The Effect of Histamine on Inward and Outward Currents in Mouse Retinal Amacrine Cells

The expression of H1 receptor has been reported in amacrine cells of mouse and rat retinae. However, we assumed that other types of histamine receptors also function in amacrine cells. In order to confirm that histamine modulates the membrane potential in mouse amacrine cells, we measured voltage-gated currents using whole-cell configuration. Under voltage-clamp conditions, the amplitude of voltage-gated outward currents was enhanced by the application of 100 µM histamine in 65% of amacrine cells. Histamine also increased the amplitudes of voltage-gated inward currents in 72% of amacrine cells. When antagonists of the histamine H1, H2, or H3 receptors were applied to histamine-sensitive amacrine cells, all three types of these inhibitors reduced the effect of histamine. Moreover, we classified recorded cells into seven types based on their morphological characteristics. Two of the seven types, diffuse multistratified cells and AII amacrine cells, responded significantly to histamine. These results indicate that histamine affected the membrane potential via three types of histamine receptors. Furthermore, there were differences in the responses to histamine among types of amacrine cells. Histamine may be one of the important neurotransmitters and/or neuromodulators in the visual processing.     

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule,N-cadherin

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2mM Ca⁺⁺ or 0.2 mM Ca⁺⁺, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca⁺⁺, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca⁺⁺ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca⁺⁺ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca⁺⁺ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca⁺⁺ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca⁺⁺, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca⁺⁺. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca⁺⁺ levels had similar effects as lowering the Ca⁺⁺ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.     

Role of Ca in stimulus-secretion coupling in the gastric oxyntic cell: Effect of A23187

The effects of Ca⁺⁺ ionophore A23187 on H⁺ secretion and histamine release were studied in the isolated gastric mucosa of the toad . A23187 added from the mucosal side stimulated H⁺ secretion. At high concentrations, A23187 also caused histamine release. This histamine was not sufficient to explain the effects of A23187 on H⁺ secretion. Metiamide, only partially inhibited the effect of ionophore. There was summation and/or potentiation of effects between A23187 and histamine. The results are consistent with the hypothesis that Ca⁺⁺ acts as a second messenger in stimulus-secretion coupling in the oxyntic cell. It is possible that Ca⁺⁺ and cAMP may interact as parallel second messengers in the control of gastric H⁺ secretion.     

MIGRATORY CELL LOCOMOTION VERSUS NERVE AXON ELONGATION : Differences Based on the Effects of Lanthanum Ion

The effects of lanthanum ions (La⁺⁺⁺) on the locomotion and adhesion of g lial cells and elongating nerve axons are reported. La⁺⁺⁺ increases adhesion of both glia and of nerve growth cones to a plastic substratum. La⁺⁺⁺ also markedly reduces glia locomotion, but it does not inhibit nerve elongation. Electron-opaque deposits are seen on the cell surface and within cytoplasmic vesicles of glia and nerves cultured in a La⁺⁺⁺-containing medium. Possible modes of action for La⁺⁺⁺ are discussed, particularly the possibilities that Ca⁺⁺ fluxes or Ca⁺⁺ involvement in adhesion are altered by La⁺⁺⁺. The results are consistent with the hypothesis that cell migration and nerve axon elongation differ in mechanism, with respect to both adhesive interactions and the activity of microfilament systems.     

Effect of cellular glutathione depletion on cadmium-induced cytotoxicity in human lung carcinoma cells

The effect of glutathione depletion on cellular toxicity of cadmium was investigated in a subpopulation (T27) of human lung carcinoma A549 cells with coordinately high glutathione levels and Cd⁺⁺-resistance. Cellular glutathione levels were depleted by exposing the cells to diethyl maleate or buthionine sulfoximine. Depletion was dose-dependent. Exposure of the cells to 0.5 mM diethyl maleate for 4 hours or to 10 mM buthionine sulfoximine for 8 hours eliminated the threshold for Cd⁺⁺ cytotoxic effect and deccreased the LD_50S. Cells that were pretreated with 0.5 mM diethyl maleate or 10 mM buthionine sulfoximine and then exposed to these same concentrations of diethyl maleate or buthionine sulfoximine during the subsequent assay for colony forming efficiency produced no colonies, reflecting an enhanced sensitivity to these agents at low cell density. Diethyl maleate was found to be more cytotoxic than buthionine sulfoximine. Synergistic cytotoxic effects were observed in the response of diethyl maleate pretreated cells exposed to Cd⁺⁺. Thus the results demostrated that depletion of most cellular glutathione in A549-T27 cells prior to Cd⁺⁺ exposure sensitizes them to the agent's cytotoxic effects. Glutathione thus may be involved in modulating the early cellular Cd⁺⁺ cytotoxic response. Comparison of reduced glutathione levels and of Cd⁺⁺ cytotoxic responses in buthionine sulfoximine-treated A549-T27 cells with those levels in other, untreated normal and tumor-derived cells suggests that the higher level of glutathione in A549-T27 is not the sole determinant of its higher level of Cd⁺⁺ resistance.Abbreviations BSO DL-buthionine-(R,S)-sulfoximine - DEM diethyl maleate - DMSO dimethyl sulfoxide - GSH reduced glutathione - MT metallothionein     

