Benzoate Hydrochloride Research Articles

Simultaneous Assessment of Alogliptin Benzoate and Pioglitazone Hydrochloride in Combined Tablet Dosage Form by UV Spectrophotometric Methods

Simultaneous Assessment of Alogliptin Benzoate and Pioglitazone Hydrochloride in Combined Tablet Dosage Form by UV Spectrophotometric Methods

RP-HPLC Method for estimation of Alogliptin and Glibenclamide in Synthetic mixture

The objective of the current study was to develop simple, accurate, precise and rapid RP-HPLC method with subsequently validate as per ICH guidelines for the determination of Alogliptin benzoate and Glibenclamide hydrochloride using mobile phase [mixture of Acetonitrile: Phosphate buffer pH-3.6 and in the ratio of 65:35.The proposed method involves the measurement of Retention time at selected analytical wavelength. 240.0 nm. The retention time of Alogliptin benzoate (ALO) and Glibenclamide was found to be 5.055 and 2.838 respectively. The linearity of the proposed method was investigated in the range of 1-5 µg/ml (r = 0.9998) for ALO and 10-50µg/ml (r = 0.9999) for MET respectively. The method was statistically validated for linearity, accuracy and precision. Both inter-day and intra-day variation was found to be showing less % RSD (Relative Standard Deviation) value indicating high grade of precision of the method.

Antimicrobial Activity of 1,2,5-Trimethylpiperidin-4-Ol Derivatives

The antimicrobial activity of 1,2,5-trimethylpiperidin-4-ols (I – IV) against 20 test microorganisms was studied. 1,2,5-Trimethyl-4-(1′,2′-dihydroxyethyl)piperidin-4-ol benzoate hydrochloride (III) had the widest spectrum of antimicrobial activity (active against 16 test microorganisms) and is suitable for further testing of antimicrobial activity.

Effect of Human Serum Albumin on Endotoxin Scattering Photometry

Laser scattering photometry (ESP) is a newly developed plasma endotoxin assay method using horseshoe crab amebocyte lysate (AL) that recognizes small particles produced by polymerization of coagulin under the stirring conditions at 1000rpm. We elucidated the effect of human serum album (HSA) in the ESP method. AL was dissolved with 630μL of the specimen and a 200-μL aliquot was used for ESP; this conventional protocol was regarded as the ESP630 method. The ESP210 method was also used, i. e. AL was dissolved with 210μL of the specimen and a 200-μL aliquot was used for ESP. Water induced the agglutination, and HSA prolonged the agglutination time depending on its concentration especially in the ESP630 method. The water-induced agglutination was not inhibited by the addition of anti-factor C monoclonal antibody, and amidinophenyl benzoate hydrochloride, used as a clotting enzyme inhibitor, intensively inhibited the water-induced agglutination. Therefore, the water-induced agglutination was suggested to be a false-positive reaction to non-specific activation of the clotting enzyme. The HSA-induced prolongation of the reaction in the national health insurance-covered turbidimetric kinetic assay was not observed. HSA or plasma protein seemed to affect the result, especially in the ESP630 method, and a non-specific reaction was found to occur in the ESP methods.

In vitro and in vivo characterization of PA01, a novel promising triple reuptake inhibitor

