The antiarrhythmic actions of bisaramil and penticainide result from mixed cardiac ion channel blockade.

Decades of focus on selective ion channel blockade has been dismissed as an effective approach to antiarrhythmic drug development. In that context many older antiarrhythmic drugs lacking ion channel selectivity may serve as tools to explore mixed ion channel blockade producing antiarrhythmic activity. This study investigated the non-clinical electrophysiological and antiarrhythmic actions of bisaramil and penticainide using in vitro and in vivo methods. In isolated cardiac myocytes both drugs directly block sodium currents with IC50 values of 13µM (bisaramil) and 60µM (penticainide). Both drugs reduced heart rate but prolonged the P-R, QRS and Q-T intervals of the ECG (due to sodium and potassium channel blockade) in intact rats. They reduced cardiac conduction velocity in isolated rat hearts, increased the threshold currents for capture and fibrillation (indices of sodium channel blockade) and reduced the maximum following frequency as well as prolonged the effective refractory period (indices of potassium channel blockade) of electrically stimulated rat hearts. Both drugs reduced ventricular arrhythmias and eliminated mortality due to VF in ischemic rat hearts. The index of cardiac electrophysiological balance (iCEB) did not change significantly over the dose range evaluated; however, different drug effects resulted when changes in BP and HR were considered. While bisaramil is a more potent sodium channel blocker compared to penticainide, both produce a spectrum of activity against ventricular arrhythmias due to mixed cardiac ion channel blockade. Antiarrhythmic drugs exhibiting mixed ion channel blockade may serve as tools for development of safer mixed ion channel blocking antiarrhythmic drugs.

Pharmacological and toxicological activity of RSD921, a novel sodium channel blocker.

BACKGROUND: RSD921, the R,R enantiomer of the kappa (k) agonist PD117,302, lacks significant activity on opioid receptors. METHODS: The pharmacological and toxicological actions were studied with reference to cardiovascular, cardiac, antiarrhythmic, toxic and local anaesthetic activity. RESULTS: In rats, dogs and baboons, RSD921 dose-dependently reduced blood pressure and heart rate. In a manner consistent with sodium channel blockade it prolonged the PR and QRS intervals of the ECG. Furthermore, in rats and NHP, RSD921 increased the threshold currents for induction of extra-systoles and ventricular fibrillation (VFt), and prolonged effective refractory period (ERP). In rats, RSD921 was protective against arrhythmias induced by electrical stimulation and coronary artery occlusion. Application of RSD921 to voltage-clamped rat cardiac myocytes blocked sodium currents. RSD921 also blocked transient (ito) and sustained (IKsus) outward potassium currents, albeit with reduced potency relative to sodium current blockade. Sodium channel blockade due to RSD921 in myocytes and isolated hearts was enhanced under ischaemic conditions (low pH and high extracellular potassium concentration). When tested on the cardiac, neuronal and skeletal muscle forms of sodium channels expressed in Xenopus laevis oocytes, RSD921 produced equipotent tonic block of sodium currents, enhanced channel block at reduced pH (6.4) and marked use-dependent block of the cardiac isoform. RSD921 had limited but quantifiable effects in subacute toxicology studies in rats and dogs. Pharmacokinetic analyses were performed in baboons. Plasma concentrations producing cardiac actions in vivo after intravenous administration of RSD921 were similar to the concentrations effective in the in vitro assays utilized. CONCLUSIONS: RSD921 primarily blocks sodium currents, and possesses antiarrhythmic and local anaesthetic activity.

Ventricular arrhythmia incidence in the rat is reduced by naloxone.

