Biological and clinical effects of interferon-beta ser at two doses.

To assess biological response, therapeutic activity, and side effects, a randomized, double-blind trial of two doses of interferon-beta ser (IFN-beta ser), differing by 20-fold 4.5 and 90 x 10(6) units), was undertaken in 64 patients with metastatic renal carcinoma. Patients were treated intravenously with injections daily for 10 days with an 11-day rest before treatment was reinitiated. The trial confirmed the relatively good toleration of IFN-beta ser; in the first cycle only 4/63 patients had anorexia of moderate or greater severity. Median weight change over the duration on study was -1.5 kg; in the first cycle only 7% of patients had performance status decline greater than 1 level. Statistically significant changes (p less than 0.05) occurred in granulocytes, lymphocytes, calcium, cholesterol, alkaline phosphatase, and aspartate transferase (AST); however, except for AST, overall clinical differences in the two doses were not great. Of 60 patients evaluated, 1 developed neutralizing antibody. When assessed 24 h after IFN-beta ser at 4.5 x 10(6) units, significant (p less than 0.05) augmentation had occurred in beta 2-microglobulin, HLA-DR, and HLA-DQ expression on monocytes, 2',5'-oligoadenylate (2-5A) synthetase in peripheral mononuclear cells, and natural killer (NK) and K cells functional activity. Although the 90 x 10(6) unit dose also resulted in stimulation of these responses, little additional augmentation of biological response occurred at the higher dose. Except for a decline in monocyte HLA-DR expression, biological responses remained increased at both doses over the 10-day period of treatment. However, no objective regressions of metastatic disease occurred. In view of objective responses in metastatic renal carcinoma in other trials with IFN-beta ser, consideration should be given to alternative schedules.

Human biologic response modification by interferon in the absence of measurable serum concentrations: a comparative trial of subcutaneous and intravenous interferon-beta serine.

The effect on a range of biologic responses of interferon-beta serine (IFN-beta ser), administered by either the sc or the iv route, was examined in 16 patients. Despite the absence of IFN in the serum of 13 of 16 patients after sc administration, biologic changes associated with IFN administration occurred. Significant increases in peripheral mononuclear cell surface proteins were evident. Monocyte human leukocyte antigen-DR (HLA-DR) showed a 23% increase in mean fluorescent intensity (P = .04) and a 9% increase in percentage of positive cells (P = .02); lymphocyte OKT10 had an 11% increase in percentage of positive cells (P less than .0001) and a 26% increase in mean fluorescent intensity (P = .002). Natural killer cell activity against the Change target increased by 125% (P = .004). Intracellular activity of 2',5'-oligoadenylate synthetase increased 297% at 24 hours and 226% at 48 hours (P less than .0001). Significant increases in serum concentrations of beta 2 microglobulin (24% at 24 hr and 27% at 48 hr, P less than .0001) and neopterin (85%, P = .0001 and 165%, P = .00001) were observed. These alterations after sc administration were similar quantitatively to those resulting from the same dose of IFN-beta ser given iv. Thus, serum IFN concentrations did not have to be measurable for IFN-beta ser to exert biologic activity. The different effects of two dose levels, 45 X 10(6) IU and 180 X 10(6) IU, also were compared independent of route. The higher dose resulted in greater increases over baseline of 2',5'-oligoadenylate synthetase activity (344% vs. 145% at 24 hr; 231% vs. 83% at 48 hr) and serum neopterin concentrations (185% vs. 99% at 24 hr; 271% vs. 153% at 48 hr). For all the other parameters, there was no significant difference between the two doses.

Dual regulation of PTH-stimulated adenylate cyclase activity by GTP.

Guanyl nucleotide regulation of parathyroid hormone (PTH)-activated adenylate cyclase was studied in membrane preparations of cultured opossum kidney cells. Guanosine triphosphate (GTP) (100 microM) decreased PTH-stimulated activity by 70%. Pertussis toxin enhanced PTH stimulation in intact cells and membranes, completely blocked the inhibitory effect of GTP, and catalyzed the [32P]ADP-ribosylation of a 38,000-dalton protein migrating in the position of the alpha-subunit of the inhibitory GTP-regulatory protein Ni. Cholera toxin was used to identify the alpha-subunit of the stimulatory GTP-binding protein Ns, a 42,000-dalton protein. We tested the idea that Ni may be involved in mediating the reduced response of opossum kidney cells to PTH after pretreatment with the hormone (desensitization). GTP inhibited PTH-stimulated activity to approximately the same degree in membranes from PTH-pretreated cells and control cells whether or not the cells had also received pertussis toxin. We conclude that GTP inhibits PTH action in opossum kidney cells through Ni but that PTH-induced desensitization is not mediated by Ni.

Solubilization of a guanine nucleotide-sensitive parathyroid hormone-receptor complex from canine renal cortex.

