Enhancing analytical accuracy of intravascular electrochemical oxygen sensors via nitric oxide release using S-nitroso-N-acetyl-penicillamine (SNAP) impregnated catheter tubing.

Implantable medical devices are an integral part of primary/critical care. However, these devices carry a high risk for blood clots, caused by platelet aggregation on a foreign body surface. This study focuses on the development of a simplified approach to create nitric oxide (NO) releasing intravascular electrochemical oxygen (O2) sensors with increased biocompatibility and analytical accuracy. The implantable sensors are prepared by embedding S-nitroso-N-acetylpenacillamine (SNAP) as the NO donor molecule in the walls of the catheter type sensors. The SNAP-impregnated catheters were prepared by swelling silicone rubber tubing in a tetrahydrofuran solution containing SNAP. Control and SNAP-impregnated catheters were used to fabricate the Clark-style amperometric PO2 sensors. The SNAP-impregnated sensors release NO under physiological conditions for 18 d as measured by chemiluminescence. The analytical response of the SNAP-impregnated sensors was evaluated in vitro and in vivo. Rabbit and swine models (with sensors placed in both veins and arteries) were used to evaluate the effects on thrombus formation and analytical in vivo PO2 sensing performance. The SNAP-impregnated PO2 sensors were found to more accurately measure PO2 levels in blood continuously (over 7 and 20 h animal experiments) with significantly reduced thrombus formation (as compared to controls) on their surfaces.

Subclasses of cyclic GMP-specific phosphodiesterase and their role in regulating the effects of atrial natriuretic factor.

Two subclasses of cyclic guanosine monophosphate (GMP)-specific phosphodiesterases were identified in vascular tissue from several beds. The activity of one subclass (phosphodiesterase IB) was stimulated severalfold by calmodulin and selectively inhibited by the phosphodiesterase inhibitor TCV-3B. The activity of the other subclass (phosphodiesterase IC) was not stimulated by calmodulin and was selectively inhibited by the phosphodiesterase inhibitor M&B 22,948. To assess the involvement of both subclasses in regulating cyclic GMP-dependent responses, the ability of TCV-3B and M&B 22,948 to potentiate the in vitro and in vivo responses to the endogenous guanylate cyclase stimulator atrial natriuretic factor (ANF) was evaluated. Both TCV-3B and M&B 22,948 relaxed isolated rabbit aortic and pulmonary artery rings and also potentiated the relaxant effect of ANF. In addition, both inhibitors produced small increases in urine flow and sodium excretion in anesthetized rats and potentiated the diuretic and natriuretic responses to exogenous ANF. M&B 22,948 (30 micrograms/kg/min) produced a threefold increase in the natriuretic response to simultaneously administered ANF, and TCV-3B (10 micrograms/kg/min) produced a twofold increase in the response to ANF. The results of the present experiments suggest that both the calmodulin-sensitive and calmodulin-insensitive subclasses of cyclic GMP-specific phosphodiesterase play a role in regulating the in vitro and in vivo response to ANF.

Nonpeptide angiotensin II receptor antagonists. 1. Synthesis and in vitro structure-activity relationships of 4-[[[(1H-pyrrol-1-ylacetyl)amino]phenyl]methyl]imidazole derivatives as angiotensin II receptor antagonists.

A novel series of non-biphenylyltetrazole angiotensin II receptor antagonists which contain a 1H-pyrrol-1-ylacetyl residue in place of the benzoyl residue in EXP 6803 have been developed. The receptor binding activity of several members of this new series was in the 10(-8) M range, which was better than that of EXP 6803. Introduction of a carboxylic acid moiety at the 2-position of the pyrrole ring enhanced the in vitro binding affinity at the receptor by 10-fold. Compounds containing an acetic acid (18) or a propionic acid residue (20) at the 5-position of the imidazole were more potent than the carboxylic acid analogue (24). The binding IC50 of the most potent compound 20 was 22 nM. Compounds 18, 20, and 24 in their best fit conformations were manually overlayed on that of the template conformation of EXP 6803 and EXP 8623, respectively. The synthesis and structure-activity relationship data are described.

Multiple binding modes for the receptor-bound conformations of cyclic AII agonists.

