Discovery and pharmacological characterization of a novel potent inhibitor of diacylglycerol-sensitive TRPC cation channels.

BACKGROUND AND PURPOSE: The cation channel transient receptor potential canonical (TRPC) 6 has been associated with several pathologies including focal segmental glomerulosclerosis, pulmonary hypertension and ischaemia reperfusion-induced lung oedema. We set out to discover novel inhibitors of TRPC6 channels and investigate the therapeutic potential of these agents. EXPERIMENTAL APPROACH: A library of potential TRPC channel inhibitors was designed and synthesized. Activity of the compounds was assessed by measuring intracellular Ca(2+) levels. The lead compound SAR7334 was further characterized by whole-cell patch-clamp techniques. The effects of SAR7334 on acute hypoxic pulmonary vasoconstriction (HPV) and systemic BP were investigated. KEY RESULTS: SAR7334 inhibited TRPC6, TRPC3 and TRPC7-mediated Ca(2+) influx into cells with IC50 s of 9.5, 282 and 226 nM, whereas TRPC4 and TRPC5-mediated Ca(2+) entry was not affected. Patch-clamp experiments confirmed that the compound blocked TRPC6 currents with an IC50 of 7.9 nM. Furthermore, SAR7334 suppressed TRPC6-dependent acute HPV in isolated perfused lungs from mice. Pharmacokinetic studies of SAR7334 demonstrated that the compound was suitable for chronic oral administration. In an initial short-term study, SAR7334 did not change mean arterial pressure in spontaneously hypertensive rats (SHR). CONCLUSIONS AND IMPLICATIONS: Our results confirm the role of TRPC6 channels in hypoxic pulmonary vasoregulation and indicate that these channels are unlikely to play a major role in BP regulation in SHR. SAR7334 is a novel, highly potent and bioavailable inhibitor of TRPC6 channels that opens new opportunities for the investigation of TRPC channel function in vivo.

Insulin-sensitizing and cardiovascular effects of the sodium-hydrogen exchange inhibitor, cariporide, in the JCR: LA-cp rat and db/db mouse.

The effects of the sodium-hydrogen (Na/H) exchange inhibitor cariporide (HOE642), on insulin sensitivity and vascular function were studied in the JCR:LA-cp rat and the db/db mouse. In the insulin-resistant rat, cariporide reduced fasting insulin levels (42%, P < 0.02) and insulin response in a meal tolerance test (50%, P < 0.01), indicating increased insulin sensitivity. The ACE inhibitor, ramipril, used as a reference agent, reduced the insulin response to the meal, but not fasting levels. The EC50 for acetylcholine-mediated relaxation of phenylephrine-precontracted aortic rings was significantly lower in cariporide-treated rats (P < 0.002), but not in ramipril-treated rats. Flow response of the coronary circulation to bradykinin was significantly greater in both cariporide- and ramipril-treated rats, (3-fold decrease in the EC50, P < 0.05). Cariporide-treated hearts were smaller, slower beating, with greater developed LVP. In the obese db/db mouse, chronic treatment with cariporide obviated vascular hypercontractility and improved endothelial function. Thus, cariporide had beneficial effects on the abnormal insulin metabolism and associated vascular dysfunction in the JCR:LA-cp insulin-resistant rat, which develops advanced cardiovascular disease and ischemic myocardial lesions. It also improved vascular function in a similar mouse model of insulin resistance. These effects were markedly greater than those of ramipril.

Regulation of calcium sparks and spontaneous transient outward currents by RyR3 in arterial vascular smooth muscle cells.

