Guinea Pig Ventricular Myocardium Research Articles

Negative Inotropic Effects of Class I Antiarrhythmics on Guinea Pig Ventricular Myocardium: Correlation with L-Type Ca<sup>2+</sup> Channel Blockade

The negative inotropic effects of nine Vaughan Williams class I antiarrhythmic drugs were examined in guinea pig ventricular tissue preparations. The drugs decreased the contractile force of papillary muscles with different potencies: the potency order was propafenone > aprindine > cibenzoline > flecainide > ranolazine > disopyramide > pilsicainide > mexiletine > GS-458967. The potency of drugs correlated with the reported IC50 values to block the L-type Ca2+ channel rather than the Na+ channel. The effects of drugs were roughly the same when examined under a high extracellular K+ solution, which inactivates the Na+ channel. Furthermore, the attenuation of the extracellular Ca2+-induced positive inotropy was strong with propafenone, moderate with cibenzoline, and weak with pilsicainide. These results indicate that the negative inotropic effects of class I antiarrhythmic drugs can be largely explained by their blockade of the L-type Ca2+ channel.

Contribution of ATP-Mediated Positive Feedback to Sympathetic Nerve-Induced Positive Inotropy in Guinea Pig Ventricular Myocardium.

The functional role of ATP released from sympathetic nerve terminals was examined in isolated guinea pig ventricular papillary muscles. The contractile force of papillary muscles was increased by field electrical stimulation of sympathetic nerve endings. This increase was attenuated by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or suramin, blockers of the P2X receptor, and was abolished by propranolol and prazosin. PPADS, suramin, and ATP affected neither the basal contractile force nor the positive inotropic effect of noradrenaline. These results provide functional evidence that ATP released from sympathetic nerve terminals enhances noradrenaline release and contributes to sympathetic nerve-induced inotropy.

Late sodium current in human, canine and guinea pig ventricular myocardium

Although late sodium current (INa-late) has long been known to contribute to plateau formation of mammalian cardiac action potentials, lately it was considered as possible target for antiarrhythmic drugs. However, many aspects of this current are still poorly understood. The present work was designed to study the true profile of INa-late in canine and guinea pig ventricular cells and compare them to INa-late recorded in undiseased human hearts. INa-late was defined as a tetrodotoxin-sensitive current, recorded under action potential voltage clamp conditions using either canonic- or self-action potentials as command signals. Under action potential voltage clamp conditions the amplitude of canine and human INa-late monotonically decreased during the plateau (decrescendo-profile), in contrast to guinea pig, where its amplitude increased during the plateau (crescendo profile). The decrescendo-profile of canine INa-late could not be converted to a crescendo-morphology by application of ramp-like command voltages or command action potentials recorded from guinea pig cells. Conventional voltage clamp experiments revealed that the crescendo INa-late profile in guinea pig was due to the slower decay of INa-late in this species. When action potentials were recorded from multicellular ventricular preparations with sharp microelectrode, action potentials were shortened by tetrodotoxin, which effect was the largest in human, while smaller in canine, and the smallest in guinea pig preparations. It is concluded that important interspecies differences exist in the behavior of INa-late. At present canine myocytes seem to represent the best model of human ventricular cells regarding the properties of INa-late. These results should be taken into account when pharmacological studies with INa-late are interpreted and extrapolated to human. Accordingly, canine ventricular tissues or myocytes are suggested for pharmacological studies with INa-late inhibitors or modifiers. Incorporation of present data to human action potential models may yield a better understanding of the role of INa-late in action potential morphology, arrhythmogenesis, and intracellular calcium dynamics.

Resistance of Fetal Guinea Pig Ventricular Myocardium to Hypoxia: Maintained Intracellular ATP Prevents the Opening of ATP-Sensitive Potassium Channels

The presence and function of the ATP-sensitive potassium channel current (IKATP) were examined in the guinea pig myocardium to clarify the mechanisms for the resistance of the fetal myocardium to hypoxia. Experimental hypoxia markedly reduced the action potential duration and contractile force in isolated ventricular myocardium from the adult, but only moderately in those from the fetus. In isolated ventricular cardiomyocytes, the density of the IKATP activated by cromakalim, as well as their sensitivity to intracellular ATP concentration, were not different between the fetus and adult. The tissue ATP content was similar between the fetal and adult myocardium under normal condition, but the hypoxia-induced decrease was smaller in the fetus. Confocal microscopic analysis revealed that the mitochondria in the fetal cardiomyocyte is less in quantity than that in the adult and is more localized to the cell center. These results indicate that IKATP in the fetal guinea pig myocardium has a current density and ATP sensitivity similar to those of the adult, but is not activated under hypoxic conditions because the energy metabolism of the fetal myocardium is less dependent on oxidative phosphorylation.

