Albert K. Urazaev

Factors necessary to produce basoapical polarity in human glandular epithelium formed in conventional and high-throughput three-dimensional culture: example of the breast epithelium

BackgroundBasoapical polarity in epithelia is critical for proper tissue function, and control of proliferation and survival. Cell culture models that recapitulate epithelial tissue architecture are invaluable to unravel developmental and disease mechanisms. Although factors important for the establishment of basal polarity have been identified, requirements for the formation of apical polarity in three-dimensional tissue structures have not been thoroughly investigated.ResultsWe demonstrate that the human mammary epithelial cell line-3522 S1, provides a resilient model for studying the formation of basoapical polarity in glandular structures. Testing three-dimensional culture systems that differ in composition and origin of substrata reveals that apical polarity is more sensitive to culture conditions than basal polarity. Using a new high-throughput culture method that produces basoapical polarity in glandular structures without a gel coat, we show that basal polarity-mediated signaling and collagen IV are both necessary for the development of apical polarity.ConclusionThese results provide new insights into the role of the basement membrane, and especially collagen IV, in the development of the apical pole, a critical element of the architecture of glandular epithelia. Also, the high-throughput culture method developed in this study should open new avenues for high-content screening of agents that act on mammary tissue homeostasis and thus, on architectural changes involved in cancer development.

Glutamate regulation of non-quantal release of acetylcholine in the rat neuromuscular junction

Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non‐quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non‐quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H‐effect) following blockade of skeletal muscle post‐synaptic nicotinic receptors by (+)‐tubocurarine and cholinesterase by armin (diethoxy‐p‐nitrophenyl phosphate). Glutamate was shown to inhibit non‐quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate‐induced decrease of the H‐effect was enhanced by glycine. Glycine alone also lowered the H‐effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition of N‐methyl‐d‐aspartate (NMDA) receptors with (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzocyclohepten‐5,10‐imine (MK801), dl‐2‐amino‐5‐phosphopentanoic acid (AP5) and 7‐chlorokynurenic acid or the elimination of Ca2+ from the bathing solution prevented the glutamate‐induced decrease of the H‐effect with or without glycine. Inhibition of muscle nitric oxide synthase by NG‐nitro‐l‐arginine methyl ester (l‐NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H‐effect. The results suggest that glutamate, acting on post‐synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non‐quantal secretion of ACh from motor nerve terminals. Non‐quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non‐quantal transmitter following muscle denervation triggers the early post‐denervation depolarization of muscle fibres.

Synthesis and release of N-acetylaspartylglutamate (NAAG) by crayfish nerve fibers: implications for axon–glia signaling

Early physiological and pharmacological studies of crayfish and squid giant nerve fibers suggested that glutamate released from the axon during action potential generation initiates metabolic and electrical responses of periaxonal glia. However, more recent investigations in our laboratories suggest that N-acetylaspartylglutamate (NAAG) may be the released agent active at the glial cell membrane. The investigation described in this paper focused on NAAG metabolism and release, and its contribution to the appearance of glutamate extracellularly. Axoplasm and periaxonal glial cell cytoplasm collected from medial giant nerve fibers (MGNFs) incubated with radiolabeled L-glutamate contained radiolabeled glutamate, glutamine, NAAG, aspartate, and GABA. Total radiolabel release was not altered by electrical stimulation of nerve cord loaded with [(14)C]glutamate by bath application or loaded with [(14)C]glutamate, [(3)H]-D-aspartate or [(3)H]NAAG by axonal injection. However, when radiolabeled glutamate was used for bath loading, radiolabel distribution among glutamate and its metabolic products in the superfusate was changed by stimulation. NAAG was the largest fraction, accounting for approximately 50% of the total recovered radiolabel in control conditions. The stimulated increase in radioactive NAAG in the superfusate coincided with its virtual clearance from the medial giant axon (MGA). A small, stimulation-induced increase in radiolabeled glutamate in the superfusate was detected only when a glutamate uptake inhibitor was present. The increase in [(3)H]glutamate in the superfusion solution of nerve incubated with [(3)H]NAAG was reduced when beta-NAAG, a competitive glutamate carboxypeptidase II (GCP II) inhibitor, was present.Overall, these results suggest that glutamate is metabolized to NAAG in the giant axon and its periaxonal glia and that, upon stimulation, NAAG is released from the axon and converted in part to glutamate by GCP II. A quisqualate- and beta-NAAG-sensitive GCP II activity was detected in nerve cord homogenates. These results, together with those in the accompanying paper demonstrating that NAAG can activate a glial electrophysiological response comparable to that initiated by glutamate, implicate NAAG as a probable mediator of interactions between the MGA and its periaxonal glia.

