Glandular kallikrein in estrogen-induced pituitary tumors: time course of induction and correlation with prolactin.

Glandular kallikrein (a trypsin-like serine protease) is an estrogen-induced and dopamine-repressed protein in the rat anterior pituitary which appears to be associated with lactotrophs. This study examined glandular kallikrein levels in diethylstilbestrol (DES)-induced pituitary tumors in F344 rats and compared it to plasma and pituitary prolactin, and pituitary wet weight. Ovariectomized F344 rats were implanted with Silastic tubes containing 0 or 5 mg DES for 1, 3, 5, 7, or 9 weeks. Glandular kallikrein was measured by microenzymatic assay using D-valylleucylarginyl-p-nitroanilide following trypsin treatment of extracts to activate latent forms of glandular kallikrein. Prolactin was measured by radioimmunoassay. DES induced steady time-dependent increases in pituitary wet weight with 7- and 16-fold increases observed by 5 and 9 weeks, respectively. Growth rates averaged 11.4 mg/week during the first 5 weeks of DES exposure, and then increased to 23.2 mg/week between weeks 5 and 9. Glandular kallikrein total activity (nmol/min/pituitary) increased 130- and 240-fold after 3 and 5 weeks of DES exposure, respectively, and then abruptly plateaued. The specific activity (nmol/min/mg protein) of glandular kallikrein peaked at 3-5 weeks (36-fold increase compared to controls) and then declined as pituitary protein but not glandular kallikrein continued to increase. Total pituitary prolactin constantly rose during DES exposure with 12- and 26-fold increases after 5 and 9 weeks, respectively. Plasma prolactin levels also continuously rose during exposure to DES with 130- and 290-fold increases after 5 and 9 weeks, respectively. No major strain differences were found with regard to sensitivity to the acute effects of estrogen or dopaminergic stimulation on glandular kallikrein induction. DES-induced pituitary tumors in F344 rats are well known to arise via lactotroph proliferation, and the striking elevation in glandular kallikrein and prolactin during the early phases of tumor growth provide further support for a localization of glandular kallikrein in lactotrophs. However, the abrupt stabilization in glandular kallikrein levels by week 5 was unexpected and may signal a biochemical transformation of the tissue during tumor progression.

Effect of diets containing different levels of linoleic acid on human breast cancer growth and lung metastasis in nude mice.

The purpose of the study was to determine the effect of three different levels of dietary linoleic acid (LA) intake on the growth of MDA-MB-435 human breast cancer cells in the mammary fat pads of nude mice, and their metastasis to the lungs. These diets were isocaloric, and contained different mixtures of safflower (LA-rich) and coconut (saturated fatty acid-rich) oils to provide 23% (w/w) total fat, with 2, 8, and 12% (w/w) LA. A fourth group was fed a low-fat, 5% (w/w) corn oil diet. There were 25 mice in each dietary group. A necropsy, 12 weeks after the tumor cell injections, the primary tumor weights in the 12% LA (4.1 +/- 2.7 g)- and 8% LA (3.5 +/- 1.7 g)-fed groups were significantly greater (P < 0.05) than those fed the 2% LA diet (2.5 +/- 1.5 g); they did not differ significantly from the weights of mammary fat pad tumors in the 5% corn oil-fed mice. The incidence of grossly visible pulmonary metastatic nodules was not significantly different between the 8 and 12% LA-fed mice, but was higher for both groups compared with the 2% LA-fed group (P < 0.05), with a similar trend in comparison with the 5% corn oil group. The mean total calculated volumes of the macroscopic metastases per tumor-bearing mouse were significantly greater in the 8 and 12% LA (157 +/- 250.7 and 99.1 +/- 140.0 mm3, respectively), compared with the 2% LA (23.3 +/- 51.8 mm3)- and 5% corn oil (24.5 +/- 35.1 mm3)-fed mice; all P < 0.05. Micrometastases were observed most frequently in the 5% corn oil and 2% LA dietary groups, but none of the differences were statistically significant. No differences were detected in the concentrations of prostaglandin E, leukotriene B4, or 5-hydroxyeicosatetraenoic acid in tumors from mice fed the four different diets.

