TY - JOUR
AU - Dietz, Nancy, J.
AB - Abstract Previous studies have documented increased K+ permeability of arterial smooth muscle in hypertension and suggested a role in altered arterial contractile function. To characterize the mechanisms responsible for these alterations, we determined the contribution of K+ current (IK) components to whole cell IK in freshly dispersed myocytes and tetraethylammonium (TEA)-induced contractile responses in mesenteric arteries of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Tetraethylammonium produced a larger tonic contractile response in SHR with a lower threshold compared to WKY (ie, 0.1 v 1 mmol/L), which was due in part to the larger Ca2+ current in SHR. Whole cell IK recorded by perforated patch methods was similar at a holding potential (HP) of −60 mV (IK60), but were larger in SHR when recorded from a HP of −20 mV (IK20). The selective blocker iberiotoxin (IbTX) was used to separate the contribution of voltage- (KV) and calcium-dependent (KCa) components of IK60. The IK60 and IK20 component inhibited by 100 nmol/L IbTX (ie, KCa) was larger in SHR than in WKY myocytes, whereas the IbTX-insensitive IK60 component (ie, KV) was larger in WKY. In the presence of IbTX, 1 and 10 mmol/L TEA inhibited a larger fraction of IK60 in SHR myocytes compared with WKY. The activation properties of the TEA-sensitive and TEA-insensitive KV components determined by fitting a Boltzmann activation function to the current-voltage data, exhibited both group and treatment differences in the half maximal activation voltage (V0.5). The V0.5 of the TEA-sensitive KV component was more positive than that of the TEA-insensitive component in both groups, and values for the V0.5 of both TEA-sensitive and TEA-insensitive components were more negative in SHR than WKY. These results show that SHR myocytes have larger KCa and smaller KV current components compared with WKY. Furthermore, SHR myocytes have a larger TEA-sensitive KV component. These differences may contribute to the differences in TEA contractions, resting membrane potential, Ca2+ influx, and KCa current reported in hypertensive arteries. Am J Hypertens 2001;14:897–907 © 2001 American Journal of Hypertension, Ltd. Calcium-dependent K+ channels, voltage-dependent K+ channels, channel activation, iberiotoxin, tetraethylammonium, TEA-induced contractions Numerous studies have documented differences in arterial smooth muscle contractile function in human and animal models of hypertension.1 Although some of the reported alterations remain controversial, there is general agreement that transmembrane ion fluxes are altered in hypertensive arterial smooth muscle.2 Beginning with the seminal studies of Jones et al,3–5 increased K+ efflux from hypertensive arterial smooth muscle cells is now universally accepted. A decade later, numerous studies have documented increased dihydropyridine-sensitive Ca2+ (ie, CaL) influx in hypertensive arterial smooth muscle.6 These differences in K+ and Ca2+ fluxes in hypertensive arterial smooth muscle are functionally significant. This conclusion is based in part on the observations that larger tonic contractile responses are produced in hypertensive compared with normal arterial smooth muscle by tetraethylammonium (TEA, a nonselective K+ channel blocker),7,8 and by Bay k 8644 (a CaL activator),9,10 both of which increase Ca2+ influx through CaL. We previously demonstrated that the larger tonic contractile responses to Bay k 8644 in the spontaneously hypertensive rat (SHR) compared with the Wistar-Kyoto (WKY) mesenteric arteries were associated with an increased CaL current amplitude and shifted voltage-dependent activation.11,12 However, the differences in CaL alone were not sufficient to explain the differences in contractile responses between the two groups. Rusch et al,7 among others, have demonstrated augmented contractile responses to K+ channel inhibition by TEA in the SHR aorta compared with the WKY. They suggested that this was due to an increased TEA-sensitive K+ current (IK) in SHR. However, they demonstrated that control values of IK and the fraction of IK inhibited by TEA were similar in aortic myocytes from WKY and SHR.7 In subsequent studies using single channel measurements, they demonstrated increased open probability of KCa channels in SHR at resting membrane potential as well as greater effectiveness (sensitivity) of Ca2+ in activating these channels.13 These results suggest that there is an increased contribution of KCa currents to whole cell IK in SHR. However, this conclusion is difficult to reconcile with the finding of similar TEA-sensitive IK between WKY and SHR myocytes. One possible explanation is that there is a greater TEA-sensitive, non-KCa component of IK (ie, KV) in WKY myocytes. Two