IONIC MECHANISMS REGULATING SODIUM ENTRY INTO VASCULAR SMOOTH MUSCLE

1. Na+/H+ exchange, an ethylisopropylamiloride (EIPA)-sensitive Na+ and HCO3- dependent system and a diisothio-cyanatostilbenedisulphonic acid (DIDS)-sensitive Na+ and HCO3- transporter, contribute to sodium influx and intracellular pH (pHi) regulation in vascular smooth muscle. 2. In cultured cells from the human internal mammary artery, Na+/H+ exchange and the EIPA-sensitive Na+ and HCO3- dependent system contribute about 80% to basal sodium influx. The residual Na+ influx is both EIPA and DIDS-insensitive. 3. Sodium influx via these mechanisms influences the ability of vascular smooth muscle to synthesize protein late in the G1 phase of the mitotic cell cycle. This, in turn, affects DNA biosynthesis. 4. These Na+ exchanges/transporters have the capability to facilitate the development of vascular hypertrophy in hypertension.     

22Na+ and 86Rb+ transport in vascular smooth muscle of SHR, Wistar Kyoto, and Wistar rats

To gain further insight into differences in cellular Na+ and K+ regulation between the spontaneously hypertensive rat (SHR), Wistar Kyoto (WKY), and American Wistar (W) rats, 22Na+ and 86Rb+ washouts were performed under steady-state conditions in cultured vascular smooth muscle cells from the three rat strains. SHR vascular smooth muscle cells showed significantly higher bumetanide sensitive 86Rb+ washout rate constant (x 10(-4)/min; mean +/- SEM) than WKY cells (-38.6 +/- 2.84 and -23.8 +/- 3.58, respectively; p less than 0.005). SHR vascular smooth muscle cells also exhibited significantly higher values than WKY cells in the total 22Na+ washout rate constant (x 10(-2)/min) (-61.0 +/- 1.57 vs. -53.8 +/- 1.24; p less than 0.005). The amiloride sensitive component of the 22Na+ washout rate constant accounted for these differences (-18.6 +/- 1.04 for SHR and -12.1 +/- 2.00 for WKY; p less than 0.05). There were no apparent differences in cellular Na+ concentrations between WKY and SHR cells. In general, the 86Rb+ and 22Na+ washout parameters of W rat cells were quite similar to those of cells from SHR. We conclude that the bumetanide-sensitive 86Rb+ washout (the Na+ K+-cotransport), the overall, and the amiloride-sensitive 22Na+ washout (the latter primarily represents the Na+/H+ antiport) are higher in SHR than WKY rat vascular smooth muscle cells. These findings indicate innate differences in cellular Na+ and K+ transport in vascular smooth muscle cells of the SHR and WKY rat. The mechanisms responsible for these differences are yet to be determined.     

ACTIVATION OF Na+/H+ EXCHANGE IS UNNECESSARY IN THE INDUCTION OF c-fos mRNA IN SERUM-STIMULATED VASCULAR SMOOTH MUSCLE CELLS

1. The effects of extracellular Na+ concentration ([Na+]o) on serum-stimulated c-fos mRNA induction in vascular smooth muscle cells (VSMC) were studied to examine whether the activation of Na+/H+ exchange and the following intracellular alkalinization are necessary in the induction of c-fos in VSMC. 2. When monitored with 2', 7'-bis (carboxylethyl)-5(6)-carboxyfluorescein, the reduction in [Na+]o in HCO(3-)-free buffer caused a dose-dependent inhibition of Na(+)-dependent intracellular pH recovery in acid-loaded VSMC. In addition, serum-stimulated intracellular alkalinization in VSMC was completely blocked by the removal of extracellular Na+. 3. By contrast, c-fos induction, quantified by slit blot hybridization of c-fos mRNA, was not fully inhibited by the reduction of [Na+]o. Furthermore, amiloride and ethylisopropyl-amiloride, inhibitors of Na+/H+ exchange, only partially blocked c-fos induction by serum. 4. We concluded from the observation above, that the intracellular alkalinization caused by the activation of Na+/H+ exchange was unnecessary in serum-stimulated induction of c-fos mRNA in VSMC.     

INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF Na+/Ca2+ EXCHANGER IN BOVINE ADRENAL CHROMAFFIN CELLS

• 1 The Na⁺/Ca²⁺ exchanger (NCX) exchanges Na+ and Ca²⁺ bidirectionally through the forward mode (Ca²⁺ extrusion) or the reverse mode (Ca²⁺ influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na⁺‐loaded cells were prepared by treatment with 100 µmol/L ouabain and 50 µmol/L veratridine. Incubation of Na⁺‐loaded cells with Na⁺‐free solution in the presence of the Ca²⁺ channel blockers nicardipine (3 µmol/L) and ω‐conotoxin MVIIC (0.3 µmol/L) caused Ca²⁺ uptake and catecholamine release.
• 2 The Na⁺‐dependent Ca²⁺ uptake and catecholamine release were inhibited by 2‐[4‐[(2,5‐difluorophenyl)methoxy]phenoxy]‐5‐ethoxyaniline (SEA0400; 1 µmol/L) and 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]isothiourea (KB‐R7943; 10 µmol/L), both NCX inhibitors. These results indicate that the Na⁺‐dependent responses are mostly due to activation of the NCX working in the reverse mode.
• 3 In addition, we examined the effects of PKC inhibitors and an activator on the NCX‐mediated Ca²⁺ uptake and catecholamine release. Bisindolylmaleimide I (0.3–10 µmol/L) and chelerythrine (3–100 µmol/L), both PKC inhibitors, inhibited NCX‐mediated responses. In contrast, phorbol 12,13‐dibutyrate (0.1–10 µmol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na⁺‐dependent catecholamine release, had a little or no effect on high K⁺‐induced catecholamine release in intact cells or on Ca²⁺‐induced catecholamine release in β‐escin‐permeabilized cells.
• 4 These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.

     

Increased 45Ca influx in response to alpha 1-adrenoceptor stimulation in spontaneously hypertensive rat caudal artery

The isometric tension development and 45Ca influx in response to norepinephrine (NE) and methoxamine stimulation were investigated in caudal arteries of spontaneously hypertensive rats (SHR) and Wistar Kyoto normotensive rats (WKY). The maximum isometric tension developed as well as 45Ca influx in response to NE and methoxamine stimulation were significantly increased (p less than 0.05) in SHR caudal arteries as compared with WKY. On the other hand, neither the isometric tension developed nor the 45Ca influx in response to K+ depolarization were different between WKY and SHR caudal arteries. Estimation of [3H]prazosin binding to the membranes isolated from caudal artery of WKY and SHR showed a single class of high-affinity binding sites with Kd values for SHR 128 +/- 14 pM and for WKY 141 +/- 19 pM, and Bmax values for SHR 108 +/- 14 fmol/mg protein and for WKY 113 +/- 21 fmol/mg protein. From these results, we conclude: (a) Increased contractile response of SHR caudal artery rings to alpha 1-adrenoceptor stimulation appears at least in part to be due to an increased Ca2+ influx through receptor-operated Ca2+ channels; (b) the affinity or density of alpha 1-adrenoceptors estimated by [3H]prazosin binding is not altered in the SHR caudal artery.     