Novel Storm-Based Quantitative Assessment of Relative Protein Localization Reveals New Role for Sodium Channel β1 Subunit in Cardiac Conduction

We recently demonstrated that cardiac sodium channels (Nav1.5) localized within gap junction (GJ) -adjacent microdomains within the intercalated disc (ID) may enable ephaptic coupling between cardiac myocytes. Further we demonstrated conduction slowing and elevated arrhythmia risk secondary to disruption of close membrane apposition (<10nm) within these microdomains. Confocal micrographs of guinea pig ventricular myocardium revealed expression of β1 (SCN1b), a sodium channel auxiliary subunit and cell adhesion molecule, throughout the interplicate regions of the ID. Therefore, we hypothesized that β1-mediated adhesion may be key to close membrane apposition within Nav1.5-rich ID microdomains and thereby, to ephaptic coupling. In order to assess β1-localization within the ID, we analyzed STORM images of guinea pig ventricular sections immunolabeled for β1 along with Cx43 and N-cadherin (N-Cad) using custom algorithms. Overall, 16±2% of Cx43 clusters overlapped with a β1 cluster while 30±3% had a β1 cluster <200nm away, with a median distance of 55±6nm between Cx43 and β1. In contrast, only 7±3% of N-Cad overlapped with β1 while 16±2% had β1 <200nm away (p<0.05 vs. Cx43-β1), with a median distance of 154±98nm between N-Cad and β1. These data suggest preferential β1 localization adjacent Cx43 GJ relative (interplicate ID regions) to N-cadherin (plicate ID regions). Next we examined β1-mediated adhesion using electric cell-substrate impedance spectroscopy (ECIS): 1610 cells heterologously overexpressing β1 revealed 3-fold higher barrier function relative to native 1610 cells. Further, βadp1, a peptide mimetic of the β1 adhesion domain, inhibited barrier function in dose dependent fashion in β1-overexpressing 1610 cells but not in native 1610 cells. Taken together our results support a role for β1-mediated adhesion in modulating ephaptic coupling in the heart and highlight it as a potential target for novel anti-arrhythmic therapy.

Temporal response of ectopic activity in guinea pig ventricular myocardium in response to isoproterenol and acetylcholine.

Both β adrenergic and muscarinic receptor stimulation independently potentiate arrhythmogenesis. However, the effect of simultaneous stimulation on arrhythmogenesis is not well known. The purpose of this study was to determine the temporal response of arrhythmia risk to individual and combined autonomic agonists. Guinea pig hearts were excised and Langendorff-perfused. The β adrenergic receptor and muscarinic receptor agonists were isoproterenol (ISO, 0.6 μM) and acetylcholine (ACh, 10 μM), respectively. All measurements with agonists occurred over 21 min. ISO induced ectopic activity for the first 8 min. ISO also transiently shortened and then prolonged R-R interval over a similar time course. ACh added after ISO transiently induced ectopic activity for 12 min, while R-R interval invariantly prolonged. ACh alone produced few ectopic beats, while invariantly prolonging R-R interval. In contrast to ISO alone, ISO following ACh significantly increased ectopic activity and shortened R-R interval for the duration of the experiment. Animals aged 17–19 months exhibited sustained arrhythmogenesis while those aged 11–14 did not. When ACh was removed in older hearts while ISO perfused, a transient increase in ectopic activity and decreased R-R interval was observed, similar to ISO alone. These data suggest that pre-treating with and maintaining ACh perfusion can sustain ISO sensitivity, in contrast to ISO perfusion alone.

Distinguishing between overdrive excited and suppressed ventricular beats in guinea pig ventricular myocardium.