recent studies have demonstrated smaller voltage-gated K+ (KV) and larger Ca2+-sensitive K+ (KCa) currents in arterial myocytes from SHR compared with WKY.14,15 However, another study concluded that delayed rectifier K+ currents (ie, KV) are not altered in hypertensive arterial myocytes.16 It is obvious that the basis for altered K+ channel function in hypertension is not completely understood. Although altered function of KCa channels have been clearly demonstrated in hypertension,3–5 the extent to which other K+ channel types (eg, KV) may be altered in hypertension is controversial. To date there have been only a limited number of studies of KV channel properties in hypertension,15–17 and their role in hypertension remains unclear. Accordingly, it was the purpose of the present study to determine whether the differences in TEA contractile responses between WKY and SHR are associated with differences in IK components. The contribution of K+ current components to whole cell IK in WKY and SHR myocytes was determined using whole cell perforated patch methods and the selective KCa blocker iberiotoxin. Methods Contractile studies Segments of mesenteric artery from 12-week-old male WKY rats and SHR were isolated and mounted as previously described.18 After a 2–3 h equilibration, duplicate responses to a 120 mmol/L K+-PSS solution were determined, in which K+ was substituted for Na+ on an equimolar basis. This response was used as a test of tissue viability and for normalization of subsequent contractile responses. After recovery from the KCl contractions, responses to the cumulative bath addition of TEA were determined. Recovery periods of at least 1 h between drug tests at baseline force were used. Electrophysiological methods Myocytes were enzymatically isolated from mesenteric arteries with collagenase and elastase treatment using methods previously described in detail by us.11,12 An aliquot of cells was placed in a 0.5-mL chamber on the stage of an inverted microscope (Nikon Diaphot, Melville, NJ), and allowed to adhere to the chamber's glass bottom. Membrane currents were recorded using conventional and perforated patch (amphotericin) whole cell configurations19,20 at room temperature (about 22–24°C). Micropipettes (2–3 Mohm resistance) were made from capillary tubing (WPI Kwik-fil, Sarasota, FL) using a programmable puller (P-80/PC, Sutter Instruments, San Rafael, CA) and fire polished. Series resistance and capacitance compensation were adjusted maximally using a voltage clamp amplifier with a 100-Mohm head stage (model 8900, Dagan, Minneapolis, MN). Experimental protocols were controlled using a computer (466/L, Dell, Austin, TX) and PCLAMP software (Axon Instruments, Foster City, CA). Current signals were converted from analog to digital format at a sampling rate of 10 kHz using a Labmaster A/D board (Axon Instruments, Foster City, CA) and stored in the computer for analysis. Multiple responses to (20 mV) hyperpolarizing voltage clamp steps (n = 5) were obtained for each protocol, averaged, and used to provide capacitance and leak compensation of the raw data where indicated. Experimental current records were analyzed using PCLAMP software. Procedures For perforated patch recordings, pipette tips were filled with amphotericin-free internal solution, then back-filled with amphotericin-containing solution as previously described.20 Junction potentials were compensated with the pipette in the bath solution before sealing. Cells with access resistances >15 Mohm were rejected. For conventional whole cell recordings, pipettes were sealed to cells with negative pressure at a −60 mV holding potential. When a gigohm seal had formed, cell break-in was accomplished by additional negative pressure. After setup, membrane potential was ramped at 20-sec intervals from −60 to +60 mV at 120 mV/sec until the current response stabilized (usually 3–5 min). Current-voltage measurements were then made from holding potentials of −60 and −20 mV in random order. A set of voltage clamp steps 1 sec long was applied from −60 to +60 mV in 10 mV increments every 20 sec. Chemicals and solutions The external solution (PSS) used for contractile studies had the following composition (in mmoles/L): 114 NaCl, 4.5 KCl, 2.5 CaCl2, 1.2 MgSO4, 24 NaHCO3, 1.2 NaH2PO4, 2.4 Na2HPO4 and 11 dextrose at pH of 7.4 (NaOH) and osmolality of 300 ± 2 mosm/L (aerated with 95% O2–5% CO2). The incubation buffer for enzymatic cell isolation and the external solution for patch clamp studies had the following composition (in mmoles/L): 140 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, and 10 glucose at pH 7.4 (NaOH), and an osmolality of 298 ± 2 mosm/L. The pipette solution for perforated patch recordings had the following composition (in mmoles/L): 100 K gluconate, 