Adenosine A(2A) and A(2B) receptor activation of erythropoietin production.

We have examined the effects of adenosine receptors and protein kinases A and C in the regulation of erythropoietin (Epo) production using hepatocellular carcinoma (Hep3B) cells in culture and in vivo in normal mice under normoxic and hypoxic conditions. CGS-21680, a selective adenosine A(2A) agonist, significantly increased levels of Epo in normoxic Hep3B cell cultures and in serum of normal mice under both normoxic and hypoxic conditions. CGS-21680 also produced a significant increase in Epo mRNA levels in Hep3B cell cultures. SCH-58261, a selective adenosine A(2A) receptor antagonist, significantly inhibited the increase in medium levels of Epo in Hep3B cell cultures exposed to hypoxia (1% O(2)). Enprofylline, a selective adenosine A(2B) receptor antagonist, significantly inhibited the increase in plasma levels of Epo in normal mice exposed to hypoxia. Chelerythrine chloride, an antagonist of protein kinase C activation, significantly inhibited hypoxia-induced increases in serum levels of Epo in normal mice. A model is presented for adenosine in hypoxic regulation of Epo production that involves kinases A and C and phospholipase A(2) pathways.

Enhancement of erythropoietin production by selective adenosine A2 receptor agonists in response to hypoxia.

The purpose of this study was to characterize the effects of two new adenosine A2 agonists, 2-(p-(2-carboxyethyl)phenethyl amino)-5'-N-ethylcarboxamidoadenosine (CGS-21680) and N6-(2(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl)-adenosine (DPMA), on erythropoietin (EPO) production in vivo and in vitro. Intravenous injections of CGS-21680 (100 to 500 nmol/kg mouse/day) and DPMA (50 to 500 nmol/kg mouse/day) for 4 days produced significant increases in serum levels of EPO in exhypoxic polycythemic mice. CGS-21680 (10(-7) to 10(-6) mol/L) and DPMA (10(-8) to 10(-5) mol/L) also produced significant increases in medium levels of EPO in a cloned EPO-producing Hep3B hepatocellular carcinoma cell line after 18 hours of incubation in 1% O2. Both compounds also increased cellular cAMP levels significantly in a dose-dependent manner after 1 hour of incubation. A2 receptor binding assays with tritiated CGS-21680 revealed a single type of adenosine receptor binding site on Hep3B cell membranes with a dissociation constant of 132.9 nmol/L and a binding capacity of 270.6 fmol/mg protein. The Ki competition binding values versus tritiated CGS-21680 were 217 nmol/L for CGS-21680 and 86.8 nmol/L for DPMA. These results indicate that adenosine A2 receptor activation amplifies EPO production in response to hypoxia, both in vivo and in vitro.

Adenosine A2 receptor modulation of erythropoietin secretion in hepatocellular carcinoma cells.

The present studies were undertaken using a cloned erythropoietin (Ep) producing hepatocellular carcinoma cell line (Hep3B) to attempt to correlate the receptor binding properties and biological activities of 5'-N-ethylcarboxamideadenosine (NECA), N6-cyclohexyladenosine (CHA) and N6-cyclopentyldenosine (CPA). Ep and cAMP levels in cultures of Hep3B cells in response to these adenosine analogues were measured by radioimmunoassay. Receptor binding affinities of the adenosine analogues were determined by measuring inhibition of binding of [3H] NECA to Hep3B cell membranes. Scatchard analysis of [3H]NECA to Hep3B cell membranes. Scatchard analysis of [3H]NECA binding to Hep3B cell membranes indicates a single class of binding sites with a dissociation constant of 431 nM and a binding capacity of 573 fmol/mg protein. The adenosine analogues tested produced a significant increase in Ep secretion and cAMP accumulation (ED50 for cAMP accumulation, NECA = 3.3 x 10(-7) M, CHA = 4.2 x 10(-6) M, CPA = 2.2 x 10(-6) M). In addition, NECA showed a higher binding affinity (Ki, 3.8 x 10(-7) M) for Hep3B cell membranes in comparison with CHA (Ki, 6.3 x 10(-6) M) and CPA (Ki, 3.9 x 10(-6) M). These results indicate that the rank order for potency for NECA, CHA and CPA in their binding to Hep3B cell membrane receptors correlates very well with their biological effects on Ep secretion and cAMP accumulation in Hep3B cells.

