Perforated Patch-clamp Configuration Research Articles

High fat diet induces change in mitochondria respiration, activation of K-ATP channel and action potential shortening in mice

Obesity is associated with an increased risk of atrial fibrillation (AF). This epidemiologic observation may not be only due to shared co-morbidities but also from direct impacts of metabolic disorders on myocardium. To examine the impact of high fat diet (HFD) induced obesity on electrical properties in mice atria. Eight weeks old C57Bl/6 J mice were subjected to 4 months of HFD (60% fat) or normal diet (ND, 4% fat). Rapid burst atrial pacing were delivered using a trans-esophageal probe to induced AF in anesthetized animals. Action potential (AP) were recorded in left atrium using glass microelectrode technique. Potassium currents were recorded in isolated atrial myocytes using whole cell or perforated patch-clamp configurations. Mitochondrial respiration was studied in saponin-permeabilized atrial muscle fibers using a Clarke electrode. HFD mice showed higher atrial vulnerability to AF as indicated by longer AF episodes. APs were shorter in HFD versus ND mice (AP duration measured at 90% of repolarization: 49.3 ± 1.6 msec in HFD vs. 56.7 ± 2.3 msec in ND, P < 0.01). Glibenclamide, a KATP channel blocker, reversed AP shortening whereas cromakalim, a KATP channel activator, had no effect. Only in perforated patch-clamp configuration, without intracellular dialysis, KATP component of potassium current was enhanced in HFD mice (at +70 mV: 0.41 ± 0.12 pA/pF in HFD vs. 0.13 ± 0.08 pA/pF in ND, P < 0.05). There was no change in mitochondria respiration capacity between ND and HFD, however the ability of palmitoyl-CoA to support mitochondrial respiration was higher in HFD (25.99 ± 4.11% vs. 46.9 ± 3.96% in ND, P = 0.001). In addition, HADHA activity was enhanced in HFD mice. Obesity in mice is associated with atrial AP shortening which appears to result from increased activity of KATP-regulated currents and be associated with a metabolic shift. Shortening of AP duration could contribute to higher vulnerability to AF during obesity.

Activation of β3‐adrenoceptors inhibits purinergic‐mediated contractions of the murine detrusor

β3‐adrenergic receptor (β3‐AR) agonists are used for treatment of overactive bladder, however the mechanisms underlying their effect are unclear. β3‐AR agonists inhibit cholinergic‐mediated responses of the detrusor, however the concentration required to exert this effect exceed the plasma levels achieved through therapeutic dosing of these drugs1. ATP is regarded as an excitatory neurotransmitter in the detrusor, and upregulation of purinergic signaling is considered as a causative factor in the pathogenesis of overactive bladder2. The purpose of the present study was 1) to examine the effect of β3‐AR activation on purinergic‐evoked contractions of the murine detrusor &amp; on P2XR1 currents recorded from isolated murine detrusor myocytes and 2) to investigate if purinergic responses in the detrusor were modulated by agents that activate exchange factor directly activated by cAMP (EPAC), a novel cAMP effector.Mice were killed by cervical dislocation and their bladder removed, in accordance with the DKIT Animal Care &amp; Use Committee and EU legislation (EU Directive 2010/63/EU). Using fine dissection, the urothelium was removed leaving the remaining detrusor layer. Isometric tension recordings were made from strips of detrusor muscle. Detrusor smooth muscle cells (DSMCs) were isolated by enzymatic digestion (Collagenase Type II, 5 minutes at 37°C) of detrusor tissue cut into fine pieces. The perforated patch clamp configuration was used to investigate inward currents in isolated DSMCs. Perforation was achieved using the antibiotic amphotericin B (600 μg/ml).In the presence of the muscarinic antagonist atropine (1 μM), electric field stimulation (EFS; 4 Hz) induced robust purinergic‐mediated contractions of murine detrusor strips. Therapeutic‐relevant concentrations (100 nM) of the β3‐AR agonists BRL37344 &amp; CL316,243, reduced the amplitude of EFS‐evoked detrusor contractions by 59% (n=8, p&lt;0.01) and 50% (n=6, p&lt;0.05), respectively. These effects were partially reversed by the β3‐AR antagonist, L748,337 (1 μM). The adenylyl cyclase stimulant forskolin and the PDE4 inhibitor rolipram, similarly inhibited EFS‐evoked purinergic contractions. Under voltage clamp conditions (‐60 mV), ATP (1 μM) evoked inward currents in isolated DSMCs. These currents were reduced from −1803 ± 418 pA to −514 ± 168 pA (n=6, p&lt;0.05) by BRL37344 (100 nM). Quantitative PCR experiments revealed that EPAC1 &amp; 2 subtypes were expressed in murine detrusor, with EPAC1 ~10‐fold more abundant. Application of the EPAC activator, 007‐AM, reduced the amplitude of purinergic contractions by ~48%, (n=6, p&lt;0.01), and this effect was partially reversed in the presence of the EPAC1 blocker, CE3F4.These data show that β3‐AR agonists inhibit purinergic‐mediated responses of the murine detrusor by inhibiting P2XR1 currents in detrusor myocytes, possibly by a mechanism involving activation of EPAC1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Minimizing Cytosol Dilution in Whole-Cell Patch-Clamp Experiments

