Role of purines on hepatic ischemia-reperfusion lesions in rabbit.

In this work, we evaluate the effects of adenosine 5' triphosphate (ATP) on hepatic lesions caused by ischemia/reperfusion (I/R) in liver rabbit. Rabbits were pretreated with ATP (15 mg/kg IV) or saline solution 0.9% (SS), before the hepatic I/R procedure. We evaluated the effects of ATP on hepatic injury before and after I/R. The warm hepatic I/R procedure caused profound acute liver injury, as indicated by elevated serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, as well as a high apoptotic cell count. All these changes were attenuate by ATP treatment before the hepatic I/R procedure. These results suggested that ATP exerted protective effects on hepatic I/R lesions in the rabbit. This ATP effect may be related to improved energy metabolism during reperfusion in ischemic livers protecting against functional damage of cellular and subcellular membranes during lipid peroxidation.

l-Arginine supplementation protects against hepatic ischemia-reperfusion lesions in rabbits.

We evaluated the effects of a substrate in the biosynthesis of nitric oxide (NO)-l-arginine (LARG)-on hepatic lesions caused by ischemia/reperfusion (I/R) injury in rabbit livers. Rabbits were pretreated with LARG (150 mg/kg IV) or saline solution 0.9% (SS) before the hepatic I/R procedure. The effects of LARG on hepatic injury were evaluated before and after I/R. The warm hepatic I/R procedure produced profound acute liver injury, as indicated by elevated values of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH), as well as a high apoptotic cell count. All changes were attenuated by treatment with LARG before the hepatic I/R procedure. These results suggested that LARG produced protective effects on hepatic I/R lesions. This protective effect of LARG was probably associated with blocking generation of superoxide anions during the hepatic I/R procedure.

Effects of allopurinol on ischemia and reperfusion in rabbit livers.

In this work, we evaluated the effects of allopurinol (ALO), an inhibitor of xanthine oxidase (XO), on hepatic lesions caused by ischemia/reperfusion (I/R) in the rabbit liver. Rabbits were pretreated with ALO (10 mg/kg IV) or saline solution 0.9% before the hepatic I/R procedure. The effects of ALO on hepatic injury were evaluated before and after I/R. A standard, warm hepatic I/R procedure caused profound acute liver injury, as indicated by elevated serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, as well as a high apoptotic cell count. All of these changes were reversed by the administration of ALO before the hepatic I/R procedure. In conclusion, ALO exerted protective effects on hepatic I/R lesions. This protective effect of ALO was probably associated with blocking the generation of superoxide anions during the hepatic I/R procedure by inhibiting XO activity.

Protective effects of heparin on hepatic ischemia and reperfusion lesions in rabbits.

Because the role of heparin (HEP) in hepatic ischemia/reperfusion (I/R) injury is still not fully understood, we investigated the effects of treatment with HEP on hepatic I/R injury in rabbits. For I/R procedures, the portal vein and hepatic artery were occluded by a metallic clamp to promote ischemia. The clamp was removed after 30 minutes to allow reperfusion. Rabbits undergoing the I/R procedure were treated with HEP (100 U/kg) or saline solution 0.9% (SS). When compared with levels before I/R, the serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, levels were increased by the hepatic I/R procedure, among rabbits treated with SS or HEP. However, the increase in these enzymes was lower among rabbits treated with HEP. Histologic analysis of hepatic tissue of rabbits undergoing I/R and treated with SS showed marked lesions in the central lobule with significant inflammatory infiltration. In contrast, a significant reduction in lesions caused by I/R was observed in the livers of rabbits treated with HEP. After starting reperfusion, we visualized apoptotic cells with nuclear staining among rabbits submitted to I/R and treated with SS, but not those treated with HEP. These results suggested that HEP was able to attenuate hepatic lesions caused by I/R in the livers of rabbits.

Evidence for the participation of calcium in non-genomic relaxations induced by androgenic steroids in rat vas deferens.