Rapid ventricular pacing rates induces two types of beats following pacing cessation: recovery cycle length (RCL) prolongation (overdrive suppression) and RCL shortening (overdrive excitation). The goals of this study were to compare common experimental protocols for studying triggered activity in whole-heart preparations and differentiate between recovery beats using a new methodology. Post-pacing recovery beat cycle length (RCL) and QRS were normalized to pre-paced R-R and QRS intervals and analyzed using a K-means clustering algorithm. Control hearts only produced suppressed beats: RCL ratio increased with rapid pacing (25 ± 4.0%, n = 10) without changing QRS duration. Rapid pacing during hypercalcemia + hypothermia (5.5 mM and 34°C) produced significantly earlier excited beats (53 ± 14%, n = 5) with wider QRS durations (58 ± 6.3%, n = 5) than suppressed beats. Digoxin + hypothermia (0.75 μM) produced the most excited beats with significantly earlier RCL (44 ± 3.2%, n = 6) and wider QRS (60 ± 3.1%, n = 6) ratios relative to suppressed beats. Increasing pacing further shortened RCL (30 ± 7.8%, n = 6). In a prospective study, TTX (100 nM) increased RCL ratio (15 ± 6.0%, n = 10) without changing the QRS duration of excited beats. The algorithm was compared to a cross-correlation analysis with 93% sensitivity and 94% specificity. This ECG based algorithm distinguishes between triggered and automatic activity.

CONTROL OF GAP JUNCTION CONDUCTANCE BY CALCINEURIN-DEPENDENT CX-43 PHOSPHORYLATION: IMPLICATIONS OF ARRHYTHMOGENESIS

Slower action potential conduction, and consequent arrhythmias, follow decreased gap junction conductance, Gj. We investigated how the Ca2+-dependent phosphatase, calcineurin, regulates Gj through altering the phosphorylation of Cx43 protein, through...

Reverse rate-dependent changes are determined by baseline action potential duration in mammalian and human ventricular preparations

Class III antiarrhythmic agents exhibit reverse rate-dependent lengthening of the action potential duration (APD). In spite of the several theories developed so far to explain this reverse rate-dependency (RRD), its mechanism has not yet been clarified. The aim of the present work was to further elucidate the mechanisms responsible for RRD in mammalian ventricular myocardium. Action potentials were recorded using conventional sharp microelectrodes from human, canine, rabbit and guinea pig ventricular myocardium in a rate-dependent manner varying the cycle length (CL) between 0.3 and 5 s. Rate-dependent drug effects were studied using agents known to lengthen or shorten action potentials, and these drug-induced changes in APD were correlated with baseline APD values. Both drug-induced lengthening (by dofetilide, sotalol, E-4031, BaCl(2), veratrine, BAY K 8644) and shortening (by mexiletine, tetrodotoxin, lemakalim) of action potentials displayed RRD, i.e., changes in APD were greater at longer than at shorter CLs. In rabbit, where APD is a biphasic function of CL, the drug-induced APD changes were proportional to baseline APD values but not to CL. Similar results were obtained when repolarization was modified by injection of inward or outward current pulses in isolated canine cardiomyocytes. In each case the change in APD was proportional to baseline APD (i.e., that measured before the superfusion of drug or injection of current). Also, the net membrane current (I (net)), determined from the action potential waveform at the middle of the plateau, was inversely proportional to APD and consequently with to CL. The results indicate that RRD is a common characteristic of all the drugs tested regardless of the modified ion current species. Thus, drug-induced RRD can be considered as an intrinsic property of cardiac membranes based on the inverse relationship between I (net) and APD.

Effects of propofol on β-adrenoceptor-mediated signal transduction in cardiac muscle; role of cAMP

BackgroundPropofol may decrease myocardial contractility via actions on the β-adrenoceptor-mediated signal transduction. The aim of this study was to evaluate the effect of propofol via β-adrenoceptor-mediated signal transduction by measuring the tissue levels of cAMP (cyclic adenosine monophosphate).MethodsThe effects of propofol on β-adrenoceptor mediated cascades were measured with cAMP concentrations, which were stimulated by agonists (l-isoproterenol, GTPγS, and forskolin) of each step of β-adrenoceptor-mediated cascades.ResultsWhile the production of cAMP stimulated by isoproterenol, GTPγS, or forskolin are increased (P < 0.05), application of each concentration of propofol (0.1, 1, 10, 100 µM) did not alter the levels of cAMP.ConclusionsConsidering that propofol did not alter the tissue cAMP levels when stimulated by isoproterenol, GTPγS, and forskolin, propofol appears to have no effect on the β-adrenoceptor signaling pathway in guinea pig ventricular myocardium.