30 KCl, 5 NaCl, 1 MgCl2, 1 CaCl2, 3 EGTA, 10 HEPES, 10 glucose, and 150 μg/mL amphotericin at pH 7.2 (titrated with KOH), and an osmolality of 303 ± 2 mosm/L. Ca2+ was included in the amphotericin pipette solution to produce cell contraction if the seal spontaneously converted from the perforated patch to whole cell configuration. Such cells were not included in the data analysis. The pipette solution for conventional recordings had the following composition (in mmoles/L): 120 KCl, 5 NaCl, 5 MgATP, 10 HEPES, and 10 BAPTA at pH 7.2 (titrated with KOH), and an osmolality of 306 ± 3 mosm/L. The [Ca2+] in this pipette solution was estimated to be about 10−9 mmol/L.21 Collagenase was purchased from Worthington Biochemical (CLS3, Freehold, NJ). Elastase was purchased from ICN Pharmaceuticals (porcine pancreas, ICN, Cleveland, OH). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Statistical analysis Statistical comparisons of membrane currents were performed using a two-way analysis of variance with repeated measures for unpaired data using the StatView application on a Macintosh computer (PowerMac 7100, Apple, Cupertino, CA). Probability values less than .05 were considered to be significant. Average values are given for individual cells studied in the two groups for each procedure as means ± 1 SEM. Force was divided by wall cross-sectional area and expressed as active stress as previously described.18 The peak values of active stress were determined at each drug concentration and expressed as percentage of the maximum response to KCl. Values of active stress and percent response were averaged at each drug concentration for the two groups (WKY and SHR). Results Ca2+ influx and TEA contractions Tetraethylammonium added to the bath solution produced a concentration-dependent tonic contractile response in mesenteric arteries from WKY and SHR, as shown in Fig. 1. However, the response in SHR was larger and occurred over a wider range of [TEA] from 0.1 to 10 mmol/L compared with WKY. Responses in WKY only occurred at [TEA] above 1 mmol/L. Differences in TEA responses between WKY and SHR were statistically significantly different (P < .05) at all concentrations of TEA. These contractile responses were totally dependent on Ca2+ influx through L-type Ca2+ channels (CaL) as they are completely inhibited by 10 nmol/L nifedipine (data not shown). In the presence of 10 mmol/L TEA, the addition of 10 nmol/L Bay k 8644 (the L-type Ca2+ channel agonist) to the bath (BK in Fig. 1A) caused a large contractile response in WKY but was without effect in the absence of TEA (Fig. 1C). In SHR, there was no additional effect of adding 10 nmol/L Bay k 8644 in the presence of 10 mmol/L TEA (data not shown). Effects of TEA on basal force. Panels on the left show typical isometric force responses to the cumulative addition of TEA to the bath for mesenteric artery from WKY (A and C) and SHR (B). The right panel shows the concentration-response curves for TEA. Peak responses at each [TEA] were normalized by the maximum response to 120 mmol/L KCl before averaging. Addition of 10 nmol/L Bay k 8644 to the WKY rings after the TEA addition results in a large increase in force (BK in A) but not in SHR rings (not shown). Addition of 10 nmol/L Bay k 8644 before the TEA responses has no effect on basal force (BK in C) but significantly increases responses to TEA at ≥1 mmol/L. Symbols represent means, whereas vertical bars are ±1 SEM (n = 12 for all three groups). TEA = tetraethylammonium; WKY = Wistar-Kyoto rats; SHR = spontaneously hypertensive rats. Figure 1. Open in new tabDownload slide Figure 1. Open in new tabDownload slide It has been suggested that the contractile responses to TEA are due to membrane depolarization secondary to decreased outward current, resulting in an increase in the open probability of CaL, and increased Ca2+ influx.22 We have shown that there is greater functional activity of CaL in mesenteric arteries of SHR versus WKY, which is associated with larger CaL currents measured by whole cell patch clamp methods.11,12 It is possible that these differences in CaL are responsible for the larger TEA responses in SHR. To test this possibility, we determined the effects of TEA on WKY mesenteric arteries after the addition of 10 nmol/L Bay k 8644 to the bath. We have shown that this amount of Bay k 8644 increases CaL currents by 50% and shifts the voltage dependence of activation by about 5 mV in the negative direction.23 That is, it makes CaL currents in WKY myocytes similar to those of SHR. Fig. 1 also shows a summary of the results of these experiments. Bay k 8644 had no direct effect on force (Fig. 1C) but produced significant (P < .05) increases in force responses at [TEA] above 0.3 mmol/L compared with untreated WKY arteries. These