Intracellular Ca(2+) levels control both contraction and relaxation in vascular smooth muscle cells (VSMCs). Ca(2+)-dependent relaxation is mediated by discretely localized Ca(2+) release events through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). These local increases in Ca(2+) concentration, termed sparks, stimulate nearby Ca(2+)-activated K(+) (BK) channels causing BK currents (spontaneous transient outward currents or STOCs). STOCs are hyperpolarizing currents that oppose vasoconstriction. Several RyR isoforms are coexpressed in VSMCs; however, their role in Ca(2+) spark generation is unknown. To provide molecular information on RyR cluster function and assembly, we examined Ca(2+) sparks and STOCs in RyR3-deficient freshly isolated myocytes of resistance-sized cerebral arteries from knockout mice and compared them to Ca(2+) sparks in cells from wild-type mice. We used RT-PCR to identify RyR1, RyR2, and RyR3 mRNA in cerebral arteries. Ca(2+) sparks in RyR3-deficient cells were similar in peak amplitude (measured as F/F(0)), width at half-maximal amplitude, and duration compared with wild-type cell Ca(2+) sparks. However, the frequency of STOCs (between -60 mV and -20 mV) was significantly higher in RyR3-deficient cells than in wild-type cells. Ca(2+) sparks and STOCs in both RyR3-deficient and wild-type cells were inhibited by ryanodine (10 micromol/L), external Ca(2+) removal, and depletion of SR Ca(2+) stores by caffeine (1 mmol/L). Isolated, pressurized cerebral arteries of RyR3-deficient mice developed reduced myogenic tone. Our results suggest that RyR3 is part of the SR Ca(2+) spark release unit and plays a specific molecular role in the regulation of STOCs frequency in mouse cerebral artery VSMCs after decreased arterial tone.

Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice.

Caveolae are plasma membrane invaginations that may play an important role in numerous cellular processes including transport, signaling, and tumor suppression. By targeted disruption of caveolin-1, the main protein component of caveolae, we generated mice that lacked caveolae. The absence of this organelle impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone. In addition, the lungs of knockout animals displayed thickening of alveolar septa caused by uncontrolled endothelial cell proliferation and fibrosis, resulting in severe physical limitations in caveolin-1-disrupted mice. Thus, caveolin-1 and caveolae play a fundamental role in organizing multiple signaling pathways in the cell.

beta(1)-Subunit of BK channels regulates arterial wall[Ca(2+)] and diameter in mouse cerebral arteries.

Mice with a disrupted beta(1) (BK beta(1))-subunit of the large-conductance Ca(2+)-activated K(+) (BK) channel gene develop systemic hypertension and cardiac hypertrophy, which is likely caused by uncoupling of Ca(2+) sparks to BK channels in arterial smooth muscle cells. However, little is known about the physiological levels of global intracellular Ca(2+) concentration ([Ca(2+)](i)) and its regulation by Ca(2+) sparks and BK channel subunits. We utilized a BK beta(1) knockout C57BL/6 mouse model and studied the effects of inhibitors of ryanodine receptor and BK channels on the global [Ca(2+)](i) and diameter of small cerebral arteries pressurized to 60 mmHg. Ryanodine (10 microM) or iberiotoxin (100 nM) increased [Ca(2+)](i) by approximately 75 nM and constricted +/+ BK beta(1) wild-type arteries (pressurized to 60 mmHg) with myogenic tone by approximately 10 microm. In contrast, ryanodine (10 microM) or iberiotoxin (100 nM) had no significant effect on [Ca(2+)](i) and diameter of -/- BK beta(1)-pressurized (60 mmHg) arteries. These results are consistent with the idea that Ca(2+) sparks in arterial smooth muscle cells limit myogenic tone through activation of BK channels. The activation of BK channels by Ca(2+) sparks reduces the voltage-dependent Ca(2+) influx and [Ca(2+)](i) through tonic hyperpolarization. Deletion of BK beta(1) disrupts this negative feedback mechanism, leading to increased arterial tone through an increase in global [Ca(2+)](i).

Sodium block and depolarization diminish P2Z-dependent Ca2+ entry in human B lymphocytes.

Despite a high Ca2+ -permeability of the P2Z receptor in human B lymphocytes, extracellular ATP4- has only a minor effect on global [Ca2+]i. The aim of this study was to reveal the mechanisms responsible for this discrepancy. We investigated the relationship between ATP4- -application, Cai 2+ -response, membrane current and membrane potential in two human B cell lines and in human tonsillar B cells. This was achieved by a combination of FACS- and voltage clamp measurements and the usage of appropriate voltage- and Ca2 -sensitive fluorescent dyes. ATP4 -induced changes in whole-cell current and [Ca2]i were blocked by extracellular as well as intracellular Na+. Under current clamp conditions, ATP4- -induced Na+ -entry diminished the Ca2+ entry via reduction of the driving force. A substantial increase in [Ca2+]iinduced by ATP4- was only observed in Na+ -free solutions. The pathway of signal transduction activated by ATP4via P2Z receptor of human B lymphocytes under physiological conditions seems not to operate by an increase in the global intracellular Ca2+ -concentration, but rather by the depolarization of the cell membrane as a result of the Na+-influx.