Dynamics of estrogen induction of glandular kallikrein in the rat anterior pituitary.

Glandular kallikrein has recently been identified as an estrogen-induced protein of the rat anterior pituitary. This study examined the dynamics of the estrogen induction of anterior pituitary glandular kallikrein in the ovariectomized rat. The estrogen induction of uterine dry weight was also examined for purposes of comparison. 17 beta-Estradiol (0.1-100 micrograms/day) produced dose-dependent increases in anterior pituitary glandular kallikrein, with the highest dose producing a 60-fold increase. Time-course studies demonstrated that a lag phase of 2-3 days was required before these estrogen effects on glandular kallikrein became evident, and levels were still rising between 7 and 10 days of treatment. The dynamics of the estrogen induction of glandular kallikrein resembled the estrogen induction of uterine dry weight with regard to estrogen sensitivity and the presence of a lag phase before estrogen-induced increases. However, uterine dry weight responded more rapidly to estrogen than did anterior pituitary glandular kallikrein, and reached a plateau after 5 days of estrogen treatment.

Prolactin proteolysis by glandular kallikrein: in vitro reaction requirements and cleavage sites, and detection of processed prolactin in vivo.

Glandular kallikrein (GK) is a trypsin-like serine protease characterized by an ability to generate bioactive peptides from inactive precursors with high specificity. In rat pituitary lactotrophs, GK is an estrogen-induced dopamine-repressed protein which is coregulated with PRL and is localized in organelles associated with precursor processing, suggesting a function to process PRL to novel forms with unique biological activities. To investigate this hypothesis we studied the ability of GK to cleave PRL in vitro and also examined rat pituitary extracts for processed forms of PRL using Western blot analysis. GK did not cleave rat PRL under routine incubation conditions. However, GK converted PRL from a 25K form to a 22K form (by gel electrophoresis) in the presence of thiols (dithiothreitol or mercaptoethanol) and Triton X-100. These reagents appear to elicit proteolysis by altering PRL's conformation; thiols were essential for proteolysis and Triton X-100 enhanced the thiol effects. Sequencing of processed PRL revealed 3 cleavage sites: Arg174-Arg175,Lys185-Phe186, and Arg188-Cys189. The cleavage sites are clustered close to the C-terminus on a 16-amino acid domain bracketed by cysteine residues. The cleavage sites in PRL resemble those of other GK substrates in their structural features and are in a highly conserved domain of PRL. Western blot analysis of rat pituitary extracts using antiserum against rat PRL revealed 22K PRL variants that were highly estrogen dependent and whose levels were markedly increased by in vivo administration of cysteamine, a biological thiol known to alter pituitary PRL conformation in vivo. The 22K PRL variants comigrated with PRL bands generated by sequential in vitro processing with GK and carboxypeptidase-B. The present findings support the hypothesis that GK may function to process PRL to novel forms in rat lactotrophs.

Characterization of phospholipase A2 activity in MDA-MB-435 human breast cancer cells.

Phospholipase A2 (PLA2) was identified and its properties characterized in MDA-MB-435 cells, a human breast cancer cell line. Cytosolic fractions, prepared in calcium-free buffer, were assayed using arachidonyl-containing phosphatidylcholine as substrate. PLA2 activity was linear as a function of both time and protein concentration. The enzyme was shown to be calcium-dependent and to require a basic pH of 9.5-10.0 for optimal activity. Activity was predominantly found in the cytosolic fraction when cells were harvested in calcium-free buffer. Phospholipase A2 may play a key role in linoleic acid-enhanced mammary tumorigenesis and metastasis.

Differential responses of pituitary kallikrein and prolactin to tamoxifen and chlorotrianisene.

