Differences in K+ current components in mesenteric artery myocytes from WKY and SHR

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 (I_K) components to whole cell I_K 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 I_K recorded by perforated patch methods was similar at a holding potential (HP) of −60 mV (I_K60), but were larger in SHR when recorded from a HP of −20 mV (I_K20). The selective blocker iberiotoxin (IbTX) was used to separate the contribution of voltage- (K_V) and calcium-dependent (K_Ca) components of I_K60. The I_K60 and I_K20 component inhibited by 100 nmol/L IbTX (ie, K_Ca) was larger in SHR than in WKY myocytes, whereas the IbTX-insensitive I_K60 component (ie, K_V) was larger in WKY. In the presence of IbTX, 1 and 10 mmol/L TEA inhibited a larger fraction of I_K60 in SHR myocytes compared with WKY. The activation properties of the TEA-sensitive and TEA-insensitive K_V 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 (V_0.5). The V_0.5 of the TEA-sensitive K_V component was more positive than that of the TEA-insensitive component in both groups, and values for the V_0.5 of both TEA-sensitive and TEA-insensitive components were more negative in SHR than WKY. These results show that SHR myocytes have larger K_Ca and smaller K_V current components compared with WKY. Furthermore, SHR myocytes have a larger TEA-sensitive K_V component. These differences may contribute to the differences in TEA contractions, resting membrane potential, Ca²⁺ influx, and K_Ca current reported in hypertensive arteries.     

Increased Ca2+ buffering function of sarcoplasmic reticulum in small mesenteric arteries from spontaneously hypertensive rats.

We compared the Ca2+ buffering function of the superficial sarcoplasmic reticulum (SR) during rest and during contraction in endothelium-denuded strips of small mesenteric arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The addition of caffeine (1-20 mM) caused a transient contraction in both strains, and the contraction was significantly larger in SHR. When the SR Ca2+ buffering function was eliminated by cyclopiazonic acid (CPA; 10 microM) or thapsigargin (100 nM), both of which inhibit SR Ca2+-ATPase, or by ryanodine (10 microM), which depletes the SR Ca2+, there was a larger contraction in SHR than in WKY, suggesting that the Ca2+ buffering function of the SR during rest is more important in SHR than in WKY. Judging from the augmenting effects of these three agents on the contractile responses to Bay k 8644 (1-300 nM), an agonist of L-type Ca2+ channels, or norepinephrine (10(-9)-10(-4) M), an alpha-adrenoceptor agonist, the effects were significantly greater in SHR than in WKY. We conclude that 1) the Ca2+ influx during rest and during stimulation with Bay k 8644 or norepinephrine is strongly buffered by Ca2+ uptake into the superficial SR in the small mesenteric arteries from SHR and WKY; and 2) these Ca2+ buffering functions are increased in SHR because of the larger capacity of SR for Ca2+ storage.     

Effect of L-carnitine and propionyl-L-carnitine on endothelial function of small mesenteric arteries from SHR

BACKGROUND:The effect of treatment with either 200 mg x kg(-1) of L-carnitine (LC) or propionyl-L-carnitine (PLC) was studied on endothelial dysfunction of small mesenteric arteries (SMA) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Methods: Systolic blood pressure (SBP) was measured and endothelial and vascular functions were assessed by the effect of carbachol (CCh) and phenylephrine (Phe). O2- produced by SMA and eNOS expression were evaluated by chemiluminescence and Western blot, respectively. Results: Although SBP was not affected, endothelial relaxation increased in both LC- and PLC-treated SHR. Nevertheless, the CCh-induced contraction remained sensitive to indomethacin in these rats. On the contrary, NO participation was increased in all the groups except for LC-treated WKY. Furthermore, high concentrations of Phe produced NO-dependent relaxation of SMA from PLC-treated rats. Both compounds decreased basal and NADPH-stimulated O2- in SHR toward values observed in WKY. Only PLC increased eNOS protein expression in SHR. Neither LC nor PLC affected endothelium-derived hyperpolarizing factor-induced relaxation. Conclusions: LC and its propionate improved endothelial responses of SMA from SHR by decreasing O2- production and thus increasing NO availability. PLC also increased NO synthesis by enhancing eNOS expression.     

