Perfused Frog Heart Research Articles

Cardiotonic activity of Parotoid gland secretion of common Indian Toad Bufo melanostictus on isolated heart of Frog

The present investigation has been undertaken to study the cardiotonic activity of parotoid gland secretion of common Indian Toad Bufo melanostictus on isolated perfused frog heart (Rana tigrina). Cardiotonic activity of parotoid gland secretion of B. melanostictus in normal as well as when induced with paraoxon (an organophosphate) was studied. For the evaluation of cardiotonic activity, Syme’s technique is being employed, digoxin and propranolol were used as standard drug and β-blocker respectively to characterize the effects on the receptors. The isolated perfused frog heart and hypodynamic frog heart showed dose dependent positive ionotrophic effects. The parotoid gland secretion exihibited cardiac stimulant activities. The propranolol was not able to block the effects of paraoxon induction on toad secretion. Thus, the present investigation reports that the parotoid gland secretion increased the force of contraction, heart beat and cardiac output in perfused frog’s heart, whereas, there was no change on hypodynamic heart, indicating that there may be existence of two components, one with β–receptor stimulating activity and other acting directly on the heart (independent of β1-adrenoreceptors).

The effect of verapamil and diltiazem on cardiac stimulant effect of adrenaline and calcium chloride on isolated frog heart

BackgroundCalcium channel blockers block voltage dependent Ltype of calcium channel and thus reduce the frequency of opening of these channels in response to depolarization. The result is a marked decrease in transmembrane calcium current associated with long lasting relaxation of vascular smooth muscle, reduction in contractility in cardiac muscle, decrease in pacemaker activity in the SA node and decrease in conduction velocity in the AV node. Among Calcium channel blockers verapamil, is cardio selective, nifedipine is vascular smooth muscle selective, while diltiazem exhibits intermediate selectivity.MethodsIn the present study, the effect of two Ca⁺⁺ channel blocker, Verapamil and Diltiazem were compared on the isolated frog heart by using adrenaline & calcium chloride as standard on frog heart contractility.Results and conclusionAdrenaline and calcium chloride increased the amplitude of contraction of isolated perfused frog heart. The Ltype of Ca²⁺ channel blockers verapamil and diltiazem produced dose dependent (2μg, 4μg, 8μg, and 16μg) reduction in the amplitude of contraction produced by calcium chloride in isolated perfused frog heart. There was no statistical significant difference (p > 0.05) between the inhibitory effect of diltiazem and verapamil on calcium chloride induced contraction of isolated frog heart.

Mn-citrate and Mn-HIDA: intermediate-affinity chelates for manganese-enhanced MRI

In this study we investigated two manganese chelates in order to improve the image enhancement of manganese-enhanced MRI and decrease the toxicity of free manganese ions. Since both MnCl₂ and a low-affinity chelate were associated with a slow continuous decrease of cardiac functions, we investigated intermediate-affinity chelates: manganese N-(2-hydroxyethyl)iminodiacetic acid (Mn-HIDA) and Mn-citrate. The T₁ relaxivity values for Mn-citrate (4.4 m m⁻¹ s⁻¹) and Mn-HIDA (3.3 m m⁻¹ s⁻¹) in artificial cerebrospinal fluid (CSF) were almost constant in a concentration range from 0.5 to 5 m m at 37 °C and 4.7 T. In human plasma, the relaxivity values increased when the concentrations of these Mn chelates were decreased, suggesting the presence of free Mn²⁺ bound with serum albumin. Mn-HIDA and Mn-citrate demonstrated a tendency for better contractility when employed with an isolated perfused frog heart, compared with MnCl₂. Only minimal changes were demonstrated after a venous infusion of 100 m m Mn-citrate or Mn-HIDA (8.3 µmol kg⁻¹ min⁻¹) in rats and a constant heart rate, arterial pressure and sympathetic nerve activity were maintained, even after breaking the blood-brain barrier (BBB). Mn-citrate and Mn-HIDA could not cross the intact BBB and appeared in the CSF, and then diffused into the brain parenchyma through the ependymal layer. The responses in the supraoptic nucleus induced by the hypertonic stimulation were detectable. Therefore, Mn-citrate and Mn-HIDA appear to be better choices for maintaining the vital conditions of experimental animals, and they may improve the reproducibility of manganese-enhanced MRI of the small nuclei in the hypothalamus and thalamus.