Adenosine A1 receptors and erythropoietin production.

N6-cyclopentyladenosine (CPA), a selective adenosine A1 receptor agonist, in a concentration range of 10(-9) to 10(-7) M, produced a significant decrease in erythropoietin (EPO) levels in a human hepatocellular carcinoma (Hep G2) cell culture (medium levels of EPO, 91.81 +/- 1.61 and 94.36 +/- 0.97% of control, respectively) after 24 h incubation in a hypoxic atmosphere. CPA, at a concentration of 10(-9) M, also produced a significant decrease in Hep G2 cell levels of adenosine 3',5'-cyclic monophosphate (cAMP; 78.13 +/- 3.89% of control) after 2 h incubation. CPA (10(-9) M) also significantly inhibited forskolin-stimulated increases in EPO production and cAMP accumulation in Hep G2 cells. On the other hand, 2-[p-(2-carboxyethyl)phenethyl-amino]-5'-N-ethylcarboxamidoadenosine (CGS-21680), a selective adenosine A2-receptor agonist, produced no significant change in EPO production in a dose range of 10(-10) to 10(-6) M but increased cAMP accumulation at 10(-6) M. A1-receptor binding assays using N6-[3H]cyclohexyladenosine revealed a single type of adenosine receptor binding site on Hep G2 cell membranes with a dissociation constant of 71.4 nM and a binding capacity of 1,530 fmol/mg protein. These results indicate that Hep G2 cells contain high-affinity adenosine A1 receptors that are linked to decreased cAMP accumulation and EPO production.

Effects of 5'-N-ethylcarboxamideadenosine (NECA) on erythropoietin production.

The present studies were undertaken to assess the effects of 5'-N-ethylcarboxamideadenosine (NECA), an adenosine analogue, on erythropoietin (Epo) production. NECA (0.05 and 0.1 mumol/kg i.v.) produced significant increases in serum Epo levels (368.8 +/- 56.1 and 384.6 +/- 45.9 mU/ml, respectively) in exhypoxic polycythemic mice after a four hour exposure to hypoxia when compared with hypoxia controls (133.2 +/- 18.2 mU/ml). The hypoxic kidney Epo levels were 46.4 +/- 13.4 mU/kg kidney which were significantly higher than normoxic kidney Ep levels (< 1.24 mU/kg kidney). Theophylline (20 mg/kg i.p.), an adenosine receptor antagonist, significantly inhibited the stimulatory effects of NECA on serum Epo levels. In vitro cultures of an Epo producing hepatocellular carcinoma (Hep3B) cell line with NECA (> or = 10(-6) M) for 20 hours under hypoxic conditions (1% O2) produced significant increases in medium levels of Epo when compared with hypoxia controls. Hepatocellular carcinoma cells treated with NECA at a concentration range of 10(-7) M to 5 x 10(-5) M for one hour in a hypoxic atmosphere also had significantly higher cAMP levels than that of hypoxia controls. Scatchard analyses of [3H]NECA binding to membrane preparations of hepatocellular carcinoma cells showed low affinity binding sites with a dissociation-constant (Kd) of 0.44 microM and a binding capacity of 863 fmol/mg protein. These findings suggest that the increase in Epo production in response to NECA under hypoxic conditions can be attributed, at least in part, to stimulation of adenosine A2 receptors which is coupled to adenylyl cyclase activation.

Interaction of nitric oxide and cyclic guanosine 3',5'-monophosphate in erythropoietin production.

