Comparative study of the effects of salinomycin and monensin on electrophysiologic and contractile properties of canine myocardium

The effects of the monocarboxylic ionophore, salinomycin (K⁺-selective), on isometric twitcches, high K⁺-induced contracture and transmembrane action potentials were compared with those of the monocarboxylic ionophore, monensin (Na⁺-selective), in isolated canine right ventricular muscle. In a concentration (5 × 10⁻⁶) which did not produce changes in resting force, salinomycin increased peak active force ( P₀, + 170 ± 36%, mean % change from control ±S.D., P< 0.01). and relaxation and maximal rates of force development (dP/dt_max, + 123 ± 33%, P < 0.01) and relaxation (−dP/dt_max, + 180 ± 40%, P < 0.01) of the isometric twitch. A similar response pattern was found for 5 × 10⁻⁶ M monensin (P₀, + 90 ± 24%, P < 0.01; dP/dt_max, + 137 ± 19%, P < 0.01; −dP/dt_max, + 145 ± 20%, P < 0.01). In contrast to their effects on isometric twitches, salinomycin reduced peak K⁺ contracture force (P_c, −35 ± 14%, P < 0.01) whereas monensin increased it (P_c, +30 ± 12%, P < 0.02). Ventricular muscle action potential duration was shortened similarly by the ionophores. β-Adrenergic receptor blockade with nadolol diminished salinomycin's effects on the isometric twitch and K⁺ contracture, but not its effect to shorten the action potential. Monensin's actions were unaffected by nadolol. These results suggest that salinomycin's effects arise from both a direct modulation of K⁺ movement and the release of endogenous catecholamine. In contrast, monensin may act to alter intracellular Na⁺ which in turn leads to Na⁺---Ca²⁺ exchange and Ca²⁺ -mediated modulation of K⁺ movement.     

Effects of drugs on ventricular fibrillation and ischaemic K loss in a model of ischaemia in perfused guinea-pig hearts in vitro

In a perfused guinea-pig heart model of myocardial isehaemia, reducing coronary flow by 95% for four successive 6 min periods caused a reproducible net loss of K⁺ into the coronary perfusate. This was reduced in a concentration-dependent manner by ATP dependent K⁺ channel blockers (glibenclamide and glipizide) and calcium channel blockers (verapamil and nifedipie). Other K⁺ channel blockers (UK-66,914, 4-aminopyridine, R56865 and phentolamine) and β₁-adrenoceptor and β₂-adrenoceptor antagonists (betaxolol and ICI 118551) did not reduce this loss significantly. A single 30 min low-flow period reliably induced K⁺ release and ventricular fibrillation in control hearts. Glibenclamide, glipizide and phentolamine suppressed ventricular fibrillation but not ischaemic K⁺ loss in this model. R56865 and 4-aminopyridine and coadministration of betaxolol and ICI 18551 yielded similar results while UK-66,914 suppressed neither. In our model, modulation of ischaemic K⁺ loss and suppression of ventricular fibrillation were not closely associated and appeared to occur via separate mechanisms.     

Sodium pump activity and contraction of renal artery from spontaneously hypertensive rats

We investigated the hypertensive changes in renal arteries isolated from 21-week-old spontaneously hypertensive rats (SHR), and from age-matched normotensive Wistar-Kyoto rats (WKY). The maximam contraction of renal arteries from SHR in response to norepinephrine (NE), serotonin (5-HT) and KCl was greater than that of arteries from WKY. The threshold and EC₅₀ concentrations of NE, 5-HT and KCl were not significantly different between SHR and WKY. Contraction induced by removal of K⁺ was inhibited by 10⁻⁸ M prazosin. Less than 10⁻⁷ M NE in K⁺-free solution did not cause contraction. Addition of 5.9 mM KCl to K⁺-free solution in the presence of 10⁻⁵ M NE induced relaxation, which was followed by contraction to about the same level as that before KCl addition. The duration of the K⁺-induced relaxation in SHR (22.4 ± 0.9 min) was slightly, but significantly shorter than that in WKY (26.6 ± 0.8 min) arteries. In K⁺-free solution with reduced Na⁺, the duration of the relaxation induced by KC1 was shorter than that in the normal solution, for both SHR (13.8 ± 0.3 min) and WKY (14.1 ± 0.5 min). Such differences could be caused by increased influx and decreased efflux of Ca²⁺, which depend on the Na⁺ concentration and are related to the Na⁺-Ca²⁺ exchange. The results suggest that enhanced renal vascular reactivity in hypertension may depend on structural changes and increased Na⁺ pump activity.     

