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Commercially available ready to use syringes (Radiometer, Sarstedt, Ciba-Corning and Portex), containing heparinate as an anticoagulant have been tested to evaluate the magnitude of induced preanalytical errors. Tonometered serum pools adjusted to four Ca2+ concentrations were sampled anaerobically.Ca2+ and pH (ICA2 with 3 digits, Radiometer Medical A/S, Denmark; Heparin: anti-Xa factor activity on a chromogenic substrate. Results were expressed as means of 10 measurements and as percentages of the reference values. Sarstedt syringes, (Li-heparinate), yielded a negative bias (-3%). However for 0.5 or 1 mL samples the bias reached -4% to -6%. Radiometer syringes (QS50 and QS90; calcium titrated heparinate) demonstrated biases below -2%. The bias in the Ciba-Corning (Gas-Lyte) syringe was below 2%. Portex (Pulsator) syringes showed biases above +4% even for nominal sampling volumes. All syringes (except Pulsator) released anticoagulant amounts corresponding to the expected values. Radiometer and Ciba-Corning were the only recommendable devices.

The angiotensin I converting enzyme inhibitor (ACEI) perindopril (2 mg/kg body weight), the peripheral vasodilator dihydralazine (DHL) (25 mg/kg body weight) or distilled water was given daily from birth to day 14 to neonatal rats. Blood pressure, plasma creatinine, plasma renin activity (PRA), substrate (PRS) and concentration (PRC) and renin content of kidney tissue sections were evaluated on days 14 and 28. By day 14, a high mortality in the ACEI group was observed. ACEI, but not DHL, led to a significant fall (P < 0.01) in blood pressure, 57 +/- 11 versus 89 +/- 25 in the DHL group and 103 +/- 24 mmHg in controls, and to a dramatic increase in plasma creatinine. PRA and PRS were undetectable in ACEI-treated rats; in contrast, PRC and renal staining with anti-renin antibody were significantly increased in ACEI rats. On day 28, the blood pressure was normal in all groups and plasma creatinine returned to the normal range in ACEI rats. PRA, PRS and PRC were not significantly different in the ACEI group and controls. These results suggest that the renin-angiotensin system (RAS) plays a major postnatal role in the neonatal rat. Inhibition of the RAS during the first 2 weeks of life leads to high mortality, severe hypotension, reversible renal failure and a defect in circulating angiotensinogen.

Dopamine decreases tubular sodium reabsorption, attributed in part to Na-K-ATPase inhibition in the proximal convoluted tubule (PCT). Because the final regulation of sodium excretion occurs in the collecting duct, where specific dopamine DA1 binding sites have been demonstrated, we examined the effects of dopamine, as well as of DA1 and DA2 receptor agonists on Na-K-ATPase activity and on the number of units in Madin-Darby canine kidney (MDCK) cells, which retain differentiated properties of the renal cortical collecting tubule epithelium. Dopamine (10(-5) M) inhibited pump activity (by 50%) and reduced the number of units. This effect was reproduced by the DA1 agonist SKF 38393, which inhibited pump activity in a dose- and time-dependent manner (maximum, 10(-5) M). The DA2 agonist quinpirole hydrochloride was without effect, either alone or in combination with SKF 38393. Inhibition of pump activity by dopamine was totally abolished by H7 (100 microM), an inhibitor of protein kinase (PK), but partially by 2',5'-dideoxy-adenosine (DDA) and H4, respective inhibitors of cAMP production and PKA, which suggests that the dopamine effect on Na-K-ATPase activity may be linked to activation of both PKC and PKA. In these cells, amiloride addition during preincubation did not alter the effect of dopamine on Na-K-ATPase activity; in contrast, furosemide increased further the inhibitory effect of dopamine on the enzyme activity. Monensin addition (10(-3) M) reversed the inhibitory effect of dopamine after a 30-min preincubation.(ABSTRACT TRUNCATED AT 250 WORDS)

To evaluate the influence of protein kinase C (PKC) activation on Na/K-ATPase activity in MDCK cells, we studied the effect of phorbol myristate acetate (PMA) and two diacylglycerol analogues, oleoylacetylglycerol and dioctanoylglycerol, on the enzyme activity. Na/K-ATPase activity was determined by cytochemistry. PMA induced a time- and dose-dependent inhibition of Na/K-ATPase activity and at 100 ng/ml decreased the enzyme activity by 55% of the initial value. These effects were mimicked by oleoylacetylglycerol and dioctanoylglycerol, and were abolished by two inhibitors of PKC, 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7) and sphingosine. A phorbol ester that does not activate PKC, 4 alpha-phorbol 12,13-didecanoate, did not inhibit Na/K-ATPase activity. PMA inhibition persisted in the presence of cycloheximide and actinomycin D but not in the presence of amiloride. Dopamine (10 microM) inhibition of Na/K-ATPase activity was abolished in a dose-dependent manner by sphingosine. Results suggest that in MDCK cells Na/K-ATPase is an effector protein for PKC and that dopamine inhibition of its activity may be mediated by PKC.

