Construction of a pseudolarynx after laryngectomy and radical neck dissection. A preliminary report

Oxytocin analogues which combine high oxytocic activities with negligible antidiuretic and pressor activities have been studied. [4-Threonine,7-glycine]oxytocin, [1-(L-2-hydroxy-3-mercaptopropionic acid),4-threonine,7-glycine]oxytocin, and [1-(L-2-hydroxy-3-mercaptopropionic acid)]oxytocin were found to possess the following specific biological activities respectively: rat uterotonic, 270 +/- 10, 337 +/- 23, 1542 +/- 0.4; rat antidiuretic, 0.002 +/- 0.0008, 0.048 +/- 0.005, 40.3 +/- 2.4. The results are analyzed from a conformation-activity viewpoint in a continued attempt to evaluate the scope and limitations of this approach in comparison to structure-activity studies.     

Oxytocin analogues with combined high smooth muscle and negligible antidiuretic activities. Investigation of position 7 in neurohypophyseal hormones

The proline residue in position 7 of oxytocin occupies one of the four corner positions in the two beta turns proposed for the preferred conformation of the pituitary hormone. It has been suggested that synthetic modifications of the residues in these corner positions will yield analogues in which one or more of the biological activities of the parent hormone is highly accentuated in terms of potency relative to other activities. In a continued effort to test this hypothesis the following analogues of oxytocin were prepared: [7-glycine]oxytocin, [1-beta-mercaptopropionic acid,7-glycine]oxytocin, [7-alanine]oxytocin, and [1-beta-mercaptopropionic acid,7-alanine]oxytocin. These peptides were found to possess the following specific activities, respectively: rat uterotonic, 65 +/- 2, 355 +/- 3, 22 +/- 1, 123 +/- 4; avian vasodepressor, 5.3 +/- 0.8, 17 +/- 0.4, 4.8 +/- 0.1, 9.8 +/- 0.5; rat antidiuretic, less than0.01, 0.062, 0.081 +/- 0.01, 0.17 +/- 0.01; rat pressor, 0.3, 0.5, 0.4, 0.5 unit/mg. Thus the analogues retain high uterotonic activity but exhibit strongly diminished renal and vascular activities relative to oxytocin. Especially noteworthy is [1-beta-mercaptopropionic acid,7-glycine]oxytocin with its high uterotonic activity but very low antidiuretic and pressor activities. The activity profile of this analogue combined with the fact that it is only slowly enzymatically degraded warrants further investigations of this peptide for clinical applications.     

Systematic substitution of an oxytocin antagonist with D-amino acids: unexpected high antagonistic potency of the D-Cys6-substituted analogue

We report twelve analogues (1-12) of [Pmp1,D-Trp2,Arg8]oxytocin, PA (parent antagonist), (Pmp = beta,beta-pentamenthylene-beta-mercaptopropionic acid), which is a potent antagonist (pA2 = 7.77) of the uterotonic effect of oxytocin (OT) in rats. The analogues were designed by replacement of each optically active amino acid residue at positions 3-8 in PA with a D-amino acid. Analogues 1-8, featuring D-amino acids in the ring portion, were weaker antagonists than PA or were inactive. Unexpectedly, replacement with D-Cys6 gave analogue 9, pA2 = 8.29, which is more than 3 times as potent as PA, and replacement with D-Pen6 gave analogue 10, pA2 = 7.98, also more potent than PA. Replacement with D-Pro7 and D-Arg8 gave analogues 11 and 12, which are approximately equipotent or somewhat more potent than PA. These data suggest that neither the orientation of the tail sequence with respect to the plane of the ring portion of an antagonist nor the configuration of individual amino acids in the tail sequence may be critical for preservation of antagonism to the uterotonic action of OT. In the antidiuretic assay, analogues 9 and 12 were very weak partial agonists and had estimated pA2 = < 6.3 and < 5.6, respectively. Analogue 9 constitutes an interesting lead for the future design of OT antagonists with different molecular requirements than those featuring L-Cys6 as a substituent.     

