Hon Chi Lee

Nitric Oxide as an Autocrine Regulator of Sodium Currents in Baroreceptor Neurons

Arterial baroreceptors are mechanosensitive nerve endings in the aortic arch and carotid sinus that play a critical role in acute regulation of arterial blood pressure. A previous study has shown that nitric oxide (NO) or NO-related species suppress action potential discharge of baroreceptors. In the present study, we investigated the effects of NO on Na+ currents of isolated baroreceptor neurons in culture. Exogenous NO donors inhibited both tetrodotoxin (TTX) -sensitive and -insensitive Na+ currents. The inhibition was not mediated by cGMP but by NO interaction with channel thiols. Acute inhibition of NO synthase increased the Na+ currents. NO scavengers (hemoglobin and ferrous diethyldithiocarbamate) increased Na+ currents before but not after inhibition of NO synthase. Furthermore, NO production in the neuronal cultures was detected by chemiluminescence and immunoreactivity to the neuronal isoform of NO synthase was identified in fluorescently identified baroreceptor neurons. These results indicate that NO/NO-related species function as autocrine regulators of Na+ currents in baroreceptor neurons. Modulation of Na+ channels may represent a novel response to NO.

Enhancement of rabbit cardiac sodium channels by beta-adrenergic stimulation.

Voltage-dependent sodium channels from a variety of tissues are known to be phosphorylated by the cAMP-dependent protein kinase, protein kinase A. However, the functional significance of sodium channel phosphorylation is not clearly understood. Using whole-cell voltage-clamp techniques, we show that sodium currents (INas) in rabbit cardiac myocytes are enhanced by isoproterenol (ISO). This enhancement of INa by ISO 1) is holding potential dependent, 2) can be mimicked by forskolin and dibutyryl cAMP, and 3) is accompanied by an increase in the rate of Na+ channel inactivation. In single-channel, inside-out patch experiments, the catalytic subunit of protein kinase A also enhances INa and increases the rate of inactivation, suggesting that cardiac Na+ channel phosphorylation may be physiologically important. Addition of the protein kinase A inhibitor to the pipette solution in whole-cell experiments blocks the stimulatory effect of forskolin without blocking the effect of ISO, suggesting that ISO also enhances INa through a cAMP-independent pathway. To determine if ISO may stimulate INa through a direct G protein pathway, single channels were recorded in the presence of the Gs-activating GTP analogue, GTP gamma S, and the stimulatory G protein subunit, Gs alpha. Both of these agents enhanced INa without affecting the rate of Na+ channel inactivation. These results suggest that ISO enhances rabbit cardiac INa through a dual (direct and indirect) G protein regulatory pathway.

Modulation of rat cardiac sodium channel by the stimulatory G protein α subunit

1 Modulation of cardiac sodium currents (INa) by the G protein stimulatory α subunit (Gsα) was studied using patch‐clamp techniques on freshly dissociated rat ventricular myocytes. 2 Whole‐cell recordings showed that stimulation of β‐adrenergic receptors with 10 μM isoprenaline (isoproterenol, ISO) enhanced INa by 68·4 ± 9·6 % (mean ±s.e.m.; n= 7, P < 0·05vs. baseline). With the addition of 22 μg ml−1 protein kinase A inhibitor (PKI) to the pipette solution, 10 μM ISO enhanced INa by 30·5 ± 7·0 % (n= 7, P < 0·05vs. baseline). With the pipette solution containing both PKI and 20 μg ml−1 anti‐Gsα IgG or 20 μg ml−1 anti‐Gsα IgG alone, 10 μM ISO produced no change in INa. 3 The effect of Gsα on INa was not due to changes in the steady‐state activation or inactivation curves, the time course of current decay, the development of inactivation, or the recovery from inactivation. 4 Whole‐cell INa was increased by 45·2 ± 5·3 % (n= 13, P < 0·05vs. control) with pipette solution containing 1 μM Gsα27‐42 peptide (amino acids 27‐42 of rat brain Gsα) without altering the properties of Na+ channel kinetics. Furthermore, application of 1 nM Gsα27‐42 to Na+ channels in inside‐out macropatches increased the ensemble‐averaged INa by 32·5 ± 6·8 % (n= 8, P < 0·05vs. baseline). The increase in INa was reversible upon Gsα27‐42 peptide washout. Single channel experiments showed that the Gsα27‐42 peptide did not alter the Na+ single channel current amplitude, the mean open time or the mean closed time, but increased the number of functional channels (N) in the patch. 5 Application of selected short amino acid segments (Gsα27‐36, Gsα33‐42 and Gsα30‐39) of the 16 amino acid Gsα peptide (Gsα27‐42 peptide) showed that only the C‐terminal segment of this peptide (Gsα33‐42) significantly increased INa in a dose‐dependent fashion. These results show that cardiac INa is regulated by Gsα via a mechanism independent of PKA that results in an increase in the number of functional Na+ channels. In addition, a 10 residue domain (amino acids 33‐42) near the N‐terminus of Gsα is important in modulating cardiac Na+ channels.