Angiotensin II, Asp-Arg-Val-Tyr-His-Pro-Phe, binds its receptor with a postulated turn centered at residue four. Analogs of angiotensin II which contain a disulfide bridge between the side chains of residues 3 and 5 retain significant activity consistent with this hypothesis. Incorporation of 4-mercaptoproline residues, a hybrid, or chimeric amino acid which combines the properties of proline and homocysteine, into either of these positions with analogous disulfide bridges allows retention of high affinity for the receptor. These more highly constrained bicyclic systems give new insight into the details of molecular recognition of residues 3-5 of angiotensin by the receptor. Retention of activity by the antiparallel dimer of [Sar1,Cys3,5]-AII in which the peptide backbone is held in an extended conformation was unexpected. Analysis of the conformational constraints imposed in these active analogs suggests that AII agonists bind to their receptor with different backbone conformations in the region of the central tyrosine residue.

Structure-activity relationships for a novel series of pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors.

Screening of a compound library for inhibitors of the fibroblast growth factor (FGFr) and platelet-derived growth factor (PDGFr) receptor tyrosine kinases led to the development of a novel series of ATP competitive pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors. The initial lead, 1-[2-amino-6-(2,6-dichlorophenyl)pyrido[2,3-d]pyrimidin-7-yl]-3- tert-butylurea (4b, PD-089828), was found to be a broadly active tyrosine kinase inhibitor. Compound 4b inhibited the PDGFr, FGFr, EGFr, and c-src tyrosine kinases with IC50 values of 1.11, 0.13, 0.45, and 0.22 microM, respectively. Subsequent SAR studies led to the synthesis of new analogs with improved potency, solubility, and bioavailability relative to the initial lead. For example, the introduction of a [4-(diethylamino)butyl]amino side chain into the 2-position of 4b afforded compound 6c with enhanced potency and bioavailability. Compound 6c inhibited PDGF-stimulated vascular smooth muscle cell proliferation with an IC50 of 0.3 microM. Furthermore, replacement of the 6-(2,6-dichlorophenyl) moiety of 4b with a 6-(3',5'-dimethoxyphenyl) functionality produced a highly selective FGFr tyrosine kinase inhibitor 4e. Compound 4e inhibited the FGFr tyrosine kinase with an IC50 of 0.060 microM, whereas IC50s for the inhibition of the PDGFr, FGFr, EGFr, c-src, and InsR tyrosine kinases for this compound (4e) were all greater than 50 microM.

Synthesis and tyrosine kinase inhibitory activity of a series of 2-amino-8H-pyrido[2,3-d]pyrimidines: identification of potent, selective platelet-derived growth factor receptor tyrosine kinase inhibitors.

Screening of a compound library led to the identification of 2-amino-6-(2,6-dichlorophenyl)-8-methylpyrido[2,3-d]pyrimidine (1) as a inhibitor of the platelet-derived growth factor receptor (PDGFr), fibroblast growth factor receptor (FGFr), and c-src tyrosine kinases (TKs). Replacement of the primary amino group at C-2 of 1 with a 4-(N,N-diethylaminoethoxy)phenylamino group yielded 2a, which had greatly increased activity against all three TKs. In the present work, variation of the aromatic group at C-6 and of the alkyl group at N-8 of the pyrido[2,3-d]pyrimidine core provided several analogues that retained potency, including derivatives that were biased toward inhibition of the TK activity of PDGFr. Analogues of 2a with a 3-thiophene or an unsubstituted phenyl group at C-6 were the most potent inhibitors. Compound 54, which had IC50 values of 31, 88, and 31 nM against PDGFr, FGFr, and c-src TK activity, respectively, was active in a variety of PDGF-dependent cellular assays and blocked the in vivo growth of three PDGF-dependent tumor lines.

Soluble 2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas. Structure-activity relationships against selected tyrosine kinases and exploration of in vitro and in vivo anticancer activity.

