Cloning and Expression of Novel Aminoglycoside Phosphotransferase Genes from Campylobacter and Their Role in the Resistance to Six Aminoglycosides.

Nine aph genes, including aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Ig, aph(2″)-If, aph(2″)-If1, aph(2″)-If3, aph(2″)-Ih, aac(6')-Ie-aph(2″)-Ia, and aac(6')-Ie-aph(2″)-If2, were previously identified in Campylobacter To measure the contribution of these alleles to aminoglycoside resistance, we cloned nine genes into the pBluescript and expressed them in Escherichia coli DH5α. The nine aph expressed in E. coli showed various levels of resistance to gentamicin, kanamycin, and tobramycin. Three genes, aac(6″)-Ie-aph(2″)-Ia, aph2″-If1, and aph2″-Ig, showed increased MICs to amikacin, and five aph genes were transferrable.

The SAGES Fundamental Use of Surgical Energy program (FUSE): history, development, and purpose.

BACKGROUND: Adverse events due to energy device use in surgical operating rooms are a daily occurrence. These occur at a rate of approximately 1-2 per 1000 operations. Hundreds of operating room fires occur each year in the United States, some causing severe injury and even mortality. The Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) therefore created the first comprehensive educational curriculum on the safe use of surgical energy devices, called Fundamental Use of Surgical Energy (FUSE). This paper describes the history, development, and purpose of this important training program for all members of the operating room team. METHODS: The databases of SAGES and the FUSE committee as well as personal photographs and documents of members of the FUSE task force were used to establish a brief history of the FUSE program from its inception to its current status. RESULTS: The authors were able to detail all aspects of the history, development, and national as well as global implementation of the third SAGES Fundamentals Program FUSE. CONCLUSIONS: The written documentation of the making of FUSE is an important contribution to the history and mission of SAGES and allows the reader to understand the idea, concept, realization, and implementation of the only free online educational tool for physicians on energy devices available today. FUSE is the culmination of the SAGES efforts to recognize gaps in patient safety and develop state-of-the-art educational programs to address those gaps. It is the goal of the FUSE task force to ensure that general FUSE implementation becomes multinational, involving as many countries as possible.

Identification and pharmacological characterization of a novel inhibitor of autotaxin in rodent models of joint pain.

OBJECTIVE: Autotaxin is a secreted lysophospholipase that mediates the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA), a bioactive lipid mediator. Autotaxin levels in plasma and synovial fluid correlate with disease severity in patients with knee osteoarthritis (OA). The goal of this study was to develop and characterize a novel small molecule inhibitor of autotaxin to inhibit LPA production in vivo and determine its efficacy in animal models of musculoskeletal pain. DESIGN: Compound libraries were screened using an LPC coupled enzyme assay that measures the amount of choline released from LPC by the action of autotaxin. Hits from this assay were tested in a plasma assay to assess inhibition of endogenous plasma autotaxin and subsequently tested for their ability to lower plasma LPA levels upon oral dosing of rats. The best compounds were then tested in animal models of musculoskeletal pain. RESULTS: Compound screening led to the identification of compounds with nanomolar potency for inhibition of autotaxin activity. Studies in rats demonstrated a good correlation between compound exposure levels and a decrease in LPA levels in plasma. The leading molecule (compound-1) resulted in a dose dependent decrease in joint pain in the mono-sodium iodoacetate (MIA) and meniscal tear models and a decrease in bone fracture pain in the osteotomy model in rats. CONCLUSION: We have identified and characterized a novel small molecule inhibitor of autotaxin and demonstrated its efficacy in animal models of musculoskeletal pain. The inhibitor has the potential to serve as an analgesic for human OA and bone fracture.

Determination of ambient ethanol concentrations in aqueous environmental matrixes by two independent analyses.

