Pseudo-noncompetitive antagonism of muscarinic receptor-mediated cyclic GMP formation and phosphoinositide hydrolysis by pirenzepine.

Pirenzepine selectively antagonized muscarinic receptor-mediated cyclic GMP formation in a noncompetitive fashion in mouse neuroblastoma cells (clone N1E-115). These effects of pirenzepine were time- and concentration-dependent and they were also reversible. Interestingly, whereas atropine elicited competitive antagonism of the cyclic GMP response at low concentrations, it also behaved like a noncompetitive antagonist at higher concentrations and its effects were partially reversible. Using additional approaches to study the mechanisms underlying this anomalous antagonistic profile of pirenzepine, we investigated whether this deviation from competition could be due to the short time of exposure to muscarinic agonists (30 sec) used in cyclic GMP measurements. Our data indicated that the mode of pirenzepine-induced antagonism of ligand binding to muscarinic receptors was different when assessed using nonequilibrium (30 sec) or equilibrium (1 hr) incubations. Thus, pirenzepine appeared to be noncompetitive and competitive under these two conditions, respectively. Furthermore, although pirenzepine blocked receptor-mediated phosphoinositide hydrolysis competitively when the response was measured at 20 min, it was clearly noncompetitive using 5-min incubations. Therefore, the noncompetitive antagonism by pirenzepine detected in cyclic GMP measurements might be only apparent and might be attributed, at least in part, to a lack of an equilibrium state under the specific conditions of these assays.

Effects of chronic exposure to ethanol on the prostaglandin E1 receptor-mediated response and binding in a murine neuroblastoma clone (N1E-115).

With the use of cultured murine neuroblastoma cells (clone N1E-115), the authors studied the effects of chronic ethanol on prostaglandin E, (PGE1)-mediated cyclic AMP formation, adenylate cyclase activity and [3H]PGE1 binding. Whereas acute exposure of these cells to ethanol potentiates the PGE1 response, exposure of cells, for as little as 1 day, to 100 mM ethanol resulted in a diminished responsiveness to PGE1 compared with that in acutely treated cells. This apparent tolerance was well developed by day 4, and, by day 7, treated cells had a diminished response to PGE1 when assayed in the absence of ethanol. To achieve the same level of PGE1-mediated cyclic AMP synthesis as acutely exposed cells, chronically exposed cells required higher concentrations of ethanol. With 7 to 10 days of treatment, there was a modest (10-13%) increase in basal, PGE1- and forskolin-stimulated adenylate cyclase activity in membranous preparations, a 28 to 40% increase in high-affinity [3H]PGE1 binding to membranes with no change in Kd or in the ability of 5'-guanylimidodiphosphate to reduce this binding and a 155% increase in [3H]PGE1 binding to intact cells with no change in Kd. Thus, chronic exposure of N1E-115 cells to ethanol resulted in tolerance to its effects on the PGE1 receptor system, and this tolerance was accompanied by apparently paradoxical changes in PGE1-stimulated cyclic AMP synthesis and [3H]PGE1 binding.

Acute effects of ethanol and other short-chain alcohols on the guanylate cyclase system of murine neuroblastoma cells (clone N1E-115).

The acute effects of ethanol were studied on the guanylate cyclase system of cultured murine neuroblastoma clone N1E-115. Using intact cells, we found that although ethanol had no effect on basal levels of cyclic GMP synthesis, it rapidly inhibited in a concentration-dependent manner cyclic GMP synthesis mediated by the agonists histamine (histamine H1 receptor) and carbachol (low-affinity muscarinic receptor) and by ionophore X537A and melittin, agents which bypass these receptors. At 200 mM ethanol, inhibition was about 40 to 50% with the agonists, X537A and melittin. Ethanol had no effect on the high-affinity muscarinic receptor, that mediates inhibition of cyclic AMP synthesis. With carbachol ethanol's inhibition was reversible and was a mixed competitive/noncompetitive type. For a series of alcohols, inhibitory potency with carbachol correlated with chain length directly. In addition, sucrose and sodium chloride, which like ethanol increases the osmolality of the incubation medium, mimicked the effects of ethanol. In a crude cellular homogenate, ethanol and other alcohols inhibited both basal and sodium nitroprusside-stimulated guanylate cyclase activity. The effect of ethanol on basal enzyme activity was noncompetitive. Thus, the inhibition by ethanol and other alcohols of receptor-mediated cyclic GMP synthesis appears to be at the level of guanylate cyclase.