Distinct effects of ω-toxins and various groups of Ca-entry inhibitors on nicotinic acetylcholine receptor and Ca channels of chromaffin cells

The effects of ω-toxins and various Ca²⁺ antagonist subtypes on the ⁴⁵Ca²⁺ entry into bovine adrenal medullary chromaffin cells stimulated via nicotinic acetylcholine receptors or via direct depolarization with K⁺, have been compared. The conditions selected to stimulate the ⁴⁵Ca²⁺ entry consisted of a 60-s period of exposure of cells to 100 μM of the nicotinic acetylcholine receptor agonist dimethylphenylpiperazinium or to 70 mM K⁺. The N-type voltage-dependent Ca²⁺ channel blockers ω-conotoxin GVIA and MVIIA (1 μM) inhibited ⁴⁵Ca²⁺ entry stimulated by dimethylphenylpiperazinium or K⁺ by around 25–30%. The P-type Ca²⁺ channel blocker ω-agatoxin IVA (10 nM) did not affect the dimethylphenylpiperazinium nor the K⁺ responses; 1 μM (Q-channel blockade) inhibited both responses by around 50%. The N/P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC (1 μM) inhibited the K⁺ evoked ⁴⁵Ca²⁺ entry by 70%, while dimethylphenylpiperazinium was blocked by 50% (P<0.001). The L-type Ca²⁺ channel blockers nifedipine, furnidipine, diltiazem or verapamil (3 μM each) inhibited much more the dimethylphenylpiperazinium than the K⁺ response. The dimethylphenylpiperazinium signal was blocked 71, 88, 89, and 53%, respectively, by nifedipine, furnidipine, diltiazem and verapamil, and the K⁺ response by 38, 29, 22, and 10%. Combined ω-conotoxin MVIIC (1 μM) and furnidipine (3 μM) blocked 100% of the K⁺ evoked ⁴⁵Ca²⁺ entry. However, combined ω-conotoxin GVIA (1 μM), and furnidipine left unblocked 50% of the K⁺ response. The ‘wide spectrum' Ca²⁺ channel antagonists flunarizine or dotarizine (3 μM each) blocked the dimethylphenylpiperazinium and the K⁺ responses to a similar extent (50%); cinnarizine (3 μM) inhibited more the dimethylphenylpiperazinium (82%) than the K⁺ response (21%). At 3 μM, the highly lipophilic β-adrenoceptor antagonist (±)-propranolol, reduced by 68% the dimethylphenylpiperazinium signal and by 23% the K⁺ signal. Other high lipophilic β-adrenoceptor antagonists such as metoprolol and labetalol, reduced little the dimethylphenylpiperazinium and the K⁺ responses. The highly lipophilic agent penfluridol blocked the dimethylphenylpiperazinium response by 30% and the K⁺ response by 50%. One of the least lipophilic compounds tested, (+)-lubeluzole, blocked by 40% the dimethylphenylpiperazinium and the K⁺ responses. These data are compatible with the idea that the various ω-toxin peptides used to separate pharmacologically the different voltage-dependent Ca²⁺ channels expressed by neurones, do not block the neuronal nicotinic acetylcholine receptor ion channel. In contrast the L-type Ca²⁺ channel blockers do block the nicotinic acetylcholine receptor ionophore. Lipophilicity of the compounds is not a requirement for Ca²⁺ channel or nicotinic acetylcholine receptor blockade.     

