Inhibitory action of lanthanum(La) on PGF2α- induced contraction of guinea-pig stomach muscle

The effects of lanthanum(La) on contractions induced by prostaglandin F_2α(PGF_2α) or isotonic K⁺ were investigated in the isolated stomach muscle of guinea-pig.Low concentrations of La(0.1–1 μM) inhibited the contraction to PGF_2α 1 μM in a dose-dependent manner, without affecting the tonic contraction to isotonic K⁺.0.1 and 1 μM La shifted the dose-response curve for PGF_2α(0.001 – 1 μM) to the right and reduced the maximum response.The IC₅₀ of La against PGF_2α and K⁺ were 0.6 μM and 30 μM, respectively.These results support the suggestion that PGF_2α -induced contraction in the stomach muscle depends mainly on the intracellular release of sequestered Ca, which would be depleted or immobilized by La.     

Different responsiveness of a variety of isolated dog arteries to prostaglandin D3

The addition of prostaglandin (PG) D₂ contracted helical strips of dog cerebral, coronary, renal and femoral arteries; the contraction was greatest in cerebral arteries. The contractile response of cerebral arteries was potentiated by aspirin and attenuated by polyphloretin phosphate. In the arterial strips contracted with PGF_2α, PGD₂ elicited a concetration-related relaxation; the relaxation was greatest in mesenteric arteries. In mesenteric arterial strips contracted with norepinephrine, a lesser degree of relaxation was induced, and in the K⁺-contracted arteries, only a contraction was induced. Treatment with PGD₂ attenuated the contractile responses of cerebral and mesentric arteries to PGF_2α or PGE₂; this inhibitory effect was approximately 10 times greater in mesenteric arteries. However, the response to serotonin (for cerebral arteries) or norepinephrine (for mesenteric) was unaffected. It may be concluded that the heterogeneity of response to PGD₂ of a variety of dog arteries is due to different contributions of vasoconstrictor and vasodilator mechanisms. PGD₂ appears top share the mechanism underlying arterial contraction with PGF_2α and PGE₂, and interferes with the effect of these PG's possibly on receptor sites.     

The effects of prostaglandins, prostaglandin inhibitors, and oxygen on the closure of the ductus arteriosus, pulmonary arteries and umbilical vessels in vitro

Three prostaglandins (PGF₂α and PGE₁, PGE₂) have been found in maternal and fetal circulation during labour. Two of these prostaglandins (PGF₂α and PGE₂) are present in elevated levels in maternal circulation during labour and their presence in fetal vessels has been shown.These three prostaglandins have been tested for their effects on fetal vessels in vitro (umbilical artery and vein, ductus arteriosus, and smaller pulmonary artery). These vessels were selected as being crucial in the conversion from fetal to extra-uterine circulation in mammalian species. Responses of these vessels to the prostaglandins under varying oxygen regimes have been examined as well as their responses to prostaglandin inhibitors. Activity of vessels of varying gestational ages exposed to PGF₂α was also examined. The following results were obtained:

1. All vessels, with the exception of pulmonary arteries, contracted in the presence of oxygen over the range 20–100mmHg pO₂. At a pO₂ of < 20mmHg the ductus arteriosus remained inactive or dilated. Pulmonary arteries dilated at high pO₂.

2. All vessels contracted in response to exogenous PGF₂α with the exception of the pulmonary arteries which dilated. In the presence of PGF₂α, the umbilical veins dilated under low (< 20mmHg) pO₂ and contracted at higher levels. Contraction also occurred at lower levels after a period of time.

3. Although PGF₂α was capable of causing contraction in the ductus arteriosus at near zero pO₂, oxygen, (or possibly the products of oxygenation), appear to be required for continued contraction in the presence of PGF₂α. A synergistic relationship between oxygen and PGF₂α responses was found as oxygen tensions increased. A synergistic response between PGF₂α and oxygen with umbilical arteries which did not increase with increased pO₂ was also found. Oxygen tension appeared to have little effect on the response of other vessels to PGF₂α.

4. PGE₁ caused dilations in all vessels examined. Such dilations appearing to be independent of the oxygen regime prevailing. However, an increase in oxygen during experiments reversed any dilation caused by the prostaglandins.

5. PGE₂ caused contractions in umbilical vessels which were independent of oxygen. PGE₂ caused contraction of pulmonary arteries. However, in the ductus arteriosus, PGE₂ caused an initial contraction followed by a strong dilation. This dilation became weaker as pO₂ increased.

