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Am J Physiol Heart Circ Physiol 289: H1265-H1276, 2005. First published May 13, 2005; doi:10.1152/ajpheart.00074.2005
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Functional alterations in cerebrovascular K+ and Ca2+ channels are comparable between simulated microgravity rat and SHR

Man-Jiang Xie, Li-Fan Zhang, Jin Ma, and Hong-Wei Cheng

Department of Aerospace Physiology, Fourth Military Medical University, Xi’an, China

Submitted 25 January 2005 ; accepted in final form 9 May 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 DISCUSSION
 GRANTS
 REFERENCES
 
Exposure to microgravity leads to a sustained elevation in transmural pressure across the cerebral vasculature due to removal of hydrostatic pressure gradients. We hypothesized that ion channel remodeling in cerebral vascular smooth muscle cells (VSMCs) similar to that associated with hypertension may occur and play a role in upward autoregulation of cerebral vessels during microgravity. Sprague-Dawley rats were subjected to 4-wk tail suspension (Sus) to simulate the cardiovascular effect of microgravity. Large-conductance Ca2+-activated K+ (BKCa), voltage-gated K+ (KV), and L-type voltage-dependent Ca2+ (CaL) currents of Sus and control (Con) rat cerebral VSMCs were investigated with a whole cell voltage-clamp technique. Under the same experimental conditions, KV, BKCa, and CaL currents of cerebral VSMCs from adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were also investigated. KV current density decreased in Sus rats vs. Con rats [1.07 ± 0.14 (n = 22) vs. 1.31 ± 0.28 (n = 16) pA/pF at +20 mV (P < 0.05)] and BKCa and CaL current densities increased [BKCa: 1.70 ± 0.37 (n = 23) vs. 0.88 ± 0.22 (n = 19) pA/pF at +20 mV (P < 0.05); CaL: –2.17 ± 0.21 (n = 35) vs. –1.31 ± 0.10 (n = 26) pA/pF at +10 mV (P < 0.05)]. Similar changes were also observed in SHR vs. WKY cerebral VSMCs: KV current density decreased [1.03 ± 0.33 (n = 9) vs. 1.62 ± 0.64 (n = 9) pA/pF at +20 mV (P < 0.05)] and BKCa and CaL current densities increased [BKCa: 2.54 ± 0.47 (n = 11) vs. 1.12 ± 0.33 (n = 12) pA/pF at +20 mV (P < 0.05); CaL: –3.99 ± 0.53 (n = 12) vs. –2.28 ± 0.20 (n = 10) pA/pF at +20 mV (P < 0.05)]. These findings support our hypothesis, and their impact on space cardiovascular research is discussed.

postspaceflight cardiovascular deconditioning; hindlimb unloading; hypertension; cerebral vasculature; vascular smooth muscle cells; voltage-gated potassium channels; large-conductance calcium-activated potassium channels; voltage-dependent calcium channels

POSTSPACEFLIGHT ORTHOSTATIC intolerance has been regarded as one of the important adverse effects of microgravity exposure, and effective countermeasures to prevent its occurrence are still lacking (2, 6, 18, 41, 52). It is now well recognized that, in addition to hypovolemia, multiple mechanisms might account for its occurrence (2, 18, 52). Findings from recent bed rest and spaceflight human studies have indicated that the inability to adequately elevate the peripheral vascular resistance and altered autoregulation of cerebral vasculature are important factors in postflight orthostatic intolerance (2, 4, 14, 41). The tail-suspended hindlimb-unloaded (TS) rat model has been widely used to simulate physiological effects of microgravity (28). In the past decade, ground-based studies with TS rats have revealed that simulated microgravity differentially alters the function and structure of cerebral and hindquarter vessels. Attenuated myogenic tone and vasoreactivity and atrophy in hindquarter arteries and arterioles and enhanced myogenic tone and vasoreactivity and hypertrophy in cerebral vessels have been demonstrated (for review, see Ref 49). These findings substantiate in general the hypothesis that redistribution of transmural pressures and flows across and within the arterial vasculature due to the removal of gravity may well initiate region-specific adaptations of vessels in different body regions (11, 18, 49).

There is a blood pressure gradient from the head to the feet in humans at 1 G in the upright posture. All blood pressure gradients are lost at microgravity. Thus blood vessels in dependent body regions are chronically exposed to lower than normal upright 1-G blood pressure, whereas vessels in upper body regions are exposed to higher than normal 1-G blood pressure (18, 41, 49). In rats, it has been postulated that elevated transmural pressures across the cerebral arterial vasculature during chronic head-down tilt by TS would induce functional and structural adaptations in these vessels similar to those induced by hypertension (26, 46, 52). Interestingly, the reported increased myogenic tone (15), enhanced vasoreactivity (54), hypertrophic remodeling (26, 46), and endothelial dysfunction (54) in cerebral vessels of simulated microgravity rats are quite similar to those changes in hypertensive rats (19, 27, 32–34, 39, 47), suggesting that these changes are mainly triggered and maintained by sustained increases in transmural pressures in cerebral vessels. However, the underlying cellular mechanisms need to be further elucidated (49). Given that cytosolic free Ca2+ concentration ([Ca2+]i) might serve as a shared critical signal transduction element in pressure-induced changes in myogenic tone, vasoreactivity, and growth of vascular smooth muscle cells (VSMCs) (5, 17, 20, 42, 48), we have speculated that ion channel mechanisms might be involved (53). Consistent with this speculation, our previous study (13, 53) revealed a smaller voltage-gated K+ (KV) current, with large-conductance Ca2+-activated K+ (BKCa) current showing no significant change, in cerebral VSMCs isolated from 4-wk TS rats.