The present study was designed to investigate whether in vivo and in vitro erythropoietin (EPO) production is modulated by nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP). Serum levels of EPO in ex-hypoxic polycythemic mice were significantly increased after injections of 200 micrograms/kg sodium nitroprusside for 4 d. One injection of NG-nitro-L-arginine methyl ester (L-NAME) produced a significant dose-related decrease in serum levels of EPO in ex-hypoxic polycythemic mice in response to hypoxia. When EPO producing Hep3B cells were incubated in 1% O2 for 30 min, cGMP levels in the Hep3B cells were significantly elevated, compared with cells incubated in 20% O2. The elevation of cGMP by hypoxia was inhibited by L-NAME (100 microM). Sodium nitroprusside (10 and 100 microM) and NO (2 microM) also significantly increased cGMP levels in Hep3B cells. L-NAME, LY 83583 (6-Anilino-5,8-quinolinedione, a soluble guanylate cyclase inhibitor), and Rp-8-Bromo-cGMPS (Rp-8-Bromo-guanosine 3',5'-cyclic monophosphothioate, a cGMP-dependent protein kinase inhibitor) significantly inhibited the hypoxia-induced increase in medium levels of EPO in Hep3B cells. 8-Bromo-cGMPS produced a dose-dependent decrease in EPO messenger RNA levels in Hep3B cells in response to hypoxia. 8-Bromo-cGMP (10(-3) M) produced significant increases in medium levels of EPO in Hep3B cell cultures incubated under normoxic conditions, which was enhanced by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (0.2 mM). These results suggest that NO and cGMP may interact in modulating hypoxic stimulation of EPO production.

In vivo and in vitro erythropoietin activities in cultures of a hepatocellular carcinoma cell line.

The present studies were undertaken to characterize erythropoietin (Ep) production in an Ep-producing hepatocellular carcinoma (Hep3B) cell line. Hep3B cells which had been maintained in culture were transplanted under the renal capsule and subcutaneously in nude mice. The Hep3B xenograft doubling time is approximately 7 days. The mean hematocrit value of the Hep3B tumor-bearing nude mice was 33.2 +/- 1.1% (n = 8), which was significantly lower than that of control nongrafted nude mice (40.8 +/- 1.7%, n = 5). The Hep3B tumor-bearing nude mice showed significantly higher Ep levels in the sera (37.5 +/- 5.5 munits/ml, n = 8) than control nude mice (13.5 +/- 2.7 munits/ml, n = 5). Ep levels in the sera were correlated (R = 0.714) with the total Ep in the tumor extracts, whereas no Ep was detectable in any of the kidney extracts. On the other hand, an inverse linear relationship (R = -0.811) between the hematocrit values and Ep levels in the sera was demonstrated in the Hep3B tumor-bearing nude mice. The Hep3B cells recultured after growing in the nude mice were capable of enhancing Ep production in response to hypoxia, very similar to the original Hep3B cells which had been maintained in culture during the same time period. In addition, 15-methyl-prostaglandin E1 at a concentration range of 4-400 ng/ml produced significant increases in Ep secretion and cAMP accumulation in Hep3B cultures under hypoxic conditions (1% O2). The Ep produced by Hep3B cells expressed 3.7 times higher in vitro bioactivity than immunoactivity. The bioactivity of Hep3B Ep was completely neutralized by an antibody to highly purified human recombinant Ep. In contrast, the in vivo bioactivity of the Hep3B Ep was less than one tenth of its immunoactivity. These results indicate that the Hep3B tumor-bearing nude mice and the in vitro Hep3B culture system may provide a reproducible model system which should be useful for studies of the mechanism of Ep production.

Characterization of erythropoietin production in a hepatocellular carcinoma cell line.

