1Altered cardiac metabolism and function (diabetic cardiomyopathy) has been observed in diabetes. We hypothesize that cardiac efficiency, the ratio of cardiac work (pressurevolume area [PVA]) and myocardial oxygen consumption (MVO 2 ), is reduced in diabetic hearts. Experiments used ex vivo working hearts from control db/؉, db/db (type 2 diabetes), and db/؉ mice given streptozotocin (STZ; type 1 diabetes). PVA and ventricular function were assessed with a 1.4-F pressure-volume catheter at low (0.3 mmol/l) and high (1.4 mmol/l) fatty acid concentrations with simultaneous measurements of MVO 2 . Substrate oxidation and mitochondrial respiration were measured in separate experiments. Diabetic hearts showed decreased cardiac efficiency, revealed as an 86 and 57% increase in unloaded MVO 2 in db/db and STZ-administered hearts, respectively. The slope of the PVA-MVO 2 regression line was increased for db/db hearts after elevation of fatty acids, suggesting that contractile inefficiency could also contribute to the overall reduction in cardiac efficiency. The end-diastolic and end-systolic pressure-volume relationships in db/db hearts were shifted to the left with elevated end-diastolic pressure, suggesting left ventricular remodeling and/or myocardial stiffness. Thus, by means of pressure-volume technology, we have for the first time documented decreased cardiac efficiency in diabetic hearts caused by oxygen waste for noncontractile purposes. Diabetes 55: 466 -473, 2006 C ardiac efficiency is the ratio between energy output (work) and energy input (myocardial oxygen consumption [MVO 2 ]) for the heart. Currently, the most accepted definition of total cardiac work is pressure-volume area (PVA), the sum of external mechanical work and the potential energy triangle (1). Importantly, MVO 2 is linearly related to PVA. Extrapolation of this linear relationship to 0 work gives unloaded (PVA independent) MVO 2 , the oxygen cost of excitation-contraction coupling and basal metabolism.Furthermore, the inverse slope of the MVO 2 -PVA relationship defines the contractile efficiency.Recently, How et al. (2) demonstrated that pressurevolume loops and resulting determinations of PVA can be obtained with ex vivo perfused working mouse hearts, using a combined micromanometer (pressure)-conductance (volume) catheter. A fiber-optic oxygen probe gave simultaneous measurements of MVO 2 . An elevation in perfusate fatty acid concentration resulted in augmented fatty acid oxidation and reduced cardiac efficiency (increased MVO 2 with no change in work), manifested as increased unloaded MVO 2 (2).Perfused hearts from db/db mice, a monogenic model of type 2 diabetes with obesity and insulin resistance, have been characterized as having an early increase in fatty acid oxidation that precedes the onset of contractile dysfunction (3). Because elevated rates of fatty acid oxidation produce a decrease in cardiac efficiency in control hearts (2), the objective of the current investigation was to test the hypothesis that cardiac efficiency will be r...

We report on the pharmacological properties of a potent and selective linear vasopressin (AVP) V_1a receptor antagonist HO-Phenylacetyl¹-D-Tyr(Me)²-Phe³-Gln⁴-Asn⁵-Arg⁶-Pro⁷-Arg⁸-NH₂ (HO-LVA). Iodinated on the phenolic substituent at position 1, [¹²⁵I]-HO-LVA displayed the highest affinity for rat liver V_1a receptors (8 pM) ever reported. Furthermore, affinities of HO-LVA and I-HO-LVA for V_1b, V₂ and oxytocin (OT) receptors was 400- to 1,000-fold lower than for V_1a receptors, rendering it a highly selective ligand. Both HO-LVA and its iodinated derivative are V_1a antagonists, they potently inhibited AVP-induced inositol-phosphate accumulation in WRK₁ cells, and also, although with a much lower potency, the AVP-induced ACTH release from freshly prepared pituitary cells. Using autoradiography [¹²⁵I]-HO-LVA appeared to be the first radioligand to successfully identify and localize the presence of V_1a receptors in rat liver and blood vessel walls. Moreover, several new brain regions expressing V_1a receptors could be identified, in addition to those brain regions that were previously identified with other radiolabelled AVP analogues.

