On the interaction of drugs with the cholinergic nervous system—IV: Tolerance to oxotremorine in mice: In vivo and in vitro studies

A new procedure was used to follow continuously and simultaneously four systemic effects induced by oxotremorine in mice: salivation, tremor, hypothermia and those measured in the rotarod test. Using an equipotent dose of methoxotremorine, it was found that apart from salivation, the other systemic effects are centrally originated. By comparing the complete dose-response curves for these systemic effects in naive and tolerant mice, it was found that: (1) Salivation is the most sensitive effect in naive mice; (2) All curves shift in parallel to the right on continuous exposure to oxotremorine, in a manner that seems to be dose-dependent; (3) the tolerance to oxotremorine is reversible. Since oxotremorine-tolerant mice were found to be cross-tolerant to various tertiary anti-cholinesterase agents and cholinergic agonists, the involvement of the muscarinic receptor in the tolerance to oxotremorine was investigated, using two different approaches. (1) Continuous blockade of the receptor by scopolamine in vivo prevented tolerance development to oxotremorine; this effect was found to depend on the time of scopolamine administration relative to oxotremorine injection, and on the systemic effect measured. (2) The amount of receptor and its affinity towards a specific ligand were determined in vitro; no significant differences were found between naive and oxotremorine-tolerant animals. The significance of these results in the elucidation of possible tolerance mechanisms is discussed.     

Denervation, supersensitivity and muscarinic receptors in the cat iris.

An attempt was made to establish the molecular basis for denervation supersensitivity in the muscarinic system of the cat iris. Several components of that system, namely cholineacetyltransferase (CAT), acetylcholinesterase (AcChE) and the muscarinic acetylcholine receptor were determined following unilateral extirpation of the ciliary ganglion in cats that resulted in supersensitivity to pilocarpine. Marked decreases in CAT activity (80–90 per cent) as well in AcChE activity (25–60 per cent) were noted, starting on the 4th day following ciliary ganglionectomy. No change occurred in the concentration of the muscarinic receptors, nor in the dissociation constant of the muscarinic antagonist N-methyl-4-piperidyl benzilate (4-NMPB), on days 4, 7, and 14 following denervation. It is concluded that the development of denervation supersensitivity is not underlain by quantitative or qualitative changes in the muscarinic receptors. These and other results suggest, rather, that the mechanism responsible for parasympathetic denervation supersensitivity at the smooth muscle of the cat iris sphincter, must be connected with post-receptorial events.     

Clinical use of network analysis for psychiatric and aged populations

Despite a recent renewal of interest in natural community support networks and self-help groups, there currently exist no systematic therapeutic approaches for working with network systems. Over the past two decades advances in the field of social network analysis have laid the groundwork for its conversion to a clinical tool. This paper illustrates how network analysis can provide quantitative and qualitative data useful in therapeutic interventions and agency planning decisions.The authors wish to thank Joan Mintz, Henry Rajkowski, Alan Laskow, Lynne Stein, John Stern, Mindy Horrow, Alissa Fox, Simeon Feinberg, the Bellevue Geriatrics Unit, and the Murray HIll SRO Project for Their assistance. This research was partially supported by grants from the Ittleson Foundation, the New York Foundation, and the vanAmeringen Foundation.     

The Cultural Context of “Successful Aging” Among Older Women Weavers in a Northern Okinawan Village: The Role of Productive Activity

Basho-fu (Musa liukiuensis) weaving has long been part of the cultural identity of Ogimi villagers and has continued in the village as a cottage industry despite its almost complete disappearance throughout the rest of the Ryukyu archipelago. It has survived largely due to the concerted efforts of a few villagers and is now carried on mainly by middle-aged and older women. Almost every elderly woman in Ogimi has at least some experience in basho-fu weaving, and most still participate in some stage of the production process. Aged women form the bulk of the core group of workers that carries out the labor-intensive u-umi (spooling) and u-biki (fiber-cleaning) activities. Without this core group of elderly women, the whole production process would be in jeopardy. Thus, older women in Ogimi play a key role as valued workers in maintaining the basho-fu production process. In exchange for their continued participation, these elderly women receive symbolic capital in the form of respect and honors, as well as wages for their labor. We argue that participating in traditional basho-fu weaving helps these older women maintain an active engagement with life as healthy and productive members of society, a role that has been culturally sanctioned and has taken on moral import in Japan's rapidly aging society. Interestingly, "doing basho" may be considered one means to help achieve successful aging in this particular cultural context.     