Role of cyclic ADP-ribose in Ca2+-induced Ca2+ release and vasoconstriction in small renal arteries

Cyclic-ADP-ribose (cADPR) has been reported to serve as a second messenger to mobilize intracellular Ca2+ independent of IP3 in a variety of mammalian cells. This cADPR-mediated Ca2+ signaling pathway importantly participates in the regulation of various cell functions. The present study determined the role of endogenous cADPR in mediating ryanodine-sensitive Ca2+-induced Ca2+ release (CICR) in vascular myocytes from small renal arteries and vasomotor response of these arteries. In freshly-isolated renal arterial myocytes, addition of CaCl2 (0.01, 0.1, and 1 mM) into the Ca2+-free bath solution produced a rapid Ca2+ release response from the sarcoplasmic reticulum (SR), with a maximal increase of 237+/-25 nM at 1 mM CaCl2. This CaCl2 response was significantly blocked by a cell-membrane permeant cADPR antagonist, 8-bromo-cADP-ribose (8-br-cADPR) (30 microM) or ryanodine (50 microM). Caffeine, a classical CICR or ryanodine receptor activator was found to stimulate the SR Ca2+ release (Delta[Ca2+]i: 253+/-35 nM), which was also attenuated by 8-br-cADPR or ryanodine. Using isolated and pressurized small renal arteries bathed with Ca2+-free solution, both CaCl2 and caffeine-induced vasoconstrictions were significantly attenuated by either 8-br-cADPR or ryanodine. Biochemical analyses demonstrated that CaCl2 and caffeine did not increase cADPR production in these renal arterial myocytes, but confocal microscopy showed that a dissociation of the accessory protein, FK506 binding protein 12.6 (FKBP12.6) from ryanodine receptors was induced by CaCl2. We conclude that cADPR importantly contributes to CICR and vasomotor responses of small renal arteries through enhanced dissociation of ryanodine receptors from their accessory protein.     

Comparison of K+ channel properties in freshly isolated myocytes from thoracic aorta of WKY and SHR

Altered function of smooth muscle cell K⁺ channels have been reported in hypertension, but the contribution of various K⁺ channel types to these changes has not been completely determined. The purpose of this study was to compare the contribution of K⁺ channel types to whole cell K⁺ currents recorded from isolated thoracic aorta myocytes of 13 to 15 week old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Cells were isolated by collagenase and elastase digestion, and K⁺ currents recorded using whole cell voltage clamp methods at room temperature. Cells were superfused with a solution containing (in mmol/L) 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose. Pipettes were filled with a solution containing (in mmol/L) 120 KCl, 5 NaCl, 5 MgATP, 20 HEPES, and 10 BAPTA. The K⁺ currents (I_K) recorded from a holding potential (HP) of −80 mV were smaller in the SHR compared to those in WKY (for example, at 20 mV: WKY = 6.1 ± 0.6 pA/pF and SHR = 3.7 ± 0.2 pA/pF). Values of cell capacitance were not different between the two groups (WKY = 25.2 ± 3.2 pF and SHR = 26.6 ± 1.9 pF). A component of I_K inhibited by voltage (K_v) over the range from −80 to −20 mV was smaller in SHR. The voltage dependence of K_v availability and activation were not significantly different between the two groups. I_K recorded from a HP = −20 mV (K_Ca) was not different between the two groups. Difference currents calculated from I_K measured at HP of −80 and −20 mV (that is, K_v) were smaller in SHR as was the fraction of I_K inhibited by 4-aminopyridine. These results suggest that under conditions of low intracellular [Ca²⁺] there are no differences in K_Ca currents, but the K_v currents are smaller in SHR. Inhibition of K_v by 4-aminopyridine (0.1 to 10 mmol/L) caused larger increases in basal tone in WKY aorta. These results suggest that K_v channels contribute to resting K⁺ conductance in both WKY and SHR aorta, but with a relatively larger contribution in the WKY.     

Pharmacological basis of Ca2+ channels and Ca2+ channel antagonists

Ca2+ plays multiple roles in muscle E-C coupling, secretion, and neural transmission, in addition to survival, proliferation, and death of cells. The voltage-dependent L-type Ca2+ channel is a transmembrane protein that selectively permeates Ca2+ on activation by membrane depolarization. Ca2+ channel blockers (or Ca2+ antagonists) selectively block this channel. The blocking action is exerted in a tissue-specific manner, which underlies the unique pharmacological properties of Ca2+ channel blockers. The later generation of slowly-acting and long-lasting Ca2+ channel blockers has been designed to overcome the side effects of classical Ca2+ channel blockers. The pharmacological and molecular basis for the unique action of Ca2+ channel blockers will be discussed.     

