CHANGES IN PLATELET FREE Ca2+ CONCENTRATION AFTER CHRONIC DIGOXIN TREATMENT

Summary— Cell Na+ and Ca²⁺ concentrations control each other by various mechanisms. In excitable cells from various origins, Ca²⁺ extrusion from the cell and its entry are dependent for a large part on the activity of the Na+, Ca²⁺-countertransport system. Cytosolic free Ca²⁺ concentration is also controlled by the Na+–H+ exchange activity. To analyze the changes in cytosolic Ca²⁺ concentration accompanying the reduction of the membrane Na+ gradient, cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured by fluorescent dyes in platelets and erythrocytes from healthy subjects, before and during digoxin treatment (0.25 mg/day for 6 days). [Ca²⁺]_i was increased in platelets from 169±30 to 321±61 nmol/l ( n = 7, P <0.02) and unchanged in erythrocytes (121±6 and 104±7 nmol/l). This increase in platelet [Ca²⁺]_i was not accompanied by a change in serotonin content (5.43±0.67 vs 5.49±0.61 10⁻⁷ mol per 10¹¹ cells) and could not be reproduced by in vitro addition of 10⁻⁴ mol/l ouabain (198±33 vs 186±73 nmol/l). The enhanced [Ca²⁺]_i in platelets is thus not a short-term consequence of a reduced membrane Na+ gradient, but reflects either the overload of intracellular Ca²⁺ stores or an enhanced in vivo stimulation by hormones or neurotransmitters.     

Imaging of Ca2+-induced cytoplasmic Ca2+ responses in normal and pathological parathyroid cells

Ca²⁺-induced changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) were studied in bovine and normal and pathological human parathyroid cells using digital image analysis of fura-2-loaded cells. When raising external Ca²⁺ from 0.5 to 3.0 mmol L⁻¹, about 95% of all cells reacted rapidly and simultaneously with sustained elevation of [Ca²⁺]_i. In approximately two out of three bovine parathyroid cells, normal human cells and cells from most patients with hyperparathyroidism (HPT) the sustained phase was preceded by an overshooting [Ca²⁺]_i transient. The proportion of cells with such a transient was decreased in cells from severe cases of uraemic parathyroid hyperplasia only. However, pathological human cells from adenomas and normal-sized glands associated with adenomas, as well as cells from primary and uraemic hyperplasias, had lower peak and sustained levels than normal human and bovine cells. The results indicate that both normal and pathological parathyroid cells exhibit heterogeneity in their [Ca²⁺]_i responses to elevation of external Ca²⁺. The Ca²⁺-induced [Ca²⁺]_i transients and the sustained elevations are attenuated in pathological human parathyroid cells. However, the presence of the overshooting transient represents physiological variability rather than being a consequence of the pathophysiology associated with HPT.     

Endothelin-1 does not modulate O2· release and [Ca2+]i variations in resting or differentiated HL-60 cells

Summary— Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing superoxide (O₂*) generation in human resting or DMSO-differentiated neutrophil-like HL-60 cells. ET-1 (0.01 – 100 nM) was not able to modulate O₂* generation stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, EC₅₀ = 4.24 ± 1.63 nM in the absence and 3.16 ± 1.95 nM in the presence of ET-1). Neither did ET-1 (0.01 – 100 nM) promote the mobilization of intracellular calcium ions or modulate fMLP-induced [Ca²⁺]_i increase in this model of human neutrophils. Phosphoramidon, a neutral endopeptidase inhibitor, was not able to reveal any biological (O₂*) or biochemical ([Ca²⁺]_i) response to ET-1 in the absence or in the presence of fMLP in these cells. These results indicate that DMSO-differentiated neutrophil-like HL-60 cells are not sensitive to ET-1 in terms of O₂* generation or [Ca²⁺]_i variations.     

Transforming growth factor β1 modulates angiotensin induced calcium influx in vascular smooth muscle

Abstract. The modulatory effects of transforming growth factor β₁ (TGF β₁) on the angiotensin II (Ang II)-induced increase in cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). [Ca²⁺]_i in VSMC was measured using the fluorescent dye fura-2. When TGF β₁ was applied 30 s prior to Ang II, the Ang II-induced [Ca²⁺]_i increase was significantly enhanced in VSMC from SHR ( P < 0.05 compared to control), whereas after the preincubation with TGF β₁ for 30 min, the Ang II-induced [Ca²⁺]_i increase was significantly reduced in VSMC from both strains. Using the manganese-quenching technique, it was confirmed that short-term exposure to TGF β₁ enhanced the Ang II-induced trans-plasma-membrane calcium influx in SHR. The inhibition of protein kinase C by calphostin C abolished the stimulatory effect of TGF β₁ on the Ang II-induced [Ca²⁺]_i increase. It is concluded that TGF β₁ modulates the Ang II-induced calcium handling in VSMC.     

