Bunazosin,a Selective α1 ‐Adrenoceptor Antagonist,as an Anti‐glaucoma Drug: Effects on Ocular Circulation and Retinal Neuronal Damage

Bunazosin hydrochloride is a potent and selective α₁‐adrenoceptor antagonist that has been clinically used both as a systemic antihypertensive as well as an ocular hypotensive drug. In a number of studies, we have examined some effects of bunazosin hydrochloride that might indicate its potential as an anti‐glaucoma drug. In normal rabbit eyes, topically instilled bunazosin hydrochloride reached the posterior retina by local penetration at concentrations sufficient to attenuate the phenylephrine‐ or endothelin‐1 (ET‐1)‐induced constriction of retinal arteries. Furthermore, bunazosin hydrochloride improved the impairment of optic nerve head (ONH) blood flow, the prolongation of visual‐evoked potentials (VEP) implicit time, the enlargement of the optic disk cup, and the decrease in the number of retinal ganglion cell layer cells induced by repeated injections of ET‐1 in rabbits. Topically instilled bunazosin hydrochloride improved the reductions in ONH capillary blood flow and VEP amplitude induced in rabbit eyes by nitric oxide synthase inhibition. In rat primary retinal cultures, bunazosin hydrochloride reduced glutamate‐induced neuronal cell death, presumably through a Na⁺ channel blocking effect. In healthy humans, topically instilled bunazosin hydrochloride reportedly increases blood velocity in the ONH, retina and choroid, without significantly altering either blood pressure or heart rate. These results indicate that bunazosin hydrochloride exerts both an improvement effect within the ocular circulation and a direct neuroprotective effect. Hence, bunazosin hydrochloride may be useful as a therapeutic drug against ischemic retinal diseases (such as glaucoma and retinal vascular occlusive diseases) that are associated with disturbances of the ocular circulation.     

Clinical L-Type CaChannel Blockade Prevents Ischemic Preconditioning of Human Myocardium

B. S. Cain, D. R. Meldrum, J. C. Cleveland, Jr, X. Meng, A. Banerjee and A. H. Harken. Clinical L-Type Ca²⁺Channel Blockade Prevents Ischemic Preconditioning of Human Myocardium. Journal of Molecular and Cellular Cardiology (1999) 31, 2191–2197. Although Ca²⁺channel blockers are commonly used to control both blood pressure and angina in patients with coronary artery disease, clinical trials have associated the use of -type Ca²⁺channel blockers with increased cardiovascular mortality. Recent evidence has implicated Ca²⁺entry through the -type Ca²⁺channel during transient ischemia as a proximal stimulus for ischemic preconditioning (IPC) in experimental animals. We therefore hypothesized that clinical -type Ca²⁺channel blockade prevents IPC in human myocardium. Human atrial trabeculae were suspended in organ baths, field simulated at 1 Hz, and force development was recorded. Following 90 min equilibration, trabeculae from control patients and patients taking -type Ca²⁺channel blockers were subjected to simulated ischemia/reperfusion (I/R: 45/120 min) with or without 5 min of simulated ischemia (IPC stimulus) prior to I/R. IPC increased post-ischemic developed force in control patients from 14.6±2.6 to 43.1±3.5% baseline developed force (%BDFP <0.05 I/R vs IPC). Whereas IPC failed to increase post-ischemic developed force in myocardium from patients taking -type Ca²⁺channel blockers (15.1±1.9 vs 16.6±1.7 %BDF,P >0.05 -type I/R v -type IPC). We conclude that: (1) atrial muscle can be preconditioned by transient ischemia; (2) atrial muscle from patients taking -type Ca²⁺channel blockers cannot be preconditioned by transient ischemia; and (3) the increased cardiovascular mortality historically associated with the use of Ca²channel blockers in patients with coronary artery disease may be, in part, due to the pharmacological inhibition of ischemic preconditioning.     

