Neuropeptide Y modulates the action of vasodilator agents in guinea-pig epicardial coronary arteries

Recently, we have demonstrated that guinea-pig epicardial coronary arteries are supplied by numerous nerve fibres containing neuropeptide Y (NPY) immunoreactivity. However, examination of vasomotor responses revealed that NPY did not elicit a contractile response in these arteries. In contrast, acetylcholine (ACh), calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) all relaxed precontracted arteries. In the present study, we have used histochemical, immunohistochemical and in vitro pharmacological techniques, in order to further investigate the possible role of NPY in guinea-pig epicardial coronary arteries. A double-immunofluorescence staining technique revealed that CGRP and substance P were co-localized in nerve fibres distinct from those displaying NPY immunoreactivity. Furthermore, using a method combining immunofluorescence and histochemical techniques, we observed that putative cholinergic nerve fibres (identified by their acetylcholinesterase content) and NPY-immunoreactive nerve fibres are two different nerve populations. An in vitro pharmacological method demonstrated that NPY markedly inhibited the relaxant responses mediated by ACh, VIP, substance P and isoprenaline but had no effect on CGRP. These results suggest that NPY-containing nerves associated with guinea-pig epicardial coronary arteries may be predominantly involved in modulating the action of vasodilator agents.     

Endothelial vasodilator production by uterine and systemic arteries. VII. Estrogen and progesterone effects on eNOS

Uterine blood flow (UBF) and uterine artery endothelial nitric oxide synthase (eNOS) expression are greatest during the follicular vs. luteal phase. 17 beta-Estradiol (E(2)beta) increases UBF and elevates eNOS in ovine uterine but not systemic arteries; progesterone (P(4)) effects on E(2)beta changes of eNOS remain unclear. Nonpregnant ovariectomized sheep received either vehicle (n = 10), P(4) (0.9 g Controlled Internal Drug Release vaginal implants; n = 13), E(2)beta (5 microg/kg bolus + 6 microg x kg(-1) x day(-1); n = 10), or P(4) + E(2)beta (n = 12). Reproductive (uterine/mammary) and nonreproductive (omental/renal) artery endothelial proteins were procured on day 10, and eNOS was measured by Western analysis. P(4) and E(2)beta alone and in combination increased (P < 0.05) eNOS expression in uterine artery endothelium (vehicle = 100 +/- 16%, P(4) = 251 +/- 59%, E(2)beta = 566 +/- 147%, P(4) + E(2)beta = 772 +/- 211% of vehicle). Neither omental, renal, nor mammary artery eNOS was altered, demonstrating the local nature of steroid-induced maintenance of uterine arterial eNOS. In the myometrial microvasculature, eNOS was increased slightly (P = 0.06) with E(2)beta and significantly with P(4) + E(2)beta. Systemic NO(x) was increased with P(4) and P(4) + E(2)beta, but not E(2)beta, suggesting differential regulation of eNOS expression and activity, since P(4) increased eNOS in uterine artery endothelium while E(2)beta and the combination further increased eNOS protein.     

Nongenomic action of progesterone in rat aorta: role of nitric oxide and prostaglandins

The mechanism of action of progesterone (Pg) on rat vascular tissue was investigated. We obtained evidence that 10-nM Pg inhibited platelet aggregation at 1-5 min. Previously, we reported that nitric oxide (NO) mediated this antiaggregatory effect. Rat aortic strips (RAS) NO synthase (NOS) activity in response to "in vitro" treatment with other sex steroids hormones was measured. The stimulatory action of Pg on NO production was specific for ovarian hormones and depends on sex. The effect was nongenomic since cycloheximide did not suppress the increment in NO induced by Pg. Finally, we demonstrated that Pg (5 min) increased prostacyclin release (42-182% above control) in a dose-dependent manner (1-100 nM). Indeed, indomethacin (10 microM) completely suppressed the increment in citrulline levels induced by the hormone. These results suggest that Pg exerts a direct nongenomic action on rat aortic metabolism, which involves NOS and cyclooxygenase (COX) activation and a cross-talk between NO- and prostacyclin (PGI(2))-dependent pathways.     

