Whole-cell Mode Research Articles

Hypoxia-induced cystic fibrosis transmembrane conductance regulator dysfunction is a universal mechanism underlying reduced mucociliary transport in sinusitis.

Hypoxia due to sinus obstruction is a major pathogenic mechanism leading to sinusitis. The objective of the current study is to define the electrophysiologic characteristics of hypoxia in vitro and in vivo. Cystic fibrosis bronchoepithelial cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and human sinonasal epithelial cells were exposed to 1% or atmospheric O2 for 24h. Time-dependent production of cytoplasmic free radicals was measured. Cells were subjected to Ussing chamber and patch clamp technique where CFTR currents were recorded in whole-cell and cell-attached mode for single channel studies. Indices of mucociliary transport (MCT) were measured using micro-optical coherence tomography. In a rabbit hypoxic maxillary sinus model, tissue oxygenation, relative mRNA expression of HIF-1α, pH, sinus potential difference (SPD), and MCT were determined. Ussing chamber (p<0.05), whole-cell (p<0.001), and single channel patch-clamp (p<0.0001) showed significant inhibition of Cl- currents in hypoxic cells. Cytoplasmic free radicals showed time-dependent elevation peaking at 4h (p<0.0001). Airway surface liquid (p<0.0001), periciliary liquid (p<0.001), and MCT (p<0.01) were diminished. Co-incubation with the free radical scavenger glutathione negated the impact of hypoxia on single channel currents and MCT markers. In sinusitis rabbits, mucosa exhibited low tissue oxygenation (p<0.0001), increased HIF1α mRNA (p<0.05), reduced pH (p<0.01), and decreased MCT (p<0.001). SPD measurements demonstrated markedly diminished transepithelial Cl- transport (p<0.0001). Hypoxia induces severe CFTR dysfunction via free radical production causing reduced MCT in vitro and in vivo. Improved oxygenation is critical to reducing the impact of persistent mucociliary dysfunction.

Optimization of Aerobic Synthesis of Succinic Acid from Glucose by Recombinant &lt;i&gt;Escherichia coli&lt;/i&gt; Strains Through the Variant Tricaboxylic Acid Cycle Mediated by the Action of 2-ketoglutarate-decarboxylase

The biosynthesis of succinic acid from glucose by the previously engineered E. coli strain SUC1.0 (pMW119-kgd) (MG1655 ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, PLglk, PtacgalP, ∆aceBAK, ∆glcB, ∆sdhAB, pMW119-kgd) was optimized. The yield of the target substance was increased, upon the activation in the strain of the variant tricarboxylic acid cycle, mediated by the action of heterologous 2-ketoglutarate decarboxylase, due to the intensification of the anaplerotic formation of oxaloacetic acid. Inactivation of the nonspecific thioesterase YciA in the strain did not considerably change the biosynthetic characteristics of the producer. The enhancement of the expression of native phosphoenolpyruvate carboxylase led to an increase in the yield of the target compound by the recombinant synthesizing succinic acid via the reactions of the native tricarboxylic acid cycle from 25 to 42%, and from 67 to 75% upon the induced expression of Mycobacterium tuberculosis 2-ketoglutarate decarboxylase. Expression in the strain of pyruvate carboxylase gene from Bacillus subtilis resulted in an increase in the yield of succinic acid up to 84%. Functioning in whole-cell biocatalyst mode, the engineered strain SUC1.0 PL-pycA (pMW119-kgd) demonstrated a substrate to target product conversion ratio reaching 93%, approaching the corresponding theoretical maximum.

Cx43 Hemichannel and Panx1 Channel Modulation by Gap19 and 10Panx1 Peptides.

Cx43 hemichannels (HCs) and Panx1 channels are two genetically distant protein families. Despite the lack of sequence homology, Cx43 and Panx1 channels have been the subject of debate due to their overlapping expression and the fact that both channels present similarities in terms of their membrane topology and electrical properties. Using the mimetic peptides Gap19 and 10Panx1, this study aimed to investigate the cross-effects of these peptides on Cx43 HCs and Panx1 channels. The single-channel current activity from stably expressing HeLa-Cx43 and C6-Panx1 cells was recorded using patch-clamp experiments in whole-cell voltage-clamp mode, demonstrating 214 pS and 68 pS average unitary conductances for the respective channels. Gap19 was applied intracellularly while 10Panx1 was applied extracellularly at different concentrations (100, 200 and 500 μM) and the average nominal open probability (NPo) was determined for each testing condition. A concentration of 100 µM Gap19 more than halved the NPo of Cx43 HCs, while 200 µM 10Panx1 was necessary to obtain a half-maximal NPo reduction in the Panx1 channels. Gap19 started to significantly inhibit the Panx1 channels at 500 µM, reducing the NPo by 26% while reducing the NPo of the Cx43 HCs by 84%. In contrast 10Panx1 significantly reduced the NPo of the Cx43 HCs by 37% at 100 µM and by 83% at 200 µM, a concentration that caused the half-maximal inhibition of the Panx1 channels. These results demonstrate that 10Panx1 inhibits Cx43 HCs over the 100-500 µM concentration range while 500 µM intracellular Gap19 is necessary to observe some inhibition of Panx1 channels.

