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Background: We investigated the effects of amitriptyline in the urinary tract smooth muscle and urolithiasis., Methods: Cats presenting with obstructive acute renal failure (ARF) received amitriptyline, and renal function and survival rates were analyzed. Isometric contractions and membrane potentials of rat, pig, or human isolated urinary tract smooth muscle were recorded in the presence or absence of amitriptyline., Results: Twenty cats with obstructive ARF caused by urethral plugs received amitriptyline. In all cases, plugs were completely eliminated, and renal function returned to normal, with a 100% survival rate in the follow-up. Amitriptyline produced potent relaxations in rat urethral strips, accompanied by significant reductions in urethral ring membrane potential. This effect was prevented by pretreatment of urethral rings with 4-aminopyridine (4-AP), a voltage-dependent potassium channel blocker. Amitriptyline abolished in a reversible manner acetylcholine-, bradykinin-, and KCl-induced contractions in rat isolated bladder, and this effect was also prevented by 4-AP. Of interest, spontaneous and KCl-induced contractions of pig and human isolated ureter were also blocked by amitriptyline., Conclusion: Our results indicate that amitriptyline is an effective and potent relaxant of urinary tract smooth muscle and this effect is mediated by opening of voltage dependent-potassium channels. We suggest that amitriptyline administration may help to promote elimination of urinary calculi.

Angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label were synthesized by solid phase methodology. Ammonium hydroxide (pH 10, 50 degrees C, l h) was the best means for reverting nitroxide protonation occurring during peptide cleavage. EPR spectra yielded rotational correlation times for internally labeled analogs that were nearly twice as large as those of N-terminally labeled analogs. Except for TOAC(1)-AngII and TOAC(0)-BK, which showed high intrinsic activities, other derivatives were inactive in smooth muscle preparations. These active paramagnetic analogs may be useful for conformational studies in solution and in the presence of model and biological membranes.

Ca2+ pathways activated by angiotensin II and carbachol were evaluated in the circular muscle of the guinea-pig ileum by recording mechanical and electrical activities. Transient contractions induced by angiotensin II were greatly reduced by Ca2+ removal from the medium whereas carbachol-induced responses were not significantly altered. Nifedipine had no effect on the responses to both agonists. A high concentration of tetrodotoxin (0.1 microM) inhibited angiotensin II-induced contractile responses without affecting the depolarization, whereas 1 mM Ni2+ inhibited the mechanical and electrical effects. Neither tetrodotoxin nor Ni2+ affected carbachol-induced effects. These results indicate that angiotensin II-induced phasic contractions depend on extracellular Ca2+ but not on voltage-dependent L-type Ca2+ channels. It is suggested that angiotensin II activates Ni2+-sensitive Na+ and non-specific cationic channels, whereas the responses to carbachol are dependent on receptor-activated Ca2+ release. Furthermore the different response of the longitudinal and circular muscles to the inhibitory effects of tetrodotoxin and Ni2+ on the angiotensin II- and carbachol-induced contractions indicates that these agonists exert their own myogenic effects on each layer and are able to trigger different Ca2+ mobilization pathways.

The increase in sensitivity of guinea pig preparations to bradykinin (BK) due to stretching occurring with time after mounting was studied by determining the time course of changes in the cell membrane potential, measured with intracellular microelectrodes. A sustained hyperpolarizing effect of BK, which was observed in recently mounted preparations, became transient after 120 min, when it was followed by depolarization, which was much more evident after 4 h of stretching. As a consequence, a parallel increase in the contractile response to BK was also observed. The hyperpolarizing effect was due to the opening of Ca(2+)-dependent K+ channels sensitive to apamin, since BK dose-response curves done within 1 h of mounting were shifted to the left, becoming similar to dose-response curves obtained 4 h after mounting of the guinea pig ileum preparation. These results were specific for BK, since the potentiating effect of apamin was not observed for acetylcholine. Our results show that the activation of B2 receptors by BK in the isolated guinea pig ileum induce a dual effect--hyperpolarization and depolarization--and that the increase in the contractile response consequent to stretching is probably due to the inactivation of apamin-sensitive Ca(2+)-dependent K+ channels.

Sodium taurocholate (NaTC), at concentrations below the critical micellar concentration, caused a transient relaxation of isolated guinea-pig ileum smooth muscle strips. The relaxation was not inhibited by previous incubation with either 10 microM ouabain, 0.4 mM d-tubocurarine or 0.5 microM apamin, ruling out the participation of hyperpolarization of the plasma membrane induced by either stimulation of Na+/K+ ATPase or by opening of Ca(++)-dependent K+ channels. In guinea-pig ileum smooth muscle cultured cells, addition of NaTC (1 mM) stimulated Na+ uptake and Ca++ efflux. The relaxation induced by NaTC was inhibited by 3',4'-dichlorobenzamil, a blocker of the Na+/Ca++ exchanger. Preincubation with NaTC, or its addition during the early stage of the tonic response of the ileum to acetylcholine, enhanced that response, whereas a relaxation was observed when NaTC was added at the late stage of the acetylcholine response. In cultured cells, NaTC potentiated the stimulation of Ca2+ influx by acetylcholine. Our results suggest that NaTC acts on the smooth muscle cell membrane causing a stimulation of the Na+/Ca++ exchange mechanism.

