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Transsynaptic and humoral regulation of adrenal catecholamine synthesis in stress
zc
Frontiers in Carecholmnine Research
We have extended this assay to include the measurement of endogenous epinephrine (E). Although NE and E are not resolved by the GC, it is possible to separate them by m~suriag specific fragments (m/e). NE~FP has its base peak at m/e 176, while the base peak for E-PFP occurs at m/e 190. NE-PFP does not have a fragment at m/e 190 and similarly E-PFP does not have a fragment at m/e 176. By applying the technique of MID, both E aad NE can be measured in a single analysis .
PFP Derivatives
GC Retention Time (Sec)
Fragment recorded For quaatitation (m/e)
a-MNE NE
125 170
190 176
E
170
190
a-MDA DA
262 350
442 428
Fragments recorded for Multiple ion detection m/e % Ratio 577 549 190 604 428 415
12 7 100 2.5 .100 5
_ 1 .7 40 20
We have used this technique to measure extremely small but meaningful concentrations of catecholamines . For example, DA pool in 14 mg of rat heart v_entriçles ._29t .009 nanomoles/g (Costa et al ., Pharmacological Reviews, 24 : 167- 190, 1972) and NE and DA in Locus Coeruleus (50-75 /lg, fresh wt.). 20 f 1.6 pmole NE and 3.2 t 0.25 pmole DA . The progress in applying this technique to the measurement of catecholamines in body fluids will be reported .
TRANSSYNAPTIC AND HUMORAL REGULATION O.F ADRENAL ~CATECHOLAMINE SYNTHESIS IN STRESS Richard Kvetâaaskß lastitute of Ezperimental Endocrinology, Slovak Academy of Sciences, Bratislava, Czechoslovakia Although as activation of sympathetic pathways during stress was already intimated by Cannon, aad Seley, in the fifties, considered catecholamiaes to be responsible for the alarm phase of the stress reaction, nevertheless, the role of catecholamines as well as the extent of their participation in adaptive reactions after repeated stress have not as yet been fully elucidated .
Frontiers in Cateehofamine Research
xci
In our experiments immobilization /IMO/ was used as the model of stress and rats were daily restrained for 150 minutes. After the first IMO a decrease in the content of adrenal epinephrine /EPI/ was observed together with as increase is the level of plasma catecholamines /CA/ and urinary excretioa /mainly EPI/. Immobilization, repeated about 40 times, produced a morphologically-confirmed increase in the activity of the adrenal medulla which was associated with a return of the adrenal EPI to control or even higher levels and with a manifold increase of urinary EPI excretion. These results are interpreted in terms of as increased biosynthesis of adrenal CA found is repeatedly IMO animals, measured in uivo after the administration of precursors-"Gtyrosiae sad 'H-dope. This technique has enabled us to ascertain that the biosynthesis of CA is accelerated mainly in the steps: tyrosine - dope and dopamine aorepinephrine . Therefore, we measured the in vitro activity of enzymes catalysing the biosynthesis of CA in these critical steps. The activity of enzymes tyrosine hydroaylase /TH/, dopamine-beta-hydroxylase /DBH/ and also phenylethanolamiae-N-methyl transferese /PNMT/ proved to be substantially increased in the adrenals of repeatedly immobilized rats . After cessation of immobilization intervals TH activity returned toward preimmobilization levels with a half-life of about 3 days . These enzyme changes produced by repeated stress represent the first evidence of a physiologically-induced elevation of adrenal TH and DBH activity . How are the biosynthesis of adrenal CA and catecholamine-synthesizing enzymes regulated in stressed animals? Repeated IMO of rats with deaervation of the left adrenal gland / severing of the splanchnic nerve/, produced no significant alteration of TH and DBH activities in the denervated adrenal, but resulted in an elevation of PNMT activity in both intact and deaervated adrenals, although the level in the denervated gland was slightly lower. Denervation did not essentially influence adrenal CA'in control animals, while in stressed animals no decrease of adrenal EPI was seen is this denervated gland. The immobilization-induced drop of adrenal EPI level was blocked also by heaamethonium administration . From these results it appears that an intact nerve supply is necessary for an increase is the enzyme activity in response to immobilization stress . Which areas of CNS may be involved in producing these adrenal medullary changes after stress? Repeated IMO of animals with septal lesions to the limbic system caused as additional decrease in adrenal EPI levels and an additional increase in the activity of adrenal TH . Amygdaloid lesions provoked and increased activity of both the enzymes TH and DHH. So, lesions of both septal and amygdaloid areas of the limbic system potentiate a stress-induced increase in the activity of CA-synthesizing enzymes is the adrenal medulla. The endocrine status also markedly influences stress-induced alterations