Quantitative changes in cardiac Na+, K+ -adenosine triphosphatase of spontaneously hypertensive rats

Sodium pumps of cardiac plasma membranes were studied in young, spontaneously hypertensive rats (SHR) and in their normotensive controls (Wistar-Kyoto; WKY) using the two following methods. The enzymatic activity and its sensitivity to ouabain were measured as the Na+, K+ -dependent ATP hydrolysis, and the number of pumps was estimated by [3H] ouabain binding. The main results of this study were the observations that (a) concentrations of ouabain as low as 10(-10) M inhibited 10-15% of the enzyme activity in both strains; (b) Na+, K+- adenosine triphosphatase (ATPase) activity in membranes from SHR was double that in membranes from WKY (16.5 +/- 3.2 mumol Pi/h/mg protein vs. 8.2 +/- 1.2 mumol Pi/h/mg protein for 10(-7) M ouabain; p less than 0.01); (c) sensitivity to three different cardiac glycosides, ouabain, digoxin, and digitoxigenin, was identical in SHR and WKY vesicles; and (d) the binding capacity of [3H] ouabain was significantly higher in SHR than in WKY vesicles, but the dissociation constant (KD) did not appear to differ between the two substrains. These studies, performed on 3-week-old rats before the appearance of hypertension, showed, on the one hand, the existence of a Na+, K+ -ATPase of very high affinity in the rat heart, and, on the other, that cardiac sarcolemmal membranes from SHR had a greater number of sodium pumps than those from WKY and thus a greater ability to extrude sodium.     

Functional role of Na+/H+ exchanger in Ca2+ influx mediated via human endothelin type A receptor stably expressed in Chinese hamster ovary cells

This study examines the functional role of Na+/H+ exchanger (NHE) in Ca2+ influx mediated by human endothelin type A receptor (ET(A)R) expressed in Chinese hamster ovary (CHO) cells. Endothelin-1 (ET-1) increased extracellular acidification rate (ECAR), which was abolished by 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an NHE inhibitor. EIPA and KB-R7943, a Na+/Ca2+ exchanger (NCX) inhibitor, inhibited ET-1-induced sustained increases in intracellular Ca2+ concentration ([Ca2+]i), and EIPA had no effect on [Ca2+]i after KB-R7943 treatment. ET-1-elicited sustained [Ca2+]i increase was suppressed by reducing extracellular Na+ concentration. These results suggest that possible coupling of NHE with NCX via Na+ transport is involved in ET(A)R-mediated sustained [Ca2+]i increase.     

KB-R7943对豚鼠心室肌细胞Na+-Ca2+交换电流的作用

目的　观察KB-R7943对豚鼠心室肌细胞Na⁺-Ca²⁺交换电流(I_Na-Ca)的内向电流成分和外向电流成分的影响。方法　采用缺血再灌时胞内Na⁺超载的细胞模型,在同时记录内向、外向电流的双向离子条件下,用膜片钳全细胞技术,记录I_Na-Ca的电流-电压关系曲线。结果　10^-6和10^-5mol·L^-1KB-R7943,在+50mV时,对I_Na-Ca的抑制率分别是29.4%和61.7%;在-80mV时抑制率分别是22.1%和56.9%。结论　KB-R7943对豚鼠心室肌细胞I_Na-Ca有抑制作用,但对外向成分和内向成分的抑制不具选择性。     

Calcium dependence of ouabain-induced contraction in aortas from spontaneously hypertensive rats

The calcium sensitivity of ouabain-induced contractions of aortic strips from spontaneously hypertensive rat (SHR) was examined using several drugs which affect Na+ and Ca2+ movements across the cell membrane, and the results were compared with those obtained with age-matched Wistar-Kyoto rat (WKY). The Ca2+ concentration-response curves (10(-3) M ouabain-treated preparations) made with aortic strips from SHR lay to the left of those made with aortic strips from WKY (Ca EC50 values: SHR, 0.51 +/- 0.16 mM, n = 6; WKY, 1.23 +/- 0.41 mM, n = 7; P less than 0.05). Amiloride (a Na+ entry blocker) and nifedipine (a Ca2+ entry blocker) attenuated the sensitivity to Ca2+ of SHR and WKY aortic strips. With 2 x 10(-4) M amiloride, WKY vessels showed a 1.3-fold increase in the Ca EC50 value and SHR a 2.1-fold increase. With 10(-6) M nifedipine. WKY vessels showed a 1.1-fold increase in the Ca EC50 value and SHR a 1.5-fold increase. Addition of monensin (Na ionophore) produced a dose-dependent potentiation in ouabain-treated aorta from WKY, but not in ouabain-treated aorta from SHR. Addition of 1.5 x 10(-5) M A23187 (Ca ionophore) eliminated the difference between the Ca2(+)-induced contractions in aortas from SHR and WKY. These results suggest that enhancement of Ca2+ influx by Na(+)-Ca2+ exchange and/or voltage-dependent Ca2+ channels in vascular smooth muscle cell membranes may be an important factor in the difference between ouabain-induced contractions in aorta from SHR and WKY.     

