Biomedical subjects

M C Fournie-Zaluski

Publications and source records attributed to M C Fournie-Zaluski.

At least 19 recordsLinked to original sources

PubMed2001

First discrete autoradiographic distribution of aminopeptidase N in various structures of rat brain and spinal cord using the selective iodinated inhibitor [125I]RB 129.

The selective and potent aminopeptidase N inhibitor [125I]RB 129 has been used for the radioautographic localization of this enzyme in rat brain, spinal cord and intestine. Brain microvessels and intestine brush-border cells were shown to present a high concentration of aminopeptidase N. Moreover, a labeling of various brain structures was observed. A very high level of binding occurred in the meninges, choroid plexus, pineal gland, paraventricular nucleus and pituitary gland. Moderate to high labeling was also observed in the cortex, caudate-putamen, subthalamic nucleus, central periaqueductal gray, thalamus, as well as in the dorsal and ventral horn of the spinal cord, which are known to contain a high concentration of enkephalins, opioid receptors and neutral endopeptidase. This co-localization confirms the physiological implication of aminopeptidase N in the inactivation of enkephalins accounting for the requirement of dual inhibition of neutral endopeptidase and aminopeptidase N to observe highly significant morphine-like effects induced by the protected endogenous opioid peptides. Aminopeptidase N was also visualized in moderate to high levels in other brain structures such as the hippocampus, nucleus accumbens, substantia nigra, hypothalamus (dorsomedial and ventromedial nuclei), raphe nucleus, pontine nucleus, inferior olive, and in high concentration in the granular layer of cerebellum. In summary, aminopeptidase N has been visualized for the first time in numerous brain areas using the selective inhibitor [125I]RB 129. This iodinated probe could allow the ex vivo and in vivo localization of aminopeptidase N in various tissues to be investigated and may also be used to evaluate quantitative changes in aminopeptidase N expression in pathological situations. Aminopeptidase N, which preferably removes NH2-terminal neutral amino acids from peptides, has probably a host of substrates. Nevertheless, a certain in vivo selectivity could be achieved by the presence of the enzyme in structures where the peptide effector and its receptors are also co-localized.

Animals↗

PubMed2000

Exploration of the S(')(1) subsite of neprilysin: a joined molecular modeling and site-directed mutagenesis study.

Based on the recently described three-dimensional model of the 507-749 region of neprilysin, which contains the catalytic site of the enzyme, experiments were performed to improve the proposed topology of its large and hydrophobic S(')(1) subsite. Docking studies, site-directed mutagenesis, and biochemical studies were combined. The mutations of various residues proposed to be part of the S(')(1) subsite (F563A, F564A, M579A, F716A, and I718A) did not induce major structural reorganization of the active site as demonstrated by the slight modification of the enzyme activity. The mutations were also analyzed by measuring the inhibitory potencies of thiol inhibitors containing P(')(1) moieties of increasing sizes. These results combined with molecular modeling studies support the proposed topology of the S(')(1) subsite. This, and the critical role of F563 and M579 in inhibitor binding, could facilitate the synthesis of new potent and selective inhibitors.

Animals↗

PubMed2000

Aminopeptidase A, which generates one of the main effector peptides of the brain renin-angiotensin system, angiotensin III, has a key role in central control of arterial blood pressure.