During a conventional whole-cell patch clamp experiment, diffusible cytosolic ions or molecules absent in the pipette solution can become diluted by a factor of one million or more, leading to diminished current or fluorescent signals. Existing methods to prevent or limit cytosol diffusion include reducing the diameter of the pipette's orifice, adding cytosolic extract or physiological entities to the pipette solution, and using the perforated patch clamp configuration. The first method introduces measurement error in recorded signals from increased series resistance and the latter two are cumbersome to perform. In addition, most perforated patch configurations, prevent investigators from using test compounds in the pipette solution. We present a method to overcome these limitations by minimizing cytosol dilution using a novel pipette holder. Cell-attached configuration is obtained with the pipette filled with pipette solution. Most of the pipette solution is then replaced with mineral oil so that cytosol dilution can be minimized in whole-cell configuration. To accomplish this requires a suction line and two Ag/AgCl electrodes inside the pipette. Testing our novel pipette holder with Chinese Hamster Ovarian cells, we demonstrate cytosol dilution factors between 76 and 234. For large cells with somas greater than 40μm, cytosol dilution factors of 10 or less are achievable.

Evoked Impulse Activity of Isolated Hippocampal Neurons in the Perforated Patch-Clamp Configuration

We describe the results obtained in studies of the impulse activity recorded from isolated neurons of the hippocampal CA1 area in the perforated patch-clamp configuration. Spiking was initiated by two modes of intracellular stimulation, namely by application of rectangular 2.5-sec-long current pulses or a ramp protocol (linear current ramp within the 10.0-sec-long interval). We proposed schemes of recording allowing experimenters to obtain reliable quantitative characteristics of the activity of isolated neurons taking into account the problem of reproducibility of the results. We also examined the rhythmicity of generation of action potentials by the studied neuron and typical phenomena of its disturbances; the techniques for selection of an optimum mode of ramp stimulation of the neuron making it possible to obtain detailed characteristics of generation of impulse activity are also described.

PKC-dependent autoregulation of membrane kainate receptors

Agonists of kainate receptors (KARs) cause both the opening of the associated ion channels and the activation of signalling pathways driven by G-proteins and PKC. Here we report the existence of an unknown mechanism of KAR autoregulation, involving the interplay of this two signalling mechanisms. Repetitive activation of native KARs evoked the rundown of the ionotropic responses in a manner that was dependent on the activation of PKC. Experiments on recombinant GluR5 expressed in neuroblastoma cells indicated that KARs trigger the activation of PKC and induce the internalization of membrane receptors. This phenomenon depends on the PKC-mediated phosphorylation of serines 879 and 885 of the GluR5-2b subunits, since mutation of these two residues abolished internalization. These results reveal that the non-canonical signalling of KARs is associated with a sensitive mechanism that detects afferent activity. Such a mechanism represents an active way to limit overactivation of the KAR system, by regulating the number of KARs in the cell membrane.