AMP579与腺苷对大鼠心室肌细胞Na~+/Ca~(2+)交换电流作用的比较(英文)

目的阐明AMP579与腺苷不同药理作用与临床效果的机制。方法采用全细胞膜片钳记录Na+/Ca2 +交换电流。结果AMP579对Na+/Ca2 +交换的外向和内向电流均呈浓度依赖性增强。灌流腺苷A1受体阻断剂PD116948 30μmol·L-1,A2受体阻断剂DMPX10μmol·L-1,蛋白激酶A特异阻断剂KT5720 0 .2μmol·L-1或蛋白激酶C特异阻断剂GF109203X0 .4μmol·L-1对AMP579 Na+/Ca2 +交换电流的激动作用均无影响,提示AMP579对Na+/Ca2 +交换电流具有直接的激动作用。结论AMP579对Na+/Ca2 +交换电流可能具有直接的激动作用。     

INTERACTION OF THE NON-STEROIDAL ANTIINFLAMMATORY DRUG FLUFENAMIC ACID WITH GASTRIC ACID SECRETION AND H+/K+ -ATPase

1. The effects of the non-steroidal anti-inflammatory drug (NSAID) flufenamic acid on H+ production in isolated and enriched guinea-pig parietal cells and on H+/K(+)-ATPase activity in ion-tight inside-out membrane vesicles from pig gastric mucosa were studied. 2. At low concentrations (0.1 and 1.0 mumol/L), flufenamic acid increased the secretory response of parietal cells to dibutyryl cyclic AMP (dbcAMP). At higher concentrations (10 and 100 mumol/L) it progressively inhibited basal and dbcAMP-stimulated acid production. 3. Flufenamic acid (10 mumol/L) increased K+ (0.5-10.0 mmol/L) and K+ (0.5-1.0 mmol/L) plus gramicidin-stimulated ATPase activity in gastric membrane vesicles. The Km value for K+ (1.6 and 1.0 mmol/L in the absence and presence of gramicidin, respectively) was decreased to 0.8 and 0.5 mmol/L, respectively. At higher concentrations (greater than or equal to 50 mumol/L), flufenamic acid inhibited K+ plus gramicidin-stimulated ATPase activity (inhibited concentration at 50% [IC50] = 186 mumol/L) and reduced the proton concentration (IC50 = 50 mumol/L). 4. It is concluded that flufenamic acid-induced enhancement of dibutyryl cyclic AMP-stimulated H+ production in the parietal cell reflects the stimulation of H+/K(+)-ATPase. We suggest that activation of the enzyme involves increased affinity of K+ towards the K(+)-binding site of the enzyme and/or increased KCl permeability at the vesicle membrane. The inhibitory action of the drug on H+ production in parietal cells results from a detergent and/or protonophoric-like action at the apical parietal cell membrane, and from inhibition of H+/K(+)-ATPase activity.     