No abstract

We report the solid phase synthesis of four pairs of L- and D-thienylalanine (Thi/D-Thi) position two modified analogues of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly(NH2)9,d (CH2)5[Tyr(Me)2,Thr4]OVT) (A); the Tyr-(NH2)9 analogue of (A), d(CH2)5[Tyr(Me)2,Thr4,Tyr-(NH2)9]OVT (B); the Eda9 analogue (where Eda = ethylenediamine) of (A), d(CH2)5[Tyr(Me)2, Thr4, Eda9]OVT (C); and the retro Tyr10 modified analogue of (C), d(CH2)5[Tyr(Me)2, Thr4, Eda9<--Tyr10]OVT (D). The eight new analogues of A-D are (1) desGly(NH2),d(CH2)5[Thi2,Thr4]OVT, (2) desGly(NH2),d(CH2)5[D-Thi2,Thr4]OVT, (3) d(CH2)5[Thi2, Thr4,Tyr-(NH2)9]OVT, (4) d(CH2)5[D-Thi2,Thr4,Tyr-(NH2)9]OVT (5) d(CH2)5[Thi2,Thr4Eda9]OVT, (6) d(CH2)5[D-Thi2,Thr4,Eda9]OVT, (7) d(CH2) [Thi2,Thr4,Eda9<--Tyr10]OVT, (8) d(CH2),[D-Thi2,Thr4,Eda9<--Tyr10]OVT. We also report the synthesis of (C). Peptides 1-8 and C were evaluated for agonistic and antagonistic activities in in vitro and in vivo OT assays, in in vivo vasopressor (V1a receptor) assays and in in vivo antidiuretic (V2 receptor) assays. None of the eight peptides nor C exhibit oxytocic or vasopressor agonism. Peptides 1-8 are extremely weak V2 agonists (antidiuretic activities range from < 0.0005 to 0.20 U/mg). Peptide C is a weak mixed V2 agonist/antagonist. Peptides 1-8 and C exhibit potent in intro (no Mg2+) OT antagonism (anti-OT pA2 values range from 7.76 to 8.05). Peptides 1-8 are all OT antagonists in vivo (estimated in vivo anti-OT pA2 values range from 6.54-7.19). With anti-V1a pA2 values of approximately 5-5.80, peptides 1-8 exhibit marked reductions in anti-V1a potencies relative to those of the parent peptides A-D (anti-V1a pA2 range from 6.48 to 7.10) and to l-deamino[D-Tyr(Et)2, Thr4]OVT (Atosiban, trade name Tractocile) (anti-V1a pA2-6.14). Atosiban has recently been approved in Europe for clinical use for the prevention of premature labour (Pharm. J. 264(7-100): 871). Peptides 1-8 exhibit striking gains in in vitro anti-OT/anti-V1a selectivities with respect to the parent peptides A, B, C and D and to Atosiban. Peptides 1-8 exhibit anti-OT (in vitro)/anti-V1a selectivities of 450, 525, 550, 450, approximately 1080, 116, 355, 227 respectively. The corresponding values for A-D and Atosiban are 30, 4.2, 4.3, 2.6 and 37. With the exception of peptide 6, the remaining seven peptides exhibit 3-18-fold gains in anti-OT (in vivo)/anti-V1a selectivity with respect to Atosiban, peptides 1-8 exhibit anti-OT (in vivo)/anti-V1a selectivities of 22, approximately 82, approximately 82, 147, approximately 83, 11, 31 and 42. By comparison, Atosiban exhibits an anti-OT (in vivo)/anti-V1a selectivity = 8. With an estimated in vivo anti-OT pA2 value = 7.19+/-0.06, peptide 4 is equipotent with Atosiban (pA2 = 7.05+/-0.05). However, with its significantly reduced anti-vasopressor potency, pA2 = approximately 5, it is approximately 18 times more selective for OT receptors with respect ...

