Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. ...

Konkuk University School of Medicine

We hypothesized that voltage-gated K+ (Kv) currents regulate the resting membrane potential (Em), and that serotonin (5-HT) causes Em depolarization by reducing Kv currents in rat mesenteric artery smooth muscle cells (MASMCs). The resting Em was about -40 mV in the nystatin-perforated patch configuration, and the inhibition of Kv currents by 4-aminopyridine caused marked Em depolarization. The inhibition of Ca2+-activated K+ (KCa) currents had no effect on Em. 5-HT (1 microM) depolarized Em by approximately 11 mV and reduced the Kv currents to approximately 63% of the control at -20 mV. Similar 5-HT effects were observed with the conventional whole-cell configuration with a weak Ca2+ buffer in the pipette solution, but not with a strong Ca2+ buffer. In the presence of tetraethylammonium (1mM), 5-HT caused Em depolarization similar to the control condition. These results indicate that the resting Em is largely under the regulation of Kv currents in rat MASMCs, and that 5-HT depolarizes Em by reducing Kv currents in a [Ca2+]i-dependent manner.

Serotonin depolarizes the membrane potential in rat mesenteric artery myocytes by decreasing voltage-gated K+ currents.

Although serotonin (5-hydroxytryptamine, 5-HT) has been found to be a potent vasoconstrictor, a pivotal role of 5-HT in the control of appetite and mood control by the modulation of neuronal synapse has also been proposed. Selective 5-HT reuptake inhibitors (SSRIs) are frequently used to suppress appetite and treat depressive disorder, and the target protein of SSRIs is the 5-HT transporter (5-HTT) in the neuronal synapse. However, SSRIs may increase the free 5-HT concentration in circulating blood because platelets and vascular smooth muscles express functional 5-HTT. In addition, enhanced vasoactive action of 5-HT and alterations in 5-HT receptor subtypes have been reported in some types of hypertension. Therefore, we can infer that the use of drugs such as SSRIs in some hypertensive patients is potentially risky. Altered functional expression of ion channels in vascular smooth muscle is suggested to be a mechanism for the enhanced vasoconstriction by vasoactive agonists, including 5-HT. In this brief review, we compared the electrophysiological properties of mesenteric artery myocytes and their modulation by 5-HT between sham-operated control and deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

Serotonin-induced ion channel modulations in mesenteric artery myocytes from normotensive and DOCA-salt hypertensive rats.

MK801 (dizocilpine), a phencyclidine (PCP) derivative, is a potent noncompetitive antagonist of the N-Methyl-D-aspartate receptor (NMDAr). Another PCP derivative, ketamine, was reported to block voltage-gated K(+) (Kv) channels, which was independent of NMDAr function. Kv currents are major regulators of the membrane potential (Em) and excitability of muscles and neurons. Here, we investigated the effect of MK801 on the Kv channels and Em in rat mesenteric arterial smooth muscle cells (RMASMCs). We used the whole-cell patch clamp technique to analyze the effect of MK801 enantiomers on Kv channels and Em. (+)MK801 inhibited Kv channels in a concentration-dependent manner (IC50 of 89.1 ± 13.1 μM, Hill coefficient of 1.05 ± 0.08). The inhibition was voltage- and state- independent. (+)MK801 didn't influence steady-state activation and inactivation of Kv channels. (+)MK801 treatment depolarized Em in a concentration-dependent manner and concomitantly decreased membrane conductance. (-)MK801 also similarly inhibited the Kv channels (IC50 of 134.0 ± 17.5 μM, Hill coefficient of 0.87 ± 0.09). These results indicate that MK801 directly inhibits the Kv channel in a state-independent manner in RMASMCs. This MK801-mediated inhibition of Kv channels should be considered when assessing the various pharmacological effects produced by MK801, such as schizophrenia, neuroprotection, and hypertension.

Blockade of voltage-gated K⁺ currents in rat mesenteric arterial smooth muscle cells by MK801.

We demonstrated the effect of nortriptyline, a tricyclic antidepressant drug and serotonin reuptake inhibitor, on voltage-dependent K (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Nortriptyline inhibited Kv currents in a concentration-dependent manner, with an apparent IC value of 2.86±0.52 µM and a Hill coefficient of 0.77±0.1. Although application of nortriptyline did not change the activation curve, nortriptyline shifted the inactivation current toward a more negative potential. Application of train pulses (1 or 2 Hz) did not change the nortriptyline-induced Kv channel inhibition, suggesting that the effects of nortiprtyline were not use-dependent. Preincubation with the Kv1.5 and Kv2.1/2.2 inhibitors, DPO-1 and guangxitoxin did not affect nortriptyline inhibition of Kv channels. From these results, we concluded that nortriptyline inhibited Kv channels in a concentration-dependent and state-independent manner independently of serotonin reuptake.

Nortriptyline, a tricyclic antidepressant, inhibits voltage-dependent K channels in coronary arterial smooth muscle cells.

