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Angiotensin II stimulates changes in the norepinephrine content of primary cultures of rat brain
Nenroscience Letters, 36 (1983) 305-309
305
Elsevier Scientific Publishers Ireland Ltd.
ANGIOTENSIN H STIMULATES CHANGES IN THE NOREPINEPHRINE CONTENT OF P ~ R Y CULTURES OF RAT BRAIN
COLIN SUMNERS, M. IAN PHILLIPS and MOHAN K. RAIZADA
Depariment o f Physiology, University o f Florida, College o f Medicine, Gainesville, FL 32610 (U.S.A.) (Received December 22rid, 1982; Accepted February 18th, 1983)
Key words: angiotensin I, - norepinephrine - primary brain cultures - saralasin
Interactions between norepiuephrine and angiotensin I! were investigated in neuron-enriched primary brain cell cultures, which have been demonstrated to contain catecholamines, angiotensin II-like immunoreactivity and specific receptors for angiotensin !1. Angiotensin I! (7.5 and 15.0 pg/ml) caused significant increases in both neuronal and growth media norepinephrine levels, which were inhibited by saralasin. These observations suggest that angiotensin acts at its specific receptors tO alter neuronal
norepinephriue levds.
The octapeptide angiotensin !I (Ang !!) is both pressor and dipsogenic following central injection into conscious rats [2, 13], and the pressor effect is thought to be due to both sympathetic activation and vasopressin release [3, 14]. Recent studies indicate that pressor doses of centrally injected Ang II or renin caused increased catecholaminergic activity in specific brainstem and hypothalamic nuclei, in viva [4, 15]. The mechanism of interaction between Ang !1 and norepinephrine (NE) in the brain is not clearly understood, and to study it we have examined Ang il-catecholamine (CA) interactions using primary cultures of brain cells. This approach avoids some of the complications of non-specific influences on CAs that may confuse the picture in an in viva situation. Levels of NE and dopamine (DA) have been quantified in 21-day-old primary brain cultures, and the effects of Ang il upon endogenous NE and DA levels in the brain cells and growth media have been examined. Primary cultures from l-day-old whole rat brains were prepared essentially as described previously [11, 16]. They were plated in poly-L-lysine-coatcd 100 m m Falcon tissue culture dishes and grown in Dulbecco's modified Eagles medium (DMEM) containing 10~0 fetal bovine serum (FBS) in a humidified incubator with 5~o C0z/95~o air for 3 days. Cultures were then treated with 10 pM cytosine arabinoside (ARC) prepared in DMEM containing 10% horse serum and 5070FBS, for 2 days. This treatment results in inhibition of cell multiplications, the majority of which are glial in origin, and causes neuron enrichment of cultures. The cultures 0304-3940t83/0000-0000/$ 03.00 © 1983 Elsevier Scientific Publishers IreMnd Ltd.
~6
were grown for a further 16 days in $ ml DMEM containing 10% horse serum prior to experiments. Cultured neurons were treated with various doses of either c~methybp-tyrosine (a-MT), Ang !I (both Sigma) or Ang II + saralasin (Calbio-
h. Ang !! ~ven ~ 7 : 5 / g / m l growth medium w o ~ d ~ ~ e r e d as 6 inoculations each of 1.25 ~g/ml, Following this treatment cells and growth media were removed from the dishes and analyzed for NIlEand DA by a sensitive radioe~ymati¢ method [12[ and for protein by the method of Lowry et al. [7]. The radioenzymatic assay for CAs utilizes [3H-CH3]S-adenosyl-g-methionine (Amersham Inc., specific activity 64 Ci/mmol} and catecho143-methyltransferase to convert NE and DA to their 3-O-methylated metabolites. Data were analyzed by a one-way analysis of variance (ANOVA). The significance of the difference between means was determined by using the pooled variance from the a~,alysis of variance and the t-test. The neuron-enriched cultJres contained approxiw,~ately 70-80% neuronal cells, as evidenced by immunocytochemical studies with neuronalspecific enolase, and electron microscopic analysis [il]. The remainder of the cells are non-nenronal in origin, based on immunocytochemical analysis [10]. Using the radioenzymatic technique it was determined that the neuronal enriched cultures contained !102 + .