First evaluation of polymerase chain reaction assays used for diagnosis of Mycoplasma genitalium in Russia

Background  Diagnosis of Mycoplasma genitalium is entirely based on nucleic acid amplification tests (NAATs). In Russia, several M. genitalium polymerase chain reaction (PCR) assays have been developed; however, any evaluation of their performance has never been performed.
Objective  To assess the performance of five PCRs developed and currently used in Russia for diagnosis of M. genitalium .
Materials and methods  Vaginal swabs and first voided urine samples (FVUs) from 281 females and urethral swabs and FVUs from 125 males were analysed using three conventional PCRs and two real-time PCRs developed by three Russian companies. As reference tests, a real-time PCR targeting the MgPa adhesin gene was used; positive results were confirmed by two conventional PCRs targeting the 16S rRNA gene and MgPa gene, respectively. For evaluation of detection limits and analytical specificities, a blinded control panel consisting of dilutions of six strains of M. genitalium and 14 other Mycoplasma species was tested.
Results  The prevalence of M. genitalium was 2.5% among females and 9.6% among males. The highest sensitivity (71.4–100% in different specimens) was exhibited by one real-time PCRs. Conventional PCRs from two manufacturers failed to detect M. genitalium in any of the seven positive female FVUs. All tests had a 100% clinical specificity; however, one cross-reacted with Mycoplasma pneumoniae .
Conclusions  Only one of the five Russian PCRs displayed reasonable sensitivity for all specimen types, but the specificities of all assays were high. Accordingly, improvements regarding sensitivity of all the tests are needed. However, larger studies, including other populations, evaluating these assays are crucial.     

Oligomeric structure of muscarinic receptors is shown by photoaffinity labeling: subunit assembly may explain high- and low-affinity agonist states.

The potent muscarinic photoaffinity reagent N-methyl-4-piperidyl p-azidobenzilate (azido-4NMPB) was used to covalently label specific muscarinic binding sites in various brain regions and in the heart. In the cortex and hippocampus, a single specifically labeled protein with an apparent molecular mass of 86,000 daltons was detected by gel electrophoresis. In the medulla pons, cerebellum, and cardiac atria, there was a 160,000-dalton band in addition to the 86,000-dalton polypeptide. Under certain conditions, alkali or hydroxylamine treatment dissociated both macromolecules into a single 40,000-dalton polypeptide. These results suggest that the muscarinic receptor exists in oligomeric forms and that a dimer and tetramer of a basic 40,000-dalton peptide may exist as interconvertible species. We propose a model to explain the biological architecture of the muscarinic receptors and suggest a possible correlation between the azido-4NMPB-labeled polypeptides and the two states of the receptor observed in agonist binding experiments.     

Affinity labeling of muscarinic receptors in rat cerebral cortex with a photolabile antagonist.

Highly potent photoaffinity probes for muscarinic binding sites were prepared by the incorporation of an azido group into the benzilic acid moiety in two compound, 3-quinuclidinyl benzilate (3QNB) and N-methyl-4-piperidyl benzilate (4NMPB). Inactivation of muscarinic sites in rat cortex depends on the formation of a reversible complex with the azides prior to their photolytic conversion to the highly reactive nitrenes. During photolysis, radiolabeled azido-4NMPB interacted specifically and with high affinity (Kd = 1.06 nM) with the muscarinic receptors, and the ligand could be covalently incorporated into a macromolecule of about 86,000 Mr, presumably the muscarinic receptor. The incorporation was almost stoichiometric when compared to determination of receptor density by reversible ligands. Atropine (10 microM) afforded specific protection (greater than 83%) of the receptor against inactivation by azido-[3H]4NMPB. This compound and the other ligands described here (i.e., amino-4NMPB, amino-3QNB, and azido-3QNB) represent powerful potential probes for the biochemical isolation and characterization of muscarinic receptors.     

Effect of Ca2+ on the binding characteristics of muscarinic receptors in rat adenohypophysis--variation during the estrous cycle

The effect of Ca2+ on the biochemical characteristics of muscarinic receptors in the adenohypophysis of male and female rats at the various stages of the estrous cycle was investigated in binding experiments using the specific muscarinic antagonist N-methyl-4-piperidyl benzylate ( [3H]-4NMPB) and the muscarinic agonist oxotremorine. By using Ca2+ chelators such as EGTA, and Ca2+ channel blockers such as D-600, we showed that Ca2+ profoundly alters the binding characteristics of both antagonists and agonists to the muscarinic receptors. In female rats the effect of Ca2+ on antagonist binding is mainly on the maximal binding capacity of the receptors, while changes in the dissociation constants are much more moderate. The effect is expressed in the ability of Ca2+ to expose or to eliminate binding sites as a function of the estrous cycle. In agonist binding, the presence of Ca2+ has a pronounced effect on the proportion of high-affinity binding sites, which parallels the changes induced in antagonist binding throughout the estrous cycle. Interestingly, the natural progression of the cycle from diestrus 2 to the estrous stage undergoes a change identical to that occurring in vitro upon Ca2+ removal. D-600 can completely block the effect of Ca2+ on the binding of both [3H]-4NMPB and oxotremorine. The concentration of D-600 required in order to induce such blocking is also dependent on the estrous cycle. It appears that the progression of the estrous cycle is accompanied by changes in the muscarinic receptors which may in turn be coupled to Ca2+ channels.