Transient Ca2+-activated Cl-currents with endothelin in isolated arteriolar smooth muscle cells of the choroid

To characterize the effects of endothelin (ET)-1 on the Ca2+-activated Cl- conductance of choroidal arteriolar smooth muscle.

Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Activation of the cGMP/PKG pathway inhibits electrical activity in rabbit urethral interstitial cells of Cajal by reducing the spatial spread of Ca2+ waves.

In the present study we used a combination of patch clamping and fast confocal Ca2+ imaging to examine the effects of activators of the nitric oxide (NO)/cGMP pathway on pacemaker activity in freshly dispersed ICC from the rabbit urethra, using the amphotericin B perforated patch configuration of the patch-clamp technique. The nitric oxide donor, DEA-NO, the soluble guanylyl cyclase activator YC-1 and the membrane-permeant analogue of cGMP, 8-Br-cGMP inhibited spontaneous transient depolarizations (STDs) and spontaneous transient inward currents (STICs) recorded under current-clamp and voltage-clamp conditions, respectively. Caffeine-evoked Cl- currents were unaltered in the presence of SP-8-Br-PET-cGMPs, suggesting that activation of the cGMP/PKG pathway does not block Cl- channels directly or interfere with Ca2+ release via ryanodine receptors (RyR). However, noradrenaline-evoked Cl- currents were attenuated by SP-8-Br-PET-cGMPs, suggesting that activation of cGMP-dependent protein kinase (PKG) may modulate release of Ca2+ via IP3 receptors (IP3R). When urethral interstitial cells (ICC) were loaded with Fluo4-AM (2 microm), and viewed with a confocal microscope, they fired regular propagating Ca2+ waves, which originated in one or more regions of the cell. Application of DEA-NO or other activators of the cGMP/PKG pathway did not significantly affect the oscillation frequency of these cells, but did significantly reduce their spatial spread. These effects were mimicked by the IP3R blocker, 2-APB (100 microm). These data suggest that NO donors and activators of the cGMP pathway inhibit electrical activity of urethral ICC by reducing the spatial spread of Ca2+ waves, rather than decreasing wave frequency.

Calcium oscillations in interstitial cells of the rabbit urethra.

Measurements were made (using fast confocal microscopy) of intracellular Ca2+ levels in fluo-4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca2+ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with D-myo-inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release using 100 microm 2-aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca2+, and frequency was directly proportional to the external Ca2+ concentration. Frequency of Ca2+ oscillation was reduced by SKF-96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca2+ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca2+ release from ryanodine-sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca2+ concentration and that conversion of the primary oscillation to a propagated Ca2+ wave depends upon IP3-induced Ca2+ release.

The alterations of Ca2+/Calmodulin/CaMKII/CaV1.2 signaling in experimental models of Alzheimer's disease and vascular dementia

The two critical forms of dementia are Alzheimer's disease (AD) and vascular dementia (VD).The alterations of Ca2+/calmodulin/CaMKII/CaV1.2 signaling in AD and VD have not been well elucidated. Here we have demonstrated changes in the levels of CaV1.2, calmodulin, p-CaMKII, p-CREB and BDNF proteins by Western blot analysis and the co-localization of p-CaMKII/CaV1.2 by double-labeling immunofluorescence in the hippocampus of APP/PS1 mice and VD gerbils. Additionally, expression of these proteins and intracellular calcium levels were examined in cultured neurons treated with Aß1–42. The expression of CaV1.2 protein was increased in VD gerbils and in cultured neurons but decreased in APP/PS1 mice; the expression of calmodulin protein was increased in APP/PS1 mice and VD gerbils; levels of p-CaMKII, p-CREB and BDNF proteins were decreased in AD and VD models. The number of neurons in which p-CaMKII and CaV1.2 were co-localized, was decreased in the CA1 and CA3 regions in two models. Intracellular calcium was increased in the cultured neurons treated with Aß1–42. Collectively, our results suggest that the alterations in CaV1.2, calmodulin, p-CaMKII, p-CREB and BDNF can be reflective of an involvement in the impairment in memory and cognition in AD and VD models.

Advanced glycation endproduct modified basement membrane attenuates endothelin-1 induced [Ca2+]i signalling and contraction in retinal microvascular pericytes

PURPOSE: To assess the effects of advanced glycation endproduct (AGE) modification of vascular basement membrane (BM) on endothelin-1 (Et-1) induced intracellular [Ca2+] ([Ca2+]i) homeostasis and contraction in retinal microvascular pericytes (RMP). METHODS: RMPs were isolated from bovine retinal capillaries and propagated on AGE modified BM extract (AGE-BM) or non-modified native BM. Cytosolic Ca2+ was estimated using fura-2 microfluorimetry and cellular contraction determined by measurement of planimetric cell surface area. ETA receptor mRNA and protein expression was assessed by real time RT-PCR and western blotting, respectively. RESULTS: Exogenous endothelin-1 (Et-1) evoked rises in [Ca2+]i and contraction in RMPs were found to be mediated entirely through ETA receptor (ETAR) activation. Both peak and plateau phases of the Et-1 induced [Ca2+]i response and contraction were impaired in RMPs propagated on AGE modified BM. ETAR mRNA expression remained unchanged in RMPs exposed to native or AGE-BM, but protein expression for ETAR (66 kDa) was lower in the AGE exposed cells. CONCLUSIONS: These results suggest that substrate derived AGE crosslinks can influence RMP physiology by mechanisms which include disruption of ETA receptor signalling. AGE modification of vascular BMs may contribute to the retinal hemodynamic abnormalities observed during diabetes.

