Effects of tricyclic antidepressants on muscarinic cholinergic receptor binding in mouse brain

Tricyclic antidepressants (TADs) were administered (10 mg/kg/day, i.p.) to mice for 2 or 4 weeks. Tolerance to the antimuscarinic effects of these agents was demonstrated by comparing their ability to supress oxotremorine-induced tremors in treated and in control animals. ED50's increased nearly three-fold after four weeks of treatment. CNS muscarinic acetylcholine receptor binding was also examined after 2 to 7 weeks of treatment by measurement of 3H-quinuclidinyl benzilate (QNB) binding. No change was found in either density or affinity of these receptors. The development of tolerance to the antimuscarinic effects of TADs is not due to alteration of either the number or the conformation of central muscarinic receptors. Evidence is presented that this phenomenon may instead be the result of an unidentified mechanism by which the post-synaptic effect of a single receptor-agonist interaction is magnified.     

Enhanced sensitivity of trisomy 21 monocytes to the maturation-inhibiting effect ot interferon

Cultured peripheral blood monocytes from subjects with trisomy 21 (Down Syndrome) demonstrated a 3.7-fold enhanced sensitivity to the maturation-inhibiting effect of leukocyte interferon. This increased sensitivity is considered to be the result of the presence of an increased concentration of the interferon receptor, which is controlled by the IfRec locus on human chromosome 21, on the surface of the trisomic monocytes. Since macrophages are important components of many immune processes and interferon is itself a product of and has regulatory functions in immune reactions, the enhanced sensitivity of trisomic monocytes to interferon may be a factor leading, paradoxically, to the greater susceptibility of trisomic individuals to viral and bacterial infections.     

"Stop and go" kinetics of receptor movements induced by anti-Ig or antigen on individual lymphocytes.

Serial observations were conducted on the time course of surface immunoglobulin (Ig) redistribution (capping) on individual mouse spleen lymphocytes. Capping of surface Ig by anti-Ig fluorescein and also antigen-induced capping of receptors on specific sheep erythrocyte antigen-binding cells were observed and the times required for individual cells to clear 90 and 180 ° of their circumference were recorded. There were striking differences between individual cells in both the onset and duration of receptor movements. Although the number of cells achieving complete clearing of first and second quadrants in successive time intervals declined, there was no correlation between the time required by a cell to clear the first quadrant and the time required by the same cell to clear the second quadrant. Thus, instead of observing gradual progressive migration of marker toward one pole of each individual cell at a rate resembling that of the whole population, we observed grossly discontinuous receptor movements, characterized by brief major shifts in receptors followed by a period of relative stability. Capping is thus viewed as a series of discrete contractile events related to the activity of membrane-associated cytoskeletal elements rather than a manifestation of “membrane flow”.     

Physiology of receptor capping induced by erythrocyte surface antigens

The capping of antigen-binding cell receptors by bound sheep erythrocytes (SRC) demonstrates that antigen mounted on a cell surface can generate a signal leading to the capping reaction. SRC-induced capping of ABC is: (a) highly dependent on both aerobic and anaerobic glycolysis, (b) unaffected by agents altering intracellular cyclic nucleotide concentrations, (c) slightly more vigorous in strain A than in CBA mice, (d) inhibited by calcium ionophore, (e) inhibited by the local anesthetic dibucaine and the tranquillizer chlorpromazine, (f) dependent on cytoskeletal activity (i.e., inhibited by the simultaneous presence of colchicine and cytochalasin B), (g) not dependent on the membrane ATPases inhibited by ouabain, (h) not dependent on motility, in that agents which inhibit motility (cytochalasin B alone) or stimulate motility (carbachol) do not alter capping behavior. These properties represent similarities between the capping of surface Ig by the cellular antigens on SRC and by proteins such as anti-Ig. SRC-induced capping is much slower than anti-Ig-induced capping, and only engages 30–40% of ABC, indicating that the nature of the crosslinking agent can influence the kinetics and extent of capping. But SRC cap with the rapid kinetics typical of anti-Ig-induced capping if the surface membrane of the ABC is first cleared of other glycoproteins with trypsin. The removal of negatively charged sialic acid residues by neuraminidase has no such effect. It is probable that the compression of bound SRC into a small area of the membrane requires more energy than does the capping of protein ligands, and that some cells cannot muster enough energy to achieve it.     

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.     

