beta2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes

Selective stimulation of beta(2)-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of beta(2)-AR stimulation on L-type Ca(2+) current (I(Ca,L)) during postnatal development. I(Ca,L) was measured (37 degrees C; either Ca(2+) or Ba(2+) as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. Ca(2+) transients were measured concomitantly by dialyzing the cell with indo-1. Activation of beta(2)-ARs (with either 100 nM zinterol or 1 microM isoproterenol in the presence of the beta(1)-AR antagonist, CGP20712A) stimulated I(Ca,L) twofold in newborns but not in adults. The beta(2)-AR-mediated increase in Ca(2+) transient amplitude in newborns was due exclusively to the augmentation of I(Ca,L). Zinterol increased the rate of inactivation of I(Ca,L) and increased the Ca(2+) flux integral. The beta(2)-AR inverse agonist, ICI-118551 (500 nM), but not the beta(1)-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 microM), PKI 6-22 amide (10 microM), and Rp-cAMP (100 microM), all failed to prevent the response to zinterol but completely blocked responses to selective beta(1)-AR stimulation of I(Ca,L) in newborns. Our results demonstrate that in addition to the conventional beta(1)-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel beta(2)-AR-mediated, PKA-insensitive pathway that stimulates I(Ca,L). This striking developmental difference plays a major role in the age-related differences in inotropic responses to beta(2)-AR agonists.     

Nerve growth factor increases L-type calcium current in pancreatic beta cells in culture

We analyzed the effect of culturing adult rat beta cells with NGF2.5 S for 5 to 7 days on macroscopic barium current (I(Ba)), and determined the role of Na and Ca channels on neurite-like process extension induced by NGF and dbcAMP, and by KCI depolarization. After five days in culture with 2.5S NGF, beta cells exhibit a 102% increase in I(Ba) density. This effect is on L-type calcium channels because most of the current is blocked by nifedipine. The application of NGF for 5 minutes to the cells deprived of the trophic factor for 24 hr further increases I(Ba) current by 91%. These results suggest that the trophic factor regulates I(Ba) by two different mechanisms, a) an increase in channel density and b) a rapid modulation of the channels already present in the membrane. Finally, we found that ion-channel activity modifies the growth of neurite-like processes. After 2 weeks in culture with high KCl, almost 14% of beta cells extend neurite-like processes and the most impressive effect is observed in the presence of KCl, NGF, and dbcAMP simultaneously, where nearly 60% of the cells extend neurite-like processes. Tetrodotoxin and nifedipine reduce the morphological changes induced by these agents.     

Citalopram inhibits L-type calcium channel current in rat cardiomyocytes in culture

Selective serotonine reuptake inhibitors (SSRI) are believed to be less dangerous in the treatment of depressive disorder in comparison with tricyclic antidepressants (TCA) due to their relative lack of cardiotoxicity. Thus, we investigated the effect of citalopram (SSRI) on membrane electrophysiology in rat cardiomyocytes in tissue culture. The results were compared with those from amitriptyline (TCA). The whole-cell configuration patch-clamp technique was used. Both citalopram and amitriptyline exhibited the concentration-dependent inhibition of the L-type calcium channel current (ICa). Citalopram in concentrations of 3 microM and 10 microM inhibited peak calcium current by 2.7% and 8%, respectively. We demonstrated the same potency of citalopram and amitriptyline to inhibit ICa. These observations led us to conclude that citalopram and amitriptyline are drugs, which exhibit a similar potency for causing concentration-dependent inhibition of ICa.     

Serotonin increases L-type Ca2+ current and SR Ca2+ content through 5-HT4 receptors in failing rat ventricular cardiomyocytes