SM-20550, a new Na+/H+ exchange inhibitor and its cardioprotective effect in ischemic/reperfused isolated rat hearts by preventing Ca2+-overload

We investigated the effect of a newly synthesized compound, SM-20550 [N-(aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonic acid] on Na+/H+ or Na+/Ca2+ exchange activity in rat cardiomyocytes, and on radioligand binding with several channels or receptors in membrane preparations, and ischemia/reperfusion injury in isolated perfused rat hearts. In myocytes, SM-20550 concentration-dependently inhibited the recovery from acidosis induced by an NH4Cl prepulse in HCO3(-)-free solution. Its IC50 was 10(-8) M, which was 10 times lower than that of ethylisopropyl amiloride (EIPA). SM-20550 (10(-6) M) did not affect the Na+-dependent Ca2+ influx (Na+/Ca2+ exchange activity) in cardiomyocytes. In the radioligand binding assay, SM-20550 did not have affinity for K+ channel, beta-adrenoceptor, adenosine, angiotensin, or endothelin receptors, and had low affinity for Na+ and Ca2+ channels and alpha-adrenoceptors, only at the concentrations of 10(-6)-10(-5) M. In perfused hearts exposed to 40 min of global ischemia and 20 min of reperfusion, SM-20550 (10(-8)-10(-7) M) significantly reduced the elevation of left ventricular end-diastolic pressure during reperfusion, improved the postischemic recovery of developed pressure, and prevented coronary perfusion pressure increase after reperfusion. Furthermore, SM-20550 reduced creatine phosphokinase release during reperfusion and prevented the abnormal gain of tissue Na+ and Ca2+ at the end of reperfusion. These results suggest that SM-20550 is a potent, highly specific Na+/H+ exchange inhibitor, which exerts a protective effect against myocardial ischemia/reperfusion injury. In addition, our data strongly support the hypothesis that Na+/H+ exchange plays an important role in the development of postischemic cardiac dysfunction, most likely by inducing Na+ and Ca2+ overload.     

Effect of serum on cell membrane Na-K transport of vascular smooth muscle in culture--a comparative study between normotensive and hypertensive rats.

In order to elucidate the effect of serum and its differential characteristics in primary hypertension, we investigated the influence of serum from spontaneously hypertensive rats (SHR) on the cell membrane Na-K pump, Na-H antiport and passive K permeability of cultured vascular smooth muscle cells (VSMC) from SHR and Wistar-Kyoto rats (WKY). In the absence of serum, the Na-K pump activity, described as ouabain-sensitive 86Rb uptake (OS), was greater in the VSMC of the SHR than that of the WKY. Addition of serum to the quiescent VSMC stimulated the OS, of which activation was significantly greater with the SHR serum than with the WKY serum. Determination of intracellular Na concentrations in the presence of 1 mM ouabain showed that Na uptake in the absence of serum of the SHR VSMC was greater than that of the WKY VSMC. Subjecting the VSMC to rat serum stimulated Na uptake, which effect was more profound with SHR serum than with WKY serum. A greater stimulation of 5-(N,N-hexamethylene) amiloride(HMA)-sensitive Na uptake by SHR serum accounted for this difference. When the Na-K pump was active, intracellular Na concentrations were unchanged in the presence of serum. Furthermore, serum from either of the two strains produced a substantial increase in the washout rate constant of 86Rb washout (Ke), which effect was also larger in SHR serum than in WKY serum. In the absence of serum, basal Ke of the SHR VSMC was greater than that of the WKY VSMC. It is concluded that serum from SHR produced a more pronounced activation of the Na-K pump, Na-H antiport and passive K permeability in cultured VSMC. The study suggests that in addition to an innate augmented activity of Na-K transporters in the VSMC of SHR, there are humoral factors in SHR serum which elicit the stimulation of the Na-H antiport, leading to either the activation of the Na-K pump or secondary stimulation of passive K permeability across the cell membrane.     

INTRACELLULAR pH AND SODIUM-PROTON EXCHANGE ACTIVITY OF LYMPHOCYTES IN STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS

1. At the age of 20 weeks, intracellular pH (pHi) of circulating lymphocytes suspended in HCO(3-)-free NaCl media was not significantly different between stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). 2. The initial recovery rate of pHi in lymphocytes tended to be greater in SHRSP than in WKY after the addition of 60 mmol/L or 120 mmol/L of NaCl, but there was no statistically significant difference. 3. The H+ equivalent efflux rate, which was a true reflection of Na(+)-H(+) activity, was significantly greater in SHRSP than in WKY (P less than 0.05). The difference in H+ equivalent efflux rate was not due to the difference in cellular buffering power between the two groups (P greater than 0.05). An increased Na(+)-H(+) exchange activity may play a partial role in the pathogenesis of hypertension.     