Na + ,K + -adenosine triphosphatase (Na + ,K + -ATPase) is an integral protein usually located in the basolateral membrane [1, 2] that actively pumps Na + out and K + in to the cell, through ATP hydrolysis [3]. The activity of Na + ,K + -ATPase is essential for the existence of water and solute transport by epithelial cells [4,5]. Measurement of Na + ,K + -ATPase activity is usually done by biochemical assays. Cytochemical methods [6] allow cellular enzyme localization and may offer an alternative approach for determination of Na + ,K + -ATPase activity in disrupted cells. We have used a cytochemical method, based on measurements of inorganic phosphate generated during ATP hydrolysis by ATPases during the incubation period and precipitated by a lead ammonium citrate acetate complex (LACA) as lead phosphate [Pb 3 (PO 4 ) 2 ]. The colorless lead phosphate was converted into a brown precipitate of lead sulphide (PbS) by treatment with ammonium sulphide. We chose to evaluate Na + ,K + -ATPase activity in MDCK (Madin-Darby canine kidney) cells because they retain many differentiated properties characteristic of distal tubule epithelia [4, 7]. In these cells, apical to basolateral salt and water transport was shown to be inhibited by ouabain and amiloride [7–10]. We show that this method is a simple, convenient, and could be easily performed to measure Na + ,K + -ATPase activity and its modulation under physiological conditions.

To test prolactin (PRL) action on osmoregulation in mammals, we evaluated in the rat the effect of this hormone on a major enzyme in renal regulation of water and electrolyte: renal Na-K-ATPase. Enzyme activity was determined by cytochemistry in medullary ascending limb (MAL) and distal convoluted tubule (DCT) from rats treated either by bromocriptine, or by PRL. Three hours after a bromocriptine injection (0.1 mg/100 g IP) a significant decrease of Na-K-ATPase activity is observed in both MAL (80% of control values, p less than 0.001) and DCT (78% p less than 0.01). Reciprocally, a significant (p less than 0.001) increase in enzyme activity is induced 3 h after a single PRL injection (140 micrograms/100 g IM), in both segments (MAL: 165%, DCT: 172% of control activities) and persists 6 h after the injection (MAL: 130%, DCT: 118%). Na-K-ATPase activity was correlated to plasma PRL levels (r = 0.78 in DCT, r = 0.89 in MAL). A direct effect of PRL on the tubule is suggested by results from experiments in which PRL, at various concentrations, is added in vitro on renal slices before Na-K-ATPase activity measurements. The increase in Na-K-ATPase activity exhibits a log-dose dependency with PRL concentration (p less than 0.01) and is still observed when AVP antagonist is added before PRL incubation, ruling out the possible role of AVP contamination of PRL. These results suggest a direct effect of PRL on renal Na-K-ATPase in MAL and DCT.

Parathyroid response to aluminum in vitro: ultrastructural changes and PTH release. The endocrine response of porcine parathyroid gland tissue slices in vitro to aluminum was studied by electron microscopy and radioimmunoassay of PTH. Medium aluminum concentrations were 20 to 500 ng/ml covering the range corresponding to concentrations reported in the plasma of aluminum-intoxicated hemodialyzed patients. Aluminum inhibited iPTH-release and caused severe cell alterations. This inhibition was incomplete and there was an aluminum–insensitive iPTH-release capacity. This phenomenon seemed to be due to heterogeneous parathyroid cell population as regards aluminum sensivity, perhaps linked to the spontaneous asynchronous cyclic parathyroid cell changes. Sensitivity to aluminum was modulated by the extra–cellular calcium concentration. Sensitivity to extra–cellular calcium concentration variations persisted in aluminum intoxicated tissues. The severity of the observed cell lesions induced by high concentrations of aluminum suggested that the recovery of an iPTH-release capacity when parathyroid tissue was withdrawn from a toxic environment and switched to aluminum–free media is more likely to be due to activation of a “less-sensitive to aluminum” cell pool than to a true reversibility of the toxic effect.