Role of ion channels and subcellular Ca2+ signalling in arachidonic acid induced dilation of pressurised retinal arterioles

Purpose: To investigate the mechanisms responsible for the dilatation of rat retinal arterioles in response to arachidonic acid (AA). Methods: Changes in the diameter of isolated, pressurized rat retinal arterioles were measured in the presence of AA alone and following pre-incubation with pharmacological agents inhibiting Ca2+ sparks and oscillations and K+ channels. Subcellular Ca2+ signals were recorded in arteriolar myocytes using Fluo-4-based confocal imaging. The effects of AA on membrane currents of retinal arteriolar myocytes were studied using whole-cell perforated patch clamp recording. Results: AA dilated pressurised retinal arterioles under conditions of myogenic tone. Eicosatetraynoic acid (ETYA) exerted a similar effect, but unlike AA, its effects were rapidly reversible. AA-induced dilation was associated with an inhibition of subcellular Ca2+ signals. Interventions known to block Ca2+ sparks and oscillations in retinal arterioles caused dilatation and inhibited AA-induced vasodilator responses. AA accelerated the rate of inactivation of the A-type Kv current and the voltage dependence of inactivation was shifted to more negative membrane potentials. It also enhanced voltage-activated and spontaneous BK currents, but only at positive membrane potentials. Pharmacological inhibition of A-type Kv and BK currents failed to block AA-induced vasodilator responses. AA suppressed L-type Ca2+ currents. Conclusions: These results suggest that AA induces retinal arteriolar vasodilation by inhibiting subcellular Ca2+ signalling activity in retinal arteriolar myocytes, most likely through a mechanism involving the inhibition of L-type Ca2+ channel activity. AA actions on K+ currents are inconsistent with a model in which K+ channels contribute to the vasodilator effects of AA.

T- and L-type Ca2+ currents in freshly dispersed smooth muscle cells from the human proximal urethra

<p>The purpose of the present study was to characterise Ca2+ currents in smooth muscle cells isolated from biopsy samples taken from the proximal urethra of patients undergoing surgery for bladder or prostate cancer. Cells were studied at 37 degreesC using the amphotericin B perforated-patch configuration of the patch-clamp technique. Currents were recorded using Cs+-rich pipette solutions to block K+ currents. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents, were present in these cells. When steady-state inactivation curves for the L current were fitted with a Boltzmann equation, this yielded a V-1/2 of -45 +/- 5 mV. In contrast, the T current inactivated with a V-1/2 of -80 +/- 3 mV. The L currents were reduced in a concentration-dependent manner by nifedipine (ED50 = 159 +/- 54 nm) and Ni2+ (ED50 = 65 +/- 16 muM) but were enhanced when external Ca2+ was substituted with Ba2+. The T current was little affected by TTX, reduction in external Na+, application of nifedipine at concentrations below 300 nm or substitution of external Ca2+ with Ba2+, but was reduced by Ni2+ with an ED50 of 6 +/- 1 mum. When cells were stepped from -100 to -30 mV in Ca2+-free conditions, small inward currents could be detected. These were enhanced 40-fold in divalent-cation-free solution and blocked in a concentration-dependent manner by Mg2+ with an ED50 of 32 +/- 16 mum. These data support the idea that human urethral myocytes possess currents with electrophysiological and pharmacological properties typical of T- and L-type Ca2+ currents.</p>

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

<p>Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months' streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy.</p>

Energy-Efficient In-Memory Data Stores on Hybrid Memory Hierarchies

Increasingly large amounts of data are stored in main memory of data center servers. However, DRAM-based memory is an important consumer of energy and is unlikely to scale in the future. Various byte-addressable non-volatile memory (NVM) technologies promise high density and near-zero static energy, however they suffer from increased latency and increased dynamic energy consumption.<br/><br/>This paper proposes to leverage a hybrid memory architecture, consisting of both DRAM and NVM, by novel, application-level data management policies that decide to place data on DRAM vs. NVM. We analyze modern column-oriented and key-value data stores and demonstrate the feasibility of application-level data management. Cycle-accurate simulation confirms that our methodology reduces the energy with least performance degradation as compared to the current state-of-the-art hardware or OS approaches. Moreover, we utilize our techniques to apportion DRAM and NVM memory sizes for these workloads.