PTK/rotein tyrosine kinase

In our experiments the tyrosine kinase inhibitors tyrphostin 23 and Tyrphostin A51 inhibited the volume-sensitive release of [3H]taurine in primary astrocyte cultures in a dose-dependent manner (Fig.1, A andB). Because there was a variation between the maximal rates of [3H]taurine release under control hyposmotic conditions in different culture preparations, in the range of 11–23%, all data were normalized to controls performed with the same cell preparation on the same day, which were very constant. Significant inhibition by Tyrphostin A51 at a concentration as low as 100 nM was observed. The maximal inhibition did not exceed 50–55% and was saturated at concentrations of 50–100 μM, with the half-maximal effect at 1.2 ± 1.3 μM (Fig. 1 B). This value is very close to the IC50 for the inhibition of EGF receptor kinase activity (IC50= 800 nM) (15). In the concentration range of 0.32–10 μM, the inhibitory potency of tyrphostin 23 was lower than that of tyrphostin A51, a finding that is consistent with the lower potency of tyrphostin 23 for inhibiting PTKs (IC50 for EGF receptor kinase = 35 μM) (4, 15). However, unlike that of tyrphostin A51, the tyrphostin 23 effect did not saturate at intermediate levels of inhibition up to a concentration of 320 μM, and computer fitting gave a maximal inhibition of ∼90% and an IC50 of 41.0 ± 39.8 μM (Fig.1 B). We think this difference in the action of these two tyrphostins might be due to nonspecific inhibition of the anion channel by high concentrations of tyrphostin 23. Indeed, tyrphostin 1, an analog of tyrphostins with minimal potency for inhibiting tyrosine kinase activity and a structure similar to that of tyrphostin 23 (4), had no effect on volume-dependent [3H]taurine release at low concentrations, whereas at 320 μM it suppressed the release by 15–20%. [1]

---

Protein tyrosyl phosphorylation is a key determinant of cell proliferation and differentiation. The aim of this study was to test the hypothesis that the signal transduction pathway(s) responsible for human bone cell proliferation may involve different groups of protein tyrosine kinase (PTKs) as compared with that for differentiation. To achieve this, we investigated the effects of two structurally different PTK inhibitors, viz, Tyrphostin A51 and genistein, on the proliferation ([3H]thymidine incorporation) and differentiation [alkaline phosphatase (ALP) specific activity and collagen synthesis] of two normal human bone cell types: mandible-derived and vertebra-derived bone cells. Tyrphostin A51 and genistein each markedly reduced cellular tyrosyl phosphorylation level (assessed by Western analysis using a commercial anti-phosphotyrosine antibody and the enhanced chemiluminescence detection assay), confirming that these two effectors are potent PTK inhibitors in human bone cells. Regarding bone cell proliferation, Tyrphostin A51 (5-30 microM) caused, a dose-dependent inhibition of basal [3H]thymidine incorporation of both human bone cell types. In contrast, genistein (5-20 microM), not only did not inhibit, but significantly stimulated [3H]thymidine incorporation of these same cell types in a dose-dependent, biphasic manner, with the optimal stimulatory dose between 10 and 20 microM. These effects on cell proliferation were confirmed by cell number counting. In addition, whereas the mitogenic activity of 10 ng/ml epidermal growth factor (EGF) on human mandible-derived bone cells was completely abolished by 5-30 microM tyrphostin A51, genistein at 5-30 microM enhanced the EGF-induced bone cell proliferation in an additive manner. With respect to bone cell differentiation, tyrphostin A51 and genistein each significantly increased basal ALP specific activity and collagen synthesis in human bone cells. In summary, (1) PTKs are involved in human bone cell proliferation and differentiation; (2) tyrphostin A51 inhibited both basal and EGF-induced cell proliferation, thus tyrphostin-sensitive PTKs are involved in basal and EGF-induced human bone cell proliferation; (3) genistein stimulated basal proliferation and enhanced EGF-mediated cell proliferation, suggesting that genistein-sensitive PTKs may play an inhibitory role in human bone cell proliferation; and (4) these differential effects of PTK inhibitors on human bone cell proliferation and differentiation are independent of basal differentiation status of the cells [2].

Efflux measurements. [1]
Amino acid efflux measurements were performed with [3H]taurine andd-[3H]aspartate, as described previously. Briefly, astrocytes grown on glass coverslips (18 × 18 mm; no. 1½; Bellco Biotechnology, Vineland, NJ) were preloaded overnight with [3H]taurine (8 μCi/ml, 264 nM) ord-[3H]aspartate (8 μCi/ml, 762 nM). The coverslips were placed in a Lucite perfusion chamber (total volume ∼100 μl) and were superfused at 1 ml/min with isosmotic or hyposmotic medium. Isosmotic medium consisted of (in mM) 122 NaCl, 3.3 KCl, 0.4 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 d-glucose, and 25 HEPES. The pH was adjusted to 7.4 by the addition of NaOH. Hyposmotic medium contained (in mM) 72 NaCl, 3.3 KCl, 0.4 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 d-glucose, and 25 HEPES (pH 7.4). The osmolarities of isotonic and hypotonic media were 285 and 190 mosM, respectively, as verified with a freezing-point osmometer. The 1-min fractions were collected and subjected to liquid scintillation counting. The rate of amino acid efflux was calculated as the fractional release (percentage of isotope remaining in the cells at the beginning of each 1-min interval) by using a custom-prepared computer program

Human Bone Cell Proliferation Assay [2]
Human bone cell proliferation was assessed by the stimulation of [ 3 H]thymidine incorporation into cell DNA as described previously. PTK inhibitors were each dissolved in DMSO. The final DMSO concentration in the assay was 0.06%. For the EGF experiment, EGF was dissolved in DMEM supplemented with 0.1% bovine serum albumin. To confirm the effects of these inhibitors on human bone cell proliferation, human mandible-derived bone cells were plated at 100 cells/mm2 in 24-well culture plates and treated with the inhibitors or solvent control as described above for 48 hours. After the incubation, cells were released from the culture well with a 5-minute trypsin treatment at 37°C. The cells were collected by centrifugation in a microfuge and resuspended in 0.1 ml DMEM containing 2% BCS. The cell number was counted with a hemocytometer in 10 separate 0.1-mm3 grids. Each treatment group had six replicates.