Bay K 8644 induces a reversible spasticity-like syndrome in rats

The dihydropyridine Bay K 8644 exerts a positive modulation of Ca²⁺ channels. Administration of Bay K 8644 3–5 mg/kg i.p. to rats induces within 15 min a severe spasticity syndrome consisting of stiff tail, arched back, stretching and twisting of forelimbs and hindlegs and backwards motility and rolling over. The syndrome was effectively antagonized by nifedipine 3–30 mg/kg but not by the other Ca²⁺ channel blockers flunarizine, diltiazem and verapamil. Diltiazem even enhanced the spasticity. Diazepam 10–30 mg/kg i.p. completely blocked the spasticity whereas the other muscle relaxants (−)-baclofen and the β-carboline ZK 93423 were completely inactive. These findings with Bay K 8644 suggest that spasticity may be caused by changed Ca²⁺ homeostasis.     

Effects of Ca channel blockers on Ca loading induced by metabolic inhibition and hyperkalemia in cardiomyocytes

The effects of the L-type (nifedipine and verapamil) and the T-type (mibefradil) Ca²⁺ channel blockers on the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by NaCN metabolic inhibition and hyperkalemia were examined in chicken cardiomyocytes using fluorescence imaging with Fura-2. NaCN induced a slow and sustained rise in [Ca²⁺]_i, which was not affected by pretreating the cells for 5 min with nifedipine, verapamil, or mibefradil at 100 nM or 10 μM. Pretreatment of the cells with 10 μM nifedipine, verapamil, or mibefradil for 5 min remarkably inhibited the K⁺-induced increase in [Ca²⁺]_i. These inhibitory effects diminished after 48-h pretreatment with nifedipine or verapamil but not with mibefradil. Ryanodine also induces an increase in [Ca²⁺]_i, and this effect was enhanced by 48-h pretreatment of the cells with 10 μM verapamil but not with 10 μM mibefradil. We conclude that the NaCN-induced increase in [Ca²⁺]_i is independent of the Ca²⁺ influx though the L-type or T-type Ca²⁺ channels. Chronic inhibition of the L-type Ca²⁺ channels but not T-type channels may enhance the ryanodine receptor-mediated Ca²⁺ release, which may be responsible for the development of tolerance to their inhibitory effects on K⁺-induced increase in [Ca²⁺]_i.     

Similarities of the in vivo and in vitro effects of mercuric chloride on H]ouabain binding and potassium activation of Na/K-ATPase in isolated rat cerebral microvessels

A previous study revealed that a single i.p. administration of 6 mg/kg body wt. of mercuric chloride (MC) durably inhibits the rat cerebral microvascular Na⁺/K⁺-ATPase activity [1]. In this study, cerebral microvessels isolated 18 h after MC treatment were compared to those obtained from control rats and subsequently treated or not treated with MC in vitro, with regard to: (a) ³H]ouabain binding to, and (b) K⁺-activation kinetics of, the Na⁺/K⁺-ATPase. Microvessels from MC-treated rats showed a decrease of ³H]ouabain binding down to 62% of the control binding, and the same degree of inhibition was attained in microvessels treated in vitro with 5 μM MC. Analysis of the K⁺-activation kinetics of Na⁺/K⁺-ATPase revealed a decrease of V_max from the control value of 13.1 to 7.67 μmol/mg/h in microvessels from MC-treated rats and 6.07 μmol/mg/h in microvessels treated in vitro with 5 μM MC, with no change in K_m in either case. The similarity of the effects of in vivo and in vitro treatments suggests that the inhibition of the cerebromicrovascular Na⁺/K⁺ATPase following in vivo administration of MC results from a direct interaction of Hg⁺ with the enzyme.     