Activation of the respiratory burst in eosinophil leucocytes—a transduction sequence decoupled from cytosolic Ca2+ rise

Abstract. The activation of the respiratory burst by complement factor 5a (C5a), platelet-activating factor (PAF), formyl-Met-Leu-Phe (fMLP) and neutrophil-activating peptide IL-8 was explored in eosinophils from patients with the hypereosinophilic syndrome. The amplitude of the response increased with increasing concentrations of C5a and PAF, but the time for its induction was unaffected by the amount of stimulus applied. Respiratory burst activity resulting from phorbol 12-myristate, 13-acetate (PMA)-mediated activation of protein kinase C (PKC) produced longer onset times, which shortened with increasing PMA concentrations. Total inhibition of the C5a- and PMA-mediated burst could be achieved with the PKC inhibitor staurosporine at concentrations of 100 and 5 nM, respectively. Calcium depletion abolished agonist-induced rises in cytosolic free calcium ([Ca²⁺]_i) and respiratory burst activity, but not PMA-mediated NADPH-oxidase activation. While PMA reduced elevations in [Ca²⁺]_i, it restored the burst response to agonists in Ca²⁺-depleted eosinophils. These results agree with the agonist-induced activation of the NADPH-oxidase via PKC, but suggest a parallel, Ca²⁺-, phospholipase C- and PKC-independent signal transduction pathway. Data obtained with B. pertussis toxin showed that the respiratory burst in eosinophils is blocked by ADP-ribosylation of G_i-proteins, but that in the presence of PMA portions of the agonist response could be recovered.     

Local and systemic effects of Na+/K+ ATPase inhibition

The positive inotropic and electrophysiological effects of cardiac glycosides on cardiac muscle are mediated through inhibition of Na⁺/K⁺ ATPase by binding to a specific extracytoplasmic site of the a-subunit of this enzyme. The inhibition of Na⁺/K⁺ ATPase affects ionic flux and produces direct local effects on cardiac contractility, electrical excitability and conduction, but also profound systemic effects mainly as a result of haemodynamic changes. These effects are responsible for beneficial therapeutic as well as toxic effects.
Inhibition of Na⁺/K⁺ ATPase results in potentiation of K⁺ loss from cells and Na⁺ entry into cells, so consequently affects action potential generation and propagation. This also underlines the potentiation of certain effects of cardiac glycosides by hypokalemia and hypomagnesaemia, and the effects of changes in calcium homeostasis on the cardiac glycoside pharmacodynamics. Furthermore, inhibition of Na⁺/Ca⁺⁺ exchange enhances Ca⁺⁺ mobilization and promotes contractility. These effects (locally and systemically) differ greatly, depending on the haemodynamic status and myocardial oxygen supply.
Cardiac glycosides have less affinity for Na⁺/K⁺ ATPases at other sites (e.g. skeletal muscle), but some extracardiac effects (vascular effects, effects on colour vision, CNS and autonomic effects, renal effects) may be related to Na⁺/K⁺ ATPase inhibition.     

Effects of K+ channel inhibitors and antagonists on NS-004 evoked relaxations in guinea-pig isolated trachea

Summary— The smooth muscle relaxant responses to NS-004, an activator of charybdotoxin-sensitive, large conductance Ca²⁺-dependent K⁺ channels (BKCa) were studied on the basal spontaneous tone in guinea-pig trachea in vitro. The sensitivity of these responses to a range of K⁺ channel inhibitors and antagonists were also evaluated. NS-004 (0.1–30 μM) evoked concentration-related relaxations (pIC₅₀ 5.48 ± 0.13) on the spontaneous tone in guinea-pig tracheal rings, suspended in Krebs bicarbonate solution, with a maximum response not different to that to aminophylline (1 μM). Charybdotoxin (0.03 and 0.1 μM) or iberiotoxin (0.1 μM) significantly displaced the NS-004 concentration-response curve to the right of control with no change in maximum response. In contrast, glibenclamide (1.0 μM), apamin (0.1 μM) and dofetilide (1.0 μM) each failed to modify the responses to NS-004 on spontaneous tone in guinea-pig trachea. These results suggest that relaxations in guinea-pig tracheal smooth muscle to the substituted benzimidazolone, NS-004, involve the activation of BK_Ca channels.     