Nongenomic actions of aldosterone and progesterone revisited

After almost 30 years of research, the existence of nongenomic steroid actions is no longer disputed. Yet, there is still a debate on the nature of receptors involved, and answers to the inherent questions are important for translational activities. In the case of aldosterone, the existence of receptors different from the classic mineralocorticoid receptors (MR) had been postulated 25 years ago as the pharmacology of about 50% of rapid actions of aldosterone reported so far is incompatible with MR involvement (insensitivity to classic MR antagonists). Candidates proposed as alternatives to MR were protein kinase C, sodium-potassium ATPase or aberrant forms of MR, none of which supported convincing evidence to represent 'the aldosterone membrane receptor'. Early in 2011, data on GPR30 showed its involvement in rapid aldosterone action, and major pharmacological aspects of this action are compatible with the landmark deviations from MR pharmacology mentioned above. GPR30, therefore, may be a receptor candidate for nongenomic aldosterone action. Similarly, a variety of promising candidates mediating rapid progesterone action has been described, including progesterone receptor membrane component 1 (PGRMC1) which seems to be associated with tumor proliferation, and membrane progesterone receptor (mPR) originally identified in fish with potential linkage to reproductive processes. So far, no candidate was unanimously convincing. In 2010, two independent groups reported that CatSper, a calcium channel, is a strong receptor candidate for the rapid action of progesterone on sperm fertilization. With these novel receptors cloned, translational activities ultimately leading to new drugs for cardiovascular protection (in the case of aldosterone) or fertilization benefits (for progesterone) are much more promising.     

Nongenomic progesterone receptor on human spermatozoa: biochemical aspects and clinical implications.

Progesterone (P) is a physiological stimulus of human sperm functions. It is present in high levels at the site of fertilization (cumulus oophorus) and has been described to affect several sperm functions, including motility, capacitation, acrosome reaction, and the ability to bind and to respond to zona proteins. The effects of the steroid are mediated essentially by an increase of intracellular calcium concentrations, stimulation of activity of phospholipases, phosphorylation of proteins, efflux of chloride. These effects are due to activation of a rapid, nongenomic pathway. Whether the effects of progesterone are mediated or not by specific interactions with sperm membrane proteins is questioned. By using an antibody directed against the C-terminal region (P-binding region) of the genomic receptor, we have recently identified two sperm proteins with molecular weights distinct from the classic genomic receptors. In addition, ligand blot analysis with peroxidase-conjugated P demonstrated that P specifically binds these two proteins. Classical ligand binding experiments demonstrated the presence of two specific binding sites with affinity in the nanomolar and in the micromolar range, respectively. The involvement of progesterone in the physiological process leading to fertilization of the oocyte is suggested by several studies. In particular, the demonstration that sperm responsiveness to progesterone is impaired in subfertile patients and that is strictly correlated to the ability of fertilizing the oocyte represents a further indication of the participation of the steroid in this process. In addition, the determination of sperm responsiveness may be predictive of fertilizing ability with a positive predictive value of 90% and can be clinically useful for the preliminary assessment of the male partner to select the appropriate assisted reproductive technique.     

Nongenomic inhibition of oxytocin binding by progesterone in the ovine uterus

Progesterone (P4) has been reported to inhibit oxytocin (OT) binding to its receptor in isolated murine endometrial membranes. The purpose of the present research was to 1). examine the in vivo and in vitro effect of P4 on the binding of OT to its receptor in the ovine endometrium and 2). determine whether the endometrial plasma membranes have high-affinity binding sites for P4. Ovariectomized ewes were pretreated with a sequence of estradiol-17beta (2 days) and P4 (5 days) before being treated with estradiol-17beta plus either vehicle (corn oil), P4, or P4 + mifepristone (RU 486) for 3 consecutive days. Treatment of ewes with 10 mg P4/day for 3 days suppressed binding of OT (P < 0.01) compared with that of controls, whereas concomitant treatment with the progestin antagonist RU 486 (10 mg/day) blocked the effect of P4. Similarly, incubation of endometrial plasma membranes with P4 (5 ng/ml) inhibited binding of OT (P < 0.05), whereas this effect of P4 was blocked by the presence of RU 486 (10 ng/ml). By radioreceptor assay, the endometrial plasma membranes were found to contain a high-affinity binding site for P4 and the progestin agonist promegestone (Kd 1.2 x 10-9 and 1.74 x 10-10M, respectively). Incubation of endometrial plasma membranes with P4 (5 ng/ml) significantly increased the concentration of progestin binding sites. Binding of labeled promegestone (R 5020) was competitively inhibited by excess unlabeled R 5020, P4, RU 486, and OT but not by estradiol-17beta, cortisol, testosterone, and arginine vasopressin. These data suggest a direct suppressive action of P4 on the binding of OT to OT receptors in the ovine endometrial plasma membrane.     

Endothelial vasodilator production by uterine and systemic arteries. VIII. Estrogen and progesterone effects on cPLA2, COX-1, and PGIS protein expression.