BACKGROUND AND PURPOSE: Androgens cause non-genomic relaxation in several smooth muscle preparations. However, such an effect has not been investigated in rat vas deferens yet. Our purpose was to study the effect of testosterone and derivatives in this tissue. EXPERIMENTAL APPROACH: The influence of androgens was tested on contraction and translocation of intracellular Ca(2+) induced by KCl in rat vas deferens in vitro. KEY RESULTS: The testosterone derivative 5alpha-dihydrotestosterone produced a rapid and reversible concentration-dependent relaxation of KCl-induced contractions. Other androgens were also effective, showing the following rank order of potency: androsterone >5beta-dihydrotestosterone >androstenedione >5alpha-dihydrotestosterone >testosterone. Calcium-induced contractions were also inhibited (about 45%) by 5alpha-dihydrotestosterone (30 microM). Moreover 5alpha-dihydrotestosterone blocked the increase of intracellular Ca(2+) induced by KCl, measured by the fluorescent dye fura-2. Relaxation to 5alpha-dihydrotestosterone was resistant to the K(+) channel antagonists glibenclamide, 4-aminopyridine and charybdotoxin. It was not affected by removal of epithelium or by L-NNA (300 microM), an inhibitor of nitric oxide biosynthesis, nor by selective inhibitors of soluble guanylate cyclase, ODQ or LY 83583, indicating that nitrergic or cGMP mediated mechanisms were not involved. The androgen-induced relaxation was also not blocked by the protein synthesis inhibitor cycloheximide (300 microM) or by the classical androgen receptor flutamide (up to 100 microM), corroborating that the effect is non-genomic. CONCLUSIONS AND IMPLICATIONS: Testosterone derivatives caused relaxation of the rat vas deferens, that did not involve epithelial tissue, K(+) channels, or nitric oxide-dependent mechanisms, but was related to a partial blockade of Ca(2+) influx.

Autonomic dysfunction of rat jejunum submitted to cold ischemic preservation is prevented by heparin.

Previous studies suggest that cold ischemic preservation (CIP) employed in small bowel transplantation promotes loss of intestinal motility due to severe lesions in autonomic enteric nerves. This autonomic dysfunction may be prevented by antioxidant agents. In this work, we investigated whether preservation with heparin prevented autonomic dysfunction of rat jejunum submitted to CIP for a long time. Jejunal segments (2 cm) of Wistar rats (12 to 16 weeks old) were preserved at 4 degrees C in Ringer's lactate solution without (-) or with (+) 100 UI/mL heparin (H). After preservation for 12 hours, H+ and H- preparations were mounted in parallel in isolated organ baths containing 10 mL Tyrode's solution at 37 degrees C for the study of neurogenic contractions evoked by electrical field stimulation (EFS; 10-30 Hz, 1-ms duration, 60 V) or by stimulation of nicotinic (NIC) or muscarinic (carbachol, CCh) cholinoceptors. The effects of NIC (hexamethonium, HEX) and muscarinic (atropine, ATR) antagonists were studied on these contractions. Contractions induced by EFS (30 Hz) were four times greater in H+ (1.02 +/- 0.12 g) versus H- (0.26 +/- 0.07 g), while contractions induced by NIC (1 mmol/L) were also four times higher in H+ (1.07 +/- 0.10 g) than H- (0.25 +/- 0.09 g) preparations. In addition, contractions induced by CCh (1 mmol/L) were two times higher in H+ (1.21 +/- 0.13 g) than in H- (0.65 +/- 0.10 g). EFS, NIC, and CCh contractions were inhibited by pretreatment of jejunum with HEX or ATR (1 mumol/L/30 min), in H+ and H-. These results indicated that addition of heparin to a preservation solution attenuated the autonomic dysfunction of rat jejunum submitted to CIP for a long time.

Atenolol attenuates autonomic dysfunction of rat jejunum submitted to cold ischemic preservation.