Inhibition by ethanol of forskolin-stimulated adenylate cyclase in a murine neuroblastoma clone (N1E-115).

Forskolin, a diterpene activator of adenylate cyclase, stimulated the formation of cyclic AMP in intact murine neuroblastoma clone N1E-115 cells and stimulated adenylate cyclase activity in a membranal preparation from these cells. Ethanol caused a concentration-dependent inhibition of the forskolin-stimulated responses in both preparations. In intact cells, the inhibition appeared to be noncompetitive. However, in the membranal preparation the inhibition was more of a competitive nature. In addition, there was also a large difference in the amount of inhibition in the two systems. Thus, the inhibition by ethanol was nearly twice as much with intact cells as with membranes. Sucrose appeared to mimic these effects of ethanol, suggesting that with intact cells the effect of this alcohol may be due, in part, to changes in cellular osmotic pressure.

Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115). Mediation of separate responses by high affinity and low affinity agonist-receptor conformations.

Murine neuroblastoma cells (clone N1E-115) possess muscarinic receptors that mediate multiple responses, including the elevation of cyclic GMP levels and the inhibition of receptor-mediated increases in cyclic AMP. Evidence is presented showing that two muscarinic agonist-receptor conformations in N1E-115 cells each separately mediate a cyclic nucleotide response. Pirenzepine inhibited the [3H]cyclic GMP response to carbachol with a KD value of approximately 6 nM, whereas it inhibited the ability of carbachol to reduce prostaglandin E1-mediated elevations in [3H]cyclic AMP levels with a KD value of 93 nM, thus differentiating between two classes of receptors involved in these responses. Ten muscarinic agonists were studied for their ability to mediate the two cyclic nucleotide responses. Six were as effective as acetylcholine in the reduction of [3H]cyclic AMP levels, but only two were as effective as acetylcholine in elevating [3H]cyclic GMP levels. Four agonists (arecoline, pilocarpine, oxotremorine, and McN-A343) were ineffective in increasing [3H]cyclic GMP levels. These four agonists and bethanecol, which could increase [3H]cyclic GMP levels only 18% as well as acetylcholine, behaved as competitive antagonists in this response to carbachol. These partial agonists, in contrast to carbachol, bound to only one class of muscarinic sites in N1E-115 cells with equilibrium dissociation constants determined by competition binding assays which agreed well with their respective EC50 values for their effect on [3H]cyclic AMP levels. The equilibrium dissociation constants for the partial agonists determined by their inhibition of carbachol in the [3H] cyclic GMP response also agreed well with their respective EC50 values for mediating the [3H]cyclic AMP response. Thus, the partial agonists bound to the same receptors at which carbachol mediated [3H]cyclic GMP formation, but with KD values about the same as their respective EC50 values for inhibition of prostaglandin E1-mediated [3H]cyclic AMP increases. The full agonists acetylcholine and methacholine, like carbachol, bound to two sites in N1E-115 cells. For the six agonists able to stimulate both responses at least to some degree, the ratio of their potencies at each response correlated with their respective efficacies at each response but with much more dependence in the [3H]cyclic GMP response.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115). Selective loss with subculturing of the low-affinity agonist site mediating cyclic GMP formation.

