In the present study, we demonstrate that the intravenous infusion of endothelin-1 (3 and 10 ng/kg/min) causes a decrease in the mean micturition volume of rats in addition to an increase in mean arterial pressure. These effects are blocked by both the ETA/ETB-non-selective and the ETA-selective endothelin antagonists SB 217242 and SB 247083 respectively (both 30 mg/kg). However, it was also observed that the ETB-selective agonist sarafotoxin 6c (3 and 10 ng/kg/min) had similar effects on both mean arterial pressure and micturition volume. Initial experiments indicated that spontaneously hypertensive rats have a much lower mean micturition volume than normal rats. Binding studies comparing the total number and ratio of ETA/ETB receptors in spontaneously hypertensive, Wister-Kyoto and Sprague-Dawley rats revealed no significant differences in receptor expression. However, the magnitude of the response to endothelin-1 was greater in spontaneously hypertensive versus normal rats.

Two endogenous receptors for the potent smooth muscle-stimulating peptide neuromedin U (NmU) have recently been identified and cloned. Pharmacological, binding, and expression studies were conducted in an attempt to determine the receptor(s) involved in the smooth muscle-stimulating effects of NmU. The NmU peptides caused a concentration-dependent contraction of canine isolated urinary bladder. NmU did not have this same effect in the urinary bladder from rat, guinea pig, rabbit, mouse, or ferret. Although NmU had no effect on canine uterus it did cause contraction of canine stomach, ileum, and colon. As well as causing contraction of canine bladder in vitro, NmU administered systemically resulted in a significant increase in urinary bladder pressure in vivo. High-affinity binding sites for NmU were identified in canine bladder. The four NmU peptides porcine NmU-8, rat NmU-23, human NmU-25, and porcine NmU-25 displaced (125)I-NmU-25 binding with similar K(i) values (0.08-0.24 nM). A different binding profile was revealed in human embryonic kidney-293 cells transiently expressed with the canine NmU-2 receptor where porcine NmU-8 (K(i) = 147.06 nM) was much less potent than the other NmU peptides. Using TaqMan, expression of NmU-1 was detected in human urinary bladder, small intestine, colon, and uterus. Expression of NmU-2 was much lower or absent in these human tissues and undetectable in canine bladder and stomach. The results of this study reveal significant species differences in the activity of NmU. The contractile activity in human and canine smooth muscle seems to be mediated by the recently cloned NmU-1 receptor.

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.

The adrenoceptor subtypes responsible for the pressor response to α1- and α2-adrenoceptor agonists have not yet been established, although gene knockout experiments in the mouse have provided evidence for a role of the α1B- and α2B-adrenoceptor. We have evaluated the blood pressure response to selective activation of postjunctional α1- and α2-adrenoceptors in the pithed mouse. The pressor response to phenylephrine was sensitive to blockade by terazosin, a selective α1-adrenoceptor antagonist, but insensitive to rauwolscine, an antagonist at α2-adrenoceptors. Phentolamine, a nonselective α-adrenoceptor antagonist, blocked the response to either phenylephrine or the selective α2-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 α1-adrenoceptor antagonist having no affinity for the α2B 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 α1-adrenoceptor subtypes suggests that this response is mediated by either α1B- or α1D-adrenoceptors. The α2B-adrenoceptor subtype is likely to take part in the response to B-HT 933. The ability of certain quinazoline α1-adrenoceptor antagonists to block the α2B adrenoceptor may contribute to their activity as antihypertensive agents.

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 μg/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

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.

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.

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)

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.

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.

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.