COVID-19 Pandemic and Dysbiosis: Can the Ivermectin Hysteria Lead to an Increase of Autoimmune Neuroinflammatory Diseases?

The pandemic caused by the SARS-CoV-2 has made new treatments a goal for the scientific community. One of these treatments is Ivermectin. Here we discuss the hypothesis of dysbiosis caused by the use of Ivermectin and the possible impacts on neuroinflammatory diseases after the end of the pandemic.

Neuroinflammation at single cell level: What is new?

Multiple sclerosis is a chronic and demyelinating disease of the central nervous system (CNS), most prevalent in women, and with an important social and economic cost worldwide. It is triggered by self-reacting lymphocytes that infiltrate the CNS and initiate neuroinflammation. Further, axonal loss and neuronal death takes place, leading to neurodegeneration and brain atrophy. The murine model for studying MS, experimental autoimmune encephalomyelitis (EAE), consists in immunizing mice with myelin-derived epitopes. APCs activate encephalitogenic T CD4 and CD8 lymphocytes that migrate mainly to the spinal cord resulting in neuroinflammation. Most of the knowledge on the pathophysiology and treatment of MS was obtained from EAE experiments, as Th17 cells, anti-alpha4 blocking Abs and the role of microbiota. Conversely, recent technology breakthroughs, such as CyTOF and single-cell RNA-seq, promise to revolutionize our understanding on the mechanisms involved both in MS and EAE. In fact, the importance of specific cellular populations and key molecules in MS/EAE is a constant matter of debate. It is well accepted that both Th1 and Th17 T CD4 lymphocytes play a relevant role in disease initiation after re-activation in situ. What is still under constant investigation, however, is the plasticity of the lymphocyte population, and the individual contribution of both resident and inflammatory cells for the progression or recovery of the disease. Thus, in this review, new findings obtained after single-cell analysis of blood and central nervous system infiltrating cells from MS/EAE and how they have contributed to a better knowledge on the cellular and molecular mechanisms of neuroinflammation are discussed.

Niflumic acid-sensitive ion channels play an important role in the induction of glucose-stimulated insulin secretion by cyclic AMP in mice.

AIMS/HYPOTHESIS: We have previously reported that glucose-stimulated insulin secretion (GSIS) is induced by glucagon-like peptide-1 (GLP-1) in mice lacking ATP-sensitive K(+) (K(ATP)) channels (Kir6.2(-/-) mice [up-to-date symbol for Kir6.2 gene is Kcnj11]), in which glucose alone does not trigger insulin secretion. This study aimed to clarify the mechanism involved in the induction of GSIS by GLP-1. METHODS: Pancreas perfusion experiments were performed using wild-type (Kir6.2(+/+)) or Kir6.2(-/-) mice. Glucose concentrations were either changed abruptly from 2.8 to 16.7 mmol/l or increased stepwise (1.4 mmol/l per step) from 2.8 to 12.5 mmol/l. Electrophysiological experiments were performed using pancreatic beta cells isolated from Kir6.2(-/-) mice or clonal pancreatic beta cells (MIN6 cells) after pharmacologically inhibiting their K(ATP) channels with glibenclamide. RESULTS: The combination of cyclic AMP plus 16.7 mmol/l glucose evoked insulin secretion in Kir6.2(-/-) pancreases where glucose alone was ineffective as a secretagogue. The secretion was blocked by the application of niflumic acid. In K(ATP) channel-inactivated MIN6 cells, niflumic acid similarly inhibited the membrane depolarisation caused by cAMP plus glucose. Surprisingly, stepwise increases of glucose concentration triggered insulin secretion only in the presence of cAMP or GLP-1 in Kir6.2(+/+), as in Kir6.2(-/-) pancreases. CONCLUSIONS/INTERPRETATION: Niflumic acid-sensitive ion channels participate in the induction of GSIS by cyclic AMP in Kir6.2(-/-) beta cells. Cyclic AMP thus not only acts as a potentiator of insulin secretion, but appears to be permissive for GSIS via novel, niflumic acid-sensitive ion channels. This mechanism may be physiologically important for triggering insulin secretion when the plasma glucose concentration increases gradually rather than abruptly.

