Histocytochemical study of Spot 35-calbindin-D28K and Ca2+-ATPase in rat kidney

Summary: We determined the distribution of Spot 35-calbindin-D28K, a vitamin-D dependent calciumbinding protein, in rat kidney using histochemical methods and compared it with the distribution of Ca²⁺-ATPase activity. Spot 35-calbindin-D28K immunoreactivity was localized in the cytosol of urinary epithelial cells in distal convoluted tubules (DCT), connecting tubules (CNT) and cortical collecting ducts (CCD), identifying the physiologically confirmed site of active transcellular calcium transport. In the cytosol, the immunoreactivity was clustered near the luminal plasma membrane and around the mitochondria. These findings indicated that Spot 35-calbindin-D28K seemed to have a cytosolic calcium buffering effect in the urinary tubular epithelial cells. Enzyme histochemical analysis showed that Ca²⁺-ATPase activity was localized at the basolateral plasma membrane of distal nephron segments and was strongest at the cortical thick ascending limb of Henle (CTAL), including the macula densa portion. Ca²⁺-ATPase activity was not evident in DCT, CNT or CCD. Strong Ca²⁺-ATPase activity and Spot 35-calbindin-D28K immunoreactivity did not coexist in a urinary tubular cell.     

Activation of the Ca2+-Activated K+ Channel via Protein Kinase A-Dependent Phosphorylation in Human Prostatic Smooth Muscle Cells

Background: We investigated the functional importance of the Ca²⁺-activated K⁺ channel (K_Ca-channel) of human prostatic smooth muscle cells in cyclic adenosine 3', 5'-monophosphate (cAMP)-induced relaxation, to clarify signal transduction pathways and intracellular mechanisms of relaxation in prostatic smooth muscle.
Methods: Using the patch-clamp technique, we characterized the K_Ca-channel of cultured human prostatic smooth muscle cells. We also investigated the effects on the K_Ca-channels of forskolin, an activator of adenylate cyclase, amrinone, a phosphodiesterase inhibitor, and protein kinase A (A-kinase)-dependent phosphorylation.
Results: Single-channel current recordings from cultured human prostatic smooth muscle cells revealed the presence of K_Ca-channels (conductance 296.7±5.67 pS, n = 7). In cell-attached patch configurations, the K_Ca-channel was activated by forskolin (1CH mol/L) and amrinone (10⁻⁴ mol/L). In inside-out patch configurations, it was activated by catalytic subunits of A-kinase (10 U/mL).
Conclusions: We conclude that the K_Ca-channel of human prostatic smooth muscle cells is regulated by intracellular cAMP levels and that A-kinase mediates the cAMP-induced activation of the K_Ca-channel. This regulation of the K_Ca-channel by cAMP may at least partially explain cAMP-induced prostatic smooth muscle relaxation and the effectiveness of certain drugs for treatment of obstruction in benign prostatic hyperplasia.     

Ca2+ is Essential for the Motility of Plasma Membrane-Intact, but not of demembranated. Hamster Spermatozoa

Extracellular Ca2+ is essential for the flagellar motility of membrane-intact hamster spermatozoa. When suspended in a medium completely free of Ca2+, most spermatozoa quickly lost their motility, and remained motionless until they were transferred back to Ca2+-containing medium. The motility could not be restored after the spermatozoa had been in Ca2+-free medium for more than 2 hr. Unlike membrane-intact spermatozoa, demembranated spermatozoa (spermatozoa without plasma membranes) exhibited active movement in Ca2+-free medium, and their motility was inhibited by Ca2+. In view of these facts, we suggest that the "hyperactivated motility" which membrane-intact spermatozoa display upon capacitation may be due to the activation of a Ca2+-dependent adenylate cyclase (and the resultant increase in intracellular cAMP), rather than being a direct effect of a rise in the intracellular Ca2+ concentration.     