In continuing our search for medicinal agents to treat proliferative diseases, we have discovered 2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas as a novel class of soluble, potent, broadly active tyrosine kinase (TK) inhibitors. An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. From the lead structure 1, several series of analogues were made that examined the C-6 aryl substituent, a variety of water solublizing substitutents at the C-2 position, and urea or other acyl functionality at the N-7 position. Compounds of this series were competitive with ATP and displayed submicromolar to low nanomolar potency against a panel of TKs, including receptor (platelet-derived growth factor, PDGFr; fibroblast growth factor, FGFr;) and nonreceptor (c-Src) classes. Several of the most potent compounds displayed submicromolar inhibition of PDGF-mediated receptor autophosphorylation in rat aortic vascular smooth muscle cells and low micromolar inhibition of cellular growth in five human tumor cell lines. One of the more thoroughly evaluated members, 32, with IC50 values of 0.21 microM (PDGFr), 0.049 microM (bFGFr), and 0.018 microM (c-Src), was evaluated in in vivo studies against a panel of five human tumor xenografts, with known and/or inferred dependence on the EGFr, PDGFr, and c-Src TKs. Compound 32 produced a tumor growth delay of 14 days against the Colo-205 colon xenograft model.

2-Substituted aminopyrido[2,3-d]pyrimidin-7(8H)-ones. structure-activity relationships against selected tyrosine kinases and in vitro and in vivo anticancer activity.

While engaged in therapeutic intervention against a number of proliferative diseases, we have discovered the 2-aminopyrido[2, 3-d]pyrimidin-7(8H)-ones as a novel class of potent, broadly active tyrosine kinase (TK) inhibitors. An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. From the lead structure 2, a series of analogues bearing variable substituents at the C-2 position and methyl or ethyl at N-8 was made. Compounds of this series were competitive with ATP and displayed submicromolar to low nanomolar potency against a panel of TKs, including receptor (platelet-derived growth factor, PDGFr; fibroblast growth factor, FGFr; epidermal growth factor, EGFr) and nonreceptor (c-Src) classes. One of the more thoroughly evaluated members was 63 with IC50 values of 0.079 microM (PDGFr), 0.043 microM (bFGFr), 0.044 microM (EGFr), and 0.009 microM (c-Src). In cellular studies, 63 inhibited PDGF-mediated receptor autophosphorylation in a number of cell lines at IC50 values of 0.026-0.002 microM and proliferation of two PDGF-dependent lines at 0.3 microM. It also caused inhibition of soft agar colony formation in three cell lines that overexpress the c-Src TK, with IC50 values of 0.33-1.8 microM. In in vivo studies against a panel of seven xenograft tumor models with known and/or inferred dependence on the EGFr, PDGFr, and c-Src TKs, compound 63 produced a tumor growth delay of 10.6 days against the relatively refractory SK-OV-3 ovarian xenograft and also displayed activity against the HT-29 tumor. In rat oral bioavailability studies, compound 63 plasma concentrations declined in a biexponential manner, and systemic plasma clearance was high relative to liver blood flow. Finally, in rat metabolism studies, HPLC chromatography identified two metabolites of 63, which were proved by mass spectrometry and synthesis to be the primary amine (58) and N-oxide (66). Because of the excellent potency of 63 against selected TKs, in vitro and in vivo studies are underway for this compound in additional tumor models dependent upon PDGFr, FGFr, and c-Src to assess its potential for advancement to clinical trials.

Two phases of contractile response in rat isolated vas deferens and their regulation by adenosine and alpha-receptors.

Contractions to transmural electrical stimulation and exogenous norepinephrine were recorded in isolated longitudinal segments of rat vas deferens. Electrical stimulation for 30 s produced a biphasic contraction in the vas deferens consisting of a rapid, transient response (Phase I), followed by a slowly developing, sustained contraction (Phase II). N6-Cyclohexyladenosine (CHA), a selective adenosine1 (A1)-receptor agonist, attenuated in a concentration-dependent manner the Phase I contractile response, while having little effect on the Phase II response. In contrast, 2-(phenylamino)adenosine (CV-1808), a selective adenosine2 (A2)-receptor agonist had little effect on either contractile phase. CHA did not inhibit the contraction to exogenous norepinephrine, suggesting that A1-receptors were located at a presynaptic site. The relatively selective alpha 2-receptor agonist clonidine produced the same pattern of contractile inhibition as CHA. The inhibitory effect of CHA on the Phase I contractile response in the vas deferens could be antagonized by the selective A1-receptor antagonist 8-cyclopentyltheophylline, while the selective alpha 2-receptor antagonist idazoxan preferentially antagonized the inhibitory effect of clonidine on the Phase I response. Both the Phase I and Phase II contractile responses were reduced by the selective alpha 1-adrenoceptor antagonist prazosin and the ATP analog alpha, beta-methylene adenosine triphosphate (alpha, beta-methylene ATP), suggesting that norepinephrine and ATP are coreleased as neurotransmitters for both responses. The results of the present study demonstrate that in the rat vas deferens the presynaptic inhibitory effects of adenosine is mediated by the A1-receptor subtype, and that both A1- and alpha 2-receptor agonists exert a selective inhibitory effect on the Phase I contractile response to electrical stimulation.

