Role Of Arterial Ion Channel Mechanism In Simulated Microgravity-Induced Vascular Adaptation

All gravitational blood pressure gradients will disappear during microgravity exposure. Thus, a redistribution of transmural pressures and flows across and within the arterial vasculature is induced by the removal of gravity. Therefore, in humans, 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. Our previous ground-based studies using hindlimb unloading model have revealed that simulated microgravity alters differentially function and structure of the cerebral and hindquarter vessels: for hindquarter arteries,attenuated myogenic tone and vasoreactivity, and atrophy; and for cerebral vessels, enhanced myogenic tone and vasoreactivity, and hypertrophy have been demonstrated. On the basis of the findings from relevant ground-based and spaceflight studies reported recently, we have raised the "peripheral effector mechanism hypothesis" to explain the inability to adequately elevate the peripheral vascular resistance and altered autoregulation of cerebral vasculature during orthostatic challenges in astronauts and cosmonauts postflight. We further showed that the cellular and molecular mechanisms regarding vascular adaptation to microgravity may involve vascular local renin-angiotensin system (L-RAS) and the ion channel mechanism of vascular smooth muscle cell (VSMC). With regard to the latter, our previous work have reported that the large conductance calcium-activated potassium channel (BKca) and voltage-dependent potassium channel (Kv) current densities of VSMCs of hindquarter (saphenous) arteries of rats increased after 1- and 4-wk simulated microgravity, BK_Ca and K_v current densities of VSMCs in small mesenteric arteries of rats also increased after 4-wk simulated microgravity. While the BK_Ca and K_v current densities of VSMCs in cerebral arteries of rats decreased after 1- and 4-wk simulated microgravity in succession. In addition, the change of Ca_L of VSMCs in small mesenteric arteries of rats may also contribute to the depressed vasoreactivity of mesenteric arteries after simulated microgravity. We have not known if BK_Ca and K_v current densities of VSMCs in small mesenteric arteries of rats change after 1 wk of simulated microgravity and if Ca_L channel function in cerebral arterial VSMCs of rats changes after simulated microgravity. It is well known that in both genetic and nongenetic hypertensive animals, sustained elevation in blood pressure isassociated with an upregulation of CaL and/or loss of Kv, and overexpression of BKca in VSMCs. In simulated microgravity rats, it remains unknown whether elevated transmural pressure across the cerebral arterial vasculature during chronic head-down tilt by hindlimb unloading would induce a change in the triad of Kv, BKca, and CaL in these vessels similar to those induced by hypertension?Three separate protocols were carried out in this study.1) The aim of protocol 1 was to elucidate the changes in BKCa and Kv channels of small mesenteric arterial VSMCs of simulated microgravity rats. BKca and Kv channels currents of small mesenteric arterial VSMCs of 1 -wk simulated microgravity rats were recorded using whole-cell patch clamp technique; Furthermore, the protein expression of BKca channel in small mesenteric arteries of 1- and 4-wk simulated microgravity rats was analyzed by Western blotting.2) The aim of protocol 2 was to elucidate the changes in BKca, Kv, and CaL channels of cerebral arterial VSMCs of simulated microgravity rats. At first, the level of cytosolic free Ca2+ ([Ca2+]j) of cerebral arterial VSMCs of 1- and 4-wk simulated microgravity rats was examined by the laser-scanning confocal microscopy with calcium-sensitive-dye Fluo-3/AM as fluorescent probe. Then BKca, Kv, and CaL channel currents were recorded by whole-cell patch clamp technique. Furthermore, the single-channel activity of BKCain cerebral arterial VSMCs of 1- and 4-wk simulated microgravity rats was recorded by both cell-attached and in-side-out modes. Finally, the protein expression of BKca channel in cerebral arteries of 1- and 4-wk simulated microgravity rats was analyzed by Western blotting.3) The aim of protocol 3 was to observe the changes of BKca, Kv and CaL channels in cerebral VSMCs of spontaneous hypertensive rat (SHR).The BKca, Kv, and CaL channel currents of cerebrovascular VSMCs of SHR and WKY rats were recorded by whole-cell patch clamp technique and then compared. Same conditions for recording were kept for both SHR/WKY and SUS/CON groups.The main findings of the present work are as follows:1. Simulated microgravity induced the changes in BKc? and Kv channels of small mesenteric arterial VSMCs of ratsAs compared with the simultaneous control rats, the VSMCs in small mesenteric arteries showed more negative membrane potentials and Kv and BKca current densities increased significantly after 1-wk simulated microgravity [BKca, 2.96 ± 0.51 (n=13) vs. 1.14 ± 0.40 (n=12) pA/pF at +20 mV, P<0.05; Kv, 3.18 ± 0.51 (n=12) vs. 1.52 ± 0.26 (n=9) pA/pF at +20 mV, P<0.05]. Furthermore, Western blotting showed that the protein expression (expressed in densitometric volume) of BKca channel in small mesenteric arteries increased significantly after 1- and 4-wk simulated microgravity [1 wk, 18595 ± 455 vs. 9406 ± 156, P<0.05; 4 wk, 18893 ± 190 vs. 10282 ± 627, P<0.05]. These findings suggest that after 1 wk of simulated microgravity, the function of Kv and BKca channel in small mesenteric arterial VSMCs of rats is enhanced and the protein expression of BKca channel is