Hiroshi Matsuura

Increase in intracellular Ca2+ and calcitonin gene-related peptide release through metabotropic P2Y receptors in rat dorsal root ganglion neurons

We examined the effects of the activation of metabotropic P2Y receptors on the intracellular Ca2+ concentration and the release of neuropeptide calcitonin gene-related peptide (CGRP) in isolated adult rat dorsal root ganglion neurons. In small-sized dorsal root ganglion neurons (soma diameter<30 μm) loaded with fura-2, a bath application of ATP (100 μM) evoked an increase in intracellular Ca2+ concentration, while the removal of extracellular Ca2+ partly depressed the response to ATP, thus suggesting that the ATP-induced increase in intracellular Ca2+ concentration is due to both the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. Bath application of uridine 5′-triphosphate (UTP; 100 μM) also caused an increase in intracellular Ca2+ concentration in small-sized dorsal root ganglion neurons and the P2 receptor antagonists suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; 10 μM) virtually abolished the response, indicating that the intracellular Ca2+ elevation in response to UTP is mediated through metabotropic P2Y receptors. This intracellular Ca2+ increase was abolished by pretreating the neurons with thapsigargin (100 nM), suggesting that the UTP-induced increase in intracellular Ca2+ is primarily due to the release of Ca2+ from endoplasmic reticulum Ca2+ stores. An enzyme-linked immunosorbent assay showed that an application of UTP (100 μM) significantly stimulated the release of CGRP and that suramin (100 μM) totally abolished the response, suggesting that P2Y receptor-mediated increase in intracellular Ca2+ is accompanied by CGRP release from dorsal root ganglion neurons. n nThese results suggest that metabotropic P2Y receptors contribute to extracellular ATP-induced increase in intracellular Ca2+ concentration and subsequent release of neuropeptide CGRP in rat dorsal root ganglion neurons.

Angiotensin II Potentiates the Slow Component of Delayed Rectifier K+ Current via the AT1 Receptor in Guinea Pig Atrial Myocytes

Background— Angiotensin II (Ang II) has diverse actions on cardiac electrical activity. Little information is available, however, regarding immediate electrophysiological effects of Ang II on cardiac repolarization. Methods and Results— The present study investigated the immediate effects of Ang II on the slow component of delayed rectifier K+ current (IKs) and action potentials in guinea pig atrial myocytes using the whole-cell patch-clamp technique. Bath application of Ang II increased the amplitude of IKs (EC50, 6.16 nmol/L) concentration dependently. The stable analogue Sar1–Ang II was also effective at increasing IKs. The voltage dependence of IKs activation and the kinetics of deactivation were not significantly affected by these agonists. The enhancement of IKs was blocked by the Ang II type 1 (AT1) receptor antagonist valsartan (1 &mgr;mol/L) and was markedly attenuated by inclusion of GDPβS (2 mmol/L) in the pipette, indicating an involvement of G protein–coupled AT1 receptor. The stimulatory effect was also significantly reduced by the phospholipase C inhibitor compound 48/80 (100 &mgr;mol/L) and the protein kinase C inhibitors bisindolylmaleimide I (200 nmol/L) and H-7 (10 &mgr;mol/L), suggesting that AT1 receptor acts through phospholipase C–protein kinase C signaling cascade to potentiate IKs. As expected from its stimulatory action on IKs, Sar1–Ang II markedly shortened the action potential duration, which could be reversed by valsartan. Conclusions— The potentiation of IKs via AT1 stimulation in atrial myocytes, accompanied by a shortening of the action potential duration, suggests a potential mechanism by which elevated levels of Ang II may promote atrial fibrillation in heart failure and warrants further investigation.

Rapidly and slowly activating components of delayed rectifier K+ current in guinea-pig sino-atrial node pacemaker cells