Cells of the murine neuroblastoma clone N1E-115 possess muscarinic receptors that influence the intracellular level of cyclic nucleotides. The stimulation of [3H]cyclic GMP levels occurs only with intact cells and has an EC50 near the "low-affinity" agonist equilibrium dissociation constant (KL) determined by radioligand binding assays. The inhibition of prostaglandin E1-stimulated [3H]cyclic AMP formation has an EC50 close to the value for the "high-affinity" agonist equilibrium dissociation constant (KH). During sequential subculturing in medium supplemented with newborn bovine serum, the inhibition of [3H]cyclic AMP was maintained, but the [3H]cyclic GMP response declined dramatically, and after 7 subculturings it was essentially absent. The time course for [3H]cyclic GMP formation in a late subculture with an 88% loss of the response was identical with the time course in early subcultures. A normal [3H]cyclic GMP response to bradykinin and histamine was demonstrated to be present in cells that had lost the [3H]cyclic GMP response to carbachol. The EC50 and KD values for the two muscarinic responses and binding sites increased 3- to 4-fold after several subculturings. A 90% loss of low-affinity binding sites was closely correlated with a similar loss of the [3H]cyclic GMP response. High-affinity binding sites did not decline significantly in concentration until the 11th subculture, where the total number of muscarinic sites was only 6% of the earliest subculture. In all subcultures, however, the ability of the muscarinic receptor to decrease [3H]cyclic AMP levels was maintained. These data, which show that the subculturing of N1E-115 cells in medium supplemented with newborn calf serum results in a selective loss of one muscarinic function, strongly support the hypothesis that these cells contain two separate muscarinic receptor-effector systems. One receptor subtype or conformation has a low affinity for the agonist and mediates cyclic GMP formation. The other receptor subtype or conformation has a higher affinity for the agonist and mediates an inhibition of prostaglandin E1-stimulated cyclic AMP formation.

A study on skin humidity in leprosy patients using a new type of humidity meter.

The Vaisala Humicap humidity meter was used to estimate the moisture of the skin. The one-minute relative evaporation (OMRE) was regarded as a manifestation of the skin humidity and assessed in areas with and without sensation. When the sweat glands were inactive, the humidity on the whole was the same in both areas. After they were activated by sun exposure, there was a great rise in humidity in the sweating areas, but the nonsweating areas also had a marked increase. However, after 20 minutes' rest indoors, the patients' humidity returned from the increased level to the level before sun exposure. Soaking the feet, followed by Vaseline rubbing, did not give more hydration than Vaseline rubbing alone. Thus foot soaks have no rational basis for restoring dry skin to normal. Application of ordinary zinc oxide adhesive tape, on the other hand, proved to be a very effective way to hydrate the skin, but for practical use only in limited areas. The urgent need of an "extra skin" for the soles and margins of the soles was stressed for two vital reasons: to prevent dryness and to provide protection.

Function and regulation of methionine5-enkephalin and its receptors in murine neuroblastoma cells.

A homogeneous class of enkephalin receptors found in murine neuroblastoma clone N1E-115 (Chang, K.-J., and Cuatrecasas, P. (1979) J. Biol. Chem. 254, 2610-2618) has been confirmed using a centrifugation assay employing cellular membranes. In intact N1E-115 cells, synthetic methionine5-enkephalin inhibited prostaglandin E1-induced intracellular cyclic AMP formation in a naloxone-sensitive manner. Upon demonstrating intracellular methionine5-enkephalin immunocytochemically (Knodel, E., and Richelson, E. (1980) Brain Res. 197, 565-570), analyses of crude N1E-115 extract were made by radioimmunoassay or opiate receptor binding assay following fractionation by molecular sieve chromatography and high pressure liquid chromatography on a mu-Bondapak C18 column. Extracted methionine5-enkephalin immunoreactive material behaved similarly to synthetic methionie5-enkephalin in these analyses. Growth curve studies of the N1E-115 cells indicated that the quantity of methionine5-enkephalin immunoreactive material synthesized per milligram of cellular protein and the maximum number of enkephalin receptor sites per milligram of membrane protein increased as the cells progressed from logarithmic to stationary phase, with no change in the apparent affinity of the enkephalin receptors for [3H]methionine5-enkephalin. These data suggest that adrenergic clone N1E-115 has functional methionine5-enkephalin membrane receptors, that this clone synthesizes methionine5-enkephalin, and that both the enkephalin receptor number and the content of stored methionine5-enkephalin are regulated with respect to cell division.

