Effects of potassium channel openers in the isolated perfused hypokalaemic murine heart

Aim: We explored the anti‐arrhythmic efficacy of K⁺ channel activation in the hypokalaemic murine heart using NS1643 and nicorandil, compounds which augment I_Kr and I_KATP respectively. Methods: Left ventricular epicardial and endocardial monophasic action potentials were compared in normokalaemic and hypokalaemic preparations in the absence and presence of NS1643 (30 μm ) and nicorandil (20 μm ). Results: Spontaneously beating hypokalaemic hearts (3 mm K⁺) all elicited early afterdepolarizations (EADs) and episodes of ventricular tachycardia (VT). Perfusion with NS1643 and nicorandil suppressed EADs and VT in 7 of 13 and five of six hypokalaemic hearts. Provoked arrhythmia studies using programmed electrical stimulation induced VT in all hypokalaemic hearts, but failed to do so in 7 of 13 and five of six hearts perfused with NS1643 and nicorandil respectively. These anti‐arrhythmic effects were accompanied by reductions in action potential duration at 90% repolarization (APD₉₀) and changes in the transmural gradient of repolarization, reflected in ΔAPD₉₀. NS1643 and nicorandil reduced epicardial APD₉₀ from 68.3 ± 1.1 to 56.5 ± 4.1 and 51.5 ± 1.5 ms, respectively, but preserved endocardial APD₉₀ in hypokalaemic hearts. NS1643 and nicorandil thus restored ΔAPD₉₀ from ?9.6 ± 4.3 ms under baseline hypokalaemic conditions to 3.9 ± 4.1 and 9.9 ± 2.1 ms, respectively, close to normokalaemic values. Conclusion: These findings demonstrate, for the first time, the anti‐arrhythmic efficacy of K⁺ channel activation in the setting of hypokalaemia. NS1643 and nicorandil are anti‐arrhythmic through the suppression of EADs, reductions in APD₉₀ and restorations of ΔAPD₉₀.     

Separation of early afterdepolarizations from arrhythmogenic substrate in the isolated perfused hypokalaemic murine heart through modifiers of calcium homeostasis

Aims: We resolved roles for early afterdepolarizations (EADs) and transmural gradients of repolarization in arrhythmogenesis in Langendorff‐perfused hypokalaemic murine hearts paced from the right ventricular epicardium. Methods: Left ventricular epicardial and endocardial monophasic action potentials (MAPs) and arrhythmogenic tendency were compared in the presence and absence of the L‐type Ca²⁺ channel blocker nifedipine (10 nm –1 μm ) and the calmodulin kinase type II inhibitor KN‐93 (2 μm ). Results: All the hypokalaemic hearts studied showed prolonged epicardial and endocardial MAPs, decreased epicardial‐endocardial APD₉₀ difference, EADs, triggered beats and ventricular tachycardia (VT) (n = 6). In all spontaneously beating hearts, 100 (but not 10) nm nifedipine reduced both the incidence of EADs and triggered beats from 66.9 ± 15.7% to 28.3 ± 8.7% and episodes of VT from 10.8 ± 6.3% to 1.2 ± 0.7% of MAPs (n = 6 hearts, P < 0.05); 1 μm nifedipine abolished all these phenomena (n = 6). In contrast programmed electrical stimulation (PES) still triggered VT in six of six hearts with 0, 10 and 100 nm but not 1 μm nifedipine. 1 μm nifedipine selectively reduced epicardial (from 66.1 ± 3.4 to 46.2 ± 2.5 ms) but not endocardial APD₉₀, thereby restoring ΔAPD₉₀ from ?5.9 ± 2.5 to 15.5 ± 3.2 ms, close to normokalaemic values. KN‐93 similarly reduced EADs, triggered beats and VT in spontaneously beating hearts to 29.6 ± 8.9% and 1.7 ± 1.1% respectively (n = 6) yet permitted PES‐induced VT (n = 6), in the presence of a persistently negative ΔAPD₉₀. Conclusions: These findings empirically implicate both EADs and triggered beats alongside arrhythmogenic substrate of ΔAPD₉₀ in VT pathogenesis at the whole heart level.     

Pharmacological separation of early afterdepolarizations from arrhythmogenic substrate in DeltaKPQ Scn5a murine hearts modelling human long QT 3 syndrome