The cytoplasmic Ca2+ response to glucose as an indicator of impairment of the pancreatic β-cell function

Abstract. The effects of glucose on the cytoplasmic Ca²⁺ concentration (Ca²⁺_i) regulating insulin release were investigated using pancreatic β-cells representative for the normal and diabetic situations. Increase of the glucose concentration resulted in a slight lowering of Ca²⁺_i followed by a rise, often manifested as high amplitude oscillations. The Ca²⁺_i-lowering component in the glucose action associated with suppression of insulin release became particularly prominent when the β-cells were already depolarized by tolbutamide. Glucose-induced inhibition of insulin release was observed also in experiments with rats made diabetic with streptozotocin or alloxan. Other studies indicated lowering of plasma insulin after intravenous glucose administration in patients with insulin- and noninsu-lin-dependent diabetes mellitus. Brief exposure of β-cells to 2–2 mmol 1^-1 streptozotocin resulted in impairment of the response to glucose, manifested as disappearance of the cyclic variation of Ca²⁺_i. The results indicate that glucose-induced depolarisation is a vulnerable process, the disturbance of which may contribute to insulin secretory defects in diabetes mellitus.     

Inhibitory effects of tacrine and physostigmine on catecholamine secretion and membrane currents in guinea-pig adrenal chromaffin cells

Summary— The effects of tacrine and physostigmine on catecholamine secretion induced by veratridine and high K⁺, and on voltage-dependent Na⁺ and Ca²⁺ currents, were investigated in guinea-pig adrenal chromaffin cells. In perfused adrenal glands, tacrine (100 μM) caused an inhibition of veratridine-induced catecholamine secretion, but physostigmine (100 μM) did not. In dispersed cells, both tacrine (1 μM-1 mM) and physostigmine (1 μM-1 mM) decreased catecholamine secretion induced by veratridine in a dose-dependent manner. The inhibitory effect of tacrine was much greater than that of physostigmine. Tacrine alone at a high concentration (such as 1 mM) caused a substantial increase in catecholamine secretion by itself and completely abolished the veratridine-induced secretory response in dispersed cells. High-concentration physostigmine showed a similar effect, but to a much lesser extent. The high K⁺ (46.2 mM)-evoked catecholamine secretion from dispersed cells was not affected by tacrine (1–100 μM) or physostigmine (1 μM-1 mM). In fura-2 loaded cells, tacrine (100 μM) almost abolished [Ca²⁺]i rise induced by veratridine, but only slightly reduced that evoked by high K⁺. In voltage-clamped cells, tacrine (300 μM) depressed the voltage-dependent Na⁺ and Ca²⁺ currents by about 93% and 69%, and physostigmine (300 μM) depressed them by about 30% and 17%, respectively. These results suggest that tacrine decreases the veratridine-induced catecholamine secretion primarily by inhibiting the voltage-dependent Na⁺ channels rather than the Ca²⁺ channels. Physostigmine acts in a manner similar to tacrine, but its potency is much lower than that of tacrine.     

Inhibited neutrophil functions in patients treated with nifedipine but not with verapamil or diltiazem

Neutrophil functions were studied in patients receiving calcium channel blockers: nifedipine, diltiazem or verapamil. Neutrophils from patients treated with nifedipine showed a significantly lower superoxide generation stimulated by phorbol myristate acetate (PMA) (50 ng mL⁻¹), opsonized zymosan (1 mg mL⁻¹) or formyl-methionyl-leucyl-phenylalanine (FMLP) (10⁻⁷  m ), whereas superoxide generation by neutrophils of patients receiving diltiazem or verapamil showed only a slight and insignificant reduction compared with controls. Similarly, chemotaxis towards 10⁻⁷  m FMLP and phagocytosis were significantly lower in patients receiving nifedipine compared with controls and were only slightly reduced in patients receiving diltiazem or verapamil. Nifedipine was the most efficient drug in inhibiting the rise in intracellular calcium ion concentration ([Ca²⁺]_i) when added in vitro and in neutrophils of patients receiving this drug, whereas verapamil had no significant effect. The correlation between the inhibitory effect of nifedipine on neutrophil function and the elevation of [Ca²⁺]_i suggests that nifedipine inhibits neutrophil functions through its effect on [Ca²⁺]_i. However, it is not the sole mechanism as superoxide generation induced by PMA, an agent that does not induce a rise in [Ca²⁺]_i, is also inhibited. The unique effect of nifedipine in reducing neutrophil functions in vivo suggests its clinical implications concerning response to acute ischaemic myocardial events.     