TEA responses, however, remained significantly smaller than responses in the SHR (P < .05) at all [TEA] except 10 mmol/L, where they were not different. Thus, differences in CaL between WKY and SHR make a partial contribution to the differences in TEA responses. KV and KCa components of IK If CaL is not the major determinant of the differences in TEA responses between WKY and SHR, it is likely that differences in K+ channels are involved. It is well known that force and membrane potential are closely coupled in arterial smooth muscle,22 and that K+ conductance (gK) plays a major role in determining membrane potential.24 To determine whether differences in gK exist in mesenteric artery myocytes between WKY and SHR, measurements of whole cell K+ currents were made from a holding potential of −60 mV (IK60) using perforated patch (amphotericin) methods.20 These results are summarized in Fig. 2A and show that some small but significant differences exist in K+ currents recorded from WKY versus SHR myocytes under these conditions. Between −20 and +20 mV, IK60 was smaller in SHR myocytes whereas > +40 mV it was larger compared with IK60 in WKY myocytes (P < .05). Although data were normalized in terms of cell capacitance before averaging, there were no significant differences in this parameter between the two groups (WKY = 26.8 ± 2.7 pF, n = 17; and SHR = 25.3 ± 2.3 pF, n = 16). Whole cell IK recorded by perforated patch methods from holding potentials of −60 mV (A) and −20 mV (B). Representative families of currents recorded in response to 1-sec voltage clamp steps from −60 to +60 mV in 10 mV steps at 20-sec intervals are shown at the top for WKY (○, 23.0 pF) and SHR (•, 24.7 pF). Calibration bars in the middle of the current traces represent 200 pA and 100 msec. Peak current was determined at each test voltage, divided by cell capacitance, and then averaged. The lower panels show a summary of whole cell K+ current density averaged from WKY (n = 17) and SHR (n = 16) for each holding potential. Symbols represent mean values, whereas vertical barsrepresent ± 1 SEM. Abbreviations as in Fig. 1. Figure 2. Open in new tabDownload slide Figure 2. Open in new tabDownload slide IK recorded from a holding potential of −60 mV is expected to include contributions from Ca2+-, voltage-, inward-rectifier (Kin), and ATP-dependent (KATP) K+ channels.24 Glibenclamide (30 μmol/L), a KATP inhibitor, and Ba2+ (100 μmol/L), a Kin inhibitor, were found to have no significant effect on whole cell IK (data not shown), suggesting that these channels do not contribute to IK in these cells under the conditions of these experiments. Because the availability of Kv channels is voltage dependent, IK was measured from a holding potential of −20 mV (IK20), where their contribution to whole cell IK is negligible and IK is primarily determined by KCa channels.25 As shown in Fig. 2B, IK recorded from a −20 mV holding potential was significantly larger (P < .05) in SHR myocytes compared with WKY at all voltages > −30 mV, suggesting a larger KCa current component in SHR myocytes. To assess the relative contribution of KV and KCa channels to whole cell IK more directly, the effects of the selective KCa inhibitor iberiotoxin (IbTX)24 on IK were determined. As shown in Fig. 3A, whole cell IK measured in the presence of 100 nmol/L IbTX (IbTX-insensitive IK) was significantly smaller at all voltages > −30 mV in SHR myocytes compared with WKY (P < .05). These data were fit with a Boltzmann activation function using least squares methods. There were no significant differences in values of the half-maximal activation voltage (V0.5) or in the slope factor (k) between the two sets of data (Table 1, control values). Table 1 Parameter of KV current activation function Group . V0.5 . k . Effects of TEA on KV current WKY Control −1.8 + 1.0 14.4 + 1.0 1 mmol/L TEA −2.5 + 0.9 14.2 + 0.9 10 mmol/L TEA −3.4 + 1.2 18.2 + 1.3 SHR Control −0.3 + 1.0 14.4 + 1.0 1 mmol/L TEA −4.6 + 1.0 15.1 + 1.0 10 mmol/L TEA −7.7 + 1.1† 15.2 + 1.1 TEA-sensitive KV current WKY 1 mmol/L TEA 10.2 + 1.5 12.2 + 1.5 10 mmol/L TEA 2.3 + 1.6 15.4 + 1.7 SHR 1 mmol/L TEA 0.7 + 1.1† 12.3 + 1.1 10 mmol/L TEA 0.3 + 0.7 12.1 + 0.7 Group . V0.5 . k . Effects of TEA on KV current WKY Control −1.8 + 1.0 14.4 + 1.0 1 mmol/L TEA −2.5 + 0.9 14.2 + 0.9 10 mmol/L TEA −3.4 + 1.2 18.2 + 1.3 SHR Control −0.3 + 1.0 14.4 + 1.0 1 mmol/L TEA −4.6 + 1.0 15.1 + 1.0 10 mmol/L TEA −7.7 + 1.1† 15.2 + 1.1 TEA-sensitive KV current WKY 1 mmol/L TEA 10.2 + 1.5 12.2 + 1.5 10 mmol/L TEA 2.3 + 1.6 15.4 + 1.7 SHR 1 mmol/L TEA 0.7 + 1.1† 12.3 + 1.1 10 mmol/L TEA 0.3 + 0.7 12.1 + 0.7 TEA = tetraethylammonium, WKY = Wistar Kyoto rats; SHR = spontaneously hypertensive rats. * Activation function is defined as: I = Go(V-EK)/[1-exp((V-V0.5)/k)] where