Effects of epoxyeicosatrienoic acids on the cardiac sodium channels in isolated rat ventricular myocytes.

1 Whole‐cell Na+ currents (holding potential, −80 mV; test potential, −30 mV) in rat myocytes were inhibited by 8,9‐epoxyeicosatrienoic acid (8,9‐EET) in a dose‐dependent manner with 22 ± 4 % inhibition at 0.5 μM, 48 ± 5 % at 1 μM, and 73 ± 5 % at 5 μM (mean ± s.e.m., n= 10, P < 0.05 for each dose vs. control). Similar results were obtained with 5,6‐, 11,12‐, and 14,15‐EETs, while 8,9‐dihydroxyeicosatrienoic acid (DHET) was 3‐fold less potent and arachidonic acid was 10‐ to 20‐fold less potent. 2 8,9‐EET produced a dose‐dependent, hyperpolarized shift in the steady‐state membrane potential at half‐maximum inactivation (V½), without changing the slope factor. 8,9‐EET had no effect on the steady‐state activation of Na+ currents. 3 Inhibition of Na+ currents by 8,9‐EET was use dependent, and channel recovery was slowed. The effects of 8,9‐EET were greater at depolarized potentials. 4 Single channel recordings showed 8,9‐EET did not change the conductance or the number of active Na+ channels, but markedly decreased the probability of Na+ channel opening. These results were associated with a decrease in the channel open time and an increase in the channel closed times. 5 Incubation of cultured cardiac myocytes with 1 μM [3H]8,9‐EET showed that 25 % of the radioactivity was taken up by the cells over a 2 h period, and most of the uptake was incorporated into phospholipids, principally phosphatidylcholine. Analysis of the medium after a 2 h incubation indicated that 86 % of the radioactivity remained as [3H]8,9‐EET while 13 % was converted into [3H]8,9‐DHET. After a 30 min incubation, 1–2 % of the [3H]8,9‐EET uptake by cells remained as unesterified EET. 6 These results demonstrate that cardiac cells have a high capacity to take up and metabolize 8,9‐EET. 8,9‐EET is a potent use‐ and voltage‐dependent inhibitor of the cardiac Na+ channels through modulation of the channel gating behaviour.

Activation of ATP-sensitive K+ channels by epoxyeicosatrienoic acids in rat cardiac ventricular myocytes

1 We examined the effects of epoxyeicosatrienoic acids (EETs), which are cytochrome P450 metabolites of arachidonic acid (AA), on the activities of the ATP‐sensitive K+ (KATP) channels of rat cardiac myocytes, using the inside‐out patch‐clamp technique. 2 In the presence of 100 μm cytoplasmic ATP, the KATP channel open probability (Po) was increased by 240 ± 60% with 0.1 μm 11,12‐EET and by 400 ± 54% with 5 μm 11,12‐EET (n= 5)–10, P < 0.05 vs. control), whereas neither 5 μm AA nor 5 μm 11,12‐dihydroxyeicosatrienoic acid (DHET), which is the epoxide hydrolysis product of 11,12‐EET, had any effect on Po. 3 The half‐maximal activating concentration (EC50) was 18.9 ± 2.6 nm for 11,12‐EET (n= 5)) and 19.1 ± 4.8 nm for 8,9‐EET (n= 5), P= n.s. vs. 11,12‐EET). Furthermore, 11,12‐EET failed to alter the inhibition of KATP channels by glyburide. 4 Application of 11,12‐EET markedly decreased the channel sensitivity to cytoplasmic ATP. The half‐maximal inhibitory concentration of ATP (IC50) was increased from 21.2 ± 2.0 μm at baseline to 240 ± 60 μm with 0.1 μm 11,12‐EET (n= 5), P < 0.05 vs. control) and to 780 ± 30 μm with 5 μm 11,12‐EET (n= 11), P < 0.05vs. control). 5 Increasing the ATP concentration increased the number of kinetically distinguishable closed states, promoting prolonged closure durations. 11,12‐EET antagonized the effects of ATP on the kinetics of the KATP channels in a dose‐ and voltage‐dependent manner. 11,12‐EET (1 μm) reduced the apparent association rate constant of ATP to the channel by 135‐fold. 6 Application of 5 μm 11,12‐EET resulted in hyperpolarization of the resting membrane potential in isolated cardiac myocytes, which could be blocked by glyburide. 7 These results suggest that EETs are potent activators of the cardiac KATP channels, modulating channel behaviour by reducing the channel sensitivity to ATP. Thus, EETs could be important endogenous regulators of cardiac electrical excitability.