In the past two decades, most of the work that has been done in the research area of vascular ion channel and hypertension has focused on the KV, BKCa, and L-type voltage-dependent Ca2+ (CaL) channels. Recent studies have revealed that hypertension is associated with a unique, disease-specific remodeling of arterial smooth muscle ion channels, with KV activity being depressed and CaL and BKCa activity enhanced (8, 10). However, patch-clamp evidence for these alterations has been obtained most definitively in VSMCs from small mesenteric arteries of the spontaneously hypertensive rat (SHR) (for review, see Ref. 10). Little of the supporting evidence for pressure-induced ion channel alterations has been obtained from the cerebral arteries of hypertensive rats (see, e.g., Refs. 25, 38, 42–44). In addition, the question of whether the increased Ca2+ influx or the elevated [Ca2+]i in SHR is a primary manifestation of hypertensive phenotype or a consequence of hypertension remains unsolved (1, 31, 38). Also, it is important to know whether the channel remodeling obtained from the SHR (8, 10) could be generalized to other forms of hypertension.

Therefore, we designed the present study to investigate changes in KV, BKCa, and CaL currents and resting membrane potential (Em) of VSMCs isolated from the cerebral arteries of 4-wk simulated microgravity (Sus) rats and to compare them with changes in cerebral VSMCs from adult SHR observed under the same experimental conditions, including the composition of the pipette solution (8, 9). In addition, global [Ca2+]i of cerebral VSMCs isolated from Sus rats was also detected by laser scanning confocal microscopy using the calcium-sensitive dye fluo-3 AM and compared with that from control (Con) rats. The goal of this study was to test the hypothesis that ion channel remodeling involving the triad of KV, CaL, and BKCa similar to that associated with hypertensive rats (8, 10, 25, 38, 43) may occur in cerebral arterial VSMCs during a prolonged exposure to the microgravity environment. Elucidating these similarities might provide further insight into the mechanism of vascular ion channel remodeling associated with genetic and nongenetic hypertension.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
DISCUSSION
 GRANTS
 REFERENCES
 
Animal models. The tail-suspended, hindlimb-unloaded rat model was used to simulate the cardiovascular effect of microgravity. The technique of tail suspension (28), with modification by our laboratory, was described in detail previously (54). The animals were maintained in an about –30° head-down tilt position with their hindlimbs unloaded. The animals received standard lab chow and water ad libitum and were caged individually in a room maintained at 23°C on a 12:12-h light-dark cycle. A total of 24 male Sprague-Dawley rats, 7–9 wk of age and weighing between 200 and 250 g, were randomly assigned into two groups (n = 12/group): 4-wk simultaneous Con and Sus groups. The Con rats in individual cages were treated similarly except for the tail suspension. At the end of a 28-day simulation period, animals from the Con and Sus groups were anesthetized with pentobarbital sodium (50 mg/kg ip) and killed by exsanguination via the abdominal aorta. Brains were rapidly removed and placed in a dissecting dish with 4°C cold physiological salt solution (PSS). The left soleus and tibia were removed, and muscle wet weight and bone length were measured to confirm the efficacy of deconditioning and to monitor any effect on growth.

Ten male SHR and ten male Wistar-Kyoto rats (WKY) 12–14 wk of age were obtained from the Animal Center of the Fourth Military Medical University. Systolic blood pressure was measured by the tail cuff method. Animals were anesthetized with pentobarbital sodium (50 mg/kg ip) and killed by exsanguination via the abdominal aorta. Brains were removed and placed in a dissecting dish with cold (4°C) PSS. All protocols and procedures were reviewed and approved by the Animal Care and Use Committee of the Fourth Military Medical University.

Isolation of arterial smooth muscle cells. PSS contained (in mM) 137 NaCl, 5.6 KCl, 1 MgCl2, 0.42 Na2HPO4, 0.44 NaH2PO4, 4.2 NaHCO3, and 10 HEPES, equilibrated with 95% O2-5% CO2, and pH was adjusted to 7.4 with NaOH. The superior, middle, and basilar cerebral arteries with the circle of Willis (diameter 300–500 µm) were dissected and cleaned of basolateral connective tissues. Enzymatic digestion was carried out essentially as previously described (13, 21). In brief, 1- to 2-mm vessel segments were digested with 4 mg/ml papain (BIB), 2 mg/ml dithioerythritol (Amresco), 1 mg/ml BSA (Sigma), and 5 mM taurine in PSS at 37°C for 18 min. Vessel segments were then transferred to enzyme-free PSS containing 1 mg/ml BSA and 5 mM taurine at room temperature for 10 min and triturated with a flame-polished pipette to disperse VSMCs. Isolated VSMCs were suspended in Ca2+-free PSS containing 1 mg/ml BSA and 5 mM taurine and stored at 4°C for use within 8 h.

