Experimental Biology and Medicine

Fibronectin is a normal glycoprotein component of plasma, interstitial fluid, and extracellular matrix which has binding sites for collagen, gelatin, actin, glycosaminoglycans, fibrin, Staphylococcus aureus, and some cells. Since it is a dimer, it can crosslink these substances to each other or to extracellular components of basement membrane, thereby affecting many physiological processes. The level of circulating fibronectin is markedly reduced following even moderate blunt or operative trauma, thermal injury, starvation, advanced cancers, hemorrhage, etc. Replacement therapy has been tried with some success in patients who become septic following multiple injuries. The reduction in plasma fibronectin has been attributed to several causes including consumption by binding to cell debris at the site of injury, binding to circulating cell debris and its subsequent removal by elements of the phagocytic system, and degradation by proteolytic cleavage. However, the amount of fibronectin removed from circulation raises some question about this. In this paper, we used indomethacin, ibuprofen, imadazole, and essential fatty acid deprivation to inhibit the synthesis of prostaglandins in young adult rats. Thirty minutes after ip administration of one of the inhibitors, the rats were subjected to a midline laparotomy and mild intestinal manipulation. Blood samples were taken at intervals following closure of the incision and analyzed for fibronectin. In all cases, the normal decline in plasma fibronectin seen in untreated rats was abrogated by inhibiting prostaglandin synthesis. Since imadazole specifically inhibits thromboxane A synthesis, this strongly suggests that thromboxanes directly or indirectly control the trauma-induced reduction in circulating fibronectin. This was confirmed by ip injection of thromboxane into the rats which resulted in a decline in plasma fibronectin levels.

Forskolin is a direct stimulant of adenylate cyclase and increases cAMP production. It also acts as a vasodilator. To study the effect of forskolin infusion on rabbit maternal vascular resistance, we instrumented 11 pregnant rabbits with catheters in the left ventricle, jugular vein, and left and right femoral arteries. After a 2-day recovery period, one of two protocols was performed. In the control period of the first protocol (N = 6), 50% ethanol in saline was infused at 0.103 ml · min^-1 for 5-min. Forskolin (10^-3 M) in 50% ethanol was then infused for 5 min at 0.103 ml · min^-1. After each infusion period, regional blood flows were measured by microsphere injection. Data are expressed as means ± SEM. Blood pressure decreased from 81 ± 3 to 79 ± 3 mm Hg, (P < 0.05, N = 10) during forskolin infusion. Total placental resistance fell from 180.3 ± 10.7 to 133.8 ± 12.0 mm Hg ± min ± ml^-1 per gram, P < 0.05. Cerebral, coronary, and renal vascular resistance fell significantly. During the second protocol (N = 5), angiotensin II (0.05 μg ± min^-1) was infused for 5 min followed by the addition of forskolin (10^-3 M at 0.103 ml ± min^-1) to the infusate. Regional blood flows, vascular resistances and blood pressures were determined. Blood pressure fell from 99 ± 6 to 92 ± 7 mm Hg (P < 0.05) when forskolin was added to the infusate. Placental resistance fell from 202.5 ± 21.6 to 158.0 ± 29.0 mm Hg · min · ml^-1 per gram (P < 0.05). While cerebral vascular resistance did not change, renal and coronary resistances fell in response to forskolin. This study demonstrates that forskolin is able to dilate rabbit placental vessels alone and in the presence of the vasoconstrictive agent angiotensin II.

We have examined the subcellular localization of doxorubicin and evaluated the effect of fatty acid modification on specific intracellular localization. L1210 leukemia cells enriched with docosahexaenoic acid (22:6) or oleic acid (18:1) were incubated with radiolabeled or unlabeled doxorubicin. After equilibration the cells were ruptured and the subcellular fractions were isolated by differential centrifugation and sucrose gradient separation. The doxorubicin localized primarily in nuclei, as expected, but appreciable amounts were also detected in mitochondria and smaller amounts in plasma membranes, microsomes, and cytoplasm. Subcellular distribution of another anticancer drug which binds to DNA, mitoxantrone, was similar. There were increased amounts of doxorubicin contained in the nuclei and all organelles of the 22:6-enriched cells. Although polyunsaturated fatty acid modification influenced the total amount of doxorubicin in fractions, the relative distribution of drug among the fractions was not different from that of the 18:1-enriched and unmodified cells. We conclude that enrichment with polyunsaturates influences total drug uptake but not proportional distribution of doxorubicin.

