Dopamine-beta-hydroxylase Activity Research Articles

Thyroid hormone-catecholamine interrelationship during cold acclimation in rats. Compensatory role of catecholamine for altered thyroid states.

Effects of hyper- and hypothyroidism on catecholamine (CA) metabolism in the brain, adrenal glands, liver, and brown adipose tissue (BAT) were studied in adult rats during cold acclimation. Hypothyroidism was induced by the administration of propylthiouracil (PTU) and hyperthyroidism by the injection of thyroxine (T4). After 2 weeks of treatment, they were exposed to cold (5 degrees C) and sacrificed after 1 or 4 weeks. Although the body weight gain of PTU-treated rats were markedly impaired, the body temperature was maintained within normal range. They had increased cerebral dopamine, adrenal CA and BAT norepinephrine (NE) contents, enhanced cerebral tyrosine hydroxylase and adrenal dopamine beta-hydroxylase (DBH) activities and elevated [3H]dihydroalprenolol (DHA) binding to liver plasma membranes (P less than 0.01 vs controls). T4-treated rats showed an increased brain and adrenal CA only after cold exposure. The BAT NE content, DHA binding to liver plasma membranes, and [3H]guanosine diphosphate binding to BAT mitochondria were reduced by 30 to 50% from control values after 4 weeks of cold exposure. These results indicate that during cold acclimation, thyroid hormone deficiency is associated with an accelerated CA synthesis and release, which results in an enhanced BAT thermogenesis, and the hyperthyroid state suppresses CA release, hepatic DHA binding, and BAT heat production. Thus, there is a close metabolic interrelationship between thyroid hormone and CA during exposure to cold. CA appears to ameliorate thyroid hormone excess or deficiency.

Effects of melatonin on adrenomedullary dopamine-beta-hydroxylase activity in golden hamsters: evidence for pineal and dose dependencies.

Pineal influence in the control of adrenomedullary function in golden hamsters was investigated by examining changes in adrenal dopamine-beta-hydroxylase (DBH) activity following pinealectomy, either alone or in combination with melatonin administration. Adult males acclimated to an LD 14:10 photoperiod were distributed in five experimental groups: intact controls (NO), sham-pinealectomized (S), sham-pinealectomized with black plastic shielding of the pineal region, pinealectomized (PX), and pinealectomized with the operated region shielded. Animals representing all of these groups were injected (between L11 and L11.75) with either vehicle, or a low dose (25 micrograms) or a high dose (2,500 micrograms) of melatonin daily for 28 days, after which they were killed, and the adrenals were collected for assay of DBH activity by means of a sensitive radioenzymatic method. We found that PX + vehicle led to increased (P less than .05) adrenal DBH activity in comparison with either NO or S groups; daily 25 micrograms of melatonin resulted in lowered DBH activity in the NO group when compared with NO + vehicle (P less than .001) or S + vehicle (P less than .001) groups; PX + 25 micrograms melatonin reversed the action of 25 micrograms melatonin in the NO + 25 micrograms group; 2,500 micrograms melatonin was without effect on adrenal DBH in any of the injected surgical groups. These results show an inhibitory pineal influence on adrenal DBH activity, and that this was dose dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Myocardial Hypertrophy in Trophic Stimulation of Indices of Sympathetic Cardiac Innervation