---

Cellular ALP-specific Activity Assay [2]
Cellular ALP-specific activity was assayed with 10 mM PNPP in 0.15 M sodium carbonate buffer (pH 10.3) in the presence of 1 mM MgCl2 [21]. One unit of ALP is the amount of enzyme required to hydrolyze 1 mmole PNPP per minute at room temperature. The specific activity was normalized against cellular protein determined according to Lowry et al.

---

Collagen Synthesis Assay [2]
Collagen synthesis was assessed by [3 H]proline incorporation into collagenase-digestible proteins. Briefly, cells were cultured in serum-free DMEM, and effectors were added for 48 hours. During the final 6 hours, [3 H]proline and b-aminoproprionitrile were added. Radioactivity in collagenase-digestible and -nondigestibleproteins was determined. Percentage of collagen synthesized was calculated after multiplying collagenase-nondigestible protein by 5.4 to correct for the relative abundance of proline in collagenasedigestible and -nondigestible proteins.

---

Determination of Cellular Steady State Phosphotyrosyl Protein Level [2]
Cellular steady state phosphotyrosyl protein level in normal human bone cells was measured as previously described. Accordingly, cells were plated at 100 cells/mm2 in 100-cm culture dishes in DMEM containing 1% BCS. After plating for 24 hours, the cells were changed to serum-free DMEM, and 30 mM of each effector or the corresponding solvent control was added an hour later, and cells were incubated at 37°C for 30 minutes. After the treatment, cell medium was removed, and the cell layer was immediately extracted with 0.3 ml of sodium dodecyl sulfate (SDS) treatment buffer [0.125 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 10% b-mercaptoethanol, 1 mM sodium orthovanadate]. The cell extracts were placed in a boiling water bath for 5 minutes and kept frozen (as aliquots) at −20°C until electrophoresis. Protein content in the extracts (after precipitation with trichloroacetic acid) was determined according to Lowry et al.

[1]. [3H]taurine andd-[3H]aspartate release from astrocyte cultures are differently regulated by tyrosine kinases. Physiology-cell physiology. 1999, 1226-1230.

[2]. Differential effects of two protein tyrosine kinase inhibitors, tyrphostin and genistein, on human bone cell proliferation as compared with differentiation. Calcif Tissue Int. 1998 Sep;63(3):243-9.

2-amino-4-(3,4,5-trihydroxyphenyl)buta-1,3-diene-1,1,3-tricarbonitrile is a benzenetriol.
Tyrphostin A51 is a kind of Protein tyrosine kinase inhibitor involved in cell proliferation and differentiation.

---

In conclusion, in primary astrocyte cultures hyposmotic medium-induced cell swelling leads to the activation of at least two anion channels permeable for amino acids with different mechanisms of volume signal transduction. One channel, which may correspond to the VSOAC/VSOR channel, is permeable to taurine (and Cl−) but not to excitatory amino acids (EAAs) and requires EGF receptor-independent, tyrphostin-sensitive tyrosine kinase activity for hyposmotic stimulation. The second channel is permeable to taurine and EAAs (and Cl−) and is not controlled by tyrosine kinases. An important implication of our study is the possibility of being able to distinguish between the volume-dependent Cl−/taurine channel and the volume-dependent channel for EAAs by using tyrphostins as a molecular tool. The former channel is required for cell volume regulation. Although the normal function of the latter channel is unclear, it can likely contribute to brain damage in the several pathologies in which astrocytic swelling occurs [1].

C13H8N4O3

268.232

268.059

C, 58.21; H, 3.01; N, 20.89; O, 17.89

126433-07-6

(Z)-Tyrphostin A51;122520-90-5

5328807

White to yellow solid powder

1.6±0.1 g/cm3

788.2±60.0 °C at 760 mmHg

430.5±32.9 °C

0.0±2.8 mmHg at 25°C

1.757

0.04

4

7

2

20

565

0

C1=C(C=C(C(=C1O)O)O)/C=C(\C#N)/C(=C(C#N)C#N)N

JKNOYWVMHPMBEL-UNXLUWIOSA-N

InChI=1S/C13H8N4O3/c14-4-8(12(17)9(5-15)6-16)1-7-2-10(18)13(20)11(19)3-7/h1-3,18-20H,17H2/b8-1+

(3Z)-2-amino-4-(3,4,5-trihydroxyphenyl)buta-1,3-diene-1,1,3-tricarbonitrile

Tyrphostin A-51; Tyrphostin A 51; tyrphostin 51; 126433-07-6; 122520-90-5; AG-183; 2-Amino-1,1,3-tricyano-4-(3',4',5'-trihydroxyphenyl)butadiene; (3Z)-2-amino-4-(3,4,5-trihydroxyphenyl)buta-1,3-diene-1,1,3-tricarbonitrile; 1,3-Butadiene-1,1,3-tricarbonitrile, 2-amino-4-(3,4,5-trihydroxyphenyl)-; Tyrphostin 51

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)