1. The dual action of bradykinin (relaxation and contraction) on the rat duodenum was investigated by studying its effect on adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in cultured duodenal smooth muscle cells, and the effects of apamin on the isolated muscle responses to agonists and antagonists of BK1 and BK2 receptors. 2. No change was observed in the cyclic AMP content of cultured cells incubated with up to 100 nM bradykinin. 3. Apamin (100-500 nM) inhibited the relaxant component and enhanced the contractile component of the responses to bradykinin and to the BK2-specific analogue [Thi5,8,D-Phe7]-bradykinin. 4. Apamin (100-500 nM) did not affect the contractile response of stretched duodenum preparation to the BK1-specific agonist des-Arg9-bradykinin. 5. The BK2 antagonist, [D-Arg0Hyp3Thi5,8,D-Phe7]-bradykinin, at a concentration which completely inhibited the relaxant response to bradykinin and to [Thi5,8,D-Phe7]-bradykinin, also prevented the contraction in response to either agonist in the presence of apamin. 6. Our results demonstrate two populations of bradykinin receptors in rat duodenum: a BK2 subtype responsible for the biphasic response of the non-stretched duodenum, and a BK1 subtype responsible for the contractile effect on the stretched tissue.

Bradykinin (BK) did not increase cyclic AMP production in cultured rat duodenum smooth muscle cells. Its relaxant effect on the tissue was inhibited by apamin and potentiated by phorbol dibutyrate (PDBU). PDBU also caused a relaxation which was inhibited by apamin. BK's relaxant effect, and its potentiation by PDBU, are due to activation of Ca(2+)-dependent K+ channels.

The effects of angiotensin II (ANG) on Na+ and Ca++ fluxes in cultured intestinal smooth muscle cells from the guinea pig ileum were studied and correlated with the contraction and desensitization observed in whole muscles. The effects of ANG were compared with those of acetylcholine (ACh), an agonist that acts at muscarinic receptors in the intestinal smooth muscle and which does not induce desensitization. Both ANG and ACh stimulated 24Na+ influx upon addition to the cells, and this stimulation persisted for at least 30 min. Both agonists also stimulated 45Ca++ uptake but ANG's effect was transient, whereas that of ACh was persistent. Short-term (30 min) treatment with PMA (phorbol-12-myristate-13-acetate) caused a fade of the tonic response of the whole muscle to ANG, and also blocked this hormone's stimulating effect on 45Ca++, but not on 24Na+ influx. Long-term (7 hr) treatment with PMA, which suppresses protein kinase C activity, restored ANG's ability to stimulate 45Ca++ influx. The stimulating effects of ACh on 24Na+ and 45Ca++ influxes were not affected by short- or long-term treatment of the cells with PMA. Our results suggest that ANG desensitization involves protein kinase C inhibition of a step in the stimulus-response chain that is subsequent to phospholipase C-activation.

The isometric responses of the longitudinal smooth muscle of the guinea-pig ileum to acetylcholine and to histamine consist of a phasic contraction followed by a fade and a tonic contraction. At high agonist concentrations the fade is more pronounced and a period of lower tonus is observed in the early stage of the tonic component of the response. Experiments done in Ca2+-free medium indicate that the events responsible for the shape of the response take place in the absence of Ca2+ and of contraction. The phasic contraction was inhibited by hyperpolarization in low-Na+ medium. Small decreases in the external Na+ concentration caused a diminution of the fade. Reduction of the Na+ concentration during the early stages of the tonic response caused contraction whereas relaxation was observed at the late stages. It is proposed that the fade is dependent on a Na+-sensitive mechanism that is predominant at early but not at late stages of the responses to agonists.

The belief that the smooth muscle contracting activity of angiotensin II (angiotensin) in the guinea pig ileum is partly mediated by release of acetylcholine was reexamined, with the following results. 1. Atropine did not reduce the maximum contraction produced by angiotensin, although it caused a shift to the right of the log dose-response curve (dose ratio = 2.2). A similar shift was observed with histamine, bradykinin and BaCl2. 2. A moderate potentiation of angiotensin by eserine was also observed, which was similarly found for the other agonists. 3. A previous report that atropine blocks the fast (phasic) component of the isometric response of the ileum to angiotensin was not confirmed. The disappearance of the phasic component was found to be due to a tachyphylactic change in the response. 4. Depolarization by high doses of nicotine, and inhibition of acetylcholine synthesis by hemicholinium, did not affect the response to angiotensin. 5. Ilei in which the intramural ganglia had been destroyed by incubation at 4 degrees 48-56 h responed maximally to angiotensin, without loss of the phasic component of the response. It is concluded that the available evidence does not support the participation of a cholinergic mechanism in the effect of angiotensin upon the guinea pig ileum.

