Penile erection and yawning induced by oxytocin and related peptides: structure-activity relationship

The potency of several oxytocin-related peptides in inducing penile erection and yawning after injection into a lateral ventricle of male rats was compared. Substitution of two amino acids in the oxytocin molecule or deletion of the C-terminal glycinamide as in des-GlyNH2-oxytocin [oxytocin(1-8)] reduced oxytocin potency in inducing both effects, the rank order being: oxytocin greater than [Thr4,Gly7]-oxytocin congruent to isotocin [( Ser4,Ile8]-oxytocin) greater than vasopressin [( Phe3,Arg8]-oxytocin) greater than des-GlyNH2-oxytocin. Oxytocin's ability to induce penile erection and yawning was abolished by permanent opening of the disulfide bridge by reduction and carboxymethylation. Oxytocin(1-6) and oxytocin(7-9) were also inactive. Penile erection and yawning induced by oxytocin-related peptides were antagonized in a dose-dependent manner by nonapeptide antagonists with a rank order of potency that follows their antioxytocic activity (d[(CH2)5Tyr(Me)Orn8]-vasotocin congruent to [Pen1,Phe(Me)2,Thr4,Orn8]-oxytocin greater than d[(CH2)5Tyr(Me)Arg8]-vasopression). Carboxymethylated oxytocin, oxytocin(1-6), and oxytocin(7-9) were devoid of antagonistic activity. The present results suggest that central oxytocin receptors mediating the expression of penile erection and yawning are structurally related to those present in the uterus and in the mammary gland.     

Prostaglandin E2 receptor in the myometrium: distribution in subcellular fractions

[³H]Prostaglandin (PG) E₂ bound specifically to several subcellular fractions from bovine myometrium. The binding was temperature dependent, rapid, and reversible. PGE₂ and PGE₁ competed for the [³H]PGE₂ binding site. The PGs inhibited in the following decreasing order: PGE₂ = PGE₁ ? PGF_2α > PGA₂ > PGF_1β > PGB₂. No competitive effect could be found for oxytocin. Scatchard analysis of the binding data were interpreted as showing a single high-affinity binding constant. There was no difference in the binding constant between the various fractions. The average molar dissociation constant was 2.74 ± 0.14 × 10^?9. Quantitative differences in the maximum number of binding sites were observed between fractions. One plasma membrane fraction contained 21.4 ± 2.3 × 10^?11 and the sarcoplasmic reticulum contained 11.2 ± 0.8 × 10^?11 mol binding sites/g. The results suggest that there is a high-affinity PGE₂ receptor present in both plasma membrane and sarcoplasmic reticulum.     

Threshold and receptor reserve in the action of 5-hydroxytryptamine on the salivary glands of Calliphora erythrocephala

The interrelationships of 5-HT receptors and the increased fluid secretion by isolated salivary glands of Calliphora have been studied using pharmacological techniques. Removal of the 5-OH group (tryptamine) or displacement to position 6 (6-HT) results in a decrease in potency but no change in intrinsic activity of the hormone whereas altering the ethyl amine side chain (5-OH tryptophan) results in a decrease in both potency and intrinsic activity. Removal of the 5-OH group and alteration of the side chain (gramine and tryptophan) results in a total loss of activity. Gramine and tryptophan behave as competitive antagonists of 5-HT.Prostaglandin E₁ (PGE₁) was found to be a non-competitive inhibitor of 5-OH tryptophan and theophylline whereas the response to 5-HT and cyclic AMP was only slightly diminished indicating a ‘receptor reserve’ for 5-HT.Saturating concentrations of gramine and tryptophan potentiate theophylline revealing a ‘threshold’ for the mediation of the response. It is concluded that 5-HT derivatives are capable of producing graded effects on adenyl cyclase both above and below the range of enzyme activity which evokes graded changes in fluid secretion.     