Measurement of resting [Ca2+]i. Isolated smooth muscle cells were placed into PSS containing 5 µM fluo-3 AM and incubated for 30 min at 37°C. The fluo-3 AM-loaded cells on coverslips were then washed with the bath solution containing (in mM) 141 NaCl, 4.7 KCl, 1.2 MgCl2, 1.8 CaCl2, 10 glucose, and 10 HEPES, equilibrated with 95% O2-5% CO2 at pH 7.4. After loading, the fluo-3 AM-loaded VSMCs were scanned under a laser confocal microscope (MRC-1024, Bio-Rad) by illuminating with a krypton/argon laser at 488-nm emitted light and capturing the emitting fluorescence at 526 nm. To ensure efficient quantum capture, the VSMCs were placed on the bottom of a recording chamber and images of VSMCs were recorded after 10–20 s when fluorescence intensity became stable. Average fluorescence intensity was used to estimate the resting [Ca2+]i of cerebral VSMCs from Sus rats compared with that of Con rats. To avoid any laser-induced changes in Ca2+ signaling, each myocyte was scanned only once.

Electrophysiological measurements. Electrophysiological measurements were performed as previously described (9, 13, 21). Whole cell currents were recorded with an amplifier (CEZ-2300, Nihon Kohden) and a version interface (Axon Instruments), using patch-clamp techniques. Command-voltage protocols and data acquisition were performed with pCLAMP software (version 8.0, Axon Instruments). Patch pipettes (tip resistance 2–6 M{Omega} when filled with a pipette solution) were fabricated on an electrode puller (Narishige) with borosilicate glass capillary tubing. After a gigaohm seal was achieved, the VSMC was perforated by applying negative suction to the surface of the cell plated onto the bottom of a 2-ml recording chamber. Cell membrane capacitance (Cm) and access resistance (Ra) were estimated from the capacitive current transient evoked by applying a 20-mV pulse for 40 ms from a holding potential of –60 mV to –40 mV. All measurements were performed at room temperature (22–24°C).

Measurement of whole cell K+ currents and Em. Resting Em was measured with the current-clamp configuration of the patch-clamp technique while the cell was held at zero membrane current (21). Whole cell K+ currents were measured with the conventional voltage-clamp configuration (13, 21). The external (bath) solution contained (in mM) 135 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, equilibrated with 95% O2-5% CO2 at pH 7.4 adjusted by NaOH (21). The pipette solution contained (in mM) 143 KCl, 1 CaCl2, 1 MgCl2, 3 EGTA, and 10 HEPES, equilibrated with 95% O2-5% CO2 at pH 7.2 titrated with KOH (9). Current-voltage (I-V) relationships were generated in voltage-clamped cells held at a membrane potential of –70 mV and then stepped in 10-mV increments to +60 mV. Voltage steps were 2 s in duration, and 10-s intervals were allowed between steps. Currents were filtered at 1 kHz and digitized at 4 kHz. Currents during the last 400 ms in each step of three voltage-clamp trials were sampled and averaged before the analysis of current amplitudes. Currents were also normalized to cell capacitance to obtain the current densities. To separate the BKCa and KV currents from the total K+ currents, the following protocol was adapted. First, whole cell currents were recorded in three trials separated by 2 min, and the currents were then recorded 3, 5, and 7 min after the perfusion fluid was changed to contain 1 mM tetraethylammonium (TEA) or 1 mM TEA + 3 mM 4-aminopyridine (4-AP). By subtracting the average currents after each perfusion changing from that before, we obtained the BKCa and KV currents in succession (13, 29).

Measurement of whole cell CaL. Whole cell CaL currents were measured with conventional voltage-clamp configuration as previously described (24, 31, 37, 43). The cell was held at –40 mV and then stepped in 10-mV increments from –30 to +60 mV. Voltage steps were 250 ms in duration, and 2-s intervals were allowed between steps. Currents were filtered at 0.5 kHz and digitized at 4 kHz. Nonspecific membrane leakage and residual capacitive currents were subtracted with the p/4 protocol (31, 37). Currents were sampled and averaged while the current amplitude was stabilized. To increase unitary currents, Ba2+ replaced Ca2+ as charge carrier and the divalent cation concentration was elevated in the bath solution (37). Two kinds of external solutions, i.e., solutions A and B, were used. Solution A was used while making a gigaohm seal between the recording pipette and cell surface. It contained (in mM) 130 NaCl, 5.4 KCl, 1 MgCl2, 10 BaCl2, 10 HEPES, and 10 glucose, equilibrated with 95% O2-5% CO2 at pH 7.4 titrated with NaOH. After a seal of 2 G{Omega} was obtained, the perfusion fluid was changed to solution B during current recording. Solution B contained (in mM) 75 Tris-Cl, 50 BaCl2, 10 HEPES, and 10 glucose, equilibrated with 95% O2-5% CO2 at pH 7.4 titrated with Tris base. To minimize outward K+ current, Cs+ instead of K+ was used in the pipette solution. It contained (in mM) 150 CsCl, 1 MgCl2, 10 EGTA, 5 HEPES, 5 Na2ATP, and 5 Na2 creatine phosphate, equilibrated with 95% O2-5% CO2 at pH 7.2 titrated with CsOH. The dihydropyridine Ca2+ channel agonist BAY K 8644 and antagonist nifedipine (both Sigma) were prepared as a stock solution in 100% ethanol and diluted to 5 or 0.1 µM in solution B before use.