A new method for the determination of ethanol in aqueous environmental matrixes at nanomolar concentrations is presented and compared to an existing method that has been optimized for low-level alcohol determinations. The new analysis is based upon oxidation of ethanol by the enzyme alcohol oxidase obtained from the yeast Hansenula sp. which quantitatively produces acetaldehyde after reaction for 120 min at 40 °C and pH 9.0. The acetaldehyde reacts with 2,4-dinitrophenylhydrazine forming a hydrazone that is separated from interfering substances and quantified by high-performance liquid chromatography (HPLC) with UV detection at 370 nm. Comparison of initial acetaldehyde concentration with that after enzymatic oxidation yields the ethanol concentration with a corresponding detection limit of 10 nM. Analytical results were verified by intercomparison with a completely independent technique utilizing a solid-phase microextraction (SPME) Carboxen/PDMS SPME fiber. A 12 mL aqueous phase sample was heated at 50 °C for 10 min prior to loading onto the SPME fiber. Extraction of ethanol was performed by introducing the fiber into the headspace above a pH 4.4 buffered sample containing 30% NaCl for 20 min. Samples were agitated during heating and extraction by magnetic stirring at a rate of 750 rpm. The fiber was thermally desorbed for 1 min at 230 °C in the injection port of a gas chromatograph equipped with a flame ionization detector (FID) set at 250 °C. The resulting ethanol detection limit is 19 nM. Results of an intercomparison study between the enzymatic and SPME analyses produced a trend line with a slope of unity demonstrating that methods produced statistically equivalent ethanol concentrations in several natural waters including rainwater, fresh surface waters, and sediment pore waters.

The effect of monoalkyl phosphates and fluoride on dissolution of hydroxyapatite, and interactions with saliva.

The aims were to investigate the effect of monoalkyl phosphates (MAPs) and fluoride on dissolution rate of native and saliva-coated hydroxyapatite (HA). Fluoride at 300 mg/l (as NaF) inhibited dissolution of native HA by 12%, while potassium and sodium dodecyl phosphates (PDP, SDP), at 0.1% or higher, inhibited dissolution by 26-34%. MAPs, but not fluoride, also showed persistence of action. MAPs at 0.5% and fluoride at 300 mg/l were then tested separately against HA pre-treated with human saliva for 2 or 18 h. Agents were applied with brushing to half the specimens, and without brushing to the other half. In control (water-treated) specimens, pre-treatment of HA with human saliva reduced dissolution rate on average by 41% (2 h) and 63% (18 h). Brushing did not have a statistically significant effect on dissolution rate of saliva-coated specimens. In brushed specimens, fluoride significantly increased the inhibition due to 2- or 18-hour saliva pre-treatment. It is hypothesised that brushing partially removes the salivary film and allows KOH-soluble calcium fluoride formation at the surfaces of HA particles. Inhibition was reduced by PDP in 2-hour/non-brushed specimens and in 18-hour/brushed specimens. PDP did not affect dissolution rates in the remaining groups and SDP did not affect dissolution rate in any group. Possible reasons for these variable results are discussed. The experiments show that pre-treatment with saliva can significantly modify results of tests on potential anti-erosive agents and it is recommended that saliva pre-treatment should be a routine part of testing such agents.

In situ randomised trial to investigate the occluding properties of two desensitising toothpastes on dentine after subsequent acid challenge.

OBJECTIVES: The aim of the study was to determine in situ the relative abilities of two desensitising toothpastes to occlude dentinal tubules with or without acid challenge. MATERIALS AND METHODS: The study design was a single centre, randomised, split mouth crossover model examining four treatments over two periods. The primary outcome was the degree of occlusion proffered by two desensitising toothpastes [Sensodyne® Rapid Relief (8% strontium acetate, 1040 ppm sodium fluoride) and Colgate® Sensitive Pro-ReliefTM daily (8% arginine, 1450 ppm sodium monofluorophosphate)], a standard toothpaste (1450 ppm sodium fluoride) and water, after acid challenge. Healthy adult volunteers wore bi-lateral lower buccal appliances each with two dentine sections, receiving two treatments per study period. Samples were brushed twice a day with treatment, with two additional 3-min extra-oral acidic challenges applied ex vivo on days 3 and 4. A secondary outcome was the degree of occlusion attained in the absence of acid challenge. Examiners blinded to the study assessed occlusion by visual score of post-treatment scanning electron microscope images. RESULTS: All 28 participants completed the study. In the absence of acid challenge, occlusion scores for both desensitising toothpastes were similar and significantly better than control scores (p < 0.02). After acid challenge both desensitising toothpastes occluded more effectively than controls; however, occlusion scores for the strontium acetate paste were significantly greater than those of the arginine paste (p < 0.02). CONCLUSIONS: The occluding properties of the strontium acetate toothpaste were significantly more robust after acid challenge than those of the arginine toothpaste. CLINICAL RELEVANCE: Patients with hypersensitivity, regularly imbibing dietary acidic drinks, should be advised that Sensodyne® Rapid Relief provides robust tubule occlusion despite repeated acidic challenges.

