T-Type and tetrodotoxin-sensitive Ca(2+) currents coexist in guinea pig ventricular myocytes and are both blocked by mibefradil

Under Na(+)-free conditions, low-voltage-activated Ca(2+) currents in cardiomyocytes from various species have been described either as Ni(2+)-sensitive T-type Ca(2+) current (I(Ca(T))) or as tetrodotoxin (TTX)-sensitive Ca(2+) current (I(Ca(TTX))). So far, coexistence of the 2 currents within the same type of myocyte has never been reported. We describe experimental conditions under which I(Ca(T)) and I(Ca(TTX)) can be separated and studied in the same cell. Rat and guinea pig ventricular myocytes were investigated with the whole-cell voltage-clamp technique in Na(+)-free solutions. Whereas rat myocytes lack I(Ca(T)) and exhibit I(Ca(TTX)) only, guinea pig myocytes possess both of these low-voltage-activated Ca(2+) currents, which are separated pharmacologically by superfusion with TTX or Ni(2+). I(Ca(T)) and I(Ca(TTX)) were of similar amplitude but significantly differed in their electrophysiological properties: I(Ca(TTX)) activated at more negative potentials than did I(Ca(T)), the potential for half-maximum steady-state inactivation was more negative, and current deactivation and recovery from inactivation were faster. I(Ca(TTX)) but not I(Ca(T)) increased after membrane rupture ("run-up"). Isolation of I(Ca(TTX)) by application of the bivalent cation Ni(2+) is critical because of possible shifts in voltage dependence. Therefore, we investigated whether the T-type Ca(2+) channel blocker mibefradil (10 micromol/L) is a suitable tool for the study of I(Ca(TTX)). However, mibefradil not only blocked I(Ca(T)) by 85+/-2% but also decreased I(Ca(TTX)) by 48+/-8%. We conclude that under Na(+)-free conditions I(Ca(T)) and I(Ca(TTX)) coexist in guinea pig ventricular myocytes and that both currents are sensitive to mibefradil. Future investigations of I(Ca(T)) will have to consider the TTX-sensitive current component to avoid possible interference.     

Ozone exposure may enhance airway smooth muscle contraction by increasing Ca(2+) refilling of sarcoplasmic reticulum in guinea pig

Ozone induces airway hyperresponsiveness, but there is controversy about effects of ozone on smooth muscle per se. We therefore investigated effects of in vivo ozone exposure on intracellular calcium mobilization in relation to tracheal smooth muscle contractility in the guinea pig in vitro. Guinea pigs underwent ozone exposure or sham exposure (3 ppm, 2 h). Then, a tracheal smooth muscle strip was mounted in an organ bath to record isometric tension. Effects of ozone exposure on acetylcholine-induced contraction of smooth muscle were as follows. Contraction was not altered in normal Krebs solution, but was increased in Ca(2+)-free solution in ozone-exposed animals. Decline of tension on repetitive application of acetylcholine in Ca(2+)-free solution was reduced, while the tension decline rate while acetylcholine was washed out with Ca(2+)-free solution was facilitated in ozone-exposed animals. Tension decline during the continuous administration of acetylcholine in Ca(2+)-free solution was slowed. Contraction occurred more quickly in Ca(2+)-free solution in ozone-exposed animals. Results suggest that ozone has a direct action on airway smooth muscle by changing Ca(2+) mobilization; Ca(2+) refilling via a Ca(2+) pump and Ca(2+) release via Ca(2+) channels in the sarcoplasmic reticulum were increased, while Ca(2+) extrusion via the plasma membrane Ca(2+) pump was unchanged.     

Sarcoplasmic reticulum Ca(2+)ATPase and cell contraction in developing rabbit heart

The purpose of the present study was to determine whether age-related changes in the expression and function of the cardiac isoform of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) play a role in SR Ca(2+)release and cell contraction. SERCA2a protein levels and subcellular localization were compared between fetal, neonatal, juvenile and adult New Zealand White rabbits. Studies of SERCA function in isolated myocytes were performed in situ by examining the rate of reloading of the SR Ca(2+)stores following caffeine-induced depletion. We found that significant quantities of SERCA2a were present early in immature heart and that SERCA2a expression reached adult levels within 15-30 days after birth. Furthermore, SERCA2a protein is present as a series of transverse striations within the cell as early as 1 day of age. In contrast to previous studies of SERCA in vitro, the SERCA protein function in situ was found to be comparable between neonatal and adult myocytes in maintaining SR Ca(2+)stores. These results indicate that the paucity of SR Ca(2+)release in immature ventricular cardiac myocytes is not the result of immaturity in SERCA2a expression.     