also increased, and within the time frame (4 wk) of our study, these enhancements would remain at this level.2. Simulated microgravity induced the changes in Kv, BKca and C3l channel in cerebral arterial VSMCs of ratsThe results from laser scanning confocal microscopy demonstrated thatthe fluorescence intensity (expressed in U) of cytosolic free Ca2+ wassignificantly higher in cerebrovascular myocytes after 1-wk and 4-wksimulated microgravity as compared with their respective control rats [1 wk,58.4 ± 7.8 (n=5) vs. 31.5 ± 5.8 (n=6), P<0.05; 4 wk, 51.7 ± 9.0 (n=6) vs. 28.6± 6.0 (n=4), P<0.05]. The whole-cell patch clamp current recording showedthat after a short-term (1 wk) and mid-term (4 wk) simulated microgravity,cerebral arterial VSMCs showed more depolarized membrane potentials andthe activities of both BKca and CaL channels current densities significantlyincreased as compared with that of respective controls [1 wk: BKca, 2.34 ±0.46 (n=15) vs. 1.16 ± 0.20 (n=13) pA/pF at +20 mV, P<0.05; CaL, -2.47 ±0.26 (n=23) vs. -1.52 ± 0.19 (n=15) pA/pF at +10 mV, P<0.05. 4 wk: 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]. CaLchannel was more sensitive to the dihydropyridine calcium channel agonistBay K 8644. However, no significant changes were found in the dynamics ofCaL activities as assessed by channel activation/inactivation experiments withBoltzmann fitting after 1- and 4-wk simulated simulate microgravity. Itshowed be noted that the Kv current density in cerebral arterial VSMCs of1-wk simulated microgravity rat showed changes depending on the testingpotential: it was larger at -20-+20 mV and became smaller at +50~+60 mV ascompared with the controls. After 4-wk simulated microgravity, the currentdensity of Kv channels in cerebral arterial VSMCs significantly decreased[1.07 ± 0.14 (n=22) vs. 1.31 ± 0.28 (n=16) pA/pF at +20 mV, P<0.05]. Thecell-attached and in-side-out mode single-channel current recording showedenhanced single-channel activities of BKCain cerebral arterial VSMCs activity: open (Po) and the mean open time (7b) of BKCa in cerebral arterial VSMCs significantly increased after 1-wk and 4-wk microgravity as compared with their control respectively [for example, in cell-attached mode and at +40 mV, 1 wk: Po, 0.19 ± 0.01 (n=9) vs. 0.09 ± 0.02 (n=8), P<0.05; To, 7.70 ± 2.60 (n=9) vs. 4.20 ± 3.70 (n=8) ms, P<0.05; 4 wk: Po, 0.21 ± 0.05 (n=10) vs. 0.11 ± 0.04 (n=8), P<0.05; To, 5.7 ± 3.6 (n=10) vs. 3.1 ± 1.8 (n=8) ms, P<0.05\ However, there were no significant differences in the unitary conductance and mean close time (7c) between the two groups. The Western blotting showed that the protein expression [expressed in densitometric volume] of BKca channel increased significantly after 1- and 4-wk simulated microgravity [1 wk, 14050 ± 910 vs. 9615 ± 157, P<0.05; 4 wk, 19604 ± 644 vs. 9870 ± 519, P<0.05]. These findings suggest that after 1-wk simulated microgravity, the function of CaL and BKca channel in cerebral arteries VSMCs of rats increased. At the same time, the single-channel activity and protein expression of BKca channel increased too, these enhancements remained with the time frame of our observation (4-wk simulated microgravity). However, the function of Kv channel of 1-wk simulated microgravity rat myocytes showed changes depending on the testing potential and decreased after 4-wk simulated microgravity. The alterations of Kv, BKca, and CaL channels in cerebral arterial VSMCs may increase the level of [Ca2+]j and regulate vascular adaptation to simulated microgravity.3. Changes of Kv, BKCa and CaL channel in cerebral VSMCs of SHR The whole cell patch clamp current recording showed that the activities of both BKca and CaL channels in cerebral arterial VSMCs significantlyincreased as compared with WKY rats [BKca, 2.54 ± 0.47 (n=ll) vs. 1.12 ± 0.33 (n=12) pA/pF at +20 mV, P<0.05; CaL, -4.0 ± 0.5 (n=12) vs. -2.28 ± 0.20 (n=10) pA/pF at +20 mV, P<0.05]. CaL channel was more sensitive to the dihydropyridine calcium channel agonist Bay K 8644. However, no significant changes were found in the dynamics of CaL activities as assessed by channel activation/inactivation experiments with Boltzmann fitting between SHR and WKY rats. The Kv current density of SHR myocytes significantly decreased [1.03 ± 0.33 (n=9) vs. 1.62 ± 0.64 (n=9) pA/pF at +20 mV, P<0.05]. These findings showed that the functional alterations in cerebrovascular K+ and Ca2+ channels are quite similar between SHR and simulated microgravity rat.Take together, our present work showed that arterial ion channel mechanism participated in the process of vascular adaptation during simulated microgravity. In small mesenteric arteries VSMCs, simulated microgravity induced a more negative membrane potential and a functional upregulation of Ky and BKca channel; whereas in cerebral arterial VSMCs, simulated microgravity induced a more depolarized membrane potential and an upregulation of CaL and BKca channel function and a downregulation in Kv channel function. The function and protein expression of BKca channel in either small mesenteric or cerebral arterial VSMCs changed after 1-wk simulated microgravity and remained at this level with the time frame of our observation (4-wk microgravity). Therefore, we speculated that ion channel mechanism appears to be an immediate, early response of vascular adaptation to microgravity and continues to play a role in the process of vascular adaptation to microgravity. This finding has provided further evidence to support our hypothesis that ion channel mechanism in VSMCs of different...