Biomedical subjects

J Hüser

Publications and source records attributed to J Hüser.

At least 19 recordsLinked to original sources

PubMed2001

Brief rapid pacing depresses contractile function via Ca(2+)/PKC-dependent signaling in cat ventricular myocytes.

The purpose of this study is to determine the effects of brief rapid pacing (RP; approximately 200-240 beats/min for approximately 5 min) on contractile function in ventricular myocytes. RP was followed by a sustained inhibition of peak systolic cell shortening (-44 +/- 4%) that was not due to changes in diastolic cell length, membrane voltage, or L-type Ca(2+) current (I(Ca,L)). During RP, baseline and peak intracellular Ca(2+) concentration ([Ca(2+)](i)) increased markedly. After RP, Ca(2+) transients were similar to control. The effects of RP on cell shortening were not prevented by 1 microM calpain inhibitor I, 25 microM L-N(5)-(1-iminoethyl)-orthinthine, or 100 microM N(G)-monomethyl-L-arginine. However, RP-induced inhibition of cell shortening was prevented by lowering extracellular [Ca(2+)] (0.5 mM) during RP or exposure to chelerythrine (2-4 microM), a protein kinase C (PKC) inhibitor, or LY379196 (30 nM), a selective inhibitor of PKC-beta. Exposure to phorbol ester (200 nM phorbol 12-myristate 13-acetate) inhibited cell shortening (-46 +/- 7%). Western blots indicated that cat myocytes express PKC-alpha, -delta, and -epsilon as well as PKC-beta. These findings suggest that brief RP of ventricular myocytes depresses contractility at the myofilament level via Ca(2+)/PKC-dependent signaling. These findings may provide insight into the mechanisms of contractile dysfunction that follow paroxysmal tachyarrhythmias.

Action Potentials↗

PubMed2001

Intracellular Ca2+ release sparks atrial pacemaker activity.

Electrical excitation of the mammalian heart originates from specialized pacemaker cells in the right atrium. Pacemaker activity depends on multiple ion channels and transport mechanisms that reside primarily within the plasma membrane. However, recent evidence indicates that intracellular Ca2+ release from the sarcoplasmic reticulum also contributes importantly to atrial pacemaker function.

Animals↗

PubMed2000

Functional coupling between glycolysis and excitation-contraction coupling underlies alternans in cat heart cells.

Electromechanical alternans was characterized in single cat atrial and ventricular myocytes by simultaneous measurements of action potentials, membrane current, cell shortening and changes in intracellular Ca2+ concentration ([Ca2+]i). Using laser scanning confocal fluorescence microscopy, alternans of electrically evoked [Ca2+]i transients revealed marked differences between atrial and ventricular myocytes. In ventricular myocytes, electrically evoked [Ca2+]i transients during alternans were spatially homogeneous. In atrial cells Ca2+ release started at subsarcolemmal peripheral regions and subsequently spread toward the centre of the myocyte. In contrast to ventricular myocytes, in atrial cells propagation of Ca2+ release from the sarcoplasmic reticulum (SR) during the small-amplitude [Ca2+]i transient was incomplete, leading to failures of excitation-contraction (EC) coupling in central regions of the cell. The mechanism underlying alternans was explored by evaluating the trigger signal for SR Ca2+ release (voltage-gated L-type Ca2+ current, ICa,L) and SR Ca2+ load during alternans. Voltage-clamp experiments revealed that peak ICa,L was not affected during alternans when measured simultaneously with changes of cell shortening. The SR Ca2+ content, evaluated by application of caffeine pulses, was identical following the small-amplitude and the large-amplitude [Ca2+]i transient. These results suggest that the primary mechanism responsible for cardiac alternans does not reside in the trigger signal for Ca2+ release and SR Ca2+ load. beta-Adrenergic stimulation with isoproterenol (isoprenaline) reversed electromechanical alternans, suggesting that under conditions of positive cardiac inotropy and enhanced efficiency of EC coupling alternans is less likely to occur. The occurrence of electromechanical alternans could be elicited by impairment of glycolysis. Inhibition of glycolytic flux by application of pyruvate, iodoacetate or beta-hydroxybutyrate induced electromechanical and [Ca2+]i transient alternans in both atrial and ventricular myocytes. The data support the conclusion that in cardiac myocytes alternans is the result of periodic alterations in the gain of EC coupling, i. e. the efficacy of a given trigger signal to release Ca2+ from the SR. It is suggested that the efficiency of EC coupling is locally controlled in the microenvironment of the SR Ca2+ release sites by mechanisms utilizing ATP, produced by glycolytic enzymes closely associated with the release channel.

