Electrophysiologic Mechanism Underlying Action Potential Prolongation by Sevoflurane in Rat Ventricular Myocytes

Background: Despite prolongation of the QTc interval in humans during sevoflurane anesthesia, little is known about the mechanisms that underlie these actions. In rat ventricular myocytes, the effect of sevoflurane on action potential duration and underlying electrophysiologic mechanisms were investigated. Methods: The action potential was measured by using a current clamp technique. The transient outward K+ current was recorded during depolarizing steps from −80 mV, followed by brief depolarization to −40 mV and then depolarization up to +60 mV. The voltage dependence of steady state inactivation was determined by using a standard double-pulse protocol. The sustained outward current was obtained by addition of 5 mm 4-aminopyridine. The inward rectifier K+ current was recorded from a holding potential of −40 mV before their membrane potential was changed from −130 to 0 mV. Sevoflurane actions on L-type Ca2+ current were also obtained. Results: Sevoflurane prolonged action potential duration, whereas the amplitude and resting membrane potential remained unchanged. The peak transient outward K+ current at +60 mV was reduced by 18 ± 2% (P < 0.05) and 24 ± 2% (P < 0.05) by 0.35 and 0.7 mm sevoflurane, respectively. Sevoflurane had no effect on the sustained outward current. Whereas 0.7 mm sevoflurane did not shift the steady state inactivation curve, it accelerated the current inactivation (P < 0.05). The inward rectifier K+ current at −130 mV was little altered by 0.7 mm sevoflurane. L-type Ca2+ current was reduced by 28 ± 3% (P < 0.05) and 33 ± 1% (P < 0.05) by 0.35 and 0.7 mm sevoflurane, respectively. Conclusions: Action potential prolongation by clinically relevant concentrations of sevoflurane is due to the suppression of transient outward K+ current in rat ventricular myocytes.

Contributions of sustained INa and IKv43 to transmural heterogeneity of early repolarization and arrhythmogenesis in canine left ventricular myocytes

The roles of sustained components of INa and IKv43 in shaping the action potentials (AP) of myocytes isolated from the canine left ventricle (LV) have not been studied in detail. Here we investigate the hypothesis that these two currents can contribute substantially to heterogeneity of early repolarization and arrhythmic risk. Quantitative data from voltage-clamp and expression profiling experiments were used to complete meaningful modifications to an existing “local control” model of canine midmyocardial myocyte excitation-contraction coupling for epicardial and endocardial cells. We include 1) heterogeneous IKv43, IKs, and ISERCA density; 2) modulation of IKv43 by Kv channel interacting protein type 2 (KChIP2) channel subunits; 3) a possible Ca2+-dependent open-state inactivation of IKv43; and 4) a sustained component of the inward Na+ current, INaL. The resulting simulations illustrate ways in which KChIP2- and Ca2+-dependent control of IKv43 can result in a sustained outward current that can neutralize INaL in a rate- and myocyte subtype-dependent manner. Both these currents appear to play significant roles in modulating AP duration and rate dependence in midmyocardial myocytes. Furthermore, an increased ratio of IKv43 to INaL is capable of protecting epicardial myocytes from the early afterdepolarizations resulting from the SCN5A-I1768V mutation-induced increase in INaL. Experimentally observed transmural differences in Ca2+ handling, including greater sarcoplasmic reticulum Ca2+ content and faster Ca2+ transient decay rates on the epicardium, were recapitulated in our simulations. By design, these models allow upward integration into organ models or may be used as a basis for further investigations into cellular heterogeneities.