To obtain the I-V curve of CaL, the current densities were plotted against the corresponding command potentials. The conductance (G) was obtained with the equation G = Ipeak/(Vrev – V), where Ipeak is the peak amplitude of current, V is the command potential, and Vrev is the reversal potential. To describe the voltage dependence of activation of CaL channels, conductance relative to its maximum value was fitted with a Boltzmann function of the form P = 1/{1 + exp[(Vh – V)/k]}, where P is the relative conductance normalized by the maximal conductance, Vh is the potential for half-maximal activation, and k is a steepness factor (Boltzmann coefficient). To gain further insight into the time- and voltage dependence of inactivation of CaL channels, steady-state inactivation was determined with a standard two-step protocol (24, 31, 37, 43). The protocol consisted of a 4.8-s conditioning prepulse to voltages from –100 mV to +40 mV in steps of 10 mV followed by a 200-ms test potential of +30 mV with a fixed 10-ms interpulse interval at the holding potential of –40 mV. Pulses were applied every 15 s. The steady-state inactivation was measured as the ratio I/Imax, where Imax is the maximum current amplitude during the test pulse after the most hyperpolarizing prepulse (–100 mV). The steady-state inactivation curve was drawn by fitting the data to a Boltzmann function of the form P = 1/{1 + exp[(V – Vh)/k]}, where P is the relative amplitude.

Statistical analysis. The data are expressed as means ± SE. A one-way ANOVA was used to determine the overall differences in K+ and Ca2+ current density between different groups. Student’s t-test was used to determine the significance of differences in body weight, soleus wet weight, tibia length, Cm, and resting Em between different groups. A value of P < 0.05 was considered to be statistically significant.

RESULTS


Physical characteristics of experimental animals. After the 4-wk tail suspension, there were no significant differences in either final body weight (Sus 356 ± 10.4 vs. Con 369 ± 9.6 g) or left tibia length (Sus 38.7 ± 1.2 vs. Con 39.8 ± 1.0 mm) between Sus and Con groups, suggesting a normal growth rate during simulated microgravity. However, after 4-wk tail suspension, the soleus muscle wet weight of Sus rats (41 ± 2.7 mg) was 70% less than that in Con rats (148 ± 5.0 mg) (P < 0.05), indicating the deconditioning effect of simulated microgravity.

The body weights of SHR (321 ± 11.0 g) and WKY (333 ± 13.5 g) were not significantly different, whereas the systolic blood pressure of SHR (205 ± 10.0 mmHg) was significantly higher than that of WKY (110 ± 8.0 mmHg) (P < 0.05).


Configuration, resting [Ca2+]i, and electrophysiological characteristics of cerebrovascular myocytes isolated from Sus and Con rats. The isolated myocytes from Sus and Con rats all showed a fusiform shape and a smooth surface. They were ~80–120 µm in length and 7–10 µm in diameter and contracted in response to superfusion with a bath solution containing 1 mM serotonin.

After a 4-wk simulated microgravity, the average fluorescence intensity of cerebral VSMCs from Sus rats (51.7 ± 9.6 U, n = 10 cells) increased by 85% (P < 0.05) compared with that of Con rats (28.6 ± 6.5 U, n = 12 cells), indicating that the resting [Ca2+]i in cerebrovascular myocytes from Sus rats was significantly higher than that in Con rats. As summarized in Table 1, there were no significant differences in Cm and Ra between Con and Sus rats, whereas the resting Em of cerebral VSMCs from Sus rats was more positive (P < 0.05), i.e., was depolarized compared with that of Con rats.


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  		Table 1. Membrane capacitance, access resistance, and resting membrane potential of cerebral VSMCs isolated from Sus and Con rats and SHR and WKY

 

Functional alterations in cerebrovascular KV and BKCa channels due to simulated microgravity are similar to those in SHR. Figure 1, A–C, illustrates typical records of whole cell K+ currents in cerebral VSMCs isolated from a Con and a Sus rat, showing the alterations due to simulated microgravity. A family of voltage-dependent outward K+ currents was elicited by depolarizing the myocyte from a holding potential of –70 mV to a series of command potentials ranging from –60 to +60 mV. Whole cell K+ currents showed time- and voltage-dependent outward currents and increased noise to higher positive potentials. Extracellular application of 1 mM TEA significantly reduced the amplitude of the K+ currents and diminished the current noise associated with higher positive command potentials. These properties suggest that these outward currents blocked by TEA were currents through BKCa channels (20, 29). Application of 3 mM 4-AP in the presence of 1 mM TEA further suppressed the currents, attenuated current decay, and depolarized the myocytes. These are the characteristics of KV currents (20, 29). The mean I-V curves for total K+ currents and those after BKCa and KV blockades of the cells are shown in Fig. 1D. The I-V relationships were further expressed in terms of current density, calculated with the estimated Cm, and summarized in Fig. 3A. Although there were no significant differences in values of total current densities between Con and Sus rats (P > 0.05), the BKCa and KV current densities showed significant differential alterations. After 4 wk of simulated microgravity, the BKCa current density was significantly larger (P < 0.05), whereas the KV current density was significantly smaller, in Sus compared with Con rats (P < 0.05).



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  		Fig. 1. K+ channel currents in cerebral arterial myocytes of control (Con) and simulated microgravity (Sus) rats. Representative families of outward currents were recorded in the absence (A) and presence of tetraethylammonium (TEA; B) or TEA + 4-aminopyridine (4-AP; C) from a holding potential of –70 mV for a Con (21.7 pF) and a Sus (21.0 pF) myocyte. Current-voltage (I-V) curves for each condition are shown in D.