~7 ( n = 7 ) p g NE/mg protein, and 858 ± 27 (n -- 7) pg DA/mg protein. Treatment with the CA synthesis inhibitor ,x-MT (1.25 ~g/ml growth medium × 6. over 24 h) significantly lowered (P<0.001) both neuronal NE and DA to 480 ± 72 (n = 6) pg NE/mg protein and 351 ± 25 (n = 6) pg DA/mg protein, suggesting that these CAs are synthesized within the cells. The NE level of the growth medium withdrawn from cultures (240 + 29 (n -~ 6) pg NE/ml) was not altered by incubation with oL-MT (211 ± 25 (n = 6) pg NE/ml), indicating that the media had no ability to synthesize CA. To examine whether Ang il alters the endogenous neuronal NE or DA contents of these cells, cultures were incubated with the peptide and then analyzed for NE and DA. At 0.75 #g/mi growth medium Ang II caused a significant fall in neuronal NE ( - 26.7%, P<0.Oi) but not DA content. This dose of peptide did not affect growth media NE levels (Table !). At 7.5 /tg/ml and 15.0/~g/ml Ang !1 both neuronal and growth media NE levels were significantly increased (+ 23.8% and + 37.5°'/o respectively, P<0.002), while neuronal DA was only affected at the higher dose of the peptide (Table !). The effects of Ang Ii given at 0.75 and 7.5/~g/ml were completely inhibited by saralasin given concomitantly with Ang 11 in doses of 7.5 and 15.0 ~g/mi, respectively (Table I). The time course of the Ang ii alteration in NE levels of cultured brain neurons was studied by seeing the effect of a single dose of Ang ii (7.5/~g/ml) given at different times over 24 h. Two hours after Ang 11 (7.5/tg/ml) there was a significant decrease ( - 21.6%) in neuronal NE levels, and after 4 h an increase in both neuronal ( + 78.3%) and media (+ 64.0%) NE contents (Table !I) was observed. An increase
307 TABLE i EFFECTS OF ANGIOTEN$1N il UPON CA LEVELS IN NEURON ENRICHED PRIMARY BRAIN CULTURES AND THE NE CONTENT OF GROWTH MEDIA Values arc mean ± S.E.M. Figures in parentheses are numbers of observations. *P<0.OI; **P<0.02; **°P
Control Aug !1 (0.75 ~,/ml) Aug !! (7.5 tzg/ml)
Aug It (15.0 ~g/ad) Ans + An8 +
II (0.75 ~g/ml) saralasin (7.5 ps/ml) !! (7.5 ~s/ml) saralasin (15.0 ~,g/ml)
ANOVA I
Neuronal NE (pg/mg protein)
Media HE (pg/ml)
Neuronal (pg/mg protein)
1102 808 1365 1516 1172
240 263 362 399 193
452 ± 432 ± 399 ± 564 ± 390___
± ± ± ± ±
57 (7) 30 (6)* 104 (6)* 90 (6)** 67 (4)
± ± ± ± +
29 (6) 19 (6) 40 (6)** 31 (6)** 31 (4)
27 (7) 23 (6) $5 (6) 48 (6)*** 47 (4)
1037 + 62 (4)
201 _+ 52 (4)
406 ~ 38 (4)
<:0.01
<:O.OI
in neuronal NE was still apparent after 12 h ( + 26.8V0), but had returned to control levels at 20 and 24 h. These results suggest that Ang 11 stimulates changes in the NE content of cultured neurons and growth media by acting on specific receptors. These findings are important for two reasons. Firstly, because brain CAs are involved in the pressor response caused by centrally injected Ang !I (15), and also central injections of this peptide or renin elicit change in NE turnover of specific brain regions [4, 15]. Secondly, because the results agree with in vivo and in vitro brain slice experiments which have shown that Ang I! stimulates increases in NE levels in cerebrospinal fluid [I], causes TABLE !i TIME COURSE FOR THE EFFECTS OF ANGIOTENSIN II UPON NE CONTENT OF NEURON ENRICHED CULTURES AND GROWTH MEDIA Values are mean ± S.E.M. Fisures in parentheses are numbers of observations. *P
Growth media NE (pg/ml)
Control Ang II (7.5 #g/ml) 2h 4 h 12 h 20 h 24h
1012 + 46 (4)
250 ± 33 (4)
795 1805 1164 1053 958
288 410 317 339 274
ANOVA!
