Ca2+-handling in heart failure--a review focusing on Ca2+ sparks

[Ca2+]i-transients have been shown to be altered in isolated ventricular myocytes from terminally failing human myocardium. It has been demonstrated that one reason for this alteration is a reduction in the Ca2+ content of the sarcoplasmic reticulum (SR). Further investigations were done to investigate, whether there may be an additional defect of the Ca2+-release mechanisms from the SR. These release mechanisms were investigated through the recording of Ca2+ sparks in single human myocytes. In cardiac myocytes, Ca2+ sparks are elementary units of Ca2+ release, which occur spontaneously, or which are triggered by Ca2+ influx through L-type Ca2+-channels (Ca2+-induced Ca2+ release). Ca2+ sparks have been investigated in various animal models of cardiac hypertrophy and cardiac failure and results were conflicting. Discrepancies may be explained by different species and also by the mechanisms underlying hypertrophy and heart failure. This review summarizes our current knowledge on Ca2+ sparks in heart failure.     

Calcium tolerance of isolated rat heart cells

Freshly isolated adult rat heart cells, which initially show the elongated, rod-shaped morphology typical of heart cells in situ, are almost quantitatively converted to rounded contracture forms by exposure to 1 m Ca²⁺. These Ca²⁺-sensitive cells became Ca²⁺-tolerant following a short period of metabolic activity in a low-Ca²⁺ medium, in that they retain their rod-shaped configuration when challenged with Ca²⁺ after this preincubation step. Tolerance to Ca²⁺ develops in parallel with the establishment of low Na⁺/K⁺ ratios in these cells and both processes are sensitive to ouabain. The initial net uptake of Ca²⁺ is greater in Ca²⁺-sensitive than in Ca²⁺-tolerant cells. These results suggest that contracture in the Ca²⁺-sensitive cells is a consequence of the rapid entry of excessive amounts of Ca²⁺ in exchange for internal Na⁺.     

Extracellulary administered lysophosphatidylcholine causes Ca2+ efflux from freshly isolated adult rat cardiomyocytes

It has previously been reported that ischemia and reperfusion of the heart cause accumulation of lyophosphatidylcholine (LPC) within the myocardium. While it is known that LPC causes the transient increase of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) during contraction of cardiac cells, little is known about the mechanism for decreasing [Ca²⁺]_i in cardiomyocytes during LPC accumulation. Since cumulative elevation in [Ca²⁺]_i leads to irreversible injury to cardiomyocytes, elevated [Ca²⁺]_i must be restored to an unstimulated level to maintain cell functions. In the present study, we therefore examined the effect of LPC on Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes. LPC stimulated the efflux of ⁴⁵Ca²⁺ from the cells in a concentration‐dependent manner (10^–7 M – 10^–5 M). Other lysophospholipids, which are generated from phopholipids of the cell membrane, failed to induce ⁴⁵Ca²⁺ efflux from the cells. Dilazep and K‐7259, which are known to inhibit the increase in [Ca²⁺]_i caused by LPC, likewise reduced ⁴⁵Ca²⁺ efflux caused by LPC addition. Furthermore, the LPC‐stimulated ⁴⁵Ca²⁺ efflux was not affected by removal of extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. On the other hand, inhibitors of Na⁺/Ca²⁺ exchange, amiloride and 5‐(N,N‐dimethyl)‐amiloride, inhibited LPC induced ⁴⁵Ca²⁺ efflux. These results suggest that LPC stimulates extracellular Na⁺‐dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through Na⁺/Ca²⁺ exchange on the plasma membrane of cells. Received: 3 March 1997, Returned for revision: 7 April 1997, Revision received: 1 August 1997, Accepted: 5 September 1997     

Alterations in properties of L-type Ca channels in aging rat heart

Previous studies of whole-cell L-type Ca currents in aging heart have demonstrated an increase in the peak Ca current magnitude in proportion to the increase in membrane area, and a slowing of the time course for inactivation. However, the single-channel mechanisms underlying this upregulation, and for the slowed inactivation are not known. We have therefore compared the properties of single L-type Ca channel currents recorded from ventricular myocytes obtained from young adult (3 month), adult (6-8 month) and aging (24 month) Wistar rats, using 5 m m Ba ions as the permeant ion. We report that the peak ensemble-averaged single Ca channel currents from aging heart (-280+/-57 fA) were enhanced compared to those from young adult (-137+/-16 fA), or adult hearts (-144+/-38 fA). This surprising result was related, in part, to an apparent increase in the number of active Ca channels per patch in aging (1.90+/-0.23) v young adult (1.33+/-0.19) or adult heart (1.50+/-0.2). Moreover, there was an increase in the time constant for inactivation of the ensemble-averaged Ca currents of aging (471+/-169 ms), compared with young adult (198+/-43 ms), or adult heart (196+/-32 ms). The aging-related changes were also traced to alterations in single Ca channel gating, including an increase in the average probability of being open, and an increase in the availability of single Ca currents in aging heart. In contrast, the unitary Ca current amplitude was unchanged with aging. These novel findings suggest that the compensatory increase in the L-type Ca currents during aging is a consequence of an apparent increase in both the number, and the activity of individual L-type Ca channels.     

