Characterization of astrocyte plasminogen activator.

We have previously shown that astrocytes produce and secrete plasminogen activator (PA) and that this function is responsive to various modulating agents. When astrocyte conditioned medium (CM) is subjected to SDS-PAGE and PA activity localized by fibrin-agar gel overlay, the activity in the CM is found to comigrate with control t-PA. On affinity chromatography CM PA specifically binds to t-PA antibody. The latter also inhibits fibrinolytic activity of CM PA. When incubated with a fibrin clot, CM PA activity can be shown to bind to fibrin. These observations help identify the enzyme in astrocyte CM as t-PA. A possible role of astrocyte PA in myelin injury could provide an explanation for the previously observed correlation between fibrin deposition and demyelination as well as inhibition of demyelination by ancrod and heparin in experimental allergic encephalomyelitis.

Divergence of ANF analogs in smooth muscle cell cGMP response and aorta vasorelaxation: evidence for receptor subtypes.

ANF analog potencies in stimulating smooth muscle cell cGMP were compared with the ability to relax histamine-constricted rabbit aorta in vitro. ANF[1-28], [5-28], [5-27] and Lys-11[5-28] elevated cGMP and were potent vasorelaxants. ANF[7-23] and Lys-11[7-23] were potent cGMP stimulators but 1000-fold weaker relaxants. Tyr-8[5-27] did not stimulate cGMP synthesis or antagonize the response of the other peptides, yet was a potent vasorelaxant. Crosslinking with 125I-ANF identified bands at 150 and 65 KD by SDS-PAGE. ANF[1-28], Lys-11[7-23] and Tyr-8[5-27] blocked crosslinking at low concentration despite disparate activities. These data support the existence of ANF receptor subtypes and suggest that cGMP elevation alone is not sufficient to promote atrial peptide-induced vasorelaxation.

Identification of the plasminogen activator(s) produced by the transformed liver cell line, SK-HEP-1.

The availability of a cell line derived from an adenocarcinoma of the liver has made it possible to study the plasminogen activator(s) (PA) biosynthesized in culture by liver cells. Conditioned cultured media purified on fibrin-celite, benzamidine-Sepharose and immunoabsorbent anti-urokinase columns have shown the presence of multiple plasminogen activators when separated on sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE). These PAs differ in molecular weight but all are urokinase-like based on their reaction with goat anti-urokinase and rabbit anti-tissue activator. Subcellular fractionation of the cultured cells shows the presence of activator in both the cytoplasmic and membrane fractions, but the higher molecular weight forms appear primarily in the cytoplasm.

Effects of growth hormone excess and deficiency on hypothalamic somatostatin content and release and on tissue somatostatin distribution.

Considerable indirect evidence now exists to suggest that hypothalamic somatostatin (SRIF) is the physiological inhibitory regulator of pituitary GH release. To support this relationship further, we studied the effect of in vivo modifications of GH homeostasis on hypothalamic SRIF content and in vitro release in an attempt to document a feedback relationship between the two peptides. GH administration to normal rats resulted in increased hypothalamic SRIF concentration and release. GH deficiency, in contrast, resulted in decreased hypothalamic SRIF concentration and release. This effect appears to be, at least in part, a direct action of GH, since a dose-related stimulation of hypothalamic SRIF release was demonstrated in the presence of GH concentrations ranging from 10(-9)-10(-5) M. The lowest dose causing stimulation (10(-9) M) is well within the normal concentration range of plasma GH in the rat, suggesting that the effect may be physiological. Specificity of the effect is suggested by a much greater sensitivity of the medial basal hypothalamus than the septum and preoptic area to the effects of GH. The perturbations of GH homeostasis studied had no effect on extrahypothalamic neural or gastrointestinal SRIF concentrations, suggesting a different regulatory mechanism in these areas.