Alexander A. Harper

Reactive oxygen species modulate neuronal excitability in rat intrinsic cardiac ganglia

Reactive oxygen species (ROS) are produced as by-products of oxidative metabolism and occur in the heart during ischemia and coronary artery reperfusion. The effects of ROS on the electrophysiological properties of intracardiac neurons were investigated in the intracardiac ganglion (ICG) plexus in situ and in dissociated neurons from neonatal and adult rat hearts using the whole-cell patch clamp recording configuration. Bath application of ROS donors, hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (t-BHP) hyperpolarized, and increased the action potential duration of both neonatal and adult ICG neurons. This action was also recorded in ICG neurons in an adult in situ ganglion preparation. H2O2 and t-BHP also inhibited voltage-gated calcium channel (VGCC) currents and shifted the current–voltage (I–V) relationship to more hyperpolarized potentials. In contrast, H2O2 increased the amplitude of the delayed rectifier K+ current in neonatal ICG neurons. In neonatal ICG neurons, bath application of either superoxide dismutase (SOD) or catalase, scavengers of ROS, prior to H2O2 attenuated the hyperpolarizing shift but not the inhibition of VGCC by H2O2. In contrast, in adult ICG neurons, application of SOD alone had no effect upon either VGCC current amplitude or the I–V relationship, whereas application of SOD prior to H2O2 exposure abolished both the H2O2-mediated hyperpolarizing shift and inhibition. These data indicate that ROS alter depolarization-activated Ca2+ and K+ conductances which underlie neuronal excitability of ICG neurons. This affects action potential duration and therefore probably modifies autonomic control of the heart during ischemia/reperfusion.

Expression and Modulation of the Intermediate- Conductance Ca2+-Activated K+ Channel in Glioblastoma GL-15 Cells

We report here the expression and properties of the intermediate-conductance Ca2+-activated K+ (IKCa) channel in the GL-15 human glioblastoma cell line. Macroscopic IKCa currents on GL-15 cells displayed a mean amplitude of 7.2±0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC50=257 nM), TRAM-34 (IC50=55 nM), and charybdotoxin (CTX, IC50=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IKCa channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a KD for Ca2+ of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 µM, for 5 days) virtually suppressed the IKCa current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17±0.75 pA/pF at 1-2 days, and 3.11±1.35 pA/pF at 5-6 days after plating. This time-dependent downregulation of the IKCa current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ~35% reduction of the IKCa channel mRNA resulting from ERK inhibition and a ~50% reduction with time in culture.

Expression and properties of hyperpolarization-activated current in rat dorsal root ganglion neurons with known sensory function

•  I h is a hyperpolarisation‐activated current that influences neuronal excitability and is present in some sensory neurons. •  The magnitude and properties of Ih in different groups of sensory neurons that respond to painful stimuli (nociceptors) or to non‐painful stimuli, such as low threshold mechanoreceptors (LTMs), were unknown. •  We found that neurons with the greatest Ih were the nociceptors and LTMs with the fastest conducting fibres. The highest Ih of all was present in LTM neurons that sense muscle stretch and length (muscle spindle afferents). •  The high levels of Ih could fundamentally influence excitability of fast conducting sensory neurons which detect muscle stretch/length, touch and pressure, and painful stimuli. Ih could thus influence sensations associated with all these. •  The properties of Ih are similar to those of HCN1‐ and HCN2‐related Ih, suggesting that these channels underlie the current.

Expression and properties of hyperpolarization-activated current (Ih) in vivo in rat DRG neurons with known sensory functions

•  I h is a hyperpolarisation‐activated current that influences neuronal excitability and is present in some sensory neurons. •  The magnitude and properties of Ih in different groups of sensory neurons that respond to painful stimuli (nociceptors) or to non‐painful stimuli, such as low threshold mechanoreceptors (LTMs), were unknown. •  We found that neurons with the greatest Ih were the nociceptors and LTMs with the fastest conducting fibres. The highest Ih of all was present in LTM neurons that sense muscle stretch and length (muscle spindle afferents). •  The high levels of Ih could fundamentally influence excitability of fast conducting sensory neurons which detect muscle stretch/length, touch and pressure, and painful stimuli. Ih could thus influence sensations associated with all these. •  The properties of Ih are similar to those of HCN1‐ and HCN2‐related Ih, suggesting that these channels underlie the current.

