Effect of K+ and Rb+ on the action of verapamil on a voltage-gated K+ channel,hKv1.3: implications for a second open state?

Background and purpose:

Verapamil blocks current through the voltage-gated K⁺ channel K_v1.3 in the open and inactivated state of the channel but not the closed state. The binding site for verapamil was proposed to be close to the selectivity filter and the occupancy of the selectivity filter might therefore influence verapamil affinity.

Experimental“ approach:

We investigated the influence of intra- and extracellular K⁺ and Rb⁺ on the effect of verapamil by patch-clamp studies, in COS-7 cells transfected with hK_v1.3 channels.

Key results:

Verapamil affinity was highest in high intracellular K⁺ concentrations ([K⁺]_i) and lowest in low [Rb⁺]_i, indicating an influence of intracellular cations on verapamil affinity. Experiments with a mutant channel (H399T), exhibiting a strongly reduced C-type inactivated state, demonstrated that part of this changed verapamil affinity in wild-type channels could be caused by altered C-type inactivation. External K⁺ and Rb⁺ could influence verapamil affinity by a voltage-dependent entry into the channel thereby modifying the verapamil off-rate and in addition causing a voltage-dependent verapamil off-rate.

Conclusions and implications:

Recovery from verapamil block was mainly due to the voltage-dependent closing of channels (state-dependent block), implying a second open state of the channel. This hypothesis was confirmed by the dependency of the tail current time course on duration of the prepulse. We conclude that the wild-type hK_v1.3 channel undergoes at least two different conformational changes before finally closing with a low verapamil affinity in one open state and a high verapamil affinity in the other open state.     

AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na(+) transport across human airway epithelial cells

BACKGROUND AND PURPOSE

Pulmonary transepithelial Na⁺ transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na⁺ channels and basolateral Na⁺K⁺ ATPase activity.

EXPERIMENTAL APPROACH

H441 human airway epithelial cells were used to examine the effects of hypoxia on Na⁺ transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS.

KEY RESULTS

AMPK was activated by exposure to 3% or 0.2% O₂ for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm⁻²) was added to the apical surface of cells grown at the air–liquid interface. Only 0.2% O₂ activated AMPK in cells grown at the air–liquid interface. AMPK activation was associated with elevation of cellular AMP : ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive I_sc (I_ouabain) and apical amiloride-sensitive Na⁺ conductance (G_Na+). Modification of AMPK activity prevented the effect of hypoxia on I_ouabain (Na⁺K⁺ ATPase) but not apical G_Na+. Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical G_Na+ (epithelial Na⁺ channels).

CONCLUSIONS AND IMPLICATIONS

Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na⁺ channels and basolateral Na⁺K⁺ ATPase activity to decrease transepithelial Na⁺ transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions.     

Vascular actions of calcimimetics: role of Ca²(+) -sensing receptors versus Ca²(+) influx through L-type Ca²(+) channels

BACKGROUND AND PURPOSE

The calcimimetic, (R)-N-(3-(3-(trifluoromethyl)phenyl)propyl)-1-(1-napthyl)ethylamine hydrochloride (cinacalcet), which activates Ca²⁺-sensing receptors (CaR) in parathyroid glands, is used to treat hyperparathyroidism. Interestingly, CaR in perivascular nerves or endothelial cells is also thought to modulate vascular tone. This study aims to characterize the vascular actions of calcimimetics.

EXPERIMENTAL APPROACH

In rat isolated small mesenteric arteries, the relaxant responses to the calcimimetics, cinacalcet and (R)-2-[[[1-(1-naphthyl)ethyl]amino]methyl]-1H-indole hydrochloride (calindol) were characterized, with particular emphasis on the role of CaR, endothelium, perivascular nerves, K⁺ channels and Ca²⁺ channels. Effects of L-ornithine, which activates a Ca²⁺-sensitive receptor related to CaR (GPRC6A), were also tested.