Randomized in situ clinical study comparing the ability of two new desensitizing toothpaste technologies to occlude patent dentin tubules.

OBJECTIVE: To compare the ability of two new desensitizing toothpaste technologies (one a 5% NovaMin-based toothpaste and the other an 8% arginine-based toothpaste) to occlude patent dentin tubules in a clinical environment relative to a negative control of water and a control toothpaste after four days of twice-daily brushing and dietary acidic challenges. METHODS: The study design was a single-center, single-blind, randomized, split-mouth, four-treatment, two-period, crossover, in situ clinical study. Healthy subjects wore two lower intra-oral appliances, retaining four dentin samples for four treatment days for each period of the clinical study. Samples were brushed twice daily with a test product (days 1-4), with an additional acidic challenge introduced on two selective days. Scanning electron microscopy (SEM) images were taken of the dentin surface, and dentinal tubule occlusion assessed using a categorical scale. RESULTS: The results demonstrated that the 5% NovaMin toothpaste was statistically superior at occluding patent dentin tubules compared to water (p = 0.009) and the control toothpaste (p = 0.02) at day 4. In contrast, the treatment effect resulting from the 8% arginine toothpaste did not demonstrate the same degree of occlusive propensity, showing no significant difference to the water and control toothpaste at the day 4 time point. CONCLUSION: Application of the 5% NovaMin toothpaste to dentin showed better dentin tubule occlusion and retention abilities in an oral environment under dietary acid challenge conditions, more so than the 8% arginine toothpaste technology. Given modern dietary habits and practices, these results highlight differences in the acid resistance properties of occlusion technologies, and a potential impact on clinical performance.

Effects of pH and acid concentration on erosive dissolution of enamel, dentine, and compressed hydroxyapatite.

The aims of this study were to determine the effects of pH and acid concentration on the dissolution of enamel, dentine, and compressed hydroxyapatite (HA) in citric acid solutions (15.6 and 52.1 mmol l(-1) ; pH 2.45, 3.2, and 3.9), using a pH-stat system. After an initial adjustment period, the dissolution rates of enamel and HA were constant, while that of dentine decreased with time. The dissolution rate increased as the pH decreased, and this was most marked for enamel. To compare substrates, the rate of mineral dissolution was normalized to the area occupied by mineral at the specimen surface. For a given acid concentration, the normalized dissolution rate of HA was always less than that for either dentine or enamel. The dissolution rate for dentine mineral was similar to that for enamel at pH 2.45 and greater at pH 3.2 and pH 3.9. The concentration of acid significantly affected the enamel dissolution rate at pH 2.45 and pH 3.2, but not at pH 3.9, and did not significantly affect the dissolution rates of dentine or HA at any pH. The variation in response of the dissolution rate to acid concentration/buffer capacity with respect to pH and tissue type might complicate attempts to predict erosive potential from solution composition.

Regulation of procollagen metabolism in the pressure-overloaded rat heart.