Cilnidipine: A New Generation Ca2+ Channel Blocker with Inhibitory Action on Sympathetic Neurotransmitter Release

Cilnidipine is a unique Ca²⁺ channel blocker with an inhibitory action on the sympathetic N‐type Ca²⁺ channels, which is used for patients with hypertension in Japan. Cilnidipine has been clarified to exert antisympathetic actions in various examinations from cell to human levels, in contrast to classical Ca²⁺ channel blockers. Furthermore, renoprotective and neuroprotective effects as well as cardioprotective action of cilnidipine have been demonstrated in clinical practice or animal examinations. After the introduction of nifedipine as an antihypertensive drug, many Ca²⁺ channel blockers with long‐lasting action for blood pressure have been developed to minimize sympathetic reflex during antihypertensive therapy, which have been divided into three groups; namely, first, second, and third generation based on their pharmacokinetic profiles. Since cilnidipine directly inhibits the sympathetic neurotransmitter release by N‐type Ca²⁺ channel‐blocking property, the drug can be expected as fourth generation, providing an effective strategy for the treatment of cardiovascular diseases.     

KN-62: A Specific Ca2+/calmodulin-dependent Protein Kinase Inhibitor as a Putative Function-searching Probe for Intracellular Signal Transduction

The relationship between changes in intracellular free calcium concentration (Ca_i²⁺) and cell functions is becoming more clear, since it has become possible to measure Ca_i²⁺ in a living cell. There are, however, still unsolved questions concerning the role of Ca²⁺ in cellular signal transmission. It is not easy to relate Ca²⁺ signalling to the functions of a living cell. It is possible to control the amount of Ca²⁺ using calcium channel blockers, but it is still almost impossible to elucidate the relationship between Ca²⁺ and cellular functions by controlling Ca²⁺ signalling. One approach to solving this problem is to develop and use protein kinase inhibitors. Inhibitors specific to Ca²⁺/calmodulin-dependent protein phosphorylation, which is one of the important network systems of the Ca²⁺ signalling, attract interest among many researchers. The substances that can control Ca²⁺ signalling include: 1) calcium channel blockers, 2) calmodulin antagonists, and 3) Ca²⁺/calmodulin-dependent protein kinase inhibitors. Each substance has a different site of action. In this article, the action of KN-62, an inhibitor specific for Ca²⁺/calmodulin protein kinases (CaM kinases) will be described.     

Effects of nicorandil on cell proliferation and cholesteryl ester accumulation in arterial smooth muscle cells in culture

Summary Ca²⁺ regulates a variety of cellular mechanisms in vascular cells as well as in platelets. Nicorandil interacts with the intracellular Ca²⁺-activated processes in vascular smooth muscle cells, while Ca²⁺ channel blockers such as verapamil and diltiazem block voltage-dependent Ca²⁺ channels. The effects of nicorandil are due to the hyperpolarization of the membrane, interference with mobilization of Ca²⁺ from the intracellular storage sites, and blockade of receptor-operated Ca²⁺ channels. In the present study, the effects of nicorandil on cell proliferation and cholesteryl ester accumulation in rat arterial smooth muscle cells in culture were compared to Ca²⁺ channel blockers. Smooth muscle cells were prepared from rat thoracic aorta, and the rate of proliferation was determined by measuring the cell number and by [³H]-thymidine incorporation into cellular DNA. The effect of nicorandil on cholesteryl ester content in smooth muscle cells was determined by thin-layer chromatography of the cell extracts. Nicorandil at concentrations of 10^–6 to 10^–4 M, as well as Ca²⁺ channel blockers (verapamil and diltiazem) inhibited the proliferation and DNA synthesis of cultured smooth muscle cells. The acute inhibitory effects on cell proliferation were observed significantly 16 hours after the addition of the three agents in serum-stimulated cells. These effects were dose dependent, both in acute and in chronic treatment with the three agents. Addition of 10^–5 M nicorandil to medium supplemented with 10% serum resulted in a decrease of the net cholesteryl ester content by 18±1%, while cellular free cholesterol content was the same as control. Similar results were also obtained in the presence of verapamil and diltiazem. These data suggest that nicorandil may suppress atheroma formation, not only by inhibiting cell proliferation but also by decreasing cholesteryl ester accumulation in arterial smooth muscle cells.     

