In situ visualization of the intracellular Ca2+ dynamics at the border of the acute myocardial infarct

Ischemic insult to the heart produces myocyte Ca²⁺ ([Ca²⁺]_i) overload. However, little is known about spatiotemporal changes in [Ca²⁺]_i within the ischemic heart in situ at the cellular level. Using real-time confocal microscopy, we successfully visualized [Ca²⁺]_i dynamics at the border zone on the subepicardial myocardium of the heart 2 h after coronary ligations followed by loading with fluo 3/AM. Three distinct regions were identified in the acute infarcted heart. In intact regions, the myocytes showed spatially uniform Ca²⁺ transients synchronously to QRS complex in the electrocardiogram. The myocytes at the infarcted regions showed no fluorescence intensity (FI). At the border zones between the intact and infarcted regions, Ca²⁺ waves emerged sporadically and randomly, instead of Ca²⁺ transients, at a mean frequency of 11.5 ± 8.5 min/cell with a propagation velocity of 151.0 ± 35.7 m/sec along the longitudinal axis of the individual myocytes. In addition, some myocytes within the border zone exhibited homogeneously high static FI, indicating severe Ca²⁺ overload. In summary, we provided the first direct evidence of abnormal [Ca²⁺]_i dynamics in acute infarcted hearts at the cellular level. The observed diversity in spatiotemporal [Ca²⁺]_i dynamics at the border zone may contribute to the arrhythmias or contractile failure in acute myocardial infarction.     

Permeability of gap junctions at the segmental border in insect epidermis

We have examined cell-cell communication between epidermal cells of fifth-instar larvae of the milkweed bug Oncopeltus fasciatus and those of maggots of the blowfly Calliphora erythrocephala. Ionic coupling and the transfer of injected Lucifer Yellow (molecular weight 450) and lead-EDTA (molecular weight 374) were used to map the pattern of communication. All epidermal cells, regardless of their position with respect to the segmental border, were ionically coupled. In both species Lucifer Yellow was transferred freely between cells lying in the same segment—that is, in the same developmental compartment as defined by cell lineage. Dye injections close to the segmental border showed that Lucifer Yellow was not transferred between cells in adjacent segments—that is, across the compartmental border. In Calliphora failure of Lucifer Yellow transfer at the segmental border was always observed; in Oncopeltus Lucifer Yellow was not transferred in 90% of preparations examined. Injections of PbEDTA^2? in Calliphora showed that this anion was transferred freely from cell to cell and did not respect the segmental boundary. Previous studies of the distribution of gap junctions at and away from the segmental border make it unlikely that the failure of Lucifer Yellow to cross from segment to segment is due to a reduced number of gap-junctional channels at the border. We conclude that gap junctions at the segmental borders may have different permeability properties from those between cells in the same segment.     

Intercellular Ca2+ signaling in alveolar epithelial cells through gap junctions and by extracellular ATP

Inter- and extracellular-mediated changes in intracellular Ca2+ concentration ([Ca2+]i) can ensure coordinated tissue function in the lung. Cultured rat alveolar epithelial cells (AECs) have been shown to respond to secretagogues with increases in [Ca2+]i and have been shown to be gap junctionally coupled. However, communication of [Ca2+]i changes in AECs is not well defined. Monolayers of AECs were mechanically perturbed and monitored for [Ca2+]i changes. Perturbation of AECs was administered by a glass probe to either mechanically stimulate or mechanically wound individual cells. Both approaches induced a change in [Ca2+]i in the stimulated cell that was propagated to neighboring cells (Ca2+ waves). A connexin mimetic peptide shown to uncouple gap junctions eliminated Ca2+ waves in mechanically stimulated cells but had no effect on mechanically wounded cells. In contrast, apyrase, an enzyme that effectively removes ATP from the extracellular milieu, had no effect on mechanically stimulated cells but severely restricted mechanically wounded Ca2+ wave propagation. We conclude that AECs have the ability to communicate coordinated Ca2+ changes using both gap junctions and extracellular ATP.     

