Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models.

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.

Activity of N-(phenethyl)phenylethanolamines at beta(1) and beta(2) adrenoceptors: structural modifications can result in selectivity for either subtype.

A series of phenylethanolamines bearing a 2-[1-phenylpropyl] substituent on the nitrogen atom was evaluated in vitro for activity at beta(1)- and beta(2)-adrenoceptors. As previously observed, the presence of 3,4-dihydroxy substitution on phenylethanolamine is required for potent activation of both subtypes, whereas the 3,5-dihydroxy analog showed selectivity for the beta(2)-subtype. Replacement by a carboxyl group of the 4-hydroxyl group on the aralkyl nitrogen substituent produced only a small reduction in beta(1) potency (5-fold), whereas beta(2) potency was reduced by more than 100-fold. Hence this structural class includes agonists having either a beta(1), nonselective beta(1)/beta(2) or beta(2) selectivity profile.

Characterization of postjunctional alpha-adrenoceptors in the pithed mouse.

The adrenoceptor subtypes responsible for the pressor response to alpha1- and alpha2-adrenoceptor agonists have not yet been established, although gene knockout experiments in the mouse have provided evidence for a role of the alpha1B- and alpha2B-adrenoceptor. We have evaluated the blood pressure response to selective activation of postjunctional alpha1- and alpha2-adrenoceptors in the pithed mouse. The pressor response to phenylephrine was sensitive to blockade by terazosin, a selective alpha1-adrenoceptor antagonist, but insensitive to rauwolscine, an antagonist at alpha2-adrenoceptors. Phentolamine, a nonselective alpha-adrenoceptor antagonist, blocked the response to either phenylephrine or the selective alpha2-adrenoceptor agonist B-HT 933, whereas rauwolscine blocked only B-HT 933. A dose of terazosin effective against phenylephrine had no effect on B-HT 933; however, the B-HT 933 response was antagonized when the terazosin dose was increased tenfold. A high dose of doxazosin, an alpha1-adrenoceptor antagonist having no affinity for the alpha2B adrenoceptor, blocked the response to phenylephrine but not B-HT 933. Comparison of the potencies of these antagonists against the pressor response to phenylephrine with their affinities for recombinant alpha1-adrenoceptor subtypes suggests that this response is mediated by either alpha1B- or alpha1D-adrenoceptors. The alpha2B-adrenoceptor subtype is likely to take part in the response to B-HT 933. The ability of certain quinazoline alpha1-adrenoceptor antagonists to block the alpha2B adrenoceptor may contribute to their activity as antihypertensive agents.

Effects of alpha1-adrenoceptor antagonists on agonist and tilt-induced changes in blood pressure: relationships to uroselectivity.

We evaluated the uroselectivity of a series of alpha1-adrenoceptor antagonists by comparing their potency against phenylephrine-induced increases in urethral perfusion pressure and diastolic blood pressure in the anesthetized rabbit and pithed rat. In the rabbit, Rec 15/2739 (N-[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]-3-methyl-4-oxo-2-phenyl -4H-1-benzopyran-8-carboxamide) as well as analogs with a chlorine substituent on the methoxyphenyl ring (Rec 15/2869) or this substituent combined with the replacement of the phenyl substituent on the pyran ring by cyclohexyl (Rec 15/3011) were 2-6-fold more potent against the urethral vs. vascular response to phenylephrine. Rec 15/2841 (N-[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]-3-methyl-4-oxo-2-cyc lohexy-4H-1-benzopyran-8-carboxamide) was only 1.5-fold more potent against the urethral response. SL 89.0591 and tamsulosin also showed selectivity for the urethral response (2-2.5-fold), while the quinazolines produced equipotent blockade of urethral and vascular responses (selectivity ratio = 0.9-1.1). The urethral selectivities of Rec 15/2739 and its derivatives were confirmed by evaluation of the response to tilt in sedated, hypovolemic rabbits. Phenylephrine challenge assays did not show any of the antagonists, with the exception of terazosin at 300 microg kg(-1), to be uroselective in the rat (selectivity ratios = 0.2-1.5); potentiation of tilt-induced hypotension in the anesthetized rat showed substantial differences from the rabbit, with Rec 15/2739, but not Rec 15/3011 and Rec 15/2841 showing orthostatic effects equivalent to that observed for prazosin. Hence, Rec 15/2739 was uroselective in the rabbit, but not in the rat, while two of its close structural analogs were highly uroselective in both species. An assay for orthostatic activity in the conscious rat yielded different results, showing prazosin and terazosin, but not Rec 15/2739, to cause a reversal of the pressor response to tilt. Hence, the apparent uroselectivity of an alpha1-adrenoceptor antagonist is both species- and assay-dependent.