Glandular kallikrein, a trypsin-like serine protease, and prolactin (PRL) are both estrogen-induced proteins in rat anterior pituitary lactotrophs. The estrogen agonist and antagonist effects of tamoxifen (TAM, a triphenylethylene antiestrogen) and chlorotrianisene (TACE, a triphenylethylene estrogen) on anterior pituitary glandular kallikrein and PRL were examined to see if TAM and TACE differentially affect these estrogen response of lactotrophs after in vivo dosing of rats. TAM and TACE acted as partial agonists on PRL and uterine weight induction. In contrast, on glandular kallikrein induction TAM acted as a pure estrogen antagonist and TACE acted as an almost pure antagonist. The results document that both TAM and TACE exhibit protein-specific estrogen agonist and antagonist efficacies in lactotrophs, with the estrogen induction of glandular kallikrein being particularly sensitive to antagonism by TAM in vivo. The marked antiestrogen character of TACE was surprising since TACE has been classified and clinically used as an estrogen.

Effects of phenobarbital and 3-methylcholanthrene induction on the formation of three glucuronide metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco specific carcinogen believed to be a causative agent for human lung cancer. To exert its carcinogenic potential, NNK must be metabolically activated, by alpha-hydroxylation, at either the methyl or methylene carbons adjacent to the N-nitroso group. We recently reported the presence of a glucuronide conjugate of 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (alpha-hydroxymethylNNK-Gluc) in the urine of Phenobarbital (PB) treated rats, and in the media of PB induced hepatocytes incubated with NNK. PB induces the alpha-hydroxylation of NNK, which generates the aglycon, as well as several UDP-glucuronosyl transferases. In the study presented here, we compared the metabolism of NNK to alpha-hydroxymethylNNK-Gluc by PB induced, 3-methylcholanthrene (3-MC) induced and control rat hepatocytes. Media was analyzed for the products of alpha-hydroxylation, N-oxidation and glucuronidation by radioflow HPLC. PB induced both N-oxidation and alpha-hydroxylation of NNK. 3-MC did not induce N-oxidation but induced alpha-hydroxylation more than 10-fold. alpha-HydroxymethylNNK-Gluc was not detected (< 0.05% total metabolites) when control hepatocytes were incubated with 1 to 100 microM NNK. When 3-MC and PB induced hepatocytes were incubated with 1-100 microM NNK alpha-hydroxymethylNNK-Gluc, expressed as the average percent of metabolites, accounted for 0.725 +/- 0.27 and 1.35 +/- 0.24% (+/-S.D.) of the NNK metabolites, respectively. The percent of NNK metabolized to alpha-hydroxymethylNNK-Gluc is small. But this glucuronide is potentially important in NNK carcinogenesis, since its formation results in the direct conjugation of an active metabolite responsible for DNA adduct formation. When PB induced rats were injected with NNK the level of NNK hemoglobin adducts, which can serve as surrogates for DNA adducts, decreased 50% compared to control rats administered NNK. Hepatic microsomal metabolism increased 2-fold and urinary alpha-hydroxymethylNNK-Gluc increased more than 10-fold in PB treated rats. One explanation for the decrease in NNK hemoglobin adducts may be a PB induced increase in the glucuronidation of alpha-hydroxymethylNNK, the metabolite responsible for adduct formation.

Comparison of linoleic acid and eicosapentaenoic acid incorporation into human breast cancer cells.

To gain some insight into the mechanisms involved in the opposing effects of linoleic acid (LA) and eicosapentaenoic acid (EPA) on the growth and invasiveness of MDA-MB-435 human breast cancer cells, the dynamics of the uptake by cells and the incorporation of [14C]LA and [14C]EPA into major lipid and phospholipid pools, as well as the effects of unlabeled EPA or LA on the uptake and distribution of [14C]LA or [14C]EPA, respectively, were examined. Cells were exposed to [14C]LA (1.28 micrograms/mL) or [14C]EPA (1.0 micrograms/mL) and unlabeled EPA or LA, respectively, at 0, 1, 4 and 16 micrograms/mL for 24 h in serum-free media. The uptake of each fatty acid (FA) was linear over time and was not affected by the presence of the opposing FA. For both FA, 80-90% was incorporated into the phospholipid fraction with the remaining 10-20% in neutral lipids. The relative distribution profile of [14C]LA among the phospholipid classes indicated a preferential incorporation into phosphatidylcholine (65%), whereas [14C]EPA was mostly found in phosphatidylethanolamine (58%). In the presence of unlabeled EPA or LA at various concentrations, corresponding dose-dependent shifts of [14C]LA or [14C]EPA from the phospholipid to the neutral lipid pool were noted, which did not alter the relative distribution of the FA among the phospholipid classes. Exogenous exposure to EPA or LA increased its content in membrane phospholipids while concurrently decreasing LA or EPA content, respectively, in a dose-dependent manner. Arachidonic acid content of membrane phospholipids remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Angle of the articular eminence in patients with temporomandibular joint dysfunction and asymptomatic volunteers.