In vitro studies have demonstrated that cold ischemic preservation (CIP) employed in small bowel transplantation produces loss of intestinal motility due to severe lesions of autonomic enteric nerves and that this autonomic dysfunction is attenuated by antioxidant agents. In this work, we investigated whether preservation with atenolol attenuated autonomic dysfunction of rat jejunum submitted to long-term CIP. Jejunal segments (2 cm) of Wistar rats (12 to 16 weeks old) were surgically isolated and preserved at 4 degrees C in Ringer's lactate solution without (-) or with (+) 1 mumol/L atenolol (AT). After preservation for 12 hours, AT+ and AT- preparations were mounted in parallel in isolated organ baths containing 10 mL Tyrode's solution at 37 degrees C for the study of neurogenic contractions evoked by electrical field stimulation (EFS; 10 to 30 Hz, 1-ms duration, 60 V) or by stimulation with nicotinic (nicotine, NIC) or muscarinic (carbachol, CCh) cholinoceptor agents as well as nicotine (hexamethonium, HEX) and muscarinic (atropine, ATR) antagonists. Contractions induced by EFS (30 Hz) were 46% higher in AT+ (0.38 +/- 0.02 g) than AT- (0.26 +/- 0.01 g), while contractions induced by NIC (1 mmol/L) were 84% higher in AT+ (0.46 +/- 0.03 g) than in AT- (0.25 +/- 0.02 g). In addition, contractions induced by CCh (1 mmol/L) were 34% higher in AT+ (0.87 +/- 0.06 g) than in AT- (0.65 +/- 0.08 g). EFS-, NIC-, and CCh-induced contractions were inhibited by pretreatment of jejunum with HEX or ATR (1 mumol/30 min), in AT+ and AT-. These results suggest that addition of atenolol in the preservation solution attenuated autonomic dysfunction of rat jejunum submitted to long-term CIP.

TTX-sensitive Na(+) and nifedipine-sensitive Ca(2+) channels in rat vas deferens smooth muscle cells.

The inward currents in single smooth muscle cells (SMC) isolated from epididymal part of rat vas deferens have been studied using whole-cell patch-clamp method. Depolarising steps from holding potential -90 mV evoked inward current with fast and slow components. The component with slow activation possessed voltage-dependent and pharmacological properties characteristic for Ca(2+) current carried through L-type calcium channels (I(Ca)). The fast component of inward current was activated at around -40 mV, reached its peak at 0 mV, and disappeared upon removal of Na ions from bath solution. This current was blocked in dose-dependent manner by tetrodotoxin (TTX) with an apparent dissociation constant of 6.7 nM. On the basis of voltage-dependent characteristics, TTX sensitivity of fast component of inward current and its disappearance in Na-free solution it is suggested that this current is TTX-sensitive depolarisation activated sodium current (I(Na)). Cell dialysis with a pipette solution containing no macroergic compounds resulted in significant inhibition of I(Ca) (depression of peak I(Ca) by about 81% was observed by 13 min of dialysis), while I(Na) remained unaffected during 50 min of dialysis. These data draw first evidence for the existence of TTX-sensitive Na(+) current in single SMC isolated from rat vas deferens. These Na(+) channels do not appear to be regulated by a phosphorylation process under resting conditions.

Audiometric findings in petrochemical workers exposed to noise and chemical agents.

This study investigated the occurrence of hearing loss among workers of a petrochemical industry during a period of five years. The records of environmental noise and solvents measurements and the results of annual audiometry performed by the company were examined. The audiometric results of workers from olefin operational areas 1 and 2 and aromatic plant areas exposed to solvents and noise and utility area workers exposed only to noise were analyzed for the standard threshold shift (STS). Despite the low exposures to solvents and a moderate exposure to noise, 45.3% of workers had hearing losses and 29.6% had STS.

Epilepsy-induced electrocardiographic alterations following cardiac ischemia and reperfusion in rats.

The present study evaluated electrocardiographic alterations in rats with epilepsy submitted to an acute myocardial infarction (AMI) model induced by cardiac ischemia and reperfusion. Rats were randomly divided into two groups: control (n=12) and epilepsy (n=14). It was found that rats with epilepsy presented a significant reduction in atrioventricular block incidence following the ischemia and reperfusion procedure. In addition, significant alterations were observed in electrocardiogram intervals during the stabilization, ischemia, and reperfusion periods of rats with epilepsy compared to control rats. It was noted that rats with epilepsy presented a significant increase in the QRS interval during the stabilization period in relation to control rats (P<0.01). During the ischemia period, there was an increase in the QRS interval (P<0.05) and a reduction in the P wave and QT intervals (P<0.05 for both) in rats with epilepsy compared to control rats. During the reperfusion period, a significant reduction in the QT interval (P<0.01) was verified in the epilepsy group in relation to the control group. Our results indicate that rats submitted to an epilepsy model induced by pilocarpine presented electrical conductivity alterations of cardiac tissue, mainly during an AMI episode.