Go is the slope conductance, EK is the K+ equilibrium potential, V0.5 is the half maximal activation voltage, and k is the slope factor at V0.5. † Statistically significant difference between WKY and SHR, P < .05. Open in new tab Table 1 Parameter of KV current activation function Group . V0.5 . k . Effects of TEA on KV current WKY Control −1.8 + 1.0 14.4 + 1.0 1 mmol/L TEA −2.5 + 0.9 14.2 + 0.9 10 mmol/L TEA −3.4 + 1.2 18.2 + 1.3 SHR Control −0.3 + 1.0 14.4 + 1.0 1 mmol/L TEA −4.6 + 1.0 15.1 + 1.0 10 mmol/L TEA −7.7 + 1.1† 15.2 + 1.1 TEA-sensitive KV current WKY 1 mmol/L TEA 10.2 + 1.5 12.2 + 1.5 10 mmol/L TEA 2.3 + 1.6 15.4 + 1.7 SHR 1 mmol/L TEA 0.7 + 1.1† 12.3 + 1.1 10 mmol/L TEA 0.3 + 0.7 12.1 + 0.7 Group . V0.5 . k . Effects of TEA on KV current WKY Control −1.8 + 1.0 14.4 + 1.0 1 mmol/L TEA −2.5 + 0.9 14.2 + 0.9 10 mmol/L TEA −3.4 + 1.2 18.2 + 1.3 SHR Control −0.3 + 1.0 14.4 + 1.0 1 mmol/L TEA −4.6 + 1.0 15.1 + 1.0 10 mmol/L TEA −7.7 + 1.1† 15.2 + 1.1 TEA-sensitive KV current WKY 1 mmol/L TEA 10.2 + 1.5 12.2 + 1.5 10 mmol/L TEA 2.3 + 1.6 15.4 + 1.7 SHR 1 mmol/L TEA 0.7 + 1.1† 12.3 + 1.1 10 mmol/L TEA 0.3 + 0.7 12.1 + 0.7 TEA = tetraethylammonium, WKY = Wistar Kyoto rats; SHR = spontaneously hypertensive rats. * Activation function is defined as: I = Go(V-EK)/[1-exp((V-V0.5)/k)] where Go is the slope conductance, EK is the K+ equilibrium potential, V0.5 is the half maximal activation voltage, and k is the slope factor at V0.5. † Statistically significant difference between WKY and SHR, P < .05. Open in new tab Effects of iberiotoxin (IbTX) on whole cell IK in WKY (○) and SHR (•) recorded from a holding potential of −60 mV. IbTX-insensitive currents summarized in panel A were recorded with 100 nmol/L IbTX added to the perfusate. IbTX sensitive currents summarized in panel B were obtained by digital subtraction of currents recorded before and during IbTX addition to the bath. Representative families of currents are shown at the top of each panel (WKY = 31.3 pF; SHR = 28.8 pF). Calibration bars represent 200 pA and 100 msec. Peak current was determined at each test voltage, divided by cell capacitance, and then averaged. The lower panels show a comparison of whole cell K+ current density averaged for WKY (n = 17) and SHR (n = 16). Symbols represent mean values, whereas vertical bars represent ± 1 SEM. Other abbreviations as in Figs. 1 and 2. Figure 3. Open in new tabDownload slide Figure 3. Open in new tabDownload slide The current inhibited by IbTX (IbTX-sensitive) was determined by digital subtraction of individual current records obtained before and after the addition of 100 nM IbTX to the bath. The results summarized in Fig. 3B show that the IbTX-sensitive IK component was significantly larger in SHR myocytes compared with WKY at all voltages from −40 to +60 mV (P < .05). The conclusions regarding the contribution of KV and KCa components to currents recorded at the two values of holding potential shown in Fig. 2 was further tested by determining the effects of IbTX on currents recorded from a −20 mV holding potential (IK20). These measurements were also expected to provide an estimate of the non-Kv, IbTX-insensitive currents. As shown in Fig. 4, 100 nmol/L IbTX inhibited nearly all of current recorded from −20 mV. The differences in IK20 recorded in the presence and absence of IbTX were statistically significant (P < .05) in WKY myocytes at voltages > +10 mV and in SHR myocytes at voltages > −10 mV. When the linear “leak” current was subtracted from the IbTX-insensitive IK20 current (lower records in Fig. 4), the remaining current was very small, averaging < 1 pA/pF at +60 mV in both WKY and SHR. Effects of IbTX on whole cell IK in WKY (A) and SHR (B) recorded from a holding potential of −20 mV. Currents were recorded before (•) and after (○) the addition of 100 n IbTX to the bath. Also shown are currents from which the “linear leak” component had been subtracted (▾). Representative families of currents are shown at the top of each panel (WKY = 24.7 pF; SHR = 32.9 pF). Calibration bars for the currents are shown at the top. Peak current was determined at each test voltage, divided by cell capacitance, and then averaged. The lower panels show a comparison of whole cell K+ current density averaged for WKY (n = 17) and SHR (n = 16). Symbols represent mean values, whereas vertical bars represent ± 1 SEM. Abbreviations as in Figs. 1–3. Figure 4. Open in new tabDownload slide Figure 4. Open in new tabDownload slide To further compare the magnitude of the Kv component of IK, the total amount of K+ current inhibited by voltage in SHR and WKY was determined. Using a standard two-step voltage protocol, steady state inactivation was determined from a holding potential of −100 mV, as shown in Fig. 5, where all of the