Laboratorio de
Bioquímica Nuclear, Comisión Nacional Depto de Ciencias Biológicas,
de
Energia Atómica;
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, and Division of Endocrinology, The Buffalo General Hospital, Buffalo, N. Y. 14203, USA
Argentina
ROLE OF NEUROTRANSMITTERS, PROSTAGLANDINS AND GLUCOSE ON PRECURSOR
INCORPORATION INTO THE RNA OF THYROID SLICES
By Guillermo J. Juvenal, Diana L. Kleiman de Pisarev, Liliana Crenovich and Mario A. Pisarev
ABSTRACT
performed
investigate the role of [3H]uridine incorporation into total RNA in beef thyroid slices. Carbamylcholine strongly stimulated RNA labelling from [3H]uridine; atropine abolished this effect. NaF, at 1 and 5 mm, progressively increased this parameter while
The present studies
were
in order to
neurotransmitters, prostaglandins and glucose
on
caused a similar effect at 10\m=-\3 but not at 10\m=-\6 M. Phenblocked the stimulatory action of TSH; propranolol and atropine did not. Glucose at concentrations between 4 and 24 mm caused a progressive increase in RNA labelling from [3H]uridine. This effect
norepinephrine
tolamine
(1
mm)
Supported by grants from the Consejo Nacional de Investigaciones Científicas y Téc¬ (CONICET), Argentina and from the Margaret Duffy and Robert Cameron Troup
nicas
Fund, Buffalo, N. Y.
0 Fellow of the Comisión Nacional de
Energía
Atómica.
2) Established investigator of the CONICET, fellow of the H. and B. Buswell Foundation.
Reprints request
Hospital,
100
to: M. A.
Pisarev, M. D., Division of Endocrinology, Buffalo General
High St., Buffalo,
N. Y. 14203, USA.
776
was
by dinitrophenol. Prostaglandins (E1, A1, F1\g=a\and F2\g=a\) \g=m\g/ml, with or without cafeffect on RNA labelling. Moreover, neither aspirin nor inhibited TSH stimulation. Under similar experimental
not inhibited
assayed
in concentrations between 5 and 25
feine, had no indomethacin conditions,
PGE1
did simulate PB125I formation.
Thyroid function is regulated by a variety of different faciors including thyrofrophin (TSH) '). This hormone inieracfs with a specific membrane receptor and activates adenylate cyclase to formation of cyclic AMP (Schell-Frederick 8c Dumonl 1970). This compound mimics the action of TSH on difieren! íhyroid parameters including [3H]uridine incorporation into RNA (Pisarev et al. 1976). Another cyclic nucleolide, cyclic GMP, also mimics TSH action on protein (Pisarev et al. 1971) and RNA synthesis (Pisarev 8c Kleiman de Pisarev 1977). Guanylate cyclase (Barmasch et al. 1973) and protein kinase sensitive to cGMP (Kuo et al. 1970) have also been demonstrated in the thyroid. However, cGMP levels can be regulated by a mechanism independent from TSH (Yamashita 8c Field 1972; van Sande et al. 1975), involving cholinergic substances. The pre¬ sent studies were performed in order to investigate the role of cholinergic and andrenergic agents and receptors, and of prostaglandins on precursor incor¬ poration inio thyroid RNA. MATERIALS AND METHODS Beel thyroids were obtained from a local slaughterhouse. Single slices (40—80 mg wet weight) were prepared with a Stadie-Riggs microtome and pre-incubated, in quadriplicate, for 30 min in 5 ml of KRB buffer, pH 7.4, containing the test substances, under 95% Og, 5"/a C02, at 37°C. After pre-incubation, a tracer dose (0.5-1 //Ci) of [5-3H]uridine (New England Nuclear), specific activity 28.3 Ci/mmol, was added and incubations continued for another 60 min. The slices were then thoroughly washed with saline, blotted on filter paper and homogenized in 1 ml of distilled water using an all-glass Potter-Elvehjem tissue homogenator. RNA and oligonucleotides were ex¬ tracted according to the method of Munro Se Fleck (1966), and the specific activity of total RNA calculated after counting the radioactivity in Bray's scintillation solution. Total RNA content was around 1 jig/mg wet weight. When the incorporation of [3H]uridine into RNA from [3H]UTP was studied, the slices were incubated in KRB containing 7 /