Coronary vascular hemodynamic and permeability changes during reperfusion after no-flow ischemia in isolated, diltiazem-treated rabbit hearts

Effects of diltiazem on coronary vascular functional integrity were assessed in isolated rabbit hearts during reperfusion after 30 min of global, no-flow ischemia. External detection of radiolabeled albumin, [125I]bovine serum albumin ([125I]BSA), and compartmental-model analysis were used to estimate the mean transit time of [125I]BSA (tBSA), vascular volume (V1), and vascular into extravascular space clearance (F21) for [125I]BSA. Perfusion pressure, left ventricular (LV) end-diastolic pressure, LV developed pressure, maximum +dP/dt, and V1 remained constant during 5 h of continuous perfusion, while tBSA and F21 gradually increased (1.5 and 2.4 times baseline, respectively). Diltiazem, 4 microM, increased total water content (8.5%) and decreased perfusion pressure (11%), LV developed pressure (22%), and +dP/dt (24%) in nonischemic control experiments, but did not significantly affect estimates of V1, extracellular space, tBSA, or albumin permeation. During reperfusion after 30 min of ischemia, V1 increased 40% and perfusion pressure increased 60%, while tBSA and F21 increased three and eight times baseline, respectively. LV developed pressure and +dP/dt returned to control levels, even though the water content and extracellular space of ischemic hearts were increased significantly. Diltiazem, 4 microM, blocked ischemia-reperfusion-induced increases in water content, extracellular space, vascular resistance, V1, and vascular permeability to [125I]BSA, without reducing LV developed pressure or +dP/dt relative to nonischemic diltiazem controls. These results suggest that protection of ischemic myocardium by diltiazem is mediated, at least in part, by preservation of vascular functional integrity.     

Reversal of acute theophylline toxicity by calcium channel blockers in dogs and rats

Theophylline, widely used in the treatment of pulmonary diseases, has a narrow therapeutic index; the recommended plasma levels being 10–20 μg/ml in humans. The misuse or abuse of theophylline can cause life-threatening central nervous system and cardiovascular effects. Increased intracellular Ca²⁺ levels are thought to play an important role in theophylline toxicity and death. The objective of this study was to determine whether Ca²⁺ channel blockers, e.g. verapamil, nifedipine, or diltiazem, prevent sudden death caused by theophylline treatment in rats and dogs. Groups of Sprague-Dawley rats were treated with theophylline alone (150 mg/kg i.p.) or with theophylline pretreatment followed by administration of verapamil (0.25 to 0.5 mg/kg i.p.), nifedipine (0.25 to 1.0 mg/kg i.p.), or diltiazem (0.5 to 1.0 mg/kg i.p.), 2.5 to 15 min later. The rats were observed for toxic signs and survival over a period of 15 days. All three calcium channel blockers significantly reduced the theophylline-induced sudden death in rats. In a separate study, neither verapamil (0.5 mg/kg i.p.) nor nifedipine (1.0 mg/kg i.p.) prevented the theophylline-induced myocardial necrosis in the rat. In beagle dogs, verapamil (0.5 mg/kg i.v.) prevented theophylline (15 mg/kg/min i.v. for 10 min)-induced hypotension, arrhythmias, and sudden death. Our results support previously reported findings that calcium plays a major role in theophylline-induced toxicity and death.     

Homologous and heterologous desensitisation of somatostatin-induced increases in intracellular Ca and inositol 1,4,5-trisphosphate in CHO-K1 cells expressing human recombinant somatostatin sst5 receptors

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst₅ receptors (CHOsst₅ cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC₅₀ 7.10) and UTP (pEC₅₀ 5.14) caused concentration-dependent increases in [Ca²⁺]_i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca²⁺]_i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P₃ but the SRIF maximum was about 30% of that to UTP. Removal of [Ca²⁺ ]_e attenuated the SRIF-induced peak rise in [Ca²⁺]_i but had no effect on the peak increases in Ins(1,4,5)P₃. UTP-induced increases in [Ca²⁺]_i and Ins(1,4,5)P₃ were attenuated in the absence of [Ca²⁺]_e. Following pre-exposure to SRIF (1 μM) or UTP (100 μM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca²⁺]_e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca²⁺]_e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca²⁺]_i in CHOsst₅ cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca²⁺ and release from an Ins(1,4,5)P₃ sensitive Ca²⁺ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca²⁺]_i could be demonstrated in the presence or absence of extracellular Ca²⁺ respectively, and the latter appeared to involve depletion of a common intracellular Ca²⁺ store.     

Cardioplegic arrest of the myocardium with calcium blocking agents.