Sarcoplasmic reticulum Ca2+ refilling controls recovery from Ca2+-induced Ca2+ release refractoriness in heart muscle

In cardiac muscle Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca2+ influx via L-type Ca2+ channels. At present, the mechanisms underlying termination of SR Ca2+ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca2+ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca2+ release, we analyzed Na+-Ca2+ exchange currents reflecting cytosolic Ca2+ signals induced by UV-laser flash-photolysis of caged Ca2+. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, beta-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by approximately 2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 micromol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca2+ refilling mediated by the SR Ca2+-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca2+ sensitivity of CICR appears to be regulated by SR Ca2+ content, possibly resulting from a change in the steady-state Ca2+ sensitivity and in the gating kinetics of the SR Ca2+ release channels (ryanodine receptors). During Ca2+ release, the concomitant reduction in Ca2+ sensitivity of the ryanodine receptors might also underlie Ca2+ spark termination by deactivation.     

Direct measurement of L-type Ca2+ window current in heart cells.

The activation and inactivation relations of several ion channel currents overlap, suggesting the existence of a steady-state or "window" current. We studied L-type Ca2+ channel window current in single cardiac Purkinje cells using a voltage-clamp protocol by which channels were first inactivated nearly completely during a long-duration depolarizing step, and then the recovery of Ca2+ current was observed during repolarizing steps into the L-type Ca2+ window voltage range. With these conditions, a small-amplitude inward Ca2+ current gradually developed after repolarization to voltages within the window but not after steps to voltages positive or negative to it. Window current was suppressed by Cd2+ (50 microM), nifedipine (1 microM), and nicardipine (1 microM), and it was augmented by isoproterenol (5 microM) and Bay K 8644 (1 microM). At voltages at which window current developed, L-type Ca2+ channels also recovered to a closed state from which they could be reopened by an additional depolarizing step. At voltages positive to the window range, channel recovery to a closed state(s) was absent, whereas at voltages negative to the window range, channel recovery to a closed state(s) increased, as expected from the "steady-state" inactivation relation. Our results provide direct measurement of L-type Ca2+ window current and distinguish it from other processes, such as slow inactivation. Our findings support the postulate that within a window there occur channel transitions from inactivated to closed states, and these channels (re)open, and this process may occur repetitively. Some physiological and pathophysiological roles for L-type Ca2+ window current are discussed.     

Increased erythrocyte content of Ca2+ in patients with Tarui's disease

OBJECTIVES: To establish by flow cytometry and fluorophores an increased calcium ion load in erythrocytes of four patients with Tarui's disease. DESIGN: Calcium ion levels were determined in erythrocytes of patients and controls under normal and energy-deprived conditions. Adenylates were measured to assess energy status of incubated erythrocytes. SETTING: The experiments were carried out at the Department of Clinical Chemistry of the University Hospital of Uppsala, Sweden. SUBJECTS: Four family members with Tarui's disease participated in the study. The proband (patient 1) was a 39-year-old male; patients (male, aged 46 years) 2 and 3 (female, 30 years) were his two siblings. Patient 4 (male, 16 years) was the son of patient 2. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Calcium ion homeostasis was measured under basic conditions and under energy-deprived conditions and related to cellular adenylate content. RESULTS: All patients showed enhanced erythrocyte calcium ion loading compared to controls under energy-deprived conditions. Under normal conditions, however, three out of the four patients showed an increased erythrocyte calcium ion level compared to controls. CONCLUSIONS: We conclude that erythrocytes from patients with Tarui's disease have an increased Ca2+ permeability, initiating compensatory mechanisms involving increased Ca2+ pump activity and increased glycolytic flux, which are not always sufficient to keep erythrocyte calcium ion concentration within physiological range.     

Experimental myocardial ischemic injury. II. Effect of in vivo ischemia on dog heart slice function in vitro.