The role of constitutive PKA‐mediated phosphorylation in the regulation of basal ICa in isolated rat cardiac myocytes

1. Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca(2+) current (I(Ca)) observed in the absence of exogenous beta-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell I(Ca) in the perforated patch-clamp configuration. 2. Calyculin A and isoprenaline (both 1 micromol l(-1)) increased basal I(Ca) (P<0.05), whereas H-89 inhibited I(Ca) in a concentration-dependent manner with an IC(50) approximately 5 micromol l(-1). H-89 also inhibited the response to 1.0 micromol l(-1) isoprenaline, although relatively high concentrations (30 micromol l(-1)) were required to achieve complete suppression of the response. 3. Double-pulse protocols were used to study the effects of 10 micromol l(-1) H-89 on time-dependent recovery of I(Ca) from voltage-dependent inactivation as well as the steady-state gating of I(Ca). T(0.5) (time for I(Ca) to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (P<0.05) but was unaffected by calyculin A or isoprenaline. 4. Steady-state activation/inactivation properties of I(Ca) were unaffected by 10 micromol l(-1) H-89 or 1 micromol l(-1) calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V(0.5) for activation and inactivation became more negative. 5. Data show that basal I(Ca) is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied beta-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to beta-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.

The SK channel blocker apamin inhibits slow afterhyperpolarization currents in rat gonadotropin‐releasing hormone neurones

Gonadotropin-releasing hormone (GnRH) neurones play an essential role in the hypothalamo-pituitary-gonadal axis. As for other neurones, the discharge pattern of action potentials is important for GnRH neurones to properly function. In the case of a luteinizing hormone (LH) surge, for example, GnRH neurones are likely to continuously fire for more than an hour. For this type of firing, GnRH neurones must have a certain intrinsic property. To address this issue, we investigated the voltage-gated Ca(2+) currents and Ca(2+)-activated voltage-independent K(+) currents underlying afterhyperpolarization, because they affect cell excitability. Dispersed GnRH neurones from adult GnRH-EGFP (enhanced green fluorescent protein) transgenic rats were cultured overnight and then used for an electrophysiological experiment involving the perforated patch-clamp configuration. The GnRH neurones showed five subtypes of voltage-gated Ca(2+) currents, i.e. the T-, L-, N-, P/Q- and R-types. The GnRH neurones also showed a slow afterhyperpolarization current (I(sAHP)), but not a medium one. It is reported that the SK channel blocker apamin (10 pm-100 nm) blocks a medium afterhyperpolarization current but not an I(sAHP). In contrast to previous reports, the I(sAHP) observed in rat GnRH neurones was potently blocked by apamin. In addition, the GnRH neurones expressed transcripts for SK1-3 channels. The results indicate that rat GnRH neurones express all five subtypes of voltage-gated Ca(2+) channels and exhibit an apamin-sensitive I(sAHP), which may allow continuous firing in response to a relatively strong depolarizing input.

Ranolazine Improves Abnormal Repolarization and Contraction in Left Ventricular Myocytes of Dogs with Heart Failure by Inhibiting Late Sodium Current

Ventricular repolarization and contractile function are frequently abnormal in ventricular myocytes from human failing hearts as well as canine hearts with experimentally induced heart failure (HF). These abnormalities have been attributed to dysfunction involving various steps of the excitation-contraction coupling process, leading to impaired intracellular sodium and calcium homeostasis. We previously reported that the slow inactivating component of the Na(+) current (late I(Na)) is augmented in myocytes from failing hearts, and this appears to play a significant role in abnormal ventricular myocytes repolarization and function. We tested the effect of ranolazine, a novel drug being developed to treat angina, on (1) action potential duration (APD), (2) peak transient and late I(Na) (I(NaT) and I(NaL), respectively), (3) early afterdepolarizations (EADs), and (4) twitch contraction (TC), including after contractions and contracture. Myocytes were isolated from the left ventricle of normal dogs and of dogs with chronic HF caused by multiple sequential intracoronary micro-embolizations. I(NaT) and I(NaL) were recorded using conventional whole-cell patch-clamp techniques. APs were recorded using the beta-escin perforated patch-clamp configuration at frequencies of 0.25 and 0.5 Hz. TCs were recorded using an edge movement detector at stimulation frequencies ranging from 0.5 to 2.0 Hz. Ranolazine significantly (P<0.05) and reversibly shortened the APD of myocytes stimulated at either 0.5 or 0.25 Hz in a concentration-dependent manner. At a stimulation frequency of 0.5 Hz, 5, 10, and 20 microM ranolazine shortened the APD(90) (APD measured at 90% repolarization) from 516+/-51 to 304+/-22, 212+/-34 and 160+/-11 ms, respectively, and markedly decreased beat-to-beat variability of APD(90), EADs, and dispersion of APDs. Ranolazine preferentially blocked I(NaL) relative to I(NaT) in a state-dependent manner, with a approximately 38-fold greater potency against I(NaL) to produce tonic block (IC(50)=6.5 microM) than I(NaT) (IC(50)=294 microM). When we evaluated inactivated state blockade of I(NaL) from the steady-state inactivation mid-potential shift using a theoretical model, ranolazine was found to bind more tightly to the inactivated state than the resting state of the sodium channel underlying I(NaL), with apparent dissociation constants K(dr)=7.47 microM and K(di)=1.71 microM, respectively. TCs of myocytes stimulated at 0.5 Hz were characterized by an initial spike followed by a dome-like after contraction, which was observed in 75% of myocytes from failing hearts and coincided with the long AP plateau and EADs. Ranolazine at 5 and 10 microM reversibly shortened the duration of TCs and abolished the after contraction. When the rate of myocyte stimulation was increased from 1.0 to 2.0 Hz, there was a progressive increase in diastolic "tension," that is, contracture. Ranolazine at 5 and 10 microM reversibly prevented this frequency-dependent contracture.