To determine the molecular events responsible for the disproportionate accumulation of myocardial fibrillar collagens during sustained hypertension, we examined the in vivo rate of procollagen synthesis, collagen accumulation, and intracellular procollagen degradation 1-16 wk after abdominal aortic banding in young rats. These measurements were correlated with tissue mRNA levels for type I and type III procollagen polypeptides. Banded animals developed moderate, sustained hypertension and mild left ventricular hypertrophy. Increased type III procollagen mRNA levels were detected early after banding and persisted for the entire observation period. Disproportionate collagen accumulation without histological evidence of fibrosis was noted within 1 wk after hypertension induction. Fibrillar collagen accumulation at this time point resulted not from a major increase in procollagen synthesis, but rather a marked decrease in the rate of intracellular procollagen degradation. Interstitial fibrosis, however, was observed 16 wk after banding. Type I procollagen mRNA levels were increased six-fold, but only after 16 wk of hypertension. These results correlated well with the results of in vivo procollagen synthesis experiments at 16 wk, which demonstrated a threefold increase in left ventricular procollagen biosynthesis. We conclude that pretranslational as well as posttranslational mechanisms regulate fibrillar collagen deposition in the myocardial extracellular matrix during sustained hypertension.

Alterations in subsidiary pacemaker function after prolonged subsidiary pacemaker dominance in the canine right atrium.

In vivo and in vitro techniques were used to study the functional characteristics of subsidiary atrial pacemaker activity 4 to 11 months after surgical excision of the sinoatrial node region of the dog heart. Characteristics of this long-term subsidiary atrial pacemaker activity were compared with those of sinoatrial node and short-term subsidiary atrial pacemaker activities. Extracellular bipolar electrodes were used to estimate the site of earliest activation and monitor spontaneous rate. Under both in vivo and in vitro conditions, long-term pacemaker activity was located in the same region of the inferior right atrium as was short-term pacemaker activity. Under in vitro conditions, long-term activity was characterized by a reduced sensitivity to acetylcholine and overdrive pacing and by a lack of dependence on beta-adrenergic stimulation compared with short-term activity. Furthermore, long-term pacemaker activity was more sensitive to acetylcholine and less sensitive to norepinephrine (greater than 10(-7) M) compared with sinoatrial node activity. It is concluded that the subsidiary atrial pacemakers that emerge soon after removal of the sinoatrial node are the same as those pacemakers that ultimately assume long-term control of the heart. In addition, after subsidiary atrial pacemakers assume dominant pacemaker function, their dependence on norepinephrine and their response to acetylcholine and overdrive pacing are reduced. These changes facilitate a more stable regulation of atrial pacemaker rhythm and, thereby, make subsidiary atrial pacemaker function more like that of the sinoatrial node.

Distinct expression patterns of insulin-like growth factor binding proteins 2 and 5 during fetal and postnatal development.

Insulin-like growth factors (IGFs), when isolated from serum or tissue fluids, are usually found as part of a protein complex which also contains one of several IGF binding proteins (IGFBPs). Although some IGFBPs have been shown to alter interactions of IGFs with their receptors in vitro and can modify the responses of cultured cells to exogenous IGFs, the in vivo functions of IGFBPs remain unclear. This study examines expression of a recently described IGFBP gene, IGFBP-5, in the rat embryo and fetus and in selected adult tissues. Embryonic IGFBP-5 messenger RNA (mRNA) can be detected as early as embryonic day 10.5 and has an mRNA expression pattern distinct from the previously characterized pattern of IGFBP-2 mRNA expression. Major sites of IGFBP-5 expression during early postimplantation stages of development include the notochord, the floor plate, regions of the surface ectoderm, muscle precursor cells, and specific axial regions of neuroepithelium. Later in development IGFBP-5 mRNA is found in several regions of the central nervous system, including the proliferative zone of the external granule layer of the cerebellum and the mitral neurons of the olfactory bulb, as well as in muscle precursor populations of the developing limb, and in most cells of the anterior pituitary. In addition, only a subset of pituicytes in the adult posterior pituitary express IGFBP-5, which provides the first evidence that this cell population is biochemically heterogeneous. Taken together, these data suggest functions for IGFBP-5 during development of several organ systems.

alpha 2-Adrenergic receptor binding in human platelets: alterations during the menstrual cycle.