Metabotropic Ca(2+) channel-induced Ca(2+) release and ATP-dependent facilitation of arterial myocyte contraction

Voltage-gated Ca(2+) channels in arterial myocytes can mediate Ca(2+) release from the sarcoplasmic reticulum and, thus, induce contraction without the need of extracellular Ca(2+) influx. This metabotropic action of Ca(2+) channels (denoted as calcium-channel-induced calcium release or CCICR) involves activation of G proteins and the phospholipase C-inositol 1,4,5-trisphosphate pathway. Here, we show a form of vascular tone regulation by extracellular ATP that depends on the modulation of CCICR. In isolated arterial myocytes, ATP produced facilitation of Ca(2+)-channel activation and, subsequently, a strong potentiation of CCICR. The facilitation of L-type channel still occurred after full blockade of purinergic receptors and inhibition of G proteins with GDPbetaS, thus suggesting that ATP directly interacts with Ca(2+) channels. The effects of ATP appear to be highly selective, because they were not mimicked by other nucleotides (ADP or UTP) or vasoactive agents, such as norepinephrine, acetylcholine, or endothelin-1. We have also shown that CCICR can trigger arterial cerebral vasoconstriction in the absence of extracellular calcium and that this phenomenon is greatly facilitated by extracellular ATP. Although, at low concentrations, ATP does not induce arterial contraction per se, this agent markedly potentiates contractility of partially depolarized or primed arteries. Hence, the metabotropic action of L-type Ca(2+) channels could have a high impact on vascular pathophysiology, because, even in the absence of Ca(2+) channel opening, it might mediate elevations of cytosolic Ca(2+) and contraction in partially depolarized vascular smooth muscle cells exposed to small concentrations of agonists.     

Ca(2+) signaling in cardiac myocytes overexpressing the alpha(1) subunit of L-type Ca(2+) channel

Voltage-gated L-type Ca(2+) channels (LCCs) provide Ca(2+) ingress into cardiac myocytes and play a key role in intracellular Ca(2+) homeostasis and excitation-contraction coupling. We investigated the effects of a constitutive increase of LCC density on Ca(2+) signaling in ventricular myocytes from 4-month-old transgenic (Tg) mice overexpressing the alpha(1) subunit of LCC in the heart. At this age, cells were somewhat hypertrophic as reflected by a 20% increase in cell capacitance relative to those from nontransgenic (Ntg) littermates. Whole cell I(Ca) density in Tg myocytes was elevated by 48% at 0 mV compared with the Ntg group. Single-channel analysis detected an increase in LCC density with similar conductance and gating properties. Although the overexpressed LCCs triggered an augmented SR Ca(2+) release, the "gain" function of EC coupling was uncompromised, and SR Ca(2+) content, diastolic cytosolic Ca(2+), and unitary properties of Ca(2+) sparks were unchanged. Importantly, the enhanced I(Ca) entry and SR Ca(2+) release were associated with an upregulation of the Na(+)-Ca(2+) exchange activity (indexed by the half decay time of caffeine-elicited Ca(2+) transient) by 27% and SR Ca(2+) recycling by approximately 35%. Western analysis detected a 53% increase in the Na(+)-Ca(2+) exchanger expression but no change in the abundance of ryanodine receptor (RyR), SERCA2, and phospholamban. Analysis of I(Ca) kinetics suggested that SR Ca(2+) release-dependent inactivation of LCCs remains intact in Tg cells. Thus, in spite of the modest cardiac hypertrophy, the overexpressed LCCs form functional coupling with RyRs, preserving both orthograde and retrograde Ca(2+) signaling between LCCs and RyRs. These results also suggest that a modest but sustained increase in Ca(2+) influx triggers a coordinated remodeling of Ca(2+) handling to maintain Ca(2+) homeostasis.     

