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Effects of aspartic acid and potassium chloride on arginine kinase from shrimp.

The aspartic acid (Asp)-induced unfolding and the salt-induced folding of arginine kinase (AK) were studied in terms of enzyme activity, intrinsic fluorescence emission spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that Asp caused inactivation and unfolding of AK with no aggregation during AK denaturation. The unfolding of the whole molecule and the inactivation of AK in different Asp concentrations were compared. Much lower Asp concentration was required to induce inactivation than to produce significant conformational changes of the enzyme molecule. However, with further addition of Asp, the molar ellipticity at 222 and 208 nm, the wavelength shift and the emission intensity of ANS hardly changed. Asp denatured AK was reactivated by dilution. In addition, potassium chloride (KCl) induced the molten globule state with a compact structure after AK was denatured with 7.5 mM Asp. These results collectively elucidate the osmotic effect of Asp anions for the molten globule formed during unfolding process. They also suggest that the effect of Asp differed from that of other denaturants such as guanidine hydrochloride or urea during AK folding. The molten globule state indicates that intermediates exist during AK folding.

Animals↗

Initial, continuous and intermittent bolus cardioplegia administration: efficacy of potassium-chloride and magnesium-sulfate as minimal additives for minimally-diluted blood cardioplegia.

AIM: The most effective delivery of blood cardioplegia (BCP) remains controversial, and a combination of initial continuous and intermittent bolus BCP seems to compensate each demerit. However, a large amount of crystalloid solution is infused into the myocardium in this method, which may nullify the advantages of BCP. We examined the hypothesis that minimally-diluted BCP resolves this issue and provides superior myocardial protective effects. METHODS: Seventy patients undergoing elective coronary revascularization between 1997-2001 (M:F=55:15, mean age 67.6+/-7.5 years) were randomly allocated into one of 2 groups: Group C (n=35) was given the standard 4:1-diluted blood-crystalloid BCP, and Group M (n=35) was given minimally-diluted BCP supplemented with potassium-chloride and magnesium-sulfate. The BCP temperature was maintained at 30 degrees C. Cardioplegic arrest was induced with 2 minutes of initial antegrade BCP infusion, followed by continuous retrograde BCP infusion. Intermittent antegrade BCP was infused every 30 minutes for 2 minutes. RESULTS: The time required for achieving cardioplegic arrest was significantly shorter in Group M (47.5+/-16.3 vs 62.5+/-17.6 s, p<0.0001) and the number of patients showing spontaneous heart-beat recovery after aortic unclamping was significantly larger in Group M (28 vs 15, p=0.0029). The number of patients suffering from atrial fibrillation during the postoperative period was significantly smaller in Group M (3 vs 11, p=0.034). The total amount of crystalloid solution infused as cardioplegia was significantly smaller in Group M (62.8+/-22.3 vs 733.6+/-382.6 mL, p<0.0001). Postoperative maximum dopamine dose (3.57+/-2.46 vs 5.44+/-2.23 mg/kg/min, p=0.0014) and peak creatine kinase-MB (19.5+/-8.5 vs 25.8+/-11.9 IU/L, p=0.0128) were significantly lower in Group M. The number of patients showing paradoxical movement of the ventricular septum by early postoperative echocardiography was significantly smaller in Group M (9 vs 24, p<0.0007). CONCLUSIONS: These results demonstrate that initial continuous and intermittent bolus administration of minimally-diluted BCP supplemented with potassium and magnesium can be a simple, reliable and effective technique of intraoperative myocardial protection.

Aged↗

Research note: sodium and potassium chloride drinking water supplementation effects on acid-base balance and plasma corticosterone in broilers reared in thermoneutral and heat-distressed environments.