Human neutrophils communicate remotely via calcium-dependent glutamate-induced glutamate release

Neutrophils are white blood cells that are critical to acute inflammatory and adaptive immune responses. Their swarming-pattern behavior is controlled by multiple cellular cascades involving calcium-dependent release of various signaling molecules. Previous studies have reported that neutrophils express glutamate receptors and can release glutamate but evidence of direct neutrophil-neutrophil communication has been elusive. Here, we hold semi-suspended cultured human neutrophils in patch-clamp whole-cell mode to find that calcium mobilization induced by stimulating one neutrophil can trigger an N-methyl-D-aspartate (NMDA) receptor-driven membrane current and calcium signal in neighboring neutrophils. We employ an enzymatic-based imaging assay to image, in real time, glutamate release from neutrophils induced by glutamate released from their neighbors. These observations provide direct evidence for a positive-feedback inter-neutrophil communication that could contribute to mechanisms regulating communal neutrophil behavior.

Group I metabotropic glutamate receptor-triggered temporally patterned action potential-dependent spontaneous synaptic transmission in mouse MNTB neurons

Rhythmic action potentials (AP) are generated via intrinsic ionic mechanisms in pacemaking neurons, producing synaptic responses of regular inter-event intervals (IEIs) in their targets. In auditory processing, evoked temporally patterned activities are induced when neural responses timely lock to a certain phase of the sound stimuli. Spontaneous spike activity, however, is a stochastic process, rendering the prediction of the exact timing of the next event completely based on probability. Furthermore, neuromodulation mediated by metabotropic glutamate receptors (mGluRs) is not commonly associated with patterned neural activities. Here, we report an intriguing phenomenon. In a subpopulation of medial nucleus of the trapezoid body (MNTB) neurons recorded under whole-cell voltage-clamp mode in acute mouse brain slices, temporally patterned AP-dependent glycinergic sIPSCs and glutamatergic sEPSCs were elicited by activation of group I mGluRs with 3,5-DHPG (200 µM). Auto-correlation analyses revealed rhythmogenesis in these synaptic responses. Knockout of mGluR5 largely eliminated the effects of 3,5-DHPG. Cell-attached recordings showed temporally patterned spikes evoked by 3,5-DHPG in potential presynaptic VNTB cells for synaptic inhibition onto MNTB. The amplitudes of sEPSCs enhanced by 3,5-DHPG were larger than quantal size but smaller than spike-driven calyceal inputs, suggesting that non-calyceal inputs to MNTB might be responsible for the temporally patterned sEPSCs. Finally, immunocytochemical studies identified expression and localization of mGluR5 and mGluR1 in the VNTB-MNTB inhibitory pathway. Our results imply a potential central mechanism underlying the generation of patterned spontaneous spike activity in the brainstem sound localization circuit.

Whole-cell patch-clamp recording and parameters.

The patch-clamp technique represents an electrophysiology type of method. This is one of several insightful approaches with five major configurations, namely a loose patch, cell-attached (also known as on-cell), whole-cell, inside-out, and outside-out modes. The patch-clamp method is more advanced compared to classical electrophysiology since it elucidates single-channel activation in a tiny portion of the membrane in addition to action potential (AP), junction potential (JP), endplate potential (EP), electrical coupling between two adjacent cells via Gap junction hemi-channels, excitatory/inhibitory postsynaptic potentials, and resting membrane potential (RMP). In fact, a malfunction of only one channel or even one component will alter AP amplitude or duration in vitro. If parameters are inferred appropriately and recordings are performed properly, the patch-clamp trace readouts and results are robust. The main hallmarks of currents via voltage-dependent calcium (Cav), hyperpolarization-activated cyclic nucleotide gated non-selective cation (HCN), inwardly rectifying potassium (Kir), voltage-dependent potassium (Kv), and voltage-dependent sodium (Nav) channels are similar and tractable among cells even when they are derived from evolutionary distinct organs and species. However, the size of the membrane area, where the functional subunits reside, and current magnitudes vary among cells of the same type. Therefore, dividing current magnitudes by cell capacitance- current density enables the estimate of functional and active channels relative to recorded cytoplasmic membrane area. Since the patch-clamp recordings can be performed in both current- and voltage-clamp modes, the action potential or spike durations can be adequately elucidated. Sometimes, optical methods are preferred to patch-clamp electrophysiology, but the obtained signals and traces are not robust. Finally, not only an alternans of AP durations, but also that of 'action potential shape' is observed with electrophysiology.

