Bladder Membranes Research Articles

In vitro and in vivo characterization of a novel naphthylamide ATP-sensitive K+ channel opener, A-151892.

1. Openers of ATP-sensitive K(+) channels are of interest in several therapeutic indications including overactive bladder and other lower urinary tract disorders. This study reports on the in vitro and in vivo characterization of a structurally novel naphthylamide N-[2-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-naphthalen-1-yl]-acetamide (A-151892), as an opener of the ATP-sensitive potassium channels. 2. A-151892 was found to be a potent and efficacious potassium channel opener (KCO) as assessed by glibenclamide-sensitive whole-cell current and fluorescence-based membrane potential responses (-log EC(50)=7.63) in guinea-pig bladder smooth muscle cells. 3. Evidence for direct interaction with KCO binding sites was derived from displacement of binding of the 1,4-dihydropyridine opener [(125)I]A-312110. A-151892 displaced [(125)I]A-312110 binding to bladder membranes with a -log Ki value of 7.45, but lacked affinity against over 70 neurotransmitter receptor and ion channel binding sites. 4. In pig bladder strips, A-151892 suppressed phasic, carbachol-evoked and electrical field stimulus-evoked contractility in a glibenclamide-reversible manner with -log IC(50) values of 8.07, 7.33 and 7.02 respectively, comparable to that of the potencies of the prototypical cyanoguanidine KCO, P1075. The potencies to suppress contractions in thoracic aorta (-log IC(50)=7.81) and portal vein (-log IC(50)=7.98) were not substantially different from those observed for suppression of phasic contractility of the bladder smooth muscle. 5. In vivo, A-151892 was found to potently suppress unstable bladder contractions in obstructed models of unstable contractions in both pigs and rats with pED(35%) values of 8.05 and 7.43, respectively. 6. These results demonstrate that naphthylamide analogs exemplified by A-151892 are novel K(ATP) channel openers and may serve as chemotypes to exploit additional analogs with potential for the treatment of overactive bladder and lower urinary tract symptoms.

Conservation of two Coptic parchment manuscript fragments

Summary The treatments of two similar parchment Coptic manuscript fragments at two different institutions are compared in this article. Both treatments involved the use of a remoistenable mending material—fish swim bladder membrane, or a fine Japanese tissue. Differences in the mending rationales are discussed and treatment innovations elaborated. Other factors impinging on treatment decision-making, beyond the conservation needs of the individual pieces, are also discussed.

Pharmacological characterization of a 1,4-dihydropyridine analogue, 9-(3,4-dichlorophenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione (A-184209) as a novelK(ATP) channel inhibitor.

1. This study reports on the identification and characterization of a 1,4-dihydropyridine analogue, 9-(3,4-dichlorophenyl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione (A-184209) as a novel inhibitor of ATP-sensitive K(+) channels. 2. A-184209 inhibited membrane potential changes evoked by the prototypical cyanoguanidine ATP-sensitive K(+) channel opener (KCO) P1075 in both vascular (A10) and urinary bladder smooth muscle cells with IC(50) values of 1.44 and 2.24 micro M respectively. 3. P1075-evoked relaxation of 25 mM K(+) stimulated aortic strips was inhibited by A-184209 in an apparently competitive fashion with a pA(2) value of 6.34. 4. The potencies of A-184209 to inhibit P1075-evoked decreases in membrane potential responses in cardiac myocytes (IC(50)=0.53 micro M) and to inhibit 2-deoxyglucose-evoked cation efflux pancreatic RINm5F cells (IC(50)=0.52 micro M) were comparable to the values for inhibition of smooth muscle K(ATP) channels. 5. On the other hand, a structural analogue of A-184209 that lacked the gem-dimethyl substituent, 9-(3,4-dichlorophenyl)-3,4,6,7,9,10-hexahydro-1,8(2H,5H)-acridinedione (A-184208), was found to be a K(ATP) channel opener, evoking membrane potential responses in A10 smooth muscle cells (EC(50)=385 nM) and relaxing aortic smooth muscle strips (IC(50)=101 nM) in a glyburide-sensitive manner. 6. Radioligand binding studies demonstrated that A-184209 displaced SUR1 binding defined by [(3)H]glyburide binding to RINm5F cell membranes with a K(i) value of 0.11 micro M whereas A-184208 was ineffective. On the other hand, both A-184209 (K(i)=1.34 micro M) and A-184208 (K(i)=1.14 micro M) displaced binding of the KCO radioligand, [(125)I]A-312110 in guinea-pig bladder membranes with similar affinities. 7. These studies demonstrate that A-184209 is a novel and structurally distinct compound that inhibits K(ATP) channels in smooth muscle with potencies comparable to glyburide. The structural overlap between DHP openers and blockers, together with their differential interaction with ligand binding sites, support the notion that both openers and blockers bind to similar or very closely coupled sites on the sulfonylurea receptor and that subtle changes in the pharmacophore itself could switch functional properties from K(ATP) channel activation to inhibition.

