Cloning and sequence of partial cDNAs encoding the human type V and VI adenylyl cyclases and subsequent RNA-quantification in various tissues.

Adenylyl cyclases (Acs) and VI are the predominant form of Acs in mammalian heart where they are part of the beta-adrenergic pathway. Up to now, the sequences for both enzymes from human tissues have not yet been reported. We investigated the mRNA expression AC V and VI in human colon, heart, liver, lung and MNL. Partial cDNAs of human types V and VI Acs were amplified by PCR using oligonucleotides derived from the cytoplasmatic domain sequences of the corresponding enzymes from dog heart. Primers derived from the human sequence were used to detect the mRNAs corresponding to both Acs. For quantification of mRNAs we constructed internal standards for competitive quantitative reverse transcriptase PCR (RT-PCR). Both types of transcripts could be found in all investigated tissues except MNL where only type VI could be detected. Further we demonstrated a more than 60 times higher amount of AC V-mRNA in human heart compared to AC VI-mRNA.

Atrial fibrillation after coronary artery bypass surgery: association with changes in G protein levels in mononuclear leukocytes.

Atrial fibrillation (AFib) is a frequent complication of coronary artery bypass grafting (CABG). Its cause, however, is unknown. As the adrenergic system is involved in some types of AFib, we hypothesized that a change in guanine nucleotide-binding protein (G protein) expression plays a role in the development of post-CABG AFib. In 28 patients undergoing CABG surgery, the G(s alpha)/G(i alpha) ratio (stimulatory/inhibitory G protein alpha) at the protein (Western blotting) and mRNA (reverse transcription polymerase chain reaction) levels was measured before and after surgery. As a suitable test system allowing multiple analysis mononuclear leukocytes (MNL) were chosen. The perioperative change of the G(s alpha)/G(i alpha) ratio of protein and mRNA was significantly different in patients who subsequently developed AFib (eight patients) and in patients who did not (20 patients; P<0.01 and <0.001, respectively). On average, the protein G(s alpha)/G(i alpha) ratio decreased from 1.79+/-1.13 (mean+/-SD) to 1.32+/-0.69 in patients without AFib (P=0.1, n.s.) whereas a significant increase from 0.86+/-0.44 to 1.62+/-0.65 (P<0.01) was observed in patients subsequently developing AFib. The mRNA G(s alpha)/G(i alpha) ratio decreased significantly from 0.53+/-0.24 to 0.36+/-0.11 in patients without AFib (P<0.01) whereas a significant increase from 0.31+/-0.14 to 0.47+/-0.13 was observed in those who subsequently developed AFib (P<0.05). These results indicate that an increase of the G(s alpha)/G(i alpha) ratio in MNL is associated with AFib after CABG surgery and possibly may be used as a prognostic indicator of this complication.

Agonist-induced down-regulation of the beta2-adrenoceptor and its mRNA in human mononuclear leukocytes.

Agonist-mediated regulation of beta2-adrenoceptors in mononuclear leukocytes has been examined at the protein but not at the mRNA level. In the present study, incubation of mononuclear leukocytes with the beta-agonist (-)-isoproterenol (10(-6) M) for up to 42 hr led to a maximum decrease in both beta2-adrenoceptor mRNA concentration and total receptor number of ca. 56 and 70%, respectively. The decrease in the mRNA level, however, was slower than for the protein level. After 4 hr of incubation with the beta-agonist, the protein level decreased to a minimum of 65% of the initial amount, while an incubation of 8 hr was necessary to reach a similar decrease in the level of mRNA (69% of the initial level). Measurements of mRNA stability revealed a reduction in the half-life of beta2-adrenoceptor mRNA from 2.7 to 1.1 hr following 4 hr of incubation with (-)-isoproterenol. Our data clearly demonstrate that treatment of human mononuclear leukocytes with (-)-isoproterenol induces a beta2-adrenoceptor down-regulation together with a slower time course of mRNA down-regulation which is partly due to a reduction of mRNA stability.

Pharmacokinetics and pharmacodynamics of the enantiomers of gallopamil.