~0.01
± ± ± ± ±
39 (4)* 63 (4)* 34 (4)** 29 (4) 52(4)
+_ 31 (4) ± 37 (4) ± 27 (4) ± 39 (4) + 24(4)
~0.01
308
release of [3HINE from hypothalamic slices [51, and possibly inhibits neuronal
renptake of lqE [81. Based on our results, neuron enriched cultures may provide a novel method for
li-like ~munoreactivity [17]; they also contain specific uptake and release mechanisms for CA, as evidenced by a number of recent studies (e.g. refs. 6 and 9). in the present experiments we have shown that CAs are synthesized within the neuron, since a-MT lowers neuronal NE and DA. Growth media CA are not altered by a-MT, demonstrating that the media does not have the ability to synthesize CA, and so it is likely that only a small amount of the neuronal .ME and DA is media derived. Saralasin abolished both the changes in neuronal and growth media HIE content caused by Ang !! (Table !), indicating that the peptide was acting at its specific receptors. The effective dose of saralasin is lower than that needed in vi,~o, probably since in the culture system this antagonist comes into immediate contact with the neurons. The results shown here with Ang !1 given as 6 inoculations over 24 h could be obtained following a single dose of Ang !! (Table !1). Twenty-four hours after a single dose of Ang II there was no change in neuronal NE levels (Table !1), which validated incubating cultures with large amounts of peptide in these studies. The incubation times and the large doses of Ang !1 used in our experiments were for two reasons. First, in experiments where endogenous CA are being measured, any alteration in cellular CA content may be neutralized by neurotransmitter resynthesis, leading to no detectable change in NE or DA levels. Hence, it was considered necessary to have a constant amount of Ang Ii present in the culture medium to cause continuous stimulation (or inhibition) of the CA rich cells, and possibly a detectable change in NE or DA levels. Second, Ang II is degraded quickly under these culture conditions (Raizada, unpublished data), and so large doses were added to the medium periodically over 24 h to keep the peptide concentration at a high level. The mechanism of Ang il-stimulated changes in neuron-enriched culture and growth media NE levels is uncertain at this time. The result may imply that Ang 11 is stimulating an increase in NE synthesis and relea~se. However, further experimentation is required to determine whether this is the case, or whether Ang !i is affecting CA metabolic enzymes (e.g. monoamine oxidase) or CA reuptake. This research was supported by grants from NIH (R-I-HL-27334A), NSF (BNS-8025969) to M.I.P. and American Heart Association to M.K.R. I Chcvillard, C., Duchene, N., Pasquier, R. and Alexandre, J.M., Relation of the centrally evoked pressor eft~t of angiotensin !! to central noradrenaline in rabbit, Europ. J. Pharmacol., 58 (1979) 203-206.
309 2 Epstein, A.N., Fitzsimons. J.T. and Rolls, B.J., Drinking induced by the intracranial injection of angiotensin into the brain of the rat, J. Physiol. (Lond.), 210 (1969) 457-474. 3 Falcon, J.E., Phillips, M.I., Hoffman, W.E. and Brody, M.J., Effects of angiotensin 11are mediated by the sympathetic nervous system, Amer. J. Physiol., 235 (1978) H392-H~99. 4 Gamten, D., Unger, T., Rascher, W.. Fuxe, K., H6kfelt, T. and Agnati. L., Peptidergic and ~ho~nergtc m ~ h ~ i s m s in blood pressure control, Contrib. Nepbrol., 23 (1980) 93-104. 5 C~rcia-.~-vi~; JiA!, ~ ~ h ~ M~L~~ d L~ger, S.Zi~ ~ o t ~ s i n !1 facilitates po~sium evok, ¢d release of 3Hmoradrenaline from the rabbit h~othalamus, Europ. J i Pha~acol,, 56 (1979) 173-176. 6 Kniglg¢, K.M., Hoffman, G., Scott, D.E. and Sladek, J.R., Identification of ;.atecholamine and luteinizing hormone releasing hormone (LHRH) containing neurons in primar~ cultures of dispersed cells of this basal hypothalamus, Brain Res., 120 (1977) 393-345. 7 Lowry, O.H., Rosebrough, N.J., Far'r, A.L. and Randall, R.J., Protein measurements with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 8 Palaic, D. and Khairallah, P.A., Effect of angiotensin on uptake and release of norepinephrine by brain, Biochem. Pharmacul., 16 (1967) 2291-2299. 