Aim: To perform an empirical, pharmacological, separation of early afterdepolarizations (EADs) and transmural gradients of repolarization in arrhythmogenesis in a genetically modified mouse heart modelling human long QT syndrome (LQT) 3. Methods: Left ventricular endocardial and epicardial monophasic action potentials and arrhythmogenic tendency were compared in isolated wild type (WT) and Scn5a+/Δ hearts perfused with 0.1 and 1 μm propranolol and paced from the right ventricular epicardium. Results: All spontaneously beating bradycardic Scn5a+/Δ hearts displayed EADs, triggered beats and ventricular tachycardia (VT; n = 7), events never seen in WT hearts (n = 5). Perfusion with 0.1 and 1 μm propranolol suppressed all EADs, triggered beats and episodes of VT. In contrast, triggering of VT persisted following programmed electrical stimulation in 6 of 12 (50%), one of eight (12.5%), but six of eight (75%) Scn5a+/Δ hearts perfused with 0, 0.1 and 1 μm propranolol respectively in parallel with corresponding alterations in repolarization gradients, reflected in action potential duration (ΔAPD₉₀) values. Thus 0.1 μm propranolol reduced epicardial but not endocardial APD₉₀ from 54.7 ± 1.6 to 44.0 ± 2.0 ms, restoring ΔAPD₉₀ from ?3.8 ± 1.6 to 3.5 ± 2.5 ms (all n = 5), close to WT values. However, 1 μm propranolol increased epicardial APD₉₀ to 72.5 ± 1.2 ms and decreased endocardial APD₉₀ from 50.9 ± 1.0 to 24.5 ± 0.3 ms, increasing ΔAPD₉₀ to ?48.0 ± 1.2 ms. Conclusion: These findings empirically implicate EADs in potentially initiating spontaneous arrhythmogenic phenomena and transmural repolarization gradients in the re‐entrant substrate that would sustain such activity when provoked by extrasystolic activity in murine hearts modelling human LQT3 syndrome.     

The contribution of refractoriness to arrhythmic substrate in hypokalemic Langendorff-perfused murine hearts

The clinical effects of hypokalemia including action potential prolongation and arrhythmogenicity suppressible by lidocaine were reproduced in hypokalemic (3.0 mM K⁺) Langendorff-perfused murine hearts before and after exposure to lidocaine (10 μM). Novel limiting criteria for local and transmural, epicardial, and endocardial re-excitation involving action potential duration (at 90% repolarization, APD₉₀), ventricular effective refractory period (VERP), and transmural conduction time (Δlatency), where appropriate, were applied to normokalemic (5.2 mM K⁺) and hypokalemic hearts. Hypokalemia increased epicardial APD₉₀ from 46.6 ± 1.2 to 53.1 ± 0.7 ms yet decreased epicardial VERP from 41 ± 4 to 29 ± 1 ms, left endocardial APD₉₀ unchanged (58.2 ± 3.7 to 56.9 ± 4.0 ms) yet decreased endocardial VERP from 48 ± 4 to 29 ± 2 ms, and left Δlatency unchanged (1.6 ± 1.4 to 1.1 ± 1.1 ms; eight normokalemic and five hypokalemic hearts). These findings precisely matched computational predictions based on previous reports of altered ion channel gating and membrane hyperpolarization. Hypokalemia thus shifted all re-excitation criteria in the positive direction. In contrast, hypokalemia spared epicardial APD₉₀ (54.8 ± 2.7 to 60.6 ± 2.7 ms), epicardial VERP (84 ± 5 to 81 ± 7 ms), endocardial APD₉₀ (56.6 ± 4.2 to 63.7 ± 6.4 ms), endocardial VERP (80 ± 2 to 84 ± 4 ms), and Δlatency (12.5 ± 6.2 to 7.6 ± 3.4 ms; five hearts in each case) in lidocaine-treated hearts. Exposure to lidocaine thus consistently shifted all re-excitation criteria in the negative direction, again precisely agreeing with the arrhythmogenic findings. In contrast, established analyses invoking transmural dispersion of repolarization failed to account for any of these findings. We thus establish novel, more general, criteria predictive of arrhythmogenicity that may be particularly useful where APD₉₀ might diverge sharply from VERP.     

Empirical correlation of triggered activity and spatial and temporal re-entrant substrates with arrhythmogenicity in a murine model for Jervell and Lange-Nielsen syndrome

KCNE1 encodes the β-subunit of the slow component of the delayed rectifier K⁺ current. The Jervell and Lange-Nielsen syndrome is characterized by sensorineural deafness, prolonged QT intervals, and ventricular arrhythmogenicity. Loss-of-function mutations in KCNE1 are implicated in the JLN2 subtype. We recorded left ventricular epicardial and endocardial monophasic action potentials (MAPs) in intact, Langendorff-perfused mouse hearts. KCNE1 ^−/− but not wild-type (WT) hearts showed not only triggered activity and spontaneous ventricular tachycardia (VT), but also VT provoked by programmed electrical stimulation. The presence or absence of VT was related to the following set of criteria for re-entrant excitation for the first time in KCNE1 ^−/− hearts: Quantification of APD₉₀, the MAP duration at 90% repolarization, demonstrated alterations in (1) the difference, ∆APD₉₀, between endocardial and epicardial APD₉₀ and (2) critical intervals for local re-excitation, given by differences between APD₉₀ and ventricular effective refractory period, reflecting spatial re-entrant substrate. Temporal re-entrant substrate was reflected in (3) increased APD₉₀ alternans, through a range of pacing rates, and (4) steeper epicardial and endocardial APD₉₀ restitution curves determined with a dynamic pacing protocol. (5) Nicorandil (20 μM) rescued spontaneous and provoked arrhythmogenic phenomena in KCNE1 ^−/− hearts. WTs remained nonarrhythmogenic. Nicorandil correspondingly restored parameters representing re-entrant criteria in KCNE1 ^−/− hearts toward values found in untreated WTs. It shifted such values in WT hearts in similar directions. Together, these findings directly implicate triggered electrical activity and spatial and temporal re-entrant mechanisms in the arrhythmogenesis observed in KCNE1 ^−/− hearts.     