We report the solid phase synthesis and some pharmacological properties of seven position two analogues (peptides 1–7) of one of our lead oxytocin antagonists. des‐9‐glycinamide[1‐(β‐mercapto‐β, β‐pentamethylenepropionic acid), 2‐O‐methyltyrosine, 4‐threonine]ornithinevasotocin(desGly‐NH2,d(CH2)5‐ [Tyr(Me)2,Thr4]OVT) (A). Peptides 1–7 have the following substituents at position two (1) d‐Tyr(Me); (2) l‐Tyr(Et); (3) d‐Tyr(Et); (4) l‐Tyr; (5) d‐Tyr; (6) d‐Phe and (7) d‐Trp. These were evaluated for agonistic and antagonistic activities in in vitro and in vitro OT assays, in vivo vasopressor (V1a‐receptor) assays and in vivo antidiuretic (V2‐receptor) assays. None of the seven peptides exhibits oxytocic or vasopressor agonism. Peptides 1,2,4,6 and 7 are extremely weak V2 agonists (V2 activities range from 0.001 to 0.02 U/mg). Peptides 3 and 5 exhibit weak V2 antagonism (pA2>6.0 and >5.5, respectively). Peptides 1–7 exhibit potent in vitro (no Mg2+) OT antagonism (anti‐OT pA2 values range from 7.66 to 8.03). Peptides 1 and 4–7 exhibit potent in vivo OT antagonism. Estimated in vivo anti‐OT pA2 values range from 7.06 to 7.79 (peptides 2 and 3 were not tested). With anti‐Vla pA2 values of 5.17‐6.25 all seven peptides exhibit reduced anti‐V1a, potencies relative to the parent peptide (A) (anti‐V1a. pA2= 6.46). Four of these peptides (4‐7) exhibit striking gains in in vitro and in vivo anti‐OT/anti‐V1a. selectivities compared to (A) which has an in vitro selectivity of 30 and an in vivo selectivity of 18. The d‐Tyr2 (5), d‐Trp2 (7), d‐Phe2 (6) and l‐Tyr2 (4) analogues of (A) exhibit anti‐OT (in vitro)/anti‐V1a selectivities = 240, 390, 404 and 540, respectively. The l‐Tyr2 (4), d‐Trp2 (7), d‐Phe2 (6)and d‐Tyr2 (5) analogues exhibited anti‐OT (in vivo)/anti‐V1a selectivities of 72, 80, 88 and 95, respectively. Peptides 4–7 appear to be the most selective peptide OT antagonists reported to date. In this regard it may be noted that they appear to be as or more potent and much more selective than the closely related OT antagonist 1‐deamino[D‐Tyr(Et)2,Thr1]OVT (Atosiban) which is currently undergoing clinical trial as a potential therapeutic agent for the prevention of premature labor. Atosiban (peptide 8) was resynthesized and pharmacologically evaluated in our laboratories. Atosiban exhibits the following antagonistic potencies. Anti‐OT (in vitro, no Mg2) pA2= 7.71; anti‐OT in vivo pA2= 7.05; anti‐V1 pA2= 6.14 and anti‐V2 pA2≅ 5.9. Its anti‐OT (in vivo)/anti‐V1a selectivity is 8. Some of these antagonists may be suitable candidates for evaluation as potential tocolytic agents for use in the treatment of pre‐term labor. They could also serve as useful new pharmacological tools for studies on the physiological roles of oxytocin. Finally, the findings presented here provide useful clues for the design of more potent and more selective OT antagonists.

Synthetic oxytocin and vasopressin agonists and antagonists have become important tools for research and were instrumental in the identification of the four known receptor subtypes, Via, V,, V,, (V,) and oxytocin, of these peptide hormones. However, the relative lack of receptor selectivity, particularly of the antagonists, has limited their usefulness as experimental probes and their potential as therapeutic agents. We now present some findings from our continuing studies aimed at the design of more selective oxytocin and vasopressin agonists and antagonists and a structure-activity relationship update on our recently discovered novel hypotensive vasopressin peptides. Bioassays have been, and continue to be, of critical importance in leading to the discovery of the novel agonists, antagonists and hypotensive peptides reported here. This paper highlights three main aspects of these studies. (1) Replacement of the tyrosine' and/or phenylalanine3 residues in the V, agonist deamino,[Va14, ~-Arg~]arginine-vasopressin (dVDAVP) by thienylalanine resulted in selective V, agonists with strikingly high potencies. However, the peptide solutions were unstable and lost activity over time. These highly potent V, agonists, which are devoid of vasopressor activity, are promising leads for improving drugs for treating diabetes insipidus, enuresis and coagulation disorders. (2) (1895) injected an extract of the pituitary gland into an anaesthetised dog and found that the extract had a potent vasopressor activity. A decade later, Dale (1906) while studying the vasopressor action of the pituitary extract discovered that the extract also possessed a powerful stimulating action on Ihe smooth muscle of the uterus. These observations were the first experimental evidence that ultimately established the biological functions of the posterior pituitary gland and its hormones: vasopressin and oxytocin. The two historical findings were incidental discoveries uncovered during an exploration of the biological activities of pituitary extracts in intact whole animal experiments. The overriding theme of this review is the discovery of a series of novel, selective, hypotensive vasopressin peptides. Like the pioneering work noted above, the hypotensive activity of these vasopressin peptides was also discovered incidentally while we were carrying out bioassay characterisations on a series of newly designed vasopressin V, receptor antagonists aimed at developing more selective and potent V, receptor antagonists.Publication of The Physiological Society

