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Anesth Analg 2007; 105:1714-1719 © 2007 International Anesthesia Research Society doi: 10.1213/01.ane.0000290334.91624.2f
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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BACKGROUND: Arginine vasopressin (AVP) inhibits ATP-sensitive potassium (KATP) channels and may help to restore vascular tone in patients with vasodilatory shock. In the present study, we investigated whether extracellular acidification modifies the inhibition of vascular KATP channels by AVP.
METHODS: We used a cell-attached patch-clamp configuration to investigate the effects of extracellular pH (pHo) on AVP-KATP channel interaction in rat aortic smooth muscle cells.
RESULTS: Bath application of AVP significantly inhibited extracellular acidification (pHo = 6.5)-induced KATP channel activity in a concentration-dependent manner, with an half-maximal inhibitory concentration (IC50) value of 16.8 pM. Furthermore, bath application of AVP significantly inhibited pinacidil-induced KATP channel activity at mild (pHo = 7.0) and severe (pHo = 6.5) extracellular acidification, with IC50 values of 266.7 and 21.4 pM, respectively, but failed to significantly inhibit at normal pH (pHo = 7.4) or under alkalosis (pHo = 9.0). Augmentation of AVP inhibition of vascular KATP channels during extracellular acidification was eliminated by pretreatment with OPC-21268, a specific blocker of the V1 receptor, but not by a V2 blocker, OPC-31260. AVP-induced inhibition was also suppressed by pretreatment with a protein kinase C inhibitor, calphostin C.
CONCLUSIONS: Our results suggest that AVP inhibits extracellular acidification-induced vascular KATP channel activity, and that the inhibitory effects of AVP on vascular KATP channels are enhanced by extracellular acidification via the V1 receptor-protein kinase C cell-signaling pathway. The potent inhibition of vascular KATP channels by AVP under acidic conditions may make it suitable for management of vasodilatory shock.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Arginine vasopressin (AVP) is an important vasopressor in vasodilatory shock; however, the detailed mechanisms of its action during shock remain largely unclear (1–3). In vascular smooth muscle cells (VSMCs), activation of ATP-sensitive potassium (KATP) channels, which leads to an increase in K+ conductance and hyperpolarization of the cell membrane, results in vascular relaxation (4,5). Previously, we found that high concentrations of AVP inhibit vascular KATP channels under physiological conditions (6). The inhibitory effects of AVP on vascular KATP channels are of interest because these channels may play a key role in the pathogenesis of vasodilatory shock and in decreased responsiveness to catecholamines (7).
One of the unique attributes of AVP compared with catecholamines is that the pressor effects of AVP are preserved under acidic conditions, which are common in vasodilatory shock (1–3,7). Several electrophysiological studies have indicated that metabolic stress, including hypoxia and acidosis, may affect the pharmacological modulation of KATP channels (8,9). These results suggest that extracellular acidification could modify the interaction between AVP and KATP channels. The present study was undertaken to examine whether extracellular acidification influences the effect of AVP on KATP channel activities in rat aortic smooth muscle cells at the single-channel level.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Cell Preparation
The study was approved by the Animal Investigation Committee of Tokushima University (Tokushima, Japan) and was conducted according to the animal use guidelines of the American Physiological Society (Bethesda, MD).
Male Wistar rats (weight, 250–300 g) were anesthetized with ether and injected intraperitoneally with 1.0 IU/g heparin 30 min before surgery. The aortas were dissected and opened longitudinally, and the endothelium and adventitia removed. The tissue was minced into small pieces in normal Tyrode solution. The pieces were explanted on glass cover slips in tissue-culture dishes filled with medium 199 (Nissui Chemicals, Tokyo, Japan), 10% fetal bovine serum (GIBCO, Grand Island, NY), 100 µg/mL streptomycin, and 100 µg/mL penicillin, and stored in a CO2 incubator (5% CO2, 37°C). Single smooth muscle cells migrated out of the tissues and adhered to the cover slips within a few days. After culturing for 6–10 days, the smooth muscle cells were used in electrophysiological recording experiments.