During ovine pregnancy, when both estrogen and progesterone are elevated, prostacyclin (PGI2) production by uterine arteries and the key enzymes for PGI2 production, phospholipase A2 (cPLA2), cyclooxygenase 1 (COX-1), and prostacyclin synthetase (PGIS), are increased. This study was conducted to determine whether exogenous estradiol-17beta (E2beta) with or without progesterone (P4) treatment would increase cPLA2, COX-1, and PGIS protein expression in ovine uterine, mammary, and systemic (renal, mental, and coronary) arteries. Nonpregnant ovariectomized sheep received vehicle (n = 10), P(4) (0.9-g controlled internal drug release vaginal implants; n = 13), E2beta (5 microg/kg bolus followed by 6 microg x kg(-1) x day(-1); n = 10), or P4 + E2beta (n = 12). Arteries were procured on Day 10, and cPLA2, COX-1, and PGIS protein were measured by Western immunoblot analysis in endothelial isolated proteins and vascular smooth muscle (VSM). The levels of cPLA2 was increased in uterine artery endothelium in ewes treated with P4 + E2beta but was not altered by any steroid treatment in renal, coronary, mammary, or omental artery endothelium or in VSM of any evaluated artery. Similarly, COX-1 was increased in uterine artery endothelium with P4 + E2beta but was not significantly altered by treatment in other endothelium or VSM. E2beta treatment increased PGIS protein in uterine and renal artery endothelium but did not alter PGIS in other endothelial tissue. P4 increased PGIS expression in the uterine, mammary, omental, and renal artery VSM, and E2beta increased PGIS expression in the uterine and omental artery VSM. Both E2beta and P4 treatments differentially alter protein expression of the key enzymes involved in PGI2 production in different artery types and may play an important role in the control of blood flow redistribution during hormone replacement therapy.     

Natural progesterone and antihypertensive action.

In a placebo controlled, double blind crossover study natural progesterone was given by mouth, in increasing doses, to six men and four postmenopausal women with mild to moderate hypertension who were not receiving any other antihypertensive drugs. When compared with values recorded before treatment and during administration of placebo progesterone caused a significant reduction in blood pressure, suggesting that progesterone has an antihypertensive action rather than a hypertensive one as has been previously thought. This possible protective effect of progesterone should be investigated further.     

Nongenomic effects of progesterone on the contraction of muscle cells from the guinea pig colon

Progesterone (PG) affects muscle cells by genomic mechanisms through nuclear receptors and by nongenomic mechanisms through unidentified pathways. This study aimed to determine the pathways mediating its nongenomic actions. Experiments were performed in dissociated muscle cells from guinea pig colons. Nongenomic actions were defined as those occurring within 10 min of PG exposure. PG blocked the contraction to CCK-8 and NKA (10(-7) M) but did not impair ACh (10(-7) M) and KCl (2.5 x 10(-2) M)-induced contraction. Both CCK-8 and NKA contract muscle cells by releasing calcium from intracellular stores, whereas ACh and KCl can utilize extracellular calcium. PG also blocked the contraction induced by inositol 1,4,5-trisphosphate, thapsigargin, and caffeine, agents that contract muscle cells by releasing calcium from storage sites. The nongenomic actions of PG were transient because they were absent 1 h after the first PG dose, remaining unresponsive after a second PG dose was administered. Furthermore, PG had no effect on the contraction induced by CCK-8 and thapsigargin in muscle cells from animals pretreated with daily intramuscular PG for 4 days. Cytosolic incorporation experiments of [(3)H]PG showed that pretreatment with unlabeled PG significantly reduced the radiolabeled PG incorporation in the cytosol. We conclude that the nongenomic actions of PG on colonic muscle cells transiently blocked calcium release from storage sites, and this response became rapidly desensitized. This effect does not appear to be specific to PG because other steroid hormones such as aldosterone and testosterone can also induce it.     

Extravascular pressure modulates responses of isolated rat coronary arteries to vasodilator, but not vasoconstrictor, stimuli

The aims of the study were to investigate whether elevated extravascular pressure modulates responses of isolated rat coronary arteries to constrictor and dilator stimuli. Isolated segments of rat coronary artery were mounted in a modified pressure myograph system that allowed independent modulation of both intra- and extravascular pressures. The influence of elevated extravascular pressure on stable levels of myogenic tone and on responses to vasoconstrictor and vasodilator stimuli was investigated at constant overall transmural pressures. Stable levels of myogenic tone were independent of the relative levels of intra- and extravascular pressure, as were responses to depolarization and to addition of the thromboxane agonist U-46619. Elevating extravascular pressure, however, significantly reduced dilatory responses to introduction of intraluminal flow and to addition of endothelium-dependent and endothelium-independent vasodilatory agonists. These results support the notion that elevated extravascular pressure may attenuate responses of coronary arteries to a variety of dilatory stimuli. This finding may be of relevance to cardiac disorders associated with elevated ventricular pressures.     