Isosteric substitution of Asn5 in antagonists of oxytocin and vasopressin leads to highly selective and potent oxytocin and V1a receptor antagonists: new approaches for the design of potential tocolytics for preterm labor

Substitution of Asn5 in oxytocin (OT) or vasopressin (VP) invariably leads to a dramatic loss of the biological activities of the peptides. Because of this observation, few structure-activity-relationship studies of OT and VP peptides have involved modifications in the 5 position. It is now recognized that peptide agonists and antagonists may use different structural and conformational features in their interactions with the receptors. Our prior studies showed that OT and VP antagonists, unlike the agonists, tolerate amino acid substitutions in the 5 position. This opens new approaches for the design of antagonists. We describe the effects of isosteric replacement of Asn5 by diaminopropionic acid (Dap) or diaminobutyric acid (Dab) in three OT and VP antagonists: (1) the V1a (vasopressor receptor) antagonist d(CH2)5[Tyr(Me)2]AVP; (2) the OT (uterine OT receptor) antagonist d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH29] OVT and (3) three selective OT antagonists, desGly-NH2,d(CH2)5[D-Tyr2, Thr4]OVT, desGly-NH2, d(CH2)5[D-Phe2, Thr4]OVT and desGly-NH2, d(CH2)5- [D-Trp2, Thr4]OVT. The Dap5 and Dab5 substitutions were tolerated remarkably well, with the less isosteric Dap5 substitution leading to a greater retention of anti-OT potency than the Dab5 substitution. Furthermore, the Dap5 and Dab5 and OT and VP antagonist analogues were surprisingly shown to be much more selective than their respective parent compounds. The Dab5 analogue of (1) was devoid of anti-OT activity. The three Dap5 analogues of (3) were devoid of anti-V1a activities. These appear to be the first single-receptor-type-selective OT and VP antagonists discovered to date. These findings could provide new leads for the development of single-receptor-type-selective receptor probes for the localization and characterization of OT and VP receptors and potential selective tocolytics for the treatment of premature labor.     

Increased pressor function of central vasopressinergic system in hypertensive renin transgenic rats

OBJECTIVE: Renin transgenic hypertensive rats [TGR(mRen2)27] have increased contents of angiotensin II and arginine vasopressin (AVP) in the cardiovascular brain regions. The aim of the present study was to evaluate the effects of centrally released AVP on the regulation of baseline blood pressure in TGR(mRen2)27 rats and to determine the interaction between AVP and angiotensin II in the central control of blood pressure in this model of hypertension. DESIGN: Three basic series of experiments were performed on 20 TGR(mRen2)27 and 20 Hannover Sprague-Dawley conscious rats, chronically instrumented with lateral cerebral ventricle (LCV) cannulae and femoral artery catheters. In series 1, blood pressure and heart rate were recorded during an LCV infusion of artificial cerebrospinal fluid before and after LCV administration of angiotensin II. In series 2, the effects of an LCV administration of angiotensin 11 (100 ng) on mean arterial pressure and the heart rate were determined during LCV infusion of a selective AVP receptor (V1) antagonist [1-(1-mercapto-4-methylcyclohexaneacetic acid)-8-arginine vasopressin (MeCAAVP) and d(CH2)5[Tyr(Me)2,Ala-NH2(9)]AVP] or a selective angiotensin II type 1 (AT1) receptor antagonist (losartan) or both. In series 3, mean arterial pressure and the heart rate were determined after an LCV injection of either AVP (10 ng) or AVP together with angiotensin II. RESULTS: The LCV infusions of antagonists to V1 and AT1 receptors caused significant comparable decreases in baseline MAP in TGR(mRen2)27 but not in Sprague-Dawley rats. Angiotensin II elicited significant pressor responses, both in TGR(mRen2)27 and in Sprague-Dawley rats. Blockade of V1 receptors significantly reduced the duration and the maximum amplitude of the central pressor response to angiotensin II in TGR(mRen2)27 rats, whereas in Sprague-Dawley rats the maximum pressor effect was not significantly altered. In both strains, the pressor response to angiotensin II was abolished by blockade of AT1 receptors. CONCLUSIONS: The results indicate that the elevated blood pressure in TGR(mRen2)27 rats is partly caused by increased function of the brain angiotensinergic AT1 and vasopressinergic V1 systems. Centrally released AVP is involved in mediation of the pressor effect exerted by centrally applied angiotensin II in TGR(mRen2)27 rats.     