One experiment utilizing 188 Vantress x Arbor Acres broilers was conducted to evaluate the effects of isomolar KCl (.5%) and NaCl (.39%) drinking water supplementation on venous pH, PO2, PCO2, HCO3-, hematocrit, and plasma corticosterone as well as rectal temperature and water consumption of broilers reared in heat-distressed and thermoneutral environments. Birds were allotted at 5 wk posthatch into either a thermoneutral (TN; 24 C) or cycling temperature (24 to 35 C) environmental chamber. Heat-distressed (HD) controls had elevated (P less than .05) body temperature (42.9 versus 41.7 C) and PO2 (144.5 versus 108.4 mm Hg). Blood pH, PCO2, and HCO3- (P greater than .1) were not affected by high ambient temperature, but hematocrit (31 versus 32%) for HD controls was reduced (P = .07) compared with TN control birds. At 35 C, drinking water NaCl supplementation decreased (P less than .05) venous PCO2 and HCO3-, increased (P less than .05) PO2, and had no effect on venous pH and water consumption relative to HD controls. Potassium chloride lowered (P = .07) venous pH, decreased (P less than .05) HCO3-, and increased water consumption at 35 C, but PO2 and PCO2 were unaffected relative to HD controls. Heat distress increased (P less than .05) plasma corticosterone by 53%. Sodium chloride failed to impact (P greater than .1) plasma corticosterone but KCl reduced (P less than .05) its concentration during heat distress and increased (P less than .05) bird survivability as compared with heat-distressed controls. The data reported herein suggests that KCl and to a lesser extent NaCl reduces HD consequences by a mechanism as yet undefined.

Acid-Base Equilibrium↗

Renal function and magnesium clearance in young and old cows given potassium chloride and sodium citrate.

Six young (3 yr old) and six old (8 to 11 yr old) cows were used to examine renal function, KCl and sodium citrate effects on Mg clearance rate and the relation of parathyroid hormone (PTH) to Mg reabsorption. The treatments were 1.5 g KCl/kg body weight (BW) and 1.5 g Na citrate/kg BW. Inulin and p-aminohippuric acid (PAH) clearance rates were used as measures of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), respectively, in control cows on each treatment day. Creatinine clearance rate was also used as a measure of GFR in all cows. Magnesium loading (2.4 mg/kg BW) through continuous intrajugular infusion of MgCl2 facilitated the measurement of changes in Mg clearance rate and reabsorption due to treatment. Inulin and creatinine clearance rates were not different as measures of GFR, and there were no differences in ERPF between young and old cows. Old cows had lower (P less than .05) GFR, creatinine to PAH clearance ratios and tubular reabsorption of Mg than young cows. Intraruminal infusion of KCl and Na citrate increased (P less than .05) the GFR. Potassium chloride increased plasma K concentration (P less than .001), plasma K clearance rate (P less than .002), plasma Na clearance rate (P less than .03), plasma Mg concentration (P less than .05), net tubular reabsorption of Mg (P less than .02), maximum tubular reabsorption of Mg (P less than .002) and plasma PTH concentration (P less than .03). Plasma Mg clearance rate was increased only by Na citrate (P less than .05). These results suggest a possible relationship between age, renal function, Mg antagonists and PTH in Mg homeostasis and in the etiology of hypomagnesemic tetany.

Animals↗

Elution of Acid Phosphatase from the Cell Surface of Saccharomyces mellis by Potassium Chloride.

Weimberg, Ralph (Northern Regional Research Laboratory, Peoria, Ill.), and William L. Orton. Elution of acid phosphatase from the cell surface of Saccharomyces mellis by potassium chloride. J. Bacteriol. 90:82-94. 1965.-Acid phosphatase of Saccharomyces mellis may be eluted from intact resting cells by 0.5 m KCl or other salts. However, the enzyme is not eluted at higher salt concentrations of about 2 m unless a thiol, such as beta-mercaptoethanol, is included in the reaction mixture. These treatments do not significantly affect viability of the cells. Neutral compounds like sorbitol or sucrose cannot substitute for ionic compounds in eluting the enzyme from resting cells. Furthermore, the neutral compounds are also inadequate for stabilizing the protoplast structure. It is suggested that the enzyme is held on the cell surface by a combination of electrostatic forces and disulfide bonds. Thiol alone dissociates protein and carbohydrate from the cell surface, but the eluate has no acid phosphatase activity. Salts also remove protein and carbohydrate from the cell surface, but the amount of protein removed is considerably less than that dissociated by thiol. A concentration of 0.5 m KCl elutes more protein than does a 2 m concentration, and enzymatic activity is present only in the 0.5 m KCl eluate. The carbohydrate eluted by either reagent has been identified as a mannan. Conditions for eluting acid phosphatase from acetonedried cells of S. mellis are essentially the same as those for resting cells. Significantly, though, thiol is required at all salt concentrations to dissociate the enzyme. Pretreatment of the cells with thiol, followed by KCl, elutes acid phosphatase, whereas the reverse procedure does not. Acid phosphatase is excreted by growing cells of S. mellis into growth media if the medium contains 0.25 m KCl. The total yield of enzymatic activity may be 8 to 10 times greater than is usually present on derepressed cells grown in a salt-free medium. The enzyme can be precipitated from the culture fluid with acetone. The acetone-precipitated fraction contains mannan and protein in a ratio of 12:1 by weight. Partial purification of the enzyme by calcium phosphate gel and elution resulted in an enzyme fraction in which the specific activity on the basis of protein increased 12-fold, and the carbohydrate-protein ratio was reduced to 1:1.