Intracellular Ca2+ Overload Induced by Extracellular Ca2+ Entry Plays an Important Role in Acute Heart Dysfunction by Tentacle Extract from the Jellyfish Cyanea capillata

The exact mechanism of acute heart dysfunction caused by jellyfish venom remains unclear for the moment. In the present study, we examined the problem caused by the tentacle extract (TE) from the jellyfish Cyanea capillata at the levels of whole animal, isolated heart, primarily cultured cardiomyocytes, and intracellular Ca²⁺. The heart indexes, including HR, APs, LVPs, and MMLs, were all decreased significantly by TE in both whole animal and Langendorff-perfused isolated heart model. Imbalance of cardiac oxygen supply and demand also took place. In both Ca²⁺-containing and Ca²⁺-free bathing solutions, TE could cause obvious cytoplasmic Ca²⁺ overload in NRVMs, but the cytoplasmic Ca²⁺ increased faster, Ca²⁺ overload peaks arrived earlier, and the morphological changes were more severe under the extracellular Ca²⁺-containing condition. L-type Ca²⁺ channel blockers, as well as the inhibitor of ryanodine receptor (ryanodine), could improve the viability of NRVMs. Moreover, diltiazem significantly inhibited the acute heart dysfunction caused by TE in both Langendorff isolated heart model and whole animal. These results suggested that intracellular Ca²⁺ overload induced by extracellular Ca²⁺ entry plays an important role in acute heart failure by TE from the jellyfish C. capillata. Inhibition of extracellular Ca²⁺ influx is a promising antagonistic alternative for heart damage by jellyfish venom.     

Endothelin-induced changes in intracellular calcium concentration in rat vascular smooth muscle cells: Effects of extracellular calcium and atrial natriuretic factor

Summary The purpose of this study was to examine whether different mechanisms might underlie the changes in intracellular calcium concentration ([Ca²⁺]_i) stimulated by high and low concentrations of endothelin, and whether atrial natriuretic factor (ANF) has an inhibitory effect on endothelin-induced [Ca²⁺]_i changes in cultured rat vascular smooth muscle cells (VSMCs). In calcium-replete buffer, cultured monolayers of rat VSMCs superfused with endothelin at a high concentration (10 nM) exhibited a marked transient rise in [Ca²⁺]_i, followed by a sustained elevation, whereas a low concentration of endothelin (0.1 nM) induced a sustained monophasic elevation. When calcium-free buffer was used, 10 nM endothelin induced a transient rise in [Ca²⁺]_i of lesser amplitude, whereas 0.1 nM endothelin did not produce a significant rise. Pretreatment of VSMCs with ANF and cosuperfusion with endothelin failed to inhibit either transient or sustained endothelin-induced changes in [Ca²⁺]_i in calcium-replete buffer.     

Prevention of Ventricular Arrhythmia and Calcium Dysregulation in a Catecholaminergic Polymorphic Ventricular Tachycardia Mouse Model Carrying Calsequestrin‐2 Mutation

Arrhythmia Prevention in CPVT . Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmic syndrome caused by mutations in genes encoding the calcium‐regulation proteins cardiac ryanodine receptor (RyR2) or calsequestrin‐2 (CASQ2). Mechanistic studies indicate that CPVT is mediated by diastolic Ca²⁺ overload and increased Ca²⁺ leak through the RyR2 channel, implying that treatment targeting these defects might be efficacious in CPVT. Method and results: CPVT mouse models that lack CASQ2 were treated with Ca²⁺‐channel inhibitors, β‐adrenergic inhibitors, or Mg²⁺. Treatment effects on ventricular arrhythmia, sarcoplasmic reticulum (SR) protein expression and Ca²⁺ transients of isolated myocytes were assessed. Each study agent reduced the frequency of stress‐induced ventricular arrhythmia in mutant mice. The Ca²⁺ channel blocker verapamil was most efficacious and completely prevented arrhythmia in 85% of mice. Verapamil significantly increased the SR Ca²⁺ content in mutant myocytes, diminished diastolic Ca²⁺ overload, increased systolic Ca²⁺ amplitude, and prevented Ca²⁺ oscillations in stressed mutant myocytes. Conclusions: Ca²⁺ channel inhibition by verapamil rectified abnormal calcium handling in CPVT myocytes and prevented ventricular arrhythmias. Verapamil‐induced partial normalization of SR Ca²⁺ content in mutant myocytes implicates CASQ2 as modulator of RyR2 activity, rather than or in addition to, Ca²⁺ buffer protein. Agents such as verapamil that attenuate cardiomyocyte calcium overload are appropriate for assessing clinical efficacy in human CPVT . (J Cardiovasc Electrophysiol, Vol. 22, pp. 316‐324, March 2011)     