This study characterized the antiarrhythmic effects of the opioid receptor antagonist naloxone in rats subject to electrically induced and ischemic arrhythmias. Naloxone (2, 8 and 32 µmol/kg/min) was examined on heart rate, blood pressure, and the electrocardiogram (EKG) as well as for effectiveness against arrhythmias produced by occlusion of the left anterior descending coronary artery or electrical stimulation of the left ventricle. Naloxone reduced blood pressure at the highest dose tested while heart rate was dose-dependently reduced. Naloxone dose-dependently prolonged the P-R and QRS intervals and increased the RSh amplitude indicative of effects on cardiac sodium (Na) channels. Naloxone prolonged the Q-T interval suggesting a delay in repolarization. Naloxone effects were comparable to the comparator quinidine. Naloxone (32 µmol/kg/min) reduced ventricular fibrillation (VF) incidence to 38% (from 100% in controls). This same dose significantly increased the threshold for induction of ventricular fibrillation (VFt), prolonged the effective refractory period (ERP) and reduced the maximal following frequency (MFF). The patterns of ECG changes, reduction in ischemic arrhythmia (VF) incidence and changes in electrically induced arrhythmia parameters at high doses of naloxone suggest that it directly blocks cardiac Na and potassium (K) ion channels.

In vitro developmental potential of macaque oocytes, derived from unstimulated ovaries, following maturation in the presence of glutathione ethyl ester.

BACKGROUND: The inadequacies of oocyte in vitro maturation (IVM) systems for both non-human primates and humans are evidenced by reduced fertilization and poor embryonic development, and may be partly explained by significantly lower glutathione (GSH) contents compared with in vivo matured (IVO) oocytes. As this influence has not been fully explored, this study investigated the effect of the GSH donor, glutathione ethyl ester (GSH-OEt), on the IVM and development of macaque oocytes as a model of human oocyte IVM. METHODS: Macaque oocytes derived from unstimulated ovaries were cultured in mCMRL-1066 alone or supplemented with 3 or 5 mM GSH-OEt. In vitro matured oocytes were subjected to the GSH assay, fixed for the assessment of spindle morphology or prepared ICSI. Embryo development of zygotes cultured in mHECM-9 was assessed up to Day 9 post-ICSI. RESULTS Supplementation of the maturation medium with GSH-OEt significantly increased oocyte maturation and normal fertilization rates compared with control oocytes, but only 5 mM GSH-OEt significantly increased the oocyte and cumulus cell GSH content. Confocal microscopy revealed significant differences in the spindle morphology between IVO and control in vitro matured metaphase II oocytes. Oocytes matured with 5 mM GSH-OEt exhibited spindle area and spindle pole width similar to that seen in the IVO oocyte. While no significant differences were observed in blastocyst rates, addition of 3 mM GSH-OEt during IVM significantly increased the proportion of embryos developing to the 5-8 cell stage while 5 mM GSH-OEt significantly increased the proportion of morula-stage embryos compared with controls. CONCLUSIONS: Supplementation of the IVM medium with GSH-OEt promotes better maturation and normal fertilization of macaque oocytes compared with non-supplemented medium. However, further improvement of the primate oocyte IVM culture system is required to support better blastocyst development of oocytes derived from unstimulated ovaries.

Oocyte glutathione and fertilisation outcome of Macaca nemestrina and Macaca fascicularis in in vivo- and in vitro-matured oocytes.

Fertilisation and development of IVM non-human primate oocytes is limited compared with that of in vivo-matured (IVO) oocytes. The present study describes the IVM of macaque oocytes with reference to oocyte glutathione (GSH). Timing of maturation, comparison of IVM media and cysteamine (CYS) supplementation as a modulator of GSH were investigated. A significantly greater proportion of oocytes reached MII after 30 h compared with 24 h of IVM. Following insemination, IVM oocytes had a significantly lower incidence of normal fertilisation (i.e. 2PN = two pronuclei and at least one polar body) and a higher rate of abnormal fertilisation (1PN = one pronucleus and at least one polar body) compared with IVO oocytes. Immunofluorescence of 1PN zygotes identified incomplete sperm head decondensation and failure of male pronucleus formation as the principal cause of abnormal fertilisation in IVM oocytes. The IVO oocytes had significantly higher GSH content than IVM oocytes. Cumulus-denuded oocytes had significantly lower GSH following IVM compared with immature oocytes at collection. Cysteamine supplementation of the IVM medium significantly increased the GSH level of cumulus-intact oocytes and reduced the incidence of 1PN formation, but did not improve GSH levels of the denuded oocyte. Suboptimal GSH levels in macaque IVM oocytes may be related to reduced fertilisation outcomes.

