The distribution of C-arachidonic acid in hamster lungs is sensitive to quinacrine

Isolated hamster lungs were labelled with ¹⁴C-arachidonic acid. When the lungs were ventillated with a respirator only a small amount of radioactivity was released to the perfusion effluent. This release was not changed significantly by pulmonary infusion of quicacrine (0.5 mM), a known inhibitor of phospholipase A₂. After the perfusion about 75% of the radioactivity in the lungs was in phospholipids, mainly in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinostil and to a lesser degree in phosphatidylserine and phosphatidic acid. About one fourth of the radioactivity was in neutral lipids (tri- and diacylglycerols) and as free unmetabolized ¹⁴C-arachiodonic acid. Pulmonary infusion of quinacrine increased the amount of radioactivity in diacylglycerols and phosphatidylinositol but had no effect on that in phosphatidylcholine, phosphatidylserine, phosphatidic acid and triacylglycerols. The amount of radioactivity in phosphatidylethanolamine was decreased by quinacrine and increased in the vicinity of an unidentified phospholipid-quinacrine complex. The present study indicates that the distribution of ¹⁴C-arachidonic acid in hamster lung lipids is sensitive to quinacrine. The detected changes can, however, not be explained by an overall inhibition of phospholipase A₂ activities.     

The distribution of 14C-arachidonic acid in hamster lungs is sensitive to quinacrine

Isolated hamster lungs were labelled with ¹⁴C-arachidonic acid. When the lungs were ventillated with a respirator only a small amount of radioactivity was released to the perfusion effluent. This release was not changed significantly by pulmonary infusion of quicacrine (0.5 mM), a known inhibitor of phospholipase A₂. After the perfusion about 75% of the radioactivity in the lungs was in phospholipids, mainly in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinostil and to a lesser degree in phosphatidylserine and phosphatidic acid. About one fourth of the radioactivity was in neutral lipids (tri- and diacylglycerols) and as free unmetabolized ¹⁴C-arachiodonic acid. Pulmonary infusion of quinacrine increased the amount of radioactivity in diacylglycerols and phosphatidylinositol but had no effect on that in phosphatidylcholine, phosphatidylserine, phosphatidic acid and triacylglycerols. The amount of radioactivity in phosphatidylethanolamine was decreased by quinacrine and increased in the vicinity of an unidentified phospholipid-quinacrine complex. The present study indicates that the distribution of ¹⁴C-arachidonic acid in hamster lung lipids is sensitive to quinacrine. The detected changes can, however, not be explained by an overall inhibition of phospholipase A₂ activities.     

Chronic cigarette smoke exposure adversely alters 14C-arachidonic acid metabolism in rat lungs, aortas and platelets

Male rats were exposed to freshly generated cigarette smoke once daily, 5 times a week for 10 weeks. Inhalation of smoke was verified by elevated carboxyhemoglobin in blood sampled immediately after smoke exposure and by increased lung aryl hydrocarbon hydroxylase activity 24 hours after the last smoke exposure. Aortic rings isolated from smoke-exposed rats synthesized less prostacyclin (PGI2) from 14C-arachidonic acid than rings from sham rats. Platelets from smoke-exposed rats synthesized more thromboxane (TXA2) from 14C-arachidonic acid than platelets from room controls but not those from sham rats. Lung microsomes from smoke-exposed rats synthesized more TXA2 and had a lower PGI2/TXA2 ratio than lung microsomes from room controls and shams. It is concluded that chronic cigarette smoke exposure alters arachidonic acid metabolism in aortas, platelets and lungs in a manner resulting in decreased PGI2 and increased TXA2, thereby creating a condition favoring platelet aggregation and a variety of cardiovascular diseases.     

The amount of arachidonic acid in the triacylglycerols of perfused hamster lungs is increased by prednisolone

Following the injection of 14C-arachidonic acid (4.1 nmol) into hamster isolated lungs about 80% of the administered radioactivity was retained by the lungs. During subsequent perfusion only a small amount of radioactivity was released to the perfusion effluent. This release was not affected by pulmonary infusion of prednisolone at 20 microM or 100 microM. In control lungs 84 +/- 1% (+/- SEM) of the retained radioactivity was recovered in the phospholipid, 13 +/- 1% in the neutral lipid and 3 +/- 1 in the free fatty acid fraction. Pulmonary infusion of prednisolone increased the amount of radiolabel in the neutral lipids. This was due to the increased amount of 14C-arachidonic acid in triacylglycerols. Prednisolone had no significant effects on the amount of 14C-arachidonate in diacylglycerols or in different phospholipids. Neither was the amount of free 14C-arachidonate in the lungs changed by prednisolone. The present study indicates that the release of arachidonic acid from triacylglycerols may be inhibited by prednisolone in hamster lungs.     