Effects of retinoic acid (RA) on the growth and phenotypic expression of several human neuroblastoma cell lines

It has been shown that retinoic acid (RA) can promote morphologic differentiation and inhibit the growth of a human neuroblastoma cell line, LA-N-1. The present study tests the histological generality of these phenomena by determining the effects of RA on seven other human neuroblastoma cell lines. Results show that RA strongly inhibited anchorage-dependent growth and induced morphologic alterations in six of seven of the cell lines. These alterations included morphologic differentiation as evidenced by formation of neurite extensions in four of the lines, cellular enlargement and vacuolization in one culture, and formation of large, flattened epithelial or fibroblastic-like cells in another culture. Although one cell line was relatively insensitive to the effects of RA in monolayer culture, all seven were strongly inhibited by RA in soft agar assays. Cellular RA-binding proteins were detected in 2/2 lines tested. These findings suggest that, as a histological group, human neuroblastoma cells are extremely sensitive to RA-induced growth inhibition and morphological alterations generally associated with reduced expression of the malignant phenotype of this type of cancer.     

Natural killer cell activity correlates with the amount of beta 2-microglobulin on human peripheral blood lymphocytes

Two cytotoxic assays, lectin-dependent cytotoxicity and natural killer (NK) cytotoxicity, were used to assess the competence of cord blood and neonatal peripheral blood mononuclear cell (PBMC) and T-cell cytotoxic reactions. The effect of exogenous interferon was also studied. Results were compared with cytotoxic capabilities of adult cells and cells from patients with primary immunodeficiency syndromes. Lectin-dependent cytotoxicity (LDCC), a property of both T and non-T cells, was assessed by lysis of chromium-labeled EL4 tumor target cells in the presence or absence of exogenous fibroblast interferon (IFN-β). Natural killer cytotoxicity was assessed by lysis of two different chromium-labeled tumor target cells, Molt 4f and K562 in the presence or absence of IFN-β. Lectin-dependent cytotoxicity (LDCC) of PBMC of cord blood (32 ± 4% SEM) and adult cells (36 ± 2% SEM) were equivalent but neonatal cells had slightly decreased LDCC (22 ± 3% lysis). T-depleted cells from cord or neonatal blood had increased LDCC but T-enriched (>95% sheep erythrocyte rosette-forming cells) from both cord (22 ± 3%) and neonatal blood (18 ± 5%) had significantly reduced LDCC compared to 55 ± 2% for adult T cells. This deficiency corrects with age and is near normal after age 2. Preincubation with IFN-β did not enhance LDCC of newborn or adult cells. The LDCC of some cord T cells was markedly reduced and was in the same low range as patients with severe combined immunodeficiency. Natural killer (NK) cytotoxicity of PBMC from cord and adult cells was equivalent at three effector:target ratios against the Molt 4f target but against the K562 target, cord PBMC had significantly less NK activity (22 ± 11 SD) compared to adult NK activity (50.5 ± 22.2 SD) at a 50:1 effector:target ratio. Similar differences were noted at 25:1 and 10:1 target:effector ratios. NK cytotoxicity against Molt 4f targets of adult cells was significantly enhanced by preincubation with IFN-β but NK of cord cells was only variably enhanced. By contrast, IFN-β enhanced NK against K562 targets of both adult and cord cells, adult greater (67.7 ± 20) than cord cells (37.8 ± 2.0). These T-cell effector deficiencies are in marked contrast to the vigorous proliferative responses of newborn T cells, and parallel deficiencies of certain neonatal lymphokines. These defects may explain the newborns' enhanced susceptibility to intracellular viruses and to congenital viral infections.     

Calcium-sensitive action potential of long duration in the fertilized egg of the ctenophore Mnemiopsis leidyi

The membrane properties of fertilized eggs of the ctenophore Mnemiopsis leidyi were studied using standard microelectrode techniques. The resting potential was approximately -80 mV, and was dependent on the extracellular K concentration. Depolarizing current injections elicited an action potential with an initial peak amplitude of +20 to +40 mV (duration about 5 sec) and a long lasting (duration 3 to 10 min) plateau phase. The depolarizing phase and the plateau phase appeared to have different ionic mechanisms. The entire action potential could be prevented by removal of extracellular Ca, but only the amplitude of the depolarizing phase, not the plateau phase, was dependent on the extracellular Ca concentration. The plateau phase was not observed in the absence of Ca, but in the presence of Ca its duration was dependent on the external Ca concentration. The data suggest that the plateau phase is activated as a consequence of Ca influx during the initial depolarizing phase. Removal of external Na resulted in only minor changes in the waveform of repolarization. The action potential was resistant to low concentrations of Mn and Cd in the presence of Ca. The role of this action potential in ctenophore development is not known, but in its waveform and duration it resembles the sperm-gated potentials that have been seen in eggs of other phyla. These experiments show ctenophore embryos to be excitable at very early stages, and suggest their utility in the study of the differentiation of cellular electrical properties.     

