Effects of newly synthetized isoquinoline derivatives on rat uterine contractility and ROCK II activity.

Protein kinases have an important role in signal transduction in the cellular system via protein phosphorylation. RhoA activated Rho-kinases have a pivotal role in the regulation of smooth muscle contraction. ROCK I and ROCK II phosphorylate myosin-phosphatase and myosin-kinase, which induces contraction in the myometrium. Several studies have investigated the affinity of isoquinoline alkaloids (HA-1077, H1152P) to Rho-kinases, and these compounds notably inhibited the Ca2+-independent process. We measured the efficiency of 25 original, newly synthesized isoquinoline derivatives for the Rho-kinase activity using Rho-associated kinase activity assay and determined their effects on the non-pregnant, 20-day pregnant and parturient rat myometrial contraction in vitro. The IC50 values of 11 from among the 25 derivatives were significantly lower on the oxytocin-induced non-pregnant rat uterine contraction compared with Y-27632 and fasudil, although their maximal inhibitory effects were weaker than those of Y-27632 and fasudil. We measured the effects of 11 isoquinoline molecules with significant IC50 values on ROCK II activity. We found two isoquinolines out of 11 compounds (218 and 852) which decreased the active ROCK II level similarly as Y-27632. Then we found that 218 and 852 relaxed the 20th-day pregnant and parturient rat uterus with greater potency as compared with fasudil. The majority of the synthesized isoquinoline derivatives have uterus relaxant effects and two of them significantly suppress the Rho-kinase mediated myosin light chain phosphorylation. Our results may suggest that the isoquinoline structure has a promising prospect for the development of new and effective inhibitors of uterine contractions in preterm birth.

Topical dura mater application of CFA induces enhanced expression of c-fos and glutamate in rat trigeminal nucleus caudalis: attenuated by KYNA derivate (SZR72).

BACKGROUND: Migraine is a debilitating neurological disorder where trigeminovascular activation plays a key role. We have previously reported that local application of Complete Freund's Adjuvant (CFA) onto the dura mater caused activation in rat trigeminal ganglion (TG) which was abolished by a systemic administration of kynurenic acid (KYNA) derivate (SZR72). Here, we hypothesize that this activation may extend to the trigeminal complex in the brainstem and is attenuated by treatment with SZR72. METHODS: Activation in the trigeminal nucleus caudalis (TNC) and the trigeminal tract (Sp5) was achieved by application of CFA onto the dural parietal surface. SZR72 was given intraperitoneally (i.p.), one dose prior CFA deposition and repeatedly daily for 7 days. Immunohistochemical studies were performed for mapping glutamate, c-fos, PACAP, substance P, IL-6, IL-1ß and TNFα in the TNC/Sp5 and other regions of the brainstem and at the C1-C2 regions of the spinal cord. RESULTS: We found that CFA increased c-fos and glutamate immunoreactivity in TNC and C1-C2 neurons. This effect was mitigated by SZR72. PACAP positive fibers were detected in the fasciculus cuneatus and gracilis. Substance P, TNFα, IL-6 and IL-1ß immunopositivity were detected in fibers of Sp5 and neither of these molecules showed any change in immunoreactivity following CFA administration. CONCLUSION: This is the first study demonstrating that dural application of CFA increases the expression of c-fos and glutamate in TNC neurons. Treatment with the KYNA analogue prevented this expression.

KYNA analogue SZR72 modifies CFA-induced dural inflammation- regarding expression of pERK1/2 and IL-1ß in the rat trigeminal ganglion.