The components and properties of the delayed rectifier K+ current (IK) in isolated guinea‐pig sino‐atrial (SA) node pacemaker cells were investigated using the whole‐cell configuration of the patch‐clamp technique. An envelope of tails test was conducted by applying depolarizing pulses from a holding potential of −50 mV to +30 mV for various durations ranging from 40 to 2000 ms. The ratio of the tail current amplitude elicited upon return to the holding potential to the magnitude of the time‐dependent outward current activated during depolarizing steps was dependent on the pulse duration, while after exposure to the selective IKr inhibitor E‐4031 (5 μm) this current ratio became practically constant irrespective of the pulse duration. These observations are consistent with the presence of the E‐4031‐sensitive, rapidly activating and E‐4031‐resistant, slowly activating components of IK (IKr and IKs, respectively) in guinea‐pig SA node cells. The activation range for IKr, defined as the E‐4031‐sensitive current (half‐maximal activation voltage (V1/2) of −26.2 mV) was much more negative than that for IKs, defined as the E‐4031‐resistant current (V1/2 of +17.2 mV). IKr exhibited a marked inward rectification at potentials positive to −50 mV, whereas IKs showed only a slight rectification. In the current‐clamp experiments, bath application of E‐4031 (0.5 and 5 μm) initially slowed the repolarization at potentials negative to approximately −30 mV and produced a significant depolarization of the maximum diastolic potential, followed by the arrest of electrical activity, thus indicating that the late phase of the repolarization leading to the maximum diastolic potential at around −60 mV in spontaneous action potentials is primarily produced by IKr in guinea‐pig SA node cells. External application of the selective IKs inhibitor 293B (30 μm) also delayed the repolarization process at potentials negative to about −20 mV and induced moderate depolarization of the maximum diastolic potential leading to the arrest of the spontaneous activity. These results provide evidence to suggest that both IKr and IKs are present and play crucial roles in the spontaneous electrical activity of guinea‐pig SA node pacemaker cells.

Effect of ATP on preadipocyte migration and adipocyte differentiation by activating P2Y receptors in 3T3-L1 cells

The effect of extracellular ATP on adipogenesis was investigated using the mouse 3T3-L1 cell line. Incubation of cells with ATP (1-100 microM) for 5 min induced actin filament reorganization and membrane ruffling mediated through P2Y receptors. Enhancement of preadipocyte migration into fat cell clusters is one of the essential processes of adipose tissue development in vivo and cell migration assays revealed that stimulation of P2Y receptors enhanced chemokinesis (migration) in a concentration dependent manner. In this cell line, growth arrest is required before initiation of differentiation and growth-arrested post-confluent cells can be converted into adipocytes by the presence of the adipogenic hormones dexamethasone, 3-isobutyl-1-methylxanthine and insulin. On the other hand, those hormones alone do not trigger differentiation in proliferating cells. ATP did not induce differentiation when applied alone to either proliferating or postconfluent cells. By contrast, proliferating cells (density <50%) preincubated with ATP for 5 min and subsequently given the adipogenic hormones in the continued presence of ATP, underwent adipocyte differentiation mediated through phospholipase C-coupled P2Y receptors. These adipocytes were found to show very similar characteristics, including morphology and intracellular triacylglycerol accumulation compared with adipocytes differentiated from post-confluent preadipocytes with those adipogenic hormones. When proliferating cells were preincubated with ATP before the addition of the adipogenic hormones, gene expression of aP2 (adipose protein 2) was markedly increased within 6 days, whereas without ATP pretreatment the expression level stayed very low. These results suggest that extracellular ATP renders preadipocytes responsive to adipogenic hormones during the growth phase.

Potentiation of slow component of delayed rectifier K+ currentby cGMP via two distinct mechanisms: inhibition of phosphodiesterase 3 and activation of protein kinase G

Regulation of the slowly activating component of delayed rectifier K+ current (IKs) by intracellular guanosine 3′5′ cyclic monophosphate (cGMP) was investigated in guinea‐pig sino‐atrial (SA) node cells using the whole‐cell patch‐clamp method. When a cell was dialyzed with pipette solution containing 100 μM cGMP, IKs started to gradually increase and reached a maximum increase of a factor of 2.37±0.39 (n=4) about 10–15 min after rupture of patch membrane. Atrial natriuretic peptide (ANP, 100 nM) also potentiated IKs, consistent with intracellular cGMP‐induced enhancement of IKs. Bath application of a selective blocker of the cGMP‐inhibited phosphodiesterase (PDE3) milrinone (100 μM) enhanced IKs by a factor of 1.50±0.09 (n=4) but failed to further enhance IKs after a maximum stimulation by intracellular cGMP (100 μM), suggesting that blockade of PDE3 activity is involved in the enhancement of IKs. A potent but nonspecific PDE inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX, 100 μM) further increased IKs stimulated by 100 μM milrinone, indicating that PDE subtypes other than PDE3 are also involved in the regulation of basal IKs in guinea‐pig SA node cells. Bath application of 100 μM 8‐bromoguanosine 3′5′ cyclic monophosphate (8‐Br‐cGMP) increased IKs by a factor of 1.48±0.11 (n=5) and this stimulatory effect was totally abolished by cGMP‐dependent protein kinase (PKG) inhibitor KT‐5823 (500 nM), suggesting that the activation of PKG also mediates cGMP‐induced potentiation of IKs. These results strongly suggest that intracellular cGMP potentiates IKs not only by blocking PDE3 but also by activating PKG in guinea‐pig SA node cells.