A key role for the subunit SUR2B in the preferential activation of vascular KATP channels by isoflurane.

BACKGROUND AND PURPOSE: It has been postulated that isoflurane, a volatile anaesthetic, produces vasodilatation through activation of ATP-sensitive K+ (KATP) channels. However, there is no direct evidence for the activation of vascular KATP channels by isoflurane. This study was conducted to examine the effect of isoflurane on vascular KATP channels and compare it with that on cardiac KATP channels. EXPERIMENTAL APPROACH: Effects of isoflurane on KATP channels were examined in aortic smooth muscle cells and cardiomyocytes of the mouse using patch clamp techniques. Effects of the anaesthetic on the KATP channels with different combinations of the inward rectifier pore subunits (Kir6.1 and Kir6.2) and sulphonylurea receptor subunits (SUR2A and SUR2B) reconstituted in a heterologous expression system were also examined. KEY RESULTS: Isoflurane increased the coronary flow in Langendorff-perfused mouse hearts in a concentration-dependent manner, which was abolished by 10 microM glibenclamide. In enzymically-dissociated aortic smooth muscle cells, isoflurane evoked a glibenclamide-sensitive current (i.e. KATP current). In isolated mouse ventricular cells, however, isoflurane failed to evoke the KATP current unless the KATP current was preactivated by the K+ channel opener pinacidil. Although isoflurane readily activated the Kir6.1/SUR2B channels (vascular type), the volatile anesthetic could not activate the Kir6.2/SUR2A channels (cardiac type) expressed in HEK293 cells. Isoflurane activated a glibenclamide-sensitive current in HEK293 cells expressing Kir6.2/SUR2B channels. CONCLUSION AND IMPLICATIONS: Isoflurane activates KATP channels in vascular smooth muscle cells and produces coronary vasodilation in mouse hearts. SUR2B may be important for the activation of vascular-type KATP channels by isoflurane.

Opening of mitochondrial K(ATP) channels attenuates the ouabain-induced calcium overload in mitochondria.

We tested whether opening of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels depolarizes mitochondrial membrane potential (DeltaPsi(m)) and thereby prevents the mitochondrial Ca(2+) overload. With the use of a Nipkow disk confocal system, the mitochondrial Ca(2+) concentration ([Ca(2+)](m)) and DeltaPsi(m) in rat ventricular myocytes were measured by loading cells with Rhod-2 and JC-1, respectively. Exposure to ouabain (1 mmol/L) for 30 minutes produced mitochondrial Ca(2+) overload, and the intensity of Rhod-2 fluorescence significantly increased to 173+/-16% of baseline (P<0.001). Treatment of myocytes with the mitoK(ATP) channel opener diazoxide (100 micromol/L) blunted the ouabain-induced mitochondrial Ca(2+) overload (131+/-10% of baseline; P<0.001 versus ouabain). Moreover, diazoxide significantly depolarized the DeltaPsi(m) and reduced the intensity of JC-1 fluorescence during application of ouabain to 89+/-2% of baseline (P<0.05). These effects of diazoxide were blocked by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 micromol/L). These results indicate that opening of mitoK(ATP) channels prevents a mitochondrial Ca(2+) overload in association with DeltaPsi(m) depolarization and thereby protects myocardium against ischemic damage.

Functional roles of cardiac and vascular ATP-sensitive potassium channels clarified by Kir6.2-knockout mice.

-ATP-sensitive potassium (K(ATP)) channels were discovered in ventricular cells, but their roles in the heart remain mysterious. K(ATP) channels have also been found in numerous other tissues, including vascular smooth muscle. Two pore-forming subunits, Kir6.1 and Kir6.2, contribute to the diversity of K(ATP) channels. To determine which subunits are operative in the cardiovascular system and their functional roles, we characterized the effects of pharmacological K(+) channel openers (KCOs, ie, pinacidil, P-1075, and diazoxide) in Kir6.2-deficient mice. Sarcolemmal K(ATP) channels could be recorded electrophysiologically in ventricular cells from Kir6.2(+/+) (wild-type [WT]) but not from Kir6.2(-/-) (knockout [KO]) mice. In WT ventricular cells, pinacidil induced an outward current and action potential shortening, effects that were blocked by glibenclamide, a K(ATP) channel blocker. KO ventricular cells exhibited no response to KCOs, but gene transfer of Kir6.2 into neonatal ventricular cells rescued the electrophysiological response to P-1075. In terms of contractile function, pinacidil decreased force generation in WT but not KO hearts. Pinacidil and diazoxide produced concentration-dependent relaxation in both WT and KO aortas precontracted with norepinephrine. In addition, pinacidil induced a glibenclamide-sensitive current of similar magnitude in WT and KO aortic smooth muscle cells and comparable levels of hypotension in anesthetized WT and KO mice. In both WT and KO aortas, only Kir6.1 mRNA was expressed. These findings indicate that the Kir6.2 subunit mediates the depression of cardiac excitability and contractility induced by KCOs; in contrast, Kir6.2 plays no discernible role in the arterial tree.