The capacity of myocardium, acutely injured in vivo by ischemia, to maintain cell volume regulation in vitro was studied in dogs with posterior papillary muscle infarcts of 60 and 120 min duration. Non-ischemic control tissue maintained mean K⁺ concentrations of about two thirds of the K⁺ found in left ventricular myocardium in vivo and had tissue H₂O volumes which were virtually identical to those observed in vivo. The extracellular space of the control slices was greater than that estimated to be present in vivo. The slices of irreversibly-injured ischemic tissue were greatly swollen, showed low K⁺ and Mg²⁺ and often exhibited structural defects in the plasma membrane of the sarcolemma. In addition, damaged tissue showed a significantly increased inulin diffusible space after 60 and a greater diffusible space after 120 min of ischemia. These findings are new evidence indicating that increased myocardial cell membrane permeability is an early feature of irreversible ischemic injury.     

Synchronous depletion of ATP in isolated adult rat heart cells

We tested the hypothesis that isolated adult rat heart cells could be depleted of most of their ATP without undergoing contracture. Two strategies for ATP depletions were employed. First, cells were exposed to a high level of rotenone plus FCCP. The cells lost 90% of their ATP within 3 min without change in sarcomere length before undergoing contracture. Even though ATP levels were so low, glycolysis from glycogen was maximally activated at this time. Second, cells exposed to repeated cycles of acidic anoxia were depleted of 77% of their ATP without change in sarcomere length and remained rod-shaped when restored to normoxia and neutral pH. The hypothesis was thus confirmed. The results support the previously developed concept that ATP decline in cells can be synchronous, with a similar decline in all cells, or asynchronous, with a sudden decline in different cells at different times. Whether the decline is synchronous or asynchronous depends on the conditions of metabolic impairment. This concept can explain the pattern of ATP decline observed in whole hearts during ischemia, and also the mechanism by which glycolytic ATP appears to protect against contracture.     

Ca2+-handling in heart failure – a review focusing on Ca2+ sparks

[Ca ²⁺] _i-transients have been shown to be altered in isolated ventricular myocytes from terminally failing human myocardium. It has been demonstrated that one reason for this alteration is a reduction in the Ca ²⁺ content of the sarcoplasmic reticulum (SR). Further investigations were done to investigate, whether there may be an additional defect of the Ca ²⁺-release mechanisms from the SR. These release mechanisms were investigated through the recording of Ca ²⁺ sparks in single human myocytes. In cardiac myocytes, Ca ²⁺ sparks are elementary units of Ca ²⁺ release, which occur spontaneously, or which are triggered by Ca ²⁺ influx through L-type Ca ²⁺-channels (Ca ²⁺-induced Ca ²⁺ release). Ca ²⁺ sparks have been investigated in various animal models of cardiac hypertrophy and cardiac failure and results were conflicting. Discrepancies may be explained by different species and also by the mechanisms underlying hypertrophy and heart failure. This review summarizes our current knowledge on Ca ²⁺ sparks in heart failure.     

Electrical and contractile consequences of Na+ or Ca2+ gradient reduction in cultured heart cells

The normal electrical and contractile activity of cultured neonatal rat ventricular cells is characterized and compared to activity seen in low [Ca²⁺]₀ and low [Na⁺]₀ solutions. In 0 Ca²⁺/0.5 mm EGTA solutions electrical activity is altered: the maximum diastolic potential (m.d.p.), maximum rate of rise ($\text{+}\text{V}\text{?}{}_{\mathrm{<mtext>max</mtext>}}$), and overshoot (o.s.) are reduced, while duration is increased. Low [Ca²⁺]₀ activity is insensitive to TTX and blocked by La³⁺. In low [Na⁺]₀ solutions electrical activity is generally absent; when present $\text{+}\text{V}\text{?}{}_{\mathrm{<mtext>max</mtext>}}$ and o.s. are decreased while duration is increased. Low [Na⁺]₀ activity is blocked by La³⁺. These data suggest the presence of one La³⁺-sensitive slow inward current channel. The absence of spontaneous electrical activity in low [Na⁺]₀ solutions suggests an inhibition of automaticity. To determine if this inhibition is due to a reduction of the Na⁺ gradient, drugs which raise [Na⁺]_i were examined. Both monensin (a Na⁺ ionophore) and ouabain inhibit the occurrence of spontaneous action potentials (cells respond to stimulation) indicating a dependence of pacemaker activity on a normal Na⁺ gradient. During Na⁺ gradient reduction, asynchronous subcellular contractile activity occurs independent of membrane potential fluctuation. This asynchronous activity is inhibited by La³⁺ or when Ca²⁺₀ is absent, but continues in the presence of verapamil (normal activity is blocked by all three conditions). The Na⁺Ca²⁺ exchange system is unaffected by verapamil but blocked by La³⁺, while both these drugs block the slow inward current. These data indicate that the Na⁺Ca²⁺ exchange system can directly supply Ca²⁺ (independent of electrical activity at the membrane) to intracellular sites that support contractile activity.     