6. Additions of prostaglandin inhibitors (Naproxen and Indomethacin) to the bathing solution in which the ductus arteriosus and umbilical arteries were contracting (in response to PGF₂α, or oxygen alone) caused a decrease in contractions, and sometimes a slight decrease when the vessel had been pretreated with PGF₂α suggesting a possible need for endogenously synthesised prostaglandins for the maintenance of oxygen mediated contractions (in vivo).

7. Vessels responsed to PGF₂α at an early gestational age. A role for prostaglandins and oxygen in the closure of fetal vessels is discussed.

     

Biphasic effect of extracellular ATP on human and rat airways is due to multiple P2 purinoceptor activation

Background

Extracellular ATP may modulate airway responsiveness. Studies on ATP-induced contraction and [Ca²⁺]_i signalling in airway smooth muscle are rather controversial and discrepancies exist regarding both ATP effects and signalling pathways. We compared the effect of extracellular ATP on rat trachea and extrapulmonary bronchi (EPB) and both human and rat intrapulmonary bronchi (IPB), and investigated the implicated signalling pathways.

Methods

Isometric contraction was measured on rat trachea, EPB and IPB isolated rings and human IPB isolated rings. [Ca²⁺]_i was monitored fluorimetrically using indo 1 in freshly isolated and cultured tracheal myocytes. Statistical comparisons were done with ANOVA or Student''s t tests for quantitative variables and χ² tests for qualitative variables. Results were considered significant at P < 0.05.

Results

In rat airways, extracellular ATP (10^-6–10^-3 M) induced an epithelium-independent and concentration-dependent contraction, which amplitude increased from trachea to IPB. The response was transient and returned to baseline within minutes. Similar responses were obtained with the non-hydrolysable ATP analogous ATP-γ-S. Successive stimulations at 15 min-intervals decreased the contractile response. In human IPB, the contraction was similar to that of rat IPB but the time needed for the return to baseline was longer. In isolated myocytes, ATP induced a concentration-dependent [Ca²⁺]_i response. The contractile response was not reduced by thapsigargin and RB2, a P2Y receptor inhibitor, except in rat and human IPB. By contrast, removal of external Ca²⁺, external Na⁺ and treatment with D600 decreased the ATP-induced response. The contraction induced by α-β-methylene ATP, a P2X agonist, was similar to that induced by ATP, except in IPB where it was lower. Indomethacin and H-89, a PKA inhibitor, delayed the return to baseline in extrapulmonary airways.

Conclusion

Extracellular ATP induces a transient contractile response in human and rat airways, mainly due to P2X receptors and extracellular Ca²⁺ influx in addition with, in IPB, P2Y receptors stimulation and Ca²⁺ release from intracellular Ca²⁺ stores. Extracellular Ca²⁺ influx occurs through L-type voltage-dependent channels activated by external Na⁺ entrance through P2X receptors. The transience of the response cannot be attributed to ATP degradation but to purinoceptor desensitization and, in extrapulmonary airways, prostaglandin-dependent PKA activation.     

Histamine releases PGI2 from human pulmonary artery

Histamine caused a triphasic response of human pulmonary artery strips in vitro, consisting of a small initial contraction followed by pronounced relaxation preceding a second contractile response. These characteristics were not seen with other contractile stimuli including 5-hdyroxytryptamine, leukotriene D₄, and KC1. The relaxant component of this response was ablated by removal of endothelium from the vascular strips or by pretreatment of the tissues with 1μM indomethacin. Measurement of the PGI₂ degradation product 6-keto-PGF_1α in supernatants from histamine-challenged tissues confirmed the synthesis of PGI₂. Supernatants from unstimulated or leukotriene-challenged tissues contained no detectable amounts of 6-keto-PGF_1α. The histamine H₁ antagonist diphenhydramine inhibited both the contractile and relaxant responses to histamine whereas the H₂ antagonist cimetidine affected neither component. The released PGI₂ significantly altered the dose-respons curve to histamine without inhibiting the maximal contractile responses. We conclude that histamine induces PGI₂ formation from pulmonary arterial endothelium via an H₁ receptor.     