Atrial Membranes Contain Nucleoside Diphosphate Kinase (NDPK) Activity: Its Role in Regulation of Muscarinic K+ Channels

Guinea pig or rabbit atrial muscarinic K⁺ channels in cell-free inside-out patches ran be activated in the absence of extracellular agonist and cytoplasmic G nucleotides by intracellular ATP-Mg²⁺,^1,2 This ATP-dependent activation is compatible with the existence of a membrane-bound nudeos de diphosphate kinase (NDPK), which directly phosphorylates G_K-bound GDP. We show that this ATP-dependent activation is also possible in frog atria] cells, and that atrail membranes of frog and guinea pig contain NDPK activity. The relative order of different nucleoside triphogphates (NTPs) as phosphate donors parallels the observed efficiency of these nucleotides in activation of the channels. Thus, atrial membranes contain NDPK activity, which can be responsible for the ATP-dependent activation o/muscarinic K⁺ channels, seen in patches of atrial cells. Under physiological conditions, NDPK can act as a GTP supply in the immediate vicinity of the G protein to ensure reliable signal transduction.     

Erythrocyte Na+–H+ exchanger kinetics and Na+–Li+ countertransport activity in essential hypertensive patients

The authors measured Na⁺–H⁺ exchanger kinetics together with Na⁺–Li⁺ countertransport V _max in the erythrocytes of 21 subjects with essential hypertension and 16 normotensive control subjects. Na⁺–H⁺ exchanger V _max appeared to be increased in patients with essential hypertension, while the Na⁺–H⁺ exchanger affinity for intracellular proton sites ( K _50%) proved to be unchanged and the index of cooperativity among intracellular proton binding sites as measured by Hill's coefficient (Hill's n ) decreased as compared with normotensive control subjects. Na⁺–Li⁺ countertransport V _max appeared to be higher in patients with essential hypertension than in control subjects. The authors were unable to find any correlations between Na⁺–H⁺ exchanger kinetic parameters and metabolic variables such as parameters of insulin resistance and plasma lipids. On the basis of the data obtained, erythrocyte Na⁺–H⁺ exchanger activity was found to be abnormal in two kinetic variables in essential hypertensive patients and showed no simple linear correlations with the main variables of glucose metabolism, plasma lipids, renin or aldosterone.     

Potentiating effects of nicorandil on the adenosine A2 receptor-mediated vasodepression in rats: potential role for KATP channels

Summary— The effects of nicorandil on systemic blood pressure (SBP) and heart rate (HR) responses to adenosine were compared with those to N⁶-cyclopentyladenosine (CPA), a selective adenosine A, receptor agonist, and 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA), a selective adenosine A₂ receptor agonist, in anesthetized rats. When injected intravenously (iv), single bolus doses of CPCA (0.01–1.0 μg/kg), like adenosine (30 μg/kg), elicited dose-dependent decreases in SBP scarcely affecting HR, while CPA (0.03–1.0 μg/kg) produced only reduction of HR without influencing SBP. The enhancement of the vasodepressor response to CPCA, like adenosine, was induced by the iv infusion of either nicorandil (10 μg/kg per min) or cromakalim (0.1 μg/kg per min), but the response to CPA in HR remained unmodified during the infusion of nicorandil as well as cromakalim. After iv treatment with glibenclamide (20 mg/kg), an adenosine triphosphate (ATP)-sensitive K⁺ channel blocker, or 3,7-dimethyl-1-propargylxanthine (DMPX) (1 mg/kg), a selective antagonist of adenosine A₂ receptor, not only CPCA action but also the enhancement of CPCA action by nicorandil and cromakalim were significantly attenuated. Similar results were obtained in the case of single bolus iv adenosine. The present result indicates that the augmentation of the adenosine action by nicorandil appears to be mediated by activation of ATP-sensitive K⁺ channels, closely linked with stimulation on A₂ receptors by adenosine.     