Essential oil of Croton zehntneri prevents electrophysiological alterations in dorsal root ganglia of streptozotocin-induced diabetes mellitus in rats

Croton zehntneri is an aromatic shrub, native to Northeast Brazil, where it is used in folk medicine. The essential oil of C. zehntneri (EOCz) has shown potent antioxidant and anti-inflammatory activities. Our aim was to investigate the beneficial effect of EOCz on electrophysiological changes in dorsal root ganglia (DRG) of rats with streptozotocin-induced diabetes mellitus (DM). DRG excitability and passive and active membrane properties were studied in control and DM-induced rats. Intracellular recording in current-clamp mode with sharp microelectrode was used. For Na+ channel investigation, patch-clamp recording in whole-cell mode was used. DM caused depolarizations (from -60.6 to -55.0 mV) of resting membrane potential without alteration of cell membrane input resistance. Also, it showed a tendency towards a decrease in rheobase and a tendency for increase in excitability, which was confirmed by increase in Na+ current (from -113.6 to -178.3 pF/pA). EOCz did not reverse the hyperglycemia of DM. Regardless of that, upon oral treatment, EOCz prevented neuronal alterations. EOCz was demonstrated to have low toxicity. The preventive effects of EOCz on neuronal changes coupled with its low toxicity make EOCz a potential candidate for a type of treatment of diabetic neuropathy complementary to therapy with hypoglycemic agents.

TRP434 and TRP435 residues are crucial for sensing calcium ions and PI(4,5)P2 molecules in TRPC5 channel.

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. Among 7 subunits, TRPC4, 5 channels are well known to be potentiated by direct bindings of calcium ion and phosphatidylinositol 4,5-biphosphate. Both calcium and PIP2 were therefore also implicated in activation and desensitization of the channel. The binding site for calcium ion was revealed but PIP2 binding site remains controversial. Previously, we found two tryptophan residues in TRPC4 channel as a candidate by which calcium sensing in S2-S3 linker is delivered to channel gating. Here, we suggest the WW site in TRPC5 channel conducts delivering of calcium sensing like in TRPC4, and also acts as a potential PIP2 binding site. We made two single mutants (W434A, W435A) and a double mutant (WW/AA) from human TRPC5 channel. Wild-type and mutant TRPC5 channels were expressed in HEK293 cells for macroscopic current recordings in whole-cell or inside-out mode. Generally, our data demonstrate W434 and W435 residues participate in PIP2 binding and calcium sensing. Interestingly, when co-expressed with human muscarinic acetylcholine receptor type 3 (mAChR3) and stimulated by carbachol, W434A channel didn’t show desensitization characteristically observed in wild-type TRPC5 channel. Loss of desensitization in W434A channel implies that it has lost the ability of sensing PIP2 hydrolysis by PLC or the ability of phosphorylation by PKC. The latter possibility is ruled out because the C-terminus of the channels we used were truncated so they do not contain the phosphorylation site. Recently, PIP2-bound ion channel structures were revealed including TRPs. Many channels had PIP2 molecules near S3, S4, and S5 helices. Our electrophysiological data also indicate that PIP2 binds near S3 helix in TRPC5 channel, and the WW site is a specific candidate for sensing PIP2 and calcium.