Lack of effect of a selective vasopressin V1A receptor antagonist SR 49,059, on potentiation by vasopressin of adrenoceptor-mediated pressor responses in the rat mesenteric arterial bed

The vasopressin receptor subtype involved in the enhancement by vasopressin of adrenoceptor-mediated vasoconstriction was investigated in rat isolated perfused mesenteric arteries. [Arg8]vasopressin (1-10 nM) dose-dependently increased the perfusion pressure and enhanced the pressor response to the adrenoceptor agonist methoxamine (40 nmol) or electrical stimulation of periarterial nerves (16 Hz), at the concentration of 10 nM of [Arg8]vasopressin up to 4 and 3 fold, respectively. During prolonged exposure (45 min) the direct vasoconstrictor effect of [Arg8]vasopressin (10 nM) rapidly declined whereas the potentiation of methoxamine-induced vasoconstriction was maintained. The selective vasopressin V1A receptor antagonist SR 49,059 (1-3 nM) and the non-selective V1A/B and oxytocin receptor antagonist [deamino-Pen1,Tyr(Me)2,Arg8]vasopressin (15-45 nM) inhibited the direct vasoconstrictor action of [Arg8]vasopressin but had no effect on the enhancement of the pressor response to methoxamine or electrical stimulation. The V1B receptor agonist [deamino-Cys1,beta-(3-pyridyl)-D-Ala2,Arg8]vasopressin (100-1000 nM) and the V2 receptor agonist [deamino-Cys1,D-Arg8]vasopressin (1-10 nM) were devoid of any pressor activity and did not potentiate methoxamine-evoked vasoconstriction. In contrast, [1-triglycyl,Lys8]vasopressin (100 - 1000 nM) potentiated the methoxamine responses without per se inducing vasoconstriction. In arteries precontracted with methoxamine (7.5 microM) pressor responses to [Arg8]vasopressin (3-10 nM) were not inhibited by a dose of SR 49,059 (3 nM) which abolished the peptide's vasoconstrictor effect under control conditions. These data show that the direct vasoconstrictor effect of [Arg8]vasopressin is mediated by V1A receptors while the enhancement of adrenoceptor-mediated pressor responses is insensitive to V1A, V1B, and oxytocin receptor antagonists and is not mimicked by selective agonists of V1B and V2 receptors. In conclusion, an unusual interaction of vasopressin with V1A receptors, or even the existence of a novel receptor subtype, has to be considered.     

Choroid plexus carcinomas in childhood: clinical features and prognostic factors

The substitution, in the human V2 vasopressin receptor, of the aspartate at position 136 by alanine leads to agonist-independent activation of this mutant V2 receptor. Pharmacological studies of the D136A V2 receptor helped us in characterizing different V2 receptor antagonists. SR-121463A and OPC-31260, two non-peptide antagonists, behaved as inverse agonists, while two cyclic peptides d(CH2)5[D-Tyr(Et)2,-Val4,Tyr-NH(2)9]AVP and d(CH2)5[D-Ile2,Ile4,Tyr-NH(2)9]AVP known to be V2 antagonists, demonstrated clear partial agonist properties. The finding of a constitutively activated human V2 receptor represents a useful tool in characterizing V2 receptor antagonist ligands.     

In vivo pharmacology of an angiotensin AT1 receptor antagonist with balanced affinity for AT2 receptors

L-163,017 (6-[benzoylamino]-7-methyl-2-propyl-3-[[2'-(N-(3-methyl-1-butoxy) carbonylaminosulfonyl)[1,1']-biphenyl-4-yl]methyl]-3H-imidazo[4,5- b]pyridine) is a potent, orally active, nonpeptide angiotensin II receptor antagonist. Conscious rats and dogs were dosed p.o. and i.v.; in both species the plasma bioequivalents are similar at the angiotensin AT1 and AT2 receptor sites indicating balanced activity is maintained in vivo. L-163,017 prevents the pressor response to intravenous (i.v.) angiotensin II in the conscious rat, dog, and rhesus monkey. L-163,017 also significantly reduces blood pressure in a renin-dependent model of hypertension, similar to an angiotensin converting enzyme inhibitor (Enalapril) and an angiotensin AT1 receptor-selective antagonist (L-159,282). These studies indicate that neither the angiotensin AT2 receptor nor bradykinin is important in the acute antihypertensive activity of angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists.     