Activation of L-type voltage-dependent Ca channels (VDCC) by membrane stretch contributes to many biological responses such as myogenic contraction of arteries. However, mechanism for the stretch-induced VDCC activation is unclear. In this study, we examined the hypothesis that caveolar remodeling and its related signaling cascade contribute to the stretch-induced activation of VDCC in rat mesenteric arterial smooth muscle cells. The VDCC currents were recorded with nystatin-perforated or with conventional whole-cell patch-clamp technique. Hypotonic (~230 mOsm) swelling-induced membrane stretch reversibly increased the VDCC currents. Electron microscope and confocal imaging analysis revealed that both hypotonic swelling and cholesterol depletion by methyl-β-cychlodextrin (MβCD) similarly disrupted the caveolae structure and translocated caveolin-1 (Cav-1) from membrane to cytosolic space. Accordingly, MβCD also increased VDCC currents. Moreover, subsequent hypotonic swelling after MβCD treatment failed to increase the VDCC currents further. Western blotting experiments revealed that hypotonic swelling phosphorylated Cav-1 and JNK. Inhibitors of tyrosine kinases (genistein) and JNK (SP00125) prevented the swelling-induced facilitation of VDCC currents. Knockdown of Cav-1 by small interfering RNA blocked both the VDCC current facilitation by stretch and the related phosphorylation of JNK. Taken together, the results suggest that membrane stretch is transduced to the facilitation of VDCC currents via caveolar structure-dependent tyrosine phosphorylation of Cav-1 and subsequent activation of JNK in rat mesenteric arterial myocytes.

Caveolar remodeling is a critical mechanotransduction mechanism of the stretch-induced L-type Ca channel activation in vascular myocytes.

We examined the effects of the PPARα activator fenofibrate on voltage-dependent K (Kv) channels using a patch clamp technique in native rabbit coronary arterial smooth muscle cells. Kv current was inhibited by application of fenofibrate in a concentration-dependent manner, with an apparent IC value of 6.39 ± 0.53μM and a slope value (Hill coefficient) of 1.63 ± 0.10. Fenofibrate accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for fenofibrate were 0.81± 0.05μMs and 4.70 ± 0.47s, respectively. Although fenofibrate did not affect the steady-state activation curves, fenofibrate shifted the inactivation curves toward a more negative potential. Application of train pulses (1 or 2Hz) progressively increased the fenofibrate-induced inhibition of the Kv channel, and the recovery time constant from inactivation was increased in the presence of fenofibrate, which suggested that the inhibitory effect of fenofibrate is use-dependent. Another PPARα activator, bezafibrate and PPARα inhibitor, GW 6471, did not affect the Kv current and also did not change the inhibitory effect of fenofibrate on the Kv current. From these results, we suggest that fenofibrate inhibited Kv current in a state-, time-, and use-dependent manner, completely independent of PPARα activation.

The PPARα activator fenofibrate inhibits voltage-dependent K channels in rabbit coronary arterial smooth muscle cells.

MK801 and ketamine, which are phencyclidine (PCP) derivative N-methyl-d-aspartate receptor (NMDAr) blockers, reportedly enhance the function of 5-hydroxytryptamine (HT)-2A receptors (5-HTRs). Both are believed to directly affect the pathogenesis of schizophrenia, as well as hypertension. 5-HTR signaling involves the inhibition of Kv conductance. This study investigated the interaction of these drugs with Kv1.5, which plays important roles in 5-HTR signaling and in regulating the excitability of the cardiovascular and nervous system, and the potential role of this interaction in the enhancement of the 5-HTR-mediated response. Using isometric organ bath experiments with arterial rings and conventional whole-cell patch-clamp recording of Chinese hamster ovary (CHO) cells ectopically overexpressing Kv1.5, we examined the effect of ketamine and MK801 on 5-HTR-mediated vasocontraction and Kv1.5 channels. Both ketamine and MK801 potentiated 5-HTR-mediated vasocontraction. This potentiation of 5-HTR function occurred in a membrane potential-dependent manner, indicating the involvement of ion channel(s). Both ketamine and MK801 rapidly and directly inhibited Kv1.5 channels from the extracellular side independently of NMDArs. The potencies of MK801 in facilitating the 5-HTR-mediated response and blocking Kv1.5 were higher than those of ketamine. Our data demonstrated the direct inhibition of Kv1.5 channels by MK801/ketamine and indicated that this inhibition may potentiate the functions of 5-HTRs. We suggest that 5-HTR-Kv1.5 may serve as a receptor-effector module in response to 5-HT and is a promising target in the pathogenesis of MK801-/ketamine-induced disease states such as hypertension and schizophrenia.

Enhancement of 5-HT receptor function and blockade of Kv1.5 by MK801 and ketamine: implications for PCP derivative-induced disease models.