Inhibition of cell growth: effects of the tyrosine kinase inhibitor CGP 53716.

The growth factors, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) play major roles in enhanced smooth muscle cells growth in rodent blood vessels after vascular injury. Tyrosine kinase inhibition has been shown to be effective in blocking tyrosine phosphorylation at the PDGF and bFGF receptors in cultured fibroblast and vascular smooth muscle cells which in turn inhibits their proliferation. Our study evaluated the PDGF selective tyrosine kinase inhibitor, CGP 53716, on serum, PDGF-BB, bFGF or epidermal growth factor-induced growth responses in cultured rat aortic smooth muscle cells (RASMC) and Balb/3T3 fibroblasts (3T3). CGP 53716 inhibited serum-induced cell growth in RASMC, but not in 3T3 cells. CGP 53716 completely blocked PDGF-BB tyrosine receptor autophosphorylation in RASMC and 3T3 cells, PDGF-BB-induced phosphorylation of mitogen-activated protein kinase at 1 microM in RASMC and inhibited PDGF-BB-induced c-Fos protein expression at 1 microM in RASMC; consistent with inhibition of PDGF-BB-induced DNA synthesis. To examine the selectivity of CGP 53716, PDGF-BB, bFGF or EGF-induced DNA synthesis was measured using thymidine incorporation. CGP 53716 inhibited PDGF-BB-, bFGF- and EGF-induced DNA synthesis in a concentration-dependent manner in each cell line. CGP 53716 showed a 2- to 4-fold selectivity for PDGF-BB-stimulated DNA synthesis over bFGF or EGF in RASMC or 3T3 cells. To rule out that bFGF induced the release of endogenous PDGF, an antibody to PDGF-AB, which binds to all three isoforms of PDGF, was coincubated with bFGF and did not suppress the DNA synthesis induced by bFGF. Based on these results, CGP 53716 is not selective for the PDGF receptor as previously reported. However, EGF-stimulated receptor autophosphorylation of mitogen-activated protein kinase phosphorylation and c-Fos protein expression were not inhibited by CGP 53716 at 1 or 10 microM in RASMC. These findings suggest that CGP 53716 may inhibit multiple growth factor pathways as indicated by inhibition of DNA synthesis. However, these effects must be downstream from the signaling for c-Fos protein expression or use an alternate signaling route. These results further suggest that CGP 53716 may have a therapeutic potential for the treatment of vascular proliferative diseases which are stimulated by not only PDGF but other growth factors such as bFGF and EGF.

Contractile responses to ouabain and K+-free solution in aorta from hypertensive rats.

This study characterizes isometric force development in response to ouabain and K+-free solution in isolated aortic strips from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. SHR aortas were more sensitive to ouabain than those from WKY (threshold: SHR, 3.1 X 10(-5) M; WKY, 25.6 X 10(-5) M), and force development in response to 10(-3) M ouabain was greater in SHR (SHR, 586 +/- 51 mg; WKY, 245 +/- 24 mg). Monensin, a Na+ ionophore, potentiated contractile responses to ouabain, whereas amiloride, a Na+ channel blocker, and low Na+ solutions depressed contractile responses to ouabain. Contractile responses of SHR aortic strips to K+-free solution were faster than those of WKY aortic strips [time to half-maximal response (t1/2): SHR, 24 +/- 5 min; WKY, 47 +/- 4 min]. Maximal force development by aortic strips from SHR in response to K+-free solution was not different from that of WKY aortic strips (SHR, 808 +/- 34 mg; WKY, 750 +/- 37 mg). Monensin (10(-5) M) increased the rate of force development to K+-free solution to a greater extent in WKY aortic strips than in those from SHR (t1/2: SHR, 3 +/- 1 min; WKY, 4 +/- 2 min). Amiloride and low Na+ solution depressed contractile responses to K+-free solution in both SHR and WKY aortic strips. These observations demonstrate that SHR aortas are more responsive to ouabain and K+-free solution compared with WKY aortas. Contractile responses to ouabain and K+-free solution were sensitive to experimental interventions that alter transmembrane Na+ movements.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for NO involvement in regulating vascular reactivity in balloon-injured rat carotid artery.

