心肌肽素对豚鼠心室肌细胞钠通道的影响

目的： 研究心肌肽素对豚鼠心室肌细胞钠通道的影响，探讨心肌肽素在离子通道水平的作用机制。 方法： 用急性酶解分离法获得豚鼠心室肌细胞，标准全细胞膜片钳技术记录钠电流(I_Na)。 结果： 心肌肽素1、5、10、50、100、500 mg/L使豚鼠心室肌细胞I_Na分别减少(0±1)%、(6±2)%、(10±2)%、(15±1)%、(22±9)%、(30±6)%，呈浓度依赖性抑制I_Na。心肌肽素50 mg/L使I_Na激活时间(TTP)从(2.8±0.7) ms延长至(3.0±0.8) ms (P<0.05)；使I_Na电流密度-电压曲线上移，但不改变激活电位、峰电位、反转电位和I-V曲线的形状；不影响稳态激活曲线、稳态失活曲线和稳态失活后恢复曲线。 结论： 心肌肽素浓度依赖性抑制豚鼠心室肌细胞I_Na，可能是其抗心律失常作用的机制之一。     

Ionic dependence of electrical activity in small mesenteric arteries of guinea-pigs

The regulation of blood vessel diameter is under the control of the autonomic nervous system (as well as hormones and metabolites), sympathetic nerve stimulation evoking depolarizing post-synaptic potentials. Excitatory junction potentials (EJPs) were recorded from vascular smooth muscle cells of guinea-pig small mesenteric arteries (pressurized) following nerve stimulation. Repetitive stimulation (>5Hz) led to summation of EJPs, which evoked spikes and vasoconstriction. Replacing extracellular Na⁺ with choline (plus atropine) resulted in a decrease in EJP amplitude, but spike amplitude and maximum rate of rise (+V_max) were unaffected. Decreasing the extracellular Ca²⁺ concentration produced decreases in EJP amplitude and spike +V_max, while increasing extracellular Ca²⁺ resulted in increased EJP amplitude and spike +V_max. Verapamil and bepridil, agents that depress Ca²⁺ influx in vascular and visceral smooth muscle, depolarized the membrane and depressed EJPs and spikes at high concentrations (10^–5 M and 5×10^–6 M, respectively). The data indicate that EJPs are dependent on external Na⁺ and Ca²⁺ ions, and that spikes are dependent on Ca²⁺. Thus, neuromuscular transmission in this muscle is similar to that in non-vascular smooth muscles, such as intestinal muscle and vas deferens.Part of this work has been presented to the Biophysical Society (Zelcer and Sperelakis 1980) and to the American Physiological Society (Zelcer and Sperelakis 1981)     

A volume-activated anion conductance in insulin-secreting cells

The whole-cell patch-clamp recording technique was used to measure volume-activated currents in K⁺-free solutions in RINm5F and HIT-T15 insulinoma cells and in dispersed rat islet cells. Cell swelling, induced by intracellular hypertonicity or extracellular hypotonicity, caused activation of an outwardly rectifying conductance which could be subsequently inactivated by hypertonic extracellular solutions. The conductance required adenosine 5-triphosphate (ATP) in the pipette solution but was Ca²⁺ independent. Na⁺ and Cl^– substitution studies suggested that the swelling-activated current is Cl^– selective with a halide permeability sequence of Br > Cl > 1. The conductance was reversibly inhibited by the anion channel inhibitors 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) and by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Further evidence for a volume-activated anion conductance was provided by studies of volume regulation in insulin-secreting cells. When RINm5F cells were exposed to a hypotonic medium, the initial cell swelling was followed by a regulatory volume decrease (RVD). This RVD response was also inhibited by DIDS and by NPPB. These data therefore provide evidence for a volume-activated anion conductance in insulin-secreting cells which could be involved in the RVD following osmotic stress. A possible role for the conductance in hypotonically induced insulin release is also discussed.     