The Electrophysiological Effects of Racemic Ketamine and Etomidate in an In Vitro Model of “Border Zone” Between Normal and Ischemic/Reperfused Guinea Pig Myocardium

Etomidate and ketamine are used during induction of anesthesia in high-risk patients. However, their effects on action potential (AP) variables and ischemia/reperfusion-induced arrhythmias and conduction blocks are unknown. Guinea pig right ventricular muscle strips were mounted in a 5-mL double chamber bath with the strips separated into two zones by an impermeable latex membrane. One-half (normal zone) was exposed to normal perfusate while the other half (altered zone) was exposed to hypoxia, hyperkalemia, acidosis, and lack of glucose. AP variables were recorded continuously in the normal and altered zones. Spontaneous arrhythmias and conduction blocks were noted. Etomidate (10(-7), 10(-6), and 10(-5) M) and ketamine (10(-6), 10(-5), and 10(-4) M) were superfused into the bath throughout the experiment and the electrophysiologic effects compared with the control group. We found that under control conditions, etomidate and ketamine did not modify resting membrane potential, maximal upstroke velocity, AP amplitude, or AP duration at 90% of repolarization (APD90). Ketamine (10(-4) M), but not weaker concentrations and none of the concentration of etomidate, reversed the ischemia-induced shortening of APD90 and APD dispersion. Etomidate and ketamine did not modify the occurrence of conduction block during simulated ischemia. In contrast, ketamine (25% at 10(-6) M, 13% at 10(-5) M, and 13% at 10(-4) M vs 90% in the control group, P < 0.05) but not etomidate (38% at 10(-7) M, 63% at 10(-6) M, and 63% at 10(-5) M vs 90% in the control group, NS) decreased the incidence of reperfusion-induced spontaneous arrhythmias. In guinea pig myocardium, our data suggest that ketamine, in clinically relevant concentrations, decreases ischemia-induced AP shortening and spontaneous reperfusion-induced ventricular arrhythmias. Further study is required to precisely determine the effect of etomidate on reperfusion-induced arrhythmias.

Class III effects of dofetilide and arrhythmias are modulated by [K +] o in an in vitro model of simulated-ischemia and reperfusion in guinea-pig ventricular myocardium

To evaluate class III effects of clinically relevant concentrations of dofetilide (5 and 10 nmol/l) and the effects of extracellular potassium [K +] o modulation of arrhythmias onset at the level of the “border zone,” we used a previously reported in vitro model whereby normoxic and ischemic/reperfused zones were studied. Guinea-pig right ventricular strips (driven at 1 Hz at 36.5 ± 0.5 °C) were superfused with Tyrode's solution in oxygenated (HCO 3 − 25 mmol/l, K + 4 mmol/l, pH 7.35 ± 0.05, glucose 5.5 mmol/l: normal zone) and ischemia-simulating conditions (HCO 3 − 9 mmol/l, pH 6.90 ± 0.05, no oxygen and no glucose: altered zone) having either [K +] o 4 ( n = 20), 8 ( n = 20) or 12 ( n = 20) mmol/l. Action potentials in normal and altered zones were recorded simultaneously during 30 min of simulated-ischemia and after 30 min of reperfusion with oxygenated Tyrode's solution. Each preparation served as control for successive phases of dofetilide studies (at 5 and 10 nmol/l) and action potential values were normalized to those present at the beginning of the experiment. During simulated-ischemia, the higher the [K +] o the worse were action potential changes, although full recovery was seen upon 30 min of reperfusion in all [K +] o groups. A high incidence of ischemia/reperfusion arrhythmias was observed in 4 and 12 mmol/l [K +] o groups as opposed to a low incidence of arrhythmias in 8 mmol/l [K +] o group. Dofetilide at 5 and 10 nmol/l with all [K +] o explored: (i) exhibited class III effects, (ii) was effective (or neutral) against ventricular arrhythmias during both simulated-ischemia and reperfusion, and (iii) did not globally increase the dispersion of action potential durations between normal and altered zones. Different arrhythmogenic mechanisms are involved in this model at different [K +] o with 8 mmol/l providing relative protection. Class III effects of dofetilide are evident in the normal zone when in the ischemic-like zone [K +] o ranges from 4 to 12 mmol/l. Thus dofetilide did not increase dispersion of repolarization and had either an antiarrhythmic or a neutral effect during ischemia/reperfusion.