The present study evaluated the influence of this newly formed intima on vascular reactivity in balloon-injured carotid arteries and the regulatory role of the vasodilator, nitric oxide (NO). Balloon injury was performed using a 2-F Fogarty catheter. After 2 and 4 wk, carotid artery segments were removed for both histomorphometric analysis and determination of in vitro contractile responses. Histomorphometric analysis showed a marked intimal thickening with an intima-to-media ratio of 126 +/- 19% (n = 5). The lack of factor VIII staining in injured carotid arteries revealed the absence of endothelium, since factor VIII-related antigen is a glycoprotein synthesized by endothelial cells. Functionally, maximal contractile responses to norepinephrine, angiotensin II (ANG II), endothelin-1, and serotonin were all attenuated in the injured vessels compared with the uninjured carotid arteries [0.38 +/- 0.11 vs. 0.73 +/- 0.10 g (n = 5), norepinephrine; 0.15 +/- 0.06 vs. 0.38 +/- 0.05 g (n = 4), ANG II; 0.60 +/- 0.14 vs. 1.05 +/- 0.12 g (n = 4), endothelin-1; 0.23 +/- 0.07 vs. 0.60 +/- 0.06 g (n = 12), serotonin]. Contractile responses induced by KCl were not affected by the balloon injury (0.62 +/- 0.10 vs. 0.64 +/- 0.09 g, n = 4). Interestingly, carbachol, a muscarinic agonist and vasodilator, caused concentration-dependent relaxations in 2- as well as 4-wk postinjured vessels despite the absence of endothelium. The NO synthase inhibitors, N omega-L-arginine methyl ester (L-NAME) and N omega-nitro-L-arginine (L-NNA), blocked the relaxation responses evoked by carbachol. Exogenously administered L-arginine reversed this blockade of the NOS inhibitors on the carbachol-induced relaxations. In addition, L-NAME partially reversed in a concentration-dependent manner the reduced maximal contractile force elicited by serotonin in the injured carotid artery.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of calcium, magnesium, lead, or cadmium on lipoprotein metabolism and atherosclerosis in the pigeon.

Epidemiological and clinical studies suggest that the incidence of atherosclerosis is higher in soft-water areas than in hard-water areas. In an attempt to discern the factor(s) in drinking water that may be associated with these observations, the current studies were performed to determine the effects of several elements associated with hard (i.e., calcium and magnesium) or soft (i.e., cadmium and lead) water in the induction and progression of atherosclerosis in the white carneau pigeon. The effect of these elements on lipoprotein metabolism was also assessed because it has been suggested that changes in the metabolism of lipoprotein may play a role in the etiology of atherosclerosis. Results show that the number and size of atherosclerotic plaques in the aorta were increased in pigeons given drinking water containing lead and/or cadmium. The effects of these elements were antagonized by the addition of calcium to drinking water containing lead and/or cadmium. Although lead and cadmium altered the profile of lipoproteins, this change did not appear to be related to an increase in the number and size of atherosclerotic plaques of the aorta. However, in pigeons treated with calcium alone the low-density lipoprotein (LDL) increased fourfold, and arteriosclerosis of the coronary arteries was observed. This result suggests that marked increases in the LDL protein may be related to arteriosclerosis of the coronary arteries. Based on these preliminary results, we suggest that lead, cadmium, and the LDL protein may be important factors in the induction and progression of atherosclerosis and arteriosclerosis in the pigeon.

Cutaneous and skeletal muscle vascular responses to hypothermia.