Purification and molecular cloning of a novel calcium-binding protein, p26olf, in the frog olfactory epithelium

The pharmacological profile and the acute and chronic aquaretic effects of OPC-41061, a novel nonpeptide human arginine vasopressin (AVP) V2-receptor antagonist, were respectively characterized in HeLa cells expressing cloned human AVP receptors and in conscious male rats. OPC-41061 antagonized [3H]-AVP binding to human V2-receptors (Ki = 0.43 +/- 0.06 nM) more potently than AVP (Ki = 0. 78 +/- 0.08 nM) or OPC-31260 (Ki = 9.42 +/- 0.90 nM). OPC-41061 also inhibited [3H]-AVP binding to human V1a-receptors (Ki = 12.3 +/- 0.8 nM) but not to human V1b-receptors, indicating that OPC-41061 was 29 times more selective for V2-receptors than for V1a-receptors. OPC-41061 inhibited cAMP production induced by AVP with no intrinsic agonist activity. In rats, OPC-41061 inhibited [3H]-AVP binding to V1a-receptors (Ki = 325 +/- 41 nM) and V2-receptors (Ki = 1.33 +/- 0. 30 nM), showing higher receptor selectivity (V1a/V2 = 244) than with human receptors. A single oral administration of OPC-41061 in rats clearly produced dose-dependent aquaresis. In treatment by multiple OPC-41061 dosing for 28 days at 1 and 10 mg/kg p.o. in rats, significant aquaretic effects were seen throughout the study period. As the result of aquaresis, hemoconcentration was seen at 4 hr postdosing although, no differences were seen in serum osmolality, sodium, creatinine and urea nitrogen concentrations at 24 hr postdosing. Furthermore, there was no difference in serum AVP concentration, pituitary AVP content or the number and affinity of AVP receptors in the kidney and liver at trough throughout the study period. These results demonstrate that OPC-41061 is a highly potent human AVP V2-receptor antagonist and produces clear aquaresis after single and multiple dosing, suggesting the usefulness in the treatment of various water retaining states.     

Absorption of an oxytocin antagonist (antocin) and a vasopressin analogue (dDAVP) through a standardized skin erosion in volunteers

PURPOSE: Transdermal administration of the peptides [Mpa1, D-Tyr (Ethyl)2, Thr4, Orn8]-oxytocin (antocin) and [Mpa1, D-Arg8]-vasopressin (dDAVP) was studied in healthy volunteers. METHODS: A standardized skin erosion was formed preliminary by suctioning. The peptides were administered in plastic reservoirs through a 5 mm erosion and the absorption was followed for a six-day period with plasma concentration determinations on days 1, 3 and 6 with refilling the reservoirs daily with 15 microns and 10 mM solutions of dDAVP and antocin, respectively. Fourteen healthy non-smoking volunteers divided equally between the sexes, participated in the study. Plasma concentrations were measured using specific radioimmunoassays. Reservoir concentrations and metabolic stability of the peptides were determined using reverse-phase HPLC. RESULTS: Both antocin and dDAVP were absorbed across the skin erosion. The absorption pattern was biphasic with a high initial absorption during days 1 and 2 followed by a lower absorption on days 3 and 6. The absorption on day 1, which was estimated at more than 50% for both peptides during a 24 h period, corresponded to a simultaneous decrease in peptide concentration in the reservoirs. The extent of absorption for antocin on days 3 and 6 was 1/3 to 1/6, respectively, of that observed on day 1. Antocin was minimally degraded in the skin reservoir while dDAVP was intact. However, accumulation of cellular material appeared in the antocin reservoirs. The absorption of antocin was reduced by exposure to intact skin surrounding the skin erosion. No pain was experienced and no scar formation was observed. CONCLUSIONS: The observed biphasic absorption may be a consequence of the mild inflammatory response occurring subsequent to eroding the skin. The standardized skin erosion may provide a route for the short-term delivery of otherwise poorly absorbable peptide and protein drugs.     