Modulation of the calcium sensitivity of the smooth muscle contractile apparatus: molecular mechanisms, pharmacological and pathophysiological implications

Summary— Smooth muscle contraction is the basis of the physiological reactivity of several systems (vascular, respiratory, gastrointestinal, urogenital…). Hyperresponsiveness of smooth muscle may also contribute to a variety of problems such as arterial hypertension, asthma and spontaneous abortion. An increase in cytoplasmic calcium concentration ([Ca²⁺]_i) is the key event in excitation-contraction coupling in smooth muscle and the relationship linking the [Ca²⁺]_i value to the force of contraction represents the calcium sensitivity of the contractile apparatus (CaSCA). Recently, it has become evident that CaSCA can be modified upon the action of agonists or drugs as well as in some pathophysiological situations. Such modifications induce, at a fixed [Ca²⁺]_i value, either an increase (referred to as sensitization) or a decrease (desensitization) of the contraction force. The molecular mechanisms underlying this modulation are not yet fully elucidated. Nevertheless, recent studies have identified sites of regulation of the actomyosin interaction in smooth muscle. Sensitization primarily results from the inhibition of myosin light chain phosphatase (MLCP) by intracellular messengers such as arachidonic acid or protein kinase C. In addition, phosphorylation of thin filament-associated proteins, caldesmon and calponin, increases CaSCA. Activation of small (monomeric) G-proteins such as rho or ras is also involved. Desensitization occurs as a consequence of phosphorylation of myosin light chain kinase (MLCK) by the calcium-calmodulin activated protein kinase II, or stimulation of MLCP by cyclic GMP-activated protein kinase. In the present review, examples of physiological modulation of CaCSA as well as pharmacological and pathophysiological implications are illustrated for some smooth muscles.     

A single isoform of the Na+/K+-ATPase α-subunit in Diptera: evidence from characterization of the first extracellular domain

The first extracellular domain of the α-subunit of the Na⁺/K⁺-ATPase (sodium/potassium pump) is functionally important, affecting sensitivity of the enzyme to cardiac glycosides (e.g. ouabain) and being implicated in the transport of K⁺. This domain is also variable among mammalian isoforms of the α-subunit. Using PCR, we have isolated from seven insect species with contrasting physiologies a DNA fragment containing this region, in order to help determine whether tissue-specific expression might be associated with isoforms encoded by a gene family, as it is in mammals. A single sequence (with one ORF) characteristic of Na⁺/K⁺-ATPase was obtained from genomic DNA of each species. Only the fragment from Manduca sexta contained an intron, but at a location different to that found in mammals. For all Diptera so far characterized, the species phylogeny is the same as the α-subunit gene phylogeny (based on the sequences of the first extracellular domain and flanking transmembrane domains). The results strongly indicate a single, ouabain-sensitive isoform of the α-subunit of Na⁺/K⁺-ATPase is present in Diptera.     

Inhibitory effects of okadaic acid on rat uterine contractile responses to different spasmogens

Summary— In the present study, we examined the effects of okadaic acid, a selective inhibitor of type 1 and 2A protein phosphatases, on the mechanical responses evoked by oxytocin, K⁺- and Na+-modified solutions and ouabain in estrogen-primed rat myometrium. Oxytocin elicited a rapid, phasic contraction followed by rhythmic oscillations. The phasic response was partially resistant to the absence of external Ca²⁺. Okadaic acid (1 μM) and the L-type calcium channel blocker nifedipine (1 μM) abolished the oscillatory component and reduced the initial, phasic response to about 80% of the control response. High K⁺ (60 mM) solution, ouabain (1 mM), K⁺-free medium and low Na⁺ (25 mM) solution induced extracellular Ca²⁺-dependent biphasic responses composed by an early rapid (KCl, ouabain and K⁺-free solution) or slower developed (25 mM Na⁺ solution) phasic contraction followed by a sustained increase in tension. Okadaic acid and nifedipine, alone or in combination, abolished or decreased similarly the contractile response evoked by these stimulants. The okadaic acid- and nifedipine-insensitive responses to ouabain, K⁺-free and low Na⁺ solution were enhanced by increasing the extracellular concentration of Ca²⁺ in the medium and were inhibited in a dose-dependent manner by amiloride (0.05–0.5 mM). These data suggest that, in estrogen-primed rat uterus, dephosphorylating mechanisms by OA-sensitive protein phosphatases play an important role in regulating myometrial contractions elicited by Ca²⁺ entry through voltage-sensitive Ca²⁺ channels.     