In vivo responses of paired giant mechanoreceptor neurons in Aplysia abdominal ganglion

Two neurons with cell bodies symmetrically located in the abdominal ganglion and giant axons in the left (L1) and right (R1) pleurovisceral connectives of Aplysia californica were examined in vivo and in vitro. Direct stimulation of R1 and L1 in the intact animal does not elicit any observable behavior, suggesting that they are neither motoneurons nor command neurons. These cells respond in vivo to sudden onset mechanical stimulation of widespread regions of the body. R1 and L1 spikes are initiated in at least three different loci: (1) the peripheral axon in the foot, (2) the neuropil of the pleural and/or pedal ganglion, and (3) the neuropil of the abdominal ganglion. Furthermore, R1 and L1 probably have two different mechanisms for spike initiation: (1) sensory (foot), and (2) synaptic (abominal and/or head ganglia). The different loci for spike initiation account for the bidirectional conduction of R1 and L1 spikes. As sensory (mechanoreceptor) neurons, R1 and L1 have peripheral axons in the ipsilateral posterior pedal nerve, show low threshold responses to stimulation of the ipsilateral posterior foot, they are rapidly adapting their responses do not decrease with repetion, and they are not blocked by high Mg⁺⁺/low Ca⁺⁺ solutions. As synaptically-driven neurons, R1 and L1 have widespread bilateral responsiveness, their responses decrease with repetition and their inputs are blocked with high Mg⁺⁺/low Ca⁺⁺ solutions. These neurons integrate sensory and synaptic inputs and conduct bidirectionally, however, their output connections must be specified before their behavioral function can be understood.     

Characterization of adenylate and guanylate cyclases in rat peritoneal mast cells

Adenylate and guanylate cyclase activities were confirmed in crude homogenates from rat peritoneal mast cells. Both enzyme activities were associated with the 105, 000 X g particulate fractions, but not detected in the supernatant fractions. The optimal pH for both cyclase activities was 8.2. Mn⁺⁺ was essentially required for guanylate cylcase activity, while adenylate cyclase activity was observed in the presence of either Mg⁺⁺ or Mn⁺⁺. The apparent Km values of adenylate cyclase for Mn⁺⁺-ATP and Mg⁺⁺-ATP were 160 μM and 340 μM, respectively, whereas the value of guanylate cyclase for Mn⁺⁺-GTP was 100 μM. Adenylate cyclase was activated by 10 mM NaF. However, both adenylate and guanylate cyclase activities were neither stimulated nor inhibited by the addition of various kinds of agents which stimulate or inhibit the release of histamine from mast cells.     

Presence and distribution of histaminergic components in rat and bovine retina

The presence of histamine and its related enzymes, histidine decarboxylase and histamine N-methyltransferase and the subcellular distribution of the amine and of H₁-receptors were studied in the retina of two mammalian species. Histamine is present in rat and bovine retinas in concentrations (113 ± 10 and 72 ± 9 ng/g wet tissue, respectively) similar to those found in the brain. Histological examination and release experiments with Compound 48/80 performed in rat retina indicate a non mast cell location for the amine. Histidine decarboxylase and histamine N-methyltransferase activities in rat and bovine retinas were also comparable to those found in brain cortex suggesting that histamine can be synthesized and catabolyzed in situ. Subcellular fractionation of bovine retina showed that both the amine and H₁-receptors are concentrated in particulate fractions where small sized synaptosomes sediment, presumably derived from horizontal and amacrine cells. These results are in agreement with a neurotransmitter or neuromodulator role for histamine in cells of the retinal inner nuclear layer.     