Flow-independent improvement by diltiazem of ischemia-induced conduction delay in porcine hearts.

The calcium channel blocking agent, diltiazem, improves ischemia-induced conduction delays in the canine heart. It is not known, however, if the improvement of myocardial blood flow caused by diltiazem participates in this response. Accordingly, ischemia-induced conduction delay was measured during brief coronary artery occlusion before and after administration of diltiazem in nine anesthetized pigs with fixed heart rate. Acute coronary occlusion prolonged subendocardial (mean +/- standard error of the mean, 39.9 +/- 3.9 ms) and subepicardial (41.6 +/- 4.1 ms) conduction times (time to peak of the bipolar electrogram in each region) by 51 +/- 4 and 58 +/- 5%, respectively. Regional myocardial blood flow at the ischemic electrode sites was 0.006 +/- 0.002 ml/min per g and was unaffected by diltiazem. Intravenous diltiazem pretreatment (0.01, 0.1, 0.3 and 1.0 mg/kg) 5 minutes before coronary occlusion significantly reduced the ischemia-induced conduction delay in both subendocardial and subepicardial regions during coronary occlusion. The pigs in which ventricular fibrillation occurred within 10 minutes showed a significantly longer conduction delay than that observed in pigs in which ventricular fibrillation occurred later (greater than 10 minutes). Thus, the data suggest that the reduction of ischemia-induced conduction delay produced by diltiazem is independent of blood flow changes and, therefore, that diltiazem may have a beneficial antiarrhythmic action.

Preferential inhibition of Ikr by MCI-154, a putative cardiotonic Ca2+ sensitizer, in guinea pig atrial cells.

OBJECTIVE: To define the electrophysiologic mechanism(s) by which MCI-154, a putative Ca2+ sensitizer, produces a positive inotropic response without a positive chronotropic response, we examined effects of MCI-154 on the action potential of atrial preparations and the membrane currents of atrial myocytes. METHODS: The action potentias were recorded from left atrial and sinoatrial node preparations of guinea pigs by the use of standard microelectrode techniques. The whole-cell membrane currents were recorded from enzymatically-dissociated guinea pig atrial myocytes using conventional patch clamp techniques. RESULTS: In isolated left atria, MCI-154 increased the developed tension in a concentration-dependent manner. MCI-154 at concentrations of 10 and 100 microM increased the action potential duration (APD) in left atria stimulated at 0.5 Hz. In sinoatrial node preparations MCI-154 at a concentration of 100 microM produced a negative chronotropic response and prolonged APD. In single right atrial myocytes, MCI-154 at concentrations of 10 and 100 microM failed to increase the inward L-type Ca2+ current, but decreased the delayed rectifier K+ current (IK) in a concentration-dependent manner. MCI-154 decreased IK elicited by short depolarizing pulses more markedly than that induced by long depolarizing pulses. In addition, MCI-154 produced only a little inhibition of IK in the presence of E-4031, a specific blocker of rapidly activating component of IK (IKr). CONCLUSIONS: MCI-154 preferentially blocks IKr and the inhibitory action on IKr may be partly involved in the negative chronotropic and positive inotropic responses in atrial preparations.

Beta 1 adrenoceptor mediated decrease in pHi in quiescent ventricular myocardium.