Triple reuptake inhibitors (TRIs) that inhibit the reuptake of serotonin (5-HT), norepinephrine (NE), and dopamine (DA) are being developed as a new class of antidepressants, which is hypothesized to produce more rapid onset and better efficacy than conventional antidepressants in part due to the addition of the DA component. 4-[2-(dimethylamino)-1-(1-hydroxycyclohexyl)-ethyl]-phenyl benzoate hydrochloride (PA01), a novel compound, potently bound to the human 5-HT, NE, and DA transporters (Ki=105, 644, and 813nM, respectively), and inhibited the reuptake of 5-HT, NE, and DA into recombinant cells (IC50=341, 427, and 753nM, respectively). In vivo, PA01 dose-dependently decreased immobility time in the forced swimming test (FST) in rats, and the tail suspension test (TST) in mice with higher efficacy than desvenlafaxine succinate (DVS), and showed no stimulatory effect on the spontaneous locomotor activity. The anti-immobility effect of PA01 in the TST was significantly prevented by the pretreatment of mice with DL-p-chlorophenylalanine (pCPA, 300mg/kg, an inhibitor of serotonin synthesis), SCH23390 (0.05mg/kg, s.c., dopamine D1 receptor antagonist), and sulpiride (50mg/kg, i.p., dopamine D2 receptor antagonist). PA01 significantly increased head-twitch response induced by 5-hydroxytryptophan (80mg/kg, i.p., a metabolic precursor to serotonin) in rats, potentiated yohimbine (25mg/kg, s.c., a α2-adrenoceptor antagonist) toxicity, and antagonized high dose apomorphine-induced hypothermia in mice. Taken together, these in vitro and in vivo results indicated that PA01 is a novel triple reuptake inhibitor, and exerts an excellent antidepressant activity in the behavioral despair animal models of depression, with more potent antidepressant activity than DVS at the same dose.

Early treatment of a child with NAGS deficiency using N-carbamyl glutamate results in a normal neurological outcome

Acute hyperammonemia has a variety of etiologies and clinical manifestations. If not treated early in neonates, it leads to irreversible brain damage or death. We present a 7-day-old female patient who was brought to the emergency department with drownsiness and vomiting. Metabolic work-up revealed a blood ammonia level of 290 μmol/L (normal <100 μmol/L in neonates) with a compensated respiratory alkalosis, normal glycaemia and lactate and absence of urinary ketones. Oral feeding was stopped, an infusion of 20 % glucose was started, and sodium benzoate and arginine hydrochloride were given. After a drop of ammonemia within 12 h of treatment, it started rising again. N-carbamylglutamate (NCG) was added resulting in a rapid normalisation of ammonemia. Feedings were progressively reintroduced, the ammonia levels remained low. The results of the metabolic work-up were compatible with carbamyl phosphate synthase 1 (CPS1) or N-acetyl glutamate synthase (NAGS) deficiency. Genetic analysis confirmed the latter diagnosis with a homozygous mutation c. 1450T > C (p.W484R) in exon 6 of the NAGS gene in the patient and a carrier state in both parents. At the age of 9 months, the child is growing well with normal neurological development, under treatment with NCG 100 mg/kg/day and a normal diet. Conclusion: This case highlights the importance of keeping a high index of suspicion and early testing for ammonia levels in neonates/children with unexplained encephalopathy. In neonates with congenital hyperammonemia, NCG should always be started together with the standard management of hyperammonemia until all laboratory investigations are complete or indicate another disease.

Synthesis and Biological Evaluation of 2-Hydroxy-3-[(2-aryloxyethyl)amino]propyl 4-[(Alkoxycarbonyl)amino]benzoates

A series of twenty substituted 2-hydroxy-3-[(2-aryloxyethyl)amino]propyl 4-[(alkoxycarbonyl)amino]benzoates were prepared and characterized. As similar compounds have been described as potential antimycobacterials, primary in vitro screening of the synthesized carbamates was also performed against two mycobacterial species. 2-Hydroxy-3-[2-(2,6-dimethoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride, 2-hydroxy-3-[2-(4-methoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride, and 2-hydroxy-3-[2-(2-methoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride showed higher activity against M. avium subsp. paratuberculosis and M. intracellulare than the standards ciprofloxacin, isoniazid, or pyrazinamide. Cytotoxicity assay of effective compounds was performed using the human monocytic leukaemia THP-1 cell line. Compounds with predicted amphiphilic properties were also tested for their effects on the rate of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. All butyl derivatives significantly stimulated the rate of PET, indicating that the compounds can induce conformational changes in thylakoid membranes resulting in an increase of their permeability and so causing uncoupling of phosphorylation from electron transport.