Fulfenamic acid sensitive, Ca 2+-dependent inward current induced by nicotinic acetylcholine receptors in dopamine neurons

Nicotinic acetylcholine receptors (nAChRs) exhibit high Ca 2+ permeabilities and the Ca 2+-influx through the nAChRs may be involved in regulation of a variety of signal processing in the postsynaptic neurons. The mesencephalic dopamine (DA) neurons receive cholinergic inputs from the brainstem and express abundant nAChRs. Here we report that the Ca 2+-influx induced by a transient pressure application of ACh activates an inward current mediated by nAChRs and subsequently an inward current component that is sensitive to fulfenamic acid (FFA) and phenytoin, presumably a Ca 2+-activated nonselective cation current in the DA neurons in the midbrain slices of the rat. The FFA- and phenytoin-sensitive current exhibits a negative slope conductance below −40 mV, suggesting its role in significant enhancement of depolarizing responses. In the current clamp recordings with perforated patch clamp configuration, bath application of carbachol markedly enhanced the glutamate-induced depolarization, which led to a long-lasting depolarizing hump. Activation of nAChRs is involved in this process, in cooperation with muscarinic receptors that suppress afterhyperpolarization caused by Ca 2+-activated K +-channels. The long-lasting depolarizing hump was suppressed by FFA. All these results suggested a potential role of the FFA-sensitive current triggered by nAChR activation in marked enhancement of the excitatory synaptic response in DA neurons.

Chapter 10 Postsynaptic GABAB receptors in supraoptic oxytocin and vasopressin neurons

Publisher Summary This chapter describes the expression and function of postsynaptic gamma-aminobutyric acid (GABA B ) receptors in magnocellular neurons, using a combination of in vitro patch-clamp recordings and immunohistochemical approaches. GABA B receptor is an unusual G-protein-coupled receptor, composed of two seven transmembrane domain subunits, GABA B R1 and GABA B R2, respectively. Heteromerization of these subunits results in a functional receptor at the cell surface. Immunohistochemical techniques were used to study the expression of GABA B receptor subunits in identified oxtytocin (OT) and vasopressin (VP) neurons. Triple fluorescent immunohistochemical experiments were performed in 30 μm coronal hypothalamic brain slices obtained from Sprague–Dawley male and female rats. Antibodies raised against GABA B R1 and/or GABA B R2 receptor subunits were combined simultaneously with antibodies raised against OT and VP neurophysins. Colocalization of the three antibodies in individual supraoptic (SON) and paraventrieular nuclei (PVN) neurons was assessed. GABAB receptor subunit immunoreactivity was quantified using an optical density analysis. The electrical activity of SON neurons was recorded using the perforated patch-clamp configuration, which allows electrical access to the cell, preventing the dialysis of the intracellular environment. GABA B receptor immunoreactivity was significantly stronger in VP, as compared to OT neurons, suggesting that the expression of this receptor in the magnocellular neuroendocrine system is cell-type dependent.