This study reports the effects of cyclic adenosine 3'-5' monophosphate (cAMP) and hypoxia on erythropoietin biosynthesis in an erythropoietin-producing hepatocellular carcinoma cell line (Hep3B). Erythropoietin levels in the medium and cell extracts of low-density Hep3B cells after 20-hour incubation under hypoxic conditions (1% O2) were 25.33 +/- 1.50 mU/ml/10(7) cells and 3.60 +/- 0.50 mU/10(7) cells, respectively. These levels were significantly higher than in the respective normoxic controls (medium, 2.51 +/- 0.31 mU/ml/10(7) cells; cell extracts, undetectable [less than 0.31 mU/10(7) cells]). Cobalt also produced a significant increase in medium and cell erythropoietin levels. However, hypoxia and cobalt alone failed to produce an increase in cAMP accumulation in the cell cultures. Erythropoietin levels in the medium and cell extracts from cells exposed to 8-bromo cAMP (1 x 10(-4) mol/L) and forskolin (4 x 10(-6) mol/L) in a hypoxic atmosphere were significantly (p less than 0.05) higher than in the respective hypoxic controls. In addition, forskolin produced a significant (p less than 0.05) increase in cAMP accumulation (180 +/- 11.5 pmol/10(6) cells) under hypoxic conditions compared with the hypoxic controls (cAMP, 2.27 +/- 0.33 pmol/10(6) cells). These results suggest that cAMP elevation is not required in vitro in Hep3B cells for the increase in medium and cell levels of erythropoietin after hypoxia, but may be involved indirectly in erythropoietin biosynthesis, secretion, or both in vivo through some synergistic action with hypoxia.

Increased erythropoietin secretion in human hepatoma cells by N6-cyclohexyladenosine.

The present studies were undertaken to assess the direct effects of N6-cyclohexyladenosine (CHA), a stable adenosine analogue, on erythropoietin (Ep) secretion in hepatocellular carcinoma cells (Hep 3B). Ep levels in the medium of low density Hep 3B cells treated with CHA in concentrations of 10(-5) and 5 x 10(-5) M for 20 h under hypoxic conditions (1% O2) were significantly higher than that of hypoxic controls. In addition, CHA at the same concentrations produced significant increases in adenosine 3',5'-cyclic monophosphate (cAMP) levels in Hep 3B cells after 1-h incubation under hypoxic conditions when compared with hypoxic controls. Dibutyryl cAMP (10(-5), 10(-4) M) also caused significant increases in Ep secretion when compared with control hypoxic cells. On the other hand, 8-phenyltheophylline, an adenosine receptor antagonist, significantly inhibited the stimulatory effects of CHA on both Ep secretion and cAMP accumulation in the Hep 3B cell cultures in response to hypoxia. These data suggest that Ep secretion may be regulated by adenosine receptor-coupled activation of adenylyl cyclase and the generation of cAMP.

Development of a new radioimmunoassay for erythropoietin using recombinant erythropoietin.

The development of a 24 hour radioimmunoassay for erythropoietin (EPO) using EPO derived from recombinant DNA as both immunogen and ligand is described in the present paper. Mixed breed rabbits immunized with 10 micrograms/kg of EPO derived from a stably transfected cell line (Elanex Pharmaceuticals Inc., Bothel, Washington, USA, through McDonnell Douglas Corp., St. Louis, Missouri, USA; "MD") produced antibodies to EPO with high titer (up to 1:896,000 final dilution in the tube), high affinity (8.4 x 10(11) liter/M), and good specificity. Purified EPO from the above source or from AmGen Biologicals (Thousand Oaks, California, USA; "AG") were successfully radioiodinated with the chloramine-T method and used as ligand in the radioimmunoassay. Standard dose-response curves prepared with EPO from both commercial sources were not significantly different and showed a sensitivity of 0.75 to 0.96 mU/tube. The dose-response curves in both systems also showed parallelism with serially diluted serum from a patient with aplastic anemia. Within-assay and between-assay precision were determined by assaying multiple replicates of a serum pool. Recovery of exogenous EPO added to a serum pool averaged 97% for both systems. The range of normal human serum EPO was determined by assaying the sera of 153 hematologically-normal adult subjects and was found to be 1.1 to 27.3 mU/ml for MD EPO and 0.5 to 16.7 mU/ml for AG EPO. Sera from several patients with hematologic abnormalities were also assayed, including those of 36 patients with anemia of end-stage renal disease (mean +/- SEM, 29.5 +/- 4.0 mU/ml; P less than 0.01). In conclusion, this new, more rapid and sensitive radioimmunoassay system can be used to measure EPO levels in sera from normal human subjects and patients with several types of anemia, and should also be very useful in therapeutic drug monitoring of patients receiving EPO from various commercial sources.