OBJECTIVE: The aims were to examine the effect of beta adrenergic stimulation on the intracellular pH (pHi) and to compare it with that of alpha adrenergic stimulation in ventricular myocardium. METHODS: Using conventional and ion selective electrodes membrane potential and pHi were measured simultaneously in quiescent papillary muscles of guinea pigs in HEPES or bicarbonate buffered solution. Isoprenaline and propranolol (1 microM) plus phenylephrine (30 microM) were used to stimulate beta and alpha adrenoceptors, respectively. In order to evaluate underlying mechanism(s) of beta adrenoceptor mediated pHi change, effects of Na(+)-H+ exchange, Cl(-)-HCO3- exchange, Na(+)-HCO3- symport, and glycolysis blockers on the pHi change were examined. RESULTS: Isoprenaline (1 microM) produced a decrease in pHi of 0.08(SEM 0.01) pH units and a transient depolarisation of the resting membrane. The isoprenaline induced intracellular acidosis was blocked by the beta 1 blocker atenolol (10 microM) but not by the beta 2 blocker ICI 118,551 (0.1 microM). Forskolin also produced a decrease in pHi of 0.06(0.03) pH units. In contrast, alpha adrenergic stimulation produced an increase in pHi, which was abolished by 1 mM amiloride, an Na(+)-H+ exchange blocker. In the presence of amiloride, the isoprenaline induced decrease in pHi was rather enhanced. 4,4'-Diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 1 mM), a blocker of Cl(-)-HCO3- exchange and the Na(+)-HCO3- symport system, failed to affect the isoprenaline induced pHi decrease in bicarbonate buffered solution. However, pretreatment with 2-deoxyglucose or iodoacetic acid abolished the isoprenaline induced pHi decrease. CONCLUSIONS: beta 1 Adrenoceptor stimulation causes intracellular acidosis via the enhanced glycolysis, and the Na(+)-H+ exchange system appears to play a compensatory role. The beta 1 adrenoceptor mediated intracellular acidosis may modulate inotropic response to adrenergic stimulation in ventricular myocardium.

Selective impairment of HCO3(-)-dependent pHi regulation by lysophosphatidylcholine in guinea pig ventricular myocardium.

OBJECTIVE: The aim was to examine the effects of lysophosphatidylcholine (LPC), an amphiphilic lipid metabolite in ischemic myocardium, on intracellular pH (pH(i)) regulatory systems in guinea pig papillary muscles. METHODS: In CO2/HCO(3-)-buffered Tyrode solution, pH(i), intracellular Na+ activity (aNai) and membrane potential of isolated guinea pig papillary muscles were measured using ion-selective microelectrode and conventional microelectrode. Standard ammonium prepulsing with 20 mM NH4Cl was used to produce an intracellular acid load, and effects of LPC on the pH(i) recovery from acidosis were evaluated in the absence and presence of a transport inhibitor. RESULTS: LPC acidified the resting pH(i) by 0.03 +/- 0.01 pH units (n = 15, p < 0.01) concomitantly with a slight decrease in resting membrane potential and an increase in aNai in quiescent preparations. The pH(i) recovery rate from an intracellular acid load was decreased to 83 +/- 4% of the control value by 30 microM LPC (n = 8, P < 0.05) but not by 30 microM phosphatidylcholine (PC). In the presence of 10 microM 5-(N,N-hexamethylene) amiloride (HMA), a Na(+)-H+ exchange inhibitor, LPC still slowed pH(i) recovery from an intracellular acid load to 77 +/- 4% of the control (n = 5, P < 0.05). However, LPC failed to alter the pH(i) recovery rate in the presence of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), a Na(+)-HCO3- symport inhibitor. CONCLUSION: LPC impairs Na(+)-HCO3- symport but not Na(+)-H+ exchange, and LPC may potentiate its arrhythmogenic action by intensifying the intracellular acidosis in ischemic myocardium.

Delayed afterdepolarisations and triggered activity in canine Purkinje fibres treated with neuraminidase.