9 Prochiantz, A., Di Porzic, U., Kato, A., Berger, B. and Cilowinski, J., In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in the presence of their striatal target cells, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5387-5391. I0 Raizada, M.K., Yang, J.W., Phillips, M.I. and Fellows, R.E., Rat brain cells in primary culture: characterization of angiotensin II bindin~ sites, Brain Res., 207 (1981) 343-355. II Raizada, M.K., Stamler, J.F., Landas, S., Quinlan, J. ant! Phillips, M.I., Identification of insulin receptor containing cells in parimary cultures of rat brain, Cell molec. Neurobiol., 2 (1982) 47-52. 12 Sailer, C.F. and Zigmond, M.J., A radioenzymatic assay for catecholamines and dihydroxyphenylacetic acid, Life Sci., 2J (1978) 1117-1130. 13 Severs, W.B., Daniels, A.E. and Buckley, J.P., On the central hypertensive effect of angiotensin I1, Int. J. Pharmacol., 6 (1967) 199-205. 14 Severs, W.B., Summy-Lonig, J., Taylor, J.S. and Connor, J.D., A Central effect of angiotensin: release of pituitary pressor material, J. PharmacoL exp. Ther., 174 (1970) 27-34. I.~ Sumners, C. and Phillips, M.I., Central injection of angiotensin II alters catecholamine activity in rat brain, Amer. J. Physiol., 244 (1983) R257 -~(,3. 16 Sumners, C., Phillips, M.I. and Raizada, M.K., Rat brain neurons in primary culture: visualization and measurement of catecholamines, Brain Res., in press. 17 Weyhenmeyer, J.A., Raizada, M.K., Phillips, M.I. and Fellows, R.I~., Presence of ansiotensin 11 in neurons cultured from fetal rat brain, Neurosci. Lett., 16 (1980) 41-46. 18 Zurgil, N. and Zisapel, N., Induced ncurotransmittcr release from primary cultures of rat brain neurons, Life Sei., 29 (1981) 2265-2271.
305
Elsevier Scientific Publishers Ireland Ltd.
ANGIOTENSIN H STIMULATES CHANGES IN THE NOREPINEPHRINE CONTENT OF P ~ R Y CULTURES OF RAT BRAIN
COLIN SUMNERS, M. IAN PHILLIPS and MOHAN K. RAIZADA
Depariment o f Physiology, University o f Florida, College o f Medicine, Gainesville, FL 32610 (U.S.A.) (Received December 22rid, 1982; Accepted February 18th, 1983)
Key words: angiotensin I, - norepinephrine - primary brain cultures - saralasin
Interactions between norepiuephrine and angiotensin I! were investigated in neuron-enriched primary brain cell cultures, which have been demonstrated to contain catecholamines, angiotensin II-like immunoreactivity and specific receptors for angiotensin !1. Angiotensin I! (7.5 and 15.0 pg/ml) caused significant increases in both neuronal and growth media norepinephrine levels, which were inhibited by saralasin. These observations suggest that angiotensin acts at its specific receptors tO alter neuronal
norepinephriue levds.
The octapeptide angiotensin !I (Ang !!) is both pressor and dipsogenic following central injection into conscious rats [2, 13], and the pressor effect is thought to be due to both sympathetic activation and vasopressin release [3, 14]. Recent studies indicate that pressor doses of centrally injected Ang II or renin caused increased catecholaminergic activity in specific brainstem and hypothalamic nuclei, in viva [4, 15]. The mechanism of interaction between Ang !1 and norepinephrine (NE) in the brain is not clearly understood, and to study it we have examined Ang il-catecholamine (CA) interactions using primary cultures of brain cells. This approach avoids some of the complications of non-specific influences on CAs that may confuse the picture in an in viva situation. Levels of NE and dopamine (DA) have been quantified in 21-day-old primary brain cultures, and the effects of Ang il upon endogenous NE and DA levels in the brain cells and growth media have been examined. Primary cultures from l-day-old whole rat brains were prepared essentially as described previously [11, 16]. They were plated in poly-L-lysine-coatcd 100 m m Falcon tissue culture dishes and grown in Dulbecco's modified Eagles medium (DMEM) containing 10~0 fetal bovine serum (FBS) in a humidified incubator with 5~o C0z/95~o air for 3 days. Cultures were then treated with 10 pM cytosine arabinoside (ARC) prepared in DMEM containing 10% horse serum and 5070FBS, for 2 days. This treatment results in inhibition of cell multiplications, the majority of which are glial in origin, and causes neuron enrichment of cultures. The cultures 0304-3940t83/0000-0000/$ 03.00 © 1983 Elsevier Scientific Publishers IreMnd Ltd.