This study was designed to compare the effects of the calcium slow channel blocking agents verapamil (0.15 mg/kg), diltiazem (0.15 mg/kg), and nifedipine (50 micrograms/kg) on the myocardium after global ischemia and reperfusion in the in situ canine model. Animals were subjected to 120-min normothermic global ischemia, followed by 45-min reperfusion. Cardioplegic arrest of the myocardium was achieved by administering one of the three calcium antagonists in a multidose fashion. Superior preservation (p less than 0.01) of left ventricular (LV) systolic function was achieved in group I (verapamil cardioplegia). dP/dt, at an intraventricular balloon volume of 25 cc, was 83% of control after reperfusion in group I. Group II (diltiazem) and group III (nifedipine) achieved only 55 and 63% of their preischemic dP/dt values. LV chamber stiffness was increased in hearts protected with nifedipine. The exponential constant m was increased from 0.04 +/- 0.01 to 0.08 +/- 0.01. Coronary blood flow after reperfusion increased from 120 to 184 cc/100 gr/min in group I (p less than 0.01). The hyperemic response in group III was negligible. The O2 consumption of the reperfused myocardium was not significantly altered in any of the treatment groups. Lactate metabolism during ischemia and after reperfusion was similar in all groups. ATP values were markedly reduced in all groups (p less than 0.05). Immediately after ischemia, ATP was 50, 28, and 44% of control in group I, II, and III, respectively. The excellent preservation of systolic function and a physiologic hyperemic response by verapamil could not be correlated with improved preservation of high-energy compounds or with significant changes in myocardial O2 consumption.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of diltiazem on the pharmacokinetics of nifedipine.

To evaluate the effect of diltiazem pretreatment (60 mg three times a day for 3 days) on pharmacokinetics and pharmacodynamic effect of nifedipine, six healthy subjects received 20 mg nifedipine orally on two occasions using a double-blind cross-over, placebo-controlled method. Diltiazem induced a marked increment of the area under the plasma concentration-time curve (AUC) for nifedipine by a mean of 140% and reduced the total body clearance (Cl) from 0.0043 +/- 0.0019 to 0.0017 +/- 0.0006 ml/min/kg (p less than 0.05, mean +/- SD). The biological half-life (t1/2) of nifedipine was prolonged from 2.46 +/- 0.65 to 3.21 +/- 0.92 h (p less than 0.05) without any changes in indocyanine green (ICG) clearance. Diltiazem did not produce significant changes of AUC, Cl, and t1/2 for the acid metabolite of nifedipine. Blood pressure (BP) after nifedipine administration with diltiazem pretreatment was more decreased than that without diltiazem. Both a decreased hepatic clearance and an increased bio-availability of nifedipine by diltiazem probably explain the significant changes in pharmacokinetics and hemodynamics of nifedipine. A clinically important drug interaction may occur with nifedipine when diltiazem is administered concurrently.     

Action of the Na ionophore monensin on vascular smooth muscle of guinea-pig aorta

The effects of the Na⁺ ionophore monensin on contractile responses were investigated in guinea-pig aorta in normal and high K⁺ solutions. In normal K⁺ (5.4 mM) solution, monensin (2 × 10⁻⁵ M) produced a rapid increase in tension followed by slow relaxation. This contraction was markedly inhibited by phentolamine (10⁻⁵ M) or prazosin (10⁻⁶ M) and was accompanied by an increase in tritium efflux from tissue preloaded with [³H]norepinephrine. In the presence of phentolamine, monensin (1–2 × 10⁻⁵ M) or ouabain (1−2 × 10⁻⁵ M) caused only a small and slowly developing contraction. Simultaneous application of these agents caused a more rapid and greater contraction. Either monensin or ouabain gradually increased cellular Na⁺ and decreased cellular K⁺ content. When monensin was applied simultaneously with ouabain, there was a rapid increase in cellular Na⁺ and loss of cellular K⁺. In high K⁺ (65.4 mM) solution, monensin (10⁻⁶ M) slightly reduced the increased tension level but when external glucose was omitted monensin markedly inhibited the contraction. A significant decrease in tissue ATP content was observed only when monensin was applied in glucose-free solution. Similarly, hypoxia (N₂ bubbling) markedly inhibited the high K⁺ contraction and decreased the tissue ATP content only in the absence of glucose. These results suggest that monensin produces a neurogenic contraction due to the release of endogenous catecholamines and also produces a myogenic contraction by a decrease in transmembrane Na⁺ and K⁺ gradients when the Na⁺ and -K⁺ pump is inhibited by ouabain, and that monensin inhibits aerobic energy metabolism of vascular smooth muscle.     