Rats with congestive heart failure (CHF) develop ventricular inotropic responsiveness to serotonin (5-HT), mediated through 5-HT(2A) and 5-HT(4) receptors. Human ventricle is similarly responsive to 5-HT through 5-HT(4) receptors. We studied isolated ventricular cardiomyocytes to clarify the effects of 5-HT on intracellular Ca(2+) handling. Left-ventricular cardiomyocytes were isolated from male Wistar rats 6 wk after induction of postinfarction CHF. Contractile function and Ca(2+) transients were measured in field-stimulated cardiomyocytes, and L-type Ca(2+) current (I(Ca,L)) and sarcoplasmic reticulum (SR) Ca(2+) content were measured in voltage-clamped cells. Protein phosphorylation was measured by Western blotting or phosphoprotein gel staining. 5-HT(4)- and 5-HT(2A)-receptor stimulation induced a positive inotropic response of 33 and 18% (both P < 0.05) and also increased the Ca(2+) transient (44 and 6%, respectively; both P < 0.05). I(Ca,L) and SR Ca(2+) content increased only after 5-HT(4)-receptor stimulation (57 and 65%; both P < 0.05). Phospholamban serine(16) (PLB-Ser(16)) and troponin I phosphorylation increased by 26 and 13% after 5-HT(4)-receptor stimulation (P < 0.05). 5-HT(2A)-receptor stimulation increased the action potential duration and did not significantly change the phosphorylation of PLB-Ser(16) or troponin I, but it increased myosin light chain 2 (MLC2) phosphorylation. In conclusion, the positive inotropic response to 5-HT(4) stimulation results from increased I(Ca,L) and increased phosphorylation of PLB-Ser(16), which increases the SR Ca(2+) content. 5-HT(4) stimulation is thus, like beta-adrenoceptor stimulation, possibly energetically unfavorable in CHF. 5-HT(2A)-receptor stimulation, previously studied in acute CHF, induces a positive inotropic response also in chronic CHF, probably mediated by MLC2 phosphorylation.     

Mechanical stretch increases L-type calcium channel stability in cardiomyocytes through a polycystin-1/AKT-dependent mechanism

The L-type calcium channel (LTCC) is an important determinant of cardiac contractility. Therefore, changes in LTCC activity or protein levels could be expected to affect cardiac function. Several studies describing LTCC regulation are available, but only a few examine LTCC protein stability. Polycystin-1 (PC1) is a mechanosensor that regulates heart contractility and is involved in mechanical stretch-induced cardiac hypertrophy. PC1 was originally described as an unconventional Gi/o protein-coupled receptor in renal cells. We recently reported that PC1 regulates LTCC stability in cardiomyocytes under stress; however, the mechanism underlying this effect remains unknown. Here, we use cultured neonatal rat ventricular myocytes and hypo-osmotic stress (HS) to model mechanical stretch. The model shows that the Cavβ2 subunit is necessary for LTCC stabilization in cardiomyocytes during mechanical stretch, acting through an AKT-dependent mechanism. Our data also shows that AKT activation depends on the G protein-coupled receptor activity of PC1, specifically its G protein-binding domain, and the associated Gβγ subunit of a heterotrimeric Gi/o protein. In fact, over-expression of the human PC1 C-terminal mutant lacking the G protein-binding domain blunted the AKT activation-induced increase in Cav1.2 protein in cardiomyocytes. These findings provide novel evidence that PC1 is involved in the regulation of cardiac LTCCs through a Giβγ-AKT-Cavβ2 pathway, suggesting a new mechanism for regulation of cardiac function.     

A glutathione deficit alters dopamine modulation of L-type calcium channels via D2 and ryanodine receptors in neurons

Synthesis of glutathione, a major redox regulator, is compromised in schizophrenia. We postulated that the resulting glutathione deficit via its effect on redox-sensitive proteins could contribute to dysfunction of some neurotransmitter systems in schizophrenia. We investigated whether a glutathione deficit, induced by a blocker of glutathione synthesis, L-buthionine-(S,R)-sulfoximine, affects intracellular pathways implicated in dopamine signaling in neurons, namely dopamine modulation of calcium responses to NMDA. Such a glutathione deficit changed the modulation of responses by dopamine, from enhanced responses in control neurons (likely via D1-type receptors) to decreased responses in low-glutathione neurons (via D2-type receptors). This difference in dopamine modulation was due to a different modulation of L-type calcium channels activated during NMDA stimulation: dopamine enhanced function of these channels in control neurons but decreased it in low-glutathione neurons. The effect of a glutathione deficit on dopamine signaling was dependent on the redox-sensitive ryanodine receptors (RyRs), whose function was enhanced in low-glutathione neurons. This suggests that enhanced RyRs in low-glutathione neurons strengthens intracellular calcium-dependent pathways following activation of D2-type receptors and causes a decrease in function of L-type channels. This represents a mechanism by which dopaminergic systems could be dysfunctional under conditions of impaired glutathione synthesis as in schizophrenia.     