Pluronic F68 (F68) is a nonionic surfactant which has been reported to inhibit the in vitro adherence and migration of polymorphonuclear leukocytes (PMN) obtained from some species. We demonstrated similar effects on PMN obtained from rats, with diminished adherence to nylon wool and diminished chemotaxis toward zymosan-activated serum. We then examined the in vivo effects of 12-hr F68 infusion on the injury induced by intratracheal bleomycin instillation (ITB) in rats. When sacrificed 24 hr following injury, rats demonstrated neutrophilia, neutrophil-prominent lung lavage cellularity, and increased lung weights. F68 decreased lavage leukocyte counts and lung weight gain in ITB-injured animals. Lung weights of ITB-injured animals correlated (r = 0.81, P < 0.001) with logarithmic values of lavage PMN. F68 also enhanced neutrophilia and decreased spleen weight gain in injured animals. The acute effects of F68 on circulating leukocyte counts, osmolality, and total complement were also examined. The data demonstrate that F68 can affect PMN traffic both in vitro and in vivo. The data also confirm the prominence of PMN in lavage fluid early in ITB injury, and suggest that an influx of relatively few PMN is associated with lung weight gain in this model.

Iron (Fe) and aluminum (Al) eliminations in bile were studied in rats after intravenous administration of Fe, Al, deferoxamine mesylate (Desferal, Ciba) (DFA), feroxamine (FeA), and aluminoxamine (AIA) at the dose of 50 μmole/kg body weight. Bile was obtained from the bile duct of anesthetized rats, and the concentrations of Fe and Al in bile were measured by an inductively coupled plasma optical emission spectrometer. The results showed an increase of Fe elimination in bile, from 10 to more than 20 μmole/liter after Fe and also after Al administration; an increase to about 350 μmole/liter after DFA administration; to 250 μmole/liter after FeA administration; and to 100 μmole/liter after AIA administration. Aluminum elimination in bile was increased only after Al and particularly after AIA administration but not after Fe and FeA administration. In conclusion, Al and AIA were able to increase Fe elimination in bile. Thus Al overload observed in hemodialyzed patients may induce an excessive iron loss in bile and partly explain microcytic anemia.

The effect of exogenous corticosterone on the level of mouse hepatic glucocorticoid receptor was monitored to ascertain whether agonist-induced glucocorticoid receptor regulation takes place in living animals as it does in isolated cell systems. Adrenalectomized male Swiss-Webster mice were given 1 mg of corticosterone ip and 24 hr later the glucocorticoid receptor binding capacity of a high-speed cytosolic extract of liver was measured. It was shown that at this time point the administered steroid had been totally cleared and thus, the decrease in binding capacity was a reflection of downregulation. Receptor binding capacity was decreased by 25%. Downregulation was not permanent; 48–72 hr after the injection receptor content returned to baseline. Multiple daily injections of corticosterone were no more effective at causing downregulation than a single injection. It is concluded that glucocorticoid agonists downregulate their own receptors in the glucocorticoid target organs of intact animals as they do in cloned cell models.

Urinary protein excretion and composition in spontaneously hypertensive rats (SHR) change dramatically with age and sex. In this study, serum proteins were analyzed by electrophoresis in male and female SHR and Wistar-Kyoto (WKY) normotensive controls aged 5 to 80 weeks. Serum albumin concentrations of SHR were significantly higher than those of WKY at 5 (4.02 ± 0.24 vs 3.60 ± 0.25 g/dl) and 20 weeks (4.30 ± 0.30 vs 3.77 ± 0.31 g/dl) and significantly lower at 73–80 weeks (2.73 ± 0.33 vs 3.45 ± 0.34 g/dl). In addition, male SHR had significantly lower albumin levels than female SHR after 40 weeks of age. These differences may contribute to the development of hypertension and reflect the appearance of pathologic proteinuria in SHR. In spite of their differences in albumin concentrations, the fractional composition of serum protein from SHR and WKY were undistinguishable. All animals, regardless of strain or sex, manifested a significant decline in the relative amounts of albumin and low molecular weight protein and a significant increase in the relative amount of high molecular weight protein with increasing age. The etiology and significance of these age related changes in the fractional composition of serum protein are unknown, but they differ from the normal developmental pattern in humans.

Cysteamine (β-mercaptoethylamine, CSH) has been reported to have various effects on the neuroendocrine system. Reports indicate CSH decreases pituitary oxytocin (OT) without affecting pituitary vasopressin (VP). However, preliminary studies from our laboratory strongly indicate that CSH has an effect on VP release. Experiments were conducted with dibenzyline-treated, urethane-anesthetized, male Sprague-Dawley (SD) rats. Rats were injected with 4 mU of standard VP and 4 mg/100 g of CSH. Administration of VP resulted in an increase in mean arterial pressure (MAP) of 23.5 ± 3.2 mm Hg. Administration of CSH resulted in a consistent. immediate decrease in MAP of 13.0 ± 2.0 mm Hg prior to an increase of 21.0 ± 2.6 mm Hg. The effects due to VP and CSH were strikingly different; the CSH-induced MAP rise took longer to peak and to return to baseline. Both the VP- and CSH-induced MAP rise were markedly inhibited by a prior administration of a specific VP antagonist d(CH₂)₅[Tyr(Me)]AVP. In addition, the typical increase in MAP observed in SD rats following CSH administration was substantially reduced when the same dose was administered in homozygous diabetes insipidus (HODI) rats. The data presented here strongly suggest that CSH-induced MAP elevation is due to the release of VP from the pituitary gland.