VPAC2 receptor mediates VIP-potentiated insulin secretion via ion channels in rat pancreatic β cells

Vasoactive intestinal peptide (VIP), a small neuropeptide composing of 28 amino acids, functions as a neuromodulator with insulinotropic effect on pancreatic β cells, in which it is of vital importance in regulating the levels of blood glucose. VIP potently agonizes VPAC2 receptor (VPAC2-R). Agonists of VPAC2-R stimulate glucose-dependent insulin secretion. The purpose of this study was to further investigate the possible ion channel mechanisms in VPAC2-R-mediated VIP-potentiated insulin secretion. The results of insulin secretion experiments showed that VIP augmented insulin secretion in a glucose-dependent manner. The insulinotropic effect was mediated by VPAC2-R rather than VPAC1 receptor (VPAC1-R), through the adenylyl cyclase (AC)/protein kinase A (PKA) signalling pathway. The calcium imaging analysis demonstrated that VIP increased intracellular Ca2+ concentration ([Ca2+]i). In addition, in the whole-cell voltage-clamp mode, we found that VIP blocked the voltage-dependent potassium (Kv) channel currents, while this effect was reversed by inhibiting the VPAC2-R, AC or PKA respectively. Taken together, these findings suggest that VIP stimulates insulin secretion by inhibiting the Kv channels, activating the Ca2+ channels, and increasing [Ca2+]i through the VPAC2-R and AC/PKA signalling pathway. These findings provide theoretical basis for the research of VPAC2-R as a novel therapeutic target.

Abstract 092: Activation Of Neuronal At 1 R Exacerbates Hypertension-associated Mci Through Vascular And Neural Mechanisms

Hypertension (HTN) has now been associated with cognitive impairment and considered as one of the risk factors for mild cognitive impairment (MCI). To investigate whether there is neuronal mechanism, HTN was induced in C57BL/6j male mice (~22 weeks) by DOCA-salt treatment (DS, 1.4mg/g+1%NaCl, 28 days), a neurogenic model. Firstly, BP was monitored by tail-cuff (sham vs. DS: SBP= 116.1±7.6 vs. 145.0±8.2 mmHg). Then, behavioral study was performed to assess the cognitive function (CFN). In whole, compared to sham, the DS mice performed worse in all 4 tasks, including object location recognition (before vs. after: NLR%= 29.34±13.73 vs. -20.81±10.70), novel object recognition (NOR%= 48.60±22.62 vs. 9.09±25.50), Barnes maze (latency T= 26.55±16.52 vs. 116.80±57.67s), and nesting (score= 4.00±0.92 vs. 2.43±1.55). Later, CBF was measured by laser doppler. The rise of CBF upon whisker-stimulation (WS) was lower in the DS mice (27.83±13.86 vs. 51.12±18.22 PU), while their basal CBF was not affected, suggesting that DS-induced MCI could be related to impairment of neuro-vascular coupling (NVC). Previously, we have showed the overactivation of brain renin-angiotensin system (RAS) in the onset of DS HTN. To identify the possible contribution from brain RAS, neuronal AT 1a R were deleted (AT1N). In AT1N mice, DS-related impairments in CFN and NVC were attenuated, as evidenced by higher nesting scores (AT1N+DS vs. NT+DS: 3.75±1.89 vs. 2.43±1.55 ), better performance in Barnes maze (61.28±54.47 vs. 116.80±57.67 s) and grater augment in CBF upon WS (41.49±10.65 vs. 27.83±13.86 PU), compared to NT. Through LTP recording in CA1 neurons using both field and whole-cell modes, we identified that activation of neuronal AT 1 R could contribute directly to the development of MCI, thereby neuronal AT 1 R was selectively knocked down in the hippocampus of NT mice (AT1hN). After DS treatment, AT1hN showed no difference in SBP compared with the sham NT (149.0±14.7 vs. 150.4±6.9 mmHg), while their CFN showed improvement in both NOR and Barnes maze (NOR%= 11.70±25.78 vs. -10.15±42.60; latency T= 71.72±47.60 vs. 113.70±65.64 s). In summary, our data suggest that neuron-expressing AT 1 R could participate in the onset of hypertension-associated MCI via both vascular and neural mechanisms.

Afterhyperpolarization potential modulated by local [K+]o in K+ diffusion-restricted extracellular space in the central clock of suprachiasmatic nucleus