The effect of cigarette smoke on the metabolism of arachidonic acid in isolated hamster lungs

The effects of cigarette smoke on the metabolism of exogenous arachidonic acid (AA) were investigated in isolated hamster lungs. Arachidonate was injected into the pulmonary circulation and the metabolites were analysed from the nonrecirculating perfusion effluent by thin layer chromatography. After the pulmonary injection of 66 nmol of ¹⁴C-AA about 20 % of the injected radioactivity appreated in the perfusion effluent mostly as metabolites in six minutes. When isolated lungs were ventilated with cigarette smoke during the perfusion, the amounts of PGF_2α, PGE₂ and two unidentified metabolite groups increased in the lung effluent. In two other experimental series hamsters were exposed to cigarette smoke before the lung perfusion either once for 30 min or during one hour daily for ten consecutive days. Neither pre-exposures caused any changes in the amounts of arachidonate metabolites in the lung effluent.     

Albumin stimulates the release of arachidonic acid from phosphatidylcholine in hamster lungs

¹⁴C-Arachidonic acid injected into the pulmonary circulation of isolated hamster lungs was effectively incorporated into lung lipids. Once retained the radiolabel was relatively stable but the release of radioactivity increased up to 10-fold when bovine serum albumin (1 %) was added to the perfusate. This efflux of radioactivity was not blocked by quinacrine, a phospholipase A₂ inhibitor. In albumin experiments the released ¹⁴C-araehidonate griginated mainly from the phospholipid fraction in which phosphatidylcholine was the main source of the released radioactivity.Pulmonary infusion of albumin had no significant effect on the amount of ¹⁴C-arachidonic acid in the neutral lipid or free fatty acid fractions of perfused lungs. In experiments with albumin about 80 % of the released radioactivity co-chromatographed with unlabelled arachidonic acid whereas in the absence of albumin only about 20 % of the released radioactivity was unmetabolized arachidonic acid. This study indicates that albumin stimulates the release of arachidonic acid from isolated hamster lungs and that the release is increased mainly from the phosphatidyl choline fraction.     

Profiles of eicosanoid production from 14C-arachidonic acid and prostaglandin metabolism by rabbit esophageal mucosa and muscularis

In an attempt to elucidate the possible involvements of eicosanoids in esophageal functions and disorders, we have investigated the formation of both cyclooxygenase and lipoxygenase metabolites from 14C-arachidonic acid by rabbit esophageal tissues. Homogenates of rabbit esophageal mucosa and muscularis were incubated with 14C-arachidonic acid and after ether extraction eicosanoids were separated and quantified by reverse phase high performance liquid chromatography. The predominant cyclooxygenase products were 6-keto-PGF1 alpha, PGF2 alpha, and PGE2 for mucosa and 6-keto-PGF1 alpha, and PGE2 for muscularis. The formation of these products was inhibited both by indomethacin and the dual pathway inhibitor, nordihydrogualaretic acid (NDGA). In mucosa the major eicosanoid was 12-HETE (12-hydroxyeicosatetraenoic acid) which was inhibited by NDGA but not by indomethacin which on the contrary enhanced its formation. Additionally four polar products were synthesized which appeared to be lipoxygenase-dependent as their formation was inhibited by NDGA but not by indomethacin. Muscularis produced as a minor lipoxygenase product only 12-HETE, which was inhibited by NDGA but unchanged in the presence of indomethacin. In addition, both tissues, but mucosa more than muscularis, possessed large prostaglandin catabolizing capacity. The present findings indicate that rabbit esophageal tissues can convert 14C-arachidonic acid into lipoxygenase as well cyclo-oxygenase products which may have a role in esophageal physiology and pathophysiology.     

Sequential incorporation of 3H- and 14C-arachidonic acid during 24 h in rabbit colonic explants cultured in vitro.