KEY RESULTS

Cinacalcet induced endothelium-independent relaxation (pEC₅₀ 5.58 ± 0.07, E_max 97 ± 6%) that was insensitive to sensory nerve desensitization by capsaicin or blockade of large-conductance Ca²⁺-activated K⁺ channels by iberiotoxin. Calindol, another calcimimetic, caused more potent relaxation (pEC₅₀ 6.10 ± 0.10, E_max 101 ± 6%), which was attenuated by endothelial removal or capsaicin, but not iberiotoxin. The negative modulator of CaR, calhex 231 or changes in [Ca²⁺]_o had negligible effect on relaxation to both calcimimetics. The calcimimetics relaxed vessels precontracted with high [K⁺]_o and inhibited Ca²⁺ influx in endothelium-denuded vessels stimulated by methoxamine, but not ionomycin. They also inhibited contractions to the L-type Ca²⁺ channel activator, BayK8644. L-ornithine induced small relaxation alone and had no effect on the responses to calcimimetics.

CONCLUSION AND IMPLICATIONS

Cinacalcet and calindol are potent arterial relaxants. Under the experimental conditions used, they predominantly act by inhibiting Ca²⁺ influx through L-type Ca²⁺ channels into vascular smooth muscle, whereas Ca²⁺-sensitive receptors (CaR or GPRC6A) play a minor role.     

Effect of sugar positions in ginsenosides and their inhibitory potency on Na+/K+-ATPase activity

Aim:

To determine whether ginsenosides with various sugar attachments may act as active components responsible for the cardiac therapeutic effects of ginseng and sanqi (the roots of Panax ginseng and Panax notoginseng) via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin.

Methods:

The structural similarity between ginsenosides and ouabain was analyzed. The inhibitory potency of ginsenosides and ouabain on Na⁺/K⁺-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of ginsenosides to Na⁺/K⁺-ATPase.

Results:

Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, and possessed inhibitory potency on Na⁺/K⁺-ATPase activity. However, their inhibitory potency was significantly reduced or completely abolished when a monosaccharide was linked to the C-6 or C-20 position of the steroid-like structure; replacement of the monosaccharide with a disaccharide molecule at either of these positions caused the disappearance of the inhibitory potency. Molecular modeling and docking confirmed that the difference in Na⁺/K⁺-ATPase inhibitory potency among ginsenosides was due to the steric hindrance of sugar attachment at the C-6 and C-20 positions of the steroid-like structure.

Conclusion:

The cardiac therapeutic effects of ginseng and sanqi should be at least partly attributed to the effective inhibition of Na⁺/K⁺-ATPase by their metabolized ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure.     

Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na^＋/K^＋- ATPase

Aim:

To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain.

Methods:

The inhibitory potency of ouabain and the identified steroid-like compounds on Na⁺/K⁺-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na⁺/K⁺-ATPase.

Results:

All the examined steroid-like compounds displayed more or less inhibition on Na⁺/K⁺-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na⁺/K⁺-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K⁺ binding sites of Na⁺/K⁺-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na⁺/K⁺-ATPase.

Conclusion:

Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na⁺/K⁺-ATPase.     

TREK-1 channels do not mediate nitrergic neurotransmission in circular smooth muscle from the lower oesophageal sphincter

Background and purpose:

The ionic mechanisms underlying nitrergic inhibitory junction potentials (IJPs) in gut smooth muscle remain a matter of debate. Recently, it has been reported that opening of TWIK-related K⁺ channel 1 (TREK-1) K⁺ channels contributes to the nitrergic IJP in colonic smooth muscle. We investigated the effects of TREK-1 channel blockers on nitrergic neurotransmission in mouse and opossum lower oesophageal sphincter (LOS) circular smooth muscle (CSM).

Experimental approach:

The effects of TREK-1 channel blockers were characterized pharmacologically in murine and opossum gut smooth muscle using conventional intracellular and tension recordings.

Key results:

In LOS, L-methionine depolarized the resting membrane potential (RMP) but did not inhibit the nitrergic IJP. Cumulative application of theophylline hyperpolarized the RMP and inhibited the nitrergic IJP concentration dependently. The induced membrane hyperpolarization was prevented by pre-application of caffeine, but not by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. 8-Br-cAMP significantly hyperpolarized membrane potential and increased the amplitude of the nitrergic IJP. In opossum LOS muscle strips, L-methionine increased resting tone but had no effect on nerve-mediated LOS relaxation. On the other hand, theophylline markedly inhibited tone. In CSM from mouse proximal colon, L-methionine caused modest inhibition of nitrergic IJPs.

