Calcium-dependent modulation of BKCa channel activity induced by plasmonic gold nanoparticles in pulmonary artery smooth muscle cells and hippocampal neurons.

AIM: Gold nanoparticles are widely used for biomedical applications, but the precise molecular mechanism of their interaction with cellular structures is still unclear. Assuming that intracellular calcium fluctuations associated with surface plasmon-induced calcium entry could modulate the activity of potassium channels, we studied the effect of 5 nm gold nanoparticles on calcium-dependent potassium channels and associated calcium signaling in freshly isolated rat pulmonary artery smooth muscle cells and cultured hippocampal neurons. METHODS: Outward potassium currents were recorded using patch-clamp techniques. Changes in intracellular calcium concentration were measured using the high affinity Ca2+ fluorescent indicator fluo-3 and laser confocal microscope. RESULTS: In pulmonary artery smooth muscle cells, plasmonic gold nanoparticles increased the amplitude of currents via large-conductance Ca2+ -activated potassium channels, which was potentiated by green laser irradiation near plasmon resonance wavelength (532 nm). Buffering of intracellular free calcium with ethylene glycol-bis-N,N,N',N'-tetraacetic acid (EGTA) abolished these effects. Furthermore, using confocal laser microscopy it was found that application of gold nanoparticles caused oscillations of intracellular calcium concentration that were decreasing in amplitude with time. In cultured hippocampal neurons gold nanoparticles inhibited the effect of EGTA slowing down the decline of the BKCa current while partially restoring the amplitude of the slow after hyperpolarizing currents. CONCLUSION: We conclude that fluctuations in intracellular calcium can modulate plasmonic gold nanoparticles-induced gating of BKCa channels in smooth muscle cells and neurons through an indirect mechanism, probably involving the interaction of plasmon resonance with calcium-permeable ion channels, which leads to a change in intracellular calcium level.

Calcium signaling in endocardial and epicardial ventricular myocytes from streptozotocin-induced diabetic rats.

AIMS/INTRODUCTION: Abnormalities in Ca2+ signaling have a key role in hemodynamic dysfunction in diabetic heart. The purpose of this study was to explore the effects of streptozotocin (STZ)-induced diabetes on Ca2+ signaling in epicardial (EPI) and endocardial (ENDO) cells of the left ventricle after 5-6 months of STZ injection. MATERIALS AND METHODS: Whole-cell patch clamp was used to measure the L-type Ca2+ channel (LTCC) and Na+ /Ca2+ exchanger currents. Fluorescence photometry techniques were used to measure intracellular free Ca2+ concentration. RESULTS: Although the LTCC current was not significantly altered, the amplitude of Ca2+ transients increased significantly in EPI-STZ and ENDO-STZ compared with controls. Time to peak LTCC current, time to peak Ca2+ transient, time to half decay of LTCC current and time to half decay of Ca2+ transients were not significantly changed in EPI-STZ and ENDO-STZ myocytes compared with controls. The Na+ /Ca2+ exchanger current was significantly smaller in EPI-STZ and in ENDO-STZ compared with controls. CONCLUSIONS: STZ-induced diabetes resulted in an increase in amplitude of Ca2+ transients in EPI and ENDO myocytes that was independent of the LTCC current. Such an effect can be attributed, at least in part, to the dysfunction of the Na+ /Ca2+ exchanger. Additional studies are warranted to improve our understanding of the regional impact of diabetes on Ca2+ signaling, which will facilitate the discovery of new targeted treatments for diabetic cardiomyopathy.

Voltage dependence of the Ca2+ transient in endocardial and epicardial myocytes from the left ventricle of Goto-Kakizaki type 2 diabetic rats.