Dopamine Modulation of Honey Bee (Apis mellifera) Antennal-Lobe Neurons

Primary olfactory centers [antennal lobes (ALs)] of the honey bee brain are invaded by dopamine (DA)-immunoreactive neurons early in development (pupal stage 3), immediately before a period of rapid growth and compartmentalization of the AL neuropil. Here we examine the modulatory actions of DA on honey bee AL neurons during this period. Voltage-clamp recordings in whole cell configuration were used to determine the effects of DA on ionic currents in AL neurons in vitro from pupal bees at stages 4–6 of the nine stages of metamorphic adult development. In ∼45% of the neurons tested, DA (5–50 × 10−5 M) reduced the amplitude of outward currents in the cells. In addition to a slowly activating, sustained outward current, DA reduced the amplitude of a rapidly activating, transient outward conductance in some cells. Both of the currents modulated by DA could be abolished by the removal of Ca2+ from the external medium or by treatment of cells with charybdotoxin (2 × 10−8 M), a blocker of Ca2+-dependent K+ currents in the cells. Ca2+ currents were not affected by DA, nor were A-type K+ currents ( IA). Results suggest that the delayed rectifier-like current ( IKV) also remains intact in the presence of DA. Taken together, our data indicate that Ca2+-dependent K+ currents are targets of DA modulation in honey bee AL neurons. This study lends support to the hypothesis that DA plays a role in the developing brain of the bee.

Effects of aging on the cardiac remodeling induced by chronic high-altitude hypoxia in rat

Effects of chronic high-altitude hypoxia on the remodeling of right ventricle were examined in three age groups of rats: 2, 6, and 18 mo. The extent of right ventricular (RV) hypertrophy (RVH) showed an age-associated diminution. RV cell size and pericellular fibrosis showed a significant increase in the 2- and 6-mo-old exposed rats but not in the 18-mo-old exposed rats compared with control. A hyperplasic response was underscored in the three exposed age groups but appeared less pronounced in the 18-mo-old rats. A significant decrease in the transient outward potassium current ( Ito) density was observed in RV cell only in the 2-mo-old exposed group compared with the control group. In the control group, there was a clear tendency for Ito density to decrease as a function of age. The sustained outward current density was modified neither by the hypoxia condition nor by the age. Neither the cytochrome c oxidase activity nor the heat shock protein 72 content in the RV was altered after hypoxic exposure regardless of age. The norepinephrine content in the RV was significantly decreased in each age group exposed to hypoxia when compared with their age-matched control group. Our findings indicate that the remodeling (at morphological and electrophysiological levels) induced by chronic hypoxia in the RV can be decreased by the natural aging process.

Comparison of a Ca(2+)-gated conductance and a second-messenger-gated conductance in rat olfactory neurons

The effect of a rise in intracellular Ca(2+) concentration was analyzed in isolated rat olfactory neurons using a whole-cell patch-clamp technique. Intracellular dialysis of 1 mmol l(−)(1) Ca(2+) in a standard-K(+), low-Cl(−) internal solution (E(Cl)=−69 mV) from the patch pipette into the olfactory neurons induced a sustained outward current of 49+/−5 pA (N=13) at −50 mV in all the cells examined. The outward currents were inhibited by external application of 100 micromol l(−)(1) 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). External application of a Ca(2+) ionophore, 3 micromol l(−)(1) ionomycin, induced an inward current in three of eight cells whose voltages were clamped using the gramicidin-perforated technique, but ionomycin elicited an outward current in the other five cells, suggesting that natural intracellular Cl(−) concentration in the olfactory neurons was heterogeneous. While intracellular dialysis of 50 micromol l(−)(1) inositol 1,4,5-trisphosphate (1,4,5-InsP(3)) in the standard-K(+), low-Cl(−) internal solution induced the NPPB-sensitive outward current in 31 % of cells, and 500 micromol l(−)(1) cAMP induced it in 21 % of cells, a large proportion of the cells displayed an inward current in response to 1,4,5-InsP(3) and cAMP. The results suggest that 1,4,5-InsP(3) and cAMP can elicit Ca(2+)-dependent Cl(−) conductance and Ca(2+)-independent cation conductance in rat olfactory neurons.

Physiological and Theoretical Analysis of K+ Currents Controlling Discharge in Neonatal Rat Mesencephalic Trigeminal Neurons