Developmental potential of bovine oocytes following IVM in the presence of glutathione ethyl ester.

Glutathione (GSH) is synthesised during oocyte maturation and represents the oocyte's main non-enzymatic defence against oxidative stress. Inadequate defence against oxidative stress may be related to poor embryo quality and viability. In the present study, bovine oocytes were matured in vitro in the presence of GSH ethyl ester (GSH-OEt), a cell permeable GSH donor, and its effects on subsequent fertilisation and embryo development were assessed. GSH-OEt significantly increased the GSH content of IVM oocytes without affecting fertilisation or Day 3 cleavage rates. Maturation in the presence of GSH-OEt did not significantly increase the blastocyst rate compared with control oocytes. However, 5 mM GSH-OEt treatment resulted in significantly higher blastocyst total cell number. The GSH level of IVM oocytes was significantly decreased in the absence of cumulus cells and when cumulus-oocyte complexes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine oocytes and restored the rate of normal fertilisation, but not embryo development, to levels seen in control oocytes. Thus, GSH-OEt represents a novel approach for effective in vitro elevation of bovine oocyte GSH and improvement in blastocyst cell number.

Bovine in vitro oocyte maturation as a model for manipulation of the gamma-glutamyl cycle and intraoocyte glutathione.

Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.

Implantation and pregnancy following in vitro fertilization and the effect of recombinant human relaxin administration in Macaca fascicularis.

Implantation and early pregnancy, and the potential effects of the reproductive-hormone relaxin, were examined in the cynomolgus macaque (Macaca fascicularis) following in vitro fertilization and embryo transfer. Mature oocytes were collected from regularly cycling, female cynomolgus monkeys subjected to ovarian superovulation using recombinant human FSH and hCG. Oocytes fertilized in vitro were cultured to the 4- to 8-cell stage, slow-cooled, and stored in liquid nitrogen before thawing and embryo transfer. Regularly cycling recipients were administered recombinant human relaxin or vehicle for 21 days through the peri-implantation period (Day 0 = pump implantation), during which time the thawed embryos were transferred (Day 7). Endometrial thickness and the number of gestational sacs were monitored by ultrasound at three time points (Days 7, 21, and 28). The number of days of placental sign (implantation bleeding) in pregnant females and menses in nonpregnant females were also recorded. Implantation (gestational sacs/embryo transferred) and multiple pregnancy (multiple gestations/ pregnant recipient) rates were slightly higher in relaxin-treated recipients compared to vehicle-treated recipients. Administration of relaxin was associated with increased implantation bleeding in pregnant females. Endometrial thickness was increased in relaxin-treated recipients at Days 7 and 28 compared to Day 0, but these differences were not observed at the same time points in vehicle-treated females. Systemic administration of recombinant human relaxin in an in vitro fertilization/embryo transfer setting was associated with effects consistent with a role for this hormone in endometrial physiology in primates.

Adenosine receptors and cardiovascular disease: the adenosine-1 receptor (A1) and A1 selective ligands.

Adenosine has often been cited as a universal retaliatory metabolite against the destructive cellular mechanisms that are initiated during metabolic/oxidative stress. Despite this billing, clinical application of adenosine has been limited to rather specific cardiovascular indications (e.g., paroxysmal supraventricular tachycardia). At least four adenosine receptor subtypes mediate the physiologic effects of adenosine, and each receptor subtype has been implicated as a target for development of agonist- and antagonist-based therapies against a wide range of disorders (cardiac arrhythmias, asthma, renal failure, and inflammation). Yet, clinical application of receptor subtype selective ligands has been very limited. The lag in clinical development of subtype selective ligands has largely been due lack of an X-ray resolved receptor structure and concomitant production of very selective agonists and antagonists. Species and tissue differences in ligand selectivity, receptor-effector coupling, and intracellular signaling also frustrate efforts in developing subtype selective therapies despite a great deal of amino acid homology across the receptor subtypes. The adenosine subtype-1 receptor (A1) has been particularly well studied in a variety of cardiovascular pathologies and a number of selective ligands for the receptor have been developed. A1 selective ligands acting as full agonists (CVT-510) or partial agonists (CVT-2759), antagonists (BG9719/CVT-124) and allosteric enhancers (PD81723) are now under preclinical scrutiny or are being developed for the clinical application in a variety of cardiovascular disorders and will be discussed herein.