Dihydroxyeicosatrienoic acids are potent activators of Ca2+‐activated K+ channels in isolated rat coronary arterial myocytes

1 Dihydroxyeicosatrienoic acids (DHETs), which are metabolites of arachidonic acid (AA) and epoxyeicosatrienoic acids (EETs), have been identified as highly potent endogenous vasodilators, but the mechanisms by which DHETs induce relaxation of vascular smooth muscle are unknown. Using inside‐out patch clamp techniques, we examined the effects of DHETs on the large conductance Ca2+‐activated K+ (BK) channels in smooth muscle cells from rat small coronary arteries (150–300 μm diameter). 2 11,12‐DHET potently activated BK channels with an EC50 of 1.87 ± 0.57 nm (n= 5). Moreover, the three other regioisomers 5,6‐, 8,9‐ and 14,15‐DHET were equipotent with 11,12‐DHET in activating BK channels. The efficacy of 11,12‐DHET in opening BK channels was much greater than that of its immediate precursor 11,12‐EET. In contrast, AA did not significantly affect BK channel activity. 3 The voltage dependence of BK channels was dramatically modulated by 11,12‐DHET. With physiological concentrations of cytoplasmic Ca2+ (200 nm), the voltage at which the channel open probability was half‐maximal (V1/2) was shifted from a baseline of 115.6 ± 6.5 mV to 95.0 ± 10.1 mV with 5 nm 11,12‐DHET, and to 60.0 ± 8.4 mV with 50 nm 11,12‐DHET. 4 11,12‐DHET also enhanced the sensitivity of BK channels to Ca2+ but did not activate the channels in the absence of Ca2+. 11,12‐DHET (50 nm) reduced the Ca2+ EC50 of BK channels from a baseline of 1.02 ± 0.07 μm to 0.42 ± 0.11 μm. 5 Single channel kinetic analysis indicated that 11,12‐DHET did not alter BK channel conductance but did reduce the first latency of BK channel openings in response to a voltage step. 11,12‐DHET dose‐dependently increased the open dwell times, abbreviated the closed dwell times, and decreased the transition rates from open to closed states. 6 We conclude that DHETs hyperpolarize vascular smooth muscle cells through modulation of the BK channel gating behaviour, and by enhancing the channel sensitivities to Ca2+ and voltage. Hence, like EETs, DHETs may function as endothelium‐derived hyperpolarizing factors.

Regulation of Coronary Arterial BK Channels by Caveolae-Mediated Angiotensin II Signaling in Diabetes Mellitus

Rationale: The large conductance Ca2+-activated K+ (BK) channel, a key determinant of vascular tone, is regulated by angiotensin II (Ang II) type 1 receptor signaling. Upregulation of Ang II functions and downregulation of BK channel activities have been reported in diabetic vessels. However, the molecular mechanisms underlying Ang II–mediated BK channel modulation, especially in diabetes mellitus, have not been thoroughly examined. Objectives: The aim in this study was to determine whether caveolae-targeting facilitates BK channel dysfunction in diabetic vessels. Methods and Results: Using patch clamp techniques and molecular biological approaches, we found that BK channels, Ang II type 1 receptor, G&agr;q/11 (G protein q/11 &agr; subunit), nonphagocytic NAD(P)H oxidases (NOX-1), and c-Src kinases (c-Src) were colocalized in the caveolae of rat arterial smooth muscle cells and the integrity of caveolae in smooth muscle cells was critical for Ang II–mediated BK channel regulation. Most importantly, membrane microdomain targeting of these proteins was upregulated in the caveolae of streptozotocin-induced rat diabetic vessels, leading to enhanced Ang II–induced redox-mediated BK channel modification and causing BK channel and coronary dysfunction. The absence of caveolae abolished the effects of Ang II on vascular BK channel activity and preserved BK channel function in diabetes. Conclusions: These results identified a molecular scheme of receptor/enzyme/channel/caveolae microdomain complex that facilitates the development of vascular BK channel dysfunction in diabetes.