Del Negro, Christopher A. and Scott H. Chandler. Physiological and theoretical analysis of K+ currents controlling discharge in neonatal rat mesencephalic trigeminal neurons. J. Neurophysiol. 77: 537–553, 1997. Whole cell voltage- and current-clamp recordings were obtained from mesencephalic trigeminal sensory (Mes 5) neurons identified visually in thin brain stem slices of neonatal rats with the use of infrared video microscopy. These cells exhibited accommodation in spike discharge responses to depolarizing current injection protocols whose duration differed as a function of holding potential (−50 vs. −65 mV). Several spikes were elicited before the membrane response accommodated from −50 mV, whereas from −65 mV only single action potentials were evoked. In response to similar protocols, application of the K+ channel blocker 4-aminopyridine (4-AP) (50 μM to 2 mM) caused sustained repetitive spiking whereas tetraethylammonium (TEA) (10–30 mM) did not cause repetitive spiking. In voltage clamp, 4-AP application (100 μM) revealed a sustained outward current ( I 4-AP) that was active between −60 and −30 mV. I 4-AP was responsible for suppressing sustained repetitive spiking behavior, producing accommodation under normal circumstances. TEA application in voltage clamp revealed a sustained outward current evoked positive to −40 mV. Two transient outward currents (TOCs) were identified by prepulse protocols typically used to characterizeA-type currents: a 4-AP-insensitive fast TOC, and a slow TOC( I TOC-S) sensitive to 4-AP (>500 μM). A Ca2+-dependent outward current that activated positive to −30 mV was also characterized. A mathematical model of a Mes 5 neuron was assembled from our voltage-clamp records to simulate the dynamic interaction of outward currents during membrane excitation. We conclude that in Mes 5 neurons, the 4-AP-sensitive currents I TOC-S and I 4-AP determine the duration of spike trains. In particular, the noninactivating I 4-AP determines whether cells exhibit sustained repetitive discharge or accommodate in response to depolarizing current. Neurotransmitter modulation of this current or modulation of the resting membrane potential could modify the output properties of Mes 5 neurons, and therefore the properties of these currents must be incorporated into our current understanding of how these cells contribute to shaping oral-motor pattern generation.

Adenosine inhibition of synaptic transmission in the substantia gelatinosa

1. We studied adenosine's action on synaptic transmission from primary afferent fibers to neurons of the substantia gelatinosa (SG) using tight-seal whole cell recordings in transverse slices of hamster spinal cord. Adenosine had two actions, hyperpolarization of the postsynaptic membrane and depression of the excitatory postsynaptic currents (EPSCs) evoked by dorsal root stimulation. 2. Under voltage clamp adenosine elicited a sustained outward current at a holding potential of -70 mV. The outward current was blocked by a combination of intracellular cesium and tetraethylammonium, an effect characteristic of potassium channels. The adenosine-induced current reversed at -97 +/- 6 (SD) mV, close to the potassium equilibrium potential. These observations suggest that adenosine activates a potassium conductance in SG neurons so as to inhibit primary afferent synaptic transmission postsynaptically. 3. Adenosine reduced the miniature EPSC frequency without significantly changing the amplitude. In contrast, the glutamate receptor competitive antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) substantially reduced the amplitudes of miniature EPSCs while producing a much smaller effect on the miniature frequency than adenosine. In evoked EPSCs adenosine reduced unitary content without reducing unitary amplitude. The effects on both miniature and evoked EPSCs suggest that adenosine inhibits synaptic currents by suppressing presynaptic transmitter release. 4. EPSCs evoked by dorsal root stimuli were subdivided into monosynaptic and polysynaptic categories. Adenosine at superfusion concentrations of 20-300 microM suppressed all polysynaptic EPSCs. Less than half of monosynaptic EPSCs were inhibited, usually those evoked by the slowest-conducting primary afferents. These observations were interpreted to indicate that a principal action of adenosine in SG is on interneuronal communication.

Electrophysiological recordings in solitary photoreceptors from the retina of squid, Loligo pealei

A protocol was developed to isolate enzymatically photoreceptors from the retina of the squid, Loligo pealei. The procedure routinely results in a high yield of intact cells. Examination of solitary photoreceptors under Nomarski optics revealed that the fine morphological features described in anatomical studies of retinal sections are retained. The distal segment is up to 250 μm long, 4–7 μm wide, covered in part by short microvilli; the inner segment and the cell body, with the initial portion of the axon, are also clearly discernible in solitary cells. Suction electrode measurements performed from the cell body confirmed that responsiveness to light survived cell isolation. Macroscopic membrane currents were measured using the whole-cell tight-seal technique, and the perforated-patch method. Step depolarizations of membrane voltage administered in the dark elicited a slowly activating, sustained outward current. Light stimulation evoked an inward current graded with stimulus intensity; the peak current could amply exceed 1000 pA. Intense photostimulation gave rise to a prolonged inward aftercurrent that lasted for tens of seconds. On-cell patch recording along the intermediate segment and most of the smooth areas of the distal segment showed a large incidence of silent patches, with the occasional presence of voltage-dependent channels. On the other hand, channel activity could be recorded more frequently from electrode placements near the apical tip of the cell, where the presence of microvilli could be confirmed visually. Some patches were unresponsive to voltage Stimulation applied in the dark but produced distinct bursts of channel openings after illumination. The feasibility of single-cell electrophysiology in isolated photoreceptors, together with the growing body of biochemical information on cephalopod preparations, makes squid an attractive model system to investigate the visual process in invertebrates using multiple experimental approaches.