Spatial heterogeneity of intracellular Ca2+ concentration in nonbeating guinea pig ventricular myocytes

The spatial distribution of intracellular Ca2+ concentration was determined by fluorescent digital imaging microscopy in fura-2-loaded quiescent cardiac myocytes isolated from guinea pig ventricle. Fluorescent ratio images revealed discrete as well as clustered bright fluorescent spots ("hot spots"), which occupied approximately 20-50% of an individual cell's area. The fluorescent intensity and the area of the hot spots were increased by agents that deplete Ca2+ in the sarcoplasmic reticulum, namely, ryanodine (20-40 nM) and caffeine (5-15 mM). However, when cells were exposed to agents that deplete mitochondrial Ca2+, such as the protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP, 100-300 nM), or the inhibitor of electron transport, antimycin A (4-40 nM), the fluorescent intensity and the area of the hot spots were reduced. These results indicate that the spatial distribution of intracellular Ca2+ concentration in the ventricular myocytes of guinea pig is quite heterogeneous. The ability of CCCP and antimycin A, but not of caffeine and ryanodine, to reduce the fluorescent intensity in the hot spots implies that Ca2+ compartmentation in the mitochondria is largely responsible for the intracellular Ca2+ heterogeneity seen in the present study.     

Mechanisms of L-type Ca(2+) current downregulation in rat atrial myocytes during heart failure

Downregulation of the L-type Ca(2+) current (I(Ca)) is an important determinant of the electrical remodeling of diseased atria. Using a rat model of heart failure (HF) due to ischemic cardiopathy, we studied I(Ca) in isolated left atrial myocytes with the whole-cell patch-clamp technique and biochemical assays. I(Ca) density was markedly reduced (1.7+/-0.1 pA/pF) compared with sham-operated rats (S) (4.1+/-0.2 pA/pF), but its gating properties were unchanged. Calcium channel alpha(1C)-subunit quantities were not significantly different between S and HF. The beta-adrenergic agonist isoproterenol (1 micromol/L) had far greater stimulatory effects on I(Ca) in HF than in S (2.5- versus 1-fold), thereby suppressing the difference in current density. Dialyzing cells with 100 micromol/L cAMP or pretreating them with the phosphatase inhibitor okadaic acid also increased I(Ca) and suppressed the difference in density between S and HF. Intracellular cAMP content was reduced more in HF than in S. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine had a greater effect on I(Ca) in HF than in S (76.0+/-11.2% versus 15.8+/-21.2%), whereas the inhibitory effect of atrial natriuretic peptide on I(Ca) was more important in S than in HF (54.1+/-4.8% versus 24.3+/-8.8%). Cyclic GMP extruded from HF myocytes was enhanced compared with S (55.8+/-8.0 versus 6.2+/-4.0 pmol. mL(-1)). Thus, I(Ca) downregulation in atrial myocytes from rats with heart failure is caused by changes in basal cAMP-dependent regulation of the current and is associated with increased response to catecholamines.     

Thyroid hormone control of contraction and the Ca(2+)-ATPase/phospholamban complex in adult rat ventricular myocytes

Thyroid hormones may have important long-term effects on cellular Ca2+ handling in the heart. We investigated isolated adult rat cardiomyocytes in a primary culture exposed (T3-cells) or not exposed to (control cells) 10(-8) M triiodothyronine (T3) for 48 h. Northern blot analysis revealed reciprocal alterations in the expression of SERCA2 and phospholamban. The ratio of the SERCA2/phospholamban signal was approximately 10 times higher in the T3-cells as compared with the control cells (P < 0.05). Phospholamban protein content was significantly reduced by 33% but SR-Ca(2+)-ATPase protein content was not significantly altered in T3-cells. These results were associated with functional alterations measured by an inverted microscope equipped to monitor fluorescence at two excitation wavelengths as well as cell shortening by a video edge detection unit. The peak calcium transients as measured by fura-2 acetoxymethyl ester (AM) were increased significantly during stimulation at 0.25 and 0.5 Hz in T3-cells compared with control cells (P < 0.05). The monoexponential decline of the fura-2 transient was significantly faster at all frequencies in the T3-cells as compared with control cells (P < 0.05). Interestingly, we observed blunted responses to both isoproterenol stimulation and post rest potentiation in the T3-cells. The intracellular level of sodium as represented by SBFI-AM was significantly lower in the T3-cells compared with the control cells (P < 0.05). The increased SR-Ca(2+)-ATPase/phospholamban ratio and decrease in phospholamban protein content in T3-treated cells was reflected in a parallel increase of contraction and calcium transients and more rapid Ca2+ reuptake, but the post-rest potentiation and response to isoproterenol were reduced.     