~6
were grown for a further 16 days in $ ml DMEM containing 10% horse serum prior to experiments. Cultured neurons were treated with various doses of either c~methybp-tyrosine (a-MT), Ang !I (both Sigma) or Ang II + saralasin (Calbio-
h. Ang !! ~ven ~ 7 : 5 / g / m l growth medium w o ~ d ~ ~ e r e d as 6 inoculations each of 1.25 ~g/ml, Following this treatment cells and growth media were removed from the dishes and analyzed for NIlEand DA by a sensitive radioe~ymati¢ method [12[ and for protein by the method of Lowry et al. [7]. The radioenzymatic assay for CAs utilizes [3H-CH3]S-adenosyl-g-methionine (Amersham Inc., specific activity 64 Ci/mmol} and catecho143-methyltransferase to convert NE and DA to their 3-O-methylated metabolites. Data were analyzed by a one-way analysis of variance (ANOVA). The significance of the difference between means was determined by using the pooled variance from the a~,alysis of variance and the t-test. The neuron-enriched cultJres contained approxiw,~ately 70-80% neuronal cells, as evidenced by immunocytochemical studies with neuronalspecific enolase, and electron microscopic analysis [il]. The remainder of the cells are non-nenronal in origin, based on immunocytochemical analysis [10]. Using the radioenzymatic technique it was determined that the neuronal enriched cultures contained !102 + .~7 ( n = 7 ) p g NE/mg protein, and 858 ± 27 (n -- 7) pg DA/mg protein. Treatment with the CA synthesis inhibitor ,x-MT (1.25 ~g/ml growth medium × 6. over 24 h) significantly lowered (P<0.001) both neuronal NE and DA to 480 ± 72 (n = 6) pg NE/mg protein and 351 ± 25 (n = 6) pg DA/mg protein, suggesting that these CAs are synthesized within the cells. The NE level of the growth medium withdrawn from cultures (240 + 29 (n -~ 6) pg NE/ml) was not altered by incubation with oL-MT (211 ± 25 (n = 6) pg NE/ml), indicating that the media had no ability to synthesize CA. To examine whether Ang il alters the endogenous neuronal NE or DA contents of these cells, cultures were incubated with the peptide and then analyzed for NE and DA. At 0.75 #g/mi growth medium Ang II caused a significant fall in neuronal NE ( - 26.7%, P<0.Oi) but not DA content. This dose of peptide did not affect growth media NE levels (Table !). At 7.5 /tg/ml and 15.0/~g/ml Ang !1 both neuronal and growth media NE levels were significantly increased (+ 23.8% and + 37.5°'/o respectively, P<0.002), while neuronal DA was only affected at the higher dose of the peptide (Table !). The effects of Ang Ii given at 0.75 and 7.5/~g/ml were completely inhibited by saralasin given concomitantly with Ang 11 in doses of 7.5 and 15.0 ~g/mi, respectively (Table I). The time course of the Ang ii alteration in NE levels of cultured brain neurons was studied by seeing the effect of a single dose of Ang ii (7.5/~g/ml) given at different times over 24 h. Two hours after Ang 11 (7.5/tg/ml) there was a significant decrease ( - 21.6%) in neuronal NE levels, and after 4 h an increase in both neuronal ( + 78.3%) and media (+ 64.0%) NE contents (Table !I) was observed. An increase
307 TABLE i EFFECTS OF ANGIOTEN$1N il UPON CA LEVELS IN NEURON ENRICHED PRIMARY BRAIN CULTURES AND THE NE CONTENT OF GROWTH MEDIA Values arc mean ± S.E.M. Figures in parentheses are numbers of observations. *P<0.OI; **P<0.02; **°P
Control Aug !1 (0.75 ~,/ml) Aug !! (7.5 tzg/ml)
Aug It (15.0 ~g/ad) Ans + An8 +
II (0.75 ~g/ml) saralasin (7.5 ps/ml) !! (7.5 ~s/ml) saralasin (15.0 ~,g/ml)
ANOVA I
Neuronal NE (pg/mg protein)
Media HE (pg/ml)
Neuronal (pg/mg protein)
1102 808 1365 1516 1172
240 263 362 399 193
452 ± 432 ± 399 ± 564 ± 390___
± ± ± ± ±
57 (7) 30 (6)* 104 (6)* 90 (6)** 67 (4)
± ± ± ± +
29 (6) 19 (6) 40 (6)** 31 (6)** 31 (4)
27 (7) 23 (6) $5 (6) 48 (6)*** 47 (4)
1037 + 62 (4)
201 _+ 52 (4)
406 ~ 38 (4)
<:0.01
<:O.OI