Functional Analysis of Natriuretic Peptide Receptors in the Bladder of the Toad, Bufo marinus

This study aimed to localize and characterize natriuretic peptide binding sites in the urinary bladder of Bufo marinus and to then examine the effect of natriuretic peptides on the bladder vascular tone and water reabsorption in isolated perfused bladder preparations. Specific 125I-rat atrial natriuretic peptide (125I-rANP) binding sites were present on blood vessels, muscle, and epithelium. In tissue sections and/or isolated membranes, the binding was completely displaced by frog ANP, rat ANP, and porcine C-type natriuretic peptide (CNP; membranes only). However, a reduction in binding was observed after incubation with 125I-rANP and 1 μM of the natriuretic peptide receptor-C (NPR-C) ligand C-ANF, but residual binding remained suggesting the presence of two distinct binding sites. Electrophoresis of bladder membranes cross-linked to 125I-rANP identified two bands at approximately 70 and 140 kDa that correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. Furthermore, the presence of natriuretic peptide receptor-A and NPR-C mRNA in the bladder was demonstrated with reverse transcription–polymerase chain reaction. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP generation in bladder membrane preparations, which indicated the presence of guanylate cyclase-linked receptors. In perfused bladder preparations, arginine vasotocin increased perfusion pressure and water permeability. The infusion of frog ANP or porcine CNP failed to alter perfusion pressure or water reabsorption in the presence or absence of arginine vasotocin. This study identified a well-developed natriuretic peptide receptor system in the urinary bladder of B. marinus but the function of the receptors remains unclear.

Effects of cholinergic agents on the vasopressin-mediated water transport in the isolated toad bladder

The aim of this work was to study the effect of some pharmacological cholinergic agents on the events that follow the interaction of arginine vasopressin with toad bladder membrane receptors related to synthesis of 3′5′ cAMP. The water flow through the membrane was measured gravimetrically in sac preparations of the membrane. In the absence of arginine vasopressin (AVP), carbachol induced a significant increase in the water flow (37%) related to the basal (Ringer's solution). On the other hand, when carbachol and AVP were associated, a significant decrease of AVP hydrosmotic activity occurred (23%). The inhibitory effect of carbachol on the AVP action was almost completely abolished by the cholinergic antagonists atropine, pirenzepine, 4-diphenylacetoxy- N-methylpiperidine methiodide (4-DAMP) and the calcium antagonist lanthanum. Similarly, when carbachol and 3′5′ cyclic adenosine monophosphate (3′5′ cAMP) were associated, a decrease of nucleotide hydrosmotic activity was observed (12.80%). This effect was partially restored by the addition of pirenzepine or 4-DAMP in the bath solution. These results suggest a role for muscarinic receptors of sub-type M 1 and M 3, which are involved in the intracellular calcium release. The increase of calcium concentration in the intracellular medium acts as a negative modulator in the hydrosmotic action of antidiuretic hormone.

Inhibitory Effect of Cortisol on the Degranulation of Eosinophilic Granular Cells in Tilapia.