 


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  		Fig. 3. Comparison of changes of K+ channel function in cerebral arterial myocytes between Sus rats (A) and SHR (B). Large-conductance Ca2+-activated K+ (BKCa) and voltage-gated K+ (KV) currents were obtained by digital subtraction of currents recorded before and after the addition of 1 mM TEA or 1 mM TEA + 3 mM 4-AP. Current density was obtained by normalization to cell capacitance. Values are means ± SE. *P < 0.05 between groups by ANOVA.

 
Of interest is that the observed alterations in electrical properties and KV and BKCa channel functions in cerebrovascular myocytes due to simulated microgravity are quite similar to those in myocytes isolated from the cerebral vasculature of SHR. Data regarding electrical properties of cerebrovascular myocytes from SHR and WKY are summarized in Table 1. There were no significant differences in Ra between SHR and WKY, whereas the resting Em was more positive (depolarized) in SHR than WKY (P < 0.05). Furthermore, Cm was significantly greater in SHR than WKY, suggesting a larger surface area of cerebral VSMCs in SHR. Figure 2 illustrates whole cell K+ currents of two cerebral VSMCs, one each from a WKY and a SHR, showing the blocking effects of TEA and 4-AP. Figure 3B, depicting the I-V relationship in terms of current density, shows that KV and BKCa current densities significantly decreased and increased (P < 0.05), respectively, in SHR compared with WKY. Current density data under +20 mV command potential are further summarized in Fig. 7 to indicate similarities in KV and BKCa functions between Sus rats and SHR.



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  		Fig. 2. K+ channel currents in cerebral arterial myocytes of Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Membrane capacitance (Cm) for the WKY and SHR myocytes recorded was 21.9 and 23.3 pF, respectively. A–D: see Fig. 1, A–D.

 


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  		Fig. 7. Similarity in functional alterations in KV, BKCa, and L-type voltage-dependent Ca2+ (CaL) channels of cerebrovascular myocytes between simulated microgravity (A) and spontaneously hypertensive (B) rats. A: K+ and Ca2+ current data were obtained under +20 and +10 mV command potential, respectively. B: K+ and Ca2+ current data were obtained under +20 mV. Values are means ± SE. *P < 0.05 compared with respective controls.

 

Augmented CaL channel current in cerebrovascular myocytes due to simulated microgravity is similar to that in SHR. Figure 4 illustrates typical records of whole cell Ca2+ currents recorded in VSMCs isolated from a Con and a Sus rat, showing augmented CaL currents due to simulated microgravity in the absence or presence of BAY K 8644. Peak inward current was recorded at +10 mV for both Con and Sus myocytes. Application of 5 µM BAY K 8644 caused the current amplitude to increase about twofold and to activate more steeply and at more negative potentials, whereas application of 0.1 µM nifedipine suppressed the inward currents almost completely. These properties suggest that the inward currents recorded were Ba2+ currents through L-type Ca2+ channels (24, 31, 37, 43). The inward currents were larger at all command potentials in Sus compared with Con myocytes, whether or not the CaL was activated by BAY K 8644 (Fig. 4D; see Fig. 6A for I-V relationship expressed in terms of current density, calculated with estimated Cm). The CaL current densities of the cerebral VSMCs from Sus rats were significantly larger than those from Con rats (P < 0.05). The mean peak current densities at +10 mV in Sus and Con rats were –2.2 ± 0.2 (n = 35 cells) and –1.3 ± 0.1 (n = 26 cells) pA/pF, respectively. Application of 5 µM BAY K 8644 caused the maximum inward current density to increase by 1.5-fold, with no shifting in maximal activation voltage. In the presence of BAY K 8644, mean peak current densities in myocytes from Sus and Con rats were –3.0 ± 0.3 (n = 25 cells) and –2.2 ± 0.3 (n = 16 cells) pA/pF, respectively, indicating an approximately twofold increase in sensitivity to BAY K 8644 in Sus over Con myocytes (P < 0.05).



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  		Fig. 4. Ca2+ channel currents in cerebral arterial myocytes of Con and Sus rats. Representative families of inward currents were recorded without Ca2+ entry modulators (A) and in the presence of BAY K 8644 (B) or nifedipine (C) from a holding potential of –40 mV for a Con (20.3 pF) and a Sus (22.2 pF) myocyte. I-V curves for each condition are shown in D.

 


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  		Fig. 6. Comparison of changes of Ca2+ channel current densities in cerebral myocytes between Sus rats (A) and SHR (B). Values are means ± SE. *P < 0.05 compared with respective control by ANOVA. For further details, see Figs. 4 (Sus vs. Con) and 5 (SHR vs. WKY).

 
It is also of great interest to note that the increase in CaL function of cerebrovascular VSMCs due to simulated microgravity is quite similar to that in cerebral artery myocytes from SHR (Figs. 5 and 6B). As previously reported for stroke-prone SHR cerebral vessels (43), we have observed augmented CaL current in cerebrovascular myocytes of SHR compared with WKY. However, the peak inward current was at +20 mV in SHR and WKY. Peak current densities at 20 mV in SHR and WKY cerebrovascular myocyte were –4.0 ± 0.5 (n = 12 cells) and –2.9 ± 0.2 (n = 10 cells) pA/pF, respectively (P < 0.05). Application of 5 µM BAY K 8644 caused the maximum inward current density to increase by twofold with no shifting in maximal activation voltage. With BAY K 8644, mean peak current densities in myocytes from SHR and WKY were –6.0 ± 0.7 (n = 11 cells) and –3.9 ± 0.4 (n = 8 cells) pA/pF, respectively (Fig. 6B), indicating an ~1.5-fold increase in sensitivity to BAY K 8644 in SHR over WKY myocytes (P < 0.05). CaL current density data under +10 and +20 mV command potential are further summarized in Fig. 7, to indicate the similarity in the alteration of CaL function between Sus rats and SHR.