Conclusions and implications:

TREK-1 channels were not involved in the nitrergic IJP in LOS CSM. Not only does L-methionine have no effect on the nitrergic IJP or LOS relaxation, but the effect of theophylline appears to be due to interruption of Ca²⁺-releasing pathways (i.e. caffeine-like effect) rather than via blockade of TREK-1 channels.     

Loss of Ca2+-mediated ion transport during colitis correlates with reduced ion transport responses to a Ca2+-activated K+ channel opener

Background and purpose:

Epithelial surface hydration is critical for proper gut function. However, colonic tissues from individuals with inflammatory bowel disease or animals with colitis are hyporesponsive to Cl⁻ secretagogues. The Cl⁻ secretory responses to the muscarinic receptor agonist bethanechol are virtually absent in colons of mice with dextran sodium sulphate (DSS)-induced colitis. Our aim was to define the mechanism underlying this cholinergic hyporesponsiveness.

Experimental approach:

Colitis was induced by 4% DSS water, given orally. Epithelial ion transport was measured in Ussing chambers. Colonic crypts were isolated and processed for mRNA expression via RT-PCR and protein expression via immunoblotting and immunolocalization.

Key results:

Expression of muscarinic M₃ receptors in colonic epithelium was not decreased during colitis. Short-circuit current (I_SC) responses to other Ca²⁺-dependent secretagogues (histamine, thapsigargin, cyclopiazonic acid and calcium ionophore) were either absent or severely attenuated in colonic tissue from DSS-treated mice. mRNA levels of several ion transport molecules (a Ca²⁺-regulated Cl⁻ channel, the intermediate-conductance Ca²⁺-activated K⁺ channel, the cystic fibrosis transmembrane conductance regulator, the Na⁺/K⁺-ATPase pump or the Na⁺/K⁺/2Cl⁻ co-transporter) were not reduced in colonic crypts from DSS-treated mice. However, protein expression of Na⁺/K⁺-ATPase α1 subunits was decreased twofold during colitis. Activation of Ca²⁺-activated K⁺ channels increased I_SC significantly less in DSS colons compared with control, as did the protein kinase C activator, phorbol 12-myristate 13-acetate.

Conclusions and implications:

Decreased Na⁺/K⁺-ATPase expression probably contributes to overall epithelial hyporesponsiveness during colitis, while dysfunctional K⁺ channels may account, at least partially, for lack of epithelial secretory responses to Ca²⁺-mediated secretagogues.     

Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes

BACKGROUND AND PURPOSE

The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes.

KEY RESULTS

In the presence of 1–10 μM AEA, suppression of both Na⁺ and L-type Ca²⁺ channels was observed. Inhibition of Na⁺ channels was voltage and Pertussis toxin (PTX) – independent. Radioligand-binding studies indicated that specific binding of [³H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in B_max values but no change in K_d. Further studies on L-type Ca²⁺ channels indicated that AEA potently inhibited these channels (IC₅₀ 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba²⁺ currents. MetAEA also inhibited Na⁺ and L-type Ca²⁺ currents. Radioligand studies indicated that specific binding of [³H]isradipine, was inhibited significantly by metAEA. (10 μM), changing B_max but not K_d.

CONCLUSION AND IMPLICATIONS

Results indicate that AEA inhibited the function of voltage-dependent Na⁺ and L-type Ca²⁺ channels in rat ventricular myocytes, independent of CB₁ and CB₂ receptor activation.     

The inhibitor of volume-regulated anion channels DCPIB activates TREK potassium channels in cultured astrocytes

Background and Purpose

The ethacrynic acid derivative, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) is considered to be a specific and potent inhibitor of volume-regulated anion channels (VRACs). In the CNS, DCPIB was shown to be neuroprotective through mechanisms principally associated to its action on VRACs. We hypothesized that DCPIB could also regulate the activity of other astroglial channels involved in cell volume homeostasis.

Experimental Approach

Experiments were performed in rat cortical astrocytes in primary culture and in hippocampal astrocytes in situ. The effect of DCPIB was evaluated by patch-clamp electrophysiology and immunocytochemical techniques. Results were verified by comparative analysis with recombinant channels expressed in COS-7 cells.