We examined the effect of cortisol on the degranulation and number of eosinophilic granular cells (EGCs) in tilapia, Oreochromis niloticus. One hour after injection of stimulants (killed Escherichia coli or substance P, a neurotransmitter) into the swim bladder, the percent of the degranulated EGCs in the swim bladder membrane was determined. Peritoneal implantation of coconut oil containing cortisol (50 mg/kg body weight) 24 h before injection of stimulants significantly reduced the percent of the degranulated EGCs, compared with the control fish administered with coconut oil without Cortisol. Cortisol also suppressed the in vitro degranulation of EGCs stimulated with substance P or with tilapia normal sera containing zymosan. To investigate the effect of cortisol on the abundance of EGCs, the number of EGCs in the swim bladder membrane was counted. Density of EGCs was decreased after implantation of cortisol, compared with that of the control group. These results suggest that cortisol suppresses the degranulating activity of EGCs and decreases the density of EGCs in the tissue.

Characterization and modulation of [ 125I]iberiotoxin-D19Y/Y36F binding in the guinea-pig urinary bladder

The radioligand binding characteristics of the Ca 2+-activated K + channel ligand [ 125I]iberiotoxin-D19Y/Y36F were examined in guinea-pig urinary bladder membranes. Saturation analysis revealed a single class of high affinity binding sites in the bladder with a K D value of 45.6 pM and a B max value of 112 fmol/mg protein. Specific binding was displaced by unlabeled iberiotoxin and penitrem A, but not by blockers of other classes of K + channels including α-dendrotoxin, margatoxin and apamin. The indole alkaloids, paxilline and verruculogen, significantly increased binding by 4.5- and 4.3-fold, respectively. Tetraacetic acid derivatives such as ethylenediamine tetraacetic acid and ethyleneglycoltetraacetic acid enhanced specific [ 125I]iberiotoxin-D19Y/Y36F binding about 2.5-fold, which was not attributable to calcium chelation. This increase was due to a significant change in ligand binding affinity ( K D=6.3 pM), but not due to a change in the B max, indicating that these compounds may enhance toxin binding via allosteric interactions. Collectively, these results demonstrate that the binding sites for [ 125I]iberiotoxin-D19Y/Y36F present in the urinary bladder shows a pharmacological profile typical of maxi-K + channels and can be modulated, not only by previously known indole alkaloids, but also by tetraacetic acid analogs.

In Vitro Degranulation of Tilapia Eosinophilic Granular Cells and its Effect on Neutrophil Migration.

Migration of neutrophils toward the supernatant of degranulated eosinophilic granular cells (EGCs) was examined using a modified Boyden chamber. EGCs of tilapia (Oreochromis niloticus) were isolated from the swim bladder membrane, and degranulated by incubation with substance P (neurotransmitter) or compound 48/80. The degranulation of the EGCs was also observed after incubation with normal tilapia sera containing zymosan, but not with heat-inactivated sera. Additionally, significantly higher neutrophil migration was observed when the degranulation of the EGCs occurred in the lower compartment of a Boyden chamber. High-speed centrifugation inhibited the degranulation of the EGCs stimulated by substance P or zymosan plus normal tilapia sera, and migration of neutrophils was inhibited. The results obtained in the present study suggest that neutrophil migration stimulating factor (s) are released by degranulation of EGCs in tilapia.

Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores

The water expulsion vacuole (WEV) in zoospores ofPhytophthora nicotianae and other members of the Oomycetes is believed to function in cell osmoregulation. We have used videomicroscopy to analyse the behaviour of the WEV during zoospore development, motility and encystment inP. nicotianae. After cleavage of multinucleate sporangia, the WEV begins to pulse slowly but soon attains a rate similar to that seen in motile zoospores. In zoospores, the WEV has a mean cycle time of 5.7 ± 0.71 s. The WEV continues to pulse at this rate until approximately 4 min after the onset of encystment. At this stage, pulsing slows progressively until it becomes undetectable. The commencement of WEV operation in sporangia coincides with the reduction of zoospore volume prior to release from the sporangium. Disappearance of the WEV during encystment occurs as formation of a cell wall allows the generation of turgor pressure in the cyst. As in other organisms, the WEV inP. nicotianae zoospores consists of a central bladder surrounded by a vesicular and tubular spongiome. Immunolabelling with a monoclonal antibody directed towards vacuolar H+-ATPase reveals that this enzyme is confined to membranes of the spongiome and is absent from the bladder membrane or zoospore plasma membrane. An antibody directed towards plasma membrane H+-ATPase shows the presence of this ATPase in both the bladder membrane and the plasma membrane over the cell body but not the flagella. Analysis of ATPase activity in microsomal fractions fromP. nicotianae zoospores has provided information on the biochemical properties of the ATPases in these cells and has shown that they are similar to those in true fungi. Inhibition of the vacuolar H+-ATPase by potassium nitrate causes a reduction in the pulse rate of the WEV in zoospores and leads to premature encystment. These results give support to the idea that the vacuolar H+-ATPase plays an important role in water accumulation by the spongiome in oomycete zoospores, as it does in other protists.

