Drug-protein binding and blood-brain barrier permeability.
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The permeability surface area (PS) product, an index of permeability of the blood-brain barrier (BBB), was measured by using the in situ perfusion method. In the cerebral circulation, the fraction of drug that permeates into the brain through the BBB is not only the unbound fraction but also the fraction dissociated from the protein in the perfusate. The sum of these two fractions, the apparent exchangeable fraction, was estimated by fitting the parameters of the BBB permeability under the condition of varying BSA concentrations in the perfusate. The unbound fraction of drugs in a buffer containing 0.5 mM BSA was measured by using the ultrafiltration method in vitro, and the apparent exchangeable fraction was measured in vivo by using the intracarotid artery injection method. The apparent exchange fraction was 100% for S-8510, 96.5% for diazepam, 90.9% for caffeine, 38.3% for S-312-d, 33.1% for propranolol, and 6.68% for (+)-S-145 Na, and each of these was higher than the corresponding unbound fraction in vitro in all drugs. The apparent exchangeable fractions, for example, were 8 times higher for diazepam and 38 times for S-312-d than the unbound fractions in vitro. The apparent exchangeable fraction of drugs was also estimated from the parameters obtained with the perfusion method. Because drugs can be infused for an arbitrary length of time in the perfusion method, substances with low permeability can be measured. The apparent exchangeable fractions obtained with this method were almost the same as those obtained with the intracarotid artery injection method. (+info)
Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets.
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Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change. (+info)
The cyclo-oxygenase-dependent regulation of rabbit vein contraction: evidence for a prostaglandin E2-mediated relaxation.
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1. Arachidonic acid (0.01-1 microM) induced relaxation of precontracted rings of rabbit saphenous vein, which was counteracted by contraction at concentrations higher than 1 microM. Concentrations higher than 1 microM were required to induce dose-dependent contraction of vena cava and thoracic aorta from the same animals. 2. Pretreatment with a TP receptor antagonist (GR32191B or SQ29548, 3 microM) potentiated the relaxant effect in the saphenous vein, revealed a vasorelaxant component in the vena cava response and did not affect the response of the aorta. 3. Removal of the endothelium from the venous rings, caused a 10 fold rightward shift in the concentration-relaxation curves to arachidonic acid. Whether or not the endothelium was present, the arachidonic acid-induced relaxations were prevented by indomethacin (10 microM) pretreatment. 4. In the saphenous vein, PGE2 was respectively a 50 and 100 fold more potent relaxant prostaglandin than PGI2 and PGD2. Pretreatment with the EP4 receptor antagonist, AH23848B, shifted the concentration-relaxation curves of this tissue to arachidonic acid in a dose-dependent manner. 5. In the presence of 1 microM arachidonic acid, venous rings produced 8-10 fold more PGE2 than did aorta whereas 6keto-PGF1alpha and TXB2 productions remained comparable. 6. Intact rings of saphenous vein relaxed in response to A23187. Pretreatment with L-NAME (100 microM) or indomethacin (10 microM) reduced this response by 50% whereas concomitant pretreatment totally suppressed it. After endothelium removal, the remaining relaxing response to A23187 was prevented by indomethacin but not affected by L-NAME. 7. We conclude that stimulation of the cyclo-oxygenase pathway by arachidonic acid induced endothelium-dependent, PGE2/EP4 mediated relaxation of the rabbit saphenous vein. This process might participate in the A23187-induced relaxation of the saphenous vein and account for a relaxing component in the response of the vena cava to arachidonic acid. It was not observed in thoracic aorta because of the lack of a vasodilatory receptor and/or the poorer ability of this tissue than veins to produce PGE2. (+info)
Internalization of the TXA2 receptor alpha and beta isoforms. Role of the differentially spliced cooh terminus in agonist-promoted receptor internalization.
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Thromboxane A2 (TXA2) potently stimulates platelet aggregation and smooth muscle constriction and is thought to play a role in myocardial infarction, atherosclerosis, and bronchial asthma. The TXA2 receptor (TXA2R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent assay and immunofluorescence microscopy that the TXA2Rbeta, but not the TXA2Ralpha, undergoes agonist-induced internalization when expressed in HEK293 cells as well as several other cell types. Various dominant negative mutants were used to demonstrate that the internalization of the TXA2Rbeta is dynamin-, GRK-, and arrestin-dependent in HEK293 cells, suggesting the involvement of receptor phosphorylation and clathrin-coated pits in this process. Interestingly, the agonist-stimulated internalization of both the alpha and beta isoforms, but not of a mutant truncated after residue 328, can be promoted by overexpression of arrestin-3, identifying the C-tails of both receptors as necessary in arrestin-3 interaction. Simultaneous mutation of two dileucine motifs in the C-tail of TXA2Rbeta did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between residues 355 and 366 of the TXA2Rbeta is essential for agonist-promoted internalization. These data demonstrate that alternative splicing of the TXA2R plays a critical role in regulating arrestin binding and subsequent receptor internalization. (+info)
Differential signaling by the thromboxane receptor isoforms via the novel GTP-binding protein, Gh.