Diabetes mellitus is a major global health disorder and, currently, over 450 million people have diabetes with 90% suffering from type 2 diabetes. Left untreated, diabetes may lead to cardiovascular diseases which are a leading cause of death in diabetic patients. Calcium is the trigger and regulator of cardiac muscle contraction and derangement in cellular Ca2+ homeostasis, which can result in heart failure and sudden cardiac death. It is of paramount importance to investigate the regional involvement of Ca2+ in diabetes-induced cardiomyopathy. Therefore, the aim of this study was to investigate the voltage dependence of the Ca2+ transients in endocardial (ENDO) and epicardial (EPI) myocytes from the left ventricle of the Goto-Kakizaki (GK) rats, an experimental model of type 2 diabetes mellitus. Simultaneous measurement of L-type Ca2+ currents and Ca2+ transients was performed by whole-cell patch clamp techniques. GK rats displayed significantly increased heart weight, heart weight/body weight ratio, and non-fasting and fasting blood glucose compared to controls (CON). Although the voltage dependence of L-type Ca2+ current was unaltered, the voltage dependence of the Ca2+ transients was reduced to similar extents in EPI-GK and ENDO-GK compared to EPI-CON and ENDO-CON myocytes. TPK L-type Ca2+ current and Ca2+ transient were unaltered. THALF decay of L-type Ca2+ current was unaltered; however, THALF decay of the Ca2+ transient was shortened in ENDO and EPI myocytes from GK compared to CON rat hearts. In conclusion, the amplitude of L-type Ca2+ current was unaltered; however, the voltage dependence of the Ca2+ transient was reduced to similar extents in EPI and ENDO myocytes from GK rats compared to their respective controls, suggesting the possibility of dysfunctional sarcoplasmic reticulum Ca2+ transport in the GK diabetic rat hearts.

Regional effects of streptozotocin-induced diabetes on shortening and calcium transport in epicardial and endocardial myocytes from rat left ventricle.

In the heart, the left ventricle pumps blood at higher pressure than the right ventricle. Within the left ventricle, the electromechanical properties of ventricular cardiac myocytes vary transmurally and this may be related to the gradients of stress and strain experienced in vivo across the ventricular wall. Diabetes is also associated with alterations in hemodynamic function. The aim of this study was to investigate shortening and Ca2+ transport in epicardial (EPI) and endocardial (ENDO) left ventricular myocytes in the streptozotocin (STZ)-induced diabetic rat. Shortening, intracellular Ca2+ and L-type Ca2+ current (ICa,L) were measured by video detection, fura-2 microfluorimetry, and whole-cell patch clamp techniques, respectively. Time to peak (TPK) shortening was prolonged to similar extents in ENDO and EPI myocytes from STZ-treated rats compared to ENDO and EPI myocytes from controls. Time to half (THALF) relaxation of shortening was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. TPK Ca2+ transient was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. THALF decay of the Ca2+ transient was prolonged in ENDO myocytes from STZ-treated rats compared to ENDO controls. Sarcoplasmic reticulum (SR) fractional release of Ca2+ was reduced in EPI myocytes from STZ-treated rats compared to EPI controls. ICa,L activation, inactivation, and recovery from inactivation were not significantly altered in EPI and ENDO myocytes from STZ-treated rats or controls. Regional differences in Ca2+ transport may partly underlie differences in ventricular myocyte shortening across the wall of the healthy and the STZ-treated rat left ventricle.

Neuraminidase inhibition primes short-term depression and suppresses long-term potentiation of synaptic transmission in the rat hippocampus.

Neuraminidase (NEU) is a key enzyme that cleaves negatively charged sialic acid residues from membrane proteins and lipids. Clinical and basic science studies have shown that an imbalance in NEU metabolism or changes in NEU activity due to various pathological conditions parallel with behavior and cognitive impairment. It has been suggested that the decreases of NEU activity could cause serious neurological consequences. However, there is a lack of direct evidences that modulation of endogenous NEU activity can impair neuronal function. Using combined rat entorhinal cortex/hippocampal slices and a specific inhibitor of NEU, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NADNA), we examined the effect of downregulation of NEU activity on different forms of synaptic plasticity in the hippocampal CA3-to-CA1 network. We show that NEU inhibition results in a significant decrease in long-term potentiation (LTP) and an increase in short-term depression. Synaptic depotentiation restores LTP in NADNA-pretreated slices to the control level. These data suggest that short-term NEU inhibition produces the LTP-like effect on neuronal network, which results in damping of further LTP induction. Our findings demonstrate that downregulation of NEU activity could have a major impact on synaptic plasticity and provide a new insight into the cellular mechanism underlying behavioral and cognitive impairment associated with abnormal metabolism of NEU.

Persistent sodium current properties in hippocampal CA1 pyramidal neurons of young and adult rats.

Persistent tetrodotoxin-sensitive sodium current (INaP) plays an important role in cellular and neuronal network excitability in physiological conditions and under different pathological circumstances. However, developmental changes in INaP properties remain largely unclear. In the present study using whole cell patch clamp technique we evaluated INaP properties in CA1 hippocampal pyramidal neurons isolated from young (postnatal day (P) 12-16) and adult (P60-75) rats. We show that the INaP density is substantially larger in the adult group. Although INaP inactivation characteristics were found to be similar in both groups, voltage dependence of INaP activation is shifted to more negative membrane potentials (young: -48.6±0.5mV vs. adult: -52.4±0.2mV, p<0.01). Our data indicates the increase of INaP contribution in the basal membrane sodium conductivity in the mature hippocampus.