Effect of dexamethasone on cyclophosphamide-induced cystitis in rats: lack of relation with bradykinin B 1 receptor-mediated motor responses

We investigated the role of bradykinin B 1 receptors in inducing urinary bladder contraction and maintaining bladder compliance in anaesthetized rats following cyclophosphamide-induced bladder inflammation and the influence of dexamethasone treatment on these responses. In the group treated with cyclophosphamide the amplitude of the contraction induced by the selective bradykinin B 1 receptor agonist des-Arg 9-bradykinin was larger than that in controls and dexamethasone prevented the up-regulation of this response induced by inflammation. The specific binding of [ 3 H ]des-Arg 10-kallidin to bladder membranes was only detected in cyclophosphamide-treated rats: this binding was prevented by dexamethasone pretreatment. The bladder contraction induced by des-Arg 9-bradykinin in cyclophosphamide-treated rats was antagonized by the bradykinin B 1 receptor antagonist des-Arg 9- d-Arg-[Hyp 3,Thi 5, d-Tic 7,Oic 8]bradykinin (des-Arg 10-Hoe 140). Cyclophosphamide treatment increased the bladder weight and dexamethasone reversed this effect. Bladder compliance was decreased in the bladder inflammation group and this effect was partially reversed by dexamethasone pretreatment. Neither des-Arg 10-Hoe 140 nor the combined administration of des-Arg 10Hoe 140 and the selective bradykinin B 2 receptor antagonist d-Arg-[Hyp 3,Thi 5, d-Tic 7,Oic 8]bradykinin (Hoe 140) affected bladder compliance, thus excluding a role of kinins in the maintenance of bladder tone during inflammation. These results indicate that: (1) dexamethasone pretreatment ameliorates cyclophosphamide-induced bladder inflammation; (2) dexamethasone pretreatment prevents cyclophosphamide-induced up-regulation of bradykinin B 1 receptors; (3) kinins do not contribute to the increased vesical tone during inflammation.

Use of a channel biosensor for the assay of paralytic shellfish toxins

Gonyautoxin (GTX), saxitoxin (STX) and tetrodotoxin (TTX), also known as paralytic shellfish poisons (PSP), block Na + channels, including those in the frog bladder membrane. A tissue biosensor has been developed, consisting of a Na + electrode covered with a frog bladder membrane integrated within a flow cell. The direction of Na + transfer, investigated in the absence of Na + channel blockers, established that active transport of Na + occurs across the frogs bladder membrane from the internal to the external face. Transfer was shown to be TTX sensitive. The tissue sensor response to each of the different PSP was recorded and the results compared with toxicities determined by the standard mouse bio-assay. Using high concentrations of TTX from the puffer fish Takifugu niphobles, a linear correlation was found between the results from the two assay systems. However, the tissue biosensor system was also able to detect very low concentrations of TTX in samples from two species of puffer fish ( Takifugu niphobles and Takifugu pardalis) at concentrations below the detection limit of the mouse bio-assay.

Anticholinergic and calcium antagonistic activities of NS-21 contribute to the inhibition of rat urinary bladder contractions

