Decay rates of Escherichia coli, Enterococcus spp., F-specific bacteriophage MS2, somatic coliphage and human adenovirus in facultative pond sludge.

The purpose of this study was to evaluate the efficacy of a waste stabilization pond (WSP) system to reduce pathogen contaminants in sludge. This included examining the factors that influence the fate and concentration of human pathogens and their indicators in two sludge layers. The decay rates of five study microorganisms were determined under in-situ conditions at a WSP. The background levels of fecal origin microorganisms were consistently detected (ranging: Escherichia coli 104 to 106, enterococci 101 to 103, F-specific bacteriophage (MS2) 101 to 103 and somatic coliphage 101 to 104 colony-forming units (CFU) mL-1, as well as 101 to 102 human adenovirus gene copies mL-1) in the primary facultative pond. Among microorganisms tested, the bacteria generally decayed faster than adenovirus and bacteriophage, particularly in the upper sludge layer. Due to the observed regrowth of E. coli, it may have a limited value as an indicator for pathogen removal in the wastewater stabilization ponds. The abundance of E. coli numbers within the pond biome followed changes in pond temperature over time. The results of the study suggest that viruses could survive for a long time, particularly in deeper layers (>1 metre) in the sludge, during winter months (T90 = 156 d). The presence of human pathogens in WSP sludge, in particular viruses, may be a barrier to its beneficial reuse in agriculture. The results indicate that additional treatment of sludge may be required to mitigate potential public health risks from reuse of sludge for agricultural purposes.

Decay of enteric microorganisms in biosolids-amended soil under wheat (Triticum aestivum) cultivation.

There is a growing need for better assessment of health risks associated with land-applied biosolids. This study investigated in-situ decay of seeded human adenovirus (HAdV), Salmonella enterica, Escherichia coli, and bacteriophage (MS2) in biosolids-amended soil under wheat cultivation. The biosolids seeded with microorganisms were placed in decay chambers which were then placed in the topsoil (10 cm depth) at three different sites. Sites were selected in arid wheat-growing regions of Australia with loamy-sand soil type (Western Australia) and sandy soil (South Australia). Seeded E. coli and S. enterica had a relatively short decay time (T90 = 4-56 days) in biosolids-amended soil compared to un-amended soil (T90 = 8-83 days). The decreasing soil moisture over the wheat-growing season significantly (P < 0.05) influenced survival time of both bacteria and MS2 at Western Australia (Moora) and South Australia (Mt Compass) sites, particularly in the un-amended soils. Increasing soil temperature also significantly (P < 0.05) influenced the decay of MS2 at these sites. In this study, no notable decline in HAdV numbers (PCR detectable units) was observed in both biosolids-amended and the un-amended soils at all three sites. The HAdV decay time (T90 ≥ 180 days) in biosolids-amended and un-amended soils was significantly higher than MS2 (T90 = 22-108 days). The results of this study suggest that adenovirus could survive for a longer period of time (>180 days) during the winter in biosolids-amended soil. The stability of adenovirus suggests that consideration towards biosolids amendment frequency, time, rates and appropriate withholding periods are necessary for risk mitigation.

Consequences of nitric oxide synthase inhibition during bovine oocyte maturation on meiosis and embryo development.

The importance of nitric oxide synthase (NOS) in bovine oocyte maturation was investigated. Oocytes were in vitro matured with the NOS inhibitor N(w)-L-nitro-arginine methyl-ester (10(-7), 10(-5) and 10(-3) m L-NAME) and metaphase II (MII) rates and embryo development and quality were assessed. The effect of L-NAME (10(-7) m) during pre-maturation and/or maturation on embryo development and quality was also assessed. L-NAME decreased MII rates (78-82%, p < 0.05) when compared with controls without L-NAME (96%). Cleavage (77-88%, p > 0.05), Day 7 blastocyst rates (34-42%, p > 0.05) and total cell numbers in blastocysts were similar for all groups (146-171 cells, p > 0.05). Day 8 blastocyst TUNEL positive cells (3-4 cells) increased with L-NAME treatment (p < 0.05). For oocytes cultured with L-NAME during pre-maturation and/or maturation, Day 8 blastocyst development (26-34%) and Day 9 hatching rates (15-22%) were similar (p > 0.05) to controls pre-matured and matured without NOS inhibition (33 and 18%, respectively), while total cell numbers (Day 9 hatched blastocysts) increased (264-324 cells, p < 0.05) when compared with the controls (191 cells). TUNEL positive cells increased when NOS was inhibited only during the maturation period (8 cells, p < 0.05) when compared with the other groups (3-4 cells). NO may be involved in meiosis progression to MII and its deficiency during maturation increases apoptosis in embryos produced in vitro. Nitric oxide synthase inhibition during pre-maturation and/or maturation affects embryo quality.