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental animal models. We have recently reported that, in the murine brain RAS, angiotensin II (AngII) is converted by aminopeptidase A (APA) into angiotensin III (AngIII),which is itself degraded by aminopeptidase N (APN), both peptides being equipotent to increase vasopressin release and arterial blood pressure when injected by the intracerebroventricular (i.c.v.) route. Because AngII is converted in vivo into AngIII, the exact nature of the active peptide is not precisely known. To delineate their respective roles in the central control of cardiovascular functions, specific and selective APA and APN inhibitors are needed to block the metabolic pathways of AngII and AngIII respectively. In the absence of such compounds for APA, we first explored the organization of the APA active site by site-directed mutagenesis. This led us to propose a molecular mechanism of action for APA similar to that proposed for the bacterial enzyme thermolysin deduced from X-ray diffraction studies. Secondly, we developed a specific and selective APA inhibitor, compound EC33 [(S)-3-amino-4-mercaptobutylsulphonic acid], as well as a potent and selective APN inhibitor, PC18 (2-amino-4-methylsulphonylbutane thiol). With these new tools we examined the respective roles of AngII and AngIII in the central control of arterial blood pressure. A central blockade of APA with the APA inhibitor EC33 suppressed the pressor effect of exogenous AngII, suggesting that brain AngII must be converted into AngIII to increase arterial blood pressure. Furthermore, EC33, injected alone i.c.v. but not intravenously, caused a dose-dependent decrease in arterial blood pressure by blocking the formation of brain AngIII but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor PC18 administered alone via the i.c.v. route increased arterial blood pressure. This pressor response was blocked by prior treatment with the angiotensin type 1 receptor antagonist losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in arterial blood pressure increase through an interaction with angiotensin type 1 receptors. These results demonstrate that AngIII is a major effector peptide of the brain RAS, exerting a tonic stimulatory control over arterial blood pressure. Thus APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors, crossing the blood-brain barrier, as central anti-hypertensive agents.

Aminopeptidases↗

PubMed1999

Aminopeptidase A inhibitors as potential central antihypertensive agents.

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental models, such as spontaneously hypertensive rats and transgenic mice expressing both human renin and human angiotensinogen transgenes. We recently reported that, in the murine brain, angiotensin II (AngII) is converted to angiotensin III (AngIII) by aminopeptidase A (APA), whereas AngIII is inactivated by aminopeptidase N (APN). If injected into cerebral ventricles (ICV), AngII and AngIII cause similar pressor responses. Because AngII is metabolized in vivo into AngIII, the exact nature of the active peptide is not precisely determined. Here we report that, in rats, ICV injection of the selective APA inhibitor EC33 [(S)-3-amino-4-mercaptobutyl sulfonic acid] blocked the pressor response of exogenous AngII, suggesting that the conversion of AngII to AngIII is required to increase blood pressure (BP). Furthermore, ICV injection, but not i.v. injection, of EC33 alone caused a dose-dependent decrease in BP by blocking the formation of brain but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol), administered alone via the ICV route, increases BP. This pressor response was blocked by prior treatment with the angiotensin type 1 (AT(1)) receptor antagonist, losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in BP increase, through interaction with AT(1) receptors. These data demonstrate that AngIII is a major effector peptide of the brain RAS, exerting tonic stimulatory control over BP. Thus, APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors as central antihypertensive agents.

Aminopeptidases↗

PubMed1999

A three-dimensional construction of the active site (region 507-749) of human neutral endopeptidase (EC.3.4.24.11).

A three-dimensional model of the 507-749 region of neutral endopeptidase-24.11 (NEP; E.C.3.4.24.11) was constructed integrating the results of secondary structure predictions and sequence homologies with the bacterial endopeptidase thermolysin. Additional data were extracted from the structure of two other metalloproteases, astacin and stromelysin. The resulting model accounts for the main biological properties of NEP and has been used to describe the environment close to the zinc atom defining the catalytic site. The analysis of several thiol inhibitors, complexed in the model active site, revealed the presence of a large hydrophobic pocket at the S1' subsite level. This is supported by the nature of the constitutive amino acids. The computed energies of bound inhibitors correspond with the relative affinities of the stereoisomers of benzofused macrocycle derivatives of thiorphan. The model could be used to facilitate the design of new NEP inhibitors, as illustrated in the paper.

Amino Acid Sequence↗

PubMed1998

Differences in transition state stabilization between thermolysin (EC 3.4.24.27) and neprilysin (EC 3.4.24.11).

Important homologies in the topology of the catalytic site and the mechanism of action of thermolysin and neprilysin have been evidenced by site-directed mutagenesis. The determination of differences in transition state stabilization between these peptidases could facilitate the design of specific inhibitors. Thus, two residues of thermolysin which could be directly (Tyr157) or indirectly (Asp226) involved in the stabilization of the transition state and their putative counterparts in neprilysin (Tyr625 and Asp709) have been mutated. The results show that Tyr157 is important for thermolysin activity while Tyr625 has no functional role in neprilysin. Conversely, the mutation of Asp226 induced a slight perturbation of thermolysin activity while Asp709 in neprilysin seems crucial in neprilysin catalysis. Taken together these data seem to indicate differences in the transition state mode of stabilization in the two peptidases.