BackgroundIntercellular coupling is essential for the suprachiasmatic nucleus (SCN) to serve as a coherent central clock. Synaptic release of neurotransmitters and neuropeptides is critical for synchronizing SCN neurons. However, intercellular coupling via non-synaptic mechanisms has also been demonstrated. In particular, the abundant perikaryal appositions with morphological specializations in the narrow extracellular space (ECS) may hinder molecular diffusion to allow for ion accumulation or depletion. MethodsThe SCN neurons were recorded in the whole-cell current-clamp mode, with pipette filled with high (26 mM)-Na+ or low (6 mM)-Na+ solution. ResultsCells recorded with high-Na+ pipette solution could fire spontaneous action potentials (AP) with peak AHP more negative than the calculated value of K+ equilibrium potential (EK) and with peak AP more positive than calculated ENa. Cells recorded with low-Na+ pipette solution could also have peak AHP more negative than calculated EK. In contrast, the resting membrane potential (RMP) was always less negative to calculated EK. The distribution and the averaged amplitude of peak AHP, peak AP, or RMP was similar between cells recorded with high-Na+ and low-Na+ solution pipette. In a number of cells, the peak AHP could increase from more positive to become more negative than calculated EK spontaneously or after treatments to hyperpolarize the RMP. TTX blocked the Na+ -dependent APs and tetraethylammonium (TEA), but not Ba2+ or Cd2+, markedly reduced the peak AHP. Perforated-patch cells could also but rarely fire APs with peak AHP more negative than calculated EK. ConclusionThe result of peak AHP negative to calculated EK indicates that local [K+]o sensed by the TEA-sensitive AHP K+ channels must be lower than bulk [K+]o, most likely due to K+ clearance from K+ diffusion-restricted ECS by the Na+/K+-ATPase. The K+ diffusion-restricted ECS may allow for K+-mediated ionic interactions among neurons to regulate SCN excitability.

Performance of Methylococcus capsulatus based microbial and enzymatic proton exchange membrane fuel cells

Activation of methane in low-temperature fuel cells has been a challenge due to its thermodynamic stability. In this study, we demonstrate the feasibility of using a pure culture of Methylococcus capsulatus microbial fuel cell in a proton exchange membrane (PEM) fuel cell in whole-cell and crude enzymatic modes. The impact of time and mediators of the microbial fuel cells (MFCs) was studied. Additionally, a mathematical model was used to predict and explain the fuel cell's electrochemical performance and mechanic details. The fuel cell generated an open-circuit voltage of 378.91 mV and a power density of 438.57 μW/m2 in the whole-cell mode, whereas an OCV of 125.62 mV and a power density of 117.94 μW/m2 in the enzymatic mode without the use of an external mediator. Although the cell was stable throughout the test duration of ten days in the whole-cell mode, the stability declined within minutes in the enzymatic mode. This work demonstrates the feasibility of generating electricity via a proton exchange membrane (PEM) fuel cell in microbial and enzymatic modes using methane as the only carbon source at room temperature with a pure culture of M. capsulatus as a direct electron-transporting biocatalyst.

Exercise Training and Chemogenetic Activation of the Dorsal Motor Nucleus of the Vagus Rescue the Changes in the Hepatic Vein Glucose Concentration by Central Action of Insulin in SHR

The Dorsal Motor Nucleus of the Vagus (DMNV) is the main parasympathetic output from CNS as part of the brain‐liver axis that participates on glucose homeostasis. Previously, we have shown that icv injection of insulin elicited a decrease the hepatic vein glucose concentration (HVGC) of Wistar, but not in SHR. Here we aimed to investigate whether the deficiency of central action of insulin to control the HVGC of SHR is due to a dysfunction in the cholinergic neurotransmission in the DMNV and which putative neurotransmitter could play a role in this circuitry. To this, we used in vivo and in vitro studies using chemogenetic tools, and the whole‐cell patch‐clamp recordings. Ethical approval: CEUA/ICB‐USP #8336131119). IN VIVO STUDIES: Male SHR (12 weeks old) received an injection of DREADD (LVV‐Gq‐hM3q‐PRSx8‐eGFP) to selectively activate de DMNV neurons with clozapine‐N‐oxide (CNO). After 10 days of CNO treatment, the animals received icv injection of insulin and the HVGC was monitored in freely moving rats. IN VITRO STUDIES: to identify liver‐projecting DMVN neurons, age‐matched (12 weeks old) of Wistar and sedentary and exercised trained SHR (SHRt) received, 5 days prior to the experiments, injections of Fluorogold (FG) on the liver parenchyma. At the day of the electrophysiological studies, the FG‐labeled DMVN neurons was visualized and recorded in whole‐cell patch‐clamp mode, before and after application of oxytocin (OT) in the bath. The results of in vivo studies have shown that in the DREADD group the icv insulin was able to reduce the HVGC when compared to the control group (Control: 113,6±14,9 mg/dL n=5; vs DREADD: 74,2±6,16mg/dL; p≤0,05). As for the in vitro studies, the application of OT in liver projecting‐DMNV depolarized the resting membrane potential (Basal: −59.4± 0.7 mV vs OT: −56.2 ± 1.2 mV, n = 10; t = 2.403, df = 9; P = 0.0397) and increased the firing frequency (baseline: 1.9±0.8 vs OT: 3.0 ± 0.9 Hz, n=10; P = 0.002) in Wistar, but not in sedentary SHR. Interestingly, in SHR trained for 4 weeks, the OT caused depolarization of resting membrane potential (baseline: −61.3 ± 1.3 vs OT: −59.6 ± 1.5 mV, n=18; P=0.0177) and an increase in the firing frequency (baseline: 3.9±0.7 vs OT: 4.7±0.6 Hz, n=18, P=0.0012). As conclusion, the selective activation of parasympathetic preganglionic neurons of the DMNV rescues the changes in the HVGC elicited by central action of insulin, which used to be defective in SHR, indicating that the circuitry involving DMNV‐liver pathway is somehow dysfunctional, which could be due to the autonomic unbalance observed in this strain. Moreover, OT elicits similar changes in the electrophysiological properties of liver‐projecting DMNV neurons of Wistar and exercise‐trained but not sedentary SHR. These results indicate that exercise training can restore the sensitivity of liver‐projecting DMNV neurones of exercise trained SHR to OT.