Proton magnetic resonance studies of bradykinin antagonists

Bradykinin (BK) is a peptide hormone with sequence Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9 and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. BK analogues having bulky, beta-branched D-aliphatic residues at position 7 combined with bulky L-aliphatic residues at position 8 have now been observed to be strong antagonists. Conformational studies based on two-dimensional nmr experiments in methanol/water (80/20 v/v) were carried out on several such active antagonists in a polar solvent. Included in this study were the very active antagonists, [D-Arg0,Hyp3,Thi5,D-Cpg7,Cpg8]-BK [Cpg: alpha-cyclo-pentyl-glycine; Hyp: trans-4-hydroxy-L-proline; Thi: beta-(2-thienyl)-L-alanine] (I), [D-Arg0,Hyp3,D-Cpg7,Cpg8]-BK (II), as well as its variant with D-Cpg7 replaced by Cpg7, namely [D-Arg0,Hyp3,Cpg7,Cpg8]-BK (III). A turn-like structure, which coexists with the extended conformation, was observed between residues 2 and 5 for the most active antagonists I and II, in direct correlation with the peptide activities. No turn-like structure was found for residues 6-9. In peptide III, a turn-like structure was not identified. The existence of a turn at the C-terminal end of bradykinin and its analogues has been predicted by empirical calculations and supported by nmr measurements. But the present nmr study on the most active antagonists (I, II) does not support this hypothesis. Instead, the data suggest that a turn-like structure between residues 2 and 5 could be important for antagonist activity. Finally, one weak inhibitor [D-Cpg7]-BK (IV) showed no defined secondary structure.     

Endopeptidase 24.11 inhibition does not modify uterotonic effects of endothelins in rat uterus

We investigated effects of the endopeptidase 24.11 inhibitor, SCH 39370, on uterotonic effects of endothelins (ETs) and sarafotoxin S6b. Responses of uteri from non-pregnant rats were inhibited by the ETA receptor antagonist, BQ123 (1 microM) but not the ETB receptor antagonist, BQ 788 (1 microM). ET-1, sarafotoxin S6b and ET-2 were more potent than ET-3 in tissues from non-pregnant and pregnant rats. SCH 39370 (10 microM) did not affect uterotonic responses to these peptides in either group, but inhibited those of big ET-1 in non-pregnant rat tissues, indicating inhibition of conversion of big ET-1 to ET-1. These data indicate that endopeptidase 24.11 does not inactivate the endothelin peptides in the rat uterus.     

Acts, omissions, intentions and motives: a philosophical examination of the moral distinction between killing and letting die

1. The exact nature of the receptor subtype(s) involved in the action of arg-vasopressin (AVP) on the rat aorta and small mesenteric artery (SMA) is controversial. Therefore, we have studied the effects of the selective V1A receptor antagonists, OPC 21268 and SR 49059, and the oxytocin (OT) receptor antagonist, atosiban, on the AVP- and OT-induced contractions of the two vessels. 2. AVP and OT displayed similar intrinsic activities in the rat aorta and SMA, but AVP was approximately 130 fold and approximately 500 fold more potent than OT, respectively. In the rat aorta, Hill slopes (nH) were similar for OT and AVP. However, in rat SMA, the OT concentration-effect (E/[A]) curve was significantly steeper than the AVP E/[A] curve (nH, = 3.3+/-0.20, 2.3+/-0.15; P<0.001). 3. In the aorta OPC 21268, SR 49059 and atosiban competitively antagonized the AVP and OT E/[A] curves. Except for atosiban and SR 49059 against AVP, competitive antagonism was also observed in the SMA. Atosiban caused concentration-dependent steepening of the AVP E/[A] curve, whereas SR 49059 decreased the upper asymptote. 4. Schild analysis yielded affinities indicative of V1A receptor involvement in both vessels: pKB/ pA2=9.20 9.48, 7.56 7.71 and 6.19 6.48 for SR 49059, OPC 21268 and atosiban, respectively. 5. Neither AVP nor OT relaxed U46619 pre-contracted aorta or SMA in the presence of SR 49059, suggesting no interference of a vasodilatory component. 6. Despite predominant involvement of V1A receptors in both vessels, the different Hill slopes of AVP and OT E/[A] curves as well as the steepening of the AVP E/[A] curves by atosiban are indicative of receptor heterogeneity in the rat SMA.     

