Margaret C. Boadle-Biber

Acceleration of noradrenaline biosynthesis in the guinea-pig vas deferens by potassium

1 . Increasing the concentration of KC1 in Krebs‐Henseleit bicarbonate solution enhanced the formation of 14C‐noradrenaline (14C‐NA) from 14C‐tyrosine in the guinea‐pig vas deferens. In 52 mm KC1 Krebs‐Henseleit solution the specific activity of the newly formed 14C‐NA was double that of controls. 2 . The rate of synthesis of 14C‐NA from 14C‐tyrosine was constant for up to 2 h in 52 mm KC1 Krebs‐Henseleit solution and for 4 h in unmodified Krebs‐Henseleit solution. 3 . There was no increase in NA formation in the presence of KC1 rich Krebs‐Henseleit solution if 14C‐DOPA was used as the starting substrate instead of 14C‐tyrosine. 4 . The specific activity of 14C‐tyrosine in the high KC1 treated vas deferens was 80% of that of control tissues. Thus the enhanced synthesis of 14C‐NA in high KC1 Krebs‐Henseleit solution did not arise from an increase in the specific activity of precursor. 5 . The effect of K+ on NA synthesis was not mimicked by ganglionic stimulants nor blocked by tetrodotoxin. 6 . Removal of Ca2+ ions or increasing the concentration of Mg2+ ions abolished the increase in synthesis of NA seen in high KC1 Krebs‐Henseleit solution but left the basal rate of NA synthesis in unmodified Krebs‐Henseleit solution unaltered. 7 . The spontaneous release of newly synthesized catecholamines (14C‐labelled) or tritiated noradrenaline (3H‐NA) from vasa deferentia was increased in 52 mm KC1 Krebs‐Henseleit solution. Removal of Ca2+ ions reduced the increased efflux of newly synthesized amine in high KC1 media to that seen in unmodified Krebs‐Henseleit solution. The efflux of 3H‐NA was reduced to one‐third of its former rate in the absence of Ca2+. 8 . High KC1 Krebs‐Henseleit solution caused a substantial contraction of the vas deferens which was not abolished by tetrodotoxin. Release of 3H‐NA paralleled the contractile response, and was likewise unaffected by tetrodotoxin. 9 . No evidence was obtained for any alterations in the activity of tyrosine hydroxylase, the rate limiting enzyme in the formation of NA from tyrosine, in homogenates of vas deferens which had been treated with 52 mm KC1 Krebs‐Henseleit solution. 10 . These results support the hypothesis that acceleration of NA synthesis occurs when tyrosine hydroxylase is freed from end‐product inhibition by the release of noradrenaline, brought about in this case, by high concentrations of KC1.

FORMATION OF DOPAMINE AND NORADRENALINE IN RAT VAS DEFERENS: COMPARISON WITH GUINEA‐PIG VAS DEFERENS

1 The formation of [14C]‐3,4‐dihydroxyphenylalanine (DOPA) from [14C]‐tyrosine, in the presence of the amino acid decarboxylase inhibitor, brocresine (3‐hydroxy‐4‐bromobenzyloxy‐amine dihydrogen phosphate), was greatly enhanced in rat vasa deferentia depolarized by a KCl‐enriched Krebs‐Henseleit solution (52 mM KCl) compared with tissues maintained in unmodified Krebs‐Henseleit solution. 2 When the conversion of tyrosine was allowed to proceed as far as catecholamine (brocresine absent) no significant difference was observed between the accumulation of [14C]‐catecholamines (CA) in depolarized rat vasa deferentia and the accumulation in control (non‐depolarized) tissues. 3 Endogenous CA levels in the depolarized rat vasa deferentia fell to 67% of the controls after a 1 h incubation period and to 53% at the end of 2 hours. 4 Chromatographic separation on Amberlite CG‐120 columns of the newly synthesized CA and catechol metabolites from the rat vas deferens revealed that a very high proportion was present as dopamine. The percentage distribution after 1 h incubation in control Krebs‐Henseleit was: noradrenaline (NA): 30.6 ± 5.2; dopamine 56.9 ± 5.9; acid metabolites: 12.8 ± 1.1; and in KCl‐rich Krebs‐Henseleit, NA: 32; dopamine: 44.7 and acid metabolites 23.3. In contrast to the newly synthesized (14C‐labelled) CA, endogenous dopamine comprises only 10% of the endogenous CA stores in rat vas deferens. 5 The distribution of newly synthesized NA and dopamine in rat vas deferens is strikingly different from that of guinea‐pig vas deferens where more than 80% of newly formed amine is present as NA. In the latter tissue depolarization with K+ causes a striking increase in CA biosynthesis.