We studied the effect of several clinically important variables on the characteristics of alpha 2-adrenergic receptors in human platelet membranes. The number and affinity of the receptor binding sites were determined from radioligand binding experiments, with [3H]yohimbine being the radioligand of choice. Platelets from female subjects had a cyclic variation in the number of alpha 2-adrenergic receptors that coincided with their menstrual cycles. The number of alpha 2-receptors was highest at the onset of menses and dropped to 74% to 79% of that value during the middle of the cycle. In concurrent experiments we did not observe comparable cyclic changes in receptor binding sites in platelets from male subjects. There was no age-dependent alteration in receptor number in a sample of 39 subjects ranging in age from 8 to 80 yr, but the number of alpha 2-receptors in platelets from male and female subjects differed. We also tested the possibility of a circadian rhythm in alpha 2-receptor number but found no cyclic changes as a function of time of day. There was no alteration in alpha 2-adrenergic receptor binding in the platelets from five subjects with Parkinson's disease. Finally, there was no change in receptor affinity as a function of any of the variables tested. These data should apply to the design of further studies on the clinical importance of platelet alpha 2-adrenergic receptors.

Phorbol ester-stimulated 6-keto-prostaglandin F1 alpha in aortas from control and aldosterone-salt rats.

OBJECTIVE: To assess the contribution of protein kinase C to the production of 6-keto-prostaglandin F1 alpha by stimulating protein kinase C directly. RESULTS: Phorbol myristate acetate caused a time-dependent increase in 6-keto-prostaglandin F1 alpha and thromboxane B2. The time course was slower than for norepinephrine-stimulated production of these metabolites, but the pattern was similar, with thromboxane B2 appearing before 6-keto-prostaglandin F1 alpha. The phorbol myristate acetate concentration-response curves for 6-keto-prostaglandin F1 alpha production for control-salt and aldosterone-salt hypertensive rats were equivalent. Staurosporine inhibited phorbol myristate acetate-stimulated 6-keto-prostaglandin F1 alpha production in control-salt and aldosterone-salt hypertensive rats concentration-dependently. The staurosporine median inhibitory concentration for phorbol myristate acetate-stimulated 6-keto-prostaglandin F1 alpha production was twofold greater in aldosterone-salt hypertensive than in control-salt rats, but was similar for norepinephrine-stimulated 6-keto-prostaglandin F1 alpha. CONCLUSION: Activation of protein kinase C results in increases in arachidonic acid metabolites, but alterations in this pathway do not seem to be responsible for the differences observed with norepinephrine-stimulated 6-keto-prostaglandin F1 alpha production. The present data offer some support for the concept of direct coupling between the alpha 1-adrenoceptor and phospholipase A2.

Design and synthesis of 1-heteroaryl-3-(1-benzyl-4-piperidinyl)propan-1-one derivatives as potent, selective acetylcholinesterase inhibitors.

Herein is described the synthesis and structure--activity relationship of a novel series of aromatic and heteroaromatic 3-(1-benzyl-4-piperidinyl)propan-1-one derivatives that display potent and selective inhibition of the enzyme acetylcholinesterase (AChE). 1-(2-Methyl-6-benzothiazolyl)-3-(N-benzyl-4-piperidinyl)propan-1-one hydrochloride, 6d, is one of the most active compounds within this series exhibiting an IC50 for the inhibition of the AChE enzyme equal to 6.8 nM. Compound 6d has shown a dose-dependent elevation of total acetylcholine (ACh) levels in the mouse forebrain with an oral ED50 = 9.8 mg/kg. In addition, in vivo microdialysis experiments in the rat demonstrate that 6d increases extracellular ACh (100% over basal) 1-3 h postdose with an oral ED50 = 4.8 mg/kg.

Novel benzisoxazole derivatives as potent and selective inhibitors of acetylcholinesterase.