The effects of adenosine on transepithelial resistance and sodium uptake in the inner medullary collecting duct

It has been previously demonstrated that adenosine induces natriuresis when administered directly into the renal circulation of the rat. It was postulated that the mechanism was inhibition of tubule Na⁺ reabsorption. In the current study, the hypothesis was tested that adenosine inhibits ion reabsorption across the inner medullary collecting duct (IMCD), a tubule segment which is rich in adenosine receptors. IMCD epithelium from rat kidney was grown in primary culture as a confluent monolayer on Costar filters, allowing selective access to the basolateral and apical surfaces of the cells. Transepithelial resistance was taken as a measure of epithelial permeability and ion conductance. Na⁺ uptake was studied using ²²Na⁺ and used to determine the permeability of the epithelial monolayer specifically to Na⁺. Exposure of the basolateral aspect of the monolayer to adenosine (10⁻⁸–10⁻⁷ M) increased transepithelial resistance in a dose- and time-dependent manner; in parallel, adenosine (10⁻⁷–10⁻⁶ M) reduced apical Na⁺ uptake from 20±5 to 10±2 nmol/cm². 1,3-Dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX, 5×10⁻⁹ M), an adenosine antagonist with selectivity for the A₁ receptor, inhibited the rise in transepithelial resistance and the decrease in Na⁺ uptake following the addition of adenosine. The effects of adenosine on transepithelial resistance were reproduced with the A₁ receptor selective adenosine analogue N ⁶-cyclohexyladenosine (CHA, 10⁻⁸ –10⁻⁷ M) but not with the A₂ selective analogues, 5′-N-ethylcarboxamidoadenosine (NECA) or CGS 21680. CHA (10⁻⁷ M) inhibited apical Na⁺ uptake by 50%, an effect abolished by PACPX. The effects of adenosine on transepithelial resistance and Na⁺ uptake were inhibited, but only in part, by amiloride. These data suggest that adenosine inhibits ion movement, specifically apical Na⁺ uptake, across the IMCD epithelium and that this effect is mediated by A₁ receptors from the basolateral aspect of the cell. The results are consistent with the hypothesis that adenosine inhibits Na⁺ reabsorption across the IMCD.     

Micropuncture studies on the filtration rate of single superficial and juxtamedullary glomeruli in the rat kidney

Summary Single nephron filtration rates of superficial and juxtamedullary nephrons were determined in high and low sodium rats. Single nephron GFR was calculated from TF/P inulin and tubular flow rate in superficial nephrons and single juxtamedullary GFR from corresponding data in long loops of Henle. In low sodium rats superficial nephron GFR was 23.5±6.4 (SD)×10^–6 ml/min×g KW, juxtamedullary nephron GFR was 58.2±13.6 and total kidney GFR (C _In) was 0.94±0.16 ml/min×g KW. Using these single nephron values, total kidney GFR and a total number of 30,000 glomeruli per kidney, the number of superficial and juxtamedullary glomeruli was calculated to be 23,267 and 6,733, respectively. During high sodium diet superficial nephron GFR increased to 38.1±11.3 and single juxtamedullary GFR decreased to 16.5±6.6, total kidney GFR increasing to 1.01±0.24. Calculation again revealed the same distribution of the two nephron types. End-proximal TF/P inulin in superficial nephrons was 2.36±0.36 in low sodium and 2.31±0.28 in high sodium rats. Loops of Henle TF/P inulin and intratubular flow rate were inversely related: the highest TF/P inulin values and lowest intratubular flow rates were found in the descending limb. These data quantify the distribution of superficial and juxtamedullary nephrons on a functional basis and suggest a mechanism by which the kidney adjusts sodium excretion by altering the contribution of each nephron type to total kidney GFR.Supported by the Deutsche Forschungsgemeinschaft and the U.S. Department of the Army, through its European Research Office.     