2-Nitrophenylcarbamoyl-(S)-prolyl-(S)-3-(2-naphthyl)alanyl-N-benzyl-N - methylamide (SDZ NKT 343), a potent human NK1 tachykinin receptor antagonist with good oral analgesic activity in chronic pain models

A lead compound which had sub-micromolar affinity for the rabbit NK1 receptor but negligible affinity for rat NK1 receptors, 3a, was discovered by directed screening. 2-Substitution in the ring of the benzylthiourea substituent in the initial lead was found to be important, and halogens (Cl, Br) in this position were found to improve affinity for the human receptor. The activity of a series of 2-halo-substituted benzylthioureas was then optimized by modification of the proline diphenylmethyl amide, guided by a simple conceptual model based on structural overlay between these early antagonists and NK1 selective peptides. In this way, aromatic amino acid amides were identified which had improved affinity with respect to the starting diphenylmethyl (DPM) amides. The first sub-nanomolar ligand for the human NK1 receptor which arose from this series, 4af, combined a 2-chlorobenzylthiourea unit with a 2-naphthylalanine amide. Contemporaneously it was discovered that the benzylthiourea unit could be simplified to a phenylthiourea providing that an appropriate 2-substituent was also incorporated. Combination of these two series gave 2-NO2 phenylthiourea analogues which led directly to the analogous urea, 5f (2-nitrophenylcarbamoyl-(S)-prolyl-(S)-3-(2-naphthyl)alanyl-N-benz yl- N-methylamide, SDZ NKT 343), a highly potent ligand for the human NK1 receptor (Ki = 0.16 nM). In addition to its high in vitro potency, 5f proved to be a potent orally active analgesic in guinea pig models of chronic inflammatory and neuropathic pain. The nature of the 2-aryl substituent was found to be critical for oral activity in this series. Clinical evaluation of 5f as a novel analgesic agent is currently underway.     

Arginine vasopressin induces contraction and stimulates growth of cultured human hepatic stellate cells

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are perisinusoidal cells believed to participate in the regulation of hepatic blood flow because of their contractile properties and presence of receptors for several vasoactive factors. It is unknown whether HSCs have receptors for vasopressin, one of the most potent endogenous vasoconstrictors. This study investigated the existence of receptors for and the effects of arginine vasopressin (AVP) on cultured human HSCs. METHODS: intracellular calcium concentration ([Ca2+]i) and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a CCD imaging system (Photometrics, Tucson, AZ). AVP-specific binding was measured with [3H]AVP. Mitogen-activated protein kinase (MAPk) activity and DNA synthesis were measured by in vitro phosphorylation of myelin basic protein and [3H]thymidine incorporation, respectively. Parallel experiments were performed in vascular smooth muscle cells. RESULTS: AVP elicited a dose-dependent increase in [Ca2+]i and contraction of HSCs. Moreover, AVP increased MAPk activity, DNA synthesis, and cell number. These effects were similar to those observed in vascular smooth muscle cells and were blocked by a V1 receptor antagonist. The existence of V1 receptors was further confirmed by binding studies. CONCLUSIONS: Human HSCs have V1-vasopressin receptors that induce effects similar to those observed in vascular smooth muscle cells. AVP may play a role in the regulation of HSC function.     