Indices of cardiac sympathetic innervation have commonly been found depressed in the failing, hypertrophied heart. In contrast, we have recently demonstrated that hemodynamically compensated, very gradually developing right ventricular hypertrophy is associated with an increase in sympathetic nervous markers. The present experiments were performed to corroborate these findings in a model of acutely induced right ventricular hypertrophy, and to further characterize changes in markers of autonomic innervation associated with cardiac hypertrophy. Male guinea pigs underwent either pulmonary artery banding (P) with an acutely constricting ligature, or bilateral stellate ganglionectomy (S), or both (PS). Appropriate sham procedures were performed in animals subjected to only one intervention; controls (C) underwent sham-S and sham-P. Groups of animals were sacrificed at 10 and 20 days after surgery. Cardiac tissues were weighed and subsequently analyzed for activities of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH), two enzymes catalyzing the biosynthesis of catecholamines (CAs), and of choline acetyltransferase (CAT), a marker of parasympathetic activity, as well as for norepinephrine (NE). S resulted in profound depletions of cardiac NE of 88-92% and in significant decreases in the activities of DBH and TH. Marked right ventricular hypertrophy developed rapidly following P, and was not modified by S. Similar to our previous results, acute right ventricular hypertrophy was associated with moderate increases (10-20%) of sympathetic markers; following S, these increases (of presumably residual sympathetic innervation) were greatly enhanced, amounting to 171% and 105% for NE at 10 and 20 days, respectively. In contrast, sympathetic markers in the left ventricle of stellatectomized animals were not affected by P. Activity of CAT remained unaltered by the experimental interventions. Our experiments indicate that increases in markers of sympathetic innervation may be a common feature of the early, compensated stage of cardiac hypertrophy, regardless of its time course. Sympathetic neural mechanisms do not appear to play a stimulatory or trophic role in the hypertrophic process. Conversely, they seem to be secondary in nature, suggesting a possible stimulatory influence of hypertrophying myocardium on sympathetic cardiac nerves.

Dopamine-beta-hydroxylase (DBH) and homovanillic acid (HVA) in autistic children.

In the present study, plasma DBH activity and urinary HVA levels were measured in 19 autistic and 15 normal children. DBH activity was significantly elevated in the 8 less retarded autistic patients. In this subgroup, a negative correlation was found between plasma DBH and urinary HVA levels. These results support the hypothesis of a possible involvement of brain catecholamine dysfunction in the production of autistic symptoms.

A comprehensive study of chronic schizophrenic patients. II: Biological, neuropsychological, and clinical correlates of CT abnormality.

There are numerous reports of lateral cerebral ventricle enlargement on computed tomography (CT) in schizophrenics, but the significance and its relationship to traditional notions of organicity remain unclear. Therefore we studied a subgroup of chronic schizophrenics who had lateral ventriculomegaly (and also cortical hyperdensity) on a battery of relevant biological, neuro-psychological, and clinical parameters such as electroencephalogram (EEG), platelet monoamine oxidase (MAO) and serum dopamine-beta-hydroxylase (DBH) activity, the Halstead Reitan Neuropsychological Battery (HRB), premorbid personality adjustment, drug response, positive and negative symptoms, employment history, and family history. Our findings support the notion that there is an "organic" subgroup of schizophrenia that has 1) CT structural abnormalities such as lateral ventricle enlargement and cortical hyperdensity; and cerebral dysfunction or deficits as evidenced by 2) an increased incidence of abnormal EEGs and also 3) greater impairment on neuropsychological tests. The biochemical measures, platelet MAO and serum DBH activity, nor any of the clinical measures could differentiate between the subgroups. The implications of these findings for the subtyping of schizophrenia are discussed.

Differential roles of raphe nuclei in the regulation of dopamine beta-hydroxylase in the adrenal gland of the rat.

The effect of reserpine on the activity of dopamine beta-hydroxylase (DBH) in the adrenal gland of the rat was determined following electrolytic lesion of the dorsal raphe nucleus (DRN) or medial raphe nucleus (MRN). In sham-operated rats, as well as in those with a lesion of the DRN, there was no significant modification of the action of reserpine on this enzyme. However, a lesion of MRN potentiated the inducing action of the drug. A specific role of MRN in the serotonergic regulation of adrenal DBH is suggested by this work.

Plasma norepinephrine, epinephrine, and dopamine beta-hydroxylase activity during exercise in man.