Contribution of the Na+/Ca2+ exchanger to rapid Ca2+ release in cardiomyocytes

Trigger Ca(2+) is considered to be the Ca(2+) current through the L-type Ca(2+) channel (LTCC) that causes release of Ca(2+) from the sarcoplasmic reticulum. However, cell contraction also occurs in the absence of the LTCC current (I(Ca)). In this article, we investigate the contribution of the Na(+)/Ca(2+) exchanger (NCX) to the trigger Ca(2+). Experimental data from rat cardiomyocytes using confocal microscopy indicating that inhibition of reverse mode Na(+)/Ca(2+) exchange delays the Ca(2+) transient by 3-4 ms served as a basis for the mathematical model. A detailed computational model of the dyadic cleft (fuzzy space) is presented where the diffusion of both Na(+) and Ca(2+) is taken into account. Ionic channels are included at discrete locations, making it possible to study the effect of channel position and colocalization. The simulations indicate that if a Na(+) channel is present in the fuzzy space, the NCX is able to bring enough Ca(2+) into the cell to affect the timing of release. However, this critically depends on channel placement and local diffusion properties. With fuzzy space diffusion in the order of four orders of magnitude lower than in water, triggering through LTCC alone was up to 5 ms slower than with the presence of a Na(+) channel and NCX.     

Role of catenins in the development of gap junctions in rat cardiomyocytes

Gap junctions are intercellular communicating channels responsible for the synchronized activity of cardiomyocytes. Recent studies have shown that the membrane-associated guanylate kinase protein, zonula occludens-1 (ZO-1) can bind to catenins in epithelial cells and act as an adapter for the transport of the connexin isotype, Cx43 during gap junction formation. The significance of catenins in the development of gap junctions and whether complexes between catenins and ZO-1 are formed in cardiomyocytes are not clear. In this study, immunofluorescence and confocal microscopy showed sequential redistribution of alpha-catenin, beta-catenin, ZO-1, and Cx43 to the plasma membrane when rat cardiomyocytes were cultured in low Ca(2+) (<5 microM) medium, then shifted to 1.8 mM Ca(2+) medium (Ca(2+) switch). Diffuse cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen in the cytoplasm when cardiomyocytes were cultured in low Ca(2+) medium. Staining of alpha-catenin, beta-catenin, and ZO-1 was detected at the plasma membrane of cell-cell contact sites 10 min after Ca(2+) switch, whereas Cx43 staining was first detected, colocalized with ZO-1 at the plasma membrane, 30 min after Ca(2+) switch. Distinct junctional and extensive cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen 2 h after Ca(2+) switch. Immunoprecipitation of Triton X-100 cardiomyocyte extracts using anti-beta-catenin antibodies showed that beta-catenin was associated with alpha-catenin, ZO-1, and Cx43 at 2 h after Ca(2+) switch. Intracellular application of antisera against alpha-catenin, beta-catenin, or ZO-1 by electroporation of cardiomyocytes cultured in low Ca(2+) medium inhibited the redistribution of Cx43 to the plasma membrane following Ca(2+) switch. These results suggest the formation of a catenin-ZO-1-Cx43 complex in rat cardiomyocytes and that binding of catenins to ZO-1 is required for Cx43 transport to the plasma membrane during the assembly of gap junctions.     

Distinct pharmacological properties of ET-1 and ET-3 on astroglial gap junctions and Ca2+ signaling