Activation of 72-kDa Type IV Collagenase/Gelatinase by Normal Fibroblasts in Collagen Lattices Is Mediated by Integrin Receptors but Is Not Related to Lattice Contraction

The matrix metalloproteinase 72-kDa type IV collagenase (also known as gelatinase A) is thought to be involved in both normal connective tissue remodeling and invasive pathological processes. Like other matrix metalloproteinases, 72-kDa type IV collagenase is secreted by fibroblast monolayers as an inactive proenzyme, but is unique among this enzyme family in that it is not activated by serine proteinases such as plasmin. However, when fibroblasts are cultured in a collagen lattice, a situation thought to better approximate in vivo conditions, we have invariably found much of the secreted 72-kDa type IV collagenase in its enzymatically active 62-kDa form. Although collagen lattice contraction appeared to be required for the activation of 72-kDa type IV collagenase, we have found that the process of contraction can be dissociated from proenzyme activation. Both cytochalasin D and α-methylmannoside completely blocked lattice contraction, but not proenzyme activation. Furthermore, the monoclonal antibody M-13, which is directed against the β₁ integrin chain, blocked collagen lattice contraction but not 72-kDa type IV procollagenase activation. At concentrations significantly higher than required to block lattice contraction or cell adhesion to collagen, M-13 was able to inhibit proenzyme activation. A second monoclonal antibody to the β₁ integrin, P5D2, had little effect on collagen lattice contraction at low concentrations, but could significantly inhibit the activation of 72-kDa type IV procollagenase. Antibodies to the integrin α₂ chain also inhibited proenzyme activation. These data show that the activation of 72-kDa type IV collagenase proenzyme, like collagen lattice contraction, is mediated by β₁ integrin receptors, possibly α₂β₁. Although both anti-β₁ antibodies used are directed to the same site on the integrin chain, the fact that each antibody preferentially blocks a different event, either lattice contraction or activation of 72-kDa type IV collagenase, suggests the existence of branch points in the receptor-mediated signal transduction pathway.     

A Long Lasting β1 Adrenergic Receptor Stimulation of cAMP/Protein Kinase A (PKA) Signal in Cardiac Myocytes

Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β₁ adrenergic receptor (β₁AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t_½ of 3.9 h) in cardiac myocytes. However, the β₁AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β₁AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β₁AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.     

Effects of prostaglandins and arachidonic acid on baboon cerebral and mesenteric arteries

Effects of prostaglandins (PGs) E₁, E₂, F_2α and I₂ in a wide range of concentration were examined in mesenteric and cerebral arteries isolated from mature baboons. PGs E₁, E₂ and F_2α at low concentrations (10⁻¹⁰ to 10⁻⁷ M) elicited relaxation in helically cut strips of cerebral arteries precontracted with phenylephrine. In contrast, the PGs did not cause relaxation in the mesentric artery. PGI₂ (10⁻⁹ to 10⁻⁶ M) produced marked relaxation in both arteries. The EC₂₅ for PGI₂ in the mesenteric artery was significantly lower than that in the cerebral artery. During baseline conditions, cerebral arteries contracted in response to high concentrations (greater than 10⁻⁷ M) of PGs E₁, E₂ and F_2α. In mesentric arteries, a large contraction was induced by PGs F_2α and E₂ but not by PGE₁. Arachidonic acid (10⁻⁶ M) produced an aspirin-inhibitable relaxation in both arteries to a similar extent, so that the vasodilator PG(s) formed in the two different arterial walls appear to exert a similar relaxant action. Thus, the baboon mesenteric artery was more sensitive to PGI₂ for the relaxant effect than was the cerebral artery, while PGs F_2α, E₁ and E₂ caused only a contraction in the mesenteric artery but both relaxation and contraction in the cerebral artery.     

Biological activities of 17-phenyl-18,19,20-trinorprostaglandins

In a number of assay ssytems, some 17-phenyl-trinor prostaglandins were similar in activity and potency to the corresponding parent prostaglandin. In others, the 17-phenyl analogs appeared several times more potent. In the hamster antifertility assay, which is considered to measure luteolytic activity, 17-phenyl-18,19,20-trinor prostaglandin F_2α was about 90-times PGF_2α in potency.Rat blood pressure responses to 17-phenyl analogs were significant. The 17-phenyl-trinor PGF_2α pressor potency was 5 times that of PGF_2α. The 17-phenyl-trinor PGE₂ blood pressure response was atypical since a pressor rebound phenomenon followed the expected depressor response. Lastly, 17-phenyl-trinor PGF_2α was more potent than PGF_2α in synchronizing the estrous cycle in beef cows.     