Effect of catecholamines on the ventricular myocyte action potential in raised extracellular potassium

We describe the relationship between catecholamines and raised extracellular potassium ([K⁺]_o) on action potential parameters and calcium currents in isolated ventricular myocytes of the guinea-pig and relate these findings to the problem of understanding how the heart is protected from exercise-induced hyperkalaemia ([K⁺]_a up to 8.5 mm ). Action potential duration (APD₉₀), amplitude and upstroke velocity were recorded in stimulated (2Hz) guinea-pig ventricular myocytes using whole-cell patch electrode recordings (37 ±C). Cells were superfused with normal K⁺Tyrode and with raised K⁺Tyrode in the presence of either noradrenaline, adrenaline or raised calcium. Inward calcium current was measured using voltage clamp. Raised K⁺(8, 12, 16 mm K⁺Tyrode) caused a significant (P < 0.01) depolarisation, shortened the APD₉₀ and decreased the action potential amplitude and upstroke velocity. In raised K⁺Tyrode addition of noradrenaline (0.08–0.1 μm ) or adrenaline (0.1–0.2 μm ) increased action potential amplitude (P < 0.01), APD₉₀ (P < 0.01) and upstroke velocity (P < 0.01) (measured only in 16 mm K⁺Tyrode). In 12 mm K⁺Tyrode raised Ca²⁺(5–6 mm ) increased action potential amplitude (P < 0.05) and shortened APD₉₀ (P < 0.05). Addition of NA (0.08–0.1 μm ) increased the inward Ca²⁺current. All effects were fully reversible. In raised [K⁺]_o increases in catecholamines and [Ca²⁺]_o cause changes in action potential parameters that would be expected to maintain propagation of the cardiac action potential in the whole heart. Thus, in the ventricular myocyte the increase in conductance to Ca²⁺caused by catecholamines may be one factor that is important in minimising the potentially adverse effects of exercise-induced hyperkalaemia.     

Epac activation, altered calcium homeostasis and ventricular arrhythmogenesis in the murine heart

The recently described exchange protein directly activated by cAMP (Epac) has been implicated in distinct protein kinase A-independent cellular signalling pathways. We investigated the role of Epac activation in adrenergically mediated ventricular arrhythmogenesis. In contrast to observations in control conditions (n = 20), monophasic action potentials recorded in 2 of 10 intrinsically beating and 5 of 20 extrinsically paced Langendorff-perfused wild-type murine hearts perfused with the Epac activator 8-pCPT-2′-O-Me-cAMP (8-CPT, 1 μM) showed spontaneous triggered activity. Three of 20 such extrinsically paced hearts showed spontaneous ventricular tachycardia (VT). Programmed electrical stimulation provoked VT in 10 of 20 similarly treated hearts (P < 0.001; n = 20). However, there were no statistically significant accompanying changes (P > 0.05) in left ventricular epicardial (40.7 ± 1.2 versus 44.0 ± 1.7 ms; n = 10) or endocardial action potential durations (APD₉₀; 51.8 ± 2.3 versus 51.9 ± 2.2 ms; n = 10), transmural (ΔAPD₉₀) (11.1 ± 2.6 versus 7.9 ± 2.8 ms; n = 10) or apico-basal repolarisation gradients, ventricular effective refractory periods (29.1 ± 1.7 versus 31.2 ± 2.4 ms in control and 8-CPT-treated hearts, respectively; n = 10) and APD₉₀ restitution characteristics. Nevertheless, fluorescence imaging of cytosolic Ca²⁺ levels demonstrated abnormal Ca²⁺ homeostasis in paced and resting isolated ventricular myocytes. Epac activation using isoproterenol in the presence of H-89 was also arrhythmogenic and similarly altered cellular Ca²⁺ homeostasis. Epac-dependent effects were reduced by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibition with 1 μM KN-93. These findings associate VT in an intact cardiac preparation with altered cellular Ca²⁺ homeostasis and Epac activation for the first time, in the absence of altered repolarisation gradients previously implicated in reentrant arrhythmias through a mechanism dependent on CaMKII activity.     