Oxytocin (OT) has a dual action in the uterus: a uterotonic action on myometrial cells and a prostaglandin (PG)-releasing action on endometrial/decidual cells. It had not been determined whether the OT-binding sites or receptors on the myometrial and the endometrial/decidual membranes are of the same type or may represent two subtypes. Our studies presented in this paper show that isolated day 19-22 pregnant rat uterine horns and myometrial tissues (uterine horns with decidual tissues removed) incubated in Kreb's buffer at 37 C released PGF2 alpha in sustained quantities into the bathing medium. OT stimulated PG release over the basal release rate in a dose-dependent manner in the whole uterine horn but not in the myometrial tissue. Two OT antagonists, P[Phe(Me)2,Thr4]ornithine vasotocin (antagonist A) and desGly-NH2(9),d(CH2)5(1)[Tyr(Me)2,Thr4]ornithine vasotocin (antagonist B) were found to have different effects on the PG-releasing action of OT. At antiuterotonic doses, antagonist A had no antagonism of the PG-releasing action of OT. On the contrary, antagonist A was found to stimulate uterine PG release. Antagonist B was a full OT antagonist. At equivalent antiuterotonic doses, antagonist B inhibited both the uterotonic action and the PG-releasing action of OT. These findings suggest that OT-sensitive PGs are synthesized/released principally in the endometrium/decidua. The myometrial uterotonic OT receptors and the endometrial/decidual PG-releasing OT receptors are two distinct subtypes and can be differentiated. The existence of two OT receptor subtypes in the uterus has important implications in the clinical application of OT antagonists as tocolytics for preterm labor. To be efficacious, OT antagonist therapy needs to block both the uterotonic and the PG-releasing action of OT.

We report the solid-phase synthesis and some pharmacological properties of 12 position three modified analogues (peptides 1-12) of the potent non-selective antagonist of the antidiuretic (V2-receptor), vasopressor (V1a-receptor) responses to arginine vasopressin (AVP) and of the uterine contracting (OT-receptor) responses to oxytocin (OT), [1(-beta mercapto-beta,beta-pentamethylenepropionic acid)-2-O-ethyl-D-tyrosine 4-valine] arginine vasopressin [d(CH2)5D-Tyr(Et)2VAVP] (A) and two analogues of (B) (peptides 13,14), the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid3 (Tic3) analogue of (A). Peptides 1-12 have the following substituents at position three in (A): (1) Pro; (2) Oic; (3) Atc; (4) D-Atc; (6) D-Phe; (7) Ile; (8) Leu; (9) Tyr; (10) Trp; (11) Hphe; (12) [HO]Tic; Peptide (13) is the Tyr-NH2(9) analogue of (B): Peptide (14) is the D-Cys(6) analogue of (B). All 14 new peptides were evaluated for agonistic and antagonistic activities in in vivo V2 and V1a assays and in vitro (no Mg2+)n oxytocic assays. With the exception of the D-Phe3 peptide (No. 6), which exhibits very weak V2 agonism (approximately 0.0017 U/mg), none of the remaining 13 peptides exhibit any agonistic activities in these assays. In striking contrast to their deleterious effects on agonistic activities in AVP, the Pro3, Oic3, Tyr3 and Hphe3 substitutions in (A) are very well tolerated, leading to excellent retention of V2, V1a and OT antagonistic potencies. All are more potent as V2 antagonists than the Ile3 and Leu3 analogues of (A). The Tyr-NH2(9) and D-Cys(6) substitutions in (B) are also well tolerated. The anti-V2 pA2 values of peptides 1-5 and 7-14 are as follows (1) 7.77 +/- 0.03; (2) 7.41 +/- 0.05; (3) 6.86 +/- 0.02; (4) 5.66 +/- 0.09; (5) approximately 5.2; (7) 7.25 +/- 0.08; (8) 6.82 +/- 0.06; (9) 7.58 +/- 0.05; (10) 7.61 +/- 0.08; (11) 7.59 +/- 0.07; (12) 7.20 +/- 0.05; (13) 7.57 +/- 0.1; (14) 7.52 +/- 0.06. All analogues antagonize the vasopressor responses to AVP, with anti-V1a pA2 values ranging from 5.62 to 7.64, and the in vitro responses to OT, with anti-OT pA2 values ranging from 5.79 to 7.94. With an anti-V2 potency of 7.77 +/- 0.03, the Pro3 analogue of (A) is surprisingly equipotent with (A), (anti-V2 pA2 = 7.81 +/- 0.07). These findings clearly indicate that position three in AVP V2/V1a antagonists, in contrast to position three in AVP agonists, is much more amenable to structural modification than had heretofore been anticipated. Furthermore, the surprising retention of V2 antagonism exhibited by the Pro3, Oic3, Tyr3, Trp3 and Hphe3 analogues of (A), together with the excellent retention of V2 antagonism by the Tyr-NH2(9) and D-Cys6 analogues of (B) are promising new leads to the design of potent and possibly orally active V2 antagonists for use as pharmacological tools and/or as radioiodinatable ligands and for development as potential therapeutic agents for the treatment of the hyponatremia caused by the syndrome of the inappropriate secretion of the antidiuretic hormone (SIADH).