Key Results

In cultured astrocytes, DCPIB promoted the activation of a K⁺ conductance mediated by two-pore-domain K⁺ (K_2P) channels. The DCPIB effect occluded that of arachidonic acid, which activates K_2P channels K_2P 2.1 (TREK-1) and K_2P 10.1 (TREK-2) in cultured astrocytes. Immunocytochemical analysis suggests that cultured astrocytes express K_2P 2.1 and K_2P 10.1 proteins. Moreover, DCPIB opened recombinant K_2P 2.1 and K_2P 10.1 expressed in heterologous system. In brain slices, DCPIB did not augment the large background K⁺ conductance in hippocampal astrocytes, but caused an increment in basal K⁺ current of neurons.

Conclusion and Implications

Our results indicate that the neuroprotective effect of DCPIB could be due, at least in part, to activation of TREK channels. DCPIB could be used as template to build new pharmacological tools able to increase background K⁺ conductance in astroglia and neuronal cells.     

Magnesium lithospermate B dilates mesenteric arteries by activating BKca currents and contracts arteries by inhibiting Kv currents

Aim:

To examine the involvement of K⁺ channels and endothelium in the vascular effects of magnesium lithospermate B (MLB), a hydrophilic active component of Salviae miltiorrhiza Radix.

Methods:

Isolated rat mesenteric artery rings were employed to investigate the effects of MLB on KCl- or norepinephrine-induced contractions. Conventional whole-cell patch-clamp technique was used to study the effects of MLB on K⁺ currents in single isolated mesenteric artery myocytes.

Results:

MLB produced a concentration-dependent relaxation in mesenteric artery rings precontracted by norepinephrine (1 μmol/L) with an EC₅₀ of 111.3 μmol/L. MLB-induced relaxation was reduced in denuded artery rings with an EC₅₀ of 224.4 μmol/L. MLB caused contractions in KCl-precontracted artery rings in the presence of N-nitro-L-arginine methyl ester (L-NAME) with a maximal value of 130.3%. The vasodilatory effect of MLB was inhibited by tetraethylammonium (TEA) in both intact and denuded artery rings. In single smooth muscle cells, MLB activated BK_Ca currents (EC₅₀ 156.3 μmol/L) but inhibited K_V currents (IC₅₀ 26.1 μmol/L) in a voltage- and concentration-dependent manner.

Conclusion:

MLB dilated arteries by activating BK_Ca channels in smooth muscle cells and increasing NO release from endothelium, but it also contracted arteries precontracted with KCl in the presence of L-NAME.     

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2＋ level in SHSY5Y cells

Aim:

To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na⁺/K⁺-ATPase, leads to the elevation of intracellular Ca²⁺ level as observed in cells treated with cardiac glycosides.

Methods:

Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca²⁺ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na⁺/Ca²⁺ exchanger inhibitor) and 2-APB (IP₃ receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na⁺/K⁺-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.

Results:

severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca²⁺ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na⁺/K⁺-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells.

Conclusion:

Comparable to ouabain, MLB leads to the elevation of intracellular Ca²⁺ level presumably via the same mechanism by inhibiting Na⁺/K⁺-ATPase. The elevated Ca²⁺ levels seem to be supplied by Ca²⁺ influx through the reversed mode of the Na⁺/Ca²⁺ exchanger and intracellular release from endoplasmic reticulum.     

Expression and function of the K+ channel KCNQ genes in human arteries

BACKGROUND AND PURPOSE

KCNQ-encoded voltage-gated potassium channels (K_v7) have recently been identified as important anti-constrictor elements in rodent blood vessels but the role of these channels and the effects of their modulation in human arteries remain unknown. Here, we have assessed KCNQ gene expression and function in human arteries ex vivo.

EXPERIMENTAL APPROACH

Fifty arteries (41 from visceral adipose tissue, 9 mesenteric arteries) were obtained from subjects undergoing elective surgery. Quantitative RT-PCR experiments using primers specific for all known KCNQ genes and immunohistochemsitry were used to show K_v7 channel expression. Wire myography and single cell electrophysiology assessed the function of these channels.