OBJECTIVE: The purpose of this study was to compare structural variations of the temporomandibular joint anatomy between symptomatic patients with temporomandibular joint dysfunction and asymptomatic controls. STUDY DESIGN: There were 74 symptomatic patients with temporomandibular joint dysfunction, 29 asymptomatic volunteers with normal joints, and 6 asymptomatic volunteers with disk displacement with reduction included in this study. All subjects had bilateral high resolution magnetic resonance imaging scans performed in the sagittal (closed and opened) and coronal (closed) positions. All subjects had right and left full profile laminagraphs, one in the centric occlusal position, and one with the incisors edge to edge. RESULTS: Student-Newman-Keuls tests demonstrated no significant angular or linear differences for depth of the articular fossa, angle of the articular eminence, horizontal and vertical overlap of the incisor teeth, and linear condylar translation. CONCLUSION: This study concludes that there are no significant differences in angular and linear measurements in the articular fossa of asymptomatic volunteers and symptomatic subjects with temporomandibular joint dysfunction.

Biochemical investigation of the subcellular localization of the estrogen-induced pro-glandular kallikrein in the rat anterior pituitary.

Glandular kallikrein (a trypsin-like serine protease) is a major estrogen-induced and dopamine-repressed protein in the rat anterior pituitary which appears to be associated with lactotrophs. In the pituitary the enzyme predominantly exists as a latent zymogen (pro-glandular kallikrein) which can be activated by trypsin. This study reports experiments employing biochemical techniques to investigate the subcellular localization of glandular kallikrein. Anterior pituitaries from estrogen-treated rats were fractionated on a discontinuous sucrose density gradient and the distribution of various organelles in the gradient was determined by conventional enzyme or protein marker assays. Each of the 8 organelle markers exhibited a unique distribution profile within the gradient. The distribution of glandular kallikrein was closely correlated (r = 0.91) with that of nucleoside diphosphatase (a marker for trans cisterna of the Golgi apparatus). For both glandular kallikrein and nucleoside diphosphatase, 35-45% of the total activity was found in Golgi zones of the gradient, and 18-22% was in the secretory vesicle fraction. In all of the subcellular fractions, 91-97% of the glandular kallikrein existed in the zymogen form (pro-glandular kallikrein). In Golgi fractions, 38% of the glandular kallikrein remained membrane-bound following freeze-thawing and two washes in hypotonic media; 94% of the nucleoside disphosphatase remained membrane-bound following such treatment. The results indicate that glandular kallikrein is most highly concentrated in trans cisternae of the Golgi apparatus with substantial activity also present in secretory vesicles. This localization is consistent with a role for glandular kallikrein as a prohormone processing enzyme in lactotrophs.

Dietary fatty acids and breast cancer invasion and metastasis.

This review presents the evidence for the hypothesis that dietary linoleic acid and its metabolic derivative arachidonic acid enhance the metastatic process in breast cancer. Key biochemical events are eicosanoid biosynthesis and protein kinase C activation, both of which are involved in tumor cell invasion and angiogenesis. It is concluded that the utilization of appropriate dietary interventions and pharmacological inhibitors offers a promising approach to suppress metastasis.

Dopaminergic regulation of the estrogen-induced glandular kallikrein in the rat anterior pituitary.

