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The depressor effect of secretin administered either centrally or peripherally in conscious rats
$146 VISUALIZATION HYBRIDIZATION.
OF
TRANSMITTER
mRNAs
IN
THE
NERVOUS
]'ISSUES
BY
in
situ
YASUHIRO MORITA:, K O U I C H I NOGUCHI ~ ' MASAYA TOHYAMA ~ , H I R O S H I KIYAMA ~, KAYOKO ISHII e', SADAO S H I O S A K A 2 , KAZUAKI YOSHIKAWA ~" and SHIGETADA NAKANISHI a' 1:Dept. Anat. 2:Dept. Neuroanat., Osaka Univ. Med. Seh., Osaka, 3:Molec. Biol., Psychiat. Res. Inst. of Tokyo, Tokyo, a:Kyoto Univ. Fac. Med., Inst. Immunol., Kyoto. In situ hybridization histochemistry is one of the new techniques based on molecular biology and gene engineering, and enables the visualization of specific mRNAs i n t i s s u e s as radioisotope or other chemical signals. Enkephalin, substance P, and substance K are neuropeptides which act as transmitters or neuroactive substances in the nervous tissues. Recently, the precursor mRNAs coding for the enkephalins (preproenkephalin A; Y o s h i k a w a et al., '82) and for the substance P and substance K (r-preprotachykinin A; K a w a g u c h i et al., '86) had been screened out from the rat brains and sequenced by molecular cloning. fhe complementary DNAs (cDNAs) against these precursor mRNAs w e r e labeled by nick translation with ~'P for macro-autoradiography and ~S for miero-autoradiography, and used for the cDNA-mRNA hybridization. Following in situ hybridization using these radioisotope-labeled eDNA p r o b e s , the hybridization signals indicating the presence of the preproenkephalin A mRNAs w e r e observed in the olfactory tubercle, septal areas, accumbens nucleus, caudate-putamen nucleus, central nucleus of the amygdala, and in several structures of the hypothalamus and the lower brain stem. Primary sensory neurons expressing the r-preprotachykinin A gene were scattered diffusely in the whole extent of the trigeminal ganglion. In the RNase treated specimens for" the control experiments, hybridization signals were totally eliminated.
THE D E P R E S S O R EFFECT OF SECRETIN A D M I N I S T E R E D IN CONSCIOUS RATS
EITHER CENTRALLY
RYUHEI H A S H I M O T O * A N D FUKUKO KIMURA. Department of Univ. School of Medicine. 3-9 Fukuura, Kanazawa-ku,
OR
PERIPHERALLY
Physiology, Yokohama Yokohama 236
City
In recent years, it has become evident that many peptides are isolated from both the central nervous system and g a s t r o i n t e s t i n a l tract, and actually mediate regulatory effects on various functions, including the cardiovascular system. This study was made to elucidate the central action of secretin, by examining its effect on systemic blood pressure (BP) when a d m i n i s t e r e d i n t r a c e r e b r o v e n t r i cularly (i.c.v.) in conscious rats. The effects of intravenous (i.v.) injection were investigated, too. The same volume of saline was injected as a control. Both i.c.v, and i.v. a d m i n i s t r a t i o n of secretin cause significant depressor effects. In i.c.v, groups, i) the secretin (0.5~g/2zl saline) injected group d e m o n s t r a t e d about 20 mmHg d e p r e s s i o n in BP 4 min after starting i.c.v., which recovered within 5 min after this effect, and when 2) the secretin (l.0~g/2~l saline) was injected, the BP began to fall as soon as i.c.v, started, and also reached about a 20 mmHg d e p r e s s i o n that continued for more than 12 min. The fact that a longer duration of h y p o t e n s i o n was seen when a higher dose secretin was a d m i n i s t e r e d suggests that this peptide has a dose-dependent depressor effect. No significant heart rate (HR) changes were seen in the former group. The latter showed tachycardia, which might be induced by the b a r o r e c e p t e r reflex m e c h a n i s m when the h y p o t e n s i o n progressed. In i.v. groups, 2pg secretin was injected in different volumes and routes, i.e. i) 30DI, intraatrial injection via the jugular vein, 2) 10DI, via the same route, 3) 25pi, intracaval injection via the femoral vein. 25-30 mmHg BP d e p r e s s i o n occured in all of the groups 1-5 min after starting i.v.. T a c h y c a r d i a was a c c o m p a n i e d with h y p o t e n s i o n as well as i.c.v, groups. In c o n s i d e r a t i o n of previous findings that many other peptides such as substance P, enkephalin, bradykinin, neurotensin and VIP cause pressor effects when a d m i n i s t e r e d centrally, but produce depressor responses after peripheral administeration, the hypotension induced as both central and peripheral effects of secretin is unique and significant.
OF
TRANSMITTER
mRNAs
IN
THE
NERVOUS
]'ISSUES
BY
in
situ
YASUHIRO MORITA:, K O U I C H I NOGUCHI ~ ' MASAYA TOHYAMA ~ , H I R O S H I KIYAMA ~, KAYOKO ISHII e', SADAO S H I O S A K A 2 , KAZUAKI YOSHIKAWA ~" and SHIGETADA NAKANISHI a' 1:Dept. Anat. 2:Dept. Neuroanat., Osaka Univ. Med. Seh., Osaka, 3:Molec. Biol., Psychiat. Res. Inst. of Tokyo, Tokyo, a:Kyoto Univ. Fac. Med., Inst. Immunol., Kyoto. In situ hybridization histochemistry is one of the new techniques based on molecular biology and gene engineering, and enables the visualization of specific mRNAs i n t i s s u e s as radioisotope or other chemical signals. Enkephalin, substance P, and substance K are neuropeptides which act as transmitters or neuroactive substances in the nervous tissues. Recently, the precursor mRNAs coding for the enkephalins (preproenkephalin A; Y o s h i k a w a et al., '82) and for the substance P and substance K (r-preprotachykinin A; K a w a g u c h i et al., '86) had been screened out from the rat brains and sequenced by molecular cloning. fhe complementary DNAs (cDNAs) against these precursor mRNAs w e r e labeled by nick translation with ~'P for macro-autoradiography and ~S for miero-autoradiography, and used for the cDNA-mRNA hybridization. Following in situ hybridization using these radioisotope-labeled eDNA p r o b e s , the hybridization signals indicating the presence of the preproenkephalin A mRNAs w e r e observed in the olfactory tubercle, septal areas, accumbens nucleus, caudate-putamen nucleus, central nucleus of the amygdala, and in several structures of the hypothalamus and the lower brain stem. Primary sensory neurons expressing the r-preprotachykinin A gene were scattered diffusely in the whole extent of the trigeminal ganglion. In the RNase treated specimens for" the control experiments, hybridization signals were totally eliminated.
THE D E P R E S S O R EFFECT OF SECRETIN A D M I N I S T E R E D IN CONSCIOUS RATS
EITHER CENTRALLY
RYUHEI H A S H I M O T O * A N D FUKUKO KIMURA. Department of Univ. School of Medicine. 3-9 Fukuura, Kanazawa-ku,
OR
PERIPHERALLY
Physiology, Yokohama Yokohama 236
City
In recent years, it has become evident that many peptides are isolated from both the central nervous system and g a s t r o i n t e s t i n a l tract, and actually mediate regulatory effects on various functions, including the cardiovascular system. This study was made to elucidate the central action of secretin, by examining its effect on systemic blood pressure (BP) when a d m i n i s t e r e d i n t r a c e r e b r o v e n t r i cularly (i.c.v.) in conscious rats. The effects of intravenous (i.v.) injection were investigated, too. The same volume of saline was injected as a control. Both i.c.v, and i.v. a d m i n i s t r a t i o n of secretin cause significant depressor effects. In i.c.v, groups, i) the secretin (0.5~g/2zl saline) injected group d e m o n s t r a t e d about 20 mmHg d e p r e s s i o n in BP 4 min after starting i.c.v., which recovered within 5 min after this effect, and when 2) the secretin (l.0~g/2~l saline) was injected, the BP began to fall as soon as i.c.v, started, and also reached about a 20 mmHg d e p r e s s i o n that continued for more than 12 min. The fact that a longer duration of h y p o t e n s i o n was seen when a higher dose secretin was a d m i n i s t e r e d suggests that this peptide has a dose-dependent depressor effect. No significant heart rate (HR) changes were seen in the former group. The latter showed tachycardia, which might be induced by the b a r o r e c e p t e r reflex m e c h a n i s m when the h y p o t e n s i o n progressed. In i.v. groups, 2pg secretin was injected in different volumes and routes, i.e. i) 30DI, intraatrial injection via the jugular vein, 2) 10DI, via the same route, 3) 25pi, intracaval injection via the femoral vein. 25-30 mmHg BP d e p r e s s i o n occured in all of the groups 1-5 min after starting i.v.. T a c h y c a r d i a was a c c o m p a n i e d with h y p o t e n s i o n as well as i.c.v, groups. In c o n s i d e r a t i o n of previous findings that many other peptides such as substance P, enkephalin, bradykinin, neurotensin and VIP cause pressor effects when a d m i n i s t e r e d centrally, but produce depressor responses after peripheral administeration, the hypotension induced as both central and peripheral effects of secretin is unique and significant.