Electrophysiological Measurements and Data Analysis
Single-KATP channel currents were recorded in the cell-attached patch-clamp configuration using a patch-clamp amplifier (CEZ 2200; Nihon Kohden, Tokyo, Japan), as described by Hamill et al. (10). The cell-attached patch-clamp configuration uses a micropipette attached to the cell membrane to allow recording from a single ion channel. AVP was applied to the bathing (extracellular) solution to examine the inhibitory effects of AVP on KATP channel activity mediated via the cell-signaling pathway. Steady-state KATP channel activation was induced by pinacidil, a selective KATP channel opener, which is less affected by pH changes (9). To evaluate the inhibitory effects of AVP on extracellular acidification-induced KATP channel activity, the inhibitory effects of AVP based on a cumulative dose response (10–12 to 10–8 M) were examined. To evaluate the effects of extracellular acidification on AVP inhibition, the inhibitory effects of AVP on pinacidil-induced KATP channel activity were examined at various extracellular pH (pHo) levels. pClamp version 7 software (Axon Instruments, Foster, CA) was used for data acquisition and analysis. The open probability (Po) was determined from current amplitude histograms and was calculated as described previously (6,11,12). Channel activity was expressed as NPo, where N was the number of channels active in the patch. The NPo in the presence of AVP was normalized to the baseline NPo value obtained in the absence of AVP and is presented as the relative channel activity.
The AVP concentration needed to induce half-maximal inhibition of pinacidil-induced KATP channel activity (IC50) and the Hill coefficients were calculated as follows
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where y is the relative NPo, [D] is the concentration of the drug, Ki is the IC50, and H is the Hill coefficient.
Solutions
In the cell-attached patch-clamp configuration, the bathing solution was composed of the following: 140 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES. The pipette solution contained 140 mM KCl, 10 mM HEPES, and 5.5 mM dextrose (pH 7.4). The pH of the extracellular solution (pHo) was adjusted with NaOH/HCl and the pH inside the bath was continuously measured with a pH meter (Model 611; Orion Research Instruments, Cambridge, MA). Recordings were made at 36°C ± 0.5°C.
Drugs
AVP, glibenclamide, and pinacidil were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Calphostin C was obtained from Calbiochem (San Diego, CA). OPC-21268 and OPC-31260 were provided by Otsuka Pharmaceutical Co. (Osaka, Japan). Glibenclamide, pinacidil, calphostin C, and OPC-21268 were dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide was <0.05%. OPC-31260 was dissolved in distilled water.
Statistics
All data are presented as means ± sd. Differences between data sets were evaluated either by repeated-measure one-way analysis of variance followed by the Scheffé F test or by Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Effects of pHo on KATP Channel Activity in VSMCs
We examined whether extracellular acidification affected the KATP channel activity in VSMCs in the cell-attached patch-clamp configuration. Figure 1A shows the single-channel characteristics under normal pHo conditions (pHo = 7.4). Spontaneous single-channel activity was observed infrequently (NPo< 0.01, n = 15); however, application of 100 µM pinacidil to the bathing solution significantly activated K+-selective channels (NPo = 0.40 ± 0.13, P < 0.05 versus baseline, n = 16). This channel activity was completely blocked by 3 µM glibenclamide, a specific KATP channel blocker.
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As shown in Figure 1B, decreasing the pHo from normal to mild (pHo = 7.0) and severe (pHo = 6.5) acidosis gradually activated KATP channel currents, with relative channel activity increasing to 0.11 ± 0.02 and 0.136 ± 0.04, respectively, within 7–10 min after decreasing the pHo. Regardless of pHo change, however, application of 100 µM pinacidil activated the KATP channel rapidly. Figure 1C shows the relationship between NPo and time for the traces in the cell-attached configurations. Although baseline KATP channel currents were slightly activated by decreasing the pHo, application of 100 µM pinacidil could induce steady-state KATP channel activity independent of changes in pHo.