Molecular Mechanisms Mediating Inhibition of Human Large Conductance Ca2+-Activated K+ Channels by High Glucose

Diabetic vascular dysfunction is associated with an increase in reactive oxygen species (ROS). In this study, we hypothesized that hyperglycemia-induced ROS generation would impair the function of large conductance Ca2+-activated K+ (BK) channels, which are major determinants in vasorelaxation. We found that when cultured in high glucose (HG) (22 mmol/L), HEK293 cells showed a reduction in expressed hSlo current densities, as well as slowed activation and deactivation kinetics. When human coronary smooth muscle cells were cultured in HG, similar findings were observed for the BK currents. HG enhanced superoxide dismutase and suppressed catalase (CAT) expression in HEK293 cells, leading to a significant increase in intracellular ROS. The effects of HG were mimicked by hydrogen peroxide (H2O2), and hSlo functions were restored by CAT gene transfer. Peroxynitrite inhibited hSlo current density but did not change channel kinetics. The hSloC911A mutant was insensitive to the effects of HG and H2O2. Hence, imbalance of antioxidant enzymes plays a critical role in ROS generation in HG, impairing hSlo functions through H2O2-dependent oxidation at cysteine 911. This may represent an important fundamental mechanism that contributes to the impairment of vasodilation in diabetes.

Reactive Oxygen Species Signaling Facilitates FOXO-3a/FBXO-Dependent Vascular BK Channel β1 Subunit Degradation in Diabetic Mice

Activity of the vascular large conductance Ca2+-activated K+ (BK) channel is tightly regulated by its accessory β1 subunit (BK-β1). Downregulation of BK-β1 expression in diabetic vessels is associated with upregulation of the forkhead box O subfamily transcription factor-3a (FOXO-3a)–dependent F-box–only protein (FBXO) expression. However, the upstream signaling regulating this process is unclear. Overproduction of reactive oxygen species (ROS) is a common finding in diabetic vasculopathy. We hypothesized that ROS signaling cascade facilitates the FOXO-3a/FBXO-mediated BK-β1 degradation and leads to diabetic BK channel dysfunction. Using cellular biology, patch clamp, and videomicroscopy techniques, we found that reduced BK-β1 expression in streptozotocin (STZ)-induced diabetic mouse arteries and in human coronary smooth muscle cells (SMCs) cultured with high glucose was attributable to an increase in protein kinase C (PKC)-β and NADPH oxidase expressions and accompanied by attenuation of Akt phosphorylation and augmentation of atrogin-1 expression. Treatment with ruboxistaurin (a PKCβ inhibitor) or with GW501516 (a peroxisome proliferator–activated receptor δ activator) reduced atrogin-1 expression and restored BK channel-mediated coronary vasodilation in diabetic mice. Our results suggested that oxidative stress inhibited Akt signaling and facilitated the FOXO-3a/FBXO-dependent BK-β1 degradation in diabetic vessels. Suppression of the FOXO-3a/FBXO pathway prevented vascular BK-β1 degradation and protected coronary function in diabetes.

The Prostacyclin Analogue Carbacyclin Inhibits Ca2+‐Activated K+ Current in Aortic Baroreceptor Neurones of Rats

1 Previous studies indicate that prostacyclin (PGI2) increases the activity of baroreceptor afferent fibres. The purpose of this study was to test the hypothesis that PGI2 inhibits Ca2+‐activated K+ current (IK(Ca)) in isolated baroreceptor neurones in culture. 2 Rat aortic baroreceptor neurones in the nodose ganglia were labelled in vivo by applying a fluorescent dye (DiI) to the aortic arch 1–2 weeks before dissociation of the neurones. Outward K+ currents in baroreceptor neurones evoked by depolarizing voltage steps from a holding potential of −40 mV were recorded using the whole‐cell patch‐clamp technique. 3 Exposure of baroreceptor neurones to the stable PGI2 analogue carbacyclin significantly inhibited the steady‐state K+ current in a dose‐dependent and reversible manner. The inhibition of K+ current was not caused indirectly by changes in cytosolic Ca2+ concentration. The Ca2+‐activated K+ channel blocker charybdotoxin (ChTX, 10−7m) also inhibited the K+ current. In the presence of ChTX or in the absence of Ca2+, carbacyclin failed to inhibit the residual K+ current. Furthermore, in the presence of high concentrations of carbacyclin, ChTX did not cause further reduction of K+ current. 4 Carbacyclin‐induced inhibition of IK(Ca) was mimicked by 8‐bromo‐cAMP and by activation of G‐protein with GTPγS. The inhibitory effect of carbacyclin on IK(Ca) was abolished by GDPβS, which blocks G‐protein activation, and by a selective inhibitor of cAMP‐dependent protein kinase, PKI5–24. 5 The results demonstrate that carbacyclin inhibits ChTX‐sensitive IK(Ca) in isolated aortic baroreceptor neurones by a G‐protein‐coupled activation of cAMP‐dependent protein kinase. This mechanism may contribute to the PGI2‐induced increase in baroreceptor activity demonstrated previously.