Ignition of calcium sparks in arterial and cardiac muscle through caveolae.

Ca(2+) sparks are localized intracellular Ca(2+) events released through ryanodine receptors (RyRs) that control excitation-contraction coupling in heart and smooth muscle. Ca(2+) spark triggering depends on precise delivery of Ca(2+) ions through dihydropyridine (DHP)-sensitive Ca(2+) channels to RyRs of the sarcoplasmic reticulum (SR), a process requiring a very precise alignment of surface and SR membranes containing Ca(2+) influx channels and RyRs. Because caveolae contain DHP-sensitive Ca(2+) channels and may colocalize with SR, we tested the hypothesis that caveolae are the structural element necessary for the generation of Ca(2+) sparks. Using methyl-ss-cyclodextrin (dextrin) to deplete caveolae, we found that dextrin dose-dependently decreased the frequency, amplitude, and spatial size of Ca(2+) sparks in arterial smooth muscle cells and neonatal cardiomyocytes. However, temporal characteristics of Ca(2+) sparks were not significantly affected. We ruled out the possibility that the decreases in Ca(2+) spark frequency and size are caused by changes in DHP-sensitive L-type channels, SR Ca(2+) load, or changes in membrane potential. Our results suggest a novel signaling model that explains the formation of Ca(2+) sparks in a caveolae microdomain. The transient elevation in [Ca(2+)](i) at the inner mouth of a single caveolemmal Ca(2+) channel induces simultaneous activation and thus opens several RyRs to generate a local Ca(2+) release event, a Ca(2+) spark. Alterations in the molecular assembly and ultrastructure of caveolae may lead to pathophysiological changes in Ca(2+) signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease.

Mice with disrupted BK channel beta1 subunit gene feature abnormal Ca(2+) spark/STOC coupling and elevated blood pressure.

Large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) sense both changes in membrane potential and in intracellular Ca(2+) concentration. BK channels may serve as negative feedback regulators of vascular tone by linking membrane depolarization and local increases in intracellular Ca(2+) concentration (Ca(2+) sparks) to repolarizing spontaneous transient outward K(+) currents (STOCs). BK channels are composed of channel-forming BKalpha and auxiliary BKbeta1 subunits, which confer to BK channels an increased sensitivity for changes in membrane potential and Ca(2+). To assess the in vivo functions of this ss subunit, mice with a disrupted BKbeta1 gene were generated. Cerebral artery VSMCs from BKbeta1 -/- mice generated Ca(2+) sparks of normal amplitude and frequency, but STOC frequencies were largely reduced at physiological membrane potentials. Our results indicate that BKbeta1 -/- mice have an abnormal Ca(2+) spark/STOC coupling that is shifted to more depolarized potentials. Thoracic aortic rings from BKbeta1 -/- mice responded to agonist and elevated KCl with a increased contractility. BKbeta1 -/- mice had higher systemic blood pressure than BKbeta1 +/+ mice but responded normally to alpha(1)-adrenergic vasoconstriction and nitric oxide-mediated vasodilation. We propose that the elevated blood pressure in BKbeta1 -/- mice serves to normalize Ca(2+) spark/STOC coupling for regulating myogenic tone. The full text of this article is available at http://www.circresaha.org.

Calcium sparks in human coronary artery smooth muscle cells resolved by confocal imaging.

OBJECTIVE: The observation of local 'elementary' Ca2+ release events (Ca2+ sparks) through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR) has changed our understanding of excitation-contraction (EC) coupling in cardiac and smooth muscle. In arterial smooth muscle, Ca2+ sparks have been suggested to oppose myogenic vasoconstriction and to influence vasorelaxation by activating co-localized Ca2+ activated K+ (K(Ca)) channels (STOCs). However, all prior studies on Ca2+ sparks have been performed in non-human tissues. METHODS: In order to understand the possible significance of Ca2+ sparks to human cardiovascular function, we used high spatial resolution confocal imaging to record Ca2+ sparks in freshly-isolated, individual myocytes of human coronary arteries loaded with the Ca2+ indicator fluo-3. RESULTS: Local SR Ca2+ release events recorded in human myocytes were similar to 'Ca2- sparks' recorded previously from non-human smooth muscle cells. In human myocytes, the peak [Ca2+]i amplitudes of Ca2+ sparks (measured as F/F0) and width at half-maximal amplitude were 2.3 and 2.27 microm, respectively. The duration of Ca2+ sparks was 62 ms. Ca2+ sparks were completely inhibited by ryanodine (10 micromol/l). Ryanodine-sensitive STOCs could be identified with typical properties of K(Ca) channels activated by Ca2+ sparks. CONCLUSION: Our data implies that modern concepts suggesting an essential role of Ca2+ spark generation in EC coupling recently derived from non-human muscle are applicable to human cardiovascular tissue. Although the basic properties of Ca2+ sparks are similar, our results demonstrate that Ca2+ sparks in coronary arteries in humans, have features distinct from non-arterial smooth muscle cells of other species.