Kv current is available. When recorded at a test potential of −10 mV, the magnitude of the current inhibited by conditioning voltage was significantly larger in WKY versus SHR myocytes (P < .05). These data were fit with a Boltzmann function and the parameters V0.5 and k were not different between the two groups. Measurements were also made to a test potential of +40 mV with similar results. That is, the magnitude of the current inhibited by voltage was larger in WKY, but no differences with found in the Boltzmann parameters between the two groups (data not shown). Steady-state inactivation (availability) of IK in WKY (•) and SHR (○) myocytes. Steady-state inactivation was determined with a standard two-step protocol consisting of a 10-sec conditioning step to voltages from −100 to −10 mV in steps of 10 mV followed by a test step to −10 mV with a 30-sec recovery between steps. Currents were measured in the presence of 100 nmol/L IbTX and were not leak corrected. They were divided by cell capacitance before averaging. Typical families of current responses are shown at the top of the fig. and the test step responses at −10 mV shown with an enlarged time scale (WKY = 26.3 pF; SHR = 34.2 pF). The lower panel shows a comparison of results for WKY (n = 15) and SHR (n = 14). Symbols represent mean values, whereas vertical bars represent ± 1 SEM. Abbreviations as in Figs. 1–4. Figure 5. Open in new tabDownload slide Figure 5. Open in new tabDownload slide TEA-sensitive KV currents In preliminary experiments to obtain insights into the basis for the differences in TEA-induced contractions, we tested the effects of TEA on whole cell currents. Because of the presence of both KV and KCa components, it was difficult to interpret these results. However, when the TEA-sensitive current were measured from holding potentials of −20 and −60 mV and compared, there were obvious differences. The TEA-sensitive components of IK20 were significant only at positive test potentials (data not shown) and had the noisy quality characteristic of KCa currents.26 TEA-sensitive IK60, on the other hand, showed the presence of significant currents at test potentials < 0 mV. These currents exhibited slow inactivation characteristic of KV currents. These characteristics of TEA-sensitive currents were qualitatively similar in myocytes from the two groups. These results suggest that TEA-sensitive KV currents are present in native mesenteric myocytes and are consistent with data on K+ channel pharmacology in the literature.27 Therefore, the effects of TEA on Kv currents were determined under conditions in which KCa currents were inhibited as completely as possible by adding 100 nmol/L IbTX to the perfusate as well as by using conventional whole cell recording methods with a pipette solution containing 10 mmol/L BAPTA to reduce intracellular free Ca2+. The results of these experiments are summarized in Fig. 6. The addition of 1 or 10 mmol/L TEA to the perfusate produced a significant reduction in IK (P < .05) in both WKY (Fig. 6A) and SHR myocytes (Fig. 6B). In WKY and SHR, the decrease in IK with 1 mmol/L TEA was statistically significant (P < .05) at voltages > −10 mV, whereas with 10 mmol/L TEA it was statistically significant (P < .05) at voltages > −30 mV. These current-voltage data were fit with a Boltzmann activation function, and the results are summarized in Table 1. The half-maximal activation voltage (V0.5) of IK60 recorded in the presence of TEA was generally shifted to more negative values in WKY and SHR myocytes by TEA. Although there were no significant differences in values of V0.5 between WKY and SHR under control conditions, values in the presence of 10 mmol/L TEA were significantly different (SHR = −7.7 ± 1.1 mV and WKY = −3.4 ± 1.2 mV, P < .05). There were no significant differences in values of slope factor (k) under any conditions. Effects of TEA on IbTX-insensitive IK recorded with 10 mmol/L pipette BAPTA. Currents were recorded from a holding potential of −60 mV in the presence of 100 nmol/L IbTX. Representative currents recorded under control conditions (•) and during the addition of 1 mmol/L (○) and 10 mmol/L TEA (▾) are shown for WKY (panel A; 29.9 pF) and SHR (panel B; 22.9 pF). Peak values of IK were determined and normalized by cell capacitance before averaging. Calibration bars represent 100 pA and 100 msec. In the lower panels, symbols represent means (n = 9 for WKY and n = 10 for SHR), whereas vertical bars represent ± 1 SEM. Solid lines represent a least-squares fit of a Boltzmann activation function to the experimental data points. Other abbreviations as in Figs. 1–5. Figure 6. Open in new tabDownload slide Figure 6. Open in new tabDownload slide The TEA-sensitive IK components were determined by digital subtraction of currents