Investigation of Drug-Receptor Interactions by Means of Irreversible Receptor Inhibitors: Binding of Oxytocin and Angiotensin II to Their Receptors in Rat Uterus

Abstract

A stimulus-response coupling model in which individual steps follow hyperbolic or Hill-type laws has been employed to mimic phenomena associated with irreversible receptor inhibition (receptor reserve) in a responding biological system. Two methods for computation of ligand-receptor dissociation constant (K_A) have been derived from this model: 1) The relation between pD₂ and maximal attainable response allows a rough estimate of K_A; 2) A generalization of the earlier Furchgott-Bursztyn procedure employing equipotent doses for noninhibited and partially inhibited systems, has been achieved by introduction of Hill equation into the model. Applied to oxytocin and angiotensin II receptors in rat uterus, these pharmacological methods indicate rather low affinity of the two receptors for the respective peptides (around 2x10^?8 for angiotensin II and 2x10^?7 mol/1 for oxytocin), in case of oxytocin much lower than values reported from binding studies. Apparently, several binding sites are present on the target tissue cells from which the methods based on cellular response can pick up those corresponding to the receptors. Biophysical methods are lacking this ability. Single pD₂ values in noninhibited systems cannot themselves deliver any relevant information about receptor binding.     

Sensitivity of the Bothrops jararaca snake uterus to oxytocin and acetylcholine

The influence of hormones on the uterine smooth muscle sensitivity has been demonstrated in mammals; however in nonmammalian species much remains to be clarified. This study investigated the sensitivity of the snake (Bothrops jararaca) uterus to oxytocin and acetylcholine. The snakes were divided into three experimental groups: snakes with uterine segments weighing ≤20.00 mg (A), snakes with uterine segments weighing between 20.01 and 35.00 mg (B) and snakes with uterine segments weighing ≥35.01 mg (C). The histomorphometric analysis of the uterus showed an increase in the smooth muscle layer thickness in groups B and C, when compared with group A, suggesting different hormonal conditions of the animals. Cumulative concentration-effect curves to oxytocin and acetylcholine were obtained in uteri of the three experimental groups and the pD₂ values determined. The sensitivity of the snake uterus to oxytocin increased in groups B and C (concentration-effect curves shifted to the left and pD₂ values increased) when compared with group A. The concentration-effect curves to acetylcholine were biphasic and shifted to the left, suggesting two binding sites (low and high affinity binding sites) in snake uteri of groups B and C. These results suggest that sex steroids may modulate the sensitivity of the snake uterus to oxytocin and acetylcholine.     

THE DEPRESSION OF PHAGOCYTOSIS BY EXOGENOUS CYCLIC NUCLEOTIDES, PROSTAGLANDINS, AND THEOPHYLLINE

The effects of agents that elevate intracellular cyclic adenosine 3',5'-monophosphate (cAMP) have been studied with respect to phagocytosis by guinea pig polymorphonuclear leukocytes. The investigation depends upon the use of a precise method for following ingestion. Theophylline, dibutyryl cAMP, and prostaglandins inhibited the phagocytosis of starch particles. The inhibitions caused by prostaglandins E₁, E₂, and F_2α (PGE₁, PGE₂, and PGF_2α) were synergistic with that due to theophylline. Inhibition by PGA₁ and PGA₂ was not. At equal concentrations the order of increasing inhibition of phagocytosis (assayed at 10 min) by the prostaglandins was PGE₁ < PGF_2α < PGE₂ < PGA₁ = PGA₂. Our results are consistent with the hypothesis that increased intracellular levels of cAMP impair the phagocyte's ability to ingest particles. The mechanism of the inhibition has not been defined. The increment in oxidation of [1-¹⁴C]glucose to ¹⁴CO₂ that normally accompanies phagocytosis was found to be depressed in the presence of PGE₁ or theophylline, together or individually as expected from the inhibition of phagocytosis. Paradoxically, oxygen consumption although depressed by theophylline or PGE₁ plus theophylline, was stimulated by PGE₁ alone.     