Ca2+ channels, 'quantized' Ca2+ release, and differentiation of myocytes in the cardiovascular system.

The application of confocal microscopy to cardiac and skeletal muscle has resulted in the observation of transient, spatially localized elevations in [Ca2+]i, termed 'Ca2+ sparks'. Ca2+ sparks are thought to represent 'elementary' Ca2+ release events, which arise from one or more ryanodine receptor (RyR) channels in the sarcoplasmic reticulum. In cardiac muscle, Ca2+ sparks appear to be key elements of excitation-contraction coupling, in which the global [Ca2+]i transient is thought to involve the recruitment of Ca2+ sparks, each of which is controlled locally by single coassociated L-type Ca2+ channels. Recently, Ca2+ sparks have been detected in smooth muscle cells of arteries. In this review, we analyse the complex relationship of Ca2+ influx and Ca2+ release with local, subcellular Ca2+ microdomains in light of recent studies on Ca2+ sparks in cardiovascular cells. We performed a comparative analysis of 'elementary' Ca2+ release units in mouse, rat and human arterial smooth muscle cells, using measurements of Ca2+ sparks and plasmalemmal K(Ca) currents activated by Ca2+ sparks (STOCs). Furthermore, the appearance of Ca2+ sparks during ontogeny of arterial smooth muscle is explored. Using intact pressurized arteries, we have investigated whether RyRs causing Ca2+ sparks (but not smaller 'quantized' Ca2+ release events, e.g. hypothetical 'Ca2+ quarks') function as key signals that, through membrane potential and global cytoplasmic [Ca2+], oppose arterial myogenic tone and influence vasorelaxation. We believe that voltage-dependent Ca2+ channels and local RyR-related Ca2+ signals are important in differentiation, proliferation, and gene expression. Our findings suggest that 'elementary' Ca2+ release units may represent novel potent therapeutic targets for regulating function of intact arterial smooth muscle tissue.

Ca2+ channels, Ca2+ sparks, and regulation of arterial smooth muscle function.

In cardiac, skeletal, and arterial muscle, transient, spatially localized elevations in [Ca2+]i, termed "Ca2+ sparks", have been observed using confocal laser scanning microscopy. Ca2+ sparks are thought to represent "elementary" Ca2+ release events, which arise from one or more ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In striated muscle, Ca2+ sparks are thought to be key elements of excitation-contraction coupling. In arterial smooth muscle, Ca2+ sparks have been suggested to oppose myogenic vasoconstriction and to influence vasorelaxation. Using a developmental model, we have investigated whether RyRs causing Ca2+ sparks and activation of Ca(2+)-activated K+ (KCa) channels (STOCs) function as "elementary" Ca2+ release units that regulate arterial myogenic tone. Whereas increases in the global [Ca2+]i induce sustained constriction of arterial smooth muscle, Ca2+ sparks induce vasodilation through the local activation of KCa channels. In cerebral arteries, the global bulk [Ca2+]i and a Ca2+ spark frequency < 10(-2) Hz/cell do not cause sufficient KCa channel activity to regulate membrane potential of smooth muscle cells and myogenic tone. The frequency of Ca2+ sparks and STOCs is regulated by agents that modulate protein kinase G and protein kinase A activity. Our findings suggest that "elementary" Ca2+ release units may represent novel, important therapeutic targets for regulating function of the intact arterial smooth muscle tissue.

Monocyte infiltration and adhesion molecules in a rat model of high human renin hypertension.