Increased secretion of erythropoietin in human renal carcinoma cells in response to atrial natriuretic factor.

The present studies were undertaken to assess the effects of atrial natriuretic factor (ANF) on erythropoietin (Ep) secretion in Ep-producing renal carcinoma (RC) cells using a sensitive radioimmunoassay for Ep. Human ANF produced a significant dose-related increase in Ep secretion at concentrations of 10(-7) and 10(-6) M when compared with vehicle controls. ANF (greater than or equal to 10(-9) M) also significantly increased the intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration after 5-min incubation with the RC cells. Scatchard analysis of the human 125I-labeled ANF binding data indicated that the RC cells contain a single class of binding sites with a dissociation constant (Kd) of 93 +/- 1 pM and a binding capacity of 2,190 +/- 750 sites/cell. Incubation of the RC cells with 8-bromo-cGMP in concentrations of 10(-7)-10(-5) M also produced a significant dose-related enhancement of Ep secretion. These findings suggest that the increase in Ep secretion in response to ANF can be attributed, at least in part, to activation of guanylate cyclase, which is coupled to specific ANF receptors on the RC cell.

Enhanced erythropoietin secretion in hepatoblastoma cells in response to hypoxia.

Erythropoietin (Ep) levels in spent culture media of a Hep G2 human hepatoblastoma cell line were measured by radioimmunoassay (RIA), fetal mouse liver erythroid colony formation (FMLC), and the exhypoxic polycythemic mouse assay (EHPCMA). The Hep G2 cells at high density produced approximately 700 mU/ml Ep when measured with the RIA. On the other hand, the Ep levels when assayed in EHPCMA and FMLC were 50 and 2,600 mU/ml, respectively. The bioactivity in FMLC was completely neutralized by an antibody to purified human recombinant Ep, indicating that the erythropoietic activity in the Hep G2 spent culture medium was immunologically equivalent to Ep. Ep levels in the medium from low-density Hep G2 cells in 5% O2 and 1% O2 were 2.5- and 4-fold greater, respectively, than that of 20% O2. In contrast, hyperoxia (40% O2) significantly inhibited Ep production. A significant increase in Ep secretion was also observed when the cells were incubated with cobaltous chloride (2 X 10(-6) -2.5 X 10(-4) M). Tunicamycin (0.5 micrograms/ml), which inhibits N-linked glycosylation, significantly reduced the enhancement of Ep secretion induced by hypoxia (1% O2) without affecting cell growth. Forskolin and cholera toxin, each of which increased the levels of cyclic AMP in the Hep G2 cells by 40-fold, produced a significant (P less than 0.05) further increase in Ep secretion in the presence of hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythropoietin production in exhypoxic polycythemic mice.