Group mating among Norway rats I. Sex differences in the pattern and neuroendocrine consequences of copulation

The copulatory pattern of groups of rats (Rattus norvegicus) was studied in the laboratory in a seminatural environment. In a given mating session, every oestrous female copulated with each male; likewise, every male copulated with each oestrous female. While individual males and females experienced similar amounts of copulation, there were dramatic sex differences in sequence and temporal pattern. Males mated in a multiple intromission pattern and had more ejaculatory series when several females were in oestrus. In contrast, females received intromissions and ejaculations in a random order, not in the sequence of a male ejaculatory series. Males copulated at shorter intervals than females did, a temporal sex difference that was determined by the pattern of female solicitations and male approaches. These sex differences are used to discuss the different units of analysis that are appropriate for male and female sexual behaviour in this species. Furthermore, the sex differences in the temporal pattern of copulation which emerged during group mating parallel the known sex differences in the temporal parameters of the neuroendocrine reflexes which mediate successful reproduction in the domestic strain.     

Reduced ability of naloxone to stimulate LH and testosterone release in aging male rats; possible relation to increase in hypothalamic met5-enkephalin

Blood luteinizing hormone (LH) and testosterone levels are lower in old than in young male rats. The specific opiate antagonist, naloxone, previously shown to increase serum LH in mature male rats, exhibited relatively little ability to raise serum LH and testosterone levels in old (18–20 mo) as compared to young (4–5 mo) male rats. The brain opiate, met⁵-enkephalin, which depresses LH, was found to be significantly higher in the hypothalamus of old than of young male rats. These observations suggest that hypothalamic opiates may be partially responsible for the lower serum LH and testosterone levels in old male rats, and for reduced release of these hormones in response to naloxone administration.     

Regulation of cyclic GMP, cyclic AMP and lactate dehydrogenase by putative neurotransmitters in the C6 rat glioma cell line.

In C6 cells norepinephrine and dopamine caused transient increases in cyclic GMP and cyclic AMP, as well as an induction of lactate dehydrogenase. All of these responses were blocked by l-propranolol, suggesting mediation by a β-receptor. Phentolamine potentiated the NE-increased cAMP levels by 5-fold when NE was used at suboptimal doses, suggesting the presence of α-adrenergic receptors in C6 cells. Carbamylcholine decreased the levels of both cyclic nucleotides, with hexamethonium partially reversing the effect on cyclic GMP. Dibutyryl-cyclic GMP or carbamylcholine reduced catecholamine-induced cyclic AMP levels. Serotonin increased cyclic GMP levels 60% and decreased cyclic AMP levels 36%. Calcium- and magnesium-free media inhibited the norepinephrine-induced levels of cyclic GMP and cyclic AMP respectively.     

Sequence analysis and evolution of sea urchin (Lytechinus pictus and Strongylocentrotus purpuratus) histone H4 messenger RNAs

The putative histone H4 (F2a₁) mRNA has been isolated from early blastula Strongylocentrotus purpuratus sea urchin embryos. Nucleotide sequences of oligonucleotides obtained by digestion of this RNA with T₁ ribonuclease have been obtained and many are found to be colinear with the amino acid sequence of histone H4 protein. The sequences obtained from the H4 mRNAs of S. pnrpuratus have been compared with those obtained from Lytechinus pictus (Grunstein & Schedl, 1976). The two mRNAs for this highly conserved protein have undergone considerable divergence of the sort that would be predicted from the degeneracy of the genetic code. 11.5% of the bases have undergone substitution at a rate calculated to be 3 × 10^?9 base changes · codon^?1 · year^?1.     

Frequency and intensity of chick distress calls: Effects on maternal foodcalling in chickens

The food call of broody domestic hens was used to measure maternal response to four frequency components found in chick distress calls (2,3,4 and 5 kHz) and to variations in distress call intensity (0—86 dB). Foodcalling increased significantly with frequency of the pure-tone test pulse; response to a taped distress call occurred between 40 dB and 86 dB intensity with a maximum at 60–65 dB. The results suggest that the mother uses the higher frequency components in recognizing the distress call, but responds maximally within a specific intensity range. The selective advantage of such behaviour is discussed.     

