HCN2 overexpression in newborn and adult ventricular myocytes: distinct effects on gating and excitability.

Ventricular pacemaker current (I(f)) shows distinct voltage dependence as a function of age, activating outside the physiological range in normal adult ventricle, but less negatively in neonatal ventricle. However, heterologously expressed HCN2 and HCN4, the putative molecular correlates of ventricular I(f), exhibit only a modest difference in activation voltage. We therefore prepared an adenoviral construct (AdHCN2) of HCN2, the dominant ventricular isoform at either age, and used it to infect neonatal and adult rat ventricular myocytes to investigate the role of maturation on current gating. The expressed current exhibited an 18-mV difference in activation (V(1/2) -95.9+/-1.9 in adult; -77.6+/-1.6 mV in neonate), comparable to the 22-mV difference between native I(f) in adult and neonatal cultures (V(1/2) -98.7 versus -77.0 mV). This did not result from developmental differences in basal cAMP, because saturating cAMP in the pipette caused an equivalent positive shift in both preparations. In the neonate, AdHCN2 caused a significant increase in spontaneous rate compared with control (88+/-5 versus 48+/-4 bpm). In adult, where HCN2 activates more negatively, the effect was evident only during anodal excitation, requiring significantly less stimulus energy than control (2149+/-266 versus 3140+/-279 mV. ms). Thus, ventricular maturational state influences the voltage dependence of expressed HCN2, resulting in distinct physiological impact of expressed channels in neonate and adult myocytes. The full text of this article is available at http://www.circresaha.org.

Muscarinic receptors mediating the positive inotropic effect of carbachol in embryonic chick ventricle have a low affinity for pirenzepine.

Electrically driven ventricular strips from 3 day old chick embryos were used to investigate the ability of pirenzepine, a muscarinic antagonist that distinguishes between subtypes of muscarinic receptors, to antagonize the positive inotropic effect of carbachol. A low pA2 value was found for pirenzepine (6.40) whereas the pA2 value for atropine was 1000-fold higher (9.36). It can be concluded that the positive inotropic effect of carbachol on this tissue is mediated by muscarinic receptors others than M1-receptors.

Mibefradil, an I(Ca,T) blocker, effectively blocks I(Ca,L) in rabbit sinus node cells.

To test the hypothesis that the Ca(2+) channel blocker mibefradil slows heart rate due to inhibition of T-type Ca(2+) current in pacemaker cells, we studied effects of mibefradil on action potentials and ionic currents of isolated rabbit sinus node cells using the patch clamp technique. Mibefradil (100 nM and 1 microM) reduced spontaneous rate, decreased action potential amplitude and finally stopped impulse initiation. This action was not due to the drug effect on hyperpolarization-activated pacemaker current, but can be explained by attenuation of both T- and L-type Ca(2+) currents, which were inhibited by mibefradil almost equally (55% and 64% inhibition with 1 microM for T- and L-types, respectively).

[The inotropic action of adrenergic agonists on the heart ventricles of the chick embryo]. / Inotropnoe deistvie adrenergicheskikh agonistov na zheludochek serdtsa kurinogo émbriona.

The effects of adrenergic drugs on the twitch tension of the electrically driven (1.2-1.5 Hz) ventricular preparations from 2-20-day old chick embryos and hatched chicks were studied. Agonists evoked positive inotropic responses of 3-day embryonic ventricles and of ventricles from older animals. 2-day embryonic ventricles were unresponsive. 5-day embryonic ventricles were most sensitive to agonists (EC50 value of adrenaline = 4.5 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-7) M). The order of agonists activity (isoproterenol greater than noradrenaline greater than adrenaline much greater than phenylephrine) and the high potency of propranolol as antagonist of adrenaline indicate that responses are mediated with beta-adrenoceptors. The role of GTP-binding protein for the regulation of adrenoreactivity in embryonic chick heart during ontogenesis is discussed.

[The inotropic action of acetylcholine on the heart ventricles during larval development in the frog Rana temporaria]. / Inotropnoe deistvie atsetilkholina na zheludochek serdtsa v khode lichinochnogo razvitiia liagushki Rana temporaria.

The effects of acetylcholine and other cholinergic drugs on the isolated electrically driven larval frog ventricles have been studied. The negative inotropic response to acetylcholine appeared as early as stage 33 of the larval development (the stages were determined according to Dabagian and Sleptsova, 1975) and persisted through all the developmental stages including metamorphosis. The response is muscarinic in origin since it was reproduced with a muscarinic agonist methylfurmetide, blocked with atropine but was not modified with tubocurarine. At the stage 41 and following stages, the sensitivity to acetylcholine was decreased while to methylfurmetide was not. The decreased sensitivity to acetylcholine is most likely due to increase of activity of cholinesterases in the myocardial tissues.