Mandibular reconstruction with bone grafts.

A total of 23 patients with partly resected mandible were repaired with autogenous bone grafts from the iliac crest and ribs. The reasons for reconstruction are presented in Table 1. The grafts healed without complications in 21 cases. With the exception of two of the cases with gunshot wounds, all patients recovered good mobility of the jaw and satisfactory mandibular contours. The patients had been folloed up for 6 months to 11 years. Radiographic examination at the last examination showed that resorption of the graft had been only slight or moderate. Through marrow-spongious bone grafts are regarded as best from an osteogenetic point of view, our cases showed that good results can be achieved also with solid block grafts. The authors discuss the use of plate osteosynthesis without IMF instead of other types of graft fixation and IMF. In six cases where the area of the graft was loaded with a prosthesis, resorption was not more extensive than in the other cases.

Maxillary growth after palatal surgery. An experimental study on dogs.

Maxillary growth after unilateral closure of surgically induced defects in the hard palate in 6- to 8-week old Beagle puppies was studied. The hard palate except for a 4 mm wide strip of bone in the midline and with its overlaying oral mucoperiosteum was removed. On one side the nasal mucoperiosteum was covered with an autogenous full-thickness skin graft (side SGS) and, on the other side the raw surface (side RS) was left for secondary epithelialization. The animals were killed at 47 to 52 weeks of age. Measurements on the dried skulls of the experimental dogs showed no difference in total maxillary length between the two sides of the maxilla. However, the posterior half of the maxilla was longer in 8 dogs on side SGS and the anterior half of the maxilla was in 7 dogs long on side RS. The palatine suture was displaced towards side RS on all dogs, and the height of the nose was in all dogs greater on side SGS. There was a small consistent tendency that the overall growth was more pronounced on the side with the full-thickness skin graft. It is concluded that reducing the amount of scar tissue by covering raw surfaces with an autogenous full-thickness skin graft is one way to reduce maxillary growth impairment after palatal surgery.

Correction of cleft lip nasal deformity. A refinement of an older method.

A method to correct the cleft lip nasal deformity is described. It consists of complete mobilization of the alar cartilage, and keeping it in position with two lifting thread loops which are anchored to the dorsal part of the septal cartilage.

S-shaped repair of the unilateral cleft lip.

A modification of Tennison's operation for closure of a cleft lip is presented. The modification entails the use of X-shaped incision lines and a piece of lead rather than a wire.

Leprosy wound healing with ordinary adhesive tape. A preliminary report.

The use of adhesive tape as wound treatment in leprosy cases is described and found to be superior to the classical dressing as regards security and facility of application, and for the rapidity and quality of healing. Its use as a "preventive" treatment is stressed. The results of this study seem to justify the following conclusions: The adhesive tape is easy to handle, it heals the leprosy wounds in about half the time necessary for the classical dressing and it costs about 50 times less.

Homologous vessel graft as substitute for flexor tendon graft. An experimental study on rabbits.

Homologous vessel grafts were tried as substitutes for flexor tendon grafts. Pieces of different lengths were excised from a flexor tendon and replaced by a homologous abdominal aorta graft which was turned inside out and then anastomised by drawing it over the tendon stumps in a cuff-like manner. The grafts became firm, homogeneous and completely tendon-like with very little adhesion but with a tendency to become extended and thus too long to be functional.