Glandular kallikrein is a major estrogen-induced protein of the rat anterior pituitary. A second kallikrein-like protease in the rat anterior pituitary (kallikrein A) is not affected by estrogens, nor is a third pituitary protease which cleaves a trypsin substrate but not kallikrein substrates. This study examined whether any of the pituitary proteases are regulated by dopaminergic mechanisms. Ovariectomized female rats were treated for 5-10 days with reserpine (a catecholamine depleting agent), haloperidol (a dopamine receptor blocker) or bromocriptine (a dopamine receptor agonist); some rats also received 1 or 2 micrograms estradiol benzoate every 48 h. Following activation of latent proteases with trypsin, anterior pituitary extracts were assayed for kallikrein activity before and after fractionation on DEAE-Sephadex to separate the two kallikrein-like proteases. Reserpine or haloperidol doubled glandular kallikrein levels in anterior pituitaries from estrogen-treated rats. Reserpine or haloperidol had little or no effect in the absence of estrogen (estrogen produced a 5- to 7-fold increase in glandular kallikrein in the absence of drug treatment). Bromocriptine markedly attenuated the ability of estrogen to induce glandular kallikrein. Further, bromocriptine blocked the ability of reserpine to increase glandular kallikrein levels, and haloperidol attenuated the effect of bromocriptine. Other anterior pituitary proteases were unaffected by either estrogen, haloperidol, reserpine or bromocriptine. The results demonstrate that the estrogen induction of glandular kallikrein in the rat anterior pituitary is modulated by inhibitory dopaminergic mechanisms. Prolactin is the only pituitary hormone which exhibits a similar profile of hormonal and neuroendocrine regulation; this suggests a possible link between glandular kallikrein and prolactin.

Development of the sex difference in glandular kallikrein and prolactin levels in the anterior pituitary of the rat.

Glandular kallikrein is a major estrogen-induced and dopamine-repressed protein of the rat anterior pituitary that appears to originate from lactotrophs. This study examined the development of glandular kallikrein levels in the anterior pituitary in both female and male rats and compared it to anterior pituitary prolactin. In addition, the development of glandular kallikrein levels in the neurointermediate lobe of the pituitary and the kidney were also examined. During puberty, a dramatic surge in glandular kallikrein occurred in female anterior pituitaries (16- to 20-fold increase) and levels remained elevated thereafter. The dynamics of the increase were biphasic--glandular kallikrein increased between Day 30 and 45, plateaued between Days 45 and 55, and then increased again between Days 55 and 65. Female anterior pituitary prolactin increased 7- to 8-fold during puberty. The rise during puberty was biphasic and was generally synchronized with increases in glandular kallikrein. However, the initial rise was proportionately less than that of glandular kallikrein, and the secondary surge was more dramatic. In contrast to females, anterior pituitary glandular kallikrein remained at low levels in male rats; prolactin levels also remained unchanged through puberty and increased moderately thereafter. Glandular kallikrein in the female neurointermediate lobe remained unchanged through Day 55, almost doubled on Day 60, and returned to prepubertal levels by Day 65; males did not exhibit the transient surge in neurointermediate lobe levels. Starting at age 60 days, renal glandular kallikrein was found to be slightly higher (15-20%) in females than in males.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological thiols elicit prolactin proteolysis by glandular kallikrein and permit regulation by biochemical pathways linked to redox control.