[The inotropic action of adrenergic substances on the heart ventricles during the ontogeny of the frog Rana temporaria]. / Inotropnoe deistvie adrenergicheskikh veshchestv na zheludochek serdtsa v khode ontogeneza liagushki Rana temporaria.

The effects of adrenaline and other adrenergic drugs on the isolated, electrically driven ventricles and ventricular strips from the tadpoles, the first year froglets and adult frogs have been studied. Adrenaline induced positive inotropic effect at all developmental stages including the earliest one, stage 32 (stages were determined according to Dabagian and Sleptsova). The pharmacological analysis revealed the beta-adrenergic origin of this effect. The sensitivity to adrenaline decreased after the stage 43 and became the lowest in adult hearts. It may partly be due to maturing of the neuronal uptake mechanism because the sensitivity to isoproterenol which is not subjected to the neuronal uptake processes was not decreased in the adults as compared to tadpoles. The adrenoceptors of the tadpole heart have the higher affinity to propranolol then the adrenoceptors of the adult heart.

[Positive inotropic action of acetylcholine on the heart ventricles of the chick embryo]. / Polozhitel'noe inotropnoe deistvie atsetilkholina na zheludochek serdtsa kurinogo émbriona.

Acetylcholine increased twitch tension in the whole ventricle or in ventricular strips from 2-10-day chick embryos. The effect of acetylcholine was mediated by muscarinic receptors, since it was prevented by atropine, but not by tubocurarine or propranolol. Prostigmine significantly increased the sensitivity to acetylcholine in the strips from 7-day embryos, being almost ineffective in the strips from 3-day embryos. The decrease in acetylcholine sensitivity in the developing chick embryo is presumably associated with the increase in cholinesterase activity of the myocardial tissue.

[The positive inotropic action of transmural electrical stimulation of the heart ventricles in the ontogeny of the frog Rana temporaria]. / Polozhitel'noe inotropnoe deistvie transmural'noi élektricheskoi stimuliatsii na zheludochek serdtsa v ontogeneze liagushki Rana temporaria.

Studies have been made of the effect of transmural electrical stimulation on twitch tension produced by atropinized ventricular preparations from tadpoles and adult frogs. In preparations from tadpoles at stage 42 and all the following stages, as well as in adult frogs, transmural electrical stimulation evoked positive inotropic responses which consisted of a slow propranolol-sensitive component or of a slow and fast components. It is highly probable that the slow component is induced by adrenergic transmitter. The fast propranolol-resistant component appears at stage 43. It may be prevented by bretilium being probably induced by a comediator which is released together with the adrenergic transmitter from the sympathetic nerve endings.

[The reflex and direct actions of novocaine on the rhythm of the heart beats in rats in early postnatal ontogeny]. / Reflektornoe i priamoe deistvie novokaina na ritm sertsebienii krys v rannem postnatal'nom ontogeneze.

In experiments on newborn rats it was shown that heart beating frequency depended on the intensity of interoceptive afferentation. Partial shutdown of the afferentation by means of intraperitoneal injection of novocaine in the dose of 25 mg/kg of body weight caused the increase of heart beating and the more complete one at higher doses (100 mg/kg of body weight) led to its decrease. The injection of 2% novocaine (150-200 mg/kg of body weight) into the peritoneum of rat puppies on the first week of life caused the transition of bradycardia into temporary or permanent stop of heart beating. The same effect was observed for isolated atrium of rat puppies on the first week of life after the addition of 0.1% novocaine to feeding solution. This effect disappeared in older animals. It was concluded that resorptive effect of novocaine on heart structures causing the disturbance of heart automatism in newborn rats occurred along with the reflex one.

[A comparative study of the stereoselectivity of cholinoreceptors]. / Sravnitel'noe izuchenie stereoselektivnosti kholinoretseptorov.

Muscarine-sensitive cholinoreceptors (M-ChR) of higher vertebrates exhibit high stereoselectivity which is also revealed with respect to enantiomers of a very potent muscarinomimetic methyldilvasen (F-2268), the stereospecific index (SSI) being about 100. M-ChRs in the neuronal membrane of the gastropod mollusc Planorbarius corneus and in the hearts of the bivalve molluscs Mercenaria stimpsoni and Anadara broughtoni are highly sensitive to methyldilvasen (10(-9)-10(-10) M), but their sensitivity to its enantiomers is identical. In heart atria of the tortoise Testudo horsfieldi, frog Rana temporaria, and fishes Siluris glanis, Cyprinus carpio, as well as in ventricles of tadpoles, high SSI was revealed. These data are consistent with a hypothesis that during evolution of vertebrates no significant changes took place in the active center of M-ChR. Possibly, the lack of stereoselectivity in the investigated molluscan M-ChRs, together with their other peculiarities (they are not blocked by atropine), indicate "immaturity" of these receptors.