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  		Fig. 5. Ca2+ channel currents in cerebral arterial myocytes of WKY and SHR. Cm for the WKY and SHR myocytes recorded was 22.7 and 21.6 pF, respectively. A–D: see Fig. 4, A–D.

 
The activation and steady-state inactivation curves of cerebrovascular CaL channels and the mean values of the Boltzmann fit parameters for both Sus-Con and SHR-WKY groups are summarized in Fig. 8 and Table 2. No significant differences were noted in the voltage dependence of either current activation or inactivation between Sus and Con or between SHR and WKY myocytes. However, the set of activation and inactivation curves for Sus and Con myocytes was more negative than the set for SHR and WKY myocytes (Fig. 8). Boltzmann fit parameters (Vh and k) were not significantly different between Sus and Con or between SHR and WKY myocytes (Table 2).



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  		Fig. 8. Steady-state inactivation and activation curves of Ca2+ currents in cerebrovascular myocytes from 4-wk Sus and Con rats (A) and SHR and WKY (B). Steady-state inactivation curves were drawn by fitting the data of relative amplitude (I/Imax) and command potential obtained through a standard double-pulse protocol to the Boltzmann function. Activation curves were drawn by fitting the data of conductance relative to its maximum (G/Gmax) and the command potential to the Boltzmann function. The mean value of the fitting parameters for inactivation and activation are summarized in Table 2. Values are means ± SE.

 

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  		Table 2. Boltzmann fit parameters for Sus and Con and SHR and WKY cerebrovascular myocytes

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of the present study are that CaL and BKCa currents are significantly larger and KV current is significantly smaller in myocytes isolated from cerebral arteries of Sus rats compared with Con rats. In addition, the larger inward Ca2+ current in cerebral VSMCs due to simulated microgravity is further supported by the increased sensitivity to the CaL agonist BAY K 8644 and the elevated resting [Ca2+]i in these cells. To our knowledge, this is the first demonstration that simulated microgravity-induced alterations in cerebral vascular ion channels involving the triad of CaL, BKCa, and KV are similar to those reported for hypertensive rats (Fig. 7). In the present study, cerebral arterial CaL, BKCa, and KV channels of adult SHR and WKY were also investigated under the same experimental conditions. The results thus obtained are consistent with the reported alterations in relevant ion channels of cerebral arterial myocytes from both genetically (25, 42, 43) and nongenetically (38, 44) hypertensive rats. These data also support our hypothesis that ion channel remodeling in myocytes similar to that associated with hypertension may occur and play a role in functional and structural adaptation of cerebral vessels during microgravity.

Ca2+ channel currents of myocytes. In the present study, we recorded Ca2+ channel currents of myocytes isolated from rat basilar, superior, and middle cerebral arteries with the circle of Willis. Because macroscopic inward currents though Ca2+ channels in cerebral arterial myocytes are relatively small, they may be obscured by leak and other outward currents. As in previous work (24, 31, 37, 43), the divalent cation Ba2+ was used as charge carrier, cells were dialyzed with 150 mM CsCl, activities of BKCa and KATP channels were reduced by buffering intracellular free Ca2+ concentration with 10 mM EGTA, and ATP and creatine phosphate were added to reduce the rate of "rundown" of CaL currents. The leak and residual capacitive currents were subtracted with a p/4 protocol (31, 37). We found no reliable evidence for the existence of a T-type Ca2+ current in rat cerebral arterial myocytes, which is in accord with the view that the expression of T-type Ca2+ channels has been primarily found in arterial myocytes from embryonic and neonatal rats (40). The voltage dependence of kinetics of activation and inactivation of CaL channels has been verified and described by the good fit to the Boltzmann function, with parameters Vh and k being close to those reported for rat cerebral VSMCs (37, 43). In the present study, no significant differences were found between Sus and Con myocytes in either activation or inactivation of CaL, suggesting that simulated microgravity-induced functional changes in CaL might not involve changes in the kinetics of these processes. This is consistent with the result that the voltage dependence of activation was not affected by blood pressure (38).