抗钠-钙交换体α-1_((106-145))抗体对大鼠离体心功能的影响及其机制

目的观察1～103nmol·L-1抗钠-钙交换体(NCX)α-1(106-145)肽段抗体对离体大鼠心功能的影响并分析其作用机制。方法用化学合成的NCXα-1(106-145)肽段主动免疫新西兰大耳白兔制备并纯化抗体,利用Langendorff离体灌流系统观察其对大鼠离体心功能的影响;利用全细胞膜片钳技术观察其对心肌细胞钠-钙交换电流、L-型钙电流、瞬时外向钾电流和内向整流钾电流的影响。结果经主动免疫后兔体内抗α-1(106-145)抗血清滴度明显升高,Protein A纯化后抗体浓度为13.1 g.L-1。与对照组相比,1～10 nmol·L-1α-1抗体可使大鼠离体心脏左室发展压(LVDP)和左室压最大上升速率(+dp/dtmax)明显增加(P<0.01),并使左室压最大下降速率(-dp/dtmax)减小(P<0.01),且KB-R7943(2μmol·L-1)可使α-1抗体(1 nmol·L-1)的上述效应进一步增强。然而,102～103nmol·L-1α-1抗体则可使大鼠离体心脏LVDP、±dp/dtmax呈不同程度地下降(P<0.01)。全细胞膜片钳实验结果表明,在1～103nmol·L-1浓度范围内,抗α-1抗体对大鼠心肌细胞外向和内向Na+/Ca2+交换电流均表现出剂量依赖性的抑制效应,对瞬时外向钾电流和内向整流钾电流均无明显影响。此外,102～103nmol·L-1α-1抗体对L-型钙电流也具有抑制作用。结论低浓度α-1抗体(1～10 nmol·L-1)可明显增强心肌收缩,这一效应与其在低浓度时专一性抑制Na+/Ca2+交换电流有关;高浓度α-1抗体(102～103nmol·L-1)则可同时抑制心肌收缩和舒张,该作用与其同时抑制Na+/Ca2+交换电流和L-型钙电流有关。     

ROLE OF EXTRACELLULAR Na+, Ca2+-ACTIVATED Cl- CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE-DEPLETED TRACHEAL SMOOTH MUSCLE

• 1 In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re‐addition of Ca²⁺ in an in vitro experimental model in which Ca²⁺ stores had been depleted and their refilling had been blocked by thapsigargin.
• 2 Mean (±SEM) contraction was diminished by: (i) inhibitors of store‐operated calcium channels (SOCC), namely 100  µ mol/L SKF‐96365 and 100  µ mol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca²⁺ channels Ca_V1.2 channels, namely 1  µ mol/L nifedipine and 10  µ mol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20  µ mol/L niflumic acid, a non‐selective inhibitor of Ca²⁺‐dependent Cl^? channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3‐fold by 100 nmol/L iberiotoxin, a blocker of the large‐conductance Ca²⁺‐activated K⁺ (BK) channels.
• 3 Furthermore, contraction was significantly inhibited when Na⁺ in the bathing solution was replaced by N‐methyl–d ‐glucamine (NMDG⁺) to 39.9 ± 7.2% of control, but not when it was replaced by Li⁺ (114.5 ± 24.4% of control). In addition, when Na⁺ had been replaced by NMDG⁺, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na⁺ had been replaced by Li⁺ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control).
• 4 In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca_V1.2 channels in the contractions induced by the re‐addition of Ca²⁺ to the solution bathing guinea‐pig tracheal rings under conditions of Ca²⁺‐depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na⁺, suggesting a role for SOCC in mediating the Na⁺ influx.