1. 1. Pharmacological characteristics of NS-21 and its main metabolite, RCC-36, in the inhibition of rat urinary bladder contractions were investigated both in vitro and in vivo 2. 2. NS-21 and RCC-36 inhibited muscarinic receptor bindings to rat bladder membranes with [ 3H]3-quinuclidinyl bezilate (pKi 6.19 and 7.24, respectively), whereas they failed to inhibit the following receptor bindings: adrenergic (α 1, α 2 and β), dopaminergic (D 1 and D 2), adenosine (A 1 and A 2), histaminergic (H 1) and opioid (μ, δ and κ) receptors. 3. 3. NS-21 and RCC-36 suppressed carbachol-induced contractions of isolated rat detrusor strips in competitive (pA 2 6.80 and 7.93, respectively) and noncompetitive (pD′ 2 5.93 and 5.77, respectively) manners. 4. 4. NS-21 and RCC-36 inhibited CaCl 2-induced contractions of rat detrusor strips in the presence of 100 mM K + (pIC 50 6.54 and 5.76, respectively), as well as the 100 mM K +-induced 45Ca influx into the isolated bladder strips at ≥1 μM 5. 5. Electrical stimulation of the peripheral end of the pelvic nerve in anesthetized rats induced bladder contractions composed of two phases: an initial phasic contraction that was weakly suppressed by atropine, and a tonic contraction that was strongly suppressed by atropine. NS-21 suppressed both contractions to the same degree (ID 50 2.65 and 2.19 mg/kg, IV, respectively). RCC-36 suppressed the tonic contraction (ID 50 1.20 mg/kg, IV) more markedly than the initial contraction (ID 50 7.43 mg/kg, IV) 6. 6. These results suggest that NS-21 and RCC-36 suppressed bladder contractions owing to their anticholinergic and calcium antagonistic activities.

Improved discriminatory properties between human and murine tachykinin NK 1 receptors of MEN 10930: a new potent and competitive antagonist

MEN 10930 (Nα{N-[(1H)indol-3-yl-carbonyl]1-amino-cyclohexane-1-carbonyl}L-3-(2-naphthyl)alanine N-(benzyl) N methyl amide) interacts with high affinity with NK 1 tachykinin receptor expressed in human IM9 (K i = 1.0 ± 0.17 nM) and U373MG (K i = 2.8 ± 0.5 nM) cells and guinea pig lung membranes (K i = 5.9 ± 0.8 nM). MEN 10930 shows no affinity for NK i sites present in rat urinary bladder membranes up to 10 μM, resulting in more than 10 000-fold selectivity for the human NK 1 receptor. In Scatchard plots performed in IM9 cells, MEN 10930 affects the substance P affinity, without changing the B max, suggesting a competitive interaction. It shows negligible affinity for calcium channels (K i = 1.6 ± 0.6 μM), NK 2 receptor (K i = 1.5 ± 0.5 μM) and for NK 3 receptor (K i > 10 μM). Furthermore, MEN 10930 inhibits in a competitive manner the SP methyl ester-induced contractions in guinea pig ileum (pA 2 = 8.7 ± 0.08). In conclusion, MEN 10930 is a potent, selective, competitive antagonist of human, but not murine, NK 1 receptor.

Effects of A-23187 and verapamil on the active transport enzymes in turtle bladder epithelial cells.

These experiments were designed to examine the effects of A-23187 (5 x 10(-4) M) and verapamil (100 microM) on membrane transport, 45Ca fluxes, and adenosine-triphosphatase (ATPase) activities in turtle bladder. In the intact membrane, the calcium ionophore decreased proton secretion and sodium transport [short-circuit current (SCC)] to approximately the same degree (by approximately 55% at 30 min). During the same period of time, verapamil decreased SCC (by approximately 58%), but proton secretion was unaffected. The turtle bladder membrane is composed predominantly of two cell types: 1) the mitochondrial-rich cells (MR cells) thought to be involved in proton (and bicarbonate) secretion containing significant H(+)-ATPase and Ca(2+)-ATPase and 2) the granular cells (G cells), postulated important in sodium reabsorption, having abundant Na(+)-K(+)-ATPase. That Na(+)-K(+)-ATPase activity was unchanged by either a calcium ionophore or a calcium channel blocker suggests that the decrease in SCC noted in the intact membrane is not directly mediated by changes in the sodium "pump." The decrease of H(+)-ATPase in MR cells, which resulted after the A-23187, suggests that it probably exerts a direct action on the proton pump, which decreases hydrogen ion secretion. The increase in ATP-dependent 45Ca transport seen after the ionophore (or the decrease in ATP-independent 45Ca transport after verapamil) most likely reflects increased (or decreased) Ca2+ availability within the cytosol, and the high (or low) cell calcium could decrease the SCC. These results thus suggest that cytosolic Ca2+ reciprocally sets, by different mechanisms, the rate of proton secretion in MR cells and the sodium reabsorption in G cells.