Our present study was undertaken to determine the serum erythropoietin concentration (radioimmunoassay), hematocrit, red cell mass, and body weight of mice exposed to hypoxia in a hypobaric chamber (0.42 atm, 22 h/day) for 14 days and during the 10 posthypoxic days at ambient pressure to clarify the correlation of the red cell mass and erythropoietin production during hypoxia. The mean serum erythropoietin titer was 326.23 +/- 77.04 mU/ml after 2 days, reached the highest level after 3 days (452.2 +/- 114.5 mU/ml), then gradually declined to a level of 36.5 +/- 11.4 mU/ml after 14 days of hypoxia, and was undetectable during the 10-day posthypoxic period. The hematocrit values were significantly increased from 41.09 +/- 0.50% at day 0 to 51.65 +/- 1.08% after 3 days and to 72.20 +/- 1.53% after 14 days of hypoxia. The red cell mass (calculated from initial body weight) increased from 3.24 +/- 0.1 ml/100 g at day 0 to 7.32 +/- 0.46 ml/100 g after 14 days of hypoxia and declined to 6.66 +/- 0.53 ml/100 g at the end of the 10-day posthypoxic period. The mice lost weight while they were in the hypobaric chamber and showed a significant increase in body weight during the 10-day posthypoxic period. These studies support the concept that chronic intermittent hypoxia causes an early increase, followed by a rapid decline, in erythropoietin production, which is correlated with the gradual increase in red cell mass.

Measurement of erythropoietin in anephric children. A report of the Southwest Pediatric Nephrology Study Group.

Serum erythropoietin (Ep) levels were measured using a highly sensitive radioimmunoassay in 69 children undergoing chronic dialysis; 31 were anephric, whereas 38 were non-nephrectomized (nephric). Twenty-nine normal children were studied as controls. Serum Ep levels in the anephric group were much higher than anticipated (mean 19.7 +/- 1.8 mU/ml), albeit significantly lower than those measured in normal children (mean 26.2 +/- 2.4 mU/ml, P less than 0.05), or in nephric children on dialysis (33.0 +/- 2.9 mU/ml, P less than 0.001). Anephric children on peritoneal dialysis (PD) had significantly (P less than 0.05) higher serum levels of Ep (22.7 +/- 2.4 mU/ml, n = 19) than anephric children on hemodialysis (HD) (15.1 +/- 2.3 mU/ml, n = 12). There was no significant difference between Ep levels in anephric patients dialyzed for less than or equal to 1 year (19.6 +/- 2.0 mU/ml, n = 20) compared with anephric patients dialyzed for more than 1 year (20.0 +/- 3.9 mU/ml, n = 11). Although serum Ep levels showed a tendency to increase with time after nephrectomy, the mean values for less than 3 months (14.7 +/- 1.9), 3 months-12 months (21.0 +/- 2.7), and greater than 12 months (21.6 +/- 6.0) were not significantly different from each other. This demonstration of relatively normal levels of serum Ep in anephric children suggests that extrarenal sites of Ep production are able to exert a significant response to severe anemia in patients who are devoid of renal parenchyma.

Effect of different modes of dialysis on serum erythropoietin levels in pediatric patients. A report of the Southwest Pediatric Nephrology Study Group.

The relative importance of erythropoietin (Ep) and inhibitors of erythropoiesis in the development of anemia in pediatric patients with end-stage renal disease (ESRD) was assessed in 82 patients: 41 treated with peritoneal dialysis (PD) and 41 with hemodialysis (HD). Serum Ep was determined with a sensitive radioimmunoassay. Potential serum inhibition of erythroid (CFU-E) and granulocytic (CFU-GM) progenitor cell growth was assessed using human bone marrow cell cultures. The mean Ep level for all 82 patients was 33.1 +/- 3.1 mU/ml, which was significantly higher (P less than 0.05) than the values obtained in 29 normal children (26.2 +/- 2.4 mU/ml). Serum Ep in the PD group (41.6 +/- 5.6 mU/ml) was significantly higher (P = 0.007) than that of the HD group (24.6 +/- 2.1 mU/ml). The mean hematocrit in the PD group (25.2 +/- 0.8%) was also significantly higher (P less than 0.002) than that of the HD group (22.2 +/- 0.5%). The mean serum parathyroid hormone (PTH) level as measured by a mid-terminal radioimmunoassay was not significantly different (P = 0.79) in the HD group (17,298 +/- 3,998 pg/ml) from that of the PD group (15,747 +/- 4,227 pg/ml). Neither serum Ep nor PTH concentration correlated with hematocrit or degree of inhibition of erythroid progenitor cell colony (CFU-E) formation in either group of dialysis patients, nor did the hematocrit correlate with the degree of serum inhibition of CFU-E formation. The higher level of Ep in the PD group may indicate more effective removal by PD of some enzymatic substance which reduces the immunologic and biologic activities of Ep.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of reactive oxygen metabolites on erythropoietin production in renal carcinoma cells.