Polymorphonuclear leucocyte rheology and cytosolic Ca2+ content after activation in chronic renal failure

SUMMARY: We evaluated polymorphonuclear leucocyte (PMN) flow properties in patients with clinically stable chronic renal failure (CRF) and in control subjects at baseline and after activation with 4-phorbol 12-myristate 13-acetate (PMA) and N -formyl-methionyl-leucyl-phenylalanine (fMLP). Initial relative flow rate (IRFR) and clogging particles (CPs) were obtained using the St. George's Filtrometer, and PMN membrane fluidity was assessed by marking PMNs with 1-(4-(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). PMN cytosolic Ca²⁺ concentration was determined by marking PMNs with Fura 2-AM. At baseline, CRF patients showed a significant increase only in PMN cytosolic Ca²⁺ content. After activation with PMA and fMLP, a decrease in IRFR and an increase in CP were observed in both control subjects and CRF patients, although the variation in IRFR present in the group of CRF patients was greater than in the control group. After activation with PMA and fMLP, we found a decrease in PMN membrane fluidity only in CRF patients, but no variation in PMN cytosolic Ca²⁺ concentration in either group was observed. These results provide evidence for PMN dysfunction in chronic renal failure.     

Abundance and activity of Ca2+-ATPase in hypercalciuric children

The plasma membrane Ca²⁺-ATPase (PMCA) is one of the main regulators of cell Ca²⁺ homeostasis. The aim of our study was to determine whether the abundance and activity of PMCA are altered in erythrocytes of children with idiopathic hypercalciuria. Twenty-four children with idiopathic hypercalciuria (13 girls and 11 boys, mean age 10.6±4.8 years; mean urinary calcium concentration 0.85±0.20 mmol/mmol creatinine) and 30 healthy age-matched children were enrolled. PMCA protein abundance was determined by Western blot analysis. Enzyme activity was determined spectrophotometrically. The abundance of PMCA did not differ in hypercalciuric patients from that of control subjects (98±22% vs 100±18%). Moreover, the activity was not different between the studied groups (3141±1494 vs 2953±780 nmol ATP/mg protein/h). The extent of hypercalciuria did not correlate with enzyme abundance or activity. Assuming that erythrocytes may reflect the renal tubular transporting processes, our data suggest that other Ca²⁺-transport mechanisms than PMCA might be involved in the development of idiopathic hypercalciuria in children. Received: 5 December 2000 / Revised: 2 May 2001 / Accepted: 3 May 2001     

Adenovirus‐delivered microRNA targeting the vitamin D receptor reduces intracellular Ca2+ concentrations by regulating the expression of Ca2+‐transport proteins in renal epithelial cells

What’s known on the subject? and What does the study add? Experimental data have shown that VDR overexpression in the duodenum and kidney cortex is a biological characteristic of genetic hypercalciuric stone‐forming rats (GHS rat), and a link between idiopathic calcium stone formation and the microstatellite marker D12S339 (near the VDR locus) has been proven in humans. Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin‐D28k and PMCA1b in NRK cell lines. VDR knockdown results in a decrease in intracellular Ca²⁺ concentration in NRK cell lines. The effect of the elevated VDR level in the kidney on hypercalciuria and the underlying mechanisms need to be further addressed.

OBJECTIVE

? To determine the effects of vitamin D receptor (VDR) on hypercalciuria and the mechanisms underlying such effects.

MATERIALS AND METHODS

? The adenovirus vector‐delivered microRNA targeting rat VDR was constructed. We infected the normal rat kidney epithelial cell line NRK (Cellbank, China) with the adenovirus and then collected the cells at 0, 48, 72, 96, 120 h after infection. The mRNA and protein levels of VDR and VDR‐dependent epithelial Ca²⁺ transport proteins were detected using real‐time polymerase chain reaction and Western blot assays, respectively. ? Fluorescent Ca²⁺ indicator Fluo‐4 NW (Fluo‐4 NW calcium assay kit, Molecular Probes, Invitrogen, USA) and laser scanning confocal microscope (Olympus, FV500‐IX71, Japan) were used to detect the cytosolic free Ca²⁺ concentration at different time points after infection.