Action Potentials↗

PubMed2000

Intracellular Ca2+ release contributes to automaticity in cat atrial pacemaker cells.

1. The cellular mechanisms governing cardiac atrial pacemaker activity are not clear. In the present study we used perforated patch voltage clamp and confocal fluorescence microscopy to study the contribution of intracellular Ca2+ release to automaticity of pacemaker cells isolated from cat right atrium. 2. In spontaneously beating pacemaker cells, an increase in subsarcolemmal intracellular Ca2+ concentration occurred concomitantly with the last third of diastolic depolarization due to local release of Ca2+ from the sarcoplasmic reticulum (SR), i.e. Ca2+ sparks. Nickel (Ni2+; 25-50 microM), a blocker of low voltage-activated T-type Ca2+ current ((ICa,T), decreased diastolic depolarization, prolonged pacemaker cycle length and suppressed diastolic Ca2+ release. 3. Voltage clamp analysis indicated that the diastolic Ca2+ release was voltage dependent and triggered at about -60 mV. Ni2+ suppressed low voltage-activated Ca2+ release. Moreover, low voltage-activated Ca2+ release was paralleled by a slow inward current presumably due to stimulation of Na+-Ca2+ exchange (INa-Ca). Low voltage-activated Ca2+ release was found in both sino-atrial node and latent atrial pacemaker cells but not in working atrial myocytes. 4. These findings suggest that low voltage-activated ICa,T triggers subsarcolemmal Ca2+ sparks, which in turn stimulate INa-Ca to depolarize the pacemaker potential to threshold. This novel mechanism indicates a pivotal role for ICa,T and subsarcolemmal intracellular Ca2+ release in normal atrial pacemaker activity and may contribute to the development of ectopic atrial arrhythmias.

Action Potentials↗

PubMed2000

Mitochondrial calcium in heart cells: beat-to-beat oscillations or slow integration of cytosolic transients?

Mitochondria have been implicated in intracellular Ca2+ signaling in many cell types. The inner mitochondrial membrane contains Ca2+-transporting proteins, which catalyze Ca2+ uptake and extrusion. Intramitochondrial (matrix) Ca2+, in turn, regulates the activity of Krebs cycle dehydrogenases and, ultimately, the rate of ATP synthesis. In the myocardium, controversy remains whether the fast cytosolic Ca2+ transients underlying excitation-contraction coupling in beating cells are rapidly transmitted into the matrix compartment or slowly integrated by the mitochondrial Ca2+ transporters. This mini-review critically summarizes the recent experimental work in this field.

Adenosine Triphosphate↗

PubMed1999

Fluctuations in mitochondrial membrane potential caused by repetitive gating of the permeability transition pore.

Confocal laser scanning microscopy and the potentiometric fluorescence probe tetramethylrhodamine ethyl ester were used to measure changes in membrane electrical potential (DeltaPsi(m)) in individual mitochondria after isolation or in the living cell. Recordings averaged over small mitochondrial populations revealed a gradual decline in DeltaPsi(m) caused by the light-induced generation of free radicals. Depolarization was attenuated by dithiothreitol or acidification. In contrast, individual organelles displayed rapid spontaneous depolarizations caused by openings of the mitochondrial permeability transition pore (MTP). Repetitive openings and closings of the pore gave rise to marked fluctuations in DeltaPsi(m) between the fully charged and completely depolarized state. Rapid spontaneous fluctuations in DeltaPsi(m) were observed in mitochondria isolated from rat heart and in mitochondria in living endothelial cells. The loss of DeltaPsi(m) of mitochondria in the living cell coincided with swelling of the organelle and the breakdown of long mitochondrial filaments. In the individual mitochondrion, oxidative stress initially triggered pore openings of shorter duration, before prolonged openings caused the complete dissipation of DeltaPsi(m) and a measurable efflux of larger solutes. Generalizing this scheme, we suggest that under conditions of prolonged oxidative stress and/or cellular Ca(2+) overload, short openings of MTP might serve as an emergency mechanism allowing the partial dissipation of DeltaPsi(m), the fast release of accumulated Ca(2+) ions and the decreased generation of endogenous oxygen radicals. In contrast, loss of matrix metabolites, swelling and other structural damage of the organelle render prolonged openings of the transition pore deleterious to mitochondria and to the cell.