Effects of cannabidiol on the function of α7-nicotinic acetylcholine receptors.

The effects of cannabidiol (CBD), a non-psychoactive ingredient of cannabis plant, on the function of the cloned α7 subunit of the human nicotinic acetylcholine (α7 nACh) receptor expressed in Xenopus oocytes were tested using the two-electrode voltage-clamp technique. CBD reversibly inhibited ACh (100 µM)-induced currents with an IC50 value of 11.3 µM. Other phytocannabinoids such as cannabinol and Δ(9)-tetrahydrocannabinol did not affect ACh-induced currents. CBD inhibition was not altered by pertussis toxin treatment. In addition, CBD did not change GTP-γ-S binding to the membranes of oocytes injected with α7 nACh receptor cRNA. The effect of CBD was not dependent on the membrane potential. CBD (10 µM) did not affect the activity of endogenous Ca(2+)-dependent Cl(-) channels, since the extent of inhibition by CBD was unaltered by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free bathing solution containing 2mM Ba(2+). Inhibition by CBD was not reversed by increasing ACh concentrations. Furthermore, specific binding of [(125)I] α-bungarotoxin was not inhibited by CBD (10 µM) in oocytes membranes. Using whole cell patch clamp technique in CA1 stratum radiatum interneurons of rat hippocampal slices, currents induced by choline, a selective-agonist of α7-receptor induced currents were also recoded. Bath application of CBD (10 µM) for 10 min caused a significant inhibition of choline induced currents. Finally, in hippocampal slices, [(3)H] norepinephrine release evoked by nicotine (30 µM) was also inhibited by 10 µM CBD. Our results indicate that CBD inhibits the function of the α7-nACh receptor.

TRPC7 is a receptor-operated DAG-activated channel in human keratinocytes.

Muscarinic and purinergic receptors expressed in keratinocytes are an important part of a functional system for cell growth. While several aspects of this process are clearly dependent on Ca(2+) homeostasis, less is known about the mechanisms controlling Ca(2+) entry during epidermal receptor stimulation. We used patch-clamp technique to study responses to carbachol (CCh) and adenosine triphosphate (ATP) in HaCaT human keratinocytes. Both agonists induced large currents mediated by cation-selective channels about three times more permeable to Ca(2+) than Na(+), suggesting that they play an important role in receptor-operated Ca(2+) entry. CCh- and ATP-induced currents were inhibited by 1-[6-([(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione, a phospholipase C (PLC) blocker. Investigation of the pathways downstream of PLC activation revealed that InsP(3) did not affect the agonist responses. In contrast, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable analog of 1,2-diacylglycerol (DAG), evoked a similar cation current. This action appears to be direct, since the effects of activators or inhibitors of protein kinase C were comparatively small. Finally, transient receptor potential canonical 7 (TRPC7) specific knockdown by antisense oligonucleotides led to a decrease in ATP- and CCh-induced calcium entry, as well as OAG-evoked current. We concluded that activation of both muscarinic and purinergic receptors via a common DAG-dependent link opens Ca(2+)-permeable TRPC7 channels.

Ca(2+)- and volume-sensitive chloride currents are differentially regulated by agonists and store-operated Ca2+ entry.

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca2+]i in a Ca(2+)-free medium followed by a secondary [Ca2+]i rise upon Ca2+ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca(2+)-activated (I(Cl(Ca))) and volume-regulated (I(Cl, swell)) chloride currents. NPPB and DIDS more efficiently inhibited I(Cl(Ca)) and I(Cl, swell), respectively. Cell swelling caused by hypotonic solution invariably activated I(Cl, swell) while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling-induced I(Cl, swell), while its inactive analogue U-73343 had no effect. I(Cl(Ca)) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca2+]i in the pipette solution at 500 nM. In contrast, I(Cl, swell) could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced I(Cl, swell) in a nonadditive manner, suggesting their convergence on a common pathway. I(Cl, swell) and I(Cl(Ca)) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated I(Cl(Ca)), but abolished I(Cl, swell), thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that I(Cl, swell) can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a "selection" switch between closely localized channels.