Acetylcholine and carbachol prevent muscle depolarization in denervated rat diaphragm

MUSCLE fibres of the rat diaphragm kept in a tissue culture medium became depolarized by 8–10 mV within 3 h after denervation. In the presence of carbachol (CB; 5 × 10−8 M), and acetylcholine (ACh; 5 × 10−8 M, the postdenervation depolarization was reduced. Both drugs were used in concentrations which mimicked the effect of non-quantal release of ACh. (+)Tubocurarine (TC) and ouabain did not prevent the protective action of CB, indicating that this effect is not mediated through ACh nicotinic receptors or the electrogenic Na+,K+pump. Addition of Mg2+, verapamil, diltiazem, nifedipine and Cd2+ in concentrations which block Ca2+ entry virtually inhibited the effect of both cholinomimetics. L-Nitroarginine methylester (NAME), an inhibitor of NO synthase, and haemoglobin, an extracellular scavenger of the NO radical, completely eliminated the protective effect of CB on post-denervation depolarization. The retrograde action of NO produced by cholinomimetics on nerve terminals is postulated.

N-acetylaspartylglutamate (NAAG) is the probable mediator of axon-to-glia signaling in the crayfish medial giant nerve fiber.

Glial cell hyperpolarization previously has been reported to be induced by high frequency stimulation or glutamate. We now report that it also is produced by the glutamate-containing dipeptide N-acetylaspartylglutamate (NAAG), by its non-hydrolyzable analog beta-NAAG, and by NAAG in the presence of 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), a potent inhibitor of the NAAG degradative enzyme glutamate carboxypeptidase II. The results indicate that NAAG mimics the effect of nerve fiber stimulation on the glia. Although glutamate has a similar effect, the other presumed product of NAAG hydrolysis, N-acetylaspartate, is without effect on glial cell membrane potential, as is aspartylglutamate (in the presence of 2-PMPA). The hyperpolarization induced by stimulation, glutamate, NAAG, beta-NAAG, or NAAG plus 2-PMPA is completely blocked by the Group II metabotropic glutamate receptor antagonist (S)-alpha-ethylglutamate but is not altered by antagonists of Group I or III metabotropic glutamate receptors. The N-methyl-D-aspartate receptor antagonist MK801 reduces but does not eliminate the hyperpolarization generated by glutamate, NAAG or stimulation. These results, in combination with those of the preceding paper, are consistent with the premise that NAAG could be the primary axon-to-glia signaling agent. When the unstimulated nerve fiber is treated with cysteate, a glutamate reuptake blocker, there is a small hyperpolarization of the glial cell that can be substantially reduced by pretreatment with 2-PMPA before addition of cysteate. A similar effect of cysteate is seen during a 50 Hz/5 s stimulation. From these results we suggest that glutamate derived from NAAG hydrolysis appears in the periaxonal space under the conditions of these experiments and may contribute to the glial hyperpolarization.