Loss of SR‐PM Contact Sites May Contribute to The Development of Hypertension in Spontaneously Hypertensive Rats

Sarcoplasmic reticulum (SR)‐plasma membrane (PM) contact sites in vascular smooth muscle (VSM) cells bring the SR Ca2+ release channels into proximity of plasma membrane large‐conductance Ca2+‐activated K+ (BKca) channels and regulate intracellular Ca2+ through BKca‐mediated membrane hyperpolarization. Loss of BKca channel activity leads to increased intracellular Ca2+ concentration, vascular hypercontractility, and activation of hypertrophic genes. We hypothesized that the loss of close contact between the SR and the PM contributes to the development of vascular hypercontractility and hypertrophy in the Spontaneously Hypertensive Rat (SHR). To evaluate this hypothesis, we compared BKca channel activity in response to the activation of inositol 1,4,5‐trisphosphate receptor (IP3R), an SR Ca2+ channel between SHR and normotensive Sprague Dawley (SD) rats. We found that in endothelium‐denuded pressurized mesenteric arteries, paxilline (1μM), a selective BKca channel blocker, significantly increased norepinephrine (NE, 0.5μM)‐induced vasoconstriction in SD arteries (n=5) by ~15% (% initial diameter from 78.58±3.93 to 66.77±3.07). However, paxilline failed to increase NE‐induced vasoconstriction in SHR arteries (Figure 1). Paxilline treatment significantly augmented the hypertrophic effect of angiotensin II (ANG II) in SD, where the total cell area of paxilline treated ANG II group was ~11% (from 3471.48±121.35 to 3854.86±129.99μm2) greater compared to the non‐paxilline‐treated ANG II group. In contrast, the paxilline‐induced increase in hypertrophic response was absent in SHR. Similarly, in WST‐1 cell proliferation assay, paxilline treatment significantly increased ANG II‐induced proliferation in SD by ~15% (Absorbance(A440nm‐A630nm) from 0.15±0.01 to 0.17±0.01) while having no effect on cell proliferation in SHR. Next, we compared the effect of 5‐HT on BKca channel activity between SHR and SD rat mesenteric VSM cells using patch‐clamp whole‐cell mode. 5‐HT activates IP3Rs by stimulating the production of IP3 in VSM cells. At +60mV, 5‐HT (0.3μM) significantly increased BKca current density at +60mV in SD VSM cells from 50.78±2.19 to 69.40±5.50 pA/pF (n=6 cells, P&lt;0.05) but the increase in current density was non‐significant in SHR (from 36.77±3.11 to 47.45±8.83 pA/pF, n=6 cells, Figure 2). We also examined the Ca2+‐ and voltage‐sensitivity of BKca channels using inside‐out patch‐clamp. BKca channels in VSM cells isolated from mesenteric arteries of SHR and SD rats displayed identical Ca2+‐ and voltage‐sensitivity. In conclusion, our data suggest a lack of BKca activation in response to IP3R activation in VSM cells ofSHR, which may be due to the loss of close contact between the SR and the PM.

Inhibition of chemically and mechanically activated Piezo1 channels as a mechanism for ameliorating atherosclerosis with salvianolic acid B.