Cecectomized rat. A model of experimental secretory diarrhea in conscious animals.

Evaluation of antisecretory antidiarrheal agents in animal models is limited primarily to extrapolations of efficacy from enteropooling studies in vivo, isolated intestinal loops in situ, and Ussing flux chamber preparations in vitro. While these standard techniques are useful, they do not mimic secretory diarrhea. Our studies indicate that in rats, the cecum may serve a "reservoir" function in response to secretagogue administration. Thus, diarrhea is not observed consistently and reliably in this species to allow valid evaluation of potential antidiarrheal agents. Therefore, we have developed a reproducible model of secretory diarrhea utilizing conscious cecectomized rats by surgical resection of the cecum, without compromising ileocecal patency, and by the use of potent intestinal secretagogues. Animals quickly recover and maintain normal growth and other physiologic parameters for as long as 60 days. After 48 hr on standard chow, secretory diarrhea can be induced by oral administration of standard intestinal secretagogues (dimethyl prostaglandin E2, cholera toxin, or carbachol). Dimethyl prostaglandin E2 (300 micrograms/kg, p.o.) induces diarrhea within 1 hr that continues for approximately 3.5 hr. Oral administration of known antidiarrheal agents chlorpromazine (10 mg/kg), clonidine (1 mg/kg), or morphine (10 mg/kg) all significantly reduce fecal output within 30-60 min following administration. These studies indicate that in the rat, the cecum may serve as a fluid reservoir during periods of small intestinal hypersecretion and that the cecectomized rat serves as a useful, accurate, and reliable tool for evaluating new compounds with proposed antidiarrheal activity.

Characterization of LTC4 effects on rabbit ileal mucosa in vitro.

The effects of peptidoleukotriene C4 (LTC4) on electrical properties and Na+ and Cl- fluxes in the presence or absence of the LTD4/LTE4 antagonist, 2(S)-hydroxy-3-(R)-carboxyethylthio)-3-[2-(8-phenyloctyl)phe nyl] propanoic acid (SK&F 104353) were investigated in stripped ileal mucosa from rabbits placed in Ussing chambers. Results from this study demonstrate that serosal addition of LTC4 produced a dose-dependent increase in short-circuit current (Isc) which is not affected by pretreatment of the tissue with SK&F 104353 (0.1 microM). The concentration of LTC4 which produced an increase in Isc of 4 mu Eq/h.cm2 was 181 nM in the absence of SK&F 104353 and this value did not differ significantly from the value of 212 nM in the presence of SK&F 104353 (0.1 microM). SK&F 104353 (0.1 microM) reduced the increase in Isc elicited by LTD4 and LTE4 by greater than 95%. Mucosal addition of LTC4 failed to alter Isc. The time course of the increase in Isc in response to LTC4 is qualitatively similar to that seen with both LTD4 and LTE4. Increases in Isc produced by LTC4 are not inhibited by removal of Ca2+ from the serosal bathing solution but are inhibited by pretreatment of the tissue with indomethacin (1 microM). The histamine receptor antagonist, mepyramine, reduced the change in Isc resulting from stimulation with LTC4 by 20% while the cholinergic antagonist, atropine, and the excitable tissue Na+ channel blocker, tetrodotoxin, were without effect. Measurement of unidirectional and net Na+ and Cl- fluxes revealed that LTC4 reduces Na+ and Cl- absorption and that subsequent addition of PGE1 produced no further decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

SK&F 104353: selective antagonism of peptidoleukotrine-induced changes in electrolyte transport by rat ileal mucosa in vitro.

Effects of the peptidoleukotriene receptor antagonist, SK&F 104353, were examined in vitro using stripped rat ileal mucosa in Ussing chambers. Changes in short-circuit current (Isc), transepithelial conductance (G1) and unidirectional and net Na+ and Cl fluxes measured in the absence or presence of SK&F 104353 (1 microM) revealed that serosal addition produced a decrease in net Na+ absorption and serosal-to-mucosal Cl- flux. Serosal addition of leukotriene (LT)D4 (10 muM) or LTE4 (10 microM) elicited transient increases in Isc and sustained decreases in G1 which were antagonized by serosal addition of SK&F 104353 in a concentration-dependent manner. Mucosal addition of SK&F 104353 (1 microM) also reduced the increase in Isc elicited by LTD4 (10 microM). LTD4 (10 microM) decreased unidirectional and net Na+ and Cl- fluxes along with G1 in the steady state. All of these changes were abolished by pretreatment with SK&F 104353 (1 microM). The intestinal secretagogues prostaglandin F2 alpha, histamine, serotonin, substance P and the thromboxane mimic, U46619, produced changes in Isc qualitatively similar to those of LTD4. However, the transient increase in Isc produced by these secretagogues was not antagonized by SK&F 104353 (1 microM). Additionally, lysbradykinin- and prostaglandin E1-induced increases in Isc were not antagonized by SK&F 104353 (1 microM). Stimulation with histamine (10 microM) or pretreatment with LTB4 (5 microM) did not alter the increase in Isc or decrease in Gt produced by the subsequent addition of LTD4 (10 microM). Pretreatment of the tissues with mepyramine (10 microM) also did not reduce the increase in Isc elicited by LTD4 (10 microM), although it inhibited completely the increase in Isc produced by histamine (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Antidiarrheal activity of alpha-2 adrenoceptor agonist SK&F 35886.