Enhanced cadmium cytotoxicity in A549 cells with reduced glutathione levels is due to neither enhanced cadmium accumulation nor reduced metallothionein synthesis

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-727) cells to the cytotoxic effects of Cd⁺⁺. The effects of GSH depletion on Cd⁺⁺ accumulation and Cd⁺-induced metallothionein (MT) content were investigated to determine the possible role of these Cd⁺⁺ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd⁺⁺-induced accumulation of exogenous³⁵s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd⁺⁺ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd⁺⁺ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd⁺⁺, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd⁺⁺-induced MT accumulation rates.Abbreviations GSH glutathione - MT metallothionein - BSO DL-buthionine-[S,R]-sulfoximine - DMSO dimethyl sulfoximine - DEM diethyl maleate - NP-40 nonidet-P40 - PBS phosphate buffered saline - HBSS Hank's balanced salt solution - DTT dithiothreitol 3. This work was presented in part at the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1–5, 1988.     

Neurotransmitter candidates in the retina of the mudpuppy,Necturus maculosus

Summary Neurons accumulating (³H)-glycine and (³H) GABA were demonstrated with the use of autoradiography. Both were accumulated by different types of amacrine cells, similar those of goldfish. (³H)-GABA was also accumulated by horizontal cells, again similar to the goldfish. These results and physiological studies from other laboratories suggest that GABA and glycine are neurotransmitter candidates in amacrine cells of the mudpuppy.Immunoreactive neuropeptide Y (NPY), glucagon, vasoactive intestinal peptide (VIP), somatostatin, substance P, and neurotensin were found in different types of stratified amacrine cells. Weakly immunoreactive enkephalin and bombesin processes were also seen in the inner plexiform layer. Gastrin-immunoreactive neurons were not detectable.Endogenous 5-hydroxytryptamine was visualized immunohistochemically in a population of diffuse amacrine cells and some cells in the ganglion cell layer. This suggests that 5-hydroxytryptamine may be a neurotransmitter in the retina of the mudpuppy.     

Gap junctions with amacrine cells provide a feedback pathway for ganglion cells within the retina.

In primates, one type of retinal ganglion cell, the parasol cell, makes gap junctions with amacrine cells, the inhibitory, local circuit neurons. To study the effects of these gap junctions, we developed a linear, mathematical model of the retinal circuitry providing input to parasol cells. Electrophysiological studies have indicated that gap junctions do not enlarge the receptive field centres of parasol cells, but our results suggest that they make other contributions to their light responses. According to our model, the coupled amacrine cells enhance the responses of parasol cells to luminance contrast by disinhibition. We also show how a mixed chemical and electrical synapse between two sets of amacrine cells presynaptic to the parasol cells might make the responses of parasol cells more transient and, therefore, more sensitive to motion. Finally, we show how coupling via amacrine cells can synchronize the firing of parasol cells. An action potential in a model parasol cell can excite neighbouring parasol cells, but only when the coupled amacrine cells also fire action potentials. Passive conduction was ineffective due to low-pass temporal filtering. Inhibition from the axons of the coupled amacrine cells also produced oscillations that might synchronize the firing of more distant ganglion cells.     

Expression and In Vitro Regulation of Integrins by Normal Human Urothelial Cells

Integrins are thought to be essential adhesion receptors for the maintenance of tissue hisr tioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed α2β1 and α3β1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, α6β4 and occasionally αvβ4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca⁺⁺serum-free medium, the monolayer cultures expressed α2β1, α3β1 and α5β1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas α6β4 was distributed in a random pattern over the substratum. Increasing exogenous Ca⁺⁺concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, α6β4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of α6β4 in high Ca⁺⁺media, and also of α3 and α5, but not α2, subunits. These results suggest that α2β1 and α3β1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas α6β4 is the principal integrin involved in substratum adhesion.     

RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells

A subset of retinal ganglion cells is intrinsically photosensitive (ipRGCs) and contributes directly to the pupillary light reflex and circadian photoentrainment under bright-light conditions. ipRGCs are also indirectly activated by light through cellular circuits initiated in rods and cones. A mammalian homologue (RdgB2) of a phosphoinositide transfer/exchange protein that functions in Drosophila phototransduction is expressed in the retinal ganglion cell layer. This raised the possibility that RdgB2 might function in the intrinsic light response in ipRGCs, which depends on a cascade reminiscent of Drosophila phototransduction. Here we found that under high light intensities, RdgB2^−/− mutant mice showed normal pupillary light responses and circadian photoentrainment. Consistent with this behavioral phenotype, the intrinsic light responses of ipRGCs in RdgB2^−/− were indistinguishable from wild-type. In contrast, under low-light conditions, RdgB2^−/− mutants displayed defects in both circadian photoentrainment and the pupillary light response. The RdgB2 protein was not expressed in ipRGCs but was in GABAergic amacrine cells, which provided inhibitory feedback onto bipolar cells. We propose that RdgB2 is required in a cellular circuit that transduces light input from rods to bipolar cells that are coupled to GABAergic amacrine cells and ultimately to ipRGCs, thereby enabling ipRGCs to respond to dim light.     