Semen collection from mice: electroejaculation.

The effects of device type (electrostimulator, function generator or computer-generated waveforms), waveform (square, triangle or sine wave), probe type (ring or strip) and anaesthetic compound (ketamine/xylazine combination or pentobarbitone sodium) were investigated on electroejaculation (EEJ) responses of C57B1 x CBA and C57Bl/6J mice. Ejaculates were analysed for total sperm count and motility variables using computer-assisted sperm analyses. Automated computer-generated waveforms delivered through a sound card were more effective and reproducible compared with waveforms generated by function generator and electrostimulator. Sine waves and triangle waves were found to be more effective in producing ejaculate than square waves. As an anaesthetic, pentobarbitone sodium tended to outperform ketamine/xylazine across waveforms and strains. Strip probes failed to produce any ejaculate regardless of the device or waveform employed. Sperm obtained by EEJ exhibited poor motility and C5B1/6J mice had lower motility variables than C57BI x CBA mice.

Comparison of slow- and rapid-cooling protocols for early-cleavage-stage Macaca fascicularis embryos.

Cryostorage of nonhuman primate embryos by time-consuming slow-cooling methods is often limited to early cleavage stages. Effective rapid-cooling methods have been developed for many species and represent valuable tools for laboratory- and field-based studies of nonhuman primate reproductive biology. However, few rapid-cooling protocols have been applied to nonhuman primate embryos in terms of comparing various developmental stages. Here we compare slow cooling vs. two- and three-step rapid cooling of two-, four-, and eight-cell Macaca fascicularis (Mf) embryos. Rapid cooling was conducted in open pulled straws (OPS) using cooling solutions containing reduced quantities of ethylene glycol (EG) and supplemented with either of two high-molecular-weight polymers, ficoll and dextran. The survival of the slow-cooled embryos, but not the rapid-cooled embryos, was independent of embryonic stage at cryostorage. Slow cooling was associated with greater cell survival (82%) post thaw compared to warming following rapid cooling (18-29%). Slow cooling resulted in a high proportion of embryo survival (18/20; 90%) and cleavage (15/18; 83%) post thaw. Rapid cooling resulted in significantly reduced percentages of embryo survival (26-32%) and embryo cleavage in culture (29-38%) after warming. Conventional slow cooling was more effective than the rapid-cooling protocols employed in this study for cryopreservation of early-cleavage-stage Mf embryos.

Sequential culture medium promotes the in vitro development of Macaca fascicularis embryos to blastocysts.

In vitro production of blastocyst stage embryos from Macaca fascicularis (Mf) has not previously been demonstrated without cell support. Historical data indicates that a large proportion of Mf embryos arrest at the morula stage in nonsequential culture medium (NSM) lacking serum supplementation and/or cell support. Here we report the application of a sequential culture system supporting in vitro production of Mf blastocysts. Mf embryos produced by in vitro fertilization (IVF; n = 69) were subjected to in vitro culture without cell support in either a commercial sequential embryo culture medium (SM) or an NSM. At 24 hr post-insemination (PI) embryos generated from in vivo and in vitro matured oocytes and cultured in the NSM cleaved to two or more cells in significantly greater proportions (15/23; 65%) compared to embryos cultured in SM (14/46; 30%). However, by day 3 PI embryo development beyond eight cells was not different in NSM (9/23; 39%) compared to SM (25/46; 54%). At day 5 PI embryo development to the morula stage was slightly lower in NSM (8/23, 35%) compared to SM (21/46, 45%), and embryo degeneration was slightly higher in NSM (9/23, 39%) compared to SM (9/46, 20%). After 7-9 days of in vitro culture, embryo development to the blastocyst stage and embryo degeneration were significantly lower and higher, respectively, in NSM (0/23, 0%; and 23/23, 100%) compared to SM (9/46, 20%; and 26/46, 56%). In this study the sequential culture system was better able to support in vitro development of Mf embryos compared to nonsequential culture systems.