BACKGROUND: Neurogenic inflammation has for decades been considered an important part of migraine pathophysiology. In the present study, we asked the question if administration of a novel kynurenic acid analogue (SZR72), precursor of an excitotoxin antagonist and anti-inflammatory substance, can modify the neurogenic inflammatory response in the trigeminal ganglion. METHODS: Inflammation in the trigeminal ganglion was induced by local dural application of Complete Freunds Adjuvant (CFA). Levels of phosphorylated MAP kinase pERK1/2 and IL-1ß expression in V1 region of the trigeminal ganglion were investigated using immunohistochemistry and Western blot. FINDINGS: Pretreatment with one dose of SZR72 abolished the CFA-induced pERK1/2 and IL-1ß activation in the trigeminal ganglion. No significant change was noted in case of repeated treatment with SZR72 as compared to a single dose. CONCLUSIONS: This is the first study that demonstrates that one dose of KYNA analog before application of CFA can give anti-inflammatory response in a model of trigeminal activation, opening a new line for further investigations regarding possible effects of KYNA derivates.

Kynurenic acid modulates experimentally induced inflammation in the trigeminal ganglion.

BACKGROUND: The trigeminal ganglion (TG) plays a central role in cranial pain. Administration of complete Freund's adjuvant (CFA) into the temporomandibular joint (TMJ) elicits activation of TG. Kynurenic acid (KYNA) is an endogenous excitatory amino acid receptor blocker, which may have an anti-inflammatory effect. We hypothesize that KYNA may reduce CFA-induced activation within the TG. METHODS: A local inflammation was induced by administration of CFA into the TMJ in rats. KYNA and kynurenic acid amide 2 (KYNAA2) were intraperitoneally administered. We investigated changes of mitogen-activated protein kinases (MAPKs as ERK1/2, p38 and SAPK/JNK), NF-κB, CaMKII and DREAM, in addition to calcitonin gene-related peptide (CGRP) and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in the TG, with immunohistochemistry and Western blot at 2 and 10 days post-CFA injection. RESULTS: We showed CFA-induces increases in pERK1/2, pp38, CaMKII, NF-κB and DREAM immunohistochemistry after 2 and 10 days. KYNAA2 displayed stronger effects on MAPKs than KYNA. Increased expression of CaMKII, NF-κB and DREAM were found in the neurons. Western blot showed significantly increase in pERK expression at 10 days post-CFA, which decreased after 10 days of KYNA treatment. Two days post-CFA, a significantly increase in pp38 expression was found, which decreased after 2 days of KYNA and KYNAA2 treatment. CONCLUSIONS: The CFA-induced inflammatory model for the TG activation provided a time-related expression of MAPK (pERK1/2, pp38) and NF-κB. It involves both the neuronal and glial activation, which points to possible neuron-glia interactions during this process. The administration of the endogenous NMDA-receptor antagonists, KYNA and its derivative KYNAA2, resulted in the inhibition of the induced signaling system of the TG, which further points the importance of the glutamate receptors in this mechanism.

Synthesis and biological effects of some kynurenic acid analogs.

The overactivation of excitatory amino acid receptors plays a key role in the pathomechanism of several neurodegenerative disorders and in ischemic and post-ischemic events. Kynurenic acid (KYNA) is an endogenous product of the tryptophan metabolism and, as a broad-spectrum antagonist of excitatory amino acid receptors, may serve as a protective agent in neurological disorders. The use of KYNA is excluded, however, because it hardly crosses the blood-brain barrier. Accordingly, new KYNA analogs which can readily cross this barrier and exert their complex anti-excitatory activity are generally needed. During the past 6 years, we have developed several KYNA derivatives, among others KYNA amides. These new analogs included one, N-(2-N,N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride (KYNA-1), that has proved to be neuroprotective in several models. This paper reports on the synthesis of 10 new KYNA amides (KYNA-1-KYNA-10) and on the effectiveness of these molecules as inhibitors of excitatory synaptic transmission in the CA1 region of the hippocampus. The molecular structure and functional effects of KYNA-1 are compared with those of other KYNA amides. Behavioral studies with these KYNA amides demonstrated that they do not exert significant nonspecific general side-effects. KYNA-1 may therefore be considered a promising candidate for clinical studies.