Mn-bicine: A low affinity chelate for manganese ion enhanced MRI

The toxicity of free Mn(2+) is a bottleneck for the in vivo application of manganese ion enhanced MRI. To reduce free Mn(2+) concentration ([Mn(2+) ]), a low affinity chelate reagent: N,N-bis(2-hydroxyethyl)glycine (bicine) was used. Considering the conditional association constant of Mn-bicine at pH 7.4 (10(2.9) M(-1) ), (i) a 100 mM Mn-bicine solution should contain about 10 mM of free manganese ion, but (ii) free manganese will make up 3/4 of the final plasma concentration (0.5 mM) with an intravenous infusion of 100 mM Mn-bicine. The T(1) relaxivity of Mn-bicine in a 5 mM Mn-bicine solution was estimated as 5 mM(-1) sec(-1) at 24°C, 7 T in a pH range of 6.8-7.5. Mn-bicine demonstrated a tendency for better contractility when employed with an isolated perfused frog heart, compared with MnCl(2) . A venous infusion of 100 mM Mn-bicine (8.3 μmol kg(-1) min(-1) ) showed a minimal decrease and maintained a constant heart rate level and arterial pressure in rats, while rats infused with 100 mM of MnCl(2) showed a significant suppression of the hemodynamic functions. Thus, Mn-bicine appears to be a better choice for maintaining the vital conditions of experimental animals, and may improve the reproducibility of manganese ion enhanced MRI.

Design, synthesis and pharmacological screening of hybrid molecules as antihypertensives

The synthesis and pharmacological profile of a series of hybrid compounds bearing the dihydropyrimidine moiety and substituted phenylthioureas joined via typical β-blocker aryloxypropanolamine group are described. Seven out of 10 new compounds tested proved to be endowed with negative inotropic and chronotropic effect as compared to Adrenaline on isolated perfused frog heart. Also ECG studies that were carried out on normotensive male rats showed that the most of the compounds exhibited significant reduction in Rmax value in the first 30 min although maintaining little change in heart rate, with hypotensive effect lasting for 1 h. We concluded that the most of the synthesized novel compounds exhibited hypotensive as well as antihypertensive effects which could be attributed to blockade of Ca2+ entry and β-adrenoreceptor blocking activities due to introduction of aryloxypropanolamine linked to N-1 substituted dihydropyrimidine moiety. Brady cardiac effects of compounds 5a–5d, 6c–6e resulting from Ca2+ entry and β-adrenoreceptor blocking attenuate the sympathetic activation-associated reflex tachycardia in the heart. We selected compound 5c as a promising lead for further detailed pharmacological and preclinical evaluation studies.

Vasoconstrictor and Inotropic Effects Induced by the Root Bark Extracts of Anthocleista schweinfurthii

The present study was undertaken to investigate the cardiovascular effect of three extracts from the root bark of Anthocleista schweinfurthii Gilg.: an aqueous extract (AE), a dichloromethane extract (DCMR) and a fraction enriched in cardiac glycoside type compounds (CARDAN). In isolated perfused frog heart, bolus injection of the extracts produced a positive inotropic effect. The responses to AE and DCMR, but not to CARDAN, were depressed by propranolol. In isolated rat aorta, DCMR produced a transient increase in contractile tension while AE and CARDAN induced a sustained constriction. AE vasoconstrictor effect was abolished by phentolamine, while contraction evoked by CARDAN was antagonized by verapamil. In aortic rings contracted in low K+ media, the addition of K+ evoked a relaxation, which was abolished by ouabain, depressed by DCMR but not affected by either A(E) or CARDAN. These observations indicate that Anthocleista schweinfurthii contains substances that promote vasoconstriction and increase cardiac contraction. The effect of DCMR was only partially mediated by inhibition of the Na+ pump while the mechanism of action of A(E) and CARDAN was distinct from the inhibition of the Na+, K+ - ATPase pump, but could involve adrenergic receptors, or either direct or indirect activation of L-type calcium channels.