Restoration of cardiac contraction by angiotensin II during raised [K+]o in the rabbit

Catecholamines restore cardiac contraction depressed by hyperkalaemia (raised [K⁺]_o) and acidosis, yet in exercise hyperkalaemia and acidosis are tolerated during βadrenergic blockade. To test whether the negative effects of raised [K⁺]_o are offset by a non-adrenergic hormone, angiotensin II (AII) was given to rabbit papillary muscle (AII 75 nm , n=9) and rabbit isolated working hearts (AII 5 nm , n=8) perfused with 8 and 10 mm K⁺ Tyrode at 37 °C. A similar protocol was also performed in a further nine isolated hearts treated with propranolol (1 μm ) and prazosin (1 μm ). AII caused a significant (P<0.01) increases in contraction and aortic flow in normal Tyrode and maintained aortic flow during high [K⁺]_o. In the papillary muscle and isolated heart treated with adrenergic blockers, high [K⁺]_o reduced the stimulatory effects of AII, but contraction and aortic flow was still significantly greater (P<0.01) than in high [K⁺]_o alone. These results show that AII can ameliorate the depressive effects of high [K⁺]_o on the heart. The local release of AII in the heart during activation of the sympathetic nervous system and the rise in circulating AII during exercise could therefore play a role in protecting the heart from hyperkalaemia.     

Effect of verapamil on restoration of cardiac performance in raised [K+]0 by adrenergic stimulation in the rabbit

Modulation of the L-type calcium channel by catecholamines improves action potential parameters in single ventricular myocytes depolarized by high [K⁺]₀ Tyrode. Whether this modulation is important in offsetting the negative effects of hyperkalaemia in the whole heart is not known. We tested the effects of the calcium channel antagonist, verapamil, on restoration of cardiac performance by adrenergic stimulation in high [K⁺]₀ in anaesthetized rabbits and isolated perfused working rabbit hearts. Raised [K⁺]₀ decreased SBP, LVP and LVdP/dt_maxin vivo ([K⁺]_a 8.6 ± 0.2 mM; n= 10) and aortic flow (AF) in the isolated heart (8 mM [K⁺]₀ Tyrode; n= 25). However, the negative effects of raised [K⁺]_a were offset by isoprenaline (Iso, 1 μg kg^-1 min^-1 i.v.) in vivo and by noradrenaline (NA, 80 nM) in the isolated heart. Verapamil (0.15 mg kg^-1 iv.; 15 nM isolated heart) markedly potentiated the negative inotropic effects of raised [K⁺]_n in both preparations. Verapamil attenuated the effect of isoprenaline in vivo but in the isolated heart, the protective effect of NA in 8 mM [K⁺] Tyrode (AF 97 ± 10 mL min¹ in 8 mM [K⁺]₀ compared with AF 141 ± 8.5 mL min^-1 in 8 mM [K⁺]₀+ NA) was offset by the drug (90±8mL min^-1 in 8 mM [K⁺]₀+ NA + V). Furthermore, verapamil abolished aortic flow in 8 mM [K⁺]₀ alone. These findings suggest that the heart may be critically dependent on modulation of intracellular calcium in order to tolerate concentrations of K⁴ similar to those seen during a short burst of intensive exercise ([K⁺]_a 8.6 mM).     

A quantitative analysis of the effect of cycle length on arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts

The clinically established proarrhythmic effect of bradycardia and antiarrhythmic effect of lidocaine (10 μM) were reproduced in hypokalaemic (3.0 mM K⁺) Langendorff-perfused murine hearts paced over a range (80–180 ms) of baseline cycle lengths (BCLs). Action potential durations (at 90% repolarization, APD₉₀s), transmural conduction times and ventricular effective refractory periods (VERPs) were then determined from monophasic action potential records obtained during a programmed electrical stimulation procedure in which extrasystolic stimuli were interposed following regular stimuli at successively decreasing coupling intervals. A novel graphical analysis of epicardial and endocardial, local and transmural relationships between APD₉₀, corrected for transmural conduction time where appropriate, and VERP yielded predictions in precise agreement with the arrhythmogenic findings obtained over the entire range of BCLs studied. Thus, in normokalaemic (5.2 mM K⁺) hearts a statistical analysis confirmed that all four relationships were described by straight lines of gradients not significantly (P > 0.05) different from unity that passed through the origin and thus subtended constant critical angles, θ with the abscissa (45.8° ± 0.9°, 46.6° ± 0.5°, 47.6° ± 0.5° and 44.9° ± 0.8°, respectively). Hypokalaemia shifted all points to the left of these reference lines, significantly (P < 0.05) increasing θ at BCLs of 80–120 ms where arrhythmic activity was not observed (∼63°, ∼54°, ∼55° and ∼58°, respectively) and further significantly (P < 0.05) increasing θ at BCLs of 140–180 ms where arrhythmic activity was observed (∼68°, ∼60°, ∼61° and ∼65°, respectively). In contrast, the antiarrhythmic effect of lidocaine treatment was accompanied by a significant (P < 0.05) disruption of this linear relationship and decreases in θ in both normokalaemic (∼40°, ∼33°, ∼39° and ∼41°, respectively) and hypokalaemic (∼40°, ∼44°, ∼50° and ∼48°, respectively) hearts. This extended a previous approach that had correlated alterations in transmural repolarization gradients with arrhythmogenicity in murine models of the congenital long QT syndrome type 3 and hypokalaemia at a single BCL. Thus, the analysis in terms of APD₉₀ and VERP provided a more sensitive indication of the effect of lidocaine than one only considering transmural repolarization gradients and may be particularly applicable in physiological and pharmacological situations in which these parameters diverge.     