Many experimental studies have utilized the activity of dopamine-beta-hydroxylase (DBH) as an index of sympathetic activity, since this enzyme is not submitted to uptake mechanisms or to enzymatic metabolism as are the circulating catecholamines norepinephrine (NE) and epinephrine (E). However, large discrepancies have been found between the results of these studies. The hypothesis which is examined in this study is that these discrepancies might arise from the different intensities in the stimuli utilized. In order to examine this possibility, plasma DBH activity was measured in seven subjects at rest and in three different conditions known to increase sympathetic activity to varying degrees, i.e., during hand-grip, standing, and supra-maximal bicycle exercise. Plasma NE and E concentrations were also measured during these stimuli. The results of this study show that DBH activity increases above resting levels only during supramaximal dynamic exercise, while plasma NE and E concentrations increase during each experimental condition. Furthermore, the increases in NE and E are related to each other and are also related to heart rate when all experimental conditions are considered. On the contrary, the variations in plasma DBH (expressed as a percentage of the resting value) are not related to other sympathetic indices or to heart rate. Since NE and E vary little from subject to subject at rest and increase discretely in response to the various stimuli, these plasma catecholamine concentrations therefore appear to represent the most accurate indices of sympathetic activity.

Serum dopamine beta-hydroxylase activity in menstrual migraine.

Migraine has been considered a manifestation of sympathetic dysfunction. Serum dopamine beta-hydroxylase (D beta H) has been shown to be an index of peripheral sympathetic activity by some workers and there are two reports of elevated activity of the enzyme during the migraine headache as well as in the headache-free interval. We studied the enzyme in seven women complaining of regular attacks of menstrual migraine and eleven controls during the mid-follicular (days 10 +/- 2) and premenstrual (days 28 +/- 2) phases of the menstrual cycle. Although levels were on average 26% and 10% higher respectively than in control subjects, the difference failed to reach statistical significance because of the large normal range for enzyme activity. However, the premenstrual results were significantly lower (p less than 0.001) than the mid-follicular measurements in the migraine group, little difference being found in controls. This finding, and the effects of successful therapy with anovulatory doses of oestradiol implants in not only significantly lowering serum D beta H but also significantly reducing the difference in enzymic activity between the early and late phases of the menstrual cycle, suggest that if this enzyme is an index of sympathetic activity, it is excessive fluctuations of the sympathetic nervous system that may be relevant in menstrual migraine.

The pH-dependent subunit dissociation and catalytic activity of bovine dopamine beta-hydroxylase.

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.

Study on the mechanism of the appearance of noise effects

This study was undertaken to investigate the dose-response relationship between the biological effect and noise exposure, and to consider the mechanism of the appearance of noise effects. Rats were exposed to noise at intensities of 60 dB (A), 80 dB (A) and 100 dB (A) for 240 min and examined for the change of activities of dopamine-beta-hydroxylase (DBH) in serum and adrenal glands. Plasma cyclic adenosine 3',5'-monophosphate (c-AMP) levels were also measured. Some rats were given 6-hydroxydopamine (6-OHDA) as a chemical sympathectomyzing agent 20 h before noise exposure in order to consider the mechanism of the appearance of noise effects. By noise exposure, serum DBH activity was significantly (P less than 0.01) increased at each intensity compared with the control group, but there were no remarkable changes in adrenal DBH activity. Plasma c-AMP level was also significantly elevated in response to the noise stress. When the rats, which had been pretreated with 6-OHDA, were exposed to noise with an intensity of 100 dB (A), the response of serum DBH activity was no longer observed. Therefore it is suggested that the effect due to noise exposure appears through the post-ganglionic sympathetic nerve fiber.