Potassium channels and pain: present realities and future opportunities

Four families of potassium channels with different structures, functional characteristics and pharmacological sensitivity, are distinguished in neurons: voltage-gated (K_v), calcium-activated (K_Ca), inward rectifier (K_ir) and two-pore (K_2P) K⁺ channels. During the last 15 years, numerous studies have demonstrated that the opening of some of these K⁺ channels plays an important role in the antinociception induced by agonists of many G-protein-coupled receptors (₂-adrenoceptors, opioid, GABA_B, muscarinic M₂, adenosine A₁, serotonin 5-HT_1A and cannabinoid receptors), as well as by other antinociceptive drugs (nonsteroidal antiinflammatory drugs [NSAIDs], tricyclic antidepressants, etc.) and natural products. Several specific types of K⁺ channels are involved in antinociception. The most widely studied are the ATP-sensitive K⁺ channels (K_ATP), members of the K_ir family, which participate in the antinociception induced by many drugs that activate them in both the central and the peripheral nervous system. The opening of G-protein-regulated inwardly rectifying K⁺ channels (GIRK or K_ir3), K_v1.1 and two types of K_Ca channels, the small- and large-conductance calcium-activated K⁺ channels (SK and BK channels, respectively), also play a role in the antinociceptive effect of different drugs and natural products. Recently, drugs that open K⁺ channels by direct activation (such as openers of neuronal K_v7 and K_ATP channels) have been shown to produce antinociception in models of acute and chronic pain, which suggests that other neuronal K⁺ channels (e.g. K_v1.4 channels) may represent an interesting target for the development of new K⁺ channel openers with antinociceptive effects.     

The effects of disopyramide phosphate on early post-coronary artery ligation dysrhythmias and on epicardial ST-segment elevation in anaesthetized dogs.

1 The antidysrhythmic, haemodynamic and metabolic effects of intravenously administered disopyramide phosphate (1 to 5 mg/kg) have been studied in greyhounds, anaesthetized with trichloroethylene. 2 In doses of 2.5 and 5.0 mg/kg, disopyramide significantly reduced the ventricular dysrhythmias that occur in the initial 30-min period following acute coronary artery ligation. None of the disopyramide-treated animals developed ventricular fibrillations. 3 The metabolic consequences of coronary artery ligation, assessed by local coronary venous sampling from the ischaemic area, were not modified by disopyramide except that K+ egress was prevented. 4 There was evidence for substantial disopyramide-induced myocardial depression (decreased cardiac output and left ventricular dP/drmax with elevated ventricular filling pressure and pulmonary oedema and shunting) and it is suggested that great care be taken when the drug is administered intravenously in conditions where cardiac function is already compromised. Disopyramide also reduced myocardial blood flow. 5 In chloralose-anaesthetized mongrel dogs, disopyramide (2.5 mg/kg) significantly reduced the ST-segment elevation (assessed from epicardial recordings) that resulted from short (3 min) coronary artery occlusions. This could indicate a reduction in the extent and severity of myocardial injury or simply reflect decreased K+ efflux (since locally administered K+ itself increased ST-segment elevation).     

Characterization of the outer pore region of the apamin-sensitive Ca2+-activated K+ channel rSK2.

