Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish.

Many Hox proteins are thought to require Pbx and Meis co-factors to specify cell identity during embryogenesis. Here we demonstrate that Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish. We find that Hoxb1b and Pbx4 act together to induce ectopic hoxb1a expression in rhombomere 2 of the hindbrain. In contrast, Hoxb1b and Pbx4 acting together with Meis3 induce hoxb1a, hoxb2, krox20 and valentino expression rostrally and cause extensive transformation of forebrain and midbrain fates to hindbrain fates, including differentiation of excess rhombomere 4-specific Mauthner neurons. This synergistic effect requires that Hoxb1b and Meis3 have intact Pbx-interaction domains, suggesting that their in vivo activity is dependent on binding to Pbx4. In the case of Meis3, binding to Pbx4 is also required for nuclear access. Our results are consistent with Hoxb1b and Meis3 interacting with Pbx4 to form complexes that regulate hindbrain development during zebrafish embryogenesis.

A novel pbx family member expressed during early zebrafish embryogenesis forms trimeric complexes with Meis3 and Hoxb1b.

pbx genes encode homeodomain-containing transcriptional regulators that interact with other proteins to control embryogenesis and tumorigenesis. We present the characterization of a zebrafish pbx CDNA that appears to encode a novel family member, pbx4. pbx4 RNA is maternally deposited and is detected throughout the zebrafish embryo during blastula stages. It becomes excluded from ventroanterior structures at late gastrula stages and is detected within the forming central nervous system during segmentation stages. pbx4 expression overlaps with that of two other homeobox genes, hoxb1b and meis3, in the region of the presumptive caudal hindbrain during gastrula stages. In vitro binding experiments revealed that protein complexes containing Pbx4/Meis3 and Pbx4/Hoxb1b, but not Meis3/Hoxb1b could be generated. A novel trimeric complex containing Pbx4, Meis3, and Hoxb1b was also formed. We speculate that complexes with different combinations of Pbx4, Meis3, and Hoxb1b specify different developmental fates during vertebrate embryogenesis. Dev Dyn 2000;217:109-119.

A multifactorial trial design to assess combination therapy in hypertension. Treatment with bisoprolol and hydrochlorothiazide.

BACKGROUND: The safety and effectiveness of different dosages and combinations of antihypertensive agents can be efficiently studied using a multifactorial trial design. In consultation with the Cardio-Renal Division of the Food and Drug Administration, we conducted a randomized, double-blind, placebo-controlled, 3 x 4 factorial trial of bisoprolol, a beta 1-selective adrenergic blocking agent, and hydrochlorothiazide. METHODS: A total of 512 patients with mild to moderate essential hypertension were randomized to once-daily treatment with bisoprolol (0, 2.5, 10, or 40 mg), hydrochlorothiazide (0, 6.25, or 25 mg), and all possible combinations. Diastolic and systolic blood pressures were monitored during this 12-week trial. RESULTS: The effects of bisoprolol and hydrochlorothiazide were additive with respect to reductions in diastolic and systolic blood pressures over the dosage ranges studied. The addition of hydrochlorothiazide (or bisoprolol) to therapy with bisoprolol (or hydrochlorothiazide) produced an incremental reduction in blood pressure. Dosages of hydrochlorothiazide as low as 6.25 mg/d contributed a significant antihypertensive effect. A hydrochlorothiazide dosage of 6.25 mg/d produced significantly less hypokalemia and less of an increase in uric acid levels than a dosage of 25 mg/d. The low-dose combination of bisoprolol, 2.5 mg/d, and hydrochlorothiazide, 6.25 mg/d, reduced diastolic blood pressure to lower than 90 mm Hg in 61% of patients and demonstrated a safety profile that compared favorably with that of placebo. CONCLUSIONS: The utility of factorial design trials to characterize dose-response relationships and to test the potential interactions between various antihypertensive agents has been demonstrated. The combination of low dosages of bisoprolol and hydrochlorothiazide may be a rational alternative to conventional stepped-care therapy for the initial treatment of patients with mild to moderate hypertension.

Bisoprolol, a once-a-day beta-blocking agent for patients with mild to moderate hypertension.