Rat glandular kallikrein (GK), a trypsin-like serine protease, cleaves rat prolactin (PRL) in vitro to novel forms detectable in vivo and likely to be of physiological significance. PRL proteolysis by GK is thiol-dependent, with thiols acting upon PRL to refold the molecule into novel conformations that are GK substrates. This study compared several natural and synthetic thiols for their ability to elicit PRL proteolysis by GK. Rat PRL was incubated with rat GK in the presence of various thiols and 0.5% Triton X-100, which enhances thiol-elicited proteolysis. Cleavage was analyzed by gel electrophoresis under reducing and nonreducing conditions. In the presence of Triton X-100, all low molecular weight thiols elicited PRL cleavage by GK. The order of potency was dithiothreitol greater than mercaptoethanol greater than lipoic acid greater than cysteamine = glutathione (GSH) = coenzyme A greater than cysteine. In the absence of Triton, however, dithiothreitol, coenzyme A, and mercaptoethanol were most effective in eliciting GK proteolysis. Triton X-100 enhanced PRL cleavage by 4-19-fold, depending upon the thiol used. Folding isomers of processed PRL observed following cleavage included disulfide-liked homodimers, oxidized monomers, reduced monomers and mixed disulfides; the folding isomers generated varied depending upon the thiol used. GSH potency in eliciting PRL proteolysis increased 10-fold in the presence of biochemical pathways shuttling reducing equivalents to GSH disulfide (GSSG). PRL cleavage by GK could be controlled by substrates, enzymes, and cofactors making up the reducing shuttle when GSSG was used. Thioredoxin (a protein disulfide oxidoreductase) potently elicited PRL proteolysis by GK in the presence of a reducing shuttle and Triton X-100.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardial salvage induced by REV-5901: an inhibitor and antagonist of the leukotrienes.

Lipoxygenase metabolites of arachidonic acid have been implicated in myocardial injury induced by coronary artery occlusion and reperfusion, as dual inhibitors of the lipoxygenase and cyclooxygenase enzymes, but not selective cyclooxygenase inhibitors, reduce infarct size. However, interpretation of these studies has been clouded by the lack of specificity of the drugs previously used. A specific 5-lipoxygenase inhibitor, REV-5901, has recently been developed. This drug inhibits A23187-induced immunoreactive leukotriene B4 generation by canine neutrophils (IC50 approximately 2.5 microM), and when given intravenously, attenuates the formation of a leukotriene D4-like material in blood ex vivo. The release of bioassayable leukotriene-like material from rabbit hearts infarcted in vivo and subsequently perfused in vitro is prevented by REV-5901. Moreover, the inhibitor also acts as an end-organ antagonist to prevent the spasmogenic effects of the peptide-containing leukotrienes in vitro with an IC50 approximately 0.1 microM. REV-5901 (10 + 2 mg/kg i.v.) reduces infarct size produced by coronary artery occlusion and reperfusion in the anesthetized dog from 56.6 +/- 2 to 28.6 +/- 3.7% of the hypoperfused zone. Salvage of the ischemic myocardium occurs independently of any apparent hemodynamic effect of the drug, but is accompanied by a diminution in neutrophil accumulation in the ischemic heart. It is proposed that inhibition of leukotriene B4 formation by REV-5901 suppresses the accumulation of neutrophils, thereby attenuating neutrophil-mediated cardiac damage.

Effects of dietary fish oil on fatty acids and eicosanoids in metastasizing human breast cancer cells.

We examined the relationships between the suppressive effects of dietary fish oil on growth and metastasis of MDA-MB-435 human breast cancer cells in female nude mice and the primary tumor phospholipid fatty acid concentrations, phospholipase A2 activity, and eicosanoid levels. Mice (n = 120) were fed a 23% (wt/wt) corn oil (CO) linoleic acid (LA)-rich diet for seven days before and after 10(6) tumor cells were injected into a mammary fat pad, and then the mice receive one of three isocaloric diets containing 23% total fat but different proportions of CO and menhaden oil (MO) (18% CO-5% MO, 11.5% CO-11.5% MO, 5% CO-18% MO) or a 23% fat diet containing 18% deodorized fish oil supplemented with tocopherol and tert-butylhydroquinone antioxidants (FAO). Primary tumor growth rate was significantly greater in mice fed the 18% CO diet than in the three diets containing higher levels of fish oil (all p < 0.05). The 18% MO diet, but not the 11.5% MO or the 18% FAO diet, suppressed the development of lung metastases compared with the 18% CO diet. Increasing the proportion of MO relative to CO in the diets produced corresponding increases in the primary tumor phospholipid eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations and reductions in LA and arachidonic acid. There was a significant positive correlation between the LA concentration in these tumors and the extent of lung metastasis (r = 0.504). Tumor phospholipase A2 activity was unaffected by dietary MO intake. Prostaglandin E2 concentration was inversely correlated with phospholipid EPA (r = -0.484) and DHA (r = -0.439), but there was no relationship with lung metastasis. Tumor leukotriene B4 and 5-hydroxyeicosatetraenoic acid levels were not reduced by dietary MO. The 18% FAO- and the 18% MO-fed mice showed similar relationships for the phospholipid fatty acids and prostaglandin E2, despite the lack of effect on metastasis. The strong correlation between phospholipid LA levels and metastasis and the lack of an association with tumor eicosanoids suggest that the 18% MO diet inhibited metastasis because dietary LA was replaced by other fatty acids.