Animals↗

PubMed1999

Focal agonist stimulation results in spatially restricted Ca2+ release and capacitative Ca2+ entry in bovine vascular endothelial cells.

1. Intracellular Ca2+ ([Ca2+]i) signals were studied with spatial resolution in bovine vascular endothelial cells using the fluorescent Ca2+ indicator fluo-3 and confocal laser scanning microscopy. Single cells were stimulated with the purinergic receptor agonist ATP resulting in an increase of [Ca2+]i due to intracellular Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores. ATP-induced Ca2+ release was quantal, i.e. submaximal concentrations mobilized only a fraction of the intracellularly stored Ca2+. 2. Focal receptor stimulation in Ca2+-free solution by pressure application of agonist-containing solution through a fine glass micropipette resulted in a spatially restricted increase in [Ca2+]i. Ca2+ release was initiated at the site of stimulation and frequently propagated some tens of micrometres into non-stimulated regions. 3. Local Ca2+ release caused activation of capacitative Ca2+ entry (CCE). CCE was initially colocalized with Ca2+ release. Following repetitive focal stimulation, however, CCE became detectable at remote sites where no Ca2+ release had been observed. In addition, the rate of Ca2+ store depletion with repetitive local activation of release in Ca2+-free solution was markedly slower than that elicited by ATP stimulation of the entire cell. 4. From these experiments it is concluded that both intracellular IP3-dependent Ca2+ release and activation of CCE are controlled locally at the subcellular level. Moreover, redistribution of intracellular Ca2+ stored within the endoplasmic reticulum efficiently counteracts local store depletion and accounts for the spatial spread of CCE activation.

Action Potentials↗

PubMed1998

High affinity regulation of cardiac Cl- and Ca2+ conductances by (13R)-spiroforskolin.

The effects of a new forskolin derivative, (13R)-spiroforskolin, on the ventricular cAMP-activated chloride current (I(Cl(cAMP))) and the atrial L-type calcium current (I(Ca,L)) were measured by means of whole-cell recording from isolated guinea-pig cardiac myocytes at 30 degrees C and 20-22 degrees C, respectively. In contrast to forskolin, the derivative contains a tetrahydrofuran rather than a tetrahydropyran moiety. (13R)-spiroforskolin activated I(Cl(CAMP)) in 58% of the ventricular myocytes studied. The concentration required for the half maximal effect (EC50 value) amounted to 9.6x10(-11) M and was lower than the EC50 value for forskolin (2.4x10(-8) M). (13R)-spiroforskolin evoked a smaller maximal I(Cl(cAMP)) amplitude than forskolin. The rundown of the (13R)-spiroforskolin-activated I(Cl(cAMP)) was faster than that of the forskolin-induced current. Neither forskolin nor (13R)-spiroforskolin in maximally effective concentrations increased I(Cl(cAMP)) in cells containing high concentrations of cAMP. Furthermore, as an activator of atrial I(Ca,L) (13R)-spiroforskolin displayed a smaller activation and a lower EC50 value (5.8x10(-10) M) than forskolin (EC50 value: 3.7x10(-7) M). The effect of (13R)-spiroforskolin was observed in only 30% of the atrial cells studied. None of the drugs exerted a stimulatory effect in atrial cells containing a high [cAMP]. The washout of the drug effect was significantly faster in (13R)-spiroforskolin- than in forskolin-treated atrial myocytes. We conclude that (13R)-spiroforskolin as a forskolin derivative displays unique characteristics. It is a more potent but less efficacious activator of cardiac ionic conductances than the parent compound. The results suggest that (13R)-spiroforskolin, like forskolin, most probably exerts its effects via stimulation of the adenylyl cyclase.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Imaging the permeability pore transition in single mitochondria.