Antithrombotic effect of topically applied 3-Oxa-Methano-prostaglandin I1 in the microcirculation and antiplatelet functions of its active form

The antithrombotic effect of topical application of the 3-oxamethano-prostaglandin (PG) I₁ analog, SM-10902 in the microcirculation and in vitro antiplatelet functions of its active form SM-10906 were estimated in comparison with PGI₂ and PGE₁. In rat platelets, SM-10906 evoked accumulation of intracellular cyclic adenosine 3′,5′-monophosphate, and exhibited antiaggregatory and disaggregatory activities, which were all enhanced by the phosphodiesterase inhibitor theophylline. Additionally, SM-10906 was shown to inhibit platelet adhesion to collagen in human platelet-rich plasma. PGI₂ and PGE₁ also showed in vitro antiplatelet effects in the order of PGI₂ > SM-10906 ≥ PGE₁. SM-10902 exhibited a dose-dependent antithrombotic effect in the guinea pig mesenteric arteriole by a topical application, and this activity might be exerted by the antiplatelet functions of SM-10906. Although SM-10906, PGI₂ and PGE₁ also showed the antithrombotic effects, SM-10902 was the most potent. In conclusion, the present studies indicate that an external topical preparation of SM-10902 may be useful for the therapy of peripheral circulatory insufficiency.     

Prostaglandin-induced enhancement of the anamnestic response

The ability of various prostaglandins (PGs) to affect the anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

Contractile effects of prostaglandin E2 in rat rectum: Sensitivity to the prostaglandin antagonists diphloretin phosphate and SC 19220

Prostaglandin E₂ (PGE₂) applied cumulatively (1 nM − 1 μM) induced concentration-dependent tonic contractions in the longitudinal muscle of isolated rat rectum. The PGE₂ effects were not altered by guanethidine (50 μM), whereas atropine (3 μM), guanethidine plus atropine or tetrodotoxin (0.1 μM) reduced them to an almost equal extent and increased the EC₅₀ values for PGE₂. The after-contractions following electrical stimulation were enhanced by PGE₂ (10 nM) and inhibited by atropine. Diphloretin phosphate (DPP, 100 μM) shifted the regression lines for PGE₂ to the right in both untreated and tetrodotoxin-treated preparations, and thereby increased the EC₅₀ values. Slopes of the concentration-effect lines for PGE₂ before and after DPP differed in the presence of tetrodotoxin. The regression line for PGE₂ with SC 19220 (100 μM) in tetrodotoxin-treated preparations was shifted to the right in a parallel fashion. It is concluded that PGE₂ exerted both a neural (cholinergic) and a smooth muscle effect. There may be a competitive antagonism between SC 19220 and PGE₂ but the block by DPP may be nonselective.     

Effect of prostaglandins F2α and E2 on milk ejection, blood pressure and blood flow through the mammary artery in the cow

The influence of prostaglandins (PG) F₂α and E₂ on milk ejection, mammary artery blood flow and arterial blood pressure was studied in lactating cows. Injections of both PG in the jugular vein or the carotid artery induced milk ejection after a relatively long latency period. The minimal effective dose amounted to 1 to 5 μg and to 100 to 300 μg for PGF₂α and PGE₂ respectively. In several cases with PGF₂α and once with PGE₂ milk ejection was accompanied with a simultaneous increase in blood flow through the mammary artery whereas arterial blood pressure remained unchanged. Both routes of administration showed the same response. It was suggested that the effect of the PG on the bovine myoepithelium is indirect, possibly secondary to a release of oxytocin from the neurohypophysis.     

