Robert S. Levy

LITHIUM PREVENTS OUABAIN-INDUCED BEHAVIORAL CHANGES : TOWARD AN ANIMAL MODEL FOR MANIC DEPRESSION

Both mania and bipolar depression have been associated with decrements in the activity of the sodium and potassium-activated adenosine triphosphatase (Na,K-ATPase) membrane pump. Although the role of this observation in the pathophysiology of bipolar illness is unclear, it has been proposed that this defect could be central to the pathogenesis of the illness. In an effort to test this hypothesis, the authors examined the efficacy of lithium pretreatment in attenuating behavioral changes secondary to acute administration of a single intracerebroventricular (i.c.v.) dose of the Na,K-ATPase-inhibiting compound, ouabain, in the Sprague-Dawley rat. Ouabain (10(-3)M) significantly decreased motor activity in automated activity monitors. Lithium pretreatment for 7 d totally prevented this effect. These preliminary data suggest that i.c.v. ouabain administration in the rat may prove to be a viable animal model for bipolar illness.

An animal model for mania: preliminary results.

1. In human bipolar patients mania and bipolar depression are both characterized by decreased membrane Na,K-AtPase activity. Additionally, digoxin neurotoxicity in patients frequently presents with symptoms of mania or depression. 2. These findings suggest that central nervous system Na,K-ATPase inhibition may play a pathophysiologic role in bipolar illness. 3. The authors tested this hypothesis by administering intracerebroventricular (i.c.v.) ouabain to rats at sublethal doses. The authors then measured behavioral activity as total square crossings in an open field. 4. Motoric activity was significantly increased by i.c.v. administration of 5 microliters of ouabain at 10(-3) M. This preliminary study suggests that i.c.v. ouabain administration may provide a useful animal model of mania that is based on observed biochemical changes in humans.

Open field is more sensitive than automated activity monitor in documenting ouabain-induced hyperlocomotion in the development of an animal model for bipolar illness.

1. Intracerebroventricular (i.c.v.) administration of ouabain to rats induces motor hyper- and hypoactivity that have been hypothesized to model the mania and depression of bipolar illness, respectively. 2. The extent of ouabain-induced change in activity may vary according to the test environment. 3. To determine the degree of differential response to i.c.v. ouabain in the open field and automated activity monitors, the authors examined a large number of animals (n=40) in both environments. 4. I.c.v. ouabain produced a four-fold increase in open field activity versus i.c.v. artificial cerebrospinal fluid (aCSF) (mean +/- SD: 258.7 +/- 316.61 vs. 84.8 +/- 86.16 squares traversed) (t = 2.648, P = 0.011), but did not alter horizontal activity in automated activity monitors (8193.5 +/- 4902.52 vs. 7088.47 +/- 3046.85 beam interruptions) (t = 0.847, P = 0.4). This increase in activity persisted for at least one week (161.0 +/- 186.35 for i.c.v. ouabain vs. 46.1 +/- 47.46 for i.c.v. aCSF, P = 0.065). 5. Open field is superior to automated activity monitors in capturing ouabain-induced hyperlocomotion response.

Persistent Hyperactivity Following a Single Intracerebroventricular Dose of Ouabain

Intracerebroventricular (i.c.v.) administration of ouabain has been shown to alter motor activity in the rat. It has been purported that this may model the behavioral abnormalities of human manic-depressive (bipolar) illness. Since manic-depression is a recurrent condition, we elected to investigate the effects of the multiple administration of i.c.v. ouabain. Male Sprague-Dawley rats were allowed to acclimate to the animal facility for 7-10 days after which time i.c.v. cannulae were placed. Animals received two i.c.v. injections of either ouabain (10[-3] M) or artificial cerebrospinal fluid (aCSF) 9 days apart, so that 6 rats received aCSF-aCSF, 6 received ouabain-aCSF, and 6 received ouabain-ouabain. Behavioral activity was evaluated in an open field (86 x 86 cm subdivided into sixteen 21.5 x 21.5-cm squares) for 20 min at baseline and immediately following each i.c.v. injection. After the last behavioral test, the animals were killed, and the brains were rapidly harvested and dissected over ice. Specific ouabain binding and sodium pump activity were determined. A single dose of ouabain produced a marked increase (297.0%, p = 0.002) in open field activity compared to both baseline behavior and to aCSF injected animals. The effects of ouabain appeared to last for 9 days. A second i.c.v. injection of either ouabain (136.5 +/- 60.4 SEM) or aCSF (108.0%, p < 0.01) had no effect on the activity level which was intermediate between the initial ouabain hyperactivity and the baseline level. Nine days after ouabain administration, hippocampal ouabain binding was increased relative to the control group (5477 +/- 485.7 vs. 3579 +/- 518.6, p < 0.05) and sodium pump activity was relatively lower (2293.8 +/- 265.5 vs. 3174.2 +/- 410.5, p < 0.05).