Ｎａ｀＋´チャネル阻害物質の活性測定用センサに用いたカエルぼうこう膜の貯蔵条件

A simple biosensor for measuring sodium channel blockers, such as tetrodotoxin (TTX) and saxitoxin (STX), and paralytic shellfish poisoning (PSP) consisted of frog bladder membrane, and Na+ electrode. Effects of storage temperature, antiseptic and species of frog on the activity of Na+ channel on the membrane were investigated from the standpoint of practical use.The results obtained were as follows:1) The maximum output of the sensor was obtained at 30 to 33°C and pH 4.8 to 6.0.These values were shown higher in the case of tropical frog than a temperate frog. This was assumed due to the effect of living the environment.2) Frog bladder was able to keep at 5°C during 7 months, but at -30°C during only 80 days.3) Antiseptic effect of NaN3 on the frog bladder membrane was recognized in the concentration of 0.003%.

Characterization and autoradiographic localization of [ 3H]α,β-methylene ATP binding sites in cat urinary bladder

1. 1. The characteristics and distribution of [ 3h]α,β-methylene ATP ([ 3H]α,β-MeATP), a radioligand for P 2x-purinoceptors, binding sites in cat urinary bladder detrusor were examined. 2. 2. Saturation analysis revealed that, in cat bladder membrane preparations, only one population of binding sites with high affinity ( K d =1.8 nM) was present, in contrast to other species where both high-and low-affinity binding sites are present. Another feature is that the density of the binding sites in the cat bladder ( B max =21.2 pmol/mg protein) is considerably higher (about 2-fold) than the high-affinity binding component in the rat bladder membrane preparations. 3. 3. Displacement experiments with unlabelled purinoceptor ligands indicate that [ 3H]α,β-MeATP mainly binds to P 2x-purinoceptors. The order of binding displacement activity was: α,β-methylene ATP, β,γ-methylene ATP>2-methylthioATP>ATP>suramin and l-β,γ-methylene ATP>adenosine. 4. 4. Autoradiographic study demonstrated dense specific binding sites of [ 3H]α,β-MeATP on detrusor smooth muscle of cat bladder. 5. 5. The results of this study are consistent with pharmacological studies for the existence of P 2x-purinoceptors in cat bladder.

Rewarming: comparison of contemporary heat-exchangers

Heat exchange methods must be efficient in order to minimize the patient's pump time. However, comparisons of heat exchangers have been rare. Therefore, the in vivo functions of the most popular, currently available heat exchangers, Sarns, Cobe, Medtronics Maxima, and an experimental model manufactured by Haemonetics were compared. Thirty-two pigs weighing between 63-74 kg were placed on cardiopulmonary bypass with right atrial and ascending aorta cannulation through a right thoracotomy. Thermocouples were placed in the pump tubing before and after the heat exchangers, in the water line before and after the heat exchangers, in the inlet and outlet line of the pump, and the esophagus, brain, bladder, rectum, liver, myocardium, and tympanic membranes of the pigs. They were cooled until the bladder temperature was reduced to 14 degrees C, and maintained at that temperature for 10 minutes. Rewarming was begun until the bladder temperature became 37 degrees C. The pump flow was maintained between 50-60 ml/kg/min with standard ventilation. Cobe, Sarns, Maxima, and Haemonetics heat exchangers were tested and their function determined by comparing the time necessary for rewarming. The Haemonetics heat exchanger required a significant shorter time than the others to rewarm the pigs to normal bladder temperature (Cobe 82.0 +/- 12.0, Sarns 80.3 +/- 15.4, Maxima 89.0 +/- 13.9 Haemonetics 68.7 +/- 13.4, p < 0.05). The principal advantage was seen at the lowest temperatures between the Haemonetics experimental heat exchanger and the other heat exchangers. No statistically significant superiority was seen at higher temperatures. The current heat exchangers are relatively comparable but improved performance is possible with available technology.