The pharmacokinetics and pharmacodynamics of the enantiomers of the calcium antagonist gallopamil have been investigated in six healthy volunteers. Each subject was studied on five occasions after receiving, in randomized order: placebo, 25 mg of (R)-gallopamil, 25 mg of (S)-gallopamil, 50 mg of pseudoracemic [25 mg of deuterated (S)-gallopamil and 25 mg of (R)-gallopamil] and 100 mg of (R)-gallopamil HCl orally. After separate administration, the apparent oral clearances of both enantiomers were similar [(R), 15.1 +/- 9.9 liters/min; (S), 11.0 +/- 6.0 liters/min], indicating that gallopamil first-pass metabolism is not stereoselective. After coadministration, the apparent oral clearance of each enantiomers decreased [(R), 5.9 +/- 2.8 liters/min; (S), 5.8 +/- 2.66 liters/min], suggesting that a partial saturation of first-pass metabolism occurs because the dose was twice as high than for the single enantiomers. Serum protein binding and renal elimination of gallopamil are stereoselective, favoring (S)-gallopamil. Analysis of urine samples revealed a marked degree of stereoselectivity in the formation of O- and N-dealkyl metabolites. Because these showed opposite stereoselectivity, canceling out each other, the net result was no or only marginal stereoselectivity. Twenty-five milligrams of (S)-gallopamil prolonged the PR interval in all subjects; however, a greater effect was elicited by 50 mg of (RS)-gallopamil. (R)-Gallopamil (100 mg) did not significantly alter the PR interval, although higher concentrations were attained than after the pseudoracemate. Based on a consideration of (S)-gallopamil serum concentrations, a comparable relationship between (S)-gallopamil level and effect occurred after (S)- and (RS)-gallopamil, indicating that the pharmacological effect produced by the racemate could be totally accounted for by the higher concentrations of (S)-gallopamil attained.

Regulation of beta-adrenoceptor density and mRNA levels in the rat heart cell-line H9c2.

The regulation of the expression of beta-adrenoceptor (beta-ARs) is not thoroughly understood. We demonstrate that the rat heart cell-line H9c2 expresses both beta 1- and beta 2-ARs. In radioligand-binding experiments, the maximal binding capacity of (-)-[125I]-iodocyanopindolol was determined as 18 +/- 0.6 fmol/mg of protein with a KD of 35.4 +/- 4.1 pM. Competitive radioligand-binding experiments with subtype-specific beta-antagonists reveal a subtype ratio of beta 1- to beta 2-ARs of 29%: 71%. With competitive reverse-transcriptase PCR we found beta 2-mRNA to be up to 1600 times more frequent than beta 1-mRNA. Treatment of the H9c2 cell-line with the beta-adrenergic agonist (-)-isoproterenol (10(-6) M), the antagonist (-)-propranolol (10(-6) M) and the glucocorticoid dexamethasone (500 nM) induces regulatory effects on both the beta-AR protein and mRNA level. Isoproterenol treatment leads to down-regulation of the total receptor number by 56 +/- 4%, due to a decrease in beta 2-ARs, while maintaining the beta 1-AR number constant. On the transcription level, both beta 1-and beta 2-mRNAs are decreased by 30% and 42% respectively. mRNA stability measurements reveal a reduced half-life of beta 2-mRNA from 9.3 h to 6.5 h after isoproterenol treatment. Incubation of cells with (-)-propranolol does not affect the amounts of beta-ARs and their mRNAs. Dexamethasone induces a 1.8 +/- 0.2-fold increase in beta-AR number over the basal level as well as a 1.9 +/- 0.2-fold increase in the amount of beta 2-mRNA. Because the half-life of beta 2-mRNA was unaffected by dexamethasone, the increased beta 2-mRNA level must be due to an enhanced transcription rate. The beta 1-mRNA levels are unchanged during dexamethasone-incubation of the cells. Our data clearly demonstrate that treatment of H9c2 rat heart cells with isoproterenol and dexamethasone induces alterations in the level of RNA stability as well as gene transcription, leading to altered receptor numbers.

Non-isotopic competitive reverse transcription polymerase chain reaction coupled with high performance liquid chromatography to measure beta 2-receptor messenger RNA in the human heart.

We describe an application of competitive reverse transcription-polymerase chain reaction (PCR) coupled with HPLC for quantification of beta 2-adrenergic receptor messenger RNA (mRNA) in human atrial tissues removed during cannulation for cardiopulmonary bypass operations. We constructed an internal standard which was reverse transcribed in different concentrations together with constant levels of cellular RNA and subsequently PCR amplified. The competitor RNA shows the same beta 2-adrenergic receptor primer sequences as the cellular mRNA but yields a different-sized product. This allows resolution of the amplified copy DNA (complementary DNA, cDNA) fragments with a specific HPLC column. The concentration of beta 2-adrenergic receptor mRNA is derived from the ratio between the peak intensities corresponding to the amplified competitor and target products. We assessed the imprecision, accuracy and sensitivity of the method. Concentrations of beta 2-adrenergic receptor mRNA of 22.7 +/- 15.2 x 10(6) molecules per micrograms total RNA in patients treated with beta 2-antagonists were not significantly different from control patients showing 16.8 +/- 9.9 x 10(6) beta 2-adrenergic receptor mRNA molecules per microgram total RNA (Mean +/- SD). Competitive reverse transcription PCR is a highly specific, non-radioactive procedure for quantification of beta 2-adrenergic receptor mRNA and simultaneously other gene expression levels of interest in atrial tissue specimens and may therefore be used to advance our understanding of heart muscle disease.