Properties of the Na+/K+ pump current in small neurons from adult rat dorsal root ganglia

The present investigation was undertaken to characterize the Na+/K+ pump current in small (25 μm in soma diameter) dorsal root ganglion (DRG) neurons isolated from lumbar L4‐6 segments of adult rats. The Na+/K+ pump current was identified as an ouabain‐sensitive current during square voltage steps to membrane potentials between +40 and −120 mV, using the whole‐cell patch‐clamp technique in which Ca2+ and K+ channel currents and Na+/Ca2+ exchange currents were minimized. The Na+/K+ pump current was practically time‐independent over the entire voltage range examined and exhibited a voltage‐dependence; its current – voltage (I–V) relationship displayed a positive slope at potentials between −120 and 0 mV but nearly plateau levels at positive membrane potentials. The concentration‐dependent block of Na+/K+ pump current (activated by 30 mM pipette Na+) by ouabain at concentrations between 0.1 μM and 5 mM was biphasic and was well described using a two‐binding site model with dissociation constants for high‐ and low‐affinity binding sites of 0.20 and 140.1 μM, respectively. The relative amplitude of the Na+/K+ pump current produced by low‐ and high‐affinity sites (probably α1β1 and α3β1 isozymes, respectively) was estimated to be 13 : 1 in the presence of 30 mM Na+ in the pipette solution. Additionally, the activation of Na+/K+ pump current by pipette Na+ at concentrations ranging from 5 to 100 mM also exhibited a biphasic concentration dependence which can be reasonably well fitted by assuming the existence of two isozymes having high and low affinities for Na+ (6.7 and 67.6 mM, respectively). Thus, the present investigation provides functional evidence to suggest that the Na+/K+ ATPase comprises two functionally distinct isozymes as expected for α1β1 and α3β1 in rat small DRG neurons.

Regulation of Cardiac IKs Potassium Current by Membrane Phosphatidylinositol 4,5-Bisphosphate

Regulation of the slowly activating component of delayed rectifier K+ current (IKs) by membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns-(4,5)P2) was examined in guinea pig atrial myocytes using the whole-cell patch clamp method. IKs was elicited by depolarizing voltage steps given from a holding potential of –50 mV, and the effect of various test reagents on IKs was assessed by measuring the amplitude of tail current elicited upon return to the holding potential following a 2-s depolarization to +30 mV. Intracellular application of 50 μm wortmannin through a recording pipette evoked a progressive increase in IKs over a 10–15-min period to 208.5 ± 14.6% (n = 9) of initial magnitude obtained shortly after rupture of the patch membrane. Intracellular application of anti-PtdIns(4,5)P2 monoclonal antibody also increased the amplitude of IKs to 198.4 ± 19.9% (n = 5). In contrast, intracellular loading with exogenous PtdIns(4,5)P2 at 10 and 100 μm produced a marked decrease in the amplitude of IKs to 54.3 ± 3.8% (n = 5) and 44.8 ± 8.2% (n = 5), respectively. Intracellular application of neomycin (50 μm) or aluminum (50 μm) evoked an increase in the amplitude of IKs to 161.0 ± 13.5% (n = 4) and 150.0 ± 8.2% (n = 4), respectively. These results strongly suggest that IKs channel is inhibited by endogenous membrane PtdIns(4,5)P2 through the electrostatic interaction with the negatively charged head group on PtdIns(4,5)P2. Potentiation of IKs by P2Y receptor stimulation with 50 μm ATP was almost totally abolished when PtdIns(4,5)P2 was included in the pipette solution, suggesting that depletion of membrane PtdIns(4,5)P2 is involved in the potentiation of IKs by P2Y receptor stimulation. Thus, membrane PtdIns(4,5)P2 may act as an important physiological regulator of IKs in guinea pig atrial myocytes.

Response to Letter Regarding Article, “Angiotensin II Potentiates the Slow Component of Delayed Rectifier K+ Current via the AT1 Receptor in Guinea Pig Atrial Myocytes”