The response of monoamines in the rat brain to local vibration exposure

An experimental study was performed to investigate the effects of local vibration on the brain monoamines of rats. The rats' hind limbs were exposed to vertical sinusoidal vibration at frequencies of 20-960 Hz under constant acceleration of 50 m/S2 for 240 min. Rats were decapitated immediately after the exposure, the brains were quickly removed from the cranium and blood was collected in a heparinized beaker. The brain was divided into seven regions on an ice plate, and the changes of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in the whole brain or regional brains were examined. Furthermore, to investigate the mechanism of the appearance of peripheral effects induced by local vibration, the response of plasma dopamine-beta-hydroxylase (DBH) activity was observed with and without pretreatment by 6-hydroxydopamine (6-OHDA), known as a drug for chemical sympathectomy. The amines were determined by fluorometry and DBH activity was by radioimmunoassay. The results obtained were as follows: NE level in the whole brain showed a tendency to decrease compared with the controls at a frequency of 120 Hz and an acceleration of 50 m/S2. Levels of DA and 5-HT in the whole brain showed no particular changes at any frequencies used in the present study. In the study of regional brains, NE showed a tendency to decrease at a frequency of 60 Hz and a significant decrease at a frequency of 120 Hz in the hypothalamus. In the hippocampus, NE showed significant decreases at frequencies of 60 Hz, 120 Hz and 240 Hz, especially at 120 Hz. DA showed a tendency to decrease in the striatum at a frequency of 20 Hz and a significant increase at a frequency of 60 Hz in the medulla oblongata and pons. 5-HT showed a significant increase in the hypothalamus at frequencies of 20 Hz and 120 Hz. The changes in brain amines induced by local vibration were compared with those by whole body vibration. By exposure to local vibration at a frequency of 20 Hz and acceleration of 50 m/S2, the amines in the whole brain were not meaningfully affected, whereas in whole body vibration at the same frequency and acceleration significant effects were observed. NE level was decreased significantly to 57% of that of the control in whole body vibration (20 Hz, 50 m/S2) and showed a tendency to decrease to 79% of that of the control in local vibration (120 Hz, 50 m/S2). Thus the effect of whole body vibration was much greater than that by local vibration.(ABSTRACT TRUNCATED AT 400 WORDS)

Recovery of function in sympathetic nerves of interscapular brown adipose tissue of rats treated with 6-hydroxydopamine.

Twenty-four hours after subcutaneous administration of 6-hydroxydopamine (6-OHDA) at a dose of 10 mg/kg body weight in warm-acclimated rats, noradrenaline (NA) content and dopamine beta-hydroxylase (DBH) activity of interscapular brown adipose tissue (IBAT) were reduced to about 10 and 35% of control values. Corresponding values for cold-acclimated rats, similarly treated, were 12 and 32%. In both groups of animals, calorigenic function in IBAT assessed by measurement of the effect of cold exposure on rate of blood flow through the tissue was lost almost completely. Twenty-four hours after treatment of rats with various doses of 6-OHDA, calorigenesis in IBAT was directly related to residual NA. Measurements of noradrenaline content and DBH activity from 1 to 10 days after 6-OHDA and the increase in IBAT blood flow of cold-exposed animals from 1 to 7 days after 6-OHDA indicated marked differences in the extent to which each of these indicators of the integrity and function of sympathetic nerve endings recovered with time and with the acclimation temperature of the animals. Regeneration of macromolecular components of noradrenergic vesicles in IBAT appeared to occur more rapidly in cold than in warm-acclimated animals. Thus, moderate doses of 6-OHDA may acutely sympathectomize IBAT, but as previously reported for other tissues, full recovery of function of IBAT occurs long before noradrenaline stores are replenished.

Renal and systemic hemodynamics in sustained essential hypertension.

Cardiac output (CO), renal blood flow (RBF), calf blood flow (CBF), and hepatic blood flow (HBF), glomerular filtration rate (GFR), and dopamine beta hydroxylase (D beta H) activity were studied in 198 men (67 normotensive controls and 131 hypertensive patients) of the same age with sustained uncomplicated essential hypertension. In the hypertensive men, the RBF and the RBF/CO ratio were significantly decreased (p less than 0.001). The RBF and RBF/CO ratio were negatively correlated with age (p less than 0.01), blood pressure (p less than 0.01), and D beta H activity (p less than 0.01). None of these relationships were observed with CBF and HBF. The observed decreases in RBF and the RBF/CO ratio in hypertensive men were reversed after administration of clonidine and alpha-methyldopa (p less than 0.01), but not after administration of propranolol. The study provides evidence that the reduction of renal perfusion in essential hypertension is partly reversible and related to an abnormality in the adrenergic system control.