Mulberry (Morus alba) has been used in traditional oriental medicine since ages. Recently, it has been reported that mulberry produces hypotensive effects through the eNOS signaling pathway. However, the mechanism underlying the hypotensive effects of mulberry is not entirely clear. Moreover, the effects of mulberry on vascular remodeling events such as hyperplasia, an important etiology in the pathogenesis of hypertension and arteriosclerosis, are also ambiguous. Here, we hypothesized that an ethanolic extract of mulberry fruit (EMF) has beneficial effects on vascular remodeling and produces hypotensive effects. The effects of a 6-week oral administration of EMF were examined in spontaneously hypertensive rats (SHRs). The animals were divided into four groups: normotensive control (Wistar Kyoto rats), non-treated SHR, low-dose (100 mg/kg) EMF-treated SHR, and high-dose (300 mg/kg) EMF-treated SHR. Our results showed that the EMF-diet normalizes hypertension in SHRs in a dose-dependent manner, by preventing smooth muscle proliferation, thickening of the tunica media, and vascular hyper-reactivity. The endothelial functions were not substantially affected by the EMF diet in our experimental setting. In conclusion, we suggest that the mulberry fruit could act as a food supplement for reducing blood pressure in hypertensive subjects through its effects on smooth muscle proliferation and vascular contractility.

Effects of an ethanolic extract of mulberry fruit on blood pressure and vascular remodeling in spontaneous hypertensive rats.

Oxidative stress is associated with many cardiovascular diseases, such as hypertension and arteriosclerosis. Oxidative stress reportedly activates the L-type voltage-gated calcium channel (VDCC) and elevates [Ca] in many cells. However, how oxidative stress activates VDCC under clinical setting and the consequence for arteries are unclear. Here, we examined the hypothesis that hydrogen peroxide (HO) regulates membrane potential (Em) by altering Na influx through cation channels, which consequently activates VDCC to induce vasoconstriction in rat mesenteric arteries. To measure the tone of the endothelium-denuded arteries, a conventional isometric organ chamber was used. Membrane currents and Em were recorded by the patch-clamp technique. [Ca] and [Na] were measured with microfluorometry using Fura2-AM and SBFI-AM, respectively. We found that HO (10 and 100 µM) increased arterial contraction, and nifedipine blocked the effects of HO on isometric contraction. HO increased [Ca] as well as [Na], and depolarized Em. Gd (1 µM) blocked all these HO-induced effects including Em depolarization and increases in [Ca] and [Na]. Although both nifedipine (30 nM) and low Na bath solution completely prevented the HO-induced increase in [Na], they only partly inhibited the HO-induced effects on [Ca] and Em. Taken together, the results suggested that HO constricts rat arteries by causing Em depolarization and VDCC activation through activating Gd-and nifedipine-sensitive, Na-permeable channels as well as Gd-sensitive Ca-permeable cation channels. We suggest that unidentified Na-permeable cation channels as well as Ca-permeable cation channels may function as important mediators for oxidative stress-induced vascular dysfunction.

Hydrogen peroxide constricts rat arteries by activating Na-permeable and Ca-permeable cation channels.

Estrogen has diverse effects on cardiovascular function, including regulation of the contractile response to vasoactive substances such as serotonin. The serotonin system recently emerged as an important player in the regulation of vascular tone in humans. However, hyperreactivity to serotonin appears to be a critical factor for the pathophysiology of hypertension. In this study, we examined the modulatory mechanisms of estrogen in serotonin-induced vasoconstriction by using a combinatory approach of isometric tension measurements, molecular biology, and patch-clamp techniques. 17β-Estradiol (E2) elicited a significant and concentration-dependent relaxation of serotonin-induced contraction in deendothelialized aortic strips isolated from male rats. E2 triggered a relaxation of serotonin-induced contraction even in the presence of tamoxifen, an estrogen receptor antagonist, suggesting that E2-induced changes are not mediated by estrogen receptor. Patch-clamp studies in rat arterial myocytes showed that E2 prevented Kv channel inhibition induced by serotonin. Serotonin increased Src activation in arterial smooth muscle required for contraction, which was significantly inhibited by E2. The estrogen receptor-independent inhibition of Src by E2 was confirmed in HEK293T cells that do not express estrogen receptor. Taken together, these results suggest that estrogen exerts vasodilatory effects on serotonin-precontracted arteries via Src, implying a critical role for estrogen in the prevention of vascular hyperreactivity to serotonin.

Young Min Bae

Department of Physiology,
KU Open Innovation Center,
Research Institute of Medical Science,
Department of Physiology, KU Open Innovation Center, Research Institute of Medical Science

Konkuk University School of Medicine

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Young Min Bae

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology,KU Open Innovation Center,Research Institute of Medical Science,Department of Physiology, KU Open Innovation Center, Research Institute of Medical Science

Konkuk University School of Medicine

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Department of Physiology,
KU Open Innovation Center,
Research Institute of Medical Science,
Department of Physiology, KU Open Innovation Center, Research Institute of Medical Science