A series of N-benzylpiperidine benzisoxazoles has been developed as potent and selective inhibitors of the enzyme acetylcholinesterase (AChE). The benzisoxazole heterocycle was found to be an appropriate bioisosteric replacement for the benzoyl functionality present in the N-benzylpiperidine class of inhibitors. The title compounds were synthesized by alkylating 3-methyl-1,2-benzisoxazoles with an iodo piperidine derivatives as the key step. Benzisoxazoles 1b-j,o displayed potent inhibition of AChE in vitro with IC50's = 0.8-14 nM. Particularly interesting were N-acetyl and morpholino derivatives 1g (IC50 = 3 nM) and 1j (IC50 = 0.8 nM), respectively, which displayed outstanding selectivity for acetyl-over butyrylcholinesterase, in excess of 3 orders of magnitude. N-Acetyl 1g also displayed a favorable profile in vivo. This analog showed a dose-dependent elevation of total acetylcholine in mouse forebrain after oral administration with an ED50 = 2.4 mg/kg. In addition, 1g was able to reverse amnesia in a mouse passive avoidance model at doses of 3.2 and 5.6 mg/kg with an average reversal of 89.7%. Molecular dynamics simulations were used to study the possible binding modes of N-benzylpiperidine benzisoxazoles to AChE from Torpedo californica. Key structural insights were obtained regarding the potency of this class of inhibitors. Specifically, Asp-72, Trp-84, Trp-279, Phe-288, and Phe-330 are implicated in the binding of these inhibitors. The N-benzylpiperidine benzisoxazoles may be suitable compounds for the palliative treatment of Alzheimer's Disease.

5,7-dihydro-3-[2-[1-(phenylmethyl)-4-piperidinyl]ethyl]-6H- pyrrolo[3,2-f]-1,2-benzisoxazol-6-one: a potent and centrally-selective inhibitor of acetylcholinesterase with an improved margin of safety.

A series of N-benzylpiperidines (2a-d, 10) with novel isoxazole-containing tricycles has been prepared. This series has shown potent in vitro inhibition of the enzyme acetylcholinesterase (AChE), with IC50S = 0.33 - 3.6 nM. Compound 2a was the most potent inhibitor with an IC50 = 0.33 +/- 0.09 nM. Derivatives 2a-d and 10 displayed weak in vitro inhibition of butyrylcholinesterase (BuChE) with IC50S = 600 - 23,000 nM. The most selective compound was 2a with a BuChE/AChE ratio in excess of 4 orders of magnitude (> 10,000). Pyrrolobenzisoxazole 2a also displayed a favorable profile in vivo. In microdialysis experiments, 2a produced a 200% increase in extracellular levels of acetylcholine (ACh) at a dose of 0.4 mg/kg in freely moving, conscious rats. Peripheral side effects (salivation ED50 = 26 +/- 1.5 mg/kg) and acute lethality (LD50[1 h] = 42 mg/kg) were observed at > 60-fold higher doses. These data indicate that 2a is an AChE inhibitor with good central selectivity and a favorable margin of safety. Compound 2a, designated as CP-118,954, is currently in clinical development for the treatment of cognitive disorders.

Glutamate-induced delta-catenin redistribution and dissociation from postsynaptic receptor complexes.

Delta-catenin (or neural plakophilin-related arm-repeat protein/neurojungin) is primarily a brain specific member of the p120(ctn) subfamily of armadillo/beta-catenin proteins that play important roles in neuronal development. Our previous studies have shown that the ectopic expression of delta-catenin induces the formation of dendrite-like extensions and that the overexpression of delta-catenin promotes dendritic branching and increases spine density. Here we demonstrate that delta-catenin displays a dendritic distribution pattern in the adult mouse brain and is co-enriched with postsynaptic density-95 (PSD-95) in the detergent insoluble postsynaptic scaffolds. Delta-catenin forms stable complexes with excitatory neurotransmitter receptors including ionotropic N-methyl-D-aspartic acid receptor 2A (NR2A), metabotropic glutamate receptor 1alpha (mGluR1alpha), as well as PSD-95 in vivo. In cultured primary embryonic neurons, delta-catenin clusters co-distribute with filamentous actin and resist detergent extraction. In dissociated hippocampal neurons overexpressing delta-catenin, glutamate stimulation leads to a rapid redistribution of delta-catenin that can be attenuated by 6-cyano-7-nitroquinoxaline-2,3-dione and dizocilpine, selective inhibitors of ionotropic glutamate receptors. Upon glutamate receptor activation, delta-catenin becomes down-regulated and its association with NR2A and mGluR1alpha in cultured neurons is diminished. These findings support a possible functional connection between delta-catenin and the glutamatergic excitatory synaptic signaling pathway during neuronal development.

