Inhibition of urothelial P2X3 receptors prevents desensitization of purinergic detrusor contractions in the rat bladder.

OBJECTIVES: To evaluate whether P2X3 receptors (P2X3R) are expressed in the bladder urothelium and to determine their possible function in modulating purinergic detrusor contractions in the rat urinary bladder. MATERIALS AND METHODS: The expression of urothelial receptors was determined using conventional immunohistochemistry in bladders from normal Sprague-Dawley rats. The urothelial layer was removed by incubation with protamine, and disruption of the urothelium was confirmed using haematoxylin and eosin staining on bladder sections. Open cystometry was used to determine the effects of both urothelial removal as well as intravesical application of a specific P2X3R antagonist on bladder properties from intact and protamine-treated rats. Isometric contractile responses to potassium chloride (KCl) depolarization, electrical field stimulation (EFS) or chemical P2X activation were determined in normal and urothelium-denuded bladder strips, with and without application of the P2X3R antagonist. RESULTS: Immunohistochemical staining showed high expression of P2X3R in the medial and basal layers of the urothelium. Removal of the urothelial layer disturbed normal bladder performance in vivo and eliminated the effects of the P2X3R antagonist on increasing the contractile interval and reducing the amplitude of voiding contractions. Removal of the urothelium did not affect bladder strip contractile responses to KCl depolarization or EFS. Pharmacological inhibition of P2X3R prevented desensitization to P2X-mediated detrusor muscle contractions during EFS only in the strips with an intact urothelium. A concentration-dependent, specific inhibition of P2X3R also prevented desensitization of purinergic contractile responses in intact bladder strips. CONCLUSIONS: In the rat bladder, medial and basal urothelial cells express P2X3R, and specific inhibition of the receptor leads to a more hyporeflexive bladder condition. This pathway may involve P2X3R driving a paracrine amplification of ATP released from umbrella cells to increase afferent transmission in the sub-urothelial sensory plexus and desensitization of P2X1-mediated purinergic detrusor contractions.

Pyruvate formate-lyase-activating enzyme: strictly anaerobic isolation yields active enzyme containing a [3Fe-4S](+) cluster.

Pyruvate formate-lyase-activating enzyme (PFL-AE) from Escherichia coli (E. coli) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S-adenosyl-l-methionine (AdoMet). Although PFL-AE is an iron-dependent enzyme, isolation of the enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center. We report here the first isolation of PFL-AE under nondenaturing, strictly anaerobic conditions. Iron and sulfide analysis as well as UV-visible, EPR, and resonance Raman data support the presence of a [3Fe-4S](+) cluster in the purified enzyme. The isolated native enzyme, but not apo-enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity.

Small angle x-ray diffraction studies on the topography of cannabinoids in synaptic plasma membranes.

In a previous publication, we have described in detail how we used small angle x-ray diffraction to determine the topography of (-)-delta 8-tetrahydrocannabinol (delta 8-THC) in dimyristoylphosphatidylcholine (DMPC) bilayers, and to deduce the conformation of the THC side chain by using the iodo-analog (5'-I-delta 8-THC) in the model membrane. We have now extended our studies to synaptic plasma membrane systems where the cannabinoids are believed to exert part of their pharmacological effects. Synaptic plasma membranes (SPM) were isolated from fresh bovine brains and delta 8-THC was incorporated into the membranes. By comparing the electron density profiles of drug free and drug-containing SPM preparations, we observed an electron density increase due to the presence of delta 8-THC in a region centered at 9.2 A from the terminal methyl groups of the membrane bilayer. In an attempt to dissect the effects of different membrane components on the topography of delta 8-THC, we carried out parallel experiments using membrane preparations from the synaptosomal membrane total lipid extract (TLX) as well as from bovine brain phosphatidyl choline extract (PCX) containing 30 mole percent cholesterol (Chol). Our results regarding the topography of delta 8-THC and 5'-I-delta 8-THC in these lipid membranes show that the TLX bilayer simulates the natural membrane environment very closely whereas in the PCX/Chol bilayer delta 8-THC resides at a location approximately 4 A closer to the membrane interface, similar to that found in our previous study using DMPC model membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

A Molecular Ferromagnet with a Curie Temperature of 6.2 Kelvin: [Mn(C5(CH3)5)2]+[TCNQ]-.

The study of magnetic phase transitions in insulating molecular solids provides new insights into mechanisms of magnetic coupling in the solid state and into critical phenomena associated with these transitions. Only a few such materials are known to display cooperative magnetic properties. The use of high-spin molecular components would enhance intermolecular spin-spin interactions and thus a series of chargetransfer (CT) salts have been synthesized that utilize the spin S = 1 molecular cation, [Mn(C(5)(CH(3))(5))(2)](+) (decamethylmanganocenium). The structure and cooperative magnetic behavior of [Mn(C(5)(CH(3))(5))(2)](+)[TCNQ(-) (decamethylmanganocenium 7,7,8,8-tetracyano-p-quinodimethanide) are reported. This salt is a bulk molecular ferromagnet with the highest critical (Curie) temperature (T(c) = 6.2 K) and coercive field (3.6 x 10(3) gauss), yet reported for such a material.

Gallbladder filling and emptying during cholesterol gallstone formation in the prairie dog. A cholescintigraphic study.