BACKGROUNDnAngiotensin II (Ang II) has diverse actions on cardiac electrical activity. Little information is available, however, regarding immediate electrophysiological effects of Ang II on cardiac repolarization.nnnMETHODS AND RESULTSnThe present study investigated the immediate effects of Ang II on the slow component of delayed rectifier K+ current (IKs) and action potentials in guinea pig atrial myocytes using the whole-cell patch-clamp technique. Bath application of Ang II increased the amplitude of IKs (EC50, 6.16 nmol/L) concentration dependently. The stable analogue Sar1-Ang II was also effective at increasing IKs. The voltage dependence of IKs activation and the kinetics of deactivation were not significantly affected by these agonists. The enhancement of IKs was blocked by the Ang II type 1 (AT1) receptor antagonist valsartan (1 micromol/L) and was markedly attenuated by inclusion of GDPbetaS (2 mmol/L) in the pipette, indicating an involvement of G protein-coupled AT(1) receptor. The stimulatory effect was also significantly reduced by the phospholipase C inhibitor compound 48/80 (100 micromol/L) and the protein kinase C inhibitors bisindolylmaleimide I (200 nmol/L) and H-7 (10 micromol/L), suggesting that AT1 receptor acts through phospholipase C-protein kinase C signaling cascade to potentiate I(Ks). As expected from its stimulatory action on IKs, Sar1-Ang II markedly shortened the action potential duration, which could be reversed by valsartan.nnnCONCLUSIONSnThe potentiation of IKs via AT1 stimulation in atrial myocytes, accompanied by a shortening of the action potential duration, suggests a potential mechanism by which elevated levels of Ang II may promote atrial fibrillation in heart failure and warrants further investigation.

Responses of the sustained inward current to autonomic agonists in guinea-pig sino-atrial node pacemaker cells

1 The present study was undertaken to examine the responses of the sustained inward current (Ist) to β‐adrenergic and muscarinic agonists in guinea‐pig sino‐atrial (SA) node cells using the whole‐cell patch‐clamp technique. Ist was detected as the nicardipine (1u2003μM)‐sensitive inward current at potentials between ∼−80 and +20u2003mV in the presence of low concentration (0.1u2003mM) of extracellular Ca2+, where the L‐type Ca2+ current (ICa,L) was practically abolished. 2 β‐adrenergic agonist isoprenaline (ISO) in nanomolar concentrations not only increased the amplitude of Ist but also shifted the membrane potential producing the peak amplitude (Vpeak) to a negative direction by ∼15u2003mV without appreciably affecting potential range for the current activation. The stimulatory effect of ISO was concentration‐dependent with an EC50 of 2.26u2003nM and the maximal effect (96.4±22.9% increase, n=6) was obtained at 100u2003nM ISO, when evaluated by the responses at −50u2003mV. 3 Bath application of acetylcholine (ACh) significantly inhibited Ist, which had been maximally augmented by 100u2003nM ISO; this inhibitory effect of ACh was concentration‐dependent with an IC50 of 133.9u2003nM. High concentration (1000u2003nM) of ACh depressed basal Ist by 10.5±2.0% (n=3). 4 In action potential clamp experiments, Ist was also detected under control conditions and was markedly potentiated by exposure to ISO. 5 These results strongly suggest that Ist not only contributes to the spontaneous action potentials of mammalian SA node cells but also plays a substantial role in mediating autonomic regulation of SA node pacemaker activity.

Inhibition of store-operated Ca2+ entry by extracellular ATP in rat brown adipocytes

1 Modulation of intracellular free Ca2+ concentration ([Ca2+]i) by extracellular ATP was investigated in cultured adult rat brown adipocytes using the fluorescent Ca2+ indicator fura‐2. 2 Bath application of ATP in micromolar concentrations caused a large increase in [Ca2+]i in cells previously stimulated with noradrenaline. This ATP‐induced [Ca2+]i increase exhibited a monotonic decline to near the resting levels within approximately 2 min, even in the continued presence of the agonist. 3 The magnitude and time course of the [Ca2+]i increase in response to ATP were not significantly affected by removal of extracellular Ca2+, suggesting that a mobilization of intracellular Ca2+ primarily contributes to the increase. 4 The [Ca2+]i increase in response to ATP was sensitive to inhibition by suramin, suggesting the involvement of P2 purinoceptors in the response. 5 Thapsigargin (100 nm) evoked a gradual and irreversible increase in [Ca2+]i which was entirely dependent upon extracellular Ca2+, providing functional evidence for the expression of store‐operated Ca2+ entry in these brown adipocytes. 6 Extracellular ATP at a concentration of 10 μm depressed this thapsigargin (100 nm)‐induced [Ca2+]i increase by 92 ± 3 % (n= 8 cells), strongly suggesting that ATP inhibits an influx of Ca2+ across the plasma membrane through the store‐operated pathway. Bath application of phorbol 12‐myristate 13‐acetate (PMA, 5 μm) did not affect the thapsigargin‐induced [Ca2+]i increase, indicating that the inhibitory action of ATP is not mediated by activation of protein kinase C (PKC). 7 These results indicate that extracellular ATP not only mobilizes Ca2+ from the intracellular stores but also exerts a potent inhibitory effect on the store‐operated Ca2+ entry process in adult rat brown adipocytes.