In vitro and in vivo multidrug resistance reversal activity by a Betti-base derivative of tylosin.

BACKGROUND: The multidrug resistance (MDR) proteins are present in a majority of human tumours. Their activity is important to understand the chemotherapeutic failure. A search for MDR-reversing compounds was conducted among various Betti-base derivatives of tylosin. METHODS: Here, we evaluate the in vitro and in vivo P-glycoprotein (P-gp)-modulating activity of the most promising compound N-tylosil-1-alpha-amino-(3-bromophenyl)-methyl-2-naphthol (TBN) using human MDR1 gene-transfected and parental L5178 mouse lymphoma cell lines. RESULTS: In vitro experiments showed that TBN dramatically increased the P-gp-mediated cellular uptake of the fluorescent substrate rhodamine 123. Similarly, TBN was found to act as a very potent enhancer of the cytotoxicity of doxorubicin on the resistant cell line. We also provide in vivo evidence using DBA/2 mice in support for an increased tumoural accumulation of doxorubicin, without affecting its tissue distribution, resulting in an enhanced antitumoural effect. CONCLUSION: Our results suggest that TBN is a potent modulator of the P-gp membrane pump and that the compound could be of clinical relevance to improve the efficacy of chemotherapy in MDR cancers.

Kynurenines in chronic neurodegenerative disorders: future therapeutic strategies.

Parkinson's, Alzheimer's and Huntington's diseases are chronic neurodegenerative disorders of a progressive nature which lead to a considerable deterioration of the quality of life. Their pathomechanisms display some common features, including an imbalance of the tryptophan metabolism. Alterations in the concentrations of neuroactive kynurenines can be accompanied by devastating excitotoxic injuries and metabolic disturbances. From therapeutic considerations, possibilities that come into account include increasing the neuroprotective effect of kynurenic acid, or decreasing the levels of neurotoxic 3-hydroxy-L-kynurenine and quinolinic acid. The experimental data indicate that neuroprotection can be achieved by both alternatives, suggesting opportunities for further drug development in this field.

Na(+)/Ca(2+) exchanger inhibition exerts a positive inotropic effect in the rat heart, but fails to influence the contractility of the rabbit heart.

BACKGROUND AND PURPOSE: The Na(+)/Ca(2+) exchanger (NCX) may play a key role in myocardial contractility. The operation of the NCX is affected by the action potential (AP) configuration and the intracellular Na(+) concentration. This study examined the effect of selective NCX inhibition by 0.1, 0.3 and 1.0 microM SEA0400 on the myocardial contractility in the setting of different AP configurations and different intracellular Na(+) concentrations in rabbit and rat hearts. EXPERIMENTAL APPROACH: The concentration-dependent effects of SEA0400 on I(Na/Ca) were studied in rat and rabbit ventricular cardiomyocytes using a patch clamp technique. Starling curves were constructed for isolated, Langendorff-perfused rat and rabbit hearts. The cardiac sarcolemmal NCX protein densities of both species were compared by immunohistochemistry. KEY RESULTS: SEA0400 inhibited I(Na/Ca) with similar efficacy in the two species; there was no difference between the inhibitions of the forward or reverse mode of the NCX in either species. SEA0400 increased the systolic and the developed pressure in the rat heart in a concentration-dependent manner, for example, 1.0 microM SEA0400 increased the maximum systolic pressures by 12% relative to the control, whereas it failed to alter the contractility in the rabbit heart. No interspecies difference was found in the cardiac sarcolemmal NCX protein densities. CONCLUSIONS AND IMPLICATIONS: NCX inhibition exerted a positive inotropic effect in the rat heart, but it did not influence the contractility of the rabbit heart. This implies that the AP configuration and the intracellular Na(+) concentration may play an important role in the contractility response to NCX inhibition.