Astrocytes represent a major target for endothelins (ETs), afamily of peptides that have potent and multiple effects on signal transduction pathways and can be released by several cell types in thebrain. In the present study we have investigated the involvement ofdifferent ET receptor subtypes on intercellular dye diffusion, intracellular Ca²⁺homeostasis, and intercellularCa²⁺ signaling in cultured ratastrocytes from hippocampus and striatum. Depending on the ETconcentration and the receptor involved, ET-1- and ET-3-inducedintracellular Ca²⁺ increases withdifferent response patterns. Both ET-1 and ET-3 are powerful inhibitorsof gap junctional permeability and intercellular Ca²⁺ signaling. The nonselectiveET receptor agonist sarafotoxin S6b and theET_B receptor-selective agonist IRL1620 mimicked these inhibitions. The ET-3 effects were only marginallyaffected by an ET_A receptorantagonist but completely blocked by anET_B receptor antagonist. However,the ET-1-induced inhibition of gap junctional dye transfer andintercellular Ca²⁺ signaling wasonly marginally blocked by ET_A orET_B receptor-selective antagonistsbut fully prevented when these antagonists were applied together. TheET-induced inhibition of gap junction permeability and intercellularCa²⁺ signaling indicates thatimportant changes in the function of astroglial communication mightoccur when the level of ETs in the brain is increased.

     

Intercellular communication: role of gap junctions in establishing the pattern of ATP-elicited Ca2+ oscillations and Ca2+-dependent currents in freshly isolated aortic smooth muscle cells

Cytosolic-free [Ca2+] was evaluated in freshly dissociated smooth muscle cells from mouse thoracic aorta by the ratio of Fura Red and Fluo 4 emitted fluorescence using confocal microscopy. The role of intercellular communication in forming and shaping ATP-elicited responses was demonstrated. Extracellular ATP (250 microM) elicited [Ca2+]i transient responses, sustained [Ca2+]i rise, periodic [Ca2+]i oscillations and aperiodic repetitive [Ca2+]i transients. Quantity of smooth muscle cells in the preparation responding to ATP with periodical [Ca2+]i oscillations depended on the density of isolated cells on the cover slip. ATP-elicited bursts of [Ca2+]i spikes in 66+/-7% of cells in dense and in 33+/-8.5% of cells in non-dense preparations. The number of cells responding to ATP with bursts of [Ca2+]i spikes decreased from 55+/-5% (n=84) to 14+/-3% (n=141) in dense preparations pretreated with carbenoxolone. Simultaneous measurement of [Ca2+]i and ion currents revealed a correlation between [Ca2+]i and current oscillations. ATP-elicited bursts of current spikes in 76% of cells regrouped in small clusters and in 9% of isolated cells. Clustered cells responding to ATP with current oscillations had higher membrane capacity than clustered cells with transient and sustained ATP-elicited responses. Lucifer Yellow (1% in 130 mM KCl) injected into one of clustered cells was transferred to the neighboring cell only when ATP-elicited oscillations. Fast application of carbenoxolone (100 microM) inhibited ATP (250 microM) elicited Ca2+-dependent current oscillations. Taken together these results suggest that the probability of ATP (250 microM) triggered cytosolic [Ca2+]i oscillations accompanied with K+ and Cl- current oscillations increased with the coupling of smooth muscle cells.     

Ca2+-calmodulin-dependent phosphorylation and passive transport of Ca2+ in the myocardial sarcolemma

Highly purified vesicles of rabbit myocardium sarcolemma with predominant inside-out orientation possess the Ca2+-calmodulin-dependent protein kinase activity. At optimal concentrations of calmodulin (0.5 microM) and Ca2+ (0.1 mM), the activity of protein kinase is 0.21 nmol 32P X min X mg of protein. The Km(app) value for ATP is 3.0 X 10(-6) M, V = 0.27 nmol 32P X mg of protein X min. Endogenous Ca2+-calmodulin-dependent protein kinase phosphorylates four protein substrates in sarcolemmal vesicles (Mr = 145, 22, 11.5, and 6-8 KD). Studies with passive efflux of Ca2+ from the SL vesicles showed that the Ca2+-calmodulin-dependent phosphorylation of protein components of sarcolemma inhibits this reaction.     