Dopamine D1 and D2 receptors in spontaneously hypertensive rat brain striatum

Since it has been reported that dopamine D2 receptors are elevated in the brain striatum of spontaneously hypertensive (SH) rats, and since both D1 and D2 receptors may interact with one another, we measured the densities of both these receptors in SH rat striatum, as well as those in the normotensive Wistar-Kyoto rat striatum. The D1 receptor density in both strains was virtually the same, 72.9 +/- 2.2 and 71.3 +/- 3.2 pmol/g, respectively (mean +/- SD). The D2 receptor densities were also almost identical, 16.3 +/- 0.6 and 16.8 +/- 1.0 pmol/g, respectively (mean +/- SD). Thus, these data do not support the concept of a dopamine receptor related role in spontaneous hypertension.     

Forskolin fails to activate L-type calcium current in hypertrophied cardiomyocytes of chronically hypoxic rats

We have shown that the contractile, cytosolic calcium ([Ca2+]i) and cyclic AMP (cAMP) responses to beta-adrenoceptor stimulation are attenuated in ventricular myocytes of chronically hypoxic (CH) rats. The aim of this study was to examine the effect of forskolin on the L-type Ca2+ current in CH hypertrophied ventricular myocytes. Patch-clamp recording of the L-type Ca2+ current was measured in right ventricular myocytes of normoxic control and CH rats exposed to 10% inspired oxygen for 4 weeks. The breadth, but not the length, of CH myocytes was significantly greater than that of the control group. Activation of beta-adrenoceptor with isoproterenol (0.1 microM) increased the peak Ca2+ current by 83% in the normoxic control but the increase of peak Ca2+ current was not significant in the CH myocytes. Forskolin (0.1 - 1 microM), an activator of adenylyl cyclase, increased the peak Ca2+ current by 49% - 102% in the normoxic controls but it did not cause significant change of the peak Ca2+ current in CH myocytes. These results suggest an absence of forskolin-induced activation of Ca2+ current in hypertrophied ventricular myocytes during chronic hypoxia. The failure of activation of the Ca2+ current is consistent with the idea that adenylyl cyclase function is down-regulated in CH hypertrophied myocytes.     

L-type calcium current in right ventricular outflow tract myocytes of rabbit heart

The mechanism of idiopathic ventricular tachycardia originating from the right ventricular outflow tract (RVOT) is not clear. Many clinical reports have suggested a mechanism of triggered activity. However, there are few studies investigating this because of the technical difficulties associated with examining this theory. The L-type calcium current (I _Ca-L), an important inward current of the action potential (AP), plays an important role in arrhythmogenesis. The aim of this study was to explore differences in the APs of right ventricular (RV) and RVOT cardiomyocytes, and differences in electrophysiological characteristics of the ICa-L in these myocytes. Rabbit RVOT and RV myocytes were isolated and their AP and I _Ca-L were investigated using the patch-clamp technique. RVOT cardiomyocytes had a wider range of AP duration (APD) than RV cardiomyocytes, with some markedly prolonged APDs and markedly shortened APDs. The markedly shortened APDs in RVOT myocytes were abolished by treatment with 4-AP, an inhibitor of the transient outward potassium current, but the markedly prolonged APDs remained, with some myocytes with a long AP plateau not repolarizing to resting potential. In addition, early afterdepolarization (EAD) and second plateau responses were seen in RVOT myocytes but not in RV myocytes. RVOT myocytes had a higher current density for I _Ca-L than RV myocytes (RVOT (13.16±0.87) pA pF⁻¹, RV (8.59±1.97) pA pF⁻¹; P<0.05). The I _Ca-L and the prolonged APD were reduced, and the EAD and second plateau response disappeared, after treatment with nifedipine (10 μmol L⁻¹), which blocks the I _Ca-L. In conclusion, there was a wider range of APDs in RVOT myocytes than in RV myocytes, which is one of the basic factors involved in arrhythmogenesis. The higher current density for I _Ca-L is one of the factors causing prolongation of the APD in RVOT myocytes. The combination of EAD with prolonged APD may be one of the mechanisms of RVOT-VT generation.     