Prostaglandin E1 or E2 inhibits an oxytocin-induced premature luteolysis in ewes when oxytocin is given early in the estrous cycle

The objective of this study was to determine whether PGE₁ or PGE₂ prevents a premature luteolysis when oxytocin is given on Days 1 to 6 of the ovine estrous cycle. Oxytocin given into the jugular vein every 8 hours on Days 1 to 6 postestrus in ewes decreased (P ≤ 0.05) luteal weights on Day 8 postestrus. Plasma progesterone differed (P ≤ 0.05) among the treatment groups; toward the end of the experimental period, concentrations of circulating progesterone in the oxytocin-only treatment group decreased (P ≤ 0.05) when compared with the other treatment groups. Plasma progesterone concentrations in ewes receiving PGE₁ or PGE₁ + oxytocin were greater (P ≤ 0.05) than in vehicle controls or in ewes receiving PGE₂ or PGE₂ + oxytocin and was greater (P ≤ 0.05) in all treatment groups receiving PGE₁ or PGE₂ than in ewes treated only with oxytocin. Chronic intrauterine treatment with PGE₁ or PGE₂ also prevented (P ≤ 0.05) oxytocin decreases in luteal unoccupied and occupied LH receptors on Day 8 postestrus. Oxytocin given alone on Days 1 to 6 postestrus in ewes advanced (P ≤ 0.05) increases in PGF_2α in inferior vena cava or uterine venous blood. PGE₁ or PGE₂ given alone did not affect (P ≥ 0.05) concentrations of PGF_2α in inferior vena cava and uterine venous blood when compared with vehicle controls or oxytocin-induced PGF_2α increases (P ≤ 0.05) in inferior vena cava or uterine venous blood. We concluded that PGE₁ or PGE₂ prevented oxytocin-induced premature luteolysis by preventing a loss of luteal unoccupied and occupied LH receptors.     

Metabolism of prostaglandins and xenobiotics by adrenal microsomal monooxygenase in the guinea pig

The possibility that prostaglandins could serve as substrates for the guinea pig adrenal microsomal monooxygenase was investigated. The binding of PGE₁ to adrenal microsomes was found to exhibit a reverse type I spectral change. Also PGE₁ diminished the magnitude of type I spectrum elicited by cortisol binding to adrenal microsomes. The incubation of [³H]PGE₁ or of [³H]PGE₂ with adrenal microsomes supplemented with NADPH yielded primarily the respective 19-hydroxy metabolite. The enzymatic activity catalyzing this hydroxylation appears to be a typical monooxygenase, requiring NADPH for activity and being strongly inhibited by metyrapone, SKF 525A, and cytochrome c. Carbon monoxide at a ratio of 9:1 to oxygen moderately inhibited the hydroxylation of PGE₁. Whereas the liver catalyzed the hydroxylation of PGE₁ and PGA₁ equally well, the adrenal microsomes preferentially catalyzed the hydroxylation of PGE₁. This finding and the observation that α-naphthoflavone is a weak inhibitor of the adrenal PGE₁ hydroxylation points to significant differences between the adrenal and liver prostaglandin hydroxylation activities. Cortisol, which is a substrate for adrenal monooxygenase, strongly inhibited PGE₁ and PGE₂ hydroxylation. By contrast, certain xenobiotics (ethylmorphine, hexobarbital, benzpyrene), which are also metabolized by adrenal microsomes, only slightly inhibited the hydroxylation of PGE₁. Similarly, PGE₁ only weakly inhibited ethylmorphine and benzphetamine demethylation and hexobarbital hydroxylation. These observations suggest that adrenal microsomes contain several monooxygenases with different affinities for prostaglandins and for the different xenobiotic substrates.     

In vitro response of relaxin-treated rat uterus to prostaglandins and oxytocin

Paired segments of rat uterus were treated $\text{in vitro}$ with relaxin (W1164-3, 150 GPU/mg) until the amplitude of contraction was reduced to at least 50% of the pre-treatment amplitude. Test segments then received 100 ng of either PGE₁, PGE₂, PGF_2α or 250 uU of oxytocin. Control segments remained untreated. There was a significant increase in contraction amplitude in response to the spasmogens (P < 0.05) but no increase was seen in controls.     