We have studied the interaction between the SK2 channel and different scorpion toxins in order to find similarity and differences to other K⁺ channels. Beside apamin, ScTX is a high affinity blocker of the SK2 channel, whereas CTX is unable to block current through SK2. In order to prove that the ScTX affinity can be explained by the character of the different residues in the outer pore of the SK channels we introduced point mutations that render SK2 K⁺ channel SK1 and SK3 like. Directed by the results of the toxin receptor on the ShakerK⁺ channel, we changed single amino acids of the SK2 K⁺ channel that should render it sensitive to other peptide toxins like CTX a blocker of the IK channel, or KTX a blocker of the voltage-dependent channel Kv1.1 and Kv1.3. Amino acids V342G, S344E, and G384D of SK2 were changed to amino acids known from ShakerK⁺ channel to improve Shaker K⁺ channel CTX sensitivity. Interestingly SK2 V342G became CTX sensitive with a K_d of 19 nM and was also KTX sensitive K_d=97 nM. SK2 S344E (K_dCTX=105 nM,K_dKTX=144 nM) and G348D (K_dCTX=31 nM,Kd KTX=89 nM) became also CTX and KTX sensitive with a lower affinity. The mutant channels SK V342G, SK2 S344E and SK2 G348D showed reduced ScTX sensitivity (K_d=6 nM,K_d=48 nM, and K_d=12 nM). Because the exchange of a single residue could create a new high affinity binding site for CTX and KTX we concluded that the outer vestibule around position V342, S344, and G348 of the SK2 K⁺ channel pore is very similar to those of voltage-gated K⁺ channels such as the Shaker K⁺ channel, Kv1.1 and Kv1.3 channels and also to the prokaryotic KcsA channel. From mutant cycle analysis of KTX position H34 and SK2 position V342G, S344E, and G348D we could deduce that KTX binds in a similar way to SK2 channel mutant pore than to the Kv1.1 pore.     

Ca dependence of the response of three adenosine type receptors in rat hepatocytes

The effect of three different receptor-specific adenosine agonists on the rate of ureagenesis by isolated rat hepatocytes and the dependence on the external free Ca²⁺ concentration ([Ca²⁺]_e) were investigated. In the presence of high [Ca²⁺]_e all adenosine receptor agonists increased ureagenesis to similar levels. However, with low [Ca²⁺]_e the effects of each agonist varied as follows: (i) the adenosine A₁ receptor agonist, 2-chloro-N⁶-cyclopentyl-adenosine, increased ureagenesis depending partially on [Ca²⁺]_e, (ii) the adenosine receptor A₂ agonist, 2-p-(-2-carboxy-ethyl) phenethylamino-5′-N-ethylcarboxyamido adenosine hydrochloride, increased ureagenesis independently of [Ca²⁺]_e and (iii) in contrast, the adenosine receptor A₃ agonist N⁶-2-(-4-aminophenyl) ethyladenosine, increased ureagenesis only in the presence of high [Ca²⁺]_e. The adenosine receptor A₁ antagonist, 1-allyl-3,7-dimethyl-8-phenyl xanthine, inhibited the effect of the adenosine receptor A₁ agonist on ureagenesis, but not the effect of the adenosine A₂ or A₃ receptor agonists. The adenosine A₂ receptor antagonist, 3,7-dimethyl-1-propargylxanthine, inhibited only the effect of the adenosine A₂ receptor agonist. Thus, in addition to A₁ and A₂ type adenosine receptors, rat hepatocytes possess an A₃-like adenosine receptor which responds to the addition of an adenosine A₃ agonist by accelerating ureagenesis a [Ca²⁺]_e dependent manner. Moreover, it was observed that in the presence of extracellular Ca²⁺ each agonist increased [Ca²⁺]_i and this effect was inhibited by the appropriate specific antagonist.     