     

Oxidative stress and alpha1-adrenoceptor-mediated stimulation of the Cl-/HCO3- exchanger in immortalized SHR proximal tubular epithelial cells

BACKGROUND AND PURPOSE: This study evaluated the signalling coupled to the alpha1-adrenoceptor-induced stimulation of the Cl-/HCO3- exchanger in hypertension. EXPERIMENTAL APPROACH: The Na+ -independent HCO3- transport system activity was assayed as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal) in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rat (SHR) and their normotensive control (Wistar Kyoto rat; WKY). KEY RESULTS: Noradrenaline increased Cl-/HCO3- exchanger activity with EC50 values of 0.6 and 5.3 microM in SHR and WKY cells, respectively. These effects were abolished by prazosin, but not by yohimbine. Phenylephrine increased Cl-/HCO3- exchanger activity in SHR and WKY cells (EC50 of 2.6 and 4.9 microM, respectively). Phenylephrine-mediated increase in Cl-/HCO3- exchanger activity in WKY and SHR cells was inhibited by protein kinase C (PKC), MAPK/ERK kinase (MEK) and p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. The expression of alpha1A- and alpha1B-adrenoceptors was identical in WKY and SHR cells. SHR cells generated more H2O2 than WKY cells. In SHR cells, the NADPH oxidase inhibitor apocynin reduced their increased ability to generate H2O2 and abolished their hypersensitivity to phenylephrine, but failed to affect basal Cl-/HCO3- exchanger activity. H2O2-dependent stimulation of Cl-/HCO3- exchange activity was significantly higher in SHR than in WKY cells. CONCLUSIONS AND IMPLICATIONS: Differences between WKY and SHR cells on their sensitivity to alpha1-adrenoceptor stimulation did not correlate with the abundance of alpha1A- and alpha1B-adrenoceptors and may be related to the increased generation of H2O2, which may amplify the response downstream of alpha1-adrenoceptor activation.     

Sodium pump activity and contraction of renal artery from spontaneously hypertensive rats

We investigated the hypertensive changes in renal arteries isolated from 21-week-old spontaneously hypertensive rats (SHR), and from age-matched normotensive Wistar-Kyoto rats (WKY). The maximam contraction of renal arteries from SHR in response to norepinephrine (NE), serotonin (5-HT) and KCl was greater than that of arteries from WKY. The threshold and EC₅₀ concentrations of NE, 5-HT and KCl were not significantly different between SHR and WKY. Contraction induced by removal of K⁺ was inhibited by 10⁻⁸ M prazosin. Less than 10⁻⁷ M NE in K⁺-free solution did not cause contraction. Addition of 5.9 mM KCl to K⁺-free solution in the presence of 10⁻⁵ M NE induced relaxation, which was followed by contraction to about the same level as that before KCl addition. The duration of the K⁺-induced relaxation in SHR (22.4 ± 0.9 min) was slightly, but significantly shorter than that in WKY (26.6 ± 0.8 min) arteries. In K⁺-free solution with reduced Na⁺, the duration of the relaxation induced by KC1 was shorter than that in the normal solution, for both SHR (13.8 ± 0.3 min) and WKY (14.1 ± 0.5 min). Such differences could be caused by increased influx and decreased efflux of Ca²⁺, which depend on the Na⁺ concentration and are related to the Na⁺-Ca²⁺ exchange. The results suggest that enhanced renal vascular reactivity in hypertension may depend on structural changes and increased Na⁺ pump activity.     