In mitochondria the opening of a large proteinaceous pore, the "mitochondrial permeability transition pore" (MTP), is known to occur under conditions of oxidative stress and matrix calcium overload. MTP opening and the resulting cellular energy deprivation have been implicated in processes such as hypoxic cell damage, apoptosis, and neuronal excitotoxicity. Membrane potential (delta psi(m)) in single isolated heart mitochondria was measured by confocal microscopy with a voltage-sensitive fluorescent dye. Measurements in mitochondrial populations revealed a gradual loss of delta psi(m) due to the light-induced generation of free radicals. In contrast, the depolarization in individual mitochondria was fast, sometimes causing marked oscillations of delta psi(m). Rapid depolarizations were accompanied by an increased permeability of the inner mitochondrial membrane to matrix-entrapped calcein (approximately 620 Da), indicating the opening of a large membrane pore. The MTP inhibitor cyclosporin A significantly stabilized delta psi(m) in single mitochondria, thereby slowing the voltage decay in averaged recordings. We conclude that the spontaneous depolarizations were caused by repeated stochastic openings and closings of the transition pore. The data demonstrate a much more dynamic regulation of membrane permeability at the level of a single organelle than predicted from ensemble behavior of mitochondrial populations.

Adenosine Triphosphate↗

PubMed1998

Subcellular properties of [Ca2+]i transients in phospholamban-deficient mouse ventricular cells.

The regulatory protein phospholamban exerts a physiological inhibitory effect on the sarcoplasmic reticulum (SR) Ca2+ pump that is relieved with phosphorylation. We have studied the subcellular properties of intracellular Ca2+ ([Ca2+]i) transients in ventricular myocytes isolated from wild-type (WT) and phospholamban-deficient (PLB-KO) mice. In PLB-KO myocytes, steady-state twitch [Ca2+]i transients revealed an accelerated relaxation and the occurrence of highly localized failures of Ca2+ release. The acceleration of SR Ca2+ uptake caused an increase in SR Ca2+ load with the frequent occurrence of spontaneous [Ca2+]i waves and Ca2+ sparks. [Ca2+]i waves in PLB-KO cells showed a marked decrease in spatial width and more frequently appeared to abort. Local Ca2+ release events (Ca2+ sparks) were larger and more variable in amplitude and [Ca2+]i declined faster in PLB-KO myocytes. Increased local buffering and reduction in the refractoriness of SR Ca2+ release caused by the increased SR pump rate led to an overall enhancement of local [Ca2+]i gradients and inhomogeneities in the [Ca2+]i distribution during spontaneous Ca2+ release, [Ca2+]i waves, and excitation-contraction coupling.

Animals↗

PubMed1997

Withdrawal of acetylcholine elicits Ca2+-induced delayed afterdepolarizations in cat atrial myocytes.

BACKGROUND: Recent experiments in atrial myocytes indicate that withdrawal of cholinergic agonist can directly increase Ca2+ influx via L-type Ca2+ current and stimulate Ca2+ uptake into the sarcoplasmic reticulum (SR), thereby increasing intracellular Ca2+. Overload of cellular Ca2+ within the SR can initiate various types of atrial dysrhythmias. The present study was designed to determine whether withdrawal of acetylcholine (ACh) can elicit Ca2+-induced delayed afterdepolarizations (DADs) in atrial myocytes. METHODS AND RESULTS: A nystatin perforated-patch whole-cell method and fluorescence microscopy (indo 1) were used to measure electrical activities and intracellular free Ca2+ ([Ca2+]i), respectively. Withdrawal of ACh (1 micromol/L) increased action potential duration, shifted plateau voltage toward positive, and generated DADs that initiated spontaneous action potentials. Voltage-clamp analysis revealed that withdrawal of ACh elicited a rebound stimulation of L-type Ca2+ current (I(Ca,L)) (+45%) and Na/Ca exchange current (I(NaCa)) (+16%) and the appearance of transient inward current (I(ti)) and spontaneous [Ca2+]i transients. Each of these changes induced by withdrawal of ACh was abolished by Rp-cAMPs (50 to 100 micromol/L) or H-89 (2 micromol/L), inhibitors of cAMP-dependent protein kinase A. Ryanodine (1 micromol/L) abolished I(NaCa) and the appearance of I(ti) without decreasing the rebound stimulation of I(Ca,L) elicited by withdrawal of ACh. CONCLUSIONS: Withdrawal of ACh can elicit cAMP-mediated stimulation of Ca2+ influx via I(Ca,L) and uptake of SR Ca2+. As a result, cellular Ca2+ overload causes enhanced SR Ca2+ release and the initiation of DADs. These mechanisms may generate triggered and/or spontaneous atrial depolarizations elicited by withdrawal of vagal nerve activity.