Electrophysiological determinants of hypokalaemia‐induced arrhythmogenicity in the guinea‐pig heart

Aim: Hypokalaemia is an independent risk factor contributing to arrhythmic death in cardiac patients. In the present study, we explored the mechanisms of hypokalaemia‐induced tachyarrhythmias by measuring ventricular refractoriness, spatial repolarization gradients, and ventricular conduction time in isolated, perfused guinea‐pig heart preparations. Methods: Epicardial and endocardial monophasic action potentials from distinct left ventricular (LV) and right ventricular (RV) recording sites were monitored simultaneously with volume‐conducted electrocardiogram (ECG) during steady‐state pacing and following a premature extrastimulus application at progressively reducing coupling stimulation intervals in normokalaemic and hypokalaemic conditions. Results: Hypokalaemic perfusion (2.5 mm K⁺ for 30 min) markedly increased the inducibility of tachyarrhythmias by programmed ventricular stimulation and rapid pacing, prolonged ventricular repolarization and shortened LV epicardial and endocardial effective refractory periods, thereby increasing the critical interval for LV re‐excitation. Hypokalaemia increased the RV‐to‐LV transepicardial repolarization gradients but had no effect on transmural dispersion of APD₉₀ and refractoriness across the LV wall. As determined by local activation time recordings, the LV‐to‐RV transepicardial conduction and the LV transmural (epicardial‐to‐endocardial) conduction were slowed in hypokalaemic heart preparations. This change was attributed to depressed diastolic excitability as evidenced by increased ventricular pacing thresholds. Conclusion: These findings suggest that hypokalaemia‐induced arrhythmogenicity is attributed to shortened LV refractoriness, increased critical intervals for LV re‐excitation, amplified RV‐to‐LV transepicardial repolarization gradients and slowed ventricular conduction in the guinea‐pig heart.     

The cytosolic chloride concentration in macula densa and cortical thick ascending limb cells

It is believed that chloride transport through the macula densa (MD) cells is a factor involved in the tubuloglomerular feedback (TGF) mechanism and in MD-mediated renin release. In this study isolated and perfused rabbit kidney cortical thick ascending limb (cTAL) segments containing MD plaques and attached glomeruli were loaded with chloride (CL-sensitive) 6 methoxy-l-fluorophore (sulphanate-propyl) quinolinium (SPQ). MD and cTAL intracellular chloride concentration ([Cl^-]_i) was determined by using image-intensified video microscopy and digital image-processing for measuring the intensity of the emitted SPQfluorescence. With 150 mM NaCl in lumen and bath the [Cl^-], in MD and cTAL cells was 58.8 ± 7.2 mm (n= 20) and 68.7 ± 9.8 mm (n= 14), respectively. When the presumed luminal Na⁺-2Cl^--K⁺ co-transporter was blocked by adding 10^--⁴m furosemide, the [Cl^-]_i was reduced in both, MD and cTAL cells from 55.5 ± 11.9 to 28.6 ± 10.0mm (n= 10) and from 43.8± 2.6 to 13.1± 4.5mm (n= 5), respectively. A reduction in luminal NaCl from 150 to 30 mm also decreased both, MD and cTAL [Cl^-]_i from 69.4± 9.1 to 36.5± 5.1 mm (n= 9) and from 82.9 ±14.5 to 49.4± 8.0 mm (n= 8), respectively. Basolateral addition of the Cl^--channel blocker NPPB increased MD [Cl^-], from 31.1± 2.0 to 100.7± 17.0 mm (n= 5) and cTAL [Cl^-]_i from 44.4 ± 12.9 to 89.7 ± 11.7 mm (n= 5). These results show the existence of a luminal Na⁺-2C1^-K⁺ cotransporter and a basolateral Cl^- conductance, and that chloride transport is directed from the luminal to the basolateral side during symmetrical conditions. They also indicate that sensing of luminal NaCl by the MD cells could be of importance in the TGF mechanism and MD mediated renin release.     

