP 3R, under the polyhedrin promoter. The recombinant protein from such a virus was expected to act as a large ligand sink for IP3, generated by stimulation of vmlAchR. Cells coinfected with recombinant BV carrying the potential dominant-negative vIP 3R construct and vmlAchR have been used to assay the modulation of IP 3R-mediated Ca 2+ release, by the ligand sink.

Inhibitory action of fatty acids on calcium fluxes in thyroid FRTL-5 cells

In the present study, we wanted to investigate the action of fatty acids on agonist-evoked changes in intracellular free calcium ([Ca 2+] i) in thyroid FRTL-5 cells. Stimulating Fura 2 loaded cells with long chain unsaturated fatty acids increased [Ca 2+] i in a dose-dependent manner. This increase was in part dependent on extracellular calcium. Long chain saturated fatty acids and short chain fatty acids had no effects on [Ca 2+] i per se. Pretreatment of the cells with long chain unsaturated fatty acids almost totally inhibited both the ATP- and thapsigargin-evoked release of sequestered calcium and the entry of extracellular calcium. Long chain saturated fatty acids also attenuated the ATP-evoked increase in [Ca 2+] i, while short chain fatty acids had no effects on the ATP-evoked change in [Ca 2+] i. The inhibitory effect of long chain unsaturated fatty acids on agonist-evoked changes in [Ca 2+] i was not dependent on activation of protein kinase C, and was not due to an enhanced efflux of calcium. These fatty acids rapidly acidified the cytosol in the cells, which could, in part, explain the inhibitory effect of the long chain unsaturated fatty acids on agonist-evoked changes in [Ca 2+] i. Addition of bovine serum albumin to the cells rapidly reversed the inhibitory effect of the fatty acids on [Ca 2+] i, and restored pH i. Thus, fatty acids could be potential modulators of calcium signaling in FRTL-5 cells, possibly by modulating calcium entry at the level of the plasma membrane.

Rapid ADP-evoked currents in human platelets recorded with the nystatin permeabilized patch technique.

"Whole-cell" patch recordings using nystatin permeabilization were made from single human platelets during application of agonists from a "puffer" pipette. In platelets clamped near the resting potential and bathed in Na+ saline, 40 microM ADP activated a transient inward current within tens of milliseconds. At -73 mV the current lasted between 0.1 and 1 s and had a peak of between 13 and 31 pA in different cells. Ion substitution experiments indicated that the channel is permeable to Na+,K+, and Ba2+ and presumably also to Ca2+, but is not permeable to Cl-. The single channel conductance was 15 pS (near the resting potential) in nominally Ca(2+)-free saline and 11 picosiemens in BaCl2 saline. Thrombin, at 1 unit/ml, did not elicit detectable currents during a 3-s application in platelets bathed in 1 mM Ca2+, Na+ saline. Under the same conditions, in fura-2-loaded cells, thrombin-evoked Ca2+ entry (monitored by Mn2+ quench) was detectable after a delay of 1.4 s. This suggests that early thrombin-evoked Ca2+ entry occurs via small conductance channels, below the resolution of the patch clamp technique, or by an electroneutral pathway. The ADP-evoked channel has the requisite speed of activation to account for the rapid Ca2+ influx observed during stopped-flow studies of agonist-evoked changes in [Ca2+]i.

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils

The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.

Ionic mechanisms of action of GABA on dorsal and ventral root myelinated fibers: effects of K+ channel blockers

Excitability changes evoked by the inhibitory neurotransmitter, GABA (gamma-aminobutyric acid) in myelinated axons of dorsal and ventral roots of the isolated bullfrog sciatic nerve were compared in the absence and presence of K+ channel blockers. Half-maximal A-fiber responses to a 0.5-Hz stimulation of the whole nerve were recorded from individual roots. Direct applications of Ringer with raised K+ levels to the site of stimulation caused increases in excitability of both dorsal and ventral root fibers, which resembled those evoked in the ventral root by the GABA agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]ol). The increases in dorsal root fiber responses produced by GABA were depressed by tetraethylammonium (TEA) (3 mM), 4-aminopyridine (4-AP) (50 microM), Cs (2 mM), and Ba (1 mM). Ventral root fibers were less consistently affected. The early component of GABA-evoked excitability increases was depressed by 4-AP, Cs, and Ba, but greatly augmented by TEA. THIP-evoked changes in the excitability of the dorsal and ventral root fibers were, respectively, depressed and enhanced by TEA. The augmenting effect of TEA on the early component of GABA agonist effects on the ventral root fibers is attributed to their high resting K+ conductance and the presence of a slowly inactivating, fast K+ current (If1). The depressant effects of K+ channel blockade on depolarizing components of agonist-evoked changes in dorsal and ventral root responses indicate interference with release and (or) sensitivity to K+ and a possible contribution from a mechanism involving voltage-dependent delayed rectifier K+ currents.

Spontaneous Oscillations and Agonist-Evoked Changes in Ca2+ in Cultured Smooth Muscle Cells

In confluent A10 aortic smooth muscle cells, at 37°C and in the presence of 1 mM extracellular Ca2+ ([Ca2+ ]0), the resting intracellular free calcium ([Ca2+]i) was found to be 111 ± 6 nM (n = 17), and in single cells it was 82.4 ± 7.6 nM. In the presence of 1 mM EGTA, in confluent cells, resting [Ca2+]i was 88.1 ± 11 nM. The confluent cells showed spontaneous transients and elevations in ([Ca2+ ]i) as well as maintained oscillations. The oscillations had a periodicity of 6–9 s and were not present in single cells. One nanomolar and 2 μM vasopressin elevated [Ca2+]i to between 0.7 and 3 μM. In the presence of extracellular Ca2+, the initial transient was followed by a plateau phase that declined slowly; in EGTA, this plateau phase was not present but could be restored by adding back Ca2+. Although A10 cells show spontaneous action potentials, oscillations in [Ca2+]i have not been reported. The plateau phase of the vasopressin response appears to be mediated by Ca2+ influx, whereas the initial transient is consistent with the release of stored Ca2+.

Spontaneous Oscillations and Agonist-Evoked Changes in Ca2+ in Cultured Smooth Muscle Cells

In confluent A10 aortic smooth muscle cells, at 37 degrees C and in the presence of 1 mM extracellular Ca2+ ([Ca2+]o), the resting intracellular free calcium [Ca2+]i) was found to be 111 +/- 6 nM (n = 17), and in single cells it was 82.4 +/- 7.6 nM. In the presence of 1 mM EGTA, in confluent cells, resting [Ca2+]i was 88.1 +/- 11 nM. The confluent cells showed spontaneous transients and elevations in [( Ca2+]i) as well as maintained oscillations. The oscillations had a periodicity of 6-9 s and were not present in single cells. One nanomolar and 2 microM vasopressin elevated [Ca2+]i to between 0.7 and 3 microM. In the presence of extracellular Ca2+, the initial transient was followed by a plateau phase that declined slowly; in EGTA, this plateau phase was not present but could be restored by adding back Ca2+. Although A10 cells show spontaneous action potentials, oscillations in [Ca2+]i have not been reported. The plateau phase of the vasopressin response appears to be mediated by Ca2+ influx, whereas the initial transient is consistent with the release of stored Ca2+.