Alpha-2 adrenoceptor agonists exhibit antidiarrheal activity in animal models and in humans. However, hypotensive and sedative side effects seriously limit the use of these agents to treat diarrhea. SK&F 35886 (2,6-dimethyl phenylamino imidazoline) is an alpha-2 adrenoceptor agonist with little central nervous system activity. In Ussing chamber preparations of rabbit ileum, SK&F 35886 produces a concentration-dependent decrease in basal short-circuit current (Isc) (EC50 0.2 microM) that is dependent on the presence of mucosal HCO3. This concentration-response curve is shifted to the right of rauwolscine, increasing the EC50 to 30 microM. Prazosin had no effect on this response. Flux studies indicate that SK&F 35886 increases net Cl absorption and enhances HCO3 absorption without altering net Na flux. After PGE2 stimulation of Isc, SK&F 35886, applied either serosally or mucosally, immediately returns the Isc to base line. This effect is due to a reversal of the PGE2-induced inhibition of Na and Cl absorption. In vivo SK&F 35886 dose-dependently inhibits PGE2-induced enteropooling when given orally (ED50 approximately 31 micrograms/kg). This effect is attenuated significantly by rauwolscine (1.0 micrograms/kg s.c.). In cecectomized rats, SK&F 35886 abolishes PGE2-induced diarrhea within 1 hr after oral administration of the drug. SK&F 35886 (500 micrograms/kg p.o.) did not alter hexobarbital sleep time or elicit piloerection or lethargy, whereas clonidine (37.3 micrograms/kg p.o.) significantly enhanced hexobarbital sleep time. These results illustrate the ability of a peripheral acting alpha-2 agonist to promote absorption and inhibit secretion and diarrhea in the mammalian intestine.

Selective alpha-2 adrenoceptor agonists alter fluid and electrolyte transport in mammalian small intestine.

The effects of the selective alpha-2 adrenoceptor agonists B-HT 920 and B-HT 933 on fluid and electrolyte transport in mammalian small intestine were assessed in vitro and in vivo. In Ussing flux chamber preparations of rabbit ileum, B-HT 920 reduces basal short-circuit current (Isc) in a concentration-dependent manner. This in vitro effect is inhibited by rauwolscine (KB = 9.7 nM) but not by prazosin. Isotope flux and ion replacement studies suggest that this decrease in Isc is due primarily to stimulation of a HCO3-dependent transport process. B-HT 920 promptly attenuates the 16,16-dimethyl prostaglandin E2 (dmPGE2)-stimulated increase in Isc and completely reverses dmPGE2-stimulated Cl secretion to absorption. Oral administration of B-HT 933 dose-dependently inhibits dmPGE2-induced enteropooling in conscious rats. This effect of B-HT 933 is likewise blocked significantly by rauwolscine but not by prazosin. Similar effects of B-HT 933 are observed on enteropooling in the pithed rat as are the effects of B-HT 920 in the conscious rat. These results indicate that selective alpha-2 adrenoceptor agonists from the azepine class of compounds have significant proabsorptive and antisecretory activities in the rabbit small intestine in vitro and in the rat intestine in vivo. This in vivo effect does not appear to be central nervous system mediated. These studies suggest that these alpha-2 adrenoceptor agonists may be useful in converting the hypersecreting mammalian small bowel to its normal absorptive state.

Effects of putative thromboxane receptor agonists and antagonists on rat small intestinal ion transport.