The kynurenate analog SZR-72 prevents the nitroglycerol-induced increase of c-fos immunoreactivity in the rat caudal trigeminal nucleus: comparative studies of the effects of SZR-72 and kynurenic acid.

Administration of nitroglycerol in a migraine model results in an increased number of c-fos-expressing secondary sensory neurons in the caudal trigeminal nucleus. Since synapses between first- and second-order trigeminal neurons are mediated by excitatory amino acids, NMDA receptors are inhibited by kynurenic acid, though this crosses the blood-brain barrier only poorly. Systemic treatment of rats with SZR-72, a newly synthetized kynurenic acid analog, diminished the nitroglycerol-induced increase of c-fos immunoreactivity in the brain stem highly significantly, while treatment with kynurenic acid resulted in a significantly smaller decrease, proving that SZR-72 is much more effective than kynurenic acid.

Salsolinol-a potential inhibitor of the gonadotropic axis in sheep during lactation.

This study tested the hypothesis that salsolinol, a derivative of dopamine, affects GnRH and LH secretion in lactating sheep. In the in vivo experiment, the structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1-MeDIQ), was infused into the infundibular nucleus-median eminence of sheep at the fifth wk of lactation to antagonize salsolinol's action. Simultaneously, cerebrospinal fluid from the third brain ventricle, to determine GnRH concentration, and plasma samples, to measure LH concentration, were collected. In the in vitro experiment, the anterior pituitary (AP) explants from weaned sheep were incubated in culture medium containing 2 doses of salsolinol, 20 and 100 µg/mL (S20 and S100, respectively). The concentration of LH in the collected media and relative expression of LHß subunit messenger RNA in the AP explants were determined. No significant difference was found in mean GnRH concentration in response to 1-MeDIQ infusion, but both mean plasma LH concentration and LH pulse frequency increased significantly (P < 0.001 and P < 0.05, respectively) compared with those in controls. Significantly higher LH concentrations occurred during the first (P < 0.001), second (P < 0.001), and fourth (P < 0.05) h of 1-MeDIQ infusion. In the in vitro study, both the S20 and S100 doses of salsolinol caused a significant decrease in the mean medium LH concentration compared with that in the control (P < 0.01 and P < 0.001, respectively). Salsolinol had no effect on the relative LHß subunit messenger RNA expression in the incubated tissue. In conclusion, salsolinol is a potential inhibitor of the secretory activity of the gonadotropic axis in lactating sheep, at least at the AP level. Although no significant changes in GnRH release were directly confirmed, an increase in the frequency of LH pulses does not allow to exclude the central action of salsolinol.

Involvement of salsolinol in the suckling-induced oxytocin surge in sheep.

During lactation, the main surge of oxytocin is induced by a suckling stimulus. Previous studies have shown that salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline), a dopamine-derived compound, stimulates both the synthesis and the release of oxytocin in lactating sheep. The objective of the present study was to verify the hypothesis that salsolinol is involved in the mechanism that generates the oxytocin surge that occurs during suckling. Thus, a structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), known to antagonize some of its actions, was infused into the third ventricle of the brain of lactating sheep nursing their offspring. Serial 30-min infusion of 1MeDIQ (4 × 60 µg/60 µL) or vehicle were administered at 30-min interval from 10 AM to 2 PM. The experimental period in every ewe consisted of a nonsuckling period (10 AM-12 PM) and a suckling period (12 PM-2 PM). Blood samples were collected every 10 min, to measure plasma oxytocin concentration by RIA. In control sheep, oxytocin surges of high amplitude were observed during the suckling period. The oxytocin surges induced by suckling were significantly (P < 0.01) diminished in sheep receiving 1MeDIQ infusions as compared to those that received control infusions. However, no significant effect of 1MeDIQ was observed on basal oxytocin release, before suckling. Furthermore, oxytocin release, as measured by the area under the hormone response curve (AUC), was significantly decreased by the administration of 1MeDIQ during the suckling period. This study shows that elimination of the effect of salsolinol within the central nervous system of lactating sheep attenuates the oxytocin surge induced by suckling. Therefore, salsolinol may be an important factor in the oxytocin-stimulating pathway in lactating mammals.