Molecular and functional characteristics of β3-adrenoceptors in late pregnant mouse uterus: A comparison with β2-adrenoceptors

β3-adrenoceptor is a potential target for uterine relaxant drugs for the treatment of preterm labor. Mouse is an ideal experimental model for preterm labor. However, there is limited information on the molecular and functional characteristics of β3-adrenoceptors in mouse uterus. Therefore, the current study was undertaken to characterize the β3-adrenoceptors in late pregnant mouse uterus by molecular and functional experiments and to compare their expression and function with the β2-adrenoceptors. Using RT-PCR, we demonstrated the presence of β3-adrenoceptor mRNA in the mouse uterus. Accordingly, selective β3-adrenoceptor agonist SAR150640 (ethyl-4-{trans-4-[((2S)-2-hydroxy-3-{4-hydroxy-3[(methylsulfonyl)amino]-phenoxy}propyl)amino]cyclohexyl}benzoate hydrochloride) caused concentration-dependent relaxation of the isolated tissue. SR59230A (1μM), a selective antagonist of β3-adrenoceptors, antagonized the relaxant response to SAR150640. Using real-time PCR we found that in comparison to β3-adrenoceptor mRNA, β2-adrenoceptor mRNA is predominantly expressed in the late pregnant mouse uterus. We then assessed the comparative efficiency of different β-adrenoceptor agonists, such as SAR150640, salbutamol and isoprenaline to relax the tissue. SAR150640 (pD2 6.64±0.21, Emax 104.9±7.95), salbutamol (pD2 8.57±0.062, Emax 103.1±3.22) and isoprenaline (pD2 9.48±0.084, Emax 102.9±5.18) caused concentration-dependent inhibition of uterine rhythmic contractions. While the maximal relaxation to these agonists was comparable, the order of potency was isoprenaline>salbutamol>SAR. These results suggest that β3-adrenoceptor mRNA is present in the pregnant mouse uterus and is functionally active. The predominance of β2- over β3-adrenoceptor expression may explain variable potency amongst the β-adrenoceptor agonists.

Cardioprotective effects of 44Bu, a newly synthesized compound, in rat heart subjected to ischemia/reperfusion injury

Excessive intracellular Na + accumulation followed by Ca 2+ overload in cardiac tissue is one of the important mechanisms leading to ischemia/reperfusion injury. In the present study, the cardioprotective effects of 44Bu, 2-hydroxy-3-(butylamino) propyl-4-{(butoxycarbonyl)amino}benzoate hydrochloride, a novel Na + channel blocker, on ischemia/reperfusion injury were investigated and compared to lidocaine. Isolated rat hearts perfused at the constant flow were exposed to global ischemia for 60 min followed by 30 min of reperfusion. In control hearts, ischemia/reperfusion markedly decreased left ventricular developed pressure and increased left ventricular end-diastolic pressure, and caused lactate dehydrogenase release and infarction. 44Bu (0.1, 0.3 and 1 µM) or lidocaine (1 and 200 µM) was administrated during the last 10 min before ischemia and the first 5 min of the reperfusion period. A significant post-ischemic functional recovery in the same degree was elicited by 0.3 and 1 μM 44Bu or 200 µM lidocaine. These effects of 44Bu and lidocaine closely correlated with the reduction in the infarct size and lactate dehydrogenase release. In contrast, 44Bu (0.1 µM) or lidocaine (1 µM) treatment did not result in a significant recovery in any of the examined parameters. In accordance with functional results, our electrophysiological data demonstrated that 44Bu was a more potent agent than lidocaine in terms of transient Na + current inhibition. On the other hand, 44Bu did not cause any change in Ca 2+ currents and on Na +/H + exchange activity. These results show that 44Bu, as a novel Na + channel blocker, has cardioprotective effects against ischemia/reperfusion injury.