Effects of a thromboxane mimic, U46619, on electrolyte transport were examined in vitro using stripped segments of rat ileal mucosa mounted in Ussing chambers. Addition of U46619 to the serosal bathing solution elicited a transient increase in short-circuit current (Isc) and decrease in transepithelial conductance (Gt). The increase in Isc was accompanied by a transient increase in Cl- secretion and decrease in Na+ absorption. In the steady-state, Isc was not increased whereas Gt remained decreased and Na+ and Cl- absorption were inhibited. Removal of Cl- or pretreatment with serosal and mucosal indomethacin (1 microM) or the thromboxane receptor antagonist, SK&F 88046, added to the serosal bathing solution, inhibited the increase in Isc stimulated by U46619 (apparent KB approximately 8 nM). The effects of U46619 on both Isc and Gt are qualitatively similar to those resulting from stimulation with leukotriene D4. However, the changes in Isc with leukotriene D4 (10 microM) are antagonized by SK&F 88046 only at high concentrations (1-10 microM). In addition, the secretagogues prostaglandin F2 alpha, lys-bradykinin, serotonin and histamine, produce qualitatively similar changes in Isc to those seen with U46619 without altering Gt. With the exception of prostaglandin F2 alpha, the effects of these secretagogues are not inhibited by SK&F 88046 (10 microM). These results indicate that U46619 acts at a thromboxane receptor to stimulate intestinal Cl- secretion and inhibit Na+ and Cl- absorption. These changes are inhibited selectively by the thromboxane receptor antagonist, SK&F 88046.

Effect of sulfidopeptide leukotrienes D4 and E4 on ileal ion transport in vitro in the rat and rabbit.

Effects of leukotrienes D4 and E4 (LTD4 and LTE4) on electrolyte transport were examined, employing stripped segments of rat and rabbit ileum mounted in Ussing chambers. Addition of LTD4 or LTE4 to the serosal but not the mucosal bathing solution elicited a transient increase in short-circuit current (Isc) with maximal responses seen at 10(-5) M and 10(-8) M in rat and rabbit respectively and a sustained decrease in transepithelial conductance (Gt) in the rat only. In the rat, Cl replacement, reduction of bathing solution [Ca2+] to 1 microM or pretreatment with 1 microM indomethacin or meclofenamic acid inhibited the LTD4- or LTE4-induced Isc changes with no effect on the decrease in Gt. LTD4 (10 microM) transiently increased net Cl secretion and produced a sustained decrease in both unidirectional and net Na transport and mucosal-to-serosal Cl flux in rat ileum. The decrease in unidirectional Na fluxes is accounted for predominantly by a change in the potential independent flux of Na. These results suggest that the increase in Isc in both rat and rabbit is mediated by arachidonic acid metabolites, whereas the decrease in Gt and net Na absorption in rat ileum is mediated by a cyclooxygenase-independent pathway.

Partial vs total esophagectomy for advanced carcinoma of the hypopharynx.

Cancer of the hypopharynx is an aggressive disease with a poor prognosis irrespective of the therapeutic regimen instituted. Controversy centers around the extent of surgery required to adequately ablate the advanced cancers, particularly related to the role of esophagectomy. A literature review and analysis of 43 cases of advanced hypopharyngeal cancer treated with total laryngopharyngectomy and partial esophagectomy support the argument that in carefully selected situations, a partial esophagectomy is oncologically an adequate operation.

Pharyngolaryngectomy reconstruction by revascularized free jejunal graft.

The results of reconstruction after total pharyngolaryngectomy using a free revascularized jejunal graft in 72 patients are presented. There was a low hospital mortality (2.8%), a short average time until swallowing (13 days) and a short average postoperative hospital stay (20 days). Twelve patients had resections more extensive than the standard total pharyngolaryngectomy. Sixteen patients (22.2%) suffered some graft complication, but only five (two early graft losses, one late graft loss, one fistula and one stricture) required further reconstructive surgery. Abdominal complications were minimal. There were no complications attributable to post-operative radiotherapy. Swallowing of solids and liquids is good and is maintained long-term. These results are compared with those reported for other methods of reconstruction. This comparison supports a contention that jejunal autograft is the reconstruction of choice after pharyngolaryngectomy.

Function of a free jejunal "conduit" graft in the cervical esophagus.

The function of an autotransplanted jejunal graft used to reconstruct the pharyngoesophagus was evaluated in 12 patients, 2-40 mo after surgery. On clinical assessment most patients swallowed liquids and solids with minimal difficulty, although several described a need for liquids to "flush" solids to the stomach. Radiologic studies demonstrated a delay in orogastric transit in some patients above the lower anastomosis. The mechanism was apparent on motility studies: swallows generally failed to induce contractions in the graft, although the esophagus below exhibited stripping peristaltic waves. Regular contractile activity, characteristic of phase III of the intestinal migrating motor complex, was identified in 11 grafts. There was no temporal association between migrating motor complexes in the graft and those recorded at the jejunal donor site. Instillation of nutrients into the gastric antrum induced a typical "fed" pattern of contractions in the intact jejunum but not in the extrinsically denervated graft. In conclusion, the graft provides a useful, though generally passive conduit. The graft maintains its intrinsic motor repertoire, which is asynchronous with that of the donor site. The findings also support the hypothesis that extrinsic nerves are required to induce a "fed" pattern of intestinal motility.