Cell affinity determining heterospecific graft intolerance in hydra

Column segments taken from hydra of different species can be grafted together; they separate after a period ranging from hours to weeks, depending on the species pair. The healing and separation processes were studied in homografts and heterografts by light and electron microscopy, using seven species of the genera Hydra, Chlorohydra and Pelmatohydra. In homografts, the edge cells show pseudopodial activity and attach and adhere rapidly. Heterografts show some normal tissue healing activities, but the poorer heterotypic cell adhesions result in the tissues eventually separating. In combinations where the tissues heal poorly initially, the tissues fall apart soon after grafting. In other heterografts, cell attachments arise which resemble septate desmosomes, and there is sufficient healing to hold the tissues together for at least several days. Later a constriction develops at the graft site and progresses until the unlike tissues are pinched apart. This constriction appears to represent not immunological rejection, but rather a replacement of the heterotypic cell junctions by homospecific attachments, leading to a gradual separation of the tissues.     

Membrane defects in the tolerant B cell. II. Mechanism of capping failure and reversal by antigen exposure.

The demonstration that TNP-binding B lymphocytes from animals whose B cells have been rendered tolerant to TNP by trinitrobenzene sulfonic acid cannot undergo antigen-induced capping of their TNP receptors for at least a year despite recovery of immune responsiveness has led to a search for the mechanism of the capping failure. Microtubule-dependent membrane “locking” analogous to that induced by concanavalin A appears to afflict the tolerant B cells, in that capping TNP receptors is restored after exposure to 10^?4M colchicine or overnight incubation at 4 °C. Assignment of the defect to the cytoskeleton rather than the receptors themselves is also supported by the observations that enzymatic stripping and regrowth of receptors does not unlock the cell and that non-Ig membrane molecules recognized by antilymphocyte serum also cannot be capped on the tolerant cells. Cells which have remained locked for 4 days to 8 months after a single tolerogen exposure become unlocked 4 days after immunogen is given. Four days after immunogen, tolerogen fails to lock the membranes of TNP-binding cells. These results suggest that tolerogen contact interferes in a much broader range of functions in the TNP-binding cell than those which affect the immune response. Among these effects is a remarkably stable “locked” configuration of the cytoskeleton which is independent of immune responsiveness or receptor turnover, but which can be reversed by exposure to immunogen whether or not an immune response ensues.     

Membrane defects of the tolerant B cell. I. Failure of antigen-induced capping.

In order to study the membrane function of tolerant B antigen-binding cells, tolerance to the trinitrophenyl (TNP) determinant was induced in mice by injecting the reactive form of the hapten, trinitrobenzene sulfonic acid (TNBS). By appropriate transfer experiments, Fidler and Golub (J. Immunol.112, 1891, 1974) had previously shown that this form of tolerance is a B-cell property, induced and expressed in the absence of T cells. Hapten inhibition demonstrated the TNP-specificity of receptors on TNP-donkey erythrocyte(TNP-D)-binding cells in tolerant and nontolerant mice. About 88% of these cells were B cells by immunofluorescence, and the remainder were T cells. In the tolerant mice, challenge with TNP-sheep erythrocytes failed to expand the TNP-binding population, but sheep erythrocyte binders and anti-sheep plaque-forming cells expanded normally. Despite little or no change in TNP-binding cell numbers after tolerance induction, the TNP-binding cells of tolerant animals could not cap their receptors, in contrast to the sheep erythrocyte-binding cells from the same animals which capped normally. Although there is no anti-TNP plaque-forming cell response when tolerogen and immunogen are given simultaneously, capping failure is not evident until 2–4 days after tolerogen exposure. By Day 7, substantial recovery of immune responsiveness had occurred, yet even 12 months after a single dose of tolerogen there was no restoration of capping. Thus despite the association of both capping failure and unresponsiveness with tolerogen exposure, these lymphocyte functional defects appeared not to be causally related.     

Tissue healing and septate desmosome formation in hydra

Tissue healing was studied in hydra tissue grafts by means of light and electron microscopy. Healing is begun by the gastrodermis: subsequently the epidermis fuses and the mesoglea is repaired. Epidermis fusion is first brought about by long processes from the basal portions of the epithelial cells bridging the wound gap and adhering to opposing cells. Irregular septate desmosomes form early in this process and continuously become more neatly organized. Concommitant with the healing process at the graft site, neighboring cells are also rearranging, their septate desmosomes undergoing transient disorganizations. We conclude that the organization of septate junctions is dynamic, and may be undergoing a balanced but continuous, steady state turnover. During the healing process the forces acting on the desmosomes, and other aspects of the cells' architecture, are not balanced, and the junctions grow and become more highly organized.