We investigated the electrophysiological alterations induced by the removal of sialic acid from the sarcolemma in canine Purkinje fibres. About 70% of total sialic acid content of Purkinje fibres was removed by 120 min of exposure to neuraminidase (1 U X ml-1). The treatment with neuraminidase did not change any of the action potential characteristics at normal Ca2+ concentration (2.7 mmol X litre-1). However, action potential duration and maximum upstroke velocity of phase zero of the action potential were reduced to a greater degree in neuraminidase-treated Purkinje fibres than in non-treated controls at high Ca2+ concentration (8.1 mmol X litre-1). At high Ca2+ concentration, delayed afterdepolarisations were induced in five out of nine neuraminidase-treated Purkinje fibres and triggered activity was observed in two, when driven in trains of 20 stimuli of different cycle length (1000 to 180 ms). No discernible delayed afterdepolarisations were observed in nine non-treated control Purkinje fibres. In addition, the amplitude of delayed afterdepolarisations induced by ouabain (0.2 mumol X litre-1) in neuraminidase-treated Purkinje fibres was larger than non-treated controls. These findings suggest that sialic acid residues of glycocalyx function as a kind of barrier to Ca2+ influx.

SD-3212, a new class I and IV antiarrhythmic drug: a potent inhibitor of the muscarinic acetylcholine-receptor-operated potassium current in guinea-pig atrial cells.

1. By use of patch-clamp techniques, the effects of SD-3212, a novel antiarrhythmic drug, on the calcium current (Ica), the sodium current (INa) and the muscarinic acetylcholine-receptor-operated potassium current (IK.ACh) were examined and compared with those of bepridil in guinea-pig single atrial cells. 2. SD-3212 inhibited ICa and INa in a concentration-dependent manner. The IC50 values of SD-3212 for inhibition of ICa and INa were 1.29 microM and 3.92 microM, respectively. The steady state inactivation curves of ICa and INa were shifted in the hyperpolarizing direction in the presence of 1 microM SD-3212. Similar inhibition of ICa and INa was also observed with bepridil. The IC50 values of bepridil for depression of ICa and INa were 1.55 microM and 4.43 microM, respectively. 3. The muscarinic acetylcholine-receptor-operated potassium current (IK.ACh) was activated by the extracellular application of 1 microM carbachol in the GTP-loaded cells or by the intracellular loading of GTP gamma S, a nonhydrolysable GTP analogue. SD-3212 potently inhibited the carbachol- and GTP gamma S-induced IK.ACh and the IC50 values were 0.38 microM and 0.20 microM, respectively. These IC50 values were very close and about 10 times lower than those for inhibiting ICa and INa. Bepridil also suppressed the carbachol- and GTP gamma S-induced IK.ACh with the IC50 values of 0.69 microM and 0.84 microM, respectively. 4. In guinea-pig atrial cells stimulated at 0.2 Hz, carbachol at a concentration of 1 microM markedly shortened action potential duration. Both SD-3212 (0.1-1 microM) and bepridil (1-10 microM) reversed the action potential shortening in a concentration-dependent manner. The antagonizing effect of SD-3212 on the carbachol-induced action potential shortening was more potent than that of bepridil. 5. These results suggest that SD-3212 inhibits IK.ACh by depressing the function of the potassium channel itself and/or associated GTP-binding proteins. SD-3212 is a unique antiarrhythmic drug, which potently inhibits IK.Ach in addition to its class I and IV effects. SD-3212 and bepridil may be useful for the termination and prevention of vagally-induced atrial flutter and fibrillation.

Electrophysiological effects of acetyl glyceryl ether phosphorylcholine on cardiac tissues: comparison with lysophosphatidylcholine and long chain acyl carnitine.

Electrophysiological effects of synthetic platelet activating factor, acetyl glyceryl ether phosphorylcholine (AGEPC), were examined and compared with those of lysophosphatidylcholine (LPC) and long chain acyl carnitine (AC) in canine Purkinje fibres and guinea-pig papillary muscles, by use of standard microelectrode techniques. In canine Purkinje fibres, AGEPC at concentrations higher than 3 X 10(-5)M, decreased maximum diastolic potential, action potential amplitude and the maximum upstroke velocity of phase 0. AGEPC also induced abnormal automaticity arising from depolarized membrane potentials. LPC and AC in concentrations higher than 3 X 10(-5)M also produced virtually identical electrophysiological alterations in Purkinje fibres. Although twitch tension was slightly decreased by low concentrations (10(-6)-10(-5)M) of these amphiphilic lipids, a transient positive inotropic response appeared at the beginning of a progressive depolarization after exposure to higher concentrations of the amphiphiles. In guinea-pig papillary muscles, AGEPC in concentrations higher than 3 X 10(-5)M produced slight decreases in resting membrane potential, action potential amplitude and action potential durations, concomitantly with a positive inotropic response. These electrophysiological and mechanical changes were also induced by LPC and AC at comparable concentrations. In guinea-pig papillary muscles depolarized with 25 mM [K+]0, AGEPC, LPC and AC all evoked slow action potentials at a concentration of 10(-4)M. It is concluded that in isolated cardiac tissues AGEPC exerts electrophysiological effects similar to those of LPC and AC only at high concentrations, and that the non-specific interaction of amphiphiles with sarcolemmal membrane may be responsible for the electrophysiological and mechanical effects.