Journal Article↗

Human potassium chloride cotransporter 1 (SLC12A4) promoter is regulated by AP-2 and contains a functional downstream promoter element.

Most K-Cl cotransport in the erythrocyte is attributed to potassium chloride cotransporter 1 (KCC1). K-Cl cotransport is elevated in sickle erythrocytes, and the KCC1 gene has been proposed as a modifier gene in sickle cell disease. To provide insight into our understanding of the regulation of the human KCC1 gene, we mapped the 5' end of the KCC1 cDNA, cloned the corresponding genomic DNA, and identified the KCC1 gene promoter. The core promoter lacks a TATA box and is composed of an initiator element (InR) and a downstream promoter element (DPE), a combination found primarily in Drosophila gene promoters and rarely observed in mammalian gene promoters. Mutational analyses demonstrated that both the InR and DPE sites were critical for full promoter activity. In vitro DNase I footprinting, electrophoretic mobility shift assays, and reporter gene assays identified functional AP-2 and Sp1 sites in this region. The KCC1 promoter was transactivated by forced expression of AP-2 in heterologous cells. Sequences encoding the InR, DPE, AP-2, and Sp1 sites were 100% conserved between human and murine KCC1 genes. In vivo studies using chromatin immunoprecipitation assays with antihistone H3 and antihistone H4 antibodies demonstrated hyperacetylation of this core promoter region.

5' Untranslated Regions↗

Effects of medetomidine on serum osmolality; urine volume, osmolality and pH; free water clearance; and fractional clearance of sodium, chloride, potassium, and glucose in dogs.

OBJECTIVE: To determine effects of i.v. medetomidine administration on selected clinicopathologic variables in dogs. ANIMALS: 6 healthy adult Beagles. PROCEDURE: Dogs were randomly assigned to each of 3 treatments in a crossover study design. Serum osmolality, urine osmolality, urine pH, and fractional clearances of sodium, chloride, potassium, and glucose were determined before and 20, 40, 60, 120, 180, 240, 300, 360, 420, and 480 minutes after i.v. administration of 0.9% NaCl (saline) solution (control) or medetomidine (10 or 20 micrograms/kg of body weight). The urinary bladder was emptied prior to saline or medetomidine administration, and urine volume was determined at the same posttreatment times as those described previously. Free water clearance was calculated for all posttreatment times. RESULTS: After medetomidine administration, serum osmolality, urine volume, free water clearance, and fractional clearance of potassium and glucose increased; urine osmolality decreased. Initially, urine pH and fractional clearance of chloride decreased, then subsequently increased. Fractional clearance of sodium did not change. CONCLUSIONS AND CLINICAL RELEVANCE: Because i.v. administration of medetomidine to dogs at dosages of 10 and 20 micrograms/kg induces a diuretic effect that lasts up to 4 hours, the drug should be used with discretion in hypovolemic or dehydrated dogs, and its use should be avoided in those with urinary tract obstruction.

Adrenergic alpha-Agonists↗

Evaluation of the i-STAT system: a portable chemistry analyzer for the measurement of sodium, potassium, chloride, urea, glucose, and hematocrit.