Prostaglandin E2: Effects on aggregation,shape change and cyclic AMP of rat platelets

PGE₂ inhibits intracellular cyclic AMP accumulation induced by PGE₁ in rat platelets. PGE₂ also counteracts PGE₁-inhibition of both platelet aggregation and shape change. However, in the presence of theophylline, PGE₂ acts like PGE₁ in inhibiting aggregation. The mechanism of interaction of the two closely related prostaglandins is discussed.     

Influence of flavin addition and removal on the formation of superoxide by NADPH-Cytochrome P-450 reductase: a spin-trap study

Several structural derivatives of the dipeptide, l (and d)-cysteinyl-l-proline were synthesized and shown to be very potent competitive and noncompetitive inhibitors of human serum angiotensin I-converting enzyme. Only if the sulfhydryl group of the cysteine was blocked with benzyl, trityl, or benzyloxycarbonyl protecting groups, was the dipeptide a noncompetitive inhibitor. Compounds with free sulfhydryl groups were competitive inhibitors with K_i values in the 10^?8m range. d-Cys-l-Pro, our most potent inhibitor (k₁ = 0.0055 μM), was an order of magnitude more potent than l-Cys-l-Pro consistent with findings of Cushman et al. (1977, Biochemistry16, 5484) that -CH₃ group substitution improves binding if the configuration is d but diminishes binding if the configuration is l. Zinc and calcium ions released inhibition by some of the noncompetitive, but only one, of the competitive inhibitors. The noncompetitive inhibitor, l-cysteinyl(benzyl)-l-proline, and the competitive inhibitor, l-cysteinyl-l-proline, were used as affinity ligands to obtain near homogenous (25 units/mg) enzyme from human plasma. The observation that compounds with a free sulfhydryl group are competitive inhibitors and those in which the sulfhydryl groups are blocked are noncompetitive inhibitors can be rationalized if the active site of the converting enzyme is an extended linear trench.     

Prostaglandin E2 produced by microsomal prostaglandin E synthase-1 regulates the onset and the maintenance of wakefulness

This study examined the effect of prostaglandin E₂ (PGE₂) produced by microsomal prostaglandin E synthase-1 (mPGES-1) on circadian rhythm. Using wild-type mice (WT) and mPGES-1 knockout mice (mPGES-1^−/−), I recorded and automatically analyzed the natural behavior of mice in home cages for 24 h and measured brain levels of PGE₂. The switch to wakefulness was not smooth, and sleepiness and the total duration of sleep were significantly longer in the mPGES-1^−/− mice. Moreover, the basal concentration of PGE₂ was significantly lower in the mPGES-1^−/− mice. These findings suggest that PGE₂ produced by mPGES-1 regulates the onset of wakefulness and the maintenance of circadian rhythm.     

Adenylate cyclase and phosphodiesterase in transitional epithelium of the urinary bladder.

Adenylate cyclase activities in cell-free preparations of isolated transitional epithelium from rabbit urinary bladders were shown to be stimulated by epinephrine, prostaglandin E₁ (PGE₁), 5-guanylyl imidodiphosphate (GMP-PNP), and NaF. ACTH, aldosterone, insulin, glucagon, oxytocin, parathyroid hormone and vasopressin were without effect at the concentrations tested. The effects of epinephrine, PGE₁, and GMP-PNP appeared to be additive. Essentially all of the adenylate cyclase activity was particulate, while approximately 70% of the cyclic nucleotide 3':5'-phosphodiesterase activity was soluble. Single reciprocal plots of the phosphodiesterase data revealed non-linear kinetics.     