Classic preconditioning decreases the harmful accumulation of nitric oxide during ischemia and reperfusion in rat hearts.

BACKGROUND: The role of NO in the mechanism of preconditioning is not understood. Therefore, we studied the effect of preconditioning and subsequent ischemia/reperfusion on myocardial NO content in the presence of an NO synthase (NOS) inhibitor. METHODS AND RESULTS: Isolated working rat hearts were subjected to preconditioning protocols of 3 intermittent periods of rapid pacing or no-flow ischemia of 5 minutes' duration each followed by a test 30 minutes of global no-flow ischemia and 15 minutes of reperfusion. Test ischemia/reperfusion resulted in a deterioration of myocardial function and a considerable increase in cardiac NO content as assessed by electron spin resonance. Preconditioning improved postischemic myocardial function and markedly decreased test ischemia/reperfusion-induced NO accumulation. In the presence of 4.6 micromol/L N(G)-nitro-L-arginine (LNA), basal cardiac NO content decreased significantly, although test ischemia/reperfusion-induced functional deterioration and NO accumulation were not affected in nonpreconditioned hearts. However, the protective effects of preconditioning on both test ischemia/reperfusion-induced functional depression and NO accumulation were abolished. When 4.6 micromol/L LNA was administered after preconditioning, it failed to block the effect of preconditioning. In the presence of 46 micromol/L LNA, ischemia/reperfusion-induced NO accumulation was significantly decreased and postischemic myocardial function was improved in nonpreconditioned hearts. CONCLUSIONS: Our results show that (1) although NO synthesis by the heart is necessary to trigger classic preconditioning, preconditioning in turn attenuates the accumulation of NO during ischemia/reperfusion, and (2) blockade of ischemia/reperfusion-induced accumulation of cardiac NO by preconditioning or by an appropriate concentration of NOS inhibitor alleviates ischemia/reperfusion injury as demonstrated by enhanced postischemic function.

Conformationally constrained deltorphin analogs with 2-aminotetralin-2-carboxylic acid in position 3.

Two approaches to the design of very active and highly selective delta opioid peptides were used to obtain new deltorpin analogs with altered hydrophobic and stereoelectronic properties. Deltorphin I and II analogs were synthesized involving the substitution of Ile instead of Val at positions 5 and 6 in the address domain and 2-aminotetralin-2-carboxylic acid (Atc) instead of Phe in the message domain. The peptides were agonists in the subnanomolar range in the MVD assay and in the micromolar or higher range in the GPI assay, showing a very high selectivity for delta receptors. A very similar trend could be observed in radioreceptor binding assays in which selective tritiated opioid ligands were used. (R)- and (S)-Atc-deltoriphins exhibited similar Ki values in the binding assay, with almost complete loss of the stereospecificity of the binding. Conformational studies provided evidence for the little disturbance of the backbone conformational equilibrium when Phe3 is replaced by (S)- or (R)-Atc. The use of the Atc constraint gives additional evidence that, during its interaction with the delta receptor, the side chain of residue 3 adopts the trans conformation at chi 1.

Direct myocardial anti-ischaemic effect of GTN in both nitrate-tolerant and nontolerant rats: a cyclic GMP-independent activation of KATP.