Endothelial and inducible nitric oxide synthases in oocytes of cattle.

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthases (NOS). The NOS/NO system appears to be involved in oocyte maturation, but there are few studies on gene expression and protein activity in oocytes of cattle. The present study aimed to investigate gene expression and protein activity of NOS in immature and in vitro matured oocytes of cattle. The influence of pre-maturation culture with butyrolactone I in NOS gene expression was also assessed. The following experiments were performed: (1) detection of the endothelial (eNOS) and inducible (iNOS) isoforms in the ovary by immunohistochemistry; (2) detection of eNOS and iNOS in the oocytes before and after in vitro maturation (IVM) by immunofluorescence; (3) eNOS and iNOS mRNA and protein in immature and in vitro matured oocytes, with or without pre-maturation, by real time PCR and Western blotting, respectively; and (4) NOS activity in immature and in vitro matured oocytes by NADPH-diaphorase. eNOS and iNOS were detected in oocytes within all follicle categories (primary, secondary and tertiary), and other compartments of the ovary and in the cytoplasm of immature and in vitro matured oocytes. Amount of mRNA for both isoforms decreased after IVM, but was maintained after pre-maturation culture. The NOS protein was detected in immature (pre-mature or not) and was still detected in similar amount after pre-maturation and maturation for both isoforms. NOS activity was detected only in part of the immature oocytes. In conclusion, isoforms of NOS (eNOS and iNOS) are present in oocytes of cattle from early folliculogenesis up to maturation; in vitro maturation influences amount of mRNA and NOS activity.

Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone I combined or not combined with roscovitine.

Cyclin dependent kinase inhibitors (CDKIs) may be used for pre-maturation culture, but can accelerate nuclear maturation. The aim of the present research was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at lesser than typically used concentrations on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100 microM BLI (B) or 6.25 microM BLI+12.5 microM ROS (BR) in TCM-199 for 24 h. Oocytes were subsequently submitted to in vitro maturation (IVM) in TCM-199+0.5 microg/ml FSH, 50 microg/ml LH and 10% FCS for another 24 h, during which oocytes were fixed every 3 h. In Experiment 2, oocytes were submitted to 24h pre-maturation treatments, with the inhibitors being diluted in TCM-199 or DMEM. IVM lasted 21 h in the culture media DMEM+0.5 microg/ml FSH, 50 microg/ml LH, 5% FCS and 50 ng/ml EGF. After IVM, oocytes from all groups were fertilized in vitro. Oocytes and sperm (2x10(6) sperm cells/ml) were co-cultured for 18 h. Embryos were co-cultured with granulosa cells in CR2aa for 8 days. All cultures were in droplets under oil, at 38.5 degrees C and 5% CO2 in air. In both experiments, control oocytes (C) were submitted only to IVM. In Experiment 1, at 0 h, C and B oocytes were all (100%) at the germinal vesicle stage (GV) of development. BR had fewer GV oocytes (89%, P<0.05). After 3 h IVM, B and BR had fewer oocytes in GV (84.7 and 79.6%, P>0.05) than C (100%, P<0.05). At 12 h, most oocytes were at intermediate stages (metaphase to telophase I) in all groups (approximately 80%, P>0.05). After 21 (77-89%) and 24 h (85-95%), all groups had similar metaphase II (MII) rates of development (P>0.05). In Experiment 2, cleavage (79-84%, P>0.05) and Day 7 blastocyst rates (26-36%, P>0.05) were similar. After 8 days, the group pre-matured with BR in DMEM had lesser blastocyst rates of development (32.3%) lower than C (40.1%, P<0.05). The other groups were similar to C (35-38%, P>0.05). Hatching rates were similar (10-15%, P>0.05) as were total cell numbers (141-170). In conclusion, BR is less effective in maintaining meiosis block; B and BR accelerate meiosis resumption; and use of pre-maturation medium may affect developmental rates.

Prematuration of bovine oocytes with butyrolactone I: effects on meiosis progression, cytoskeleton, organelle distribution and embryo development.

