Outgrowth of chondrocytes from human articular cartilage explants and expression of alpha-smooth muscle actin.

The objectives of this study were to investigate the effect of various enzymatic treatments on the outgrowth of chondrocytes from explants of adult human articular cartilage and the expression of a specific contractile protein isoform, alpha-smooth muscle actin, known to facilitate wound closure in other connective tissues. Explants of articular cartilage were prepared from specimens obtained from patients undergoing total joint arthroplasty. The time to cell outgrowth in vitro was determined and the expression of alpha-smooth muscle actin shown by immunohistochemistry. Treatment of the explants with collagenase for 15 minutes reduced the time to outgrowth from more than 30 days to 3 days. Hyaluronidase, chondroitinase ABC, and trypsin applied for the 15-minute period had no effect on the time to cell outgrowth when compared with untreated controls. Pretreatment with hyaluronidase prior to collagenase reduced the time to outgrowth. A notable finding of this study was that the majority of chondrocytes in the adult human articular cartilage specimens and virtually all of the outgrowing cells contained alpha-smooth muscle actin. We conclude that human articular chondrocytes have the capability to migrate through enzymatically degraded matrix and express a contractile actin isoform. Collagenase treatment reduces the time required for cell outgrowth.

Vasodilator effect of scoparone (6,7-dimethoxycoumarin) from a Chinese herb.

A possible mechanism of the vasodilator effect of scoparone was investigated. Scoparone (10(-6)-3 x 10(-5) M) dilated rat aortic rings precontracted with phenylephrine in a dose-dependent manner. The presence of endothelium facilitated the vasodilator effect. Scoparone depressed the contractile responses to phenylephrine and serotonin, but not that to potassium chloride. Both the vasoconstriction and O2- production induced by alloxan, a diabetogenic compound, were depressed by scoparone. It appears that scoparone exhibited a free radical scavenger-like effect. The dilatation elicited by acetylcholine was potentiated by scoparone. The dilator activity of scoparone was markedly inhibited by methylene blue and hemoglobin, guanylate cyclase inhibitors. Furthermore, the basal guanosine 3',5'-cyclic monophosphate (cGMP) level was elevated in the presence of scoparone. The dilator activity of scoparone was also inhibited by quinacrine (inhibitor of phospholipase A2) and indomethacin (inhibitor of cyclooxygenase). Our results showed further that the output of 6-keto-prostaglandin F1 alpha, a stable metabolite of prostacyclin, was enhanced by scoparone. It is suggested that the vasodilator effect of scoparone in rat aorta may be mediated through the enhancement of prostacyclin release, protecting against EDRF inactivation, and activating guanylate cyclase.

Vasorelaxant effect of emodin, an anthraquinone from a Chinese herb.

The possible mechanism underlying the vasorelaxant effect of emodin isolated from a Chinese herb, was investigated in this study. Emodin dose dependently relaxed isolated vascular rings of human internal mammary artery and saphenous vein, rabbit thoracic aorta, abdominal aorta and mesenteric artery, and rat thoracic aorta. There were no differences in the sensitivity (IC50) and maximal relaxation between intact and endothelium-denuded preparations of rat aorta. In the presence of emodin (10 microM), the contractile responses of rat aorta to phenylephrine, serotonin and potassium chloride were depressed. The relaxation response to acetylcholine was attenuated by emodin, whereas that to isoproterenol was unaffected. The relaxation response to emodin was inhibited by free radical scavengers, superoxide dismutase, catalase and mannitol, and guanylate cyclase inhibitors, methylene blue and hemoglobin. Catalase was the most effective scavenger. Quinacrine (phospholipase A2 inhibitor), indomethacin (cyclooxygenase inhibitor) and nordihydroguaiaretic acid (NDGA, lipoxygenase inhibitor) potentiated the relaxation induced by emodin. NDGA was the most effective potentiator. Exposure of aortic rings to emodin (10 microM) increased the basal level of guanosine 3',5'-cyclic monophosphate (cGMP). It is suggested that the vasorelaxant effect of emodin may be mainly due to cGMP accumulation as a result of guanylate cyclase activation by free radicals and/or hydrogen peroxide generated from semiquinone.

Effect of dietary fat and cholesterol supplements on glucagon receptor binding and adenylate cyclase activity of rat liver plasma membrane.

We have examined the effect on rat liver glucagon receptors and adenylate cyclase activity of a high saturated fat diet (butter fat), a high n-6 polyunsaturated fat diet (corn oil), and a high n-3 polyunsaturated fat diet (menhaden fish oil) with or without additional cholesterol. The number and affinity of the glucagon receptors were unaffected by diet. The glucagon-stimulated adenylate cyclase activity from fish oil-fed animals exhibited the greatest stimulation, followed by corn oil-fed animals. Butter fat-fed and all cholesterol-supplemented groups showed a depression in stimulation. The pattern of adenylate cyclase activity with fluoride stimulation was similar to that observed with glucagon. The effect of dietary fat on forskolin stimulation was similar to glucagon and fluoride for the groups without added cholesterol. However, the cholesterol-supplemented groups did not exhibit a decreased activity. It is suggested that the effect of dietary lipid on glucagon-stimulated adenylate cyclase is not due to changes in the glucagon receptor, but rather due to changes in signal transduction, the Gs-protein or the catalytic unit.

The use of arginine analogues for investigating the functional organization of the arginine-binding site in lobster muscle arginine kinase. Role of the 'essential' thiol group.

1. The nature of arginine binding to lobster arginine kinase and the extent of its possible involvement with the ;essential' thiol group of the enzyme has been investigated with some inhibitory analogues of arginine. 2. Most of the analogues inhibit competitively, although mixed inhibition may occur if the alpha-carboxy group or alpha-amino group is absent. 3. The K(i) values indicate that strength of binding depends on the length of the carbon chain (l-isoleucine>l-valine>l- alpha-aminobutyrate>l-alanine) and the integrity of the substituents on the alpha-carbon atom (l-arginine>agmatine and l-ornithine>putrescine). The guanidino group probably contributes little to substrate binding, but a positive charge near the delta-nitrogen atom appears to be important (l-ornithine>l -citrulline>l-alpha-aminobutyrate). A cyclic analogue, 2-carboxymethyl-3-oxo-2,3,5,6,7,8-hexahydro-1H-imidazo [1,2-a][1,3]diazepine-8-carboxylic acid, has a low K(i) value similar to that of an equivalent straight-chain form, suggesting that arginine probably binds in a folded configuration. 4. The aliphatic l-amino acids give enzyme difference spectra similar to that with l-arginine and the integrity of the alpha-carboxy and alpha-amino groups appears to be a minimal but not sufficient requirement for this, as l-ornithine gives an atypical difference spectrum. A difference spectrum is interpreted as indicating an enzyme conformational change. No difference spectrum was observed with methylguanidine. 5. The ability of aliphatic alpha-l-amino acids to protect against inhibition by 5,5'-dithiobis-(2-nitrobenzoic acid) is proportional to the number of atoms in the carbon chain and inversely proportional to K(i). Ornithine gives greater protection than citrulline; analogues lacking the alpha-amino groups also protect. Agmatine, lacking the alpha-carboxy group, did not protect. 6. It is concluded that it is unlikely that the ;essential' thiol group in the enzyme interacts with any part of the arginine molecule during catalysis except, possibly, the alpha-carboxyl group.