The ability of angiotensin II (AII) and of seven N-terminally modified peptide analogs to induce tachyphylaxis in isolated preparations of the rat uterus and the guinea-pig ileum was found to correlate well with the degree of amino group protonation. No correlation was found between tachyphylaxis and the affinity for the rat uterus receptors, as measured by the reciprocals of ED50 values. Dissociation between the tachyphylactic property and receptor affinity in the rat uterus was also evident by lowering the calcium concentration in the medium, which resulted in increased tachyphylaxis without significant changes in ED50 values. In the guinea-pig ileum, tachyphylaxis correlated with the reciprocal of ED50, but there was a better correlation with amino group protonation. [6-Pyrazolylalanine] AII was approximately as tachyphylactic as AII in both smooth muscles studied, indicating that protonation of the histidine side-chain may not be important for the manifestation of tachyphylaxis. The recovery of the guinea-pig ileum for AII tachyphylaxis was not affected by repeated treatments with BaCl2, but it was partially impaired by [Suc1]AII, des-Asp-AII, bradykinin and histamine.

The role of Ca++ in the stimulus-contraction coupling of the response of the isolated rabbit aorta to angiotensin II was investigated. Angiotensin was found to have lower intrinsic activity than epinephrine and to be more sensitive to acute exposure of the organ to Ca++-free medium. Two minutes after removal of Ca++, the maximal responses to angiotensin and epinephrine were reduced by 40% +/- 8% and 7% +/- 5%, respectively. Further loss of response for the two agonists followed parallel time courses. In another series of experiments, angiotensin tachyphylaxis was obtained in the rabbit aorta by administration of either [1-sarcosine]angiotensin or betainyl-angiotensin. The intrinsic activity of [1-sarcosine]angiotensin was lower than that of angiotensin and was not affected by removal of Ca++. It is concluded that the low intrinsic activity and the tachyphylaxis may be dependent on a strong binding of the molecule's positively charged N-terminus to sites responsible for release of Ca++ into the cell.

1. Bradykinin produces a biphasic response in the rat duodenum that consists of a relaxation (pD2 = 8.44) followed by a contraction (pD2 = 6.91). 2. The B1 agonist des-Arg9-bradykinin produced a contraction (pD2 = 7.16) but no relaxation. Des-Arg9-[Leu8]-bradykinin, which is a B1 antagonist in other systems produced contraction (pD2 = 7.65) in the rat duodenum. 3. Four bradykinin analogues that are preferential B2 agonists in other tissues had a biphasic effect with pD2 values in the range 7.22-8.68 for relaxation and 6.26-6.91 for contraction. 4. [Thi5,8,D-Phe7]-bradykinin, which is a B2 antagonist in most other systems produced relaxation in the rat duodenum, with a pD2 of 7.49. 5. It is concluded that the contractile component of the response to bradykinin in rat duodenum may be mediated by a subtype of the B1 receptor and the relaxant component by a receptor of the B2 subtype.

The biphasic (relaxation-contraction) response of the isolated duodenum was used to study the reactivity of non-vascular smooth muscles in genetic (SHR) and renal hypertensive rats compared to their respective controls (WKY and Wistar). For the contractile component of the response to bradykinin, the duodenum from WKY rats was more sensitive, whereas the duodenum from SHR was both more sensitive and hyperreactive, compared to that from Wistar rats. The relaxant component of the response to bradykinin was present in the duodenum of both WKY rats and SHR, but was concentration-dependent only in the WKY group. The relaxant response to K+ was very small in SHR, and was not concentration-dependent. The concentration-response curves for relaxant responses to adrenaline and for contractile responses to acetylcholine did not differ in the SHR and WKY groups. Ca2+/Mg2+-ATPase activity was found to be markedly reduced in the SHR group. No qualitative or quantitative differences were observed between the responses of the duodenum of renal hypertensive rats and those of their normotensive controls. It is proposed that the altered reactivity of the SHR duodenum is due to changes in ion handling by the smooth muscle cell membrane.