Effects of AVP on Extracellular Acidification-Induced KATP Channel Activity
We first examined whether AVP affected the extracellular acidification (pHo = 6.5)-induced KATP channel activity in the cell-attached patch-clamp configuration. Figure 2A shows representative examples of effects of AVP on extracellular acidification-induced KATP channel activity. Application of 10–12, 10–11, and 10–10 M AVP to the outside of the membrane surface inhibited these KATP channel currents, with relative channel activities decreasing to 0.97 ± 0.11 (n = 8), 0.57 ± 0.14 (n = 8), and 0.18 ± 0.06 (n = 8), respectively. Figure 2A also shows that the channel currents recovered after washout of AVP.
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The concentration-dependent effects of AVP on extracellular acidification-induced KATP channel activities in the cell-attached patch-clamp configuration are shown in Figure 2B. AVP inhibited KATP channel activity significantly at concentrations of 10–11 M or larger. The IC50 value and Hill coefficients were 16.8 pM and 1.07, respectively. AVP did not change the single-channel conductance (data not shown).
Effects of pHo on Pinacidil-Induced KATP Channel Inhibition by AVP
We next examined whether pHo modifies the AVP-KATP channel interaction in intact VSMCs by use of the cell-attached patch-clamp configuration. The inhibitory effects of AVP on the steady-state KATP channel activity induced by pinacidil (100 µM) were evaluated at various pHo levels. Representative examples of the effects of AVP are shown in Figure 3A. Application of a physiological concentration of AVP (10–11 M) at normal pHo failed to significantly inhibit the pinacidil-induced KATP channel activity, with a relative channel activity of 0.97 ± 0.05 (n = 15). However, switching from a pHo of 7.4 to 6.5 significantly inhibited the pinacidil-induced KATP channel currents, with relative channel activities decreasing to 0.51 ± 0.12 of the control. Channel activity recovered when AVP was superfused in solution at a normal pHo (Fig. 3A).
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The concentration-dependent effects of AVP on pinacidil-induced KATP channel currents in the cell-attached patch-clamp configuration at various pHo levels are shown in Figure 2B. At pHo 7.0 and pHo 6.5, AVP inhibited KATP channel currents in a concentration-dependent manner at concentrations of 10–12 to 10–8 M. The IC50 values were 266.7 pM (pHo 7.0) and 21.4 pM (pHo 6.5), and the Hill coefficients were 1.06 (pHo 7.0) and 1.16 (pHo 6.5). The average recovery of the KATP channel activity after AVP washout was 97% ± 8% (pHo 7.0) and 95 ± 12% (pHo 6.5) of the NPo obtained before application of AVP. In contrast, at pHo 7.4 and pHo 9.0, even high concentrations of AVP had no significant inhibitory effects on the KATP channel currents (Fig. 3B).
Involvement of a Vasopressinergic Receptor in AVP Inhibition During Extracellular Acidification
The pHo-dependent effectiveness of bath-applied AVP in cell-attached patches suggests that the AVP effect is mediated by intracellular signaling systems. AVP exerts its effects via interaction with a family of membrane-bound G protein-coupled AVP-specific receptors, V1 (formally known as V1a) and V2 (1,2). To test whether these receptors were involved in the augmentation of AVP inhibition of KATP channels during extracellular acidification, the effects of selective antagonists of the V1a (OPC-21268) and V2 (OPC-31260) receptors on AVP inhibition of KATP channels at pHo 6.5 in the cell-attached patch-clamp configuration were examined.