Axonal Synapses Utilize Multiple Synaptic Ribbons in the Mammalian Retina

In the mammalian retina, bipolar cells and ganglion cells which stratify in sublamina a of the inner plexiform layer (IPL) show OFF responses to light stimuli while those that stratify in sublamina b show ON responses. This functional relationship between anatomy and physiology is a key principle of retinal organization. However, there are at least three types of retinal neurons, including intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells, which violate this principle. These cell types have light-driven ON responses, but their dendrites mainly stratify in sublamina a of the IPL, the OFF sublayer. Recent anatomical studies suggested that certain ON cone bipolar cells make axonal or ectopic synapses as they descend through sublamina a, thus providing ON input to cells which stratify in the OFF sublayer. Using immunoelectron microscopy with 3-dimensional reconstruction, we have identified axonal synapses of ON cone bipolar cells in the rabbit retina. Ten calbindin ON cone bipolar axons made en passant ribbon synapses onto amacrine or ganglion dendrites in sublamina a of the IPL. Compared to the ribbon synapses made by bipolar terminals, these axonal ribbon synapses were characterized by a broad postsynaptic element that appeared as a monad and by the presence of multiple short synaptic ribbons. These findings confirm that certain ON cone bipolar cells can provide ON input to amacrine and ganglion cells whose dendrites stratify in the OFF sublayer via axonal synapses. The monadic synapse with multiple ribbons may be a diagnostic feature of the ON cone bipolar axonal synapse in sublamina a. The presence of multiple ribbons and a broad postsynaptic density suggest these structures may be very efficient synapses. We also identified axonal inputs to ipRGCs with the architecture described above.     

Stromal Interaction Molecule 1 (STIM1) and Orai1 Mediate Histamine-evoked Calcium Entry and Nuclear Factor of Activated T-cells (NFAT) Signaling in Human Umbilical Vein Endothelial Cells

Histamine is an important immunomodulator involved in allergic reactions and inflammatory responses. In endothelial cells, histamine induces Ca²⁺ mobilization by releasing Ca²⁺ from the endoplasmic reticulum and eliciting Ca²⁺ entry across the plasma membrane. Herein, we show that histamine-evoked Ca²⁺ entry in human umbilical vein endothelial cells (HUVECs) is sensitive to blockers of Ca²⁺ release-activated Ca²⁺ (CRAC) channels. RNA interference against STIM1 or Orai1, the activating subunit and the pore-forming subunit of CRAC channels, respectively, abolishes this histamine-evoked Ca²⁺ entry. Furthermore, overexpression of dominant-negative CRAC channel subunits inhibits while co-expression of both STIM1 and Orai1 enhances histamine-induced Ca²⁺ influx. Interestingly, gene silencing of STIM1 or Orai1 also interrupts the activation of calcineurin/nuclear factor of activated T-cells (NFAT) pathway and the production of interleukin 8 triggered by histamine in HUVECs. Collectively, these results suggest a central role of STIM1 and Orai1 in mediating Ca²⁺ mobilization linked to inflammatory signaling of endothelial cells upon histamine stimulation.     

Histamine deficiency delays ischaemic skeletal muscle regeneration via inducing aberrant inflammatory responses and repressing myoblast proliferation

Histidine decarboxylase (HDC) catalyses the formation of histamine from L‐histidine. Histamine is a biogenic amine involved in many physiological and pathological processes, but its role in the regeneration of skeletal muscles has not been thoroughly clarified. Here, using a murine model of hindlimb ischaemia, we show that histamine deficiency in Hdc knockout (Hdc^?/?) mice significantly reduces blood perfusion and impairs muscle regeneration. Using Hdc‐EGFP transgenic mice, we demonstrate that HDC is expressed predominately in CD11b⁺Gr‐1⁺ myeloid cells but not in skeletal muscles and endothelial cells. Large amounts of HDC‐expressing CD11b⁺ myeloid cells are rapidly recruited to injured and inflamed muscles. Hdc^?/? enhances inflammatory responses and inhibits macrophage differentiation. Mechanically, we demonstrate that histamine deficiency decreases IGF‐1 (insulin‐like growth factor 1) levels and diminishes myoblast proliferation via H3R/PI3K/AKT‐dependent signalling. These results indicate a novel role for HDC‐expressing CD11b⁺ myeloid cells and histamine in myoblast proliferation and skeletal muscle regeneration.     