Desensitization of the Neurokinin-1 Receptor (NK1-R) in Neurons: Effects of Substance P on the Distribution of NK1-R, Gαq/11, G-Protein Receptor Kinase-2/3, and β-Arrestin-1/2

Observations in reconstituted systems and transfected cells indicate that G-protein receptor kinases (GRKs) and β-arrestins mediate desensitization and endocytosis of G-protein–coupled receptors. Little is known about receptor regulation in neurons. Therefore, we examined the effects of the neurotransmitter substance P (SP) on desensitization of the neurokinin-1 receptor (NK1-R) and on the subcellular distribution of NK1-R, G_αq/11, GRK-2 and -3, and β-arrestin-1 and -2 in cultured myenteric neurons. NK1-R was coexpressed with immunoreactive G_αq/11, GRK-2 and -3, and β-arrestin-1 and -2 in a subpopulation of neurons. SP caused 1) rapid NK1-R–mediated increase in [Ca²⁺]_i, which was transient and desensitized to repeated stimulation; 2) internalization of the NK1-R into early endosomes containing SP; and 3) rapid and transient redistribution of β-arrestin-1 and -2 from the cytosol to the plasma membrane, followed by a striking redistribution of β-arrestin-1 and -2 to endosomes containing the NK1-R and SP. In SP-treated neurons G_αq/11 remained at the plasma membrane, and GRK-2 and -3 remained in centrally located and superficial vesicles. Thus, SP induces desensitization and endocytosis of the NK1-R in neurons that may be mediated by GRK-2 and -3 and β-arrestin-1 and -2. This regulation will determine whether NK1-R–expressing neurons participate in functionally important reflexes.     

The influence of xanthines on the contractile responses of PGF2α and PGD2 in human parenchymal strips

Prostaglandins (PGs) F_2α and D₂ are bronchoconstrictor agents which are released under allergic conditions such as asthma. The efficacy and potency of PGF_2α and PGD₂ differ in some tissues. We compared the effects of these two PGs in sensitized human parenchymal strips. In six experiments, PGF_2α 0.1 and 0.3 μM produced greater contractions than PGD₂ at the same concentrations. There were no significant differences between the contractions from the two PGs at concentrations of 0.01, 0.03, 1.0–10 μM and the two PGs appeared to be equipotent.We studied the effects of the anti-asthmatic drug theophylline, and its analogue enprofylline, on the contraction caused by these PGs. Theophylline 100 μM caused no change to the cumulative concentration response curves. However, enprofylline 100 μM reduced the PGF_2α-induced contractions.     

β1 Integrin-mediated collagen gel contraction is stimulated by PDGF

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent β₁ integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-β1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin β₁-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-β₁ integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the β₁ integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.     

Prostaglandin E1 inhibits the pulmonary vascular pressor response to hypoxia and prostaglandin F2α

In the anesthetised dog an infusion of exogenous prostaglandin E₁ (100μG/min) inhibits the pulmonary vascular pressor response to hypoxia. Both 25 and 100 μG/min PGE₁ can reduce the transient pulmonary hypertension caused by a bolus of prostaglandin F_2α. This suggests that hypoxia and PGF₂α may share a final common pathway in producing pulmonary vasoconstriction. These results may help to explain the mechanism by which endotoxin inhibits the pulmonary vascular response to hypoxia. This effect is probably achieved by stimulating the production of an endogenous dilator prostaglandin. Exogenous PGE₁ can mimic this effect.     

Cyclic Nucleotide-Gated Channels Contribute to Thromboxane A2-Induced Contraction of Rat Small Mesenteric Arteries

Background

Thromboxane A₂ (TxA₂)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can be partly attributed to TxA₂-induced Ca²⁺ influx, which resulted in vascular contraction via Ca²⁺-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA₂-induced Ca²⁺ influx in vascular smooth muscle cells.

Methodology/Principal Findings

Application of U-46619, a thromboxane A₂ mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relies on the presence of extracellular Ca²⁺, because removal of extracellular Ca²⁺ abolished the constriction. This constriction was partially inhibited by an L-type Ca²⁺ channel inhibitor nifedipine (0.5–1 µM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner. Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In the primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca²⁺ ([Ca²⁺]_i), which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence of CNGA2 protein in vascular smooth muscle cells.