Acetylcholine↗

PubMed1997

Sarcoplasmic reticulum Ca2+ release flux underlying Ca2+ sparks in cardiac muscle.

Discrete events of Ca2+ release from the sarcoplasmic reticulum (SR) have been described in cardiac, skeletal, and smooth muscle. In skeletal muscle these release events originate at individual channels. In cardiac muscle, however, it remains a question of debate whether localized Ca2+ release transients, termed Ca2+ sparks, originate from single release channels or multiple channels clustered in close vicinity. Generalizing methods used earlier to describe cell-averaged Ca2+ release, we derived, as a function of space and time, the flux of Ca2+ release that underlies Ca2+ sparks. Using the method to analyze spontaneous sparks recorded with confocal microscopy in dissociated cat atrial cells, we obtained in most cases single sparks of Ca2+ release that appear to originate from approximately 1-microm-wide regions. In many cases, doublets, triplets, and greater groups of release sparks were observed. This multiplicity, the estimated release flux magnitude, and existing data on the structure of junctions between SR and plasmalemma suggest that individual release sparks result from the opening of multiple Ca2+ release channels clustered within discrete SR junctional regions.

Animals↗

PubMed1997

Elementary events of agonist-induced Ca2+ release in vascular endothelial cells.

The subcellular spatial and temporal organization of agonist-induced Ca2+ signals was investigated in single cultured vascular endothelial cells. Extracellular application of ATP initiated a rapid increase of intracellular Ca2+ concentration ([Ca2+]i) in peripheral cytoplasmic processes from where activation propagated as a [Ca2+]i wave toward the central regions of the cell. The average propagation velocity of the [Ca2+]i wave in the peripheral processes was 20-60 microns/s, whereas in the central region the wave propagated at < 10 microns/s. The time course of the recovery of [Ca2+]i depended on the cell geometry. In the peripheral processes (i.e., regions with a high surface-to-volume ratio) [Ca2+]i declined monotonically, whereas in the central region [Ca2+]i decreased in an oscillatory fashion. Propagating [Ca2+]i waves were preceded by small, highly localized [Ca2+]i transients originating from 1- to 3-micron-wide regions. The average amplitude of these elementary events of Ca2+ release was 23 nM, and the underlying flux of Ca2+ amounted to approximately 1-2 x 10(-18) mol/s or approximately 0.3 pA, consistent with a Ca2+ flux through a single or small number of endoplasmic reticulum Ca(2+)-release channels.

Adenosine Triphosphate↗

PubMed1996

Spatially non-uniform Ca2+ signals induced by the reduction of transverse tubules in citrate-loaded guinea-pig ventricular myocytes in culture.

1. Ratiometric confocal microscopy and the whole-cell patch clamp technique were used to simultaneously record intracellular Ca2+ transients and membrane currents from guinea-pig ventricular myocytes. Intracellular dialysis with the low-affinity Ca2+ buffer citrate enabled us to record and analyse Ca2+ transients caused by Ca2+ influx alone and by additional Ca2+ release from the sarcoplasmic reticulum (SR) in the same cell. 2. In freshly isolated adult myocytes (used within 1-4 h of isolation) both types of Ca2+ transients ('Ca2+ entry' and 'Ca2+ release' transients) were spatially uniform regardless of the Ca2+ current (ICa) duration. In contrast, Ca2+ transients in short-term cultured (1-2 days) myocytes exhibited marked spatial inhomogeneities. ICa frequently evoked Ca2+ waves that propagated from either or both ends of the cardiac myocyte. Reduction of the ICa duration caused Ca2+ release that was restricted to one of the two halves of the cell. 3. Analysis of the Ca2+ entry signals in freshly isolated and short-term cultured myocytes indicated that the spatial properties of the Ca2+ influx signal were responsible for the spatial properties of the triggered Ca2+ release from the SR. In freshly isolated ventricular myocytes Ca2+ influx was homogeneous while in short-term cultured cells pronounced Ca2+ gradients could be found during Ca2+ influx. Spatial non-uniformities in the amplitude of local Ca2+ entry transients were likely to cause subcellularly restricted Ca2+ release. 4. The changes in the spatial properties of depolarization-induced Cai2+ signals during short-term culture were paralleled by a decrease (to 65%) in the total cell capacitance. In addition, staining the sarcolemma with the membrane-selective dye Di-8-ANEPPS revealed that, in cultured myocytes, t-tubular membrane connected functionally to the surface membrane was reduced or absent. 5. These results demonstrate that the short-term culture of adult ventricular myocytes results in the concomitant loss of functionally connected t-tubular membrane. The lack of the t-tubular system subsequently caused spatially non-uniform SR Ca2+ release. Evidence is presented to show that in ventricular myocytes lacking t-tubules non-uniform SR Ca2+ release was, most probably, the result of inhomogeneous Ca2+ entry during ICa. These findings directly demonstrate the functional importance of the t-tubular network for uniform ventricular Ca2+ signalling.