The 24-h blood pressure control of bisoprolol, a new beta-selective, beta-blocking agent, was studied in 240 mild to moderate hypertensive patients in this 4-week, randomized, double-blind, placebo-controlled trial. A once-daily dosing schedule was evaluated by comparing bisoprolol's antihypertensive effectivness and safety at 24 h postdose and 3 h postdose, the latter time intended to correspond to peak effectiveness. Results from this trial demonstrated the antihypertensive effectiveness of once-daily bisprolol at doses ranging from 5-20 mg. Mean reductions from baseline diastolic blood pressure, measured 24 h postdose, were 6.3, 8.8, and 10.1 mmHg for patients receiving bisoprolol 5, 10, and 20 mg, respectively, compared with 1.6 mmHg for placebo-treated patients (p < 0.01); mean reductions from baseline systolic blood pressure for the bisoprolol groups were 8.6, 8.6, and 10.9 mmHg, respectively, versus 3.3 mmHg for placebo (p < or = 0.01); and mean reductions from baseline heart rate for the bisoprolol groups were 5.1, 7.1, and 10.2 beats/min, respectively, compared with a 0.9 beats/min increase in heart rate for the placebo group (p < 0.01). The response rates for bisoprolol-treated patients ranged from 47 to 70% compared with 18% for patients on placebo (p < 0.01). Antihypertensive effects were dose-related and sustained over the 24-h dosing interval. Near maximal antihypertensive effects were achieved within 1 week of initiation of therapy with bisoprolol and were sustained over the course of the trial.(ABSTRACT TRUNCATED AT 250 WORDS)

Double-blind evaluation of the dose-response relationship of amlodipine in essential hypertension.

To determine the dose-response efficacy of once-daily administration of placebo or a new long-acting calcium channel blocker amlodipine in patients with mild to moderate hypertension, a randomized, multicenter, placebo-controlled, double-blind trial was conducted. The study included 210 patients with diastolic hypertension (blood pressure 95 to 114 mm Hg) without major hematologic, renal, hepatic, cardiac, or endocrine abnormalities. After a 4-week single-blind placebo run-in period, patients were given placebo or amlodipine (1.25, 2.5, 5, or 10 mg) daily for 4 weeks. To assess the antihypertensive effect of amlodipine over a 24-hour period, blood pressure and pulse rate at weeks 0 and 4 were recorded for 12 hours after the dose and then again at 24 hours. At the end of the study patients treated with all doses of amlodipine greater than 1.25 mg daily had significantly reduced diastolic blood pressure in both supine and standing than 1.25 mg daily had significantly reduced diastolic blood pressure in both supine and standing positions. Amlodipine, 1.25 mg daily, was also associated with a decrease in standing diastolic blood pressure. Response to treatment was greater in all amlodipine-treated patients than in those receiving placebo. Pulse rate in both the supine and standing positions was not significantly affected by amlodipine. At doses of 2.5, 5.0, or 10.0 mg daily, amlodipine maintained blood pressure below values obtained with placebo throughout the 24-hour period. Treatment with amlodipine was well tolerated and the incidence of side effects was low.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term open evaluation of amlodipine vs hydrochlorothiazide in patients with essential hypertension.

The long-term efficacy and safety of amlodipine (2.5 to 10 mg) once daily was compared with that of hydrochlorothiazide (HCTZ) (25 to 100 mg) daily in 139 patients with mild-to-moderate hypertension. The study was a randomized, open-label, parallel comparison of 50 weeks' duration. Patients were randomized in a 2:1 ratio (amlodipine n = 92: HCTZ n = 47). Atenolol was added at week 12 if monotherapy was inadequate. At week 12, the mean reductions for supine and standing systolic and diastolic blood pressure values with amlodipine were found to be -15.2/-12.3 mmHg and -14.0/-11.6 mmHg respectively, as compared to -15.5/-11.1 mmHg and -16.1/-10.1 mmHg after treatment with HCTZ. The percentage of patients responding to treatment at week 12 was 74% on amlodipine and 70% on HCTZ. The addition of atenolol in those patients not adequately controlled on monotherapy produced additional mean reductions in supine and standing systolic and diastolic pressures in both the amlodipine-atenolol group and in the HCTZ-atenolol group. The incidence of adverse effects was 47% with amlodipine and 26% with HCTZ at 12 weeks. Overall, six patients were discontinued because of side effects while receiving amlodipine monotherapy and one from the HCTZ monotherapy group; none were discontinued because of side effects on combination therapy. Laboratory test abnormalities were reported by 16% of amlodipine-treated patients compared with 63% of patients on HCTZ. The antihypertensive effects of amlodipine and hydrochlorothiazide appeared to be comparable and were maintained during long-term therapy.