Role of K+ channels in EDHF-dependent relaxation induced by acetylcholine in canine coronary artery

Summary To identify the K⁺ channels responsible for endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation, we studied the effects of various K⁺ channel blockers on acetylcholine-induced relaxation, which persists even in the presence of both an inhibitor of nitric oxide synthase and that of cyclooxygenase, in canine coronary artery rings. A nonselective K⁺ channel blocker, tetrabutylammonium (TBA), a large and intermediate conductance Ca²⁺-activated K⁺ channel blocker, charybdotoxin (CTX), and a voltage-dependent K⁺ channel blocker, 4-aminopyridine (4-AP), significantly inhibited this residual relaxation. A combined treatment with CTX and 4-AP almost completely blocked the relaxation. Neither a large (iberiotoxin) nor a small (apamin) conductance Ca²⁺-activated K⁺ channel blocker blocked the relaxation. We also investigated effects of K⁺ channel blockers on basal tone to determine whether or not EDHF is involved in regulating basal tone. TBA and CTX substantially raised basal tone to a greater degree in endothelium-intact preparations than in endothelium-denuded preparations. These results indicate that EDHF may exert its relaxing action through intermediate conductance Ca²⁺-activated and voltage-dependent K⁺ channels in canine coronary arteries. In addition, EDHF may play a role in maintaining basal vascular tone. This study was supported in part by a Grant-in-Aid for Scientific Research (B07457167) from the Ministry of Education, Science and Culture of Japan.     

Dependence of prolactin release on coupling between Ca(2+) mobilization and voltage-gated Ca(2+) influx pathways in rat lactotrophs

Two Ca²⁺-mobilizing receptors expressed in lactotrophs, endothelin-A (ET_A) and thyrotropin-releasing hormone (TRH), induce a rapid Ca²⁺ release from intracellular stores and prolactin (PRL) secretion but differ in their actions during the sustained stimulation; TRH facilitates and ET-1 inhibits voltage-gated calcium influx (VGCI) and PRL secretion. In pertussis toxin (PTX)-treated cells, ET-1-induced inhibition of VGCI was abolished and the pattern of Ca²⁺ signaling was highly comparable with that observed in TRH-stimulated cells. The addition of Cs⁺, a relatively specific blocker of inward rectifier K⁺ channels, mimicked the effect of PTX on the pattern of ET-1-induced sustained Ca²⁺ signaling, but only in about 50% of cells, and did not affect agonist-induced inhibition of PRL secretion. Extracellular Cs⁺ was also ineffective in altering the TRH-induced facilitation of VGCI and PRL secretion. Furthermore, apamin and paxilline, specific blockers of Ca²⁺-activated SK-and BK-type K⁺ channels, respectively; E-4031, a blocker of ether a-go-go K⁺ channel; and linopirdine, a blocker of M-type K⁺ channel, did not affect the agonist-specific patterns of calcium signaling and PRL secretion. These results suggest that ET-1 inhibits VGCI through activation of Cs⁺-sensitive channels, presumably the G_i/o-controlled inward rectifier K⁺ channels, and that this agonist also inhibits PRL release, but downstream of Ca²⁺ influx. Further studies are required to identify the mechanism of sustained TRH-induced facilitation of VGCI and PRL secretion.     