Differential Calcium Requirements For Growth of Mouse Skin Epithelial and Fibroblast Cells

Concentrations of extracellular Ca⁺⁺ optimum for growth of cell types of mesodermal origin have been reported to be up to 100-fold higher than concentrations optimal for epidermal or other epithelial lining cells. In order to examine Ca⁺⁺ requirements of epithelial v. fibroblastic cells derived from a common tissue source, prior to prolonged culture, freshly isolated mouse epidermal keratinocytes, hair follicle cells and dermal fibroblasts were plated at high density or at clonal density in medium ranging from 0.014 to 1.4 mM Ca⁺⁺. Epithelial skin cells grew best at Ca⁺⁺ levels below 0.1 mM while dermal fibroblasts grew best at a Ca⁺⁺ concentration of 1.4 mM. the epithelial cell types exhibited marked morphologic changes in response to Ca⁺⁺, while the fibroblasts did not. These results suggest that the variations in Ca⁺⁺ response between lining epithelium and mesenchymal cells resulted from inherent differences in these cell types, but a mechanism for such differential effects has not yet been defined.     

Histamine deficiency facilitates coronary microthrombosis after myocardial infarction by increasing neutrophil-platelet interactions

Neutrophil-platelet interactions are responsible for thrombosis as well as inflammatory responses following acute myocardial infarction (AMI). While histamine has been shown to play a crucial role in many physiological and pathological processes, its effects on neutrophil-platelet interactions in thromboinflammatory complications of AMI remain elusive. In this study, we show a previously unknown mechanism by which neutrophil-derived histamine protects the infarcted heart from excessive neutrophil-platelet interactions and redundant arterial thrombosis. Using histamine-deficient (histidine decarboxylase knockout, HDC^−/−) and wild-type murine AMI models, we demonstrate that histamine deficiency increases the number of microthrombosis after AMI, in accordance with depressed cardiac function. Histamine-producing myeloid cells, mainly Ly6G⁺ neutrophils, directly participate in arteriole thrombosis. Histamine deficiency elevates platelet activation and aggregation by enhancing Akt phosphorylation and leads to dysfunctional characteristics in neutrophils which was confirmed by high levels of reactive oxygen species production and CD11b expression. Furthermore, HDC^−/− platelets were shown to elicit neutrophil extracellular nucleosomes release, provoke neutrophil-platelet interactions and promote HDC-expressing neutrophils recruitment in arteriole thrombosis in vivo. In conclusion, we provide evidence that histamine deficiency promotes coronary microthrombosis and deteriorates cardiac function post-AMI, which is associated with the enhanced platelets/neutrophils function and neutrophil-platelet interactions.     

Calcium regulation of normal human mammary epithelial cell growth in culture

Summary The concentration of Ca⁺⁺ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca⁺⁺ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca⁺⁺ to less than 0.08 mM. The reduction of Ca⁺⁺ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca⁺⁺ on growth and differentiation were specific (Mg⁺⁺ and Mn⁺⁺ variations were without effect) and reversible and in many respects resembled Ca⁺⁺ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca⁺⁺-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca⁺⁺ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.     

Properties of 3H-cimetidine binding in rat brain membrane fractions

In an attempt to characterize the brain histamine H₂ receptor, experiments were undertaken to study the binding properties of (N-methyl-³H) -cimetidine, an H₂ receptor antagonist, in rat brain membranes. Using a centrifugation assay, ³H-cimetidine binding having a K_d of 0.40μM and a Bmax of 3.9 pmoles/mg protein was detected. Of fourteen anions and cations tested, one, Cu⁺⁺, dramatically increased specific ³H-cimetidine binding, the increase being due mainly to a change in Bmax. Studies of substrate specificity for ³H-cimetidine binding revealed that Cu⁺⁺, while not significantly affecting the potency of H₂ receptor agonists and antagonists, dramatically decreases the potency of H₁ receptor substances on the ³H-cimetidine binding site. In addition, both the relative and absolute potencies of various H₂ receptor agonistsv and antagonists in displacing the ligand in the presence of Cu⁺⁺ parallels their potencies in biological systems. These findings suggest that, under these conditions, ³H-cimetidine may be labelling a biologically relevant H₂ binding site in brain and that Cu⁺⁺ may regulate the substrate specificity for this site. The brain regional distribution and kinetic analysis of the binding suggest that it is not localized solely to the synaptic receptor for histamine, but may also be associated with histamine receptors at other neuronal, glial or vascular sites.