Structural remodelling of cardiomyocytes in the border zone of infarcted rabbit heart

Cardiomyocyte dedifferentiation, as detected in hibernating myocardium of chronic ischemic patients, is one of the characteristics seen at the border of myocardial infarcts in small and large animals. Our objectives were to study in detail the morphological changes occurring at the border zone of a rabbit myocardial infarction and its use as model for hibernating myocardium. Ligation of the left coronary artery (LAD) was performed on rabbit hearts and animals were sacrificed at 2, 4, 8 and 12 weeks post-infarction. These hearts together with a non-infarcted control heart were perfusion-fixed and tissue samples were embedded in epoxy resin. Hibernating cardiomyocytes were mainly distributed in the non-infarcted region adjacent to the border zone of infarcted myocardium but only in a limited number. In the border zone itself vacuolated cardiomyocytes surrounded by fibrotic tissue were frequently observed. Ultrastructural analysis of these vacuolated cells revealed the presence of a basal lamina inside the vacuoles adjacent to the surrounding membrane, the presence of pinocytotic vesicles and an association with cisternae of the sarcoplasmatic reticulum. Myocyte quantitative analyses revealed a gradual increase in vacuolar area/total cell area ratio and in collagen fibril deposition inside the vacuoles from 2 to 12 weeks post-infarction. Related to the remote zone, the increase in cell width of myocytes located in and adjacent to the border zone demonstrated cellular hypertrophy. These results indicate the occurrence of cardiomyocyte remodelling mechanisms in the border zone and adjacent regions of infarcted myocardium. It is suggested that the vacuoles represent plasma membrane invaginations and/or dilatations of T-tubular structures.     

Dynamic remodeling of K+ and Ca2+ currents in cells that survived in the epicardial border zone of canine healed infarcted heart

Action potentials (APs) of the epicardial border zone (EBZ) cells from the day 5 infarcted heart continue to be altered by day 14 postocclusion, namely, they shortened. However, by 2 mo, EBZ APs appear "normal," yet conduction of wave fronts remains abnormal. We hypothesize that the changes in transmembrane APs are due to a change in the distribution of ion channels in either density or function. Thus we focused on the changes in Ca2+ and K+ currents in cells isolated from the 14-day (IZ14d) and 2-mo (IZ2m) EBZ and compared them with those occurring in cells from the same hearts but remote (Rem) from the EBZ. Whole cell voltage-clamp techniques were used to measure and compare Ca2+ and K+ currents in cells from the different groups. Ca2+ current densities remain reduced in cells of the 14-day and 2-mo infarcted heart and the kinetic changes previously identified in the 5-day heart begin to, but do not recover to, cells from noninfarcted epicardium (NZ) values. Importantly, I(Ca,L) in both the EBZ and Rem regions still show a slowed recovery from inactivation. Furthermore, during the remodeling process, there is an increased expression of T-type Ca2+ currents, but only regionally, and only within a specific time window postmyocardial infarction (MI). Regional heterogeneity in beta-adrenergic responsiveness of I(Ca,L) exists between EBZ and remote cells of the 14-day hearts, but this regional heterogeneity is gone in the healed infarcted heart. In IZ14d, the transient outward K+ current (Ito) begins to reemerge and is accompanied by an upregulated tetraethylammonium-sensitive outward current. By 2-mo postocclusion, Ito and sustained outward K+ current have completed the reverse remodeling process. During the healing process post-MI, canine epicardial cells downregulate the fast Ito but compensate by upregulating a K+ current that in normal cells is minimally functional. For recovering I(Ca,L) of the 14-day and 2-mo EBZ cells, voltage-dependent processes appear to be reset, such that I(Ca,L) "window" current occurs at hyperpolarized potentials. Thus dynamic changes in both Ca2+ and K+ currents contribute to the altered AP observed in 14-day fibers and may account for return of APs of 2 mo EBZ fibers.     