AH‐1058: A Novel Cardioselective Ca2+ Channel Blocker

The pharmacologic profile of a cyproheptadine‐related compound, 4‐(5H‐dibenzo[a,d]cyclohepten‐5‐ylidene)‐1‐[(E)‐3‐(3‐methoxy‐2‐nitro)phenyl‐2‐propenyl]piperidine hydrochloride (AH‐1058), was assessed in various in vivo and in vitro models. In guinea pig cardiomyocytes, AH‐1058 effectively suppressed L‐type Ca²⁺ channel currents without affecting other ion channel or ion exchange currents. In rat cerebral cortical membranes AH‐1058 appears to bind preferentially to L‐type Ca²⁺ channels at phenylalkylamine‐ and benzothiazepine‐binding sites. In canine isolated, blood‐perfused heart preparations, AH‐1058 exerted negative inotropic, dromotropic, and chronotropic and weak coronary vasodilator effects. In halothane‐anesthetized dogs, AH‐1058 suppressed ventricular contractility and decreased blood pressure and cardiac output. Total peripheral vascular resistance was hardly affected by the drug, suggesting that in vivo AH‐1058 can selectively suppress cardiac, as compared to peripheral vascular, function. In conscious dogs, by intravenous administration AH‐1058 reduced systolic blood pressure and maximal upstroke velocity of the left ventricular pressure, while it increased heart rate in a dose‐dependent manner. The drug did not affect diastolic blood pressure, which is quite different from cardiovascular properties of well‐known Ca²⁺ channel blockers, verapamil and diltiazem. This unique cardiovascular profile of AH‐1058 is expected to be useful in the treatment of certain pathological processes such as the obstructive hypertrophic cardiomyopathy, vasovagal syncope, dissecting aortic aneurysm, and ventricular arrhythmias, in which selective inhibition of the ventricular Ca²⁺ channels is essential for drug therapy.     

Verapamil, a phenylalkylamine Ca2 + channel blocker, inhibits ATP-sensitive K + channels in insulin-secreting cells from rats

Summary Radioisotopic and electrophysiological techniques were used to assess the effects of verapamil, a phenylalkylamine Ca^{2 +} channel blocker, on K ⁺ permeability of insulin-secreting cells. Verapamil provoked a concentration-dependent inhibition of ⁸⁶Rb (⁴²K substitute) outflow from prelabelled and perifused rat pancreatic islets. This property appears to be inherent to the phenylalkylamine Ca^{2 +} channel blockers since gallopamil, a methoxyderivative of verapamil, but not nifedipine, a 1,4-dihydropyridine Ca^{2 +} channel blocker, inhibited ⁸⁶Rb outflow. The experimental data further revealed that verapamil interacted with a Ca^{2 +} -independent, glucose- and glibenclamide-sensitive modality of ⁸⁶Rb extrusion. Moreover, verapamil prevented the increase in ⁸⁶Rb outflow brought about by BPDZ 44; a potent activator of the ATP-sensitive K ⁺ channel. Single-channel current recordings by the patch clamp technique confirmed that verapamil elicited a dose-dependent inhibition of the ATP-dependent K ⁺ channel. Lastly, under experimental conditions in which verapamil clearly inhibited the ATP-sensitive K ⁺ channels, the drug did not affect ⁴⁵Ca outflow, the cytosolic free Ca^{2 +} concentration or insulin release. It is concluded that the Ca^{2 +} entry blocker verapamil inhibits ATP-sensitive K ⁺ channels in pancreatic beta cells. This effect was not associated with stimulation of insulin release [Diabetologia (1997) 40: 1403–1410]. Received: 21 May 1997 and in final revised form: 28 August 1997     

Mechanism underlying catecholaminergic polymorphic ventricular tachycardia and approaches to therapy