Electrophysiological effects of azimilide in an in vitro model of simulated-ischemia and reperfusion in guinea-pig ventricular myocardium

There are few investigations on azimilide effects during ischemia/reperfusion. We have therefore investigated low concentrations of azimilide (0.1 and 0.5 μmol/l) versus Controls on action potential parameters and occurrence of repetitive responses during simulated ischemia and reperfusion. An in vitro model of “border zone” in guinea-pig ventricular myocardium (n=30) was used. Azimilide 0.5 μmol/l lengthened action potential duration in normoxic but not in ischemic-like conditions. Therefore an increased dispersion of action potential duration at 90% of repolarization during simulated ischemia in presence of azimilide was seen. Upon reperfusion, both normal and reperfused myocardium showed azimilide-induced action potential duration increase. There was a neutral effect on the occurrence of arrhythmias during simulated ischemia; however azimilide showed significant (P=0.033) antiarrhythmic properties following reperfusion. To mimic IKr and IKs blocking properties of azimilide we further used dofetilide 10 nmol/l with HMR 1556 1 nmol/l (N=9), which was accompanied by less severe shortening (P<0.05) of action potential duration at 90% of repolarization at 30 min of ischemic-like conditions (−43±9%), as compared with azimilide 0.5 μmol/l (−64±5%) but similar to what seen with azimilide 0.1 μmol/l (−53±5%) and Controls (−52±6%). During reperfusion, 2/9 (22%) preparations had sustained activities, which was less than what observed in Controls (5/10, 50%) and with azimilide 0.5 μmol/l (0/10, 0%), although not statistically different (respectively, P=0.35 and P=0.21). Lack versus homogenous class III effects of azimilide in respectively simulated ischemia and reperfusion may explain its different efficacy on arrhythmias, although prevention of reperfusion arrhythmias calls for other than just its IKr and IKs blocking properties.

Propofol Decreases Reperfusion-Induced Arrhythmias in a Model of ???Border Zone??? Between Normal and Ischemic-Reperfused Guinea Pig Myocardium

We examined the effect of propofol on the main mechanisms involved in ischemia/reperfusion-induced arrhythmias (i.e., spontaneous arrhythmias, conduction blocks, and dispersion of repolarization) in vitro. In a double-chamber bath, guinea pig right ventricular muscle strips were subjected to 30 min of simulated ischemia followed by 30 min of reperfusion (altered zone; AZ) and to standard conditions (normal zone; NZ). Action potential (AP) parameters were recorded in the NZ and AZ. We studied the effects of Intralipid(R) and of propofol at 10(-6), 10(-5), and 2 x 10(-5) M on the occurrence of spontaneous sustained arrhythmias, conduction blocks, and the dispersion of repolarization. In NZ, Intralipid and propofol did not significantly modify the AP parameters. Propofol, but not Intralipid, lessened the ischemia-induced decrease in AP duration (APD) at 90% of repolarization (APD(90)) and attenuated the APD dispersion around the "border zone." Propofol did not modify the occurrence of ischemia-induced arrhythmias. Propofol 10(-6) M, but not Intralipid or propofol at 10(-5) and 2 x 10(-5) M, decreased the occurrence of ischemia-induced conduction blocks. Propofol decreased the occurrence of reperfusion-induced spontaneous sustained arrhythmias. We conclude that, in vitro, propofol attenuated the ischemia-induced APD(90) dispersion around the "border zone" and decreased the occurrence of spontaneous arrhythmias related to myocardial reperfusion injury. In isolated guinea pig ventricular myocardium propofol, but not Intralipid(R), attenuated the ischemia-induced shortening of action potential and, thus, the dispersion of repolarization and decreased the occurrence of spontaneous ventricular arrhythmia related to reperfusion injury. This result may be important for propofol-based anesthesia in patients at high risk for intraoperative ischemia.

Pharmacological, Pharmacokinetic and Clinical Properties of Olopatadine Hydrochloride, a New Antiallergic Drug