Inhibition of Na/Ca exchange stimulates insulin release from isolated rat pancreatic islets

Summary— Na/Ca exchange was recently shown to regulate cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic B-cell. The aim of the present study was to provide direct evidence that inhibition of the activity of the exchange may also increase insulin release. In the presence of extracellular Na+, caffeine stimulated ⁴⁵Ca outflow but did not increase insulin release from islets perifused in the presence of 2.8 mM glucose. By contrast, in the absence of extracellular Na+, caffeine almost failed to increase ⁴⁵Ca outflow and reversibly stimulated insulin release despite the fact that the absence of extracellular Na+ per se reduced basal insulin release. Similar findings were observed in islets perifused at a higher glucose concentration (8.3 mM) except that, in the presence of extracellular Na+, caffeine more markedly increased ⁴⁵Ca outflow and stimulated insulin release. Our data provide direct evidence that inhibition of Na/Ca exchange with resulting blockade of Ca²⁺ outflow may increase insulin release from the pancreatic B-cell under suitable experimental conditions.     

Electrophysiological Remodeling in Human Atrial Fibrillation

Atrial fibrillation (AF) is a progressive disease characterized by cumulative electrophysiological and structural remodeling of the atria. Cellular electrophysiological studies have revealed marked reductions in the densities of the L-type voltage-gated Ca²⁺ current, I_Ca,L, the transient outward K⁺ current, I_TO, and the ultra-rapid delayed rectifier K⁺ current, I_Kur, in atrial myocytes from patients in persistent or permanent AF. The density of the muscarinic K⁺ current (I_KACh) is also reduced, however the inward rectifier K⁺ current (I_K1) density is increased. The net shortening or lengthening of the action potential is dependent on the balance between changes in inward and outward currents. The prominent reduction in I_Ca,L appears to be sufficient to explain the observed decreases in action potential duration and effective refractory period that are characteristic of the fibrillating atria. Earlier studies have shown that calcium overload and perturbations in calcium handling play prominent roles in AF induced atrial remodeling. More recently, we have shown that AF is associated with evidence of oxidative injury to atrial tissue, and suggested that oxidative stress may directly contribute to the pathophysiology of AF. It is anticipated that insights gleaned from mechanistic studies will facilitate the development of improved pharmacological approaches to treat AF and to prevent the progression of arrhythmia. (PACE 2003; 26[Pt. II]:1572–1575)     

Comparative inotropic effect of calcium, endothelin-1, and forskolin on ferret papillary muscles during acidosis

Summary— Acidosis affects multiple steps in the excitation-contraction coupling pathway of myocardium, producing decreased calcium sensitivity of myofibrils and modification of the function of the sarcoplasmic reticulum. Our aim was to evaluate the effectiveness of three different classes of inotropic agents under acidotic conditions: 1) forskolin, an adenylate cyclase activator that enhances cellular cyclic AMP concentrations, 2) elevated extracellular Ca²⁺ and 3) endothelin-1, an activator of the inositol triphosphate, diacylglycerol pathway. Ferret papillary muscles were mounted in organ baths containing normal physiological solution (pH = 7.4). After baseline tension was measured, the muscles were bathed in an acidotic solution (pH = 6.98) that decreased tension to 40% of the control; subsequently, the muscles were washed with normal physiological solution until they returned to baseline. Each inotropic agent was added to the bathing solution in a concentration sufficient to increase tension by 40% above the baseline. Then the solution was made acidotic (pH = 6.98) in the continuous presence of that concentration of inotropic agent and the resultant steady-state developed tension measured. The increases in tension induced by each inotropic agent at normal pH were adjusted to be similar; in contrast, the response to each drug in acidosis was significantly different. Under acidotic conditions, endothelin-1 was the most effective inotropic agent in restoring the depressed developed tension. This was possibly due to enhancement of the myofilament sensitivity to Ca²⁺, which was more effective than increasing [Ca²⁺]_i through elevating extracellular Ca²⁺ or the addition of forskolin which increased [Ca²⁺]_i but desensitized the myofilaments to Ca²⁺.