As shown in Figure 4A, OPC-21268, a specific blocker of the V1 receptor, had no effect on channel activity at pHo 6.5 at 8 µM, 20 times the IC50 for V1 receptor blockade. When AVP (10–11 M) was added in the presence of OPC-21268, AVP failed to inhibit pinacidil-induced KATP channel activity. This failure could be explained by the blockage of a specific receptor on the cell membrane, because AVP-induced inhibition reappeared when OPC-21268 was washed out (Fig. 4A). In contrast, OPC-31260, a specific blocker of the V2 receptor, failed to suppress the AVP-induced inhibition of KATP channels at pHo 6.5 at 1 µM, >1000 times the IC50 for V2 receptor blockage (Fig. 4B). The AVP-induced changes in relative channel activities during receptor blockage are summarized in Figure 4C. The values at pHo 6.5 were 0.42 ± 0.05 (n = 15) without blockers (P < 0.05), 0.91 ± 0.12 (n = 12) with OPC-21268 (P = NS), and 0.48 ± 0.07 (n = 14) with OPC-31260 (P < 0.05).
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Effects of Protein Kinase Inhibitors on AVP Inhibition During Extracellular Acidification
The KATP channel is modulated by protein kinases, including protein kinase A (PKA)- and protein kinase C (PKC)-dependent mechanisms (5,13). We observed that pHo-dependent AVP-induced inhibition took place via the V1 vasopressinergic receptor but not via the V2 receptor. Although the V2 receptor is coupled to increased adenylate cyclase activity, leading to the activation of PKA, the V1 receptor exerts its effect through phosphatidylinositol hydrolysis, leading to the mobilization of intracellular Ca2+ and the activation of PKC. This suggests that PKC may be involved in a possible signal-transduction pathway. Therefore, we examined whether augmentation of AVP inhibition of KATP channels during extracellular acidification was affected by the suppression of PKC.
Calphostin C inhibits PKC by competing for the diacylglycerol binding site on PKC with a reported half-inhibition constant (Ki) of 50 nM. Pretreatment (10 min) of rat VSMCs with calphostin C (500 nM) added to the bath solution greatly reduced AVP (10–11 M) inhibition of KATP channel activity at pHo 6.5 (Fig. 5A). Calphostin C alone did not have any effect on KATP channel activity induced by pinacidil. The AVP-induced inhibitions in relative channel activity during treatment with calphostin C are summarized in Figure 5B. The values at pHo 6.5 were 0.51 ± 0.11 (n = 15) without calphostin C (P < 0.05) and 0.92 ± 0.14 (n = 15) with calphostin C (P = NS).
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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In the current study, the effects of pHo on the inhibition of vascular KATP channels by AVP were tested using the cell-attached patch-clamp configuration. Our results demonstrate that AVP inhibits extracellular acidification-induced KATP channel activity in a concentration-dependent manner. Furthermore, the inhibitory effects of AVP on pinacidil-induced KATP channel activity are enhanced by extracellular acidification in a pHo-dependent manner: bath application of AVP significantly inhibits pinacidil-induced KATP channel activity under the extracellular mild (pHo = 7.0) and severe (pHo = 6.5) acidosis conditions, with IC50 values of 266.7 and 21.4 pM, respectively, but fails to inhibit activity under normal (pHo = 7.4) conditions. Our results also show that this augmentation of the AVP inhibition of KATP channels by extracellular acidification is mediated by the vasopressinergic V1 receptor-PKC cell-signaling pathway.
Our previous study indicated that a supraphysiologic concentration (10–8 M) of AVP inhibits vascular KATP channels under physiological conditions in the cell-attached patch-clamp configuration (6). The same set of experiments here further demonstrate that lower concentrations of AVP are able to inhibit extracellular acidification-induced vascular KATP channels. In addition, the present results provide evidence that the inhibitory action of AVP on pinacidil-induced vascular KATP channel activity can be modulated by decreasing the pHo. Relative to the response recorded at pHo 7.4, extracellular acidification enhances the inhibitory efficacy of AVP in a pHo-dependent manner.