Bioquímica Nuclear, Comisión Nacional Depto de Ciencias Biológicas,
de
Energia Atómica;
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, and Division of Endocrinology, The Buffalo General Hospital, Buffalo, N. Y. 14203, USA
Argentina
ROLE OF NEUROTRANSMITTERS, PROSTAGLANDINS AND GLUCOSE ON PRECURSOR
INCORPORATION INTO THE RNA OF THYROID SLICES
By Guillermo J. Juvenal, Diana L. Kleiman de Pisarev, Liliana Crenovich and Mario A. Pisarev
ABSTRACT
performed
investigate the role of [3H]uridine incorporation into total RNA in beef thyroid slices. Carbamylcholine strongly stimulated RNA labelling from [3H]uridine; atropine abolished this effect. NaF, at 1 and 5 mm, progressively increased this parameter while
The present studies
were
in order to
neurotransmitters, prostaglandins and glucose
on
caused a similar effect at 10\m=-\3 but not at 10\m=-\6 M. Phenblocked the stimulatory action of TSH; propranolol and atropine did not. Glucose at concentrations between 4 and 24 mm caused a progressive increase in RNA labelling from [3H]uridine. This effect
norepinephrine
tolamine
(1
mm)
Supported by grants from the Consejo Nacional de Investigaciones Científicas y Téc¬ (CONICET), Argentina and from the Margaret Duffy and Robert Cameron Troup
nicas
Fund, Buffalo, N. Y.
0 Fellow of the Comisión Nacional de
Energía
Atómica.
2) Established investigator of the CONICET, fellow of the H. and B. Buswell Foundation.
Reprints request
Hospital,
100
to: M. A.
Pisarev, M. D., Division of Endocrinology, Buffalo General
High St., Buffalo,
N. Y. 14203, USA.
776
was
by dinitrophenol. Prostaglandins (E1, A1, F1\g=a\and F2\g=a\) \g=m\g/ml, with or without cafeffect on RNA labelling. Moreover, neither aspirin nor inhibited TSH stimulation. Under similar experimental
not inhibited
assayed
in concentrations between 5 and 25
feine, had no indomethacin conditions,
PGE1
did simulate PB125I formation.
Thyroid function is regulated by a variety of different faciors including thyrofrophin (TSH) '). This hormone inieracfs with a specific membrane receptor and activates adenylate cyclase to formation of cyclic AMP (Schell-Frederick 8c Dumonl 1970). This compound mimics the action of TSH on difieren! íhyroid parameters including [3H]uridine incorporation into RNA (Pisarev et al. 1976). Another cyclic nucleolide, cyclic GMP, also mimics TSH action on protein (Pisarev et al. 1971) and RNA synthesis (Pisarev 8c Kleiman de Pisarev 1977). Guanylate cyclase (Barmasch et al. 1973) and protein kinase sensitive to cGMP (Kuo et al. 1970) have also been demonstrated in the thyroid. However, cGMP levels can be regulated by a mechanism independent from TSH (Yamashita 8c Field 1972; van Sande et al. 1975), involving cholinergic substances. The pre¬ sent studies were performed in order to investigate the role of cholinergic and andrenergic agents and receptors, and of prostaglandins on precursor incor¬ poration inio thyroid RNA. MATERIALS AND METHODS Beel thyroids were obtained from a local slaughterhouse. Single slices (40—80 mg wet weight) were prepared with a Stadie-Riggs microtome and pre-incubated, in quadriplicate, for 30 min in 5 ml of KRB buffer, pH 7.4, containing the test substances, under 95% Og, 5"/a C02, at 37°C. After pre-incubation, a tracer dose (0.5-1 //Ci) of [5-3H]uridine (New England Nuclear), specific activity 28.3 Ci/mmol, was added and incubations continued for another 60 min. The slices were then thoroughly washed with saline, blotted on filter paper and homogenized in 1 ml of distilled water using an all-glass Potter-Elvehjem tissue homogenator. RNA and oligonucleotides were ex¬ tracted according to the method of Munro Se Fleck (1966), and the specific activity of total RNA calculated after counting the radioactivity in Bray's scintillation solution. Total RNA content was around 1 jig/mg wet weight. When the incorporation of [3H]uridine into RNA from [3H]UTP was studied, the slices were incubated in KRB containing 7 /