Effects of cytosolic ATP on Ca(2+) sparks and SR Ca(2+) content in permeabilized cardiac myocytes

Confocal imaging was used to study the influence of cytosolic ATP on the properties of spontaneous Ca(2+) sparks in permeabilized ventricular myocytes. Cells were perfused with mock intracellular solutions containing fluo 3. Reducing [ATP] to <0.5 mmol/L decreased the frequency but increased the amplitude of spontaneous Ca(2+) sparks. In the presence of 20 micromol/L ATP, the amplitude increased by 48.7+/-10.9%, and the frequency decreased by 77.07+/-3.8%, relative to control responses obtained at 5 mmol/L ATP. After exposure to a solution containing zero ATP, the frequency of Ca(2+) sparks decreased progressively and approached zero within 90 seconds. As ATP washed out of the cell, the sarcoplasmic reticulum (SR) Ca(2+) content increased, until reaching a maximum after 3 minutes. Subsequent introduction of adenylyl imidodiphosphate precipitated a burst of large-amplitude Ca(2+) sparks. This was accompanied by a rapid decrease in SR Ca(2+) content to 80% to 90% of the steady-state value obtained in the presence of 5 mmol/L ATP. Thereafter, the SR Ca(2+) content declined much more slowly over 5 to 10 minutes. The effects of ATP withdrawal on Ca(2+) sparks may reflect reduced occupancy of the adenine nucleotide site on the SR Ca(2+) channel. These effects may contribute to previously reported changes in SR function during myocardial ischemia and reperfusion, in which ATP depletion and Ca(2+) overload occur.     

Voltage dependence of intracellular [Ca2+]i transients in guinea pig ventricular myocytes

[Ca2+]i transients, elicited by voltage-clamp depolarization of single guinea pig cardiac ventricular cells, were observed through use of the fluorescent Ca2+ indicator, fura-2. Individual cells, loaded with fura-2 either by internal perfusion or by exposure to fura-2/AM, were studied with the use of an inverted microscope that was equipped with ultraviolet epifluorescence illumination, an intensified silicon intensifier target camera, and a photomultiplier tube. Variation of membrane voltage and exposure of cells to verapamil (a Ca2+ channel blocker) and ryanodine (which was assumed to abolish selectively the release of Ca2+ from the sarcoplasmic reticulum) were used to investigate the cellular processes that determine the [Ca2+]i transient. The principal results of the study are: When appropriate methods are used, the properties of cytosolic fura-2 inside guinea pig cells are similar to those of fura-2 in solution, irrespective of the method of loading. The amplitude (at 100 msec) of verapamil-sensitive fluorescence transients elicited by pulse depolarization (range -30 to 80 mV) has a bell-shaped dependence on membrane voltage (maximum at 10 mV). Rapid, ryanodine-sensitive and verapamil-sensitive "tail transients" are elicited on repolarization from membrane potentials greater than 30 mV; their amplitude increases as the amplitude of the preceding pulse increases. The amplitude of slow fluorescence transients that are insensitive to verapamil and ryanodine increases continuously with membrane potential throughout the range -20 to 80 mV. The voltage dependence and pharmacology of the rapid transients elicited by pulse depolarization or by repolarization are consistent with their having arisen from Ca2+ released from the sarcoplasmic reticulum, via Ca2+-induced Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dyssynchronous Ca(2+) sparks in myocytes from infarcted hearts

The kinetics of contractions and Ca(2+) transients are slowed in myocytes from failing hearts. The mechanisms accounting for these abnormalities remain unclear. Myocardial infarction (MI) was produced by ligation of the circumflex artery in rabbits. We used confocal microscopy to record spatially resolved Ca(2+) transients during field stimulation in left ventricular (LV) myocytes from control and infarcted hearts (3 weeks). Compared with controls, Ca(2+) transients in myocytes adjacent to the infarct had lower peak amplitudes and prolonged time courses. Control myocytes showed relatively uniform changes in [Ca(2+)] throughout the cell after electrical stimulation. In contrast, in MI myocytes [Ca(2+)] increased inhomogeneously and localized increases in [Ca(2+)] occurred throughout the rising and falling phases of the Ca(2+) transient. Ca(2+) content of the sarcoplasmic reticulum did not differ between MI and control myocytes. Peak L-type Ca(2+) current density was reduced in MI myocytes. The macroscopic gain function was not different in control and MI myocytes when calculated as the amplitude of the Ca(2+) transient/peak I:(Ca). However, when calculated as the peak rate of rise of the Ca(2+) transient/peak I:(Ca), the gain function was modestly decreased in the MI myocytes. Application of isoproterenol (100 nmol/L) improved the synchronization of Ca(2+) release in MI myocytes at both 0.5 and 1 Hz. The poorly coordinated production of Ca(2+) sparks in myocytes from infarcted rabbit hearts likely contributes to the diminished and slowed macroscopic Ca(2+) transient. These abnormalities can be largely overcome when phosphorylation of Ca(2+) cycling proteins is enhanced by ss-adrenergic stimulation.     