Amino Acid Sequence↗

PubMed1998

Aminophosphinic inhibitors as transition state analogues of enkephalin-degrading enzymes: a class of central analgesics.

Inhibition of aminopeptidase N and neutral endopeptidase-24.11, two zinc metallopeptidases involved in the inactivation of the opioid peptides enkephalins, produces potent physiological analgesic responses, without major side-effects, in all animal models of pain in which morphine is active. Dual inhibitors of both enzymes could fill the gap between opioid analgesics and antalgics. Until now, attempts to find a compound with high affinity both for neutral endopeptidase and aminopeptidase N have failed. We report here the design of dual competitive inhibitors of both enzymes with KI values in the nanomolar range. These have been obtained by selecting R1, R2, and R3 determinants in aminophosphinic-containing inhibitors: NH2---CH(R1)P(O)---(OH)CH2---CH(R2)CONH---CH(R3)COOH, for optimal recognition of the two enkephalin inactivating enzymes, whose active site peculiarities, determined by site-directed mutagenesis, have been taken into account. The best inhibitors were 10x more potent than described dual inhibitors in alleviating acute and inflammatory nociceptive stimuli in mice, thus providing a basis for the development of a family of analgesics devoid of opioid side effects.

Analgesics, Non-Narcotic↗

PubMed1997

Cooperative exosite-dependent cleavage of synaptobrevin by tetanus toxin light chain.

The light chain (L chain) of tetanus neurotoxin (TeNT) has been shown to have been endowed with zinc endopeptidase activity, selectively directed toward the Gln76-Phe77 bond of synaptobrevin, a vesicle-associated membrane protein (VAMP) critically involved in neuroexocytosis. In previous reports, truncations at the NH2 and COOH terminus of synaptobrevin have shown that the sequence 39-88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH2- and COOH-terminal peptides of synaptobrevin, S 27-55 (S1) and S 82-93 (S2), to the synaptobrevin fragment S 56-81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S1 and S2 with complementary sites present on TeNT as shown by surface plasmon resonance experiments and the determination of kinetic constants. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT, probably involving a conformational change of the toxin. This could account for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins.

Amino Acid Sequence↗

PubMed1997

Aminopeptidase A: distribution in rat brain nuclei and increased activity in spontaneously hypertensive rats.

Aminopeptidase A is a membrane-bound zinc metalloprotease which cleaves angiotensin II into angiotensin III. Using a new specific aminopeptidase A inhibitor, EC33, we evaluated its enzymatic activity in several microdissected brain nuclei involved in the control of cardiovascular functions and in the pituitary. We compared this distribution with that of the angiotensin I-converting enzyme which converts angiotensin I to angiotensin II. Aminopeptidase A activity was heterogenously distributed with a 150-fold difference between the lowest and the highest levels. The pituitary and the circumventricular organs were the richest source of enzyme, followed by the median eminence, the arcuate nucleus, the area postrema, the choroid plexus and the supraotic and paraventricular nuclei. We did not find any close parallel between aminopeptidase A and angiotensin I-converting enzyme distributions. We examined both enzymatic activities in brain nuclei of spontaneously hypertensive rats. Aminopeptidase A activity was higher in the spontaneously hypertensive rats than in age-matched Wistar Kyoto control rats. The difference was up to 2.5-fold in several brain nuclei involved in the blood pressure regulation; in contrast, no differences in angiotensin I-converting enzyme activity were found in the same regions. The close correspondence between the distribution of aminopeptidase A activity and angiotensin receptors and nerve terminals in the brain associated with the observation that aminopeptidase A activity was overactivated in the spontaneously hypertensive rats suggest that this enzyme may contribute, at least in part, to the regulation of cardiovascular functions by its ability to convert angiotensin II to angiotensin III.