Inadequate response to H2-receptor antagonists. Absence of parietal cell cAMP-stimulating autoantibodies.

In peptic ulcer patients with adequate (AR; N = 16) and inadequate response (IR; N = 20) to H2-receptor antagonists, the presence of parietal cell cAMP-stimulating autoantibodies was studied. Serum Ig fractions from these patients and 10 control subjects were examined to test whether they could stimulate cAMP production in a gastric cell line model. The human cell line HGT-1 was found to be a sensitive in vitro model for the cAMP stimulation assay as histamine (10 microM) increased by 11-fold the production of cAMP. Neither IgG (4 mg/ml) nor IgG-free Ig fractions (1 mg/ml) isolated from the blood of AR or IR affected cAMP production in the HGT-1 cells. The results obtained with the cAMP stimulation assay were confirmed by indirect immunofluorescence measurements based on frozen sections of rat stomach and kidney. No specific staining of rat parietal cells could be observed with patient sera. In addition, human gastric biopsies of the oxyntic mucosa from the same patients were studied for immunoreactive cell populations to assess organ-specific autoimmune processes. Biopsy specimens from AR and IR showed increased lymphocytic infiltrates, usually associated with gastritis. However, no significant differences in location of various cell populations between AR and IR could be observed. Our findings do not support a recent hypothesis that poor response to treatment with H2-receptor antagonists is due to the presence of autoantibodies.

Stereoselectivity in cardiovascular and biochemical action of calcium antagonists: studies with the enantiomers of the dihydropyridine nitrendipine.

OBJECTIVES: The cardiovascular and biochemical effects of R- and S-nitrendipine were studied in six healthy subjects in a single-blind placebo-controlled study. METHODS: After received oral doses of placebo, 20 mg R-, 80 mg R- (n = 5), 20 mg S-, and 20 mg racemic nitrendipine, heart rate, systolic, diastolic, and mean arterial blood pressure, leg blood flow, peripheral vascular resistance, plasma renin activity, norepinephrine, epinephrine, dopamine, and aldosterone plasma levels were measured before and up to 3 hours after administration. RESULTS: Neither placebo nor 20 or 80 mg R-nitrendipine caused significant changes of cardiovascular and biochemical parameters. After 20 mg S-nitrendipine and 20 mg racemic nitrendipine, significant changes in diastolic blood pressure (-9.1/-7.4 mm Hg), heart rate (+21.9/+17.3 beats/min), leg blood flow (+6.8 ml.min-1.gm tissue-1), peripheral vascular resistance (-16.9 mm Hg.min.gm tissue.ml-1), norepinephrine (+476/+281 ng.L-1), and plasma renin activity (+9.5/+3.6 ng.ml-1.hr-1) were observed. The changes in cardiovascular and biochemical parameters were closely related to the serum S-nitrendipine concentrations. CONCLUSIONS: It can be concluded that, after administration of the racemate, the S-enantiomer is responsible for the cardiovascular and biochemical effects observed and that S-nitrendipine is at least an order of magnitude more potent than the R-enantiomer.

Detection of hepatitis C virus antibodies and hepatitis C virus RNA in patients with alcoholic liver disease.