Induction by endogenous noradrenaline of an alpha 1-adrenoceptor-mediated positive inotropic effect in rabbit papillary muscles.

1. The possible involvement of alpha 1-adrenoceptors in the inotropic and electrophysiological responses to endogenous noradrenaline released by tyramine was examined in rabbit papillary muscles. 2. A concentration-dependent positive inotropic effect was produced by tyramine. This effect of tyramine was not observed in muscles from rabbits pretreated with reserpine. 3. The positive inotropic effect of tyramine was greatly inhibited by propranolol, but not altered by prazosin. However, when beta-adrenoceptors were blocked by pretreatment with propranolol, tyramine still produced a positive inotropic effect, an effect which was antagonized by prazosin. 4. Tyramine caused a decrease in action potential duration (APD) and an increase in action potential amplitude in a concentration-dependent manner. Isoprenaline also produced the same electrophysiological effects. These electrophysiological effects of both agents were inhibited by propranolol. 5. When beta-adrenoceptors were blocked by propranolol, the observed prazosin-sensitive positive inotropic effect of tyramine was not accompanied by any change in APD. In contrast, APD was markedly prolonged by alpha 1-adrenoceptor stimulation with phenylephrine in the presence of propranolol, in association with the positive inotropic effect. 6. It is concluded that in rabbit papillary muscles, endogenous noradrenaline causes a positive inotropic effect predominantly mediated by beta-adrenoceptors, but can still evoke a positive inotropic effect through alpha 1-adrenoceptors when beta-adrenoceptor stimulation is eliminated. This suggests that the alpha 1-adrenoceptor-mediated positive intropic mechanism(s) may be masked by simultaneous activation of beta-adrenoceptors. In addition, this study indicates that APD prolongation is not involved in the alpha 1-adrenoceptor-mediated inotropic responses to endogenous noradrenaline.

Effects of ATP-sensitive K+ channel blockers on the action potential shortening in hypoxic and ischaemic myocardium.

1. In order to determine whether activation of adenosine triphosphate (ATP)-sensitive K+ channels exclusively explains the hypoxia- and ischaemia-induced action potential shortening, effects of tolbutamide and glibenclamide on changes in action potential duration (APD) during hypoxia, metabolic blockade or experimental ischaemia were examined in guinea-pig and canine isolated myocardium by standard microelectrode techniques. 2. With use of patch clamp techniques, activity of ATP-sensitive K+ channels was recorded from open cell-attached patches of guinea-pig isolated ventricular myocytes. The probability of opening of the K+ channels was decreased by 2 mM tolbutamide and 20 microM glibenclamide to almost the same extent, whereas it was increased by 100 microM pinacidil. 3. In guinea-pig papillary muscles a marked shortening of the action potential produced by 100 microM pinacidil was completely antagonized by 2 mM tolbutamide or 20 microM glibenclamide. 4. In guinea-pig papillary muscles exposed to hypoxic, glucose-free solution or dinitrophenol (10 microM)-containing, glucose-free solution, APD declined gradually and twitch tension decreased. Pretreatment with glibenclamide partially but significantly inhibited the action potential shortening, whereas tolbutamide failed to improve it during hypoxia or metabolic blockade. 5. When in canine isolated myocardium, experimental ischaemia was produced by the cessation of coronary perfusion, APD was gradually shortened. The action potential shortening was partially but not completely inhibited by pretreatment with 20 microM glibenclamide. 6. These results suggest that changes in membrane current(s) other than the outward current through ATP-sensitive K+ channels also contribute to the action potential shortening in hypoxic or ischaemic myocardium.