Respiratory compliance during laparoscopic hiatal and inguinal hernia repair

BACKGROUND: Arginine vasopressin (AVP) not only acts directly on blood vessels through V1 receptor stimulation but also may modulate adrenergic-mediated responses in animal experiments in vivo and in vitro. The aim of the present study was to investigate whether AVP can contribute to an abnormal adrenergic constrictor response of human saphenous veins. METHODS AND RESULTS: Saphenous vein rings were obtained from 32 patients undergoing coronary artery bypass surgery. The vein rings were suspended in organ bath chambers for isometric recording of tension. AVP (3x10[-9] mol/L) enhanced the contractions elicited by electrical field stimulation at 1, 2, and 4 Hz (by 80%, 70%, and 60%, respectively) and produced a leftward shift of the concentration-response curve to norepinephrine (half-maximal effective concentration decreased from 6.87x10[-7] to 1.04x10[-7] mol/L; P<.05). The V1 vasopressin receptor antagonist d(CH2)5Tyr(Me)AVP (10[-6] mol/L) prevented the potentiation evoked by AVP. The selective V1 receptor agonist [Phe,2 Orn8]-vasotocin (3x[-10]-9 mol/L) induced potentiation of electrical stimulation-evoked responses, which was also inhibited in the presence of the V1 receptor antagonist (10[-6] mol/L). In contrast, the V2 receptor agonist desmopressin (10[-9] to 10[-7] mol/L) did not modify neurogenic responses, and the V2 receptor antagonist [d(CH2)5, D-Ile,2 Ile,4 Arg8]-vasopressin (10[-8] to 10[-6] mol/L) did not prevent the potentiation induced by AVP. The dihydropyridine calcium antagonist nifedipine (10[-6] mol/L) did not affect the potentiating effect of AVP. CONCLUSIONS: The results suggest that low concentrations of AVP facilitate sympathetic neurotransmission and potentiate constrictor effects of norepinephrine in human saphenous veins. These effects appear to be mediated by V1 receptor stimulation and are independent of calcium entry through dihydropyridine calcium channels. Thus, AVP may contribute to vascular mechanisms involved in acute ischemic syndromes associated with venous grafts, particularly if the sympathetic nervous system is activated.     

Interference of S-alkyl derivatives of glutathione with brain ionotropic glutamate receptors

The effects of glutathione, glutathione sulfonate and S-alkyl derivatives of glutathione on the binding of glutamate and selective ligands of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors were studied with mouse synaptic membranes. The effects of glutathione and its analogues on 45Ca2+ influx were also estimated in cultured rat cerebellar granule cells. Reduced and oxidized glutathione, glutathione sulfonate, S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione inhibited the Na+-independent binding of L-[3H]glutamate. They strongly inhibited also the binding of (S)-2-amino-3-hydroxy-5-[3H]methyl-4-isoxazolepropionate [3H]AMPA (IC50 values: 0.8-15.9 microM). S-Alkylation of glutathione rendered the derivatives unable to inhibit [3H]kainate binding. The NMDA-sensitive binding of L-[3H]glutamate and the binding of 3-[(R)-2-carboxypiperazin-4-yl][1,2-(3)H]propyl-1-phosphonate ([3H]CPP, a competitive antagonist at NMDA sites) were inhibited by the peptides at micromolar concentrations. The strychnine-insensitive binding of the NMDA coagonist [3H]glycine was attenuated only by oxidized glutathione and glutathione sulfonate. All peptides slightly enhanced the use-dependent binding of [3H]dizocilpine (MK-801) to the NMDA-gated ionophores. This effect was additive with the effect of glycine but not with that of saturating concentrations of glutamate or glutamate plus glycine. The glutamate- and NMDA-evoked influx of 45Ca2+ into cerebellar granule cells was inhibited by the S-alkyl derivatives of glutathione. We conclude that besides glutathione the endogenous S-methylglutathione and glutathione sulfonate and the synthetic S-alkyl derivatives of glutathione act as ligands of the AMPA and NMDA receptors. In the NMDA receptor-ionophore these glutathione analogues bind preferably to the glutamate recognition site via their gamma-glutamyl moieties.     

Solid phase synthesis and some hormonal activities of 1-deamino-4-L-valine-8-D-homolysine-and 1-deamino-4-L-valine-8-D-homoarginine-vasopressin

1-Deamino-4-L-valine-8-DL-homolysine-vasopressin and protected 1-deamino-4-l-valine-8-D-lysine-vasopressin were synthesized by the solid phase method and were then converted into the title compounds (dVDHLVP and dVDHAVP) by tryptic digestion and epsilon-guanidination, respectively. The new hormone analogues exhibit only moderate antidiuretic potency, dVDHLVP 21 units/mg and dVDHAVP 31 units/mg, but since they are essentially devoid of pressor activity (o.o1 units/mg/ the A/P ratios are very high. In fact, dVDHLVP is the most specific antidiuretic agent in the lysine series known so far.     