K+ channel currents of myocytes. With respect to the alterations in K+ channel function, the present study showed that the KV current is smaller and the BKCa current is larger in cerebral arterial myocytes of rats after a 4-wk simulated microgravity. These changes are similar to those of SHR observed under the same experimental conditions in the present study and to those in previous work with different vascular beds (9, 10, 23, 25, 27). However, in our previous study (13), the BKCa current of cerebral artery myocytes did not show significant change after a 4-wk simulated microgravity. The reason for this discrepancy is apparently related to the difference in composition of pipette solution used. Because the aim of the present study was to examine whether ion channel remodeling involving the triad of KV, BKCa, and CaL channels may occur in cerebral arterial myocytes after a midterm simulated microgravity, we had to choose a pipette solution basically according to that used by Cox et al. (9), so as to make comparisons of changes in both KV and BKCa currents under a very close experimental condition, whereas in our previous work (13) the aim was just to test the hypothesis that the function of K+ channels of cerebral and hindquarter arterial VSMCs are differentially activated during simulated microgravity. Thus, in that experiment (13), we used a pipette solution according to Ref. 21. As pointed out by Cox (7, 8), the K+ currents recorded are influenced by the composition of the pipette solution, particularly the Ca2+ concentration. Ca2+ influx and/or intracellular Ca2+ per se influences K+ currents of the arterial myocytes from SHR. Cox further pointed out that "it appears that under conditions that reflect physiological ones, BKCa currents are larger and KV currents are smaller in arterial SMCs from hypertensive animals" (7). Examples showing differences in BKCa results associated with the methods used are listed and compared in Table 2 of Ref. 7. Evidence supporting the idea that cytosolic Ca2+ concentration in arterial smooth muscle cells is a critical regulator of the levels of BKCa and KV currents has been provided in well-designed experiments (7–10). Using the Maxchelator program (http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm), we have calculated that the Ca2+ concentration in the pipette solution we used was ~ 200 nM, which approximates the physiological Ca2+ concentration within VSMCs, whereas in our previous study (13), the pipette solution did not contain CaCl2 and its Ca2+ concentration was very low. Thus it seems that differences in the composition of the pipette solution can account for the discrepancy in results regarding BKCa channels between the present study and our previous study (13). The activation of BKCa channels by [Ca2+]i is well established. It involves the Ca2+-binding domain in the carboxy terminus of BKCa channels that transduces increased Ca2+ to an increase in open probability of BKCa channels (8). Although it has been shown that Ca2+ influx, intracellular Ca2+ release, and direct application of Ca2+ to the intracellular surface of KV channels in excised patches can inhibit KV currents, the Ca2+-dependent mechanism for the inhibition of KV channels remains less clear (8). After 4 wk of simulated microgravity, the mean resting Em of cerebral arterial myocytes was more positive than that of Con myocytes, which is similar to that for the cerebral artery myocytes of SHR observed in the present study as well as in previous work (3, 16, 23, 42). It has been speculated that a larger inhibitory effect on KV channels and a greater activating effect on BKCa channels by Ca2+ concentration might account for the new steady-state Em of cerebral artery myocytes during adaptation to chronic hypertension. Although our previous study (13) and the present study did not show significant changes in Cm of Sus myocytes, SHR myocytes exhibited a significantly larger Cm, which is consistent with that reported in Ref. 43.

Physiological significance. Cerebrovascular changes during real or simulated microgravity have been postulated to be basically an upward autoregulation in function and structure of cerebral vessels to adapt to altered local circumferential wall stress (18, 49). In addition to cerebral vascular changes (15, 26, 46, 49, 54) similar to those in hypertensive rats (19, 32–34, 47), simulated microgravity also induces an increase in cerebral vascular resistance from the beginning of exposure, which further suggests the autoregulatory nature of the cerebral vascular adaptation (45). The significance of the present findings should be considered at first in light of recent progress in elucidating the pivotal role of the triad of CaL, BKCa, and KV channels in mediating and modulating vascular autoregulation under physiological conditions and during the pathogenesis of vascular diseases (3, 8, 10, 16, 23, 29, 48). Cerebral arterial tone and vascular resistance are maintained and regulated by the influx of Ca2+ through a small number of CaL channels (37). In cerebral arteries, the open state probability of such channels is very sensitive to Em. A small change of Em in this range will have a profound effect on Ca2+ influx into VSMCs (24). The development of myogenic tone in response to an increase in transmural pressure is associated with a graded depolarization of the membrane of VSMCs. Thus Em is a key determinant that regulates Ca2+ influx through CaL channels, and K+ channels are in turn one of the primary determinants that maintain and regulate Em. Among the four types of K+ channels expressed in vascular myocytes, at least voltage-gated KV and high-conductance, voltage- and Ca2+-sensitive BKCa channels are highly implicated in the regulation of vascular tone (10, 29). KV channels are important regulators of smooth muscle Em (20, 29). One important role is to dampen excitation by providing a mechanism of hyperpolarization in response to depolarizing stimuli. A second important role of KV is its contribution to the maintenance of resting Em (29). BKCa channels are activated by membrane depolarization, particularly by focal increases of subsarcolemmal Ca2+ (i.e., calcium sparks and waves) (22). BKCa channels are thought to represent a negative-feedback mechanism that limits active vasoconstriction to pressure increase and other vasoconstrictor stimuli (20, 22, 25, 29, 35).