Aminopeptidases↗

PubMed1996

Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release.

Angiotensin (Ang) II and Ang III are two peptide effectors of the brain renin-angiotensin system that participate in the control of blood pressure and increase water consumption and vasopressin release. In an attempt to delineate the respective roles of these peptides in the regulation of vasopressin secretion, their metabolic pathways and their effects on vasopressin release were identified in vivo. For this purpose, we used recently developed selective inhibitors of aminopeptidase A (APA) and aminopeptidase N (APN), two enzymes that are believed to be responsible for the N-terminal cleavage of Ang II and Ang III, respectively. Mice received [3H]Ang II intracerebroventricularly (i.c.v.) in the presence or absence of the APN inhibitor, EC33 (3-amino-4-thio-butyl sulfonate) of the APN inhibitor, EC27 (2-amino-pentan-1,5-dithiol). [3H]Ang II and [3H]Ang III levels were evaluated from hypothalamus homogenates by HPLC. EC33 increased the half-life of [3H]Ang II 2.6-fold and completely blocked the formation of [3H]Ang III, whereas EC27 increased the half-life of [3H]Ang III 2.3-fold. In addition, the effects of EC33 and EC27 on Ang-induced vasopressin release were studied in mice. Ang II was injected i.c.v. in the presence or absence of EC33, and plasma vasopressin levels were estimated by RIA. While vasopressin levels were increased 2-fold by Ang II (5 ng), EC33 inhibited Ang II-induced vasopressin release in a dose-dependent manner. In contrast, EC27 injected alone increased in a dose-dependent manner vasopressin levels. The EC27-induced vasopressin release was completely blocked by the coadministration of the Ang receptor antagonist (Sar1-Ala8) Ang II. These results demonstrate for the first time that (i) APA and APN are involved in vivo in the metabolism of brain Ang II and Ang III, respectively, and that (ii) the action of Ang II on vasopressin release depends upon the prior conversion of Ang II to Ang III. This shows that Ang III behaves as one of the main effector peptides of the brain renin-angiotensin system in the control of vasopressin release.

Amino Acid Sequence↗

PubMed1996

Design of orally active dual inhibitors of neutral endopeptidase and angiotensin-converting enzyme with long duration of action.

Mercaptoacyl dipeptides, containing a glycine linked to a C-terminal 5-phenylproline, have been synthesized in order to obtain new highly efficient dual inhibitors of the two zinc metallopeptidases, neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), which are involved in the control of blood pressure and fluid homeostasis. These compounds have been designed (i) to fit optimally the ACE pharmacophore previously described (Fournié-Zaluski, M. C.; et al. J. Med. Chem. 1994, 37, 1070-1083), through interaction with the S1, S1', and S2' subsites of this enzyme, (ii) and to interact with the S1' and S2' subsites of NEP with the 5-phenylproline moiety outside the catalytic domain (Coric, P.; et al. J. Med. Chem. 1996, 39, 1210-1219). Replacement of Gly by Ala in these mercaptoacyl dipeptides induced an about 100-fold decrease in ACE inhibition. This shows that, in agreement with molecular modeling studies, a steric constraint as weak as a methyl group hinders optimal ACE active site recognition. Among these compounds, the dual inhibitor 26 (RB 106) (Ki, ACE = 0.35 nM; NEP = 1.6 nM) showed excellent pharmacokinetic properties with an almost complete in vivo inhibition of NEP and ACE for more than 4 h after oral administration in mice of a low dose (2.6 x 10(-5) mol/kg) of the inhibitor. Moreover, RB 106 remained active 12 h after oral administration. In spontaneous hypertensive rats, a chronic treatment of orally administered RB 106 (25 mg/kg/day) induced a prolonged hypotensive effect (-28 mmHg) still significant 2 days after the end of the treatment. In DOCA salt rats, a hypotensive response and a significant natriuresis were observed after i.v. administration. RB 106, which is one of the most potent dual inhibitors described to date, could have interesting clinical applications in long term treatment of congestive heart failure and myocardial ischemia.