Cyclic GMP-mediated inhibition of L-type Ca2+ channel activity by human natriuretic peptide in rabbit heart cells.

1. Effects of atrial natriuretic peptide (ANP) on the L-type Ca2+ channels were examined in rabbit isolated ventricular cells by use of whole-cell and cell-attached configurations of the patch clamp methods. ANP produced a concentration-dependent decrease (10-100 nM) in amplitude of a basal Ca2+ channel current. 2. The inactive ANP (methionine-oxidized ANP, 30 nM) failed to decrease the current. 3. 8-Bromo-cyclic GMP (300 microM), a potent activator of cyclic GMP-dependent protein kinase (PKG), produced the same effects on the basal Ca2+ channel current as those produced by ANP. The cyclic GMP-induced inhibition of the Ca2+ channel current was still evoked in the presence of 1-isobutyl-3-methyl-xanthine, an inhibitor of phosphodiesterase. ANP failed to produce inhibition of the Ca2+ channel current in the presence of 8-bromo-cyclic GMP. 4. In the single channel recording, ANP and 8-bromo-cyclic GMP also inhibited the activities of the L-type Ca2+ channels. Both agents decreased the open probability (NPo) without affecting the unit amplitude. 5. The present results suggest that ANP inhibits the cardiac L-type Ca2+ channel activity through the intracellular production of cyclic GMP and then activation of PKG.

Effects of class III antiarrhythmic drugs on the Na(+)-activated K+ channels in guinea-pig ventricular cells.

1. Class III antiarrhythmic drugs are known to block the outward currents through voltage-gated K+ channels. However, effects of class III antiarrhythmic drugs on the ligand-gated K+ channels have not been thoroughly examined. In this study effects of amiodarone and newer class III antiarrhythmic drugs, E-4031 and MS-551, on the Na+-activated K+ (KNa) current were examined in inside-out membrane patches and in whole cells isolated from guinea-pig ventricle. 2. The KNa channel current was activated by increasing [Na+]i from 0 mM to 30-100 mM with 150 mM [K+]o in inside-out membrane patches of ventricular myocytes. The channel current showed a larger slope conductance (210 pS), inward-going rectification and subconductance levels of various amplitudes. 3. E-4031 and MS-551 at high concentrations (300 microM) inhibited the K+ current by decreasing the open time (flickering block). On the other hand, amiodarone at relatively low concentrations (0.1-10 microM) inhibited the KNa channel current by decreasing the open probability rather than by decreasing the open time. The IC50 value of amiodarone for inhibiting the KNa channel current was 1.0 microM. 4. These drugs also inhibited the whole-cell outward current activated by intracellular loading of 50 mM [Na+]i and extracellular application of 10 microM ouabain. 5. These results indicate that class III antiarrhythmic drugs inhibit the KNa channel current in cardiac cells. However, there are sharp differences in the effective concentrations and the mode of inhibition between amiodarone and the newer class III antiarrhythmic drugs.

Voltage-dependence of Ca2+ agonist effect of YC-170 on cardiac L-type Ca2+ channels.

1. We investigated the voltage-dependence of the agonist actions of YC-170, a dihydropyridine (DHP) derivative, on cardiac L-type Ca2+ channels in rabbit ventricular cells, using the patch clamp technique. The characteristics of YC-170 were compared with those of other DHP Ca2+ agonists (Bay K 8644, CGP 28392). Ca2+ channel activities were elicited by depolarizing pulses to 0 mV from a holding potential (HP) of either -80 mV or -40 mV. 2. YC-170 (10 microM) increased Ca2+ channel activities when HP was set at -80 mV. However, decreasing HP to -40 mV abolished the agonist action. The agonist effect of Bay K 8644 (1 microM) on Ca2+ channels was elicited to the same extent at either HP. CGP 28392 (10 microM) also increased Ca2+ channel activities at both HPs, but its agonist effect was significantly larger at an HP of -80 mV than at -40 mV. 3. All of the three DHP Ca2+agonists prolonged open times of Ca2+ channels, but the prolongation did not correspond to the voltage-dependence of Ca2+ agonist effects of the three DHPs. 4. YC-170 markedly reduced the closed time of the Ca2+ channel when the HP was at -80 mV, but prolonged it at HP of -40 mV. Bay K 8644 reduced closed times at an HP of -80 mV. At an HP of -40 mV, Bay K 8644 slightly reduced closed times. CGP 28392 reduced closed times at an HP of -80 mV and prolonged those at an HP of -40 mV. Thus the voltage-dependence of the agonist effects of these agents was in good agreement with the voltage-dependence of changes in closed times of Ca2+ channel. 5. Two mechanisms may be involved in the agonist action of YC-170; a prolongation of open times, and a reduction of closed times of Ca2+ channels, i.e. an increase in reopening. The former mechanism is not dependent on Hp and the latter mechanism is highly dependent on HP. Thus, the voltage-dependence of the agonist action may be attributed to the voltage-dependence of their enhancing effect on reopening of Ca2+ channels.