Dopamine receptors D1 and D2 are related to observed maternal behavior

The dopamine pathway and especially the dopamine receptors 1 and 2 (DRD1 and DRD2) are implicated in the regulation of mothering in rats. Evidence for this in humans is lacking. Here, we show that genetic variation in both DRD1 and DRD2 genes in a sample of 187 Caucasian mothers predicts variation in distinct maternal behaviors during a 30-min mother-infant interaction at 6 months postpartum. Two DRD1 single-nucleotide polymorphisms (SNPs rs265981 and rs686) significantly associated with maternal orienting away from the infant (P = 0.002 and P = 0.003, respectively), as did DRD1 haplotypes (P = 0.03). Two DRD2 SNPs (rs1799732 and rs6277) significantly associated with maternal infant-directed vocalizing (P = 0.001 and P = 0.04, respectively), as did DRD2 haplotypes (P = 0.01). We present evidence for heterosis in DRD1 where heterozygote mothers orient away from their infants significantly less than either homozygote group. Our findings provide important evidence that genetic variation in receptors critical for mothering in non-human species also affect human maternal behaviors. The findings also highlight the importance of exploring multiple dimensions of the complex human mothering phenotype.     

Conditioning of beta(1)-adrenoceptor effect via beta(2)-subtype on L-type Ca(2+) current in canine ventricular myocytes

We investigated the roles of beta(1)- and beta(2)-receptors (beta-AR) in adrenergic enhancement of L-type Ca(2+) current (I(CaL)) in canine ventricular myocytes. Isoproterenol and l-norepinephrine produced a monophasic and a biphasic concentration-I(CaL) relationship (CR), respectively. alpha(1)-AR inhibition with prazosin and beta(2)-AR stimulation with zinterol or l-epinephrine shifted the CR of l-norepinephrine leftward. Zinterol (50 nM) and l-epinephrine (10 nM), but not prazosin, altered the biphasic CR of l-norepinephrine to a monophasic CR. Zinterol and l-epinephrine applied after l-norepinephrine had no effect on I(CaL). beta(2)-AR inhibition with ICI-118551 reduced the E(max) of isoproterenol and l-norepinephrine by 60% and abolished the augmentation of l-norepinephrine by zinterol and l-epinephrine. Carbachol (100 nM) modestly reduced the I(CaL) response to beta(1)-AR stimulation but abolished the enhancement via beta(2)-AR. Zinterol augmented the enhancement of I(CaL) by forskolin, IBMX, and theophylline, but not in the presence of CGP-20712A. We conclude that selective beta(2)-AR stimulation does not increase I(CaL) but enhances adenylyl cyclase activity when stimulated via beta(1)-AR and with forskolin. beta(2)-AR activity preconditions adenylyl cyclase for beta(1)-AR stimulation.     

L-type channel inactivation balances the increased peak calcium current due to absence of Rad in cardiomyocytes

The L-type Ca²⁺ channel (LTCC) provides trigger calcium to initiate cardiac contraction in a graded fashion that is regulated by L-type calcium current (I_Ca,L) amplitude and kinetics. Inactivation of LTCC is controlled to fine-tune calcium flux and is governed by voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). Rad is a monomeric G protein that regulates I_Ca,L and has recently been shown to be critical to β-adrenergic receptor (β-AR) modulation of I_Ca,L. Our previous work showed that cardiomyocyte-specific Rad knockout (cRadKO) resulted in elevated systolic function, underpinned by an increase in peak I_Ca,L, but without pathological remodeling. Here, we sought to test whether Rad-depleted LTCC contributes to the fight-or-flight response independently of β-AR function, resulting in I_Ca,L kinetic modifications to homeostatically balance cardiomyocyte function. We recorded whole-cell I_Ca,L from ventricular cardiomyocytes from inducible cRadKO and control (CTRL) mice. The kinetics of I_Ca,L stimulated with isoproterenol in CTRL cardiomyocytes were indistinguishable from those of unstimulated cRadKO cardiomyocytes. CDI and VDI are both enhanced in cRadKO cardiomyocytes without differences in action potential duration or QT interval. To confirm that Rad loss modulates LTCC independently of β-AR stimulation, we crossed a β₁,β₂-AR double-knockout mouse with cRadKO, resulting in a Rad-inducible triple-knockout mouse. Deletion of Rad in cardiomyocytes that do not express β₁,β₂-AR still yielded modulated I_Ca,L and elevated basal heart function. Thus, in the absence of Rad, increased Ca²⁺ influx is homeostatically balanced by accelerated CDI and VDI. Our results indicate that the absence of Rad can modulate the LTCC without contribution of β₁,β₂-AR signaling and that Rad deletion supersedes β-AR signaling to the LTCC to enhance in vivo heart function.     