Presynaptic Ca2+ dynamics, Ca2+ buffers and synaptic efficacy

In synapses neurotransmitter release is triggered by elevation of Ca2+ concentration at a Ca2+ sensor of the release machinery. The Ca2+ concentration at the release site at the given time point is determined by Ca2+ dynamics within presynaptic terminal. It depends on a source of Ca2+ (usually voltage-gated Ca2+ channels), diffusional distance between the source of Ca2+ and the Ca2+ sensor and Ca2+ buffering by endogenous Ca2+ buffers. In many synapses transmitter release can be enhanced (facilitated) during repetitive activity of neurons. The main source of facilitation is activity-dependent increase of Ca2+ concentration at the release site. Several mechanisms of facilitation have been proposed, namely, accumulation of residual Ca2+, multi-site (X receptor) mechanism and partial Ca2+ buffer saturation mechanism. In this review we discuss theoretical and experimental evidence in favor of one or the other of proposed mechanisms.     

Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries

Increases in global Ca(2+) in the endothelium are a crucial step in releasing relaxing factors to modulate arterial tone. In the present study we investigated spontaneous Ca(2+) events in endothelial cells, and the contribution of smooth muscle cells to these Ca(2+) events, in pressurized rat mesenteric resistance arteries. Spontaneous Ca(2+) events were observed under resting conditions in 34% of cells. These Ca(2+) events were absent in arteries preincubated with either cyclopiazonic acid or U-73122, but were unaffected by ryanodine or nicotinamide. Stimulation of smooth muscle cell depolarization and contraction with either phenylephrine or high concentrations of KCl significantly increased the frequency of endothelial cell Ca(2+) events. The putative gap junction uncouplers carbenoxolone and 18alpha-glycyrrhetinic acid each inhibited spontaneous and evoked Ca(2+) events, and the movement of calcein from endothelial to smooth muscle cells. In addition, spontaneous Ca(2+) events were diminished by nifedipine, lowering extracellular Ca(2+) levels, or by blockers of non-selective Ca(2+) influx pathways. These findings suggest that in pressurized rat mesenteric arteries, spontaneous Ca(2+) events in the endothelial cells appear to originate from endoplasmic reticulum IP(3) receptors, and are subject to regulation by surrounding smooth muscle cells via myoendothelial gap junctions, even under basal conditions.     

Mitochondrial dynamics and Ca2+ signaling

Recent data shed light on two novel aspects of the mitochondria-Ca2+ liaison. First, it was extensively investigated how Ca2+ handling is controlled by mitochondrial shape, and positioning; a playground also of cell death and survival regulation. On the other hand, significant progress has been made to explore how intra- and near-mitochondrial Ca2+ signals modify mitochondrial morphology and cellular distribution. Here, we shortly summarize these advances and provide a model of Ca2+-mitochondria interactions.     

Cochlear gap junctions coassembled from Cx26 and 30 show faster intercellular Ca2+ signaling than homomeric counterparts

The importance of connexins (Cxs) in cochlear functions has been demonstrated by the finding that mutations in Cx genes cause a large proportion of sensorineural hearing loss cases. However, it is still unclear how Cxs contribute to the cochlear function. Recent data (33) obtained from Cx30 knockout mice showing that a reduction of Cx diversity in assembling gap junctions is sufficient to cause deafness suggest that functional interactions of different subtypes of Cxs may be essential in normal hearing. In this work we show that the two major forms of Cxs (Cx26 and Cx30) in the cochlea have overlapping expression patterns beginning at early embryonic stages. Cx26 and Cx30 were colocalized in most gap junction plaques in the cochlea, and their coassembly was tested by coimmunoprecipitation. To compare functional differences of gap junctions with different molecular configurations, homo- and heteromeric gap junctions composed of Cx26 and/or Cx30 were reconstituted by transfections in human embryonic kidney-293 cells. The ratio imaging technique and fluorescent tracer diffusion assays were used to assess the function of reconstituted gap junctions. Our results revealed that gap junctions with different molecular configurations show differences in biochemical coupling, and that intercellular Ca²⁺ signaling across heteromeric gap junctions consisting of Cx26 and Cx30 was at least twice as fast as their homomerically assembled counterparts. Our data suggest that biochemical permeability and the dynamics of intercellular signaling through gap junction channels, in addition to gap junction-mediated intercellular ionic coupling, may be important factors to consider for studying functional roles of gap junctions in the cochlea. cochlea; coassembly; deafness     