Dual effect of alpha-adrenoceptor antagonists in rat isolated vas deferens.

1 In rat isolated vas deferens, the isotonic contractile responses to low doses of noradrenaline or adrenaline were antagonized, and those to high doses were potentiated, by yohimbine, piperoxan, phentolamine and tolazoline. Effects due to intermediate doses were not affected, or were potentiated within about 30 min, following an initial inhibition. 2 The alpha-adrenoceptor blockers thus caused a shift to the right and an increase of the maximum height of log dose-response curves of alpha-adrenoceptor stimulants. For a given dose of antagonist, the onset was slower for the potentiating than for the blocking effect. 3 The shift to the right induced by piperoxan and yohimbine on dose-response curves of noradrenaline and adrenaline was analysed with the Schild plot, and the slopes obtained, around 0.3, were lower than expected from receptor theory. When cocaine was used to block neuronal uptake, the slopes were close to 1.0. 4 The increase in maximum response to noradrenaline and adrenaline induced by alpha-adrenoceptor blockers was dependent on the time of incubation, on the dose of antagonist, and on the initial height of responses to the agonist. A less pronounced potentiation was also obtained when acetylcholine was used as agonist. 5 The findings are explained in terms of receptor theory as being due to a dual effect of alpha-adrenoceptor antagonists; competitive antagonism proper, which may be disclosed after blockade of neuronal uptake, and an interaction at a different locus, which results in potentiation of the effects of noradrenaline and adrenaline.

Different mechanisms of action of agents acting on beta-adrenoceptors in barium-stimulated and electrically-stimulated rat vas deferens.

1. The relaxation induced by beta-adrenoceptor agonists in rat vas deferens was examined under two different experimental conditions: on electrically-induced twitch responses (35 V, 3 ms, 0.07 Hz), and on contractions induced by single doses of barium chloride (300 microM). The experiments were performed in vasa of reserpine-treated rats, after blockade of alpha-adrenoceptors and extraneuronal uptake with dibenamine (10 microM, 30 min), and neuronal uptake with cocaine (10 microM). 2. When twitch responses were used, the values of pD2, interpolated from cumulative concentration-response curves for isoprenaline (Iso), adrenaline (Ad), and noradrenaline (NA) showed a rank order of potency consistent with the presence of beta 2-adrenoceptors (Iso greater than Ad much greater than NA). 3. When twitch responses were used, the non-selective beta-antagonist, propranolol, caused a concentration-dependent parallel shift to the right of Iso concentration-response curves. Similar shifts were obtained by use of the beta 2-antagonist, isopropylmethoxamine (IMA), and higher doses of the beta 1-antagonist, practolol, according to the expectations from receptor occupation theory. Practolol presented the lowest value of pKB, 5.03, corroborating the presence of beta 2-adrenoceptors. 4. When twitch responses were used, and Ad or NA employed instead of Iso, the antagonists produced shifts of concentration-response curves which were smaller than expected from theory, precluding the determination of pKB values. This indicates that other mechanisms are involved besides an interaction with a single population of postsynaptic beta 2-adrenoceptors. 5. When barium chloride was used instead of twitch responses, although the potencies of Iso and Ad were increased respectively by about 30 fold and 5 fold, the rank order of potency was still consistent with an interaction with beta 2-adrenoceptors. In addition, the antagonists produced parallel and concentrationdependent shifts of the curves of all the agonists, as expected from receptor theory. The values of pKB for a given antagonist were not modified by interchanging the agonists used, indicating a typical interaction with a single population of beta 2-adrenoceptors. When compared to the field-stimulated vas, the values of pKB for propranolol and IMA against isoprenaline were respectively 1.3 and 0.6 log units larger. These results suggest the beta l-adrenoceptor agents act by different mechanisms of action in barium-stimulated and electrically-stimulated vas. 6. It is suggested that when barium is used, the effects of agents acting on beta l-adrenoceptors are mediated only by postsynaptic beta 2-receptors, while other complicating factors, probably nerve-dependent presynaptic mechanisms, may be involved with electrical stimulation.