In Vitro and in Vivo Pharmacological Characterization of Ethyl-4-{trans-4-[((2S)-2-hydroxy-3-{4-hydroxy-3[(methylsulfonyl)amino]-phenoxy}propyl) Amino]cyclohexyl}benzoate Hydrochloride (SAR150640), a New Potent and Selective Human β3-Adrenoceptor Agonist for the Treatment of Preterm Labor

Ethyl-4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl)amino] phenoxy]propyl) amino]cyclohexyl]benzoate hydrochloride (SAR150640) was characterized as a new potent and selective beta(3)-adrenoceptor agonist for the treatment of preterm labor. SAR150640 and its major metabolite, the corresponding acid 4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl) amino] phenoxy]propyl)amino]cyclohexyl]benzoic acid (SSR500400), showed high affinity for beta(3)-adrenoceptors (K(i) = 73 and 358 nM) and greater potency than (-)-isoproterenol in increasing cAMP production in membrane preparations from human neuroblastoma cells (SKNMC), which express native beta(3)-adrenoceptors (pEC(50) = 6.5, 6.2, and 5.1, respectively). SAR150640 and SSR500400 also increased cAMP production in membrane preparations from human uterine smooth muscle cells (UtSMC), which also express native beta(3)-adrenoceptors (pEC(50) = 7.7 and 7.7, respectively). In these cells, SAR150640 dose-dependently inhibited oxytocin-induced intracellular Ca(2+) mobilization and extracellular signal-regulated kinase 1/2 phosphorylation. SAR150640 and SSR500400 had no beta(1)- or beta(2)-agonist or antagonist activity in guinea pig atrium and trachea, or in human isolated atrium and bronchus preparations. Both compounds concentration-dependently inhibited spontaneous contractions in human near-term myometrial strips, with greater potency than salbutamol and 4-[3-[(1,1-dimethylethyl)-amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP12177) (pIC(50) = 6.4, 6.8, 5.9, and 5.8, respectively), but with similar potency to (-)-isoproterenol and atosiban (oxytocin/vasopressin V(1)a receptor antagonist). SAR150640 also inhibited the contractions induced by oxytocin and prostaglandin F(2alpha). In vivo, after intravenous administration, SAR150640 (1 and 6 mg/kg), but not atosiban (6 mg/kg), dose-dependently inhibited myometrial contractions in conscious unrestrained female cynomolgus monkeys, with no significant effects on heart rate or blood pressure. In contrast, salbutamol (50 and 250 microg/kg) had no inhibitory effect on uterine contractions, but it dose-dependently increased heart rate. These findings indicate a potential for the therapeutic use of SAR150640 in mammals during preterm labor.

Liquid Chromatography and Chemometric-Assisted Spectrophotometric Methods for the Analysis of Two Multicomponent Mixtures Containing Cough Suppressant Drugs

Three methods were applied for the analysis of 2 multicomponent mixtures containing dextromethorphan hydrobromide, phenylephrine hydrochloride, chlorpheniramine maleate, methylparaben, and propylparaben, together with either sodium benzoate (Mix 1) or ephedrine hydrochloride and benzoic acid (Mix 2). In the first method, liquid chromatography was used for their simultaneous determination using an ODS column with a mobile phase consisting of acetonitrile-phosphate buffer, pH 2.7 (40 + 60, v/v), containing 5mM heptanesulfonic acid sodium salt and ultraviolet (UV) detection at 214 nm. Also, 2 chemometric methods, principal component regression, and partial least squares were used. For both chemometric calibrations, a concentration set of the mixture consisting of each compound in each mixture was prepared in distilled water. The absorbance data in the UV spectra were measured for the 76 or 71 wavelength points in the spectral region 210-240 or 210-224 nm considering the intervals of deltagamma = 0.4 or 0.2 nm for Mix 1 and Mix 2, respectively. The 2 chemometric methods did not require any separation step. These methods were successfully applied for the analysis of the 2 multicomponent combinations in synthetic mixtures and in commercial syrups, and the results were compared with each other.