Previous reports have demonstrated that KATP channels contain their pH-sensitive sites on the inside of the cell membrane (14). Thus, our results suggest that enhanced action of AVP during extracellular acidification is related to a direct effect of H+ modulating the AVP-channel interaction rather than the pHo-channel interaction. These results indicate that vascular KATP channels are regulated by AVP under acidic conditions through receptor-mediated signaling pathways. Indeed, our results show that the pHo-dependent effectiveness of bath-applied AVP in cell-attached patches is antagonized by the V1 receptor antagonist, OPC-21268, which indicates that the effects of AVP take place through the V1 receptor stimulation. It has been reported that the AVP V1 receptor couples to PKC via activation of phospholipase C (1,2). Recently, phosphorylation by protein kinases, including PKC and PKA, has been shown to directly modulate vascular KATP channel activity (5,13). Some smooth-muscle constrictors (e.g., angiotensin II, serotonin, and acetylcholine) that act through stimulation of PKC have been shown to inhibit vascular KATP channels (5,13). Similarly, the abolition of pHo-dependent AVP inhibition of vascular KATP channels by a specific PKC inhibitor, calphostin C, suggests that the AVP-induced PKC activation via the V1 receptor is likely to affect vascular KATP channel protein phosphorylation and hence the channel activity.
KATP channels are present in a wide range of tissues and are believed to be involved in the coupling of cellular metabolic status and excitability (15). In VSMCs, KATP channels regulate membrane potential, which controls calcium entry through voltage-dependent calcium channels, thereby controlling the contractility through changes in intracellular calcium (5,13). Recent studies provide increasing evidence that excessive activation of vascular KATP channels is associated with the catastrophic vasodilation and vascular hyporeactivity to catecholamine in circulatory shock (7). AVP is well recognized as a direct vasoconstrictor of the systemic vasculature mediated by the V1 receptor, and is becoming established as an important vasopressor in vasodilatory shock (1–3). The rationale for the use of vasopressin is its relative deficiency in plasma and hypersensitivity to its vasopressor effects during septic shock (7). Dosing guidelines indicate that low-dose exogenous AVP infusion (0.01–0.04 U/min) increases AVP concentrations to approximately 30–100 pM and restores arterial blood pressure and the response to catecholamine (1–3). Our results indicate that AVP inhibits vascular KATP channels at these clinical concentrations under acidic conditions but has no effect under physiological conditions (Fig. 3B). Thus, it is likely that when exogenous AVP is used as a vasoconstrictor under acidic conditions, which is common in shock, it may inhibit vascular KATP channel activity. These results support the idea that low-dose AVP infusion increases sensitivity to the vasoactive effects of AVP during vasodilatory shock by closing vascular KATP channels and compensating for acute AVP deficiency.
The inherent limitations of this study model must be addressed. First, although we studied the cell-signaling effects of AVP on KATP channels under normal pHo and low pHo, there is a possibility that the inhibitory action of AVP is influenced by alkalotic pHo. To confirm this possibility, it is necessary to study the effects of AVP under alkalotic pHo conditions. Second, the vascular KATP channels are regulated by multiple metabolic-effector pathways during pathophysiological conditions such as hypoxia and vasodilatory shock (5,13). However, in our experimental model, the synthetic KATP channel opener, pinacidil, was used to activate the channels, and thus their activities may have been altered as compared with in vivo conditions. Third, our experiments were not conducted under controlled oxygen tension, and thus may be subject to differences in metabolic states between individual cells. Therefore, it may be difficult to directly extrapolate our results to in vivo conditions.
In conclusion, our results suggest that the inhibitory effects of AVP on vascular KATP channels are enhanced by extracellular acidification via the V1 receptor-PKC cell-signaling pathway. Thus, extracellular acidification during vasodilated shock will modulate the AVP inhibition of vascular KATP channels and may result in increased sensitivity to the vasoconstrictive effects of AVP.
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Footnotes |
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Accepted for publication August 13, 2007.
Supported in part by a Grant-in-Aid for Scientific Research (C, 15591636) from the Japan Society for the Promotion of Science, Tokyo, Japan.
This study is attributed to the Department of Anesthesiology, The University of Tokushima Graduate School, Tokushima, Japan.
There are no conflicts of interest.
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