Cardiac defibrillator neurones

The cardiac vagal motor system exerts a myriad of actions upon the beating mammalian heart and its nutrient vascular supply, but one of its most fascinating actions is to quell potentially lethal arrhythmias. It has been recognised for a long time that the amount of nervous vagal tone to a heart has a direct bearing on the outcome of potentially fatal cardiac arrhythmias following a heart attack (La Rovere et al. 1998). Life and death in a coronary care unit is sometimes governed by cardiac vagal tone. In 2007, Brack et al. provided evidence that nitric oxide mediates the protective effect of the vagi on ventricular fibrillation. In this issue of The Journal of Physiology this group has succeeded in measuring nitric oxide release in the beating left ventricle in response to vagal stimulation (Brack et al. 2009). One interpretation of this work is that there is a population of cardiac nitrergic postganglionic neurones which project to the left ventricle and protect it from fibrillation. This is the conception that I am drawn to, being richer in possibilities than other hypotheses. For almost 10 years this group of physiologists and cardiologists has employed the century old method of Oscar Langendorff but with a twist; they leave the cardiac autonomic nerves attached. Like countless investigators before them, they have shown that there is no vagal inotropic effect on the left ventricle, provided the heart rate is maintained constant. Interestingly they have reported that the vagus can affect dispersion of ventricular repolarisation (Mantravadi et al. 2007). This was followed by experiments that showed vagal stimulation could also raise the ventricular threshold to fibrillation. Pharmacological experimentation provided evidence that nitric oxide (NO) mediates this vagal protection via effects on action potential duration restitution (Brack et al. 2007). The next logical step is described in this issue of The Journal of Physiology: to detect an increase of NO in the wall of the left ventricle during vagal stimulation (Brack et al. 2009). The question arises: what is the source of this NO? Is it nitrergic nerves or nitrergic myocytes? The burst of NO is probably not due to constriction and ischaemia causing increased NO production due to disturbed energy metabolism (because vagus nerve stimulation did not increase perfusion pressure under constant flow conditions i.e. did not increase coronary resistance). One caveat is that the researchers measured pressure a little remotely from the ostia of the coronary arteries. Of note, the NO increase also occurred in response to acetylcholine infusion, but this was not blocked by an antagonist of the neuronal form of NO synthase. In contrast, the vagally released NO was sensitive to this antagonist. If the NO is from nitrergic nerves then the heart should contain these NO-producing postganglionic neurones. There is no difficulty in finding supportive evidence for this proposal in many species e.g. 37% of human cardiac ganglion neurones contain NO synthase (Singh et al. 2009). The work of Brack et al. (2009) should act as a stimulus to trace the trajectory of these NOS-positive neurones within atrial ganglionated plexuses. The question of vagal innervation of mammalian ventricles may have become embroiled in controversy in the past, partly because there has been too much emphasis on cholinergic markers. The paper is a testament to the ingenuity of its authors and an enduring method of Carl Ludwig who in 1866, together with Elias Cyon created the first isolated perfused frog heart.

The first recordings of pharmacological effects

The first recordings of pharmacological effects

Positive inotropic effect of the leaf extracts of parent and tissue culture plants of Coleus amboinicus on an isolated perfused frog heart preparation

The leaves of the indigenous plant Coleus amboinicus Lour (Lamiaceae) possess a characteristic aroma and are used in many food preparations to enhance the flavour. Apart from this, they also exhibit medicinal properties. Aqueous extracts of fresh leaves of C. amboinicus Lour. parent (CAP), as well as tissue culture-raised plants (CAT), showed a dose-dependent positive inotropic effect on isolated frog heart. The extracts of CAT showed higher activity. The responses to CAP and CAT (50–800 μg) were not affected in either way by propranolol, atropine, verapamil or imidazole. The responses to both CAP and CAT were inhibited after lowering sodium chloride concentration (from 111 to 55.6 mM) and potentiated after increasing sodium chloride concentration (from 111 to 222 mM). Lignocaine was found to inhibit the responses significantly. Thus, C. amboinicus induces a positive inotropic effect, possibly by increasing sodium influx, leading to greater intracellular availability of calcium.