Hypertension and kidney damage in the double transgenic rat (dTGR) harboring both human renin and human angiotensinogen genes are dependent on the human components of the renin angiotensin system. We tested the hypothesis that monocyte infiltration and increased adhesion molecule expression are involved in the pathogenesis of kidney damage in dTGR. We also evaluated the effects of long-term angiotensin-converting enzyme (ACE) inhibition, AT1 blockade, and human renin inhibition on monocyte recruitment and inflammatory response in dTGR. Systolic blood pressure and 24-hour albuminuria were markedly increased in 7-week-old dTGR as compared with age-matched normotensive Sprague Dawley rats. We found a significant monocyte/macrophage infiltration in the renal perivascular space and increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the interstitium, intima, and adventitia of the small renal vessels. alphaLbeta2 integrin and alpha4beta1 integrin, the corresponding ligands for ICAM-1 and VCAM-1, were also found on infiltrating monocytes/macrophages. The expression of plasminogen activator inhibitor-1 and fibronectin in the kidneys of dTGR were increased and distributed similarly to ICAM-1. In 4-week-old dTGR, long-term treatment with ACE inhibition (cilazapril), AT1 receptor blockade (valsartan), and human renin inhibition (RO 65-7219) (each drug 10 mg/kg by gavage once a day for 3 weeks) completely prevented the development of albuminuria. However, only cilazapril and valsartan were able to decrease blood pressure to normotensive levels. Interestingly, the drugs were all equally effective in preventing monocyte/macrophage infiltration and the overexpression of adhesion molecules, plasminogen activator inhibitor-1, and fibronectin in the kidney. Our findings indicate that angiotensin II causes monocyte recruitment and vascular inflammatory response in the kidney by blood pressure-dependent and blood pressure-independent mechanisms. ACE inhibition, AT1 receptor blockade, and human renin inhibition all prevent monocyte/macrophage infiltration and increased adhesion molecule expression in the kidneys of dTGR.

[Liposomal DNA transfection of human sarcoma cells with p53 alterations]. / Liposomale DNA-Transfektion humaner Sarkomzellen mit p53-Alterationen.

An vital assay allows to optimize liposomal transfection for human tumor cells via FACS. Various cationic lipids were tested to analyse the reporter gene expression (green fluorescent protein, GFP) in different soft tissue sarcoma (STS) cells with known genetic alterations. Furthermore, the cellular uptake of fluorescence-labeled oligodeoxynucleotides (ODN's) was determined. The results obtained with two self-established sarcoma cell lines (LMS6-93, US8-93) were compared with ATCC sarcoma cell lines (Saos-2, A-204, RD) and fibroblast cells. We found maximal 37% cells expressing GFP 24 h post-transfection. All mesenchymal (tumor) cells but not fibroblast cells could be transfected in a cell-specific and lipid-dependent manner. In kinetic studies highest transfection rates were determined between 24 and 48 h, whereas the GFP expression is downregulated after 72 h. Furthermore, we found transfectability is p53 mutation-independent and a relative low toxicity of the new lipids (Lipotaxi and Clonfectin) in comparison to other lipids (Lipofectin, Lipofectamine). By a cell sorting system sarcoma cell lines expressing the reporter gene could be enriched up to 84% of the living cell population. Labeled ODN's were taken up more efficiently (> 90%) when they were mixed with lipids before, but ODN's alone were incorporated into sarcoma cells only in a low percentage (< 10%) and concentration. STS cell cultures showed also a relative high ODN uptake compared with cell lines. We propose the liposomal transfection strategy as an efficient method which can be applied to adherent-growing tumor cells. The method allows simultaneously to study transfection rates, apoptosis and cell cycle alterations in vitro. Furthermore, in future, extension on ex vivo and in vivo transgene expression (xenotransplanted sarcomas) will be evaluated.

Induction of aminopeptidase N/CD13 on human lymphocytes after adhesion to fibroblast-like synoviocytes, endothelial cells, epithelial cells, and monocytes/macrophages.