Studies on the mechanism of the vasodilator action of nicorandil

Nicorandil, a compound having structural similarities to some of the organic nitrates, was studied for its mechanism of vasodilation. Nicorandil is thought to be a K+ channel opening agent. However, little is known about its receptor activation profile, its endothelial dependence, and its effects in atherosclerotic vessels. Nicorandil, at 0.2 to 5 x 10(-6) M, relaxed norepinephrine precontracted rabbit aortic rings in a concentration-dependent manner. Moreover, nicorandil relaxed aortic rings to the same extent in the presence and absence of an intact endothelium. However, nicorandil's effect was diminished in aortic rings from atherosclerotic rabbits. The vasorelaxation action of nicorandil was unaffected by the cyclooxygenase inhibitor ibuprofen or the lipoxygenase inhibitor propyl gallate, suggesting that nicorandil does not act via the release of a vasodilator eicosanoid. Although the nicorandil effect was not influenced by atropine, a muscarinic receptor antagonist, it was significantly attenuated by methylene blue, a guanyl cyclase inhibitor. Thus, nicorandil has some properties in common with organic nitrates and with K+ channel activators but appears to be a unique type of vasodilator.     

Role of progesterone on peri-implantation stage endometrium-embryo interaction in the primate

Progesterone secretion during the luteal phase influences oviductal and endometrial functions which are essential for embryo viability and implantation in a number of species including primates. Luteal phase estrogen is not essential for progesterone-dependent endometrial receptivity towards implantation and pregnancy in the rhesus monkey and in the human. However, synchronous development of embryo and endometrium is an essential prerequisite for evolutive implantation. Progesterone helps to maintain synchronous development of preimplantation embryo through its action on maternal uterus. The anti-nidatory action of mifepristone, a potent progesterone receptor modulator (PRM) with pronounced antiprogestagenic activity, is known to be associated with desynchronization of endometrium along with repression of glandular secretory differentiation and vascular maturation. Thus, it is likely that early luteal phase administration of mifepristone affects paracrine action of the secretory stage endometrium on the preimplantation stage embryo, and thereby inhibits embryonic development and viability. We shall examine this hypothesis using the rhesus monkey as a primate model.     

Prostacyclin increases cAMP in coronary arteries.

This study was designed to determine whether prostacyclin (PGI2)-induced relaxation in circular strips of coronary arteries might be mediated by cAMP. Partially depolarized circular strips of bovine coronary arteries were used and PGI2-induced changes in length were compared with tissue concentrations of cAMP and cGMP, measured by RIA. It was found that PGI2 produced significant and concentration dependent increases in cAMP levels which were closely associated with the relaxant effects produced by the same concentrations (0.3 - 26.7 muM). Cyclic GMP was not changed by these concentrations. The relaxant effects of PGI2 were not antagonized by propranolol. There was a significant linear correlation between log increases in cAMP and percent relaxation produced by PGI2 which was almost identical with similar correlations obtained with either isoprenaline or adenosine, indicating that the relaxant effects of PGI2 are in analogy to those of isoprenaline and adenosine likely to be mediated by cAMP.     

Vasodilator action of docosahexaenoic acid (DHA) in human coronary arteries in vitro

Coronary arterial tissues obtained from mammalian hearts are known to develop spontaneous phasic contractions. The aim of the present study was to investigate the vasodilatory effects of docosahexaenoic acid (DHA) on the rhythmic contractions of isolated human coronary arterial (HCA) preparations obtained from the recipient hearts of patients undergoing cardiac transplantation. Results from 8 hearts show that: (i) most HCA tissues displayed spontaneous rhythmic phasic contractions with a cycle length around 10 min in the absence or presence of PGF2alpha or elevated [K+]0 (20 mM); (ii) the rhythmic activity could be suppressed by a free fatty acid DHA (30 microM); (iii) high [K+]0 (20 and 80 mM) could induce sustained tonic contraction in addition to phasic contractions in HCA tissues, the tonic contraction could be antagonized by L-type Ca(2+) channel blockers or by DHA (depending on [K+]0); (iv) a digitalis substance ouabain also could induce tonic contraction and suppress phasic contraction; (v) in isolated HCA vascular smooth muscle cells, DHA increased the magnitude of outward voltage-gated K+ (IKV) currents and the inwardly rectifying IK1 currents. Enhancement of K+ currents could be related to vasorelaxation induced by DHA in HCA preparations. Further studies on the effects of DHA on various ionic currents and intracellular Ca(2+) transient are needed to clarify the Ca(2+)-dependent and the Ca(2+)-independent actions of DHA in HCA.