Vasopressin stimulates Ca2+ spiking activity in A7r5 vascular smooth muscle cells via activation of phospholipase A2

[Arg8]-vasopressin (AVP) is both a potent vasoconstrictor and a mitogen for vascular smooth muscle cells. AVP binds to a single class of receptors (V1a) in the A7r5 rat aortic smooth muscle cell line (Kd approximately 2 nmol/L). Stimulation of these cells with AVP results in an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) by releasing intracellular Ca2+ stores and increasing Ca2+ influx; the EC50 for these effects is approximately 5 nmol/L. AVP has recently been reported to stimulate arachidonic acid release in primary cultures of rat aortic smooth muscle over a much lower concentration range (EC50 approximately 0.05 nmol/L). The present study examined the effects of varying concentrations of AVP on spontaneous Ca2+ spiking activity in fura 2-loaded A7r5 cells. Frequency of CA2+ spiking increased with increasing [AVP] in the range of 10 to 500 pmol/L. Higher concentrations of AVP inhibited spiking but elicited the characteristic [Ca2+]i changes ascribed to the release of Ca2+ stores and increased Ca2+ entry. The effects of both low and high concentrations of AVP were inhibited by [1-(beta-mercapto-beta,beta,-pentamethylenepropionic acid),2-0-methyltyrosine]arginine vasopressin, a selective V1a vasopressin antagonist. Nimodipine (50 nmol/L), a blocker of L-type voltage-sensitive Ca2+ channels, abolished the Ca(2+)-spiking activity without inhibiting a maximal [Ca2+]i response to AVP (1 mumol/L). AVP-stimulated Ca2+ spiking, but not release of intracellular Ca2+ stores, was also abolished by ONO-RS-082 (1 mumol/L), an inhibitor of phospholipase A2. These results suggest that occupation of a small fraction of V1a vasopressin receptors by AVP results in stimulation of phospholipase A2 and leads to increased Ca(2+)-spiking activity. This effect may be important for fine tuning of vascular tone, whereas maximal stimulation by AVP (full receptor occupancy) may be required for more vigorous or sustained vasoconstriction or mitogenesis.     

Amino acid side chain conformation in angiotensin II and analogs: correlated results of circular dichroism and 1H nuclear magnetic resonance

[1-Sarcosine,8-isoleucine]angiotensin II (Sar-Arg-Val-Tyr-Ile-His-Pro-Ile) has been shown to be a potent antagonist of the pressor action of angiotensin II. With a view to increase half-life in vivo of this peptide, the amino acid residue at position 4 (tyrosine) or position 5 (isoleucine) was replaced with the corresponding N-methylated residue. This change drastically reduced the antagonistic properties of this analog. The present work was therefore undertaken to investigate the effect of N-methylation on overall conformation of these peptides and to determine the conformational requirements for maximum agonistic or antagonistic properties. Conformation studies were carried out by circular dichroism and proton nuclear magnetic resonance spectroscopy in aqueous solution as a function of pH. The results indicated that: (i) angiotensin II and [1-sarcosine,8-isoleucine]angiotensin II gave practically identical spectroscopic data; and (ii) N-methylation in either position 4 or position 5 resulted in remarkable changes in the peptide backbone and a severe limitation in rotational freedom of side chains in tyrosine, isoleucine, and histidine residues. However, rotational restriction of the tyrosine side chain was found to be less pronounced in [1-sarcosine,4-N-methyltyrosine,8-isoleucine]angiotensin II than in [1-sarcosine,5-N-methylisoleucine,8-isoleucine]angiotensin II. Thus, these results suggest that: (i) the backbone and side chain structure of a potent angiotensin II antagonist should resemble that of the hormone, angiotensin II, so that it can mimic the hormone in recognizing and binding with the receptor on the cell membrane; and (ii) greater impact of N-methylation in position 5 on the overall conformation of these peptides points to the controlling influence of position 5 (isoleucine) in aligning the residues in the central segment (tyrosine-isoleucine-histidine) of angiotensin II and its potent agonist and antagonist analogs in a nearly extended structure. Any change in this arrangement may lead to reduced biological activity.