[Comparative findings on the stereoselectivity of cholinoreceptors]. / Sravnitel'nye dannye o stereoselektivnosti kholinoretseptorov

The potency of the optical isomers of the muscarinomimetic agent 2-methyl-4-dimethylaminomethyl-1.3-dioxolane methiodide (F-2268) was compared on cholinoreceptors, (ChR) of different animals. The greatest difference between optical isomers was observed on the muscarinic ChR of guinea pig ileum smooth muscle cis-L(+)isomer being more than hundred times as potent as cis-D(-)isomer. On the ChR of muscarinic type in the holothuria Cucumaria japonica retractor muscle, cis-L(+)isomer was 25 times as efficient as cis-D(-)isomer. On the ChR of sea urchin and sipunculid locomotor muscles, optical isomers differ only 3 to 5 times. There was no difference between the effect of optical isomers on the ChR of muscarinic type which mediate hyperpolarization in the neurones of the gastropod mollusc Planorbarius corneus. This suggest that some changes in ChR stereoselectivity may occur in the course of evolution.

Positive and negative inotropic effects of carbachol on the embryonic chick atrium.

The effect of carbachol on twitch tension of atrial preparations from chick embryos of different incubation ages (3-14 days) was studied. At every age carbachol evoked negative (at low concentrations) and positive (at higher concentrations) inotropic responses. Maximal response values for both effects increased with age; in 3- and 5-day atria the positive inotropic response prevailed. The muscarinic antagonist pirenzepine inhibited the positive (on 5-day atria) and negative (on strips of 14-day atria) inotropic effects of carbachol with pA2 values of 6.8 and 8.0, respectively, suggesting that muscarinic receptors mediating these effects belong to different receptor subtypes.

Ontogeny of cholinergic and adrenergic mechanisms in the frog (Rana temporaria) heart.

Histochemical techniques, field stimulation, and application of autonomic drugs were used to study neurotransmission in the heart during ontogenesis of Rana temporaria. Cholinesterase (ChE)-containing fibers, fluorescent chromaffinlike cells, and fluorescent fibers were found first in the heart at tadpole stages 40, 40, and 50, respectively. Inhibitory cholinergic and stimulatory adrenergic responses to field stimulation first appeared at stages 39-40 and 42, respectively. Inhibitory responses to acetylcholine (ACh) and stimulatory responses to epinephrine (Epi) were observed as early as stages 31 and 32. The concentrations producing half-maximal response values for both neurotransmitters increased during development. Indirect evidence was obtained that the subsensitivity of tadpole hearts to ACh was due to increased hydrolysis of ACh by tissue ChE and that the subsensitivity of adult frog heart to Epi could be connected with a maturation of the neuronal uptake mechanism. The pA2 values for atropine and propranolol were 10 times greater in tadpoles than in adults. The main conclusion is that the cholino- and adrenoreactive systems appear in the frog heart cells before they become innervated and the sensitivity of these systems to neurotransmitters does not increase with innervation.

Neuropeptide Y contributes to innervation-dependent increase in I(Ca, L) via ventricular Y2 receptors.

The developmental increase in L-type Ca current (I(Ca,L)) density in the rat ventricle is reproduced in vitro by culturing neonatal myocytes with sympathetic neurons. We tested whether this effect of sympathetic innervation results from a chronic or sustained action of neurally released neuropeptide Y (NPY). Ventricular myocytes from newborn rats were cultured in serum-free medium with or without sympathetic neurons, NPY, or NPY analogs. Ca currents were measured in single myocytes at room temperature using the perforated patch clamp. In all cell groups (control, innervated, or NPY treated), the current-voltage relation for I(Ca,L) was represented by a bell-shaped curve with maximal value near 0 mV. The current density at 0 mV normalized to that of corresponding mean control values was 1.63 +/- 0.12 and 1.52 +/- 0.16 for innervated and NPY-treated myocytes, respectively. Both groups differed significantly from control (P < 0.05). NPY analogs exhibited the following rank order of effectiveness: NPY >/= NPY-(13-36) >/= PYY >> [Leu31Pro34]NPY, suggesting that the NPY effect occurs via a Y2-receptor subtype. In confirmation, chronic treatment of innervated cultures with a Y2-selective NPY antagonist prevented the innervation-dependent increase in I(Ca,L). These results indicate that sympathetic innervation contributes to the developmental increase in I(Ca,L) via neurally released NPY acting at Y2 receptors on the ventricular myocytes.

L-type but not T-type calcium current changes during postnatal development in rabbit sinoatrial node.