Olopatadine hydrochloride (olopatadine, 11-[(Z)-3-(dimethylamino)propylidene]-6,11-dihydrodibenz[b,e]oxepin-2-acetic acid monohydrochloride) is a novel antiallergic/histamine H1-receptor antagonistic drug that was synthesized and evaluated in our laboratories. Oral administration of olopatadine at doses of 0.03 mg/kg or higher inhibited the symptoms of experimental allergic skin responses, rhinoconjunctivitis and bronchial asthma in sensitized guinea pigs and rats. Olopatadine is a selective histamine H1-receptor antagonist possessing inhibitory effects on the release of inflammatory lipid mediators such as leukotriene and thromboxane from human polymorphonuclear leukocytes and eosinophils. Olopatadine also inhibited the tachykininergic contraction in the guinea pig bronchi by prejunctional inhibition of peripheral sensory nerves. Olopatadine exerted no significant effects on action potential duration in isolated guinea pig ventricular myocytes, myocardium and human ether-a-go-go-related gene channel. Olopatadine was highly and rapidly absorbed in healthy human volunteers. The urinary excretion of olopatadine accounted for not less than 58% and the contribution of metabolism was considerably low in the clearance of olopatadine in humans. Olopatadine is one of the few renal clearance drugs in antiallergic drugs. Olopatadine was shown to be useful for the treatment of allergic rhinitis and chronic urticaria in double-blind clinical trials. Olopatadine was approved in Japan for the treatment of allergic rhinitis, chronic urticaria, eczema dermatitis, prurigo, pruritus cutaneous, psoriasis vulgaris and erythema exsudativum multiforme in December, 2000. Ophthalmic solution of olopatadine was also approved in the United States for the treatment of seasonal allergic conjunctivitis in December, 1996 (Appendix: also in the European Union, it was approved in February 2002).

Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels during reoxygenation in guinea-pig ventricular myocytes.

ATP-sensitive K+ (KATP) channels activated by glucose-free anoxia close immediately upon reoxygenation in single guinea-pig ventricular myocytes, while KATP channels open persistently during reperfusion in coronary-perfused guinea-pig ventricular myocardium. To investigate the reasons behind this discrepancy, we investigated whether protein kinase C (PKC) modulates the opening of KATP channels during anoxia-reoxygenation and ischaemia-reperfusion. Exposure of guinea-pig ventricular cells to glucose-free anoxia shortened the action potential duration at 90% repolarisation (APD90) and evoked the glibenclamide-sensitive robust outward current (IK,ATP). Subsequent reoxygenation caused an immediate prolongation of APD90 and a decrease in IK,ATP within approximately 20 s. When the novel (Ca2+-independent) PKC was activated by applying 1,2-dioctanoyl-sn-glycerol (1,2DOG, 20 M) with EGTA (20 mM) in the pipette, the APD90 restored gradually after reoxygenation and the extent of recovery was appoximately 80% of the pre-anoxic value. Moreover, IK,ATP decreased slowly and remained opened for up to approximately 4 min after reoxygenation. These results suggest persistent opening of KATP channels during reoxygenation. The persistent activation of KATP channels was augmented when both novel and conventional (Ca2+-dependent) isoforms of PKC were activated by applying 1,2DOG without EGTA in the pipette. In coronary-perfused right ventricular myocardium, APD90 remained shortened for up to approximately 30 min of reperfusion. The gradual restoration of APD90 after ischaemia-reperfusion was facilitated by the KATP channel blocker glibenclamide and by the potent PKC inhibitor chelerythrine. Our results provide the first evidence that PKC activation contributes to the persistent opening of KATP channels during reoxygenation and reperfusion. We also conclude that both novel and conventional PKC isoforms co-operatively modulate the opening of KATP channels during the early phase of reoxygenation.

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

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

Pharmacological properties of cardiovascular histamine H1 receptor binding sites: characterisation with 2-phenylhistamines.