血漿中 (1-3)-β-D-グルカン測定法における非特異反応出現に関する検討

We detected non-specific reactions in the measurement of (1-->3)-beta-D-glucan levels (beta-glucan) in plasma, and the influences of the non-specific reactions on sensitivity and specificity of measurement methods were examined. In this study, 460 plasma samples from 174 patients at Kawasaki Medical School Hospital were used, and the plasma beta-glucan levels were measured by different four methods. The methods included the dilution-heating-endpoint (DHE), dilution-heating-turbidimetric (DHT), alkaline-kinetic (AK), and alkaline-endpoint method (AE) with and without 4-amidinophenyl benzoate hydrochloride (APB) of a protease inhibitor blocking the Limulus reaction. Non-specific reactions were detected from the calculated value under conditions with APB. Therefore, both of the actual values and the values equivalent to non-specific reactions were calculated. The incidence of non-specific reactions was 2.4% in DHE method, 0% in DHT, 53.3% in AK, and 99.3% in AE. The sensitivity and specificity in the methods were 35.7% and 96.0%, 28.6% and 96.0%, 78.6% and 80.1%, and 57.1% and 84.1%, respectively. When subtracted the non-specific reaction values from the actual values in AK and AE method, the specificity was increased by 91.4% and 94.0%, respectively. In these two methods, the non-specific reaction was considered to be a major cause of the low specificity. Finally, to measure plasma beta-glucan levels accurately, non-specific reactions should be excluded as possible by further improvement of measurement methods.

Unexpected Reactions of Dithiodiglycolic Acid: Isolation of a Novel Seven‐Membered Ring System Based on a 2‐Amino Derivative of 7‐Oxo‐6,7‐dihydro‐[1,4,5]oxadithiepine.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Unexpected reactions of dithiodiglycolic acid: isolation of a novel seven-membered ring system based on a 2-amino derivative of 7-oxo-6,7-dihydro-[1,4,5]oxadithiepine

A novel seven-membered ring was unexpectedly isolated from the coupling of dithiodiglycolic acid with 2.2 equiv. of N-hydroxysuccinimide, followed by addition of 0.25 equiv. of methyl (4-aminomethyl)benzoate hydrochloride ( 2 ) in the presence of 2 equiv. of triethylamine.

An investigation of non-specific reactions in measurement of plasma (1-->3)-beta-D-glucan

We investigated the detection of non-specific reaction in measurement of plasma (1-->3)-beta-D-glucan (beta-glucan) by alkaline treatment, chromogenic automated kinetic assay (alkaline-kinetic assay) and dilution and heating method, chromogenic endpoint assay (dilution heating endpoint assay). In this study, we reexamined the values of beta-glucan by both methods with and without 4-amidinophenyl benzoate hydrochloride (APB) as protease inhibitor that blocks Limulus reaction in the 142 serum samples from 142 patients who had been treated and measured beta-glucan in Kawasaki medical school hospital between January 1999 and May 1999. Non-specific reactions were judged by the calculated value under APB additive condition. The non-specific reactions were found in 135 of total 142 samples (95.1%) in the alkaline-kinetic assay while no non-specific reactions were recognized in dilution heating endpoint assay. The alkaline-kinetic assay has been used widely and been evaluated it's usefulness because of good sensitivity. However, we found very high frequency of nonspecific reaction in this method. Further studies are needed to define the reasons of non-specific reaction. On the other hand, although non-specific reactions were not detected in dilution heating end-point assay, it's clinical utilities should be evaluated in future clinical studies.

Chemotaxis of the Nematode Caenorhabditis elegans toward Cycloheximide and Quinine Hydrochloride.