Vascular resistance and capacitance were studied in innervated or denervated canine forelimbs. Hypothermia (38-28 degrees C) was induced systemically, by external cooling of blood which returned to the right heart, or locally, by cooling blood perfusing the forelimb. Systemic cooling to 33 and then to 28 degrees C elicited significant decreases in limb weight with substantial increases in both skin and skeletal muscle vascular resistances. Acute denervation of the forelimbs attenuated both the fall in limb weight and increase in skin vascular resistance associated with cooling. These data support the contention that cutaneous vasoconstriction during systemic cooling is mediated primarily by sympathetic nerves, whereas skeletal muscle vasoconstriction is mediated primarily by circulating hormones. Local cooling elicited skin and skeletal muscle vascular dilation at 33 degrees C in both innervated and denervated forelimbs whereas either no change or a slight increase in skin and skeletal muscle vascular resistance resulted upon local cooling to 28 degrees C, perhaps due to the inhibition of Na+ - K+ - ATPase activity and/or a rise in blood viscosity. The locally induced vasodilation was found to override the powerful vasoconstrictor response caused by systemic cooling. The vasodilation is considered active rather than passive, because the increase in forelimb weight and decreases in forelimb vascular resistances occurred in the denervated as well as innervated limbs.

Effects of quinapril, a new angiotensin-converting enzyme inhibitor, on vasoconstrictor activity in the isolated, perfused mesenteric vasculature of hypertensive rats.

Previously, we had reported that 7-day administration of the angiotensin-converting enzyme inhibitor quinapril markedly reduced electrically evoked pressor responses in the isolated, perfused mesenteric vascular bed of the spontaneously hypertensive rat (SHR). In the present study, we investigated the possibility that quinapril alters postsynaptic vasoconstrictor activity to a variety of vasoconstrictive agents. Quinapril (10 mg/kg/day), administered orally to SHR for 7 days, significantly reduced the potency and the maximal vasopressor response to phenylephrine (2.5-fold and 40%, respectively) and the maximal response to serotonin (50%) compared with the responses from vehicle treated SHR. In contrast, quinapril had no significant effect on the pressor responses to KCl or phorbol ester. Furthermore, an equipotent antihypertensive dose of hydralazine (5 mg/kg/day, p.o., for 7 days) exerted no inhibitory effect on the pressor responses elicited by phenylephrine, KCl phorbol ester, but significantly reduced the maximal response to serotonin. In addition, vasopressor responses to phenylephrine were not affected by an acute (i.e., 75 min) infusion of quinaprilat, the active metabolite of quinapril. The results suggest that 7-day quinapril administration, and not acute treatment, reduces alpha 1 adrenoceptor and S2-serotonergic receptor-mediated vasoconstriction. However, quinapril did not reduce the vasoconstrictor responses induced by KCl or phorbol ester, indicating that those pressor responses that are due to depolarization or protein kinase C activation are, in part, independent of angiotensin-converting enzyme inhibition. This inhibition of vascular alpha 1 adrenoceptor and S2-serotonergic receptor pressor activity may underlie, in part, the long-term antihypertensive activity of quinapril in the SHR.

Functional studies but not receptor binding can distinguish surmountable from insurmountable AT1 antagonism.

Our study demonstrated that inhibition of angiotensin II- (Ang II) mediated contractions of rabbit aorta by structurally diverse nonpeptide AT1 antagonists could distinguish surmountable from insurmountable AT1 antagonism. CI-996, L158809, EXP 3174 and SKF 108834 produced concentration-related rightward shifts in Ang II response curves and reduced the maximal contraction to Ang II, characteristic of insurmountable antagonism. In contrast, DuP 753 and SKF 108566, produced parallel rightward shifts in Ang II contractile curves without affecting the maximal response which is consistent with the definition of surmountable or competitive antagonism. In addition, CI-996 demonstrated potent inhibition of Ang II-stimulated inositol phosphate accumulation in rat aortic smooth muscle cells, behaving as an insurmountable antagonist. However, DuP 753 was a surmountable antagonist of Ang II-stimulated inositol phosphate accumulation. Repeated washing of rabbit aorta preincubated with either CI-996 or EXP 3174 did not restore the blunted Ang II contractions. In contrast, both DuP 753 and the structurally dissimilar SKF 108566 at a concentration of 100 nM showed complete recovery of Ang II responses within 2 hr of repeated washing. Surprisingly, repeated rinsing of rabbit aorta for up to 5 hr after incubation with 1 microM DuP 753 failed to restore responses to Ang II. In addition, Scatchard analysis of [125I] Ang II saturation binding experiments revealed a competitive and rapidly reversible nature of AT1 receptor antagonism for all the AT1 antagonists examined. Taken together, the results of this study provide evidence for a competitive and rapidly reversible binding interaction of structurally diverse non-peptide antagonists at the AT1 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin and structurally related analogs distinguish between endothelin receptor subtypes.