RESULTS

? The mRNA and protein level of VDR, transient receptor potential vanilloid receptor subtype 5 (TRPV5), calbindin‐D_28k and plasma membrane Ca²⁺‐ATPase (PMCA1b) in infected NRK cells was significantly lower at 72 and 96 h after infection than that in control cells. ? There was no significant difference between the two groups in the mRNA and protein level of TRPV6 and the Na⁺/Ca²⁺‐exchanger (NCX1). ? Furthermore, VDR knockdown results in a decrease in intracellular Ca²⁺ concentration ([Ca²⁺]i) in NRK cell lines.

CONCLUSIONS

? Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin‐D28k and PMCA1b, but not of TRPV6 or NCX1, in NRK cell lines. VDR knockdown results in a decrease in [Ca²⁺]i in NRK cell lines. ? The effect of the elevated VDR level in the kidney on hypercalciuria and the mechanisms underlying need to be further addressed.     

七氟醚后处理对大鼠心肌缺血再灌注时Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性的影响

目的 评价七氟醚后处理对大鼠心肌缺血再灌注时Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性的影响,探讨其减轻心肌缺血再灌注损伤的机制.方法 健康雄性Wistar大鼠45只,体重250～280 g,随机分为3组(n=15):假手术组(S组)、缺血再灌注组(I/R组)和七氟醚后处理组(Spo组).I/R组和Spo组采用结扎左冠状动脉前降支30 min时进行再灌注的方法制备心肌缺血再灌注模型,S组仅在左冠状动脉前降支下穿线.Spo组再灌注前1 min开始吸入2.5%七氟醚持续5 min.于再灌注2 h时取心肌组织,测定心肌梗死体积、Na+-K+-ATP酶活性和Ca2+-Mg2+-ATP酶活性.结果 与S组比较,I/R组心肌梗死体积扩大,心肌组织Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶的活性降低(P＜0.05);与I/R组比较,Spo组心肌梗死体积缩小,心肌组织Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶的活性升高(P＜0.05).结论七氟醚后处理可提高Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶的活性,从而减轻大鼠心肌缺血再灌注损伤.     

氯胺酮对大鼠大脑皮质和丘脑突触体ATP酶活性的影响

目的 动态观察氯胺酮对大鼠大脑皮质和丘脑Na+-K+ATP酶和Ca2+-ATP酶活性的影响。方法 SD大鼠32只,随机分为四组:对照组、麻醉组、恢复I组和恢复Ⅱ组。对照组给予腹腔注射生理盐水10 ml·kg-1,10 min后断头;其它三组均为腹腔注射氯胺酮100 mg·kg-1。其中麻醉组在大鼠翻正反射消失后断头;恢复I组在大鼠翻正反射恢复后断头;恢复Ⅱ组在大鼠完全清醒后断头。在生理盐水冰面上分离双侧大脑皮质和丘脑,以分光光度法测大脑皮质和丘脑Na+-K+-ATP酶和Ca2+-ATP酶活性。结果大鼠腹腔注射氯胺酮后,其大脑皮质的Na+-K+-ATP酶和Ca2+-ATP酶活性分别较对照组降低32.8％和26.2％(P<0.05);丘脑的Na+-K+-ATP酶和Ca2+-ATP酶活性也分别较对照组降低31.4％和24.5％(P<0.05);大鼠翻正反射恢复后和动物完全清醒后两种ATP酶活性的变化与对照组相比差异均无统计学意义(P>0.05)。结论 Na+-K+-ATP酶和Ca2+-ATP酶活性的改变在氯胺酮全麻作用机制中可能起重要作用。     

Purification and characterization of phospholipase A2 from bovine prostate

Phospholipase A2 (PLA2) was purified from bovine prostate by ammonium sulphate precipitation and fractionation by anion exchange chromatography, chromatofocusing and gel filtration. The purified enzyme was Ca(2+)-dependent and had a pH-optimum of 8.0. Ba2+, Fe2+, Hg2+, Mg2+, Pb2+, Sr2+ and Zn2+ as well as lysophosphatidylcholine, lysophosphatidylethanolamine and p-bromophenacyl bromide (p-BPB) inhibited the enzyme strongly. The enzyme had an estimated molecular weight of 12,000 +/- 1,000 daltons on SDS-PAGE. Isoelectric focusing showed one PLA2 activity-containing band at pl 5.3. The purified enzyme hydrolysed linoleic acid at the sn-2 position of phosphatidylcholine and phosphatidylethanolamine with high selectivity, compared to arachidonic acid.     