Antihypertensive and metabolic effects of concomitant administration of terazosin and methyclothiazide for the treatment of essential hypertension.

The efficacy and safety of once-daily 2.5- or 5.0-mg methyclothiazide (MCTZ) added to once-daily 5.0-mg terazosin (TRZ) versus 5.0-mg TRZ alone was evaluated in this double-blind, multicenter study. All patients received TRZ during a 6-week titration period. Hypertensive patients (222) (mean blood pressure of 159/104 mm Hg) were randomized to one of three treatment groups: TRZ alone (N = 76); TRZ+MCTZ-2.5 mg (N = 74); and TRZ+MCTZ-5.0 mg (N = 72) for the 8-week double-blind period. Changes in the supine and standing SBP/DBP from preTRZ period were: TRZ alone (-4.8/-8.1 and -2.6/-6.1 mm Hg); TRZ+MCTZ-2.5 mg (-17.3/-12.4 and -16.0/-11.2 mm Hg); and TRZ+MCTZ-5.0 mg (-20.6/-14.4 and -23.3/-14.6 mm Hg). Blood pressure changes in the combination groups were significantly greater than those in the TRZ alone group. However, there were no statistically significant differences between the TRZ+MCTZ-2.5-mg and TRZ+MCTZ-5.0-mg groups. The combination of TRZ and MCTZ tends to mitigate the adverse effects on serum glucose, uric, potassium and lipids usually associated with thiazide diuretics. Thus, combination treatment that begins with TRZ and adds MCTZ is effective in lowering blood pressure without any significant adverse metabolic effects.

Influence of beta 2 agonism and beta 1 and beta 2 antagonism on adverse effects and plasma lipoproteins: results of a multicenter comparison of dilevalol and metoprolol.

Dilevalol combines vasodilation due to selective beta 2 agonism and nonselective beta antagonism. We studied 311 patients randomized to dilevalol and 138 to metoprolol in a multicenter trial. After a 4-week placebo washout, dilevalol was titrated from 200 to 1,600 mg once daily and metoprolol from 100 to 400 mg to a goal supine diastolic blood pressure less than 90 and greater than or equal to 10 mm Hg decrease from baseline. Responders were followed for 1 year. The average age of patients was 51 years; 72% were men and 54% were white. Both drugs reduced blood pressure effectively to a similar level. Fewer patients discontinued dilevalol than did those taking metoprolol (9 vs 16%; p less than 0.03). More metoprolol-treated patients withdrew because of depression (6 vs less than 1%; p = 0.03) and impotence (5 vs less than 1%; p = 0.03). Lipoprotein levels before and after treatment were measured in 99 patients treated for 53.5 weeks with dilevalol (mean dose 438 mg). Dilevalol increased high-density lipoprotein (HDL) cholesterol by 2.5 mg/dl to 47.2 (p = 0.05), reduced low-density lipoprotein (LDL) cholesterol by 2.5 mg/dl, increased HDL/LDL by 0.03, and decreased total cholesterol/HDL cholesterol by 0.18. Triglycerides increased by 21 mg/dl (p = 0.06). In patients with an initial HDL cholesterol less than 35 mg/dl, dilevalol increased it by 9 mg/dl. In patients treated with metoprolol, the only significant change (p = 0.02) was a 41.9-mg/dl increase in triglyceride levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicardipine and propranolol in the treatment of essential hypertension.

Two hundred thirty-four patients with supine diastolic blood pressure of between 95 and 114 mm Hg were enrolled into a double-blind, randomized, parallel, multicenter trial. The patients were randomized to either nicardipine 30 mg tid, propranolol 40 mg tid, or nicardipine 30 mg tid and propranolol 40 mg tid for six weeks. Two hundred six patients yielded data for analyses. Of the 28 not included, seven had missing data, whereas the remaining 21 were excluded because they either failed to meet inclusion criteria or were noncompliant at endpoint. Both nicardipine and propranolol as monotherapies and in combination achieved statistically significant, (P less than .01), supine diastolic blood pressure reduction relative to baseline. The combination of nicardipine and propranolol showed a greater reduction in supine diastolic and systolic measurements than either of the monotherapies. Nicardipine produced greater blood pressure reductions one hour after dosing, whereas the propranolol treatment tended to produce slightly greater blood pressure decreases eight hours after dose. The combination always resulted in the greatest blood pressure reduction, independent of time after dose. Adverse experiences were reported by 26% of patients in the nicardipine-treated group, most often transient vasodilatory effects, by 17% of the propranolol-treated patients, and by 18% of the combination-treated group. This study demonstrated at the doses studied that nicardipine alone produced equivalent blood pressure reductions to those obtained by propranolol alone, but that the combination of these two drugs produced greater reductions in blood pressures than either of the monotherapies.