We investigated the chemotaxis of the nematode Caenorhabditis elegans toward cycloheximide, denatonium benzoate, 6-n-propyl-2-thiouracil, and quinine hydrochloride. Interestingly, C. elegans was strongly attracted to cycloheximide, while avoiding quinine hydrochloride. This is the first report thus far to describe the chemotactic responses of C. elegans toward bitter tastants for humans.

HL 752: A potent and long-acting antispasmodic agent

Ester analogues of methyl-2-(4-(2-piperidinoethoxy)benzoyl)-benzoate hydrochloride (pitofenone) ( 2) were prepared with an aim to find a more potent and metabolically stable antispasmodic compound. The compounds were evaluated for their in vitro and in vivo antispasmodic activity, and stability to in vitro enzymatic hydrolysis. Of the compounds synthesised, HL 752 ( 21) showed the most potent and long-lasting antispasmodic activity and was selected as the candidate for clinical development.

14C]-labelling of benzisothiazolone based inhibitors of human leukocyte elastase

The [14C] labelled synthesis of two benzisothiazolone based inhibitors of Human Leukocyte Elastase is described. These are [3-14C]-[6-methoxy-4-(1-methylethyl)-1,1,3-trioxo-1,2-benzisothianol-2(2H)-y]methyl [carboxy-14C]-2,6-dichlorobenzoate, [14C2]-WIN 63394, 2, and [3-14C]-[6-methoxy-4-(1-methylethyl)-1,1,3-trioxo-1,2-benzisothiazol-2(2H)-yl]methyl [carboxy-14C]-2,6-dichloro-3-[(2-pyrrolidin-1-yl)ethoxy]benzoate hydrochloride, [14C2]-WIN 64733, 3. The synthesis of the intermediate [3-14C]-2-(chloromethyl)-6-methoxy-4-(1-methylethyl)-1,2-benzisothiazol-3(2H)-one 1, 1-dioxide, 8, common to both these syntheses, was achieved in 8 steps in 37% radiochemical yield.

RS 23597-190: a potent and selective 5-HT4 receptor antagonist.

1. The pharmacological properties of RS 23597-190 (3-(piperdine-1-yl)-propyl-4-amino-5-chloro-2-methoxy benzoate hydrochloride) have been studied in vitro and in vivo. 2. RS 23597-190 competitively antagonized 5-HT4 receptor-mediated relaxations of rat, carbachol precontracted oesophageal muscularis mucosae, (pA2 = 7.8 +/- 0.1; Schild slope = 1.2 +/- 0.2). Affinity estimates (-log KB) at 5-HT4 receptors using either renzapride or SC-53116 as agonists yielded a -log KB value of 8.0 +/- 0.01. In contrast, RS 23597-190 failed to antagonize contractile responses to 5-HT of guinea-pig ileal 5-HT3 receptors, even at concentrations up to 10 microM. 3. Increases in short-circuit current, induced by 5-HT, were studied in guinea-pig ileal mucosal sheets. Concentration-response curves to 5-HT were biphasic, with the high potency phase to 5-HT inhibited by RS 23597-190 and mimicked by 5-methoxytryptamine. The -log KB value for RS 23597-190 at the high potency phase was 7.3 confirming that 5-HT4 receptors mediated the high potency phase. 4. In rat isolated vagus nerve, 5-HT elicited a slow, maintained depolarization at low concentrations and a rapid, transient depolarization at higher concentrations. The high potency, slow depolarizing phase to 5-HT was abolished selectively in the presence of 1 microM RS 23597-190 and the low potency phase was abolished selectively in the presence of 1 microM ondansetron. These data confirm that 5-HT4 and 5-HT3 receptors mediated slow and fast depolarization responses, respectively. 5. At 5-HT3 binding sites in membranes from NG 108-15 cells, labelled by [3H]-quipazine, RS 23597-190 exhibited an apparent affinity (- log Ki) of 5.7 +/- 0.1. At 5-HT3 receptors in membranes from rat cerebral cortex, labelled by [3H]-RS 42358-197, the apparent affinity (- log Ki) of RS 23597-190 was also 5.7 +/- 0.1. In both studies, Hill coefficients were not significantly different from unity. At 5-HT1A, 5-HT2,muscarinic M1, M2, M3, M4 and dopamine D1 and D2 receptors, RS 23597-190 exhibited low apparent affinities, with all - log Ki values less than 5.5.6. Intravenous infusion of RS 23597-190 in the conscious, restrained rat antagonized the von Bezold Jarisch reflex induced by 2-methyl 5-HT, with an ID50 of 300 microg kg-1 min-1, i.v. In the anaesthetized,bilaterally vagotomized micropig, RS 23597-190 (6 mg kg-1, i.v.) antagonized 5-HT-induced tachycardia with a half-life of 77 (63-99) min. Transient arrhythmic effects were noted after administration of the compound.7. In conclusion, RS 23597-190 acts as a high affinity, selective competitive antagonist at 5-HT4 receptors. Thus, the compound appears to be a useful tool for 5-HT4 receptor identification in vitro. In vivo, the compound is rapidly metabolized in pigs such that 5-HT4 blockade is not maintained. However,in the rat, when given by infusion, RS 23597-190 antagonizes 5-HT3 mediated responses, at doses consistent with a low affinity 5-HT3 receptor. These data suggest that, under appropriate experimental conditions, RS 23597-190 may also be used in vivo to characterize further 5-HT4 receptor function.