Although the neonatal sinus node beats at a faster rate than the adult, when a sodium current (I(Na)) present in the newborn is blocked, the spontaneous rate is slower in neonatal myocytes than in adult myocytes. This suggests a possible functional substitution of I(Na) by another current during development. We used ruptured [T-type calcium current (I(Ca,T))] and perforated [L-type calcium current (I(Ca,L))] patch clamps to study developmental changes in calcium currents in sinus node cells from adult and newborn rabbits. I(Ca,T) density did not differ with age, and no significant differences were found in the voltage dependence of activation or inactivation. I(Ca,L) density was lower in the adult than newborn (12.1 +/- 1.4 vs. 17.6 +/- 2.5 pA/pF, P = 0.049). However, activation and inactivation midpoints were shifted in opposite directions, reducing the potential contribution during late diastolic depolarization in the newborn (activation midpoints -17.3 +/- 0.8 and -22.3 +/- 1.4 mV in the newborn and adult, respectively, P = 0.001; inactivation midpoints -33.4 +/- 1.4 and -28.3 +/- 1.7 mV for the newborn and adult, respectively, P = 0.038). Recovery of I(Ca,L) from inactivation was also slower in the newborn. The results suggest that a smaller but more negatively activating and rapidly recovering I(Ca,L) in the adult sinus node may contribute to the enhanced impulse initiation at this age in the absence of I(Na).

Carbachol increases contractions and intracellular Ca++ transients in guinea pig ventricular myocytes.

We tested hypotheses concerning the muscarinic receptor subtype and the involvement of L-type Ca current (ICa) in the stimulation of contractions by carbachol (CCh) in single guinea pig ventricular myocytes. When superfused with Tyrode's solution (36 degrees C, 5.4 mM [Ca++]o) and stimulated at 0.2 Hz, CCh (EC50 approximately 18 microM) increased the early component of isotonic contractions by acting at muscarinic receptors indistinguishable from the M2 subtype because AF-DX 116 (M2-selective) was more potent than pirenzepine (M1-selective) as an antagonist of the CCh effect. Action potential duration decreased slightly and ICa was not increased when CCh increased contractions. Carbachol increased intracellular Ca++ transients and contractions reversibly, which indicated an effect via sarcoplasmic reticulum (SR) Ca stores. Ryanodine (1-10 microM) blocked the early contraction component increased by CCh, another indication that CCh action depends on SR Ca stores. We previously found that CCh increased a background Na+ current by occupancy of M2 receptors. We now report that the increased contractions by CCh can also originate at M2 receptors and that SR Ca stores are involved in the CCh effect. Because CCh did not significantly increase ICa, the initial increase of intracellular Na+ by CCh may eventually act through Na-Ca exchange to enhance excitation-contraction coupling.

[Occurrence of atypical histamine H2 receptors in the wall of the frog subclavian vein]. / O sushchestvovanii atipichnykh H2-gistaminovykh retseptorov v stenke podkliuchichnoi veny liagushki.

Selective H2-histamine agonist dimaprit was shown to produce relaxation of the isolated frog subclavian vein, with it persisting under the effect of selective H2-histamine antagonist cimetidine. Possible nonspecific mechanisms of relaxation produced by histamine are discussed. The data presented do not exclude that there are atypical H2-histamine receptors in subclavian vein of frogs, the activation of which initiates the attenuation of the active tension.

[Effect of activation of alpha- and beta-adrenergic receptors, M-cholinergic receptors and H1-histamine receptors on mechanical tension in the frog subclavian vein]. / Vliianie aktivatsii alpha- i beta-adreno-, M-kholino- i H1-gistaminovykh retseptorov na mekhanicheskoe napriazhenie podkliuchichnoi veny liagushki.

The effects of adrenaline, noradrenaline, isoproterenol, dopamine, histamine, acetylcholine, and serotonin on the wall tension in the isolated frog subclavian vein and modification of these effects by appropriate antagonists were examined. The existence of alpha- and beta-adreno-, M-choline- and H1-histamine receptors was demonstrated. Stimulation of alpha-adreno- and H1-histamine receptors augments and that of beta-adreno- and M-cholinoreceptors diminishes active wall tension in the vein.

[Action of polymethylene-bis-quaternary compounds on the muscarinic and nicotinic cholinoreceptors]. / Deistvie nekotorykh polimetilen-bis-chetvertichnykh soedinenii na muskarinovye i nikotinovye cholinoretseptory.

The action of compounds with general formula (formula: see text) on the frog heart ventricle, cat blood pressure, guinea pig ileum and frog rectus abdominis was studied. With dioxolane radicals (type F-2268) a strong muscarinomimetic action on the cat arterial blood pressure and guinea pig ileum was observed, with maximum marked action at n = 10, which was more pronounced at an even than at odd number of methylene groups. On the frog heart the compounds with an odd number of "n" elicited an atropine-like action. The compounds with pentyl radicals produced no effect on blood pressure and a weak cholinolytic effect on the frog heart. On the ileum they exhibited a cholinomimetic effect. All compounds studied acted as noncompetitive cholinolytics on the frog rectus.