Effects of strophanthidin on intracellular Ca2+ concentration and cellular morphology of guinea pig myocytes

To study the role of cytosolic calcium concentration [( Ca2+]i) in the pathophysiology of Ca2+ overload, we used a digital image of calcium indicator signals (fura-2 fluorescence) from guinea pig myocytes. [Ca2+]i increased from 69(SEM 5) nmol.litre-1 (n = 15) to 204(45) nmol.litre-1 (n = 14) after 10 min perfusion with 100 mumol.litre-1 strophanthidin. The effects of strophanthidin on cellular morphology were associated with variable increases in [Ca2+]i of myocytes. [Ca2+]i was 263(9) nmol.litre-1 in the spontaneous contracting cells with "Ca2+ wave", and 784(103) nmol.litre-1 in rounded cells. There was a significant relationship (r = 0.77, p less than 0.001) between elevation of [Ca2+]i and shortening of longitudinal length of the cells. After 20 min pretreatment with 10 mumol.litre-1 ryanodine, strophanthidin (100 mumol.litre-1) did not cause spontaneous contractile activity, and induced a slower increase in [Ca2+]i than found previously with strophanthidin alone. We suggest that [Ca2+]i played a primary role in mediating irreversible injury in this study, and that the absolute value of [Ca2+]i was correlated with morphological changes. We also suggest that the Ca2+ release from the sarcoplasmic reticulum was related to the increase in [Ca2+]i and the spontaneous contracting activity.     

Microtubule disruption modulates Ca(2+) signaling in rat cardiac myocytes

Microtubules have been shown to alter contraction in cardiac myocytes through changes in cellular stiffness. However, an effect on excitation-contraction coupling has not been examined. Here we analyze the effects of microtubule disruption by 1 micromol/L colchicine on calcium currents (I(Ca)) and [Ca(2+)](i) transients in rat ventricular myocytes. I(Ca) was studied using the whole-cell patch-clamp technique. Colchicine treatment increased I(Ca) density (peak values, -4.6+/-0.4 and -9.1+/-1.3 pA/pF in 11 control and 12 colchicine-treated myocytes, respectively; P<0.05). I(Ca) inactivation was well fitted by a biexponential function. The slow component of inactivation was unchanged, whereas the fast component was accelerated after colchicine treatment (at -10 mV, 11.8+/-1.0 versus 6.7+/-1.0 ms in control versus colchicine-treated cells; P<0.005). [Ca(2+)](i) transients were analyzed by fluo-3 epifluorescence simultaneously with I(Ca). Peak [Ca(2+)](i) transients were significantly increased in cardiac myocytes treated with colchicine. The values of F/F(0) at 0 mV were 1.1+/-0.02 in 9 control cells and 1.4+/-0.1 in 11 colchicine-treated cells (P<0.05). beta-Adrenergic stimulation with 1 micromol/L isoproterenol increased both I(Ca) and [Ca(2+)](i) transient in control cells. However, no significant change was induced by isoproterenol on colchicine-treated cells. Colchicine and isoproterenol effects were similar and not additive. Inhibition of adenylyl cyclase by 200 micromol/L 2'-deoxyadenosine 3'-monophosphate blunted the colchicine effect. We suggest that beta-adrenergic stimulation and microtubule disruption share a common pathway to enhance I(Ca) and [Ca(2+)](i) transient.     

