Aldosterone stimulates superoxide production in macula densa cells

Two major factors which regulate tubuloglomerular feedback (TGF)-mediated constriction of the afferent arteriole are release of superoxide (O(2)(-)) and nitric oxide (NO) by macula densa (MD) cells. MD O(2)(-) inactivates NO; however, among the factors that increase MD O(2)(-) release, the role of aldosterone is unclear. We hypothesize that aldosterone activates the mineralocorticoid receptor (MR) on MD cells, resulting in increased O(2)(-) production due to upregulation of cyclooxygenase-1 (COX-2) and NOX-2, and NOX-4, isoforms of NAD(P)H oxidase. Studies were performed on MMDD1 cells, a renal epithelial cell line with properties of MD cells. RT-PCR and Western blotting confirmed the expression of MR. Aldosterone (10(-8) mol/l for 30 min) doubled MMDD1 cell O(2)(-) production, and this was completely blocked by MR inhibition with 10(-5) mol/l eplerenone. RT-PCR, real-time PCR, and Western blotting demonstrated aldosterone-induced increases in COX-2, NOX-2, and NOX-4 expression. Inhibition of COX-2 (NS398), NADPH oxidase (apocynin), or a combination blocked aldosterone-induced O(2)(-) production to the same degree. These data suggest that aldosterone-stimulated MD O(2)(-) production is mediated by COX-2 and NADPH oxidase. Next, COX-2 small-interfering RNA (siRNA) specifically decreased COX-2 mRNA without affecting NOX-2 or NOX-4 mRNAs. In the presence of the COX-2 siRNA, the aldosterone-induced increases in COX-2, NOX-2, and NOX-4 mRNAs and O(2)(-) production were completely blocked, suggesting that COX-2 causes increased expression of NOX-2 and NOX-4. In conclusion 1) MD cells express MR; 2) aldosterone increases O(2)(-) production by activating MR; and 3) aldosterone stimulates COX-2, which further activates NOX-2 and NOX-4 and generates O(2)(-). The resulting balance between O(2)(-) and NO in the MD is important in modulating TGF.     

Regulation by GTP of a Ca2+-activated K+ channel in the apical membrane of rabbit cortical collecting duct cells

Ca²⁺-activated K⁺ channels play an important role in Ca²⁺ signal transduction and may be regulated by mechanisms other than a direct effect of Ca²⁺. Inside-out patches of the apical membrane of confluent transformed rabbit cortical collecting duct cells cultured on collagen were subjected to patch clamp analysis. Two types of K⁺ channel, of medium and high conductance, were observed. The latter channel was characterized by a K⁺/Na⁺ permeability ratio of 10, an inwardly rectified current, a conductance of 80 pS at 0 mV, and an open probability dependent on both voltage and Ca²⁺. Guanosine 5-triphosphate (GTP) but not a guanosine 5-diphosphate (GDP) analogue, adenosine 5-triphosphate (ATP), cytidine 5-triphosphate (CTP), or inosine 5-triphosphate (ITP), inhibited the activity of this Ca²⁺-activated K⁺ channel. The inhibitory effect of GTP was dose dependent, with a 50% inhibitory concentration of 10^–5 m in the absence of Mg²⁺. In the presence of Mg²⁺ (1 mm), which is required for the binding of GTP to G proteins, the 50% inhibitory concentration decreased to 3×10^–12 m. Pertussis toxin or cholera toxin (each at 10 ng/ml) did not prevent the inhibitory effect of GTP. After removal of GTP from the medium bathing an inhibited channel, subsequent application of Ca²⁺ failed to activate the channel. Ca²⁺-activated K⁺ channels of smooth muscle cells and proximal tubule cells did not respond to GTP. Thus, the Ca²⁺-activated K⁺ channel in the apical membrane of collecting duct cells is inhibited by GTP, which appears to exert its effect via a G protein that is insensitive to both cholera and pertussis toxins.     

The regeneration of the cells of the macula densa after subtotal nephrectomy in the rat.

An autoradiographic study of the proliferative response of the cells of the Macula Densa days or months after the removal of 5/6 of the rat's kidney has shown that these cells are capable of division. The index of labelling of these cells is considerably lower than that of the other cells of the distal tubules. This applies of the kidney of normal controls and to the remnant after partial nephrectomy, shortly after operation as well as several months later. The cells of the Macula Densa therefore seem to represent a more stable population in the nephron. They therefore differ from the other cells of the distal tubules not only in appearance and function, but also in the pattern of cell proliferation.     

