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Characterization of intracellular opioid binding sites and G regulatory proteins in neonatal and adult rat brain
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Characterization of intracellular opioid binding sites and G reg~atory proteins in neom~t~ and adult rat brain SzUcs *'* *, M., Bem *, W.T., Yeung *, S-C.J. and Coscia, C.J. * Dept. of Biochen~, St. Louis Univ. Sch. Meal 1402 $. Grandblvd, St. Louis, MO 63104, U.S.A. and ** Inst. of Biochem., Biol. Res. Centre Hung. Acod. $ci., 6701 Szegeg PO Box 521, Hungary
Opioid binding sites have been p:eviously demonstrated both in light membrane fractions (LMs) which are enriched in smooth endoplasmic reticulum and Golgi as well as in synaptic plasma membranes (SPMs). Binding characteristics of mu and delta opioid receptors were studie~d with highly selective radioligands: 3H-Tyr-D-Ala-GlyMePhe-Glyol (3H-DAGO) and 3H-D-Pen2-D-PenS-enkephalin (3H-DPDPE), respectively. In adult rat brain, SPMs constitute about 85~$ of the total opioid receptor population, while 155 were found in LMs. The most prominent feature of LM opioid binding is a GTP sensitivity which is significantly less than that displayed in SPMs. The alpha-subunit of pertussis toxin-sensitive guanine nucleotide (G) regulatory proteins were demonstrated in both LMs and SPMs. Immunoblotting experiments with anti-G-peptide antibodies suggest that the ratio of Go protein alpha subunits for SPM: LM is 3:1. Inability of opioid agonists to stimulate the low K m GTPase activity and the decreased GTP sensitivity of opioid binding suggest that many opioid receptors are not coupled to G proteins in adult LMs. However, whee. LMs were solubilized with 1~$ digitonin, the potency of morphine to compete for 3H-naloxone oinding was significantly decreased by 100 uM Gpp(NH)p. These experiments suggest that intracellular opioid binding sites and G proteins might be physically uncoupled, but undergo functional coupling upon solubilization. In parallel experiments forebrains of 1-day-old rat pups (P-l) were also resolved into heavy membranes (HMs) and LMs. Both mu opioid receptor and G protein content of P-1 LMs are elevated over those in adult LMs. P-1 LMs contained about 45~$ of the total mu sites wich were inhibited 40~$ by 50 uM Gpp(NH)p. The ratio of G O protein alpha-subunits for HM: LM in P-1 neonates was 0.24:1. Thus P-1 neonatal forebrain may contain more intracellular newly synthesized mu opioid binding sites and G proteins than the adult.
~ .fr.2311 An analysis of the functional relations of various (6-substituted) morphine derivatives to multiple opiate receptors Ftirst, S., Friedmann *, T. and Hosztafi * *, S. • Department of Pharmacology, Semmelweis University of Medicine, P.O.B. 370, Budapest 1445 and * * Alkaloida Chemical Works, 4440 Tiszavasvdri, Hungary
Among the important properties of the opioids that can be altered by structural modifications are their affinity for various classes of opiate receptors, spectrum of their agonistic versus antagonistic activity their lipid solubility etc. Our studies were designed to determine the importance of position 6 of the ring C of morphine in receptor sensitivity and selectivity, focusing our attention to the analgesic, sedative anticonvulsive and neurochemical actions. There was a dramatic change in analgesic activity, when dihydroisomorphine has been converted via its 6-tosylate into 6-deoxy-6azidodihydroisomorphine (azidomorphine, AM) as it was previously reported by us (Bognar and Maldeit, 1968; Knoll, Ftirst and Kelemen, 1973). AM and its 14-OH derivative are extremely potent analgesics, as potent as fentanyi, a fully synthetic agent. Analogs in this family were synthetized with varir,,,~ N-substituents: methyl, allyl, cyclopropylmethyl. These compounds were evaluated for receptor agonism and antagonism in various animal assays. The results obtained show, that while the formal inversion of the 6-OH group of morphine to a-isomorplune or a-isocodeine does not
