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Smooth muscle cells were grown from thoracic aortas of rats. The effect of insulin on the proliferation of these cells was studied by comparing the growth of cells in culture medium containing insulin and 1% fetal calf serum with growth of cells in culture medium containing only 1% serum and in culture medium containing 10% serum. Insulin in concentrations of 10, 50, 100, 1000 and 10 000 microunits/ml induced smooth muscle cells to stationary growth more rapidly than basal medium. This could be demonstrated as well in the logarithmic growth as in confluent cells grown in medium with 1% serum. However, the highest concentration of insulin did not stimulate growth to the same degree as medium containing 10% serum. Cells that were older in culture life (11th passage) did not show a growth response to insulin.

Serum contains various growth factors which regulate the proliferation of cells. We investigated the growth of cultured arterial smooth muscle cells under the influence of insulin-like growth factor I (IGF I), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), and examined the effect of these growth factors on the binding of [125I] IGF I and on the binding of [125I]PDGF to these cells. IGF I, FGF, and PDGF stimulated [6-3H]thymidine incorporation into DNA of confluent cultures of cells which were incubated in modified Dulbecco's modified Eagle medium. However, the effect of these growth factors on DNA synthesis was much more potent in Dulbecco's modified Eagle medium with 1% fetal calf serum. FGF and PDGF potentiated the growth-promoting effect of IGF I. The binding of [125I]IGF I to the cells was increased after a preincubation with FGF and PDGF. The binding was potently increased by FGF (100 ng/ml) after a preincubation time of 30 min. There was an increase in binding during the first 3 h of preincubation followed by a decrease after 4-5 h. PDGF (10-1000 ng/ml) stimulated [125I]IGF I binding only after 2 h of preincubation. The stimulation was dose dependent. Maximal stimulation of the binding was observed after 3 h of preincubation followed by a decrease after 4-5 h of preincubation. Specific binding sites for PDGF on smooth muscle cells could be demonstrated too. A preincubation of confluent cells with IGF I caused a dose-dependent increase in [125I]PDGF binding. These results support the hypothesis that the regulation of the binding of a specific growth factor by a second growth factor is important for the control of cell growth.

The effect of thyroid hormones (thyroxine and triiodothyronine) on catecholamine receptors in isolated rat fat cells was investigated. Binding of (3H)isoproterenol and (3H)norepinephrine were increased by thyroid hormones. (3H)isoproterenol binding was more enhanced than (3H)norepinephrine binding. Triiodothyronine had a more potent effect than thyroxine. (3H)isoproterenol was used to estimate the number or affinity of beta-adrenergic receptors in rat fat cells treated with thyroid hormones. The binding sites for (3H)isoproterenol were the same in untreated and with triiodothyronine treated fat cells. The equilibrium dissociation constants (KD) for the interaction of receptors with (3H)isoproterenol were altered in thyroid hormone treated cells. There was a significant difference between the untreated and triiodothyronine treated fat cells in the affinity of beta-adrenergic receptor binding sites for (3H)isoproterenol. Thyroid hormone could alter negative cooperative site-to-site interaction among the binding sites for (3H)isoproterenol.

The role of thyroid hormones on lipolysis in human subcutaneous adipose tissue was investigated. Incubation of subcutaneous fat pads with thyroxine (0.1--10 000 nM) augmented the subsequent isoproterenol stimulation of lipolysis, measured by glycerol release. The basal lipolysis could not by stimulated by thyroxine. The theophylline- and dibutyryl-cyclic AMP stimulated lipolysis also could not be increased by thyroxine at these concentrations. In separate studies, the effect of thyroxine (0.01 pM--1 microM) and triiodothyronine (0.01 pM--1 microM) on cyclic AMP accumulation was examined. No effect of thyroid hormones on cyclic AMP accumulation was seen in non-isoproterenol stimulated tissue. Fat pads stimulated by isoproterenol and then treated with thyroid hormones showed marked increases in accumulation of cyclic AMP as compared to control tissue in the presence of isoproterenol alone.

Insulin and insulinlike growth factors (IGF) are related polypeptides that have similar biological activities. Both factors produce metabolic effects as well as growth effects. Most cells have separate receptors for IGF and insulin. In the present study we have demonstrated specific IGF receptors in cultured smooth muscle cells of rat aorta. The properties and specificity of these receptors were compared with those of the insulin receptor in the same cell system. The specific binding of both 125I-IGF and 125I-insulin binding in the order of potency: insulin proinsulin IGF. The potency of IGF in displacing 125I-insulin was rapid and reversible. Maximal 125I-IGF binding occurred at 20 degrees C with a specific binding of 10%. At this temperature specific 125I-insulin binding was 1.3% and occurred in biphase. The pH optimum for 125I-IGF binding was between pH 7 and 8. Both receptors show a high degree of specificity. IGF, insulin and proinsulin competed for 125I-IGF binding in the order of potency: IGF proinsulin insulin. The potency of insulin in displacing 125I-IGF was about 2000 times lower than that of IGF itself. In addition, IGF, insulin and proinsulin competed for 125I-insulin was about 450 times lower than that of insulin. These results indicate two separate binding sites for insulin and IGF on arterial smooth muscle cells.