Similar efficacy of nitrendipine in young and elderly hypertensive patients.

Calcium channel blockers have been postulated to be more effective as monotherapeutic antihypertensive agents in the elderly than in younger patients. To determine if a new dihydropyridine derivative, nitrendipine, is more effective in the elderly (older than 60 years) than in younger hypertensive subjects (younger than 60 years), nitrendipine was administered in a multicentered study to 21 elderly and 33 younger subjects with essential hypertension. After gradual discontinuation of previous antihypertensive therapy and 2 weeks of placebo, the daily dose of nitrendipine (10 to 40 mg) was titrated over 3 weeks to achieve a 10 mm Hg decrease in supine diastolic blood pressure (BP) for patients entering with 90 to 99 mm Hg. For patients entering with at least 100 mm Hg, the dose was titrated to diastolic BP no greater than 90 mm Hg. Titrated dose of nitrendipine was maintained for 4 additional weeks. Propranolol was added for "symptomatic" tachycardia. Nitrendipine reduced BP in 90% of patients completing all phases of the study (n = 49). The proportion of responders was 47% among the elderly and 44% among young subjects. Change in heart rate was similar in both groups (-0.1 +/- 9.9 and +2.9 +/- 8.8 beats/min, mean +/- standard deviation). Two elderly and 1 younger subject required addition of propranolol (difference not significant). There was no correlation between the age of patients and changes in supine systolic and diastolic BP or heart rate (r = 0.21, -0.15 and -0.21, respectively). Adverse effects occurred with equal frequency in older and younger subjects (19 of 21 vs 23 of 33 patients, difference not significant).(ABSTRACT TRUNCATED AT 250 WORDS)

Hypokalemia in thiazide-treated systemic hypertension.

Potassium supplementation in diuretic-induced hypokalemia (serum potassium less than 3.5 mmol/liter) in patients being treated for hypertension is a common event. In a previous study 40 mmol/day of orally administered potassium was not effective in preventing diuretic-induced hypokalemia in patients who had previously developed hypokalemia while being treated for hypertension with hydrochlorothiazide. In the study reported here dosages as high as 60 to 80 mmol/day of orally administered potassium failed to prevent hypokalemia in 7 of 19 hypertensive patients who were receiving hydrochlorothiazide. Potassium supplementation was compared with the potassium-sparing diuretic amiloride. The study design was open label and subject matched with crossover of therapeutic regimens.

Pharmacodynamics of intravenous labetalol and follow-up therapy with oral labetalol.

The effectiveness, safety, and pharmacodynamics of repeated doses of intravenous labetalol for rapid reduction of severe hypertension and of subsequent oral labetalol dosing were studied. Twelve patients with severe hypertension were admitted to the hospital after the withholding of antihypertensive therapy for 2 to 14 days. Thirty minutes after an injection of vehicle only, labetalol, 0.25 mg/kg body weight, was injected and followed by repeat injections of 0.5 mg/kg every 15 minutes until the supine diastolic blood pressure (BP) was reduced to less than 90 mm Hg or a total of 3.25 mg/kg had been administered. Twenty-four hours after the last injection, oral labetalol was started at an initial dosage of 100 or 200 mg b.i.d., then increased every 2 days until the standing diastolic BP was less than 90 mm Hg or a maximum daily dosage of 2400 mg was reached. The initial injection achieved mean falls in supine systolic/diastolic BPs of 11/7 mm Hg. Subsequent injections produced additional falls in a dose-related fashion; the mean falls after the last injection (total cumulative dose 2.7 mg/kg) were 40/20 mm Hg. The effect lasted for 12 hours or more in most patients and tended to be biphasic, with one peak at approximately 5 minutes and another much less pronounced peak at about 4 hours. There was no evidence of precipitous falls in BP. All patients were able to ambulate 6 hours after the last injection without symptoms of postural hypotension. Oral labetalol effectively and safely restored and maintained the BP reductions achieved with intravenous labetalol.

Catecholamines in plasma and erythrocytes of rats with transplantable pheochromocytoma.

