PKA

To investigate whether the stirnulatory action of H89 is mediated through an inhibitory action of PKA, we applied other types of PKA inhibitors and an inactive form of H89. H85, an inactive form of H89 (no inhibitory action on PKA), stimulated the amiloride-sensitive Isc (Fig. 3). H8, a PKA inhibitor, also stimulated the amiloride-sensitive Isc (Fig. 3). However, H8 required a higher concentration (100 j&l) to show the stimulatory action identical to that of 5 p.M H89, and 5 pM H8 had no significant action on the amiloride-sensitive Isc (Fig. 3). H7, a non-specific inhibitor of protein kinases including PKA and PKC, also required a high concentration, 100 pM, to show the PL-112 PKA-inhibition Independent Action of H89 Vol. 65, No. 10, 1999 stimulatory action on the amiloride-sensitive Isc. KT5720 (0.5 @VI), an inhibitor of PKA (14), did not stimulate the amiloride-sensitive Isc, but rather diminished it (Fig. 3). Further, another type of PICA inhibitor, Myr-PIU (3 @I) (15), also diminished the amiloride-sensitive Isc (Fig. 3). These observations suggest that all PKA inhibitors used in the present study do not have stimulatory action on the amiloride-sensitive Na+ transport, but that the stimulatory action is only seen in H-compounds. We also studied the effects of H8 and H7 on the stimulatory action of terbutaline on the amiloridesensitive Isc. Even in the presence of H8 or H7 of low concentration (5 pM) which did not stimulate the Isc, terbutaline still stimulated the amiloride-sensitive Isc; i.e., the stimulatory action of terbutaline was not blocked by H8 or H7 of a low concentration (5 pM). Although we need further experiments to confirm the signaling pathway of terbutaline and stimulatory mechanisms of H-compounds in regulation of the amiloride-sensitive Isc, we report here that H-compounds activate amiloride-sensitive Na+ channels resulting in stimulation of the amiloride-sensitive Na+ transport [1].

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Preincubation of Dunn osteosarcoma cells for 1 h with both 100 nM of staurosporine and 10 micrograms/ml of genistein resulted in a significant decrease in the motility stimulated by autocrine motility factor (AMF), whereas these reagents did not affect the basal motility and proliferation at these concentrations. The effect of the agents on the stimulated motility was both dose- and time-dependent. The motility stimulated by the anti-AMF receptor mAb was also inhibited. In contrast, H8 had a negligible effect upon the stimulated motility. These data suggest that both kinase C and tyrosine kinase play a role in AMF-stimulated cell motility, while protein kinase A, which is selectively associated with the adenylate cyclase pathway, may not be required for the stimulation [2].

Cell harvest and culture: [1]
Alveolar type II epithelial cells were isolated from the fetuses of pregnant Wistar rats (20 days’ gestational age; term = 22 days) which were completely anesthetized with inhalational ether (over dose) for 15 min. The epithelial cell was harvested from the fetuses and grown in primary culture according to the method previously described. In brief the lung fragments minced into 1 mm3 pieces obtained from fetuses were incubated at 37“C with 0.125% trypsin and 0.002% DNase and dissociated cells were then passed through a Nitex 100 mesh filter. The cells were then incubated with collagenase (0.1%) and purified by a differential adhesion technique. The majority of these cells are known to have morphologic and biochemical characteristics of alveolar type II epithelial cells. The cells were seeded at 3 x lo5 cells/well onto polycarbonate porous membranes in filter cups (Tissue culture-treated Transwell with 6.5 mm diameter) for Isc measurement or at 1 x lo6 cells/cm2 onto translucent porous Nunc filter inserts for single channel recording. All cells were grown in MEM with 10 % fetal bovine serum and penicillin-streptomycin at 37OC in a humidified 95 % air / 5 % CO:! environment. These epithelia were subsequently used 3 days after seeding under confluent conditions for short circuit current measurements or single channel recording. Cells plated on permeable supports formed polarized monolayers with their apical surfaces upward.

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Short circuit current measurement: [1]
Monolayers were transferred to a modified Ussing chamber designed to hold the filter cup. Short-circuit currents were measured with an amplifier VCC-600. A positive current represents a net flow of cation from apical to basolateral solutions. Transepithelial voltage was measured with a pair of calomel electrodes which were immersed in a saturated KC1 solution and bridged to Ussing chamber by a pair of polyethylene tubes filled with a solution of 2% agarose in 2 M KCl.

[1]. Effects of PKA inhibitors, H-compounds, on epithelial Na+ channels via PKA-independent mechanisms. Life Sci. 1999;65(10):PL109-14.

[1]. Effects of protein kinase inhibitors on the cell motility stimulated by autocrine motility factor. Biochim Biophys Acta. 1994 Jul 21;1222(3):395-9.

N-[2-(methylamino)ethyl]isoquinoline-5-sulfonamide is a member of isoquinolines and a sulfonamide.

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The Na+ transport in alveolar type II epithelial cells of rat fetal lung was stimulated by cAMP, which is generally thought to act through activation of protein kinase A (PKA). PKA inhibitors (H8, H89 and H7) stimulated amiloride-sensitive Na+ transport in the alveolar type II epithelial cells. H85, an inactive form of H89 as a PKA inhibitor, had also mimicked the stimulatory action of H89 on the Na+ transport. On the other hand, another type of PKA inhibitor, KT5720 or myristoylated PKA inhibitory peptide [14-22] amide, did not stimulate the Na+ transport, but inhibited the Na+ transport unlike H-compounds. These observations suggest that H-compounds act on the Na+ transport depending on the structure.[1]

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Taken together, these observations indicate that the stimulatory action of H-compounds on the __. . amiloride-sensitive Na+ transport is based upon their structures. Figure 4 shows the structure ot compounds used in the present study. The H-compounds (H89, H85, H8 and H7) contains 5- isoquinolinesulfone. Other PKA inhibitors, KT5720 and Myr-PKI, have completely different structures (Fig. 4). Although the power (sensitivity) of the H-compound in stimulation of the amiloride-sensitive Na+ transport depends upon the structure of side chain, the structure of 5- isoquinolinesulfone is strongly suggested to play a role in stimulation of the Na+ transport.[1]

C12H15N3O2S

265.33

265.088

84478-11-5

113276-94-1

3540

Typically exists as solid at room temperature

1.268g/cm3

473.9ºC at 760 mmHg

238-246ºC dec.

240.4ºC

1.605

2.595

2

5

5

18

353

0

CNCCNS(=O)(=O)C1=CC=CC2=C1C=CN=C2

PJWUXKNZVMEPPH-UHFFFAOYSA-N

InChI=1S/C12H15N3O2S/c1-13-7-8-15-18(16,17)12-4-2-3-10-9-14-6-5-11(10)12/h2-6,9,13,15H,7-8H2,1H3

N-[2-(methylamino)ethyl]isoquinoline-5-sulfonamide

84478-11-5; N-(2-(Methylamino)ethyl)-5-isoquinolinesulfonamide; N-[2-(methylamino)ethyl]isoquinoline-5-sulfonamide; Protein kinase inhibitor H-8; H-8 Protein kinase inhibitor; H8 protein kinase inhibitor; N-[2-(METHYLAMINO)ETHYL]-5-ISOQUINOLINESULFONAMIDE; Protein kinase inhibitor H8;

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.

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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).

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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)

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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).

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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

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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.

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