Primary Cultures of Muscle from Embryonic Locusts (Locusta Migratoria, Schistocerca Gregaria): Developmental, Electrophysiological and Patch-Clamp Studies

Primary cultures of muscle tissue from 10-day-old embryos of Locusta migratoria and 11-day-old embryos of Schistocerca gregaria have been grown and maintained in 5+4 insect medium and Grace's insect medium. Myofibres grown in 5+4 medium reached maximum size after about 5 weeks in culture and could be maintained for 2–3 months. They were often branched and rarely striated in appearance. Those rown in Grace's medium reached maximum size within 3 weeks and could be aintained for about 4 weeks in toto. They were striated and resembled, at least superficially, locust myofibres in vivo. Patch-clamp recordings from myofibre cultures grown in 5+4 medium, either on the myofibres or from excised membrane patches, indicated the presence of a diffusely-distributed population of receptors for L-glutamate. 10−6 moll−1 concanavalin A blocked the desensitization of these receptors. The glutamate receptors gated large conductance channels which had reversal potentials of about 0mV. The amplitude of the channel current was sensitive to the concentration of calcium in the membrane environments.

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Primary Cultures of Ovarian Tissue from Locusts, Locusta migratoria and Schistocerca gregaria (Orthoptera: Acrididae)

Invertebrate and Fish Tissue Culture ◽

10.1007/978-3-642-73626-1_1 ◽

1988 ◽

pp. 3-6 ◽

Cited By ~ 1

Author(s):

S. K. Raina ◽

A. M. Khurad

Keyword(s):

Schistocerca Gregaria ◽

Locusta Migratoria ◽

Ovarian Tissue ◽

Primary Cultures

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Glutamate and GABA Activate Different Receptors and Cl− Conductances in Crab Peptide-Secretory Neurons

Journal of Neurophysiology ◽

10.1152/jn.2000.83.1.31 ◽

2000 ◽

Vol 83 (1) ◽

pp. 31-37 ◽

Cited By ~ 12

Author(s):

Shumin Duan ◽

Ian M. Cooke

Keyword(s):

Patch Clamp ◽

Concanavalin A ◽

Response Curve ◽

Ibotenic Acid ◽

Aminobutyric Acid ◽

Hill Coefficient ◽

Channel Blockers ◽

Whole Cell Patch Clamp ◽

Secretory Neurons ◽

Reversal Potentials

Responses to rapid application of glutamic acid (Glu) and γ-aminobutyric acid (GABA), 0.01–3 mM, were recorded by whole-cell patch clamp of cultured crab ( Cardisoma carnifex) X-organ neurons. Responses peaked within 200 ms. Both Glu and GABA currents had reversal potentials that followed the Nernst Cl− potential when [Cl−]i was varied. A Boltzmann fit to the normalized, averaged dose-response curve for Glu indicated an EC50 of 0.15 mM and a Hill coefficient of 1.05. Rapid ( t 1/2 ∼ 1 s) desensitization occurred during Glu but not GABA application that required >2 min for recovery. Desensitization was unaffected by concanavalin A or cyclothiazide. N-methyl-D-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, quisqualate, and kainate (to 1 mM) were ineffective, nor were Glu responses influenced by glycine (1 μM) or Mg2+ (0–26 mM). Glu effects were imitated by ibotenic acid (0.1 mM). The following support the conclusion that Glu and GABA act on different receptors: 1) responses sum; 2) desensitization to Glu or ibotenic acid did not diminish GABA responses; 3) the Cl−-channel blockers picrotoxin and niflumic acid (0.5 mM) inhibited Glu responses by ∼90 and 80% but GABA responses by ∼50 and 20%; and 4) polyvinylpyrrolydone-25 (2 mM in normal crab saline) eliminated Glu responses but left GABA responses unaltered. Thus crab secretory neurons have separate receptors responsive to Glu and to GABA, both probably ionotropic, and mediating Cl− conductance increases. In its responses and pharmacology, this crustacean Glu receptor resembles Cl−-permeable Glu receptors previously described in invertebrates and differs from cation-permeable Glu receptors of vertebrates and invertebrates.

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In vivo patch-clamp analysis of the actions of 5-HT on excitatory synaptic transmission evoked in rat substantia gelatinosa neurons by cutaneous mechanical stimuli

Neuroscience Research ◽

10.1016/s0168-0102(00)81385-1 ◽

2000 ◽

Vol 38 ◽

pp. S90

Author(s):

H Furue

Keyword(s):

Patch Clamp ◽

Synaptic Transmission ◽

Excitatory Synaptic Transmission ◽

Substantia Gelatinosa ◽

Mechanical Stimuli

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Continuous Quantitative Monitoring of Mural, Platelet-Dependent, Thrombus Kinetics in the Crushed Rat Femoral Vein

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648165 ◽

1991 ◽

Vol 65 (04) ◽

pp. 425-431 ◽

Cited By ~ 29

Author(s):

F Stockmans ◽

H Deckmyn ◽

J Gruwez ◽

J Vermylen ◽

R Acland

Keyword(s):

Thrombus Formation ◽

Femoral Vein ◽

Maximum Size ◽

Digital Analysis ◽

Video Recordings ◽

Quantitative Monitoring ◽

Set Up ◽

Kinetics Of ◽

Quantitative Nature

SummaryA new in vivo method to study the size and dynamics of a growing mural thrombus was set up in the rat femoral vein. The method uses a standardized crush injury to induce a thrombus, and a newly developed transilluminator combined with digital analysis of video recordings. Thrombi in this model formed rapidly, reaching a maximum size 391 ± 35 sec following injury, after which they degraded with a half-life of 197 ± 31 sec. Histological examination indicated that the thrombi consisted mainly of platelets. The quantitative nature of the transillumination technique was demonstrated by simultaneous measurement of the incorporation of 111In labeled platelets into the thrombus. Thrombus formation, studied at 30 min interval in both femoral veins, showed satisfactory reproducibility overall and within a given animalWith this method we were able to induce a thrombus using a clinically relevant injury and to monitor continuously and reproducibly the kinetics of thrombus formation in a vessel of clinically and surgically relevant size

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Synthesis and Evaluation of Novel α-Aminoamides Containing Benzoheterocyclic Moiety for the Treatment of Pain

Molecules ◽

10.3390/molecules26061716 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1716

Author(s):

Kun Tong ◽

Ruotian Zhang ◽

Fengzhi Ren ◽

Tao Zhang ◽

Junlin He ◽

...