KEY RESULTS

KCNQ4 was expressed in all arteries assessed, with variable contributions from KCNQ1, 3 and 5. KCNQ2 was not detected. K_v7 channel isoform-dependent staining was revealed in the smooth muscle layer. In functional studies, the K_v7 channel blockers, XE991 and linopirdine increased isometric tension and inhibited K⁺ currents. In contrast, the K_v7.1-specific blocker chromanol 293B did not affect vascular tone. Two K_v7 channel activators, retigabine and acrylamide S-1, relaxed preconstricted arteries, actions reversed by XE991. K_v7 channel activators also suppressed spontaneous contractile activity in seven arteries, reversible by XE991.

CONCLUSIONS AND IMPLICATIONS

This is the first study to demonstrate not only the presence of KCNQ gene products in human arteries but also their contribution to vascular tone ex vivo.

LINKED ARTICLE

This article is commented on by Mani and Byron, pp. 38–41 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01065.x     

Mechanisms of actions of hydrogen sulphide on rat distal colonic epithelium

BACKGROUND AND PURPOSE

The aim of this study was to clarify the mechanisms by which hydrogen sulphide (H₂S) affects ion secretion across rat distal colonic epithelium.

EXPERIMENTAL APPROACH

Changes in short-circuit current induced by the H₂S-donor, sodium hydrosulphide (NaHS; 10 mmol·L⁻¹), were measured in Ussing chambers after permeabilization of the apical membrane with nystatin. Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺ in intracellular stores were measured with fluorescent dyes. Changes in mitochondrial membrane potential were estimated with rhodamine 123.

KEY RESULTS

NaHS had a biphasic effect on overall currents across the basolateral membrane: an initial inhibition followed by a secondary stimulation. Both a scilliroside-sensitive action on the Na⁺-K⁺-ATPase and modulation of glibenclamide-sensitive and tetrapentylammonium-sensitive (i.e. ATP-sensitive and Ca²⁺-dependent) basolateral K⁺ channels were involved in this action. Experiments with rhodamine 123 revealed that NaHS induced a hyperpolarization of the mitochondrial membrane. NaHS evoked a biphasic change in [Ca²⁺]_i, an initial decrease followed by a secondary increase, known to be mediated by the release of stored Ca²⁺. Initial falls in [Ca²⁺]_i were not mediated by a sequestration of Ca²⁺ in intracellular Ca²⁺ storing organelles, as the Mag-Fura-2 signal was unaffected by NaHS. Falls in [Ca²⁺]_i were inhibited by 2′,4′-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺-exchanger, and attenuated in Na⁺-free buffer, suggesting a transient stimulation of Ca²⁺ outflow by this transporter, directly demonstrated by Mn²⁺ quenching experiments.

CONCLUSIONS AND IMPLICATIONS

ATP-sensitive and Ca²⁺-dependent basolateral K⁺ conductances, the basolateral Na⁺-K⁺-pump as well as Ca²⁺ transporters were involved in the action of H₂S in regulating colonic ion secretion.     

L-tryptophan ethyl ester dilates small mesenteric arteries by inhibition of voltage-operated calcium channels in smooth muscle

BACKGROUND AND PURPOSE

L-tryptophan (L-W) is a precursor of the vasoconstrictor, 5-HT. However, acute administration of L-W ethyl ester (L-Wee) lowered blood pressure. The mechanism of action is unknown. This study compares the vascular effects of L-W and L-Wee in intact animals, isolated aortic rings, small mesenteric arteries (MA) and explores possible mechanisms by studies in vascular smooth muscle cells (VSMC) of MA.

EXPERIMENTAL APPROACH

Effects of L-W or L-Wee (5–50 mg kg⁻¹, i.v.) on mean arterial pressure (MAP) and heart rate (HR) were determined in male Sprague-Dawley rats. The effects of L-W and L-Wee on basal tone and of phenylephrine- or KCl-induced contractions of aortic and MA rings were assessed. Effects of L-Wee and L-W on voltage-operated calcium channels (VOCC) of VSMC of MA were also examined in patch-clamp studies.

KEY RESULTS

Administration of L-Wee, but not L-W, evoked a rapid and transient dose-dependent decrease in MAP and HR. While both agents failed to affect basal tone, L-Wee decreased, concentration-dependently, (I_max > 98%) tension responses to phenylephrine and KCl in an endothelium-independent manner in aorta (IC₅₀ 2 mM) and MA (IC₅₀ 17 µM). L-Wee evoked concentration-dependent inhibition of VOCC currents (IC₅₀ 12 µM; I_max 90%) in VSMC of MA.