A highly sensitive and specific radioimmunoassay for the measurement of insulin-like growth factor I (IGF I) has been developed. The IGF I concentrations were measured in sera of normal subjects, patients with acromegaly, hypophysectomized patients and patients with hyperprolactinaemia. The results were compared with the results obtained after measurement of IGF I in the sera of the same patients with two commercially available radioimmunoassays for somatomedin-C. IGF I was separated from its carrier protein using Sep-Pak C18 cartridges. The total recovery of IGF I by this method was about 100%. The in-house assay shows a high specificity for IGF I and a high sensitivity. As little as 0.1 microgram/l of IGF I can be detected. Only a small amount of serum (25 microliter) is necessary for the IGF I determination and a great number of serum samples (more than 100 per day) can be processed. The mean immunoreactive IGF I concentration was 379 +/- 159 micrograms/l in normal adult subjects, 3340 +/- 1094 micrograms/l in acromegalic patients, and 52 +/- 11 micrograms/l in growth-hormone deficient patients. Patients with hyperprolactinaemia had an IGF I concentration of 433 +/- 112 micrograms/l.

Insulin-like growth factors (IGF) were isolated from human serum and compared with some biological actions of IGF supplied by Dr. J. Hapf, Zurich. Both factors were potent mitogens. They stimulated DNA-, RNA- and protein synthesis in cultivated human arterial smooth muscle cells. Furthermore, they enhanced the aminoacid transport. Our protein fraction (IGF Ulm) had a more potent biological activity than IGF (Zurich). Specific binding receptors for IGF (Zurich) on human arterial smooth muscle cells could be demonstrated. Specific binding of 125I-IGF (Zurich) was 10%. Half-maximal displacement was achieved by 250 ng/ml of unlabeled IGF (Zurich), by 1.2 micrograms/ml of IGF (Ulm), by 6.3 micrograms/ml of pro-insulin and by 17.8 micrograms/ml of insulin. In separate studies we could demonstrate that sera of normal adults, diabetic, acromegalic and hypophysectomized patients showed different growth-promoting activity in human arterial smooth muscle cells.

The specific binding of (3H) isoproterenol to isolated fat cells of human and rat was characterized. Binding of (3H) isoproterenol to isolated fat cells of rat was saturable with 420 pmol of (3H) isoproterenol bound/100 mg of lipid. Half-maximal saturation occurred at 5 microM providing an estimate of the equilibrium dissociation constant, KD, for the interaction of (3H) isoproterenol with its binding sites. Kinetic analysis of (3H) isoproterenol binding provided a value of 2.01 x 10(4) min-1. M-1 for the forward bimolecular rate constant, k1. Dissociation of (3H) isoproterenol was a first order reaction with a rate constant, k2, of 0.62 x 10(-1) min-1. The ratio k2/k1 = 3.07 microM provides an independent measurement of the KD for the interaction of (3H) isoproterenol with its binding sites which is in agreement with the values obtained by steady state analysis (3 to 5 microM). The apparent equilibrium dissociation constant, KD, for the interaction of (3H) isoproterenol with its receptor in human fat cells obtained by steady state analysis was 1 to 0.9 microM. Scatchard- and Hill-analysis suggest the possibility of different negatively cooperative interactions among the binding sites in human and rat. beta-Adrenergic agonists competed for the binding sites. The order of potency was isoproterenol greater than epinephrine greater than norepinephrine. Compounds such as DOPA, dopamine and (m-Hydroxyphenyl)2-methyl-aminoethanol which are structurally related to catecholamines had little or no affinity for (3H) isoproterenol binding sites.

There are indications for the existence of an intrinsic renin angiotensin system in vascular walls, which is assumed to participate in blood pressure regulation and in pathogenesis of arterial hypertension. It was evaluated if and to what extent the decapeptide angiotensin (A) I, one of the natural substrates of A I converting enzyme (ACE), is degraded by other peptidases than ACE in rat vascular tissues. A I and A II degradation was studied in arterial and venous vascular wall extracts. The activities ranged between 0.068 ± 0.025 U and 0.044 ± 0.025 U. The enzymes involved were biochemically characterized by determination of isoelectric points (pI), pH optima, molecular weights and by investigation of their inhibition behavior in vitro. One potent A I degrading enzyme (AIDE) was identified with pI between 3.6 and 3.9, and pH optimum at 7.75. In vitro studies revealed that AIDE activity was not blocked by the specific ACE inhibitors MK 421 or MK 422 (both 11 nMol/ml). The molecular weight of AIDE ranged between 440,000 and 457,000. The results indicate that AIDE is not identical to ACE (pI 4.2–5.0; pH optimum 8.3). AIDE was also observed in aortic smooth muscle cells cultured in vitro. AIDE decreased following bilateral nephrectomy or administration of aldosterone combined with sodium chloride loading, whereas it was elevated in spontaneously hypertensive rats (Okamoto strain). Since AIDE metabolizes A I, one of the substrates of ACE, it may indirectly affect A II formation and bradykinin inactivation as well.