Fura-2 fluorescent technique for the assessment of Ca2+ homeostasis in cardiomyocytes

Ca2+ homeostasis plays a pivotal role in maintaining cell growth and function. Many heart diseases are related to the abnormalities in Ca2+ mobilization and extrusion. Ca2+-sensitive fluorescent dyes have been used successfully to estimate intracellular free Ca2+ ([Ca2+]i) level and the mechanisms of Ca2+ movements in living cells. This article is focused on the methodology involving the use of Fura-2/AM or free Fura-2 to measure agonist-induced Ca2+ mobilization as well as the mechanisms of changes in [Ca2+]i in cardiomyocytes. Methods involving Fura-2 technique for the measurement of Ca2+ extrusion from the cells and Ca2+ reuptake by sarcoplasmic reticulum (SR) are also described. The prevention of KCl-induced increase in the intracellular Ca2+ is shown by chelating the extracellular Ca2+ with EGTA or by the presence of Ca2+-channel inhibitors such as verapamil and diltiazem. The involvement of SR in the ATP-induced increase in intracellular Ca2+ is illustrated by the use of Ca2+-pump inhibitors, thapsigargin and cyclopiazonic acid as well as ryanodine which deplete the SR Ca2+ storage. The use of 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate (NCDC), an inhibitor of inositol 1,4,5-trisphosphate (IP3) production, is described for the attenuation of phosphatidic acid (PA) induced increase in Ca2+-mobilization. The increase in intracellular Ca2+ in cardiomyocytes by PA, unlike that by KCl or ATP, was observed in diabetic myocardium. Thus, it appears that the Fura-2 method for the measurement of Ca2+ homeostasis in cardiomyocytes is useful in studying the pathophysiology and pharmacology of Ca2+ movements.     

Anisotropic propagation of Ca2+ waves in isolated cardiomyocytes.

Digital imaging microscopy of fluor-3 fluorescence was used to study the propagation of intracellular Ca2+ waves in isolated adult rat cardiomyocytes from 17 to 37 degrees C. Ca2+ waves spread in both transverse and longitudinal direction of a myocyte. Transverse propagation was pronounced in waves starting from a focus at the edge of a myocyte and in waves following an irregular, curved path (spiral waves). For the former type of waves, propagation velocities were determined. Both transverse and longitudinal wave components propagated at constant velocity ranging from 30 to 125 micron/s. Myocytes were anisotropic with respect to wave propagation: waves propagated faster in the longitudinal than in the transverse direction. The ratio between longitudinal and transverse velocity increased from 1.30 at 17 degrees C to 1.55 at 37 degrees C. Apparent activation energies for transverse and longitudinal wave propagation were estimated to be -20 kJ/mol, suggesting that these processes are limited by diffusion of Ca2+. Direction-dependent propagation velocities are interpreted to result from the highly ordered structure of the myocytes, especially from the anisotropic arrangement of diffusion obstacles such as myofilaments and mitochondria.     

Association between mitochondria and gap junctions in mammalian myocardial cells

In ventricular myocardial cells of mouse, guinea-pig, dog, and monkey, mitochondria frequently form close associations with gap junctions, the two structures being separated by a space of 20 nm or less. Similar appositions are found in both the mature atria and the developing myocardium of the mouse. The gap junctions assume a variety of configurations with respect to the apposed mitochondria. These include profiles in which the gap junctions conform closely to the contours of mitochondria, as well as profiles in which finger-like sarcolemmal evaginations, composed entirely of gap junctions, extend longitudinally or transversely into an adjoining cell to envelop mitochondria. In mouse ventricular wall, over 40% of the length of gap junctions is juxtaposed to mitochondria, and strands of connecting material are often present in the interspace between the two structures. In addition, in freeze-fracture replicas, portions of mitochondria are found attached to areas of myocardial sarcolemma that contain gap-junctional particles. Since mitochondria are known to sequester Ca²⁺ ion, it is possible that the close association between mitochondria and gap junction may function to buffer the intracellular Ca²⁺ concentration near the gap junctions, and thereby regulate the ionic permeability of the gap junctions.     