Postnatal rat nigrostriatal dopaminergic neurons exhibit five types of potassium conductances

1. We have investigated the electrical properties of neurons acutely dissociated from the substantia nigra zona compacta (SNZC) of the postnatal rat with whole cell patch-clamp recordings. Retrogradely labeled nigrostriatal neurons were identified with the use of rhodamine-labeled fluorescent latex microspheres. Over 90% of the rhodamine-labeled neurons in the SNZC demonstrated formaldehyde/glutaraldehyde-induced catecholamine fluorescence, indicating that they were dopaminergic (DA) neurons. 2. DA neurons had 15-20 microns ovoid or fusiform-shaped cell bodies with 2-3 thick proximal processes. Labeled neurons generated spontaneous action-potential activity in both regular and irregular patterns. These cells exhibited input resistances of 300-600 M omega and action-potential amplitudes of 60-80 mV. Locally applied dopamine inhibited the spontaneous activity of these neurons by hyperpolarizing the cells. 3. Outward currents were examined with voltage-clamp recordings using a tetrodotoxin (TTX)-containing medium. In all DA cells, depolarizing voltage commands activated several components of outward current depending on the holding potential of the cell. When cells were held at -40 mV (or more positive), voltage steps activated a sustained outward current. If the membrane potential was held more negative than -50 mV, a rapidly activating and inactivating component of outward current response could also be detected. 4. From a hyperpolarized holding potential (-90 mV) the transient outward current activated with depolarizing commands to -55 mV, peaking within 5 ms. The current inactivated with a monoexponential time constant of 53 +/- 4 (SE) ms. At more positive holding potentials (-40 mV) the steady-state inactivation of the current could be removed by applying a conditioning hyperpolarizing prepulse. In response to a fixed depolarizing voltage step, half-maximal inactivation occurred at about -65 mV. The transient current was blocked by 4-aminopyridine (4-AP). 5. The sustained outward currents were isolated by holding the cells at -40 mV. Two components of sustained outward current were distinguished by their sensitivity to the calcium channel blockers Co2+ (5 mM) and/or Cd2+ (200 microM). The current remaining in the presence of Co2+/Cd2+ was activated by depolarizing voltage commands more positive than -40 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular calcium regulates outward currents in rabbit intestinal smooth muscle cell

The nature of transient and oscillatory outward currents (ITO and IOO) in fragmented smooth muscle cells (smooth muscle ball, SMB) from the longitudinal muscle layer of the rabbit ileum, was studied using a single electrode voltage clamp technique. With a high K+ solution containing 0.3 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) in the pipette and physiological salt solution (PSS) in the bath, the Ca inward current was followed by a large transient outward current (ITO) and spontaneous oscillations of the outward current (IOO) on the sustained outward current (ISO) were elicited by a depolarizing pulse, positive to -30 mV (holding potential of -60 mV). When the internal fluid of the SMB was replaced with Cs+-tetraethylammonium+ (TEA+) solution, or when the concentration of EGTA in the pipette was increased to 4 mM, using the intracellular perfusion technique, both ITO and IOO were abolished. In Mn2+ solution both currents were also inhibited. Bath application of TEA+, procaine or A23187 completely blocked both ITO and IOO. Caffeine (0.3-1 mM) enhanced the amplitude of ITO and generations of IOO, and concentrations of caffeine over 3 mM transiently enhanced, but finally suppressed both these currents. These results suggest that the generation of ITO is closely related to the Ca2+ influx, whereas the generation of IOO may be initiated by an increment in the intracellular concentration of Ca2+, possibly released from store sites.