Hypertonic cell shrinkage reduces the K+ conductance of rat colonic crypts

 It has previously been shown in studies of a renal epithelial cell line that nonselective cation (NSC) channels are activated by exposure to hypertonic solution. We have also found such channels in excised patches of colonic crypt cells. They require high Ca²⁺ activities on the cytosolic side and a low ATP concentration for their activation and have not been recorded from cell-attached patches of colonic crypts. We examine here whether this type of channel is activated by hypertonic cell shrinkage. Bath osmolality was increased by addition of 25, 50 or 100 mmol/l mannitol. Cell-attached and whole-cell patch recordings were obtained from rat base and mid-crypt cells. In whole-cell recordings we found that addition of 50 or 100 mmol/l mannitol depolarized these cells significantly from –78±2.0 to –66±3.8 mV (n=22) and from –78±1.3 to –56±2.6 mV (n=61), respectively, and reduced the whole-cell conductance from 20±8.0 to 14±6.6 nS (n=7) and from 20±3.0 to 9.8±1.6 nS (n=19), respectively. In cell-attached patches K⁺ channels with a single-channel conductance of ≈16 pS were found in most recordings. The activity of these channels (N×P _o, N=number, P _o=open channel probability) was reduced from 2.08±0.37 to 0.98±0.23 (n=15) by the addition of 50 mmol/l mannitol and from 1.75±0.26 to 0.77±0.20 (n=12) by 100 mmol/l mannitol. No NSC channel activity was apparent in any of these recordings. Previously we have shown that the 16-pS K⁺ channel is controlled by cytosolic Ca²⁺ ([Ca²⁺]_i). Therefore we measured [Ca²⁺]_i by the fura-2 method and found that hypertonic solution reduced [Ca²⁺]_i significantly (n=16). These data indicate that exposure of rat colonic crypts to hypertonic solutions does not activate NSC channels; [Ca²⁺]_i falls in hypertonic solution leading to a reduction in the value of K⁺ channel N×Po, a reduced whole-cell conductance and depolarization of mid-crypt cells. These processes probably assist volume regulation inasmuch as they reduce KCl losses from the cell. Received: 21 July 1997 / Received after revision: 24 November 1997 / Accepted: 15 December 1997     

Exacerbated potassium-induced paralysis of mouse soleus muscle at 37°C vis-à-vis 25°C: implications for fatigue. K+ -induced paralysis at 37°C

The main aim was to investigate the effects of raised [K⁺]_o on contraction of isolated non-fatigued skeletal muscle at 37°C and 25°C to assess the physiological significance of K⁺ in fatigue. Mouse soleus muscles equilibrated at 25°C had good mechanical stability when temperature was elevated to 37°C. The main findings at 37°C vis-à-vis 25°C were as follows. When [K⁺]_o was raised from 4 to 7?mM, there was greater twitch potentiation, but no significant difference in peak tetanic force. At 10?mM [K⁺]_o there was (1) a faster time course for the decline of peak tetanic force, (2) a greater steady-state depression of twitches and tetani, (3) an increase of peak force over 50?C200?Hz (whereas it decreased at 25°C), (4) significant tetanus restoration when stimulus pulse duration increased from 0.1 to 0.25?ms and (5) greater depolarisation of layer-2 fibres, with no repolarisation of surface fibres. These combined data strengthen the proposal that a large run-down of the K⁺ gradient contributes to severe fatigue at physiological temperatures via depolarisation and impaired sarcolemmal excitability. Moreover, terbutaline, a ??₂-adrenergic agonist, induced a slightly greater and more rapid, but transient, restoration of peak tetanic force at 10?mM [K⁺]_o at 37°C vis-à-vis 25°C. A right shift of the twitch force?Cstimulation strength relationship at 10?mM [K⁺]_o was partially reversed with terbutaline to confer the protective effect. Thus, catecholamines are likely to stimulate the Na⁺?CK⁺ pump more powerfully at 37°C to restore excitability and attenuate, but not prevent, the detrimental effects of K⁺.     

Na+ dependence of single-channel current and channel density generate saturation of Na+ uptake in A6 cells

 In high-resistance, salt-absorbing epithelia the apical amiloride-sensitive Na⁺ channel is the key site for regulation of salt and water balance. The saturation of macroscopic Na⁺ transport through these channels was investigated using A6 epithelial monolayers. The relation between transepithelial Na⁺ transport (I _Na) and apical Na⁺ concentration ([Na⁺]_ap) under short-circuit conditions was studied. Michaelis-Menten analysis of the saturable short-circuit current (I _sc) yielded an apparent Michaelis-Menten constant (K _m ^I ) of 5 mmol/l and a maximal current (I _max) of 8 μA/cm². The microscopic parameters underlying I _Na, namely the single-channel current (i) and the open channel density (N _o), were investigated by the analysis of current fluctuations induced by the electroneutral amiloride analogue CDPC (6-chloro-3,5-diaminopyrazine-2-carboxamide). A two-state model analysis yielded the absolute values of i (0.18 ± 0.01 pA) and N _o (65.38 ± 9.57 million channels/cm² of epithelium) at [Na⁺]_ap = 110 mmol/l containing 50 μmol/l CDPC. Our data indicate that in A6 cells both i and N _o depend on [Na⁺]_ap. Between 3 and ≈ 20 mmol/l the density of conducting pores, N _o, decreases sharply and behaves again as an almost [Na⁺]_ap-independent parameter at higher [Na⁺]_ap. The single-channel current clearly saturates with an apparent Michaelis-Menten constant, K _m ⁱ , of ≈ 17 mmol/l. Thus, the [Na⁺]_ap dependence of N _o as well as the limited transport capacity of the amiloride-sensitive Na⁺ channel are both responsible for the saturation of I _Na. Received: 2 June 1997 / Received after revision: 12 November 1997 / Accepted: 10 December 1997     