Enhanced contractile responsiveness to cytosolic Ca(2+) by delta-2 opioid agonist deltorphin in intact guinea pig hearts

Opioid receptor subtypes, delta and kappa, are found in cardiac tissue and may play a role in cardiac function. We explored if the synthetic opioid delta(2)[D-Ala(2)]-deltorphin (DTP) and mu peptide agonist [D-Ala(2)]-enkephalin (DAMGO) alter the left ventricular pressure (LVP) [Ca(2+)](i) relationship in isolated guinea pig hearts. LV phasic [Ca(2+)](i) was measured from dual fluorescence signals using indo 1. Ca(2+) transients were corrected and calibrated to nM [Ca(2+)](i). Diastolic (d), systolic (s) [Ca(2+)](i), and s-d[Ca(2+)](i) were plotted v LVP at 0.3 to 6.8 mM [CaCl(2)](e)to assess the association of contractility to Ca(2+). Also given were naltriben (NTB) and CTOP, delta(2) and mu antagonists, and nifedipine (NIF) and thapsigargin (THAP). From a control of 880+/-95 nM (SEM), DTP decreased s-d[Ca(2+)](max) to 525+/-82 nM after DTP and to 405+/-84 nM after NIF, whereas THAP increased s-d[Ca(2+)](max)to 1605+/-275 nM. NTB, 795+/-33 nM, NTB+DTP, 820+/-98 nM, DAMGO, 970+/-82 nM, and DAMGO+CTOP, 830+/-93 nM, gave values similar to controls. From a control value of 61+/-4 mm Hg, LVP(max)was increased by DTP to 73+/-3 mmHg and by THAP to 77+/-2 mmHg, was unchanged by DAMGO at 48+/-6 mmHg, and was decreased by NIF to 24+/-2 mmHg. Compared to the control value of 594+/-18 nM, less s-d[Ca(2+)](i) was required to attain 50% s-dLVP(max)(curve left shift) with increasing [CaCl(2)](e) for DTP, 407+/-17 nM, and more was required for THAP, 737+/-35 nM. DTP raised the slope max of s-dLVP(max)(100%) v. s-d[Ca(2+)](i)by 2.7-fold. This indicates DTP enhances cardiac performance by enhancing responsiveness to cytosolic Ca(2+)rather than by raising diastolic Ca(2+) and subsequently released Ca(2+), as does THAP.     

Diversity of early afterdepolarizations in guinea pig myocytes: Spatial characteristics of intracellular Ca2+ concentration

Summary We classified early afterdepolarizations (EADs) into subgroups according to the spatial features of the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Myocytes were enzymatically isolated from guinea pig ventricles. When fura-2 salt was applied through a whole cell patch pipette after the formation of a gigaohm seal, the membrane potential was measured using the current, clamp technique. When myocytes were loaded with fura-2 AM, the membrane potential was recorded with a conventional microelectrode technique. Spatio-temporal changes in fura-2 fluorescence and cell length were recorded simultaneously, using a digital TV system. EADs were induced after superfusion with potassium-free Tyrode solution. Irrespective of the fura-2 loading procedure, EADs could be classified into those with spatially synchronous fluorescence changes (n = 26 from eight hearts) and those with heterogeneous changes (n = 20 from three hearts). EADs with synchronous features took off from a higher membrane potential (–34mV) than EADs with heterogeneous features (–57 mV). These results suggest that EADs have at least two constituents.     

Loss of beta-adrenoceptor response in myocytes overexpressing the Na+/Ca(2+)-exchanger

Increased Na+/Ca(2+)-exchanger (NCX) and altered beta-adrenoceptor (betaAR) responses are observed in failing human heart. To determine the possible interaction between these changes, we investigated the effect of NCX overexpression on responses to isoproterenol in adult rat ventricular myocytes. Responses to isoproterenol were largely mediated through the beta1AR in control myocytes. Adenovirally-mediated overexpression of NCX, at levels, which did not alter basal contraction of myocytes, markedly depressed the isoproterenol concentration-response curve. Responses to isoproterenol could be restored to normal by beta2AR blockade, suggesting a beta2AR-mediated inhibition of beta1AR signalling. Pertussis toxin normalised isoproterenol responses in NCX cells, indicating that beta2AR effects were mediated by Gi. Negative-inotropic effects of high concentrations of ICI 118,551, previously shown to be due to beta2AR-Gi coupling, were increased in NCX cells. We conclude that NCX upregulation can markedly alter the consequences of betaAR stimulation and that this may contribute to the alterations in betaAR response seen in failing human heart.