Arachidonic acid (AA) might be released from a number of different intracellular pools. This aspect was studied by sequential incorporation of 3H- and 14C-AA during 24 h in rabbit colonic biopsies in culture. Mucosal explants were subjected to 3H-AA (0-6 h) and 14C-AA (6-24 h) followed by a 2-h washout. Lipid extraction of biopsies revealed a distinct pattern where 3H, in relative terms, dominated in phosphatidylinositol (PI) and phosphatidylethanolamine (PE) and 14C was more abundant in neutral lipids, whereas the remaining lipids had equal proportions of labellings. When stimulating double labelled biopsies with 1 microM phorbol 12-myristate 13-acetate (PMA), 10 micrograms/ml A23187 and a combination thereof, the 14C-labelling was in general released in larger amounts, i.e. a maximum of 7.5% with the combined drugs vs 5% for 3H. However, the relative release of 3H increased upon stimulation especially for PMA + A23187. The release of labelling was directly coupled to a stimulation dependent production of PGE2 in the order PMA less than A23187 less than PMA + A23187, maximum release = 8.2 +/- 2.4 ng/h and 3 biopsies. This study shows the existence of different AA-pools, where PI and PE (3H-labelling) appears to have a slow turnover and representing a terminus in a redistribution chain and where neutral lipids (14C-labelling) have a more rapid turnover. Upon stimulation more AA appears to be recruited from pools with slow turnover.     

The effect of cigarette smoke on the metabolism of arachidonic acid to myotropic compounds in rat and hamster isolated lungs

Perfusion effluent from isolated rat and hamster lungs caused a relaxation of superfused strip of bovine coronary artery (BCA). This relaxation was abolished by pulmonary infusion of indomethacin. Pre-exposure of rats and hamsters to cigarette smoke during half an hour before the lung perfusion did not change the degree of this initial relaxation of BCA. Injection of 10 μg of sodium arachidonate (AA) into the pulmonary circulation of isolated hamster lungs caused a contraction of BCA, which was not changed by cigarette smoke pre-exposure. When AA (10 μg) was injected into the pulmonary circulation of isolated hamster lungs during cigarette smoke ventilation the contractions of superfused BCA and rat stomach strip (RSS) were not significantly different from those during the preceding and following air ventilation. In experiments with isolated rat lungs the initial relaxation of superfused BCA was accompanied by a contraction of superfused RSS. AA injection (10 μg) into rat lungs caused a further relaxation of BCA and contraction of RSS, which were abolished by pulmonary infusion of indomethacin. Cigarette smoke ventilation of isolated rat lungs caused a relaxation of superfused BCA, which was not abolished by indomethacin. During cigarette smoke ventilation injection of AA (10 μg) into the pulmonary circulation of rat lungs caused a relaxation of BCA and a contraction of RSS.The present study indicates that neither cigarette smoke ventilation nor pre-exposure to cigarette smoke has a drastic effect on the metabolism of arachidonic acid to myotropic compounds in isolated hamster and rat lungs.     

The use of fluorescence microscopy and microfluorimetry to study the distribution of quinacrine in mice

Summary A simple method was used to prepare cryostat-cut sections in which quinacrine-induced fluorescence could be seen by fluorescence microscopy. Such sections were used in preliminary studies of the distribution of this drug in mice. Micro-fluorimetry was used to quantify the fluorescence. The method may be of value in the detection of drugs in various fields.     

Aspirin inhibits arachidonic acid metabolism via lipoxygenase and cyclo-oxygenase in hamster isolated lungs

The effect of aspirin on the fate of exogenous arachidonic acid (AA) was investigated in isolated perfused lungs of female hamsters. During pulmonary infusion of aspirin (10 μM, 100 μM or 1 mM) 45 nmol of ¹⁴C-AA was infused in two minutes into the pulmonary circulation. The nonrecirculating perfusion effluent was collected for 6 minutes after the beginning of the AA infusion. Arachidonate infusion increased the perfusion pressure. This pressor response was completely abolished by 1 mM aspirin. When aspirin was infused into the pulmonary circulation, the amount of radioactivity was increased in the perfused lungs and decreased dose dependently in the nonrecirculating perfusion effluent. The amount of unmetabolized free arachidonate was not changed significantly by aspirin in the perfused lungs or in the perfusion effluent. In the effluent the amounts of all arachidonate metabolites, which were extracted with ethyl acetate first at pH 7.4 and then at pH 3.5 and analysed by thin layer chromatography, were decreased quite similarly by aspirin. The formation of arachidonate metabolites was completely inhibited by 1 mM aspirin. In the perfused lung tissue the amount of ¹⁴C-AA was increased by aspirin in phospholipids and neutral lipids. The present study indicates that the metabolism of arachidonic acid is inhibited by aspirin in hamster lungs not only via cyclo-oxygenase but also via other lipoxygenases.     

