Clinton N. Corder

Variable Levels of Plasma Catecholamines and Dopamine β-Hydroxylase in Hemodialysis Patients

Plasma norepinephrine (NE), epinephrine (EPI), dopamine (DPM), dopamine beta-hydroxylase (DBH), and renin were measured in patients undergoing maintenance hemodialysis. The predialysis NE, EPI, and DPM concentrations were lower than normal at 29, 7.3 and 4.8 pg/ml, respectively. The NE level increased in the erect posture to 100 pg/ml at 30 min. Levels were also measured during the postdialysis period, and were approximately the same as in normal subjects, but lower than previously published values for patients undergoing hemodialysis. In the predialysis period, NE and renin correlated upon assumption of the upright posture. It is concluded that maintenance hemodialysis can be carried out without a major distortion of the plasma catecholamine levels.

Studies on rat kidney 15-hydroxy-prostaglandin dehydrogenase☆

Abstract Rat kidney 15-hydroxy-prostaglandin dehydrogenase (PGDH) was isolated, and its characteristics and the effects of various drugs upon it were examined. The enzyme was found in the cell cytosol; was labile when unfrozen; and most active at alkaline pH, at 41°, and with the E prostaglandins. Additionally, the enzyme was inhibited by furosemide ( K i = 0.019 mM), ethacrynic acid ( K i = 0.27 mM, phenylbutazone ( K i = 0.16 mM), acetylsalicylic acid ( K i = 3.8 mM), and potassium cyanide ( K i = 1.03 mM). Inhibition of PGDH may play a role in the mechanism of action of the diuretic and anti-inflammatory drugs. Little or no inhibition was seen with amobarbital, hydralazine, alpha-methyldopa, bethanidine and guanethidine. Amobarbital inhibits NADH oxidase ( K i = 0.5 mM), but does not inhibit PGDH. This drug, therefore, may be useful in permitting the use of the fluorometric assay for PGDH in preparations of PGDH contaminated by NADH oxidase.

Purification of the (Na+ + K+)-adenosine triphosphatase from human renal tissue

(Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was purified from human cadaver renal tissue and exhibited a linear reaction rate with time. 100 g of whole kidney would yield 1--3.5 mg protein with a specific activity of 50--200 mol - kg-1 - h-1 for (Na+ + K+)-ATPase. The preparation was completely inhibited by 100 micronM ouabain with a Ki of 1.8 micronM. K+-dependent phosphatase increased during purification of (Na+ + K+)-ATPase to 7.8 mol - kg-1 - h-1. There was no detectable Mg2+-ATPase in the final preparation. Sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis yielded three protein peaks of 117 000, 92 500, and 56 000 daltons. The peptide band corresponding to 92 500 daltons underwent an Na+-dependent phosphorylation with [gamma-32P]-ATP. The band at 56 000 daltons stained for glycoprotein. The Km for ATP was 0.38 mM and that for Mg2+ was 0.5 mM. The formation of ADP and inorganic phosphate from ATP was stoichiometric. The Km for Na+ in the presence of 20 mM K+ was 16 mM and the Km for K+ in the presence of 100 mM Na+ was 1.5 mM. The temperature optimum was 51degrees C and the pH optimum was 7.0. (Na+ + K+)-ATPase in whole homogenate, microsomes, and NaI-treated microsomes exhibited a slowing of reaction rate (non-linearity) with time such that the enzyme was inactive by 10--15 min of reaction. This non-linearity was eliminated during purification. The significance is discussed.

Reversible inactivation of purified (Na+ + K+)-ATPase from human renal tissue by cyclic AMP-dependent protein kinase

Human renal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations which exhibited a non-linear reaction rate, contained high levels of membrane-bound cyclic AMP-dependent protein kinase, while this latter activity was much less or absent in purified preparations. A non-linear reaction rate was observed in a purified preparation of (Na+ + K+)-ATPase by reconstituting the enzyme into lipid vesicles with cyclic AMP-dependent protein kinase. The addition of cyclic AMP to the ATPase assay of these lipid vesicles inactivated the (Na+ + K+)-ATPase. The cytoplasmic fraction of the cell contained a non-dialyzable factor, which prevented (or reversed) the cyclic AMP-mediated inactivation of the enzyme.