Relationship between phenolamines and catecholamines during rat brain embryonic development in vivo and in vitro.

p-Octopamine and phenylethanolamine are present in the embryonic rat brain earlier than catecholamines. These phenolamines are localized mainly in the hypothalamus, where the level of p-octopamine is very high. The parallel developmental study of the activities of dopamine beta-hydroxylase, 3,4-dihydroxyphenylalanine decarboxylase, tyrosine hydroxylase, and monoamine oxidase shows that phenolamines are present in significant amounts in the hypothalamus until tyrosine hydroxylase and monoamine oxidase become catalytically active. The culture of embryonic hypothalamus at different ages shows that no tyrosine hydroxylase and monoamine oxidase activities can be detected if the tissue is cultured before 15 days. This clearly indicates that all the enzymes related to catecholamine biosynthesis are not triggered at the same time during the development of the rat brain. These results are discussed on the basis of the physiological importance of phenolamines in mammals and of the use of the developing rat brain as a model for the study of the onset of the catecholaminergic system and the decline of the octopamine.

Orthograde, retrograde, and turnaround axonal transport of dopamine-beta-hydroxylase: response to axonal injury.

Reversal of the direction (turnaround) of orthograde axonal transport of dopamine-beta-hydroxylase (DBH) activity was studied at a ligature placed on rat sciatic nerve. DBH was allowed to accumulate at a ligature in vivo for selected intervals, at which time a second ligature was placed proximal to the first and turnaround transport measured just distal to the second tie after incubation in vivo or in vitro. Orthograde accumulation of DBH activity proximal to a ligature peaked at 2 days, and then rapidly decreased as a result of turnaround transport and injury-induced reduction of orthograde transport. Destruction of postganglionic sympathetic axon terminals in vivo with 6 hydroxydopamine resulted in a decrease in orthograde transport similar to that seen after axotomy and turnaround at or proximal to the site of chemical injury. Turnaround transport of DBH in vitro was blocked by incubation in the cold and in the presence of NaCN and vinblastine. Orthograde transport of DBH appeared to reverse direction within a few millimeters of a ligature.

Changes in tyrosine hydroxylase and dopamine-beta-hydroxylase activities but not in phenylethanolamine- N-methyltransferase activity within central adrenaline neurons after 6-hydroxydopamine administration

By using a new microdissection procedure allowing the noradrenaline (NA) and adrenaline (A) cell groups of the A2-C2 region to be sampled preferentially, it was possible to study the biochemical response of these two neuronal populations after 6-hydroxydopamine (6-OHDA) administration. Five days after an intraventricular 6-OHDA injection, tyrosine hydroxylase (TH) activity increased (+ 104%, P s 0.01) in the adrenergic C2 region, in the locus coeruleus (LC) and in the A1-C1 104 region, while the NA A2 region exhibited no significant increase. Twenty-one days after 6-OHDA administration, dopamine-beta-hydroxylase (DBH) activity had decreased in both the noradrenergic regions (LC, A1-C1 and A2 regions) and in the C2 adrenergic region. Conversely, phenylethanolamine- N-methyltransferase (PNMT) activity was not modified either in the cell bodies or in the terminals located in the tractus intermediolateralis of the spinal cord and in the hypothalamic nuclei. These data suggest: (i) that adrenaline-containing neurons could be sensitive to the neurotoxic action of 6-OHDA since they exhibit changes in TH and DBH activities; and (ii) that the determination of PNMT activity may not be sensitive enough to estimate the functional integrity of the A cell bodies or terminals.

Adrenomedullary catecholaminergic activity increases with age in male laboratory rodents.

In this report, we have examined the effects of aging on adrenomedullary catecholaminergic activity by evaluating the levels of activity of adrenal dopamine-beta-hydroxylase (DBH) in three species of laboratory rodents. Groups of male Fischer 344 rats (3-, 18-, and 28-month-old), Chinese hamsters (4- and 30-month-old), and Mongolian gerbils (3- and 26-month-old) were killed, and their adrenal DBH activities were measured by a sensitive radioenzymatic method. The results indicated that adrenomedullary DBH activities showed a progressive age-related increase in all three species investigated. Specifically, (a) adrenal DBH activity in the 18-month-old rats was significantly (p less than .001) higher than that of 3-month-old rats, and the enzyme activity in the 28-month-old rats was significantly (p less than .001) higher than that of the 18-month-old rats; (b) adrenal DBH activity in the 30-month-old hamsters showed a significant (p less than .001) increase over that found in the 4-month-old hamsters; and (c) DBH activity in the 26-month-old gerbils was significantly (p less than .05) higher than that noted in 3-month-old gerbils. These results suggest that an age-related increase in adrenal DBH activity may, at least in part, contribute to increased levels of circulating catecholamines.