The effects of prematuration (PM) of bovine oocytes with butyrolactone I (BLI) for 24h on meiosis progression, cell structures and embryo development were assessed. Germinal vesicle (GV) rates decreased (97.4-65.1%, P<0.05) with decreasing BLI concentrations (100-25microM). Without BSA in PM medium, GV rates were similar (98.7-97.2, P>0.05) with low BLI (10-25microM). After in vitro maturation (IVM) for 24h, metaphase II (MII) rates for controls (IVM only) were similar (91.1%, P>0.05) to PM with 10microM BLI in BSA-free medium (B10=91.5%) and 100microM BLI in medium with BSA (B100=92.4%). Meiosis resumption occurred earlier in treated oocytes (71.4-74.3% in GV for B10 and B100, respectively, after 6h IVM compared with 97.3% in controls, P<0.05). By 18h of IVM, most oocytes reached MII (72.0-78.9%, P>0.05). Microtubules and microfilaments were unaffected by BLI. Cortical granules (CG) migration was reversibly blocked by BLI. Mitochondria translocation was partially blocked by PM culture and after IVM more oocytes in B10 and B100 (95.2 and 98.2%, respectively) had mitochondria translocated to a mature pattern (all cytoplasm) than controls (81.5%, P<0.05). Cleavage rates were similar (81-87%, P>0.05), but blastocysts (day 7) decreased in B100 (33.0%, P<0.05) compared with controls and B10 (38.3 and 41.6%, respectively). Day 8 hatching rates (11.0-19.2%) and mean total cell numbers (136-150) were similar (P>0.05). PM did not improve oocyte competence but also did not cause major structural alterations, suggesting that PM may be improved and used to study the mechanisms involved in oocyte differentiation.

Impaired cardiac muscarinic receptor function in dogs with heart failure.

Prior physiological studies have suggested that parasympathetic control is altered in heart failure. The goal of our studies was to investigate the influence of heart failure on the muscarinic receptor, and its coupling to adenylate cyclase. Ligand binding studies using [3H]quinuclidinyl benzilate and enriched left ventricular (LV) sarcolemma, demonstrated that muscarinic receptor density in heart failure declined 36% from a control of 5.6 +/- 0.6 pmol/mg, with no change in antagonist affinity. However, agonist competition studies with both carbachol and oxotremorine showed that it was a loss of high affinity agonist binding sites in the sarcolemma from failing LV that accounted for this difference. The functional efficacy of the muscarinic receptor was also examined. When 1 microM methacholine was added to 0.1 mM GTP and 0.1 mM isoproterenol, adenylate cyclase stimulated activity was inhibited by 15% in normal LV but only 5% in LV sarcolemma from animals with heart failure even when the reduced adenylate cyclase in these heart failure animals was taken into account. Even at 100-fold greater concentrations of methacholine, significantly less inhibition of adenylate cyclase activity was observed in LV failure as compared with normal LV sarcolemma. Levels of the GTP-inhibitory protein known to couple the muscarinic receptor to adenylate cyclase, as measured with pertussis toxin labeling, were not depressed in LV failure. Thus, the inhibitory pathway regulating LV adenylate cyclase activity is defective in heart failure. The decrease in muscarinic receptor density, and in particular the specific loss of the high affinity agonist binding component of this receptor population, appears to be the major factor underlying this abnormality.

Coupling of the alpha 1-adrenergic receptor to a guanine nucleotide-binding regulatory protein by a discrete domain distinct from its ligand recognition site.

At rat hepatic membrane alpha 1-adrenergic receptors, the nonhydrolyzable GTP analogue p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding. This p[NH]ppG effect is consistent with the involvement of a guanine nucleotide-binding regulatory protein (G-protein) in alpha 1-adrenergic receptor signalling. Although readily apparent in membranes prepared to avoid retention of endogenous nucleotides and activation of Ca2+-sensitive proteinases (+pi), this p[NH]ppG effect is not observed in membranes prepared without proteinase inhibitors (-pi), or in -pi membranes treated with Ca2+ (-pi, +Ca2+). In these various membrane preparations, different Mr forms of the receptor are also identified by photoaffinity labeling with [125I]CP65526, an aryl azide analog of the alpha 1-selective antagonist, prazosin, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. Whereas a predominant Mr = 80,000 subunit is identified in +pi membranes, in -pi membranes a proteolytic Mr = 59,000 fragment is also observed. In -pi, +Ca2+ membranes, only this latter peptide is detected. To evaluate the ability of each of these forms of the receptor to couple with a G-protein, the effect of p[NH]ppG on the agonist-inhibition of [125I]CP65526 labelling was determined by laser densitometry scanning and computer analysis. At the Mr = 80,000 subunit, p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding, even in -pi membranes. By contrast, agonist-binding at the Mr = 59,000 subunit is of low-affinity and was not affected by p[NH]ppG. These data indicate that the cleaved Mr = 59,000 fragment, while retaining hormone binding activity is unable to undergo G-protein coupling. Thus, the alpha 1-adrenergic receptor appears to contain a discrete domain necessary for G-protein coupling that is distinct from its ligand recognition site.