measured before and during the addition of TEA to the perfusate. The current voltage relation of this current component is summarized in Fig. 7. The TEA-sensitive KV component was significantly larger (P < .05) in SHR myocytes compared with WKY over the entire voltage range from −30 to +60 mV at both 1 (Fig. 7A) and 10 mmol/L TEA (Fig. 7B). These data were also fit with a Boltzmann activation function and the results are summarized in Table 1. At 1 mmol/L, the V0.5 of the TEA-sensitive component was significantly more negative in SHR compared with WKY (SHR = 0.7 ± 1.1 mV and WKY = 10.2 ± 1.5 mV; P < .05) but no other group-related differences were present. When measured with 1 and 10 mmol/L TEA in the perfusate, the V0.5 for the TEA-sensitive component was significantly more positive (P < .05) than values of V0.5 for the TEA-insensitive component for WKY and SHR. TEA-sensitive IK for WKY (○) and SHR (•). TEA-sensitive currents were determined by digital subtraction of currents recorded under control conditions (in the presence of 100 nmol/L IbTX) and after the addition of TEA to the perfusate. Representative families of currents inhibited by 1 mmol/L (panel A) and 10 mmol/L TEA (panel B) are shown at the top of each panel for WKY (29.9 pF) and SHR (22.9 pF), and average current-voltage relations are shown at the bottom. Peak values of current were determined and normalized by cell capacitance before averaging. Calibration barsrepresent 100 pA and 100 msec. Symbols represent means (n = 9 for WKY and n = 10 for SHR), whereas vertical bars represent ± 1 SEM. Solid lines in the lower panels represent a least-squares fit of a Boltzmann activation function to the experimental data points. Other abbreviations as in Figs. 1–6. Figure 7. Open in new tabDownload slide Figure 7. Open in new tabDownload slide Discussion These experiments were performed to test the hypothesis that differences in the contribution of K+ current components to whole cell K+ conductance are responsible for the differences in the contractile effect of TEA reported in SHR arteries compared with WKY.7,8 Our results indicate that KCa currents are larger and KV currents smaller in SHR myocytes, and that SHR myocytes have a larger TEA-sensitive KV current compared with WKY. The conclusions concerning the relative contribution of KV and KCa channels to whole cell K+ currents in WKY and SHR myocytes come from two sets of experiments: 1) measurements at different holding potentials, and 2) the effects of iberiotoxin. IK measured under control conditions from a holding potential of −20 mV were larger in SHR myocytes (Fig. 2B). At this holding potential, KV channels are largely inactivated and KCa channels dominate IK.15,25 Thus, IK20 measurements support the conclusion that KCa currents are larger in SHR. This conclusion is also further supported by the observation that the current component inhibited by the KCa selective blocker iberiotoxin is also larger in SHR myocytes (Fig. 3B). The conclusion concerning KV currents is based on the finding that the iberiotoxin-insensitive current (Fig. 3A), and the current inhibited by holding potential (Fig. 5) are both smaller in SHR myocytes. Although KV channels exhibit a lower TEA sensitivity compared with KCa channels24,27 inhibition of KV channels by TEA could also contribute to TEA contractile responses. We modified our basic experimental approach to ensure that KCa currents were inhibited as completely as possible by using the whole cell patch clamp configuration with 10 mmol/L pipette BAPTA in conjunction with iberiotoxin. Under these conditions we found a larger TEA-sensitive KV component in SHR compared with WKY mycoytes. We did not perform measurements over a sufficiently wide range of [TEA] to determine KI values for the two groups, as this was not an original goal of these studies. It should be noted that there were differences in the relative magnitude of Kv currents shown in Fig. 3 and 6. There are several reasons why the data in Fig. 3 and 6 are different. The data in the two figures were not obtained from the same cells because they were recorded using different methods. The data in Fig. 3 were recorded with perforated patch recording methods, whereas those in Fig. 6 were recorded using whole cell recording methods with 10 mmol/L BAPTA. This was done to reduce intracellular Ca2+. We believe that intracellular Ca2+ has a significantly larger inhibitory effect on Kv currents25 in SHR compared with WKY, contributing to the apparent inconsistency in the results. This issue is not addressed further in this study. The above conclusions assume that no other iberiotoxin sensitive current of similar magnitude to maxiK currents are present in these myocytes including small (SK) and intermediate (IK) conductance KCa