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by VT induced by adrenergic stress in the absence of structural heart disease and high incidence of sudden cardiac death. The diagnosis is made based on reproducible ventricular tachyarrhythmias including bidirectional VT and polymorphic VT during exercise testings. Two causative genes of CPVT have been identified: RYR2, encoding the cardiac ryanodine receptor (RyR2) Ca²⁺ release channel, and CASQ2, encoding cardiac calsequestrin. A mutation in RYR2 or CASQ2 is identified in approximately 60% of patients with CPVT. Mutations in these two genes destabilize the RyR2 Ca²⁺ release channel complex in sarcoplasmic reticulum and result in spontaneous Ca²⁺ release through RyR2 channels leading to delayed after depolarization, triggered activity, and bidirectional/polymorphic VT. Implantable cardioverter defibrillators (ICDs) are recommended for prevention of sudden death in patients with CPVT.1. A.E. Epstein, J.P. DiMarco, K.A. Ellenbogen, et al., ACC/AHA/HRS 2008 Guidelines for Device-Based Therapy of Cardiac Rhythm Abnormalities: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the ACC/AHA/NASPE 2002 Guideline Update for Implantation of Cardiac Pacemakers and Antiarrhythmia Devices): developed in collaboration with the American Association for Thoracic Surgery and Society of Thoracic Surgeons. Circulation. 2008;117:e350 However, painful shocks can trigger further adrenergic stress and arrhythmias, and deaths have occurred despite appropriate ICD shocks. Treatment with β-adrenergic blockers reduces arrhythmia burden and mortality, but is not completely effective. The beneficial effects of Ca²⁺ channel blocker verapamil in combination with β-blocker have been reported, but the role of verapamil has not been well assessed. Because Ca²⁺ leakage through ryanodine channel is a common mechanism of CPVT, ryanodine channel block may have a therapeutic effect. We discovered that flecainide directly inhibits RyR2 channels and prevent CPVT. Left cardiac sympathetic denervation may be an effective alternative treatment in combination with ICD, especially for patients whose arrhythmias are not controlled by drug therapies.     

Cardiac-Specific Knockout of ET<Subscript>A</Subscript> Receptor Mitigates Paraquat-Induced Cardiac Contractile Dysfunction

Paraquat (1,1’-dimethyl-4-4’-bipyridinium dichloride), a highly toxic quaternary ammonium herbicide widely used in agriculture, exerts potent toxic prooxidant effects resulting in multi-organ failure including the lung and heart although the underlying mechanism remains elusive. Recent evidence suggests possible involvement of endothelin system in paraquat-induced acute lung injury. This study was designed to examine the role of endothelin receptor A (ET_A) in paraquat-induced cardiac contractile and mitochondrial injury. Wild-type (WT) and cardiac-specific ET_A receptor knockout mice were challenged to paraquat (45 mg/kg, i.p.) for 48 h prior to the assessment of echocardiographic, cardiomyocyte contractile and intracellular Ca²⁺ properties, as well as apoptosis and mitochondrial damage. Levels of the mitochondrial proteins for biogenesis and oxidative phosphorylation including UCP2, HSP90 and PGC1α were evaluated. Our results revealed that paraquat elicited cardiac enlargement, mechanical anomalies including compromised echocardiographic parameters (elevated left ventricular end-systolic and end-diastolic diameters as well as reduced factional shortening), suppressed cardiomyocyte contractile function, intracellular Ca²⁺ handling, overt apoptosis and mitochondrial damage. ET_A receptor knockout itself failed to affect myocardial function, apoptosis, mitochondrial integrity and mitochondrial protein expression. However, ET_A receptor knockout ablated or significantly attenuated paraquat-induced cardiac contractile and intracellular Ca²⁺ defect, apoptosis and mitochondrial damage. Taken together, these findings revealed that endothelin system in particular the ET_A receptor may be involved in paraquat-induced toxic myocardial contractile anomalies possibly related to apoptosis and mitochondrial damage.     