Conversion of Angiotensin I to Angiotensin II by cathepsin A isoenzymes of porcine kidney

We have reported the existence of a carboxypeptidase in a human renal extract that converts Angiotensin I (AI) to Angiotensin II (AII) in two steps with des-leu-AI (dl-AI) being formed as an intermediate. Since this carboxypeptidase had properties similar to cathepsin A, the ability of cathepsin A to metabolize AI was studied. Cathepsin A was purified from hog kidney with enzyme activity being monitored using both benzyloxycarbonyl-glutamyl-tyrosine (ZGT) and AI as substrates. The procedure separated the expected large and small molecular weight forms of cathepsin A as well as two additional isoenzymes. All of the isoenzymes had carboxypeptidase activity with ZGT, AI, and dl-AI. No detectable cleavage of AII was observed. Cathepsin A,S (small) activity with ZGT or AI as substrate was inhibited to a similar extent by diisopropylfluorophosphate, mersalyl acid, and a decapeptide renin inhibitor. It is concluded that the renal angiotensin carboxypeptidase activity is catalyzed by cathepsin A. By its ability to convert AI to AII, cathepsin A may be a component of the intrarenal renin-angiotensin system.

Angiotensin II generated by a human renal carboxypeptidase

Angiotensin II, the potent hypertensive octapeptide, can be generated by a sequential cleavage of the carboxyl-terminal leucine and histidine from angiotensin I by a human renal extract. This extract does not hydrolyze further the resulting octapeptide. The more widely recognized biosynthetic pathway is by the extracellular dipeptide cleavage of angiotensin I by an enzyme which also degrades bradykinin, i.e., angiotensin converting enzyme. The presence of a carboxypeptidase activity capable of generating but not further hydrolyzing angiotensin II was observed in an ammonium sulfate fraction of a human renal extract. This novel enzymatic activity is distinct from angiotensin converting enzyme activity in that it is not dependent upon calcium and is not inhibited by known angiotensin converting enzyme inhibitors.

Molecular structure of serum lipoproteins

Abstract Recent data from electron microscopy permits the subdivision of the serum lipoproteins into two groups: those molecules with visible substructure and invariable particle size (group I) and those with no visible substructure and a wide variation in particle size (group II). Using data from the literature a concept has been developed to explain the molecular structure of the group I lipoproteins. To allow for the fact that these lipoproteins display evidence both of helical and non-helical peptide chains, a system of small repeating units called “lipotides” has been proposed. These units consist of two helical peptide chains enclosing a lipid core, each helix attached to another by a non-helical peptide connector. The conformation of the actual lipoprotein molecule is dependent upon how these lipotides fold upon one another which is not yet known. The model is based upon the ability of the helical peptide chains to bind to phospholipid, which in turn binds to cholesterol and its esters and to glycerides. Data to support this model have been gathered from the literature, but final proof for the proposal will depend upon the sequence of the amino acids composing the peptide chains, particularly the spatial interrelationships of the ionic and hydrophobic amino acids.

Des-Leu angiotensin I: biosynthesis and drinking response

The crude rat and bovine synaptosomal lysate from brain can hydrolyze angiotensin I (AI) to des-Leu angiotensin I (AI-dL) and no further. This cytosolic enzyme has a specificity for angiotensin-related sequences, R-His-Pro-Phe-His-Leu and therefore named angiotensin-related carboxypeptidase (ARC). These studies led to the biosynthesis and purification of AI-dL in order to determine if it can provoke a drinking response. This nonapeptide is a potent dipsogen when injected into the cerebroventricles of rats. The drinking response probably requires a second hydrolysis to angiotensin II (AII) since both captopril and saralasin can inhibit this response.

Angiotensin carboxypeptidase activity in urine from normal subjects and patients with kidney damage

Angiotensin carboxypeptidase (ACP) activity has been detected in urine samples from normal subjects and patients with hypertension and diabetes by determining the enzymes ability to convert angiotensin I to des-Leu angiotensin I. Gel filtration chromatography of a concentrated urine sample indicated that about equal amounts of the enzyme exist as 100 kDa and 500 kDa molecular weight forms, respectively. This ACP activity co-eluted with activity that cleaved histidine from des-Leu angiotensin I to form angiotensin II and activity that cleaved tyrosine from benzyloxycarbonyl-glutamyl-tyrosine (ZGT). These results suggest that the urinary ACP activity is due to cathepsin A as we have reported previously for the porcine kidney enzyme. Analysis of sequential urine samples from a single individual over a 6-day period revealed as much as a 6-fold fluctuation in creatinine-normalized ACP activity. Of five male healthy adult subjects, the creatinine-normalized urinary ACP activity ranged from 1.7 to 3.7 mU/mL with a mean of 2.8 mU/mL. However, five male patients with renovascular hypertension had elevated levels of ACP activity with a mean of 11.6 mU/mL. Of five male patients with diabetic nephropathy, all had elevated ACP activity levels with a mean of 21.0 mU/mL. It is concluded that ACP activity in the urine is due to cathepsin A probably derived from kidney tissue, and that the release is increased in patients with kidney damage. We suggest that urinary ACP activity should be evaluated further for a possible relationship to renal hypertension and as a potentially early marker for diabetic nephropathy.

Elimination of low-density lipoprotein-polyanion interaction by amino modifications

Lowdensity lipoprotein (LDL) is selectively precipitated from human serum at physiological pH by macromolecular polyanions. Several studies on the mechanism of this interaction have been carried out [l-6] , and it appears that it is an electrostatic phenomenon. To test this hypothesis further we changed the charge profile of the native LDL molecule by chemical modification of its free amino groups. LDL was acylated with succinic, maleic and acetic anhydrides and ethyl thioltrifluoroacetate, and the interaction of the modified LDL with three polyanions was studied over a wide range of polyanion/LDL ratios. None of these modified lipoproteins would precipitate with the polyanions amylopectin sulfate (AI’S), dextran sulfate (DS) or heparin (F’).