Increased susceptibility to Ca2+‐induced permeability transition and to cytochrome c release in rat heart mitochondria with aging: effect of melatonin

Abstract: Aging is associated with a decline of cardiac function. The mitochondrial permeability transition (MPT) may be a factor in cardiac dysfunction associated with aging. We investigated the effect of aging and long‐term treatment with melatonin (approximately 10 mg/kg b.w./day for 2 months), a known natural antioxidant, on the susceptibility to Ca²⁺‐induced MPT opening and cytochrome c release in rat heart mitochondria. The mitochondrial content of normal and oxidized cardiolipin as a function of aging and melatonin treatment was also analyzed. Mitochondria from aged rats (24 month old) displayed an increased susceptibility to Ca²⁺‐induced MPT opening, associated with an elevated release of cytochrome c, when compared with young control animals (5 month old). Melatonin treatment counteracted both these processes. Aging was also associated with an oxidation/depletion of cardiolipin which could be counteracted as well by melatonin. It is proposed that the increased level of oxidized cardiolipin could be responsible, at least in part, for the increased susceptibility to Ca²⁺‐induced MPT opening and cytochrome c release in rat heart mitochondria with aging. Melatonin treatment counteracts both these processes, most likely, by preventing the oxidation/depletion of cardiolipin. Our results might have implications in the necrotic and apoptotic myocytes cell death in aged myocardium, particularly in ischemia/reperfusion injury.     

Mechanism of non-capacitative Ca2+ influx in response to bradykinin in vascular endothelial cells

Bradykinin is a potent vasoactive nonapeptide. It elicits a rise in cytosolic Ca(2+) (Ca(2+))(i) in endothelial cells, resulting in Ca(2+)-dependent synthesis and release of endothelial vasodilators. In the present study, we investigated the mechanism of bradykinin-induced Ca(2+) influx in primary cultured rat aortic endothelial cells and in a mouse heart microvessel endothelial cell line (H5V). Bradykinin-induced Ca(2+) influx was resolved into capacitative Ca(2+) entry (CCE) and non-CCE. The non-CCE component was inhibited by a B2 receptor antagonist (HOE140; 1 microM) and a phospholipase C (PLC) inhibitor (U73122; 10 microM). The action of bradykinin could be mimicked by 1-oleoyl-2-acetyl-glycerol, an analogue of diacylglycerol (DAG), and by RHC80267, a DAG-lipase inhibitor. Immunoblots showed that TRPC6 was one of the main TRPC channels expressed in endothelial cells. Transfection of H5V cells with two siRNA constructs against TRPC6 both markedly reduced the TRPC6 protein level and, at the same time, decreased the percentage of cells displaying bradykinin-induced non-CCE. siRNA transfection also reduced the magnitude of non-CCE among the cells responding to bradykinin. Taken together, our data suggest that bradykinin-induced non-CCE is mediated via the B2-PLC pathway, and that DAG may be involved in this process. Further, TRPC6 is one of the important channels participating in bradykinin-induced non-CCE in endothelial cells.     

Plasma membrane Ca2+ release-activated Ca2+ channels with a high selectivity for Ca2+ identified by patch-clamp recording in rat liver cells

