Positive inotropic effect of Murraya koenigii (Linn.) Spreng extract on an isolated perfused frog heart

Ethanolic extract of fresh leaves of M. koenigii (MKEE) showed a dose dependent positive inotropic effect on isolated frog heart. The responses to MKEE (62.5-1000 microg) were not affected in either way by theophylline, imidazole, propranolol and sildenafil. The change in potassium and sodium concentration did not alter MKEE-induced positive inotropic effect. Lignocaine did not alter the responses to MKEE significantly. Responses to MKEE were significantly inhibited when calcium concentration was reduced to half (from 1.58 to 0.79 mM) and were significantly potentiated when calcium concentration was doubled (from 1.58 to 3.16 mM). Verapamil was found to inhibit the responses significantly. The results suggest that M. koenigii induced positive inotropic effect possibly by increasing availability of calcium from extra cellular sites.     

Effect of potassium channel modulators on toxicity of Cleistanthus collinus

The study was conducted to determine the effects of boiled extract of Cleistanthus collinus on rats by observing ECG changes and electrolyte levels in serum and urine. Influence of minoxidil and glibenclamide on Cleistanthus collinus induced toxicity was determined. ED50 for arrhythmia, changes in contractility and heart rate were recorded using the isolated frog heart. Cleistanthus at low doses caused transient tachycardia and increase in contractility and at high dose caused arrhythmia and cardiac arrest in rat. LD50 was found to be 1690 mg/kg. Minoxidil potentiated cardiac toxicity, whereas glibenclamide did not produce any significant change. High concentration of potassium in Cleistanthus extract hindered comparison of its levels. There was excretion of sodium even in the presence of hyponatraemia. Cleistanthus at low dose caused transient tachycardia and increase in contractility and at high dose caused arrhythmia and cardiac arrest in isolated frog heart. ED50 for arrhythmia was found to be 1406 mg/kg. Acute toxicity was mainly due to depressive cardiac activity of Cleistanthus. It also caused renal failure. Potassium channel modulators did not have important role in acute cardiac toxicity treatment. Probably in chronic toxicity, electrolyte level changes are involved and potassium channel modulators might have a role.     

Action potential-like responses due to the inward rectifying potassium channel

Summary This paper describes experiments carried out in the absence of sodium and calcium in the external solution. Frog atrial trabeculae were stimulated in current clamp with the double sucrose gap technique. The voltage responses looked like slow action potentials with a clear threshold. These responses were not suppressed in the presence of EGTA, in the presence of sodium or calcium channel blockers, or when sulfate ions replaced chloride. Guinea pig isolated ventricular myocytes were studied in whole cell clamp mode with a pathch pipette. Under current clamp, they displayed also voltage responses with a threshold. These responses were resistant to cadmium (5mm), and were suppressed by barium (0.5mm). A negative slope conductance is required to take into account these results. The membrane current in current clamp can be estimated by plotting the response in the phase plane. This analysis shows that on both types of preparations, the current responsible for the negative slope is not time dependent. This current is suppressed by barium. It can be concluded that it is the outward current flowing through the inward rectifying potassium channels. To confirm this hypothesis, data obtained in voltage clamp on the same preparations were introduced into a computer model to predict the response in current clamp. The results were in agreement with the experiments. Similar responses could be recorded and analyzed on skeletal muscle in isotonic potassium solution. These results show that the inward rectifier can induce by itself properties looking like excitability on different preparations. The physiological significance of this effect in normal conditions is discussed. The voltage responses described in this paper look similar to the slow action potentials on heart, which are sensitive to modifications of the calcium channels, but also of the potassium channels. Some implications in cardiac pharmacology are discussed.     

Cardiotonic activity of aqueous extract of heartwood of Pterocarpus marsupium

The present study was undertaken to evaluate cardiotonic activity of aqueous extract of heartwood of P. marsupium. This plant species contains 5,7,2-4 tetrahydroxy isoflavone 6-6 glucoside which are potent antioxidant and are believed to prevent cardiovascular diseases. Cardiotonic effect of aqueous extract of heartwood of P. marsupium was studied by using isolated frog heart perfusion technique (IFHP). Calcium free Ringer solution was used as vehicle for administration of aqueous extract of P. marsupium as a test extract and digoxin as a standard. A significant increase in height of force of contraction (positive inotropic effect) and decrease in heart rate (negative chronotropic effect) at a very low concentration (0.25 mg/ml) was observed with test extract as compared to the same dose of a standard digoxin. The present results indicated that a significant increase in height of force of contraction with decrease in heart rate was observed as the dose of test extract increased. The test extract produced cardiac arrest at 4 mg/ml, a higher concentration, as compared to standard, digoxin (0.5 mg/ml). Compared to digoxin, a drug with narrow therapeutic window, P. marsupium showed wide therapeutic window.     