Temporomandibular joint axiography and MRI findings: a comparative study.

Axiography may be useful in the evaluation of condyle motion and the effects of internal derangements on this motion. Fifty-one patients were selected by one investigator (RHT) to provide a representative sample of asymptomatic and symptomatic subjects for the following categories: (1) normal disk position, (2) disk displacement with reduction, (3) disk displacement without reduction and (4) disk displacement without reduction associated with degenerative joint disease. Axiography was performed by a separate investigator (KGP) in a blinded fashion. The diagnosis of the presence of internal derangement was based on the tracings only. All subjects had bilateral magnetic resonance imaging scans to evaluate for the presence or absence of internal derangement. The diagnostic sensitivity was 0.64, which indicated that axiography is marginal at identifying disease when present. The negative predictive value was 0.78, which indicated that axiography is accurate in the detection of normal disk condyle relationship.

Na,K-ATPase in several tissues of the rat: tissue-specific expression of subunit mRNAs and enzyme activity.

The relative contents of Na,K-ATPase subunit mRNAs in rat renal cortex, ventricular myocardium, skeletal muscle (hind limb), liver and brain (cerebrum) were measured. Expressed per unit DNA, mRNA alpha 1 content was approximately 2-fold greater in the kidney and brain as compared to either heart, skeletal muscle or liver. The hierarchy of mRNA alpha 2 expression was brain > skeletal muscle > heart, whereas mRNA alpha 3 was restricted to brain. Beta 1 subunit mRNA content in both kidney and brain exceeded the abundance of liver mRNA beta 1 by approximately 7-fold. In all tissues examined, the combined abundances of the alpha subunit mRNAs exceeded the content of mRNA beta 1. The hierarchy of Na,K-ATPase activity expressed per unit DNA was brain > kidney > skeletal muscle = heart > liver. The sum of mRNA alpha as well as mRNA beta 1 content, expressed per g of tissue, was highest in brain and kidney. A statistically significant correlation between mRNA beta 1 content and Na,K-ATPase activity was evident.

A neutrophil-derived cytochrome P450-dependent metabolite of arachidonic acid modulates neutrophil behavior.

Recently, the metabolism of arachidonic acid to two unidentified products (Peak 1 and Peak 2) by a cytochrome P-450 dependent mixed function oxidase has been described in canine polymorphonuclear leukocytes (PMNs). This study assessed the biologic activity of one of these metabolites, Peak 2, on PMN function. Peak 2 was formed biologically following addition of exogenous arachidonic acid to canine PMNs pretreated with BW755c to inhibit lipoxygenase and cyclooxygenase enzymes, and purified by high performance liquid chromatography following separation by column chromatography. Peak 2 (20-200 ng/ml) inhibited calcium ionophore A23187-induced aggregation and the second phase of LTB4-induced aggregation. Additionally, Peak 2 inhibited A23187-induced PMN adhesion to columns of Sephadex G-25. BW755c (94 microM), which increased the formation of Peaks 1 and 2 by almost 300%, also inhibited A23187-induced PMN adhesion. In contrast, Peak 2 did not inhibit the release of superoxide anions or immunoreactive LTB4, after stimulation of the PMNs with A23187. Thus, Peak 2 may modulate some activities of canine PMNs. Because the biologic activity of Peak 2 is opposite to that of LTB4, which promotes PMN aggregation and adhesion, and because LTB4 may be metabolized by a cytochrome P-450-dependent mixed function oxidase to less active metabolites, this enzyme system may play a central role in the control of PMN function.