Accumulation of catecholamines in erythrocytes (RBC) was compared to rising plasma levels of catecholamines at weekly intervals following transplantation of pheochromocytoma (line P-259) in the New England Deaconess Hospital rat strain. Additionally changes were investigated during a 12 hour interval after tumor was established in PHEO rats. Starting 2 weeks after tumor implant, the concentrations of norepinephrine (NE) and dopamine (DA) in RBC paralleled and correlated strongly with rising levels of plasma NE and DA which were maximum by 4 weeks. Four to 6 weeks after implant, the RBC to plasma (L/P) concentration ratio of NE was 30% higher in PHEO rats than controls (p less than 0.05) indicating a shift in distribution of NE between the 2 circulating pools. Three measurements, 6 hours apart, showed that mean arterial pressure, plasma and RBC NE and DA concentrations were highest in AM in both PHEO and control groups. Shifts in DA were smaller and did not rise in PM as did NE suggesting DA may reflect tumor secretion and NE, tumor secretion plus sympathetic neuronal activity.

Effect of phosphate depletion on blood pressure and vascular reactivity to norepinephrine and angiotensin II in the rat.

Phosphate depletion (PD) adversely affects myocardial function but its influence on blood pressure is not well elucidated. In this study we evaluated whether PD influences blood pressure and/or affects its hormonal regulation or the peripheral vascular response to pressor agonists. Mean arterial pressure (MAP) in PD rats was lower than in normal animals, whereas heart rate was not significantly different between the two groups. Cardiac index (CI) in PD rats was lower and systemic vascular resistance (SVR) was higher than in controls. Plasma norepinephrine (NE) in the resting state and during the stress of immobilization was significantly greater in PD rats than in controls. Base-line plasma renin activity was also significantly higher in PD rats and increased similarly in the two groups of rats after administration of isoproterenol. Bolus injections of NE or angiotensin II produced a smaller rise in MAP in PD rats than in controls. Reduced responsiveness to NE was also demonstrated in isolated hind-limb preparation from PD rats. When NE was infused to achieve a rise in MAP of 30 mmHg, the dose required in PD was higher than in controls. Treatment with indomethacin did not affect the response in MAP to NE. The content and affinity of both alpha- and beta-receptors in PD hearts were not different from those of control hearts. The contents of PiATP, and AMP in the mesenteric vessels of PD rats were significantly lower than in control animals. These data show that PD leads to reductions in MAP, arteriolar response to pressor agents, and CI with appropriate compensatory rise in NE but with inadequate compensatory increase in SVR.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral potassium chloride and amiloride in hydrochlorothiazide-induced potassium loss.

To compare the effect of amiloride with that of oral potassium chloride (KCl) in hypokalemia, metabolic balance studies were carried out in hospitalized subjects with mild hypertension without edema who developed negative potassium balance after 4 days on hydrochlorothiazide (HCTZ). Subjects' diets contained measured amounts of sodium and potassium. While HCTZ treatment continued, oral preparations of either KCl solution or amiloride was added for 5 additional days. Potassium balance in the KCl-treated group further decreased by -44.9 +/- 32.3 mEq K+, while subjects on amiloride went into positive balance that averaged +51.7 +/- 24.1 mEq K+. Hypokalemia after HCTZ did not respond to KCl, while K+ levels rose from 3.32 +/- 0.22 to 3.67 +/- 0.26 mEq/l after amiloride.

Sympathetic activity and cardiac adrenergic receptors in one-kidney, one clip hypertension in rats.

The activity of the sympathetic nervous system, as measured by levels of plasma and cardiac catecholamines and catecholamine metabolites and the function of cardiac alpha- and beta-adrenergic receptors, was evaluated at 3 days and 4 weeks after induction of one-kidney, one clip hypertension (1K1C) in the rat. At 3 days, the plasma level of norepinephrine (NE) was lower in the 1K1C group than the control group (p less than 0.01), whereas epinephrine (E) and the metabolites dihydroxymandelic acid (DOMA), dihydroxyphenylglycol (DOPEG), and normetanephrine (NMN) were similar in both groups. In addition, cardiac content of catecholamines, their metabolites, and adrenergic receptors were similar in both groups. At 4 weeks, plasma levels of NE and DOPEG were lower (p less than 0.01), whereas levels of DOMA and NMN were higher (p less than 0.02 and p less than 0.001, respectively) in the 1K1C group than the control group. Cardiac content of NE (p less than 0.01), and DOPEG (p less than 0.05) was significantly lower, whereas DOMA and NMN were significantly higher (p less than 0.01) in the 1K1C group as compared to controls. In addition, cardiac density of both alpha- and beta-adrenergic receptors was reduced in the 1K1C group, whereas receptor affinities were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Circadian variation in plasma dopamine levels in man.