The prevalence of hepatitis C virus (HCV) antibody was determined in 130 patients with alcoholic liver disease using a second-generation anti-HCV enzyme immunoassay (ELISA 2) and confirmed by a sensitive polymerase chain reaction procedure measuring HCV RNA. Hepatic disease was evaluated by clinical and biochemical studies and, whenever possible, by liver biopsy. Seventy-one patients were diagnosed as having cirrhosis, and 59 alcoholic hepatitis (n = 33) or fatty liver (n = 26). The prevalence of anti-HCV in the total group was 9.2% and did not differ significantly in the cirrhotics (11.3%) as compared with the non-cirrhotics (6.8%). HCV RNA was detected in six out of eight cirrhotics and three out of four non-cirrhotics who were ELISA 2 positive. A positive test for antibodies to hepatitis core antigen (anti-HBc) was more frequent in anti-HCV-positive patients (75%) than in the anti-HCV-negative group (14%, P < 0.001). Anti-HBc was also found more frequently in the cirrhotics (25.4%) than in the alcoholics without cirrhosis (11.9%). However, the prevalence of hepatitis B surface antigen was equally low in both groups (cirrhotics 1.4%, non-cirrhotics 1.7%). No correlation was observed between the prevalence of anti-HCV antibodies and the severity of liver dysfunction. These results indicate that HCV, and especially HCV-viraemia, is less frequent in alcoholics in southern Germany than suspected in previous studies, and that the prevalence of HCV markers in alcoholics has been overestimated by ELISA 1 used alone.

Development and evaluation of a reagent carrier with a new reaction sequence for the determination of creatinine in blood, plasma, serum and urine.

After a short outline of the history of creatinine determination methods we describe the development of a dry-reagent-carrier system for the reflometric determination of the creatinine concentration in blood, plasma, serum and urine (Reflotron Creatinine (new)). The method is based on a sequence of enzymatically catalyzed reactions producing H2O2, but which in contrast to the previously used procedure do not lead to the formation of creatine as an intermediate. Hence, pretreatment of sample material to eliminate endogenous creatine is no longer necessary. In the indicator reaction, use is made of an imidazole derivative as the chromogen. The dye formed in the presence of peroxidase can be measured by reflectance photometry beyond the long-wave absorption bands of haemoglobin and bilirubin at 642 nm. We present in detail the results of the multicentre evaluation of the analytical properties of this new test principle. The data obtained show that Reflotron Creatinine (new) correlates well with the routine method Creatinine PAP, which was used as a comparison method, with respect to accuracy and precision and even surpasses it with respect to specificity. Advantages over the first generation of Reflotron Creatinine are: shorter reaction time, longer stability of the reagent carrier, no interference by bilirubin and reduced interference by haemoglobin.

In vitro study on histamine-induced cyclic adenosine monophosphate production in ulcer patients with different antisecretory responses to ranitidine.

Failure of acid suppression by H2 receptor antagonists has been observed in some patients with peptic ulcer diseases. The reasons for the drug resistance are unknown. In the present study, the effects of histamine and the H2 receptor antagonist ranitidine (CAS66357-35-5) on cyclic adenosine monophosphate (AMP) production was investigated using intact cells from gastric mucosal biopsies derived from patients with adequate (AR) and inadequate (IR) antisecretory response to ranitidine. The classification of AR and IR was performed by intragastric nocturnal pH-monitoring. Parietal cell content was increased in IR (14.5 +/- 5.3%; mean +/- SD) compared to AR (11.2 +/- 4.5%; p < 0.05). The histamine-induced cyclic AMP production was decreased in IR compared to AR whereas the EC50-values where similar (22-27 mumol/l). Inhibitory activity of ranitidine (Ki; 105-133 nmol/l) did not differ significantly in samples of both patient groups. Duration of clinical pretreatment with ranitidine did not affect the cyclic AMP production. These data suggest that the inadequate response may be rather due to lower H2 receptor density or non-competitive impairment of the receptor in gastric mucosal cells than to a decreased affinity of the antagonist to the receptor.

Quantitative determination of natriuretic peptides in human biological samples with a bioassay using cultured cells.

Up to now, members of the natriuretic peptide family have usually been determined by radioimmunoassays using antibodies more or less specific for the distinct peptides so far identified. However, natriuretic peptides differing significantly in their amino acid sequence from the one against which the antibody has been raised cannot be determined by this means, and still unknown natriuretic peptides cannot be detected. We therefore developed a new bioassay system sensitive to all members of the natriuretic peptide family by taking advantage of the biological activity of these hormones, the activation of the guanylate cyclase/cyclic GMP system. In this assay, cultured cells are incubated with the natriuretic peptides, and the amount of cyclic GMP produced by the cells is determined by radioimmunoassay. From the relative stimulation of the cellular cyclic GMP production, the concentration of natriuretic peptides in the sample is determined after calibration with synthetic atrial natriuretic peptide. For a qualitative identification of the various peptides, the bioassay is combined with a reversed-phase HPLC step. Using cultured bovine aortic endothelial or bovine kidney epithelial cells for the bioassay, we achieved detection limits of 1 fmol or 50 fmol, respectively, for human atrial as well as brain natriuretic peptide. Intra-assay coefficients of variation of 4.3% (aortic endothelial cells, at 0.65 nmol/l peptide) and 5.8% (kidney epithelial cells, at 6.5 nmol/l peptide) were obtained. The total content of natriuretic peptides as well as the amounts of the individual natriuretic peptides following HPLC separation were determined in extracts of human atria obtained at aortocoronary bypass operations.(ABSTRACT TRUNCATED AT 250 WORDS)

A method to simultaneously investigate histamine-induced cyclic AMP and aminopyrine accumulation in isolated gastric mucosal cells from human biopsies.