1. We have recently demonstrated that glyceryl trinitrate (GTN) exerts a direct myocardial anti-ischaemic effect in both GTN-tolerant and nontolerant rats. Here we examined if this effect is mediated by GTN-derived nitric oxide (NO) and involves guanosine 3'5' cyclic monophosphate (cyclic GMP) and ATP-sensitive K+ channels (KATP). 2. Rats were treated with 100 mg kg-1 GTN or vehicle s.c. three times a day for 3 days to induce vascular GTN-tolerance or nontolerance. Isolated working hearts obtained from either GTN-tolerant or nontolerant rats were subjected to 10 min coronary occlusion in the presence of 10-7 M GTN or its solvent. 3. GTN improved myocardial function and reduced lactate dehydrogenase (LDH) release during coronary occlusion in both GTN-tolerant and nontolerant hearts. 4. Cardiac NO content significantly increased after GTN administration in both GTN-tolerant and nontolerant hearts as assessed by electron spin resonance. However, cardiac cyclic GMP content measured by radioimmunoassay was not changed by GTN administration. 5. When hearts from both GTN-tolerant and nontolerant rats were subjected to coronary occlusion in the presence of the KATP-blocker glibenclamide (10-7 M), the drug itself did not affect myocardial function and LDH release, however, it abolished the anti-ischaemic effect of GTN. 6. We conclude that GTN opens KATP via a cyclic GMP-independent mechanism, thereby leading to an anti-ischaemic effect in the heart in both GTN-tolerant and nontolerant rats.

Effects of N-substitution on the fragmentations of some cyclohexene-fused 2-N-phenyliminoperhydro-3,1-oxazines and related thiazines.

Six cyclohexene-fused 2-N-phenyliminoperhydro-3,1-oxazines and four related thiazines were prepared and their mass spectrometric behavior was studied by means of metastable ion analysis and exact mass measurement. In most of the fragmentations, extensive rearrangements took place. The decompositions through the retro-Diels-Alder reaction initiated by the double bond dominated in the case of the unsubstituted compounds. The effect of the double bond, however, was greatly outweighed by the effect of the substituent on the ring nitrogen atom. In comparison with the unsubstituted compounds, both electron-releasing (methyl) and electron-withdrawing (benzyl) substituents increased the contribution of the ring cleavage reactions in the heterocyclic part of the molecule; in the case of the benzyl group the loss of the substituent also became important. For isomeric compounds, the relative peak intensities were so different that such compounds were easy to differentiate.

Stress- as well as suckling-induced prolactin release is blocked by a structural analogue of the putative hypophysiotrophic prolactin-releasing factor, salsolinol.

Prolactin is secreted from the anterior lobe of the pituitary gland in response both to suckling and to stress. We recently observed that 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), produced in the neurointermediate lobe of the pituitary gland, as well as in the medial basal hypothalamus, can selectively release prolactin from the anterior pituitary. Therefore, it has been proposed that salsolinol is a putative endogenous prolactin-releasing factor (PRF). Here, we report that one structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), can block salsolinol-induced release of prolactin, but does not affect prolactin release in response to thyrotropin releasing hormone (TRH), alpha-methyl-p-tyrosine (alpha MpT) (an inhibitor of tyrosine hydroxylase), domperidone (a D(2) dopamine receptor antagonist), or 5-hydroxytryptophan (5-HTP), a precursor of serotonin). 1MeDIQ profoundly inhibited suckling-, immobilization-, as well as formalin-stress induced prolactin release without any influence on corticosterone secretion. The 1MeDIQ-induced reduction in prolactin response to immobilization stress was dose-dependent. These results suggest that salsolinol can play a pivotal role in the regulation of prolactin release induced by either physiological (suckling) or environmental (stress) stimuli.

Salsolinol is a putative endogenous neuro-intermediate lobe prolactin-releasing factor.