遗传高钙尿结石大鼠肾小管上皮细胞基底膜钙转运蛋白的异常表达及意义

目的 观察肾小管上皮细胞基底膜钙转运蛋白细胞膜钙泵1b(PMCA1b)和钠钙交换体.(NCX1)在遗传性高钙尿结石(GHS)大鼠肾组织的表达及其在特发性高钙尿症(IH)发病中的作用.方法 雄性遗传高钙尿结石大鼠和正常野生型SD大鼠各6只,采用实时荧光定量PCR检测PMCA1b和NCX1 mRNA表达,westem blot方法 检测其蛋白表达.结果 GHS大鼠与正常对照(NC)组大鼠的NCX1在mRNA水平和蛋白水平比较差异均无统计学意义(P＞0.05);GHS大鼠与NC大鼠的PMCA1b在mRNA水平比较差异无统计学意义(P＞0.05),GHS大鼠的PMCA1b蛋白平均相对吸光度值(A)为(0.18±0.05),NC组为(0.43±0.07),差异有统计学意义(P＜0.01).结论 PMCA1b蛋白表达下降可能是特发性高钙尿症形成的发病机制之一.     

安氟醚麻醉大鼠不同时期脑Ca2+-ATP酶活性变化

目的动态观察安氟醚吸入麻醉大鼠不同时期各脑区Ca2+-ATP酶活性变化和行为学变化,探讨安氟醚的麻醉作用机制.方法 40只SD雌性大鼠随机均分为五组诱导期组、麻醉期组、恢复期组、清醒期组和对照组.分光光度法测定不同时期大脑皮层、脑干、海马Ca2+-ATP酶活性.结果在诱导期各脑区Ca2+-ATP酶活性即开始下降(P＜0.05);麻醉期降至最低(P＜0.01);恢复期又开始升高,但仍低于对照组水平.其中,仅脑干Ca2+-ATP酶活性降低有显著性差异(P＜0.05);清醒期基本恢复至对照组水平(P＞0.05).且行为学变化与脑Ca2+-ATP酶活性变化有关.结论安氟醚的麻醉效应可能与其抑制脑Ca2+-ATP酶活性相关,麻醉深浅与抑制的程度一致.     

Effect of calmodulin-like protein from buffalo (Bubalus bubalis) seminal plasma on Ca2+, Mg2+-ATPase of purified plasma membrane of buffalo spermatozoa

Summary. Buffalo sperm heads and tails were cleaved by sonication and isolated in relatively pure proportions i.e. 95% and 98% respectively, by discontinuous sucrose density-gradient centri-fugation. Purified plasma membranes from the isolated sperm heads and tails were obtained by hypotonic treatment and brief sonication followed by discontinuous sucrose density-gradient centrifugation. Ca²⁺, Mg²⁺-ATPase activity was evident in plasma membrane from sperm heads and tails, although activity was greater in the latter. A calmodulin-like protein isolated from buffalo seminal plasma increased the Ca²⁺, Mg²⁺-ATPase of plasma membrane from the sperm heads and tails by 128 and 136% respectively. Based upon the data obtained here and elsewhere (Sidhu & Guraya, 1989a) a model is proposed which explains regulation of Ca²⁺ in buffalo spermatozoa and implicates calmodulin-like protein and Ca²⁺, Mg²⁺-ATPase in sperm acrosome reaction.     