Passive properties of neostriatal neurons during potassium conductance blockade

Voltage recordings from neostriatal projection neurons were obtained using in vitro intracellular techniques before and during K⁺-conductance blockade. Neurons were stained with the biocytin technique. Somatic surface area (A _S) was determined by both whole-cell recordings in isolated somata and by measuring stained somata recorded in slices. Dendritic measurements were done in reconstructed neurons. Average determinations of dendritic (A _D) and neuronal (A _N) surface areas coincided with previously reported anatomical data. Thus: A _S≈ 6.5 × 10^–6 cm²; A _D≈ 1.9 × 10^–4 cm²; A _N≈A _D + A _S≈ 2 × 10^–4 cm²; A _D/A _S≈ 30. Measurements were done before and after superfusion with K⁺-conductance blockers (K⁺-blockers). Cells whose neuronal morphology was not obviously distorted by K⁺-blockade were chosen for the present study. Electrotonic transients were matched to a somatic shunt equivalent cylinder model adjusted with the generalized correction factor (F _dga) that constrains the parameters for neuronal anatomy. Neuronal input resistance (R _N; mean ± SEM) increased when it was corrected for somatic shunt, from 49 ± 2 MΩ (n = 80) to 179 ± 7 MΩ (n = 32). A difference was also obtained between the slowest time constant, τ₀ = 16 ± 0.9 ms (n = 49), and the dendritic membrane time constant, τ_mD = 33 ± 1.6 ms (n = 36). When these electrophysiological measurements were used to calculate A _N, the value obtained was similar to the anatomical measurements. Combining anatomical and electrophysiological data, somatic and dendritic input resistances were determined: R _D = 182 ± 7 MΩ; R _S (with shunt) = 74 ± 4 MΩ (n = 32). The generalized correction factor, F _dga = 0.91 ± 0.007 (n = 10), implied a short effective electrotonic length for dendrites: L _D = 0.46 ± 0.014 (n = 32). Saturating concentrations of the K⁺-blockers tetraethylammonium, Cs⁺, and Ba²⁺ increased R _N and induced charging curves well fitted by single exponential functions in 56% of neostriatal neurons. Ba²⁺ greatly decreased the somatic shunt (n = 5): (R _N = 216 ± 21 MΩ, τ₀ = 46 ± 2 ms, R _D = 239 ± 25 MΩ, and R _S = 3.2 ± 0.5 GΩ), rendering values similar to those obtained with whole-cell recordings (e.g., R _N≈ 198 MΩ, R_S≈ 2.62 GΩ) (n = 52). Cs⁺ (n = 5) had less effect on the somatic shunt (R _N = 115 ± 19 MΩ, τ₀ = 49 ± 13 ms, R _S = 161 ± 8 MΩ), although dendritic conductance was equally blocked (R _D = 261 ± 16 MΩ). The Cs⁺-sensitive conductance exhibited inward rectifying properties not displayed by the Ba²⁺-sensitive conductance, suggesting that Cs⁺ preferentially acted upon inward rectifier conductances. In contrast, Ba²⁺ significantly acted upon linear conductances making up the somatic shunt. This suggests a differential action of different K⁺-blockers on the somato-dendritic membrane, implying a differential distribution of membrane conductances. Another action of K⁺-blockers, in about 40% of the cells, was to induce dye and probably electrical coupling between neighboring neurons. Received: 30 April 1997 / Accepted: 14 October 1997     

Extracellular ionic variations during spreading depression

Changes in the concentrations of K⁺, Ca²⁺, Na⁺ and Cl^? were measured during spreading depression in the exposed lissencephalic cerebellar molecular layer of the catfish,Corydoras aneus. Liquid ion exchanger ion-selective microelectrodes were used in pairs to monitor simultaneously changes in the concentrations of two ionic species in the extracellular space. Normothermic spreading depression in the catfish cerebellum consists of a slow negative potential shift that develops at a rate of 2–3mV/s, reaches an amplitude of ?25 mV, lasts 1–6 min and propagates at a rate of 0.5–1.5 mm/min. [K⁺]_o rises from a resting level of 2.3–35 mM at the peak of spreading depression. Between 20 and 40s later [Ca²⁺]_o falls from 2.2 mM to 0.8 mM and [Na⁺]_o and [Cl^?]_o decrease from 149 to 57 mM and 137 to 47 mM respectively at approximately the same time. [Ca²⁺]_o, [Na⁺]_o and [Cl^?]_o decreases begin when [K⁺]_o exceeds 10 mM.These results establish the magnitude and temporal sequence of the major ion concentration changes in extracellular space during spreading depression. The earliest extracellular precursor of spreading depression is a rise in [K⁺]_o. In light of the equality of [Na⁺]_o and [Cl^?]_o changes and the rise in [K⁺]_o, electroneutrality in extracellular space involved may be maintained by the net accumulation of some unidentified anion equivalent to the rise in [K⁺]_o. Alternatively, no net accumulation is needed if some extracellular anions are impermeant and the volume of extracellular space decreases. The sum of the extracellular ion concentrations suggests that the ionic strength of extracellular space decreases by greater than one-third during spreading depression. These results demonstrate that the brain is capable of establishing and recovering from local ionic inhomogenejties.     