The present studies were undertaken to determine the effects of reactive oxygen metabolites on erythropoietin (Ep) biosynthesis in Ep-producing renal carcinoma (RC) cells using a sensitive radioimmunoassay for Ep. Xanthine (10-5M) and increasing concentrations of xanthine oxidase (8 x 10(-7) to 5 x 10(-4) units/ml) produced a significant dose-related increase in Ep production at a concentration of greater than or equal to 4 x 10(-6) units/ml, whereas xanthine alone had no effect. Catalase, a scavenger of hydrogen peroxide (H2O2), in concentrations of 50 to 500 micrograms/ml produced a significant inhibition of the increase in Ep production induced by xanthine-xanthine oxidase; while no effect was seen on basal levels of Ep production and the growth of RC cells. Glucose oxidase (greater than or equal to 0.032 mU/ml), a direct H2O2 generator, and exogenous H2O2 (greater than or equal to 4 x 10(-6)M) added to the incubation mixture, caused a significant enhancement of Ep production in a dose-dependent manner. Xanthine-xanthine oxidase, glucose oxidase, and H2O2 in the above concentrations did not produce significant cytotoxicity (51Cr release or trypan blue dye exclusion). The present data suggests that H2O2, a reactive oxygen metabolite may play a significant role in Ep production.

Pharmacokinetics of erythropoietin in intact and anephric dogs.

The present studies were performed to determine the pharmacokinetic parameters of erythropoietin in intact and anephric dogs by use of unlabeled crude native erythropoietin (nEp) and iodine 125-labeled purified recombinant erythropoietin (rEp) given by intravenous infusion for 15 minutes. Sephadex G-75 gel filtration was used to confirm that the 125I-rEp molecule remained iodinated in dog plasma during the 24-hour period of these studies. The plasma disappearance of erythropoietin conformed to a biexponential equation for both nEp and 125I-rEp, with the central compartment being larger than the peripheral compartment. The mean distribution half-life of 75.3 +/- 21.2 minutes for nEp was significantly (p less than 0.05) longer than that of 125I-rEp (23.7 +/- 5.0 minutes) in intact dogs. The intercompartmental clearance (CIic) for nEp (0.018 +/- 0.006 L/kg/hr) was significantly smaller than that of 125I-rEp (0.068 +/- 0.018 L/kg/hr) in intact dogs (p less than 0.05). There were no significant differences in apparent volume of distribution, elimination half-life, and elimination clearance (CIe) for nEp and rEp in intact dogs. The mean elimination half-life for 125I-rEp in intact dogs (9.0 +/- 0.6 hours) and anephric dogs (13.8 +/- 1.4 hours) was significantly different (p less than 0.05). The CIe for 125I-rEp in anephric dogs (0.008 +/- 0.001 L/kg/hr) was significantly (p less than 0.05) smaller than that of 125I-rEp in intact dogs (0.011 +/- 0.001 L/kg/hr). There were no significant differences in apparent volume of distribution, distribution half-life, and CIic for 125I-rEp in intact and anephric dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

A1 and A2 adenosine receptor regulation of erythropoietin production.