Animals↗

PubMed1996

Subcellular properties of triggered Ca2+ waves in isolated citrate-loaded guinea-pig atrial myocytes characterized by ratiometric confocal microscopy.

1. Spatiotemporal aspects of subcellular Ca2+ signalling were studied in cultured adult guinea-pig atrial myocytes. A mixture of the Ca2+ indicators fluo-3 and Fura Red in combination with laser-scanning confocal microscopy was used for [Ca2+]i measurements while membrane currents were recorded simultaneously. 2. In citrate-loaded atrial myocytes not every Ca2+ current (ICa) could trigger Ca2+ release from the sarcoplasmic reticulum (SR). Two types of Ca2+ signals could be observed: Ca2+ transients resulting from (i) Ca2+ influx alone and (ii) additional Ca2+ release. 3. Ca2+ release elicited by voltage steps of 100-150 ms duration was either apparently homogeneous or propagated as Ca2+ waves through the entire cell. With brief ICa (50-75 ms), Ca2+ waves with limited subcellular propagation were observed frequently. These waves always originated from either end of the myocyte. 4. The time course of changes in Na(+)-Ca2+ exchange current (INaCa) depended on the subcellular properties of the underlying Ca2+ transient and on the particular cell geometry. Apparently homogeneous Ca2+ release was accompanied by an inward change of INaCa the onset phase of which was fused with ICa. Changes in INaCa caused by a Ca2+ wave propagating through the entire cell showed a W shape, which could be attributed to differences of the fractional surface-to-volume ratio in different cell segments during propagation of the Ca2+ wavefront. Those waves with limited spreading only activated a small component of INaCa. 5. The different subcellular patterns of Ca2+ release signals can be explained by spatial inhomogeneities in the positive feedback of the SR. This depends on the local SR Ca2+ loading state under the control of the local Ca2+ influx during activation of ICa. Due to the higher surface-to-volume ratio at the two ends of the myocyte, SR loading and therefore the positive feedback in Ca(2+)-induced Ca2+ release may be higher at the ends, locations where Ca2+ waves are preferentially triggered. 6. We conclude that the individual cell geometry may be an important determinant of subcellular Ca2+ signalling not only in cardiac muscle cells but presumably also in other types of cells that depend on Ca2+ signalling. In addition, the cell geometry in combination with varying subcellular Ca2+ release patterns can greatly affect the time course of Ca(2+)-activated membrane currents.

Animals↗

PubMed1996

Calcium gradients during excitation-contraction coupling in cat atrial myocytes.

1. Confocal microscopy in combination with the calcium-sensitive fluorescent probe fluo-3 was used to study spatial aspects of intracellular Ca2+ signals during excitation-contraction coupling in isolated atrial myocytes from cat heart. 2. Imaging of [Ca2+]i transients evoked by electrical stimulation revealed that Ca2+ release started at the periphery and subsequently spread towards the centre of the myocyte. 3. Blocking sarcoplasmic reticulum (SR) Ca2+ release with 50 microM ryanodine unmasked spatial inhomogeneities in the [Ca2+]i was higher in the periphery than in central regions of the myocyte. 4. Positive (or negative) staircase or postrest potentiation of the 'whole-cell' [Ca2+] transients were paralleled by characteristic changes in the spatial profile of the [Ca2+]i signal. With low SR Ca2+ load [Ca2+]i transients in the subsarcolemmal space were small and no Ca2+ release in the centre of the cell was observed. Loading of the SR increased subsarcolemmal [Ca2+]i transient amplitude and subsequently triggered further release in more central regions of the cell. 5. Spontaneous Ca2+ release from functional SR units, i.e. Ca2+ sparks, occurred at higher frequency in the subsarcolemmal space than in more central regions of the myocyte. 6. Visualization of the surface membrane using the membrane-selective dye Di-8-ANEPPS demonstrated that transverse tubules (t-tubules) were absent in atrial cells. 7. It is concluded that in atrial myocytes voltage-dependent Ca2+ entry triggers Ca2+ release from peripheral coupling SR that subsequently induces further Ca2+ release from stores in more central regions of the myocyte. Spreading of Ca2+ release from the cell periphery to the centre accounts for [Ca2+]i gradients underlying the whole-cell [Ca2+]i transient. The finding that cat atrial myocytes lack t-tubules demonstrates the functional importance of Ca2+ release from extended junctional (corbular) SR in these cells.