Agonist interactions at hepatic alpha 1- and beta-adrenergic receptors: affinity-state regulation by guanine nucleotides and temperature.

We investigated the binding characteristics of agonists to alpha 1- and beta-adrenergic receptors of intact liver cells, broken rat liver cell membranes, and detergent-solubilized preparations under varying experimental conditions, focusing on the different "states" of the receptor for agonists and the regulation of these states by temperature and guanine nucleotides. While only low-affinity binding of agonists to both receptor subtypes was evident in studies performed at 37 degrees C with solubilized preparations, biphasic competition curves for agonists were observed in both intact cells and membrane preparations; the majority of sites were of low affinity. In membrane preparations, the nonhydrolyzable GTP analogue Gpp(NH)p caused a rightward shift of agonist competition curves and a loss of high-affinity binding. These results are consistent with the involvement of guanine nucleotide binding proteins in both alpha 1- and beta-adrenergic transduction pathways. When competition studies were performed at 4 degrees C, receptor sites existed predominantly in the high-affinity configuration, in intact cells and membranes, as well as in soluble preparations. In contrast to the studies conducted at 37 degrees C, no Gpp(NH)p-induced conversion to the lower affinity state could be demonstrated in studies performed with membrane preparations at 4 degrees C. Thus, the high-affinity state of alpha 1- and beta-adrenergic receptors is stabilized at 4 degrees C in intact cells, membranes, and soluble preparations. After incubations had been performed at 37 degrees C, high-affinity binding of agonists could not be restored by subsequent incubation at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum progesterone levels in women treated with human menopausal gonadotropin and human chorionic gonadotropin for in vitro fertilization.

For evaluation of the adequacy of luteal function after in vitro fertilization-embryo transfer (IVF-ET), serum progesterone (P) levels were measured on days 3, 7, and 10 after laparoscopic follicle aspiration. Fifty-six infertile patients were treated during 86 cycles with human menopausal gonadotropin-human chorionic gonadotropin (hMG-hCG) for stimulation of follicular development. Serum estradiol (E2) levels were measured daily during hMG-hCG treatment. P levels were determined in 67 cycles. The mean (+/- standard deviation [SD]) of the sums of 3 P levels was 55.63 +/- 24.13 ng/ml. There were 11 pregnancies. The mean of the sums of 3 P levels of pregnant patients was 64.45 +/- 26.23 ng/ml and of 56 nonpregnant cycles was 53.90 +/- 23.35 ng/ml. The duration of luteal phase varied from 9 days to 15 days. The mean of the sums of 3 P values of patients with different luteal phase lengths ranged from 28.8 ng/ml to 60.51 +/- 25.68 ng/ml. The mean of the sums of 3 P levels of women with normal luteal phase and that of women with luteal phase defect by endometrial biopsy study were used as controls for comparison. There was poor correlation (r = 0.3441) between E2 peak levels and P levels; the sum of 3 P levels did not indicate luteal phase inadequacy in IVF-ET patients; and the majority of the nonpregnant cycles (32/56) showed a luteal phase of 11 days or less, in spite of adequate P levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist-induced isomerization of the alpha 1-adrenergic receptor: kinetic analysis using broken-cell and solubilized preparations.