Ca2+-transients induced by K+ channel openers in isolated coronary capillaries

 To study the role of endothelial ATP-sensitive K⁺ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca²⁺]_i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from the guinea-pig heart and [Ca²⁺]_i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K⁺ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid, transient rise in [Ca²⁺]_i followed by a sustained elevation of [Ca²⁺]_i (19 of 40 experiments). [Ca²⁺]_i in the endothelial cells increased from 32 ± 7 nM at rest to 66 ± 11 nM at the peak (n = 19). One third of the [Ca²⁺]_i-transients showed irregular oscillations of [Ca²⁺]_i. No significant difference in the [Ca²⁺]_i-response induced by 100 nM or 1 μM diazoxide was found. Similar results were obtained with the K⁺ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca²⁺]_i with fluorometric methods indicated that the hyperpolarization but not the [Ca²⁺]_i-transient could be repeatedly induced in a single capillary by the K⁺ channel openers. Electrophysiological recordings of the membrane potential using the ”perforated patch” method (n = 4), showed that rilmakalim (1 μM) induced hyperpolarization of capillaries towards the K⁺ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that K⁺ channel openers induce [Ca²⁺]_i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly, only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of K_ATP-channel in coronary capillaries characterised by high sensitivity to diazoxide. Received: 22 August 1997 / Received after revision and accepted: 7 November 1997     

Contribution of pH,diprotonated phosphate and potassium for the reflex increase in blood pressure during handgrip

The relative importance of pH, diprotonated phosphate (H₂PO₄^?) and potassium (K⁺) for the reflex increase in mean arterial pressure (MAP) during exercise was evaluated in seven subjects during rhythmic handgrip at 15 and 30% maximal voluntary contraction (MVC), followed by post-exercise muscle ischaemia (PEMI). During 15% MVC, MAP rose from 92 ± 1 to 103 ± 2 mmHg, [K⁺] from 4.1 ± 0.1 to 5.1 ± 0.1 mmol L^?1, while the intracellular (7.00 ± 0.01 to 6.80 ± 0.06) and venous pH fell (7.39 ± 0.01 to 7.30 ± 0.01) (P < 0.05). The intracellular [H₂PO₄^?] increased 8.4 ± 2 mmol kg^?1 and the venous [H₂PO₄^?] from 0.14 ± 0.01 to 0.16 ± 0.01 mmol L^?1 (P < 0.05). During PEMI, MAP remained elevated along with the intracellular [H₂PO₄^?] as well as a low intracellular and venous pH. However, venous [K⁺] and [H₂PO₄^?] returned to the level at rest. During 30% MVC handgrip, MAP rose to 130 ± 3 mmHg, [K⁺] to 5.8 ± 0.2 mmol L^?1, the intracellular and extracellular [H₂PO₄^?] by 20 ± 5 mmol kg^?1 and to 0.20 ± 0.02 mmol L^?1, respectively, while the intracellular (6.33 ± 0.06) and venous pH fell (7.23 ± 0.02) (P < 0.05). During post-exercise muscle ischaemia all variables remained close to the exercise levels. Analysis of each variable as a predictor of blood pressure indicated that only the intracellular pH and diprotonated phosphate were linked to the reflex elevation of blood pressure during handgrip.     

马桑内酯致痫大鼠海马神经细胞外Na+、K+活度的变化

目的和方法：应用Na⁺、K⁺选择性微电极检测马桑内酯致痫大鼠海马及海马脑片神经细胞外Na⁺、K⁺活度的改变。结果：海马内注射马桑内酯（5 μL,5×10^-4 mol/L）致痫大鼠30 s、1 min和2 min后,海马神经细胞外Na⁺活度分别低于对照组27.7 mmol/L、50.3 mmol/L和57.8 mmol/L,而K活度则分别高于对照组2.3 mmol/L、2.4 mmol/L和2.9 mmol/L（P<0.01）。3 min后,K⁺活度基本恢复至对照水平,而Na⁺活度仍持续低于对照水平（P<0.01）。海马脑片的实验结果与在体实验相似。结论：海马神经细胞处于癫痫状态时,存在Na⁺内流、K⁺外流现象。