Estimation of Blood Flow in Transcutaneous PO2 Measurements

A method for estimation of the cutaneous blood flow in transcutaneous Po₂ measurements is presented. Recordings of electrode and skin temperature make it possible to compute the effect dissipated to the circulating blood. Eighteen measurements were performed on three healthy volunteers at electrode temperature settings from 37.0°C to 45.0°C. The blood-flow estimates ranged from 0.07 to 0.19 ml·cm^-2·min^-1. At an electrode temperature of 45.0°C the investigations showed a tc-Po₂ value as low as 7 mmHg (0.9 kPa) which, however, corresponded well to the lowest blood-flow estimate determined. The temperature-corrected (37°C) a-Po₂-tc-Po₂ gradient ranged from 50 mmHg to 95 mmHg (6.7–12.6 kPa). The investigations confirm the importance of simultaneous determinations of cutaneous blood flow, capillary temperature and cutaneous oxygen consumption in order to describe the connection between arterial and cutaneous oxygen tension. The cutaneous blood How seems in this connection to be the most important parameter.     

The effect of flow on the neutrophil-mediated Ca2+ responses in human vascular endothelial cells stimulated by endotoxin

2+ levels ([Ca²⁺]_i). Cultured human umbilical vein ECs stimulated by endotoxin were labeled with Fura-2 and exposed to fluid flow with neutrophils. The individual changes in [Ca²⁺]_i were monitored. The application of flow with neutrophils to stimulated ECs led to an increase in [Ca²⁺]_i although either flow without neutrophils or neutrophils without flow rarely induced a rise in [Ca²⁺]_i. Furthermore, flow application with neutrophils to unstimulated ECs also rarely promoted a rise in [Ca²⁺]_i. These findings suggest that the flow might thus induce or enhance the inflammatory process by the induction of Ca²⁺ signaling in endotoxin-stimulated endothelium facing neutrophils in the blood flow. (Received for publication on Sept. 28, 1998; accepted on Mar. 11, 1999)     

PGE1 and hCG Responsive Adenylyl Cyclases in Interstitial Cells from Rat Testis: Biphasic Effects of Calcium

The effects of PGE₁ (10 μg/ml), hCG (10 mIU/ml) and Ca²⁺ (0–10 mM) on rat interstitial cell adenylyl cyclase (AC) activity have been investigated. Like hCG, PGE₁ also stimulated membrane bound AC, and both hormonal effects proved to be influenced by Ca²⁺. Low concentrations of Ca²⁺ (0.3 mM) enhanced hormonal stimulation, while high concentrations (up to 10 mM) suppressed AC activity. Relative stimulations caused by PGE₁ and hCG were maximal in the presence of 0.6 mM Ca²⁺. Ovine FSH (NIH-S12), at concentrations causing maximal activation of AC in seminiferous tubules (12.5 μg/ml), had no effect on AC in interstitial cells. Germinal cell membranes displayed insignificant AC activity. We therefore conclude that the AC activity stimulable by PGE₁ is located on Leydig cell membranes and that both PGE₁ and hCG activities are susceptible to regulation by Ca²⁺.     

Muscarinic M2 receptors inhibit Ca2+-activated K+ channels in rat bladder smooth muscle.

PURPOSE: To clarify the functional relationship between M2 muscarinic receptor and Ca2+-activated K+ channel, we investigated the effect of carbachol (CCh) on the membrane current of rat bladder smooth muscle cells. METHODS: Rat bladder single smooth muscle cells were patch clamped with whole-cell configuration. RESULTS: CCh (10 micro mol/L) transiently induced an outward current in the presence of K+ in the pipette solution. A high Ca2+ concentration in the pipette solution persistently induced an outward current, which was inhibited by CCh. In the presence of M2 inhibitor, AFDX-384, CCh induced the outward current persistently, indicating that M2 was involved in the current inhibition. In pertussis toxin pretreated cells, CCh did not apparently inhibit the outward current. The CCh-induced outward current was inhibited by iberiotoxin, a selective inhibitor of large-conductance Ca2+-activated K+ channels (BKCa). CONCLUSION: CCh induces BKCa, which is inhibited by M2- and Gi-mediated signal transduction pathway. This M2-mediated pathway may enhance contraction which is initiated by M3-stimulation in rat bladder smooth muscle.