Chronic hypoxia suppresses pregnancy-induced upregulation of large-conductance Ca2+-activated K+ channel activity in uterine arteries

Our previous study demonstrated that increased Ca(2+)-activated K(+) (BK(Ca)) channel activity played a key role in the normal adaptation of reduced myogenic tone of uterine arteries in pregnancy. The present study tested the hypothesis that chronic hypoxia during gestation inhibits pregnancy-induced upregulation of BK(Ca) channel function in uterine arteries. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (≈ 300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia during gestation significantly inhibited pregnancy-induced upregulation of BK(Ca) channel activity and suppressed BK(Ca) channel current density in pregnant uterine arteries. This was mediated by a selective downregulation of BK(Ca) channel β1 subunit in the uterine arteries. In accordance, hypoxia abrogated the role of the BK(Ca) channel in regulating pressure-induced myogenic tone of uterine arteries that was significantly elevated in pregnant animals acclimatized to chronic hypoxia. In addition, hypoxia abolished the steroid hormone-mediated increase in the β1 subunit and BK(Ca) channel current density observed in nonpregnant uterine arteries. Although the activation of protein kinase C inhibited BK(Ca) channel current density in pregnant uterine arteries of normoxic sheep, this effect was ablated in the hypoxic animals. The results demonstrate that selectively targeting BK(Ca) channel β1 subunit plays a critical role in the maladaption of uteroplacental circulation caused by chronic hypoxia, which contributes to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia.     

Engineered calmodulins reveal the unexpected eminence of Ca2+ channel inactivation in controlling heart excitation

Engineered calmodulins (CaMs), rendered Ca2+-insensitive by mutations, function as dominant negatives in heterologous systems, and have revealed mechanisms of ion channel modulation by Ca2+/CaM. The use of these CaMs in native mammalian cells now emerges as a strategy to unmask the biology of such Ca2+ feedback. Here, we developed recombinant adenoviruses bearing engineered CaMs to facilitate their expression in adult heart cells, where Ca2+ regulation may be essential for moment-to-moment control of the heartbeat. Engineered CaMs not only eliminated the Ca2+-dependent inactivation of native calcium channels, but exposed an unexpectedly large impact of removing such feedback: the unprecedented (4- to 5-fold) prolongation of action potentials. This striking result recasts the basic paradigm for action-potential control and illustrates the promise of virally delivered engineered CaM to investigate the biology of numerous other CaM-signaling pathways.     

Evoked and spontaneous purinergic junctional Ca2+ transients (jCaTs) in rat small arteries

Confocal microscopy of fluo-4 fluorescence in pressurized rat mesenteric small arteries subjected to low-frequency electrical field stimulation revealed Ca2+ transients in perivascular nerves and novel, spatially localized Ca2+ transients in adjacent smooth muscle cells. These muscle Ca2+ transients occur with a very brief latency to the stimulus pulse (most <3 ms). They are wider (approximately 5 micro m) and last longer (t(1/2), 145 ms) than Ca2+ sparks. They are abolished by the purinergic receptor (P2X) antagonist suramin, but they are totally unaffected by the alpha1 adrenoceptor antagonist prazosin or by capsaicin (which inhibits the function of perivascular sensory nerves). We conclude that these novel Ca2+ transients represent Ca2+ entering smooth muscle cells through P2X receptors activated by ATP released from sympathetic nerves, and we therefore call them "junctional Ca2+ transients" or jCaTs. As expected from spontaneous neurotransmitter release, jCaTs also occur spontaneously, with characteristics identical to evoked jCaTs. Visualization of sympathetic neurotransmission shows that purinergic components dominate at low frequencies of sympathetic nerve fiber activation.     