Nocturnal and daytime recumbent secretory patterns of norepinephrine, epinephrine and dopamine were determined at 30 min intervals in 9 normotensive males. Plasma levels of dopamine as well as those of norepinephrine and epinephrine followed a circadian pattern in these recumbent males. Plasma levels of dopamine were more closely related to clock time (r = -0.54, p less than 0.001) than either norepinephrine or epinephrine levels. As for norepinephrine and epinephrine, the sleep period in all 9 males was characterized by a paucity of dopamine secretory peaks as well as lower mean dopamine levels than during the awake state. There was a strong relationship between the circadian changes in plasma dopamine levels and those of norepinephrine (r = 0.92) and epinephrine (r = 0.78) throughout the 24-h period of recumbency. These interrelationships are consistent with a common regulatory mechanism governing the sleep/wake and/or rest/activity in plasma catecholamine levels.

Role of conjugation and red blood cells for inactivation of circulating catecholamines.

We investigated the extent of catecholamine (CA) conjugation in plasma and accumulation inside red blood cells (RBCs) after forced immobilization of the rat. A control blood sample was obtained from undisturbed rats resting in home cages via an indwelling aortic catheter. Then rats were immobilized for 2 h, and blood samples were taken during stress at 15 min and 2 h and at 30 min poststress. Both sulfate and glucuronide conjugates were deconjugated by enzymatic hydrolysis and measured by radioenzymatic thin-layer chromatographic methods. In plasma of resting rats, the major conjugate of norepinephrine (NE) is sulfate and that of epinephrine (E) and dopamine (DA) is glucuronide; total conjugates in plasma are 53, 57, and 97% of total NE, E, and DA, respectively. During stress, sulfate conjugates of NE, E, and DA increased significantly along with free NE, E, and DA. DA glucuronide unexpectedly declined at 2 h of stress and 30 min poststress. Concentrations of free CAs rose in RBCs during stress, as did NE sulfate, the only conjugate present in RBC lysate. We conclude that both CA conjugation and RBC accumulation are enhanced when large amounts of free CA enter the circulation as a result of immobilization stress and that the relative importance of these inactivation pathways is different for the three CAs.

Conjugated normetanephrine in human and rat plasma and erythrocytes.

Enzymatic hydrolysis was applied to the deconjugation of normetanephrine (NMN) in plasma and red blood cell lysate. By this procedure, in human plasma 77% of total NMN circulated in sulfate-conjugated form, while in rat plasma 63% was in glucuronidated form. Total NMN in human lysate was significantly higher than in plasma (P less than 0.001) and was mostly in the free form, indicating that red blood cells may play an important role in metabolism of norepinephrine. Enzymatic hydrolysis is superior to the standard method by acid hydrolysis plus heat since: (1) more conjugated NMN is hydrolyzed in human plasma and (2) a smaller sample is needed for hydrolysis.

Role of conjugation and red blood cells for inactivation of circulating normetanephrine.

Plasma and red blood cell (RBC) concentrations of normetanephrine (NMN), in both free and glucuronide-conjugated forms, were measured before, during, and after forced immobilization, an intense stressor of the sympathoadrenal system of rats. In this study NMN glucuronide was deconjugated by enzymatic hydrolysis; free and total NMN were assayed by radioenzymatic, thin-layer chromatographic procedures. In plasma, free NMN and NMN glucuronide are 777 +/- 99 and 792 +/- 74 pg/ml, respectively, when rats are at rest. Both free NMN and NMN glucuronide increased about 200% after 15 min of stress; in absolute amounts, increases were equivalent to that of the simultaneous increase in norepinephrine (NE). At 2 h of stress, NMN glucuronide, but not free NMN, increased further and significantly. The mean concentration of RBC-free NMN is about 50 times higher than that of plasma-free NMN, and it did not change significantly during stress; RBCs do not contain conjugated NMN. RBC NMN levels showed a strong correlation with RBC catechol methyltransferase activity. The latter seems to operate under conditions of substrate saturation; an acute release of NE leads to temporary storage of NE in RBCs but not conversion to NMN. The results indicate that conjugation of NMN with glucuronic acid is an important route for inactivation of plasma NMN formed during forced immobilization stress, whereas free NMN does not accumulate in RBCs during stress.