Administration, Oral↗

PubMed1996

Optimal recognition of neutral endopeptidase and angiotensin-converting enzyme active sites by mercaptoacyldipeptides as a means to design potent dual inhibitors.

An interesting approach for the treatment of congestive heart failure and chronic hypertension could be to avoid the formation of angiotensin II by inhibiting angiotensin converting enzyme (ACE) and to protect atrial natriuretic factor by blocking neutral endopeptidase 24.11 (NEP). This is supported by recent results obtained with potent dual inhibitors of the two zinc metallopeptidases, such as RB 105, HSCH2CH(CH3)PhCONHCH(CH3)COOH (Fournié-Zaluski et al. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4072-4076), which reduces blood pressure in experimental models of hypertension, independently of the salt and renin angiotensin system status. In order to develop new dual inhibitors with improved affinities, long duration of action, and/or better bioavailabilities, various series of mercaptoacyldipeptides corresponding to the general formula HSCH(R1)CONHCH(R1')CON(R)CH(R2')COOH have been synthesized. The introduction of well-selected beta-branched chains in positions R1 and R1', associated with a tyrosine or a cyclic amino acid in the C-terminal position, led to potent dual inhibitors of NEP and ACE such as 21 [N-[(2S)-2-mercapto-3-methylbutanoyl]-Ile-Tyr] and 22 [N-[(2S)-2-mercapto-3-phenylpropanoyl]Ala-Pro] which have IC50 values in the nanomolar range for NEP and subnanomolar range for ACE. These compounds could have different modes of binding to the two peptidases. In NEP, the dual inhibitors seem to interact only with the S1' and S2' subsites, whereas additional interactions with the S1 binding subsite of ACE probably account for their subnanomolar inhibitory potencies for this enzyme. The localization of the Pro residue of 22 outside the NEP active site is supported by biochemical data using (Arg102,Glu)NEP and molecular modeling studies with thermolysin used as model of NEP. One hour after oral administration in mice of a single dose (2.7 x 10(-5) mol/kg), 21 inhibited 80% and 36% of kidney NEP and lung ACE, respectively, while 22 inhibited 40% of kidney NEP and 56% of lung ACE.

Amino Acid Sequence↗

PubMed1996

Effects of angiotensin-converting enzyme and neutral endopeptidase inhibitors: influence of bradykinin.

These experiments compare the effects of a neutral endopeptidase inhibitor, retrothiorphan, 1-[(1-mercaptomethyl-2-phenyl)ethyl]amino-1-oxopropanoic acid, a converting enzyme inhibitor, enalaprilat, and the combination of the two inhibitors on changes in blood pressure and renal function induced by exogenous and endogenous bradykinin in deoxycorticosterone acetate (DOCA)-salt rats. Enalaprilat potentiated the exogenous bradykinin-induced hypotensive responses while retrothiorphan potentiated the effects on urinary cyclic-GMP (cGMP) and bradykinin. The combination potentiated the exogenous bradykinin-induced hypotensive effects and the bradykinin-induced urinary excretion of cGMP, bradykinin and prostaglandin. The bradykinin B2 receptor antagonist, Hoe 140, had no effect on the enalaprilat- and retrothiorphan-induced changes in blood pressure and renal function. In conclusion, while angiotensin-converting enzyme and neutral endopeptidase are involved in the vascular and renal catabolism of exogenous bradykinin, the effects of the peptidase inhibitors do not appear to depend on the protection of endogenous bradykinin under acute conditions in DOCA-salt rats.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1996

The CCKB antagonist PD-134,308 facilitates rewarding effects of endogenous enkephalins but does not induce place preference in rats.