Aminopeptidase N (APN/CD13) is a transmembrane ectoenzyme occurring on a wide variety of cells. In contrast to monocytes and granulocytes, lymphocytes of peripheral blood do not express CD13 Ag. However, tumor-infiltrating T cells in renal cell cancer as well as synovial fluid T cells from patients suffering from various forms of arthritis can be CD13 positive. To learn more about expression of CD13 in these tissues, we cocultured lymphocytes with different adherent cell lines. CD13 expression was induced in T and B lymphocytes upon adhesion to fibroblast-like synoviocytes, HUVEC, renal tubular epithelial cells, and monocytes/macrophages but not always upon interaction with different tumor cell lines. Induction of APN was rapid, occurring as early as 1 h after coincubation. Expression persisted for >3 days and partially resisted inhibition by cycloheximide. Fixation of adherent cells with paraformaldehyde could not prevent induction of CD13 in lymphocytes. Soluble APN from human kidneys or placenta could not induce CD13 expression on lymphocytes. Induction of CD13 Ag on lymphocytes required direct cell-to-cell contact as shown in experiments using dual chambers. Lymphocytes exhibited an induction not only in CD13 protein but also in Ala-pNA-cleaving enzyme activity and in CD13 mRNA. Lymphocytic expression of CD13 represents a potentially increased cellular ability to inactivate inflammatory mediators. Furthermore, CD13 could be involved in adhesion, in lymphocytic migration, or in the Ag processing of peptides bound in the groove of MHC class II molecules.

Purinoceptor-operated cationic channels in human B lymphocytes.

1. Using the patch clamp method in the outside-out configuration, purinoceptor-dependent unitary currents were measured in tonsillar and transformed tonsillar human B lymphocytes. 2. Single channel currents were evoked by ATP4-, the free-acid form of ATP, and by 2',3' O-benzoyl-4-benzoyl-ATP (BzATP) in the micromolar concentration range, but not by 10 mM ADP3- or 0.5 mM Mg(2+)-bound ATP. 3. The channels could be activated and deactivated several times for as long as 30 min even in the absence of intracellular ATP, GTP, or glucose. 4. The channels were selective for small cations and had a conductance of 9 pS with Cs+ as the intracellular and Na+ as the extracellular monovalent cation. 5. The half-maximal activation of the channels was obtained by 114 microM ATP4- and by 16 microM BzATP. The increase in the open probability after raising the ATP4- concentration was mainly due to a decrease in the times the channels spend in the closed state. 6. It is concluded that human B lymphocytes possess cationic channels directly gated by extracellular ATP4-. Their agonist binding characteristics are typical for P2z purinoceptors, but their permeation behaviour is different from the large non-specific pores formed by ATP4- in fibroblasts, macrophages and mast cells.

Deformation of the Bowditch staircase in Ca(2+)-overloaded mammalian cardiac tissue--a calcium phenomenon?

Concentrations of 1-4 mumol l-1 isoproterenol cause both in right ventricular papillary muscles and in enzymatically isolated myocytes of the guinea-pig a Ca2+ overload-induced state which is functionally characterized by biphasic (multiphasic) twitches and biphasic (multiphasic) intracellular calcium transients, respectively, during excitation-contraction coupling. This state was stabilized in the in vitro experiments for some hours by a co-ordination of the interstimulus interval, the temperature of the superfusion fluid and the addition of calcium agonists. The functional stability is the precondition for the reproducibility of the experimental results particularly after the application of long-lasting stimulation programmes. Changes in the shape of biphasic contractions were compared with changes in the time course of biphasic intracellular calcium transients using three manipulations of a different kind: (1) the interruption of the steady pacing rhythm, (2) the variation of the interstimulus interval, (3) the addition of ryanodine. It was shown that: (1) The BOWDITCH staircase in calcium overloaded multicellular preparations is changed in that each individual component of the twitch passes through its own staircase. A homologous behaviour can be observed in the configuration of the phasic and tonic component of biphasic intracellular calcium transients. (2) At different driving frequencies the relative proportion of the two components of a biphasic twitch corresponds to the time integrals of the two components of biphasic intracellular calcium transients. (3) Ryanodine suppresses both the first component of the biphasic twitch and the phasic component of the biphasic intracellular calcium transient. The SR Ca(2+)-ATPase inhibitor thapsigargin increases the second component of the biphasic calcium transient. This supports the hypothesis that the size of the tonic component is in part determined by intracellular calcium reuptake. The results of both kinds of experiments would be compatible with the assumption that in calcium overloaded mammalian cardiac cells calcium reaches the contractile system directly as well as via two intracellular stores ('extended two-Ca-store concept').