Keyword(s):

Ion Channels ◽

Patch Clamp ◽

Formalin Test ◽

Sodium Ion ◽

Inhibitory Potency ◽

Voltage Gated ◽

Patch Clamp Electrophysiology ◽

Sodium Ion Channels

Novel α-aminoamide derivatives containing different benzoheterocyclics moiety were synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain. Compounds 6a, 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also exhibited potent in vitro anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Nav targets, could serve as new leads for the development of analgesic medicines.

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In Vitro Culture of Human Thyroid Cells: Preliminary Findings on the Role of the Pathological Condition of the Donors

Alternatives to Laboratory Animals ◽

10.1177/026119299702500208 ◽

1997 ◽

Vol 25 (2) ◽

pp. 153-160

Author(s):

Francesca Mattioli ◽

Marianna Angiola ◽

Laura Fazzuoli ◽

Francesco Razzetta ◽

Antonietta Martelli

Keyword(s):

Pathological Condition ◽

Primary Cultures ◽

S Phase ◽

Human Thyroid ◽

Thyroid Cells ◽

Toxicological Studies ◽

Predictive Indicator

Although primary cultures of human thyroid cells are used for endocrinological and toxicological studies, until now no attention has been paid toward verifying whether the hormonal conditions to which the gland was exposed in vivo prior to surgery could influence in vitro responses. Our findings suggest that the hormonal situation in vivo cannot be used as a predictive indicator of triiodothyronine and thyroxine release and/or S-phase frequency in vitro, either with or without the addition of bovine thyrotropin.

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A capsaicinoid-based soft drug, AG1529, for attenuating TRPV1-mediated histaminergic and inflammatory sensory neuron excitability

Scientific Reports ◽

10.1038/s41598-020-80725-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Magdalena Nikolaeva-Koleva ◽

Laura Butron ◽

Sara González-Rodríguez ◽

Isabel Devesa ◽

Pierluigi Valente ◽

...

Keyword(s):

Transient Receptor Potential ◽

Primary Cultures ◽

Receptor Potential ◽

Neuronal Firing ◽

In Vivo Model ◽

Drg Neurons ◽

Trpv1 Channels ◽

Soft Drug ◽

Transient Receptor

AbstractTRPV1, a member of the transient receptor potential (TRP) family, is a nonselective calcium permeable ion channel gated by physical and chemical stimuli. In the skin, TRPV1 plays an important role in neurogenic inflammation, pain and pruritus associated to many dermatological diseases. Consequently, TRPV1 modulators could represent pharmacological tools to respond to important patient needs that still represent an unmet medical demand. Previously, we reported the design of capsaicinoid-based molecules that undergo dermal deactivation (soft drugs), thus preventing their long-term dermal accumulation. Here, we investigated the pharmacological properties of the lead antagonist, 2-((4-hydroxy-2-iodo-5-methoxybenzyl) amino)-2-oxoethyl dodecanoate (AG1529), on heterologously expressed human TRPV1 (hTRPV1), on nociceptor excitability and on an in vivo model of acute pruritus. We report that AG1529 competitively blocked capsaicin-evoked activation of hTRPV1 with micromolar potency, moderately affected pH-induced gating, and did not alter voltage- and heat-mediated responses. AG1529 displays modest receptor selectivity as it mildly blocked recombinant hTRPA1 and hTRPM8 channels. In primary cultures of rat dorsal root ganglion (DRG) neurons, AG1529 potently reduced capsaicin-evoked neuronal firing. AG1529 exhibited lower potency on pH-evoked TRPV1 firing, and TRPA1-elicited nociceptor excitability. Furthermore, AG1529 abolished histaminergic and inflammation mediated TRPV1 sensitization in primary cultures of DRG neurons. Noteworthy, dermal wiping of AG1529, either in an acetone-based formulation or in an anhydrous ointment, dose-dependently attenuated acute histaminergic itch in a rodent model. This cutaneous anti-pruritic effect was devoid of the normal nocifensive action evoked by the burning sensation of capsaicin. Taken together, these preclinical results unveil the mode of action of AG1529 on TRPV1 channels and substantiate the tenet that this capsaicinoid-based soft drug is a promising candidate for drug development as a topical anti-pruritic and anti-inflammatory medication.

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Expression of laminin γ 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter

Biochemical Journal ◽

10.1042/bj3130745 ◽

1996 ◽

Vol 313 (3) ◽

pp. 745-752 ◽

Cited By ~ 5

Author(s):

Françoise LEVAVASSEUR ◽

Jocelyne LIÉTARD ◽

Kohei OGAWA ◽

Nathalie THÉRET ◽

Peter D. BURBELO ◽

...

Keyword(s):

Transcriptional Activation ◽

Hepatoma Cell ◽

Primary Cultures ◽

Regulatory Elements ◽

Hepatoma Cells ◽

Basement Membranes ◽

Major Protein ◽

Cultured Hepatocytes

Laminin γ1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin γ1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin γ1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5´ flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin γ1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a Mr 60000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

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