Synthesis and cardiac effects of 3,4-dihydropyrimidin-2(1H)-one-5 carboxylates

A series of 4-(substituted)-3,4-dihydropyrimidinone derivatives have been synthesized by heating 1,3 dicarbonyl compounds, urea, and aromatic aldehydes in acetic acid under microwave irradiation conditions. The cardiovascular effects of 3,4-dihydropyrimidinones were studied on isolated perfused frog heart at different dose levels and compared with the activity of digoxin. The interaction of 3,4-dihydropyrimidinones with β-blocker and calcium channel blocker was also investigated. Compound 4d emerged as the most interesting compound in this series with potential cardiotonic activity.

Isolated heart perfusion according to Langendorff—Still viable in the new millennium

The isolated perfused mammalian heart preparation was established in 1897 by Oscar Langendorff. The method was developed on the basis of the isolated perfused frog heart established by Elias Cyon at the Carl Ludwig Institute of Physiology in Leipzig, Germany in 1866. Observations made using both methods at the end of the 19th and at the beginning of the 20th century led to important discoveries, forming the basis for our understanding of heart physiology. This included the role of temperature, oxygen and calcium ions for heart contractile function, the origin of cardiac electrical activity in the atrium, the negative chronotropic effect of vagus stimulation and the chemical transmission of impulses in the vagus nerve by acetylcholine. Langendorff himself demonstrated that the heart receives its nutrients and oxygen from blood via the coronary arteries and that cardiac mechanical function is reflected by changes in the coronary circulation. The method underwent many modifications but its general principle remains the same today. Blood, or more commonly crystalloid perfusates, are delivered into the heart through a cannula inserted in the ascending aorta, either at constant pressure or constant flow. Retrograde flow in the aorta closes the leaflets of the aortic valve and as a consequence, the entire perfusate enters the coronary arteries via the ostia at the aortic root. After passing through the coronary circulation the perfusate drains into the right atrium via the coronary sinus. The simplicity of the isolated mammalian heart preparation, the broad spectrum of measurements which can be done using this method, its high reproducibility and relatively low cost make it a very useful tool in modern cardiovascular and pharmacological research, in spite of a few shortcomings. In the last decade the method has brought many important advances in many areas including ischemia–reperfusion injury, cell-based therapy and donor heart preservation for transplant.

Who discovered the Frank-Starling mechanism?

In 1866 at Carl Ludwig's Physiological Institute at Leipzig, Elias Cyon described the influence of diastolic filling of the isolated perfused frog heart on ejection volume. A study performed at the institute of the effect of filling pressure on contraction amplitude was published in 1869 by Joseph Coats, based on a recording made by Henry P. Bowditch.

The Isolated Perfused Heart and Its Pioneers.

In 1866, Carl Ludwig together with Elias Cyon created the first isolated perfused frog heart preparation. Perfusion systems for the isolated mammalian heart were developed by H. Newell Martin in 1883 and by Oscar Langendorff in 1895. In its working mode, the isolated perfused rat heart was established in the 1960s.

Investigation of the cardiotoxic peptides of the venom of the cobraNaja oxiana

Six populations of basic polypeptides have been found in the venom of the Central Asian cobraNaja oxiana Eichwald, and four of them — V″c-1, V″c-2, V″c-5, and V″c-6 — have been obtained in the electrophoretically homogeneous state. On a perfused frog heart preparation, all the basic polypeptides proved to be cardiotoxic. However, only some of them (V′c-2, V″c-3, and V″c-4) were characterized by arrhythmic activity as well as by the negative ino- and chronotropic effects, common for all of them. An enhancement of the specific cardiotoxic effect of each of the polypeptides by pure phospholipase A-2 has been shown.

Thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) as a sodium-23 shift reagent for the in vivo rat liver

The use of thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate (TmDOTP5-) as an in vivo 23Na NMR shift reagent for rat liver was evaluated by collecting interleaved 23Na and 31P spectra. Infusion of 80 mM TmDOTP5- without added Ca2+ produced baseline-resolved peaks from intra- and extracellular sodium without producing any changes in phosphate metabolite resonances or intracellular pH. Several key physiological parameters measured in parallel groups of animals confirmed that liver physiology is largely unaffected by this shift reagent. A direct comparison of TmDOTP5- versus DyTTHA3- showed that after infusion of 5-8 times more DyTTHA3-, the extracellular sodium peak shifted by the same amount as with TmDOTP5-, but the two 23Na resonances were very broad and not resolved. The baseline-resolved peaks with TmDOTP5- allowed us to measure the in vivo T1 and T2 relaxation characteristics of intra- and extracellular Na+. The measured T1, T2s, and T2f values and the relative contributions from the slow and fast T2 components for intracellular Na+ in liver did not differ significantly from the values reported for perfused frog heart. The T1 and T2 relaxation curves of the extracellular Na+ resonances fit a monoexponential function. Analysis of the relative contribution of the fast- and slow-relaxing T2 components from intracellular Na+ resulted in a calculated visibility factor of 69 +/- 4% and the intracellular Na+ concentration calculated from the NMR peak intensity ratio, the measured visibility factor, and literature values of intra- and extracellular volume was 19 mM. These results indicate that TmDOTP5- promises to be quite useful as an in vivo shift reagent for liver and other organs.

Continuous read-out of cytochrome b, flavin and pyridine nucleotide oxido-reduction processes in the perfused frog heart and contracting skeletal muscle

The time-sharing instrument for continuous read-out of the fluorescence levels of NAD(P)H and oxidized-flavoproteins, which also makes simultaneous dual beam differential absorption measurements of cytochrome b, has been applied to the study of the metabolic control of the frog heart, perfused in vitro with normal and variously modified Ringer solutions, and to the frog skeletal muscle in situ. In the ionically depolarized resting heart, the long term kinetic analysis of the redox processes during oxygen/anoxia transitions has proved to be an adequate method for identifying and following three fundamental intracellular redox compartments: (1) the respiratory system, (2) the microsomal system, and (3) the glycolytic system. The provision of substrates and/or suitable inhibitors has allowed the description of functional interrelationships within the mitochondria) and the cytoplasmic spaces. The ouabain major site of response has been located on the cytosolic pyridine nucleotides, with a very small response in the mitochondria) space. The switching on and off of substantial fractions of the NAD(P)+/NAD(P)H and Fp oxidized/Fp reduced pools has been observed, which helps to clarify the large phase shift between the overall pyridine nucleotide and flavin oxidation-reduction processes, during the cycle of the spontaneously beating heart and during skeletal muscle contraction and recovery, here originally prompted out.

Alterations in the energy metabolism of the isolated perfused frog heart during calcium depletion and subsequent repletion

The changes in myocardial energy metabolism of isolated perfused Rana ridibunda hearts subjected to prolonged calcium depletion and reperfusion with calcium-containing medium were studied. Calcium-free perfusion resulted in an increase in the concentrations of glucose, glucose-6-phosphate, alpha-ketoglutarate and malate. The myocardial contents of high-energy phosphates were maintained while concentrations of key amino acids were significantly altered. During the reperfusion period the tissue high-energy phosphate content fell abruptly. A marked increase in glycolytic flux and lactate production was observed. The tissue contents of citric acid cycle intermediates and key amino acids decreased. Examination of the activities of marker enzymes during the calcium-free and reperfusion periods showed that only cytoplasmic enzymes are lost during reperfusion, while the activities of other enzymes remained unchanged. The results suggest that the fluxes of both glycolysis and the citric acid cycle are significantly altered during calcium depletion and following repletion in the amphibian heart. The major characteristics of calcium paradox-induced damage in Rana ridibunda heart are the depletion of high-energy stores, the impairment of mitochondrial oxidative metabolism, and a significant increase in anaerobic metabolism.