xcü
Frontiers in Catecholamine Research
in the levels of adrenal CA and CA-synthesizing enzymes. In hypophysectomized /hypox ./ animals adrenal EPI levels are below normal values and are further depressed by repeated IIVIO. In such repeatedly immobilized hypophysectomized rats the total plasma catecholamine levels are also significantly decreased. The activities of adrenal TH, DBH and PNMT decreased following hypophysectomy. After repeated IMO of hypos . rats, TH and DHH activities significantly increased but failed to reach control levels, while PNMT activity remained considerably depressed. When ACTH was administered to hypox. rats before each IMO period adrenal EPI was less depleted and TH, DBH and PNMT activities approached those found is sham-operated IMO rats . When dexamethasone was given prior to IMO there was again less depletion of adrenal EPI, an increase in PMT activity, but no change in TH activity. Adrenal denervation of hypox. rats completely blocked the inuaobilizationinduced increase in TH and DBH activity . ACTH administered to hypox. rats before each IMO, induced a marked increase in TH activity in the intact adrenal and a small, though significant increase of TH is the denervated gland. DBH activity in the intact adrenal was also increased but there was no rise of this enzyme in the denervated adrenal. PNMT activity increased in both intact and denervated adrenals of hypox. rats given ACTH prior to IMO. Thus, increases is TH activity induced by IMO stress require both intact neural and pituitary-adrenal systems. Adrenal DBH activity is also under both neural and humoral control, but the neural regulation appears to be the more important. PNMT activity likewise appears to be controlled by both the systems, but here the pituitary-adreaocortical system is the more important. Our results with dexamethasone or dibutyryl cyclio-AMP administration support the view that TH activity does not appear to be influenced by glucocortiooids . To answer the question whether or not the adrenal TH is regulated by a direct affect of ACTH on the adrenal medullary cells, or by some unknown factor released from the adrenal cortex, studies have been initiated in this laboratory designed to clarify these regulatory mechanisms on isolated cells of bovine adrenal medulla. Ia summary, animals subjected to repeated immobilization stress have shown an increase of adrenal medullary catecholamine biosynthesis influenced by both transsynaptic and humoral /pituitary-adrenal/ regulation .
Frontiers in Carecholmnine Research
We have extended this assay to include the measurement of endogenous epinephrine (E). Although NE and E are not resolved by the GC, it is possible to separate them by m~suriag specific fragments (m/e). NE~FP has its base peak at m/e 176, while the base peak for E-PFP occurs at m/e 190. NE-PFP does not have a fragment at m/e 190 and similarly E-PFP does not have a fragment at m/e 176. By applying the technique of MID, both E aad NE can be measured in a single analysis .
PFP Derivatives
GC Retention Time (Sec)
Fragment recorded For quaatitation (m/e)
a-MNE NE
125 170
190 176
E
170
190
a-MDA DA
262 350
442 428
Fragments recorded for Multiple ion detection m/e % Ratio 577 549 190 604 428 415
12 7 100 2.5 .100 5
_ 1 .7 40 20
We have used this technique to measure extremely small but meaningful concentrations of catecholamines . For example, DA pool in 14 mg of rat heart v_entriçles ._29t .009 nanomoles/g (Costa et al ., Pharmacological Reviews, 24 : 167- 190, 1972) and NE and DA in Locus Coeruleus (50-75 /lg, fresh wt.). 20 f 1.6 pmole NE and 3.2 t 0.25 pmole DA . The progress in applying this technique to the measurement of catecholamines in body fluids will be reported .
TRANSSYNAPTIC AND HUMORAL REGULATION O.F ADRENAL ~CATECHOLAMINE SYNTHESIS IN STRESS Richard Kvetâaaskß lastitute of Ezperimental Endocrinology, Slovak Academy of Sciences, Bratislava, Czechoslovakia Although as activation of sympathetic pathways during stress was already intimated by Cannon, aad Seley, in the fifties, considered catecholamiaes to be responsible for the alarm phase of the stress reaction, nevertheless, the role of catecholamines as well as the extent of their participation in adaptive reactions after repeated stress have not as yet been fully elucidated .