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Thromboxane A2 acts via G protein-coupled receptors; two splice variants of the thromboxane A2 receptor (TPalpha and TPbeta) have been cloned. It is unknown whether they differ in their capacity to activate intracellular signaling pathways. Recently, a high molecular weight G protein, Gh, that can also function as a tissue transglutaminase, has been described. We investigated whether Gh functions as a signaling protein in association with thromboxane receptors. First, we sought Gh expression in cells known to express TPs. Reverse transcription-polymerase chain reaction and immunoblotting demonstrated Gh expression in platelets, megakaryocytic cell lines, and endothelial and vascular smooth muscle cells. Second, immunoprecipitation of both TPalpha and TPbeta in transfected COS-7 cells resulted in the co-immunoprecipitation of Gh, indicating that TPs may associate Gh in vivo. Finally, agonist activation of TPalpha, but not of TPbeta, resulted in stimulation of phospholipase C-mediated inositol phosphate production in cells cotransfected with Gh. By contrast, agonist activation of both TP isoforms resulted in Gq-mediated inositol phosphate signaling. Gh is expressed in platelets and vascular cells and may associate with both TP isoforms. However, stimulation of TP isoforms results in differential activation of downstream signaling pathways via this novel G protein. (+info)
Acetylcholine-induced endothelium-derived contracting factor in hypoxic pulmonary hypertensive rats.
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We determined the role of an endothelium-derived contracting factor in the impaired relaxation response to ACh of conduit pulmonary arteries (PAs) isolated from rats with hypoxic pulmonary hypertension (PH). A PGH2/thromboxane A2 (TxA2)-receptor antagonist (ONO-3708) partially restored the impairment of ACh-induced relaxation, whereas TxA2 synthase inhibitors (OKY-046 and CV-4151) did not affect the impaired relaxation in phenylephrine-precontracted hypertensive PAs. Endothelium-denuded hypertensive PA rings showed no difference in the response to ACh between preparations with and without ONO-3708. In both endothelium-denuded control and hypertensive PAs, exogenous PGH2 induced contractions, and the magnitude of the contractions was greater in the control than in hypoxic PH preparations. An endothelin A-receptor antagonist (BQ-485), an endothelin B-receptor antagonist (BQ-788), and a superoxide anion scavenger (superoxide dismutase) did not restore the impaired response to ACh in hypertensive PAs. These findings suggest that PGH2 produced from the conduit PAs of rats with chronic hypoxic PH may be the endothelium-derived contracting factor responsible for the impairment of ACh-mediated vasorelaxation. (+info)
Identification of Galpha13 as one of the G-proteins that couple to human platelet thromboxane A2 receptors.
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Previous studies have shown that ligand or immunoaffinity chromatography can be used to purify the human platelet thromboxane A2 (TXA2) receptor-Galphaq complex. The same principle of co-elution was used to identify another G-protein associated with platelet TXA2 receptors. It was found that in addition to Galphaq, purification of TXA2 receptors by ligand (SQ31,491)-affinity chromatography resulted in the co-purification of a member of the G12 family. Using an antipeptide antibody specific for the human G13 alpha-subunit, this G-protein was identified as Galpha13. In separate experiments, it was found that the TXA2 receptor agonist U46619 stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) incorporation into G13 alpha-subunit. Further evidence for functional coupling of G13 to TXA2 receptors was provided in studies where solubilized platelet membranes were subjected to immunoaffinity chromatography using an antibody raised against native TXA2 receptor protein. It was found that U46619 induced a significant decrease in Galphaq and Galpha13 association with the receptor protein. These results indicate that both Galphaq and Galpha13 are functionally coupled to TXA2 receptors and dissociate upon agonist activation. Furthermore, this agonist effect was specifically blocked by pretreatment with the TXA2 receptor antagonist, BM13.505. Taken collectively, these data provide direct evidence that endogenous Galpha13 is a TXA2 receptor-coupled G-protein, as: 1) its alpha-subunit can be co-purified with the receptor protein using both ligand and immunoaffinity chromatography, 2) TXA2 receptor activation stimulates GTPgammaS binding to Galpha13, and 3) Galpha13 affinity for the TXA2 receptor can be modulated by agonist-receptor activation. (+info)
Role of thromboxane in the altered vascular reactivity of pregnant rats with adriamycin nephropathy.
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BACKGROUND: Pregnant rats with adriamycin nephropathy (ADRP rats) develop hypertension and have an increased vascular reactivity to noradrenaline in the isolated mesenteric bed in vitro. We have shown previously that the administration of daltroban, a specific thromboxane receptor antagonist, prevented hypertension in ADRP rats. METHODS: We measured the effect of daltroban (10(-5) mol/l) on the vasoconstrictory response to noradrenaline (1-10 micromol/l) in the isolated mesenteric bed of ADRP rats at the end of pregnancy, as compared with normal pregnant and adriamycin-treated virgin rats. In further experiments, we measured the changes of flow induced by increasing concentrations of the thromboxane analogue, U46619 (10(-7)-10(-6) mol/l). Finally, changes of flow were assessed in arteries maximally constricted with U46619 (10(-6) mol/l), during perfusion in the presence of increasing concentrations of daltroban (10(-7)-10(-5) mol/l). RESULTS: Daltroban diminished the response to noradrenaline in all groups, shifting the concentration-effect curve to the right. However, at maximal concentrations of noradrenaline, daltroban was ineffective in all rats, except in ADRP animals. The vasoconstrictory response to U46619 was significantly reduced in all pregnant rats, both normal and adriamycin-treated. Daltroban progressively released the vasoconstriction induced by U46619 in all groups. However, this vasodilator response was attenuated in the adriamycin-treated rats, the slopes of their curves being smaller than those of the respective untreated groups (0.038 +/- 0.006 in virgin rats vs 0.063 +/- 0.011 in controls, P < 0.05; and 0.015 +/- 0.005 in ADRP vs 0.028 +/- 0.008 in normal pregnancy, P < 0.05). CONCLUSIONS: The findings could be explained by enhanced occupancy of thromboxane receptors by an endogenous agonist, possibly PGH2, as a consequence of either increased levels of the autacoid or increased number of affinity receptors. (+info)