CONCLUSIONS AND IMPLICATIONS

Esterified L-W (L-Wee), but not L-W, preferentially relaxed resistance vessels rather than conduit vessels. These effects were associated with blockade of VOCC by L-Wee. Our findings suggest that the falls in MAP and HR induced by L-Wee were due to blockade of VOCC by L-Wee.     

Pals-associated tight junction protein functionally links dopamine and angiotensin II to the regulation of sodium transport in renal epithelial cells

Background and purpose:

Dopamine inhibits renal cell Na⁺,K⁺-ATPase activity and cell sodium transport by promoting the internalization of active molecules from the plasma membrane, whereas angiotensin II (ATII) stimulates its activity by recruiting new molecules to the plasma membrane. They achieve such effects by activating multiple and distinct signalling molecules in a hierarchical manner. The purpose of this study was to investigate whether dopamine and ATII utilize scaffold organizer proteins as components of their signalling networks, in order to avoid deleterious cross talk.

Experimental approach:

Attention was focused on a multiple PDZ domain protein, Pals-associated tight junction protein (PATJ). Ectopic expression of PATJ in renal epithelial cells in culture was used to study its interaction with components of the dopamine signalling cascade. Similarly, expression of PATJ deletion mutants was employed to analyse its functional relevance during dopamine-, ATII- and insulin-dependent regulation of Na⁺,K⁺-ATPase.

Key results:

Dopamine receptors and components of its signalling cascade mediating inhibition of Na⁺,K⁺-ATPase interact with PATJ. Inhibition of Na⁺,K⁺-ATPase by dopamine was prevented by expression of mutants of PATJ lacking PDZ domains 2, 4 or 5; whereas the stimulatory effect of ATII and insulin on Na⁺,K⁺-ATPase was blocked by expression of PATJ lacking PDZ domains 1, 4 or 5.

Conclusions and implications:

A multiple PDZ domain protein may add functionality to G protein-coupled and tyrosine kinase receptors signalling during regulation of Na⁺,K⁺-ATPase. Signalling molecules and effectors can be integrated into a functional network by the scaffold organizer protein PATJ via its multiple PDZ domains.     

Insulin induced translocation of Na^＋/K^＋-ATPase is decreased in the heart of streptozotocin diabetic rats

Aim:

To investigate the effect of acute insulin administration on the subcellular localization of Na⁺/K⁺-ATPase isoforms in cardiac muscle of healthy and streptozotocin-induced diabetic rats.

Methods:

Membrane fractions were isolated with subcellular fractionation and with cell surface biotinylation technique. Na⁺/K⁺-ATPase subunit isoforms were analysed with ouabain binding assay and Western blotting. Enzyme activity was measured using 3-O-methylfluorescein-phosphatase activity.

Results:

In control rat heart muscle α1 isoform of Na⁺/K⁺ ATPase resides mainly in the plasma membrane fraction, while α2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of α1 isoform (25 %, P<0.05) in plasma membrane and α2 isoform (50%, P<0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of α2- but not α1-subunits was detected. α1-Subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of α2 increased by 3.3-fold, α1 by 1.37-fold and β1 by 1.51-fold (P<0.02) in the plasma membrane of control, and less than 1.92-fold (P<0.02), 1.19-fold (not significant) and 1.34-fold (P<0.02) in diabetes. The insulin-induced translocation was wortmannin sensitive.

Conclusion:

This study demonstrate that insulin influences the plasma membrane localization of Na⁺/K⁺-ATPase isoforms in the heart. α2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. α1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates Na⁺/K⁺-ATPase α1- and α2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.     

N-arachidonoyl glycine suppresses Na+/Ca2+ exchanger-mediated Ca2+ entry into endothelial cells and activates BKCa channels independently of GPCRs

Background and Purpose

N-arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly''s action on unstimulated and agonist-stimulated endothelial cells.

Experimental Approach

The effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity.