i
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Characterization of intracellular opioid binding sites and G reg~atory proteins in neom~t~ and adult rat brain SzUcs *'* *, M., Bem *, W.T., Yeung *, S-C.J. and Coscia, C.J. * Dept. of Biochen~, St. Louis Univ. Sch. Meal 1402 $. Grandblvd, St. Louis, MO 63104, U.S.A. and ** Inst. of Biochem., Biol. Res. Centre Hung. Acod. $ci., 6701 Szegeg PO Box 521, Hungary
Opioid binding sites have been p:eviously demonstrated both in light membrane fractions (LMs) which are enriched in smooth endoplasmic reticulum and Golgi as well as in synaptic plasma membranes (SPMs). Binding characteristics of mu and delta opioid receptors were studie~d with highly selective radioligands: 3H-Tyr-D-Ala-GlyMePhe-Glyol (3H-DAGO) and 3H-D-Pen2-D-PenS-enkephalin (3H-DPDPE), respectively. In adult rat brain, SPMs constitute about 85~$ of the total opioid receptor population, while 155 were found in LMs. The most prominent feature of LM opioid binding is a GTP sensitivity which is significantly less than that displayed in SPMs. The alpha-subunit of pertussis toxin-sensitive guanine nucleotide (G) regulatory proteins were demonstrated in both LMs and SPMs. Immunoblotting experiments with anti-G-peptide antibodies suggest that the ratio of Go protein alpha subunits for SPM: LM is 3:1. Inability of opioid agonists to stimulate the low K m GTPase activity and the decreased GTP sensitivity of opioid binding suggest that many opioid receptors are not coupled to G proteins in adult LMs. However, whee. LMs were solubilized with 1~$ digitonin, the potency of morphine to compete for 3H-naloxone oinding was significantly decreased by 100 uM Gpp(NH)p. These experiments suggest that intracellular opioid binding sites and G proteins might be physically uncoupled, but undergo functional coupling upon solubilization. In parallel experiments forebrains of 1-day-old rat pups (P-l) were also resolved into heavy membranes (HMs) and LMs. Both mu opioid receptor and G protein content of P-1 LMs are elevated over those in adult LMs. P-1 LMs contained about 45~$ of the total mu sites wich were inhibited 40~$ by 50 uM Gpp(NH)p. The ratio of G O protein alpha-subunits for HM: LM in P-1 neonates was 0.24:1. Thus P-1 neonatal forebrain may contain more intracellular newly synthesized mu opioid binding sites and G proteins than the adult.
~ .fr.2311 An analysis of the functional relations of various (6-substituted) morphine derivatives to multiple opiate receptors Ftirst, S., Friedmann *, T. and Hosztafi * *, S. • Department of Pharmacology, Semmelweis University of Medicine, P.O.B. 370, Budapest 1445 and * * Alkaloida Chemical Works, 4440 Tiszavasvdri, Hungary
Among the important properties of the opioids that can be altered by structural modifications are their affinity for various classes of opiate receptors, spectrum of their agonistic versus antagonistic activity their lipid solubility etc. Our studies were designed to determine the importance of position 6 of the ring C of morphine in receptor sensitivity and selectivity, focusing our attention to the analgesic, sedative anticonvulsive and neurochemical actions. There was a dramatic change in analgesic activity, when dihydroisomorphine has been converted via its 6-tosylate into 6-deoxy-6azidodihydroisomorphine (azidomorphine, AM) as it was previously reported by us (Bognar and Maldeit, 1968; Knoll, Ftirst and Kelemen, 1973). AM and its 14-OH derivative are extremely potent analgesics, as potent as fentanyi, a fully synthetic agent. Analogs in this family were synthetized with varir,,,~ N-substituents: methyl, allyl, cyclopropylmethyl. These compounds were evaluated for receptor agonism and antagonism in various animal assays. The results obtained show, that while the formal inversion of the 6-OH group of morphine to a-isomorplune or a-isocodeine does not