We have investigated whether the androgen-induced masculine differentiation of the sex organs involves an induction of phospholipases. We have measured phosphatidylinositol-specific phospholipase C and phosphatidylcholine-specific phospholipase A₂ in the reproductive tract of male and female mouse (CD-I) fetuses at the 18th day of gestation. We report here that (1) the activity of these two enzymes is higher in the male genitalia than in the female genitalia; (2) exogenous testosterone at the 13th to 17th day of pregnancy induces both phospholipase A₂ and phospholipase C in the female fetal genitalia; and (3) prenatal administration of cyproterone acetate, an antiandrogen, known to produce feminized males, completely prevents the stimulation of phospholipase A₂ and C by testosterone in the female fetuses. In the male fetuses, however, cyproterone acetate inhibits the PLC activity but is unable to alter phospholipase A₂ activity. These findings provide evidence that the mechanism by which testosterone organizes the genitalia may involve a modification of phospholipases A₂ and C.

Adriamycin-treated rats were monitored for survivorship while consuming a normal diet adequate in riboflavin, a normal diet and receiving daily high-dose injections of riboflavin-5′-phosphate (flavin mononucleotide, FMN), or a riboflavin-deficient diet. Each animal was compared to a corresponding pair-fed, saline-treated control. In Adriamycin-treated rats fed the normal chow diet alone, survivorship declined within 7 days and remained constant after 12 days to about 50%. Adriamycin-treated rats consuming the normal diet and injected with FMN initially showed similar survivorship; however, after 20 days survival fell to 14%. Adriamycin-treated, riboflavin-deficient rats showed within 5 days a precipitous decline in survivorship which leveled to 5%. These results suggest that during Adriamycin treatment, proper riboflavin nutriture may be a crucial determinant of survival.

To provide insight into the mechanism of wolffian duct virilization the androgen receptor has been characterized in pooled epididymal tissue from Day 27 rabbit embryos. Although the receptor is present at levels about a tenth that of other androgen target tissues in rabbit embryos, it appears to be a typical androgen receptor in regard to sedimentation characteristics and preferential binding of 5α-dihydrotestosterone over testosterone. Thus, the mechanism by which testosterone virilizes the wolffian duct cannot be explained by unique binding characteristics of the androgen receptor in tissues derived from the wolffian ducts.

Thromboxane A₂ (TXA₂) receptor antagonists can limit infarct size in models of coronary occlusion and reperfusion, but it was unknown if these compounds can mitigate reperfusion injury. Anesthetized open chest dogs were subjected to left circumflex coronary (LCX) occlusion for 90 min. Two minutes before reperfusion, the dogs were given iv saline (0.9% NaCl) or the TXA₂ antagonist SQ 29,548 (0.2 mg/kg + 0.2 mg/kg/hr). Reperfusion was instituted for 5 hr at which time infarct size was determined. Regional myocardial blood flow was determined before, during, and after occlusion. SQ 29,548 treatment resulted in a significant reduction in infarct size (57 ± 7 and 34 ± 8% of the left ventricular area at risk infarcted in the saline and SQ 29,548 groups, respectively). No differences in collateral flow during occlusion were observed between groups, but SQ 29,548 treatment resulted in a significantly higher subendocardial reperfusion flow (54 ± 10 and 93 ± 14 ml/min/100g for the saline and SQ 29,548 groups, respectively). Thus, TXA₂ seems to play a role in exacerbating reperfusion injury and TXA₂ receptor blockade may have potential as a mode of therapy for ischemia-reperfusion damage.

An experiment was conducted to determine the effect of feeding ornithine in combination with α-aminoisobutyric acid (AIB), an inhibitor of arginase, on the regulation of polyamine synthesis in chicks. A total of 48 chicks with genetically elevated renal arginase activity was fed diets containing crystalline amino acids and 1% AIB with or without 2% ornithine. Feeding AIB reduced renal arginase activity, while renal and hepatic ornithine decarboxylase (ODC) activity increased. Feeding AIB plus ornithine caused no further reduction in renal arginase activity compared with that in chicks fed the AIB-supplemented diet. Renal and hepatic ODC activities, however, fell to below control levels. Renal, hepatic, and breast muscle ornithine concentrations increased substantially when ornithine was fed. AIB plus ornithine increased renal putrescine and spermidine concentrations. It was concluded that AIB could partially overcome the ornithine-induced inhibition of ODC activity. These findings support the hypothesis that dietary manipulation of precursor amino acids of polyamines in the presence of metabolites that induce ODC activity can influence tissue polyamine concentrations.