Effects of MS-551, a new class III antiarrhythmic drug, on action potential and membrane currents in rabbit ventricular myocytes.

1. Electrophysiological effects of MS-551, a new class III antiarrhythmic drug, were examined and compared with those of (+)-sotalol in rabbit ventricular cells. 2. In rabbit ventricular muscles stimulated at 1.0 Hz, MS-551 (0.1-10 microM) and (+)-sotalol (3-100 microM) prolonged action potential duration (APD) and effective refractory period without affecting the maximum upstroke velocity of phase 0 depolarization (Vmax). The class III effect of MS-551 was approximately 30 times more potent than that of (+)-sotalol. 3. Class III effects of MS-551 and (+)-sotalol showed reverse use-dependence, i.e., a greater prolongation of APD at a longer cycle length. 4. In rabbit isolated ventricular cells, 3 microM MS-551 and 100 microM sotalol inhibited the delayed rectifier potassium current (IK) which was activated at more positive potentials than -50 mV and saturated around +20 mV. 5. MS-551 at a higher concentration of 10 microM decreased the transient outward current (Ito) and the inward rectifier potassium current (IK1) although 100 microM sotalol failed to inhibit these currents. 6. MS-551 is a non-specific class III drug which can inhibit three voltage-gated K+ channels in rabbit ventricular cells.

Effects of nicorandil on the recovery of reflex potentials after spinal cord ischaemia in cats.

1. The pathophysiological significance of ATP-sensitive K+ (KATP) channels in the central nervous system is not fully understood. In this study the effects of nicorandil (a hybrid vasodilator having a dual mechanism of action as a K+ channel opener and a nitrate) on the recovery of the spinal cord reflex potentials after spinal cord ischaemia were examined and compared with those of pinacidil and nitroprusside in anaesthetized spinal cats. 2. Spinal cord ischaemia was produced by occlusion of the thoracic aorta and the bilateral internal mammary arteries for 10 min. Regional blood flow in the spinal cord was continuously measured with a laser-Doppler flow meter. The monosynaptic (MSR) and polysynaptic reflex (PSR) potentials, elicited by electrical stimulation of the tibial nerve, were recorded from the lumbo-sacral ventral root. The recovery process of spinal reflex potentials was reproducible when the occlusion was repeated twice at an interval of 120 min. 3. Pretreatment with nicorandil (30-100 micrograms kg-1) accelerated the recovery of PSR potentials after spinal cord ischaemia. Such an accelerating effect on the recovery of PSR potentials was also shared by pinacidil (100 micrograms kg-1), another K+ channel opener. In addition, the accelerating effect of nicorandil (100 micrograms kg-1) on the recovery of PSR potentials was abolished by co-administration of glibenclamide (3 mg kg-1), a sulphonylurea KATP channel blocker. Nitroprusside (8 micrograms kg-1min-1) retarded rather than improved the recovery of PSR potentials after spinal cord ischaemia. All of these drugs failed to improve the spinal cord blood flow during ischaemia and reperfusion. 4 These results suggest that nicorandil promotes the recovery of polysynaptic reflex potentials after spinal cord ischaemia by opening the KATP channels of neurones rather than by increasing local bloodflow. K+ channel openers may exert a salutary effect on the functional recovery of the ischaemic spinal cord.