Acceleration of catecholamine biosynthesis in sympathetically innervated tissues by angiotensin‐II‐amide

1 . The effect of angiotensin‐II‐amide on the biosynthesis of catecholamines (CA) has been studied in a number of isolated tissues in vitro. 2 . Angiotensin increased the synthesis of CA from 14C‐tyrosine in guinea‐pig atria and portal vein, in rat vasa deferentia and the rabbit portal vein. 3 . Angiotensin had no effect on synthesis of CA from 14C‐labelled dl‐DOPA. 4 . The conditions required to demonstrate an increased synthesis were critical with respect to incubation time and angiotensin concentration. Effects were most readily apparent after incubation for 1 h with concentrations of angiotensin ranging from 10−9 to 10−7m. Higher concentrations caused a significant reduction in synthesis. 5 . An increased release of newly synthesized CA into the incubation medium was sometimes seen in the presence of angiotensin. However, there was no correlation between increased synthesis and release of CA. 6 . Angiotensin was rapidly destroyed when incubated with guinea‐pig or rat tissues in Krebs solution. The increase in CA synthesis was only apparent at a time when the incubation medium could have contained only a fraction of the original angiotensin activity. 7 . It is concluded that the effect of angiotensin is not due to increased release of noradrenaline (NA) or to inhibition of NA uptake into nerves. It is possible that angiotensin may influence the activity of tyrosine hydroxylase or its cofactors by an as yet unknown mechanism.

Effect of drugs on the synthesis of noradrenaline in guinea-pig vas deferens

1 . Reserpine in vitro (10−5m) caused a profound inhibition (>85%) of the formation of both 14C‐catecholamine (14C‐CA) and 14C‐dihydroxyphenylalanine (14C‐DOPA) (in the presence of the amino acid decarboxylase inhibitor brocresine) from 14C‐tyrosine in guinea‐pig vas deferens. The magnitude of the inhibition was similar for both 14C‐CA and 14C‐DOPA suggesting that the inhibition occurred primarily at the tyrosine hydroxylase step. 2 . One hour after in vivo treatment with reserpine (1 mg/kg) when tissue stores of noradrenaline (NA) were depleted by 50%, there was a significant inhibition of the formation of 14C‐DOPA. Twenty‐four hours after such treatment, when endogenous NA could no longer be detected, synthesis of 14C‐DOPA was indistinguishable from untreated controls. However a 45% inhibition of 14C‐DOPA synthesis from 14C‐tyrosine could be produced in tissues which had been depleted of NA for 24 h or 48 h by the addition of reserpine, 10−5m, to the incubation medium. 3 . Addition of pteridine cofactor, 2‐amino‐6,7,‐dimethyl‐4‐hydroxy‐5,6,7,8‐tetrahydropteridine, to the incubation medium in a concentration of 5 × 10−3m enhanced the formation of both 14C‐CA and 14C‐DOPA from 14C‐tyrosine in guinea‐pig vas deferens. In 52 mm KCl Krebs‐Henseleit medium 14C‐CA formation increased from 2·58 ± 0·20 (nmol/g)/h to 6·35 ± 0·47 (nmol/g)/h whilst 14C‐DOPA formation increased from 5·04 ± 0·88 (nmol/g)/h to 11·29 ± 0·59 (nmol/g)/h. 4 . Pteridine cofactor (5 × 10−3m) did not reverse the inhibition of 14C‐DOPA formation seen with reserpine (10−5m) in previously untreated tissues or in vasa deferentia from animals pretreated with reserpine 1 mg/kg for 24 hours. However, the inhibition did disappear in the presence of pteridine cofactor when treatment with reserpine was prolonged to 48 h and included two doses of reserpine of 2 mg/kg. 5 . Tyramine (5·8 × 10−5m) and bretylium (10−5m) in vitro inhibited the formation of 14C‐CA and 14C‐DOPA from 14C‐tyrosine to the same extent in guinea‐pig vas deferens again indicating that their major site of action is on tyrosine hydroxylase. The inhibitory effects were reversed by pteridine cofactor. 6 . Synthesis of 14C‐NA from 14C‐tyrosine in calf splenic nerve was not increased by incubating the tissue in 52 mm KCl‐Krebs‐Henseleit solution.

Factors modifying the synthesis of dopamine from tyrosine in pedal ganglia of Mercenaria mercenaria (mollusca)

1. 1. The dopamine (DA) and 5-hydroxytryptamine (5-HT) contents of the ganglia of Mercenaria mercenaria expressed as ng. per ganglion were as follows: Pedal ganglion: 236±16 ng. DA 101± NG. 5-HT. Visceral ganglion: 76±3 ng. DA 116±7 NG. 5-HT. Cerebral ganglion: 37±5 ng. DA 107±15 ng. 5-HT. 1. 2. Pedal ganglia synthesize [14C]DA from [14C]tyrosine but do not form significant amounts of [14C]noradrenaline (NA). [14C]5-HT is also formed from [14C]tryptophan in pedal ganglia. 2. 3. Incubation of pedal ganglia in KCl-rich sea water accelerates synthesis of [14C]DA from [14C]tyrosine two- to threefold compared with controls. The specific activity of DA isolated from the K+-stimulation ganglia is corresponding increased. 3. 4. The acceleration of DA synthesis appears to take place at the tyrosine hydroxylation step. 4. 5. Synthesis of DA from tyrosine in pedal ganglia is inhibited up to 60 per cent by 10−4M DA in the medium.