Repetitive waves of increased cytoplasmic Ca2+ concentration play a central role in the process by which hormones regulate liver function. Maintenance of these Ca2+ waves requires Ca2+ inflow through store-operated Ca2+ channels. The properties and mechanism(s) of activation of these channels are not well understood. Store-operated Ca2+ channels (SOCs) in the H4-IIE rat liver cell line were studied by whole-cell patch clamping. Depletion of Ca2+ in intracellular stores by intracellular perfusion with either inositol 1,4,5-trisphosphate (InsP(3)) or thapsigargin in the presence of 10 mmol/L ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid (EGTA), or with 10 mmol/L EGTA alone, activated an inward current that reversed at a membrane potential above +40 mV. In physiologic extracellular medium, this inward current was carried exclusively by Ca2+ and was blocked by a variety of di- and trivalent cations. In the absence of extracellular Ca2+ and Mg2+, the inward current was carried by monovalent cations. This current was 10 to 30 times larger than that observed in the presence of extracellular Ca2+, and permitted the detection of single-channel events that corresponded to a single-channel conductance of about 40 pS. Both the Ca2+ and Na+ inward currents were blocked by the calmodulin antagonist, N-(6-amino hexyl)-5-chloro-1-naphthalenesulphonamide (W7), but not by calmidazolium or calmodulin-dependent protein kinase II fragment 290-309. It is concluded that liver cells possess plasma membrane Ca2+ channels that have a high selectivity for Ca2+, are activated by a decrease in the concentration of Ca2+ in intracellular stores through a mechanism that is unlikely to involve calmodulin, and are involved in re-filling intracellular Ca2+ stores during Ca2+ signaling.     

Dual dependence on both Ca2+ and Mg2+ for electrical stability in cells of canine false tendon.

When Ca²⁺ was omitted from the physiologic superfusing solution or when 3.0 mmol/l EGTA was added, cells in 20 canine right ventricular false tendons depolarized from −84±5 mV to −48±9 mV (mean ± 1 s.d.). A similar response (from −83±5 mV to −43±9 mV) omission followed of Mg²⁺ alone or addition of 3.0 mmol/l calcium EDTA. In all trials the degree of depolarization reached a stable level within 30 min. Omission of both Ca²⁺ and Mg²⁺ from the superfusing solution resulted in depolarization from −83±5 mV to −38±11 mV within 19 min. Complete recovery of transmembrane potential from each of these several manipulations was seen within 30 min after resuming superfusion with control solution. In contrast to these observations, addition of 3.0 mmol/l disodium EDTA brought about depolarization from −84±5 mV to −1±5 mV within 5 min. Further-more, superfusion with disodium EDTA for 30 min resulted in permanently depolarized false tendon cells. Studies with electron microscopy demonstrated that La³⁺ was able to cross the sarcolemma of these cells. Thus, the removal of either Ca²⁺ or Mg²⁺ alone, or omission of both, produces a consitent but readily reversible depression of electrical activity. On the other hand, the chelation of both divalent cations causes more rapid and complete depolarization within 5 min and which was not reversible after 30 min. Physiological irreversibility was associated with abnormal permeability of the cells by lanthanum. Electrical stability in canine false tendon cells appears to be dependent on ready availability of both Ca²⁺ and Mg²⁺.     

Annexins and Ca2+ handling in the heart

Annexins are a family of 13 proteins known to bind phospholipids (PL) in a Ca(2+)-dependent way. They are ubiquitous proteins and share a similar structure characterized by a conserved C-terminal domain with Ca(2+) binding sites and a variable N-terminal domain. Depending on Ca(2+) concentration, they have been reported to participate in a variety of membrane-related events such as exocytosis, endocytosis, apoptosis and binding to cytoskeletal proteins. They have also been reported to regulate protein activities. This review will focus on annexins in the heart, and particularly on annexins A2, A5, A6 and A7. Annexin A2 has been found in endothelial cells and reported to play a central role in control of plasmin-mediated processes. Annexin A5 is mainly localized in cardiomyocytes. However, it could be relocated to interstitial tissue in ischemic and failing hearts or it could be externalized and exhibit a proapoptotic effect in cardiomyocytes. Annexin A6 is the most abundant annexin in the heart, and has been localized in various cell types including myocytes. Overexpression of annexin A6 has underlined physiological alterations in contractile mechanics leading to dilated cardiomyopathy, whereas knockout has been found to induce faster changes in Ca(2+) transient and increased contractility, suggesting a negative inotropic role for annexin A6. Annexin A7 is expressed in heart and skeletal muscle. In annexin A7 null mutant mice decreases in the force-frequency relationship were observed in adult cardiomyocytes, consistent with regulation of Ca(2+) handling. In conclusion, while annexin A2 was involved in regulation of fibrin homeostasis, alterations in expression and activity of annexins A5, A6 and A7 have been associated with regulation of Ca(2+) handling in the heart, but the target of each annexin has not yet been identified.     