Verapamil Block of Large-Conductance Ca-Activated K Channels in Rat Aortic Myocytes

Abstract. The effects of verapamil on the large conductance Ca-activated K (BK) channel from rat aortic smooth muscle cells were examined at the single channel level. Micromolar concentrations of verapamil produced a reversible flickering block of the BK channel activity. Kinetic analysis showed that verapamil decreased markedly the time constants of the open states, without any significant change in the time constants of the closed states. The appearance of an additional closed state — specifically, a nonconducting, open-blocked state — was also observed, whose time constant would reflect the mean residence time of verapamil on the channel. These observations are indicative of a state-dependent, open-channel block mechanism. Dedicated kinetic (group) analysis confirmed the state-dependent block exerted by verapamil. D600 (gallopamil), the methoxy derivative of verapamil, was also tested and found to exert a similar type of block, but with a higher affinity than verapamil. The permanently charged and membrane impermeant verapamil analogue D890 was used to address other important features of verapamil block, such as the sidedness of action and the location of the binding site on the channel protein. D890 induced a flickering block of BK channels similar to that observed with verapamil only when applied to the internal side of the membrane, indicating that D890 binds to a site accessible from the cytoplasmic side. Finally, the voltage dependence of D890 block was assessed. The experimental data fitted with a Langmuir equation incorporating the Woodhull model for charged blockers confirms that the D890-binding site is accessed from the internal mouth of the BK channel, and locates it approximately 40% of the membrane voltage drop along the permeation pathway.

Increased (Na+K+Cl) Cotransport in Rat Arterial Smooth Muscle in Deoxycorticosterone (DOCA)/Salt-Induced Hypertension

The inhibition by loop diuretics of K efflux (tracer 86Rb) from the rat femoral arterial smooth muscle was measured in normotension and in DOCA-salt hypertension. The sensitivity sequence (bumetanide > piretanide > furosemide) was the characteristic pharmacological profile of (Na+K+Cl) cotransport. In hypertension, cotransport activity was 46% greater than in normotension and the sensitivity to loop diuretics was threefold less. Intracellular [K] and the Na, K and Cl permeability ratios and electrogenic Na pump activity were assessed electrophysiologically in normotension and hypertension. [K]i was lower in hypertension (173 mM) than normotension (198 mM) but the other parameters (PNa/Cl = 0.14, PCl/PK = 0.19 and electrogenic pump = –8.3 mV in normotension) were not significantly different. Ionic permeabilities to Na, K and Cl were significantly lower in hypertension than normotension. Plasma [Na], but not [K], was higher in hypertension than normotension. The conclusion is that increased activation of (Na+K+Cl) cotransport in hypertension plays a major role in the elevation of [Cl]i and depolarisation of the membrane potential in vascular smooth muscle in DOCA-salt hypertension. The role of (Na+K+Cl) cotransport in vascular smooth muscle in this model of hypertension is discussed in relation to [Cl]i, depolarisation of the membrane potential and contraction and in relation to cell growth.

Stimulation of intracellular chloride accumulation by noradrenaline and hence potentiation of its depolarization of rat arterial smooth muscle in vitro

1 Double‐barrelled ion‐selective microelectrodes were used to examine the effects of exogenous noradrenaline upon the membrane potential (Em) and intracellular chloride concentration ([Cl]i) of arterial smooth muscle from the saphenous branch of the femoral artery of the rat. 2 After treatment with 0.6 mM 6‐hydroxydopamine (to functionally denervate the tissue), exogenous noradrenaline (5 nM) caused repeatable depolarization of Em from −63.7±2.4 mV (s.d., n=18) to −53.8±3.4 mV (P<0.0001) and increases in [Cl]i from 31.0±0.5 mM to 42.5±2.2 mM (P<0.0001). 3 In the presence of 10 μM bumetanide (an inhibitor of (Na‐K‐Cl) cotransport), 5 nM noradrenaline caused a depolarization of Em of 3.0±3.2 mV, and a rise in [Cl]i of 4.5±2.5 mM. 4 In the presence of bumetanide and 1 mM acetazolamide (used as an inhibitor of a Na‐independent inward Cl pump), noradrenaline had no effect on Em or [Cl]i. 5 In the absence of extracellular chloride, the rise in apparent [Cl]i in response to 5 nM noradrenaline was abolished but there was a depolarization of 2.0±3.9 mV. 6 These results are consistent with the stimulation of (Na‐K‐Cl) cotransport and a Na‐independent Cl pump by exogenous noradrenaline and with the consequent increase in [Cl]i and shift in ECl potentiating the depolarization caused by noradrenaline. The possibility that modulation of [Cl]i may be a general mechanism of Em regulation is discussed.