The Effect of Vanadate on Human Kidney Potassium Dependent Phosphatase

SummaryThis study examined the effects of vanadate on the potassium dependent phosphatase activity present in purified human kidney microsomal (Na++K+)-adenosine triphosphatase. Vanadate anion inhibited the K+-dependent phosphatase at a KI of 35 nM. This inhibition, was noncompetitive with the substrate, p-nitrophenylphosphate. The inhibition by vanadate at 1 mM K+ was only 45% of the inhibition that was observed at 10 mM K+. Neither preincubation of the enzyme with vanadate, nor changing the pH of the assay from 8.2 to 7.2 had any effect on the KI for vanadate. The inclusion of 2.5 mM isoproterenol, to complex the yanadate, reversed the inhibition, as did diluting the enzymatic reaction. Vanadate also inhibited the overall (Na++K+)-ATPase reaction at a KI of 1.91 μM. This inhibition was also reversible upon inclusion of isoproterenol in the assay. Increasing the level of magnesium from 6 mM to 30 mM lowered the KI of vanadate to 0.25 μM. The possible role of vanadate as a physiological mediator of (Na++K+)-ATPase activity is discussed.

Postural hypotension Adrenergic responsivity and levodopa therapy

Four subjects with orthostatic hypotension were given intravenous infusions of methoxamine and isoproterenol. Methoxamine caused an elevation in systolic blood pressure. isoproterenol resulted in a fall in blood pressure in three of the subjects. The heart rate decreased with methoxamine, but increased with isoproterenol. The responsivity in orthostatic hypotension was compatible with denervation supersensitivity. These effects were compared with the responsivity to methoxamine and isoproterenol of five labile hypertensives. Two patients with severe orthostatic hypotension were treated with regimens including levodopa. Levodopa alone would further aggravate postural hypotension. But in one subject given levodopa, ephedrine, and fludrocortisone and in the other managed on levodopa, tranylcypromine, and fludrocortisone, symptomatic orthostatic hypotension was successfully eliminated. These results support the usefulness of levodopa, in combination with adrenergic agents, as a therapeutic measure for advanced forms of orthostatic hypotension.

Non-linearity of human renal (Na+ + K+)-ATPase; elimination by purification and correlation with cyclic AMP-dependent protein kinase activity.

Abstract Human renal (Na + + K + )-ATPase exhibits a non-linear reaction rate which can be eliminated by purification of the enzyme. The activity in the microsomes and the first several stages of purification is nearly zero after 8 minutes of assay. The reaction of the purified enzyme, however, is linear with assay time. This change to a linear reaction rate correlates with the loss of cyclic AMP-dependent protein kinase activity from the (Na + + K + )-ATPase preparation.

Alpha and Beta Blockers: Effects on Renin Release

Publisher Summary This chapter presents the analysis of alpha- and beta-receptors, their role in renin release, and the efficacy of receptor blockade. A growing body of evidence demonstrates the intimate relationship between the adrenergic nervous system and renin release. Changes in the activity of either appear to be reflected in the activity changes of the other. Current evidence suggests that such a relationship exists within certain areas of the central nervous system, within the kidney, and at peripheral adrenergic vascular termination. A variety of modes of interaction has been proposed, but none has been confirmed. Adrenergic receptor blocking agents have been used in an attempt to clarify mechanisms of action. Because of the nuances in experimental design, results appear equivocal. Stimulation of renin release can be evoked by both alpha and beta adrenergic agonists, but beta blockade appears to be far more consistent in modification of renin release. Dissection of the interrelationship is dependent upon elucidation of each of the feedback loops involved and specification of receptor types.

Bethanidine Dose, Plasma Levels, and Antihypertensive Effects

Bethanidine dose, plasma levels, and hypotensive effects were evaluated in twelve hypertensive patients. Diuretic therapy with benzthiazide was started two weeks before and continued during the study. Bethanidine was given three times daily. After two to 12 months of therapy in 11 of the patients, the mean standing diastolic pressure was reduced from 112 to 91 mm Hg. The mean total daily bethanidine dose was 79 mg (range 30--150 mg). The mean plasma bethanidine was 0.65 micrometer (range 0.1--2.8 micrometer), which correlated with the bethanidine dose. There was less correlation between dose and antihypertensive effect. The effects of sudden withdrawal from chronic bethanidine dosing were observed in six patients. The orthostatic effect of bethanidine was lost within 12 hours, but the half-time of elimination of bethanidine from plasma was 39 hours. Based upon the present findings, recommendations are presented for initiation and maintenance of antihypertensive therapy with bethanidine.

Effect of mercaptoethanol in the radioactive thin layer chromatography assay of NAD+-15-hydroxyprostagland in dehydrogenase

The use of mercaptoethanol in the assay of rat kidney 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to have minimal effect on activity assayed with the spectrophotometric and substrate loss assays. However, mercaptoethanol appeared to inhibit PGDH when assayed by thin-layer chromatography, based upon conversion of 3H-PGE1 to 15-keto-3H-PGE1. Mercaptoethanol reacted with 15-keto-PGE1 to alter its chromatographic mobility and to suppress the U.V. absorption spectrum of 15-keto-PGE1. The implication of the use of ME in radiometric assays is discussed.