Restoration of osmotically inhibited twitch force in rat cardiac trabeculae: role of Na+-H+ exchange

1. When rat cardiac muscle is subjected to an increase of osmolality, its peak twitch force is immediately inhibited. Subsequently, over a period of several minutes, twitch force undergoes restoration, the extent of which is determined by the osmolality. The aim of the present study was to determine the factors that contribute to this restorative phenomenon. 2. Trabeculae were isolated from the right ventricles of rat hearts and mounted in an organ bath at 37 degrees C. The osmolality of the bathing solution was increased by 100 mOsmol (to 400 mOsmol) by the addition of various proportions of NaCl and sucrose while recording twitch force production. The role of Na+-H+ exchange in restoring twitch force was examined by use of the specific inhibitor cariporide (HOE 642). The role of Na+-Ca2+ exchange was examined by reducing [Ca2+]o (from 2 mmol/L to 0.5 mmol/L) or by substituting LiCl for NaCl. 3. Cariporide (25 micro mol/L) completely abolished twitch force restoration, thereby implicating a central role for the Na+-H+ exchanger. At constant [Na+]o, the extent of restoration was [Ca2+]o dependent, suggesting an independent contribution by the Na+-Ca2+ exchanger. This suggestion was supported by the finding that Li+, which substitutes for Na+ on the Na+-H+ exchanger, but not on the Na+-Ca2+ exchanger, also reduced the extent of restoration of hyperosmotically inhibited twitch force. 4. We conclude that the immediate inhibition of peak twitch force of rat cardiac muscle by hyperosmotic solutions reflects, in part, elevation of [H+]i, subsequent to reduction of cell volume. Hyperosmotic activation of Na+-H+ exchange then progressively relieves the inhibitory effect of protons on force development. The accompanying increase in [Na+]i in turn enhances Ca2+ influx on the Na+-Ca2+ exchanger, with the result that twitch force undergoes further restoration.     

Role of Na+/H+ exchanger in acetylcholine-mediated pulmonary artery contraction of spontaneously hypertensive rats

Compared to sympathetic nervous system, the role of parasympathetic innervation on tone development, especially under diseased conditions, of the pulmonary artery is relatively unknown. In this study, the contractile effect of acetylcholine and the type(s) of muscarinic (M) receptor involved in the pulmonary artery (1st intralobar branch; endothelium-denuded, under resting tension) of the normotensive Wistar-Kyoto (WKY) and age-matched (male, 22-26 weeks old) Spontaneously hypertensive rats (SHR) were investigated. Cumulative administration of acetylcholine (> or =0.1 microM) caused a concentration-dependent increase in tension (antagonised by p-fluoro-hexahydro-sila-difenidol and 4-diphenylacetoxy-N-methylpiperidine, both are selective muscarinic M(3) receptor antagonists) and the magnitude of maximum contraction (expressed as % of 50 mM [K(+)](o)-induced contraction) was markedly enhanced in the presence of neostigmine (10 microM, an anti-cholinesterase) (acetylcholine 30 microM, SHR: 72% vs. 35%; WKY: 32% vs. 20%). In SHR only, acetylcholine-elicited contraction was suppressed by 1-[beta-[3-(4-Methoxyphenyl)-propoxyl]-4-methoxyphenethyl]-1H-imidazole (SK&F 96365, 1 microM), amiloride (500 microM), ethyl-isopropyl-amiloride (EIPA, 10 microM), 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R 7943, 5 microM), 2,4-dichlorobenzamil (10 microM), and an equal molar substitution of [Na(+)](o) (< or =30 mM) with choline or N-methyl-D-glucamine. In nominally [Ca(2+)](o)-free, EGTA (0.5 mM)-containing Krebs' solution, acetylcholine (> or =3 microM) only elicited a small contraction. In conclusion, muscarinic M(3) receptor activation is responsible for the pulmonary artery contraction induced by acetylcholine, with a greater magnitude observed in SHR. The exaggerated contraction in SHR is probably due to an influx of [Na(+)](o) through the Na(+)/H(+) exchanger and the store-operated channels (SOC) into smooth muscle cells. Elevation of cytosolic [Na(+)](i) subsequently leads to an influx of [Ca(2+)](o) through the reverse mode of the Na(+)/Ca(2+) exchanger seems to play a permissive role in mediating the exaggerated contractile response of acetylcholine recorded in the SHR.