Key Results

In EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na⁺/Ca²⁺ exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs⁺-based Na⁺-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na⁺ withdrawal and inhibited by bepridil. NAGly (0.3–30 μM) suppressed NCX currents in a URB597- and guanosine 5′-O-(2-thiodiphosphate) (GDPβS)-insensitive manner, [Ca²⁺]_i elevation evoked by Na⁺ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in a GDPβS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BK_Ca channel activity.

Conclusion and Implications

Our data showed that NCX is a Ca²⁺ entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca²⁺ entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BK_Ca channels.     

Action potential bursts in central snail neurons elicited by paeonol: roles of ionic currents

Aim:

To investigate the effects of 2′-hydroxy-4′-methoxyacetophenone (paeonol) on the electrophysiological behavior of a central neuron (right parietal 4; RP4) of the giant African snail (Achatina fulica Ferussac).

Methods:

Intracellular recordings and the two-electrode voltage clamp method were used to study the effects of paeonol on the RP4 neuron.

Results:

The RP4 neuron generated spontaneous action potentials. Bath application of paeonol at a concentration of ≥500 μmol/L reversibly elicited action potential bursts in a concentration-dependent manner. Immersing the neurons in Co²⁺-substituted Ca²⁺-free solution did not block paeonol-elicited bursting. Pretreatment with the protein kinase A (PKA) inhibitor KT-5720 or the protein kinase C (PKC) inhibitor Ro 31-8220 did not affect the action potential bursts. Voltage-clamp studies revealed that paeonol at a concentration of 500 μmol/L had no remarkable effects on the total inward currents, whereas paeonol decreased the delayed rectifying K⁺ current (I_KD) and the fast-inactivating K⁺ current (I_A). Application of 4-aminopyridine (4-AP 5 mmol/L), an inhibitor of I_A, or charybdotoxin 250 nmol/L, an inhibitor of the Ca²⁺-activated K⁺ current (I_K(Ca)), failed to elicit action potential bursts, whereas tetraethylammonium chloride (TEA 50 mmol/L), an I_KD blocker, successfully elicited action potential bursts. At a lower concentration of 5 mmol/L, TEA facilitated the induction of action potential bursts elicited by paeonol.

Conclusion:

Paeonol elicited a bursting firing pattern of action potentials in the RP4 neuron and this activity relates closely to the inhibitory effects of paeonol on the I_KD.     

Ca2+ sparks promote myogenic tone in retinal arterioles

Background and Purpose

Ca²⁺ imaging reveals subcellular Ca²⁺ sparks and global Ca²⁺ waves/oscillations in vascular smooth muscle. It is well established that Ca²⁺ sparks can relax arteries, but we have previously reported that sparks can summate to generate Ca²⁺ waves/oscillations in unpressurized retinal arterioles, leading to constriction. We have extended these studies to test the functional significance of Ca²⁺ sparks in the generation of myogenic tone in pressurized arterioles.

Experimental Approach

Isolated retinal arterioles (25–40 μm external diameter) were pressurized to 70 mmHg, leading to active constriction. Ca²⁺ signals were imaged from arteriolar smooth muscle in the same vessels using Fluo4 and confocal laser microscopy.

Key Results

Tone development was associated with an increased frequency of Ca²⁺ sparks and oscillations. Vasomotion was observed in 40% of arterioles and was associated with synchronization of Ca²⁺ oscillations, quantifiable as an increased cross-correlation coefficient. Inhibition of Ca²⁺ sparks with ryanodine, tetracaine, cyclopiazonic acid or nimodipine, or following removal of extracellular Ca²⁺, resulted in arteriolar relaxation. Cyclopiazonic acid-induced dilatation was associated with decreased Ca²⁺ sparks and oscillations but with a sustained rise in the mean global cytoplasmic [Ca²⁺] ([Ca²⁺]_c), as measured using Fura2 and microfluorimetry.

Conclusions and Implications

This study provides direct evidence that Ca²⁺ sparks can play an excitatory role in pressurized arterioles, promoting myogenic tone. This contrasts with the generally accepted model in which sparks promote relaxation of vascular smooth muscle. Changes in vessel tone in the presence of cyclopiazonic acid correlated more closely with changes in spark and oscillation frequency than global [Ca²⁺]_c, underlining the importance of frequency-modulated signalling in vascular smooth muscle.