Red blood cell catecholamine levels in normotensive and DOCA-salt hypertensive rats

Under basal conditions in anesthetized rats, significant concentrations of free norepinephrine (NE), epinephrine (E), and dopamine (DA) were detected in red blood cell (RBC) lysate. These concentrations were not proportional to their respective plasma concentrations and thus RBC-to-plasma concentration ratios were different for each catecholamine (CA). DA was by far the most concentrated amine inside the RBC. An acute increase in plasma NE and E levels, induced by hemorrhagic hypotension in normotensive (NT) rats, did not result in any modification of the RBC CA content. However, chronic elevation of the NE plasma levels in bilaterally adrenalectomized rats and in deoxycorticosterone acetate (DOCA)-salt hypertensive rats (DOCA-salt HT) were associated with increased NE levels in the RBC. In addition, the large elevation in plasma E concentrations following hemorrhagic hypotension in DOCA-salt HT rats, as well as the greater plasma NE response to hypotension in adrenalectomized animals, were accompanied by increases in the respective RBC amine concentrations. During a steady-state intravenous infusion of tritiated NE, we observed a slow accumulation of radioactivity inside the RBC, indicating that CA can enter the RBC from the plasma. Moreover, catechol methyltransferase activity was measured in the cytosolic fraction of the RBC of both NT and DOCA-salt HT rats suggesting that, once inside the RBC, the catecholamines can be metabolized.

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.

Regulation of norepinephrine levels in synaptic clefts of dog vascular tissue

The overflow of endogenous norepinephrine (NE) from superfused dog pulmonary artery and saphenous vein in response to electrical stimulation and the content of NE remaining in the tissue after stimulation were measured using liquid chromatography with electrochemical detection. Overflow of NE was 0.15-0.22 and 0.14 to 0.30 ng/min in pulmonary artery and saphenous vein, respectively, during stimulation. Release of NE (measured in the presence of cocaine and corticosterone to block removal of NE from the synaptic cleft) was greater in the pulmonary artery (0.50-0.87 ng/min) than in saphenous vein (0.39-0.51 ng/min). NE overflow decreased after the first 4 min of continuous stimulation in saphenous vein but not in pulmonary artery. As only 2.26% of the NE content of vein overflowed during stimulation, depletion of NE or deterioration of the preparation seemed unlikely. Presynaptic alpha- or beta-receptor blockade did not change the pattern of NE overflow. Thus in the saphenous vein, but not in the pulmonary artery, removal of NE from the synaptic cleft appears to be facilitated during electric stimulation. This suggests the existence of a new mechanism, possibly related to enhanced catechol methyltransferase activity, which regulates NE levels in the synaptic cleft in saphenous vein but not in pulmonary artery.

Immunological Studies of Catechol Methyltransferase from the Yeast Candida tropicalis

Immunization of rabbits with purified catechol methyltransferase from Candida tropicalis yielded a potent antiserum. Ouchterlony double diffusion analysis showed a single precipitin line. There was no cross reactivity with the catechol methyltransferase from rat and bovine liver. Specific antigen-antibody interaction was demonstrated by a potent inhibitory effect of the antibody on the yeast enzyme. Immunological titration and quantitative precipitin reaction of the enzyme showed that the maximum amount of precipitable complex occurred at the equivalence point where enzyme activity was completely inhibited.