Central effects of muscarinic agonists and antagonists on hippocampal theta rhythm and blood pressure in the anaesthetised rat.

The in vivo central effects of a range of full and partial muscarinic receptor agonists have been investigated on hippocampal theta rhythm and blood pressure. In the isoflurane-anaesthetised rat, pretreated with N-methylscopolamine, i.v. administration of arecoline, oxotremorine, arecaidine propargyl ester, aceclidine and pilocarpine produced dose-dependent increases in the frequency of hippocampal theta rhythm and blood pressure, with an order of potency of arecoline = oxotremorine = arecaidine propargyl ester greater than aceclidine greater than or equal to pilocarpine. To increase theta wave frequency, pilocarpine showed a low maximum response and possessed antagonist activity against arecoline, indicating that pilocarpine was acting as a partial agonist. AF102B failed to alter blood pressure or theta rhythm. Intraventricular injections of scopolamine and the M1 receptor-selective antagonist, pirenzepine, produced dose-dependent antagonism of the enhanced theta wave frequency and hypertensive response produced by arecoline. The differences in antagonist potency for the two responses was less than 6-fold, which indicated that both the increase in hippocampal theta wave activity and increase in blood pressure may have been mediated through muscarinic receptors of the M1 subtype. Further studies using a wider range of antagonists will be required to confirm this conclusion.

Characterization of muscarinic M4 binding sites in rabbit lung, chicken heart, and NG108-15 cells.

We have carried out an extensive pharmacological characterization of muscarinic binding sites in rabbit lung and chicken heart in parallel with M1, M2, and M3 sites, [3H]Pirenzepine, a selective antagonist at M1 receptors, bound saturably and reversibly to membranes from chicken heart and rabbit lung. These binding sites were not M1 receptors, however, because the cardioselective antagonist himbacine had 10-fold higher affinity at these sites than at [3H]pirenzepine sites in rat and rabbit cortex (true M1 sites). We measured the inhibitory potency of 28 antagonists at [3H]N-methylscopolamine-labeled sites in chicken heart, rabbit lung, rat heart (M2 sites), and rat submandibular gland (M3 sites) and at M1 sites in rat cortex. The sites in rabbit lung were different from M1, M2, and M3 sites, because they had moderate to high affinity for M1-selective compounds (pirenzepine and telenzepine), M2-selective compounds (himbacine and methoctramine), and M3-selective compounds (hexahydrosiladifenidol and 4-diphenylacetoxy-N-methylpiperidine methiodide). The sites in chicken heart resembled most those in rabbit lung, with similar high affinity for secoverine, but they were not the same because tropicamide, diphenylacetoxybutynyl dimethylamine, and [3H]-N-methylscopolamine were more potent in rabbit lung. In a further series of experiments, we compared the affinity of six of the most discriminating antagonists in membranes from rabbit lung and NG108-15 cells, a neuroblastoma-glioma cell line reported to express the muscarinic m4 receptor gene. The antagonists had very similar affinities in the two tissues, the largest discrepancy being that pirenzepine was twice as potent in rabbit lung as in NG108-15 cells. Northern blots using probes designed to discriminate between five species of muscarinic receptor RNA detected only m4 mRNA in rabbit lung. We conclude that rabbit lung contains a muscarinic M4 binding site with a quite distinctive pharmacology and that chicken heart contains a receptor with similarities to the M4 sites. This is the first report to characterize native M4 binding sites in a nonneuronal mammalian tissue.

Functional and binding studies with muscarinic M2-subtype selective antagonists.

1. The potency of a series of selective muscarinic antagonists has been measured on two functional isolated tissue preparations (rat ileum and atria) and these compared with their potency on a range of binding preparations in order to determine whether the subtypes of M2 receptor measured functionally are the same as those measured in binding studies. 2. On the functional preparations pirenzepine, hexahydrosiladiphenidol (HSD) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were more potent on the ileum than on the atrium (3 fold, 29 fold and 5 fold respectively), whereas himbacine, AF-DX 116 and methoctramine showed the opposite selectivity (5 fold, 3 fold and 56 fold respectively). Atropine had a similar potency on the ileum and atrium. 3. [3H]-N-methyl scopolamine was used to study M2 binding sites on membranes from rat heart and rat submandibular gland. Each preparation appeared to contain a homogeneous binding site population. The potencies of the five M2 selective antagonists (and pirenzepine) in binding studies to heart membranes were very similar to those observed in functional studies of rat atria (correlation coefficient = 0.98). Similarly the binding to submandibular gland membranes was very similar to that observed in functional studies on rat ileum (correlation coefficient = 0.97). 4. [3H]-pirenzepine was used to examine the binding of these antagonists to M1 binding sites on membranes from rat cerebral cortex. The affinities of 4-DAMP, HSD, AF-DX116 and himbacine at M1 sites were similar to their affinities on the gland. Only pirenzepine and methoctramine had higher affinity on M1 sites than on the gland. 5. Himbacine had a 20 fold lower affinity at M1 binding sites than at heart sites, and it should therefore be an important tool in identifying M1 sites. 6. Inhibition of [3H]-N-methyl scopolamine binding to rat ileum and rat brainstem by M2-selective antagonists was best described by a two-site model. In both cases the major population of sites (70-90%) appeared to be similar to sites found on the heart (correlation coefficients = 0.95 and 0.97). The other site appeared to be similar to that on the submandibular gland (correlation coefficients = 0.96 and 1.00). 7. The correlations observed in these studies in which a range of selective muscarinic antagonists was used lend weight to previous studies indicating the presence of three functionally important muscarinic receptor subtypes, typified by the binding sites studied in the cerebral cortex, submandibular gland and heart. 8. We propose that the sub-classification of the M2 muscarinic receptor into M2 and M3 subtypes on the basis of ligand binding studies should be extended to cover functionally-defined receptors as well.

