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As the most prototypical G protein-coupled receptor, [beta]-adrenergic receptor ([beta]AR) regulates the pace and strength of heart beating by enhancing and synchronizing L-type channel (LCC) [Ca.sup.2+] influx, which in turn elicits greater sarcoplasmic reticulum (SR) [Ca.sup.2+] release flux via ryanodine receptors (RyRs). However, whether and how [beta]AR-protein kinase A (PKA) signaling directly modulates RyR function remains elusive and highly controversial. By using unique single-channel [Ca.sup.2+] imaging technology, we measured the response of a single RyR [Ca.sup.2+] release unit, in the form of a [Ca.sup.2+] spark, to its native trigger, the [Ca.sup.2+] sparklet from a single LCC. We found that acute application of the selective [beta]AR agonist isoproterenol (1 [micro]M, [less than or equal to] 20 min) increased triggered spark amplitude in an LCC unitary current-independent manner. The increased ratio of [Ca.sup.2+] release flux underlying a [Ca.sup.2+] spark to SR [Ca.sup.2+] content indicated that EAR stimulation helps to recruit additional RyRs in synchrony. Quantification of sparklet-spark kinetics showed that [beta]AR stimulation synchronized the stochastic latency and increased the fidelity (i.e., chance of hit) of LCC-RyR intermolecular signaling. The RyR modulation was independent of the increased SR [Ca.sup.2+] content. The PKA antagonists Rp-8-CPT-cAMP (100 [micro]M) and H89 (10 [micro]M) both eliminated these effects, indicating that [beta]AR acutely modulates RyR activation via the PKA pathway. These results demonstrate unequivocally that RyR activation by a single LCC is accelerated and synchronized during [beta]AR stimulation. This molecular mechanism of sympathetic regulation will permit more fundamental studies of altered [beta]AR effects in cardiovascular diseases. [beta]-adrenergic receptor | calcium signaling | excitation-contraction coupling | isoproterenol | protein kinase A doi/ 10.1073/pnas.0906560106

Objective: To express the human HCN2 and HCN4 genes in HEK293 cells and investigate the electrophysiology of the expressed channel protein., Methods: cDNA encoding human HCN2 or HCN4 gene was ligated into a shuttle vector pAdTrack-CMV. Homologous recombination was performed in Escherichia coli of the line BJ5183. Human embryonic kidney cells of the line 293 (HEK293 cells) were cultured and transfected with the positive recombinant adenovirus plasmid. Then the HEK293 cells were infected by AdhHCN2 or AdhHCN4 and the whole cell hyperpolarization-activated currents were recorded in HEK293 cells transfected with hHCN2 and hHCN4., Results: If-like currents could be found in the HEK293 cells transfected with hHCN2 and hHCN4. The channels were activated by hyperpolarized potentials. Boltzmann equation showed that the half-activation voltage of the hHCN2 and hHCN4 channels were -114.8 mV +/- 3.3 mV and -125.9 mV +/- 2.9 mV respectively (P = 0.024). The reversal slope factors of the hHCN2 and hHCN4 channels were 11.1 mV +/- 1.2 mV and 13.7 mV +/- 1.3 mV respectively (P = 0.22). The activation kinetics was faster in hHCN2 than in hHCN4, with the activation constants at -110 mV being 0.99 s +/- 0.21 s and 8.47 s +/- 2.85 s respectively. The relative permeation ratio for sodium and potassium were 0.40 and 0.34 respectively in these two channels. Caesium chloride of the concentration of 2 mmol/L prominently inhibited both currents., Conclusion: The target genes hHCN2 and hHCN4 are successfully expressed in HEK293 cells, and the expressed functional channels have profoundly different activation kinetics.

Aim: To assess the blockade by CPU 86017 on the L-type calcium channels in the myocardium and on the Ca(2+)-related contractions of vascular smooth muscle., Methods: The whole-cell patch-clamp was applied to investigate the blocking effect of CPU 86017 on the L-type calcium current in isolated guinea pig myocytes and contractions by KCl or phenylephrine (Phe) of the isolated rat tail arteries were measured., Results: Suppression of the L-type current of the isolated myocytes by CPU 86017 was moderate, in time- and concentration-dependent manner and with no influence on the activation and inactivation curves. The IC(50) was 11.5 micromol/L. Suppressive effect of CPU 86017 on vaso-contractions induced by KCl 100 mmol/L, phenylephrine 1 micromol/L in KH solution (phase 1), Ca(2+) free KH solution ( phase 2), and by addition of CaCl(2) into Ca(2+)-free KH solution (phase 3) were observed. The IC(50) to suppress vaso-contractions by calcium entry via the receptor operated channel (ROC) and voltage-dependent channel (VDC) was 0.324 micromol/L and 16.3 micromol/L, respectively. The relative potency of CPU 86017 to suppress vascular tone by Ca(2+) entry through ROC and VDC is 1/187 of prazosin and 1/37 of verapamil, respectively., Conclusion: The blocking effects of CPU 86017 on the L-type calcium channel of myocardium and vessel are moderate and non-selective. CPU 86017 is approximately 50 times more potent in inhibiting ROC than VDC.

Aim: To study the effect of puerarin (Pue) on Na+ channel in rat ventricular myocytes., Methods: Whole-cell patch-clamp technique was applied on isolated cardiomyocytes from rats., Results: Pue inhibited cardiac INa in a positive rate-dependent and dose-dependent manner, with an IC(50) of 349 micromol/L. The kinetics of blockage of cardiac sodium channel by Pue resembled the ClassIa/Ic of antiarrhythmic agents. Pue 300 micromol/L did not alter the shape of the I-V curve of INa, but markedly shifted the steady-state inactivation curve of INa towards more negative potential by 15.9 mV, and postponed the recovery of INa inactivation state from (21.9+/-1.6) ms to (54.4+/-3.4) ms (P<0.01). It demonstrated that the steady state of inactivation was affected by Pue significantly., Conclusion: Pue protected ventricular myocytes against cardiac damage and arrhythmias by inhibiting recovery from inactivation of cardiac Na+ channels.

Aim: To study the blockade of paeoniflorin (Pae) on I(Na) in the acutely isolated hippocampus neurons of mice., Methods: The whole-cell patch clamp technique was used., Results: Pae inhibited I(Na) in frequency-dependent and concentration-dependent manners, with an IC50 of 271 micromol/L. Pae 0.3 mmol/L shifted the activation potential of the maximal I(Na) from -40 mV to -30 mV, shifted the steady-state activation and inactivation curves toward more positive and negative potentials by 10.8 mV, and 18.2 mV, respectively, and postponed the recovery of I(Na) inactivation state from (4.2+/-0.7) ms to (9.8+/-1.2) ms., Conclusion: Pae inhibited I(Na) in mouse hippocampus neurons.