Salvianolic acid B (SalB) is effective for treating cardiovascular diseases. However, the molecular mechanisms underlying its therapeutic effects remain unclear. Mechanosensitive Piezo1 channels play important roles in vascular biology, although their pharmacological properties are poorly defined. Here, we aimed to identify novel Piezo1 inhibitors and gain insights into their mechanisms of action. Intracellular Ca2+ ions were measured in HUVECs, murine liver endothelial cells (MLECs), THP-1 and RAW264.7 cell lines and bone marrow-derived macrophages (BMDMs). Isometric tensions in mouse thoracic aorta were recorded. Shear-stress assays with HUVECs were conducted. Patch-clamp recordings with mechanical stimulation were performed with HUVECs in whole-cell mode. Foam cell formation was induced by treating BMDMs with oxidised LDL (oxLDL). Atherosclerotic plaque assays were performed with Ldlr-/- and Piezo1 genetically depleted mice on a high-fat diet. Salvianolic acid B inhibited Yoda1-induced Ca2+ influx in HUVECs and MLECs. Similar results were observed in macrophage cell lines and BMDMs. Furthermore, we demonstrated that salvianolic acid B inhibited Yoda1- and mechanically activated currents. Salvianolic acid B suppressed Yoda1-induced aortic ring relaxation and inhibited HUVECs alignment in the direction of shear stress. Additionally, Yoda1 enhanced the formation of foam cells, which was reversed by salvianolic acid B. Salvianolic acid B also inhibited formation of atherosclerotic plaques and was insensitive to Piezo1 genetic depletion. Our study provides novel mechanistic insights into the inhibitory role of salvianolic acid B against Piezo1 channels and improves our understanding of salvianolic acid B in preventing atherosclerotic lesions.

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

Carbon monoxide activation of delayed rectifier potassium currents of human cardiac fibroblasts through diverse pathways.

To identify the effect and mechanism of carbon monoxide (CO) on delayed rectifier K+ currents (IK) of human cardiac fibroblasts (HCFs), we used the whole-cell mode patch-clamp technique. Application of CO delivered by carbon monoxide-releasing molecule-3 (CORM3) increased the amplitude of outward K+ currents, and diphenyl phosphine oxide-1 (a specific IK blocker) inhibited the currents. CORM3-induced augmentation was blocked by pretreatment with nitric oxide synthase blockers (L-NG-monomethyl arginine citrate and L-NG-nitro arginine methyl ester). Pretreatment with KT5823 (a protein kinas G blocker), 1H-[1,-2,-4] oxadiazolo-[4,-3-a] quinoxalin-1-on (ODQ, a soluble guanylate cyclase blocker), KT5720 (a protein kinase A blocker), and SQ22536 (an adenylate cyclase blocker) blocked the CORM3 stimulating effect on IK. In addition, pretreatment with SB239063 (a p38 mitogen-activated protein kinase [MAPK] blocker) and PD98059 (a p44/42 MAPK blocker) also blocked the CORM3’s effect on the currents. When testing the involvement of S-nitrosylation, pretreatment of N-ethylmaleimide (a thiol-alkylating reagent) blocked CO-induced IK activation and DL-dithiothreitol (a reducing agent) reversed this effect. Pretreatment with 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)-21H,23H porphyrin manganese (III) pentachloride and manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (superoxide dismutase mimetics), diphenyleneiodonium chloride (an NADPH oxidase blocker), or allopurinol (a xanthine oxidase blocker) also inhibited CO-induced IK activation. These results suggest that CO enhances IK in HCFs through the nitric oxide, phosphorylation by protein kinase G, protein kinase A, and MAPK, S-nitrosylation and reduction/oxidation (redox) signaling pathways.

Abstract 37: Loss Of BK Ca - IP3R Functional Coupling Underlies Vascular Hypercontractility In Spontaneously Hypertensive Rats