Modulation of L-type Ca2+ current by fast and slow Ca2+ buffering in guinea pig ventricular cardiomyocytes.

Free Ca2+ near Ca2+ channel pores is expected to be lower in cardiomyocytes dialyzed with bis-(o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) than with ethyleneglycol-bis-(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) because BAPTA chelates incoming Ca2+ more rapidly. The consequences of intracellular Ca2+ buffering by BAPTA (0.2-60 mM) and by EGTA (0.2-67 mM) on whole-cell L-type Ca2+ current (ICa,L) were investigated in voltage-clamped guinea pig ventricular cardiomyocytes; bulk cytoplasmic free Ca2+ (Cac2+) was monitored using the fluorescent Ca2+ indicator indo-1. ICa,L was augmented by approximately 12-fold when BAPTA in the cell dialysate was increased from 0.2 to 50 mM (half-maximal stimulation at 31 mM), whereas elevating internal EGTA from 0.2 to 67 mM increased ICa,L only by approximately 2-fold. Cac2+ was < 20 nM with internal BAPTA or EGTA > or = 20 mM. While EGTA up to 67 mM had only an insignificant inhibitory effect on the stimulation of ICa,L by 3 microM forskolin, ICa,L in 50 mM BAPTA-dialyzed myocytes was insensitive to forskolin-induced elevation of adenosine 3',5'-cyclic monophosphate (cAMP); conversely, ICa,L in cAMP-loaded cells was unresponsive to BAPTA dialysis. Cell dialysis with BAPTA, but not with EGTA, accelerated the slow component of ICa,L inactivation (tau S) without affecting its fast component (tau F), resembling the effects of cAMP-dependent phosphorylation. BAPTA-stimulated ICa,L was inhibited by acetylcholine and by the cAMP-dependent protein kinase (PKA) blocker H-89. These results suggest that BAPTA-induced lowering of peri-channel Ca2+ stimulates cAMP synthesis and channel phosphorylation by disinhibiting Ca(2+)-sensitive adenylyl cyclase.     

Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics

The Ca(2+) dissociation constant (K(d)) of Fluo-3 was determined using confocal fluorescence microscopy in two different situations: (i) within the cytosol of a permeabilised cardiomyocyte; and (ii) in an intact cardiomyocyte after incubation with the acetoxymethyl ester form of Fluo-3 (AM). Measurements were made on isolated rabbit ventricular cardiomyocytes after permeabilisation by a brief treatment with beta-escin (0.1mg/ml) and equilibration with 10 microM Fluo-3. The K(d) of Fluo-3 within the cytosol was not significantly different from that in free solution (558 +/- 15 nM, n=6). Over a range of cytoplasmic [Ca(2+)], the minimum [Ca(2+)] values between Ca(2+) waves was relatively constant despite changes in wave frequency. After loading intact cardiomyocytes with Fluo-3 by incubation with the -AM, spontaneous Ca(2+) waves were produced by incubation with strophanthidin (10 microM). By assuming a common minimum [Ca(2+)] in permeabilised and intact cells, the intracellular K(d) of Fluo-3 in intact myocytes was estimated to be 898 +/-64 nM (n=6). Application of this K(d) to fluorescence records shows that Ca(2+) waves in intact cells have similar amplitudes to those in permeabilised cells. Stimulation of cardiac myocytes at 0.5 Hz in the absence of strophanthidin (room temperature) resulted in a Ca(2+) transient with a maximum and minimum [Ca(2+)] of 1190 +/- 200 and 158 +/- 30 nM (n=11), respectively.