Effect of N‐acetylaspartylglutamate (NAAG) on non‐quantal and spontaneous quantal release of acetylcholine at the neuromuscular synapse of rat

N‐Acetylaspartylglutamate (NAAG), known to be present in rat motor neurons, may participate in neuronal modulation of non‐quantal secretion of acetylcholine (ACh) from motor nerve terminals. Non‐quantal release of ACh was estimated by the amplitude of the endplate membrane hyperpolarization (H‐effect) caused by inhibition of nicotinic receptors by (+)‐tubocurarine and acetylcholinesterase by armin (diethoxy‐p‐nitrophenyl phosphate). Application of exogenous NAAG decreased the H‐effect in a dose‐dependent manner. The reduction of the H‐effect by NAAG was completely removed when N‐acetyl‐β‐aspartylglutamate (βNAAG) or 2‐(phosphonomethyl)‐pentanedioic acid (2‐PMPA) was used to inhibit glutamate carboxypeptidase II (GCP II), a presynaptic Schwann cell membrane‐associated ectoenzyme that hydrolyzes NAAG to glutamate and N‐acetylaspartate. Bath application of glutamate decreased the H‐effect similarly to the action of NAAG but N‐acetylaspartate was without effect. Inhibition of NMDA receptors by dl‐2‐amino‐5‐phosphopentanoic acid, (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzocyclohepten‐5,10‐imine (MK801), and 7‐chlorokynurenic acid or inhibition of muscle nitric oxide synthase (NO synthase) by NG‐nitro‐l‐arginine methyl ester and 3‐bromo‐7‐nitroindazole completely prevented the decrease of the H‐effect by NAAG. These results suggest that glutamate, produced by enzymatic hydrolysis of bath‐applied NAAG, can modulate non‐quantal secretion of ACh from the presynaptic terminal of the neuromuscular synapse via activation of postsynaptic NMDA receptors and synthesis of nitric oxide (NO) in muscle fibers. NAAG also increased the frequency of miniature endplate potentials (mEPPs) generated by spontaneous quantal secretion of ACh, whereas the mean amplitude and time constants for rise time and for decay of mEPPs did not change.

Increased non-quantal release of acetylcholine after inhibition of endocytosis by methyl-β-cyclodextrin: the role of vesicular acetylcholine transporter.

We investigated the role of the vesicular acetylcholine transporter in the mechanism of non-quantal (non-vesicular) secretion of neurotransmitter in the neuromuscular synapse of the rat diaphragm muscle. Non-quantal secretion was estimated electrophysiologically by the amplitude of end-plate hyperpolarization after inhibition of cholinesterase and nicotinic receptors (H-effect) or measured by the optical detection of acetylcholine in the bathing solution. It was shown that 1 mM methyl-β-cyclodextrin (MCD) reduced both endocytosis and, to much lesser extent, exocytosis of synaptic vesicles (SV) thereby increasing non-quantal secretion of acetylcholine with a concurrent decrease in axoplasm pH. During high-frequency stimulation of the motor nerve, that substantially increases vesicles exocytosis, the non-quantal secretion was further enhanced if the endocytosis of SV was blocked by MCD. In contrast, non-quantal secretion of acetylcholine did not increase when the MCD-treated neuromuscular preparations were superfused with either vesamicol, an inhibitor of vesicular transporter of acetylcholine, or sodium propionate, which decreases intracellular pH. These results suggest that the proton-dependent, vesamicol-sensitive vesicular transporters of acetylcholine, which become inserted into the presynaptic membrane during SV exocytosis and removed during endocytotic recycling of SV, play the major role in the process of non-quantal secretion of neurotransmitter.

Regulation of glutamate carboxypeptidase II hydrolysis of N-acetylaspartylglutamate (NAAG) in crayfish nervous tissue is mediated by glial glutamate and acetylcholine receptors