If blood pressure remains high, the myogenic response may amplify the initial rise in blood pressure by increasing vascular tone and may ultimately lead to structural remodeling of vessels (12). In addition to its role in excitation-contraction coupling, [Ca2+]i also serves as a critical signal transduction element in the regulation of growth and/or proliferation of arterial myocytes (5, 17, 42, 48). For example, in Dahl salt-sensitive hypertensive rats, membrane depolarization, decreased KV currents, and elevated [Ca2+]i in cerebral arterial myocytes are coupled to the activation of the transcription factor (cAMP response element bending protein) and an increased expression of the immediate-early gene c-fos (42). Furthermore, K+ channel expression programs correlated with distinct patterns of Ca2+ signaling in proliferating VSMCs have been suggested (30). Therefore, when blood pressure is at a sustained high level, complex interactions among CaL, BKCa, and KV channels in arterial VSMCs may be disturbed and remodeled, and a new equilibrium will be achieved again. Patch clamp evidence demonstrating augmented activity of CaL and BKCa channels and depressed activity of KV channels has been provided in arterial myocytes from mesenteric (8, 9, 31), renal (27), and cerebral (25, 35, 43) arteries. Increased expression of the CaL protein and/or increased open state probability of the CaL channel have been suggested to be responsible for the functional upregulation of CaL (8, 10). Membrane depolarization has been suggested to be a potential signal involved in high-blood pressure-induced upregulation of the vascular CaL channel protein {alpha}1C (36). The KV1.x genes are differentially expressed between WKY and SHR. Upregulation of mRNA encoding KV{alpha}1.2 and increases in KV{alpha}1.3 and KV{beta}1.1 at the transcript level were found in SHR systemic arteries (10). The paradox of increased gene expression of KV with a depressed current of KV has been explained by the greater inhibitory effect of Ca2+ influx and/or [Ca2+]i per se on KV currents in arterial myocytes of SHR (7, 8, 10). Ion channel remodeling has also been thought to be involved in pulmonary hypertension (48). The defect in K+ channel expression has been proposed as a unique mechanism involved in the pathogenesis of primary pulmonary hypertension (48). It has been speculated that a greater BKCa channel expression and an increased open state probability in response to Ca2+ sparks in arterial myocytes are among the mechanisms underlying the augmented activity of BKCa channels in VSMCs of hypertensive rats (7, 8, 10, 22). A major question of whether the ion channel alterations in VSMCs are the cause or the consequence of hypertension remains to be solved. Some findings suggest that channel alterations are secondary to changes in blood pressure (27, 38). However, increased activity of CaL channels in mesenteric VSMCs isolated from young but not adult SHR has been reported, suggesting that altered CaL channel function is genetically regulated and occurred in the prehypertensive stage (31). Nevertheless, the present study supports the idea that simulated microgravity-induced changes in channel function appear to be a consequence of the sustained elevation in transmural pressure across the cerebral vasculature during simulated microgravity. Although the present comparative study used different strains, it seems that the main findings might not be influenced by the strain factor. From the relevant references, we know that main conclusions regarding vascular adaptation, including ion channel remodeling, are drawn from experiments using various strains of rats, and even other mammalian species (3, 12, 16, 23). For example, similar alterations in K+ and Ca2+ channel functions in deoxycorticosterone acetate-hypertensive and SHR vascular myocytes have been shown by a comparison using Sprague-Dawley and WKY rats as their respective controls (27).

Practical implications. As in the case of hypertension, adaptational changes in cerebral vessels during microgravity have been speculated to play a role in preventing pressure-induced damage to the microvasculature, particularly cerebral congestion and edema and possibly stroke (19, 34, 46, 51, 54). However, adaptational changes in the cerebral arterial vasculature might also contribute to postspaceflight orthostatic intolerance due to an impairment in the autoregulation of cerebral vasculature (14, 41, 49, 54). In addition, cerebrovascular ion channel remodeling during microgravity exposure, particularly during a prolonged exposure to microgravity, would be expected to have a possible adverse effect on cerebral vasculature. A sustained increase in functional Ca2+ channel activity could lead to the hypertension-associated vasculopathic condition called arteriolosclerosis (see Ref. 38). This might be further aggravated by a possible increase in activities of the local renin-angiotensin system in arterial wall tissues of cerebral vessels in responding to the sustained high transmural pressure during a prolonged existence in microgravity (50). An increase in gene and protein expression for both angiotensinogen and angiotensin II type 1 receptor (AT1R) has been demonstrated in cerebrovascular wall tissue of the simulated microgravity rat (50). Recent evidence also suggests that in SHR the pathophysiological alterations are secondary to changes in blood pressure or distal to an angiotensin-triggered event (27). It has been shown that retrovirally mediated delivery of AT1R antisense into prehypertensive rat pups may prevent the onset of pathophysiological alterations, including K+ channel alterations observed in hypertension (27). It seems that this potential adverse effect should not be overlooked, because an Earth-like distribution of transmural pressure cannot be restored and maintained by current exercise-based countermeasures and some additional proposals (6, 18, 49). In this regard, the genetic aspect of astronaut hypertension perhaps also merits attention, because studies with SHR have raised the problem of a genetic link of vascular ion channel remodeling (1, 31).

In conclusion, previous work has provided evidence indicating that simulated microgravity-induced adaptational changes in the cerebral arterial vasculature are quite similar to those reported for genetically and nongenetically hypertensive rats (26, 46, 49, 54). In the present study, simulated microgravity induced increased CaL and BKCa currents and decreased KV current associated with membrane depolarization in cerebral arterial myocytes, which are comparable with those observed in SHR myocytes under the same experimental conditions. These data suggest that cerebrovascular adaptation to microgravity is mediated or modulated by channel remodeling in vascular myocytes. The impact of these findings on the health of astronauts and countermeasures in future spaceflight warrants further investigation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 DISCUSSION
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REFERENCES
 
This study was supported by the National Natural Science Foundation of China (Grant Nos. 30170355 and 30200090) and a grant from the Graduate School of the Fourth Military Medical University (to M.-J. Xie).


   ACKNOWLEDGMENTS
 
We thank Deng Jing-Mao for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Li-Fan Zhang, Dept. of Aerospace Physiology, Fourth Military Medical Univ., Xi’an 710032, China (e-mail: zhanglf{at}fmmu.edu.cn)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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J.-H. Xue, L.-F. Zhang, J. Ma, and M.-J. Xie
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