Therapeutic potential of chlorotoxin-like neurotoxin from the Chinese scorpion for human gliomas

Chlorotoxin, one of the key toxins in scorpion Leiurus quinquestriatus venom, has been shown to bind specifically to glioma cell surface as a specific chloride channel blocker. In this study, a purified, recombinant chlorotoxin-like peptide from the scorpion Buthus martensii Karsch (named rBmK CTa) was characterized by in vivo and in vitro studies. The results from cell proliferation assay with human glioma (SHG-44) cells showed that rBmK CTa inhibits the growth of glioma cells in a dose-dependent manner, with an IC₅₀ value of approximately 0.28 μM. Under the same conditions, the IC₅₀ value for normal astrocytes increased to 8 μM. This clearly indicated that rBmK CTa had specific toxicity against glioma cells but not astrocytes. Results from whole-cell patch-clamp recording showed that chloride current in SHG-44 was inhibited by rBmK CTa in a voltage-dependent manner and percent inhibitions for the blocking action of rBmK CTa (0.07 and 0.14 μM) on I_Cl was 17.64 ± 3.06% and 55.86 ± 2.83%, respectively. Histological analysis of rBmK CTa treated mice showed that brain, leg muscle and cardiac muscle were the target organs of this toxin. These results suggest that rBmK CTa may have potential therapeutic application in clinical treatment of human glioma. It represents an approach for developing a novel therapeutic agent.     

Characteristics of the transport of oxalate and other ions across rabbit proximal colon

In order to characterize oxalate handling by the P2 segment of the rabbit proximal colon, the fluxes of [¹⁴C]oxalate, ²²Na⁺, and ³⁶Cl^– were measured in vitro using conventional short-circuiting techniques. In standard buffer the proximal colon exhibited net secretion of Na⁺ (–2.31±0.64 equiv cm^–2 h^–1), negligible net Cl^– transport, and net secretion of oxalate (–12.7±1.6 pmol cm^–2 h^–1). Replacement of buffer Na⁺ or Cl^– abolished net oxalate secretion, while HCO ₃ ^– -free media revealed a net absorption of oxalate (19.3±4.2 pmol cm^–2 h^–1) and stimulated NaCl absorption. Mucosal amiloride and dimethylamiloride (1 mM) significantly reduced the unidirectional fluxes of oxalate and enhanced sodium secretion by decreasing J _ms ^Na . The anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS; 0.1 mM, both sides) reduced the unidirectional fluxes of oxalate and chloride. Serosal epinephrine (50 M) stimulated oxalate absorption (21.3±6.3 pmol cm^–2 h^–1) and sodium absorption (5.71±1.20 equiv cm^–2 h^–1), whereas dibutyryl-cAMP enhanced oxalate secretion (–43.4±6.9 pmol cm^–2 h^–1) and stimulated chloride secretion (–7.27 ±0.64 equiv cm^–2 h^–1). These results indicate that the P2 segment of the proximal colon possesses (a) secretory as well as absorptive capacities, (b) oxalate fluxes that are mediated by pathways involving Na⁺, Cl^–, HCO ₃ ^– transport and (c) a net oxalate flux that is sensitive to absorptive and secretory stimuli.     

In situ characterization of oxalate transport across the basolateral membrane of the proximal tubule

 Oxalate transport across the contraluminal membrane of the proximal tubule was studied in vivo using the ”capillary stopped flow microperfusion method” (Pflügers Arch 400:250–256, 1984). Cellular uptake of oxalate was characteristic of a carrier-mediated transport process (J _max = 1.6 ± 0.6 pmol/s per cm proximal tubular length, K _m = 2.03 ± 0.77 mmol/l). Sulphate inhibited oxalate transport in a dose-dependent manner (K _i-value = 1.53 ± 0.38 mmol/l). Sulphate transport across the basolateral membrane was also characteristic of a carrier- mediated transport process (J_max = 1.83 ± 0.56 pmol/s per cm proximal tubular length, K _m = 1.37 ± 0.57 mmol/l). Oxalate inhibited the sulphate transport in a dose-dependent manner (K _i = 2.06 ± 0.82 mmol/l). No significant differences were found between the K _i values and the K _m values of the two substances, indicating that oxalate and sulphate are transported by the same carrier across the basolateral membrane of the proximal tubule. Oxalate transport was not dependent on the extracellular sodium or potassium concentration. Bicarbonate competitively inhibited the oxalate transport. Chloride significantly inhibited the oxalate transport, but not dose dependently. It is, therefore, suggested that oxalate is transported into the cell of the proximal tubule in exchange for sulphate or bicarbonate. The dose-independent inhibition by chloride is suggested to be mediated by the coupling of the sulphate (bicarbonate)/oxalate exchanger with the chloride/bicarbonate exchanger at the basolateral membrane of the proximal tubule. This, furthermore, suggests that the transport of oxalate or sulphate across the basolateral membrane might be indirectly coupled with the reabsorption of chloride at this membrane side. Received: 5 August 1997 / Accepted: 8 January 1998     