Enhanced Ca2+-induced contractions and attenuated alpha-adrenoceptor responses in resistance arteries from rats with congestive heart failure

AIM: The aim of the present study was to examine the role of Ca2+-mediated contractile responses in isolated mesenteric resistance arteries from rats with congestive heart failure (CHF). METHODS: Heart failure was induced by ligation of the left coronary artery. Rats exposed to the same surgical procedure except ligation served as controls (Sham). The following experiments were conducted: (1) passive increase in radial stretch (the length-tension relationship) in Ca2+-free and in depolarizing high K+-solution; (2) the contractile responses to external application of Ca2+ and high K+-solutions in the presence of nifedipine and phentolamine; and (3) a histological evaluation of CHF and Sham vessels. RESULTS: The length-tension induced response in Ca2+-free buffer solution was significantly lower in arteries from CHF rats, starting at a very low tension (0.9+/-0.2 mN/mm for heart failure and 1.7+/-0.2 mN/mm for Sham). This difference, but at a higher degree of stretch, was also present in K+-activated vessels. The external application of Ca2+ in K+-depolarized vascular segments in the presence of phentolamine (1 microM) induced an enhanced contractile response in arteries from CHF rats compared with Sham (4.8+/-0.3 mN/mm and 3.6+/-0.6 mN/mm, respectively, P=0.059). In the absence of phentolamine the reverse response was found (4.0+/-0.4 mN/mm and 5.7+/-0.3 mN/mm for CHF vs. Sham respectively, P=0.035). Application of increasing concentrations of K+-solution induced a stronger contractile response in Sham compared with CHF arteries (Sham 4.9+/-0.4 and heart failure 4.0+/-0.3, P=0.04). Microscopic examination of vessels yielded no difference in gross morphology, media thickness or wall to lumen ratio between CHF and Sham arteries. CONCLUSION: The results indicate an attenuation of alpha-adrenoceptors and a difference of Ca2+-mediated vascular contractility in resistance arteries of congestive heart failure rats.     

Effects of Ca2+ agonists on cytosolic Ca2+ in isolated hepatocytes and on bile secretion in the isolated perfused rat liver.

The effects of increases in cytosolic Ca2+ on hepatocyte bile secretion are unknown. A number of agents that alter levels of cytosolic Ca2+ in the hepatocyte also produce hepatic vasoconstriction and activate protein kinase C, which complicates interpretations of their effects on bile secretion. To better understand the role of cytosolic Ca2+ in bile secretion, we examined the effect of the Ca2+ ionophore A23187 (0.1 mumol/L), the Ca2+ agonist vasopressin (10 nmol/L) and the Ca(2+)-mobilizing agent, 2,5-di(tert-butyl)-1,4-benzohydroquinone (25 mumol/L) on cytosolic Ca2+ in isolated hepatocytes and on bile flow in the isolated perfused rat liver, using vasodilators and inhibitors of protein kinase C and Ca2+ influx. Single-pass perfused livers were used, and cytosolic Ca2+ was measured by luminescent photometry in isolated hepatocytes loaded with the Ca(2+)-sensitive photoprotein aequorin. After A23187 perfusion, a sustained 74% +/- 10% (mean +/- S.D.) decrease in bile flow and a sustained 271% +/- 50% increase in perfusion pressure was observed. Simultaneous pretreatment with the vasodilator papaverine (25 mumol/L) and the protein kinase C inhibitor H-7 (50 mumol/L) abolished the pressure increase but not the decrease in bile flow, whereas pretreatment with Ni2+ (25 mumol/L) to block the influx of extracellular Ca2+ markedly reduced both the pressure increase and the decrease in bile flow. Vasopressin produced a transient (mean = 6 min) 75% +/- 4% decrease in bile flow and a sustained 7% +/- 4% increase in perfusion pressure. Pretreatment with H-7 alone corrected the vasopressin-induced pressure increase but also failed to eliminate the decrease in bile flow, whereas pretreatment with Ni2+ decreased the magnitude of the decrease by two-thirds without affecting the increase in perfusion pressure, 2,5'-di(tert-butyl)-1,4-benzohydroquinone produced a transient 65% +/- 20% decrease in bile flow and a transient 56% +/- 15% increase in perfusion pressure. In isolated hepatocytes, bromo-A23187, the nonfluorescent form of the ionophore, produced a sustained 56% +/- 32% increase in the cytosolic Ca2+ signal, whereas vasopressin resulted in a transient 241% +/- 75% increase and 2,5-di(tert-butyl)-1,4-benzohydroquinone resulted in a sustained 149% +/- 66% increase. The ionophore-induced increase in Ca2+ was abolished completely by pretreatment of the hepatocytes with Ni2+, whereas the vasopressin-induced increase was reduced by 38%.(ABSTRACT TRUNCATED AT 400 WORDS)