Development of an ultra high sensitive tissue biosensor for determination of swellfish poisoning, tetrodotoxin

A simple tissue biosensor for measuring Na + channel blockers such as tetrodotoxin (TTX) and saxitoxin (STX) has been developed. The membrane of frog bladder has Na + channels which control the passage of Na +. It is well known that TTX blocks Na + channels. The tissue biosensor consists of a Na + electrode integrated within a flow cell. The tip of the electrode was covered with frog bladder membrane sandwiched between two sheets of cellulose acetate membrane, and the electrode was set in a flow cell. A solution of 8% NaCl was carried in the cell and the output of the electrode allowed to stabilize. TTX was injected into the sensor system and measured from the inhibition ratio of the sensor peak output. One assay took approximately 5 min. The lower limit of detection was 86 fg. The continuous determination of TTX was feasible for 250 h in the presence of 0·003% NaN 3. A Linear correlation was obtained between TTX activities of F-niphobles and F-parudale determined by the methods of TTX sensor and mouse assay.

604 Regulation of baroreceptor reflex by vasopressin in the hypertensive rats

We synthesized a novel ligand [4,5-3H-Leu9]-Neurokinin A (3H-NKA, S.A 117–144 Ci/mmol), and evaluated its binding to hamster urinary bladder membranes (HUBM). The ligand bound to HUBM in a highly-specific (94 ± 4%) and protein-dependent manner. Binding was rapid (k1 = 0.037 nM−1∗min−1) and saturable (Bmax = 1210 ± 177 fmol/mg protein), to a single population of high-affinity sites (KD = 2.41 ± 0.15 nM, nH = 0.99 ± 0.02). Binding was inhibited by non-hydrolyzable GTP analogs. Competition experiments with HUBM demonstrated the following rank order of potency: NKA > Kassinin > [β-Ala8]-NKA(4–10) > [Nle10]-NKA(4–10) = Eledoisin = NKB > Physaelamin > Substance P. The selective NK-1 and NK-3 ligands, [Sar9-Met (O2)11]-SP, (±) CP96,345 and Senktide respectively, did not inhibit binding at 10 μM, whereas, the selective NK-2 antagonists: (±) SR-48,968 ⪢ L-659,877 > R396 ⪢ MEN-10,207 > MEN-10,376, inhibited binding in a competitive manner. In contrast, the low specific binding (<30%) detected in guinea pig lung membranes, was not inhibited by selective NK-2 ligands. Over 30 ligands (0.1–10 μM) from other receptor classes, were not inhibitory. The data suggest that this new ligand binds with high-affinity and selectivity to homogeneous population of NK-2 receptors on HUBM but not on lung membranes, and is a suitable ligand to study NK-2 receptors.

3H][β-Ala 8]neurokinin A-(4–10): a novel, selective radioligand for the tachykinin NK 2 receptor

The binding characteristics of the novel radioligand [ 3H][β-Ala 8]neurokinin A-(4–10) were assessed in hamster urinary bladder membranes. This labelled compound bound in a reversible, highly specific and concentration-dependent manner to a single class of high affinity binding sites with a K d of 1.8 ± 0.2 nM and a B max of 139 ± 21 fmol/mg of protein. Specific binding of [ 3H][β-Ala 8]neurokinin A-(4–10) was displaced only by NK 2, but not by NK 1 or NK 3, tachykinin receptor agonists and antagonists. Neurokinin A, [β-Ala 8]neurokinin A-(4–10), L 659877 [cyclo(Leu-Met-Gln-Trp-Phe-Gly)], MEN 10376 (H-Asp-Tyr- d-Trp-Val- d-Trp- d-Trp-Lys-NH 2), MEN 10627 [cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2β-5β)]and SR 48968 [( S)- N-methyl- N-[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide]displaced the binding with K i values of 0.4 ± 0.1 nM, 1.9 ± 0.36 nM, 3.05 ± 0.1 nM, 7.9 ± 0.4 μM, 0.36 ± 0.02 nM and 2.5 ± 0.9 nM, respectively. Functional data, obtained in isolated hamster urinary bladder strips with the newly developed tachykinin NK 2 receptor antagonists (MEN 10627 and SR 48968), showed a good agreement with binding data. This novel radioligand could represent a new useful tool for the assessment of tachykinin NK 2 receptor antagonists.