Frontiers in Cateehofamine Research
xci
In our experiments immobilization /IMO/ was used as the model of stress and rats were daily restrained for 150 minutes. After the first IMO a decrease in the content of adrenal epinephrine /EPI/ was observed together with as increase is the level of plasma catecholamines /CA/ and urinary excretioa /mainly EPI/. Immobilization, repeated about 40 times, produced a morphologically-confirmed increase in the activity of the adrenal medulla which was associated with a return of the adrenal EPI to control or even higher levels and with a manifold increase of urinary EPI excretion. These results are interpreted in terms of as increased biosynthesis of adrenal CA found is repeatedly IMO animals, measured in uivo after the administration of precursors-"Gtyrosiae sad 'H-dope. This technique has enabled us to ascertain that the biosynthesis of CA is accelerated mainly in the steps: tyrosine - dope and dopamine aorepinephrine . Therefore, we measured the in vitro activity of enzymes catalysing the biosynthesis of CA in these critical steps. The activity of enzymes tyrosine hydroaylase /TH/, dopamine-beta-hydroxylase /DBH/ and also phenylethanolamiae-N-methyl transferese /PNMT/ proved to be substantially increased in the adrenals of repeatedly immobilized rats . After cessation of immobilization intervals TH activity returned toward preimmobilization levels with a half-life of about 3 days . These enzyme changes produced by repeated stress represent the first evidence of a physiologically-induced elevation of adrenal TH and DBH activity . How are the biosynthesis of adrenal CA and catecholamine-synthesizing enzymes regulated in stressed animals? Repeated IMO of rats with deaervation of the left adrenal gland / severing of the splanchnic nerve/, produced no significant alteration of TH and DBH activities in the denervated adrenal, but resulted in an elevation of PNMT activity in both intact and deaervated adrenals, although the level in the denervated gland was slightly lower. Denervation did not essentially influence adrenal CA'in control animals, while in stressed animals no decrease of adrenal EPI was seen is this denervated gland. The immobilization-induced drop of adrenal EPI level was blocked also by heaamethonium administration . From these results it appears that an intact nerve supply is necessary for an increase is the enzyme activity in response to immobilization stress . Which areas of CNS may be involved in producing these adrenal medullary changes after stress? Repeated IMO of animals with septal lesions to the limbic system caused as additional decrease in adrenal EPI levels and an additional increase in the activity of adrenal TH . Amygdaloid lesions provoked and increased activity of both the enzymes TH and DHH. So, lesions of both septal and amygdaloid areas of the limbic system potentiate a stress-induced increase in the activity of CA-synthesizing enzymes is the adrenal medulla. The endocrine status also markedly influences stress-induced alterations
xcü
Frontiers in Catecholamine Research
in the levels of adrenal CA and CA-synthesizing enzymes. In hypophysectomized /hypox ./ animals adrenal EPI levels are below normal values and are further depressed by repeated IIVIO. In such repeatedly immobilized hypophysectomized rats the total plasma catecholamine levels are also significantly decreased. The activities of adrenal TH, DBH and PNMT decreased following hypophysectomy. After repeated IMO of hypos . rats, TH and DHH activities significantly increased but failed to reach control levels, while PNMT activity remained considerably depressed. When ACTH was administered to hypox. rats before each IMO period adrenal EPI was less depleted and TH, DBH and PNMT activities approached those found is sham-operated IMO rats . When dexamethasone was given prior to IMO there was again less depletion of adrenal EPI, an increase in PMT activity, but no change in TH activity. Adrenal denervation of hypox. rats completely blocked the inuaobilizationinduced increase in TH and DBH activity . ACTH administered to hypox. rats before each IMO, induced a marked increase in TH activity in the intact adrenal and a small, though significant increase of TH is the denervated gland. DBH activity in the intact adrenal was also increased but there was no rise of this enzyme in the denervated adrenal. PNMT activity increased in both intact and denervated adrenals of hypox. rats given ACTH prior to IMO. Thus, increases is TH activity induced by IMO stress require both intact neural and pituitary-adrenal systems. Adrenal DBH activity is also under both neural and humoral control, but the neural regulation appears to be the more important. PNMT activity likewise appears to be controlled by both the systems, but here the pituitary-adreaocortical system is the more important. Our results with dexamethasone or dibutyryl cyclio-AMP administration support the view that TH activity does not appear to be influenced by glucocortiooids . To answer the question whether or not the adrenal TH is regulated by a direct affect of ACTH on the adrenal medullary cells, or by some unknown factor released from the adrenal cortex, studies have been initiated in this laboratory designed to clarify these regulatory mechanisms on isolated cells of bovine adrenal medulla. Ia summary, animals subjected to repeated immobilization stress have shown an increase of adrenal medullary catecholamine biosynthesis influenced by both transsynaptic and humoral /pituitary-adrenal/ regulation .