Oxytocin Receptors in Bovine Mammary Tissue

Abstract

Oxytocin receptors were identified and characterized in bovine mammary tissue. [³H]-oxytocin was specifically bound to the 105,000 × g particulate fractions from 5 lactating cows and 5 non-lactating cows. Binding reached equilibrium by 50 min at 20°C and by 8 hr at 4°C. The half-time of displacement at 20°C was approximately 1 hr. ACTH, TRH, angiotensin I, angiotensin II, pentagastrin, bradykinin, xenopsin and L-valyl-histidyl-L-leucyl-L-threonyl-L-prolyl-L-valyl-L-glutamyl-L-lysine were not competitive in the dose range tested at 20°C. The ability of other peptides to inhibit ³H-oxytocin binding was as follows: oxytocin > vasotocin > arginine - vasopressin >lysine - vaso-pressin > Pen¹ Phe² Thr⁴ - oxytocin. The Kd of the oxytocin receptor averaged 1.66 ± 1.19 nMol/L for lactating cows and 0.97± 0.49 nMol/L for non-lactating cows, respectively. The maximum number of binding sites was 0.14 ± 0.12 nM/mg protein and 0.15 ± 0.08 nM/mg protein for lactating cows and non-lactating cows, respectively. Identification and characterization of these receptors now makes it possible to study the dynamics of hormonal binding throughout various physiological states of the animal.     

Pseudohypericin is necessary for the light-activated inhibition of prostaglandin E2 pathways by a 4 component system mimicking an Hypericum perforatum fraction

Hypericum perforatum (Hp) has been used medicinally to treat a variety of conditions including mild-to-moderate depression. Recently, several anti-inflammatory activities of Hp have been reported. An ethanol extract of Hp was fractionated with the guidance of an anti-inflammatory bioassay (lipopolysaccharide (LPS)-induced prostaglandin E₂ production (PGE₂)), and four constituents were identified. When combined together at concentrations detected in the Hp fraction to make a 4 component system, these constituents (0.1 μM chlorogenic acid (compound 1), 0.08 μM amentoflavone (compound 2), 0.07 μM quercetin (compound 3), and 0.03 μM pseudohypericin (compound 4)) explained the majority of the activity of the fraction when activated by light, but only partially explained the activity of this Hp fraction in dark conditions. One of the constituents, light-activated pseudohypericin, was necessary, but not sufficient to explain the reduction in LPS-induced PGE₂ of the 4 component system. The Hp fraction and the 4 component system inhibited lipoxygenase and cytosolic phospholipase A₂, two enzymes in the PGE₂-mediated inflammatory response. The 4 component system inhibited the production of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), and the Hp fraction inhibited the anti-inflammatory cytokine interleukin-10 (IL-10). Thus, the Hp fraction and selected constituents from this fraction showed evidence of blocking pro-inflammatory mediators but not enhancing inflammation-suppressing mediators.     

Cyclic AMP inhibits synthesis of prostaglandin endoperoxide (PGG2) in human platelets.

Dibutyryl-cAMP but not dibutyryl-cGMP inhibited platelet aggregation and release of ¹⁴C-serotonin and ADP when induced by collagen and arachidonate but not when induced by the endoperoxide PGG₂^* (TXB₂) induced by addition of collagen to platelet rich plasma (PRP) was decreased by dibutyryl-cAMP and agents known to increase the concentration of cAMP (PGE₁, PGD₂, theophylline and acetyl choline).PGE₂ in concentrations known to decrease cAMP levels increased the formation of TXB₂ whereas concentrations of PGE₂ known to increase cAMP levels decreased the amount of TXB₂ formed. That this was due to an effect on the cyclooxygenase was indicated by inhibition of the transformation of arachidonic acid by DB-cAMP and by high concentrations of PGE₂. Additional support for regulation of the cyclo-oxygenase by cAMP and its relevance to platelet aggregation was obtained by demonstrating stimulation of PGG₂ induced aggregation by low concentrations of PGE₂ and the absence of this effect in the presence of a cyclo-oxygenase inhibitor.