The effect of pirenzepine on spatial learning in the Morris Water Maze.

The effects of the selective M1-muscarinic antagonist, pirenzepine, were studied on the Morris Water Maze, a test of spatial learning in the rat. Pirenzepine (0, 10 or 30 micrograms) was administered into lateral ventricle during acquisition of this task. Although 30 micrograms of pirenzepine impaired acquisition of the spatial aspects of the task, treated animals still appeared to be able to acquire a taxon strategy. A low dose of pirenzepine (10 micrograms) produced a slight deficit but this was only visible in a "spatial probe" trial. Although these results are consistent with the belief that muscarinic M1-receptors are involved in spatial learning, it cannot be excluded that the effects recorded were mediated by muscarinic M2-receptors, due to the low selectivity of pirenzepine.

The anaerobic decomposition of benzoic acid during methane fermentation. III. The fate of carbon four and the identification of propanoic acid.

Anaerobic rupture of the benzoic acid ring was investigated. Carbon 4 was converted primarily to carbon dioxide. Following ring rupture during methane fermentation, propanoic acid is an intermediate, and carbon 4 of benzoate becomes its carboxyl.

Routine oblique radiography of the pediatric lumbar spine: is it necessary?

A series of 86 pediatric lumbar spine abnormalities was evaluated to determine the diagnostic benefit of radiography in oblique projection as compared to frontal-lateral projections alone. In only four patients was an abnormality apparent on the oblique view which had not already been demonstrated by the frontal-lateral series; each of these represented an isolated spondylolysis. Because the diagnostic yield was low at a patient cost of more than double the gonadal radiation dose, it is recommended that oblique views be eliminated in the routine radiography of the pediatric lumbar spine.

Osteolytic lesions following traumatic pancreatitis.

We present a case of osteolytic lesions secondary to medullary fat necrosis associated with pancreatitis. The bone lesions are usually secondary to pancreatic trauma and evaluation of possible child abuse is indicated. The roentgenographic findings may not be seen until three to four weeks after the onset of pancreatic disease, and must be differentiated from osteomyelitis and traumatic periostitis. The presence of multiple lesions in many bones without substantial clinical signs of infection should suggest the pancreas as the primary problem.

Familial neuroblastoma presenting as multiple tumors.

Multiple primary tumors are a common mode of presentation in familial neuroblastoma but must be differentiated from metastatic disease. The cases of 2 siblings with multiple neuroblastomas are presented and 44 additional cases of neuroblastoma reviewed. It was found that primary tumors localize to the posterior mediastinum, the adrenals, and the paravertebral ganglia whereas metastatic disease is found in the anterior and middle mediastinum, bones and para-aortic nodes. Based on these observations, the diagnosis of multiple primary tumors can be made and should lead to early consideration of familial neuroblastoma.

Some singular properties of bacterial flagella, with special reference to monotrichous forms.

Roberts, F. F., Jr. (University of Maryland, College Park), and R. N. Doetsch. Some singular properties of bacterial flagella, with special reference to monotrichous forms. J. Bacteriol. 91:414-421. 1966.-Heat (60 C for 30 min), 10 m acetamide, and 8 m urea all brought about rapid and complete dissolution of flagella from monotrichous bacteria; hence, these flagella respond similarly to those of peritrichous forms. Chloramphenicol (10(3) mug/ml) inhibited regeneration of flagella in all peritrichously flagellated cultures; however, monotrichous forms were able to regenerate their flagella in a concentration 10(2) times that required to inhibit multiplication. Peritrichous bacteria did not synthesize flagella when infected by lytic bacteriophages. In these experiments, the time from infection to lysis was sufficient for uninfected controls to resynthesize their flagella. Monotrichous bacteria, however, in all but one instance, were able to resynthesize their flagella before lysis occurred. A study of flagella resynthesis in a non-nutritive milieu indicated that only a small amount of flagellum precursor is present in any given cell. The effect of temperature on synthesis of flagella indicated that, although some bacteria multiply and are motile at a given temperature, they are unable to resynthesize their flagella at that same temperature. This strongly suggests that initial flagellum synthesis and flagellum regeneration are not necessarily identical processes.