Electrophysiology of the flounder heart (Platichthys flesus)—the effect of agents which modify transmembrane ion transport

1. A sucrose gap system was used to record action potentials and mechanical responses of flounder heart.2. Diltiazem eliminated mechanical responses and strongly inhibited the action potential plateau while nifedipine only slightly reduced cardiac contractions without significantly changing the action potential.3. Verapamil slightly hyperpolarized flounder heart but was without effect on either the action potential or mechanical activity except at very high concentrations.4. Lanthanum was ineffective at 2 mM on flounder heart, but manganese at 3 mM substantially inhibited electrical and mechanical responses accompanied by a small hyperpolarization. Substitution of manganese for calcium abolished all flounder cardiac activity.5. BAY K 8644 enhanced cardiac force and enhanced the action potential plateau while depolarizing the preparations. Calcium-free salines abolished heart contractions and the action potential plateau while the spike phase persisted.6. Low sodium salines enhanced while sodium-free salines abolished all heart activity as did tetrodotoxin above I μM. Tetrodotoxin abolished the action potential spike leaving only a small plateau phase.7. Substituting lithium for sodium hyperpolarized the heart, enhanced contractions and prolonged the action potential plateau. Ouabain enhanced cardiac activity and depolarized the heart but ferosemide was without effect on either electrical or mechanical activity.8. TEA at 6 mM had a modest positive inotropic effect and negative chronotropic effect on the heart while the action potential plateau phase was enhanced.9. These results indicate that extracellular sodium and calcium are crucial in flounder heart electrogenesis but such a major role for potassium could not be established.     

Properties of a halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. Preliminary studies of the salt relations and kinetics of the crude enzyme

The effects of chlorides on NADP-specific isocitrate dehydrogenase from Halobacterium salinarium were investigated. The enzyme is stabilized by potassium chloride and sodium chloride and this effect is discussed in relation to the Hill (1913) equation. Kinetics of the enzyme were studied within a range of concentrations of potassium chloride and sodium chloride. Apparent Michaelis constants for both substrates were affected by salt concentration, the effect being greater in sodium chloride than in potassium chloride. Minimal apparent Michaelis constants for both substrates were similar to the corresponding constants reported for yeast isocitrate dehydrogenase. V(max.) was maximal in each salt at a concentration of about 1m. The maximum was higher in sodium chloride than in potassium chloride. At salt concentrations above about 2.3m, the apparent V(max.) was lower in sodium chloride than in potassium chloride, and at salt concentrations below 0.75-1.0m, each salt behaved as a linear activator of the enzyme. Within this concentration range salt and NADP(+) acted competitively; the activation by salt was overcome at finite concentrations of NADP(+). At concentrations above about 1m, potassium chloride was a linear non-competitive inhibitor of the enzyme. Within the range 1.0-2.5m, sodium chloride was also a linear non-competitive inhibitor, but above 2.5m it caused more pronounced inhibition.     

Salt Secretion in Aeluropus litoralis (Willd.) Parl.

The effect of ion composition and concentration in the rootmedium on salt secretion by Aeluropus litoralis was investigated.The presence of a high ionic concentration in the medium stimulatedthe secretion process. The sodium concentration in the secretedfluid was found to be always higher than its concentration inthe medium. A positive correlation was found between the outersodium chloride concentration and the amount of sodium secretedand/or leaf contents. Sodium secretion exhibited a high efficiencyin excluding excess sodium from leaftissues. Sodium retentionin the leaves occurred in relatively low rates. The secretion mechanisms were found to be highly selective tosodium, opposing potassium and calcium. In contrast, potassiumand calcium were retained in the leaves to a greater degreethan sodium. Antagonistic relationships between sodium and potassiumand sodium and calcium were observed in secretion. The secreted fluid contains also various organic substances.Several interpretations to the results in connection with theproposed hypotheses to the mechanism of salt secretion werediscussed.     