The effects of adenosine (ADE) and ADE agonists on erythropoietin (Ep) production were determined using percent (%) 59Fe incorporation in red cells of exhypoxic polycythemic mice. The hemisulfate salt of ADE produced a significant increase in % 59Fe incorporation in response to hypoxia in concentrations of 400 to 1600 nmol/kg/day (i.v.). 5'-N-ethyl-carboxamideadenosine (NECA), a selective A2 receptor agonist, increased radioiron incorporation in a dose-dependent manner (10-100 nmol/kg/day, i.v.). In contrast, N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, did not affect radioiron incorporation in concentrations up to 1600 nmol/kg/day (i.v.). Albuterol, a beta 2-adrenergic agonist, enhanced % 59Fe incorporation in polycythemic mice and low doses of CHA (50 and 100 nmol/kg/day), which were not effective alone on % 59Fe incorporation in polycythemic mice exposed to hypoxia, inhibited the enhancement in radioiron induced by albuterol (25 and 100 micrograms/kg/day, i.p.) plus hypoxia. Theophylline (20 and 80 mg/kg/day, i.p.), a well-known antagonist of ADE receptors, blocked the ADE and NECA enhancement in radioiron incorporation at a dose of theophylline alone which produced only a slight enhancement of % 59Fe incorporation. These results suggest that ADE may both inhibit through A1 receptor activation and increase via A2 receptor stimulation the production of Ep.

Effects of low calcium levels on erythropoietin production by human renal carcinoma cells in culture.

Recent investigations have shown that calcium entry blockers enhance the effects of hypoxia on erythropoietin (Ep) production in vivo. To determine whether deprivation of calcium increases Ep production and/or release, studies were carried out to determine the effects of low levels of extracellular calcium on Ep (radioimmunoassay) secretion in human renal carcinoma cells in culture. Low extracellular calcium levels (0.3 mM) in culture medium significantly (P less than 0.01) enhanced Ep secretion (64-145% increase per day) by renal carcinoma cells in culture when compared with a concentration of 1.9 mM calcium in the control culture medium (23-68% increase per day) incubated for 24 h or more. A 53% increase per day in Ep secretion was also produced by the calmodulin inhibitor trifluoperazine. To determine whether the effects of low calcium levels on Ep production could be due to nonspecific leakage of large intracellular molecules caused by a permeabilization of the cell membrane, the effect of low calcium levels in the cultures of the renal carcinoma cells on lactate dehydrogenase release into the culture medium was studied. Low calcium concentrations failed to significantly enhance lactate dehydrogenase secretion by the renal carcinoma cells. In conclusion, our results indicate a possible involvement of the calcium ion and calmodulin in the biosynthetic pathway for Ep and that calcium may exert a suppressive effect on Ep production.

Enhanced erythropoietin production by calcium entry blockers in rats exposed to hypoxia.

In order to determine the influence of calcium on erythropoietin release in response to hypoxia, the effects of the calcium entry blocker verapamil on erythropoietin production were investigated. Hypoxia was induced in male Sprague-Dawley rats by placing them in a hypobaric chamber at 0.42 atmospheres for 6 and 12 hr. Serum levels of erythropoietin were measured by the exhypoxic polycythemic mouse bioassay and a sensitive radioimmunoassay for erythropoietin. Verapamil (5, 10 and 20 mg/kg i.p.) produced significant increases in mean serum erythropoietin levels after 6 and 12 hr of hypoxia, which was significantly higher than those of saline-injected hypoxic control rats. Mean arterial blood pressure values in rats injected with 5 and 10 mg/kg of verapamil were not significantly different from the preinjection controls when measured at ambient pressure over a 12-hr period. A dosage of 20 mg/kg of verapamil i.p. in rats produced a significant decrease in mean arterial pressure between 5 and 30 min after injection but was not significantly different from the vehicle controls between 30 min and 12 hr postinjection. In conclusion, the calcium entry blocker verapamil enhanced erythropoietin production in response to hypoxia. Thus, it is postulated that calcium plays a significant role in erythropoietin production and/or release.