水孔蛋白2（AQP2）在SHR和WKY大鼠肾脏的不同表达

目的　本研究旨在探讨SHR和WKY大鼠肾脏中 ,AQP2mRNA是否存在差异表达及其和精氨酸加压素 (AVP)的关系。方法　成年SHR和WKY大鼠各 9只 ,12周龄 ,体重 2 0 0～ 3 0 0g。断头取血 ,放免法测定血浆AVP含量。取肾脏近髓组织 10 0mg ,RT PCR法检测大鼠肾脏AQP2mRNA的表达量 ,数据以 x±s表示。结果　SHR肾脏AQP2mRNA表达水平显著高于WKY( 0 80 1± 0 0 8vs 0 5 71± 0 0 6,P <0 0 5 ) ,SHR血浆中AVP的浓度显著高于WKY( 90± 17 83vs60 82± 12 5 9) pg/mL。结论　SHR肾脏AQP2mRNA表达量上调 ,可能在SHR高血压的发生发展中发挥着一定的作用 ,AVP可能介导了AQP2mRNA表达     

水孔蛋白2(AQP2)在SHR和WKY大鼠肾脏的不同表达

目的本研究旨在探讨SHR和WKY大鼠肾脏中,AQP2 mRNA是否存在差异表达及其和精氨酸加压素(AVP)的关系.方法成年SHR和WKY大鼠各9只,12周龄,体重200～300 g.断头取血,放免法测定血浆AVP含量.取肾脏近髓组织100 mg,RT-PCR法检测大鼠肾脏AQP2mRNA的表达量,数据以-x±s表示.结果 SHR肾脏AQP2mRNA表达水平显著高于WKY(0.801±0.08vs0.571±0.06,P＜0.05),SHR血浆中AVP的浓度显著高于WKY(90±17.83 vs60.82±12.59)pg/mL.结论 SHR肾脏AQP2 mRNA表达量上调,可能在SHR高血压的发生发展中发挥着一定的作用,AVP可能介导了AQP2 mRNA表达.     

Higher Ca2+ sensitivity of triton-skinned guinea pig mesenteric microarteries as compared with large arteries.

Small mesenteric resistance arteries and the main branch of the mesenteric artery (outer in situ diameter 115 +/- 3 microns [n = 76] and greater than 1,000 microns, respectively) were skinned with 1% Triton X-100. Both preparations were mounted as rings for circumferential force measurement in an EGTA solution (free Ca2+, less than 10 nM; calmodulin, 0.3 microM; pH 6.7). Force-pCa curves were obtained by increasing free Ca2+ (0.05 to 30 microM). The resulting dose-dependent contractions, after normalization to maximal force development (arteries, 21.4 +/- 2.4 [n = 3]; arterioles, 15.3 +/- 2.1 mN/mm2 [n = 5]) were fitted to sigmoidal force-pCa curves. Values of ED50 and of the cooperativity factor h were 6.08 and 2.39 in arterioles and 5.64 and 1.64 in arteries. The higher Ca2+ sensitivity of arteriolar preparations remained at pH 7.0 at higher calmodulin concentrations and after inhibition of smooth muscle phosphatase with okadaic acid. Total myosin light chain kinase activity in crude arteriolar extracts (using [gamma-32P] ATP and isolated gizzard light chains as substrates) was approximately 25% of arterial kinase. Both kinase preparations had identical Ca2+ sensitivities. Likewise, total arteriolar phosphatase activity (using 32P-labeled gizzard light chains) was approximately 25% of the arterial activity; both phosphatases had an identical sensitivity toward okadaic acid. The ratio of kinase/phosphatase activities was identical in both tissues. Extracts of both tissues contained two isozymes of the myosin heavy chain as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular distribution of cardiac beta-adrenoceptors in SHR and WKY

To compare the intracellular distribution of beta-adrenoceptors in isolated myocytes of SHR with that of age-matched WKY and to examine changes in the distribution with aging, we measured the number of cell surface and total beta-adrenoceptors in 6-(young groups) and 37-week old (old groups) SHR and WKY. The number of surface beta-adrenoceptors was significantly lower in 6-week old SHR compared with that in age-matched WKY. But there was no difference in the number of total beta-adrenoceptors between the two groups. The number of surface beta-adrenoceptors in old groups was significantly reduced compared with that in the young groups, in both SHR and WKY. However, the number of total beta-adrenoceptors in the old groups did not show any difference from that in the young groups. Isoproterenol-stimulated c-AMP formation in 6-week old SHR was significantly lower than that of WKY of the same age. Isoproterenol-stimulated c-AMP formation in the old groups was significantly reduced than that of the young groups in both SHR and WKY. These results suggested that (1) there may be a difference in the intracellular distribution of cardiac beta-adrenoceptors between 6-week old SHR and WKY, (2) the intracellular distribution of cardiac beta-adrenoceptors may be changed with aging, and (3) intracellular distribution might be related to the difference in c-AMP formation to beta-agonist.