Wayne Hoss

Selectivity of pirenzepine in the central nervous system. II. Differential effects of pirenzepine and scopolamine on performance of a representational memory task

The behavioral effects of the two muscarinic antagonists scopolamine and pirenzepine were examined using a representational memory task for rats in a T-maze. Rats were pretrained to a criterion of 100% correct responses for daily sessions of 10 paired-run trials. The training procedures eliminated all neophobic or wary responses, and response times were invariably short (less than 3 s). Following the initial training sessions, guide cannulae were surgically implanted bilaterally over the hippocampus of each animal. Following recovery from surgery, animals were injected with saline (0.5 microliter to each hippocampus), scopolamine hydrobromide (0.5 microliter of a 60 mg/ml solution (30 micrograms) to each side), or pirenzepine (0.5 microliter of a 69.1 mg/ml solution (34.6 micrograms) to each side) according to a fixed schedule. Saline injections aimed between the blades of the dorsal dentate gyrus failed to produce any change in the performance of the memory task. Initial doses of scopolamine, applied to the same area, produced a decrease in the percentage of correct responses as did the initial dose of pirenzepine. In contrast to pirenzepine, scopolamine also produced increases in response times even to the point of defaulted trials (response times greater than 90 s) in some animals following drug injections. Saline injections failed to produce significant impairments on the days following scopolamine injections, although animals receiving pirenzepine injections were still impaired on the two days immediately following the initial pirenzepine injection. Subsequent doses of pirenzepine were ineffective in producing an impairment of performance while scopolamine injections were less effective than the initial dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a noncholinergic nicotine receptor on human phagocytic leukocytes

SummaryA noncholinergic nicotine receptor on human phagocytic leukocytes has been characterized using the binding of3H-(d,l)-nicotine. The average affinity +/-standard deviation of (d,l)-nicotine for the receptor on neutrophils is 36 ± 18 nM (n=6). The binding is saturable with an average of 8.7 x 104 sites per neutrophil. Monocytes and to a lesser extent lymphocytes but not erythrocytes also display specific binding. Bound nicotine is dissociable from the receptor and is not metabolized. Only close structural analogs of nicotine bind to the receptor, which is stereoselective for the (d)-isomer. The receptor can be occupied by (l)-nicotine at concentrations present in the blood of smokers. It is suggested that some of the adverse effects of smoking on leukocyte functions may be mediated by a specific nicotine receptor.

Conversion between configurational states of the muscarinic receptor in rat brain

Reductive alkylation of neural membranes by N-ethyl maleimide (NEM) converts muscarinic acetylcholine receptors from a state of low to high affinity for receptor agonists. Interactions of muscarinic antagonists with the receptor are unaffected by this treatment. Muscarinic receptors from the rat telencephalon in the high agonist affinity state are increased from 34.2 to 53.4% of the total receptor population and the Ki for carbamylcholine inhibition of 3-quinuclidinyl benzilate binding is decreased from 1.2 X 10(-4) to 6.9 X 10(-5) M by NEM treatment.

Competitive interaction of gallamine with multiple muscarinic receptors

Gallamine, a cholinergic antagonist at the (nicotinic) neuromuscular junction possesses antimuscarinic potency in several systems. We report here that gallamine inhibited the binding of [3H] quinuclidinyl benzilate (QNB) in a competitive manner in the brainstem and forebrain of the rat. The occupancy curves derived from these studies suggest that gallamine has widely varying affinities for different subpopulations of muscarinic receptors, a finding which sets gallamine apart from classical muscarinic antagonists such as atropine and QNB. The greatest difference in affinities for gallamine occurred in the brainstem, where the data could be satisfactorily fitted to a two-site model, with 77% of the receptors having high affinity (Kd = 25 nM) and 23% low affinity (93 microM). Further, these affinities displayed rank order correlation with those of carbachol (an agonist), although gallamine has not, so far, displayed agonist (or partial agonist) activity. The finding that antagonists as well as agonists can display multiple affinities for muscarinic receptors suggests that there are fundamental differences among subpopulations of these receptors.

Selectivity of pirenzepine in the central nervous system. I. Direct autoradiographic comparison of the regional distribution of pirenzepine and carbamylcholine binding sites

The binding capacities of the novel antagonist pirenzepine and the agonist carbamylcholine were examined autoradiographically to compare their abilities to reduce the binding of 1-[3H]quinuclidinyl benzilate ([3H]-1-QNB). This technique, which is applicable to any muscarinic ligand, permits a direct comparison between the binding of carbamylcholine and pirenzepine in the same assay. Analysis of the binding curves generated by standard scintillation counting of whole-brain slices indicated that the ligands bound heterogeneously to muscarinic receptors in the brain. Following apposition of slides to tritium-sensitive film, the binding profile for each ligand was examined visually and by microdensitometry. Regional analyses indicated that the agonist carbamylcholine displayed highest potency for thalamic nuclei, lower potency for cortical regions, and the lowest affinity for layers of the hippocampus. The M1-selective ligand pirenzepine displayed the highest potency for the dentate gyrus of the hippocampus, with lower inhibition levels in the cortex, and the lowest levels of inhibition found in the thalamus. The distribution of high affinity agonist sites was found to be distinct from the distribution of high-affinity antagonist binding sites. In a separate assay, the regional inhibition of pirenzepine and scopolamine was compared for the hippocampus and the forebrain. While scopolamine did not distinguish between muscarinic receptor sites in the hippocampus and cortex, pirenzepine inhibited [3H]-1-QNB labeling in the hippocampus significantly greater than in the cerebral cortex, providing additional evidence for the hypothesis that pirenzepine is a selective antagonist.