ATP induces intracellular calcium increases and actin cytoskeleton disaggregation via P2x receptors

The consequences of purinoceptor activation on calcium signalling, inositol phosphate metabolism, protein secretion and the actin cytoskeleton were demonstrated in the WRK-1 cell line. Extracellular ATP was used as a secretagogue to induce a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)), acting via P2x purinergic receptors, which causes actin skeleton disaggregation and protein secretion. ATP bound specifically to purinergic receptors, with Ki of 0.8 microM. The magnitude order for binding of different nucleotides was alpha beta-Met-ATP >or= dATPalphaS > ATP >or= ADP > UTP > AMP > suramin. No increase in inositol phosphates (IPs) was observed after ATP application suggesting that the purinergic sites in WRK-1 cells are not of a P2y type. ATP (1-100 microM) caused a concentration-dependent increase in [Ca(2+)](i)(EC(50)= 30 microM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca(2+)](e)) was reduced to 100 nM. A non-specific purinergic antagonist, suramin, reversibly inhibited the ATP-response suggesting that ATP is able to bind to P2x purinergic sites to trigger Ca(2+) entry and increase of [Ca(2+)](i). ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca(2+)](e). Similarly, alpha,beta-Met-ATP, a potent P2x agonist also stimulated Ca(2+) mobilization, actin network destructuration, and protein release. In the isolated rat neurohypophysial nerve terminals, ATP was shown to act as a physiological stimulus for vasopressin release via Ca(2+) entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is also able to induce actin disaggregation by a Ca(2+) dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis.     

L-type but not T-type calcium current changes during postnatal development in rabbit sinoatrial node

Although the neonatal sinus node beats at a faster rate than the adult, when a sodium current (I(Na)) present in the newborn is blocked, the spontaneous rate is slower in neonatal myocytes than in adult myocytes. This suggests a possible functional substitution of I(Na) by another current during development. We used ruptured [T-type calcium current (I(Ca,T))] and perforated [L-type calcium current (I(Ca,L))] patch clamps to study developmental changes in calcium currents in sinus node cells from adult and newborn rabbits. I(Ca,T) density did not differ with age, and no significant differences were found in the voltage dependence of activation or inactivation. I(Ca,L) density was lower in the adult than newborn (12.1 +/- 1.4 vs. 17.6 +/- 2.5 pA/pF, P = 0.049). However, activation and inactivation midpoints were shifted in opposite directions, reducing the potential contribution during late diastolic depolarization in the newborn (activation midpoints -17.3 +/- 0.8 and -22.3 +/- 1.4 mV in the newborn and adult, respectively, P = 0.001; inactivation midpoints -33.4 +/- 1.4 and -28.3 +/- 1.7 mV for the newborn and adult, respectively, P = 0.038). Recovery of I(Ca,L) from inactivation was also slower in the newborn. The results suggest that a smaller but more negatively activating and rapidly recovering I(Ca,L) in the adult sinus node may contribute to the enhanced impulse initiation at this age in the absence of I(Na).     

Dopamine D1 and D2 receptor Co-activation generates a novel phospholipase C-mediated calcium signal

Although dopamine D1 and D2 receptors belong to distinct subfamilies of dopamine receptors, several lines of evidence indicate that they are functionally linked. However, a mechanism for this linkage has not been elucidated. In this study, we demonstrate that agonist stimulation of co-expressed D1 and D2 receptors resulted in an increase of intracellular calcium levels via a signaling pathway not activated by either receptor alone or when only one of the co-expressed receptors was activated by a selective agonist. Calcium signaling by D1-D2 receptor co-activation was abolished following treatment with a phospholipase C inhibitor but not with pertussis toxin or inhibitors of protein kinase A or protein kinase C, indicating coupling to the G(q) pathway. We also show, by co-immunoprecipitation from rat brain and from cells co-expressing the receptors, that D1 and D2 receptors are part of the same heteromeric protein complex and, by immunohistochemistry, that these receptors are co-expressed and co-localized within neurons of human and rat brain. This demonstration that D1 and D2 receptors have a novel cellular function when co-activated in the same cell represents a significant step toward elucidating the mechanism of the functional link observed between these two receptors in brain.     