The distribution of quinacrine on chromosomes as determined by X-ray microanalysis

The distribution of quinacrine and protein sulphur has been compared with that of DNA in euchromatic and heterochromatic regions of mouse chromosomes stained with the fluorescent dye quinacrine, using X-ray microanalysis. Heterochromatin tends to bind relatively more quinacrine than euchromatin, and contains a greater concentration of sulphur. Measurements of quinacrine fluorescence, when compared with quinacrine binding, show that the excitation of fluorescence is more efficient when the dye is bound to euchromatin than when it is bound to heterochromatin. Although this observation is consistent with the hypothesis that the dull quinacrine fluorescence of mouse centromeres is due to quenching by guanine residues, two other factors should also be considered: the lower absolute amount of dye bound to the centromeres, and a concentration-dependent quenching of fluorescence.     

Heart arachidonic acid is uniquely sensitive to dietary arachidonic acid and docosahexaenoic acid content in domestic piglets

This study determined the sensitivity of heart and brain arachidonic acid (ARA) and docosahexaenoic acid (DHA) to the dietary ARA level in a dose–response design with constant, high DHA in neonatal piglets. On day 3 of age, pigs were assigned to 1 of 6 dietary formulas varying in ARA/DHA as follows (% fatty acid, FA/FA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3–D3) 0.69/1.0; (A4) 1.1/1.0; (D2) 0.67/0.62; and (D1) 0.66/0.33. At necropsy (day 28) higher levels of dietary ARA were associated with increased heart and liver ARA, while brain ARA remained unaffected. Dietary ARA had no effect on tissue DHA accretion. Heart was particularly sensitive, with pigs in the intermediate groups having different ARA (A2, 18.6±0.7%; A3, 19.4±1.0%) and a 0.17% increase in dietary ARA resulted in a 0.84% increase in heart ARA. Further investigations are warranted to determine the clinical significance of heart ARA status in developing neonates.     

The distribution of quinacrine on chromosomes as determined by X-ray microanalysis

The distribution of quinacrine in relation to Q-banding on CHO chromosomes has been investigated using X-ray microanalysis. Technical problems involved in this type of experiment were studied in detail. It was necessary to use a solution of quinacrine acetate in acetic acid to ensure that the only chlorine detectable in quinacrine-stained chromosomes was in the quinacrine molecule. Electron irradiation during analysis rapidly destroys quinacrine fluorescence, but the chlorine is not lost from the chromosomes, and there are several reasons for supposing that a reliable distribution of quinacrine on the chromosome can be obtained by the method. — Small variations along the chromosome in the amounts of chlorine (representing quinacrine) and of phosphorus (mainly DNA) occur. The distribution patterns for chlorine and phosphorus show a good resemblance to each other for each homologous chromosome; quinacrine fluorescence patterns (Q-bands) do not resemble chlorine distribution patterns, however. The results of this study therefore support the view that Q-bands result from the differential quenching of fluorescence along chromosomes to which the quinacrine is essentially uniformly bound, and do not reflect differential binding of quinacrine along the chromosome.With an Appendix by A. D. Carothers and D. Rutovitz     

Hybridization of Chinese hamster cells sensitive to ultraviolet irradiation

Resistance to UV-light was studied in two UV-sensitive aneuploid Chinese hamster cell clones to different origin and degree of sensitivity, their respective polyploids and somatic cell hybrids. The karyotype of the parental clones, cell hybrids and polyploids was analyzed in parallel. A great variability of karyotypes was detected in hybrid cells. Serial cultivation of hybrids was accompanied by chromosome loss. Soon after fusion the hybrid clones proved to be more resistant to UV than the parental sensitive cells. However, their sensitivity increased with passages. The comparison of UV-sensitivity with data on karyotype analysis allowed to assume that the increase in sensitivity was correlated with the loss of particular chromosomes or chromosome regions. The results obtained indicated the existence of a polygenic control of UV-sensitivity, the multiple genes being assigned to different chromosomes. A reverse effect of ploidy was detected, i.e. a decrease in the resistance to the lethal action of UV-light in polyploids as compared to the parental clones.     

Studies on a Chinese hamster line that is temperature sensitive for the commitment to DNA synthesis

The Chinese hamster cell line K12 is temperature sensitive for the initiation of DNA synthesis and for the programming of rounding up to enter mitosis. Viability is lost expontentially after twelve hours at the non-permissive temperature. This temperature sensitivity is reversed following exposure of K12 cells to SV40 virus.     

Identification of Chinese hamster chromosome bivalents at diakinesis by quinacrine mustard fluorescence

The eleven chromosome bivalents of the Chinese hamster at diakinesis can be identified on account of their morphology, in combination with the fluorescence pattern. Comparison of the fluorescence pattern of the sex-chromosomes in both mitosis and meiosis shows that the distal part of the short arm of the X chromosome is homologous with part of the Y chromosome. It is not possible, however, to decide with certainty which part of the Y chromosome is involved.