The endothelin (ET) analog ET-1[1,3,11,15-Ala] was compared with ET-1, ET-2, ET-3 and sarafotoxins (SRTX) S6b and S6c for receptor binding and function. All the peptides exhibited high affinity binding and contracted rabbit pulmonary artery with near equal potency. In rat aorta both ET-3 and ET-1 [1,3,11,15-Ala] bound with much lower affinity than ET-1 while ET-3 displayed weak contractile potency and ET-1 [1,3,11,15-Ala] and SRTX-c were inactive. In rat left atria, ET-1 [1,3,11,15-Ala] and SRTX-c were weak inhibitors of binding and were also functionally inactive, whereas ET-1, ET-2, ET-3, and SRTX-b were equipotent in producing contractile responses. The data support the idea of there being a predominance of ETA receptors in rat aorta and ETB receptors in rabbit pulmonary artery. In rat left atria, the ET receptor could not be readily classified into ETA or ETB and suggests the existence of a new receptor subtype.

CI-906 and CI-907: new orally active nonsulfhydryl angiotensin-converting enzyme inhibitors.

CI-906, [3S-[2[R*(R*)]], 3R*]-2-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]-amino]-1-oxopropyl] 1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid, monohydrochloride, and CI-907, [2S-[1[R*(R*)]], 2 alpha, 3a beta, 7a 7a beta]-1-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]amino] 1-oxopropyl]octahydro-1H-indole-2-carboxylic acid, monohydrochloride, are two new nonsulfhydryl-type angiotensin-converting enzyme (ACE) inhibitors. Monoester (prodrug) and diacid forms produced concentration-related ACE inhibition in guinea pig serum (IC50 for CI-906 = 8.3 X 10(-9) M, diacid = 2.8 X 10(-9) M; CI-907 = 1.0 X 10(-7) M, diacid = 2.6 X 10(-9) M). In isolated rabbit aortic rings and in in vivo rat and dog autonomic studies, both compounds were highly specific in suppressing the contractile or pressor responses to angiotensin I. In two-kidney, one-clip Goldblatt (renin-dependent) hypertensive rats there was a good correlation between the inhibition of vascular converting enzyme and blood pressure lowering and a poor correlation between blood pressure lowering and plasma and brain converting enzyme inhibition. Cardiovascular, pulmonary, and central nervous system performance evaluations showed no side effects or gross toxicity. The preclinical profile shows CI-906 and CI-907 to be specific, potent, orally active ACE inhibitors. They are expected to have therapeutic utility in hypertension and in any other condition where converting enzyme inhibition would be useful.

Evidence for a functional tissue renin-angiotensin system in the rat mesenteric vasculature and its involvement in regulating blood pressure.

Electrical stimulation of the isolated rat mesenteric vascular bed resulted in a frequency-dependent pressor response, which could be potentiated by increasing concentrations of renin substrate (synthetic tetradecapeptide). This potentiating effect appeared to be mediated by tissue conversion of renin substrate to angiotensin II because the response 1) could be mimicked by angiotensin II, 2) was accompanied by an increase in angiotensin II production and 3) was blocked by the angiotensin converting enzyme (ACE) inhibitor quinaprilat and the angiotensin II receptor antagonist saralasin ([Sar1,Ile5,Ala8]angiotensin II). To assess the role of this tissue renin-angiotensin system in contributing to blood pressure regulation, spontaneously hypertensive rats were administered the prodrug ACE inhibitor quinapril at a dose of 10 mg/kg/day for 7 days. Such administration resulted in a reduction in systolic blood pressure of 48 +/- 3 mm Hg, a greater than 95% inhibition of serum ACE activity, and a significant attenuation of the potentiating effect of renin substrate on electrically evoked contractions of isolated mesenteric beds. Significant reductions in blood pressure and the potentiating effect of renin substrate on the isolated mesenteric vasculature were still observed 24 and 48 hr after the last dose of quinapril. In contrast, serum ACE activity returned to normal levels within 48 hr after the last dose of quinapril. These results suggest that the changes in tissue renin-angiotensin system, and not the circulating system, are closely related to the blood pressure lowering effect of the ACE inhibitor, quinapril.