Differences in Ca-Handling and Sarcoplasmic Reticulum Ca-Content in Isolated Rat and Rabbit Myocardium

We made novel measurements of the influence of rest intervals and stimulation frequency on twitch contractions and on sarcoplasmic reticulum (SR) Ca²⁺-content (using rapid cooling contractures, RCCs) in isolated ventricular muscle strips from rat and rabbit hearts at a physiological temperature of 37 °C. In addition, the frequency-dependent relative contribution of SR Ca²⁺-uptake and Na⁺/Ca²⁺-exchange for cytosolic Ca²⁺-removal was assessed by paired RCCs. With increasing rest intervals (1–240 s) post-rest twitch force and RCC amplitude decreased monotonically in rabbit myocardium (after 240 s by 45±10% and 61±11%, respectively P<0.05, n=14). In contrast, rat myocardium (n=11) exhibited a parallel increase in post-rest twitch force (by 67±16% at 240 sP <0.05) and RCC amplitude (by 20±14%P<0.05). In rabbit myocardium (n=11), increasing stimulation frequency from 0.25 to 3 Hz increased twitch force by 295±50% (P<0.05) and RCC amplitude by 305±80% (P<0.05). In contrast, in rat myocardium (n=6), twitch force declined by 43±7% (P<0.05), while RCC amplitude decreased only insignificantly (by 16±7%). The SR Ca²⁺-uptake relative to Na⁺/Ca²⁺-exchange (based on paired RCCs) increased progressively with frequency in rabbit, but not in rat myocardium (66±2% at all frequencies). We conclude that increased SR Ca²⁺-load contributes to the positive force–frequency relationship in rabbits and post-rest potentiation of twitch force in rats. Decreased SR Ca²⁺-load contributes to post-rest decay of twitch force in rabbits, but may play only a minor role in the negative force–frequency relationship in rats. SR Ca²⁺-release channel refractoriness may contribute importantly to the negative force-frequency relationship in rat and recovery from refractoriness may contribute to post-rest potentiation.     

Experimental diabetes enhances Ca2+ mobilization and glutamate exocytosis in cerebral synaptosomes from mice

The present study was conducted to investigate the effects of the diabetic condition on the Ca²⁺ mobilization and glutamate release in cerebral nerve terminals (synaptosomes). Diabetes was induced in male mice by intraperitoneal injection of streptozotocin. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in synaptosomes were determined using fura-2 and enzyme-linked fluorometric assay, respectively. Diabetes significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. In addition, diabetes significantly enhanced K⁺- and 4-AP-evoked Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) inhibited K⁺- or 4-AP-induced increases in [Ca²⁺]_i and Ca²⁺-dependent glutamate release in synaptosomes from the control and diabetic mice to a similar extent, respectively. These results indicate that diabetes enhances a K⁺- or 4-AP-evoked Ca²⁺-dependent glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through both P- and N-type Ca²⁺ channels.     

Moderation of myocardial ischemia reperfusion injury by calcium channel and calmodulin receptor inhibition

Summary Intracellular Ca²⁺ accumulation is implicated in the pathogenesis of myocardial reperfusion injury. To study approaches designed to modify Ca²⁺ uptake during coronary revascularization after acute infarction, a pig heart surgical infarct model (left anterior descending artery occlusion for 60 min) was subjected to 60 min hypothermic potassium cardioplegic arrest, followed by 60 min of global reperfusion. Four groups of six hearts each were studied in a randomized manner, i.e., cardioplegia alone (control), cardioplegia + 10 µM diltiazem (Ca²⁺ slow channel blocker), cardioplegia + 10 µM trifluoperazine (TFP), (a Ca²⁺-calmodulin antagonist), and cardioplegia + diltiazem (10 µM) + TFP (10 µM). Left ventricular contractility (global and segmental), metabolism (coronary blood flow and O₂ consumption), and creatine kinase generation were measured during reperfusion. Both the Ca²⁺ channel blocker, diltiazem, and the calmodulin antagonist, TFP, improved myocardial global and regional function as well as myocardial metabolism. While diltiazem better restored global and regional contractility, trifluoperazine had a greater effect on coronary blood flow and myocardial oxygen consumption. Enzyme release and lipid peroxidation were equally moderated by both drugs. From this study it can be concluded that Ca²⁺ influx does play a role in ischemic and reperfusion injury. The mechanisms of its effect are complex, but can be successfully antagonized by Ca²⁺ blockers as well as by calmodulin antagonists, with improved myocardial preservation.Supported in part by grants NIH HL 22559 and HL 34360     