The affinity of agonists but not antagonists at hepatic membrane alpha 1-adrenergic receptors is temperature dependent; a 100-fold higher affinity is observed at 4 degrees C than at 37 degrees C. The relationship between these two agonist affinity states was investigated by using a strategy that allows the kinetics of this transition to be examined under equilibrium conditions. When competition assays are performed at 37 degrees C for varying intervals and the reaction mixture is then rapidly cooled by freezing, allowed to thaw, and further equilibrated at 4 degrees C, a rapid and progressive decrease (t1/2 of 1-2 min) in agonist affinity occurs, the extent of which is directly related to the incubation time at 37 degrees C. This decrease in agonist affinity is sustained as long as agonist is present but can be reversed by its subsequent removal. In contrast, no change in affinity is seen in identical experiments when antagonists are employed as the competing ligand. High-affinity binding of agonists is also demonstrated in short-term nonequilibrium experiments, indicating that the low-temperature incubations do not induce, but rather stabilize, a receptor conformation of high affinity for agonists. These findings suggest that the predominantly low-affinity binding of agonists to alpha 1-adrenergic receptors demonstrated in equilibrium studies at physiological temperatures may be the result of a ligand-driven decrease in affinity. Since the transition in receptor affinity for agonists occurs not only in broken-cell preparations but also after detergent solubilization of the membrane receptor, it most likely is due to an agonist-induced change in the conformation of the receptor protein per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol esters inhibit alpha 1-adrenergic effects and decrease the affinity of liver cell alpha 1-adrenergic receptors for (-)-epinephrine.

4 beta-Phorbol 12-myristate 13-acetate (PMA) modified the metabolic actions of three calcium-dependent hormones in different ways. The stimulations of glycogenolysis ureogenesis and phosphatidylinositol labeling produced by alpha 1-adrenergic agonist was blocked by the phorbol ester. In contrast, PMA slightly increased the stimulation of ureogenesis produced by low concentration of angiotensin II without modifying the maximal response. No effect of PMA was observed on the stimulation of ureogenesis induced by vasopressin. The stimulation of phosphatidylinositol labeling induced by vasopressin was decreased by PMA, whereas that induced by angiotensin II was not affected. In intact freshly isolated hepatocytes, [3H]prazosin binds with high affinity to a site which displays the characteristics of alpha 1-adrenergic receptor. Competitive inhibition studies with (-)-epinephrine reveal two different sites for this agonist: a high affinity site (Kd 9 nM) and a low affinity site (Kd 2 microM). In the presence of phorbol esters, (-)-epinephrine binding data now show the presence of a single class of low affinity sites, with similar affinity to those present in control cells. Thus, the inhibition of hepatocyte alpha 1-adrenergic action by PMA may be related to the loss of high affinity binding sites caused by the tumor promoter.

Transient high-affinity binding of agonists to alpha 1-adrenergic receptors of intact liver cells.

At alpha 1-adrenergic receptors in isolated rat liver parenchymal cells, (-)-epinephrine is potent in eliciting a maximal increase in glycogenolysis (Kact = 24 nM). This contrasts with a 100-fold lower affinity for the agonist at alpha 1-adrenergic receptors of intact hepatocytes determined from equilibrium competition assays with the alpha 1-adrenergic antagonist [3H]prazosin. We demonstrate here that agonists bind to alpha 1-adrenergic receptors of intact liver cells initially with a markedly higher affinity than under equilibrium conditions. When incubations are performed for 15 s at 37 degrees C, the affinity is more than 100-fold higher than that obtained in equilibrium (45 min) assays (IC50 = 28 +/- 3 vs 5300 +/- 400 nM for (-)-epinephrine and 32 +/- 3 vs 6100 +/- 500 nM for (-)-norepinephrine). When incubations are performed at 4 degrees C (150 min), high-affinity binding similar to that obtained in short-term incubations can also be demonstrated. In contrast, antagonist compete with similar affinities in 15 s and 45 min assays, and their dissociation constants are not affected by changes in the incubation temperature. These results indicate that agonists bind to native alpha 1-adrenergic receptors transiently with high affinity. The conversion of receptors to a state of predominantly low affinity for agonists, which occurs rapidly and irreversibly with increasing incubation at 37 degrees C, is inhibited at low incubation temperatures. It is suggested that the high-affinity configuration of the alpha 1-adrenergic receptor for agonists observed in nonequilibrium experiments or at reduced incubation temperatures represents the physiologically relevant state of the alpha 1-adrenergic receptor.

[Chronic therapy of congestive cardiomyopathy: effects of prenalterol and digoxin]. / Chronische Therapie der kongestiven Kardiomyopathie: Wirkungen von Prenalterol und Digoxin.