Peripheral catecholamine enzyme function and cognitive impairment of elderly schizophrenics and controls.

Alterations of peripheral and central catecholamine systems are found in both schizophrenic and demented subjects. Reported here are the results of bivariate and multivariate analyses of cognitive function and peripheral catecholamine enzyme activities in a group of elderly schizophrenic patients and controls. The entire sample shows an inverse relationship of platelet monoamine oxidase (MAO) activity with cognitive impairment after controlling for the effects of age, race, and gender. Schizophrenic subjects also demonstrated an inverse relationship of plasma dopamine beta hydroxylase (DBH) activity with cognitive impairment. Demented subjects were characterized in both groups as having increments in platelet MAO activity. Demented schizophrenic subjects also were characterized by increments in plasma DBH activity. Results from this sample are discussed with respect to findings from other studies and future research.

Central and peripheral catecholamine synthesizing enzymes during the development of two-kidney, one clip hypertension in rats.

The activities of the catecholamine synthesizing enzymes have been determined in discrete brain areas and in peripheral tissues of rats, at different times after clipping the left renal artery (two-kidney Goldblatt hypertension, 2KGH) and in sham operated animals. Three days after clipping the only enzymatic change was a slight decrease in plasma dopamine-beta-hydroxylase (DBH) activity. Ten days after clipping no change in enzymatic activity was found at the central level. However, the DBH and the phenylethanolamine-N-methyltransferase (PNMT) activities were increased in the adrenal medulla (+49.0%, P less than 0.001 and +36.6%, P less than 0.001, respectively) and DBH activity was also increased in the superior cervical ganglia (+22.8%, P less than 0.01). These data suggest that sympathetic hyperactivity is present in 2KGH rats when hypertension is established. In addition, as this type of hypertension does not alter the PNMT activity in brainstem areas, it seems that the alterations in PNMT activity reported for genetically hypertensive rats are unlikely to be secondary to the elevated blood pressure.

Effects of collagenase on the release of [3H]-noradrenaline from bovine cultured adrenal chromaffin cells.

Bovine isolated adrenal chromaffin cells maintained in culture at 37 degrees C for 1-7 days become polygonal and bipolar, with typical varicosity-like extensions. Catecholamine levels and dopamine beta-hydroxylase activity decreased after 24-48 h of culture, but recovered to normal levels 3-7 days later. Incubation of 1-7 day-old cells in the presence of increasing concentrations of [3H]-noradrenaline (3.91 to 125 nM) resulted in the retention by the cells of amounts of radioactivity directly proportional to the amine present in the media. One day-old cells took up and retained only one third of the radioactivity found in 2-7 day-old cells. The addition of collagenase to cultured cells caused a decrease in the uptake of tritium. However, the enzyme treatment did not affect the amine taken up by the cell before collagenase treatment. Release of tritium from cultured cells evoked by nicotine, acetylcholine (ACh) or 59 mM K+ was very poor in 24 h-old cells; the secretory response to nicotine, ACh or K+ was dramatically increased after 2-7 days of culture. Bethanecol did not cause any secretory response. When treated with collagenase, cultured cells which had recovered fully their secretory response, lost again the ability to release tritium evoked by ACh or nicotine. However, the responses to high K+, veratridine or ionophore X537A were not affected. The nicotinic response was recovered two days after collagenase treatment. The data suggest that the use of collagenase to disperse the adrenomedullary tissue during the isolation procedure might be responsible for the lost secretory response of young cultured chromaffin cells. Since collagenase specifically impairs the nicotinic cholinoceptor-mediated catecholamine release, it seems likely that the enzyme is exerting its action on the ACh receptor complex. It is unlikely that either voltage-sensitive Na+ or Ca2+ channels are affected by collagenase as the responses induced by high K+ or veratridine were unaffected by this enzyme.