Modulation of L-type Ca2+ currents and intracellular calcium by agmatine in rat cardiomyocytes

It is shown that agmatine inhibits L-type Ca²⁺ currents in isolated cardiomyocytes of rats in a dose-dependent manner. The inhibitory analysis indicates that imidazoline receptors of type I (I₁Rs) rather than α₂-adrenoceptors (α₂-ARs) are implicated in mediating the effects of agmatine. Agmatine affects the dynamics of intracellular Ca²⁺ concentration changes in spontaneously active cardiomyocytes. The averaged intracellular Ca²⁺ concentration ([Ca²⁺]_in) varied biphasically, depending on the agmatine dose: at 1–500 μM, agmatine decreased [Ca²⁺]_in; at 500 μM-2 mM, [Ca²⁺]_in remained unchanged, and at concentrations above 2 mM agmatine caused an increase of [Ca²⁺]_in. The effects of low agmatine concentrations were inhibited by 7NI, an inhibitor of NO synthases (NOS), as well as by the inhibitors of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) thapsigargin and cyclopiazonic acid. In contrast, ODQ, a blocker of NO-sensitive guanylate cyclase, and the antagonist of I₁Rs efaroxan were ineffective. At low concentrations agmatine did not affect the increase in [Ca²⁺]_in induced by stimulating doses of ryanodine (40 nM). In addition, agmatine at low doses was found to markedly stimulate NO production. When efaroxan (10 μM) or ryanodine (200 μM) were added to the bath to inhibit I₁Rs and ryanodine receptors (RyRs), respectively, [Ca²⁺]_in became much less sensitive to millimolar agmatine. In contrast to low concentrations (100 μM), high agmatine doses (10–15 mM) did not stimulate the NO synthesis but were effective as NOS inducer in cells pretreated with efaroxan. The selective I₁R agonist rilmenidine increased [Ca²⁺]_in in a dose-dependent manner. The effect of rilmenidine was similar to that of agmatine at high doses and was abolished by RyRs inhibition. Our findings indicate that in spontaneously active cardiomyocytes agmatine at low concentrations decreases [Ca²⁺]_in, does not stimulate I₁Rs but most likely enhances NO synthase followed by an increase in SERCA activity due to the direct nitrosylation of SERCA and/or phospholamban. The effects of high agmatine doses are apparently mediated by I₁Rs and involve RyRs.     

S100A1 enhances the L-type Ca2+ current in embryonic mouse and neonatal rat ventricular cardiomyocytes

S100A1 is an EF-hand type Ca2+-binding protein with a muscle-specific expression pattern. The highest S100A1 protein levels are found in cardiomyocytes, and it is expressed already at day 8 in the heart during embryonic development. Since S100A1 is known to be involved in the regulation of Ca2+ homeostasis, we tested whether extracellular S100A1 plays a role in regulating the L-type Ca2+ current (I(Ca)) in ventricular cardiomyocytes. Murine embryonic (day 16.5 postcoitum) ventricular cardiomyocytes were incubated with S100A1 (0.001-10 microM) for different time periods (20 min to 48 h). I(Ca) density was found to be significantly increased as early as 20 min (from -10.8 +/- 1 pA/pF, n = 18, to -22.9 +/- 1.4 pA/pF; +112.5 +/- 13%, n = 9, p < 0.001) after the addition of S100A1 (1 microM). S100A1 also enhanced I(Ca) current density in neonatal rat cardiomyocytes. Fluorescence and capacitance measurements evidenced a fast translocation of rhodamine-coupled S100A1 from the extracellular space into cardiomyocytes. S100A1 treatment did not affect cAMP levels. However, protein kinase inhibitor, a blocker of cAMP-dependent protein kinase A (PKA), abolished the S100A1-induced enhancement of I(Ca). Accordingly, measurements of PKA activity yielded a significant increase in S100A1-treated cardiomyocytes. In vitro reconstitution assays further demonstrated that S100A1 enhanced PKA activity. We conclude that the Ca2+-binding protein S100A1 augments transsarcolemmal Ca2+ influx via an increase of PKA activity in ventricular cardiomyocytes and hence represents an important regulator of cardiac function.