We studied gallbladder bile flow before, during, and after cholesterol gallstone formation in the prairie dog using infusion cholescintigraphy with 99mTc-diethyl iminodiacetic acid. In 18 fasting animals partitioning of bile between gallbladder and intestine was determined every 15 min for 140 min, and gallbladder response to cholecystokinin (5 U/kg X h) was calculated from the gallbladder ejection fraction. Ten prairie dogs were then placed on a 0.4% cholesterol diet and 8 on a regular diet, and the studies were repeated 1, 2, and 6 wk later. The proportion of hepatic bile that entered the gallbladder relative to the intestine varied from one 15-min period to the next, and averaged 28.2% +/- 5.1% at 140 min. Partial spontaneous gallbladder emptying (ejection fraction 11.5% +/- 5.6%) was intermittently observed. Neither the number nor the ejection fraction of spontaneous gallbladder contractions changed during gallstone formation. By contrast, the percent of gallbladder emptying in response to cholecystokinin decreased from 72.1% +/- 5% to 25.9% +/- 9.3% (p less than 0.025) in the first week and was 14.3% +/- 5.5% at 6 wk (p less than 0.01 from prediet values, not significant from first week). Gallbladder filling decreased from 28.2% +/- 5.1% to 6.7% +/- 3% (p less than 0.01), but this change was only observed after 6 wk, when gallstones had formed. This study shows that bile flow into the gallbladder during fasting is not constant; the gallbladder contracts intermittently; gallbladder emptying in response to exogenous cholecystokinin is altered very early during gallstone formation; and gallbladder filling remains unaffected until later stages, when gallstones have formed.

Pigment sludge and stone formation in the acutely ligated dog gallbladder.

We studied the effects of stasis of gallbladder bile in a dog model. Three days after cystic duct ligation, all gallbladders contained sludge, and the mucosa was covered by densely adherent mucus with solid particles 1-4 mm in diameter (gravel). Thirty percent of the animals developed stones (greater than 4 mm), which appeared grossly like human pigment stones and microscopically like condensed biliary sludge. Centrifugation of bile yielded colorless pellets (3.8 +/- 3.2 mg/ml) at day 0 and pigmented pellets (33.1 +/- 11.0 mg/ml) at day 3 (p less than 0.05). Pellets contained 73 +/- 8% mucin by weight. Dissolved mucin in supernatant bile increased from 7.46 +/- 1.19 mg/ml (day 0) to 27.36 +/- 3.05 mg/ml (day 3) (p less than 0.001), while bilirubin concentration decreased from 127 +/- 12 mg/dl (day 0) to 71 +/- 16 mg/dl (day 3) (p less than 0.001). Cholesterol concentration increased but did not reach saturation, while the concentration of bile salt and phospholipid did not change. Mucin-bilirubin complexes formed and remained suspended as sludge initially. As bile mucin content increased, sludge particles coalesced, precipitated, and eventually formed gravel and stones. We suspect that stone formation in this setting occurs because of sequestration of biliary lipids by mucin.

Diagnosis and treatment of gastric emptying disorders. Clinical usefulness of radionuclide measurements of gastric emptying.

We studied 53 patients with severe gastrointestinal symptoms thought to be due to a gastric motility disorder. Sixty-six percent had had a previous operation on the stomach, and 21 percent had insulin-dependent diabetes mellitus. Based on clinical, radiographic, and endoscopic findings, 48 patients were thought to have gastroparesis, 3 were thought to have dumping, and 2 had no diagnosis. Measurement of gastric emptying of solids showed that gastric emptying was normal in 12 patients, rapid in 15 patients, and slow in 26 patients. Further evaluation showed that half of the patients with normal gastric emptying, and one third of those with rapid gastric emptying had other diseases of the gastrointestinal tract that responded well to surgery. Of those patients with dumping, diet modification was effective in 40 percent, and half of those who did not respond to dietary manipulations did well after reoperation. Nineteen patients with delayed gastric emptying were treated with metoclopramide. Sixty percent of those without previous gastric surgery responded, whereas only 25 percent of those with previous gastric surgery had good results. The rate of gastric emptying improved following reoperation in 9 (90 percent) of 10 patients with delayed gastric emptying (4 who had not responded to metoclopramide). Gastric emptying was measured again in 15 patients after treatment. The changes after treatment paralleled the clinical response. These studies indicate that gastroparesis cannot be reliably diagnosed on the basis of clinical findings and standard tests. Gastric emptying studies are essential to diagnose and treat patients thought to have gastric motility disorders, and to evaluate the results of therapy.

Development of instruction in hospital electrical safety for medical education.

Although hospital electrical safety is receiving increased attention in the literature of engineers, it is not, at present, reflected in the curricula of medical schools. A possible reason for this omission is that biomedical and/or clinical engineers knowledgeable in electrical safety are not usually trained to teach. One remedy for this problem is to combine the knowledge of engineers with that of instructional developers to design a systematic curriculum for a course in hospital electrical safety. This paper describes such an effort at the University of Texas Health Science Center at San Antonio (UTHSCSA). A biomedical engineer and an instructional developer designed an instructional module in hospital electrical safety; the engineer taught the module, and both evaluated the results. The process and outcome of their collaboration are described. This model was effectively applied in the classroom as a four-hour segment in hospital electrical safety for first-year medical students at UTHSCSA. It is hoped that an additional benefit of this system will be that it offers an opportunity for continuing improvement in this kind of instruction at other medical schools and hospitals.