Simultaneous measurement of contractile effects in the circular and longitudinal smooth muscle of the rat vas deferens by drugs perfused externally or via the lumen.

The effects of noradrenaline and barium chloride were studied in the rat isolated vas deferens by perfusion of drugs either externally or through the lumen of the organ. Two effects were recorded simultaneously in the same preparation: (a) isometric contractions, due to the tension elicited by drugs on the external (longitudinal) smooth muscle layer and (b) pressure of internal perfusion, due to contractions of the internal (circular) smooth muscle layer. It was found with the longitudinal muscle that: (a) the potency, expressed as pD2 values, and the maximum response to noradrenaline were lower if the drug was perfused internally rather than externally; (b) the differences in maximum effects were pronounced on the prostatic half but were not observed on the epididymal half; (c) the maximum response obtained by internal perfusion could be increased by simultaneously adding the same dose of drug externally; (d) when barium chloride was used instead of noradrenaline no significant differences were observed on pD2 values, but differences on maximal responses were similar to that observed for noradrenaline; (e) it was possible to block completely the effect of internal or external noradrenaline on the longitudinal muscle, by perfusing external phenoxybenzamine. In these conditions the responses of the circular muscle to the agonist were only partly blocked. With the circular muscle, the differences related to internal and external perfusion were less marked than in the longitudinal muscle. However, unlike the latter, the circular layer was slightly more sensitive to drugs applied internally, in relation to pD2 values. It is suggested that the difference in pD2 values may be due to the removal of noradrenaline by the neuronal uptake process, whereas the difference in maximal effect is due to the inaccessibility of part of the receptor population when drugs are added through the lumen.

In vitro denervation of the rat vas deferens through hypothermic storage.

1. The rat vas deferens was excised, stored at 4-6 degrees C and tested after 24, 48, 72 or 96 h for its contractile activity and for the presence of innervation. 2. The maximal contractile capacity of the vas, tested through cumulative concentrations of barium chloride (3 x 10(-2) M) was progressively reduced from about 110 mm to about 63 mm after 72 h, without further decay after 96 h. Spontaneous rhythmic contractions were practically absent. 3. A loss of endogenous pools of catecholamines was indicated by four parameters: (a) a decline of about 80% after 24 h and of more than 95% after 48 h of the contractile effect of the indirect sympathomimetic agonist tyramine; (b) a fall of about 20%, 50% and 85% on the concentration of noradrenaline, respectively after 24, 48 and 72 h; (c) a fall of about 25% and 90% after respectively 24 and 48 h, of the activity of dopamine-beta-hydroxylase (DBH); (d) a decline of noradrenaline-induced histofluorescence on cross sections of the vas. 4. A loss of neuronal uptake capacity was indicated by: (a) a progressive variation of the apparent affinity for adrenaline, expressed as pD2 values, that increased by about 1.5 log units (corresponding to a 30 fold potentiation) after 72 h, and (b) a reduction of the ability of cocaine to potentiate the contractile effects of adrenaline. 5. The pD2 values for barium chloride, 5-hydroxytryptamine (5-HT) and histamine were not significantly changed, while the corresponding value for acetylcholine was slightly but significantly reduced by about 0.8 log units. 6. The maximal heights of concentration-response curves for noradrenaline, acetylcholine, histamine and 5-HT were reduced by 42-66% in relation to controls. However, when this reduction was measured in relation to the corresponding barium effect, by means of the relative responsiveness ratio (p), a small though significant increase was observed for noradrenaline, and a fall for the other drugs.7. It is concluded that: (1) the values for the various biochemical and pharmacological parameters decline at different rates, though revealing altogether that denervation is completed by at least 85% after 72 h of hypothermic storage; (2) two of the results, i.e., the lack of spontaneous rhythmic contractions and the lack of increased contractile effects for acetylcholine, 5-HT and histamine, indicate that in these conditions the vas is devoid of the so-called nonspecific signs of denervation.

Effects of indoramin in rat vas deferens and aorta: concomitant alpha1-adrenoceptor and neuronal uptake blockade.