The effects of 5HT(1) agonists on erection in rats in vivo and rabbit corpus cavernosum in vitro.

We have examined the effects of the selective 5-HT(1a) and 5-HT(1b) agonists 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) and 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline (CGS12066b), respectively on erection in rats in vivo and rabbit corpus cavernosum in vitro. Apomorphine (0.1 mg/kg) induced 3.1+/-0.4 erections in vehicle-pretreated animals. At the highest doses tested 8-OHDPAT (0.4-0.64 mg/kg) and CGS12066b (1.0-10.0 mg/kg) significantly reduced apomorphine erection to 0.9+/-0.3 erections and 0.5+/-0.2 erections respectively. The nonselective 5-HT agonist metachlorophenylpiperazine (m-CPP; 0.1 mg/kg) elicited characteristic increases in cavernous nerve activity (CNA) and intracavernous pressure responses (ICP) in anesthetized rats. 8-OHDPAT (0.64 mg/kg) and CGS12066b (1.0 mg/kg) failed to elicit CNA or ICP responses. CGS12066b reduced ICP responses resulting from the direct stimulation of the cavernous nerve whereas 8-OHDPAT did not. CGS12066b reduced the CNA and ICP responses to m-CPP administration whereas 8-OHDPAT potentiated m-CPP induced CNA and ICP responses. In isolated rabbit corpus cavernosum (CC) 8-OHDPAT and CGS12066b both failed to alter noradrenergic induced contraction and non-adrenergic non-cholinergic relaxation. Our results indicate that selective 5-HT(1a) and 5-HT(1b) agonists have different effects in different models of erection.

Metachlorophenylpiperazine (m-CPP) induced intracavernous pressure responses in anaesthetized rats.

Here we have recorded the effects of metachlorophenylpiperazine (m-CPP) on intracavernous pressure (ICP) in anesthetized rats pretreated with various pharmacological agents in an attempt to determine the mechanism and relevance of the m-CPP induced ICP response to other models of erection. m-CPP elicited consistent and significantly greater increases in ICP (71.5+/-6.6 mmHg) compared with the mixed 5-HT(2a/2c) agonists trifluoromethylphenylpiperazine (3.4+/-1.3 mmHg) and quipazine (10.9+/-1.8 mmHg). Blockade of 5-HT(2a) receptors with ketanserin failed to unmask any stimulatory effect of quipazine (7.2+/-1.0 mmHg). m-CPP induced ICP responses (71+/-7.0 mmHg) were unaffected in the presence of mianserin (63+/-5 mmHg) and ketanserin (51+/-12 mmHg). Spiperone significantly reduced the m-CPP induced increase in ICP (8.0+/-1.0 mmHg). Naloxone, yohimbine and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) failed to elicit increases in ICP on their own. All three drugs significantly reduced the latency to the first m-CPP induced ICP response compared to saline. Yohimbine increased the duration of m-CPP induced ICP responses whereas 8-OHDPAT increased the mean number of m-CPP induced ICP responses compared to saline. The effects of m-CPP on ICP in anesthetized rats may not be mediated by 5-HT(2c) receptors and appears to be similar to erection in copula, but not erection elicited by other drugs or penile sheath retraction.

Cryopreservation of epididymal spermatozoa collected by needle biopsy from cynomolgus monkeys (Macaca fascicularis).