The interaction between cholecystokinin and endogenous opioid systems on rewarding responses was examined. Motivational effects induced by peripheral administration of a complete inhibitor of enkephalin catabolism, RB 101 or the CCKB antagonist PD-134,308, and by both compounds in combination were evaluated in the conditioned place preference test in rats. RB 101 (5, 10, 20, 40 and 80 mg/kg, IP, and 20 mg/kg, IV) given alone produced a bell-shaped dose-effect function. A significant increase of the preference for the drug-associated compartment was only observed at doses of 10 and 20 mg/kg (IP). The effect observed with morphine was stronger, and all the doses used of this compound (1.25, 2.5 and 5 mg/kg, SC) were found to be active. These results suggest that the inhibitor of enkephalin catabolism has weak rewarding properties. Pretreatment with the CCKB antagonist PD-134,308 (0.1, 0.3, 1 and 3 mg/kg, IP) alone failed to produce a reliable aversion or preference on the paradigm studied. When PD-134,308 (0.3 mg/kg, IP) was coadministered with a subthreshold dose of morphine (0.6 mg/kg, SC) or RB 101 (5 mg/kg, IP), a conditioned place preference was observed, indicating that the CCKB antagonist facilitated the motivational responses induced by endogenous enkephalins as compared to morphine. This suggests that endogenous cholecystokinin, acting through CCKB receptors, modulates the rewarding effects of endogenous enkephalins.

Animals↗

PubMed1996

Opposite role of delta 1- and delta 2-opioid receptors activated by endogenous or exogenous opioid agonists on the endogenous cholecystokinin system: further evidence for delta-opioid receptor heterogeneity.

Using the mouse caudate-putamen, where delta-opioid receptor subtypes have been shown to regulate adenylyl cyclase activity, we show in this study that endogenous enkephalins inhibit enzyme activity through activation of delta 1- and delta 2-opioid receptors. Thus, naltriben or 7-benzylidenenaltrexone as well as the delta-selective antagonist naltrindole (mixed delta 1 and delta 2 antagonist) antagonized inhibition of adenylyl cyclase activity induced by methionine- or leucine-enkephalin, while the micro-antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) was without effect. Furthermore, we have previously shown that activation of delta-opioid receptors increases cholecystokinin release in the central nervous system, resulting in a potentiation of micro-opioid antinociceptive responses, and the respective role of delta 1- and delta 2-opioid receptors in this facilitatory effect has now been evaluated. Activation of delta 2-opioid receptors, either by endogenous enkephalins protected from catabolism by the complete enkephalin-degrading enzyme inhibitor N-((R,S)-2-benzyl-3((S)(2-amino-4-methyl-thio) butyldithio)-1-oxopropyl)-L-phenyl-alanine benzyl ester (RB 101), or by the delta 2-selective agonist Tyr-D-Ser(O-tert-butyl)-Gly-Phe-Leu-Thr(O-tert-butyl) (BUBU), potentiated micro-opioid antinociceptive responses in the hot-plate test in mice. This effect was antagonized by a selective cholecystokinin-A antagonist. Activation of delta 1-opioid receptors by endogenous opioid peptides decreased the micro-opioid responses. These results suggest that stimulation of delta 2-opioid receptors potentiates micro-opioid analgesia in the hot-plate test in mice through an increase in endogenous cholecystokinin release, while activation of delta 1-opioid receptors could decrease it. Thus, the pre-existing physiological balance between opioid and cholecystokinin systems seems to be modulated in opposite directions depending on whether delta 1- or delta 2-opioid receptors are selectively activated. This is the first demonstration that endogenous enkephalins, methionine- and leucine-enkephalin, are the natural ligands of delta-opioid receptor subtypes, and that delta 2-opioid receptor activation may facilitate the endogenous cholecystokinin-related modulation of micro-opioid analgesia, while the delta 1-opioid receptors may have an inhibitory role. These results could have important applications for the characterization of opioid delta 1 and delta 2 as subtypes or subsites and in pain alleviation.

Adenylyl Cyclases↗

PubMed1995

Opposite role of CCKA and CCKB receptors in the modulation of endogenous enkephalin antidepressant-like effects.