OBJECTIVE: To evaluate the analytical performance of the i-STAT system, which is designed for point of care testing and employs a hand-held chemistry analyzer and disposable cartridges, which in the configuration tested, are capable of measuring sodium, potassium, chloride, urea, glucose, and hematocrit in approximately 65 microL of blood in 90's. METHODS: Linearity and imprecision in hematocrit measurement were assessed using whole blood, while that for the other analytes were evaluated with aqueous solutions. The accuracy of the i-STAT system was judged by assay of patient specimens obtained both by venipuncture and fingerprick and correlated with the Kodak Ektachem 700XR and the microhematocrit methods. RESULTS: Linearity was obtained over the clinically relevant range for all analytes. Total imprecision as expressed by the coefficient of variation (CV) was less than 3.5% for all analytes at both high and low concentration except for a low concentration of urea where a CV of 9.4% was obtained. Linear regression analysis revealed minimal systematic errors. The standard error about the regression line (Sy/x) ranged from 0.017 for hematocrit to 2.262 for chloride in the assay of venous blood, whereas in the assay of capillary blood the Sy/x ranged from 0.018 for hematocrit to 0.755 for glucose. CONCLUSIONS: The analytical performance of the i-STAT was deemed acceptable by calculation of total error and comparison with published performance standards. Our study has shown the i-STAT system to be reliable, robust, and simple to operate. Moreover, the compactness of the analyzer and the requirement for only small volumes of whole blood will make it a valuable diagnostic tool in point of care settings.

Blood Chemical Analysis↗

Sodium-potassium-chloride cotransport.

Obligatory, coupled cotransport of Na(+), K(+), and Cl(-) by cell membranes has been reported in nearly every animal cell type. This review examines the current status of our knowledge about this ion transport mechanism. Two isoforms of the Na(+)-K(+)-Cl(-) cotransporter (NKCC) protein (approximately 120-130 kDa, unglycosylated) are currently known. One isoform (NKCC2) has at least three alternatively spliced variants and is found exclusively in the kidney. The other (NKCC1) is found in nearly all cell types. The NKCC maintains intracellular Cl(-) concentration ([Cl(-)](i)) at levels above the predicted electrochemical equilibrium. The high [Cl(-)](i) is used by epithelial tissues to promote net salt transport and by neural cells to set synaptic potentials; its function in other cells is unknown. There is substantial evidence in some cells that the NKCC functions to offset osmotically induced cell shrinkage by mediating the net influx of osmotically active ions. Whether it serves to maintain cell volume under euvolemic conditons is less clear. The NKCC may play an important role in the cell cycle. Evidence that each cotransport cycle of the NKCC is electrically silent is discussed along with evidence for the electrically neutral stoichiometries of 1 Na(+):1 K(+):2 Cl- (for most cells) and 2 Na(+):1 K(+):3 Cl(-) (in squid axon). Evidence that the absolute dependence on ATP of the NKCC is the result of regulatory phosphorylation/dephosphorylation mechanisms is decribed. Interestingly, the presumed protein kinase(s) responsible has not been identified. An unusual form of NKCC regulation is by [Cl(-)](i). [Cl(-)](i) in the physiological range and above strongly inhibits the NKCC. This effect may be mediated by a decrease of protein phosphorylation. Although the NKCC has been studied for approximately 20 years, we are only beginning to frame the broad outlines of the structure, function, and regulation of this ubiquitous ion transport mechanism.

Animals↗

Effects of protein kinase C activation on sodium, potassium, chloride, and total CO2 transport in the rabbit cortical collecting tubule.

Several hormones induce phosphatidylinositol turnover in cell membranes and thus activate protein kinase C. Activation of protein kinase C can, in turn, have effects on epithelial transport. These experiments were designed to investigate the effects of two activators of protein kinase C, phorbol 12-myristate,13-acetate (PMA) and L-alpha-1,2-dioctanoylglycerol (L-alpha-1,2-DOG), and two inactive analogues, 4 alpha-phorbol and 4-O-methyl phorbol 12-myristate,13-acetate, on sodium, potassium, chloride, and total CO2 transport in the rabbit cortical collecting tubule. Utilizing in vitro microperfusion techniques, we found that activation of protein kinase C with either PMA or L-alpha-1,2-DOG significantly inhibited net sodium absorption, net potassium secretion and transepithelial voltage in a dose-dependent manner. There was no effect on net chloride or total CO2 transport. In contrast, the inactive phorbol analogues did not alter either sodium or potassium transport. These studies demonstrate that in the rabbit cortical collecting tubule sodium and potassium transport can be inhibited by compounds known to activate proteins kinase C. Thus, hormones that induce phosphatidylinositol turnover in the rabbit cortical collecting tubule may lead to inhibition of sodium transport by activation of protein kinase C.

Animals↗