Characterization of the calcium paradox in the isolated perfused frog heart: enzymatic, ionic, contractile and electrophysiological studies

The effect of perfusion temperature and duration of calcium deprivation on the occurrence of the calcium paradox was studied in the isolated frog heart. Loss of electrical and mechanical activity, ion fluxes, creatine kinase and protein release were used to define cell damage. Perfusion was performed at 22, 27, 32, and 37 degrees C, and calcium deprivation lasted 10, 20, 30, or 40 min. At 22 degrees C and 27 degrees C even a prolonged calcium-free perfusion failed to induce a calcium paradox. After 30 min of calcium-free perfusion at 37 degrees C ventricular activity ceased and a major contraction occurred followed by an increase in resting tension. During the 15-min re-perfusion period the release of creatine kinase was 158.24 +/- 2.49 IU.g dry wt-1, and the total amount of protein lost was 70.37 +/- 0.73 mg.g dry wt-1, while lower perfusion temperatures resulted in a decreased loss of protein and creatine kinase. Ion fluxes in the perfusion effluent indicate that during re-perfusion a massive calcium influx accompanied by a potassium and a magnesium efflux, and an apparent sodium efflux, occur at a perfusion temperature of 37 degrees C after 30 min of calcium deprivation. The results suggest that the basic principles and damaging effects of calcium overloading are common to both mammalian and frog hearts.

The role of phospholipase A2 in calcium-induced damage in cardiac and skeletal muscle.

This study compares the action of inhibitors of the eicosanoid cascade on calcium-induced myofilament damage in cardiac muscle of the perfused frog heart and incubated frog ventricle slices, and in skeletal muscle of incubated mammalian diaphragm and isolated and saponin-skinned amphibian pectoris cutaneous muscle. Mepacrine (10(-5) M) and indomethacin (3 x 10(-6) M) protected completely against myofilament damage induced by entry of calcium in the 'calcium-paradox' in frog heart. However, inhibition of phospholipase A2 (PLA2) (with chlorpromazine, 2 x 10(-4) M, or mepacrine, 10(-5) M, 5 x 10(-5) M), of cyclo-oxygenase enzymes (with indomethacin, 3 x 10(-6) M to 10(-5) M or BW755C, 3.8 x 10(-4) M), or of lipoxygenase enzymes (with BW755C, 3.8 x 10(-4) M or nordihydroguaiaretic acid, 2 x 10(-6) M or 5 x 10(-6) M) all failed in intact cardiac or skeletal muscle cells to prevent the myofilament damage that is rapidly triggered by 10(-2) M caffeine, 6 x 10(-6) M ruthenium red, 10(-4) M DNP or 5 micrograms ml-1 A23187. These agents also failed completely to protect against myofilament damage in saponin-skinned amphibian skeletal muscle when [Ca]i was raised to 8 x 10(-6) M. Thus, inhibition of PLA2 does not protect the myofilament apparatus against calcium released intracellularly, and it is suggested that mepacrine and indomethacin can block entry of calcium in the calcium-paradox in the amphibian heart. Chlorpromazine (2 x 10(-4) M) and mepacrine (10(-3) M) at zero [Ca] caused severe myofilament damage in skinned muscle, possibly due to an effect on membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear magnetic resonance studies of intracellular ions in perfused frog heart.

Intracellular sodium, potassium, and lithium were observed in a perfused frog heart by nuclear magnetic resonance (NMR) spectroscopy. A perfusate buffer containing the shift reagent, dysprosium tripolyphosphate, was used in combination with mathematical filtering or presaturation of the extracellular resonance to separate the intra- and extracellular sodium NMR signals. Addition of 10 microM ouabain to the perfusate, perfusion with a zero potassium, low-calcium buffer, and replacement of 66% of the perfusate sodium with lithium resulted in the following percent changes in the intracellular sodium levels (mean +/- SD): ouabain + 460 +/- 60% (n = 6), zero potassium + 300 +/- 30% (n = 3), and lithium - 51 +/- 6% (n = 3). An increase of 45% in the intracellular sodium was observed when changing the pacing rate from 0 to 60 beats/min (with proportional changes for intermediate pacing rates). The ratio of intracellular potassium to sodium concentration was determined to be 2.3 by NMR, indicating that a substantial amount of the intracellular potassium is undetectable with these NMR methods. In addition, intracellular lithium was observed during perfusion with a lithium-containing perfusate.