in neuronal NE was still apparent after 12 h ( + 26.8V0), but had returned to control levels at 20 and 24 h. These results suggest that Ang 11 stimulates changes in the NE content of cultured neurons and growth media by acting on specific receptors. These findings are important for two reasons. Firstly, because brain CAs are involved in the pressor response caused by centrally injected Ang !I (15), and also central injections of this peptide or renin elicit change in NE turnover of specific brain regions [4, 15]. Secondly, because the results agree with in vivo and in vitro brain slice experiments which have shown that Ang I! stimulates increases in NE levels in cerebrospinal fluid [I], causes TABLE !i TIME COURSE FOR THE EFFECTS OF ANGIOTENSIN II UPON NE CONTENT OF NEURON ENRICHED CULTURES AND GROWTH MEDIA Values are mean ± S.E.M. Fisures in parentheses are numbers of observations. *P
Growth media NE (pg/ml)
Control Ang II (7.5 #g/ml) 2h 4 h 12 h 20 h 24h
1012 + 46 (4)
250 ± 33 (4)
795 1805 1164 1053 958
288 410 317 339 274
ANOVA!
~0.01
± ± ± ± ±
39 (4)* 63 (4)* 34 (4)** 29 (4) 52(4)
+_ 31 (4) ± 37 (4) ± 27 (4) ± 39 (4) + 24(4)
~0.01
308
release of [3HINE from hypothalamic slices [51, and possibly inhibits neuronal
renptake of lqE [81. Based on our results, neuron enriched cultures may provide a novel method for
li-like ~munoreactivity [17]; they also contain specific uptake and release mechanisms for CA, as evidenced by a number of recent studies (e.g. refs. 6 and 9). in the present experiments we have shown that CAs are synthesized within the neuron, since a-MT lowers neuronal NE and DA. Growth media CA are not altered by a-MT, demonstrating that the media does not have the ability to synthesize CA, and so it is likely that only a small amount of the neuronal .ME and DA is media derived. Saralasin abolished both the changes in neuronal and growth media HIE content caused by Ang !! (Table !), indicating that the peptide was acting at its specific receptors. The effective dose of saralasin is lower than that needed in vi,~o, probably since in the culture system this antagonist comes into immediate contact with the neurons. The results shown here with Ang !1 given as 6 inoculations over 24 h could be obtained following a single dose of Ang !! (Table !1). Twenty-four hours after a single dose of Ang II there was no change in neuronal NE levels (Table !1), which validated incubating cultures with large amounts of peptide in these studies. The incubation times and the large doses of Ang !1 used in our experiments were for two reasons. First, in experiments where endogenous CA are being measured, any alteration in cellular CA content may be neutralized by neurotransmitter resynthesis, leading to no detectable change in NE or DA levels. Hence, it was considered necessary to have a constant amount of Ang Ii present in the culture medium to cause continuous stimulation (or inhibition) of the CA rich cells, and possibly a detectable change in NE or DA levels. Second, Ang II is degraded quickly under these culture conditions (Raizada, unpublished data), and so large doses were added to the medium periodically over 24 h to keep the peptide concentration at a high level. The mechanism of Ang il-stimulated changes in neuron-enriched culture and growth media NE levels is uncertain at this time. The result may imply that Ang 11 is stimulating an increase in NE synthesis and relea~se. However, further experimentation is required to determine whether this is the case, or whether Ang !i is affecting CA metabolic enzymes (e.g. monoamine oxidase) or CA reuptake. This research was supported by grants from NIH (R-I-HL-27334A), NSF (BNS-8025969) to M.I.P. and American Heart Association to M.K.R. I Chcvillard, C., Duchene, N., Pasquier, R. and Alexandre, J.M., Relation of the centrally evoked pressor eft~t of angiotensin !! to central noradrenaline in rabbit, Europ. J. Pharmacol., 58 (1979) 203-206.