The cellular mechanisms underlying enhanced vascular contractility in hypertension are still not fully clear. Recent studies have demonstrated that functional coupling between the large-conductance Ca 2+ -activated K + (BK Ca ) channel in the cell membrane and inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum is involved in the regulation of intracellular Ca 2+ in vascular smooth muscle cells (VSMC) and vascular contractility. Here, we examined the role of BK Ca -IP3R coupling in vascular hypercontractility in Spontaneously Hypertensive Rats (SHR), as compared with Sprague Dawley (SD) rats. In pressurized mesenteric arteries, paxilline (1μM), a selective BK Ca channel blocker, significantly increased norepinephrine (1μM)-induced vasoconstriction in both SHR (% initial diameter: 71.6±6.8% [95.9±13 of 132.8±6.2 μm] and 65.0±4.8% [80.6±7.7 of 123.6±3.9 μm] in arteries treated with vehicle and paxilline respectively, n=4, P&lt;0.05) and in SD rats (% initial diameter: 84.2±7.9% [108.4±12.3 of 128.5±2.5 μm] and 60.5±0.2% [77.4±0.7 of 127.9±0.6 μm] in arteries treated with vehicle and paxilline respectively, n=3, P&lt;0.05). Paxilline-induced increases in vasoconstriction were significantly greater in arteries of SD as compared with SHR rats, suggesting that BK Ca channels are involved in hypercontractility in SHR. In inside-out patch-clamp, BK Ca channels in VSMCs isolated from mesenteric arteries of SHR and SD rats displayed identical calcium and voltage sensitivity. We next examined the effect of the selective IP3R-agonist, Adenophostin A, on BK Ca current density in VSMCs, using patch-clamp whole-cell mode. The results demonstrated that Adenophostin A (5μM) significantly increased BK Ca current density in VSMCs from both SHR (from 41.9± 5.7 to 61.5±9.1 pA/pF; n=6 cells, p&lt;0.05) and SD rats (from 29.9 ± 8.0 to 92.6 ± 9.1 pA/pF, n=5 cells, P&lt;0.01). There was no significant difference in basal BKca current density between SHR and SD rats; however, the Adenophostin-induced elevation in BK Ca current density was significantly diminished in VSMCs of SHR as compared with SD rats. In conclusion, the results indicate that loss of functional coupling between BKca and IP3R is involved in vascular hypercontractility in SHR arteries.

Heterogeneity and Remodeling of Ion Currents in Cultured Right Atrial Fibroblasts From Patients With Sinus Rhythm or Atrial Fibrillation.

Cardiac fibroblasts express multiple voltage-dependent ion channels. Even though fibroblasts do not generate action potentials, they may influence cardiac electrophysiology by electrical coupling via gap junctions with cardiomyocytes, and through fibrosis. Here, we investigate the electrophysiological phenotype of cultured fibroblasts from right atrial appendage tissue of patients with sinus rhythm (SR) or atrial fibrillation (AF). Using the patch-clamp technique in whole-cell mode, we observed steady-state outward currents exhibiting either no rectification or inward and/or outward rectification. The distributions of current patterns between fibroblasts from SR and AF patients were not significantly different. In response to depolarizing voltage pulses, we measured transient outward currents with fast and slow activation kinetics, an outward background current, and an inward current with a potential-dependence resembling that of L-type Ca2+ channels. In cell-attached patch-clamp mode, large amplitude, paxilline-sensitive single channel openings were found in ≈65% of SR and ∼38% of AF fibroblasts, suggesting the presence of “big conductance Ca2+-activated K+ (BKCa)” channels. The open probability of BKCa was significantly lower in AF than in SR fibroblasts. When cultured in the presence of paxilline, the shape of fibroblasts became wider and less spindle-like. Our data confirm previous findings on cardiac fibroblast electrophysiology and extend them by illustrating differential channel expression in human atrial fibroblasts from SR and AF tissue.

Patch‐Seq Analysis of Dbx1‐derived Respiratory Neurons of the preBötzinger Complex in Neonatal Mice

Inspiratory breathing movements depend on neurons in the preBötzinger complex (preBötC) whose progenitors express the transcription factor Dbx1 (henceforth Dbx1 neurons). These Dbx1 preBötC neurons generate both the rhythm and the rudimentary motor output pattern. Electrophysiological properties may differentiate rhythmogenic and pattern-related neurons, but whether two discrete functional classes of Dbx1 preBötC neurons exist remains to be demonstrated. Here, we use the Patch-Seq technique to measure the transcriptomes of individual Dbx1 preBötC neurons. We aim to develop a comprehensive understanding of the molecular components of the inspiratory core oscillator and to investigate whether discrete neuron classes exist therein. We recorded Dbx1 inspiratory neurons in whole-cell patch-clamp mode to assess their electrophysiological properties and then aspirated their cellular contents into the patch electrode for single-cell RNA-sequencing. We sequenced a total of 18 samples: 7 putative rhythm generators, 9 putative output neurons, and 1 that we could not definitely characterize physiologically. We obtained an average of 11,371,215 uniquely aligned paired-end reads per sample with an average length of 151 bp. More than a dozen differentially expressed genes distinguish putatively rhythmogenic and pattern-related Dbx1 preBötC neurons. These data make testable predictions about the composition and function of the mammalian preBötC that advance understanding at the molecular genetic level.