Effects of prostaglandin E2 on membrane voltage of the connecting tubule and cortical collecting duct from rabbits.

1. Effects of prostaglandin E2 (PGE2) on ion transport were examined by observing the transmural (VT) and basolateral membrane voltage (VB) in the in vitro perfused rabbit connecting tubule (CNT) and the cortical collecting duct (CCD). 2. Addition of 1 microM PGE2 to the bath induced a biphasic response of transmural voltage (VT), with initial negative VT deflection followed by positive deflection in the CNT, but monophasic negative deflection in the CCD. Because PGE2 had no affect on the basolateral membrane voltage (VB), PGE2 mainly causes changes in the apical membrane voltage. 3. Elimination of Na+ from the lumen abolished the PGE2-induced VT response in the CNT. In the presence of 10 microM luminal amiloride, PGE2 caused only an initial negative deflection without causing later positive deflection. The positive VT deflection induced by PGE2 in the CCD was also blocked by luminal amiloride. 4. Addition of ouabain (0.1 mM) to the bath completely abolished the PGE2-induced VT changes in the CNT, indicating that an intact Na(+)-K+ pump is a prerequisite for the VT response to PGE2. 5. Addition of 2 mM Ba2+ to the lumen did not affect biphasic VT response to PGE2, indicating that Ba(2+)-sensitive K+ conductance is not involved. 6. Basolateral addition of 0.1 mM 8-(4-chlorophenylthio)-cAMP inhibited only the negative VT deflection induced by PGE2. 7. The positive VT deflection was blocked by basolateral addition of 50 microM 8-(N,N-diethylamino)octyl 3,4,5-trimethoxy benzoate hydrochloride (TMB-8), an inhibitor of intracellular Ca2+ release. But elimination of luminal Ca2+ did not affect the biphasic response to PGE2. 8. These findings suggest that the initial negative VT deflection is caused by an increase in Na+ influx across the luminal membrane through an amiloride-insensitive Na+ conductive pathway, whereas the later positive deflection is caused by the inhibition of Na+ influx through the amiloride-sensitive Na+ conductive pathway. The cAMP messenger system may be responsible for the initial negative deflection, whereas an increased intercellular Ca2+ release from the store is necessary for the later positive deflection caused by PGE2. The response in the CCD is comparable to the later response in the CNT.