Ca2+ protection from the negative inotropic effect of contraction frequency on teleost hearts

Summary Isometric tension development by ventricular strips of 9 species of teleosts, a frog and a turtle was assessed at varying contraction frequencies and Ca_o (external calcium concentration). With teleost hearts an increase in contraction frequency at constant Ca_o was always associated with a decrease in tension development; however, under comparable conditions a positive staircase was exhibited by the frog and turtle heart preparations. The reaction of the teleost heart was thus very different from the well established response of the hearts of higher vertebrates. Elevations in Ca_o always resulted in an increase in tension development such that the positive inotropic effect of Ca_o could compensate for the negative effect of a high contraction frequency. Perfused isolated cod hearts exhibited an increase in cardiac output and pressure development as a result of increases in Ca_o. At 30 contractions min⁻¹ a transition from 1–2 mM Ca_o led to a 68% increase in performance defined as the product of cardiac output times pressure development. The response was in excess of that of ventricular strips. At low Ca_o increases in rate from in situ resting levels to the high end of the physiological range resulted in a decrease in performance. Increases in Ca_o were able to ameliorate the detrimental effect of high imposed contraction frequency. In conclusion, both ventricular strip and perfused heart experiments show that a positive inotropic effect of increased Ca_o can compensate for or even surpass the negative effect of high contraction frequency when both variables are at physiological levels. This finding could have relevance to the maintenance of cardiac performance during/or following intense swimming when both heart rate and plasma calcium may be elevated.     

Dog Red Blood Cells : Adjustment of salt and water content in vitro

Dog red blood cells (RBC) lack a ouabain-sensitive sodium pump, and yet they are capable of volume regulation in vivo. The present study was designed to find in vitro conditions under which dog RBC could transport sodium outward, against an electrochemical gradient. Cells were first loaded with sodium chloride and water by preincubation in hypertonic saline. They were then incubated at 37°C in media containing physiologic concentrations of sodium, potassium, chloride, bicarbonate, glucose, and calcium. The cells returned to a normal salt and water content in 16–20 h. Without calcium in the medium the cells continued slowly to accumulate sodium. Removal of glucose caused rapid swelling and lysis, whether or not calcium was present. The net efflux of sodium showed a close relationship to medium calcium over a concentration range from 0 to 5 mM. Extrusion of salt and water was also demonstrated in fresh RBC (no hypertonic preincubation) when calcium levels in the media were sufficiently raised. The ion and water movements in these experiments were not influenced by ouabain or by removal of extracellular potassium. Magnesium could not substitute for calcium. It is concluded that dog RBC have an energy-dependent mechanism for extruding sodium chloride which requires external calcium and is quite distinct from the sodium-potassium exchange pump.     

Electrolyte distribution and active salt uptake in frog skin

1. The "chloride space" in frog skin was determined and found to be 69.7 per cent by weight of wet skin. The chloride space occupies about 94 per cent of the total water space of skin. From this and other information, it appears that the "non-chloride space" measures only a part of the space occupied by the structural elements of skin. This space is referred to here as the intracellular compartment and the remainder as the extracellular compartment of frog skin. On this basis, potassium and sodium in skin are distributed as follows: total sodium, 60 to 75 µeq./gm. of wet skin; all sodium is probably extracellular; total potassium, 39 to 49 µeq./gm.; intracellular potassium, 37 to 47 µeq./gm. 2. Skins were immersed in solutions differing from each other in their sodium and potassium concentrations. Three levels of NaCl were studied: 48, 119, and 169 µeq./ml. For each of these solutions (referred to below as diluted, physiological, and concentrated saline), the potassium levels were varied from 0.1 to 20 µeq./ml. For skins in solutions low in potassium and high in sodium, it was found that an exchange of intracellular potassium against extracellular sodium occurs. The ratio for the number of potassium ions lost/number of sodium ions gained was 4:1,4:6, and 4:8 for skin in K⁺-free diluted, physiological, and concentrated saline, respectively. 3. Uptake of NaCl by the epithelium of frog skin is dependent on the potassium concentration of the environment. For skins in physiological saline, net uptake of NaCl was optimal (0.90 µeq. x cm.^–2 x hr.^–1) at 1 to 5 µeq. K₊/ml. For skins in diluted and concentrated saline optimal NaCl uptake was seen at potassium concentrations of approximately 5 and 10 µeq. K₊/ml., respectively. Net uptake of NaCl by the skin is also discussed, with relation to the potassium balance of skin. 4. Skin potentials decreased with increasing extracellular potassium concentration when diluted saline solutions were used. The opposite of this was found for skins in concentrated saline. For skins in physiological saline, skin potentials rose sharply from rather low values, when placed in solutions very low in potassium, to relatively high values, when immersed in solutions containing 1 to 5 µeq. K⁺/ml. Further increase in potassium concentration of the bath led to slight reductions in skin potentials. The highest potentials observed were of the order of 40 mv. In all cases studied, the inside was positive with relation to the outside. 5. It can be shown that values for intracellular potassium concentration as a function of extracellular potassium concentration satisfy, at a first but good approximation, Freundlich's isotherm. A modification of Freundlich's isotherm, recently introduced by Sips, may also be used to correlate the experimental data quantitatively. Since the latter isotherm has a rational interpretation, it is suggested that this be used, rather than Freundlich's isotherm, to express quantitatively the dependence of intracellular on extracellular potassium in frog skin.     