Conclusions/Significance

These data suggest a functional role of CNG channels in U-46619-induced Ca²⁺ influx and contraction of smooth muscle cells.     

In vitro ovulation of trout oocytes : Effect of prostaglandins on smooth muscle-like cells of the theca

Ovulation (active expulsion of oocyte from the mature follicle) of trout follicles matured can be induced by adding PGF_2α at doses of 1 and 5 μg/ml. PGE₂ is ineffective.The induction of ovulation by PGF_2α is inhibited in a calcium free medium or by inhibitors of calcium influx, particularly by Mn⁺⁺ and La⁺⁺⁺, suggesting that ovulation process implies active contraction of the smooth muscle cells of the theca.A significant but partial inhibition is also observed with cytochalasin B (1 and 5 μg/ml) demonstrating that contraction of other cell types than muscle, containing actin-like filaments, may also participate in the process.     

The Signaling Mechanism of Contraction Induced by ATP and UTP in Feline Esophageal Smooth Muscle Cells

P2 receptors are membrane-bound receptors for extracellular nucleotides such as ATP and UTP. P2 receptors have been classified as ligand-gated ion channels or P2X receptors and G protein-coupled P2Y receptors. Recently, purinergic signaling has begun to attract attention as a potential therapeutic target for a variety of diseases especially associated with gastroenterology. This study determined the ATP and UTP-induced receptor signaling mechanism in feline esophageal contraction. Contraction of dispersed feline esophageal smooth muscle cells was measured by scanning micrometry. Phosphorylation of MLC₂₀ was determined by western blot analysis. ATP and UTP elicited maximum esophageal contraction at 30 s and 10 μM concentration. Contraction of dispersed cells treated with 10 μM ATP was inhibited by nifedipine. However, contraction induced by 0.1 μM ATP, 0.1 μM UTP and 10 μM UTP was decreased by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, chelerythrine, ML-9, PTX and GDPβS. Contraction induced by 0.1 μM ATP and UTP was inhibited by Gαi₃ or Gαq antibodies and by PLCβ₁ or PLCβ₃ antibodies. Phosphorylated MLC₂₀ was increased by ATP and UTP treatment. In conclusion, esophageal contraction induced by ATP and UTP was preferentially mediated by P2Y receptors coupled to Gαi₃ and G q proteins, which activate PLCβ₁ and PLCβ₃. Subsequently, increased intracellular Ca²⁺ and activated PKC triggered stimulation of MLC kinase and inhibition of MLC phosphatase. Finally, increased pMLC₂₀ generated esophageal contraction.     

Physiological concentrations of ADP stimulate the release of prostacyclin from bovine aortic endothelial cells

ADP (0.2−200 μN) stimulated the synthesis of prostacyclin (PGI₂), as reflected by the release of 6-keto-prostaglandin F_1α (6-K-PGF_1α), in endothelial cells cultured from bovine orta. This effect of ADP was mimicked by ATP, whereas AMP and adenosine were completely inactive. The release of 6-K-PGF_1α triggered by ADP was rapid in onset (within 5 min), transient (10 min) and followed by a period of refractoriness to a new ADP challenge. Growing and confluent cells were equally responsive to ADP. ADP stimulated the release of free arachidonic acid from the endothelial cells. ADP could be thus exert two opposite actions on platelet aggregation in vivo: a direct stimulation and an inhibition mediated by PGI₂. This last action might contribute to limit thrombus formation to areas of endothelial cell damage.     

Effects of PGD2 and PGF2α on longitudinal and circular muscles of guinea-pig isolated proximal colon

The mechanical effects of PGD₂ and PGF_2α on longitudinal and circular muscles of the guinea-pig isolated proximal colon were investigated. PGD₂ and PGF_2α (1 nM – 10 μM) produced a dose-dependent contraction in longitudinal and circular muscles. The contractile action of PGD₂ was more potent than that of PGF_2α in circular muscle and was less potent in longitudinal muscle.Contractions induced by PGD₂ or PGF_2α(1 μM) were unaffected by atropine (1 μM) in both muscles, but tetrodotoxin (1 μM) slightly inhibited these contractions in longitudinal muscle.The results suggest that in longitudinal muscle PGD₂ and PGF_2α have largely a direct action on the muscle cells and a partial neuronal action on the non-cholinergic intrinsic nerves, whereas in circular muscle these PGs have only a direct action on the muscle cells.