Analysis of regional variations in the affinities of muscarinic agonists in the rat brain

Abstract The brain stem of the rat has a higher affinity toward muscarinic agonists than does the forebrain. Receptor occupancy curves of both regions of the brain deviate from simple mass-action binding. The characteristics of the binding in each region are compatible with the existence of two non-interacting binding sites, and are not attributable to desensitization or to negatively cooperative binding within a small oligomer; however, the possibility of large oligomers remains to be excluded. The agonist binding data were analyzed by a linear transformation of Scatchard-like inhibition curves of the binding of the antagonist [ 3 H]quinuclidinyl benzilate (QNB). Such analysis, based on a model of two subpopulations of receptors in each area, shows the subpopulations of the brain stem and the forebrain to be distinct. Brain stem: 44% of receptors possess high affinity with dissociation constant for carbachol, K H = 2.8 × 10 −8 M , dissociation constant of low-affinity receptor, K L = 2.3 × 10 −6 M ; forebrain: 41% high affinity, K H = 2.1 × 10 −7 M, K L = 1.7 × 10 −5 M . The data suggest that whole brain contains at least three major muscarinic receptors, which can be distinguished on the basis of their affinities for agonists.

Effect of 2450 MHz microwave energy on the blood—brain barrier to hydrophilic molecules. A. Effect on the permeability to sodium fluorescein

Significantly elevated levels of sodium fluorescein (MW 376) were found only in the brains of conscious rats made considerably hyperthermic (colonic temperatures greater than 41.0 degrees C) by exposure to ambient heat (42 +/- 2 degrees C) for 90 min or 2450 MHz CW microwave energy at 65 mW/cm2 (SAR approximately equal to 13.0 W/kg) for 30 or 90 min. For microwave-exposed rats, fluorescein levels within the cortex and hypothalamus appeared to increase with increasing duration of exposure. This trend was not apparent in the cerebellum or medulla. Exposure to ambient heat resulted in increased fluorescein with the cortex, hypothalamus and medulla, but not the cerebellum, and, in general, ambient heat was not as effective as microwave energy in raising tracer concentrations within the brain. By far the greatest elevation of fluorescein dye in the brain occurred in those animals whose blood-brain barrier had been opened osmotically by intracarotid injection of 10 M urea. It is suggested that increased levels of sodium fluorescein found in the brain tissue of ambient heat and microwave-exposed rats most likely represent technically derived artifact and not a breakdown of the blood-brain barrier.

Opiates Stimulate Low Km GTPase in Brain

Abstract: Low Km GTP hydrolysis in rat brain is stimulated in a concentration‐dependent manner by the opiate alkaloid etorphine, and by the opioid peptide d‐Ala2‐leucine‐enkephalinamide. The opiate antagonist naloxone inhibits the maximal d‐Ala2‐leucine‐enkephalinamide stimulation of the GTPase, also with concentration dependency. The magnitude of maximally stimulated, opioid‐sensitive, GTP hydrolysis is differentially distributed across brain regions. Opioid‐stimulated GTPase may represent one means of identifying a specific type of opioid receptor.

Raman spectral analysis of the 1300 cm−1 region for lipid and membrane studies

Abstract The Raman spectra of fatty acids, fatty acid methyl esters and several membrane lipids are analyzed in the 1300 cm−1 region. The ratio of peak intensities at 1303/1267 cm−1 varies linearly with the ratio of methylene to vinyl groups in the hydrocarbon chain. This parameter should be useful for estimating the degree of unsaturation in isolated lipids and lipids in membranes.

Regional distribution of muscarinic receptors preferring gallamine in the rat brain

The regional distribution of muscarinic receptors recognized by the antagonist gallamine was determined autoradiographically by the ability of gallamine to reduce the binding of [3H]quinuclidinyl benzilate in rat brain slices. The inhibition data obtained from indirect binding assays on whole slices indicated that gallamine distinguished at least two sites with differing affinities. Analysis using a two-site model gave Kh = 0.6 microM, K1 = 10 microM. The regions of highest and lowest affinity for gallamine were apparent qualitatively by visual inspection of the autoradiograms. A number of regions in coronal sections at three different levels were compared by microdensitometry. Gallamine possessed greater overall affinity for the diencephalon and brainstem than for the forebrain. Within the forebrain, the septal nucleus was unique in that it displayed high affinity for gallamine. Within the brainstem, the superior colliculus had the greatest proportion of sites with high affinity for gallamine. In general, the binding profile of gallamine was opposite to that of the antagonist pirenzepine and similar to that of the agonist carbachol, suggesting that gallamine is selective for M2 muscarinic receptors.