1. The actions of the alpha1-adrenoceptor antagonist indoramin have been examined against the contractions induced by noradrenaline in the rat vas deferens and aorta taking into account a putative neuronal uptake blocking activity of this antagonist which could result in self-cancelling actions. 2. Indoramin behaved as a simple competitive antagonist of the contractions induced by noradrenaline in the vas deferens and aorta yielding pA2 values of 7.38+/-0.05 (slope=0.98+/-0.03) and 6.78+/-0.14 (slope=1.08+/-0.06), respectively. 3. When the experiments were repeated in the presence of cocaine (6 microM) the potency (pA2) of indoramin in antagonizing the contractions of the vas deferens to noradrenaline was increased to 8.72+/-0.07 (slope=1.10+/-0.05) while its potency remained unchanged in the aorta (pA2=6.69+/-0.12; slope=1.04+/-0.05). 4. In denervated vas deferens, indoramin antagonized the contractions to noradrenaline with a potency similar to that found in the presence of cocaine (8.79+/-0.07; slope=1.09+/-0.06). 5. It is suggested that indoramin blocks alpha1-adrenoceptors and neuronal uptake in rat vas deferens resulting in Schild plots with slopes not different from unity even in the absence of selective inhibition of neuronal uptake. As a major consequence of this double mechanism of action, the pA2 values for this antagonist are underestimated when calculated in situations where the neuronal uptake is active, yielding spurious pKB values.

Low dihydropyridine receptor density in vasa deferentia of castrated rats.

Radioligand binding studies in crude membrane preparations of vasa deferentia of normal rats, with the 1,4-dihydropyridine (+)-[3H]-PN200-110 (isradipine) showed typical saturation isotherms. The binding exhibited a KD of 259 +/- 60 pM and Bmax of 144 +/- 20 fmol mg-1 protein. The low KD and the stereoselective displacement of (+)-[3H]-PN200-110 binding by (+)- and (-)-PN200-110 and by nifedipine suggests that these tissues contain dihydropyridine receptors probably coupled to voltage-sensitive, L-type calcium channels. In membrane preparations from vasa deferentia from rats castrated 30 days previously the maximum specific binding was 25 +/- 10 fmol mg-1 protein, representing only 11% of total binding; thus, the calculation of reliable KD values was not feasible. These findings suggest that a testicular hormone, possibly testosterone, plays an important role in the regulation of dihydropyridine-sensitive, voltage-dependent calcium channels in the rat vas deferens.

Down-regulation of Na(+)/K(+)-ATPase alpha(2) isoform in denervated rat vas deferens.

In the rat vas deferens, an organ richly innervated by peripheral sympathetic neurons, we have demonstrated recently the expression of alpha(1) and alpha(2), but not alpha(3) isoforms of the alpha subunit of Na(+)/K(+)-ATPase (EC 3.6.1.37), a membrane-bound enzyme of vital function for living cells (Noël et al., Biochem Pharmacol 55: 1531-1535, 1998). In the present work, we characterized, qualitatively and quantitatively, Na(+)/K(+)-ATPase alpha isoforms in denervated rat vasa deferentia. [(3)H]Ouabain binding at concentrations defined for high-affinity isoforms (alpha(2) and/or alpha(3)) detected only one class of specific binding sites in control (C) and denervated (D) vas deferens. Although the dissociation constant was similar for both groups [K(d) = 138 +/- 14 nM (C) and 125 +/- 8 nM (D)], a marked decrease in density was observed after denervation [716 +/- 81 fmol.mg protein(-1) (C) and 445 +/- 34 fmol.mg protein(-1) (D), P < 0.05]. In addition, western blotting revealed that denervated vasa deferentia produce the alpha(1) and alpha(2) isoforms but not alpha(3), just as we reported for the controls previously (Noël et al., Biochem Pharmacol 55: 1531-1535, 1998). Densitometric analysis showed a decrease of the alpha(2) isoform by about 40% in denervated organs, in very good agreement with what was shown with the [(3)H]ouabain binding technique, but no significant change in alpha(1) isoform density. Truncated alpha(1) (alpha(1)T), an isoform suggested to exist in the guinea pig vas deferens, was not detected. Altogether, our results demonstrated that Na(+)/K(+)-ATPase alpha(2) is down-regulated after sympathetic denervation of the rat vas deferens.