Glutamate carboxypeptidase II (GCPII), a glial ectoenzyme, is responsible for N‐acetylaspartylglutamate (NAAG) hydrolysis. Its regulation in crayfish nervous tissue was investigated by examining uptake of [3H]glutamate derived from N‐acetylaspartyl‐[3H]glutamate ([3H]NAAG) to measure GCPII activity. Electrical stimulation (100 Hz, 10 min) during 30 min incubation with [3H]NAAG increased tissue [3H]glutamate tenfold. This was prevented by 2‐(phosphonomethyl)‐pentanedioic acid (2‐PMPA), a GCPII inhibitor, suggesting that stimulation increased the hydrolysis of [3H]NAAG and metabolic recycling of [3H]glutamate. Antagonists of glial group II metabotropic glutamate receptors (mGLURII), NMDA receptors and acetylcholine (ACh) receptors that mediate axon–glia signaling in crayfish nerve fibers decreased the effect of stimulation by 58–83%, suggesting that glial receptor activation leads to stimulation of GCPII activity. In combination, they reduced [3H]NAAG hydrolysis during stimulation to unstimulated control levels. Agonist stimulation of mGLURII mimicked the effect of electrical stimulation, and was prevented by antagonists of GCPII or mGLURII. Raising extracellular K+ to three times the normal level stimulated [3H]NAAG release and GCPII activity. These effects were also blocked by antagonists of GCPII and mGLURII. No receptor antagonist or agonist tested or 2‐PMPA affected uptake of [3H]glutamate. We conclude that NAAG released from stimulated nerve fibers activates its own hydrolysis via stimulation of GCPII activity mediated through glial mGLURII, NMDA and ACh receptors.

Properties of glutaminase of crayfish CNS: implications for axon–glia signaling

Glutaminase of crayfish axons is believed to participate in recycling of axon-glia signaling agent(s). We measured the activity and properties of glutaminase in crude homogenates of crayfish CNS, using ion exchange chromatography to separate radiolabeled product from substrate. Crayfish glutaminase activity is cytoplasmic and/or weakly bound to membranes and dependent on time, tissue protein, and glutamine concentration. It resembles the kidney-type phosphate-activated glutaminase of mammals in being stimulated by inorganic phosphate and alkaline pH and inhibited by the product glutamate and by the glutamine analog 6-diazo-5-oxo-L-norleucine. During incubation of crayfish CNS fibers in Na(+)-free saline containing radiolabeled glutamine, there is an increased formation of radiolabeled glutamate in axoplasm that is temporally associated with an increase in axonal pH from about 7.1 to about 8.0. Both the formation of glutamate and the change in pH are reduced by 6-diazo-5-oxo-L-norleucine. Our results suggest that crayfish glutaminase activity is regulated by cellular changes in pH and glutamate concentration. Such changes could impact availability of the axon-glia signaling agents glutamate and N-acetylaspartylglutamate.

UPTAKE AND METABOLISM OF GLUTAMATE AT NON-SYNAPTIC REGIONS OF CRAYFISH CENTRAL NERVE FIBERS: IMPLICATIONS FOR AXON-GLIA SIGNALING

In crayfish and squid giant nerve fibers, glutamate appears to be an axon-glia signaling agent. We have investigated glutamate transport and metabolism by crayfish central nerve fibers in order to identify possible mechanisms by which glutamate could subserve this non-synaptic signaling function. Accumulation of radiolabeled L-glutamate by desheathed cephalothoracic nerve bundles was temperature and Na(+) dependent, linear with time for at least 8h and saturable at about 0.5-1mM L-glutamate. Most accumulated radiotracer was associated with the periaxonal glial sheath and remained as glutamate. Compounds known to block glutamate transport in invertebrate peripheral nerves or mammalian brain slices or cell cultures were also effective on crayfish central nerve fibers. Tissue radiotracer levels were only 3% of control levels when 1mM p-chloromercuriphenylsulfonate was present, and 13%, 20%, 26%, 38% and 42% of control levels, respectively, when L-cysteate, L-cysteine sulfinate, L-aspartate, D-aspartate or DL-threo-beta-hydroxyaspartate was present. L-Glutamine, GABA, N-methyl-DL-aspartate, alpha-aminoadipate and D-glutamate were without inhibitory effect on tissue tracer accumulation. Radiolabeled D-aspartate was an equivalent non-metabolized substitute for radiolabeled L-glutamate. D-Aspartate, p-chloromercuriphenylsulfonate and GABA had comparable effects on isolated medial giant nerve fibers.These studies indicate that L-glutamate is taken up primarily by the periaxonal glia of crayfish central nerve fibers by a low-affinity, saturable, Na(+)-dependent transport system and is retained by the fibers primarily in that form. Our results suggest that the glia are not only the target of the glutamate signal released from non-synaptic regions of the crayfish medial giant axon during high-frequency stimulation, but that they are also the primary site of its inactivation.