channels. There have been no reports of SK or IK channels in arterial myocytes and apamin, a selective blocker of some SK channels,28 has no effect on K+ current in mesenteric artery myocytes (data not shown). In addition, single channel studies of KCa in arterial myocytes have reported the presence of only large conductance (maxi) KCa channels.24 An implicit assumption in the interpretation of the patch clamp results is that K+ currents dominate the IK60 and IK20 data. Based on the results of our previous experiments and results in the literature, the only other significant currents likely to be present in mesenteric myocytes are Cl− and nonselective cation currents.25,29 The magnitude of these “background” currents can be estimated from the current remaining after inhibition of KV and KCa currents. The IbTX-insensitive IK20 current, which includes contributions from these background currents as well as from leak currents, was found (Fig. 4) to be quite small and to be similar in WKY and SHR (eg, at +60 mV: WKY = 0.9 ± 0.1 pA/pF, n = 17; and SHR = 0.8 ± 0.1 pA/pF; n = 16). Furthermore, the high input impedance of the smooth muscle cell membrane at resting membrane potential suggests that the K+ counter conductances must be very small.30 Because there are no selective blockers for these “background” currents, we believe that it is reasonable to assume that the IbTX-sensitive and -insensitive currents are good approximations of KCa and KV currents, respectively. The above conclusions regarding the relative contributions of K+ current components to resting IK are consistent with the isometric force responses to TEA summarized in Fig. 1. The KI for TEA inhibition of KCa channels has been reported to be about 0.2 mmol/L, whereas the KI for KV channels has been reported to be about 10 mmol/L.24 Thus, the TEA mediated responses <1 mmol/L in SHR are likely due to the effects of inhibition of the larger KCa current, whereas responses in WKY and SHR >1 mmol/L are likely due to the effects of inhibition of KV channels, again with a larger contribution in SHR. The TEA-sensitive and -insensitive KV components were found to have different V0.5 values for their activation functions. If TEA only inhibited a single class of KV channels, it would be expected that the V0.5 values of the KV-sensitive and -insensitive components would be the same. That they were different suggests that they may represent different KV classes (homotetramers) or different KV combinations (heterotetramers). It is known that KV channels form an extensive gene family with individual members and groups exhibiting differences in gating and pharmacologic properties.27,31 Thus, it is likely that multiple transcripts of KV genes are expressed in these myocytes. This conclusion is supported by published results in several types of smooth muscle that have been shown to express multiple KV transcripts.32–34 A greater KCa current has been observed previously by several investigators in hypertensive myocytes and has been suggested to be a compensatory response to increased blood pressure.7 Also, an increase in maxiK protein expression has been reported in other hypertensive arteries.35 The cause, significance, or relation to hypertension in the SHR of the smaller KV current in this animal model is not clear. A scenario could be envisioned in which the smaller KV current leads to membrane depolarization, an increase in L-type Ca2+ current, and augmented KCa current. Acutely blocking KV current with 4-aminopyridine has been shown to produce these effects.36 A similar situation under chronic conditions could explain several properties previously described in arterial smooth muscle of hypertensives including membrane depolarization at rest,30 increased L-type Ca2+ influx,37 and increased sensitivity to L-type Ca2+ channel blockers.38 It is clear that more information is necessary on the mechanisms associated with the reduced KV current to understand the contribution of these channels to altered excitation-contraction coupling in hypertensive arterial smooth muscle. In conclusion, the larger and more sensitive contractile responses of hypertensive (SHR) arterial smooth muscle to TEA are due to a larger KCa and a smaller total KV channel contribution to resting K+ conductance, and to a larger TEA-sensitive KV current compared with that of normotensive WKY. References 1. 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TI - Differences in K+ current components in mesenteric artery myocytes from WKY and SHR
JO - American Journal of Hypertension
DO - 10.1016/S0895-7061(01)02145-8
DA - 2001-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/differences-in-k-current-components-in-mesenteric-artery-myocytes-from-BWMpxQAhNB
SP - 897
EP - 907
VL - 14
IS - 9
DP - DeepDyve
ER -