The responses of rabbit mesenteric or coeliac artery rings to angiotensin II or adrenaline (but not to K+) were enhanced by endothelium destruction (by rubbing). Potentiation by indomethacin of the response to the agonists was observed in rubbed rings but not in intact ones. Both angiotensin II and adrenaline (in the presence of propranolol and prazosin) induced endothelium-dependent relaxation of the arteries. Rubbed rings, but not intact ones, contracted when Ca2+ was added to a previously Ca2+-free medium containing angiotensin II or adrenaline. The vasoconstrictor response appears to be modulated by the regulation of receptor-operated Ca2+ channels through EDRF released by the endothelium and by some cyclo-oxygenase product at the level of the smooth muscle cell membrane.

Indomethacin had an equal inhibitory effect on the response of the guinea-pig isolated ileum to angiotensin II (angiotensin), bradykinin, histamine and acetylcholine. This effect did not seem to result from inhibition of prostaglandin synthesis, as it did not depend on the time of treatment with indomethacin. Prostaglandin E2 (prostaglandin) potentiated the responses of the guinea-pig ileum to angiotensin, bradykinin, histamine and acetylcholine without significant differences in the effects observed. In the rabbit isolated mesenteric and coeliac arteries, indomethacin had an equal potentiating effect on the responses to angiotensin and to adrenaline. In these organs pre-incubation with indomethacin was necessary for the effect to be observed, and this effect lasted for 2 h or more after that drug was removed from the medium. No cross-tachyphylaxis between angiotensin and adrenaline was observed in the rabbit mesenteric and coeliac arteries. It is concluded that the effects of indomethacin and prostaglandin on the response of the guinea-pig ileum to the four agonists result from an action on the smooth muscle contractile mechanism per se rather than from an inhibitory action on the release of endogenous prostaglandin produced by the four agonists. The results with the rabbit isolated arteries indicate that tachyphylaxis to angiotensin in these organs is not caused by prostaglandin release.

We have examined the responsiveness of the guinea-pig ileum and taenia coli to angiotensin (ANG) analogues that were previously shown to be either able ([1-sarcosine]-ANG, Sar1-ANG) or unable ([2-lysine]-ANG, Lys2-ANG) to induce tachyphylaxis in the ileum. The taenia coli, in which tachyphylaxis had not been previously shown to occur, was strongly tachyphylactic to Sar1-ANG, but not to Lys2-ANG. The contractile responses of the ileum, as well as the contractile and electrical (sucrose-gap) events in the taenia coli, in response to the two ANG analogues, were used to investigate the tachyphylactic phenomenon and the role of Na+ in the manifestation. Relaxation of the ileum and repolarizations of the taenia coli were faster after treatment with Lys2-ANG than after Sar1-ANG. In the tachyphylactic state, relaxation and repolarization after Sar1-ANG became as fast as after Lys2-ANG. In "low-Na+" (80 mmol/l) medium, as well as in ouabain-treated preparations, the responses of the ileum to ANG analogues were similar to those of tissues in the tachyphylactic state. Addition of Ca2+ to taenia coli preparations previously treated with the two ANG analogues in Ca2+-free medium, caused contractile and electrical responses only in the case of Sar1-ANG. It is proposed that ANG tachyphylaxis is due to changes at the level of the receptor causing increased Na+ permeability which leads to a decreased Na+ gradient across the cell membrane.

The homologous desensitization induced by angiotensin II analogues in the guinea-pig isolated ileum was studied. Desensitization assessed by the loss of response on repeated treatment showed [Sar1]angiotensin II to be a strong desensitizer whereas no desensitization to [Lys2]angiotensin II was detected. However, prolonged treatment with either analogue desensitized the tissue, indicating that [Lys2]angiotensin II-induced desensitization was reversed faster. A correlation was found between the degree of desensitization caused by repeated treatment and the time for half-relaxation after washout of the first treatment, but the relaxation after washout became faster in the desensitized state. In experiments designed to study competition between the agonistic and desensitizing properties of angiotensin II analogues, high concentrations of [Lys2]angiotensin II blocked the agonistic but not the desensitizing effect of lower concentrations of [Sar1]angiotensin II. It is concluded that desensitization is due to the interaction of angiotensin II with a regulatory site on the receptor.

Na/K-ATPase from the duodenum of spontaneously hypertensive rats (SHR) is inhibited compared to that of the Wistar-Kyoto (WKY) controls. The present study investigates depolarization of smooth muscle cell membranes through direct determination of the membrane potential in the longitudinal duodenal smooth muscle isolated from the two rat strains. Membrane potentials were not different in the two groups: -64.0 +/- 0.5 mV (N = 86) (means +/- SEM) in SHR and -62.0 +/- 3.0 mV (N = 95) in the WKY group. However, when the electrogenic contribution of Na/K-ATPase was abolished by removing potassium from the extracellular medium, the membrane potentials differed significantly: -35.0 +/- 1.0 mV (N = 45) and -28.0 +/- 1.0 mV (N = 48) for SHR and WKY, respectively. The lower depolarization observed in the duodena isolated from SHR is a further indication that the sodium pump is inhibited in these animals.