Interaction of sodium with the sarcolemmal calcium system.

Sarcolemma isolated from guinea pig heart binds calcium in an ATP-dependent manner. Sodium ions decrease the total amount of calcium bound by the membranes. ATP-dependent calcium binding is more sensitive to sodium than the non-ATP-dependent calcium binding. The ATPase active during calcium binding is affected by sodium ions to the same extent as the ATP-dependent calcium binding process. The inhibition of the calcium binding process and of ATPase activity by sodium was more pronounced when the membranes were preincubated with sodium. The effect of sodium on calcium binding is dependent on both the time of contact between sodium and the membranes and the concentration of sodium. It is suggested that the effect of sodium on the calcium binding system in the sarcolemma may be a link between the inhibition of Na+K+-ATPase (EC 3.6.1.3) by cardiac glycosides and the subsequent increase in intracellular calcium.     

Complex-formation between triphosphoinositide and experimental allergic encephalitogenic protein

1. Reactions between triphosphoinositide and the basic experimental allergic encephalitogenic (EAE) protein were examined in aqueous solution and in a biphasic solvent system (chloroform-methanol-water, 8:4:3, by vol.). 2. In the absence of salt an insoluble complex (I) is formed containing triphosphoinositide and EAE protein in proportions that represent complete neutralization of lipid and protein at the pH concerned. 3. In the presence of a low concentration (0.05m) of sodium chloride an insoluble positively charged complex (II) forms. It contains triphosphoinositide and EAE protein in a lower concentration ratio than complex I. This complex, which has a constant composition between pH7.5 and pH10, can take up additional micellar triphosphoinositide producing complex I, which can then be solubilized by excess of triphosphoinositide. 4. The complexes are dissociated by more concentrated sodium chloride solutions and low concentrations of calcium chloride, suggesting that they are largely stabilized by electrostatic bonds. The protein recovered after dissociation is immunologically active and has the same electrophoretic mobility as the original. 5. Water-insoluble ternary complexes containing triphosphoinositide, EAE protein and large amounts of phosphatidylcholine can be prepared. From these, chloroform-methanol (2:1, v/v) extracts only phosphatidylcholine. 6. An insoluble ternary complex of Ca(2+) ion, EAE protein and triphosphoinositide can be prepared by adding calcium chloride to a complex I preparation solubilized by excess of triphosphoinositide. 7. EAE protein will also form complexes with other acidic phospholipids, e.g. phosphatidic acid, phosphatidylserine and phosphatidylinositol, but not with phosphatidylcholine or phosphatidylethanolamine. The phosphatidylinositol and phosphatidylserine complexes are chloroform soluble, i.e. proteolipids. 8. The possibility that complexes between EAE protein and acidic phospholipids occur in vivo is discussed. Triphosphoinositide and EAE protein occur in ox brain myelin in approximately the same concentration ratios as they do in complex II, formed at physiological salt concentration and pH.     

Potassium- and ionophore A23187-induced discharge of secretory protein in guinea pig pancreatic lobules. Role of extracellular calcium

Elevated concentrations of potassium chloride (50 to 120 mM) in the incubation medium stimulated in vitro discharge of secretory protein from guinea pig pancreatic lobules. The effect of potassium was not inhibited by 10(-4) M atropine, sodium substitutes, or 10(-5) M tetrodotoxin. Exposure of lobules to elevated concentrations of potassium chloride did not increase the release of tissue lactic dehydrogenase and resulted in the appearance of exocytotic images detected by electron microscopy. The time course and extent of discharge due to 75 mM KCl were similar to those caused by the ionophore A23187 and the secretory effect of both agents depended on extracellular calcium and intracellular energy reserves. Potassium chloride stimulation of 75 mM increased the influx of extracellular calcium by 49%, as measured by net 45Ca uptake. Optimal carbamylcholine chloride or pancreozymin stimulation consistently showed a greater effect on discharge than optimal KCl or A23187 stimulation and the additional effect depended on the ability of these physiological secretagogues to recruit calcium from intracellular sources. Potassium chloride stimulation did not result in cyclic GMP elevations in the presence of atropine and those elevations due to A23187 stimulation were small (21 to 30%) and dissimilar both in character (calcium dependence) and time course compared to those resulting from the physiological secretagogues. These findings allow us to define two interrelated pathways which couple hormonal stimulation and discharge of secretory protein in the exocrine pancreas.     