A method has been developed to simultaneously measure two parameters of histamine-induced gastric secretion, cyclic AMP and aminopyrine accumulation, in gastric mucosal cells from human biopsies. Histamine stimulated cyclic AMP and aminopyrine accumulation with different time courses. Cyclic AMP production reached a maximum at 30 min, whereas maximal aminopyrine accumulation was obtained after the cells had been incubated for 90 min in presence of 100 mcM histamine. Histamine was more potent in stimulating aminopyrine than cyclic AMP accumulation. The ED50 values for histamine-induced aminopyrine accumulation were estimated to be 0.88 +/- 0.02 and 25.53 +/- 8.75 mcM for cyclic AMP production, respectively. The aminopyrine accumulation was more than half-maximally increased at 1 mcM histamine without significant effects on the cyclic AMP basal level. It remains to be elucidated whether this finding indicates, besides cyclic AMP, the involvement of calcium in histamine stimulus. The histamine H2-receptor antagonists cimetidine and ranitidine inhibited both in vitro parameters, whereas the gastric proton pump inhibitor omeprazole only affected aminopyrine accumulation. Our method might be a valuable tool for in vitro pharmacological and clinical investigations on histamine-induced gastric secretion in human biopsies.

Distribution of free and conjugated catecholamines between plasma, platelets and erythrocytes: different effects of intravenous and oral catecholamine administrations.

Plasma, platelet and erythrocyte contents of free and conjugated norepinephrine, epinephrine and dopamine were determined by radioenzymatic assay in 12 resting healthy volunteers. Mean platelet/plasma concentration ratios were 533 for free norepinephrine, 502 for free epinephrine and 149 for free dopamine. Corresponding erythrocyte/plasma ratios were 1.04, 1.13 and 4.5, respectively. The presence of conjugated catecholamines in platelets and erythrocytes could be confirmed; however, their relative proportion within these cells, particularly in platelets, was lower than that in plasma. Upon intravenous infusion of dopamine for 3 hr at 5 micrograms kg-1 min-1, concentrations of free dopamine in plasma increased rapidly (280-970-fold), whereas conjugated dopamine only reached maximal values (14-19-fold increase) at 30 to 60 min after cessation of the infusion. The relative distribution of unconjugated dopamine in whole blood between plasma, platelets and erythrocytes changed from mean values of 1:0.33:3.7 at rest to 1:1.1:0.5 at the end of the infusion. As a result of the subsequent rapid decrease of dopamine in plasma and erythrocytes, this distribution was 1:17:1 shortly thereafter and remained constant up to the end of the investigation period. The relative distribution for conjugated dopamine of 1:0.001:0.5 at rest changed to about 1:0.2:0.1 at the termination of the infusion. Oral administration of norepinephrine and dopamine led to increases in the plasma concentrations of these amines in their conjugated forms only, whereas epinephrine concentrations remained constant. These elevations were not accompanied by corresponding increases in platelet and erythrocyte norepinephrine, epinephrine and dopamine contents.(ABSTRACT TRUNCATED AT 250 WORDS)

A method to assess histamine-induced cyclic AMP production in isolated gastric mucosal cells from human biopsies.

Human gastric mucosal cells were isolated by digestion of fundic biopsies with pronase and collagenase. The mean value of gastric cells per milligram biopsy specimen +/- SEM was 59,000 +/- 4,300 (n = 31) with a viability of 90 +/- 5%. With the cell yield of 1 patient a series of approximately 70-80 cyclic AMP measurements was possible. Histamine stimulated intracellular cyclic AMP production with an EC50 value of 35 +/- 25 mumol/l (SEM; n = 4). In the presence of 100 mumol/l histamine the Ki values (mumol/l) for the histamine H2 receptor antagonists averaged 1.45 (cimetidine), 0.10 (ranitidine), and 0.02 (famotidine). No significant inhibition of histamine-induced cyclic AMP production was obtained with the histamine H1 receptor antagonist triprolidine. With the new method histamine-induced cyclic AMP production can be measured in intact human gastric mucosal cells from fundic biopsy samples.