Atrial natriuresis under the condition of a constant renal perfusion pressure

An experimental elevation of left atrial pressure (eLAP ) by means of a reversible mitral stenosis is accompanied with an increase in sodium excretion (UNa—) and arterial blood pressure (by about 20 mm Hg, 2.7 kPa), and by a decrease in plasma renin activity.It is well established that an increase in renal perfusion pressure (Pren) can augment UNa—. Therefore the present study was undertaken to examine whether the eLAP -induced natriuresis was caused by the increased Pren. — Four female beagle dogs were kept under controlled environmental conditions. They received asodium rich diet (14.5 mmol/Na/kg/d). The dogs were chronically instrumented: purse string around the mitral annulus, catheter in the left atrium, carotid loop, pneumatic cuff above the renal arteries, pressure transducer below the renal arteries. Pren was kept constant by means of a digital servofeedback control circuit. The dogs served as their own controls (13 experiments without and 15 experiments with a controlled renal perfusion pressure were performed).After eLAP(+1.0 kPa), UNa— rose from 4.1±2.6 to 10.3±3.9 mol Na/min/kg. If Pren was kept constant, the corresponding values were 4.2±2.8 and 9.3±2.9 mol/min/kg. These data clearly indicate that the atrial natriuresis is not mediated by an augmentation of renal perfusion pressure. Therefore these results support the hypothesis that atrial natriuresis probably is due to an eLAP-induced suppression of the renin-angiotensin-system or other natriuretic mechanisms.Abbreviations ADP 3×20 min After distension period - AN Atrial natriuresis - bw kg Body weight - CP 3×20 min Control period - DP 3×20 min Distension period - eLAP kPa Experimental increase of left atrial pressure (during all DP's about +1.0 kPa) - HR l/min Heart rate - LAP kPa Left atrial pressure - n ₁ Number of dogs used - n ₂ Number of experiments (1 expt/day) - n ₃ Number of collection periods (urine) or number of samples (HR) - Part kPa Mean systemic arterial blood pressure (carotid artery) - Pren kPa Mean renal perfusion pressure (aorta, below the renal arteries) - UCI— mol/min/kg Chloride excretion - UK— mol/min/kg Potassium excretion - UNa— mol/min/kg Sodium excretion - Uosm— osm/min/kg Osmolar excretion - — l/min/kg Urine volume Preliminary parts of this work have been presented in Kiel (Spring meeting of the Deutsche Physiologische Gesellschaft, March 1979) and in Stockholm, Sweden (III. European Colloquium on Renal Physiology, June 1979).     

Inhibition of sodium transport by angiotensin II in the main duct of the rabbit mandibular gland isolated and perfused in vitro

Summary The effect of angiotensin II on nett electrolyte transport by the main duct of the rabbit mandibular gland was investigated in vitro using a perfused duct preparation bathed in a Haemaccel^®-nutrient salt solution. In a bath concentration of 4×10^–10 M, angiotensin reduced nett absorption of Na⁺ and Cl^– by about 8% and depolarized the transepithelial electrical potential difference (P.D.) by about 13%; the drug had no effect on ductal transport of K⁺ and HCO ₃ ^– . In both lower (4×10^–11 M) and higher (4×10^–9 M) concentrations, angiotensin had qualitatively similar effects. After exposure to the hormone for about 30 min, Na⁺ transport and P.D. became unstable and gradually fell away towards zero. It is concluded that angiotensin in physiological concentrations has a specific inhibitory effect on Na⁺ absorption by salivary duct cells which could arise either from a change in the Na⁺ pump rate or from a conductance change in the apical or basal membrane of the epithelial cell.