Duration of protection of calcium channel blockers against exercise-induced bronchospasm: Comparison of oral diltiazem and inhaled gallopamil

Summary The present study was conducted to determine the duration of the positive effect of oral diltiazem and inhaled gallopamil in mild asthmatic volunteers, ages 18–37 years, with a history of exercise-induced asthma and a 25–56% decrease in FEV₁ after a standardized exercise challenge. Oral diltiazem 120 mg, inhaled gallopamil 10 mg, and placebo were administered in a double blind, randomized, crossover manner on different days 48 h apart. Diltiazem was administered 90 min and gallopamil 30 min before the first exercise challenge. Challenges were then repeated 3 and 6 h later.Neither diltiazem nor gallopamil significantly altered baseline FVC, FEV₁, or FEF_25–75. The mean maximum decrease in FEV₁ after the first challenge was 16.8% after gallopamil, 25.2% after diltiazem and 30.1% after placebo. The mean post-exercise decrease in FEV₁ after gallopamil was significantly smaller than after placebo. There were no significant differences in the post-exercise decreases in FEV₁ between the three treatment regimens 3 and 6 h later.Thus, inhaled gallopamil provided significant protection against exercise-induced bronchospasm, but the beneficial effect was modest and short in duration.From the Department of Pharmacy Practice, College of Pharmacy, the Division of Clinical Pharmacology/Toxicology, Department of Pediatrics and the Division of Pulmonary Medicine, Department of Internal Medicine, University of Florida, Gainesville, Florida, 32610, USAAt the time of this study, Dr. Massey was a Fellow in the College of Pharmacy and Division of Clinical Pharmacology/Toxicology, Department of Pediatrics, University of Florida     

Fantofarone (SR 33557): effect on post-ischaemic functional recovery in perfused rat hearts

Fantofarone (SR 33557) is a novel, highly potent calcium channel antagonist representative of a new class of slow channel blockers. In this study, we have assessed its ability to influence cardiac function in two, isolated, perfused heart models and then assessed its ability to modify post-ischacmic functional recovery. In isolated, rat hearts perfused in the Langcndorff mode, fantofarone increased coronary flow by 25% at 100 and 1000 nM with no effect on left ventricular pressure or heart rate below 100 nM. In working hearts, fantofarone again increased coronary flow within a similar concentration range. A significant reduction (approximately 40%) was observed in peak systolic pressure and dP/dt_max when hearts were perfused with 1000 nM fantofarone. Working rat hearts were also subjected to a 30 min period of global, low-flow (0.1 ml/min) ischaemia, followed by a 30 min period of repcrfusion. Perfusion with 1 or 10 nM fantofarone, began 20 min prior to the onset of ischaemia and continued throughout the ischaemic and repcrfusion periods. The addition of 1 nM fantofarone did not cause a significant increase in the recovery of ardiac function during the repcrfusion phase. In contrast, perfusion with 10 nM fantofarone resulted in a substantial increase in the recovery of several indices of cardiac function such as aortic output, dP/dt_max and peak systolic pressure. Thus, in the working rat heart, at concentrations which cause minimal alterations to normal cardiac function, fantofarone can improve significantly functional recovery following an ischaemic insult.     

The role of potassium channels in antihistamine analgesia

The effect of the administration of pertussis toxin as well as modulators of different subtypes of K⁺ channels on the antinociception induced by the H₁-antihistamines pyrilamine, diphenhydramine and promethazine was evaluated in the mouse hot plate test. Pretreatment with pertussis toxin (0.25 μg/mouse i.c.v.) prevented pyrilamine, diphenhydramine and promethazine antinociception. The K_ATP channel openers minoxidil and pinacidil potentiated the antinociception produced by the H₁-antihistamines whereas the K_ATP channel blocker gliquidone prevented the anti H₁-induced analgesia. The Ca²⁺-gated K⁺ channel blocker apamin antagonized pyrilamine, diphenhydramine and promethazine analgesia. Pretreatment with an antisense oligonucleotide (aODN) to mKv1.1, a voltage-gated K⁺ channel, at the dose of 3.0 nmol/single i.c.v. injection, never modified the antinociception induced by the H₁-antihistamines in comparison with degenerate oligonucleotide (dODN)-treated mice. At the highest effective doses, none of the drugs used modified animals’ gross behaviour nor impaired motor coordination, as revealed by the rota rod test. The present data demonstrate that both K_ATP and Ca²⁺-gated K⁺ channels, contrary to voltage-gated K⁺ channel Kv1.1, represent an important step in the transduction mechanism underlying central antinociception induced by H₁-antihistamines.