Quantitative analysis of the high-affinity binding sites for [3H]ouabain in the rat vas deferens and their immunological identification as the alpha 2 isoform of Na+/K(+)-ATPase.

Binding assays were performed with [3H]ouabain to investigate the presence of, and to characterize, a Na+/K(+)-ATPase isoform with high affinity for cardiac glycosides in the rat vas deferens. Nonlinear regression analysis of equilibrium experiments carried out with crude preparations in a Mg-Pi medium indicated the presence of high-affinity sites characterized with good precision (individual coefficients of variation = 11-35%) by their density (Bmax = 0.42 to 0.72 pmol/mg protein) and dissociation constant (Kd = 0.069 to 0.136 microM) values. The values of the dissociation rate constant (kappa-1) and the association rate constant (kappa+1) for these sites were 0.151 to 0.267 min-1 and 2.87 to 3.60 microM-1.min-1, respectively. A higher number of low-affinity sites (Kd around 15 microM), supposed to correspond to the alpha 1 isoform, was also identified, but their Kd and Bmax values were not quantified precisely in this crude preparation. Western blot assays indicated hybridization with specific anti-alpha 1 and anti-alpha 2 isoform antibodies but not with anti-alpha 3 isoform antibody. Taken together, the present results indicate the existence of a low proportion of the alpha 2 isoform of Na+/K(+)-ATPase in the rat vas deferens that can be quantified precisely by [3H]ouabain binding even in a crude membrane preparation that is suitable for studies under conditions of plasticity.

Dissociation constants and relative efficacies estimated from the functional antagonism of beta-adrenoceptor agonists on transmural stimulation in rat vas deferens.

In rat vas deferens, the beta-adrenoceptor agonists, isoprenaline, salbutamol, terbutaline and fenoterol, inhibited the contractions elicited by transmural electrical stimuli to an extent which depended on voltage magnitude. The dose-response curves for these agonists were shifted slightly to the right when the amplitude of the twitch was increased up to twice its original size. Consequently, the values of pD2 for the respective agonists obtained with a '50% twitch' and a '100% twitch' were: 7.6 and 7.5, 6.8 and 6.6, 6.2 and 6.0, 7.6 and 7.4. In addition, the maximal effects of the agonists were significantly reduced (by 13-18%). The values of other parameters for the same agonists, calculated according to a model for functional antagonism, were: negative log of dissociation constants (pKa): 7.2, 6.3, 6.2 and 7.2; percent receptors occupied to induce a half-maximal effect (y50): 42, 46, 63, and 53; relative intrinsic efficacy (er): 1.0, 0.9, 0.7, and 0.8. The usefulness of this type of functional antagonism as an alternative method for the estimation of drug-receptor parameters is discussed in the light of receptor theory.

Increased density of alpha-adrenoceptors in vas deferens of spontaneously hypertensive rats (SHR), indicated by functional and receptor binding studies.

Pharmacological parameters were determined from contractile responses mediated by alpha-adrenoceptors in vas deferens from spontaneously hypertensive rats (SHR) and corresponding normotensive controls, Wistar Kyoto rats (WKY), and compared with data obtained from radioligand binding assays. Contractile responses induced in longitudinal and circular muscle layers by the alpha-adrenoceptor agonist noradrenaline (NA) and by barium chloride were recorded as described previously. In both muscle layers the maximal effects induced by NA, but not by BaCl2, were significantly greater in SHR. As a consequence, the relative responsiveness ratio (rho) for the alpha-adrenoceptor was also larger for SHR than for WKY. NA-induced contractions of both muscle layers were competitively antagonized by indoramine. The pA2 values for indoramine and pD2 values for NA were the same in SHR and WKY, indicating that alpha-adrenoceptor affinity was not changed in SHR. Additionally, binding studies with the alpha-adrenoceptor ligand [3H]WB4101 revealed that Bmax values were greater in the vas deferens of SHR, whereas Kd values were not significantly different from those of WKY controls. In summary, although differences could not be detected for affinity-related parameters, a greater density of alpha-adrenoceptors was shown for SHR in receptor binding studies and this was corroborated by functional studies.