On the effects of divalent cations and ethylene glycol-bis-(beta- aminoethyl ether) N,N,N'',N''-tetraacetate on action potential duration in frog heart

Resting and action potentials were recorded from superfused strips of frog ventricle. Reducing the bathing calcium concentration ([Ca2+]0) with or without ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) prolongs the action potential (AP). The change in the duration of the AP extends over many minutes, but is rapidly reversed by restoring calcium ions. Other changes (e.g., in resting potential and overshoot) are, however, only more slowly reversed. Reducing [Ca2+]0 with 0.2, 2, or 5 mM EGTA produces progressively greater prolongation of AP; maximum values were well in excess of 1 min. This prolongation can be reversed by other divalent cations in EGTA (Mg2+, Sr2+) or Ca-free (Mn2+) solutions, or by acetylcholine. Barium ions increase AP duration in keeping with their known effect on potassium conductance. D600, which blocks the slow inward current in cardiac muscle, is without effect on the action potentials recorded in EGTA solutions, or on the time course and extent of the recovery to normal duration upon restoring calcium ions. It is concluded that divalent cations exert an influence on membrane potassium conductance extracellularly in frog heart. The cell membrane does not become excessively "leaky" in EGTA solutions.     

The inhibitory effect of strophanthidin on secretion by the isolated gastric mucosa

The unidirectional fluxes of Na⁺ and Cl^- were measured across the isolated gastric mucosa of the bullfrog (R. catesbiana). The addition of strophanthidin, a cardiac aglycone, resulted in marked reductions of the spontaneous potential and short-circuit current. Associated with these changes, the isolated gastric mucosa ceased secreting chloride and hydrogen ion. Although the active component of chloride transfer was inhibited, the exchange diffusion component seemed to increase. No significant changes in membrane conductance or sodium flux were noted. Possible mechanisms of strophanthidin inhibition were discussed in view of its effect on chloride transport across the gastric mucosa and on sodium and potassium transfer in other tissues. It was concluded that the cardiac glycosides may not be specific inhibitors of sodium and potassium transport. This non-specific inhibition suggests that active chloride transport is affected by strophanthidin directly and/or anion secretion is dependent upon normal functioning of cation transport systems in the tissue.     

THE EFFECT OF POTASSIUM ON THE ACID METABOLISM OF SURVIVING SKELETAL AND CARDIAC MUSCLES OF THE FROG

The potassium contraction of skeletal muscle and relaxation of cardiac muscle have been correlated with the carbon dioxide and total acid production of these tissues. 1. The immersion of surviving sartorius muscles of the frog in isotonic potassium chloride solution causes a marked increase in the rate of acid production. 2. It is probable that carbon dioxide is the principal acid involved in the above effect. 3. The immersion of surviving cardiac muscle of the frog in isotonic potassium chloride solution causes a pronounced depression in the rate of survival acid production. 4. Reasons are given for believing that these changes in metabolism may be independent of the stimulation and inhibition of contraction which potassium simultaneously produces in these tissues.     

Effect of various chloride salts on the utilization of phosphorus, calcium, and magnesium

Only part of the effect of dietary protein on urinary calcium excretion can be ascribed to sulfur amino acids. We hypothesized that chloride, another factor often associated with isolated proteins, and another amino acid, lysine, affect utilization of calcium. The effects of supplemental dietary chloride, inorganic or organic, on calcium, phosphorus, and magnesium utilization were studied in two rat studies. Weanling Sprague-Dawley rats were fed semi-purified diets that contained moderate (1.8 mg Cl/g diet) or supplemental (15.5 mg Cl/g diet) chloride as sodium chloride, potassium chloride, or lysine monohydrochloride with or without calcium carbonate for 56 or 119 days. Rats fed supplemental sodium chloride or potassium chloride had higher urinary phosphorus excretion, more efficient phosphorus absorption, but unchanged tissue phosphorus levels after 7 and 16 weeks of dietary treatment as compared to rats fed moderate chloride. Rats fed supplemental sodium chloride or potassium chloride excreted more calcium in urine at 7 weeks and absorbed calcium less efficiently at 16 weeks. Tissue calcium concentrations were unaffected, but total tibia magnesium and plasma magnesium concentrations were lower in rats fed supplemental sodium chloride or potassium chloride than those fed moderate chloride. Lysine chloride with or without additional calcium elevated urinary calcium excretion even more than sodium chloride and potassium chloride ingestion. Rats fed lysine chloride with supplemental calcium had smaller apparent absorption and urinary losses of phosphorus and magnesium after 16 weeks and lower tibia and plasma magnesium concentrations than rats fed lysine chloride.     