Antibodies as a source of analytical errors.

This review discusses the interference in clinical chemical analysis caused by autoantibodies and by antibodies to foreign antigens. The nature of these disturbances, their occurrence, prevalence, and detection, the different ways in which they are manifested, the clinical consequences of the failure to recognize such interference, and finally methods for avoiding these disturbances are discussed. Interference by cold agglutinins in the automatic determination of the erythrocyte count, interference by cryoglobulins in the determination of the leukocyte count, and EDTA-induced thrombocyte agglutination are well documented as sources of error in the analysis of haematological parameters. Enzyme determinations may be affected by the occurrence of macro-complexes of the measured enzyme with immunoglobulins. This type of interference, for example, may occur in the determination of amylase and creatine kinase. Immunoassays for the determination of hormones or tumour markers are sensitive to autoantibodies and heterophilic antibodies. The former are particularly important in the determination of thyroid hormones, where the interference is method-dependent. In several immunoassays, interference by heterophilic antibodies can be abolished by the addition of non-immune serum. Finally, rheumatoid factors, antibodies administered for therapeutic purposes, and monoclonal gammopathies are possible sources of interference in the determination of various analytes.

Magnitude and kinetics of alterations in plasma catecholamines and leukocyte beta-adrenergic receptors in response to anaesthesia and surgery.

We studied the response of the sympatho-adrenal system to varying intensities of different stimuli. Concentrations of norepinephrine and epinephrine in plasma as well as densities of beta 2-adrenergic receptors on mononuclear leukocytes were determined in patients subjected to operations of varying complexity and different types of anaesthesia. In patients undergoing hysterectomy (n = 9), the maximal increases in plasma norepinephrine and epinephrine were 2.7- and 2.8-fold, respectively, corresponding to a post-operative decrease of the mononuclear leukocyte beta 2-adrenergic receptors of 27% after 4 hours. Patients with coronary revascularization (n = 17) were randomly selected to receive either enflurane/N2O or neurolept anaesthesia. During intraoperative periods of stress, such as cardiopulmonary bypass and hypothermia, norepinephrine and epinephrine levels were 2-3 times higher in the neurolept patients, compared with the enflurane patients. In the former group, the respective maximal norepinephrine and epinephrine concentrations were 9.7 and 28 times the vasal values of the non-anaesthetized patients. One day postoperatively, the mononuclear leukocyte beta 2-receptor density decreased maximally by 45 +/- 11% in the enflurane patients, and by 53 +/- 6% in the neurolept patients. As early as two to five days after cardiac surgery, beta 2-receptor densities were no longer distinguishable from the preoperative values. Significant correlations between the increases in catecholamine concentrations and the decreases in beta 2-receptor densities did not exist. It is concluded that enflurane blocks the sympatho-adrenal response to surgical stress more effectively than neurolept anaesthesia.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine infusion in healthy subjects and critically ill patients.

1. Little is known about the metabolism and the pharmacokinetics of dopamine (DA) in critically ill patients. To study the influence of the total administered DA dose on the disposition of free (i.e. unconjugated) and sulfoconjugated DA, plasma levels of free and sulfoconjugated DA were measured following infusion of 5 micrograms DA/kg per min for 0.5 and 3 h in six healthy volunteers and in eight critically ill patients receiving DA at the same infusion rate for 6.5 to 329 h. 2. In patients and volunteers steady state concentrations of free DA showing fairly large inter-individual variations (12.4-73.4 micrograms/L) were reached within 10 min of the beginning of the infusion. 3. DA sulfate was generated immediately. In volunteers peak values of the sulfoconjugate were observed 15-60 min after the termination of the DA infusion. In patients steady state concentrations of conjugated DA (63-80 micrograms/L) were reached within 5-10 h of DA infusion. 4. The initial half-life (t1/2 alpha), the terminal elimination half life (t1/2) and the distribution volume of free DA in the volunteers were significantly higher after 3 h of the DA infusion as compared to the shorter infusion. These parameters as well as the total plasma clearance of free DA were independent of the length of the DA infusion period in patients. The large distribution volumes of 19.8-75 L/kg indicate that DA has been taken up by peripheral tissues. 5. Substantial inter-individual variations in the patients' clearance of free DA (3.9-16.5 L/kg per h) may partly explain the variability in haemodynamic responses to DA infusion reported in clinical studies.(ABSTRACT TRUNCATED AT 250 WORDS)