Complications associated with free jejunal graft reconstruction of the pharyngoesophagus--a multiinstitutional experience with 52 cases.

Fifty-two patients who underwent reconstruction of the pharynx and esophagus using the free jejunal graft were retrospectively reviewed. The complications were categorized into those associated with the resection, those associated with the harvesting of the graft, and those related to the reconstruction per se. In this series, the graft failure rate was 7.6% with an overall success rate of 90.3%. Graft necrosis was found to be the most serious complication occurring in four patients. Methods of detection of graft necrosis and management of these complications are discussed.

Characterization of alpha adrenoceptors on vascular smooth muscle: electrophysiological differentiation in canine saphenous vein.

The effects of selective alpha adrenoceptor agonists on resting transmembrane potential (Em) and contractile responses of vascular smooth muscle of the canine saphenous vein (CSV) were investigated using microelectrode and isometric tension recording techniques. The Em of the smooth muscle cells of the CSV was -59.5 mV +/- 0.3 (mean +/- S.E., n = 363). The cells were electrically quiescent and did not show spontaneous electrical activity. Norepinephrine (a mixed alpha-1/alpha-2 adrenoceptor agonist), applied in concentrations of 1 X 10(-9) to 1 X 10(-7) M did not depolarize the CSV cell membrane. However, 50% of the maximum contractile response to norepinephrine occurred over this concentration range. At concentrations greater than 1 X 10(-7) M, a dose-dependent contraction and depolarization was observed with norepinephrine. The contractile and depolarization effects of norepinephrine were antagonized by the selective alpha-1 antagonist, prazosin. The selective alpha-1 agonists methoxamine and SK&F l-89748 (l-[1,2,3,4-tetrahydro-8-methoxy-5-(methylthio)-2-naphthalenamine] ) caused a dose-dependent depolarization and contraction of the CSV. In contrast, the alpha-2 adrenoceptor agonists, BHT-920 and M-7, could be distinguished from the alpha-1 adrenoceptor agonists by their lack of effect on Em. M-7, at concentrations up to 1 X 10(-6) M, failed to produce a depolarization; however, at 10(-6) M, M-7 produced 80% of its maximum contractile response.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chemical sympathectomy by 6-hydroxydopamine on alpha-adrenoceptor-mediated pressor responses in pithed rat.

The effects of 6-hydroxydopamine (6-OHDA) treatment of neonatal and adult rats on vasopressor responses to postsynaptic alpha-adrenoceptor activation were studied in adult pithed rats. The pressor response to electrical stimulation of the thoracosympathetic outflow (T7-T9) was markedly reduced after neonatal or adult 6-OHDA treatment, indicating a functional destruction of the sympathetic input to the vasculature. Pressor response curves to injected noradrenaline (a nonselective alpha-agonist) and phenylephrine (a selective alpha 1-agonist) were shifted to the left in all 6-OHDA-treated animals. However, the pressor response to the selective alpha 2-agonist M-7 (5,6-hydroxy-2-dimethylamino tetralin) was unaltered in all the 6-OHDA-treated animals. It is concluded that functional destruction of peripheral sympathetic nerve endings in vascular smooth muscle by treatment of neonatal or adult rats with 6-OHDA produces an increased pressor response to alpha 1-adrenoceptor agonists, but not to alpha 2-adrenoceptor agonists in the pithed rat.

Correlation of [14C]muscimol concentration in rat brain with anticonvulsant activity.

Muscimol, an in vivo and in vitro GABA agonist, has anticonvulsant activity against bicuculline-induced seizures when given systemically to rats. To determine whether parent compound or a metabolite possessed the anticonvulsant activity, experiments were performed with [14C]muscimol. Anticonvulsant activity was determined by the percent of animals protected against tonic forelimb extension induced by bicuculline. Brain and urine were analyzed for unchanged [14C]muscimol by thin-layer chromatography. The time course of anticonvulsant activity and [14C]muscimol concentration in brain after intravenous injection were similar. Peak brain concentration of [14C]muscimol and maximal protection against bicuculline-induced seizures occurred simultaneously. These data suggest that intravenously administered [14C]muscimol rapidly penetrates brain tissue and parent compound is responsible for antagonism of bicuculline-induced convulsions.