Effects of alpha 2-adrenergic agonist preincubation on subsequent forskolin-stimulated adenylate cyclase activity and [3H]forskolin binding in membranes from HT29 cells.

alpha 2-Adrenergic agonist preincubation resulted in a leftward shift in the subsequent concentration-response curve to forskolin-stimulated adenylate cyclase activity in membranes from HT29 cells, a human colon adenocarcinoma cell line. This effect was much less pronounced than the effect seen in the intact cell cyclic AMP production assays. Removal of GTP from the assay caused a further slight leftward shift in the concentration-response curve. In [3H]forskolin binding experiments, alpha 2-adrenergic agonist preincubation caused a doubling of the maximal number of binding sites (80 vs 31 fmol/mg protein) compared to control. The addition of MgCl2 and NaF to the assay buffer increased control binding 5-fold. With agonist preincubation, there was a further increase in binding in the presence of MgCl2 and NaF which was not significantly different from the appropriate control. Pertussis toxin pretreatment blocked both the leftward shift in the forskolin concentration-response curve and the increase in maximal number of binding sites, indicating that a pertussis toxin sensitive protein is involved in these changes. Activation of cyclic AMP production in the intact cell by cholera toxin followed by norepinephrine preincubation and then stimulation by forskolin resulted in a degree of sensitization similar to that seen in the membrane adenylate cyclase and binding assays. Pertussis toxin also blocked this sensitization. It appears that if the cyclase system is highly activated, then the degree of sensitization is similar in the membrane and intact cell assay.

Ascorbic acid inhibition of alpha-adrenergic receptor binding.

Relatively low concentrations of ascorbic acid inhibited the binding of the alpha-1 adrenergic antagonist [125I]HEAT [DL-[beta(3-iodo-4-hydroxyphenyl)-ethyl-aminomethyl]-tetralone) in rat submandibular gland and rat aorta. However, no inhibition was observed with this ligand in several other tissues, nor with several other ligands in these tissues. The inhibition observed was dependent on the concentration of both the ascorbic acid and the tissue. Maximal inhibition of [125I]HEAT occurred in submandibular gland at 10 microM ascorbic acid with Bmax values reduced 65% and no change in affinity. Ascorbic acid had a greater effect in assays in which less tissue was used, causing a 22% decrease in binding at 46 micrograms/ml, but a 48% decrease in binding at a tissue concentration of 12 micrograms/ml. EDTA prevented the loss of binding normally seen with ascorbic acid at a tissue concentration of 17 micrograms/ml. We suggest that, if an antioxidant is thought to be necessary in an assay system, its effects be carefully examined before routine use.

Effects of dihydropyridine derivatives and anticonvulsant drugs on [3H]nitrendipine binding and calcium and sodium fluxes in brain.

The binding of [3H]nitrendipine to rat cortical membranes was reduced by phenytoin, phenobarbital, and pentobarbital. The IC50 values were 0.09, 0.40, and 0.76 mM respectively. The drugs reduced the apparent binding affinity of [3H]nitrendipine with little effect on the maximum number of binding sites. The inhibitory effects of the drugs were similar in the absence and presence of calcium (4.5 mM). Neither nimodipine (10(-8) to 10(-5) M) nor nifedipine (10(-8) to 10(-7) M) altered the voltage-dependent uptake of 45Ca2+ by synaptosomes from rat cortex. Phenytoin inhibited 45Ca2+ influx, and this inhibition was not altered by nifedipine. Nimodipine and nifedipine (10(-6) M) produced a small inhibition of the voltage-dependent uptake of 24Na+ by synaptosomes. Ethanol, phenytoin or pentobarbital reduced 24Na+ influx, and this action was not altered by nimodipine. Thus, sedative-anticonvulsant drugs reduced the binding of dihydropyridines to brain membranes, but these interactions did not appear to involve either calcium or sodium channels.