L-type calcium currents in atrial myocytes from patients with persistent and non-persistent atrial fibrillation

Objective. In patients with persistent atrial fibrillation (AF), the atrial myocardium is characterized by a reduced contractile force, by a shortened duration of the action potential and a recently demonstrated reduction of the L-type Ca²⁺ currents. We analyzed potential effects on L-type Ca²⁺ currents of the patients' medication and of the duration of AF. Methods and results. Human atrial myocytes were prepared from the right auricles of patients undergoing open-heart surgery. Three groups of patients were studied: a control group with sinus rhythm (SR, n = 26 patients) and a group with persistent AF (> 3 months duration; n = 10), a group with non-persistent AF (3 patients with SR but with documented episodes of AF in their history). L-type Ca²⁺ currents were measured during depolarizing pulses from a holding potential of −70mV to a test potential of +10mV and are given as mean ±SEM of current densities (currents normalized to the cell capacitance). Ca²⁺ current densities were significantly (p < 0.0001) smaller in cells from patients with persistent AF than in control cells (0.54 ± 0.08 pA/pF vs. 1.96 ± 0.12 pA/pF). No indication was found that these changes were caused by medication with Ca²⁺ channel antagonists, β blockers, or digitalis. Stimulation with the dihydropyridine Bay K 8644 (1 μM) or with isoproterenol (0.1 μM) increased Ca²⁺ currents in control cells 3.5 ± 0.2 and 3.5 ± 0.3-fold. In persistent AF, this increase was significantly larger (6.0 ± 0.5 and 5.2 ± 0.6-fold) but stimulated currents were still significantly lower than in control cells. Patients with non-persistent AF exhibited Ca²⁺ currents well within the control range. Conclusion. A reduction in Ca²⁺ currents, due to a reduction in number as well as a depression of L-type channels, is a characteristic and pathophysiologically important part of the myocardial remodeling during long-lasting atrial fibrillation. It is not present in patients with non-persistent AF and not caused by medication. Received: 27 September 2000, Returned for revision: 9 October 2000, Revision received: 8 November 2000, Accepted: 9 November 2000     

The effects of Ca-free perfusion and the calcium paradox on [I] endothelin-1 binding to rat cardiac membranes

The binding characteristics of [¹²⁵I]endothelin-1 (ET-1) to cardiac membranes isolated from rat hearts subjected to Ca²⁺-free perfusion or the Ca²⁺ paradox were examined. The effect of treatment with 2, 3 butanedione monoxime (BDM), which inhibits the tissue damage associated with the calcium paradox, was also investigated. Membranes from rat hearts perfused under control conditions bound [¹²⁵I]ET-1 to a single population of sites with a B_max of 107·7 ± 3.7 fmol/mg protein and an affinity (K_D) of 153 ± 12 pM. Ten minutes of Ca²⁺-free perfusion resulted in a significant (P < 0.01) increase in B_max to 167.5 ± 8.3 fmol/mg protein without change in K_D. Ca²⁺ repletion following Ca²⁺-free perfusion tended to increase further the B_max (180.6 ± 10.4 fmol/mg protein) without change in K_D. Treatment with BDM attenuated but did not prevent the rise in B_max following Ca²⁺-free perfusion. Following Ca²⁺ repletion, however, B_max returned to control levels in the BDM treated group. These changes were not associated with changes in the ability of ET-1 and ET-3 to inhibit [¹²⁵I]ET-1 binding. The results demonstrate that Ca²⁺-free perfusion is associated with an increase in the binding site density of [¹²⁵I]ET-1 which is maintained or further increased upon Ca²⁺ repletion. If, however, the tissue damage associated with the Ca²⁺ paradox is prevented with BDM, Ca²⁺ repletion is associated with a reversal of the increase due to Ca²⁺-free perfusion.