Na+/amino acid coupling stoichiometry of rheogenic system B0,+ transport in Xenopus oocytes is variable.

Using electrophysiological and radiotracer studies in parallel, we have investigated the characteristics of the endogenous Na+-dependent amino acid transporter (system B0,+) in Xenopus oocytes with regard to ion dependence, voltage dependence and transport stoichiometry. In voltage-clamped oocytes (−60 mV) superfusion with saturating concentrations of amino acids (1 mM) in 100 mM NaCl resulted in reversible, inward currents (mean±SEM): alanine, 1.83±0.09 nA (n=21); arginine, 2.54±0.18 nA (n=17); glutamine, 1.73±0.10 nA (n=19). Only arginine evoked a current in choline medium (0.50±0.13 nA, n=10), whereas Cl− replacement had no effect on evoked currents. The glutamine-evoked current was saturable (Imax=1.73 nA, glutamine Km=0.12 mM) and linearly dependent upon voltage between −90 and −30 mV. Using direct and indirect (activation) methods, we found that transport can proceed with Na+/amino acid coupling stoichiometry of either 1∶1 or 2∶1, but coupling was the same for each amino acid tested (alanine, arginine and glutamine) within a batch of oocytes (i.e. from a single toad). Despite the net single positive charge on arginine, the magnitude of the net transmembrane charge movement during Na+-coupled arginine transport was identical to that for the zwitterionic neutral amino acids glutamine and alanine; this may be explained by a concomitant stimulation of K+ efflux during arginine transport with a putative coupling of 1 K+∶1 arginine.

Reactive oxygen species alters the electrophysiological properties and raises [Ca2+]i in intracardiac ganglion neurons

We have investigated the effects of the reactive oxygen species (ROS) donors hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (t-BHP) on the intrinsic electrophysiological characteristics: ganglionic transmission and resting [Ca2+]i in neonate and adult rat intracardiac ganglion (ICG) neurons. Intracellular recordings were made using sharp microelectrodes filled with either 0.5 M KCl or Oregon Green 488 BAPTA-1, allowing recording of electrical properties and measurement of [Ca2+]i. H2O2 and t-BHP both hyperpolarized the resting membrane potential and reduced membrane resistance. In adult ICG neurons, the hyperpolarizing action of H2O2 was reversed fully by Ba2+ and partially by tetraethylammonium, muscarine, and linopirdine. H2O2 and t-BHP reduced the action potential afterhyperpolarization (AHP) amplitude but had no impact on either overshoot or AHP duration. ROS donors evoked an increase in discharge adaptation to long depolarizing current pulses. H2O2 blocked ganglionic transmission in most ICG neurons but did not alter nicotine-evoked depolarizations. By contrast, t-BHP had no significant action on ganglionic transmission. H2O2 and t-BHP increased resting intracellular Ca2+ levels to 1.6 ( ± 0.6, n = 11, P < 0.01) and 1.6 ( ± 0.3, n = 8, P < 0.001), respectively, of control value (1.0, ∼60 nM). The ROS scavenger catalase prevented the actions of H2O2, and this protection extended beyond the period of application. Superoxide dismutase partially shielded against the action of H2O2, but this was limited to the period of application. These data demonstrate that ROS decreases the excitability and ganglionic transmission of ICG neurons, attenuating parasympathetic control of the heart.

Neural control of the heart: developmental changes in ionic conductances in mammalian intrinsic cardiac neurons

The expression and properties of ionic channels were investigated in dissociated neurons from neonatal and adult rat intracardiac ganglia. Changes in the hyperpolarization-activated and ATP-sensitive K+ conductances during postnatal development and their role in neuronal excitability were examined. The hyperpolarization-activated nonselective cation current, Ih, was observed in all neurons studied and displayed slow time-dependent rectification. An inwardly rectifying K+ current, IK(IR), was present in a population of neurons from adult but not neonatal rats and was sensitive to block by extracellular Ba2+ Using the perforated-patch recording configuration, an ATP-sensitive K+ (KATP) conductance was identified in > or = 50% of intracardiac neurons from adult rats. Levcromakalim evoked membrane hyperpolarization, which was inhibited by the sulphonylurea drugs, glibenclamide and tolbutamide. Exposure to hypoxic conditions also activated a membrane current similar to that induced by levcromakalim and was inhibited by glibenclamide. Changes in the complement of ion channels during postnatal development may underlie observed differences in the function of intracardiac ganglion neurons during maturation. Furthermore, activation of hyperpolarization-activated and KATP channels in mammalian intracardiac neurons may play a role in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion.