Biomedical subjects
Publications and source records attributed to J Bockaert.
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Washed membrane particles from pig Graafian follicles larger than 6 mm in diameter were found to contain an adenylyl cyclase system that is highly responsive to luteinizing hormone (LH). Under the assay conditions used (1.5 mM ATP, 5 mM MgCl2, 1.4 mM EDTA, and 1.0 mM ethylene glycol bis(beta-aminoethyl)-N,N'-tetraacetic acid, pH 7.0) LH stimulated enzyme activity up to 7-fold. Half-maximal activation was obtained between 3 and 6 X 10(-10) M of added LH, provided the reaction was carried out under equilibrium conditions. In contrast to findings in large follicles, adenylyl cyclase activity was found to be unresponsive to LH in small immature follicles (1 to 2 mm in diameter) and partially responsive to LH (2- to 3-fold stimulation over basal) in follicles of medium size (3 to 5 mm in diameter). Washed membrane particles from follicles larger than 6 mm were found to contain also an ATP- and Mg2+-dependent process which, upon incubation at 30 degrees, causes the adenylyl cyclase system to lose its susceptibility to respond to LH. Because this loss of LH-stimulated activity was not accompanied by a concomitant loss of basal activity, it was concluded to be the expression of a desensitization reaction. Desensitization was almost undetectable at 5 mM total added MgCl2 and required, to be readily detectable, addition of MgCl2 concentrations in excess of 10 mM. LH was found to stimulate desensitization in the presence of 1 mM cyclic adenosine 3':5'-monophosphate by diminishing the requirement for MgCl2. Loss of LH-stimulated adenylyl cyclase activity was readily detectable at 5.0 mM total added MgCl2 in the presence of 10 mug per ml of LH. Half-maximal stimulation of desensitization by LH was found to occur at about 25 X 10(-10) M, i.e. at about 4 to 6 times higher concentrations than needed for half-maximal stimulation of adenylyl cyclase activity. Half-maximal LH-stimulated desensitization was obtained with 0.5 to 0.7 mM ATP in the incubation. Neither GTP, nor adenyl-5'-yl imidodiphosphate, the nonphosphorylating imidodiphosphate analogue of ATP, supported the desensitization reaction, suggesting that this process may be mediated via phosphorylation of one or more of the components of the adenylyl cyclase system. LH-stimulated desensitization was detected also in washed membrane particles derived from dissected rabbit Graafian follicles, whose adenylyl cyclase becomes desensitized in vivo within minutes after human chorionic gonadotropin injection (Hunzicker-Dunn, M., and Birnbaumer, L., Endocrinology, in press), but not in membrane particles from corpora lutea of pregnant or pseudopregnant rabbits, whose adenylyl cyclase becomes desensitized in vivo only several hours or days after human chorionic gonadotropin injection (Hunzicker-Dunn, M., and Birnbaumer, L., Endocrinology, in press). It is suggested that both actions of LH (stimulation of adenylyl cyclase and stimulation of desensitization) are receptor-mediated, and that neither of them is mediated by cyclic adenosine 3':5'-monophosphate...
The characteristics of dopamine adenylate cyclase in the rat striatum were first studied on homogenates of fresh tissues. In the assay conditions used, dopamine (10(-4) M) stimulated the enzyme activity by 250%. This effect was completely blocked by fluphenazine (10(-5) M; Ki=9X10(-9) M) and by phentolamine (10(-5) M; Ki=3 X 10(-7) M). D-LSD stimulated the adenylate cyclase activity (Km=1.4 X 10(-7) M) by interacting with dopamine receptors; indeed the dopamine effect on the enzyme activity was competitively reduced in presence of D-LSD. L-Isoproterenol (Km=10(-6) M) activated an adenylate cyclase through a receptor distinct form the dopaminergic receptor; this stimulation was not affected by fluphenazine or phentolamine but suppressed by DL-propranolol (10(-4) M). The topographical distribution of the dopamine, D-LSD and L-isoproterenol adenylate cyclase activities were examined in homogenates prepared from discs punched out on serial frozed (--7C) slices of the striatum. Under this condition, tge dioanube naxunak stunykatuib was if 150%. A 4.8-fold progressive decrease in the amount of cyclic AMP produced in presence of dopamine (10(-4) M) was observed in the rostrocaudal plane of the structure; the decline of the basal activity was 3.6-fold. The topographical curves of maximal activation of adenylate cyclase by dopamine and D-LSD were superimposable confirming that D-LSD acts on dopaminergic receptors. This topographical distribution of dopamine sensitive adenylate cyclase is comparable on one hand to that of endogenous dopamine and on the other hand to that of the dopamine high affinity uptake activity measured in simultaneous experiments. In contrast to that observed with dopamine or D-LSD, the topographical distribution of the adenylate cyclase sensitive to L-isoproterenol was homogenous within the striatum.
Since quipazine is a potent 5-HT agonist in peripheral organs, its possible stimulatory effects on serotoninergic receptors in the rat brain were investigated. Quipazine administration (10 mg/kg, i.p.) induced a significant decrease in the synthesis and turnover rates of serotonin in the brain stem as well as in the forebrain. It is not likely that these changes were mediated by a negative feed-back mechanism triggered by a direct action of quipazine on central 5-HT postsynaptic receptors. Indeed, in contrast to LSD and 5-methoxy-N,N-dimethyltryptamine, this compound failed to activate the 5-HT sensitive adenylate cyclase in colliculi homogenates of newborn rats. However, quipazine exerted direct effects on serotoninergic terminals. It inhibited competitively the reuptake process in synaptosomes (Ki=1.38 X 10(-7) M) and stimulated the K+ evoked release of newly synthesized 3H-5-HT in slices of the brain stem. Injected in vivo in a dose which affected 5-HT uptake and release, quipazine did not modify MAO activity. However, this activity was non-competitively inhibited by high concentratin of the drug in vitro (Ki=3.0 X 10(-5) M). These actions are very likely indirectly responsible for the stimulation of central 5-HT receptors.
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Responsiveness of ovarian adenylyl cyclases to luteinizing hormone (LH), found to be 5 to 10-fold in cell-free preparations under optimal conditions, required gentle homogenizations and storage in sucrose-containing media. Assay conditions required the use of an ATP-regenerating system consisting of creatine kinase, creatine phosphate, and myokinase for the preservation of ATP levels. LH-stimulated adenylyl cyclase (AC) in rabbit CL showed the following properties: 1) The pH optimum of basal activity was about 8.0; that of LH-stimulated activity was about 7.5. 2) The relative response to LH was low (1.5 to 2-fold) at 0.1 mM ATP and increased with increasing ATP, but not with increasing GTP. At low (0.1 mM) ATP, GTP increased catalytic efficacy of the system, both in the absence and in the presence of LH (no effect on relative stimulation). 3) The optimal relative stimulation by LH was obtained at about 1.0 mM MgCl2 in excess of added magnesium-binding ingredients. 4) The sensitivity to stimulation by LH (about 0.2 mug/ml NIH-LH-B8) was unaffected by either pH, nucleotides (ATP and GTP), or MgCl2 concentration. 5) Under the assay conditions used, activity was stimulated by prostaglandin E1 (PGE1) about 1.5 to 2-fold, and by epinephrine about 3 to 4-fold. In all aspects tested, LH-stimulated AC in rat CL resembled that in rabbit CL, except that about 5-fold higher concentrations of NIH-LH-B8 were needed for half-maximal stimulation. The AC activity in pig Graafian follicles, however, differed from that in rabbit CL in that 1) the ATP concentration needed for optimal stimulation by LH was lower (in the micromolar rather than the millimolar range); 2) catecholamines elicited only a 1.3 to 1.4-fold stimulation; and 3) NIH-LH-B8 elicited half-maximal stimulation at 0.008 to 0.020 mug/ml. We were unable to detect LH-responsive AC activity in either homogenates or washed particles of CL from either cycling or pregnant pigs. LH fractions of three origins (human, bovine, and ovine) and of varying specific activities (from 0.041 to 2.0 NIH-LH-S18 units/mg) were tested and the relative potencies by OAAD assay were found to correlate well with the relative potencies in the adenylyl cyclase assays (rat CL, rabbit CL, and pig follicles), consistent with the possibility that AC receptors are responsible for biologic actions of LH.
The characteristics of dopamine, 1-isoproterenol, and d-LSD stimulated adenylate cyclases were studied in homogenates of fresh or frozen tissues. In rat striatum, when the assay was done in the presence of 1 mM MgSO4, dopamine (10(-4) M) stimulated the enzyme activity by 3.5-fold. This effect was completely blocked by fluphenazine (10(-5) M; Ki = 9 X 10(-9) M) and by phentolamine (Ki = 3 X 10(-7) M). d-LSD stimulated the adenylate cyclase activity (Km = 1.4 X 10(-7) M) by interacting with the dopaminergic receptors. Maximal adenylate cyclase stimulation by d-LSD was 1.4-fold; as a matter of fact, this compound acted as a partial agonist on the dopaminergic receptors. l-Isoproterenol (Km = 10(-6) M) activated an adenylate cyclase present in rat striatum homogenates through a receptor distinct from the dopaminergic receptor; this stimulation was not affected by addition of fluphenazine or phentolamine but suppressed by dl-propranolol (10(-4) M). The topographical distributions of dopamine adenylate cyclase activity and endogeneous dopamine content were examined in homogenates prepared from discs punched out from serial frozen (-7 degrees C) slices of the striatum. A 4.8-fold progressive decrease in the amount of cyclic AMP produced in the presence of dopamine (10(-4) M) was observed from the rostral to the caudal part of the structure. The d-LSD-sensitive adenylate cyclase followed a similar distribution. It should be noted that the topographic distribution of endogeneous dopamine is quite comparable to the distribution of the dopamine-sensitive adenylate cyclase, suggesting that this enzyme is an integral part of the dopamine synapses. We also reported that the frontal cortex contains a dopamine-sensitive adenylate cyclase. In conclusion, we trust that the micromethod described for adenylate cyclase assay will be of some use in the study of the precise topographic distribution of catecholamine sensitive adenylate cyclases in different structures of brain.
Adenylate cyclase and the [8-lysine]vasopressin receptor were solubilized from pig kidney medulla membranes using the nonionic detergent Triton X-100. Optimal conditions for solubilization were under continuous stirring in a medium containing 0.5% (/v) Triton X-100, 100 mM Tris-HCl, pH 8, and 10 mM MgCl2. Both adenylate cyclase activity and [3H][8-lysine]vasopressin binding activity were recovered in a -26,000 X g supernatant of detergent-treated membranes. The yield of solubilized adenylate cyclase was nearly 100%. The soluble enzyme was no longer sensitive to antidiuretic hormone but was slightly activated by sodium fluoride. The affinity of the soluble receptor for [8-lysine]vasopresin was les than that of the membrane-bound receptor (mean apparent Km values, respectively 10(-7) M and 2 X 10(-8) M), however binding cooperativity was preserved. Hill coefficients were 1.42 for the soluble receptor and 1.50 for the membrane receptor. The soluble receptor discriminated as efficiently as did the membrane receptor between [8-lysine-a1vasopressin and oxytocin. The yield of spolubilized receptor was only 30% despite the fact that all binding activity had disappeared from the residual pellet of detergent-treated membranes. When the membranous receptors were occupied before solubilization and the latter was performed under conditions in which dissociation of the hormone-receptor comples is slow, i.e. at low temperature, 65% to 100% of the hormone-receptor complex was recovered in the soluble fraction. The soluble hormone-receptor complex partially dissociated on rewarming whereas the free hormone concentration was kept unchanged in the medium. The residual binding capacity, which was 30% of the initial value, was identical with that determined when the receptor was solubilized in free form before incubation with labeled hormone. It was concluded that (a) solubilization of the receptor molecules was complete, (b) during solubilization two forms of the receptor appear, of which only one is accessible to the hormone, (c) occupancy of the receptor by the hormone prevented the formation of the nonaccessible form, and (d) some component or components of the soluble fraction might be responsible for the loss in apparent affinity.
Recent data on the effects of neurohypophysial peptides at the cellular level are discussed with respect to the two basic processes involved in peptide hormone action--i.e., specific recognition of the information contained in the hormonal molecule and the transformation of this information into a stimulus leading to the final biological response. Four main aspects of this general problem are considered. A. Hormone-Receptor Interaction: Recent contributions in this field concern partial analysis of the three-dimensional conformation of oxytocin and vasopressin moleculal cells of the mammalian kidney. Conformational analysis of oxytocin and vasopressin molecules leads to the conclusion that, in solution, these peptides probably have a compact and highly stabilized three-dimensional configuration. Models have been proposed that provide a valuable clue to the interpretation of structure-activity relationships among natural hormones and many structural analogues. Binding studies with tritiated oxytocin and vasopressin have permitted determination of the kinetic parameters of hormone-receptor interaction in amphibian epithelial cells and mammalian kidney. B. Stimulus Generation: The nature of the primary stimulus generated by hormone-receptor interaction is still unknown. In the epithelial target cells of the amphibian skin and bladder and of the mammalian kidney, one of the first consequences of hormone-receptor interaction is the activation of membrane-bound adenylate cyclase. Analysis of the correlations between hormonal binding and adenylate cyclase activation suggests that activation is a function of receptor occupation rather than of the number of hormonal molecules interacting with the receptor per unit of time. On medullary adenylate cyclase of pig kidney, the relation between receptor occupancy and enzyme activation was found to be complex and nonlinear. The effects of several agents (calcium, nucleotides) on receptor occupancy and adenylate cyclase activation have been described. In mammalian uterus and other smooth muscle target cells, there is no evidence for direct involvement of cyclic AMP in the contractile response to oxytocin and other neurohypophysial peptides.
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