We have examined the motility, morphology, and cryopreservation of epididymal spermatozoa collected by needle biopsy from cynomolgus monkeys (Macaca fascicularis). At collection, epididymal sperm (23 x 10(6) +/- 4 x 10(6) sperm/sample; 611 x 10(6) +/- 116 x 10(6) sperm/ ml; n = 18) were alive (79 +/- 2%), motile (67 +/- 2%), and exhibited intact membranes (65 +/- 2%). Sperm maintained at room temperature in handling medium exhibited decreased motility over time, but head-to-head agglutination was limited. Tris egg-yolk extender containing 6% glycerol and dimethylsulfoxide (DMSO) did not significantly affect functional morphology, whereas extender containing propanediol significantly reduced motility, survival, and membrane integrity. Cryostorage reduced all measures of functional morphology independent of cryoprotectant. Post-thaw motility was superior for glycerol and DMSO compared to propanediol. Variation in glycerol concentration (4, 6, and 8%) produced equivocal effects on sperm functional morphology post-thaw. Needle biopsy may be a useful technique for laboratory and field-based collection of spermatozoa from nonhuman primates.

Ischaemia selectivity confers efficacy for suppression of ischaemia-induced arrhythmias in rats.

Eight novel and three reference antiarrhythmics were investigated in anaesthetised rats for antiarrhythmic actions, as well as for effects on the electrocardiogram (ECG) under normal and "simulated ischaemic" conditions. In rats subjected to coronary artery occlusion lidocaine, (+/-)-trans-[2-(4-morpholinyl)-cyclohexyl]naphthyl-1-acetate, RSD1000 and (+/-)-trans-[2-(4-morpholinyl)-cyclohexyl]-2-(1-naphthyl)propionate, RSD1030, (Group A) produced dose-related and complete antiarrhythmic protection. Group B compounds, such as (+/-)-trans-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-3, 4-dichlorocinnamamide, RSD995, produced complete antiarrhythmic protection but had aberrant dose-response curves. Group C compounds, such as quinidine and flecainide, failed to give full antiarrhythmic protection and had shallow dose-response curves. The potency of Group A compounds, but not Group B or C compounds, for ECG actions indicative of Na(+) channel blockade (prolongation of PR and QRS intervals) were significantly increased under "simulated ischaemic" conditions ([K(+)] 10 mM and pH 6.4) in isolated rat hearts. Thus, compounds with ischaemia-selective actions provided superior protection against ischaemia-induced arrhythmias in rats.

5-HT4 receptors in isolated human corpus cavernosum?

The novel serotonin subtype-4 (5-HT4) receptor agonist, SC53116 (SC), produced a limited relaxation of noradrenaline (NA) pre-contracted human corpus cavernosum (CC) smooth muscle in vitro. This effect was not significantly attenuated by the 5-HT4 antagonist SDZ250557 (SDZ). In the presence of (+/-) pindolol (1 microM) and methysergide (1 microM), employed to mask 5-HT1 and beta-adrenergic, and 5-HT2 receptors respectively, SC failed to relax NA pre-contracted CC strips to a greater extent than saline. Functional cAMP dependent relaxation pathways were demonstrated by a significant reduction in NA induced tone by prostaglandin E1 (PGE1) and isopropylnoradrenaline (IPNA), the action of the latter compound was effectively eliminated in the presence of (+/-) pindolol. Relaxation of NA induced tone caused by the nitric oxide donor nitro-glycerine (NTG) was significant and similar in the absence and presence of the 5-HT and beta-adrenergic antagonists. The results of this present study indicate that human corporal smooth muscle does not contain 5-HT4 receptors and that, although compounds like SC act to relax non-vascular smooth muscle via cAMP dependent mechanisms, 5-HT4 receptor agonists may be expected to be of limited utility in triggering cAMP dependent relaxation responses in human CC.

Sodium channel-blocking properties of spiradoline, a kappa receptor agonist, are responsible for its antiarrhythmic action in the rat.