Systemic administration of RB 101, a complete inhibitor of the enkephalin degrading enzymes, has been reported to induce naltrindole-reversed antidepressant-like effects in the conditioned suppression of motility (CSM) test in mice. The selective CCKB antagonist L-365,260 also elicits the same naltrindole-blocked responses on CSM. The aim of this study was therefore to investigate the possible modulation of RB 101 induced behavioral responses by activation or blockade of CCK receptors. Thus, the effects induced by RB 101 administered alone or associated with an ineffective dose of a selective CCKB agonist (BC 264), a CCKB antagonist (L-365,260) or a CCKA antagonist (L-364,718), were evaluated on the CSM in mice. RB 101 alone decreased the stress-induced loss of motility, as previously reported. The antidepressant-like effect of RB 101 was potentiated by L-365,260, and suppressed by BC 264 and to a lesser extent by L-364,718. The facilitatory effect induced by L-365,260 on RB 101 responses was blocked by the delta selective antagonist naltrindole. All these effects occurred only in shocked animals. The present results suggest that the activation of CCKA and CCKB receptors by endogenous CCK, could play an opposite role in the control of behavioral responses induced by endogenous enkephalins. Delta opioid receptors seem to be selectively involved in this interaction.

Analgesics↗

PubMed1995

Inhibition of morphine withdrawal by the association of RB 101, an inhibitor of enkephalin catabolism, and the CCKB antagonist PD-134,308.

1. The effects induced in rats on naloxone-precipitated morphine withdrawal syndrome by the new mixed inhibitor of enkephalin catabolism able to cross the blood-brain barrier RB 101 (N-((R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyl dithio]-1-ox-opropyl-L-phenylalanine benzyl ester) given alone or associated with the selective CCKB antagonist, PD-134,308, were investigated. 2. The systemic administration of RB 101 (5, 10 and 20 mg kg-1, i.v.) elicited a significant decrease in 8 of the 14 withdrawal signs evaluated. PD-134,308 (3 mg kg-1, i.p.) did not modify the expression of morphine abstinence when given alone, but induced a strong facilitation of RB 101 responses (12 of 14 withdrawal signs were decreased). This potentiation was particularly intense in peripherally mediated withdrawal signs. 3. In order to clarify the biochemical mechanisms implicated in these responses, the effects induced by the association of RB 101 and PD-134,308 on the occupation of brain opioid receptors by endogenous enkephalins were also investigated in mice. PD-134,308, as well as RB 101, inhibited [3H]-diprenorphine binding to opioid receptors. These results suggest that an increase in endogenous enkephalin levels induced by PD-134,308 could participate in the facilitation of RB 101 behavioural responses. 4. RB 101 has a promising potential role in the management of the opiate withdrawal syndrome. CCKB antagonists, such as PD-134,308 may be useful in potentiating this anti-withdrawal effect.

Analgesics↗

PubMed1995

Effect of endopeptidase-24.11 inhibitors and C-ANP receptor ligand on responses evoked in arterioles of rat cremaster muscle by atrial natriuretic peptide.

1. The present study examined the effect of exogenous atrial natriuretric peptide (ANP), alone or in presence of inhibitors of the two major mechanisms for clearing ANP, metabolism by neutral endopeptidase-24.11 (NEP) and internalization by C-ANP receptors, on arteriolar responses using intravital microscopy on the rat cremaster muscle after intravenous or topical administration of the peptide. 2. Topical application of ANP (3 x 10(-10) to 3 x 10(-8) M) produced a gradual increase in arteriolar diameter. NEP inhibitors, thiorphan (30 mg kg-1, i.v.), kelatorphan (10 mg kg-1, i.v.) and retrothiorphan (25 mg kg-1, i.v.) alone, did not significantly affect vascular tone but caused significant potentiation of the arteriolar responses to topically applied ANP. 3. When given as an i.v. bolus, ANP dilates skeletal arterioles at a high dose (20 micrograms kg-1). At a lower dose (10 micrograms kg-2), ANP alone or with retrothiorphan or the C-ANP receptor ligand C-ANP (4-23) did not produce any arteriolar responses, while after the combined administration of the two inhibitors, an increase in arteriolar diameter was induced. 4. These results indicate that low doses of topically applied ANP dilate rat cremaster arterioles and that the vasodilator responses can be potentiated by NEP inhibition. When given as an i.v. bolus, a high dose of ANP can also dilate skeletal arterioles. However at a lower dose the rapid metabolism of the peptide prevents it from producing its action.

Abdominal Muscles↗