309 2 Epstein, A.N., Fitzsimons. J.T. and Rolls, B.J., Drinking induced by the intracranial injection of angiotensin into the brain of the rat, J. Physiol. (Lond.), 210 (1969) 457-474. 3 Falcon, J.E., Phillips, M.I., Hoffman, W.E. and Brody, M.J., Effects of angiotensin 11are mediated by the sympathetic nervous system, Amer. J. Physiol., 235 (1978) H392-H~99. 4 Gamten, D., Unger, T., Rascher, W.. Fuxe, K., H6kfelt, T. and Agnati. L., Peptidergic and ~ho~nergtc m ~ h ~ i s m s in blood pressure control, Contrib. Nepbrol., 23 (1980) 93-104. 5 C~rcia-.~-vi~; JiA!, ~ ~ h ~ M~L~~ d L~ger, S.Zi~ ~ o t ~ s i n !1 facilitates po~sium evok, ¢d release of 3Hmoradrenaline from the rabbit h~othalamus, Europ. J i Pha~acol,, 56 (1979) 173-176. 6 Kniglg¢, K.M., Hoffman, G., Scott, D.E. and Sladek, J.R., Identification of ;.atecholamine and luteinizing hormone releasing hormone (LHRH) containing neurons in primar~ cultures of dispersed cells of this basal hypothalamus, Brain Res., 120 (1977) 393-345. 7 Lowry, O.H., Rosebrough, N.J., Far'r, A.L. and Randall, R.J., Protein measurements with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 8 Palaic, D. and Khairallah, P.A., Effect of angiotensin on uptake and release of norepinephrine by brain, Biochem. Pharmacul., 16 (1967) 2291-2299. 9 Prochiantz, A., Di Porzic, U., Kato, A., Berger, B. and Cilowinski, J., In vitro maturation of mesencephalic dopaminergic neurons from mouse embryos is enhanced in the presence of their striatal target cells, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5387-5391. I0 Raizada, M.K., Yang, J.W., Phillips, M.I. and Fellows, R.E., Rat brain cells in primary culture: characterization of angiotensin II bindin~ sites, Brain Res., 207 (1981) 343-355. II Raizada, M.K., Stamler, J.F., Landas, S., Quinlan, J. ant! Phillips, M.I., Identification of insulin receptor containing cells in parimary cultures of rat brain, Cell molec. Neurobiol., 2 (1982) 47-52. 12 Sailer, C.F. and Zigmond, M.J., A radioenzymatic assay for catecholamines and dihydroxyphenylacetic acid, Life Sci., 2J (1978) 1117-1130. 13 Severs, W.B., Daniels, A.E. and Buckley, J.P., On the central hypertensive effect of angiotensin I1, Int. J. Pharmacol., 6 (1967) 199-205. 14 Severs, W.B., Summy-Lonig, J., Taylor, J.S. and Connor, J.D., A Central effect of angiotensin: release of pituitary pressor material, J. PharmacoL exp. Ther., 174 (1970) 27-34. I.~ Sumners, C. and Phillips, M.I., Central injection of angiotensin II alters catecholamine activity in rat brain, Amer. J. Physiol., 244 (1983) R257 -~(,3. 16 Sumners, C., Phillips, M.I. and Raizada, M.K., Rat brain neurons in primary culture: visualization and measurement of catecholamines, Brain Res., in press. 17 Weyhenmeyer, J.A., Raizada, M.K., Phillips, M.I. and Fellows, R.I~., Presence of ansiotensin 11 in neurons cultured from fetal rat brain, Neurosci. Lett., 16 (1980) 41-46. 18 Zurgil, N. and Zisapel, N., Induced ncurotransmittcr release from primary cultures of rat brain neurons, Life Sei., 29 (1981) 2265-2271.