Spiradoline (U-62,066E), a selective kappa (kappa) receptor agonist, was examined for actions on the cardiovascular system and on myocardial ionic currents in rats. We initially characterized cardiac, hemodynamic, and antiarrhythmic actions of spiradoline in isolated perfused rat hearts and pentobarbital-anesthetized rats. Electrophysiologic studies in isolated myocytes were used to elucidate the mechanism for changes observed in vivo in the ECG, as well as for antiarrhythmic actions against electrical and ischemia-induced arrhythmias. In isolated rat hearts, spiradoline reduced heart rate and cardiac contractility and increased the PR interval and QRS width of the ECG in a concentration-dependent manner. In anesthetized rats, spiradoline dose-dependently reduced blood pressure and heart rate and prolonged the PR interval and QRS width. At slightly higher doses, it increased the QaT interval of the ECG. RSh, an index of sodium channel blockade in the rat, also was dose-dependently increased. Electrical stimulation of the left ventricle suggested that spiradoline may exert its antiarrhythmic action by blockade of myocardial sodium currents. The electrophysiologic actions of spiradoline on sodium currents, the transient outward (i(to)) and sustained plateau potassium (ik(sus)) currents were studied in isolated cardiac rat myocytes by whole-cell patch-clamp techniques. Spiradoline (15-500 microM) reduced peak sodium current in a rapid, reversible, and concentration-dependent manner; it also increased the rate of decay of I(to) and reduced the amplitude of Ik(sus). At a concentration of 150 microM, spiradoline produced a 24 +/- 2 mV hyperpolarizing shift in sodium current inactivation kinetics but did not alter activation processes. Spiradoline showed both tonic and frequency-dependent components of sodium current block. Thus spiradoline produced its antiarrhythmic actions via sodium channel blockade in myocardial tissue, although higher doses also block potassium currents. This combined ion channel-blocking property may be of added clinical benefit in the setting of myocardial ischemia.

Effects of halothane and isoflurane on rat ventricular action potentials recorded in situ.

The effects of halothane and isoflurane on ventricular intracellular action potentials recorded in situ in pentobarbital anesthetized rats were studied. Halothane (0.5, 1 and 2 vol.%) and isoflurane (0.75, 1.5 and 3 vol.%) did not have identical effects on rat epicardial action potentials recorded by floating microelectrodes. However, over the concentration range tested, both anesthetics reduced blood pressure and heart rate to a similar extent. Isoflurane did not effect the maximum rate of rise of the action potential amplitude. However, 3 vol.% isoflurane reduced the resting membrane potential from -72+/-2 to -65+/-3 mV (mean+/-SEM, p<0.05) while the highest concentration of halothane had no effect. Halothane (2 vol.%) reduced action potential amplitude from 74+/-4 to 65+/-3 mV (p<0.05) and reduced the maximum rise rate of action potential from 175+/-21 to 133+/-8 V/s (p<0.05). Both isoflurane and halothane prolonged action potential duration at 10, 25 and 50% repolarization while only halothane significantly shortened action potential duration at 75% repolarization, Thus the effects of halothane and isoflurane on ventricular transmembrane action potentials were similar, but not identical. The relevance of such observations to the antiarrhythmic actions of halothane, but not isoflurane in this species is not clear.

Lack of selectivity for ventricular and ischaemic tissue limits the antiarrhythmic actions of lidocaine, quinidine and flecainide against ischaemia-induced arrhythmias.

The antiarrhythmic effectiveness, electrocardiographic and haemodynamic properties of three representative class I antiarrhythmics have been investigated in anaesthetized rats. Quinidine, lidocaine and flecainide were chosen as representatives of class Ia, Ib and Ic, respectively. Lidocaine showed the greatest frequency and 'ischaemia' dependency and a high dose provided complete protection against ischaemic arrhythmias induced by coronary artery occlusion. Flecainide showed the least frequency and ischaemia dependency and the least antiarrhythmic effectiveness. Quinidine was only slightly more effective than flecainide. The three drugs were approximately equi-potent in lowering blood pressure which limited the maximum dose that could be tested. The highest dose of lidocaine also caused convulsions in conscious animals. Thus, while lidocaine had selectivity for ischaemic tissue, and for high frequencies, the central nervous system and cardiovascular toxicity limited its usefulness against ischaemia-induced arrhythmias. Quinidine and flecainide's lack of selectivity for ischaemia, and/or high frequencies, probably accounted for their limited antiarrhythmic actions against ischaemia-induced arrhythmias. This study emphasizes that class I drugs can only provide useful protection against ischaemia-induced arrhythmias if they have marked cardiac selectivity as well as selectivity for ischaemic cardiac tissue.