Induction of growth-related metabolism in human vascular smooth muscle cells by low density lipoprotein.

Human vascular smooth muscle cells (hVSMC) rendered quiescent by maintenance under serum-free culture conditions for 48 h exhibited several metabolic responses, normally associated with proliferation, following exposure to low density lipoprotein (LDL). LDL induced a time- and dose- (half-maximally effective concentration, ED50 25.0 +/- 8 nM) dependent activation of S6 kinase which could be negated following pretreatment of hVSMC with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 48 h. In myo-[3H]inositol-prelabeled hVSMC, LDL caused a rapid (maximum within 1 min) decrease in phosphatidylinositol 4,5-bisphosphate (35% p less than 0.001) and phosphatidylinositol 4-phosphate (20%, p less than 0.01) with a return to prestimulated levels within 5-10 min. LDL induced a concomitant increase in [3H]inositol phosphates for which the order of generation was inositol-tris greater than -bis greater than -mono phosphate and which reached threshold levels of significance (p less than 0.05) above control values within 1, 2, and 10 min, respectively. The effect of LDL on hVSMC phosphoinositide metabolism was dose-dependent (half-maximally effective concentration, ED50 32.1 +/- 5.0 nM). This concentration, like that for S6 kinase, approximates with the KD (5-21 nM) for high affinity binding of 125I-LDL to specific receptors (1.5 x 10(4) sites/cell) on hVSMC. LDL induced a rapid but transient translocation of protein kinase C from the cytosol to membranes as assessed using both immunoblotting and [3H] 4-beta-phorbol-12-13-dibutyrate-binding procedures. Exposure of quiescent hVSMC to LDL elevated intracellular pH (delta pH 0.30 +/- 0.03, p less than 0.001). Such alkalinization was prevented in the presence of Na+/K+ exchange inhibitors such as amiloride, dimethylamiloride, and ethylisopropylamiloride. In an investigation of the nuclear action of LDL, a time-dependent induction of both c-myc and c-fos was observed. Such LDL-induced expression of these nuclear proto-onco-genes was not detectable in protein kinase C down-regulated hVSMC. Nevertheless, in spite of the cascade of "growth-promotional" responses elicited by LDL in quiescent hVSMC, this lipoprotein alone (under serum-free conditions) was neither mitogenic in nuclear labeling experiments, nor could it support growth of hVSMC in culture. We demonstrate that LDL might function in a complementary/synergistic fashion with other weakly mitogenic (to VSMC) growth factors and suggest that activation of protein kinase C (vis à vis intrinsic tyrosine kinase characteristic of other growth factor receptors) may be crucial to the signal transduction pathway for LDL.

Quantitative comparison of bromoacetylated derivatives of alprenolol and pindolol on beta-adrenergic receptor binding and mobility in S49 cells.

The reactivities with beta-adrenergic receptors of the bromoacetyl derivatives of the beta-adrenergic antagonists alprenolol and pindolol, BAAM and BIM, respectively, were compared in intact S49 mouse lymphoma cells. Both compounds caused irreversible blockade of receptors and changes in the mobility of the remaining non-modified receptors. BIM proved to be an irreversible blocker of high potency, with an IC50 of 40-60 nM at 4 degrees C, whereas the IC50 for BAAM was 600-900 nM. Moreover, treatment with both compounds resulted in an inhibition of internalization of non-modified receptors (IC50 = 200-300 nM for both). After treatment of desensitized cells which have internalized 50-60% of their surface receptors, with BIM or BAAM, receptor reappearance was found to be slowed down (IC50 about 100 nM for both compounds). The effects of the bromoacetylated antagonists on receptor internalization were apparently selective for beta-adrenergic receptors, since binding and internalization of transferrin or low-density lipoprotein were not affected.

Similarities in cAMP responses between murine lymphoid cell lines and subsets of mouse lymphocytes.

Intracellular cAMP responses to forskolin (which stimulates independently of hormone receptors) and to several hormones were studied in mouse lymphoid cell lines of B- and T-cell origin and in subpopulations of mouse lymphocytes. In the T-cell lines, isoproterenol caused an increase in cAMP amounting at most to 28% of that caused by 100 microM forskolin. In contrast, in the B-cell lines the increase ranged from 50% to over 100%. In 11 of 13 T-cell lines, forskolin at 1 microM concentration potentiated the isoproterenol response five- to tenfold. Forskolin potentiation was also found in thymocytes and peripheral T-cells but not in B-cell lines. The relative increases in intracellular cAMP evoked by isoproterenol, prostaglandin E2 (PGE2) and histamine varied markedly from cell line to cell line. For instance, three lines carrying the T-helper cell marker L3T4 responded only to PGE2 and not to isoproterenol, whereas two cell lines carrying the Ly2 marker gave a higher response to isoproterenol than to PGE2. The results indicate that subsets of lymphocytes respond differently to various hormones and that these differences are maintained in continuous cell lines.

Bromoacetylalprenololmenthane binding to beta-receptors modulates the rate of hormone-induced internalization but not desensitization in S49 cells.

Bromoacetylalprenololmenthane was found to inhibit hormone-induced beta-adrenergic receptor internalization in a dose-dependent fashion in S49 lymphoma cells, besides its known ability to bind to beta-receptors irreversibly. This new found property of BAAM+ was taken advantage of in studying whether receptor internalization is a necessary step in the desensitization of adenylate cyclase. BAAM-treated cells showed functional desensitization even when receptor internalization had been blocked substantially by 50-65%. This finding suggests that receptor internalization is not directly involved in desensitization.