Effect of calcium on the potassium flux into the exudate of excised maize roots

Summary The effect of varying calcium concentration in the medium on the potassium flux into the exudate has been studied. In media of low ionic strength (o.1 mM KCl) the potassium flux, J _K, was significantly increased by increasing the calcium concentration of the medium. But in higher ionic strength media (10 mM) KCl) there was no increase in J _K as the calcium concentration of the medium was increased. The effect of external sodium concentration on J _K was also studied. These results are discussed in relation to present theories of salt and water movement into the plant root. It is concluded that two pathways potentially exist for movement of salts to the exudate stream: firstly, via a symplasm and secondly, through the cell wall pathway. But is is further concluded that the cell wall pathway, at normal physiological ionic strengths, is not available for salt transport due to co-ion exclusion by the fixed negative charges.     

Salinity perception by larvae of the crab Rhithropanopeus harrisii (gould)

The mechanism of salinity perception was measured in Stage I zoeae of the estuarine crab Rhithropanopeus harrisii. The behavioral assay for perception of a salinity decrease was the loss of positive phototaxis while the reversal in geotactic sign from positive to negative was used for studying a salinity increase. A change in sodium chloride concentration is the primary environmental cue signalling a salinity change. However, neither sodium nor chloride per se is required for detection, since other salts such as lithium nitrate, can substitute for sodium chloride. The minor ions of seawater (calcium, magnesium, potassium and sulfate) have a modulating effect upon larval behavior, but play no role in salinity perception. The exact mechanism for perception of an increase and a decrease in salinity is different. Substitution experiments indicate that in the perception of a salinity decrease, only salts having a cation similar in size to sodium can mimic salinity changes whereas in the detection of a salinity increase, cation size is not an important parameter. The stimulation effectiveness of anions is correlated with neither size nor valence. Though osmolality is probably not involved in perceiving a decreased salinity, it may serve a role in the detection of a salinity increase. A charge in pH has no effect upon perception of a salinity decrease, but does affect perception of a salinity increase. A classical salt receptor appears to be involved in sensing a salinity decrease, while a different type of receptor is used for detecting an increase in salinity.     

The theaflavin fraction is responsible for the facilitatory effect of black tea at the skeletal myoneural junction

The effect of various fractions of black tea [(Camellia Sinensis) (L) O. Kuntze (Theaceae)] on the function of mammalian skeletomotor apparatus was studied. The theaflavin fraction (Tfs) produced a concentration- dependent facilitation of indirect twitch responses of the rat phrenic nerve diaphragm preparation and the facilitation was dependent on the amount of calcium present in the bathing fluid. Nifedipine reduced the facilitatory effect of Tfs as a function of its concentration. Tfs failed to produce facilitation when the twitch height was reduced to about 50% of the control value in presence of magnesium chloride. Tfs completely antagonized the submaximal paralytic effect of d- tubocurarine and decamethonium bromide. Tfs did not have any effect on direct twitch responses or on acetylcholine (Ach) and potassium chloride (KCl) induced contractures of denervated diaphragm. The results revealed that the site of action of Tfs is on the contractile mechanism of the voluntary muscle and point to a critical role of calcium in the mechanism of action of Tfs. N omega-nitro-L-arginine-methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, antagonized both the facilitatory and inhibitory effects on indirect twitch responses of rat diaphragm induced by L-arginine and Tfs when the phrenic nerve was stimulated at 5 Hz and 50 Hz respectively. The thearubigin (Trs) fraction of black tea and the aqueous part which is completely devoid of Tfs, did not potentiate the twitch responses. The findings suggest that Tfs have a potentiating effect on the contractile mechanism of skeletal muscle and that calcium and nitric oxide may modulate this action of Tfs.