The isolation and identification of a prolactin-releasing factor (PRF) from the neuro-intermediate lobe of the pituitary gland has been pursued for over a decade. Using high-pressure liquid chromatography with electrochemical detection (HPLC-ECD) and gas chromatography/mass spectrometry (GC/MS) (R)-salsolinol (SAL) (a dopamine-related stereo-specific tetrahydroisoquinoline) was found to be present in neuro-intermediate lobe as well as median eminence extracts of male, intact-, and ovariectomized female rats. Moreover, analysis of SAL concentrations in neuro-intermediate lobe revealed parallel increases with plasma prolactin in lactating rats exposed to a brief (10 min) suckling stimulus following 4-h separation. SAL appears to be a selective and potent stimulator of prolactin secretion in vivo and it was without effect on the secretion of other pituitary hormones. We have also found that SAL can elevate prolactin release, although to a lesser extent, in pituitary cell cultures as well as in hypophysectomized rats bearing anterior lobe transplants under the kidney capsule. Lack of interference of SAL with [3H]-spiperone binding to AP homogenates indicates that SAL does not act at the dopamine D2 receptor. Moreover, [3H]-SAL binds specifically to homogenate of AL as well as neuro-intermediate lobe obtained from lactating rats. Taken together, our data clearly suggest that SAL is synthesized in situ and this compound can play a role in the regulation of pituitary prolactin secretion.

Pivotal role of an endogenous tetrahydroisoquinoline, salsolinol, in stress- and suckling-induced release of prolactin.

In mammals, the role of a prolactin-releasing factor (PRF) in the acute changes of prolactin (PRL) secretion that usually occur after challenges (e.g., suckling stimulus or stress) of homeostasis has been suspected for a long time. We have recently observed that 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, salsolinol (SAL), produced by the hypothalamus and the neuro-intermediate lobe (NIL) of the pituitary gland, can selectively release PRL from the anterior lobe (AL). Moreover, binding sites for SAL have been detected in areas like median eminence, NIL, and AL. It has been proposed that SAL is a putative endogenous PRF. We have also found that a structural analogue of SAL, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), is able to block dose-dependently SAL-, suckling-, and immobilization (IMO) stress-induced release of PRL without having any influence on alpha-methyl-p-tyrosine (alphaMpT)-induced PRL responses. Neither SAL nor 1MeDIQ has any effect on alpha-melanocyte-stimulating hormone (alphaMSH), adrenocorticotrophic hormone (ACTH), beta-endorphin (beta-END) and arginine-vasopressin (AVP) secretion. Moreover, SAL-induced PRL response was attenuated in male rats pretreated with dexamethasone (DEX). These results strongly suggest that SAL has an important role in the regulation of PRL release induced by physiologic and environmental stimuli; therefore, it can be considered as the strongest candidate for being the PRF in the hypothalamo-hypophysial system. Our findings also indicate that the adrenal steroids may play an inhibitory feedback role in SAL-mediated PRL response.

In vivo effects of 2-substituted [Mpa1Sar7Arg8]-oxytocin antagonists on postpartum rat.

A set of oxytocin antagonists consisting of [Mpa1Sar7Arg8]-oxytocin substituted by various conformationally restricted or bulky D amino acids at position 2 were synthetized and biologically tested. In in vivo pharmacological investigations, the effects of these peptides were examined on the spontaneous motor activity of postpartum rat. Three of the newly prepared peptides proved at least as effective in inhibiting uterine contractions as clinically investigated atosiban.

Noncompetitive nature of oxytocin antagonists with general structure Mpa(1)Xxx(2)Sar(7)Arg(8).

Eight oxytocin (OT) antagonists with general structure Mpa(1)Sar(7)Arg(8), substituted at position 2 with conformationally constrained and bulky amino acids, were synthesized and pharmacologically tested. Binding affinities and selectivities of compounds for OT, and vasopressin receptor subtypes were investigated. In vitro effects of antagonists were evaluated via inhibition of OT-induced contractions of isolated guinea-pig uterus. The abilities of OT antagonists to inhibit spontaneous contractility in 24 h postpartum rat uterus were investigated. These peptides exhibited pseudoirreversible pharmacological properties, and comprise a novel group of OT antagonists for potential clinical use. Their noncompetitive pharmacological nature can be of therapeutic benefit through a sustained effect on myometrium.