Canine renal cortical PTH receptors were solubilized after occupancy of membrane-associated receptors with the agonist ligand [125I]bovine (b) PTH-(1-34). Stabilization of binding during solubilization required the use of high concentrations of BSA (optimally 5%) and appropriate detergents (0.5% 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfonate, 0.5% 3-[(3-cholamidopropanyl)dimethylammonio]1-propanesulfonate, or 0.5-1.0% digitonin). The soluble fraction (240,000 X gav supernatant) contained [125I]bPTH-(1-34) associated with macromolecular components as well as unbound [125I]bPTH-(1-34) that dissociated during solubilization. The soluble macromolecular complex had functional properties expected of a ternary complex consisting of [125I]bPTH-(1-34) receptor stimulatory guanine nucleotide-binding protein (Ns). Thus, the dissociation of labeled PTH at 30 C was slow (t1/2 = 75 min); in the presence of GTP (10(-4) M), 75% of the sites displayed rapid dissociation kinetics (t1/2 = 2.3 min). This effect was nucleotide specific, with GTP approximately equal to GTP gamma S approximately equal to GDP greater than GDP beta S greater than ITP approximately equal to guanylylimidodiphosphate much greater than GMP approximately equal to App(NH)p. ATP was ineffective. GTP produced a half-maximal response at a concentration of 200 nM. These results are consistent with the reported nucleotide specificity and affinity of purified Ns. Treatment of membranes with N-ethylmaleimide during the binding reaction rendered the solubilized complex refractory to GTP. Gel filtration chromatography (Sepharose 6B) revealed a GTP-sensitive complex that eluted in the position expected of a detergent-free spherical protein of 180,000 daltons. This complex may consist of the 60,000 to 70,000-dalton PTH-binding subunit (previously identified by photoaffinity labeling) together with Ns.

Down-regulation of parathyroid hormone (PTH) receptors in cultured bone cells is associated with agonist-specific intracellular processing of PTH-receptor complexes.

Exposure of cultured embryonic chicken bone cells to the PTH agonists bovine (b) PTH-(1-34) and [8Nle, 18Nle, 34Tyr]bPTH-(1-34)amide [bPTH-(1-34)A] reduces the subsequent cAMP response to the hormone and decreases the specific binding of 125I-labeled PTH to these cultures. To determine whether PTH receptor down-regulation in cultured bone cells is mediated by cellular internalization of PTH-receptor complexes, we measured the uptake of [125I]bPTH-(1-34) into an acid-resistant compartment. Uptake of radioactivity into this compartment was inhibited by incubating cells at 4 C with phenylarsineoxide and unlabeled bPTH-(1-34). Tracer uptake into the acid-resistant compartment at any time was directly proportional to total cell binding at 22 C. Thus, it is likely that PTH-receptor complexes are internalized by bone cells. This mechanism may explain the loss of cell surface receptors after PTH pretreatment. To determine whether internalized PTH-receptor complexes are reinserted into the plasma membrane, we measured PTH binding and PTH stimulation of cAMP production after cells were exposed to monensin, a known inhibitor of receptor recycling. Monensin (25 microM) had no effect on PTH receptor number or affinity and did not alter PTH-stimulated cAMP accumulation. However, monensin (25 microM) incubated with cells pretreated with various concentrations of bPTH-(1-34) for 1 h potentiated the effect of the hormone to reduce subsequent [125I]bPTH-(1-34) binding and PTH-stimulated cAMP accumulation by more than 2 orders of magnitude. Chloroquine also potentiated PTH-induced down-regulation of PTH receptors. By contrast, neither agent influenced PTH binding or PTH-stimulated cAMP production in cells pretreated with the antagonist bPTH-(3-34)A. Thus, monensin potentiated PTH receptor loss only in cells pretreated with PTH agonists, indicating that antagonist-occupied receptors may be processed differently from agonist-occupied receptors in bone cells. The data further suggest that the attenuation of PTH stimulation of cAMP production in treated bone cells may be, at least in part, due to receptor-mediated endocytosis of the hormone.

Parathyroid hormone receptors coupled to cyclic adenosine monophosphate formation in an established renal cell line.

We examined the relationship between PTH binding and stimulation of cAMP formation in a cell line derived from opossum kidney (OK). In the presence of isobutylmethylxanthine (1 mM) bovine PTH(1-34) [bPTH(1-34)] (244 nM) stimulated cAMP accumulation in confluent cultures up to 40-fold over basal; this response to PTH was stable for 35 passages. The concentration of bPTH(1-34) required to raise cell cAMP levels half-maximally was 5-12 nM. Binding of [125I]bPTH(1-34) to OK cells was saturable; Scatchard analysis of competitive binding data yielded a dissociation constant (KD) = 6 +/- 2 nM, with 1.0 pmol binding sites/mg cell protein. Under steady state binding conditions 89% of labeled PTH remained precipitable by 10% trichloroacetic acid, suggesting minimal metabolism of the hormone. The PTH antagonist (8Nle, 18Nle, 34Tyr)bPTH(3-34)amide competed for [125I]bPTH(1-34) binding sites and inhibited the action of bPTH(1-34) to raise cAMP levels. The intact PTH molecule, bPTH(1-84), and the weak agonist hPTH(1-34) synthesized by Brewer were both less potent than bPTH(1-34) (6 times and 30 times, respectively) with regard to binding and cAMP production. Calcitonin and arginine vasopressin did not bind to PTH receptors but raised cAMP levels in OK cell cultures 3- and 10-fold, respectively; neither glucagon nor ACTH(1-24) influenced PTH binding of cAMP in OK cells. Varying the extracellular calcium concentration in the medium bathing cells did not influence basal or PTH-stimulated cAMP generation. These data suggest that PTH receptors in OK cells are of high affinity, are selective for PTH, and are coupled to adenylate cyclase. This established epithelial cell line provides a model in which to study the mechanism of action of PTH in the kidney.

Tissue deposition and metabolism of 125I-labeled synthetic amino-terminal parathyroid hormone bPTH(1-34).

The tissue deposition and metabolism of 125I-labeled synthetic amino-terminal parathyroid hormone, bPTH(1-34), were studied in rats. In comparison with the intact hormone molecule bPTH(1-84), the synthetic fragment was (a) cleared more rapidly from serum; (b) degraded more rapidly in peripheral tissues; (c) deposited to a greater extent in kidney; (d) deposited to a much smaller extent in liver. In bone, the accumulation of total radioactivity was approximately the same with both labeled hormones. The possible physiological significance of these patterns of distribution and metabolism is discussed.