Animals↗

PubMed1993

Serum contains a potent factor that decreases beta-adrenergic receptor-stimulated L-type Ca2+ current in cardiac myocytes.

L-type Ca2+ current (ICa) was measured in cultured atrial myocytes from hearts of adult guinea-pigs using whole-cell voltage clamp. Potentiation of ICa induced by beta-adrenergic stimulation (isoprenaline 2.10(-7) M) could be completely antagonized by diluted sera (1:100 v/v). Half-maximal inhibition of beta-receptor-stimulated ICa occurred at about 1:1000. Basal ICa was not affected by serum. Atropine in a concentration (10(-6) M) that completely antagonized the anti-adrenergic effect of acetylcholine (ACh, 2.10(-6) M) did not interfere with the effect of serum. In cells dialysed with cyclic adenosine monophosphate (cAMP)-containing (10(-4) M) pipette solution, potentiated ICa was insensitive to both ACh and serum. Preincubation of the myocytes with pertussis toxin almost completely abolished the anti-adrenergic effects of both ACh and serum. The potency of serum was not reduced by dialysis. It is concluded that serum contains a factor which, like ACh, inhibits beta-receptor-stimulated adenylyl cyclase via Gi-protein.

Acetylcholine↗

PubMed1993

Activation of muscarinic K+ current in guinea-pig atrial myocytes by a serum factor.

1. Atrial myocytes obtained by enzymatic perfusion of hearts from adult guinea-pigs and cultured for 0-14 days were studied using the whole-cell voltage-clamp technique. 2. Superfusion of the myocytes with diluted sera (1:100 to 1:10,000) from different species (human, horse, guinea-pig) evoked an inward rectifying K+ current. The voltage-dependent properties of this current were identical to those of the K+ current activated by acetylcholine (IK(ACh)). Current density in the presence of horse serum (1:100) approximately corresponded to the non-desensitizing fraction of IK(ACh) during superfusion with 1-2 x 10(-6) M ACh. 3. During a maximal serum-evoked current, application of ACh (10(-6) M) failed to evoke additional K+ current. After switching superfusion from serum-containing to serum-free solution, the K+ current decayed 1-2 orders of magnitude slower than ACh-activated IK(ACh). During the decay of the serum-evoked current, a proportional increase in responsiveness to ACh was recorded. During submaximal activation of K+ current by serum, a saturating concentration of ACh resulted in a total current that was identical to the current evoked by ACh alone minus the desensitizing component. Thus, activation of K+ current by serum caused desensitization of IK(ACh). From these results it is concluded that sera contain a factor that activates the same population of K+ channels as ACh. 4. Irreversible activation of IK(ACh) by ACh in myocytes dialysed with the GTP-analogue GTP-gamma-S abolished sensitivity to serum and vice versa. 5. The effect of serum was not modified by atropine (10(-6) M) which completely blocked the response to 2 x 10(-6) M ACh. Furthermore, theophylline (1 mM), which completely inhibited IK(ACh) activation by adenosine (100 microM), failed to inhibit the effect of serum. Thus, neither muscarinic nor purinergic (A1) receptors are involved. 6. The peptide somatostatin (10(-6) M) and the alpha 1-agonist phenylephrine (1 microM) which previously have been shown to cause activation of IK(ACh) channels, in the present study failed to evoke any measurable current, which excludes the involvement of the corresponding receptors. 7. Pre-incubation of the cells with pertussis toxin completely abolished IK(ACh) evoked by ACh, adenosine and serum, suggesting that the activating factor, like the classical agonists, causes opening of IK(ACh) channels via a G protein (Gi, GK). 8. The potency of serum to activate IK(ACh) was not reduced by dialysis, suggesting the molecular mass of the unknown factor to be > or = 5 kDa. No activating potency was found in the dialysing solutions.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