Noise analysis reveals K+ channel conductance fluctuations in the apical membrane of rabbit colon

Summary In this paper we describe current fluctuations in the mammalian epithelium, rabbit descending colon. Pieces of isolated colon epithelium bathed in Na⁺ or K⁺ Ringer's solutions were studied under short-circuit conditions with the current noise spectra recorded over the range of 1–200 Hz. When the epithelium was bathed on both sides with Na⁺ Ringer's solution (the mucosal solution contained 50 m amiloride), no Lorentzian components were found in the power spectrum. After imposition of a potassium gradient across the epithelium by replacement of the mucosal solution by K⁺ Ringer's (containing 50 m amiloride), a Lorentzian component appeared with an average corner frequency,f _c=15.6±0.91 Hz and a mean plateau valueS _o=(7.04±2.94)×10^–20 A² sec/cm². The Lorentzian component was enhanced by voltage clamping the colon in a direction favorable for K⁺ entry across the apical membrane. Elimination of the K⁺ gradient by bathing the colon on both sides with K⁺ Ringer's solutions abolished the noise signal. The Lorentzian component was also depressed by mucosal addition of Cs⁺ or tetraethylammonium (TEA) and by serosal addition of Ba²⁺. The one-sided action of these K⁺ channel blockers suggests a cellular location for the fluctuating channels. Addition of nystatin to the mucosal solution abolished the Lorentzian component. Serosal nystatin did not affect the Lorentzian noise. This finding indicates an apical membrane location for the fluctuating channels. The data were similar in some respects to K⁺ channel fluctuations recorded from the apical membranes of amphibian epithelia such as the frog skin and toad gallbladder. The results are relevant to recent reports concerning transcellular potassium secretion in the colon and indicate that the colon possesses spontaneously fluctuating potassium channels in its apical membranes in parallel to the Na⁺ transport pathway.     

微动脉平滑肌细胞K+通道的研究进展

K+通道维持着血管平滑肌细胞的静息膜电位.目前发现血管微动脉平滑肌细胞上主要表达内向整流型K+通道、ATP敏感型K+通道、电压依赖型K+通道和大电导钙激活型K+通道等四种K+通道.本文对微动脉平滑肌细胞K+通道最新进展做一综述.     

Conduction properties of the cloned Shaker K+ channel.

The conduction properties of the cloned Shaker K+ channel were studied using electrophysiological techniques. Single channel conductance increases in a sublinear manner with symmetric increases in K+ activity, reaching saturation by 0.6 M K+. The Shaker K+ channel is highly selective among monovalent cations; under bi-ionic conditions, its selectivity sequence is K+ > Rb+ > NH+4 > Cs+ > Na+, whereas, by relative conductance in symmetric solutions, it is K+ > NH+4 > Rb+ > Cs+. In Cs+ solutions, single channel currents were too small to be measured directly, so nonstationary fluctuation analysis was used to determine the unitary Cs+ conductance. The single channel conductance displays an anomalous molefraction effect in symmetric mixtures of K+ and NH+4, suggesting that the conducting pore is occupied by multiple ions simultaneously.     

Stability of the Shab K+ channel conductance in 0 K+ solutions: the role of the membrane potential

Shab channels are fairly stable with K⁺ present on only one side of the membrane. However, on exposure to 0 K⁺ solutions on both sides of the membrane, the Shab K⁺ conductance (G_K) irreversibly drops while the channels are maintained undisturbed at the holding potential. Herein it is reported that the drop of G_K follows first-order kinetics, with a voltage-dependent decay rate r. Hyperpolarized potentials drastically inhibit the drop of G_K. The G_K drop at negative potentials cannot be explained by a shift in the voltage dependence of activation. At depolarized potentials, where the channels undergo a slow inactivation process, G_K drops in 0 K⁺ with rates slower than those predicted based on the behavior of r at negative potentials, endowing the r-V_m relationship with a maximum. Regardless of voltage, r is very small compared with the rate of ion permeation. Observations support the hypothesized presence of a stabilizing K⁺ site (or sites) located either within the pore itself or in its external vestibule, at an inactivation-sensitive location. It is argued that part of the G_K stabilization achieved at hyperpolarized potentials could be the result of a conformational change in the pore itself.     

Toxin-induced K+ efflux through the Na+ channel of neuroblastoma cells

Neurotoxins which modify the gating system of the Na+ channel in neuroblastoma cells and increase the initial rate of 22Na+ influx through this channel also give rise to the efflux of 86Rb+ and 42K+. These effluxes are inhibited by tetrodotoxin and are dependent on the presence in the extracellular medium of cations permeable to the Na+ channel. These stimulated effluxes are not due to membrane depolarization or increases in the intracellular content of Na+ and Ca2+ which occur subsequent to the action of neurotoxins. The relationships of 22Na+ influx and 42K+ (or 86Rb+) effluxes to both the concentration of neurotoxins and the concentration of external permeant cations strongly suggest that the open form of the Na+ channel stabilized by neurotoxins permits an efflux of K+ ions. Our results indicate that for the efflux of each K+ ion there is a corresponding influx of two Na+ ions into the Na+ channel.     

Single channel K+ currents from HeLa cells

The extracellular patch-clamp technique was used in order to investigate the presence of ionic channels in HeLa cells, a well-known cultured cell type obtained from an epidermoid carcinoma of the cervix. Under Gigohm-seal conditions, discrete current jumps could be observed with patch electrodes containing KCl. These channels were found to be mainly permeable to K+ and showed multiple levels of conductance. From single-channel I-V curve measurements, a strong rectification effect, characterized by a large inward and no detectable outward current, was observed. For negative membrane potentials (0 to -90 mV), the measured current-voltage relationship was found to be mostly linear, corresponding to a single-channel conductance of 40 pS. An analysis of some selected time records has revealed in addition that the probability of the channel to be in the open state was a function of the KCl concentration in the patch pipette.