UNLABELLED: Prenalterol (P), a partial adrenergic agonist with functional beta 1-specificity, has been shown to have inotropic effects when given orally and thus represents a potential substitute or adjunct to conventional digitalis therapy (D) in the long-term management of congestive cardiomyopathy (COCM). A direct comparison between both drugs has not been reported. In a blind controlled trial, 15 patients with COCM (NYHA II-III) with sinus rhythm and a left ventricular ejection fraction (LV-EF) of 34.5 +/- 2.6% received consecutively D (0.25-0.5 mg/d), placebo (PLAC), P (slow releases = SR) (80 mg/d SR) and both drugs combined in respective doses. After 4 weeks of therapy with each drug, effects were assessed by gated blood pool scintigraphy at rest (R) and during graded bicycle exercise (EX), systolic time intervals (STI), Holter monitoring and a clinical score. Plasma levels of both drugs and of catecholamines and lactate were also determined. Compared to PLAC, LV-EF was not significantly altered by D at R (34.5 +/- 2.6 vs. 31.9 +/- 2.3%, p = ns), but a shortening of the QS2-interval could be demonstrated (533 +/- 7 vs. 550 +/- 6 msec, p less than 0.05). In contrast, during EX an improvement of LV-EF was observed (34.5 +/- 3 vs. 31.3 +/- 2.8%, p less than 0.05). P alone showed no significant alterations in LV-EF and STI, along with a lack of symptomatic improvement. The addition of D (D + P) resulted in improved left ventricular performance both at R (LV-EF 37.9 +/- 3.3 vs. 31.9 +/- 2.3%, p less than 0.01, QS2 530 +/- 8 vs. 550 +/- 6 msec, p less than 0.01) and during EX (LV-EF 35.3 +/- 2.5 vs. 31.1 +/- 2.8%). Values between D and D + P were not significantly different. No drug or combination improved maximal working capacity. CONCLUSIONS: Beneficial effects of chronic treatment with D could be demonstrated in patients with COCM, particularly during EX. Further studies are needed to determine why the acute effects of P are not fully sustained during long-term therapy.

Rapid reciprocal changes in adrenergic receptors in intact isolated hepatocytes during primary cell culture.

In hepatocytes freshly isolated from adult female rat livers, catecholamine-stimulated glycogenolysis is mediated predominantly by alpha 1-adrenergic receptors, and to only a minimal extent by beta 2 receptors. Primary cell culture of these hepatocytes results in a switch in the adrenergic control of glycogenolysis from an alpha 1 to a predominant beta 2 type of response. To investigate whether this switch is due to an alteration in the plasma membrane receptor composition, we characterized alpha 1 and beta 2-adrenergic receptors in both freshly isolated and cultured hepatocytes, using radioligand-binding techniques. Binding of the selective alpha 1-adrenergic antagonist [3H]prazosin and the beta-adrenergic antagonist [125I]pindolol to intact freshly isolated hepatocytes was of high affinity, saturable, and of appropriate specificity for an alpha 1- and beta 2-adrenergic receptor, respectively. Equilibrium binding studies evaluated by a computer-assisted curve-fitting procedure indicated interaction with a single class of high affinity sites for radiolabeled prazosin (KD = 126 +/- 10 pM; Bmax = 93,000 +/- 5,500 sites/cell) and pindolol (KD = 66 +/- 6 pM; Bmax = 2,000 +/- 700 sites/cell). In intact hepatocytes and in membranes prepared from these hepatocytes, competitive inhibition curves revealed the coexistence of two different sites with high and low affinities for agonists at both alpha 1- and beta 2-adrenergic receptors. When isolated hepatocytes were kept in monolayer cell culture for up to 72 hr, the switch in adrenergic control of glycogenolysis (phosphorylase a activation) from an alpha to a beta pathway was confirmed and was associated with a progressive decrease in the number of alpha 1 receptors and an increase in beta 2-adrenergic receptor density, without marked change in the affinity of agonists or antagonists. To investigate the mechanism(s) of this reciprocal change, a number of perturbations were examined including alterations in the composition of the culture medium and the influence of various hormones and inhibitors of cellular function. De novo protein synthesis is implicated in both receptor alterations as the inhibitors cycloheximide and actinomycin D prevented the increase in beta- and attenuated the decrease in alpha-adrenergic sites. The other perturbations were without effect. Thus, these studies provide evidence for a coupling of the functional alteration in glycogenolysis to changes at the receptor level per se. The mechanism underlying the reciprocal changes in hepatocyte adrenergic receptors during culture remains undefined.(ABSTRACT TRUNCATED AT 400 WORDS)