We determined and compared the molecular properties of histamine H1 receptor binding sites in bovine thoracic aorta smooth muscle and guinea pig myocardial membranes from ventricles with saturation and inhibition binding assay, using 3H-mepyramine to label the receptor and specific and selective H1 receptor agonists of the 2-phenylhistamine group as displacers of specific 3H-mepyramine binding. 3H-mepyramine binds in a saturable manner to a homogenous population of binding sites with a K(D) of 5.6 nM and a Bmax of 57 fmol/mg of protein in bovine aorta vascular smooth muscle membranes, whereas in the guinea pig myocardium high and low affinity 3H-mepyramine binding sites exist having the following molecular characteristics: a K(D) of 1.6 nM and a Bmax of 99 fmol/mg of protein (high affinity site) and a K(D) 15.0 nM and a Bmax of 466 fmol/mg of protein (low affinity site). Halogenated 2-phenylhistamines: 2-(3-fluoro-) (2-FPH), 2-(3-trifluoromethyl-) (2-triFMPH), 2-(3-chloro-) (2-CPH), 2-(3-bromo-) (2-BPH) and 2-(3-iodophenyl)histamine (2-IPH), which showed high selectivity and potency for H1 receptors in the functional pharmacological studies, were potent inhibitors of specific radioligand binding in comparison with histamine and parent nonhalogenated 2-phenylhistamine (2-PH). Their rank order of potencies and affinities differ significantly for the vascular and cardiac H1 receptor binding sites: Specific 3H-mepyramine binding to H1 receptors in bovine vascular smooth muscle membranes was displaced in a biphasic manner by 2-(3-fluoro-), 2-(3-trifluoromethyl-), 2-(3-chloro-), 2-(3-bromo-), 2-(3-iodophenyl)histamine and histamine. In guinea pig ventricular myocardium the rank order was 2-(3-iodo-), 2-(3-bromo-), histamine, 2-(3-chloro-), and 2-(3-fluorophenyl)histamine showing better correlation with the lipophilicity of the derivatives than in vascular tissue (order of lipophilicity: 2-triFMPH >2-IPH >2-BPH >2-CPH >2-FPH >>2-PH). Displacement of the radioligand binding to myocardial H1 receptor by the above drugs is (except for 2-(3-fluorophenyl)histamine), better fitted to a two-site model. 2-phenylhistamine, which acted as a moderate agonist in functional studies, displaced the radioligand in a monophasic manner and was the weakest displacer of specific radioligand binding in both model systems (pKi = 5.76--vascular and 5.57--cardiac tissue). The agonistic nature of the halogenated 2-phenylhistamine derivatives was confirmed on the molecular level, since their interaction with the H1 receptor is regulated by guanine nucleotides. GTP (0.1 mM) significantly lowered the affinities of all tested halogenated 2-phenylhistamines and histamine for H1 receptor binding site converting biphasic displacement curves, to monophasic ones, whereas GTP had no effect on the affinity of 2-PH. The results of this study support the conclusions that bovine vascular and guinea pig myocardial histamine H1 receptors differ in their molecular properties. Selective and potent H1 receptor agonists of 2-phenylhistamine class can discriminate between vascular and cardiac receptor.

Influence of ryanodine on the mechanical restitution and on the post-extrasystolic potentiation of the guinea-pig ventricular myocardium.

This paper records the results of an investigation into potentiation and staircase phenomena in rightventricular guinea-pig papillary muscles with particular reference to the sarcoplasmic Ca(2+)-channel. As a tool to isolate the second ('late', 'tonic') component of isoproterenol-induced biphasic contractions ryanodine was used. On the evidence at present available the monophasic ryanodine-resistant component of the twitch represents that portion of the activator calcium which reaches the troponin C directly, that is, not taking the roundabout way through the intracellular storage structures. In order to avoid functional instabilities of the isolated muscle preparation a short-time double rest stimulation programme was used which combines a number of different tests and gives information on (1) the post-rest potentiation, (2) the post-extrasystolic potentiation, (3) the mechanical post-rest recovery, (4) the interval-strength relationship, and (5) the mechanical restitution. The results of the present work show that under the influence of ryanodine (1) the Bowditch staircase, a typical feature of normodynamic mammalian ventricular preparations as well as of hypodynamic frog heart preparations, does not exist, (2) the post-extrasystolic potentiation disappears, (3) the curve reflecting the mechanical restitution, under normal in vitro conditions a monotonically increasing function, becomes biphasic within the relative refractory period, (4) the conspicuous depression of the isometric post-rest contraction for long lasting pauses interrupting the regular pacing rhythm, a typical feature of isolated guinea-pig ventricular tissue, is clearly diminished, and (5) the characteristic curve, reflecting the potentiation of the post-extrasystolic post-rest contraction as a function of the delay time preceding the extrastimulus, becomes displaced to the premature interstimulus interval. The concept of an 'extended 2-calcium-store model' is supported by this work.

Functional evidence against the existence of beta 2-adrenoceptors mediating positive intropic effects in guinea-pig right ventricular myocardium.

In guinea-pig isolated papillary muscles we studied the positive inotropic effects of the beta-adrenoceptor agonists isoprenaline, fenoterol and noradrenaline in the presence and absence of the beta 1-selective antagonist atenolol and the beta 2-adrenoceptor antagonist ICI 118.551. In Schild regression analysis pA2 values, for either atenolol (7.14-7.16) and ICI 118.551 (6.79-6.84) were independent of the agonists used. These results support the view that the inotropic response in guinea-pig ventricular myocardium is mediated by beta 1-adrenoceptors only.